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1 MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND ECHNOLOGY Lai Hop Hieu STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES FROM THE LEAVES OF EXCOECARIA AGALLOCHA L AND EXCOECARIA COCHINCHINENSIS Lour Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi – 2021 This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Adviser 1: Prof Dr Ngo Dai Quang Adviser 2: Dr Nguyen Van Thanh 1st Reviewer: …………………………………………………… 2nd Reviewer: : …………………………………………………… 3rd Reviewer: : …………………………………………………… The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at hour date month 2021 Thesis can be found in: - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology - National Library INTRODUCTION The urgency of the thesis Throughout human history, marine microorganisms and natural plants have become potential sources in the discovery of novel drugs for the treatment of human diseases Nowadays, more than 70% of anticancer drugs in the market are derived from natural products or synthesized based on the structure of natural compounds Besides cancer, which is a major issue of concern for scientists, the emergence of antibiotic drug resistance is also a big threat to human health worldwide Antibiotic drug resistance occurs when microorganisms such as viruses, fungi or parasites change their mechanism of action in response to the existing antimicrobial treatments Several factors contribute to antibiotic resistance such as the overuse/misuse of antibiotics and the self-medication with antibiotics The important role of natural bioactive compounds has been investigated from traditional medicine to modern medicine Their value is not only for direct use as a medicine but also as a structure lead compound for the discovery and development of new drugs In an attempt to investigate and research medicinal materials for public health care programs, the study on natural compounds which exhibit several biological activities such as cytotoxicity, anti-cancer, antimicroorganisms for treatment of cancer and antibiotic multidrugresistance is one of the main goals of scientists around the world Marine organisms and mangrove plants raise much attention to the scientists in the field of biomedicine and pharmacology Several studies have been carried out to investigate new bioactive compounds derived from mangrove plants Therefore, the thesis namely “Study on chemical constituents and biological activities from the leaves of Excoecaria agallocha L and Excoecaria cochinchinensis Lour.” was conducted to investigate potential bioactive compounds from E agallocha and E cochinchinensis in order to demonstrate more clearly the therapeutic uses in traditional medicine and increase the scientific value of these plants in Vietnam The objectives of the thesis Isolation and determination of chemical structures of the isolated compounds from the leaves of Excoecaria agallocha L and Excoecaria cochinchinensis Lour Studied the cytotoxic, anti-inflammatory, and antimicrobial activities of the isolated compounds to find the bioactive compounds The main contents of the thesis Isolation of compounds from the leaves of Excoecaria agallocha and E cochinchinensis using various chromatographic separations Determination of chemical structures of the isolated compounds Evaluation of the cytotoxic, anti-inflammatory, and antimicrobial activities of the isolated metabolites to find out potential compounds CHAPTER I OVERVIEW This chapter presents the overview of domestic and international studies related to the chemical compositions and biological activities of E agallocha and E cochinchinensis CHAPTER II RESEARCH OBJECTIVE AND RESEARCH METHODOLOGY II.1 Research objective Figure II.1 E agallocha Figure II.2 E cochinchinensis The leaves of E agallocha were collected in Xuan Thuy, Nam Dinh, Vietnam in July 2013 The leaves of E cochinchinensis were collected in Van Giang, Hung Yen, Vietnam in April 2016 Two samples were identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST The voucher specimens were deposited at the Institute of Ecology and Biological Resources and Institute of Marine Biochemistry, VAST, Vietnam II.2 Research methodology II.2.1 Methods for extraction The samples were cut into pieces and extracted three times with MeOH at room temperature (for days) or in an ultrasonic bath (three times, each time 45 min) Evaporation of the solvent in vacuo obtained a residue, which was suspended in distilled water and partitioned in turn with n-hexane, CH2Cl2, and EtOAc 2.2.2 Methods for metabolites isolation Combining a number of chromatographic methods including thinlayer chromatography (TLC), column chromatography (CC), silica gel, RP-18, and Sephadex LH-20 II.2.2 Methods for determination of the chemical structure of compounds The general method used to determine the chemical structure of compounds is the combination between physical parameters and modern spectroscopic including optical rotation ([α]D), electrospray ionization mass spectrometry (ESI-MS), and high-resolution ESI-MS (HR-ESI-MS), one/twodimension nuclear magnetic resonance (NMR) spectra II.2.3 Methods for evaluation of biological activities Cytotoxic activity was evaluated against three human cancer cell lines, MCF-7 (human breast cancer cells), LU-1 (human lung adenocarcinoma), and KB (human epidermoid carcinoma) by the MTT and SRB assays Anti-inflammatory activity of isolated compounds was assessed based on inhibiting NO production in lipopolysaccharide (LPS) activated RAW264.7 cells The antimicrobial activity of the isolated metabolites against a selected panel of the Gram-positive (Bacillus subtillis ATCC11774 and Staphylococcus aureus ATCC11632) and Gram-negative (Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC27853) bacteria, as well as a set of yeast molds (Aspergillus niger 439, Fusarium oxysporum M42, Candida albicans ATCC7754, and Saccharomyces cerevisiae SH 20), were also determined CHAPTER III EXPERIMENT AND EMPIRICAL RESULTS III.1 Isolation of compounds III.1.1 Isolation of compounds from E agallocha This part showed the extraction and isolation experiments of the compounds isolated from the leaves of E agallocha The leaves of Excoecaria agallocha (2.5 kg dried) A: Acetone CC: Chromatography column D: Dichloromethane Extraction with ultrasonic bath MeOH, times, 45min, 45-60oC M: Methanol H: n-Hexane MeOH extract (A, 200 g) W: water Add water (1L) Add CHCl3 (1L×3 times) CHCl3/H2O 1:1 CHCl3 fraction (94 g) H2O layer Add EtOAc (1L×3 times) n-Hexane/60% Aq MeOH n-Hexane fraction (H, 80 g) MeOH fraction (C, 14 g) H2O layer (W) EtOAc fraction (E, g) n-Hexane, n-Hexane-acetone gradien 100:1, 70:1, 0:100 C-1 C-2 (2.6 g) ( 1.1 g) C-3 CC, Silica gel HA 3:1, DA 4:1 C-3A (10.1 g) C-3B RP-18, CC, MW 1:1 C-3C RP-18, CC, MW 1:2 EA-5 EA-3 (5,8 mg) (8 mg) Figure III.1 Isolation of compounds from the CHCl3 fraction of E agallocha Cặn ethylfraction acetate EtOAc (E, g) RP-C18, CC, H2O/MeOH :2/1 E-1 E-2 E-3 E-4 (0,45 g) (1,5 g) (2,7g) (2,24 mg) Sephadex H2O/MeOH:2/1 E-2B E-2B CC, CH2Cl2/MeOH: 2/1 v/v EA-9 (8,2 mg) E-1 E-2 E-3 (0,45 g) (1,5 g) (2,7g) Sephadex H2O/MeOH:1/1 Sephadex H2O/MeOH:1/1 EA-4 EA-6 EA-7 (12 mg) (7 mg) (10,2 mg) Figure III.2 Isolation of compounds from the EtOAc fraction of E agallocha Water layer Diaion HP-20 MeOH-H2O (gradient 0:100, 25:75, 50:50, v/v) W-1 W-2A W-2B Silica gel CC, CHCl3-MeOH (30:1, 20:1, v/v) W-3A W-3C RP-18 CC MeOH-H2O (3:3, v/v) W-3A1 Silica gel CC, n-Hexane-acetone (5:2) W-4 W-3 W-2 Silica gel CC, CHCl3-MeOH (50:1, 25:1, v/v) W-3A2 W-3B W-3D RP-18 CC Acetone-H2O (1:3,5, v/v) W-3B1 W-3B2 W-3B3 Sephadex LH-20, MeOH-H2O (1:2) Sephadex LH-20, MeOH-H2O (1:2) EA-8 EA-1 EA-2 (4 mg) (3.5 mg) (5 mg) Figure III.3 Isolation of compounds from the water layer of E agallocha III.1.2 Isolation of compounds from E cochinchinensis This section presents the process of isolating 13 compounds from the leaves of E cochinchinensis The leaves of Excoecaria cochinchinensis (3 kg dried) Extraction with ultrasonic bath MeOH, time, 45min, 45-55oC MeOH extract (M, 450 g) n-Hexane/H2O 1:1 Add water (1L) Add n-hexane (1.5L × time) H2O layer n-Hexane fraction (H, 120 g) add EtOAc (1.5L × time) EtOAc/H2O 1:1 EtOAc fraction (E, 100 g) H2O layer (W) Figure III.4 The partitioned MeOH extract of E cochinchinensis Water layer Diaion HP-20 MeOH-H2O (gradient 0:100, 25:75, 50:50, v/v) W-1 W-3 W-2 Silica gel CC, CHCl3-MeOH (50:1, 25:1, v/v) W-2A W-2B W-4 (85 g) Silica gel CC, CHCl3-MeOH (30:1, 20:1, v/v) W-3B W-3A W-3C W-3D RP-C18 CC Acetone-H2O (1:3,5, v/v) W-3B1 W-3B3 W-3B2 Sephadex LH-20 (MeOH-H2O, 1:2) EC-6 EC-2 (3 mg) (8 mg) Silica gel CC, n-Hexane-acetone (5:2); Sephadex LH-20, MeOH-H2O (1:1) EC-11 EC-10 EC-9 EC-8 (2,7 mg) (6,6 mg) (3 mg) (3 mg) Silica gel CC, CH2Cl2-MeOH-H2O (5:1:0,1); RP-C18, MeOH-H2O (1:1) EC-5 EC-4 EC-3 EC-1 (5,5 mg) (2 mg) (2 mg) (2,2 mg) Silica gel CC, CH2Cl2-MeOH-H2O (6:1:0,05); Sephadex LH-20, MeOH-H2O (1:1) EC-13 EC-12 EC-7 (20 mg) (21 mg) (3 mg) Figure III.7 Isolation of compounds from the water layer of E cochinchinensis III.1.3 Physical properties and spectroscopic data of the isolated compounds III.1.3.1 Physical properties and spectroscopic data of the isolated compounds from E agallocha This section presents physical properties and spectroscopic data of 09 compounds from E agallocha III.1.3.2 Physical properties and spectroscopic data of the isolated compounds from E cochinchinensis This section presents physical properties and spectroscopic data of 13 compounds from E cochinchinensis III.2 Results on cytotoxic activities of isolated compounds III.2.1 Results on cytotoxic activity of extract from E agallocha Table III.1 The effects of the MeOH extract from E agallocha Cell line LU-1 % inhibition IC50 (µg/mL) % inhibition IC50 (µg/mL) MCF7 % IC50 inhibition (µg/mL) MeOH extract 81.90 19.77 85.03 15.23 65.38 57.57 Ellipticine 10 µg/mL 97.18 0.39 96.35 0.50 95.73 0.48 Sample KB III.2.2 Results on antimicrobial activity of compounds from E agallocha Table III.3 The effects of isolated compounds from E agallocha Minimum inhibitory concentration (MIC, g/mL) Samples Gram (-) Ec Streptomycin (57,5 g/mL) Nystatin (92,5 g/mL) Tetracyclin (44 g/mL) * Gram (+) Pa * Bc * Sa Fungus * * An Fo * Sc* Ca* - - 7.188 14.375 - - - - - - - - 23.125 11.563 5.781 11.563 5.5 11 - - - - - - EA-1 >50 - - - - - - >50 EA-2 - - >50 - >50 - - - EA-3 - - - - >50 - - - EA-4 - - >50 - - - - - EA-5 - >50 - - - 50 - - EA-6 - - - - - - - - EA-7 - - - - - - - - EA-8 - - - - - - - - EA-9 >50 - - - >50 - >50 - MeOH extract - - 200 - - - - - Streptomycin, nystatin, and tetracyclin were used as the positive control Ec (Escherichia coli), Pa (Pseudomonas aeruginosa), Bc (Bacillus subtillis), Sa (Staphylococcus aureus), An (Aspergillus niger), Fo (Fusarium oxysporum), Sc (Saccharomyces cerevisiae), and Ca (Candida albicans) (-) No detection III.2.3 Results on anti-inflammatory activity of isolated compounds from E cochinchinensis Table III.4 Effects of compounds on the LPS-induced NO production on RAW264.7 cells from E cochinchinensis Compounds Concentration (µM) Inhibition (%) Growth of cell (%) EC-1 38.72 ± 0.56 76.12 ± 1.36 EC-2 46.78 ± 0.35 78.89 ± 1.32 EC-3 75.83 ± 0.77 86.65 ± 1.54 EC-4 31.09 ± 1.60 56.49 ± 0.97 EC-5 42.02 ± 1.01 75.51 ± 1.52 EC-6 68.18 ± 0.67 73.59 ± 0.67 39.22 ± 1.07 78.60 ± 2.18 EC-8 94.96 ± 0.26 80.41 ± 1.66 EC-9 82.91 ± 1.03 83.19 ± 2.37 EC-10 27.87 ± 0.81 84.55 ± 0.98 EC-11 30.47 ± 0.69 82.91 ± 1.43 EC-12 38.38 ± 0.19 70.16 ± 1.77 EC-13 35.01 ± 0.76 55.50 ± 2.54 0.3 33.89 ± 0.51 95.35 ± 0.75 88.80 ± 0.51 86.00 ± 1.55 100 EC-7 Cardamonin Cardamonin was used as a positive control Data are presented as the mean ± standard deviation (SD) of at least three independent experiments performed in triplicate Table III.5 The IC50 values of selected compounds Compounds IC50 values (µM) EC-3 13.80 ± 1.23 EC-6 58.10 ± 2.04 EC-8 6.17 ± 0.25 EC-9 12.02 ± 0.73 Cardamonin 1.57 ± 0.24 Cardamonin was used as a positive control Data are presented as the mean ± standard deviation (SD) of at least three independent experiments performed in triplicate 11 Figure VI.6 HMBC spectrum of EA-1 Figure VI.7 COSY spectrum of EA-1 Detailed analysis of correlations provided by COSY and HMBC experiments (Fig IV.1) revealed that the planar structure of EA-1 was similar to that of agallochin I, previously isolated from the same species, except for the presence of an additional hydroxy group at C-17 In fact, the HMBC cross-peaks from H-17 to C-12, C-13, C-14, and C-16 placed the hydroxy group at C-17, whereas the other hydroxy group and the methyl group were placed at C-6 and C-4, respectively, due to the COSY correlations of H-18/H-4/H-5/H6/H-7 The downﬁeld chemical shift of the quaternary carbon at δC 98.6 (C-3) in conjunction with the HMBC correlations from H-20 to C-1, C-3, C-5, and C-10 indicated that the ether bridge was positioned between C-20 and C-3, and the last hydroxy group was located at C-3 12 Figure VI.8 Keys NOESY correlations of EA-1 Figure IV.9 NOESY spectrum of EA-1 The relative stereochemistry of EA-1 was obtained through analysis of 1H NMR coupling constants and NOESY experiment Speciﬁcally, the large J-values (J = 11.0 - 12.5 Hz) of H-5, H-6, Ha-7, and H-9 indicated the axial orientation of these protons The NOE correlations between H-5/H-9, Ha-1, Ha-7; Ha-7/Ha-14, H-9; Hb-20/H15; H-15/H-16 and Ha-20/Ha-11, Hb-1, Ha-2 conﬁrmed the structure of beyer-15-ene diterpenoid skeleton Finally, the conﬁgurations at C-4 and C-6 were determined on the basis of the NOE correlations between H6/Hb-20, H-15, H-4 and between H3-18/Hb-2 (Fig IV.8-IV.9) Therefore, compound EA-1 was elucidated as 3β,20-epoxy-3,6α,17trihydroxy-19-nor-beyer-15-ene (excoecarin L) 13 Table IV.1 The NMR data of EA-1 and reference compound δCa,b  δCc,d δCc,e (mult., J in Hz) 31.2 32.7 26.9 28.2 1.27 (1H, m) 2.09 (1H, ddd, 3.5, 12.5, 12.5) 1.71 (1H, m) 2.02 (1H, ddd, 3.5, 12.0, 13.5) 98.3 98.6 - 41.5 42.7 1.96 (1H, m) 56.9 57.9 1.03 (1H, dd, 5.0, 11.0) 69.5 44.7 71.1 46.2 3.75 (1H, ddd, 4.0, 11.0, 11.5) 1.43 (1H, dd, 11.5, 13.0) 1.85 (1H, dd, 4.0, 13.0) 49.4 50.2 - 44.5 46.4 1.20 (1H, dd, 4.5, 12.5) 10 36.3 37.5 - 11 20.7 21.4 1.08 (1H, m)/1.72 (1H, m) 12 31.9 28.0 1.28 (1H, m)/1.36 (1H, m) 13 14 43.7 60.4 51.1 56.6 1.09 (1H, m) 1.67 (1H, dd, 2.5, 9.5) 15 133.3 135.5 5.73 (1H, d, 6.0) 16 17 137.9 24.4 135.2 68.6 5.66 (1H, d, 6.0) 3.40 (1H, d, 11.0) 3.45 (1H, d, 11.0) 18 20 19.3 68.5 19.6 69.4 1.12 (3H, d, 7.0) 3.80 (1H, dd, 5.0, 9.5) 3.89 (1H, dd, 3.5, 9.5) No # CDCl3, b75MHz; cCD3OD, d125MHz, e500MHz #δC of agallochin I  a 14 Figure IV.26 The structures of compounds isolated from E agallocha IV.2 Determination of chemical structure of isolated compounds from E cochinchinensis VI.2.1 6α,7α-Epoxy-4β,5β,9α,13α-tetrahydroxy-rhamnofola-1,15-dien3-one 20-O-β-D-glucopyranoside (EC-1, new compound) Figure IV.27 Structure of EC-1 and reference compound Compound EC-1 was isolated as a white, amorphous powder Its molecular formula was determined to be C26H38O12 by the negative HR-QTOF-MS ion peaks at m/z 541.2297 [M - H]– (calcd for C26H37O12–, 541.2291), 577.2063 [M + Cl]– (calcd for C26H38ClO–, 577.2057), and 587.2346 [M + HCOO]– (calcd for C27H39O–, 587.2345), indicating eight degrees of unsaturation (Fig IV.28) 15 Figure IV.28 HR-ESI-MS spectrum of EC-1 Figure IV.28 1H NMR spectrum of EC-1 Figure IV.29 13C NMR spectrum of EC-1 16 Figure IV.30 HSQC spectrum of EC-1 13 The C NMR and HSQC spectra revealed the presence of 26 carbon atoms including non-protonated carbons, 13 methines, methylenes, and methyls Among them, a typical α,β-unsaturated carbonyl moiety [δC 209.9 (C-3), 134.8 (C-2), 163.1 (C-1)], two other olefinic carbons [δC 145.9 (C-15), and 117.3 (C-16)], three oxygenated tertiary carbons [δC 74.8 (C-4), 64.6 (C-6), and 77.3 (C-9)], three oxymethines [δC 71.4 (C-13), 68.1 (C-5), and 62.3 (C-7)], and an oxymethylene [δC 74.2 (C-20)], along with a glucopyranosyl unit [δC 104.8 (C-1′), 75.2 (C-2′), 78.0 (C-3′), 71.7 (C-4′), 78.0 (C-5′), and 62.8 (C-6′)] were observed (Table IV.9) Since one carbonyl group and two double bonds accounted for three degrees of unsaturation, EC-1 was determined to be a pentacyclic compound Accordingly, the 1H NMR spectrum showed the existence of three methyls [δH 1.70 (3H, s, H-17), 0.96 (3H, d, J = 7.0 Hz, H-18), and 1.76 (3H, d, J = 2.0 Hz, H-19)], one terminal double bond [δH 4.98 (1H, d, J = 2.0 Hz, H-16a)/5.04 (1H, br s, H-16b)], and one trisubstituted double bond [δH 7.66 (1H, br s, H-1)] (Fig IV.28-IV.29) The large coupling constant of the anomeric proton [δH 4.33 (1H, d, J = 7.5 Hz, H-1′) confirmed the β-glucosidic linkage (Fig IV.26) Careful comparison of the 1H and 13C NMR spectroscopic data for diterpenoidal nucleus of (Table IV.9) with those of venenatin, a daphnane-type diterpenoid, revealed that they were very similar and these compounds had the same structure of A and B rings 17 Figure IV.30 Keys COSY, HMBC, and NOESY correlations of EC-1 Figure IV.32 HMBC spectrum of EC-1 This deduction was also confirmed by COSY and HMBC correlations as shown in Fig IV.30 Besides, the COSY cross-peaks of H-7/H-8/H-14/H-13/H-12/H-11/H-18 in combination with the HMBC correlations from H3-18 to C-9, C-11, C-12, from H-7 to C-9 and C-14, from H-8 to C-9, C-11, C-13, C-14, C-15, from H3-17 to C-14, C-15, C16, and from H2-16 to C-14, C-17 established structure of C ring, which was fused to the B ring at the C-8 and C-9, and substituted with two hydroxy groups at C-9 and C-13, a methyl group at C-11, and an 18 Figure III.33 COSY spectrum of EC-1 Figure IV.34 NOESY spectrum of EC-1 isopropenyl moiety at C-14 The downfield chemical shift of the oxymethylene carbon at δC 74.2 (C-20) along with the HMBC correlation from H-1′ to C-20 indicated the position of glucosyl moiety Thus, EC-1 was established as a rhamnofolane diterpene glucoside The relative configuration of EC-1 was assigned by analysing proton-proton coupling constants and NOESY data The H-14 signal (dd, J = 10.0, 12.5 Hz) exhibited two large coupling with the H-8 (br d, J =12.5 Hz) and H-13 (ddd, J = 4.5, 10.0, 10.5 Hz) revealed the transdiaxial orientation of these protons The equatorial orientation of Ha-12 (ddd, J = 4.5, 4.5, 12.5 Hz) was deduced by the small coupling constants with its vicinal protons In the NOESY spectrum, the correlations from 19 Table IV.9 The NMR data of EC-1 and reference compound No Venenatin  δCa,b δHa,c (mult., J in Hz) 10 11 12 13 14 15 16 17 18 19 20 162.2 135.5 209.9 74.6 70.5 64.0 65.4 39.1 79.8 51.0 39.5 39.1 75.4 79.4 147.2 114.7 19.3 18.3 9.9 65.0 163.0 134.7 209.9 74.7 68.1 64.6 62.3 40.0 77.3 51.5 39.4 41.1 71.4 54.2 145.9 117.2 18.9 18.9 9.9 74.2 1' 2' 3' 4' 5' 6' - 104.8 75.2 78.0 71.7 78.0 62.8 7.66 (1H, br s) 4.30 (1H, br s) 3.19 (1H, br s) 3.10 (1H, br d, 12.5) 4.15 (1H, dd, 2.5, 3.0) 2.10 (1H, m) 1.60 (1H, m)/1.73 (1H, m) 3.52 (1H, m) 2.80 (1H, dd, 10.0, 12.5) 4.98 (1H, d, 2.0)/5.04 (1H, br s) 1.70 (3H, s) 0.96 (3H, d, 7.0) 1.76 (3H, d, 2.0) 3.45 (1H, d, 11.0) 4.27 (1H, d, 11.0) 4.33 (1H, d, 7.5) 3.20 (1H, *) 3.36 (1H, *) 3.27 (1H, *) 3.26 (1H, m) 3.64 (1H, dd, 5.5, 12.0) 3.86 (1H, dd, 1.0, 12.0) a CD3OD, b125 MHz, c500 MHz *Overlapped signals 13C NMR of venenatin  H-8 to H-11, H-13, H3-17 and H-7, and from H-7 to Ha-20 and H3-17, and from H3-17 to H-13 and H-16b, from H3-18 to H-1 established the β-configuration for H-8, H-11, H-13, H-7, and C-6-C-20 bond On the other hand, the NOESY cross-peaks from H-14 to Hb-12 and Ha-16, and from H-5 to H-10 suggested that these protons were α-oriented The configuration of OH-4 and OH-9 was assigned as β and α-orientation, respectively, by comparing the 13C NMR data with those of venenatin as well as the coexistence of two compounds in this plant Furthermore, 20 most related [5-7-6]tricyclic diterpenoids (daphnanes and tiglianes) of Euphorbiaceae family have been found to possess trans-A/B ring and trans-B/C ring Therefore, compound EC-1 was determined as 6α,7αepoxy-4β,9α,13α,20β-tetrahydroxy-rhamnofola-1,15-dien-3-one 20-Oβ-D-glucopyranoside Figure IV.53 The structures of 13 compounds isolated from E cochinchinensis 21 IV.3 Biological activities of isolated compounds IV.3.1 Cytotoxic activity of extracts and compounds isolated from Excoecaria agallocha Cytotoxicity testing method is carried out to evaluate the ratio of living cells and dead cells after treatment cells with tested samples This is a basic method to screening new compounds for the development of anti-cancer agents All isolated compounds were examined in three cancer cells line: human breast cancer (MCF-7), human lung cancer (LU) and human epithelial cancer The results showed that MeOH extract inhibited more than 50% of the growth of all three cancer cell lines The positive compound Ellipticine was used to determine the stability of the experiment The values had high accuracy with r2 ≥ 0,99 (Table II.1) However, all isolated compounds exhibited weak or no cytotoxic activity against tested cell lines (IC50 > 100 µM) III.3.2 Anti-microorganism activity of extracts and compounds isolated from Excoecaria agallocha Screening results showed that MeOH extract inhibited the growth of only one Gram-positive bacteria B subtillis strain (MIC = 200 µg/mL) Isolated compounds were tested the anti-microorganism in bacterial strains including Gram-negative bacteria strains: E coli, P aeruginosa, Gram-positive bacteria strains: B subtillis, S aureus, fungal strains: A niger, F oxysporum, yeast strains: S cerevisiae C albicans Results demonstrated that compound blumenol A (EA-6) exhibited good inhibition activity against the growth of F oxysporum strain (MIC = 50 µg/mL) III.3.3 Anti-inflammatory activity of compounds isolated from Excoecaria cochinchinensis NO production process is one of the body's self-protective responses, but NO overproduction leads to cell and tissue damage, promotes inflammation, and causes acute and chronic inflammatory diseases Therefore, the level of NO production is considered as one of the indicators for the inflammatory process Compounds that have the ability to inhibit the production of NO are anti-inflammatory agents 13 compounds (EC-1 – EC-13) isolated from E cochinchinensis were tested the anti-inflammatory effect in LPS-induced RAW264.7 macrophages cells Results showed that compounds EC-3, EC-8, and EC-9 inhibited the NO production significantly (Table II.2-II.3) at the concentration of 100 µM The IC50 of three compounds EC-3, EC-8 and 22 EC-9 were 13.80 ± 1.23, 6.17 ± 0.25, and 12.02 ± 0.73 µM, respectively compared with the IC50 of positive control Cardamonin (1.57 ± 0.24 µM) At the concentration of 100 µM, compound EC-6 showed medium inhibitory activity, other compounds exhibited weak activity (IC50 > 50 µM These findings indicated high similarity with the previously published results of active compounds from the genus Excoecaria This contributes to demonstrate more clearly the therapeutic uses in traditional medicine and increase the scientific value of these plants in Vietnam 23 CONCLUSIONS Chemical composition investigations: By using various chromatographic methods, 22 compounds were isolated from Excoecaria agallocha L and E cochinchinensis Lour Their chemical structures were determined by NMR, electrospray ionization (ESI)-MS, and as well as by comparison with those reported in the literature - 09 compounds were isolated and identified from E agallocha, including two new compounds, named excoecarin L (EA-1) and excoecarin O (EA-2), as well as seven known compounds: aquillochin (EA-3), (+)-isolariciresinol (EA-4), (+)-epipinoresinol (EA-5), blumenol A (EA-6), blumenol B (EA-7), kaempferol (EA-8), and methyl gallate (EA-9) - 13 compounds were isolated and identified E cochinchinensis, including two new compounds, named 6α,7α-epoxy-4β,5β,9α,13αtetrahydroxy-rhamnofola-1,15-dien-3-one 20-O-β-D-glucopyranoside (EC-1) and acid 3-(2-O-β-D-glucopyranosyl-3-hydroxyphenyl) propanoic (EC-9), as well as 11 known compounds: venenatin (EC-2), glochionionol A (EC-3), (6R,9S)-roseoside (EC-4), isofraxoside (EC5), pinoresinol-4'-O-β-D-glucoside (EC-6), liriodendrin (EC-7), rhamnocitrin 3-glucoside (EC-8), sinapyl alcohol 4-O-β-Dglucopyranoside (EC-10), 2,3-dihydroxypropyl-benzoate 3-O-α-(4''methoxyglucuronide) (EC-11), phenethyl rutinoside (EC-12, and benzyl-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside (EC-13) Investigation of biological activity - The cytotoxic effects of MeOH extract were investigated in vitro in three human cancer cell lines: KB, LU-1, and MCF7 The results showed that MeOH extract showed the strong cytotoxic effect in three tested cell lines KB, LU-1, and MCF-7 - Anti-microorganism activity testing showed that MeOH extract from E agallocha inhibited the development of only one Gram-positive bacteria B subtilli strain with the MIC of 200 µg/mL Compound blumenol A (EA-6) revealed an inhibitory effect against the growth of F oxysporum strains (MIC = 50 µg/mL) - 13 compounds from E cochinchinensis were used to measure the anti-inflammatory activity in LPS-induced RAW264.7 macrophage cells Three compounds EC-3, EC-8, and EC-9 inhibited the NO production significantly with the IC50 of 13.80 ± 1.23, 6.17 ± 0.25, and 12.02 ± 0.73 µM, respectively 24 RECOMMENDATIONS All findings of chemical compositions and biological activity of E agallocha and E cochinchinensis suggested that compound EC-8 might be a potential candidate to study further the mechanism of action in vivo in the treatment of inflammatory diseases NEW CONTRIBUTIONS OF THE THESIS From two Excoecaria species: Excoecaria agallocha L and E cochinchinensis Lour were isolated and identified 04 new compounds, including: - Compounds excoecarin L (EA-1) and excoecarin O (EA-2) were isolated for the first time from the leaves of E agallocha Compounds 6α,7α-epoxy-4β,5β,9α,13α-tetrahydroxyrhamnofola-1,15-dien-3-one 20-O-β-D-glucopyranoside (EC-1) acid 3-(2-O-β-D-glucopyranosyl-3-hydroxyphenyl) propanoic (EC-9) were isolated for the first time from the leaves of E agallocha The antimicrobial activity of the isolated metabolites from the leaves of E agallocha against a selected panel of the Gram-positive and Gram-negative bacteria, as well as a set of yeast molds, was determined The results showed that blumenol A (EA-6) exhibited selective activities against the fungus F oxysporum with a MIC of 50 µg/mL The present study is the first report for antimicrobial activity of the chemical components isolated from E agallocha in Vietnam and warrants further studies concerning the potential of this plant for medicinal purposes The isolated compounds (EC-1 – EC-13) from E cochinchinensis were tested for their ability to inhibit NO production in LPS-stimulated RAW264.7 cells Among them, Compounds EC-3, EC8 EC-9 were the most active compounds with the IC50 values of 13.80 ± 1.23, 6.17 ± 0.25, and 12.02 ± 0.73 µM, respectively The in vitro cytotoxic activity of MeOH extract and the isolated compounds from E agallocha were investigated on three human cancer cell lines, MCF-7 (human breast cancer cells), LU-1 (human lung adenocarcinoma), and KB (human epidermoid carcinoma) by the MTT and SRB assays The results indicated that MeOH extract exhibited potent cytotoxic effects against three tested human cancer cell lines (MCF-7, LU-1, and KB), relative to the effects of the positive control, ellipticine PUBLICATIONS WITHIN THE SCOPE OF THESIS Nguyen Van Thanh, Lai Hop Hieu, Phan Thi Thanh Huong, Le Thi Vien, Tran My Linh, Nguyen The Cuong, Nguyen Xuan Cuong, Nguyen Hoai Nam, Ngo Dai Quang, Chau Van Minh Excoecarins L and O from the mangrove plant Excoecaria agallocha L Phytochemistry Letters, 25, 52-55, 2018 Lai Hop Hieu, Nguyen Phuong Thao, Nguyen Van Thanh, Do Hoang Anh, Tran Thi Hong Hanh, Nguyen Duy Cong, Nguyen The Cuong, Nguyen Van Thanh, Nguyen Xuan Cuong, Nguyen Hoai Nam, Ngo Dai Quang, and Chau Van Minh Metabolites of Excoecaria cochinchinensis Lour Phytochemistry Letters, 37, 116120, 2020 ... mangrove plants Therefore, the thesis namely ? ?Study on chemical constituents and biological activities from the leaves of Excoecaria agallocha L and Excoecaria cochinchinensis Lour.? ?? was conducted to... objectives of the thesis Isolation and determination of chemical structures of the isolated compounds from the leaves of Excoecaria agallocha L and Excoecaria cochinchinensis Lour 2 Studied the. .. and antimicrobial activities of the isolated compounds to find the bioactive compounds The main contents of the thesis Isolation of compounds from the leaves of Excoecaria agallocha and E cochinchinensis - 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Xem thêm: Tóm tắt luận án Tiến sĩ Study on chemical constituents and biological activities from the leaves of Excoecaria agallocha L. and Excoecaria cochinchinensis Lour., Tóm tắt luận án Tiến sĩ Study on chemical constituents and biological activities from the leaves of Excoecaria agallocha L. and Excoecaria cochinchinensis Lour.