Biochemical Systematics and Ecology 38 (2010) 1065–1068 Contents lists available at ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Mangiferonic acid, 22-hydroxyhopan-3-one, and physcion as specific chemical markers for Alnus nepalensis Minh Giang Phan a, *, Thi To Chinh Truong a, Tong Son Phan a, Katsuyoshi Matsunami b, Hideaki Otsuka 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 Article history: Received 10 July 2010 Accepted 26 September 2010 Available online 20 October 2010 Keywords: Alnus nepalensis Betulaceae Triterpenoid Flavonoid Anthraquinone Phytosterol Subject and source Recently the Betulaceae family was divided into two families: Betulaceae with Betula and Alnus genera and Corylaceae with Corylus and Carpinus Alnus is a genus of flowering plants which comprises about 30 species of deciduous trees and shrubs, few reaching large size, mainly distributed throughout the northern hemisphere (Daniere et al., 1991) There are two Betulaceae plant species recorded in the Flora of Vietnam Alnus nepalensis D Don and Betula alnoides Buch -Ham (Pham, 1993) A nepalensis D Don (Betulaceae) (Vietnamese name: Tống quán sủi) is a woody plant that reaches up to 10–15 m in height In the Chinese traditional medicine A nepalensis is used to treat diarrhoea, bacillary dysentery, and inflammatory diseases (Vo, 1997) For the present study the fresh leaves, twigs, and stem bark of A nepalensis were collected two times from mountainous areas in district Dong Van, province Ha Giang, Vietnam by a botanist, Dr Tran Ngoc Ninh of the Institute of Biological Resources and Ecology, Vietnam Academy of Science and Technology, Hanoi, Vietnam (collection in July 2006 and collection in June 2007) Voucher specimens of the plant (voucher number: 10.999) were deposited at the same Institute Previous work The bark of A nepalensis was reported to contain 7% tannin (Vo, 1997) * Corresponding author Tel.: ỵ84 38351439 E-mail address: phanminhgiang@yahoo.com (M.G Phan) 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd All rights reserved doi:10.1016/j.bse.2010.09.020 1066 M.G Phan et al / Biochemical Systematics and Ecology 38 (2010) 1065–1068 Present study The dried powdered leaves, dried twigs, and dried stem barks of the two collections of A nepalensis were extracted separately with MeOH at room temperature (seven times, each time for three days) The combined MeOH extract was concentrated under reduced pressure and the resultant MeOH extract was successively partitioned between water and organic solvents of increasing polarities After removal of the organic solvents in vacuo the collection (leaves: 432.7 g, twigs: 433 g, and stem bark: 145 g) gave n-hexane- (22.8 g, 1.3 g, and 1.46 g from the leaves, twigs, and stem bark, respectively), CH2Cl2- (not extracted, 1.21 g, and 1.4 g), and EtOAc- (8.1 g, 0.69 g, and 1.65 g) soluble fractions, and the collection (leaves: 1.48 kg, twigs: 451 g, and stem barks: 1.1 kg) gave n-hexane- (56.6 g, 18.9 g, and 13.3 g), CH2Cl2- (20.5 g, 12.9 g, and 9.4 g), and EtOAc- (48.9 g, 15.6 g, and 8.94 g) soluble fractions The soluble fractions of the leaves were treated as followed Part of the n-hexane-soluble fraction (22 g) of the collection was chromatographed by silica gel open-column chromatography (CC) using a n-hexane–EtOAc gradient solvent system (n-hexane, n-hexane–EtOAc 7:1, 4:1, 2:1, and 1:1, and EtOAc) to give fifteen fractions Separation of fraction (0.38 g) by silica gel CC (n-hexane–EtOAc 90:1) gave (21.3 mg) Separation of fraction (3.71 g) by silica gel CC (n-hexane–EtOAc 90:1, 70:1, and 49:1) gave (50 mg), (22 mg) after recrystallization from n-hexane–EtOAc 9:1, and 15 (30 mg) Fraction (0.88 g) was chromatographed by silica gel CC (n-hexane–EtOAc 30:1, 15:1, 7:1, and 4:1) followed by recrystallization from CH2Cl2 to give 16 (8 mg) Part of the EtOAc-soluble fraction (8 g) of the collection was subjected to silica gel CC and eluted with a CH2Cl2– MeOH gradient solvent system (CH2Cl2, CH2Cl2–MeOH 90:1, 70:1, 19:1, 9:1, 7:1, 4:1, 2:1, and 1:1, and MeOH) to give ten fractions Fraction (0.98 g) gave crystals on standing at room temperature, which were purified by silica gel flash chromatography (FC) (n-hexane–EtOAc 1:1) to give 11 (26 mg) or washed with (CH3)2CO and then recrystallized from a CHCl3– MeOH mixture to give (25 mg) Compound 12 (141.5 mg) was obtained from fraction (3.29 g) on repeated separation by silica gel CC using a CH2Cl2–(CH3)2CO gradient (2:1 and 1:7) and FC (CH2Cl2–EtOAc 1:9) followed by recrystallization from (CH3)2CO Since the n-hexane-soluble fraction of the collection was similar in TLC pattern to the n-hexane-soluble fraction of the collection only the CH2Cl2- and EtOAc-soluble fractions of the collection were subjected to chromatographic separation Part of the CH2Cl2-soluble fraction (17.5 g) of the collection was chromatographed on silica gel using a CH2Cl2– EtOAc gradient solvent system (49:1, 29:1, 19:1, 9:1, 4:1, 2:1, and 1:1) to give nine fractions Separation of fraction (0.19 g) by silica gel CC (n-hexane–EtOAc 70:1 and 19:1) gave (5 mg) and 10 (1 mg) Repeated separation of fraction (0.6 g) by silica gel CC (n-hexane–EtOAc 7:1 and 2:1) and FC (n-hexane–EtOAc 15:1) gave (19.7 mg) and (10.7 mg) Compound (6 mg) was obtained from fraction (0.67 g) by silica gel CC (n-hexane–EtOAc 15:1, 9:1, and 4:1) followed by recrystallization from (CH3)2CO Fractions (0.51 g) and (1.74 g) were combined and separated by silica gel CC (n-hexane–EtOAc 15:1 and 4:1) to give (1.2 mg) and (7 mg) Part of the EtOAc-soluble fraction (40 g) of the collection was chromatographed on silica gel using a CH2Cl2–MeOH gradient solvent system (19:1, 9:1, 2:1, 1:1, and 1:2) to give six fractions Fraction (1.41 g) was separated twice by silica gel FC using a n-hexane–EtOAc gradient 19:1, 9:1, 4:1, 2:1, and 1:1 for the first column and a n-hexane–EtOAc gradient 9:1, 4:1, and 2:1 for the second column to give 14 (5.5 mg) Separation of fraction (6.67 g) by silica gel FC (n-hexane–EtOAc 7:1, 4:1, 2:1, 1:1, 1:2, and 1:3) and fraction (6.95 g) by silica gel FC (CH2Cl2–EtOAc 9:1, 4:1, 2:1, 1:1, and 1:2) followed by recrystallization from (CH3)2CO gave 11 (35 mg and 15 mg, respectively) Fraction (11.9 g) was separated by silica gel CC (CH2Cl2–(CH3)2CO 9:1, 4:1, 2:1, 1:1, and 1:2); the fractions obtained were recrystallized from (CH3)2CO or subjected to silica gel FC (CH2Cl2–(CH3)2CO 6:1) to give 12 (10 mg) and 13 (5 mg) Fraction (7.7 g) was successively separated by silica gel CC (CH2Cl2–(CH3)2CO 4:1, 2:2, 1:1, and 1:2) and FC (CH2Cl2–CH3OH 4:1) to give 17 (5 mg) The n-hexane-soluble fractions of the twigs of the two collections (1.3 g and 18.9 g) were treated separately in the same manner to yield the same compounds The n-hexane-soluble fractions were chromatographed on a silica gel open column using a n-hexane–EtOAc gradient solvent system 19:1, 9:1, 6:1, 4:1, and 2:1, then the fractions obtained were washed with n-hexane to give (33 mg and 20 mg from the collections and 2, respectively), (10 mg and 160 mg), and 15 (21.6 mg and 62.6 mg) The CH2Cl2-soluble fractions of the twigs of the two collections (1.21 g and 12.9 g) were combined based on TLC analysis and separated by silica gel CC (CH2Cl2–EtOAc 29:1, 19:1, 9:1, 6:1, 4:1, 2:1, 1:1, and 1:2), then the fractions obtained were washed with n-hexane to give (6 mg) and (47 mg) The EtOAc-soluble fractions of the twigs of the two collections (0.69 g and 15.6 g) were combined based on TLC analysis and successively separated by silica gel CC (CH2Cl2–(CH3)2CO 19:1, 9:1, 6:1, 4:1, 2:1, 1:1, and 1:2) and FC (CH2Cl2–(CH3)2CO 9:1 and 4:1), then the fractions obtained were washed with (CH3)2CO and recrystallized from CH2Cl2–(CH3)2CO to give (6 mg) The n-hexane-soluble fractions of the stem bark of the two collections (1.46 g and 13.3 g) were combined based on TLC analysis and separated by silica gel CC (n-hexane–EtOAc 29:1, 19:1, 9:1, 6:1, and 4:1) to give (0.14 g) The structures of the isolated compounds (Fig 1) were determined as taraxeryl acetate (1) (Goad and Akihisha, 1997; Jin et al., 2007b), taraxerol (2) (Goad and Akihisha, 1997; Jin et al., 2007b), b-sitosterol (3) (Goad and Akihisha, 1997), b-sitosterol 3-O-b-D-glucopyranoside (4) (Goad and Akihisha, 1997), betulinic acid (5) (Jin et al., 2007b), betulin (6) (Jin et al., 2007b), mangiferonic acid (7) (Silva et al., 2005), 22-hydroxyhopan-3-one (8) (Wilkins et al., 1987; Tanaka and Matsunaga, 1992), 2-hydroxydiploterol (9) (Wang et al., 2009), physcion (10) (Choi et al., 1996), quercetin (11) (Ternai and Markham, 1976), quercitrin (quercetin 3-O-a-L-rhamnopyranoside) (12) (Jung et al., 2007), gallic acid (13) (Wang et al., 2007), hirsutenone (14) (Jin et al., 2007b), 1-nonacosanol (15), heptacosanoic acid (16), and quercetin 3-O-b-D-galactopyranoside (17) (Harborne, 1994) by superimposing their spectroscopic data (EI-MS, ESI-MS, 1H NMR, and 13C NMR) with the reported literature values We report herein full unambiguous 1H and 13C NMR assignments for compound based on 2D NMR techniques (1H-1H COSY, HSQC, and HMBC) (Supplementary Data) M.G Phan et al / Biochemical Systematics and Ecology 38 (2010) 1065–1068 1067 21 22 18 23 24 26 20 19 R O HO RO R = COOH R = CH2OH R = Ac 2R=H 28 10 12 11 17 13 14 25 16 COOH 27 15 30 29 OH OH OH O OH HO O OH R1 H3CO R2 R3 R1 = H, R2,R3 = O R1 = OH, R2 = R3 = H O CH3 10 OR OH O 11 R = H 12 R = α-L-Rha 17 R = β-D-Gal Fig Chemical structures of compounds 1, 2, 5–12, and 17 Chemotaxonomic significance This is the first systematic report of the secondary metabolites in A nepalensis Compounds 1, 2, 4, 5, and 7–17 were isolated from the leaves of A nepalensis, compounds 1, 2, 4, 6, and 15 from the twigs, and compound from the stem bark Previous reports have indicated the predominance of diarylheptanoids in various plant parts of the Alnus species; they have been isolated from the aerial parts of A maximowiczii (Tori et al., 1995), bark of A hirsuta var sibirica (Lee et al., 2000b), bark of A rubra (Chen et al., 2000), bark, stem bark, and leaves of A hirsuta (Lee et al., 2000a,b; Cho et al., 2002; Jin et al., 2007b), bark, leaves, and wood of A japonica (Nomura et al., 1981; Lee et al., 2005; Kim et al., 2005; Kuroyanagi et al., 2005; Choi et al., 2008), and seeds of A glutinosa (O’Rourke et al., 2005) The occurrence of oregonin in a large number of Alnus species such as A glutinosa, A incana, A viridis, A.cordata, A formosana, A hirsuta, A serrulatoides, and A japonica is very noticeable (Guz et al., 2002; Kuo et al., 2008) In this study a diarylheptanoid hirsutenone (14) was isolated, however, the compound was already found in several Alnus species such as A japonica and A hirsuta (Jin et al., 2007a, 2007b; Choi et al., 2008) Two flavonoids quercetin (11) and quercitrin (12) were also reported from A firma (Yu et al., 2007) Flavonoids and flavonoid glycosides have previously been reported from the buds of A crispa, A japonica, A koehnei, A sinuata, and A viridis, leaves and seeds of A glutinosa, aerial parts of A maximowiczii, and leaves of A firma (Wollenweber, 1974; Favre-Bonvin et al., 1978; Gonnet and Daniere, 1989; Daniere et al., 1991; Tori et al., 1995; O’Rourke et al., 2005; Yu et al., 2007), however, the occurrence of quercetin 3-O-b-D-galactopyranoside (17) may be reported for the first time Two phytosterols b-sitosterol (3) and b-sitosterol 3-O-b-D-glucopyranoside (4), a simple phenolic acid gallic acid (13), and two aliphatic hydrocarbon derivatives 1-nonacosanol (15) and heptacosanoic acid (16) are common in higher plants Secodammarane triterpenoids were essentially found in the flowers of A japonica, A serrutilatoides, A pendula, and A siebodiana (Sakamura et al., 1985; Suga et al., 1986; Aoki et al., 1988, 1990) Among the isolated triterpenoids from the leaves and twigs of A nepalensis taraxeryl acetate (1), taraxerol (2), betulinic acid (5), and betulin (6) were previously isolated from the bark and stem bark of A hirsuta (Chung et al., 2006; Jin et al., 2007b) However, the distribution of cycloartane (mangiferonic acid) and hopane (22-hydroxyhopan-3-one) triterpenoids as well as an anthraquinone (physcion) has not been detected in any Alnus species The hopane triterpenoid 2-hydroxydiploterol (9) has never been isolated from a plant source till this study; was reported as a constituent from fungus Aspergillus variecolor B-17 (Wang et al., 2009) Therefore, from a chemotaxonomical point of view, mangiferonic acid (7), 22-hydroxyhopan-3-one (8), and physcion (10) were disclosed by this study as specific chemical markers of A nepalensis Acknowledgement This work was supported by the National Foundation for Science and Technology Development (NAFOSTED, Hanoi, Vietnam) Appendix Supplementary data Supplementary data related to this article can be found online at doi:10.1016/j.bse.2010.09.020 1068 M.G Phan et al / Biochemical Systematics and Ecology 38 (2010) 1065–1068 References Aoki, T., Ohta, S., Suga, T., 1988 Phytochemistry 27, 2915 Aoki, T., Ohta, S., Suga, T., 1990 Phytochemistry 279, 3611 Chen, J., Gonzalez-Laredo, R., Karchesy, J.J., 2000 Phytochemistry 53, 971 Cho, S.M., Kwon, Y.M., Lee, J.H., Yon, K.H., Lee, M.W., 2002 Arch Pharm Res 25, 885 Choi, J.S., Jung, J.H., Lee, J.H., Kang, S.S., 1996 Arch Pharm Res 19, 302 Choi, S.E., Kim, K.H., Kwon, J.H., Kim, S.B., Kim, H.W., Lee, M.W., 2008 Arch Pharm Res 31, 1287 Chung, M.Y., Rho, M.C., Lee, S.W., Park, R.P., Kim, K., Lee, I.A., Kim, D.H., Jeune, K.H., Lee, H.S., Kim, Y.K., 2006 Planta Med 72, 267 Daniere, C., Gonnet, J.-F., Moiroud, A., 1991 Biochem Syst Ecol 19, 587 Favre-Bonvin, J., Jay, M., Wollenweber, E., 1978 Phytochemistry 17, 821 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365 Wang, W.L., Liu, P.P., Zhang, Y.P., Li, J., Tao, H.W., Gu, Q.Q., Zhu, W.M., 2009 Arch Pharm Res 32, 1211 Wilkins, A.L., Ronaldson, K.J., Jager, P.M., Bird, P.W., 1987 Aust J Chem 40, 1713 Wollenweber, E., 1974 Phytochemistry 13, 2318 Yu, Y.B., Miyashiro, H., Nakamura, N., Hattori, M., Park, J.C., 2007 Arch Pharm Res 30, 820 ... full unambiguous 1H and 13C NMR assignments for compound based on 2D NMR techniques (1H-1H COSY, HSQC, and HMBC) (Supplementary Data) M.G Phan et al / Biochemical Systematics and Ecology 38 (2010)... view, mangiferonic acid (7), 22-hydroxyhopan-3-one (8), and physcion (10) were disclosed by this study as specific chemical markers of A nepalensis Acknowledgement This work was supported by the National... 9:1, 6:1, 4:1, and 2:1, then the fractions obtained were washed with n-hexane to give (33 mg and 20 mg from the collections and 2, respectively), (10 mg and 160 mg), and 15 (21.6 mg and 62.6 mg)