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.
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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.
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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 (HR-ESI-MS), (ESI-MS) and high-resolution 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
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This section presents the process of isolating ten compounds from G.
glomerulatum.
Figure 2.4. Isolation of compounds from G. glomerulatum
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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 OVCAR HT-29
A-549 97,54 ± 2,06 94,66 ± 1,22 71,24 ± 0,52 94,67 ± 1,62 96,21 ± 0,72 72,89 ± 0,56 97,43 ± 1,02 54,68 ± 0,21 69,54 ± 1,08 75,11 ± 0,96 MCF-7 82,28 ± 1,42 79,69 ± 1,30 83,25 ± 1,26 79,86 ± 2,34 80,34 ± 2,80 72,15 ± 0,38 74,38 ± 4,60 54,89 ± 0,30 71,02 ± 1,24 61,34 ± 4,20 90,64 ± 1,28 95,22 ± 2,38 88,18 ± 0,84 93,12 ± 2,92 97,12 ± 2,04 92,34 ± 0,20 83,89 ± 2,06 91,98 ± 0,53 91,56 ± 1,16 96,89 ± 3,20 78,03 ± 1,86 77,21 ± 0,12 92,08 ± 3,46 99,32 ± 4,44 80,11 ± 2,82 81,11 ± 3,96 82,13 ± 0,92 87,23 ± 1,36 85,67 ± 1,04 79,52 ± 1,76 Comp. GG1 GG2 GG3 GG4 GG5 GG6 GG7 GG8 GG9 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.
*)Mitoxantrone is used as a positive control (PC).
A-549 9,3± 1,4 10,2± 2,3 41,0 ± 3,5 9,7 ± 1,2 7,9 ± 0,8 58,2 ± 2,4 8,2 ± 1,0 94,9 ± 4,1 58,1 ± 4,6 51,7 ± 3,1 7,2 ± 0,5 MCF-7 50,1± 3,2 56,1± 4,3 58,4 ± 3,7 60,7 ± 5,2 42,8 ± 5,2 69,3 ± 5,2 63,5 ± 3,6 86,3 ± 5,2 67,5 ± 4,8 77,2 ± 5,5 10,3 ± 1,2 OVCAR 8,9± 2,2 10,6± 3,3 6,6 ± 0,7 41,5 ± 3,1 9,8 ± 2,1 59,4 ± 6,8 8,6 ± 3,1 34,1 ± 3,4 45,8 ± 2,5 27,7 ± 4,6 8,4 ± 0,9 HT-29 7,8± 1,2 9,5± 1,6 7,3 ± 1,4 7,5 ± 1,7 5,9 ± 0,5 49,3 ± 3,1 5,9 ± 0,7 45,0 ± 2,4 49,1 ± 4,1 58,7 ± 3,9 3,1 ± 0,3 GG1 GG2 GG3 GG4 GG5 GG6 GG7 GG8 GG9 GG10 ĐC*
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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
A-549 MCF-7 SW-626 HepG2
90,10 ± 2,80 91,33 ± 1,12 90,22 ± 3,14 92,17 ± 1,38 98,60 ± 1,64 98,28 ± 2,14 98,21 ± 3,72 99,09 ± 1,76 97,44 ± 4,28 99,49 ± 0,98 96,98 ± 3,34 99,83 ± 2,38 98,69 ± 2,32 96,86 ± 1,28 94,14 ± 2,66 99,39 ± 3,64 96,06 ± 2,24 96,74 ± 3,12 95,18 ± 1,80 96,77 ± 4,90 Comp. GH1 GH2 GH3 GH4 GH5
Table 2.4. The effects of compounds GH1-GH5 on the growth of
A-549, MCF-7, SW-626, HepG2 cells
IC50 (µM) Comp.
*) Ellipticine is used as a positive control (PC).
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 MCF-7 9,2 ± 0,5 4,7 ± 0,6 51,9 ± 3,7 8,8 ± 1,3 10,2 ± 2,4 2,0 ± 0,3 SW-626 8,5 ± 1,3 6,6 ± 1,0 54,4 ± 1,5 9,1 ± 1,1 10,1 ± 1,9 2,1 ± 0,3 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 GH1 GH2 GH3 GH4 GH5 ĐC*
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.
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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
resolution electrospray (HR-ESI)-MS ionization (m/z
amorphous powder. Its molecular formula is determined to be C55H84O20 by high 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
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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 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 1H-, 13C-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 1H- spectroscopic, 13C-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 1H-, 13C- 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 the HSQC corralations at (H-1/C-1, H-25/C-25). Similarly,
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.
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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 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/
(1→3)-[β-D-glucopyranosyl
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→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 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
a,d (mult., J, Hz) H 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)
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
#C a,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
a,c 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
3 3.68 (d, 10.8)/ 4 4.02 (d, 10.8)
34.48 27.65 132.31 130.61 129.79 134.24 167.33
34.32 27.49 132.10 130.43 129.62 134.09 167.18
CH3 CH3 C CH CH CH C
0.93 (s) 1.04 (s) - 8.05 (d, 7.6) 7.49 (t, 7.6) 7.60 (t, 7.6) -
29 30 22-O-Bz 1 2. 6 3. 5 4 7
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a,d (mult., J, Hz)
#C
DEPT CH CH CH CH CH CH2
a,c C Glc 105.83 76.40 85.25 69.97 75.92 63.76
H 4.46 (d, 8.0) 4.00 (m) 3.81 (m) 4.12 (br s) 3.55 (m) 3.54 (m)/3.83 (m)
107.25 72.24 84.00 69.66 66.81
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)
105.53 75.47 77.80 71.33 78.04 62.52
105.24 75.28 78.32 71.17 78.07 62.40
4.62 (d, 7.6) 3.32 (m) 3.32 (m) 3.30 (m) 3.33 (m) 3.73 (m)/3.84 (m)
CH CH CH CH CH CH2
a,b C 3-O- Ara 1 2 3 4 5 6 2-O- Glc 1 2 3 4 5 6 3-O- Glc 1 2 3 4 5 6 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 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.
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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 and 6 methyl
carbons. Those signals suggested the structure of an olean-12-ene
aglycone with 6 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-
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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 H- 2 (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/C- 6). 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
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The 1H-NMR at H 4,43 (1H, d, J = 8,0 Hz, H-1), 13C-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 α- orientations 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 21β-
benzoyloxy-3,16,23,28-tetrahydroxyolean-12-ene 3-O--D-
glucopyranoside. This is a new compound and named as Hirsutoside A. The 1H and 13C-NMR spectroscopic data of GH1 were summarized in table 3.2.
Figure 3.20. The key COSY, HMBC and NOESY correlations of GH1
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Table 3.2. NMR spectroscopic data for GH1 and reference compound
C 1 2 3 4 5 6 7 8 9 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
a,b C 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
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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
CH3 CH3
29.6 19.3 132.0 130.4 129.4 133.7 166.7
29.43 18.85 134.23 130.43 129.63 131.87 167.85
C CH CH CH C
a.c (mult., J. Hz) H 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) - 8.04 (d, 8.0) 7.51 (dd, 8.0, 8.0) 7.62 (t, 8.0) -
29 30 21-O-Bz 1 2. 6 3. 5 4 7
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a,b
a.c (mult., J. Hz)
#C
C 105.72 75.63 77.72 71.56 78.32 62.73
DEPT CH CH CH CH CH CH2
C 3-O-Glc 1 2 3 4 5 6
H 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)
a 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]
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 HT- 29 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
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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 HCT- 116 [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.
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CONCLUSIONS
This research is the first study on chemical constituents and biological activities of Glochidion glomerulatum and Glochidion hirsutum in Vietnam.
1. 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.
(GG6), Glomeruloside E
- 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 (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).
2. 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.
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- The cancer cytotoxic activity of 5 new compounds from G.hirsutum on four human cancer cell lines was evaluated: A-529, MCF- 7, 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
1. This is the first study of chemical constituents and biological
activities of G. glomerulatum and G. hirsutum growing in Vietnam.
2. 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.
- 5 new compounds Hirsutoside A-E were isolated and
determined from leaves of the G. hirsutum.
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3. 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 A-
549, 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.
4. The cytotoxic effects of five isolated compounds from leaves of
the G. hirsutum. were evaluated against four human cancer cell lines A-
549, 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
1. 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.
2. 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.
3. 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.
