Biochemical Systematics and Ecology 44 (2012) 205–207 Contents lists available at SciVerse ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Natural phenoxazine alkaloids from Peristrophe bivalvis (L.) Merr Trinh Thi Thuy, Tran Hoai Lam, Nguyen Thi Thanh Huong, Le Thi Hong Nhung, Pham Thi Ninh, Nguyen Thi Hoang Anh, Tran Thi Phuong Thao, Tran Van Sung* Institute of Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam a r t i c l e i n f o Article history: Received 20 December 2011 Accepted 19 May 2012 Available online Keywords: Peristrophe bivalvis Acanthaceae Phenoxazine Peristrophine Perisbivalvine A 1. Subject and source Peristrophe, a genus of the Acanthaceae family, is distributed in Southern Asia and Africa (Balkwill, 1996; Balkwill et al., 1988). According to the literature there are four Peristrophe species in the Vietnamese flora. One of them Peristrophe bivalvis (L.) Merr., local name “Cẩm” [syn. P. roxburghiana (Schult.) Bremek.; Peristrophe tinctoria (Roxb.) Nees], is growing wildly and cultivated for dying foods as sticky rice and other dishes by the ethnic minorities (Chi, 1999). This species appears in four forms, namely red, purple, purple-magenta, and yellow (the rare form) according to the colours of aqueous extract (Chi, 1999; Trinh et al., 2003). Besides the utilization as food dyestuff, this plant has been used in traditional medicine for the treatment of pulmonary tuberculosis, haemoptysis, congestion, bronchitis and wrench (sprain) (Yang et al., 2002). We investigated the leaves of red and purple P. bivalvis (L.) Merr., two common cultivated forms as a plant to dye food. The plant materials were collected in July 2009 in “Moc Chau steppe”, Son La province of Vietnam and determined by Dr. Ninh Khac Ban, Institute of Ecology and Biological Resources (IEBR), Vietnam Academy of Science and Technology (VAST). A voucher specimen (No. HN35415) was deposited at the Herbarium of the IEBR, VAST. 2. Previous work Previous phytochemical studies on species of the genus Peristrophe have revealed the presence of aliphatics, 14-methyltritriacont-14-en-15-ol, 35-hydroxynonatriacontanal from Peristrophe bicalyculata (Singh et al., 2000); cholest-5-en-3b-oxyl hexadecanoate, octadecanoic acid, palmitic acid, steroids from Peristrophe japonica (Pi et al., 2008) and two alkaloids, peristrophamide and peristrophine (1) from aerial parts of Peristrophe roxburghiana growing in China (Qin et al., 1999). * Corresponding author. Tel.: þ84 4 37564794; fax: þ84 4 38361283. E-mail address: tranvansungvhh@gmail.com (T. Van Sung). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2012.05.009 206 T.T. Thuy et al. / Biochemical Systematics and Ecology 44 (2012) 205–207 3. Present study The fresh leaves of red P. bivalvis (5 kg) were washed with distilled water, cut into small pieces, crushed, extracted with distilled hot water and allowed to boil for 15–20 min (2  8 L). The colorants of aqueous extracts (CAE) was combined, filtered and dried in open air at a temperature of 60-70  C until ca one third volume left, then successively extracted with EtOAc and n-BuOH. The BuOH extract residue was subjected to a Sephadex LH-20 column, eluted with MeOH to give compound 1 (15 mg). The fresh leaves of purple P. bivalvis (5 kg) was treated in the same way as described above to give purple colorant of aqueous extract (PCAE). The PCAE was successively extracted with EtOAc and n-BuOH. The BuOH extract residue was subjected to a Sephadex LH-20 column (MeOH) to yield 2 fractions (F1–F2). Fraction F2 was chromatographed over LiChroprep RP18 (MeOH–H2O, 1:1) and then purified again by Sephadex LH-20 column (MeOH) to afford compound 2 (20 mg). Although compound 1 has been identified as peristrophine from aerial parts of P. roxburghiana (Qin et al., 1999), its NMR data contained some mistakes, because of the lack of 2D-NMR. Herein, its completed spectroscopic data are elucidated by analysis of 2D-NMR (HMQC, HMBC) (Table 1) and ESI-FTICR-MS (m/z 273.0936 [M þ H]þ, calc. for C14 H13 N2 Oþ 4 273.0875). The molecular formula of compound 2, C15H12N2O5, was deduced from the ESI-FTICR-MS (negative ions) at m/z 299.0674 [M H] (calc. for C15 H11 N2 O5 299.0673), 1H and 13C DEPT NMR spectra Table 1. The 13C NMR and DEPT spectra of 2 displayed 15 carbons (2  CH3, 4  CH, 9xCq). The 1H- and 13C NMR spectra (in CD3OD) revealed the presence of a methoxy group (dH 3.93/dC56.3), one acetyl group (dH 2.25, dC 171.5) and NH signal in the lower field (dH 9.02, 1H, br s, DMSO-d6). The low field region displayed four aromatic protons at dH 6.46, 6.51, 6.92, and 8.51 ppm (each 1H, singlet). The HMBC correlations between C-7 (dC155.8) with OCH3, H-6 and H-9 revealed a methoxy group at C-7 (Table 1). The linkage between acetylamino group (AcNH) and phenoxazine unit was confirmed by HMBC correlations between NH (dH 9.02) with C-2 and C-3. The structure of 2 was elucidated from the above data as 2-acetylamino-7-methoxy-8-hydroxy-3Hphenoxazine-3-one, a novel natural phenoxazine alkaloid, named as perisbivalvine A, isolated and identified for the first time (Fig. 1). Considering the widespread utilization of the decoction of P. bivalvis, we evaluated the acute oral toxicity of CAE and PCAE. Based on the results in an acute toxicity study in mice, the acute median lethal dose (LD50) of CAE and PCAE were 9.10  0.29 and 26.50  1.56 g/kg body weight, respectively have been found. 4. Chemotaxonomic significance Peristrophine (1) has been identified as the first phenoxazine alkaloid from P. roxburghiana (Qin et al., 1999), whereas the isolation and identification of perisbivalvine A (2) is now firstly reported from this plant. The phenoxazine alkaloids have been before reported to found from arthropods and fungi (Okafor, 1986; Fomsgaard et al., 2004). The phenoxazine derivatives, pycnosanguin, cinnabarine and cinnabarinic acid were found from Pycnoporus sanguineus (Polyporaceae), a white red saprobic fungus, usually growing on dead hardwoods. It grows in the form of a thin dry conk with a lateral attachment to its substrate (Achenbach and Blümm, 1991). Because P. bivalvis is a herb, the contamination of this plant by this fungus is not possible. Moreover, the plant material has been carefully treated before the extraction. Therefore, the occurrence of phenoxazine 1 and 2 in P. bivalvis (P. roxburghiana) might be a special valuable chemotaxonomic marker for the classification for Table 1 13 C and 1H NMR spectral data of peristrophine (1) and perisbivalvine A (2) (125/500 MHz, d ppm, DMSO-d6). C 1, DMSO-d6 d H 98.6 146.7 179.4 102.5 148.7 137.4 99.1 150.4 147.0 108.8 127.9 145.4 56.3 55.9 – – – – 6.40 – – 6.33 – – 7.21 – – 7.26 – – 3.89 3.85 – – 6.55 – d 1 2 3 4 4a 5a 6 7 8 9 9a 10a 7-OMe 8-OMe CH3CO CH3CO NH OH a 2 13 C Weak correlation. 1 s s s s s s (2H) br s d 13C DMSO-d6 d 1H DMSO-d6 d 13C CD3OD d 1H CD3OD HMBC 1H/13C 114.0 146.3 171.2 99.6 147.2 126.7 106.4 153.4 174.2 101.9 130.8 131.8 55.2 – 168.2 24.3 – – 8.23 – – 6.06 – – 6.69 – – 6.12 – – 3.92 – – 2.12 9.02 8.36 116.9 148.5 173.0 102.1 149.6 130.6 107.3 155.8 177.1 103.6 134.6 133.6 56.3 – 171.5 24.3 – – 8.51 – – 6.51 – – 6.92 – – 6.46 – – 3.93 – – 2.25 – – C-3, C-2 – – C-2a, C-10a – – C-8, C-7, C-9a, C-5aa – – C-7, C-9aa, C-5a – – C-7 – – CO C-3, C-2 – s s s s s s (1H) br s br s br s s s s s s T.T. Thuy et al. / Biochemical Systematics and Ecology 44 (2012) 205–207 207 Fig. 1. Structure of compounds 1 and 2. this species, P. bivalvis. Additionally, the presence of peristrophine (1) and perisbivalvine A (2) in red and purple forms, respectively, could be valuable taxonomic marker for red and purple P. bivalvis forms. Our results might contribute to the further knowledge about the phytochemical data and circumscription of Peristrophe species. Acknowledgements We would like to thank the National Foundation for Science and Technology Development of Vietnam (NAFOSTED) for financial support (Reference code 104.01.130.09). References Achenbach, H., Blümm, E., 1991. Arch. Pharm. (Weinheim) 324, 3. Balkwill, K., 1996. Bothalia 26, 83. Balkwill, K., Getliffffe, N.F., Balkwill, M.-J., 1988. J. Bot. 54, 47. Chi, V.V., 1999. Tu dien Cay thuoc Vietnam (A Dictionary of Vietnamese Medicinal Plant). Medicine Publication, p. 191. Fomsgaard, I.S., Mortensen, A.G., Carlsen, S.C., 2004. Chemosphere 54, 1025. Okafor, C.O., 1986. Dyes and Pigments 7, 103. Pi, H., Yang, X., Ruan, H., Zhang, Y., Wu, J., 2008. Nat. Prod. Res. 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