CN112062716A - Hyperforin derivatives, use thereof and antiviral preparations containing the same - Google Patents
Hyperforin derivatives, use thereof and antiviral preparations containing the same Download PDFInfo
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Abstract
The invention discloses hyperforin derivatives, application and an antiviral preparation containing the hyperforin derivatives, belonging to the technical field of antiviral preparations, wherein the hyperforin derivatives have the structure shown in the formula (I),
Description
Technical Field
The invention belongs to the technical field of antiviral preparations, and particularly relates to a hyperforin derivative, application and an antiviral preparation containing the hyperforin derivative.
Background
The Transmissible gastroenteritis of pig (TGE) is a highly contact infectious disease which is caused by Transmissible gastroenteritis virus (TGEV) and is characterized by serious diarrhea, vomiting and dehydration as clinical features, the TGEV is an RNA virus, pigs of all ages are susceptible, the death rate of newborn piglets is high (almost up to 100%), the death rate of piglets of more than 5 weeks is low, but the production performance of the infected piglets is reduced, the feed conversion rate is reduced, and great economic loss is caused to the pig industry. At present, no effective therapeutic medicine exists for the disease, and vaccine immunization is adopted as a main preventive measure, but the existing TGEV vaccine has certain defects in the aspects of immune effect and safety, so the disease can be only treated symptomatically. At present, no effective therapeutic medicine exists for the disease, and vaccine immunization is adopted as a main preventive measure, but the existing TGEV vaccine has certain defects in the aspects of immune effect and safety, so the disease can be only treated symptomatically. The application of antibiotics is easy to generate drug resistance and residue, the antibiotic residue not only causes the increase of the drug resistance, but also threatens the life safety of human beings, and the problem of antibiotic residue is taken as a technical trade barrier abroad, thereby restricting the export of animal food in China. Therefore, the development of natural, low-toxicity, drug-residue-free, green biologics is one of the directions for preventing and treating the disease.
Hyperforin (HF) is a fat-soluble component in Hypericum perforatum flowering branches, belongs to anthrone compounds in chemical structure, has various physiological activities, but is extremely sensitive to light, heat and oxygen, and is completely oxidized after being stored for several weeks or even days at normal temperature. The high instability of hyperforin has created a certain degree of difficulty in the intensive research and pharmaceutical development of this compound. Therefore, the structural modification and the reconstruction of the material can keep stable chemical characteristics under normal conditions, and the material has important significance.
Disclosure of Invention
One of the purposes of the invention is to provide a hyperforin derivative which has good antiviral capacity, has the effects of preventing and treating TGEV viral diseases and can obviously reduce the generation of TGEV infected pigs and the death rate of the TGEV infected pigs.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a hyperforin derivative has the structure shown in formula (I),
preferably, the hyperforin derivative inhibits transcription of the TGEV genome and expression of TGEV specific proteins in the cell.
Preferably, the hyperforin derivatives can up-regulate the expression levels of the relevant factors IFN-alpha, IFN-beta, IFN-gamma and IRF-3.
The hyperforin derivative has good antiviral capacity, can obviously inhibit the transcription of TGEV genome and the expression of TGEV specific protein in cells, and can increase the expression quantity of relevant factors IFN-alpha, IFN-beta, IFN-gamma and IRF-3, thereby playing a role in inhibiting the proliferation of TGEV, having obvious blocking effect on TGEV adsorbed cells and finally inhibiting TGEV from infecting the cells. Therefore, the hyperforin derivative has the effects of preventing and treating TGEV viral diseases, can obviously reduce the generation of TGEV infected pigs and the death rate of the TGEV infected pigs, and has good application prospect.
The invention also discloses a preparation method of the hyperforin derivative, which comprises the following steps,
s1: reacting nicotinamide with chloroacetyl chloride to generate an intermediate compound;
s2: the intermediate compound and hyperforin are subjected to substitution reaction to generate the hyperforin derivative.
Preferably, the molar ratio of nicotinamide to chloroacetyl chloride is 1: 2.8-3.2.
Preferably, the molar ratio of hyperforin to intermediate compound is 1: 1.05-1.15.
Preferably, the process for the preparation of hyperforin derivatives comprises,
s1: stirring nicotinamide, potassium carbonate and DMF until the nicotinamide, the potassium carbonate and the DMF are completely dissolved, adding dichloromethane, stirring at normal temperature, slowly dropwise adding chloroacetyl chloride under an ice bath condition, reacting overnight, and after the dichloromethane is dried in a spinning mode, pouring reactants into ice water to obtain an intermediate compound, wherein the intermediate compound is directly subjected to the next reaction without purification;
s1: heating and stirring the intermediate compound, potassium carbonate and DMF, slowly dropwise adding DMF containing the intermediate compound, heating and stirring, pouring the reactant into water, adding 6mol/L hydrochloric acid under stirring to adjust the pH to acidity, separating out a large amount of solids, performing suction filtration, washing with secondary water, and recrystallizing to obtain the hyperforin derivative.
More preferably, the process for the preparation of hyperforin derivatives comprises,
s1: stirring 1mmol of nicotinamide, 2.8-3.2mmol of potassium carbonate and 10-15mL of DMF until the nicotinamide, the potassium carbonate and the DMF are completely dissolved, adding 5-10mL of dichloromethane, stirring at normal temperature for 30-60min, slowly dropwise adding 2.8-3.2mmol of chloroacetyl chloride under an ice bath condition, reacting overnight, after the dichloromethane is dried in a spinning mode, pouring reactants into ice water to obtain an intermediate compound, wherein the yield is 50.73-52.24%, and the next reaction is directly carried out without purification;
s2: stirring 1mmol of hyperforin, 2.8-3.2mmol of potassium carbonate and 17-23mL of DMF at 80-90 ℃ for 45-75min, slowly dropwise adding 9-11mL of DMF containing 1.05-1.15mmol of intermediate compound, stirring at 100 ℃ for reaction for 4-6h, pouring the reactant into 280-320mL of deionized water, adding 6mol/L of hydrochloric acid under stirring to adjust the pH to acidity, separating out a large amount of solids, performing suction filtration, washing with deionized water for 2-4 times, and recrystallizing the DMF and methanol to obtain the hyperforin derivative, wherein the yield is 49.52-51.45%.
The invention also discloses the application of the hyperforin derivative in preparing antiviral preparations.
The invention also discloses the application of the hyperforin derivative in preparing an anti-TGEV preparation.
The invention also discloses an antiviral preparation containing the hyperforin derivative. The antiviral preparation has the effects of preventing and treating TGEV viral diseases, can remarkably reduce the generation of TGEV infected pigs and the death rate of the TGEV infected pigs, and has good application prospect.
In order to solve the technical problems, isosteviol is also added into the antiviral preparation, and can up-regulate PK-15 cell Bcl-2 gene transcription together with hyperforin derivatives, down-regulate BAX gene transcription and inhibit PK-15 cell apoptosis caused by TGEV, thereby protecting PK-15 cells from being damaged by TGEV, improving the resistance of the antiviral preparation to TGEV and reducing the death rate of pigs infected by TGEV. In addition, it together with hyperforin derivatives can further inhibit the proliferation of TGEV. Preferably, the ratio of hyperforin derivative to isosteviol is 100g to 1.5-4.5 g. More preferably, the hyperforin derivative and isosteviol act synergistically to achieve at least one of the following when the antiviral formulation is administered to a subject pig: up-regulates PK-15 cell Bcl-2 gene transcription, and down-regulates BAX gene transcription.
More preferably, the hyperforin derivative and isosteviol act synergistically to achieve at least one of the following when the antiviral formulation is administered to a subject pig: up-regulates PK-15 cell Bcl-2 gene transcription, and down-regulates BAX gene transcription.
More preferably, the hyperforin derivative and isosteviol act synergistically to achieve at least one of the following: inhibiting the relative expression of TGEV genome and the expression of TGEV specific protein in cells.
Preferably, the hyperforin derivative and isosteviol act synergistically to achieve at least one of the following: the expression levels of IFN-alpha, IFN-beta, IFN-gamma and IRF-3 as the up-regulation correlation factors.
The invention also discloses the application of the hyperforin derivative and isosteviol in preparing antiviral preparations.
The invention also discloses the application of the hyperforin derivative and isosteviol in preparing anti-TGEV preparations.
The invention also discloses application of the antiviral preparation in preparing feed additives.
The invention also aims to provide the traditional Chinese medicine additive for the feed, which has the advantages of improving the immunity, improving the feed conversion rate, improving the daily gain of growing and fattening livestock, along with low cost, meat fragrance of the livestock and capability of meeting the national green inspection standard.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a Chinese medicinal additive for feed comprises radix Codonopsis, radix Paeoniae alba, Poria, semen Pruni, rhizoma Atractylodis, Bulbus Allii, herba Artemisiae Annuae, and fructus crataegi.
In the traditional Chinese medicine additive, the codonopsis pilosula has the effects of tonifying qi and nourishing blood, strengthening spleen and stomach and removing free radicals; white peony root, radix paeoniae alba, liver softening, blood harmonizing, blood nourishing; poria cocos, poria cocos; cang Zhu eliminates dampness and strengthens spleen, dispels wind and dispels cold. The garlic powder has the functions of sterilizing and detoxifying and harmonizing spleen and stomach. The bunge cherry seeds have the functions of removing dampness, transporting water, strengthening spleen and stomach; the hawthorn can promote digestion and stimulate appetite. Increase appetite and promote absorption. Sweet wormwood herb, herba Artemisiae Annuae, antibacterial, anticancer, cooling and fire fighting. The eight Chinese herbal medicines are used for strengthening the spleen and stomach, promoting absorption, increasing palatability, increasing appetite, increasing absorption, improving immunity and removing various harmful germs and virus microorganisms in a broad spectrum through monarch, minister, assistant and guide; and the required advantages and functions are enhanced, and simultaneously, the functions are complemented, and the defects of heat bias, compensation bias and insufficient blood circulation can be overcome. In a word, the traditional Chinese medicine additive has the advantages of improving immunity, improving feed conversion rate, improving daily gain of growing and fattening pigs, along with low cost, mellow meat flavor and capability of reaching the national green inspection standard.
Preferably, the traditional Chinese medicine additive for the feed comprises, by weight, 0.8-1.2 parts of codonopsis pilosula, 0.7-1.3 parts of white paeony root, 1.3-1.7 parts of poria, 0.8-1.2 parts of bunge cherry seed, 1.5-2.1 parts of rhizoma atractylodis, 0.1-0.5 part of garlic, 1.5-2.7 parts of sweet wormwood herb and 1.2-2.1 parts of hawthorn.
Preferably, the Chinese medicinal additive is sterilized. More preferably, the Chinese medicinal additive is 7-9kGy Co60And (5) radiation sterilization.
Preferably, the traditional Chinese medicine additive also comprises the antiviral preparation. The addition of the antiviral preparation does not affect the original performance of the traditional Chinese medicine additive, and the traditional Chinese medicine additive has the effects of preventing and treating TGEV virus diseases, can obviously reduce the generation of TGEV infected pigs, and further improves the growth state of the pigs.
More preferably, the amount of antiviral agent added is 0.2-5g per 1000g of the pharmaceutical additive.
The invention also discloses a premix which contains 5-10 wt% of the traditional Chinese medicine additive.
Preferably, the premix also comprises 12-20 parts of bone meal, 1-2 parts of chromium picolinate, 1-2 parts of amino acid zinc, 0.8-1.5 parts of amino acid copper, 1-2 parts of amino acid iron, 0.3-0.6 part of yeast selenium, 2.2-3.1 parts of phytase, 4-9 parts of multivitamin, 3-5 parts of magnesium sulfate, 0.3-0.5 part of phytase and 30-40 parts of soybean meal according to parts by weight. The premix of the invention has six major balances of protein, energy, vitamins, trace elements, calcium, phosphorus, acid and alkali, and the like, and achieves the aims of strengthening the spleen of a fattening pig, low feed conversion ratio, fast weight gain, mellow meat flavor, low cost and income increase of pig farmers by overlapping traditional Chinese medicine additives which can strengthen the stomach and promote absorption and conversion, thereby achieving the national green inspection standard.
The invention also discloses a traditional Chinese medicine ecological feed, wherein each 1000 kg of the traditional Chinese medicine ecological feed comprises 50-100 kg of premix, 180 kg of bean pulp, 700 kg of corn meal, 40-60 kg of bran and 3-7 kg of soybean oil or grease.
The traditional Chinese medicine ecological feed is fed from 80 jin to the age of slaughtering pigs, the feed-meat ratio is 2.2-3.0, the lean meat percentage is 62-64%, and the daily gain is 2.2-2.7 jin. According to the traditional Chinese medicine ecological feed, the old feed 1/3 is adopted for changing materials, the materials are gradually added for transition, the 1 st to 2 nd are matched according to the ratio 1/3, the ratio 1/3 is increased at the 3 rd after the material change under the condition that the feed intake of pigs is normal and the feces are normal and not diluted, the 4 th d is good in feeding and not diluted, the materials are completely changed, and the whole process needs a 4d transition period. If the phenomenon that the excrement of the pork chop is thin is found in the process of changing the feed, the feed changing transition can be prolonged by 1-2 days until the excrement is formed and is completely changed.
The invention adopts nicotinamide to modify hyperforin, thereby having the following beneficial effects: the hyperforin derivative has good antiviral capacity, can obviously inhibit the transcription of TGEV genome and the expression of TGEV specific protein in cells, and can increase the expression quantity of relevant factors IFN-alpha, IFN-beta, IFN-gamma and IRF-3, thereby playing the role of inhibiting the proliferation of TGEV, having obvious blocking effect on TGEV adsorbed cells, finally inhibiting TGEV infected cells, being used for preparing antiviral preparations and having good application prospect. Therefore, the hyperforin derivative has good antiviral capacity, has the effects of preventing and treating TGEV viral diseases, and can obviously reduce the generation of TGEV infected pigs and the death rate of the TGEV infected pigs.
Drawings
FIG. 1 shows the inhibition of TGEV by hyperforin derivatives of example 2;
FIG. 2 shows the qRT-PCR results of TGEV N gene in example 2;
FIG. 3 shows the results of Westernblot assay of TGEV-specific proteins in example 2;
FIG. 4 shows the qRT-PCR results of IFN- α, IFN- β, IFN- γ and IRF-3 genes in example 2;
FIG. 5 shows the results of inhibition of TGEV by the antiviral agent in example 5;
FIG. 6 shows the qRT-PCR results of TGEV N gene in example 5;
FIG. 7 shows the qRT-PCR results for Bcl-2 and BAX genes in example 5.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
example 1:
a process for the preparation of hyperforin derivatives, comprising,
s1: stirring 1mmol of nicotinamide, 3mmol of potassium carbonate and 12mL of DMF until the nicotinamide, the potassium carbonate and the DMF are completely dissolved, adding 8mL of dichloromethane, stirring for 45min at normal temperature, slowly dropwise adding 3mmol of chloroacetyl chloride under an ice bath condition, reacting overnight, drying the dichloromethane by spinning, and pouring reactants into ice water to obtain an intermediate compound, wherein the yield is 51.47%, and the next reaction is directly carried out without purification;
s2: stirring 1mmol of hyperforin, 3mmol of potassium carbonate and 20mL of DMF at 85 ℃ for 60min, slowly dropwise adding 10mL of DMF containing 1.1mmol of intermediate compound, stirring at 110 ℃ for reaction for 5h, pouring the reaction product into 300mL of deionized water, adding 6mol/L hydrochloric acid under stirring to adjust the pH value to acidity, separating out a large amount of solid, performing suction filtration, washing with deionized water for 3 times, and recrystallizing DMF and methanol to obtain the hyperforin derivative, wherein the yield is 51.26%.
Process for the preparation of hyperforin derivatives1Compared with hyperforin, the H NMR spectrum shows that 9.89ppm of chemical shift of amido hydrogen and 7.75-8.86ppm of chemical shift of benzene ring hydrogen show that the hyperforin derivative is successfully synthesized.
Example 2:
determination of anti-TGEV Effect of hyperforin derivatives
1. Material
Porcine kidney cells (PK-15) were purchased from the type culture Collection of the Chinese academy of sciences, accession number: GDC 061. Transmissible gastroenteritis virus (TGEV) was purchased from ATCC, accession No.: VR-763.
DMEM liquid medium purchased from Hyclone, USA, stored at 4 ℃ in refrigerator; fetal bovine serum, trypsin, purchased from Gibco, usa; 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) are all products of Sigma company of America; RNA extraction reagent TransZol and RT-PCR reverse transcription kit are purchased from Beijing all-style gold biotechnology limited company; ultra SYBR texture available from Beijing kang, century Biotechnology Ltd; corresponding secondary antibodies (goat anti-rabbit IgG, rabbit anti-goat IgG) labeled with horseradish peroxidase were purchased from beijing kang, a century biotechnology limited.
2. Instrument for measuring the position of a moving object
TI-DH fluorescence inversion microscope (Nikon, Japan); medical decontamination benches (suzhou decontamination plant); ultra pure water instruments (milopore corporation, usa);full-automatic enzyme labeling instrument and CO2A constant temperature incubator (Thermo corporation, usa); cell culture plates (corning, usa); eight-way tubes (Orcugen corporation, usa); refrigerated centrifuge (Eppendorf, germany); general PCR machine, iQ5 fluorescent quantitative PCR machine (Bio-Rad, USA). Gel electrophoresis apparatus (model DYY-III, manufactured by six instruments, Beijing), transfer electrophoresis tank (VE-186, Shanghai Tianneng).
TGEV half-cell infectious amount (TCID)50) Measurement of (2)
Using Reed-Muench method to determine, TGEV virus liquid is respectively diluted 10 times with cell maintenance liquid (DMEM culture liquid containing 2% serum) to 10 times-1,10-2,10-3,…,10-7A total of 7 dilutions. Each diluted virus suspension was inoculated into 100. mu.L of a 96-well plate in which a monolayer of porcine kidney (PK-15) cells was grown, and 8 wells were provided for each suspension. 37 ℃ and 5% CO2Incubating for 2 hr, collecting virus solution, adding cell growth maintenance solution, standing at 37 deg.C and 5% CO2Culturing in incubator, observing and recording cytopathic effect (CPE) every 12h, continuously observing for 96h, and calculating Titer (TCID) of half cytopathic effect of virus according to Reed-Muench method50)。
lgTCID50=L-D(S-0.5);
In the formula: l-logarithm of highest dilution, difference between D-logarithms of dilutions, sum of S-positive well ratios.
Calculating TCID of TGEV according to Reed-Muench method50Is 10-6.3100 μ L of diluted virus solution.
4. Determination of maximum nontoxic concentration of hyperforin derivative on porcine kidney PK-15 cells
The cell suspension was diluted to 1X 10 using the MTT assay5Inoculating each/mL into 96-well cell culture plate, growing a monolayer, adding hyperforin derivative substance with concentration of 10-1-10-8g/mL maintenance solution (serum concentration 2%), 100 μ L per well, repeating 6 wells per concentration, setting normal cell control, culturing in incubator, observing cell morphology change every day, detecting light absorption value (OD value) of cell by MTT method after 72 hr, calculating PK-15 cell survival rate, and screening hyperforin derivativeMaximum non-toxic concentration of organisms to PK-15 cells.
Cell survival (%) — OD value of each concentration drug group/OD value of blank group × 100%.
As shown in Table 1, it can be seen from Table 1 that the concentration of the hyperforin derivative is 10-8-10-7g/L is OD570The value is higher than that of the blank group, and the cell life is maintained or the cell growth is promoted; the difference between the subsequent mass concentration groups is not significant until the mass concentration is 10-3g/L is OD570The value and cell survival rate are close to those of the control group, the cell survival rate of PK-15 cells reaches more than 94 percent, and the maximum nontoxic mass concentration of the coptis extract is determined to be 10-3g/L。
TABLE 1 toxicity of hyperforin derivatives on PK-15 cells at different concentrations
Drug concentration (g/mL) | OD570 | Cell survival rate (%) |
Blank space | 0.68±0.04 | 100.00 |
10-8 | 0.69±0.05 | 101.53 |
10-7 | 0.69±0.03 | 101.47 |
10-6 | 0.67±0.06 | 98.53 |
10-5 | 0.66±0.02 | 97.06 |
10-4 | 0.65±0.04 | 95.59 |
10-3 | 0.64±0.05 | 94.12 |
10-2 | 0.53±0.03 | 77.83 |
10-1 | 0.47±0.02 | 69.12 |
5. In vitro inhibition of TGEV by hyperforin derivatives
Dilution of PK-15 cell suspension to 1X 105each/mL cell is inoculated into a 96-well cell culture plate, after the cell abundance reaches 80%, a blank group, a virus group, a drug control group, 3 test groups and 2 comparison groups are respectively arranged, each group has 6 wells, and 200 mu L of maintenance liquid is added into a normal cell control group to serve as a control; adding 100 mu L of virus solution into virus group cells for 2h, and adding maintenance solution for culturing for 72h as virus control; adding 200 μ L of the maximum nontoxic concentration drug as drug control group; test 1 group (drug administration after drug administration)) Firstly, viruses and drugs with the maximum non-toxic concentration are acted for 2 hours at 4 ℃, and then 200 mu L of the viruses and the drugs are added to cells to be cultured for 72 hours; test 2 (mixed drug and toxin infection) cells are infected with virus for 2h, then virus liquid is discarded, and 200 mu L of drug with the maximum non-toxic concentration is added to be cultured for 72 h; test 3 groups (first administration and then inoculation) of cells are firstly added with 200 muL of medicine with the maximum nontoxic concentration for 48h, then 100 muL of virus is added for infection for 2h, virus solution is discarded, and 200 muL of maintenance solution is added for culture for 24 h; comparative group 1 cells were first added to a final concentration of 10-3Allowing 200 μ L of hyperforin in g/L to act for 48h, adding 100 μ L of virus to infect for 2h, discarding virus solution, adding 200 μ L of maintenance solution, and culturing for 24 h; comparative 2 group cells were first added to a final concentration of 10-3Allowing 200 mu L of ribavirin of g/L to act for 48h, adding 100 mu L of virus to infect for 2h, discarding virus solution, adding 200 mu L of maintenance solution, and culturing for 24 h. After the experiment is finished, the culture solution of each group of cells is discarded, 20 mu L of MTT is added in the dark, the cells are incubated for 4h at 37 ℃, 50 mu L of dimethyl sulfoxide is added, the mixture is fully mixed and dissolved, the OD value is detected at the wavelength of 570nm, and the PK-15 cell viability is calculated.
FIG. 1 shows the inhibition results of hyperforin derivatives on TGEV, and it can be seen that the cell survival rates of PK-15 in the test groups 1-3 are higher than those in the virus group, and the cell survival rates of the comparison group 1 and the comparison group 2 are far lower than those in the test group 3, which indicates that the hyperforin derivatives have the inhibition effect on TGEV and the effect is better than that of hyperforin and ribavirin; the survival rate of cells in the test 3 group (drug administration followed by drug administration) is highest, the survival rate of cells in the test 1 group (drug administration followed by drug administration) is lowest, and the survival rate of cells in the test 2 group (drug and toxin mixed induction) is lowest, which preliminarily shows that the drug administration mode of drug administration followed by drug administration has the best effect and the effect of preventing TGEV from infecting PK-15 cells is good, namely, the hyperforin derivative can inhibit the adsorption effect of TGEV on PK-15 cells and keep the activity of the cells, thereby playing the effect of resisting TGEV.
6. Inhibition of TGEV by hyperforin derivatives
Grouping and testing 5 hyperforin derivative for inhibiting TGEV in vitro, extracting virus RNA, electrophoretically detecting total RNA integrity, measuring RNA purity with ultraviolet spectrophotometer, reverse transcribing into cDNA,RT-PCR amplification was performed using TGEV gene primers (as in Table 2) to detect TGEV transcript levels in 25. mu.L reaction: cDNA2.0 μ L, Mix 12.5 μ L, upstream and downstream primers 1.0 μ L each, RNase free dH2Supplementing O to 25 mu L; reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 95 ℃ for 45s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 45s for 35 cycles; further extension at 72 ℃ for 10min and reaction at 4 ℃ was completed. Beta-actin is used as an internal reference, 3 replicates are set for each sample, and finally, the results are analyzed by taking the average value. The gene expression intensity in the RT-PCR result is expressed by absolute copy number/beta-actin absolute copy number respectively. The expression amount of the virus group was set to 1 ×, and the expression change level of each gene from the normal group was calculated.
TABLE 2 primer sequences of TGEV N genes
FIG. 2 is the qRT-PCR result of TGEV N gene, it can be seen that the transcription factor of TGEV N gene of test 1-3 groups is lower than that of virus group, and the transcription factor of TGEV N gene of comparative 1 group and comparative 2 groups is far lower than that of test 3 group, which shows that hyperforin derivative can significantly reduce TGEV N mRNA relative expression level at transcription level, and the effect is better than that of hyperforin and ribavirin; the transcription multiple of the TGEV N genes in the test 3 groups is the lowest, the transcription multiple of the TGEV N genes in the test 1 group is the lowest, and the transcription multiple of the TGEV N genes in the test 2 groups is the highest, which shows that the effect of inhibiting the proliferation of the TGEV by a drug administration mode of firstly adding drugs and then receiving toxins is the best.
7. Inhibition of TGEV-specific proteins by hyperforin derivatives
Grouping and testing the in-vitro inhibition effect of the hyperforin derivative on TGEV, collecting virus supernatant after the test, extracting total protein, placing in 100 ℃ water bath for 3min, adding 15% SDS-PAGE, and performing electrophoresis at 120V for about 2 h. Proteins were transferred to nitrocellulose membranes at 4 ℃ under a voltage of 90V. The membrane is placed in a plate, PBST is washed for 3 times, then the membrane is blocked by 5% skimmed milk solution for 2h, then PBST is washed for 3 times, then primary antibody (anti-TGEV polyclonal antibody, diluted by 1: 200) skimmed milk powder solution (with the concentration of 10g/L) is added into the plate, the membrane is incubated overnight at 4 ℃, secondary antibody (diluted by 10g/L skimmed milk powder 1: 1000) is incubated for 1h at room temperature, luminescence is carried out in a dark room, after images are scanned, the absorbance analysis result of each strip is determined by using Quantity One software, and the expression intensity of the protein is expressed by using the ratio of the LRP strip to the GAPDH absorbance value. The experiment was repeated 3 times.
FIG. 3 shows the results of Westernblot assay of TGEV-specific proteins, wherein, the a-blank group, the b-virus group, the c-test 1 group, the d-test 2 group, the e-test 3 group, the f-comparison 1 group, and the g-comparison 2 group, it can be seen that the TGEV-specific proteins in the test 1-3 groups are all expressed in amounts lower than those in the virus group, and the TGEV-specific proteins in the test 3 groups are expressed in amounts far lower than those in the comparison 1 group and significantly lower than those in the comparison 2 group, which indicates that hyperforin derivatives can express TGEV-specific proteins in cells at a transcription level significantly better than hyperforin and have better effects than ribavirin; the expression level of the TGEV-specific protein in the test 3 group is the lowest, the expression level of the TGEV-specific protein in the test 1 group is the second lowest, and the expression level of the TGEV-specific protein in the test 2 group is the lowest, which shows that the effect of inhibiting the proliferation of the TGEV is the best in a drug administration mode of adding drugs first and then poisoning.
8. Modulation of cellular antiviral associated factors by hyperforin derivatives
Grouping and testing treatment are carried out on the in-vitro inhibition effect of the hyperforin derivative on TGEV, after the test is finished, cell RNA is extracted and is reversely transcribed into cDNA, and the expression quantity change of related factors IFN-alpha, IFN-beta, IFN-gamma and IRF-3 is detected. The transcription level of IFN-alpha, IFN-beta, IFN-gamma and IRF-3 gene primers (shown in the table 3) is detected by performing RT-PCR amplification, and the reaction system is 25 mu L: 2.0 μ L cDNA, 12.5 μ L Mix, 1.0 μ L upstream and downstream primers, respectively, adding RNase free dH2Supplementing O to 25 mu L; reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 95 ℃ for 45s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 45s for 35 cycles; further extension at 72 ℃ for 10min and reaction at 4 ℃ was completed. Beta-actin is used as an internal reference, 3 replicates are set for each sample, and finally, the results are analyzed by taking the average value. The gene expression intensity in the RT-PCR result is respectively expressed as absolute copy number/beta-actin absolute copyAnd (4) representing the number. The expression amount of the virus group was set to 1 ×, and the expression change level of each gene from the normal group was calculated.
TABLE 3 primer sequences of FN-alpha, IFN-beta, IFN-gamma and IRF-3 genes
FIG. 4 shows the qRT-PCR results of IFN-alpha, IFN-beta, IFN-gamma and IRF-3 genes, and it can be seen that the transcription factor of IFN-alpha, IFN-beta, IFN-gamma and IRF-3 in the test groups 1-3 is higher than that of the virus group, and the transcription factor of IFN-alpha, IFN-beta, IFN-gamma and IRF-3 in the comparison group 1 is much lower than that in the test group 3, which indicates that hyperforin derivatives can up-modulate the expression level of the relevant factors IFN-alpha, IFN-beta, IFN-gamma and IRF-3, thereby exerting the effect of inhibiting TGproliferation, while hyperforin EV has little effect on the expression level of the relevant factors IFN-alpha, IFN-beta, IFN-gamma and IRF-3; the expression quantity of the related factors IFN-alpha, IFN-beta, IFN-gamma and IRF-3 in the test group 3 is higher than that of the test group 1 and the test group 2, which shows that the effect of up-regulating the expression quantity of the related factors IFN-alpha, IFN-beta, IFN-gamma and IRF-3 in a drug administration mode of firstly adding drugs and then receiving the drugs is the best, and the method can be used for preparing antiviral preparations.
Example 3:
an antiviral preparation comprising the hyperforin derivative of example 1.
Example 4:
an antiviral preparation comprising the hyperforin derivative of example 1 and isosteviol in an amount ratio of 100g to 2.2 g.
Example 5:
1. in vitro inhibition of TGEV by antiviral agents
Dilution of PK-15 cell suspension to 1X 105each/mL of the cells is inoculated into a 96-well cell culture plate, after the cell abundance reaches 80%, a blank group, a virus group, a drug control group and 2 test groups are respectively arranged, each group has 6 wells, and 200 mu L of maintenance liquid is added into a normal cell control group to serve as a control; adding 100 mu L of virus solution into virus group cells for 2h, and adding maintenance solution for culturing for 72h as virus control; drug controlsGroup addition 10-3200 μ L of drug at g/L concentration as drug control; test 1 group cells were first added to a final concentration of 10-3Treating the antiviral preparation of example 3 with concentration of g/L at 200 μ L for 48h, adding virus at 100 μ L for infection for 2h, discarding virus solution, adding maintenance solution at 200 μ L, and culturing for 24 h; experiment 2 group cells were first added to a final concentration of 10-3The antiviral agent of example 4 at a concentration of g/L, i.e., 200. mu.L, was allowed to act for 48 hours, then 100. mu.L of the virus was added to infect the cells for 2 hours, the virus solution was discarded, and 200. mu.L of the maintenance solution was added thereto and cultured for 24 hours. After the experiment is finished, the culture solution of each group of cells is discarded, 20 mu L of MTT is added in the dark, the cells are incubated for 4h at 37 ℃, 50 mu L of dimethyl sulfoxide is added, the mixture is fully mixed and dissolved, the OD value is detected at the wavelength of 570nm, and the PK-15 cell viability is calculated.
FIG. 5 shows the inhibition results of antiviral agent on TGEV, and it can be seen that the survival rate of PK-15 cells in test 2 group is higher than that in test 1 group, which shows that hyperforin derivative and isosteviol together can further inhibit the adsorption of TGEV on PK-15 cells, and maintain the activity of cells, thereby playing the effect of anti-TGEV.
2. Inhibition of TGEV by antiviral agents
Grouping and testing treatment are carried out in the same way as the step 1, the in vitro inhibition effect of the antiviral preparation on the TGEV, and after the test is finished, RT-PCR amplification is carried out to detect the transcription level of the TGEV by referring to the method 6, the inhibition effect of the hyperforin derivative on the TGEV in the example 2.
FIG. 6 is the qRT-PCR results of TGEV N gene, and it can be seen that the transcription fold of TGEV N gene in test 2 group is lower than that in test 1 group, which indicates that hyperforin derivative and isosteviol together can further reduce the relative expression level of TGEV N mRNA significantly at the transcription level.
3. Modulation of Bcl-2 and BAX genes in cells following TGEV action by antiviral agents
Grouping and testing treatment are carried out on the in-vitro inhibition effect of the antiviral preparation on TGEV, after the test is finished, 0.25% pancreatin is used for digestion, DMEM culture solution containing 10% calf serum is used for stopping digestion, cells are collected by centrifugation, cell RNA is extracted and is reversely transcribed into cDNA, and the expression quantity change of Bcl-2 and BAX genes is detected. RT-PCR amplification Using Bcl-2 and BAX Gene primers (see Table 4)Their transcription level was measured in a reaction system of 25. mu.L: 2.0 μ L cDNA, 12.5 μ L Mix, 1.0 μ L upstream and downstream primers, respectively, adding RNase free dH2Supplementing O to 25 mu L; reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 95 ℃ for 45s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 45s for 35 cycles; further extension at 72 ℃ for 10min and reaction at 4 ℃ was completed. Beta-actin is used as an internal reference, 3 replicates are set for each sample, and finally, the results are analyzed by taking the average value. The gene expression intensity in the RT-PCR result is expressed by absolute copy number/beta-actin absolute copy number respectively. The expression amount of the virus group was set to 1 ×, and the expression change level of each gene from the normal group was calculated.
TABLE 4 primer sequences for Bcl-2 and BAX genes
FIG. 7 shows the qRT-PCR results of Bcl-2 and BAX genes, and it can be seen that the transcription multiples of Bcl-2 gene in test 2 group are all higher than that in test 1 group, and the transcription multiples of BAX gene in test 2 group are all lower than that in test 1 group, which indicates that hyperforin derivative and isosteviol can up-regulate the transcription of PK-15 cell Bcl-2 gene, down-regulate the transcription of BAX gene, and inhibit the PK-15 cell apoptosis caused by TGEV, thereby protecting PK-15 cell from TGEV damage, and improving the resistance of antiviral agent to TGEV.
Example 6:
1. therapeutic test of antiviral agent for transmissible gastroenteritis of swine
Test animals and groups: 150 sick pigs suffering from porcine transmissible gastroenteritis are selected, the number of the sick pigs is about 30kg, the 150 sick pigs are randomly divided into 2 treatment groups and a control group by adopting a complete random digital table method, each group comprises 50 sick pigs, the comparison difference of general data of the sick pigs in each group has no statistical significance (P is more than 0.05), and the sick pigs have comparability. Treatment 1 group was treated with the antiviral preparation of example 3, treatment 2 group was treated with the antiviral preparation of example 4, and the control group was not administered.
The treatment method comprises the following steps: treatment 1 group: feeding 1.2g of the antiviral preparation of example 3 to each pig every day for 5 days; treatment 2 groups: each pig was fed 1.2g of the antiviral preparation of example 4 daily for 5 consecutive days; control group: no drug was administered.
And (3) test results: the results of the test for treating transmissible gastroenteritis of swine with the antiviral agent are shown in table 5, and it can be seen that the treatment of 1 group and the treatment of 2 groups have the treatment effect on transmissible gastroenteritis of swine, wherein the treatment of 2 groups has a better effect on transmissible gastroenteritis of swine. This indicates that hyperforin derivatives have a better therapeutic effect on transmissible gastroenteritis of swine, and hyperforin derivatives and isosteviol used together have a better therapeutic effect on transmissible gastroenteritis of swine.
TABLE 5 therapeutic test results of antiviral preparations on transmissible gastroenteritis of swine
2. Prevention test of antiviral preparation against transmissible gastroenteritis of swine
Test animals and groups: 150 healthy pigs of about 30kg are selected, a complete random digital table method is adopted to randomly divide the 150 sick pigs into 2 prevention groups and control groups, each group comprises 50 pigs, the comparison difference of general data of the pigs in each group has no statistical significance (P is more than 0.05), and the pigs have comparability. Wherein, the group for prevention 1 was fed the antiviral preparation of example 3, the group for prevention 1 was fed the antiviral preparation of example 4, and the control group was not administered.
The treatment method comprises the following steps: prevention group 1: feeding 1.2g of the antiviral preparation of example 3 to each pig every day for 5 days; prevention 2 groups: each pig was fed 1.2g of the antiviral preparation of example 4 daily for 5 consecutive days; control group: no drug was administered.
And (3) test results: the incidence rate of preventing the transmissible gastroenteritis of the pigs in the group 1 is 4.6 percent, the incidence rate of preventing the transmissible gastroenteritis of the pigs in the group 2 is 0.7 percent, the incidence rate of the control group is 77.3 percent, the spirit and the appetite of the pigs in the prevention group are better than those of the control group, and the disease symptoms are lighter than those of the control group. The cure rate of the prevention group is 99.9 percent after the treatment is given after the disease attack. This shows that hyperforin derivatives have a better preventive effect on transmissible gastroenteritis of swine, and hyperforin derivatives and isosteviol used together have a better preventive effect on transmissible gastroenteritis of swine.
Example 7:
a traditional Chinese medicine additive for feed comprises, by weight, 1 part of radix codonopsis pilosulae, 1 part of white peony root, 1.5 parts of poria, 1 part of bunge cherry seed, 1.7 parts of rhizoma atractylodis, 0.3 part of garlic, 2 parts of sweet wormwood herb and 1.5 parts of hawthorn. The Chinese medicine additive is processed by 8kGy Co60And (5) radiation sterilization.
The premix comprises 16 parts by weight of bone meal, 1.5 parts by weight of chromium picolinate, 1.5 parts by weight of zinc amino acid, 1.1 parts by weight of copper amino acid, 1.4 parts by weight of ferric amino acid, 0.4 part by weight of selenium yeast, 2.8 parts by weight of phytase, 7 parts by weight of multivitamin, 4 parts by weight of magnesium sulfate, 0.5 part by weight of phytase and 38 parts by weight of soybean meal, and contains 7 wt% of the traditional Chinese medicine additive.
A Chinese medicine ecological feed, every 1000 kg of which comprises 80 kg of premix, 195 kg of soybean meal, 670 kg of corn flour, 50 kg of bran and 5 kg of soybean oil.
Example 8:
a traditional Chinese medicine additive for feed comprises 1 part of radix codonopsitis, 1 part of white peony root, 1.5 parts of poria, 1 part of bunge cherry seed, 1.7 parts of rhizoma atractylodis, 0.3 part of garlic, 2 parts of sweet wormwood herb and 1.5 parts of hawthorn by weight, and the addition amount of the antiviral preparation in the embodiment 3 in each 1000g of the traditional Chinese medicine additive is 0.5 g. The Chinese medicine additive is processed by 8kGy Co60And (5) radiation sterilization.
The premix comprises 16 parts by weight of bone meal, 1.5 parts by weight of chromium picolinate, 1.5 parts by weight of zinc amino acid, 1.1 parts by weight of copper amino acid, 1.4 parts by weight of ferric amino acid, 0.4 part by weight of selenium yeast, 2.8 parts by weight of phytase, 7 parts by weight of multivitamin, 4 parts by weight of magnesium sulfate, 0.5 part by weight of phytase and 38 parts by weight of soybean meal, and contains 7 wt% of the traditional Chinese medicine additive.
A Chinese medicine ecological feed, every 1000 kg of which comprises 80 kg of premix, 195 kg of soybean meal, 670 kg of corn flour, 50 kg of bran and 5 kg of soybean oil.
Example 9:
a Chinese medicinal additive for feed comprises radix Codonopsis 1 part, radix Paeoniae alba 1 part, Poria 1.5 part, semen Pruni 1 part, rhizoma Atractylodis 1.7 parts, Bulbus Allii 0.3 part, herba Artemisiae Annuae 2 parts, and fructus crataegi 1.5 parts by weightThe antiviral preparation of example 4 was added in an amount of 0.5g per 1000g of the pharmaceutical additive. The Chinese medicine additive is processed by 8kGy Co60And (5) radiation sterilization.
The premix comprises 16 parts by weight of bone meal, 1.5 parts by weight of chromium picolinate, 1.5 parts by weight of zinc amino acid, 1.1 parts by weight of copper amino acid, 1.4 parts by weight of ferric amino acid, 0.4 part by weight of selenium yeast, 2.8 parts by weight of phytase, 7 parts by weight of multivitamin, 4 parts by weight of magnesium sulfate, 0.5 part by weight of phytase and 38 parts by weight of soybean meal, and contains 7 wt% of the traditional Chinese medicine additive.
A Chinese medicine ecological feed, every 1000 kg of which comprises 80 kg of premix, 195 kg of soybean meal, 670 kg of corn flour, 50 kg of bran and 5 kg of soybean oil.
Example 10:
feeding test of traditional Chinese medicine ecological feed
400 healthy pigs of about 40kg are selected, the 400 sick pigs are randomly divided into 3 test groups and control groups by adopting a complete random digital table method, each group has 100 pigs, the comparison difference of the general data of the sick pigs in each group has no statistical significance (P is more than 0.05), and the sick pigs have comparability. Wherein, the experiment 1 group is fed with the traditional Chinese medicine ecological feed of the embodiment 7, the experiment 2 group is fed with the traditional Chinese medicine ecological feed of the embodiment 8, the experiment 3 group is fed with the traditional Chinese medicine ecological feed of the embodiment 9, and the control group is fed with the common feed (each 1000 kg of the feed comprises 80 kg of premix (by weight, 16 parts of bone meal, 1.5 parts of chromium picolinate, 1.5 parts of zinc amino acid, 1.1 parts of copper amino acid, 1.4 parts of ferric amino acid, 0.4 part of selenium yeast, 2.8 parts of phytase, 7 parts of vitamin complex, 4 parts of magnesium sulfate, 0.5 part of phytase, 38 parts of soybean meal), 195 kg of soybean meal, 670 kg of corn flour, 50 kg of bran and 5 kg of soybean oil). Feeding till slaughtering. Wherein, the old feed 1/3 is adopted to change the feed gradually, the 1 st to the 2 nd are mixed according to the ratio of 1/3, when the feed intake of the pigs is normal and the feces are not diluted normally, the 3 rd after the feed change is added with the ratio of 1/3, the 4 th has good feed and is not diluted, the whole process needs a 4d transition period. If the phenomenon that the excrement of the pork chop is thin is found in the process of changing the feed, the feed changing transition can be prolonged by 1-2 days until the excrement is formed and is completely changed. After the feeding test is finished, fasting is carried out for 24 hours under free drinking water, slaughtering is carried out, the left half carcass is stripped and divided into four tissues of bones, skin, muscles and fat, weighing is carried out respectively, and then the lean meat percentage is calculated; meanwhile, the pork samples of the group 1 of the test are qualified according to the NY/T2799-2015 standard.
The feeding results are shown in table 6, and the detection results of the pork samples in the group of experiment 1 are shown in table 7, so that the traditional Chinese medicine ecological feed in the examples 7-9 can effectively improve the daily gain of growing-finishing pigs, enables the pigs to grow faster, enables the pigs to enter and exit from the pigsty in advance, is low in feed consumption, low in feed conversion ratio and high in lean meat percentage, is qualified according to the NY/T2799 plus 2015 standard, and belongs to green food pork.
TABLE 6 feeding test of Chinese medicinal ecological feed
Test 1 | Test | 2 groups | Test 3 groups | Control group | |
Example number (head) | 100.00 | 100.00 | 100.00 | 100.00 | |
Initial weight (kg) | 39.86 | 40.05 | 39.27 | 40.42 | |
Ending weight (kg) | 112.35 | 114.36 | 115.29 | 107.33 | |
Weight gain (kg) | 72.49 | 74.31 | 76.02 | 66.91 | |
Days of rearing (d) | 60 | 60 | 60 | 78 | |
Daily gain (kg) | 1.21 | 1.24 | 1.27 | 0.86 | |
Feed consumption (kg) | 178.00 | 178.00 | 178.00 | 205.00 | |
Meat ratio of materials | 2.46 | 2.40 | 2.34 | 3.06 | |
Lean meat percentage (%) | 63.42 | 63.85 | 63.73 | 43.25 |
TABLE 7 pork test results
Remarking: sulfonamides (in total) (μ g/kg) were not detected (sulfamethazine <20.0 sulfamonomethoxine <5.0 sulfamonomethoxine <10.0 sulfamethoxazole <5.0 sulphamoacetyl <5.0 sulphamethodiazole <2.5 sulfamethoxazole <5.0 sulphachloropyridazine <5.0 sulphathiazole <10.0 sulphanilazole <5.0 sulphadiazine-6-methoxazine <5.0 sulphamethoxazine <5.0 sulphadiazine <5.0 sulphamethoxazine <20.0 sulphamethoxazine <10.0 sulphatopyrazole < 40.0).
Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (10)
2. the process for preparing a hyperforin derivative according to claim 1, comprising,
s1: reacting nicotinamide with chloroacetyl chloride to generate an intermediate compound;
s2: the intermediate compound and hyperforin are subjected to substitution reaction to generate the hyperforin derivative.
3. The method for preparing hyperforin derivatives according to claim 2, wherein: the mol ratio of the nicotinamide to the chloroacetyl chloride is 1: 2.8-3.2.
4. The method for preparing hyperforin derivatives according to claim 2, wherein: the mol ratio of the hyperforin to the intermediate compound is 1: 1.05-1.15.
5. Use of hyperforin derivatives according to claim 1 in the preparation of antiviral preparations.
6. An antiviral preparation comprising the hyperforin derivative of claim 1.
7. Use of the antiviral preparation according to claim 6 for the preparation of an additive for feed.
8. A Chinese medicinal additive for feed comprises radix Codonopsis, radix Paeoniae alba, Poria, semen Pruni, rhizoma Atractylodis, Bulbus Allii, herba Artemisiae Annuae, and fructus crataegi.
9. The traditional Chinese medicine additive for feed as claimed in claim 8, which is characterized in that: the herbal additive further comprises the antiviral formulation of claim 6.
10. The traditional Chinese medicine additive for feed as claimed in claim 9, which is characterized in that: the addition amount of the antiviral preparation in each 1000g of the traditional Chinese medicine additive is 0.2-5 g.
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CN1300623A (en) * | 1999-12-20 | 2001-06-27 | 张发平 | Chinese patent medicine |
CN103859168A (en) * | 2014-04-03 | 2014-06-18 | 常熟理工学院 | Avian influenza resistant feed additive for large-scale ecological chicken breeding and preparation and use methods thereof |
CN111228402A (en) * | 2020-04-02 | 2020-06-05 | 广州广力莱生物科技有限公司 | Chinese herbal medicine composition for preventing and controlling African swine fever, preparation method and pig feed |
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