Isolation, characterization and pharmacological screening of active constituents from Stevia rebaudiana and Rhizophora mucronata

Abstract

The current study was designed to isolate and characterize some bioactive secondary metabolites from two plants of Stevia rebaudiana Bertoni. (Family- Asteraceae) and Rhizophora mucronata Lam. (Family- Rhizophoraceae) by using repeated chromatographic and spectroscopic techniques, targeting their antioxidant, analgesic, antihyperglycemic, antimicrobial, and anticancer properties through in vitro, in-vivo and in silico approaches. The leaves from two plants were extracted with methanol and then fractioned by organic solvents nhexane, dichloromethane, and ethyl acetate to obtain three fractions which were HSR, DSR and

ESR

from Stevia rebaudiana (SR) and three fractions HRM, DRM and ERM from Rhizophora mucronata (RM). All the fractions from Stevia rebaudiana gave strong antioxidant, analgesic, and antidiabetic activities whereas, fractions from Rhizophora mucronata gave prominent antioxidant effect but exhibit mild analgesic, antimicrobial and antidiabetic effects. A total fourteen (14) compounds were isolated from the two plants. A total nine known compounds were isolated from Stevia rebaudiana and five components from Rhizophora mucronata. The structure of the isolated compounds were identified by studying 1D and 2D spectral feature with comparin the published data and characterized as 5-O-caffeoyl quinic acid (1), trans- syringin (2), luteolin (3), apigenin (4), quercitrin (5), 1,8-dihydroxy-3,6-dimethylanthracene-9,10-dione (6), jhanol (7) and jhanidiol (8), decanoic acid (9) and compounds from Rhizophora mucronata were β-amyrin (10), 2-oxo-14,15-bisnor-3,11 E-Kalavadien-10, 13, 17-triol (11), β-sitosterol (12) , rutin (13), and N-trans-para-caffeoyl-tyramine (14). Among the five compounds isolated from Rhizophora mucronata, compound 11 was a new compound characterized by 1D, 2D and Mass spectroscopy. A promising antioxidant effects was observed by the compounds 1, 3, 4, 7, 9 of Stevia rebaudiana except compound 2 when compared to the standard drug with IC value 6.10 μg/mL. Antimicrobial assay was done by disc diffusion method where the isolated compounds (1, 2, 3, 4, 7 and 8) from Stevia rebaudiana gave mild to moderate antibacterial activity against selected both Gram (+)ve and Gram (-)ve strains. Among the isolates, compound 1 exhibited highest antibacterial activity with zone of inhibition 12-15mm with comparison of standard drug ciprofloxacin. The in-vitro cytotoxic effect of each isolate from Stevia rebaudiana was done by MTT assay in HeLa cell line and compound 1 (5-O-caffeoyl quinic acid) showed a higher anti-proliferative effect (IC value was 181.3 μg/ ml) than other compounds 2, 4, 5, and 6 from Stevia. Finally, the in vitro bioactivities of the isolates from Stevia rebaudiana were also supported by molecular docking studies. The computational study demonstrated that the isolated compounds exerted stronger affinity compared to the standard drugs towards the binding sites of dihydrofolate reductase (DHFR), glutathione reductase, and urase oxidase. The in-vitro cytotoxicity activity of the isolates 10 (β-amyrin), 11 (2-oxo-14,15-bisnor-3,11 E-Kalavadien-10, 13, 17-triol), and 13 (rutin) from Rhyzophora mucronta was also done by MTT assay in Ehrlich:s Ascites carcinoma (EAC cell) cell line and the compounds showed inhibitory activity in dose dependent manner having IC 50 50 value of the three compounds 10, 11 and 13 were 139.8, 112.01 and 144.92 µg/ml respectively. Based on this in-vitro cytotoxicity result, in-vivo anticancer activity of the three compounds 10, 11 and 13 was evaluated against EAC cell line in mice model and bleomycin was used as standard drug. It was observed that the maximum cell growth inhibition was given by the compounds 11 which was 58.7% compared to standard drug bleomycin which showed 84.83% cell growth inhibition. A promising apoptotic cell morphological changes were observed using optical and fluorescence technique by these three compounds. EAC cells treated with the three compounds for five consecutive days to examine expression pattern of apoptosis regulatory gene showed upregulation of Bax, P , Cyt C, TNFalpha,

Cas-3, Cas-8, Cas-9 and down regulation of Bcl-2 and NF-κB which indicated that the apoptosis induced by compounds was mediated by extrinsic and mitochondrial pathway. In the current study, we also examine blood, liver, and kidney parameters treated by the compounds especially compounds 10 and 11 regain the healing process as well as these compounds have no toxic effects to this organ.