Summary of Chemistry Doctoral thesis: Study on chemical constituents and cytotoxic activities of Glochidion Glomerulatum and Glochidion Hirsutum growing in study on chemical constituents

Objectives of the thesis: Research to clarify the main chemical composition of two species of G. glomerulatum, G. hirsutum in Vietnam. Evaluation of toxic activity cells of isolated compounds to search for bioactive compounds, as a scientific basis for further research to create care products health for the community and contribute to explaining the curative effect of these species.

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY - NGUYEN VAN THANG STUDY ON CHEMICAL CONSTITUENTS AND CYTOTOXIC ACTIVITIES OF GLOCHIDION GLOMERULATUM AND GLOCHIDION HIRSUTUM GROWING IN VIETNAM Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi - 2018 This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Advisors 1: Asc Prof Dr Phan Van Kiem Advisors 2: Dr Vu Kim Thu 1st Reviewer: Prof Dr Nguyen Van Tuyen 2nd Reviewer: Asc Prof Dr Tran Thu Huong 3rd Reviewer: Asc Prof Dr Nguyen Thi Mai 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 INTRODUCTION The rationale of the thesis According to The World Health Organization (WHO), there are approximate 80 percent of population relied on traditional medicines, especially the medicinal plants in initial health care In the research and development process of drugs, experience of using traditional medicines is one of the most important factors that create the increasing in the success rate of searching for leading compounds through reducing time consuming, saving costs and being less harmful to living bodies Therefore, medicinal plants are always considered as an attractive subject that significantly stimulates the attention of scientists worldwide According to the Dictionary of Vietnamese medicinal plants, Glochidion in Vietnam has many species used as drugs and medicine for treatment of diseases such as: Glochidion daltonii cures bacillary dysentery; Glochidion eriocarpum Champ cures inflammatory bowel and dysentery, allergic contact dermatitis, itching, psoriasis, urticarial (hives), and eczema; At the Institute of Medicinal Materials, Leaves of Glochidion hypoleucum are used to strengthen tendons and bones and recover wound; Glochidion hirsutum is often used to cure diarrhea, indigestion, abdominal bloating, and its leaves are used for snake bites, etc Researches on chemical compositions show that Glochidion contains many layers of interested substances such as terpenoids, steroids, megastigmane, flavonoid, lignanoid and some other phenolic forms Biological evaluation studies show that the extracts and compounds isolated from these species have interested activities such as cancer cytotoxic, antifungal, antimicrobial, antioxidant,… Therefore, the thesis title was chosen to be "Study on chemical constituents and cytotoxic activities of Glochidion glomerulatum and Glochidion hirsutum growing in Vietnam" The objectives of the thesis Study on chemical constituents of two Glochidion species including Glochidion glomerulatum and Glochidion hirsutum in Vietnam Evaluation of biological activities of isolated metabolites to find out potential compounds The main contents of the thesis Isolation of compounds from the leaves of Glochidion glomerulatum and Glochidion hirsutum; Determination of chemical structures of the isolated compounds; Evaluation of the cytotoxic activity of the isolated compounds; CHAPTER 1: OVERVIEW This chapter presents the overview of domestic and international studies related to the chemical compositions and biological activities of Glochidion CHAPTER 2: EXPERIMENT AND EMPIRICAL RESULTS 2.1 Research objective - The leaves, branches and fruits of G glomerulatum were collected in Phuc Yen, Vinh Phuc, Vietnam in September, 2012 - The leaves, branches and fruits of G hirsutum were collected in Son Dong, Bac Giang, Vietnam in December, 2012 2.2 Research Methodology 2.2.1 Methods for metabolites isolation Combining a number of Chromatographic methods including thin layer chromatography (TLC), column chromatography (CC), highperformance liquid chromatography (HPLC) 2.2.2 Methods for determination of chemical structure of compounds The general method used to determine the chemical structure of compounds is the combination between physical parameters and modern spectroscopic including optical rotation ([]D), electrospray ionization mass spectrometry (ESI-MS) and high-resolution ESI-MS (HR-ESI-MS), one/two-dimention nuclear magnetic resonance (NMR) spectra 2.2.3 Methods for evaluation of biological activities - Cytotoxic activity is determined by the MTT and SRB assay 2.3 Isolation of compounds 2.3.1 Isolation of compounds from G glomerulatum This section presents the process of isolating ten compounds from G glomerulatum Figure 2.4 Isolation of compounds from G glomerulatum 2.3.2 Isolation of compounds from G hirsutum This section presents the process of isolating five compounds from G hirsutum Figure 2.2 Isolation of compounds from G hirsutum 2.4 Physical properties and spectroscopic data of the isolated compounds 2.4.1 Physical properties and spectroscopic data of the isolated compounds from G glomerulatum This section presents physical properties and spectroscopic data of 10 compounds from G glomerulatum 2.4.2 Physical properties and spectroscopic data of the isolated compounds from G hirsutum This section presents physical properties and spectroscopic data of compounds from G hirsutum 2.5 Results on cytotoxic activities of isolated compounds 2.5.1 Results on cytotoxic activity of compounds from G glomerulatum - 10 compounds (GG1-GG10) are evaluated for their cytotoxic activities against A-549, MCF-7, OVCAR, HT-29 cells by MTT assay Table 2.1 % inhibition on cells of compounds GG1-GG10 at concentration of 100 μM Comp A-549 MCF-7 OVCAR HT-29 97,54 ± 2,06 82,28 ± 1,42 90,64 ± 1,28 95,22 ± 2,38 GG1 94,66 ± 1,22 79,69 ± 1,30 88,18 ± 0,84 93,12 ± 2,92 GG2 71,24 ± 0,52 83,25 ± 1,26 97,12 ± 2,04 92,34 ± 0,20 GG3 94,67 ± 1,62 79,86 ± 2,34 83,89 ± 2,06 91,98 ± 0,53 GG4 96,21 ± 0,72 80,34 ± 2,80 91,56 ± 1,16 96,89 ± 3,20 GG5 72,89 ± 0,56 72,15 ± 0,38 78,03 ± 1,86 77,21 ± 0,12 GG6 97,43 ± 1,02 74,38 ± 4,60 92,08 ± 3,46 99,32 ± 4,44 GG7 54,68 ± 0,21 54,89 ± 0,30 80,11 ± 2,82 81,11 ± 3,96 GG8 69,54 ± 1,08 71,02 ± 1,24 82,13 ± 0,92 87,23 ± 1,36 GG9 75,11 ± 0,96 61,34 ± 4,20 85,67 ± 1,04 79,52 ± 1,76 GG10 Table 2.2 The effects of compounds GG1-GG10 on the growth of A-549, MCF-7, OVCAR, HT-29 cells IC50 (µM) Comp A-549 MCF-7 OVCAR HT-29 9,3± 1,4 50,1± 3,2 8,9± 2,2 7,8± 1,2 GG1 10,2± 2,3 56,1± 4,3 10,6± 3,3 9,5± 1,6 GG2 41,0 ± 3,5 58,4 ± 3,7 6,6 ± 0,7 7,3 ± 1,4 GG3 9,7 ± 1,2 60,7 ± 5,2 41,5 ± 3,1 7,5 ± 1,7 GG4 7,9 ± 0,8 42,8 ± 5,2 9,8 ± 2,1 5,9 ± 0,5 GG5 58,2 ± 2,4 69,3 ± 5,2 59,4 ± 6,8 49,3 ± 3,1 GG6 8,2 ± 1,0 63,5 ± 3,6 8,6 ± 3,1 5,9 ± 0,7 GG7 94,9 ± 4,1 86,3 ± 5,2 34,1 ± 3,4 45,0 ± 2,4 GG8 58,1 ± 4,6 67,5 ± 4,8 45,8 ± 2,5 49,1 ± 4,1 GG9 51,7 ± 3,1 77,2 ± 5,5 27,7 ± 4,6 58,7 ± 3,9 GG10 7,2 ± 0,5 10,3 ± 1,2 8,4 ± 0,9 3,1 ± 0,3 ĐC* *) Mitoxantrone is used as a positive control (PC) 2.5.2 Results on cytotoxic activity of compounds from G hirsutum - compounds (GH1-GH5) are evaluated for their cytotoxic activities against A-549, MCF-7, SW-626, HepG2 cells by SRB assay Table 2.3 % inhibition on cells of compounds GH1-GH5 at concentration of 100 μM Comp GH1 GH2 GH3 GH4 GH5 A-549 90,10 ± 2,80 98,60 ± 1,64 97,44 ± 4,28 98,69 ± 2,32 96,06 ± 2,24 MCF-7 91,33 ± 1,12 98,28 ± 2,14 99,49 ± 0,98 96,86 ± 1,28 96,74 ± 3,12 SW-626 90,22 ± 3,14 98,21 ± 3,72 96,98 ± 3,34 94,14 ± 2,66 95,18 ± 1,80 HepG2 92,17 ± 1,38 99,09 ± 1,76 99,83 ± 2,38 99,39 ± 3,64 96,77 ± 4,90 Table 2.4 The effects of compounds GH1-GH5 on the growth of A-549, MCF-7, SW-626, HepG2 cells Comp A-549 9,3 ± 0,3 4,4 ± 0,7 49,3 ± 4,1 8,0 ± 2,2 8,6 ± 1,3 1,8 ± 0,3 IC50 (µM) MCF-7 SW-626 9,2 ± 0,5 8,5 ± 1,3 4,7 ± 0,6 6,6 ± 1,0 51,9 ± 3,7 54,4 ± 1,5 8,8 ± 1,3 9,1 ± 1,1 10,2 ± 2,4 10,1 ± 1,9 2,0 ± 0,3 2,1 ± 0,3 GH1 GH2 GH3 GH4 GH5 ĐC* *) Ellipticine is used as a positive control (PC) HepG2 8,2 ± 1,3 3,4 ± 0,3 47,0 ± 5,6 7,6 ± 0,8 9,9 ± 3,1 1,4 ± 0,2 CHAPTER 3: DISCUSSIONS 3.1 Chemical structure of compounds from G glomerulatum This section presents the detailed results of spectral analysis and structure determination of 10 new isolated compounds from G glomerulatum Figure 3.1 The structure of 10 compounds from G glomerulatum The detailed methods for determination of chemical structure of a new compound are introduced in the following section 3.1.1 Compound GG1: Glomeruloside I (new compound) Compound isolated GG1 was obtained as a white amorphous powder. Its molecular formula is determined to be C55H84O20 by high resolution electrospray ionization (HR-ESI)-MS (m/z 545.1995 [M+Cl] ; Calcd for [C55H84O20Cl] , 1099,5250 u) The 1H-NMR spectrum of compound GG1 shows proton signals for seven singlet methyl groups at H 0.89 (3H, s), 0.93 (3H, s), 0.99 (3H, s), 1.04 (3H, s), 1.07 (3H, s), 1.10 (3H, s) and 1.30 (3H, s); one olefinic proton at H 5,35 (1H, br s); five aromatic protons at H 8.05 (2H, d, J = 7.6 Hz), 7.49 (2H, t, J = 7.6 Hz) and 7.60 (1H, t, J = 7.6 Hz) suggest the - - existence of a phenyl group; three anomeric protons at 4.46 (1H, d, 8.0 Hz), 4.62 (1H, d, 7.6 Hz), 4.86 (1H) indicate there is an appearance of three sugar moieties The 1H NMR data of anomeric protons, seven singlet methyl groups in aglycone and the presence of multiple protons at upfield (δH 0.81 ~ 2.46) can be suggested that this is an oleane-type saponin Figure 3.2 Chemical structures of compound GG1 and reference compoud GG1A Figure 3.3 HR-ESI-MS spectrum of GG1 Figure 3.4 1H-NMR spectrum of GG1 The 13C-NMR and DEPT spectra of GG1 revealed signals of 55  carbons which is divived into carbonyl group, quaternary carbons, 27 methines, 12 methylenes and methyl carbons Among them, 30 carbons belong to triterpene skeleton, 18 carbons belong to hexose sugar 11 Figure 3.9 GC analysis of standard sugar samples and sugar moieties after acid hydrolysis of GG1 a) GC analysis of L – glucose c) GC analysis of D – glucose b) GC analysis of L – galactose d) GC analysis of D – galactose e) GC analysis of sugar moieties after acid hydrolysis of GG1 Next, the spectroscopic data of sugar moieties in compound GG1 were done by 13C-NMR, COSY, HSQC, HMBC experiments and acid hydrolysis of GG1 was analyzed by GC The result of acid hydrolysis and GC analysis showed that GG1 contained two sugar units with retention time at tR1 = 14.098 and tR2 = 18.713 (fig 3.9e), which is similar with that of reference D-glucose at tR = 14,106 (fig 3.9b) and D-galactose reference at tR = 18.706 (fig 3.9d), suggested the presence of D-glucose and D-galactose sugar moieties The HMBC correlation between Gal H-1 (δH 4.46, d, J = 8.0 Hz) and aglyone C-3 (δC 91.90), the COSY correlations at Gal H-1/ Gal H-2/ Gal H-3/ Gal H-4/ Gal H-5 were observed The results indicated that the sugar unit to be galactose with the location of sugar moiety being at C-3 The HMBC correlations between Glc I H-1 (δH 4.86) and Gal C-2 (δC 76.40), and COSY correlations at Glc I H-1/Glc I H-2/Glc I H-3/ 12 Glc I H-4/ Glc I H-5/ Glc I H-6 indicate that the sugar unit to be Glc I and the linkage of sugar moities to be Glc I-(1→2)-Gal Spectroscopic data of carbon at Glc II (δC 105.24, 75.28, 77.32, 71.17, 78.07, 62.40) and HMBC correlations between Glc II H-1 (δH 4.62) and Gal C-3 (δC 85.25) indicate that sugar linkage to be Glc II-(1→3)-Gal From above evidence, the trisaccharide linkages were confirmed to be 3-O-β-Dglucopyranosyl (1→3)-[β-D-glucopyranosyl (1→2)]-β-Dgalactopyranoside Figure 3.10 The key COSY, HMBC and ROESY correlations of GG1 The configurations of functional groups of aglycone of GG1 were further confirmed by ROESY experiments The β-orientation of protons H-25, H-26, H-18, H-30 were determined from observation of ROESY correlations between H-25/H-26, H-18/H30 Similarly, the αorientation of protons H-5/H-9/H-27 were deterined from ROESY observations The α-orientation of H-3, H-5 were determined by observation of ROESY correlations between H-3 (δH 3.22) and H-5 (δH 0.81) Morever, the α-orientation of H-16, H-22 were confirmed by observation of ROESY correlations between H-22 (δH 5.91) and H-16 (δH 4.32), and without observation of ROESY correlation between H-18 (δH 2.46) and H-22 (δH 5.91)/H-16 (δH 4.32) From above evidence, the chemical structure of GG1 was elucidated to be 22β-benzoyloxy3β,16β,28-trihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1→3)-[β-Dglucopyranosyl (1→2)]-β-D-galactopyranoside This is a new compound 13 and named as Glomeruloside I The 1H and 13C-NMR spectroscopic data of GG1 were summarized in table 3.1 Table 3.1 NMR spectroscopic data for GG1 and reference compound C 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 # Ca,b 40.08 27.20 90.50 40.40 57.08 19.44 33.77 41.36 48.31 37.87 24.83 124.41 143.61 44.38 37.72 69.60 44.97 43.59 47.31 31.15 38.49 72.20 28.64 17.14 16.31 17.44 28.08 64.86 Ca,c 39.94 27.09 91.90 40.54 56.87 19.28 33.61 41.18 48.10 37.66 24.67 124.23 143.40 44.20 37.55 69.44 44.80 43.41 47.13 30.98 38.33 72.04 28.32 16.80 16.12 17.29 27.49 64.69 DEPT CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH C CH CH2 C CH2 CH CH3 CH3 CH3 CH3 CH3 CH2 29 30 22-O-Bz 1 2 6 3 5 4 7 34.48 27.65 34.32 27.49 CH3 CH3 132.31 130.61 129.79 134.24 167.33 132.10 130.43 129.62 134.09 167.18 C CH CH CH C Ha,d (mult., J, Hz) 1.02 (m)/1.65 (m) 1.76 (m)/1.96 (m) 3.22 (br d, 11.2) 0.81 (d, 11.2) 1.47 (m)/1.62 (m) 1.41 (m)/1.63 (m) 1.60 (m) 1.95 (m) 5.35 (br s) 1.52 (m)/1.98 (m) 4.32 (br d, 10.0) 2.46 (d, 12.4) 1.22 (m)/1.90 (m) 1.78 (m) 5.91 (br s) 1.10 (s) 0.89 (s) 0.99 (s) 1.07 (s) 1.30 (s) 3.68 (d, 10.8)/ 4.02 (d, 10.8) 0.93 (s) 1.04 (s) 8.05 (d, 7.6) 7.49 (t, 7.6) 7.60 (t, 7.6) - 14 C 3-O1 2 3 4 5 6 2-OGlc 1 2 3 4 5 6 3-OGlc 1 2 3 4 5 6 Ca,b Ara 107.25 72.24 84.00 69.66 66.81 # 105.53 75.47 77.80 71.33 78.04 62.52 Ca,c Glc 105.83 76.40 85.25 69.97 75.92 63.76 DEPT Ha,d (mult., J, Hz) CH CH CH CH CH CH2 4.46 (d, 8.0) 4.00 (m) 3.81 (m) 4.12 (br s) 3.55 (m) 3.54 (m)/3.83 (m) 103.51 76.05 78.32 72.53 77.86 62.34 CH CH CH CH CH CH2 4.86(m) 3.14 (t, 8.0) 3.33 (m) 3.08 (t, 8.0) 3.32 (m) 3.70 (m)/3.84 (m) 105.24 75.28 78.32 71.17 78.07 62.40 CH CH CH CH CH CH2 4.62 (d, 7.6) 3.32 (m) 3.32 (m) 3.30 (m) 3.33 (m) 3.73 (m)/3.84 (m) a CD3OD, b measured at 200 MHz, c at 100 MHz, d at 400 MHz # C for GG1A (Glochierioside A [14]) Figure 3.11 ROESY spectrum of GG1 15 3.2 Determination of chemical structure of isolated compounds from G hirsutum This section presents the detailed results of spectral analysis and structure determination of new compounds from G hirsutum Figure 3.12 The structure of 10 compounds from G hirsutum The detailed method for determination chemical structure of Hirsutoside A (GH1) is presented in the following section 3.2.1 Compound GH1: Hirsutoside A GH1 compound is isolated as white amorphous powder Its molecular formula is determined as C43H64O11 by high resolution electrospray ionization (HR-ESI)-MS at (m/z 779.4370 [M+Na]+; Calcd for [C43H64O11Na]+: 779.4346) The 1H-NMR spectrum of GH1 shows signals of six singlet methyl groups at 0.75 (3H, s), 0.96 (3H, s), 1.04 (3H, s), 1.06 (3H, s), 1.17 (3H, s) and 1.34 (3H, s); one olefinic proton at H 5.37 (1H, t, J = 3.0 Hz); five aromatic protons at H 8.04 (2H, d, J = 8.0 Hz), 7.51 (2H, dd, J = 8.0 and 8.0 Hz) and 7.62 (1H, t, J = 8.0 Hz) suggested a phenyl group; an anomeric proton at H 4.43 (1H, d, J = 8.0 Hz) suggests the appearance of a sugar unit 16 Figure 3.13 Chemical structure of compound GH1 and reference compoud GH1B Figure 3.14 HR-ESI-MS of GH1 The 13C-NMR and Figure 3.15 1H-NMR spectrum of GH1 DEPT spectra of GH1 revealed signals of 43 carbons which were divived into one carbonyl group, quaternary carbons, 17 methines, 11 methylenes and methyl carbons Those signals suggested the structure of an olean-12-ene aglycone with methyl groups at C 13.39 (H 0.75, 3H, s), 16.60 (H 1.04, 3H, s), 17.45 (H 1.06, 3H, s), 18.85 (H 1.17, 3H, s), 27.41 (H 1.34, 3H, s) and 29.43 (H 0.96, 3H, s); two olefinic carbons at C 124.93 (H 5.37, 1H, t, 3.0 Hz) and 142.99 Further, resonance signals at C 129.63; 130.43; 131.87; 134.23 and 167.85 demonstrated the presence of a benzoyl group Through analysing the 1H-NMR and 13C-NMR spectroscopic data of GH1, it reveals a similar result to those of reported reference compound 21β-benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene (GH1B) [9], except for the addition of a sugar unit The location of substitued groups and assignments were further confirmed by two- 17 dimensional nuclear magnetic resonance spectroscopic methods such as HSQC, HMBC, COSY Figure 3.16 13C-NMR spectrum of GH1 Figure 3.17 HSQC spectrum of GH1 The spectroscopic data of C-1, C-2, C-3, C-4, C-5, C-6, C-7 in benzoyl group were determined from the HMBC correlations from H2 (δH 8.04)/ H-6 (δH 8.04) to C-7 (δC 167.85), C-1 (δC 134.23), COSY correlations of H-2/H-3, H-3/H-4, H-4/H-5, H-5/H-6 and direct correlation in HSQC (H-2/C-2, H-3/C-3, H-4/C-4, H-5/C-5, H-6/C6) The location of a benzoyl group at C-21 was assigned based on the HMBC cross-peak from H-29 (δH 0,96)/H-30 (δH 1,17) to C-19 (δC 47.95)/C-20 (δC 36.61)/C-21 (δC 78.17), and from H-21 (δH 5.16) to C-7 (δC 167.85) Additionally, the HMBC correlations between H-24 (δH 0.75) to C-3 (δC 83.33)/ C-4 (δC 43.89)/ C-5 (δC 48.11)/ C-23 (δC 64.82), and chemical shifts of C-3 and C-23 suggested the location of oxygenated-carbon group at C-3 and the hydroxyl group at C-23 Figure 3.18 HMBC spectrum of GH1 Figure 3.19 COSY spectrum of GH1 18 The 1H-NMR at H 4,43 (1H, d, J = 8,0 Hz, H-1), 13 C-NMR at (δC 105.72, 75.63, 77.72, 71.56, 78.32, 62.73), acid hydrolysis and GC analysis showed the sugar unit to be D-glucose In addition, the HMBC correlation between H-1 (δH 4.43) and aglycone C-3 (δC 83.33), and the COSY correlation sequences of H-1/ H-2/ H-3/ H-4/ H-5/ H-6 further confirmed the sugar component to be D-glucose, with the location of suger moiety being at C-3 of aglycone The α-orientation of H-3, H-5 and the hydroxyl methylene group were determined by observation of NOESY correlations between H-3 (δH 3.67), H-5 (δH 1.66) and H-23 (δH 3.31 and 3.67) Futhermore, the αorientations of both H-16 and H-21 were also confirmed by NOESY correlations between H-16 (δH 4.36) and H-27 (δH 1.34)/Hα-19 (δH 2.10), and H-21 (δH 5.16)/Hα-19 (δH 2.10)/H-29 (δH 0.96) (Fig 3.20) Based on the above evidence, compound GH1 was determined to be 21βbenzoyloxy-3,16,23,28-tetrahydroxyolean-12-ene 3-O- -D- glucopyranoside This is a new compound and named as Hirsutoside A The 1H and 13 C-NMR spectroscopic data of GH1 were summarized in table 3.2 Figure 3.20 The key COSY, HMBC and NOESY correlations of GH1 19 Table 3.2 NMR spectroscopic data for GH1 and reference compound C 10 11 12 13 14 15 16 17 18 19 20 21 22 # C 39.4 28.2 73.6 43.4 48.9 19.0 33.2 40.6 47.7 37.5 24.4 124.1 143.1 44.2 37.2 67.2 44.7 43.5 47.7 36.4 77.7 31.0 Ca,b 39.62 26.31 83.33 43.89 48.11 18.81 33.29 41.06 48.14 37.51 24.72 124.93 142.99 44.55 36.48 67.88 44.74 43.64 47.95 36.61 78.17 30.21 DEPT CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH C CH CH2 C CH CH2 23 68.1 64.82 CH2 24 25 26 27 28 13.7 16.7 17.5 27.4 66.9 13.39 16.60 17.45 27.41 66.58 CH3 CH3 CH3 CH3 CH2 29 30 21-O-Bz 1 2 6 3 5 4 7 29.6 19.3 29.43 18.85 CH3 CH3 Ha.c (mult., J Hz) 1.00 (m)/1.66 (m) 1.73 (m)/1.98 (m) 3.67 (dd, 3.5, 13.0) 1.66 (m) 1.44 (m)/1.57 (m) 1.36 (m)/1.74 (m) 1.27 (m) 1.96 (m) 5.37 (t, 3.0) 1.44 (m)/1.82 (m) 4.36 (dd, 5.0, 12.0) 2.51 (dd, 4.5, 14.0) 1.33 (m)/2.10 (m) 5.16 (dd, 5.0, 12.0) 1.73 (dd, 12.0, 13.5) 2.39 (dd, 5.0, 13.5) 3.67 (d, 13.0)/ 3.31 (d, 13.0) 0.75 (s) 1.04 (s) 1.06 (s) 1.34 (s) 3.42 (d, 11.0)/ 3.73 (d, 11.0) 0.96 (s) 1.17 (s) 132.0 130.4 129.4 133.7 166.7 134.23 130.43 129.63 131.87 167.85 C CH CH CH C 8.04 (d, 8.0) 7.51 (dd, 8.0, 8.0) 7.62 (t, 8.0) - 20 C 3-O-Glc 1 2 3 4 5 6 C # Ca,b DEPT Ha.c (mult., J Hz) 105.72 75.63 77.72 71.56 78.32 62.73 CH CH CH CH CH CH2 4.43 (d, 8.0) 3.20 (t, 8.0) 3.36 (m) 3.31 (m) 3.30 (m) 3.67 (dd 4.5, 12.0) 1.86 (dd 2.0, 12.0) measured in CD3OD, b at 125MHz, c at 500 MHz, # C of GH1B (21β-benzoyloxy 3β,16β,23,28-tetrahydroxyolean-12-ene) measured in pyridine-d5, at 100MHz [9] a Figure 3.21 NOESY spectrum of GH1 3.3 Biological activities of isolated compounds 3.3.1 Cytotoxic activity of compounds from G glomerulatum The results of cytotoxic activities of ten compounds GG1-GG10 on four human cancer cell lines A-549, MCF-7, OVCAR, HT-29 (table 2.2) demonstrates that compounds GG1, GG2, GG5 and GG7, which have benzoyl group at C-22, show significant cytotoxic activities against the A-549, HT-29, and OVCAR cancer cell lines with IC50 values ranging from 5.9 to 10.6 µM, which is similar with mitoxantrone, an anticancer agent was used as a positive control with IC50 values ranging from 3.1 to 10.3 µM In addition, compound GG3 displayed cytotoxicity against HT29 and OVCAR cell lines with IC50 values of 7.3 and 6.6 µM, respectively Compounds GG8–GG10 without the benzoyloxy group at C-22 showed only moderate cytotoxic activity lines with IC50 values ranging from 27.7 to 94.9 µM Compound GG4, which dose not have any 21 functional group at C-16 and C-22, exhibited significant cytotoxicity, the IC50 values of 9.7 and 7.5 µM, against A-549 and HT-29 cancer cell lines even with no benzoyloxy group at C-22, respectively On the other hand, all ten compounds also exhibited moderate cytotoxic activity on the MCF-7 cancer cell line These results are consistent with previous studies reporting the cytotoxicity of the oleanane-type saponins with acyl groups at C-21 and C-22 against various cancer cell lines including A-549, HL-60, and HCT116 [43, 46, 84-86] The current study demonstrates that the cytotoxic activity of compounds GG1, GG2, GG5 and GG7 against A-549, HT-29, and OVCAR cell lines comparable to those of mitoxantrone 3.3.2 Cytotoxic activity of compounds from G hirsutum The results of cytotoxic effects of five isolated compounds GH1-GH5 on four human cancer cell lines A-549, MCF-7, SW-626, HepG2 (table 2.4) demonstrated that compounds GH1, GH2, GH4 and GH5, which have benzoyl group at C-21, displayed significant cytotoxic activities against the four A-549, MCF-7, SW-626, HepG2 cancer cell lines with IC50 values ranging from 3.4 to 10.2 µM Ellipticine, an anticancer agent, was used as a positive control with IC50 values ranging from 1.4 to 2.1 µM for all the human cancer cell lines This work has thus provided a further example of the importance of oleanane-type saponins contain a benzoyloxy group at C-21 as potential anticancer agents Compound GH3 containing acetyl group at glc C-6″ exhibited weak cytotoxic activity with IC50 values ranging from 47.0 to 54.4 μM In the structure-activity relationship of isolated compounds GH1-GH3, when additional sugar moiety at glc C-3″ (compound GH2), the cytotoxic activity exhibited stronger, however, when acetyl group at glc C-6″ (compound GH3) the cytotoxic activity decreased The current study demonstrates that the cytotoxic activity of compound GH2 on all tested human cancer cell lines comparable to those of ellipticine 22 CONCLUSIONS This research is the first study on chemical constituents and biological activities of Glochidion glomerulatum and Glochidion hirsutum in Vietnam Chemical composition investigations By using various chromatographic methods, 15 compounds were isolated from Glochidion glomerulatum and Glochidion hirsutum Their chemical structures were determined by NMR, electrospray ionization (ESI)-MS and as well as by comparison with those reported in the literature - Ten new compounds were isolated and identified from G glomerulatum: Glomeruloside I (GG1), Glomeruloside II (GG2), Glomeruloside A (GG3), Glomeruloside B (GG4), Glomeruloside C (GG5), Glomeruloside D (GG6), Glomeruloside E (GG7), Glomeruloside F (GG8), Glomeruloside G (GG9), Glomeruloside H (GG10) - Five new compounds were isolated and identified from G hirsutum: Hirsutoside A (GH1), Hirsutoside B (GH2), Hirsutoside C (GH3), Hirsutoside D (GH4), Hirsutoside E (GH5) Investigation of biological activity - The cancer cytotoxic activity of 10 compounds from G glomerulatum against four human cancer cell lines was evaluated: A-529, HT-29, OVCAR, MCF-7 Results showed that Glomeruloside I, II, Glomeruloside C and E compounds exhibited strong cytotoxic activity against A-549, HT-29 and OVCAR cancer cell lines with IC50 values ranging from 5.9 μM to 10.6 μM Glomeruloside A exhibited strong cytotoxic activity on HT-29 and OVCAR cell lines with IC50 values of 7.3 μM and 6.6 μM, respectively; Glomeruloside F-H exhibits weak toxic activity against all four test cell lines; Glomeruloside B exhibited strong toxicity with an IC50 value of 9.7 μM and 7.5 μM for A-549 and HT-29 cancer cell lines All ten compounds have potent cytotoxic activity on the MCF-7 cancer cell line 23 - The cancer cytotoxic activity of new compounds from G.hirsutum on four human cancer cell lines was evaluated: A-529, MCF7, HepG2, SW-626 Results showed that Hirsutoside A, B, D, E showed strong cytotoxic activity against all four A-549, MCF-7, SW-626 and HepG2 cancer cell lines with IC50 values ranging from 3.4 μM to 10.2 μM Hirsutoside C has weak cytotoxic activity on all four test cell lines with IC50 values ranging from 47.0 μM to 54.4 μM RECOMMENDATIONS Compound Glomeruloside B presents a strong toxicity with IC50 values of 9.7 μM and 7.5 μM for cancer cell lines A-549 and HT-29; The compounds Glomeruloside I, II and Glomeruloside C, E present a strong cytotoxic activity against the A-549, HT-29 and OVCAR cancer cell lines with IC50 values ranging from 5.9 μM to 10.6 μM The compounds Hirsutoside A, B, D, E compounds represent strong cytotoxic activity against all four cancer cell lines A-549, MCF-7, SW-626 and HepG2 with an IC50 ranging from 3.4 μM to 10.2 μM Therefore, there should be further in-depth studies of cytotoxic mechanisms and pharmacological effects of these compounds conducted in the future NEW CONTRIBUTIONS OF THE THESIS This is the first study of chemical constituents and biological activities of G glomerulatum and G hirsutum growing in Vietnam 15 new compounds were isolated and identified from G glomerulatum and G hirsutum, including: - 10 new compounds Glomeruloside I, Glomeruloside II, Glomeruloside A – H were isolated and determined from leaves of the G Glomerulatum - new compounds Hirsutoside A-E were isolated and determined from leaves of the G hirsutum 24 The cytotoxic activities of ten compounds were evaluated against four human cancer cell lines A-549, MCF-7, OVCAR, HT-29 The results demonstrated that compounds Glomeruloside I, II and Glomeruloside C, E showed significant cytotoxic activities against the A549, HT-29, and OVCAR cancer cell lines with IC50 values ranging from 5.9 to 10.6 µM In addition, Glomeruloside A displayed cytotoxicity against HT-29 and OVCAR cell lines with IC50 values of 7.3 and 6.6 µM, respectively Compounds Glomeruloside F-H showed only moderate cytotoxic activity against four human cancer cell lines Compound Glomeruloside B exhibited significant cytotoxicity with the IC50 values of 9.7 and 7.5 µM against A-549 and HT-29 cancer cell lines The cytotoxic effects of five isolated compounds from leaves of the G hirsutum were evaluated against four human cancer cell lines A549, MCF-7, SW-626, HepG2 The result demonstrates that compounds Hirsutoside A, B, D, E showed significant cytotoxic activities against the four A-549, MCF-7, SW-626, HepG2 cancer cell lines with IC50 values ranging from 3.4 to 10.2 µM Compound Hirsutoside C exhibited weak cytotoxic activity with IC50 values ranging from 47.0 to 54.4 μM against all four human cancer cell lines PUBLICATIONS WITHIN THE SCOPE OF THESIS Vu Kim Thu, Nguyen Van Thang, Nguyen Xuan Nhiem, Bui Huu Tai, Nguyen Hoai Nam, Phan Van Kiem, Chau Van Minh, Hoang Le Tuan Anh, Nanyoung Kim, Seonju Park, Seung Hyun Kim Oleane-type saponins from Glochidion glomerulatum and their cytotoxic activities Phytochemistry, 2015, 116, 213-220 Vu Kim Thu, Nguyen Van Thang, Nguyen Xuan Nhiem, Hoang Le Tuan Anh, Phạm Hải Yến, Chau Van Minh, Phan Van Kiem, Nan Young Kim, Seon Ju Park and Seung Hyun Kim Oleane-type saponins from Glochidion glomerulatum Natural Product Communications, 2015, 10(6), 875-876 Nguyen Van Thang, Vu Kim Thu, Nguyen Xuan Nhiem, Duong Thi Dung, Tran Hong Quang, Bui Huu Tai, Hoang Le Tuan Anh, Pham Hai Yen, Nguyen Thi Thanh Ngan, Nguyen Huy Hoang, and Phan Van Kiem Oleane-type Saponins from Glochidion hirsutum and Their Cytotoxic Activities Chemistry and Biodiversity, 2017, 14(5), 1-9 ... be "Study on chemical constituents and cytotoxic activities of Glochidion glomerulatum and Glochidion hirsutum growing in Vietnam" The objectives of the thesis Study on chemical constituents of. .. research is the first study on chemical constituents and biological activities of Glochidion glomerulatum and Glochidion hirsutum in Vietnam Chemical composition investigations By using various chromatographic... contents of the thesis Isolation of compounds from the leaves of Glochidion glomerulatum and Glochidion hirsutum; Determination of chemical structures of the isolated compounds; Evaluation of