ISOLATION AND IDENTIFICATION OF ISOQUERCETIN - A FLAVONOID FROM BRYONIA LACINIOSA LINN.

Bryonia laciniosa Linn. (Family: Cucurbitaceae), is an annual climbing herb. Traditionally, it is mentioned in the Ayurvedic text in Vrishya Rasayana category and highly valued as a female fertility booster. The present study involves the isolation and identification of isoquercetin from methanolic extract of Bryonia laciniosa by using TLC, UV, FTIR, 1 HNMR and HR-LCMS techniques. The structure of the isolated compound was established by High-Resolution Liquid Chromatogram Mass Spectrometry and proton-NMR. The mass spectra of compound produced molecular ion peak (m/z) at 469.07. Proton- NMR showed two proton signals of H-6 and H-8 of ring ‘A’ and three aromatic proton signals of H-6’, H-2’ and H-5’ of ring ‘B’ confirming 3’,4’ dihydroxy structure in the isolated compound corresponding to the aromatic ring ‘B’ of quercetin nucleus. The signal of glucose moiety at δ 5.299 indicated the isolated compound has β- linked glucose. Thus, confirming the isolated compound as isoquercetin, a flavonoid glucoside. This is the first paper to report a flavonoid from Bryonia laciniosa .

The traditional system of medicine consists of numerous medicinal plants, which have their potential therapeutic utilities. Bryonia laciniosa Linn. (Syn. Diplocyclos palmatus (Linn.) Jeffrey, Family: Cucurbitaceae), is an annual climbing herb, belongs to the genus Bryonia. It is commonly known as Shivlingi and found growing throughout India.
Traditionally, in Ayurveda, B. laciniosa is considered in Vrishya Rasayana category and is highly valued as a female fertility booster (Kirtikar et al., 1988;Dwivedi et al., 2007). It is also used in the treatment of skin diseases, paralysis of the tongue, snakebite. Extensive pharmacological activity has been reported which include antiinflammatory (Gupta et al., 2003), antidiabetic (Patel et al., 2012) , analgesic, antipyretic (Sivakumar et al., 2004), antimicrobial (Bonyadi et al.,), anti-asthmatic, anticonvulsant and antioxidant activities (Jayaram et al., 2010). Preliminary phytochemical analysis of B. laciniosa reveals the presence of alkaloids, triterpenoids, steroids, saponins, flavonoids, proteins, and fixed oils and fats. Punicic acid (Gowrikumar et al., 1981), goniothalamin (Mosaddik et al., 2000) and glucomannan (Vandana et al., 2006) were reported from seeds of the plant. But there are fewer reports on extraction and isolation of secondary metabolites from this plant. Flavonoids constitute an ISSN: 2320-5407 Int. J. Adv. Res. 7(10), 780-786 782 Results and discussion:-PTLC: -PTLC separates the compounds on a microscale using a thick layer of adsorbent, sufficient for characterization and identification (Harborne et al., 1975). Isoquercetin was isolated by Preparative TLC using flavonoid-rich ethyl acetate extract in the solvent system containing ethyl acetate: glacial acetic acid: formic acid: water. The extract showed a fluorescent band under UV at 366 nm and yellowish-brown under visible light which was collected and purified. The compound appeared as yellowish-white crystals, soluble in water and insoluble in alcohol and nonpolar solvents. It gave positive tests for flavonoids, the Shinoda test and 10% NaOH test. It gave positive test for reducing sugars (Fehling's test) and confirms the presence of sugar moiety ( Fig.1 A) (Harborne, 1998).

TLC studies:-
The isolated compound showed sharp single fluorescent spot on precoated silica gel GF 254 corresponding to the spot in the methanolic extract using ethyl acetate: glacial acetic acid: formic acid: water as solvent system. The Rf value of the isolated compound was matched with the Rf value of the spot in extract (Fig.1 B).

UV/ Visible and FTIR Spectroscopy:-
The methanol spectra of flavonoids exhibit two major absorption peaks in the region of 240-400 nm. These two peaks are commonly known as Band I (300-380 nm) and Band II (240-280). The isolated compound gave two main absorption peaks (Fig. 2), band I at 327.5 nm due to B-ring of cinnamoyl system and band II was observed at 270.2 nm due to A-ring benzoyl system of flavonoid moiety. UV Spectral shifts were also studied, the details of which are represented in Table 1 (Mabry et al., 1970).
FTIR spectra mainly used to identify the types of functional group present in the compound. The isolated compound exhibited a broad peak at 3327.32 cm -1 indicates the presence of hydroxylic (-OH) group, 1396.51 cm -1 due to aromatic ring, 1654.98 cm -1 was due to the carbonyl (C=O) group and -OH bending vibrations at 1288.49 cm -1 , 1159.26 cm -1 ,1080.17cm 1 (Fig. 3).

HR-LCMS study:-
HR-LCMS helps in the identification of unknown compounds. The spectrum of an unknown compound is compared with the spectrum of the known compounds stored in the software library. It gives information about the name, retention time, molecular weight, molecular formula and structure of compounds of the sample under the study. It can also be used when the standard is not available (Asha et al., 2016). HR-LCMS of isolated compound was conducted using the database of Sophisticated Analytical Instrument Facility (SAIF) IIT Bombay, having more than 60,000 patterns. According to HR-LCMS, the mass of isolated compounds was found to be 464.09 and manufactured formula was found to be C 21 H 20 O 12 . The spectrum produced molecular ion peak (m/z) at 469.07 corresponds to [(M+Na) +(H2O) and signal at (ESI, EIC) 464.092 corresponds to molecular ion peak (M + ) (Fig. 4). Thus, confirming the mass of isolated compound.  (Fig. 5). The signal of glucose moiety was observed as triplet at δ 5.299 (1H'') indicating the compound to contain βlinked glucose (Maria et al., 2000). Multiplet signals at δ 3.0 -3.70 (6 H, m) showed six sugar protons and signals at δ 12.601 (H-13), 10.93 (H-12), 9.70 (H-15) and signals at 9.1 (H-14) for four -OH groups attached to the flavonoids core structure (Fig. 6). This confirmed the isolated compound is a flavonoid glycoside.

H NMR study:-
Based on TLC and spectral study, viz., UV, FTIR, HR-LCMS, 1 H NMR and comparison with previously reported data (Agus et al., 2014;Maria et al., 2000), the isolated compound was identified as a flavonoid glycoside and structure was established as Quercetin-3-O-β-D-glucopyranoside. It is commonly known as isoquercetin and the molecular formula was found to be C 21 H 20 O 12 . This is being the first report on the isolation of phytoconstituent from this plant.