New Triterpenoids with Cytotoxic Activity from Actinidia Valvata

Abstract

Two new triterpenoids, 30-O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (1) and 2α,3β,3,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (2) were isolated from roots of Actinidia valvata Dunn. Their structures were elucidated by means of extensive spectroscopic studies. Both these two new compounds showed moderate cytotoxic activity in vitro against BEL-7402 and SMMC-7721 tumor cell line.

1. Introduction

Actinidia valvata Dunn is a shrub mainly growing in eastern China (Zhejiang and Jiangxi province) [1]. Its roots known as “mao ren shen” in traditional Chinese medicine exhibit anti-tumor and anti-inflammatory activity, and have been used for many years in the treatment of hepatoma, lung carcinoma and myeloma [2,3]. As we all know, hepatoma is very difficult to treat, and active components from medicinal herbs may be effective for research and development of new drugs [4]. In a previous study, we have carried out screening for cytotoxic activity of “mao ren shen”, and two new polyoxygenated triterpenoids, 2β,3α,6α,20α,24,30-hexahydroxyurs-12-en-28-oic acid and 2β,3α,20β,23,24,30-hexahydroxyurs-12-en-28-oic acid O-β-d-glucopyranosyl ester were separated [5]. In this paper, two new triterpenoid saponins with cytotoxic activity against BEL-7402 and SMMC-7721 tumor cell line are reported.

2. Results and Discussion

The roots of Actinidia valvata Dunn were extracted with 80% EtOH. The concentrated extract was suspended in H₂O and successively extracted with petroleum ether (60°–90°), AcOEt, and n-BuOH. The n-BuOH-soluble extract was repeatedly subjected to column chromatography to yield compound 1,2Figure 1. Both these compounds were triterpenoid saponins with 12,18-diene-urs skeleton. Their structures were elucidated by detailed spectroscopic analysis.

Compound 1 was a white amorphous powder, displayed positive Liebermann–Burchard test, was optically active with [α]_D²⁵ = 5.89 (c = 0.1, MeOH), and had the molecular formula C₄₂H₆₆O₁₆, with ten degrees of unsaturation, as determined according to a pseudo-molecular-ion peaks at 849.4240 ([M + Na]⁺; calc. 849.4249) in the HR-ESI-MS.

¹³C-NMR (DEPT) spectra of compound 1 revealed 42 carbon signals, including five CH₃, two C=C bonds (tri-, four-substituted) and one C=O group. Assuming compound 1 has skeleton of urs-triterpenoid, the assignment of the two C=C bonds should be highly concerned. In ¹H-NMR spectra of compound 1, five singlets at δ(H) 0.80–1.89 consisting of five CH₃, and proton signal at δ(H) 5.53 (br) was assigned to 12-position. In HMBC spectra, clear correlation of 19–CH₃ with two quaternary C-atoms at δ(C) 126.17 (s) and δ(C) 136.92 (s) was observed, and the correlation signals of 12-H with these two quaternary C-atoms were also observed. Thus, the two C=C bonds can be rightly assigned to 18-, 19-position, respectively. The HMBC correlation signal of 20-H with C-atom at δ(C) 69.68(t), was observed, then the oxygenation of 30-C may be deduced successfully. In NOSEY spectra, as the correlations of 2-H with 3-H, 2-H with 10-Me, 8-Me with 10-Me were observed, and by comparing with reference data [6,7], the configuration of 2α-OH, 3α-OH, and the 24-OH can be confirmed. ¹H-NMR and ¹³C-NMR data of glycon moiety of compound 1 indicated it featured two glucopyranosyls. The C-atom at δ(C) 95.82(d) and H (5.39, d, J = 8.0 Hz), C-atom at δ(C) 101.85(d) and H (4.20, d, J = 8.0 Hz) were assigned as anomeric C-atoms and prontons, respectively. The linkage of two glucopyranosyls with aglycone maybe deduced by correlation of anomeric pronton with glycosidated C-atoms in HMBC spectra [H (5.39, d, J = 8.0 Hz) to C=O, H (4.20, d, J = 8.0 Hz) to C-atom at 69.68(t)].

Basing on above analysis, in combination with the ¹H-, ¹³C-NMR spectra (

Table 1. ¹H and ¹³C-NMR Data of 1 and 2 (in C₅D₅N). δ in ppm, J in Hz.

Position	1		2
	δ(C) a	δ(H) b	δ(C) a	δ(H) b
1	42.81 (t)	1.31 (dd, J = 12,4.2), 2.19 (dd, J = 12,4.2)	48.03 (t)	1.30 (m), 2.29 (m)
2	66.98 (d)	3.88 (m)	68.93 (d)	4.25 (m)
3	74.90 (d)	3.70 (d, J = 2.0)	78.18 (d)	4.19 (m)
4	45.36 (s)	—	43.61 (s)	—
5	49.94 (d)	1.79 (m)	48.31 (d)	1.73 (m)
6	19.15 (t)	1.33 (m)	18.41 (t)	1.04 (m), 1.15 (m)
7	33.23 (t)	1.28 (m), 1.30 (m)	33.86 (t)	1.31 (m), 1.75 (m)
8	40.67 (s)	—	39.80 (s)	—
9	48.96 (d)	1.71 (m)	48.03 (d)	1.83 (m)
10	39.07 (s)	—	38.31 (s)	—
11	24.25 (t)	1.74 (m), 1.93 (m)	23.64 (t)	1.74 (m), 2.01 (m)
12	129.19 (d)	5.53 (br)	127.98 (d)	5.60 (br)
13	137.97 (s)	—	137.53 (s)	—
14	44.69 (s)	—	43.90 (s)	—
15	29.09 (t)	1.09 (m), 1.88 (m)	28.58 (t)	1.06 (m), 2.25 (m)
16	24.72 (t)	1.74 (m), 1.98 (m)	32.97 (t)	1.79 (m), 1.98 (m)
17	48.83 (s)	—	47.25 (s)	—
18	126.17 (s)	—	129.59 (s)	—
19	136.92 (s)	—	131.27 (s)	—
20	51.45 (s)	3.30 (br)	50.74 (d)	3.55 (br)
21	24.67 (t)	1.97 (m)	23.87 (t)	1.99 (m)
22	35.19 (t)	1.28 (m), 1.53 (m)	24.41 (t)	2.06 (m)
23	22.56 (q)	1.07 (s)	66.52 (t)	3.66 (d, J = 10.2), 4.16 (d, J = 10.2)
24	65.72 (t)	3.36 (d, J = 8.0), 3.68 (d, J = 8.0)	14.42 (q)	1.03 (s)
25	17.86 (q)	0.94 (s)	17.95 (q)	1.08 (s)
26	18.21 (q)	0.85 (s)	18.27 (q)	1.15 (s)
27	23.17 (q)	1.89 (s)	22.23 (q)	0.97 (s)
28	178.07 (s)	—	176.24 (s)	—
29	17.44 (q)	1.68 (s)	16.83 (q)	1.75 (s)
30	69.68 (t)	4.09 (d, J = 20.0), 4.45 (d, J = 20.0)	62.20 (t)	4.38 (d, J = 10.2)
28-glc-1	95.82 (d)	5.39 (d, 8)	95.82 (d)	5.60 (d, J = 8.0)
2	73.75 (d)	3.31 (m)	74.19 (d)	3.31 (m)
3	77.91 (d)	3.33 (m)	78.84 (d)	3.33 (m)
4	71.46 (d)	3.33 (m)	71.12 (d)	3.33 (m)
5	78.54 (d)	3.38 (m)	79.25 (d)	3.38 (m)
6	62.49 (t)	3.64 (dd, J = 12.0,1.8), 3.77 (dd, J = 12.0, 1.8)	62.99 (t)	3.65 (dd, J = 12.0, 1.8), 3.78 (dd, J = 12.0, 1.8)
30-glc-1	101.85 (d)	4.20 (d, J = 8.0)
2	74.53 (d)	3.31 (m)
3	77.83 (d)	3.33 (m)
4	70.97 (d)	3.33 (m)
5	77.95 (d)	3.38 (m)
6	62.30 (t)	3.63 (dd, J = 12.0, 1.8), 3.76 (dd, J = 12.0, 1.8)

^aRecorded at 150 MHz, multiplicity by DEPT;

^bRecorded at 600 MHz.

), HMQC, HMBC, and NOSEY data (Figure 2), established the structure of compound 1 as 30-O-β-d-glucopyranosyloxy- 2α,3α,24-trihydroxyurs-12,18-diene-28-oic acid O-β-d-glucopyranosyl ester (1).

The compound 2 was obtained as white amorphous powder, displayed positive Liebermann–Burchard test, was optically active with [α]_D²⁵ = 7.35 (c = 0.1, MeOH), and had the molecular formula C₃₆H₅₆O₁₁ with nine degrees of unsaturation, as determined according to a pseudo-molecular-ion peaks at 687.3717 ([M + Na]⁺; calc. 687.3720) in the HR-ESI-MS.

Comparing the ¹H-NMR and ¹³C-NMR (Table 1) spectra, compound 2 has only a 28-glucopyranosyl. The configuration of 2α-OH, 3β-OH, and 23-OH can be confirmed by analyzing the correlations of NOSEY spectra and comparing with reference data [6,7]. In combination with the ¹H-, ¹³C-NMR spectra (Table 1), HMQC, HMBC, and NOSEY data (Figure 2), established the structure of compound 2 as 2α,3β,23,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (2).

Compounds 1 and 2 showed moderate in vitro cytotoxic activity against BEL-7402 (IC₅₀ value of 92.2 and 89.7 μg/mL, resp.) and SMMC-7721 (IC₅₀ value of 58.1 and 89.7 μg/mL, resp.), as determined by classical MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) colorimetric assay.

3. Experimental Section

3.1. General

Silica-gel plates (Sinopharm Chemical Reagent Co., Ltd.) were used for TLC analysis. mp: WRS-1A micro-melting-point apparatus; uncorrected. Optical rotations: JASCO P-1300 spectropolarimeter. IR: Spectra: BRUKER VECTOR-22 spectrophotometer; in cm^{−1. 1}H-, ¹³C-, 2D-NMR spectra: BRUKER AVANCE 600 spectrometer; chemical shifts δ in ppm rel. to (CH₃)₄Si, coupling constant J in Hz. ESI-MS: Finnigan LCQ mass spectrometer; in m/z. HR-ESI-MS: Q-Tof micro YA019 mass spectrometer.

3.2. Material

The roots of Actinidia valvata Dunn were collected in Changshan County, Zhejiang Province, China, in October 2006, and identified by Zheng Han-Chen, Department of pharmacognosy, School of pharmacy, Second military medical university. A voucher specimen (No. 20061005) was deposited at Department of pharmacognosy, School of pharmacy, Second military medical university.

3.3. Extraction and Isolation

The powdered plant material of roots of Actinidia valvata Dunn 30 kg was refluxed with 8 times of 80% EtOH solution for 3 times, 1.5 h each time. The extract was concentrated under reduced pressure to brown syrup, which was partitioned between H₂O and petroleum ether (PE), AcOEt, and BuOH, successively. The n-BuOH soluble fraction (280.6 g) was subjected to column chromatography (CC) on silica gel (SiO₂), eluting with CHCl₃/MeOH/H₂O (10:1:0.1 to 2:1 0.1) to afford 9 fraction 1–9. Fraction 5 were repeatedly subjected to CC (Pharmadex LH-20 and RP C-18) to yield compound 1 (10.4 mg) and 2 (14.6 mg).

30-O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (1): white amorphous powder, mp 140°–142°, [α]_D²⁵ = 5.89 (c = 0.1, MeOH). IR (KBr): 3432, 2920, 2852, 1641, 1380, 1038. ¹H-NMR (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N): Table 1. ESI-MS: 849.48 ([M + Na]⁺), HR-ESI-MS: 849.4240 ([M + Na]⁺, C₄₂H₆₆N_aO₁₆⁺, calc. 849.4249).

2α,3β,23,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (2): white amorphous powder. mp 220° (carbonification), [α]_D²⁵ = 7.35 (c = 0.1, MeOH). IR (KBr): 3448, 2963, 1681, 1644, 1381, 1278, 1080. ¹H-NMR (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N): Table 1 ESI-MS: 687.89 ([M + Na]⁺), HR-ESI-MS: 687.3717 ([M + Na]⁺, C₃₆H₅₆N_aO₁₁⁺, calc. 687.3720).

4. Conclusions

In the present research, two new triterpenoids, 30-O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester and 2α,3β,23,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester were isolated from roots of Actinidia valvata Dunn, and their structures were elucidated by means of extensive spectroscopic studies. Both these new compounds showed moderate cytotoxic activity in vitro against BEL-7402 and SMMC-7721 tumor cell line.