Chinese Journal of

Natural Chinese Journal of Natural Medicines 2016, 14(3): 02270231 Medicines

doi: 10.3724/SP.J.1009.2016.00227

Two novel resin glycosides isolated from cairica with α-glucosidase inhibitory activity

LI Jie-Hong, PAN Jie-Tao, YIN Yong-Qin*

Department of Traditional Chinese Medicinal Chemistry, Guangdong Pharmaceutical University, Guangzhou 510006, China

Available online 20 Mar., 2016

[ABSTRACT] In the present study, two new compounds from were identified and demonstrated to have α-glucosidase inhibitory activity. They were isolated by column chromatography on silica gel and sephadex LH-20 and finally purified by prep-HPLC, with their structures being elucidated by spectroscopic methods, such as 1D- and 2D-NMR and HR-TOF-MS, and chemical methods. Compounds 1 and 2, named cairicoside A and cairicoside B, were evaluated for α-glucosidase inhibitory activity −1 by the MTT method, with the IC50 values being 25.3 ± 1.6 and 28.5 ± 3.3 μmol·L , respectively.

[KEY WORDS] Ipomoea cairica; Resin glycoside; Simonic acid A; α-Glucosidase [CLC Number] R284 [Document code] A [Article ID] 2095-6975(2016)03-0227-05

[11-12]  anti-diabetic activity . In this paper, we present the Introduction results from elaboration of the structures of two new The structure of resin glycosides is composed with a long compounds and the evaluation of their α-glucosidase chain fatty acid aglycone and oligosaccharide, and some inhibitory activities. modifiable fatty acids are acylated in different sites in the Results and Discussion oligosaccharide [1]. Many different kinds of resin glycosides have been found from genera of the family , Compound 1 was obtained as a white, amorphous powder, [2] [3] [4] including Ipomoea , Operculina , Quamoclit , and and the molecular formula C69H110NaO26 as determined with Merremia [5]. Various activities have been reported, including HR-TOF/MS at m/z [M + Na]+ 1 377.786 6 (Calcd. as cytotoxicity[5] and inhibition of multidrug efflux pumps [6]. 1 377.718 3) [M + Na]+. Its IR spectrum gave peaks of hydroxyl Ipomoea. cairica (L.) Sweet (Convolvulaceae) is an (3 424 cm1), carbonyl (1 737 cm1), and aromatic (1 640 cm1) invasive species widely distributed from tropical to groups. Alkaline hydrolysis of compound 1 afforded simonic subtropical regions, and used as a folk medicine all over the acid A (compound 3) and organic acid fractions, which were world [7-9]. Several kinds of compounds have been found. identified as isobutyric acid methyl ester (tR = 3.60 min) m/z [10] Some resin glycosides have been found in I. cairica . In 101 [M]+ (28), 88 (100), 57(70), 41 (35), n-decanoic acid order to find other different lactone site of the resin  (tR = 12.37 min): m/z 172 [M] (4), 155 (5), 143 (30), 129 (5), glycosides in I. cairica, as a part of our ongoing chemical 87 (59), 74 (100), 55 (18), and transcinnamic acid methyl studies on the resin glycosides from Ipomoea species, a + ester (tR =13.29 min) m/z 162 [M] (40), 131 (100), 103 (66), chemical investigation of I. cairica has been undertaking in 77 (32) by GC-MS experiment. Acid hydrolysis of compound our laboratory. Some from convolvulaceae family are 3 afforded the monosaccharides mixture, which was further shown to have significant inhibition against α-glucosidase or derivatized and detected with GC-MS experiment. In the acid hydrolysate of operculinic acid A, methyl ester L-rhamnose, and D-glucose were confirmed by comparison of their  [Received on] 25-Apr.-2015 retention times of their derivatives with those of authentic [Research funding] This work was supported by Chinese L-rhamnose (t =30.14 min) and D-glucose (t = 31.65 min) National Natural Science Fund (No. 81202885) and the Natural R R Science Foundation of Guangdong province of China (No. derivatives prepared in the same way, respectively. 1 2015A030313585). The H data of 1 (Table 1) exhibited two trans-coupled * [ Corresponding author] Email: [email protected] olefinic protons at δH 6.54 (d, J = 16.0 Hz, H-2 of Cna) and These authors have no conflict of interest to declare. 7.81 (d, J = 16.0 Hz, H-3 of Cna), and a multiplet due to five

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13 1 Table 1 C and H NMR data for compounds 1 and 2 in Pyridine-d5 1 2 Position c H c H Glc 1 104.3 4.98 d (7.2) 104.3 4.92 d (7.2) 2 81.8 3.87* 81.8 3.88* 3 76.3 4.17* 76.3 4.18* 4 71.3 4.19 dd (9.0, 9.0) 71.4 4.13 dd (9.0, 9.0) 5 77.8 3.90* 77.8 3.84* 4.51−4.53* 4.41* 6 62.6 62.6 4.29* 4.32* Rha 1 99.0 5.65 br s 98.5 5.58 br s 2 73.4 6.13 br s 71.6 6.13 br s 3 69.6 5.14 dd (9.6, 3.0) 69.5 5.07 dd (9.6, 3.0) 4 78.2 4.28* 79.2 4.23* 5 68.1 4.47* 68.1 4.32* 6 19.2 1.56 d (6.0) 19.2 1.59 d (6.0) Rha′ 1 98.5 6.20 br s 99.0 6.19 br s 2 73.0 6.09 br s 72.4 6.02 br s 3 79.3 4.71 dd (9.0, 2.4) 79.7 4.65 dd (10.0, 2.4) 4 80.2 4.27* 80.2 4.21* 5 68.1 4.30* 68.0 4.48* 6 18.4 1.64 d (6.0) 18.4 1.64 d (6.0) Rha′′ 1 103.4 6.04 br s 103.3 5.97 br s 2 69.8 5.00 br s 72.3 4.77 br s 3 73.3 5.95 dd (3.0, 10.0) 73.0 5.89 dd (3.0, 10.0) 4 73.4 6.11 t (10.0) 73.4 6.13 dd (10.0, 10.0) 5 68.5 4.56* 68.5 4.30* 6 17.6 1.36 d (6.5) 17.6 1.43 d (6.0) Rha 1 104.3 5.70 br s 104.4 5.63 br s 2 72.4 4.85 br s 69.8 4.95 br s 3 72.3 4.54* 72.3 4.47* 4 73.4 4.26* 73.2 4.19* 5 70.6 4.37* 70.5 4.41* 6 18.6 1.58 d (6.0) 18.7 1.61 d (6.0) Ag 1 173.2 173.0 2 34.2 2.44 m; 2.34 m 34.3 2.40, m; 2.28, m 11 82.6 3.90* 82.6 3.90* 16 14.0 0.88 t (7.0) 14.0 0.84 t (7.0) Cna 1 166.2 166.2 2 118.1 6.54 d (16.0) 118.1 6.61 d (16.0) 3 145.3 7.81 d (16.0) 145.3 7.87 d (16.0) 1′ 134.4 134.5 2′ and 6′ 128.4 7.43 m 128.4 7.50 m 3′ and 5′ 129.1 7.33 m 129.0 7.38 m 4′ 130.6 7.33 m 130.6 7.38 m Deca 1 172.7 172.7 2 34.2 2.47 m 34.0 2.30 m 12 14.0 0.85 t (7.0) 14.0 0.80 t (7.0) Iba 1 176.2 2 34.0 2.67 m 3 19.0 1.16 d (7.0) 3′ 18.7 1.15 d (7.0) Hexa 1 173.2 2 34.3 2.43 m 6 13.8 0.67 t (7.0) Chemical shifts marked with an asterisk (*) indicate overlapped signals and all assignments are based on 1H-1H TOCSY experiments; Abbreviations: Glc: glucose; Rha: rhamnose; Ag: 11-hydroxyhexadecanoyl; Cna: trans-cinnamoyl; Deca: n-decanoyl; Hexa: n-hexanoyl; Iba: isobutyryl

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protons at δH 7.33–7.43 (m, C6H5 of Cna). A methyl triplet signal at 0.85 and a triplet-like signal for a methylene group at

CH2-2 (2.47, m) of a decanoyl group were indicated. Two methyl signals at 1.15 (1H, d, J = 7.0 Hz) and 1.16 (1H, d, J =

7.0 Hz) and a methylene group at CH2-2 (2.67, m) gave an isobutyryl group. The 13C NMR data of compound 1 exhibited five signals at δC 104.3, 103.4, 99.0, and 98.5 assigned to anomeric carbons of five sugar units, and δC 176.2, 173.2, 172.7, and 166.2 were four ester carbonyl carbons. Then the anomeric protons at 4.98 (d, J = 7.2 Hz), 5.70 (br s), 6.04 (br s), 5.65 (br s) and 6.20 (br s) were defined by HSQC data, respectively. All protons in each saccharide system were assigned by 2D NMR TOCSY, HMBC, and HSQC experiments, leading to the identification of one glucopyranosyl Fig. 2 Key HMBCs from H to C for compound 1 unit and four rhamnopyranosyl units as the monosaccharides 1 405.749 6) [M + Na]+. Its IR spectrum gave peaks of present in compound 1. The interglycosidic connectivity was hydroxyl (3 443 cm1), carbonyl (1 724 cm1), and aromatic determined from the following HMBC (Fig. 2): C-2 (81.8) of (1 641 cm1) groups. Alkaline hydrolysis of compound 2 glucose with H-1 (5.65) of rhamnose; C-4 (78.2) of rhamose with H-1 (6.20) of rhamnose′; C-4 (80.2) of rhamnose′ with afforded organic fractions, which were identified as  H-1 (6.04) of rhamnose′′; and C-3 (79.3) of rhamnose′ with n-hexanoic acid methyl ester (tR = 12.37 min): m/z 131 [M] H-1 (5.70) of rhamnose′′′. Esterification positions were (4), 99 (30), 87 (42), 74 (100), 43 (40) , n-decanoic acid (tR = 12.37 min): m/z 172 [M] (4), 155 (5), 143 (30), 129 (5), 87 determined by HMBC data between H-4 of rhamnose′′ (δH (59), 74 (100), 55 (18) and trans-cinnamic acid methyl ester 6.11) and δC 176.2 (C-1 of Iba); H-3 of rhamnose′′ (δH 5.95) (t = 13.29 min) m/z [M]+ 162 (40), 131 (100), 103 (66), 77 and δC 166.2 (C-1 of Cna); H-2 of rhamnose′ (δH 6.09) and δC R (32) by GC-MS experiment with the same chromatographic 172.7 (C-1 of Deca); and H-2 of rhamnose (δH 6.13) and δC 173.2 (C-1 of aglycone), respectively. The position of the conditions as compound 1 and simonic acid A (compound 3). jalapinolic acid unit was finally determined by HMBC to So L-rhamnose and D-glucose were identified in compound 2 be between jalapinolic acid H-11 (3.90) and glucose C-1 by the same program detection as compound 1. (104.3), which correlations established the structure of The NMR spectra (Tables 1) of compound 2 were similar compound 1 (Fig. 2). From these observations, the to that of compound 1; the interglycosidic connectivities and structure of cairicoside A (compound 1) was elucidated as esterification sites were all the same though by 2D NMR (11S)-jalapinolic acid 11-O- α-L-rhamnopyranosyl-(1→3)- TOCSY, HMBC and HSQC, with just one group of hexanoyl O-[3-O-trans-cinnamoyl-4-O-isobutyryl-α-L-rhamnopyran cairicoside A being different by GC-MS experiments. osyl-(1→4)]-O-[2-O-n-decanoyl]-α-L-rhamnopyrano-syl- Accordingly, the structure of compound 2 (cairicoside B) was (1→4)-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyran elucidated as (11S)-jalapinolic acid 11-O-α-L-rhamnopyranosyl- oside-(1, 2′′-lactone) (Fig. 1). (1→3)-O-[3-O-trans-cinnamoyl-4-O-hexanoyl-α-L-rhamnopy rano-syl-(1→4)]-O-[2-O-n-decanoyl]-α-L-rhamnopyranosyl-(1 →4)-O-α-L-rhamnopyrano-syl-(1→2)-O-β-D-glucopyranoside- (1, 2′′-lactone) (Fig. 1). The α-glucosidase inhibitory activities of the two compounds were analyzed using acarbose as a cotnrol.

Compounds 1 and 2 exhibited strong inhibitory activity (IC50 25.3 ± 1.6 and 28.5 ± 3.3 μmol·L−1, respectively (Table 2).

Table 2 α-Glucosidase inhibition of compounds 1 and 2 α-Glucosidase inhibition contstanta Compound −1 IC50/(μmol·L ) 1 25.3 ± 1.6

2 28.5 ± 3.3 Fig. 1 Structures of compounds 1 and 2 Acarboseb 385.0 ± 9.3 Compound 2 was obtained as a white, amorphous powder; a IC50 is defined as the concentration that resulted in a 50% α-glucosidase the molecular formula C71H114NaO26 was determined with inhibition and the results are means ± SD, n = 3; b Positive control + HR-TOF-MS at m/z [M + Na] 1 405.818 8 (Calcd. as substance

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Experimental chromatography, and then subjected to preparative HPLC to afford compound 2 (21 mg). General experimental procedures Alkaline hydrolysis of compounds 1 and 2 The IR spectra were measured on a Shimadzu ftir (FTIR) Compounds 1 and 2 (8 mg each) in 5% KOH (3 mL) spectrophotometer (Shimadzu, Tokyo, Japan) and UV on a were refluxed at 90 ºC for 2 h, respectively. The reaction Shimadzu UV-2550 spectrophotometer. The NMR spectra mixture was acidified to pH 4.0 with 2 mol·L−1 HCl and were recorded on INOVA 500 spectrometers (Bruker, Bremen, extracted with hexane (3 mL × 2) and n-BuOH (3 mL × 2). Germany, 1H NMR, HSQC and HMBC at 500 MHz; 13C The organic layer was washed with H2O, dried over NMR at 125 MHz) using C5D5N as solvent, with anhydrous Na2SO4, and then methylated by following method tetramethylsilane (TMS) as internal reference. The chemical [13]. The hexane extract was combined in 0.1 mL of 0.5 shifts were given in δ and coupling constants in Hz. The −1 mol·L of CH3ONa solution and then rocked at room HRTOF-MS experiments were performed on Triple TOF temperature for 5 min; after adding 5 μL CH3COOH and 1 g 5600 plus MS spectrometer (AB SCIEX, Framingham, MA, anhydrous CaCl2 powder and stewing 1 h, the mixture was USA). GC-MS experiment was performed on a TRACE GC centrifuged at 2 000−3 000 r·min−1 for 2−3 min, and the Ⅱ ULTRA DSQ instrument (Thermo Scientific, Shanghai, supernatant was collected and then analyzed by GC-MS on a China). The optical rotations were measured with an Anton TRACE GC ULTRA DSQⅡ instrument under the following Paar-MCP600 polarimeter (Anton Paar, Maharastra, ) in conditions: 30 m × 0.25 mm × 0.25 m, TG-5MS (Thermo) MeOH solution. The centrifugation was done with column; He, 0.8 mL·min−1; 40 ºC, 3 min; 50310 ºC, 10 ALLEGRA X-12 (Beckman, Shanghai, China). The ºC·min−1, 70 eV. preparative HPLC was performed using an Shimadzu Acid hydrolysis and sugar analysis LC-6AD series instrument (Shimadzu, Tokyo, Japan) The glycosidic acid (compound 3 [10], 5 mg, from alkaline equipped with a UV detector at 280 nm and Shim-Park RP-C18 hydrolysis) was methylated with MeOH and catalyzed column (Shimadzu, Tokyo, Japan) (20 mm × 200 mm i.d.). with 0.5 mol·L−1 HSO to give simonic acid A methyl Adsorbents for column chromatography were silica gel 2 4 ester (compound 4). Compound 4 was hydrolyzed with (200−300 μm, Qingdao Marine Chemical Co., Ltd., China), 1 mol·L−1 HSO and extracted with ether to obtain Sephadex LH-20 (75−150 μm, Pharmacia, Uppsala, 2 4 11-hydroxyhexadecanoic acid methyl ester (compound 5). Sweden), ODS (40−63 μm, FuJi, Japan). The thin-layer The aqueous layer of acidic hydrolysis was concentrated chromatography was performed on pre-coated silica gel GF254 under reduced pressure to give a residue of the sugars. The plates (Qingdao Marine Chemical Co., Ltd., China) and residue was dissolved in pyridine (0.1 mL), to which 0.08 mol·L−1 detected by spraying with 10% H2SO4-EtOH. α-Glucosidase L-cysteine methyl ester hydrochloride in pyridine (0.15 mL) was monitored continuously with an auto multi-functional was added. The mixture was kept at 60 ºC for 1.5 h. After the microplate reader ELX800 (BioTek Instruments, Inc). reaction mixture was dried in vacuo, the residue was Acarbose was obtained from Aladdin company (No. l1424006, trimethylsilylated with 1-trimethylsilylimidazole (0.1 mL) for Aladdin Company, Shanghai, China), α-Glucosidase (No. 2 h. The mixture was partitioned between n-hexane and H O M0035-15) and p-nitrophenyl-α-D-glucopy-ranoside (PNPG) (No. 2 (0.3 mL each) and then-hexane extract was analyzed by M0103) were purchased from Sigma Company (San Ⅱ Francisco, CA, USA). GC-MS on a TRACE GC ULTRA DSQ instrument under materials the following conditions: 30 m × 0.25 mm × 0.25 m, −1 The aerial parts of I. cairica were collected at Guangzhou, TG-5MS (Thermo) column; He, 0.8 mL·min ; 60 ºC, 3 min; −1 Guangdong Province, China, in Agust 2012, and identified by 60180 ºC, 10 ºC·min keep 3 min, 180205 ºC, 3 Prof. LIU Ji-Zhu. A voucher specimen (No. 2012-8) was ºC·min−1 keep 5 min, 205300 ºC, 20 ºC·min−1 keep 5 min, deposited at Department of Traditional Chinese Medicinal 70 eV. In the acid hydrolysate of simonic acid A, methyl ester Chemistry, Guangdong Pharmaceutical University. L-rhamnose and D-glucose were confirmed by comparison of Extraction and isolation their retention times of their derivatives with those of

The aerial parts (5 kg) of I. cairica were cut to pieces and authentic L-rhamnose (tR = 30.14 min) and D-glucose (tR = extracted twice with 95% EtOH under reflux for 2 h each. 31.65 min) derivatives prepared in the same way, respectively. The extract (230 g) was suspended in water and partitioned Preparation of Mosher’s esters with CHCl3. The crude CHCl3 extract (107 g) was first The procedures for preparation of Mosher’s esters for chromatographed on a silica gel packed column and eluted determination of absolute configuration of 11S of the with CH2Cl2−MeOH (98 : 2 to 0 : 100) to give fractions 1 to 7. aglycone were same as described previously for resin Fraction 2 was chromatographed on silica gel eluted with glycosides from Ipomoea batatas [14].

CHCl3−MeOH (100 : 0 to 0 : 100), then purified by ODS and Enzyme inhibition assay preparative HPLC to obtain compound 1 (15 mg). Fraction 3 The α-glucosidase inhibition assay was performed [15] was subjected to a silica gel column eluted with CH2Cl2− according to the slightly modified method of Pierre et al. . MeOH (20 : 1 to 2 : 1), purified by Sephadex LH-20 (MeOH) The inhibition was measured spectrophotometrically at pH

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−1 6.8, 37 ºC, for 10 min, using 0.01 mol·L p-nitrophenyl Purgin II, a resin glycoside ester-type dimer and inhibitor of −1 α-D-glucopyranoside (PNPG) as a substrate and 1 U·mL of multidrug efflux pumps from [J]. J Nat Prod, −1 enzyme, in 0.067 mol·L of KH2PO4-Na2HPO4 buffer. 2013, 76(1): 64-71. Acarbose was used as positive control. The increment in [7] Song LY, Wu JR, Li CH, et al. Different responses of invasive absorbance at 410 nm due to the hydrolysis of PNPG by and native species o elevated CO2 concentration [J]. Acta a-glucosidase was monitored continuously with an auto Oecologica, 2009, 35(1): 128-135. multi-functional microplate reader ELX800 (BioTek [8] Ferreira AA, Amaral FA, Duarte IDG, et al. Antinociceptive Instruments, Inc., Winooski, VT, USA). effect from Ipomoea cairica extract [J]. J Ethnopharmacol, 2006, 105(2): 148-153. References [9] Li ZY, Xie Y, Zhang JS, et al. Invasive species in China [M]. China Forest Press, 2001, 6: 187-188. [1] Lu SF, Guo ZW, Ouyang QQ, et al. Recent progress on the [10] Yu BW, Luo JG, Wang JS, et al. Pentasaccharide resin research of resin glycosides [J]. Chin J Org Chem, 1997, 17(6): glycoside from Ipomoea cairica and their cytotoxic activities 488-497. [J]. Phytochemistry, 2013, 95 (11): 421-427. [2] Yin YQ, YiL, Kong LY. Pentasaccharide glycosides from the [11] Gong J, Ni SF, Zhao T, et al. Study on medical functionality tubers of (Ipomoea batatas) [J]. Agric Food Chem, and health benefits of [J]. J Anhui Arti Sci, 2008, 56(7): 2363-2368. 2010, 38(21): 11124-11125. [3] Ding WB, Jiang ZH, Wu P, et al. Resin glycosides from the [12] Erum A, Itrat A, Saeed A, et al. α-Glucosidase inhibitory aerial parts of Operculina turpethum [J]. Phytochemistry, 2012, constituets from Cuscuta reflexa [J]. Chem Pharm Bull, 2002, 81(9): 165-174. [4] Ono M, Akiyama K, Kishida M, et al. Two new glycosidic 50(1): 112-114. acids multifidinicacids F and G, of the ether-insoluble resin [13] She ZH. Oil fatty acid derivatization method and choose in gas glycoside (convolvulin) from the seeds of Quamoclit multifida chromatography [J]. Grain Proc, 2004, 29(6): 64-66. [J]. J Nat Med, 2013, 67(4): 822-826. [14] Yin YQ, Huang XF, Kong LY, et al. Three new pentasaccharide [5] Wang WQ, Song WB, Lan XJ, et al. Merremins A-G, resin resin glycosides from the roots of sweet potato (Ipomoea glycosides from Merremia hederacea with multidrug batatas) [J]. Chem Pharm Bull, 2008, 56(12): 1670-1674. resistance reversal activity [J]. J Nat Prod, 2014, 77(10): [15] Pierre C, Roland R, Tremblay D. P-Nitrophenol-α- 2234-2240. lucopyramoside as substrate for measurement of maltase [6] Castañeda-Gómez J, Figueroa-González G, Jacobo N, et al. activity in human semen [J]. J Clin Chem, 1978, 24: 208-211.

Cite this article as: LI Jie-Hong, PAN Jie-Tao, YIN Yong-Qin. Two new resin glycosides isolated from Ipomoea cairica with α-glucosidase inhibitory activity [J]. Chin J Nat Med, 2016, 14(3): 227-231.

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