Biological Screening and Phytochemical Studies of Sterculla Diversifolia (G. Don) Stem Bark and Leaves

Abstract

This Ph.D. project is based upon the biological screening and phytochemical studies of medicinally important plant Sterculia diversifolia, belonging to Sterculaceae family. The present study was designed to scrutinize and provide scientific rationale in the light of modern sophisticated technologies and to accredit the activities of the crude extract with the isolated pure chemical entities. In the preliminary phytochemical tests, the crude extracts of Sterculia diversifolia stem bark and leaves revealed the presence of carbohydrates, alkaloids, saponins, sterols, steroids, glycosides, flavonoids, phenols, tannins, phalobotannins, terpenoides and vitamin C. Coumarins are present in leaves but absent in stem bark. Sterculia diversifolia stem bark and leaves are rich source of various micro and macro nutrients. The extracts were tested for their micronutrients (Fe, Cu, Ni, Sb, Zn, Co, Mg, Cr, Mn, Cd, Ag, Au, Pb) and macronutrients (Na, K and Ca) accumulation. The results demonstrated the accumulation of reasonable concentrations of these nutrients within recommended limit in plants. Gas chromatography-mass spectrometry (GC-MS) of n-hexane fractions of both stem bark and leaves revealed 32 and 62 compounds respectively. Following the principle of bioactivity guided isolation; stem bark and leaves of the plant were subjected to column chromatography for the isolation of pure moieties. The structures of isolates were elucidated using physical and comprehensive spectral analysis. These techniques were, 1H-NMR, 13C-NMR, DEPT, 1H-1H COSY, NOESY, HMBC, HSQC and mass spectrometry (EI-MS, HREI-MS, FAB-MS). The column chromatography led to the isolation of a new compound named Stercularin (1), along with eleven known compounds but from a purely new source, including Gossypetin (2), Summary XV Quercetin (3), Kaempferol (4), Luteolin (5), Taxifolin (6), Benzoic acid (7), Methyl 4- hydroxycinnamate (8), Ursolic acid (9), p-hydroxy benzoic acid (10), β-sitosterol-Dglucoside (11) and Quercetin 3-D-glucoside (Isoquercitrin) (12). The Methanolic extracts of Sterculia diversifolia (MESD) and its fractions showed very poor antibacterial potential. However, mild activity was exhibited by stem bark, n-hexane and ethyl acetate fractions against S. aureus. Moreover the extraction of leaves with nhexane and DCM fractions showed same effect against S. aureus. Other fractions showed no antibacterial activity. There was no antifungal, leishmanicidal and anthelmintic activity shown by the MESD and its fractions. Prominent scavenging activity was observed on stable free radical (DPPH) by extracts as well as subsequent fractions of both stem bark and leaves, therefore showing its antioxidant potential. Nevertheless, the protein antiglycation, insecticidal and genotoxic activities were not prominent. In crude MESD stem bark and subsequent solvent fractions, ethyl acetate fraction showed maximum ROS inhibition followed by DCM and n-butanol fractions. However MESD, n-hexane and aqueous fractions showed no significant activity. In case of crude MESD leaves and their subsequent fractions, aqueous fraction showed maximum ROS inhibition followed by ethyl acetate and DCM fractions while the MESD, n-hexane and n-butanol fractions showed no significant activity. Significant cytotoxicity of stem bark against brine shrimp was observed for n-hexane and DCM fractions respectively. Similarly significant cytotoxicity of leaves against brine shrimp was observed for ethyl acetate and DCM fractions respectively. The rest of samples of both stem and leaves produced mild to moderate cytotoxic behavior. The Summary XVI phytotoxic potential observed against Lemna minor was significant at dose of 1000 μg/ml and in order of ethyl acetate > n-butanol > MESD > n-hexane > DCM > aqueous fractions. Similarly the phytotoxic potential observed against Lemna minor was significant at dose of 1000 μg/ml and in order of ethyl acetate > n-butanol > n-hexane > MESD > aqueous > DCM fractions. Anticancer potential against PC-3 cell lines of stem bark extract and its various fractions was observed. A significant potential was observed in case of DCM followed by ethyl acetate fraction and MESD, while crude MESD leaves extract and its fractions showed comparatively better results. DCM fraction showed significant results followed by ethyl acetate, n-hexane and MESD. Larvicidal activity of stem bark extract, leaves extract and its various fractions was observed. A significant potential was observed on higher doses in case of DCM followed by ethyl acetate fraction. A significant anticancer activity was observed for Gossypetin (5.42 ± 0.19 μg/ml) and Isoquercitrin (8.27 ± 0.28 μg/ml) against PC-3 cell lines. The maximum immunomodulatory activity was exhibited by Gossypetin followed by Methyl 4- hydroxycinnamate with IC50 values 13.6 ± 3.2 and 16.1 ± 2.5 μg/ml respectively. Maximum antiglycation activity was exhibited by Taxifolin followed by Gossypetin with percent inhibition values 48.74 and 46.98% respectively. Methyl 4-hydroxycinnamate and β-sitosterol-D-glucoside showed low activity with percent inhibition values 18.63 and 10.59% respectively. Gossypetin showed moderate anti-leishmanial activity results with IC50 values 16.21 ± 0.21, while methyl 4-hydroxycinnamate and β-sitosterol-D-glucoside did not show appreciable inhibition. Summary XVII The crude methanolic extracts of both stem bark and leaves elicited significant antinociceptive activity in different animal models at test doses (100, 200 and 300 and 400 mg/kg). The inhibition of pain perception was dose dependent. Anti-inflammatory profile of the stem bark and leaves extracts was tested in carrageenan induced paw edema model and significant (p < 0.05) attenuation of the inflammatory reflux provoked by carrageenan at test doses (100, 200 and 300 mg/kg) was observed. Brewer’s yeast induced pyrexia test was employed for the assessment of antipyretic character. The intra-peritoneal administration of extracts (100, 200 and 300 mg/kg) demonstrated marked and significant (p < 0.05) attenuation of infectious pyrexia during various assessment times (1 - 5 hour). Crude MESD stem bark and leaves significantly (p < 0.05) prolonged the onset of action of convulsion and shortened the duration of action of convulsion seizure. At a dose of 500 mg/kg of both stem bark and leaves extracts, produced highly significant (p < 0.05) prolongation of onset of convulsion. All the animals were protected from death at a dose of 500 mg/kg of both stem bark and leaves. The crude MESD stem bark and leaves were found to exhibit hypnotic property. Both the extracts prolonged the duration of sleeping time in dose dependent manner. The onset and duration of sleep of MESD stem bark and leaves at a dose of 500 mg/kg was more significant as compared to 300 and 400 mg/kg respectively. Keeping in view the folkloric use of Sterculia diversifolia as laxative, the current study was designed to evaluate the laxative and anti-diarrheal potential of the Sterculia diversifolia stem bark and leaves. It is evident from the results that MESD enhance the impetus movement of charcoal meal in the small intestine and also raised the production Summary XVIII of wet faeces, hence showing pro-kinetic and laxative activities which is almost similar to Carbachol. The methanol extracts of both stem bark and leaves at 50, 100 and 200 mg/kg body weight doses, significantly lowered several typical parameters of diarrhea. The anti-diabetic activity of the crude methanol extract of Sterculia diversifolia stem bark and leaves were evaluated in alloxan-induced diabetic mice. Admirable decrease in the blood glucose levels was seen at second and third hour respectively by using stem bark and leaves crude extracts. The highest decline occurred at the fourth hour by the action of the extracts in case of acute study. The extracts exhibited extended duration of hypoglycemic activity. A dose of 150 mg/kg of both stem bark and leaves crude extracts showed optimum pharmacological effect. The study demonstrated significant scientific evidences in favour of Sterculia diversifolia in the indigenous system of treatment and also opened a new avenue for researchers to isolate new pharmacologically active compounds. Further detailed studies on the extracts as well as isolated entities may lead to the discovery of revolutionary new therapeutic molecule(s) for the management of different diseased conditions.