Phytochemical and Biological Studies on the Plants Calotropis gigantea (Linn) and Amoora rohituka Roxb

Abstract

The universal role of medicinal plants in the treatment of diseases is established by their employment in all important systems of medicine. Many active drugs have been derived from different medicinal plants, and the process is going on. As a preliminary approach along this direction, this study was designed to carry out phytochemical and biological studies on the two important medicinal plants, Calotropis gigantea (Linn.) and Amoora rohituka (Roxb.). The plant Calotropis gigantea (Linn.) (Bengali Name: Boro Akanda) belonging to the Asclepiadaceous family has wide folk medicinal uses and various parts of this plant possess different pharmacological properties. As a part of phytochemical investigation of this study, the root bark and flower powder of Calotropis gigantea was extracted with methanol and ethyl acetate, respectively, at room temperature to get methanol (ME) and ethyl acetate (EECF) extracts. I was fractionated with petroleum ether and chloroform successively to yield petroleum ether (PEF) and chloroform (CF) soluble fractions, respectively. EECF was applied on silica gel column chromatography using n-hexane with a gradient of ethyl acetate. Fractions 21~30 afforded white crystals as compound-2 whereas fractions 40~48 were combined and subjected on preparative thin layer chromatography (PTLC) to find colorless oily liquid as compound-1. Based on TLC profile, fractions 7~13 were combined and rechromatographed on silica gel column eluting with n-hexane and ethyl acetate (19 : 1) and three compounds were purified as compound-3 (white crystals), compound-4 (white amorphous powder) and compound-5 (colorless crystals) from different fractions of this second column. Amoora rohituka (Roxb) (Bengali Name: Pithraj) another plant of this study, belongs to the Meliaceae family and according to ayurvedic classical texts, the stem bark of Amoora ruhituka is being prescribed in liver and spleen diseases, oedema, anaemia, intestinal worms, urinary disorders, internal tumours and abdominal complaints. As a part of phytochemical study of this research work, the stem bark powder of Amoora rohituka was successively extracted with ethyl acetate and dichloromethane at room temperature to have ethyl acetate (EAEAR) and dichloromethane (DMEAR) extracts. Preparative thin layer chromatography (PTLC) was then applied on DMEAR and an UV active band afforded orange oily liquid as compound-6. Finally based on analysis and comparison of spectroscopic evidences (Mass, IR, 1H- and 13C-NMR data) of each compound with the literature, compound-1, -2, -3, -4, -5 and -6 were identified as di-(2-ethylhexyl) phthalate, anhydrosophoradiol-3-acetate, taraxasteryl acetate, lup-12,20(29)-dien-3β,28-diol, β-boswellic acid and 2-methoxy-14-calamenenone, respectively. Except taraxasteryl acetate, all of the five isolated compounds are reported here for the first time from the corresponding plant. In brine shrimp lethality bioassay, the cytotoxicity exhibited by EECF, compound-1, compound-2, compound-5 and DMEAR was promising with the LC50 values of 14.61, 9.19, 15.55, 15.26 and 17.67 µg/mL, respectively. But in comparison to ampicillin trihydrate (LC50: 7.21 µg/mL), compound-3, compound-4 and EAEAR demonstrated moderate activity with the LC50 values of 45.46, 30.58 and 26.59 µg/mL, respectively. EECF and compound-1 showed a better broad spectrum of antibacterial activity against pathogenic bacteria In vitro than the other isolated compounds and extracts. The intensity of antibacterial activity was found in the order of EECF > compound-1 > EAEAR > compound-5 > DMEAR > compound-2 > compound-4. The zone of inhibition produced by these extracts and compounds was found in the range 06 to 24 mm whereas the lowest minimum inhibitory concentration (MIC) values for these samples were in the range 16 to 64 μg/ml. In antifungal activity test, only EECF and compound-1 exhibited activity against the test fungi and produced zone of inhibition between 07 to 15 mm. In vivo antineoplastic effect of EECF (50, 100 and 200 mg/kg), compound-1 (10, 20 and 40 mg/kg), compound-2 (10 and 20 mg/kg), ME (10 and 20 mg/kg), PEF (40 and 80 mg/kg), CF (20 and 40 mg/kg), EAEAR (20 and 40 mg/kg) and DMEAR (20 and 40 mg/kg) was assessed by evaluating the viable tumour cell count, survival time, body weight gain due to tumour burden, heamatological (hemoglobin content, RBC and WBC count) and biochemical (Glucose, cholesterol, triglyceride, blood urea, SALP, SGPT and SGOT) parameters of Ehrlich ascites carcinoma (EAC) bearing mice. Treatment with the all above samples dose dependently and significantly (P < 0.05; P< 0.01 and P < 0.001) decreased the viable EAC cells and body weight gain thereby increasing the life span of EAC bearing mice and also brought back the altered hematological (Hb, total RBC and total WBC) and biochemical parameters more or less to normal level. Among the test samples, EECF, CF and DMEAR and compound-2 showed prominent antineoplastic activity as compared with standard drug bleomycin. In addition, treatment of normal mice with EECF did not cause any extreme abnormality at the three doses used in this study. In vitro cytotoxicity assay against A431 cell line (human vulval-derived epidermoid carcinoma), compound-1 (IC50: 0.34 μg/mL) and compound-5 (IC50: 0.36 μg/mL) showed strong cytotoxic effect than the others in comparison with doxorubicin (IC50: 0.31 μg/mL). In insecticidal activity against both larvae and adults of Tribolium castaneum (Herbst), EECF caused the highest mortality of the 1st instars larvae in comparison with other larval instars indicating high susceptibility to the newly hatched larvae with lowest LD50 value (0.134 mg/cm2) and less susceptibility to the adult (after 72 hr exposure) with highest LD50 value (1.371 mg/cm2). From the overall results of this study, it is concluded that extracts and purified compounds from flower of Calotropis gigantea and stem bark of Amoora rohituka have noteworthy antibacterial, cytotoxic and antineoplastic effects that might be a source of herbal drugs in respective therapeutic area.