CN114052039A

CN114052039A - Liquid composition

Info

Publication number: CN114052039A
Application number: CN202010745211.3A
Authority: CN
Inventors: 团克昭
Original assignee: Tuanlong Biotechnology Co ltd
Current assignee: Tuanlong Biotechnology Co ltd
Priority date: 2020-07-29
Filing date: 2020-07-29
Publication date: 2022-02-18

Abstract

Disclosed is a liquid composition having antibacterial and antiviral activities, which is suitable for use in disinfection of fingers, articles, and the like. The liquid composition contains 0.1ppm to 200ppm of potassium tungstate heteropoly acid salt and polyaminopropyl biguanide; the content of the polyaminopropyl biguanide is 1ppm to 10000 ppm; the ratio of potassium tungstophosphate to polyaminopropyl biguanide was in the range of 1: 1-1: 10000 in the range.

Description

Liquid composition

Technical Field

The present invention relates to a liquid composition which can be used for disinfection of fingers, articles, and the like, and has antibacterial activity and antiviral activity.

Background

In recent years, the prevalence of emerging viruses (SARS, MERS, african swine fever, influenza, novel coronavirus, etc.) has had a great impact on the international society. For viral infections, humans repeat the following operations: the type of virus is identified, and a vaccine is made to cope with the outbreak of infection with the virus and to suppress the virus. Although the prevention of viral infections with vaccines is an extremely important technology in public health, the response to new epidemic viral infections is always passive.

Polyacids (polyoxometalates) are a group of substances having the following structure: the basic unit is a tetrahedral or octahedral structure with 4 or 6 oxygen atoms coordinated to the metal atoms (mainly Mo, W, V, Nb), which are bonded by edges or vertices. The polyacid has a cluster structure having an oxide having oxygen coordinated around the metal element as a basic skeleton. It was also found that polyacids have structural activity dependency with different biological effects by changing transition elements or changing the steric structure type of clusters. Also, a polyacid showing antitumor activity or antiviral activity has been reported.

On the other hand, Polyaminopropyl Biguanide (PHMB) has a bactericidal effect on fungi including a wide range of molds, and is also used as a bactericide for contact lens cleaning solutions or wet tissues. Since PHMB is a highly safe bactericide that has little effect on the human body, it is used in many countries or regions.

[ Prior art document ]:

[ patent document ]: [ patent document 1] Japanese patent laid-open No. Hei 1-38022; [ patent document 2] Japanese patent laid-open No. Hei 5-320059 publication.

[ non-patent document ]: "study on the effect of wiping with a quick-drying disinfectant of a virus added to a finger, dry-wiping with a wet tissue, and cleaning and deactivating with functional water" using Feline Calicivirus (Feline Calicivirus) as a surrogate indicator for Norovirus (Norovirus) (journal of infectious diseases 81: 249-255, 2007).

Disclosure of Invention

[ problems to be solved by the invention ]

Although there are a large number of substances having killing ability against bacteria and viruses, such as antibiotics and antiviral agents, there are few substances that are not likely to develop resistant strains or drugs having a broad spectrum that can cope with a wide range of bacteria and viruses. The material has the effects of sterilization or disinfection for pathogenic microorganisms existing in living environment without selecting the types of the pathogenic microorganisms, has low cytotoxicity and high safety, can be used before the epidemic of infection diseases, and has high demand and universality.

To solve the above technical problems, the present invention aims to: provided is a liquid composition which is suitable for use in disinfection of fingers, articles, and the like, and which has antibacterial activity and antiviral activity.

[ means for solving problems ]

Among the polyacids, a compound showing antitumor activity and antiviral activity has been reported. Among them, potassium tungstate heteropolyacid (K) is known₇[PTi₂W₁₀O₄₀]·6H₂O: hereinafter abbreviated as DR-1) has high antiviral activity against herpes viruses. DR-1 is a substance developed for use as a pharmaceutical, but it has been found that it is not a pharmaceutical because it has a high excretory action in the human body and does not easily maintain blood levels, but it has been found that it does not have the problems of acute toxicity and subacute toxicity in the human body at all.

The present inventors have continued to study the antiviral activity of DR-1, and as a result, have found that DR-1 has antiviral activity against various viruses in addition to herpes viruses. The present inventors have also found that a liquid composition (solvent for formulation) which retains antibacterial and antiviral effects and is excellent in safety can be obtained by blending PHMB having a high antibacterial activity and DR-1 having a high antiviral activity at a certain blending ratio, and have completed the present invention.

Specifically, in order to achieve the above object, the technical solution adopted by the present invention is: a liquid composition having antibacterial and antiviral activity, which comprises a liquid composition comprising DR-1 (potassium tungstoheteropolyate) and PHMB (polyaminopropyl biguanide); the content of the potassium tungstate heteropolyacid salt is 0.1 ppm-200 ppm; the content of the polyaminopropyl biguanide is 1ppm to 10000 ppm; the ratio of the DR-1 to the PHMB is in the range of 1: 1 to 1: 10000 in the range.

The liquid composition of the present invention contains DR-1 having antiviral activity and PHMB having antibacterial activity as active ingredients in a specific ratio, has an activity inhibitory effect on both viruses and bacteria, is highly safe to the human body, and has excellent stability. The liquid composition of the present invention is suitable for the following uses: the use of a dispenser or sprayer, etc. for disinfecting fingers, or for disinfecting the surface of an object that a human body contacts, such as a table, a handrail, or a door handle.

The liquid composition of the present invention is preferably an aqueous solution, and the pH value thereof is in the range of 3 to 8; from the viewpoint of the stability of DR-1 and PHMB, the pH of the aqueous solution is preferably 3 to 8, and is preferably set within the range of 5 to 8 when considering the use method such as direct dropping or spraying onto fingers.

[ Effect of the invention ]

According to the liquid composition of the present invention, disinfection of fingers, articles, and the like can be easily and safely performed.

Drawings

FIG. 1 is a graph of the titer of test solutions 1-5 and control solutions 1-5 against influenza virus (type A; H1N 1);

fig. 2 is a photograph taken of petri dishes in which e.coli colonies appeared in standard agar media of the test solutions 1 to 5 and the control solutions 1 to 5.

Detailed Description

The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent from the description.

Production of < DR-1 >

6g of sodium dihydrogen phosphate and 30g of sodium tungstate were placed in a glass flask, and 100ml of purified water was added thereto and dissolved, and then 1.8ml of titanium tetrachloride was slowly added dropwise thereto while stirring the aqueous solution in the glass flask. After the aqueous solution in the glass flask was refluxed for 20 minutes, the aqueous solution was immediately filtered with a filter paper, 30g of potassium chloride was added to the filtrate, and then the resulting precipitate was separated by filtration, recrystallized 2 times with heated purified water, and the obtained crystal was air-dried to obtain DR-1.

Study of antiviral Activity of < DR-1 >

Cultured cells sensitive to various viruses (Polio virus)/Hela cells (Hela cell), EMC virus/DBA/2-generation 2-fetal mouse cells, Influenza virus (Influenza virus)/MDCK cells, other viruses as Vero cells) were seeded in a 96-well plate at 5 ten thousand per well, and cultured cells sensitive to each virus (Polio virus)/Hela cells, and Vero cells were seeded at 37 ℃ in 5% CO₂The culture was carried out in the culture apparatus under the conditions for 24 hours. Before the experiment, the previous culture solution was removed, the cell surface was washed with PBS (Phosphate buffered saline), and immediately, dilutions of each virus and DR-1 at various concentrations, which was obtained by continuously diluting 10-fold each of the virus dilutions from a stock solution of 1000 μ g/ml, were added to each well in a state of being added by one percent to the culture solution, and the culture was continued.

Cell degeneration caused by the virus was observed under a microscope 48 hours after the addition of DR-1, and TCID50 (Median tissue culture infection dose, or 50% tissue culture infection titer; the virus concentration of 50% infected cells) was calculated from the dilution concentration at which cell degeneration was confirmed in 4 (50%) of 8 wells under the same conditions. Then, the concentration at which a cytotoxic effect was confirmed in 4 (50%) of 8 wells (lowest cytotoxic concentration; IC50) when only DR-1 was added to the culture solution was calculated. Then, the MIC (Minimum inhibitory concentration) is calculated again.

Using axixibPyrrosia (Acyclovir: manufactured by Cayman chemical Co., Ltd.), HPA-23 (ammonium-21-tungsto-9-azonate)), and K₁₈[KSbW₂₁O₈₆]By Institute Pasteur, France) as a control antiviral, the same procedure as for DR-1 was performed. TCID50, IC50 and MIC were calculated in the same manner as DR-1.

Table 1 shows the antiviral activities of DR-1 and acyclovir against a herpes simplex virus type I standard strain (HSV-1), a herpes simplex virus type II standard strain, and a herpes simplex virus type II anti-acyclovir resistant strain (anti-ACV strain), which are DNA (deoxyribonucleic acid) viruses.

[ Table 1 ]:

with regard to the value of IC50/MIC, higher values indicate more effective and safe agents. It was confirmed that DR-1 has an antiviral effect on herpes simplex virus, which is a DNA virus, to the same extent as that of acyclovir, which is a special anti-herpes agent, and also has an antiviral effect on acyclovir-resistant strains unchanged from standard strains. However, while no safe pharmaceutical agent having an antiviral effect against an acyclovir-resistant strain of herpes simplex virus has been found so far, it has been confirmed that DR-1 has a useful antiviral effect against the acyclovir-resistant strain.

This is presumably because the mechanism of action of the antiviral activity of DR-1 is not that it is introduced into cells like acyclovir and acts as a terminator during phosphorylation, but acts on viruses during adsorption and invasion of viruses on the cell surface.

Table 2 shows the antiviral activity of DR-1 and HPA-23 against 4 types of picornaviruses (Picorna viruses: Polio type 1, Coxsackie type B, Echo type 9, and Encephalomyocarditis virus (Enphalomyocardia virus) (EMC) DK-27), which are RNA (Ribonucleic Acid) viruses.

[ Table 2 ]:

table 3 shows the antiviral activity of DR-1 and HPA-23 against myxoviruses (Myxo viruses/influenza A (H2N2), A (H3N2), 3 types of B/Lee) and enveloped viruses (Toga viruses/VEE TC-83) which are RNA viruses. Wherein "ND" in Table 3 means "Not Done (Not Done)".

[ Table 3 ]:

when the results shown in tables 1 to 3 were collectively judged, it was confirmed that DR-1 exhibited antiviral effects against a wide range of viruses and also exhibited an effect superior to HPA-23, which is a control antiviral agent recognized as an anti-HIV agent, depending on the virus. Furthermore, HPA-23 is known to be unable to expect antiviral activity against influenza virus.

Furthermore, even after the culture was continued for 14 days, the same results as those shown in tables 1 to 3 were obtained.

< antibacterial and antiviral Activity of PHMB >

PHMB has excellent odorless, low-irritation, safety, heat stability, etc., and has antibacterial activity against Escherichia coli, Legionella defecans, Blakeslea melanomyces and Mycobacterium tuberculosis. Furthermore, PHMB has an antibacterial effect against Bacillus cereus (Bacillus cereus) which cannot be sterilized by sodium hypochlorite, and also has an antiviral activity against norovirus or influenza virus. The MICs of PHMB against bacteria and viruses are reported below.

[ Table 4]

Bacteria species	MIC(ppm)	Viruses	MIC(ppm)
				Staphylococcus aureus	6	Influenza (hong Kong)	300
Intestinal bacteria	4	HSV-1	300
				Escherichia coli	5	Rotavirus virus	200
Bacillus of Legionella of refuge	150	Swine influenza virus	80
				Bacillus bacteria	33	Foot and mouth disease virus	2000
Sabdellosis sp	33	Feline calicivirus	400
				Serratia bacteria	33	Norovirus	200
o-157	16	Avian influenza (H7N1)	6000
				Black mould	150	Avian influenza (H5N1)	6000

< verification of antiviral Activity of aqueous solution (formulated solution) containing DR-1 and PHMB >

A commercially available product (trade name "spaalux" from Islands store Co., Ltd.) was used for PHMB. DR-1 and PHMB were dissolved in purified water. A10-fold dilution series of 0.1ppm to 1000ppm was prepared for DR-1, and a 10-fold dilution series of 1ppm to 10000ppm was prepared for PHMB. Then, the DR-1 aqueous solution and the PHMB aqueous solution were mixed in equal amounts at the ratios shown below to prepare test solutions for measuring antiviral activity. And an aqueous solution containing only DR-1 was used as a control solution.

In addition, the numbers in table 5 represent ppm values. The relationships between the numerical values on the horizontal axis of FIG. 1 and the test solutions 1 to 5 and the control solutions 1 to 5 are shown in Table 5.

[ Table 5]

Transverse axis	DR-1&PHMB	DR-1 concentration	PHMB concentration	DR-1 alone	DR-1 concentration
						1	Test solution 1	500	5000	Comparative solution 1	1000
2	Test solution 2	50	500	Comparative solution 2	100
						3	Test solution 3	5	50	Comparative solution 3	10
4	Test solution 4	0.5	5	Comparative solution 4	1
						5	Test solution 5	0.05	0.5	Comparative solution 5	0.1

The antiviral activity against influenza virus (type A; H1N1) was measured by the Plaque assay method (Plaque assay method) described below using the test solutions 1 to 5 and the control solutions 1 to 5. 35mm Petri dishes were seeded with 10 ten thousand MDCK cells and incubated at 37 ℃ with 5% CO₂The culture was carried out in the culture apparatus under the conditions for 24 hours. Before the experiment, the culture medium was removed, and the cell surface was washed with PBS (Phosphate-buffered saline). Then, 100ml of DR-1 was added to each well at each concentration, which was obtained by serially diluting 10-fold from a stock solution of 1000. mu.g/ml. Immediately, 100. mu.l (10) of each virus solution was added⁶PFU) into each well and incubation continued for 1 hour for virus adsorption.

After 1 hour of infection, the non-adsorbed virus was aspirated off, 1.5ml of agar medium was added thereto, and the petri dish was turned upside down at the stage of agar solidification to continue the culture. After 24 hours, 1ml of a second agar medium (2nd agar over medium) containing Neutral Red (Neutral Red) was placed on the agar to form an upper layer, and the culture was continued. After 6 to 8 hours, the site where the virus died due to the virus was regarded as a viral plaque, and it was not stained with neutral red and appeared transparent, so that the site was counted as a colony.

FIG. 1 is a graph showing the titer (Virus titer/PFU) of test solutions 1 to 5 and control solutions 1 to 5 against influenza Virus (type A; H1N 1). In FIG. 1, the correspondence between the numbers on the horizontal axis and the test solutions (designated as DR-1& PHMB) and the control solution (designated as DR-1 alone) is shown in Table 5. That is, when the horizontal axis represents the same number, the DR-1 concentration of the control solution is 2 times that of the test solution.

As shown in FIG. 1, DR-1 was confirmed to have an antiviral activity of completely inhibiting influenza virus (type A; H1N1) at 0.1ppm even when used alone, and was confirmed to have an antiviral activity of completely inhibiting influenza virus at 0.05ppm half of that of PHMB when used together with PHMB.

< verification of antibacterial Activity of aqueous solution (formulated solution) containing DR-1 and PHMB >

The test solutions 1 to 5 and the PHMB aqueous solution used for the verification of the antiviral activity were verified for the antibacterial activity against escherichia coli (e.coli) by the following methods.

Luria-Bertani medium (LB medium) (about 10 ten thousand CFU/ml) of E.Coli, and 1ml of an aqueous solution containing PHMB alone or mixed with DR-1 after being serially diluted were cultured in an incubator at 25 ℃ for 1 hour. 0.1ml of a mixed solution of an LB medium and an aqueous solution was added to a petri dish of a standard agar medium, diffused by a cell-coated rod in such a manner that the inside of the petri dish became uniform, and cultured again in an incubator for 24 hours. Thereafter, the colonies appeared were observed, and photographs of the petri dish were taken.

Fig. 2 shows photographs taken of petri dishes in which colonies of e.coli were present in standard agar media of test solutions 1 to 5 (expressed as DR-1& PHMB) and PHMB aqueous solutions (expressed as 10-fold dilution series of 10000ppm to 1ppm of PHMB alone). According to fig. 2, it was confirmed that PHMB had an antibacterial activity of completely inhibiting e.coli by only 1ppm even when used alone, and exhibited an antibacterial activity of completely inhibiting e.coli by only half 0.5ppm when used together with DR-1.

Thus, it was confirmed that DR-1 and PHMB, when combined, exert a potent inhibitory effect on representative influenza viruses and escherichia coli, which are closely related to the living environment, even at a final concentration of 1/2, which is the minimum effective concentration.

The liquid composition of the present invention is preferably an aqueous solution, and may contain a water-soluble alcohol such as ethanol or propanol, or a dissolving agent such as glycerin. In addition to DR-1 and PHMB as active ingredients, the composition may further contain auxiliary ingredients such as perfumes, preservatives, and excipients that do not affect the antibacterial and antiviral activities of the composition. When the liquid composition of the present invention is an aqueous solution, the pH is preferably adjusted to 3 or more and 8 or less by means of distilled water, a water-soluble alcohol, or the like.

The ratio of DR-1 to PHMB in the liquid composition of the present invention is more preferably 1: 10-1: 1000. the liquid composition of the present invention preferably has a concentration of DR-1 and PHMB of 1ppm to 1000ppm when used, and a higher concentration of the liquid composition can be prepared and diluted with purified water or the like so that the concentration of the liquid composition becomes 1ppm to 1000ppm when used.

[ industrial applicability ]

The liquid composition of the present invention is free from volatility or rancidity such as hypochlorous acid and has low irritation, and therefore can be used in various applications for the purpose of disinfecting living environments such as fingers and surfaces of articles. The liquid composition of the present invention is particularly useful in the field of public health.

The foregoing description and examples are exemplary only, and are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

1. A liquid composition having antibacterial and antiviral activity, comprising: the composition comprises a potassium tungstate heteropolyacid salt and polyaminopropyl biguanide, wherein the content of the potassium tungstate heteropolyacid salt is 0.1-200 ppm, the content of the polyaminopropyl biguanide is 1-10000 ppm, and the ratio of the potassium tungstate heteropolyacid salt to the polyaminopropyl biguanide is 1: 1-1: 10000 in the range.

2. The liquid composition as set forth in claim 1, wherein: the liquid composition is an aqueous solution, and the pH value is within the range of 3-8.