Tóm tắt Luận án Study on chemical constituents and biological activity of balanophora laxiflora hemsl. and ficus hirta vahl

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND 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 (HR- ESI-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.   3225 D (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.   4125 D (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). 1 H-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. 1 H-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). 13 C-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. 1 H-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). 13 C-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). 13 C-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 (C- 8), 115,7(C-3, C-5). * Compound BL-14 (isolariciresinol 4-O-β-D-glucopyranoside) Compound BL-14 (2,5 g), white amorphous powder. 1 H- NMR (500 MHz, DMSO-d6 ), δH (ppm): 6,68 (1H, d, J = 1,5 HZ, H- 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 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 (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 * 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. 1 H- NMR (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; H- 3'); 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). 13 C- NMR (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'). * Compound BL-20 (lariciresinol 4-O-β-D-glucopyranoside) Compound BL-20 (23 mg), white amorphous powder. 1 H- NMR (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), 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). 13 C-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 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 Concentration (µg/ml) % NO inhibitory activity n-hexane EtOAc n-BuOH L-NMMA 100 91,21 95,91 42,33 102,54 20 20,88 36,96 16,46 70,08 4 14,51 7,9 8,75 35,91 0,8 5,93 1,48 3,09 14,02 IC50 65,39 ± 3,46 27,35 ± 1,53 >100 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 Concentration (µg/ml) % surviving cells 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-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). 1 H-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, H- 4), 2,75 (2H, t, J = 7,0 Hz, H-3). 13 C-NMR (125 MHz, CDCl3), δC (ppm): 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). * 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). 13 C-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). 1 H-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). 13 C-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. 1 H-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). 13 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. 1 H-NMR (500 MHz, CD3OD), δH (ppm): 4,28 (1H; d; J = 8,0 Hz), 1,25 (3H; t; J = 7,0 Hz; -CH3). 13 C-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-D- glucopyranosyl]bergaptol) (new) Compound F-6 (10 mg), white amorphous powder. HR-ESI- MS 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). 1 H-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′′), 3,25- 3,22 (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 (C- 6′′), 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-ESI- MS m/z 268,1046 [M+H] + (Calcd for C10H14N5O4 268,0968), molecular formula F-7 C10H13N5O4. 1 H-NMR (500 MHz, DMSO- d6), 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-ESI- MS m/z 229,0104 [M+H] + (Calcd for C10H7O5 229,0215), molecular formula F-8 C10H6O5. 1 H-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). 13 C-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-ESI- MS 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 (C- 7), 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 (C- 3′′), 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). 1 H-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; C