Inhibitory effect of different eupatoriumfortunei turcz extracts on the growth of microcystis aeruginosa - Pham Thanh Nga

Vietnam Journal of Science and Technology 55 (4C) (2017) 103-108 INHIBITORY EFFECT OF DIFFERENT EUPATORIUMFORTUNEI TURCZ EXTRACTS ON THE GROWTH OF MICROCYSTIS AERUGINOSA Pham Thanh Nga 1, 2 , Pham Huu Dien 1 , Nguyen Van Quyen 1 , Tran HoaiThuong 1 , Le Thi Phuong Quynh 3 , Nguyen Tien Dat 4 , Duong ThiThuy 5 , Dang Dinh Kim 5, * 1 Hanoi National University of Education,136 Xuan Thuy, Cau Giay, Ha Noi, Viet Nam 2 Graduate University of Science and Technology, VAST, 18 Hoàng Quốc Việt, Ha Noi, Viet Nam 3 Institute of Natural Products Chemistry, VAST, 18 Hoang Quoc Viet, Ha Noi, Viet Nam 4 Center for Research and Technology Transfer, VAST, 18 Hoang Quoc Viet, Ha Noi, Viet Nam 5 Institute of Environmental Technology, VAST, 18 Hoang Quoc Viet, Ha Noi, Viet Nam * Email: dangkim.iet@gmail.com Received: 30 June 2017; Accepted for publication: 20 October 2017 ABSTRACT Blooms of freshwater cyanobacteria have caused increasing eco-environmental problems in recent years. Recently, plant extracts have been considered as attractive alternative algicides for algal bloom treatment. The present work presented the effect of three different extracts (ethanol, water and methanol) prepared from Eupatorium fortunei Turcz at concentrations of 500 and 200 µg/mL on the growth of Microcystisaeruginosain a 10-day experiment. By using optical density method, the ethanol extract at concentration of 500 µg/mL indicated the highest toxicity with the inhibition efficiency reaching 95.5 % after 10 days which was significantly greater than those of the water, methanol extracts at the same concentration and that of CuSO4 at 5 µg/mL (59.7 %, 66.5 % and 81,7 %, respectively). In addition, the pH of the algal suspensions was fluctuated from 6.0 to 7.0 for the control and extract-treated samples. In contrast, the pH of the CuSO4-treated suspension decreased to 5.0 - 5.5. These results proved that the E. fortunei Turcz ethanol extract had the best properties at 500 µg/mL in comparision with other extracts. Keywords: Eupatorium fortunei Turcz, algal bloom, Microcystisaeruginosa inhibition, plant extracts. 1. INTRODUCTION 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 algae, Microcystis aeruginosa, one of the most Pham Thanh Nga, Pham Huu Dien, Nguyen Van Quyen, Tran HoaiThuong, Le Thi Phuong Quynh 104 common representative specie responsible for the water blooming, can produce hepatotoxin and neurotoxins which may lead to headache, fever, abdominal pain, nausea, vomiting and even cancer [1]. 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 [2]. Chemical treatments can effectively and rapidly remove algal bloom. However, some algicidal chemicals can cause secondary pollution of aquatic environments 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 devicesor 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 bacteriahave 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. In recent years, chemical secretions or extracts of many plants have been reported to inhibit the growth of algae. For example, barley straw has been reported for having algal-inhibiting properties by authors [3, 4]. Other natural compounds have also been screened to control algal bloom, including extracts of banana and mandarin skin [5], rice straw [2] and the family Papaveraceae [2]. In 2013, our previous studies [6] revealed that some plant extracts from Cyperus rotundus, Chromolaena odorataand Callisia fragrans inhibited the growth of M.aeruginosa and showed the dose-concentration dependence. Eupatorium fortunei Turcz (synonym Eupatorium staechadosmum Hance) belonging to the family Asteraceae, has been used in Vietnamese traditional medicine as diuretic, antiseptic, antipyretic agent or for treatment of edema, dropsical swelling. Note that, this plant is readily available and inexpensive in North Vietnam. Therefore, the main objective of this study was to confirm and estimate the algal inhibiting effects of some different E. fortunei extracts (ethanol, water and methanol) at concentrations of 200 and 500 g/mL in comparision with CuSO4-5 g/mL sample. The results of this work may be useful for controlling the toxic cyanobacterium M.aeruginosa in natural aquatic ecosystem. 2. MATERIAL AND METHOD 2.1. Algal species and cultivation A culture of M. aeruginosa Kutzing was isolated from the Nui Coc lake using theShirai method (which was available at the Environmental Hydrobiology Department, Institute of Environmental Technology). M. aeruginosa was grown in the CB-culture medium [6]. 2.2. Preparation of different extracts from E. fortunei The aerial parts (leaves and stem) of E. fortunei were collected in January 2016 from Hoa Binh province and Soc Son district, Ha Noi, Viet Nam. The cleaned material was dried on trays Inhibitory effect of different euptoriumfortunei turcz extracts on the growth of 105 at room temperature to constant weight, cut into small pieces and then ground topowder. Then, the powdered material was immersed separately inthree different solvents including methanol, 96 % ethanol and water (5L×3 times) and subsequently macerated for two days at room temperature. The combined extracts were concentrated under vacuum to obtain crude residue and kept at -5 0 C until use. 2.3. Experimental design To study the inhibitory effects of different plant extracts on the growth of M.aeruginosa the test was conducted in 250-ml Erlenmeyer flasks that contained 150 mL CB medium. Flasks were covered with plastic foil to avoid evaporation and placed on a sterile at room temperature (25 o C) with1000 lux light intensity under a 12-h light:12-h dark cycle. The glassware used in the test was sterilized with steam for 30 minutes at 120 o C in advance and the flasks were shaken twice a day during the experiment. Thecrude methanol (E-Me), ethanol (E-Eth) and water (E-W) E. fortuneiextracts were added to the flasks at concentrations of 200 and 500 µg/mL. The sample CuSO45 µg/mL was used as a reference. The control with no addition of any extract as well as chemicals was set. The results were recorded after 10 days. Experiments were conducted in triplicate under the same environmental conditions. 2.4. Analytical methods, analysis data and statistical analysis Growth of cyanobacteria M.aeruginosawas assessed by optical density (OD) at 680 nm wavelength using UV-Vis spectrophotometer (Shimadzu) and the efficiency of growth inhibitionwas calculated using the following equation: Inhibition efficiency (IE)(%) = [(control- treatment)/control] × 100 The growth dynamics of M.aeruginosawas monitored at 1, 3, 7 and 10 days. The data was expressed as the mean value ± SE of triplicate experiments. The data was analyzed and drawn by the software GraphPad Prism 6 (one – way ANOVA). The validity of investigation was expressed as probability value of p < 0.05. 3. RESULTS AND DISSCUSSION 3.1. Effect of different plant extracts at concentration of 200 µg/mL on the growth of M.aeruginosa Figure 1. Effect of plant extracts at 200 µg/mL on the growth of M.aeruginosa. Figure 1. Effect of plant extracts at 200 µg/mL on the growth of M.aeruginosa. Pham Thanh Nga, Pham Huu Dien, Nguyen Van Quyen, Tran HoaiThuong, Le Thi Phuong Quynh 106 Table 1. Inhibition efficiency of plant extracts at 200 µg/mL on the growth of M.aeruginosa after 10 day treatment. E-Eth- 200 E-Me- 200 E-W- 200 IE (%) 49.0 -0.2 -18.9 Three different extracts from E. fortunei, at the concentration of 200 µg/mL, showed differentinhibitory effect on the growth of M.aeruginosa after 10 days (Figure 1).The ethanol extracts had the highest toxicity with IE value of 49.0 %, while two other extracts did not have any inhibition effect at that concentration (Table 1). The OD values of methanolextract were similar to that of the control, which increased from 0.03 at initial time to about 0.17, whereas the OD of the water extract slightly higher than 0.20 at the end of the experiment (T = 10). 3.2. Effect of different plant extracts at concentration of 500 µg/mLon the growth of M.aeruginosa The results of OD 680nm values (Figure 2) indicated that E. fortunei extracts affected to the growth of M.aeruginosadifferently. In contrast tothe extracts at 200 µg/mL (Figure 1), at the concentration of 500 µg/mL all three extracts showed clear algal inhibition properties after ten experimental days. Cell densities of the ethanol, methanol and water extracts were reduced to 95.5 %, 66.5 % and 59.7 % in comparison with that of the control, respectively (Table 2). Table 2. Inhibition efficiency of plant extracts at 500 µg/mL and CuSO4- 5 µg/mL on the growth of M. aeruginosa after 10 day treatment. E-Eth- 500 E-Me- 500 E-W- 500 CuSO4-5 IE (%) 95.5 66.5 59.7 81.7 Applications of extraction solvents may have a significant impact on the yield of phenolic compounds from plant materials [7]. 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 [2, 4]. 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 [8]. CuSO4 5 µg/mL significantly inhibited Figure 2. Effect of plant extracts at 500 µg/mL on the growth of M.aeruginosa. Inhibitory effect of different euptoriumfortunei turcz extracts on the growth of 107 growth of M.aeruginosa with the IE of 81.7 %. This result agreed with those of many authors [2, 9] in term of the use of plant extracts and CuSO4 as algicidesto control growth of M.aeruginosa. 3.3. Effect of different plant extracts and CuSO4 5 µg/mL on pH value of the culture medium Table 3. pH- value of the culture medium of M.aeruginosa during the 10 day experiment. Treatment Experimental Time (Days) 1 3 7 10 Control 5.96 6.44 6.63 6.97 E-Eth-200 6.11 6.04 6.37 6.95 E-Eth-500 5.88 5.63 6.33 6.40 E-W-200 6.03 6.53 6.76 7.08 E-W-500 6.07 6.56 6.87 6.87 E-Me-200 6.24 6.33 6.55 7.10 E-Me-500 6.01 6.20 6.56 6.55 CuSO4 -5 5.56 5.40 5.20 5.07 The pH value increased in both treatments and the control except the CuSO4 sample during ten-day experiment (Table 3). For the control sample, it ranged from an initial value of about 6.0 to the highest value of slightly 7.00 at the end. That pattern was observed similarly to three plant extracts at 200 and 500 µg/mL. Previous study also indicated that plant extracts might alter the pH of the culture solution [8]. Depending the chemical composition of the plant, the pH of the sample-treated solutions could increase (alkaloid components for example) or decrease (in case of phenolic compounds). During the treatment, several chemical processes such as oxidation or reduction may occur, which can explain for the different change in pH values observed in E-Eth- 200 and E-Eth-500 in the first three days as compared with other samples. In contrast, pH value of CuSO4 sample was decreased gradually from 5.6 to 5.0 because CuSO4itseft made the medium slightly more acidic. The results of previous researches [9, 10] showed that pH of the investigated culture medium has not only shown great effect on the growth of M.aeruginosa but also on the toxicity of the species.The decrease in the cytoplasm, chloroplast and the PS II damage at pH of 5.5 and 6.0 led to M. aeruginosa death.The possible mechanism of inhibiting the growth of M.aeruginosaby using plant extracts is due to the presence of a number of biologically active compounds, including alkaloids, allelochemicals, and phenol compounds [4, 11]. Effective doses of extracts from different plant species against M.aeruginosa range from milligrams to hundreds of milligrams per liter. The most commonly studied natural material was barley straw with a recommended effective dose against cyanobacteria at 6–28 g/mL [12]. Other extracts act at similar concentrations: mandarin skin extract at—70 g/mL [5], rice straw extract - from 0.1 to 10 g/mL [13]. But some other extracts showing effective inhibition on the growth of M.aeruginosa were in high concentrations like we have used in the present study such as extracts from Ailanthus altissimawith dose from 25 to 200 g/mL [1]. Our research presents the first evidence of inhibitory properties ofE.fortunei extracts against the growth of M.aeruginosa. 4. CONCLUSION The present work indicated that the ethanol extract of Eupatorium fortunei Turcz at 500 µg/mL with inhibition efficiency of 95.5 % showed higher potential ability to inhibit the growth of M.aeruginosa than those of the water and methanol extracts with inhibitionefficiencyof 59.7 % and 66.5 %, respectively. In addition, pH level of the control and plant extracts increased from 6.0 to 7.0 during the experiment while that of CuSO4 5 µg/mL decreased from 5.5 to 5.0. Pham Thanh Nga, Pham Huu Dien, Nguyen Van Quyen, Tran HoaiThuong, Le Thi Phuong Quynh 108 Specific compounds in the extracts and inhibitory mechanism should be further investigated in our near future studies. Acknowledgement. This study was funded by the Ministry of Education and Training of Viet Nam, under the grant number B 2016-SPH-19. This work forms part of the PhD thesis requirement of Pham Thanh Nga. REFERENCES 1. Panpan M., Haiyan P., Wenrong H., Zhongde L., Xiuqing L., Hangzhou X. - Allelopathic effects of Ailanthus altissima extracts on Microcystis aeruginosa growth, physiological changes and microcystins release. Chemosphere 141 (2015) 219–226. 2. Shao.J, Renhui L., Joe E. L., Ji-Dong G. - Potential for control of harmful cyanobacterial blooms using biologically derived substances problems and prospects.Journal of Environmental Management 125 (2013) 149- 155. 3. Barrett, P.R.F., Littlejohn, J.W., Curnow, J. - Long-term algal control in a reservoir using barley straw. Hydrobiologia 415 (1999) 309–313. 4. Pillinger J. M. Cooper, J. A. Ridge, I. - Role of phenolic compounds in the antialgal activity of barley straw. Journal of ChemicalEcology 20 (7) (1994), 1557- 1569. 5. Chen JZ, Liu ZL, Ren GJ, Li PF, Jiang YW. - Control of Microcystis aeruginosa TH01109 with batangas mandarin skin and dwarf banana peel. Water SA 30 (2004) 279–282. 6. NguyễnTiến Đạt, DươngThịThuỷ, Lê Thị Phương Quỳnh, Hồ Tú Cường, Vũ Thị Nguyệt, Phạm Thanh Nga, Đặng Đình Kim. - Nghiên cứu tác dụng diệt vi khuẩn lam độc Microcystis aeruginosa của một số dịch chiết thực vật. Tạp chí Hóa học 51 (2C) (2013) 737- 739, (in Vietnamese). 7. Quy Diem Do, Artik Elisa Angkawijaya, Phuong Lan Tran Nguyen, Lien Huong Huynh – Felycia Edi Soetaredjo, SuryadiIsmadji, Yi-Hsu Ju - Effect of extraction solvent on total phenol content, total flavonoids content, and antioxidant activity of Limnophilaaromatica. Journal of Food and Drug Analysis 22 (3) (2014) 296-302. 8. Li Jiang, Yunguo Liu, Pingyang Zhang, Guang ming Zeng, Xiaoxi Cai, Shaobo Liu, Yicheng Yin, Xinjiang Hu, Xi Hu, Xiaofei Tan -Growth inhibition and oxidative damage of Microcystis aeruginosa induced by crude extract of Sagittaria trifolia tubers. Journal of environmenta Science 43 (2016) 40-47. 9. Tsai K. P. - Effects of two copper compounds on Microcystis aeruginosa cell density, membrane integrity, and microcystin release, Ecotoxicology and Environmental Safety 120 (2015) 428–435. 10. Westhuizen A.J. van de, J.N. Eloff. - Effect of Culture Age and pH of Culture Medium on the Growth and Toxicity of the Blue-green Alga Microcystis aeruginosa. Zeitschrift für Pflanzenphysiologie 110 (2) (1983) 157-163. 11. Trần Hồng Hạnh, NguyễnTrườngTuấn, Đan Thúy Hằng, ChâuVăn Minh, Trần Huy Thái, Nguyễn Tiến Đạt - Bướcđầunghiêncứuthànhphầnhóahọccủacâymầntưới (Eupatorium fortune). TạpchíHóahọc 49 (2ABC) (2011) 264-266, (in Vietnamese). 12. Barrett, P.R.F., Littlejohn, J.W., Curnow, J. - Long-term algal control in a reservoir using barley straw. Hydrobiologia 415 (1999) 309–313. 13. Park MH, Han MS, Ahn CY, Kim HS, Yoon BD, Oh HM. - Growth inhibition of bloom- forming cyanobacterium Microcystis aeruginosa by rice straw extract. Lett Appl Microbiol. 43 (2006) 307-312.