KR20240023286A - Chitosan composition having antibacterial lasting and moisturizing effect and manufacturing method thereof - Google Patents

Abstract

The purpose of the present invention is to provide a composition having sustainable antibacterial and moisturizing effects by providing a composition containing chitosan, allantoin, and centella asiatica extract. The present invention provides a chitosan mixed composition comprising chitosan, allantoin, and centella asiatica extract.

Description

Chitosan mixture composition having antibacterial persistence and moisturizing effect and method for producing the same {CHITOSAN COMPOSITION HAVING ANTIBACTERIAL LASTING AND MOISTURIZING EFFECT AND MANUFACTURING METHOD THEREOF}

The present invention relates to a chitosan mixed composition having antibacterial persistence and moisturizing effects and a method for producing the same.

Antibiotics are generally a general term for antimicrobial agents. In particular, substances that have an antibacterial effect on bacteria, specifically substances that have an excellent antibacterial effect by inhibiting the system by which bacteria synthesize cell walls or proteins, or substances manufactured from such substances. it means. The ingredients of antibiotics are mainly extracted from fungi, and are widely used today to treat diseases caused by bacterial infections. The most representative antibiotic is penicillin, manufactured by British physician Alexander Fleming in 1928. A representative antibiotic developed after penicillin is methicillin, which is recognized as being more effective than penicillin. Methicillin was manufactured by partially changing the chemical structure of penicillin.

Antibiotic-resistant bacteria are bacteria that are resistant to specific antibiotics and have no effect on them. For example, this includes penicillin-resistant Staphylococcus aureus, which does not respond to the efficacy of penicillin at all. In addition, there is Methicillin-Resistant Staphylococcus aureus (MRSA), which was first reported to academic circles in 1961 and has since become a major nosocomial infection worldwide. MRSA has been found to have a unique gene that can show resistance to antibiotics such as penicillin and methicillin. It is reported that MRSA does not mainly infect healthy people, but mainly infects patients with weak immune systems or patients who have undergone surgery, and in cases of infection, it causes sepsis or pneumonia and causes death.

As infections caused by pathogenic microorganisms resistant to antibiotics derived from existing chemicals increase, the side effects and resistance of antibacterial agents derived from existing chemicals have recently become a problem, and the demand for the development of antibacterial substances derived from natural products has increased. , attempts to develop various drugs or antibiotics using natural products are actively underway.

As such an antibacterial material derived from natural products, a composition containing chitosan is being developed. Chitosan is a natural, non-toxic biopolymer obtained by deacetylating chitin [polyβ-(1→4)-N-acetyl-D-glucosamine], a major component of crustacean shells such as crabs, shrimp, and crayfish. Over the past decades, chitosan has received much attention due to its various biological activities, such as antimicrobial, antitumor, antioxidant, and blood cholesterol-lowering activities. Chitosan is also reported to have properties such as water and fat absorption, emulsification, and pigment binding.

Since chitosan is very difficult to mix with other active ingredients in its polymer state, it must be decomposed into a form with a low molecular weight. However, when chitosan is decomposed using enzymes, the glucosamine that makes up chitosan is usually decomposed into low molecular weight oligosaccharides containing 5 or less. Such very low molecular weight oligosaccharides do not have significant antibacterial activity and are used as nutritional sources such as carbon or nitrogen sources. Therefore, there was a problem in that it was difficult to prepare a composition containing chitosan of a specific molecular weight size with various physiological activities.

Registered Patent No. 10-1120161

The purpose of the present invention is to provide a composition having sustainable antibacterial and moisturizing effects by providing a composition containing chitosan, allantoin, and centella asiatica extract.

The present invention provides a chitosan mixed composition comprising chitosan, allantoin, and centella asiatica extract.

The chitosan mixed composition of the present invention has excellent antibacterial persistence and moisturizing effect.

Figure 1 is a diagram showing part of the results of an MRSA antibacterial test using the chitosan mixed composition of the present invention.
Figure 2 is a diagram showing part of the results of a VRSA antibacterial experiment using the chitosan mixed composition of the present invention.
Figure 3 is a diagram showing part of the results of an E. coli antibacterial test using the chitosan mixed composition of the present invention.
Figure 4 shows a product filled with the wound coating material of the present invention in a charger.

Below, the present invention will be described in more detail.

Allantoin has the chemical formula C ₄ H ₆ N ₄ O ₃ and is a natural ingredient mainly extracted from the perennial grasses of comfrey, oak, and wheat. It helps drugs penetrate well and helps heal by supplying moisture to wound tissue.

Centella asiatica is a perennial plant that commonly grows in the mountains and fields of the southern islands of the Korean Peninsula. The main stem extends to the side and has two degenerated scale-shaped leaves near the node where the root comes from. “Centella asiatica extract” contains Asiatic acid, Madecassic acid, and Asiaticoside as its main sexual properties. Centella asiatica extract plays a role in skin regeneration, anti-inflammatory/sedative effects, alleviating pigmentation, and strengthening skin elasticity. In this specification, “centella extract” may be a substance extracted using an extraction solvent such as water, lower alcohol with 1 to 4 carbon atoms, butylene glycol, propylene glycol, glycerin, hexane, methylene chloride, ethyl acetate, and n-butanol. Preferably, the use of ethanol extract is superior in terms of antibacterial persistence and moisturizing effect.

Aurea Helianthus is a rare medicinal plant that is an annual herb and is also called wild bouillon. It is said that the flowers and leaves of Geumhwagyu, as well as the flower stalks and roots, are rich in collagen and are used to treat various malignant pains, including antipyretic, detoxification, anti-inflammatory, and analgesic effects. It is also rich in natural plant estrogen, which helps reduce or prolong menopausal syndrome. “Geumhwagyu Extract” is known to contain a lot of collagen, an important tissue component of the skin, and contains a large amount of complex polysaccharides composed of vegetable collagen and polysaccharides that can suppress or block the destruction of skin tissue due to external physical influences. It is known that it is being done. In this specification, “Geumhwagyu extract” refers to substances extracted using extraction solvents such as water, lower alcohols with 1 to 4 carbon atoms, butylene glycol, propylene glycol, glycerin, hexane, methylene chloride, ethyl acetate, and n-butanol. Preferably, using ethanol extract is superior in terms of antibacterial persistence and moisturizing effect.

Hereinafter, with reference to experimental examples and drawings, a composition containing hyaluronic acid, Geumhwagyu extract, and chitosan and a method for producing the same will be described in detail.

(a) mixing chitosan, allantoin, centella asiatica extract, hydroxyethylcellulose, and acetic acid;

Mix chitosan, allantoin, centella asiatica extract, hydroxyethylcellulose, and acetic acid. To enhance the antibacterial persistence and moisturizing effect, Geumhwagyu ethanol extract can be additionally mixed. 10 to 200 parts by weight of allantoin and 10 to 200 parts by weight of Centella asiatica extract are mixed with 100 parts by weight of chitosan. Additionally, 10 to 150 parts by weight of Geumhwagyu ethanol extract can be mixed with 100 parts by weight of chitosan. For 100 parts by weight of chitosan, 10 to 150 parts by weight of hydroxyethyl cellulose and 1 to 10 parts by weight of acetic acid can be mixed. These ingredients can be mixed using a rotating stirrer and stirred sufficiently to form a uniform mixture.

(b) dispersing and dissolving the mixture in organic acid;

For the enzyme reaction, the mixture prepared in step (a) is dispersed and dissolved in organic acid. The organic acid may be any one selected from the group consisting of lactic acid, formic acid, and ascorbic acid. The dispersing and dissolving steps are performed at room temperature to 70°C and are not particularly limited.

To increase mixing uniformity, a vortex or rotating stirrer can be used, and chitosan is dispersed and dissolved with allantoin, centella asiatica extract, or Geumhwagyu extract until sufficient coagulation occurs. Here, coagulation means the formation of a precipitate in a non-uniform form due to electrical, ionic, or hydrophobic bonds between substances, and the formed precipitate precipitates after a certain period of time.

(c) adding chitosanase;

Chitosanase is added as an enzyme to decompose polysaccharides to the reaction solution produced in step (b). The amount of chitosanase added is not particularly limited as long as it can sufficiently decompose chitosan contained in the mixture, and is preferably 0.1 to 2.0 Unit/L. Chitosan is broken down into oligosaccharide form by chitosanase. In other words, the glucosamine that makes up chitosan is broken down into about 7 to 15 linked pieces by chitosanase.

(d) reacting at 25-70°C until agglomeration by chitosan disappears;

The reaction is induced between 25 and 70°C, and the reaction is continued until the agglomeration caused by chitosan disappears. The end point of the reaction is when no agglomeration occurs. Here, aggregation by chitosan means aggregation of chitosan and allantoin, centella asiatica extract, or Geumhwagyu extract. Chitosan decomposed into oligosaccharide form has a smaller molecular size than chitosan, so it becomes water-soluble without being affected by pH, and can be disentangled through enzymatic reactions.

(e) adjusting the pH to 4.5-6.5 using sodium hydroxide;

For application to the skin, the pH is adjusted to 4.5-6.5 using sodium hydroxide.

(f) filtering;

The reaction product is filtered through a filtration filter to prepare a chitosan mixed composition. The filtration filter is not particularly limited, and is preferably filtered using a 0.22-0.45 um vacuum filtration filter.

The chitosan mixture composition prepared through the above steps can be manufactured into a wound coating material through the following steps: filling a prefilled syringe (PFS) using a filling machine; Sterilization step at 100~141℃ for 20~40 minutes to kill microorganisms.

Figure 4 shows a product filled with the wound coating material of the present invention in a charger.

Example 1.

1 ml of 2% (w/v) water-soluble chitosan (purchased from Sokcho Products), 0.5 ml of allantoin (commercially available), 0.5 ml of Centella asiatica extract (commercially available Centella asiatica was placed in methanol and extracted for 2 hours at room temperature, then extracted at 13,000 rpm. Centrifuge for 5 minutes and take 0.5ml of supernatant), mix 0.6ml of hydroxyethylcellulose (commercially available), 20ml of water, and 0.05ml of acetic acid.

The above mixture is added to 27 ml of 0.5% lactic acid as an organic acid and stirred to disperse and dissolve. Additionally, 0.01 unit/2uL of chitosanase (purchased from Sigma Aldrich) was added, and the reaction was continued at 37°C until agglomeration by chitosan disappeared.

After completion of the reaction, the resulting reaction solution is centrifuged (13,000 rpm, 5 minutes), and 25 ml of the supernatant is taken and used as the reaction stock solution.

Add about 0.5 ml of sodium hydroxide to the reaction solution and adjust the pH to about 5. The chitosan mixture composition is prepared by filtration with a vacuum filtration filter.

Example 2.

In Example 1, instead of 0.5ml of Centella asiatica extract, 0.25ml of Centella asiatica extract and 0.25ml of Geumhwagyu methanol extract (commercially available Geumhwagyu were added to methanol and extracted for 2 hours at room temperature, then centrifuged at 13,000 rpm for 5 minutes to obtain the supernatant) A chitosan mixture composition was prepared in the same manner as in Example 1, except that 0.25 ml was used.

Comparative Example 1.

A chitosan mixed composition was prepared in the same manner as Example 1, except that 2 ml of chitosan was used instead of allantoin and Centella asiatica extract.

Comparative Example 2.

In Example 1, a chitosan mixed composition was prepared in the same manner as Example 1, except that 1 ml of Centella asiatica extract was used instead of allantoin.

Comparative Example 3.

A chitosan mixed composition was prepared in the same manner as Example 1, except that 1 ml of allantoin was used instead of Centella asiatica extract.

Experimental Example 1. Antibacterial test results

The strains to be tested (MRSA, VRSA, E coli) were each inoculated into sterilized LB medium and cultured for 18 to 24 hours. Measure the absorbance of the pre-culture medium at 600 nm to check the degree of bacterial culture. The initial culture of the test strain was diluted with LB medium to a viable cell count of 1×10 ⁵ CFU/ml and used as a test bacterial solution. 1ml of bacterial solution (1×10 ⁵ CFU/ml) was placed in a new tube, centrifuged (13,500rpm, 1 minute), and the supernatant was removed. The chitosan mixture composition prepared in Examples 1 and 2 and Comparative Examples 1 to 3 was prepared as an external disinfectant.

For the test group (Examples 1 and 2 and Comparative Examples 1 to 3), 1 ml of external disinfectant was added to the test bacterial solution from which the supernatant was removed, suspended well, and left at room temperature for 10 minutes for 24 hours. For the control group, 1 ml of PBS was added to the test bacterial solution from which the supernatant was removed, suspended well, and left at room temperature for 10 minutes for 24 hours. After centrifugation (13,500 rpm, 1 minute) of the test and control groups left for 10 minutes and 24 hours, the supernatant was removed, and 1 ml of medium or PBS was added again to suspend the precipitate well. After centrifugation again under the same conditions, the washing process of removing the supernatant was repeated twice, and finally, 1 ml of medium or PBS was added to the precipitate to suspend it well. 0.1 ml of each suspended bacterial solution was spread on LB Agar medium and cultured at 37°C for 24 hours.

The antibacterial test results for MRSA, VRSA, and E.Coli are shown in Tables 1 to 3 below, respectively, and pictures of the test results are shown in Figures 1 to 3. 1 to 3, test group I, test group II, and test group III are Example 1, Example 2, and Comparative Example 1, respectively.

division Viable bacteria count after 10 minutes (CFU/ml) Viable bacterial count after 24 hours (CFU/ml) control group 34,200 66,160 Example 1 25 <10 Example 2 15 <10 Comparative Example 1 330 50 Comparative Example 2 530 132 Comparative Example 3 830 185

division Viable bacteria count after 10 minutes (CFU/ml) Viable bacterial count after 24 hours (CFU/ml) control group 57,390 34,630 Example 1 <10 20 Example 2 <10 <10 Comparative Example 1 42 22 Comparative Example 2 65 32 Comparative Example 3 57 41

division Viable bacteria count after 10 minutes (CFU/ml) Viable bacterial count after 24 hours (CFU/ml) control group 37,030 52,110 Example 1 390 <10 Example 2 <10 <10 Comparative Example 1 1,880 35 Comparative Example 2 2,123 56 Comparative Example 3 2,228 75

As shown in Experimental Example 1, it can be confirmed that the chitosan mixed composition of Example 1 of the present invention has an excellent antibacterial persistence effect, and in particular, the chitosan mixed composition of Example 2 has a better antibacterial persistence effect.

Experimental Example 2. Film formation test

A film formation test was conducted using the compositions of Examples 1 and 2 and Comparative Examples 1 to 3. Apply 2ml of each sample to an area of 7x7 cm on a glass plate and dry at 35°C for 20 minutes. Keep it in an inverted state for 12 hours at room temperature and then observe it with the naked eye. If no deformation (flowing, falling) of the sample is observed at all, it is judged acceptable, if some deformation of the sample is observed, it is judged to be partially unsuitable, and if the sample completely falls off, it is judged to be unsuitable. The test results are shown in Table 4 below.

division result Example 1 fitness Example 2 fitness Comparative Example 1 some unsuitable Comparative Example 2 some unsuitable Comparative Example 3 some unsuitable

As shown in Experimental Example 2, the chitosan mixed composition of the present invention formed a coating film and no deformation was observed, confirming that it had an excellent moisturizing effect when forming a coating film on a skin wound.

Claims (9)

A chitosan mixed composition comprising chitosan, allantoin, and centella asiatica extract. According to claim 1,
Based on 100% by volume of the composition, 3-7% by volume of chitosan, 1-5% by volume of allantoin, 1-5% by volume of Centella asiatica extract, 0.02-0.1% by volume of sodium hydroxide, 1.5-4.5% by volume of hydroxyethylulose. Chitosan mixed composition containing 0.01 to 0.1% by volume of acetic acid. According to claim 2,
A chitosan mixed composition further comprising 1 to 3 vol% of Geumhwagyu ethanol extract based on 100 vol% of the composition. A wound coating material comprising the chitosan mixture composition of claim 1 or 2. A method for preparing the chitosan mixture composition of claim 1, comprising the following steps in this order:
(a) mixing chitosan, allantoin, centella asiatica extract, hydroxyethylcellulose, and acetic acid;
(b) dispersing and dissolving the mixture obtained above in organic acid;
(c) adding chitosanase;
(d) reacting at 25-70°C until agglomeration by chitosan disappears;
(e) adjusting the pH to 4.5-6.5 using sodium hydroxide;
(f) filtering step. According to claim 5,
In step (a), 10 to 200 parts by weight of allantoin and 10 to 200 parts by weight of Centella asiatica extract are mixed with 100 parts by weight of chitosan. According to claim 6,
In step (a), 10 to 150 parts by weight of Geumhwagyu methanol extract is additionally mixed with 100 parts by weight of the hyaluronic acid. According to claim 5,
In step (b), the organic acid is any one selected from the group consisting of lactic acid, formic acid, and ascorbic acid. According to claim 5,
In step (c), the chitosanase is added in an amount of 0.1 to 2.0 Unit/L.