JP2010090066A - Anti-sars coronavirus agent and product containing anti-sars coronavirus agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-SARS coronavirus agent having excellent anti-SARS coronavirus action and a product containing the anti-SARS coronavirus agent. <P>SOLUTION: The anti-SARS coronavirus agent contains, as active components, a guava extract extracted by using an extraction solvent containing at least one kind of solvent selected from water and hydrophilic organic solvents as a main component, and ascorbic acids. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an anti-SARS coronavirus agent excellent in anti-SARS coronavirus action and a product containing an anti-SARS coronavirus agent.

  SARS (Severe Acute Respiratory Syndrome) is an infection caused by the SARS virus, which was recently discovered as a new coronavirus. Major symptoms include respiratory symptoms such as fever above 38 ° C., cough, shortness of breath, and difficulty breathing. Symptoms such as diarrhea, headache, chills, loss of appetite, general malaise, and consciousness may be observed. At present, an effective curative treatment for SARS has not been established, and symptomatic treatment is the center.

There are also reports that therapeutic effects can be expected by combination therapy of antiviral Ribavirin and steroids, treatment with interferon α, β, γ, glycyrrhizin and the like. There is also a report that proanthocyanidins and catechins derived from natural products such as grapes and cranberries exert an antiviral action against SARS coronavirus (see Patent Document 1). However, an effective treatment method has not yet been established.
JP 2005-314316 A

  The present invention has been made based on the finding that guava extract and ascorbic acid exhibit antiviral action against SARS coronavirus as a result of intensive studies by the present researchers. The object is to provide an anti-SARS coronavirus agent and an anti-SARS coronavirus agent-containing product having an excellent anti-SARS coronavirus action.

  In order to achieve the above object, the anti-SARS coronavirus agent according to claim 1 comprises a guava extract extracted with an extraction solvent containing at least one of water and a hydrophilic organic solvent as a main component, and ascorbine. It contains acids as an active ingredient.

The anti-SARS coronavirus agent according to claim 2 is characterized in that, in the invention according to claim 1, the guava extract is derived from a guava leaf or stem.
The anti-SARS coronavirus agent according to claim 3 is characterized in that, in the invention according to claim 1 or claim 2, the pH is adjusted to 5.4 or less.

  The anti-SARS coronavirus agent according to claim 4 is an extraction solvent further comprising at least one of water and a hydrophilic organic solvent as a main component in the invention according to any one of claims 1 to 3. It contains the koji extract extracted by the above.

  The anti-SARS coronavirus agent-containing product according to claim 5 contains the anti-SARS coronavirus agent according to any one of claims 1 to 4.

  ADVANTAGE OF THE INVENTION According to this invention, the anti- SARS coronavirus agent excellent in the anti- SARS coronavirus action and the anti-SARS coronavirus agent containing product can be provided.

Hereinafter, an embodiment embodying the present invention will be described in detail.
The anti-SARS coronavirus agent of this embodiment contains guava extract and ascorbic acid extracted with an extraction solvent containing at least one of water or a hydrophilic organic solvent as a main component as active ingredients. Guava (Psidium Guajava Linn), which is a raw material for the extract of guava, is a plant that is also called bunjiro or banzakuro, and is an evergreen small tree in the tropics belonging to the genus Phylumaceae and the genus Vanjiro. Guava grows naturally in the Caribbean coast, northern Central America, North America, Southeast Asia, and is cultivated in warm regions such as Okinawa in Japan. In addition, the production area of the guava used in this embodiment is not specifically limited.

  The raw material of the guava extract contained in the anti-SARS coronavirus agent of the present embodiment is not particularly limited, and any of guava organs and their constituent components can be used. Each of these organs and components may be used alone as a raw material, or two or more kinds may be mixed and used. However, from the viewpoint of improving the anti-SARS coronavirus action, the raw material preferably contains at least leaves or stems. Such raw materials can be collected, crushed, crushed or ground after collection, dried after collection, crushed, crushed or ground after collection / dry, or crushed, crushed or ground after collection, The extraction operation can be performed in the dried state. In addition, it is preferable to use the crushed raw material from a viewpoint of efficiency of extraction operation. Moreover, it is preferable to use a raw leaf as a raw material from a viewpoint of an anti-SARS coronavirus action improvement.

  As an extraction solvent for extracting the guava extract from the raw material, an extraction solvent containing at least one of water and a hydrophilic organic solvent as a main component is used. Here, the extraction solvent containing at least one of water and a hydrophilic organic solvent as a main component means a solvent in which the component having the highest abundance ratio (volume%) in the solvent is water or a hydrophilic organic solvent. is doing. And the content of water or hydrophilic organic solvent in the solvent (when both water and hydrophilic organic solvent are contained, the total value of both) is preferably 50% by volume or more. Examples of the hydrophilic organic solvent include lower alcohols such as methanol and ethanol, acetone, and ethyl acetate. In addition, as the extraction solvent, a single kind of water and various hydrophilic organic solvents may be used, or a mixed solvent obtained by mixing a plurality of kinds may be used. The extraction solvent may contain a small amount of a solvent other than water and a hydrophilic organic solvent, and other additives such as organic salts, inorganic salts, buffers, and emulsifiers are dissolved. Also good.

  The extraction operation is performed by immersing the raw material in an extraction solvent for a predetermined time. In that case, it is desirable that the extraction solvent is added so as to have a bath ratio of 2 to 100, preferably 5 to 50, more preferably 20 to 40 with respect to the raw material. The bath ratio here means the volume (L) of the extraction solvent relative to 1 kg of the raw material. Moreover, you may perform processes, such as stirring operation, heating, and pressurization, as needed. The extraction conditions during the extraction operation vary depending on the state of the raw material and the extraction solvent used, but when the extraction temperature is high, the extraction can be performed in a shorter time. For example, the guava extract can be sufficiently extracted if it is 10 days at room temperature under a pressure of 1 to 2 atmospheres. Further, at a temperature of 50 to 130 ° C. under a pressure of 1 to 2 atmospheres, the guava extract can be extracted by an extraction treatment for 1 to 200 minutes, preferably 3 to 60 minutes, more preferably 5 to 30 minutes. .

  Then, by performing a solid-liquid separation operation after the extraction operation, the guava extract and the raw material residue are separated. As a method of such solid-liquid separation operation, for example, a known separation method such as filtration or centrifugation can be used. The obtained guava extract may be used as it is as a guava extract, or a guava extract obtained by further concentration and drying may be used as a guava extract.

  Examples of ascorbic acids contained in the anti-SARS coronavirus agent of this embodiment include at least one selected from ascorbic acid, ascorbate and ascorbate. Examples of ascorbate include sodium ascorbate, potassium ascorbate, and calcium ascorbate. Moreover, ascorbic acid ester includes ascorbic acid phosphate and ascorbic acid stearate. In the anti-SARS coronavirus agent of this embodiment, ascorbic acids are preferably blended at a mass ratio of 1: 0.05 to 1: 5 with respect to the guava extract (dry mass). More preferably, it is blended at a ratio of 0.1 to 1: 2.

  In addition, the anti-SARS coronavirus agent of this embodiment is preferably blended with an extract derived from a plant material. Examples of the plant material include berries such as blueberries, cranberries, and elderberries, cassis, grapes, grapes, tea leaves, turmeric, perilla, plantains, hops, and propolis. Among these plant materials, it is preferable to use a koji-derived extract from the viewpoint of improving the promoting action of the anti-SARS coronavirus action. In addition, these plant material-derived extracts can be obtained by the same method as the guava extract.

  In addition, the anti-SARS coronavirus agent of the present embodiment is preferably adjusted so that its pH is not more than a specified value. The pH of the anti-SARS coronavirus agent is preferably 5.4 or less, and more preferably 4.5 or less, from the viewpoint of enhancing the action of the anti-SARS coronavirus action. The lower limit of the pH is not particularly limited, but is preferably pH 2.0 or more from the viewpoint of ease of adjustment. The pH is adjusted by adding an organic acid. Examples of such organic acids include citric acid, malic acid, tartaric acid, and acetic acid. Moreover, you may use fruit juices (for example, fruit juice derived from citrus fruits, plums, berries, apples, and grapes) and vinegars (for example, grain vinegar and fruit vinegar) containing these organic acids.

  The anti-SARS coronavirus agent of the present embodiment can be used by applying to a product intended to exert an anti-SARS coronavirus action. Examples of such anti-SARS coronavirus-containing products include medical agents (including drugs), cleaning agents, cosmetics, medical equipment, sanitary materials, sanitary products, food and drinks, and the like. Examples of the medical agent include pharmaceuticals. Examples of the detergent include laundry softeners, laundry bleaches, soaps, bathing agents, detergents, shampoos, and toothpastes. Examples of the cosmetics include face washing soaps, hand soaps, hand creams, perfumes, skin lotions, milky lotions, beauty essences, beauty creams, lipsticks, lip balms, and funny. Examples of the medical device include a mask, bandage, gauze, glove, adhesive bandage, cotton ball, diagnostic mechanical device, surgical mechanical device, therapeutic mechanical device, hospital mechanical device, dental mechanical device, and medical device. Auxiliary instruments, orthodontic instruments, etc. are mentioned. Examples of the sanitary material include gauze, absorbent cotton, nonwoven fabric, and cotton swab. Examples of the sanitary product include a humidifier, an air conditioner, and an air purifier. Examples of the food and drink include health drinks and health foods. In addition, quasi-drugs such as gargle, throat cake, throat spray, medicated soap, disinfectant, etc. are included in the medical agent or the cosmetic.

  When the anti-SARS coronavirus agent of this embodiment is applied to pharmaceuticals or quasi-drugs, all administration methods such as intravascular administration and transdermal administration other than administration by oral administration It is possible to employ a method of applying to the skin or article. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, an application agent etc. are mentioned. In addition, excipients, bases, emulsifiers, solvents, stabilizers and the like as additives may be blended as long as the object of the present invention is not impaired.

  When the anti-SARS coronavirus agent of this embodiment is applied to food and drink, it is added to various food materials or beverage materials, for example, powder, tablet, granule, liquid (drink Preparations), capsules, syrups, candies and the like, and can be used as health food preparations, dietary supplements and the like. Specific examples of the food and drink include sports drinks, tea beverages extracted from tea leaves and herbs, dairy products such as milk and yogurt, foods containing gelling agents such as pectin and carrageenan, hard candy and soft candy, Beverages containing candies such as gummi, chewing gum, sugars such as glucose, sucrose, fructose, lactose, dextrin, flavorings, sweeteners such as stevia, aspartame, sugar alcohols, and fats such as vegetable oils and animal fats Goods and groceries. In addition, a substrate, an excipient, an additive, a secondary material, a bulking agent, and the like may be added as appropriate within a range that does not impair the object of the present invention.

Next, operational effects in the present embodiment will be described below.
(1) The anti-SARS coronavirus agent of this embodiment contains, as active ingredients, guava extract and ascorbic acid extracted with an extraction solvent containing at least one of water or a hydrophilic organic solvent as a main component. Yes. Thereby, the excessive proliferation of SARS coronavirus can be suppressed and the outstanding anti- SARS coronavirus is exhibited.

  (2) The anti-SARS coronavirus agent of the present embodiment is preferably formulated with an extract derived from a plant material. Thereby, the anti-SARS coronavirus action of the anti-SARS coronavirus agent can be further enhanced.

  (3) The pH of the anti-SARS coronavirus agent of this embodiment is preferably adjusted to 5.4 or less. Thereby, the anti-SARS coronavirus action of the anti-SARS coronavirus agent can be further enhanced.

(4) Since the guava extract contained in the anti-SARS coronavirus agent of this embodiment is derived from a natural product, it is highly safe and can be easily applied to pharmaceuticals and foods and drinks.
In addition, this embodiment can also be changed and embodied as follows.

-You may use the anti- SARS coronavirus agent of this embodiment for animals other than humans, such as a horse and a cow, for example.
-The anti-SARS coronavirus agent of this embodiment can be used not only as a therapeutic agent for infected or diseased patients, but also for healthy people to prevent infection.

Next, the embodiment will be described in more detail with reference to each test example.
<Test for raw materials>
(Preparation of Test Examples 1 to 6)
First, as a preliminary test, the anti-SARS coronavirus action for each site of guava was evaluated. The guava was separated into fruits, seeds, roots, stems, and leaves (tea leaves), and these were extracted as raw materials (100 g). A solid-liquid separation process was performed, and the resulting extract was concentrated to dryness to obtain a powdery extract. Each obtained extract was dissolved in PBS (−), and these were designated as Test Examples 1 to 6. The extraction conditions and the yield of each extract during the extraction process for each part are shown in Table 1 below.

(Test on therapeutic effect)
Next, with respect to Test Examples 1 to 6, the anti-SARS coronavirus action (therapeutic effect) was evaluated.

  First, the SARS-CoV (human isolated Frankfurt FFM-1 strain) stock solution (108 PFU / ml) was diluted with a 1% BSA-containing phosphate buffer solution (Mg2 +, Ca2 + -free: PBS (-), pH 7.0). , 103 PFU / ml virus solution was prepared.

  Next, Vero cells (derived from African green monkey kidney cells) were cultured in 6-well culture plates using MEM-10% FBS medium. When Vero cells formed a monolayer of 70-80%, 200 μl of the virus solution was added to each cell, and SARS-CoV was inoculated into Vero cells. After infecting at 22 ° C. for 1 hour, 200 μl of a sample solution obtained by diluting each of Test Examples 1 to 6 to a predetermined concentration (twice the target treatment concentration) was added to each cell, and then at 22 ° C. for 1 hour. Processed.

After the treatment, each cell was overlaid with 0.9% methylcellulose-containing D-MEM medium (non-immobilized 5% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin). After culturing at 37 ° C. for 6 to 7 days, the medium is removed and washed with PBS, and cells are fixed and stained with 2.5% crystal violet solution (containing 30% ethanol and 1% ammonium oxalate). It was. Thereafter, the cells were washed with PBS (−) and irradiated with UV, and the number of plaques formed in each cell was measured. Then, based on the measurement data E obtained for each test example 1-6, the concentration of reducing the number of plaques by 50% (50% inhibitory concentration: _{IC 50)} was calculated. Further, from the yield of the extract and the IC ₅₀ value as the therapeutic effect, the amount of raw material (raw material converted value) necessary for preparing 1 l of the sample solution showing IC ₅₀ was calculated. The results are shown in Table 1 below.

  From the therapeutic effects and raw material conversion values of Test Examples 1 to 5, it was confirmed that active ingredients can be efficiently extracted in the order of leaf> stem >> seed> root = fruit. From this result, it was confirmed that stems and leaves are suitable as raw materials. In addition, from the results of Test Examples 5 and 6, it was confirmed that the raw state was more suitable as a raw material than the dry state. Furthermore, although data are not shown, the therapeutic effect was increased when hot water extraction was used as the extraction condition.

<Test on anti-SARS coronavirus action>
(Preparation of anti-SARS coronavirus agent)
The dried guava tea leaves were extracted with hot water at a bath ratio of 30 and 95 ° C. for 8 minutes, and then subjected to solid-liquid separation to obtain a guava extract. Similarly, extraction / separation processing was performed on bamboo leaf tea leaves (dried) to obtain bamboo leaf extract. In this test, guava extract and persimmon leaf extract were used as guava extract and persimmon extract, respectively. And the said guava extract, the said koji extract, and ascorbic acid were mixed so that it might become the ratio shown in following Table 1, and the pH was adjusted so that it might become a value shown in following Table 2. The pH was adjusted by appropriately adding anhydrous citric acid and sodium bicarbonate. This was heat sterilized at 95 ° C. to obtain Examples 1 to 7. Moreover, what contains only the said guava extract was prepared, and this was used as the comparative example 1.

(Test on therapeutic effect)
The therapeutic effect of anti-SARS coronavirus agents against SARS virus was evaluated.
The evaluation of the therapeutic effect was performed by the same method as the test for the raw material (test for the therapeutic effect). In addition, the sample solution used the solution which diluted Examples 1-7 and the comparative example 1 to 2xn times (n is the target dilution rate), respectively. Then, based on the measurement data E obtained for each of Examples 1-7 and Comparative Example 1, the concentration of reducing the number of plaques by 50% (50% inhibitory concentration: IC ₅₀₎ was calculated. The results are shown in Table 2 below.

(Examination on preventive effect)
The preventive effect of anti-SARS coronavirus agents against SARS virus was evaluated.
First, SARS-CoV (human isolated Frankfurt FFM-1 strain) stock solution (10 ⁸ PFU / ml) was added to 1% BSA-containing phosphate buffer (Mg ²⁺ , Ca ²⁺ not contained: PBS (−), pH 7.0). 2 × 10 ³ PFU / ml virus solution was prepared.

  Next, Vero cells were cultured in 6-well culture plates using MEM-10% FBS medium. In addition, a mixed solution was prepared by mixing a sample solution obtained by diluting each of Examples 1 to 6 and Comparative Example 1 2 × n times (n is a target dilution factor) and the virus solution at a ratio of 1: 1. This was treated on ice for 1 hour. When 70 to 80% monolayers of Vero cells were formed, 200 μl of the mixed solution was added to each cell, and SARS-CoV was inoculated into Vero cells.

After infection at 22 ° C. for 1 hour, each cell was overlaid with 0.9% methylcellulose-containing D-MEM medium (non-immobilized 5% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin). After culturing at 37 ° C. for 6 to 7 days, the medium is removed and washed with PBS, and cells are fixed and stained with 2.5% crystal violet solution (containing 30% ethanol and 1% ammonium oxalate). It was. Thereafter, the cells were washed with PBS (−) and irradiated with UV, and the number of plaques formed in each cell was measured. Then, based on the measurement data E obtained for each of Examples 1-6 and Comparative Example 1, the concentration of reducing the number of plaques of 100% (100% inhibitory _{concentration: IC 100)} was calculated. The results are shown in Table 2 below.

  As shown in Table 2, compared with Comparative Example 1 containing only guava extract, Examples 1 to 6 containing guava extract and ascorbic acid showed three times or more high therapeutic and preventive effects. It was. And it was confirmed that each effect changes depending on the concentration of guava extract and ascorbic acid (see Examples 1 to 6). Moreover, it was confirmed that the anti-SARS coronavirus agent of the present invention is further improved in therapeutic effect and preventive effect by lowering its pH. (See Examples 1, 5 and 6).

  In addition, although not shown in Table 2, the therapeutic effect and the preventive effect with respect to SARS coronavirus were evaluated similarly to the above about the sample solution which consists of ascorbic acid which does not contain a guava extract. As a result, even when the concentration of ascorbic acid was 300 μM, no anti-SARS coronavirus activity was shown (the ascorbic acid concentration in the 15-fold diluted solution of Example 1 was 150 μM).

  Furthermore, Example 7 containing a koji extract in addition to guava extract and ascorbic acid showed a higher therapeutic effect than Example 1 containing guava extract and ascorbic acid. Since the sputum extract alone does not show a therapeutic effect (data not shown), it is considered that the sputum extract promotes the therapeutic effect of guava extract and ascorbic acid.

  From these results, it was confirmed that the anti-SARS coronavirus agent containing guava extract and ascorbic acid exhibits high anti-SARS coronavirus activity both before and after infection with SARS coronavirus. In addition, from the viewpoint of anti-SARS coronavirus activity, it was confirmed that it is effective to combine guava extract, ascorbic acid and sputum extract, or to lower the pH.

Next, a technical idea that can be grasped from the above embodiment and another example will be described.
The anti-SARS coronavirus agent is characterized in that the guava extract is extracted from raw guava leaves with hot water or a hydrophilic organic solvent at 50 ° C or higher.

Claims (5)

An anti-SARS coronavirus agent characterized by containing a guava extract extracted with an extraction solvent containing at least one of water and a hydrophilic organic solvent as a main component, and ascorbic acids as active ingredients. The anti-SARS coronavirus agent according to claim 1, wherein the guava extract is derived from guava leaves or stems. The anti-SARS coronavirus agent according to claim 1 or 2, wherein the pH is adjusted to 5.4 or less. Furthermore, it contains the soot extract extracted with the extraction solvent which contains at least 1 type of water and a hydrophilic organic solvent as a main component, The anti-resistance as described in any one of Claims 1-3 characterized by the above-mentioned. SARS coronavirus agent. An anti-SARS coronavirus agent-containing product comprising the anti-SARS coronavirus agent according to any one of claims 1 to 4.