The dried flowers of Hibiscus sabdariffa L (family Malvaceae) were purchased at the local market and further dried at 40 °C until a constant weight was obtained. The material was authenticated at the Forestry Research Institute of Nigeria (FRIN), Ibadan. A herbarium species (reference number FHI 107622) was submitted for future reference. The dried calyx was pulverized to obtain a coarsely powdered material. One liter 50% ethanol (water/ethanol -50:50) was used to infuse 100g of the powdered plant material for four hrs. The extract obtained was decanted and the material was re-extracted with another one L. The extracts obtained were pooled, filtered and dried in vacuo using a rotary evaporator. The extract was further 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). The fractions obtained were dried in vacuo and the yield was noted.
Sub-acute toxicity tests
Male swiss albino mice obtained from the animal house of Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, with weight ranging from 18 – 24 g (16.25 ± 9.85 g) were kept in laboratory cages. Feed and water were allowed ad libitum and the animals were maintained in standard environmental conditions (temperature 27 ± 1.5 °C; humidity 73 ± 2.3%) throughout the period of the study.
Different concentrations of residual water-soluble fraction (RWSF) and the ethyl acetate-soluble fractions (EAC) were prepared in 20% Tween 80. To three groups of four male swiss albino mice each were administered 50, 100 mg/kg of the ethyl acetate-soluble fraction or 100 mg/kg body weight of the residual water-soluble fraction daily for seven days with oral feeding needle. Another group of four male mice were administered 0.8 ml of 20% Tween 80 as control. The weight of each animal was monitored over a 7-day observation period. The animals were closely monitored the first 24 hr post administration of the fractions. Thereafter, a daily observation was kept for seven days. Blood (0.7 ml) was obtained through the medial canthus of the mice seven days after administration of the fractions. Erythrocyte, total leukocyte and differential leukocyte count were determined using standard methods. Plasma was obtained by centrifuging heparinised blood at 2500 rpm for 10 minutes. The plasma obtained was analysed for glucose, triglycerides and creatinine using a RA-50 standardised Clinical Chemistry System (RA232C, Serial No 30650, Bayer Diagnostics Mfg. Ltd., Swords, Co. Dublin, Ireland). All experimental protocols were in compliance with CIMAP institutional Ethical Committee Guidelines as well as internationally accepted principles for laboratory animal use and care as found in US guidelines (NIH publication #85-23, revised in 1985
This activity was evaluated using red blood cell-induced immunostimulation in swiss albino mice weighing 18–24 g (16.25 ± 9.85 g). Residual water soluble fraction (RSWF) at 100 mg/kg body weight and ethyl acetate-soluble fraction (EAC) at 50 and 100 mg/kg body weight were administered to groups of mice each with oral gavage needle daily for 28 days. Levamisole (0.6818 mg/kg) administered orally was used as positive control, while the vehicle-control group was administered 0.5 ml of water daily. The negative control group was administered intraperitoneal cyclophosphamide (200 mg/kg) on day 5. On day 7, 200 µl of 10% of freshly prepared New Zealand rabbit red blood cell (RRBC) suspension in normal saline was administered intraperitoneally. This was repeated 14 days later as a booster dose. On day 28, all the animals were bled and the serum collected was stored at −20 °C for further studies. Body weights of the animals were monitored weekly.
To evaluate the ability of the fractions to enhance or diminish the formation of antibodies to the RRBC, haemaglutination test was performed by adding 100 µl of rabbit RBC (6 × 103 cells per ml) to 25 µl of the serial two fold dilutions of the serum in Alsever's solution in U-bottom microtitre plates. This was shaken and allowed to stand for 4 hr at 25 °C. Rabbit RBC-setting patterns were then read. The haemaglutination (HA) titer was expressed as the reciprocal of the highest dilution of the serum showing definite agglutination formation as opposed to smooth dot in the center of the well.
Previous studies have showed that extracts (Chen et al., 2003
) and some of the fractions of the dried calyx possess anti-inflammatory activity in carrageenan-induced rat paw model, and were able to reduce production of tissue necrosis factor-α (TNF-α) and increase interleukin-10 (IL-10) production (implicated as pro-inflammatory and anti-inflammatory interleukins respectively). Enzyme-linked immunosorbant assay (ELISA) for the quantitative measure of mouse TNF-α, and IL-10 (Endogen® Mouse ELISA kit, Pierce Biotechnology, Inc, Rockford) was used to evaluate the effects of the fractions on the production of the two cytokines. Sera from animals given the same treatment were pooled. A 50 µl of the pooled sera (from animals which had received the same treatment) or standard concentrations of lyophilized recombinant mouse tissue necrosis factor-alpha was added to biotin-labelled detecting antibody reagent in TNF-α precoated wells. Incubation was done for 2 hr at 37 °C. Thereafter, a 100 µl dilution of 1:400 streptavidin conjugated with highly purified horseradish peroxidase was added to each well and further incubated for 30 mins.
For determination of IL-10, 50 µl assay buffer was added to each endogen mouse interleukin-10 pre-coated well. The same amount of standards (containing known concentrations of lyophilized recombinant mouse IL-10) or samples from the pooled sera was added to the wells and incubated at 37 °C for 3 hr. After washing the plates, 50 µl of biotin-labelled detecting antibody reagent was added to each well, incubated for 1 hr, after which 100 µl of 1:400 diluted streptavidin-horse radish peroxidase was added and incubated for 30 mins.
To both IL-10 and TNF-α plates, 100 µl of premixed 3,3/,5,5/-tetremethylbenzidine substrate solution was added to each well and developed in the dark for 30 mins. The blue color obtained was stopped with 100 µl of 0.18M sulphuric acid. The difference in absorbance was measured using a spectrophotometer plate reader (Versamax, Molecular Devices, USA) at 450 nm and 550 nm. The difference in absorbance was read off the calibration curve obtained from the standard concentrations. The concentration of each cytokine was determined in picogram per milliliter of serum.