The thesis aims to study the chemical composition and survey the biological activity of two species: the lizard (B. laxiflora) and the cow breast (F. hirta). - Isolate and determine the chemical structure of two species of whole lizard (B. laxiflora) and the root of cow breast (F. hirta). Evaluation of some cytotoxic activity, anti-inflammatory activity, anti-proliferation activity on acute bone marrow cell line (OCI-AML) of extracting extracts and of some isolated compounds to orient for Next application studies.
Trang 1GRADUATE 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
Trang 2This thesis is completed at: Vietnam Academy of Science and
Technology
Scientific instructors:
Assoc Dr TRINH THI THUY
Dr NGUYEN QUYET TIEN
Trang 3INTRODUCTION
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
Trang 4Evaluation 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
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
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 (HR-ESI-MS), one/two-dimention nuclear magnetic resonance spectra (NMR)
2.2.4 Biological activities
2.2.4.1 Method for cytotoxic activity
Trang 5The 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
Trang 6* Compound BL-5, (7-hydroxy-6-methoxycoumarin)
Compound BL-5 (10 mg), crystalline
* Compound BL-6 (+)-lariciresinol
Compound BL-6 (30mg), White amorphous powder
25D 32(c 0,1; MeOH) (+)-ESI-MS m/z 383,1 [M+Na]+
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-HO]+ (Calcd for C H O, 289,0740), molecular
Trang 7formula BL-10 C15H14O7 IR (KBr, νmax, cm ): 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
HR-ESI-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-6,9,10-trihydroxybenzo[kl]xanthene-1,2-dicarboxylat)
Compound BL-12 (7 mg), white amorphous powder
(-)-ESI-MS 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 (C-11), 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
Trang 89), 146,6 4, C-7), 131,1 2, C-6), 127,3 1), 116,8 8), 115,7(C-3, C-5)
(C-* Compound BL-14 (isolariciresinol 4-O-β-D-glucopyranoside)
Compound BL-14 (2,5 g), white amorphous powder 1
H-NMR (500 MHz, DMSO-d 6 ), δH (ppm): 6,68 (1H, d, J = 1,5 HZ, 2) 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, H- 9), 6,67 (1H, s, H-2′), 6,31 (1H, s, H-5′), 2,7 (1H, dd, J = 5,0; 4,5
H-Hz, H-7′), 1,70 (1H, m, H-8′), 3,56 (2H, m, H-9′), 3,71 (3H, s, OCH3), 3,69 (3H, s, 5-OCH3), 5,0 (1H, d, J = 4,5 Hz), 3,1 -1,8 m
3′-13
C NMR (125 Hz, DMSO-d 6, ), 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 (C-4′), 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
Trang 9* 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 1NMR (500 MHz, CD3OD), δH (ppm): 7,42 (2H; H-2/H-6); 5,72
(1H; d; J = 7,5 Hz; 1'); 3,95 (1H; m; 2'); 3,46 (1H; m; 3'); 3,87 (1H; m; H-4'); 3,54 (1H; m; H-5'); 3,97/3,81 (2H; dd; J
H-= 1,5/2,0 Hz; H-6'a/H-6'b); 3,92 (6H, s, 3-OCH3/5-OCH3) 13NMR (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-OCH3/5-OCH3) 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')
C-* Compound BL-20 (lariciresinol 4-O-β-D-glucopyranoside)
Compound BL-20 (23 mg), white amorphous powder 1NMR (500 MHz, CD3OD), δH (ppm): 7,01 (1H, d, J = 1,0 Hz, H- 2), 7,14 (1H, d, J = 1,0 Hz, H-5), 6,91 (1H, d, J = 1,5 Hz, H-6),
H-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 (C-1'), 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
Trang 10Figure 2.4 Isolation of fraction from F hirta
Trang 11Test 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
2.3.2.3 Isolation of compounds from ethyl acetate fraction
Figure 2.5 Isolation of compounds from EtOAc fraction
Trang 12 Spectral data of isolated compounds
* Compound F-1 (6,7-furano-hydrocoumaric acid methyl ester) Compound F-1 (10 mg), white amorphous powder HR-ESI-
MS 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, 4), 2,75 (2H, t, J = 7,0 Hz, H-3) 13C-NMR (125 MHz, CDCl3),
H-δC (ppm):176,05 2), 154,83 7), 152,24 9), 144,13 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)
(C-* 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 (C-10), 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 (C-10), 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
Trang 13= 7,0 Hz, H-2) C-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
F-5 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