JPH042574B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antiviral active substance, and more specifically, it is derived from the Hypericum erectum plant of the family Hypericumaceae, has a broad antiviral spectrum, has both virucidal action and virus growth suppressing action, and has cellular Concerning antiviral active substances with low toxicity. Various antiviral agents (chemotherapy agents) have been reported, but these conventional antiviral agents have high virus specificity and are only effective against specific viral infections. Basically, the mechanism of action is to inhibit some process of proliferation within the host cell. However, at the time of onset of the disease, virus proliferation often reaches its maximum, and the reality is that conventional antiviral agents based on the inhibition of viral proliferation alone cannot be expected to have much therapeutic effect. In the process of conducting intensive research in search of an antiviral agent with a wide antiviral spectrum and having not only a viral growth inhibiting effect but also a virucidal effect, the present inventor happened to find a drug that has a hemostasis, astringent, and gargling effect. The present inventors have discovered that a substance with extremely strong antiviral activity exists in the extracted components of Hypericum erectum, a member of the Hypericum family that is used in the private sector as a herbal medicine, and has completed the present invention. That is, the antiviral active substance provided by the present invention is a water-soluble component present in Hypericum perforatum plants of the family Hypericumaceae, and is a substance having the following physical and chemical properties. (1) Molecular weight is 1000 or less. Here, "molecular weight" is estimated by obtaining the active substance in the external dialysis fluid using a dialysis tube that cuts off the molecular weight of 1000 in Spectrapore Membrane Chewing manufactured by Spectrum Medical Industries, Inc. in the United States. This is what I did. (2) Solubility in solvents Easily soluble in water and methanol, and ethanol, n
-butanol, ethyl acetate, 2-butanone,
Slightly soluble to insoluble in 1,4-dioxane, chloroform, petroleum ether and ether. (3) Rf value on paper chromatogram (using Toyo Paper No. 526) (a) Rf value when using pyridine/n-butanol/distilled water (1:1:1) as the developing solvent
The Rf value is 0.75 to 0.78 (at 22 to 25℃), but (b) n-butanol/acetic acid/water (4:1:5) and n-butanol/ammonia/water (6:1 :2), n-butanol/ethanol/water/ammonia (40:10:49:
1), it stays at the origin in both cases. (4) Column chromatogram Column packed with cellulose support (e.g. Cellulofine GLL-90m) (e.g.
2.6 cm x length 72 cm) and elute with distilled water as the mobile phase at a flow rate of 1 ml/min, approximately 180 ml of effluent
The active substance is obtained in fractions ranging from ~270 ml. (5) Adsorption characteristics for ion exchange resin
IRA400) is also not substantially adsorbed. Therefore,
The active substance is considered to be a substantially neutral substance. (6) Stability against acids and bases An aqueous solution of the substance of the present invention is adjusted to pH 3 or 5 with hydrochloric acid, or pH 9 with sodium hydroxide.
Antiviral activity was retained even after treatment at ℃ for 60 minutes. (7) Stability against oxygen An aqueous solution of the substance of the present invention was prepared using β-Glucosidase.
(sigma NOG-8625) at 25℃ for 30 minutes,
The antiviral activity was maintained even after 12 hours of treatment. The buffer used at this time was acetic acid receiving buffer PH5. The above antiviral active substance is derived from Hypericum erectum Thun-
berg) can be produced by extraction and purification. Hypericum perforatum is a perennial plant that grows wild in the Gifu and Toyama regions, and in the present invention, the whole plant of Hypericum perforatum is harvested during the flowering period and extracted as is or after drying. Details of the extraction and purification operations will be described in Examples below, but the outline thereof will be as follows. Extraction is first performed with 50-100% ethanol. This ethanol extraction can be carried out by immersing Hypericum per 1 kg (dry weight) in 5 to 10 ethanol per kg (dry weight) for 3 to 5 days at a temperature of about 0 to about 100°C. Appropriate stirring may be performed during this time. After repeating the extraction 2 to 3 times, the ethanol extract was then dried under reduced pressure, and the solid residue was dissolved in a heterogeneous mixed solvent of chloroform/methanol/water (2.5 to 3 volumes of chloroform and 1 volume of methanol per 1 volume of water). After thorough mixing and stirring at about 4 DEG to about 30 DEG C., the methanol/water layer is separated and evaporated to dryness. The resulting residual solid content is further divided between n-butanol (saturated with water) and water at about 4 to about 30°C by thorough mixing and stirring, and the n-butanol (saturated with water) layer is separated and evaporated to dryness. harden A crude extract with antiviral activity is thus obtained, which can be further purified if necessary. Purification can be carried out by a column chromatography method known per se.
For example, the above crude extracted substance is dissolved in a 1-5% by weight aqueous sodium chloride solution (appropriate concentration is about 1-3% by weight), and the resulting solution is washed at least once with 1-2 times the volume of ethyl acetate. After that, the heavy sodium chloride solution is treated with a strongly acidic ion exchange resin, such as Amberlite.
IR120, Dowex 50, etc., and a strong basic ion exchange resin such as Amberlite IRA400, Amberlite IRA410, Dowex-1, etc. column at least once, preferably once or twice, to collect the antiviral active fraction. do. The fraction thus collected may be subjected to dialysis using a dialysis membrane with a molecular weight of 1000 and a cut-off ability, and a cellulose-based gel filter, such as Cellulofine GCL-90-m manufactured by Chitsuso Co., Ltd., as necessary.
Further purification can be achieved by using an appropriate combination of column chromatography using GCL-25m, GC-15m, etc. In this way, an antiviral active substance having the above-mentioned physicochemical properties can be obtained. The antiviral active substance provided by the present invention has the following biological activities (in addition,
(Specific activity data are shown in Reference Examples below), and can be used as a chemotherapeutic agent for the prevention and treatment of various viral infections. (1) The active substance of the present invention has an extremely broad antiviral spectrum. For example, the active substance exhibits strong antiviral activity against viruses such as herpes simplex virus, influenza virus, vesicular stomatitis virus, and rabies virus. In particular, although no substance capable of inhibiting the proliferation of rabies virus has yet been found, the active substance of the present invention is extremely unique in that it also has the ability to effectively inhibit the proliferation of rabies virus. Viral diseases are generally thought to have significantly altered cells at the time of onset, so it is extremely desirable to be able to administer chemotherapeutic agents with a wide spectrum of action early, without waiting for the identification of the causative virus, for the treatment and prevention of viral diseases. This is the case, and the active substance of the present invention is an innovative drug that can meet this need. (2) The active substance of the present invention has both virucidal and viral growth-inhibiting effects. No conventional antiviral agent is known to have both of these effects, and the active substance of the present invention is a novel antiviral agent that is completely different from conventional antiviral agents in this respect. Conventional antiviral agents basically have a mechanism of action that inhibits some process of virus multiplication within host cells. However, at the onset of illness, the proliferation of viruses is often at its maximum, and therefore, the reality is that conventional antiviral agents cannot be expected to have much therapeutic effect even if administered after onset of illness. In contrast, the active substance of the present invention not only has a viral growth-inhibitory effect but also has a strong virucidal effect, so even if administered after the onset of illness, it will not inactivate infectious virus particles outside the cells. This can help prevent the spread of lesions. (3) less cytotoxic than known antivirals; Since the virus multiplication process is dependent on the metabolic system of the host cell, suppression of virus multiplication is linked to damage to normal host cells, and conventional antiviral agents that inhibit virus multiplication inevitably suffer from side effects. . However, the active substance of the present invention is characterized by having extremely little toxic effect on normal cells. (4) The active substance of the present invention also has an immunostimulatory effect. The present invention will be further explained below using Examples and Reference Examples. Example 1 1 kg of dried Hypericum erectum was ground with a mixer and immersed in 80% ethanol 10 for 3 days to extract the components at room temperature. After removing the solvent with a rotary evaporator, chloroform/methanol/distilled water = 1000:500:375 was added to perform distribution, and the methanol-distilled water layer was taken. After drying this under reduced pressure, 500 ml of n-butanol was added. distilled water 500
ml, the liquid is separated, and the n-butanol layer is freeze-dried to obtain 25-30 g of crude extract. Dissolve 5 g of the above crude extract in 500 ml of 2.5% sodium chloride water, add an equal amount of ethyl acetate to separate the liquids, and take the sodium chloride water portion. This is a strongly acidic ion exchange resin,
Amberlite IR120 (manufactured by Rohm and Haas)
800 ml was passed through and the passed liquid was further treated with a strong basic ion exchange resin, Amberlite IRA400 (ROHM & Co., Ltd.).
(manufactured by Haas) onto a 300ml column. After concentrating the permeate using a rotary evaporator, it was transferred to a dialysis tube and Spectrapor memb-rane tubing (MW
cutoff: 1000, Spectrum Medical Industries
Inc.) and dialyzed against distilled water for several days, and the external solution is freeze-dried before proceeding to the next step. Approximately 30 mg of the freeze-dried product was dissolved in 0.5 ml of distilled water and placed on a column (diameter 2.6 cm x length 72 cm) filled with Cellulofine GCL-90m (manufactured by Chitsuso Co., Ltd.).
When elution is performed using distilled water as a mobile phase at a flow rate of 1 ml/min, the target substance flows out in a fraction of about 180 ml to about 270 ml of the effluent. This is lyophilized to obtain approximately 5 mg of active extract. This active extract exhibits the physical properties shown in (1) to (7) above, and further shows the infrared absorption spectrum and visible-ultraviolet absorption spectrum shown in FIGS. 1 and 2. Furthermore, the active extract was positive in coloring due to ferric chloride, and negative in the nitrogen detection reaction using metallic sodium. Reference Example Experiments conducted on the antiviral activity, toxicity to host cells, and immunostimulatory ability of the substance obtained in Example 1 above are shown below as a reference example.
In addition, in the following, the unit of virus concentration is
TCID ₅₀ (50% cell culture infectivity titer) was used, and the following viruses were used as sample viruses. (1) Herpes simplex virus: type 1 (HF strain): type 1 (miyama strain) Both of the above two strains were grown in Vero cells as hosts. (2) Influenza virus: A/Aichi/2/68 strain: A/RI/5 ^- (H ₂ N ₂ ) strain: A/PR/8/34 strain: B/Gifu/2/73 strain Except for the PR/8 strain grown in embryonated chicken eggs.
MDCK cells were used as hosts to grow. (3) Vesicular stomatitis virus: Propagated using Indiana strain X cells as hosts. (4) Rabies virus: PV strain: CV strain PV strain grows using BHK ₂₁ cells as a host.
For the CV strain, the brain emulsion of a mouse with rabies was used as a virus solution. (5) S antigen (HBs) of hepatitis B virus was isolated and purified from HBs-positive serum. Throughout all experiments, MEM medium (manufactured by Nissui) with 10% fetal bovine serum was used for cell growth, and 2% of the same serum was used for cell maintenance and virus dilution. 1 Virus inactivation test This was conducted according to the general disinfectant assay method. Mix 1 ml of virus at 10 ⁷ TCID ₅₀ /ml with 1 ml of a solution obtained by dissolving the extract of Example 1 at the concentration shown in Table 1 or 2 below, and immediately (0 minutes) take out 0.2 ml from the mixture and quickly add 10 ml of virus. Perform serial dilutions. Challenge a monolayer culture of host cells prepared in advance in a 24-well plastic tube with 0.5 ml/well of each dilution, then hold for 3 days like a mold to check for the appearance of cytopathic effect (CPE). Ru. Similarly, measure the amount of inactivated virus at 30 and 60 minutes. The mixed solution was maintained at room temperature or 37°C, and the temperature dependence of inactivation was also investigated. Results The inactivation status of herpes simplex virus (HF strain) is shown in Table 1 below.

[Table] The inactivation status of influenza virus is also shown in Table 2 below.

[Table] Both A/Aichi strain and B/Gifu strain had similar inactivation results. Furthermore, there was no difference in the inactivation status of any virus at room temperature or at 37°C. In addition, herpes simplex virus Miyama strain, influenza virus PR/8 strain, and rabies virus.
The virus that was inactivated by mixing each of the CV strains with the substance shown in Example 1 was inactivated in vivo (mouse, mouse, etc.).
Even when administered to ICR (5 animals in each group, 4 to 5 weeks old), the toxicity did not recover. The survival status is shown in Table 3 below.

[Table] Discussion In this test, a mixture of inactivating agent and virus is added to the host cell maintenance solution, so a large amount of virus is used to ensure that the inactivating agent has a concentration that does not affect virus proliferation within the cells. On the other hand, the substance was tested at a concentration that did not inhibit the intracellular growth of the virus when diluted 10 times. Therefore, it can be said that what was obtained here is the result of extracellular virus inactivation. It was also found that the strength of the inactivated state varies depending on the concentration of the substance. However, no temperature dependence is observed. It has been confirmed that viruses that have been partially inactivated with the extract of the present invention do not exhibit pathogenicity even when administered in vivo, and this is considered to be of great significance in this respect as well. 2 Hemagglutination inhibition test 50μ of purified HBs antigen (10μg/ml) and 50μ of the substance of the present invention (500μg/ml) are mixed and allowed to stand at room temperature for 30 minutes. Take 25μ of this mixture and perform 2-fold serial dilution on a 96-well U-shaped microplate, add 25μ of 1% HBs antibody-sensitized sheep red blood cells, stir, and react at room temperature for 60 minutes. judge accordingly. Results The substance of the present invention inhibited the agglutination of HBs-sensitized sheep red blood cells in 5 to 6 wells. This is a blocking rate of over 90% when converted to the amount of antigen. Of course, this substance alone does not agglutinate red blood cells. Discussion Possible phenomena that inhibit the hemagglutination reaction include (1) steric inhibition, (2) nonspecific inhibition, and (3) inhibition based on denaturation of viral antigens. Therefore, it is necessary to examine which of the above mechanisms (1) to (3) is responsible for the inhibition phenomenon of the substance of the present invention. So first (1)
Considering this, it is found that the substance of the present invention has a molecular weight of 1000
Since the following is considered, it can be ruled out that steric hindrance occurs. Regarding (2), when the presence or absence of inhibition of aggregation in the reaction system was examined based on an anti-antibody measurement method that does not involve viral antigens, this substance had no effect in the anti-antibody measurement method. Therefore, the hemagglutination inhibition reaction of the substance of the present invention is thought to act specifically on the reaction system involving viral antigens. 3 Suppression of virus proliferation (1) For herpes simplex virus, influenza virus, and vesicular stomatitis virus, incubate each host cell (Vero, MDCK,
Remove the culture medium, challenge with 10 ² TCID ₅₀ viruses, and after adsorption for 60 minutes, add 1 part of the maintenance solution.
Pour through the ml/hole. The time when this maintenance solution is added is set as 0 minutes, and after that, a substance is added at an arbitrary time to challenge the virus, and the infectious virus in the maintenance solution is quantified 72 hours later. Yield measurement is 96
Judgment is made by CPE using a hole microplate. (2) For rabies virus, BHK ₂₁ (1×10 ⁶ /
ml) and 0.1 ml of PV strain (10 ⁴ TCID ₅₀ ) were mixed, and 0.1 ml of the substance obtained in Example 1 (500-125 μg/ml) dissolved in serum-free medium was added to the mixture. were planted in Laptek (manufactured by Laptek) and kept in an incubator containing 5% carbon dioxide gas at 37°C. 48 years after planting
After 72 hours, perform fluorescent antibody staining using a direct method to check for the presence of viral antigens.
The amount of virus is also quantified when the culture is maintained for a certain period of time. The suppression status of these virus proliferation is shown in Table 4 below. Note that this result was obtained when the substance was added 0 minutes after the virus challenge. Result

[Table] In the case of herpes simplex virus, the results when the substance was added only once at 2, 4, 8, and 12 hours after virus challenge were the same as at 0 minutes. Commercially available Adenine Arabinoside (Ara
-a) (Warner-Lambert/Parke Davis,
Manufactured by Clinical Research Department) as a control
When the growth inhibition test of HF strain was conducted in the same way, Ara−
a 100% inhibition was observed with the addition of 50 μg/ml. Discussion The proliferation of viruses begins with adsorption to cells and is completed through several processes thereafter. Although it is still unclear at which stage the substance of the present invention acts, it is assumed that the minimum inhibitory concentration will be relatively small. In addition, it is considered effective against respiratory diseases such as influenza, for which vaccines are not very effective, and persistently infectious viruses such as herpes simplex, as well as against rabies virus, for which there have been no reports of substances that can inhibit its proliferation. The substance of the present invention is very unique. 4 Effect on cell proliferation Sow the cells in a 12-well plastic jar so that they form a monolayer within 3 days. The state of cell proliferation 24 hours, 48 hours and 72 hours after the addition of the substance at the time of implantation can be examined in accordance with a conventional method for incorporation of ^6-3H -thymidine into the acid-insoluble fraction. The isotope was added at 1 microcury/well and allowed to be taken up into the cells for 24 hours in each case. The results of comparison with the material obtained in Example 1 using Ara-a as a control for this experiment are shown in FIGS. 3 to 6. Discussion Substances reported as antiviral agents have difficulty in use due to toxicity issues. Currently, the clinical antiviral agent is Ara-a.
However, when comparing this with the substance of the present invention, as described in Reference Example 3, the substance of the present invention has
Approximately half the amount of Ara-a inhibits the proliferation of the same amount of herpes simplex virus. Furthermore, as shown in Figures 5 and 6 of this experiment, regarding cytotoxicity,
Less results were obtained than Ara-a. As mentioned above, since there have been no reports of substances that have a lower effective dose and lower toxicity than currently used antiviral agents, it is thought that the effectiveness of the substance of the present invention is high. 5 Antiviral effect in living organisms Mouse (ICR, male, 6.5-7
Weekly age, average 30g (introduced from Japan Claire),
The Miyama strain of herpes simplex virus (brain-tropic strain) was used as the virus. Divide into groups of 10 and first inject all groups with the Mriyama strain.
Administer 10LD ₅₀ intraperitoneally. Thereafter, the substance of the present invention was administered according to the experimental schedule shown below, and comparisons were made with a group administered with distilled water, which was used as a control. The presence or absence of an effect was determined based on whether the drug was alive or dead.
The results are shown in Table 5 below. Result

【table】

[Table] Discussion When mice are administered herpes simplex virus, which has affinity for the brain, they usually die on the 5th to 7th day. However, by intravenously administering 100 μg of the substance of the present invention three times, about half of the life-saving effects were obtained.
As for the route of administration, intravenous injection appears to be preferable to subcutaneous injection. Furthermore, although there remains a possibility that the effect will further increase by increasing the dosage, the issue of side effects should also be examined in detail. However, 100μ
No change in appearance was observed in sample g.

[Brief explanation of drawings]

Figure 1 is an infrared absorption spectrum of the active extract obtained in Example 1, Figure 2 is a visible-ultraviolet absorption spectrum of the active extract obtained in Example 1, and Figure 3 is an Ara FIG. 4 is a graph showing the influence of Ara-a on Vero cell proliferation.
5 is a graph showing the influence on cell proliferation;
The figure is a graph showing the influence of the substance of the present invention on Vero cell proliferation, and Figure 6 is a graph showing the effect of the substance of the present invention on Vero cell proliferation.
It is a graph showing the influence on cell proliferation.

Claims (1)

[Scope of Claims] 1 (a) present in Hypericum perforatum plants of the family Hypericumaceae, (b) having a molecular weight of 1000 or less, (c) easily soluble in water and methanol, and containing ethanal, n.
-butanol, ethyl acetate, 2-butanone,
It is sparingly soluble or insoluble in 1,4-dioxane, chloroform, petroleum ether, and ether; (d) Paper chromatogram [Toyo Paper No. 526;
Developing solvent = pyridine/n-butanol/distilled water (1:1:1)] Rf value is 0.75 to 0.78 (22 to 25
°C), and (e) a substantially neutral antiviral active substance.