Anti-tumor and antioxidant activity of kaempferol-3-O-alpha-L-rhamnoside (Afzelin) isolated from Pithecellobium dulce leaves

Abstract

Background: Pithecellobium dulce (Roxb.), an evergreen medium-sized, spiny tree which have vast nutritional values and widely used in ayurvedic medicines and home remedies. The plant has also been a rich source of biologically active compounds. The present study was designed to isolate pure compound from ethyl acetate fraction of methanol extract of leaves and to know the efficacy as antioxidant as well as its anti-tumor activity on Ehrlich ascites carcinoma cell (EAC). METHODS: The leaves were extracted with methanol and fractionated with different solvents. The isolation of the compound was carried out by column chromatography from ethyl acetate fraction (EAF) and structure was revealed by ¹H-NMR and ¹³C NMR. The antioxidant activity was investigated by the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals as well as the inhibition of oxidative damage of pUC19 plasmid DNA, hemolysis and lipid peroxidation induced by a water-soluble free radical initiator 2,2'-azo (2-asmidinopropane) dihydrochloride (AAPH) in human erythrocytes. In vivo anti-tumor activity of the compound was also evaluated by determining the viable tumor cell count, hematological profiles of experimental mice along with observing morphological changes of EAC cells by fluorescence microscope.

Results: The isolated compound kaempferol-3-O-alpha-L-rhamnoside effectively inhibited AAPH induced oxidation in DNA and human erythrocyte model and lipid per oxidation as well as a stronger DPPH radical scavenging activity. In anti-tumor assay, at a dose of 50 mg/kg body weight exhibit about 70.89 ± 6.62% EAC cell growth inhibition, whereas standard anticancer drug vincristine showed 77.84 ± 6.69% growth inhibition.

Conclusion: The compound may have a great importance as a therapeutic agent in preventing oxidative damage of biomolecules and therapeutic use in chemotherapy.

Keywords: AAPH; Anti-tumor; Antioxidant; EAC cell; EAF; NMR analysis.

Fig. 1

TLC profile at UV 254 nm of methanolic extract and its fraction (CHF, EAF and AQF) of leaf of P. dulce. M-Crude methanol extract; C-Chloforform fraction; E-Ethylacetate fraction; A-Aqueous fraction

Fig. 2

Time course effects of kaempferol-3-O-alpha-L-rhamnoside (Sz-02) and ascorbic acid on AAPH-induced hemolysis on erythrocytes. Erythrocyte suspension at 5% hematocrit was incubated with 50 mmol/l AAPH at 37⁰C in the absence or presence of compound or ascorbic acid at the indicated concentrations. Values are expressed as mean ± standard deviation

Fig. 3

Effect of kaempferol-3-O-alpha-L-rhamnoside (Sz-02) and ascorbic acid on MDA formation in erythrocytes. Erythrocyte suspension at 5% hematocrit was incubated with 50 mmol/l AAPH at 37⁰C in the absence or presence of compound or ascorbic acid at the indicated concentrations. Values are expressed as mean ± standard deviation

Fig. 4

Agarose electrophoretic pattern of pUC19 plasmid DNA with and without the treatment of pure compound and standard gallic acid. Lane 1: untreated DNA; Lane 2: DNA treated with AAPH; Lane 3: DNA + AAPH + 5 µg compound; Lane 4: DNA + AAPH + 10 µg compound; Lane 5: DNA + AAPH + 20 µg compound; Lane 6: DNA + AAPH + 40 µg compound; Lane 7: DNA + AAPH + 50 µg compound; Lane 8: DNA + AAPH + 50 µg gallic acid

Fig. 5

(a-b) Fluorescence and (c-d) optical microscopic observation of EAC cells for control mice and treated mice. (a) and (b) represent as fluorescence microscopic view of control and pure compound treated mice cell where (c) and (d) express the optical microscopic view of control and pure compound treated cells, respectively. Normal cell with round shape nucleuses appeared in control group indicated by white and red arrow in (a) and (c) whereas the mice treated with pure compound, condensed nucleus and fragmentation of cells (apoptotic characteristics) were found (b) and (d) indicated by white and red arrow, respectively