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-d