Biochemical Systematics and Ecology 45 (2012) 115–119 Contents lists available at SciVerse ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Two new sesquiterpene lactones and other chemical constituents of Artemisia roxbughiana Minh Giang Phan a, *, Thi Thanh Nhan Tran a, Tong Son Phan a, Hideaki Otsuka b, Katsuyoshi Matsunami b a b Faculty of Chemistry, College of Natural Science, Vietnam National University, Hanoi, 19 Le Thanh Tong Street, Hanoi, Viet Nam Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan a r t i c l e i n f o a b s t r a c t Article history: Received 20 March 2012 Accepted 14 July 2012 Available online 11 August 2012 Two new sesquiterpene lactones, named roxbughianins A and B, were isolated together with thirteen other compounds from the leaves of Artemisia roxbughiana Bess (Asteraceae) Their structures were determined by spectroscopic methods Ó 2012 Elsevier Ltd All rights reserved Keywords: Aretemisia roxbughiana Asteraceae Sesquiterpene lactone Guaianolide Subject and source The genus Artemisia comprises over 500 species worldwide with fifteen species described in the Flora of Vietnam and is one of the largest of 1535 genera in the family Asteraceae (Vallès et al., 2003; Tariku et al., 2010) The genus Artemisia is usually presented by small herbs and shrubs with aromatic and bitter taste Many species of the genus are of economic values because of their importance in the pharmaceutical and food industries The genus has been divided into five sections, Absinthium (Tournefort) de Cand., Artemisia Tournefort (¼section Abrotanum Besser), Dracunculus Besser, Seriphidium Besser (Hayat et al., 2009a), and Tridentatae (Rydb.) McArthur, which is endemic to North America (McArthur et al., 1981) The biology of Artemisia is diversified because of the high number of taxa and species are known to be difficult to identify (Kelsey, 1984) Chemical studies of Artemisia have been extensively published in the last 50 years (Tan et al., 1998) and compounds isolated can be utilized as an important aid in the systematic classification of Artemisia For instance, in the Asteraceae sesquiterpene lactones have been used as chemical characteristics to understand the systematic relationships of the genera Centaurea (Bruno et al., 1998), Scalesia (Spring et al., 1999), and Artemisia (Kelsey and Shafizadeh, 1979) Our systematic study of Artemisia roxbughiana was carried out to evaluate whether the profile of sesquiterpene lactones in the species could be used as a chemical tool for differentiating species from other in the genus The leaves of A roxburghiana Bess (Vietnamese name: Ngải rừng) were collected in Ha Giang province, Viet Nam at an altitude of 600 m above sea level in November 2008 The plant was identified by Dr Nguyen Quoc Binh, a botanist of the * Corresponding author Tel.: ỵ84 38351439 E-mail address: phanminhgiang@yahoo.com (M.G Phan) 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.bse.2012.07.027 116 M.G Phan et al / Biochemical Systematics and Ecology 45 (2012) 115–119 Institute of Biological Resources and Ecology, Vietnam Academy of Science and Technology, Hanoi, Viet Nam Voucher specimen of the plant (voucher number: VMN-B0000302) was deposited at the same Institute Previous work The chemistry of the genus Artemisia is diversified and characterized by the occurrence of essential oils (Güvenalp et al., 1998; Lopes-Lutz et al., 2008), sesquiterpene lactones (Kelsey and Shafizadeh, 1979; Lee et al., 2003), sesquiterpene alkaloids (Su et al., 2010), diterpenoids (Li et al., 1990), triterpenoids (Zheng, 1994; Sharma and Ali, 1996; Hu and Feng, 2000), polyacetylenic compounds (Marco et al., 1994; Lee et al., 2003), alkamides (Saadali et al., 2001), and phenolic compounds (Tan et al., 1998; Sheu and Tan, 1999; Lee et al., 2003) The common skeletal types of sesquiterpene lactones found in Astemisia are germacranolide, guaianolide, and eudesmanolide The only report on the constituents of the essential oil obtained by hydrodistillation of the aerial parts of A roxbughiana Bess revealed the presence of mono- and sesquiterpenoids as its main constituents (Bicchi et al., 1998) Oxygenated sesquiterpenoids are very minor compounds in this oil of which several compounds are of eudesmane and eremophilane structures Present study 3.1 Extraction and isolation The powder of dried A roxbughiana leaves (6 kg) was extracted with MeOH at room temperature (three times, each time for three days) The combined MeOH extract was successively partitioned between water and n-hexane, CH2Cl2, and EtOAc to give the corresponding soluble fractions Part of the n-hexane-soluble fraction (45 g) was subjected to silica gel open-column chromatography (CC) using n-hexane–acetone 19:1, 9:1, 6:1, and 3:1 to give eleven fractions Fractions (0.15 g) and (61 mg) were purified by silica gel CC eluting with n-hexane–acetone 99:1, 49:1, and 30:1 to give compounds (20 mg) and (15 mg), respectively Fraction (1.1 g) was washed with n-hexane to give compound (1 g) Compounds (30 mg), (0.1 g), and a mixture of compounds and (8 mg) were obtained from fraction (0.8 g) and (5 mg) from fraction (2 g) by silica gel CC eluting with n-hexaneÀEtOAc 9:1, 6:1, and 3:1 Fraction (0.4 g) was chromatographed on a silica gel column eluting with n-hexaneÀCH2Cl2 1:4, 1:9, and 1:15, which yielded, compound (8 mg) Fraction (1.9 g) was fractionated by silica gel CC eluting with n-hexaneÀacetone 6:1, 3:1, and 1:1 to afford a mixture of compounds 10 and 11 (65 mg) Part of the CH2Cl2soluble fraction (42 g) was subjected to silica gel CC using n-hexane–acetone 49:1, 29:1, 19:1, 6:1, 3:1, and 1:1 to give eleven fractions Silica gel CC of fraction (3.8 g) eluting with n-hexaneÀacetone 19:1, 9:1, 6:1, 3:1, and 1:1 gave compounds 12 (3 mg), 13 (4 mg), and a mixture of compounds and 14 (15 mg) The EtOAc-soluble fraction (3.0 g) was separated by silica gel CC eluting with n-hexaneÀEtOAc–HCOOH 20:19:1, 20:20:1, and 10:20:1 to give five fractions Fraction was purified by silica gel CC eluting with CH2Cl2–EtOAc 3:1 and 1:1 to give compound 15 (6 mg) The structures of the isolated compounds (Fig 1) were determined as friedelin (1) (Akihisa et al., 1992), friedelan-3b-ol (2) (Monkodkaew et al., 2009), tetracosanoic acid (4), b-sitosterol (5) (Goad and Akihisha, 1997), docosanoyl and tetracosanoyl p-coumarates (6 and 7) (Martínez et al., 1997), achillin (8) (Martínez et al., 1988), eicosanoic acid (9), 1-O-(tricosanoyl) and 1-O-(pentacosanoyl)glycerols (10 and 11) (Qi et al., 2004), palmitic acid (12), (23Z)-cycloart-3b,25-diol-23-ene (13) (Pei et al., sínský and Saman, 1995) by comparing their spectroscopic data (IR, MS, 1H NMR and 13C 2007), 1b,10b-epoxyachilin (14) (Bude NMR including 2D NMR) with the reported literature values Compounds and 15 are new compounds 14 O 10 H R1 15 R2 O O O 13 11 HO 12 n = 21 n = 23 O R1,R2 = O R1 = H, R2 = OH OH O H O OH HO O H O O (CH2)nCH3 OH O H O O O RO 13 14 Fig Chemical structures of compounds 1–3, 6–8, and 13–15 15 M.G Phan et al / Biochemical Systematics and Ecology 45 (2012) 115–119 117 Compound was isolated as colorless needles (ẵa24 D ỵ 172, CHCl3), and its molecular formula was determined as C15H20O3 on the basis of HRÀESIÀTOFÀMS analysis The 1H and 13C NMR and HSQC spectroscopic data of revealed the presence of three methyl groups, three methylenes, five methines, and four quaternary carbons of an epoxide ring [dC 72.4 (s) and 62.5 (s)], an olefinic carbon [dC 140.8 (s)], and a lactone carbonyl group [dC 180.0 (s); IR: nmax 1764 cmÀ1] Considering the sesquiterpene lactones of the guaiane type and HMBC correlations, compound was considered to have structure similarity 13 to arborescin (ẵa25 D ỵ 60, CHCl3) and its epimer, 1,10-arborescin (Wong and Brown, 2002) The C NMR chemical shifts at C-7 (dC 49.9), C-11 (dC 39.6), C-12 (dC 180.0), and C-13 (dC 10.1) clearly indicated the epimeric relationship at C-11 between sínský and Saman, arborescin and (Bude 1995; Martínez et al., 1988) Further stereochemical confirmation was provided by NOESY correlations between H3-13 (dH 1.14) and H-6b (dH 4.21), and between H-5a (dH 2.82) and H-7a (dH 1.82); no correlation was observed between H3-13 and H-5a/H-7a (Fig 2) Therefore, the structure of was determined to be 11-epiarborescin, which was given a trivial name roxbughianin A Compound 15 was obtained as a white amorphous powder (ẵa24 D ỵ 33.6, CHCl3) The molecular formula C15H22O5 of 15 was determined by HRÀESIÀTOFÀMS The 1H and 13C NMR spectroscopic data of 15 were close to those of 1a,4a,10a-trihydroxy5a,11bH-guai-2-en-12,6a-olide and 1b,4a,10a-trihydroxy-5a,11bH-guai-2-en-12,6a-olide from Artemisia adamsii (Bohlmann et al., 1985) and 4b,10b-dihydroxy-1a-methoxy-5a,11aH-guaia-2-en-12,6a-olide from Ursinia nudicaulis (Jakupovic et al., 1992) The stereochemistry H-11a was well-established on the basis of the chemical shifts of C-11 (dC 10.9) and sínský and Saman, 1995) The stereochemistry at C-5, C-6, and C-7 could be determined from the C-13 (dC 40.8) (Bude proton coupling constants (Appendino and Gariboldi, 1982) and was further supported by the observation of NOESY correlations between H3-13 (dH 1.20) and H-6b (dH 4.87) and between H-5a (dH 2.17) and H-7a (dH 2.80) and no NOESY correlations between H3-13 and H-5a/H-7a (Fig 2) NOESY correlation between H-5a and H-15 (dH 1.44) and no NOESY correlations observed between H-6b and H-14 (dH 1.25) and between H-6b and H-15 proved b-orientation of the hydroxyl groups at C-4 and C-10, respectively The presence of H-5 at dH 2.17 supported the b-orientation of the hydroxyl group at C-1 (Bohlmann et al., 1985) Therefore, the structure of compound 15 was determined to be 1b,4b,10b-trihydroxy-5a,11aH-guai-2-en12,6a-olide, which was given a trivial name roxbughianin B 3.2 Roxbughianin A (3) À1 Colorless needles, m.p 145146 C, ẵa24 D ỵ 172 (c 0.16, CHCl3) IR (film): nmax (cm ) 1764, 1650 HRESITOFMS (positive mode): m/z 249.1488, [M ỵ H]ỵ (calc for C15H21O3: 249.1485) 1H NMR (500 MHz, CDCl3): d 1.14 (3H, d, J ¼ 8.0 Hz, H3-13), 1.33 (3H, s, H3-14), 1.49 (2H, m, 2H-8), 1.82 (1H, m, H-7), 1.93 (3H, m, H3-15), 1.94 (1H, m, H-9a), 2.11 (1H, m, H-2a), 2.14 (1H, m, H-9b), 2.54 (1H, quint., J ¼ 8.0 Hz, H-11), 2.75 (1H, dd, J ¼ 16.0 Hz, 1.5 Hz, H-2b), 2.82 (1H, d, J ¼ 10.5 Hz, H-5), 4.21 (1H, t, J ¼ 10.5 Hz, H-6), 5.56 (1H, br s, H-3) 13C NMR (125 MHz, CDCl3): d 10.1 (q, C-13), 18.2 (q, C-15), 20.4 (t, C-8), 22.7 (q, C-14), 33.5 (t, C-9), 39.6 (t, C-2), 39.6 (d, C-11), 49.9 (d, C-7), 52.8 (d, C-5), 62.5 (s, C-10), 72.4 (s, C-1), 82.1 (d, C-6), 124.7 (d, C-3), 140.8 (s, C-4), 180.0 (s, C-12) 3.3 Roxbughianin B (15) À1 White amorphous powder, ẵa24 D ỵ 33.6 (c 0.11, CHCl3) IR (lm): nmax (cm ) 3412, 1740, 1651 HRESITOFMS (positive ỵ mode): m/z 305.1361, [M ỵ Na] (calc for C15H22O5Na: 305.1359) H NMR (500 MHz, CD3OD): d 1.20 (3H, d, J ¼ 8.0 Hz, H3-13), 1.25 (3H, s, H3-14), 1.44 (3H, s, H3-15), 1.50 (1H, ddd, J ¼ 3.5 Hz, 4.0 Hz, 14.5 Hz, H-9a), 1.68 (1H, m, H-8a), 1.73 (1H, m, H-8b), 2.17 (1H, d, J ¼ 9.0 Hz, H-5), 2.32 (1H, dddd, J ¼ 5.5 Hz, 12.5 Hz, 14.5 Hz, H-9b), 2.64 (1H, quint., J ¼ 8.0 Hz, H-11), 2.80 (1H, m, OH O HO H H H OH O H H H O O O HMBC NOESY 15 Fig Key HMBC and NOESY correlations of compounds and 15 118 M.G Phan et al / Biochemical Systematics and Ecology 45 (2012) 115–119 H-7), 4.87 (1H, overlapped with solvent signal, H-6), 5.80 (1H, d, J ¼ 6.0 Hz, H-2), 5.93 (1H, d, J ¼ 6.0 Hz, H-3) 13C NMR (125 MHz, CD3OD): d 10.9 (q, C-13), 23.7 (t, C-8), 26.0 (q, C-15), 28.5 (q, C-14), 36.7 (t, C-9), 40.8 (d, C-11), 41.5 (d, C-7), 65.9 (d, C-5), 75.9 (s, C-10), 80.8 (s, C-4), 84.3 (d, C-6), 89.8 (s, C-1), 138.3 (d, C-2), 140.2 (d, C-3), 183.2 (s, C-12) Chemotaxonomic significance This study is the first report of the compounds 1–15 in A roxbughiana Previous reports indicated the predominance of guaianolides in the aerial parts of the genus Artemisia The isolation of sesquiterpene lactones of the guaiane type from the leaves of A roxbughiana is consistent with the chemical constituents of other species of Artemisia Guaiane-type sesquiterpene lactones are considered a more biosynthetically advanced class of sesquiterpenoids because of the high level of cyclization and oxidation reactions in their biosynthesis The newly-isolated guaianolides from A roxbughiana may be chemosystematically useful in furthering our knowledge about the distribution of these compounds in the genus The occurrence of C-11 epimeric guaianolides such as achillin (H-11a) and leukodin (H-11b) (Martínez et al., 1988) illustrates two different biosynthetic lactonization pathways of guaianolides in Artemisia All the guaianolides in A roxbughiana, roxbughianin A (3), achillin (8), 1b,10b-epoxyachilin (14), and roxbughianin B (15) have H-11a stereochemistry Roxbughianins A and B were isolated for the first time from plants The triterpenoids isolated from A roxbughiana are of friedelane and cycloartane types; friedelin (1) and friedelan-3b-ol (2) which commonly occur in plants were isolated from Artemisia annua (Zheng, 1994), but so far there was no report on (23Z)-cycloart-3b,25-diol-23-ene (13) from Artemisia species Alkyl p-coumarates were found in some Artemisia species such as Artemisia campestris (Vajs et al., 1975) and Artemisia assoana (Martínez et al., 1997) The classification of the genus Artemisia has been established based on pollen morphology, floral and capitular morphology, chromosome counts, or molecular phylogeny techniques However, the study results have led to controversial phylogenic relationships within the genus After various taxonomic rearrangements the genus Artemisia was divided into five sections or subgenera including Artemisia, Absinthium, Seriphidium, Tridentatae, and Dracunculus (Hayat et al., 2009a) Even so, this classification is not accepted by all authors (Vallès and Garnatje, 2005) The presence and absence of receptacle hair is the only morphological characteristic that separates the species of the subgenera Artemisia and Absinthium (Watson et al., 2002) and several authors combined the subgenera Artemisia and Absinthium to form the only subgenus Artemisia (Hayat et al., 2009b) Tridentatae was originally created as a section within the subgenus Seriphidium, but it was segregated from Seriphidium as a subgenus in Artemisia by McArthur et al (McArthur et al., 1981; Vallès et al., 2003) Poljakov (Poljakov, 1961) and Ling (Ling, 1995) separated Seriphidium from Artemisia as an independent genus This segregation is not accepted by several authors (Hayat et al., 2009b; Watson et al., 2002) Sesquiterpene lactones have been used as a chemical tool to understand the systematics of Artemisia species The data on sesquiterpene lactones supported the segregation of Seriphidium, which produced mainly eudesmanolides and the combination of Artemisia and Absinthium on the basis of the chemical similarity of their eudesmanolide- and guaianolide-type sesquiterpene lactones (Kelsey and Shafizadeh, 1979) The common occurrence of 5a,11bH-guaian-12,6a-olides and 11,13-guaiaen-12,6a-olides in the genus Artemisia made the presence of guaianolides with the exclusive H-11a stereochemistry (compounds 3, 8, 14, and 15) in A roxbughiana chemotaxonomically relevant for the species According to the morphology-based classification A roxbughiana belongs to the subgenus Artemisia (Hayat et al., 2009a) Since the occurrence of achillin (8) and 1,10-epoxyachillin (14) was reported from both the subgenera Artemisia (Artemisia ludoviciana) and Absinthium (Artemisia lanata) (Kelsey and Shafizadeh, 1979) the two new guaianolides roxbughianins A (3) and B (15) are considered useful chemical markers for A roxbughiana Furthermore, the guaianolides and 15 may be the key markers for the placement of A roxbughiana in the subgenus Artemisia Considering the high number of taxa in Artemisia more information on sesquiterpene lactones in Artemisia species is needed to evaluate the separation between the subgenera Artemisia and Absinthium Acknowledgment This work was supported by the National Foundation for Science and Technology Development (NAFOSTED, Hanoi, Viet Nam) (Grant No 104.01.137.09) Appendix A Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bse 2012.07.027 References Akihisa, T., Yamamoto, K., Tamura, T., Kimura, Y., Iida, T., Nambara, T., Chang, C.F., 1992 Chem Pharm Bull 40, 789 Appendino, G., Gariboldi, P., 1982 Phytochemistry 21, 2555 Bicchi, C., Rubiolo, P., Marschall, H., Weyerstahl, P., 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Nat Prod 65, 481 Zheng, G.Q., 1994 Planta Med 60, 54 ... eudesmanolides and the combination of Artemisia and Absinthium on the basis of the chemical similarity of their eudesmanolide- and guaianolide-type sesquiterpene lactones (Kelsey and Shafizadeh,... leaves of A roxbughiana is consistent with the chemical constituents of other species of Artemisia Guaiane-type sesquiterpene lactones are considered a more biosynthetically advanced class of sesquiterpenoids... 2002) Sesquiterpene lactones have been used as a chemical tool to understand the systematics of Artemisia species The data on sesquiterpene lactones supported the segregation of Seriphidium, which