ISSN: Volume 11 Number 1 January March 2016 CENTRAL COUNCIL FOR RESEARCH IN UNANI MEDICINE

Transcription

1 ISSN: Volume 11 Number 1 January March 2016 CENTRAL COUNCIL FOR RESEARCH IN UNANI MEDICINE

2 HIPPOCRATIC JOURNAL OF UNANI MEDICINE Volume 11, Number 1, January - March 2016 Hippocratic J. Unani Med. 11(1): 1-172, 2016 CENTRAL COUNCIL FOR RESEARCH IN UNANI MEDICINE Ministry of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy (AYUSH) Government of India

3 Chief Patron Minister of AYUSH, Government of India Patron Secretary, Ministry of AYUSH, Government of India International Advisory Board Prof. G.N. Qazi, New Delhi, INDIA Prof. Ranjit Roy Chaudhury, Delhi, INDIA Dr. Fabrizio Speziale, Paris, France Mrs. Sadia Rashid, Karachi, PAKISTAN Prof. Ikhlas A. Khan, USA Prof. Abdul Hannan, Karachi, PAKISTAN Prof. Rashid Bhikha, Industria, SOUTH AFRICA Prof. Ram Vishwakarma, Jammu, INDIA Prof. Talat Ahmad, New Delhi, INDIA Hakim Syed Khaleefathullah, Chennai, INDIA Dr. Suraiya H. Hussein, Kuala Lumpur, MALAYSIA Prof. Allauddin Ahmad, Patna, INDIA Dr. Maarten Bode, Amsterdam, THE NETHERLANDS Prof. Usmanghani Khan, Karachi, PAKISTAN Dr. S.S. Handa, Gurgaon, Haryana, INDIA Prof. Irfan Ali Khan, Hyderabad, INDIA Editorial Board Prof. Wazahat Husain, Aligarh Dr. (Mrs.) Nandini Kumar, New Delhi Dr. O.P. Agarawal, New Delhi Prof. Y.K. Gupta, New Delhi Prof. A. Ray, Delhi Dr. S. Asad Pasha, New Delhi Prof. S. Shakir Jamil, New Delhi Prof. Mansoor Ahmad Siddiqui, Bengaluru Prof. V.H. Talib, Dehradun Prof. K.M.Y. Amin, Aligarh Dr. A.B. Khan, Aligarh Dr. (Mrs.) Neena Khanna, New Delhi Dr. (Mrs.) Yasmeen Shamsi, New Delhi Dr. Mohammad Khalid Siddiqui, Faridabad Dr. Ghufran Ahmad, Aligarh Dr. M.A. Waheed, Hyderabad Editor-in-Chief Prof. Rais-ur-Rahman Director General Central Council for Research in Unani Medicine (CCRUM) Associate Editor Dr. Khalid M. Siddiqui, Deputy Director General, CCRUM Assistant Editors Dr. Wasim Ahmed Azmi, Deputy Director, CRIUM, Lucknow Dr. Shariq Ali Khan, Deputy Director, RRIUM, Aligarh Mr. Shamsul Arfin, Research Officer (Chemistry), CCRUM Dr. Munawwar Hussain Kazmi, Deputy Director, CRIUM, Hyderabad Mr. Aminuddin, Research Officer (Botany), CCRUM Mr. Mohammad Niyaz Ahmad, Research Officer (Publication), CCRUM Managing Editor Dr. V.K. Singh, Consultant (Botany), CCRUM Editorial Office CENTRAL COUNCIL FOR RESEARCH IN UNANI MEDICINE Ministry of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy (AYUSH), Government of India 61-65, Institutional Area, Janakpuri, New Delhi , India Telephone: , , , /52/62/83/97, , Fax: Website: Annual Subscription: ` 300/- (India) US $ 100/- (Other Countries) Single Issue: ` 150/- (India) US$ 50/- (Other Countries) Payments in respect of subscription may be sent by bank draft marked payable to Director General, CCRUM, New Delhi. On behalf of Central Council for Research in Unani Medicine (CCRUM) published and printed by Prof. Rais-ur-Rahman Director General, CCRUM at CCRUM headquarters, Institutional Area (Opposite D Block), Janakpuri, New Delhi and printed at Rakmo Press Pvt. Ltd., C-59; Okhla Industrial Area (Phase I), New Delhi

4 Contents 1. Anxiety Assessment in Individuals of Bilious and Phlegmatic Temperament... 1 Sartaj Ahmad, Ferasat Ali and Akbar Husain 2. Antibacterial Potential of Fruits of Bakayin (Melia azedarach Linn.): A Unani Drug of Repute Sumbul Rehman and Abdul Latif 3. Combating Trichomoniasis Through a Unani Drug Marham Dakhliyun Fahmeeda Zeenat and Azhar Hasan 4. Therapeutic Efficacy of a Dietary Formulation in Chronic Tonsillitis: A Case Study Ferasat Ali 5. Safety Study of Majoon-e-Gul A Unani Pharmacopoieal Compound Formulation Fozia Yaqub and Naeem Ahmad Khan 6. Scientific Validation of Unani Formulation: Majoon-e-Hamal Ambari Alwi Khani Rampratap Meena, S.C. Verma, S. Mageswari, P. Meera Devi Sri, A.S. Khan, S.A. Ansari, Shamsul Arfin and Aminuddin 7. Standardization and Evaluation of Antibacterial Activity of Some Unani Drugs Tariq Rahim Malik, Tajuddin and Aziz ur Rahman 8. Pharmaco-botanical Studies on Some Powdered Herbal Drugs for Their Diagnostic Characterization-III Nitin Rai and Rajeev Kr. Sharma 9. Standardization and HPTLC Fingerprinting of a Unani Compound Formulation Qurs-e-Luboob with Modern Techniques N.M.A. Rasheed, Rakesh Kumar Negi, Juveria Mahmood, Aslam Siddiqui, M.H. Kazmi, Shamsul Arfin, Aminuddin and Javed Inam Siddiqui 10. Traditional Phytotherapy of Jajpur Forests of Eastern Ghat, Odisha Usha Devi, Himanshu Dwivedi, Aminuddin, Mohammad Zakir and Hakimudin Khan 11. Preparation and Physico-chemical Evaluation of an Anti-diarrhoeal Safoof Ubaidul Hai, Shariq Shamsi, Roohi Zaman and Peerzada Mohd Younis 12. Standardization of a Unani Drug Habb-e-Banafsha Asma Sattar Khan, R.P. Meena, Shoeb A. Ansari, Afzal Hashmi, Shams-ul-Arfin and Aminuddin 13. Standardization and Phyto-chemical Screening of a Unani Drug Berg-e-Sem (Dolichos lablab L.) with Modern Analytical Techniques N.M.A. Rasheed, Rakesh Kumar Negi, Juveria Mahmood, Aslam Siddiqui, M.H. Kazmi, S. Arfin, Aminuddin and Javed Inam Siddiqui 14. Pharmacognostical Standardization of Mushkdana seed (Abelmoschus moschatus Medic) Kiran Negi, Akhlaq Mustafa, Mahesh Chandra and S.M. Asim Instructions to Contributors

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6 Editorial Currently there is a global interest in the study and use of medicinal plants because such investigations provide important new leads on novel, active molecules of therapeutic importance. And, with the world-wide growing demand of traditional drugs, issues of their quality, safety and efficacy have received renewed attention of scientists. All these ongoing investigations in India and abroad have generated lot of new research data in recent times and there is an enormous need for exchange of this vital information amongst academicians and researchers engaged in the scientific validation of traditional drugs, particularly the Unani medicine. In this context, Central Council for Research in Unani Medicine, through its clinical, drug research, literary research, survey & cultivation of medicinal plants programme is contributing significantly for over three decades. Vitiligo, sinusitis, filariasis, eczema, malaria, infective hepatitis, asthma are some of the conditions where Unani therapies have earned recognition. The Council has been publishing the peer reviewed Hippocratic Journal of Unani Medicine (HJUM), mainly to bring out fundamental and applied aspects of Unani Medicine. The journal also publishes recent advances in other related sciences and traditional medicines as well as different streams of medical sciences, which have bearing on validation and scientific interpretation of various concepts and strengths of Unani medicine. In view of an overwhelming response, the journal earlier published twice a year, its periodicity had been changed to quarterly w.e.f. January 2008 to accommodate more articles for quick dissemination of research data among scientific community. The journal has sufficient room for invited articles from luminaries of modern medicine and sciences as well as scholars of Unani medicine. The broad areas being covered include clinical research on single and compound Unani drugs, validation of regimental therapy, experimental pharmacological studies, standardization of single and compound drugs, development of standard operating procedures, ethnobotanical studies, experimental studies on medicinal plants and development of agro-techniques thereof, and literary research on classics of Unani medicine. The journal is also open for studies on safety evaluation of Unani and other herbo-mineral drugs, nutraceuticals, cosmotherapeutics, aromatics, oral health, life style disorders, sports medicine etc. and such other newer areas which are the outcome of modern day living. The current issue of this journal provides 14 original and review papers in the areas of Clinical research, Experimental pharmacology, Drug standardization, Pharmacy, Ethnopharmacology and allied disciplines contributed by eminent scholars in their respective fields. It is hoped that data presented will contribute significantly in R&D sector of traditional drugs and prove to be an excellent exposition of current research efforts of scientists in this direction. Council acknowledges the authors for their contributions included in this issue and hope for their continued support in this endeavor. We wish to ensure the readers to bring out the future issues of the journal on time. We at the CCRUM have been constantly striving to reach to higher standards and make HJUM the leading journal of Unani medicine and related sciences. In this context, we thank our learned reviewers for their invaluable inputs in improving the manuscripts. We sincerely hope and trust that the mission can be accomplished with active partnership of quality-conscious individuals and institutions. Through these lines we seek your cooperation and support in materializing our dreams about the HJUM. In this regard, we request you for your as well as your colleagues contributions for publication in and subscription to the journal. Further, we will appreciate if the journal is introduced far and wide. We would also welcome esteemed suggestions for achieving the highest standards of quality for the journal. February 28, 2016 (Prof. Rais-ur-Rahman) Editor-in-Chief

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8 Anxiety Assessment in Individuals of Bilious and Phlegmatic Temperament 1 *Sartaj Ahmad, 1 Ferasat Ali and 2 Akbar Husain 1 Department of Kulliyat, Faculty of Unani Medicine, 2 Department of Psychology, Faculty of Social Sciences, Aligarh Muslim University, Aligarh AAbstract nxiety is a physiological state characterized by somatic, cognitive, emotional and behavioral components. As per the literature of Unani Medicine, anxiety belongs to infia lat-e-nafsaniyah which is a part of ajnas-eashrah (ten parameters) for the assessment of mizaj (temperament) of an individual. According to ancient physicians, infia lat-e-nafsaniyah are higher in the persons of bilious temperament than the persons of phlegmatic temperament. The present study was designed to assess the level of anxiety during normal and stressful conditions in persons of bilious and phlegmatic temperament by using Anxiety State Test (AST). Volunteers were selected randomly from the student fraternity of Aligarh Muslim University and Aligarh city. 100 male and female volunteers of years of age of both the temperaments were selected for the study and the AST score was determined. In normal condition the mean AST score was found to be ± and ± in bilious and phlegmatic subjects respectively. During stressful condition, the mean AST score was recorded as 44.40±7.149 and 35.64±3.729 of bilious and phlegmatic subjects, respectively. A significant difference between the two groups in both the conditions was recorded. The individuals of bilious temperament have higher anxiety than their phlegmatic counterparts both in normal as well as stressful conditions. Keywords: Anxiety, Infia lat-e-nafsaniyah, Ajnas-e-ashrah, Mizaj, Temperament, Bilious, Phlegmatic Introduction Anxiety is a normal phenomenon which is characterized by a state of apprehension or unease arising out of anticipation of danger. It is an alerting signal that warns of impending danger and enables the person to take measures to deal with a threat. Normal anxiety becomes pathological when it causes significant subjective distress and/or impairment in functioning of an individual. Basically anxiety is of two types i.e. trait anxiety and state anxiety. Trait anxiety is related with the personality of an individual while the state anxiety is related with a specific state or condition (Ahuja, 2006). The concept of mizaj (temperament) is a pillar of Unani Medicine on which the states of health and disease of human being and the entire Unani therapeutics including diagnosis, prevention and treatment of diseases are based. According to Unani system of medicine, there are four types of temperament viz. safravi 1 *Author for correspondence Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages

9 (bilious), damvi (samguine), balghami (phlegmatic) and saudavi (melancholic) indicating the dominance of safra (bile), dam (blood), balgham (phlegm) and sauda (black bile), respectively (Majoosi, 1902). For the diagnosis of the temperament of an individual, a number of parameters have been devised by different Unani physicians. However, Ajnas-e-Ashrah (ten parameters) suggested by Ibn Sina (1993) to diagnose the temperament of an individual are widely accepted. It includes psychological (infia lat-e-nafsaniyah), morphological and physiological parameters. Unani physicians have been applying the theory of mizaj in their practice with a fair degree of success since hundreds of years. They did their best despite having limited instrumental and technical facilities, to imply the theory of temperament in clinical and therapeutic practices but at present an evidence based data is required to prove its validity and relevance (Ahmad, 1980). The present study was conducted on two groups of individuals having two different temperaments i.e. Bilious (Safravi) and Phlegmatic (Balghami). The two temperaments show different types of signs and symptoms as their physiological, physical, and psychological features are different from each other because of having har (hot) and barid (cold) temperament, respectively. The study was started with following objectives: 1. To find the correlation between anxiety and temperament, if any. 2. To find out whether the level of anxiety of bilious and phlegmatic subjects is same or there exists some variation within the physiological limits. 3. To institute anxiety as a parameter of temperament assessment. In view of the importance of psychological parameters in the diagnosis of temperament of an individual and also in view of the variation of the characteristics of individual of Safravi (Bilious) and Balghami (Phlegmatic) temperament, it was hypothesized that anxiety will be comparatively higher in the persons of bilious temperament than the persons of phlegmatic temperament because anxiety has a direct relationship with infia lat-e-nafsaniyah which has been described to be higher in the individuals of bilious temperament as compared to those having phlegmatic temperament (Majoosi, 1902; Masihi, 1963; Ibn Sina, 1993; Ferasat, 2005). The participants were categorized on the basis of temperament which was determined on the basis of ajnase ashra and then the level of anxiety was measured to know the difference between the two groups. The level of anxiety was also determined in male and fenale volunteers also. Material and Methods This study was carried out in the Department of Kulliyat, Ajmal Khan Tibbiya College, Aligarh Muslim University, Aligarh. Hippocratic Journal of Unani Medicine 2

10 Selection of volunteers Volunteers were selected randomly from different batches of the students of Ajmal Khan Tibbiya College and from few other faculties of Aligarh Muslim University and other colleges of Aligarh city. Sample One hundred (100) healthy male and female volunteers having Bilious and Phlegmatic temperaments were randomly selected. Inclusion criteria Only healthy volunteers of Bilious and Phlegmatic temperaments of both the sexes in the age group of years were included in the study. The reason for selecting this age group is that this age lies in Sin-e-Namu (growing age) and Sin-e-Shabab (adulthood) and the individuals in this age group mostly remain healthy and fit. Exclusion criteria The volunteers having Saudavi (Melancholic) and Damvi (Sanguine) temperament were excluded from the study. Volunteers below 18 years and above 35 years of age. Volunteers suffering from any physical, mental or psychiatric disorder. For the selection of healthy volunteers, detailed clinical history, physical, general and local examinations were done. Informed consent An informed consent form was provided to the volunteers during study prior to starting the measurement. The purpose of the informed consent form was to obtain permission from each of the volunteers for their willingness to take part in this study. The form indicated exactly what the study demands, what the volunteers expect from the study, the minimal risks and benefits of their participation, and guarantees of confidentiality. It had also stated the volunteer is free to withdraw from the study at any time without giving any specific reason. Determination of temperament The assessment of temperament (mizaj) of the volunteers was made on the basis of Ajnas-e-Ashra (ten determinants), mentioned in classical Unani literature. The proforma of the temperament was given in tabulated form to the volunteers (Mizaj Assessment Proforma). Hippocratic Journal of Unani Medicine 3

11 Categorization of the participants After determination of the temperament, the volunteers selected for study were divided into two groups according to their temperament. Group A: Bilious temperament (Safravi Mizaj) Group B: Phlegmatic temperament (Balghami Mizaj) After categorization of volunteers, the anxiety level and various physiological parameters were determined during two conditions i.e. (i) when the volunteers had no sessional test or examination (normal condition) and (ii) when they had sessional test or the examination (stressful condition). Assessment of anxiety For the assessment of anxiety the Anxiety State Test (AST) which is actually a part of Sensation Seeking and Anxiety State Test (SSAST) developed by Neary and Zuckerman (1976) was used. The tool consists of 36 statements in which 15 items were regarding Sensation Seeking, and 15 items for Anxiety. The remaining 6 items were the items from anxiety scale that did not meet the factor analysis criteria. For Anxiety State Test (AST), the scoring varies from 1 (not at all) to 5 (very much) for each item (range= 15 to 75). The item number 5, 9, 14, and 25 are scoring reversed, i.e. 1 (very much) to 5 (not at all). Neary (1975) has shown high internal consistency and low retest reliabilities of the SSAST (Anxiety State Test). Observations and Results Out of 100 volunteers, 50 were bilious (safrawi mizaj) and remaining 50 were of phlegmatic temperament (balghami mizaj). There was equal distribution of male and female volunteers in each group. During the normal condition the mean AST score of the individuals of bilious temperament was found to be 28.24± while that of the persons of phlegmatic temperament was 25.30± During stressful condition the mean AST score of the persons of bilious temperament was recorded to be 44.40±7.149 while that of the phlegmatic volunteers it was 35.64±3.729, indicating that the persons of bilious temperament have comparatively more anxiety than the persons of phlegmatic temperament in both normal as well as stressful condition (Table 1, Fig. 1). During normal condition, the mean of AST score of males of bilious temperament was found to be 26.92±3.402, while that of females of the same temperament it was recorded to be 29.56± During normal condition the mean of AST score Hippocratic Journal of Unani Medicine 4

12 Table 1: AST Score in Volunteers of Bilious and Phlegmatic Temperament (Mean ±SD) Temperament No. of Volunteers Mean ± SD Normal Condition Stressful Condition Bilious ± ± Phlegmatic ± ± Total ± ± Fig. 1: AST Score in Volunteers of Bilious and Phlegmatic Temperament of males of phlegmatic temperament was 24.44±2.770 which increased to 26.16±2.853 in female candidates (Table 2, Fig. 2). Table 2: AST Score in Two Genders of Bilious and Phlegmatic Temperament Temperament No. of No. of Mean ± SD of Mean ± SD of Male Females Males Females Volunteers Volunteers Normal Stressful Normal Stressful Condition Condition Condition Condition Bilious ± ± ± ± Phlegmatic ± ± ± ± Total ± ± ± ± Hippocratic Journal of Unani Medicine 5

13 Fig. 2: AST Score in two Genders of Bilious and Phlegmatic Temperament Expressed as Mean During stressful condition, the mean of AST score of males of bilious temperament was 40.92±3.785, while that of females it increased to 47.88± During stressful condition the mean of AST score of males of phlegmatic temperament was 35.20±3.884 while that of females of the same temperament it was found to be 36.08±3.593 (Table 3, Fig. 3). Statistical significance The unpaired t-test was applied to determine the significance of the findings, if any. A significant difference in AST score of the individuals of bilious and phlegmatic temperament was found during normal as well as stressful condition. It was also found that the findings in respect of the volunteers of two temperaments were significantly different in normal and stressful condition (Table 3). Table 3: Significance of AST Score in Volunteers of Bilious and Phlegmatic Temperament AST Score between Significance Bilious vs Phlegmatic during normal condition t = 4.202, p < 0.01 Bilious vs Phlegmatic during stressful condition t = 7.682, p < 0.01 Bilious in normal condition vs Bilious in stressful condition t = , p < 0.01 Phlegmatic in normal condition vs Phlegmatic in t = , p < 0.01 stressful condition Hippocratic Journal of Unani Medicine 6

14 During normal condition the AST score between the males and females of bilious temperament and also between the males and females of phlegmatic temperament was significant indicating that females have higher AST score. It also revealed that during normal condition the AST score between males of bilious and phlegmatic temperament and also between the females of bilious and phlegmatic temperament is significant (Table 4). Table 4: AST Score in Two Genders of Bilious and Phlegmatic Temperament During Normal Condition AST Score between Significance Bilious Male vs Bilious Female t = 2.451, p= Phlegmatic Male vs Phlegmatic Female t = 2.163, p= Bilious Male vs Phlegmatic Male t = 2.826, p= Bilious Female vs Phlegmatic Female t = 3.362, p = During stressful condition the AST score between the males and females of bilious temperament was found significant while between male and female volunteers of phlegmatic temperament it was not found to be significant. A significant difference was also recorded in the AST score between males of bilious and phlegmatic temperament and also between the females of bilious and phlegmatic temperament (Table 5). Table 5: AST Score in Two Genders of Bilious and Phlegmatic Temperament During Stressful Condition AST Score between Significance Bilious Male vs Bilious Female t = 3.913, p= Phlegmatic Male vs Phlegmatic Female t = , p= Bilious Male vs Phlegmatic Male t = 5.274, p< Bilious Female vs Phlegmatic Female t = 6.694, p< Hippocratic Journal of Unani Medicine 7

15 Mizaj Assessment Proforma Name of Volunteer: Father s Name: Age: Gender: Height: Weight: Occupation: Mobile No.: Address: Parameter Damwi Balghami Safrawi Saudawi (Evidence) (Sanguineous) (Phlegmatic) (Bilious) (Melancholic) Morphological 1. Skin texture/ Warm and Soft and Hard and Rough and Temperature smooth moist hot cold Score: Complexion Reddish Whitish Pale Blackish Score: Body built Muscular Fatty Moderate Lean and thin Score:5 4. Texture of Thick and Thin and Curly Straight hairs lusty smooth Score: Growth and Rapid, Slow, Moderate/ Excessive distribution Average Scanty Profuse of hairs Score: Colour of Blackish Brownish Yellow-black Black and hairs (Golden) white (Mixed) Score:0.5 Physiological 7. Urine Moderate in White, more Yellow, less Turbid, less Score:01 quantity in quantity in quantity in quantity 8. Tolerate Well Dryness Summer Cold Dampness Score:01 9. Remains Spring Summer Winter Autumn well in Score: Appetite Strong appetite Less appetite Strong appetite Irregular Score:01 (can skip a (feel heaviness (can t skip appetite meal) after eating) a meal) Hippocratic Journal of Unani Medicine 8

16 Parameter Damwi Balghami Safrawi Saudawi (Evidence) (Sanguineous) (Phlegmatic) (Bilious) (Melancholic) 11. Thirst Average Poor Increased Low Score:01 (+++) (+) (++++) (++) 12. Digestion Average Slow Strong Irregular Score: Movements Average in Dull Brisk, Less and activities physical activity laziness Hyper active Score: Sleep Average Excess sleep Disturbed sleep Insomnia Score:01 Psychological 15. Dream Blood, Water, Fire, Yellow Black, Fearful Score:01 Red objects Snow objects dreams 16. Anger/Joy Comes on Comes on Frequent, Severe Infrequent Score:01 easily and hardly and persists but persist easily lost for long 17. Responsee Aggressively Weakly Bravely Cowardly to external respond respond respond respond stimuli in adverse condition Score: Decision Take Hesitate in Take Afraid in taking power boldly taking quickly taking Score:01 decisions decisions 19. Memory Good Not Good Don t learn Score:01 retention good but can t easily but also good retain for excellent long retention Total Collection : Damvi : Balghami : Safravi : Saudavi : Diagnosed Temperament : Hippocratic Journal of Unani Medicine 9

17 10 Anxiety State Test (AST) S.1- I feel afraid. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.2- I feel secure. (1) Very much (2) Definitely (3) Somewhat (4) Slightly (5) Not at all S.3- I feel desperate. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.4- I feel steady. (1) Very much (2) Definitely (3) Somewhat (4) Slightly (5) Not at all S.5- I feel upset. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.6- I feel contented. (1) Very much (2) Definitely (3) Somewhat (4) Slightly (5) Not at all S.7- I feel nervous. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.8- I feel frightened. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.9- I feel tense. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.10- I feel shaky. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.11- I feel calm. (1) Very much (2) Definitely (3) Somewhat (4) Slightly (5) Not at all S.12- I feel fearful. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.13- I f terrified. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.14- I feel panicky. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much S.15- I feel worried. (1) Not at all (2) Slightly (3) Somewhat (4) Definitely (5) Very much AST Score:.

18 11 Discussion AST score was recorded in the volunteers having bilious and phlegmatic temperament during normal and stressful condition. It was found that the mean AST score during normal condition was higher in bilious group (28.24±3.998) than phlegmatic group (25.30 ± 2.915). Similarly the mean AST score during stressful condition was higher in bilious temperament ±7.149 than phlegmatic temperament35.64 ± The results of comparison of AST score between the persons of bilious and phlegmatic temperament during normal as well as during stressful condition were extremely significant.the findings also showed that AST score of bilious subjects during stressful condition was higher as it amounted to ± from the normal value of ± The mean AST score of phlegmatic temperament during stressful condition was more (35.64 ± 3.729) than the normal condition (25.30 ± 2.915). The AST score between the stressful and normal conditions of the persons of both bilious and phlegmatic temperament were found to be significant statistically. AST score was also recorded according to sex of the volunteers during both normal as well as stressful condition. In this regard our analysis showed that mean of AST score during normal condition was lower in bilious males (26.92±3.402) than bilious females (29.56±4.174). A comparison of AST score between the males and females of bilious temperament was found significant during the normal condition. The AST score during stressful condition was also found lower in bilious males (40.92±3.785) than bilious females (47.88±8.048). During the stress the AST score of the two genders of bilious temperament was found significantly different and was found increased in female volunteers. The AST score during normal condition was lower in phlegmatic males than phlegmatic females. However, during stressful condition the AST score of two genders of phlegmatic temperament was found non-significant. The findings further suggested that the AST score of the same gender of opposite temperaments during normal condition was higher in bilious males than the phlegmatic males. Almost similar pattern was observed in case of the female volunteers indicating that individuals of bilious temperament and the females are more susceptible to be afflicted with anxiety. The findings of the study clearly indicated that persons with bilious temperament have a larger tendency to get affected with anxiety than their phlegmatic counterparts in normal conditions. However, during the stressful condition both have almost equal chance of being afflicted with anxiety. Similarly the blious females have more anxiety than their male counterparts during both normal and stressful conditions. The level of anxiety in phlegmatic females was comparatively higher than the phlegmatic males during normal condition but there was no significant difference in the level of anxiety between phlegmatic females and

19 12 males during stressful condition. The findings as a whole suggested that biliousness may induce both trait anxiety and state anxiety (Ahuja, 2006) while the phlegmatic temperament do not have the direct relationship with anxiety. Since anxiety is related with infia lat-e-nafsaniyah which is found in higher concentration in bilious individual, therefore, the chances of anxiety are high. This study also indicated that anxiety may also be included as an important parameter for the assessment of temperament along with Ajnase Ashra. Conclusion It can be concluded that the level of anxiety in bilious males is comparatively higher that phlegmatic males and that the bilious females have more chances to be afflicted with anxiety than the phlegmatic females during both normal and stressful conditions. The study validated the Unani concept that biliousness is directly associated with anxiety. References Ahmad, S.I Introduction to Al Umoor Al Tabi yah, 1 st edn. Saini Printers, New Delhi, pp Ahuja, N A Short Textbook of Psychiatry. Jaypee Brothers, New Delhi, pp Ali, Ferasat, Mizaj-e-Insani Aqleemi Tanazur Main. Universal Book House, Aligarh, pp. 21, 25, 52. Galen, Kitab Fil Anasir (Edited and Translated by Zillur Rehman). International Printing Press, Aligarh, pp Ibn Sina, Al Qanoon fit Tib, Book-I. English Translation of the Critical Arabic Text). Jamia Hamdard, New Delhi, pp. 7-13, 65, Majoosi, A.A., Kamil Al Sana, Part- 1. (Urdu translation by Ghulam Husain Kantoori), Matba Munshi Nawal Kishore, Lucknow, pp. 17, 23, 102. Masihi Abu Sehal, Kitab-al-Meyat Fil Tibb, Part- I. Islamic Publication Society, Hyderabad, pp. 82, 93. Neary, As cited in Beharioural expression and Biosocial Bases of Sensation Seeking by Marvin Zukerman, Syndicate Press of the University of Cambridge, p. 49. Neary, Zukerman, Sensation Seeking, Trait and State Anxiety and the Electrodermal Orienting Response. Journal of Psychophysiology 13 (3):

20 Antibacterial Potential of Fruits of Bakayin (Melia azedarach Linn.): A Unani Drug of Repute *Sumbul Rehman and Abdul Latif Department of Ilmul Advia (Unani Pharmacology & Pharmaceutical Sciences), A.K. Tibbiya College, Aligarh Muslim University, Aligarh Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages BAbstract akayin (Melia azedarach L.; Family Meliaceae) has been used in Traditional Systems of Medicine since long time to treat many diseases including infectious diseases. However, information to revalidate its use on scientific parameters is limited. Therefore, present study has been done to screen antibacterial activity of ethanolic extracts of fruits of Bakayin against pathological bacterial strains using disk diffusion method and its efficacy was compared with the standard drug Ciprofloxacin for gram positive and Gentamycin for gram negative bacterial strains. Qualitative analysis of the extracts was also done to confirm the presence of various phytoactive constituents. It was found that Bakayin contains alkaloids, glycosides, flavonoids, carbohydrates, tannins, starch, saponin, resins and terpenes. This study scientifically validates the antibacterial potential of Bakayin and reconfirms the claims of Unani physicians for its use in various infectious diseases as leprosy, scrofula, scabies, diarrhoea, dysentery, typhoid, suppurating wounds etc. The findings can usefully be applied by researchers elsewhere for comparison of its bioactive constituents, and this might be a helpful lead in the rational use of Bakayin. 13 Keywords: Infectious diseases, Bakayin (Melia azedarach), Antibacterial screening Introduction Unani system of medicine provides a large number of drugs of natural origin which can be used today to combat many dreadful diseases which are difficult to be treated with the western medicine along with the emerging health hazards nowadays. Ethno-botanical, medical and phytochemical literature available in our classical books has abundant remedies from natural resources as mentioned by eminent Unani physicians that need to be re-explored now for different health hazards. Unani medicine claims to possess a large number of effective and safe drugs to combat infectious diseases that are in use since centuries (Rehman and Latif, 2015). Bacterial strains have developed resistance to almost all the antibiotics, further some antibiotics have serious undesirable effects (Rehman et al., 2011). This is an alarming situation and calls for serious consideration including drugs available in indigenous system of medicines. There appears no effort made to investigate the antimicrobial potential of bakayin fruits through in-vitro testing. Present study is based on this rationale and presents results of phyto-chemical and antibacterial *Author for correspondence; dr.sumbulrehman@gmail.com

21 14 screening of this important Unani drug in an effort to revalidate its medical efficacy to combat infectious diseases. Bakayin (Melia azedarach Linn.) is a medium sized deciduous tree; flowers liliacpurple; fruit drupe 4-seeded, yellowish with very hard endocarp. Wild in the sub- Himalayan tract at m; cultivated and naturalized throughout India upto 1800 m; grows as a hedge plant also; known as Pride of India, Chinaberry, Persian liliac, Umbrella tree, Bead tree. Most of the parts of Bakayin have been used in various diseases as root bark, fruit or berry, seeds, flowers, leaves oils and gum. Root decoction or fluid extract used as anthelmintic while root bark is used as vermifuge, in intermittent fevers and dysentery, as a cathartic and emetic. In India, before quinine, its root bark was also used in malaria as infusion of bark used as febrifuge especially for periodic fevers; also, for thirst and nausea. Poultice of bark is used in leprosy and scrofulous ulcers (Pal and Bose, 2011; Chopra et al., 1958; Panda et al., 2008; Sindhia and Bairwa, 2010; Nadkarni, 2002; Kiritikar and Basu, 1935). Material and Methods The study was undertaken in the department of Ilmul Advia, A.K. Tibbiya College, Aligarh, during Plant Material The berries of Bakayin were collected from the campus of Ajmal Khan Tibbiya College, Aligarh Muslim University, Aligarh, and was identified by the available literature. Preparation of Berry Extracts The test drug was dried at room temperature in a ventilated room, milled to fine powder and stored in a close air tight container in dark until use. Extraction was done according to the method described by Afaq et al. (1994) and Peach and Tracey (1955) with some minor modifications, keeping in mind that the thermo labile elements present in the drug are destroyed when exposed to a higher temperature beyond 55 C, so the heat wherever needed was kept as low as possible to prevent the loss of thermo-labile substances present in the drugs from destruction. The coarse drug material was extracted with 95% ethanol as a solvent at 50 C for 6 hours and dried under reduced pressure in the Lypholizer. Strict aseptic precautions were followed throughout the process. The stock solutions for extract were prepared from the dried extract so obtained in the Dimethyl Sulphoxide (DMSO) as a solvent for use. The respective stock solutions so prepared were refrigerated till further use.

22 15 Phytochemical Analysis Phytochemical studies of the plant preparations are necessary for standardization, which helps in understanding the significance of phytoconstituents in terms of their observed activities. Phytochemistry also helps in standardizing the herbal preparations so as to get the optimal concentrations of known active constituents, and in preserving their activities. Qualitative analysis of the chemical constituents present in the drug sample. Standard tests and methodology has been followed to confirm the presence of chemical constituents (Bhattacharjee and Das, 2005 and Afaq et al., 1994) (Table-1). Table 1: Qualitative Analysis of the Phytochemicals S.No. Chemical Test Reagents Inference Constituents Bakain (M.azedarach Linn.) 1. Alkaloids Dragendorff s reagent + Wagner s reagent + Mayer s reagent + 2. Carbohydrates Molish Test + Fehling Test + Benedict Test + 3. Flavonoids Mg Ribbon and dil. Hcl _ 4. Glycosides NaOH Test + 5. Tannins/Phenols Ferric Chloride Test _ Liebermann s test + Lead Acetate test + 6. Proteins Xanthoproteic test _ Biuret test + 7. Starch Iodine Test _ 8. Saponins Frothing with NaHCO Steroids/Terpenes Salkowski Reaction _ 10. Resins Acetic anhydride test + Indications: - Absence and + Presence of constituents

23 16 Antibacterial Susceptibility Testing Antimicrobial susceptibility testing was done by Kirby Bauer s disk diffusion method (Bauer et al., 1996) as was performed on biofilm isolates by Kirby-Bauer on Muller Hinton Agar (Barry et al., 1999). Bacterial strains used for the study are listed in Table-2. The standard medium Mueller Hinton Agar, was poured to a depth of 4 mm in a 90 mm petridish (PW008, Himedia Labs Pvt. Ltd., Mumbai, India). The plate was inoculated by Table 2: Microbial Strains Used S.No. Bacterial Strains Type 1. Streptococcus mutans Gram Positive 2. Staphylococcus epidermidis 3. Streptococcus pyrogenes 4. Bacillus cereus 5. Staphylococcus aureus 6. Corynebacterium xerosis 7. Escherichia coli Gram Negative 8. Proteus vulgaris 9. Pseudomonas aeruginosa 10. Klebseilla pneuomoniae Fig. 1: Fruit (Berry of Bakayin) Melia azedirach Linn.

24 17 streaking the entire surface in three planes with a sterile cotton swab (PW041, Himedia Labs Pvt. Ltd., Mumbai, India) dipped into standardized inoculums, spreaded evenly with the help of L-Spreader (PW1085, Himedia Labs Pvt. Ltd., Mumbai, India). The bacterial inoculum was prepared from an 18 hour broth culture of the microbe to be tested and was standardized with sterile physiologic saline to contain 10 6 cfu/ml. Standardized commercial paper disk containing amounts of the antimicrobial agents to be tested were placed on the surface of the agar. The plate was incubated in an inverted position at 37 0 C for 18 hours. The diameter of zone of inhibition produced by the drug was measured with the help of Antibiotic zone scale (PW297, Himedia Labs Pvt. Ltd., Mumbai, India) from each disk (Kingsbury and Wagner, 1990). Statistical Analysis The statistical analysis was done using gpaid software, One way ANOVA and the post test named Bonferroni: Selected pairs of column with multiple comparison was performed with p-value <0.05. Results and Discussion Qualitative analysis of the phyto-chemicals of fruits of Bakayin was carried out for the determination of presence of alkaloids, amino acids, proteins, carbohydrates, flavonoids, glycosides, tannins, sterols, phenols, resins and volatile oil. As the therapeutic properties of the crude drugs are mainly due to physiologically active chemical constituents present in the drugs and the lower percentage of chemical constituents may cause lesser therapeutic values of the drugs and therefore, they are considered as low standard drugs. Moreover, the chemical constituents present in the drug have documented antimicrobial activities. Tannins have the property of precipitating proteins. By an analogous process they prevent the development of bacteria and since the proteins are necessary for their growth and development so, they not only stop their nutrition but also precipitate their own proteins (Said, 1996). The phytochemical analysis of the chemical constituents present in the test drug revealed that it contains alkaloids, phenol, resins, saponins, sugars and tannins. These were the encouraging results for our study as such types of chemical compounds are usually responsible for the therapeutic efficacy of that drug. As can be realized from the fact that alkaloids possess antimicrobial and anti-inflammatory activity, this effect has been confirmed by us in our in-vitro study of antimicrobial screening that the drugs were found to be sensitive to many microbes. This study also confirms the presence of saponin by qualitative test, as they are generally considered as the soapy substances that are general cleansers, having

25 18 antiseptic properties (Hirat and Suga, 1983). Sterols either decrease the activity of S.aureus, E.coli, P.vulgaris and Pseudomonas pyocyanea or have no effect in case of Klebseilla and S. dysentrica (Anuradha and Goyal, 1995). Flavonoids possess anti-inflammatory, anti-spasmodic, antibacterial, antifungal and antitumor activity, they act as an anti-oxidant and are also thought to protect plants from UV radiations and micro-organisms (Cushnie and Lamb, 2005).These are few evidences in support of the therapeutic activity of the Unani drugs. Most of these findings were found to be helpful in showing its biological activity. In an effort to validate the antibacterial efficacy of selected test drug Bakayin all ethno- pharmacological knowledge was found to be in favour of our selection of the drug as per the guidelines by Cos et al. (2006) in which he described that how we can develop a stronger in vitro proof-of-concept in determining antiinfective potential of natural products. The literature survey revealed that an ample data is available regarding the authentication of Unani drugs to be used in infectious diseases, however, very little assay has been done on Unani drugs regarding their antimicrobial activity. Present study is a step ahead in this regard to scientifically validate the antimicrobial activity of selected test drug viz. Bakayin using Kirby Bauer s disk diffusion and Agar well diffusion method against different Gram positive and Gram Table-2 (a): Antibacterial activity of Bakayin against Gram positive bacterial strains S. Test strains Zone of Inhibition (in mm) No. expressed as Mean ± S.E.M (S.D) Probability of error Drug Extract Control Standard (μg/ml) (DMSO- 50μl) (Ciprofloxacin 30μg) 1. S.mutans 9.4±0.67(1.51)* 6.6±0.24(0.54) 21.2±0.37(0.83) (ATCC 25175) (S) (R) (S) 2. S.epidermidis 9.2±0.96(2.16)* 6.4±0.24(0.54) 21.6±0.24(0.54) (ATCC 155) (S) (R) (S) 3. S.pyrogenes 11.8±1.02(2.28)*** 6.4±0.24(0.54) 21.2±0.37(0.83) (ATCC 14289) (S) (R) (S) 4. B.cereus 9.2±0.96(2.16)* 6.6±0.24(0.54) 21.4±0.24(0.54) (ATCC 11778) (S) (R) (S) 5. S.aureus 22.8±1.02(2.28)* 6.6±0.24(0.54) 26.8±0.20(0.44) (ATCC 29213) (S) (R) (S) 6. C.xerosis 6.6±0.24(0.54) 6.6±0.24(0.54) 21.2±0.37(0.83) (ATCC 373) (R) (R) (S) (S) Sensitive (R) Resistant

26 19 Table-2 (b): Antibacterial activity of Bakayin against Gram negative bacterial strains S. Test strains Zone of Inhibition (in mm) No. expressed as Mean ± S.E.M (S.D) Probability of error Drug Extract Control Standard (μg/ml) (DMSO- 50μl) (Gentamicin 30μg) 1. E.coli 11.2±0.37(0.83)*** 6.4±0.24(0.54) 14.8±0.20(0.44) (ATCC 25922) (S) (R) (S) 2. P.vulgaris 9.4±0.67(1.51)* 6.4±0.24(0.54) 14.0±0.54(1.22) (ATCC 6380) (S) (R) (S) 3. P.aeruginosa 9.6±0.24(0.54)* 6.4±0.24(0.54) 14.8±0.20(0.44) (ATCC 25619) (S) (R) (S) 4. K.pneuomoniae 8.2±0.48(1.09)*** 6.4±0.24(0.54) 14.8±0.20(0.44) (ATCC 15380) (S) (R) (S) (S) Sensitive Fig. 2: (R) Resistant Antibacterial activity of ethanolic extracts of the test drugs against Gram positive strains Negative bacterial strains. A total volume of 40 μl of test drugs from concentrations viz μg/ml was used and compared with the standard drug Ciprofloxacin (30μg) for Gram positive and Gentamicin (30 μg) for Gram Negative bacteria and Plane control i.e. DMSO- (Dimethyl Sulphoxide) the solvent used (to exclude out any activity due to the solvent used to dissolve the plant extract).

27 20 Fig. 3: Antibacterial activity of ethanolic extracts of the test drugs against gram negative strains There was an increased inhibitory activity against most of the strains. Among Gram positive strains S.aureus (22.8±1.02) > S.pyrogenes (11.8±1.02) > S.mutans (9.4±0.67)> B.cereus (9.2±0.96) = S.epidermidis (9.2±0.96) while it was completely resistant to C.xerosis ATCC 373 at all concentration. For Gram negative strain used it showed moderate sensitivity to all strains and there was a significant inhibitory activity in the order of E.coli (11.2±0.37) > P.aeruginosa (9.6±0.24) = P.vulgaris (9.4±0.67) > K.pneuomoniae (8.2±0.48) > All showed a significant inhibition as compared to Gentamicin (ZOI mm) (table 2a & b) and (fig. 2 & 3). Conclusion The present study provides the first report to ameliorate the efficacy of Bakayin in infectious diseases on scientific parameters. A large number of active phytoconstituents present in the drug have been found obviously to check the growth of bacteria and treat the disease. The study concludes Bakayin s fruit has a potent antibacterial activity against many pathogenic bacterial strains and thus could be used to derive antibacterial agents to fight against number of infectious diseases. Among the gram positive strains Bakayin s fruit was found to be sensitive to all the tested bacterial strains viz. S.aureus, S.mutans,

28 21 S.epidermidis, S.pyogenes, B.cereus, E.coli, K.pneuomoniae, P.aeruginosa and P.vulgaris while it showed a complete resistance to C.xerosis. Most significant sensitivity was shown towards S.aureus and E.coli. Acknowledgement Authors are thankful to DRS-I (SAP-II) UGC, Department of Ilmul Advia, Ajmal Khan Tibbiya College Hospital, A.M.U., Aligarh, for providing all the financial assistance for this study. References Afaq, S.H., Tajuddin., Siddiqui, M.M.H., Standardization of Herbal Drugs. AMU Publication Division, Aligarh Muslim University Press, Aligarh. Anuradha, V. and Goyal, M.M., Phytochemical study on the leaves of Alstonia scholaris and their effects on pathogenic organisms. Ancient Science of Life 15 (1): Barry, L.A., Craig, A.W., Nadler, H., Reller, B.L., Sanders, C.C. and Swensor, J.M., Methods for determining bacteriacidal activity of antimicrobial agents. Approved Guidelines. Clinical and Laboratory Standard Institute (CLSI), September, Vol. 19 (18): M26-A:1-19. Bauer, K., Sherris and Turck, Performance standards for antimicrobial disk susceptibility tests. CLSI (formerly NCCLS). Am. J. Cl. Path.45: 493. Bhattacharjee, S.K., Handbook of aromatic plants. Pointer Publishers, Jaipur, p.283. Bhattacharjee, S.K. and Das, L.C., Medicinal Herbs and Flowers. Aavishkar Publications, Jaipur. Chopra, R.N., Chopra, J.C., Handa, K.L. and Kapur,L.D., Indigenous drugs of India, pp Cos, P., Vlietinck, A.J., Berghe, D.V. and Maes, L., Anti-infective potential of natural products: How to develop a stronger in vitro proof of concept. Journal of Ethnopharmacology 106: Cushnie, T.P. and Lamb, A.J., Antimicrobial activity of flavonoids. Int. J. Antimicrob. Agents 26 (5): Hirat, T. and Suga, T., The efficiency of aloe plants, chemical constituents and biological activities. Cosmetics and Toiletries 98: Kirtikar, K.R. and Basu, B.D., Indian Medicinal Plants, Vol.1; Edn 2. Lalitmohan Basu Allahabad, India, pp

29 22 Kingsbury, D.T. and Wagner, G.E., Microbiology, 2 nd edition. Harvard Publishers, U.S.A, pp Nadkarni, A. K., Indian Materia Medica. Bombay Prakashan Pvt. Ltd. Vol- I. pp Pal, P and Bose, S., Phytopharmacological and Phytochemical Review of Melia azadirachta. International Journal of Research in Pharmaceutical and Biomedical Sciences 2 (3): Panda, S., Jafri, M., Kar, A. and Meheta, B.K., Thyroid inhibitory, antiperoxidative and hypoglycemic effects of stigmasterol isolated from Melia azadirachta. Fitoterapia 80: Peach, K. and Tracey, M.V., Modern methods of Plant analysis. Springer- Verlag, pp Rehman, S., Latif, A., Ahmad, S. and Khan, A.U., In-vitro Antibacterial screening of Swertia chirayita Linn. against MRSA (Methicillin Resistant Staphylococcus aureus). International Journal of Current Research and Review 03 (6): Rehman, S. and Latif, A., Antibacterial Screening of Karanjwa Seeds (Caesalpinia bonducella Roxb.): An effective Unani Medicine for Infectious diseases. Hippocratic Journal of Unani Medicine 10 (2): Said, H.M., Medicinal herbs. Hamdard Foundation Pakistan, Pakistan, p Sindhia, V.R. and Bairwa, R., Plant Review Melia azadirachta. International Journal of Pharmaceutical and Clinical Research 2(2):

30 Combating Trichomoniasis Through a Unani Drug Marham Dakhliyun 1 *Fahmeeda Zeenat and 2 Azhar Hasan 1 Z.V.M. Unani Medical College & Hospital, Azam Campus, Camp, Pune Mohammadia Tibbia College, Malegaon, Nashik Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract T 23 richomoniasis is a common infection worldwide. It is found in approximately 50% of the patients who complain of vaginal discharge and over 200 million people worldwide become infected every year with it. Literature reports show highly divergent prevalence rate of trichomoniasis in different countries and even in different geographical regions. The World Health Organization has estimated that this infection accounts for almost half of all curable infections worldwide. In spite of the wide prevalence of this disease, the choice of treatment available in western is limited and the recurrence rate is very high after the treatment and symptomatic relief. Tibb-e-Unani claims to possess a number of effective and safe therapeutic agents which are used commonly in the treatment of trichomoniasis however the scientific data validating their efficacy are very few. Therefore, in the present study an attempt has been made to evaluate a pharmacopoeial Unani preparation viz. Marham Dakhliyun on 60 patients for its efficacy in the management of trichomoniasis. The ointment was locally applied over vaginal mucosa and fornices in a dose of 5 gm, at bed time, for 14 days. Patients treated with test drug showed 86.6% reduction in important clinical features such as abnormal vaginal discharge, pruritus vulva and trichomonas in wet mount examination. The findings suggested that Unani drug Marham Dakhliyun is effective in the treatment of trichomoniasis. Keywords: Unani drug Marham Dakhliyun, Trichomoniasis, Vaginal discharge, Pruritus vulva. Introduction In clinical practice, trichomoniasis is the most common and important vaginal infection in women. It is found in approximately 50% of the patients who complain of vaginal discharge. It occurs at any age from birth onwards but most often in the young adults (Kumar and Malhotra, 2008). Scientific reports show highly divergent prevalence rates of trichomonas in different countries and even in different geographical regions (Say and Jacyntho, 2005). The World Health Organization has estimated that this infection accounts for almost half of all curable infections worldwide (Schwebke and Burgess, 2004). The complications associated with trichomoniasis in women include various inflammatory conditions, cervical erosion, cervical cancer and infertility. Premature rupture of the placental membranes, contributing to premature labour, and low birth weight babies are known perinatal complications. Increased risk of HIV infection due to trichomoniasis is seen in both sexes (Cudmore et al., 2004). It is caused by an ovoid/pear shaped, actively motile, flagellated parasite i.e. Trichomonas vaginalis 1 *Author for correspondence; fahmeedazeenat@gmail.com

31 24 that is a protozoon, slightly larger than a leucocyte and is anaerobic (Dutta, 2001; Dawn, 2001). Unani medicine postulates that it occurs following the changes in the quality or/and quantity of Phlegm (Balgham) in the body especially in pelvic region. Phlegm is described to be synthesized in liver and used by the organs as it is one of the important components of the humour, necessary to maintain the homeostasis. However in case its quality or quantity is compromised anyhow it becomes a source of diseases and may also serve as a medium for infection (Ahmad et al., 2011). Trichomoniasis in Unani literature has been further described to be a type of Sailanur Rahem characterized with excessive uterine discharge. It has been described that ufunat (infection) in the uterus leads to weakening of Quwate Hazema (digestive faculty) of urooqe haiz and predominance of Akhlate Arba are responsible for Sailanur Rahem (Ibn Sina, 2007). Majoosi (1889) described that Zofe Quwate Jazeba (weak retentive power), excess of waste in the body and predominance of Akhlat Arba are the causative factors of Sailanur Rahem. Some other Unani physicians have described that Sailanur Rahem is caused by Zofe Quwate Ghazia of Rahem along with predominance of Akhlate Arba and accumulation of waste material in the body (Ibn Hubal, 2007; Jurjani, 1903; Kabiruddin, 2003). Two approaches of treatment are usually adopted by Unani physicians. In case of chronic and recurrent cases of trichomoniasis both systemic and local drugs are used to normalize the quality of phlegm in the body and to improve the symptoms however in acute day to day cases local application is considered sufficient. If treatment with local application of medicament fails then both oral and local treatment is given simultaneously. The choice of treatments available in modern medicine are comparatively few. Even the drugs available for the purpose are reported to cause side effects and often fail to cure the disease completely. The disease is usually treated with Metronidazole, a 5-nitroimidazole drug derived from the antibiotic azomycin. Common adverse reactions of Metronidazole are usually mild, although some patients do have reactions severe enough to necessitate halting Metronidazole therapy (Cudmore et al., 2004). Such a situation warrants some alternative arrangement for the treatment of trichomoniasis. There are a number of safe and effective drugs in Unani system of medicine that have been described to be effective in trichomoniasis, vaginitis and Sailanur Rahem etc. However, many important drugs used extensively in trichomoniasis by Unani physicians have not been scientifically evaluated for their efficacy and safety. One such Unani pharmacopoeial preparation Marham Dakhliyun (ointment), is described to be effective in the conditions mentioned above (Kabiruddin, 1978) and is commonly used by Unani physicians in gynecological practice. Therefore present study was designed to study the efficacy of Marham Dakhliyun in the management of Sailanur Rahem (Trichomoniasis).

32 25 Materials and Methods Marham Dakhliyun, manufactured and marketed by Dehlavi Remedies, New Delhi (Kabiruddin, 1978), was procured from local agency of Malegaon. The ingredients of Marham Dakhliyun are given in Table 1. The patients who visited the OPD of Department of Ilmul Qabalat wa Amraz-e- Niswan, Mohammadia Tibbia College and Assayer Hospital, Mansoora, Malegaon during , were screened for the presence of Trichomonas vaginalis on the basis of clinical signs and symptoms. The diagnosis of screened patients was however confirmed after pathological investigation. After taking the informed consent, 60 diagnosed patients of years of age were included in the study. They were informed about the disease, examination to be performed and type of treatment. The patients suffering from candidiasis, chlamydial vaginitis, diphtheritic vaginitis, granular vaginitis, bacterial vaginitis, senile vaginitis, emphysematous vaginitis, vaginitis adhaesiva, neoplasm of cervix or vagina or any other systemic disease were excluded from the study. The permission of Institutional Ethics Committee (IEC) was taken prior to the initiation of the study. The patients were divided into two groups of 30 patients each with the help of computer randomized tables/ numbers. The patients in group I, serving as standard control were treated with Metronidazole in a dose of 400 mg, twice a day for one week, orally. While the patients in group II, were treated with Marham Dakhliyun in a dose of 5 gm, intravaginally, once a day at bed time for two weeks. Before and after the treatment, specific investigations such as Vaginal ph Determination, Amine (Whiff) test and Saline Wet Mount Examination of Vaginal Discharge were done to confirm the diagnosis and to use as objective parameters for the assessment of the efficacy. The ph was measured (Khan, Table 1: Ingredients of Marham Dakhliyun S.No. Name of Ingredients Scientific Name Quantity 1 Roghan Zaitoon Olea europaeae 140 gm 2 Murdarsang Mono Oxide of Lead 70 gm 3 Tukhm-e-Khitmi Althea officinalis 23.3 gm 4 Tukhm-e-Marwa (Kanocha) Phyllanthus maderaspatensis 23.3 gm 5 Tukhm-e-Katan Linum usitatissimum 23.3 gm 6 Aspaghol Plantago ovata 23.3 gm 7 Methi Trigonella foenum-graecum 23.3 gm

33 ) by using a Ranbaxy ph indicator paper with a range of 4.5 to 7 with distinct colour keys to 4.5, 5, 5.5, 6, 6.5 and 7. The paper was applied to the anterior vaginal fornix to avoid contamination with cervical mucus. The colour developed on the moistened paper was matched with the colour scale provided. The amine test (Egan and Lipsky, 2000) was performed by adding 2 to 3 drops of 10% KOH directly to swab or to the discharge on the speculum; release of fishy or amine odour was recorded as positive Amine or Whiff test. Differentiation between bacterial vaginosis and trichomoniasis depends on microscopic evaluation of material recovered from the vagina. The most useful approach for evaluation in the clinical setting is the wet mount. A drop of vaginal discharge was collected by a pipette or swab from the posterior vaginal fornix and placed on a clean warm glass slide. Two drops of normal saline were added and mixed with the vaginal discharge. A glass cover slip was placed over it. The wet film was immediately examined under the microscope (HPF 45X). The Trichomonas vaginalis were recognized by their shape, size and their nozing and rotatory movements. The presence of large numbers of polymorphonuclear eucocytes (PMNs) supports the diagnosis of trichomoniasis (Rein and Liang, 1999). The presence of parasite was recorded in the Case Record Form. The clinical features were graded on point scales and the changes were noted in CRF on every follow up. The patients were advised for weekly follow up and abstinence was advised and no concomitant therapy was allowed during the period of treatment. At each visit the patients were examined carefully to assess the different parameters. Each patient underwent per vaginal examination in lithotomy position after general and systemic examination. Tenderness of vaginal wall and straw berry appearance of vagina and cervix was recorded by per speculum examination. Scoring system for overall evaluation of each patient was done. The vaginal discharge was graded (Mirza et al., 2011) as none (-) for no discharge, mild (+) for normal moistness of vagina without staining or moistening the underclothes, moderate (++) for undeniably soiled the underclothes that requires frequent changing and washing, and severe (+++) that requires the use of absorbent pad. Pruritus (Akhyani et al., 2005), burning micturition and dysuria (Marickar and Salim, 2009) were classified as none (-), mild (+), moderate (++) and severe (+++). Dyspareunia, backache and lower abdominal pain were assessed by visual analogue scale (Lin et al., 2005) as none (-), mild (+), moderate (++) and severe (+++). Tenderness and congestion of vaginal wall and straw-berry appearance of vagina and cervix were also graded as none (-), mild (+), moderate (++) and severe (+++). The percentage decrease in scores was determined by comparing the baseline scores with that of post treatment. Finally, recorded findings were statistically analyzed using Chi square test to determine the significance.

34 27 Results and Discussion The test drug was studied for its efficacy in the management of trichomoniasis by observing clinical features and laboratory investigations. The subjective (Table 2) and objective (Table 3) findings were tabulated, analyzed and compared with the standard drug. On the day of registration abnormal vaginal discharge was found in all (100%) the patients included in the study, while after treatment it remained only in 16.66%, and 13.33% of the patients indicating an improvement of 83.33% and 86.66% of the cases in group I and II, respectively. Malodour on day zero was found in 93.33% and 86.66% of the cases in group I and II, respectively, whereas after treatment it reduced and was found only in 14.28% and 11.53% of the cases. Pruritus vulvae on day zero was found in 100% of the patients in each group, whereas after treatment it reduced and was found only in 16.66% and 13.33% of the patients thus 83.33% and 86.66% improvement was observed in group I and II, respectively. On day zero, dyspareunia was found in 76.66% and 73.33% of the cases in group I and II, respectively, whereas it was found absent in 86.95% and 90.9% of the cases and found present only in 13.04% and 9.09% of the cases, respectively. Prior to the treatment low backache was found in 60% and 56.66% of the patients in group I and II, respectively, whereas after treatment it reduced and found only in 11.11% and 11.76% in group I and II, respectively. The improvement was observed in 88.88% and 88.23%, of the patients, respectively. On day zero 53.33% of the patients of both the groups reported pain in lower abdomen, whereas after treatment it continued only in 12.5% and Table 2: Effect of Marham Dakhliyun on subjective parameters Symptoms Group I (Standard Control) Group II (Test) (Subjective Parameters) Baseline Post Improvement Baseline Post Improvement treatment treatment No % No % No % No % No % No % Abnormal vaginal discharge Malodour Pruritus vulva Dyspareunia Low backache Pain in lower abdomen Burning micturition & Dysuria

35 % of the patients in group I and II, respectively. Prior to the treatment burning micturition and dysuria were found in 70% and 60% of the cases in group I and II, respectively. The symptoms were found to be reduced significantly as only in 14.28% and 16.66% of the patients in group I and II, respectively reported these symptoms. On examination, prior to the treatment tenderness of vaginal wall was found in 76.66% and 60% of the patients in group I and II, respectively, which reduced significantly after the treatment and was found in 13.04% and 11.11% of the cases only. On day zero straw berry appearance of cervix and vagina was found in 26.66% and 23.33% of the patients in group I and II, respectively. While after treatment it disappeared totally, showing 100% improvement in both groups. The ph > 4.5 on day zero, was found in all the patients in each group. 80 % improvement was observed after the treatment in both the groups. On day zero, positive amine test was found in 100% of the patients in each group, whereas after treatment it was decreased and found only in 10% and 16.66% of the patients indicating an improvement of 90% and 83.33% in group I, II, respectively. The examination of slides prepared from vaginal discharge of the patients prior to the treatment showed Trichomoniasis in 100% of the patients, whereas after treatment it reduced and was found only in 16.66% and 13.33% of the patients, respectively indicating significant improvement. Relief in clinical symptoms along with reduction in microbiological count was considered as the criteria of efficacy. The cases having relief from abnormal vaginal discharge along with absence of trichomonas in slide after treatment were rated as cured, while the patients having no relief in these two findings were rated as not cured. Complete cure was observed in 83.33% cases in group I and 86.66% cases in group II (Table 4). Table 3: Effect of Marham Dakhliyun on objective parameters Signs Group I (Standard Control) Group II (Test) (Objective Parameters) Baseline Post Improvement Baseline Post Improvement treatment treatment No % No % No % No % No % No % Tenderness of Vagina Strawberry Nil Nil appearance ph > Positive Amine Test Trichomonas in slide

36 29 Table 4: Cure rate after the treatment Response Group I (Standard Control) Group II (Test) No. % No. % Cured Not cured The findings in respect of different parameters indicated that there was no statistical difference between the two groups suggesting that the two drugs produced almost equal degree of response. The findings suggested that local application of test drug is equally effective as the oral administration of Metronidazole. Although in Unani medicine both local and oral treatment is suggested simultaneously to treat the patients of trichomoniasis with the aims to improve the quality of phlegm that has undergone through derangement and to improve the local pathology. But only local treatment is also advised commonly to treat such a condition. Present study indicated that only local application is also sufficient to cure the majority of the patients. About 13% of the patients were not cured with the test drug probably because few patients may have required the local as well as the systemic intervention as the only local treatment may not be sufficient to negotiate the severity of the disease. The result is in consonance with the Unani principle of treatment that suggests that the disease arises following the disturbance in the quality, quantity or composition of the phlegm (Majoosi, 2005). Trichomoniasis may develop as sequelae of systemic derangement of phlegm but it may also develop because of its local overthrow that is frequently compounded with other factors that trigger the pathology. The cure rate of only 83% may be because of the demand of the nature of pathological condition for systemic intervention. Since the trichomoniasis is a disease of Khilt Balgham having the symptoms of cold and wet temperament therefore it is obvious to treat it with the drugs that have hot and dry temperament. Most of the ingredients of Marham Dakhliyun used to treat the disease are attributed to be hot and dry therefore are suitable for the treatment of trichomoniasis. Its ingredients have been described in Unani and ethnopharmacological literature to be Qabiz (astringent), Mohallil (antiinflammatory), Mufatteh (deobstruent), Mulattif (demulscent), Mujaffif (dessicative), Munzij (concoctive) Musakkin (analgesic), stimulant and mentioned to be effective in vaginal discharge and leucorrhoea (Anonymous, 1962; Anonymous, 2000; Ghani, YNM; Hakeem, 1999; Khan, 1313H; Kirtikar & Basu, 1991; Nabi, 2007; Nadkarni, 1954). Due to these medicinal properties, Marham Dakhliyun produces constriction in the vaginal wall, absorbs the vaginal secretions and resolves the discharge and improves the inflammatory condition.

37 30 These symptoms are common in a disease that arises following the ascendance of phlegm. Marham Dakhliyun was used intravaginally because the vagina not only secretes; it absorbs water, electrolytes and the substances of low molecular weight. Absorption and re-absorption are believed to occur mainly in the lateral recesses of the lower vagina (Jeffcoate, 1975) therefore local application appears to be appropriate measure to relieve the symptoms locally. The study clearly showed that the Marham Dakhliyun is very effective in trichomoniasis, which is evidenced by decrease in the amount of abnormal vaginal discharge, pruritus, dyspareunia, backache, pain in lower abdomen, burning micturition and dysuria, tenderness and congestion of vaginal wall, strawberry appearance, ph and trichomonas in slide. It was further strengthened by negative amine test. The efficacy of the test drug may be attributed to its antiprotozoal activity which was almost equal to that of Metronidazole. Thus, the study validated the therapeutic regimen proposed by Unani physicians and their age-old practice with the test drug Marham Dakhliyun in the management of trichomoniasis. Conclusion In the light of findings and the discussion it can be concluded that Unani drug Marham Dakhliyun possesses significant effect against trichomoniasis. Therefore, it may be used effectively in patients afflicted with trichomoniasis (Sailanur Rahem) and associated condition. References Ahmad, W., Hasan, A., Ansari, A., Tarannum, T., Efficacy of a combination of Unani drugs in patients of Trichomonal vaginitis. Indian J. Traditional Knowledge 10(4): Akhyani, M., Ganji, M.R., Samadi, N., Khamesan, B., Daneshpazhooh, M., Pruritus in haemodialysis patients. BMC Dermatology 5:7doi: / Anonymous, PDR for Herbal Medicines (New Jersey): Medicals Economics Company; Montvale, pp Anonymous, The Wealth of India - A dictionary of Indian raw materials industrial ptoducts. First supplement series. Vol. VI: L-M. (New Delhi): CSIR, Cudmore, S.L., Delgaty, K.L., Hayward-McClelland, S.F., Petrin, D.P., Garber, G.E., Treatment of infections caused by metronidazole-resistant trichomonas vaginalis. Clinical Microbiology Review 17 (4):

38 31 Dawn, C.S., Undergraduate and postgraduate textbook of gynaecology and contraception. 13 th ed. Dawn Books, Kolkata, pp Dutta, D.C., Text book of gynaecology. 3 rd ed. New Central Book Agency, Kolkata, pp Egan, M.E., Lipsky, M.S., Diagnosis of vaginitis, Am. Fam Physician 62: Ghani N., YNM. Khazainul Advia. Idara Kitabul Shifa, New Delhi, pp , , Hakeem, M.A., Bustanul Mufradat. Zafar Book Depo, Delhi, pp. 180, 327, 362. Ibn Hubal., Kitabul mukhtarat fit tib. Vol. IV. (Urdu translation by CCRUM), New Delhi, p. 37. Ibn Sina., Al-Qanoon fit tib. Vol. II. (Urdu translation by Kintoori, G.H.). Idara Kitabus Shifa, New Delhi, p Jeffcoate, N., Principles of gynaecology. 4 th ed. Butterworth and Company, Edinburgh, pp , Jurjani, I., Zakheera khawarizm shahi. Vol. 6. (Urdu translation by Khan, A.H.). Matba Munshi Nawal Kishore, Lucknow, p Kabiruddin, M., Bayaz-e-Kabeer. Vol. II. Hikmat Book Depo, Hyderabad, p Kabiruddin, M., Al-Akseer. Vol. II. Aijaz Publishing House, New Delhi, Khan, M., Clinical study of Sailanur Rahem and its management with Unani formulation. MD Dissertation. National Institute of Unani Medicine, Bangalore, pp Khan, M.A., 1313H. Muheet-e-Azam. Vol. 1V. Part 2. Maktaba Nizami, Kanpur, pp Kirtikar, K.R. & Basu, B.D., Indian Medicinal Plants. Vol. I. 2 nd Edn. Bishen Singh Mahendra Pal Singh, Dehradun, pp , Vol. III, pp Kumar, P., Malhotra, N., Jeff coate s Principles of gynaecology, 7 th Ed. Jaypee Brothers Medical Publishers, New Delhi, pp. 27, Majoosi, AIA., Kamilus Sana at al-tabia, Vol I, CCRUM, New Delhi, p Majoosi, AIA Kamilus sana at. (Urdu translation by Kantoori). Matba Munshi Naval Kishore, Lucknow, pp ,

39 32 Marickar, YMF, Salim, A., Temporary risk identification in urolithiasis. Urological Research 37(6): Mirza, S., Begum, W., Mubeen, U., Efficacy of Irsa (Iris ensata) in the management of Iltehabe Unqur Rehm (Cervititis): a clinical trial with standard chemotherapeutic regimen as control reatment. Unani Medicus 1(2): Nabi, M.G., Makhzan-e-Mufradat-wa-Murakkabat. 2 nd Edn. CCRUM, New Delhi, pp. 44, 124, 139, 224. Nadkarni, A.K., Indian Materia Medica. Vol.I. 3 rd Edn. Bombay Popular Prakashan, Mumbai, pp , Rein, M.F., Liang, B.A., Diagnosis and treatment of infectious vaginitis. Hospital Physician, pp Say, P.J., Jacyntho, C., Difficult to manage vaginitis. Clinical Obstetrics and Gynaecology 48 (4): Schwebke, J.R., Burgess, D., Trichomoniasis. Clinical Microbiology Review 17 (4):

40 Therapeutic Efficacy of a Dietary Formulation in Chronic Tonsillitis: A Case Study *Ferasat Ali Department of Kulliyat, A.K. Tibbiya College, Aligarh Muslim University, Aligarh Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract I 33 n this paper an attempt has been made to construct a hypothesis on the basis of author s personal experience treating tonsillitis that occurs mainly due to allergy mostly in winter season, and its management with local application of a preparation containing few common dietary objects. Initially both the allopathic and Unani medicines were used separately to ameliorate the disease. However, since the recurrence of disease is very common and frequent use of antibiotics is not advisable, therefore, based on Unani concept of Ilaj-bil- Ghiza author used a combination of pop gram, mixed with salt and turmeric powder at bed time after cleaning the mouth with brush and toothpaste or Neem Miswak. The symptoms of tonsillitis improved after few days of application. A review of literature was conducted to explore the effect of the test combination and its likely mechanism of action so as do develop a valid hypothesis for future clinical studies. Keywords: Cicer arietinum Linn., Salt, Turmeric, Tonsillitis, Sore throat, Temperament, Ilaj-bil-Ghiza. Introduction Present work is based on author s personal experience of management of tonsillitis and prevention of its recurrence with the help of Ilaj-bil-Ghiza (dietotherapy). According to the principles described in Unani System of Medicine (USM), the methodology of treatment of a disease is based on the following four fundamental therapeutic principles: (i) Ilaj-bil-Ghiza (Dietotherapy) (ii) Ilaj-bil-Tadbeer (Regimenal therapy) (iii) Ilaj-bil-Dawa (Drug therapy) (iv) Ilaj-bil-Yad (Surgical intervention/therapy) Dietotherapy/Dietary therapy is practiced as a first line treatment in the management/control of diseases. It is considered an essential part of natural therapy to provide health to the individuals. Since diet (Makool wa Mashroob) is one of the six essential factors responsible for maintaining the health, therefore, it s deficiency may prove to be a causative factor for the diseases. The diet may also play a role in the management of some of the diseases as Ibn Sina (1932) described that illness is treated with diet and drugs attributed to have opposite temperament to that of the temperament of a disease. For example, Yarqan (hepatitis) is considered a disease of hot temperament, hence, it has been named *Author for correspondence; drfirasatalikhan@gmail.com

41 34 as warm-e-kabid har. It is treated with the medicines having cold temperament such as Tamar Hindi (Tamarindus indica), Mako (Solanum nigrum) and Kasani (Cichorium intybus) etc. As a dietary therapy, patient is also advised to have low protein diet and a lot of citrus fruits including boiled rice, vegetables, salad and the juices of pomegranate, orange etc. These above mentioned foods, vegetables and fruits are considered to have cold temperament and thereby are able to interfere with the hot temperament of the disease. Sometimes, the dietary therapy is applied as a second line of treatment to help the primary treatment in which drugs are prescribed mainly to ameliorate the disease (Gruner, 1930; Ibn Rushd, 1980). Similarly, there is also a concept of abstinence (parhez) or avoiding some of the foods in different diseases. Since a combination of certain dietary supplement was used to manage the chronic tonsillitis, therefore, a brief review of tonsillitis and the drugs used in the case study to improve was also undertaken. Tonsillitis Tonsils are the part of the immune system. They are believed to produce antibodies that protect the body from infection. They are located at both sides of throat and act as a filter to all the germs, bacteria, virus and other organisms which may enter inside the body. In the process, they get inflamed occasionally and injured due to infection and allergy but they continue to guard the body effectively. However, sometimes causative factors dominate over the defence mechanism causing tonsillitis (Warm-e-Louztain) both in children and adults and giving rise to specific symptoms. Etiology Tonsillitis commonly occurs in younger people under the influence of cold. Polluted air (Kaseef Hawa) is believed to cause tonsillitis (Warm-e-Louztain). Use of too cold food and beverages mainly in extremely cold and hot season frequently cause the disease. The cold items may cause local vasoconstriction causing a compromised state of local immunity and allowing the bacteria and other organisms to cause infection. Sometimes, the disease occurs in patients afflicted with diphtheria, rheumatic fever and epidemic diseases (Samarqandi, ynm). According to Maqbool (1994) it may occur as a primary infection of tonsil itself, but may occur secondarily as a result of upper respiratory tract infection following the viral infection. Poor orodental hygiene, poor nourishment and congested surroundings are important predisposing factors for this disease. Signs and Symptoms Red, swollen tonsils, white or yellow coating or patches on the tonsils, sore throat, painful blister or ulcer in the throat, difficult or painful swallowing, fever, enlarged,

42 35 tender glands (lymph nodes) in the neck, a scratchy, muffled or throaty voice and bad breath are considered to be important signs and symptoms of tonsillitis. Acute tonsillitis is very common which is mostly treated successfully or subsides by its own. However, in many patients it transforms into chronic form; such patients experience recurrence of the symptoms (Dhingra, 2007). Case Presentation The author used to get affected frequently with tonsillitis especially in cold season for many years. Use of the antibiotics was effective to the extent of temporary relief as after sometimes there had been the recurrence of tonsillitis and related symptoms. Since frequent use of antibiotics is not advisable, therefore, he decided to take some home remedies based on the principles of Unani medicine. As the tonsillitis is characterized by inflammation, pain and infection of tonsils, therefore, it was decided to try the medicaments prepared from commonly used dietary objects that have the opposite temperament of tonsillitis. He initially started gargle with luke warm water mixed with turmeric powder and salt, and there was a relief. However, it was realized later that the use of gargle was a bit inconvenient, therefore, author switched to use solid dosage form with a view to generalize the therapy, if found effective, and started to use popped gram mixed with salt and turmeric powder at bed time after cleaning the mouth with brush and tooth paste or neem miswak. The combination was chewed well and swallowed. Water was not used because the dietary constituents that stuck to throat to induce local effect will be washed out with water. There was tremendous response. Now the author takes this combination at the start of winter season for one week as a preventive measure. Tonsillitis usually does not appear thereafter. However, in case of its occurrence author repeats it for one more week. The experience suggests that this combination of dietary supplement may be used for therapeutic purposes especially in the management of tonsillitis and sore throat. In view of the above mentioned experience a review of Unani and ethnobotanical literature was also conducted so as to explore the medicinal properties of the ingredients of the combination and their correlation with tonsillitis, with special reference to the likely mechanism of action. Management with the dietary combination To avoid regular use of antibiotic or surgical removal of the tonsils the dietary formulation was used. It included parched/pop gram sprinkled and mixed with turmeric and sodium chloride. Application of dietary therapy is considered a natural way of management of any disease. Chronic tonsillitis requires long period of time to be treated, apart from providing relief to the patient from tonsillitis and sore throat its therapeutic regimen also aims at enhancing the immunity so that

43 36 the burden of tonsils may be reduced. The main properties and actions of the ingredients of the dietary formulation are described below: (i) Sodium chloride (Salt): Being hot & dry in second degree of temperament and possessing antiseptic, anti inflammatory and diuretic activity the gargle with saline water in the diseases of throat has been described to be very useful (Kabiruddin, 2007; Nadkarni, 2000). (ii) Turmeric: Haldi (Curcuma longa Linn): acts as analgesic and antiinflammatory agent and used in a number of diseases. Externally, it is used as anti-inflammatory, analgesic and wound healer to relieve swelling, sprain, stretch, ulcer, toothache and sore throat etc. It is also used as general tonic and to provide a glow to the skin (Anonymous, 1950). Its fomentation has been described to relieve the pain (Hakim, 2002). (iii) Gram (Cicer arietinum Linn): It is called Hums in Unani Medicine but commonly known as Chana. It is hot and dry in 1 st degree of temperament. In bronchial catarrh the seeds in a parched condition are given at night followed by a cup of warm milk; it gives tremendous relief (Nadkarni, 2000). It is also used in ulcer due to its astringent and antiseptic properties. It is laxative in nature, increases Hararat-e-Ghariza (innate heat), gives strength to the lungs and produces blood of good quality. Liquid obtained from macerating the seeds works as a tonic; its gargle also gives relief in toothache and produce anti inflammatory effect. It is harmful, however, to drink water after having it. Likely Mechanism of Action of the formulation The preparation was taken at bed time after cleaning the mouth with brush and tooth paste or using the Neem Miswak. Since these cleansing agents are basic or alkaline in nature which neutralizes the acidic medium of the mouth produced by bacteria; therefore, apart from washing out the bacteria they also minimized the chances of bacterial growth because of the adverse medium. Since, sodium chloride is basic in nature and also possesses antiseptic and osmotic properties, therefore, it helps in neutralizing the acidic nature of mouth which causes irritation and infection to the tonsils. Its strong alkaline nature actually does not allow bacteria to grow. Moreover, because of having osmotic property it absorbs intracellular fluid causing relief to tonsils and nearby tissues by inducing anti inflammatory effect. Curcumin isolated from turmeric is considered to get absorbed into the affected organ and provides immunity and strength to the organ that helps improve its inflammatory condition. Curcumin is absorbed through the mucus membrane of tonsils and surrounding tissues which in turn reduces the

44 37 inflammatory condition. Besides, it keeps the organ protected from allergy because it changes the cold temperament to the hot one. Gram due to its astringent (Qabiz), antiseptic and obstruent properties reduces the production of fluid. It also absorbs extra fluid and helps in cleaning the allergens present in throat and its surroundings area. Discussion The food supplements are used as dietotherapy (Ghiza-e-Dawai) in many diseases including hepatitis, hypertension, diabetes, nephritis and many metabolic diseases etc. However, in most of the cases these are used orally to ameliorate the diseases or act as an adjuvant with certain drugs which are used specifically for a particular disease. In the present case efficacy of a combination of three dietary supplements was determined after its local application. The parched gram (Cicer arietinum Linn) flavoured with turmeric and sodium chloride is the part of our dietary habit and has been in use as a diet from the ages in India. However, its use in a specific mode may be useful in the management tonsillitis and sore throat. It should be properly prepared as described above and packed in such a manner so that the cracking quality and adequate quantity of turmeric and sodium chloride remains intact with it. For getting maximum advantage it must be taken after getting the buccal cavity cleaned because the mode of application and its timings are considered to be important for its optimal effect. Water is not used after taking the combination. The constituents of the combination especially curcumin of turmeric and sodium cholride thus find enough time to be absorbed into tissues of tonsils and its surrounding tissues to render maximum local effect. Apart from this it also changes the local cold temperament to the hot one and allows sodium chloride to extract water from intercellular space through osmotic process thus reducing the swelling. Besides, it neutralizes the acidic medium of the mouth produced by bacteria which in turn inhibit the growth of bacteria and the influence of the allergens. The local anti inflammatory and analgesic activity also helps in relieving the symptoms and improving the pathological condition. Thus, the said combination of dietary supplements helps to improve the tonsillitis and relives other symptoms of throat and buccal cavity. Conclusion The findings suggested that the dietary combination is effective in chronic tonsillitis. It helps in improving the symptoms and prevents the recurrence of tonsillitis. However, it warrants further investigation and larger clinical study to ascertain and generalize its efficacy.

45 38 References Anonymous, The Wealth of India (Raw Materials). Council of Scientific and Industrial Research, New Delhi, Vol. II, pp Dhingra, Diseases of Ear, Nose and Throat. Jaypee Brothers, New Delhi, pp Gruner, O Cameron, A Treatise on the Canon of Medicine of Avicenna. LUZAC & Company, London, pp Hakim, A. M., Al-Mufridat. S H Offset Press, New Delhi, p Ibn Rushd, Kitabul-Kulliyat. CCRUM, New Delhi, p.32 Ibn Sina, Al-Qanoon Fi-al-tibb. Matb a, Tamar-e-Hind, Lucknow, pp Kabiruddin, M., Makhzan al-mufridat. S H Offset Press, New Delhi, pp Maqbool, Text Book of Ear, Nose and Throat Diseases. Jaypee Brothers Medical Publishers, Daryaganj, New Delhi, pp Nadkarni, A.K., Indian Materia Medica. Popular Prakashan, Bombay, pp Samarqandi, ynm. Sharah-e-Asbab (Urdu translation by Kabiruddin). Hikmat Book Depo, pp

46 Safety Study of Majoon-e-Gul A Unani Pharmacopoieal Compound Formulation *Fozia Yaqub and Naeem Ahmad Khan Dept. of Ilmul Advia, A.K. Tibbiya College, Aligarh Muslim University, Aligarh Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract S 39 afety study of herbal drugs and food items is now mandatory as per WHO guidelines. It includes determination of aflatoxin, heavy metals, pesticidal residue and microbial load. Plants are more vulnerable to be contaminated with these components during the agricultural practices and thereafter. Therefore, studies to determine the presence / absence of these components to decide the toxicity / safety of drug is necessary. In the present study, the ingredients of Majoon-e-Gul (MG) except its base i.e. sugar and vinegar were studied on safety parameters. All the safety parameters were found below the permissible limit as per WHO guidelines, indicating that Majoon-e-Gul is free from toxicity. Keywords: Majoon-e-Gul, Safety study, Heavy metals, Aflatoxins, Pesticide residue, Microbial load. Introduction Unani System of Medicine possess a number of formulations and single drugs which are used in the management of liver disorders for a long time. Majoon-e- Gul (MG) is one of the important compound preparations mentioned in National Formulary of Unani Medicine (Anonymous, 2007) and other Qarabadeen which is used in liver ailments like Hepatitis (Warm-e-jigar), Jaundice (Yarqaan) etc. (Anonymous, 2007). It has been shown to produce hepatoprotective and antihepatitis effect against Carbontetrachloride (CCl 4 ) induced hepatic damage. It contains 8 ingredients (Table 1) including sugar and vinegar. However, the study was conducted on the ingredients other than sugar and vinegar in powder form. Table 1: Ingredients of Unani Pharmacopoeial Compound Formulation Majoon-e-Gul S.No. Scientific name Unani name 1. Rosa damascena Mill Gul-e-Surkh 2. Crocus sativus Linn Zafran 3. Rheum emodi Wall ex Meissn Rewand Chini 4. Irsa ensata Thunb. Irsa 5. Cinnamomum cassia Blume Saleekha 6. Coccus lacca Laak 7. Sugar 8. Vinegar *Author for correspondence

47 40 These ingredients are mixed in a base prepared with sugar to make a semi solid preparation (Majoon). However, due to increasing incidence of diabetes and dietary restrictions warranted in a number of metabolic and other diseases the use of the drugs prepared in sugar bases is not desirable. Therefore, MG s hepatoprotective, curative and hepatocytes regenerative effects are attributed to its antioxidant activity (Parveen et al., 2009). Cause of contamination of foods & water and herbal materials All the ingredients of MG have been described to have hepatoprotective, resolvent, anti-inflammtory, astringent and diuretic activity and few have also shown anti-cancerous activity (Nadkarni, 2010). Herbal materials normally carry a large number of bacteria and moulds, often originating in soil or derived from manure. Current practices of harvesting, production, transportation and storage may cause additional contamination and microbial growth. Proliferation of microorganisms may result from failure to control the moisture levels of herbal medicines during transportation and storage. (Anonymous, 2007). Aflatoxins B 1, G 1, B 2 and G 2 are fungal secondary toxic metabolites produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius. Aflatoxins are the strongest natural carcinogens and their main target organ is the liver. The International Agency for Research on Cancer (IARC) has classified aflatoxin B 1 in the group 1 as a human carcinogen and aflatoxins G 1, B 2 and G 2 in the group B 2 as possible carcinogens to humans (Meritxell Ventura, 2004). Contamination of herbal materials with toxic substances such as arsenic can be attributed to many factors. These include environmental pollution (i.e. contaminated emissions from factories, leaded petrol, and contaminated water including runoff water which finds its way into rivers, lakes and the sea, and some pesticides), soil composition and fertilizers. The contamination of the herbal material leads to contamination of the products during various stages of the manufacturing process (Anonymous, 2007). The worldwide consumption of herbal medicines is enormous, so in terms of population exposure alone, it is essential to identify the risks associated with their use as safety of herbal medicines is an important public health issue (Anonymous, 2004). In view of the above, the safety study of test drug Mazoone-Gul, has been conducted so as to prepare its safety profile. Material and Methods The study was conducted in the Department of Ilmul Advia, A.K. Tibbiya College, Aligarh, during Sample preparation The ingredients of MG were procured from Dawakhana Tibbiya College, Aligarh. After the confirmation of purity and identity of each of the ingredients from

48 41 pharmacognosy section, all these, except Zafran, were powdered in an electric grinder, separately. Zafran was powdered in dry mortar (kharal) with slow and light movement of pestle to avoid sticking of the drug material with the mortar and there after all the ingredients were powdered and mixed together in a proportion as mentioned in the Unani Pharmacopeia. After mixing of all the ingredients the drug was passed through the sieve no. 80 to confirm its fineness and uniformity of particle size. Finally the powder was stored in air tight container for experimental study. Powdered test drug was studied to evaluate the presence of microbial load, pesticides residue, aflatoxins and heavy metals at Delhi Test House, Azadpur, Delhi Microbiological determination tests Total viable aerobic count (TVC) For detection of the anti-bacterial activity of the test drug, total viable aerobic count (TVC) of the compound formulation was determined, as specified in the test procedure, using plate count. Pre-treatment of the compound formulation Depending on the nature of the compound sample used, it was dissolved using a suitable method and any antimicrobial property present in the sample was eliminated by dilution or neutralization. Buffered Sodium Chloride-Peptone Solution, ph 7.0 (MM G), Himedia Labs, Mumbai, India) was used to dilute the test sample. Test procedures 1. Plate count for bacteria and fungi For bacteria: 1 ml of the pretreated test sample was added to about 15 ml of the liquefied casein-soybean digest agar in a petridish of 90 mm diameter at a temperature not exceeding 45 0 C. Alternatively the test sample was spread on the surface of the solidified medium. Two dishes were prepared with the same dilution, they were inverted and incubated at C for hrs, unless a more reliable count was obtained in a short period of time. The number of colonies so formed were counted and the results were calculated using the plates with the largest number of colonies, up to a maximum of 300. For fungi: 1 ml of the pretreated test sample was added to about 15 ml of the liquefied Sabouraud glucose agar with antibiotics in a petridish of 90 mm diameter at a temperature not exceeding 45 0 C. Alternatively the test sample was spread on the surface of the solidified medium. Two dishes were prepared with

49 42 the same dilution; they were inverted and incubated at C for 5 days, unless a more reliable count was obtained in a short period of time. The number of colonies so formed was counted and the results were calculated using the plates with not more than 100 colonies (Lohar, 2007). 2. Pesticidal residue The test for the assessment of specific pesticide residues like Organochlorine compounds, Organophosphorous compounds and Pyrethroids compounds were conducted using GCMS-Ms (Ramkrishanan, 2015). 3. Aflatoxins The test for determination of the aflatoxins was performed using LCMS-MS. 4. Heavy metals Heavy metals including Arsenic, Mercury, Cadmium and Lead were determined in the test sample using Atomic Absorption Spectroscopy. Table 2: Heavy Metals in MG S.No. Test Parameter Result LOQ Permissible limit (mg/ kg) (mg/kg) (mg/kg) 1 Lead (Pb) Not detected 1.25 Not more than 10 2 Mercury (Hg) Not detected 0.5 Not more than 1 3 Arsenic (As) Not detected 0.5 Not more than 3 4 Cadmium (Cd) Not detected 0.25 Not more than 3 LOQ = Limit of quantification BLQ = Below the limit of quantification Table 3: Microbial load in MG S.No. Microbes Result Permissible Limit 1. Total Bacterial Count cfu/g Not more than 10 5 cfu/g 2. Total Fungal Count <10 cfu/g Not more than 10 3 cfu/g Table 4: Aflatoxin in MG S.No. Aflatoxin Result LOQ Permissible Limit (mg/kg) (mg/kg) 1 Aflatoxin B 1 BLQ Not more than Aflatoxin G 1 BLQ Not more than Aflatoxin G 2 BLQ Not more than Aflatoxin B 2 BLQ Not more than 0.10 LOQ = Limit of quantification BLQ = Below the limit of quantification

50 43 Table 5: Pesticidal residue in MG S.No. Pesticide Residue Result LOQ Permissible (mg/kg) (mg/kg) Limits (mg/kg) 1 Alachor (mg/kg) Not detected Aldrin & dieldrin (mg/kg) Not detected Aziphos-methyl (mg/kg) Not detected Bromopropylate (mg/kg) Not detected Cholordane (mg/kg) Not detected Chlorpyriphos (mg/kg) Not detected Chlorpyriphos-methyl (mg/kg) Not detected Cypermethrin ( mg/kg) Not detected DDT (Sum of p.p-ddt, Not detected p.p-dde and p.p-tde) 10 Deltamethrin (mg/kg) Not detected Diazinon (mg/kg) Not detected Dichlorvos (mg/kg) Not detected Dithiocarbamates (mg/kg) Not detected Endosulfan (Sum of Isomer Not detected and Endosulfan Sulphate) 15 Endrin (mg/kg) Not detected Ethion (mg/kg) Not detected Fenitrothion (mg/kg) Not detected Fenvalerate (mg/kg) Not detected Fonofos (mg/kg) Not detected Heptachlor (mg/kg) Not detected Hexachlorobenzene (mg/kg) Not detected Malathion (mg/kg) Not detected Parathion (mg/kg) Not detected Parathion Methyl (mg/kg) Not detected Permethrin (mg/kg) Not detected Phosalone (mg/kg) Not detected

51 44 Discussion and Conclusion All the four parameters undertaken in the study are considered instrumental to determine the safety /toxicity of a drug. They serve as important tools of quality control and standardization and have been proposed as mandatory requirement for plant drugs by WHO. The findings of the study demonstrated that four heavy metals namely arsenic, mercury, cadmium and lead and four different aflotoxins were not found to be present. Presence of heavy metals in a drug beyond the permissible limits cause serious side effect on brain, kidney, developing foetus and vascular and immune system (Moses et al., 2012). Similarly, aflotoxin have emerged as a major threat to human health because a number of serious side effects such as hepatotoxicity, carcinogenecity and immuno suppression etc. are associated with them. The absence of these toxic elements in the test drug make it safe and free from serious toxic effect. The bacterial and fungal counts were although found to be present but their concentration was found to be far below the permissible limits that can not produce any toxicity. The pesticide residue were not found at all suggesting that the drug is free from contamination. The results of the study revealed that all the safety parameters carried out on Majoon-e-Gul (MG) are within the permissible limits which indicated that the MG in powdered form is quite safe and can be used as such in the management of different ailments. It can also be used to prepare some other non sugar based dosage form such as pills, tablets and capsules etc. References Anonymous, WHO guidelines on safety monitoring of herbal medicines in pharmacovigilance systems. WHO, Geneva. Anonymous, National Formulary of Unani Medicine (Urdu Edition). Ministry of Health and Family Welfare, Dept of Ayush, New Delhi. Anonymous, WHO guidelines for assessing quality of herbal medicines with reference to contaminants and residues, World Health Organization, Geneva, pp Lohar, D.R., Protocol for testing of Ayurvedic, Siddha & Unani Medicines. Pharmacopoeial Laboratory for Indian Medicines, Ghaziabad, pp Meritxell Ventura, Determination of Aflatoxin B 1,G 1,B 2,G 2 in medicinal herbs by liquid chromatography- tandem mass spectrometry. Journal of chromatography 1048(2): Moses, A.G., Maobe, Erastus Gatebe, Leonard Gitu and Henry Rotich, Profile of Heavy Metals in Selected Medicinal Plants Used for the Treatment

52 45 of Diabetes, Malaria and Pneumonia in Kisii Region, Southwest Kenya. Global Journal of Pharmacology 6(3): Nadkarni, K.M., Indian Plants and Drugs. Ajay Book Service, New Delhi, pp. 299,109 Parveen, Asia, Naeem, A. Khan, Aijaz A. Khan, Mohammad Nasiruddin, Evaluation of Unani Pharmacopeial Compound Formulation Majoon-e-Gul in experimentally induced Hepatic Demage in rats. MD Thesis submitted to the Department of Ilmul Advia, Faculty of Unani Medicine, AMU, Aligarh, p. 124 Ramkrishanan, G., Physicochemical and phytochemical standardization of Thraatchathi chooranam-a polyherbal formulation. Journal of Pharmaceutical Sciences and Research 7(6):

53 46

54 Scientific Validation of Unani Formulation: Majoon-e- Hamal Ambari Alwi Khani 1 *Rampratap Meena, 2 S.C. Verma, 3 S. Mageswari, 3 P. Meera Devi Sri, 1 A.S. Khan, 1 S.A. Ansari, 4 Shamsul Arfin and 4 Aminuddin 1 Drug Standardisation Research Institute, PLIM Campus, Kamla Nehru Nagar, Ghaziabad Pharmacopoeia Commission of Indian Medicine & Homoeopathy, PLIM Campus, Ghaziabad Regional Research Institute of Unani Medicine, 1 West Mada Church Street, Chennai Central Council for Research in Unani Medicine, Institutional Area, Janakpuri, New Delhi Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract T 47 he quality and safety of the Unani formulations have to be scientifically validated in order to produce quality medicines and protect the health of people suffering from various kinds of diseases. For the validation or standardization of Unani formulations a systematic scientific protocol is very much needed. Though the Unani medicines are an important healthcare option, but due to lack of scientific standards, these traditional medicines are hindering us from capitalizing on the global opportunities provided by increasing interests all over the world. The drug Majoon-e-Hamal Ambari Alwi Khani is one of the ancient most commonly used Unani formulations in the ailments of atony to the uterus and habitual abortion. For the validation of this drug the parameters such as Standard Operating Procedure (SOP s) for the preparation of drug, powder microscopy to find out the characteristic botanical characters of raw drugs added in the formulation were worked out. Evaluation of physico-chemical, TLC/HPTLC, finger prints, analysis of WHO parameters like heavy metals, microbial loads, aflotoxins and pesticide residues were also carried out to ascertain the quality of drug. The evaluated data will help to lay down the scientific standards for the drug studied to contribute mateial for in Unani pharmacopoeia of India. Keywords: Majoon-e-Hamal Ambari Alwi Khani, Microscopy, TLC/HPTLC, Scientific validation. Introduction Plenty of modern medicines are derived from higher plants (Anonymous, 2005). All medicines, either synthetic or plant origin, have to fulfill the basic requirements of safety and efficacy (EMEA, 2005; Anonymous, 2002). Standardization of traditional medicines is the process of evaluation of a set of pharmacopoeial standards and definitive qualitative and quantitative values which gives an assurance of quality, efficacy, safety and reproducibility. Quality of raw materials, good laboratory practices and good manufacturing processes plays the important roles for providing the quality and efficacious herbal preparations for the needy mass (Anonymous, 2000). Validation of pharmacopoeial standards by experimentation and observations provides a set of characteristics to a particular herbal medicine. Therefore, Scientific Validation of Unani Formulations is an important tool used in the standardization process (Kunle, 2012). Majoon-e- Hamal Ambari Alwi Khani is used in the ailments of Atony to the Uterus and Habitual abortion. Present research studies deals the pharmacognostical, physicochemical, TLC/HPTLC finger print, heavy metals, microbial load, aflatoxins and pesticide residues. 1 *Author for correspondence

55 48 Materials and Methods All the ingredients were procured from the local market and identified. Specimens of all ingredients of the formulation were deposited in the museum of Drug Standardization Research Unit at Regional Research Institute of Unani Medicine, Chennai, Tamil Nadu, India The drug Majoon-e-Hamal Ambari Alwi Khani was prepared as per the formulation composition given in NFUM, Part-I using 20 ingredients showed in Table 1 (Anonymous, 1981). Table 1: Formulation composition of Majoon-e-Hamal Ambari Alwi Khani 1. Ambar Ash-hab Ambra grasea (Animal origin) Part Used 30g 2. Tukhm-e-Khurfa Portulaca oleracea Linn. Seed 25g 3. Maghz-e-Tukhm-e- Benincasa hispida (Thunb.) Kernel 25g Petha Cogn. 4. Marwarreed Mytilus margaritiferus Pearl 10g (Animal origin) 5. Kahruba Pinus succinifera Linn. Resin 10g 6. Busud Mohraq Corallium ruburum Coral roots 10g (Animal origin) 7. Sandal Surkh UPI-V Pterocarpus santalinus Linn. Heart wood 10g 8. Sandal Safaid UPI-VI Santalum album Linn. Heart wood 10g 9. Tabasheer Bambusa bambos Druce. Bamboo 10g Manna 10. Mazu UPI-III Quercus infectoria Oliv. Excrescence/ 10g Gall 11. Darunaj Aqrabi API-VI Doronicum hookeri Hook.f. Rhizome 10g 12. Ood-e-Saleeb UPI-III Paeonia emodi Wall. Tuber 10g 13. Abresham Bombyx mori (Animal origin) Silk Cocoon 10g 14. Beikh-e-Anjabar Polygonum bestorta Linn. Rhizome 10g UPI-III 15. Gil-e-Armani Silicate of Alumina, 10g Magnesia and oxide of Iron 16. Sharbat-e-Angoor Vitis vinefera Linn. Fruit 400 ml 17. Waraq-e-Nuqra Gold leaf 20 Nos. 18. Waraq-e-Tila Silver leaf 20 Nos. 19. Asal Honey 200g 20. Qand Safaid Sugar 700g

56 49 Method of Preparation All the ingredients were taken of pharmacopoeial quality. Grinded the ingredient number 1 of the formulation composition in mortar & pestle and kept separately. Cleaned, dried and powdered the ingredient number 2 to 3, 5 to 12, 14 and 15 of the formulation composition and sieved through 80 meshes. Made the coarse powder of ingredient number 4 of the formulation composition and soaked in Arq-e-Gulab for 5 days and grinded it daily about 4 hours using mortar and pestle till becomes nice powder. Dissolved the ingredient number 20 in 800 ml of water on slow heat, at the boiling stage 0.1% citric acid was added and mixed thoroughly. Boiled the content and prepared the quiwam of 72% consistency and added 0.1% sodium benzoate. At this stage added the ingredient number 16, 19 and extract of ingredient number 13, mixed well and made the quiwam of 76% consistency. Then vessel was removed from the fire, while hot condition ingredient number 1 was added and mixed well, finally mixed powders of ingredient number 2 to 12, 14 and 15 were added, mixed thoroughly and obtained the homogenous product. Allowed it to cool to room temperature and added the required quantity of ingredient number 17 and 18 and mixed thoroughly and was packed in tightly closed containers to protect from light and moisture. Powder Microscopy Drug (5g) was weighed, mixed with 50ml of water in a beaker and warmed gently inorder to make complete dispersion in water. Then mixture was centrifuged and decanted supernatant. The sediment was washed several times with distilled water, centrifuged again and decanted the supernatant. Small quantity of the sediment was taken and mounted in glycerin, out of which another small quantity was taken in watch glass and a few drops of phloroglucinol and concentrated hydrochloric acid were added, mounted in glycerin to locate lignified cells. The following characters in different mounts were observed (Wallis, 1987; Johansen, 1940). Physico-chemical Analysis The Physico-chemical data viz., moisture content, ash values, alcohol and water soluble extractives, ph value, bulk density and estimation of sugar were evaluated as per the standard method (Anonymous, 1987; 1998).

57 50 TLC/HPTLC finger print analysis Preparation of extracts for TLC Three batch samples of the drug (5g each) were extracted with 20 ml of chloroform and 20 ml of alcohol separately. Both the extracts were filtered and concentrated separately up to 10 ml in volumetric flask and were used for the TLC/HPTLC finger print analysis. TLC/HPTLC finger print studies of chloroform and alcohol extracts of the drug were carried out using aluminum plate precoated with silica gel 60 F 254 (E. Merck) with CAMAG Linomat IV sample applicator. The chromatograms of both the extracts were taken using the solvent systems toluene: ethyl acetate (9: 1) and toluene: ethyl acetate (6 : 4) for chloroform and alcohol extracts respectively. The plates were dried at room temperature and observed the spots at various wavelengths. The plates were scanned at 254 nm to record the finger print spectrum after that same plates were visualized at UV-366 nm and derivatized with spraying of vanillin-sulphuric acid reagent and heated at 105 C till appeared coloured spots (Wagner and Bladt, 1984; Sethi, 1996). Estimation of microbial load The microbial load viz. total bacterial count (TBC), total fungal count (TFC), Enterobacteriaceae, Escherichia coli, Salmonella spp and Staphylococcus aureus were estimated as per standard method (WHO, 1998). Estimation of heavy metals The method used for the analysis of heavy metals like lead, cadmium, mercury and arsenic was as per Guidelines & WHO (Anonymous, 1998) and AOAC (Anonymous, 2005). Details of the Instrument and operating parameters Thermo Fisher M Series, V1.27 model Atomic Absorption Spectrometer (AAS) was used for the analysis. The operating parameters: Lead and Cadmium: Instrument technique - Flame technique; wavelength (Lead) nm; wavelength (Cadmium) nm; slit width mm; lamp current (Pb) ma; lamp current (Cd) ma; carrier gas and flow rate - air and acetylene, 1.1 L/min; sample flow rate - 2 ml/min. Mercury: Instrument technique - Cold vapour technique; wavelength nm; slit width mm; lamp current ma; carrier gas and flow rate - argon, 1.1 L/min; sample flow rate - 5ml/ min. Arsenic: Instrument technique - Flame vapor technique; wavelength

58 51 nm; slit width mm; lamp current ma; carrier gas and flow rate - acetylene, argon, 1.1 L/min; sample flow rate - 5ml/min. The Hallow cathode lamp for Pb, Cd, Hg and As analysis were used as light source to provide specific wavelength for the elements to be determined. Analysis of aflatoxins Aflatoxins B 1, B 2, G 1 and G 2 were analyzed as per Official Analytical Methods of the American Spice Trade Association (ASTA), Details of instrument and operating parameters High Performance Liquid Chromatography (Thermo Fisher) was used for the analysis of aflatoxins. Column - Ultra C18, 250 X 4.6 mm, 5 μm particles; Mobile phase: Water: Acetonitrile: Methanol (65: 22.5: 22.5); Flow rate: 1 ml/min; Temperature: 35º C; Detector: Fluorescence detector at 360 nm; Injection: 20 μl (Aflatoxins mixture and sample) Analysis of pesticide residue The method used for the analysis of pesticide residues was as per AOAC (Anonymous, 2005). Pesticide residues were analyzed by Gas Chromatography- Mass Spectra (GC-MS) (Instrument-Agilent, detector-mass selective detector, column specification-db5ms, carrier gas - helium, flow rate - 1ml/min, column length - 30 m, internal diameter mm, column thickness ìm). Results and Discussion The prepared drug was obtained in brownish colour semi-solid, shows characteristic of its own odour and gives sweetish taste. Identification Microscopy Parenchyma cells with wavy margin in surface view filled with dark reddish brown contents from the epidermis of the seed, perisperm cells in surface view filled with starch grains (Tukhm-e-Khurfa); elongated palisade like parenchyma and polygonal parenchyma cells from the cotyledons (Maghz-e-Tukhm-e-Petha); vessels with pitted thickening, transverse to oblique perforations with tail like projections at one or both ends (Sandal Safaid / Sandal Surkh); clusters of calcium oxalate crystals (Ood-e-Saleeb / Beikh-e-Anjabar) (upto 100μ in Beikhe-Anjabar); prismatic crystals of calcium oxalate upto 500μ (Darunaj Aqrabi);

59 52 lignified elongated sclereids upto 260μ long (Mazu); lignified sclereids / stone cells of polygonal in shape upto 150μ (Ood-e-Saleeb) (Fig. 1). Physico-chemical parameters Physico-chemical parameters of Majoon-e-Hamal Ambari Alwi Khani are tabulated in Table 2. Quantitatively evaluated data revealed that the moisture content was %, ash content was 2.61 % and acid insoluble ash 1.44 % Outer epidermal cells Tukhm-e-Khurfa Perisperm cells Sandal Safaid / Sandal Surkh Darunaj Aqrabi Prismatic calcium oxalate crystals Pitted vessels Ood-e-Saleeb Stone cells Fig. 1: Powder Microscopy Maghz-e-Tukhm-e-Petha Palisade like cotyledonary parenchyma cells Polygonal cotyledonary parenchyma cells Beikh-e-Anjabar / Ood-e-Saleeb Druses of calcium oxalate crystals Mazu Elongated sclereids / stone cells

60 53 Table 2: Physico-chemical Parameters of studied drug S.No. Parameters Majoon-e-Hamal Ambari Alwi Khani indicated the negligible amount of siliceous matter present in the drug. The water soluble extractive value of the drug % indicated the presence of inorganic and more polar organic content and the alcohol soluble extractive value % indicated the extraction of polar constituents. TLC studies of chloroform extract The TLC studies of chloroform extract are tabulated in Table 3. All the three batch samples showed identical spots in UV-254 nm, UV-366 nm and visible light (after derivatized with vanillin sulphuric acid reagent). In UV 254 nm, 366 nm and visible light it shows 06, 10 and 11 spots respectively with different R f values (Fig. 2). HPTLC finger print studies of chloroform extract Batch - I Batch - II Batch - III 1 Moisture (% w/w) Extractive values (% w/w) Alcohol soluble matter Water soluble matter Ash values (% w/w) Total ash Acid insoluble ash ph values 1% Aqueous solution % Aqueous solution Sugar estimation Reducing sugar (% w/w) Non reducing sugar (% w/w) Bulk Density All values are mean of three determinations The finger print of the chloroform extract shows 14 peaks of which peaks at R f 0.11, 0.22 and 0.48 were the major peak whereas peaks at R f 0.03, 0.06, , 0.43, 0.60, 0.67, 0.77, 0.83, 0.89 and 0.99 were moderately smaller peaks (Fig. 3). The HPTLC densitometry chromatogram of chloroform extract of three batch samples of Majoon-e-Hamal Ambari Alwi Khani formulation were found to be same when scanned at 254 nm (Fig. 4)

61 54 Table 3: R f values of the chloroform extract of Majoon-e-Hamal Ambari Alwi Khani Solvent Rf Values System UV- 254 nm UV 366 nm Visible light (after derivatization with vanillin sulphuric acid reagent) Toluene: 0.82 Green 0.89 Blue 0.90 Violet Ethyl 0.73 Green 0.73 Blue 0.73 Violet acetate 0.41 Green 0.67 Yellowish green 0.69 Yellowish green (9:1) 0.31 Green 0.59 Violet 0.59 Gray 0.19 Green 0.54 Brown 0.54 Grey 0.10 Green 0.45 Blue 0.41Violet 0.41 Violet 0.32 Violet 0.21 Blue 0.26 Yellowish green 0.17 Blue 0.23 Yellowish green 0.12 Blue 0.16 Violet 0.12 Grey UV- 254 nm UV- 366 nm Visible Light (After derivatisation with vanillinsulphuric acid reagent) Fig 2: TLC photos of chloroform extract

62 55 Fig. 3: HPTLC finger print profile of chloroform extract at 254 nm of Majoon-e-Hamal Ambari Alwi Khani formulation Fig. 4: HPTLC densitometry chromatogram of chloroform extracts of three batch of Majoon-e-Hamal Ambari Alwi Khani at 254 nm TLC studies of alcohol extract The TLC studies of alcohol extract are tabulated in Table 4. All the three batches of Majoon-e-Hamal Ambari Alwi Khani formulation showed identical spot in UV- 254 nm, UV-366 nm and visible light (after derivatized with vanillin sulphuric acid reagent). In UV 254 nm, 366 nm and visible light it shows 5, 10 and 6 spots respectively with different R f values (Fig. 5).

63 56 Table 4: R f values of the alcohol extract of Majoon-e-Hamal Ambari Alwi Khani Solvent R f Values System UV- 254 nm UV 366 nm Visible Light (After derivatisation with vanillin sulphuric acid reagent) Toluene: 0.83 Green 0.89 Fluorescent blue 0.90 Grey Ethyl 0.72 Green 0.83 Fluorescent blue 0.82 Red acetate 0.68 Green 0.74 Blue 0.69 Gray (1:1) 0.37 Green 0.70 Blue 0.44 Violet 0.10 Green 0.66 Blue 0.19 Grey 0.57 Fluorescent blue 0.11 Grey 0.48 Fluorescent blue 0.41 Blue 0.21 Blue 0.12 Blue UV-254nm UV-366nm Visible Light (After derivatisation with vanillin sulphuric acid reagent) Fig. 5: TLC photos of alcohol extract

64 57 HPTLC finger print studies of alcohol extract of Majoon-e-Hamal Ambari Alwi Khani formulation The finger print of the chloroform extract shows 10 peaks of which peaks at R f 0.02, 0.44, 0.77 and 0.94 were the major peak whereas peaks at R f 0.05, 0.11, 0.19, 0.47, 0.50 and 0.65 were moderately smaller peaks (Fig. 6). The HPTLC densitometry chromatogram of alcohol extract of three batch samples were found to be same when scanned at 254 nm (Fig. 7) Fig. 6: Fig. 7: HPTLC finger print profile of alcohol extract of Majoon-e-Hamal Ambari Alwi Khani formulation at 254 nm HPTLC densitometry chromatogram of alcohol extracts of Majoon-e-Hamal Ambari Alwi Khani formulation at 254 nm

65 58 Detection of quality parameters In order to assess the quality of drug samples the estimation of microbial load viz. total bacterial count (TBC), total fungal count (TFC), Enterobacteriaceae, Escherichia coli, Salmonella spp and Staphylococcus aureus were analyzed and found to be in permissible limit. The results are shown in (Table 5). The heavy metal such as lead was present within the permissible limit where as cadmium; mercury and arsenic were not detected from the drug samples (Table-6). The studies of other parameters like estimation of afltoxins such as B 1, B 2, G 1 and G 2 and pesticide residue such as organo chlorine group, organo phosphorus group, alachlor, aldrin, chlordane, DDT, endosulfan, heptachlor, lindane and malathion were not detected from the drug. Table 5: Microbial load reported in Majoon-e-Hamal Ambari Alwi Khani formulation Parameters Results WHO Limits for internal use Total Bacterial Count (TBC) 4 x 10 3 cfu/g 1x10 5 cfu/g Total Fungal Count (TFC) <10CFU 2 x 10 2 cfu/g 1x10 3 cfu/g Enterobacteriaceae Absent 1x10 3 cfu/g Escherichia coli Absent 1x10 1 cfu/g Salmonella spp Absent Absent Staphylococcus aureus Absent Absent Table 6: Analysis of heavy metals in Majoon-e-Hamal Ambari Alwi Khani formulation Sl.No. Parameters Values 1. Lead ppm 2. Cadmium Not detected 3. Arsenic Not detected 4. Mercury Not detected Conclusion Standardization is an essential part for the evaluation of scientific standards to justify the quality of Unani herbal formulations. To maintain the batch-to-batch consistency and quality of the drug, each plant material used in preparation of Majoon-e-Hamal Ambari Alwi Khani was identified and evaluated for their pharmacopoeial standards. The evaluated pharmacognostical and physico-

66 59 chemical parameters will be helpful for fixing-up pharmacopoeial standards of the drug. TLC/HPTLC finger print profile of chloroform and alcohol extracts provided a suitable method for monitoring the identity and purity and also standardization of the drug. Analyzed data of quality parameters viz. heavy metals, aflatoxins, pesticide residues and microbial load were found within permissible limit of WHO, which indicate that the studied drug Majoon-e-Hamal Ambari Khani is free from toxic materials and can be used as uterine tonic, and also in the ailments of atony to the uterus and habitual abortion. Acknowledgement The authors are extremely thankful to the Director General, and Dy. Director General, CCRUM, New Delhi, for valuable guidance, encouragement and providing necessary research facilities to carry out the present studies. Reference Anonymous, National Formulary of Unani Medicine, Part-I. Central Council for Research in Unani Medicine, Ministry of Health and Family Welfare, Govt. of India, p Anonymous, Physico-chemical standards of Unani Formulations, Part II. CCRUM, Ministry of Health & Family Welfare, New Delhi, pp Anonymous, Quality Control Methods for Medicinal Plant Materials. World Health Organization, Geneva, pp Anonymous, The Unani Pharmacopoeia of India, Part-I, Vol.-III. Ministry of Health & Family Welfare, New Delhi, pp. 3-4, and Anonymous, The Unani Pharmacopoeia of India, Part-I, Vol.-V. Ministry of Health & Family Welfare, New Delhi, pp Anonymous, The Ayurvedic Pharmacopoeia of India, Part-I, Vol.-VI. Ministry of Health & Family Welfare, New Delhi, pp Anonymous, The Unani Pharmacopoeia of India, Part-I, Vol.- VI. Ministry of Health & Family Welfare, New Delhi, pp Anonymous, Official Analytical Methods of the American Spice Trade Association (ASTA). Inc. 4 th edn., New Jersey, pp Anonymous, General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine, WHO, Geneva. Anonymous, General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine, WHO, Geneva.

67 60 Anonymous, Official Methods of Analysis of AOAC International, Horwitz W, Latimer G W. (edn). 18 th Edn. AOAC International: Maryland, chapter 3, p , chapter 10 p and chapter-26, p.17. Anonymous, Global Atlas of Traditional, Complementary and Alternative Medicine, Vol.1 and Vol. 2. WHO, Geneva. EMEA, Guidelines on Quality of Herbal Medicinal Products/Traditional Medicinal Products. EMEA/CVMP/814OO Review. London: European Agency for the Evaluation of Medicinal Products (EMEA). Johansen, D.A., Plant Microtechnique. Mc. Graw Hill Book Company Inc., New York and London, pp Kunle, O.F., Egharevba, H.O., Ahmadu, P.O., Standardization of Herbal Medicines: The Review. Int. J Biodivers. Conserve 4: Sethi, P.D., High Performance Thin Layer Chromatography, 1st Edn., Vol. X. CBS Publishers and Distributers, New Delhi, pp Wagner, H. and Bladt, S., Plant Drug Analysis - A Thin Layer Chromatography Atlas. Springer-Verlag, 2nd Edn., Germany. Wallis, R.E., Text Book of Pharmacognosy, 5 th Edition. CBS Publisher & Distributors, Delhi, pp

68 Standardization and Evaluation of Antibacterial Activity of Some Unani Drugs 1 Tariq Rahim Malik, 2 Tajuddin and 2 *Aziz ur Rahman 1 Department of Ilmul Advia; 2 Department of Saidla, A.K. Tibbiya College, Aligarh Muslim University, Aligarh Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract M icroorganisms have developed resistance due to indiscriminate use of many antibiotics which are also associated with severe adverse effects. To overcome these problems plants and their products provides a better substitute for treating infectious diseases. Afsanteen (Artemisia absenthium), flowers of Banafshah (Viola odorata) and Bisheri booti (Aerva lanata) are used in Unani System of Medicine since long time either as single drug or in compound formulation. All the three drugs were screened for antibacterial activity against different strains of gram positive and gram negative bacteria such as Bacillus sp. Staphylococcus aureus, Streptococcus pyogenes Escherichia coli, Citrobacter sp., Klebsiella sp. Salmonella typhimurium and Proteus sp. etc. The Aqueous and alcoholic extracts of these crude drugs were used to determine antibacterial activity by disc-agar diffusion technique at four concentration levels (100, 50, 25 and 12.5μg/disc) and compared with standard drug ciprofloxacin at concentration of 5 μg/disc. Zone of Inhibition was taken as the parameter for the assessment of antibacterial activity. Of all the drugs tested, the alcoholic extract of Viola odorata was found to be the most active against Salmonella typhimurium at the doses of 50 and 100μg/disc. Furthermore, before testing these drugs for antimicrobial activity, their samples were also checked for authenticity based on certain physico-chemical parameters. Keywords: Artemisia absenthium, Viola odorata, Aerva lanata, Antibacterial, Physico-chemical Introduction It is well known that infectious diseases account for a high proportion of the health problems in the developing countries, hence it becomes necessary to search for quick, effective and safe remedies for these health problems and this gave a stimulus to the search for new anti-microbial substances. World Health Organization noted that majority of the world s population depends on traditional medicine for primary healthcare. Plants which are widely used as medicines constitute a major source of natural organic compounds (Prabuseenivasan et al., 2006). During the recent past there has been a growing interest in traditional and alternative systems of medicine in industrialized countries. Now greater attention has been given to the plant and their products for use in therapy due to hazards and side effects of synthetic drugs. Nature has been a source of medicinal agents for thousands of years and a good number of modern drugs have been isolated from natural sources, many of these isolations were based on the use of the agents in traditional medicine (Owolabi et al., 2007). In India 2 *Author for correspondence; rahman.mdi@gmail.com

69 62 medicinal plants are a source of great economic value. Nature has bestowed on us a very rich botanical wealth and a large number of diverse types of medicinal plants grow in different parts of the country (Parekh et al., 2005). Microorganisms have developed resistance due to indiscriminate use of many antibiotics, thus creating an immense clinical problem in the treatment of infectious diseases (Davis 1994). In addition, antibiotics are also sometimes associated with adverse effects on patients, which include hypersensitivity, depletion of beneficial gut mucosal flora, immune-suppression and allergic reactions (Ahmad et al., 1998). Thus there is an urgent and constant need to develop new and effective therapeutic agents for the treatment of infectious diseases (Khan and Jain, 2003; Monroe and Polk, 2000; Bhavnani and Ballow, 2000). Several screening studies have been carried out in different parts of the world which aim at knowing the different phytochemical constituents of medicinal plants possessing antimicrobial activity so as to use them for the treatment of microbial infections as a possible alternative to chemically synthetic drugs, to which many infectious microorganisms have become resistant (Akinpelu and Onakoya, 2006). There are several reports on the antimicrobial activity of different herbal extracts in different regions of the world (Chung et al., 2004; Nair and Chanda, 2004; De Boer et al., 2005; Nair et al., 2005). Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, recently much attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine (Essawi and Srour, 2000). Plant-based antimicrobials represent a vast untapped source of medicines and further exploration of plant antimicrobials needs to occur. Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials (Iwu et al., 1999).Unani medicine also offers a number of single and compound preparations that are used successfully in the management of various infectious diseases (Rahman et al., 2013). Therefore, present study was undertaken to evaluate the anti-microbial potential of aqueous and alcoholic extract of Afsanteen (Artemisia absinthium) - whole herb, Banafshah (Viola odorata) - flowers and Bisehri Booti (Aerva lanata) - whole herb. These Unani medicinal plants were selected on the basis of their actions mentioned in classical as well as modern medico-ethno-botanical literatures as antiseptic, anti-microbial, anti-fungal or as anti- infective and blood purifier, or are in clinical practices of Unani physicians for treatment of various infectious diseases.

70 63 Since the efficacy, safety and global acceptance of medicinal plants depends upon its authenticity and quality, therefore, standardization of these drugs is mandatory to make them less controversial and more efficacious to use (Rahman et al., 2013). Hence, these drugs were also tested for their originality based on certain physico-chemical parameters before evaluating for antimicrobial activity. Materials and Methods The study was carried out at the Department of Ilmul Advia, A.K. Tibbiya College, A.M.U., during Collection of plants: The plant materials were procured from different sources. Afsanteen and Banafshah were purchased from the local market of Srinagar (Jammu and Kashmir), Bisehri booti was obtained from the herbarium of Department of Ilmul Advia, Faculty of Unani Medicine, Aligarh Muslim University, Aligarh. The samples were authenticated in Pharmacognosy section of the Department of Ilmul Advia and found within the range of standards (Anonymous, 1984, 1992, 2007; Khory and Katrak, 1985). The samples have been submitted to the Department of Ilmul Advia for future reference. All the drugs were dried in shade and then powdered. Physico-chemical parameters It included successive extractive values, ash values (total ash, acid insoluble ash, water soluble ash), moisture content, loss of weight on drying, ph (1% and 10% solution), water and alcohol soluble contents (Anonymous, 1968; Jenkins et al.,1967; Anonymous, 1987), chemical treatment and fluorescence analysis (Anonymous, 1992). Qualitative estimation of chemical constituents present in the powder form of test drugs was carried out according to the scheme proposed by Bhattacharjee and Das (1969) depicted in figure 1. Anti-bacterial studies Extracts preparation Alcoholic extracts of the coarsely powdered drugs were prepared by the method of Ibrahim and Osman (1995) with some modifications. Coarse powdered drugs were extracted using a Soxhlet s apparatus with 95% ethanol as solvent, at 55 C for 06 hours or until the extracting return in the Siphon was colorless. The resultant extract was then concentrated in a water bath, at 40 C. The, stock solutions were prepared in 95% ethanol. Strict aseptic precautions were followed throughout the process and the heat wherever needed was kept as low as

71 64 possible to prevent the thermolabile substances present in the drugs from destruction. Same procedure, as mentioned above, was carried out to prepare aqueous extracts using distilled water as solvent at 60 C and concentrated at 50 C. Figure 1: Scheme for qualitative analysis of chemical constituents present in the test drugs

72 65 Bacterial strains used in the study Various Gram positive and Gram negative bacteria were used for detection of the anti-bacterial activity of the extracts (Table 1). Standard and clinical bacterial strains were obtained from the Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh. Antibacterial susceptibility testing Serial dilutions of the alcoholic extracts were prepared in ethyl alcohol so that 01 ml contained 100 times the amount of the extracts required per disc. Aliquots (01 ml) of these dilutions were transferred to bottles containing batches of 100 discs (06mm in diameter) of filter paper (Whatmann No.1, Whatmann, England). Bottles were placed in a water bath at 50 C with occasional shaking to allow an even distribution of the extracts between discs until complete evaporation of the alcohol had been achieved. Anti-bacterial activity was determined using the disc-agar, diffusion technique according to the method described by Finegold and Martin, Overnight cultures of the bacterial isolates were prepared in Mueller-Hinton Broth (M 173, HiMedia, India) standardized to match 0.5 McFarland Barium sulphate tubes and, used to inoculate Mueller-Hinton agar plates. Discs impregnated with different amounts of the extracts were placed in the surface of these plates and incubated at 37 0 C overnight. Diameters of zones of inhibition were recorded. For fastidious organisms such as Streptococci, the agar supplemented with 05% sterile, defibrinated blood was used. The cultures of Streptococci were incubated Table 1: Standard and clinical bacterial strains used for the detection of antibacterial activities of the drug S.No. Gram Negative bacterial strains S.No. Gram Positive bacterial strains 1. Citrobacter sp. 1. Bacillus sp. 2. Escherichia coli 2. Staphylococcus aureus 3. Klebsiella sp 3. Staphylococcus aureus ATCC Proteus sp. 4. Streptococcus pyogenes 5. Pseudomonas aerugenosa 6. Pseudomonas aerugenosa ATCC Salmonella typhimurium 8. Shigella sp.

73 66 in an atmosphere enriched with 10% CO 2, The whole experiment was performed in triplicate. The concentration levels used to determine antibacterial activity of test drug was 50, 25 and 12.5μg/disc for Afsanteen while concentration levels for Gul-e- Banfshah and Bisehri Booti were 100, 50, 25 and 12.5μg/disc. Commercial diagnostic sensitivity disc (Hi Media) of Ciprofloxacin was used for comparison at concentration of 5μg/disc. Furthermore, results were analyzed statistically by Student s t test and P value d 0.05 were considered statistically significant. Results Physico-chemical parameters The analytical values of different physicochemical parameters such as solubility, moisture content, loss of weight on drying, ash values and ph values of the test drugs were determined and are depicted in table 2. Successive extractive values of the powdered test drugs in different solvents viz. petroleum ether, diethyl ether, chloroform, benzene, alcohol and distilled water were also determined with Soxhlet s apparatus. The extractive values were expressed in percentage and depicted in table 3. Table 2: Physico-chemical values of the test drugs Physico-chemical parameters Afsanteen Gul-e-Banfshah Bisehri Booti (Mean±SE) (Mean±SE) (Mean±SE) Water soluble contents 10.10± ± ±0.05 (1% solution) Water soluble contents 6.34± ± ±0.02 (10% solution) Alcohol soluble contents 8.08± ± ±0.04 (1% solution) Alcohol soluble contents 0.06± ± ±0.02 (10% solution) Moisture content (%) 8.83± ± ±0.14 Loss on drying at C (%) 12.07± ± ±0.09 Total Ash (%) 4.70± ± ±0.03 Acid Insoluble Ash (%) 2.63± ± ±0.19 Water Soluble Ash (%) 4.32± ± ±0.01 ph (1% solution) 1.60± ± ±0.03 ph (10% solution) 1.70± ± ±0.00

74 67 Table 3: Successive extractive values of the test drugs Solvent Extractive values in % (Mean±SE) Afsanteen Gul-e-Banfshah Bisehri Booti (Mean±SE) (Mean±SE) (Mean±SE) Petroleum Ether 2.95± ± ±0.15 Diethyl Ether 2.64± ± ±0.02 Chloroform 1.79± ± ±0.03 Benzene 0.21± ± ±0.00 Alcohol 22.36± ± ±0.06 Distilled Water 12.47± ± ±0.03 Qualitative estimation The therapeutic properties of the crude drugs are mainly due to presence of physiologically active chemical constituents present in the drugs. The qualitative phytochemical analysis of all the three test drug extracts was done systematically and the presence or absence of active compounds is shown in table 4. Chemical treatment and fluorescence analysis The powdered test drugs were treated with different chemicals and the changes in color were observed in day light and shown in table 5. For fluorescence Table 4: Qualitative analysis of chemical constituents S.No. Tested for Afsanteen Gul-e-Banfshah Bisehri Booti 1. Alkaloids +ve +ve +ve 2. Amino acids +ve +ve +ve 3. Flavonoids +ve +ve -ve 4. Phenols +ve +ve -ve 5. Proteins +ve +ve +ve 6. Resins -ve -ve -ve 7. Saponins -ve -ve +ve 8. Sterols / Terpenes +ve +ve +ve 9. Sugar (Reducing) +ve +ve +ve 10. Sugars (Non-reducing) -ve -ve +ve 11. Tannins -ve -ve +ve

75 68 analysis powder of all the three drugs were treated with different reagents and change in color was observed in Ultra-violet (UV) radiations alongwith day light, results are depicted in table 6. Table 5: Treatment of test drugs with chemicals Chemical Treatment Afsanteen Gul-e-Banfshah Bisehri Booti Change in color Change in color Change in color Concentrated HNO 3 Golden Brown Dark Brown Yellow Concentrated HCL Green Brownish Green Green Concentrated H 2 SO 4 Black Black Black 2% Iodine Solution Yellow Brown Green Acetic Acid Brown Brown Green 10% NaOH Solution Brown Brown Green Acetic Acid + H 2 SO 4 Brownish Yellow Brown Green 10% NaOH + few drops Black Black Green of CuSO 4 sol. 10% NaOH + few drops Golden Brown Golden Brown Green of 5% lead acetate Acetic Acid + few drops Yellowish Brown Yellowish Brown Yellowish Green of 5% ferric chloride + Concentrated H 2 SO 4 5% ferric chloride Fluorescent Green Fluorescent Green Fluorescent Green Table 6: Fluorescence analysis of test drugs Reagents Afsanteen Gul-e-Banfshah Bisehri Booti Change in color Change in color Change in color Day light UV Light Day light UV Light Day light UV Light 1N HCL Dull Yellow Green Light Brown Brown Dull Green Green Concentrated Golden Green Dark Brown Brown Yellow Green HNO 3 Brown Concentrated Black Green Black Blackish Black Green H 2 SO 4 Green Water Straw Dull Green Brown Brownish Parrot Green Green Green Acetic Acid Straw Green Brown Black Green Green 5% Ferric Chloride Straw Dark Fluorescent Green Fluorescent Green Green Green Green 10% NaOH Straw Dark Green Brown Dark Green Green Parrot Green 2N HCL Light Brown Green Brown Black Dull Green Green 1N H 2 SO 4 Straw Green Brown Black Dull Green Brown 4N H 2 SO 4 Straw Green Brown Black Yellowish Green Green

76 69 Anti-bacterial studies Aqueous and alcoholic extracts of Afsanteen, Gul-e-Banfshah and Bisehri Booti were screened against Gram positive and Gram negative bacterial strains. Results are depicted in table 7 (a,b,c). Table 7 (a): Antibacterial activity of Afsanteen (Artemisia absinthium) Microbial Test Drug Zone of inhibition (ZOI) in mm (Mean ± S.E.) Strains Concentration (μg/disc) Alcoholic Aqueous Ciprofloxacin extract Extract (5μg/disc) Bacillus sp. 50 No Effect No Effect 17.10± No Effect No Effect Not Tested Not Tested Staphylococcus 50 No Effect No Effect 25.16±0.02 aureus 25 No Effect No Effect Not Tested Not Tested Streptococcus 50 No Effect No Effect 10.13±0.01 pyogenes 25 No Effect No Effect Not Tested Not Tested Escherichia 50 No Effect No Effect 19.10±0.00 coli 25 No Effect No Effect Not Tested Not Tested Klebsiella sp 50 No Effect No Effect 25.03± No Effect No Effect Not Tested Not Tested Pseudomonas 50 No Effect No Effect 23.16±0.17 aerugenosa 25 No Effect No Effect Not Tested Not Tested Proteus sp. 50 No Effect No Effect 13.16± No Effect No Effect Not Tested Not Tested Salmonella 50 No Effect No Effect 10.13±0.02 typhimurium 25 No Effect No Effect Not Tested Not Tested

77 70 Table 7 (b): Antibacterial activity of Banafshah (Viola odorata) Microbial Test Drug Zone of inhibition (ZOI) in mm (Mean ± S.E.) Strains Concentration (μg/disc) Alcoholic Aqueous Ciprofloxacin extract Extract (5μg/disc) Staphylococcus 100 No Effect No Effect 25.16±0.02 aureus 50 No Effect No Effect ATCC Not Tested Not Tested Not Tested Not Tested Streptococcus 100 No Effect No Effect 10.13±0.02 pyogenes 50 No Effect No Effect 25 Not Tested Not Tested Not Tested Not Tested Escherichia 100 No Effect No Effect 19.10±0.00 coli 50 No Effect No Effect 25 Not Tested Not Tested Not Tested Not Tested Shigella sp. 100 No Effect No Effect 16.10± No Effect No Effect 25 Not Tested Not Tested Not Tested Not Tested Pseudomonas 100 No Effect No Effect 23.16±0.17 aerugenosa 50 No Effect No Effect ATCC Not Tested Not Tested Not Tested Not Tested Salmonella ±0.15(P<0.01) No Effect 10.13±0.02 typhimurium ±0.02(P<0.01) No Effect 25 Not Tested Not Tested Not Tested Not Tested Citrobacter sp. 100 No Effect No Effect 22.00± No Effect No Effect 25 Not Tested Not Tested Not Tested Not Tested Discussion and Conclusion Standardization of the test drugs An impressive number of modern drugs have been isolated from natural sources; many of these isolations were based on the uses of the agents in traditional medicine (Rahman et al., 2011). This plant-based, traditional medicine system

78 71 Table 7 (c): Antibacterial activity of Bisehri Booti (Aerva lanata) Microbial Test Drug Zone of inhibition (ZOI) in mm (Mean ± S.E.) Strains Concentration (μg/disc) Alcoholic Aqueous Ciprofloxacin extract Extract (5μg/disc) Staphylococcus 100 No Effect No Effect 25.16±0.02 aureus 50 No Effect No Effect ATCC Not Tested Not Tested Not Tested Not Tested E. Coli 100 No Effect No Effect 19.10± No Effect No Effect 25 Not Tested Not Tested Not Tested Not Tested Shigella sp. 100 No Effect No Effect 16.10± No Effect No Effect 25 Not Tested Not Tested Not Tested Not Tested Pseudomonas 100 No Effect No Effect 23.16±0.17 aerugenosa 50 No Effect No Effect ATCC Not Tested Not Tested Not Tested Not Tested Salmonella 100 No Effect No Effect 10.13±0.02 typhimurium 50 No Effect No Effect 25 Not Tested Not Tested Not Tested Not Tested Citrobacter sp. 100 No Effect No Effect 22.00± No Effect No Effect 25 Not Tested Not Tested Not Tested Not Tested continues to play an essential role in health care, with about 80% of the world s inhabitants relying mainly on traditional medicines for their primary health care (Doughari et al., 2008). Therefore, with the ever-increasing use of herbal medicines worldwide and the rapid extension of the global market for these products, the safety and quality of medicinal plant materials and finished products have become a major concern for health authorities and pharmaceutical industries. From the time of collection of drug to its storage and up to the production of medicine, chances of deterioration in quality are quite frequent, resulting in the decline of the efficacy of drug. To overcome these problems of Unani drugs it is almost inevitable to standardize the drugs for their rational therapeutic use. A disease cannot be managed comprehensively until the delivery

79 72 of genuine samples of drug is ensured (Jahan et al., 2008; Alam and Khan, 2010). These physico-chemical parameters are considered as tools of checking quality, identity, purity and strength of the Unani drugs. The solubility and extractive values sometimes are used as an index of purity for formulations. The amount of the extract that the drug yields in a solvent is often an approximate measure of the amount of certain constituents that the drug contains. Therefore, for establishing the standards of any drug these extractive values and solubility play an important role, as the adulterated or exhausted drug material will give different values rather than the extractive percentage of the genuine one (Jenkins et al.,1967). The Moisture content of the test drugs determines the release of their active ingredients as well as their chemical, physical, microbial, shelf-life properties and adulterations (Jacob et al., 2006). Loss in weight on drying at C also indicates towards the loss of volatile substances along with water. ph value of the drug determines whether the drug absorb in stomach or intestine because the drugs in the opposite ph are unionized and absorbed rapidly from stomach or intestine. The ph of all the three test drugs were found to be acidic therefore, test drugs gets ionized in stomach and absorbs somewhere in intestines. Adulteration of herbal drugs with unwanted materials like earthy matters resulting in higher ash percentage. Therefore, Estimation of Ash values are important criterion for judging impurities along with identity and quality of the drugs (Jenkins et al.,1967). The fluorescence analysis under UV light and treatment of test drugs with chemicals are very characteristic and helps in the identification of the drugs (Chase and Pratt, 1949) from the adulterated ones. Many compounds show distinct colours under UV-light after treatment e.g. aconitine (light blue), berberine (yellow) and emetine (orange). Hence, the fluorescence analysis provides valuable information regarding the genuineness of drugs. The efficacy of plant drugs is mainly because of their active chemical constituents therefore, it is necessary to determine active compounds in the test drugs at least qualitatively. Alkaloids, tannins, sterols, phenols, flavonoids, oils etc possesses antibacterial property. If all or some of these are present in drugs then drug may have antimicrobial efficacy (Said 1996; Katzung, 2001; Anuradha and Goyal, 1995; Evans, 2003). Antimicrobial studies Many diseases are being spread in the world by microorganisms and the discovery of microorganisms as the causative agent of many infectious diseases of man naturally created an interest in substances toxic to these organisms and this gave a stimulus to the search for new antimicrobial substances. The

80 73 antibiotics in modern therapeutic system have tremendous effect in controlling the infectious diseases (Finland, M. 1978). However, the advent of escape mechanism (cf.drug resistance) adopted by most of the pathogens certainly needs a suitable replacement of the presently available antibiotics (Conly et al., 1992; Threlfall et al., 1996). As plant drugs which constitute the major chunk of Unani therapeutics are considered important because they are physiologically innocuous and safe and also because they may be useful against resistant microorganisms (Rahman et al., 2013). According to Unani literature in infectious diseases the blood has become impure and the treatment is attempted with a class of drugs described as blood purifiers. In all the conditions like bacteraemia, septicaemia, viraemia, toxaemia, pyrexia, pyaemia, uraemia, etc., the blood contains such substances that should not be normally present in it or an abnormally high concentration of normal constituents. There may be bacteria, bacterial toxins, viruses, allergens, pus, metabolic products, bile pigments, etc. They are listed as impurities that have rendered the blood impure (Vohola, 1985).The antibiotics and other chemotherapeutic agents purify the blood by destroying the infective organisms. In the present study antimicrobial activity of the alcoholic and aqueous extracts of Unani drugs was quantitatively assessed using agar well method on the basis of Zone of Inhibition (ZOI) which was expressed in mm. Extracts of Afsanteen and Bisehri Booti at given concentration level doesn t show any significant activity against any of the bacterial species tested. While, the alcoholic extract of Gule-Banfshah showed significant antibacterial activity against Salmonella typhimurium at the concentration level of 50 and100 μg/disc. As bacterial strains were the clinical isolates and may develop resistance. So Afsanteen didn t show any effect at the particular test doses against any of the test species. This also correlates partially with the studies carried out by Anesini and Perez, 1993 and Perez and Anesini, 1994 in which Afsanteen extracts doesn t show any effect against Salmonella typhimurium and penicillin G resistant Staphylococcus aureus, Escherichia coli and Aspergillus niger. Gul-e-Banafshah doesn t depict effect against any of the other test strains except Salmonella typhimurium. This result is totally in contrast to the study carried out by Afaq et al., 2004 where they have shown the aqueous extract of the drug effective against Escherichia coli K-12, Staphylococcus aureus, Bacillus subtilis ATCC 6051, but no effect was seen against Salmonella typhimurium ATCC The difference in the results may be due to a number of reasons as antibacterial activity of different medicinal plants shows variability due to different chemicals found in them as well as the quality and quantity produced during different seasons, at different stages and in different species of plants. Variation in results may also be related to extraction methodology (Prasad et al., 1999). The difference may

81 74 also be due to the differences in the bacterial strains tested. No effect was shown by Bisehri booti at any dose against any of the bacterial strain. Although it is usually recommended in urinary tract infections may be because of other actions like anti-inflammatory and diuretic. There are many documented, scientifically proved and globally accepted Unani drugs having striking and significant antimicrobial effects. But the present study has partially revealed the activity of test drugs to control antibiotic resistant bacteria, which are a major threat to human health. However the alcoholic extracts of Gul-e-Banafshah could be a possible source to obtain new and effective herbal medicines to treat infections specially caused by multi-drug resistant strains of Salmonella typhimurium. Further, it is necessary to determine the toxicity of the test drugs, their side effects and pharmaco-kinetic properties. References Afaq, S.H., Shehbaz, A., Masood, A.K., Owais, M. and Nadeem, A., Antibiotic screening of certain Unani medicinal plants. Indian Drugs 41(4): Ahmad, I., Mehmood, Z., Mohammad, F., Screening of some Indian medicinal plants for their antimicrobial properties. J Ethnopharmacol. 62: , Akinpelu, D.A. and Onakoya, T.M., Antimicrobial activities of medicinal plants used in folklore remedies in south-western. African Journal of Biotechnology 5 (11), Alam, S. and Khan, N.A., A Physicochemical Study of Qurs-e-Rewand. Unani Medicus 1(1): Anesini, C., and Perez, C., Screening of plants used in Argentine folk medicine for antimicrobial activity, Journal of Ethnopharmacology 39: Anonymous, British Pharmacopoea. General Medicine Council. Pharmaceutical Press, Bloomsbury square, London, pp , , , Anonymous, Medicinal Plants of Gwalior Forest Division, Madhya Pradesh, CCRUM, New Delhi, p. 6. Anonymous, Standardization of single drugs of Unani Medicine, CCRUM, New Delhi, Part-2: pp.1-6, Anonymous, The Unani Pharmacopoeia of India. New Delhi: Ministry of Health and Family Welfare, Deptt. of AYUSH, Govt. of India; Part-I, (1 and 3), pp. 3-4,

82 75 Anonymous Physiochemical Standards of Unani Formulations. New Delhi: Central Council for Research in Unani Medicine (CCRUM), Part-1 & 2, pp Anuradha, V. and Goyal, M.M., Phytochemical study on the leaves of Alstonis scholaris and their effects on pathogenic organism. Ancient science of life 15 (1): Bhattacharjee, A.K. and Das, A.K., Phyto-chemical screening of Some Indian Plants. Quart. Jour. Crude Drug. Res. 9: Bhavnani, S.M. and Ballow, C.H., New agents for Gram-positive bacteria. Curr Opin Microbiol 3: Chase, C.R. and Pratt, R., Fluorescence of powdered vegetable drugs with particular reference to develop of a system of identification. J. Amer. Pharm. Asso. 38: 324. Chung, P.Y., Chung, L.Y., Ngeow, Y.F. et al., Antimicrobial activities of Malaysian plant species. Pharm Biol 42: Conly, J., Rennie, R., Johnson, J., Farah, S. and Hellman, L, Disseminated candidiasis due to Amphotericin B resistant Candida albicans, J. Infect. Dis, 165: Davies, J., Inactivation of the antibiotics and the dissemination of resistance genes. Science 264: De Boer, H.J., Kool, A., Broberg, A., et al., Antifungal and antibacterial activity of some herbal remedies from Tanzania. J Ethnopharmacol. 96: Doughari, J.H., EI-Mahmood, A.M., Antimicrobial activity of leaf extracts of Senna obtusifolia (L). Indian Journal of Pharmacology 2 (1):7-13. Essawi, T., Srour, M., Screening of some Palestinian medicinal plants for antibacterial activity. J Ethnopharmacol 70: Evans, W.C., Trease and Evan s pharmacognosy, WB Saunders Company Ltd. London, UK 14: pp , 251,257. Finegold, S.M., and Martin, H.J., Diagnostic Microbiology, C.V. Mosby Company, London (Ed.) 6, pp Finland, M., The new development in antibiotics, Ann. Intern.Med. 89: Ibrahim, D., and Osman, H., Antimicrobial activity of Cassia alata from Malaysia, J. Ethnopharmacology 45:

83 76 Iwu, M.W., Duncan, A.R., Okunji, C.O., New antimicrobials of plant origin In: Janick, J. (Ed), Perspectives on New Crops and New Uses. ASHS Press, Alexandria, VA, p Jacob, S., Shirwaikar, A.A. and Srinivasan, K.K. et al., Stability of Proteins in Aqueous Solution and Solid State. Indian Journal of pharmaceutical Sciences 68 (2): Jahan, N., Afaq, S.H. and Khan, N.A., et al., Physico-Chemical studies of the Gum Acacia. Natural Product Radiance 7(4): Jenkins, G.L., Knevel, A.M. and Digangi, F.E., Quantitative Pharmaceutical Chemistry, The McGraw Hill Book Company Limited, London, pp. 225, , , 336. Katzung, B.G., Basic and clinical pharmacology. McGraw-Hill Company, USA 8: p Khan, K.H. and Jain, S.K., Medicinal plants a retrospective. Hamdard Medicus XLVI (3): Khory, R.N. and Katrak, N.N., Materia medica of India and their therapeutics, Neeraj publishing house, Delhi, pp , and 505. Monroe, S. and Polk, R., Antimicrobial use and bacterial resistance. Curr Opin Microbiol 3: Nair, R. and Chanda, S.V., Antibacterial activity of some medicinal plants of Saurashtra region. J. Tissue Res 4: Nair, R., Kalariya, T., Chanda, S., Antibacterial activity of some selected Indian medicinal flora. Turk J Biol 29: Owolabi, J., Omogbai, E.K.I. and Obasuyi, O., Antifungal and antibacterial activities of the ethanolic and aqueous extract of Kigelia Africana (Bignoniaceae) stem bark, Afr. J. Biotechnol. 6 (14), Perez, C. and Anesini, C., In vitro antibacterial activity of Argentine folk medicinal plants against Salmonella typhi. Journal of Ethnopharmacology 44: Prabuseenivasan, S., Jayakumar, M., and Ignacimuthu, S., In vitro antibacterial activity of some plant essential oils. BMC Complementary and Alternative Medicine 6(39): 1-8. Prasad, G., Pandey, S. and Gupta, K.K., Antibacterial activity of certain indigenous drugs and drug plants against certain bacterial species. Hamdard Medicine XLII (3):

84 77 Rahman, A., Tajuddin, Amin, K.M.Y., Physico-chemical assay alongwith HPLC profile of a Pharmacopeial preparation containing colchicum, ginger and aloe. International Journal of Pharmaceutical Research 5 (4): Rahman, A., Tajuddin, Amin K.M.Y., Rehman, S., Antimicrobial assay of alcoholic and hydroalcoholic extract of Unani formulation by Agar well method, Hippocratic Journal of Unani Medicine 8 (1): Rahman A., Tajuddin and Amin, K.M.Y., Identification and standardization of a Pharmacopoeial Unani formulation: estimation of marker compounds, Unani Medicus: An international Journal 01 (2): Said, H.M., Medicinal herbal, Hamdard foundation, Karachi, Pakistan, 1: pp. 27, 30. Threlfall, E.J., Frost, J.A., Ward, L.R. and Rowe, B., Increasing spectrum of resistance in multi-resistant salmonella typhimurium. Lancet 347: Vohola, S.B., Skin diseases: Two Unani concepts, Proceedings of the symposium on Dermatology and Unani System of Medicine. Hamdard National Foundation, New Delhi, pp

85 78

86 Pharmacobotanical Studies on Some Powdered Herbal Drugs for Their Diagnostic Characterization- III Nitin Rai and *Rajeev Kr. Sharma Pharmacopoeia Commission for Indian Medicine & Homoeopathy, (Ministry of AYUSH, Govt. of India) PLIM Campus, Kamla Nehru Nagar, Ghaziabad (U.P.) Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract Q 79 uality of herbal formulations primarily depends upon the use of authenticated ingredients. Adulteration and substitution of herbal drugs in commerce leads to problem for quality of formulations. It can be duly addressed by use of identified herbal ingredients. The identification of herbal drugs involves organoleptic, macro and microscopic evaluation based on diagnostic characteristics of drug. In the present studies pharmaco-botanical studies were carried on powdered herbal drugs viz. Nardostachys jatamansi DC., Picrorhizakurroa Royle ex Benth., Plumbago zeylanica Linn and Withania somnifera Dunal to establish their diagnostic characters for identification.these diagnostic characters can be employed for the identification of powdered herbal drugs in the composition of a formulation or dosage form. Keywords: Nardostachys jatamansi DC., Picrorhiza kurroa Royle ex Benth., Plumbago zeylanica Linn., Withania somnifera Dunal, Powdered herbal drug. Introduction In manufacturing process herbal drugs are pulverized to convert into powdered form. Powdered herbal drugs are used as such in various classical dosages form in Ayurveda (Churna and Kvatha Churna), Unani (Sufoof )and Siddha (Churnam and Kudineer Churnam). Vati, Gutika (in ayurveda), Huboob, Aqras (in unani) Mathirai,Vadagam (in siddha),tablets and capsules are other dosages forms wherein powdered ingredients are intact and identifiable. To ensure the identity of herbal ingredients used in a dosages form pharmaco-botanical studies (organoleptic, macroscopic and microscopic evaluation) is important tool. In the present studies pharmaco-botanical studies are carried on powdered herbal drugs derived from of Nardostachys jatamansi DC. (rhizome), Picrorhiza kurroa Royle ex Benth. (Rhizome), Plumbago zeylanica Linn. (root), Withania somnifera Dunal. (root) are studied. Regulatory status of herbal drugs studied in presented in Table 1. Nardostachys jatamansi DC. (Family-Valerianaceae) has been held in great esteem as Jatamansi in commerce. It is an official drug of ayurveda, unani and siddha systems of medicine. The official tile of the drug is based on the eternal appearance of drug and refers to its bearded appearance. The drug has been mentioned in the classical treatises of Charak, Sushruta, Bhav Prakash, Vag Bhatta etc. It is an indigenous drug which has been mentioned by Dioscorides in his work. In modern medicine Jatamansi is treated as a good substitute for Valarian (Valeriana officinalis Linn.).The drug is often adulterated with Selinum *Author for correspondence

87 80 vaginatum Clarke of the family Umbelliferae (Watt, ; Srivastava, 1954; Mehra and Jolly, 1963; Anonymous, 1966, 1978; Chunkar, 1972). Picrorhiza kurroa Royle ex Benth. (Family-Scrophulariaceae) has been used in Ayurveda, Unani and Siddha systems of medicine. It is an official drug in all these systems of medicines and titles as Katuka, Kutki and Katukurohini.The drug referred in common parlance as Kutki appears to have originated from its sanskrit names Katuka, Tikta, Katurohini and many other synonyms all of which literally described its bitter taste. This drug is spoken as Dhanvantarigrasta ; the plant is eaten by Dhanvantri.It is mentioned in classical test of BhavPrakash,Charak, Dhanvantri, Sushruta etc. The drug is considered to have similar medicinal properties as Gentian viz. improves appetite and stimulates gastric secretion. The drug is useed either as an adulterant or as a substitute for Indian Gentian (Gentiana kurroaroyle) and true Gentian (G. lutealinn.) (Watt, ; Anonymous, 1969 and Chunekar, 1972). Plumbago zeylanica Linn. (Family-Plumbaginaceae) constitutes the drug Chitraka, Sheetraj Hindi and Kodiveli which are official drug in ayurveda, unani and siddha systems of medicine. It is an erect or straggling perennial undershrub and native of East Indies. The generic name of the plant is based on Plumbum - a disorder in the eye, which some species of this genus were formerly used to cure. The drug is described in Ashtanh Hridaya, Bhav Prakash Nighantu, Charak Samhita, Dhanvantri Nighantu, Hridya Priyam, Navanitake, Raj Nighantu, Sushruta Samhita etc. of ayurvedicliterature.in south India, the white flowered P. zeylanica Linn. which grows wild, as well as the red flowered P. rosea Linn. which is cultivated are used. The drugs is substituted with P. rosa Linn. (Herman, 1868; Aiyer and Kolamal, ; Anonymous, 1969 and Chunekar, 1972). Withania somnifera Dunal. (Family-Solanaceae) is botanical source of Ashwagandha which has been used in Ayurvedasince a long time. It is an official drug and one of the ingredients of a number of important Ayurvedic, Unani and Siddha formulations. It is an evergreen perennial undershrub. The generic name of the plant is in honour of H. Withan, a British geologist and writer on Paleobotany, in the nineteenth century. The drug finds mention in various literature of Ayurveda viz.ashtang Hridaya,Bhav Prakash Nighantu, Charak Samhita, Raj Nighantu, Sushruta Samhita etc. The tender shoots of the plant are also used as vegetable and seeds are used as masticatory. The green berries are bruised and rubbed on ringworm in human beings and on animal sores and girth-golls in horse. They are also employed to curdle milk. The drug is substituted with W. coaguland Dunal. (Chopra et al., 1949; Watt, ; Aiyer and Kolamal, ; Anonymous, 1976; Chunekar, 1972).

88 81 Table 1: Regulatory Status of Herbal drugs Sl.No. Botanical Name Official Name Pharmacopoeia Formulary References 1. Nardostachys Jatamansi Ayurvedic Pharmacopoeia of Ayurvedic Formulary of India, Anonymous, 1978; 1986 jatamansi DC. India, Part-I, Vol.-I Part-I Sumbul-ut-Teeb Unani Pharmacopoeia of India, Anonymous, 1998 Part-I, Vol.-I Balchhar National Formulary of Unani Anonymous, 1981 Medicine, Part-I Catamancil Siddha Pharmacopoeia of India, Anonymous, 2008 Part-I, Vol.- I Sadamanjil Siddha Formulary of India, Part-I Anonymous, Picrorhizakurroa Katuka Ayurvedic Pharmacopoeia of India, Ayurvedic Formulary of India, Part-I Anonymous, 1978; 1999 Royle ex Benth. Part-I, Vol.-II Katukurohini Siddha Pharmacopoeia of India, Siddha Formulary of India, Part-I Anonymous, 1984; 2008 Part-I, Vol.- I Kutki Unani Pharmacopoeia of India, National Formulary of Unani Anonymous, 1981; 2007 Part-I, Vol.-IV Medicine, Part-I 3. Plumbago Citraka Ayurvedic Pharmacopoeia of India, Ayurvedic Formulary of India, Part-I Anonymous, 1978;2004 zeylanica Linn. Part-I, Vol.-IV Kodiveli Siddha Formulary of India, Part-I Anonymous, 1984 Sheetraj Hindi Unani Pharmacopoeia of India, National Formulary of Unani Anonymous, 1981; 1998 Part-I, Vol.-I Medicine, Part-I 4. Withania Asvagandha Ayurvedic Pharmacopoeia of India, Ayurvedic Formulary of India, Part-I Anonymous, 1978;1986 somnifera Dunal. Part-I, Vol.-I Asgand Unani Pharmacopoeia of India, National Formulary of Unani Anonymous, 1981; 1998 Part-I, Vol.-I Medicine, Part-I Amukkara Siddha Pharmacopoeia of India, Siddha Formulary of India, Part-I Anonymous, 1984; 2008 Part-I, Vol.- I

89 82 Table 2: Organoleptic Characteristics of Herbal Drugs Sl. Drug Organoleptic Characteristics No. Entire Drug Powdered Drug 1. Nardostachys The drug consists of dried rhizomes which are brown in colour, cylindrical and covered with brown fibres. The rhizome is Dark brown in colour jatamansi DC. completely encircled by fibrous remain of leaf bases which arise at the nodes of rhizome. The rhizomes are cm withstrong aromaticodour long and cm in dimeter including the fibres. Thefibres are skeleton of leaf bases matted together forming a net and acrid taste which hasappearance of tail of sable. The remains of older leaves are almost splitted into fibres. The scars or remains of adventitious roots are present on the rhizome which are hidden by fibres. The naked rhizome has rough surface showing annulatednodes, scars of leaf bases, aerial shoots and adventitious roots. Rhizomes are sometimes dichotomously and laterallybranched. Drug is easily breakable and fracture is splitting. 2. Picrorhiza The drug consists of dried rhizome which are greyish brown in colour, cylindrical and surrounded by a tufted crown of Blackish in colour with kurroa Royle withered leaf bases.the drugs are longitudinal pieces of the rhizome measuring cm long and cm in pleasant odour and ex Benth. dimeter. The surface of the rhizome have longitudinal wrinkles, transverse cracks, dotted scars and annulation of bud bitter taste. scales and stem remnants. The rhizome brakes with short fracture exhibiting black lacunar surface with whitish xylem ring on transverse plane of broken ends. 3. Plumbago The drug consist of dried matured tap roots, dressed in pieces of varying dimension. The roots are cylindrical, hard and Brown in colour with zeylanica Linn. deep brown in colour. These are generally cm in length and cm in dimeter. The surface is irregular slightly disagreeable fissured. The roots have small projections of secondary roots and scars of rootlets; fairly thick, shrivelled, smooth and odour and acrid taste. brittle bark. The fracture of the root is short and the broken ends exhibit concentrically striated transverse plane 4. Withania The drug consists of dried and matured tap roots which are more or less straight and buff to grey yellow in colour. In Yellowish brown in somnifera commerce, roots are cut into pieces of varying length of cm.the dimeter of the roots vary with the age, ranging colour with mucilaginous, Dunal. from cm.the surface of the roots are smooth except a few occasional lenticels and longitudinal wrinkles. Some of acrid and bitter taste, the pieces of roots bear card like secondary roots or parts therof. The root pieces which are cut with stem base bear root having no characteristic break with short and starchy fracture. The broken ends exhibit white transvers plane with scattered pores on them. odour.

90 83 Table 3: Diagnostic microscopic characteristics of powdered herbal drugs Sl. Drug Diagnostic Microscopic Characters No. Cellular Elements Ergastic Contents Starch grains Calcium oxalate crystals 1. Nardostachys Fragments of plellem comprising thick walled cells containing yellowish oil globules, abundant Absent Absent jatamansi DC. thin walled parenchymatous cells of phelloderm and pith, occasional parenchymatous cells of phloem with small patches of sieve elements and fairly numerous fragments of interxylary cork. Vessels are fairly present and are usually fragmented, singly or in small groups or associated with parenchymatous cells and have scalariform or spiral or reticulate thickening. 2. Picrorhiza kurroa Fragments of phellem comprising thick walled tangentially elongated cells containing granular The occasional starch Absent Royle ex Benth. resinous mass, usuallyattached to the part of phelloderm,abudent parenchyma of cortex and grains are found pith, composed of cells varying from rounded to polygonal or rectangular in outline and scattered and are simple contain starch grains, oil globules and occasional groups of sieve tissue embedded in and compound with two phloem parenchyma. The vessels occur singly or in small groups, associated with xylem to four component parenchymatous cells and have pitted or annular or spiral or scalairiform thickening. showing distinct hilum. 3. Plumbago Fragmented phellem constituting the thin walled somewhat rectangular cells, some of the cells Starch grains are abundantly Absent zeylanica Linn. are filled with brown content; abundant thin walled polygonal parenchymatouscells of phelloderm, present mostly simple, phloem and medullary ray. Most of the cells of varying shape and size are filled with starch grains. round and concentric, a few Some of the cells also contain yellow content, fairly common lignified xylem parenchyma cells of the starch grains are and non-lignified and lignified thick walled fibres usually in group of two to four with narrow lumen. compound with much more The vessels are found singly or in small groups, often fragmented and have pitted thicking. Some components forming clusters groups of sieve tissue composed of thin walled cells with faint sieve areas are also present. of twelve to twenty or more starch grains. 4. Withania Fragmented phellem cells composed of thin walls and or rectangular shape, abundant Starch grains are varying in Scattered microsomnifera Dunal parenchymatous cells of phelloderm, phloem and medullaryray. Some of these are packed with shape and sizes and are sphenoidal crystals of starch grains or microsphenoidal crystals of calcium oxalate, very occasional fragments of sieve ovoid to circular or reniform calcium oxalate, which tissue composed of small elongated elements and vessels in group, mostly fragmented showing or club shaped. Compound are not abundant as bordered pit or spiral or annular thickening.lignified xylem parenchyma cells are also scattered starch grains are with two to most of the crystals and a few fragmented patches are associated with the vessels and fibres. A few elements of three or more components. are found enclosed in tracheid with pitted thickening and fibres havening pointed or bifurcated ends are also present. parenchyma. Abundantly present starch grains are generally simple and have characteristic marking of fissured and eccentric hilum.

91 84 Material and Methods Nardostachys jatamansi DC. (rhizome), Picrorhiza kurroa Royle ex Benth. (rhizome), Plumbago zeylanica Linn. (root) and Withania somnifera Dunal. (root) selected for present study were procured from the natural habitats and authenticated by complying the macroscopoical characteristics of these drugs with that of standard reference drug samples available in the museum-cumherbarium of the Pharmacopoeial Laboratory for Indian Medicine, Ghaziabad, India. To study the powder microscopy, the drugs were first washed thoroughly under running tap water to remove any dust or soil particles and then air dried for few days at room temperature or in shade. The dried drugs were then powdered and pass through 120 μm sieve. The fine powder obtained through sieve 120 μm was then subjected to various histo-chemical tests and the temporary mounts of powder prepared to observe under light microscope (Jackson and Snowdon 1968; Johansen, 1940; Youngken, 1951). Results and Conclusion Powdered herbal drugs derived fromnardostachys jatamansi DC. (rhizome), Picrorhiza kurroa Royle ex Benth. (Rhizome), Plumbago zeylanica Linn. (root) and Withania somnifera Dunal. (root) were subjected to pharmaco-botanical studies to draw out diagnostic characteristics in respect of organoleptic characters (Table-2) and microscopic characters (Table-3).The diagnostic characters observed are to be taken as a reference for the identification of powdered ingredients [Nardostachys jatamansi DC. (rhizome), Picrorhiza kurroa Royle ex Benth. (Rhizome), Plumbago zeylanica Linn. (root) andwithaniasomnifera Dunal. (root)]as such or in different dosage forms. Microscopic examination of powdered drug also entails the presence of foreign matter, biological contamination, adulterated or substituted ingredients in a dosage or formulation. References Aiyer, K.N. and M., Kolamal, Pharmacognosy of Ayurvedic Drugs (Travancore-Cochin), Series 1, No. 1 to 9.University of Travancore, Trivandrum Anonymous, 1966.The Wealth of India (Raw Materials), Vol. VII (N-Pe). C.S.I.R., New Delhi Anonymous, 1969.The Wealth of India (Raw Materials), Vol.VIII (Ph-Re). C.S.I.R., New Delhi Anonymous, 1976.The Wealth of India (Raw Materials), Vol. X (Sp-W). C.S.I.R., New Delhi

92 85 Anonymous, The Ayurvedic Formulary of India, Pt. I. Ministry of Health & Family Welfare, New Delhi Anonymous, National Formulary of Unani Medicine, Part-I, (English ed.), Govt. of India, Ministry of Health & Family Welfare, New Delhi Anonymous, Siddha Formulary of India, Part-I, (Tamil ed.), Govt. of India, Ministry of Health & Family Welfare, New Delhi Anonymous, The Ayurvedic Pharmacopoeia of India, Part- I, Volume I, First edition, Govt. of India, Ministry of Health & Family Welfare, New Delhi Anonymous, The Unani Pharmacopoeia of India, Part-I, Vol.-I, Govt. of India, Ministry of Health & Family Welfare, New Delhi Anonymous, The Ayurvedic Pharmacopoeia of India, Part- I, Volume II, First edition, Govt. of India, Ministry of Health & Family Welfare, New Delhi Anonymous, The Ayurvedic Pharmacopoeia of India, Part- I, Volume IV, First edition, Govt. of India, Ministry of Health & Family Welfare, New Delhi Anonymous, The Unani Pharmacopoeia of India, Part-I, Vol. -IV, Govt. of India, Ministry of Health & Family Welfare, New Delhi Anonymous, The Siddha Pharmacopoeia of India, Part-I, Vol.-I, Govt. of India, Ministry of Health & Family Welfare, New Delhi Chopra, R.N., R.L. Badhwar and S. Ghosh, Poisonous Plants of India.Manager of Publication, New Delhi Chunekar, K.C., Glossary of Vegetable drugs in Brahattrayi. Chowkhambha Sanskrit Series Office, Varanasi Herman, Samuel, Paxton s Botanical Dictionary-comprising the names, history and culture of all plants known in Britain. Bradury, Evans & Co., Bouverie, London Jackson, B.P. and D.W. Snowdon, Powdered Vegetable Drug. Churchill Ltd., London Johansen, D.A., Plant Microtechnique, MC Graw Hill Book Co., New York Mehra, P.N. and S.S. Jolly, 1963.The identity and pharmacognosy of the adulterant of Nardostachys jatamansi DC. Planta Medica. 11 (1): 8-15 Srivastava, G.P., History of the Indian Pharmacy, Vol. I. Pindars Ltd., Calcutta

93 86 Watt, G., A Dictionary of Economic Products of India, 6 Vols. (Index 1896). Govt. Printing Press, Calcutta Youngken, H.W., Pharmaceutical Botany, 7 th ed., The Blackistan Company, Toronto

94 Standardization and HPTLC W Fingerprinting of a Unani Compound Formulation Qurs-e- Luboob with Modern Techniques 1 *N.M.A. Rasheed, 1 Rakesh Kumar Negi, 1 Juveria Mahmood, 1 Aslam Siddiqui, 1 M.H. Kazmi, 2 Shamsul Arfin, 2 Aminuddin and 1 Javed Inam Siddiqui 1 Central Research Institute of Unani Medicine, Opp. E.S.I. Hospital, A.G. Colony Road, Erragadda, Hyderabad Central Council for Research in Unani Medicine, 61-65, Institutional Area, Janakpuri, New Delhi Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract ith global realization that use of synthetic drugs is not safe on the long run, the medical fraternity at large is looking at alternatives from natural sources to combat diseases particularly those in which conventional modern system of medicine has little to offer. This realization on the one hand has increased demand for herbal drugs and on the other hand need for quality standardization of drugs has gone up. Central Research Institute of Unani Medicine, Hyderabad being engaged in multidisciplinary research in Unani Medicine, working on standardization of herbal drugs used in this system of medicine. One such drug Qurs-e-Luboob which is prescribed in Unani system for therapeutic use as Zof-e-Bah (Sexual Debility), Qillat-e-Mani (Oligospermia), and has action as Muqawwi-e-Bah (Aphrodisiac), Mughalliz-e-Mani (Inspissant to Semen), has been taken up for standardization by modern techniques, so as to ascertain its quality. The parameters carried out are pharmacognostic studies, physico-chemical parameters, phytochemical screening, High performance thin layer chromatography, microbial load, aflatoxins, and heavy metals revealing specific identities for the particular drug and to evaluate pharmacopoeial standards. Results suggest that the drug is safe for therapeutic use and its batch to batch identification for quality control is possible on the basis of present study. Keywords: Qurs-e-Luboob, Standardization, Physico-chemical analysis, HPTLC. Introduction Recently, there has been a shift in universal trend from synthetic to herbal medicine, which can be said Return to Nature. Medicinal plants have been known for millennia and are highly esteemed all over the world as a rich source of therapeutic agents for the prevention of diseases and ailments (Sharma et al., 2008). The global resurgence of interest in herbal medicines has led to an increase in their demand leading to a decline in their quality, primarily due to a lack of adequate regulations pertaining to drugs (Rajini and Kanaki, 2008). WHO has emphasized the need to ensure quality control of medicinal plant products by using modern techniques (Imam et al., 2009; Rasheed et al., 2010a; 2010b; 2010c;2010d; 2011; 2012; 2014a; 2014b) and (Naikodi et al., 2011) by applying suitable parameters and standards, In order to overcome certain inevitable shortcomings of the pharmacopoeial monographs other quality control measures must be explored (Shinde, 2009; Singh and Soni, 2004; Street et al., 2008). Curative efficacies of compound herbal medicine are reliant on the quality and 1 *Author for correspondence

95 88 the quantity of the constituent single drugs as they contain specific bio-active marker species with specific pharmacological actions. Though, it is very difficult to identify the ingredients after the formulation is prepared and the organoleptic parameters like taste, odour, colour etc. will not establish the standard quality of the medicine. A Unani formulation, Qurs-e-Luboob (figure 1) was taken for the present study. The drug is a compound formulation mentioned in National formulary of Unani medicine of India, Part-IV. and prescribed for the treatment of Zof-e-Bah (Sexual debility), Qillat-e-Mani (Oligospermia), and Muqawwi-e-Bah (Aphrodisiac), Mughalliz-e-Mani (Inspissant to Semen). In order to standardize and to lay down the standard operating procedures (SOP s) and pharmacopoeial standards, the formulation was prepared in three batches at laboratory scale. It was subjected to analysis for microscopic study, physico-chemical parameters, microbial load, heavy metals, aflatoxins, and high performance thin layer chromatographic studies (Anonymous, 2009). The present paper describes the salient features of preparation, phytochemical screening, safety evaluation studies and High performance thin layer chromatographic studies for the drug. Materials and Methods Collection of material Ingredients of formulation were procured from the pharmacy of Central Research Institute of Unani Medicine, Hyderabad, authenticated and identified by the expert botanist. Fig. 1: Formulation Qurs-e-Luboob (tablets).

96 89 Preparation of the formulation as per Sharif Khan (1921 A.D.). It is prepared according to the composition of the formulation given in national formulary which is as follows: S.No. Name of the drug Botanical Name Part Used Qty 1. Maghz-e-Funduq Corylus avellana Linn. Cotyledons 100g 2. Maghz-e-Pista Pistacia vera Linn. Cotyledons 100g 3. Maghz-e-Badam Prunus amygdalus Cotyledons 100g shireen Batsch. 4. Maghz-e-Behidana Cydonia oblonga Mill. Cotyledons 100g 5. Maghz-e-Tukhm-e- Cucumis melo Linn. Cotyledons 100g Kharbuza 6. Maghz-e-Tukhm-e- Cucumis sativus Linn. Cotyledons 100g Khiyarain 7. Maghz-e-Tukhm-e- Cucurbita moschata Cotyledons 100g Kadu Duchesne. 8. Maghz-e-Tukhm-e- Citrullus vulgaris Cotyledons 100g Hindu dana Schrad. 9. Tukhm-e-Khashkhash Papaver somniferum Seed 100g safaid Linn. 10. Maghz-e-Habb-e- Prunus mahaleb Linn. Cotyledons 100g Mahlab 11. Nishasta Triticum aestivum Linn Starch 100g 12. Tukhm-e-Khatmi Althaea officinalis Linn. Seed 100g 13. Tukhm-e-Khubbazi Malva sylvestris Linn Seed 100g 14. Rubbus-soos Glycyrrhiza glabra Linn. Root extract 100g 15. Gil-e-Armani Armenian bole Aluminium Silicate, 100g Silicate of Alumina, Magnesia andiron Oxide 16. Maghz-e-Chilgoza Pinus gerardiana Wall. Cotyledons 100g 17. Duqu Peucedanum grand Seed 100g C.B. Clark 18. Badiyan Foeniculum vulgare Fruit 100g Mill. 19. Tukm-e-Karafs Apium graveolens Linn. Seed 100g 20. Luab-e-Tukhm-e- Linum usitatissimum Seed Mucilage Q.S. Katan Linn.

97 90 Processing of raw material It is prepared according to the composition of the formulation given above is as follows; Take all the ingredients of pharmacopoeial quality. Clean all the ingredients from 1-10 and 16 and make them free from all foreign matters and make fine powder separately. This forms Part A with the help of pulveriser and pass through 80 mesh sieve. Similarly ingredients from 11-15, 17, 18 and 19 are powdered separately with the help of pulveriser and pass through 80 mesh sieve. This form Part B. Soak Tukm-e-katan in water to make Luab and add part A and part B in it to make granules and prepare the Qurs through mechanical process. Store the Qurs in a sealed container protected from light and moisture. Preparation of the Qurs (Tablets) The tablets were prepared as per the procedure described by Hm. Mohd. Sharif Khan, (1921). The granules were made into 500mg tablets (excluding binding material weight) using rotary tablet punching machine (Cadmach-GMP model). Chemical analysis Physico-Chemical parameters of the prepared compound formulation Qurs-e- Luboob were studied such as total ash, acid insoluble ash, water soluble ash, solubility matter in alcohol and water, loss on drying at C, microbial load, aflatoxins, pesticide residue and GBC-908 AA model Atomic Absorption Spectrophotometer (AAS) was used to determine the concentration of heavy metals as per the methods described in WHO guidelines (Anonymous, 1998). Phytochemical screening was carried out in different solvents extracts such as Petroleum ether, Chloroform, Ethyl acetate, Ethanol, Acetone and Aqueous extracts as per the methods described by Trease and Evans, (1972). HPTLC analysis DESAGA Sarstedt Gruppe system is used for analysis along with Automatic TLC applicator and UV visible cabinet as imaging system, the instrument had Proquant 1.6 version as software system for documentation. Preparation of extract of the drug for HPTLC analysis Five grams fine powder of Qurs-e-Luboob was reflux on water bath for 30 min in Petroleum ether ( C) through sohxlet apparatus. Later the contents were removed and filtered through Whattmann No. 41 filter paper and evaporated the solution to 20 ml. Thus the solution so obtained was used as sample for the determination of components.

98 91 Development and determination of the solvent system Sample Applied : Sample drug solution of about 10μl. Solvent system : Toluene: Ethyl Acetate (9: 1) The sample was spotted with the help of Automatic TLC applicator system of the DESAGA Sarstedt Gruppe on Precoated Aluminium Sheets of Silica Gel 60 F 254 (Merck) After trying with various solvent systems with variable volume ratios, the suitable solvent system as stated above is selected in its proportional ratio and developed in the Twin through chamber of TLC to the maximum height of the plate so that it can be able to separate the components on the polar phase of silica gel and that of mobile phase of solvent system. The drug extract was spotted on TLC plate and developed the TLC plate. Development of HPTLC technique After developing, TLC plates were dried completely and detected with the suitable detection system like UV Cabinet system for detection of spots at 254, 366nm and also under iodine vapours and after derivatizing with anisaldehyde sulphuric acid reagent as shown in the figure 2. Further it was scanned with the Densitometer CD60 of DESAGA Sarstedt Gruppe system under the UV range of 366nm, under exposure to Iodine vapours at 410nm and after derivatization with anisaldehyde sulphuric acid reagent at 580nm showing corresponding densitogram in the figure 3 in which peaks appeared for the corresponding spots being detected in the densitometer while scanning and the peaks areas under the curve correspond to the concentration of the component in the sample. The separation of the components in the compound formulation and its ingredients are corresponding compared with respect to Rf values. Results and Discussion Analytical Profile Organoleptic Characters Light brown colour tablet having characteristic smell. Identification Microscopy Fine powder of six tablets was immersed in the water and kept for half an hour. Material was stirred with a glass rod and supernatant was discarded. Residue

99 92 a) At UV 366nm b) At UV 254nm c) Expose to Iodine vapours d) Derivatized with Anisaldehyde sulphuric acid Fig. 2: TLC plates of alcoholic extract of Qurs-e-Luboob a) At UV 366nm, b) At UV 254nm, c) Under Iodine vapours d) At visible region after derivatizing with Anisaldehyde sulphuric acid reagent. was taken in a glass jar and it was treated with chloral hydrate solution for an hour. After that residue was treated with iodine solution, safranin and mounted with glycerine. Fatherly slide was subjected for microscopical studies.

100 93 Fig. 3: HPTLC Densitogram of Qurs-e-Luboob at A) UV 366nm B) Under Iodine vapours at 410nm C) derivatized with anisaldehyde sulphuric acid at 580nm. Qurs-e-Luboob Microscopical observations expose the presence of unicellular pointed trichomes, thin walled parenchyma cells, spherical shaped parenchyma cells with crystals and starch grains.

101 94 Parenchyma cells with reddish tannin content Physico-Chemical Standards The Physico-Chemical Parameters data as given in table 1 is expressed as mean values of the three readings calculated. Total ash was found to be , and acid insoluble ash gm%; whereas Alcohol soluble matter in terms of %w/w is found to be and water soluble matter as ; The moisture content i.e., Loss of weight on drying at C found to be gm%. P H of the 1% aqueous solution observed as and 10% aqueous solution observed as ; and Disintegration time of tablet was 35min. Phytochemical screening for the phytoconstituents were carried out and Table 1: Physico-chemical parameters of the compound formulation Qurs-e-Luboob. S.No. Parameters Sample I Sample II Sample III 1 Ash values Total Ash (%) Acid insoluble ash (%) Alcohol Soluble matter (%w/w) Water soluble matter(%w/w) ph of 1% aq. Solution ph of 10% aq. Solution Disintegration time in min Loss of wt. on drying at 1050C Table 2: TLC profile of alcoholic extract of Qurs-e-Luboob along with R f values and detection system. S. Name of Solvent Detection No.of R f values No. the extract system spots 1. Alcoholic Tol: Ethyl UV 366nm six 0.60 (Blue), 0.16 (Blue), extract acetate=9:1 spots 0.24 (Fluorescent Blue), 0.40 (Red), 0.44 (light blue) and 0.53 (Fluorescent Blue) UV 254nm two 0.58, 0.90 (All black) spots Under exposure five 0.29, 0.37, 0.53, 0.76, to Iodine vapours spots 0.91 (All brown) After derivatizing five 0.29, 0.37, 0.53, 0.76, with anisaldehyde spots 0.91 (All Grey) sulphuric acid

102 95 are represented in the table 3. Powdered drug was screened for fluorescence characteristic with or without chemical treatment. The observations pertaining to their colour in daylight i.e, visible region and under ultra-violet light were noticed and are presented in the table 4. Fluorescence analysis of powdered drug extracts in different solvents was observed and reported in the table 5. The results Table 3: Phytochemical screening of the nature of compounds present in different solvent extracts of Qurs-e-Luboob. S. Phyto Pet. CHCl 3 E.A. ethanol Acetone Aqueous No. constituent ether ext. ext. ext. ext. ext. ext. 1. Alkaloid Carbohydrates Glycosides Phenols Saponins Proteins Starch Steroids Flavonoids Tannins Table 4: Fluorescence analysis of powdered drug UV light S. Reagents Visible Short Long No. light 254nm 366nm 1. Powder as such Brown Black Pale yellow 2. Powder treated with 1N HCl Brown Black Light blue 3. Powder treated with 50% H 2 SO 4 Dark Brown Black Blue aqueous 4. Powder treated with 50% HNO 3 Brown Black L. Blue aqueous 5. Powder treated with Glacial Brown Black L. Blue Acetic acid 6. Powder treated with 1N NaOH Brown Black Pale yellow in Water 7. Powder treated with 1N NaOH in Yellow Black Indigo Methanol

103 96 of total bacterial load and total fungal count of the microbial studies were within the permissible limits and the other parameters were found to be absent in the drug. The analysis of aflatoxins and heavy metal analysis showed that the drug was free from any contaminations. These findings as observed for microbial load, aflatoxin contamination and heavy metal analysis are given in table 6 respectively. Table 5: Fluorescence analysis of powdered drug extracts in different solvents S.No. Extraction Solvent Visible light UV light Short 254nm Long 366nm 1. Petroleum ether extract Light yellow Black Light pink 2. Chloroform Extract Light brown Black Blue 3. Ethyl Acetate Yellow Black Blue 4. Alcoholic Extract Yellow Greenish Black Light blue 5. Acetone Extract Yellow Black Blue 6. Distilled water Brown Black Light blue Table 6: Heavy Metal Analysis, Aflatoxins, Microbial and fungal contamination in the drug. Heavy Metal Analysis Parameter analyzed Results Permissible limits as per WHO 1 Arsenic Nil Not more than 3.0 ppm 2 Cadmium Nil Not more than 0.3 ppm 3 Lead Nil Not more than 10.0 ppm 4 Mercury Nil Not more than 1.0 ppm Aflatoxin Contamination Parameter analyzed Results Permissible limits as per WHO 1 B1 Nil Not more than 0.50 ppm 2 B2 Nil Not more than 0.10 ppm 3 G1 Nil Not more than 0.50 ppm 4 G2 Nil Not more than 0.10 ppm Microbial and fungal Contamination Parameter analyzed Results Permissible limits as per WHO 1 Total Bacterial Load 10 x 10 2 Not more than 10 5 / g 2 Salmonella Spp Nil Nil 3 Escherichia Coli Nil Nil 4 Total Fungal count 4 x 10 Not more than 10 3 / g

104 97 HPTLC analysis HPTLC fingerprint studies of alcoholic extract of Qurs-e-Luboob was carried out and TLC plate developed and detected using the UV visible chamber which clearly showed various spots at UV 254nm and 366nm in the densitogram and also under iodine vapours and after derivatizing with anisaldehyde sulphuric acid reagent. The corresponding Rf values under each detection is illustrated in the table 2. Which showed shows six spots under 366nm at Rf values 0.06 (Blue), 0.16 (Blue), 0.24 (Fluorescent Blue), 0.40 (Red), 0.44(light blue) and 0.53(Fluorescent Blue); and under UV 254nm shows two spots at Rf values 0.58, 0.90 (All black); and upon exposure to Iodine vapours shows five spots at Rf values 0.29, 0.37, 0.53, 0.76, 0.91 (All brown) and under visible region after derivatizing with anisaldehyde sulphuric acid shows five spots at Rf values 0.29, 0.37, 0.53, 0.76, 0.91 (All Grey). Thus established HPTLC fingerprinting profile helps to authenticate the formulation in batch to batch consistency and quality control analysis of formulation as a reference. Conclusion The drug under study was subjected to Physico-chemical analysis, which is helpful in establishing the standard along with the other parameters such as phytochemical screening, microscopic study, HPTLC analysis. Safety evaluation of drug such as Heavy metal analysis, aflatoxins contamination analysis was done and found absent; microbial load was found within the permissible limits of WHO guidelines. Modern technique of HPTLC analysis was employed in respect to standardization and to separate the compounds which can be isolated for further studies to generate marker for the formulation. Consequently the drug was brought up in determining and ascertaining its quality standard. The study is likely to help in the quality assurance of drug used in the Unani System of Medicine and in development of standard parameters. The development of this traditional system of medicines with the perspectives of safety, efficacy and quality will help not only to preserve the traditional heritage but also to rationalize the use of natural herbal products in the healthcare. Acknowledgements The authors would like to record their gratitude to the Director General, CCRUM, New Delhi, for providing necessary facilities and also Dr. Khalid M. Siddiqui, Deputy Director General, CCRUM, for support and encouragement. We are greatly indebted to the Deputy Director Incharge, Central Research Institute of Unani Medicine, Hyderabad, for giving constant guidance and support.

105 98 References Sharma, Alok, Shanker C, Lalit Kumar Tyagi, Mahendra Singh and Rao Ch V., Herbal Medicine for Market Potential in India: An Overview. Academic Journal of Plant Sciences 1(2): Anonymous, Quality Control Methods for Medicinal Plant Materials. World Health Organization, Geneva, pp Anonymous, The Unani Pharmacopoeia of India, Part-I, Vol.VI. Ministry of Health & Family welfare, Govt. of India, New Delhi, pp Giorgio Pifferi, Paola Santoro, Massimo Pedrani, Quality and functionality of excipients, II. Farmaco 54:1-2, Imam, S., Rasheed, N., Ayesha, M., Shareef, M.A., Khan, S.A. and Arfin, S Role of Chromatography in the Identification and Quality Control of Herbal Drugs 1. - HPTLC Finger Prints of Qurs-e-Istisqa. Hippocratic Journal of Unani Medicine 4(3):41-58 Khan, Mohd. Sharif, Ilajul Amraz (Persian). Barqi Press, Delhi, p.233. Naikodi, M.A.R., Waheed, M.A., Shareef, M.A., Ahmad, M., Nagaiah, K., Standardization of the Unani drug Myristica fragrans Houtt. (Javetri) with modern analytical techniques. Pharmaceutical Methods 2(2): Rajini, M. and Kanaki, N.S Phytochemical Standardization of Herbal Drugs and Polyherbal Formulations: Bioactive Molecules and Medicinal Plants. Ramawat, K.G., Merillon J.M. (eds.), Springer, pp Rasheed, N.M.A., Ayesha, M., Shareef, M.A., Alam, M.D., Gupta, V.C., Khan, S.A., Arfin, S. and Aminuddin, 2010a. Role of Chromatography in the Identification and Quality Control of Herbal Drugs 1.HPTLC Finger Prints Of Qurs-e-Kundur - a Unani Compound Formulation. Hippocratic Journal of Unani Medicine 5(3): Rasheed, N.M.A., Ayesha, M., Waheed, M.A., Alam, M.D., Khan, S.A. and Arfin, S., 2010b. A Chemical Standardization of a Unani single drug -1 : Ood-e- Saleeb (Paeonia emodi Wall.) and Evaluation of its Antimicrobial Activity Against Bacterial Strains. Hippocratic Journal of Unani Medicine 5(3): Rasheed, N.M.A., Rehana, A., Gupta, V.C., Ahmad, M. and Aminuddin, Chemical Standardization of a Unani Herbal drug - Cordia dichotoma Forst.f. (Sapistan). Hippocratic Journal of Unani Medicine 6(1): Rasheed, N.M.A., Rehana, A., Maqbool Ahmed, Husain, K., Waheed, M.A., Arfin, S. and Aminuddin, 2014b. Standardization and HPTLC fingerprinting of a

106 99 Unani Compound Formulation Habb-e-Paan with Modern Techniques. Hippocratic Journal of Unani Medicine 9(2): Rasheed, N.M.A., Shareef, M.A., Ahmad, M., Gupta, V.C., Arfin, S. and Shamshad, A.K., 2010c. HPTLC Finger Print Profile of Dried Fruit of Physalis alkekengi Linn. Pharmacognosy Journal 12(3): Rasheed, N.M.A., Shareef, M.A., Mateen, A., Rehana, A., Waheed, M.A., Arfin, S., Shamshad, A.K., and Aminuddin, Physico-chemical and Phytochemical evaluation of Cocoon of Bombyx mori Linn. (Abresham). Hippocratic Journal of Unani Medicine 7(4): Rasheed, N.M.A., Shareef, M.A., Rehana, A., Waheed, M.A., Arfin, S. and Aminuddin. 2014a. Phytochemical and HPTLC fingerprint analysis of Terminalia arjuna (Roxb) Wight & Arn. in different solvent extracts. Indo- American Journal of Pharmaceutical Research 4(1): Rasheed, N.M.A., Shareef, M.A., Waheed, M.A., Ahmad, M., Shamshad, A.K., Arfin. S and Nageshwar Rao, 2010d. Standardization of Herbal Unani Drug- Aegle marmelos Corr. with modern analytical equipment Herbal Tech Industry. Journal of Herbal Science and Technology 6(9): Shinde, V.M., Dhalwal, K., Potdar, M. and Mahadik, K.R., Application of Quality Control Principles to Herbal Drugs. International Journal of Phytomedicine 1: 4-8. Singh, S. and Soni, G.R., WHO Expert committee on biological Standardization. Indian J Med. Res. 120: Street, R.A., Stirk, W.A. and Van Staden, J., South African traditional medicinal plant trade-challenges in regulating quality, safety and efficacy. Journal of Ethnopharmacology 119(3): Trease, G.E. and Evans, W.C., Pharmacognosy, 10th edn. Edn. Bailliere Tindel, London.

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108 Traditional Phytotherapy of E Jajpur Forests of Eastern Ghat, Odisha 1 *Usha Devi, 1 Himanshu Dwivedi, 2 Aminuddin, 1 Mohammad Zakir and 1 Hakimudin Khan 1 Regional Research Institute of Unani Medicine, Bhadrak , Odisha 2 Central Council for Research in Unani Medicine, 61-65, Institutional Area, Janakpuri, New Delhi Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract thnopharmacological surveys conducted in Jajpur forests of Odisha during 2014 and 2015 have resulted into collection of first-hand data on 68 folk medicinal species belonging to 60 genera and 40 families for treatment and cure of many diseases and conditions. The information on therapeutic uses of plants have been recorded through interviews of forest ethnics and traditional folk healers commonly known as Vaidyas. Each species enumerated has been provided with the information on plants scientific name, family, habitat, local name, locality with voucher specimen number, recipe and part(s) used in folk medicine, medical efficacy claimed, mode of administration, name of informant(s) and occurrence. Need for conservation and protection of such species under threat on account of their over-exploitation has been re-stressed. It s likely that data presented will contribute significantly to discover new drugs of natural origin for many of the diseases having no satisfactory cure in modern medicine, thus far. Keywords: Traditional phythotherapy, Jajpur forests, Medicinal plants, Odisha. Introduction Since time immemorial traditional herbal medicines are using to cure various ailments. Besides playing a crucial role in the primary health care, medicinal plants have been also one of the important sources of modern drug discoveries (Balunas and Kinghorn, 2005). Many modern medicine drugs were initially used in crude form in traditional or folk healing practice and other potentially useful biological activity (Iwu et al., 1999 and Maitera et al., 2011). This can be explained by the fact that 80% of 122 plant-derived drugs developed between 1981 and 2001 were related to their original ethnopharmacological uses (Fabricant & Farnsworth, 2001 and Wangchuk & Tobgay, 2015). Therefore documentation of indigenous knowledge is not only useful in highlighting locally important plant species but it is also helping to discover a new crude drug sources (Cox, 2000, Lahlou, 2013 and Newman, 2008). Presently, the study of indigenous knowledge of plants has become imperative facet of ethno medical research which is increasingly valuable in the development of healthcare and conservation programs in different parts of the world (Kunwar and Bussmann, 2008 and Shukla et al., 2010.). Medicinal plants are an integral part of indigenous system of Indian medicine and form a rich source of knowledge (Nadkarni, 1982, and Singh et al., 2013). 1 *Author for correspondence; usharudhyan18@gmail.com

109 102 India harbours about 15 percent ( ) medicinal plants out of 20,000 globally listed medicinal plants by World Health Organization (WHO) (Ramya et al., 2012 and Thaware and Saoji, 2013). It is worthwhile to note that, about 53.8 million tribal people with 427 different tribal communities are still dependent on traditional medicinal plant therapy for meeting their health care needs (Ghimire et al., 2004). Odisha is regarded as the homeland of the tribals having a total tribal population of about 7 million of 62 different ethnic groups, inhabiting in the dense and thick tropical forest areas and possesses rich empiric knowledge about plants wealth (Prusti, and Behera, 2007). Jajpur, a district of Odisha, is situated towards the north eastern region of the state. It lies between N latitudes and E longitudes. According to census (2011) about 95.5% populations of the district are rural and live in villages and the rest 4.5% live in urban areas. The district harbors a number of rural and tribal communities such as Munda, Shabar, Kolha, Jaung, Gond, Santal, Saora etc. which are living in remote hills of Jajpur district and a rich reservoir of natural resources of medicinal plants in its forests, wastelands, and grazing grounds, hedges and as agricultural weeds in crop fields. Many rural and tribal villages on the hills are without proper roads and primary health centers. They largely depend upon the ethno-botanical resources for their livelihood and primary healthcare and possess vast and important knowledge about many plants and their uses. However, traditional knowledge of plants is being constantly eroding due to urbanization and loss of traditional cultural systems. Therefore, many traditionally used medicinal species which can lead to new drug discovery may be lost in absence of proper documentation and identification. Keeping this in view, the present study was conducted with a view to identify knowledgeable resource persons and document their knowledge on the utilization of medicinal plants in the study area. Material and Methods Field surveys were conducted during August September of 2014 and 2015 to collect the information on ethnomedicinal plants from the study area. The plant specimens are collected, dried, mounted on herbarium sheets and preserved for further analysis. Standard procedures were adopted for collection, preserving and identifying the specimens (Jain and Rao, 1977). Plant specimens were identified on the basis of field notes, with the help of flora of Odisha (Saxena and Brahmam, 1996), Botany of Bihar & Orissa (Haines, ), other regional floras and online literature as well as through comparison with previous authenticated herbarium specimens of Survey of Medicinal Plant Unit (SMPU) of Bhadrak and deposited in the Herbarium of the SMPU of Regional Research Institute of Unani Medicine (RRIUM), Bhadrak, Odisha, for future reference.

110 103 Data on ethnomedicinal information were collected from the rural pockets of Tomka, Sukinda, Dalijoda, and Jajpur road forest ranges of Jajpur district through semi-structured interviews with the local knowledgeable rural and tribal people and traditional healer Vaidyas who were familiar with traditional uses of medicinal plants. Total 41 knowledgeable people, (of which 33 are male and 8 are female, between age group 20 to 76 years), were identified and interviewed. Details for local names of the plants, parts used, ailments treated, mode of administration, curative properties, source of the information and status were recorded. Status of plant species were noted on the bases of their occurrence in the nature. The species which found frequently are considered common (C); species found after wider distance in small patches were considered scattered (S); and thinly distributed species found in a few places were noted rare (R). Enumeration A total of 68 ethnomedicinal plants species collected and identified are used in the study area to cure various ailments. The listing represents botanical name with family, habit, local name, locality with voucher number, part(s) used, medical efficacy claimed, mode of application with informant(s) name and occurrence (C-Common, S-Scattered, R-Rare): Abrus precatorius L. (Fabaceae); Climber; Runjo; Amiyajhari-10416; Fruit; Hair loss; Fruits of Abrus precatorius L., leaves of Gritkunvari (Aloe barbadensis Mill.), fruit of Badi ambla (Phyllanthus amarus Schum. & Thonn.), leaves of Dhoob grass (Cynodon dactylon (L.) Pers.), leaves of Nimbu (Citrus aurantifolia (Christm. & Panz.) Swingle), leaves of Brahmi (Bacopa monnieri (L.) Pennell.), and rice (seed of Oryza sativa L.), all are mixed, grinded and made into paste. The paste is applied on scalp for hair loss (Smt. Mandiri Nayak/Gond) C. Abutilon indicum (L.) Sweet. syn. Abutilon albidum (Willd.) Sweet (Malvaceae); Shrub; Pidpidika; Bairi-10406; Leaf; Stomacheache and jaundice; Leaves of Pirpidika are mixed with termeric powder (Curcuma longa L.) and made into paste. The paste is consumed for stomacheache. Powder of leaves is used for jaundice (Shri. Dhamodar Dass) C. Achyranthes aspera L. (Amaranthaceae); Herb; Apamarango; Sukinda-10364; Whole plant; Kidney stone; Whole plant dried, burned and made into ash. Leaves of Gangosuli (Nyctanthes arbor-tristis L.), leaves of Basongo (Justicia adhatoda L.), leaves of Pasaruni (Paederia foetida L.), sonth (dried rizhome of Curcuma longa L.), leaves of pepal (Ficus religiosa L.), Kali mirchi (Piper nigrum L.), leaves of Bel patra (Aegle marmelos Corr.), all are soaked in water for whole night and water is filtered in next morning. One table spoon ash of Apamarang is taken with filtered water for kidney stone (Shri. Sudershan Mahnto) C.

111 104 Acorus calamus L. (Araceae); Herb; Goda-Vacha; Sansailu-10384; Rhizome; Join pain and diarrhea; About one table spoon of rhizome powder is given for joint pain. Decoction of rhizome is used for diarrhea (Shri. Sudershan Mahnto) S. Adiantum lunulatum Burm. syn. Adiantum philippense L. (Adiantaceae); Herb; Chhata gass, Kachandu; Gobarghati-10393; Leaf; Skin problem (boils, eczema); Leaves of Adiantum lunulatum Burm, rhizome of kachi haldi (Curcuma longa L.), jada tel (seeds oil of Ricinus communis L.) are made into paste. The paste is applied topically to cure skin problem (boils and eczema) (Shri. Saro Patro) S. Aerva lanata (L.) Juss. ex Schults. (Amaranthaceae); Herb; Pichhudi Sago; Ashokajhar-10367; Leaf and whole plant; Eye complaint, joint pain/joint inflammation.; Fresh leaves crushed and squeezed and the juice of crushed leaves is used as eye drop for running eye complaint. Whole plant dried and made into fine powder. One table spoon of powder is given for joint pain and joint inflammation (Shri. Palu Murmur/ Santal and Shri. Sudershan Mahnto) C. Ageratum conyzoides L. (Asteraceae); Herb; Puru; Kuila-10349; Leaf; Cut/wound; Extraction of fresh leaves is applied on cuts and wounds for healing purpose (Shri. Kulo Ram Munda) C. Ampelocissus latifolia (Roxb.) Planch. (Vitaceae); Climber; Kanzi; Kauliholi ; Leaf; Cut/wound and boil; Leaves paste is applied on cuts/wounds and boils (Smt. Jashoda Mahnto and Shri. Sudershan Mahnto) S. Andrographis paniculata (Burm.f.) Wall. ex Nees (Acanthaceae); Herb; Bhuieneem; Rankia-10348; Whole plant; Diabetes; Decoction of whole plant is used for diabetes (Shri. Budhu Ram Munda) C. Annona reticulata L. (Annonaceae); Tree; Atto; Kurkora-10011; Root; Boils; Paste prepared from the fresh root is use as poultice to cure boils (Shri Jayant Kumar Mishra) C. Annona squamosa L. (Annonaceae); Tree; Atto, Badhal; Burudisahi-10317; Root and Leaf; Snake bite, skin problem (itching, boils) and wound; Root paste is applied on snake bite. Extraction of fresh leaves is topically applied on wounds and skin problem (itching and boil) as antiseptic (Smt. Arti Patra and Shri Jayant Kumar Mishra) C. Arisaema tortousum (Wall.) Schott. (Araceae); Herb; Ulo; Saruabilli-10318; Bulb; Bloody dysentery; Boiled bulbs of plant are consumed by local people to stop bloody dysentery (Smt. Kunti Mahnto) S. Atylosia scarabaeoides Benth. (Fabaceae); Climber; Bankulthi; Gobarghati ; Seed; Kidney stone; Decoction of seeds is taken for kidney stone (Shri. Sudershan Mahnto) C.

112 105 Bacopa monnieri (L.) Pennell. (Scrophulariaceae); Herb; Brahmi; Gobarghati ; Leaf and whole plant; Headache and to increase memmory; Leaves of Brahmi (Bacopa monnieri (L.) Pennell.), leaves of Nimbu (Citrus aurantifolia (Christm. & Panz.) Swingle), Dhoob grass (Cynodon dactylon (L.) Pers.), Mehandi leaves (Lawsonia inermis L.), Gritkunvari leaves (Aloe barbadensis Mill.) and rice (seed of Oryza sativa L.), all are mixed and made into paste and the paste is applied for headache. Whole plant cooked with cow ghee and consumed to increase the memmory (Shri. Saro Patro and Shri. Sudershan Mahnto) S. Bryophyllum calycinum Salisb. syn. Kalanchoe pinnata Pers. (Crassulaceae); Herb; Amarpoi; Mirgichara-10396; Leaf; Dysentery; Leaves powder of the plant is mixed with fruit powder of kali mirchi (Piper nigrum L.); about one table spoon is given to cure dysentery (Shri. Niranjan/ Munda) C. Calotropis gigantea R. Br. (Asclepiadaceae); Shrub; Ark; Raighati-10314; Leaf; Joint pain; Warmed leaves are applied on painful joint to get relief (Shri. Ratan Chhatar/Munda) C. Cardiospermum helicacabum L. (Sapindaceae); Climber; Bishpatka; Bairi-10410; Leaf; Boils; Leaves paste is used for boils and on the scalp of children for suppuration (Sh. Dhamodar Dass) C. Cassia tora L. (Caesalpiniaceae); Shrub; Chakundi; Raighati-10313; Leaf and whole plant; Eczema, diabetes and diarrhea; Leaves are made into paste and the paste is fixed with thin cloth on affected part of eczema daily till cured. Young plant is consumed as vegetable by diabetic patient to control the blood sugar. Leaves extract is used to cure diarrhea (Shri. Ratan Chhatar/Munda and Shri. Sudershan Mahnto) C. Centella asiatica (L.) Urban (Apiaceae); Herb; Thalkudi; Kushi-10007; Whole plant, leaf, and root; Fever, brain tonic, dysentery and wounds; Decoction of whole plant is used for fever and as brain tonic. Leaves extraction is taken to cure dysentery. Paste of leaves and root is applied on wounds to kill germs (Shri. Surat Malik and Shri Ratana Kaurdass) S. Chloroxylon swietiana DC. (Rutaceae); Tree; Bheru; Khodihudi-10327; Bark; Wound; Paste of bark is used as antiseptic on wounds (Smt. Jashoda Mahnto) S. Chromolaena odorata (L.) King. & Robins. (Asteraceae); Herb; Poksunga, Gandhuri; Kurkora-10016; Leaf; Skin problem (acne) and cuts; Fresh leaves paste is used to cure skin problem (acne). Extraction of fresh leaves used on cuts to stop bleeding (Shri. Mangal Mohan Jaung/Jaung and Shri Bikari Charan Dass) C.

113 106 Cissampelos pareira L. (Menispermaceae); Climber; Akalbindi; Kunsai-10412; Root; Dysentery; Roots paste is used for dysentery (Shri. Mangal Mohan Jaung/ Jaung) S. Cleome viscosa L. (Capparidaceae); Herb; Janglisarson; Gobarghati-10475; Fruit; Malaria; About one table spoon fruit powder of the plant is given to cure malaria (Shri. Sudershan Mahnto) C. Clitoria ternatea L. (Fabaceae); Climber; Aparajita; Kushi-10004; Leaf; Skin problem (scabies and itching), swelling; Leaves paste is applied for skin problem (scabies and itching). Leaves are grinded into paste with water and applied on swelling (Shri. Ratan Chhatar/Munda and Shri Ratana Kaurdass) C. Corchorus capsularis L. (Tiliaceae); Herb; Naudita; Kalakundiya-10390; Leaf; Boils; Leaves paste is applied to cure boils (Shri. Roymuni Mahnto and Shri. Ram Dass/Besara) C. Cheilocostus speciosus (J.Koenig) C.D.Specht syn Costus speciosus (J.Koenig) Sm; (Costaceae); Herb; Keokonda, Ban makka; Satmano-10420; Rhizome; Joint pain; Paste of rhizome is applied for joint pain (Shri. Mohan Dass) S. Croton bonplandianus Baill. syn. Croton sparsiflorus Morong. (Euphorbiaceae); Herb; Banmirich; Ashokajhar-10374; Latex; Cut/wound; The latex is used on cuts and wounds for healing (Shri. Palu Murmur/Santal) C. Cymbopogon citratus (DC.) Stapf. (Poaceae); Herb; Dhanastri; Ponasia-10378; Leaf; Veterinary use (Stomachache); Leaves are given to domestic animal for stomachache (Shri. Bejanti Murmur/Santal) C. Datura metel L. syn. Datura fastuosa L. (Solanaceae); Herb; Koda Dudura; Kurkora-10015; Leaf; Joint pain, boils and sores; Leaves paste is applied for joint pain. Extract of leaves is used on boils and sores (Shri Bikari Charan Dass and Shri. Budhu Ram/Munda) C. Dioscorea bulbifera L. (Dioscoreaceae); Climber; Pita Allu; Saruabilli-10320; Bulb and tuber; Stomach problem (stomachache, indigestion, gastric problem) and cut/wound; Bulb of the plant is cooked and consumed for all type of stomach problem (stomachache, indigestion, gastric problem). Paste of tuber is applied on cuts and wounds (Smt. Kunti Mahnto and Shri. Sudershan Mahnto) S. Dioscorea oppositifolia L. (Dioscoreaceae); Climber; Pani Aloo; Ramthenga ; Rhizome, tuber and leaf; Fever, stomachache and wound; Decoction of rhizome is taken internally for fever. Tuber is consumed for stomachache. Leaves paste is used as antiseptic on wound (Smt. Jashoda Mahnto) S. Ficus hispida L.f. syn. Ficus oppositifolia Willd. (Moraceae); Tree; Dimuri; Raighar10430; Leaf; Skin infection; Paste of leaves is used for skin infection (Shri. Anand Kumar/Santal) S.

114 107 Gloriosa superba L. (Liliaceae); Climber; Langlangalia; Kaliapani-10298; Rhizomes; Joint pain; Rhizome crushed and boiled in mustard oil. After cooling, a lotion made and lotion is massaged on body for joint pain (Shri. Baldev Nayak) R. Helicteres isora L. (Sterculiaceae); Shrub; Murmuri; Bondhopoli-10427; Fruit; Diarrhea and dysentry; Fruit powder is used to cure diarrhea and dysentery (Shri. Hari Dass) S. Holarrhena pubescens (Buch. - Ham.) Wall. ex. G. Don syn. Holarrhena antidysentrica Wall. (Apocynaceae); Tree; Kurai; Kansa-10324; Seeds and root; Dysentery, cut/wound and boil; Seeds are made into paste. One table spoon paste is taken with one table spoon honey to cure dysentery in children. Roots paste is applied to cure cuts/wounds and boils (Shri. Vipro Deori) C. Justicia adhatoda L. (Acanthaceae) syn. Adhatoda zeylanica Medik.; Shrub; Basang; Ashokajhar-10368; Leaf and stem; Dysentery, skin infections, cough and fever; About 20 ml extraction of leaves is used to cure dysentery. Young leaves are made into paste and applied as poultice for skin infections. Decoction of leaves and stem are used for curing cough and fever (Shri. Palu Murmur/ Santal and Shri Jayant Kumar Mishra) C. Leucas cephalotes (Roth) Spreng. syn. Phlomis cephalotes Roth. (Lamiaceae); Herb; Gaso; Tomka-10307; Whole plant; Snake bite; Whole plant is dried in shade and made into powder. One table spoon of powder is given for snake bite with one glass of water for 7 days (Shri. Kumar Pradhan/Gond) C. Limonia acidissima L. syn. Feronia elephantum Corr. (Rutaceae); Tree; Kaitho; Jauranasahi-10360; Fruit; Dysentery; Fruit pulp is consumed to cure dysentery (Smt. Nagi Machhi/Santal) C. Mallotus philippensis (Lam.) Muell. - Arg. (Euphorbiaceae); Tree; Sinduri; Naya Bagopatiya-10337; Fruit; Vermifuge, skin problem (ring worm, scabies, burn); Decoction of fruit is used as vermifuge. Fruits crushed and mixed with coconut oil and applied externally for treatment of skin problem such as ringworm, scabies and burn (Shri. Sudershan Mahnto and Smt. Kunti Mahnto) S. Martynia annua L. (Martyniaceae); Herb; Baghnakha, Gomukhi; Singhapur ; Fruit; Joint pain; Fruits paste is applied for joint pain. Fruit boiled in mustad oil and the oil is massaged for joint pain (Shri. Vipro Deori) C. Mimusops elengi L. (Sapotaceae); Tree; Boalosree; Snakebite; Poichandia ; Stem bark; Paste of stem bark of Mimusops elengi L. and stem bark of Ficus religiosa L. is applied on snakebite (Shri Naranjan Dass) S.

115 108 Murraya koenigii (L.) Spreng. (Rutaceae); Shrub; Bhursunga; Barabati-10470; Leaf; Stomach problem (Diarrhea, dysentery and indigestion); Leaves are cooked with local dishes to cure all type of stomach problem such as diarrhea, dysentery and indigestion (Shri. Surender/Munda and Shri. Sudershan Mahnto) C. Nyctanthes arbor-tristis L. (Oleaceae); Gangosuli/Harsingar/Saringar; Shrub; Kushi-10008; Leaf; Fever and dysentery, common cold and cough; About ml leaves extraction is given daily for three days to cure fever. Leaves decoction is used for dysentery. Three to five leaves of Nyctanthes arbor-tristis L.and 2-3 black pepper seeds are boiled in one glass of water till half left. The decoction is given for common cold and cough (Shri. Jeevan Ram and Shri Narender) S. Ocium canum Sims. (Lamiaceae); Herb; Bantulsi/Gandhtulsi; Kushi-9997; Leaf; Boil and Dysentery; Fresh leaves paste is used on boils. Decoction of leaves is given for dysentery (Shri K.C. Jaina) C. Oxalis corniculata L. (Oxalidaceae); Herb; Sunsuniya; Kushi-9996; Whole Plant, Leaf; Indigestion and wound; Whole plant is consumed in raw form for indigestion. Leaves juice is applied as antiseptic on wounds (Shri K.C. Jaina) C. Paederia foetida L. syn. Paedaria scandens (Lour.) Merr. (Rubiaceae); Climber; Pasaruni; Balukantiya-10391; Leaf; Joint pain; About 10 ml extraction of leaves is given for joint pain for two times in a day morning/evening (Shri. Ram Dass/ Besara) R. Pergularia daemia (Forssk.) Chiov. syn. Asclepias daemia Forssk. (Asclepiadaceae); Climber; Utradi; Talapatia-10387; Latex and whole plant; Boils and joint pain; Latex of plant is applied on skin topically to cure boils. Whole plant is made into paste with jada oil (seed oil of Ricinus communis L.) and the paste is applied for joint pain (Shri. Pratap Mahnto and Shri. Ram Dass/Besara) C. Phyllanthus fraternus Webster (Euphorbiaceae); Herb; Bhuiamla; Kushi-9999; Leaf, whole plant and Fruit; Jaundice, diabetes, wounds, sores and leucorrhoea; The fresh leaves are grinded into paste. This paste is made into a tablet which is swelled in the morning with one glass of water to cure jaundice. Whole plant is kept in water for whole night and in the morning the water is drink to cure diabetes. The fruits paste is used to cure wounds and sores. Whole plant is boiled in one glass of water when half glass of water left the decoction is given to treat leucorrhoea (Shri. K.C. Jaina, Shri. Mongu Champia Munda and Shri. Sudershan Mahnto) C.

116 109 Phyllanthus reticulatus Poir. syn. Kirginelia reticulata (Poir.) Baill. (Euphorbiaceae); Shrub; Janju; Kushi-10009; Stem; Diarrhea; Decoction of stem is given to cure diarrhea (Shri Narender) C. Physalis minima L. syn. Physalis indica Lamk (Solanaceae); Herb; Phutka; Mirgichara-10397; Leaf; Fever; Leaves extraction is used to cure fever (Shri. Niranjan/Munda) C. Plumbago zeylanica L. (Plumbaginaceae); Herb; Rangchita; Ponasia-10379; Root; Rheumatoid arthritis and wound/cut; Dried roots are grinded into powder and about gm powder are given for rheumatoid arthritis. A handful of roots made into paste and applied to heal on cuts and wounds (Smt. Bejanti Murmur/ Santal and Smt. Nagi Machhi/Santal) S. Rauvolfia serpentina (L.) Benth. Ex Kurz. (Apocynaceae); Shrub; Patalgaruda; Burudisahi-10316; Root; Snake bite and stomachache; Fresh roots are made into paste and paste is feed slowly to the patient for snake bite. Extraction of fresh roots is used for stomachache (Shri. Sudershan Mahnto) R. Rauvolfia tetraphylla L. (Apocynaceae); Shrub; Patalgrud; Tomka-10306; Leaf and root; Stomachache; About 8-10 ml extraction of fresh leaves and roots is used for stomachache (Shri. Kumar Pradhan/Gond) S. Sida acuta Burm.f. (Malvaceae); Herb; Bajramuli; Kushi-10006; Leaf; Gastric problem, fever and wound; A handful of leaves are boiled with full glass of water, when half glass of water left, the decoction is used for gastric problem and fever. Paste of leaves applied on wounds for quick healing (Shri. Surender Kumar Tiria and Shri Ratana Kaurdass) C. Streblus asper Lour. (Moraceae); Tree; Shahda; Champajharo-10309; Leaf; Piles, diarrhea, dysentery; One table spoon of leave powder is taken with water twice in a day for piles. Decoction of leaves is used for diarrhea and dysentery (Shri.Surender Kumar Tiria and Shri. Sudershan Mahnto) C. Strychnos nux-vomica L. (Strychnaceae); Tree; Kochila; Kurkora-10014; Leaf; Boils; Leaves paste is applied externally to cure boils (Shri Bikari Charan Dass) S. Tephrosia purpurea (L.) Pers. (Fabaceae); Herb; Chakundo; Kaliapani-10300; Root and whole plant; Stomachache and skin problem (boils, acne); About 10 gm root powder is taken with water to get relief from stomachache. Paste of whole plant is applied to cure skin problem such as boils and acne (Shri. Baldev Nayak and Shri. Kumar Pradhan/Gond) C. Terminalia arjuna Wight. &Arn. syn. Pentaptera arjuna Roxb. ex DC. (Combretaceae); Tree; Arjun; Chandia-10467; Bark; Fever; Decoction of bark is used for fever (Shri. Mangat Ram/Santal) C.

117 110 Terminalia bellirica (Gaertn.) Roxb. (Combretaceae); Tree; Baheda; Baliapal ; Fruit; Indigestion and piles; Dried fruit powder is used to cure indigestion and piles (Shri. Chaitan Pradhan/Gond) C. Terminalia catappa L. (Combretaceae); Tree; Pestabadam; Darapan-10404; Bark; Dysentery; Decoction of bark is used for dysentery (Shri. Jeevan Ram) C. Terminalia chebula Retz. (Combretaceae); Tree; Harad; Patharbondo-10330; Fruit; Stomachache and indigestion; Dried fruits of Harad (Terminalia chebula Retz.), Ambla (Phyllanthus emblica L.), and Baheda (Terminalia bellirica (Gaertn.) Roxb.) are taken in equal quantity, and grinded into powder. One tablespoon of powder is taken for stomachache and indigestion (Smt. Lakshmi Chatar/Munda) C. Terminalia tomentosa (Roxb. ex DC.) Wight. & Arn. syn. Terminalia alata Heyne ex Roth (Combretaceae); Tree; Asan; Patabali-10331; Bark; Itching; Paste of bark is applied on itching (Shri. Geda Suagan/Santal) C. Tragia involucrata L. (Euphorbiaceae); Climber; Bichati; Ashokajhar-10375; Whole plant; Cough; The whole plant mixed with wheat flour and Roti (bread) made of flour is consumed to cure cough (Shri. Palu Murmur/Santal) C. Trewia nudiflora L. (Euphorbiaceae); Tree; Pithal/Jandakhai; Kalana-10461; Fruit; Wound healing; Paste of fruit is used for wound healing (Sh. Surat Malik) S. Trichosanthes tricuspidata Lour. (Cucurbitaceae); Climber; Mahakala; Amiyajhari ; Leaf; Joint point; Leaves of Mahakala (Trichosanthes tricuspidata Lour.), leaves of Begonia (Vitex negundo L.), and leaves of Ark (Calotropis gigantea R. Br.) are mixed with jada oil (seed oil of Ricinus communis L.) and made into paste. The paste is applied for joint point (Smt. Mandiri Nayak/Gond) S. Vanda roxburghii R. Br. syn. Vanda tessellata (Roxb.) Hook. ex G. Don (Orchidaceae); Epiphyte; Madang; Patabali-10332; Whole plant; Joint pain; Fresh plant is made into paste and the paste applied for joint pain (Sh. Geda Suagan Santal) S. Vitex negundo L. (Verbenaceae); Shrub; Begonia; Burudisahi-10315; Leaf; Joint pain, cut/wound, cold and cough; Leaves of Vitex negundo L. and leaves of Ark (Calotropis gigantea R. Br.) are made into paste. The paste is applied to cure joint pain. Fresh leaves extraction is applied for healing on cuts/wounds. Fresh leaves are boiled in water till vaporization and the vapors are inhaled to get relief from cold and cough (Smt. Arti patra, Sh. Sudershan Mahnto, and Sh. Geda Suagan/Santal) C. Xanthium indicum Koenig syn. Xanthium strumarium L. (Asteraceae); Herb; Mendasiuli; Darapan-10403; Root; Eczema; Paste of root is used topically on eczema (Sh. Jeevan Ram) C.

118 111 Results and Discussion The study area is rich with plant resources and their traditional knowledge. Most people are poor and lack the basic health care facilities. They largely depend upon the forest products for various herbal remedies. A total of 68 plant species belonging to 60 genera and 40 families were collected and are used by the rural and tribal people to cure various ailments (Figure 1). The most commonly (a) Abrus precatorius L.; (b) Abutilon indicum (L.) Sweet.; (c) Achyranthes aspera L.; (d) Acorus calamus L.; (e) Ageratum conyzoides L.; (f) Bacopa monnieri (L.) Pennell.; (g) Calotropis gigantea R. Br.; (h) Cardiospermum helicacabum L.; (i) Cissampelos pareira L.; (j) Cleome viscosa L.; (k) Clitoria ternatea L.; (l) Corchorus capsularis L.; (m) Costus spesiosus (J.Koenig.) Sm.; (n) Cymbopogon citratus (DC.) Stapf.; (o) Gloriosa superba L.; (p) Helicteres isora L.; (q) Pergularia daemia (Forssk.) Chiov.; (r) Rauvolfia serpentina (L.) Benth. Ex Kurz.; (s) Terminalia arjuna Wight. &Arn.; (t) Terminalia tomentosa (Roxb. ex DC) Wight. & Arn. Figure 1 (a-t): Some ethnomedicinal plants of Jajpur Forests

119 112 represented families are Euphorbiaceae (6 sps.), Combretaceae (5 sps.), Fabaceae (4 sps.), Apocynaceae, Asteraceae, Rutaceae (3 sps. each), Acanthaceae, Amaranthaceae, Araceae, Asclepiadaceae, Dioscoreaceae, Lamiaceae, Malvaceae, Moraceae, Solanaceae, Annonaceae (2 sps. each). Rest of the families are represented by one species each. The results of growth form analysis of medicinal plants showed that herbs made up the highest proportion with 26 species followed by trees (16 sps), climbers (13 sps), shrubs (12 sps.) and epiphyte (1 sps.) (Figure 2). A diverse use of these medicinal plants has been identified for the treatment of various diseases; among these, highest recorded number of species are used for Joint pain (12 sps.), followed by boil, dysentery (11 sps. each), wound (10 sps.), cuts (8 sps.), stomachache (7 sps.), diarrhea (6 sps.), fever (5 sps.), indigestion, snake bite (4 sps. each), cough, diabetes, eczema, itching (3 sps. each), brain tonic, cold, gastric problem, jaundice, kidney stone, piles, scabies, sore, acne (2 sps. each), bloody dysentery, burn, eye complaint, hair loss, headache, kill lice, leucorrhoea, malaria, rheumatoid arthritis, ring worm, skin infection, swelling, vermifuge (1 sps. each). Different parts of plant are used widely to treat these health problems included root, stem, leaves, latex, seeds etc. The most commonly used plant parts for herbal preparations are leaves (32 sps.) followed by whole plant (14 sps.), fruit, root (10 sps. each), bark (5 sps.), rhizome (4 sps.), bulb, latex, seed, tuber (2 sps. each) and stem (1 sps.) (Figure 3). Herbal remedies have been prepared by various methods. Most of them are prepared in paste form (34 sps.) followed by decoction (14 sps.), extraction (14 sps.), powder (12 sps.), juice (2 sps.), ash, lotion, vaporous (one sps. each). Some species are used in cooked form and consumed to cure the ailment (e.g Arisaema tortousum (Wall.) Schott., Bacopa monnieri (L.) Pennell., Cassia tora L., Dioscorea bulbifera L., D. oppositifolia L., Tragia involucrata L.). The preparation and application method of herbal medicine Figure 2: Pie diagram showing habit of different plant species

120 113 Figure 3: Different plant parts used for herbal medicine preparation varies and is based on the type of disease treated e.g. diseases such as dysentery, stomachache, diarrhoea, fever, indigestion, diabetes, jaundice, kidney stone, leucorrhoea, malaria etc. are mostly treated by oral administration in form of decoction, powder, juice, extraction etc. Paste and lotion mostly used topically for joint pain, wound, cuts, eczema, itching, scabies, sore, acne, headache, ring worm, skin infection etc. Leaves of Oxalis corniculata L. and fruit pulp of Limonia acidissima L. are consumed in raw form to cure disease and warm leaves of Calotropis gigantea R. Br. directly applied for joints pain. Generally fresh plant parts are preferred to prepare the paste, extraction, decoction, and juice, however, to prepare powder mostly the plant parts are collected, dried and preserved for future use. Herbal medicines prescribed by local healers are either the preparations based on single plant part or some times a combination of several plant parts are used to cures diseases rapidly; e.g. Abrus precatorius L., Abutilon indicum (L.) Sweet., Achyranthes aspera L., Adiantum lunulatum Burm., Bacopa monnieri (L.) Pennell., Bryophyllum calycinum Salisb., Mimusops elengi L., Pergularia daemia (Forssk.) Chiov., Terminalia chebula Retz., Trichosanthes tricuspidata Lour., Vitex negundo L etc. are used in combination of many plant parts of different species. Comparing the present data on folk medicines with previous published literature (Ali et al., 2010; Aminuddin et al., 2013; Aminuddin and Girach, 1996; Anonymous, 2001; Behera et al., 2006, 2008; Brahmam. & Saxena, 1990; Devi et al., 2014a & 2014b; Dhal et al., 2014; Girach et al., 2008; Girach et al., 2011; Jain, 1991; Kandari et al., 2012; Mallik et al., 2012; Mukesh et al., 2011, 2012; Mukesh et al., 2014a & 2014b; Murty et al., 1997; Panda, and Das, 1999; Panda et al., 2013; Pandey and Rout, 2006; Patra et al., 2014; Prusti, 1998; Raut et al., 2013; Rout et al, 2009a & 2009b; Sahu et al., 2010, 2013a & 2013b; Sarkar et al., 1999; Satapathy, 2010, 2015; Satapathy and

121 114 Brahmam, 1999; Satapathy & Chand, 2003; and Satapathy, et al., 2012), it is found that most of the folk medicinal plants recorded from the study area are either new or less known. However, their mode of administration, ingredients and parts used are sometimes different. Therefore, present study represents the contemporary uses of medicinal plants in the study area. The data presented contribute material for the discovery of new drugs of plant origin. During the study it has been also observed that traditional knowledge of plants in many tribal and rural communities of the area is changing because of rapid urbanization, socio-economic and cultural changes. In the present study 41 resources people (33 male and 8 female) were identified who possessed good knowledge of herbal therapy. However, it has been observed that the tradition knowledge is mostly confined with the older people above age 50 years (Figure 4). The questionnaires data indicated that the number of people with sound knowledge of herbal drugs is declining due to lack of interest among the younger generation as well as their tendency to migrate to cities for lucrative jobs, therefore, and there is a possibility of losing this ancient wealth of knowledge in the near future. As indigenous knowledge of medicinal plants is transmitted orally from one generation to next generation orally without any manuscript, hence the documentation of this valuable wealth will help to preserve it for future use. Moreover, local people are largely dependent on wild plants for herbal preparations but unsustainable collection of many medicinal plants has placed them in threatened and vulnerable categories. Many mountainous regions of Jajpur forests such as Sukinda and Tomka hills are rich plant resources but with the shrinking of the forests especially due to degradation of the forest areas, mining activities, industrialization and over-exploitation, their occurrence has been badly affected. Analyzing the distribution status in the study area, it is revealed Figure 4: Demography of informants of different age group

122 115 that majority of species are common (43 sps.) followed by scattered (22 sps.) and rare (3 sps.). Most of the common species are found in vicinity of the villages and are easily available for herbal preparation. However, some species are scattered and occurred at distance in few patches. The over-exploitation and unscientific extraction of these species could lead their scarcity in nature for future use. Three species such as Gloriosa superba, Rauvolfia serpentina, Paederia foetida are thinly distributed in the study area and are considered rare. Gloriosa superba and Rauvolfia serpentina are also in endangered category at state level (Ved et al., 2008). However, these species are largely extracted for herbal preparations, so diversity and distribution of these rare and endangered species and their traditional knowledge needs to be tapped and preserved for future use. Efforts must also be made to protect the medicinal plants resources by involving the local communities in their preservation and conservation before these are lost permanently. Germplasm collection, cultivation and propagation through modern agronomical techniques are suggested for these valuable species. Furthermore, the extensive field studies may also help in the discovery of new plant species and also new plant uses for health care needs. Acknowledgements The authors are extremely grateful to the Director-General, Central Council for Research in Unani Medicine, New Delhi, for providing all necessary facilities to carry out this work. We are also thankful to the local people of the study area to share their useful knowledge of traditional plants willingly. References Ali, Z.A., Hussaini, S.A. and Mukesh, K., Traditional Phytoremedies in Health Care among the Forest Ethnics of Balasore District, Orissa. Hippocratic J. Unani Med. 5(1): Aminuddin and Girach, R.D., Native phytotherapy among the Paudi Bhuinya of Bonai Hills. Ethnobotany 8: Aminuddin, Hussaini, S.A., Mukesh, K. and Samiulla, L Ethnobotanical Survey of Konark Forest of District Puri, Odisha. Hippocratic J. Unani Med. 8(2): Anonymous, Medicinal plants in Folklores of Orissa and Bihar. Central Council for Research in Unani Medicine, New Delhi. Balunas, M.J. and Kinghorn, A.D., Drug discovery from medicinal plants. Life Sci. 78:

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128 Preparation and Physicochemical Evaluation of an Antidiarrhoeal Safoof Ubaidul Hai, *Shariq Shamsi, Roohi Zaman and Peerzada Mohd Younis Department of Ilmul Saidla, National Institute of Unani Medicine, Kottigepalya, Magadi Main Road, Bengaluru Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract D 121 iarrhoea is one of the global public health concerns now a days. In Unani System of Medicine (USM) various anti-diarrhoeal formulations are used for the treatment of diarrhoeal diseases. For the assurance of safety, efficacy and reproducibility, it is mandatory that every herbal product should pass the tests of identification, authentication and physico-chemical stanardization. In the present study, a Unani anti-diarrhoeal Safoof (ADS) was prepared and its quality standards were established. After preparation of powder dosage form (ADS), it was evaluated on pysico-chemical parameters which included organoleptic properties; alcohol soluble matter; water soluble matter; successive extractives; ash values; moisture content; loss of weight on drying; ph in 1% solution and 10% solution; angle of repose; bulk density; tapped density; carr s index; hausner s ratio; volatile oils; qualitative estimation; quantitative estimation and TLC finger printing. The results of standard parameters of ADS have shown alcohol soluble matter ( ± 0.03%), water soluble matter ( ± 0.06%), moisture content (3.661 ± 0.33%), loss of weight on drying (7.156 ± 0.04%), ph in 1% solution and 10% solution (6.83 ± 0.04 and 5.67 ± 0.03 respectively), angle of repose ( ± 0.50), bulk density ( ± 0.00 gm/ml), tapped density ( ± 0.00 gm/ml), carr s index ( ± 1.02), hausner s ratio ( ± 0.01), volatile oils (3.066 ± 0.13%) and tannins (2.54 ± 0.04%). Various chemical constituents were also found positive. The TLC finger printing showed five spots on silica gel plate. These developed physicochemical standards of ADS may be used for future reference. Keywords: Anti-diarrhoeal, Physico-chemical standards, Safoof, Powder. Introduction The disease Is hal which strongly simulates with diarrhoea in symptoms and pathogenesis is a global public health concern. According to the World Health Organization (WHO) and UNICEF, about two billion cases of diarrheal disease occur worldwide every year, and 1.9 million children younger than five years of age perish from diarrhoea annually, mostly in developing countries. This amounts to 18% of all the deaths of children under the age of five years. 78% of all child deaths from diarrhoea occur in the African and South-East Asian regions (Anonymous, 2012). In Unani literature, there are number of powder formulations, which are used to treat diarrhoea such as Safoofe Kharnoob, Safoofe Sumaq, Safoofe Ood etc. However, a powdered drug (hence forth ADS anti-diarrhoeal Safoof.) suggested *Author for correspondence; shariqaligs@gmail.com

129 122 by Azam Khan is commonly used in the management of diarrhoea of diverse orgin in patients of all age groups. It contains Belgiri, Zeera Safaid, Kishneez Khushk, Zanjabeel, Badiyan and Raal safaid and used by the physicians for the treatment of Is hal (Khan, 2011) in common practice. In view of the fact that there are reports of wide-spread use of adulterated raw material leading to the production of inferior medicines (both single and compound), there is dire need for developing their pharmacopoeial standards. Present study on anti-diarrhoeal drug Safoof is based on this rationale and presents data on its physicochemical standards for future reference in an efforts to check adulteration in the finished product. Materials and Methods Materials The ingredients of ADS Belgiri, Zeera Safaid, Kishneez Khushk, Zanjabeel, Badiyan and Raal safaid (Table 1) were procured from the authorised dealer at Bengaluru. Prof. K. Ravikumar of Centre for Repository of Medicinal Resources (C-RMR), Trans-Disciplinary University (TDU), Attur, Bengaluru authenticated the drug samples. The voucher specimens of all the ingredients have been deposited in the museum of Institute of Trans-Disciplinary Health Sciences and Technology, Bengaluru. Preparation of Safoof All crude drugs were cleaned manually from impurities and allowed to dry in shade. Zeera safaid was roasted in a pan on low flame separately. Then all crude drugs were ground in an electrical grinder and passed through mesh No. 80 (Said, 1997) separately. The required quantity of each powder drug was mixed Table 1: Ingredients of ADS S.No. Unani Name Scientific Name Part used Quantity 1. Zeera safaid Cuminum cyminum L. Roasted dried 100gm biryan fruits 2. Badiyan Foeniculum vulgare Mills Dried fruits 100gm 3. Zanjabeel Zingiber officinale Rosc. Dried rhizome 100gm 4. Belgiri Aegle marmelos Corr. Dried fruit pulp 100gm 5. Kishneez khushk Coriandrum sativum (L.) Dried fruits 100gm Corr. 6. Raal safaid Vateria indica L. Resin 100gm

130 123 together to get homogenous powder and stored in an air tight glass container at room temperature (Fig. 1). Physico-chemical Evaluation The physicochemical studies of this anti-diarrhoeal Safoof were carried out in the laboratory of dept. of Ilmul Saidla, NIUM, Bengaluru, which included; (a) organoleptic properties like appearance, colour, smell and taste; (b) alcohol soluble matter; (c) water soluble matter; (d) successive extractives; (e) ash values; (f) moisture content; (g) loss of weight on drying; (h) ph in 1% solution and 10% solution; (i) angle of repose; (j) bulk density; (k) tapped density; (l) carr s index; (m) hausner s ratio; (n) volatile oils; (o) qualitative estimation; (p) quantitative estimation and; (q) TLC finger printing. Determination of organoleptic properties Appearance: Small quantity of ADS was taken and uniformity of particle size was observed. Colour: Five gram of ADS was taken into watch glass and placed against white background in white tube light. The colour of ADS was noted by using Pantone colour chart. Odour: A small quantity of the ADS was rubbed between thumb and index finger and inhaled. First, the strength of the odour like none, weak, distinct, strong etc was determined and then the odour sensation like aromatic, fruity, musty, mouldy, rancid, etc was evaluated. Fig. 1: Prepared sample of ADS

131 124 Taste: A pinch of ADS was examined for the taste on the upper surface of the tongue and one minute time was given to decide the taste. Determination of alcohol-soluble matter Five gm of anti-diarrhoeal Safoof was placed in a glass-stopper conical flask. Macerated with 100 ml of Ethyl alcohol for six hours shaking and then allow standing for 18 hours. Shake well and filtered rapidly through a dry filter paper. 25 ml of the filtrate was transferred to a previously weighed and tarred flat-bottom petridish and evaporate to dryness on a water bath. This filtrate was dried at 105 C for six hours, cooled in a desiccator for 30 minutes and weighed without delay. The percentage of alcohol soluble matter was calculated with reference to the amount of drug taken (Anonymous, 1998; 2009). Determination of water-soluble matter The percentage of water soluble matter was determined as above by using chloroform water instead of ethanol (Anonymous, 1998; 2009). Determination of successive extractive values The extractive values of ADS in different solvent viz. petroleum ether, chloroform and ethanol were carried out in soxhlet extractor. Five gm of Safoof was successively extracted with 150 ml of each solvent for 6 hours. The extracts were filtered with filter paper and transferred to a previously weighed and tarred flatbottom petridish and evaporated for complete drying on a water bath. After complete drying, the successive extractive values were determined with reference to the weight of Safoof (% w/w) (Anonymous, 2009). Determination of total ash Two gm accurately weighed air dried Safoof was incinerated in tarred silica dishes at a temperature not exceeding 450 C until free from carbon. Thereafter proper cooling was done in desiccators and weighed. The percentage of total ash was calculated with reference to the air-dried Safoof (Anonymous, 1998; 2006). Determination of acid-insoluble ash Total ash of ADS was boiled with 25 ml of diluted hydrochloric acid for five minutes and filtered. The insoluble matter was collected on an ash less filter paper (Whatman), washed with hot water and ignited at a temperature not exceeding 450 C for one hour and weighed after cooling in desiccator. The percentage of acid-insoluble ash was calculated with reference to the air dried Safoof (Anonymous, 1998; 2006).

132 125 Determination of water-soluble ash The total ash was boiled with 25 ml of distilled water for five minutes and filtered. The insoluble matter was collected on an ash less filter paper (Whatman), washed with hot water and ignited at a temperature not exceeding 450 C for one hour. The weight of insoluble ash was subtracted from the weight of total ash, giving the weight of the water soluble ash. The percentage of water soluble ash was calculated with reference to the air dried Safoof (Anonymous, 1998; 2006). Determination of moisture content The moisture content of Safoof was determined by Toluene Distillation method. 10 gm of Safoof was taken in a flask and 75 ml of Toluene was added to it. Distillation was carried out for 5 hours. The volume of water collected in the receiver tube (graduated in ml) was noted and the percentage of moisture was calculated with reference to the weight of the air dried Safoof (Afaq, 1994). Determination of loss on drying (gravimetric determination) Four gm of Safoof was taken, spread uniformly and thinly in a shallow petridish. It was heated at a regulated temperature of 105 C for five hours, cooled in desiccator and weighed. The process was repeated many times till two consecutive weights were constant. The percent loss in weight was calculated with reference to initial weight of Safoof (Anonymous, 1998). Determination of ph ph value of 1% solution One gm of Safoof was dissolved in accurately measured 100 ml of distilled water, filtered and ph measured with digital ph meter (Anonymous, 2006). ph value of 10% solution Ten gm of Safoof was dissolved in accurately measured 100 ml of distilled water, filtered and ph measured with digital ph meter (Anonymous, 2006). Determination of angle of repose The angle of repose was determined by using fixed funnel method. The height of the tip of funnel was fixed two cm above the horizontal surface. A graph paper was placed below the funnel on the table. The Safoof was allowed to flow through the funnel freely on to the surface until the apex of the conical pile just touches

133 126 the tip of the funnel. The diameter of the powder cone base was measured and the angle of repose was calculated by using this formula: (Anonymous, 2007). height of funnel tan θ = 0.5 base Determination of bulk density An accurately weigh ADS was introduced into a dry 100 ml graduated glass cylinder. The test sample was carefully levelled without compacting and the unsettled apparent volume (Vo), was observed. The bulk density was calculated by using the following formula: Where; ρb = M ρb = M = V o = V o gm/ml Apparent bulk density Weight of sample Apparent (untapped) volume of sample (Anonymous, 2006 and Gupta, 2013). Determination of tapped density After carrying out the bulk density by above procedure, sample containing graduated cylinder was tapped firstly for 500 times, followed by an additional taps of 750 and 1250 times in Tapped Density Apparatus (TD 1025) Lab India, until the difference between the two succeeding measurement is less than 2% and then tapped volume (Vf), was measured, to the nearest graduated unit. The tapped density was calculated, in gm per ml, using the following formula: M ρtap = Vf gm/ml Where; ρtap = Tapped density M = Weight of sample Vf = Tapped volume of sample (Anonymous, 2006 and Gupta, 2013). Determination of Carr s index Carr s index was calculated by using the following formula: Tapped Density (ρtap) Bulk Density (ρb) Compressibility index = 100 Tapped Density (ρtap)

134 127 Where; ρb = Bulk Density ρtap = Tapped Density (Anonymous, 2006; Gupta, 2013). Determination of Hausner s ratio Hausner s ratio is an indirect index of ease of powder flow. It was calculated by the following formula (Anonymous, 2006; Gupta, 2013): Tapped Density (ρtap) Hausner s Ratio = Bulk Density (ρb) Qualitative Estimation Alkaloids Mayer s test: One ml of acidic aqueous extract of ADS was taken in a test tube and few drops of Mayer s reagent were added. If pale yellow precipitate is formed then it is the indication of presence of alkaloids (Anonymous, 2006). Resin Small quantity of ethanolic extract of ADS was dissolved in five ml of acetic anhydride solution and gently heated. After cooling, 0.05 ml of sulphuric acid was added. If bright purplish red colour rapidly changed into violet then it is indication of presence of resin (Anonymous, 2006). Tannins Five ml of aqueous extract of ADS was taken in a test tube and few drops of 1% solution of lead acetate were added. If yellow precipitate is formed then it is indication of presence of tannins (Anonymous, 2006). Flavonoids 0.5 ml of alcoholic extract of ADS was taken in a test tube, 5-10 drops of diluted hydrochloric acid was added, after that small piece of zinc was added in this solution. This solution was boiled for few minutes. If pink colour is produced then it is indication of presence of flavonoids (Anonymous, 2006). Glycosides Small amount of alcoholic extract of ADS was dissolved in one ml of water, and sodium hydroxide solution was added in it. Yellow colour is produced in presence of glycosides (Anonymous, 2006).

135 128 Phenols A small quantity of alcoholic extract of Safoof was dissolved in five ml of distilled water, and 5-8 drops of 1% solution of lead acetate was added. If yellow precipitate is formed then it is indication of presence of phenols (Anonymous, 2006). Steroids Salkowski Reaction: One ml of conc. sulphuric acid was added in two ml of chloroform extract of ADS carefully from the side of test tube. Red colour is produced in chloroform layer, in the presence of steroids (Anonymous, 2006). Terpenoids Five ml of aqueous extract of ADS was taken in a test tube and two ml chloroform was added, then three ml conc. sulphuric acid was mixed in solution. If reddish brown interface is formed then it is indication of presence of terpenoids (Pandey and Mishra, 2011). Quantitative Estimation Determination of volatile oils 50 gm of ADS was mixed with the 30 ml of glycerol and 300 ml of water in the flask and few pieces of earthenware was added. Clavenger s apparatus was attached to the flask and heated on heating mantle with frequent agitation for five hours. The flask was rotated occasionally to wash down the adhering material to the upper part of the flask wall. After five hours, distillation was discontinued. After five minutes of cooling, volume of volatile oil was observed in Clavenger s apparatus graduated tube. This process was repeated few times for obtaining constant reading (Anonymous, 2006; 2009 and Afaq, 1994). Tannin Assay Preparation of drug infusion: Three gm of Safoof was extracted in 250 ml DD water for four hours at room temperature and then filtered. Quantitative estimation of Tannin: For the analysis of tannin content in test drugs, 25 ml of drug infusion, prepared by the method given above, was taken into one litre conical flask, then 25 ml of indigo solution and 750 ml distilled deionised water were added. 0.1 N aqueous solution of KMnO 4 was used for titration until the blue coloured solution changed

136 129 to green colour. Then few drops at time were added until solution turned into golden yellow colour. The volume of 0.1 N KMnO4 solution required for titration was recorded. For blank test (without drug infusion), the mixture of 25 ml Indigo carmine solution and 750 ml DD H 2 O was titrated with 0.1 N KMnO4 solution and the volume required for titration until solution turned into golden yellow colour was recorded. All samples were analyzed in triplicates. Calculations The tannin content (T %) in the sample is calculated as follows: (V V 0 ) x x 250 x 100 T (%) = g x 25 Where; T (%) V V 0 = Tannin quantity in percentage = Volume of 0.1 N aq. solution of KMnO 4 for the titration of the test sample in ml. = Volume of 0.1 N aq. solution of KMnO 4 for the titration of the blank sample in ml = Tannin equivalent in 1 ml of 0.1 N aqueous solution of KMnO 4 g = Mass of the sample taken for the analysis in gm 250 = Volume of the volumetric flask in ml. (Atanassova et al., 2009) TLC finger printing For the separation of different phytochemical constituents in the ADS, the methanolic extract was spotted manually using a capillary tube on pre-coated silicagel TLC plates 60 F 254 (layer thickness 0.25). The spotted plates were put into a solvent system- Toluene : Ethyl acetate : Formic acid (6 : 3 : 1). After the separation of phytochemical constituents, the plate was taken out and placed in oven at 110 C for drying. After drying, iodine vapours were used to visualize the spots. The colour of the spots were noted and R f values were calculated by using the following formula (Karthika et al., 2014): Distance travelled by spot Retention time (R f ) = Distance travelled by solvent Results and Discussion The organoleptic properties of ADS were found to be greenish yellow in colour, amorphous uniform particles with aromatic pungent taste (Table 2). These are

137 130 Table 2: Organoleptic Properties of ADS Appearance Amorphous powder Colour Goldenrod 3 (greenish yellow) Odour Aromatic pungent Taste Spicy very important for drug identification and quality assurance and also necessary for patient compliance; the acceptance among the consumers automatically gets increased if these properties are good. The alcohol and water soluble matter of ADS was found to be ± 0.03 and ± 0.06 respectively (Table 3). This shows that the constituents of the drug are more soluble in alcohol than water (Fig. 2). Table 3: Physicochemical parameters of ADS Parameters Samples Mean ± SEM Alcohol soluble matter (%) ± 0.03 Water soluble matter (%) ± 0.06 Successive Extractive Values: Petroleum ether (%) ± 0.60 Chloroform (%) ± 0.05 Ethyl alcohol (%) ± 0.18 Ash Values: Total ash (%) ± 0.02 Acid insoluble ash (%) ± 0.07 Water soluble ash (%) ± 0.07 Moisture content (%) ± 0.33 Loss of weight on drying (%) ± 0.04 ph Value: 1% solution ± % solution ± 0.03 Angle of repose ± 0.50 Bulk density (gm/ml) ± 0.00 Tapped density (gm/ml) ± 0.00 Carr s index ± 1.02 Hausner s ratio ± 0.01

138 131 The mean percentage of successive extractive values of ADS were found to be ± 0.60, ± 0.05 and ± 0.18 in petroleum ether, chloroform and ethanol respectively (Table 3). This is an important parameter to evaluate the quality, purity and adulteration of the drugs. The chemical constituents of the drugs are extractable in different solvent systems. These values are high in particular solvent in which the drug constituents are maximally soluble (Fig. 3). The mean percentage of total ash, acid insoluble ash and water soluble ash were found to be ± 0.02, ± 0.07 and ± 0.07 respectively (Table 3) (Fig. 4). Total ash includes both physiological ash, which is derived from the plant tissue itself and non-physiological ash which is residue of the extraneous matter adhering to the plant surface. Acid insoluble ash includes the amount of silica present, especially as sand and siliceous earth (Bele et al., 2011). The mean percentage of moisture content by toluene distillation was found to be ± 0.33 (Table 3) (Fig. 5). Low moisture content is always desirable for higher stability of drugs (Bhargava et al., 2013). An excess of water in medicinal plant materials provide good media for microbial growth, and deterioration following hydrolysis. The mean percentage of loss of weight on drying was found to be ± 0.04 (Table 3) (Fig. 5). Higher value than the moisture content by toluene method shows the presence of volatile oil in the ADS. It indicates the loss of volatile substances along with the water. Fig. 2: Alcohol and Water soluble matter Fig. 3: Successive extractive values of of ADS ADS

139 132 Fig. 4: Ash values of ADS Fig. 5: Moisture content & Loss of wt. on drying of ADS Angle of repose was found to be ± 0.50 (table 3). The result shows that inter-particular friction of particles of ADS is very high which shows its low flow ability. The mean values of bulk density and tapped density of ADS were found to be ± 0.00 and ± 0.00 respectively (Table 3) (Fig. 7). Bulk and tapped density are very important while deciding the size of containers needed for handling, shipping, and storage of raw material and blend. Fig. 6: ph values of ADS Fig. 7: Bulk density and Tapped density of ADS

140 133 The mean values of carr s index and hausner s ratio were found to be ± 1.02 and ± 0.01 respectively (Table 3) (Fig. 8) which is graded as passable flow ability. In theory, the less compressible a material the more flowable it is (Anonymous, 2006). In qualitative estimation of ADS; alkaloids, resin, trannins, flavonoids, glycosides, phenols, steroids and terpenoids were examine for their presence which are responsible for the therapeutic effects and these were found to be positive (Table 4). Volatile oils are plant s secondary metabolites that are known for their fragrance and food flavour properties. They consist of a complex mixture of mono and sesqui-terpenes, phenyl propanoids and oxygenated compounds (Bazaid et al, 2013). The mean percentage of volatile oil was found to be ± 0.13 (Table 5) (Fig. 9). Tannins are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins. The astringency from the tannins is that which causes the dry and puckery feeling in the mouth on consumption. Tannins may be employed medicinally in anti-diarrheal, haemostatic, and anti-haemorrhoidal compounds. Tannins have been used for immediate relief of sore throats, diarrhoea, dysentery, haemorrhage, and skin ulcers (Ashok, 2012). The mean percentage of tannin was found to be 2.54 ± 0.04 (Table 5) (Fig. 9). Table 4: Qualitative estimation of ADS S.No. Medicinal constituents Presence 1. Alkaloids + 2. Flavonoids + 3. Glycosides + 4. Phenols + 5. Resin + 6. Steroids + 7. Tannins + 8. Terpenoids + Table 5: Volatile oil and Tannins of ADS S.No. Volatile oil (%) Tannins (%) Mean ± SEM ± ± 0.04

141 134 Fig. 8: Carr s index and Hausner s ratio of ADS Fig. 9: Volatile oil and Tannins of ADS TLC is one of the important parameters used for detecting the adulteration for evaluating the quality of drugs. If the drug is adulterated there might be appearance of the other compounds present in adulterant, in turn may increase the number of spots. On the other hand the exhausted or deteriorated drugs may lose the component and the number of spots appeared might be less. Five spots were found on TLC silica plate with the methanolic extract of ADS. The R f values of five spots were found to be 0.50, 0.58, 0.66, 0.80 and 0.86 and the colour of spots were brown, light brown, light yellow, dark yellow and dark yellow respectively (Table 6) (Fig. 10). Under visible light Fig. 10: TLC of ADS Under UV light

142 135 Table 6: TLC of ADS Extract Solvent No. of spots R f Value Colour Methanol Toluene: , Brown, Ethyl acetate: 0.58, light brown, Formic acid 0.66, light yellow, (6 : 3 : 1) 0.80 and dark yellow and 0.86 dark yellow Conclusion The developed physicochemical standards of ADS may be used for future reference to check adulteration (if any) in the drug studied. Acknowledgement The authors are highly thankful to Prof. M.A. Siddiqui, Director, National Institute of Unani Medicine, Bengaluru, for providing necessary facilities for the present study. References Afaq, S.H., Tajuddin, Siddiqui, M.M.H., Standardization of herbal drugs. AMU Press, Aligarh, pp , 100. Ahsan, A., 2006, Qarabadeene Ahsani. CCRUM, Ministory of Health & Family Welfare, Govt. of India, New Delhi, p.3. Anonymous, Quality control methods for medicinal plant materials. WHO, Geneva, pp Anonymous, Physicochemical standards of Unani formulations. Part IV, CCRUM, New Delhi, pp. 142, , 148, Anonymous, The Japanese Pharmacopoeia. Ministry of Health, Labour and Welfare, Japan. Anonymous, The United States Pharmacopoeia. 30-NF 25 (e-book). Anonymous, National formulary of Unani medicine. Part V, Ministry of Health and Family Welfare, Govt of India, New Delhi, p Anonymous, The Unani pharmacopoeia of India. Part II (I). Ministry of Health and Family Welfare, Department of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy (Ayush), Government of India, New Delhi, pp

143 136 Anonymous, Acute diarrhea in adults and children: a global perspective. World Gastroenterology Organisation Global Guidelines. org/assets/export/userfiles/acute%20 Diarrhea_long_FINAL_ pdf. [cited on ] Ashok, P.K., Upadhyaya, K., Tannins are astringent. Journal of Pharmacognosy and Phytochemistry 1(3): Atanassova, M., Bagdassarian, V.C., Determination of tannins content by titrimetric method for comparison of different plant species. Journal of the University of Chemical Technology and Metallurgy 44(4): Bazaid, S.A., El-Amoudi, M.S., Ali, E.F., Abdel-Hameed, E.S., Volatile oil studies of some aromatic plants in Taif region. Journal of Medicinal Plants Studies 1(5): Bele, A.A., Khale, A., Standardization of herbal drugs: an overview. IRJP 2(12): Bhargava, V.V., Saluja, A.K., Dholwani, K.K., Detection of heavy metal contents and proximate analysis of roots of Anogeissus latifolia. Journal of Pharmacognosy and Phytochemistry 1(6): Gupta, R., Sharma, P., Garg, A., Soni, A., Sahu, A., Rai, S., et al., Formulation and Evaluation of Herbal Effervescent Granules Incorporated with Calliandra haematocephala Leaves Extract. Indo-American Journal of Pharmaceutical Research 3(6): Karthika, K., Jamuna, S., Paulsamy, S., TLC and HPTLC fingerprint profiles of different bioactive components from the tuber of Solena amplexicaulis. Journal of Pharmacognosy and Phytochemistry 3(1): Khan, A., Iksea Azam Ida Kitabul Shifa, New Delhi, p Pandey, A., Mishra, R., Antibacterial properties of Aegle marmelos leaves, fruits and peels against various pathogens. JPBMS 13(13): 1-3. Said, M., Hamdard pharmacopoeia of eastern medicine. Sri Satguru Publications, Delhi, p. 152.

144 Standardization of a Unani Drug Habb-e- Banafsha 1 *Asma Sattar Khan, 1 R.P. Meena, 1 Shoeb A. Ansari, 2 Afzal Hashmi, 2 Shams-ul-Arfin and 2 Aminuddin 1 Drug Standardization Research Institute, PLIM Campus, Kamla Nehru Nagar, Ghaziabad Central Council for Research in Unani Medicine, 61-65, Institutional Area, Janakpuri, New Delhi Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract T 137 raditional medicines are being used extensively all over the world for primary health care. Nowadays, the needy mass requires safe and efficacious herbal medicines due to their lesser side effects. Hence there is an urgent need for standardization and development of standard operative procedures (SOPs) of traditional medicine to provide the quality medicines in the market. To ensure the genuineness and quality of drug, work on evaluation of pharmacopoeial standards was carried out on Habb-e-Banafsha which is used to cure Asthma, Bronchitis & Caphalagra. The Standard Operative Procedure (SOP) for the preparation of quality medicine was developed at the laboratory scale, using botanically identified and standardized ingredients. The samples of the formulation were subjected to evaluate the pharmacopoeial parameters such as Microscopy, Physico-chemical analysis, TLC, HPTLC and WHO parameters viz. microbial contamination, pesticide residues, aflatoxins level and the presence of heavy metals to ascertain the quality of the drug. Keywords: SOP, Microscopy, TLC, HPTLC, UV, Heavy metals. Introduction Habb-e-Banafsha is a Unani poly-herbal formulation, categorized under Huboob in the National Formulary of Unani Medicine, Part III (Anonymous, 2001). Habbe-Banafsha is reputed for its expectorant (Munaffis-e-Balgham), sedative (Musakkin), Coctive, concoctive and Maturative (Munzij) action and is used in the treatment of Suda (Cephalagia), sual (Bronchitis) and Zeeq-un-Nafas (Asthma) (Kabiruddin, 1938). The present study was aimed to standardize this important Unani drug in order to ensure its authenticity, quality and efficacy. Habb-e-Banafsha was prepared according to the formula as described in NFUM-III using four ingredients of plant origin (UPI, Part I, Vol. I, II, V) (Table 1). In order to lay down SOPs and pharmacopoeial standards, this drug was prepared in laboratory at DSRI, Ghaziabad. The present paper describes the salient features of preparation, microscopical characters, physico-chemical parameters, TLC, HPTLC, UV spectroscopy, heavy metals estimations, Aflatoxins and pesticide estimation (Anonymous, 2000) not reported so far. Materials and Methods Preparation of Drug: All the ingredients were procured from local raw drug dealer and were identified 1 *Author for correspondence

145 138 Table 1: Formulation Composition S.No. Ingredients Botanical Name Part used 1. Banafsha Viola odorota L. Flower 2. Turbud Operculina turpethum (L.) Root S. Manso 3. Rubb-us-Soos Glycyrrhiza glabra L. Root 4. Arq-e-Barg-e-Kasni Sabz Chichorium intybus L. Fresh leaf juice botanically (Wallis, 1967; Trease & Evans, 1972) using pharmacognostical methods. The ingredients were further validated by comparing with a monograph available in UPI (Part I), Vol. I, II & V. All the ingredients were taken of pharmacopoeial quality. The ingredients were cleaned and dried under shade to remove the moisture if any. The ingredients (S. No. 1-3, Table 1) were crushed separately in an iron-mortar to obtain their coarse powders. The coarse powders were further ground in a mixer grinder to get the fine forms. The fine powders were mixed thoroughly and sieved through mesh No. 80. Arq-e-Barg-e-Kasni sabz was then added to the mixture and again mixed thoroughly to obtain the lubdi mass. Huboob was prepared from the lubdi mass by mechanical process and dried under shade. The prepared Huboob were stored in tightly closed glass container free from moisture and kept in a cool and dry place. Microscopy 5 g of the powdered drug was taken and stirred gently with hot water in a beaker. The supernatant was discarded and the residue was washed with the distilled water. A little residue was stained with Iodine solution and mounted in 50% glycerine. Some of the residue was heated in chloral hydrate solution and mounted in 50% glycerine and a little residue was boiled in 2% potassium hydroxide solution, washed with distilled water and mounted in 50% glycerine (Johansen, 1940; Wallis, 1967). Chemical analysis Physico-chemical parameters of Habb-e-Banafsha such as removal of foreign matters, solubility in water, ethanol and petroleum ether (60-80 o ), total ash, acid insoluble ash and water soluble ash, loss on drying at 105 o C, ph values of 1% and 10% aqueous solution and volatile oil estimation were analysed by standard methods (Anonymous, 1987; 1998). Detection of microbial load, Aflatoxins and analysis of pesticide residue (Anonymous, 2000) and heavy metal were carried out as per standard methods (Sahito et al., 2001).

146 139 Preparation of extract for TLC/HPTLC Samples of all the three batches of the formulation were extracted separately with chloroform and ethanol. The extracts were concentrated and made up to 10ml in a volumetric flask separately. These solutions were used for the TLC/ HPTLC finger print analysis by employing CAMAG Linomat IV sample applicator on aluminium TLC plate pre-coated with silica gel 60 F 254 (E. Merck). The chromatograms were developed using the solvent system toluene: ethyl acetate in the ratio 9:1 & 1:1 respectively for chloroform and ethanol extracts. The plates were dried at room temperature and observed the spots at UV-254 and UV-366. Further the plates were dipped in 1% vanillin-sulphuric acid reagent and heated at C till coloured spots appeared. (Wagner et al., 1984; Sethi, 1996; Stahl, 1996) Preparation of extract for U.V. Spectroscopic studies 1g of the drug was extracted with 100 ml. Petroleum ether (60-80 o ) by refluxing for 15 minutes on water bath and filtered. The solution was made up to 100 ml. in volumetric flask. This solution was used for U.V. analysis and pure petroleum ether (60-80 o ) was used as blank solution (Willard et. al, 1965). Observations Habb-e-Banafsha is small brown pill having hard texture, characteristic odour and slightly sweetish taste. The drug did not show any filth, fungus or objectionable matter while the sample was spread in a Petri dish (Fig. 1). The microscopy shows xylem vessels with pitted thickenings, cork cells, few crystal sheath of parenchymatous cells containing a prism of calcium oxalate (Rubb-us-soos), parenchyma having rosettes of prismatic calcium oxalate crystals uni and biseriate medullary rays, resin cells, tubular and polygonal epidermal cells, cellulosic fibres with pointed tips (Turbud). Thin walled trichomes with printed tips arising from epidermal cells with radially oriented subsidiary cells. Vessels narrow, seleriform, tailed at both end, round shaped pollen grains (Banafsha). The results observed for the physico-chemical data, microbial load, aflatoxins, pesticide residues, heavy metals, TLC and HPTLC finger printing are shown in Table 2,3,4,5,6,&7 respectively. Results and Discussion The physico-chemical data of the drug are shown in table-2. The low value of water soluble extractive ( %) shows the absence of any inorganic

147 140 Fig. 1 : Unani drug Habb-e-Banafsha Table2: Physico-chemical Parameters S.No. Parameters Results 1. Water solubility % 2. Alcohol solubility % 3. Pet ether ( ) solubility % 4. Loss in weight on drying at C % 5. Total Ash % 6. Acid Insoluble Ash % 7. Water Soluble Ash % 8. ph of 1% Solution ph of 10 % Solution Volatile Oil Traces constituents. The moisture content in the drug was also low as the loss in weight on drying at C occurred below 10%. The low value of acid insoluble ash of the drug indicates that the drug is free from siliceous matter. The results of microbial studies viz. TBC and TFC are within the permissible limits while the other microbes are absent (Table-3). The results of Aflatoxins studies (Table-4) and pesticide residues (Table-5) show that the drug is free from Aflatoxins as

148 141 Table 3: Microbial Load S.No. Parameter Analyzed Results Permissible limit as per WHO 1. Total Bacterial load 2x10 3 cfu/g 10 5 cfu/g 2. Total fungal count < 10 cfu/g 10 3 cfu/g 3. Enter obacteriaceae Absent Nil 4. Escherichia coli Absent Nil 5. Salmonella cpp. Absent Nil 6. Staphoilococcus aureus Absent Nil Table 4: Aflatoxins Level S.No. Parameter Analyzed Results Detection limits 1. B-1 Not detected 0.50 ppm 2. B-2 Not detected 0.10 ppm 3. G-1 Not detected 0.50 ppm 4. G-2 Not detected 0.10 ppm Table 5: Pesticide Residue S.No. Parameter Analyzed Results Limit 1. Chlorpyriphos Not detected 0.20 mg/kg 2. DDT Not detected 1.00 mg/kg 3. Endosulfan Not detected 3.00 mg/kg 4. Malathon Not detected 1.00 mg/kg 5. Parathion Not detected 0.50 mg/kg Table 6: Heavy Metals S.No. Heavy Metal Analyzed Results Permissible limit as per WHO 1. Arsenic Not detected 3.00 ppm 2. Cadmium Not detected 0.30 ppm 3. Mercury Not detected ppm 4. Lead Not detected ppm well as pesticide residues. The content of heavy metals are below detection limits (Table-6) suggests that drug is free from any type of heavy metal contamination.

149 142 HPTLC profile HPTLC methods are reliable and convenient means in identification of crude drugs as well as compound formulations as each plant species produces a distinct chromatogram. TLC of all the three batches of Habb-e-Banafsha were observed under UV 254nm, UV 366nm and after derivatization (Table-7). Chromatogram of chloroform extract shows 08 spots under UV 254nm (Fig. 2), 12 spots under UV 366nm (Fig. 3) and 09 spots after derivatization (Fig. 4). The finger print of chloroform extract shows 13 peaks out of which peaks at R f 0.09, 0.61, 0.70 and 0.77 were major peaks whereas peaks at R f 0.04, 0.17, 0.39, 0.42, 0.49, 0.55, 0.85, 0.92 and 0.96 are relatively smaller peaks (Fig. 5). HPTLC Chromatogram of chloroform extract is shown in Fig. 6. Table 7: TLC Results S. Extract Solvent Developing Rf Values with colour No. System reagent UV254nm UV366nm After derivatisation 1. Chloroform Toluene : Vanillin 0.95 Black 0.95 Red 0.91 Grey Ethyl Sulphuric 0.66 Black 0.91 Red 0.83 Grey acetate (9:1) acid 0.60 Black 0.67 Red 0.66 Green 0.53 Black 0.60 Red 0.60 Yellowish green 0.47 Black 0.55 Blue 0.53 Violet 0.42 Black 0.52 Red 0.40 Violet 0.33 Black 0.47 Red 0.31 Grey 0.10 Black 0.31 Red 0.14 Violet 0.26 Red 0.10 Grey 0.18 Red 0.13 Red 0.11 Blue 2. Ethanol Toluene : Vanillin 0.95 Black 0.95 Red 0.85 Violet Ethyl Sulphuric 0.89 Black 0.87 Pink 0.74 Violet acetate (1:1) acid 0.74 Black 0.74 Pink 0.71 Grey 0.69 Black 0.71 Red 0.58 Yellow 0.58 Black 0.61 Brown 0.46 Grey 0.45 Black 0.51 Red 0.39 Grey 0.30 Black 0.43 Violet 0.35 Violet 0.14 Black 0.35 Red 0.14 Yellowish green 0.11 Black 0.19 Violet 0.11 Grey 0.11 Red

150 143 B1 B2 B3 B1 B2 B3 B1 B2 B3 UV254nm UV366nm After derivatization *B1 Batch1, B2 Batch2, B3 Batch3 Fig. 2, 3 4: TLC of Chloroform extract of Habb-e-Banafsha Fig. 5: HPTLC Finger printing of Chloroform extract Fig. 5: R f Values of HPTLC Finger prints of Chloroform extract

151 144 Fig. 6: HPTLC Chromatogram of Chloroform extract Similarly TLC of ethanol extract shows 09 spots under UV 254nm (Fig. 7), 10 spots under UV 366nm (Fig. 8) and 09 spots after derivatization (Fig. 9). The finger print of ethanol extract shows 13 peaks out of which peaks at R f 0.03, 0.07, 0.17, 0.34, 0.53, 0.68 and 0.87 were major peaks whereas peaks at R f 0.22, 0.40, 0.45, 0.59, 0.78 and 0.82 are relatively smaller peaks (Fig. 10). HPTLC Chromatogram of ethanol extract is shown in Fig. 11. The HPTLC densitometry chromatograms of chloroform and ethanol extract of all the three batches were found to be similar when scanned at 254nm. It indicates batch to batch consistency of the compound formulation. B1 B2 B3 B1 B2 B3 B1 B2 B3 UV254nm UV366nm After derivatization Fig. 7, 8, 9: TLC of Ethanol extract of Habb-e-Banafsha

152 145 Fig. 10: HPTLC Finger Printing of Ethanol extract Fig. 10: R f Values of HPTLC Finger prints of Ethanol extract Fig. 11: HPTLC Chromatogram of Ethanol extract

153 146 UV Spectroscopic Studies The study of the U.V. spectra of Habb-e-Banafsha and its ingredients shows that a broad spectrum at 208nm with an absorbance of 1.609% appears in the spectrum of Habb-e-Banafsha (Fig. 12). This single broad spectrum indicates the merger of characteristic peaks of its ingredients as its ingredients show a sharp peak at 206nm with an absorbance of 1.090%(Banafsha), a sharp peak at 206nm with an absorbance of 1.158%(Rubb-us-soos) and a sharp peak at 208nm with an absorbance of 1.540%(Turbud) (Fig.13, 14 & 15).It is clearly evident that each drug has its specific data regarding maximum absorption peak and also has characteristic spectra which can be applied for the prediction of authenticity of the drug. Habb-e-Banafsha Fig. 12: UV Spectrum of Habb-e-Banafsha

154 147 Banafsha Fig. 13: UV Spectrum of Banafsha Conclusion It can be concluded that organoleptic parameters are not much reliable in identification of herbal drugs once the ingredients are powdered and mixed together for preparing compound formulation. The present study, therefore, holds

155 148 Rubb-us-soos Fig. 14: UV Spectrum of Rubb-us-Soos high significance as the microscopic features, various Physico-chemical parameters, HPTLC profile, UV spectrum etc. provide criteria for easy identification of the drug Habb-e-Banafsha and ensure the authenticity, quality and efficacy of the medicine.

156 149 Turbud Fig. 15: UV Spectrum of Turbud Acknowledgement The authors are extremely thankful to Director-General, CCRUM, New Delhi, for his constant encouragement and valuable guidance. We are also thankful to Incharge DSRI, Ghaziabad, for providing necessary facilities and support.

157 150 References Anonymous, 1987, Physico-Chemical Standards of Unani Formulations, Part-II. CCRUM, Govt. of India, New Delhi, pp Anonymous, WHO Quality Control Methods for Medicinal Plants Materials. World Health Organization, Geneva, pp Anonymous, Official Methods of Analysis Association of Official Analytical Chemist (AOAC), 17 th ed. Arlington, USA, pp Anonymous, 2001, National Formulary of Unani Medicine, Part-III. Ministry of Health and Family Welfare, Govt. of India, p.11. Johansen, D.A., 1940, Plant Microtechniques. Mc-Graw Hill book company Inc., New York and London, pp Kabiruddin, 1938, Al-qarabadeen (Urdu). Dafter Almasih, Karol Bagh, Delhi, p.27. Sahito, S.R., Kazi, T.G., Kazi, G.H., Jakhrani, M.A. and Shaikh, M.S., Trace Elements in Two Varieties of an Indigenous Medical Plant Catharanthus roseus (Vinca rosea). The Sciences 1(2): Sethi, P. D., 1996, High Performance Thin Layer Chromatography, 1 st ed, Vol. X. CBS Publishers and Distributors, New Delhi, pp Stahl, E., 1996, Thin layer Chromatography-A laboratory Hand book. George Allen and Unwin Ltd., London, p Trease, G.E. and Evans, W.C., Pharmacognosy, 10 th ed. Bailliere Tindall, London, pp Unani Pharmacopoeia of India, Part I, Vol. I, II, V. Ministry of Health & Family Welfare, Govt. of India, pp. 09, 41, 105. Wagner, H., Bladt, S. and Zgainski, E.H., 1984, Plant Drug Analysis- A Thin Layer Chromatography Atlas, 2 nd ed. Springer Verlag, Germany, p. 76. Wallis, T.E., Text Book of Pharmacognosy, 3 rd ed. J & A Churchill Ltd., London, p Willard, H.H., Merit, L.L. and Dean, J.A., 1965, Instrumental Methods of Analysis, 4 th ed. Affiliated East-West Press Pvt. Ltd., New Delhi.

158 Standardization and Phytochemical Screening of a Unani Drug Berg-e-Sem (Dolichos lablab L.) with Modern Analytical Techniques 1 *N.M.A. Rasheed, 1 Rakesh Kumar Negi, 1 Juveria Mahmood, 1 Aslam Siddiqui, 1 M.H. Kazmi, 2 S. Arfin, 2 Aminuddin and 1 Javed Inam Siddiqui 1 Central Research Institute of Unani Medicine, Opp. E.S.I. Hospital, A. G. Colony Road, Erragadda, Hyderabad Central Council for Research in Unani Medicine, 61-65, Institutional Area, Janakpuri, New Delhi Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract B 151 erg-e-sem (Dolichos lablab L.), a Unani drug of repute has been standardized by modern techniques. The pharmacopoeial parameters studied include : physico-chemical, phyto-chemical, HPTLC, microbial load and aflatoxins etc. which reveal that it is safe for medicinal use. The study will serve as standard reference for quality check of the drug investigated. Keywords: Barg-e-Sem, Standardization, Phyto-chemical screening, HPTLC. Introduction Standardization of herbal drugs direct towards the confirmation of its identity and determination of its quality and purity. At present due to advancement in the chemical knowledge of crude drugs, various methods like botanical, chemical, spectroscopic and biological are used for estimating active constituents present in the crude drugs. Every crude drug and herbal product is only as good as its quality. And highest quality standards are the key driver of success. Drugs must be marketed as safe and therapeutically active formulations whose performance is consistent and predictable. New and better medicinal agents are being produced at an accelerated rate. At the same time more exacting and sophisticated analytical methods are being developed for their evaluation. The medical fraternity at large is looking at alternatives from natural sources to combat diseases particularly those in which conventional modern system of medicine has little to offer. This realization on the one hand has increased demand for herbal drugs and on the other hand need for their quality standardization has gone up. Barg-e-Sem, Dolichos lablab L. belongs to family Leguminosae. Synonyms: Dolichos lablab L.; Dolichos purpureus L.; Lablab niger Medikus; Lablab lablab (L.) Lyons; Lablab vulgaris (L.) Savi. An herbaceous, climbing, warm seasonal, annual or short-lived perennial herb with a vigorous taproot. It has a thick, herbaceous stem that can grow up to 3 feet, and the climbing vines stretching up to 25 ft from the plant (Valenzuela and Smith, 2002). It has trifoliate, long-stemmed leaves. Each egg-shaped leaflet widens in the middle and 3 6 in. ( cm) long. The surface of the leaflet is smooth above and short haired below. Known as: Sanskrit-Nispava; Hindi-Sem, Bhatvas, Shimi; English-Lablab bean etc. Within India, Lablab, a field crop is mostly confined to the peninsular region and cultivated widely in Andhra Pradesh and adjoining districts of Tamil Nadu, Karnataka and Maharashtra. 1 *Author for correspondence

159 152 The global acceptance and interest in herbal medicines has led to an increase in their demand leading to a decline in their quality, primarily due to a lack of adequate regulations pertaining to these drugs (Rajini and Kanaki, 2008). WHO emphasized the necessity to ensure standards of medicinal plants drugs by using modern techniques (Imam et al., 2009; Rasheed et al., 2010a; 2010b; 2010c;2010d; 2011; 2012a; 2012b; 2012c; 2014a; 2014b; Naikodi et al., 2011). The hyacinth bean is an old domesticated pulse and multi-purpose crop (Smartt and John, 1985; Shivashankar and Kulkarni, 1992). In order to overcome certain shortcomings of the pharmacopoeial monograph, other quality control measures must be addressed (Pifferi et al., 1999; Shinde, 2009; Singh and Soni, 2004; Street et al., 2008). The organoleptic parameters like taste, odour, colour etc. are not adequate to establish the standard quality of the medicine. Therefore, with a view to standardize and lay down the pharmacopoeial standards as per the standard operating procedures (SOP s), the leaf of the Unani drug Dolichos lablab was subjected to analysis for phytochemical, physico-chemical parameters, microbial load, aflatoxins, and high performance thin layer chromatographic studies. The paper presents the results of the studies carried out on this drug to ensure its quality for commercial use. Materials and Methods Collection of material The leaf material of the Dolichos lablab L. (Barg-e-sem) was obtained from the locality or surrounding region of Hyderabad and was authenticated and identified by the botanist Dr. V.C. Gupta, at Central Research Institute of Unani Medicine, Hyderabad. Chemical analysis Physico-Chemical parameters of the Dolichos lablab was studied such as total ash, acid insoluble ash, solubility matter in alcohol and water, loss on drying at C, microbial load and aflatoxins contamination as per the methods described in WHO guidelines (Anonymous, 1998). Phytochemical screening was carried out in different solvents extracts such as Ethanol, methanol, Ethyl acetate, Chloroform, Petroleum ether, acetone and aqueous extracts as per the methods described by Trease and Evans (1972). HPTLC analysis: DESAGA Sarstedt Gruppe system was used for analysis along with Automatic TLC applicator and UV visible cabinet as imaging system, the instrument had Proquant 1.6 version as software system for documentation.

160 153 Preparation of extract for HPTLC analysis Five grams fine powder of dried Barg-e-sem was reflux on water bath for 30 min in Ethanol, Ethyl acetate, Chloroform and Petroleum ether ( C) separately through soxhlet apparatus. Later the contents were removed and filtered through Whattmann No. 41 filter paper and evaporated the solution to 20 ml. Thus the solution so obtained was used as sample for the determination of components. Chromatography HPTLC was carried out on the Precoated Aluminium Sheets of Silica Gel 60 F 254 (Merck) dimensions of 20 cm 10 cm. Different solvent extracts of about 10ìl were applied as 4 mm width bands using Automatic TLC applicator system of the DESAGA Sarstedt Gruppe (Germany). A Linear ascending development with Toluene: Ethyl acetate: methanol (6:3:1) (v/v) as mobile phase was carried out in a twin trough glass chamber previously saturated with mobile phase vapour for 20 min. at room temperature (25 ± 2 C). The development of solvent distance was 86 mm. After development plates were air- dried. Scanning was performed using densitometer of DESAGA Sarstedt Gruppe (Germany) under UV 254nm and 366nm and after derivatizing with anisaldehyde sulphuric acid and observed under visible range, at Rf values and operated by ProQuant 1.06 version software. The source of radiation was a deuterium lamp emitting a continuous UV spectrum in the range nm. The slit dimensions were 4 mm 6mm. Development of HPTLC technique After the development, TLC plate was then removed dried completely and detected with under UV Cabinet system for detection of spots. Further it was scanned with the Densitometer CD60 of DESAGA Sarstedt Gruppe system under UV 254nm and 366nm and after derivatizing with anisaldehyde sulphuric acid and observed under visible range, at Rf values mentioned in the table as shown in the figure 2. A corresponding densitogram was then obtained in which peaks are appeared for the corresponding spots being detected in the densitometer while scanning and the peaks area under the curve corresponds to the concentration of the component in the sample for the concentration that applied on the TLC plate. Results and Discussion Organoleptic characters: The drug consists of leaves which are dark green colour, on drying changes to light green having characteristic smell (figure 1). The physico-chemical parameters data are given in table 1; total ash gm%;

161 154 Table 1: Physico-chemical parameters of the compound formulation Barg-e-Sem. S.No. Parameters %w/w 1 Total Ash (%) gm% 2 Acid insoluble ash (%) gm% 3 Alcohol soluble matter (%w/w) gm% 4 Water soluble matter(%w/w) gm% 5 Loss of wt. on drying at 1050C gm% Fig. 1: Barg-e-Sem; Dolichos lablab L. acid insoluble gm%, alcohol soluble matter gm%, and water soluble matter gm%; The moisture content i.e., Loss of weight on drying at C was also determined and found to be gm%. Phytochemical screening for phytoconstituents was carried out in the aqueous extract. This revealed the presence of carbohydrates, glycosides, tannins, flavonoids. Whereas in ethyl acetate and chloroform extract revealed the presence of proteins and tannins; in petroleum ether it reveals the presence of glycosides, proteins, tannins; in ethanol and acetone extracts revealed the presence of tannins only as shown in the table 3. Powdered drug was screened for fluorescence characteristic with or without chemical treatment such as powder as such, powder treated with 1N NaOH in Methanol, powder treated with 1N NaOH in water, powder treated with 1N HCl, powder treated with 50% HNO3 aqueous, powder treated with 50% H 2 SO 4 aqueous, powder treated with glacial acetic acid and the observations pertaining to their colour in daylight i.e, visible region and under ultra-violet light were noticed and are presented in the table 4. Fluorescence analysis of powdered drug

162 155 Table 2: TLC profile of alcoholic, ethyl acetate, chloroform and pet. ether extract of Barge-Sem along with R f values. Mobile phase: Toluene: ethyl acetate: methanol (6:3:1) Peak list & densitogram of Barg-e-Sem ethanolic extract at UV 366nm with Rf values Peak no Y-Pos Area Area (%) Height Rf values Peak list & densitogram of Barg-e-Sem ethyl acetate extract at UV 366nm with Rf values Peak no Y-Pos Area Area (%) Height Rf values Peak list & densitogram of Barg-e-Sem chloroform extract at UV 366nm with Rf values Peak no Y-Pos Area Area (%) Height Rf values Peak list & densitogram of Barg-e-Sem pet ether extract at UV 366nm with Rf values Peak no Y-Pos Area Area (%) Height Rf values

163 156 Table 3: Phytochemical screening of the nature of compounds present in different solvent extracts of Barg-e-Sem. S.No. Phytoconstituent Acetone ethanol Meth E.A. Pet. Aqueous CHCl 3 ext. ext. ext. ext. ether ext. ext. ext. 1. Alkaloid Carbohydrates Fixed oil Glycosides Phenols Proteins Steroids Tannins Flavonoids Saponins Table 4: Fluorescence analysis of powdered drug: S.No. Reagents UV light Visible light Short 254nm Long 366nm 1. Powder as such Black Green Green 2. Powder treated with 1N NaOH Black Pink Green in Methanol 3. Powder treated with 1N NaOH Black Reddish pink Dark green in Water 4. Powder treated with 1N HCl Black Black Dark brown 5. Powder treated with 50% HNO3 Black Dark black Brown aqueous 6. Powder treated with 50% H 2 SO 4 Black Black Dark brown aqueous 7. Powder treated with Glacial Black Reddish pink Dirty green Acetic acid extracts in seven different solvents was observed and has been reported in the table 5. Safety evaluation of drug reveals that the total bacterial load and total fungal count of the microbial studies were found to be as 36 x 10 3 /g and7 x 10

164 157 Table 5: Fluorescence analysis of powdered drug extracts in different solvents: S.No. Extraction Solvent UV light Visible light Short 254nm Long 366nm 1. Acetone Extract Black Black Dark green 2. Alcoholic Extract Black Reddish pink Dark green 3. Chloroform Extract Dark green Pink Dark green 4. Petroleum ether extract Black Reddish pink Light green 5. Methanol Dark green Reddish brown Dark green 6. Ethyl Acetate Black Red Dark green 7. Distilled water Dark brown Dark green Dark brown a) At UV 366nm b) At UV 254nm c) At Visible range d) Derivatized with Anisaldehyde sulphuric acid Fig. 2: TLC chromatogram of Barg-e-Sem Ethanolic (Track 1,2); Ethyl acetate (Track 3,4); Chloroform (Track 5,6), Pet ether extract (Track 7,8) at UV 254nm and 366nm respectively. a) At UV 366nm, b) At UV 254nm, c) At Visible range d) At visible region after derivatizing with Anisaldehyde sulphuric acid reagent.

165 158 Fig. 3: HPTLC Densitogram of Barg-e-Sem at A) ethanolic extract B) ethyl acetate extract C) chloroform extract D) pet ether extract at UV 366nm respectively which is within the permissible limits and the other parameters studied were found to be absent in the drug. The analysis of aflatoxins showed that the drug was free from any contaminations. These findings as observed for microbial load and aflatoxin contamination analysis are given in table 6.

166 159 Table 6: Aflatoxins, Microbial and fungal contamination in the drug. Aflatoxin Contamination Parameter analyzed Results Permissible limits as per WHO 1 B1 Nil Not more than 0.50 ppm 2 B2 Nil Not more than 0.10 ppm 3 G1 Nil Not more than 0.50 ppm 4 G2 Nil Not more than 0.10 ppm Microbial and fungal Contamination Parameter analyzed Results Permissible limits as per WHO 1 Total Bacterial Load 36 x 10 3 Not more than 10 5 / g 2 Salmonella Spp Nil Nil 3 Escherichia Coli Nil Nil 4 Total Fungal count 7 x 10 Not more than 10 3 / g HPTLC analysis HPTLC fingerprint studies of different solvent extract of Barg-e-sem was carried out and TLC plate developed and detected using the UV visible chamber which clearly showed various spots at UV 254nm and 366nm in the densitogram and also under visible region and after derivatizing with anisaldehyde sulphuric acid reagent. TLC profile of alcoholic, ethyl acetate, chloroform and petroleum ether extract of Barg-e-Sem along with R f values under UV 366nm detection is illustrated in the table 2 and the corresponding HPTLC densitogram of Barg-e- Sem at A) ethanolic extract B) ethyl acetate extract C) chloroform extract D) pet ether extract was shown in figure 3. It was observed that there are eight spots in the Tlc plate of Barg-e-Sem Ethanolic extract at UV 366nm with Rf values 0.04, 0.27, 0.37, 0.44, 0.49, 0.62, 0.70, 0.93; it was observed that there are seven spots in the Tlc plate of Barg-e-Sem Ethyl acetate extract at UV 366nm with Rf values 0.04, 0.35, 0.42, 0.48, 0.62, 0.71, 0.93; it was observed that there are eight spots in the Tlc plate of Barg-e-Sem chloroform extract at UV 366nm with Rf values 0.04, 0.35, 0.42, 0.47, 0.61, 0.80, 0.87, 0.93; it was observed that there are four spots in the Tlc plate of Barg-e-Sem petroleum ether extract at UV 366nm with Rf values 0.04, 0.61, 0.69, Thus established HPTLC fingerprinting profile will help to authenticate the leaves of the studied drug lablab and serve as a tool for the quality control analysis. Conclusion With a view to develop standard reference, the Unani drug Berg-e-Sem (Dolichos lablab L. - leaf) has been studied for physico-chemical, phytochemical screening,

167 160 HPTLC analysis and safety evaluation such as aflatoxins, contamination analysis, microbial load etc. which was found within permissible limits as per WHO guidelines. The data provided will serve as quality control standards for the drug investigated and help in the manufacture of genuine medicines and detect adulterations. Acknowledgement The authors would like to record their gratitude to Director General, CCRUM, New Delhi for providing necessary facilities; to the laboratory staff of CCRIUM, Hydeabad, for help in this study. References Anonymous, Quality Control Methods for Medicinal Plant Materials. World Health Organization, Geneva, pp Anonymous, The Unani Pharmacopoeia of India, Part-I, Vol.VI. Ministry of Health & Family welfare, Govt. of India, New Delhi, pp Imam, S., Rasheed, N., Ayesha, M., Shareef, M.A., Khan, S.A. and Arfin. S., Role of Chromatography in the Identification and Quality Control of Herbal Drugs 1. HPTLC Finger Prints of Qurs-e-Istisqa. Hippocratic Journal of Unani Medicine 4(3):41-58 Naikodi, M.A.R., Waheed, M.A., Shareef, M.A., Ahmad, M., Nagaiah, K., Standardization of the Unani drug Myristica fragrans Houtt. (Javetri) with modern analytical techniques. Pharmaceutical Methods 2(2): Pifferi, G., Paola Santoro, Massimo Pedrani, Quality and functionality of excipients. II. Farmaco 54 (1-2): Rajini, M. and Kanaki, N.S., Phytochemical Standardization of Herbal Drugs and Polyherbal Formulations: Bioactive Molecules and Medicinal Plants. Ramawat, K.G., Merillon, J.M. (eds.). Springer pp Rasheed, N.M.A., Ayesha, M., Shareef, M.A., Alam, M.D., Gupta, V.C. Khan, S.A., Arfin, S. and Aminuddin, 2010a. Role of Chromatography In The Identification And Quality Control Of Herbal Drugs 1.HPTLC Finger Prints Of Qurs-e- Kundur a Unani Compound Formulation. Hippocratic Journal of Unani Medicine 5(3): Rasheed, N.M.A., Ayesha, M., Waheed, M.A., Alam, M.D., Khan, S.A. and Arfin, S., 2010b. A Chemical Standardization of a Unani single drug -1.Ood-e- Saleeb (Paeonia emodi Wall.) and Evaluation of its Antimicrobial Activity Against Bacterial Strains. Hippocratic Journal of Unani Medicine 5(3):

168 161 Rasheed, N.M.A., Rehana, A., Gupta, V.C., Ahmad, M. and Aminuddin, Chemical Standardization of a Unani Herbal drug - Cordia dichotoma Forst.f. (Sapistan). Hippocratic Journal of Unani Medicine 6(1): Rasheed, N.M.A., Rehana, A., Maqbool Ahmed, Husain, K., Waheed, M.A., Arfin, S. and Aminuddin, 2014b. Standardization and HPTLC fingerprinting of a Unani Compound Formulation Habb-e-Paan with Modern Techniques. Hippocratic Journal of Unani Medicine 9(2): Rasheed, N.M.A., Shareef, M.A., Ahmad, M., Gupta, V.C., Arfin, S. and Shamshad, A.K., 2010c. HPTLC Finger Print Profile of Dried Fruit of Physalis alkekengi Linn. Pharmacognosy Journal 12(3): Rasheed, N.M.A., Shareef, M.A., Mateen, A., Rehana, A., Waheed, M.A., Arfin, S., Shamshad, A.K., and Aminuddin. 2012a. Physicochemical and Phytochemical evaluation of Cocoon of Bombyx mori Linn. (Abresham). Hippocratic Journal of Unani Medicine 7(4): Rasheed, N.M.A., Shareef, M.A., Rehana, A., Waheed, M.A., Arfin, S. and Aminuddin, 2014a. Phytochemical and HPTLC fingerprint analysis of Terminalia arjuna (Roxb) Wight & Arn. in different solvent extracts Indo American Journal of Pharmaceutical Research 4(1): Rasheed, N.M.A., Shareef, M.A., Waheed, M.A., Ahmad, M., Shamshad, A.K., Arfin. S and Nageshwar Rao, R., 2010d. Standardization of Herbal Unani Drug-Aegle marmelos Corr. with modern analytical equipment Herbal Tech Industry. Journal of Herbal Science and Technology 6(9): Rasheed, N.M.A.; Nagaiah, K.; Goud, P.R. and Sharma, U.V.M., 2012b. Chemical marker compounds and their essential role in quality control of herbal medicines. Annals of phytomedicine 1(1):1-8. Rasheed, N.M.A.; Nagaiah, K.; Mehveen, A.; Rehana, A.; Waheed, M.A. and Shareef, M.A., 2012c. Phytochemical evaluation and quantification of Betasitosterol in geographical variation of Withania coagulans Dunal by HPTLC analysis. Annals of phytomedicine 1(2): Shinde, V.M., Dhalwal, K., Potdar, M. and Mahadik, K.R., Application of Quality Control Principles to Herbal Drugs. International Journal of Phytomedicine 1: 4-8. Shivashankar, G. and Kulkarni, R. S van der Maesen, ed. Plant Resources of South-East Asia, No. 1, Pulses. Wageningen, The Netherlands, Pudoc., pp Singh, S. and Soni, G.R., WHO Expert Committee on Biological Standardization. Indian J Med Res 120:

169 162 Smartt and John, Evolution of grain legumes. II. Old and new world pulses of lesser economic importance. Experimental Agriculture 21 (3): Trease, G.E. and Evans, W.C., Pharmacognosy, 10th edn. Edn. Bailliere Tindel, London. Valenzuela, H., and J. Smith Sustainable agriculture green manure crops. SA-GM-7. Cooperative Extension Service, College of Tropical Agric. and Human Resources, Univ. of Hawaii at Manoa.

170 Pharmacognostical Standardization of Mushkdana Seed (Abelmoschus moschatus Medic) *Kiran Negi, Akhlaq Mustafa, Mahesh Chandra and S.M. Asim Drug Standardization Research Unit (CCRUM), PLIM Building, Kamla Nehru Nagar, Ghaziabad Hippocratic Journal of Unani Medicine January - March 2016, Vol. 11 No. 1, Pages Abstract M 163 ushkdana which is botanically identified as Abelmoschus moschatus Medic. (syn. Hibiscus abelmoschus Linn.) is an important medicinal plant of Indian traditional systems of medicine. The plant is cultivated for its muskscented seeds, useful in perfumery and medicine. The seeds also known as Ambrette seeds are valued for the volatile oil present in the seed coat. They have been used as a tonic, stimulant, carminative, as flavouring agent for food and as a substitute for musk. Present paper deals with the pharmacognostical standardization of Muskhkdana seed, unstudied so far, in order to lay down the standards for its quality assurance. The study includes macro and microscopic features of the drug, besides maceration, powder analysis, measurement of cells/ tissues, reaction with different chemical reagents and fluorescence analysis of the crude drug powder. Other parameters worked out are physiochemical constants such as ash value, alcohol and water soluble matter and thin layer chromatography. Keywords: Abelmoschus moschatus Medic., Seeds, Pharmacognostical standardization Introduction In view of the fact that there still exists controversial status of many of the Unani and Ayurvedic drugs, there is a need to fix-up their pharmacopoeial standards in order to ensure authenticity of pharmaceutical materials used in therapeutic formulations. In this direction some 250 such Unani drugs have been earlier investigated for their pharmacognostical standardization (Anonymous, 1987; 1992; 1997; 2006a & 2006b). However, there appears no work done on Mushkdana seeds thus far, whereas, leaves & roots of this drug have been investigated (Anonymous, 2006b). Hence, the present studies. Abelmoschus moschatus Medic. (Family : Malvaceae) is an aromatic and medicinal plant native to India. It is commonly known as Mushkdana in Hindi; Ambrette plant and Musk mellow in English; Habbulmishk or Habbulmushk in Arabic; Mushkdana in Persian; Latakasturika in Sanskrit and Mushkadanah in Urdu. The plant is an erect, herbaceous annual, grown in the hotter parts of India. Leaves polymorphous, lower ovate, acute, upper palmately 3-7 lobed, flowers bright yellow, large, axillary, capsules cm. long, ovate, acute, hispid (Anonymous, 1985; Chopra & Chopra, 1969; Purohit &Vyas, 2004; Verma et al., 1993; Agharkar, 1991; Lindley, 1985). The plant is generally cultivated for their musk-scented seeds useful in perfumery and medicine(singh et al., 1996).Seed analysis report 11.1% moisture, 31.5% *Author for correspondence

171 164 crude fiber; 14.5% lipids, 13.4% starch, 2.3% protein, volatile oil ( %) and 5% resin (Srivastava, 1995). The crushed seeds on steam-distillation yield a volatile oil of commerce noted for their rich, sweet, floral-mushkyodour. Nee and Carts Pollard (1986) showed that the essential oil of these seeds is localized in the outer layer of the seed coat, whereas fatty oil is concentrated in the embryo and the endosperm. The oil from the seeds is rich in linoleic acid and contains a-cephalin, phosphatidylserine, its plasmalogen and phosphatidylcholine plasmalogen (Ghani, 2003). The characteristic musk-like odor of the seed oil is mainly due to the presence of a ketone, ambrettelide, a lactone of ambrettolic acid. The chemical constituents of the essential oil of the seeds are: trans-2- trans-6-farnesyl acetate and ambrettolide, cis-2-cis-6- farnesyl acetate, cis-2- trans-6-farnesyl acetate, ethyl hexadecanate, ethyl laurate and trans-2-trans-6- farnesol (Du et al., 2008). The seeds are said to be stimulant, antiseptic, cooling, tonic, carminative and aphrodisiac. They are effective to treat intestinal complaints, skin diseases, stomatitis, dyspepsia, alley thirst and checking vomiting (James et al., 1993; Peter, 2007; Rival et al., 2009). Powdered seeds are inhaled for dryness and hoarness of throat. The paste of pulverized seed used to treat leucoderma, heat and itch (Panda, 2004). A decoction or infusion or tincture of the seeds is useful in nervous disability, hysteria and other nervous disorders. In Unani system of medicine the seeds are valued for their diuretic, demulcent and stomachic properties. (Kirtikar & Basu, 1988; Nadkarni, 1986) Considering the medicinal importance, it was felt desirable to undertake pharmacognostic standardization of Mushkdana seed in detail so as to find out the reliable data for distinguishing the drug from its possible adulterants. Material and Method Seeds were collected from local market, New Delhi; identified and authenticated with the help of standard floras (Hooker, 1875; Cooke, 1903). Its macroscopical characters were studied. For microscopical studies, slides were prepared and stained in different reagents like saffranine, iodine solution, ferric chloride solution etc. and mounted in glycerine to study various cells/tissues/cell contents (Johansen, 1940; Trease and Evans, 1983). The representative diagrams were drawn using camera lucida. Measurement of the individual cell / tissues of the various parts of the seed were recorded. The powder and its behaviour on treatment with different chemical reagents were studied and the physico-chemical constants were determined. Fluorescence characters of the powdered drug were observed under U.V. according to the method described by Kokoski et al. (1958). Standard analytical methods were followed for chemical analysis (Anonymous, 1998).

172 165 Observations Macroscopic characters Seeds greyish brown, reniform, compressed, 0.4 mm. long with striations concentric about the hilum. Odour - delicate musk like; Taste bitter and sharp. Average wt. of 100 seeds is 1.45gm. Microscopic characters (Fig. 1 3) Ovule campylotropus; bitegmic; testaregulose. In T.S. seed shows following characters:- Seed coat Outermost single layered epidermis, thick walled cells, lignified on the ridges followed by a palisade of malpighian cells which are radially elongated and lignified in inner part. Then a few layers of somewhat longitudinally elongated cells with thickened, brownish often pitted radial walls, somewhat lignified. Perisperm with thin parenchyma cells. Endosperm Thin walled, several layered, hexagonal to polygonal parenchyma cells. Cotyledon Outermost single layered epidermis followed by single layer palisade cells and few layers of spongy parenchyma cells and then inner epidermis. All the cells of endosperm and cotyledon are filled with very minute, spherical starch grains; aleurone grains and oil globules. Powder analysis Greyish brown powder with delicate musk like odour and sharp, bitter taste. Microscopic examination of the powder in 40 mesh shows epidermal cells of testa, malpighian cells 112.5μ in length and 13.5μ in width, sclerenchymatous cells, fragment of cotyledon,oil globules, very minute, spherical starch grains 11.25μ μ in diameter. Maceration Maceration of the seed with conc. Nitric acid shows epidermal cells, elongated cells lignified in inner part, thin walled parenchyma and palisade cells of cotyledon.

173 166 Fig. 1 3 : Abelmoschus moschatus Medic. (seed) Fig. 1: L.S. of seed (Diagrammatic) Fig. 2: T.S. of seed Fig. 3: Surface view in powder and macerate (i) Parenchyma cells of endosperm with oil globules and starch grains (ii) Malpighian cells of seed coat (iii) Lignified cells of seed coat Abbreviations: AG : Aleurone grains; COT : Cotyledon; END : Endosperm; IEP : Inner Epidermis; L : Lignified cells; MC : Malpighian cells; OEP : Outer Epidermis; OG : Oil Globules; P : Parenchyma; PAL : Palisade cells; PER : Perisperm; SC : Seed coat; SG : Starch grains