Study on chemical constituents and cytotoxic activities of glochidion glomerulatum and glochidion hirsutum growing in Vietnam

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 1 st Reviewer: Prof. Dr. Nguyen Van Tuyen 2 nd Reviewer: Asc. Prof. Dr. Tran Thu Huong 3 rd 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. 1 INTRODUCTION 1. 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". 2. The objectives of the thesis Study on chemical constituents of two Glochidion species including Glochidion glomerulatum and Glochidion hirsutum in Vietnam. 2 Evaluation of biological activities of isolated metabolites to find out potential compounds. 3. The main contents of the thesis 1. Isolation of compounds from the leaves of Glochidion glomerulatum and Glochidion hirsutum; 2. Determination of chemical structures of the isolated compounds; 3. 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), high- performance 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 3 This section presents the process of isolating ten compounds from G. glomerulatum. Figure 2.4. Isolation of compounds from G. glomerulatum 4 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. 5 2.4.2. Physical properties and spectroscopic data of the isolated compounds from G. hirsutum This section presents physical properties and spectroscopic data of 5 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 GG1 97,54 ± 2,06 82,28 ± 1,42 90,64 ± 1,28 95,22 ± 2,38 GG2 94,66 ± 1,22 79,69 ± 1,30 88,18 ± 0,84 93,12 ± 2,92 GG3 71,24 ± 0,52 83,25 ± 1,26 97,12 ± 2,04 92,34 ± 0,20 GG4 94,67 ± 1,62 79,86 ± 2,34 83,89 ± 2,06 91,98 ± 0,53 GG5 96,21 ± 0,72 80,34 ± 2,80 91,56 ± 1,16 96,89 ± 3,20 GG6 72,89 ± 0,56 72,15 ± 0,38 78,03 ± 1,86 77,21 ± 0,12 GG7 97,43 ± 1,02 74,38 ± 4,60 92,08 ± 3,46 99,32 ± 4,44 GG8 54,68 ± 0,21 54,89 ± 0,30 80,11 ± 2,82 81,11 ± 3,96 GG9 69,54 ± 1,08 71,02 ± 1,24 82,13 ± 0,92 87,23 ± 1,36 GG10 75,11 ± 0,96 61,34 ± 4,20 85,67 ± 1,04 79,52 ± 1,76 Table 2.2. The effects of compounds GG1-GG10 on the growth of A-549, MCF-7, OVCAR, HT-29 cells Comp. IC50 (µM) A-549 MCF-7 OVCAR HT-29 GG1 9,3± 1,4 50,1± 3,2 8,9± 2,2 7,8± 1,2 GG2 10,2± 2,3 56,1± 4,3 10,6± 3,3 9,5± 1,6 GG3 41,0 ± 3,5 58,4 ± 3,7 6,6 ± 0,7 7,3 ± 1,4 GG4 9,7 ± 1,2 60,7 ± 5,2 41,5 ± 3,1 7,5 ± 1,7 GG5 7,9 ± 0,8 42,8 ± 5,2 9,8 ± 2,1 5,9 ± 0,5 GG6 58,2 ± 2,4 69,3 ± 5,2 59,4 ± 6,8 49,3 ± 3,1 GG7 8,2 ± 1,0 63,5 ± 3,6 8,6 ± 3,1 5,9 ± 0,7 GG8 94,9 ± 4,1 86,3 ± 5,2 34,1 ± 3,4 45,0 ± 2,4 GG9 58,1 ± 4,6 67,5 ± 4,8 45,8 ± 2,5 49,1 ± 4,1 GG10 51,7 ± 3,1 77,2 ± 5,5 27,7 ± 4,6 58,7 ± 3,9 ĐC* 7,2 ± 0,5 10,3 ± 1,2 8,4 ± 0,9 3,1 ± 0,3 *) Mitoxantrone is used as a positive control (PC). 6 2.5.2. Results on cytotoxic activity of compounds from G. hirsutum - 5 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. A-549 MCF-7 SW-626 HepG2 GH1 90,10 ± 2,80 91,33 ± 1,12 90,22 ± 3,14 92,17 ± 1,38 GH2 98,60 ± 1,64 98,28 ± 2,14 98,21 ± 3,72 99,09 ± 1,76 GH3 97,44 ± 4,28 99,49 ± 0,98 96,98 ± 3,34 99,83 ± 2,38 GH4 98,69 ± 2,32 96,86 ± 1,28 94,14 ± 2,66 99,39 ± 3,64 GH5 96,06 ± 2,24 96,74 ± 3,12 95,18 ± 1,80 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. IC50 (µM) A-549 MCF-7 SW-626 HepG2 GH1 9,3 ± 0,3 9,2 ± 0,5 8,5 ± 1,3 8,2 ± 1,3 GH2 4,4 ± 0,7 4,7 ± 0,6 6,6 ± 1,0 3,4 ± 0,3 GH3 49,3 ± 4,1 51,9 ± 3,7 54,4 ± 1,5 47,0 ± 5,6 GH4 8,0 ± 2,2 8,8 ± 1,3 9,1 ± 1,1 7,6 ± 0,8 GH5 8,6 ± 1,3 10,2 ± 2,4 10,1 ± 1,9 9,9 ± 3,1 ĐC* 1,8 ± 0,3 2,0 ± 0,3 2,1 ± 0,3 1,4 ± 0,2 *) Ellipticine is used as a positive control (PC). 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. 7 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 1 H-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 8 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 1 H 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 1 carbonyl group, 8 quaternary carbons, 27 methines, 12 methylenes and 7 methyl carbons. Among them, 30 carbons belong to triterpene skeleton, 18 carbons belong to 3 hexose sugar 9 moieties and the rests belong to benzoyl group. The assignments were done by HSQC. The spectroscopic data analysis of 1 H-, 13 C-NMR and HSQC spectra suggested the presence of an olean-12-ene type aglycone with 7 methyl groups at C 16.12 (H 0.99, 3H, s), 16.80 (H 0.89, 3H, s), 17.29 (H 1.07, 3H, s), 27.49 (H 1.30, 3H, s), 27.49 (H 1.04, 3H, s), 28.32 (H 1.10, 3H, s) and 34.32 (H 093, 3H, s); 2 olefinic carbons at C 124.23 (H 5.35, 1H, br s) and 143.40 suggest the presence of C=C bond. Furthermore, the observation of resonance signals at C 132.10 (C-1), 130.43 (C-2 and C-6), 129.62 (C3 and C-5), 134.09 (C-4) and 167.33 (C-7) showed the presence of a benzoyl group. Figure 3.5. 13C-NMR spectrum of GG1 Figure 3.6. HSQC spectrum of GG1 It can be seen that the NMR spectroscopic data of GG1 is similar to those of GG1A (Glochierioside A) [14] in aglycone part, except for sugar units (table 3.1). The location of substitued groups and the 1 H- spectroscopic, 13 C-NMR of compound GG1 are conducted by comparing with reference compound GG1A, and further confirmed by two- dimensional nuclear magnetic resonance spectroscopic method such as HSQC, HMBC, COSY. The HMBC correlations from H-24 (δH 0.89) to C-3 (δC 91.90)/ C-4 (δC 40.54)/ C-5 (δC 56.87)/ C-23 (δC 28.32) and chemical shifts of C-3 suggest the conjunction of C-O at C-3. Simultaneously, the assignments of 1 H-, 13 C- NMR at H-3/C-3, H-24/C- 24, H-23/C-23 were done by HSQC. Furthermore, the assignments of C- 1, C-9, C-10 and C-25 were done by HMBC correlations from H-25 (δH 0.99) to C-1 (δC 39.94)/ C-5 (56.87)/ C-9 (δC 48.10)/ C-10 (δC 37.66) and the HSQC corralations at (H-1/C-1, H-25/C-25). Similarly, the 10 assignments of C-7, C-8, C-14 and C-26 were done by HMBC corrleations from H-26 (δH 1.07) to C-7 (δC 33.61)/ C-8 (δC 41.18)/ C-9 (48.10)/ C-14 (δC 44.20) and HSQC correlations (H-7/C-7, H-26/C-26). Figure 3.7. HMBC spectrum of GG1 Figure 3.8. 1H– 1H COSY spectrum of GG1 Moreover, the HMBC correlations from H-27 (δH 1.30) to C-8/ C- 13 (δC 143.40)/ C-14/ C-15 (δC 37.55) and a quaternary carbon suggested the presence of a double bond C=C at C-12/C-13, the assignments at C- 12, C-13, C-15 and C-27 were determined from HSQC correlations (H- 27/C-27, H-15/C-15, H-12/C-12). Furthermore, the assignments at C-18, C-19, C-20, C-21, C-29 and C-30 were done based on the HMBC correlations from H-29 (δH 0.93) and H-30 (δH 1.04) to C-19 (δC 47.13), C-20 (30.98), C-21 (38.33), and HSQC correlations (H-29/C-29, H-30/C- 30, H-19/C-19, H-21/C-21). The assignments at C-2, C-6, C-11, C-16, C- 18 and C-22 were done based on the COSY correlations between H-2/H- 3, H-5/H-6, H-11/H-12, H-15/H-16, H-18/H-19, H-21/H-22. Similarly, the assignment of C-28 was done based on the HMBC correlations from H-28 (δH 3.68 and 4,02) to C-16 (δC 69.44), C-18 (43.41), C-22 (72.04), and HSQC correlations at (H-28/C-28). The signal at carbon δC 44,80 was assiged to C-17 and further confirmed by HMBC correlations between H-16 (δH 4.32), H-18 (δH 2.46) and H-22 (δH 5.91) to C-17. 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 13 C-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 min and tR2 = 18.713 min (fig. 3.9e), which is similar with that of reference D-glucose at tR = 14,106 min (fig. 3.9b) and D-galactose reference at tR = 18.706 min (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-D- glucopyranosyl (1→3)-[β-D-glucopyranosyl (1→2)]-β-D- galactopyranoside. 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 α- orientation 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-benzoyloxy- 3β,16β,28-trihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1→3)-[β-D- glucopyranosyl (1→2)]-β-D-galactopyranoside. This is a new compound 13 and named as Glomeruloside I. The 1 H and 13 C-NMR spectroscopic data of GG1 were summarized in table 3.1. Table 3.1. NMR spectroscopic data for GG1 and reference compound C #C a,b C a,c DEPT H a,d (mult., J, Hz) 1 40.08 39.94 CH2 1.02 (m)/1.65 (m) 2 27.20 27.09 CH2 1.76 (m)/1.96 (m) 3 90.50 91.90 CH 3.22 (br d, 11.2) 4 40.40 40.54 C - 5 57.08 56.87 CH 0.81 (d, 11.2) 6 19.44 19.28 CH2 1.47 (m)/1.62 (m) 7 33.77 33.61 CH2 1.41 (m)/1.63 (m) 8 41.36 41.18 C - 9 48.31 48.10 CH 1.60 (m) 10 37.87 37.66 C - 11 24.83 24.67 CH2 1.95 (m) 12 124.41 124.23 CH 5.35 (br s) 13 143.61 143.40 C - 14 44.38 44.20 C - 15 37.72 37.55 CH2 1.52 (m)/1.98 (m) 16 69.60 69.44 CH 4.32 (br d, 10.0) 17 44.97 44.80 C - 18 43.59 43.41 CH 2.46 (d, 12.4) 19 47.31 47.13 CH2 1.22 (m)/1.90 (m) 20 31.15 30.98 C - 21 38.49 38.33 CH2 1.78 (m) 22 72.20 72.04 CH 5.91 (br s) 23 28.64 28.32 CH3 1.10 (s) 24 17.14 16.80 CH3 0.89 (s) 25 16.31 16.12 CH3 0.99 (s) 26 17.44 17.29 CH3 1.07 (s) 27 28.08 27.49 CH3 1.30 (s) 28 64.86 64.69 CH2 3 3.68 (d, 10.8)/ 4 4.02 (d, 10.8) 29 34.48 34.32 CH3 0.93 (s) 30 27.65 27.49 CH3 1.04 (s) 22-O-Bz 1 132.31 132.10 C - 2. 6 130.61 130.43 CH 8.05 (d, 7.6) 3. 5 129.79 129.62 CH 7.49 (t, 7.6) 4 134.24 134.09 CH 7.60 (t, 7.6) 7 167.33 167.18 C - 14 C #C a,b C a,c DEPT H a,d (mult., J, Hz) 3-O- Ara Glc 1 107.25 105.83 CH 4.46 (d, 8.0) 2 72.24 76.40 CH 4.00 (m) 3 84.00 85.25 CH 3.81 (m) 4 69.66 69.97 CH 4.12 (br s) 5 66.81 75.92 CH 3.55 (m) 6 63.76 CH2 3.54 (m)/3.83 (m) 2-O- Glc 1 103.51 CH 4.86(m) 2 76.05 CH 3.14 (t, 8.0) 3 78.32 CH 3.33 (m) 4 72.53 CH 3.08 (t, 8.0) 5 77.86 CH 3.32 (m) 6 62.34 CH2 3.70 (m)/3.84 (m) 3-O- Glc 1 105.53 105.24 CH 4.62 (d, 7.6) 2 75.47 75.28 CH 3.32 (m) 3 77.80 78.32 CH 3.32 (m) 4 71.33 71.17 CH 3.30 (m) 5 78.04 78.07 CH 3.33 (m) 6 62.52 62.40 CH2 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 5 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 1 H-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 Figure 3.15. 1H-NMR spectrum of GH1 The 13C-NMR and DEPT spectra of GH1 revealed signals of 43 carbons which were divived into one carbonyl group, 8 quaternary carbons, 17 methines, 11 methylenes