Two pentasaccharide resin glycosides from Argyreia acuta

Two new compounds of acutacosides 1 and 2, pentasaccharide resin glycosides were isolated from the aerial parts of Argyreia acuta. The core of the two compounds was operculinic acid A, and they were esterfied at the same position, just one substituent group was linked at C-2 of Rha. The absolute configuration of the aglycone in the two compounds was established by Mosher's method, which was (11S)-hydroxyhexadecanoic acid (jalapinolic acid). Their structures were established by a combination of spectroscopic and chemical methods.


Introduction
Argyreia acuta L. is a member of the genus Argyreia (Convolvulaceae; Editorial Committee of China flora of Chinese Academy of Sciences 1997). A. acuta is a common folk herbal medicine, with the efficacies of dispelling wind and eliminating dampness, relieving cough and reducing sputum, stopping the bleeding and promoting tissue regeneration, relaxing and activating the tendons, removing toxicity for eliminating carbuncles. Now a few compounds were found, and recent research found it has hemostasis properties (Cai & Xu 2013). The resin glycoside showed characteristics of Convolvulaceae; however, so far no resin glycoside has been reported from A. acuta. In this study, two new pentasaccharide resin glycosides, designated as acutacosides A (1) and acutacosides B (2), were isolated from the aerial parts of A. acuta. The new compounds are maclactones of operculinic acid A, partially esterified with Mba, Cna, Dodeca and Deca of different fatty acids. The lactone esterification site of the aglycone, jalpinolic acid, was attached to the second saccharide at C-2 in 1 and 2 ( Figure 1). Their structures were elucidated on the basis of extensive spectroscopic data interpretation and chemical degradation.

Results and discussion
A 95% EtOH extract of the dried aerial parts of A. acuta was partitioned between CHCl 3 and H 2 O to afford a resinous fraction which was chromatographed over Si gel, Rp-18, Sephadex LH-20 and purified by preparative HPLC to afford compounds 1 and 2, which were hydrolysed with alkaline and acid to afford organic acid and operculinic acid A (3). Peaks in the chromatograms were detected from the alkaline hydrolysis mixtures and identified by comparison with authentic samples as 2-methylbutyric acid methyl ester (t R 4.39 min) m/z [M þ H] þ 117 (5) (20), 41 (18) from 2 was identified. The 2-methylbutanoic acid was proved to be S-configuration by comparing the specific rotation with that of authentic 2S-methylbutanoic acid (Yin et al. 2009). Acidic hydrolysis of operculinic acid A liberated the aglycone, 11-hydroxyhexadecanoic acid, which was identified S-configuration (Yin et al. 2008) and the monosaccharides mixture was derivatised and detected with GC -MS (gas chromatography-mass spectrometer) by comparison with those of authentic samples to improve as D-fucose, L-rhamnose and D-glucose (Luo et al. 2008).
Acutacosides A (1) and B (2), obtained as a white, amorphous powder. In HR-TOF-MS, a pseudomolecular ion peak at m/z 1391.7322 (100%) [M þ Na] þ (calculated for 1391.7339) was corresponding to the molecular formula C 70 H 112 O 26 (1) and a pseudomolecular ion peak at m/z 1419.7686 (100%) [M þ Na] þ (calculated for 1419.7652) was determined as the molecular formula C 72 H 116 O 26 (2). Its IR spectrum gave peaks of hydroxyl (3443 cm 21 ), carbonyl (1723 cm 21 ) and aromatic (1637 cm 21 ) groups from 1 and peaks of hydroxyl (3442 cm 21 ), carbonyl (1722 cm 21 ) and aromatic (1637 cm 21 ) groups from 2. Alkaline hydrolysis of 1 and 2 afforded operculinic acid A (3) and dodecanoic, decanoic, 2-methylbutyric and trans-cinnamic acids. 2-Methylbutyric acid was found to have the S-configuration by comparison of its optical rotation value with that of an authentic sample. Acid hydrolysis afforded the monosaccharides mixture, which was derivatised and detected with GC -MS by comparing with those of authentic samples to improve as D-fucose, L-rhamnose and D-glucose. The 13 C NMR data of 1 and 2 were all the same from 177 to 41 ppm. All these evidences suggested that 1 and 2 were the substituents attaching the same sites, the only difference was in the decanoic group in 1 and dodecanoic group in 2. The 1 H NMR data of 1 (Table S1)   protons at d H 7.27 -7.45 (m, C 6 H 5 of Cna) and at d H 0.81 (t, J ¼ 7.0 Hz, H-4 of Mba), 1.15 (d, J ¼ 7.4 Hz, CH 3 -2 of Mba) and 2.48 (m, H-2 of Mba). Also a methyl triplet signal at 0.83 ppm and a triplet-like signal in a methylene group at CH 2 -2 (2.32 ppm) of a decanoyl group, and two signals at 2.28 (1H, m) and 2.44 (1H, m) ppm of the non-equivalent protons of the methylene group at C-2 in the aglycone moiety were observed, suggesting a macrocyclic lactone-type structure.
The NMR spectra (Table S1) of 2 were similar to those of 1, the interglycosidic connectivities and esterification sites were all the same though by 2D NMR TOCSY, HMBC and HSQC, just one group of decanoyl was different by GC -MS experiments. Accordingly, the structure of 2 was elucidated as (

Experimental 3.1. General
NMR spectra were recorded on INOVA 500 spectrometers ( 1 H NMR, HSQC and HMBC at 500 MHz; 13 C NMR at 125 MHz) using C 5 D 5 N as solvent with tetramethylsilane as internal reference. The chemical shifts were given in d (ppm) and coupling constants in Hz. HR-TOF-MS experiments were performed on AB SCIEX Triple TOF 5600 plus MS spectrometer. UV on a Shimadzu UV-2550 spectrophotometer and IR spectra were measured on a Shimadzu FTIR Bruker-TENSOR 37 spectrophotometer. GC -MS experiment was performed on a TRACE GC ULTRA DSQII intrument. Optical rotations were measured with an Anton Paar-MCP600 polarimeter in MeOH solution. The centrifugation was applied with D05 (Hunan Hexi Instrument Co., Ltd, Changsha, China). Adsorbents for column chromatography were silica gel (200 -300 mm, Qingdao Marine Chemical Co., Ltd, Qingdao, China), Sephadex LH-20 (75 -150 mm, Pharmacia, Uppsala, Sweden), ODS (octa decylsilyl silicion) (40 -63 mm, FuJi, Tokyo, Japan). Preparative HPLC was performed using a Shimadzu LC-6AD series instrument equipped with a UV detector at 280 nm and Shim-Park RP-C 18 column (20 £ 200 mm i.d.). Thin-layer chromatography was performed on pre-coated silica gel GF 254 plates (Qingdao Marine Chemical Co., Ltd) and detected by spraying with 10% H 2 SO 4 -EtOH.

Plant material
The aerial parts of A. acuta L. were collected at Guilin City, Guangxi Province, China, in August 2013, and identified by Prof. Jizhu Liu. A voucher specimen (no. 2013-2) is deposited at Department of Traditional Chinese Medicinal Chemistry, Guangdong Pharmaceutical University.

Acid hydrolysis and sugar analysis
The glycosidic acid (3, 3 mg, from alkaline hydrolysis) was methylated with MeOH and catalysed with 0.5 N H 2 SO 4 to give operculinic acid A methyl ester (4). Compound 4 was hydrolysed with 1 N H 2 SO 4 and extracted with ether to obtain 11-hydroxyhexadecanoic acid methyl ester (5). The aqueous layer of acidic hydrolysis was concentrated under reduced pressure to give a residue of the sugars. The residue was dissolved in pyridine (0.1 mL), to which 0.08 M L-cysteine methyl ester hydrochloride in pyridine (0.15 mL) was added. The mixture was kept at 608C for 1.5 h. After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.1 mL) for 2 h. The mixture was partitioned between n-hexane and H 2 O (0.3 mL each) and then the hexane extract was analysed by GC -MS on a TRACE GC ULTRA DSQII instrument under the following conditions: 30 m £ 0.25 mm £ 0.25 mm, TG-5MS (Thermo) column; He, 0.8 mL min 21 ; 608C, 3 min; 60 -1808C, D108C min 21 keep 3 min, 180 -2058C, D38C min 21 keep 5 min, 205 -3008C, D208C min 21 keep 5 min, 70 eV. In the acid hydrolysate of operculinic acid A methyl ester, D-fucose, L-rhamnose and D-glucose were confirmed by comparing the retention times of their derivatives with those of authentic D-fucose (t R 30.35 min), L-rhamnose (t R 30.14 min) and D-glucose (t R 31.65 min) derivatives prepared in the same way, respectively.

Preparation of Mosher's esters
The procedures for the preparation of Mosher's esters, which was determined the absolute configuration of 11S of the aglycone, were same as described previously for resin glycosides from Ipomoea batatas.

Conclusion
In conclusion, investigation of the aerial parts of A. acuta afforded two new compounds. Their structures were established by different spectroscopic analyses.