JPH01265023A - Agent for inhibiting infection with diarrhetic virus - Google Patents

Abstract

PURPOSE:To obtain the objective agent effective in inhibiting the infection with diarrhetic virus, easily applicable mainly to human and animal infant, enabling long-term administration and having low toxicity, by using a polyphenol compound which is a component of tea as an active component. CONSTITUTION:The objective agent can be prepared by using a polyphenol compound of formula I and formula II (R1 is H or hydroxyl; R2 is H or 3,4,5- trihydroxybenzoyl) as an active component, mixing with conventional additives and forming in the form of oral drug by conventional process. The dose of the active component is 0.1mug/kg. The viruses to be inhibited are rotavirus, norowkvirus, calicivirus, astrovirus, enteroadenovirus, coronavirus, bredavirus, etc. The compound of formula I and II can be extracted from a plant of family Cameliaceae, especially from the component of green tea used as ordinary drink.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a diarrheal virus infection inhibitor.

[Conventional technology]

Acute viral gastroenteritis (diarrhea) is a very common disease, but the virus that causes it has been isolated.

It has only recently been identified, and the development of therapeutic agents has just begun research. So far, rotavirus, Noriou virus, calicivirus, astrovirus, enteric adenovirus, coronavirus, and predavirus have been detected as the causative viruses of viral diarrhea. Among them, rotavirus is widely distributed throughout the world and infects most mammals and birds, from mice to humans, and causes overt diarrhea in infants and young animals.

Moreover, worldwide, mainly in developing countries, millions of
An estimated 10,000,000 infants and children die from diarrhea, many of which are known to be caused by rotavirus. Therefore, in an effort to control rotavirus, which is a pathogenic virus,
To date, numerous antibiotics and vaccines have been developed. However, no effective antibiotic has yet been found, and since the affected individuals are infants and young animals, it has been reported that live vaccines are completely ineffective in administration tests (DeMo
l,P,et alHLancet,I[:108,
1986. Ebina Kuruma Part 3; History of medicine, 140, 32
．． 1987).

In the livestock industry, diarrhea caused by rotavirus in calves can cause calf death or growth retardation.
This is a big problem. Although various preventive methods have been attempted against this problem, the use of antibiotics has many problems that need to be resolved, such as the presence of residue in meat, and the problem has not yet been completely overcome.

[Problem that the invention aims to solve]

Based on the above-mentioned current situation, it is possible to effectively inhibit the infection of diarrheal virus, to be easy to administer to infants and young animals, which are the main targets, to be able to be administered for a long period of time, to have no problems such as residue, and to be able to be mass-produced at low cost. The development of a possible diarrheal virus infection inhibitor is desired.

[Means for solving problems]

The present inventors have conducted intensive research to find a substance that has a high antiviral effect against viruses that cause viral diarrhea and is highly safe for long-term use. In particular, the tea we drink every day (Ca
mellia 5inensis, (L,)O,Ku
The present invention was completed based on the discovery that polyphenol compounds contained in A.

That is, the present invention provides formulas (1) and (2) (wherein R2 represents a hydrogen atom or a hydroxyl group, and R
2 represents a hydrogen atom or a 3,4.5-trino

The polyphenol compound of the present invention refers to the six types of catechin analog compounds shown in Table 1 represented by general formulas (1) and (2).

Table 1 Formula R, R, Generic name Compound I (1) HH(+)-Chicken compound n(2) HH(-)-Epicabuquin compound I [(1)
Ot(H(+)-Mrocatechin Compound IV
(2) OHH(-)-epigalocachkin The polyphenol compound of the present invention can be obtained from the ethyl acetate soluble fraction of tea water or a water-soluble organic solvent extract, but it may also be obtained from other raw materials or chemically synthesized products. But it doesn't matter.

A typical method for preparing the polyphenol compound of the present invention is as follows.

First, tea is extracted with a sufficient amount of water, aqueous acetone, or aqueous alcohol. After extraction, the residue is separated by a known method to obtain an extract. The solvent was distilled off from the extract, and the residue was dissolved in water and then dissolved in hexane.

Partitioning was performed using chloroform and ethyl acetate sequentially.
A hexane soluble fraction, a chloroporum soluble fraction and an ethyl acetate soluble fraction are obtained. Partitioning with hexane and chloroform in this operation can be omitted depending on the coloring degree and viscosity of the tea extract, but in order to increase the purity of the ethyl acetate soluble fraction, partitioning with hexane and chloroform can be omitted. is desirable.

As the alcohol that can be used for extraction, lower alcohols such as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, and butyl alcohol are preferable from the viewpoint of operability and extraction effect.

Furthermore, the ethyl acetate soluble fraction obtained above was subjected to silica gel column chromatography and added to the solvents of chloroform-methanol (20:1.V/V) and chloroform-methanol (10:1.V/V). By sequentially eluting with the following steps, the six compounds shown in Table 1 can be obtained. If necessary, it may be further subjected to Sephadex LH-20 and eluted with a suitable solvent such as methanol, or recycled HPLC (Japan Analytical Industrial Bag).

LC-908, G5-320 column, solvent methanol)
By using this, it is possible to obtain the 6 compounds of Table 1 with higher purity.

[Effect]

Viruses that can be inhibited by the diarrheal virus infection inhibitor of the present invention include rotavirus, Noriou virus, calicivirus, astrovirus, monogenic adenovirus,
The polyphenol compound, which is the active ingredient of the inhibitor of the present invention, has a direct action to directly act on and inactivate these viruses, such as coronavirus and predavirus, and also prevents the viruses from binding to the target small chest. It is presumed to have an effect.

The details will be explained below using test examples.

(Test Example 1) Ethyl acetate soluble fraction of hot water extract of green tea (purity 74.1% as a mixture of polyphenol compounds shown in Table 1)
2μg/mj2.1μg/m 12 and 0.5μ
g/m Dissolved in physiological saline at 16 degrees,
Human rotavirus Wa strain (serotype 1) 1.5X10'
After reacting with FCFU/mN for 1 hour at 37°C, MA
104 cells were infected, and the infection inhibition rate (%) was calculated by measuring FCFU (fluorescent antibody positive focus units) 20 hours later in comparison with the control.

As shown in Table 2, infection was inhibited in a dose-dependent manner.

Table 2 Ethyl acetate soluble fraction of green tea extract (virus control average 60.2 (±5.64)) (Test Example 2) Compound ■ shown in Table 1 was added at 1 μg/mp, 0. .

Human rotavirus Wa was dissolved in physiological saline to a concentration of 5 μg/mN and 0.
After reacting with 1.5X 10' FCFU/ml of the strain (serotype 1) for 1 hour at 37°C, MA104 cells were infected, and the infection inhibition rate (%) was determined by measuring FCFU 20 hours later compared to the control. ) was calculated.

As shown in Table 3, infection was inhibited in a dose-dependent manner.

Table 3 Compound ■ (Virus control average 60.2 (±5.64)
) (Test Example 3) Compound V listed in Table 1 was tested in the same manner as Compound ■ in (Experiment Example 2), and as a result, infection was inhibited in a dose-dependent manner as shown in Table 4.

Table 4 Compound V (virus control average 60.2 (±5.64)
) (Test Example 4) Ethyl acetate soluble fraction of hot water extract of green tea (purity 74.1% as a mixture of polyphenol compounds listed in Table 1)
Dissolve it in physiological saline and add 100μ of it to 5 days after birth B.
Orally given to ALB/C mice. After 5 to 10 minutes, the same mice were infected with human rotavirus strains Wa, Kun, and Mo.

1 of each, 100μ of virus solution containing OXI O' FCFU! A challenge test was conducted by orally infecting the animals.

Thereafter, the presence or absence of diarrhea was observed every day for 5 days, and the results were evaluated based on the diarrhea incidence (number of mouse strokes with diarrhea/number of test mice). As a control, a similar challenge test was performed on mice given 100 μl of physiological saline.

Table 5 (Test Example 5) Acute Toxicity Experiment A group of 10 DDY mice was used, and each group was given a polyphenol compound (compound described in Test Example 1) suspended in physiological saline at a constant temperature (23 ± 1°). , orally administered under conditions of constant humidity (55 ± 5%).
As a result of determining LD by the method, it was 3.1g/Kg for females.
In males, it was 5 g/Kg or more.

(Test Example 6) Cytotoxicity test MA104 cells (monkey kidney cells) were
The cells were added to a 10% FC8-containing BHK cell medium (no antibiotics added) at a concentration of 11/l cube. Add to it 5 μg/m of polyphenol compounds shown in Table 1.
j! , 1 μg/m R and 0.5 μg/m 4
2, cultured at 37°C for 4 days, and examined cell proliferation. As a result, the growth curve was similar to the control in which only physiological saline was added, and no cytotoxicity was observed.

Next, the present invention will be explained in detail using application examples, but the present invention is not limited thereby.

Application example 1. Powdered milk for infants Commercially prepared powdered milk 100 parts by weight Compounds listed in Table 1 vo, oootloo, 0001 The above formulation is mixed according to a conventional method.

Application example 2. Milk substitute for young livestock Whole blood powder 26 Parts by weight Bloodless milk powder 40 Casein 9 Beef tallow 5 Coconut oil 1 Lactose 18 Vitamin/mineral mix 1 Compound listed in Table 1 VI O,0000510
0,00005 The above formulation is mixed according to a conventional method.

Application example 3. Beverage composition glucose 4.8 parts by weight fructose
0.776 Powdered citric acid 0.144 Sodium citrate 0.102 Calcium lactate
0.012 Magnesium Chloride 0.012 Powder Natural Flavor 0.120 Vitamin CO,00
18 Mixture of compounds ■ to ■ listed in Table 1 Add 0.0001 water to 10
0 parts by weight.

The product of the present invention can exert its effects when administered orally, and can be used by being added to the drinking water or feed of infants and young animals. Although the amount of the original polyphenol compound to be added varies depending on the mode of use, it is generally desirable to administer it at an effective amount of 0.1 μg/Kg body weight or more.

〔Effect of the invention〕

The polyphenol compound, which is the active ingredient of the present invention, exhibits a strong infection-inhibiting effect against the virus that causes acute viral gastroenteritis (diarrhea). In addition, this ingredient, which is effective in extremely small amounts, is an ingredient of tea that has been used for drinking since ancient times, so it is extremely safe, and it is possible to supply large quantities of the diarrhea virus infection inhibitor. It is thought that it will not only contribute to the eradication of infant mortality due to diarrheal diseases in developing countries, but also be extremely useful industrially.

Claims (1)

[Claims] (1), general formulas (1) and (2) ▲There are mathematical formulas, chemical formulas, tables, etc.▼----(1) ▲There are mathematical formulas, chemical formulas, tables, etc.▼----(2) (In the formula R_1 represents a hydrogen atom or a hydroxyl group,
R_2 is a hydrogen atom or a 3,4,5-trihydroxybenzoyl group ▲ Numerical formula, chemical formula, table, etc. ▼) A diarrheal virus infection inhibitor containing a polyphenol compound as an active ingredient.