Antioxidant lignan glucosides from Strychnos vanprukii

Fitoterapia 75 (2004) 623 – 628 www.elsevier.com/locate/fitote

Antioxidant lignan glucosides from Strychnos vanprukii Piyanuch Thongphasuka, Rutt Suttisria,*, Rapepol Bavovadaa, Robert Verpoorteb a

Department of Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand b Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden 2300 RA, The Netherlands Received 2 July 2003; accepted 3 April 2004 Available online 27 July 2004

Abstract From the stem of Strychnos vanprukii, two new lignan glucosides, vanprukoside (1) and strychnoside (2), were isolated together with the known lignan glucoside, (+)-lyoniresinol-3a-O-hglucopyranoside (3). The structures of these compounds were elucidated on the basis of their spectroscopic data. All three compounds exhibited antioxidant activity. D 2004 Elsevier B.V. All rights reserved. Keywords: Strychnos vanprukii; Lignan glucosides; Antioxidant

1. Introduction Strychnos vanprukii Craib (Loganiaceae), locally called bThao changQ, is a large woody climber found in northern Thailand [1]. The quaternary alkaloids of this plant were reported as exhibiting muscle relaxant activity [2]. Plants of the genus Strychnos have been employed both as folk medicine and as poison in many parts of the world [3]. The

* Corresponding author. Tel.: +66 2 218 8353; fax: +66 2 254 5195. E-mail address: [email protected] (R. Suttisri). 0367-326X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.04.013

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Fig. 1. Compounds 1–3.

alkaloid strychnine found in several of these plants can cause paralysis of the respiratory system, resulting in death. Non-alkaloidal constituents such as lignans [4], iridoid [5], benzenoids [6] and triterpenoids [7] have also been isolated from this genus. In this paper, we report the isolation of two new lignan glucosides, vanprukoside (1) and strychnoside (2), together with a known lignan, (+)-lyoniresinol-3a-O-h-glucopyranoside (3), from the stem of S. vanprukii Fig. 1.

2. Experimental setup 2.1. General UV spectra were obtained on a Milton Roy Spectronic 3000 Array Spectrometer. IR spectra were recorded on a Perkin Elmer FT-IR 1760 spectrophotometer. MS spectra were recorded on an Applied Biosystems Q-STAR (quadrupole-tof) mass spectrometer. NMR spectra were recorded in CD3OD using a Varian DPX-300 FT-NMR

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Spectrometer (300 MHz for 1H NMR and 75 MHz for 13C NMR). TLC was carried out using precoated Kieselgel 60 F254 plates (Merck). The spray reagents used for TLC were 10% H2SO4 in 95% EtOH and FeCl3/HClO4 reagent. The microplate was read by a Bio-Rad microplate reader model 3550 UV (Bio-Rad Laboratories, Richmond, CA, USA). 2.2. Plant material S. vanprukii stem—collected from Chaiyapum, Thailand, in December 1998 and identified by Dr. Rapepol Bavovada. A voucher specimen (No. RB9824) was deposited in the Herbarium of the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok. 2.3. Extraction and isolation Dried and powdered stem of S. vanprukii (100 g) was extracted with 95% EtOH. The EtOH extract (4.1 g) was dissolved in aqueous MeOH and partitioned with hexane, followed by CHCl3, to give hexane (258 mg), CHCl3 (328 mg) and aqueous MeOH (3.5 g) extracts. The CHCl3 extract was subjected to Si-gel CC eluting with CHCl3/MeOH 10:1 to yield vanprukoside (1, 15 mg) and strychnoside (2, 15 mg). The MeOH extract was Si-gel CC eluting with CHCl3/MeOH 4:1 to give seven fractions (M1–M7). Fraction M2 (100 mg) was subjected to further Si-gel CC eluting with CHCl3/Me2CO/MeOH 10:2:3 to afford (+)-lyoniresinol-3a-O-h-glucopyranoside [8] (3, 24 mg). Vanprukoside (1). Amorphous powder; [a]D25 3.28 (c 0.1, MeOH); UVmax (MeOH): 208 and 277 nm; IR bands (MeOH): 3365, 2941, 1706, 1613, 1516 cm 1; positive ESIMS-MS: m/z [M+Na]+ 785, [M]+ 762, 737, 711, 685; 1H NMR (CD3OD, 300 MHz): d 2.12 (1H, m , H-2), 2.27 (1H, m, H-3), 2.74 (2H, m, H-1), 3.20–3.26 (4H, m, H-2- H-5 Glc), 3.34 (3H, s, 5-OCH3), 3.57 (1H, dd, J 9.9, 5.4 Hz, H-3a, a), 3.69 (1H, m, H-6 Glc), 3.71 (6H, s, 3V-OCH3, 5V-OCH3), 3.82 (6H, s, 3W-OCH3, 5W-OCH3), 3.84 (3H, s, 7OCH3), 3.85 (1H, m, H-6 Glc), 3.98 (1H, dd, J 9.9, 4.3 Hz, H-3a, b), 4.27 (1H, d, J 8.9 Hz, H-1 Glc), 4.30 (1H, dd, J 12.9, 6.3 Hz, H-2a, a), 4.38 (1H, d, J 6.1 Hz, H-4), 4.43 (1H, dd, J 12.9, 4.8 Hz, H-2a, b), 6.38 (2H, s, H-2V, H-6V), 6.59 (1H, s, H-8), 7.22 (2H, s, H-2W, H-6W). For 13C NMR data, see Table 1. Strychnoside (2). Amorphous powder; [a]D25 8.48 (c 0.1, MeOH); UVmax (MeOH): 206 and 277 nm; IR bands (MeOH): 3369, 2928, 1706, 1613, 1516, 1462 cm 1; positive ESI-MS-MS: m/z [M+Na]+ 965, 767, 737; 1H NMR (CD3OD, 300 MHz): 1.84 (1H, m, H2), 2.14 (1H, m, H-3), 2.59 (2H, br d, J 7.5 Hz, H-1), 3.16 (3H, s, 5-OCH3), 3.32 (1H, m, H-5 Glc), 3.34 (1H, m, H-4 Glc), 3.48 (1H, m, H-3a, a), 3.67 (6H, s, 3VVV-OCH3, 5VVVOCH3), 3.72 (6H, s, 3V-OCH3, 5V-OCH3), 3.74 (1H, m, H-3 Glc), 3.75 (1H, m, H-6 Glc), 3.81 (9H, s, 7-OCH3, 3VV-OCH3, 5VV-OCH3), 3.90 (1H, m, H-6 Glc), 4.01 (1H, m, H-3a, b), 4.08 (1H, m, H-2a, a), 4.13 (1H, m, H-2a, b), 4.24 (1H, d, J 6.1 Hz, H-4), 4.61 (1H, d, J 8.1 Hz, H-1 Glc), 5.02 (1H, dd, J 9.5, 8.1 Hz, H-2 Glc), 6.27 (2H, s, H-2V, H-6V), 6.48 (1H, s, H-8), 7.12 (2H, s, H-2VVV, H-6VVV), 7.21 (2H, s, H-2VV, H-6VV).For 13C NMR (CD3OD, 75 MHz), see Table 1.

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Table 1 13 C NMR data of compounds 1–3a 1 1 2 2a 3 3a 4 5 6 7 8 9 10 1V 2V 3V 4V 5V 6V 1U 2U 3U 4U 5U 6U 7U Glc1 2 3 4 5 6 1VVV 2VVV 3VVV 4VVV 5VVV 6VVV 7VVV 5-OCH3 7-OCH3 3V,5V-OCH3 3VV,5VV-OCH3 3VVV,5VVV-OCH3 a

33.5 38.0 68.7 46.6 71.8 42.5 147.6 139.2 148.8 107.9 129.5 125.8 139.1 106.9 149.0 134.7 149.0 106.9 121.5 108.2 149.0 142.1 149.0 108.2 168.2 104.7 75.2 78.1 71.6 77.9 62.8 – – – – – – – 60.3 56.6 56.9 56.9 –

2 t d t d t d s s s d s s s d s s s d s d s s s d s d d d d d t

q q q q

33.4 37.8 68.3 47.0 71.3 42.2 147.6 138.9 148.6 107.5 129.4 125.4 139.2 106.6 148.9 134.5 148.9 106.6 121.3 108.1 148.9 141.9 148.9 108.1 167.9 102.9 75.8 76.0 71.8 78.1 62.6 121.3 108.3 148.6 141.7 148.6 108.3 167.4 60.0 56.5 56.6 56.8 56.8

3 t d t d t d s s s d s s s d s s s d s d s s s d s d d d d d t s d s s s d s q q q q q

33.8 40.6 66.2 46.7 71.5 42.8 147.6 – 148.6 107.8 130.2 126.4 139.3 106.9 149.0 – 149.0 106.9 – – – – – – – 104.8 75.1 78.2 71.7 77.9 62.8 – – – – – – – 60.2 56.6 56.9 – –

t d t d t d s s d s s s d s s d

d d d d d t

q q q

75 MHz, CD3OD.

2.4. Antioxidant activity Reduction of the purple-colored 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was used as the assay for the antioxidant activity of the isolated compounds according to Cavin

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et al. [9]. Rapid screening of these lignans was done by developing TLC of the compounds, and upon drying, the plate was sprayed with a 0.2% DPPH solution in MeOH. Thirty minutes later, the plate was examined. Active compounds appeared as yellow spots against the purple background. Evaluation of the antioxidant activity as EC50 was performed spectrophotometrically [9]. Different concentrations of the test compounds were mixed with 50 Al of 0.022% DPPH solution in MeOH and left standing for 30 min. Absorbance of the solutions was then determined at 550 nm and the percentage of activity was calculated.

3. Results and discussion The CHCl3 extract of the stem of S. vanprukii yielded compounds 1 and 2 after being subjected to Si-gel CC, whereas repeated CC of the MeOH extract gave a known lignan glycoside, (+)-lyoniresinol-3a-O-h-glucopyranoside 3 [8]. The ESI-MS-MS gave a molecular ion peak of compound 1 at m/z 762, corresponding to the molecular formula C37H46O17, together with another ion peak at m/z 785 for [M+Na]+. The NMR spectra of compound 1 displayed signals characteristic of an aryl tetralin-type lignan glucoside with an additional acyloxy moiety. In the 1H NMR spectrum, three signals due to five aromatic protons appeared at d 6.38 (2H, s, H-2V, H-6V), 6.59 (1H, s, H-8) and 7.22 (2H, s, H-2VV, H-6VV), suggesting the presence of two tetra- and one penta-substituted benzene rings. For the aliphatic part of the compound, 1H NMR spectrum exhibited signals of three methine protons at d 2.12 (1H, m, H-2), 2.27 (1H, m, H-3) and 4.38 (1H, d, J 6.1 Hz, H-4), together with the signals of methylene protons at d 2.74 (2H, m, H-1), 3.57 (1H, dd, J 9.9, 5.4 Hz, H-3a, a), 3.98 (1H, dd, J 9.9, 4.3 Hz, H3a, b), 4.30 (1H, dd, J 12.9, 6.3 Hz, H-2a, a) and 4.43 (1H, dd, J 12.9, 4.8 Hz, H-2a, b). The APT spectrum of compound 1 displayed 37 carbon signals (Table 1), including those of one glucose and one acyloxy units. Among the 22 signals of the aglycone, those corresponding to 9 quaternary carbon atoms appeared at d 147.6 (C-5), 139.2 (C-6), 148.8 (C-7), 129.5 (C-9), 125.8 (C-10), 139.1 (C-1V), 149.0 (C-3V, C-5V), 134.7 (C-4V) and 6 methine carbon atoms at d 38.0 (C-2), 46.6 (C-3), 42.5 (C-4), 107.9 (C-8), 106.9 (C-2V, C6V). Three methylene carbon signals at d 33.5, 68.7 and 71.8 could be assigned as those of C-1, C-2 and C-3, respectively. The NMR data also showed the presence of four methoxy groups located at C-5, C-7, C-3V and C-5V. The carbon signals of acyloxy moiety detected at d 121.5 (C-1VV), 108.2 (C-2VV, C-6VV), 149.0 (C-3VV, C-5VV), 142.1 (C-4VV), 168.2 (C7VV) and 56.9 (3VV-OCH3, 5VV-OCH3) were indicative of a syringyl unit [10]. Chemical shift analysis indicated the glucose moiety as located at C-3a, similar to compound 3. The location of the syringyl moiety was determined by comparison of the 13C NMR data of 1 with that of 3 in which the downfield shift of C-2 (+2.5 ppm) and upfield shift of C-2 ( 2.4 ppm) were evident, indicating the attachment of the additional ester unit at C-2a. Consequently, the structure of 1 was assigned as 2a-[(3,5-dimethoxy-4-hydroxy)benzoyl]-(+)-lyoniresinol-3a-O-h-d-glucopyranoside. The molecular formula of compound 2 was determined as C46H54O21 from the [M+Na]+ ion peak at m/z 965 in the ESI-MS-MS spectrum. Both 1H and 13C NMR data of 2 were very similar to those of 1 except for the signals of one more syringyl unit. The

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Table 2 Antioxidant activity of compounds 1–3 Compounds

EC50 (AM)

1 2 3 Ascorbic acid

18.7 12.5 31.2 57.2

downfield shift of C-2 signal (+0.6 ppm) and the upfield shift of both C-1 and C-3 signals ( 1.8 and 2.1 ppm, respectively) of the glucosyl moiety in 2 compared to those of 1 indicated the attachment of this second syringyl group to C-2 of the glucose moiety. Moreover, HMBC showed correlation between the signals of H-2 (d 5.02) of the glucosyl moiety and carbonyl carbon (d 167.4) of this syringyl unit. The structure of compound 2 was therefore elucidated as 2a-[(3,5-dimethoxy-4-hydroxy)-benzoyl]-(+)-lyoniresinol-3a[2-[(3,5-dimethoxy-4-hydroxy)-benzoyl]-O-h-glucopyranoside. A number of lignans have been demonstrated as possessing antioxidant property [11,12]. All three lignan glucosides 1, 2, 3, isolated from S. vanprukii were tested for their radical scavenging activity against DPPH and compared with that of ascorbic acid, which is a well-known antioxidant (Table 2). All three lignans exhibited stronger antioxidant activity than ascorbic acid.

Acknowledgements Financial support from the Thailand Research Fund through the Royal Golden Jubilee PhD Program to P.Thongphasuk and R.Bavovada is acknowledged.

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