Summary Of Chemistry Doctoral Thesis: Study on chemical constituents and biological activities from the tubers of Ophiopogon Japonicus (L.F.) KER-GAWL

The research objectives of the dissertation isolate and determine the structure of clean compounds from mondo grass root roots. Evaluate cytotoxic effects and anti-inflammatory activity based on the effect of inhibiting NO production of isolated compounds.

1 MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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 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 1st Reviewer: 2nd Reviewer: 3rd 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 INTRODUCTION 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, 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 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 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 OVERVIEW Overview of internal and international researches related to our study CHAPTER 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 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/twodimention 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 SKMel-2 (human melanoma ) by the MTT assay; - Anti-inflammatory activity of isolated compounds was assessed on the basis of inhibiting NO lipopolysaccharide (LPS) activated RAW264.7 cells production in 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) Warter layer OJW CHCl3 fraction OJC1.2 (8.6 g) Diaion HP-20 CC, Water/Methanol (100:00:100) 75:25 100:0 OJW 1.3 0:100 OJW 1.5 OJW 1.4 Figure Isolation and extraction from the tubers of O japonicus CHCl3 fraction OJC1.2 (8.6 g) Silica gel CC, n-Hexane:EtOAc (100:0-0:100) … OJC17.1 OJC17.3 OJC17.5 OJC17.4 … OJC17.9 (986 mg) (2.46 g) Silica gel CC, n-Hexane:EtOAc (20:1) OJC21.1 … OJC21.4 … c (116.4 mg) YMC RP-18 CC, Acetone:H2O (2:1) OJC21.7 (264.9 mg) YMC RP-18 CC, Acetone:H2O (1.5:1) OJ-5 (69.5 mg) … OJC21.9 (50.5 mg) Silica gel CC, n-Hexane:acetone (8:1) OJ-4 (18.2 mg) OJ-2 (20.8 mg) Figure OJ-2, OJ-4, and OJ-5 compounds isolated from CHCl3 fraction OJC 17.3 (986 mg) Silica gel CC, n-Hexane:EtOAc (9:1) OJC19.1 OJC19.2 OJC19.3 (351.9 mg) (60 mg) (197 mg) OJ-8 (31.5 mg) OJC20.1 OJC20.2 (70.9 mg) (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) YMC RP-18 CC, Acetone:H2O (1.5:1) YMC RP-18 CC, Acetone:H2O (1:1) OJC19.4 OJ-6 (40.4 mg) Kết tinh, Rửa tủa nHexane:CH2Cl2 (3:1) OJ-7 (125.7 mg) Figure 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) … c OJW2.4 OJW2.5 OJW2.6 (455 mg) (585 mg) (3.6 g) Silica gel CC, n-Hexane:EtOAc (12:1) Silica gel CC, n-Hexane:CH2Cl2:Acetone (15:1:0.1) … OJW2.9 Silica gel CC, CH2Cl2:MeOH (10:1) OJ-1 (30.1 mg) OJW9.1 OJW9.2 (1.39 g) Sephadex LH-20 CC, Methanol:H2O (1:1) YMC RP-18 CC, Methanol:H2O (1:2) OJ-12 (45.7 mg) OJW9.3 (868 mg) Sephadex LH-20 CC, Methanol:H2O (1:1) OJ-15 (365.7 mg) Figure OJ-1, OJ-12, and OJ-15 compounds isolated from OJW1.5 OJW2.4 (455 mg) YMC RP-18 CC, MeOH:H2O (1:1) OJW4.2 OJW 4.1 OJW 4.3 OJW 4.4 (152.5 mg) (49.3 mg) Sephadex LH-20 CC, Methanol:H2O (1:1) Silica gel CC, CH2Cl2:Acetone (10:1 6:1) OJ-3 (20 mg) OJ-10 (35.5 mg) OJ-11 (15.5 mg) Figure 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) Sephadex LH-20 CC, Methanol:H2O (1.5:1) OJ-13 (13.9 mg) OJW12.5 OJW12.6 (26.6 mg) Silica gel CC, CH2Cl2:Methanol (20:1) OJ-14 (10.1 mg) Figure OJ-13 and OJ-14 compounds isolated from OJW2.5 subfraction 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-Mel2) 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 - 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 OJ-1 OJ-2 OJ- OJ- OJ- 29,1 >30 >30 >30 22,5 19,3 OJ- OJ- OJ- OJ- OJ- OJ- 10 11 12 13 14 15 >30 >30 >30 >30 >30 >30 chất IC50 (μM) Hợp chất IC50 (μM) 11,4 OJ-9 >30 * Cardamonin was used as a positive control OJ-6 OJ- OJ-8 >30 Card.* 2,80 11 5′)], an aromatic singlet [δH 6.67 (1H, s, H-4)], an oxygenated methine proton [δH 4.86 (partially overlapped with HDO signal, H2)], 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 13C 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 NMR spectroscopic data (CD3OD, δ ppm) of OJ-1 Positions Ref [2] δCa δHb, mult (J = Hz) 85.3 85.1 4.86, m 34.4 35.8 3.07, dd (8.5, 15.0) 2.82, dd (7.5, 15.0) 3a 118.4 118.0 - 121.3 123.9 6.67, s 118.4 117.3 - 152.2 153.8 - 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 6-Me 56.4 (OMe) - 7-Me 60.5 (OMe) 9.15 2.05, s 55.1 55.6 3.78, s 4'-OMe a 2.12, s b 125 MHz, 500 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 1H 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 1H 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 1H 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 13 C NMR spectrum of OJ-7 In the 13C NMR and DEPT spectra, a methyl, two methoxyls, three methylenes, an aliphatic methine, five aromatic methines, and 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 1H and 13C NMR spectroscopic data (CDCl3, δ ppm) of OJ-7 Positions Ref [4] δCa 69.9 69.2 δHb, mult (J = Hz) 4.06, dd (2.0, 11.0) 3.83, dd (6.0, 11.0) 34.1 33.2 2.25, m 30.3 30.2 2.80, dd (5.5, 16.0) 2.44, dd (6.5, 16.0) 4a 113.8 112.4 - 130.5 131.4 6.76, s 107.8 119.1 - 155.3 156.7 - 103.0 98.9 6.34, s 8a 154.7 152.7 - 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 55.2 3.74, s 55.3 3.73, s 7-OCH3 4'-OCH3 56.0 a 125 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 OJ1, 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 C2′, was inactive against all tested cells 18 % apoptotic cells 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 2benzylbenzofuran 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% 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 ability to inhibit NO production in LPS-stimulated RAW264.7 cells Compound OJ-1 was the most active compound with an IC50 of 11.4 μM, while compound OJ-2 had a moderate effect (IC50 = 29.1 μM) 2,3-Dihydrobenzofurans have been known as potent antiinlammatory powerful compounds anti-inlammatory 2,3-Dihydrobenzofuran-2-one activity in vivo, and had 5-chloro-6- cyclohexyl-2,3-dihydrobenzofuran-2-one was significantly more potent than the reference compound, diclofenac, in all testing models [6] More recently, a series of dihydrobenzofurans was isolated from the seeds of Prunus tomentosa, some of which strongly inhibited NO production in LPS-stimulated BV-2 cells [7] Consistently, my results suggest that O japonicus is a potential natural source of antiinlammatory dihydrobenzofurans Homoisopogon A (OJ-6) and homoisopogon B (OJ-7) showed moderate effects with the IC50 of 22.5 19.3 μM, respectively Other compounds showed weak or inactive up to the highest concentration tested (30 μM) 20 CONCLUSIONS Chemical investigations Using various chromatographic methods, from the tubers of O japonicus, 15 compounds were isolated and identified 09 new compounds, 02 compounds were isolated for the first time from a natural source, and 04 know compounds, including: Benzofuran derivatives: (2R)-(4-methoxybenzyl)-5,7- dimethyl-6-hydro xyl-2,3-dihydrobenzofuran compound), 2-(2-hydroxyl-4-methoxy-benzyl)-5-methyl-6- methoxyl-2,3-dihydrobenz ofuran (OJ-1, new (OJ-2, new compound), 2-(4- hydroxy-benzyl)-5,6-dihydroxy lbenzofuran (OJ-3, new compound), 2-(4-methoxy-benzyl)-6,7-dimethoxyl-2,3-dihydrobenzofuran (OJ-4, the first time from a natural source), and 2-(4-methoxy-benzyl)-6,7methylenedioxy-2,3-dihydrobenzofuran (OJ-5, the first time from a natural source) Homoisoflavonoid derivatives: homoisopogon A (OJ-6, new compound), homoisopogon B (OJ-7, new compound), homoisopogon C (OJ-8, new compound), and homoisopogon D (OJ-9, new compound) Flavonoid derivatives: 8-C-methyl-3',5,5',7-tetrahydroxy3,4′-dimethoxy flavone (OJ-10, new compound) and myricetin 3,4'dimethyl ether (3',5,5',7-tetrahydroxy-3,4'-dimethoxyflavone, OJ11) Eudesmane sesquiterpenoid derivatives: 1α,4β,6β- trihydroxy-5,10-bis-epi-eudesmane-6-O-β-D-glucopyranoside 12), (OJ- 1α,6β-dihydroxy-5,10-bis-epi-eudesm-4(15)-ene-6-O-β-D- 21 glucopyranoside (OJ-13), and 1α,6β-dihydroxy -5,10-bis-epi- eudesm-3-ene-6-O-β-D-glucopyranoside (OJ-14) Steroidal glycoside: (25R)-ruscogenin 1-O-(4-O-sulfo)-β-Dfuco pyranoside (OJ-15, new compound) 2-Benzylbenzofuran derivatives are rare in that the 2-benzyl2,3-dihydrobenzofuran derivatives are almost exclusively isolated from nature The thesis have isolated and identified the chemical structures of the five compounds of this class Along with the new compounds belonging to other groups, these are also the new highlights of the thesis contributing to the database of chemical compounds of natural products Biological activity The in vitro cytotoxic activity of the isolated compounds from O japonicus was investigated on four human cancer cell lines, human lung carcinoma (A549), human lung adenocarcinoma (LU-1), human epidermoid carcinoma (KB), and human melanoma (SK-Mel2) The results indicated that homoisopogon A (OJ-6) exhibited potent cytotoxic effects against three tested human cancer cell lines (LU-1, KB, and SK-Mel-2) with the IC50 values ranging from 0.51 to 0.66 µM, relative to the effects of the postitive control, ellipticine Additionally, these results showed that this compound inducted the apoptosis on A549 cells at the concentrations 25 and 50 μM Compound OJ-1 exhibited potent cytotoxic activity on all four tested cell lines with IC50 values ranging from 8.86 to14.0 µM The isolated compounds were tested for their ability to inhibit NO production in LPS-stimulated RAW264.7 cells 22 Interestingly, the benzofuran derivatives were the most active compounds Among them, compound OJ-1 was the most active compound with an IC50 = 11.4 μM, while OJ-2 had a moderate effect with an IC50 = 29.1 μM Two homoisoflavonoids, homoisopogon A (OJ-6) and homoisopogon B (OJ-7) inhibited NO production with the IC50 of 22.5 and 19.3 μM, respectively RECOMMENDATIONS The results indicated that compounds isolated from O japonicus may be a potential material for the development of anticancer and anti-inflammatory agents Homoisopogon A (OJ-6) showed potent anti-proliferation on cancer cell line Further studies to clarify activity mechanism and pharmacoglogical study of this compound should be carried NEW FINDINGS OF THE THESIS From the tubers of O japonicus, 15 compounds were isolated and identified including: - 09 new compounds: (2R)-(4-methoxybenzyl)-5,7-dime thyl-6-hydroxyl-2,3-dihydrobenzofuran (OJ-1), 2-(2-hydroxyl-4-me thoxy-benzyl)-5-methyl-6-me thoxyl-2,3-dihydrobenzofuran (OJ-2), 2-(4-hydroxy-benzyl)-5,6-dihydroxyl benzofuran (OJ-3), homoisopogon A (OJ-6), homoisopogon B (OJ-7), homoisopogon C (OJ-8), homoisopogon D (OJ-9), 8-C-methyl-3',5,5',7-tetrahydroxy3,4′-dimethoxyflavone (OJ-10), and (25R)-ruscogenin 1-O-(4-Osulfo)-β-D-fucopyranoside (OJ-15) 23 - 02 compounds were isolated for the first time from a natural source: 2-(4-methoxy-benzyl)-6,7-dimethoxyl-2,3-dihydro benzofuran (OJ-4) and 2-(4-methoxy-benzyl) -6,7-methylenedioxy2,3-dihydrobenzofuran (OJ-5) The in vitro cytotoxic activity of the isolated compounds from O japonicus was investigated on four human cancer cell lines, human lung carcinoma (A549), human lung adenocarcinoma (LU-1), human epidermoid carcinoma (KB), and human melanoma (SK-Mel2) The results indicated that homoisopogon A (OJ-6) exhibited potent cytotoxic effects against three tested human cancer cell lines (LU-1, KB, and SK-Mel-2) with the IC50 values ranging from 0.51 to 0.66 µM, relative to the effects of the postitive control, ellipticine These results indicate that this compound inducted the apoptosis on A549 cells at different concentrations (25 and 50 μM) Compound OJ-1 exhibited potent cytotoxic activity on all four tested cell lines with the IC50 values ranging from 8.86 to14.0 µM The isolated compounds were tested for their ability to inhibit NO production in LPS-stimulated RAW264.7 cells Among them, the benzofuran derivatives were the most active compounds Compound OJ-1 was the most active compound with an IC50 = 11.4 μM, while OJ-2 had a moderate efect with an IC50 = 29.1 μM Two homoisoflavonoids, homoisopogon A (OJ-6) and homoisopogon B (OJ-7) inhibited NO production with the IC50 of 22.5 and 19.3 μM, respectively 24 REFERENCES Ortega, N.; Urban, S.; Beiring, B.; Glorius, F., Ruthenium NHC catalyzed highly asymmetric hydrogenation of benzofurans Angewandte Chem Int Edition 2012, 51, 1710−1713 Qi, P.; Jiang, J.; Qi, H.; Jin, Y.; Shen, Q.; Wu, Y.; Song, H.; Zhang, W., Synthesis and antiproliferative activity of new polyoxo 2-benzyl-2,3-dihydrobenzofurans and their related compounds Lett Drug Des Dis 2013, 10, 886−894 Kwon, J.; Hiep, N T.; Kim, D W.; Hong, S.; Guo, Y.; Hwang, B Y.; Lee, H J.; Mar, W.; Lee, D., Chemical constituents isolated from the root bark of Cudrania tricuspidata and their potential neuroprotective effects J Nat Prod., 2016, 79, 1938−1951 Masaoud, M.; Ripperger, H.; Porzel, A.; Adam, G., Flavonoids of dragon's blood from Dracaena cinnabari Phytochemistry 1995, 38(3), 745-749 Awale, S.; Miyamoto, T.; Linn, T Z.; Li, F.; Win, N N.; Tezuka, Y.; Esumi, H.; Kadota, S., Cytotoxic Constituents of Soymida febrifuga from Myanmar J Nat Prod., 72(9), 1631–1636 Closse, A.; Haefliger, W.; Hausser, D.; U.Gubler, H.; Dewald, B.; Baggiolini, M., 2,3-Dihydrobenzofuran-2-ones: A new class of highly potent antiinflammatory agents J Med Chem., 1981, 24, 1465−1471 Liu, Q.-B.; Huang, X.-X.; Bai, M.; Chang, X.-B.; Yan, X.-J.; Zhu, T.; Zhao, W.; Peng, Y.; Song, S.-J., Antioxidant and antiinflammatory active dihydrobenzofuran neolignans from the seeds of Prunus tomentosa J Agri Food Chem., 2014, 62, 7796– 7803 25 PUBLICATIONS WITHIN THE SCOPE OF THESIS Nguyen Dinh Chung, Nguyen Phuong Thao, Ha Manh Tuan, Nguyen Van Thanh, Nguyen Hai Dang, Nguyen Tien Dat New steroidal glycoside and flavonoid constituents from Ophiopogon japonicus Nat Prod Commun., 2017, 12(6), 905–906 Nguyen Hai Dang, Nguyen Dinh Chung, Ha Manh Tuan, Nguyen Tuan Hiep, Nguyen Tien Dat Cytotoxic homoisoflavonoids from Ophiopogon japonicus tubers Chem Pharm Bull., 2017, 65(2), 204–207 Nguyen Hai Dang, Nguyen Dinh Chung, Ha Manh Tuan, Nguyen Van Thanh, Nguyen Tuan Hiep, Dongho Lee, and Nguyen Tien Dat 2-Benzyl-benzofurans from the tubers of Ophiopogon japonicus Chem Cent J., 2017, 11:15 Nguyen Dinh Chung, Nguyen Tien Dat, Le Thi Van Anh, Jeong-Hyung Lee, Nguyen Hai Dang Homoisopogon A from Ophiopogon japonicus induced apoptosis in A549-a non small cell lung cancer cell line Vietnamese J Biotechnol., 2017, 15(1), 35–39 Nguyen Dinh Chung, Nguyen Hai Dang, Nguyen Van Thanh, Nguyen Tien Dat Eudesmane sesquiterpene glucosides from Ophiopogon japonicus Vietnamese J Sci Technol., 2015, 53(4C), 56–62 ... to the clarification of traditional medicine and increase the scientific value of this plant in Vietnam The objectives of the thesis  Study on chemical constituents from the tubers of O japonicus; ... 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... 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)