Uricosuric activity of Tinospora cordifolia

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Introduction
Tinospora cordifolia (Willd) Miers Ex Hook is a large, glabrous, climbing shrub indigenous to tropical Indian subcontinent and other Asian countries from family Menispermaceae.It is widely used especially for immunomodulatory (Desai et al., 2007), cardioprotective (Rao et al., 2005) and hepatoprotective (Adhvaryu et al., 2008) activity.In Ayurveda, it has shown antirheumatic and antigout activity (Gogte, 2000).In humans, it has shown beneficial effect in diabetic foot ulcers (Purandare and Supe, 2007) and allergic rhinitis (Badar et al., 2005).Prolonged and untreated hyperuricemia results into gout, a severe inflammatory condition.Sustained hyperuricemia leads to impaired blood pressure control, renal impairment and nephropathy (Feig et al., 2006).The drugs used in treatment of hyperuricemia and for prophylaxis of gout include xanthine oxidase inhibitors viz.allopurinol and uricosuric agents like probenecid and benzbromarone (Chohan and Becker, 2009).Allopurinol is contraindicated in patients with compromised renal function (Perez-Ruiz et al., 2005) and frequently causes severe hypersensitivity reactions (Halevy et. al., 2008).Febuxostat is contraindicated in liver failure (Rider et. al., 2010).Uricosuric agents furthermore can't be administered in patients with renal stones (Perez-Ruiz et al., 1999).So, it is the need of the today to find out novel drug with minimal adverse effects.Galo satwa is the sedimented starch of T. cordifolia, the most widely used preparation in Indian system of medicine.
In this study, we tried to evaluate the uricosuric activity of aqueous, hydro-alcoholic, dichloromethane extracts and galo satwa of T. cordifolia.
cm; approximately 5 kg) were collected in summerrainy season (June to August, that is, flowering season) from the botanical garden of K. B Institute of Pharmaceutical Education and Research, Gandhinagar, Gujarat, India.Plant was authenticated (Taxonomist: Dr. Hitesh Solanki, Department of Botany, University School of Sciences, Ahmedabad, Gujarat, India) and a herbarium specimen (PH/09/0015) was preserved at the Department of Pharmacognosy, K. B. Institute of Pharmaceutical Education and Research, Gandhinagar, India, for future reference.

Preparation of extracts
Stems of T. cordifolia were shade dried (weight loss 60%) and reduced into coarse powder with help of ball mill.Aqueous extract, hydro-alcoholic extract and dichloromethane extract from dried powdered stems; galo satwa from fresh stems were prepared in following manner:

Preparation of aqueous extract
Powder (100 g) was boiled in distilled water in 1:3 ratio at 100⁰C for 30 min with gradual shaking.After 30 min, the mixture was filtered and the filtrate was evaporated to dryness at 100⁰C on water bath in a tared flatbottomed petridish with occasional shaking.Residue was weighed (yield 16% w/w).

Preparation of hydro-alcoholic extract
Powder (100 g) was defatted with petroleum ether and then extracted with mixture of ethanol (95% v/v) and distilled water (80:20) using a Soxhlet continuous extraction apparatus.The filtrate was evaporated to dryness at 100⁰C on water bath in a tared flat-bottomed petridish with gradual shaking, and residue was weighed (yield 13.6% w/w).

Preparation of dichloromethane extract
Powder (100 g) was defatted with petroleum ether followed by chloroform and finally extracted with dichloromethane using a Soxhlet continuous extraction apparatus for 1 week.The filtrate was evaporated to dryness at 100⁰C on water bath in a tared flat-bottomed petridish, and residue was weighed (yield 2.4 % w/w).

Preparation of galo satwa
The fresh stems (250 g) were cut, crushed to coarse and crush was socked in distilled water in sufficient amount for 8 hours.It was properly macerated and filtered with nylon cloth.The filtrate was kept to allow the starch to settle down the supernatant was decanted and the sediment was further washed with fresh distilled water till it becomes whitish.Finally it was dried and residue was weighed (yield 10.2% w/w).

Phytochemical analysis of T. cordifolia extracts
Phytochemical tests, for the presence of alkaloids, redu-cing and non-reducing sugars (polysaccharides), tannins, flavonoids, sterols and terpenoids were performed for all four extracts (Harborne, 1998).Dragendorff's test was carried out for presence of alkaloids.Orange colored precipitates were observed.For the presence of polysaccharides, Molisch's test (purple color ring) and Fehling test (yellow and then brick red precipitates) were performed.For tannins, lead acetate was added and white precipitates were observed.In Shinoda test for flavonoid, to the extract solutions, small piece of magnesium ribbon and 3 to 4 drops of concentrated sulfuric acid were added and red color was observed.For phenolic compounds, drop of freshly prepared FeCl3 solution was added to extracts and brownish green color precipitates were observed.For sterols and triterpenoids, Liberman Buchardt test was performed.
To the extracts, acetic anhydride and 2 drops of sulfuric acid were added.Purple to violet color was observed.

Animals
Female albino Wistar rats (body weight 200 to 250 g) were used.The animals were acclimatized to standard condition of temperature (20-22⁰C) with relative humidity (30 to 70%) and 12 hours alternate light and dark cycle in poly propylene cages.Animals were kept on free access to food and water ad libitum during the course of experiment.

Uricosuric activity in oxonate induced hyperuricemia
Hyperuricemia was induced using potassium oxonate, a uricase inhibitor to study in vivo uricosuric effect of extracts.Food and water were withheld overnight prior to study.Hyperuricemia was induced as described by Murugaiyah and Chan, 2009.Based on pilot study previously performed, the dose of each extract was decided.The doses for extracts were aqueous (500 mg/ kg), hydro-alcoholic (100 mg/kg), dichloromethane (100 mg/kg) and galo satwa (1,000 mg/kg).Animals were divided into 7 groups of 6 animals in each.All the animals received either extract or standard via oral route.Extracts and potassium oxonate were administered via two different routes to avoid any possible interaction between them.Animals in all the groups except control Group I were administered potassium oxonate (250 mg/kg, i.p, oid) and uric acid (UA) (1 g/kg, p. o, oid), 1 hour prior to administration of test or standard, on day 1, 3 and 7. Group II served as disease group (received potassium oxonate and UA).Standard group (III) received probenecid (50 mg/kg, oid).Group IV, V, VI and VII received, aqueous extract (500 mg/kg, oid), hydro-alcoholic extract (100 mg/kg, oid), dichloromethane extract (100 mg/kg, oid) and galo satwa (1,000 mg/kg, oid) respectively.Extracts and probenecid were administered from day 1 to 7. Probenecid, aqueous extract and galo satwa were suspended in distilled water using carboxymethyl cellulose (1% w/v).Hydro-alcoholic extract was dissolved in distilled water.Dichloromethane extract was suspended using 20% tween 20 aqueous solution.After 2 hours of administration of either extract or probenecid, blood was collected using retro orbital puncture.Urine was collected at 5 hours and volume was measured.Blood was centrifuged at 2817 xg using cooling centrifuge and serum was separated.Serum uric acid and urinary uric acid levels were estimated (on day 1, 3 and 7) using kits based on uricase method (Fossati et al., 1980).Serum creatinine (mg/dL) and urine creatinine (mg/dL) were measured (on day 1, 3 and 7) using kits based on Jaffe's method (Murray, 1989) respectively.Fractional excretion of uric acid (FEUA) was calculated using given equation to assess the uricosuric effect of the extracts (Dan et al., 1994).FEUA = ([urine urate]/[plasma urate])/([urine creatinine]/[plasma creatinine]).

Uricosuric activity in phenol red model
Extracts were studied further for evaluation of uricosuric activity in phenol red model as described by Turner, 1965.Animals were divided into 6 groups of 6 animals in each.All the animals received either extracts or probenecid orally (one time only) 30 min prior to intravenous injection of phenol red.Aqueous solution of phenol red (3%, 2.5 mL/kg) was injected via tail vein followed by 0.5 mL normal saline.Group I (control) received saline and Group II (standard) received probenecid (50 mg/kg, oid).Test Group III, IV, V and VI received aqueous extract (500 mg/kg, oid), hydroalcoholic extract (100 mg/kg, oid), dichloromethane extract (100 mg/kg, oid) and galo satwa (1000 mg/kg, oid) respectively.Extracts and probenecid were dispensed as mentioned previously.By retro-orbital puncture, blood was withdrawn at interval of 1 hour and 3 hours after administration of phenol red.Blood (0.2 mL) was diluted with 2 mL of 0.9 % NaCl solution, centrifuged and supernatant was collected.To 1 mL of the supernatant, 1 mL of 1% sodium carbonate solution and 8 mL of saline were added.Absorbance was measured at 546 nm using spectrophotometer.Concentration (μg/mL) of phenol red in blood sample was determined from standard graph.At each time interval the values in treated rats were compared statistically with those of controls.

Statistical analysis
All the data were expressed as mean ± SEM (n=6).p<0.05 was considered as statistically significant.Data were analyzed using one-way ANOVA followed by post hoc Tukey test using SPSS computer software version 7.5.

Phytochemical study
Aqueous extract and galo satwa showed the presence of polysaccharides.Hydro-alcoholic extract showed the presence of polysaccharides and terpenoids.In dichloromethane extract, alkaloids and terpenoids were found.

A B
Figure 2: Effect of T. cordifolia extracts on FEUA Data expressed as mean ± SEM (n=6).One-way ANOVA followed by post Tuckey test; @ significant difference from control group, # significant difference from disease group, & significant difference from dicholomethane ex., $ significant difference from hydro-alcoholic ex.(p<0.05) Figure 3: Effect of T. cordifolia extracts on phenol red excretion (μg/mL) Data expressed as mean ± SEM (n=6).One-way ANOVA followed by post Tuckey test; @ significant difference from control group (p<0.05)

Discussion
In hyperuricemic animals, serum uric acid level was significantly increased as compared to control.Probenecid and all the extracts significantly decreased serum uric acid level.Urinary uric acid level was significantly increased in hyperuricemic animals as well as animals treated with probenecid or either of the extracts.Urinary uric acid level was significantly higher in animals treated with galo satwa and aqueous extract as compared to hydro-alcoholic and dichloromethane extract treated groups.There was no significant difference in uric acid excretion between galo satwa and aqueous extract treated animals.Fractional excretion of uric acid was significantly increased in hyperuricemic animals as compared to normal controls.Probenecid, aqueous extract and galo satwa significantly increased FEUA as compared to hyperuricemic group while hydro-alcoholic and dichloromethane extract did not.In phenol red excretion model, probenecid, aqueous extract and galo satwa treatment significantly increased plasma concentration of phenol red in blood.Concentration of phenol red was higher in hydro-alcoholic and dichloromethane extract group but not statistically significant.
Uricosuric like probenecid increased urinary uric acid level and decreased elevated serum uric acid level in oxonated induced hyperuricemia (Yonetani and Iwaki, 1983).The higher dose of potassium oxonate may result in a much higher increase in urinary uric acid level overshadowing the uricosuric effects being investigated (Sugino and Shimada, 1995).Yu et al., (2006) also reported increased excretion of uric acid level in hyperuricemic animals.Our study showed similar results.Fractional excretion of UA was not increased in hydroalcoholic and dichloromethane extract treated groups.This simply can be explained by function of urine creatinine and urine volume excreted by each animal.Most of the uricosurics inhibit excretion of phenol red and increase its blood level which is an indirect indication of uricosuric activity.Probenecid, aqueous extract and galo satwa increased phenol red concentration in blood but not hydro-alcoholic extract and dichloromethane extract.This indicates inhibition of organic anion transport in renal brush border responsible for excretion of phenol red and uric acid by aqueous extract and galo satwa.
There are several different carriers with overlapping specificities.The classical view of a single carrier transporting many different organic acids is probably an oversimplification (Tanner et al., 1979).Four complementary DNAs have recently been cloned which are expressed as proteins transport urate.Few proteins have been localized to the basolateral membrane of proximal tubular cells and others in apical membrane of proximal tubular cells (Rafey et al., 2003).

Conclusion
Extracts of T. cordifolia show potent uricosuric action.Uricosuric activity of hydro-alcoholic and dichloro methane extract may be attributed to other transporter involved in secretion of uric acid and probably extracts may be working in different pattern.Out of all four, aqueous extract and galo satwa were found more efficacious.Both of these contain higher amount of polysaccharide and galo satwa is the pure starch of T. cordifolia.They may used for treatment of hyperuricemia and prophylactic treatment of gout.

Figure 1 :
Figure 1: Effect of T. cordifolia extracts on serum (A) and urinary (B) uric acid levels (mg/dL)