Four New Pentasaccharide Resin Glycosides from Ipomoea cairica with Strong α-Glucosidase Inhibitory Activity

Six pentasaccharide resin glycosides from Ipomoea cairica, including four new acylated pentasaccharide resin glycosides, namely cairicoside I–IV (1–4) and the two known compounds cairicoside A (5) and cairicoside C (6), were isolated from the aerial parts of Ipomoea cairica. Their structures were established by a combination of spectroscopic, including two dimensional (2D) NMR and chemical methods. The core of the six compounds was simonic acid A, and they were esterfied the same sites, just differing in the substituent groups. The lactonization site of the aglycone was bonded to the second saccharide moiety at C-2 in 1–4, and at C-3 in 5–6. Compounds 1 and 5, 4 and 6 were two pairs of isomers. The absolute configuration of the aglycone in 1–6 which was (11S)-hydroxyhexadecanoic acid (jalapinolic acid) was established by Mosher’s method. Compounds 1–4 have been evaluated for inhibitory activity against α-glucosidase, which all showed inhibitory activities.


Introduction
Ipomoea cairica (L.) Sweet (Convolvulaceae) is widely distributed in the tropical and subtropical regions and is an invasive species in Southern China.I. cairica grows quickly and can then climb up the nearby trees and thus block the light, killing these trees, and damaging small ecological environments [1][2][3].I. cairica was used in folk medicine to treat sores and so on [4].Many different compounds have been found in the plant, including resin glycosdes, lignans, benzenoids, coumarins, flavonoids, steroids, and fatty acids [5,6].There were five reported resin glycosides from I. cairica, in which the lactone was attached at C-3 of Rha.In this paper six compounds were found, including two pairs of isomers, which were 1 and 5, and 4 and 6.The lactone attachment sites in four new resin glycosides 1-4 were assigned to C-2 of Rha and the lactone attachment sites of the two known resin glycosides 5-6 were assigned to C-3 of Rha.Some plants from the Convolvulaceae family showed strengthened inhibition against α-glucosidase or anti-diabetes activity [7,8], so this paper will elaborate on the structures and α-glucosidase inhibitory activities of compounds 1-4.

Results and Discussion
Cairicoside I (1) obtained as a white, amorphous powder, which gave a quasi-molecular ion at m/z 1321.7212[M+Na] + in HR-TOF-MS, which suggested the molecular formula C65H102O26 (calcd.for C65H102O26Na, 1321.6557).Its IR spectrum gave a hydroxyl group absorption band at 3442 cm −1 , carbonyl group at 1724 cm −1 , and aromatic group at 1641 cm −1 .Alkaline hydrolysis of 1 afforded a glycosidic acid (compound 7) and organic acids.The organic acids were identified as 2-methylbutyric, and Cna (trans-cinnamic acids), with a ratio of 2:1 by GC-MS.Mba a (2-Methylbutyric acid) was found to have the S-configuration by comparison of its optical rotation value with that of an authentic sample [3].Acid hydrolysis the glycosidic acid afforded Rha (L-rhamnose) and Glu (D-glucose), with a ratio of 4:1.The monosaccharides was derivatized and identified as Rha and Glu by GC-MS by comparison with authentic samples.Taken together the hydrolysis information and NMR data identified the glycosidic acid as simonic acid A (7).
Cairicoside A (5) and cairicoside C (6) appeared as white, amorphous powders, and gave quasi-molecular ions at m/z 1333 [M+Cl] − , and 1403 [M+Cl] − in ESI-MS, which suggested molecular weights of 1298 and 1368, so these two compounds were isomeric with 1 and 4, respectively.Alkaline hydrolysis of 5 afforded the same substituent groups as 1, and alkaline hydrolysis of 6 afforded the same substituent groups as 4. The groups' esterfication sites were suggested by HMBC data, and just the lactone sites were different between 1 and 4, which were bonded at C-3 of Rha.The structures of 5-6 are shown in Figure 1.
Some plants from the Convolvulaceae family have been reported to exert anti-diabetes activities or potent α-glucosidase inhibitory activities, so compounds 1-4 have been evaluated for inhibitory activities against α-glucosidase.As shown in Table 2, new compounds 1-4 exhibited more potent α-glucosidase inhibitory activities compared to acarbose, a widely used clinically useful drug, used as a positive control (IC50 = 385.0± 9.3 μM).To our knowledge, these are the first examples of resin glycosides with α-glucosidase inhibitory activities.The IC50 values of the four compounds are almost the same, probably due to the similarity of their structures, and every compound with strong inhibitory activity has four α-anomeric monosaccharides in the structure, which is the same relative configuration as α-glucosidase.a IC 50 is defined as the concentration that resulted in a 50% α-glucosidase inhibition and the results are means ± standard deviation of three independent replicates; b Positive control substance.

General Information
1 H-and 13 C-NMR spectra were recorded on an INOVA 600 spectrometer using tetramethylsilane (TMS) as internal standard.The chemical shifts were given in δ (ppm) and coupling constants in Hz.The ESIMS experiments were performed on an Agilent 1100 Series LC/MSD ion-trap mass spectrometer.HR-TOF-MS experiments were performed on AB SCIEX Triple TOF 5600 plus MS spectrometer.UV were measured on a Shimadzu UV-2550 spectrophotometer and IR spectra were measured on a Shimadzu FTIR spectrophotometer.GC-MS experiments were performed on a TRACE GC ULTRA DSQ II instrument.Optical rotations were measured with an Anton Paar-MCP600 polarimeter in MeOH solution.The centrifugation was performed with a Beckman Allegra X-12).Adsorbents for column chromatography were silica gel (200-300 μm, Qingdao Marine Chemical Co., Ltd., Qingdao China), Sephadex LH-20 (75-150 μm, Pharmacia, Uppsala, Sweden), ODS (40-63 μm, FuJi, Tokyo, Japan).Preparative high-performance liquid chromatography (HPLC) was performed using a Shimadzu LC-6ADseries instrument equipped with a UV detector at 280 nm and Shim-Park RP-C18 column (20 × 200 mm i.d.).TLC (Thin-layer chromatography) was performed on pre-coated silica gel GF254 plates (Qingdao Marine Chemical Co., Ltd.) and detected by spraying with 10% H2SO4-EtOH.α-Glucosidase was monitored continuously with an auto multi-functional microplate reader ELX800 (BioTek Instruments, Inc., Winooski, VT, USA).

Plant Material
The aerial parts of I. cairica were collected at Guangzhou City, Guangdong Province, China, in August 2012, and identified by Prof. Ji-Zhu Liu.A voucher specimen (No. 2012-8) was deposited at Department of Traditional Chinese Medicinal Chemistry, Guangdong Pharmaceutical University.

Acid Hydrolysis and Sugar Analysis
The glycosidic acid (7, 4 mg, from alkaline hydrolysis) was methylated with MeOH catalyzed by 1.0 mol/L H2SO4 to give simonic acid A methyl ester (8).Compound 7 was hydrolyzed with 2 mol/L H2SO4 and extracted with ether to obtain 11-hydroxyhexadecanoic acid methyl ester (9) [12].The aqueous layer of acidic hydrolysis was concentrated under reduced pressure to give a sugar residue.The protocols described in [13] were applied to determine the stereochemistry of sugars, which allowed the identification of the components of the mixture of sugars as L-rhamnose and D-glucose by comparison their derivatives with those of authentic samples.GC-MS was performed on a TRACE GC ULTRA DSQ II intrument under the following conditions: 30 m × 0.25 mm × 0.25 μm, TG-5MS (Thermo)column; He, 0.8 mL/min; 60 °C, 3 min; 60-180 °C, ∆10 °C/min keep 3 min, 180-205 °C, ∆3 °C/min keep 5 min, 205-300 °C, ∆20 °C/min keep 5 min, 70 eV.In the acid hydrolysate of operculinic acid A methyl ester L-rhamnose, and D-glucose were confirmed by comparison of their retention times of their derivatives with those of authentic L-rhamnose (tR 30.14 min) and D-glucose (tR 31.65 min) derivatives prepared in the same way, respectively.

Figure 2 .
Figure 2. Key HMBC correlations from H to C for cairicoside I (1).
a Chemical shifts (δ) are in ppm relative to TMS.The spin coupling (J) is given in parentheses (Hz).Chemical