Chemical constituents of the leaves of alnus nepalensis d. don. (betulaceae) - Truong Thi To Chinh

521 Journal of Chemistry, Vol. 46 (4), P. 521 - 525, 2008 CHEMICAL CONSTITUENTS OF THE LEAVES OF ALNUS NEPALENSIS D. DON. (BETULACEAE) Received 28 March 2008 TRUONG THI TO CHINH1,2, PHAN MINH GIANG1, PHAN TONG SON1 1Laboratory of Chemistry of Natural Products, Faculty of Chemistry, College of Natural Science, Vietnam National University, Hanoi, Vietnam 2Vietnam Institute of Industrial Chemistry, Hanoi, Vietnam Summary Taraxeryl acetate (1), physcion (2), 1-nonacosanol (3), β-sitosterol (4), quercetin (5), β- sitosterol 3-O-β-D-glucopyranoside (6), and quercitrin (7) were isolated from the leaves of Alnus nepalensis D. Don. (Betulaceae). Their structures were determined by spectroscopic methods. Keywords: Alnus nepalensis; Betulaceae; flavonoid; anthraquinone; phytosterol; triterpenoid. I - INTRODUCTION Woody plants have been known to produce many biologically active metabolites. In addition to the studies on chemical constituents of herbal medicinal plants, our phytochemical program also targets woody plants as potential sources of useful natural compounds. Alnus nepalensis D. Don. (Betulaceae) (Vietnamese name: Tống quán sủi) is a woody plant that reaches up to 10 - 15 m in height. The bark of A. nepalensis is used to treat diarrhoea, bacillary dysentery, and inflammatory diseases [1]. The study on A. nepalensis should be of interest since the constituents of Alnus species have been demonstrated to possess antioxidant [2], anti-inflammatory [3 - 5], anticancer [6] and hepatoprotective effects [2, 7]. In this paper the isolation and structural elucidation of seven compounds, taraxeryl acetate (1), physcion (2), 1-nonacosanol (3), β-sitosterol (4), quercetin (5), β-sitosterol 3-O-β-D-glucopyranoside (6), and quercitrin (7) (Fig. 1) from the MeOH extract of the leaves of A. nepalensis D. Don. (Betulaceae) collected in mountainous areas of northern Vietnam were reported. II - EXPERIMENTAL General Procedure Electron-impact (EI) mass spectra (70 eV) were measured on a Hewlett-Packard 5989B mass spectrometer. Electrospray Ionization (ESI) mass spectra were recorded on a LC/MSD Trap Agilent Series 1100 system with an ESI source. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) with DEPT program spectra were obtained on a Bruker Avance 500 NMR spectrometer. Tetramethyl silane (TMS) was used as zero reference. Silica gel 60 (63-200 μm, Merck, Darmstadt, Germany) was used for open column (CC) and silica gel 60 (15-40 and 40-63 μm, Merck, Darmstadt, Germany) for flash column (FC) chromatography. TLC was performed on precoated DC Alufolien 60 F254 sheets (Merck, Darmstadt, Germany) and detected by UV light (λ 254 nm) or by spraying with 1% vanillin in conc. H2SO4. 522 Plant Material The fresh leaves of A. nepalensis were identified and collected by Dr. Tran Ngoc Ninh, Institute of Biological Resources and Ecology, Vietnamese Academy of Science and Technology, Hanoi, Vietnam in Dong Van, province Ha Giang, Vietnam in June 2007. Extraction and Isolation The air-dried leaves of A. nepalensis (432.7 g) were oven-dried at 40oC, then powdered and extracted with MeOH by percolation (6 times) at room temperature. The combined MeOH extract was concentrated under reduced pressure. The resultant MeOH extract was suspended in H2O and partitioned successively with n-hexane and EtOAc. After removal of solvents n-hexane- (AH, 22.8 g) and EtOAc- (AE, 8.1 g) soluble fractions were obtained. Part of the n-hexane-soluble fraction (22 g) was chromatographed on silica gel CC using a gradient n-hexane-EtOAc solvent system (n- hexane; n-hexane-EtOAc 7:1, 4:1, 2:1, and 1:1; and EtOAc). Fifteen pooled fractions were collected on the basis of the volumes of eluents and TLC analysis; fraction AH0 (0.41 g) was eluted with n-hexane; fractions AH1 (0.38 g), AH2 (3.71 g), AH3 (0.44 g), and AH4 (0.84 g) with n-hexane-EtOAc 7:1; fractions AH5 (1.45 g), AH6 (0.88 g), AH7 (0.29 g), and AH8 (0.7 g) with n-hexane-EtOAc 4:1; fractions AH9 (0.79 g) and AH10 (0.7 g) with n-hexane- EtOAc 2:1; fractions AH11 (0.77 g), AH12 (0.31 g), AH13 (0.86 g), and AH14 (0.54 g) with n-hexane-EtOAc 1:1; and fraction AH15 (0.66 g) with EtOAc. Separation of fr. AH1 on silica gel CC (n-hexane-EtOAc 90:1) gave 1 (21.3 mg). Separation of fr. AH2 on silica gel CC (gradient n-hexane-EtOAc 90:1, 70:1, and 49:1) gave 1 (50 mg), 2 (22 mg) after recrystallization from n-hexane-EtOAc 9:1, and 3 (30 mg). 3 (28 mg) was also obtained from fr. AH3 on column separation on silica gel (gradient n-hexane-EtOAc 30:1, 15:1, and 7:1) and recrystallization (n-hexane-EtOAc 7:1). Fr. AH4 was washed with EtOAc and then recrystallized from CH2Cl2 to afford 3 (49 mg). Fr. AH6 was chromatographed by silica gel CC (gradient n-hexane-EtOAc 30:1, 15:1, 7:1 and 4:1) and one of the fractions obtained was recrystallized from CH2Cl2 to give 4 (8 mg). Part of the EtOAc-soluble fraction (8 g) was subjected to silica gel column chromatography and eluted with a gradient CH2Cl2-MeOH solvent system (CH2Cl2; CH2Cl2-MeOH 90:1, 70:1, 19:1, 9:1, 7:1, 4:1, 2:1, and 1:1; and MeOH). Based on TLC profile, ten fractions were collected, fraction AE1 (70 mg), AE2 (20 mg), AE3 (60 mg), AE4 (0.27 g), AE5 (0.7 g), AE6 (0.88 g), AE7 (0.98 g), AE8 (0.98 g), AE9 (3.29 g), and AE10. Fr. AE5 was rechromatographed on silica gel CC (gradient CH2Cl2-(CH3)2CO 19:1 and 1:1). Two of the fractions obtained gave crystalls on standing at room temperature, one of which was purified by FC on silica gel (n-hexane-EtOAc 1:1) to give 5 (26 mg) and the other was washed with (CH3)2CO and recrystallized from a CHCl3- MeOH mixture to give 6 (25 mg). 7 (141,5 mg) was obtained from fr. AE9 on repeated separation on silica gel CC (gradient CH2Cl2- (CH3)2CO 2:1 and 1:7) and FC (CH2Cl2-EtOAc 1:9) followed by recrystallization from (CH3)2CO. Taraxeryl acetate (1): White rods. Rf 0.46 (n-hexane-EtOAc 4:1). EI-MS: m/z 468 (M+., C32H52O2). 1H-NMR (CDCl3): δ 0.82 (3H, s, 17- CH3), 0.86 (3H, s, 4-CH3), 0.88 (3H, s, 4-CH3), 0.90 (3H, s, 20-CH3), 0.91 (3H, s, 13-CH3), 0.95 (6H, s, 10-CH3, 20-CH3), 1.09 (3H, s, 8-CH3), 2.04 (3H, s, 3-OAc), 4.46 (1H, dd, J = 11.0 Hz, 5.0 Hz, H-3), 5.53 (1H, dd, J = 8.0 Hz, 3.0 Hz, H-15). 13C-NMR (CDCl3): δ 15.5 (q, C-25), 16.6 (q, C-24), 17.5 (t, C-11), 18.7 (t, C-6), 21.3 (q, C-30), 23.5 (t, C-2), 25.9 (q, C-26), 27.9 (q, C- 23), 28.8 (s, C-20), 29.8 (q, C-27), 29.9 (q, C- 28), 33.1 (t, C-7), 33.4 (q, C-29), 33.7 (t, C-16), 35.1 (t, C-21), 35.8 (s, C-17), 36.7 (t, C-12), 37.4 (t, C-22), 37.6 (s, C-10), 37.7 (t, C-1), 37.7 (s, C-13), 37.9 (s, C-4), 39.0 (s, C-8), 41.3 (t, C- 19), 48.8 (d, C-18), 49.2 (d, C-9), 55.7 (d, C-5), 81.0 (d, C-3), 116.9 (d, C-15), 158.0 (s, C-14), 21.3 (q) and 170.9 (s, 3-OAc). Physcion (2): Orange needles. Rf 0.54 (n- hexane-EtOAc 15:1). EI-MS: m/z 284 (M+., 523 C16H12O5). 1H-NMR (CDCl3): δ 2.45 (3H, s, 6- CH3), 3.94 (3H, s, 3-OCH3), 6.69 (1H, d, J = 2.0 Hz, H-7), 7.08 (1H, br s, H-2), 7.37 (1H, d, J = 2.0 Hz, H-5), 7.63 (1H, br s, H-4), 12.1 (1H, s, 1-OH), 12.3 (1H, s, 8-OH). 13C-NMR (CDCl3): δ 22.2 (q, C-15), 56.1 (q, 3-OCH3), 106.8 (d, C- 2), 108.2 (d, C-4), 110.3 (s, C-13), 113.7 (s, C- 7), 121.3 (d, C-12), 124.5 (d, C-5), 133.3 (s, C- 14), 135.3 (s, C-11), 148.5 (s, C-6), 162.5 (s, C- 8), 165.2 (s, C-1), 166.6 (s, C-3), 182.1 (s, C- 10), 190.8 (s, C-9). 1-Nonacosanol (3): White amorphous powder. Rf 0.5 (n-hexane-EtOAc 4:1). EI-MS: m/z 364 (M+., C29H60O, − 60). 1H-NMR (CDCl3): δ0.88 (3H, t, J = 7.0 Hz, H-29), 1.26 50H, br s), 1.58 (4H, m (2H-2 → 2H-28), 3.64 (2H, t, J = 6.5 Hz, H-1). β-Sitosterol (4): White amorphous powder. Rf 0.37 (n-hexane-EtOAc 4:1). The co-TLC analysis is superimposable with that of our authentic sample. Quercetin (5): Yellow needles. Rf 0.54 (CH2Cl2-(CH3)2CO 2:1). ESI-MS: m/z 302.9 [M+H]+ (positive mode), m/z 301.0 [M−H]− (negative mode). 1H-NMR (CD3OD): δ 6.2 (1H, d, J = 2.0 Hz, H-8), 6.4 (1H, d, J = 2.0 Hz, H-6), 6.90 (1H, d, J = 8.5 Hz, H-5′), 7.64 (1H, dd, J = 8.5 Hz, 2.5 Hz, H-6′), 7.75 (1H, d, J = 2.5 Hz, H-2′). 13C-NMR (CD3OD): δ 94.4 (d, C-8), 99.2 (d, C-6), 104.5 (s, C-10), 116.0 (d, C-2′), 116.2 (d, C-5′), 121.7 (s, C-6′), 124.1 (s, C-1′), 137.2 (s, C-3), 146.2 (s, C-3′), 148.0 (s, C-2), 148.7 (s, C-4′), 158.2 (s, C-9), 162.5 (s, C-5), 165.5 (s, C- 7), 177.3 (s, C-4). β-Sitosterol 3-O-β-D-glucopyranoside (6): White amorphous powder. Rf 0.54 (CH2Cl2- (CH3)2CO 1:3). The 1H-NMR (CD3OD) is identical with that of our authentic sample. Quercitrin (7): Yellow needles. Rf 0.57 (CH2Cl2-(CH3)2CO 1:3). EI-MS: m/z 302 (M +., C21H20O11, − 146). 1H-NMR (CD3OD): δ 0.96 (3H, d, J = 6.0 Hz, 5″-CH3), 3.33 (1H, m, H-4″), 3.44 (1H, m, H-3″), 3.77 (1H, dd, J = 8.0 Hz, 3.5 Hz, H-2″), 4.24 (1H, m, H-5″), 5.37 (1H, d, J = 1.0 Hz, H-1″), 6.22 (1H, d, J = 2.0 Hz, H-8), 6.39 (1H, d, J = 2.0 Hz, H-6), 6.93 (1H, d, J = 8.5 Hz, H-5′), 7.32 (1H, dd, J = 8.5 Hz, 2.0 Hz, H-6′), 7.36 (1H, d, J = 2.0 Hz, H-2′). 13C-NMR (CD3OD): δ 17.6 (q, C-6″), 71.9 (d, C-5″), 72.0 (d, C-3″), 72.1 (d, C-2″), 73.3 (d, C-4″), 94.7 (d, C-8), 99.8 (d, C-6), 103.6 (d, C-1″), 105.9 (s, C- 10), 116.4 (d, C-2′), 117.0 (d, C-5′), 122.9 (s, C- 6′), 123.0 (s, C-1′), 136.3 (s, C-3), 146.4 (s, C- 3′), 149.8 (s, C-4′), 158.5 (s, C-2), 159.3 (s, C- 9), 163.2 (s, C-5), 165.8 (s, C-7), 179.6 (s, C-4). III - ReSULTS AND DISCUSSION The dried leaves of A. nepalensis were extracted with MeOH, and the resultant MeOH extract was partitioned between H2O and solvents of increasing polarity to give n-hexane- and EtOAc-soluble fractions. Fractionation of the n-hexane- and EtOAc-soluble fractions by silica gel open column (CC) and flash column (FC) chromatography resulted in the isolation of seven compounds 1-7 (Fig. 1). Compounds 1-7 have so far not been reported from the genus Alnus (Betulaceae). Compound 1 was isolated as white rods [Rf 0.46 (n-hexane-EtOAc 4:1)] from the n-hexane- soluble fraction. The electron impact mass spectrum (EI-MS) of 1 showed the molecular ion peak at m/z 468 (M+., C32H52O2). The 1H- NMR spectrum of 1 indicated the presence of eight tertiary methyl groups (all singlets) [[δH 0.82 (3H), 0.86 (3H), 0.88 (3H), 0.91 (6H), 0.95 (6H), 1.09 (3H)], an oxygenated methine [[δH 4.46 (1H, dd, J = 11.0 Hz, 5.0 Hz)], and an olefinic proton [δH 5.53 (1H, dd, J = 8.0 Hz, 3.0 Hz)]. The 13C-NMR and DEPT spectra of 1 supported the 1H-NMR data; the occurence of 30 13C signals was suggestive for a triterpenoid structure. The presence of an acetoxy group was shown by the NMR signals at δH 2.04 (3H, s) and δC 21.3 (s) and 170.9 (s). Thus, the structure of 1 was determined to be taraxeryl acetate by comparing their 1H- and 13C-NMR spectroscopic data with those reported in literature [8]. 524 O OH HO 5 R=H 7 R=Rha RO CH2CH3 H H H H 4 R=H 6 R=Glc 7 2 345 6 1' 4' 8 9 10 O AcO 1 O O OH OCH3 OH H3C 2 OH OH OR 2' 3' 5' 6' 1 2 3 45 6 7 8 9 1011 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 21 22 23 24 25 26 27 28 29 30 Fig. 1: Chemical Structures of Compounds 1-7 Compound 2 was isolated as orange needles [Rf 0.54 (n-hexane-EtOAc 15:1)] from the n- hexane-soluble fraction. 2 was suggested to have a molecular formula C16H12O5 from its EI- MS spectrum. The 1H-, 13C-NMR, and DEPT spectra of 2 exhibited the presence of two meta- coupled proton pairs [δH 6.69 (1H) and 7.37 (1H) (each d, J = 2.0 Hz)] and [δH 7.08 (1H) and 7.63 (1H) (each br s)], a methoxy group [δH 3.94 (3H, s), δC 56.1 (q)], and a methyl group bonded to an aromatic ring [δH 2.45 (3H, s), δC 22.2 (q)]. In addition, the 13C NMR chemical shifts were indicative of the presence of two substituted benzene rings and two carbonyl groups [δC 190.8 (s) and 182.1 (s)]. Taken together, a 9,10-anthraquinone skeleton of emodin-type compounds was suggested for 2 [9]. The chemical shifts of two peri-hydroxyl protons at C-1 [δH 12.1 (s)] and C-8 [δH 12.3 (s)] and two carbonyl groups at C-9 [δC 190.8 (s)] and C-10 [δC 182.1 (s)] [9] were used to unequivocally determine the structure of 2 to be 1,8-dihydroxy-3-methoxy-6- methylanthraquinone (physcion). Compound 3 was isolated as a white amorphous powder [Rf 0.5 (n-hexane-EtOAc 4:1)] from the n-hexane-soluble fraction. 3 was determined to be 1-nonacosanol from its 1H- NMR spectroscopic data. In the 1H-NMR spectrum of 3 the terminal methyl group δH 0.88 (3H, t, J = 7.0 Hz)], methylene chains [δH 1.26 (50 H, br s) and 1.58 (4H, m)], and a methylene group bearing a hydroxy group [δH 3.64 (2H, t, J = 6.5 Hz)] were observed. The number of methylene groups was deduced to be 28 from the 1H-NMR integration. The EI-MS spectrum of 3 showed the highest peak at m/z 364, which was probably derived from simutlaneous loss of H2O and ethylene, and a methylene group (M +., C29H60O, − 18 − 28 − 14). 1-Nonacosanol was found as constituent of several species, Agave, Sisalana, Citrulus, and Rhizophora [10]. Compound 5 was isolated as yellow needles [Rf 0.54 (CH2Cl2-(CH3)2CO 2:1)] from the EtOAc- soluble fraction and was suggested to to have a molecular formula C15H10O7 from its electrospray ionization mass spectrum (ESI-MS) (m/z 302.9 [M+H]+, m/z 301.0 [M−H]−). The 1H-NMR 525 spectrum of 5 exhibited a flavonoid pattern; two meta-coupled protons [δH 6.2 (1H) and 6.4 (1H) (each d, J = 2.0 Hz)] of the flavonoid A ring and a 1,3,4-trisubstituted benzene ring [δH 6.9 (1H, d, J = 8.5 Hz), 7.64 (1H, dd, J = 8.5 Hz, 2.5 Hz), and 7.75 (1H, d, J = 2.5 Hz)] of the flavonoid B ring. Furthermore, the 13C-NMR signals [δC 137.2 (s, C- 3), 148.0 (s, C-2), and 177.3 (s, 4-CO)] were indicative for a flavonol skeleton. Comparison of the 1H- and 13C-NMR spectroscopic data of 5 with literature data [11] determined the structure of 5 to be quercetin. Compound 7 was isolated as yellow needles [Rf 0.57 (CH2Cl2-(CH3)2CO 1:3)] from the EtOAc-soluble fraction. The 1H- and 13C-NMR spectra of 7 indicated that quercetin was the aglycon of 7. The presence of an α-L- rhamnopyranosyl unit was evidenced by the anomeric NMR signal at δH 5.37 (1H, d, J = 1.0 Hz), δC 103.6 (d) and the secondary methyl group at δH 0.96 (3H, d, J = 6.0 Hz), δC 17.6 (q). Comparison of the 13C-NMR signals of 7 and 5 showed the upfield shift at C-3 (ΔδC −0.9) and pronounced downfield shift at C-2 (ΔδC +10.5) suggesting the attachment of the rhamnopyranosyl unit at C-3. The EI-MS spectrum of 7 showed the peak at m/z 302 which was resulted from the loss of a deoxyhexose unit (M+., C21H20O11, − 146). Thus, the structure of 7 was determined to be quercetin 3-O-α-L- rhamnopyranoside (quercitrin) by comparison of its spectroscopic data with literature data [11]. Compound 4, β-sitosterol, was identified on the basis of co-TLC analysis. Compound 6, β- sitosterol 3-O-β-D-glucopyranoside, was identified by comparison of its 1H-NMR spectrum with that of our authentic sample. 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