JPH0778021B2 - Diarrhea virus infection inhibitor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a diarrhea virus infection inhibitor.

[Conventional technology]

Acute viral gastroenteritis (diarrhea) is a very common disease, but the causative virus has been isolated and identified only recently, and the development of therapeutic agents is just under study. So far, rotavirus, noroku virus, calicivirus, astrovirus, enteric adenovirus, coronavirus, predavirus and the like have been detected as causative viruses of viral diarrhea. Among them, rotavirus is widely distributed throughout the world and infects almost all mammals and birds from mouse to human, and diarrhea is overtly developed in infants and young animals. In addition, it is known that diarrhea causes death in infants estimated to be in the millions to ten millions a year, mainly in developing countries, and most of them are caused by rotavirus. Therefore, many antibiotics and vaccines have been developed to control the rotavirus, which is a pathogenic virus. However, effective antibiotics have not been found yet, and since vaccines affect infants and juveniles, it has been reported to be completely ineffective in live vaccine administration studies (DeMol). , P. Et al; Lance
t.II: 108,1986. Ebina Takusaburo; History of Medicine. 140 , 32,1
987).

In the livestock industry as well, diarrhea caused by rotavirus in calves causes death of calves, delay in growth, etc.
It's a big problem. Various preventive measures against this have been tried, but the use of antibiotics and the like has not yet been completely overcome because there are many problems to be solved such as residual in meat.

[Problems to be solved by the invention]

From the above-mentioned current situation, effective inhibition of diarrhea virus infection, easy and long-term administration to infants and young animals, which are the main targets, and no problems such as residue, and inexpensive mass production Development of possible diarrhea virus infection inhibitors is desired.

[Means for solving problems]

The present inventors show a high antiviral action against the causative virus of viral diarrhea, and as a result of repeated studies to find a highly safe substance in long-term continuous use, a plant of theaceae family, In particular, the tea we use for daily drinking (Came
The present inventors have found that the polyphenol compound contained in Llia sinensis, (L.) O. Kuntze) has excellent diarrhea virus infection inhibitory activity and has extremely low toxicity, and completed the present invention.

That is, the present invention relates to general formulas (1) and (2) (In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, R ₂ represents a hydrogen atom or a 3,4,5-trihydroxybenzoyl group. Is a diarrhea virus infection inhibitor containing a polyphenol compound represented by

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

The polyphenol compound of the present invention can be obtained from an ethyl acetate-soluble fraction of tea water or an extract of a water-soluble organic solvent, but it may be derived from other raw materials or a chemically synthesized product.

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

First, tea is extracted with a sufficient amount of water, hydrous acetone or hydrous alcohol. After extraction, the residue is separated by a known method to obtain an extract. The solvent was distilled off from the extract, water was added to the residue to dissolve it, and the mixture was partitioned using hexane, chloroform and ethyl acetate in that order, and the hexane soluble fraction, chloroform soluble fraction and ethyl acetate soluble fraction were separated. To get The distribution 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, distribution with hexane and chloroform should be performed. 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 to obtain chloroform-methanol (20: 1, V / V) and chloroform-methanol (1
The six compounds shown in Table 1 can be obtained by sequentially eluting with a solvent of 0: 1, V / V). If necessary, attach it to Sephadex LH-20 and elute with a suitable solvent such as methanol, or recycle.
By using HPLC (manufactured by Nippon Analytical Industry Co., Ltd., LC-908, GS-320 column, solvent methanol), it is possible to obtain higher purity six compounds of Table 1.

[Action]

Viruses to be inhibited by the diarrhea virus infection inhibitor of the present invention include rotavirus, norok virus, calicivirus, astrovirus, enteric adenovirus, coronavirus, and Bredavirus. It is presumed that the polyphenol compound, which is an active ingredient of the agent, has a direct action of directly acting on these viruses to inactivate them and also has an action of preventing the viruses from binding to target cells.

Hereinafter, detailed description will be made with reference to test examples.

(Test Example 1) 5 parts of the ethyl acetate-soluble fraction of a hot water extract of green tea (purity 74.1% as a mixture of the polyphenol compounds shown in Table 1) were used.
Human rotavirus Wa strain (serotype 1) 1.5 was dissolved in physiological saline so that the concentration becomes μg / ml, 1 μg / ml and 0.5 μg / ml.
After reacting with × 10 ⁴ FCFU / ml for 1 hour at 37 ℃, MA104 cells were infected and FCFU (fluorescent antibody positive focus unit) measurement was performed 20 hours later, and infection inhibition rate (%) compared to the control. Was calculated.

As the result is shown in Table 2, infection was blocked in a dose-dependent manner.

(Test Example 2) Compound VI shown in Table 1 was added at 1 μg / ml, 0.5 μg / ml and 0.01
Hydrotavirus Wa strain (serotype 1) 1.5 × 10 ⁴ FCFU / ml was dissolved in physiological saline to a concentration of μg / ml for 1 hour, 37
After reacting at 0 ° C, MA104 cells were infected and F after 20 hours
Infection inhibition rate (%) was calculated by CFU measurement compared with the control.

As the result is shown in Table 3, infection was blocked in a dose-dependent manner.

Test Example 3 As a result of testing the compound V described in Table 1 in the same manner as the compound VI of (Experimental example 2), infection was blocked in a dose-dependent manner as shown in Table 4.

(Test Example 4) An ethyl acetate-soluble fraction of a hot water extract of green tea (purity 74.1% as a mixture of polyphenol compounds shown in Table 1) was dissolved in physiological saline, and 100 μ thereof was dissolved on the 5th day after birth BALB / C mice were given orally. After 5 to 10 minutes, 1.0 × each of the human rotavirus Wa strain, Kun strain, and Mo strain against the same mouse.
Oral infection was carried out with 100 μl of virus solution containing 10 ⁵ FCFU, and an attack test was conducted. Thereafter, the presence or absence of diarrhea was observed every day for 5 days, and the result was evaluated based on the incidence of diarrhea (number of diarrhea mice / number of test mice). Control is saline 100μ
The same challenge test was carried out on the mice given with.

(Test Example 5) Acute toxicity experiment A group of 10 ddy mice was used, and a polyphenol compound (compound described in Test Example 1) suspended in physiological saline was kept at a constant temperature (23 ± 1 °) and constant humidity in each group. Orally administered under conditions of (55 ± 5%) Litchfield Wilcockson (Litc
hfield-Wilcoxon) result of obtaining LD ₅₀ by method 3 females.
It was 1 g / Kg, and male was more than 5 g / Kg.

(Test Example 6) Cytotoxicity test MA104 cells (monkey kidney cells) were added to 10% FCS-containing BHK cell medium (without addition of antibiotics) at 1.2 × 10 ⁵ cells / tube. Then, add the polyphenol compounds shown in Table 1 to 5 μg / ml, 1 μg / ml and 0.5 μg / ml, and add 3
After culturing at 7 ° C for 4 days, cell proliferation was examined. As a result, the growth curve was similar to that of the control containing only physiological saline, and no cytotoxicity was observed.

Next, the present invention will be described in more detail with reference to application examples, but the present invention is not limited thereby.

Application Example 1. Infant milk powder Commercially available milk powder 100 parts by weight Compound V described in Table 1 0.0001 100.0001 The above ingredients are mixed according to a conventional method.

Application example 2. Milk substitute for livestock larvae Full-fat milk powder 26 parts by weight Non-fat dry milk 40 Casein 9 Beef tallow 5 Coconut oil 1 Lactose 18 Vitamin / mineral mix 1 Compounds listed in Table VI 0.00005 100.00005 The above ingredients are 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 Powdered natural flavor 0.120 Vitamin C 0.0018 Mixture of compounds I to VI in Table 1 0.0001 Add water 100 parts by weight.

The product of the present invention can exhibit its effect by oral administration, and can be used by adding it to drinking water and feed of infants and young animals. The amount of the original polyphenol compound to be added varies depending on the mode of use, but it is desirable to administer the polyphenol compound so that the usual effective amount is 0.1 μg / Kg body weight or more.

〔The invention's effect〕

The polyphenol compound, which is an active ingredient of the present invention, exhibits a strong infection inhibitory action against the causative virus of acute viral gastroenteritis (diarrhea). Moreover, since this ingredient, which is effective in a very small amount, is a tea ingredient that has been used for drinking since ancient times, its safety is extremely high and it is possible to supply a large amount of diarrhea virus infection inhibitor. In addition, contributing to the eradication of infant mortality due to diarrhea in developing countries is of course very useful industrially.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takehiko Yamamoto 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Sun Kagaku Co., Ltd. (72) Inventor Nagataka Yamazaki 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Solarization Gaku Co., Ltd. Examiner Shinichi Naito

Claims (1)

[Claims] 1. General formulas (1) and (2) (In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, R ₂ represents a hydrogen atom or a 3,4,5-trihydroxybenzoyl group. A diarrhea virus infection inhibitor comprising a polyphenol compound represented by