A Novel Triterpene from Centella asiatica

Abstract

A novel triterpene, 2α,3β,20,23-tetrahydroxyurs-28-oic acid (1), was isolated from the aerial part of Centella asiatica. Its structure was elucidated by spectroscopic methods, including 2D-NMR spectra. It displayed no activity against Hela and A549 cell lines at concentrations of 10 and 30 μg/mL, respectively.

Introduction

The perennial subshrub Centella asiatica (L.) Urban (Umbelliferae/Apiaceae family, commonly known as Gutu kola, Asiatic pennywort, Indian pennywort or Spadeleaf) has been widely cultivated as a vegetable or spice in China, Southeast Asia, India, Sri Lanka, Africa and Oceania. It has been used in Southeast Asia for the treatment of a wide variety of afflictions such as skin diseases, rheumatism, inflammation, syphilis, mental illness, epilepsy, hysteria, dehydration and diarrhea [1]. It was also used in Europe for treatment of wounds and ulcers. Earlier work on this plant has led to the isolation of more than 70 constituents, such as triterpenoid saponins [2,3,4], polyacetylenes [5], flavones [6], sterols and lipids [7]. A systematic study of the chemical constituents and antitumor activities of C. asiatica led us to isolate a new urs-type triterpene compound 1, together with ten known compounds, namely asiatic acid, madecassic acid, indocentoic acid, bayogenin, kaempferol, quercetin, euscaphic acid, terminolic acid, 3β-6β-23-tri-hydroxyolean-12-en-28-oic acid, and 3β-6β-23-trihydroxyurs-12-en-28-oic acid. This paper deals with the structural elucidation of the new triterpene 1.

Results and Discussion

Compound 1 was a white powder, +26.6 (c 0.1, MeOH). Its HRFABMS showed a [M-H₂O]⁺ peak at m/z 489.7028, corresponding to the molecular formula C₃₀H₅₀O₆ (calcd. 489.7033). Its IR spectrum showed absorption bands at 3433 and 1722 cm^-1, ascribable to hydroxyl and carboxyl functions, respectively. ¹³C- and DEPT 135°NMR spectra showed six signals for Me carbons, ten methylenes, seven methines, and six quaternary carbons, together with a carboxyl group. A total of 30 carbon resonances were observed, which confirmed its triterpenic nature. The following NMR data suggested the structural features of urs-28-oic acid for compound 1: a methyl doublet (δ 1.03, d, J = 6.9 Hz, Me-29), and the carbonyl carbon resonance at δ 180.1 (C-28). The spectrum also showed signals at δ 3.68 and 3.34 (J = 9.4 Hz) ascribable to the 2β- and 3α-protons on carbons bearing a hydroxyl function, respectively. An AB doublet, δ 3.50 (J = 11.0 Hz) and 3.26 (J = 11.0 Hz), indicated the presence of a –CH₂OH function. The chemical shifts of C-4 and Me-24 led to placement of the -CH₂OH at the C-23 position. The A and B ring proton and carbon signals matched those reported for asiatic acid (2α,3β,23-trihydroxyurs-12-en-28-oic acid) [8], but the ¹H-NMR spectrum of 1, compared with that of asiatic acid, lacked a methyl doublet (Me-30) and contained a signal corresponding to a methyl singlet at δ 1.32 in the ¹H-NMR, as well as a quaternary hydroxylated carbon (δ 86.2) in the ¹³C-NMR spectrum. The carbon signals of the E ring were in agreement with those reported for 3β-O-(β-D-xylopyranosyl-(1-3)-α-L-arabinopyranosyl)-2α,20β,23-trihydroxyurs-12-en-28-O-[β-D-glucopyranosyl-(1-6)-β-D-glucopyranosyl] ester [9]. The ¹H- and ¹³C-NMR spectra were completely assigned by detailed 2D-NMR experiments (Table 1), which showed the HMBC correlations between H-30 and C-19, C-20, C-21, H-29 and C-18, C-19, C-20, H-3 and C-2, C-4, C-23, C-24. NOESY correlation of H-2 and H-25, H-3 and H-23 further corroborated the above conclusions. In summary, compound 1 was identified as 2α,3β-20,23-tetrahydroxyurs-28-oic acid (Figure 1).

Figure 1. Structure of Compound 1.

Figure 1. Structure of Compound 1.

Table 1. ¹H-NMR (300 MHz), and ¹³C-NMR (75 MHz) data of 1 (CD₃OD, TMS, δ ppm).

Carbon No.	δH	δC
1		48.3 (t)
2	3.68 (1H, m)	70.0 (d)
3	3.34 (1H, d, J = 9.4 Hz)	78.2 (d)
4		44.2 (s)
5		48.5 (d)
6		19.1 (t)
7		34.6 (t)
8		41.7 (s)
9		51.9 (d)
10		39.3 (s)
11		22.4 (t)
12		28.6 (t)
13		44.5 (d)
14		42.4 (s)
15		28.1 (t)
16		33.2 (t)
17		49.7 (s)
18		49.5 (d)
19		43.5 (d)
20		86.2 (s)
21		28.5 (t)
22		26.4 (t)
23	3.50 (1H, d, J = 11.0 Hz)	66.3 (t)
	3.26 (1H, d, J = 11.0 Hz)
24	0.67 (3H, s)	13.8 (q)
25	0.95 (3H, s)	18.5 (q)
26	0.96 (3H, s)	16.3 (q)
27	0.98 (3H, s)	14.7 (q)
28		180.1 (s)
29	1.03 (3H, d, J = 6.9 Hz)	19.0 (q)
30	1.32 (3H, s)	24.4 (q)

Table 1. ¹H-NMR (300 MHz), and ¹³C-NMR (75 MHz) data of 1 (CD₃OD, TMS, δ ppm).

Carbon No.	δH	δC
1		48.3 (t)
2	3.68 (1H, m)	70.0 (d)
3	3.34 (1H, d, J = 9.4 Hz)	78.2 (d)
4		44.2 (s)
5		48.5 (d)
6		19.1 (t)
7		34.6 (t)
8		41.7 (s)
9		51.9 (d)
10		39.3 (s)
11		22.4 (t)
12		28.6 (t)
13		44.5 (d)
14		42.4 (s)
15		28.1 (t)
16		33.2 (t)
17		49.7 (s)
18		49.5 (d)
19		43.5 (d)
20		86.2 (s)
21		28.5 (t)
22		26.4 (t)
23	3.50 (1H, d, J = 11.0 Hz)	66.3 (t)
	3.26 (1H, d, J = 11.0 Hz)
24	0.67 (3H, s)	13.8 (q)
25	0.95 (3H, s)	18.5 (q)
26	0.96 (3H, s)	16.3 (q)
27	0.98 (3H, s)	14.7 (q)
28		180.1 (s)
29	1.03 (3H, d, J = 6.9 Hz)	19.0 (q)
30	1.32 (3H, s)	24.4 (q)

Biological Activity

The EtOH extract of C. asiatica and the individual compounds were screened for anti-cancer activity against Hela and A549 cell lines. The MTT method was used to determine cytotoxic activity. No activity was observed at concentrations of 10 and 30 μg/mL, respectively.

Experimental

General

NMR spectra were run on a Bruker AVANCE 300 instrument using TMS as internal standard. MS data was obtained on a JEOL JMS D-300 instrument. Column chromatography was performed on silica-gel (Qingdao Haiyang Chemical Co., Ltd), and Toyopearl HW-40 (Tosoh). The HPLC instrument was a JASCO Gulliver Series equipped with a PU-1580 (pump), RI-1530 and UV-1575 (detectors). Semi-Preparative HPLC was performed using a YMC-Pack ODS-A, SH-343-5 column. IR spectra were recorded on a Nicolet 380 FT-IR spectrophotometer (Thermo Electron Corporation). Optical rotation was measured with a MC 241 digital polarimeter (Perkin-Elmer).

Plant material and product isolation

Aerial parts of C. asiatica were collected in September 2003, in Hebei province, P.R. China. A voucher specimen, identified by Dr. Wen-Yuan Gao, was deposited under registration No. TJU-03928 at the herbarium of the Department of Natural Products and Traditional Chinese Medicine, Tianjin University. The plant material (3 kg) was refluxed three times with 95% EtOH. The extract was concentrated under reduced pressure to give a residue (700 g) which was partitioned between ethyl acetate and H₂O. The EtOAc extract (160 g) was chromatographed on a silica gel column with an eluent of increasing polarity and eluates of similar composition, according to TLC analysis, were pooled to yield 19 fractions. Fraction 16 (7.7 g, R_f = 0.5, eluted with 9:1 CHCl₃-MeOH) was chromatographed on Toyopearl HW-40, and then further purified by reverse phase HPLC (8:2 MeOH-H₂O) and GPC (MeOH) to give compound 1 (6 mg).