Two New Sphingolipids from the Leaves of Piper betle L.

Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.


Introduction
The stems and/or leaves of Piper betle L., an evergreen perennial creeper plant widely distributed in the south of China, are used as Chinese Traditional Medicines to cure stomachache, wind-cold cough, sore and furuncle, and eczema [1]. This plant has been known to contain chemical compounds, such as alkaloids, steroids, phenols, and terpenes, with a wide range of bioactivities, like antioxidant, antifungal, antiulcerogenic, antiplatelet, antidiabetic, anti-inflammatory, antifilarial, and antimicrobial activity [2][3][4].
In a previous study, we extracted P. betle stems collected from western China with acetone. This extract, from which eight piper amides and two lignans were isolated, showed antioxidant activity in a 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), scavenging assay and antibacterial activity in a disc diffusion test [5]. Our continuing studies on the leaves of the plant has now led to the isolation of two new sphingolipids, pipercerebrosides A (1) and B (2) and other five known sphingolipids, 3-octadecenamide, 3-eicosenamide, agelasphin-11, agelasphin-7a and agelasphin- 13. Sphingolipids have been found to have OPEN ACCESS anti-tumor, immunostimulatory, neuritogenic, antiviral, antifungal, and nematicidal activities [6][7][8].
In this paper we present the isolation and structural elucidation of the new compounds.

Structure Analysis of Pipercerebroside A
Compound 1 was isolated as a white amorphous powder. Its molecular formula was determined to be C 35 H 67 NO 10  0.83, 6H, t, J = 6.5 Hz) and two long-chain aliphatic moieties appearing as a multiplets (δ H 1.20-1.30). All of the above spectral data revealed that 1 was a glycosphingolipid [9,10]. Analysis of the 1 H-1 H COSY, HMQC, and HMBC spectra led to the assignment of proton and carbon signals for 1. Methanolysis of 1 yielded a fatty acid methyl ester 1a and a long-chain base 1b (Scheme 1). Compound 1a was identified as 2-hydroxydodecanoic acid methyl ester [α] 23 D −1.2 (c 0.07, CHCl 3 ) by means of GC/MS analysis, and the absolute configuration of C-2′ was determined to be R from the specific rotation [11]. The phytosphingosine part is thus a C 17 aliphatic amino alcohol unit with three hydroxyls, an amino group, and an olefinic bond. The 2S, 3S, and 4R configurations of the ceramide moieties were assigned by comparing the specific rotation [α] 23 D +9.6 (c 0.11, pyridine)], 1 H-NMR, and 13 C-NMR data of compound 1 with those of the known synthetic ceramide (2S,3S,4R)-2-[(2′R)-2′-hydroxytetracosanoylamino]-1,3,4-hexadecanetriol [12] and the natural ceramide [13].
To determine the position of the olefinic bond in the dihydrosphingosine moiety, KMnO 4 oxidation [14] was performed on compound 1b to yield nonanoic acid (1a) (Scheme 1), which was methylated and detected by GC/MS. This indicated the location of the olefinic bond between C-8 and C-9. The ∆ [8,9] olefinic bond was confirmed to have a (Z)-configuration as evidenced by the vicinal coupling constants (J = 10.0 Hz), together with the chemical shifts of C-7 (δ 27.4) and C-10 (δ 27.2). Analysis of the 1 H-1 H COSY, HMQC, and HMBC spectra led to the assignment of proton and carbon signals for 1.
To determine the position of the olefinic bond in the dihydrosphingosine moiety, KMnO 4 oxidation [14] was performed on compound 1b to yield nonanoic acid (1a) (Scheme 1), which was methylated and detected by GC/MS. This indicated the location of the olefinic bond between C-8 and C-9. The ∆ [8,9] olefinic

Structure Analysis of Pipercerebroside B
Pipercerebroside B was isolated as a white amorphous powder. Its molecular formula was determined to be C 46 H 91 NO 5 by HRESIMS (m/z 738.6966 [M + H] + , calcd. for 738.6975). The IR spectrum of 2 showed absorption bands for hydroxyl groups at 3,480 cm −1 and for a secondary amide at 1,640 cm −1 . The 1 H-and 13 C-NMR (Table 1)  , and Two terminal methyls (δ H 0.87, 6H, t, J = 6.5 Hz) and two long-chain aliphatic moieties appearing as a multiplets (δ H 1.26-1.32). All of the above spectral data revealed that 2 was also a sphingolipid [17,18]. Analysis of the 1 H-1 H COSY, HMQC, and HMBC spectra assigned the proton and carbon signals for 2. Methanolysis of 2 yielded a fatty acid methyl ester (FAME) and a long chain base (LCB). The FAME compound was identified as 2-hydroxydodecanoic acid methyl ester by means of GC/MS analysis {EI: m/z 398, [[α] 23 D −1.4 (c 0.09, CHCl 3 )]} and the absolute configuration of C-2′ was determined to be R as in compound 1. Thus, the LCB part is a C 23 aliphatic amino alcohol unit containing three hydroxyls, an amino group and a double bond. The dihydrosphingosine (LCB) moiety was oxidized to yield undecanoic acid, which was methylated and detected by GC/MS. This indicated that the double bond was located at C-11. The 11,12 alkene bond was shown to be trans by the large vicinal coupling constants (J = 15.0). The trans geometry of this double bond was also supported by the chemical shift of C-10 (δ 34.2) and C-13 (δ 33.8).

Cytotoxic Activity of Compounds
The two compounds were evaluated for their cytotoxic activity. None of them showed considerable inhibitory cytotoxic activity against cancer cell lines K562 and HL-60 at the concentration of 10 μM in a MTT assay.

General
All reagents were analytical grade and water was distilled twice. TLC was preformed with silica gel GF254 (Marine Chemical Industry Factory, Qingdao, China), and the spots were visualized by spraying with 10% H2SO4/EtOH reagent. Column chromatography was performed using silica gel (Marine Chemical Industry Factory, Qingdao, China), reverse-phase C18 silica gel (Merck, Darmstadt, Germany) and Sephadex LH-20 (Sigma). Melting points were measured with an X-4 melting point apparatus and are uncorrected. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter. UV spectra were obtained on a Perkin-Elmer Lambda 900 UV/VIS/NIR spectrophotometer. IR spectra were obtained on a Perkin-Elmer 577 spectrometer with KBr pellets.-NMR Spectra were recorded on a Bruker AV 500 spectrometer ( 1 H, 500 MHz; 13 C, 125 MHz), and chemical shifts are presented as values relative to tetramethylsilane as an internal standard. Low-resolution electrospray-ionization mass spectrometry (ESI-MS) and HR-ESI-MS were recorded on a Finnigan LCQ-Advantage mass spectrometer and a VG Auto-Spec-3000 mass spectrometer. Gas chromatography-mass spectrometer (GC-MS) experiments were performed on a Shimadzu GC-17A gas chromatograph apparatus (DB-5 capillary column: 30 m × 0.25 mm × 0.25 μm, helium flow rate: 0.8 ml/min) attached to a Shimadzu GCMS-QP5050A mass spectrometry equipped with an electron impact (EI) ion source (70 eV). The column was maintained at 100 °C for 1 min and then ramped from 100 to 260 °C at a rate of 4 °C/min with a final hold at 260 °C for 15 min.

Plant Resource
The leaves of Piper betle were collected from Baoshan city of Yunnan Province in China, in October 2010. It was identified by Prof. Shaobin Ma (Department of Biology, Yunnan University). The stems were harvested and air-dried at room temperature in shade. A voucher specimen (No. 20101020) was deposited in School of Chemistry and Biotechnology, Yunnan University of Nationalities, China.

Methanolysis, Oxidation and Methylation of Pipercerebroside A
Pipercerebroside A (3 mg) was refluxed with 0.9 mol L −1 HCl in 82% aqueous MeOH (5 mL) for 18 h [19]. The resulting solution was extracted three times with n-hexane. The n-hexane solution was washed with water (5 mL) and dried over anhydrous Na 2 SO 4 then concentrated to yield compound 1a (1.1 mg). Compound 1b (0.8 mg) was dissolved in 10% H 2 SO 4 and acetone (2.0 mL each). KMnO 4 (50 mg) was then added to the solution and stirred overnight at room temperature [16,20]. The reaction was then quenched with aqueous Na 2 S 2 O 3 (5%). After removal of acetone, the reaction mixture was extracted with Et 2 O. The Et 2 O layer was dried over Na 2 SO 4 and concentrated to yield the residue, which was methylated using CH 2 N 2 in Et 2 O at 0 °C [20]. The reaction mixture was kept in ice for 30 min before being allowed to stand at room temperature overnight. This procedure resulted in compound  (7), 55 (19), 43 (23).

Methanolysis, Oxidation and Methylation of Pipercerebroside B
Pipercerebroside B (3 mg) was treated under the same conditions mentioned above to afford 2-hydroxytetracosanoic acid methyl ester (0.9 mg) and methyl undecanoate (0.3 mg).

Bioassay
Inhibition of cell-growth activity was determined by a MTT assay using human chronic myelogenous leukemia cells (K562) and human promyelocytic leukemia cells [21]. cis-Diamminedichloroplatinum (DDP) was used as a positive control. None of the compounds showed any obvious cytotoxic effect against the cancer cell lines K562 and HL-60.

Conclusions
Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle. L. Their structures including absolute configurations were determined by spectroscopic analysis 1D-NMR, 2D-NMR and MS experiment and chemical degradation. None of the compounds showed any significant cytotoxic activity against the cancer cell lines K562 and HL-60 at a concentration of 10 μM in the MTT assay.