JP2006096684A - Pathogen infection inhibitory agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new pathogen infection inhibitory agent by utilizing an extract of a plant of Cypressaceae. <P>SOLUTION: This pathogen infection inhibitory agent is obtained by incorporating the extract of the plant of Cypressaceae such as Khiva oil, as an active ingredient. Preferably, the extract is the extract oil of the plant of the Cypressaceae. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a pathogen infection inhibitor that is taken to prevent infection by pathogens such as influenza viruses.

    Every year, a large number of humans are infected with the influenza virus, are ill, and deaths occur. It is human influenza virus that infects humans, but new human influenza virus is generated every year and causes great damage. In order to prevent influenza virus infection, prior vaccine administration, hand washing and gargle enforcement are effective. In addition, as it became a big problem from the end of 2003 to 2004, it was pointed out that there is a possibility that highly pathogenic avian influenza virus was directly transmitted to humans in Southeast Asia, East Asia, North America, etc. For those who need to enter, etc., infection prevention measures such as wearing protective clothing and subsequent cleaning are very important (see Non-Patent Document 1).

On the other hand, there is knowledge that the extracted oils of Hinoki family plants such as Aomori Hiba and Taiwan Hinoki (referred to as cypress oil, hiba oil, etc.) have antibacterial activity. In a device for removing suspended particulate matter such as microorganisms, viruses, pollen, house dust, harmful gases, etc. present in indoor air by discharging the air after spraying A technique of using cypress oil or the like has been considered (for example, see Patent Document 1).
Harada Okada, “The Threat of Avian Influenza-The Real Scary is Coming!”, Kawade Shobo Shinsha, May 20, 2004 JP 2000-210521 A

  In Patent Document 1, it is effective against influenza virus when tea oil (tea extract oil containing catechin) is used as a drug, but MRSA (methicillin-resistant Staphylococcus aureus) when cypress oil is used as a drug. ) And pathogenic E. coli, and the basis for this difference is that the spectrum of sterilization and control differs depending on the type of plant essential oil. However, the specific test results show that the aqueous solution containing cypress oil has low toxicity to animals (mouse), and that the insects mainly consisting of mites were found after 2 weeks by the operation test of the device. It was present in a chemical solution (an aqueous solution containing cypress oil) and mold spores had disappeared. From the same document, whether cypress oil has anti-MRSA activity or anti-Escherichia coli activity, whether it is tea oil It is not always clear if there is anti-influenza virus activity. Vaccination, administration of antibiotics, hand washing and gargle, which are conventional infection prevention methods, are useful, but vaccines and antibiotics are generally expensive, and there is a shortage of vaccines when influenza is pandemic. And there is a risk that the price will rise, and as a result, infection may spread. In addition, the problem also entails the danger of even greater damage in poor areas.

  On the other hand, the present inventor has so far focused on the termite control action of the cypress family extract oil (Hiba oil) or the Hinokithiol contained therein, and controls termites in consideration of the environment and health using the natural Hiba oil. We have researched and developed drugs, etc., and in the process, we found that the extracts of Cypressaceae plants have the potential to suppress pathogen infection, especially anti-influenza virus.

  Then, in view of the above problems, this invention aims at provision of the novel pathogen infection inhibitor using the extract of a cypress family plant.

  That is, the pathogen infection inhibitor according to the present invention comprises an extract of a cypress family plant as an active ingredient.

  Cupressaceae plants are evergreen conifers distributed all over the world, including Aomori Hiba (Hinokisunaro (Asunaro)), Taiwan Cypress, Hinoki, Alaska Hinoki (Hinokiceae), Cypress (Cyprus), Nezu (Mususus). Genus), Japanese cedar, Japanese cypress (Nezuko genus), Japanese cypress (genus Shonanboku), and the like. The extraction method of the cypress plant is not particularly limited, but it is preferable to apply the steam distillation method in order to efficiently obtain the extracted water containing the hydrophilic component and the extracted oil containing the lipophilic component. When using the steam distillation method, the cypress plant extract oil (hereinafter sometimes referred to as “hiba oil”) is separated into two layers from the extracted water by steam distillation of sawdust and waste materials generated during sawmilling. Distilled in a separated state, mainly containing phenols (hinokitiol, β-drabrin, l-rosinic acid, carvacrol, etc.) as an acidic oil (about 3 to 4% by weight), and a neutral oil ( About 96 to 97% by weight) mainly contains sesquiterpenes (Tyuopsen, paracymene, dihydrocymene, cedrol, widrol, etc.). The acidic oil is also slightly contained in the extracted water.

  When the active ingredient that suppresses infection of pathogens is roughly classified, extracted water and extracted oil can be mentioned. From the viewpoint of infection suppression efficiency, it is preferable to apply cypress plant extract oil as the extract. Desirably, the extract oil contains a lipophilic component in an extract obtained by steam distillation of a crushed or powdered cypress plant.

  Preferred dosage forms of pathogen infection inhibitors include powders or fine granules, granules, tablets, capsules, lozenges, instant-dissolving preparations in the mouth, quick-disintegrating tablets in the mouth, chewable preparations, oral solution or dry syrup. Any oral agent selected may be mentioned. In particular, as a dosage form capable of preventing infection more effectively by dissolving or disintegrating in the mouth and bringing the extract of the cypress plant into contact with the oral mucosa or pharyngeal mucosa, lozenges, instant oral dissolution preparations, oral cavity Internally disintegrating tablets, chewable agents, and liquids for internal use are preferred. Other useful dosage forms include liquid preparations for external use such as gargles.

  Moreover, it is a pathogen for which it is desired to suppress infection widely because it repeats every year, and in particular, a pathogen that is an infection suppression target for which infection suppression action is obtained by the present invention is an influenza virus.

  According to the present invention, an extract of a cypress family that exhibits an infection-suppressing action against pathogens causing infectious diseases typified by influenza virus, particularly an extract oil is used as an active ingredient. New pathogen infection inhibitors can be created and provided. And by orally ingesting this pathogen infection inhibitor, the extract or extracted oil is brought into contact with the mucous membrane of the body to exert the infection suppression effect of the pathogen, thereby contributing to the prevention of infectious diseases of the human body.

  Hereinafter, embodiments of the present invention will be described.

  The pathogen infection inhibitor described here uses a pathogen to be suppressed as an influenza virus. Therefore, hereinafter, the pathogen infection inhibitor is referred to as an anti-influenza virus agent.

  Here, the anti-influenza virus agent of this embodiment applies a candy type tablet as a kind of oral agent. Such tablets mainly consist of cured chickenpox, contain cypress plant extract oil as an effective ingredient for suppressing influenza virus infection, and further, if necessary, sweeteners such as sugar, oxidation of vitamin C and the like An inhibitor, a refreshing agent such as mint oil and L-menthol can be added as appropriate.

  As a specific example of the cypress plant extract oil in this embodiment, Aomori hiba is applied as the cypress plant, and hiba oil is used as the extract oil. Such Hiba oil is, for example, an oil-soluble component in a liquid extracted by collecting the sawdust produced during the ordinary steam distillation method, that is, sawing Aomori Hiba into a steam kettle and applying steam heated by a boiler. It is obtained as In addition, about 1 kg of hiba oil is obtained from about 100 kg of Aomori hiba wood. In addition, the liquid obtained by the steam distillation method contains hiba extract water in which water-soluble components are dissolved in addition to hiba oil, and the weight ratio of hiba oil and hiba extract water is about 1: 100. The thus obtained hiba oil contains about 3 to 4% acid oil and about 96 to 97% neutral oil by weight. Acidic oils include compounds such as carvacrol, l-rosinic acid, hinokitiol, β-drabrin, and neutral oils include compounds such as tyuopsen, paracymene, dihydrocymene, cedrol, and widrol. Yes. And in the anti-influenza virus agent of this embodiment, Hiba oil is added by the volume ratio with respect to the whole quantity at the density | concentration of about 1 / 1,000-1 / 10,000 times. However, the concentration of hiba oil can be appropriately set within a range that is sufficient for preventing influenza virus infection and is not toxic and that is appropriate in terms of cost.

  If a user licks and takes such an anti-influenza virus agent, cypress oil that exudes at the same time as the main varicella dissolves in the mouth and adheres to the oral mucosa, pharyngeal mucosa, nasal mucosa, etc. Influenza virus infection that can be inhaled through the nose can be prevented.

  Moreover, as an anti-influenza virus agent which concerns on another embodiment of this invention, the mouthwash as a liquid for external use can be mentioned. As such a mouthwash, Hiba oil was dissolved in water by mixing an aspect in which Hiba oil was uniformly suspended in water and a substance that functions as an edible surfactant (for example, a small amount of edible ethanol). Appropriate modes are selected, such as a mode in which Hiba oil is suspended in Hiba extract water instead of water or in a mixture of water and Hiba extract water, or Hiba oil is dissolved by adding a surfactant as described above can do. Moreover, the content of the Hiba oil in the said mouthwash is diluted and added by the density | concentration of about 1,000 to 10,000 times in the volume ratio with respect to the whole quantity. However, the dilution rate of Hiba oil can be appropriately set within a range that can provide sufficient influenza virus infection-preventing activity and does not cause toxicity and is appropriate in terms of cost. Further, depending on the content of hiba oil, the mouthwash may be diluted with water at the time of use, or may be used as it is without being diluted.

  If the user gargles using such an anti-influenza virus agent (gargle), Hiba oil can be attached to the oral mucosa and pharyngeal mucosa, so that infection with influenza virus can be suitably prevented. .

  Combinations of pathogens other than influenza viruses that are expected to prevent infection by the pathogen infection inhibitor of the present invention including this embodiment and diseases caused thereby are as follows. [Class 1 infection] Ebola virus; Ebola hemorrhagic fever, Crimea congo virus; Crimea congo hemorrhagic fever, plague bacteria; plague, Marburg virus; Marburg fever, Lassa virus; Lassa fever; [Class 2 infection] Cholera toxin (CT) production Vibrio cholerae; Cholera, disentiri, flexinel, void, and sonne; bacterial dysentery, Salmonella typhi, paratyphoid; typhoid fever, paratyphoid, poliovirus; [Class 3 infections] Morning newspaper hemorrhagic Escherichia coli (O157, etc.); Enterohemorrhagic Escherichia coli infection, [Class 4 infections] Shigella amoeba cysts; Amoeba dysentery, Echinococcus (multi-clad worms, single-encrusted worms); Disease, yellow fever virus; yellow fever, chlamydia sittan; parrot fever, spirochete; recurrent fever, liver Flame virus; acute viral hepatitis, cocciella burnetti (rickettsia); Q fever, rabies virus; rabies, Cryptosporidium protozoa; cryptosporidiosis, infectious prion protein; Creutzfeldt-Jakobprion disease, group A streptococci; Fulminant hemolytic streptococcal infection, HIV virus; acquired immune deficiency syndrome, coccidioidomythitis; coccidioidomycosis, rumble flagellate; giardiasis, hantavirus; nephrogenic hemorrhagic fever, meningococcus; Membranitis, rubella virus; congenital rubella syndrome, anthrax; anthrax, tsutsugamushi disease rickettsia; tsutsugamushi disease, dengue virus; dengue fever, rickettsia japonica; Japanese erythema fever, Japanese encephalitis virus; Japanese encephalitis, botulinum; infant botulism, syphilis Treponema; syphilis, tetanus Tetanus, vancomycin-resistant enterococci; vancomycin-resistant enterococcal infection, anti-rainy illness; hantavirus lung syndrome, monkey-derived B virus; B virus disease, Brucella; brucellosis, rash typhoid rickettsia; rash typhoid, malaria parasite; Lime fever Borrelia; Lime fever, Legionella spp .; Legionellosis, West Nile virus; West Nile fever, adenovirus type 3-7, etc .; Pharyngeal conjunctivitis, Group A hemolytic streptococcus; Group A hemolytic streptococcal pharyngitis, Virus (rotavirus, small spherical virus, etc.), bacteria (Salmonella, Campylobacter, etc.); infectious gastroenteritis, chickenpox / zoster virus; chickenpox, coxsackie virus, etc .; hand-foot-and-mouth disease, human parvovirus; apple disease, human herpes virus; Idiopathic rash, Bordetella pertussis; Pertussis, Rubella Rus; rubella (3 days), coxsackievirus A group; herpangina, measles virus; measles, mumps virus; mumps, enterovirus 70, etc .; acute hemorrhagic conjunctivitis, adenovirus 8 etc .; epidemic conjunctivitis (habit) (Herpes simplex virus, etc.), secondary (measles virus, etc.); acute encephalitis, Haemophilus influenzae, pneumococcus, meningococcus; bacterial meningitis, coxsackie virus, etc .; aseptic meningitis, pneumonia mycoplasma; mycoplasma pneumonia Chlamydia pneumonia, acute measles virus; adult measles, Chlamydia trachomatis; genital chlamydial infection, herpes simplex virus; genital herpesvirus infection, human papilloma virus; Resistant Staphylococcus aureus Infectious diseases, penicillin-resistant pneumococci; penicillin-resistant pneumococcal infections, multidrug-resistant Pseudomonas aeruginosa; drug-resistant Pseudomonas aeruginosa infections, tuberculosis bacteria; In addition to the above, new infections such as SARS (severe acute respiratory syndrome) caused by SARS coronavirus. Moreover, it is thought that drinking of the functional drink of this embodiment is effective also in the prevention with respect to the secondary infection to the human of the infection derived from an animal. In particular, according to the examples described later, it is considered that infection of pathogens that are highly likely to be infected when a person such as influenza virus inhales through the mouth or nose can be effectively prevented.

  The present invention is not limited to the embodiment described above. As an extract of cypress family plants, for example, cypress extract water can be applied instead of cypress oil, or a mixture thereof can be applied, and one or more contained compounds can also be applied. Other plants can be used in addition to Aomori Hiba. The present invention is not limited to the various dosage forms described above, and can be provided in appropriate dosage forms.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited by the following description. The following tests were conducted by entrusting to the Osaka Prefectural Institute of Public Health and the Infectious Diseases Department.

<Test 1: Anti-influenza virus effect comparison test of Hiba derived products>
(1) Purpose: To investigate whether the water-soluble or oil-soluble fraction in the water extract of Aomori Hiba has anti-influenza virus activity.
(2) Test sample: (a) Hiba extract water; water-soluble component (lower layer) of the extract obtained by steam distillation of Aomori Hiba wood sawdust, specifically 100% Hiba extract water (product) Name “Forest power”, manufactured by Topics Co., Ltd., (b) Hiba oil solution (60% Hiba oil); oil-soluble component (upper layer) of the extract, dissolved in water together with a predetermined amount of emulsifier, Hiba The volume ratio of oil is 60% of the total amount. Specifically, natural termite control agent (trade name “water-soluble Hiba oil HB-60”, manufactured by Topics Co., Ltd.), (c) hinokitiol copper complex; purified hinokitiol Copper complex. Each preparation is diluted with a culture solution at several dilution rates shown in Table 1 below.
(3) Test method: An influenza virus infection test is performed by FFU assay (Focus Forming Unit Assay). Specifically, a predetermined amount of canine kidney-derived MDCK cells (culture medium: EAGLE MEM) cultured in a 96-well microplate were infected with influenza virus (H3N2 human vaccine New Caledonia strain), and 16 hours later, immunization Infected cells (1 FFU = 1 infectious virus) are stained and stained. The concentration of the standard in which the number of infected cells is reduced to 50% is defined as 50% Effective Concentration (EC50). In addition, 0.1 ml (the number of viable viruses is about 200) is supplied to a microplate containing MDCK cells as influenza virus suspended in the culture medium.
(4) Test results: Table 1 shows the results of determining the average value of FFU tested three times for each standard.

  From the results of Test 1, the EC50 of Hiba oil aqueous solution is a dilution rate of 625,000 times or more (dilution rate of 1,041,667 times or more when converted to Hiba oil stock solution (100% Hiba oil)), and the anti-influenza virus activity is remarkably high. Conceivable. On the other hand, EC50 of Hiba extract water is about 10 times diluted, and hinokitiol copper complex is ineffective. The reason why anti-influenza activity is observed in the Hiba extract water is not necessarily clear, but it is thought that the acid oil is dissolved in the Hiba extract water in a small amount.

<Test 2: Antiviral effect test of Hiba oil>
(1) Objective: In Test 1, since Hiba oil was found to have a very high anti-influenza virus activity, the difference in influenza virus infection on cells depending on the timing of Hiba oil administration was examined.
(2) Test sample: Hiba oil (the same Hiba oil aqueous solution as in Test 1) is used at each dilution rate as shown in Table 2 below.
(3) Test method: Influenza virus infection test by FFU assay substantially the same as Test 1. . However, (i) a predetermined amount of cultured MDCK cells (0.1 ml) was added to a microplate containing a medium (EAGLE MEM), and (ii) influenza virus solution (the same as in Test 1, hereinafter “ 0.1 ml of virus solution ") is added (approximately 200 viable viruses / culture solution). Then, (iii) the influenza virus is adsorbed to MDCK cells at 37 ° C. for 1 hour, (iv) the virus solution is removed by suction, and the medium is added and aspirated to perform washing 2-3 times, and further at 37 ° C. for 16 hours. MDCK cells are cultured. Here, the case where the aqueous solution of Hiba oil at each dilution rate is added to the microplate in the stage (i) and a predetermined time (10 minutes) is left before the stage (ii) is referred to as “pre-infection pretreatment”, (ii) When adding the aqueous solution of Hiba oil to the microplate together with the virus solution at the stage “at the time of adsorption”, when adding the aqueous Hiba oil solution at the stage of (iv) after 16 hours of incubation, “after the adsorption” ( The case where the aqueous solution of Hiba oil at each dilution ratio is added to the microplate in both stages ii) and (iv) is referred to as “at the time of adsorption / after”. Infected cells (1 FFU = 1 infectious virus) are stained and measured by immunostaining in 4 patterns of “at and after adsorption”.
(4) Test results: Table 2 shows the results of determining the average value of FFU tested three times for each test category.

  From the results of Test 2, high anti-influenza virus activity was observed in “at adsorption” and “at and after adsorption”, and their EC50 was about 12,500,000 times that of Hiba oil aqueous solution (about 20,833,333 times in terms of Hiba oil stock solution) Dilution. On the other hand, almost no anti-influenza virus activity is observed after “adsorption”. In addition, “pre-infection pretreatment” showed almost the same effect as “at and after adsorption”. Based on the above, it is recognized that Hiba oil exerts an inhibitory activity against influenza virus cells when adsorbed to cells before or after adsorbing influenza virus to cells. It is considered to have an adsorption inhibiting action on Although its mechanism of action is not always clear, it is possible that Hiba oil acts on the cell side and inhibits the steps after adsorption of influenza virus to cells, and the main infection inhibition mechanism is thought to be adsorption inhibition .

<Test 3: Examination of infection pretreatment time>
(1) Objective: Based on the results of Test 2, examine the pretreatment time for infection in which Hiba oil has an inhibitory effect on infection of influenza virus cells.
(2) Test sample: Hiba oil (the same Hiba oil aqueous solution as in Test 1 and Test 2) is used at each dilution rate as shown in Table 3 below.
(3) Test method: Follow the test 2 method. However, in the case of this test, Hiba oil is added to the cell culture medium before the influenza virus is added to the cell culture medium, and the time from the addition of Hiba oil to the addition of influenza virus is defined as the “pretreatment time”. Change in multiple ways as shown in Table 3 below.
(4) Test results: Table 3 shows the results of determining the average value of FFU tested three times for each test category. For comparison, the FFU value at the time of adsorption (see the test method in Test 2) in which influenza virus and Hiba oil solution are simultaneously added to the cell culture medium is also measured.

  From the results of Test 3, the pretreatment time for the infection (infection) inhibitory effect of Hiba oil on cells was not sufficient for 2 minutes, and 30 minutes was almost the same as “at the time of adsorption”. The effect is recognized. When infection pretreatment time is 5 minutes, EC50 is about 500,000 times (about 833,333 times equivalent to Hiba oil stock solution) diluted with Hiba oil aqueous solution, and when diluted with 100,000 times (about 166,667 times) The effect is almost the same as this effect. From the above, the mechanism of inhibition of influenza virus cell infection by Hiba oil is not necessarily clear, but Hiba oil may bind to the cell membrane (probably at or near the influenza virus receptor) and inhibit the adsorption of virus. However, it is also possible that Hiba oil penetrates into the cell and inhibits the influenza virus growth step in the cell.

<Test 4: Cytotoxicity Evaluation Test of Hiba Oil>
(1) Purpose: To evaluate the cytotoxicity of Hiba oil, considering the use of Hiba oil in anti-influenza virus agents for animals including humans.
(2) Test sample: Hiba oil (Hiba oil aqueous solution identical to Test 1, Test 2, and Test 3) is used at appropriate dilution ratios.
(3) Test method: After adding Hiba oil to the culture solution and culturing MDCK cells for 16 hours (however, the culture solution and cells are the same as those used in Tests 1 to 3), Measure enzyme activity.
(4) Test results: Enzyme activity decreased to 72% at 25,000 times the Hiba oil aqueous solution (about 41,667 times in terms of Hiba oil stock solution), but cytotoxic at 125,000 times (about 208,333 times) dilution It is not allowed. In addition, when pre-infection with Hiba oil solution for 30 minutes, cytotoxicity is not observed even when diluted 1,000 times (about 1,667 times).

Claims (6)

A pathogen infection inhibitor comprising a cypress plant extract as an active ingredient. The pathogen infection inhibitor according to claim 1, wherein the extract is an extract oil of a cypress family plant. The pathogen infection inhibitor according to claim 2, wherein the extracted oil contains a lipophilic component in an extract obtained by steam distillation of a crushed or powdered cypress plant. Any oral dosage form selected from powders or fine granules, granules, tablets, capsules, lozenges, instant-dissolving preparations in the mouth, quick-disintegrating tablets in the oral cavity, chewables, oral liquids or dry syrups The pathogen infection inhibitor according to claim 1, 2 or 3, which is an agent. The pathogen infection inhibitor according to claim 1, 2, or 3, wherein the dosage form is a liquid for external use. The pathogen infection inhibitor according to claim 1, 2, 3, 4 or 5, wherein the pathogen to be infected is an influenza virus.