Terpenoids from Vitex Negundo Seeds

Biochemical Systematics and Ecology 38 (2010) 247–249

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Biochemical Systematics and Ecology

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Terpenoids from Vitex negundo seeds

Cheng-Jian Zheng a, Bao-Kang Huang a, Yan-Bin Wu b, Ting Han a, Qiao-Yan Zhang a, Hong Zhang a, Lu-Ping Qin a,* a Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, P.R. China b Department of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350108, P.R. China article info

Article history: Received 17 November 2009 Accepted 1 January 2010

Keywords: Vitex negundo vitexilactone B diterpenoid triterpenoids

1. Subject and source

Vitex negundo Linn (Verbenaceae) is a small aromatic plant that ﬂourishes abundantly in wastelands and is widely distributed in tropical to temperate regions, being a native of South Asia, China, Indonesia, and the Philippines (Dharmasiri et al., 2003). The seeds of V. negundo (Chinese name ‘‘Huang-Jing-Zi’’), which were identiﬁed by Prof. Han-Chen Zheng, School of Pharmacy, Second Military Medical University, were obtained from the Wanglang National Nature Reserve, Sichuan province in Oct 2006. A voucher specimen (#2006-168) has been deposited in the herbarium of the Department of Phar- macognosy, School of Pharmacy, Second Military Medical University, Shanghai, China.

2. Previous work

Several species of the genus Vitex have been extensively studied and more than 60 diterpenoids, most of which are of labdane- type, have been isolated from them, together with triterpenoid, ecdysteroid, lignan, and ﬂavonoid compounds (Li et al., 2005; Ono et al., 2001a, b, 2004; Rimpler, 1972; Chawla et al., 1992; Ono et al., 2004). Previous phytochemical investigations on V. negundo led to the isolation of ten diterpenoids, including six labdanes (vitedoin B, negundoins A–E), three abietanes (5b-hydro-8,11,13-abietatrien-6a-ol, 3b-hydroxy-8,11,13-abietatrien-7-one, negundoin F), and one sandaracopimarane (negundoin G) (Chawla et al., 1991; Ono et al., 2004; Zheng et al., 2010).

3. Present study

The air-dried and powdered seeds (25 kg) of V. negundo were extracted with aqueous ethanol (80% v/v) three times for 2 h each time. After removal of the solvent under reduced pressure, the residue was partitioned sequentially with petroleum

* Corresponding author. Tel./fax: þ86 21 81871300. E-mail address: [email protected] (L.-P. Qin).

16 12 O 11 OH OH 13 15 OH 20 1 OH 14 O OH 2 9 17 O 10 8 4 5 OH O 3 7 1’ 6 OCOCH3 2‘ 18 19 OCOCH3

1 2 3 4

HOH2C

COOH

CH2OH HO HO HO

567

COOH COOH COOH HO

HO HO HO

8 9 10

Fig. 1. Terpenoids from Vitex negundo seeds.

ether, CH2Cl2, EtOAc, and n-BuOH, respectively. The CH2Cl2 extract (260 g) was subjected to silica gel chromatography using mixtures of petroleum ether-EtOAc mixtures (50:1, 20:1,10:1, 5:1, 3:1,1:1, 0:1 v/v) to afford fractions A-G. Further purification was submitted to silica gel, ODS, Sephadex LH-20 chromatography and preparative TLC. Fraction A (petroleum ether-EtOAc, 50:1) yielded vitetrifolin D (4, 5 mg, Ono et al., 2001a, b). Fraction B (petroleum ether-EtOAc, 20:1) gave obtusalin (5, 20 mg, Siddiqui et al., 1989), betulinic acid (7, 10 mg, Patra et al., 1988), and oleanolic acid (9, 40 mg, Tori et al., 1974). From Fraction C (petroleum ether-EtOAc, 10:1), viteagnusin C (2, 12 mg, Ono et al., 2008), 8-epi-sclareol (3, 15 mg, Torrenegra et al., 1992), and ursolic acid (10, 36 mg, Poehland et al., 1987) was purified. Fraction D yielded vitexilactone B (1), lup-20(29)-en-3b, 30-diol (6, 4 mg, Abdel-Mogib, 1999), and maslinic acid (8, 15 mg, Tori et al., 1974). The structures of the known compounds (Fig. 1) were identified based on spectroscopic analysis (1HNMR, 13C NMR, 2D NMR, MS, and IR) and by comparison of their spectral data with those reported previously in the literature. Compound 1, trivially named vitexilactone B, was obtained as a colorless syrup and analyzed for the molecular formula þ C22H34O5 by positive HRESIMS [M þ Na] at m/z 401.2307 (calcd 401.2304), which was supported by its NMR data. The IR spectrum revealed absorption bands of an a, b-unsaturated-g-lactone group (1778, 1749 cm1). The 1H and 13C NMR spectra of 1 together with DEPT and 1H–13C COSY analysis indicated the presence of general structural features in common with vitexilactone, which was previously isolated from the leaves of Vitex cannabifolia (Taguchi, 1976) and the fruits of Vitex rotundifolia (Kondo et al., 1986; Ono et al., 2001b). The only differences were the splitting pattern and chemical shift of an oxygenated methane proton (dH 4.46, dd, J ¼ 4.2, 12.0 Hz in 1; dH 5.39, ddd, J ¼ 3.0, 3.0, 3.0 Hz in vitexilactone) and the chemical shift corresponding to the oxygenated methane carbon signal (dC 80.2 in 1; dC 69.8 in vitexilactone). These data led to the assumption that 1 was a regioisomer of vitexilactone with an acetoxyl group at C-3 instead of C-6, which was confirmed by detailed HMBC and 1H–1H COSY spectroscopic analysis (Fig. 2). In the NOESY spectra of 1, correlations were observed

O O H C H OH O 3 H O CH3 AcO O CH3 CH O H 3 H OH

Fig. 2. Key HMBC ( ), 1H–1H COSY ( ), and NOSEY ( ) correlations of vitexilactone B (1). C.-J. Zheng et al. / Biochemical Systematics and Ecology 38 (2010) 247–249 249

Table 1 1 13 H NMR (600 MHz) and C NMR (150 MHz) data of compound 1 in CDCl3 (d, ppm; J, Hz)

Positions. dH (J) dC Positions. dH (J) dC 1 1.50, 1.73 (2H, m) 29.8 t 12 2.48 (2H, m) 23.5 t 2 1.63, 1.72 (2H, m) 23.2 t 13 171.2 s 3 4.46 (1H, dd, J ¼ 4.2, 12.0) 80.2 d 14 5.82 (1H, s) 114.9 d 4 37.7 s 15 174.0 s 5 1.58 (1H, m) 45.9 d 16 4.75 (2H, br s) 73.2 t 6 1.38, 1.65 (2H, m) 20.9 t 17 0.79 (3H, d, J ¼ 6.6) 16.1 q 7 1.36, 1.53 (2H, m) 31.0 t 18 0.89 (3H, s) 28.3 q 8 1.75 (1H, m) 36.8 d 19 0.86 (3H, s) 16.6 q 9 76.4 s 20 0.96 (3H, s) 16.9 q 10 42.9 s 10 170.7 s 11 1.73, 1.96 (2H, m) 31.7 t 20 2.05 (3H, s) 21.2 q

Multiplicity was determined by DEPT experiments (s ¼ quaternary, d ¼ methine, t ¼ methylene, q ¼ methyl). between H-8/H-20, H-19/H-20, H-20/H-11, H-3/H-18, and H-5/H-3, indicating a b-orientation for H-8, H-19, and H-20 and an a- orientation for H-3, H-5, H-17, H-18, and OH-9. Compound 1 was therefore characterized as (rel 3S, 5S, 8R, 9R, 10S)-3- acetoxy-9-hydroxy-13(14)-labden-15, 16-olide. 25 1 1 Vitexilactone B (1): colorless syrup; ½aD 14.8 (c 1.26, MeOH); IR nmax 3525, 1778, 1749, 1728, 1710, 1633 cm ; H(CDCl3, 13 þ 600 MHz) and C NMR (CDCl3, 150 MHz) data (see Table 1); (þ)-HRESIMS m/z [M þ Na] 401.2307 (calcd for C22H34O5Na, 401.2304).

4. Chemotaxonomic signiﬁcance

Vitex species are known to contain terpenoids metabolites, especially different types of diterpenoids (Li et al., 2005). Our study has afforded a new labdane diterpenoid (1) with two known ones (2–3), a halimane diterpenoid (4), three lupane triterpenoids (5–7), and two oleanane triterpenoids (8 and 9) as well as an ursane triterpenoid (10) from V. negundo seeds. To the best of our knowledge, the occurrence of compounds 2–6 and 8 was reported for the ﬁrst time in this plant. In addition, compounds 5 and 6 have not been reported in any species of the genus Vitex. Investigations on other Vitex species, such as Vitex altissima (Sridhar et al., 2005), Vitex peduncularis (Li et al., 2005), Vitex trifolia (Ono et al., 2001a), V. rotundifolia (Ono et al., 2001b), and Vitex agnus-castus (Ono et al., 2008), also gave different kinds of terpenoids. Labdane diterpenoids viteagnusin C (2) and 8-epi-sclareol (3) have been recently isolated from the fruits of V. agnus-castus (Ono et al., 2008). Halimane diterpenoid vitetrifolin D (4) has been previously separated from the fruits of V. trifolia and V. agnus-castus (Ono et al., 2001a; Hoberg et al., 1999; Hoberg et al., 2003). Besides, oleanane triterpenoid maslinic acid (8) was formerly obtained from V. altissima (Sridhar et al., 2005). Among all those terpenoids metabolites isolated from Vitex plants, labdane diterpenoids structurally characterized by a furan ring from C13 to C16 have been regarded as the characteristic constituents in this genus (Li et al., 2005). The data suggests that the presence of vitexilactone B (1), a new labdane diterpenoid, could be considered as a chemosystematic marker for V. negundo.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 30772783).

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