Chemical Characterization of the Flavonoid Constituents of Cuscuta reflexa

Article Information Received 12 April 2014 Received in revised form 25 June 2014 Accepted 02 July 2014 Abstract The two new compounds were isolated by column chromatography from ethanol extract of the entire plant of Cuscuta reflexa. On the basis of IR, NMR and ES-MS spectroscopic analysis and by chemical transformation, structures of two compounds were elucidated as 3’-methoxy-3,4’,5,7tetrahydroxy flavone and 3’-methoxy-4’,5,7-trihydroxy flavone-3-glucoside respectively. These isolated compounds were characterized by UV, IR, NMR and mass spectral studies.


Introduction
Flavonoids and derivatives are among the most ubiquitous of polyphenolic natural products, occurring widely within the plant kingdom.These compounds are important constituents of fruits, vegetables and many herbal preparations.Epidemiological evidence indicates that flavonoids and their derivatives are important for treatment of various diseases such as cancer, hypertension, liver necrosis, etc, due to their anti-oxidant properties and ability to reduce reactive oxygen species (ROS) production.Importantly, flavonoids can reduce oxidative stress in the mitochondria which may contribute to their usefulness in the prevention of diseases.
They are rootless, leafless twining annual parasites with yellowish stems, distributed on tropical and temperate regions, and in India about 6 species found.It grows on thorny or other shrubs, sometimes completely covering bushes and trees 1,2 .C. reflexa spread from one host to another, on each victim they twine and cling tightly with special branching organs called houstoria, penetrating the host and connecting to the host xylem as well as to the host phloem and absorbing from it both water and elaborated food stuffs such as sugars and amino acids 3 .Various parts of this plant are used in tribal medicine for the disease like impotence, premature ejaculation, sperm leakage, frequent urination, ringing in the ears, lower back pain, sore knees, leucorrhea, dry eyes, blurred vision, and tired eyes.
Cuscuta is one of nine herbs included in the manufacture of Equiguard, a Chinese herbal medicine recommended for kidney and prostate disorders.Research performed at New York Medical College indicates that the combination of ingredients in Equiguard may well be effective in the treatment of prostate cancer.The preparation inhibited the growth of cancer cells, increased the rate of self-destruction (apoptosis) of cancer cells, and prevented the surviving cells from forming colonies 4 .Phytochemical investigated on Cuscuta reflexa have reported the presence of kaempferol-3-Oglucoside, astragallin 5 , myrecetin, benzopyrones 6 , glucopyranosides 7 , propenamide, flavonols

Preparation of extract
1 Kg of powdered drug was packed in soxhlet apparatus and extracted with petroleum ether (60-80ºC) to defat the drug.Defatted powdered drug was then extracted with chloroform.The chloroform extract was separated and the marc was further extracted with ethanol (95%).The solvents were removed by distillation and the last traces of solvent being removed under reduced pressure.

Fractionations and isolation of the ethanol extract C. reflexa
The ethanol extract of C. reflexa was subjected to column chromatography using silica gel (60-120 mesh size), and the extract was eluted with solvent ratios of Hexane: ethyl acetate, 100:0, 80:20,       Furthermore, the IR spectrum revealed absorptions attributable to free hydroxyl (3353.4cm -1 ) and ketone (1701.4cm -1 ) groups.An examination of its NMR data and a comparison with the literature suggested that compound A was a flavone.The most downfield shifted peak was 181.52 ppm which was assigned ketone group (C-4).The downfield shifted peak 158.32 ppm and 121.36 ppm were C-2 and C-1' respectively due absence of proton, and it indicates that C-2 and C-1' was attached by ring.This may further confirmed by the presence of C-2' and C-6' shifted peaks at 115.62 and 123.53 ppm respectively.This was further supported by the doublet peak of 1 H NMR spectrum for H-2' and H-6' at 6.52 and 6.72 respectively due to methine group.Thus, its 1 H NMR spectrum revealed characteristic resonances of aromatic protons such as H6 and H8 (δ 5.91, s), and this aromatic proton is confirmed by the 13 C NMR peaks of C-6 and C-8 produces at δ 92.29 and δ 94.18 ppm.Here decrease in downfield peak is due to resonance of hydroxyl group present at neighboring carbon.The multiplicities and the weak coupling constants of H6 and H8 were in agreement with the existence of the hydroxyl group at C7 (δ 164.17

Compound
60:40, 40:60, 20:80, 0:100 at in column.After that extract was eluted with ethyl acetate: Methanol gradient beginning with 100:10 and further eluted with increment of 5% of methanol and was finally with 100% methanol.The fractions (25 ml) were collected from the column.The collected elute were monitored by thin layer chromatography (eluent: ethyl acetate-MeOH, 9:1 and 3:2) for homogeneity and the similar fraction were pooled together.The eight different fractions were collected and dried.The fraction F1 and F2 were containing waxy material; the fractions F3, F5, F7 and F8 were powder but quantity was very little.The fraction F4 was purified by column chromatogram and eluted with solvent ethyl acetate-MeOH, 9:1.The compound was repeatedly washed with warm methanol and acetone mixture, which resulted in leaving behind brown amorphous powder of compound A. The fraction F6 was purified by column chromatogram and eluted with solvent ethyl acetate-MeOH, 9:1.The collected eluted of F6 was washed with methanol and it produces compound B. These compounds were further analyzed by different physicochemical methods to determine the nature of compound.The general experimental procedures used were as follows: melting points, UV spectra, IR spectra, NMR spectra and Mass spectra were recorded.

UK Journal of Pharmaceutical and Biosciences Available at www.ukjpb.com
Chatterjee et al.Chemical Characterization UK J Pharm & Biosci, 2014: 2(3); 14 Sagar (M.P.), India.Whole plant was shade dried, reduced to coarse powder and stored in airtight container till further use.