Cerebrosides from the Roots of Serratula Chinensis

A new cerebroside,


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, CHCl 3 and n-BuOH.The CHCl 3 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).
Compound 1 was isolated as a white amorphous powder, [α] 25 D + 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 C 40 H 77 NO 9 .The IR spectrum showed strong absorption bands for hydroxyl (3430 cm -1 ), amide (1646 and 1540 cm -1 ) and (CH 2 ) n (721 cm -1 ) groups.The 1 H-and 13 C-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 1 H-1 H 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 13 C-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 KMnO 4 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 CD 3 OD.This was supported by two carbon signals at δ 33.2/33.1 for the carbons next to the double bond in the 13 C-NMR spectrum [8][9][10], which were assigned by the aid of 1 H-1 H COSY and HMQC.In conclusion, 1 was established to be 1 The positive ESI-MS of 3 showed a [M + Na] + peak at m/z 838, consistent with the composition C 46 H 89 NO 10 .The IR spectrum of 3 was similar to that of 1.The 1 H-and 13 C-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 C 22 , and the positive ESI-MS data of the LCB from the methanolysis showed that the chain length of the phytosphingosine moiety was C 18 .The double bond was located at C-8, as seen in 1, by the KMnO 4 oxidation method.The presence of the two double triplets (J = 14.8, 4.8 Hz) at δ 5.35 and 5.32 in the 1 H-NMR spectrum of 3 (CD 3 OD) and the chemical shifts of the carbons next to the double bond (δ 33.4,33.1) in the 13 C-NMR spectrum also indicated a trans double bond in the phytosphingosine moiety.Thus, compound 3 was identified as 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.

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 H 2 O and the aqueous suspension was successively extracted three times each with petroleum ether, CHCl 3 , and n-BuOH.The CHCl 3 extract, on concentration, yielded a brown syrup (15 g).This syrup was subjected to CC on silica gel, eluting with CHCl 3 -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 CHCl 3 -MeOH was further subjected to silica gel CC, using 92:8 CHCl 3 -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-H 2 O, to yield 1 (20 mg), 2 (18 mg), and 3 (8 mg).

Oxidation of the LCB from 1
The LCB resulting from methanolysis of 1 (0.3 mg) was dissolved in a mixture of 10 % H 2 SO 4 and acetone (5 mL each).KMnO 4 (50 mg) was added and the reaction mixture was stirred overnight at room temperature.The reaction was then quenched with aqueous Na 2 S 2 O 3 (5%

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;