CN114392283A - anti-IPNV kelp extract and application thereof - Google Patents
anti-IPNV kelp extract and application thereof Download PDFInfo
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- CN114392283A CN114392283A CN202210095312.XA CN202210095312A CN114392283A CN 114392283 A CN114392283 A CN 114392283A CN 202210095312 A CN202210095312 A CN 202210095312A CN 114392283 A CN114392283 A CN 114392283A
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
Abstract
The invention belongs to the field of extracts, and particularly relates to an IPNV-resistant kelp extract and application thereof. The invention discloses a kelp extract, which is prepared by the following steps: 1) preparation of crude extract of kelp: removing protein in the kelp leaching liquor to obtain kelp crude extract; the kelp extract is an extract obtained by carrying out enzymolysis and water extraction on kelp, 2) ethanol precipitation dialysis retentate is collected and precipitated to obtain a kelp extract, and the dialysis retentate is an in-bag liquid obtained by carrying out dialysis purification on the kelp crude extract by using a dialysis bag method. The kelp extract with the obvious resistance to Infectious Pancreatic Necrosis Virus (IPNV) is prepared, and the inhibition rate of the kelp extract with the concentration of 300 mu g/mL to the IPNV reaches 68.84%. The herba Zosterae Marinae extract can exert antiviral effect by blocking IPNV adsorption and invasion, and is expected to be a novel medicine/immunopotentiator for resisting IPNV.
Description
Technical Field
The invention belongs to the field of extracts, and particularly relates to an IPNV-resistant kelp extract and application thereof.
Background
Infectious Pancreatic Necrosis (IPN) is a viral disease which seriously harms salmon and trout and can cause hemorrhage and Necrosis of internal parenchymal organs of the salmon and trout. The disease is caused by Infectious Pancreatic Necrosis Virus (IPNV), is susceptible to juvenile salmon, can cause a death rate of fry of more than 90% once the fry is burst, and survivors of the infected fries are infected for the lifetime, so that the disease becomes a potential infection source. Due to the characteristics of wide prevalence and strong infectivity of the IPN, huge economic loss is caused to the salmon and trout breeding industry, and the IPN is a first-class quarantine object for fish port quarantine.
The IPN virus mainly infects the pancreas, causing pancreatic necrosis, and pancreatic acini, pancreatic islets and all cells are almost abnormal, most of which are necrotic. Currently, no effective treatment method for IPNV exists, and the prevention is still mainly performed. Although drugs such as ribavirin and pyrazolofuranidin inhibit the replication of IPNV, their pharmacological effects are unclear and drug residues are prohibited. Based on the actual situation that the IPN medicine for preventing and controlling salmon and trout is lacked at present, a novel IPNV (infectious bronchitis Virus) resisting medicine needs to be developed urgently.
Disclosure of Invention
The technical problem to be solved by the invention is how to control the serious harm of Infectious Pancreatic Necrosis Virus (IPNV) and reduce the loss of IPNV to the rainbow trout breeding industry.
In order to solve the above technical problems, the present invention provides, in a first aspect, a kelp extract, which is prepared according to a method comprising the steps of:
1) preparation of crude extract of kelp: removing protein in the kelp leaching liquor to obtain kelp crude extract; the kelp extract is an extract obtained by carrying out enzymolysis and water extraction on kelp,
2) precipitating with ethanol, dialyzing the retentate, collecting the precipitate to obtain herba Zosterae Marinae extract, and purifying the herba Zosterae Marinae crude extract with dialysis bag method to obtain in-bag liquid.
Further, in the above kelp extract, the kelp extract is prepared according to a method comprising 1-1) and 1-2):
1-1) mixing the kelp with water to obtain kelp soaking liquid;
1-2) treating the kelp soak solution with cellulase, pectinase and papain to obtain water extract, filtering the water extract, collecting filtrate, centrifuging the filtrate, and collecting supernatant to obtain kelp extract.
Further, in the above kelp extract, the step of treating the kelp soak solution with cellulase, pectinase and papain in the step 1-2) to obtain an aqueous extract solution comprises:
adjusting the pH value of the kelp soaking solution to 5.0, adding cellulase, and performing enzymolysis at 55 ℃ for 20min to obtain a cellulase hydrolysate; adjusting the pH value of the cellulose enzymolysis liquid to 4.2, adding pectinase, and carrying out enzymolysis for 2h at 50 ℃ to obtain pectin enzymolysis liquid; adjusting the pH value of the pectin enzymolysis liquid to 6.0, adding papain, carrying out enzymolysis at 50 ℃ for 2h, and then leaching at 75 ℃ for 3h to obtain the water extract.
Further, in the above kelp extract, the centrifugation conditions in the above 1-2) are 3500g centrifugation for 15 min.
Further, in the kelp extract, the step 1) of removing the protein in the kelp extract by using a sevage method comprises centrifuging at 3500g for 10 min.
In the invention, the removing of the protein in the kelp leach liquor by the sevage method further comprises:
s1) before removing protein in herba Zosterae Marinae extract, concentrating the herba Zosterae Marinae extract at 60 deg.C under reduced pressure to 1/10 of original volume to obtain concentrated herba Zosterae Marinae extract;
s2) adding chloroform and n-butanol in a ratio of 4: 1 to obtain a mixed reagent. Mixing the mixed reagent with the concentrated kelp leaching liquor according to the ratio of 1: 2, placing the mixture in an air oscillator, and violently oscillating for 30min at room temperature;
s3) and then subjecting the shaken mixture to centrifugation under the above conditions.
Further, in the above kelp extract, the cut-off molecular weight of the dialysis in 2) is 3500 Da.
Further, in the above kelp extract, the step of collecting the precipitate from the ethanol precipitation dialysis retentate in 2) comprises the step of centrifuging at 3500g for 10min to collect the precipitate.
Further, in the above kelp extract, the step 2) includes a step of freeze-drying the precipitate to prepare a dry powder.
In order to solve the above technical problems, in a second aspect, the present invention provides the use of the above kelp extract for the preparation of a virus inhibitor or an antiviral drug, wherein the virus is infectious pancreatic necrosis virus.
In order to solve the above technical problems, in a third aspect, the present invention provides a use of kelp in the preparation of a virus inhibitor or an antiviral drug, wherein the virus is an infectious pancreatic necrosis virus.
In order to solve the above technical problems, in a fourth aspect, the present invention provides a viral inhibitor and/or an antiviral drug, the viral inhibitor and the antiviral drug containing the above kelp extract.
The virus inhibitor and antiviral drug may contain a suitable carrier or excipient in addition to the above extract of Laminaria japonica. The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. In order to prepare the unit dosage form into preparations for injection, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.
In the present invention, the cellulase may specifically be a cellulase derived from Trichoderma reesei ATCC26921 having CAS number 9012-54-8. The cellulase can be a product of Allandin reagent company with the product number of C128647, and the addition amount can be 10U/g dried kelp.
The pectinase is specifically pectinase derived from Aspergillus niger, and the CAS number is 9032-75-1. The pectinase can be a product with the product number of P116864 of Aladdin reagent company, and the addition amount of the pectinase can be 10U/g of dried kelp.
The CAS number of papain is 9001-73-4. The papain can be a product of No. P164463 of Aladdin reagent company, and the addition amount can be 10U/g dried kelp.
In the present invention, the freeze-drying may be vacuum freeze-drying.
In the present invention, the infectious pancreatic necrosis virus may be the infectious pancreatic necrosis virus IPNV-ChRtm213 strain.
In the present invention, the kelp may be Laminaria japonica. The kelp may be composed of a frond and a stalk.
The invention has the following beneficial effects:
the kelp extract with obvious IPNV resistance is prepared, and the inhibition rate of the kelp extract with the concentration of 300 mu g/mL to IPNV reaches 68.84%. Through research and analysis on IPNV (infectious bronchitis Virus) resistance mechanism of the kelp extract, the kelp extract can play an antiviral role by blocking the adsorption and invasion of IPNV. The herba Zosterae Marinae extract is expected to be used as novel medicine/immunopotentiator for resisting IPNV.
The following examples were processed using SPSS19.0 statistical software and the results were expressed as mean. + -. standard deviation and significant differences were indicated by One-way ANOVA test with P < 0.05.
Drawings
FIG. 1 shows the safety analysis of Laminaria japonica extract.
FIG. 2 shows the inhibition of IPNV by Laminaria japonica extract.
FIG. 3 shows the results of the measurement of the effect of Laminaria japonica extract on the cell-adsorbing capacity of IPNV.
FIG. 4 is a measurement of the ability of Laminaria japonica extract to block IPNV-invading cells.
FIG. 5 shows the results of measurement of the ability of Laminaria japonica extract to inhibit IPNV replicating cells.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The IPNV-ChRtm213 strain of infectious pancreatic necrosis virus was isolated from a rainbow trout farm and is disclosed in the literature "Feng Ji, jin-Zhuang ZHao, Miao Liu, Tong-Yan Lu, Hong-Bai Liu, Jiansheng Yin, Li-Ming xu.Complex genetic sequence of an infectious pancreatic necrosis virus from free raw wind trail tout (Oncorhynchus mykiss) in China. Virus genes.2016-11-20" and is available from the applicant under national biosafety regulations, and is used only for experiments in which the present invention was repeated and is not otherwise applicable.
Salmon embryonic cells (Salmon Embryo cell line, CHSE-214 cells) are from China Center for Type Culture Collection (CCTCC) with the serial number of GDC 0168.
The kelp finger (Laminaria japonica) described in the following examples was purchased from the market.
The cellulase in the following examples has CAS number 9012-54-8, which is a product of Allantin reagent company with catalog number C128647; the CAS number of the pectinase is 9032-75-1, and the CAS number is a product of the Aladdin reagent company with the catalog number of P116864; the papain has CAS number 9001-73-4, and is a product of Allantin reagent company with catalog number P164463.
The following examples were processed using SPSS19.0 statistical software and the results were expressed as mean. + -. standard deviation and significant differences were indicated by One-way ANOVA test with P < 0.05.
Example 1 preparation of Laminaria japonica extract
1.1 preparation of crude Laminaria extract
1.1.1 preparation of Laminaria japonica leach liquor
The preparation method of the kelp leaching liquor comprises the following steps:
step 1, crushing dried kelp (Laminaria japonica) (consisting of fronds and stalks) and sieving the crushed kelp with a 20-mesh sieve to obtain dry kelp powder; mixing distilled water with the dry kelp powder to ensure that the liquid-material ratio reaches 30: 1 (namely the mass ratio of distilled water to the dry kelp powder is 30: 1), and soaking for 6 hours to obtain kelp soaking liquid.
Step 2, enzymolysis: placing the kelp soaking solution obtained in the step 1 in an ultrasonic oscillator (with ultrasonic power of 360W) for ultrasonic treatment for 30min, adjusting the pH value of the kelp soaking solution to 5.0 by using HCl or NaOH, adding cellulase, and performing enzymolysis at 55 ℃ for 20min to obtain a cellulase hydrolysate; adjusting pH of the cellulose enzymolysis solution to 4.2 with HCl or NaOH, adding pectinase, and performing enzymolysis at 50 deg.C for 2 hr to obtain pectin enzymolysis solution; adjusting pH of the pectin enzymolysis solution to 6.0 with HCl or NaOH, adding papain, performing enzymolysis at 50 deg.C for 2 hr, and leaching at 75 deg.C for 3 hr to obtain water extractive solution.
The cellulase can be specifically cellulase derived from Trichoderma reesei ATCC26921, and the CAS number of the cellulase is 9012-54-8. The cellulase can be a product of Allandin reagent company with the product number of C128647, and the addition amount can be 10U/g dried kelp.
The pectinase is specifically pectinase derived from Aspergillus niger, and the CAS number is 9032-75-1. The pectinase can be a product with the product number of P116864 from Aladdin reagent company, and the addition amount of the pectinase is 10U/g of dried kelp.
The CAS number of papain is 9001-73-4. The papain can be a product of No. P164463 of Aladdin reagent company, and the addition amount is 10U/g dried kelp.
Step 3, centrifugation: and (3) carrying out vacuum filtration (Buchner funnel) on the water extract obtained in the step (2), collecting filtrate, centrifuging 3500g of filtrate for 15min, and collecting supernatant and precipitate.
The Buchner funnel is a Shanghai Huake product, the specification of the Buchner funnel is 150mm, the filter paper is quantitative filter paper of Jiaojie brand, and the specification is medium speed 15 cm.
And 4, mixing the distilled water with the precipitate obtained in the step 3 according to the mass ratio of 30: 1, repeating the step 2 and the step 3, and collecting supernatant.
And 5, combining the supernatants obtained in the step 3 and the step 4 to obtain the kelp leaching liquor.
1.1.2 preparation of protein-removed Laminaria japonica leach liquor
The kelp leaching liquor obtained by purifying 1.1.1 by adopting a sevage method comprises the following steps:
step 1: vacuum concentrating herba Zosterae Marinae leaching solution at 60 deg.C to 1/10 of original volume to obtain concentrated herba Zosterae Marinae leaching solution.
Step 2: chloroform and n-butanol were mixed as 4: 1 to obtain a mixed reagent. Mixing the mixed reagent with the concentrated kelp leaching liquor according to the ratio of 1: 2, placing the mixture in an air oscillator, and violently oscillating for 30min at room temperature;
and step 3: 3500g, centrifuging for 10min, collecting supernatant, and removing protein;
repeating the step 1 and the step 2 until no protein appears, wherein the obtained liquid is a kelp leaching liquor without protein, namely the kelp crude extract.
1.2 dialyzing to obtain Laminaria japonica extract
Step 1: dialyzing the crude extract of herba Zosterae Marinae obtained at 1.1.2 with dialysis bag (molecular weight cutoff is 3500Da) in flowing deionized water for 5 hr, and collecting liquid in the dialysis bag, i.e. dialysis retentate;
step 2: mixing the dialysis retention solution obtained in the step 1 with absolute ethyl alcohol with the volume 3 times that of the dialysis retention solution, and standing for 24 hours at 4 ℃; centrifuging 3500g of the standing mixed solution for 10min, collecting precipitate, and vacuum drying to obtain herba Zosterae Marinae extract.
Example 2 analysis of safety of Laminaria japonica extract against cells
2.1 measurement method
CHSE-214 cells were plated at 1.0X 10 per well4Cell safety analysis was performed after culturing cells at a rate of 100. mu.L/cell in a 96-well plate for 24 hours. The kelp extracts of example 1 were dissolved with the cell maintenance solution to contents of 100, 200, 300, 400, 500, 1000 and 2000. mu.g/mL, respectively, to obtain cell maintenance solutions containing kelp extracts of different concentrations. Experimental groups: after discarding the culture medium of 96-well plate (10% FBS, 100U/ml penicillin and 100U/ml streptomycin in MEM medium), 100. mu.L of cell-maintaining solution containing kelp extract at different concentrations was added to the CHSE-214 cells in 8 replicates each and cultured in a carbon dioxide incubator at 15 ℃. A normal control group (inoculated with cells, without herba Zosterae Marinae extract, i.e. cultured with cell maintenance solution) and a blank group (not inoculated with cells, without herba Zosterae Marinae extract, i.e. only cell maintenance solution) are provided. Cell activity was measured by MTS method after 7 days of culture.
MTS method: mu.L of MTS diluent (Promega, G3582) was added to each well, mixed well and placed at 37 ℃ in CO2Incubate at 5% concentration for 2h and read absorbance at 490 nm.
Wherein the cell maintenance solution is obtained by adding FBS, penicillin and streptomycin into MEM culture medium, and the cell maintenance solution contains FBS 2%, penicillin 100U/ml and streptomycin 100U/ml.
Cell survival rate (cell viability) (%) ═ aExperimental group-ABlank group)/(ANormal control group-ABlank group)×100％。
2.2 measurement results
The measurement results are shown in fig. 1 and table 1. As can be seen from FIG. 1 and Table 1, the activity of CHSE-214 cells was maintained at 80% or more in the case of the concentration of the Laminaria japonica extract in the cell culture broth of 0 to 500. mu.g/mL, and at 90% or more in the case of the concentration of the Laminaria japonica extract of 300. mu.g/mL. The above results indicate that the concentration of the Laminaria japonica extract is in the range of 300. mu.g/mL, which is safe for the CHSE-214 cells.
TABLE 1 safety of Laminaria japonica extracts against cells
Note: the different letter represents the remarkable difference
Example 3 protective Effect of Laminaria japonica extract on IPNV infected cells
3.1 measurement method
1.0X 10 CHSE-214 cells per well4anti-IPNV effect studies were performed after 24h of culture in 96-well plates after seeding each 100. mu.L of cells. The experimental group dissolved the Laminaria japonica extract in a cell maintenance medium (2% FBS, 100U/mL penicillin and 100U/mL streptomycin in MEM medium), and mixed with IPNV virus suspension to prepare mixed solutions containing 0, 100, 200, 300, 400 and 500. mu.g/mL, respectively. The 96-well plate cell culture medium (10% FBS, 100U/ml penicillin and 100U/ml streptomycin in MEM medium) was discarded, 100. mu.L of each of the above-mentioned mixtures was aspirated and added to CHSE-214 cells so that the MOI of IPNV in each well was 0.01, and each set was repeated 8 times, and cultured in a carbon dioxide incubator at 15 ℃ to observe Cytopathic (CPE) conditions. The experiment was performed with positive group (inoculated cells, inoculated with IPNV (MOI, 0.01) without kelp extract), normal control group (inoculated cells, without kelp extract, without virus) and blank group (not inoculated cells, without kelp extract, without virus).
When the positive control group CPE reached 75% or more, the cell activity was measured according to the MTS method in example 2, and the virus inhibition rate was calculated.
Viral inhibition ratio (%) ═ aExperimental group-APositive group-ABlank group)/(ANormal control group-APositive group-ABlank group)×100％。
The criterion of cytopathic effect is that the cells are in the shape of a reticulum, the nuclei are solidified and condensed, and the plaques are obvious.
3.2 measurement results
The measurement results are shown in fig. 2 and table 2. As can be seen from FIG. 2 and Table 2, the concentration of the Laminaria japonica extract in the range of 0-300. mu.g/mL gradually increases in IPNV resistance with the increase in concentration. The highest IPNV resistance was achieved at 68.84% when the concentration of Laminaria japonica extract was 300. mu.g/mL. When the concentration of the kelp extract is more than 300 mug/mL, the inhibition capability of the kelp extract on IPNV is obviously reduced.
TABLE 2 inhibitory Activity of Laminaria japonica extracts on IPNV
Note: the different letter represents the remarkable difference
Example 4 Studies on IPNV-resistant mechanism of Laminaria japonica extract
4.1 Virus adsorption test
1.0X 10 CHSE-214 cells per well5cells/mL were plated in 6-well plates and cultured for 24 h. Adding cell maintenance solution (1mL) containing 300 μ g/mL herba Zosterae Marinae extract into CHSE-214 cells, incubating at 15 deg.C in 5% carbon dioxide incubator for 12h, discarding maintenance solution, inoculating IPNV suspension to make MOI reach 1, incubating at 4 deg.C in 5% carbon dioxide incubator for 1h, washing CHSE-214 cells with precooled PBS for 3 times, and collecting cells. The CHSE-214 cells (infected group) inoculated with IPNV without treatment with the Laminaria japonica extract under the same conditions were used as a control.
Extracting total RNA of cells by Trizol method, detecting the content of IPNV structural protein gene IPNV-VP2 in the cells by RT-qPCR technology, correcting the difference between individuals by internal reference beta-actin, and adopting 2－△△CtThe method performed data analysis, 3 replicates per sample.
The RT-qPCR detection method comprises the following specific steps: using One Step
PLUS RT-PCR Kit (Perfect Real Time) (Takara, RR096A) Kit, the content of IPNV-VP2 gene was analyzed by the method according to the instruction, and the RT-qPCR primer sequence is shown in Table 3.
TABLE 3 primer information for related genes
The results are shown in FIG. 3. As can be seen from FIG. 3, the relative content of IPNV-VP2 gene on CHES-214 cells was 0.599 after 300. mu.g/mL of the kelp extract-treated cells compared to the infected group, indicating that the kelp extract had the ability to block IPNV-adsorbing cells.
4.2 Virus entry test
1.0X 10 CHSE-214 cells per well5cells/mL were plated in 6-well plates and cultured for 24 h. Adding cell maintenance solution (1mL) containing 300 μ g/mL herba Zosterae Marinae extract into CHSE-214 cells, incubating at 15 deg.C in 5% carbon dioxide incubator for 12h, discarding maintenance solution, inoculating IPNV suspension to make MOI reach 1, incubating at 4 deg.C in 5% carbon dioxide incubator for 1h, washing CHSE-214 cells with precooled PBS, adding fresh cell maintenance solution, incubating at 15 deg.C in incubator for 2h, and collecting cells. The CHSE-214 cells (infected group) inoculated with IPNV without treatment with the Laminaria japonica extract under the same conditions were used as a control.
Extracting total RNA of cells by Trizol method, detecting IPNV-VP2 content in cells by RT-qPCR technology, correcting difference between individuals by internal ginseng beta-actin, and adopting 2－△△CtThe method performed data analysis, 3 replicates per sample.
The results are shown in FIG. 4. As can be seen from FIG. 4, the relative content of IPNV-VP2 gene in CHE-214 cells was 0.757 after 300. mu.g/mL of laminarin extract treated cells compared to the infected group, indicating that the Laminaria japonica extract has the ability to block the invasion of IPNV into cells.
4.3 replication of viruses
1.0X 10 CHSE-214 cells per well5cells/mL were seeded in 6-well platesAnd culturing for 24 h. Adding cell maintenance solution (1mL) containing 300 μ g/mL herba Zosterae Marinae extract into CHSE-214 cells, incubating at 15 deg.C in 5% carbon dioxide incubator for 12h, discarding maintenance solution, inoculating IPNV suspension to make MOI reach 1, incubating at 15 deg.C in 5% carbon dioxide incubator for 1h, replacing fresh cell maintenance solution, incubating at 15 deg.C in 15% carbon dioxide incubator for 8h, and collecting cells. The CHSE-214 cells (infected group) inoculated with IPNV without treatment with the Laminaria japonica extract under the same conditions were used as a control.
Extracting total RNA of cells by Trizol method, detecting IPNV-VP2 content in cells by RT-qPCR technology, correcting difference between individuals by internal ginseng beta-actin, and adopting 2－△△CtThe method performed data analysis, 3 replicates per sample.
The results are shown in FIG. 5. As can be seen from FIG. 5, the relative content of IPNV-VP2 gene in CHSE-214 cells was 1.106 after 300. mu.g/mL of the kelp extract treated cells compared to the infected group, indicating that the kelp extract had no significant ability to inhibit intracellular replication of IPNV.
4.4 Release and Assembly of viruses
1.0X 10 CHSE-214 cells per well5cells/mL were plated in 6-well plates and cultured for 24 h. The culture medium was discarded and IPNV suspension was inoculated to an MOI of 1, and incubated at 15 ℃ in a 5% strength carbon dioxide incubator for 12 h. After discarding the virus suspension, the cell maintenance solution (1mL) containing 300. mu.g/mL of the Laminaria japonica extract was added to the CHSE-214 cells and incubated in a carbon dioxide incubator at 15 ℃ and 5% concentration for 30min, 60min, and after 90min, the supernatants were collected and treated under the same conditions without the Laminaria japonica extract and inoculated with IPNV of the CHSE-214 cells (infected group) as a control.
Determination of supernatant Virus Titers (TCID) Using the Reed-Muench method50). Namely, after the supernatant was continuously diluted 10 times, 100. mu.L of the above-mentioned diluted solution was added to each well containing 1.0X 10 of the supernatant4The cells were incubated in a 96-well plate containing 100. mu.L of CHSE-214 cells at 15 ℃ in a 5% carbon dioxide incubator for 7 days, and then the pathological changes of the cells were observed to calculate TCID50。
TABLE 4 Effect of Laminaria japonica extracts on IPNV Release
The results are shown in Table 4. As can be seen from Table 4, the virus titer in the supernatant was 10 when the 300. mu.g/mL kelp extract-treated CHSE-214 cells were incubated for 30min, 60min and 90min1.15TCID50/mL、102.2TCID50/mL、102.90TCID50mL, while the virus titer in the supernatant was 10 when the infected group was incubated without addition of the Laminaria japonica extract for 30min, 60min and 90min1.58TCID50/mL、102.66TCID50/mL、103.12TCID50and/mL. This result indicates that the kelp extract can inhibit the release of IPNV to some extent.
The kelp extract with obvious IPNV resistance is prepared, and the inhibition rate of the kelp extract with the concentration of 300 mu g/mL to IPNV reaches 68.84%. Through research and analysis on IPNV (infectious bronchitis Virus) resistance mechanism of the kelp extract, the kelp extract can play an antiviral role by blocking the adsorption and invasion of IPNV. The herba Zosterae Marinae extract is expected to be used as novel medicine/immunopotentiator for resisting IPNV.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
1. The application of the kelp in preparing virus inhibitors or antiviral drugs, wherein the virus is infectious pancreatic necrosis virus.
2. A kelp extract characterized by: the kelp extract is prepared by the method comprising the following steps:
1) preparation of crude extract of kelp: removing protein in the kelp leaching liquor to obtain kelp crude extract; the kelp extract is an extract obtained by carrying out enzymolysis and water extraction on kelp,
2) precipitating with ethanol, dialyzing the retentate, collecting the precipitate to obtain herba Zosterae Marinae extract, and purifying the herba Zosterae Marinae crude extract with dialysis bag method to obtain in-bag liquid.
3. The kelp extract according to claim 2, characterized in that: the kelp leaching liquor is prepared by the method comprising 1-1) and 1-2):
1-1) mixing the kelp with water to obtain kelp soaking liquid;
1-2) treating the kelp soak solution with cellulase, pectinase and papain to obtain water extract, filtering the water extract, collecting filtrate, centrifuging the filtrate, and collecting supernatant to obtain kelp extract.
4. The kelp extract according to claim 3, characterized in that: the step 1-2) of treating the kelp soak solution with cellulase, pectinase and papain to obtain a water extract comprises the following steps:
adjusting the pH value of the kelp soaking solution to 5.0, adding cellulase, and performing enzymolysis at 55 ℃ for 20min to obtain a cellulase hydrolysate; adjusting the pH value of the cellulose enzymolysis liquid to 4.2, adding pectinase, and carrying out enzymolysis for 2h at 50 ℃ to obtain pectin enzymolysis liquid; adjusting the pH value of the pectin enzymolysis liquid to 6.0, adding papain, carrying out enzymolysis at 50 ℃ for 2h, and then leaching at 75 ℃ for 3h to obtain the water extract.
5. The kelp extract according to any one of claims 2 to 4, wherein: the centrifugation condition in the 1-2) is 3500g centrifugation for 15 min.
6. The kelp extract according to any one of claims 2 to 5, which is characterized in that: the step 1) is to remove the protein in the kelp leaching liquor by adopting a sevage method, and the step of removing the protein in the kelp leaching liquor by adopting the sevage method comprises the step of centrifuging at 3500g for 10 min.
7. The kelp extract according to any one of claims 2 to 5, characterized in that: 2) the cut-off molecular weight of the dialysis solution in (1) is 3500 Da.
8. The kelp extract according to any one of claims 2 to 5, characterized in that: 2) the ethanol precipitation dialysis retentate collection of (1) comprises a step of centrifuging at 3500g for 10min to collect the precipitate.
9. Use of the kelp extract according to any one of claims 2 to 8 for the preparation of a virus inhibitor or an antiviral drug, characterized in that: the virus is infectious pancreatic necrosis virus.
10. A viral inhibitor and/or antiviral drug characterized by: the virus inhibitor and the antiviral drug contain the kelp extract according to any one of claims 2 to 8.
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| US6194192B1 (en) * | 1996-02-29 | 2001-02-27 | Takara Shuzo Co., Ltd. | Method of purifying and removing viruses |
| US20180289759A1 (en) * | 2015-04-08 | 2018-10-11 | Maqui New Life S.A. | Veterinary composition of marine algae and andrographis sp extracts, which can be used to treat infections in fish |
| CN108530552A (en) * | 2018-04-26 | 2018-09-14 | 河北中医学院 | The preparation of laminarin and application in preparation of anti-tumor drugs |
| CN110240660A (en) * | 2019-06-21 | 2019-09-17 | 福建福瑞康信息技术有限公司 | The extraction process of one Polysaccharides From Laminaria Japonica |
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