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

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

MINISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADEMY

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-

Nguyen Dinh Chung

STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES FROM THE TUBERS

OF OPHIOPOGON JAPONICUS (L.F.) KER-GAWL

Major: Organic chemistry

Code: 62.44.01.14

SUMMARY OF CHEMISTRY DOCTORAL THESIS

Hanoi - 2018

This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology

Adviser 1: Assoc Prof Dr Nguyen Tien Dat

Adviser 2: Dr Nguyen Van Thanh

1st Reviewer:

2nd Reviewer:

3rd Reviewer:

The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at

hour date month 2018

Thesis can be found in:

- The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology

- National Library

INTRODUCTION

1 The urgency of the thesis

The important role of active compounds from natural products from various sources especially derived from plants, has been confirmed from the folk medicine to modern medicine Their effects are not only used directly as a folk medicine but as a prototype or an inportant role for the discovery and development of new drugs Vietnam is rich a country in medicinal resources, which has a high potential of medicinal plants and has a lot of experience using this source of medication by its long tradition of medicine According to Dictionary of Vietnamese medicinal plants, in Vietnam, there were

13000 species, in which 5000 species were used to folk medicine This is the suggestions for us to study this source of medicine for the life In the course of screening program of extracts from Vietnam medicinal plants with cytotoxic and anti-inflammatory activities, the

methanolic extract of the tubers of O japonicus showed significant

cytotoxic and anti-inflammatory effects and was chosen for further in continuing studies of this thesis

Ophiopogon japonicus (L.f) Ker-Gawl (Convallariaceae) has

been widely cultivated in several areas of Vietnam and is commonly used as an or namental flower and in traditional medicine The tubers

of O japonicus are used as folk medicine and many studies have

reported that they have been used to treat cough, fever, epistaxis, inlammation, respiratory disease, constipation, and gastrointestinal disorders Previous phytochemical investigations have revealed that

O japonicus contains steroidal saponins, homoisoflavonoids,

polysaccharides, phenolic acids, and sesquiterpenes

Anti-inflammatory, antitumor, antidiabetic, anti-oxidant activities of O

japonicus have been reported Therefore, thesis title was chosen to be

“Study on chemical constituents and biological activities from the

tubers of Ophiopogon japonicus (L.f.) Ker-Gawl.”The aim of this

study was to identify the potential active compounds from O

japonicus that could contribute to the clarification of traditional

medicine and increase the scientific value of this plant in Vietnam

2 The objectives of the thesis

 Study on chemical constituents from the tubers of O

japonicus;

 Evaluation of biological activities of isolated compounds to find potential compounds

3 The main contents of the thesis

 Isolation and determination of chemical structures of

compounds isolated from the tubers of O japonicus;

 Evaluation on the cytotoxic and anti-inflammatory activities of the isolated compounds

The tubers of O japonicus were collected in Feb 2014 at

Me Linh, Hanoi and identified by Prof Tran Huy Thai, Institute of

Ecology and Biological Resources, Vietnam Academy of Science and Technology The voucher specimens were deposited at the Department of Bioactive Products, Institute of Marine Biochemistry,

Vietnam Academy of Science and Technology

2.2 Methods

2.2.1 Methods for isolation of secondary metabolites

Chromatographic methods such as thin layer chromatography (TLC), column chromatography (CC)

2.2.2 Methods for determination of chemical struture of compounds

Physical parameters and modern spectroscopic methods such

as optical rotation ([α]D), electrospray ionization mass spectrometry (ESI-MS) and high-resolution ESI-MS (HR-ESI-MS), one/two-dimention nuclear magnetic resonance (NMR) spectra, and circular dichroism spectrum (CD)

2.2.3 Methods for evaluation of biological activities

- Cytotoxic activity was evaluated against four human cancer cell lines, including A549 (human lung carcinoma), LU-1 (human lung adenocarcinoma), KB (human epidermoid carcinoma), and SK-Mel-2 (human melanoma ) by the MTT assay;

- Anti-inflammatory activity of isolated compounds was assessed on the basis of inhibiting NO production in

lipopolysaccharide (LPS) activated RAW264.7 cells

2.3 Extraction and isolation of compounds from O japonicus

The air-dried and powdered tubers of

O japonicus (2.4 kg)

Extract with MeOH (5L×3 times)

Remove solvent MeOH extract (360 g)

Partition with CHCl 3 (3L×3 time)

CHCl 3 fraction OJC1.2 (8.6 g)

YMC RP-18 CC, Acetone:H2O (1.5:1)

OJC 17.3

(986 mg) Silica gel CC,

Acetone:H2O (2:1)

OJ-9 (46.3 mg)

YMC RP-18 CC, Acetone: H 2 O (1:1) Silica gel CC,

OJ-15 (365.7 mg)

Figure 6 OJ-1, OJ-12, and OJ-15 compounds isolated from OJW1.5

OJW2.4

(455 mg) YMC RP-18 CC, MeOH:H2O (1:1)

2.4 Physical properties and spectroscopic data of the isolated compounds

This section presents physical properties and spectroscopic

data of 15 compounds isolated from O japonicus

2.5 Results on biological activities of isolated compounds

2.5.1 Results on cytotoxic activity of compounds

15 compounds (OJ-1 ‒ OJ-15) were evaluated for their

cytotoxic activity against four human cancer cell lines, including human lung carcinoma (A549), human lung adenocarcinoma (LU-1), human epidermoid carcinoma (KB), and human melanoma (SK-Mel-2) by MTT assay

Table 14 Cytotoxic effects of compounds OJ-1 ‒ OJ-15 (IC50, μM)

Ellipticine and camptothecin were used as the positive controls

Table 15 Effects of compounds OJ-1 – OJ-15 on the LPS-induced

NO production on RAW264.7 cells (IC50, μM)

IC50

(μM) 11,4 29,1 >30 >30 >30 22,5 19,3 >30 Hợp

CHAPTER 3 DISCUSSIONS

3.1 Chemical structure of compounds from the tubers of O

japonicus

This section presents the detailed results of spectral analysis

and structure determination of 15 compounds isolated from O

japonicus

Figure 9 Structures of compounds 1–15 isolated from O japonicus

Detailed methods for determination of chemical structure of

a new compound was showed as bellowing:

3.1.1 Compound OJ-1: (2R)-(4-methoxybenzyl)-5,7-dimethyl-6-hy droxyl-2,3-dihydrobenzofuran (New compound)

Figure 10 Structure of OJ-1 and the important HMBC correlations

Figure 12 1H NMR spectrum of OJ-1 Compound OJ-1 was obtained as a brown solid Its

molecular formula was determined to be C18H20O3 from highresolution electrospray ionisation mass spectrometry (HRESIMS

m/z 283.1365 [M − H]−) Its 1H NMR spectrum showed the

characteristic resonance of an AA′BB′ aromatic ring [δH 7.19 (2H, d,

J = 8.5 Hz, 2′ and 6′), and 6.86 (2H, d, J = 8.5 Hz, 3′ and

H-5′)], an aromatic singlet [δH 6.67 (1H, s, H-4)], an oxygenated

methine proton [δH 4.86 (partially overlapped with HDO signal,

H-2)], one methoxyl group [δH 3.78 (3H, s, 4′-OMe)], two methylene

groups [δH 3.07 (1H, dd, J = 15.0, 8.5 Hz, H-3a), 2.82 (1H, dd,

J = 15.0, 7.5 Hz, H-3b), 3.02 (1H, dd, J = 14.0, 7.0 Hz, H-7′a), 2.84

(1H, dd, J = 14.0, 6.5 Hz, H-7′a)] and two aromatic methyl groups [δH 2.12 (3H, s, Me-5) and 2.05 (3H, s, Me-7)] (Table 3).

Figure 13 13C NMR spectrum of OJ-1

The 13C NMR and DEPT spectra revealed the presence of

two methyl carbons at δC 9.2 (7-Me) and 16.5 (5-Me), two methylene

carbons at δC 35.9 (C-3) and 42.0 (C-7′), a methoxy carbon at δC 55.7

(4′-OMe), an oxygenated methine carbon at δC 85.1 (C-2), five

methine carbons at δC 123.9 (C-4), 131.4 (C-2′ and C-6′), and 114.7

(C-3′ and H-5′), and seven quaternary carbons at δC 153.8 (C-6, observed from HMBC spectrum), 158.1 (C-7a), and 159.8 (C-4′), 118.0 (C-3a), and 117.3 (C-5) [1,2] The HMBC correlations from aromatic singlet H-4 to C-3, C-3a, C-5, C6, C-7a, and from Me-5 to

Table 3 NMR spectroscopic data (CD3OD, δ ppm) of OJ-1

located at C-6 and two methyl groups located at C-5 and C-7 The methoxyl group was placed on C-4′ based on the HMBC correlation

of the proton of this group with C-4′ (Figure 10) From these data,

OJ-1 was identified as 2-(4-methoxybenzyl)-5,7-dimethyl-6-hydro

xyl-2,3-dihydrobenzofuran

Figure 17 Experimental and calculated CD spectrum for OJ-1

The quantum chemical electronic circular dichroism (ECD) calculation method, based on time-dependent density functional theory (TDDFT), was used to determine of the absolute coniguration

at C-2 [3] The predicted ECD patterns for 2R were consistent with

the experimentally measured ECD of OJ-1 (Figure 17) Thus, compound OJ-1 was assigned as (2R)-(4-methoxybenzyl)-5,7-dime

thyl-6-hydroxyl-2,3-dihydrobenzofuran

3.1.2 Compound OJ-7: Homoisopogon B

Figure 55 Structure of OJ-7 and the important HMBC correlations

Figure 57 1H NMR spectrum of OJ-7 Compound OJ-7 was obtained as a yellow powder with the

molecular formula C19H22O4, which was established from the

HRESIMS data (m/z 315.1602 [M + H]+) The 1H NMR spectrum

showed characteristic resonances at δH 4.06 (1H, dd, J = 2.0, 11.0 Hz and 3.83 (1H, dd, J = 6.0, 11.0 Hz) corresponding to H-2 protons, δH

2.25 (1H, m) corresponding to H-3, δH 2.80 (1H, dd, J = 5.5, 16.0 Hz) and 2.44 (1H, dd, J = 6.5, 16.0 Hz) corresponding to H-4 protons, and δH 2.64 (1H, dd, J = 9.0, 14.0 Hz) and 2.52 (1H, dd, J =

6.5, 14.0 Hz) corresponding to H-9 protons The 1H NMR spectrum

also showed signals at δH 6.38 (1H, d, J = 2.5 Hz, H-3′), 6.40 (1H, d,

J = 2.5, 8.0 Hz, H-5′), and 6.98 (1H, d, J = 8.0 Hz, H-6′) suggesting a

1,2,4-trisubstituted pattern for the B ring Additionally, two aromatic

singlet protons at δH 6.76 (1H, s, H-5) and 6.34 (1H, s, H-8) were detected, indicating the presence of a tetrasubstituted A ring

In the 13C NMR and DEPT spectra, a methyl, two methoxyls, three methylenes, an aliphatic methine, five aromatic methines, and 7 aromatic quaternary carbons were observed These data suggested

that OJ-7 possesses a homoisoflavane skeleton [5] The HMBC

correlations from the aromatic methyl group at δH 2.10 (3H, s) to C-5

(δC 131.4), C-6 (δC 119.1), and C-7 (δC 156.7), and from the methoxy

signals δH 3.74 to C-7, and δH 3.73 to C-4′ (δC 159.3), indicated that the methyl and methoxyl groups attached to C-6, C-7, and C-4, respectively The absolute configuration of C-3 was determined to be

R based on the Cotton effect at 230 nm (negative) and 285 nm

(positive) in the CD analysis [5] Accordingly, the structure of OJ-7

Table 9 1H and 13C NMR spectroscopic data (CDCl3, δ ppm) of OJ-7

was elucidated as (3R)-4′,7-dimethoxy-2′-hydroxy-6-methylhomoiso

flavane, named homoisopogon B

3.2 Biological activities of isolated compounds

3.2.1 Cytotoxic activity of compounds 1–15

Compounds 1–15 were evaluated for their cytotoxic effect

against LU-1, KB, and SK-Mel-2 cells As the results showed in

Table 14, compounds OJ-1, OJ-6, OJ-7, and OJ-8 showed

significant cytotoxic activity on LU-1, in which OJ-6 had the strongest cytotoxic activity with an IC50 = 0.66 µM Compounds OJ-

1, OJ-6, and OJ-15 showed significant cytotoxic activity on KB cells, in which OJ-1 showed the hightest cytotoxic activity with an

IC50 = 0.51 µM Moderate cytotoxic activities were observed with

OJ-1, OJ-3, OJ-6, OJ-7, OJ-10, and OJ-15 on SK-Mel-2 cells Interestingly, homoisopogon A (OJ-6) exhibited a strong cytotoxic

effect on all tested cell lines with the IC50 values of 0.51–0.66 µM

The activity is comparable to that of the positive control, ellipticine

The cytotoxic effect of homoisoflavonoids has been indicated elsewhere, and the structure-activity relationship has been investigated This is the first time to evaluate cytotoxic effects of series of homoisoflavonoids was reported on human cell lines at low

concentrations Compound OJ-7 showed moderate cytotoxic activity

on all tested cell lines with the IC50 values ranging 17.14 to 32.94

µM Additionally, homoisopogon C (OJ-8) exhibit significant

cytotoxicity toward LU-1 cells, with an IC50 value of 27.66 µM

Accordingly, the 2′-hydroxy and 4′-methoxy groups seem to have a contribution to the activity In my study, compounds possessing 2′-hydroxy and 4′-methoxy substituent showed positive effect on at

least one cancer cell line Homoisopogon D (OJ-9) with a

methylenedioxy group at 3′–4′ and lack of hydroxyl group at 2′, was inactive against all tested cells

C-The results showed that weak or no effects of benzofuran

derivatives were evident on all three cancer cell lines, but OJ-1

exhibited cytotoxic activity on all three tested cell lines (Table 14) These results indicate that the presence of 2R configuration in 2-

benzylbenzofuran skeleton may significantly activity

Figure 115 Apoptotic effect of homoisopogon A in A549 cells

analyzed with Annexin V-FTIC/PI assay after 24h treatment

Due to homoisopogon A (OJ-6) showed strongly cytotoxic

activity on LU-1 cells, thus, we continued to further investigate the mechanism of action of this compound in A549 cells As the results, homoisopogon A exhibited strong cytotoxic effect to the wild type of EGFR-TKI-resistant A549 cells (IC50 = 6.26 ± 0.79 μM), which was

more potent than to the positive control, camptothecin (IC50 = 12.42

± 0.56 μM) Also, homoisopogon A exhibited strong cytotoxicity

toward two other cell lines NCI-H1975 and NCI-H1650

As showed in Figure 115, homoisopogon A induced

apoptosis potently at two investigated concentrations after 24h of treatment Homoisopogon A treatment of A549 cells at the

concentration of 25 μM generated apoptosis in 27.5% of cells (7%

early apoptosis and 20.5% late apoptosis) The effect increased

significantly at the concentration of 50 μM, the homoisoflavanone

generated apoptosis in 83.8% of cells (23.5% early apoptosis and 60.3% late apoptosis) The movement of cells strongly suggested the cells underwent the apoptosis by treatment of homoisopogon A The results strongly suggested that homoisopogon A induces apoptosis in EGFR and TKI-resistant-A549 cells, thus resulting in the cytotoxicity

3.2.2 Anti-inflammatory activity of compounds

The isolated compounds 1–15 were tested for their ability to

inhibit NO production in LPS-stimulated RAW264.7 cells

Compound OJ-1 was the most active compound with an IC50 of

11.4 μM, while compound OJ-2 had a moderate effect (IC50 =

29.1 μM) 2,3-Dihydrobenzofurans have been known as potent

anti-inlammatory compounds 2,3-Dihydrobenzofuran-2-one had

powerful anti-inlammatory activity in vivo, and

5-chloro-6-cyclohexyl-2,3-dihydrobenzofuran-2-one was significantly more potent than the reference compound, diclofenac, in all testing models [6] More recently, a series of dihydrobenzofurans was isolated from

the seeds of Prunus tomentosa, some of which strongly inhibited NO

production in LPS-stimulated BV-2 cells [7] Consistently, my

results suggest that O japonicus is a potential natural source of

anti-inlammatory dihydrobenzofurans

Homoisopogon A (OJ-6) and homoisopogon B (OJ-7)

showed moderate effects with the IC50 of 22.5 và 19.3 μM,

respectively Other compounds showed weak or inactive up to the

highest concentration tested (30 μM)