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14Chemotypic Variation among Different

Accessions of Centella asiatica (L.)Urban from Central Zone of India and

Strategies for their ConservationSharad Srivastava, Shikhar Verma, Abhishek Gupta and

A.K.S. Rawat

ABSTRACT

Chemical diversity among the plant species provides great opportunityfor identification of new and elite sources for further bio-prospection andalso plays an important role in plant conservation. Centella asiatica (L.) Urban(Apiaceae) is claimed to possess various healing effects and antioxidantproperties. However, there has been very less focus on investigation ofchemotypic variations of C. asiatica found in different geographical zones ofthe country. In order to conserve elite accessions of C. asiatica as it is anindustrially valuable herb and over exploited from wild, thus, differentdistinct accessions of C. asiatica from central zones of India were comparedin relation to the levels of triterpenoid saponins. Physico-chemical parameterswere evaluated in all the accessions. The metabolites investigated includemadecassoside, asiaticoside and its sapogenin, asiatic acid. The threetriterpenoids of interest were analyzed and quantified by HPTLC. Acomparison and evaluation of the triterpenoid content in these accessions

Natural Products: Recent Advances Pages 276–285Editors: Ashok K. Chauhan, Palpu Pushpangadan and Varughese GeorgePublished by: Write & Print Publications

Chemotypic Variation among Different Accessions of Centella asiatica (L.) 277

are reported. The highest concentration of asiaticoside and madecassosidewas found in the CA-15 accession that was asiaticoside (1.35 per cent),madecassoside (2.15 per cent) and asiatic acid was found higher in CA-17(0.29 per cent). It can be concluded that the geographical conditions (soiltype and altitude) of CA-15 and CA-17 are comparatively favourable forproduction of higher levels of triterpenoid saponins in C. asiatica. Thereported data will contribute to the establishment of knowledge about thetriterpenoid saponin composition of different chemotypes of C. asiatica foundin central zones of India in comparison to other geographical areas, andlays a foundation for the conservation of this plant from over exploitation.

Keywords: Asiatic acid, Asiaticoside, Madecassoside, Chemotypes,Centella asiatica, HPTLC, Conservation

Introduction

Chemical diversity and its role in taxonomy and plant biodiversityconservation have much relevance and plays important role in planningfuture strategies for their bioprospection. Traditional knowledge and theirunderstanding are still very limited. Further many plants and animals aresteadily becoming extinct through the influence of humans interventions(Dung et al. 1995). Taxonomy is a cornerstone discipline for the delineationand protection of biotic diversity (Lozano et al. 2007). Centella asiatica (L.)Urban belongs to the family of Apiaceae (Umbelliferae) and in India,commonly known as ‘Mandukaparni’. In classical Indian Ayurvedic literature,it is considered to be one of the best ‘Rasayana’ (rejuvenator) drugs (Jayashreeet al., 2003). It has been reported for treatment of ulcers and vein diseases(Brinkhaus 2000), memory improvement (Rao et al., 2005), wound healing(Maquart et al., 1999), antidepressant (Chen et al., 2003). Based on thenumerous studies, the biologically active ingredients are believed to be itstriterpenes (Srivasta et al., 1997) and the medicinal values of this plant aremainly attributed to the presence of several triterpenes, like asiatic acid,madecassic acid, asiaticoside and madecassoside (Ling et al., 2000; Zhengand Qin, 2007). Quantification of triterpenes of C. asiatica has been successfullyestablished by several researchers using TLC and HPTLC (Zainol et al.,2008; James and Dubery, 2011). However, the triterpene components inC. asiatica are known to vary depending on its growth location and thediverse environmental conditions it is subjected to (James and Dubery,2009). This study was done to investigate the behaviour of active componentsin different accessions commercially grown in central zones of India.Analytical studies have shown that C. asiatica contains triterpenoids, essentialoils and amino acids. The plant contains asiaticoside, centelloside,madecassoside, brahmoside, brahminoside, thankuniside, centellose, andterminolic, asiatic, brahmic, centic, centoic, centellic and madecassic acids(Jamil et al., 2007). Previous chemotype studies have been performed on

278 Natural Products: Recent Advances

accessions from south India and Andaman Islands (Thomas, 2010). The aimof this study was to reveal the elite chemotypes collected from untouchedgeographical zones of central India. Precise HPTLC analysis was performedfor the estimation of triterpenoid saponins madecassoside, asiaticoside andits sapogenin, asiatic acid.

Materials and methods

Asiatic acid, asiaticoside and madecassoside were procured from SigmaAldrich. All other chemicals used were of analytical grade.

Plant Material

Different accessions i.e. whole plants of C. asiatica were collected fromits natural habitat from different locations of central zone of India whereit was available in abundance. The plant was authenticated by Dr. AKSRawat, NBRI, Lucknow. A voucher specimen of each has been submittedin Institute’s repository.

Preparation of Plant Extracts

The collected plant material were thoroughly washed with water toremove all debris and then shade dried, the dried material was thenpowdered using electric grinder at 100 mesh size. Extraction was performedby soxhlation process in two steps. Firstly, the powdered material wasdefatted under soxhlet assembly using 250mL of 98 per cent ether for 6hours. This is followed by 9 hours soxhlation of defatted powder by using250mL of methanol as solvent. The final extracts were passed throughWhatman No. 1 filter paper. The filtrates obtained were concentrated undervacuum in a rotary evaporator at 40 °C and stored at 4 °C for further use.

Physicochemical and Phytochemical Studies

Physicochemical and Phytochemical studies like extractive values, totalash, acid insoluble ash, total sugar, starch, tannin, and phenols werecalculated from the shade-dried and powdered (60 mesh) plant material(Peach and Tracy, 1955; Anonymous, 2007; Anonymous, 2004).

HPTLC Studies

Extract prepared above were redissolved in methanol, filtered andfinally made up to100 ml with methanol prior to HPTLC analysis. Reagentsused were from Merk (Germany) and standard asiatic acid, asiaticosideand madecassoside was procured from Sigma-Aldrich (Steinheim).

Chromatographic Conditions

Chromatography was performed on Merk HPTLC precoated silica gel60GF

254 (10X10 cm) plates. Methanolic solution of samples and standard


compounds asiatic acid, asiaticoside and madecassoside of knownconcentrations were applied to the layers as 6 mm-wide bands positioned10 mm from the bottom and 15 mm from side of the plate, using CamagLinomat V automated TLC applicator with nitrogen flow providing a deliveryspeed of 150nl/s from application syringe. These conditions were keptconstant throughout the analysis of samples. Following sample application,layers were developed in a Camag twin trough glass chamber which waspre-saturated with mobile phase of toluene: ethyl acetate: formic acid (5:5:1)for asiatic acid and n-butanol: ethyl acetate: water (4:1:5) for asiaticosideand madecassoside till proper separation of bands up to 8 cm height. Afterdevelopment, layers were dried with a hair dryer. Asiatic acid, asiaticosideand madecassoside were simultaneously quantified using Camag TLCscanner model 3 equipped with Camag Wincats IV software. Followingscan conditions were applied: slit width, 6 mm x 0.45 mm; wavelength 600nm; and absorption-reflection mode. In order to prepare calibration curvesof peak area vs. concentration, stock solution of asiatic acid, asiaticosideand madecassoside (0.1 μg/mL) were prepared and various volumes of thesolution were analyzed through HPTLC.

Results

Phytogeographical studies

Altitude and soil type of the collection sites of these accessions wererecorded in passport data sheet and details are summarized (Table 14.1;Figs. 14.1 and14.2).

Fig. 14.1. Distribution pattern of Centella asiatica in Central zone


Table 14.1. Passport data sheet of collected accession from central India

Collection No.

Collection Site

District & State

Alt.

(Mtr)

Latitude Longitude Associated species

CA--12 Jabalpur Jabalpur &

Madhya Pradesh

411 23° 10' N 79° 59' E Hemidesmus indicus Oxalis

CA-13 Bhedaghat Jabalpur &

Madhya Pradesh

411 23.1.N 79.8E Hemidesmus indicus

Oxalis; Blumea

CA-15 Amarkantak forest

Anuppur

Madhya Pradesh

1060.70 20.5937N 78.9629 Oxalis; Rumex sp

Convolvulus sp.

CA-16 Amarkantak river side

Anuppur

Madhya Pradesh

1060.70 20.5937N 78.9629 Oxalis Rumex sp

Convolvulus sp.

CA-17 Panchamarahi

(bee fall)

Hosangabad &

Madhya Pradesh

1000 22° 28' 0" N

78° 26' 0" E Hemidesmus indicus

Oxalis

CA-18 Panchamarahi

(forest)

Hosangabad &

Madhya Pradesh

1000 22° 28' 0" N

78° 26' 0" E Hemidesmus indicus

Oxalis Rumex sp

CA-19 Tamia Chhindwara &

Madhya Pradesh

364.84 22.9734N 78.6569 Hemidesmus indicus

Oxalis

CA-20 Mandla Mandla & Madhya Pradesh

268 22º 2'- 23º 22'N

80º 18'- 81º 50'

E

Oxalis sp. Rumex sp


Fig. 14.2: Different accessions of C. asiatica from central zone of India

CA-16

CA-12 CA-13

CA-1

CA-1

CA-15

CA-18-CA 17

CA-1

CA-19 CA-20


Physicochemical Studies

Parameters such as extractive values (Water and alcohol soluble), totalash, acid insoluble ash, total sugar, starch, and tannins and phenolics weredetermined. Total phenol was found in higher concentrations in CA-18,CA-19 and CA-20. Total Tannins was higher in CA-17 (1.95 per cent). Totalsugar was higher in CA-17 (4.78 per cent). Total starch was higher in CA-20 (9.5 per cent). Results are shown in Figs. 14.3 and 14.4.

Fig. 14.3. Extractive and Ash Values

Fig. 14.4. Total Sugar, Starch, Tannin and Phenol


HPTLC Studies

The calibration plots were obtained after densitometric analysis of thethree authentic standards i.e., asiatic acid, asiaticoside and madecassoside.Quantification of asiatic acid, asiaticoside and madecassoside in the samplesof C. asiatica has been performed and shown in Table 14.2. HPTLC bandingpattern and chromatograms obtained from extracts are shown in Fig. 14.5to 14.8.

Table 14.2. Quantitative analysis of different markers in C. asiatica samples fromcentral zone of India ( per cent in dry wt.)

AccessionNo.

Collection Place

Altitude (m)

Soil Type

Madeca- ssoside

Asiat- icoside

Asiatic acid

CA-12 Jabalpur 411 Lateritic 0.27±0.03 0.14±0.01 0.06±0.01

CA-13 Bedhaghat 623 Lateritic 0.74±0.02 0.45±0.02 0.12±0.02

CA-15 Amarkantak 1065 Black Soil 2.15±0.21 1.35±0.03 0.19±0.02

CA-16 Amarkantak 1083 Black Soil 1.02±0.06 0.13±0.01 0.12±0.04

CA-17 Panchmarhi 1000 Red Soil 1.24±0.04 0.26±0.02 0.29±0.01

CA-18 Panchmarhi 985 Red Soil 1.46±0.08 1.01±0.04 0.27±0.03

CA-19 Tamia 870 Black Soil 1.67±0.05 1.07±0.04 0.03±0.00

CA-20 Mandala 443 Lateritic 1.79±0.11 0.54±0.03 0.03±0.01

Note: Each percentage value is an average of six values ± SD

Fig. 14.5: HPTLC profile of C.asiatica extracts with asiatic

acid

Fig. 14.6: HPTLC chromatograms ofC. asiatica extracts with asiatic acidSpectra from top: CA-19; CA-20; CA-12; CA-17; Asiatic acid; CA-15; CA-

16; CA-18; CA-13


Fig. 14.7: HPTLC profile of C.asiatica extracts with asiaticoside

and madecassoside

Fig. 14.8: HPTLC chromatograms of C.asiatica extracts with asiaticoside and

madecassosideSpectra from top: Madecassoside; CA-19; CA-20; CA-12; CA-17; Asiaticoside;

CA-15; CA-16; CA-18; CA-13Conclusion

Quantificaton of asiatic acid, asiaticoside and madecassoside present indifferent accessions of C. asiatica, has been studied through HPTLC (Table14.2). From the above studies it is concluded that ecological factors such assoil type (black soil) of CA-15 are comparatively favourable for biosynthesisof asiaticoside and madecassoside while asiatic acid was found in higherconcentrations in CA-17 with red soil. Thus, it can be easily depicted thatthe ecological factors such as soil type and altitude play an important rolein the biosynthesis of triterpenoidal saponins in C. asiatica. It is worth tomention here as an observation that accessions of C. asiatica growing inshade flourished well as compared to those that were grown under directsunlight. The reported data will contribute to the knowledge about thetriterpenoid saponin composition of different chemotypes of C. asiatica foundin central zones of India in comparison to other geographical areas, andlays a foundation for conservation of this plant from overexploitation.

Acknowledgements

The authors are thankful to Director, CSIR-NBRI for providing all thefacilities (under OLP-0077) to conduct this research work. Authors are alsothankful to CSIR for financial grant under CSIR-EMPOWER scheme.

Conflict of Interest

The authors declare no conflict of interest

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