Antibacterial Activity of Extracts from Dried and Fresh Herbal Plant (Phyllanthus amarus) Against Pathogens Causing Acute Hepatopancreatic Necrosis Disease (Ahpnd) in White Leg Shrimp (Litopenaeus vannamei) at Thua Thien Hue Province, Vietnam
Phuong TV1, Hai Yen PT2, Linh NQ1,3*
1Institute of Biotechnology, Hue University, Road No.10, Phu Vang, Thua Thien Hue, 530000, Vietnam
2Faculty of Fisheries, University of Agriculture and Forestry, Hue University, 102 Phung Hung St., Hue, 530000, Vietnam
3Hue University, 3 Le Loi St., Hue, 530000, Vietnam
Corresponding Author: Nguyen Quang Linh
Address: Institute of Biotechnology, Hue University, Road No.10, Phu Vang, Thua Thien Hue, 530000, Vietnam; E-mail: email@example.com; firstname.lastname@example.org
Received date: 24 October 2019; Accepted date: 16 November 2019; Published date: 22 November 2019
Citation: Phuong TV, Hai Yen PT, Linh NQ. Antibacterial Activity of Extracts from Dried and Fresh Herbal Plant (Phyllanthus amarus) Against Pathogens Causing Acute Hepatopancreatic Necrosis Disease (Ahpnd) in White Leg Shrimp (Litopenaeus vannamei) at Thua Thien Hue Province, Vietnam. Asp Biomed Clin Case Rep. 2019 Nov 22;2(3):120-128.
Copyright © 2019 Phuong TV, Hai Yen PT, Linh NQ. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited.
Keywords: EMS; AHPND; Herbs; Extract; Phyllanthus amarus; MIC/MBC
The study aimed to determine extract yield (%), antibacterial activity, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of plant extracts from dried and fresh herbal plants (Phyllanthus amarus) against Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (AHPND) in white leg shrimp (L. vannamei). The result showed that the extract yields of dry and fresh herbs reached 11.50% and 2.75%, respectively and the antibacterial activity of the two extracts both are good at concentrations from 250 to 1,000 mg/mL at the same bacterial density of 106 CFU/mL. Specifically, the diameter of the inhibition zone at 250; 500; 750 and 1,000 mg/mL concentration of dried herbal extracts reached 16.75±0.96; 18.50±1.29; 20.75±0.96 and 21.25±0.50 mm, while that of fresh herbal extracts reached 14.50±1.29; 16.25±0.50; 16.75±0.50 and 17.00±0.00 mm, respectively, with a statistically significant difference p<0.05. The result also showed that MIC values of dried and fresh extracts were defined at 125 mg/mL and 250 mg/mL, respectively and that MBC values of the extracts were 500 and 1,000 mg/mL respectively. The GC-MS analysis revealed that there were 19 natural compounds in the dried extract, in which Ethyl Linoleolate (C20H36O2) compound occupied the highest ratio (22.43%), while 2.3-Dihydro-3.5-dihydroxy-6-methy-4H-pyran-4-one (C6H8O4) was the lowest (0.24%).
Vietnam shrimp farming industry has brought great economic efficiency and has also become one of the key economic sectors of the country. The revenue of the industrial brackish shrimp export was estimated to reach $4.2 billion in 2019 (DOF, 2019). White leg shrimp (Litopenaeus vannamei) is one of species which has big harvest and high economic value . Acute Hepatopancreatic Necrosis Disease (AHPND), also called Early Mortality Syndrome (EMS) in shrimp is caused by Vibrio bacteria. This disease can lead to death rate up to 100 % in the habitats of both white leg shrimp (L. vannamei) and black tiger shrimp (Penaeus monodon), which causes considerable losses to shrimp farming. Clinical signs of the disease include empty gastrointestinal tracts, opaque white stomachs, white atopic livers, lethargic shrimp, anorexia, and soft shells (Leano và Mohan, 2012). AHPND disease was first noticed in China in 2009, and then found in other parts of Asia, such as: Vietnam (2010), Malaysia (2011), Thailand (2012), [2,3] and Mexico (2013) [4-6]. The causative agent of AHPND/EMS was indentified to be Vibrio parahaemolyticus strain  that harbors a pVA plasmid encoding toxins PirAVp and PirBVp . As also reported by Giang et al., (2016), three bacterial strains were determined, including: V. parahaemolyticus; V. alginolyticus and V. vulnificus with highest encountering frequency of more than 60% in disease shrimp samples. In which, the PCR results determined the presence of V. parahaemolyticus bacterial which is considered a causative agent of acute hepatopancreatic necrosis disease . Meanwhile, the new analysis results of Jee Eun Han et al, (2017) showed that all four Vibrio strains (16-902/1, 16-903/1, 16-904/1 and 16-905/1) were isolated from either stomaches of diseased shrimp or sediment samples from AHPND-affected farms in Latin America during 2016. Bacterial identifications were carried out using 16S rRNA sequencing and Vibrio-specific PCR assays targeting hly gene. By the PCR assays, these 4 strains were identified to be V. campbellii by 16S rRNA sequence analysis and hly gene PCR. These V. campbellii strains had both pirAvp and pirBvp genes .
According to Linh NQ (2014), V. parahaemolyticus V1 strain was isolated from EMS disease juvenile shrimp samples in Thua Thien Hue province with density from 103 – 104 CFU/mL in the incubation period . According to Citarasu (2010), the herbs have beneficial properties such as growth stimulant, immune booster, antibacterial and anti-fugus). Many other researches also proved that there are many kind of herbs such as: guava leaves (Psidium guajava), betel leaves (Piper betel L.), Phyllanthus amarus and seeds of Myrtle (Rhodomyrtus tomentosa), which have antibacterial properties against bacteria causing AHPND in white leg shrimps including V. parahaemolyticus KC12.020; V. parahaemolyticus KC13.14.2 and V. harveyi KC13.17.5 strain [12,13]. Herbs are also used for flavoring, stimulating gastrointestinal secretion, thus increasing feed intake as well as decreasing feed conversation rate (Venketramalingam et al, 2007). Besides, not only this herb is studied for disease prevention and treatment for white-leg shrimp (L. vannamei), but many other herbs are also studied for disease prevention and treatment for Indian white shrimp (Fenneropenaeus indicus), specifically: the three methanolic antibacterial extracts, Psoralea corylifolia, Murraya koenigii and Quercus infectoria effectively suppressed the shrimp bacterial pathogens isolated from infected F. indicus gut. The average zone of inhibition was observed ranging between 9.00 to 14.00 mm against the selected bacterial pathogens including Pseudomanas aeruginosa, Staphylococus aureus and V. harveyi strains . Another research illustrated that using Phyllanthus amarus extracted by ethanol to enrich Artemia nauplii produced the best SR, WG and SGR (96.6%, 1.01g, and 4.1%, respectively against the control 82%, 0.6g and 3.9%, respectively) in Macrobrachium rosenbergii PL .
Material and Research Methods
Herbal Plant: The P. amarus (Fig-1) was collected from January to March 2019 at the mountainous area of Thua Thien Hue province, Vietnam. The chosen herbal plants were mature ones of which average weight reached 3,25g and average height 28,82cm. The samples had to be fresh and not crushed, have green colour. They were washed using fresh water.
Bacterial Strain: Vibrio parahaemolyticus strain identified to carry two PirAVp, PirBVp toxin gene was isolated from disease shrimp samples (AHPND) in farms at Thua Thien Hue, Vietnamand labeled. The samples’ average weight was 0.36g. The experiment was carried out in a sterile laboratory (Labcaire VLF-R) at the Institute of Biotechnology of Hue University. Bacterial strain was kept at -800C and then restored in enrichment culture medium, specifically Tryptic Soy Brorth (TSB) supplemented with 2% NaCl in 2-tray shaking incubator (GFL 3032) at the temperature of 370C, with the shaking frequency of 180 rpm for 24 hours to collect bacterial outbreak. Subsequently, the bacterial density was determined using optical density (OD) measuring method by UV-VIS spectroscopy (U2900, Hitachi, Japan) at 600 nm wavelength. The bacterial density was adjusted to 106 CFU/mL (OD = 1, equivalent to a bacterial density of 108 CFU/mL) to test antibacterial activity compared to initial bacterial density.
Antimicrobial Preparation: Herbal extracts were mixed in sterile distilled water (diluted at 1g/1 mL ratio) into concentrations of 1,000, 750, 500 and 250 mg/mL. Negative control was sterile distilled water.
Herbal Extraction Method:
The powdered herbal (400 g, d=1 mm) and fresh herbal (400 g, pureed) were soaked in 70% ethanol (1:5 ratio of herbal plant:ethanol) and stirred for 48 hours, then filtered through vacuum filtration system (Rocker 300-LF31) on Whatman filter paper No. 4 (code: 1004042, diameter of 20-25 μm). After the primary herbal extract was separated, the remaining were soaked in 40% ethanol (1:5 ratio) for 48 hours, and another batch of herbal extract was collected. Two batches of extracts were mixed to obtain total extract. They were then taken into rotation at 60oC by Heidolph vacuum evaporation system (Germany). The filtrates were evaporated and dried at 50oC until there was no more change in weight. The extract yields were stored at 4oC and their yield percentages were calculated using the following formula (Turker et al, 2009):
Antibacterial Activities of the Herbal Extract:
The bactericidal activity of the extracts was tested by disk diffuse test, also known as Kirby-Bauer method. Testing was done in sterile Labcaire VLF-R. The suspension containing bacterial strain (100 μL, 106 CFU/mL) was evenly distributed on agar plates containing a solid alkaline peptone medium. The sterile paper disc (d = 0.6 mm) was put on agar plates surface (including 4 samples and 1 negative control (sterile distilled water). 50 μL of plant extracts with 1,000; 750; 500 and 250 mg/mL concentration was put on paper discs. After that they were stored at 4oC for 8 hours for extracts to spread over the surface of the paper discs with 4 replications. The diameter of inhibition zone was measured after 24; 48; 72 and 96 hours.
Determination of Minimum Inhibitory Concentration of Plant Extract:
Minimum Inhibitory Concentration (MIC) was defined using Satyajit method . 100 μL of bacterial solution was added into each well of a 96-well plate containing 100 μL of the plant extract that was diluted to different concentrations with an initial stock extract of 1,000 mg/mL from 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256 concentration. Control wells contain 100 μL of sterile distilled water which was used to dissolve plant extracts. The plates was then incubated at 37°C for 24 hours, after that each well was added 20 μL resazurin 0,1% (Fig-2). MIC value was defined as the lowest concentration of plant extracts (no change resazulin color) that inhibits the bacterial growth.
Determination of Minimum Bactericidal Concentration of Plant Extract:
All test solution from wells with no discoloration of 0.01% resazurin, which is spread on plates of solid alkaline agar medium was absorbed and incubated at 37 °C. Bacterial survival after 24 hours was observed. Minimum Bactericidal Concentration (MBC) was defined as the lowest concentration of plant extract that did not exhibit any bacterial growth on the agar plates (no colonies appeared on the agar plate), while the control plate had bacteria colonies .
Analysis of Natural Compounds of Extracts:
Based on the results of the antibacterial test of the above experiments, we selected the extract that gave the optimal results which had the best antibacterial activity. The presence of natural compounds of extract from herbal powder was analyzed using Gas Chromatography Mass Spectrometry.
Data were expressed as mean ± standard error (mean ± SEM). One-way ANOVA was followed by SPSS 16.0 software. LSD test was used to compare the diameter of inhibitory zones induced by different extract concentrations. In all analysis, significance was established when probability level was ≤ 0.05.
Results and Discussion
Extract yield of the herbal tree (P. amarus):
Extracts from the herbal plant (P. amarus) was obtained using vacuum filtration system. After complete ethanol solvent removal, extracts yield was identified as the percentage of the weight of extracted residue and weight of initial raw plant. The results of dried and fresh herbal extraction yield (P. amarus) were presented in Table-1.
The average weight of extract obtained out of 400 g fresh material was 11.0 g, equivalent extract yield was 2.75 %. Meanwhile, the average weight of extract obtained out of 400 g dried material (dried from 2 kg of fresh herbal plants) was 46.0 g, equivalent extract yield was 11.50 % (p<0.05). The research result showed that the dried herbal plant (P. amarus) had higher extraction yield than fresh one. According to the research result of Hong Mong Huyen et al., (2018), extraction yield of dried basket plant (Callisia fragrans or Golden Comb in Vietnam) was only 10.8 %, while extraction yield of dried Moringa (Moringa oleifera) was 15.0 % . The research by Ashraf A. Mostafa indicated that the highest extraction yield of the plants under experiment belonged to dried powder of Punica granatum which was 4.87 g, equivalent to 9.74% extraction yield .
Antibacterial Activity of Dried and Fresh Extracts Against V. parahaemolyticus:
As can be seen in Table-2, Fig-4 and Fig-5, all extract concentrations from lowest to highest (250 – 1,000 mg/L) of dried extracts exhibited higher diameters of inhibition zone than those of fresh extracts at bacterial density of 106 CFU/mL, (p<0.05); specifically diameters of inhibition zone of dried extracts at the concentration of 250; 500; 750 and 1,000 mg/mL were 16.75 ± 0.96, 18.50 ± 1.29, 20.75 ± 0.96, 21.25 ± 0.50 mm, respectively (Fig-3), higher than the diameters of inhibition zone of fresh extracts 14.50 ± 1.29, 16.25 ± 0.50, 16.75 ± 0.50 and 17.00 ± 0.00 mm, respectively (Fig-4). The difference were statistically significant (p<0.05). Of which, at extract concentration of 1,000 mg/mL, highest antibacterial activity against V. parahaemoliticus was observed, both in dried (21.25 ± 0.50 mm) and fresh extracts (17.00 ± 0.00). The research by Lua Thi Dang et al, (2018) revealed that diameters of inhibition zone of P. amarus at the concentration of 1,000 (μg/plate) against the following bacteria: V. parahaemolyticus KC12.020, V. parahaemolyticus KC13.14.2, and V. harveyi KC13.17.5 were 12.0 ± 1.0, 13.3 ± 1.5 and 13.7 ± 0.6 mm, respectively . In addition, the study of Hong Mong Huyen et al. (2018) illustrated that among the tested 7 herbal plants, antibacterial activity of Ricinus communis L at the concentration of 40 mg had the largest antibacterial zone against 2 types of bacteria V. harveyi and V. parahaemolyticus causing AHPND on white-leg shrimp (L. vannamei) antibacterial zone 18,0 ± 1,4 và 17,5 ± 0,70 mm , respectively.
Identification of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) of Dried and Fresh Extract:
Minimum inhibitory concentration MIC is the lowest level of concentration in the series of tested concentration of the extracts that exhibited growth inhibitory of bacteria did not change the colour of resazurin (Fig-5A and Fig-5B). Therefore, the lower minimum inhibitory concentration was, the more affective antibacterial activity was. Minimum bactericidal concentration MBC is the lowest level of concentration in the series of tested concentration of the extracts that could eliminate all bacteria in the wells, there appeared no colonies in the agar environment on the plates. Analysis result of MIC and MBC of the dried and fresh herbal plant (P. amarus) was presented in Table-3.
As can be seen in Table-3, extracts of dried herbal plants (P. amarus) exhibited MIC at 125 mg/mL, which was lower than that of fresh herbal plants at 250 mg/mL, equivalent ratio of MBC/MIC of both dried and fresh extracts was 4, however MBC of dried extract was 500 mg/mL while MBC of fresh extract was as high as 1.000 mg/mL. MBC of dried extract was optimal than that of fresh extract. According to the report of Canillac and Mourey (2001), if the ratio of MBC/MIC is lower than or equal to 4, the extract is considered bactericidal; in contrast, if this ratio is higher than 4, the extract is bacteriostatic . Therefore, based on the result in Table-3, it can be concluded that extract from the herbal plant (P. amarus) in both dried and fresh conditions exhibited bactericidal activity against V. parahaemolyticus (AHPND) causing disease on white-leg shrimps. Similarly, in the research of Lua Thi Dang et al, (2018), extract from the herbal plant (P. amarus) exhibited MIC at 312 mg/mL and MBC at 625 mg/mL against 2 strains of bacteria V. parahaemolyticus and V. harveyi . Meanwhile, extract of Ricinus communis L has bactericidal activity against V. harveyi và V. parahaemolyticus causing disease on shrimps (MBC/MIC = 2) .
Natural compounds in the extract from the herbal plant (P. amarus):
After antibacterial activity of extract from dried herbal plant (P. amarus) proved superior to fresh one, presence of natural compounds in dried herbal plant (P. amarus) was analyzed. The result of compound analysis in 1 g of dried extract was presented in Fig-6.
As can be seen in Fig-6, there were 19 natural bioactive components in 1g of extract: of which the compound Ethyl Linoleolate (C20H36O2) occupied the highest percent at 22,43 %, Methy linalelate (C19H34O2) followed at 11,72 % while 2,3-Dihydro-3,5-dihydroxy-6-methy-4H-pyran-4-one (C6H8O4) only occupied 0,24 %, the lowest percentage among the identified 19 compounds. Arun’s Gas Chromatography-Mass Spectrum (GC-MS) Analysis  showed that in the extract of P. amarus using Acetone there existed 9 bioactive compounds while using ethanol there existed only 7 bioactive compounds which are lower than in our research results.
Extract from both dried and fresh herbal plant (P. amarus) exhibited antibacterial activity against V. parahaemolyticus causing AHPND on white-leg shrimp (L. vannamei) under experimental conditions. Dried extract has higher extraction yield and antibacterial activity than fresh extract (p < 0,05).
Minimum inhibitory concentration (MIC) of extract of dried herbal plant (P. amarus) was 125 mg/mL, and of fresh herbal plant was 250 mg/mL, inhibiting bacteria at the concentration of 106 CFU/mL.
The ratio of MBC/MIC of both dried and fresh extracts was 4, however MBC of dried extract was 500 mg/mL while MBC of fresh extract was 1.000 mg/mL.
There were 19 natural components in the extracts of the herbal plant (P. amarus), of which Ethyl Linoleolate (C20H36O2) occupied the highest percent at 22,43 % while 2,3-Dihydro-3,5-dihydroxy-6-methy-4H-pyran-4-one (C6H8O4) occupied the lowest percent at 0,24 %.
This article was supported by Hue University and Ministry of Education and Traning project with code number: CT-2018-DHH-07 and students participated in the research.
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