Summary of doctoral thesis: Study on chemical constituents and cytotoxic activity of 3 species allophylus livescens gagnep, chirita halongensis kiew& t.h.nguyen and oldenlandia pinifolia (wa

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.....


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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


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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
1.2.1. Botanical introduction
1.2.2. Use in traditional medicine
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
1.3.1. Botanical introduction
1.3.2. Use in traditional medicine
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


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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
Extraction with n-hexane
Extraction with n-BuOH

ALH (9.0 g)
n-hexane/ EtOAc, 95/5

0/100

ALE (8.0 g)
n-hexane/ EtOAc, grad.

H4 (1.2 g)

H3 (1.7 g)
n-hexane/ DCM, 95/5
AL1
1.3 g

ALB
26.3 g

H2 O
aqueous

H8 (1.7 g)

n-hexane/ DCM, 9/1

n-hexane/MeOH, 95/5

AL3 + AL 4
30 mg
AL2
63 mg

AL5
35 mg

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). 13C-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 (C15); 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’).13C-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, CH319); 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, CH328); 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, CH319); 0.85 (t, 7.2 Hz, CH3-24); 0.83 (d, 6.5 Hz, CH3-26); 0.81 (d, 6.6 Hz, CH327); 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
Extraction with n-hexane
Extraction with n-hexane
Extraction with n-BuOH

CHH (6.0 g)
CHE (9.1 g)
1. n-hexane/ EtOAc, grad.
2. n-hexane/ DCM, grad.
3. Sephadex LH-20, DCM/MeOH, 1/9

DCM/MeOH, grad.

CH1
12 mg

CH2
15 mg

CHB
26.0 g

CH4
700 mg

CH3
330 mg

H2O
aqueous

DCM/MeOH, grad.

B3
360 mg

B10
540 mg

B3
360 mg

DCM/MeOH, grad.

Sephadex LH-20, MeOH

DCM/MeOH, grad.

CH5
18 mg
CH6
25 mg

CH7
35mg

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).13C 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);

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164.7 (C-7); 113.5 (C-8); 137.1 (C-8a); 184.6 (C-9); 134.9 (C-9a); 183.3 (C10); 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). (-)-ESIMS 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). 13C-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 (C12); 139.6 (C-13); 43.3 (C-14); 29.2 (C-15); 26.1 (C-16); 54.4 (C-18); 40.5 (C19); 40.4 (C-20); 30.7 (C-21); 38.1 (C-22); 22.8 (C-23); 66.3 (C-24); 16.3 (C25); 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). (-)-ESIMS m/z: 455.2 [M-H]-, molecular formula: C30H48O3. 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). 13C-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 (C19); 38.5 (C-20); 27.5 (C-21); 36.3 (C-22); 28.3 (C-23); 16.91 (C-24); 16.1 (C25); 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). (-)-ESIMS m/z: 455.2 [M-H]-, molecular formula: C30H48O3. 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). 13C-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 (C13); 41.7 (C-14); 27.2 (C-15); 23.4 (C-16); 46.5 (C-17); 41.1 (C-18); 45.9 (C19); 30.7 (C-20); 33.8 (C-21); 32.5 (C-22); 28.1 (C-23); 15.6 (C-24); 15.3 (C25); 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). 13C-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).
1
H-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”’).13C-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 (C8’); 148.0 (C-7’); 104.1 (C-1”); 76.1 (C-2”); 81.6 (C-3”); 70.4 (C-4”); 75.9 (C5”); 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).
1
H-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”’). 13C-NMR (125 MHz, CD3OD): C (ppm) 131.4 (C-1); 117.1 (C2); 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 (C6’); 169.2 (C-9); 114.8 (C-8’); 147.2 (C-7’); 104.3 (C-1”); 75.3 (C-2”); 84.0 (C3”); 70.0 (C-4”); 75.6 (C-5”); 64.6 (C-6”); 102.7 (C-1”’); 72.2 (C-2”’); 72.3 (C3”’); 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, H1”); 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 13C-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 (C8); 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 (C5”’); 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). 1HNMR (500 MHz, CD3OD): H (ppm) 6.73 (d, 2.0 Hz, H-2); 6.76 (d, 8.0 Hz, H5); 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”’, H3”’, H-4”’, H-5”’); 1.12 (d, 6.0 Hz, H-6”’). 13C-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 (C1”’); 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
Extraction with n-hexane
Extraction with n-hexane

HPH (9.0 g)

Extraction with n-BuOH

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

HP1
12 mg

HP2
5 mg

HP3
6 mg

HP5
11 mg

HPE (34.2 g)
1. n-hexane/ EtOAc, 100/0 1/1
2. Sephadex LH-20, MeOH
HPB
32.0 g

HP6
30 mg

HP7
200 mg

HP8
30 mg

HP4
10 mg

HP9
11 mg

H2 O
aqueous

1. DCM/MeOH/H2O, 4/1/0 3/1/0.1
2. Sephadex LH-20, MeOH

HP10
10 mg

HP11
16 mg

HP12
10 mg

HB13
10 mg

HP14
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 13C-NMR (125 MHz, CDCl3 + CD3OD and DMSOd6,)  (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). 13C 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). 13C-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, DMSOd6), 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). 13C 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); 6.586.67 (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, H2’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). 13C 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 (1Me); 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 (C9’); 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). 13C NMR (125 MHz,
CD3OD): C (ppm) 93.3 (C-1); 150.3 (C-3); 106.1 (C-4); 37.4 (C-5); 86.3 (C6); 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 (Glc4); 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). 13C NMR (125 MHz,
CD3OD): C (ppm) 93.3 (C-1); 150.2 (C-3); 106.5 (C-4); 37.5 (C-5); 86.6 (C6); 125.7 (C-7); 149.8 (C-8); 45.0 (C-9); 60.1 (C-10); 172.8 (C-11); 99.9 (Glc1); 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). 13C 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). 13C 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”). 13C-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 (C9); 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). 13C-NMR (125 MHz,
CD3OD): C (ppm) 158.5 (C-2); 135.5 (C-3); 179.3 (C-4); 162.9 (C-5); 100.0 (C6); 166.0 (C-7); 94.9 (C-8); 158.9 (C-9); 105.7 (C-10); 123.0 (C-1’); 114.6 (C2’); 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”’). 13C-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 (C5’); 123.6 (C-6’); 104.8 (C-1”); 75.5 (C-2”); 78.0 (C-3”); 71.3 (C-4”); 77.1 (C5”); 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.
6'

19

20

17

18

15

16

10

12

OH

HO

3

6

9 O

7

1

2

1'

4'

OH

3'

OH

3
10

5

AL1

AL2

OH

4
OH

12 29
28
1

11

3
4

10

22

21

26

25

12 29

27

16

14

28

3

HO

4

5
19
12
28

11

1
9
4

10

18
17

13
8

21 22

16

14
7

3

GlcO

29

23

20

10

16

14

7
6

24

20
18
17

13
8

9

AL3

6

11

1

15

7

9

HO

18
17

23

19

20

13
8

24

23

19

21

22

25

26
27

15

AL4

5

24
25 26
27

15

6
5

AL5

Figure 4.1. The chemical structure of compounds from Allophylus livescens


13
4.1.2. Cytotoxic activity
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
4.2.1. Chemical structures
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.
30 2

O
HO 7

8

6
5

8a

9

9a

R

1

2

R

12

3

10a 10 4a 4

25

O
CH1

11

1

OH
3
O

HOHO

1

4

OH

R

OH

1

5
23

OH

R

19
18

17

14

7

4

21

26

9

3

O

R

29 3

22
28

COOH
16

27

5

24

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

CH5
OR 2
R3

3

O
O

OR 1 O

OH

OH

1

OH

4

1"

OH

O

HO

OH

1"'

CH6
CH7
CH8
CH6

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

Figure 4.9. The chemical structure of compounds from Chirita halongensis

4.2.2. Cytotoxic activity
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
4.3.1. Chemical structures
Fourteen compounds were isolated from the n-hexane, ethyl acetate and nbutanol 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-methylanthraquinone (HP1), 2-hydroxy-1-methoxy-anthraquinone (HP2), 1,6dihydroxy-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.
R

O
8

9

8a

7

9a

1

1

R

2

5'

2

1

7
6

2

6

R

4

10a
5
10

4a 4

R

O

HP1
HP2
HP3
HP4

1

3

3

8

HO

4

4

12'

14'

12
11
25
1
9
2

26
14
8

10
5

3

19
1318

21

COOH
2

10
5

3

HO

6
24

O

8

30

R

H

10

1
O

1' O 5'
2'

HO

3'
OH
HP8 R = OAc

8

7
6

6'

5

OH

5'

9

O

OH

3' 4' OH
OH

4'

O

5''

3

6

10

5
OH
6"'

OH
2''

3'' OH
4''
OH

HP10 R1 = COOH, R2 = OAc

1'

2

O

4
O

1"
O

1''

O

9 O

7

5'

3 O

8

HO

OH

2 1' 6'

4

10

OH
4'

2'
HO

OH

OH

3'
3'

HO H R
4
6
3
5
7
9
O
8
1
2
R
H O 1' O
10

OH

HP9 R = OH

HP7

1

6'

4'

24

HP6

2'

3

5

6

4

23

HO

H

11
CO

9

20 21
19
12 18
22
13

26
17
COOH
14
16 28
8
15
7 27

11
25
9

1

28

1'

4

7

7 27

4

7'

6

15

9'

11'

O

22
17
16

OH
3'
2'

6'

HP5

29

20

8'

10'

13'

15'

14

R = R = R = OH, R = Me
R1 = OMe, R2 = OH, R3 = R4 =H
R1 = R4 = OH, R2 = Me, R3 = H
R1 = OH, R2 = CH2OH, R3 = R4 = H

29

23

15

2

30

HO

13
12

10

5

3

3

11

9

4'

O 1"'

5"'
4"'
HO 3"'

O
6"

2"'
OH
OH

5"
OH

OH
2''
3"
4" OH

HP13 R = OH

HP12

HP14 R = OMe

HP11 R1 = COOMe, R2 = OH

Figure 4.23. The chemical structure of compounds from Oldenlandia pinifolia
Compound HP1: 2-methyl-1,4,6-trihydroxy-anthraquinone
OH

O
8
8a

7
6

HO

5

9

9a

10a
10
O

4a

1

O

OH

CH3

2
3

4

OH

HO
O

OH

HMBC


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 δ H 2.37 due to an
aromatic methyl group. Besides carbonyl carbons and carbons, which are
suitable for proton signals, the 13C 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 (H5), 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 (C3) 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
1
2
3
4
4a
5
6
7
8
8a
9
9a
10
10a
2-CH3
1-OH
4-OH

CDCl3 + CD3OD
1
13
H
C
156.7
141.2
7.12 (s)
128.0

157.4
111.7
7.64 (d, 2.5)
112.6
163.6
7.21 (dd, 2.5; 8.5)
121.9
8.22 (d, 8.5)
130.1
125.9
186.7
111.9
187.0
136.1
2.37 (s)
16.6
-

DMSO-d6
H
7.29 (s)
7.51 (d, 2.5)
7.23 (dd, 2.5; 8.5)
8.14 (d, 8.5)
2.3 (s)
13.51 (s)
12.73 (s)
1

13

C
155.9
140.6
127.8
156.6
111.1
112.4
164.5
122.1
130.4
124.1
185.8
111.4
186.3
135.4
16.3
-

4.3.2. Cytotoxic activity
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 nbutanol 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

23

19
12 29
28
1

11

4

10

6
5

16

14
7

9
3

HO

18
17

13
8

24

20

15

21

22

25

26
27


19
AL4

-sitosterol

20

12 29

AL5

-sitosterol glucoside

28

11

1

CH1
HP1

HP2
HP3
HP4

27

6

10

4

26

25

22

15

7

9

21

16

14

3

RO

18
17

13
8

24

23

19

5

AL4: R = H
AL5: R = Glc
Anthraquinones
1
R
7-hydroxytectoquinone
O
2
5
8
1
9
1,4,6-trihydroxy-2R
R 7
9a
2
8a
methyl-anthraquinone
6
3
(novel)
4

10a
4a
R
4
5
10
3
2-hydroxy-1-methoxyR
O
anthraquinone
CH1 R1 = R3 = R4 = H, R2 = Me, R5 = OH
1,6-dihydroxy-2HP1 R1 = R3 = R4 = OH, R2 = Me, R5 = H
methylanthraquinone
HP2 R1 = OMe, R2 = OH, R3 = R4 = R5 = H
HP3 R1 = R4 = OH, R2 = Me, R3 = R5 = H
digiferruginol
HP4 R1 = OH, R2 = CH2OH, R3 = R4 = R5 = H

Carotenoid
HP5

Lutein

5'
1

7
6

2

8

AL1

CH3
HP6
CH4
HP7

15

14'

12'
13'

15'

14

10'

8'
9'

11'

4'

7'

OH
3'
2'

6'
1'

4

Terpenoids
19
1,6,10,14-phytatetraen3-ol
20

CH2

13
12

10

5

3

HO

11

9

17

18

15

10

12

3α, 24-Dihydroxy-urs12-ene-28-oic acid
Ursolic acid

6

30 2

R

R

29 3

R

12
25

11

1

Oleanolic acid
3

R

1

5
23

R

19
18

14

7

OH
3

4

21

17

26

9

16

22
28

COOH
16

27

5

24

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

1


20
Iridoid glycosides
HP8

HP9

Asperuloside
Deacetyl asperuloside

11
CO

O
6

4

5

3

7
8
R

O

9

1

10

2'

Asperulosidic acid
Scandoside methyl
ester

OH

3'
OH

1

R
5 4

HO
6
7
8
R

OH

4'

HO
HP8 R = OAc
HP9 R = OH

HP10

HP11

6'

1' O 5'

O

9

2

3
O

1

6'

O 1' O

10

OH

5'

2'
HO

3' 4' OH
OH

HP10 R1 = COOH, R2 = OAc
HP11 R1 = COOMe, R2 = OH

Flavonoids
AL2

Catechin

6'
HO

7

O

9

2

1'

4'

OH

3'

OH

3
10

5

OH

4
OH

HP12

3'

Afzelin
HO

8

7
6

9

O

OH

4'
5'

2 1' 6'

4

10

5

3 O
1''

O

OH

O

2''

5''

HP13

OH

2'

3'' OH
4''
OH
R
3'

Isorhamnetin-3-O-rutinoside

OH
4'

8

HO

9 O

1'

2

7
3

6

10

5
OH

O

4
O

1"
O

6"'

O 1"'

5"'
4"'
HO 3"'

O
6"

2"'
OH
OH

5"
OH

OH
2''
3"

4" OH

HP13 R = OH
HP14 R = OMe


21
Rutin

HP14

Phenylethanoid glycosides
OH

2-(3,4dihydroxyphenyl)ethyl
-β-D-glucopyranoside

CH5

3

O
O

HOHO

OH

1

4

OH

OH

OR 2

Acteoside
Isoacteoside
Decaffeoylacteoside
β-hydroxy acteoside

CH6
CH7
CH8
CH9

R3

3

O
O

OR 1 O

OH

OH

1

4

1"

OH

CH6
CH7
CH8
CH6

1"'

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

O

5

2'

4'
HO

5

1'

3'

6'

O

6

OH

OH
4

5'

1

O

O

3
2

OH

1"'

OH

HO

11
CO

O
6

4

5

3

7
8
10

O

9

HO

1
O

1' O 5'
2'

HO
HP9

6'
OH

4'
3'
OH

OH

HO
6
7
8

COOMe
5 4
3

10

O 1' O

9

1"'

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

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

HO

OH

O

O

HO

3
2

OH 1"

OH

OH 1"

OH

4

1

O

O

OH

OH

6

O

HO

O

HO

OH

O

HO

HO

OH

R
3'
8

HO

9 O

1'

2

7

O
1

6'
5'

2'
HO

OH
4'

4' OH

3'
OH

HP11

6

OH

3
10
4
5
OH
O

O
1"
O

6"'

O 1"'

5"'
4"'
HO 3"'

O
6"

2"'
OH
OH

5"
OH

OH
2''
3"
4" OH

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-methylanthraquinone (HP1), 2-hydroxy-1-methoxy-anthraquinone (HP2), 1,6dihydroxy-2-methyl-anthaquinone (HP3) and digiferruginol (HP4),
1
carotenoid: lutein, 5 phenylethanoid glycoside: 2-(3,4-dihydroxyphenyl)ethylβ-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.