Chemical Fingerprint Analysis of Two Centella Species, Quantification of Triterpenoids and its Glycosides by using HPTLC Method

Centella asiatica (L.) Urb. (Family Apiaceae commonly known as Gotu Kola or Indian Pennywort) has long been used in the Ayurvedic system of medicine for improving memory and for the treatment of a variety of ailments [1]. The triterpenoid compounds purportedly represent the chief pharmacologically active constituents. The triterpenoids, especially asiticoside, triterpine trisaccharide, are reported as the most active compounds in the plant [2]. A simple and fast method was developed for the quantitative determination of four triterpenes and their glycosides i.e. asiatic acid (AA), madecassic acid (MA), asiaticoside (AS) and madecoside (MS) in Centella asiatica and Centella erecta by using high performance thin layer chromatographic method. The separation was achieved with chloroform: methanol: water: 13.0:6.5:0.5 v/v/v on silica gel ⁶⁰F₂₅₄ HPTLC plates. Quantitation was performed with densitometry in absorption-reflection mode at 600 nm by scanning the HPTLC plates after a color development by anisaldehyde reagent. The linear regression data for the calibration plots showed a good linear relationship with r = 0.999, 0.998, 0.997 and 0.998 for asiatic acid, madecassic acid, asiaticoside and madecoside, respectively. The established method was validated in terms of LOD and LOQ, linearity. Acknowledgements: This research is funded in part by The United States Department of Agriculture Specific Cooperative Research Agreement Number 58-6408-6-067 and the FDA/CFSAN grant entitled Science Based Authentication of Dietary Supplements Number 2 U01 FD 002071-07 References: [1] Shakir JS, et al. (2007), Nat. Prod. Radiance, 6 (2): 158–170. [2] de Paula Reis, et al. (1996), Revista Brasileira de Farmácia, 77(2): 71–72.

Fig. 1 (A) Centella asiatica, (B) Standard mix, (C) Centella erecta.

Table 1 Validation Parameters.

	Parameter	AA	MA	AS	MS
1	Linearity range (ng/spot)	200–600	200–600	100–500	100–500
2	Correlation coefficient	0.999	0.998	0.997	0.998
4	LOD (ng/spot)	30	60	30	30
5	LOQ (ng/spot)	180	200	100	100
6	Specificity	Specific	Specific	Specific	Specific
7	Regression equation	Y=94.580 +8.961 X	Y=61.937 +3.124 X	Y=22.600 +0.495 X	Y=12.773 +0.113 X
8	Rf	0.72	0.61	0.17	0.11

Table 2 Percentage (w/w) of asiatic acid, madecassic acid, asiticoside, and madecoside in plant sample.

Sample name (Percentage in dry plant material)	AA	MA	AS	MS
C. asiatica	0.2	0.2	3.6	2.0
C.erecta	0.1	0.1	4.5	3.1