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.

AND TRAINING 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

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

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

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

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 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

Table 2.2 The effects of compounds GG1-GG10 on the growth of

A-549, MCF-7, OVCAR, HT-29 cells

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

Table 2.4 The effects of compounds GH1-GH5 on the growth of

A-549, MCF-7, SW-626, HepG2 cells

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 1 H-NMR spectrum of GG1

The 13

C-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

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 C124.23 (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 13 C-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

H-24/24, H-23/23 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 (δH0.99) 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

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 1 H– 1 H COSY spectrum of

GG1

Moreover, the HMBC correlations from H-27 (δH 1.30) to 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 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-

(H-30, H-19/19, H-21/21) The assignments at 2, 6, 11, 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

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/

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 (δC85.25) 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 (δH0.81) 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 (δH4.32), 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 glucopyranosyl (1→2)]-β-D-galactopyranoside This is a new compound

(1→3)-[β-D-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 #C a,b C a,c DEPT H a,d (mult., J, Hz)

C #C a,b C a,c DEPT H a,d (mult., J, Hz) 3-O- Ara Glc

# C for GG1A (Glochierioside A [14])

Figure 3.11 ROESY spectrum of GG1

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