Application of star anise water extract in preparation of inhibitor of carp herpesvirus type II
The drug prepared by using star anise water extract has solved the problems of prevention and treatment of carp herpesvirus type II infection and fish viral hematopoietic organ necrosis, achieving effective virus inhibition and low-toxicity drug application, reducing virus replication and mortality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI OCEAN UNIV
- Filing Date
- 2023-12-29
- Publication Date
- 2026-06-09
AI Technical Summary
There is a lack of effective methods in the current technology to inhibit the infection of carp herpesvirus type II (CyHV-2) and to prevent and treat viral hematopoietic organ necrosis in fish, which leads to high mortality and economic losses.
Anise extract was used as the antiviral active ingredient. The drug was prepared by soaking, decocting and centrifuging. It was used to prepare a drug to inhibit carp herpesvirus type II at a concentration of 0.1-1 mg/mL. It was applied to prevent or treat carp herpesvirus type II infection and fish viral hematopoietic organ necrosis.
Star anise water extract can significantly reduce intracellular CyHV-2 virus titers, showing obvious anti-CyHV-2 activity, providing a green and safe antiviral drug for fisheries. It can produce significant effects at low doses, preventing and treating CyHV-2 virus infection and related diseases.
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Figure CN118045120B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to aquaculture disease prevention and control technology, specifically to the application of star anise extract in the preparation of carp herpesvirus. Background Technology
[0002] Cyprinid herpesvirus 2 (CyHV-2) primarily infects crucian carp and goldfish, and is a significant pathogen causing viral diseases in farmed crucian carp. CyHV-2 belongs to the order Herpesvirales, family Alloherpesviridae, and genus Cyprinivirus. It is a linear double-stranded DNA virus that causes herpesvirus-related hematopoietic necrosis disease (HVHND). This disease results in a mortality rate of 50-100% in affected fish; therefore, measures are urgently needed to reduce the economic losses caused by CyHV-2.
[0003] Star anise (Illicium verum Hook.f.) is the dried fruit of a plant in the Magnoliaceae family. It is mostly composed of 8 follicles, hence the name "star anise". It mainly contains volatile oils, organic acids, flavonoids, glycosides, triterpenoids, minerals and other physiologically active substances.
[0004] There are currently no reports on the inhibition of CyHV-2 by star anise. Summary of the Invention
[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide the application of star anise extract in the preparation of inhibitors of carp herpesvirus type II.
[0006] To achieve the above objectives, this invention proposes the application of star anise extract in the preparation of inhibitors of carp herpesvirus type II.
[0007] On the other hand, the present invention proposes the application of star anise extract in the preparation of drugs for the prevention or treatment of carp herpesvirus type II infection.
[0008] In another aspect, the present invention also proposes the application of star anise water extract in the preparation of drugs for the prevention or treatment of viral hematopoietic organ necrosis in fish.
[0009] It should be noted that the fish mentioned can be crucian carp, goldfish, or other fish susceptible to carp herpesvirus.
[0010] Preferably, the method for preparing the star anise water extract comprises the following steps:
[0011] Star anise is ground into powder, soaked in water (preferably ultrapure water) for 8-12 hours, decocted and brought to a constant volume, and then centrifuged and filtered to obtain the star anise water extract.
[0012] More preferably, the method for preparing the star anise extract satisfies at least one of the following conditions:
[0013] Soaking conditions: 0–4℃;
[0014] The decoction should be prepared at 90–100℃ for 1–2 hours.
[0015] Centrifuge at 11000–12000 r / min for 10–15 min;
[0016] Filtration is performed using a 0.22–0.4 μm filter membrane.
[0017] Preferably, the star anise water extract is an antiviral active ingredient, and its application dose is 0.1-1 mg / mL, more preferably 0.3-1 mg / mL.
[0018] It should be noted that in the technical solution described in this invention, the applied dosage is the final concentration of star anise extract when added to the culture medium. However, as those skilled in the art will know, when it is used as a fish medicine, those skilled in the art can also adjust it based on this concentration condition, for example, by mixing it into feed.
[0019] Furthermore, the present invention proposes a drug for the prevention or treatment of carp herpesvirus type II infection, wherein the active component of the drug against carp herpesvirus type II is anise extract.
[0020] Preferably, the drug further includes pharmaceutically acceptable excipients.
[0021] More preferably, the dosage form of the drug includes tablets, powders, granules, capsules, or sustained-release formulations.
[0022] Preferably, the method for preparing the star anise water extract comprises the following steps:
[0023] Star anise is ground into powder, soaked in water overnight (soaking time is 8-12 hours), decocted and brought to a constant volume, and then centrifuged and filtered to obtain the star anise water extract.
[0024] It should be noted that, in the technical solution described in this invention, the use of star anise water extract in the preparation of carp herpesvirus type II (i.e., CyHV-2) inhibitor drugs aims to provide an alternative solution for the preparation of drugs for the prevention and treatment of crucian carp hematopoietic organ necrosis.
[0025] This is because, on the one hand, star anise extract can effectively inhibit the replication of CyHV-2 virus in GiCF cells, reducing the copy number and titer of CyHV-2 virus.
[0026] By applying star anise in vitro, it was found that the drug could effectively reduce the titer of carp herpesvirus type II in GiCF cells in a dose-dependent manner. This indicates that star anise can effectively inhibit the infection and replication of carp herpesvirus type II in GiCF cells, thereby hindering viral replication. Therefore, star anise can effectively prevent and treat carp herpesvirus type II infection and the diseases it causes. It can be used to prepare drugs for the prevention and treatment of carp herpesvirus type II infection, as well as drugs for viral hematopoietic organ necrosis in fish.
[0027] On the other hand, the extract obtained through a simple and convenient extraction method can be effectively used to inhibit carp herpesvirus, so as to achieve the goal of developing green, safe and efficient antiviral fish drugs.
[0028] It should be noted that the various natural active ingredients in star anise extract work synergistically to effectively inhibit the replication of CyHV-2 virus and have a relatively obvious anti-CyHV-2 activity effect.
[0029] The technical solution of this application has the following beneficial effects:
[0030] 1. This invention is the first to apply star anise and its water extract to the prevention and treatment of carp herpesvirus type II (CyHV-2) infection. At a concentration of 0.1 to 1 mg / mL (especially 0.3 mg / mL), it can significantly reduce the intracellular titer of CyHV-2 virus, thereby inhibiting viral replication and providing a new solution for the prevention and treatment of CyHV-2 virus infection and viral hematopoietic organ necrosis in fish.
[0031] 2. Star anise can produce significant effects at low application concentrations and commonly used dosages. It has low cytotoxicity and is expected to become a candidate drug for the prevention and treatment of carp herpesvirus type II. Attached Figure Description
[0032] Figure 1 The results of virus titer detection in cell supernatant after treatment with star anise water extract in Example 1 of the present invention are shown.
[0033] Figure 2 The results of virus titer detection in cell supernatant after treatment with star anise ethanol extract in Example 1 of the present invention are shown.
[0034] Figure 3 The results of virus titer detection in cell supernatant after shikimic acid treatment in Example 1 of this invention are shown. Detailed Implementation
[0035] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.
[0036] The present invention will be further described below with reference to the embodiments:
[0037] Experimental materials
[0038] (I) Experimental strains and cells
[0039] The caudal fin cell line GiCF (Carassius auratus gibelio) was constructed in our laboratory. The construction method can be found in Lu, JF; Xu, D; Lu, LQ. A novel cell line established from caudal fin tissue of Carassius auratus gibelio is susceptible to cyprinid herpesvirus 2 infection with the induction of apoptosis[J]. Virus Research. 2018; 258:19-27. The cells were cryopreserved in liquid nitrogen. The cell resuscitation medium was M199 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the cells were cultured in a constant temperature incubator at 27℃. CyHV-2 isolate YC-01 (hereinafter referred to as CyHV-2, GenBank: MN593216.1) was used to infect cells at 25°C. After all cells died, the cell supernatant was collected and filtered through a 0.22μm syringe filter to obtain pure virus solution, which was then stored at -80°C for later use.
[0040] (II) Experimental Reagents
[0041] M199 culture medium, fetal bovine serum, and penicillin-streptomycin 100× were purchased from Gibco; DMSO was purchased from Sigma; viral genomic DNA / RNA extraction kit (DP315) was purchased from Tiangen; RNase-free double-distilled water was purchased from Sangon Biotech (Shanghai) Co., Ltd.; TB Premix Ex Taq TM II (RR820A) was purchased from TaKaRa; star anise (brown powder, store at room temperature): 250 mg / mL.
[0042] Example 1
[0043] 1. An in vitro inhibition experiment was conducted using star anise aqueous extract as an inhibitor. The control example in this case used an in vitro inhibition experiment with star anise alcohol extract and shikimic acid as inhibitors.
[0044] The star anise extract was prepared using the following method:
[0045] Star anise was pulverized into powder, and 2.5g was soaked in 50mL of ultrapure water at 4℃ overnight (soaking time was 8-12h). The next day, the star anise soaking solution and the herb packet were placed in a pot and decocted to 10mL at 100℃ for 2h. The volume was adjusted to obtain a 250mg / mL star anise water extract mother liquor. After centrifugation at 12000r / min for 10min, the extract was filtered through a 0.22μm filter membrane to obtain the star anise water extract, which was then aliquoted and stored at -80℃ for later use.
[0046] The star anise ethanol extract was dissolved in DMSO at a concentration of 100 mg / mL and diluted to the specified concentration with 2% M199 medium before administration. The extraction method of the star anise ethanol extract adopted conventional techniques in the art. For example, the specific operation was to weigh dried star anise powder, add 60% ethanol by volume, extract at 80°C for 2 hours, and then filter through a 0.45 μm membrane for later use. The final concentration of the star anise ethanol extract was 100 mg / mL.
[0047] Shikimic acid was dissolved in DMSO at a concentration of 25 mg / mL and diluted to the specified concentration with 2% M199 medium before administration.
[0048] Anise extract: The experimental concentrations were 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL or 1 mg / mL, with the H2O group serving as the control;
[0049] Anise extract: The experimental concentrations were 1 μg / mL, 10 μg / mL, 50 μg / mL and 100 μg / mL, with the DMSO group serving as the control;
[0050] Shikimic acid: The experimental concentrations were 0.01 μg / mL, 0.1 μg / mL, 0.3 μg / mL and 0.5 μg / mL, respectively, with the DMSO group serving as the control.
[0051] It should be noted that since the solvent in the water extract of star anise is water, H2O is used as a control, while in the alcohol extract of star anise and shikimic acid, the solvent is DMSO, so DMSO is used as a control group.
[0052] In addition, shikimic acid was chosen as the comparative ratio because it is a component of star anise extract and an important biochemical intermediate found in plants, which has antioxidant and antiviral effects.
[0053] GiCF cells were first pretreated with an inhibitor for 2 hours, and then infected with CyHV-2 (MOI = 0.1). Cells were collected 72 hours after infection, and the CyHV-2 viral titer was measured in triplicate for each concentration.
[0054] 2. DNA extraction
[0055] Collect the cell supernatant after drug treatment and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min at room temperature.
[0056] Take 200 μL of the supernatant and place it in a new centrifuge tube. Extract viral DNA from the cell supernatant using the Viral Genomic DNA / RNA Extraction Kit (DP315). Dissolve the DNA in 20 μL of RNase-free double-distilled water.
[0057] 3. Quantitative Real-Time PCR Detection (RT-qPCR)
[0058] The RT-qPCR reaction system was based on the TB Green Premix Ex Taq II (Tli RNaseH Plus) Kit.
[0059] The primer sequences used for RT-qPCR are shown in Table 1. The reaction system and reaction conditions are shown in Tables 2 and 3.
[0060] Table 1 RT-qPCR primers
[0061]
[0062]
[0063] Table 2 RT-qPCR reaction system
[0064]
[0065] Table 3 RT-qPCR reaction conditions
[0066]
[0067] 4. Virus titer detection
[0068] The recombinant plasmid pDsRed-express-N1-ORF79 was constructed in our laboratory, following the construction method described in Chinese patent document CN116064511A, published on May 5, 2023, entitled "An RNA interference sequence for inhibiting the immediate early gene ORF121 of carp herpesvirus type II and its application," which outlines the construction of the pDsRed-express-ORF121 recombinant plasmid. The plasmid concentration was measured using NanoDrop 2000. The copy number (copies / μL) was 6.02 × 10⁻⁶. 23 (copies / mol) × plasmid concentration (ng / μL) × 10 -9 / (recombinant plasmid base number × 660 g / mol). RT-qPCR analysis was performed using plasmids diluted 10-fold serially as templates. Our laboratory constructed a standard curve for the CyHV-2ORF79 gene, with cycle number (Ct) and logarithm of copy number as the x and y axes, respectively. The equation of this curve is: y = -3.1394x + 38.9R 2 =0.9974.
[0069] Using cellular DNA as a template, RT-PCR was performed using the same primers as the standard curve. The Ct value was substituted into the standard curve to obtain the viral copy number, and the viral copy number was then substituted into the formula to calculate the viral titer.
[0070] The formula for calculating titer (integration units per mL, IU / mL) is as follows:
[0071] IU / mL=(C×N×D×1000) / V
[0072] Where: C = average number of viral copies integrated per genome; N = number of cells at infection (approximately 1 × 100,000); D = dilution factor of the viral vector; V = volume of diluted virus added.
[0073] GiCF cells were treated with star anise water extract, star anise alcohol extract, and shikimic acid for 2 hours, then infected with CyHV-2 at MOI = 0.1. Cells were collected 72 hours after infection, and CyHV-2 copy number was measured. The effects of each drug at different concentrations are shown below. Figures 1-3 As shown in Tables 4-6, Tables 4-6 show the antiviral test results of star anise water extract, star anise alcohol extract and shikimic acid, respectively. A concentration of 0 indicates that no reagents were added, i.e., the initial viral titer.
[0074] like Figure 1As shown in Table 4, compared with the control group H2O, the fennel extract significantly reduced the CyHV-2 virus copy number and intracellular titer after treatment. At a concentration of 0.1 mg / mL, it significantly inhibited CyHV-2 virus replication in cells, and the results were statistically significant. However, after 72 hours of treatment with fennel alcohol extract and shikimic acid, the binding... Figure 2 , Figure 3 As shown in Tables 5 and 6, neither the ethanol extract of star anise nor shikimic acid reduced the copy number and titer of CyHV-2 virus. This indicates that the ethanol extract of star anise can effectively inhibit the replication of CyHV-2 virus. However, the ethanol extract of star anise and shikimic acid had no inhibitory effect on CyHV-2.
[0075] Table 4
[0076]
[0077]
[0078] Table 5
[0079]
[0080] Table 6
[0081]
[0082]
[0083] It should be noted that the scope of protection of the prior art in this invention is not limited to the embodiments given in this application. All prior art that does not contradict the solution of this invention, including but not limited to prior patent documents, prior publications, prior public uses, etc., can be included in the scope of protection of this invention.
[0084] Furthermore, the combination of the technical features in this case is not limited to the combination methods described in the claims of this case or the combination methods described in the specific embodiments. All technical features described in this case can be freely combined or combined in any way, unless they contradict each other.
[0085] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.
Claims
1. Application of star anise water extract in the preparation of inhibitors of carp herpesvirus type II.
2. Application of star anise water extract in the preparation of drugs for the prevention or treatment of carp herpesvirus type II infection.