Biflavones and megastigmane glycosides from the leaves of Antidesma bunius - Do Thi Trang

Vietnam Journal of Chemistry, International Edition, 54(4): 434-438, 2016 DOI: 10.15625/0866-7144.2016-00342 434 Biflavones and megastigmane glycosides from the leaves of Antidesma bunius Do Thi Trang 1 , Le Thi Huyen 2 , Dan Thi Thuy Hang 1 , Nguyen Xuan Nhiem 1 , Pham Hai Yen 1 , Bui Huu Tai 1 , Hoang Le Tuan Anh 1 , Chau Van Minh 1 , Phan Van Kiem 1* 1 Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST) 2 Faculty of Chemistry, University of Science, Vietnam National University Received 3 September 2015; Accepted for publication 12 August 2016 Abstract Two biflavones, podocarpusflavone A (1) and amentoflavone (2) and two megastigmane glycosides, byzantionoside B (3) and (6S,9R)-roseoside (4) were isolated from the methanol extract of the leaves of Antidesma bunius. Their structures were determined by spectroscopic methods and in comparison with the published data. Keywords. Antidesma bunius, Euphorbiaceae, biflavone, megastigmane. 1. INTRODUCTION Antidesma bunius (L.) Spreng belongs to Euphorbiaceae family and widely distributes throughout Vietnam and China. The fruits of A. bunius are edible and have been used to prepare drink supplement or healthy foods. In traditional medicine, A. bunius was used for the treatment of inflammation and infection diseases [1]. Phytochemical analysis of this plant indicated the presence of flavonoids, phenolics and organic acids [2]. In addition, this plant exhibited antioxidant [3] and antimicrobial activities [4]. Herein, we report the isolation and structure elucidation of two biflavones and two megastigmane glycosides from the methanol extract of A. bunius leaves. 2. MATERIAL AND METHODS 2.1. Plant material The leaves of Antidesma bunius (L.) Spreng were collected in Daklak province, Vietnam, in March 2013, and identified by Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature. A voucher specimen (AB1303) was deposited at Institute of Marine Biochemistry, VAST. 2.2. General experimental procedures All NMR spectra were recorded on a Bruker AM500 FT-NMR spectrometer (500 MHz for 1 H- NMR and 125 MHz for 1 3 C-NMR) . NMR measurements, including 1 H-NMR, 13 C-NMR, HSQC, and HMBC experiments, were carried out using 5-mm probe tubes at temperature of 22.2 o C. Optical rotations were determined on a Jasco DIP- 1000 polarimeter. Column chromatography was performed using a silica-gel (Kieselgel 60,70-230 mesh and 230-400 mesh, Merck) or RP-18 resins (30-50 µm, Fujisilisa Chemical Ltd.), thin layer chromatography (TLC) using a pre-coated silica-gel 60 F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm, Merck). 2.3. Extraction and isolation The dried powder leaves of A. bunius (2.0 kg) were sonicated in methanol (MeOH) three times to yield 110.0 g of a dark solid extract, which was then suspended in water and successively partitioned with CH2Cl2 and ethyl acetate (EtOAc) to give CH2Cl2 (AD1, 17.0 g), EtOAc (AD2, 20.0 g), and water layers (AD3, 73.0 g) after removing solvent in vacuo. The EtOAc layer (AD2, 20.0 g) was chromatographed on a RP-18 column eluting with MeOH/water (3/1, v/v) to give three fractions, AD2A-AD2C. The AD2B fraction was applied to a silica gel column eluting with CH2Cl2/MeOH/water (5/1/0.1, v/v/v) to yield compounds 2 (30.0 mg) and 1 (40.0 mg). The water layer (AD3, 73.0 g) was subjected to a Diaion HP-20 column eluting with water to remove sugar, then increase concentration of methanol in water (25, 50, 75, and 100 %) to obtain 4 fractions, AD3A-AD3D. The AD3B fraction VJC, 54(4) 2016 Phan Van Kiem, et al. 435 Figure 1: Chemical structures of compounds 1-4 was chromatographed on a silica gel column eluting with CH2Cl2/MeOH (8/1, v/v) to give three fractions, AD3B1-AD3B3. The AD3B1 fraction was further purified by a RP-18 column eluting with MeOH/water (1/2, v/v) to yield compounds 3 (20.0 mg) and 4 (15.0 mg). Podocarpusflavone A (1): Yellow powder, C31H20O10, ESI-MS m/z 551 [M−H]−, 1 H- and 13 C-NMR (DMSO- d6), see table 1. Amentoflavone (2): Yellow amorphous powder, C30H18O10, ESI-MS m/z 537 [M−H] − , 1 H- and 13 C- NMR (CD3OD), see table 1. Byzantionoside B (3): White powder, C19H32O7, 25][ D : +50.0 (c = 0.1, MeOH), 1 H- and 13 C-NMR (CD3OD), see table 1. (6S,9R)-roseoside (4): White powder, C19H30O8, 25][ D : +65.0 (c = 0.1, MeOH), 1 H- and 13 C-NMR (CD3OD), see table 1. 3. RESULTS AND DISCUSSION Compound 1 was obtained as a yellow amorphous powder. The molecular formula of 1 was determined to be C31H20O10, by the combination of ESI-MS ion at m/z 551 [M−H]− and 13C-NMR data. The 1 H-NMR spectrum of compound 1 showed the following signals: three aromatic protons of the ABX system in B ring at δH 7.15 (1H, d, J = 8.0 Hz), 7.99 (1H, d, 1.5 Hz), and 8.01 (1H, dd, J = 1.5, 8.0 Hz); four aromatic protons of para-substituted aromatic ring at δH 6.92 (2H, d, J = 9.0 Hz), and 7.67 (2H, d, J = 9.0 Hz); three singlet protons at δH 6.41 (1H, s), 6.82 (1H, s), 6.88 (1H, s); two meta-protons of aromatic ring at δH 6.18 (1H, d, J = 2.0 Hz), and 6.45 (1H, d, J = 2.0 Hz). The 13 C-NMR and DEPT spectra of compound 1 showed the presence of two carbonyl at δC 181.74 and 182.15, sixteen non-protonated at δC 2 × 103.72, 104.01, 121.01, 119.97, 122.98, 154.54, 157.37, 159.54, 160.54, 161.45, 162.00, 162.20, 163.22, 163.79, and 164.11, twelve methine carbons at δC 94.03, 98.70, 98.83, 103.02, 103.24, 2×114.48, 116.18, 127.84, 2×127.98, 132.76, and one methoxy group at δC 55.50, assigned to a biflavone. The 1 H- and 13 C-NMR spectra of 1 were identical to those of podocarpusflavone A [5]. The position of methoxy group at C-4' was confirmed by the HMBC correlation from methoxy (δH 3.75) to C-4' (δC 162.20). The HMBC correlations from H-6 (δH 6.18)/H-8 (δH 6.45) to C-7 (δC 164.11); from H-6 (δH 6.18) to C-5 (δC 161.45)/C-7 (δC 164.11); from H-2′ (δH 7.99)/H-5′ (δH 7.15)/H-6′ (δH 8.01) to C-4′ (δC 159.54) suggested the positions of hydroxyl groups at C-5, C-7, and C-4′ of flavone unit I. The HMBC correlations between H-6″ (δH 6.41) and C-5″ (δC 160.54)/C-7″ (δC 162.00)/C-8″ (δC 104.01)/C-10″ (δC 103.72), H-2' (δH 7.67)/H-3’ (δH 6.92) and C-4’ (δC 162.20) suggested that the hydroxyl groups were at C- 5″, C-7″, and C-4' of flavone unit II. In addition, the HMBC cross peaks from H-2′ (δH 7.99)/H-5′ (δH 7.15) to C-3′ (δC 119.97) and H-2′ (δH 7.99)/H-6′′ (δH 6.41) to C-8′′ (δC 104.01) indicated the linkages between the two flavone units at C-3′ and C-8′′. Consequently, the structure of 1 was determined to be podocarpusflavone A [5]. This compound was reported from the genus Antidesma for the first time. The molecular formula of 2 was determined to be C30H18O10, by ESI-MS ion at m/z 537 [M−H] − and 13 C-NMR data. The 1 H-NMR spectrum of 2 showed the following signals: three aromatic protons of the ABX system in aromatic ring at δH 7.09 (1H, d, J = 8.5 Hz), 7.83 (1H, dd, J = 1.5, 8.5 Hz), and 7.96 (1H, d, J = 1.5 Hz), four aromatic protons of para- substituted aromatic ring at δH 6.73 (2H, d, J = 8.5 Hz), and 7.49 (2H, d, J = 8.5 Hz), five aromatic protons at δH 6.16 (1H, s), 6.36 (1H, s), 6.43 (1H, s), 6.57 (1H, s), and 6.58 (1H, s). The 13 C-NMR and DEPT spectra revealed the signals of 30 carbons, including two carbonyl at δC 183.59 and 184.02, sixteen non-protonated at δC 105.15, 105.28, 105.37, 121.44, 123.13 × 2, 156.34, 159.21, 160.78, 162.39, 162.44, 163.09, 163.26, 165.78 × 2, and 165.92, twelve methine carbons at δC 95.19, 99.89, 100.15, 436 Table 1: The 1 H- and 13 C-NMR data for compounds 1-4 and reference compounds Pos. 1 2 Pos. 3 4 δC @ δC a δH a (mult., J, Hz) δC b δH b (mult., J, Hz) δC # δC a δH a (mult., J, Hz) δC $ δC a δH a (mult., J, Hz) Aglycone 2 163.8 163.79 - 165.92 - 1 37.2 37.31 - 42.2 42.42 - 3 103.0 103.02 6.82 (s) 103.45 6.57 (s) 2 48.0 48.07 1.97 (d, 17.5) 2.49 (d, 17.5) 50.5 50.71 2.54 (d, 17.5) 2.17 (d, 17.5) 4 181.7 181.74 - 184.02 - 3 202.3 204.42 - 201.3 201.20 - 5 161.4 161.45 - 163.09 - 4 125.3 125.37 5.82 (s) 127.1 127.18 5.89 (d, 1.5) 6 98.8 98.83 6.18 (d, 2.0) 100.15 6.16 (d, 1.5) 5 170.0 170.13 - 167.2 167.24 - 7 164.1 164.11 - 165.78 - 6 52.3 52.38 2.02 (m) 79.9 80.00 - 8 94.0 94.03 6.45 (d, 2.0) 95.19 6.43 (d, 1.5) 7 26.7 26.82 1.53 (m)/1.98 (m) 134.9 135.29 5.88* 9 157.3 157.37 - 159.21 - 8 37.7 37.80 1.66 (m) 131.4 131.56 5.88* 10 103.7 103.72 - 105.15 - 9 75.0 75.48 3.91 (m) 77.0 77.28 4.44 (m) 1′ 121.0 121.01 - 121.44 - 10 19.8 19.87 1.21 (d, 6.0) 20.8 21.18 1.31 (d, 6.5) 2′ 131.3 131.38 7.99 (d, 1.5) 132.76 7.96 (d, 1.5) 11 27.5 27.53 1.11 (s) 19.2 19.54 1.03 (s) 3′ 120.0 119.97 - 123.13 - 12 29.0 29.08 1.03 (s) 23.0 23.42 1.02 (s) 4′ 159.6 159.54 - 160.78 - 13 25.0 24.97 2.07 (s) 24.4 24.68 1.94 (d, 1.5) 5′ 116.2 116.18 7.15 (d, 8.0) 117.27 7.09 (d, 8.5) 9-OGlc 6' 127.8 127.84 8.01 (dd, 1.5, 8.0) 128.96 7.83 (dd, 1.5, 8.5) 1′ 102.0 102.11 4.35 (d, 8.0) 102.5 102.75 4.36 (d, 8.0) 2′′ 163.2 163.22 - 165.78 - 2′ 75.4 75.14 3.16 (dd, 8.0, 9.0) 75.1 75.26 3.18 (dd, 8.0, 9.0) 3′′ 103.2 103.24 6.88 (s) 104.06 6.58 (s) 3′ 78.0 78.15 77.9 78.13 4′′ 182.1 182.15 - 183.59 - 4′ 71.7 71.83 71.3 71.68 5′′ 160.5 160.54 - 162.44 - 5′ 77.7 77.89 77.8 78.04 6′′ 98.7 98.70 6.41 (s) 99.89 6.36 (s) 6' 62.8 62.91 3.66 (dd, 5.5, 11.5) 3.87 (dd, 2.0, 11.5) 62.3 62.2.85 3.64 (dd, 5.5, 11.5) 3.87 (dd, 2.0, 11.5) 7′′ 161.0 162.00 - 163.26 - 8′′ 104.1 104.01 - 105.28 - 9′′ 154.5 154.54 - 156.34 - 10′′ 103.6 103.72 - 105.37 - 1′ 123.0 122.98 - 123.13 - 2′, 6′ 128.0 127.98 7.67 (d, 8.5) 129.31 7.49 (d, 8.5) 3′, 5′ 114.5 114.48 6.92 (d, 8.5) 116.87 6.73 (d, 8.5) 4′ 162.2 162.20 - 162.39 - 4′-OCH3 55.5 55.50 3.75 (s) a) Recorded in CD3OD, b) recorded in DMSO-d6,*overlapped signals, @δC of podocarpusflavone A in CD3OD [5], #δC of byzantionoside B in CD3OD [7], $δC of (6S,9R)-roseoside in CD3OD [8]. V JC , 5 4 (4 ) 2 0 1 6 B ifla vo n es a n d m eg a stig m a n e g lyco sid es 4 3 6 VJC, 54(4) 2016 Phan Van Kiem, et al. 437 Figure 2: The important HMBC correlations of compounds 1 and 3 103.45, 104.06, 116.87 × 2, 117.27, 128.96, 129.31 × 2, 132.76, indicating of the presence of two flavone units. The 1 H- and 13 C-NMR spectra of 2 were almost similar to those of 1 except for disappearance of methoxy group at C-4. The positions of the remaining functional groups were based on the HSQC and HMBC spectra. Thus, the structure of compound 2 was elucidated to be amentoflavone, it was previously isolated from A. laciniatum [6]. The 1 H-NMR spectrum of 3 showed the signals of one olefinic proton at δH 5.82 (1H, s), one secondary methyl group at 1.21 (3H, d, J = 6.0 Hz), three tertiary methyl groups at δH 1.03 (3H, s), 1.11 (3H, s), and 2.07 (3H, s), assigned to a megastigmane aglycone; one anomeric proton at δH 4.35 (1H, d, J = 8.0 Hz) assigned to one sugar moiety. The 13 C-NMR and DEPT spectra of compound 3 displayed the signals of 19 carbons, including one carbonyl at δC 204.42, two non-protonated at δC 37.1 and 170.13; eight methine at δC 52.38, 71.83, 75.14, 75.48, 77. 89, 78.15, 102.11, and 125.37; four methylene at δC 26.82, 37.80, 48.07, and 62.91; four methyl carbons at δC 19.87, 24.97, 27.53 and 29.08. Analysis of 1 H- and 13 C-NMR data indicated that structure of 3 was identical to byzantionoside B [7]. The 13 C-NMR data of sugar moiety (δC 102.11, 78.15, 77.89, 75.14, 71.83, and 62.91) and coupling constant of glc H-1′ and glc H-2′, J = 8.0 Hz proved the presence of β-D- glucopyranosyl moiety in 3. The position sugar unit at C-9 of aglycone was confirmed by HMBC correlations between glc H-1′ (δH 4.31) and C-9 (δC 75.48). 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