JP2022055977A - Inhibitor for new coronavirus growth - Google Patents

Abstract

To provide an inhibitor for new coronavirus growth, which is capable of strongly inactivating new coronavirus.SOLUTION: Provided is an inhibitor for new coronavirus growth, comprising an extract of a Meliaceae plant. The inhibitor is prepared by, for example, adding 9 volumes of distilled water to 1 volume of Meliaceae leaves (Leaf of Melia azedarach L.), subjecting the mixture to heat treatment at 115°C for 35 minutes, grinding the mixture with a food processor and centrifuging it at 7, 500 x g for 13 minutes, and adding an equal amount of alcohol to the supernatant, letting the supernatant stand overnight, filtering the supernatant after centrifugation at 7, 500 x g for 13 minutes, and adding phytoncide to this at a rate of 2.5% to prevent precipitation.SELECTED DRAWING: Figure 1

Description

There is an application for application of Article 30, Paragraph 2 of the Patent Act. February 14, 2nd year of Reiwa Website https: // gendai. ismedia. jp / articles // 70375 https: // gendai. ismedia. jp / articles /-/ 70375? page = 2 https: // gendai. ismedia. jp / articles /-/ 70375? page = 3 https: // gendai. ismedia. jp / articles /-/ 70375? Published on page = 4

The present invention relates to a new type coronavirus growth inhibitor.

Vaccines have been developed as a preventive measure against influenza, which represents respiratory infections, and have been widely used in developed countries including Japan to this day. At the same time, research on exploration of useful disinfectants in parallel with vaccines has been carried out, and physical substances against the H5N1 virus of avian influenza (Non-Patent Documents 1 and 2) and Iodine-based chemical agents (Non-Patent Documents 3 and 4, 5) etc. have also been reported as influenza disinfectants. However, it has not been used as a full-fledged disinfectant. On the other hand, there is no vaccine against parainfluenza and RS virus, which are representative of the paramyxoviridae family, and no effective antiseptic solution has been developed yet. More than these respiratory viruses, the new coronavirus is currently a problem, but full-scale vaccine development is underway in the United States, Europe, and Japan. However, as for the effective disinfectant solution for the new coronavirus, scientifically verified disinfectant solution has not been reported to date even after examining about 500 latest papers.

Looking back on this situation, the use of vaccines and treatment with antiviral agents are ideal measures to protect against infectious viruses. As for measures against infectious diseases, it is important to take harmonious measures by defense and attack. Thinking in this way, it is an inevitable logic that weapons, which are the main axis of attack, are needed. Therefore, the present inventor discovered a Sendan leaf extract that strongly kills influenza virus in the process of searching for a disinfectant that effectively kills the virus from biological resources (Non-Patent Document 6). However, it is not known whether the Chinaberry leaf extract is effective against the new coronavirus.

Wanaratana S, Tantilertcharoen R, Sasipreeyajan J, Pakpinyo S (2010) The inactivation of avian influenza virus subtype H5N1 isolated from chckens in Thailand by chemical and physical treatments. Vet. Microbiol 140: 43-48 Shahid MA, Abubakar M, Hameed S, Hassan S (2009) Avian influenza virus (H5N1); effects of physico-chemical factors on its survival. Virol J 6:38 Sabracos L, Romanou S, Dontas I et al (2007) The in vitro effective antiviral action of povidone-iodine (PVP-I) may also have therapeutice potential by its intravenous administration diluted with Ringer's solution. Me. Hypotheses 68: 272-274 Hidari KIPJ, Tsujii E, Hiroi J et al (2004) In vitro and in vivo inhibitory effects of disodium cromoglycate in influenza virus infection. Biol Pharm Bull 27: 825-830 Zhao L, Huo CH, Shen LR et al (2010) Chemical constituents of plants from the genus Melia. Cehm Biodivers 7: 839-859 Nerome K, Shimizu F, Zukeran S, Igarashi Y, Kuroda K, Sugita S, Shibata T, Ito Y, Nerome R (2018). Functional growth inhibition of influenza A and B viruses by liquid and powder components of leaves from the subtropical plant Melia Azedarach L. Arch. Virol. 163: 2099-2109

The present inventor investigated whether this Sendan solution is also effective against the new coronavirus. As a result, it was unexpectedly found that the Sendan extract can strongly inactivate the new coronavirus, and the present invention was completed.

That is, the present invention relates to the following.
[1] A new type coronavirus growth inhibitor containing an extract of a mahogany plant,
[2] The new coronavirus growth inhibitor according to [1], wherein the plant of the family Meliaceae is Sendan (Melia azedarach).
[3] The new type coronavirus growth inhibitor according to [2], wherein the mahogany plant is a leaf of the mahogany plant.

The family Meliaceae is a family of plants belonging to the order Sapindales of the dicotyledonous family, and about 700 species of 46 genera are distributed from tropical to subtropical and temperate zones. In Japan, Chinaberry grows naturally in warm regions, and Chinese native toona sinensis is cultivated as a garden tree.

Chinaberry (Melia azedarach L.) is a species of deciduous tree that belongs to the genus Melia of the family Melia. Also known as one of the medicinal plants, it was useful for medicinal purposes, such as chilblains for fruits, insect repellent for bark, and insect repellent for leaves. It grows naturally in the Himalayas, China, Taiwan, the southern part of the Korean Peninsula, and the tropical and subtropical regions of Japan. In Japan, it is distributed in Honshu (west of the Izu Peninsula), the Izu Islands, Shikoku, Kyushu, and Okinawa.

The mahogany plant can be used as an aqueous extract by extracting the dry powder as it is or after drying it into a solid state such as powder or granules with water. The amount of water used for extraction can be arbitrary, but it is preferable to use it in an amount of 1/5 to 10 times, and particularly preferably in an amount of about 9 times. Heat treatment can be performed after water addition. The heat treatment can be, for example, at 115 ° C. for 35 minutes. Extraction can be carried out by pulverizing with stirring, in particular, with a mixer or the like. The stirring time in the mixer can be appropriately determined by those skilled in the art. After stirring, the pulverized solution can be centrifuged to collect the supernatant to obtain an extract. The number of revolutions and the time for centrifugation can be appropriately determined by those skilled in the art, and may be, for example, 7,500 xg for 13 minutes. Alternatively, if necessary, 10,000 xg may be used for 60 minutes. An equal amount of alcohol can be added to the obtained supernatant and left overnight. The extract may be used as it is, or it may be cryopreserved and sterilized by filtration with a filtration sterilization filter before use. In addition, phytoncide can be added to prevent precipitation.

The new coronavirus is SARS coronavirus 2 (severe acute respiratory syndrome 2, abbreviated as SARS-CoV-2), which is a cause of the new coronavirus infection (COVID-19). It is a coronavirus belonging to (SARSr-CoV). The Japanese state institution calls it the new coronavirus, which is sometimes abbreviated as the new corona.

SARS coronavirus 2 belongs to the coronavirus, which has a single-strand plus-strand RNA as a genome. This family includes a large number of species that infect mammals and birds, but the SARS coronavirus (SARS-CoV) and MERS coronavirus (MERS) that cause severe pneumonia, even those that cause infectious diseases in humans, are included. -CoV), human coronavirus 229E (HCoV-229) that causes seasonal colds, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and the like. SARS-CoV-2 emerged in 2019 as the seventh coronavirus pathogenic to humans.

Virus particles (virions) have a size of about 50 to 200 nm (nanometers). Like a common coronavirus, it is composed of four proteins known as S (spike) protein, N (nucleocapsid) protein, M (membrane) protein, and E (envelope) protein, and RNA. Of these, N proteins bind to RNA to form nucleocapsids, and S, E and M proteins bound to lipids surround them to form an envelope. The outermost S protein in the envelope binds to the ACE2 receptor on the cell surface, allowing infection of the cell. The point that ACE2 receptor is used for human infection is common with SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV) that uses DPP-4 receptor, and aminopeptidase N (APN). ) Is different from human coronavirus 229E. The viral genome is 29,903 bases and is a single-strand plus-strand RNA virus.

In the present invention, the growth inhibitory effect on the new coronavirus was evaluated using the TCID50 value (50% tissue culture cell infectious titer).

The novel coronavirus growth inhibitor of the present invention includes various substances such as excipients, diluents, disintegrants, binders, coating agents, lubricants, lubricants, lubricants, flavoring agents, sweeteners, and the like. A solubilizer or the like can be included as an auxiliary agent. Specific examples thereof include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycol, glycerol and the like.

The dilution ratio of the extract of the family Meliaceae can be appropriately determined by those skilled in the art according to the purpose of use, conditions, etc., but is preferably 2 to 20000 times, more preferably 10 to 5000 times, for example. , More preferably 500 to 2000 times.

The novel coronavirus growth inhibitor of the present invention may be sprayed, but is not limited thereto.

The amount of spray can be appropriately determined by those skilled in the art depending on the purpose of use, conditions, etc., but for example, it is preferably 0.001 mL / m ² to 50 mL / m ² based on the sprayed area, and more preferably 0. It is 0.01 mL / m ² to 20 mL / m ² , more preferably 0.1 mL / m ² to 10 mL / m ² .

The new coronavirus growth inhibitor of the present invention can suppress the growth of the new coronavirus.

It is a growth inhibitory effect of Sendan's solution against the new coronavirus. The X-axis is an exponential value of the amount of virus used for the attack in the infection experiment with respect to the tissue culture cells, and the y-axis is the dilution factor of log ₁₀ of the Sendan solution at that time.

Hereinafter, the present invention will be described in detail with reference to Examples.

Materials and Methods 9 volumes of distilled water was added to 1 volume of Leaf of Melia azedarach L. and heat-treated at 115 ° C. for 35 minutes, then ground with a food processor and centrifuged at 7,500 xg for 13 minutes. An equal amount of alcohol was added to Qing. After allowing this to stand overnight, the supernatant after centrifugation was filtered at 7,500 xg for 13 minutes, and phytoncide was added to the supernatant at a ratio of 2.5% to prevent precipitation.

Cells and Viruses The new coronavirus SARS-COV-2 was commissioned to Dr. Morikawa of the Faculty of Veterinary Medicine, Okayama University of Science. The Vero E6 / TMPRSS-2 cells used at that time were cultured in DMEM medium containing 10% fetal bovine serum (FCS).

Reaction of Sendan solution and virus 10 volumes of each Sendan diluted solution diluted with DMEM and 1% FCS medium and 1 volume of SARS-COV-2 were mixed and reacted at 25 ° C. for 1 hour. As a control, 1 volume of virus solution was mixed with 10 volumes of medium in which DMEM and 1% FCS were mixed, and the mixture was reacted at room temperature for 1 hour. The mixed reaction solution described above was diluted 10-fold and inoculated into a 96-well plate at 40 μl per well, and after 4 days, the appearance of CPE was observed to obtain a 50% TCID value. TCID50 is a tissue culture cell infectious titer of 50%.

Results and Discussion Figure 1 shows the experimental results. When the viral load of the new coronavirus SARS-COV-2 infecting Vero cells was 5.11 × ¹⁰⁶ , the virus in the culture solution was treated by diluting the Sendan extract 100 times. Was ¹ × 101 or less, and it was revealed that virus growth was 100% blocked. As a matter of course, it was estimated that the CPE was negative even at the 10-fold dilution, and the virus growth was ¹ × 101 or less. Since the plaque inhibition rate for influenza virus A / PR-8 / 34 (H1N1) virus was 10 ^-4 in the same Sendan solution, the growth inhibition rate for the new corona was 5.11 × 10 ⁶ with a dilution of 10 ^-4 . It was estimated that 50% of the coronaviruses were killed.

It is known that alcohol disinfection is generally effective for enveloped viruses having a lipid component as an outer membrane, such as influenza virus and paramyxovirus such as Hashika mumps. Even if the influenza virus is treated with alcohol concentrations of 10%, 20%, 40%, and 50%, the virus cannot be inactivated. In addition, hypochlorite water, which is widely used in Japan at present, has strong cytotoxicity, and all cells are killed by its 10-fold diluted solution, but the essential virus-killing effect is 1/1000 of that of Sendan. It was about.

From this, it was considered that the Sendan extract has a strong killing effect on the new coronavirus and is a promising disinfectant. A major feature of the new coronavirus is that there are many asymptomatic infected people, and there are also many patients with mild upper respiratory tract, and in the meantime, the infection spreads to many people. The disinfectant is more practically important than the disinfectant. In that sense, we believe that this discovery is worth the development of a vaccine.

The new coronavirus growth inhibitor of the present invention can suppress the growth of the new coronavirus in the environment. The growth inhibitor of COVID-19 of the present invention is a spray (spray, etc.), a coating agent (cream, etc.), a cleaning agent (liquid, semi-solid, solid soap, etc.) for cleaning spaces and object surfaces. It can be used as a component of.

Claims (3)

A new type of coronavirus growth inhibitor containing an extract of a mahogany plant. The novel coronavirus growth inhibitor according to claim 1, wherein the plant of the family Meliaceae is Sendan (Melia azedarach). The new coronavirus growth inhibitor according to claim 2, wherein the mahogany plant is a leaf of the mahogany plant.

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