Evaluation of antioxidant and hepatoprotective effects of Khamira Gaozaban Ambri Jadwar Ood Saleeb Wala (KGA)

Materials and Methods

The polyherbal formulation of KGA manufactured by Hamdard Laboratories, Karachi, a reputed Unani Pharmaceutical concern was purchased from local market of Sargodha, Pakistan. Folin‐Ciocalteu reagent, diagnostic kit, ascorbic acid, rutin, silymarin, carbon tetrachloride and other chemicals were purchased from Merck, Germany. Wister albino rats of both sexes were used for hepatoprotective study.

Estimation of total phenolic contents

Total phenolic contents of the test formulations were determined by Folin‐ Ciocalteu reagent. To 0.5 mL samples of each formulation added 1 mL of 10:100 μg/mL ethanolic gallic acid solution which was added to 9 mL of distilled water in a 25 mL flask. A blank reagent was prepared by taking 10 mL distilled water and 1 mL Folin‐ Ciocalteu’s phenol reagent with shaken vigorously. After 5 min, 10 mL 7.5% w/v sodium carbonate solution was added and volume was made with distilled water. The absorption was read after 90 min, at room temperature at 750 nm with a spectrophotometer, and calibration curve was drawn (Pal and Shukla, 2003). Total phenolic contents in KGA formulation was calculated from the graph.

Estimation of total flavonoids

It was carried out by aluminum trichloride colorimetric method (Marinova et al., 2005) and 0.5 mL KGA was extracted with 50 mL of 80% aqueous methanol on an ultrasonic bath for 20 min. An aliquot (2 mL) of the extract was centrifuged for 5 min at 14000 rpm. 1 mL of aliquot was mixed with 2 mL aluminum trichloride in methanol (2% w/v). Blank was prepared from 1 mL of standard solution and diluted to 25 mL with methanolic acetic acid (0.5% v/v). The absorbance of probe solution against standard solution was determined at 420 nm after 30 min. All the determinations were carried out in triplicate. Results were express as total (%) flavonoids contents (TFC) in formulations as quercetin equivalent was calculated by following formula:    

TFC (%)=     (Absorbance x Dilution factor)/( Weight of sample (mL))*100* E^1% _{1 cm}

E^1% _1cm = Specific absorption of the quercetin ALCl₃ complex (500)

DPPH radical scavenging activity

The scavenging activity of KGA was measured in vitro by 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) assay. About 0.1 mM solution of DPPH in 100% ethanol was prepared and 1 mL of this solution was added to 3 mL of each KGA dissolved in ethanol at different concentrations (10–100 μg/mL). The mixture was shaken and allowed to stand at room temperature for 30 min and the absorbance was measured at 517 nm with a spectrophotometer. The IC₅₀ value of the drug was compared with that of ascorbic acid, which was used as the standard (Roy and Burdon, 1994). The ability to scavenge the DPPH radicals was calculated using the following formula:

DPPH scavenged (%)= (Absorbance (control - test)/ Absorbance control) × 100                                                     

Nitric oxide scavenging activity

The reaction mixture (3 mL) containing sodium nitroprusside (10 mM, 2 mL), phosphate buffer saline (0.5 mL) and extract or standard solution (0.5 mL) was incubated at 25ºC for 150 min. After incubation, 0.5 mL of the reaction mixture containing nitrite was pipetted and mixed with 1 mL of sulphanilic acid reagent (0.3% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 mL of naphthylethylene diamine dihydrochloride (1%) was added to it, mixed and allowed to stand for 30 min. A pink colored chromophore was formed in diffused light. The absorbance of these solutions was measured at 547 nm against the corresponding blank solutions. The IC₅₀ value is the concentration of sample required to inhibit 50% of nitric oxide radical (Salma et al., 2004). All determinations were performed in triplicates.

Hepatoprotective activity

The hepatoprotective study was conducted in albino rats (150-250 g) against carbon tetrachloride induced toxicity and the study was got approved by the institutional ethics committee. The rats were kept by maintaining standard housing conditions and 12 hours light/dark cycle. They were maintained on standard diet in polypropylene cages and supplied with water ad libitum. Albino rats of either sex were divided into following group with six animals each: Group I: Normal, received normal rat fed and water; Group II: Control, CCl₄ hepatotoxic (0.7 mL/kg intraperitoneally); Group III: Standard drug treated, (Silymarin, 100 mg/kg, orally); Group IV: Polyherbal treated group (150 mg/kg disperse in 1% Tween 80 p.o); Group V: Polyherbal treated group (300 mg/kg disperse in 1% Tween 80 p.o).

The normal levels of serum liver enzymes were determined by withdrawing blood samples directly by puncturing the Retro-orbital plexus on the first day of study. Collected blood was centrifuged at 2,500 rpm for separation of serum. Then to the tested animals 0.7 mL per kg body weight, carbon tetrachloride (CCl₄) was administered i.p for five days. On the sixth day enzymatic levels were recorded. After intoxication with CCl₄, tested formulation and standard silymarin were administered for five days. On the 11^th day the serum levels were determined by using semi-autoanalyser against commercially available kits (Randox).

Statistical analysis

Data obtained from this work were analyzed statistically using Students’ t-test and ANOVA (One- or Two-way) followed by a post test (Tukey-Kramer multiple comparison test). Differences between means will be considered significant at 5% level of significance (p<0.05).