Immunomodulatory effect of extracts of Hibiscus ... - ResearchGate

IMMUNOMODULATORY EFFECT OF HIBISCUS SABDARIFFA 665Extracts from the 50% ethanol/water mixture werefurther partitioned successively into dichloromethane(Merck KGaA, Darmstadt, Germany), ethyl acetate(Thomas Baker Chemicals PVT Ltd, Mumbai, India)and butanol (Thomas Baker Chemicals PVT Ltd,Mumbai, India).Anthocyanin determination. The anthocyanin contentof the dried calyx was determined calculating forcyanindin-3-glucoside using a colorimetric method basedon the ability of anthocyanins to produce a color at pH1.0 that disappears at pH 4.5. This characteristic reactionis produced by a pH dependent structural transformationof the chromophore. The colored oxoniumion predominates at pH 1.0, while the non-coloredhemiketal is present at pH 4.5 allowing the accurateand fast determination of total anthocyanins, still withthe presence of polymeric pigments and other compounds(Wrolstad et al., 2005). Briefly, 1 g of the plantmaterial was extracted with the appropriate solvent anddiluted with buffer solutions at pH 1.00 and pH 4.5.The difference in the absorbance at 510 and 700 nm atthe different pH was used in calculating the monomericanthocyanin pigment present in 1 g of the plant materialusing the following equation:Total anthocyanins (mg/L)= A × MW × DF × ∑ × l (1)where A, absorbance =(A 510nm − A 700nm )pH 1.0 − (A 510nm − A 700nm )pH 4.5;MW, molecular weight; DF, dilution factor;∑ = molar extinction coefficient( L × mol −1 × cm −1 ).Immunomodulatory activity. This activity was evaluatedusing red blood cell-induced immunostimulationin a mouse model. Swiss albino mice weighing between18 g and 24 g consisting of five animals per group wereused for the study. Aqueous (A), 50% ethanol (AE) orabsolute ethanol (E) extracts were administered at 5,50 or 300 mg/kg body weight dose to albino mice withan oral gavage needle daily for 28 days. In a separateexperiment, the insoluble fraction (EAC) obtained fromthe 50% ethanol extract were administered at 50 and100 mg/kg and the residual water-soluble fraction at100 mg/kg body weight. Levamisole (0.6818 mg/kg) orallywas used as a positive control, while the vehicle-controlgroup was administered 0.5 mL of water daily. Thenegative control group was administered intraperitonealcyclophosphamide (200 mg/kg) on day 5.On day 7, 200 μL of 10% of New Zealand rabbitred blood cell (RRBC) suspension in normal salinewas administered intraperitoneally. This was repeated14 days later as a booster dose. The body weight ofthe animals was recorded weekly. On day 28, all theanimals were bled and the serum collected was storedat −20 °C for further studies.Haemagglutination test was performed by adding100 μL of rabbit RBC (6 × 10 3 cells per mL) to 25 μL ofthe serial two fold dilutions of the serum in Alsever’ssolution in U-bottom microtitre plates, shaken andallowed to stand for 4 h at 25 °C. Rabbit RBC-settingpatterns were then read. The haemagglutination (HA)titer was expressed as the reciprocal of the highestdilution of the serum showing definite agglutinationformation as opposed to smooth dot in the center ofthe well.Cytokine determination. Due to a prior study (Ogundipeet al., 1998) showing that extract of the dried calyxpossess antiinflammatory activity in carrageenaninducedrat paw model, two cytokines, namely tumornecrosis factor-α (TNF-α) and interleukin-10 (IL-10)implicated as proinflammatory and antiinflammatoryinterleukins, respectively, were evaluated using enzymelinkedimmunosorbant assay (ELISA) for the quantitativemeasurement of mouse TNF-α, and IL-10 (Endogen (R)Mouse ELISA kit, Pierce Biotechnology, Inc, Rockford).Sera from animals in the same group were pooled forthe assay.To 50 μL of pooled serum or standard concentrationsof lyophilized recombinant mouse tissue necrosisfactor-alpha, biotin-labelled detecting antibody reagentwas added in tumor necrosis factor-alpha precoatedwells. Incubation was done for 2 h at 37 °C. Thereafter,a 100 μL dilution of 1:400 streptavidin conjugated withhighly purified horseradish peroxidase was added toeach well and further incubated for 30 min.For determination of IL-10, to each endogen mouseinterleukin-10 pre-coated well was added 50 μL assaybuffer. The same amount of standards (containingknown concentrations of lyophilized recombinant mouseIL-10) or samples from the pooled sera was added tothe wells and incubated at 37 °C for 3 h. After washingthe plates, 50 μL of biotin-labelled detecting antibodyreagent was added to each well, incubated for 1 h, afterwhich 100 μL of 1:400 diluted streptavidin-horseradishperoxidase was added and incubated for 30 min. Toboth IL-10 and TNF-α plates, 100 μL of premixed3,3′,5,5′-tetremethylbenzidine substrate solution wasadded to each well and developed in the dark for 30 min.The blue color reaction obtained was stopped with100 μL of 0.18 M sulphuric acid. The difference inabsorbance was measured using a spectrophotometerplate reader (Versamax, Molecular Devices, USA)at 450 nm and 550 nm. The difference in absorbancewas read off the calibration curve obtained from thestandard concentrations. The concentration of eachcytokine was determined in picogram per milliliterof serum.Statistical analysis. The effects of the extracts and fractionson haemagglutination titer and other parameterswere compared with the controls using the one-wayanalysis of variance with Dunnett’s post hoc test. Thelevel of significance was set at p < 0.05 using GraphPadInstat (R) statistical analytical software.RESULTS AND DISCUSSIONThe yield of the extracts was found to be 18.54% withethanol (E), 15.18% with water (A) and 15.0% in 50%ethanol (AE). Ethanol was observed to extract the leastanthocyanin (as cyanidine-3-glucoside) of 1.23 mg/gplant material, while the highest was found with 50%ethanol (3.83 mg/g) and water (3.22 mg/g).The summary of the results for the immunomodulationtests are shown in Tables 1 and 2. The extracts didnot cause significant weight changes in the animals whencompared with the vehicle group. Cyclophosphamidecaused a reduction in the weight of the animals ascan be seen in Table 1. As expected, there was a greatCopyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 664–668 (2008)DOI: 10.1002/ptr