KR101651948B1 - Anti-Coronavirus Composition - Google Patents

Abstract

The present invention relates to a composition for antiviral therapy against coronaviruses, and more particularly, to a composition for antiviral therapy that is safe for use with no side effects even when taken for a long period of time and has an efficacy for prevention and treatment of coronavirus infection diseases, And more particularly, to a health food composition for mammals which is characterized by extracting a guinea purpurea, a windbreak or a mixture thereof with one or more selected from the group consisting of water and a C1 to C3 alcohol An antiviral pharmaceutical composition for coronavirus and a health food composition for mammals.

Description

Anti-Coronavirus Composition < RTI ID = 0.0 >

The present invention relates to a composition for antiviral therapy against coronaviruses, and more particularly, to a composition for antiviral therapy that is safe for use with no side effects even when taken for a long period of time and has an efficacy for prevention and treatment of coronavirus infection diseases, To a health food composition for mammals.

Coronavirus is an RNA virus that causes respiratory diseases and digestive diseases in mammals and birds. Mutations easily occur in the process of replicating genetic information in the nature of RNA viruses. The mutation of the coronavirus is more important because it does not share the mutation immunity and vaccine development is difficult and the common treatment is also unknown.

Cat infectious peritonitis is one year old It predominantly occurs in younger feline animals, with a mortality rate of nearly 100%, with a mortality rate of about 17%, the most fatal and difficult to cure.

Feline infectious peritonitis is caused by the Feline Infectious Peritonitis Virus (FIPV) caused by the genetic mutation of Feline enteric coronavirus (FECV), and FIPV binds strongly to cells and tissues, And is infected through contaminated food, bedding, urine, or feces. Infected individuals are asymptomatic for a considerable period of time and act as disease mediators, leading to a high risk of transmission that can spread the virus over a long period of time. At the onset, symptoms such as anorexia, fever, jaundice, diarrhea, weight loss, neurological symptoms, and uveitis show clinical symptoms, gradually become vigorous and dyspnea, but the symptoms progress very slowly due to slow progression of the disease. It is difficult. In addition, once the outbreak is not known, proper treatment is a highly fatal disease with a high mortality rate. The effectiveness of the vaccine is still controversial.

Recently, many researchers have been actively studying the phytochemical efficacy (antiviral, anti-inflammation, immunostimulating agent, etc.) derived from natural products. Especially, antiviral efficacy has been studied in many fields . Examples include Enteric coronavirus (Rosa nukana), respiratory syncytial virus (RSV) (Sambucus racemose extract), parainfluenza virus type 3 (ipsomosyl extract) and rotavirus (Lomatium dissectum root extract) According to domestic research, Ajou University School of Medicine has reported that ginseng, ogapi, jiuhe, and casualty extract have an antiviral effect that inhibits coronavirus proliferation. However, there are not many studies on plants that have antiviral activity against coronavirus. Since extracts derived from natural materials have been used for a long period of time by folk remedies, safety has been established, and if the efficacy is proved, it can be taken for a long time without side effects, so that it can be used more effectively in terms of prevention as well as treatment.

Bupleurum Chinese is a perennial herb that is also known as the North Sea shore and the common weeping crab, and is distributed in Korea, Japan, China, Mongolia, Siberia and Europe. The roots contain saponin, fatty oil, and alkaloids, and they are used as medicines for fever, pain, tonic, respiratory, digestive and circulatory diseases.

Lepidoptera (Ledebouriellas Seseloides) is a perennial herbaceous herbaceous perennial herbaceous plant, native to northeast China, Faye Bay, and Inner Mongolia, and used as a medicinal crop. The name "windblown" means to prevent wind, and it is very important medicine to treat headache, chills, fever, paralysis caused by paralyzed whole body, paralytic pain (wind sickness). The main ingredient is coumarin, which contains rutin and tannin. Pharmacological actions include antipyretic, antipyretic, immune activation, antiallergic, anti-ulcer, antimicrobial, and skin anti-bacterial inhibition.

An object of the present invention is to provide an antiviral pharmaceutical composition for coronavirus useful for preventing and treating coronavirus infectious diseases in order to solve the problems of the prior art.

It is another object of the present invention to provide a health food composition for mammals which is capable of reducing the risk of infection by coronaviruses and enhancing resistance to coronaviruses so as to reduce the induction rate upon contact with viruses For other purposes.

In order to accomplish the above object, the present invention provides an antiviral agent for coronavirus, which comprises extracts of Seahorse, windshield or a mixture thereof, which is extracted with water or a mixture of one or more selected from the group consisting of C1 to C3 alcohols ≪ / RTI >

The extract of the present invention has an antiviral activity against coronavirus which is a virus causing respiratory diseases, gastrointestinal diseases, liver diseases, brain diseases and the like in mammals such as cats, dogs, cows, pigs and humans.

According to the preliminary experiments on the feline infectious peritonitis virus (FIPV), a kind of corona virus, when extracted with a mixture of C1 to C3 alcohol or C1 to C3 alcohol and water in addition to water, it exhibits the same or similar effect as the water extract . In addition, although no separate results were described in the following examples, the antiviral efficacy was exhibited even when the extract of Shiho or windblown was used alone. However, since the efficacy was more excellent in the mixed preparation, only the results of the mixed prescription were shown in the following examples Respectively. In the case of using a mixture of sea urchins and windshields, a synergistic effect was exhibited by mixing prescription when the weight ratio was 1: 5 to 5: 1 by weight.

In order to use the composition of the present invention as a medicament for prevention and treatment, it may be mixed with a carrier or a pharmaceutically acceptable carrier, a forming agent, a diluent or the like by a known method in the pharmaceutical field to prepare tablets, capsules, The composition may be formulated into oral preparations such as troches, liquids and suspensions, or parenteral administration preparations such as injectable preparations and dentifrices, and may be used as antiviral agents against coronaviruses. The dosage of the active ingredient according to the present invention is determined by the prescriber according to the degree of absorption of the active ingredient in the body, the form of the preparation, the route of administration, the species of the mammal, age, weight and the specific disease or pathological condition to be treated, The dosage may be administered once a day or divided into several doses. Typical dosages are 0.001 mg / kg · day to 10 g / kg · day. Since the composition of the present invention has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

Wherein the extract is obtained by extracting Shiho, windshield or a mixture thereof with one or more selected from the group consisting of water and a C1 to C3 alcohol and enhancing resistance to coronavirus will be.

The composition of the present invention shows excellent efficacy against coronavirus infection as well as prevention, and can be safely administered without side effects even when taken for a long time, so that it can be effectively used as a health food composition.

The health food composition of the present invention may be a tablet, a capsule, a pill, a powder or a liquid, and may be used by drinking itself or by adding 0.01 to 100% by weight to various foods or beverages. When used in mammals other than humans, they may be added to feeds.

As described above, according to the composition of the present invention, composition using antibiotic activity against coronavirus, which is a virus causing respiratory diseases, gastrointestinal diseases, liver diseases, brain diseases and the like, Which can be used safely without worrying about toxicity or side effects because of its long-term stability, and can be effectively used for the prevention and treatment of diseases caused by coronavirus infection since it does not cause drug resistance.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the antiviral potency of FIPV of the composition of the present invention.
Figure 2 shows RT-PCR results and graphs showing antiviral potency against FIPV of the composition of the present invention.
Figure 3 shows RT-PCR results and graphs showing the effect of the composition of the present invention on the expression of inflammatory cytokines.
FIG. 4 is a graph showing changes in blood leukocyte count and A / G ratio in an animal experiment with the composition of the present invention. FIG.
Figure 5 is an image and graph showing the results of imaging analysis in an animal experiment with the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these embodiments are merely examples for explaining the content and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

Example 1: Preparation of extract

Dried roots were purchased from the Korean Medicines Agency in Daejeon. The mixture of sesame oil and windblown roots or each mixture having a weight ratio of 5: 1 to 1: 5 is put into a high-temperature high-pressure extractor, After 10 times of water was added, it was immersed at room temperature and normal pressure for 24 hours, and then extracted at 100 to 110 ° C for 48 hours. When extraction is complete, the solid is removed by filtration and the supernatant And concentrated. The yield of the extract was 70% by weight of the dry weight .

Example 2: Antiviral activity measurement

1) Inhibition of virus inhibition in vitro

The virus inhibitory activity of the extracts prepared in Example 1 was analyzed using the extracts of Seiko: windblown 3: 1 (w / w). FIP virus and FIP virus (FIPV, VR-2108) were purchased from the American Cell Line Bank, and RPMI 1640 (Invitrogen, UK) containing 10 strains of Felis catus whole fetus cell line (Fcwf-4, ATCC No. CRL-2787) Cells were cultured using 2% non-inactivated Fetal Bovine Serum (FBS, BioWest, MO, USA) and 2 mM L-glutamine.

More specifically, Fcwf-4 cells were injected into each well of a 24-well plastic incubator at a concentration of 2 × 10 ⁵ cells / well and washed twice with 1 × PBS buffer.

First, the pretreatment group of the sample (Pre) was treated with the extract prepared in Example 1 at a concentration of 20, 50, and 100 μg / ml in wells with Fcwf-4 cells for 1 hour and washed twice with 1 × PBS buffer And washed. Subsequently, FIPV was inoculated at a concentration of 1.0 × 10 ⁴ TCID ₅₀ / ml at a concentration of at least TCID ₅₀ / ml and infected for 1 hour. After 1 hour of FIPV inoculation, the cells were cultured in 10% FBS medium.

Simultaneous treatment of the samples was performed by administering the extract prepared in Example 1 at a concentration of 20, 50 and 100 μg / ml in wells to which (Co) Fcwf-4 cells were attached and at the same time, FIPV was added at a concentration of a minimum TCID ₅₀ / ml At a concentration of 1.0 x 10 < ⁴ > TCID ₅₀ / ml and treated for 1 hour in 1% FBS medium. After 1 hour of FIPV inoculation, the cells were cultured in 10% FBS medium.

The posttreatment group (Post) was inoculated with FIPV at a concentration of 1.0 × 10 ⁴ TCID ₅₀ / ml at a concentration of at least TCID ₅₀ / ml in wells to which Fcwf-4 cells were attached and infected in 1% FBS medium for 1 hour . After 1 hour of FIPV inoculation, the cells were changed to 10% FBS medium and the extract (LBE) prepared in Example 1 was administered at a concentration of 20, 50 and 100 μg / ml.

Fcwf-4, which did not receive FIPV inoculation and extraction, was used as a control, and Fcwf-4, which was inoculated only with FIPV and no extract, was used as a virus.

The concentration of virus in each experimental group and the secretion amount of inflammatory cytokine were measured to verify that the extract according to the present invention had an antiviral effect.

First, Fcwf-4 cells in each experimental group were cultured in 5% CO ₂ , 37 ° C, and 10% FBS medium for 3 days, and viral titer was measured by a cytopathic assay. After 3 days, 500 μl of Fcwf-4 cells were injected at a concentration of 1 × 10 ⁴ cells / well, and the cell culture was incubated with a serum-free medium X 10 < ^{9 &} gt ;, injected with the cells in a 96 well plastic incubator and monitored for 3 days. Then, TCID ₅₀ of the extract was calculated using the TCID50 calculation method developed by Hierholzer & Killington (Virology Methods Manual, Academic Press, pp24-32, 1996), and the results are shown in FIG. 1 Respectively. From FIG. 1, it can be seen that the extract of the present invention greatly reduces the number of viruses and also shows an effect on prevention as well as treatment.

ViralRNAextractkit(Qiagen,Valencia,CA,USA)과 TRIZOL (Invitrogen, CA)를 이용하여 제작사의 프로토콜에 따라 총 RNA를 추출하였다. 추출된 총 RNA로부터 QuantiTect

reverse transcriptional kit(Qiagen, Valencia, CA, USA)와 ReverTraAce

qPCR RT kit (TOYOBO, Osaka, Japan)를 사용하여 제작사의 프로토콜에 따라 하기 서열번호 1과 2의 프라이머를 사용하여 rt-PCR에 의해 FIPV-N 바이러스를 정량하였다. Separately, the concentration of FIP virus in each experimental group was measured by PCR, and the results are shown in FIG. Fcwf-4 cells of each experimental group were cultured in 5% CO ₂ , 37 ° C, 10% FBS medium for 3 days, and then QiAamp

Total RNA was extracted according to the manufacturer's protocol using Viral RNA Extract Kit (Qiagen, Valencia, CA, USA) and TRIZOL (Invitrogen, CA). From the extracted total RNA, QuantiTect

reverse transcriptional kit (Qiagen, Valencia, CA, USA) and ReverTraAce

The FIPV-N virus was quantified by rt-PCR using the qPCR RT kit (TOYOBO, Osaka, Japan) according to the manufacturer's protocol using the primers of SEQ ID NOS: 1 and 2 below.

SEQ ID NO: 1: 5'-CAACTGGGGAGATGAACCT-3` (forward)

SEQ ID NO: 2: 5'-GGTAGCATTTGGCAGCGTTA-3` (reverse direction)

The PCR conditions were denaturation and annealing at 95 ° C for 5 minutes, followed by PCR for 30-40 cycles at 55 ° C and reaction for 1 minute and 30 seconds at 72 ° C. Virus was quantitatively analyzed by PCR with Image-Lab, a software from Bio-rad. It was also confirmed that the number of viruses was reduced by the treatment of the extract of the present invention, as in the case of the virus titer, in the quantification of virus by PCR. Especially, the inhibitory effect was excellent at a low concentration of 20 ㎍ / ㎖ of extract, and the higher the treatment concentration, the better the therapeutic effect.

To predict the effect on inflammation, the concentration of the inflammatory cytokine in the culture medium of the pretreatment group (FIG. 3A) and posttreatment group (FIG. 3B) of the extract was measured by RT-PCR The results are shown in Fig. Inflammatory cytokines were quantified by the same method as the above-mentioned rt-PCR of FIPV-N, except that the primers shown in Table 1 were used. For comparison, the amount of inflammatory cytokine secretion in the treated group of chloroquine, which is known to help relieve the symptoms of cat infectious peritonitis, was also measured.

As shown in FIG. 3, the extract of the present invention had the effect of reducing the expression of TNF-alpha and IL-6, which are inflammatory cytokines, as well as Chloroquine, which is known to be effective in alleviating the symptoms of feline infectious peritonitis. Chloroquine or better.

2) Animal experiments

The efficacy of the extract against cat-infectious peritonitis was verified by using the extract of LBE of Seishi: Wind 3: 1 (w / w) prepared in Example 1. Nine cats under 12 ~ 14 months of age were purchased from the Yuseong market in Daejeon and used for the experiment. The cats were divided into three groups: control group, virus group, and LBE group. The cats in the virus group and LBE group were inoculated with FIPV (3.6 × 10 ⁴ TCID ₅₀ ) after inducing sedation. Immediately after inoculation, the LBE group was orally administered daily with 3 ml / kg of the extract of Seishi: wind 3: 1 (w / w) of the extract prepared in Example 1. After that, we observe progress with free feeding and collect blood once a week. Imaging was performed by Computed Tomography (CT, Alexion ^™ , Toshiba, Japan) and Ultrasonography (iU22, Philips, Netherland) 27 days after the inoculation of the virus and the degeneration of each organ was confirmed by autopsy on the 28th day after sacrifice .

In the behavioral observations for the clinical evaluation, clinical symptoms such as vomiting, diarrhea, anorexia and hypothyroidism started to appear from the 14th day of inoculation in the virus group, but in the LBE group, Clinical findings were rarely seen .

FIG. 4 shows the results of measurement of leukocyte count and A / G (albumin / globulin) ratio in the collection of cats in each experimental group. Leukocyte counts were significantly increased by inoculation of viruses, which was highest at 14 days in the virus group, and leukocyte increase was slower in the LBE group than in the virus group. However, there was no statistical significance after 14 days due to the large difference between individuals. The A / G ratio, which is an indicator of chronic inflammation, was significantly decreased by the inoculation of the virus, but the degree of decrease was markedly reduced in the LBE group and gradually decreased with the passage of time in the virus group, Showing a tendency to relieve inflammation.

Figure 5 shows the results of CT and ultrasound imaging. As a result of the radiological analysis of FIG. 5, it is possible to confirm the mesenteric lymph node enlargement in the cats of the virus group. As a result of autopsy, pleural fluid and ascites observed in the exudative FIP were not found but pyuric granulomas, . Lymph node enlargement was less in the LBE group than in the virus group.

<110> LEE, Hyun Gwang          The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Anti-Coronavirus Composition <130> P1015-691 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 caactgggga gatgaacct 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggtagcattt ggcagcgtta 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggagaaagct gccaaatacg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcacaagtgt gttgggtgac 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atcactactg gtcttcagga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttacattatc cgaacagccc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cccatgtagt agcaaacccc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gtctggtagg aaacggcaaa 20

Claims (7)

An antiviral pharmaceutical composition for a feline infectious peritonitis virus, which comprises an extract obtained by extracting a mixture of seaweed and windshield with one or more selected from the group consisting of water and C1 to C3 alcohols.
delete delete The method according to claim 1,
Wherein the mixture of sea urchins and windshields is mixed at a weight ratio of 1: 5 to 5: 1.
Wherein the mixture contains an extract of a mixture of seaweed and windshield with one or more selected from the group consisting of water and C1 to C3 alcohols and enhances resistance to feline infectious peritonitis virus.
delete 6. The method of claim 5,
Wherein the mixture of sea urchins and windshields is mixed at a weight ratio of 1: 5 to 5: 1.

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