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1 ISSN International, Peer reviewed, Open access, Monthly Online Journal Research article PHARMACOGNOSTICAL AND PRELIMINARY PHYSICOCHEMICAL PROFILE OF ALBIZZIA LEBBECK BENTH. STEM BARK Harmeet Kaur 1*, CR Harisha 2, Galib R 3, PK Prajapati 4 1,3,4 Department of Rasashastra and Bhasihajya Kalpana, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India 2 Pharmacognosy Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India *Corresponding Author: E mail- harmeetkaur134@yahoo.com; Mobile: ABSTRACT Received: 09/10/2015; Revised: 25/10/2015; Accepted: 10/11/2015 Albizzia lebbeck Benth., is one of the important herbal source, useful in many diseases. Different fractions of this plant are reported to have anti inflammatory, anti histaminic, anti microbial etc. activities. Pharmacognostical profiles of flower, leaf and heartwood of this plant are reported but the same for stem bark is not available. Considering this, an attempt has been made to evaluate pharmacognostical, phytochemical profile and to validate preliminary physicochemical profile of stem bark of the drug. Stem bark was collected in the month of February, 2013; shade dried; pulverized; sieved through 80# and preserved in an airtight glass container. Transverse section of stem bark and its powder, histochemical tests and preliminary physicochemical investigations were done to validate methods for quality control of drug and its botanical evaluation. Results showed stone cells with tannin, sclereids, rhomboidal prismatic crystals, cork cells, crystal fibre and oil globules. Preliminary physicochemical analysis revealed loss on drying (7.05%w/w), ash value (7.59%w/w), acid insoluble ash (0.68%w/w), alcohol soluble extractive (14.40%w/v) and water soluble extractive (16.30%w/v). Preliminary analysis for the presence of various functional groups revealed the presence of alkaloids, saponins, phenols and steroids. High Performance Thin Layer chromatography of alcoholic extract showed the presence of five and four spots in short UV and long UV respectively. The information generated in this study will provide relevant pharmacognostical and physicochemical data needed for proper identification, authentication and validation of pharmacopoeial characters of stem bark of Albizzia lebbeck Benth. KEYWORDS: Albizzia lebbeck, Ayurveda, Shirisha, Stem bark. Cite this article: Harmeet Kaur, CR Harisha, Galib R, PK Prajapati (2015), PHARMACOGNOSTICAL AND PRELIMINARY PHYSICOCHEMICAL PROFILE OF ALBIZZIA LEBBECK BENTH. STEM BARK, Global J Res. Med. Plants & Indigen. Med., Volume 4(11):

2 INTRODUCTION: Herbal medicine is a part of the knowledge of indigenous cultures and marginal societies across the globe which has stood the test of time (Prakash et al., 2014). During the last decade, use of traditional medicine has expanded globally and has gained popularity. Studies reveal that there are more traditional medicine providers than the allopathic providers especially in the rural areas (WHO, 2002). Development of these traditional systems of medicines with perspectives of safety, efficacy and quality control will help not only to preserve the traditional heritage but also to rationalize the use of natural products in health care (Mukherjhee et al., 2006). Majority of the world population still relies on herbal medicines to meet its health needs. It has not only continued to be used for primary health care of the poor in developing countries, but has also been used in countries where conventional medicine is predominant in the national health care systems. WHO is engaged to establish definitive guidelines for methodology of research and the appraisal of effectiveness of traditional medicine (Fabio Firenzuoli and Luigi Gori, 2007). Accurate identification and guarantee of purity through pharmacognosy and pharmaceutical chemistry measures is first step for the quality assurance and standardization of any of the herbal medicine. Albizzia lebbeck Benth., is one of such beneficial herbs. It is large erect unarmed deciduous, spreading tree common all over India, from the plains up to 900m in the Himalayas, and also in the Andamans (Wealth of India., 2006). Bark is expectorant (Tripathi et al., 1974), aphrodisiac, anti inflammatory, anti allergic (Tripathi et al., 1979), antianaphylactic (Johri et al., 1985), anti-asthmatic (Bhattathri et al., 1997), anti histaminic (Kumar et al., 2010), analgesic (Achinto et al., 2009), antioxidant (Resmi et al., 2006), immunomodulatory (Shyamlal et al., 2011.a), anticonvulsant (Kasture et al., 1996) and anti spermatogenic (Gupta et al., 2004). Pharmacognosy of heartwood (Shyamlal et al., 2011.b), flower (Shyamlal et al., 2011.c) and leaf (Shyamlal et al., 2011.d) were reported, but was not available for stem bark. Considering these, detailed investigation of powdered stem bark of Albizzia lebbeck Benth. had been carried out using various pharmacognostical and phytochemical parameters. MATERIALS AND METHODS: Plant material: Stem bark of Albizzia lebbeck Benth. was collected from the Botanical garden of the Institute for Post Graduate Teaching & Research in Ayurveda (IPGT & RA), Gujarat Ayurved University, Jamnagar in the month of Feburary, 2013 [Figure 1]. Voucher specimen along with crude drug sample is preserved in the Pharmacognosy Lab, IPGT & RA, Gujarat Ayurved University, Jamnagar. Botanical identification was carried out by using various floras (Gamble., 1967). Macroscopic Characterization of stem bark: Macroscopic characters of stem bark were done by naked eye observations like shape, colour, taste and odour (Khandelwal, 2008). Microscopic Characterization of stem bark: For microscopical studies, free hand transverse sections of stem bark were taken and examined. Surface preparation was done and both the surfaces were observed. Powder microscopy of shade-dried powder (80#) was carried out using suitable method (Kokate, 2005.a). The powder was uniformly spread on glass slides and observed under microscope at different magnifications. For the detection of lignified tissues (stone cells, sclereids, xylem vessel, etc.), the powder was stained with phloroglucinol and hydrochloric acid and to observe the starch grains, the powder was stained with iodine solution (Trease, 1983). Photomicrographs were taken by using Carl zeiss binocular microscope attached with camera (Kokate, 2005.b). Physico-chemical Parameters of stem bark: Physico-chemical parameters were determined as per Ayurvedic Pharmacopoeia of

3 India. Moisture content (The Ayurvedic Pharmacopoeia of India, 2001.a), total ash value (The Ayurvedic Pharmacopoeia of India, 2001.b), acid insoluble ash (The Ayurvedic Pharmacopoeia of India, 2001.c), alcohol soluble extractive value (The Ayurvedic Pharmacopoeia of India, 2001.d) and water soluble extractive value (The Ayurvedic Pharmacopoeia of India, 2001.e) were determined. Preliminary Phyto-chemical screening of stem bark: The methanolic extract of stem bark was prepared and subjected to detect the presence of various functional groups like alkaloids, tannins, phenols, carbohydrates, glycosides, flavonoids, steroids, saponins by using relevant reagents. (Kokate, 2005.c) [Table-3]. HPTLC : For High Performance Thin Layer Chromatography (HPTLC), methanol extract was prepared by taking 5 g of drug in 100 ml of methanol, it was shaken for some time; mild heat was provided to it for half an hour and then filtered on cooling. The filtrate is evaporated on water bath to approximately 20 ml and used. A CAMAG (Switzerland) HPTLC system equipped with a sample applicator Linomat V was used for application of samples. CAMAG TLC Scanner 3, Reprostar and Wincats 4.02 were used for scanning the plates. CAMAG twin through glass chamber was used for developing the plates. Pre-coated silica gel GF 254 plate was used as stationary phase. Toluene : Ethyl acetate : formic acid ( 7 : 2 : 1) v/v was used as mobile phase. After 30 minutes of chamber saturation, plate was developed, and then scanned under short UV (254 nm) and long UV (366 nm). RESULTS AND DISCUSSION: Macroscopic Characters of stem bark: Macroscopically, bark pieces are slightly curved. External surface is very rough due to longitudinal and transverse fissures, exfoliation of rhytidoma and characteristic peeling of cork near the lenticels, colour of bark is buff with grayish white and blackish brown patches. Internal surface of bark is yellowish to grayish brown. Fracture in outer part of bark is laminated and inner part is fibrous and taste is astringent [Table-1, Figure 1:1 3]. Microscopic characteristics: Transverse section of stem bark of Albizzia lebbeck Benth. The transverse sections of the mature bark are made up of alternate layers of hard and woody suberous tissues. The suberous tissues are broad and consist of several easily separable rows of thin walled rectangular cells some of which are empty, while others are filled with yellowish to brown contents. The secondary cortex or mid bark which is situated immediately beneath the rind is composed of large thin walled oblong cells. Most of them are loaded with starch grains while a number of others contain solitary prismatic crystals or yellowish brown contents which give the tissue its characteristic reddish or reddish purple colour. In this region there are numerous scattered groups of sclereids and stone cells of varies sizes and shapes. The inner bark is a fairly broad zone composed of thin walled phloem alternating with tangential strips of sclerenchyma and medullary rays. The medullary rays are mostly biseriate while uni and triseriate rays are also present. The living tissue of the bark is mainly composed of wide secondary phloem consists of groups of tangentially running sieve tissues frequently getting obliterated companion cells and phloem parenchyma alternating with phloem fibre are associated with idioblast containing prisms of calcium oxalate crystals, groups of stone cells occasionally associated with fibres. The elements of wood are arranged in series. Extended xylem fibres form the mass of the wood [Figure 1:4 8 and Figure 2:1 4].

4 FIGURE 1- Morphology and transverse section of stem bark and its powder 1.Whole plant; 2. Stem bark of shirisha; 3. Powder of shirisha; 4. TS of bark with cork; 5.TS showing cortical region; 6. TS with pericyclic fibres and stone cells; 7. Parenchyma cells with tannin content; 8. Cork zone with tannin content

5 FIGURE 2- Transverse section of stem bark and its powder 1.Parenchyma cells with simple and compound starch grains; 2. Solid cork with stone cells layers; 3. Group of stone cells; 4. Sclereids

6 Powder microscopy of stem bark: Organoleptic characters are shown in [Table-1]. Plenty of stone cells with tannin, parenchymatous cells, sclereids, fragment of fibres, rhomboidal prismatic crystals, brown content, oil globule, cork with tannin content, cork cells, fibres passing through medullary rays, lignified stone cells, lignified parenchymatous cells, corky surface and crystal fibre were observed under the microscope [Figure 3:1 8]. Table-1: Organoleptic characters of stem bark powder of Albizia lebbeck Benth. Sr. no. Character Observation 1 Colour Brownish 2 Odour Sweetish to aromatic 3 Texture Rough 4 Taste Sweetish astringent FIGURE 3- Powder microscopy of stem bark of shirisha 1.Cork in surface view; 2. Oil globule; 3. Tannin content; 4. lignified stone cells; 5. stone cells with tannin content; 6. rhomboidal crystals; 7. lignified pitted parenchyma; 8. lignified fibres

7 Physicochemical Parameters of stem bark: Results of physicochemical parameters of stem bark are provided in [Table -2] Preliminary Phytochemical Screening of stem bark: Qualitative analysis for the presence of various functional groups was carried out in methanol soluble extractive [Table-3]. HPTLC: Alcoholic extract of stem bark showed the presence of five and four spots in short UV (254 nm) and long UV (366 nm) respectively [Table-4] [Figure 4:1 2]. Table-2: Physicochemical evaluations of stem bark of Albizia lebbeck Benth. Parameters Values obtained (%) Values in API (%)Part I, Vol.III Loss on drying 7.05 w/w Ash Value 7.59 w/w Not more than 8 percent Acid insoluble ash 0.68 w/w Not more than 1 percent Water soluble extractive w/v Not less than 6 percent Alcohol soluble extractive w/v Not less than 12 percent Table-3: Qualitative analysis for presence of functional groups in Albizia lebbeck Benth. stem bark Sr. no. Material Reagents Functional groups Observation Result 1 Dragendorff s reagent Alkaloids No Brown ppt. Present 2 Dil. FeCl 3 Tannins Blue brownish color Present 3 Neutral FeCl 3 Phenols Violet color Present 4 Benedict s reagent Carbohydrates Yellow ppt. Present 5 Conc.H 2 SO 4 Glycosides color change Present Methanolic extract of stem bark powder brown disappears colour 6 Lead acetate flavonoid Yellow color Present 7 Chloroform, Steroids Brown ring with Present Conc.H 2 SO 4, distilled water rinse 8 Shaking in test-tube Saponins Frothing with honeycomb appearance Present Table-4: HPTLC of Shirisha stem bark Chromatogram No. of spots Max. Rf value 254 nm , 0.15, 0.53, 0.78, nm , 0.23, 0.35,0.78

FIGURE 4- HPTLC of shirisha stem bark powder CONCLUSION: Pharmacognostical and physicochemical evaluation of stem bark of Albizzia lebbeck Benth.

8 FIGURE 4- HPTLC of shirisha stem bark powder CONCLUSION: Pharmacognostical and physicochemical evaluation of stem bark of Albizzia lebbeck Benth. provided specific parameters that will be helpful in proper identification, scientific evaluation and authentication of the drug. Externally bark is whitish, crackled with rhomboidal crystals of calcium oxalate, crystal fibres and tannin content that are the specific characteristic features to identify Shirisha bark and its powder. Stone cells with tannin, parenchymatous cells, sclereids, brown content, oil globule, cork cells, lignified 1. AT 254nm 2. AT 365nm parenchymatous cells are other identification characters of stem bark. The physico-chemical profile observed in the current study was complying with the standards laid down in the Ayurvedic Pharmacopoeia of India, revealing its genuinity. As no published reports are available on Pharmacognostical aspects of the stem bark; the results obtained may be referred as standard in future studies. ACKNOWLEDGEMENTS: Authors are thankful to Dr. VJ Shukla, Head, Pharmaceutical Chemistry Lab for providing basic facilities during Chemical analysis.

9 REFERENCES: Achinto Saha, Ahmad Muniruddin (2009). The analgesic and anti-inflammatory activities of the extract of Albizia lebbeck in animal model. Pak. J. Pharm. Sci. 22(1): Bhattathri PPN, Rao PV, Acharya MV, Bhikshapathi T, Swami JK (1997). Clinical Evaluation of Shirisha Twak Kwatha in the management of Tamaka Shwasa. Journal of Research of Ayurveda and Siddha 18 (1 2): Fabio Firenzuoli and Luigi Gori (2007). Herbal Medicine Today: Clinical and research Issues. Evidence- based Complementary and Alternative Medicine 4(1): Gamble JS (1967). The Flora of Presidency of Madras. Vol. 1. London: Adlard and Sons. p Gupta RS, Kachhawa JB and Chaudhary R (2004). Antifertility effects of methanolic pod extract of Albizia lebbeck Benth. in male rats. Asian J. Androl 6(2): Johri RK, Zutshi U, Kameshwara L, Atal C K (1985). Effect of quercetin and Albizzia saponins on rat mast cell. Indian J. Physiol Pharmacol 29(1): Kasture VS, Pal SC (1996). Anticonvulsant activity of Albizzia lebbeck leaves. Indian journal of Experimental Biology 34(1): Khandelwal KR (2008). Practical and Pharmacognosy: Techniques and Experiments. 17th ed. Pune: Nirali Prakashan. p Kokate CK (2005.a). Practical Pharmacognosy. 4th edition. Delhi: Published by Vallabh prakashan. p. 28. Kokate CK (2005.b). Practical Pharmacognosy. 4th edition. Delhi: Published by Vallabh prakashan. p Kokate CK, Purohit AP, Gokhale SB (2005.c). Pharmacognosy: CBS Publishers and distributors. p Kumar S, Bansal P, Gupta V, Sannd R, Rao MM (2010). Clinical efficacy of Albizia lebbeck stem bark decoction on Bronchial asthma. International Journal of Pharmaceutical Science and Drug Research 2(1): Mukherhee PK, Wahile A (2006). Integrated approach towards drug development from Ayurveda and other system of medicines. J Ethanopharmacology 103: Prakash CP, Rakesh KM, Krishna GS (2014). The efficacy of herbal system of medicine in the context of allopathic system in Indian Central Himalaya. Journal of Herbal Medicine 4(3): Resmi CR. Antioxidant activity of albizia lebbeck in alloxan diabetic rats (2006). Indian J Physiol Pharmacol 50(3): Shyamlal SY, Galib, Ravishankar, Prajapati PK, Ashok BK (2011.a). Evaluation of Immunomodulatory activity of Shirishavaleha - An Ayurvedic Compound formulation in albino rats. Journal of Ayurveda and Integrative Medicine 2(4): Shyamlal SY (2011.b). Pharmacognostical Screening and Phytochemical Evaluation of Albizia lebbeck Benth. Heartwood. Asian Journal of Biomedical and Pharmaceutical Sciences 1(5),

10 Shyamlal SY (2011.c). Pharmacognostical and Physico-Chemical Investigations of Albizia lebbeck Benth. Flower. International Journal of Pharmaceutical & Biological Archives 2(5): Shyamlal SY (2011.d). Pharmacognostical and physicochemical investigations of Albizia Lebbeck Benth. Leaf. International Journal of Science Innovations and Discoveries 1 (2), The Ayurvedic Pharmacopoeia of India (2001.a). Reprinted 1st ed. Part 1, Vol. I. Appendix-2(2.2.9) Govt. of India: Ministry of Health and Family Welfare. New Delhi. p The Ayurvedic Pharmacopoeia of India (2001.b). Part 1, Vol. 5. Appendix- 2(2.2.3.) Govt. of India: Ministry of Health and Family Welfare. New Delhi. p The Ayurvedic Pharmacopoeia of India (2001.c). Part 1, Vol. 5. Appendix- 2(2.2.4.) Govt. of India: Ministry of Health and Family Welfare. New Delhi. p.213. The Ayurvedic Pharmacopoeia of India (2001.d). Reprinted 1st ed. Part 1, Vol. I. Appendix-2(2.2.6) Govt. of India: Ministry of Health and Family Welfare. New Delhi. p The Ayurvedic Pharmacopoeia of India (2001.e). Reprinted 1st ed. Part 1, Vol. I. Appendix-2(2.2.7) Govt. of India: Ministry of Health and Family Welfare. New Delhi. p Trease GE, Evans WC (1983). Pharmacognosy. 12th ed. U.K: Baillier Tindall Can Macmillan Publishers. p. 95 9, Tripathi RM, Sen PC, Das PK (1979). Studies on the mechanism of action of Albizzia lebbeck, an Indian indigenous drug used in the treatment of atopic allergy. J Ethnopharmacol 1(4): Tripathi VJ, Ray AB, Das Gupta B (1974). Neutral constituents of Albizzia lebbeck. Curr Sci 43:46 8. Wealth of India: A dictionary of Indian Raw Materials and Industrial Products (2006).1st ed. Vol. I: A. New Delhi. CSIR. p.126. WHO. WHO Traditional Medicine Strategy (2002). Geneva: World Health Organization. Source of Support: NIL Conflict of Interest: None Declared

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