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MINISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADEMY

OF SCIENCE AND

TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

----------------------------

KHIEU THI TAM

STUDY ON CHEMICAL CONSTITUENTS AND CYTOTOXIC

ACTIVITY OF 3 SPECIES ALLOPHYLUS LIVESCENS GAGNEP,

CHIRITA HALONGENSIS KIEW& T.H.NGUYEN AND

OLDENLANDIA PINIFOLIA (WALL. EX G.DON) KUNTZE] IN

VIET NAM

Major: Organic chemistry

Code: 62.44.01.14

SUMMARY OF DOCTORAL THESIS

HA NOI - 2018

This thesis is completed at: Vietnam Academy of Science and

Technology

Scientific instructors:

Assoc. Dr. NGUYEN THI HOANG ANH

Assoc. Dr. TRAN VAN LOC

Thesis reviewer 1:

Thesis reviewer 2:

Thesis reviewer 3:

The thesis will be presented to the scientific council at the Vietnam

Academy of Science and Technology at ......, date........, month.......,

year.....

1

INTRODUCTION

1. The urgency of the thesis

Today, the economic development, the need for care and protection of the health of the community becomes more urgent. Thus, the demand for medicine in preventing and treatment of incurable diseases especially cancer is increasing. The plant resources in Viet Nam are rich and multiple with over 12000 species of 309 plant families. Among them, 3900 species have been used for medicine. However, the relationship between the chemical structure and pharmacological activity in many cases has been not clarified. Allophylus livescens and Chirita halongesis, two endemic species of Ha Long Bay, have not yet studied on chemical compositions and biological activities. Oldenlandia pinifolia is used in traditional medicine for treatment of inflammation, pain and other diseases and there has been only one report on the phytochemistry of this species.

To contribute to understanding, research and systematic evaluation of biologically active compounds from coastal plants and mangroves, and to propose directions for the exploitation and conservation of genetic resources of the rare plant species in Vietnam, we conducted research on the chemical constituents and cytotoxic activity of 3 species: Allophylus livescens Gagnep., Chirita halongesis Kiew & T. H. Nguyen and Oldenlandia pinifolia (Wall. ex G. Don) Kuntze growing in Viet Nam.

2. The objectives of the thesis

Study on the chemical compositions of three species: Allophylus livescens, Chirita halongesis and Oldenlandia pinifolia growing in Viet Nam to find bioactive compounds as the basis for further research. 3. The main contents of the thesis

- Isolation of compounds from three species Allophylus livescens, Chirita halongesis and Oldenlandia pinifolia collected in Viet Nam by column chromatography.

- Determination of chemical structure of isolated compounds based on IR, MS, 1D-NMR, 2D-NMR spectroscopic analysis.

- Evaluation of cytotoxic activity of extracts and some isolated compounds. 4. The contribution of the thesis

This is the first time the chemical constituents and cytotoxic activities of Allophylus livescens and Chirita halongenis species have been studied in Vietnam as well as in the world. Two compounds were first isolated from Allophylus genus: 1,6,10,14-phytatetraen-3-ol and catechin. Oleanolic acid, decaffeoylacetoside and β-hydroxy acteoside were found for the first time in Chirita genus.

This is the first study on the cytotoxic activity of Oldenlandia pinofolia

2

species in Vietnam and in the world. From this species, 14 compounds have been isolated and structurally elucidated, consisting of a novel compound

(1,4,6-trihydroxy-2-methyl-anthraquinone) and two compounds (lutein và afzelin) isolated for first time from Oldenlandia genus. The n-butanol extract and four isolated compounds inhibited the proliferation of chronic myelogenous leukemia cells, induced apoptosis, and activated caspase 3 (p<0.05). 5. The structure of the thesis

The thesis includes preface (2), contents (131 pages, divided into 4 charptes), conclusion and recommendation (2 pages). There are also list of

abbreviations, list of tables, list of figures, published scientific works related to this

thesis and references and appendices.

THE CONTENT OF THE THESIS

CHAPTER 1: OVERVIEW

1.1. Overview of Allophylus genus

1.1.1. Botanical introduction

1.1.2. Use in traditional medicine

1.1.3. Studied results on the chemical composition and biological activity

1.1.4. Allophylus livescens

Until now, there have been no phytochemical and biological activity work about this species. 1.2. Overview of Chirita genus



1.2.3. Study results on the chemical composition and biological activity

1.2.4. Chirita halongensis

Chirita halongensis, an endemic species of Ha Long Bay, detected in the year 2000 and until now has not been chemically and biologically investigated. 1.3. Overview of Oldenlandia genus



1.3.3. Studied results on the chemical composition and biological activity

1.3.4. Oldenlandia pinifolia

Oldenlandia pinifolia (Wall. Ex G. Don) Kuntze (synonym Hedyotis pinifolia Wall. Ex G. Don) is a small herb growing in sandy areas from Hue to the south of Vietnam. To the best of our knowledge, there has been only one report on the phytochemistry of this species. 1.4. Iridoid compounds

1.4.1. Introduction

1.4.2. Classification

3

1.4.3. Biological activity

CHAPTER 2: PLANT METERIALS AND STUDY METHODS

2.1. Plant material and determination of scientific name

- Allophylus livescens Gagnep. was collected in Ha Long bay in April 2013 and determined by Dr. Tran Thi Phuong Anh, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology (VAST). A voucher specimen (No. VHH.HL 4.2013.1) was deposited in Institute of Chemistry-Vietnam Academy of Science and Technology (VAST). - Chirita halongensis Kiew & T.H.Nguyen was collected in Ha Long bay, Quang Ninh province in October 2013 and determined by Dr. Tran Thi Phuong Anh, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology (VAST). A voucher specimen (No. VHH.HL 10.2013.2) was deposited in Institute of Chemistry-Vietnam Academy of Science and Technology (VAST). - Oldenlandia pinifolia Wall. Ex G. Don was collected in Phu Vang, Thua Thien Hue province in October 2014 and determined by Dr. Do Xuan Cam, Hue university. A voucher specimen (No. VHH.TTH 10.2014.1) was deposited in Institute of Chemistry-Vietnam Academy of Science and Technology (VAST). 2.2. Methods for treatment and extraction

Leaves, twigs of Allophylus livescens and whole plants of Chirita halongensis, Oldenlandia pinifolia were extracted by using a general procedure. 2.3. Methods for isolation of secondary metabolites

Chromatographic methods: thin layer chromatography (TLC), column

chromatography (CC). 2.4. Methods for determination of chemical structure

Spectroscopic methods: Electron Spray Ionisation Mass Spectroscopy (ESI-MS) and High Resolution Electron Spray Ionisation Mass Spectroscopy (HR-ESI-MS), one/two-dimention nuclear magnetic resonance spectra (NMR). 2.5. Methods for biological assays

2.5.1. Cytotoxic activity

The test determined the total cell protein content based on the optical density measured when the cellular protein component was stained with Sulforhodamine B (SRB) 2.5.2. Apoptotic activity

The test determined by the Hoechst 33342 staining method and nuclei staining method with propidium iodide and the analysis by flow cytometry. CHAPTER 3: EXPERIMENT

3.1. Isolation of compounds

3.1.1. Isolation of compounds from Allophylus livescens

4

Allophylus livescens's leaves and twigs

0.6 kg

Extraction with MeOH 95 %(4 x 2.0L)

Total extract

Extraction with n-hexane

ALH (9.0 g)

n-hexane/ EtOAc, 95/5 0/100

H3 (1.7 g) H4 (1.2 g)

n-hexane/ DCM, 95/5

AL1

1.3 g

n-hexane/ DCM, 9/1

AL3 + AL 4

30 mg


ALE (8.0 g)

n-hexane/ EtOAc, grad.

H8 (1.7 g)

n-hexane/MeOH, 95/5

AL2

63 mg

AL5

35 mg

Extraction with n-BuOH

ALB

26.3 g

H2O

aqueous

Figure 3.1. Isolation of compounds from Allophylus livescens

3.1.2. Physical and spectroscopic data of isolated compounds from Allophylus

livescens

3.1.2.1. 1,6,10,14 Phytatetraen-3-ol (AL1)

Oil, Rf = 0.48 (n-hexane:CH2Cl2 = 95:5, v/v). ESI-MS, m/z = 291

[M+H]+,

1H-NMR (500 MHz, CD3OD): H (ppm) 5.21 (d, 17.5, H-1); 5.05 (d,

11.0, H-1); 5.91 (dd, 10.5 & 17.5; H-2); 1.55 (m, H-4); 1.95 – 2.09 (m, H-5);

5.14 (t, 7.0; H-6); 1.95 – 2.09 (m, H-8, H-9, H-12, H-13); 5.11 (t, 6.5; H-10);

5.11 (t, 6.5, H-14); 1.68 (s, H-16); 1.59 (s, H-17); 1.27 (s, H-18); 1.60 (s, H-19,

H-20). 13

C-NMR (125 MHz, CD3OD): C (ppm) 111.6 (C-1); 145.1 (C-2); 73.4

(C-3); 42.1 (C-4); 22.7 (C-5); 124.4 (C-6); 135.5 (C-7); 39.7 (C-8); 26.5 (C-9);

124.1 (C-10); 135.0 (C-11); 39.7 (C-12); 26.7 (C-13); 124.2 (C-14); 131.2 (C-

15); 25.6 (C-16); 17.6 (C-17); 27.8 (C-18); 16.0 (C-19, C-20).

3.1.2.2. Catechin (AL2)

Orange power, Rf = 0.56 (CHCl3:MeOH:H2O = 6.5:3.5:0.5, v/v). 1H-NMR

(500 MHz, CD3OD): H (ppm) 4.60 (d, 7.5 Hz, H-2); 4.00 (m, H-3); 2.53 (dd,

16.0, 8.1 Hz, H-4a); 2.87 (dd, 16.0; 8.1 Hz, H-4b); 5.88 (d, 2.3 Hz, H-6); 5.96 (d,

2.3 Hz, H-8); 6.86 (d, 1.9 Hz, H-2’); 6.79 (d, 8.1 Hz, H-5’); 6.74 (dd, 8.1; 1.9 Hz,

H-6’).13

C-NMR (125 MHz, CD3OD): C (ppm) 82.8 (C-2); 68.8 (C-3); 28.5 (C-4);

156.9 (C-5); 95.5 (C-6); 157.5 (C-7); 96.3 (C-8); 157.8 (C-9); 100.8 (C-10); 132.2

(C-1’); 115.3 (C-2’); 146.2 (C-3’); 146.2 (C-4’); 116.1 (C-5’); 120.0 (C-6’).

3.1.2.3. Stigmasterol(AL3)

White needles, Rf = 0.55 (n-hexane:EtOAc = 3:1, v/v). 1H-NMR (500

5

MHz, CDCl3): H (ppm) 3.51 (m, H-3); 5.34 (m, H-5); 0.92 (d, 6.5 Hz, CH3-

19); 5.01 (dd, 8.5; 15.0 Hz, H-20); 5,15 (dd, 8.5; 15.0 Hz, H-21); 0.84 (t, 7.2

Hz, CH3-24); 0.83 (d, 6.5 Hz, CH3-26); 0.80 (d, 6.6 Hz, CH3-27); 0.70 (s, CH3-

28); 1.03 (s, CH3-29).

3.1.2.4. -sitosterol(AL4)

White needles, Rf = 0.55 (n-hexane:EtOAc = 3:1, v/v). 1H-NMR (500

MHz, CDCl3): H (ppm) 3.51 (m, H-3); 5.34 (m, H-5); 0.92 (d, 6.5 Hz, CH3-

19); 0.85 (t, 7.2 Hz, CH3-24); 0.83 (d, 6.5 Hz, CH3-26); 0.81 (d, 6.6 Hz, CH3-

27); 0.68 (s, CH3-28); 1.02 (s, CH3-29).

3.1.2.5. -sitosterol glucoside (AL5)

White needles, Rf = 0.45 (CH2Cl2:MeOH = 9:1, v/v)

3.2. Isolation of compounds from Chirita halongensis

The whole palnt of Chirita halongensis

0.64 kg

Extraction with MeOH 95% (4 x 2.0L)

Total extract


CHH (6.0 g)

1. n-hexane/ EtOAc, grad.

2. n-hexane/ DCM, grad.3. Sephadex LH-20, DCM/MeOH, 1/9

CH1

12 mg


CHE (9.1 g)

DCM/MeOH, grad.

CH3

330 mg

CH4

700 mg


CHB

26.0 g

H2O

aqueous

CH2

15 mg DCM/MeOH, grad.

B3

360 mg

CH5

18 mg

DCM/MeOH, grad.

B3

360 mg

DCM/MeOH, grad.

CH6

25 mg

CH7

35mg

B10

540 mg

Sephadex LH-20, MeOH

CH8

20 mg

CH9

15 mg

Figure 3.2. Isolation of compounds from Chirita halongensis

3.2.2. Physical and spectroscopic data of isolated compounds from Chirita

halongensis

3.2.2.1. 7-Hydroxytectoquinone (CH1):

Yellow needles, Rf = 0.30 (CH2Cl2:MeOH = 4.8:0.2, v/v). 1H NMR (500

MHz, CD3OD): H (ppm) 8.07 (brs, H-1); 7.68 (dd, 1.0 & 8.0 Hz, H-3); 8.16 (d,

8.0 Hz; H-4); 8.17 (d, 8.5 Hz, H-5); 7.21 (dd, 2.5 & 8.5 Hz, H-6); 7.59 (d, 2.5

Hz, H-8); 2.54 (s, 2-CH3).13

C NMR (125MHz, CD3OD): C (ppm) 128.2 (C-1);

146.3 (C-2); 136.1 (C-3); 128.2 (C-4); 132.8 (C-4a); 130.9 (C-5); 122.3 (C-6);

6

164.7 (C-7); 113.5 (C-8); 137.1 (C-8a); 184.6 (C-9); 134.9 (C-9a); 183.3 (C-

10); 127.2 (C-10a); 21.8 (2-Me).

3.2.2.2. 3,24-Dihydroxy-urs-12-ene-28-oic acid (CH2)

Amorphous white power, Rf = 0.3 (n-hexane:EtOAc = 1:1, v/v). (-)-ESI-

MS m/z: 471.0 [M-H]-, molecular formula: C30H48O4.

1H-NMR (500 MHz,

CDCl3 & CD3OD): H (ppm) 3.83 (br s, H-3); 2.19 (d, 11.0 Hz, H-18); 3.70 (d,

11.0 Hz, Ha-24); 3.46 (d, 11.0 Hz, Hb-24). 13

C-NMR (125 MHz, CD3OD): C

(ppm) 34.4 (C-1); 24.5 (C-2); 71.3 (C-3), 44.0 (C-4); 50.6 (C-5); 19.5 (C-6);

34.6 (C-7); 40.9 (C-8); 48.8 (C-9, C-17); 37.9 (C-10); 25.3 (C-11); 126.9 (C-

12); 139.6 (C-13); 43.3 (C-14); 29.2 (C-15); 26.1 (C-16); 54.4 (C-18); 40.5 (C-

19); 40.4 (C-20); 30.7 (C-21); 38.1 (C-22); 22.8 (C-23); 66.3 (C-24); 16.3 (C-

25); 17.6 (C-26); 24.1 (C-27); 181.6 (C-28); 17.7 (C-29); 21.6 (C-30).

3.2.2.3. Ursolic acid (CH3)

Amorphous white power, Rf = 0.5 (CH2Cl2:MeOH = 94:6, v/v). (-)-ESI-


1H-NMR (500 MHz,

CDCl3): H (ppm) 2.98 (m, H-3), 5.11 (m, H-12); 2.09 (d, 11.3 Hz, H-18); 0.88

(s, Me-23); 0.66 (s, Me-24); 0.85 (s, Me-25); 0.73 (s, Me-26); 1.02 (s, Me-27);

0.79 (d, 6.4 Hz, Me-29); 0.89 (d, 8.7 Hz, Me-30). 13

C-NMR (125 MHz, CDCl3):

C (ppm) 38.2 (C-1); 26.9 (C-2); 76.9 (C-3); 38.4 (C-4); 54.8 (C-5); 18.0 (C-6);

30.2 (C-7); 39.1 (C-8); 47.0 (C-9); 36.5 (C-10); 23.8 (C-11); 124.6 (C-12); 138.2

(C-13); 41.6 (C-14); 32.7 (C-15); 22.8 (C-16); 46.83 (C-17); 52.4 (C-18); 38.4 (C-

19); 38.5 (C-20); 27.5 (C-21); 36.3 (C-22); 28.3 (C-23); 16.91 (C-24); 16.1 (C-

25); 15.2 (C-26); 23.3 (C-27); 178.3 (C-28); 17.0 (C-29); 21.1 (C-30).

3.2.2.4. Oleanolic acid (CH4)

Amorphous white power, Rf = 0.48 (CH2Cl2:MeOH = 94:6, v/v). (-)-ESI-


1H-NMR (500 MHz,

CDCl3): H (ppm) 5.27 (1H, t, 3.5 Hz, H-12); 3.20 (1H, dd, 4.0; 11.0 Hz, H-3);

2.81 (1H, dd, 4.0; 13.5 Hz, H-18); 1.12; 0.97; 0.91; 0.90; 0.89; 0.76; 0.74 (each

signal 3H, s, Me-23, 24, 25, 26, 27, 29, 30). 13

C-NMR (125 MHz, CDCl3): C

(ppm) 38.4 (C-1); 27.7 (C-2); 79.1 (C-3); 38.8 (C-4), 55.3 (C-5); 18.3 (C-6); 32.7

(C-7); 39.3 (C-8); 47.7 (C-9); 37.1 (C-10); 23.0 (C-11); 122.7 (C-12); 143.6 (C-

13); 41.7 (C-14); 27.2 (C-15); 23.4 (C-16); 46.5 (C-17); 41.1 (C-18); 45.9 (C-

19); 30.7 (C-20); 33.8 (C-21); 32.5 (C-22); 28.1 (C-23); 15.6 (C-24); 15.3 (C-

25); 17.1 (C-26); 25.9 (C-27); 181.6 (C-28); 33.1 (C-29); 23.6 (C-30).

3.2.2.5. 2-(3,4-Dihydroxyphenyl)ethyl--D-glucopyranoside (CH5)

Amorphous pale yellow power, Rf = 0.3 (EtOAc:MeOH:H2O = 4:0.5:0.1,

v/v). (-)-ESI-MS m/z: 315.0 [M-H]-, molecular formula C14H20O8.

1H NMR

(500 MHz, CD3OD): H (ppm) 6.71 (d, 1.5 Hz, H-2); 6.69 (d, 8.5 Hz, H-5); 6.57

(dd, 1.5 & 8.5 Hz, H-6); 2.80 (m, H-7); 3.72 (m, H-8); 4.05 (m, H-8); 4.31 (d,

8.0 Hz, Glc-1); 3.20 (t, 8.0 Hz, Glc-2); 3.28 – 3.39 (m, Glc-3, 4, 5); 3.68 (dd,

7

5.0 & 12.0 Hz, Glc-6); 3.88 (dd, 1.5 & 12.0 Hz, Glc-6). 13

C-NMR (125 MHz,

CD3OD): C (ppm) 131.6 (C-1); 117.1(C-2); 146.1 (C-3); 144.6 (C-4); 116.3

(C-5); 121.3 (C-6); 36.6 (C-7); 72.1 (C-8); 104.4 (Glc-1); 75.1(Glc-2); 77.9

(Glc-3); 71.6 (Glc-4); 78.1 (Glc-5); 62.7 (Glc-6).

3.2.2.6. Acteoside (CH6)

Amorphous power, Rf = 0.56 (CH2Cl2:MeOH:H2O = 3.75:1.0:0.1, v/v). 1H-NMR (500 MHz, CD3OD): H (ppm) 6.73 (d, 2.0 Hz, H-2); 6.71 (d, 8.0 Hz,

H-5); 6.58 (dd, 2.0; 8.5 Hz, H-6); 3.75 (m, H-8); 4.06 (m, H-8); 2.81 (m, H-7);

7.09 (d, J = 1.5 Hz, H-2’); 6.81 (d, 8.0 Hz, H-5’); 6.98 (dd, 1.5 & 8.0 Hz, H-6’);

6.30 (d, 16.0 Hz, H-8’); 7.62 (d, 16.0 Hz, H-7’); 4.40 (d, 8.0 Hz, H-1”); 3.3 –

4.1 (m, H-2”, 3”); 4.95 (t, 9.0 Hz, H-4”); 3.3 – 4.1 (m, H-5”); 5.22 (brs, H-1”’);

3.3 -4.1 (m, H-2”’, H-3”’, H-4”’, H-5”’); 1.12 (d, 6.5 Hz, H-6”’).13

C-NMR (125

MHz, CD3OD): C (ppm) 131.5 (C-1); 116.5 (C-2); 144.6 (C-3); 146.0 (C-4);

117.1 (C-5); 121.3 (C-6); 72.3 (C-8); 36.5 (C-7); 127.6 (C-1’); 114.7 (C-2’);

149.7 (C-3’); 146.7 (C-4’); 116.3 (C-5’); 123.2 (C-6’); 168.3 (C-9’); 115.3 (C-

8’); 148.0 (C-7’); 104.1 (C-1”); 76.1 (C-2”); 81.6 (C-3”); 70.4 (C-4”); 75.9 (C-

5”); 62.3 (C-6”); 103.0 (C-1”’); 72.0 (C-2”’); 72.2 (C-3”’); 73.8 (C-4”’); 70.6

(C-5”’); 18.4 (C-6”’).

3.2.2.7. Isoacteoside (CH7)

Amorphous power, Rf = 0.44 (CH2Cl2:MeOH:H2O = 3.75:1.0:0.1, v/v). 1H-NMR (500 MHz, CD3OD): H (ppm) 6.70 (d, 1.5 Hz, H-2); 6.67 (d, 8.0 Hz,

H-5); 6.55 (dd, 1.5; 8.0 Hz, H-6); 3.73 (m, H-8); 4.05 (m, H-8); 2.80 (m, H-7);

7.06 (d, 2.0 Hz, H-2’); 6.79 (d, 8.0 Hz, H-5’); 6.90 (dd, 2.0 & 8.5 Hz, H-6’);

6.30 (d, 16.0 Hz, H-8’); 7.58 (d, 16.0 Hz, H-7’); 4.33 (d, 8.0 Hz, H-1”); 3.4 –

4.0 (m, H-2”, H-3”, H-4”, H-5”); 4.39 (brt, 5.5 Hz, H-6”); 4.52 (dd, 1.5; 11.5

Hz, H-6”); 5.21 (brs, H-1”’); 3.4 – 4.0 (m, H-2”’, H-3”’, H-4”’,H-5”’); 1.28 (d,

6.0 Hz, H-6”’). 13

C-NMR (125 MHz, CD3OD): C (ppm) 131.4 (C-1); 117.1 (C-

2); 146.0 (C-3); 144.6 (C-4); 116.4 (C-5); 121.3 (C-6); 72.4 (C-8); 36.6 (C-7);

127.7 (C-1’’); 115.1 (C-2’); 146.7 (C-3’); 149.5 (C-4’); 116.6 (C-5’); 123.2 (C-

6’); 169.2 (C-9); 114.8 (C-8’); 147.2 (C-7’); 104.3 (C-1”); 75.3 (C-2”); 84.0 (C-

3”); 70.0 (C-4”); 75.6 (C-5”); 64.6 (C-6”); 102.7 (C-1”’); 72.2 (C-2”’); 72.3 (C-

3”’); 74.0 (C-4”’); 70.4 (C-5”’); 17.9 (C-6”’).

3.2.2.8. Decaffeoylacteoside (CH8)

Amorphous power, Rf = 0.56 (CH2Cl2:MeOH:H2O = 3.5:1.0:0.1, v/v). (-)-

ESI-MS m/z: 461.0 [M-H]-, molecular formula: C20H30O12.

1H-NMR (500 MHz,

CD3OD): H (ppm) 6.71 (d, 1.5 Hz, H-2); 6.69 (d, 8.0 Hz, H-5); 6.57 (dd, 2.0;

8.0 Hz, H-6); 3.72 (m, H-8); 4.01 (m, H-8); 2.80 (m, H-7); 4.31 (d, 8.0 Hz, H-

1”); 3.3 – 4.1 (m, H-2”, H-3”, H-4”, H-5”); 3.37 (brt, 9,5 Hz, H-6”); 3.42 (brt,

9.5 Hz, H-6”); 5.17 (d, 1.0 Hz, H-1”’); 3.3 – 4.1 (m, H-2”’, H-3”’, H-4”’, H-

8

5”’); 1.26 (d, 7,0 Hz, H-6”’) và 13

C-NMR (125 MHz, CD3OD): C (ppm) 131.5

(C-1); 116.3 (C-2); 144.6 (C-3); 146.1(C-4); 117.1 (C-5); 121.2 (C-6); 72.1 (C-

8); 36.5 (C-7); 104.2 (C-1”); 75.6 (C-2”); 84.5 (C-3”); 70.1 (C-4”); 77.8 (C-5”);

62.7 (C-6”); 102.8 (C-1”’); 72.2 (C-2”’); 72.3 (C-3”’); 74.0 (C-4”’); 70.2 (C-

5”’); 17.9 (C-6”’).

3.2.2.9. -hydroxyacteoside (CH9)

Amorphous power, Rf = 0.38 (CH2Cl2: MeOH: H2O = 3.5:1.0:0.5, v/v). 1H-

NMR (500 MHz, CD3OD): H (ppm) 6.73 (d, 2.0 Hz, H-2); 6.76 (d, 8.0 Hz, H-

5); 6.87 (dd, 2.0; 8.0 Hz, H-6); 3.57 (m, H-8); 4.00 (m, H-8); 4.77 (m, H-7);

7.07 (d, 2.0 Hz, H-2’); 6.80 (d, 8.0 Hz, H5’); 6.98 (dd, 2.0 & 8.0 Hz, H-6’);

6.29 (d, 16.0 Hz, H-8’); 7.62 (d, 16.0 Hz, H-7’); 4.40 (d, 8.0 Hz, H-1”); 3.5 –

4.0 (m, H-2”, H-3”); 4.96 (t, 9.0 Hz, H-4”); 3.5 – 4.0 (m, H-5”); 3.42 (brt, 8.5

Hz, H-6”); 3.84 (brt, 9.0 Hz, H-6”); 5.22 (brs, H-1”’); 3.5 – 4.0 (m, H-2”’, H-

3”’, H-4”’, H-5”’); 1.12 (d, 6.0 Hz, H-6”’). 13

C-NMR (125 MHz, CD3OD): C

(ppm) 133.7 (C-1); 114.6 (C-2); 146.3 (C-3); 146.1 (C-4); 116.5 (C-5); 119.0

(C-6); 76.7 (C-8); 74.2 (C-7); 127.7 (C-1’’); 115.3 (C-2’); 146.9 (C-3’); 149.8

(C-4’); 116.2 (C-5’); 123.2 (C-6’); 168.3 (C-9’); 114.7 (C-8’); 148.0 (C-7’); 104.6

(C-1”); 76.4 (C-2”); 81.3 (C-3”); 70.5 (C-4”); 76.1 (C-5”); 62.3 (C-6”); 102.9 (C-

1”’); 72.1 (C-2”’); 72.4 (C-3”’); 73.8 (C-4”’); 70.4 (C-5”’); 18.4 (C-6”’).

3.3. Isolation of compounds from Oldenlandia pinifolia

The whole palnt of Oldenlandia pinifolia

2.1 kg

Extraction with MeOH 95% (4 x 3.0L)

Total extract


HPH (9.0 g)

1. n-hexane/ EtOAc, grad.2. Sephadex LH-20, DCM/MeOH, 1/9

HP1

12 mg


HPE (34.2 g)

1. n-hexane/ EtOAc, 100/0 1/12. Sephadex LH-20, MeOH

HP6

30 mg

HP4

10 mg


HPB

32.0 g

H2O

aqueousHP5

11 mg

HP8

30 mg

HP2

5 mgHP3

6 mgHP7

200 mg 1. DCM/MeOH/H2O, 4/1/0 3/1/0.1

2. Sephadex LH-20, MeOH

HP9

11 mgHP10

10 mg

HP11

16 mgHP12

10 mg

HB13

10 mgHP14

40 mg

Figure 3.3. Isolation of compounds from Oldenlandia pinifolia

3.3.2. Physical and spectroscopic data of isolated compounds from

Oldenlandia pinifolia

3.3.2.1. 1,4,6-Trihydroxy-2-methyl-anthraquinone (HP1)

9

Orange powder, Rf = 0.45 (n-hexane:CH2Cl2 = 4:1, v/v). HR-ESI-MS: m/z =

269.0464 [M-H]-. Molecular formula C15H10O5.

1H-NMR (500 MHz, CDCl3 +

CD3OD and DMSO-d6,) và 13

C-NMR (125 MHz, CDCl3 + CD3OD and DMSO-

d6,) (ppm) (Table 4.3).

3.3.2.2. 2-Hydroxy-1-methoxy-anthraquinone (HP2)

Orange-red needles, Rf = 0.52 (n-hexane: EtOAc = 4.5: 1, v/v). 1H NMR

(500 MHz, CDCl3): H (ppm) 7.36 (d, 9.0 Hz, H-3); 8.14 (1H, d, 9.0 Hz, H-4); 8.27

(m, H-5, H-8); 7.74 (m, H-6, H-7); 4.04 (s, OMe); 6.69 (s, OH). 13

C NMR (125

MHz, CDCl3): C (ppm) 146.6 (C-1); 155.6 (C-2); 120.3 (C-3); 125.8 (C-4); 127.1

(C-5); 133.9 (C-6, C-7); 126.9 (C-8); 182.7 (C-9); 182.1 (C-10); 133.0 (C-11);

134.5 (C-12); 125.7 (C-13); 127.6 (C-14); 62.3 (OMe).

3.3.2.3. 1,6-Dihydroxy-2-methylanthraquinone (HP3)

Orange powder, Rf = 0.47 (n-hexane:CHCl3:EtOAc = 1.0:1.5:1.0, v/v).(-)-

ESI-MS m/z: 253.0 [M-H]-, molecular formula: C15H10O4.

1H-NMR (500 MHz,

DMSO-d6): H (ppm) 7.61 (d, 7.5 Hz, H-3); 7.55 (d, 7.5 Hz, H-4); 7.44 (d, 2.5

Hz, H-5); 7.21 (dd, 2.5; 8.5 Hz, H-7); 8.08 (d, 8.5 Hz, H-8); 13.08 ( s, 1-OH);

2.27 (s, 2-CH3). 13

C-NMR (125 MHz, DMSO-d6): C (ppm) 159.9 (C-1); 114.6

(C-2); 136.8 (C-3); 118.6 (C-4); 112.5 (C-5); 163.9 (C-6); 121.4 (C-7); 129.8

(C-8); 187.6 (C-9); 181.7 (C-10); 131.1 (C-4a); 124.4 (C-8a); 134.2 (C-9a);

135.6 (C-10a); 15.7 (2-CH3).

3.3.2.4. Digiferruginol (HP4)

Orange-yellow needles, Rf = 0.5 (CH2Cl2: MeOH = 9:1, v/v). (-)-ESI-MS

m/z: 253.0 [M-H]-, molecular formula: C15H10O4.

1H-NMR (500 MHz, DMSO-

d6), δH (ppm) 7.77 (d, 7.5 Hz, H-3); 7.92 (d, 8.0 Hz, H-4); 8.20 (m, H-5); 7.95

(m, H-6, H-7); 7.92 (m, H-7); 8.25 (m, H-8); 4.66 (d, 5.0 Hz, 2 -CH2OH); 5.46

(t, 5.5 Hz, 2-CH2OH); 12.77 (s, 1-OH). 13

C NMR (125 MHz, DMSO- d6), C

(ppm) 158.4 (C-1); 138.2 (C-2); 131.3 (C-3); 118.8 (C-4); 126.8 (C-5); 134.5

(C-6); 135.1 (C-7); 126.6 (C-8); 188.7 (C-9); 181.8 (C-10); 133.6 (C-5a); 133.2

(C-8a); 114.9 (C-9a); 132.8 (C-10a); 57.4 (CH2OH).

3.3.2.5. Lutein(HP5)

Orange-red powder, Rf = 0.44 (n-hexane: EtOAc = 3.75:1.25, v/v). (+)-

ESI-MS m/z: 569.3 [M+H]+, molecular formula: C40H56O2.

1H NMR (500 MHz,

CDCl3): H (ppm) 1.48 (t, 12.0 Hz, H2-2); 4.00 (m, H-3); 2.04 (dd, 17.0, 10.0

Hz, H-4ax); 2.33 – 2.42 ( m, H-4eq); 6.12 (s, H-7, H-8); 6.15 (m, H-10); 6.58-

6.67 (m, H-11, H-15, H-11’, H-15’); 6.36 (d, 15.0 Hz, H-12); 6.25 (brd, 9.0 Hz,

H-14); 1.07 (s, 1-(CH3)2); 1.97 (s, 9-CH3, 13-CH3); 1.37 (dd, 13.0; 7.0 Hz, H-

2’ax); 1.84 (dd, 13.0; 6.0 Hz, H-2’eq); 4.25 (m, H-3’); 5.54 (brs, H-4’); 2.33 –

2.42 (m, H-6’); 5.43 (dd, 10.0; 15.5 Hz, H-7’); 6.15 (m, H-8’, H-10’); 6.36 (d,

15.0 Hz, H-12’); 6.25 (brd, 9.0 Hz, H-14’); 0.85 (s, 1’-CH3); 1.00 ( s, 1’-CH3);

10

1.63 (s, 5’-CH3); 1.91 (s, 9’-CH3); 1.97 (s, 13’-CH3). 13

C NMR (125 MHz,

CDCl3): C (ppm) 37.1 (C-1); 48.5 (C-2); 65.1 (C-3); 42.6 (C-4); 126.2 (C-5);

137.6 (C-6); 125.6 (C-7); 138.5 (C-8); 135.7 (C-9); 131.3 (C-10); 124.8 (C-11);

137.7 (C-12); 136.5 C-13); 132.6 (C-14); 130.1 (C-15); 28.7 (1-Me); 30.3 (1-

Me); 21.6 (5 –Me); 12.8 (9-Me, 13-Me); 34.0 (C-1’); 44.6 (C-2’); 65.9 (C-3’);

125.6 (C-4’); 137.8 (C-5’); 55.0 (C-6’); 128.7 (C-7’); 138.0 (C-8’); 135.1 (C-

9’); 130.8 (C-10’); 124.5 (11’); 137.6 (C-12’); 136.4 (C-13’); 132.6 (C-14’);

130.0 (C-15’); 24.3 (1’-Me); 29.5 (1’-Me); 22.9 (5’-Me); 14.1 (9’-Me); 13.1

(13’-Me).

3.3.2.6. Ursolic acid (HP6)

Amorphous white power, Rf = 0.5 (CH2Cl2:MeOH = 94:6, v/v).

3.3.2.7. Oleanolic acid (HP7)

Amorphous white power, Rf = 0.48 (CH2Cl2:MeOH = 94:6, v/v).

3.3.2.8. Asperuloside (HP8)

White power, Rf = 0.54 (CH2Cl2: MeOH: H2O = 4.0:1.0:0.1, v/v). (+)-

ESI-MS m/z: 437.0 [M+Na]+, molecular formula: C18H22O11.

1H NMR (500

MHz, CD3OD): H (ppm) 5.97 (d, 1.0 Hz, H-1); 7.32 (d, 2.0 Hz, H-3); 3.70 (m,

H-5); 5.59 (brd, 6.5 Hz, H-6); 5.75 (brs, H-7); 3.32 (m, H-9); 4.69 (dd, 14.0 Hz,

1.0 Hz, H-10a); 4.80 (dd, 14.0, 1.0 Hz, H-10b); 2.10 (s, CH3CO); 4.71 (d, 8.0

Hz, Glc-1); 3.22 (dd, 9.0, 8.0 Hz, Glc-2); 3.31 – 3.40 (m, Glc-3, 4, 5); 3.94 (dd,

12.0, 2.0 Hz, Glc-6); 3.70 (dd, 12.0; 6.0 Hz, Glc-6). 13

C NMR (125 MHz,

CD3OD): C (ppm) 93.3 (C-1); 150.3 (C-3); 106.1 (C-4); 37.4 (C-5); 86.3 (C-

6); 128.9 (C-7); 144.2 (C-8); 45.2 (C-9); 61.9 (C-10); 172.3 (C-11); 172.6

(CH3CO); 20.6 (CH3CO); 100.0 (Glc-1); 74.6 (Glc-2); 78.3 (Glc-3); 71.6 (Glc-

4); 77.8 (Glc-5); 62.8 (Glc-6).

3.3.2.9. Deacetyl asperuloside (HP9)

White power, Rf = 0.67 (CH2Cl2:MeOH = 4:1, v/v). (-)-ESI-MS m/z:

370.9 [M-H]-, molecular formula: C16H20O10.

1H NMR (500 MHz, CD3OD): H

(ppm) 5.97 (d, 1.5 Hz, H-1); 7.31 (d, 2.0 Hz, H-3); 3.70 (m, H-5); 5.58 (dd, 1.5;

6.5 Hz, H-6); 5.66 (brs, H-7); 3.32 (m, H-9); 4.21 (brs, H-10); 4.70 (d, 8.0 Hz,

Glc-1); 3.21 (dd, 9.0; 8.0 Hz, Glc-2); 3.30 – 3.60 (m, Glc-3, Glc-4, Glc-5); 3.94

(dd, 12.0; 2.0 Hz, Glc-6); 3.84 (dd, 12.0; 6.0 Hz, Glc-6). 13

C NMR (125 MHz,

CD3OD): C (ppm) 93.3 (C-1); 150.2 (C-3); 106.5 (C-4); 37.5 (C-5); 86.6 (C-

6); 125.7 (C-7); 149.8 (C-8); 45.0 (C-9); 60.1 (C-10); 172.8 (C-11); 99.9 (Glc-

1); 74.7 (Glc-2); 78.4 (Glc-3); 71.6 (Glc-4); 77.9 (Glc-5); 62.8 (Glc-6).

3.3.2.10. Asperulosidic acid (HP10)

White power, Rf = 0.56 (CH2Cl2:MeOH = 4:1, v/v). 1H NMR (500 MHz,

CD3OD): H (ppm) 5.07 (d, 9.0 Hz, H-1); 7.63 (s, H-3); 3.05 (m, H-5); 4.85 (m,

H-6); 6.04 (brs, H-7); 2.65 (t, 8.0 Hz, H-9); 4.97 (brd, 14.5 Hz, H-10a); 4.83

11

(brd, 14.5 Hz, H-10b); 2.03 (s, CH3CO); 4.75 (d, 8.0 Hz, Glc-1); 3.41 (brt, 8.5

Hz, Glc-2); 3.25 – 3.33 (m, Glc-3, Glc-4, Glc-5); 3.87 (brd, 10.0 Hz, Glc-6);

3.64 (dd, 5.0; 12.0 Hz, Glc-6). 13

C NMR (125 MHz, CD3OD): C (ppm) 101.2

(C-1); 154.6 (C-3); 109.0 (C-4); 42.7 (C-5); 75.5 (C-6); 131.9 (C-7); 14.9 (C-8);

46.4 (C-9); 63.8 (C-10); 170.0 (C-11); 172.5 (CH3CO-); 20.8 (CH3CO), 100.6

(Glc-1); 74.9 (Glc-2); 78.5 (Glc-3); 71.6 (Glc-4); 77.9 (Glc-5); 63.0 (Glc-6).

3.3.2.11. Scandoside methyl ester (HP11)

White power, Rf = 0.48 (CH2Cl2:MeOH = 4.5:1, v/v).(-)-ESI-MS m/z:

403.0 [M-H]-, molecular formula: C17H24O11.

1H NMR (500 MHz, CD3OD): H

(ppm) 5.21 (t, 6.0 Hz, H-1); 7.53 (brs, H-3); 3.04 (m, H-5); 4.57 (brs, H-6); 5.83

(brs, H-7); 3.23 (brt, 8.0 Hz, H-9); 4.21 (brd, 15.0 Hz, H-10a); 4.36 (brd, 15.0,

H-10b); 3.77 (s, OCH3); 4.69 (d, 8.0 Hz, Glc-1); 3.20 – 3.34 (m, Glc-2 – Glc-5);

3.88 (brd, 11.5 Hz, Glc-6); 3.66 (brd, 11.5 Hz, Glc-6). 13

C NMR (125 MHz,

CD3OD): C (ppm) 98.3 (C-1); 153.9 (C-3); 110.8 (C-4); 45.6 (C-5); 82.6 (C-6);

130.1 (C-7); 147.5 (C-8); 47.1 (C-9); 61.0 (C-10); 170.3 (C-11); 52.0 (OCH3);

100.3 (Glc-1); 74.8 (Glc-2); 78.4 (Glc-3); 71.5 (Glc-4); 77.9 (Glc-5); 62.7 (Glc-6).

3.3.2.11. Afzelin (HP12)

Yellow power, Rf = 0.51 (CH2Cl2:MeOH = 4.25:0.75, v/v).(-)-ESI-MS m/z:

430.9 [M-H]-, công thức phân tử: C21H20O10.

1H-NMR (500 MHz, CD3OD): H

(ppm) 6.22 (d, 2.0 Hz, H-6); 6.40 (d, 2.0 Hz, H-8); 7.79 (d, 9.0 Hz, H-2’, 6’);

6.96 (d, 9.0 Hz, H-3’, 5’); 5.40 (d, 1.5 Hz, H-1”); 3.73 (dd, 3.0; 9.0 Hz, H-2”);

3.36 (d, 5.0 Hz, H-3”); 3.35 (d, 5.0 Hz, H-4”); 4.24 (dd, 2.0; 4.0 Hz, H-5”); 0.94

(d, 6.0 Hz, H-6”). 13

C-NMR (125 MHz, CD3OD): C (ppm) 159.3 (C-2); 136.2

(C-3); 179.6 (C-4); 163.2 (C-5); 99.9 (C-6); 166.1 (C-7); 94.8 (C-8); 158.6 (C-

9); 105.9 (C-10); 122.7 (C-1’); 131.9 (C-2’, C-6’); 116.5 (C3’, C-5’); 161.6 (C-4’);

103.5 (C-1”); 72.0 (C-2”); 72.2 (C-3”); 73.2 (C-4”); 71.9 (C-5”); 17.6 (C-6”).

3.3.2.13. Isorhamnetin-3-O--rutinoside(HP13)

Yellow power, Rf = 0.63 (CH2Cl2:MeOH:H2O = 3.75:1:0.1, v/v).(-)-ESI-MS

m/z: 623.2 [M-H]-, molecular formula: C28H32O16.

1H-NMR (500 MHz, CD3OD):

H (ppm) 6.22 (d, 1.5 Hz, H-6); 6.41 (d, 1.5 Hz, H-8); 7.95 (d, 2.0 Hz, H-2’); 6.93

(d, 8.5 Hz, H-5’); 7.64 (dd, 2.0, 8.5 Hz, H-6’); 5.24 (d, 7.5 Hz, H-1”); 4.55 (d, 1.0

Hz, H-1”’); 1.12 (d, 7.5 Hz, H-6”’); 3.96 (s, OMe). 13

C-NMR (125 MHz,

CD3OD): C (ppm) 158.5 (C-2); 135.5 (C-3); 179.3 (C-4); 162.9 (C-5); 100.0 (C-

6); 166.0 (C-7); 94.9 (C-8); 158.9 (C-9); 105.7 (C-10); 123.0 (C-1’); 114.6 (C-

2’); 148.3 (C-3’); 150.8 (C-4’); 116.1 (C-5’); 124.0 (C-6’); 104.4 (C-1”); 75.9

(C-2”); 78.1 (C-3”); 71.6 (C-4”); 77.3 (C-5”); 68.5 (C-6”); 102.5 (C-1”’); 72.0

(C-2”’); 72.3 (C-3”’); 73.8 (C-4”’); 69.8 (C-5”’); 17.9 (C-6”’); 56.8 (-OMe).

3.3.2.14. Rutin (HP14)

Yellow power, Rf = 0.4 (CH2Cl2:MeOH:H2O = 2.75:1:0.1, v/v). ESI-MS

12

m/z: 609.2 [M-H]-, 633.1 [M+Na]

+ molecular formula: C27H30O16.

1H-NMR

(500 MHz, CD3OD): H (ppm) 6.22 (brs, H-6); 6.42 (brs, H-8); 7.71 (brs, H-2’);

6.91 (d, 8.0 Hz, H-5’); 7.64 (brd, 8.5 Hz, H-6’); 5.07 (d, 7.5 Hz, H-1”); 3.53 (t,

9.0 Hz, H-2”); 3.81 (brd, 9.5 Hz, H-6”); 4.54 (brs, H-1”’); 3.69 (brs, H-2”’);

3.58 (dd, 3.5, 9.5 Hz, H-3”’); 3.31 (m, H-4”’); 3.44 (m, H-5”’); 1.14 (d, 6.5 Hz,

H-6”’). 13

C-NMR (125 MHz, CD3OD): C (ppm) 158.4 (C-2); 135.6 (C-3);

179.3 (C-4); 162.7 (C-5); 100.0 (C-6); 166.0 (C-7); 95.0 (C-8); 159.4 (C-9);

105.6 (C-10); 123.1 (C-1’); 117.7 (C-2’); 145.6 (C-3’); 149.7 (C-4’); 116.1 (C-

5’); 123.6 (C-6’); 104.8 (C-1”); 75.5 (C-2”); 78.0 (C-3”); 71.3 (C-4”); 77.1 (C-

5”); 68.6 CH2 (C-6”); 102.3 (C-1”’); 72.0 (C-2”’); 72.1 (C-3”’); 73.9 (C-4”’);

69.6 (C-5”’); 17.8 (C-6”’).

CHAPTER 4: RESULTS AND DISCUSSIONS

4.1. The results of Allophylus livescens

4.1.1. Chemical structures

This section presents the detailed results of spectral analysis and structure

determination of 5 compounds isolated from Allophylus livescens’s leaves and

twigs including: 1 diterpene (AL1), 1 flavonoid (AL2), 3 steroid (AL3-AL5).

1,6,10,14-Phytatetraen-3-ol (AL1) and catechin (AL2) have been found for the

first time in Allophylus genus. This is the first study on chemical constituents

and cytotoxic activity of this species.

OH

13

6101215

16171819

20

O

OH

OH

OH

OH

OH

2

3

45

7 9

10

1' 3'

4'6'

OH

1

3

4 5

6

7

8

9

10

11

12

13

1415

16

17

18

1920

2122

2324

25 26

2728

29

GlcO

OH

1

3

4 5

6

7

8

9

10

11

12

13

1415

16

17

18

1920

2122

2324

25 26

2728

29

AL1AL2

AL3AL4

1

3

4 510

9

6

7

8 14

11

12

13

15

16

1718

1920

2122

23

25 26

27

24

29

28

AL5

Figure 4.1. The chemical structure of compounds from Allophylus livescens

13


The cytotoxic assay on five human cancer cell lines (KB, Hep G2, LU-1,

MCF-7, SK) of compound 1,6,10,14-phytatetraene-3-ol showed negative result.

4.2. The results of Chirita halongensis


The chemical investigation of the whole plant of Chirita halongensis led

to the isolation of nine compounds. Their structures were elucidated by mass,

NMR spectroscopy and comparison with published data, including one

anthraquinone (CH1), three triterpenoids (CH2, CH3, CH4) and five

phenylethanoid glycosides (CH5-CH9). Three of them (oleanolic acid,

decaffeoylacteoside and -hydroxy acteoside) have been found for the first time

in Chirita genus. It is the first study on chemical constituents and cytotoxic

activity of this species. O

O

OH1

2

45

6

8 9

10

7 8a 9a

10a 4a

CH1

3

CH2 R1 = -OH, R2 = R3 = CH3, R4

= H, R5

= CH2OH

CH3 R1

= -OH, R2

= R3 = R5 = CH3, R4

= H

CH4 R1

= -OH, R3

= H, R2 = R4

= R5 = CH3

COOH

R5

R2

R3

R1

R4

1

3

57

9

11

12

14 16

1718

1921

22

2324

25 26

27

28

29

30

OOH

OH

OH

O

OH

OH

OH

1

3

4

CH5

O

OH

OH

OR1

O

OH

O

OR2

O

OH

OH

OH

R3

1"'

1"

1

3

4

CH6 R1 = Caffeoyl, R2 = R3 = H


CH8 R1

= R2 = R3

= H

CH6 R1 = Caffeoyl, R2 = H, R3 = OH

Figure 4.9. The chemical structure of compounds from Chirita halongensis


Three isolated compounds (CH2, CH7, CH8) exhibited weak activity

against all four tested cancer cell lines KB (human epidermic carcinoma),

HepG2 (human hepatocellular carcinoma), Lu (human lung carcinoma) and

MCF7 (human breast carcinoma).

4.3. The results of Oldenlandia pinifolia


Fourteen compounds were isolated from the n-hexane, ethyl acetate and n-

butanol extracts of the whole plant O. pinifolia by chromatography method. Their

14

structures were elucidated using MS and NMR analysis and compared with

reported data. They are four anthraquinones: 1,4,6-trihydroxy-2-methyl-

anthraquinone (HP1), 2-hydroxy-1-methoxy-anthraquinone (HP2), 1,6-

dihydroxy-2-methylanthraquinone (HP3), digiferruginol (HP4), a carotenoid: :

lutein (HP5), two triterpenes: ursolic acid (HP6), oleanolic acid (HP7), four

iridoid glycosides: asperuloside (HP8), deacetyl asperuloside (HPB3, HP9),

asperulosidic acid (HP10), scandoside methyl ester (HPB4, HP11) and three

flavonoid glycosides: afzelin (HP12), isorhamnetin-3-O--rutinoside (HP13),

rutin (HP14). Among them, 2-methyl-1,4,6-trihydroxy-anthraquinone is a new

one, and two compounds (HP5, HP12) were found for the first time in this genus.

O

O

R1

R2

R4

R3

12

4

3

5

7

8 9

10

6

9a

4a

8a

10a

OH

COOH1

3 5

6

7

2

4

8

9

10

1112

13

14

15

16

23 24

25 17

18

1920 21

2226

27

28

29

30

OH

OH

1

2

3

4

5

67

8

9

10

11

12

13

14

15

1'

2'

3'

4'5'

6'

7'

8'9'

10'

11'

12'13'

14'

15'

HP1 R1 = R3 = R4 = OH, R2 = Me

HP2 R1 = OMe, R2 = OH, R3 = R4 =H

HP3 R1 = R4 = OH, R2 = Me, R3 = H

HP4 R1 = OH, R2 = CH2OH, R3 = R4 = H

OH

COOH1

3 5

6

7

2

4

8

9

10

1112

13

14

15

16

23 24

25 17

18

1920 21

2226

27

28

29 30O

O CO

O O

OH

OH

OH

OH

R

H

H1

3

4

579

10

11

1'

2'

3'

4'

5'6'

6

8

HP6 HP7

HP8 R = OAc

HP9 R = OH

HP5

O

R1

OH

R2

O O

OH

OH

OH

OH

H

H1

3

4

56

7

89

10 1'

2'

3' 4'

5'

6'

HP10 R1 = COOH, R2 = OAc

HP11 R1 = COOMe, R2 = OH

O

O

OH

OH

OH

OO

OH

OH

OH

3'

1'

4'

6'2

34

9

10

7

5

1''

5''

2'

5'

2''

3''4''

6

8

OOH

OH O

OH

OH

OOH

O

O

OH

OH

O

OH

OH

OH

1"'

2"'

3"'

5"'

4"'

6"'

2

3

410

5

6

7

89

1'

3'

4'

1"

2''

3"4"

6" 5"

HP12HP13 R = OH

HP14 R = OMe

Figure 4.23. The chemical structure of compounds from Oldenlandia pinifolia

Compound HP1: 2-methyl-1,4,6-trihydroxy-anthraquinone O

O

OH

CH3

OH

OH HMBC

O

O

OH

OH

OH

12

4

3

5

7

8 9

10

6

9a

4a

8a

10a

15

Figure 4.24. The structure and the important HMBC (→) correlations of HP1

Compound HP1, obtained as an orange powder, showed the [M-H]- peak

at m/z 269.0464 in the HR-ESI-MS (Calcd for C15H9O5 269.0450). The NMR

spectra (table 4.3) showed characteristic signals of an 9,10-anthraquinone,

revealing two carbonyl carbons at δC 187.0 and 186.7, as well as signals of two

aromatic rings. The 1H NMR spectrum indicated a singlet aromatic proton at δH

7.12; typical aromatic protons of the 1,3,4-substituted ring at δH 8.22 (d, 8.5

Hz), 7.21 (dd, 2.5, 8.5 Hz) and 7.64 d (2.5 Hz) and a singlet at δH 2.37 due to an

aromatic methyl group. Besides carbonyl carbons and carbons, which are

suitable for proton signals, the 13

C NMR spectrum revealed eight aromatic

quaternary carbons comprising three hydroxy-carbons at δC 156.7, 157.4 and

163.6; a carbon connected to a methyl group at δC 141.2 and four other carbons.

The connections of two hydroxy groups at C-1 and C-4 were confirmed based

on two chelated hydroxy protons in 1H NMR (measured in DMSO-d6) at δH

13.52 and 12.73, as well as the downshifted carbonyl carbons. The position of

the hydroxy group at C-6 was deduced from HMBC correlations among signals

at δH 8.22 (H-8), δC 186.7 (C-9) and 163.6 (C-6); among signals at δH 7.64 (H-

5), δC 187.0 (C-10) and 121.9 (C-7); and among signals at δH 7.21 (H-7), 112.6

(C-5) and 125.9 (C-8a). The correlations among signals at 2.37 ppm, 128.0 (C-

3) and δC 156.7 (C-1) suggested that a methyl group was attached at C-2. Thus,

the structure of HP1 was 2-methyl-1,4,6-trihydroxy-anthraquinone, a novel

compound.

Figure 4.26.

1H-NMR spectrum of HP1 (CDCl3 + CD3OD)

16

Figure 4.27.

1H-NMR spectrum of HP1 (DMSO-d6)

Figure 4.28.

13C-NMR spectrum of HP1 (CDCl3 + CD3OD)

Figure 4.29. HMBC spectrum of HP1

17

Table 4.3. NMR spectral data of HP1

C CDCl3 + CD3OD DMSO-d6 1H

13C

1H

13C

1 - 156.7 - 155.9

2 - 141.2 - 140.6

3 7.12 (s) 128.0 7.29 (s) 127.8

4 - 157.4 - 156.6

4a - 111.7 - 111.1

5 7.64 (d, 2.5) 112.6 7.51 (d, 2.5) 112.4

6 - 163.6 - 164.5

7 7.21 (dd, 2.5; 8.5) 121.9 7.23 (dd, 2.5; 8.5) 122.1

8 8.22 (d, 8.5) 130.1 8.14 (d, 8.5) 130.4

8a - 125.9 - 124.1

9 - 186.7 - 185.8

9a - 111.9 - 111.4

10 - 187.0 - 186.3

10a - 136.1 - 135.4

2-CH3 2.37 (s) 16.6 2.3 (s) 16.3

1-OH - - 13.51 (s) -

4-OH - - 12.73 (s) -


4.3.2.1. Results of apoptotic activity of n-butanol extract against acute myeloid

leukemia cells OCI-AML

Results show that the most effective concentration was 300 μg/mL,

although all HBP concentrations were effective in specifically increased the cell

death. The low impact in decreasing the cell number was possibly due to the

lack of effect in blocking the proliferation of cells (no effect on the cell cycle).

4.3.2.2. Results of cytotoxic activity of n-butanol extract and some isolated

compunds

MTT assay resulted that the n-butanol extract and four its isolated

compounds (HP9, HP11, HP13, HP14) inhibited the proliferation of chronic

myelogenous leukaemia cells, among them n-butanol extract is the strongest.

4.3.2.3. Results of apoptotic activity of of n-butanol extract and some isolated

compunds

Based on Hoechst 33,342 staining, these compounds significantly induced

apoptosis in the following order: compound HP13 > n-butanol extract > HP14

> HP9 > HP11.

The effect on caspase 3 in K562 cells that were untreated or treated with

18

the reported compounds was shown in Figure 4.69.

Compound 8: HP9, compound 10: HP11, compound12: HP13, compound13: HP14

Figure 4.69. The fold-change relevant caspase 3-inducing activities of n-

butanol extract and compounds HP9, HP11, HP13, HP14 on K562 at 24 h.

Cultured cells (5 × 104 cells/well) were treated with 300 μg/mL of either extract

or compound 8; 10; 12; 13 (respectively, with 806.45 μM of 8; 742.57 μM of

10; 480.77 μM of 12 and 491.80 μM of 13). Camptothecin (1.44 μM) was

served as reference control. Notes: *p<0.05, **p<0.01

As shown in Figure 4.69, chronic myelogenous leukemic (K562) cells

treated with compounds HP13 and HP9 showed significantly increased caspase

3 activity (p < 0.05), whereas n-butanol activated caspase 3 similar to those of

compound HP11 and HP14 but lower than those of compound HP13 and HP9.

The results from Hoechst 33343 staining and caspase 3-inducing

exhibited that n-butanol extract and those four tested compounds induced

apoptosis and activated caspase 3 (p <0.05).

The phytochemical and cytotoxic activity results from three species:

Allophylus livescens, Chirita halongensis and Oldenlandia pinifolia are shown

in Table 4.13 and Figure 4.70.

Symbol Compounds Structure

Steroids

AL3 Stigmasterol

OH

1

3

4 5

6

7

8

9

10

11

12

13

1415

16

17

18

1920

2122

2324

25 26

2728

29

19

AL4 -sitosterol

RO

1

3

4 5

6

7

8

9

10

11

12

13

1415

16

17

18

1920

2122

2324

25 26

2728

29

AL4: R = H

AL5: R = Glc

AL5 -sitosterol glucoside

Anthraquinones

CH1 7-hydroxytectoquinone O

O

R1

R2

R4

R3

R5 1

2

4

3

5

78 9

10

6

9a

4a

8a

10a

CH1 R1 = R3 = R4 = H, R2 = Me, R5 = OH

HP1 R1 = R3 = R4 = OH, R2 = Me, R5 = H

HP2 R1 = OMe, R2 = OH, R3 = R4 = R5 = H

HP3 R1 = R4 = OH, R2 = Me, R3 = R5 = H

HP4 R1 = OH, R2 = CH2OH, R3 = R4 = R5 = H

HP1 1,4,6-trihydroxy-2-

methyl-anthraquinone

(novel)

HP2 2-hydroxy-1-methoxy-

anthraquinone

HP3 1,6-dihydroxy-2-

methylanthraquinone

HP4 digiferruginol

Carotenoid

HP5 Lutein

OH

OH

1

2

3

4

5

67

8

9

10

11

12

13

14

15

1'

2'

3'

4'5'

6'

7'

8'9'

10'

11'

12'13'

14'

15'

Terpenoids

AL1 1,6,10,14-phytatetraen-

3-ol

OH

13

6101215

16171819

20

CH2 3α, 24-Dihydroxy-urs-

12-ene-28-oic acid

HP6 = CH3 R1

= -OH, R2

= R3 = R5 = CH3, R4

= H

HP7 = CH4 R1

= -OH, R3

= H, R2 = R4

= R5 = CH3

CH2 R1 = -OH, R2 = R3 = CH3, R4

= H, R5

= CH2OH

COOH

R5

R2

R3

R1

R4

1

3

57

9

11

12

14 16

1718

1921

22

2324

25 26

27

28

29

30

CH3

HP6

Ursolic acid

CH4

HP7

Oleanolic acid

20

Iridoid glycosides

HP8 Asperuloside

O

O CO

O O

OH

OH

OH

OH

R 1

3

45

7

9

10

11

1'

2'

3'

4'

5'6'

6

8

HP8 R = OAc

HP9 R = OH

HP9 Deacetyl asperuloside

HP10 Asperulosidic acid

O

R1

OH

R2

O O

OH

OH

OH

OH

1

3

456

7

8 9

10 1'

2'

3' 4'

5'

6'

HP10 R1 = COOH, R2 = OAc

HP11 R1 = COOMe, R2 = OH

HP11 Scandoside methyl

ester

Flavonoids

AL2 Catechin

O

OH

OH

OH

OH

OH

2

3

45

7 9

10

1' 3'

4'6'

HP12 Afzelin

O

O

OH

OH

OH

OO

OH

OH

OH

3'

1'

4'

6'2

34

9

10

7

5

1''

5''

2'

5'

2''

3''4''

6

8

HP13

Isorhamnetin-3-O--

rutinoside OOH

OH O

OH

R

OOH

O

O

OH

OH

O

OH

OH

OH

1"'

2"'

3"'

5"'

4"'

6"'

2

3

410

5

6

7

89

1'

3'

4'

1"

2''

3"4"

6" 5"

HP13 R = OH

HP14 R = OMe

21

HP14 Rutin

Phenylethanoid glycosides

CH5 2-(3,4-

dihydroxyphenyl)ethyl

-β-D-glucopyranoside O

OH

OH

OH

O

OH

OH

OH

1

3

4

CH6

Acteoside

O

OH

OH

OR1

O

OH

O

OR2

O

OH

OH

OH

R3

1"'

1"

1

3

4



CH8 R1

= R2 = R3

= H

CH6 R1 = Caffeoyl, R2 = H, R3 = OH

CH7 Isoacteoside

CH8 Decaffeoylacteoside

CH9 β-hydroxy acteoside

O

O

OH

O

O

O

OHOH

OH

OHOH

OH

OH

OH

O

1"'

1"

12

3

4

5

61'

2'3'

4'6'

5'

CH7

Cytotoxic activity against human

cell lines (KB, HepG2 and LU-1)

with IC50 value of 51.04±1.86;

57.61±0.51; 125.71±1.84 μM

O

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

1"'

1"

12

3

4

5

6

CH8

Cytotoxic activity against human cell

lines (KB, HepG2 and LU-1) with IC50

value of 48.67±3.38; 78.80±3.34;

132.82±0.95 μM

O

O CO

O O

OH

OH

OH

OH

OH 1

3

45

7

9

10

11

1'

2'

3'

4'

5'6'

6

8

HP9

O

COOMeOH

OHO O

OH

OH

OH

OH

1

3

456

7

8 9

10 1'

2'

3' 4'

5'

6'

HP11

OOH

OH O

OH

R

OOH

O

O

OH

OH

O

OH

OH

OH

1"'

2"'

3"'

5"'

4"'

6"'

2

3

410

5

6

7

89

1'

3'

4'

1"

2''

3"4"

6" 5"

HP13 R = OH

HP14 R = OMe

The n-butanol extract and its four isolated compounds inhibited the

proliferation of chronic myelogenous leukemia cells, induced apoptosis, and

activated caspase 3.

22

CONCLUSIONS AND RECOMMENDATIONS

1. Conclusion

This is the first study on the chemical constituents and cytotoxic activities

of Allophylus livescens and Chirita halongensis species in Vietnam and in the

world.

The cytotoxic activity of Oldenlandia pinofolia species was studied for

the first time in Vietnam and in the world. The n-butanol extract and its four

isolated compounds inhibited the proliferation of chronic

myelogenous leukemia cells, induced apoptosis, and activated caspase 3.

26 compounds from these three species were isolated and structurally

elucidated using spectroscopic analysis. They contain 3 steroids: stigmasterol

(AL3), β-sitosterol (AL4) and β-sitosterol glucoside (AL5), 4 terpenes:

1,6,10,14-phytatetraen-3-ol (AL1), 3α, 24-dihydroxy-urs-12-ene-28-oic acid

(CH2), ursolic acid (CH3, HP6), oleanolic acid (CH4, HP7), 5

anthraquinones: 7-hydroxytectoquinone (CH1), 1,4,6-trihydroxy-2-methyl-

anthraquinone (HP1), 2-hydroxy-1-methoxy-anthraquinone (HP2), 1,6-

dihydroxy-2-methyl-anthaquinone (HP3) and digiferruginol (HP4), 1

carotenoid: lutein, 5 phenylethanoid glycoside: 2-(3,4-dihydroxyphenyl)ethyl-

β-D-glucopyranoside (CH5), acteoside (CH6), isoacteoside (CH7),

decaffeoylacteoside (CH8), β-hydroxyacteoside (CH9), 4 iridoid glycosides:

asperuloside (HP8), deacetyl asperuloside (HP9), asperulosidic acid (HP10),

scandoside methyl ester (HP11), 4 flavonoid: catechin (AL2), afzelin (HP12),

isorhamnetin-3-O--rutinoside (HP13), rutin (HP14). Among them, HP1 is a

novel compound and seven compounds were found for the first time in studied

genuses

2. Recommendations

Further research on the biological activity of some active compounds

from the three species to find out the correlations between chemical structure

and biological activity.

LIST OF PUBLISHED WORKS

1. Khieu Thi Tam, Dao Duc Thien, Nguyen Thi Hoang Anh, Trinh Thi Thuy,

Tran Van Loc, Tran Van Sung, Chemical constituents and biological activity

of Allophylus livescens Gagnep. collected in Halong bay, Vietnam Journal

of Chemistry - Vol 53, No 6 (2015).

2. Nguyen Thi Hoang Anh, Khieu Thi Tam, Nguyen Van Tuan, Dao Duc

Thien, Tran Duc Quan, Nguyen Thanh Tam, Nguyen Chi Bao, Thi Thao

Do, Nguyen Thi Nga, Trinh Thi Thuy, Tran Van Sung & Domenico V.

23

Delfino, Chemical constituents of Oldenlandia pinifolia and their

antiproliferative activities, Natural Product Research, Pages 1-7 |

Received 27 Sep 2017, Accepted 26 Nov 2017, Published online: 06 Dec

2017, doi.org/10.1080/14786419.2017.1410806.

3. Khieu Thi Tam, Nguyen Thi Hoang Anh, Nguyen Van Tuan, Đao Đuc

Thien, Nguyen Thanh Tam, Tran Duc Quan, Le Quoc Thang, Trinh Thi

Thuy, Tran Van Sung, Chemical constituents of Chirita halongensis Kiew &

T.H.Nguyen collected in Halong bay, Quang Ninh province, Viet Nam,

Accepted by Vietnam Journal of Chemistry.

4. Khieu Thi Tam, Nguyen Thi Hoang Anh, Nguyen Van Tuan, Dao Duc

Thien, Tran Duc Quan, Nguyen Thanh Tam, Nguyen Chi Bao, Trinh Thi

Thuy, Tran Van Sung, chemical constutuents of Oldenlandia pinifolia Wall.

collected in Thua Thien Hue, accepted in Journal Science and Technology.

ministry of education and training …gust.edu.vn/media/26/uftai-ve-tai-day26216.pdflivescens,...

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