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.
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 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." The objectives of the thesis Study on chemical constituents and biological activity of two species: B Laxiflora and F hirta 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 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 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 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 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, 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 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 = 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) 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 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), 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: new compound 5-O-[β-D-apiofuranosyl(1→2)-β-D-glucopyranosyl]bergaptol (F-6), compound new isolated from nature 6,7-furano-hydrocoumarate methyl ester (F1), 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 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) 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 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 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 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: new compound balanochalcone (BL-10), compound first time from this species B laxiflora 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: new compound 5-O-[β-D-apiofuranosyl(1→2)-β-D-glucopyranosyl]bergaptol (F-6), the first time isolated from nature 6,7-furano-hydrocoumarate methyl ester (F1), compound (F-3 -> F-11) first time from this species F hirta 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 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 Further research on biological activity as well as the mechanism of substances action THE CONTRIBUTION OF THE THESIS Study on chemical constituents and biological activity of B laxiflora Hemsl B laxiflora has been studied on chemical constituents and biological activity for the first time in Vietnam 20 compounds have been isolated and structures of the compounds were characterized using spectroscopic techniques: a new compound named balanochalcone (BL-10) and 03 compounds were isolated for the first time from B laxiflora Compound BL-4 isolated from 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 were tested for the cytotoxic activity on 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, BL-11, BL-12) isolated from B.laxiflora, compound BL4 and compoud BL-12 had expressed activities on KB cell with IC50 at 107,06 and 80,37 µM Study on chemical constituents and biological activity of F hirta F.hirta has been studied on chemical constituents and biological activity for the first time in Vietnam 11 compounds have been isolated and structures of the new compounds were characterized using spectroscopic techniques a new compound named 5-O-[β-D-apiofuranosyl-(1→2)-β-Dglucopyranosyl]bergaptol (F-6), a compound the was isolated from the first time nature 6,7-furano-hydrocoumarate methyl ester (F-1), compound (F-3 -> F-11) was isolated first time from F hirta LIST OF PUBLISHED WORKS Trần Đức Đại, Nguyễn Quyết Tiến, Nguyễn Ngọc Tuấn, Nguyễn Quảng An, Trương Thị Thanh Nga, Trịnh Thị Thủy, Đặng Ngọc Quang Thành phần hóa học ngọc cẩu (Balanophora laxiflora Hemsl) thu Tuyên Quang Phần hợp chất glucoside Tạp chí hóa học Việt Nam, 2016, vol 54 (6e2), 48-52 Trần Đức Đại, Nguyễn Quyết Tiến, Nguyễn Ngọc Tuấn, Nguyễn Quảng An, Trương Thị Thanh Nga, Trịnh Thị Thủy, Nguyễn Thị Tuyết, Đặng Ngọc Quang Thành phần hóa học ngọc cẩu (Balanophora laxiflora Hemsl) thu Tuyên Quang Phần Thành phần hóa học dịch chiết phân cực Tạp chí hóa học Việt Nam, 2017, 55 (1), 48-51 Dang Ngoc Quang, Tran Cong So, Nguyen Thi Phuong Thanh, Le Thi Phuong Hoa, Pham Huu Dien, Truong Minh Luong, Nguyen Quang Tung, Le Duc Long, Tran Duc Dai and Nguyen Quyet Tien Balanochalcone, a new chalcone from Balanophora laxiflora Hemsl Natural Product Researc, (2018), 32 (7), 767-772 Tran Duc Dai, Trinh Thi Thuy, Nguyen Ngoc Tuan, Nguyen Quang An, Truong Thi Thanh Nga, Hoang Duy Cuong, Nguyen Quyet Tien Chemical constituents of (Balanophora laxiflora) collected in Tuyen Quang Par Polyphenol compounds Vietnam Journal of chemistry accepted 6/2/2018 Tran Duc Dai, Nguyen Thanh Tam, Dao Duc Thien, Nguyen Hoang Sa, Trinh Thi Thuy, Nguyen Thi Hoang Anh, Tran Duc Quan A new furanocoumarin glycoside from the roots of Ficus hirta Letters in organic chemistry Submit 11/2017 ... 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. "... objectives of the thesis Study on chemical constituents and biological activity of two species: B Laxiflora and F hirta The main contents of the thesis Isolation and determination of chemical structure... cell with IC50 at 107,06 and 80,37 µM Study on chemical constituents and biological activity of F hirta F .hirta has been studied on chemical constituents and biological activity for the first time