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 cru
Trang 1TRAINING 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
Trang 2Graduate University of Science and Technology, Vietnam Academy
of Science and Technology
Supervisors: Prof Dr Dang Dinh Kim
Dr Le Thi Phuong Quynh
This doctoral thesis can be found at:
Library of the Graduate University of Science and Technology, VAST
Trang 3INTRODUCTION
1 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 do 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”
2 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
3 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)
4 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
Trang 4aeruginosa and Ch vulgaris under the exposure of plant extracts (TEM); 6) Standard methods in water
analysis (physical and chemical parameters)
5 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 do not harm to other non-target organisms in aquatic ecosystems
6 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
7 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 1 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 2 presents research objectives, methods and the design of experiments Chapter 3 shows the
reaserch results and gives discussion The chapter 3 will be presented in more detail in the next section
Trang 5CHAPTER 3 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
Table 3.2 Effeciency of fraction production from crude ethanol extracts of E fortunei
Fractions Gram fractions/100 gram crude ethanol
extract (%)
Table3.3 Effeciency of isolating 7 chemical compounds from E fortunei
Compounds Mg compound/100 g EtOAc fractions of E fortunei
Trang 6Figure 3.1 Process of isolating chemical compounds from the ethyl acetate fraction
2 new compounds
Figure 3.2 7,8,9-trihydroxythymol (EfD4.7) Figure 3.3
8,10-didehydro-7,9-dihydroxythymol(EfD4.8)
Trang 7EfD4.7 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) và 13C NMR (125 MHz, CD3OD) [Table 3.4]
Actually, the 1H and 13C-NMR spectra data of EfD4.7 are very similar to that for the dihydroxythymol compound, except for the appearance of the hydroxymethyl group instead of the methyl group at C- 7 The data of the HMBC spectra also showed interactions from H-7 (δH 4.52) to C-1, C-2 and C-
8,9-6, 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) và 13C NMR (125 MHz, CD3OD) [Table 3.4] The 1H và 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
Trang 8Firuge 3.6.HSQC spectra of EfD4.7 Figure 3.7.HMBC of EfD4.7
Trang 93 8,9,10- Trihydroxythymol
(EfD5.1):
5 4-(2-hydroxyethyl)benzaldehyde (EfD10.1):
6 Kaempferol (EfD10.3):
7
10-Acetoxy-8,9-dihydroxythymol (EfD14.1)
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
Figure 3.8 Growth of M aeruginosa under the exposure of crude ethanol extract at the concentration of 200 (A)
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 5 µg/mL significantly inhibited
growth of M aeruginosa with the IE of 81.7%
Figure 3.9.Growth of M aeruginosa under the exposure of crude ethanol extract at the concentration of 200 (A)
0.00 0.10 0.20 0.30 0.40 0.50
0.00 2.00 4.00 6.00 8.00
B
A
Trang 10In the treatment samples exposed to ethanol and methanol extracts at the concentration of 500 μg.mL-1 cyanobacteria biomass were lower than that of the control at T3, T6 and T10 (p <0.05) with growth inhibitory effect at T10 of 88.28% and 69.10%, respectively The treatment of water crude extract
at 500 μg.mL-1 had inhibitory effect of 52.51% with biomass at T10 of 3.12 ± 0.37 μg.L-1 However, the treatment at the concentration of 200 μg.mL-1 slightly stimulated growth compared with the control (p
<0.05).Applications of extraction solvents may have a significant impact on the yield of phenolic compounds from plant materials The extract obtained by 96 % ethanol had highest total antioxidant activity as well as phenolic content compared with those of the methanol and water solvents It was noted that phenolic compounds have demonstrated anti-algal inhibitory effect It may be the reason why the ethanol extract had shown the most effective cyanobacteria growth inhibition in our study However, plant extracts at lower
concentration sometimes slightlystimulated the growth of M aeruginosa
3.2.2 Inhibitory effect of ethanol crude extracts from E fortunei on the growth of M aeruginosa
and Ch vulgaris
Figure3.10 Growth of M aeruginosa under the
exposure of crude ethanol extract determined by
optical density (A), chlorophyll a content (B) and cell
density (C)
Figure 3.11 Growth of Ch vulgaris under the exposure of crude ethanol extract determined by optical density (A), chlorophyll a content (B) and cell
0.00 0.10 0.20 0.30 0.40 0.50
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
Trang 11The results clearly indicated that ethanol crude extract from E fortunei at the both 200 and 500 μg.mL
-1 concentration showed effective inhibition on the growth of M aerguinosa
Table 3.5 shows that the ethanol extracts had selective inhibitory effect between M auruginosa and
Ch vulgaris with growth inhibitory values (IE%) on C vulgaris recorded lower than M aeruginosa in all three
analytical methods (optical density, chlorophyll a concentration and cell density) (p <0.05)
Table.3.5 Inhibition efficiency (IE) of ethanol crude extract from E fortunei on the growth of M.aeruginosa
3.2.3 Inhibitory effect of ethyl acetate and water fractions from E fortunei ethanol extract on the
growth of M aeruginosa and Ch vulgaris
Figure 3.12 Growth of M aeruginosa under the exposure of ethyl acetate (A) and water fractions (B) determined
by optical density
Figure 3.13 Growth of M aeruginosa under the exposure of ethyl acetate (A) and water fractions (B)
determined by chlorophyll a content
0.00 0.10 0.20 0.30 0.40 0.50 0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Trang 12Figure 3.14 Growth of M aeruginosa under the exposure of ethyl acetate (A) and water fractions (B)
determined by cell density
The results shown in Figures 3.13 and 3.14 by optical density and chlorophyll a methods both reflect the same trend It was clearly demonstrated that the ethyl acetate fraction inhibited more strongly on the
growth of M auruginosa compared to the water fraction after 10 days of experiment At the concentrations
of 200 and 500 μg.mL-1, the water fractionation was slightly inhibited M aeruginosa at the last day of
experiment, measured with the optical values of 0.354 ± 0.015 and 0.199 ± 0.016; with chlorophyll a contents
of 5.76 ± 0.38 and 3.96 ± 0.223 μg / L, respectively The inhibitory effect on M aeruginosa growth at 200
μg.mL-1 was 18-20% and 45-60% at 500 μg.mL-1
In term of ethyl acetate fraction, it indicated high toxicity to M aeruginosa at the concentrations of
200 and 500 μg.mL-1 after 10 days of exposure The optical values were 0.102 ± 0.03 and 0.031 ±0.001 và
chlorophyll a contents were 1.78± 0.018 và 0.27 ± 0.019 µg/L, respectively The inhibitory effect on M
aeruginosa growth was over 90% at the concentration of 500 µg/mL
Figure 3.15 Growth of Ch.vulgaris under the exposure of ethyl acetate (A) and water fractions (B)
determined by optical density
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
0.00 0.10 0.20 0.30 0.40 0.50
E-W-100 E-W-200 E-W-500
0.00 10.00 20.00 30.00 40.00 50.00 60.00
E- W-100 E- W-200
Trang 13Figure 3.16 Growth of Ch vulgaris under the exposure of ethyl acetate (A) and water fractions
(B) determined by chlorophyll a content
Figure 3.17 Growth of Ch vulgaris under the exposure of ethyl acetate (A) and water fractions (B)
determined by cell density
Compared with the harmful effect of the extracts on the growth of M aeruginosa, the extract showed less toxic to Ch vulgaris The sample exposed to water fraction from E fortunei at 500 μg / mL after 10 days
had the slight lower optical values (0.260 ± 0.013) than that of the control (OD of 0.391 ± 0.0228) The inhibitory effeciency (IE) of 32.33% by optical density and 40.16% by chlorophyll a concentration The ethyl
acetate faction showed stronger toxicity than the water fraction to Ch vulgaris with the IE values of 76.98%
and 78.40%, respectively
Bảng 3.6 Inhibition efficiency (IE) of ethyl acetate and water fractions from E fortunei on the growth of
IE % (TB)
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
E-W-100 E-W-200 E-W-500
Trang 14Figure 18 B Effect of plant extracts on the
growth of M.aeruginosa
after 72 hours treatment
by Chlorophyll a concentration
Table 3.7 Inhibition efficiency