CN118252770A - Salicylic acid chitosan co-enzymolysis compound and preparation method and application thereof - Google Patents
Salicylic acid chitosan co-enzymolysis compound and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 20
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a preparation method of a salicylic acid chitosan co-enzymolysis compound. Compared with the prior art, the method directly utilizes the acid-base interaction between salicylic acid and chitosan and/or chitosan derivatives, and uses hydrolytic enzyme to carry out enzymolysis on the salicylic acid, and other acid sources (such as hydrochloric acid and acetic acid) are not introduced in the whole preparation process to assist dissolution of the chitosan and/or chitosan derivatives, so that the introduction of potential irritant substances is reduced, the reaction economy is improved, and the method is more suitable for industrial application; the prepared salicylic acid chitosan co-enzymolysis compound not only has the advantages of high water solubility and high temperature resistance stability and expands the application range of salicylic acid, but also has the advantages that the salicylic acid chitosan co-enzymolysis compound does not influence the performance of salicylic acid raw materials, can be creatively found to remarkably reduce the irritation of salicylic acid, has obviously better antibacterial performance and also has good oil control effect.
Description
Technical Field
The invention belongs to the technical field of skin care products, and particularly relates to a salicylic acid chitosan co-enzymolysis compound and a preparation method and application thereof.
Background
Salicylic acid, also known as o-hydroxybenzoic acid, is a fat-soluble organic acid. As a raw material of the pharmaceutical industry, salicylic acid can be used for preparing aspirin, sodium salicylate and the like. The salicylic acid is widely applied to external application painting skin care products besides a certain preservative effect, and can play roles in softening cutin, sterilizing, treating acne and the like. However, salicylic acid has poor water solubility and strong irritation, so that the application effect of the salicylic acid is limited, and the salicylic acid is particularly strictly limited in addition to skin-contact skin care products.
The chitosan is a product of removing partial acetyl of natural polysaccharide chitin, has multiple physiological functions of biodegradability, biocompatibility, no toxicity, bacteriostasis and the like, and has been widely applied to industries of foods, cosmetics, medicines and the like. Meanwhile, when the chitosan is used for cosmetic, a film is easily formed on the surface of the skin, and the chitosan has the functions of moisture absorption and water retention, so that the skin becomes soft, moist and good in extensibility. In addition, the chitosan amino cationic polysaccharide has a certain antibacterial effect due to the property. However, chitosan alone cannot be dissolved in water and needs to be solubilized with acetic acid, hydrochloric acid, or the like.
Chinese patent publication No. CN104622710a discloses a complex composition comprising salicylic acid and chitosan, which is obtained by mixing salicylic acid and chitosan, adding ethanol or polyalcohol, stirring and dissolving agent, dispersing, and then adding water, but the complex composition still has a problem of low solubility due to the high molecular nature of chitosan.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a salicylic acid chitosan co-enzymolysis compound with high water solubility and low irritation, and a preparation method and application thereof.
The invention provides a preparation method of a salicylic acid chitosan co-enzymolysis compound, which comprises the following steps:
s) mixing chitosan and/or chitosan derivative, salicylic acid and water, then adding a pH regulator to regulate the pH value, and then adding hydrolase to react to obtain the salicylic acid chitosan co-enzymolysis compound.
Preferably, the mass ratio of the chitosan and/or chitosan derivative to the salicylic acid is (1.1-1.5): 1.
Preferably, the chitosan and/or chitosan derivative has a degree of deacetylation of 75% or more;
the chitosan derivative is carboxymethyl chitosan;
The pH regulator is selected from basic amino acid and/or inorganic base; the basic amino acid is selected from one or more of lysine, arginine and betaine; the inorganic base is selected from one or more of sodium hydroxide, potassium carbonate, sodium carbonate and sodium bicarbonate;
The hydrolase is selected from one or more of cellulase, protease and chitosanase.
Preferably, the pH is adjusted to 4.5 to 6.5.
Preferably, the mass ratio of the hydrolase to the chitosan and/or chitosan derivative is (2-5): (11-15); the enzyme activity of the hydrolase is 5000-15000U/g.
Preferably, the step S) specifically includes:
mixing chitosan and/or chitosan derivative with water, adding salicylic acid, mixing, adding pH regulator to regulate pH value, and adding hydrolase to react to obtain salicylic acid chitosan co-enzymolysis complex;
The temperature of the mixing and stirring is 45-55 ℃; the mixing and stirring time is 5-15 min; the temperature for continuous mixing is 45-55 ℃; the continuous mixing time is 20-40 min; the temperature of the reaction is 45-55 ℃; the reaction time is 10-20 h.
Preferably, after the reaction, heating to terminate the reaction, filtering and drying to obtain the salicylic acid chitosan co-enzymolysis compound;
The temperature of the heating termination reaction is 75-85 ℃; the heating termination reaction time is 30-60 min.
The invention also provides the salicylic acid chitosan co-enzymolysis compound prepared by the preparation method.
Preferably, the relative molecular weight of the salicylic acid chitosan co-enzymolysis complex is 1000-3000 g/mol.
The invention also provides application of the salicylic acid chitosan co-enzymolysis compound serving as a raw material with antibacterial and/or oil control effects.
The invention provides a preparation method of a salicylic acid chitosan co-enzymolysis compound, which comprises the following steps: s) mixing chitosan and/or chitosan derivative, salicylic acid and water, then adding a pH value regulator to regulate the pH value, then adding hydrolase, and heating for reaction to obtain the salicylic acid chitosan co-enzymolysis compound. Compared with the prior art, the method directly utilizes the acid-base interaction between salicylic acid and chitosan and/or chitosan derivatives, enhances the water solubility of the salicylic acid and the chitosan and/or chitosan derivatives, uses hydrolytic enzymes to carry out enzymolysis on the salicylic acid and the chitosan and/or chitosan derivatives, prepares the low molecular weight salicylic acid chitosan co-enzymolysis compound, does not introduce other acid sources (such as hydrochloric acid and acetic acid) to carry out dissolution assisting on the chitosan and/or chitosan derivatives in the whole preparation process, reduces the introduction of potential irritant substances, improves the reaction economy, and is more suitable for industrialized application; the prepared salicylic acid chitosan co-enzymolysis compound has excellent performance, has the advantages of high water solubility and high temperature resistance stability, expands the application range of salicylic acid, and discovers that the salicylic acid chitosan co-enzymolysis compound does not influence the performance of salicylic acid raw materials, creatively discovers that the salicylic acid chitosan co-enzymolysis compound can obviously reduce the irritation of the salicylic acid, has obviously better antibacterial performance and also shows good oil control effect.
Drawings
FIG. 1 is a molecular weight distribution diagram of a chitosan salicylate co-enzyme complex obtained in example 1 of the present invention;
FIG. 2 is a graph showing the comparison of the high temperature stability of the salicylic acid chitosan co-enzymatic hydrolysis complex obtained in examples 1 to 3 and comparative example 1 of the present invention;
FIG. 3 is a graph showing the solubility of the chitosan salicylate co-enzyme complex obtained in example 1 of the present invention and the chitosan salicylate complex obtained in comparative example 3;
FIG. 4 is a graph showing the results of the mildness test of salicylic acid-chitosan co-enzymolysis complex and salicylic acid on zebra fish embryos obtained in example 1 of the present invention;
FIG. 5 is a graph showing the statistical results of the mildness test of the salicylic acid chitosan co-enzymolysis complex obtained in examples 1 to 3 and comparative examples 1 to 2 on zebra fish embryos;
fig. 6 is a graph showing the experimental results of fluorescence staining and oil control of human sebaceous gland cells of arachidonic acid and isotretinoin, which are obtained in example 1 of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a preparation method of a salicylic acid chitosan co-enzymolysis compound, which comprises the following steps: s) mixing chitosan and/or chitosan derivative, salicylic acid and water, then adding a pH value regulator to regulate the pH value, then adding hydrolase, and heating for reaction to obtain the salicylic acid chitosan co-enzymolysis compound.
The source of all the raw materials is not particularly limited and may be commercially available.
Mixing chitosan and/or chitosan derivative, salicylic acid with water; the chitosan derivative is a chitosan hydroxyl modified derivative well known to those skilled in the art, and is not particularly limited, and carboxymethyl chitosan is preferred in the present invention; the average molecular weight of the chitosan and/or chitosan derivative is preferably 10 to 80W, more preferably 20 to 50W, still more preferably 20 to 40W, most preferably 30W; the viscosity of the chitosan and/or chitosan derivative is preferably 200-800 mPa.s; the degree of deacetylation of the chitosan and/or chitosan derivative is preferably 75% or more, more preferably 75% to 90%; the mass ratio of the chitosan and/or chitosan derivative to the salicylic acid is preferably (1.1-1.5): 1, more preferably (1.1 to 1.3): 1, more preferably (1.1 to 1.2): 1, a step of; the mass ratio of the chitosan and/or chitosan derivative to water is preferably (1.1-1.5): (500 to 800), more preferably (1.1 to 1.5): (500 to 700), more preferably (1.1 to 1.5): 600, more preferably (1.1 to 1.3): 600; the temperature of the mixing is preferably 45-55 ℃; the mixing time is preferably 25-45 min; in the invention, the method specifically comprises the following steps: mixing chitosan and/or chitosan derivative with water, stirring, adding salicylic acid, and mixing; the temperature of the mixing and stirring is preferably 45-55 ℃; the mixing and stirring time is preferably 5-15 min, more preferably 8-12 min, and still more preferably 10min; the temperature for continuing mixing is preferably 45-55 ℃; the time for continuing the mixing is preferably 20 to 40 minutes, more preferably 25 to 35 minutes, still more preferably 30 minutes.
Then adding a pH value regulator to regulate the pH value; the pH regulator is preferably a basic amino acid and/or an inorganic base; the basic amino acid is preferably one or more of lysine, arginine and betaine; the inorganic base is preferably one or more of sodium hydroxide, potassium carbonate, sodium carbonate and sodium bicarbonate; in the present invention, the pH is preferably adjusted to 4.5 to 6.5, more preferably 5.0 to 6.5, still more preferably 5.0 to 6.0, and most preferably 5.0 to 5.5.
After regulating the pH value, adding hydrolase to react; the hydrolase is a specific enzyme and/or a non-specific enzyme known to those skilled in the art for hydrolyzing chitosan and/or chitosan derivatives, and is not particularly limited, and in the present invention, preferably a polysaccharide hydrolase, more preferably a cellulase, and one or more of a protease and a chitosanase; the mass ratio of the hydrolase to the chitosan and/or chitosan derivative is preferably (2-5): (11 to 15), more preferably (2 to 5): (11 to 13), more preferably (2 to 5): (11 to 12), more preferably (2 to 4): (11 to 12), more preferably (2.5 to 3.5): (11-12), most preferably (2.5-3): (11-12); the enzyme activity of the hydrolase is preferably 5000-15000U/g, more preferably 8000-12000U/g, and still more preferably 10000U/g; the temperature of the reaction is preferably 45-55 ℃; the reaction time is preferably 8 to 16 hours, more preferably 9 to 16 hours, still more preferably 10 to 14 hours, still more preferably 10 to 13 hours, and most preferably 10 to 12 hours.
After the reaction is finished, preferably heating to terminate the reaction, filtering and drying to obtain the salicylic acid chitosan co-enzymolysis compound; the heating temperature is preferably 75-85 ℃, more preferably 80 ℃; the heating time is preferably 30 to 60 minutes, more preferably 40 to 60 minutes, and still more preferably 50 to 60 minutes; after heating to terminate the reaction, preferably cooling and filtering; the filtration is preferably carried out by adopting a nano-filtration membrane and an ultrafiltration membrane in sequence; the pore diameter of the nanofiltration membrane is preferably 200-250 nm, more preferably 220nm; the ultrafiltration membrane is preferably an ultrafiltration membrane of 10-20 kDa, more preferably an ultrafiltration membrane of 15 kDa; the drying method is a method well known to those skilled in the art, and is not particularly limited, and spray drying is preferred in the present invention.
The invention directly utilizes the acid-base interaction between salicylic acid and chitosan and/or chitosan derivatives, enhances the water solubility of the salicylic acid and chitosan and/or chitosan derivatives, and uses hydrolytic enzyme to carry out enzymolysis on the salicylic acid and chitosan derivatives, thus obtaining the low molecular weight salicylic acid chitosan co-enzymolysis compound, and other acid sources (such as hydrochloric acid and acetic acid) are not introduced in the whole preparation process to carry out dissolution assisting on the chitosan and/or chitosan derivatives, thereby not only reducing the introduction of potential irritant substances, but also improving the reaction economy, and being more suitable for industrialized application; the prepared salicylic acid chitosan co-enzymolysis compound has excellent performance, has the advantages of high water solubility and high temperature resistance stability, expands the application range of salicylic acid, and discovers that the salicylic acid chitosan co-enzymolysis compound does not influence the performance of salicylic acid raw materials, creatively discovers that the salicylic acid chitosan co-enzymolysis compound can obviously reduce the irritation of the salicylic acid, has obviously better antibacterial performance and also shows good oil control effect.
The invention also provides the salicylic acid chitosan co-enzymolysis compound prepared by the method.
According to the invention, the relative molecular weight of the salicylic acid chitosan co-enzymolysis complex is preferably 1000-3000 g/mol, more preferably 1000-2500 g/mol, and even more preferably 1400-2500 g/mol.
The invention also provides application of the salicylic acid chitosan co-enzymolysis compound serving as a raw material with antibacterial and/or oil control effects.
Specifically, the salicylic acid chitosan co-enzymolysis compound is applied to a cosmetic raw material with antibacterial and/or oil control effects.
The invention also provides a cosmetic, which comprises the salicylic acid chitosan co-enzymolysis compound.
Specifically, the salicylic acid chitosan co-enzymolysis compound can improve the antibacterial and oil control effects of cosmetics.
In order to further illustrate the invention, the following embodiment is used for describing the salicylic acid chitosan co-enzymolysis compound, and the preparation method and application thereof.
The reagents used in the examples below are all commercially available; the chitosan used in the examples had an average molecular weight of 30W and a degree of deacetylation of 85%.
Example 1
Weighing 11g of chitosan, dispersing in 600mL of purified water, setting the temperature to 50 ℃ and stirring, adding 10g of salicylic acid powder after 10min, maintaining stirring for 30min, and adding a pH regulator to regulate the pH value of the reaction solution to 5.0-5.5. After the pH value of the solution reaches a set value, 2.5g of cellulase (cellulase activity 10000U/g) is added, and the solution is stirred for 10 hours at a set temperature.
Heating the reaction kettle to 80 ℃ and stirring, continuously maintaining the temperature and stirring for 60min after the reaction kettle reaches the set temperature, and cooling. Filtering the reaction solution by a 220nm filter membrane, filtering by a 15kDa ultrafiltration membrane, collecting filtrate, concentrating, and spray-drying to obtain the salicylic acid chitosan co-enzymolysis complex product.
Example 2
The mass ratio of salicylic acid to chitosan was 1:1.2, otherwise as in example 1.
Example 3
The mass ratio of salicylic acid to chitosan was 1:1.5, otherwise as in example 1.
Comparative example 1
The mass ratio of salicylic acid to chitosan was 1:1.8, otherwise as in example 1.
Comparative example 2
9G of chitosan is weighed and dispersed in 600mL of purified water, the temperature is set to 50 ℃ and the mixture is stirred for 10min, 10g of salicylic acid powder is added, the stirring is maintained for 30min, and then a pH regulator is added to regulate the pH value of the reaction solution to 5.5+/-0.5. After the pH value of the solution reaches a set value, 2.5g of cellulase (cellulase activity 10000U/g) is added, and the solution is stirred for 10 hours at a set temperature.
Heating the reaction kettle to (80+/-5 ℃) and stirring, continuously maintaining the temperature and stirring for 60min after the temperature reaches the set temperature, and cooling. Filtering the reaction liquid by a 220nm filter membrane, filtering by a 15kDa ultrafiltration membrane, collecting filtrate, concentrating, and spray drying to obtain a finished product.
Comparative example 3
Weighing 11g of chitosan, dispersing in 600mL of purified water, setting the temperature to (50 ℃ +/-5 ℃) and stirring, adding 10g of salicylic acid powder after 10min, maintaining stirring for 30min, and drying to obtain a salicylic acid chitosan compound finished product.
Molecular weight detection of Compounds
The molecular weight of the chitosan salicylate co-enzyme complex obtained in example 1 was measured by GPC gel permeation chromatography, and the obtained molecular weight distribution diagram is shown in fig. 1, and the obtained results are shown in table 1. As can be seen from the molecular weight distribution chart 1, the relative molecular weight range of the tested samples is mainly concentrated between 1000 and 3000.
TABLE 1 detection results of GPC gel permeation chromatography molecular weight
High temperature stability contrast test
0.5G of the product powders of examples 1,2 and 3 and comparative example 1 were weighed and dissolved in 24.5g of water to prepare a 2% aqueous solution, which was divided into two parts and stored in an environment of 25℃and an environment of 41℃for two months, respectively, and the state change of the solutions was observed to obtain photographs as shown in FIG. 2.
As can be seen from FIG. 2, the products obtained in examples 1 and 2 showed no significant change in color after two months of heating, example 3 showed a slight change in color, and comparative example 1 showed a significant change from near colorless to yellowish-brown.
Solubility contrast test
The products of example 1 and comparative example 3 were prepared as aqueous solutions of 20%, 10%, 5% and 2.5% by mass concentration, respectively, heated to 35-40 ℃ and stirred for 30min for reconstitution. The sample to be measured is placed in a 2ml EP tube, centrifuged to the bottom of the tube, inverted and the flow condition of the solution is observed.
As shown in FIG. 3, the 20% aqueous solution of the salicylic acid chitosan co-enzyme complex (example 1) had good solubility, and the solution was clear and high in fluidity. In the comparative example implementation process, the 20% concentration solution cannot be completely dissolved, and the solubility and fluidity of the 10%, 5% and 2.5% concentration aqueous solutions are sequentially improved along with the concentration reduction, but the overall appearance is very high in viscosity, serious in wall hanging and poor in fluidity.
From the experimental results, it can be seen that the solution of the present invention has higher dispersibility and solubility in aqueous solutions than the comparative examples, can achieve rapid dissolution even under high concentration conditions, and exhibits good fluidity. Therefore, when the product is added into the skin care product as an efficacy raw material, the scheme of the invention can realize a higher adding proportion under the condition of ensuring the skin feel of the product compared with a comparative example.
Efficacy validation test
1. Mildness test of zebra fish embryo
Principle of: zebra fish embryos are sensitive to external irritants and are commonly used for detection of external contaminants. The zebra fish embryo develops for 24 hours to produce spontaneous spin movements. The spin movement frequency of zebra fish embryos is directly related to the external stimulus to which they are subjected. Therefore, the established zebra fish embryo type is often used for evaluating whether the raw material is mild or not by taking the spin movement frequency of the zebra fish embryo as an index.
The testing steps are as follows: and (3) dissolving the compound to be tested in Holt-Buffer culture solution to prepare mother solution, and diluting the mother solution to the concentration of the experimental group by using the Holt-Buffer culture solution. Holt-buffer is formulated as follows: 1900mL of ddH 2 O were measured, placed in a 2L beaker, stirred with a stirrer, and added in this order: 7.000g NaCl,0.400gNaHCO3,0.100g KCl and 0.235g CaCl 2,ddH2 O were fixed to 2000mL and filtered using a disposable vacuum filter (pore size 0.22. Mu. Mol/L) and stored at room temperature for further use.
Zebra fish embryos which are healthy in development, consistent in state and 24hours (24 hourspost-fertilization, 24hpf for short) are randomly selected, placed in 96-well plates, 5 zebra fish embryos are placed in each well, 200 mu L of sample diluent with the concentration is added into each well, 3 compound wells are arranged in each concentration, the mixture is placed in a temperature of 28+/-0.5 ℃ for culturing for 60 minutes, and a blank control group (Holt-Buffer culture solution treatment) is arranged.
The number of spin movements of the zebra fish embryos within 30s was recorded using a split microscope video.
Data statistical analysis is performed by GRAPHPAD PRISM 8.0.0 software, a chart is drawn, and inter-group differences are analyzed by Two-way ANOVA, and P < 0.05 is used as the difference to have statistical significance.
The test results are shown below:
As can be seen from the test results of FIG. 4, the obtained salicylic acid-chitosan co-enzymolysis complex example 1 has significantly reduced irritation to zebra fish embryos compared with salicylic acid.
In addition, as can be seen from fig. 5, at the test concentrations of 0.1% (mass concentration), the irritation to the zebra fish embryo was close to that of example 1 and example 3, but the irritation to the zebra fish embryo was very high in comparative example 2, and few embryos were dead, compared to examples 1 and 2.
Therefore, in combination with the zebra fish mildness test and the raw material high-temperature stability test, the reaction mass ratio is preferably that of salicylic acid: chitosan = 1: (1.1-1.5).
2. Minimum inhibitory concentration assay
In order to verify the antibacterial effect of the scheme of the invention, the following experiments are carried out for verification:
The Minimum Inhibitory Concentration (MIC), i.e., the minimum concentration that can inhibit bacterial growth in the medium, can be expressed in the dilution method using inhibition of bacterial growth as an evaluation criterion. 100. Mu.L of a sample for test (100. Mu.L of a bacterial suspension 10 8 CFU/mL) was added to 12 wells of a 96-well plate, and the concentration of the bacteriostatic substance in each well was 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.195, 0.09, 0.049, 0.025mg/mL, respectively, and the culture medium was used as a negative control. The 96-well plate is placed under 37 ℃ (bacteria) for culturing for 48 hours, and the concentration corresponding to the last well without turbidity is the MIC value of the corresponding bacteriostatic agent for the bacteria to be tested.
Test strain: staphylococcus aureus ATCC6538, escherichia coli ATCC25922, propionibacterium acnes ATCC6919.
The operation steps are as follows:
(1) Sample configuration: the products obtained in example 1, example 2, example 3 and comparative example 3 were respectively prepared into 50mg/ml with sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and pH value was 5.5-6.0;
(2) Preparing staphylococcus aureus, escherichia coli and propionibacterium acnes suspension;
(3) Preparing a culture medium: LB medium (large intestine, staphylococcus aureus): 5g of beef extract, 10g of fish peptone and 10g of sodium chloride are dissolved in 1L of deionized water, the pH value is regulated to 7-7.2, and the mixture is packaged and sterilized for standby.
Propionibacterium acnes medium: 10g of fish peptone, 13g of beef extract, 2g of diammonium citrate, 5g of sodium acetate, 5g of glucose, 5g of sodium chloride, 0.5g of cysteine hydrochloride, 1g of soluble starch, and dissolving in 1L of deionized water, adjusting the pH value to 6.6-7, packaging and sterilizing for later use.
(4) Preparation of diluted antibacterial drugs and bacterial liquid inoculation: 100 mu L of culture medium (staphylococcus aureus, LB culture medium for escherichia coli and Propionibacterium acnes) is added into each well of a 96-well plate, 100 mu L of liquid to be tested is added into A1-H1 of the 96-well plate and mixed uniformly, and then 100 mu L of liquid is sucked out from the A1-H1 well and added into A2-H2 well and mixed uniformly. 100. Mu.L of the solution was then aspirated from A2-H2 and added to the A3-H3 wells and mixed well. And so on, until wells A11-H11, well 12 (i.e., A12-H12) was a negative control sample (not used as a growth control).
(5) Inoculating 0.1ml of bacterial suspension with the bacterial content of about 10 8 cfu/ml into a 96-well plate containing a culture medium of an anti (bacteriostatic) bacterial agent to be used as a test group sample;
(6) Placing the 96-well plate in a 37 ℃ incubator, culturing for 48 hours, and observing the result; the method is only meaningful when the bacteria in the negative control hole (i.e. the hole without the sample solution) obviously grow, and the concentration corresponding to the last hole without turbidity (obviously without colony in a clear state) is the MIC value of the corresponding bacteriostat for the bacteria to be tested. (7) In the test, the bacterial suspension for the test is subjected to viable bacteria culture counting, and the action concentration is 5X 10 6cfu/mL～5×107 cfu/mL.
Test results: when the positive control hole has bacterial growth (turbidity), the negative control tube has sterile growth (transparency), and the action concentration of the bacterial suspension for test is 5 multiplied by 10 6cfu/mL～5×107 cfu/mL, the concentration of the anti-bacterial agent corresponding to the highest dilution of the sterile growth of the test group is the MIC of the sample to the test bacteria.
The experimental results are shown in table 2: examples 1-3 are salicylic acid chitosan co-enzyme complexes of different reaction ratios, while comparative example 3 is a salicylic acid chitosan complex. As can be seen from the bacteriostasis test of three common skin bacteria, the bacteria inhibition effect of the examples 1 to 3 on three common epidermic bacteria is good, and the effect is far better than that of the comparative example 3.
Table 2 minimum inhibitory concentration assay
| Test bacteria/minimum inhibitory concentration (mg/ml) | Example 1 | Example 2 | Example 3 | Comparative example 3 |
| Propionibacterium acnes | 0.39 | 0.195 | 0.39 | 1.56 |
| Staphylococcus aureus | 0.78 | 0.39 | 0.78 | 1.56 |
| Coli bacterium | 0.39 | 0.195 | 0.39 | 3.12 |
3. Fluorescent staining and oil control experiment for human sebaceous gland cells
Experimental principle: BODIPY 493/503 is a lipophilic fluorescent probe that localizes to polar lipids and can be used to label neutral lipid levels in cells, particularly lipid levels localized to lipid droplets. The BODIPY dye is a bright green fluorescent dye and is a small-molecule dye with strong ultraviolet absorption capacity, the fluorescence peak is relatively sharp, the quantum yield is high, and the BODIPY dye is relatively insensitive to the polarity and the pH value of the environment, so that the BODIPY dye is relatively stable under different physiological conditions. BODIPY has a variety of derivative structural products due to its structural asymmetry. The BODIPY lipid drop dye can well penetrate through cell membranes and enter the inside of cells, and is positioned on polar lipids in the cells for specific staining.
The experimental steps are as follows:
(1) Cell inoculation: the density of the suspension of the SZ95 cells of the human sebaceous gland cells was adjusted to 1.4X10 5 cells/mL, and the suspension was inoculated into a 12-well plate, 1 mL/well, and cultured in a 5% CO 2 incubator at 37℃for 24 hours.
(2) A blank, a 100. Mu.M Arachidonic Acid (AA) group, a 100. Mu.M Arachidonic Acid (AA) +100. Mu.M isotretinoin group (13-cRA), a 100. Mu.M AA+1.6g/L example 1 (i.e., HT-001 in the figure) group were set; each group of cells was dosed under light-protected conditions and then incubated at 37℃in a 5% CO 2 incubator for 24 hours.
(3) The supernatant was discarded, washed 1 time with PBS, fixed with 4% paraformaldehyde for 15min, then stained with PBS in 2uM bodipy solution under dark conditions, 1 mL/well, placed in a 37℃5% CO 2 incubator for 30min, removed, discarded, washed twice with PBS, and then infiltrated with PBS.
(4) Collecting pictures: observing and photographing under an inverted fluorescent microscope.
(5) ImageJ mean fluorescence intensity analysis: after adding the fluorescence Image, extracting a single channel (Image-Color-SPLIT CHANNELS); after the operation, the original image is divided into an 8-bit black-and-white image with three channels of red, green and blue, the corresponding picture needing to be analyzed for fluorescent color is selected, and the other two pictures can be closed. Adjusting the Threshold value, selecting an appropriate region (Image-Adjust-Threshold); manually selecting a threshold value to ensure that all regions are characterized by red; measurement (analysis-Measure).
(6) Statistical analysis: data statistical analysis is carried out by adopting GRAPHPAD PRISM 8.0.0 software, a chart is drawn, metering data are expressed by x+/-s, the difference between groups is analyzed by adopting one-way ANOVA, P <0.05 is taken as the difference, the statistical significance is achieved, and the average fluorescence intensity detection result is shown in figure 5.
Analysis of results: isotretinoin, having a chemical formula of C 20H28O2, has effects of reducing sebaceous gland tissue, inhibiting sebaceous gland activity, reducing sebaceous gland secretion, reducing the number of propionibacterium acnes, and the like, and has remarkable therapeutic effects for treating acne conglobata, nodulocystic acne, acne fulminans, and the like, and thus is used as a positive reference for oil control effect, and in the test, example 1 was administered at a concentration of 1.6g/L and positive control 13-cRA (isotretinoin) was administered at a concentration of 0.3g/L, and as can be seen from the results of fig. 6, example 1 (i.e., HT-001 in the figure) at a concentration of 1.6g/L has an equivalent or more excellent oil control effect with isotretinoin at 0.3 g/L.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the salicylic acid chitosan co-enzymolysis compound is characterized by comprising the following steps of:
s) mixing chitosan and/or chitosan derivative, salicylic acid and water, then adding a pH regulator to regulate the pH value, and then adding hydrolase to react to obtain the salicylic acid chitosan co-enzymolysis compound.
2. The preparation method according to claim 1, wherein the mass ratio of the chitosan and/or chitosan derivative to salicylic acid is (1.1-1.5): 1.
3. The method according to claim 1, wherein the chitosan and/or chitosan derivative has a degree of deacetylation of 75% or more;
the chitosan derivative is carboxymethyl chitosan;
The pH regulator is selected from basic amino acid and/or inorganic base; the basic amino acid is selected from one or more of lysine, arginine and betaine; the inorganic base is selected from one or more of sodium hydroxide, potassium carbonate, sodium carbonate and sodium bicarbonate;
The hydrolase is selected from one or more of cellulase, protease and chitosanase.
4. The method according to claim 1, wherein the pH is adjusted to 4.5 to 6.5.
5. The preparation method according to claim 1, wherein the mass ratio of the hydrolase to chitosan and/or chitosan derivative is (2-5): (11-15); the enzyme activity of the hydrolase is 5000-15000U/g.
6. The preparation method according to claim 1, wherein the step S) is specifically:
mixing chitosan and/or chitosan derivative with water, adding salicylic acid, mixing, adding pH regulator to regulate pH value, and adding hydrolase to react to obtain salicylic acid chitosan co-enzymolysis complex;
The temperature of the mixing and stirring is 45-55 ℃; the mixing and stirring time is 5-15 min; the temperature for continuous mixing is 45-55 ℃; the continuous mixing time is 20-40 min; the temperature of the reaction is 45-55 ℃; the reaction time is 10-20 h.
7. The preparation method of claim 1, wherein after the reaction, heating to terminate the reaction, filtering, and drying to obtain the salicylic acid chitosan co-enzymolysis complex;
The temperature of the heating termination reaction is 75-85 ℃; the heating termination reaction time is 30-60 min.
8. A chitosan salicylate co-enzyme complex prepared by the method of any one of claims 1 to 7.
9. The chitosan salicylate co-enzyme complex of claim 8, wherein the relative molecular weight of the chitosan salicylate co-enzyme complex is 1000-3000 g/mol.
10. The use of the salicylic acid chitosan co-enzymolysis complex prepared by the preparation method of any one of claims 1 to 7 or the salicylic acid chitosan co-enzymolysis complex of claim 8 or 9 as a raw material with antibacterial and/or oil control effects.
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