Study on chemical constituents and biological activities from the tubers of ophiopogon japonicus (l.f.) ker - Gawl

1 MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ---------------------------- Nguyen Dinh Chung STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES FROM THE TUBERS OF OPHIOPOGON JAPONICUS (L.F.) KER-GAWL Major: Organic chemistry Code: 62.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi - 2018 2 This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Adviser 1: Assoc. Prof. Dr. Nguyen Tien Dat Adviser 2: Dr. Nguyen Van Thanh 1 st Reviewer:................................................................. 2 nd Reviewer:................................................................ 3 rd Reviewer:................................................................. The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at hour date month 2018. Thesis can be found in: - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology - National Library 1 INTRODUCTION 1. The urgency of the thesis The important role of active compounds from natural products from various sources especially derived from plants, has been confirmed from the folk medicine to modern medicine. Their effects are not only used directly as a folk medicine but as a prototype or an inportant role for the discovery and development of new drugs. Vietnam is rich a country in medicinal resources, which has a high potential of medicinal plants and has a lot of experience using this source of medication by its long tradition of medicine. According to Dictionary of Vietnamese medicinal plants, in Vietnam, there were 13000 species, in which 5000 species were used to folk medicine. This is the suggestions for us to study this source of medicine for the life. In the course of screening program of extracts from Vietnam medicinal plants with cytotoxic and anti-inflammatory activities, the methanolic extract of the tubers of O. japonicus showed significant cytotoxic and anti-inflammatory effects and was chosen for further in continuing studies of this thesis. Ophiopogon japonicus (L.f) Ker-Gawl (Convallariaceae) has been widely cultivated in several areas of Vietnam and is commonly used as an or namental flower and in traditional medicine. The tubers of O. japonicus are used as folk medicine and many studies have reported that they have been used to treat cough, fever, epistaxis, inlammation, respiratory disease, constipation, and gastrointestinal disorders. Previous phytochemical investigations have revealed that O. japonicus contains steroidal saponins, homoisoflavonoids, 2 polysaccharides, phenolic acids, and sesquiterpenes. Anti- inflammatory, antitumor, antidiabetic, anti-oxidant activities of O. japonicus have been reported. Therefore, thesis title was chosen to be “Study on chemical constituents and biological activities from the tubers of Ophiopogon japonicus (L.f.) Ker-Gawl.” The aim of this study was to identify the potential active compounds from O. japonicus that could contribute to the clarification of traditional medicine and increase the scientific value of this plant in Vietnam. 2. The objectives of the thesis  Study on chemical constituents from the tubers of O. japonicus;  Evaluation of biological activities of isolated compounds to find potential compounds. 3. The main contents of the thesis  Isolation and determination of chemical structures of compounds isolated from the tubers of O. japonicus;  Evaluation on the cytotoxic and anti-inflammatory activities of the isolated compounds. CHAPTER 1. OVERVIEW Overview of internal and international researches related to our study. CHAPTER 2. EXPERIMENT AND RESULTS 2.1. Plant materials The tubers of O. japonicus were collected in Feb. 2014 at Me Linh, Hanoi and identified by Prof. Tran Huy Thai, Institute of 3 Ecology and Biological Resources, Vietnam Academy of Science and Technology. The voucher specimens were deposited at the Department of Bioactive Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology. 2.2. Methods 2.2.1. Methods for isolation of secondary metabolites Chromatographic methods such as thin layer chromatography (TLC), column chromatography (CC). 2.2.2. Methods for determination of chemical struture of compounds Physical parameters and modern spectroscopic methods such as optical rotation ([α]D), electrospray ionization mass spectrometry (ESI-MS) and high-resolution ESI-MS (HR-ESI-MS), one/two- dimention nuclear magnetic resonance (NMR) spectra, and circular dichroism spectrum (CD). 2.2.3. Methods for evaluation of biological activities - Cytotoxic activity was evaluated against four human cancer cell lines, including A549 (human lung carcinoma), LU-1 (human lung adenocarcinoma), KB (human epidermoid carcinoma), and SK- Mel-2 (human melanoma ) by the MTT assay; - Anti-inflammatory activity of isolated compounds was assessed on the basis of inhibiting NO production in lipopolysaccharide (LPS) activated RAW264.7 cells. 4 2.3. Extraction and isolation of compounds from O. japonicus The air-dried and powdered tubers of O. japonicus (2.4 kg) Extract with MeOH (5L×3 times) Remove solvent MeOH extract (360 g) Partition with CHCl3 (3L×3 time) CHCl3 fraction OJC1.2 (8.6 g) Warter layer OJW Diaion HP-20 CC, Water/Methanol (100:0- 0:100) OJW 1.3 OJW 1.5 OJW 1.4 100:0 75:25 0:100 Figure 3. Isolation and extraction from the tubers of O. japonicus OJC21.7 (264.9 mg) YMC RP-18 CC, Acetone:H2O (1.5:1) CHCl3 fraction OJC1.2 (8.6 g) Silica gel CC, n-Hexane:EtOAc (100:0-0:100) OJC17.1 (2.46 g) OJC17.3 (986 mg) OJC17.4 OJC17.5 OJC17.9 Silica gel CC, n-Hexane:EtOAc (20:1) OJ-5 (69.5 mg) OJC21.1 OJC21.4 (116.4 mg) OJC21.9 (50.5 mg) YMC RP-18 CC, Acetone:H2O (2:1) OJ-4 (18.2 mg) Silica gel CC, n-Hexane:acetone (8:1) OJ-2 (20.8 mg) c Figure 4. OJ-2, OJ-4, and OJ-5 compounds isolated from CHCl3 fraction 5 YMC RP-18 CC, Acetone:H2O (1.5:1) OJC 17.3 (986 mg) Silica gel CC, n-Hexane:EtOAc (9:1) OJC19.1 (351.9 mg) OJC19.2 (60 mg) OJC19.3 (197 mg) OJC19.4 YMC RP-18 CC, Acetone:H2O (1:1) OJ-7 (125.7 mg) OJC20.1 (70.9 mg) OJC20.2 (162.3 mg) YMC RP-18 CC, Acetone:H2O (2:1) OJ-9 (46.3 mg) YMC RP-18 CC, Acetone:H2O (1:1) Silica gel CC, n-Hexane:acetone (7:1) OJ-6 (40.4 mg) OJ-8 (31.5 mg) Kết tinh, Rửa tủa bằng n- Hexane:CH2Cl2 (3:1) Figure 5. OJ-6 - OJ-9 compounds isolated from OJC17.3 fraction OJW1.5 (11.8 g) Silica gel CC, CHCl3:MeOH (100:0-0:100) OJW2.1 (200 mg) OJW2.4 (455 mg) OJW2.5 (585 mg) OJW2.6 (3.6 g) OJW2.9 c 1. Silica gel CC, n-Hexane:EtOAc (12:1) 2. Silica gel CC, n-Hexane:CH2Cl2:Acetone (15:1:0.1) OJ-1 (30.1 mg) Silica gel CC, CH2Cl2:MeOH (10:1) OJW9.1 OJW9.2 (1.39 g) OJW9.3 (868 mg) 1. Sephadex LH-20 CC, Methanol:H2O (1:1) 2. YMC RP-18 CC, Methanol:H2O (1:2) OJ-12 (45.7 mg) Sephadex LH-20 CC, Methanol:H2O (1:1) OJ-15 (365.7 mg) Figure 6. OJ-1, OJ-12, and OJ-15 compounds isolated from OJW1.5 6 OJW2.4 (455 mg) YMC RP-18 CC, MeOH:H2O (1:1) OJW 4.1 OJW4.2 (49.3 mg) OJW 4.3 OJW 4.4 (152.5 mg) Silica gel CC, CH2Cl2:Acetone (10:1 và 6:1) OJ-3 (20 mg) Sephadex LH-20 CC, Methanol:H2O (1:1) OJ-11 (15.5 mg) OJ-10 (35.5 mg) Figure 7. OJ-3, OJ-10, and OJ-11 compounds isolated from OJW2.4 OJW2.5 (585 mg) YMC RP-18 CC, Methanol : H2O (1:1) OJW12.1 OJW12.4 (40 mg) OJW12.5 OJW12.6 (26.6 mg) Sephadex LH-20 CC, Methanol:H2O (1.5:1) OJ-13 (13.9 mg) Silica gel CC, CH2Cl2:Methanol (20:1) OJ-14 (10.1 mg) Figure 8. OJ-13 and OJ-14 compounds isolated from OJW2.5 subfraction 7 2.4. Physical properties and spectroscopic data of the isolated compounds This section presents physical properties and spectroscopic data of 15 compounds isolated from O. japonicus. 2.5. Results on biological activities of isolated compounds 2.5.1. Results on cytotoxic activity of compounds 15 compounds (OJ-1 ‒ OJ-15) were evaluated for their cytotoxic activity against four human cancer cell lines, including human lung carcinoma (A549), human lung adenocarcinoma (LU-1), human epidermoid carcinoma (KB), and human melanoma (SK-Mel- 2) by MTT assay. Table 14. Cytotoxic effects of compounds OJ-1 ‒ OJ-15 (IC50, μM) Compounds LU-1 KB SK-Mel-2 A549 OJ-1 10,90 8,86 14,01 - OJ-2 >30 >30 >30 - OJ-3 >30 >30 29,00 - OJ-4 >30 >30 >30 - OJ-5 >30 >30 >30 - OJ-6 0,66 0,51 0,66 6,26 OJ-7 17,14 >30 28,29 - OJ-8 27,66 >30 >30 - OJ-9 >30 >30 >30 - 8 OJ-10 >30 >30 20,38 - OJ-11 >30 >30 >30 - OJ-12 >30 >30 >30 - OJ-13 >30 >30 >30 - OJ-14 >30 >30 >30 - OJ-15 >30 28,84 24,29 - Ellipticine 0,43 0,51 0,27 - Camptothecin - - - 12,4 Ellipticine and camptothecin were used as the positive controls. Table 15. Effects of compounds OJ-1 – OJ-15 on the LPS-induced NO production on RAW264.7 cells (IC50, μM). Hợp chất OJ-1 OJ-2 OJ- 3 OJ- 4 OJ- 5 OJ-6 OJ- 7 OJ-8 IC50 (μM) 11,4 29,1 >30 >30 >30 22,5 19,3 >30 Hợp chất OJ-9 OJ- 10 OJ- 11 OJ- 12 OJ- 13 OJ- 14 OJ- 15 Card.* IC50 (μM) >30 >30 >30 >30 >30 >30 >30 2,80 * Cardamonin was used as a positive control. 9 CHAPTER 3. DISCUSSIONS 3.1. Chemical structure of compounds from the tubers of O. japonicus This section presents the detailed results of spectral analysis and structure determination of 15 compounds isolated from O. japonicus. Figure 9. Structures of compounds 1–15 isolated from O. japonicus 10 Detailed methods for determination of chemical structure of a new compound was showed as bellowing: 3.1.1. Compound OJ-1: (2R)-(4-methoxybenzyl)-5,7-dimethyl-6-hy droxyl-2,3-dihydrobenzofuran (New compound) Figure 10. Structure of OJ-1 and the important HMBC correlations Figure 12. 1 H NMR spectrum of OJ-1 Compound OJ-1 was obtained as a brown solid. Its molecular formula was determined to be C18H20O3 from highresolution electrospray ionisation mass spectrometry (HRESIMS m/z 283.1365 [M − H]−). Its 1H NMR spectrum showed the characteristic resonance of an AA′BB′ aromatic ring [δH 7.19 (2H, d, J = 8.5 Hz, H-2′ and H-6′), and 6.86 (2H, d, J = 8.5 Hz, H-3′ and H- 11 5′)], an aromatic singlet [δH 6.67 (1H, s, H-4)], an oxygenated methine proton [δH 4.86 (partially overlapped with HDO signal, H- 2)], one methoxyl group [δH 3.78 (3H, s, 4′-OMe)], two methylene groups [δH 3.07 (1H, dd, J = 15.0, 8.5 Hz, H-3a), 2.82 (1H, dd, J = 15.0, 7.5 Hz, H-3b), 3.02 (1H, dd, J = 14.0, 7.0 Hz, H-7′a), 2.84 (1H, dd, J = 14.0, 6.5 Hz, H-7′a)] and two aromatic methyl groups [δH 2.12 (3H, s, Me-5) and 2.05 (3H, s, Me-7)] (Table 3). Figure 13. 13 C NMR spectrum of OJ-1 The 13 C NMR and DEPT spectra revealed the presence of two methyl carbons at δC 9.2 (7-Me) and 16.5 (5-Me), two methylene carbons at δC 35.9 (C-3) and 42.0 (C-7′), a methoxy carbon at δC 55.7 (4′-OMe), an oxygenated methine carbon at δC 85.1 (C-2), five methine carbons at δC 123.9 (C-4), 131.4 (C-2′ and C-6′), and 114.7 (C-3′ and H-5′), and seven quaternary carbons at δC 153.8 (C-6, observed from HMBC spectrum), 158.1 (C-7a), and 159.8 (C-4′), 118.0 (C-3a), and 117.3 (C-5) [1,2]. The HMBC correlations from aromatic singlet H-4 to C-3, C-3a, C-5, C6, C-7a, and from Me-5 to 12 Table 3. NMR spectroscopic data (CD3OD, δ ppm) of OJ-1 Positions Ref [2] δC a δH b , mult. (J = Hz) 2 85.3 85.1 4.86, m 3 34.4 35.8 3.07, dd (8.5, 15.0) 2.82, dd (7.5, 15.0) 3a 118.4 118.0 - 4 121.3 123.9 6.67, s 5 118.4 117.3 - 6 152.2 153.8 - 7 104.4 108.1 - 7a 158.4 158.0 - 1' 130.4 131.2 - 2' 129.3 131.4 7.19, d (8.5) 3' 113.9 114.7 6.86, d (8.5) 4' 158.4 159.7 - 5' 113.9 114.7 6.86, d (8.5) 6' 129.3 131.4 7.19, d (8.5) 7' 40.9 42.0 3.02, dd (7.0, 14.0) 2.84, dd (6.5, 14.0) 5-Me - 16.4 2.12, s 6-Me 56.4 (OMe) - 7-Me 60.5 (OMe) 9.15 2.05, s 4'-OMe 55.1 55.6 3.78, s a125 MHz, b500 MHz. δC of 6,7-dimethoxy-2-(4-methoxylbenzyl)-2,3-dihydrobenzo furan theo [2]. C-4, C-5, C-6, as well as from Me-7 to C-6, C-7, and C-7a indicated the presence of a dihydrobenzofuran skeleton with a hydroxyl group 13 located at C-6 and two methyl groups located at C-5 and C-7. The methoxyl group was placed on C-4′ based on the HMBC correlation of the proton of this group with C-4′ (Figure 10). From these data, OJ-1 was identified as 2-(4-methoxybenzyl)-5,7-dimethyl-6-hydro xyl-2,3-dihydrobenzofuran. Figure 17. Experimental and calculated CD spectrum for OJ-1 The quantum chemical electronic circular dichroism (ECD) calculation method, based on time-dependent density functional theory (TDDFT), was used to determine of the absolute coniguration at C-2 [3]. The predicted ECD patterns for 2R were consistent with the experimentally measured ECD of OJ-1 (Figure 17). Thus, compound OJ-1 was assigned as (2R)-(4-methoxybenzyl)-5,7-dime thyl-6-hydroxyl-2,3-dihydrobenzofuran. 14 3.1.2. Compound OJ-7: Homoisopogon B Figure 55. Structure of OJ-7 and the important HMBC correlations Figure 57. 1 H NMR spectrum of OJ-7 Compound OJ-7 was obtained as a yellow powder with the molecular formula C19H22O4, which was established from the HRESIMS data (m/z 315.1602 [M + H] + ). The 1 H NMR spectrum showed characteristic resonances at δH 4.06 (1H, dd, J = 2.0, 11.0 Hz and 3.83 (1H, dd, J = 6.0, 11.0 Hz) corresponding to H-2 protons, δH 2.25 (1H, m) corresponding to H-3, δH 2.80 (1H, dd, J = 5.5, 16.0 Hz) and 2.44 (1H, dd, J = 6.5, 16.0 Hz) corresponding to H-4 protons, and δH 2.64 (1H, dd, J = 9.0, 14.0 Hz) and 2.52 (1H, dd, J = 6.5, 14.0 Hz) corresponding to H-9 protons. The 1 H NMR spectrum also showed signals at δH 6.38 (1H, d, J = 2.5 Hz, H-3′), 6.40 (1H, d, 15 J = 2.5, 8.0 Hz, H-5′), and 6.98 (1H, d, J = 8.0 Hz, H-6′) suggesting a 1,2,4-trisubstituted pattern for the B ring. Additionally, two aromatic singlet protons at δH 6.76 (1H, s, H-5) and 6.34 (1H, s, H-8) were detected, indicating the presence of a tetrasubstituted A ring. Figure 58. 13C NMR spectrum of OJ-7 In the 13 C NMR and DEPT spectra, a methyl, two methoxyls, three methylenes, an aliphatic methine, five aromatic methines, and 7 aromatic quaternary carbons were observed. These data suggested that OJ-7 possesses a homoisoflavane skeleton [5]. The HMBC correlations from the aromatic methyl group at δH 2.10 (3H, s) to C-5 (δC 131.4), C-6 (δC 119.1), and C-7 (δC 156.7), and from the methoxy signals δH 3.74 to C-7, and δH 3.73 to C-4′ (δC 159.3), indicated that the methyl and methoxyl groups attached to C-6, C-7, and C-4, respectively. The absolute configuration of C-3 was determined to be R based on the Cotton effect at 230 nm (negative) and 285 nm (positive) in the CD analysis [5]. Accordingly, the structure of OJ-7 16 Table 9. 1 H and 13 C NMR spectroscopic data (CDCl3, δ ppm) of OJ-7 Positions Ref [4] δC a δH b , mult. (J = Hz) 2 69.9 69.2 4.06, dd (2.0, 11.0) 3.83, dd (6.0, 11.0) 3 34.1 33.2 2.25, m 4 30.3 30.2 2.80, dd (5.5, 16.0) 2.44, dd (6.5, 16.0) 4a 113.8 112.4 - 5 130.5 131.4 6.76, s 6 107.8 119.1 - 7 155.3 156.7 - 8 103.0 98.9 6.34, s 8a 154.7 152.7 - 9 37.3 31.0 2.64, dd (9.0, 14.0) 2.52, dd (6.5, 14.0) 1' 132.6 118.0 - 2' 115.1 155.0 - 3' 145.0 102.0 6.38, d (2.5) 4' 145.5 159.3 - 5' 116.6 106.1 6.40, dd (2.5, 8.0) 6' 120.4 131.5 6.98, d (8.0) 6-CH3 - 15.3 2.10, s 7-OCH3 55.2 3.74, s 4'-OCH3 56.0 55.3 3.73, s a125 MHz, b500 MHz. δC of 7-hydroxy-3-(3-hydroxy-4-methoxybenzyl)chroman [4]. was elucidated as (3R)-4′,7-dimethoxy-2′-hydroxy-6-methylhomoiso flavane, named homoisopogon B. 17 3.2. Biological activities of isolated compounds 3.2.1. Cytotoxic activity of compounds 1–15 Compounds 1–15 were evaluated for their cytotoxic effect against LU-1, KB, and SK-Mel-2 cells. As the results showed in Table 14, compounds OJ-1, OJ-6, OJ-7, and OJ-8 showed significant cytotoxic activity on LU-1, in which OJ-6 had the strongest cytotoxic activity with an IC50 = 0.66 µM. Compounds OJ- 1, OJ-6, and OJ-15 showed significant cytotoxic activity on KB cells, in which OJ-1 showed the hightest cytotoxic activity with an IC50 = 0.51 µM. Moderate cytotoxic activities were observed with OJ-1, OJ-3, OJ-6, OJ-7, OJ-10, and OJ-15 on SK-Mel-2 cells. Interestingly, homoisopogon A (OJ-6) exhibited a strong cytotoxic effect on all tested cell lines with the IC50 values of 0.51–0.66 µM. The activity is comparable to that of the positive control, ellipticine. The cytotoxic effect of homoisoflavonoids has been indicated elsewhere, and the structure-activity relationship has been investigated. This is the first time to evaluate cytotoxic effects of series of homoisoflavonoids was reported on human cell lines at low concentrations. Compound OJ-7 showed moderate cytotoxic activity on all tested cell lines with the IC50 values ranging 17.14 to 32.94 µM. Additionally, homoisopogon C (OJ-8) exhibit significant cytotoxicity toward LU-1 cells, with an IC50 value of 27.66 µM. Accordingly, the 2′-hydroxy and 4′-methoxy groups seem to have a contribution to the activity. In my study, compounds possessing 2′- hydroxy and 4′-methoxy substituent showed positive effect on at least one cancer cell line. Homoisopogon D (OJ-9) with a methylenedioxy group at C-3′–C-4′ and lack of hydroxyl group at C- 2′, was inactive against all tested cells. 18 The results showed that weak or no effects of benzofuran derivatives were evident on all three cancer cell lines, but OJ-1 exhibited cytotoxic activity on all three tested cell lines (Table 14). These results indicate that the presence of 2R configuration in 2- benzylbenzofuran skeleton may significantly activity. Figure 115. Apoptotic effect of homoisopogon A in A549 cells analyzed with Annexin V-FTIC/PI assay after 24h treatment. Due to homoisopogon A (OJ-6) showed strongly cytotoxic activity on LU-1 cells, thus, we continued to further investigate the mechanism of action of this compound in A549 cells. As the results, homoisopogon A exhibited strong cytotoxic effect to the wild type of EGFR-TKI-resistant A549 cells (IC50 = 6.26 ± 0.79 μM), which was more potent than to the positive control, camptothecin (IC50 = 12.42 ± 0.56 μM). Also, homoisopogon A exhibited strong cytotoxicity toward two other cell lines NCI-H1975 and NCI-H1650. As showed in Figure 115, homoisopogon A induced apoptosis potently at two investigated concentrations after 24h of treatment. Homoisopogon A treatment of A549 cells at the concentration of 25 μM generated apoptosis in 27.5% of cells (7% % a p o p to ti c ce ll s 19 early apoptosis and 20.5% late apoptosis). The effect increased significantly at the concentration of 50 μM, the homoisoflavanone generated apoptosis in 83.8% of cells (23.5% early apoptosis and 60.3% late apoptosis). The movement of cells strongly suggested the cells underwent the apoptosis by treatment of homoisopogon A. The results strongly suggested that homoisopogon A induces apoptosis in EGFR and TKI-resistant-A549 cells, thus resulting in the cytotoxicity. 3.2.2. Anti-inflammatory activity of compounds The isolated compounds 1–15 were tested for their ab