Essence containing chlorella extract and preparation method thereof

Abstract

The present invention provides an essence containing chlorella extract and a preparation method thereof. The chlorella extract process comprises the following steps: raw material pulverization; enzymatic hydrolysis with cellulase and trypsin; primary fermentation using a mixture of Bacillus natto and Bacillus pumilus; addition of bamboo leaf extract, corn steep liquor, and sucrose, followed by inoculation with Rhodotorula glutinosus for secondary fermentation to obtain the chlorella extract. The mixed fermentation of multiple strains exhibits a synergistic effect, resulting in greater enrichment of natural products during the fermentation process compared to single-strain fermentation. Furthermore, the present invention provides an essence containing chlorella extract, glycerin, sodium hyaluronate, honey, and water, which exhibits significant moisturizing, antioxidant, and soothing effects.

Description

Technical Field

[0001] The invention belongs to the field of cosmetic preparation, and particularly relates to an essence containing chlorella extract and a preparation method thereof. Background Art

[0002] Chlorella, a microalgae rich in protein, vitamins, minerals, and bioactive substances, has been widely studied and applied in the food, pharmaceutical, and cosmetic industries. However, traditional Chlorella extraction processes suffer from low efficiency, high cost, and insufficient retention of active ingredients.

[0003] Currently, Chlorella extraction methods primarily include physical and chemical methods. While physical methods, such as mechanical and ultrasonic disruption, can preserve the active ingredients, they have low extraction efficiency. Chemical methods, such as organic solvent extraction, while highly efficient, may introduce solvent residues, impacting product safety. Furthermore, existing extraction processes often overlook microbial fermentation, which can significantly enhance the release and conversion of active ingredients in Chlorella. Summary of the Invention

[0004] The present invention provides an essence containing chlorella extract and a preparation method thereof, which has the effects of anti-oxidation, moisturizing and soothing the skin through the natural ingredients extracted from plants.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for extracting a Chlorella extract, wherein the specific preparation steps are as follows:

[0007] S1: Cleaning and removing impurities from Chlorella vulgaris, drying to constant weight, breaking the cell wall, and grinding to obtain product A1;

[0008] S2: A1 is enzymatically hydrolyzed using cellulase and trypsin to obtain enzymatic hydrolysis product A2;

[0009] S3: Inoculate Bacillus natto and Bacillus pumilus into A2, ferment for 4-6 hours, and then sterilize and filter to obtain filtrate A3;

[0010] S4: Add bamboo leaf extract, corn steep liquor, and sucrose to A3, stir evenly, inoculate with red yeast rice, ferment for 3-4 hours, and then ultrasonically disrupt and filter to obtain filtrate A4;

[0011] S5: A4 is freeze-dried to obtain the final product A5.

[0012] The amount of cellulase and trypsin added in S2 is 1-3 wt % of that in A1, the mass ratio of cellulase to trypsin is 1:0.4-0.7, and the enzymatic hydrolysis is carried out for 30-60 minutes.

[0013] The added amounts of Bacillus natto and Bacillus pumilus in S3 are 3-5wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.5-0.8; the fermentation starting temperature is 32-34°C, and the fermentation starting pH is 7±0.5.

[0014] The total amount of bamboo leaf extract, corn steep liquor, and sucrose added in S4 is 10-15wt% of A3, and the mass ratio of the bamboo leaf extract, corn steep liquor, and sucrose is 1:0.7:0.1-0.3; the amount of red yeast added is 2-3wt% of A3; the fermentation starting temperature is 29±1°C, and the fermentation starting pH is 6.5±0.5.

[0015] In a second aspect, the present invention provides an essence composition, the specific components of which are as follows:

[0016] Chlorella extract 1-5wt%;

[0017] Glycerol 20-25wt%;

[0018] Sodium hyaluronate 10-12wt%;

[0019] Honey 1-5wt%;

[0020] The balance was made up with sterile deionized water.

[0021] In the present invention, three different bacteria are used to ferment Chlorella. First, Bacillus natto and Bacillus pumilus are used for fermentation. Then, bamboo leaf extract, corn steep liquor and sucrose are added to the fermentation product. Then, red yeast rice is used for fermentation again to obtain the final product. During the first fermentation (i.e., fermentation with Bacillus natto and Bacillus pumilus), the intervention of Bacillus pumilus can effectively enrich the products of Bacillus natto such as nattokinase, polymyxin and bacitracin. During the second fermentation (i.e., fermentation with red yeast rice), the base raw materials are formulated to induce red yeast rice to selectively secrete astaxanthin, carotenoids and B vitamins. Experimental results show that the secretion of astaxanthin, carotenoids and B vitamins by red yeast rice can be effectively increased by adding a specific proportion of base raw materials. The results show that the chlorella extract obtained after the first and second fermentations of Chlorella has significant antioxidant, moisturizing and soothing effects.

[0022] Compared with the prior art, the present invention has the following beneficial effects: the extract obtained by fermenting and extracting Chlorella using Bacillus natto, Bacillus pumilus and Rhodotorula glutinosus has significant antioxidant, moisturizing and soothing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1: The cumulative percentage of single-bacteria fermentation products to the cumulative percentage of mixed-bacteria fermentation products.

[0024] Among them, the secretion amount of each product of the mixed fermentation of Bacillus natto and Bacillus pumilus was used as a positive control, and the secretion amount of each product of the single bacteria fermentation was mapped as a percentage of the secretion amount of each product of the mixed fermentation of Bacillus natto and Bacillus pumilus; the fermentation experimental steps and the amount of each component were the same as in Example 1, and the detection of various secretions was carried out using conventional laboratory methods or kits.

[0025] Figure 2 : The cumulative percentage of fermentation products without adding (corn steep liquor, bamboo leaf extract, sucrose) to the cumulative percentage of fermentation products with adding (corn steep liquor, bamboo leaf extract, sucrose).

[0026] Note: With the addition of (corn steep liquor, bamboo leaf extract, sucrose) as the positive control, the percentage of fermentation products without (corn steep liquor, bamboo leaf extract, sucrose) to fermentation products with (corn steep liquor, bamboo leaf extract, sucrose) was mapped; the fermentation experimental steps and the dosage of each component were the same as in Example 1; all kinds of secretions were detected by routine laboratory methods or kits. DETAILED DESCRIPTION

[0027] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples. The terms used in the examples are for describing specific embodiments and do not constitute a limitation on the scope of protection of the present invention.

[0028] The experimental methods in the following examples, where specific conditions are not specified, are generally based on conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0029] Some of the raw materials and their sources are as follows:

[0030] Bacillus subtilis natto was purchased from Ningbo Testo Biotechnology Co., Ltd., product number TS288161.

[0031] Bacillus pumilus strain: purchased from Ningbo Testo Biotechnology Co., Ltd., catalog number TS290749;

[0032] Rhodotorula glutinosus strain: purchased from Ningbo Testo Biotechnology Co., Ltd., product number TS324223;

[0033] Chlorella: The Chlorella is Xinjiang Tianshan Chlorella JY-QWWZ-1900386, which was deposited in the General Microbiology Center of China Culture Collection Administration on June 7, 2021, with the deposit number CGMCC No. 22396.

[0034] Bamboo leaf extract: purchased from Shaanxi Pinhong Biotechnology Co., Ltd.

[0035] The extraction process of Chlorella extract is as follows:

[0036] S1: Cleaning and removing impurities from Chlorella vulgaris, drying to constant weight, breaking the cell wall, and grinding to obtain product A1;

[0037] S2: A1 is enzymatically hydrolyzed using cellulase and trypsin to obtain enzymatic hydrolysis product A2;

[0038] S3: Inoculate Bacillus natto and Bacillus pumilus into A2, ferment for 4-6 hours, and then sterilize and filter to obtain filtrate A3;

[0039] S4: Add bamboo leaf extract, corn steep liquor, and sucrose to A3, stir evenly, inoculate with red yeast rice, ferment for 3-4 hours, and then ultrasonically disrupt and filter to obtain filtrate A4;

[0040] S5: A4 is freeze-dried to obtain the final product A5.

[0041] The amount of cellulase and trypsin added in S2 is 1-3 wt % of that in A1, the mass ratio of cellulase to trypsin is 1:0.4-0.7, and the enzymatic hydrolysis is carried out for 30-60 minutes.

[0042] The added amounts of Bacillus natto and Bacillus pumilus in S3 are 3-5wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.5-0.8; the fermentation starting temperature is 32-34°C, and the fermentation starting pH is 7±0.5.

[0043] The total amount of bamboo leaf extract, corn steep liquor, and sucrose added in S4 is 10-15wt% of A3, and the mass ratio of the bamboo leaf extract, corn steep liquor, and sucrose is 1:0.7:0.1-0.3; the amount of red yeast added is 2-3wt% of A3; the fermentation starting temperature is 29±1°C, and the fermentation starting pH is 6.5±0.5.

[0044] Example 1: Following the preparation methods S1-S5, the addition amount of S2 cellulase and trypsin was 2 wt % of A1, the mass ratio of cellulase to trypsin was 1:0.5, and the enzymatic hydrolysis was carried out for 45 min.

[0045] The addition amount of Bacillus natto and Bacillus pumilus in S3 is 4wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.6; the fermentation starting temperature is 33°C, and the fermentation starting pH is 7.

[0046] The total amount of bamboo leaf extract, corn steep liquor and sucrose added in S4 is 13wt% of A3, and the mass ratio of bamboo leaf extract, corn steep liquor and sucrose is 1:0.7:0.2; the amount of red yeast added is 3wt% of A3; the fermentation starting temperature is 30°C, and the fermentation starting pH is 6.5.

[0047] Example 2: Following the preparation methods S1-S5, the addition amount of S2 cellulase and trypsin was 1 wt % of A1, the mass ratio of cellulase to trypsin was 1:0.4, and the enzymatic hydrolysis was carried out for 30 min.

[0048] The added amount of Bacillus natto and Bacillus pumilus in S3 is 3wt% of that of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.8; the fermentation starting temperature is 32°C, and the fermentation starting pH is 6.5.

[0049] The total amount of bamboo leaf extract, corn steep liquor, and sucrose added in S4 is 10 wt % of A3, and the mass ratio of the bamboo leaf extract, corn steep liquor, and sucrose is 1:0.7:0.1; the amount of red yeast added is 2 wt % of A3; the fermentation starting temperature is 31° C., and the fermentation starting pH is 6.0.

[0050] Example 3: Following the preparation methods S1-S5, the addition amount of S2 cellulase and trypsin was 3 wt % of A1, the mass ratio of cellulase to trypsin was 1:0.7, and the enzymatic hydrolysis was carried out for 60 min.

[0051] The added amount of Bacillus natto and Bacillus pumilus in S3 is 5wt% of that of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.5; the fermentation starting temperature is 34°C, and the fermentation starting pH is 7.5.

[0052] The total amount of bamboo leaf extract, corn steep liquor and sucrose added in S4 is 15wt% of A3, and the mass ratio of bamboo leaf extract, corn steep liquor and sucrose is 1:0.7:0.3; the amount of red yeast added is 2wt% of A3; the fermentation starting temperature is 29°C, and the fermentation starting pH is 7.0.

[0053] Comparative Example 1: The difference from Example 1 is that only Bacillus natto is used in S3, and the rest is the same as Example 1.

[0054] Comparative Example 2: The difference from Example 1 is that only Bacillus pumilus is used in S3, and the rest is the same as Example 1.

[0055] Comparative Example 3: The difference from Example 1 is that step S3 is not included, and the rest is the same as Example 1.

[0056] Comparative Example 4: The difference from Example 1 is that different amounts of bamboo leaf extract, corn steep liquor and sucrose in a mass ratio of 1:2:1 are used in S4, and the rest are the same as Example 1.

[0057] Comparative Example 5: The difference from Example 1 is that no bamboo leaf extract, corn steep liquor, or sucrose is added to S4, and the rest is the same as Example 1.

[0058] Comparative Example 6: The difference from Example 1 is that step S4 is not included, and the rest is the same as Example 1.

[0059] Comparative Example 7: The difference from Example 1 is that the order of steps S3 and S4 is changed.

[0060] Comparative Example 8: Following steps S1-S3 in Example 1, the strains added in S3 were Bacillus natto, Bacillus pumilus, and Rhodotorula glutinosus, and bamboo leaf extract, corn steep liquor, and sucrose were added before inoculating S3 in the same amounts as in Example 1. The fermentation product of S3 was then sterilized, filtered, and freeze-dried to obtain the final product, Chlorella extract.

[0061] An essence composition, the specific mass percentage ratio of which is as follows:

[0062] Table 1

[0063]

[0064]

[0065] Note: The remainder is made up of sterile deionized water.

[0066] Safety trials

[0067] Compositions 1-5 were evaluated for cosmetic irritation using the 2015 Technical Specifications for Safety of Cosmetics. The test method was a skin patch test on 50 randomly distributed subjects aged 16-65 years.

[0068] Test Method: Place the test substance in a patch tester at a dosage of 0.020-0.025g. Cover the patch tester with non-irritating cloth-based tape on the subject's back or forearm. Use the palm of your hand to gently press the patch tester to evenly adhere to the skin surface. Continue for 24 hours. Remove the patch tester and observe the skin reaction 30 minutes after the indentation disappears. If the result is negative, observe again 24 hours and 48 hours after the patch test.

[0069] Evaluation criteria:

[0070] Grade 0: negative reaction;

[0071] Grade 1: Suspicious reaction, only slight erythema;

[0072] Grade 2: weak positive reaction, erythema, infiltration, edema, and papules may be present;

[0073] Grade 3: Strong positive reaction, with erythema, infiltration, edema, papules, and the reaction may extend beyond the test area;

[0074] Grade 4: Very strong positive reaction, obvious erythema, severe infiltration, edema, confluent blister, and reaction beyond the test area.

[0075] Test results: All subjects had negative skin reactions.

[0076] The above test results show that the composition provided by the present invention is mild and non-irritating to the skin and is safe to use.

[0077] DPPH detection

[0078] The antioxidant capacity of Examples 1-3 and Comparative Examples 1-8 was verified using DPPH free radicals.

[0079] Prepare a DPPH solution with a concentration of 0.1 mmol / L. Take Examples 1-3 and Comparative Examples 1-8 to prepare 10 g / L sample solutions, 1 mg / mL positive control Vc solutions, and blank control solutions. Take 0.1 ml of the sample solution and 4.9 ml of the DPPH solution, respectively, and place them in a dark room for reaction for 20 minutes. Then, measure the absorbance at a wavelength of 517 nm, and calculate the clearance rate DVC%.

[0080]

[0081] Table 2

[0082] experimental group Clearance rate / % Example 1 87.42 Example 2 85.17 Example 3 86.23 Comparative Example 1 65.42 Comparative Example 2 41.52 Comparative Example 3 35.41 Comparative Example 4 74.23 Comparative Example 5 64.14 Comparative Example 6 42.25 Comparative Example 7 66.51 Low ratio 8 56.52 VC 100％

[0083] According to the results of Examples 1-3 in Table 2, the Chlorella extract provided by the embodiments of the present invention has a good antioxidant effect, especially the effect of Example 1 is better.

[0084] Comparison of Example 1 with Comparative Examples 1-3 shows that Bacillus natto and Bacillus pumilus have significant synergistic effects.

[0085] By comparing the results of Example 1 with Comparative Examples 4-5, it can be seen that bamboo leaf extract, corn steep liquor and sucrose can effectively increase the secretion of astaxanthin, carotenoids and B vitamins by Rhodotorula mucilaginosa. Due to the addition of a specific ratio of bamboo leaf extract, corn steep liquor and sucrose, the antioxidant efficacy of Example 1 is significantly improved compared with Comparative Examples 4-5.

[0086] Comparing the results of Example 1 with Comparative Examples 3 and 6, it can be seen that the extract prepared by the secondary fermentation has a significant antioxidant effect compared with the extract prepared by the primary fermentation.

[0087] Comparing the results of Example 1 with Comparative Examples 7-8, it can be seen that the Chlorella extract obtained by the mixed single fermentation has a poorer antioxidant effect than the Chlorella extract obtained by the process of Example 1, indicating that the number of fermentations and the order of feeding are specific and cannot be changed.

[0088] Zebrafish tail moisturizing test

[0089] The zebrafish epidermis has a certain osmotic pressure tolerance range, exceeding which it will lose water. Treating zebrafish with a hyperosmotic pressure solution causes their tails to shrink and shrink due to water loss. Therefore, an inductive model for hydration and moisturizing in zebrafish can be established to evaluate the effectiveness of moisturizing products.

[0090] (1) Breeding: Pair zebrafish at a male-to-female ratio of 2:1, collect the eggs and incubate them overnight (incubator at 28°C).

[0091] (2) Experimental groups: The experiment set up a blank control group and an experimental group. The experimental groups included a model group, a positive group, and a sample group, with 8 2-dpf juvenile fish in each group.

[0092] (3) Solution Preparation: Accurately weigh 1 mg of the samples of Examples 1-3 and Comparative Examples 1-8 and prepare the test sample solutions. Urea (0.05%) was used as a positive control for the experiment, and 1 mg of sample was added to each well. After administration, the 6-well plate was placed in a 28°C incubator in the dark and incubated for 24 h.

[0093]

[0094] Test results: as shown in Table 3 below.

[0095] Table 3

[0096] experimental group Zebrafish tail area reduction inhibition rate Positive group 1 blank 0 Example 1 0.89 Example 2 0.84 Example 3 0.83 Comparative Example 1 0.64 Comparative Example 2 0.31 Comparative Example 3 0.24 Comparative Example 4 0.63 Comparative Example 5 0.58 Comparative Example 6 0.42 Comparative Example 7 0.68 Low ratio 8 0.57

[0097] According to the results analysis in Table 3: the results of Examples 1-3 show that the Chlorella extract provided by the embodiments of the present invention has a good moisturizing effect, especially the effect of Example 1 is better.

[0098] Comparison of Example 1 with Comparative Examples 1-3 shows that Bacillus natto and Bacillus pumilus have significant synergistic effects.

[0099] By comparing the results of Example 1 with Comparative Examples 4-5, it can be seen that bamboo leaf extract, corn steep liquor and sucrose can effectively increase the secretion of astaxanthin, carotenoids and B vitamins by Rhodotorula mucilaginosa. Due to the addition of a specific ratio of bamboo leaf extract, corn steep liquor and sucrose, the moisturizing effect of Example 1 is significantly improved compared with Comparative Examples 4-5.

[0100] Comparing the results of Example 1 with Comparative Examples 3 and 6, it can be seen that the extract prepared by secondary fermentation has a significant moisturizing effect compared with the extract prepared by primary fermentation.

[0101] Comparing the results of Example 1 with Comparative Examples 7-8, it can be seen that the Chlorella extract obtained by the mixed one-time fermentation has a poorer moisturizing effect than the Chlorella extract obtained by the process of Example 1, indicating that the number of fermentations and the order of feeding are specific and cannot be changed.

[0102] Human body 30s instant soothing effect test

[0103] Volunteer selection criteria: a) healthy volunteers aged 18 to 60 years; b) subjects without skin lesions / dermatitis or other factors that affect measurement in the test area; c) those with sensitive or dry faces; d) those who test positive for lactic acid stinging.

[0104] The number of volunteers is guaranteed to be more than 30, and the subjects should mainly engage in indoor activities to avoid long-term exposure to light and high temperature or cold environments.

[0105] 140 volunteers aged 22-55 years with sensitive facial skin (lactic acid stinging test score ≥ 3 points on both sides of the nose) and dry East Asian skin were selected. A sample group and a blank group (i.e., model control group: no sample was applied) were set up, and the test areas were randomly distributed on the left and right sides of the volunteers' faces. After using the composition 1-14 of the present invention, the redness image was collected by the skin facial image analyzer VISIA7, and the skin redness a* value was analyzed by Image-Pro Plus (IPP) analysis software to evaluate its moisturizing and soothing effects. The laboratory temperature was 21±1°C; the relative humidity was 50%±10%.

[0106] The experiment adopted the self-before and after control method and the blank group control method. The test period was 30s, and the skin image was collected twice, 0s and 30s respectively. The sample group (compositions 1-3) and the blank group (composition 9) were set up, and the test areas were randomly distributed on the left and right faces of the volunteers. The sample was applied at a dosage of 0.25g / half face. Qualified subjects were selected according to the requirements. When the volunteers visited, they signed an informed consent form with them, collected skin images and questionnaire data after using the product for 30s. When visiting, after washing the face with the designated cleanser, sit quietly in the test environment for about 20-30 minutes. After the meditation, the test began.

[0107] With the help of facial image analyzer VISIA 7 ( Company, USA), the instrument uses three different light sources to capture facial images, which can intuitively observe changes in facial skin condition.

[0108] The redness image a* value was analyzed using Image-Pro Plus software.

[0109] Skin redness a* value: The a* value represents the redness of the skin. An increase in the a* value indicates increased vascular reactivity of the skin.

[0110] The data were analyzed using SPSS19.0 data analysis software.

[0111] Table 4

[0112]

[0113] From the analysis of the results in Table 4: Comparing compositions 1-3, the results show that the skin redness a* of compositions 1-3 decreased, and composition 1 showed the highest degree of decrease, indicating that the skin soothing effect of the composition prepared using the process of the present invention does not show a linear trend with the addition amount.

[0114] Comparison of composition 1 and compositions 6-13 shows that the use of the Chlorella extract prepared by the present invention can maximize the efficacy of the composition in reducing the a* value of skin redness, and the human efficacy experiments corroborate the previous experiments.

[0115] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of the present application.

Claims

1. A process for extracting chlorella extract, characterized in that: The extraction process of the chlorella extract is specifically as follows: S1: washing, drying, and crushing Chlorella to obtain product A1; S2: A1 is enzymatically hydrolyzed using cellulase and trypsin to obtain enzymatic hydrolysis product A2; S3: Inoculate Bacillus natto and Bacillus pumilus into A2, ferment for 4-6 hours, and then sterilize and filter to obtain filtrate A3; S4: Add bamboo leaf extract, corn steep liquor, and sucrose to A3, stir evenly, inoculate with red yeast rice, ferment for 3-4 hours, crush, and filter to obtain filtrate A4; S5: freeze-drying A4 to obtain the final product A5; The total amount of bamboo leaf extract, corn steep liquor and sucrose added in S4 is 10-15wt% of A3, and the mass ratio of bamboo leaf extract, corn steep liquor and sucrose is 1:0.7:0.1-0.3; the amount of red yeast rice added is 2-3wt% of A3.

2. The extraction process according to claim 1, characterized in that The Chlorella is Xinjiang Tianshan Chlorella JY-QWWZ-1900386, which was deposited in the General Microbiology Center of China Culture Collection Administration on June 7, 2021, with the deposit number CGMCC No. 22396.

3. The extraction process according to claim 1, characterized in that The amount of cellulase and trypsin added in S2 is 1-3 wt % of that in A1, the mass ratio of cellulase to trypsin is 1:0.4-0.7, and the enzymolysis time is 30-60 min.

4. The extraction process according to claim 1, characterized in that The added amount of Bacillus natto and Bacillus pumilus in S3 is 3-5wt% of A1, and the mass ratio of Bacillus natto to Bacillus pumilus is 1:0.5-0.

8.

5. The extraction process according to claim 1, characterized in that The S3 fermentation starting temperature is 32-34° C., and the fermentation starting pH is 7±0.

5.

6. The extraction process according to claim 1, characterized in that: The S4 fermentation starting temperature is 29±1° C., and the fermentation starting pH is 6.5±0.

5.

7. A Chlorella extract, characterized in that The chlorella extract is prepared by the chlorella extract extraction process according to any one of claims 1 to 6.

8. An essence, characterized in that: The invention comprises the chlorella extract according to claim 7.

9. The essence according to claim 8, characterized in that The following components are included in mass percentage: Chlorella extract 1-5wt%; Glycerol 20-25wt%; Sodium hyaluronate 10-12wt%; Honey 1-5wt%; The balance is sterile deionized water.

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