Isolation and Identification of Anticancer Apigenin Glycosides Flavonoids from Plantation White Sugar

Apigenin and its derivatives are biological active flavonoids that are useful in a variety of dietary constituents. These flavonoids may exert some influence over the transition from normal to cancerous, and have value as chemopreventive substance. In this study, a new purification method of three apigenin glycosides flavonoids from Indian plantation white sugar extracts was developed. Three unknown sugar flavonoids were isolated from sugar by using resin based column chromatography. After filtration, the colorant solution was adsorbed on to the gel column at a flow rate of 1 mL/3 min and elution was done with water at the same rate. 10 mL fractions were collected which were then chromatographed on cellulose TLC plates. The pure fractions were completely evaporated and investigated for identification. The detected flavonoids were: apigenin-8-C-b-Dglucopyranoside, apigenin 6-C-b-glucopyranoside and apigenin-7-O-b-glucopyranoside. Ultraviolet and nuclear magnetic resonance spectroscopy introduces an additional analytical dimension for the identification of sugar flavonoids. keywords: Flavonoid, Sugarcane Plant Extract, Extraction, Resin.

Sugarcane flavonoids may interact with protein molecule and be eliminating protein those are broken during the digestion process 26 . Apigenin derived from sugarcane has been used to treat various diseases such as inflammatory, neuralgia, and shingles 27 . An Apigenin derivative has been reported as cancer chemopreventive agents and appears to confer protection against a large variety of cancer as reviewed 28 .
These flavonoids suppress cell cycle progression, including those of oral squamous carcinoma, esophageal, gastric, and cancer of organs associate with the gastrointestinal tract 29 . Additional clinical uses include antiviral and antihepatotoxic effects. The antioxidant activity of sugarcane flavonoids leads to the place and sequence of the OH group on the benzenoid ring that inhibits superoxide radicals [30][31][32] .
Rare features of flavonoids in sugar cane to develop flower color for entomophilic pollination. Sugarcane flavonoids (Flavonol, flavonone, chalcones) are mostly water soluble. Some flavonoids were identified in mill syrup, bagasse and sugarcane leaves [33][34] .
Many studies have shown that the sugarcane flavonoids possess antioxidant activities. Individual recovery of flavonoids from sugar has not been done yet. Thus, in this study, individual flavonoids components from plantation white sugar were separated by gel permeation technique and characterized by retardation factor, ultraviolet and nuclear magnetic resonance spectroscopy.

EXPERIMENTAL
1 H spectra of flavonoids were recorded using JEOL AL 500 MHz spectrometer in DMSO-d 6 containing TMS as internal standard reference. The UV-Vis measurements in the range of 200-800 nm were recorded using the Shimadzu UV-1601 spectrophotometer. Plantation white sugar was supplied by different sugar factories. Analytical grade solvents were used for sample preparation, purchased from Merck (Mumbai, India). For recovering of sugar flavonoids a XAD-4 macroporous adsorption resin (polystyrene resin, 20-60 mesh particle size, pore diameter 40 A0, surface area =725 m 2 /g) was used.

Preparation of Plantation white sugar
A 25 0 Bx solution of plantation white sugar was filtered and the pH was adjusted to about 4 with concentrated HCl.

Extraction and Isolation
A glass chromatography column (300×20 mm ID), filled with XAD-4 resin was used for flavonoids adsorption. The column was activated with 4 BV of 5% (v/v) HCl and followed by 4 BV of 5% (v/v) NaOH, and redistilled water to a neutral pH. Initial concentration of plantation white sugar extract was 0.8 mg/mL, pH of sugar solution was 7 (10 bed volume feeding solution; flow rate 2.5 bed volume per hour). For flavonoids recovery a mixture of methanol: ammonia: water (50:5:45) was used. The desorbed solution of colorants was completely evaporated under vacuum. The solid colorants were completely dried over P 2 O 5 and weighed. The solid colorant was dissolved in about 100 mL water and 1-2 drops of concentrated HCl were added to precipitate any polymeric colorant 35 . After filtration, the colorant solution was adsorbed on to the gel column at a flow rate of 1 mL/3 min and elution was done with water at the same rate. 10 mL fractions were collected which were then chromatographed on cellulose TLC plates. The pure fractions were completely evaporated and investigated for identification.
Compound 3 shows UV-Vis maxima at 266, 332 (Fig. 8). This compound was tentatively assigned as apigenin-7-O-b-glucopyranoside 11 . The 1 H NMR characterization of compound 3 shows an anomeric proton at δ H 5.08 (1H, d, J=6.85).The compound 3 showed H-3 and H-6 at δ H 6.41 and 6.61. 1 HNMR spectrum of compounds shows doublet at 6.9 (1H, d, J = 2.3Hz) for C-3' and C-5' proton. Doublet at 7.81 (2 H, d, J = 8.4) for 2' and C-6' proton. Shift at δ H 6.77 for C-8' proton (Fig. 9). The data allowed us to establish apigenin-7-O-β-glucopyranoside as the structure of the compound by comprising of the spectral data with literature value 36 . Various methods have been developed for the identification of apigenin and its glucosides in different plants by spectroscopic and chromatographic techniques like high-performance thin-layer chromatography [37][38] , HPLC [39][40][41][42] and UHPLC-DAD 43 . Color concentration and high performance liquid chromatographic (HPLC) methods have been used to measure the approximate levels of major flavonoid colorants in sugar mill and refinery products using apigenin as an internal standard.

ACkNOWLEDGMENT
The author wishes to acknowledge the support of Prof. V. K. Agrawal, Awadhesh Pratap Singh University, Rewa for this work.