Chinese Journal of Natural Medicines 2014, 12(3): 0218−0221

Chinese Journal of Natural Medicines

A new triterpenoid glycoside from Vitex negundo CHEN Jiao1#, FAN Chun-Lin2, 3#, WANG Ying2, 3*, YE Wen-Cai1, 2, 3* 1

Department of Phytochemistry, China Pharmaceutical University, Nanjing 210009, China;

2

Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou 510632, China;

3

JNU-HKUST Joint Laboratory for Neuroscience & Innovative Drug Research, Jinan University, Guangzhou 510632, China Available online 20 Mar. 2014 [ABSTRACT] AIM: To investigate the chemical constituents of Vitex negundo. METHOD: Compounds were isolated by different chromatographic methods and their structures were elucidated on the basis of NMR spectroscopy. RESULTS: Four compounds were isolated and identified as 2α, 3α, 24-trihydroxyurs-12, 20(30)-dien-28-oic acid-28-Oβ-D-glucopyranosyl ester (1), corosolic acid (2), vulgarsaponin A (3) and 2α, 3α, 24-trihydroxyurs-12-en-28-oic acid-28-O-β-Dglucopyranosyl ester (4), respectively. CONCLUSION: Compound 1 is a new triterpenoid glycoside.

[KEY WORDS] Vitex negundo; Triterpenoid glycosides; 2α, 3α, 24-Trihydroxyurs-12, 20(30)-dien-28-oic acid-28-O-β-D-glucopyranosyl ester

[CLC Number] R284.1

[Document code] A

[Article ID] 2095-6975(2014)03-0218-04

Introduction The plant Vitex negundo L. (Verbenaceae) is mainly distributed in southern Asia, for example in India and the southwest of China [1]. The leaves and seeds of V. negundo have been used as traditional medicine in China for their analgesic [2], antifungal [3], and anti-inflammatory [4] activities. Previous phytochemical investigations on this plant revealed the presence of flavonoids [5], diterpenes [6], triterpenes [6], iridoid glycosides [7], and steroids [8]. In a continuing investigation on the medicinal plants in southern China [9-10] , the chemical constituents of the aerial part of V. negundo were invetigated which resulted in the isolation of a new triterpenoid glycoside, 2α, 3α, 24-trihydroxyurs-12, [Received on] 28-Jan.-2013 [Research funding] This project was supported by the Program for Changjiang Scholars and Innovative Research Team in the University (No. IRT0965), the Team Project of Natural Science Foundation of Guangdong Province (No. 8351063201000003), and the Science and Technology Planning Project of Guangdong Province (No. 2012A080204005). [*Corresponding author] YE Wen-Cai: Prof., Tel: 86-20-8522 1559, E-mail: [email protected]; WANG Ying: Associate Prof., Tel: 86-20-85223553, E-mail: [email protected] # Co-first author These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

20(30)-dien-28-oic acid-28-O-β-D-glucopyranosyl ester (1) (Fig. 1), together with three known ones, corosolic acid (2), vulgarsaponin A (3), and 2α, 3α, 24-trihydroxyurs-12-en28-oic acid-28-O-β-D-glucopyranosyl ester (4). In this paper, the isolation and structural elucidation of the new triterpenoid glycoside are reported.

Fig. 1 Chemical structure of 1

Results and Discussion Compound 1 was obtained as an amorphous powder. The HR-ESI-MS spectrum of 1 exhibited a quasi-molecular ion at m/z 671.376 9 [M + Na]+ (Calcd. for C36H56O10Na: 671.376 6), corresponding to the molecular formula of C36H56O10. The IR spectrum showed the characteristic absorptions of hydroxyl (3 412 cm−1) and carbonyl (1 734 cm−1) groups. The 1H NMR spectrum of 1 displayed resonances for five methyl groups at δH 1.03 (3H, d, J = 5.1 Hz), 1.04 (3H,

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HMBC, and NOESY experiments, all of the 1H and 13C NMR signals of 1 were assigned (Table 1). The aglycone of compound 1 was identified as 2α, 3α, 24-trihydroxyurs-12, 20(30)-dien-28-oic acid by comparison of the NMR data with those published [11]. In addition, in the NOESY spectrum of 1, correlations between H-2 (δH 4.45) and H-3 (δH 4.59)/H3-25 (δH 1.04), and between H-3 (δH 4.59) and H-24 (δH 4.11) were observed (Fig. 2), which further confirmed the relative configuration of the aglycone of 1.

s), 1.06 (3H, s), 1.13 (3H, s), and 1.67 (3H, s), two oxymethines at δH 4.45 (1H, m) and 4.59 (1H, br s), an exocyclic double bond at δH 4.70 (1H, br s) and 4.73 (1H, br s), as well as an olefinic proton at δH 5.41 (br s). The 13C NMR and DEPT spectra of 1 showed the signals for 36 carbons, including five methyls (δC 16.4, 17.3, 17.5, 23.5 and 23.8), three oxygenated carbons (δC 65.2, 66.2 and 74.2), four olefinic carbons (δC 105.2, 126.4, 138.0 and 153.2), and an ester carbonyl carbon (δC 175.6). With the assistance of 1H-1H COSY, HSQC, Table 1

1

H and 13C NMR spectral data of 1 (pyridine-d5) a, b

Position

δC

1

43.2

δH

Position

δC

δH 2.38

α 1.84

19

37.5

β 1.93 (dd, 2.7, 0.8)

20 21

153.2 32.4

2

66.2

4.45

3

74.2

4.59 (br s)

4

45.1

22

38.9

α 1.77 1.67 (s)

5

49.4

1.81

6

18.9

α 1.48

23

23.8

β 1.70

24

65.2

7

33.9

α 1.58

8

40.3

9

48.1

10

38.5

α 2.13 β 2.28 (m) β 2.04 a 3.81 (d, 11.3) b 4.11 (d, 11.3)

β 1.39 (d, 11.1)

25

17.3

1.04 (s)

1.84

26

17.5

1.13 (s)

27

23.5

28

175.6

1.06 (s)

11

23.9

2.04

29

16.4

1.03 (d, 5.1)

12

126.4

5.41 (br s)

30

105.2

a 4.70 (br s)

13

138.0

14

42.5

1′

95.7

6.24 (d, 8.0)

15

28.5

16

24.6

17

48.5

18

55.3

b 4.73 (br s) α 1.31

2′

73.9

4.18

β 2.44

3′

78.8

4.26

α 2.04

4′

71.1

4.31

β 2.15

5′

79.2

4.01

6′

62.3

4.37 (dd, 10.9, 4.2)

2.62 (d, 11.1)

4.44 (dd, 10.9, 4.2)

a

Data were recorded on a Bruker AV-300 spectrometer; assignments were confirmed by 1H-1H COSY, HSQC, HMBC and NOESY; b Overlapped signals are reported without designating multiplicity.

Fig. 2 Selected HMBC and NOESY correlations of 1

The 1H and 13C NMR spectra of 1 also showed the presence of a sugar unit with an anomeric proton at δH 6.24 (d, J = 8.0 Hz) and an anomeric carbon at δC 95.7 (Table 1).

Acid hydrolysis of 1 afforded D-glucose, which was identified by HPLC analysis [11]. The β-configuration of D-glucose was determined by the 3JH1,H2 coupling constant (J = 8.0 Hz) of the anomeric proton. In the HMBC spectrum of 1, the correlation between H-1′ (δH 6.24) of the glucose and C-28 (δC 175.6) of the aglycone indicated that the glucose was attached to the C-28 position of the aglycone (Fig. 2). Therefore, the structure of 1 was determined to be 2α, 3α, 24-trihydroxyurs-12, 20(30)-dien-28-oic acid-28-O-β-Dglucopyranosyl ester. Compounds 2−4 were identified as corosolic acid (2) [12], vulgarsaponin A (3) [13], and 2α, 3α, 24-trihydroxyurs-12-en28-oic acid-28-O-β-D-glucopyranosyl ester (4) [14], respectively, by comparison of their spectral data with the literature values.

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Experimental General experimental procedures Melting points were determined on an X-5 micromelting point detector (uncorrected). Optical rotation values were obtained on a JASCO P-1020 digital polarimeter at room temperature. IR spectra were recorded on a JASCO FT/IR-480 Plus Fourier Transform infrared spectrometer with KBr pellets. ESIMS spectra were obtained on a Finnigan LCQ Advantage MAX spectrometer. HRESIMS spectra were obtained on an Agilent 6210 ESI/TOF mass spectrometer. 1D- and 2D-NMR spectra were carried out on Bruker AV-300 spectrometer and a Bruker AV-400 spectrometer using TMS as internal standard. Analytical and preparative HPLC were carried out using an Agilent 1260 series with Cosmosil 5C18-MS-II reversed-phase column (4.6 mm × 250 mm i.d., 20 mm × 250 mm i.d., 5.0 μm). Column chromatography was carried out on silica gel (200−300 mesh; Qingdao Marine Chemical Group Co. Ltd, Qingdao, China), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) and ODS (50 μm, 120 Å; YMC). TLC was performed using precoated silica gel GF254 plates (Yantai Chemical Industry Research Institute, Yantai, China) and Rp-18 F254 plates (Merck). The standards D-glucose and L-glucose were purchased from Alfa Aesar. L-Cysteine methyl ester and O-Tolyl isothiocyanate were purchased from Sigma (St. Louis, MO, USA). Plant material The aerial parts of V. negundo were purchased from Qingping medicinal market, Guangzhou city, Guangdong province of China, in April 2012, and authenticated by Prof. ZHOU Guang-Xiong (College of Pharmacy, Jinan University). A voucher specimen (No. 20120406) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, China. Extraction and isolation The air-dried aerial parts of V. negundo (10.0 kg) were powdered and percolated with 75% (V/V) EtOH at room temperature three times. The solution was evaporated under vacuum to obtain a crude extract (625 g). The extract was suspended in water and partitioned successively with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract (224 g) was chromatographed on a silica gel column (20 cm × 70 cm, 200−300 mesh, 2 kg), and eluted with CHCl3−MeOH (100 : 0 to 0 : 100, V/V, each 30 L) to give 13 fractions (Fr. 1−Fr. 13). Fr. 3 (4 g) was subjected to a Sephadex LH-20 column (4 cm × 60 cm, MeOH) to obtain compound 2 (5.8 mg). Fr. 10 (11 g) was chromatographed over an ODS column (4 cm × 60 cm, MeOH−H2O, 10 : 90 to 100 : 0, V/V, each 1.5 L) to afford 17 subfractions (A−Q). Subfraction O was then re-purified by preparative HPLC (CH3CN−H2O, 35 : 65, V/V) to obtain compounds 1 (11.2 mg) and 3 (14.8 mg). Subfraction P was chromatographed over preparative HPLC (CH3CN−H2O, 32 : 68, V/V) to afford 4 (11.7 mg).

Acid hydrolysis of compound 1 and absolute configuration determination of sugar [15] Compound 1 (5 mg) was hydrolyzed with 2 mol·L−1 HCl (10 mL) for 5 h at 80 °C. The reaction mixture was concentrated and dried by N2. Then, anhydrous pyridine (1 mL) and L-cysteine methyl ester (5 mg) were added to the residue. The mixture was heated at 60 °C for 1 h. O-Tolyl isothiocyanate (5 μL) was added and the mixture kept at 60 °C for 1 h. Finally, the solution was filtered through 0.45 μm millipore and analyzed by HPLC under the following conditions: column: Cosmosil 5C18-MS-II (4.6 mm × 250 mm i.d., 5.0 μm); mobile phase: CH3CN−H2O (25 : 75, V/V) with 0.05% acetic acid; column temperature: 25 °C; detection wavelength: 250 nm; detection time: 40 min; flow-rate: 0.8 mL·min−1; injection volume: 10 μL. The standard D-glucose and L-glucose were treated in the same manner (tR: 20.78 min of D-glucose and 18.97 min of L-glucose). D-glucose (tR: 20.74 min of D-glucose) was detected from the hydrolyzate of 1.

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Cite this articls as: CHEN Jiao, FAN Chun-Lin, WANG Ying, YE Wen-Cai. A new triterpenoid glycoside from Vitex negundo [J]. Chinese Journal of Natural Medicines, 2014, 12(3): 218-221

Corrigendum In Chinese Journal of Nateral Medicines, 2014, 12(1): 8-14 Incorrect

Correct

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A new triterpenoid glycoside from Vitex negundo.

To investigate the chemical constituents of Vitex negundo...
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