Use of methanol extract of Bidens pilosa in combating white spot virus infection

Abstract

This invention discloses a methanol extract of Bidens pilosa that can be used to combat white spot virus infection.

Description

Technical Field

[0001] This invention relates to the use of a methanol extract of Bidens pilosa to combat white spot syndrome virus infection. Prior Technology

[0002] White spot syndrome virus (WSSV) is a double-stranded DNA virus belonging to the genus Whispovirus in the family Nimaviridae. WSSV primarily infects farmed aquatic animals such as shrimp and crabs, and transmission occurs through ingestion of contaminated food and exposure to water containing WSSV particles. When a host is infected with WSSV, white spots ranging from 0.5 to 3 mm in diameter typically appear on its exoskeleton, appendages, and inside the epidermis. Abnormal behaviors such as lethargy and loss of appetite also occur, leading to death within hours or days; this condition is known as white spot syndrome.

[0003] Currently, there is no effective method to combat WSSV infection; prevention strategies are the only options to prevent WSSV from invading aquaculture farms. These include: careful management of feeding and water quality, maintaining hygiene and cleanliness of the farm, and strengthening disinfection procedures for the rearing ponds before stocking. However, the effectiveness of these strategies in preventing WSSV infection is not ideal. Therefore, there is a need to develop drugs that can effectively combat WSSV infection for use by the industry.

[0004] Bidens is a genus in the family Asteraceae, containing about 230 species, which are usually annual or perennial herbs and are widely distributed throughout the world. Previous studies have found that extracts from plants of the genus *Bidens* possess antibacterial activity. For example, the methanol extract of *Bidens pilosa* has been found to be effective against *Salmonella typhimurium*, *Shigella boydii*, *Vibrio parahaemolyticus*, *Escherichia coli*, and *Klebsiella pneumoniae* (Shandukani PD et al. (2018), BMC Complement Altern. Med., doi: 10.1186 / s12906-018-2230-9), and the water extract of *Bidens alba* has been found to be effective against *Vibrio alginolyticus* in *Litopenaeus vannamei* (Huang HT et al. (2021), Aquaculture, doi: 10.1016 / j.aquaculture.2021.737306).

[0005] To the best of the applicant's knowledge, no literature or prior patent has yet revealed that extracts from the genus Bidens have efficacy against WSSV infection. Summary of the Invention

[0006] [Invention Summary]

[0007] In this invention, the applicant unexpectedly discovered that the methanol extract of Bidens pilosa can effectively inhibit the replication of white spot syndrome virus (WSSV) in Litopenaeus vannamei and prevent hepatopancreas lesion, thereby effectively slowing down mortality in Litopenaeus vannamei caused by WSSV infection. Therefore, the methanol extract of Bidens pilosa of this invention is expected to be used to combat WSSV infection.

[0008] Therefore, in a first aspect, the present invention provides the use of a methanol extract of Bidens pilosa for the preparation of a composition for use against WSSV infection in an aquatic animal.

[0009] In a second aspect, the present invention provides a method for combating WSSV infection, comprising administering to an individual in need a methanol extract of Bidens pilosa as described above. Simple Explanation of the Diagram

[0010] The above and other objects, features and advantages of the present invention will become apparent upon reference to the following detailed description and preferred embodiments and the accompanying drawings, wherein: Figure 1 shows the high-performance liquid chromatography spectrum of the methanol extract of Bidens pilosa, where peaks a1 to a7 represent the seven major components that appeared during the retention period from 10 to 60 minutes. Figure 2 shows the survival rate of Litopenaeus vannamei in each group in Example 3 over time, where "**" indicates that when compared with the pathological control group, p<0.01; Figure 3 shows the WSSV virus load measured in the gills of Litopenaeus vannamei in each group in Example 3, where "***" indicates a p < 0.001 when compared with the pathological control group; and Figure 4 shows the percentage of normal cells in the hepatopancreas of each group of Litopenaeus vannamei in Example 3, where “***” indicates that when compared with the pathological control group, p<0.001. Implementation

[0011] [Detailed Description of the Invention]

[0012] It should be understood that if any prior publication is cited herein, such prior publication does not constitute an admission that it forms part of the common general knowledge in the art in Taiwan or any other country.

[0013] For the purposes of this instruction manual, it will be clearly understood that the word “comprising” means “including but not limited to”, and the word “comprises” has a corresponding meaning.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. One skilled in the art will recognize many similar or equivalent methods and materials that can be used to practice this invention. Of course, this invention is by no means limited to the methods and materials described.

[0015] This invention provides the use of a methanol extract of Bidens pilosa for the preparation of a composition for use in aquaculture animals to combat white spot syndrome virus (WSSV) infection.

[0016] As used herein, the terms “against white spot syndrome virus infection” and “anti-white spot syndrome virus infection” may be used interchangeably and are intended to encompass prevention of infection caused by white spot virus, inhibition of white spot virus replication, and treatment and / or prevention of infectious diseases caused by white spot virus.

[0017] According to the present invention, the methanol extract can be obtained from Bidens pilosa or its varieties. The Bidens pilosa varieties applicable to the present invention include, but are not limited to: Bidens pilosa minor, Bidens pilosa var. pilosa, and Bidens pilosa var. radiata.

[0018] According to the present invention, the methanol extraction method can be carried out using techniques well known and commonly used by those skilled in the art. In this regard, reference can be made, for example, to Shandukani PD et al. (2018) (ibid.).

[0019] It is understood that the operating conditions for methanol extraction can be further varied depending on factors such as the type of Bidens pilosa used, the processing method, and the ratio of methanol to Bidens pilosa, in order to achieve the best extraction results. These operating conditions are routinely determined by those skilled in this technique.

[0020] According to the present invention, the extractable material of Bidens pilosa can be fresh flowers, stems, leaves, seeds, whole plant, or pre-processed from a group selected from the following: drying, heating, grinding, chopping, pulverizing, and combinations thereof. Preferably, the extractable material of Bidens pilosa is fresh whole plant.

[0021] According to the present invention, the methanol extraction can be carried out at a temperature of 15°C to 25°C. In a preferred embodiment of the present invention, the methanol extraction is carried out at a temperature of 25°C for 24 hours.

[0022] According to the present invention, the aquaculture animal may be selected from the group consisting of shrimp, crabs, and combinations thereof.

[0023] According to the present invention, the shrimp species may be selected from the group consisting of the following: *Litopenaeus* {e.g., *L. vannamei* [also known as Pacific white shrimp and whiteleg shrimp] and *L. setiferus*, etc.}, *Metabenaeus ensis*, *Procambarus clarkii*, *Cherax quadricarinatus*, *Macrobrachium* {e.g., *M. rosenbergii* [also known as giant river prawn]}, *Penaeus* {e.g., *P. monodon* [also known as tiger shrimp], *P. japonicus*, *P. semisulcatus*, *P. amazonicus*, *P. aztec*, etc.} The following are listed: *Penaeus aztecus*, *Penaeus barmerensis*, *Penaeus bolcensis*, *Penaeus brasiliensis*, *Penaeus brevirostris*, *Penaeus californiensis*, *Penaeus chinensis*, *Penaeus cornappensis*, *Penaeus duorarum*, *Penaeus esculentus*, *Penaeus glaessneri*, *Penaeus gracilis*, *Penaeus hamleti*, *Penaeus hathor*, *Penaeus indicus*, *Penaeus isabelae*, *Penaeus kapurdii*, *Penaeus kerathurus*, *Penaeus konkani*, *Penaeus latisulcatus*, and *Penaeus longipes*. Longistylus, Crazy Shrimp (P. maddeni), Edge Shrimp (P. marginatus), Merguiensis Shrimp (P. merguiensis), Pink Shrimp (P. notalis), Obtusus Shrimp (P. obtusus), Western Shrimp (P. occidentalis), Pauline Shrimp (P. occidentalis).The text lists various species including *Penaeus paulensis*, *Penaeus penicillatus*, *Penaeus plebejus*, *Penaeus pubescens*, *Penaeus pulchricaudatus*, *Penaeus schmitti*, *Penaeus silasi*, *Penaeus similis*, *Penaeus simplex*, *Penaeus smyrnacus*, *Penaeus sorbinii*, *Penaeus stylirostris*, *Penaeus subtilis*, *Penaeus telsodecacanthus*, *Penaeus tenuis*, *Penaeus vanzii*, and *Penaeus villosus*, as well as North American lobsters (*Homarus americanus*), ornate lobsters (*Panulirus ornatus*), interrupted lobsters (*Panulirus interruptus*), and combinations thereof. In a preferred embodiment of the invention, the shrimp is the whiteleg shrimp (L. vannamei).

[0024] According to the present invention, the crab species may be selected from the group consisting of: *Scylla serrata*, *Charybdis feriatus*, *C. granulata*, *C. natator*, *Portunus sanguinolentus*, *P. pelagicus*, *P. haanii*, *Calappa philargius*, *Ranina ranina*, *Paralithodes camtschaticus*, *Chionoecetes opilio*, *Birgus latro*, and combinations thereof.

[0025] Alternatively, the aquatic animal can also be a large isopod (Bathynomus doederleinii).

[0026] According to the present invention, the composition may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may comprise one or more reagents selected from the group consisting of: solvents, buffers, emulsifiers, suspending agents, decomposers, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, wetting agents, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents fall within the scope of expertise and routine practice of those skilled in the art.

[0027] According to the present invention, the composition can be directly added to a body of water in which aquatic animals are raised, or it can be added to the feed of aquatic animals using a standard technique known to those skilled in the art, and then added to the water. For example, the composition can be added directly to the feed of aquatic animals, or it can be used to generate one or more intermediates [such as feed additives or premixes], which are then added to the feed. In a preferred embodiment of the invention, the composition is added to the water after being mixed with the feed of aquatic animals.

[0028] According to the present invention, the composition may be added to and mixed into aquaculture feed at an amount ranging from 0.2% (w / w) to 10% (w / w). In a preferred embodiment of the present invention, the composition is added to and mixed into aquaculture feed at an amount of 1% (w / w). [Detailed Description of Preferred Embodiments]

[0029] The present invention will be further described with reference to the following embodiments, but it should be understood that these embodiments are for illustrative purposes only and should not be construed as limiting the implementation of the present invention. [Example] [, General experimental methods: , ] 1. Statistical analysis:

[0030] The experimental data obtained in the following examples are presented as mean ± standard deviation (SD). All data were evaluated using one-way analysis of variance (ANOVA) to assess differences between groups. A p-value < 0.05 indicates statistical significance. [Example 1.] [Preparation of Bidens pilosa ( , Bidens pilosa , ) ] [Methanol extract]

[0031] First, the whole plant of Bidens pilosa collected from Yunlin County was washed with deionized water, then dried and pulverized. Next, 1,000 g of the dried Bidens pilosa powder was soaked in 10 L of 95% methanol at room temperature and thoroughly mixed. The resulting mixture was then allowed to stand for 24 hours. Afterward, the mixture was filtered using ADVANTEC No.2 Qualitative Filter Papers with a pore size of 5 µm. The filtrate was collected and concentrated using a rotary evaporator (LABOROTA 4000, Heidolph) to remove methanol. The filtrate was then freeze-dried to obtain a lyophilized powder of the methanol extract of Bidens pilosa. [Example 2.] High-performance liquid chromatography of methanol extract of Bidens pilosa [(high performance liquid chromatography, HPLC)] [analyze]

[0032] To understand the main component distribution of the methanol extract of Bidens pilosa according to the present invention, the methanol extract of Bidens pilosa obtained according to Example 1 above was subjected to high performance liquid chromatography analysis. [, Experimental materials: , ] [, , ]

[0033] For HPLC analysis, the methanol extract of Bidens pilosa obtained in Example 1 above was dissolved in methanol to prepare a test solution sample with a concentration of 1 mg / mL. This test solution sample was then used for the following HPLC analysis. [, Experimental methods: , ] [, , ]

[0034] The HPLC instruments used in this experiment were as follows: a high-performance liquid chromatography system (model 1100 Series, Agilent), a pump (model G1311A, Agilent), and a diode array detector (model G1315B, Agilent); the analytical column was an RP-18 (Phenomenex), with a length of 250 mm × 4.6 mm. The HPLC operating conditions are shown in Table 1 below. Table 1. Operating conditions for HPLC Operating parameters condition Detection wavelength 240 nm Moving phase 0.05 vol% trifluoroacetic acid (TFA) aqueous solution / acetonitrile containing 0.05 vol% TFA, 90:10 (v / v) Gradient elution of the mobile phase From 0 to 5 minutes, acetonitrile changed from 10% to 19%; from 5 to 17 minutes, acetonitrile changed from 19% to 21%; from 17 to 23 minutes, acetonitrile changed from 21% to 30%; from 23 to 40 minutes, acetonitrile changed from 30% to 35%; and from 40 to 60 minutes, acetonitrile changed from 35% to 10%. Flow rate (mL / min) 0.2 [, result: , ] [, , ]

[0035] Figure 1 shows the HPLC spectrum obtained by performing high performance liquid chromatography on the methanol extract of Bidens pilosa prepared according to Example 1 above. As can be seen from Figure 1, the methanol extract of Bidens pilosa showed 7 main elution peaks (labeled as peaks a1, a2, a3, a4, a5, a6 and a7) during the retention period from 10 to 60 minutes. [Example 3.] [Methanol extract of Bidens pilosa is effective against white spot syndrome virus (WSSV)] [Utility assessment in infection control] [, Experimental materials: , ] 1. Laboratory animals:

[0036] The Litopenaeus vannamei shrimp (89 days old, weighing approximately 10 g) used in this embodiment were purchased from the National Taiwan Ocean University's aquaculture farm. All experimental animals were housed in a water tank with 12 hours of light and 12 hours of darkness, and the water temperature was maintained at 25±5°C, with ample feed provided. 2. Preparation of WSSV suspension:

[0037] In this embodiment, the WSSV suspension used was prepared from the muscle of shrimp infected with WSSV. In short, muscle was extracted from WSSV-infected crayfish [obtained from a shrimp farm in Dawen area, Yilan County] and cut into pieces. Then, 1 mL of PBS buffer (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄, pH 7.4) was added for grinding to homogenize the mixture. The mixture was then centrifuged at 2,000 rpm for 10 minutes at 4°C. The supernatant was collected and diluted 100-fold with PBS. The solution was then filtered through 0.45 µm filter paper, and the filtrate was collected to obtain the WSSV suspension [virus count: 5 × 10⁶ copy number / mL]. This WSSV suspension was stored at -80°C for later use. 3. Preparation of water extract of Bidens pilosa:

[0038] For comparison, the applicant prepared a water extract of Bidens pilosa in accordance with the steps described in Example 1 above, except that boiling water was used instead of methanol and the extraction was carried out at 100°C. 4. Preparation of methanol extract of Bidens alba:

[0039] For comparison, the applicant prepared a methanol extract of Bidens pilosa by generally following the steps described in Example 1 above, except that Bidens pilosa collected from Yunlin County was used instead of Bidens pilosa. 5. Preparation of water extract of Bidens pilosa:

[0040] For comparison, the applicant prepared an aqueous extract of Bidens pilosa in accordance with the steps described in Example 1 above, except that: Bidens pilosa was used instead of Bidens pilosa, and boiling water was used instead of methanol and the extraction was carried out at 100°C. [, Experimental methods: , ] [A.] [Farming of Litopenaeus vannamei:]

[0041] Pacific white shrimp were randomly divided into 6 groups (n=20 per group), including 1 normal control group, 1 pathological control group, 1 experimental group, and 3 control groups (i.e., control groups 1 to 3). Each group of Pacific white shrimp was then fed a standard diet (brand: Marine Feed, trade name: No. 1 Shrimp Feed) (dosage: 0.05 g / g body weight / day). Before feeding, the experimental group and control groups 1 to 3 were pre-mixed with the extract shown in Table 2 at a 1% (w / w) addition rate. During the 14-day trial, each group was fed once daily. Table 2. Extracts added to each group of feeds Group extract normal control group - Pathological control group - experimental group methanol extract of Bidens pilosa Comparison Group 1 Bidens pilosa water extract Comparison Group 2 Methanol extract of Bidens pilosa Comparison Group 3 Water extract of Bidens pilosa [B.] [WSSV] [Injection of suspension:]

[0042] On day 7 of the experiment, the pathological control group, experimental group, and each comparison group of Litopenaeus vannamei were intramuscularly injected with WSSV suspension (viral load of 1.65 × 10⁴ replicates / g). The normal control group of Litopenaeus vannamei was intramuscularly injected with PBS. [C.] Survival rate of Litopenaeus vannamei (survival rate) [The determination:]

[0043] On days 8, 9, 10, 11, 12, 13 and 14 of the experiment, the number of surviving whiteleg shrimp in each group was counted and substituted into the following formula (1) to calculate the survival rate of each group. [formula] [(1)] [:] [A] [=] [(B / 20)] [×]

[0100] Where: A = Survival rate (%) B = Number of surviving whiteleg shrimp in each group

[0044] Then, the obtained experimental data were analyzed according to the method described in item 1 "Statistical Analysis" of "General Experimental Methods" above. [D.] [WSSV] [Viral load measurement:]

[0045] On day 12 of the experiment, five shrimp (30 in total) were randomly selected from each group of Litopenaeus vannamei. Their gills were removed, and DNA was extracted using the EZNA®SQ Tissue DNA Kit (Omega Bio-tek) according to the manufacturer's instructions. The obtained DNA was then used as a template for real-time PCR, employing the WSSV-specific primers and probes shown in Table 3 below, as described in Nunan LM and Lightner DV (2011), J. Virol. Method., 171:318-321, to determine the WSSV viral load in each group. Table 3. Primers and probes used for real-time PCR Primer / Probe Sequence (5'→3') Forward Introduction ttggtttcagcccgagatt (Sequence Identification Number: 1) Reverse pull ccttggtcagccccttga (Sequence identification number: 2) probe tgctgccgtctccaa (Sequence identification number: 3) Note: The 5' and 3' ends of the probe are labeled with fluorescein amidite (FAM) and tetramethylrhodamine (TAMRA), respectively.

[0046] Real-time PCR was performed using a real-time PCR system (model Biometra TRIO, manufacturer: Analytik Jena GmbH+Co.) and following the manufacturer's operating instructions. The operating and reaction conditions for real-time PCR are shown in Table 4 below. Table 4. Reaction conditions for instant PCR Contents Volume (µL) DNA (100 ng / µL) 5 Forward inductor (2.5 µM) 1 Reverse primer (2.5 µM) 1 Probe (2.5 µM) 1 TaqMan® PCR Master Mix Premixed Reagent (The label is ThermoFisher) 10 sterile water 2 Operating conditions: Pre-denaturation was performed at 95°C for 1 minute; followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 30 seconds.

[0047] Then, the obtained experimental data were analyzed according to the method described in item 1 "Statistical Analysis" of "General Experimental Methods" above. [E.] Histopathology of the hepatopancreas [(histopathology of hepatopancreas)] [:]

[0048] At the end of the 14-day experiment, the normal control group, the pathological control group, and the experimental group of Litopenaeus vannamei were sacrificed. The hepatopancreas tissue was then removed with a scalpel and fixed at room temperature with 4% paraformaldehyde (prepared in PBS) for 48 hours. The tissue was then embedded in paraffin and sectioned to obtain tissue sections with a thickness of 5 μm.

[0049] Subsequently, the obtained tissue sections were stained with hematoxylin-eosin according to the techniques known and commonly used by those skilled in this art. Then, an optical microscope (model AXIO Imager Z1, brand ZEISS) was used at a magnification of 200x to randomly select a region from the tissue section for photographing and counting of normal and abnormal hepatopancreatic cells. The percentage of normal cells in each group was then calculated by substituting the following formula (2). [formula] [(2)] [:] [C] [=] [(D / E)] [×]

[0100] Where: C = percentage of normal cells (%) D = Number of normal cells in each group E = Total number of cells in the normal control group

[0050] Then, the obtained experimental data were analyzed according to the method described in item 1 "Statistical Analysis" of "General Experimental Methods" above. [, , ] [, result: , ] [A.] [Survival rate determination of Litopenaeus vannamei:]

[0051] Figure 2 shows the survival rate of Litopenaeus vannamei in each group over time from day 8 to day 14 of the experiment. As can be seen from Figure 2, the survival rate of Litopenaeus vannamei in the normal control group remained at 100%, while the survival rate of Litopenaeus vannamei in the pathological control group gradually decreased over time, dropping to approximately 10% at the end of the experiment. This indicates that WSSV successfully infected Litopenaeus vannamei and caused its death. The survival rate of Litopenaeus vannamei in the experimental group remained above 50% at the end of the experiment, while the survival rates of Litopenaeus vannamei in comparison groups 1 to 3 all decreased to levels similar to those in the pathological control group. These results demonstrate that the methanol extract of Bidens pilosa can effectively combat WSSV infection and slow down mortality caused by the infection. [B.] [WSSV] [Viral load measurement:]

[0052] Figure 3 shows the WSSV virus load measured in the gills of Litopenaeus vannamei in each group. As can be seen from Figure 3, compared with the normal control group, the WSSV virus load in the pathological control group was significantly increased, indicating that WSSV successfully infected Litopenaeus vannamei and replicated extensively within the body. Compared with the pathological control group, the WSSV virus load in both the experimental group and control group 1 was significantly decreased. In particular, the WSSV virus load in the experimental group was not only reduced to less than half of that in control group 1, but also approached that of the normal control group. There was no significant difference in the WSSV virus load between control groups 2 and 3 and the pathological control group. These results show that the methanol extract of Bidens pilosa is significantly more effective than the water extract of Bidens pilosa and the methanol and water extracts of Bidens pilosa in combating WSSV infection and inhibiting viral replication. [C.] Histopathology of the hepatopancreas:

[0053] Figure 4 shows the percentage of normal cells in the hepatopancreas of Litopenaeus vannamei in each group. As can be seen from Figure 4, compared with the normal control group, the percentage of normal cells in the pathological control group was significantly decreased, indicating that WSSV successfully infected Litopenaeus vannamei and caused hepatopancreas lesion. In contrast, compared with the pathological control group, the percentage of normal cells in the experimental group was significantly increased, even approaching that of the normal control group. This experimental result shows that the methanol extract of Bidens pilosa can effectively combat WSSV infection and prevent infection-induced damage.

[0054] Based on the above experimental results, the applicant believes that the methanol extract of Bidens pilosa of the present invention is expected to be used to combat WSSV infection in various aquatic animals.

[0055] All patents and documents cited in this specification are incorporated herein by reference in their entirety. In the event of any conflict, the detailed description herein (including its definitions) shall prevail.

[0056] Although the present invention has been described with reference to the specific examples described above, it is evident that many modifications and variations can be made without departing from the scope and spirit of the invention. Therefore, it is intended that the invention be limited only to those shown in the appended claims.

[0057] none

[0058] TW202535252A_113117732_SEQL.xml

Claims

1. The use of a methanol extract of Bidens pilosa for the preparation of a composition for use against white spot disease virus infection in aquatic animals.

2. As claimed in claim 1, wherein the aquaculture animal is a shrimp species selected from the group consisting of: Litopenaeus vannamei, Litopenaeus setiferus, Metapenaeus ensis, Penaeus monodon, P. japonicus, P. semisulcatus, P. amazonicus, P. aztecus, P. barmerensis, P. bolcensis, P. brasiliensis, P. brevirostris, P. californiensis, P. chinensis, Litopenaeus cornappensis, P. duorarum, P. esculentus, and P. gracile. glaessneri), gracilis, hamleti, hathor, indicus, isabelae, kapurdii, kerathurus, konkani, latisulcatus, longistylus, maddeni, marginatus, merguiensis, notalis, obtusus, occidentalis, paulensis, penicillatus, plebejus, and hairy shrimp. *Prunus pubescens*, *P. pulchricaudatus*, *P. schmitti*, *P. silasi*, *P. similis*, *P. simplex*, *P. smyrnacus*, *P. sorbinii*, *P. stylirostris*, *P. subtilis*, *P. telsodecacanthus*, *P. squalidus*.Prawns (Penaeus tenuis), Penaeus vanzii, Penaeus villosus, and combinations thereof.

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