A New Triterpenoid Saponin from Abrus precatorius Linn

Abstract

A new triterpenoid saponin, 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl subprogenin D (1), together with six known triterpenoids: subprogenin D (2), abrusgenic acid (3), triptotriterpenic acid B (4), abruslactone A (5), abrusogenin (6) and abrusoside C (7) were isolated from the leaves and stems of Abrus precatorius. Their structures were elucidated on the basis of physical and NMR analysis, respectively. Compounds 5 and 6 showed moderate cytotoxicity against MCF-7, SW1990, Hela, and Du-145 cell lines. Compounds 1, 2 and 4 were isolated from this plant for the first time.

1. Introduction

Abrus precatorius Linn belongs to the family Leguminosae. Its seeds, known as Xiang-si-zi, have been used in China as an insecticide and for treatment of some skin diseases since ancient times [1]. Besides, the leaves and roots are sweetish and traditionally used to cure fever, stomatitis, asthma and bronchititis [2]. Several groups of biologically active secondary compounds including alkaloids [3], flavones [4], triterpenoids [5] and isoflavano-quinones [6] have been isolated from this plant, some of which possess anti-inflammatory [7], antibiosis [8], antiplatelet [9], and anti-implantation [10] properties. In our research on bioactive compounds from Abrus precatorius collected from the mangrove wetlands of Hainan Island, China, a new 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl subprogenin D (1), as well as six known ones (compounds 2–7) were obtained. The structure of the new compound was elucidated using 1D, 2D NMR and MS experiments, while the configuration of 1 was defined by NOESY spectroscopy. Compounds 2–7 were identified as subprogenin D (2) [11], abrusgenic acid (3) [12], triptotriterpenic acid B (4) [13], abruslactone A (5) [14], abrusogenin (6) [15] and abrusoside C (7) [15], respectively, by comparison of their spectroscopic data with those reported in the literature. Compounds 1, 2 and 4 were isolated from this plant for the first time.

2. Results and Discussion

The aqueous EtOH extract of the Abrus precatorius was suspended in water, and then partitioned with petroleum ether, EtOAc, and n-butanol by liquid-liquid extraction respectively. The EtOAc and n-butanol fractions were successively subjected to repeated silica gel column, Sephadex LH-20 to yield compounds 1–7 (Figure 1).

Figure 1. Structures of compounds 1–7.

Figure 1. Structures of compounds 1–7.

Compound 1 had the molecular formula C₄₂H₆₆O₁₄ as deduced from HRESI-MS m/z 817.4345 [M+Na]⁺ (calcd for C₄₂H₆₆O₁₄Na, 817.4350) and NMR data (Table 1). The IR spectrum exhibited absorption bands at 3458 (OH), 1727(C=O), 1693(C=O) and 1624 (C=C) cm^–1. Seven methyl groups (δ_H 1.42, 1.32, 1.27, 1.23, 1.14, 0.91 and 0.86), one oxygenated methine proton (δ_H 3.29, dd, J = 5.0, 11.0 Hz), and one olefinic proton (δ_H 5.31, br s) were observed in the ¹H-NMR spectrum. The ¹³C-NMR and DEPT data confirmed the presence of seven methyl carbons (δ_C 28.1, 25.5, 21.6, 20.9, 16.8, 16.7, 15.6), two olefinic carbons (δ_C 124.7, 141.4), one oxygenated methine carbons (δ_C 89.0), a carbonyl carbon (δ_C 214.9) and a carboxylic carbon (δ_C 178.6) (Table 1). The ¹H- and ¹³C-NMR spectra of 1 has the characteristic of Δ¹² oleanene skeleton [16]. Comparison the ¹³C-NMR data of 1 with 2 except for the C-3 signal (δ_C 89.0) which shifted down field by 11 ppm, others were in accordance with that of 3β-hydroxy-22-oxo-12-oleanen-29-oic acid (2) [11]. The locations of carbonyl carbon and carboxylic carbon could be confirmed by the HMBC correlations from δ_H 1.23 (Me-28) to δ_C 214.9, 26.6 (C-16), and δ_H 1.42 (Me-30) to δ_C 178.6, 41.6 (C-19), 46.5 (C-21) (Figure 2). Moreover, the ¹H and ¹³C-NMR spectra of 1 showed two sugar anomeric protons at δ_H 5.26 (1H, d, J = 7.0 Hz) and δ_H 5.03 (1H, d, J = 7.0 Hz) and carbons at δ_C 107.2 and 105.3 (Table 1). The monosaccharides were analysed as β-D-glucose with acetylated alditols derivatives by GC using authentic samples as references after hydrolysis of 1. This also could be validated by a combination of the coupling constants (J = 7.0 Hz for H-1″ and J = 7.0 Hz for H-1′) and 1D, 2D-NMR experiments. The signal at δ_C 89.0 in the ¹³C-NMR suggesting that the β-D-glucose moieties are linked to the oxygen at C-3 of the aglycon [17].This deduction and sequence of inter-glycosidic linkages were deduced from the following HMBC correlations: H-1′ (δ_H 5.03) of inner glucose with C-3 (δ_C 89.0) of sapogenin, H-1′′ (δ_H 5.26) of terminal glucose with C-2′ (δ_C 83.9) (Figure 2). The relative configuration of the hydroxylated carbon (C-3) was assigned as β form mainly on the basis of ¹H-NMR coupling (1H, dd, J = 5.0, 11.0 Hz, H-3) [17] and by comparison with 2. In NOESY spectrum, the key NOE correlations of H-28/H-21β, H-28/H-18 and H-30/H-18, showed that H-30, H-28, H-21β, H-18 were on the same face, so the relative stereochemistry were determined (Figure 3).

Table 1. ¹H- (500 MHz) and ¹³C-NMR (125 MHz) data of compound 1 (in Pyr-d₅, δ in ppm, J in Hz).

No.	δC	δH	Key HMBC (H to C)
1	38.7 CH2	1.40 (1H, m, H-1a)0.82 (1H, m, H-1b)	C-2,C-3
2	27.3 CH2	2.24 (2H, m, H-2)	C-1,C-3
3	89.0 CH	3.29 (1H, dd, J = 5.0, 11.0 Hz, H-3)	C-1′, 23, 24
4	39.5 qC
5	55.6 CH	0.71 (1H, br d, J = 11.5 Hz, H-5)	C-23,24,25
6	18.4 CH2	1.64 (1H, m, H-6a),	C-24
		1.46 (1H, m, H-6b)
7	32.8 CH2	1.48 (1H, m, H-7a),
		1.29 (1H, m, H-7b)
8	39.9 qC
9	47.6 CH	1.56 (1H, m, H-9)
10	36.8 qC
11	23.8 CH2	1.84 (2H, m, H-11)
12	124.7 CH	5.31 (1H, br s, H-12)
13	141.4 qC
14	41.9 qC
15	25.5 CH2	1.68 (1H, m, H-15a),
		0.98 (1H, m, H-15b)
16	26.6 CH2	1.89 (1H, m, H-16a),	C-28
		1.28 (1H, m, H-16b)
17	48.2 qC
18	47.0 CH	2.54 (1H, m, H-18)
19	41.6 CH2	2.88 (1H, t, J = 13.5 Hz, H-19a),	C-30
		1.97 (1H, br d, J = 12.0 Hz, H-19b)
20	44.6 qC
21	46.5 CH2	3.46 (1H, d, J = 14.5 Hz, H-21a),	C-30
		2.71 (1H, br d, J = 14.0 Hz, H-21b)
22	214.9 qC
23	28.1 CH3	1.32 (3H, s, Me-23)	C-3
24	15.6 CH3	0.86 (3H, s, Me-24)	C-3
25	16.7 CH3	0.91 (3H, s, Me-25)
26	16.8 CH3	1.14 (3H, s, Me-26)
27	25.5 CH3	1.27 (3H, s, Me-27)	C-13
28	20.9 CH3	1.23 (3H, s, Me-28)	C-22,C-16
29	178.6 qC
30	21.6 CH3	1.42 (3H, s, Me-30)	C-29,C-19,C-21
glu
1′	105.3 CH	5.03 (1H, d, J = 7.0 Hz, H-1′)	C-3
2′	83.9 CH	4.33 (1H, t, J = 8.0 Hz, H-2′)	C-1′,C-1″,C-4′
3′	77.7 CH	4.40 (1H, br d, J = 8.0 Hz, H-3′)
4′	73.1 CH	4.64 (3H, m, H-4′, 2″, 3″)
5′	74.7 CH	4.20 (1H, br d, J = 9 Hz, H-5′)
6′	61.3 CH2	4.44 (2H, m, H-6′)
glu
1″	107.2 CH	5.26 (1H, d, J = 7.0 Hz, H-1″)	C-2′,C-3″
2″	74.9 CH	4.64 (3H, m, H-4′, 2″, 3″)	C-4″
3″	77.4 CH	4.64 (3H, m, H-4′, 2″, 3″)
4″	69.5 CH	4.73 (1H, m, H-4″)
5″	76.9 CH	4.09 (1H, t, J = 6.1 Hz, H-5″)
6″	61.3 CH2	4.67 (2H, m, H-6″)

Table 1. ¹H- (500 MHz) and ¹³C-NMR (125 MHz) data of compound 1 (in Pyr-d₅, δ in ppm, J in Hz).

No.	δC	δH	Key HMBC (H to C)
1	38.7 CH2	1.40 (1H, m, H-1a)0.82 (1H, m, H-1b)	C-2,C-3
2	27.3 CH2	2.24 (2H, m, H-2)	C-1,C-3
3	89.0 CH	3.29 (1H, dd, J = 5.0, 11.0 Hz, H-3)	C-1′, 23, 24
4	39.5 qC
5	55.6 CH	0.71 (1H, br d, J = 11.5 Hz, H-5)	C-23,24,25
6	18.4 CH2	1.64 (1H, m, H-6a),	C-24
		1.46 (1H, m, H-6b)
7	32.8 CH2	1.48 (1H, m, H-7a),
		1.29 (1H, m, H-7b)
8	39.9 qC
9	47.6 CH	1.56 (1H, m, H-9)
10	36.8 qC
11	23.8 CH2	1.84 (2H, m, H-11)
12	124.7 CH	5.31 (1H, br s, H-12)
13	141.4 qC
14	41.9 qC
15	25.5 CH2	1.68 (1H, m, H-15a),
		0.98 (1H, m, H-15b)
16	26.6 CH2	1.89 (1H, m, H-16a),	C-28
		1.28 (1H, m, H-16b)
17	48.2 qC
18	47.0 CH	2.54 (1H, m, H-18)
19	41.6 CH2	2.88 (1H, t, J = 13.5 Hz, H-19a),	C-30
		1.97 (1H, br d, J = 12.0 Hz, H-19b)
20	44.6 qC
21	46.5 CH2	3.46 (1H, d, J = 14.5 Hz, H-21a),	C-30
		2.71 (1H, br d, J = 14.0 Hz, H-21b)
22	214.9 qC
23	28.1 CH3	1.32 (3H, s, Me-23)	C-3
24	15.6 CH3	0.86 (3H, s, Me-24)	C-3
25	16.7 CH3	0.91 (3H, s, Me-25)
26	16.8 CH3	1.14 (3H, s, Me-26)
27	25.5 CH3	1.27 (3H, s, Me-27)	C-13
28	20.9 CH3	1.23 (3H, s, Me-28)	C-22,C-16
29	178.6 qC
30	21.6 CH3	1.42 (3H, s, Me-30)	C-29,C-19,C-21
glu
1′	105.3 CH	5.03 (1H, d, J = 7.0 Hz, H-1′)	C-3
2′	83.9 CH	4.33 (1H, t, J = 8.0 Hz, H-2′)	C-1′,C-1″,C-4′
3′	77.7 CH	4.40 (1H, br d, J = 8.0 Hz, H-3′)
4′	73.1 CH	4.64 (3H, m, H-4′, 2″, 3″)
5′	74.7 CH	4.20 (1H, br d, J = 9 Hz, H-5′)
6′	61.3 CH2	4.44 (2H, m, H-6′)
glu
1″	107.2 CH	5.26 (1H, d, J = 7.0 Hz, H-1″)	C-2′,C-3″
2″	74.9 CH	4.64 (3H, m, H-4′, 2″, 3″)	C-4″
3″	77.4 CH	4.64 (3H, m, H-4′, 2″, 3″)
4″	69.5 CH	4.73 (1H, m, H-4″)
5″	76.9 CH	4.09 (1H, t, J = 6.1 Hz, H-5″)
6″	61.3 CH2	4.67 (2H, m, H-6″)

Figure 2. Key HMBC and COSY correlations of 1.

Figure 2. Key HMBC and COSY correlations of 1.

Figure 3. Key NOESY correlations of 1.

Figure 3. Key NOESY correlations of 1.

All the above data identified 1 as 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl subprogenin D. The structures of known compounds 2–7 were confirmed by detailed NMR data comparison with those in the literature [11,12,13,14,15].

The cytotoxicity of 1–7 against MCF-7, SW1990, Hela, Du-145 cancer cell lines were evaluated with 5-FU (5-Fluorouracil) and DOX (doxorubicine) as positive controls. Compound 5 showed moderate cytoxicity against SW1990, Hela, Du-145 cancer cell lines, and compound 6 showed moderate cytoxicity against MCF-7, SW1990, Du-145 cancer cell lines, whereas other compounds had no significant activity (Table 2).

Table 2. Cytotoxicity of 1–7 against four cancer cell lines.

	Cytotoxicity (IC50 [μg/mL])(mean ± SD%)
	MCF-7	SW1990	Hela	Du-145
1	-a	-	-	-
2	-	-	-	-
3	-	-	-	-
4	-	-	-	-
5	-	5 ± 0.32	10 ± 0.89	5 ± 0.40
6	4 ± 0.18	2 ± 0.09	-	2 ± 0.08
7	-	-	-	-
DOX	1 ± 0.06		2 ± 0.16	1 ± 0.05
5-Fu		10 ± 0.95

^a No significant activity at 10 μg/mL.

Table 2. Cytotoxicity of 1–7 against four cancer cell lines.

	Cytotoxicity (IC50 [μg/mL])(mean ± SD%)
	MCF-7	SW1990	Hela	Du-145
1	-a	-	-	-
2	-	-	-	-
3	-	-	-	-
4	-	-	-	-
5	-	5 ± 0.32	10 ± 0.89	5 ± 0.40
6	4 ± 0.18	2 ± 0.09	-	2 ± 0.08
7	-	-	-	-
DOX	1 ± 0.06		2 ± 0.16	1 ± 0.05
5-Fu		10 ± 0.95

^a No significant activity at 10 μg/mL.

3. Experimental

3.1. General

1D and 2D NMR spectra were recorded on a Bruker-AV-500 spectrometer with TMS as internal standard. HRESIMS were measured with MAT 95XP mass spectrometer. IR were recorded on FT-IR Nicolet 6700. UV spectra were obtained on a Beckman DU-640 UV spectrophotometer. Optical rotations were measured with a Perkin-Elmer 341 plus. GC were run on a QP2010PLUS (Shimadzu Corporation) equipped with an ACQ mass spectrometer. For column chromatography (CC), silica gel (200–300 mesh) and GF₂₅₄ for TLC were obtained from the Qingdao Marine Chemical Factory, Qingdao, China.

3.2. Plant Material

The leaves and stems of Abrus precatorius were collected in October 2010 from the mangrove wetlands of Hainan Island, China. The identification of the plant was performed by Professor Si Zhang. A voucher sample (No. 20101001) is maintained in the Key Laboratory of Marine Bio-Resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, China.

3.3. Extraction and Isolation

The air-dried leaves and stems of Abrus precatorius (8 kg) were extracted with EtOH (95%, 20 L) three times (7 days each time) at room temperature. The combined extract was evaporated in vacuo, suspended in water, and then successively partitioned with petroleum ether, EtOAc, and n-butanol (800 mL × 3). The EtOAc and n-butanol fractions were concentrated to afford 53.7 g and 108 g of residues, resp. The EtOAc extract was subjected to silica gel CC using a gradient elution of CHCl₃-MeOH (100:0–1:1) to afford ten fractions (Frs. A–J). Compound 3 (11 mg) was crystallizated in the bottle when eluted with the solvent CHCl₃-MeOH 90:10 (Frs. B) and then purified with MeOH. Frs. B (7 g) was subjected to CC and eluted with CHCl₃-acetone (30:1, 20:1, 15:1, 10:1, 5:1, each 1500 mL) to give six fractions (B1–B6), B3 was purified by Sexphadex LH-20 (CHCl₃-MeOH 1:1) to yield 2 (8 mg). Compound 4 (10 mg) was isolated from B1 by repeated chromatographic on Sephadex LH-20 (CHCl₃-MeOH 1:1) and silica gel column (CHCl₃-MeOH 80:1). Frs. C (4.4 g) was subjected to CC with gradient eluting of CHCl₃-acetone (30:1, 20:1, 15:1, 10:1, 5:1, each 1,000 mL) to give six fractions (C1–C6). Compound 5 (7 mg) was crystallizated from C2 when eluted with the solvent CHCl₃-acetone 20:1, and then recrystallizated with MeOH. C4 was purified by Sexphadex LH-20 (CHCl₃-MeOH 1:1) to give compound 6 (7 mg). Frs. G (2.36 g) was subjected to CC with gradient eluting of CHCl₃-MeOH (30:1, 20:1, 15:1, 10:1, 5:1, each 500 mL) and purification on Sephadex LH-20 to yield 7 (20 mg). The n-BuOH extract (108 g) was subjected to CC on Amberlite XAD using MeOH-H₂O (20%, 40%, 60% and 95%). The 40% extract part (7.23 g) was fractioned on silica gel column eluting with CHCl₃-MeOH-H₂O 9:1:0.1 to give ten fraction H–Q. Fraction Q (0.4265 g) was purified by Sexphadex LH-20 (CHCl₃-MeOH 1:1) to give compound 1 (5 mg). Yellow powder: = −10 (c = 0.04, MOH), UV (MeOH) λ_max 255 nm, IR (KBr) ν_max: 3458, 2946, 1727, 1693, 1624, 1466, 1383, 1211, 1042 cm⁻¹; HRESI-MS m/z 817.4345 [M+Na]⁺ (calcd for C₄₂H₆₆O₁₄Na, 817.4350). ¹H and ¹³C-NMR data see Table 1.

3.4. Acid Hydrolysis of 1

Compound 1 (2 mg) was added into 3 N HCl (0.5 mL) and refluxed for 5 h in a water bath (100 °C). The solution was neutralized and extracted with EtOAc to afford the aglycon. The aglycon of 1 found to be identical with 2 by TLC. The sugars released were converted into acetylated alditols by reduction with NaBH₄ followed by acetylation with acetic anhydride-pyridine mixture. The alditol acetates derivatives obtained were analyzed by GC using a GCMS-QP2010 Plus: The injector temperature was set at 250 °C and the column temperature program was as follows: The initial temperature of 200 °C was held constant for 2.5 min and then increased by 5 °C min to the final temperature of 250 °C. The detector temperature was set at 280 °C. MS-Scan: ACQ mode, event Time: 0.50 s with 1,000 scan speed. Alditol acetates were identified by comparison of their retention times with those of authentic samples [16].

3.5. Cytotoxicity Assays

Cytotoxicity was evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide] method using MCF-7, SW1990, Hela and Du-145 cell lines. Details of the assays were described in a previous report [18].

4. Conclusions

The new compound 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl subprogenin D (1) was isolated from Abrus precatorius, together with six known triterpenoids. Compounds 5 and 6 showed moderate cytoxicities against MCF-7, SW1990, Hela and Du-145. However, the new compound 1 and the other known ones had no significant activity.