Chinese Journal of Natural Medicines 2014, 12(4): 0300−0304
Chinese Journal of Natural Medicines
Chemical constituents from the stems of Gymnema sylvestre LIU Yue 1, XU Tun-Hai 2, ZHANG Man-Qi 3, LI Xue 1, XU Ya-Juan 1, 4*, JIANG Hong-Yu 5*, LIU Tong-Hua 2, XU Dong-Ming 1 1
Jilin Academy of Chinese Medicine Sciences, Changchun 130012, China;
2
Department of Traditional Chinese Medicine Chemistry, School of Traditional Chinese Medicine, Beijing University of Chinese Medi-
cine, Beijing 100102, China; 3
School of Pharmacy, China Pharmaceutical University, Nanjing 211198, China;
4
School of Pharmacy, Jilin University, Changchun 130021, China;
5
Department of General Internal Medicine, The First Hospital of Jilin University, Jilin University, Changchun 130021, China Available online 20 Apr. 2014
[ABSTRACT] AIM: To study the chemical constituents of stems of Gymnema sylvestre (Retz.) Schult. METHODS: Chromatographic techniques using silica gel, C18 reversed phase silica gel, and prep-HPLC were used. The structures were elucidated on the basis of MS and spectroscopic analysis (1D and 2D NMR), as well as chemical methods. RESULTS: Seven compounds were isolated and their structures were elucidated as conduritol A (1), stigmasterol (2), lupeol (3), stigmasterol-3-O-β-D-glucoside (4), the sodium salt of 22α-hydroxy-longispinogenin-3-O-β-D-glucopyranosyl-(1→3)-β-D-glucurono-pyranosyl-28-O-α-L-rhamnopyranoside (5), oleanolic acid-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (6), and the sodium salt of 22α-hydroxy-longispinogenin 3-O-β-D-glucuronopyranosyl-28-O-α-L-rhamnopyranoside (7). The inhibition activities of compounds 1, 5−7 on non-enzymatic glycation of protein in vitro were evaluated. CONCLUSION: Compound 7 is a new triterpenoid saponin. It was shown that compounds 1, 5−7 have weak inhibition activities for non-enzymatic glycation of protein in vitro. [KEY WORDS] Gymnema sylvestre; Triterpenoid saponin; Non-enzymatic glycation-inhibiting activity
[CLC Number] R284.1
[Document code] A
[Article ID] 2095-6975(2014)04-0300-05
Gymnema sylvestre (Retz.) Schult. (Asclepiadaceae), which is distributed in India and southwestern China, has been used as anti-diabetic remedy. Total saponins of its leaves have an antisweet effect, inhibiting glucose absorption in the small intestine and suppressing elevated glucose levels in blood following the administration of sucrose in rats [1]. In addition, it has hypolipidemic, anti-atherosclerosis, anticaries, and anti-obesity effects [2-7]. In contrast, the constituents of its stems and their pharmacological effects were seldom studied. The nature and contents of the chemical constituents in the [Received on] 03-Feb.-2013 [Research funding] This project was supported by Program for the Development of Scientific and Technological Plan of Jilin Province (No. 20100122). [*Corresponding author] XU Ya-Juan: Prof., Tel: 86-431-8605 8690, E-mail: [email protected]; JIANG Hong-Yu: Associate Prof., E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
stems are different from those in the leaves [8]. In order to elucidate the feasibility as an anti-diabetic medicinal part and the reasonable development and utilization of the stems, this investigation of the constituents of the stems led to the isolation of a new triterpenoid saponin (7), along with six known constituents: conduritol A (1), stigmasterol (2), lupeol (3), stigmasterol-3-O-β-D-glucoside (4), the sodium salt of 22α-hydroxy-longispinogenin-3-O-β-D-glucopyranosyl-(1→ 3)-β-D-glucuronopyranosyl-28-O-α-L-rhamnopyranoside (5), and oleanolic acid-3-O-β-D-glucopyranosyl-(1→6)-β-Dglucopyranoside (6). Compounds 3 and 6 were obtained from the genus Gymnema and compound 5 was obtained as the sodium salt form for the first time. Inhibition activities of compounds 1, 5−7 on the non-enzymatic glycation of protein in vitro were evaluated.
Results and Discussion Compound 7, obtained as a white amorphous powder, mp 247.4−247.7 °C. Its HR-MS offered a [M + H]+ ion peak at m/z
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LIU Yue, et al. / Chin J Nat Med, 2014, 12(4): 300−304
819.446 7 (C42H68O14Na, Calcd. 819.450 1) in positive-ion mode. MALDI-TOF-MS showed molecular ion peak m/z 819 [M + H]+. The occurrence of a weak quasi-molecular ion peak m/z 841 [M + Na]+ inferred that 7 has a formula of C42H67O14Na. MALDI-TOF-MS/MS showed fragment ion peaks at m/z 643 [M + H − 176]+, 625 [M + H − 194]+ and 479 [M + H −194 − 146]+ due to the loss of a terminal glucuronic acid and a deoxyhexose unit. In addition, fragment ion peaks at m/z 217 and 199 also inferred the existence of a GlcA unit. In 13C NMR spectrum (100 MHz, pyridine-d5), thirty carbon signals were assigned to a triterpenoid aglycone and twelve to sugar units. Seven signals of the aglycone part were assigned to methyl carbons at δ 15.9, 17.3, 17.5, 25.0, 27.8, 28.5, and 33.7. Three oxygen-bearing methine carbon signals at δ 66.6, 69.3, and 89.5, a hydroxymethyl carbon signal at δ 64.6, and a pair of olefinic carbon signals at δ 123.5 and 142.9 were observed. Accordingly, in the 1H NMR spectrum (400 MHz, pyridine-d5), seven methyl group signals at δ 0.73 (3H, s), 0.76 (3H, s), 0.88 (3H, s), 0.89 (3H, s), 1.06 (3H, s), 1.16 (3H, s), and 1.29 (3H, s), three oxymethine signals at δ 3.22 (1H, dd, J = 11.7, 4.0 Hz), 4.95 (1H, br s), 4.75 (1H, dd, J = 12.1, 5.5 Hz), one oxymethylene at δ 4.18 (1H, d, J = Table 1
10.0 Hz) and 4.90 (1H, d, J = 11.0 Hz), and an olefinic proton signal at δ 5.37 (1H, m) were observed. All of the 1H- and 13C NMR data (Table 1) were as- signed 1 by H-1H COSY, HMQC, and HMBC experiments. By comparison of the NMR data with those of alternosides II, IV, and V reported previously [9-10], the aglycone of 7 was deduced to a 3, 16, 22, 28-tetrahydroxy- olean-12-ene. The oxymethylene proton signals at δ 4.18 and 4.90 were diagnostic for H-28 [9]. The oxymethine proton signal at δ 4.95 correlated with C-15 (δ 36.9) and C-28 (δ 64.6) in the HMBC spectrum, and could be assigned to H-16. The correlation between H-16 (δ 4.95) and H-27 (δ 1.92) in the NOESY spectrum suggested an α- configuration of H-16. The proton signal at δ 4.75 could be assigned to H-22 on the basis of its correlations with C-18 (δ 43.8), C-20 (δ 32.3), C-21 (δ 44.3), and C-28 (δ 64.6) in the HMBC spectrum. The correlation between H-22 (δ 4.75) and H-30 (δ 0.89) in the NOESY spectrum indicated a β-configuration of H-22, which was also supported by the splitting pattern of H-22 (1H, dd, J = 12.1, 5.5 Hz). Thus, the alglycone of 7 could be assigned as 3β, 16β, 22α, 28β-tetrahydroxyolean-12-ene, i.e., 22α-hydroxy-longispinogenin, from its NOESY, HMQC, and HMBC spectra.
1
H NMR and 13C NMR data for compound 7 (400 MHz and 100 MHz, pyridine-d5)
C
δc
δH (J, Hz)
C
δc
δH (J, Hz)
1
39.0
0.80 (m), 1.37 (m)
23
28.5
1.16 (s)
2
26.5
1.78 (m), 1.97 (m)
24
17.3
0.76 (s)
3
89.5
3.22 (dd, 11.7, 4.0)
25
15.9
0.73 (s)
4
39.7
-
26
17.5
1.06 (s)
5
56.0
0.73 (m)
27
27.8
1.29 (s)
64.6
4.18 (d, 10.0), 4.90 (d,11.0)
19.2
1.48 (m), 161 (m)
7
33.3
1.32 (m), 1.57 (m)
29
33.7
0.88 (s)
8
40.5
-
30
25.0
0.89 (s)
9
47.3
1.52 (m)
C-3
10
36.9
-
GlcA-1′
106.7
4.67 (d, 7.6)
11
24.3
1.86 (m)
2′
75.4
3.93
12
123.5
5.37 (m)
3′
78.4
4.19
13
142.9
-
4′
73.6
4.12
14
42.8
-
5′
76.7
4.14
15
36.9
1.53 (m), 1.96 (m)
6′
176.9
16
66.6
4.95 (br s)
C-28
17
44.8
-
Rha-1″
102.0
5.17 (br s)
18
43.8
2. 90 (m)
2″
72.7
4.38
19
46.8
1.10 (m),1.90 (m)
3″
73.2
4.36
20
32.3
-
4″
74.0
4.16
21
44.3
1.75 (m), 2.03 (m)
5″
70.1
4.12
22
69.3
4.75 (dd, 12.1, 5.5)
6″
19.2
1.57 (d, 6.0)
6
28
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LIU Yue, et al. / Chin J Nat Med, 2014, 12(4): 300−304
Acid hydrolysis of 7 afforded a mixture of D-glucuronic acid and L-rhamnose by high-performance thin-layer chromatography (HPTLC). 1H- and 13C NMR resonances of the sugar units indicated the presence of glucuronic acid and rhamnose. Anomeric carbon signals at δC 106.7 and 102.0 and anomeric proton signals at δH 4.67 (1H, d, J = 7.6 Hz) and 5.17 (1H, br s) were observed in 1H and 13 C NMR spectra, respectively. The coupling constant (7.6 Hz) of the anomeric proton of GlcA, along with the correlation between H-1′ (δ 4.67) and H-5′ (δ 4.14) in the NOESY experiment, indicated a β configuration for the glucuronic acid moiety. The coupling pattern of the anomeric proton and the existence of a NOESY cross peak between H-1″ and H-2″ indicated an α configuration for the rhamnose unit [11]. The respective locations of the sugar moieties were subsequently deduced from the 2D NMR data. In the HMBC spectrum (Fig. 2), correlations between H-1′ (δ 4.76) of glucuronic acid and C-3 (δ 89.5) of the aglycone, and between H-1″ (δ 5.17) of rhamnose and C-28 (δ 64.6) of the aglycone inferred that the glucuronopyranosyl and rhamnopyranosyl units were linked at C-3 and C-28, respectively. Thus, the skeleton structure of 7 was 3-O-β-D-glucuronopyranosyl-3β, 16β, 22α, 28β-tetrah-
ydroxy-olean-12-ene 28-O-α-L-rhamnopyranoside (22α-hydroxy-longispinogenin 3-O-β-D-glucuronopyranosyl-28-Oα-L-rhamnopynoside), and is a new isolate. In addition, the carboxylic carbon signal (δ 176.9) of the glucuronic acid residue in 7 showed a significant downfield shift (+ 4.3) in comparison to that (δ 172.6) in the non-ionized state in the literature [12-13]. The isolated compound was therefore inferred to be a sodium salt. Some similar constituents as sodium (e.g., the sodium salt of alternoside II) or potassium salt forms with close carboxylic carbon signal values of GlcA were isolated previously from G. sylvestre [9]. It was also supported by the presence of a [(C42H67O14·Na) + H]+ ion at m/z 819 and the ion [(C 42 H 67 O 14 ·Na) + Na] + at m/z 841 in its MALDI-TOF MS and [C 42 H 67 O 14 ·Na + H] + at m/z 819.446 7 in its HR-MS. All of the available evidence led to the conclusion that 7 was the sodium salt of 3-O-βD-glucuronopyranosyl-3β, 16β, 22α, 28β-tetrahydroxyolean-12-ene 28-O-α-L-rhamnopyranoside (the sodium salt of 22α-hydroxy-longispinogenin 3-O-β-D-glucuronopyranosyl-28-O-α-L-rhamnopynoside). The structure is shown in Fig. 1, and the 13C- and 1H NMR data are shown in Table 1.
Fig. 1 Compounds 1−7 isolated from G. sylvestre
The known compounds, conduritol A (1) [14], stigmasterol (2) , lupeol (3) [17], stigmasterol-3-O-β-D-glucoside (4) [18] , the sodium salt of 22α-hydroxy-longispinogenin-3-O-β-Dglucopyranosyl-(1→3)-β-D-glucuronopyranosyl-28-O-α-Lrhamnopyranoside (5) [10], and oleanolic acid-3-O-β-Dglucopyranosyl-(1→6)-β-D-glucopyranoside (6) [1, 19] were identified by comparing their physical and spectroscopic data with reported data. An inhibition assay of compounds 1 and 5−7 on the non-enzymatic glycation of protein in vitro was carried out. Compounds 1 and 5−7 had much weaker inhibition activi[15-16]
Fig. 2 Key HMBC correlations of compound 7
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LIU Yue, et al. / Chin J Nat Med, 2014, 12(4): 300−304
ties than that of aminoguanidine hydrochloride. Changes of inhibition activities were apparently independent of the concentrations of samples. Table 2 Inhibition of compounds 1 and 5−7 on the non-enzymatic glycation of protein in vitro Sample/Control
Inhibition ratio/% −1
50 μg·mL
5 μg·mL−1
0.5 μg·mL−1
1
10.42
9.71
8.99
5
10.78
25.25
28.09
6
23.63
25.41
18.27
7 aminoguanidine hydrochloride (0.015 mol·L−1)
3.82
3.64
5.07
97.86
Experimental Apparatus and reagents MS spectra were recorded on a Finnigan MAT LCQ mass spectrometer and a MALDI/TOF-MS. The HR-MS was recorded on Autoflex III MALDI/TOF/TOF-MS. NMR spectra were obtained on Bruker AV400 and AV600 instruments. Melting points were obtained on an X-6 apparatus and are uncorrected. A RF-540 fluorescence spectrometer (Shimadzu), an LHP-160E intelligent constant temperature and humidity incubator (Nanbei Equipment Co., Ltd., Zhengzhou, China) and a SHA-C water bath constant-temperature oscillator (Jingbo Experimental Instrument Factory, Jintan, China) were applied in the activity assay. Prep-HPLC was performed using an ODS column (Zorbax PrepHT Eclipse XDB-C18, 21.2 mm × 250 mm, 7 μm) on Waters-600E HPLC. Column chromatographic separations were performed on silica gel (200-300 mesh, Qingdao Oceanic Chemical Industry, Qingdao, China) and reversed phase silica gel (50 μm) (YMC). Macroporous resin D21 was purchased from Shandong Lukang Pharmaceutical Co., Ltd. Bovine serum albumin and D-glucose were from Solarbio Science Co., Beijing, China aminoguanidine hydrochloride from Sigma-Aldrich, St. Louis, MO, USA, sodium azide from Huadong Reagent Factory, Tianjin, China, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and other reagents of analytical grade from Beijing Chemical Works, Beijing, China and those of chromatographic grade were from Tedia, USA were applied. Plant material The stems of G. sylvestre were purchased from a company in the Chinese Medicinal Materials market in Yulin, Guangxi Province, China, in September 2009, and authenticated by Prof. Guojun Xu, Jilin Academy of Chinese Medicine Sciences. The voucher specimen has been deposited in Jilin Academy of Chinese Medicine Sciences. Extraction and isolation The dried stems of G. sylvestre (9.5 kg) were extracted twice with 75% EtOH (100 and 80 L, respectively; each 8 h)
by percolation extraction processes. An aqueous solution of the extract was partitioned with EtOAc. The lower water solution was chromatographed on D21 porous resin (1.5 kg), eluting with water and 50% EtOH, successively, to give water-eluting part (81 g) and a 50% EtOH part (84 g). The water-eluting part (27 g) was chromatographed over a silica gel column eluted with CHCl3−MeOH gradients (30 : 1 to 5 : 1). Compound 1 was obtained from Frs. 85−232. The EtOAc part (132 g) was subjected to silica gel column chromatography with a CHCl3−MeOH gradient (2 : 0 to 2 : 1). Compounds 2 (65 mg) and 3 (40 mg) were isolated from Frs. 24-57, and compound 4 (34 mg) was isolated from Frs. 75−162 by repeated silica gel column chromatography. The 50% EtOH part was subjected to silica gel column chromatography with a CHCl3−MeOH gradient (20 : 1 to 1 : 1.5). Combined Frs. 110−166 was repeatedly chromatographed over a silica gel column (CHCl3−MeOH−n-BuOH gradients, 9 : 1 : 0.1 to 6 : 4 : 1). The Frs. 1−37 were successively chromatographed over a Rp-18 column (MeOH−H2O gradients, 50 : 50 to 80 : 20) and prep-HPLC (MeOH−H2O, 80 : 20) to give compound 6 (28 mg). The Frs. 90−115 were subjected to a Rp-18 column (MeOH−H2O gradients, 50 : 50 to 80 : 20) and then prep-HPLC (CH3CN−H2O, 20:80) to give compound 7 (18 mg). The Frs. 116−167 were subjected to a Rp-18 column (MeOH−H2O gradients, 50 : 50 to 80 : 20) and then prep-HPLC (CH3CN− H2O, 20 : 80) to give compound 5 (60 mg). Acid hydrolysis Compound 7 (10 mg) were heated with 1 mol·L−1 HCl in MeOH (10 mL) under reflux for 2 h. The reaction mixture was neutralized with NaHCO3. The water phase was chromatographed on the silica gel HPTLC with the system of n-BuOH−i-PrOH−H2O (10 : 5 : 4, homogenous), then the brown colored spots were visualized by spraying with phenylamine-o-benzenedicarboxylic acid reagent followed by heating. D-Glucuronic acid and L-rhamnose were detected by comparison with authentic samples. Non-enzymatic glycation of protein inhibition assay in vitro Inhibition activities in vitro of the non-enzymatic glycation of protein of the candidate compounds were evaluated by determination of the fluorescence intensity of advanced glycosylation end products (AGEs) of proteins [20]. Bovine serum albumin (20 mg) and D-glucose (90.1 mg) were added into 5 mL phosphate solutions (pH 7.4, NaN3 3 mmol·L−1) of the control and each sample. Aminoguanidine hydrochloride was used as the positive control. Corresponding blank solutions were also prepared. Every sample was prepared in triplicate. All sample solutions were incubated for 7 days at 37 °C, in a constant temperature and humidity environment. The fluorescence intensities were determined at the excitation/emission wavelength of 370/440 nm. The inhibition rate (I) was calculated by the following formula: I (%) = [1 − (S − S0) / (Nc − Nc0)] × 100. S: fluorescence intensity in positive control or sample
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LIU Yue, et al. / Chin J Nat Med, 2014, 12(4): 300−304
groups; S0: fluorescence intensity in positive control or sample blank groups without D-glucose; Nc: fluorescence intensity in negative control groups without sample; Nc0: fluorescence intensity in negative control blank groups without sample and D-glucose
[11]
[12]
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Ye WC, Zhang QW, Liu X, et al. Oleanane saponins from Gymnema sylvestre [J]. Phytochemistry, 2000, 53 (8): 893-899. [2] Wang XH. Progress on diabetes treatment of Gymnema sylvestre [J]. J Med Res, 2009, 38(1): 15-16. [3] Qin JJ, Zhen HS, Fang H, et al. Study on hypoglycemic effect of gymnemic acid [J]. Chin J Inf Tradit Chin Med, 2000, 7(3): 28-31. [4] Baskaran K, Kizar AB, Radha SK, et al. Antidiabetic effect of a leaf extract from Gymnema sylvestre in non-insulindependent diabetes mellitus patients [J]. J Ethnopharmacol, 1990, 30(3): 295-300. [5] Bishayee A, Chatterjee M. Hypolipidaemic and antiatherosclerotic effects of oral Gymnema syIvestre R. Br. leaf extract in albino rats fed on a high fat diet [J]. Phytother Res, 1994, 8(2): 118-120. [6] Wang LF, Luo H, Miyoshi M, et al. Inhibitory effect of gemnemic acid on intestinal absoprtion of oleic acid in rats [J]. Can J Physiol Pharmacol, 1998, 76(10-11): 1017-1023. [7] Zhu JG, Chen YG, Li H, et al. Study on hypoglycemic and hypolipidemic effects of compound Gymnema syIvestre Granules [J]. Chin Tradit Pat Med, 1997, 19(8): 38-40. [8] Zhen HS, Ma LF, Tang BL, et al. Pharmacognosy identification of Gymnema syIvestre [J]. J Chin Med Mater, 1997, 20(2): 70-72. [9] Ye WC, Liu X, Zhang QW, et al. Antisweet saponins from Gymnema sylvestre [J]. J Nat Prod, 2001, 64(2): 232-235. [10] Yoshikawa K, Ogata H, Arihara S, et al. Antisweet natural
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Cite this article as: LIU Yue, XU Tun-Hai, ZHANG Man-Qi, LI Xue, XU Ya-Juan, JIANG Hong-Yu, LIU Tong-Hua, XU Dong-Ming. Chemical constituents from the stems of Gymnema sylvestre [J]. Chinese Journal of Natural Medicines, 2014, 12(4): 300-304
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Chinese Journal of Natural Medicines
Chemical constituents from the stems of Gymnema sylvestre LIU Yue 1, XU Tun-Hai 2, ZHANG Man-Qi 3, LI Xue 1, XU Ya-Juan 1, 4*, JIANG Hong-Yu 5*, LIU Tong-Hua 2, XU Dong-Ming 1 1
Jilin Academy of Chinese Medicine Sciences, Changchun 130012, China;
2
Department of Traditional Chinese Medicine Chemistry, School of Traditional Chinese Medicine, Beijing University of Chinese Medi-
cine, Beijing 100102, China; 3
School of Pharmacy, China Pharmaceutical University, Nanjing 211198, China;
4
School of Pharmacy, Jilin University, Changchun 130021, China;
5
Department of General Internal Medicine, The First Hospital of Jilin University, Jilin University, Changchun 130021, China Available online 20 Apr. 2014
[ABSTRACT] AIM: To study the chemical constituents of stems of Gymnema sylvestre (Retz.) Schult. METHODS: Chromatographic techniques using silica gel, C18 reversed phase silica gel, and prep-HPLC were used. The structures were elucidated on the basis of MS and spectroscopic analysis (1D and 2D NMR), as well as chemical methods. RESULTS: Seven compounds were isolated and their structures were elucidated as conduritol A (1), stigmasterol (2), lupeol (3), stigmasterol-3-O-β-D-glucoside (4), the sodium salt of 22α-hydroxy-longispinogenin-3-O-β-D-glucopyranosyl-(1→3)-β-D-glucurono-pyranosyl-28-O-α-L-rhamnopyranoside (5), oleanolic acid-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (6), and the sodium salt of 22α-hydroxy-longispinogenin 3-O-β-D-glucuronopyranosyl-28-O-α-L-rhamnopyranoside (7). The inhibition activities of compounds 1, 5−7 on non-enzymatic glycation of protein in vitro were evaluated. CONCLUSION: Compound 7 is a new triterpenoid saponin. It was shown that compounds 1, 5−7 have weak inhibition activities for non-enzymatic glycation of protein in vitro. [KEY WORDS] Gymnema sylvestre; Triterpenoid saponin; Non-enzymatic glycation-inhibiting activity
[CLC Number] R284.1
[Document code] A
[Article ID] 2095-6975(2014)04-0300-05
Gymnema sylvestre (Retz.) Schult. (Asclepiadaceae), which is distributed in India and southwestern China, has been used as anti-diabetic remedy. Total saponins of its leaves have an antisweet effect, inhibiting glucose absorption in the small intestine and suppressing elevated glucose levels in blood following the administration of sucrose in rats [1]. In addition, it has hypolipidemic, anti-atherosclerosis, anticaries, and anti-obesity effects [2-7]. In contrast, the constituents of its stems and their pharmacological effects were seldom studied. The nature and contents of the chemical constituents in the [Received on] 03-Feb.-2013 [Research funding] This project was supported by Program for the Development of Scientific and Technological Plan of Jilin Province (No. 20100122). [*Corresponding author] XU Ya-Juan: Prof., Tel: 86-431-8605 8690, E-mail: [email protected]; JIANG Hong-Yu: Associate Prof., E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
stems are different from those in the leaves [8]. In order to elucidate the feasibility as an anti-diabetic medicinal part and the reasonable development and utilization of the stems, this investigation of the constituents of the stems led to the isolation of a new triterpenoid saponin (7), along with six known constituents: conduritol A (1), stigmasterol (2), lupeol (3), stigmasterol-3-O-β-D-glucoside (4), the sodium salt of 22α-hydroxy-longispinogenin-3-O-β-D-glucopyranosyl-(1→ 3)-β-D-glucuronopyranosyl-28-O-α-L-rhamnopyranoside (5), and oleanolic acid-3-O-β-D-glucopyranosyl-(1→6)-β-Dglucopyranoside (6). Compounds 3 and 6 were obtained from the genus Gymnema and compound 5 was obtained as the sodium salt form for the first time. Inhibition activities of compounds 1, 5−7 on the non-enzymatic glycation of protein in vitro were evaluated.
Results and Discussion Compound 7, obtained as a white amorphous powder, mp 247.4−247.7 °C. Its HR-MS offered a [M + H]+ ion peak at m/z
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LIU Yue, et al. / Chin J Nat Med, 2014, 12(4): 300−304
819.446 7 (C42H68O14Na, Calcd. 819.450 1) in positive-ion mode. MALDI-TOF-MS showed molecular ion peak m/z 819 [M + H]+. The occurrence of a weak quasi-molecular ion peak m/z 841 [M + Na]+ inferred that 7 has a formula of C42H67O14Na. MALDI-TOF-MS/MS showed fragment ion peaks at m/z 643 [M + H − 176]+, 625 [M + H − 194]+ and 479 [M + H −194 − 146]+ due to the loss of a terminal glucuronic acid and a deoxyhexose unit. In addition, fragment ion peaks at m/z 217 and 199 also inferred the existence of a GlcA unit. In 13C NMR spectrum (100 MHz, pyridine-d5), thirty carbon signals were assigned to a triterpenoid aglycone and twelve to sugar units. Seven signals of the aglycone part were assigned to methyl carbons at δ 15.9, 17.3, 17.5, 25.0, 27.8, 28.5, and 33.7. Three oxygen-bearing methine carbon signals at δ 66.6, 69.3, and 89.5, a hydroxymethyl carbon signal at δ 64.6, and a pair of olefinic carbon signals at δ 123.5 and 142.9 were observed. Accordingly, in the 1H NMR spectrum (400 MHz, pyridine-d5), seven methyl group signals at δ 0.73 (3H, s), 0.76 (3H, s), 0.88 (3H, s), 0.89 (3H, s), 1.06 (3H, s), 1.16 (3H, s), and 1.29 (3H, s), three oxymethine signals at δ 3.22 (1H, dd, J = 11.7, 4.0 Hz), 4.95 (1H, br s), 4.75 (1H, dd, J = 12.1, 5.5 Hz), one oxymethylene at δ 4.18 (1H, d, J = Table 1
10.0 Hz) and 4.90 (1H, d, J = 11.0 Hz), and an olefinic proton signal at δ 5.37 (1H, m) were observed. All of the 1H- and 13C NMR data (Table 1) were as- signed 1 by H-1H COSY, HMQC, and HMBC experiments. By comparison of the NMR data with those of alternosides II, IV, and V reported previously [9-10], the aglycone of 7 was deduced to a 3, 16, 22, 28-tetrahydroxy- olean-12-ene. The oxymethylene proton signals at δ 4.18 and 4.90 were diagnostic for H-28 [9]. The oxymethine proton signal at δ 4.95 correlated with C-15 (δ 36.9) and C-28 (δ 64.6) in the HMBC spectrum, and could be assigned to H-16. The correlation between H-16 (δ 4.95) and H-27 (δ 1.92) in the NOESY spectrum suggested an α- configuration of H-16. The proton signal at δ 4.75 could be assigned to H-22 on the basis of its correlations with C-18 (δ 43.8), C-20 (δ 32.3), C-21 (δ 44.3), and C-28 (δ 64.6) in the HMBC spectrum. The correlation between H-22 (δ 4.75) and H-30 (δ 0.89) in the NOESY spectrum indicated a β-configuration of H-22, which was also supported by the splitting pattern of H-22 (1H, dd, J = 12.1, 5.5 Hz). Thus, the alglycone of 7 could be assigned as 3β, 16β, 22α, 28β-tetrahydroxyolean-12-ene, i.e., 22α-hydroxy-longispinogenin, from its NOESY, HMQC, and HMBC spectra.
1
H NMR and 13C NMR data for compound 7 (400 MHz and 100 MHz, pyridine-d5)
C
δc
δH (J, Hz)
C
δc
δH (J, Hz)
1
39.0
0.80 (m), 1.37 (m)
23
28.5
1.16 (s)
2
26.5
1.78 (m), 1.97 (m)
24
17.3
0.76 (s)
3
89.5
3.22 (dd, 11.7, 4.0)
25
15.9
0.73 (s)
4
39.7
-
26
17.5
1.06 (s)
5
56.0
0.73 (m)
27
27.8
1.29 (s)
64.6
4.18 (d, 10.0), 4.90 (d,11.0)
19.2
1.48 (m), 161 (m)
7
33.3
1.32 (m), 1.57 (m)
29
33.7
0.88 (s)
8
40.5
-
30
25.0
0.89 (s)
9
47.3
1.52 (m)
C-3
10
36.9
-
GlcA-1′
106.7
4.67 (d, 7.6)
11
24.3
1.86 (m)
2′
75.4
3.93
12
123.5
5.37 (m)
3′
78.4
4.19
13
142.9
-
4′
73.6
4.12
14
42.8
-
5′
76.7
4.14
15
36.9
1.53 (m), 1.96 (m)
6′
176.9
16
66.6
4.95 (br s)
C-28
17
44.8
-
Rha-1″
102.0
5.17 (br s)
18
43.8
2. 90 (m)
2″
72.7
4.38
19
46.8
1.10 (m),1.90 (m)
3″
73.2
4.36
20
32.3
-
4″
74.0
4.16
21
44.3
1.75 (m), 2.03 (m)
5″
70.1
4.12
22
69.3
4.75 (dd, 12.1, 5.5)
6″
19.2
1.57 (d, 6.0)
6
28
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Acid hydrolysis of 7 afforded a mixture of D-glucuronic acid and L-rhamnose by high-performance thin-layer chromatography (HPTLC). 1H- and 13C NMR resonances of the sugar units indicated the presence of glucuronic acid and rhamnose. Anomeric carbon signals at δC 106.7 and 102.0 and anomeric proton signals at δH 4.67 (1H, d, J = 7.6 Hz) and 5.17 (1H, br s) were observed in 1H and 13 C NMR spectra, respectively. The coupling constant (7.6 Hz) of the anomeric proton of GlcA, along with the correlation between H-1′ (δ 4.67) and H-5′ (δ 4.14) in the NOESY experiment, indicated a β configuration for the glucuronic acid moiety. The coupling pattern of the anomeric proton and the existence of a NOESY cross peak between H-1″ and H-2″ indicated an α configuration for the rhamnose unit [11]. The respective locations of the sugar moieties were subsequently deduced from the 2D NMR data. In the HMBC spectrum (Fig. 2), correlations between H-1′ (δ 4.76) of glucuronic acid and C-3 (δ 89.5) of the aglycone, and between H-1″ (δ 5.17) of rhamnose and C-28 (δ 64.6) of the aglycone inferred that the glucuronopyranosyl and rhamnopyranosyl units were linked at C-3 and C-28, respectively. Thus, the skeleton structure of 7 was 3-O-β-D-glucuronopyranosyl-3β, 16β, 22α, 28β-tetrah-
ydroxy-olean-12-ene 28-O-α-L-rhamnopyranoside (22α-hydroxy-longispinogenin 3-O-β-D-glucuronopyranosyl-28-Oα-L-rhamnopynoside), and is a new isolate. In addition, the carboxylic carbon signal (δ 176.9) of the glucuronic acid residue in 7 showed a significant downfield shift (+ 4.3) in comparison to that (δ 172.6) in the non-ionized state in the literature [12-13]. The isolated compound was therefore inferred to be a sodium salt. Some similar constituents as sodium (e.g., the sodium salt of alternoside II) or potassium salt forms with close carboxylic carbon signal values of GlcA were isolated previously from G. sylvestre [9]. It was also supported by the presence of a [(C42H67O14·Na) + H]+ ion at m/z 819 and the ion [(C 42 H 67 O 14 ·Na) + Na] + at m/z 841 in its MALDI-TOF MS and [C 42 H 67 O 14 ·Na + H] + at m/z 819.446 7 in its HR-MS. All of the available evidence led to the conclusion that 7 was the sodium salt of 3-O-βD-glucuronopyranosyl-3β, 16β, 22α, 28β-tetrahydroxyolean-12-ene 28-O-α-L-rhamnopyranoside (the sodium salt of 22α-hydroxy-longispinogenin 3-O-β-D-glucuronopyranosyl-28-O-α-L-rhamnopynoside). The structure is shown in Fig. 1, and the 13C- and 1H NMR data are shown in Table 1.
Fig. 1 Compounds 1−7 isolated from G. sylvestre
The known compounds, conduritol A (1) [14], stigmasterol (2) , lupeol (3) [17], stigmasterol-3-O-β-D-glucoside (4) [18] , the sodium salt of 22α-hydroxy-longispinogenin-3-O-β-Dglucopyranosyl-(1→3)-β-D-glucuronopyranosyl-28-O-α-Lrhamnopyranoside (5) [10], and oleanolic acid-3-O-β-Dglucopyranosyl-(1→6)-β-D-glucopyranoside (6) [1, 19] were identified by comparing their physical and spectroscopic data with reported data. An inhibition assay of compounds 1 and 5−7 on the non-enzymatic glycation of protein in vitro was carried out. Compounds 1 and 5−7 had much weaker inhibition activi[15-16]
Fig. 2 Key HMBC correlations of compound 7
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LIU Yue, et al. / Chin J Nat Med, 2014, 12(4): 300−304
ties than that of aminoguanidine hydrochloride. Changes of inhibition activities were apparently independent of the concentrations of samples. Table 2 Inhibition of compounds 1 and 5−7 on the non-enzymatic glycation of protein in vitro Sample/Control
Inhibition ratio/% −1
50 μg·mL
5 μg·mL−1
0.5 μg·mL−1
1
10.42
9.71
8.99
5
10.78
25.25
28.09
6
23.63
25.41
18.27
7 aminoguanidine hydrochloride (0.015 mol·L−1)
3.82
3.64
5.07
97.86
Experimental Apparatus and reagents MS spectra were recorded on a Finnigan MAT LCQ mass spectrometer and a MALDI/TOF-MS. The HR-MS was recorded on Autoflex III MALDI/TOF/TOF-MS. NMR spectra were obtained on Bruker AV400 and AV600 instruments. Melting points were obtained on an X-6 apparatus and are uncorrected. A RF-540 fluorescence spectrometer (Shimadzu), an LHP-160E intelligent constant temperature and humidity incubator (Nanbei Equipment Co., Ltd., Zhengzhou, China) and a SHA-C water bath constant-temperature oscillator (Jingbo Experimental Instrument Factory, Jintan, China) were applied in the activity assay. Prep-HPLC was performed using an ODS column (Zorbax PrepHT Eclipse XDB-C18, 21.2 mm × 250 mm, 7 μm) on Waters-600E HPLC. Column chromatographic separations were performed on silica gel (200-300 mesh, Qingdao Oceanic Chemical Industry, Qingdao, China) and reversed phase silica gel (50 μm) (YMC). Macroporous resin D21 was purchased from Shandong Lukang Pharmaceutical Co., Ltd. Bovine serum albumin and D-glucose were from Solarbio Science Co., Beijing, China aminoguanidine hydrochloride from Sigma-Aldrich, St. Louis, MO, USA, sodium azide from Huadong Reagent Factory, Tianjin, China, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and other reagents of analytical grade from Beijing Chemical Works, Beijing, China and those of chromatographic grade were from Tedia, USA were applied. Plant material The stems of G. sylvestre were purchased from a company in the Chinese Medicinal Materials market in Yulin, Guangxi Province, China, in September 2009, and authenticated by Prof. Guojun Xu, Jilin Academy of Chinese Medicine Sciences. The voucher specimen has been deposited in Jilin Academy of Chinese Medicine Sciences. Extraction and isolation The dried stems of G. sylvestre (9.5 kg) were extracted twice with 75% EtOH (100 and 80 L, respectively; each 8 h)
by percolation extraction processes. An aqueous solution of the extract was partitioned with EtOAc. The lower water solution was chromatographed on D21 porous resin (1.5 kg), eluting with water and 50% EtOH, successively, to give water-eluting part (81 g) and a 50% EtOH part (84 g). The water-eluting part (27 g) was chromatographed over a silica gel column eluted with CHCl3−MeOH gradients (30 : 1 to 5 : 1). Compound 1 was obtained from Frs. 85−232. The EtOAc part (132 g) was subjected to silica gel column chromatography with a CHCl3−MeOH gradient (2 : 0 to 2 : 1). Compounds 2 (65 mg) and 3 (40 mg) were isolated from Frs. 24-57, and compound 4 (34 mg) was isolated from Frs. 75−162 by repeated silica gel column chromatography. The 50% EtOH part was subjected to silica gel column chromatography with a CHCl3−MeOH gradient (20 : 1 to 1 : 1.5). Combined Frs. 110−166 was repeatedly chromatographed over a silica gel column (CHCl3−MeOH−n-BuOH gradients, 9 : 1 : 0.1 to 6 : 4 : 1). The Frs. 1−37 were successively chromatographed over a Rp-18 column (MeOH−H2O gradients, 50 : 50 to 80 : 20) and prep-HPLC (MeOH−H2O, 80 : 20) to give compound 6 (28 mg). The Frs. 90−115 were subjected to a Rp-18 column (MeOH−H2O gradients, 50 : 50 to 80 : 20) and then prep-HPLC (CH3CN−H2O, 20:80) to give compound 7 (18 mg). The Frs. 116−167 were subjected to a Rp-18 column (MeOH−H2O gradients, 50 : 50 to 80 : 20) and then prep-HPLC (CH3CN− H2O, 20 : 80) to give compound 5 (60 mg). Acid hydrolysis Compound 7 (10 mg) were heated with 1 mol·L−1 HCl in MeOH (10 mL) under reflux for 2 h. The reaction mixture was neutralized with NaHCO3. The water phase was chromatographed on the silica gel HPTLC with the system of n-BuOH−i-PrOH−H2O (10 : 5 : 4, homogenous), then the brown colored spots were visualized by spraying with phenylamine-o-benzenedicarboxylic acid reagent followed by heating. D-Glucuronic acid and L-rhamnose were detected by comparison with authentic samples. Non-enzymatic glycation of protein inhibition assay in vitro Inhibition activities in vitro of the non-enzymatic glycation of protein of the candidate compounds were evaluated by determination of the fluorescence intensity of advanced glycosylation end products (AGEs) of proteins [20]. Bovine serum albumin (20 mg) and D-glucose (90.1 mg) were added into 5 mL phosphate solutions (pH 7.4, NaN3 3 mmol·L−1) of the control and each sample. Aminoguanidine hydrochloride was used as the positive control. Corresponding blank solutions were also prepared. Every sample was prepared in triplicate. All sample solutions were incubated for 7 days at 37 °C, in a constant temperature and humidity environment. The fluorescence intensities were determined at the excitation/emission wavelength of 370/440 nm. The inhibition rate (I) was calculated by the following formula: I (%) = [1 − (S − S0) / (Nc − Nc0)] × 100. S: fluorescence intensity in positive control or sample
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groups; S0: fluorescence intensity in positive control or sample blank groups without D-glucose; Nc: fluorescence intensity in negative control groups without sample; Nc0: fluorescence intensity in negative control blank groups without sample and D-glucose
[11]
[12]
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Cite this article as: LIU Yue, XU Tun-Hai, ZHANG Man-Qi, LI Xue, XU Ya-Juan, JIANG Hong-Yu, LIU Tong-Hua, XU Dong-Ming. Chemical constituents from the stems of Gymnema sylvestre [J]. Chinese Journal of Natural Medicines, 2014, 12(4): 300-304
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