Evaluation of quantitative variation of secondary metabolites in Bergenia ciliata (Haw.)using high performance thin layer chromatography

Evaluation of quantitative variation of secondary metabolites in Bergenia ciliata (Haw.)using high performance thin layer chromatography

Dear Editor:

Therapeutically active metabolite contents in a med–icinal plant vary in nature,which may impact on its therapeutic efficacy.Bergenia(Saxifragaceae)is an evergreen perennial herb widely distributed in Central and East Asia with about 30 species reported world–wide.It grows at a range of altitudes from the Khasia hills at 400 feet to the temperate Himalayas from Kashmir to Bhutan at 7,000–10,000 feet[1].Its distribu–tion over a wide range of altitudinal zones makes it a good candidate for studying variations in its metabolic profiles under different climatic conditions.Bergenin (C–glycoside of 4–O–methyl gallic acid)has been identified as a potent active secondary metabolite in Bergenia and other therapeutically active constituents including,among others,gallic acid(3,4,5 trihydroxy–benzoic acid),(+)catechin,and gallicin(Fig.1). Here,we analyzed three accessions of Bergenia ciliata collected from different altitudes of Uttrakhand,India, to study the quantitative variation in bergenin,gallic acid,(+)catechin,and gallicin by high performance thin layer chromatography(HPTLC).For accurate quantifi–cation,different hydrolysis conditions were employed and their effects on individual compound were assessed.

We found significant variations in the content of these compounds in the collected rhizomes of Bergenia ciliata ranging from 0.756±0.3 to 0.85±0.2 for bergenin, 0.145±0.2 to 0.27±0.2 for(+)catechin,0.027±0.2 to 0.166±0.3 for gallic acid and 0.135±0.3 to 0.186±0.1 for gallicin.Such variability is expected due to variation in environmental conditions which var–ies on varying altitudes.Fig.2illustrates the effect of climatic and altitudinal variations on the distribution of these four compounds in Bergenia rhizomes,showing that even a small altitudinal variation is capable of indu–cing quantitative variation in the contents of these com– pounds.It has been established that biosynthesis of polyphenolic compounds increases on higher altitude in response to lower environmental stress.But the pre–sent study revealed that it was not the case for every phenolic compound.Bergenin content showed no sig–nificant positive correlation with altitude while gallic acid showed significant correlation with altitude. However,it is difficult to judge whether this is due to a significant shift in the biosynthetic pathways or whether it is merely a mathematical artifact resulting from the fact that one variable varies with altitude, whereas another does not.

Fig.1Chemical structure of A:Bergenin;B:(+)Catechin;C:Gallic acid;D:Gallicin.

Phenolic compounds exist in glycoside and aglycone forms[2].In most cases,the aglycone form is absorbed at a greater rate and has higher antioxidant activity than the glycoside form[3–6].Hydrolysis of flavonol glyco–sides to their corresponding aglycones offer a practical method for the quantification of flavonoids in foods[7]. Hydrolysis is also used for quantification of phenolic acids in fruits or other plant part[8].We exposed Bergenia extract to different hydrolysis conditions.Fig.3shows concentration variations of the four com–pounds under the different hydrolysis conditions.(+) Catechins are unglycosylated and naturally occur in the aglycone form.Catechin hydrolysis is pH dependent and it decomposes very rapidly in alkaline and neutral solution while remaining stable in acidic condition[9]. Processing methods of catechin rich food and beverages cause catechin degradation;acid hydrolysis may be recommended.Hydrolysis contributes not only to the cleavage of glycosides into their corresponding agly–cones but also prevents their decomposition into other moieties.Bergenin is enriched in the hydrolyzed extract, suggesting that bergenin may be present in the O–glyco–side form.These glycosides are cleaved into aglycone and aglycone moiety in a pH dependent manner.Free aglycone moiety enhances the content of naturally occurring bergenin aglycone content.Hydrolysis is also important in enrichment of biologically active aglycone from their natural glycosides at the industrial level. Gallic acid is simultaneously converted into gallicin (methyl ester of gallic acid)and gallicin into gallic acid. Among all hydrolysis conditions,acid hydrolysis is overall valuable;it prevents decomposition of(+)cate–chin and cleavage of bergenin glycosides into their ber–genin aglycone and simultaneously esterifies gallic acid into gallicin.

Fig.2Altitudinal variation in bergenin,(+)catechin,gallic acid and gallicin

Fig.3Variation in bergenin,(+)catechin,gallic acid and gallicin quantity induced by different hydrolysis conditions

Table 1Geographical details ofBergenia ciliatacollection

Table 2Statistical analysis of calibration curves in HPTLC determination of bergenin,gallic acid,(+)catechin, and gallicin

In conclusion,understanding variations of therapeu–tically active metabolite contents in medicinal plants in nature are for better utilization of natural resources. High bergenin,(+)catechin,gallic acid and gallicin bearing rhizome environmental conditions,which accelerate the biosynthesis of these compounds,need to be conserved for better utilization.Altitudinal varia– tion correlation patterns in more phenolic compounds in a larger population of Bergenia samples needs to be further studied.Neutral hydrolysis is the optimum condition to increase the aglycone content of bergenin in Bergenia ciliata,but this will lead to decomposition of(+)catechin.On the other hand,acid hydrolysis is the best hydrolysis method as it not only increases aglycone content but also prevents decomposition of (+)catechin.

Collection conditions of rhizomes of Bergenia ciliata are shown inTable 1and HPTLC data are provided inTable 2.Detailed methods are available from authors upon request.

Yours Sincerely, CSIR–SRF Nishi Srivastava

Pharmacognosy and Ethnopharmacology Division

CSIR–National Botanical Research Institute;and Chemistry Division, Integral University, Lucknow 226001,

India.

Dr.Sharad Srivastava,Project assistant Shikhar Verma and Dr.Ajay Kumar Singh Rawat Pharmacognosy and Ethnopharmacology Division, CSIR–National Botanical Research Institute, Lucknow 226001, India.

Dr.Abdul Rahman Khan Chemistry Division, Integral University, Lucknow 226001, India.

Tel:91-522-2297816;

Fax:91-522 2207219;

E-mail:pharmacognosy1@rediffmail.com

The authors reported no conflict of interests.

Acknowledgement

The authors thank to the Director Dr.C.S.Nautiyal CSIR–NBRI,Lucknow for his kind support and to make available required resources during this study.We are also thankful to Central Instrument facility(CIF)CSIR–NBRI Lucknow.First author(NS)is thankful to CSIR (New Delhi)for the award of Senior Research fellowship.

[1] Anonymous 1948.The Wealth of India.Raw materials, Council of Scientific and Industrial Research(CSIR) New Delhi 1:179.

[2] Ogbuewu IP,Omedo AA,Chukwuka OK,Iheshiulor, Uehegbu MC,et al.The overview of the chemistry,health benefits and the potential threats associated with prolonged exposure to the dietary soy isoflavones.Int J Agric 2010; 75:23–7.

[3] Izumi T,Piskula MK,Osawa S,Obata A and Tobe K, et al.Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans.J Nutr 2000;130:1695–1699.

[4] Setchell KD,Brown NM,Desai P,Zimmer–Nechemias L, Wolfe BE,Brashear WT,Kirschner AS,Cassidy A and Heubi JE.Bioavailability of pure isoflavones in health humans and analysis of commercial soy isoflavone sup–plements.J Nutrit 2001a;131:1362S–75S.

[5] Chen CY,Holtzman GI,and Bakhit RM.High–genistin isoflavone supplementation modulated erythrocytes anti–oxidant enzymes and increased running endurance in rats undergoing one session of exhausting exercise–A pilot study.Pak J Nutr 2002;1:1–7.

[6] Amadou I,Yong–Hui S,Sun J and Guo–Wei,L. Fermented soybean products:Some methods,antioxidantscompound extraction and their scavenging activity.Asain J Biochem 2009;4:68–76.

[7] Hertog MGL,Hollman PCH,van de Putte,B.Content of potentially anticarcinogenic flavonoids of tea infusions, wines,and fruit juices.J Agric Food Chem 1993;B41: 1242–6.

[8] Lee HS,Widmer BW.Phenolic compounds,In:Nollet LML,ed.Handbook of Food Analysis,New York, USA,Marcel Dekker,Inc.1996;1:821–94.

[9] Su YL,Huang Y,Chen ZY.Stability of tea theaflavins and catechins.Food Chem 2003;83:189–95.

Received 29 November 2013,Revised 26 December 2013,Accepted 07 April 2014,Epub 27 March 2014

?2014 by the Journal of Biomedical Research.All rights reserved.

10.7555/JBR.28.20130190