Summary of Doctoral thesis: Study on chemical constituents and biological activity of Balanophora Laxiflora hemsl. and Ficus Hirta Vahl

MINISTRY OF

VIETNAM ACADEMY

EDUCATION AND

OF SCIENCE AND

TRAINING

TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND
TECHNOLOGY
----------------------------

TRAN DUC DAI

STUDY ON CHEMICAL CONSTITUENTS AND
BIOLOGICAL ACTIVITY OF BALANOPHORA
LAXIFLORA HEMSL. AND FICUS HIRTA VAHL.

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. TRINH THI THUY
Dr. NGUYEN QUYET TIEN

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 2018


INTRODUCTION
1. The urgency of the thesis
Vietnam has 54 ethnic groups such as: Kinh, Tay, Dao, San
Chay, Mong, Nung, San Diu, E de.... Some ethnic groups have
precious medicinal plants, valuable traditional treatment and
therapeutic remedies trusted by the people and recognized by the
Oriental Medicine Association of Vietnam. However people's
medicine has not been proven in science. Vietnam is located in
tropical monsoon climate zone, so the country’s vegetation is rich
and diversified, Vietnam has many natural conservations that are
home to thousands of species of rare plants and animals, and
rich medicinal herbs and various resources.
Species Balanophora laxiflora Hemsly and Ficus hirta Vahl,
are precious medicinal plants in the treasure herbs, medicinal
Vietnam, species B.laxiflora and species F. hirta has been used in

traditional medicine Vietnam for treatment of various diseases such
as: a tonic for blood circulation improvement, recovery, antipyretic,
antidote, appetite stimulation, Recent researchers have discovered
various compounds and bioactivities of B. laxiflora. For instance,
antioxidant hydrolysable tannins with a phenylacrylic acid
derivative such as caffeoyl, coumaroyl,
anti-inflammatory
metabolites, hypouricemic activity....Study on chemical
constituents and biological activity of two species Balanophora
laxiflora Hemsl and Ficus hirta Vahl are necessary, in order to
elucidate biochemical and bioactive significance as well as extend
the use of species Balanophora laxiflora Hemsl and Ficus hirta
Vahl, we carry out the topic:"Study on chemical constituents
and biological activity of Balanophora laxiflora Hemsl. and
Ficus hirta Vahl."
2. The objectives of the thesis
Study on chemical constituents and biological activity of two
species: B. Laxiflora and F. hirta.
3. The main contents of the thesis
Isolation and determination of chemical structure of
compounds of two species: B. Laxiflora and F. hirta roots by
column chromatography.
Determination of chemical structure of compounds isolated
by IR, MS, 1D-NMR, 2D-NMR spectroscopy.


Evaluation of some biological activity of extracts and isolated
compounds: anti-inflammatory activity, in vitro, apoptosis.
CHAPTER 1. OVERVIEW
1.1. Introduction of B. laxiflora Hemsl

1.2. Introduction of genus Ficus
1.2.1. Genus Ficus
1.2.2. Species F. hirta
CHAPTER 2. EXPERIMENT
2.1. Plant material
2.1.1. Plant material B. laxiflora
The B. laxiflora was collected in Yen Son district, Tuyen
Quang province, Vietnam in December, 2016 and were identified
by Assoc. Prof. Do Huu Thu, Institute of Ecology and Biological
Resources, Vietnam Academy of Science and Technology
(VAST). A voucher specimen has been kept in Laboratory of
Natural Products Research, Institute of Chemistry, VAST, Hanoi,
Vietnam.
2.1.2. Plant material F. hirta
The roots of Ficus hirta was collected in Yen Son district,
Tuyen Quang province, Vietnam in December, 2016 and were
identified by Assoc. Prof. Do Huu Thu, Institute of Ecology and
Biological Resources, Vietnam Academy of Science and
Technology (VAST). A voucher specimen has been kept in
Laboratory of Natural Products Research, Institute of Chemistry,
VAST, Hanoi, Vietnam.
2.2. Methods
2.2.1. Extraction
2.2.2. Isolation
Chromatographic methods such as thin layer chromatography
(TLC), column chromatography (CC).
2.2.3. Spectroscopic means
Physical parameters and modern spectroscopic methods such
as optical rotation ([α]D), Infrared Spectroscopy (IR), Electron
Spray Ionisation Mass Spectroscopy (ESI-MS) and High

Resolution Electron Spray Ionisation Mass Spectroscopy (HRESI-MS), one/two-dimention nuclear magnetic resonance spectra
(NMR).
2.2.4. Biological activities
2.2.4.1. Method for 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)
2.2.4.2. The apoptosis (Programmed Cell Death) in Italy
2.2.4.3. The nitric oxide inhibition (NOs inhibition) in Vietnam
The test determines the NO production potential of RAW
macrophage 264,7.
2.3. Extraction and isolation
2.3.1. B. laxiflora
2.3.1.1. Extraction
2.3.1.2. Isolation of compounds from dichloromethane extraction

Figure 2.1. Isolation of compounds from dichloromethane extraction
 Spectral data of isolated compounds
* Compound BL-1 (4-hydroxy-3-methoxycinnamandehyde)
Compound BL-1 (40 mg), white crystalline.
* Compound BL-2 (methyl 4-hydroxycinnamate)
Compound BL-2 (53 mg), white crystalline.
* Compound BL-3 (pinoresinol)
Compound BL-3 (45 mg), crystalline.
* Compound BL-4 (methyl 3,4-dihydroxycinnamate)
.
Compound BL-4 (210 mg), crystalline.

* Compound BL-5, (7-hydroxy-6-methoxycoumarin).
Compound BL-5 (10 mg), crystalline.
* Compound BL-6 (+)-lariciresinol
Compound BL-6 (30mg), White amorphous powder.
25
 D  32 (c 0,1; MeOH). (+)-ESI-MS m/z 383,1 [M+Na]+
Molecular formula C20H24O6
* Compound BL-7 (+)-isolariciresinol
Compound BL-7 (210 mg), White amorphous powder.
25
 D  41 (c 0,1, MeOH). (-)-ESI-MS m/z 359 [M-H]-.
* Compound BL-8 (quercetin)
Compound BL-8 (10 mg), yellow powder.
2.3.1.3. Isolation of compounds from ethyl acetate extraction

Figure 2.2. Isolation of compounds from ethyl acetate extraction
 Spectral data of isolated compounds
* Compound BL-9 (methyl gallate)
Compound BL-9 (63 mg), white amorphous powder.
* Compound BL-10 (new)- balanochalcone
Compound BL-10 (7 mg), light yellow oil. HR-ESI-MS m/z
289,0696 [M+H-H2O]+ (Calcd for C15H13O6, 289,0740), molecular


formula BL-10 C15H14O7. IR (KBr, νmax, cm-1): 3200 (-OH), 1633
(>C=O), 1601-1530 (C=C, benzen). 1H-NMR (500 MHz, CD3OD),
δH (ppm): 6,94 (1H; s; H-4), 6,81 (2H; s; H-2 và H-6), 5,92 (1H; d;
J = 2,0 Hz; H-5′), 5,90 (1H; d; J = 2,0 Hz; H-3′), 5,34 (1H; dd; J =
3,0 Hz; 7,5 Hz; H-β), 3,90 (1H; dd; J = 7,5; 17,0 Hz; H-α), 3,72

(1H; dd; J = 3,0 Hz; 17,0 Hz; H-α); 13C-NMR (125 MHz, CD3OD),
δC (ppm): 197,8 (>C = O), 168,4 (C-4′), 165,5 (C-6′), 164,8 (C-2′),
146,9 (C-3), 146,5 (C-5), 131,8 (C-1), 119,3 (C-6), 116,3 (C-2),
114,7 (C-4), 103,4 (C-1′), 97,0 (C-3′), 96,2 (C-5′), 80,5 (C-β), 44,1
(C-α).
* Compound BL-11 (β-hydroxydihydrochalcone)
Compound BL-11 (20 mg), white amorphous powder. HRESI-MS m/z 291,2671 [M+H]+ (Calcd for C15H15O6, 291,0790),
molecular formula BL-11 C15H14O6. 1H-NMR (500 MHz, CD3OD),
δH (ppm): 2,72 (1H, dd, J = 17,0 Hz; 3,0 Hz), 3,13 (1H; dd; J = 17,0
Hz; 13,0 Hz), 5,36 (1H; dd; J = 13,0 Hz; 2,5 Hz), 5,90 (1H; d; J = 2.5
Hz), 5,92 (1H; d; J = 2.0 Hz), 6,84 (2H; d; J = 8,5 Hz), 7,33 (2H; d; J
= 8,5 Hz). 13C-NMR (125 MHz, CD3OD), δC (ppm): 44,0 (C-α) , 80,5
(C-β), 96,2 (C-5′), 97,1 (C-3′), 103,3 (C-1′), 116,3 (C-2, C-6), 129,0
(C-3, C-5), 131,1 (C-1), 159,0 (C-4), 164,9 (C-2′), 165,5 (C-6′), 168,5
(C-4′), 197,8 (> C=O).
* Compound BL-12 (dimethyl-6,9,10trihydroxybenzo[kl]xanthene-1,2-dicarboxylat)
Compound BL-12 (7 mg), white amorphous powder. (-)-ESIMS m/z 381,0684 [M-H]- Calcd for C20H14O8. 1H-NMR (500
MHz, CD3OD), δH (ppm): 3,94 (3H; s), 4,04 (3H; s), 6,73 (1H; s),
7,08 (1H; s), 7,25 (1H; d; J = 8,5 Hz), 7,40 (1H; d; J = 8,5 Hz),
8,11 (1H; s). 13C-NMR (125 MHz, CD3OD), δC (ppm): 173,5
(>C=O), 168,2 (>C=O), 105,0 (C-8), 110,9 (C-11a), 112,3 (C11), 120,9 (C-5), 121,2 (C-2), 122,4 (C-4), 124,7 (C-3a1), 125,7,
125,9 (C-11b), 128,1 (C-3a), 130,1 (C-3), 138,3, 143,2 (C-6),
143,1 (C-10), 148,4 (C-9), 149,8 (C-7a), 53,5 (-OCH3), 52,9 (OCH3).
* Compound BL-13 (p-cumaric acid)
Compound BL-13 (20 mg), white amorphous powder der.
1
H-NMR (500 MHz, CD3OD), δH (ppm): 6,30 (1H; d; J = 16,0
Hz), 6,83 (2H; d; J = 8,5 Hz), 7,47 (2H; d; J = 8,5 Hz), 7,62 (1H;
d; J = 16,0 Hz). 13C-NMR (125 MHz, CD3OD), δC (ppm): 161,1

(C-9), 146,6 (C-4, C-7), 131,1 (C-2, C-6), 127,3 (C-1), 116,8 (C8), 115,7(C-3, C-5).
* Compound BL-14 (isolariciresinol 4-O-β-D-glucopyranoside)
Compound BL-14 (2,5 g), white amorphous powder. 1HNMR (500 MHz, DMSO-d6 ), δH (ppm): 6,68 (1H, d, J = 1,5 HZ, H2) 6,69 (1H, d, J = 9,0 Hz, H-5), 6,50 (1H, dd, J = 8,1; 1,7 Hz, H-6),
3,79 (2H, d, J = 10, 0 Hz, H-7), 1,78 (1H, m, H-8), 3,43 (2H, m, H9), 6,67 (1H, s, H-2′), 6,31 (1H, s, H-5′), 2,7 (1H, dd, J = 5,0; 4,5
Hz, H-7′), 1,70 (1H, m, H-8′), 3,56 (2H, m, H-9′), 3,71 (3H, s, 3′OCH3), 3,69 (3H, s, 5-OCH3), 5,0 (1H, d, J = 4,5 Hz), 3,1 -1,8 m.
13
C NMR (125 Hz, DMSO-d6,), δC (ppm): 13,6 (C-1), 113,3 (C-2),
147,3 (C-3), 144,1 (C-4), 115,2 (C-5), 121,4 (C-6), 45,9 (C-7), 38,0
(C-8), 59,4 (C-9), 130,2 (C-1′), 112,2 (C-2′), 144,7 (C-3′), 146,8 (C4′), 116,6 (C-5′), 132,6 (C-6′), 32,2 (C-7′), 45,3 (C-8′), 63,5 (C-9′),
55,71 (3′-OCH3), 55,67 (5-OCH3), 100,2 (C-1′′), 73,0 (C-2′′), 76,5
(C-3′′), 68,6 (C-4′′), 76,8 (C-5′′), 60,0 (C-6′′).
* Compound BL-15 (daucosterol)
2.3.1.3. Isolation of compounds from methanol extraction

Figure 2.3. Isolation of compounds from methanol extraction
 Spectral data of isolated compounds


* Compound BL-16 (5-hydroxymethylfurfural)
Compound BL-16 (40 mg), crystalline.
* Compound BL-17 (methyl β-D-glucopyranoside)
Compound BL-17 (60 mg), crystalline.
* Compound BL-18 (methyl 4-O-β-D-glucopyranosylconiferyl ether)
Compound BL-18 (30 mg), white amorphous powder.
*
Compound
BL-19
4-hydroxy-3,5-dimethoxybenzoyl
glucopyranoside

Compound BL-19 (27 mg), white amorphous powder. 1HNMR (500 MHz, CD3OD), δH (ppm): 7,42 (2H; H-2/H-6); 5,72
(1H; d; J = 7,5 Hz; H-1'); 3,95 (1H; m; H-2'); 3,46 (1H; m; H3'); 3,87 (1H; m; H-4'); 3,54 (1H; m; H-5'); 3,97/3,81 (2H; dd; J
= 1,5/2,0 Hz; H-6'a/H-6'b); 3,92 (6H, s, 3-OCH3/5-OCH3). 13CNMR (125 MHz, CD3OD), δC (ppm): genin: 119,4 (C-1); 106,6
(C-2/C-6); 147,2 (C-3/C-5); 141,0 (C-4); 56,3 (3-OCH3/5OCH3). glucopyranose: 96,2 (C-1'); 74,1 (C-2'); 78,9 (C-3');
71,1 (C-4'); 78,1 (C-5'); 62,3 (C-6').
* Compound BL-20 (lariciresinol 4-O-β-D-glucopyranoside)
Compound BL-20 (23 mg), white amorphous powder. 1HNMR (500 MHz, CD3OD), δH (ppm): 7,01 (1H, d, J = 1,0 Hz, H2), 7,14 (1H, d, J = 1,0 Hz, H-5), 6,91 (1H, d, J = 1,5 Hz, H-6),
4,8 (2H, m, H-7), 2,38 (1H, m, H-8), 3,67-3,90 (2H, m, H-9),
6,81 (1H, d, J = 1,0 Hz, H-2''), 6,74 (1H, d, J = 8,0 Hz, H-5'),
6,66 (1H, dd, J = 8,0; 1,0 Hz, H-6'), 2,52 (1H, dd, J = 13,0; 11,5
Hz, H-7'a), 2,93 (1H, dd, J = 13,5; 5,0 Hz, H-7'b), 2,74 (1H, m,
H-8'), 4,02 (2H, dd, J = 6,5; 8,0 Hz, H-9'), 3,88 (3H, s, 3'-OCH3),
3,85 (3H, s, 5-OCH3), 4,91 (1H, d, J = 7,5 Hz, H-1''), 3,4-4,2
(1H, m, H-4''), 3,86 (1H, dd, J = 12,0; 5,0 Hz, H-6''a), 3,91 (1H,
br d, J = 12,0 Hz, H-6''b). 13C-NMR (500 MHz, CD3OD), δC
(ppm): 139,5 (C-1), 114,1 (C-2), 150,9 (C-3), 147,3 (C-4), 118,0
(C-5), 119,6 (C-6), 83,8 (C-7), 54,1 (C-8), 60,5 (C-9), 133,5 (C1'), 113,5 (C-2'), 149,0 (C-3'), 145,8 (C-4'), 116,2 (C-5'), 122,2
(C-6'), 33,6 (C-7'), 43,8 (C-8'), 73,7 (C-9'), 56,8 (3-OCH3), 56,4
(3'-OCH3), 102,9 (C-1''), 74,9 (C-2''), 77,8 (C-3''), 71,4 (C-4''),
78,2 (C-5''), 62,5 (C-6'').
2.3.2. F. hirta
2.3.2.1. Extraction


Figure 2.4. Isolation of fraction from F. hirta
2.3.2.2. Biological activity
Table 2.1. Effect of NO inhibitory reproductive of semple stady
% NO inhibitory activity

Concentration

(µg/ml)
100
20
4
0,8

n-hexane
91,21
20,88
14,51
5,93

IC50

65,39 ± 3,46

EtOAc
95,91
36,96
7,9
1,48
27,35 ±
1,53

n-BuOH
42,33
16,46
8,75
3,09

>100

L-NMMA
102,54
70,08
35,91
14,02
7,81 ±
0,74


Test results of NO inhibitory activity: EtOAc and n-hexane
extract was able to inhibit NO production with good IC50 values of
27,35 ± 1,5 và 65,39 ± 3,46 µg/ml. n-BuOH extract show weak
inhibition activity. These experimental results are the basis for the
direction of the isolation of the compounds from the corresponding
extractor.
Table 2.2. The ability to inhibit the growth of RAW cells 264,7
% surviving cells
Concentration
(µg/ml)
n-hexane
EtOAc
n-BuOH L-NMMA
100
104,25
33,29
99,67
95,45
20

103,53
100,26
98,43
96,65
4
100,92
99,65
99,52
98,43
2.3.2.3. Isolation of compounds from ethyl acetate fraction

Figure 2.5. Isolation of compounds from EtOAc fraction


 Spectral data of isolated compounds
* Compound F-1 (6,7-furano-hydrocoumaric acid methyl ester)
Compound F-1 (10 mg), white amorphous powder. HR-ESIMS m/z 243,0631 [M+Na]+ (Calcd for C12H12O4Na, 243,0736),
molecular formula F-1 C12H12O4. IR (KBr, νmax, cm-1): 3250 (–
OH), 2853 (-OCH3), 1710 (>C=O, C=C-Ar), 1623-1539 (C=C,
benzen). 1H-NMR (500 MHz, CDCl3), δH (ppm): 7,48 (1H, d, J =
2,5 Hz, H-2′) 7,28 (1H, s, H-5), 7,04 (1H, s, H-8), 6,63 (1H, d, J
= 2,5 Hz, H-3′), 3,68 (3H, s, OCH3), 2,99 (2H, t, J = 7,0 Hz, H4), 2,75 (2H, t, J = 7,0 Hz, H-3). 13C-NMR (125 MHz, CDCl3),
δC (ppm): 176,05 (C-2), 154,83 (C-7), 152,24 (C-9), 144,13 (C2′), 123,91 (C-10), 121,67 (C-5), 121,09 (C-6), 106,03 (C-3′),
99,90 (C-8), 52,24 (OCH3), 35,56 (C-3), 24,83 (C-4).
* Compound F-2 (umbelliferone)
Compound F-2 (15 mg), crystalline. IR (KBr, νmax, cm-1):
3158 (–OH), 1681 (>C=O), 1603-1508 (C=C, benzene). 1H-NMR
(500 MHz, CD3OD), δH (ppm): 7,86 (1H; J = 9,5 Hz; H-4), 7,47 (1H;
d; J = 8,5 Hz; H-5), 6,81 (1H; d; J = 8,5 Hz; 2,5 Hz; H-6), 6,73 (1H;
d; J = 2,5 Hz; H-8), 6,20 (1H; d; J = 9,5 Hz; H-3). 13C-NMR (125

MHz, CD3OD), δC (ppm): 163,71 (C-7), 163,15 (C-2), 157,26 (C-9),
146,05 (C-4), 130,66 (C-5), 114,53 (C-6), 113,17 (C-3), 112,36 (C10), 103,43 (C-8).
* Compound F-3 (bergapten)
Compound F-3 (3 g), yellow crystalline. IR (KBr, νmax, cm-1):
3088-3013 (>C=CH), 2959 (-OCH3), 1732 (>C=O), 1606-1542
(C=C, benzene). 1H-NMR (500 MHz, CDCl3), δH (ppm): 8,16 (1H,
d, J = 10 Hz, H-4), 7,59 (1H, d, J = 2,5 Hz, H-9), 7,14 (1H, s, H-8),
7,02 (1H, d, J = 2,5 Hz, H-10), 6,27 (1H, d, J = 10,0 Hz, H-3), 4,27
(3H, s, 5-OCH3). 13C-NMR (125 MHz, CDCl3), δC (ppm): 161,34
(C-2), 158,53 (C-7), 152,87 (C-5), 149,72 (C-8a), 144,92 (C-9),
139,36 (C-4), 112,86 (C-6), 112,73 (C-3), 106,59 (C-4a), 105,15 (C10), 94,02 (C-8), 60,24 (-OCH3).
2.3.2.2. Isolation of compounds from n-butanol fraction
 Spectral data of isolated compounds
* Compound F-4 (ethyl β-D-fructofuranoside)
Compound F-4 (8 mg), oil. HR-ESI-MS m/z 231,0836
[M+Na]+ (Calcd for C8H16O6Na, 231,0947), molecular formula F-4
C8H16O6. 1H-NMR (500 MHz, CD3OD), δH (ppm): 4,12 (1H; d; J =
8,0 Hz, H-4′), 3,98-3,95 (1H, m, H-3′), 3,79-3,53 (m), 1,17 (3H, t, J


= 7,0 Hz, H-2). 13C-NMR (125 MHz, CD3OD), δC (ppm): 105,29
(C-2′), 83,41 (C-5′), 78,50 (C-3′), 77,33 (C-4′), 64,92 (C-6′), 62,01
(C-1′), 57,88 (C-1), 16,02 (C-2).
* Compound F-5 (ethyl β-D-glucopyranoside)
Compound F-5 (7 mg), oil. HR-ESI-MS m/z 231,0835
[M+Na]+ (Calcd for C8H16O6Na, 231,0947), molecular formula F5 C8H16O6. 1H-NMR (500 MHz, CD3OD), δH (ppm): 4,28 (1H; d;
J = 8,0 Hz), 1,25 (3H; t; J = 7,0 Hz; -CH3). 13C-NMR (125 MHz,
CD3OD), δC (ppm): 104,11 (C-1), 78,12 (C-5), 77,91 (C-3), 75,10
(C-2), 71,68 (C-4), 62,79 (C-6), 66,16 (C-1′), 15,43 (C-2′).

Figure 2.6. Isolation of compounds from n-butanol fraction
* Compound F-6 (5-O-[β-D-apiofuranosyl-(1→2)-β-Dglucopyranosyl]bergaptol) (new)
Compound F-6 (10 mg), white amorphous powder. HR-ESIMS m/z 497,1295 [M+H]+ (Calcd for C22H25O13, 497,1217),
molecular formula F-6 C22H24O13. IR (KBr, νmax, cm-1): 3438 (OH), 1687 (>C=O), 1613-1534 (C=C, benzene). 1H-NMR (500
MHz, DMSO-d6), δH (ppm): 6,45 (1H; d; J = 9,5 Hz, H-3), 8,31
(1H; d; J = 10,0 Hz, H-4), 7,37 (1H, s, H-8), 8,04 (1H, d, J = 2,5


Hz, H-2′), 7,18 (1H, d, J = 1,5 Hz, H-3′), 5,19 (1H, d, J = 8,0 Hz,
H-1′′), 3,58-3,55 (1H, m, H-2′′), 3,50-3,46 (3H, m, H-3′′), 3,253,22 (3H, m, H-4′′), 3,50-3,46 (3H, m, H-5′′), 3,74 (1H, m, 6′a),
3,50-3,46 (1H, m, 6′′b), 5,34 (1H, d, J = 2,5 Hz, H-1′′′), 3,80 (1H,
br s, H-2′′′), 3,82 (1H, d, J = 9,0 Hz, H-4a), 3,61 (1H, d, J = 9,0
Hz, H-4b), 3,25-3,22 (3H, m, H-5′′′). 13C-NMR (125 MHz,
DMSO-d6), δC (ppm): 159,70 (C-2), 112,55 (C-3), 140,03 (C-4),
151,26 (C-5), 110,30 (C-6), 156,95 (C-7), 94,85 (C-8), 147,67
(C-9), 105,89 (C-10), 146,24 (C-2′), 103,47 (C-3′), 99,03 (C-1′′),
78,48 (C-2′′), 76,67 (C-3′′), 69,79 (C-4′′), 76,97 (C-5′′), 60,59 (C6′′), 109,68 (C-1′′′), 76,0 (C-2′′′), 78,73 (C-3′′′), 73,29 (C-4′′′),
63,0 (C-5′′′).
* Compound F-7 (adenosine)
Compound F-7 (15 mg), white amorphous powder. HR-ESIMS m/z 268,1046 [M+H]+ (Calcd for C10H14N5O4 268,0968),
molecular formula F-7 C10H13N5O4. 1H-NMR (500 MHz, DMSOd6), δH (ppm): 8,34 (1H, s, H-8), 8,13 (1H, s, H-2), 5,88 (1H, d, J =
6,0 Hz, H-1′), 4,61 (1H, dd, J = 6,0; 5,5 Hz, H-2′), 4,14 (1H, dd, J =
4,5; 3,0 Hz, H-3′), 3,96 (1H, dd, J = 3,5; 3,0 Hz, H-4′), 3,68-3,66
(1H, m, H-5a′), 3,57-3,54 (1H, m, H-5b′). 13C-NMR (125 MHz,
DMSO-d6), δC (ppm): 156,11 (C-6), 152,32 (C-2), 149,04 (C-4),
139,86 (C-8), 119,33 (C-5), 87,88 (C-1′), 85,84 (C-4′), 73,41 (C-2′),
70,61 (C-3′), 61,64 (C-5′).
* Compound F-8 (6-carboxy-umbelliferone)
Compound F-8 (10 mg), white amorphous powder. HR-ESIMS m/z 229,0104 [M+H]+ (Calcd for C10H7O5 229,0215), molecular
formula F-8 C10H6O5. 1H-NMR (500 MHz, CD3OD): δH (ppm): 8,11

(1H; s; H-5), 7,89 (1H; d; J = 9,5 Hz, H-4), 6,70 (1H; s; H-8), 6,19
(1H; d; J = 9,5 Hz, H-3). 13C-NMR (125 MHz, CD3OD), δC (ppm):
174,31 (-COOH), 167,22 (C-7), 163,46 (C-2), 158,76 (C-9), 146,46
(C-4), 132,34 (C-5), 118,47 (C-6), 112,23 (C-3), 112,01 (C-10),
103,90 (C-8).
* Compound F-9 (picraquassioside A)
Compound F-9 (10 mg), white amorphous powder. HR-ESIMS m/z 421,1108 [M+Na]+ (Calcd for C18H22O10Na 421,1213),
molecular formula F-9 C18H22O10. IR (KBr, νmax, cm-1): 3381 (OH), 2937 (-OCH3), 1708 (>C=O), 1619-1509 (C=C, benzene).
1
H-NMR (500 MHz, CD3OD), δH (ppm): 7,61 (1H; d; J = 2,0 Hz;
H-2′), 7,12 (1H; s; H-8), 6,98 (1H; dd; J = 2,0 Hz; 1,0 Hz; H-3′),


4,95 (1H; d; J = 7,5 Hz; H-1′′), 4,07 (3H; -OCH3), 3,95 – 3,44
(CH-OH&CH2-OH), 3,10-3,07 (2H; m; H-4), 2,55 (2H; br s; H-3).
13
C-NMR (125 MHz, CD3OD), δC (ppm): 174,0 (C-2), 156,97 (C7), 155,62 (C-9), 152,36 (C-5), 144,64 (C-2′), 117,07 (C-10),
114,10 (C-6), 105,53 (C-3′), 103,24 (C-1′′), 94,75 (C-8), 78,20 (C3′′), 78,10 (C-5′′), 75,01 (C-2′′), 71,40 (C-4′′), 62,57 (C-6′′), 60,67
(OCH3), 35,22 (C-3), 20,51 (C-4).
* Compound F-10 (rutin)
Compound F-10 (15 mg), white amorphous powder. IR
(KBr, νmax, cm-1): 3427 (-OH), 1654(>C=O), 1600-1504 (C=C,
benzene). 1H-NMR (500 MHz, CD3OD), δH (ppm): 7,69 (1H; d; J
= 2,5 Hz; H-2′), 7,65 (1H; dd; J = 8,5 Hz; 2,0 Hz; H-6′), 6,90
(1H; d, J = 8,5 Hz; H-5′), 6,43 (1H; d; J = 2,0 Hz; H-8), 6,24
(1H; d; J = 2,0 Hz; H-6), 5,13 (1H; d; J = 7,0 Hz; H-1′′), 4,54
(1H; d; J = 1,0 Hz; H-1′′′), 3,83-3,23 (CH-OH), 1,15 (1H; d; J =
6,5 Hz; CH3-6′′′). 13C-NMR (125 MHz, CD3OD), δC (ppm):
179,45 (C-4), 166,08 (C-7), 163,01 (C-5), 159,63 (C-9), 158,55
(C-2), 149,82 (C-4′), 145,86 (C-3′), 135,63 (C-3), 123,56 (C-6′),

123,16 (C-1′), 117,70 (C-5′), 116,08 (C-2′), 105,66 (C-10),
104,70 (C-1′′), 102,43 (C-1′′′), 99,9 (C-6), 94,9 (C-8), 78,2 (Cglc-3′′′), 77,3 (C-glc-5′′′), 75,7 (C-glc-2′′′), 73,9 (C-glc-4′′), 72,3
(C-glc-3′′), 72,2 (C-rha-3′′′), 72,1 (C-rha-2′′′), 71,4 (C-rha-4′′′),
69,7 (C-rha-5′′′), 68,5 (C-rha-6′′′) and 17,9 (-CH3).
* Compound F-11 (aspartic acid)
Compound F-11 (2,5 g), white amorphous powder. 1H-NMR
(500 MHz, D2O ), δH (ppm): 2,82 (1H; dd; J = 17,0 Hz; 7,5 Hz),
2,95 (1H; dd; J = 17,0 Hz; 4,5 Hz), 4,0 (1H; dd; J = 8,0 Hz; 4,5
Hz). 13C-NMR (125 MHz, D2O), δC (ppm): 34,5, 51,4, 173,4,
174,5.
CHAPTER 3. RESULTS AND DISCUSSIONS
3.1. B. laxiflora
This section presents the detailed results of spectral analysis and
structure determination of 20 compounds (BL-1 -> BL-20) isolated
from species B. laxiflora include: a new compound
balanochalcone (BL-10), 3 compounds first time from this
species B. laxiflora (BL-11, BL-12, BL-16).


Figure 3.1. Chemical structure of compounds


4-hydroxy-3-methoxycinnamandehyde (BL-1), methyl 4hydroxycinnamate (BL-2), pinoresinol (BL-3), methyl 3,4dihydroxycinnamate (BL-4), scopoletin (BL-5), lariciresinol
(BL-6), isolariciresinol
(BL-7), quercetin (BL-8), methyl
gallat (BL-9), balanochalcone new compound (BL-10), βhydroxydihydrochalcone
(BL-11),
dimethyl
6,9,10trihydroxybenzo[kl]xanthene-1,2-dicarboxylat
(BL-12),

pcumaric acid (BL-13), isolariciresinol 4-O-β-D-glucopyranoside
(BL-14), daucosterol (BL-15), 5-hydroxymethylfurfural (BL16), methyl β-D-glucopyranoside (BL-17), methyl 4-O-β-Dglucopyranosylconiferyl
ether
(BL-18),
4-hydroxy-3,5dimethoxybenzoylglucopyranoside (BL-19), lariciresinol 4-O-βD-glucopyranoside (BL-20).
Compound BL-10 (new compound)- Balanochalcone.

Figure 3.2. Chemical structure and major HMBC correlation of
compound BL-10

Figure 3.3. HMBC spectrum of BL-10


Compound BL-10 was obtained as an oil. The molecular
formula was established as C15H14O7 by positive HR-ESI-MS,
which showed a quasi-molecular ion peak [M + H−H2O]+ at
289,0696 (Calcd for C15H13O6, 289,0712). Its UV spectrum
absorption maxima of 225 and 288 nm together with IR
absorption bands for hydroxyl (3200 cm−1), carbonyl (1633 cm−1)
and aromatic rings (1601 and 1530 cm−1) revealed the βhydroxydihydrochalcone skeleton for compound BL-10 (Muiva
et al. 2009; Özbek et al. 2016). Then, the structure of BL-10 was
deduced from analysis of its 1D and 2D NMR spectra. Its 1HNMR spectrum shows the presence of five olefinic protons, one
carbinol and one methylene group. Analysis of its 13C-NMR
spectrum indicates the presence of 15 carbon signals, including
one conjugated ketone (197,8 ppm), two aromatic rings. Three
singlet protons resonanced at 6,81 (2H) and 6,94 (1H) ppm are
assigned at C-2, C-4 and C-6 of the first aromatic ring due to the
HBMC correlations between (1) H-2 and H-6/C-1, C-3, C-4 and
C-5; (2) H-4/ C-1, C-2, C-3, C-5. This ring is connected to C-β is
proved by the long-range correlations from C-β to H-2, H-6. In

addition, two doublet protons at 5,92 and 5,90 ppm which both
have a small coupling constant (J = 2,0 Hz), therefore, they are
characterised at meta-position (H-3′ and H-5′) of the second ring.
The hydroxyl group is placed at C-β due to its low field shift in
13
C-NMR spectrum and HMBC correlation from (i) H-β to C=O;
(ii) H-α to C-β, C=O, C-1 (Muiva et al. 2009; Özbek et al. 2016).
From above discussion, compound BL-10 is found to be a new βhydroxydihydrochalcone and given a trivial name as
balanochalcone. This appears to be the first report on the
occurrence of a β-hydroxydihydrochalcone in the B. laxiflora.
3.2. F. hirta
This section presents the detailed results of spectral analysis and
structure determination of 11 compounds (F-1 -> F-11) isolated from
species F. hirta include: 1 new compound 5-O-[β-D-apiofuranosyl(1→2)-β-D-glucopyranosyl]bergaptol (F-6), 1 compound new
isolated from nature 6,7-furano-hydrocoumarate methyl ester (F1), 8 compounds (F-3 -> F-11) first time from this species F.
hirta.


3.2.1. Compound F-1

Figure 3.4. Chemical structure and major HMBC (→) correlations
of compound F-1
Compound F-1 was obtained as a white solid. HR-ESI-MS
of compound F-1 showed [M+Na]+ peaks at m/z 243,0631 (calcd
for C12H12O4Na, 243,0736), which established a molecular
formula of F-1 (C12H12O4). Its IR spectrum showed absorption
bands for hydroxyl groups at 3250 cm-1, a carbonyl group at 1710
cm–1, -OCH3 group at 2853 cm–1 and a C=C group at 1623 to 1539
cm–1.

Figure 3.5. HMBC spectrum of F-1
1

The H-NMR spectrum exhibited signals characteristic for a
linear bezofuran resonances at δH 7,48 (1H, d, J = 2,5 Hz; H-2'),
6,63 (1H, d, J = 2,5 Hz; H-3'), 7,28 (1H; s; H-5), 7,04 (1H; s; H8). Ở 3,68 (3H; s; -OCH3), 2,99 (2H; t; J = 7,0 Hz; H-4), 2,75
(2H; t; J = 7,0 Hz; H-3). The 13C-NMR and HSQC spectra of F-1
displayed the signals of 12 carbons at δC: 176,05 (C-2), 154,83
(C-7), 152,24 (C-9), 144,13 (C-2′), 123,91 (C-10), 121,67 (C-5),
121,09 (C-6), 106,03 (C-3′), 99,90 (C-8), 52,24 (-OCH3), 35,56
(C-3), 24,83 (C-4), including a carbonyl carbon (>C=O) at


176,05 (C-2), two ankan carbon at 35,56 (C-3), 24,83 (C-4), four
methine carbon (CH) ở 121,67 (C-5), 99,90 (C-8), 144,13 (C-2'),
106,03 (C-3') and at 52,24 (2-OCH3).
Further HMBC correlations between the proton δH 2,99 (H-4)
and the carbon (C-5, C-9, C-10, C-2, C-3), between the proton
2,75 (H-3) and the carbon (C-10, C-4, C-2), between the proton
(-OCH3) and the carbon C-2 confirmed that the position of the
esther moiety at C-10 of bezofuran. The proton 7,28 (1H, s, H-5)
and the carbon (C-7, C-9, C-4, C-3'), between the proton 7,04
(1H, s, H-8) and the carbon (C-6, C-10, C-7, C-9). The key
HMBC correlations have been given in Fig. 2. From all the above
evidence comparison of NMR spectroscopic data with those
reported in the literature (Shinsuke et 2011) the structure of F-1
was determined as 6,7-furano-hydrocoumarate methyl ester.
compound F-1 new isolated from nature.
3.2.2. Compound F-6
Compound

F-6 (5-O-[β-D-apiofuranosyl-(1→2)-β-Dglucopyranosyl]bergaptol) (new compound)
.

Figure 3.6. Chemical structure and major HMBC (→) correlations
of compound F-6
Compound F-6 was obtained as a white solid. Its IR spectrum
showed absorption bands for hydroxyl groups at 3438 cm–1, a
carbonyl group at 1687 cm–1, and a C=C group at 1634 and 823
cm–1. The molecular formula of F-6 was determined to be
C22H24O13 by the molecular ion peak at m/z [M+H]+ 497,1295 in
HR-ESI-MS spectrum and NMR data. The 1H-NMR spectrum
exhibited signals characteristic for a linear furanocoumarin
resonances at δH 8,30 (1H, d, J = 10,0 Hz, H-4), 8,04 (1H, d, J =
2,5 Hz, H-2’), 7,37 (1H, s, H-8), 7,18 (1H, d, J = 1,5 Hz, H-3’),
6,45 (1H, d, J = 9,5 Hz, H-3), two sugar units with two anomeric
protons at δH 5,34 (1H, d, J = 2,5 Hz), 5,19 (1H, d, J = 8,0 Hz)


and eleven protons in the range δH 3,83-3,22. The 13C-NMR and
HSQC spectra of F-6 displayed the signals of 22 carbons,
including three methylene, twelve methine and seven quaternary
carbons. Of which, 11 carbons were assigned to a
furanocoumarin and 11 carbons to the sugar moieties. The carbon
signals of the furanocoumarin nucleus of F-6 were in good
agreement with those of bergaptol glucoside in the literature [9].
This suggested that F-6 is a C-5-glycosylated furanocoumarin.
The sugar moieties included a D-glucopyranose moiety at δC
99,03; 78,48; 76,97; 76,67; 69,79; 60,59 and a D-apiofuranose
moiety at δC 109,68; 78,73; 76,00; 73,29; 63,00. Both glycosidic
units were determined as β-configurations based on J1,2 values of

the anomeric protons H-1′′ and H-1′′′ (J = 8,0 Hz and 2,5 Hz,
respectively) (Matsuda N et al 1995, Nguyen. M.C et al 2016).

Figure 3.7. HMBC spectrum of F-6
The HMBC correlations observed between the anomeric
proton H-1′′′ (δH 5,34) and carbon C-2′′ (δC 78,48), between the
proton H-2′′ (δH 3,57) and carbon C-1′′′ (δC 109,68), as well as
the downfield shift of C-2′′ (δC 78,48) indicated the linkage of
sugar
moiety
as
O-β-D-apiofuranosyl
(1→2)-O-β-D-


glucopyranoside. Further HMBC correlations between the proton
H-1′′ (δH 5,19) and the carbon C-5 (δC 151,26) confirmed that the
position of the sugar moiety at C-5 of furanocoumarin. The key
HMBC correlations have been given in (Figure. 3.6). From all
the above evidence, the structure of F-6 was determined as 5-O[β-D-apiofuranosyl-(1→2)-β-D-glucopyranosyl]bergaptol.

F-1

F-7

F-3

F-2

F-4

F-6

F-5

F-9

F-10
F-11
Figure 3.8. Chemical structure of compounds

Compound 6,7-furano-hydrocoumarate methyl ester, the first
isolated from nature (F-1), umbelliferone (F-2), bergapten (F-3),
ethyl β-D-fructofuranoside (F-4), ethyl β-D-glucopyranoside (F5), adenosine (F-7), 5-O-[β-D-apiofuranosyl-(1→2)-β-Dglucopyranosyl]bergaptol,
new
compound
(F-6),
6carboxyumbelliferone (F-8), picraquassioside A (F-9), rutin (F10), acid aspartic (F-11).
3.3. Results of biological activity test


3.3.1. Results of in vitro test (in Vietnam)
Results of cytotoxic activity tests on 4 lines of human
epidermic carcinoma (KB), human hepatocellular carcinoma
(HepG2), human lung carcinoma (Lu) and human breast
carcinoma (MCF7): Compound methyl caffeate (BL-04) and
compound dimethyl 6,9,10-trihydroxybenzo[kl]xanthene-1,2dicarboxylate (BL-12) had IC 50 : 107,06 µM and 80,37 µM.
Bảng 3.1. Biological in vitro activities of some isolated
compounds from species B. Laxiflora.
Compounds

IC50 (µM)
KB

Hep-G2

Lu-1

MCF-7

BL-10

> 200

> 200

> 200

> 200

BL-04

107,06

> 200

> 200

> 200

BL-11

> 200

> 200

> 200

> 200

BL-12

80,37

146,3

145,03

178,5

Ellipticine

1,01

2,72

1,10

1,18

3.3.2. Results of apoptosis (Programmed Cell Death) (in
Italy)
The apoptosis activity on OCI-AML cells: BL-1, BL-2,
BL-4, BL-6, BL-7, BL-9. Result compound BL-2 activity at
1685 μM (300 μg/ml) and compound BL-9 activity at 815 μM
(150 μg/ml), results show that most effective was the higher
concentration through an influence on apoptosis and cell cycle.
Compound BL-1 activity at 421,6 μM (75 μg/ml) and 210,7 μM
(37,5 μg/ml) , results show that changes induced by BL-1 in cell
death and in the cell cycle were not sufficient to alter the OCI
cell number. Compound BL-4 is a very powerful compound
since decreased the OCI cell number even with a concentration as
low as 80,5 μM (15,62 μg/ml) and this is the result of increased
apoptosis and decreased proliferation. Compounds BL-6 and BL7 seems to not bio-active in this particulat test even when used at
a concentration as high as 500 μg/ml. It would be interesting to
test this substance on other cell lines.
.


CONCLUSIONS AND RECOMMENDED FURTHER
WORK
❖ Conclusion
F. hirta grouing in Vietnam was studied on chemical
constituents and biological activity for first time.
First time reported on biological activity of isoleted
compounds from B. Laxiflora in Vietnam.
1. Chemical constituents of B. laxiflora
Using chromatographic methods thin layer chromatography
(TLC) and column chromatography (CC) compounds 20 were
isoleted. Their structures were determined by Spectroscopic

method such as: ([α]D), Infrared Spectroscopy (IR), Electron
Spray Ionisation Mass Spectroscopy (ESI-MS) and High
Resolution Electron Spray Ionisation Mass Spectroscopy (HRESI-MS), one/two-dimention nuclear magnetic resonance spectra
(NMR) determination of chemical structure of compounds 20: 1
new compound balanochalcone (BL-10), 3 compound first time
from this species B. laxiflora.
2. Chemical constituents of F. hirta
Use chromatographic methods thin layer chromatography
(TLC) and column chromatography (CC) isolation compounds
11. Use optical rotation ([α]D), Infrared Spectroscopy (IR),
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) determination of chemical structure of
compounds 11: 1 new compound 5-O-[β-D-apiofuranosyl(1→2)-β-D-glucopyranosyl]bergaptol (F-6), 1 the first time
isolated from nature 6,7-furano-hydrocoumarate methyl ester (F1), 8 compound (F-3 -> F-11) first time from this species F. hirta
3. Biological activity
First time apoptosis activity test on OCI-AML cells:
Compounds BL-1, BL-2, BL-4, BL-6, BL-7, BL-9, from species
B. laxiflora.
First time public active of compound BL-4 extraction from
species B. laxiflora is a very powerful compound since decreased
the OCI cell number even with a concentration as low as 80,5 μM


(15,62 μg/ml) and this is the result of increased apoptosis and
decreased proliferation.
First time in Vietnam in vitro activity test of 04 cancer cell
lines: human epidemic carcinoma (KB), hepatocellular
carcinoma (Hep-G2); human lung carcinoma (LU) and human

breast carcinoma (MCF-7) of 04 compounds (BL-4, BL- 10, BL11, BL-12) isolation from species B. laxiflora, compound BL-4
and compoud BL-12 had expressed activities on KB cell with
IC50 at 107,06 and 80,37 µM.
❖ In summary
The results have fully answered the aimed and questions
posted in this thesis. Among 31 isolated compounds, 02 news
compounds, 01 compound the first time isolated from nature,
there are 09 compounds were isolated for the first time from
species B.laxiflora and species F.hirta, 02 compounds had
biological activity in vitro.
❖ Recommendations
1. Continue to study on the chemical composition some
species B.laxiflora in Vietnam, continue to study parts:
leaf, fruit, truck of species F.hirta in Vietnam.
2. Further research on biological activity as well as the
mechanism of substances action.