Bioactive Amento flavone isolated from Cassia fistula L. leaves exhibits therapeutic efficacy

Plant material

Leaves of C. fistula Linn. were collected from Madhurantagam village, Kanchipuram District, Tamil Nadu, India and subsequently maintained as a herbarium sample with a voucher number PARC/2015/3071. The samples were cleansed thoroughly in water, air dried and ground into semi-granulated powder to be used for further processing.

Preparation of extract and isolation

The fresh leaves about 60 g was subjected to exhaustive extraction in a soxhlet apparatus using 100% ethanol (250 ml) as a solvent. This extracts, referred to as the crude extract here onwards, yielded an extract of 2.5 g. Thereafter, 1 g of dried ethanolic crude extract was subjected to column chromatography with 30 g silica gel (60–120 mesh). In column, hexane with silica gel was poured into the column and gradually added slurry. The fraction was eluted from the crude extract with hexane and ethyl acetate (4:1). Fractions obtained from column chromatography were checked individually, and identical fractions were pooled together based upon the TLC band. This process continued until we obtain minimum compound with maximum purity.

Phytochemical characterization

The yielded compound is semisolid (20 mg), and the absorbance wavelength of isolated molecules was analyzed using UV–Vis spectrophotometer (Systronics AU-2401 UV–Vis double beam spectrophotometer). The spectral values of the isolated compound were identified with the peak obtained in the spectral range of 250–400 nm. FTIR analysis was carried out using a Shimadzu IR Affinity-1 Fourier transform infrared spectrophotometer. Spectra were collected and treated using the OMNIC software. ¹H and ¹³C NMR spectra were determined in CDCl₃ solution at 400 MHz using Bruker Ascend Model. All spectra were recorded on a Bruker AC-250 spectrometer at 5.87 T (250.133 and 62.896 MHz). The compound was dissolved in CD3OD (99.8% D) and estimated as per the standard protocol. Amentoflavone present in the sample was identified by matching the retention time against those of the standard, and the content of Amentoflavone was determined using calibration curve. The data analysis was done using the Empower 2 software.

DPPH scavenging activity

The ability of the isolate to scavenge free radicals was confirmed by estimation of its DPPH radical scavenging activity, as described by Ohinishi et al. (1994). Five different concentrations (6, 12, 25, 50, and 100 µg/ml) of the pure compound were prepared. To each, 3 ml of DPPH (0.1 mM/ml in ethanol) solution was added. The entire reaction mixture was incubated in complete darkness for 30 min, and thereafter, its absorbance was read at 517 nm. The results were compared with ascorbic acid, which is a standard antioxidant equivalent. Percentage radical scavenging (%RS) activity was calculated using the formula:\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$\% {\text{RS }} = \, \left( {\left[ {A_{\text{br}} - \, A_{\text{ar}} } \right]/A_{\text{br}} } \right) \, \times \, 100$$\end{document}%RS=Abr-Aar/Abr×100where A_br is the absorbance before reaction and A_ar is the absorbance after the reaction has taken place. The results of the comparative analysis of the standard and Amentoflavone were shown in Fig. 3.

Cytotoxicity by MTT assay

HepG2 cells were cultured in Dulbecco’s modified medium (DMEM) supplemented with glutamine (0.6 g/l), gentamicin (25 mg/ml), and 10% fetal calf serum at 37 °C and humidified with 5% CO₂. Cells were plated in a 96-well plate (10⁵ cells/well for adherent cells or 0.3 × 10⁶ cells/well for suspended cells in 100 µl of medium). After 24 h, the standard and compound (6, 12, 25, 50, and 100 µg/ml) dissolved in DMSO (1%) was added to each well and incubated for 96 h. At the end of 96 h incubation, the medium in each well was replaced with fresh medium containing 0.5 mg/ml of MTT. The growth of HepG2 cells was quantified by the ability of living cells to reduce the yellow dye MTT to a blue formazan product. Four hours later, the formazan product of MTT reduction was dissolved in DMSO and absorbance was measured at 570 nm (Ferrari et al. 1990). The change in color indicates the cell viability and toxicity. The absorbance was noted down at 570 nm, and the % cell survival was calculated using the following formula:\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$\% {\text{ Cell survival }} = \, \left( {{\text{OD}}_{\text{T}} /{\text{OD}}_{\text{C}} } \right) \, \times { 1}00.$$\end{document}%Cell survival=ODT/ODC×100.

OD_T and OD_C are the mean absorbance of the treated and the control cells, respectively. The concentration of the extract that caused a half-maximal inhibition of cell proliferation (IC₅₀) was determined obtained from a semi-log plot of the concentrations against the percentage of cell survival.