Evaluation of antispasmodic activity of different Shodhit guggul using different shodhan process

According to ayurvedic texts shodhan vidhi is an important process which enhances the biological activity of a compound and reduces the toxicity at the same time. Before incorporating into formulations, guggul is processed using Shodhan vidhi involving different shodhan dravyas like gulvel, gomutra, triphala, dashmul . We have evaluated the antispasmodic activity of guggul on ileum of guinea pig and Wistar rats. The animals were sacriﬁ ced and ileum tissue of guinea pig and rat was isolated and tested for antispasmodic activity using different spasmogens like acetylcholine, histamine and barium chloride. It was observed that the different shodhit guggul ( shudha guggul ) i.e. processed using different shodhan vidhi , showed good antispasmodic activity as compared to Ashudha guggul . When acetylcholine was used as spasmogen, gulvel and triphala shodhit guggul showed good antispasmodic activity than other shodhit guggul. Thus shodhan vidhi enhances the therapeutic properties of guggul . simple, reproducible and efﬁ cient reverse phase high performance liquid chromatographic method developed for simultaneous determination of valsartan and hydrochlorothiazide in tablets. A column having 200 × 4.6 mm i.d. in isocratic mode with mobile phase containing methanol:acetonitrile:water:isopropylalcohol (22:18:68:2; adjusted to pH 8.0 using triethylamine; v/v) was used. The ﬂ ow rate was 1.0 ml/min and efﬂ uent was monitored at 270 nm. The retention time (min) and linearity range (µg/ml) for valsartan and hydrochlorothiazide were (3.42, 8.43) and (5-150, 78-234), respectively. The developed method was found to be accurate, precise and selective for simultaneous determination of valsartan and hydrochlorothiazide in tablets.

changing its position. The percentage RSD was found to be within the limits. The recovery study was carried out at two levels, 50% and 100%. The complete validation parameters are shown in Table 2.
Hence the developed HPTLC technique is simple, precise, specific and accurate, statistical analysis proved that the method is repeatable and selective for simultaneous analysis of rabeprazole and itopride hydrochloride as bulk drugs and in pharmaceutical dosage forms without any interference from the excipients.

ACKNOWLEDGMENTS
The authors are grateful to M/s SNR and Sons Charitable Trust, Coimbatore, India, for providing the facilities to carry the experiment and Grandix using Shodhan vidhi involving different shodhan dravyas like gulvel, gomutra, triphala, dashmul. We have evaluated the antispasmodic activity of guggul on ileum of guinea pig and Wistar rats. The animals were sacrifi ced and ileum tissue of guinea pig and rat was isolated and tested for antispasmodic activity using different spasmogens like acetylcholine, histamine and barium chloride. It was observed that the different shodhit guggul (shudha guggul) i.e. processed using different shodhan vidhi, showed good antispasmodic activity as compared to Ashudha guggul. When acetylcholine was used as spasmogen, gulvel and triphala shodhit guggul showed good antispasmodic activity than other shodhit guggul. Thus shodhan vidhi enhances the therapeutic properties of guggul. Healthy Wistar Rats weighing 150-200 g or guinea pigs (weighing 300-500 g) were procured for the study (Haffkines Institute, Mumbai). Animals of either sex were fasted 24 h before the study. Then the animals were sacrifi ced to isolate the ileum pieces. In case of rats, ether was used as anaesthetic agent, until death and guinea pigs were sacrificed by stunning or exsanguination as per CPCSEA recommended guidelines. Two methods were used to evaluate the Antispasmodic activity.
The in vitro method was performed using rat or guinea pig ileum using matching and interpolation method. Guinea pig ileum was used for acetylcholineand histamine-induced contractions as it is very sensitive to both whereas rat ileum was used for barium chloride-induced contractions. The abdominal cavity was quickly opened and a piece of ileum was isolated. It was placed in a beaker containing Tyrode solution maintained at 37°C. Tissue was cut into pieces of 2-3 cm in length. The distal piece was most preferred and used, being the most sensitive to different spasmogens. The remaining study was carried out on an assembly for isolated tissue i.e. Sherrington revolving drum and an organ bath. The spasms were induced using acetylcholine, histamine and barium chloride.
Submaximal doses of acetylcholine, histamine and barium chloride were selected and different doses of different shodhit guggul (like gulvel, dashmul, triphala, gomutra shodhit, ashodhit) were administered. The responses were recorded on the Sherrington recording drum. The same protocol was followed for different processed gugguls to evaluate their respective antispasmodic activity. A dose response curve was obtained and then percent inhibition of the contractions produced by submaximal Guggul, Commiphora mukul (Indian bdellium Tree) is a small tree or shrub with spinescent branches. It is a gum resin obtained from incision of the bark. Ayurvedic literature is full of praises for guggul and its divine action 1 . The explanation of word guggul is Gunjo vyadhegurdti rakshati which means to give relief against different diseases. Indians know guggul from ancient age as it is used in the treatment of many diseases 2 . Guggul is a resin and hence before incorporating it into different formulations it is required to be purified and detoxified. This process is called as Shodhan vidhi. Shodhan vidhi is important to remove unwanted and harmful effects of the resin and to increase useful therapeutic effects. Guggul is purifi ed by two processes, Samanya shudhi (general detoxifi cation) and Vishesh Shudhi (special detoxification). Vishesh shodhit guggul is purified using special substances (vishesh dravyas) like gulvel, dashmul, triphala and gomutra. The present study has been carried out using guggul processed using different Shodhan vidhi, to evaluate its antispasmodic activity on guinea pig and rat ileum.
Guggul was manufactured and supplied by Shri Dhootpapeshwar Ltd., Panvel, India. In order to compare the activity of various processed guggul with a marketed preparation, Entostal was used as a standard drug, which is a polyherbal, ayurvedic preparation used as antispasmodic marketed by Om Pharmaceuticals Ltd, Bangalore. Shodhan vidhi was carried out according to ayurvedic texts. First Samanya shodhan was done using distilled water and then Vishesh shodhan was done using different Shodhan dravyas 3 .
Study was performed using healthy Wistar rats and guinea pigs of average weight of either sex. Approval for the use of animals was obtained from the of vagal stimulation in the body. So mostly all endogenous colic pain like biliary, gastrointestinal, ureter arise due to such acetyl choline-induced spasms. Histamine-induced spasms are mediated by H 1 receptor activation which is characteristic of allergy producing substances leading to abdominal pain e.g. lead poisoning, uremia, excessive gastric acid or even bile secretion. Barium chloride-induced spasms are not mediated by any receptor but they are mediated by increased Ca ++ channel entry due to spasmogen or increased phosphodiaesterase activity leading to calcium channel activation.
Percent inhibition of induced spasms by different spasmogens was taken as the parameter to evaluate the antispasmodic activity. Various processed guggul namely gulvel, dashmul and triphala shodhit guggul showed considerable antispasmodic activity against spasms induced by all three different spasmogens used. This indicates that processed guggul possessed antispasmodic activity against spasms of different origin. This explains the possible utility of processed gugguls for variety of spasms. Ashodhit guggul failed to inhibit the spasms.
Results indicated that Ashudha guggul was not effective in inhibiting the spasms induced by acetylcholine, histamine or barium chloride (Table 1, fi g. 1). On the contrary, processed gugguls inhibited the spasms induced by these spasmogens. Gulvel shodhit guggul showed maximum inhibition of acetylcholine-induced spasms whereas dashmul shodhit guggul showed maximum inhibition of histamineinduced spasms. Triphala shodhit guggul showed maximum inhibition of barium chloride-induced spasms. Gulvel shodhit, triphala shodhit and dashmul shodhit guggul have a considerable inhibitory activity against spasms induced by different spasmogens compared to other processed gugguls. Entostal, the marketed polyherbal formulation, also exhibited a highly significant inhibitory activity but only in acetylcholine induced spasms. It produced negligible inhibitory activity against barium chloride-induced spasms. When different processed gugguls were subjected to charcoal transit along with Entostal, most of the vishesh shodhit guggul showed significantly lower charcoal transit indicating that processed guggul defi nitely exhibited better antispasmodic activity than ashodhit or even samanya shodhit guggul ( Table 2, fi g. 2). It also showed higher activity than Entostal, which is a marketed antispasmodic preparation.
dose of the spasmogen was reported. Results are expressed as mean±standard error mean (SEM), n=4, Student t test The effect of different processed guggul was noted on the length of the intestine travelled by orally administered charcoal in an in vivo test. This was expressed as % of small intestine length reached by lower limit of charcoal. The animals were kept fasting for 24 h before the experiment. Ashodhit guggul and different processed guggul were fed orally 30 min prior to the oral administration of charcoal meal. A 10% w/v solution of animal charcoal in 5% gum acacia was used. 0.5 ml of this suspension was administered orally per mouse irrespective of weight. After 20 min mice were sacrificed by severing the carotids. The stomach and intestine were excised from gastro-esophageal junction to the ileocecal junction. The distance the charcoal meal travelled from the pylorus was measured. As the intestines of the mice used were all of similar length, it was considered justifi able to use the distance travelled by the charcoal meal as an index of intestinal transit. In this way, the intestinal transit was measured for different groups of mice.
Properties of guggul have been described as hridya, medoghna and mehaghna 4 . The two important pharmacological properties of guggul are antiinflammatory action 5 and antihypolipidemic action 6 . Ayurvedic physicians most widely prescribe guggul formulations for the treatment of arthritis. Guggul is also used for healing bone fractures and infl ammations, in cardiovascular disease, obesity and lipid disorders.
Spasms are very common in human beings. Spasms are continuous smooth muscle contractions, may be induced due to endogenous acetyl choline and histamine. They can lead to discomfort, uneasiness and could result into irritation and infl ammation of the gastrointestinal tract posing a major health problem to the human being. It could even lead to threatening conditions such as gastritis and infl ammatory bowel disorders. Antispasmodics are used to treat such conditions successfully, though they show various side effects such as dry mouth, narrow angle glaucoma, tachycardia, obstructive disease of GI tract.
Acetyl choline-induced spasms are due to muscarinic M 3 receptor activation, which is a characteristic histamine H 1 receptors or it may be simply inhibiting phosphodiaesterase enzyme and consequently inactivating Ca ++ channels responsible for spasms. The shodhit guggul can be thus explored for its activity as antispasmodic agents. Also, Ashodhit guggul exhibited very weak antispasmodic activity, indicating that the process of Shodhan vidhi is very important to increase the therapeutic activity of a drug. So while manufacturing of ayurvedic medicines, this concept of Shodhan vidhi must be considered for safer and better utilization of therapeutic activity of a drug.
When Entostal was evaluated for its antispasmodic activity, it exhibited inhibition of only Ach induced spasms indicating anticholinergic activity against different spasmogens indicating its narrow range antispasmodic activity. As guggul processed by different ways exhibited antispasmodic activity both by in vitro and in vivo methods and against different spasmogens used; further exploration of antispasmodic activity would help guggul establish itself as a valuable antispasmodic in therapeutics.    2'-(1H-tetrazol-5yl)[1,1'-biphenyl]-4-yl]methyl]-L-valine, is an orally active specifi c angiotensin II receptor blocker effective in lowering blood pressure in hypertensive patients 1 . A number of high performance liquid chromatographic (HPLC) methods are available for separation and quantifi cation of valsartan from pharmaceutical dosage forms 2 . Hydrochlorothiazide is a diuretic of the class of benzothiadiazines widely used in antihypertensive pharmaceutical formulations, alone or in combination with other drugs, which decreases active sodium reabsorption and reduces peripheral vascular resistance 3 . It is chemically 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide, and was successfully used as one content in association with other drugs 4-9 in the treatment of hypertension. Simultaneous determination of both drugs is highly desirable as this would allow more effi cient generation of clinical data and could be more cost-effective than separate assays. There are very few methods appearing in the literature for the simultaneous determination of valsartan and hydrochlorothiazide in tablets. Since these methods were based on HPLC and UV-derivative spectrophotometry 10-11 , the procedure was inconvenient for determination and the run times were rather long. The aim of this study was to develop a simple, precise and accurate reverse-base high performance liquid chromatographic method to estimate valsartan and hydrochlorothiazide in tablets. This method was simple and rapid and provides accurate and precise results, as compared with other