Cerebrosides from the Roots of Serratula chinensis

Abstract

A new cerebroside, 1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(2’R)-2-hydroxy- palmitoylamino]-8-octadecene-1,3-diol, along with aralia cerebroside and 1-O-β-D- glucopyranosyl-(2S,3S,4R,8E)-2-[(2’R)-2-hydroxybehenoylamino]-8-octadecene-1,3,4-triol were isolated from the roots of Serratula chinensis S. Moore. The structure of the new cerebroside was established by spectroscopic and chemical means. Occurrence of cerebrosides in Serratula is reported here for the first time.

Introduction

Serratula chinensis S. Moore (Compositae) is a perennial herbaceous plant growing mainly in South China [1]. Its roots have long been used as a folk medicine in China for treatment of pharyngitis and morbilli [2]. During the course of our continuing investigation of bioactive natural products found in folk medicinal plants from the southern part of mainland China, we have investigated the chemical constituents of S. chinensis roots and have reported in previous papers [3,4] the isolation of seven ecdysteroids and a mixture of five ceramides from that source. This paper deals with the isolation and structure elucidation of some cerebrosides, a novel class of constituents for this plant.

Results and Discussion

The EtOH extract of the powdered dry roots of S. chinensis was successively fractionated with petroleum ether, CHCl₃ and n-BuOH. The CHCl₃ fraction was separated by a combination of silica gel, Sephadex LH-20, and RP-18 silica gel column chromatography (CC) to yield compounds 1, 2, and 3 (Figure 1).

Figure 1. Structures of compounds 1-3.

Figure 1. Structures of compounds 1-3.

Compound 1 was isolated as a white amorphous powder, + 8.8 (c 0.17, MeOH). Its positive ESI-MS showed a [M + K]⁺ peak at m/z 754, a [M + Na]⁺ peak at m/z 738 and a [M + H]⁺ peak at m/z 716, all in accordance with the molecular formula C₄₀H₇₇NO₉. The IR spectrum showed strong absorption bands for hydroxyl (3430 cm^-1), amide (1646 and 1540 cm^-1) and (CH₂)_n (721 cm^-1) groups. The ¹H- and ¹³C-NMR spectra of 1 (Table 1) indicated the presence of a β-D-glucopyranosyl moiety (δ_H 4.86, 1H, d, J = 7.6 Hz, anomeric proton; δ_C 105.6, 75.1, 78.6, 71.7, 78.5, and 62.8), an amide linkage (δ_H 8.40, 1H, d, J = 9.2 Hz; δ_C 175.7), an amidomethine (δ_H 4.69, δ_C 54.6), an oxygenated methylene (δ_H 4.70 and 4.18; δ_C 70.4), two oxygenated methines (δ_H 4.16 and 4.59; δ_C 71.3 and 72.5), and two long chain aliphatic moieties. The above structural features indicated a dihydrosphingosine type cerebroside [5]. Methanolysis of 1 afforded a fatty acid methyl ester (FAME) and a long chain base (LCB) [6]. The FAME was identified as methyl 2-hydroxypalmitate by GC-MS analysis. The dihydrosphingosine moiety of 1 was derived as 2-amino-octadecene-1,3-diol by analysis of the ¹H-¹H COSY of 1 and the positive ESI-MS of the LCB. The absolute configuration of C-2′ was determined to be R form from the specific rotation of the FAME [7]. The 2S,3R stereochemistry was determined by comparison of the ¹³C-NMR chemical shifts of C-2 and C-3 with those of plakosides C and D [5,6]. In order to determine the position of the double bond in the dihydrosphingosine moiety, the KMnO₄ oxidation was performed on the LCB [4]. The oxidation afforded n-decanoic acid which was determined by GC-MS analysis. This allowed the location of the double bond at C-8. The trans (E) configuration of the double bond in 1 was indicated by the olefinic proton signals which appeared as two double triplets (J = 14.4, 5.6 Hz) at δ 5.36 and 5.33 in CD₃OD. This was supported by two carbon signals at δ 33.2/33.1 for the carbons next to the double bond in the ¹³C-NMR spectrum [8,9,10], which were assigned by the aid of ¹H-¹H COSY and HMQC. In conclusion, 1 was established to be 1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(2’R)-2-hydroxylpalmitoylamino]-8-octadecene-1,3-diol.

The positive ESI-MS of 3 showed a [M + Na]⁺ peak at m/z 838, consistent with the composition C₄₆H₈₉NO₁₀. The IR spectrum of 3 was similar to that of 1. The ¹H- and ¹³C-NMR data of 3 (Table 1) were essentially identical with those of poke-weed cerebrosides [6], indicating a cerebroside comprised of monounsaturated (2S,3S,4R)-phytosphingosine, (2R)-2-hydroxy fatty acid and β-D-glucopyranose moieties. GC-MS analysis of the FAME obtained from the methanolysis of 3 indicated that the chain length of the fatty acid moiety was C₂₂, and the positive ESI-MS data of the LCB from the methanolysis showed that the chain length of the phytosphingosine moiety was C₁₈. The double bond was located at C-8, as seen in 1, by the KMnO₄ oxidation method. The presence of the two double triplets (J = 14.8, 4.8 Hz) at δ 5.35 and 5.32 in the ¹H-NMR spectrum of 3 (CD₃OD) and the chemical shifts of the carbons next to the double bond (δ 33.4, 33.1) in the ¹³C-NMR spectrum also indicated a trans double bond in the phytosphingosine moiety. Thus, compound 3 was identified as 1-O-β-D-glucopyranosyl-(2S,3S,4R,8E)-2-[(2’R)-2-hydroxybehenoylamino]-8-octadecene-1,3,4-triol [11]. Cerebroside 2 was identified as aralia cerebroside {1-O-β-D-glucopyranosyl-(2S,3S,4R,8E)-2- [(2’R)-2-hydroxypalmitoylamino]-8-octadecene-1,3,4-triol} by the methods described for 1 and 3, and by direct comparison of its spectral data with the reported literature values [12].

Conclusions

The present study provides the first report on the presence of cerebrosides in Serratula spp., in addition to the identification of the new cerebroside 1. Cerebrosides have a wide range of biological functions, all potentially related to the amphipathic nature of the molecule [13]. The broad bioactivity spectrum of cerebrosides suggests the further potential utilization of S. chinensis roots as a valuable crude drug.

Experimental

General

Optical rotations were measured with a Perkin Elmer 343 spectropolarimeter. The IR spectra were taken in KBr on a WQF-410 FT-IR spectrophotometer. The ¹H-NMR (400 MHz), ¹³C-NMR (100 MHz), and 2D-NMR spectra were recorded on a Bruker DRX-400 instrument. Chemical shifts were expressed in ppm (δ) with TMS as an internal standard. The positive ESI-MS data were obtained with a MDS SCIEX API 2000 LC/MS/MS system by direct inlet using MeOH as solvent. The GC-MS analyses were performed with a Shimadzu QP-5000 instrument [GC conditions: DB-1 capillary column (30 m × 0.25 mm); column temperature, 60→260°C for 1-2, and 40→260 °C for 3; rate of temperature increase, 10°C/min; injector temperature, 270°C; He at 15 mL/min]. Silica gel 60 (200-300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, People’s Republic of China), Develosil ODS (5 μm, Nomura Chemical Co. Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences, Sweden) were used for column chromatography (CC). TLC was performed on precoated plates (Kieselgel 60GF₂₅₄, Merck) with detection effected by exposure to iodine vapor.

Plant material

Roots of S. chinensis were collected in Lechang County, Guangdong Province, China, in the autumn of 2001, and identified by Prof. Zexian Li, South China Botanical Garden, Chinese Academy of Sciences. A voucher sample (No. 621633) was deposited at the Herbarium of South China Botanical Garden, Chinese Academy of Sciences.

Extraction and isolation

Ground dry roots of S. chinensis (8 kg) were extracted with 95% EtOH by percolation at room temperature. The EtOH percolate was concentrated in vacuo to a syrup (500 g). This syrup was suspended in H₂O and the aqueous suspension was successively extracted three times each with petroleum ether, CHCl₃, and n-BuOH. The CHCl₃ extract, on concentration, yielded a brown syrup (15 g). This syrup was subjected to CC on silica gel, eluting with CHCl₃-MeOH mixtures of increasing polarities (from 98:2 to 9:1), to obtain three fractions A-C. Fraction B, obtained on elution with 96:4 CHCl₃-MeOH was further subjected to silica gel CC, using 92:8 CHCl₃-MeOH as eluant, to afford two subfractions B1 and B2. Subfraction B2 was separated by CC on Sephadex LH-20 with MeOH as eluant, followed by CC over RP-18 silica gel eluting with 9:1 MeOH-H₂O, to yield 1 (20 mg), 2 (18 mg), and 3 (8 mg).

Cerebroside 1

White amorphous powder. + 8.8 (c 0.17, MeOH). IR (KBr) cm⁻¹: 3430, 2921, 2852, 1646, 1540, 1467, 1278, 1164, 1078, 721. ¹H- and ¹³C-NMR data, see Table 1. Positive ESI-MS m/z: 754 [M + K]⁺, 738 [M + Na]⁺, 716 [M + H]⁺, 554 [M - Hexose + H]⁺.

Methanolysis of 1

Compound 1 (2.0 mg) was refluxed with 0.9 N HCl in 82 % aqueous MeOH (15 mL) for 18 h. The resulting solution was extracted three times with n-hexane. The n-hexane solution was dried over anhydrous Na₂SO₄ and then concentrated to yield the FAME 2R-hydroxypalmitic acid methyl ester (0.6 mg), as a white amorphous powder, -4.2 (c 0.01, CHCl₃), GC-MS: GC t_R 13.13 min, EI-MS m/z: 286 [M]⁺ (4), 268 [M - H₂O]⁺ (0.2), 227 [M - CH₃OCO]⁺ (9), 182 [M - CH₃OCO - CH₂OHCH₂]⁺ (0.4), 159 (3), 145 (2), 127 [C₉H₁₉]⁺ (5), 125 (3), 111 (7), 97 (20), 90 [CH₃OC(OH)=CHOH]⁺ (19), 83 (26), 69 (30), 57 (85). The H₂O layer, after evaporation of MeOH, was adjusted to pH 9 with aqueous ammonia and extracted with Et₂O. The Et₂O layer was dried over anhydrous Na₂SO₄ and evaporated to yield the LCB 2-aminooctadec-8-ene-1,3-diol (0.7 mg); positive ESI-MS m/z: 300 [M + H]⁺, 282 [M - H₂O + H]⁺.

Oxidation of the LCB from 1

The LCB resulting from methanolysis of 1 (0.3 mg) was dissolved in a mixture of 10 % H₂SO₄ and acetone (5 mL each). KMnO₄ (50 mg) was added and the reaction mixture was stirred overnight at room temperature. The reaction was then quenched with aqueous Na₂S₂O₃ (5%). The reaction mixture, after removal of acetone, was extracted with Et₂O. The Et₂O layer was dried over anhydrous Na₂SO₄, and concentrated, to give n-decanoic acid, GC-MS: GC t_R 12.34 min, EI-MS m/z 172 [M]⁺ (4), 155 [M - OH]⁺ (0.5), 143 (3), 129 (27), 73 (70), 60 [CO₂H₂]⁺ (98), 55 (100).

Table 1. ¹H- and ¹³C-NMR Data of 1 and 3 (in pyridine-d₅).

Position			1			3
			δH (J in Hz)	δC		δH (J in Hz)	δC
1a			4.70 m	70.4		4.71 dd (10.4, 6.8)	70.4
1b			4.18 m			4.51 dd (10.4, 4.0)
2			4.69 m	54.6		5.30 m	51.7
3			4.16 m	71.3		4.29 m	76.0
4			2.10 m	34.9		4.20 m	72.5
5			1.78 m	25.9		2.10 m	34.0
6			1.27-1.60	29.7-30.5		1.70 m	25.9
7 and 10			2.10 m	33.2 33.1		2.10 m	33.4 33.1
8 and 9			5.47 m a)	130.8		5.48 m b)	130.9 130.7
11-15			1.27-1.60	29.7-30.5		1.23-1.32	29.7-30.1
16			1.27-1.60	32.3		1.23-1.32	32.2
17			1.27-1.60	23.1		1.23-1.32	23.0
18			0.88 t (6.8)	14.4		0.86 t (6.8)	14.4
NH			8.40 d (9.2)			8.58 d (9.2)
1′				175.7			175.8
2′			4.59 dd (6.8, 3.6)	72.5		4.58 dd (7.6, 3.2)	72.5
3′			2.10 m	35.7		2.10 m	35.6
4′			2.10 m, 1.78 m	26.3		2.10 m, 1.70 m	26.9
5′-(n - 3)′			1.27-1.60	29.7-30.5		1.23-1.32	29.7-30.1
(n - 2)′			1.27-1.60	32.3		1.23-1.32	32.2
(n - 1)′			1.27-1.60	23.1		1.23-1.32	23.0
n′			0.88 t (6.8)	14.4		0.86 t (6.8)	14.4
1″			4.86 d (7.6)	105.6		4.94 d (7.6)	105.5
2″			3.98 t (8.0)	75.1		4.00 t (8.0)	75.2
3″			4.18 m	78.6		4.20 m	78.6
4″			4.18 m	71.7		4.20 m	71.6
5″			3.87 m	78.5		3.87 m	78.5
6″a			4.48 br d (11.6)	62.8		4.48 dd (11.6, 2.0)	62.7
6″b			4.30 dd (11.6, 5.6)			4.30 dd (11.6, 6.8)
a): δ 5.36 and 5.33 (each dt, J = 14.4, 5.6 Hz) when measured in CD3OD. b): δ 5.35 and 5.32 (each dt, J = 14.8, 4.8 Hz) when measured in CD3OD. n = 16 for 1; n = 22 for 3.

Table 1. ¹H- and ¹³C-NMR Data of 1 and 3 (in pyridine-d₅).

Position			1			3
			δH (J in Hz)	δC		δH (J in Hz)	δC
1a			4.70 m	70.4		4.71 dd (10.4, 6.8)	70.4
1b			4.18 m			4.51 dd (10.4, 4.0)
2			4.69 m	54.6		5.30 m	51.7
3			4.16 m	71.3		4.29 m	76.0
4			2.10 m	34.9		4.20 m	72.5
5			1.78 m	25.9		2.10 m	34.0
6			1.27-1.60	29.7-30.5		1.70 m	25.9
7 and 10			2.10 m	33.2 33.1		2.10 m	33.4 33.1
8 and 9			5.47 m a)	130.8		5.48 m b)	130.9 130.7
11-15			1.27-1.60	29.7-30.5		1.23-1.32	29.7-30.1
16			1.27-1.60	32.3		1.23-1.32	32.2
17			1.27-1.60	23.1		1.23-1.32	23.0
18			0.88 t (6.8)	14.4		0.86 t (6.8)	14.4
NH			8.40 d (9.2)			8.58 d (9.2)
1′				175.7			175.8
2′			4.59 dd (6.8, 3.6)	72.5		4.58 dd (7.6, 3.2)	72.5
3′			2.10 m	35.7		2.10 m	35.6
4′			2.10 m, 1.78 m	26.3		2.10 m, 1.70 m	26.9
5′-(n - 3)′			1.27-1.60	29.7-30.5		1.23-1.32	29.7-30.1
(n - 2)′			1.27-1.60	32.3		1.23-1.32	32.2
(n - 1)′			1.27-1.60	23.1		1.23-1.32	23.0
n′			0.88 t (6.8)	14.4		0.86 t (6.8)	14.4
1″			4.86 d (7.6)	105.6		4.94 d (7.6)	105.5
2″			3.98 t (8.0)	75.1		4.00 t (8.0)	75.2
3″			4.18 m	78.6		4.20 m	78.6
4″			4.18 m	71.7		4.20 m	71.6
5″			3.87 m	78.5		3.87 m	78.5
6″a			4.48 br d (11.6)	62.8		4.48 dd (11.6, 2.0)	62.7
6″b			4.30 dd (11.6, 5.6)			4.30 dd (11.6, 6.8)
a): δ 5.36 and 5.33 (each dt, J = 14.4, 5.6 Hz) when measured in CD3OD. b): δ 5.35 and 5.32 (each dt, J = 14.8, 4.8 Hz) when measured in CD3OD. n = 16 for 1; n = 22 for 3.

Cerebroside 3

White amorphous powder; + 9.4 (c 0.11, MeOH); IR (KBr) cm⁻¹: 3400, 2923, 2854, 1722, 1643, 1537, 1466, 1381, 1261, 1167, 1078, 720; ¹H- and ¹³C-NMR data, see Table 1; positive ESI-MS m/z: 854 [M + K]⁺, 838 [M + Na]⁺, 816 [M + H]⁺, 654 [M - hexose + H]⁺ .

Methanolysis of 3

Methanolysis of 3 (2.0 mg), performed by the same method as described for 1, also yielded a FAME (0.6 mg) and a LCB (0.5 mg). The FAME, 2-hydroxybehenic acid methyl ester, was a white amorphous powder; - 3.7 (c 0.01, CHCl₃); GC-MS: GC t_R 27.03 min; EI-MS m/z 370 [M]⁺ (6), 352 [M - H₂O]⁺ (0.5), 311 [M - CH₃OCO]⁺ (7), 266 [M - CH₃OCO - CH₂OHCH₂]⁺ (1), 159 (2), 145 (3), 127 [C₉H₁₉]⁺ (8), 125 (5), 111 (13), 97 (28), 90 [CH₃OC(OH)=CHOH]⁺ (25), 83 (30), 69 (31), 57 (88). The LCB, 2-amino-octadec-8-ene-1,3-diol, had the following ESI-MS: m/z 316 [M + H]⁺, 298 [M - H₂O + H]⁺, 280 [M - 2H₂O + H]⁺, 262 [M - 3H₂O + H]⁺.

Oxidation of the LCB from 3

The LCB from 3 (0.3 mg) was oxidized with KMnO₄ by the same method as described for that from 1 to give n-decanoic acid, GC-MS: GC t_R 14.07 min, EI-MS m/z 172 [M]⁺ (5), 155 [M - OH]⁺ (0.1), 143 (5), 129 (34), 115 (10), 101 (5), 73 (72), 60 [CO₂H₂]⁺ (100), 55 (55).