Project Incharge: 
Dr. Asoke Banerji
Co-Project Incharge: 
Dr. Bipin Nair
Wednesday, July 1, 2009
School of Biotechnology
Funding Agency: 
Amrita Vishwa Vidyapeetham

After broad separation of tannins as lead complex from the SBT extract , flavonoid glycosides (in the supernatant) were subjected to column chromatography using SiO2 and LH-20. Partially purified fractions were put for preparative HPLC and glycosides were isolated. The amounts of individual compounds were small. The UV spectra of the isolated fractions before and after the hydrolysis show a bathochromic shift of 16 nm (in Band II) suggesting that positions 3 of flavonoids were blocked. Therefore it was inferred that the 3-hydroxyls were blocked by glycosidation or conjugation. Use of UV shift reagents (AlCl3, NaOAc, H3BO3) gave information on the positions of other hydroxyls in the molecules. LC-MS/MS gave the molecular weights of the glycosides. Acid hydrolysis of the glycosides furnished information on the aglycones and the carbohydrates. The qualitative information on the carbohydrates could be obtained by TLC/paper chromatography. A careful analysis of MS/MS data gave quantitative data on the sequence of attachment of carbohydrates and structure of the aglycone. As an illustration of this strategy, analysis of peak at m/e 771 is given below: The peak at 771 (M+ +1) on MS loses a mass of 146 to give an ion at 625 .This ion on further MS/MS show loss of another fragment of 146 to give an ion at 479. Successive loss of two 146 moieties suggest the presence of two consecutiverhamnosyl residues. Ion 479 give 317 with the loss of 162 which represents a glucosyl entity. The remaining ion 317 was identified as isorhamnetin by MS/MS. Taking consideration of the fragmentations of the peak at 771 it is concluded that it represents isorhamnetin 3-glucodirhamnoside Similarly the structures of other glycosides were derived as given in the following



Table : 1

1 Kaempferol -3-diglucoside MW=611, fragment 611-162-162=287.
2 Isorhamnetin -3-rhamnodiglucoside MW=787, fragment 787-162-162-146=317
3 Kaempferol-3- rhamnoglucoside MW=595,  fragment 595-162-146=287.
4 Isorhamnetin-3- glucorhamnoside MW=595,  fragment 595-162-146=287.
5 Kaempferol-3- glucorhamnoside MW=595, fragment 595-162-146=287.
6 Isorhamnetin-3- rhamnoglucoside MW=625, fragment 625-146-162=317
7 Quercetin-3- diglucoside MW= 627, fragment 627-162-162=303.
8 Isorhamnetin-3- rhamnoglucoside MW= 641, fragment 641-162-146=317
9 Quercetin-3- diglucoside MW= 627, fragment 627-162-162=303.
10 Quercetin -3-diglucorhamnoside MW=773, fragment 773-162-146-162=303.

Following similar strategy, tannins were also characterized. For example one of the fractions from Sephadex LH-20 colunm, on LCMS gave an ion M++1 at 751 which lost 152 to give 599 ion indicating loss of galloyl unit. Further loss of 162 from 599 suggests the loss of glucosyl moiety to give 303 which was identified as ellagic acid by the analysis of its MS/MS fragments. Thus 751 ion represent :Ellagi-gluco- gallate. About half a dozen tannins were thus identified.