Triterpenes from Vitex limonifolia - Nguyen Thi Kim Thoa

Vietnam Journal of Chemistry, International Edition, 55(6): 715-719, 2017 DOI: 10.15625/2525-2321.2017-00531 715 Triterpenes from Vitex limonifolia Nguyen Thi Kim Thoa 2 , Ninh Khac Ban 1 , Do Thi Trang 1 , Tran My Linh 1 , Vu Huong Giang 1 , Nguyen Xuan Nhiem 1 , Phan Van Kiem 2* 1Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST) 2Faculty of Basic Science, Hanoi University of Mining and Geology Received 2 December 2017; Accepted for Publication 29 December 2017 Abstract Using combined chromatographic methods, four triterpenoids, 2α,3α-dihydroxyurs-12-en-28-oic acid (1), euscaphic acid (2), 2α,3α-dihydroxy-19-oxo-18,19-seco-urs-11,13(18)-dien-28-oic acid (3), and maslinic acid (4) were isolated from the methanol extract of the leaves of Vitex limonifolia. Their structures were elucidated by 1D-, 2D-NMR spectra as well as by comparison with those reported in the literature. Compound 3 was reported from Vitex genus for the first time. Keywords. Vitex limonifolia, triterpenoid, ursane, oleanane. 1. INTRODUCTION Vitex is the genus of shrubs and trees and mainly distributed in tropics and subtropics [1]. Since ancient times, civilization used Vitex plants for treating many diseases such as malaria, herpes, itches, dermatitis or controlling menstruation [1]. Phytochemical study of the genus Vitex revealed the presence of flavonoids, terpenoids, ecdysteroids, iridoid glucosides, etc. [2]. However, phytochemistry study of this plant has not been studied yet. This paper reported the isolation and structure elucidation of four triterpenes from the methanol extract of the leaves of V. limonifolia. 2. MATERIAL AND METHODS 2.1. Plant materials The leaves of Vitex limonifolia Wall. ex C.B.Clarke were collected in Bachma National Park, Thua Thien Hue, Vietnam in September 2015, and identified by one of the authors, Prof. Dr. Ninh Khac Ban. A voucher specimen was deposited at the Herbarium Institute of Marine Biochemistry, VAST. 2.2. General experimental procedures Optical rotations were determined on a Jasco DIP- 370 automatic polarimeter. The NMR spectra were recorded using a Bruker DRX 500 spectrometer (1H- NMR, 500 MHz; 13C-NMR, 125 MHz). Column chromatography was performed using silica-gel (Kieselgel 60, 230-400 mesh, Merck) or RP-18 resins (30-50 μm, Fujisilisa Chemical Ltd.), and thin layer chromatography (TLC) was performed using a precoated silica gel 60 F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm, Merck). 2.3. Extraction and isolation The dried leaves of V. limonifolia (4.2 kg) were extracted with hot MeOH three times (3 × 5 L) under reflux for 12 h to yield 350 g extract after evaporation of the solvent. This extract was suspended in H2O and successively partitioned with CH2Cl2 and EtOAc to obtain the CH2Cl2 (VIL1, 130.0 g), EtOAc (VIL2, 27.0 g), and H2O (VIL3, 190.0 g) extracts after removal of the solvents in vacuo. The VIL1 fraction was chromatographed on a silica gel column eluting with a gradient of n- hexane:acetone (100:0 → 0:1, v/v) to give six fractions, VIL1A – VIL1F. The VIL1C fraction was chromatographed on an RP-18 column eluting with MeOH:water (5:1, v/v) to give three fractions, VIL1C1-VIL1C3. VIL1C3 was chromatographed on a silica gel column eluting with n-hexane:EtOAc (1.4:1, v/v) to yield 1 (16.0 mg). The VIL1D fraction was chromatographed on an RP-18 column eluting with MeOH:water (4:1, v/v) to give five fractions, VIL1D1-VIL1D5. VIL1D2 was chromatographed on an RP-18 column eluting VJC, 55(6), 2017 Phan Van Kiem et al. 716 Figure 1: The chemical structures of compounds 1−4 with acetone:water (1.8:1, v/v) to give three smaller fractions, VIL1D2A-VIL1D2C. Compounds 2 (18.0 mg) and 3 (4.5 mg) were obtained from VIL1D2A fraction using silica gel column (n-hexane:EtOAc, 1.4:1, v/v as eluent solvent). Compound 4 (7.0 mg) was obtained from VIL1D2C using silica gel column with solvent of n-hexane:EtOAc (1.4:1, v/v). 2α,3α-Dihydroxyurs-12-en-28-oic acid (1): white amorphous powder; 25][ D : +30.0 (c 0.1, MeOH); ESIMS m/z 473 [M+H]+, C30H48O4; MW: 472; 1H- and 13C-NMR (DMSO-d6), see table 1. Euscaphic acid (2): white amorphous powder; 25][ D : +20.0 (c 0.1, MeOH); ESIMS m/z 489 [M+H] +, C30H48O5; MW: 488; 1H- and 13C-NMR (DMSO-d6), see table 1. 2α,3α-Dihydroxy-19-oxo-18,19-seco-urs- 11,13(18)-dien-28-oic acid (3): white amorphous powder; 25][ D : –40.0 (c 0.1, MeOH); ESIMS m/z 487 [M+H]+, C30H46O5; MW: 486; 1H- and 13C-NMR (CD3OD), see table 1. Maslinic acid (4): white amorphous powder; 25][ D : –64.1 (c 0.1, MeOH); ESIMS m/z 473 [M+H] +, C30H48O4; MW: 472; 1H- and 13C-NMR (CD3OD), see table 1. 3. RESULTS AND DISCUSSION Compound 1 was obtained as a white powder. The 1H-NMR spectrum of 1 showed the signals of one olefinic proton at H 5.14 (1H, t, J = 3.0 Hz) and seven methyl groups at H 0.70 (3H, s), 0.78 (3H, s), 0.82 (3H, d, J = 6.5 Hz), 0.88 (3H, s), 0.89 (3H, s), 0.91 (3H, d, J = 7.0 Hz), 1.04 (3H, s), two oximethine protons at H 3.15 (br s) and 3.77 (br d, J = 11.0 Hz). The 13C-NMR and DEPT spectra of 1 displayed the signals of thirty carbons, including seven methyls, eight methylenes, eight methines, and seven quaternary carbons in which two olefinic carbons at C 124.5 and 138.2. Analysis the 1H- and 13C-NMR spectra of 1 suggested the presence of an ursan-12-ene-type triterpene skeleton [3]. In addition, the NMR data of 1 was found to be similar to those of 2α,3α-dihydroxyurs-12-en-28-oic acid methyl ester [4] except for the disappearance of methyl ester group. The HMBC correlations between H-23 (δH 0.89)/H-24 (δH 0.78) and C-3 (δC 77.8)/C-4 (δC 38.0)/C-5 (δC 47.6) indicated the position of the hydroxyl group at C-3. The HMBC correlations between H-1 (δH 1.13 and 1.41)/H-3 (δH 3.15) and C-2 (δC 64.7) indicated the hydroxyl group was located at C-2. Beside, the large coupling constant of H-1 and H-2, J = 11.0 Hz [H-2: δH 3.77 (br d, J = 11.0 Hz)] and small coupling constant of H-2 and H-3 (J~0 Hz) [H-3: δH 3.15 (br s)] suggested the configurations of two hydroxyl groups at C-2/C-3 as equatorial and axial, respectively. The HMBC correlations between H-27 (δH 1.04) and C-8 (δC 39.1)/C-13 (δC 138.2)/C-14 (δC 41.7)/C-15 (δC 28.2); between H-12 (δH 5.14) and C-9 (δC 46.8)/C- 14 (δC 41.7)/C-18 (δC 52.4) proved the position of the double bond at C-12/C-13. The ESIMS of 1 exhibited an ion peak at m/z 473 [M+H]+, corresponding to the formula of C30H48O4. Consequently, compound 1 was identified as 2α,3α- dihydroxyurs-12-en-28-oic acid and previously reported from V. altissima [5] and V. negundo [6]. Analysis the NMR spectra of 2 exhibited the structure was similar to those of 1 except for the additional hydroxyl group at C-19. This was confirmed by the HMBC correlations between H-29 (δH 1.08) and C-18 (δC 53.2)/C-19 (δC 71.6)/C-20 (δC 41.4); between H-30 (δH 0.84) and C-19 (δC 71.6)/C- 20 (δC 41.4)/C-21 (δC 25.9). The molecular formular of 2 was futher confirmed by the exhibition of an ESIMS ion peak at m/z 489 [M+H]+, corresponding to the formula of C30H48O5. Thus, the structure of 2 was determined to be euscaphic acid [3]. This compound was already isolated from V. altissima [5], V. negundo [6]. VJC, 55(6), 2017 Triterpenes from Vitex limonifolia. 717 Table 1: The 1H- and 13C-NMR data for compounds 1-4 C 1 2 3 4 C # δC a,c δH a,d (J in Hz) δC ¥ δC a,c δH a,d (J in Hz) δC $ δC b,c δH b,d (J in Hz) δC £ δC b,c δH b,d (J in Hz) 1 42.2 41.7 1.13 (m) 1.41 (m) 42.6 41.6 1.15 (m) 1.39 (m) 43.0 42.3 1.32 (m) 1.85 (m) 46.8 48.2 0.92 (m) 1.95 (m) 2 66.7 64.7 3.77 (br d, 11.0) 66.2 64.7 3.77 (br d,11.0) 66.3 67.0 4.00 (dt, 4.0, 11.0) 68.8 69.5 3.64 (dt, 4.5, 10.0) 3 79.2 77.8 3.15 (br s) 79.3 77.9 3.15 (d, 1.0) 79.8 80.1 3.37 (br s) 83.8 84.4 2.93 (d, 10.0) 4 39.3 38.0 - 38.9 38.0 - 39.3 39.5 - 39.1 40.6 - 5 48.3 47.6 1.11 (m) 48.8 47.6 1.15 (dd, 4.0, 13.5) 48.7 49.0 1.36 (m) 55.3 56.7 0.86 (m) 6 18.2 17.6 1.27 (m) 1.35 (m) 18.8 17.7 1.28 (m) 1.35 (m) 18.6 19.0 1.45 (m) 1.54 (m) 18.3 19.6 1.45 (m) 1.58 (m) 7 33.0 32.6 1.25 (m) 1.42 (m) 33.7 32.6 1.21 (m) 1.43 (m) 32.9 33.2 1.30 (m) 1.42 (m) 32.6 33.8 1.52 (m) 1.57 (m) 8 40.0 39.1 - 40.8 39.5 - 41.6 41.9 - 39.1 40.5 - 9 47.6 46.8 1.54 (m) 47.8 46.5 1.66 (m) 55.0 55.3 2.19 (br s) 47.5 49.0 1.65 (m) 10 38.4 37.8 - 38.8 37.8 - 38.8 39.1 - 38.3 39.3 - 11 23.4 22.9 1.85 (m) 24.3 23.1 1.89 (m) 128.0 128.2 5.71 (dd, 1.5, 10.5) 23.5 24.6 1.92 (m) 1.96 (m) 12 125.8 124.5 5.14 (t, 3.0) 128.7 126.8 5.17 (br s) 130.9 131.3 6.01 (dd, 3.0, 10.5) 122.0 123.5 5.27 (t, 3.5) 13 138.7 138.2 - 139.6 138.6 - 142.9 144.0 - 143.6 145.3 - 14 42.4 41.7 - 42.3 41.2 - 41.9 42.4 - 41.7 42.9 - 15 28.2 27.4 0.98 (m) 1.80 (m) 29.1 28.0 0.89 (m) 1.69 (m) 26.9 27.1 1.16 1.82 27.6 28.8 1.10 (m) 1.79 (m) 16 24.4 23.8 1.52 (m) 1.93 (m) 26.3 25.2 1.39 (m) 2.49 (m) 27.8 27.9 1.48 (m) 2.20 (m) 23.1 24.0 1.62 (m) 2.03 (m) 17 48.3 46.9 - 48.3 46.9 - 47.9 48.5 - 46.6 47.6 - 18 53.2 52.4 2.11 (d, 11.5) 54.4 53.2 2.37 (s) 129.2 128.4 5.41 (s) 41.3 42.7 2.87 (dd, 4.0, 14.0) 19 39.1 38.5 1.31 (m) 73.2 71.6 - 211.8 215.1 - 45.8 47.2 1.16 (m) 1.51 (m) 20 38.5 38.4 0.94 (m) 42.5 41.4 1.13 (m) 47.8 48.7 2.56 (m) 30.7 31.6 - 21 30.8 30.2 1.28 (m) 1.43 (m) 27.0 25.9 1.12 (m) 1.61 (m) 28.6 28.7 1.34 (m) 1.70 (m) 33.8 34.9 1.22 (m) 1.42 (m) 22 36.8 36.3 1.51 (m) 1.59 (m) 38.4 37.3 1.50 (m) 1.59 (m) 39.4 39.4 1.42 (m) 1.69 (m) 32.3 33.9 1.36 (m) 1.76 (m) 23 28.6 28.9 0.89 (s) 29.5 28.9 0.88 (s) 29.7 29.1 1.01 (s) 28.6 29.3 1.04 (s) 24 22.0 21.9 0.78 (s) 22.3 21.8 0.78 (s) 22.1 21.8 0.88 (s) 16.8 17.7 0.84 (s) 25 16.5 16.2 0.88 (s) 16.7 16.1 0.88 (s) 19.6 20.3 0.99 (s) 16.8 17.5 0.83 (s) 26 17.1 17.0 0.70 (s) 17.4 16.6 0.68 (s) 17.2 17.1 0.75 (s) 16.8 17.1 1.03 (s) 27 23.9 23.2 1.04 (s) 24.7 24.1 1.29 (s) 20.4 19.4 1.00 (s) 26.0 26.4 1.18 (s) 28 178.4 178.3 - 179.4 179.0 - 178.3 178.5 - 178.0 181.8 - 29 17.1 16.9 0.82 (d, 6.5) 27.2 26.4 1.08 (s) 28.4 28.3 2.16 (s) 33.1 33.6 0.93 (s) 30 21.2 21.1 0.91 (d, 7.0) 16.6 16.3 0.84 (d, 6.5) 16.7 16.5 1.10 (d, 7.0) 23.5 24.0 0.96 (s) arecorded in DMSO-d6, bCD3OD, c125MHz, d500MHz, #C of methyl 2α,3α-dihydroxy-urs-12-en-28-oate [4], ¥δC of euscaphic acid [3], $δC of 2α,3α-dihydroxy-19-oxo-18,19-seco-urs-11,13(18)-diene-28-oic acid [7], £δC of maslinic acid [8]. VJC, 55(6), 2017 Phan Van Kiem et al. 718 Compund 3 was obtained as a white powder. The 1H-NMR spectrum of 3 showed the signals of three olefinic protons at H 5.41 (s), 5.71 (dd, J = 1.5, 10.5 Hz), and 6.01 (dd, J = 3.0, 10.5 Hz), two hydroxymethine protons at H 3.37 (br s) and 4.00 (dt, J = 4.0, 11.0 Hz), and six methyl groups at H 0.75 (s), 0.88 (s), 0.99 (d, J = 6.5 Hz), 1.00 (s), 1.01 (s), 1.10 (d, J = 7.0 Hz), and 2.16 (s). The 13C-NMR and DEPT spectra of 3 displayed the signals of 30 carbons, including seven methyls at δC 16.5, 17.1, 19.4, 20.3, 21.8, 28.3, 29.1 and four olefinics at δC 128.2, 128.4, 131.3, 144.0 and one carbonyl carbon at δC 178.5. Analysis the 1H- and 13C-NMR spectra of 3 suggested 3 was very similar to those of 2α,3α- dihydroxy-19-oxo-18,19-seco-urs-11,13(18)-dien- 28-oic acid [7]. The HMBC correlations between H- 23 (δH 1.01)/H-24 (δH 0.88) and C-3 (δC 80.1); between H-3 (δH 3.37) and C-2 (δC 67.0) indicated the position of the hydroxyl group at C-3. The HMBC correlations indicated the hydroxyl group was located at C-2. The HMBC correlations from H- 27 (δH 1.00) to C-8 (δC 41.9)/C-13 (δC 144.0)/C-14 (δC 42.4)/C-15 (δC 27.1); from H-12 (δH 6.01) to C-9 (δC 55.3)/C-11 (δC 128.2)/C-13 (δC 144.0)/C-18 (δC 128.4); and from H-18 (δH 5.41) to C-12 (δC 131.3)/C-14 (δC 42.4)/C-16 (δC 27.9)/C-17 (δC 48.5)/ C-22 (δC 39.4) proved the location of two the double bond at C-11/C-12 and C-13/C-18. The HMBC correlations between H-29 (δH 2.16) and C-19 (δC 215.1)/C-20 (δC 48.7); between H-30 (δH 1.10) and C-19 (δC 215.1)/C-20 (δC 47.8)/C-21 (δC 28.7) proved the oxo group at C-19. All NMR assignments of 3 were confirmed by detailed analyses of HSQC and HMBC spectra (table 1), which are in good agreement with those reported for 2α,3α- dihydroxy-19-oxo-18,19-seco-urs-11,13(18)-dien- 28-oic acid in the literature [7], and further confirmed by the exhibition of an ESIMS ion peak at m/z 487 [M+H]+, corresponding to the formula of C30H46O5. Thus, compound 3 was identified as 2α,3α-dihydroxy-19-oxo-18,19-seco-urs-11,13(18)- dien-28-oic acid. Analysis the NMR and mass spectra of 4 indicated that the structure of 4 was similar to those of maslinic acid [8]. The HMBC correlations between two methyl groups H-23 (H 1.04)/H-24 (H 0.84) and C-3 (δC 84.4); between H-3 (H 2.93) and C-2 (C 69.5) indicated two hydroxyl groups located at C-3 and C-2. The HMBC correlations between two methyl groups H-29 (H 0.93)/H-30 (H 0.96) and C-19 (δC 47.2)/C-20 (δC 31.6)/C-21 (δC 34.9) confirmed the location of two methyl groups at C-20. The HMBC correlations between H-27 (δH 1.18) and C-8 (δC 40.5)/C-14 (δC 42.9)/C-15 (δC 28.8) proved the double bond at C-12/C-13. Consequently, 4 was elucidated as maslinic acid [8] and previously reported from V. altissima [5], and V. negundo [9]. Figure 2: The key HMBC correlations of 1 and 3 Acknowledgment. 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