ANVESHANA PHARMACEUTICO ANALYTICAL STUDY OF MALLA YOGA W.S.R. TO ITS TOXICITY STUDY IN RATS

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1 ANVESHANA Research Article AYURVEDA MEDICAL JOURNAL ISSN: PHARMACEUTICO ANALYTICAL STUDY OF MALLA YOGA W.S.R. TO ITS TOXICITY STUDY IN RATS Vineeth P. K. 1, Rajalakshmi S. 2 1 Asst. Prof. of Rasashastra and Bhaishajya Kalpana, 2 Research Assistant, Amrita School of Ayurveda, Kollam, Kerala, India Corresponding Author: vinubrocha@gmail.com ABSTRACT Rasashastra is one of the subjects in Ayurveda which specially deals with minerals, herbal and herbo-mineral preparations along with their therapeutic uses and is known for its quick action and has been proved to be effective in treating various ailments. Malla Yoga is a preparation mentioned in Sidhabheshaja manimala Swasa adhikara 4/8, which contains Shodhita Shankha and Shoditha Malla as its main ingredients. This article points towards pharmaceutical study, physico-chemical study, analytical study and acute toxicity study of Malla yoga. The study was done for 14 days in Albino rats to find out the minimum therapeutic dose, maximum tolerated dose and median lethal dose (LD50 value) and toxic effect in the organ system. Thus this paper highlights the importance of toxicity study of a drug before administration to prove its safety profile, acute toxicity and pharmaceutical study of Malla yoga and its efficacy to cure various disorders whose incidence has been increasing tremendously in the present world with begrimed surroundings. Key words: Rasashastra, Ayurveda, herbo-mineral preparations, Malla Yoga, toxicity study, safety profile, acute toxicity INTRODUCTION Rasa Shastra mainly deals not only with the mineralogy aspects of metals and minerals but also preparation of medicines. It has great therapeutic efficacy as it possess innate qualities like quick action, less dose, tastelessness, prolonged shelf life and a better palatability. Rasa Shastra has a glorious past regarding Good Manufacturing Practices (G.M.P.), standardization, Standard Operating Procedure (S.O.P) etc. This is evident from its fossils which reveal the description of Rasa Sala, which lays down the clear-cut picture of a planned and fully equipped Pharmacy cum Research Institute. Malla Yoga 1 is one such preparation mentioned in Sidhabheshaja manimala Swasa adhikara 4/8, which contains Shodhita Shankha and Shoditha Malla as its main ingredients. An article in JAMA 2 dated on 15 th December 2004, created a strong feel for necessity of safety data on the metallo mineral and Herbo-mineral formulations used in Ayurveda. Conventionally modern toxicology and pharmacology consider that heavy metals are toxic for the body. This has been a major threat to the development of this ancient science to a great extent. But this toxicity i.e the vishathva was tamed by

2 our Acharyas by means of Shodhana 3 which includes the specific purificatory processes for each metal and mineral. On internal administration of metals and minerals i.e. Rasausadhis, in unprocessed or misprocessed form, they may be toxic but when Shodhana and Marana of these substances are done systematically, scientifically and sometimes subjecting to some special processes, they became non-toxic and gains therapeutic potency. Hence toxicology and purification methods play a key role in the manufacture and efficacy of any mineral or metallic preparations. Further there is a classic toxicology maxim "All things are poison and nothing is without poison; only the dose makes a thing not a poison." This is often condensed to: "The variation of dose makes the medicine poisonous or non-poisonous". Determining scientifically consistent toxicological profile of Ayurvedic drugs complying with current regulatory practices can be a fillip to their prospects of wide spread acceptance. Malla Yoga is a preparation mentioned in Sidhabheshaja manimala - Swasa adhikara, which contains Shankha and Malla as its main ingredients. This is indicated for management of many acute disorders like Svasa, Kasa and Jwara. Shankha 4 is tridoshagna, lekhana, vishagna and agnideepana. Malla 5 is vata kaphahara and is specially indicated in swasa roga and jwara. Hence a positive hypothesis (H1) was made, to study the safety of Malla Yoga when administered in classical therapeutic dosage, and to prove its safety use through acute toxicity studies. So the present work was taken up in the interest of producing experimental base for safe clinical use of Malla Yoga. Pharmaceutical Study: Shodhana of Malla was done by Swedana with Karavellaka swarasa for 6 hours and Shodhana of Shanka by Swedana with Nimbu swarasa for 3 hours. Table 1: Changes in characteristics of Karavellaka swarasa and Malla before and after Shodhana Particulars Before Shodhana After Shodhana Color of Karavellaka swarasa Light green Brownish green Consistency of swarasa Liquid and Clear Increased with suspending particles Odour of the Swarasa Natural fresh odour of swarasa Very obsessive odour ph of swarasa 6.84 (Acidic) 6.72 (More acidic) Colour of Malla White White Appearance Shiny/glassy Opaque dull Weight of Malla 100 g 90 g Observations during Shankha Shodhana: Within few minutes after starting procedure, froth and air bubbles appeared. The Nimbu swarasa attained curd like consistency after the shodhana procedure. After shodhana the hard nature, dirty brown colour and smooth surface of Shankha turned to slightly brittle, pure white colour and rough surface. Preparation of Malla yoga Shuddha Shankha was taken after proper drying. 5 tola (50 g) of Shudha Malla was powdered and kept inside Shankha. Then Arka ksheera was poured till Malla gets soaked completely. The remaining space in Shankha was filled with Arka patra kalka. The opening of the Shankha was covered with mud smeared cloth, dried and was kept in a sharava. Another sharava having the same dimensions was placed over it in such a way that the mouth of both sharava come in contact and sandhibandhana was done with a cloth smeared with multanimitti. It was allowed for drying. Then it was subjected to Gaja puta. Complete procedure was keenly observed AAMJ / Vol. 1 / Issue 5 / Sep Oct

3 and the temperature pattern was noticed with digital pyrometer, half an hourly. Sharava was collected after self-cooling. Sandhi bandhana was removed carefully and the drug was collected. Maximum temperature attained in Gaja puta 9 was 1003 c. Maximum temperature was attained after 210 minutes. Drug attained is powdered in a khalva yantra 10 and was light grey in colour. Analytical Study Analytical study is the key part of any scientific research. It tells us about the correlation between pre-determined hypothetical values and actual results obtained. It gives us valuable information about safety, efficacy, stability, and contraindications etc. of any formulation. Following analysis were carried out. 1) Organoleptic tests 2) Physico -Chemical tests a. ph value b. Moisture value c. Total ash value d. Acid insoluble ash Value e. Water soluble ash Value 3) X-RAY Diffraction Studies (XRD) 4) Scanning Electron Microscope Studies (SEM) with EDX Table 2: Showing the Organoleptic tests of Malla Yoga Sl. No. Parameter Observation 1 Colour Greyish white 2 Taste Tasteless 3 Odour Odourless 4 Touch Smooth Table 3: Showing the Physico- chemical tests of Malla Yoga ph value Moisture value Total ash value Acid insoluble ash Value Water soluble ash Value 2.37 % w/w 91.53% w/w 89.38% w/w 24.56% w/w X-RAY Diffraction Studies (XRD): Details about identified compounds are listed below. Sample 1: Raw material Malla Sample 2: Malla Yoga AAMJ / Vol. 1 / Issue 5 / Sep Oct

4 Table 4: Showing the Quantity of all the elements in Malla Yoga by EDAX ELEMENT WEIGHT % ATOMIC% C O Ca As Total Animal study: Methods: Inclusive criteria: Adult healthy albino rats from both the genders. Rat weighing g. Albino rats between days were included. Exclusive criteria: Unhealthy albino rats. Weight below 150gms and above 200g. Albino rats of below 90 days and above 120 days were excluded. Fixation and preparation of rat dose: The normal human adult dose of Test drug (Malla Yoga) is 1 Ratti, which is equal to 125mg. This was converted into animal dose based on Paget and Barner s surface area ratio. i.e. Rat dose / kg body wt. = x Human dose x 5 = x 1 rattii x 5 = x 125mg x 5 Therefore Rat dose of Test drug = mg/kg The OECD-423 method 11 uses pre-defined doses and the results allow a substance to be ranked and classified according to the Globally Harmonized System for classification of chemicals, which causes acute toxicity. For toxicity study the limit dose was fixed as 2000mg/kg body weight. Hence, that rat dose is 180mg/kg body weight (16 times of therapeutic dosage). Vehicle for Administration of Drug: As the drugs are insoluble, the gum acacia 2% solution (by mixing 2 g of gum acacia in 100ml of distilled water) is taken as the vehicle and the same as control. The 2% gum acacia is calculated for rat dose of 5 ml /kg body weight. Procedure: Administration of Drugs: Drug was administered through intra gastric tube using 2 ml syringe fitted with 18 gauze needle made of steel provided with 5 number infant feeding tube to avoid injury to the rats during drug administration. Prescribed dose of suspended drug was loaded in syringe and the tube was inserted into the esophagus. After confirming that the tube was inside the esophagus, drug was pushed slowly to reach the gastrum. Fixation: The tissues were excised out immediately after sacrificing, cleaned of extraneous tissue cut into pieces of 3-5 mm thickness and transferred to 10% formalin solution. The tissues were allowed to remain in it till they are taken up for processing. Tissue Processing: Tissues were thoroughly washed by placing them under running tap water and placed in 70% alcohol. The tissues were subjected to dehydration, clearing and paraffin infiltration by passing them through 80, 90 and 95% alcohol (2 changes), isopropylalcohol, acetone (2 changes) chloroform (3 changes) paraffin (2 changes) (3 each). Next the tissues were embedded in paraffin to prepare tissue block in paraffin. Tissue blocks were fixed to metal object holder after trimming them to suitable size. Section Cutting: The tissue sections (5 μm) were cut with help of a Spencer type rotary microtome and floated in a water bath between C. Then they were mounted on clean glass slides with a drop of Mayer s egg albumin, dried on hot plate at about 50 C for 30 min. AAMJ / Vol. 1 / Issue 5 / Sep Oct

5 Staining Procedure (Haematoxylin Eosin Stain): The sections were stained by serially placing them in xylol, acetone, 95% alcohol, running water, haematoxylin stain, running water again, eosin solution 95%.alcohol (3 changes), acetone (2 changes), xylol - 2 changes and mounted with D.P.X. The slides were viewed under a microscope at various magnifications to note down the microscopic features. Statistical Analysis: The data generated during the study was subjected to student t test for unpaired data to assess the statistical significance. 'p value less than 0.05 were considered as statistically significant. Experimental Design for Acute Toxicity Study (14 days): Rats were divided in to normal control group and two different groups containing three animals each as follows. Group 1: Control Group Group 2: Treated group Main Study Procedure: The animals were observed individually every 30 minutes after dosing the first 24hrs and thereafter daily for a total of 14 days. The time at which signs of toxicity appear and disappear was observed systematically and recorded for each animal. Additional signs of toxicity such as changes in bodyweight, skin and fur, eyes and mucus membranes, respiratory system, circulatory system, autonomous nervous system and central nervous system, somatomotor activity and behavior pattern were also recorded. Attention was given to observe the tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. The absence or presence of compound-related mortality of the animals dosed at one step was determine the next step. Any mortality during the experiment for 14 days were observed and recorded. Part 1: Clinical Observations 1. Rats were placed on the flat surface to observe the posture, movements and behaviour. Gently feel from head to tail to check the condition of the fur and damaged areas of skin, areas of tenderness. All natural orifices were checked for any discharges, blockages, blood, mucus etc., the color and consistency of the faeces. The mouth examined for excessive salivation, breathing abnormalities etc. 2. Body weight recorded: The body weight of all the rats are recorded on initial day, 7 th day and 14 th day to rule out any significant changes. Part 2: Haematology & Biochemical Investigations I. Haematology:- The following haematological parameters were determined using blood samples collected on Initial day, 7 th and 14 th day using microhaematocrit capillary tubes. Haemoglobin levels (Hb) Total Leucocyte Count (TLC) Red Blood Corpuscles (RBC) II. Serum biochemical parameters:- Serum biochemical parameters were estimated from the serum samples collected from the animals on 14 th day.the following humoral parameters were estimated. Total protein Total cholesterol Serum Albumin Serum Creatinine SGOT (Serum glutamic oxalacetate transaminase)/ast SGPT (Serum glutamic pyruvate transaminase)/ ALT AAMJ / Vol. 1 / Issue 5 / Sep Oct

6 Master Chart of Different Parameters after 14 days treatment with Malla Yoga in rats Parameters Control Treated Group t value p value Significance Body weight ± ± NS Liver 8.24 ± ± NS Organ Kidney 1.69 ± ± NS weights Spleen 0.61 ± ± NS Haemoglobin (%) ± ± NS Red blood cell(mm 3 ) 4.53 ± ± NS Total Leukocyte count (*10 6 /ml) ± ± NS SGOT (U/L) ± ± NS SGPT (U/L) ± ± NS Serum Creatinine (mg/dl) 0.82 ± ± NS Total Cholestrol (mg/dl) 101 ± ± NS Serum Albumin (mg/dl) 2.27 ± ± NS Total proteins (g/dl) 7.05 ± ± NS **Values are mean of 6 animal s MEAN } S.D. (Unpaired t test. *P<0.05; **P<0.01.vs. control group N=10, NS- Not significant Part 3: Histopathological Studies At the end of experimental period of 14 days, the animals from all the groups were sacrificed and observed for gross lesions of internal organs. The main organs like Heart, Liver, Kidney, Spleen, Intestine and Lungs were collected separating it from adhering tissues using saline and collected by placing on blotting paper and gently pressed to remove excess of saline. The tissues were also collected from the rats which are dead due to escalated doses on particular days post-mortem however, severely autolysed were only necropsied and inspected for obvious changes. The histopathological findings were more or less similar in the treated group when compared to control group. The histopathological findings observed in toxicity studies showed no changes in the structure of vital organs. This infers that the drug Malla Yoga is safe and not toxic for the vital organs of Albino rats. CONCLUSION Malla Yoga reference for present study was adopted from Sidhabheshaja manimala Swasa adhikara 4/8. Malla Yoga was prepared by classical Putapaka method. Malla Yoga is prepared in a unique pattern in which Malla is kept inside Shankha and subjected to Gaja puta. XRD of Malla showed As2O3 peaks which was cubic in structure. XRD of the final product showed major peaks of CaCO3 (Calcite) which was rhombohedral in crystalline structure and few minor peaks of As3ClO12Pb5 (Mimetite). Malla Yoga did not show any significant toxic effects on vital organs in albino rats in Acute Toxicity study. Biochemically Malla Yoga did not show any significant change in various normal physiological variations in albino rats. In Histopathological Study no adverse effects were observed in the vital organs of Albino rats. Therefore, it can be concluded that Malla Yoga can be used safely for therapeutic purposes. The LD50 of Malla Yoga falls under Category 4 with no signs of acute toxicity at the maximum dose of 2000mg/kg. Any change in normal behavioral pattern or signs and symptoms of toxicity and mortality were not observed up to this dose level. REFERENCES 1. Siddha Bhaishajya Manimala of Shri Krishna Ram Bhatta Chapter 4, Sloka 8 2. Heavy metal content of Ayurvedic herbal medicine products authored by Dr Robert Saiper on Journal of American AAMJ / Vol. 1 / Issue 5 / Sep Oct

7 Medical Association Vol 292,No.23,Dec 15, Rasa Tarangini of Sadananda Sharma Chapter 2, Sloka Rasa Tarangini of Sadananda Sharma Chapter 12, Sloka Rasa Tarangini of Sadananda Sharma Chapter 11, Sloka Rasa Ratna Samuchchaya of Pandit Dharmananda Sharma Chapter 3, Sloka Rasa Tarangini of Sadananda Sharma Chapter 12, Sloka Sharangdhara Samhita Madhyama khanda Chapter 1, Sloka 2 9. Rasa Ratna Samuchchaya of Pandit Dharmananda Sharma Chapter 10, Sloka Rasa Ratna Samuchchaya of Pandit Dharmananda Sharma Chapter 9, Sloka OECD (2001), Test Guideline 423, Acute Oral Toxicity Acute Toxic Class Method. In: OECD Guidelines for the Testing of Chemicals. Organisation for Economic Cooperation and Development, Paris. How to cite this article: Vineeth & Rajalakshmi: Pharmaceutico Analytical Study of Malla Yoga w.s.r. to its Toxicity Study in Rats. AAMJ 2015; 1: Source of Support: Nil. Conflict of Interest: None declared AAMJ / Vol. 1 / Issue 5 / Sep Oct