New antioxidant and antiglycation active triterpenoid saponins from the root bark of Aralia taibaiensis

Fitoterapia 83 (2012) 234–240

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New antioxidant and antiglycation active triterpenoid saponins from the root bark of Aralia taibaiensis Linlin Bi a, b, Xiangrong Tian b, c, Fang Dou b, Liangjian Hong b, Haifeng Tang b,⁎, Siwang Wang a,⁎ a b c

Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi'an 710032, PR China Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, PR China Research & Development Center of Biorational Pesticide, College of Plant Protection, Northwest A & F University, Yangling 712100, PR China

a r t i c l e

i n f o

Article history: Received 31 August 2011 Accepted in revised form 25 October 2011 Available online 9 November 2011 Keywords: Aralia taibaiensis Triterpenoid saponin Antioxidant Antiglycation Diabetes mellitus

a b s t r a c t Four new oleanane type triterpenoid saponins (1–4) and a known saponin (5) were isolated from the root bark of Aralia taibaiensis Z.Z. Wang et H.C. Zheng. The structures of the four new compounds were elucidated as 3-O-{β-D-glucopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 3)]β-D-glucurono-pyranosyl}-olean-11,13(18)-diene-28-oic acid 28-O-β-D-glucopyranosyl ester (1), 3-O-{β-D-gluco-pyranosyl-(1 → 3)-[α-L-arabinofuranosyl-(1→ 4)]-β-D-glucuronopyranosyl}olean-11,13(18)-diene-28-oic acid 28-O-β-D-glucopyranosyl ester (2), 3-O-{β-D-glucopyranosyl(1 → 2)-[α-L-arabinofuranosyl-(1 → 4)]-β-D-glucuronopyranosyl}-oleanolic acid 28-O-β-Dglucopyranosyl ester (3) and 3-O-{β-D-glucopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl}-oleanolic acid 28-O-β-D-glucopyranosyl ester (4), on the basis of extensive spectral analysis and chemical evidence. Compounds 1–5 exhibited moderate effects on antioxidant and antiglycation activities, which correlated with treatment of diabetes mellitus. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Aralia taibaiensis Z.Z. Wang et H.C. Zheng (Araliaceae) is an abundant species distributed widely in the northwest of China [1]. The root barks of several species belonging to genus Aralia have been used in folk medicine for the treatment of diabetes, hepatitis, stomach ulcer, etc. [2]. Triterpenoid saponins characterized as oleanane acid aglycone with an oligosaccharide moiety at C-3, and with or without a sugar moiety at C-28 are the predominant chemical constituents of genus Aralia [3]. In our previous studies focused on A. taibaiensis collected in Yuzhong County, Gansu Province of China, seven new and several known oleanane type triterpenoid saponins were isolated [4–6]. In our ongoing search for new antidiabetic constituents from Traditional Chinese Medicine (TCM), we found that total saponins from this species in Taibai Mountain, Shaanxi Province of China, showed notable antioxidant and antiglycation activities [7]. However, different geographical environment of the same species may ⁎ Corresponding authors. Tel./fax: + 86 29 84775471. E-mail addresses: [email protected] (H. Tang), [email protected] (S. Wang). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.11.002

produce different metabolites. Further studies in antidiabetic activities of the pure compounds from the root bark of A. taibaiensis in Taibai Mountain led to the isolation of three new and nine known saponins [8], most of which were not identical with the previous isolated constituents, i.e., saponins in A. taibaiensis from Yuzhong County possessed the characteristic aldyl ester in the carboxy group of the glucuronic acid moiety, whereas saponins in this species from Taibai Mountain were not possessed. We report herein the isolation and structural elucidation of the three new saponins (1, 2 and 4) and a later identified new saponin (3) (Fig. 1). In addition, diabetes mellitus related antioxidant and antiglycation activities of new compounds 1–4 and a known saponin (5) are reevaluated to illustrate their structure-active relationship. 2. Experimental procedure 2.1. General The ESI-MS and HR-ESI-MS were obtained on a Micromass Quattro mass spectrometer. The melting points were determined on an XT5-XMT apparatus and uncorrected. The optical rotations were measured on a Perkin-Elmer 343 polarimeter.

L. Bi et al. / Fitoterapia 83 (2012) 234–240

235

Fig. 1. Structures of compounds 1−5 from Aralia taibaiensis.

GC-MS was performed on an Agilent 6890 GS/5973 MS apparatus using an HP-1 capillary column (25 m × 0.32 mm i.d., 0.25 μm, Agilent Tech., USA) with an initial temperature of 130 °C for 2 min and then temperature programming to 300 °C at the rate of 15 °C/min. Separation and purification were performed by column chromatography (CC) on silica gel H (10–40 μm, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Pharmacia Inc., NJ, USA). HPLC was carried out on a Dionex P680 liquid chromatograph equipped with a UV 170 UV/Vis detector at 206 nm using a Sino Chrom ODS-BP column (250 × 10 mm i.d., 5 μm, Elite Inc., China) for semi-preparation. TLC detection was achieved by spraying the silica gel plates (Qingdao Marine Chemical Inc., Qingdao, China) with 20% H2SO4 in EtOH followed by heating. 1D and 2D NMR spectra experiments were measured in C5D5N on Bruker INOVA-400 NMR spectrometer with tetramethylsilane (TMS) as an internal standard. 2.2. Plant material The root bark of A. taibaiensis Z.Z. Wang et H.C. Zheng were collected in Taibai Mountain, Shaanxi Province of China in September 2008, and identified by one of the authors (Prof. H. Tang). A voucher specimen (FMMUDP-Voucher No: SAP012) was deposited in the Herbarium of the Department of Pharmacy, Xijing Hospital, Fourth Military Medical University. 2.3. Extraction and isolation The air-dried root bark (1.2 kg) was powdered and then extracted with 70% EtOH (5 × 1.0 L) under reflux. The organic

solvent was concentrated to afford a crude extract (340 g). The extract was suspended in H2O and then partitioned successively with petroleum ether (3 × 0.4 L) and n-BuOH (4 × 0.4 L). The n-BuOH extract was concentrated in vacuo to yield a yellowish residue (110 g), which was dissolved in MeOH (550 mL) and precipitated with acetone (5.5 L) to give crude saponins (60 g). A portion of the crude saponins (10 g) was subjected to CC over silica gel eluting with CHCl3–MeOH–H2O gradient (from 7:1:1 to 6.5:3.5:1, lower phase) to give 6 fractions (Fr.A1–Fr.A6) based on TLC analysis. By TLC and HPLC comparison with authentic samples, some known oleanane type triterpenoid saponins were identified as the main constituents of Fr.A1–Fr.A4 [8]. Fr.A6 (1.4 g) was eluted with CHCl3–MeOH (1:1) on Sephadex LH-20 to remove pigments, and then further purified by semi-preparative HPLC using MeOH–H2O (50:50) as the mobile phase at a flow rate of 2.0 mL/min to afford saponin 1 (227.0 mg, tR = 35.8 min) and 4 (123.1 mg, tR = 33.1 min). Fr.A5 (1.7 g) was subjected to CC over silica gel eluting with CHCl3– MeOH–H2O (8.5:2.5:0.5, lower phase), and further purified by semi-preparative HPLC with MeOH–H2O (53:47) as the mobile phase at a flow rate of 2.0 mL/min to afford saponins 2 (110.0 mg, tR = 23.8 min), 3 (69.6 mg, tR = 32.7 min), and 5 (240.0 mg, tR = 34.1 min). 2.3.1. 3-O-{β-D-glucopyranosyl-(1→ 2)-[β-D-glucopyranosyl(1 → 3)]-β-D-glucuronopyranosyl}-olean-11,13(18)-diene-28-oic acid 28-O-β-D-glucopyranosyl ester (1) White amorphous powder, mp 201–203 °C; [α] 23D −4.1° (c 0.74, MeOH); 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data, see Table 1; positive ESI-MS m/z 1161

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L. Bi et al. / Fitoterapia 83 (2012) 234–240

Table 1 1 H and 13C NMR data for compounds 1–4 (C5D5N, 1H NMR 400 MHz, 1

13

C NMR 100 MHz)a.

2

3

4

No.

δC

δH Mult. (J in Hz)

δC

δH Mult. (J in Hz)

δC

δH Mult. (J in Hz)

δC

δH Mult. (J in Hz)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

39.4 28.6 91.7 40.3 56.6 17.3 33.8 42.5 55.2 37.6 126.7 128.0 137.2 42.9 33.8 24.5 48.6 134.0 40.3 33.2 37.6 34.7 26.9 16.1 18.2 17.3 19.2 178.1 31.4 24.4

0.76 m, 1.38 m 1.22 m, 2.12 m 3.22 brd (8.4) – 0.70 m 1.16 m, 1.48 m 1.30 m, 1.40 m – 2.10 m – 5.59 d (11.0) 6.64 d (11.0) – – 1.10 m, 1.30 m 1.82 m, 1.94 m – – 1.82 m, 1.92 m – 1.16 m, 1.22 m 1.45 m, 1.76 m 1.22 s 0.78 s 1.01 s 1.13 s 0.99 s – 0.91 s 0.88 s

39.3 28.8 90.4 40.0 56.4 17.5 33.8 42.4 55.3 37.5 126.6 128.1 137.2 42.8 33.8 24.3 48.6 133.4 40.5 33.1 37.5 34.6 26.7 16.0 18.1 17.5 19.1 177.8 31.3 24.0

0.80 m, 1.34 m 1.20 m, 2.10 m 3.20 brd (8.0) – 0.71 m 1.16 m, 1.40 m 1.30 m, 1.38 m – 2.08 m – 5.65 d (11.6) 6.63 d (11.6) – – 1.08 m, 1.30 m 1.80 m, 1.92 m – – 1.78 m, 1.90 m – 1.12 m, 1.26 m 1.45 m, 1.78 m 1.23 s 0.74 s 0.97 s 1.16 s 0.90 s – 0.89 s 0.87 s

39.0 26.6 90.4 39.8 56.1 18.8 33.5 40.2 48.3 37.2 23.7 123.3 144.4 42.5 28.5 24.1 47.4 42.1 46.6 31.1 34.3 32.8 28.3 17.0 15.7 17.8 26.4 177.1 33.5 24.0

0.52 m, 1.14 m 0.94 m, 1.90 m 3.00 m – 0.48 m 1.04 m, 1.24 m 1.16 m, 1.24 m – 1.38 m – 1.66 m, 1.74 m 5.25 m – – 0.92 m, 2.06 m 1.66 m, 1.94 m – 2.96 m 1.10 m, 1.60 m – 0.94 m, 1.24 m 1.54 m, 1.68 m 0.97 s 0.85 s 0.61 s 0.85 s 1.10 s – 0.75 s 0.73 s

39.3 26.8 91.1 40.1 56.5 19.1 33.7 40.4 48.5 37.4 23.9 123.5 144.6 42.7 28.8 24.3 47.7 42.3 46.9 31.2 34.5 33.1 28.5 17.3 16.0 18.0 26.6 177.6 33.7 24.2

0.74 m, 1.18 m, 3.20 m – 0.69 m 1.10 m, 1.27 m, – 1.50 m – 1.86 m, 5.40 m – – 1.10 m, 1.84 m, – 3.08 m 1.18 m, – 1.06 m, 1.68 m, 1.18 s 1.06 s 0.76 s 0.98 s 1.20 s – 0.88 s 0.86 s

28-Glc 1 2 3 4 5 6

96.2 74.2 79.3 71.5 79.3 62.7

6.08 d (8.0) 4.10 m 4.18 m 4.12 m 3.95 m 4.10 m, 4.25 m

96.2 74.2 79.3 71.5 79.3 62.6

6.07 d (8.4) 4.06 m 4.18 m 4.10 m 3.94 m 4.18 m, 4.20 m

95.9 74.1 79.1 71.3 79.2 62.5

6.00 d (8.0) 3.96 m 4.05 m 4.06 m 3.82 m 4.00 m, 4.12 m

96.1 74.2 79.3 71.4 79.3 62.6

6.08 d (8.0) 4.06 m 4.18 m 4.12 m 3.94 m 4.10 m, 4.28 m

3-GluA 1′ 2′ 3′ 4′ 5′ 6′

105.4 78.8 86.6 72.6 78.3 176.1

4.83 d (7.2) 4.45 m 4.36 m 4.28 m 3.78 m –

106.3 74.2 82.4 75.9 75.8 176.0

4.80 d (8.0) 4.06 m 4.84 m 4.58 m 3.98 m –

105.0 80.9 76.5 78.7 78.0 175.7

4.57 d (7.6) 4.20 m 3.75 m 4.36 m 4.10 m –

105.3 79.3 86.3 72.4 78.3 176.1

4.78 d (7.2) 4.42 m 4.30 m 4.24 m 3.82 m –

2′-Glc 1 2 3 4 5 6

103.5 76.4 78.6 71.8 78.5 62.7

5.60 d (7.6) 3.78 m 4.18 m 3.93 m 4.30 m 4.10 m, 4.37 m

104.8 77.9 78.4 72.1 78.4 63.2

5.10 d (7.4) 3.70 m 4.00 m 3.84 m 4.06 m 4.00 m, 4.30 m

104.1 76.4 78.6 71.6 78.4 62.4

5.57 d (7.6) 3.80 m 4.20 m 3.94 m 4.28 m 4.08 m, 4.40 m

3′-Glc 1 2 3 4 5 6

104.2 75.6 78.6 72.8 78.5 63.7

5.35 d (7.4) 3.80 m 4.18 m 3.70 m 4.15 m 4.06 m, 4.45 m

104.3 75.5 78.6 72.7 78.4 62.6

5.32 d (7.4) 3.84 m 4.20 m 3.71 m 4.12 m 4.08 m, 4.40 m

4′-Ara(f) 1 2 3

105.0 76.3 78.6 71.5 78.5 62.6

5.49 d (7.6) 4.15 m 4.10 m 4.00 m 3.84 m 4.04 m, 4.20 m

108.7 82.9 78.2

6.04 s 4.44 m 4.12 m

109.2 82.8 76.5

5.83 s 4.70 m 4.04 m

1.35 m 2.14 m

1.32 m 1.36 m

1.97 m

2.14 m 2.04 m

1.72 m 1.32 m 1.82 m

L. Bi et al. / Fitoterapia 83 (2012) 234–240

237

Table 1 (continued) 1 No.

δC

2 δH Mult. (J in Hz)

4 5 a

3

4

δC

δH Mult. (J in Hz)

δC

δH Mult. (J in Hz)

87.2 63.1

5.00 d (4.0) 3.96 m, 4.04 m

86.4 62.9

4.65 m 3.90 m, 4.05 m

δC

δH Mult. (J in Hz)

Assignments aided by the DEPT, DQCOSY, HMQC, HMBC, TOCSY, and NOESY experiments.

[M+ 2Na −H]+, 1139 [M +Na]+; negative ESI-MS m/z 1115 [M− H]−, 953 [M−162− H]−, 453 [M−3 × 162− 176 − H]−; positive HR-ESI-MS m/z 1139.5264 [M+ Na]+ (calcd. for C54H84O24Na, 1139.5250). 2.3.2. 3-O-{β-D-glucopyranosyl-(1→3)-[α-L-arabinofuranosyl(1→4)]-β-D-glucuronopyranosyl}-olean-11,13(18)-diene-28-oic acid 28-O-β-D-glucopyranosyl ester (2) White amorphous powder, mp 207–209 °C; [α] 23D −15.0° (c 1.10, MeOH); 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data, see Table 1; positive ESI-MS m/z 1131 [M+ 2Na − H]+, 1109 [M+ Na]+; negative ESI-MS m/z 1085 [M− H]−, 953 [M− 132 − H]−, 923 [M− 162 − H]−; positive HR-ESI-MS m/z 1109.5137 [M+ Na] + (calcd. for C53H82O23Na, 1109.5145). 2.3.3. 3-O-{β-D-glucopyranosyl-(1 → 2)-[α-L-arabinofuranosyl(1 → 4)]-β-D-glucuronopyranosyl}-oleanolic acid 28-O-β-Dglucopyranosyl ester (3) White amorphous powder, mp 194–196 °C; [α] 23D −22.5° (c 1.05, MeOH); 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data, see Table 1; positive ESI-MS m/z 1133 [M+ 2Na − H]+, 1111 [M+ Na]+; negative ESI-MS m/z 1087 [M− H]−, 955 [M− 132 − H]−, 925 [M− 162 − H]−; positive HR-ESI-MS m/z 1111.5284 [M+ Na] + (calcd. for C53H84O23Na, 1111.5295). 2.3.4. 3-O-{β-D-glucopyranosyl-(1 → 2)-[β-D-glucopyranosyl(1 → 3)]-β-D-glucuronopyranosyl}-oleanolic acid 28-O-β-Dglucopyranosyl ester (4) White amorphous powder, mp 189–191 °C; [α] 23D −3.9° (c 1.14, MeOH); 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data, see Table 1; positive ESI-MS m/z 1163 [M+ 2Na − H]+, 1141 [M+ Na]+; negative ESI-MS m/z 1117 [M− H]−, 955 [M− 162 − H]−, 793 [M− 2 × 162 − H]−; 455 [M− 3 × 162 − 176 − H]−; positive HR-ESI-MS m/z 1141.5419 [M+ Na]+ (calcd. for C54H86O24Na, 1141.5407). 2.4. Acidic hydrolysis of compounds 1–4 Each saponin (5 mg) was heated in an ampule with 2 mol/L CF3COOH (5 mL) at 120 °C for 2 hours. The reaction mixture was poured into CH2Cl2–H2O (1:1). The aqueous phase was evaporated under vacuo, 1 mL pyridine and 2 mg NH2OH⋅ HCl were added to the dried residue, and the mixture was stirred at 90 °C for 1 hour. After the reaction mixtures were cooled, 1.5 mL of Ac2O was added and the mixtures were heated at 90 °C for 1 hour. The reaction mixtures were evaporated under reduced pressure, and the resulting aldononitrile peracetates were analyzed by GC-MS.

The carbohydrates were determined by comparing the retention times and MS behavior with standard aldononitrile peracetates prepared from authentic sugars by the same procedure performed for the sample. The D-glucose (Glc) and Dglucuronic acid (GluA) were identified in a ratio of 3:1 for saponins 1 and 4, whereas D-glucose, D-glucuronic acid and L-arabinose (Ara) were identified in a ratio of 2:1:1 for saponins 2 and 3. 2.5. Antioxidant and antiglycation activities of compounds 1–5 The antioxidant activities of compounds 1–5 were evaluated by studying on the inhibition of lipid peroxidation in rat liver microsomes induced by ascorbate/Fe 2 +, cumine hydroperoxide (CHP) or CCl4/reduced form of nicotinamide– adenine dinucleotide phosphate (NADPH), using butylated hydroxytoluene (BHT) as positive control. The antiglycation activities of compounds 1–5 were evaluated by hemoglobin-δ-gluconolactone (δ-Glu) assay, bovine serum albumin (BSA)-glucose assay and N-acetyl–glycyl–lysine methyl ester (GK peptide)-ribose assay with aminoguanidinehydrochloride (AG) as positive control. The detailed experiments procedure has been described in our previous published literature [7,8]. Statistical analysis was performed by analysis of variance (ANOVA) followed by LSD's test for multiple comparisons. Data were analyzed by SPSS v.11.0 software package and expressed as mean ± SD. Differences were considered significant at P b 0.05. 3. Results and discussion Compound 1 was obtained as a white amorphous powder and showed positive to Liebermann–Burchard and Molish tests. The positive ion mode HR-ESI-MS showed a pseudomolecular ion peak at m/z 1139.5264 [M + Na] + (calcd. for C54H84O24Na, 1139.5250), which, together with the pseudomolecular ion peak at m/z 1115 [M − H] − in the negative ion mode ESI-MS, enabled the determination of the molecular formula as C54H84O24, with the help of NMR spectral data. Seven tertiary methyl proton signals at δH 1.22 (3H, s, H3-23), 1.13 (3H, s, H3-26), 1.01 (3H, s, H3-25), 0.99 (3H, s, H3-27), 0.91 (3H, s, H3-29), 0.88 (3H, s, H3-30), 0.78 (3H, s, H3-24), and a cis-olefinic group signal at δH 5.59 (1H, d, J = 11.0 Hz, H-11) and δH 6.64 (1H, d, J = 11.0 Hz, H-12) in the 1H NMR spectrum, together with four olefinic carbon signals at δC 126.7 (C-11), 128.0 (C-12), 134.0 (C-18) and 137.2 (C-13) in the 13C NMR spectrum, suggested the aglycone of compound 1 to be olean-11,13(18)-diene-28-oic acid [9,10], which had been isolated from the same genus plant Aralia subcapitata Hoo [11]. The correctness of the

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conclusion was also confirmed by careful analysis of HSQC and HMBC spectra (Fig. 2). The chemical shifts of C-3 (δC 91.7) and C-28 (δC 178.1) in the 13C NMR spectrum indicated that 1 was a bidesmosidic glycoside [12]. The correlations of H-3 (δH 3.22) with H3-23 and H-5 (δH 0.70) in the NOESY spectrum indicated the βconfiguration for the 3-O-sugar moiety. The presence of Dglucose and D-glucuronic acid in a ratio of 3:1 was confirmed by acidic hydrolysis and preparation of the corresponding aldononitrile peracetates which were analyzed by GC-MS [13]. The 1H, 13C NMR and HSQC spectra of 1 suggested the presence of a β-glucuronopyranosyl and three β-glucopyranosyl moieties on the basis of four anomeric proton signals at δH 4.83 (1H, d, J = 7.2 Hz, 3-GluA H-1′), 6.08 (1H, d, J = 8.0 Hz, 28-Glc H-1), 5.60 (1H, d, J = 7.6 Hz, 2′-Glc H-1) and 5.35 (1H, d, J = 7.6 Hz, 3′-Glc H-1), as well as the corresponding anomeric carbons at δC 105.4 (3-GluA C-1′), 96.2 (28-Glc C-1), 103.5 (2′-Glc C-1) and 104.2 (3′-Glc C-1). The β-configuration for the four carbohydrate units of 1 was also confirmed by careful analysis of NOESY experiment (Fig. 2). All of the proton signals due to the sugar moieties were identified by careful analysis of DQCOSY, TOCSY, and NOESY spectra, and the carbon signals were assigned by HSQC and further confirmed by HMBC spectra (Table 1). After mapping all of the signals for each sugar moiety, the sequence and binding sites for the sugar moieties of 1 were determined by careful inspection of HMBC spectrum. In brief, long range correlations were observed between H-1 of 28-Glc and C-28, H-1′ of GluA and C-3, H-1 of 2-Glc and C-2′ (δC 78.8) of GluA, along with H-1 of 3-Glc and C-3′ (δC 86.6) of GluA in the HMBC spectrum (Fig. 2). Accordingly, compound 1 was elucidated as 3O-{β-D-glucopyranosyl-(1→ 2)-[β-D-glucopyranosyl-(1→ 3)]β-D-glucuronopyranosyl}-olean-11,13(18)-diene-28-oic acid 28-O-β-D-glucopyranosyl ester. Compound 2 was obtained as a white amorphous powder, which was also determined to be triterpenoid saponin due to positive results for the Liebermann–Burchard and Molish tests. The molecular formula was established as C53H82O23 according to the positive HR-ESI-MS from the pseudomolecular ion peak at m/z 1109.5137 [M + Na] + (calcd. for C53H82O23Na, 1109.5145). The chemical shifts of C-3 (δC 90.4) and C-28 (δC 177.8) in the 13C NMR spectrum were indicative of 2 also being a bidesmoside. Detailed

analysis of the 1H and 13C NMR data of 2 (Table 1) indicated that compound 2 had the same 3β-hydroxy-olean-11,13 (18)-diene-28-oic acid aglycone as 1, but a difference in the category and linkage of the sugar moieties at C-3. GCMS analysis after acid hydrolysis of 2 as the same method with 1 gave D-glucose, D-glucuronic acid and L-arabinose a ratio of 2:1:1. Careful inspection of 13C NMR data of the sugar moieties indicated that the D-glucose and D-glucuronic acid were in their pyranose forms, and L-arabinose in its furanose forms. The 1H NMR spectrum of 2 showed four anomeric proton signals at δH 6.07 (1H, d, J = 8.4 Hz, 28-Glc H-1), 4.80 (1H, d, J = 8.0 Hz, 3-GluA H-1′), 5.49 (1H, d, J = 7.6 Hz, 3′-Glc H-1) and 6.04 [1H, s, 4′-Ara(f) H-1], which correlated with the corresponding carbon signals at δC 96.3 (28-Glc C-1), 106.3 (3-GluA C-1′), 105.0 (3′-Glc C-1) and 108.7 [4′-Ara(f) C-1] , respectively, in the HMQC spectrum. According to the anomeric proton coupling constants, the relative configurations of glucuronopyranosyl, glucopyranosyl and arabinofuranosyl units were deduced as β, β and α, respectively. The sequence of the bidesmosidic residue in 2 was determined by HMBC experiment. The observations of long range correlations from GluA H-1′ to C-3, and 28-Glc H-1 to C-28 determined the linkages of sugar moieties to the aglycone of 2, and correlations from 3′-Glc H-1 to C-3′ (δC 82.4), and 4′-Ara(f) H-1 to C-4′ (δC 75.9) determined the terminal sugar linkages of 2. Therefore, compound 2 was deduced as 3-O-{β-D-glucopyranosyl-(1 → 3)-[α-Larabinofuranosyl-(1 → 4)]-β-D-glucuronopyranosyl}-olean-11, 13(18)-diene-28-oic acid 28-O-β-D-glucopyranosyl ester. Compound 3 was also obtained as a white amorphous powder, and showed positive results to Liebermann–Burchard and Molish tests. The molecular formula was established as C53H84O23 on the basis of its positive HR-ESI-MS (m/z 1111.5284 [M+ Na] +, calcd. for C53H84O23Na, 1111.5295). The 1H and 13C NMR spectral data of 3 (Table 1) showed characteristic signals for common oleanane triterpenoid saponin with a double bond between C-12 (δC 123.3) and C-13 (δC 144.4) in the aglycone and four monosaccharides by comparison with literature data [14]. Acidic hydrolysis of 3 also yield four sugar units, D-glucose, D-glucuronic acid and Larabinose in a ratio of 2:1:1 by GC-MS analysis. Detailed comparison of the 1H and 13C NMR data of 3 with those of reported tarasaponin IV methyl ester [15] showed that the

Fig. 2. Key HMBC and NOESY correlations of compound 1.

L. Bi et al. / Fitoterapia 83 (2012) 234–240

239

Table 2 Effects of compounds 1–5 on lipid peroxidation of rat liver microsomes. Ascorbate/Fe2 + MDA Control 1 2 3 4 5 BHT

a

7.24 ± 0.17 4.28 ± 0.13c 5.72 ± 0.22c 5.91 ± 0.24c 4.60 ± 0.18c 5.61 ± 0.21c 0.24 ± 0.02c

CHP I%

b

– 40.88 21.00 18.37 36.46 22.51 96.69

CCl4/NADPH

MDA

I%

MDA

I%

3.72 ± 0.17 2.74 ± 0.06c 2.82 ± 0.05c 2.89 ± 0.12c 2.62 ± 0.07c 2.81 ± 0.12c 0.12 ± 0.02c

– 26.34 24.19 22.31 29.57 24.46 96.77

5.17 ± 0.08 1.86 ± 0.05c 2.14 ± 0.06c 4.54 ± 0.07c 1.68 ± 0.04c 2.12 ± 0.07c 0.27 ± 0.02c

– 64.02 58.61 12.19 67.50 59.00 94.78

a The values are expressed as mean ± SD (n = 3). Thiobarbituric acid reactive substances (TBARS) were expressed by malondialdehyde (MDA) produced in the presence of 0.1 mM of compounds 1–5 in the condition assays. b Percentage of inhibition (I%) due to compounds 1–5 was calculated after deducting the basal level of peroxidation. c P b 0.01 compared with the control group.

signals for the aglycone and sugar residues of the two compounds were in good agreement, except for the absence a methyl of 3 in the carboxyl group of the glucuronopyranosyl unit. Furthermore, long range correlations between the aromeric proton at δH 6.00 (1H, d, J = 8.0 Hz, 28-Glc H-1) with the carbon at δC 177.1 (C-28), between the anomeric proton at δH 4.57 (1H, d, J = 7.6 Hz, 3-GluA H-1′) with the carbon at δC 90.4 (C-3), between the anomeric proton at δH 5.10 (1H, d, J = 7.4 Hz, 2′-Glc H-1) with the carbon at δC 80.9 (3-GluA C-2′), and the anomeric proton at δH 5.83 [1H, s, 4′-Ara(f) H-1] with the carbon at δC 78.7 (3-GluA C-4′) in the HMBC spectrum also confirmed the correctness of the inference. On the basis of above analysis, saponin 3 was elucidated as a new compound and named 3-O-{β-D-glucopyranosyl-(1→ 2)-[α-Larabinofuranosyl-(1 → 4)]-β-D-glucuronopyranosyl}-oleanolic acid 28-O-β-D-glucopyranosyl ester. Compound 4 was obtained as a white amorphous powder with a molecular formula of C54H86O24 determined by HR-ESI-MS and 13C NMR analyses. The presence of the same D-glucose and D-glucuronic acid in a ratio of 3:1 was established by acidic hydrolysis followed by GC-MS analysis of the corresponding aldononitrile peracetates. Comparison of the NMR spectra of 4 with 1 and extensive 2D NMR studies indicated that 4 and 1 possess the same disaccharide chain, but differ in their aglycones. Comparison of the NMR spectra of 4 with 3 indicated that they possess the same oleanane aglycone with a characteristic double bond between C-12

(δC 123.5) and C-13 (δC 144.6). Thus, saponin 4 was elucidated as 3-O-{β-D-glucopyranosyl-(1 → 2)-[β-D-glucopyranosyl(1 → 3)]-β-D-glucuronopyranosyl}-oleanolic acid 28-O-β-Dglucopyranosyl ester. Compound 5 was determined to be stipuleanoside R2 by comparing its NMR data with those published in the literature [16]. Stipuleanoside R2 was first isolated from the rhizomes of Panax stipuleanatus, and had been later found in the genus of Aralia [6,17]. Our previous studies have showed that the twelve triterpenoid saponins isolated from A. taibaiensis all displayed certain diabetes mellitus related antioxidant and antiglycation activities [8]. However, their further structure-activity relationship (SAR) was not discussed. Reevaluating the antioxidant and antiglycation activities of the representative compounds 1–5 is to illustrate their SAR, and the results are listed in Tables 2 and 3, respectively. The results showed that all of the saponins exhibited antioxidant and antiglycation activities, saponins 1 and 4 were more potent than 2 and 5, while 3 was only marginally active. In view of their distinct structures compared with each other, it revealed that the structural features, i.e., the β-D-glucopyranosyl(1→ 2)-[β-D-glucopyranosyl-(1→ 3)]-β-D-glucuronopyranosyl oligosaccharide moiety at C-3, are responsible for the remarkable antioxidant and antiglycation activities. Our results suggested that the structural differences such as the category and the sequence of the oligosaccharide chain at

Table 3 Effects of compounds 1–5 on non-enzymatic protein glycation. δ-Glu assay

Control 1 2 3 4 5 AG a

BSA-glucose assay

GK peptide-ribose assay

HbA1ca

I%b

AGEsc

I%

AGEs

I%

16.39 ± 0.52 14.38 ± 0.51d 14.49 ± 0.50d 14.52 ± 0.45d 14.35 ± 0.45d 14.44 ± 0.35d 10.59 ± 0.34d

– 17.19 16.25 15.99 17.45 17.38 49.62

44.97 ± 2.57 31.74 ± 2.12d 35.12 ± 1.59d 42.74 ± 2.35 30.52 ± 1.89d 35.15 ± 1.97d 15.38 ± 0.45d

– 29.42 21.90 4.96 32.13 21.84 65.80

189.51 ± 28.06 132.75 ± 17.34e 167.36 ± 24.98 172.34 ± 22.09 125.23 ± 15.12d 170.26 ± 26.87 84.57 ± 13.53d

– 29.95 11.69 9.06 33.92 10.16 55.37

HbA1c means the percentage of glycated hemoglobin, and the values are expressed as mean ± SD (n = 3). Percentage of inhibition {I% = [control group–experimental group]/[control group–baseline control group (4.7)] × 100%} due to compounds 1–5 at a concentration of 1 mM. c Percentage of advanced glycation end products' (AGEs) formation, which analyzed by fluorescence. d P b 0.01 compared with the control group. e P b 0.05 compared with the control group. b

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C-3 in such saponins play an important role in terms of diabetes mellitus related antioxidant and antiglycation activities. Besides, there were no significant differences between the antioxidant and antiglycation effects of 1 and 4, and between the effects of 2 and 5, indicated that the double bond in the aglycon, i.e., Δ 12 in 3, 4 and 5, together with Δ 11,13 in 1 and 2 are less important for their antidiabetic activity. It was envisioned that, based on the data available, the antidiabetic effects of triterpenoid saponins from this species are very sensitive to their precise functionalization. Although more studies are needed to determine the clear SAR, and to determine the molecular mechanism responsible for the diabetes mellitus related antioxidant and antiglycation activities, the present studies provided valuable leads for further development of this TCM in the treatment of diabetes mellitus. Acknowledgements This work was financially supported by grants from National Natural Science Foundation of China (No. 30671788), the Administration of Traditional Chinese Medicine of Shaanxi Province, PR China (No. 41), and the “13115” Technology Innovation Project of Shaanxi Province, PR China (No. 2010ZDKG-62). References [1] Wang ZZ, Zheng HC. A new species of Aralia L. from China. J Plant Res Environ 1994;3:60. [2] College Jiangsu New Medical. Chinese materia medica dictionary. Shanghai: Shanghai People Press; 1977. p. 2439–40.

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