Experimental Study. Experimental Study 174

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1 Experimental Study Man is in continuous interaction with universe. Every change in universe is reflected in human body. Medical research is to establish karya karana sambandha between various changes happening in human body. It is very well known that any karya will invariably have many karanas. While evaluating effect of one karana it is necessary to maintain all remaining karanas in the unchanged state for precisely assessing effect of the selected karana. This is probably the most challenging task while dealing with research in human subjects. Human beings have large diversity in their nature, different likes and dislikes, behavioural changes, working atmospheres, mental setups and so on. Changes in socio economical status, education and living conditions contribute to this diversity. So finding a population of similar status and maintaining the experimental conditions akin for all the subjects in a research work is next to impossible in human subjects. In vitro studies are an option available for this which has its own limitations. It is very well established that in-vitro studies cannot replace in-vivo studies as artificial environment created in external environment can t be exactly similar to one which exists inside human body. In this scenario, animal experimentation provides a good option where maintenance of similar husbandry conditions can neutralize these hurdles up to a great extent. Science which deals with animal experiments is called as pharmacology. Moreover pharmaco-kinetics of drugs in human body is to be understood on the basis of anumana as direct visualization of this is not possible. In the case of animal experimentation same can be observed directly by pratyaksha through sacrifice of animals. Animal experiments also provide a good source to study effect of unknown drugs specifically related to safety such as acute and chronic toxicity. Thus animal experiments can be adopted in Ayurveda as a substitute to human models in certain conditions. At the same time it is necessary to keep in mind that the conventional models in pharmacology are to be assessed on Ayurvedic principles and those suit the Ayurvedic lines of thinking can be adopted. Where ever such models are not available there newer models should be designed as per principles of Ayurveda keeping the Ethics issues in to consideration. Experimental Study 174

2 Pharmacology is the science of the drugs (Greek: Pharmacon-drug: logosdiscourse in). In a broad sense, it deals with interaction of exogenously administered chemical molecules (drugs) with living systems. It encompasses all aspect of knowledge about drugs, but most importantly those that are relevant to effective and safe use for medicinal purposes. Samskara one among the paradi gunas plays a vital role in bheshaja nirmana and chikitsa. Three divisions of samskara are vega, bhavana and sthitisthapakatva. Among these sthitisthapakatva play a major role in determining the saviryata avadhi (potency of medicine) of the drug. Potency of Ayurvedic formulations has been explained in Sharangadhara Samhita on the basis of formulations and not on the basis of contents in it. This is a completely different approach compared to that of conventional medicine. It appears that there is quantitative difference of sthitisthapakatva guna present in different formulations. Selection of Drug : Haritaki was selected as trial drug and was assessed for its anulomana activity. In order to find out effect of sthitisthapakatva samskara on two different formulations of Haritaki, churna and vati were selected for present study. Both these preparations were evaluated for anulomana karma twice with gap of one year newly prepared for first time and after completing one year for second time for sthitisthapakatva guna present in them. Preparation of Drug: Preparation of vati needed external binding agent which may alter its sthitisthapakatva as compared to churna. To overcome this problem of tablet preparation bhavana of Haritaki kashaya was given to Haritaki churna for three times. Bhavita Haritaki churna was divided in two equal parts half of which was stored as churna whereas remaining half was used to prepare vati. Both the formulations churna and vati were stored in airtight HDPE containers in normal environmental conditions avoiding direct exposure to sunlight or air. These drugs were assessed for Anulomana karma in experimental study. Hypothesis : Here experimental study was undertaken with following hypothesis that Does two different formulations Churna and Vati of the same medicine (Bhavita Haritaki) when stored for one year show any difference in sthitisthapakatva guna expressed in terms of their potency (saviryata)? Experimental Study 175

3 Aims and Objectives : 1. To compare the Anulomana effect of two different formulations of the same drug (Bhavita Haritaki) with water control and between the groups expressed in terms of intestinal transit time. 2. To evaluate sthitisthapakatva guna in two samples (newly prepared, and stored for one year) of churna and vati formulations for Anulomana karma expressed in terms of intestinal transit time. Materials and Methods: Procurement of Animals and Husbandry Condition: Animals were procured from Animal house attached to pharmacology laboratory, I P G T & R A Jamnagar. Swiss albino mice of either sex weighing between 30 ± 4g were procured from the animal house attached to pharmacology laboratory. They were housed in large spacious polypropylene cages and fed with Amrut brand rat pellet feed supplied by Pranav Agro Industries and tap water given ad libitum. The animals were acclimatized for at least one week in lab condition before commencement of the experiment in standard laboratory conditions 12 ± 01 hour day and night rhythm, maintained at 25 ± 3 o C and 40 to 60 % humidity. Ethics Committee approval : Institutional Animal Ethics Committee had approved the experimental protocol (Approval number; IAEC/09/11/07) and the care of animals was taken as per the CPCSEA guidelines. Sample Nomenclature:: AHC- Bhavita Haritaki churna newly prepared AHV- Bhavita Haritaki vati newly prepared C1 - Control 1 BHC - Bhavita Haritaki churna stored for one year BHV - Bhavita Haritaki vati stored for one year C2 - Control 2 Grouping Grouping was done as follows Newly Prepared Formulations (Phase I) : Group 1 - Control (C1) Group 2 - Bhavita Haritaki Churna newly prepared (AHC) Group 3 - Bhavita Haritaki Vati newly prepared (AHV) Experimental Study 176

4 Formulations Stored for One year (Phase II): Group 4 - Control (C2) Group 5 Bhavita Haritaki Churna stored for One year (BHC) Group 6 - Bhavita Haritaki Vati stored for one year (BHV) Dosage : Dosage was fixed on the basis of body surface area ratio of standard table of Paget and Barens (1961) Experimental Model : Anulomana karma comprises of many facets such as digestion of faecal matter, fluid mechanics and enhanced motility. Simultaneous assessment of all these was difficult hence in this study only one parameter of increased motility expressed in terms of intestinal transit time was selected as experimental model. To assess the action of test drug on the intestinal motility, latency of onset of kaolin expulsion in faecal matter was selected as a parameter. As it was difficult to assess in vivo movement of the drug, it was thought useful to administer a marker (kaolin), which causes color change of faecal matter and doesn t alter the effect of drug. To compare effects of churna and vati newly prepared (Phase I) and stored for one year (Phase II). Same model was repeated after one year maintaining all other conditions in similar manner with same protocol. Dose Fixation: The dose of Haritaki for the purpose of anulomana in human is one Karsha 1 which is about ten grams. The dose for the mouse was calculated on the basis of body surface area ratio by referring to the standard table of Paget and Barnes (1964) 2. On this basis the mouse dose was found to be 550 mg/kg. The test drugs were suspended in deionized water with suitable concentration depending up on body weight of animals and administered orally to overnight fasted animals with the help of oral catheter. Method of administration: (Phase I) The selected animals were divided in to three groups of six animals each. First group served as control and deionized water was administered in the volume of 0.1ml/10g. Second and third groups were administered with Bhavita Haritaki Churna newly prepared (AHC) and Bhavita Haritaki vati newly prepared (AHV) at the dose Experimental Study 177

5 of 550 mg/kg. The test formulations and vehicle (deionized water) were administered to overnight fasted animals and in equal volume. One hour after drug administration 0.1 ml of 40% Kaolin solution was administered with the help of oral catheter to all the animals. Animals were placed in a transparent arena and were carefully observed for beginning of the Kaolin expulsion which begins in the form of white colored faecal pellets. Time required for passage of first normal pellet, first kaolin pellet along with reappearance of normal pellet (if any) were noted. Reappearance of normal pellet or no excretion for 60 min was considered as completion of activity. (Phase II) Same experiment was repeated after one year where first group served as control and second and third groups were administered with Bhavita Haritaki Churna stored for one year (BHC) and Bhavita Haritaki vati stored for one year (BHT) respectively. (Phase II) Statistical Analysis: Data was expressed as mean ± standard error mean (SEM). The significance of differences among the groups was assessed using one-way analysis of variance followed by Dunnett s test. Among the groups t test was applied to find out statistical significance. P values less than 0.05 were considered as significant. Observations and Results: Observations - (Phase I) a. Defecation of faecal matter was seen early in AHC and AHV groups as compared to C1. b. Increased frequency of defecation was seen in both AHC and AHV groups; which was absent in C1. c. Consistency of faecal matter was also changed; it lost its well formed normal shape of a pellet and was turned to semisolid form in few pellets in both AHC and AHV groups but was unchanged in C1. d. Mean time for completion of activity was 500 min in AHC and 523 minutes in AHV which was 680 minutes in C1. (Phase II) a. Defecation of faecal matter was seen early in BHV groups as compared to BHC and C2. Experimental Study 178

6 b. Marked increase in frequency of defecation was seen in BHV group which was comparatively less in BHC group and was absent in C2. c. Consistency of faecal matter was also changed; it lost its well formed normal shape of a pellet and was turned to semisolid form in few pellets in both BHC and BHV groups but was unchanged in C2. d. Mean time for completion of activity was 513 minutes in BHV and 581 minutes in AHV which was 760 minutes in C2. Results: A) Phase I Table 1 Effect of AHC and AHV on intestinal transit time Groups Kaolin pellet expulsion time (Minutes) % Change C ± AHC ± 4.82* AHV ± 3.54* [Data: Mean ± SEM; - Decrease; *One Way ANOVA df-17, F = , p<0.05] The result of the present study showed that, both the dosage forms of Haritaki significantly decreased the intestinal transit time in comparison to control group (Table1). Further between the two dosage forms the decrease observed in intestinal transit time of AHC (37.94%) form was found to be comparatively better than AHV (35.85%) but this difference was statistically insignificant. B) Phase II Table 2 Effect BHC and BHV on intestinal transit time Groups Kaolin pellet expulsion time (Minutes) % Change C BHC BHV [Data: Mean ± SEM; - Decrease; *One Way ANOVA df-18, F = , p<0.05] The difference in the mean values of the two groups was statistically significant (P = <0.001). The data revealed that both the drugs shortened the intestinal transit time which was statistically significant. Further between the two dosage forms the decrease observed in intestinal transit time of BHV (50.58 %) form was found to be comparatively better than BHV (22.18 %) and this difference was statistically significant. (Table2). Experimental Study 179

7 t test 1) Control Vs BHC Table 3 Effect of Control (C) and BHC on intestinal transit time Groups Kaolin pellet expulsion time (Minutes) % Change C BHC [t = with 10 degrees of freedom. (P = 0.063)] The difference in the mean values of the two groups was statistically insignificant (p = 0.063) (Table 3). 2) Control Vs BHV Table 4 Effect of Control (C2) and BHV on intestinal transit time Groups Kaolin pellet expulsion time (Minutes) % Change C BHV % [t = with 10 degrees of freedom. (p = <0.001)] The difference was statistically significant (p = <0.001) (Table 4). Comparison of results : Table 5 Change in Sthitisthapakatva Percentage wise No. Formulation Sample % change Difference % 1. AHC Churna BHC Vati AHV BHV Formulation Churna Vati Table 6 Change in Sthitisthapakatva Time wise Sample Kaolin 1 st Pallet (minutes) AHC ± 4.82 BHC AHV ± 3.54 BHV Difference (minutes) Total Time (minutes) Difference (minutes) Experimental Study 180

8 Comparative results of two phases : a. On administration of both the newly prepared formulations AHC and AHV significantly decreased the intestinal transit and among them the observed decrease in intestinal transit time of AHC dosage form is found marginally better. (Phase I) b. On administration both the formulations stored for one year BHC and BHV decreased the intestinal transit. Among the groups BHV vati form was found better which was also statistically significant. (Phase II) c. After one year vati form showed increase in potency in comparison to newly prepared where as churna form showed decrease. Discussion on Results : 1. In phase I newly formed churna showed better results than vati which indicated that it possesses better anulomana property when compared to vati this supports the claim of Ayurveda that Haritaki in churna form is having better anulomana effect than vati In phase II one year stored vati showed better results than churna which indicate that it possesses better anulomana property when compared to churna this was because of lesser sthitisthapakatva present in churna as compared to vati. 3. Churna formulation lost anulomana property over a period of one year on contrary in vati form enhancement in anulomana property was observed. The increase in result may be because of following reasons a. Newly formed vati had more hardness and less solubility. b. One year stored vati has reduced hardness which may improve solubility. c. In the internal metabolism pathway of drug vati form might have produced secondary metabolites which led to augmentation of anulomana property. Possible mode of action : Anulomana Karma : The mechanism of observed effect may be due to interference with local stimulant effect on motility or acceleration of gastric emptying. Haritaki helps in proper absorption of water and other liquids in undigested material so as to help separation of faecal matter and maintaining its normal consistency by virtue of its ruksha, ushna gunas 4. Another mechanism may be breaking of any lineage between faecal matter and intestinal walls which may be a result of constipation. It may also Experimental Study 181

9 increase stimulation of the enteric nervous system so as to accelerate the intestinal motility. It may be affecting the fluid dynamics because the test drug also changed the consistency of the expelled faecal matter to significant extent. It has been reported that the fruits of Haritaki contain phyto constituents like tannins, anthraquinones and polyphenolic compounds 5. It is possible that one or the combination of these phyto constituents may be responsible for the observed effect. The neural regulation of gastric motility involves stimulation by cholinergic neurons and inhibition by adrenergic neurons. Antagonist of D 2 and 5-HT 3 receptors as well as agonists of 5-HT 4 receptors can stimulate gastric motility. Some of the drugs increase the motility of intestine by modifying the fluid dynamics of the mucosal wall and may cause fluid accumulation in lumen. In the light of the above it can be suggested that the test drug may enhance the intestinal motility by cholinergic stimulation or stimulation of 5-HT 4 receptors; it may be antagonizing the effect of sympathetic system; it may be stimulating the enteric nervous system. Further studies on other animal models will throw some light on exact mechanism involved in observed activity. Sthitisthapakatva: Sthitisthapakatva is a guna responsible for maintaining its own state. Any changes which may originate from the drug itself or from external factors are to be avoided completely if not possible; are to be reduced so as to maintain its own state. Churna formulation of drug was having less sthitisthapakatva as compared to vati formulation. Churna has more surface area, less compactness (sanghata), no hardness (upachaya) by virtue of which it is more prone for effects of external factors such as moisture, temperature variations. Regarding the factors in the drug itself churna form is produced by kuttana (grinding) which increases vayu mahabhuta predominance in it. Larger particles which were aggregated together (bandhana) in raw drug reflect comparative predominance of jala mahabhuta in it; which is reduced in churna. Vati formulation on contrary has increased compactness (sanghata), hardness (upachaya) which reflects predominance of prithvi mahabhuta by virtue of which it is more resistant to effects of external factors such as moisture, and temperature variations. Regarding the internal factors vati form is produced by binding (bandhana) which increases jala mahabhuta predominance in it. Smaller particles are Experimental Study 182

10 aggregated together in a vati which reflect comparatively increased predominance of jala mahabhuta in it. Conclusion: I. Newly formed both the dosage forms of Haritaki AHC and AHT possess significant intestinal motility enhancing effect, indicating towards some of the working mechanisms of Anulomana drug as described in Ayurveda. Among them AHC form has slightly stronger effect as compared to AHT but the difference is statistically insignificant. II. Among both formulations stored for one year BHT possess significant intestinal motility enhancing effect, whereas this effect is also present in BHC but is statistically insignificant. III. After storing for one year churna dosage form show decrease in its potency which is not seen in vati. On the contrary vati form showed increase in activity. Newly formed churna which showed better result as compared to control as well as vati lost its potency and showed negligible increase in comparison to control whereas was less effective than vati. IV. From this study it is concluded that among two formulations churna and vati namely, vati form possesses more sthitisthapakatva as compared to churna and its altered panchabhautika composition i.e. predominance of jala and prithvi mahabhuta is responsible for it. Experimental Study 183

11 References: Sharangadhara, Sharangadhara Samhita Madhyamakhanda Churnakalpana Adhyaya 6/1, with Deepika and Gudhartha Deepika commentary of Adhamalla and Kashirama Vaidya Varanasi: Chaukhambha Orientalia ; Fourth Edition, 2000,pp. 35 KD Tripathi, Medical Pharmacology, 1985, sixth edition 2008, Jaypee Brothers Medical Publishers (P)LTD, New Delhi, 1985, sixth edition 2008,P-3 Bhavamishra, Bhava Prakasha, Haritakyadi, varga, 19, including Bhavaprakasha Nighantu portion, Edited with Vidyotini Hindi Commentary by Sri Brahmashankara Mishra and Sri Rupalaji Vaishya, Ninth Edition, Vol. 1, Chaukhamba Sanskrit Sansthan, Varanasi, 1999; pp. 5. Bhavamishra, Bhava Prakasha, Haritakyadi, varga, 30, including Bhavaprakasha Nighantu portion, Edited with Vidyotini Hindi Commentary by Sri Brahmashankara Mishra and Sri Rupalaji Vaishya, Ninth Edition, Vol. 1, Chaukhamba Sanskrit Sansthan, Varanasi, 1999; pp. 7. Anonymous, The Ayurvedic Pharmacopeia of India, Vol 1, Part I, New Delhi, Ministry of Health & Family Welfare, Government of India. Reprint Edition, 2001, pp Experimental Study 184