KR20170130945A - A Coxsackie virus proliferation inhibitory composition extracted from Amomi Cardamomi Fructus - Google Patents

Abstract

The present invention relates to an inhibitor for proliferation of coxsackievirus and, more specifically, to an Amomum cardamomum extract which is effective in inhibiting proliferation of coxsackievirus. The Amomum cardamomum of the present invention, when treated with infection of coxsakievirus, inhibits proliferation of coxsackievirus infected by cells by adjusting cell growth signals of a compound. Also, the Amomum cardamomum extract has an antiviral effect against coxsackievirus causing myocarditis, thereby being usefully used by being treated to cardiac inflammation occurring by coxsackievirus.

Description

A Coxsackie virus proliferation inhibitory composition extracted from Amomi Cardamomi Fructus having antoxokinetic activity

The present invention relates to a coxsackievirus antiproliferative agent, and more particularly, to a coxsackievirus antiproliferative effect of an extract of Baekduguk.

The Paecil extract of the present invention inhibits the proliferation of the infected Coxsackie virus through the regulation of the cell growth signal of the compound when it is treated together with the coxsackievirus infection. In addition, the subject substance of the present invention has anti-viral efficacy against coxsacki virus that causes myocarditis, and can be effectively used by treatment of cardiac inflammation caused by coxsack virus.

Diseases caused by bacteria (bacteria) have been prevented to some extent due to the development of various types of preventive vaccines and antibiotics. However, treatment and prevention of various diseases caused by viruses have not been effective yet, And viral diseases are increasing.

In addition to the existing diseases caused by influenza virus, herpes virus and hepatitis virus that cause influenza virus and vesicular stomatitis, avian influenza (AI), severe acute respiratory syndrome (SARS) The emergence of new viruses that are triggered is seriously threatening the lives of humans and livestock. As a result, interest in the prevention and treatment of various viral diseases is increasing throughout the world.

Most antiviral agents currently on the market are proteins such as nucleoside derivatives and interferon (IFN), but these substances exhibit various side effects such as cytotoxicity, hepatotoxicity and over-tolerance. Therefore, there is a continuing need for the development of new antiviral substances that are low in side effects, low in cost and safe, and research to search for substances with antiviral activity from safe natural substances with few side effects in order to avoid the side effects of existing chemical synthetic drugs It is actively proceeding.

The Coxsackie viruses associated with the present invention are non-enveloped RNA viruses that have a single-stranded positive sense RNA genome (about 7.4 kb) Lt; / RTI > Coxsackieviruses belong to the family Picornaviridae and belong to Enterovirus together with poliovirus.

Coxsackie virus B3 (CVB3) is a cardiotropic virus that binds to the coxsackie-adenovirus receptor that is weakly expressed in the heart of a normal person and is a virus that causes myocarditis and dilated cardiomyopathy. It also has the characteristic of causing general cold. Therefore, the development of coxsackievirus inhibitors is very important and has a large ripple effect.

The present inventors previously confirmed the inhibitory effect of ORI-2 compounds extracted from duckweed on the coxsakievirus type B3 and B4 (CVB3, CVB4), and this natural extract was developed as a novel coxsakievirus infection inhibitor without cytotoxicity And the like.

In this study, we obtained extracts from hundreds of other natural plants and tested the cell survival assay using HeLa cells infected with coxsakievirus B3 (CVB3), and obtained plant extracts of Amomi Cardamomi Fructus I could.

Baekdugu, which has attracted the attention of the present inventors, is grown in Cambodia, Vietnam, Thailand and Indonesia and grown in the southern part of China. It removes fruit sack and fruit husk (peel) from mature fruit with yellowish green and uses it in the sun. It removes the moisture from the nose, and it is effective for dryness, digestion and abdominal pains, vomiting, pregnancy vomiting, and chick embryo. Pharmacological actions have been reported to promote gastric secretion, excitement of the intestines, and directional structure. In the present invention, Baekdugu treatment proved the possibility of being developed as a therapeutic agent for intestinal virus by delaying proliferation of viruses infected with cells and proliferation to surrounding cells.

As described above, the object of the present invention is to provide a substance that can be safely used without side effects, and also has excellent inhibitory effect against cocksaki virus, while solving the problems of conventional antiviral agents.

Another object of the present invention is to provide a composition for treating and preventing cold, which contains the above-mentioned substance as an active ingredient.

Another object of the present invention is to provide a composition for treating and preventing cardiac inflammation containing the above-mentioned substance as an active ingredient.

The present inventors purified the extract of Baekdukwu through freeze-drying method and observed the activity of coxsakievirus proliferating cell signal Akt using HeLa cell and the effect of inhibiting virus proliferation and the mechanism of action. The virus concentration of the cell supernatant treated with Baekdugu extract after viral infection And intracellular viral RNA were measured to confirm the virus infection and proliferation inhibitory effect.

The survival rate of HeLa cells infected with CVB3 was significantly improved with increasing concentration of Baekdugu extract. The activity of Akt, which is known to be a cell signal activated upon proliferation after coxsakievirus infection, was strongly inhibited by Baekdugu treatment and effectively suppressed viral membrane protein VP1 expression and eIF4G1 cleavage. These results demonstrate that Baekduguk extract has an important antiviral effect to inhibit virus proliferation and confirms the possibility of developing Baekdugu extract as a therapeutic agent for coxsakievirus infection inhibition.

As described above, Coxsakievirus B3 (CVB3) is a picornavirus and belongs to the intestinal virus as well as poliovirus. CVB3 is a cardiotropic virus that binds to a coxsackie-adenovirus receptor that infects host cells. Although most intestinal virus infections are symptomless, acute myocarditis due to these infections causes severe arrhythmias and sudden cardiac death, and also leads to the development of chronic myocarditis and dilated cardiomyopathy.

Previous studies have reported several antiviral drugs made of chemical compounds. The survival rate of mice infected with CVB3 was dramatically increased by treatment with enterovirus protease 3C inhibitor (3CPI). These 3CPIs demonstrated strong anti-viral effects in the murine myocarditis model, demonstrating significant reduction of viral replication and myocardial damage in the heart. These results suggest that inhibiting viral replication may be an effective method for intestinal virus treatment.

In order to discover new anti-viral agents, we examined the ability of natural compounds to inhibit intestinal viral replication and regulate cell survival signaling molecules such as Akt. Thus, it was confirmed that the natural extract isolated from Baekdugu (Amomi Cardamomi Fructus) inhibited the replication of CVB3 in HeLa cells and had a strong antiviral effect particularly in the HeLa cell survival assay.

The present invention provides a coxsackievirus growth inhibitor comprising at least one selected from the group consisting of Baekdugu extract and pharmaceutically acceptable salts thereof as an active ingredient.

Also provided is a health food for inhibiting the growth of coxsackievirus comprising at least one selected from the group consisting of Baekdugu extract and pharmaceutically acceptable salts thereof as an effective ingredient.

The present invention also provides a pharmaceutical composition for the treatment and prevention of cold which contains the coxsackievirus inhibitor.

Also provided is a pharmaceutical composition for the treatment and prevention of myocardial infarction containing the above cocksaki virus antiproliferative agent.

The extract of Baekduguchi according to the present invention has no toxicity and exhibits excellent coxsackievirus proliferation inhibitory effect, so that it can be effectively used as an antiviral therapeutic agent or a health food.

The extract of Baekduk according to the present invention can be used in the form of a pharmaceutically acceptable salt. For example, as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The Baekdugu extract may form acid addition salts which are pharmaceutically acceptable according to the methods conventional in the art. Examples of the free acid include free acid and inorganic acid. Examples of the inorganic acid include hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid. Examples of the organic acid include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid or aspartic acid Can be used.

As a result of measuring the inhibitory effect on the coxsack virus proliferation and the inhibitory effect on VP1 expression of the extract of Paekdukuga or its pharmaceutically acceptable salt of the present invention, it has been confirmed that it has excellent inhibitory activity against cocksakivirus proliferation.

Therefore, the Baekdugu extract of the present invention can be used for preparing a therapeutic composition and a health food for an inhibitory effect against an abnormally induced cockskill virus proliferation.

The composition may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, etc. in clinical administration, and may be used in the form of general medicines and health foods.

The compositions may also be formulated for oral administration, for example as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs, ≪ / RTI >

Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation in tablets, capsules and the like; Excipients such as dicalcium phosphate; Disintegrating agents such as corn starch or sweet potato starch; Lubricating oil such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intramuscular injection. For formulation into a non-oral dosage form, it may be formulated as a solution or suspension in water or a unit dosage form of an ampoule or vial by mixing it with water in combination with a stabilizer or buffer.

In addition, the active ingredient of the present invention generally has an effective dose of 1 to 50 mg / day, preferably 5 to 20 mg / day, per kg body weight of an adult patient and is administered at a predetermined time interval It may be administered several times a day, preferably two to three times.

The health food described above is a food prepared by adding the active ingredient to a food material such as a beverage, a tea, a spice, a gum or a confection, or encapsulating, pulverizing or suspending the food. However, unlike general medicine, there is an advantage that there is no side effect that can occur when a food is used as a raw material for a long period of taking the medicine.

Hereinafter, the present invention will be described in more detail with reference to the following examples. It is to be understood, however, that the scope of the present invention is not limited by the examples.

<Baekduguk Extraction Method>

The Baekdugu extract was obtained as follows.

In other words, the dried 200 g Baekduk was crushed and extracted with 95% ethanol for 2 weeks and then concentrated under reduced pressure to obtain 1.08 g of ethanol extract to obtain a yield of 0.54%. Dissolve the product in DMSO to obtain a high concentration of 100 mg / ml of reagent And diluted in cell culture broth to be used for the experiment.

EXPERIMENTAL EXAMPLE 1 : Measurement of Inhibitory Effect on the Growth of Coxsackievirus B3 of White Dug Extract

In order to confirm the potency and cytotoxicity inhibitory effect of the compound represented by Baekdugu extract of coxsackievirus B3, experiments were carried out using HeLa cells as follows.

The above-described Baekduguk extract was added to the cell culture medium by adding 100 μg / ml of the extract of Baekduguchi to HeLa cells cultured on a 96-well cell culture dish and treated with 10 ⁵ pfu / ml of Coxsackie virus B3 (CVB3) for 18 hours After infection, 10 μl of Cell Counting Kit 8 (CCK-8), a cell proliferation detection reagent, was added and further cultured for 2 hours. Then, the absorbance was measured at 450 nm using a microplate reader (Molecular device, USA) to confirm the viability of the cells and to confirm the antiviral efficacy.

Fig. 1, the survival rate of HeLa cells was increased dependently upon the treatment of Baekduguk extract after infection with Coxsackie virus B3 (CVB3). Thus, it is possible to confirm the excellent inhibition of Coxsackie B3 proliferation and cytotoxicity of Baekdugu extract.

Experimental Example 2: Confirmation of VP1 expression inhibition effect of Baekdugu extract>

The inhibitory effect of the Baekdugu extract on VP1 expression was examined by Western blot analysis as follows.

Treating the extract in the one hundred and two in HeLa cells 100-10 ug / ml concentration of the coxsackie virus ^{B3 (CVB3) 10 4 pfu /} ml were infected 37 ℃, in 5% CO ₂ and incubated for 18 hours. Proteins were extracted from the cultured cells, and cell lysates were prepared using a buffer solution and subjected to Western blotting.

Fig. 2 shows the Western blot analysis of the inhibitory effect of the protein VP1 of the cocktail virus B3 (CVB3) on the protein extracted from the cell extract of Baekduguchi extract. From this, it was confirmed that the direct coxsackievirus B3 CVB3), and the growth of Coxsackie virus was controlled by the concentration of Baekdugu extract. In particular, the transcription factor protein eIF4GI, which is cleaved by the protease 2A enzyme of the virus produced when the virus grows, is a very important protein for the production and survival of the cell. In order to inhibit the protein production of the virus, 2A. According to the treatment concentration of the compound, the destruction of eIF4GI abruptly decreased to leave much of the normal protein on the upper part. When the compound was not treated, the normal protein was destroyed by the production of protease 2A due to virus multiplication, Only partially confirmed. These results indicate that Baekdugu extract is effective in inhibiting CVB3 replication in Hela cells infected with CVB3.

Experimental Example 3 : Antiviral B3 efficacy test using an animal model of myocarditis infected with coxsackievirus>

A group (n = 20) in which 100 ug / ml of Baekdugu extract was injected into a 5-week old mouse and a group (n = 20) in which injection of Baekdugu extract was not injected intraperitoneally were prepared. ³ pfu / ml was infused through abdominal injection. The survival of mice with viral infection was observed until 20 days after the viral infection, and the inhibitory effect of the compounds on the growth of the cockskirus virus was verified. The survival rate of mice treated with Baekdugu extract was significantly higher than that of the group not administered with 50% (Fig. 3).

As a result, it was confirmed that the inhibitory effect of the compound of Chemical Formula 1 on the growth of coxsackievirus and the anticancer toxicity by infection were significantly observed in the experimental animal models as well as the cultured cells.

As can be seen from the above experimental results, the extract of Baekduguchi according to the present invention can show an excellent effect as an antiviral treatment agent.

Claims (4)

A method for inhibiting the growth of a coxsack virus, which comprises, as an active ingredient, at least one selected from the group consisting of Baekdugu extract and a pharmaceutically acceptable salt thereof. A health food for inhibiting the growth of coxsackievirus comprising at least one selected from the group consisting of Baekdugu extract and pharmaceutically acceptable salts thereof as an active ingredient. A pharmaceutical composition for the treatment and prevention of cold, which comprises the coxsackievirus inhibitor of claim 1. A pharmaceutical composition for treating and preventing myocarditis comprising the coxsackievirus inhibitor of claim 1.