Nghiên cứu tác dụng ức chế của cao chiết cây mần tưới (eupatorium fortunei turcz ) lên sinh trưởng của vi khuẩn lam độc microcystis aeruginosa kutzing trong các thủy vực nước ngọt tt tiếng anh

MINISTRY OF EDUCATION AND VIETNAM ACADEMY OF SCIENCE TRAINING AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ********************** PHAM THANH NGA INHIBITORY EFFECT OF EUPATORIUM FORTUNEI TURCZ EXTRACTS ON THE GROWTH OF A TOXIC CYANOBACTERIAL SPECIES Microcystis aeruginosa IN FRESH WATERBODIES Major: Environmental Engineering Code: 9.52.03.20 SUMMARY OF DOCTORAL THESIS IN ENVIRONMENTAL ENGINEERING Hanoi - 2019 The doctoral thesis was completed at Institute of Environmental Technology (IET), Graduate University of Science and Technology, Vietnam Academy of Science and Technology Supervisors: Prof Dr Dang Dinh Kim Dr Le Thi Phuong Quynh Reviewer Reviewer Reviewer This doctoral thesis will be defended at Graduate University of Science and Technology, Vietnam Academy of Science and Technology at …… on …… 2019 This doctoral thesis can be found at: Library of the Graduate University of Science and Technology, VAST INTRODUCTION NECESSITY OF DOCTORAL THESIS Eutrophication is a widespread problem in aquatic ecosystems around the world due to sewage and surface run-off It significantly affects water quality and induces off-flavor problem Moreover, cyanobacterial blooms usually break out along with release of cyanotoxins, which cause a series of adverse effects such as decreasing water quality and biodiversity, and illness in animals and humans Among all sorts of microalgae, Microcystis aeruginosa, one of the most common representative species responsible for the water blooming, can produce hepatotoxins and neurotoxins which may lead to headache, fever, abdominal pain, nausea, vomiting and even cancer Therefore, it is of great importance to inhibit the growth of cyanobacteria, especially M aeruginosa in eutrophic waters Basically, there are three short-term approaches to control harmful algal blooms such as chemical, physical and biological approaches Chemical treatments can effectively and rapidly remove algal bloom However, some algicidal chemicals can cause secondary pollution of aquatic environment or persistence in the environment and the inhibitory effects of most chemicals not selectively target harmful cyanobacteria; leading to the collapse of aquatic ecosystems Physical methods like mixing lake water using an air compressor, pressure devices or ultraviolet irradiation indicate less subsequent secondary pollution However, the disadvantages of physical treatments of algal removal are energy intensive and tend to be low efficiency as well as injury to non-target species In recent years, biological methods including using algicidal bacteria have received much more attention as alternatives to chemical agents These approaches tend to be environmental friendly and promising methods for controlling toxic cyanobacteria However, the efficiency of biological method is influenced by many biotic and abiotic factors in the environment For these limitations of the above approaches, the discovery and use of natural compounds that feature selective toxicity towards phytoplankton communities and are nontoxic to other aquatic species, have been a significant advance in the management of aquatic ecosystems Eupatorium fortunei Turcz, a species of Asteraceae, is a perennial herb used in folk medicine as a medicinal and has been demonstrated antibacterial activity in various scientific studies In 2008, Nguyen Tien Dat and et al carried out the experiments of using plant extracts to inhibit the growth of M aeruginosa The results showed that the extract from E fortunei indicated the highest inhibitory effect on the species This conclusion was confirmed by the publication of Pham Thanh Nga in the following years However, these are only preliminary studies investigating the using of the plant extract to control toxic cyanobacterial bloom By wishing to inherit, develop previous research results and solve several reaserch questions related to the issue, author chose topic: “Inhibitory effect of Eupatorium fortunei Turcz extracts on the growth of a toxic cyanobacterial species Microcystis aeruginosa Kützing in fresh waterbodies” RESEARCH PROPOSE OF THE DOCTORAL THESIS Research to create effective plant extracts from E fortunei to inhibit growth of Microcystis aeruginosa Kützing in the laboratory and outdoor larger scale TASKS OF THE DOCTORAL THESIS - Develop the process of producing crude extracts, fractions and pure chemical compounds isolated from E fortunei - Study of inhibitory effect of the crude ethanol extracts from E fortunei on the growth of M.aeruginosa and evaluating their ecological safety to non-target aquatic organisms - Study of inhibitory effect of the fraction extracts from E fortunei on the growth of M aeruginosa and evaluating their ecological safety to non-target aquatic organisms - Study of bioactive properties of chemical compounds isolated from E fortunei - Research on the application of plant extracts to control cyanobacterial bloom in natural water samples (in the laboratory and outdoor scales) METHODOLOGY OF RESEARCH The author uses different modern research methods which provide the scientific reliable results and suitable to Vietnam's conditions The methods include 1) Methods of plant sample treatment, production of plant extraction and isolation of pure chemical compounds; 2) Method of identifying the chemical structure of pure compounds (1H, 13C-NMR, DEPT, HMBC, HR- ESI-MS); 3) Methods of evaluating the growth of cyanobacterial M aeruginosa, Ch vulgaris and phytoplanktons; 4) Method of evaluating the toxicity of plant extracts to non-target aquatic organisms (Daphnia magna and Lemna minor); 5) Morphology of M aeruginosa and Ch vulgaris under the exposure of plant extracts (TEM); 6) Standard methods in water analysis (physical and chemical parameters) SCIENTIFIC AND PRACTICAL MEANINGs OF THE DOCTORAL THESIS Water pollution, especially the eutrophication that caused the cyanobacterial bloom including mainly M aeruginosa which releases microcystin toxins, has received much attention and research in recent times Using plant extracts to control this phenomenon indicates more advantages than other traditional methods used previously The results of the doctoral thesis provide a scientific basis of the feasibility of using plant extracts as a selective inhibitor to the growth of M aeruginosa in order to control the toxic cyanobacterial bloom while not harm to other non-target organisms in aquatic ecosystems NEW CONTRIBUTION OF THE DOCTORAL THESIS Isolation of 02 pure new chemical compounds from Eupatorium fortunei which have not been published in international scientific journals Investigation of the biological activity of these compounds to control M aeruginosa at the concentrations from 1.0 µg.mL-1 to 50 µg.mL-1 Growth inhibitory effect (IE) was recorded from 10 to 45% after 72 hours of exposure Application of the innovative method to control the growth of toxic microalga (M aeruginosa) by using extracts from Eupatorium fortunei Turcz The experiment was carried out from the laboratory scale in 150- mL flashes with IE of over 90%, then in the 5L aquarium and in the outdoor scale (3 m3) with IE around of 60 % for evaluating the different efficiency between the theoretical value and practical application The ethanol extract proved to be more toxic to M aeruginosa than to Daphnia magna and Lemna minor STUCTURE OF THE THESIS The thesis is organized in the introduction, three chapters and concluding section with 143 pages, 18 tables and 45 figures and graphs The thesis uses 182 references with more than 40% of the papers published in the last five years (from 2013 to 2018) Chapter presents an overview about researches related to eutrophication and the toxic cyanobacterial bloom in aquatic ecosystem and the methods used to control these problems Chapter presents research objectives, methods and the design of experiments Chapter shows the reaserch results and gives discussion The chapter will be presented in more detail in the next section CHAPTER RESULTS AND DISCUSSION 3.1 The process of producing crude extracts, fractions and pure chemical compounds isolated from E fortunei Turcz Table 3.1 Effeciency of crude extract production in various solvents Solvent Gram crude plant extract/100gram dried materials Ethanol 9.17 Methanol 12.75 W (Water) 8.75 Table 3.2 Effeciency of fraction production from crude ethanol extracts of E fortunei Fractions Gram fractions/100 gram crude ethanol extract (%) n-hexan 18.97 EtOAc 10.57 W 60.27 Table3.3 Effeciency of isolating chemical compounds from E fortunei Compounds EfD5.1 EfD14.1 EfD1.8 EfD10.1 EfD10.3 EfD4.7 EfD4.8 Mg compound/100 g EtOAc fractions of E fortunei 71.69 20.80 13.34 4.56 3.91 2.61 1.56 Figure 3.1 Process of isolating chemical compounds from the ethyl acetate fraction new compounds Figure 3.2 7,8,9-trihydroxythymol (EfD4.7) Figure 3.3 8,10-didehydro-7,9dihydroxythymol(EfD4.8) EfD4.7 White powders; []D24 = +0,2 (c 0.1, MeOH) The HR-ESI-MS (positive) revealed a peak [M + Na]+ at m/z 221,0783 (C10H14NaO4) In the 1H NMR spectra of EfD4.7 compound, the presence of aromatic signalsABX at δH 6,79 (1H, d, J = 2,0 Hz, H-2), 7,20 (1H, d, J = 7,5 Hz, H-5), and 6,81 (1H, dd, J = 7,5, 2,0 Hz, H-6)], one group ethyl at δH 1,58 (3H, s, H-10) 1H NMR (500 MHz, CD3OD) 13C NMR (125 MHz, CD3OD) [Table 3.4] Firuge 3.4.HSQC spectra of EfD4.7 Figure 3.5.HMBC of EfD4.7 Actually, the 1H and 13C-NMR spectra data of EfD4.7 are very similar to that for the 8,9dihydroxythymol compound, except for the appearance of the hydroxymethyl group instead of the methyl group at C- The data of the HMBC spectra also showed interactions from H-7 (δH 4.52) to C-1, C-2 and C6, from H-9 (δ H 3.76 and 3.65) to C-4, C-8 and C-10, and from H-10 (δH 1.58) to C-4, C-8 and C-9 It can be concluded that EfD4.7 is 7,8,9-trihydroxythymol, a new compound that was first published EfD4.8 is white powder HR-ESI-MS (positive): m/z181.0864 [M + H]+ (C10H13O2).1H NMR (500 MHz, CD3OD) 13C NMR (125 MHz, CD3OD) [Table 3.4] The 1H 13C-NMR speactras of EfD4.8 was similar to that of EfD4.7 compound, excep for the appearance of one methylene group (δC/δH 114,8/5,41 and 5,20) instead of the methyl group as in the EfD4.7 structure This is also confirmed by HR-ESI-MS spectra with chemical formular of C10H13O2 The HMBC speactra was also confirmed the structure of EfD4.8 Firuge 3.6.HSQC spectra of EfD4.7 Table3.4.1H NMR and 13 C NMR spectra of EfD4.7 EfD4.8 compounds STT EfD4.7 δH (m, J in Hz) 10 Figure 3.7.HMBC of EfD4.7 6.79 (1H, d, 2.0) 7.20 (1H, d, 7.5) 6.81 (1H, dd, 7.5, 2.0) 4.52 (2H, s) 3.76 (1H, d, 11.0) 3.65 (1H, d, 11.0) 1.58 (3H, s) EfD4.8 δC δH (m, J in Hz) 140.1 116.3 156.8 133.5 129.5 120.5 64.9 76.9 72.4 6.82 (1H, d, 2.0) 7.12 (1H, d, 7.5) 6.80 (1H, dd, 7.5, 2.0) 4.55 (2H, s) 4.39 (2H, s) δC 143.6 115.1 155.8 127.8 131.1 119.0 64.8 149.3 65.8 26.1 5.41 (1H, d, 2.0), 5.20 (1H, d, 2.0) 114.8 Chemical structures of 07 chemical compounds isolated from E fortunei 7,8,9-trihydroxythymol(EfD4.7) 8,10-didehydro-7,9dihydroxythymol(EfD4.8) o-Caumaric acid (EfD1.8) 8,9,10- Trihydroxythymol (EfD5.1): 4-(2-hydroxyethyl)benzaldehyde (EfD10.1): Kaempferol (EfD10.3): 10-Acetoxy-8,9dihydroxythymol (EfD14.1) 0.50 Control - M.a E- Eth-200 E- Me-200 E-W-200 CuSO4-1 0.40 0.30 A Optical Density, (λ= 680 nm) Optical Density (λ= 680 nm) 3.2 Inhibitory effect of plant extracts and pure chemical compounds from E fortunei on the growth of M aeruginosa and Ch vulgaris 3.2.1 Inhibitory effect of different crude extracts from E fortunei on the growth of M aeruginosa 0.20 0.10 0.00 T0 T3 T6 Time (days) 0.50 B Control- M.a E- Eth-500 E- Me-500 E-W-500 CuSO4-5 0.40 0.30 0.20 0.10 0.00 T10 T0 T3 T6 Time (days) T10 Figure 3.8 Growth of M aeruginosa under the exposure of crude ethanol extract at the concentration of 200 (A) and 500 µg.mL- (B) determined by optical density The ethanol extract of Eupatorium fortunei Turcz at 500 µg/mL with inhibition efficiency of 91.5 % showed higher potential ability to inhibit the growth of M.aeruginosa than those of the water and methanol extracts with inhibition efficiencyof 61.7 % and 78.5%, respectively CuSO4 µg/mL significantly inhibited growth of M aeruginosa with the IE of 81.7% 6.00 8.00 A Control- M.a E- Eth-200 E- Me-200 E-W-200 CuSO4-1 Chlorophyll a Concentration , µg/L Chlorophyll a Concentration, µg/L 8.00 4.00 2.00 Control - M.a E- Eth-500 E- Me-500 E-W-500 CuSO4-5 6.00 B 4.00 2.00 0.00 0.00 T0 T3 T6 T i me ( d a ys ) T10 T0 T3 T6 T10 Time (days) Figure 3.9.Growth of M aeruginosa under the exposure of crude ethanol extract at the concentration of 200 (A) and 500 µg.mL- (B) determined by chlorophyll a content In the treatment samples exposed to ethanol and methanol extracts at the concentration of 500 Control- M.a E-Ethanol 50 E-Ethanol- 100 E-Ethanol-200 E-Ethanol-500 Chlorophyll a Concentration , µg/L T0 T3 A T6 5.00 4.00 B 3.00 2.00 1.00 0.20 0.10 0.00 35.00 T3 T6 B Control- Chlorella E-Ethanol -50 E-Ethanol-100 E-Ethanol-200 E-Ethanol-500 30.00 25.00 20.00 T10 15.00 10.00 5.00 0.00 0.00 40.00 T3 T6 C Control- M.a E-Ethanol-50 E-Ethanol-100 E-Ethanol-200 E-Ethanol-500 30.00 20.00 T0 T10 10.00 0.00 Cell Density, × 105 TB/mL T0 Cell Density x 105 TB/mL 0.30 T0 Control- M.a E-Ethanol 50 E-Ethanol- 100 E-Ethanol-200 E-Ethanol-500 A Control-Chlorella E-Ethanol-50 E-Ethanol-100 E-Ethanol- 200 E-Ethanol- 500 0.40 T10 7.00 6.00 Optical Density (λ= 680 nm) 0.50 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 Chlorophyll a Concentration, ug/L Optical Density (λ= 680 nm) μg.mL-1 cyanobacteria biomass were lower than that of the control at T3, T6 and T10 (p