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To extract, isolate, determine the structure of compounds from Huperzia serrata (Thunb.) Trevis. Subjects and methods: The samples were collected in Tamdao National Park. The structure of substances was determined by spectral methods.
Journal of military pharmaco-medicine no8-2019 TWO TRITERPENOIDS ISOLATED FROM ETHYL ACETATE FRACTION OF Huperzia Serrata (Thunb.) Trevis Le Dinh Manh1; Nguyen Van Thu2; Trinh Nam Trung2 Nguyen Duy Bac2; Pham Duc Thinh2 SUMMARY Objectives: To extract, isolate, determine the structure of compounds from Huperzia serrata (Thunb.) Trevis Subjects and methods: The samples were collected in Tamdao National Park The structure of substances was determined by spectral methods Results and conclusions: compounds of terpenoid group were isolated, namely: 3β, 21β, 29-trihydroxyserrat-14-en-24-oic acid-3β-yl- (7’-hydroxycinnamate); 16-oxo-3α-hydroxyserrat-14-en-21β-ol, the substances were confirmed by the structure of NMR, MS, HMBC, HSQC * Keywords: Huperzia serrata (Thunb.) Trevis; Terpenoid; 3β, 21β, 29-trihydroxyserrat-14en-24-oic acid-3β-yl-(7’-hydroxycinnamate); 16-oxo-3α-hydroxyserrat-14-en-21β-ol INTRODUCTION Huperzia serrata (Thunb.) Trevis belongs to the family of Lycopodiaceae, which was used as traditional medicinal plant in treatment of types of lesions, hematemesis, hematuria, hemorrhoids [1, 2] In the 1980s, scientists discovered an alkaloid compound, huperzine A The alkaloid compound has been proven to be a potent, highly specific, and reversible inhibitor of acetylcholinesterase, and potential to develop as a drug, which is used in treatment of Alzheimer symptoms [3] At Tamdao National Park (Vinhphuc province), some plants belong to genus of Huperzia were discovered, one of them is identified as Huperzia serrata (Thunb.) Trevis Moreover, this genus is distributed in wide area from north western midland and mountainous, central, to central highlands of Vietnam This study was conducted: To extract, isolate, determine the structure of compounds from Huperzia serrata (Thunb.) Trevis Results of our study provided more information about phytochemical of Huperzia serrata (Thunb.) Trevis MATERIALS AND METHODS Materials The sample was collected at Tamdao National Park (Vinhphuc province) and was identified by Dr Do Van Hai, Department of Botany, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology The sample was deposited at the same Institute o Military Medical College N Vietnam Military Medical University Corresponding author: Le Dinh Manh (manh.le40@gmail.com) Date received: 08/09/2019 Date accepted: 10/10/2019 115 Journal of military pharmaco-medicine no8-2019 Solvents and equipments - Ethanol was purchased from Vietnam as n-hexane/EtOAc (15/1 to 5/1, v/v) as eluent in column chromatography Analytical solvent such as n-hexane, HSE extract was applied to silica gel methanol, dichloromethane, ethyl acetate column was purchased from Taian Health Co Ltd, n-hexane/EtOAc (15/1 to 5/1, v/v), and China and used without further purification; monitored by TLC analysis Five sub- NMR solvents: pyridine-d5 fractions (HSEI - HSEV) were collected - The 1D and 2D nuclear magnetic chromatography, eluted with based on their TLC profiles resonance (NMR) spectra were obtained Fraction HSEII (3.6 g) was fractionated by using a Bruker Advance 500 MHz by silica gel chromatography eluted with spectrometer (Germany) ESI-MS of the n-hexane/acetone (25/1 to 0/1, v/v) to compound was recorded on AGILENT yield three sub-fraction HSEII-1 (320 mg), 1,200 series LC-MSD Ion Trap Evaporation HSEII-2 (1.2 g), and HSEII-3 (528 mg), was performed on Rotavapor R-200 respectively, based on their TLC profiles (BUCHI) Automatic collector was performed on Eyela DC-1200 from Eyela, Japan Extraction and isolation of compounds Fraction HSEII-2 was applied to silica gel chromatography, eluted with CH2Cl2/EtOAc HSE-1 (20/1, v/v) to give compound (11.2 mg) Fraction HSEII-3 The air-dried whole plants (300 g) of was subjected into MIC gel column Huperzia serrata were powdered and chromatography, eluted with 90% to extracted by Soxhlet extraction method 100% MeOH to give two sub-fraction with ethanol 96% (3 × 500 mL, hours HSEII-3-1 and HSEII-3-2, respectively each) The solvent was evaporated and Fraction HSEII-3-1 was further purified by crude extract was suspended in hot water silica gel chromatography, eluted with o (70 C) The solution was partitioned with CH2Cl2/MeOH n-hexane and ethyl acetate, respectively compound HSE-2 (9.8 mg) (75/1, v/v) to obtain Ethyl acetate layer was evaporated to give 9.0 g of crude ethyl acetate extract RESULTS AND DISCUSSIONS (HSE) 10 mg of HSE extract was dissolved into mL of methanol to determine the content by thin layer chromatography (TLC) The best solvent system was selected as n-hexane/ethyl acetate 3/1 (v/v) Thus, solvent system was chosen 116 Extraction and isolation of compounds Compound HSE-1 was isolated as a white powder, molecule formula C39H54O7, molecular weight 634.2 1H NMR and NMR data were showed in table 13 C Journal of military pharmaco-medicine no8-2019 Table 1: 1H and 13C NMR data for compound HSE-1 and reference compound a a δC δH (J in Hz) C δC 39.4 39.31 1.11, m; 1.92, m 21 27.1 27.00 1.99, m; 2.20, m 22 80.1 80.01 5.11, dd (12.0, 4.0) 49.0 48.86 57.1 57.01 21.6 45.9 37.8 37.69 63.1 62.99 10 39.6 39.51 11 27.1 12 28.2 b,≠ C δC b δC δH (J in Hz) 70.2 70.11 4.61, br s 44.0 43.89 b,≠ b 1.61, s 23 23.5 23.39 24 177.5 178.0 1.20, m 25 14.6 14.47 21.51 1.99, m; 2.15, m 26 20.3 20.18 0.91, s 45.81 1.25, m; 1.45, m 27 57.1 57.01 1.87, m; 2.33, m 28 14.6 14.47 0.87, s 0.85, m 29 65.6 65.41 3.99, d (10.8) 27.00 1.15, m; 1.85, m 30 23.5 23.39 28.07 1.94, m; 2.01, m 1’ 168.2 168.1 2.10, m 13 58.0 57.87 14 139.3 139.22 15 124.5 124.12 16 28.5 28.07 17 45.3 18 36.9 19 20 1.15, s 4.22, d (10.8) 1.60, s 2’ 116.4 116.28 6.77, d (16.0) 3’ 145.7 145.6 8.12, d (16.0) 5.51, br s 4’ 126.7 126.6 2.14, m; 2.25, m 5’ 131.2 131.05 7.56, d (8.4) 45.12 2.40, m 6’ 117.3 117.15 7.16, d (8.4) 36.74 1.85, m; 2.11, m 7’ 161.9 161.76 32.3 32.20 1.70, m; 2.05, m 8’ 117.3 117.15 7,16, d (8,4) 25.0 24.84 1.85, m; 2.11, m 9’ 131.2 131.05 7.56, d (8.4) (a: 1H NMR measured in pyridine-d5; b: ±: Deference compound) 13 C NMR measured in pyridinre-d5; Compound HSE-2 was obtained as a white powder; molecule formula C30H48O3, molecular weight 457.2 1H NMR and 13C NMR data were showed in table Table 2: 1H and 13C NMR data for compound HSE-2 and reference compound a b,≠ b C δC δC 33.8 δH a b,≠ b C δC 33.8 16 201.3 201.85 26.7 26.5 17 59.0 58.7 75.9 76.3 18 44.7 44.3 38.6 36.7 19 32.0 31.4 49.4 50.4 20 25.9 22.9 19.1 18.5 21 75.0 78.3 (J in Hz) δC δH (J in Hz) 3.05 (1H, s) 117 Journal of military pharmaco-medicine no8-2019 45.3 44.7 22 37.6 36.8 38.2 38.2 23 29.3 27.9 62.5 62.4 24 22.6 21.7 10 38.2 38.0 25 16.0 14.8 0.83 (3H, s) 11 25.2 24.9 26 20.1 20.0 0.91 (3H, s) 12 26.7 26.4 27 56.1 55.8 13 59.5 58.5 28 15.2 15.3 1.06 (3H, s) 14 163.6 164.05 29 22.2 22.4 1.39 (3H, s) 15 128.9 129.48 30 29.1 28.0 1.72 (3H, s) 5.97 (1H, s) (a: 1H NMR measured in pyridine-d5; b: Deference compound) Structural identification of isolated compounds * Compound HSE-1: The ESI-MS (positive mode) spectrum of compound HSE-1 showed a quasimolecular ion at m/z 635.1 [M+H]+, which was consistent with the molecular formula C39H54O7 In 1H NMR analysis, signal for five methyl groups at δH 0.87 (3H, s), 0.91 (3H, s), 1.15 (3H, s), 1.60 (3H, s), and 1.61 (3H, s), two oxygenated methine protons at δH 4.61 (1H, s), and 5.11 (1H, dd, J = 12.0, 4.0 Hz), and three olefinic protons at δH 8,11 (1H, d, J = 16 Hz), 7.55 (2H, d, J = 8.4 Hz), 7.15 (2H, d, J = 8.4 Hz), 6.77 (1H, d, J = 16 Hz), and 5.51 (1H, brs) were observed The 13C NMR data of compound HSE-1 showed 39 carbon signals including five methyls, ten methylene, five methines carbons, six quaternary carbons, two oxymethines carbons at δC 80.01 (C-3) and δC 70.11 (C-21), one oxymethylene at δC 65.41 (C29), and one carbonyl group at δC 178.0 (C-24), together with signals of eight 118 13 1.24 (3H, s) C NMR measured in pyridinre-d5; ± aromatic carbons, which belong to parahydroxycinnamoyl moiety (table 1) The NMR data of HSE-1 were similar with those of lycernuic acid A [4, 5], except the data at position C-3 The parahydroxycinnamate moiety was located at C-3 in HSE-1 instead of hydroxyl group To confirm the structure of HSE-1, 2D-NMR analysis (HMQC and HMBC) were performed The HMQC data were indicated all carbons bearing proton Detailed of HMBC spectrum showed the correlation between proton at δH 3.99 (1H, d, 10.8) and 4.22 (1H, d, 10.8) and carbon at δC 70.2 (C-21) This correlation indicated the location of -CH2OH moiety at carbon C-22 Moreover, the correlation from proton at δH 5.11 (H-3α) to carbon at δC 168.2 (C-1’) confirmed the location of parahydroxycinnamate moiety at C-3 Thus, the structure of HSE-1 was identified as 3β, 21β, 29-trihydroxyserrat-14-en-24-oic acid-3β-yl-(7’-hydroxycinnamate) [6], which was isolated from Lycopodiella cernua (L.) Pie.-Serm, and the structure was shown as in figure Journal of military pharmaco-medicine no8-2019 CONCLUSION * Compound HSE-2: The molecular formula was determined to be C30H48O based on positive ESI-MS data at m/z 457.2 [M + H]+ In 1H NMR analysis, signal for seven methyl groups at δH 0.74 (3H, s), 0.83 (3H, s), 0.91 (3H, s), 1.06 (3H, s), 1.24 (3H, s), 1.39 (3H, s), and 1.72 (3H, s), an oxygenated methine proton at δH 3.05 (1H, s), and an olefinic proton at δH 5.97 (1H, s) were observed The 13C NMR data of HSE-2 showed the presence of thirty carbons including a ketone carbon at δC 201.9, two olefinic carbons at δC 164.1, and 129.5, and two oxygenated methine carbons at δC 76.3, and 78.3 The NMR data of HSE-2 were similar with those of 16-oxo-3αhydroxyserrat-14-en-21β-ol [7] Thus, the structure of HSE-2 was identified as 16oxo-3α-hydroxyserrat-14-en-21β-ol, and the structure was shown in figure 21 12 O 3' O 2' 7' HO 14 24 HOOC OH CH2 30 28 16 15 H 1' 4' H 26 25 H H 23 In this study, we successed in isolation and purification of two triterpenoid compounds from the EtOAc extract of Huperzia serrata (Thunb.) Trevis by using column chromatography Structures of isolated compounds were studied using spectroscopic methods and were compared with the literatures Two compounds were identified as 3β, 21β, 29-trihydroxyserrat14-en-24-oic acid-3β-yl-(7ʹ-hydroxycinnamate) (HSE-1), and 16-oxo-3α-hydroxyserrat14-en-21β-ol (HSE-2) Compound 3β, 21β, 29-trihydroxyserrat-14-en-24-oic acid-3βyl-(7ʹ-hydroxycinnamat) (HSE-1) was first isolated from this plant REFERENCES Vo V.C Dictionary of Vietnam medicinal plants Medical Publishing House Hanoi 1997, p.1119 Pham H.H Vietnam Tre Publishing House 1999, Vol I, p.22 Ma X, Tan C, Zhu D et al Huperzine A from Huperzia species: An ethnopharmacology review J Ethnopharmacol 2007, 113, pp.15-34 Zhang Z, Elsohly H.N, Jacob M.R et al Natural products inhibiting Candida albicans secreted aspartic proteases from Lycopodium cernuum J Nat Prod 2002, 65, pp.979-985 Tsuda Y, Isobe K, Sano T Triterpenoid chemistry IX Lycopodium triterpenoid (6) The structures of three new tetra-ols, lycocryptol, 21-epilycocryptol, and diepilycocryptol, and two new acids, lycernuic acid-a and -b Chem Pharm Bull 1975, 23, pp.264-271 Nguyen V.T, To D.C, Tran M.H et al Isolation of cholinesterase and β-secretase inhibiting compounds from Lycopodiella cernua Bioorg Med Chem 2015, 23, pp.3126-3134 Figure 1: Chemical structure of isolated compounds (HSE-1 and HSE-2) Zhou H, Tan C.H, Jiang S et al Serratene-type triterpenoids from Huperzia serrata J Nat Prod 2003, 66 (10), pp.1328-1332 119 ... in isolation and purification of two triterpenoid compounds from the EtOAc extract of Huperzia serrata (Thunb.) Trevis by using column chromatography Structures of isolated compounds were studied... ethyl acetate, respectively compound HSE-2 (9.8 mg) (75/1, v/v) to obtain Ethyl acetate layer was evaporated to give 9.0 g of crude ethyl acetate extract RESULTS AND DISCUSSIONS (HSE) 10 mg of. .. pp.3126-3134 Figure 1: Chemical structure of isolated compounds (HSE-1 and HSE-2) Zhou H, Tan C.H, Jiang S et al Serratene-type triterpenoids from Huperzia serrata J Nat Prod 2003, 66 (10), pp.1328-1332