JP6960707B1 - New coronavirus inactivating agent - Google Patents
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Abstract
【課題】新型コロナウイルスに対して有効な感染予防剤、感染抑制剤が未だ存在しないことに対応することを課題とする。【解決手段】本発明は、柿の果実エキスを主原料としている。【効果】本発明によれば、新型コロナウイルスの感染価を１／１００００程度まで下げることができた。【選択図】図１PROBLEM TO BE SOLVED: To deal with the fact that an infection preventive agent and an infection suppressant effective against a new type of coronavirus do not yet exist. The present invention uses persimmon fruit extract as a main raw material. [Effect] According to the present invention, the infectious titer of the new coronavirus could be reduced to about 1/10000. [Selection diagram] Fig. 1
Description
本発明は、新型コロナウイルス（SARS-CoV-2）不活性化剤に関する。 The present invention relates to a novel coronavirus (SARS-CoV-2) inactivating agent.
昨今、猛威を振るう新型コロナウイルス（SARS-CoV-2ウイルス）に対しては、しだいにウイルス特性が判明し、ワクチンが開発されつつある。しかし、今度は変異種が登場し、ワクチンも効果が不安定で、現状では副作用が危惧されているものもある。 Recently, the viral characteristics of the new coronavirus (SARS-CoV-2 virus), which is rampant, have been gradually revealed, and vaccines are being developed. However, mutants have appeared this time, and the effects of vaccines are unstable, and there are some that are currently concerned about side effects.
感染の予防剤、抑制剤としては、例えば特許文献１（特開２０００−４４４７３号公報）には茶ポリフェノールを有効成分としたコロナウイルス感染予防剤が、特許文献２（特開２０１２−１３６４３６号公報）には松樹皮、ピーナッツ渋皮、ぶどう種子、小豆からポリフェノール成分を水抽出した乳酸菌発行物が抗コロナウイルス活性を向上させると、それぞれ記載されている。 As the infection preventive agent and suppressor, for example, Patent Document 1 (Japanese Patent Laid-Open No. 2000-44673) describes a coronavirus infection preventive agent containing tea polyphenol as an active ingredient, and Patent Document 2 (Japanese Patent Laid-Open No. 2012-136436). ) Describes that lactic acid bacteria publications obtained by water-extracting polyphenol components from pine bark, peanut astringent skin, grape seeds, and red beans improve anti-coronavirus activity.
しかしながら、上記はいずでもコロナウイルス(SARSやMERS)に対するものであり、新型コロナウイルスに対するものではなかった。したがって、新型コロナウイルスに対して有効な感染予防剤、感染抑制剤は存在しない。 However, all of the above are for coronaviruses (SARS and MERS), not for the new coronavirus. Therefore, there are no effective infection preventive agents or infection suppressants against the new coronavirus.
解決しようとする問題は、新型コロナウイルスの感染予防、感染抑制に対して有効な不活性化剤が未だ存在しないことである。
AND SUMMARY problems, infection of the new coronavirus is to infect effective inactivating agent for the suppression is not yet there.
上記課題を解決するため、本発明の新型コロナウイルス不活性化剤は、柿の果実から柿タンニンを搾汁した残滓を柿の果実エキスとし、この柿の果実エキスを主原料とした。
In order to solve the above problems, the new coronavirus inactivating agent of the present invention uses the residue of persimmon tannin squeezed from persimmon fruit as a persimmon fruit extract, and uses this persimmon fruit extract as a main raw material.
本発明によれば、新型コロナウイルスの感染価を１／１００００程度まで下げることができた。 According to the present invention, the infectious titer of the new coronavirus could be reduced to about 1/10000.
本発明は、新型コロナウイルスを不活性化するという目的を、柿の果実から柿タンニンを搾汁した残滓を柿の果実エキスとし、この柿の果実エキスを主原料とすることで達成した。
The purpose of inactivating the new type of coronavirus has been achieved by using the residue of persimmon tannin squeezed from persimmon fruit as a persimmon fruit extract and using this persimmon fruit extract as a main raw material.
以下、図１を参照して、本発明の試験結果について説明する。試験材料及び被験物質など各種条件、試験手法は次のとおりである。 Hereinafter, the test results of the present invention will be described with reference to FIG. Various conditions such as test materials and test substances, and test methods are as follows.
（ウイルス）
SARS-CoV-2/JP/Hiroshima-46059T/2020
事前にDMEM(※Dulbecco's Modified Eagle Medium)で調整した冷凍保管ウイルス溶液（血清含まず）を使用。※ダルベッコ・フォークト変法イーグル最小必須培地(virus)
SARS-CoV-2 / JP / Hiroshima-46059T / 2020
Use frozen storage virus solution (without serum) prepared in advance with DMEM (* Dulbecco's Modified Eagle Medium). * Dulbecco Vogt's modified Eagle's minimum essential medium
（細胞）
VeroE6/TMPRSS2 ※アフリカミドリザルの腎臓細胞(cell)
VeroE6 / TMPRSS2 * Kidney cells of African green monkeys
（被験物質）
・実施例１：柿の果実から柿タンニンを搾汁した残滓つまり柿の果実エキスの５％水溶液
・実施例２：実施例１を酸性にｐＨ調整した果実エキスの１％水溶液
・比較例１：緑茶エキスの５％水溶液
・比較対象：滅菌水
(Test substance)
-Example 1: Persimmon tannin squeezed residue from persimmon fruit, that is, a 5% aqueous solution of persimmon fruit extract-Example 2: 1% aqueous solution of fruit extract whose pH was adjusted to acidity in Example 1-Comparative Example 1: 5% aqueous solution of green tea extract ・ Comparison target: Sterilized water
（ウイルスとの反応条件）
被験物質：ウイルス ＝ ９：１（容量比）
室温で３０分
※血清非存在下(Reaction conditions with virus)
Test substance: virus = 9: 1 (volume ratio)
30 minutes at room temperature * In the absence of serum
（試験方法）
被験物質とウイルス溶液を混合し、所定の反応を行った。
細胞毒性確認用に、被験物質とEMEM（Eagle's minimum; essential medium:イーグル最小必須培地）を混合し、所定の反応を行った。(Test method)
The test substance and the virus solution were mixed and a predetermined reaction was carried out.
For confirmation of cytotoxicity, the test substance and EMEM (Eagle's minimum; essential medium) were mixed and a predetermined reaction was carried out.
本反応液５０μリットルを４５０μリットルのEMEMと混合した。
EMEMで１０倍希釈を繰り返し、８段階希釈系を作成した。
細胞毒性確認用として、４段階希釈系を作成した。50 μliters of this reaction was mixed with 450 μliters of EMEM.
10-fold dilution was repeated with EMEM to create an 8-step dilution system.
A 4-step dilution system was prepared for confirmation of cytotoxicity.
細胞に各希釈液１００μリットルを加え、３７℃、５％Ｃｏ2 下で１時間の感染処理を行った。ウイルス感染後、２０％FCS(※)含有EMEMを５０μリットル加え、Ｃｏ2 インキュベーター内で２日間培養した。※(FCS＝Fetal Calf Serum：ウシ胎児血清)
経日的に顕微鏡下で細胞変性効果（cytopathic effect；CPE）を確認した。100 μliters of each diluted solution was added to the cells, and the cells were infected at 37 ° C. under 5% Co 2 for 1 hour. After virus infection, 50 μl of EMEM containing 20% FCS (*) was added, and the cells were cultured in a Co 2 incubator for 2 days. * (FCS = Fetal Calf Serum: Fetal bovine serum)
The cytopathic effect (CPE) was confirmed under a microscope over time.
細胞がウェルからはがれたことを確認後、１００μリットルのイソプロパノールを加え、１００μリットル固定液（エタノール：酢酸 ＝ ５：１）を加え、一晩静置した。 After confirming that the cells had detached from the wells, 100 μliters of isopropanol was added, 100 μliters of fixative (ethanol: acetic acid = 5: 1) was added, and the cells were allowed to stand overnight.
固定液を捨て、染色液（０．５％アミドブラック入りエタノール：酢酸：水 ＝ ４５：１０：４５）を各ウェルに３０μリットル分注し、ウェル全体に行きわたらせ、１分程度おいて流水ですすぎ、ウイルス感染の有無を目視で判定した。 Discard the fixative, dispense 30 μliters of staining solution (ethanol with 0.5% amide black: acetic acid: water = 45:10:45) into each well, spread throughout the wells, and leave for about 1 minute to run water. Too much, the presence or absence of virus infection was visually determined.
５０％感染量(TCID50／ｍリットル)は、Behrens-karber法で算出した。
TCID：Tissue Culture Infectious Dose＝組織培養感染量
Behrens-karber法による算出
CID50＝（最も低い希釈倍率）×（段階希釈した倍率）Σ-0.5
Σ＝（各希釈段階における細胞変性が認められたウェル数）／（検体数）の総和The 50% infection rate (TCID 50 / ml) was calculated by the Behrens-karber method.
TCID: Tissue Culture Infectious Dose = Tissue Culture Infectious Dose
Calculation by Behrens-karber method
CID 50 = (lowest dilution factor) x (stepwise dilution factor) Σ-0.5
Σ = (number of wells in which cytopathic effect was observed at each dilution stage) / sum of (number of samples)
（結果）
上記試験の結果を図１と下記表１に示す。図１において斜線ハッチングはウイルス感染価(SARS-CoV-2添加)、ドットハッチングは細胞毒性(SARS-CoV-2無添加)を示す。図１において、本発明の細胞毒性は確認されなかった。
また、下記、表１においては、本発明は、比較対象に対して常用対数で３．８（実施例１）、４．８（実施例２）のSARS-CoV-2の不活性効果を有した。さらに、本発明は、比較例１に対して常用対数で０．５（実施例１）、１．５（実施例２）のSARS-CoV-2の不活性効果を有した。(result)
The results of the above tests are shown in FIG. 1 and Table 1 below. In FIG. 1, diagonal hatching indicates viral infectivity (SARS-CoV-2 added), and dot hatching indicates cytotoxicity (SARS-CoV-2 not added). In FIG. 1, the cytotoxicity of the present invention was not confirmed.
Further, in Table 1 below, the present invention has the inactivating effect of SARS-CoV-2 of 3.8 (Example 1) and 4.8 (Example 2) in common logarithm with respect to the comparison target. bottom. Furthermore, the present invention had the inactive effect of SARS-CoV-2 of 0.5 (Example 1) and 1.5 (Example 2) in common logarithm with respect to Comparative Example 1.
Claims (1)
A new type of coronavirus (SARS-CoV-2) inactivating agent that uses the residue of persimmon tannin squeezed from persimmon fruit as the persimmon fruit extract and uses this persimmon fruit extract as the main raw material.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2021005690 | 2021-01-18 | ||
| JP2021005690 | 2021-01-18 | ||
| PCT/JP2021/016897 WO2022153572A1 (en) | 2021-01-18 | 2021-04-28 | New coronavirus infection inhibitor |
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| JP6960707B1 true JP6960707B1 (en) | 2021-11-05 |
| JPWO2022153572A1 JPWO2022153572A1 (en) | 2022-07-21 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009007323A (en) * | 2007-05-29 | 2009-01-15 | Earth Chem Corp Ltd | Virus inactivation agent, method for preventing viral infection and container holding the virus inactivation agent |
| JP2010090066A (en) * | 2008-10-08 | 2010-04-22 | Pokka Corp | Anti-sars coronavirus agent and product containing anti-sars coronavirus agent |
| WO2010067873A1 (en) * | 2008-12-12 | 2010-06-17 | 国立大学法人広島大学 | Anti-viral fiber products for use in sanitary applications |
| WO2010067869A1 (en) * | 2008-12-12 | 2010-06-17 | 国立大学法人広島大学 | Anti-viral agent and anti-viral composition |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009007323A (en) * | 2007-05-29 | 2009-01-15 | Earth Chem Corp Ltd | Virus inactivation agent, method for preventing viral infection and container holding the virus inactivation agent |
| JP2010090066A (en) * | 2008-10-08 | 2010-04-22 | Pokka Corp | Anti-sars coronavirus agent and product containing anti-sars coronavirus agent |
| WO2010067873A1 (en) * | 2008-12-12 | 2010-06-17 | 国立大学法人広島大学 | Anti-viral fiber products for use in sanitary applications |
| WO2010067869A1 (en) * | 2008-12-12 | 2010-06-17 | 国立大学法人広島大学 | Anti-viral agent and anti-viral composition |
Non-Patent Citations (3)
| Title |
|---|
| J. FOOD BIOCHEM., vol. 44, JPN6021027398, 2020, pages 13432 - 1, ISSN: 0004584410 * |
| PLOS ONE, vol. 8(1), JPN6021027400, 2013, pages 55343 - 1, ISSN: 0004584409 * |
| 福井大学 繊維・マテリアル研究センター年報, vol. 13, JPN6021027396, 2020, pages 12 - 16, ISSN: 0004584411 * |
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