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Journal of Chemistry, Vol. 45 (6), P. 791 - 794, 2007 A NEW FLAVONOID FROM OPHIOPOGON CONFERTIFOLIUS Received 2 July 2007 NGUYEN THI VINH HUE , NGUYEN DUY THUAN2, NGUYEN TRONG THONG3, CHAU VAN MINH4, PHAN VAN KIEM4 1 Traphaco Joint Stock Company, Ministry of Health 1 2 National Institute of Medicinal Materials, Ministry of Health 3 Hanoi Medical University, Ministry of Health 4 Institute of Natural Products Chemistry, VAST SUMMARY From the methanolic extract of the roots of Ophiopogon confertifolius N. Tanaka (Convallariaceae) a new flavonoid named ophiofolius A (1) and two known compounds (2, 3) were isolated. Their structures were elucidated as 6,8-dimethyl-4’methoxy-5,7,3’,5’tetrahydroxyflavanone (1), 6,8-dimethyl-3’,4’-dimethoxy-5,7,5’-trihydroxyflavanone (2), and 6methoxy-5,7-dihydroxyflavanone (3) by the spectroscopic evidences (1D NMR, 2D NMR, ESIMS). I - INTRODUCTION “Cao c ng” is a traditional medicinal plant of the people who lives in some mountainous areas of Bac Giang province. It has been used to treat osteocopic pain, dispel swelling and blood clotting in ecchymosis, renal failure…The scientific name of this plant has been identified as Ophiopogon confertifolius N. Tanaka (Convallariaceae). This is a new species of Vietnamese flora [1]. Up to date, no studies on the chemical and bioactivities of this plant were carried out. As a part of our study on this plant, we report herein the isolation and the structural elucidation of 8-dimethyl-4’methoxy-5,7,3’,5’tetrahydroxyflavanone (1), 6,8-dimethyl-3’,4’dimethoxy-5,7,5’-trihydroxyflavanone (2), and 6-methoxy-5,7-dihydroxyflavanone (3) by the spectroscopic evidences (1D NMR, 2D NMR, ESI-MS). Compound 1 was isolated for the first time from nature, and compounds 2 and 3 were first isolated from the Ophiopogon species. II - EXPERIMENTAL 1. Plant material The roots of Ophiopogon confertifolius N. Tanaka (Convallariaceae) were collected in Yen The, Bac Giang province, Vietnam, and the plant was identified by Dr Nguyen Thi Do, Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology. A voucher specimen was deposited at the National Institute of Medicinal Materials, Ministry of Health. 2. General experimental procedures Melting points were determined using an Electro thermal IA-9200. The IR spectra were obtained on a Hitachi 270-30 type spectrometer with KBr discs. Optical rotations were determined on a Jasco DIP-1000 KUY polarimeter. The electrospray ionization (ESI) mass spectra were obtained using an AGILENT 1100 LC-MSD Trap spectrometer. The 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra 791 were recorded on a Bruker AM500 FT-NMR spectrometer and TMS was used as an internal standard. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) and YMC RP18 resins. 3. Extraction and isolation Dried roots of O. confertifolius were powdered and then extracted three times with MeOH. The MeOH extract (50 g) was suspended in water and partitioned in turn with n-hexane, chloroform, ethyl acetate, and nBuOH to obtain n-hexane (5.8 g), chloroform (10,2 g), ethyl acetate (20 g), and n-BuOH fractions (13,0 g). The ethyl acetate fraction (20 g) was chromatographed on silica gel column using CHCl3-MeOH-H2O (80:20:2) and on YMC column using MeOH-H2O (3:1) to yield compounds 1 (75 mg), 2 (15 mg), and 3 (21 mg) as yellow crystals. 6,8-Dimethyl-4’methoxy-5,7,3’,5’tetrahydroxyflavanone (1): Yellow crystals; mp. 223-224oC; IR (KBr) max cm-1: 3400 (OH), 1716 (C=O), 1445 (C=C); ESI-MS m/z: 347 [M+H]+; 345 [M-H]- (C18H18O7); 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6): see Table 1. 6,8-Dimethyl-3’,4’-dimethoxy-5,7,5’trihydroxyflavanone (2): Yellow crystals, mp. 234-235oC; ESI-MS m/z: 361 [M+H]+; 359 [MH]- (C19H20O7); 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6), see table 1. 6-Methoxy-5,7-dihydroxyflavanone (3): Yellow crystals; mp. 176-177oC; ESI-MS m/z: 287 [M+H]+; 285 [M-H]- (C16H14O5); 1H-NMR (500 MHz, CD3OD) and 13C-NMR (125 MHz, CD3OD), see table 1. OR 3' 3' CH 3 10 H3C 6 2 4 5' OH 3 O 1R=H 2 R = CH3 III - RESULTS AND DISSCUSSION Compound 1 was isolated as yellow crystals from the ethyl acetate fraction suggesting a flavonoid compound. The 1H-NMR spectrum of 1 showed only a singlet at 6.43 (2H) of the aromatic ring suggesting that the A ring was full substituted, and the B ring was substituted at C1, C-3, C-4, C-5 positions. The signals at 5.34 (dd, J = 3.0, 12.5 Hz), 2.76 (dd, J = 3.0, 12.5 Hz) and 3.02 (dd, J = 12.5, 17.0 Hz) confirming the presence of a flavanone compound [2], two methyl groups resonance at 1.98 (3H, s) and 1.95 (3H, s) suggested that they were directly 792 2 10 H3CO 6 1' O 9 7 6' 5 OH 8 HO 1' O 9 7 4' 4' 8 HO 2' OCH3 2' 4 5' 6' 3 5 OH O 3 attached to the A ring. In addition, the signal at 12.33 displayed the hydroxyl group at C-5 [3], and a methoxyl group was at 3.68 (3H, s). The 13C-NMR of 1 displayed signals of 18 carbon atoms, including 15 signals of the flavonoid and 3 signals of the two methyl groups ( 7.55 and 8.26) and of the methoxyl group at 59.63. The carbonyl group was assigned at 196.51, the flavanone compound was confirmed by the signals of the oxymethine at 77.58 and of the methylene group at 42.06. In the HSQC spectrum, protons at 6.43, 5.34, 3.68, 1.98 and 1.95 had cross-peaks to carbons at 105.33, 77.58, 59.63, 8.26 and 7.55, respectively. While protons at 2.76 and 3.02 had cross-peaks to carbon at 42.06. In the HMBC, proton H-2’ at 6.43 correlated with carbon C-2 ( 77.58)/C-1’ ( 135.25)/C-3’ ( 150.70) and carbon C-4’ ( 134.36), methoxyl proton at 3.68 correlated with C-4’ ( 134.36), methyl proton at 1.98 correlated with C-7 ( 162.39)/C-8 ( 102.54)/C-9 ( 157.09), methyl proton at 1.95 correlated with C-5 ( 158.34)/C-6 (103.21)/C-6 (162.39). This evidence confirmed the position of the two hydroxyl groups at C-3’ and C-5’, and methoxyl group at -4’, and two methyl groups at C-6 and C-8 of the A ring. In addition, H-C long range correlations were observed between H-2 ( 5.34) and C-1’ ( 135.25)/C-3 ( 42.06)/C-4 ( 196.51), between proton H-3 ( 2.67/3.02) and carbons C-2 ( 77.58)/C-4 ( 196.51) in the HMBC confirming again the flavanone skeleton of 1. Comparing the chemical shifts ( C and H) and proton coupling-constants (J) at C-2 and C3 of 1 (JH-2/Ha-3 = 12.5 Hz, JH-2/Hb-3 = 3.0 Hz, JH-3gem = 17.0 Hz) with those of naringerin [4] led to determine the absolute configuration at C-2 as R. Furthermore, the ESI-MS of 1 exhibited the quasi ion peaks at m/z 347 [M+H]+ (positive) and 345 [M-H]- (negative), corresponding to the molecular formula of C18H18O7. From the above data, compound 1 was determined to be new natural product 6,8-dimethyl-4’methoxy5,7,3’,5’-tetrahydroxyflavanone, which we named ophiofolius A. Table 1: NMR data of 1 - 3 C 1 a,c C 2 77.58 3 42.06 4 5 6 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’ 6-Me 8-Me 4’-OMe 3’-OMe 6-OMe 5-OH 196.51 158.34 103.21 162.39 102.54 157.09 101.74 135.25 105.33 150.70 134.36 150.70 105.33 7.55 8.26 59.63 2 ,b,c H 5.34 dd (3.0, 12.5) 2.76 dd (3.0, 17.00) 3.02 dd (12.5, 17.0) 6.43 s 6.43 s 1.95 s 1.98 s 3.68 s 12.33 a,c C 77.92 42.19 196.56 158.46 103.37 162.47 102.62 157.15 102.62 134.61 107.40 153.19 136.2 150.57 101.75 7.59 8.29 55.75 55.89 3 b,c H 5.36 dd (3.0, 12,0) 2.70 dd (3.0, 17.00) 3.12 dd (12.5, 17.0) 6.64 s 6.64 1.96 s 1.99 s 3.68 s 3.79 s a,d C 80.54 b,d H 198.05 160.85 130.53 159.98 96.27 140.38 103.52 140.38 127.32 130.53 129.60 130.53 127.32 5.42 dd (3.0, 12.0) 2.78 dd (3.0, 17.00) 3.08 dd (12.0, 17.0) 6.02 s 7.38-7.50 7.38-7.50 7.38-7.50 7.38-7.50 7.38-7.50 60.99 3.80 s 44.19 12.35 a 125 MHz, b500 MHz, cIn DMSO-d6, dIn CD3OD. Chemical shifts are given in ppm; coupling constant J (in parentheses) in Hz. 793 The NMR data of 2 were very similar to those of 1 except for the additional signals of one methoxyl group at C 59.89/ H 3.79. This suggested that compound 2 was a methoxyl derivative of 1 with the suggested molecular formula as C19H20O7, which was further confirmed by the appearance of the quasi ion peaks at m/z 361 [M+H]+ (positive) and 359 [MH]- (negative) in the ESI-MS spectrum. To determine the position of the additional methoxyl group, the HSQC and HMBC spectra OH HO OCH 3 OCH 3 CH 3 O H3 C were taken. All HMBC correlations in the HMBC spectrum were shown in Fig. 2, and the NMR data of this compound were summarized in Table 1. Consequently, the structure of 2 was determined as 6,8-dimethyl-3’,4’,5’-trimethoxy5,7-dihydroxyflavanone, which was isolated from Alluaudiposis marnieriana. However, this is the first report of 2 from Ophiopogon species, and the NMR data (1D and 2D) of this compound have been reported here for the first time. OH OCH 3 CH 3 HO O O O OH H3CO H3 C OH HO OH OH O O Figure 2: H-C correlations in the HMBC of 1 - 3 The 13C-NMR spectrum of 3 displayed the resonances of 16 carbon atoms including a benzene ring (B ring) and a methoxyl group at 80.54 (CH) C 60.99/ H 3.80. The signals at and 44.19 (CH2) suggest a flavanone compound. In the 1H-NMR, signal at 5.42 (1H, dd, J = 3.0, 12.0 Hz), 2.78 (1H, dd, J = 3.0, 17.0 Hz) and 3.08 (1H, dd, J = 12.0, 17.0 Hz) suggesting the 2R configuration of 2 [4] as 1. The position of the methoxyl and the two hydroxyl groups were determined from the analysis of the HSQC and HMBC as shown in table 1 and Fig. 2. In addition, the ESI-MS of 3 showed the quasi ion peaks at m/z 287 [M+H]+ (positive) and 285 [MH]- (negative) corresponding to the molecular formula of C16H14O5. Thus, compound 3 was determined to be 6-methoxy-5,7dihydroxyflavanone, which was the first isolated from Ophiopogon species. To the best of our knowledge, the NMR data (1D and 2D) of this compound have been reported here for 794 the first time. Acknowledgments: The authors would like to thank Dr Tran Nguyen Thi Do, Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology for the plant identification. REFERENCES 1. N. T. Do, N. T. V. Hue, N. D. Thuan, N. V. Trai, N. N. Thin. Vietnamese Journal of Pharmacy, Vol. 369, 19 - 21 (2007). 2. C. Kamperdick, N. H. Van, T. V. Sung. Phytochemistry, Vol. 61, 991 - 994 (2002). 3. P. K. Agrawal. Carbon 13 NMR of flavonoids, Elsevier Science Publishers B. V. 1989. 4. C. C. Shen, Y. S. Chang, and L. K. Ho. Phytochemistry, Vol. 34, 843 - 845 (1993).