Application of tetrandrine in preparation of medicine for preventing and treating porcine coronavirus

Abstract

The application discloses application of tetrandrine in preparation of a medicine for preventing and treating porcine coronavirus, and the effective component of the medicine is tetrandrine / tetrandrine and methylhesperidin. It is found by the application that tetrandrine or tetrandrine and methylhesperidin can significantly inhibit replication of the porcine coronavirus at a low concentration, and the medicine prepared by using tetrandrine or tetrandrine and methylhesperidin at a low concentration can effectively prevent and treat porcine transmissible gastroenteritis virus or porcine epidemic diarrhea virus.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly relates to application of tetrandrine in preparation of a medicine for preventing and treating porcine coronavirus. BACKGROUND

[0002] Porcine coronaviruses (COV) belong to the alpha, beta and delta coronavirus genera of the Coronavirinae subfamily. The effects on the gastrointestinal tract, respiratory system, peripheral nerves and central nervous system are usually visible. At present, porcine coronaviruses include transmissible gastroenteritis virus of swine and porcine epidemic diarrhea virus; and the characteristics of the two viruses are as follows:

[0003] Transmissible gastroenteritis of swine (TGE) is an acute, highly contagious digestive tract infectious disease caused by transmissible gastroenteritis virus (TGEV). The TGEV is a single-stranded positive-sense RNA virus with a capsule, and the virus belongs to the Coronaviridae family and the alpha coronavirus genus. The virus was first reported in the United States in 1945 and then spread globally. The virus can infect pigs of any age, and the infection of piglets before weaning is particularly serious, and the morbidity rate in some areas is as high as 87.97%, and the mortality rate is as high as 89.90%. After the pig is infected, the virus will rapidly destroy the gastrointestinal flora and mucosal structure of the host, and the infected pig will show clinical symptoms such as vomiting, diarrhea and dehydration. Because it seriously endangers the development of the pig industry, TGE was listed as a severe infectious disease by the World Organization for Animal Health (OIE) in 2019.

[0004] Porcine epidemic diarrhea (PED) is an infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). The PEDV is a single-stranded positive-sense RNA virus with a capsule, and the virus belongs to the Coronaviridae family and the alpha coronavirus genus. PEDV can infect pigs of different ages, and the infected pigs show symptoms such as severe enteritis, diarrhea, vomiting, anorexia and dehydration, and the morbidity rate and mortality rate of piglets can reach 100%.

[0005] At present, vaccination is still the preferred measure for preventing and controlling porcine coronavirus infection. However, the coronavirus mutates rapidly and has many epidemic strains, and the protection provided by inactivated vaccines and attenuated vaccines is not sufficient.

[0006] A large amount of research work has been done on TGEV and PEDV drug development at home and abroad, but no specific medicine for preventing and controlling TGEV and PEDV has been developed. SUMMARY

[0007] The present application aims to overcome the deficiencies of the prior art, and provides an application of tetrandrine in preparation of a medicine for preventing and treating porcine coronavirus.

[0008] To achieve the above-mentioned object, the technical scheme of the present application is as follows:

[0009] The present application provides an application of tetrandrine in preparation of a medicine for preventing and treating porcine coronavirus; the porcine coronavirus is porcine transmissible gastroenteritis virus or porcine epidemic diarrhea virus.

[0010] The present application also provides an application of tetrandrine and methyl hesperidin in combined preparation of a medicine for preventing and treating porcine coronavirus.

[0011] The present application also provides a medicine for preventing and treating porcine coronavirus, wherein the effective component in the medicine is tetrandrine.

[0012] Further, the effective component in the medicine comprises tetrandrine and methyl hesperidin.

[0013] Further, the concentration of the effective component is 1-6 muM.

[0014] Further, the concentration of the effective component is 6 muM.

[0015] Further, the weight ratio of the tetrandrine and the methyl hesperidin is 1:1-3.

[0016] Further, the weight ratio of the tetrandrine and the methyl hesperidin is 1:1.

[0017] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0018] Further, the dosage form of the medicine is powder, granules, tablets or capsules but is not limited to powder, granules, tablets or capsules, and is any one of the dosage forms acceptable in veterinary clinics.

[0019] The principle of selection of raw materials of the present application is as follows:

[0020] 1. Fangchinoline is a bisbenzylisoquinoline compound isolated from the plant of the family of Ranunculaceae, which has a wide range of pharmacological activities, such as anti-inflammatory, antioxidant, inhibition of platelet formation, anti-thrombosis, reduction of blood pressure, reduction of blood sugar, etc. In addition, Fangchinoline can also play an anti-tumor effect. Fangchinoline (Fan) has a molecular weight of 608.72 g / mol, a molecular formula of C 37 H 40 N2O6, as shown in the molecular structure formula I:

[0021]

[0022] Fangchinoline has a variety of biological functions, such as anti-inflammatory analgesic, antioxidant, non-specific blocking of calcium ion channels, inhibition of histamine release, dilation of coronary arteries, reduction of myocardial oxygen consumption, etc. It is often used as an analgesic, anti-rheumatic and antihypertensive drug in traditional Chinese medicine. In recent years, people have begun to pay attention to its anti-cancer effect. Literature shows that Fangchinoline can inhibit tumor cells through various ways, such as inducing apoptosis, blocking cell cycle progression, inhibiting tumor cell invasion, etc.

[0023] 2. Methyl hesperidin (MEH) is a natural flavonoid compound, which is an important derivative of hesperidin and is also an effective therapeutic and preventive agent for vascular diseases caused by hypertension and arteriosclerosis. Compared with hesperidin, MEH is more soluble in water and more easily absorbed by the human body, so it can also be used as a fortifying food additive and has a wide application in clinical medicine and food science. In addition, MEH has strong antiviral and antibacterial effects, and a large dose can inhibit the replication of influenza virus. MEH also has the functions of maintaining the normal permeability of blood vessels, improving the resistance of capillaries, enhancing the elasticity and toughness of capillaries, preventing and treating capillary bleeding, gum bleeding, etc. The molecular weight of MEH is 624.59 g / mol, the molecular formula is C 29 H 36 O 15 , as shown in the molecular structure formula II:

[0024]

[0025] Advantages of the present application:

[0026] The present application finds that Fangchinoline or Fangchinoline and methyl hesperidin can significantly inhibit the replication of porcine coronavirus (porcine transmissible gastroenteritis virus or porcine epidemic diarrhea virus) at a low concentration, and the use of Fangchinoline or Fangchinoline and methyl hesperidin at a low concentration to prepare a medicine for preventing and treating porcine coronavirus can effectively prevent and treat porcine transmissible gastroenteritis virus or porcine epidemic diarrhea virus. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1Figure of the influence of different drugs for preventing and treating porcine coronavirus on the cell activity after treating PK-15 cells for 36 hours;

[0028] Figure 2 Figure of the influence of different drugs for preventing and treating porcine coronavirus on the cell activity after treating Vero-E6 cells for 36 hours;

[0029] Figure 3 Figure of the influence of drugs 9, 10, 11 and 13 for preventing and treating porcine coronavirus on the copy number of TGEV in PK-15 cells at the transcription level;

[0030] Figure 4 Figure of the influence of drugs 9, 10, 11 and 13 for preventing and treating porcine coronavirus on the copy number of TGEV in PK-15 at the protein level;

[0031] Figure 5 Figure of the influence of different drugs for preventing and treating porcine coronavirus on the inhibition effect of Vero-E6 cells infected with PEDV virus at the transcription level;

[0032] Figure 6 Figure of the time addition effect of drug 13 for preventing and treating porcine coronavirus; Figure 7 Figure of the influence of different traditional Chinese medicine prescriptions on the cell activity after treating PK-15 cells for 36 hours;

[0033] Figure 8 Figure of the influence of drugs 13, methyl hesperidin and traditional Chinese medicine prescription 7 on the copy number of TGEV in PK-15 cells at the transcription level;

[0034] Figure 9 Figure of the influence of drugs 13, methyl hesperidin and traditional Chinese medicine prescription 7 on the copy number of TGEV in PK-15 cells at the protein level. DETAILED DESCRIPTION

[0035] The application will be further described in detail below with specific examples, so that those skilled in the art can understand.

[0036] Example 1 Preparation of drug 1 for preventing and treating porcine coronavirus

[0037] Drug 1 for preventing and treating porcine coronavirus is prepared by the following steps:

[0038] First, tetrandrine is dissolved in DMSO to prepare a mother liquor with a concentration of 1 mM; and then the mother liquor is diluted to a solution with a final concentration of 1.25 μM.

[0039] Examples 2-6 Preparation of drugs 2-6 for preventing and treating porcine coronavirus with different concentrations

[0040] The drugs 2-6 for preventing and treating porcine coronavirus are respectively diluted from the mother liquor to solutions with concentrations of 2.5, 5, 10, 20 and 40 μM.

[0041] Toxic effects of the above different drugs for preventing and treating porcine coronavirus on PK-15 cells

[0042] 1. CCK-8 method for determining the cytotoxicity of different drugs for preventing and treating porcine coronavirus on PK-15

[0043] Logarithmic growth phase PK-15 cells are collected, diluted to a cell suspension of 10 6 cells / mL with DMEM complete medium containing 10% fetal bovine serum, mixed, inoculated into 96-well plates (with blank wells without added cells), 100 μL of the cell suspension is added to each well, the periphery of the 96-well plate is gently tapped to make the cells uniformly distributed, and then the plate is placed in a cell incubator at 37°C and 5% CO2 for culture after the cells are precipitated at the bottom of the plate. When the cells grow to 70%-80%, the medium is removed, the cells are washed once with sterile PBS, and then the PK-15 cells are treated in groups, with 6 parallel wells in each group; specifically as follows:

[0044] Drug-added groups 1-6: the above drugs 1-6 (same volume) are added;

[0045] Blank group: no cells are added;

[0046] Control group: no drug is added;

[0047] After the drugs are added, the 96-well plate is placed in an incubator for incubation for 36 h, 10 uL of CCK-8 solution is added to each well, and the plate is further incubated in the incubator for 1.5 h. After the incubation, the absorbance value (OD 450 ) at 450 nm is read by an enzyme label instrument.

[0048] Cell survival rate = (OD 450 of the drug-added group - OD 450 of the blank group) / (OD 450 of the control group - OD 450 of the blank group).

[0049] The results are shown in Table 1. Figure 1 The drugs 1-4 have little effect on PK-15 cells and have no obvious cytotoxicity, and drug 5 has relatively obvious cytotoxicity on PK-15.

[0050] 2. CCK-8 method for determining the cytotoxicity of different drugs for preventing and treating porcine coronavirus on Vero-E6

[0051] Vero-E6 cells in logarithmic growth phase were collected, diluted to 10 6 cells / mL of cell suspension with DMEM complete medium containing 10% fetal bovine serum, and inoculated into a 96-well plate after mixing the cells. 100 μL of the cell suspension was added to each well, and the 96-well plate was gently tapped around to evenly distribute the cells. After the cells were precipitated at the bottom of the culture plate, the plate was placed in a cell incubator at 37°C and 5% CO2. When the cells grew to 70%-80%, the culture medium was removed, and the cells were washed with sterile PBS. The Vero-E6 cells were then treated in groups, with 6 parallel wells in each group. The specific treatments were as follows:

[0052] Drug groups 1-6: the above drugs 1-6 (same volume) were added;

[0053] Blank group: no cells were added;

[0054] Control group: no drug was added;

[0055] After the addition of the drugs, the 96-well plate was placed in an incubator for 36 h, and then 10 uL of CCK-8 solution was added to each well. The plate was further incubated in the incubator for 1.5 h. After the incubation, the absorbance value (OD 450 ) at 450 nm was read by a microplate reader.

[0056] Cell survival rate = (drug group OD 450 - blank group OD 450 ) / (control group OD 450 - blank group OD 450 .

[0057] The results are shown in Figure 2 . The above drugs 1-3 had little effect on Vero-E6 cells and showed no obvious cytotoxicity. Drug 4 had relatively obvious cytotoxicity on Vero-E6 cells.

[0058] Examples 7-14

[0059] The above mother liquor was diluted to a concentration of 0.05, 0.1, 0.5, 1, 2, 4, 6, and 8 μM to prepare drugs 7-14 for preventing and treating porcine coronavirus.

[0060] I. Inhibition effect of the above different drugs for preventing and treating porcine coronavirus on TGEV in PK-15

[0061] 1. Virus inoculation and drug treatment for preventing and treating porcine coronavirus

[0062] PK-15 cells were diluted to 10 6The cells were inoculated with TGEV virus liquid when they grew to 70-80%. 1.5 hours after the inoculation, the same volume of drugs 9, 10, 11 and 13 were added to each well respectively. The cells were observed 36 hours later, and the cell supernatant and cells were collected respectively.

[0063] 2. Effect of different drugs for preventing and treating porcine coronavirus on inhibition of TGEV copy number

[0064] The RNA of the cell supernatant was extracted, and cDNA was reversely transcribed. Absolute fluorescent quantification was performed under the following reaction conditions: initial template pre-denaturation at 95°C for 1 minute; denaturation at 95°C for 20 seconds, annealing at 58°C for 30 seconds; extension at 68°C for 30 seconds; 35 cycles, and all detections were repeated three times.

[0065] The average cycling threshold (Ct) was taken, and the virus standard curve was substituted:

[0066] y = -3.8003x + 38.945,

[0067] wherein y is Ct, and copy number = 10 x Calculation was performed.

[0068] The total protein of the collected cells was extracted, and protein quantification was performed by BCA method. Western Blot was used to verify the protein level of the virus content.

[0069] The results are shown in Figure 3 , 4 Drugs 9, 10, 11 and 13 had significant inhibitory effect on TGEV in PK-15 cells, and the inhibitory effect was enhanced with the increase of tetrandrine concentration.

[0070] II. Inhibitory effect of different drugs for preventing and treating porcine coronavirus on PEDV in Vero-E6 cells

[0071] 1. Virus inoculation and drug treatment for preventing and treating porcine coronavirus

[0072] Vero-E6 cells were added to 12-well cell culture plates at 10 5 cells / well, and the cells were inoculated with PEDV virus liquid when they grew to 70-80%. 1.5 hours after the inoculation, the same volume of drugs 7-14 was added to each well respectively. The cells were observed 36 hours later, and the cell supernatant was collected.

[0073] 2. Effect of different drugs for preventing and treating porcine coronavirus on inhibition of PEDV copy number

[0074] The cell supernatant RNA was extracted, reverse transcribed into cDNA, and absolute fluorescence quantification was performed, and the reaction conditions were as follows: initial template pre-denaturation 95°C, 1 min; denaturation 95°C, 20 s, annealing 58°C, 30 s; extension 68°C, 30 s; 35 cycles, all detections were repeated 3 times;

[0075] The average cycling threshold (Ct) was taken, and the virus calibration curve was as follows:

[0076] y = -3.3039x + 39.2267,

[0077] wherein y is Ct, and the copy number = 10 x was calculated;

[0078] The results are shown in Figure 5 The drug containing 2.243 μM tetrandrine can inhibit 50% of the PEDV virus in the infected Vero-E6 cells, and the inhibitory effect is enhanced with the increase of the concentration of tetrandrine in the drug.

[0079] As shown above: the drug containing tetrandrine can significantly inhibit TGEV in PK-15 cells and PEDV in Vero-E6 cells, and the drug 13 can achieve the ideal inhibitory effect without obvious toxic side effects, and has potential therapeutic value for TGE and PED, providing a new scheme for the treatment of TGE and PED in clinical production.

[0080] III. Time addition effect of drug 13 on the prevention and treatment of porcine coronavirus

[0081] The following three groups were treated in this experiment (same volume of drug 13):

[0082] (1) Pretreatment with drug 13 1 h before cell infection with virus, remove the culture medium, add the virus-containing basic culture medium with MOI = 0.1, wash with PBS for 3 times after 1 h, and add the maintenance culture medium.

[0083] (2) Co-treatment with drug 13 containing tetrandrine and virus-containing basic culture medium for 30 min, then add to the cells, wash with PBS for 3 times after 1 h, and add the maintenance culture medium.

[0084] (3) After incubating the cells with virus-containing basic culture for 1 h, remove the culture medium, treat with drug 13-containing basic culture for 1 h, wash with PBS for 3 times, and add the maintenance culture medium.

[0085] Finally, the supernatant of the three groups of cells was collected, and the RNA of the supernatant was extracted, reverse transcribed into cDNA, and subjected to absolute fluorescent quantification, and the reaction conditions were as follows: initial template pre-denaturation at 95°C for 1 min; denaturation at 95°C for 20 s, annealing at 58°C for 30 s; extension at 68°C for 30 s; 35 cycles, all detections were repeated three times;

[0086] The average cycling threshold (Ct) was taken as the result, and the virus calibration was as follows:

[0087] y = -3.3039x + 39.2267,

[0088] where y is Ct, and the copy number = 10 x were calculated; and were used for further research.

[0089] The results are shown in Table 1: Figure 6 Drug 13 containing 6 μM tetrandrine can significantly inhibit the infection of PEDV in three ways of pre-infection (Pre-), co-incubation with virus (Co-), and post-infection (Post-).

[0090] Preparation of drug 15 (Chinese medicine formula) for preventing and treating porcine coronavirus

[0091] The preparation method of drug 15 (Chinese medicine formula 1) for preventing and treating porcine coronavirus is as follows:

[0092] Tetrandrine and methyl hesperidin were weighed according to a weight ratio of 1:1 and added to DMSO to prepare Chinese medicine formula 1 at a concentration of 1.25 μM (total concentration of tetrandrine and methyl hesperidin).

[0093] Preparation of drugs 16-21 (Chinese medicine formulas 2-6) for preventing and treating porcine coronavirus

[0094] The preparation methods of drugs 16-21 are basically the same as those of Example 15, except that:

[0095] The concentrations of drugs 16-21 (Chinese medicine formulas 2-6) are 2.5, 5, 10, 20, and 40 μM, respectively.

[0096] I. Toxic effect of Chinese medicine formula on PK-15 cells

[0097] 1. CCK-8 method for determining the cytotoxicity of Chinese medicine formula on PK-15

[0098] Logarithmic growth period PK-15 cells were collected and diluted to 10 6After mixing the cells with a cell suspension of 100 μL / mL, seed them into 96-well plates. Gently tap the area around the 96-well plate to distribute the cells evenly. Once the cells have settled to the bottom of the culture plate, incubate them in a cell culture incubator at 37°C and 5% CO2.

[0099] Once the cells have grown to 70%-80%, remove the culture medium, wash the cells once with sterile PBS, and then process the PK-15 cells into groups, with 6 parallel wells per group; details are as follows:

[0100] Groups 16-21: Add the above-mentioned Chinese herbal formulas 1-6 (same volume);

[0101] Blank group: No cells added;

[0102] Control group: No drugs added;

[0103] After adding the drug, the cells were incubated in an incubator for 36 hours. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 1.5 hours. After incubation, the absorbance (OD) was read at 450 nm using a microplate reader. 450 The cell viability rate of the drug-treated group relative to the control group was calculated.

[0104] Cell viability = (OD of drug-treated group) 450 - Blank group OD 450 ) / (control group OD 450 - Blank group OD 450 ).

[0105] The results are as follows Figure 7 As shown: Traditional Chinese medicine formulas 1-5 had little effect on PK-15 cells and no obvious cytotoxicity, while traditional Chinese medicine formula 6 had more obvious cytotoxicity to PK-15.

[0106] Example 22: Preparation of Drug 22 (Traditional Chinese Medicine Formula 7) for the Prevention and Treatment of Swine Coronavirus

[0107] The preparation method of drug 22 is basically the same as that of Example 15, except that:

[0108] The concentration of drug 22 (traditional Chinese medicine formula 7) was 6 μM.

[0109] The above-mentioned traditional Chinese medicine formula 7 showed an inhibitory effect of TGEV on PK-15.

[0110] 1. Virus inoculation and drug treatment

[0111] PK-15 cells were divided into 10 6Individuals / hole are added to 6-hole cell culture plates, and when the cells grow to 70-80%, TGEV virus liquid is inoculated, and 1.5h after virus inoculation, the PK-15 cells are treated in groups, and the specific treatments are as follows:

[0112] Positive control group: adding sinomine with a concentration of 6 μM;

[0113] Control group 1: adding the above drug 13;

[0114] Control group 2: adding methylhesperidin with a concentration of 6 μM;

[0115] Experimental group: adding traditional Chinese medicine prescription 7;

[0116] After 36h, observation is carried out, and cell supernatant and cells are collected.

[0117] 2. Effect of drug on inhibition of TEGV copy number

[0118] RNA in the cell supernatant is extracted, reverse transcribed into cDNA, absolute fluorescence quantification is carried out, and the reaction conditions are as follows: initial template pre-denaturation 95℃, 1min; denaturation 95℃, 20s, annealing 58℃, 30s; extension 68℃, 30s; 35 cycles, all detections are repeated for 3 times;

[0119] The average cycling threshold (Ct) is taken, and the virus standard curve is substituted:

[0120] y=-3.8003x+38.945,

[0121] Wherein, y is Ct, and copy number=10 x Calculation is carried out;

[0122] Total protein of the collected cells is extracted, protein quantification is carried out by BCA method, and Western Blot is used to verify the protein level of the virus content.

[0123] The results are shown in the following table: Figures 8-9 It is shown that drug 13 and traditional Chinese medicine prescription 7 have significant inhibitory effects on TGEV in PK-15 cells, and the virus inhibitory effect of traditional Chinese medicine prescription 7 is the best.

[0124] In summary, the present application finds that the traditional Chinese medicine prescription containing sinomine and methylhesperidin can significantly inhibit TGEV in PK-15 cells, and the traditional Chinese medicine prescription 7 can achieve ideal inhibitory effect without obvious toxic side effects, and has potential therapeutic value for TGE, thereby providing a new scheme for the treatment of TGE in clinical production.

[0125] Other parts not described in detail are prior art. Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiment of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. The application of tetrandrine and methyl hesperidin in the combined preparation of a drug for preventing and treating porcine transmissible gastroenteritis virus, characterized in that: The concentrations of tetrandrine and methyl hesperidin were both 6 μM.

Images