CN115645455B

CN115645455B - High-concentration ethanol/chitosan derivative washing-free disinfection gel composition and application thereof

Info

Publication number: CN115645455B
Application number: CN202211356521.1A
Authority: CN
Inventors: 郑暘
Original assignee: Zhongshan Natural Science And Technology Co ltd
Current assignee: Zhongshan Natural Science And Technology Co ltd
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2023-08-25
Anticipated expiration: 2042-11-01
Also published as: CN115645455A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a washing-free disinfection gel composition of a high-concentration ethanol/chitosan derivative and application thereof. The antibacterial effect of the disposable sterilizing composition is far higher than that of other disposable sterilizing gels, and the gel can form a chitosan derivative gel antibacterial film with good biocompatibility on the surface of human skin after ethanol volatilizes, so that the long-term and efficient antibacterial and sterilizing effects are achieved. Meanwhile, the chitosan derivative and the kudzuvine root extract are mutually cooperated, so that a remarkable synergic anti-inflammatory effect can be achieved, the stimulation of alcohol to skin can be reduced, and the long-term use is safer.

Description

High-concentration ethanol/chitosan derivative washing-free disinfection gel composition and application thereof

Technical Field

The invention belongs to the technical field of medicines, and in particular relates to a washing-free disinfection gel composition of a high-concentration ethanol/chitosan derivative and application thereof

Background

With the increase of medical level, advanced medical means are continuously intervened in clinic, and the large amount of antibiotics are used to increase drug-resistant bacteria, so that the probability of infection is increased. In recent years, infections have become a public health concern worldwide, receiving increasing attention. Infection occurs primarily due to contact transmission of pathogenic bacteria, the hands being the most important transmission medium during transmission. Hand sanitizers are the simplest, convenient, and effective method of hand cleaning and preventing infection. The hand sanitizer in the market mainly comprises three types of solution type, foam type and gel type, wherein the gel type sanitizer is free of water washing, is convenient and quick, is safe and effective, and can be added with moisturizing and skin care components, so that the compliance of hand hygiene is greatly improved, and the disinfection gel is favored by people. The existing disinfection gel mostly uses carbomer, cellulose and sodium alginate as gel matrix, uses low concentration ethanol water solution as solvent, uses trichlorohydroxydiphenyl ether, chlorhexidine acetate and benzalkonium bromide as sterilization components. The practice shows that the chemical sterilization components in the disinfectant inevitably bring adverse skin reactions and harm the health of people after long-time use.

Among a plurality of bactericidal components, ethanol is a bactericidal agent which has the advantages of quick action, wide bactericidal spectrum, best effect and minimum toxic and side effects, and is widely applied to the medical industry and the life production of people. Ethanol has the best sterilization effect only at higher concentration, and ethanol gel with lower concentration is very easy to obtain and is the main formula of the conventional disinfection gel, but the gel can be layered, agglomerated, precipitated and the like when the concentration of the ethanol is increased. Therefore, the invention obtains the stable wash-free disinfection gel of the ethanol/chitosan derivative with high concentration by screening the gel matrix material and the antibacterial agent.

Disclosure of Invention

The invention aims to solve the technical problems of providing the washing-free disinfection gel of the high-concentration ethanol/chitosan derivative aiming at the technical defects of the existing products on the market, which has extremely strong bactericidal effect, has the killing rate of more than 99.8 percent on pathogenic bacteria such as escherichia coli, staphylococcus aureus, candida albicans and the like, has long bactericidal duration, relieves the symptoms such as dry skin and the like caused by the rapid volatilization of the ethanol, and can solve the problems such as skin irritation and the like caused by using the disinfectant.

In order to achieve the above purpose, the invention is realized by the following technical scheme:

a high-concentration ethanol/chitosan derivative washing-free disinfection gel composition comprises the following components in parts by mass: 2-4% of chitosan derivative, 2.3-5.0% of cosolvent, 0.2-0.3% of radix puerariae extract, 0.015-2.0% of emollient, 0.02-0.15% of pH regulator, 60-80% of ethanol and the balance of water.

Preferably, the chitosan derivative is one or more of carboxymethyl chitosan, chitosan quaternary ammonium salt, beta-cyclodextrin grafted chitosan and tetracaine hydrochloride chitosan.

Preferably, the cosolvent is one or more of lactic acid, tween-20, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil and alkyl glycoside.

Preferably, the emollient is one or more of sodium lactate, glycerol, natural moisturizing factor, xylitol, sorbitol, and polypropylene glycol.

Preferably, the pH regulator is triethanolamine.

The preparation method of the high-concentration ethanol/chitosan derivative washing-free disinfection gel comprises the following steps:

(1) Weighing chitosan derivative according to a proportion, adding cosolvent and water into a stainless steel reaction pot, heating and stirring until the chitosan derivative is completely dissolved, adding ethanol into the mixture to prepare solution A, and sealing and preserving;

(2) Weighing the components according to a certain proportion, adding radix puerariae extract and emollient into a stainless steel reaction pot, mechanically stirring at a speed of 90-120r/min for 60-150 min, dispersing to a uniform turbid dispersion, and standing overnight to obtain solution B;

(3) Pouring the solution B into the solution A, adding the rest ethanol, stirring uniformly at a speed of 60-80r/min, regulating the pH to be close to neutral by using a pH regulator, stirring and standing for 3-5h, and thus obtaining the high-concentration ethanol/chitosan derivative washing-free disinfection gel.

The high-concentration ethanol has excellent sterilization capability, has low air boiling point, is quickly volatilized when being coated on skin, can play a role in quick drying, and has no residue after the ethanol is volatilized. In addition, the ethanol has low price and can well reduce the cost.

Chitosan (CS), a natural cationic antibacterial polymer, has good solubility and reactivity, and has antibacterial activity against various common bacteria, but has slightly poorer antibacterial performance. Therefore, the chitosan derivative with better antibacterial effect is used as an antibacterial agent and also used as a gel-based material.

The radix puerariae extract contains a large amount of isoflavone, puerarin and other substances, so that the radix puerariae has remarkable antibacterial effect, and preclinical experiments prove that the radix puerariae extract can kill helicobacter pylori and propionibacterium acnes which cause gastric ulcer and acne.

The beneficial effects are that:

(1) The concentration of ethanol in the prepared no-clean disinfection gel is high, and compared with the low-concentration no-clean disinfection gel in the market, the no-clean disinfection gel has better antibacterial effect.

(2) After the ethanol volatilizes, the prepared wash-free disinfection gel can form a layer of kudzuvine root extract/chitosan derivative gel antibacterial film with excellent biological safety on the surface layer of the skin, so that the prepared disinfection gel has longer antibacterial effect and smaller toxic and side effects.

(3) In the preparation process, the chitosan quaternary ammonium salt can play a synergistic effect with the radix puerariae extract, so that the gel prepared by the invention can treat the inflammation of the skin surface layer and has a good treatment effect on acne.

(4) The non-washing disinfection gel prepared by the invention has an anti-inflammatory effect, so the non-washing disinfection gel prepared by the invention has wider application range.

(5) The no-clean disinfection gel prepared by the invention is more stable, has moderate flowing speed when being smeared on the back of the hand, has comfortable hand feeling, and has no caking phenomenon after being stored for a long time.

Drawings

FIG. 1 is a scan of a xerogel of example 1 prepared according to the invention.

FIG. 2 is a scan of a xerogel of the sample of example 2 prepared according to the invention.

FIG. 3 is a scan of a xerogel of example 3 prepared according to the invention.

FIG. 4 is a scan of xerogel of a sample of comparative example 1 prepared according to the invention.

Fig. 5 is a three-dimensional atomic force microscope photograph of example 2.

FIG. 6 is a graph showing the results of the samples prepared in examples 1 to 3 and comparative examples 1 to 3 after being left for 30 days.

Fig. 7 shows the inhibition zones of the blank group and the sample group.

Detailed Description

Preparation and characterization of wash-free disinfection gel

Example 1

A high-concentration ethanol/chitosan derivative washing-free disinfection gel comprises the following components in parts by mass: 2.0% of chitosan quaternary ammonium salt, 2.5% of cosolvent, 0.2% of radix puerariae extract, 0.015% of natural moisturizing factor, 0.02% of triethanolamine, 60% of ethanol and the balance of water.

Preparation of radix puerariae extract: 100g of kudzuvine root is weighed, and is added with ethanol 2L with the mass fraction of 70% for heating reflux, the mixture is heated for reflux extraction for 5 times, and is cooled for 2 hours, and after the volume of the mixture is fixed to 5L, the mixture is filtered, and finally the kudzuvine root extract is obtained.

(1) Weighing chitosan derivative according to a proportion, adding lactic acid and water into a stainless steel reaction pot, heating and stirring until the chitosan quaternary ammonium salt is completely dissolved, adding ethanol into the mixture to prepare solution A, and sealing and preserving the solution A;

(2) Weighing the components according to a certain proportion, adding radix Puerariae extract and natural moisturizing factor into a stainless steel reaction kettle, mechanically stirring at 90r/min for 150 min, dispersing to obtain uniform turbid dispersion, and standing overnight to obtain solution B;

(3) Pouring the solution B into the solution A, adding the rest ethanol, stirring uniformly at the speed of 80r/min, regulating the pH to 6.7 with triethanolamine, stirring and standing for 5h to obtain the high-concentration ethanol/chitosan derivative washing-free disinfection gel.

Example 2

The procedure of example 2 was essentially the same as in example 1, except that: the mass ratio of each component is 2.5 percent of chitosan quaternary ammonium salt, 2.8 percent of lactic acid, 0.25 percent of kudzuvine root extract, 0.015 percent of natural moisturizing factor, 75 percent of triethanolamine, and the balance of water.

Example 3

The procedure of example 3 was essentially the same as in example 1, except that: 4% of chitosan derivative, 2.5% of lactic acid, 0.3% of radix puerariae extract, 0.2% of emollient, 0.15% of triethanolamine, 73% of ethanol and the balance of water.

Comparative example 1

The procedure of comparative example 1 was substantially the same as that of example 2, except that: the gel base material chitosan quaternary ammonium salt in example 2 was replaced with cellulose; the mass ratio of each component is as follows: cellulose 0.8%, cosolvent 2.8%, radix Puerariae extract 0.25%, emollient 0.015%, pH regulator 0.02%, ethanol 75%, and water in balance.

Comparative example 2

Procedure of comparative example 2 the procedure of example 2 was substantially the same except that: replacing the antibacterial agent chitosan quaternary ammonium salt in the example 2 with beta-cyclodextrin grafted chitosan; the mass ratio of each component is as follows: beta-cyclodextrin grafted chitosan, 2.8% of cosolvent, 0.25% of radix puerariae extract, 0.015% of emollient, 0.02% of pH regulator, 75% of ethanol and the balance of water.

Comparative example 3

Procedure of comparative example 2 the procedure of example 2 was substantially the same except that: comparative example 2 was not added with pueraria extract; the mass ratio of each component is 2.5 percent of chitosan quaternary ammonium salt, 2.8 percent of lactic acid, 0.015 percent of natural moisturizing factor, 75 percent of triethanolamine, ethanol and the balance of water.

Morphology study

The gels prepared in examples 1-3 and comparative examples 1-3 were freeze-dried for 72 hours to give xerogels, and then sprayed with gold under vacuum to give the scans shown in FIGS. 1-4. From the figure, we can see that the gel membrane surface prepared by the invention has a uniform, porous and interconnected three-dimensional network structure. Furthermore, as can be seen from the scan, the pore size in example 2 is the smallest, which may be related to the high concentration of ethanol in example 2, and the pore size gradually becomes smaller as the concentration of ethanol increases, which makes the water absorption of the gel prepared by the present invention worse and can well retain the moisture on the surface layer of the skin.

Roughness of

The atomic force microscope can further provide surface morphology structure information and reflect the surface roughness of the sample, the roughness can directly influence the hand feeling of the disinfection gel, and the disinfection gel with smaller roughness is more comfortable to smear. Fig. 5 is a three-dimensional atomic force microscope photograph of example 2, from which we can see that the gel surface is highly heterogeneous, which may be related to high ethanol concentration. In the chitosan gel system, a large number of good solvent water molecules can be wrapped by chitosan molecular chains, and the xerogel after freeze drying forms larger holes. When the poor solvent ethanol is added, the water molecules wrapped by the chitosan are reduced, and when the ethanol content is increased, the water molecules wrapped by the chitosan are fewer and fewer, which also causes the dissolubility of the chitosan quaternary ammonium salt to be poor, and the surface of the gel film becomes rough.

TABLE 1 roughness of xerogel (Rq)

Stability of

The gel prepared by the invention is placed for 30 days at room temperature under the same environment, and the condition of each sample is observed.

FIG. 6 is a photograph of a sample prepared according to the present invention after being left for 30 days. From the graph in FIG. 6, it can be seen that the high-concentration ethanol/chitosan quaternary ammonium salt gel prepared by the method does not have the phenomena of layering, caking, floccules and the like, but has the phenomena of layering, floccules and the like in comparative examples 1 and 2, which shows that the chitosan quaternary ammonium salt gel prepared by the method has better stability. The reason may be that the network structure of the high concentration ethanol/chitosan quaternary ammonium salt gel is more stable, so that the prepared gel can be stored for a long time, and thus, the gel does not deteriorate.

Antibacterial experiments

Preparing escherichia coli seed liquid: the alcohol lamp is firstly ignited on the ultra-clean bench, and the inoculating needle is burned on the alcohol lamp to two times, so that the sterilizing effect is achieved. Standing for 20 seconds, after the inoculating needle is cooled, lightly scraping the refrigerated bacterial strain by the inoculating needle, taking out the inoculating needle, putting into 50ml of nutrient broth which is prepared in advance and sterilized, and taking out after stirring for a few times. Sealing the nutrient broth with sealing film, placing in a gas bath constant temperature oscillator at 37deg.C, shaking at 160r/min for 12 hr, taking out, and refrigerating in refrigerator.

Preparation of bacterial suspension: taking 1ml of cold-stored bacterial seed liquid in a refrigerator by a liquid-transferring gun, putting the liquid into 50ml of nutrient broth, sealing by a sealing film, and putting into a gas bath constant temperature oscillator with the temperature of 37 ℃ and 160r/min for oscillation until the OD600 value of the bacterial liquid is between 0.4 and 0.5.

Bacterial suspensions were uniformly spread on an agar medium, round filter paper sheets having a diameter of 5mm, coated with the gels of examples 1 to 3 and comparative examples 1 to 3, were attached to a plate, and filter paper sheets coated with distilled water were used as a negative control, and cultured in a constant temperature incubator at 37℃for 16 hours. After incubation, the diameter of the inhibition zone of the growing bacterial colony was measured.

Fig. 6 shows the inhibition zones of the blank and sample groups, and it can be seen from the figure that the inhibition zones of examples 1-3 are larger than those of comparative examples 1 and 2. The gel prepared by taking chitosan quaternary ammonium salt as a gel base material has better disinfection effect than gel prepared by taking cellulose and beta-cyclodextrin grafted chitosan as the gel base material, and probably because the chitosan quaternary ammonium salt can have synergistic effect with the pueraria extract, so that the antibacterial performance of the chitosan quaternary ammonium salt and the pueraria extract is better, and therefore, the antibacterial circle is larger, which is not possessed by cellulose and beta-cyclodextrin grafted chitosan. Meanwhile, the bacteriostasis area of the example 2 is larger, which is related to the high ethanol concentration in the example 2, which shows that the high-concentration ethanol gel prepared by the invention is successful.

Anti-inflammatory experiment

Observing the growth state of the cells until the cells grow to a concentration of about 4×10 per milliliter ⁵ Cells were plated onto 24-well plates at 500 μl per well overnight. The experiments were performed in groups, i.e. blank, lipopolysaccharide and sample. The sample set was added to the gel prepared according to the present invention and filled to 250. Mu.L with DMEM to a final concentration of 2. Mu.g/ml. Other groups ofEqual amounts of DMEM were added while three parallel wells were made for each experimental group. One hour after administration, 250. Mu.l of lipopolysaccharide (1. Mu.g/ml) was added to the blank, lipopolysaccharide and sample groups, and after 12 hours of incubation, the supernatant was split-packed and stored at-80℃for each cytokine measurement without statistical differences between groups.

The reaction strips were removed from the sealed bags, 20-fold dilutions of primary antibodies were added to the corresponding wells with coating solution, 100 μl per well, the plates sealed with a sealing plate membrane, and overnight at 4 ℃. The liquid in the plate is thrown off, and the plate is washed four times by washing liquid. 200. Mu.L of the sample diluent was added, the plate was sealed, and the plate was allowed to stand at room temperature and shaken for 1 hour. The liquid in the plate is thrown off, and the plate is washed four times by washing liquid. 100 μl of the diluted IL-1β standard and the sample to be tested were added, respectively, and the mixture was shaken at room temperature for two hours. The liquid in the plate is thrown off, and the plate is washed four times by washing liquid. 200-fold dilutions of IL-1β secondary antibody were added, 100 μl per well, and shaken at room temperature for one hour. The liquid in the plate is thrown off, and the plate is washed four times by washing liquid. Adding 100 μl of horseradish peroxidase, shaking for 30min at normal temperature to remove liquid in the plate, and washing the plate with washing solution for five times. 3,3', 5' -tetramethyl benzidine substrate chromogenic solution was added, and developed for 15 minutes in a dark place at 100ml per well. 100. Mu.L of stop solution was added. OD (450 nm) of each reaction well was measured by an enzyme-labeled instrument. And (5) making a standard curve according to the standard substance of each concentration and the corresponding OD value. And calculating the concentration of the cytokines in each sample to be tested according to the standard curve.

TABLE 2 IL-1 beta secretion

Sample of	IL-1 beta secretion amount ((μg/ml))
		Blank control group	3.12±0.23
Lipopolysaccharide	13.22±1.52
		Example 1	8.35±1.51
Example 2	7.61±1.32
		Example 3	7.95±0.84
Comparative example 1	12.56±1.08
		Comparative example 2	13.25±0.98
Comparative example 3	13.56±1.25

TNF-alpha, IL-6, IL-1 beta are pro-inflammatory factors that in turn induce the secretion of a number of inflammatory mediators, such as prostaglandins, nitric oxide, leukotrienes, etc. Lipopolysaccharide, in turn, can enter the body, causing the expression of related protein genes in the body's inflammatory signaling pathway. Table 2 shows the secretion amount of IL-1. Beta. Pro-inflammatory factor by the samples prepared according to the present invention and the blank control, lipopolysaccharide control. When lipopolysaccharide enters the body, it can induce the release of many active substances such as cytokine, proinflammatory factor, etc., such as IL-1β, TNF- α, IL-6, etc. High concentrations of IL-1β can lead to epithelial cell damage and have the potential to lead to pulmonary fibrosis, and can also increase vascular permeability, cause alveolar surfactant abnormalities, and exacerbate pulmonary edema.

As can be seen from Table 2, the content of IL-1β in the lipopolysaccharide group reached 13.22. Mu.g/ml, but the content of IL-1β in examples 1-3 was greatly reduced, which suggests that the anti-inflammatory effect of the example group was remarkable. The reason is probably that the chitosan quaternary ammonium salt and the kudzuvine root extract in the embodiment have synergistic effect, and can inhibit the expression of some proteins, thereby inhibiting the secretion of IL-1 beta pro-inflammatory factors. The cellulose and beta-cyclodextrin grafted chitosan of the comparative example did not have such a synergistic effect with the pueraria extract.

Claims

1. The washing-free disinfection gel composition of the high-concentration ethanol/chitosan derivative is characterized by being prepared from the following raw materials in parts by mass: 2-4% of chitosan quaternary ammonium salt, 2.3-5.0% of cosolvent, 0.2-0.3% of radix puerariae extract, 0.015-2.0% of emollient, 0.02-0.15% of pH regulator, 60-80% of ethanol and the balance of water;

the kudzuvine root extract is extracted by ethanol, and the specific steps are as follows: 100g of kudzuvine root is weighed, and is added with ethanol 2L with the mass fraction of 70% for heating reflux, the mixture is heated for reflux extraction for 5 times, and is cooled for 2 hours, and after the volume of the mixture is fixed to 5L, the mixture is filtered, and finally the kudzuvine root extract is obtained.

2. A high concentration ethanol/chitosan derivative no-wash disinfecting gel composition according to claim 1, wherein: the cosolvent is one or more of lactic acid, tween-20, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil and alkyl glycoside.

3. A high concentration ethanol/chitosan derivative no-wash disinfecting gel composition according to claim 1, wherein: the emollient is one or more of sodium lactate, glycerol, natural moisturizing factor, xylitol, sorbitol, and polypropylene glycol.

4. A high concentration ethanol/chitosan derivative no-wash disinfecting gel composition according to claim 1, wherein: the pH value regulator is triethanolamine.

5. A high concentration ethanol/chitosan derivative no-wash disinfecting gel composition according to claim 1, wherein: the mass ratio is as follows: 2.0% of chitosan quaternary ammonium salt, 2.5% of cosolvent, 0.2% of radix puerariae extract, 0.015% of natural moisturizing factor, 0.02% of triethanolamine, 60% of ethanol and the balance of water.

6. A method for preparing a high concentration ethanol/chitosan derivative no-clean disinfecting gel composition as claimed in claim 1, comprising the steps of: (1) Weighing chitosan quaternary ammonium salt according to a proportion, adding cosolvent and water into a stainless steel reaction pot, heating and stirring until the chitosan quaternary ammonium salt is completely dissolved, adding ethanol into the solution to prepare solution A, and sealing and preserving; (2) Weighing the components according to a certain proportion, adding radix puerariae extract and emollient into a stainless steel reaction pot, mechanically stirring at a speed of 90-120r/min for 60-150 min, dispersing to a uniform turbid dispersion, and standing overnight to obtain solution B; (3) Pouring the solution B into the solution A, adding the rest ethanol, stirring uniformly at the speed of 60-80r/min, regulating the pH to be close to neutral by using a pH regulator, stirring and standing for 3-5h, and obtaining the high-concentration ethanol/chitosan derivative washing-free disinfection gel.

7. The method of claim 6 provides a high concentration ethanol/chitosan derivative of the gel composition.