CN113198008B

CN113198008B - Disinfectant and preparation method and application thereof

Info

Publication number: CN113198008B
Application number: CN202110507852.XA
Authority: CN
Inventors: 张晋玮; 唐齐玉
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-05-10
Filing date: 2021-05-10
Publication date: 2022-08-26
Anticipated expiration: 2041-05-10
Also published as: CN113198008A

Abstract

The application relates to the technical field of biological medicines, and particularly discloses a disinfectant, and a preparation method and application thereof. The disinfectant comprises the following raw materials in parts by weight: 1000 parts of water, 0.01-10 parts of MPO, 7-11 parts of halide and H ₂ O ₂ 28-52 parts of iron salt, 14.5-23.5 parts of organic acid, wherein the pH value of the disinfectant after the organic acid is added is 5-6; the preparation method comprises the following steps: mixing MPO, a halide and H ₂ O ₂ Respectively adding the mixture into water, then adding iron salt and organic acid, and uniformly stirring. The disinfectant can be used for disinfecting skin and mucous membrane and preparing preparations for promoting the healing of skin and mucous membrane wounds, and has the characteristics of small tissue damage, excellent sterilizing effect and no generation of drug resistance.

Description

Disinfectant and preparation method and application thereof

Technical Field

The application relates to the technical field of biological medicines, in particular to a disinfectant, and a preparation method and application thereof.

Background

Myeloperoxidase (MPO) is an important granular protein in phagosome, belongs to the family of mammalian heme peroxidase, and can have the capability of sterilization and disinfection by combining with hydrogen peroxide, a disinfectant as well as a preparation method and application thereof, namely water and chloride. MPO is mainly present in neutrophils and also a small amount in monocytes, but once monocytes differentiate into macrophages, MPO disappears. When mammals have no inflammation, MPO does not exist in extravascular tissues, and once inflammation occurs, MPO can escape from incompletely closed phagocytes to the outside of cells or be released to the outside of cells under the stimulation of antibodies or complement coated larger foreign matters which cannot be phagocytized, and plays a role in sterilization. The research shows that: MPO can bind to bacteria, fungi, etc. in an appropriate amount in the presence of hydrogen peroxide and chloride to inhibit the growth of target microorganisms without damaging the host cells.

MPO can play an anti-inflammatory role in the development of a variety of inflammatory diseases. For example, when acute otitis media occurs, the expression level of MPO is increased, and the bactericidal effect on streptococcus pneumoniae can be achieved. For another example, in a patient with vasculitis associated with anti-neutrophil cytoplasmic myeloperoxidase antibody, the expression level of MPO is also significantly increased, and the MPO exerts its bactericidal action in combination with other substances (ceruloplastin).

When MPO is used as a sterilization and disinfection product, MPO is usually used together with chloride and hydrogen peroxide. For wound infection (most of bacterial infection), when sterilization is performed by using a substance containing MPO, in US20090280102A1, the amount of MPO is in the range of 0.001-50 mg/g, but when the amount of MPO is too high, there is a risk of causing tissue damage. When the amount of MPO is reduced, the effects of sterilization and growth inhibition on microorganisms are reduced. Therefore, it is necessary to provide a disinfectant solution which has a low MPO amount and can efficiently sterilize and inhibit the growth and propagation of bacteria.

Disclosure of Invention

In order to ensure the excellent sterilization effect of the disinfectant under the condition of low MPO consumption, the application provides the disinfectant as well as a preparation method and application thereof.

In a first aspect, the present application provides a disinfection solution, which adopts the following technical scheme:

the disinfectant comprises the following raw materials in parts by weight:

1000 parts of water, 0.01-10 parts of MPO, 7-11 parts of halide and H ₂ O ₂ 28-52 parts of iron salt, 14.5-23.5 parts of organic acid; the pH value of the disinfectant after the organic acid is added is 5-6.

By adopting the technical scheme, MPO, halide and H ₂ O ₂ When the two materials coexist, a substance HOCl with strong oxidizing property can be generated through chemical reaction, and a free radical with an oxidizing function is formed, so that an excellent sterilization effect is achieved. However, when the body does not produce inflammation, MPO is not produced, and the MPO is produced when the inflammation is produced, so that the effect of inhibiting bacteria is exerted; but in the normal case as well as above,the presence of excess MPO can also cause physical damage. The application comprehensively considers the risk of tissue damage caused by the fact that the content of MPO outside cells is too high, and reduces the using amount of MPO by using iron salt, MPO, halide and H ₂ O ₂ The components are mutually combined to finally obtain the disinfectant with excellent sterilizing effect. After the MPO dosage is reduced, although the sterilizing capability of the sterilizing liquid is obviously reduced; however, after adding iron salt to the disinfectant, it was found that: after the ferric salt and the organic acid are compounded, the disinfectant has stronger sterilization capability. The addition of the organic acid enables the disinfectant to be an acidic solution, and the bactericidal effect of the disinfectant and the inhibition effect on the growth and the propagation of microorganisms can be effectively improved under an acidic environment; and the ferric salt also has certain oxidability, so that the sterilizing effect of the disinfectant can be further improved. The ferric salt and the organic acid are added into the disinfectant, so that MPO, halide and H are effectively improved ₂ O ₂ The prepared disinfectant has the sterilization effect. The sterilizing rate of the sterilizing liquid to staphylococcus aureus reaches 94-99.6%; the sterilization rate of the pseudomonas aeruginosa reaches 95 to 100 percent; the sterilization rate of the escherichia coli is as high as 91.6% -99.8%. In addition, compared with the common disinfectant, the disinfectant disclosed by the application takes MPO as a main acting substance, and bacteria, fungi and the like cannot generate drug resistance to the disinfectant.

When the disinfectant is used, MPO, halide and H are required to be prepared ₂ O ₂ Separately added to water, followed by the addition of iron salt and organic acid to make a homogeneous solution ready for use.

Preferably, the halide is chloride or bromide.

Further preferably, the chloride is sodium chloride.

Preferably, 1000 parts of water, 0.01-10 parts of MPO, 7-11 parts of halide and H ₂ O ₂ 28-52 parts of iron salt, 18-20.5 parts of organic acid.

By adopting the technical scheme, the more obvious sterilization effect can be obtained by further optimizing the matching proportion relation among the raw materials.

Preferably, the iron salt is at least one of ferric chloride and ferric nitrate.

When selecting the iron salt, various substances can be selected, such as iron carbonate, iron sulfate and the like. To avoid the generation of gases and other precipitates after the addition of the iron salt, ferric chloride and ferric nitrate were chosen here. In addition, the two iron salts and the organic acid, MPO, halide and H ₂ O ₂ The components are compounded with each other, so that the oxidability of the disinfectant is further improved, and the sterilization performance of the disinfectant is obviously improved.

Preferably, the iron salt is a mixture of ferric chloride and ferric nitrate.

Experiments show that when the iron salt is a mixture of ferric chloride and ferric nitrate and the iron salt is only selected to be ferric chloride or ferric nitrate, the sterilizing effect of the prepared sterilizing liquid is more excellent.

Preferably, the mass ratio of the ferric chloride to the ferric nitrate is 1 (4-6).

By adopting the technical scheme, the selection of the types of the iron salts and the addition proportion among the selected iron salts are further optimized, and the disinfectant with more excellent sterilizing effect is obtained. When the selected ferric salt is a mixture of ferric nitrate and ferric chloride, the prepared disinfectant has the sterilization rate of 99.4 percent on staphylococcus aureus, 100 percent on pseudomonas aeruginosa and 98.6 percent on escherichia coli.

Preferably, the organic acid is selected from one of citric acid, acetic acid, tartaric acid, malic acid and lactic acid.

By adopting the technical scheme, the added organic acid is common natural organic acid, and the added organic acid provides an acidic environment for the disinfectant and has small damage to non-target substances (namely, cells or tissues except bacteria to be inhibited). When the citric acid is selected as the organic acid, the organic acid can well interact with the ferric nitrate, so that the disinfection solution has more excellent sterilization effect after the acidity and the oxidability of the disinfection solution are improved.

In a second aspect, the present application provides a preparation method of the above disinfectant, which adopts the following technical scheme:

the preparation method of the disinfectant comprises the following steps: mixing MPO, a halide and H ₂ O ₂ Respectively adding the mixture into water, then adding ferric salt and organic acid, and uniformly stirring.

Preferably, the organic acid is added until the pH of the solution is 5-6.

By adopting the technical scheme, the prepared disinfectant has excellent sterilization effect under the pH condition. Because MPO has better sterilization performance under weak alkali and neutral conditions, the pH value of the solution is not suitable to be too low; meanwhile, in order to effectively enter the disinfectant to exert better capability of inhibiting the growth and the propagation of microorganisms after the ferric salt and the organic acid are compounded, the pH value of the disinfectant is adjusted to be within the range of 5-6, so that the disinfectant has excellent sterilization performance.

In a third aspect, the application provides an application of the disinfectant, which adopts the following technical scheme: the application of the disinfectant can be used for disinfecting skin and mucous membrane and preparing a preparation for promoting the healing of skin and mucous membrane wounds.

The disinfectant prepared by the application is relatively wide in application. Such as for inhibiting the growth and proliferation of gram-negative and gram-positive bacteria, spores, fungi, viruses and prions. The microorganism can be bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans, Enterobacter aerogenes, Enterobacter cloacae, Lactobacillus acidophilus, Propionibacterium acnes, Bacillus cereus, Clostridium sporogenes, Gardnerella vaginalis, Salmonella choleraesuis, Mycobacterium bovis, Mycobacterium geotrichum, and Mycobacterium smegmatis. Prokaryotes such as: chlamydia trachomatis and chlamydia psittaci. Molds and yeasts such as: aspergillus fumigatus, Aspergillus flavus, Trichophyton rubrum, Fusarium moniliforme, Candida albicans and Cryptococcus neoformans. Viruses such as rhinovirus, HIV, papilloma virus, herpes simplex and neocoronavirus. The disinfecting liquid of the present application may act on the above microorganisms but is not limited to the above-listed microorganisms.

Preferably, the using mode of the disinfection solution comprises the following steps: directly applied to wound or prepared into dressing and then applied to wound.

The disinfectant can be used for inhibiting the growth and reproduction of microorganisms such as bacteria, fungi and the like at wounds, and is also suitable for inhibiting the growth and reproduction of microorganisms such as bacteria, fungi and the like of skin and mucous membrane tissues such as eyes, respiratory tracts, esophagus, urethra, vagina, anus and the like.

In summary, the present application has the following beneficial effects:

1. according to the application, the use amount of MPO is reduced, and the ferric salt and the organic acid are added, so that the high-efficiency sterilization disinfectant is obtained on the premise of low use amount of MPO.

2. The preferred ferric salt in the disinfectant is ferric nitrate and ferric chloride, and the disinfectant prepared under the same condition has a better sterilization effect than the disinfectant only added with single ferric chloride.

Drawings

FIG. 1 is a schematic view of a burn wound after the disinfectant of example 5 is applied to rats in an experimental group and the disinfectant of comparative example 5 is applied to rats in a control group, respectively.

Detailed Description

The present application will be described in further detail with reference to examples.

Examples

The starting materials are, unless otherwise specified, commonly commercially available. MPO was purchased from Sigma-Aldrich. MH agar medium is commercially available from Changsha, Sandline Biotech Co., Ltd, under the brand name of cyclokey, model number 028050. Staphylococcus aureus (Staphylococcus aureus) is ATCC 25923 strain purchased from China General Microbiological Culture Collection Center (CGMCC); pseudomonas aeruginosa (Pseudomonas aeruginosa) was ATCC 27853 strain purchased from CGMCC; escherichia coli (Escherichia coli) is a standard strain of Escherichia coli available from Highai, Xuan, Biotechnology, Inc. under the trade designation XY-WSW-1699.

Example 1

A disinfectant liquid comprises the following raw materials: 100g of water, 0.001g of MPO, 0.7g of sodium chloride and H ₂ O ₂ 2.8 g, 0.29g of ferric chloride, 1.16g of ferric nitrate and citric acid.

Adding the above-mentioned amounts of MPO, sodium chloride and H ₂ O ₂ Respectively adding into water, and stirring; and then adding ferric nitrate, ferric chloride and citric acid, and uniformly stirring, wherein the pH value of the solution after adding the citric acid is 5.5.

Examples 2 to 15

Examples 2 to 13 differ from example 1 in the fact that the starting materials for the preparation of the disinfecting liquid differ from those in examples 2 to 13, as shown in Table 1.

TABLE 1

Detailed description of the preferred embodiments	Water/g	MPO/g	Sodium chloride/g	H ₂ O ₂ /g	Ferric chloride/g	Ferric nitrate per gram
							Example 1	100	0.001	0.7	2.8	0.29	1.16
Example 2	100	1	1.1	5.2	0.34	2.01
							Example 3	100	0.65	0.9	3.6	0.3	1.5
Example 4	100	0.65	0.9	3.6	0.25	1.25
							Example 5	100	0.65	0.9	3.6	0.32	1.58
Example 6	100	0.65	0.9	3.6	0.34	1.71
							Example 7	100	0.65	0.9	3.6	0.36	1.79
Example 8	100	0.65	0.9	3.6	0.38	1.52
							Example 9	100	0.65	0.9	3.6	0.27	1.63

Example 10

This example differs from example 5 in that the ferric nitrate is replaced by an equal weight of ferric chloride, i.e. the iron salt is selected to be ferric chloride only, otherwise it is identical to example 9.

Comparative examples 1 to 3

Comparative examples 1 to 3 differ from example 5 in the amount and/or kind of the raw materials used for preparing the disinfecting solution, as shown in Table 2.

TABLE 2

Comparative example 4

Comparative example 4 is different from example 5 in that citric acid is not added to the sterilizing solution of comparative example 4.

Comparative example 5

Comparative example 5 is different from example 5 in that citric acid, ferric chloride and ferric nitrate are not added to the sterilizing solution in comparative example 5.

Performance test

The prepared disinfectant is used for a sterilization experiment, and the inhibition effects of the disinfectant in each embodiment and each proportion on staphylococcus aureus, pseudomonas aeruginosa and escherichia coli are respectively detected.

The method comprises the following specific steps:

activation and culture of Staphylococcus aureus (Staphylococcus aureus): inoculating Staphylococcus aureus preserved at-80 deg.C on inoculating loop, inoculating on sterile MH agar culture medium, and culturing in 37 deg.C constant temperature incubator for 24 hr to activate Staphylococcus aureus. Then the bacteria were picked up to 0.9% sterile saline and adjusted to 1000CFU/mL before use.

Activation and culture of Pseudomonas aeruginosa (Pseudomonas aeruginosa): inoculating the pseudomonas aeruginosa preserved at-80 ℃ to an inoculating loop, inoculating the inoculating loop to a sterile MH agar culture medium, and culturing in a constant-temperature incubator at 37 ℃ for 24h to activate the pseudomonas aeruginosa. Then the bacteria were picked up to 0.9% sterile saline and adjusted to 1000CFU/mL before use.

Activation and culture of Escherichia coli (Escherichia coli): escherichia coli preserved at-80 deg.C is inoculated onto inoculating loop, and inoculated onto sterile MH agar medium, and cultured in 37 deg.C incubator for 24 hr to activate Escherichia coli. Then the bacteria were picked up to 0.9% sterile saline and adjusted to 1000CFU/mL before use.

The disinfectant prepared in example 1 and a staphylococcus aureus suspension having a bacterial content of 1000CFU/mL were mixed at a volume ratio of 1:1 (the bacterial content in the mixture at this time was 500CFU/mL), and after incubation at room temperature for 3 hours, 1mL of the mixture was spread on an MH agar medium, and after incubation for 24 hours in an incubator at 37 ℃, Colony Forming Units (CFU) were calculated. The disinfectant prepared in example 1 was then mixed with each of a suspension of Escherichia coli and a suspension of Pseudomonas aeruginosa in the same manner, followed by incubation and plating on MH agar medium, and Colony Forming Units (CFU) were calculated after incubation.

Then, the disinfectant liquids prepared in examples 2 to 16 and comparative examples 1 to 6 were treated in the same manner as in example 1, and then Colony Forming Units (CFU) were calculated, respectively. The specific results are shown in Table 3.

TABLE 3

As can be seen from the data results in table 3, the disinfectant of the present application has an excellent bactericidal effect on bacteria. After the disinfectant is added into a bacterial suspension containing 1000CFU/mL of staphylococcus aureus for 4 hours (the disinfectant: the bacterial suspension is 1:1, v/v), the sterilization rate of the disinfectant on the staphylococcus aureus reaches 94% -99.6%, which is higher than that of a comparative example of 85.2% -93%. After the disinfectant is added into a bacterial suspension containing 1000CFU/mL of pseudomonas aeruginosa for 4 hours (the disinfectant: the bacterial suspension is 1:1, v/v), the sterilization rate of the pseudomonas aeruginosa reaches 95-100 percent, which is higher than that of a comparative example of 80.8-85.8 percent. After the disinfectant is added into a suspension containing 1000CFU/mL of Escherichia coli for 4 hours (the disinfectant: the suspension is 1:1, v/v), the sterilization rate of the disinfectant on the Escherichia coli is as high as 91.6% -99.8%, which is far higher than that of the disinfectant in the comparative example of 78.8% -86.4%.

As can be seen from the data results of examples 3-7 and comparative examples 1-2, wherein the total amount of iron salts (ferric chloride and ferric nitrate) has an effect on the disinfection rate of the disinfectant with the addition of citric acid: wherein, when the total dosage of the ferric salt (ferric chloride and ferric nitrate) is 1.9g, the sterilizing rate of the sterilizing liquid is the best.

The data results of comparative examples 5 and 8-9 show that the sterilizing rate of the prepared disinfectant is highest when the weight ratio of the ferric chloride to the ferric nitrate is in the range of 1 (4-6).

As can be seen from the data results of comparative examples 3-4, the excellent sterilizing ability of the disinfectant of the present application is achieved by the synergy among ferric chloride, ferric nitrate, citric acid and MPO systems (MPO, hydrogen peroxide and sodium chloride), which are indispensable to each other.

The disinfectant can be used for preparing a preparation for promoting the healing of mucosal wounds or skin surface wounds. The using mode of the disinfectant comprises the following steps: directly applied to wound or prepared into dressing and then applied to wound.

The disinfectant prepared in the embodiment 5 and the comparative example 5 of the application is used for animal experiments, and the specific experimental steps are as follows:

male SD rats (8 weeks old) were purchased from slagoday. Four rats per experimental group were divided into two groups: experimental and control groups. All animals used in the experiment were individually housed and housed for 1 week to accommodate laboratory conditions. The back hair was trimmed and combed 24h before surgery. On the day of surgery, rats were first anesthetized: injecting 1% sodium pentobarbital (6mL/kg) into the abdominal cavity; a burn wound is then created: a cylindrical glass container (1 cm in diameter, filled with 94 ℃ hot water) was placed on the dorsal skin of the rat for 15s, followed by inoculation of 5X 10 cells per wound ⁸ Staphylococcus aureus in CFU (ATCC 43300). Then, the disinfectant of example 5 was applied to the wounds of rats in the experimental group, and the disinfectant of comparative example 5 was applied to the wounds of rats in the control group. The wounds were treated three times a week with the corresponding disinfectant.

The rats were then anesthetized with isoflurane and photographed at standard height. All rats were fed until after wound healing, and the rats were euthanized. An area of skin 0.5cm around the wound of all experimental rats was then collected for further observation. In particular, the results are shown in fig. 1, which is a graph of the wound size after the first day (i.e., the picture shown as d0 in fig. 1) of wound preparation for rats, and the seventh day (i.e., the picture shown as d7 in fig. 1) and the twentieth day (i.e., the picture shown as d20 in fig. 1) of the disinfectant solution of example 5 and comparative example 5 for rats with wounds, respectively.

The results in the figure show that: the wounds of rats were recovered by applying the disinfectant solutions of example 5 and comparative example 5 to the wounds of rats, but it was apparent that the wounds of rats were recovered more rapidly by applying the disinfectant solution of example 5, and the wound areas of rats to which the disinfectant solution of example 5 was applied were smaller after the 7 th and 20 th days.

The specific embodiments are only for explaining the present application and are not limiting to the present application, and those skilled in the art can make modifications to the embodiments without inventive contribution as required after reading the present specification, but all the embodiments are protected by patent law within the scope of the claims of the present application.

Claims

1. The disinfectant is characterized by comprising the following raw materials in parts by weight:

1000 parts of water, 0.01-10 parts of MPO, 7-11 parts of halide and H ₂ O ₂ 28-52 parts of iron salt, 14.5-23.5 parts of organic acid; the pH value of the disinfectant after the organic acid is added is 5-6;

the iron salt is a mixture of ferric chloride and ferric nitrate.

2. The disinfectant liquid according to claim 1, which comprises the following raw materials in parts by weight: 1000 parts of water, 0.01-10 parts of MPO, 7-11 parts of halide and H ₂ O ₂ 28-52 parts of iron salt, 18-20.5 parts of organic acid.

3. The disinfectant liquid according to claim 1, wherein the mass ratio of the ferric chloride to the ferric nitrate is 1 (4-6).

4. The disinfecting solution of claim 1, wherein the organic acid is selected from one of citric acid, acetic acid, tartaric acid, malic acid and lactic acid.

5. The method for preparing the disinfectant liquid as claimed in any one of claims 1 to 4, which comprises the following steps: reacting MPO, a halide and H ₂ O ₂ Respectively adding the mixture into water, then adding ferric salt and organic acid, and uniformly stirring.

6. The use of the disinfectant liquid as claimed in any one of claims 1 to 4, wherein the disinfectant liquid can be used for disinfecting skin and mucous membrane and preparing preparations for promoting the healing of skin and mucous membrane wounds.