New glycosylsphingolipids from Psychotria serpens L

Abstract In clinical, Psychotria serpens L. was often substitute for Caulis trachelospermi to treat cancer in China. Meanwhile, EtOAc and n-BuOH fractions of MeOH extract of P. serpens L. show power activity against H460, HepG2, Hela, and PC9/GR cell lines, and no toxic effects against normal 16HBE cell lines. In our ongoing search for bioactive novel compounds from Chinese material medica, one new type of glycosylsphingolipids Psychotramide (1a–1c) were isolated from P. serpens L., and their structures were identified through spectroscopic techniques including NMR (1D and 2D) and MS (LC-MS, and GC-MS). Graphical Abstract


Introduction
In clinical, Psychotria serpens L. was often substitute for Caulis trachelospermi to treat cancer in China (Shen 1993). However, the chemical constituents of it were few reported. Meanwhile, P. serpens L. as one of four specimens which were as folk medicines to treat swelling and pain (Editorial board of Chinese flora of the Chinese Academy of Sciences 2004; Guangdong Food and Drugs Administration 2004), has ability to relax jinluo, strengthen bones, relieve rheumatic pains and colds, cool the blood, and disperse swelling. It was used to treat arthralgia due to wind-dampness, sciatica, swollen or pain of acne and throat. Moreover, Psychotria (Rubiaceae) is widespread in tropical and subtropical regions, and in China, 17 species and one variation have been found (Editorial board of Chinese flora of the Chinese Academy of Sciences 2004; Guangdong Food and Drugs Administration 2004; Schultes and Raffauf 1990). The most important is that EtOAc and n-BuOH fractions of MeOH extract of P. serpens L. show power activity against H460, HepG2, and Hela cell lines with IC 50 of 45 lg/ml, reversal of tumor multidrug resistance activity to PC9/GR cell lines with IC 50 of 45 lg/ ml, and no toxic effects against normal 16HBE cell lines with IC 50 of 150 lg/ml in the preliminary pharmacological experiment. As a continuation of our anticancer active chemical investigation on genus of Psychotria (Lin et al. 2015;Zhang et al. 2011Zhang et al. , 2012Zhang et al. , 2013Ye et al. 2014) from China, a new type of cerebroside Psychotramide (1a-1c) were isolated from P. serpens L according to NMR (1D and 2D) and MS (LC-MS, and GC-MS).
All protons and carbons were assigned according to HSQC experiment (see Table 1 of supporting information). Detailed analysis of the 1 H-1 H COSY spectrum of 1 exhibited three long coupling relationships (see Figure S1 of supporting information). In order to determine the plan structure of 1, HMBC experiment was carried out (see Figure 1). The correlation ship between anomeric proton at d H 4.27 (H-1 00 ) and d C 70.1 (C-1) indicated the saccharide residues connected with C-1. Meanwhile, correlations between d H 7.81 (-NH) and 4.26 (H-2) with d C 177.3 (C-1 0 ), d H 4.02 (H-2 0 ) and 1.63 (H-3 0 ) with d C 177.3 (C-1 0 ) also revealed the position of carbonyl group connected with amino-group and a-OH. Above all conformed the structure of 1 was a glycosphingolipids substituted by monosaccharides.
The cis configuration of the double bond was an E configuration from the coupling constant between the olefinic H-atom (J ¼ 6.5 Hz) and the chemical shifts of the allylic C-atom at d C 28.5 (t) and 28.4 (t), which could be more than 30 ppm if the configuration was trans (Ishii et al. 2006). In order to determine the stereo-configuration of 1, which was hydrolyzed in methanol solution with 1 N HCl, and hydrolysates were LCB, FAM and sugar (see Figure S2 of supporting information). The known natural key structure (Kim et al. 1997;Zhang et al. 2012) of LCB was D-erythro configuration, that is 2S, 3R, and 4R, and the specific rotation of known natural cemcines was þ 18.9 (c, 0.01, CHCl 3 ). Because the specific rotation of 1 was [a] 20 D þ 16 (c 0.02, MeOH), and 18.0 (c, 0.02, CHCl 3 ), the configurations of C-2, C-3, C-4, and C-2 0 were S, R, R, and R. Meanwhile, the absolute configurations at C-2, C-3, C-4, and C-2 0 were also determined to be S, S, R, and R, respectively, by comparison of 1 H and 13 C NMR data with those obtained from the literature (Ishii et al. 2006;Sandjo et al. 2008). Moreover, the coupling constant of anomeric proton was 7.8 Hz indicted the configuration of C-1 00 was b.
In order to determine the molecular formulas of Psychotramide (1), 1 was hydrolyzed in 1 N HCl with methanol solution. Meanwhile, the hydrosoluble amine products were examined by ESI-MS, and two signal of LCB at m/z 303, and 289 [M þ H] þ was observed (see Figure S3 of supporting information). The methyl esterification of FAM was analyzed by GC-MS, and two compounds were determined as 17-carbon acid and 19-carbon acid respectively (see Figure S4 of supporting information). According to molecular weight at m/z [M þ H] þ 816, 830, and 844, the molecular formulas of 1 were determined as C 46 H 89 NO 10 , C 47 H 91 NO 10 , C 48 H 93 NO 10 , named as psychotramide E, psychotramide F and psychotramide G, respectively.

General experimental procedures
Melting points were recorded on an X-6 micro-melting point apparatus, which was uncorrected. Optical rotations were measured on a Schmidt þ Haensch polaptronic

Plant material
Specimens of Psychotria serpens L. were collected in Luofu Mountain, Zhaoqing City, Guangdong province, Peoples 0 Republic of China and identified by Dr. Guang-Tian Peng, Guangzhou University of Chinese Medicine. A voucher specimen (No. PSS-1) was deposited at the Lab of Natural Products Research, Guangzhou University of Chinese Medicine.

Extraction and isolation
The whole Psychotria serpens L. (10.5 kg) was extracted with MeOH at room temperature. The combined methanol extracts were concentrated in vacuum to give a light brown residue (2.0 kg). The residue was suspended in H 2 O, and then partitioned with PE (200 g), EtOAc (350 g), and n-BuOH (350 g), successively.
The EtOAc fraction (350 g) was subjected to column chromatography on silica gel (200-300 mesh, 2500 kg, / ¼ 13 cm, l ¼ 80 cm) eluting with PE followed by increasing amounts of EtOAc in PE, and finally eluting with MeOH. The eluent was combined to 8 fractions according to the result of TLC. Fr4 (5.8 g) was subjected to column chromatography on SiO 2 with eluent of CH 3 Cl and MeOH (V CH3Cl :V MeOH ¼ 1:1), then Fr 4-3 (50 mg) was carried out with Sephadex LH-20 to obtain 1 (22.0 mg).

Acid hydrolysis of compounds psychotramide (1)
Psychotramide 1 (20 mg) in a mixture of 1 N HCl (4 ml) and MeOH (15 ml) was heated under reflux temperature for 15 h (Zhang et al. 2012). Then, 25 ml H 2 O was added, and the reaction mixture was extracted with hexane. The combined organic layer was concentrated under N 2 to yield a white solid FAM, which was identified by ESI-MS and GC-MS analysis. The water layer was neutralized by 5% NaHCO 3 , then the solution was freeze-dried to yield a white solid LCB, which was identified by ESI-MS.

Conclusion
Psychotramide (1) was a new type of glycosphingolipids substituted by monosaccharide, and composed of the same monosaccharide: glucose, and different carboxylic chains and sphingosine chains, named as psychotramide E (C 46 H 89 NO 10 ), psychotramide F (C 47 H 91 NO 10 ) and psychotramide G (C 48 H 93 NO 10 ). Psychotramide E and psychotramide G had same sphingosine chains with total 22 carbons, and different carboxylic chains with total 18 or 20 carbons, while, Psychotramide F and Psychotramide G had same carboxylic chains with total 20 carbons, and different sphingosine chains with total 21 or 22 carbons. According to literatures, glycosphingolipids showed significant anticancer (Jin et al. 1994;Carter and Rinehart 1978).