阅读说明：本技术 低分子量硫酸软骨素在制备日化产品和外用制剂中的用途 (Use of low molecular weight chondroitin sulfate in preparation of daily chemical products and external preparations ) 是由 田志清 谭岸 朱水芳 丛浩龙 姜帆 朱松阳 于 2020-01-20 设计创作，主要内容包括：本发明涉及制药技术,具体涉及低分子量硫酸软骨素在制备日化产品或外用制剂中的用途。所述低分子量硫酸软骨素的分子量范围为500-15000,优选为1000-5000。所述低分子量硫酸软骨素在所述日化产品或外用制剂中的重量百分比含量为0.0001-99％,优选为0.001-5％。所述日化产品和外用制剂可有效缓解和预防皮肤或口腔粘膜炎症,改善相关红肿、瘙痒、疼痛等症状,促进人体皮肤痤疮消除,促进痘肌愈合,增强皮肤细胞活性,抗氧化或减少头发脱落。(The invention relates to a pharmaceutical technology, in particular to application of low molecular weight chondroitin sulfate in preparation of daily chemical products or external preparations. The molecular weight range of the low molecular weight chondroitin sulfate is 500-15000, and preferably 1000-5000. The content of the low molecular weight chondroitin sulfate in the daily chemical product or the external preparation is 0.0001-99% by weight, and preferably 0.001-5% by weight. The daily chemical product and the external preparation can effectively relieve and prevent skin or oral mucosa inflammation, improve related symptoms such as redness, itching, pain and the like, promote the elimination of acne on human skin, promote the healing of acne muscle, enhance the activity of skin cells, resist oxidation or reduce hair loss.)

1. Use of low molecular weight chondroitin sulfate in the preparation of a daily chemical product or an external preparation.

2. Use according to claim 1, characterized in that: the molecular weight range of the low molecular weight chondroitin sulfate is 500-15000, 500-14000, 500-13000, 500-12000, 500-11000, 500-10000, 500-9000, 500-8000, 500-7000, 500-6000, 1000-6000, 1500-6000 or 2000-5000.

3. Use according to claim 1 or 2, characterized in that: the content of the low molecular weight chondroitin sulfate in the daily chemical product or the external preparation is 0.0001-99%, 0.0001-90%, 0.0001-80%, 0.0001-70%, 0.0001-60%, 0.0001-50%, 0.0001-40%, 0.0001-30%, 0.0001-20%, 0.0001-10%, 0.0001-9%, 0.0001-8%, 0.0001-7%, 0.0001-6%, 0.0001-5%, 0.001-5%, 0.01-5%, 0.1-5% or 1-5% by weight percentage.

4. A daily chemical product or an external preparation, characterized in that: comprises low molecular weight chondroitin sulfate.

5. The daily chemical product or external preparation according to claim 4, wherein: the molecular weight range of the low molecular weight chondroitin sulfate is 500-15000, 500-14000, 500-13000, 500-12000, 500-11000, 500-10000, 500-9000, 500-8000, 500-7000, 500-6000, 1000-6000, 1500-6000 or 2000-5000.

6. The daily chemical product or external preparation according to claim 4 or 5, characterized in that: the content of the low molecular weight chondroitin sulfate in the daily chemical product or the external preparation is 0.0001-99%, 0.0001-90%, 0.0001-80%, 0.0001-70%, 0.0001-60%, 0.0001-50%, 0.0001-40%, 0.0001-30%, 0.0001-20%, 0.0001-10%, 0.0001-9%, 0.0001-8%, 0.0001-7%, 0.0001-6%, 0.0001-5%, 0.001-5%, 0.01-5%, 0.1-5% or 1-5% by weight percentage.

7. The daily chemical product or external preparation according to claim 6, wherein: the daily chemical products comprise shampoo, hair conditioner, skin cream, mouthwash, hand sanitizer, shower gel, emulsion and facial mask; the dosage forms of the external preparation include sprays, ointments, creams, gels and solutions.

8. The daily chemical product or external preparation according to claim 6, wherein: the daily chemical product or the external preparation is used for applying on the skin surface of a subject or gargling.

9. A method for producing a daily chemical product or an external preparation according to any one of claims 4 to 8, wherein: adding low molecular weight chondroitin sulfate into daily chemical products or external preparations.

10. The method of claim 9, wherein: the preparation method of the low molecular weight chondroitin sulfate comprises the following steps: dissolving chondroitin sulfate in water, adding copper sulfate, heating to 50 ℃, adding a hydrogen peroxide solution, reacting for 4 hours at 50 ℃, stopping heating, dialyzing the reaction solution overnight by using a dialysis bag with the molecular weight cutoff of 500-15000, and freeze-drying the solution to obtain the white low-molecular-weight chondroitin sulfate.

Technical Field

The invention relates to a pharmaceutical technology, in particular to application of low molecular weight chondroitin sulfate in preparation of daily chemical products and external preparations.

Background

Chondroitin Sulfate (CS) is a polysaccharide composed mainly of repeating disaccharide units connecting β (1-3) N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA). At present, commercial chondroitin is mainly extracted from cartilage tissues of animals such as pigs, cattle, chickens, ducks, sharks, sturgeons and the like. Chondroitin sulfate is thought to have a variety of pharmacological activities such as relieving osteoarthritis, reducing blood fat and blood sugar, resisting cancer metastasis, promoting cell growth, promoting skin healing, and the like. At present, the chondroitin sulfate is mainly used for moisturizing in daily cosmetics and is common chondroitin sulfate which is not subjected to low molecular weight treatment, and the molecular weight is relatively large and is between 20k and 100 k. In the pharmaceutical preparation field, chondroitin sulfate is used for lowering blood lipid, preventing arthritis and the like, or as external eye drops for treating keratitis in the prior art.

At present, the application of the low molecular weight chondroitin sulfate in daily chemical products or external preparations is not found.

Disclosure of Invention

In one aspect, the present invention provides the use of a low molecular weight chondroitin sulfate in the preparation of a daily chemical product or an external preparation.

The molecular weight range of the low molecular weight chondroitin sulfate is 500-15000, 500-14000, 500-13000, 500-12000, 500-11000, 500-10000, 500-9000, 500-8000, 500-7000, 500-6000, 1000-6000, 1500-6000 or 2000-5000.

The content of the low molecular weight chondroitin sulfate in the daily chemical product or the external preparation is 0.0001-99%, 0.0001-90%, 0.0001-80%, 0.0001-70%, 0.0001-60%, 0.0001-50%, 0.0001-40%, 0.0001-30%, 0.0001-20%, 0.0001-10%, 0.0001-9%, 0.0001-8%, 0.0001-7%, 0.0001-6% or 0.0001-5% by weight percentage.

In a preferred embodiment, the content of the low molecular weight chondroitin sulfate in the daily chemical product or the external preparation is 0.001-5%, 0.01-5%, 0.1-5% or 1-5% by weight.

The low molecular weight chondroitin sulfate can be prepared by degrading common chondroitin sulfate by mainly acid hydrolysis, enzymolysis, catalytic oxidation and the like.

The chondroitin sulfate is extracted from cartilage tissue of animals such as pig, cattle, chicken, duck, shark, and sturgeon.

In the preparation of daily chemical products, the low molecular weight chondroitin sulfate is used for preparing hair washing products, hair care products, skin cream, mouth wash, hand sanitizer, bath products, emulsion, mask or other daily chemical product forms.

In the preparation of the external preparation, the low molecular weight chondroitin sulfate is used for preparing a spray, an ointment, a cream or other external preparation forms.

The administration mode of the low molecular weight chondroitin sulfate in the preparation of external preparations and daily chemicals is skin administration.

In another aspect, the present invention provides a daily chemical product or an external preparation, characterized in that: comprises low molecular weight chondroitin sulfate.

The molecular weight range of the low molecular weight chondroitin sulfate is 500-15000, 500-14000, 500-13000, 500-12000, 500-11000, 500-10000, 500-9000, 500-8000, 500-7000, 500-6000, 1000-6000, 1500-6000 or 2000-5000.

In a preferred embodiment, the molecular weight range of the low molecular weight chondroitin sulfate is 1000-5000, 1000-4000, 1000-3000 or 1000-2000.

The content of the low molecular weight chondroitin sulfate in the daily chemical product or the external preparation is 0.0001-99%, 0.0001-90%, 0.0001-80%, 0.0001-70%, 0.0001-60%, 0.0001-50%, 0.0001-40%, 0.0001-30%, 0.0001-20%, 0.0001-10%, 0.0001-9%, 0.0001-8%, 0.0001-7%, 0.0001-6% or 0.0001-5% by weight percentage.

In a preferred embodiment, the content of the low molecular weight chondroitin sulfate in the daily chemical product or the external preparation is 0.001-5%, 0.01-5%, 0.1-5% or 1-5% by weight.

The daily chemical products include shampoo products (e.g., shampoo), hair care products (e.g., hair conditioner), skin cream, mouth wash, hand sanitizer, bath products (e.g., body wash), lotion, and mask; the dosage forms of the external preparation include sprays, ointments, creams, gels and solutions. The daily chemical product or the external preparation is used for applying on the skin surface of a subject or gargling.

The formulation of the gel is as follows: according to weight percentage, carbomer 0.7%, glycerin 5%, propylene glycol 5%, phenoxyethanol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate 0.01-2% or 1%, and the balance of deionized water.

The formulation of the spray is as follows: according to weight percentage, hyaluronic acid 0.1%, phenoxyethanol 0.3%, hexylene glycol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate 0.1-5% or 1.2%, and the balance of deionized water.

The formulation of the solution is as follows: according to weight percentage, 0.3 percent of phenoxyethanol, 0.5 percent of hexanediol, 0.05 percent of essence, 0.05 percent of citric acid, 0.1 to 2 percent or 0.5 percent of low molecular weight chondroitin sulfate and the balance of deionized water.

The formula of the mouthwash is as follows: 0.5 percent of low molecular weight chondroitin sulfate aqueous solution.

In still another aspect, the present invention provides a method for producing any of the aforementioned daily chemical products or external preparations, characterized in that: adding low molecular weight chondroitin sulfate into daily chemical products or external preparations, wherein the molecular weight range of the low molecular weight chondroitin sulfate is 500-15000, 500-14000, 500-13000, 500-12000, 500-11000, 500-10000, 500-9000, 500-8000, 500-7000, 500-6000, 1000-6000, 1500-6000 or 2000-5000.

In some embodiments, the low molecular weight chondroitin sulfate is prepared by the following method: dissolving chondroitin sulfate in water, adding copper sulfate, heating to 50 ℃, adding a hydrogen peroxide solution, reacting for 4 hours at 50 ℃, stopping heating, dialyzing the reaction solution overnight by using a dialysis bag with the molecular weight cutoff of 500-15000, and freeze-drying the solution to obtain the white low-molecular-weight chondroitin sulfate.

The invention provides application of low molecular weight chondroitin sulfate in the field of preparation of daily chemical products such as daily chemical cleaning products, disinfection products, personal care products, sanitary products, medicinal preparations, cosmetic products and the like and external preparations. The daily chemical product and the external preparation are especially suitable for relieving and preventing skin or oral mucosa inflammation and improving related symptoms such as redness, itching, pain and the like, or promoting acne elimination of human skin, promoting acne muscle healing, or enhancing skin cell activity, eliminating free radical oxidation resistance, or reducing hair loss.

Drawings

FIG. 1 is a comparison of the transdermal permeation of a low molecular weight chondroitin sulfate with a conventional chondroitin sulfate, wherein the abscissa is permeation time and the ordinate is permeation rate, LCS: low molecular weight chondroitin sulfate, CS: common chondroitin sulfate.

FIG. 2. Effect of low molecular weight chondroitin sulfate on the inhibition of the inflammatory factor TNF- α (U937 cell assay), wherein the ratio of-LPS: control group of cells without LPS stimulation, Control: control group of cells without drug treatment after LPS stimulation, DXMS: control group of cells treated with dexamethasone (final concentration 0.6mg/ml) after LPS stimulation, LCS: experimental groups of cells treated with low molecular weight chondroitin sulfate (final concentration 0.6mg/ml) after LPS stimulation, CS: experimental groups in which cells (final concentration 0.6mg/ml) were treated with normal chondroitin sulfate after LPS stimulation; the ordinate is TNF-. alpha.level (ng/L).

FIG. 3 shows the effect of low molecular weight chondroitin sulfate on the inhibition of the inflammatory factor TNF- α (RAW 264.7 cell assay), wherein LPS, Control, DXMS, LCS, CS are represented in the same manner as in FIG. 2; the ordinate is TNF-. alpha.level (ng/L).

FIG. 4 shows the effect of low molecular weight chondroitin sulfate on the inhibition of interleukin-6 (IL-6) (U937 cell assay), wherein-LPS, Control, DXMS, LCS, CS are represented in the same manner as in FIG. 2; the ordinate is IL-6 level (ng/L).

FIG. 5 shows the effect of low molecular weight chondroitin sulfate on the inhibition of interleukin-6 (IL-6) (RAW 264.7 cell assay), wherein-LPS, Control, DXMS, LCS, CS are represented in the same manner as in FIG. 2; the ordinate is IL-6 level (ng/L).

FIG. 6 shows the effect of low molecular weight chondroitin sulfate on the inhibition of interleukin-8 (IL-8) (U937 cell assay), wherein-LPS, Control, DXMS, LCS, CS are represented in the same manner as in FIG. 2; the ordinate is IL-8 level (ng/L).

FIG. 7 shows the effect of low molecular weight chondroitin sulfate on the inhibition of interleukin-1 (IL-1) (U937 cell assay), wherein-LPS, Control, DXMS, LCS, CS are represented in the same manner as in FIG. 2; the ordinate is IL-1 level (ng/L).

FIG. 8 enhancing effect of low molecular weight chondroitin sulfate on HSF cell activity, wherein, Control: control group without low molecular weight chondroitin sulfate or common chondroitin sulfate in the medium, LCS: experimental group with low molecular weight chondroitin sulfate (final concentration 0.3mg/ml) added to the medium, CS: an experimental group in which ordinary chondroitin sulfate (final concentration of 0.3mg/ml) was added to the medium; the cell viability (%) is plotted on the ordinate.

FIG. 9 is a graph showing the enhancement of Hacat cell activity by low-molecular-weight chondroitin sulfate, wherein Control, LCS, and CS are the same as those shown in FIG. 8; the cell viability (%) is plotted on the ordinate.

FIG. 10 shows the antioxidant effect of low molecular weight chondroitin sulfate, wherein the abscissa is the final concentration (0.3mg/ml, 0.6mg/ml, 1.2mg/ml, 2.4mg/ml) of low molecular weight chondroitin sulfate and normal chondroitin sulfate in the medium, and the ordinate is the active oxygen level, LCS: experimental group with low molecular weight chondroitin sulfate added to the medium, CS: the experimental group of normal chondroitin sulfate was added to the medium.

Detailed Description

The present invention is further described below in conjunction with specific examples, which are to be understood to be illustrative and explanatory only and not limiting in any way of the scope of the invention.

Biological material

Human macrophage U937 cell is provided by the institute of microbiology, academy of sciences of China; the mouse mononuclear macrophage system RAW264.7 is provided by China institute of quarantine science; the immortalized normal keratinocyte Hacat of human is purchased from a selfish organism, and the product number is CL 0114; human immortalized normal skin fibroblasts HSF are purchased from a selfing organism, and the product number is CL 0159. The biological material is also stored in the laboratory and can be issued to the public for verification experiments within twenty years from the filing date.

Experimental reagent

Chondroitin sulfate (CS, otherwise known as "common chondroitin sulfate") was purchased from Hainan five star Biotechnology, Inc. and has a weight average molecular weight of 4.2 ten thousand.

Dexamethasone (DXMS) is a drug on the market, is a well-known anti-inflammatory drug and has a strong inhibiting effect on inflammatory factors such as TNF-alpha, IL-6, IL-8 and the like, so dexamethasone is set as a control in an anti-inflammatory experiment. Dexamethasone for the following experiments was purchased from Sigma.

DMEM medium, 1640 medium, FBS (fetal bovine serum), pancreatin were purchased from LIFE TECH.

0.25% pancreatin formulation: 100ml of PBS solution, 0.25g of pancreatin. The preparation method comprises the following steps: first, 8g of NaCl and 2.9g of Na were weighed₂HPO₄，0.2gKCl，0.2g KH₂PO₄Dissolving the PBS in distilled water, and fixing the volume to 1L to obtain a PBS solution; then weighing 0.25g of pancreatin (trypsin), adding into 100ml of PBS solution, and putting into a refrigerator at 4 ℃ overnight; after dissolution, pH7.4 was adjusted in ice bath, and filtered for sterilization.

Copper sulfate, hydrogen peroxide solution, carbomer, glycerin, propylene glycol, phenoxyethanol, citric acid, hyaluronic acid and hexylene glycol are all purchased from Chinese medicine reagents.

In the following examples, unless otherwise specified, the experimental reagents are conventional in the art, and can be prepared according to conventional methods in the art or obtained commercially; unless otherwise stated, the experimental methods are all routine in the field, and reference is made to relevant experimental manuals.

Example 1 preparation of Low molecular weight chondroitin sulfate

The method for preparing the low molecular weight chondroitin sulfate by adopting the catalytic oxidation method comprises the following steps: dissolving chondroitin sulfate in water, adding copper sulfate (Chinese medicine reagent), heating to 50 deg.C, adding hydrogen peroxide solution (Chinese medicine reagent), reacting at 50 deg.C for 4 hr, and stopping heating. The reaction solution was dialyzed overnight using a dialysis bag (Shanghai-derived leaf Biotechnology Co., Ltd.) having a molecular weight cut-off of 500-.

Example 2 identification of biological Activity of Low molecular weight chondroitin sulfate

First, a low molecular weight chondroitin sulfate is prepared, comprising the following steps: 0.5g chondroitin sulfate was dissolved in 100ml water, 0.032g copper sulfate (national medicine reagent) (0.2mmol) was added, heating was carried out to 50 ℃, and 5ml hydrogen peroxide 30% solution (national medicine reagent) was added. After reacting for 4h at constant temperature of 50 ℃, stopping heating. The reaction solution was dialyzed overnight using a dialysis bag (Shanghai-derived leaf Biotech Co., Ltd.) having a molecular weight cut-off of 1000. The solution was lyophilized to give 0.2g of white low molecular weight chondroitin sulfate. Gel Permeation Chromatography (GPC) of the obtained low molecular weight chondroitin sulfate was conducted by entrusting the institute of metals of Chinese academy of sciences to use a gel permeation chromatograph (Shimadzu LC-20AT) and a gel permeation chromatography column (Viscotek, A3000, Col 300 x 8.0mm), and the results showed that the weight average molecular weight of the prepared low molecular weight chondroitin sulfate was about 3600.

Then, the following experiments show that the low molecular weight chondroitin sulfate has better biological activity in the aspects of skin absorption and penetration, anti-inflammation, skin cell activity enhancement, active oxygen generation inhibition and the like, and is obviously superior to the common chondroitin sulfate.

1. Transdermal penetration of low molecular weight chondroitin sulfate

Solution preparation: the normal saline is used as a solvent, and a low molecular weight chondroitin sulfate (LCS) solution and a common Chondroitin Sulfate (CS) solution with the mass percentage concentration of 0.3% are respectively prepared.

Skin preparation: the pig, Guangxi breed, male, with a weight of 8-10kg, purchased from the market is killed, the breast hair is shaved off immediately, the skin is peeled off, the subcutaneous adipose tissue is scraped off, and the pig breast skin in vitro is obtained by cleaning, with a thickness of 0.8-1 mm.

The penetration test method comprises the following steps: the Franz diffusion cell method was used. The prepared breast skin of the in vitro pig is fixed between diffusion cells, and constant temperature water bath at 37 ℃ is carried out on the diffusion cells. After the temperature is constant, 1.5mL of each of the LCS solution with the mass percentage concentration of 0.3% and the CS solution with the mass percentage concentration of 0.3% is taken, the LCS solution and the CS solution with the mass percentage concentration of 0.3% are respectively added into a supply room of a Franz diffusion cell (Shanghai Yuyan scientific instruments Co., Ltd., model TT-6), the breast skin (with the thickness of 0.8-1mm) of an isolated pig is taken as a skin model, physiological saline (sodium chloride solution with the mass fraction of 0.9%) is taken as a receiving liquid, the receiving liquid in the receiving cell is respectively extracted at the time points of 2h, 6h and 12h, the concentration of D-glucuronic acid in the receiving liquid is measured, the cumulative permeation amount is calculated, the permeation rate is the cumulative permeation amount/dose, and the transdermal permeation effect of the chondroitin sulfate with the two molecular weights is investigated.

The experimental results are as follows: as shown in FIG. 1, it can be seen that the transdermal absorption rate and the cumulative permeation amount of the low molecular weight chondroitin sulfate are significantly improved. At 2h, the normal chondroitin sulfate is not substantially transdermal, and after 12h, the penetration amount is only about 20%. And the low molecular weight chondroitin sulfate has a penetration amount of 19% within 2h and reaches a penetration amount of more than 90% after 12 h.

2. Effect of low molecular weight chondroitin sulfate on inhibiting inflammatory factor TNF-alpha

Anti-inflammatory effects were measured by inhibition of TNF-. alpha.production by LPS (Lipopolysaccharide) stimulated human macrophage U937 and mouse monocyte macrophage line RAW 264.7.

U937 cell experimental method：

(1) Cell recovery and preparation: removing frozen U937 cells from liquid nitrogen, rapidly standing at 37 deg.C for 5min, centrifuging at 800rpm for 5min, removing supernatant, adding 10ml 1640 medium (LIFE TECH) containing 10% (v/v) FBS (LIFE TECH) into 10cm culture dish, standing at 37 deg.C and 5% CO₂Culturing in an incubator. Adjusting the cell state to logarithmic growth phase. Then, the cells were aspirated, centrifuged at 800rpm to collect the cells, and the cells were resuspended and counted. The cells were divided into 24-well plates at 10 ten thousand per well. Three replicate wells were set for each sample and incubated for three hours.

(2) LPS-stimulated cells: 50 microliters of PMA (phorbol myristate ethyl ester, Sigma) was added per well to a final concentration of 20 nM. Standing at 37 deg.C for 5% CO₂The culture was carried out for 24 hours. LPS (lipopolysaccharide, Sigma) was added to each well to a final concentration of 1 microgram per ml and incubated with the cells.

(3) And (3) drug treatment: a cell control group without LPS stimulation, a cell control group without drug treatment after LPS stimulation, a control group in which cells were treated with dexamethasone after LPS stimulation (dexamethasone was added to the cell culture well at a final concentration of 0.6mg/ml), an experimental group in which cells were treated with low-molecular-weight chondroitin sulfate after LPS stimulation (low-molecular-weight chondroitin sulfate was added to the cell culture well at a final concentration of 0.6mg/ml), and an experimental group in which cells were treated with normal chondroitin sulfate after LPS stimulation (normal chondroitin sulfate was added to the cell culture well at a final concentration of 0.6mg/ml) were set. Each of the control and experimental groups was cultured for 24 hours, and then the cells and the supernatant were aspirated and centrifuged at 800rpm for 8 minutes at 4 ℃.

(4) ELISA test: the supernatant was transferred to a new tube, and the TNF-. alpha.content in the supernatant was assayed using a human tumor necrosis factor-. alpha.ELISA kit (Jiangsu enzyme immunoassay, product No. MM-0122H1) according to the kit instructions. The kit was used to measure a human TNF-a standard (purchased from Jiangsu enzyme immunoassay) and to plot a standard curve, and the TNF-a content in the sample supernatant was calculated from the standard curve. The standard deviation was calculated. Plotted against the calculated data (fig. 2).

RAW 264.7 cell assay：

(1) Cell recovery and preparation: removing frozen RAW264.7 cells from liquid nitrogen, rapidly standing at 37 deg.C for 5min, centrifuging at 800rpm for 5min, removing supernatant, adding 10ml DMEM medium (LIFE TECH) containing 10% (v/v) FBS (LIFE TECH) into 10cm culture dish, standing at 37 deg.C and 5% CO₂Culturing in an incubator. Adjusting the cell state to logarithmic growth phase. Cells were digested with 0.25% trypsin, neutralized with serum FBS, harvested by centrifugation at 800rpm, and counted after resuspension. The cells were divided into 24-well plates at 5 ten thousand per well. Three replicate wells were set for each sample and incubated for 24 hours.

(2) LPS stimulates the cells as described above.

(3) The drug treatment method is the same as above.

(4) The supernatant was transferred to a new tube and assayed for TNF- α content in the supernatant using the mouse tumor necrosis factor α (TNF- α) ELISA kit (jiangsu enzyme immunoassay, product No. MM-0132M1) according to the kit instructions. The kit was used to measure mouse TNF-a standards (purchased from Jiangsu enzyme immunoassay) and to plot a standard curve, and the TNF-a content in the sample supernatant was calculated from the standard curve. The standard deviation was calculated. Plotted against the calculated data (fig. 3).

The experimental results are as follows: u937 cell experiments demonstrated that both common Chondroitin Sulfate (CS) and low molecular weight chondroitin sulfate (LCS) have inhibitory effects on TNF- α (p <0.001 compared to control), but low molecular weight chondroitin sulfate has higher synergistic inhibition (p <0.001) than common chondroitin sulfate, and it is noteworthy that low molecular weight chondroitin sulfate has close to the inhibitory effect of dexamethasone. Similar results are obtained in RAW264.7 cell experiments, and the low molecular weight chondroitin sulfate has obvious inhibition effect on TNF-alpha, is obviously superior to common chondroitin sulfate (p is less than 0.001) and is only slightly inferior to dexamethasone.

3. Low molecular weight chondroitin sulfate has effect of inhibiting interleukin-6 (IL-6)

Anti-inflammatory effects were tested by inhibition of IL-6 production by LPS-stimulated human macrophage U937 and mouse monocyte macrophage line RAW 264.7.

The experimental method comprises the following steps: methods of cell recovery and preparation, LPS stimulation and drug treatment are as described above. In ELISA test, human interleukin 6(IL-6) ELISA kit (Jiangsu enzyme immunoassay, product number MM-0049H1) is adopted in ELISA test of human macrophage U937, and the content of human IL-6 is determined according to the kit specification; ELISA experiment of RAW264.7 cells mouse interleukin-6 (IL-6) ELISA kit (Jiangsu enzyme immunoassay, product number MM-0163M1) was used, and the content of mouse IL-6 was determined according to the kit instructions.

The experimental results are as follows: as shown in FIG. 4, U937 cell assay demonstrated that both chondroitin have strong inhibitory effects on IL-6. Wherein, the inhibiting effect of the low molecular weight chondroitin sulfate is even obviously stronger than that of dexamethasone (p is less than 0.001), and the effect of the common chondroitin sulfate is equivalent to that of the dexamethasone. As shown in FIG. 5, RAW264.7 cell experiments also prove that the inhibitory effect of low molecular chondroitin is obviously stronger than that of common chondroitin sulfate (p < 0.001).

4. Low molecular weight chondroitin sulfate has effect of inhibiting interleukin-8 (IL-8)

Anti-inflammatory effects were measured by inhibition of IL-8 production by LPS-stimulated human macrophage U937.

The experimental method comprises the following steps: methods of cell recovery and preparation, LPS stimulation and drug treatment are as described above. The ELISA test adopts human interleukin 8(IL-8/CXCL8) ELISA kit (Jiangsu enzyme immunoassay, product number MM-1558H1), and the content of human IL-8 is determined according to the kit instruction.

The experimental results are as follows: as shown in figure 6, U937 cell test proves that the low molecular weight chondroitin sulfate has good inhibition effect on IL-8, is equivalent to the effect of dexamethasone, and is remarkably superior to the common chondroitin sulfate (p is less than 0.001). As can be seen, the normal chondroitin sulfate has little inhibitory effect on IL-8.

5. Low molecular weight chondroitin sulfate has effect of inhibiting interleukin-1 (IL-1)

The anti-inflammatory effect was examined by inhibition of IL-1 production by LPS-stimulated human macrophage U937.

The experimental method comprises the following steps: methods of cell recovery and preparation, LPS stimulation and drug treatment are as described above. The ELISA test adopts human interleukin 1(IL-1) ELISA kit (Jiangsu enzyme immunoassay, product number MM-0179H1), and the content of human IL-1 is determined according to the kit specification.

The experimental results are as follows: as shown in FIG. 7, U937 cell test proves that the low molecular weight chondroitin sulfate has obvious inhibition effect on IL-1 and is obviously superior to the common chondroitin sulfate (p < 0.001). While the inhibition effect of common chondroitin sulfate on IL-1 is weaker.

6. Function of low molecular weight chondroitin sulfate for enhancing skin cell vitality

Detecting the activity influence of the low molecular weight chondroitin sulfate and the common chondroitin sulfate on human immortalized normal keratinocyte Hacat and human immortalized normal skin fibroblast HSF.

The experimental method comprises the following steps: 10ml of DMEM medium (LIFE TECH) containing 10% (v/v) FBS (LIFE TECH) was added to a 10cm dish, and the mixture was placed at 37 ℃ and 5% CO₂Cells were cultured in an incubator. The cell state was adjusted to logarithmic growth phase and the cells were digested with 0.25% pancreatin at 37 ℃. Cells were counted and plated into 96 well cell culture plates, 2000 cells per well. The LCS or CS was added to a final concentration of 0.3 mg/ml. Incubated at 37 ℃ for 24 hours. The total volume was controlled to 200. mu.l. Add 10. mu.l of CCK-8 solution (Shanghai Biyuntian Biotech Co., Ltd.) to each well. A control group without LCS or CS added was set. Further incubation in cell incubator for 0.5h, followed by measuring absorbance at 450nmLuminosity, 630nm is the reference wavelength. The percentage of cell proliferation was calculated and plotted against the survival rate of the control group without LCS or CS as 100%.

The experimental results show that: the low molecular weight chondroitin sulfate has obvious enhancement effect on the activities of HSF cells (figure 8) and Hacat cells (figure 9), and is obviously superior to the common chondroitin sulfate (p is less than 0.001), and the common chondroitin sulfate has no enhancement effect on the activities of the two cells.

7. Antioxidation effect of low molecular weight chondroitin sulfate

The antioxidant effect is detected by the active oxygen inhibition effect of low molecular weight chondroitin sulfate and common chondroitin sulfate on human immortalized normal keratinocyte Hacat cells.

Cell culture: hacat cells were cultured to logarithmic growth phase and split into 96-well plates, 5000 cells per well. Culturing at 37 deg.C overnight, adding LCS or CS at different ratios to final concentrations of 0.3mg/ml, 0.6mg/ml, 1.2mg/ml and 2.4mg/ml respectively. Incubated at 37 ℃ for 24 hours.

The reactive oxygen species level of cells stimulated by different concentrations of LCS and CS was measured using a reactive oxygen species detection kit (Shanghai Biyuntian Biotechnology Co., Ltd., product No. S0033) according to the kit instructions. The method comprises the following steps: DCFH-DA (fluorochrome 2,7-Dichlorodi-hydrofluorescein diacetate, Shanghai Biandongan, S0033-1) was diluted with serum-free DMEM medium (LIFE TECH) to a final concentration of 10. mu.M. Cells are stimulated by LCS and CS with different concentrations, a positive control is set at the same time, an active oxygen positive control reagent is Rosu (Shanghai Biyuntian biology, S0033-2), and the active oxygen level can be obviously improved after the cells are stimulated for 20-30 minutes. The cell culture was removed, washed twice with serum-free DMEM medium (LIFE TECH), and 50. mu.l of diluted DCFH-DA solution (Shanghai Biyunnan biosciences) was added. Incubate at 37 ℃ for 30 minutes. The unbound probes were washed thoroughly with three serum-free DMEM media (LIFE TECH) washes. The 488nm excitation wavelength and the 525nm emission wavelength are used for detecting the intensity of fluorescence before and after stimulation in real time. The active oxygen level of the control group without LCS or CS was taken as 100%, and plotted as a percentage of active oxygen.

The experimental results are as follows: as shown in FIG. 10, the low molecular weight chondroitin sulfate has a significant inhibitory effect on active oxygen, and the inhibitory effect is gradually enhanced as the concentration is increased. The common chondroitin sulfate has a certain inhibiting effect on active oxygen, but is obviously weaker than the low-molecular-weight chondroitin sulfate, and the inhibiting effect is not obviously enhanced along with the increase of the concentration.

Example 3 preparation of anti-acne gel

The formula of No. 1 gel is as follows: according to weight percent, carbomer is 0.7 percent, glycerol is 5 percent, propylene glycol is 5 percent, phenoxyethanol is 0.5 percent, essence is 0.05 percent, citric acid is 0.05 percent, low molecular weight chondroitin sulfate (molecular weight is 3000) is 1 percent, and the balance is deionized water.

No. 2 gel formulation: according to weight percent, carbomer is 0.7 percent, glycerol is 5 percent, propylene glycol is 5 percent, phenoxyethanol is 0.5 percent, essence is 0.05 percent, citric acid is 0.05 percent, low molecular weight chondroitin sulfate (molecular weight is 800) is 1 percent, and the balance is deionized water.

The formula of No. 3 gel is as follows: according to weight percent, carbomer 0.7%, glycerol 5%, propylene glycol 5%, phenoxyethanol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate (molecular weight 15000) 1%, and the balance of deionized water.

No. 4 gel formulation: according to weight percent, carbomer is 0.7 percent, glycerol is 5 percent, propylene glycol is 5 percent, phenoxyethanol is 0.5 percent, essence is 0.05 percent, citric acid is 0.05 percent, chondroitin sulfate (molecular weight 20000) is 1 percent, and the balance is deionized water.

The preparation method comprises the following steps: mixing carbomer, glycerol, propylene glycol, phenoxyethanol, essence, and deionized water, heating to 70 deg.C, dissolving completely to transparent state, stirring and cooling to 40 deg.C, adding essence and low molecular weight chondroitin sulfate (or chondroitin sulfate), adjusting pH to 6-7 with citric acid, stirring and cooling slowly to room temperature to obtain the final product.

Evaluation of gel Effect of acne treatment

Number of sexes (per experimental group): 10 women; 10 men.

Age: between 20 and 50 years old.

Skin condition: the skin has obvious acne and pox, has no allergic history of the skin disease, and meets the voluntary selection standard of the subject.

The test method comprises the following steps: applying appropriate amount of test sample on cleaned affected part 2 times per day, 1 time each in the morning and evening, and trying for 1 week; the subjects kept the former daily life and rest time and diet during the trial period, and no other medicine was taken on the affected parts. Specific results are shown in table 1.

The evaluation result criteria are as follows:

the method is good: has obvious effects of detumescence, itching relieving and pain relieving, and no obvious cyst or pustule on the affected part observed by naked eyes. When the touch is performed actively or not, the discomfort such as itching, pain, burning and the like does not exist.

Preferably: the detumescence/itching/pain-relieving effect is obvious, and the effect of detumescence is obvious when the affected part is observed by naked eyes. When the hands are not touched actively, the discomfort of itching, pain, burning and the like is avoided.

In general: basically has no detumescence/itching/pain relieving effect, and has obvious red and swollen, and obvious discomfort such as itching, pain, burning and the like when the affected part is observed by naked eyes.

Difference: without any detumescence/itching/pain relief effect, without any reduction or even aggravation of the red swelling or itching sensation.

TABLE 1 evaluation of the acne-removing Effect of the gel

Example 4 preparation of spray for preventing alopecia

No. 1 spray composition: according to weight percent, hyaluronic acid 0.1%, phenoxyethanol 0.3%, hexanediol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate (molecular weight 3000) 1.2%, and the balance of deionized water.

No. 2 spray composition: according to weight percent, hyaluronic acid 0.1%, phenoxyethanol 0.3%, hexanediol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate (molecular weight 800) 1.2%, and the balance of deionized water.

No. 3 spraying composition: according to weight percent, hyaluronic acid 0.1%, phenoxyethanol 0.3%, hexanediol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate (molecular weight 15000) 1.2%, and the balance of deionized water.

No. 4 spraying composition: according to weight percent, hyaluronic acid 0.1%, phenoxyethanol 0.3%, hexanediol 0.5%, essence 0.05%, citric acid 0.05%, chondroitin sulfate (molecular weight 20000) 1.2%, and the balance of deionized water.

The preparation method comprises the following steps: mixing hyaluronic acid, hexanediol, phenoxyethanol, essence and deionized water uniformly, heating to 70 deg.C, dissolving completely to transparent state, stirring and cooling to 40 deg.C, adding essence and low molecular weight chondroitin sulfate (or chondroitin sulfate), adjusting pH to 6-7 with citric acid, stirring and cooling slowly to room temperature to obtain the final product.

Evaluation of spray Effect against alopecia

Number of sexes (per experimental group): 8 women; 12 men.

Age: between 30 and 70 years old.

Hair condition: the hair is soft and lackluster, the daily hair loss is higher than the normal shedding level, and the voluntary selection standard of the subject is met.

The test method comprises the following steps: after the hair is cleaned and wiped dry, a proper amount of the sample to be tested is sprayed on the surface of the scalp, and the scalp is dried by blowing or naturally dried for 1 time every day for 4 weeks. The specific results are shown in Table 2.

The evaluation result criteria are as follows:

the method is good: compared with the hair before use, the hair is obviously blackened and glossy, and the toughness and the strength are obviously improved. The number of hair drops was found to be significantly reduced by combing or actively grabbing the hair with a light hand, or after rinsing the hair.

Preferably: compared with the hair before use, the hair has improved lackluster or withered and yellow condition to a certain extent, and the toughness and the strength are increased to a certain extent. The number of hair falling is found to be reduced to some extent after combing or actively grabbing the hair with a hand, or after washing the hair.

In general: compared with the prior art, the hair loss hair cream has the advantages that the hair luster, the toughness and the strength are not obviously improved, and the hair loss condition is not obviously improved.

Difference: the hair had no effect, even worse, on shine, tenacity, etc. than before use. The condition of hair loss is more obvious.

TABLE 2 evaluation of spray alopecia prevention Effect

EXAMPLE 5 preparation of anti-inflammatory desensitizing solutions

The composition of the No. 1 solution is as follows: calculated by weight (%), phenoxyethanol 0.3%, hexanediol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate (molecular weight 3000) 0.5%, and the balance of deionized water.

The composition of the No. 2 solution is as follows: calculated by weight (%), phenoxyethanol 0.3%, hexanediol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate (molecular weight 800) 0.5%, and the balance of deionized water.

The composition of the No. 3 solution is as follows: calculated by weight (%), phenoxyethanol 0.3%, hexanediol 0.5%, essence 0.05%, citric acid 0.05%, low molecular weight chondroitin sulfate (molecular weight 15000) 0.5%, and the balance of deionized water.

The composition of No. 4 solution is as follows: calculated by weight (%), phenoxyethanol 0.3%, hexanediol 0.5%, essence 0.05%, citric acid 0.05%, chondroitin sulfate (molecular weight 20000) 0.5%, and the balance of deionized water.

The preparation method comprises the following steps: mixing hexanediol, phenoxyethanol, essence, and deionized water, heating to 40 deg.C, dissolving completely to transparent state, adding essence and low molecular weight chondroitin sulfate (or chondroitin sulfate), adjusting pH to 6-7 with citric acid, stirring, and slowly cooling to room temperature.

Evaluation of Effect of anti-inflammatory desensitizing solution

Number of sexes (per experimental group): 10 women; 10 men.

Age: between 20 and 60 years old.

Inflammatory conditions: the skin local inflammation caused by allergy, mosquito bite and other factors is red and itchy in appearance, and may have slight swelling.

The test method comprises the following steps: spraying appropriate amount of test sample on cleaned affected part, using once, and observing effect after 3 hr. The specific results are shown in Table 3.

Evaluation result criteria:

the method is good: has obvious detumescence/itching relieving effect, and the red and swollen phenomenon is obviously reduced by visual observation. Under the condition of active touch or inactive touch, the discomfort such as itching, pain and the like is not obvious.

Preferably: the detumescence/itching relieving effect is obvious, and the red and swollen phenomenon is obviously reduced by visual observation. When the patient is not touched actively, the discomfort of itching, pain and the like is obviously relieved.

In general: basically has no or less detumescence/itching relieving effect, has no obvious reduction of red swelling phenomenon observed by naked eyes, and still has pain and itch feeling.

Difference: has no detumescence/itching relieving effect, and has no reduction or even aggravation of red swelling or itching feeling.

TABLE 3 evaluation of the anti-inflammatory desensitizing Effect of the solutions

Example 6 preparation of mouthwash for alleviating dental ulcer and evaluation effects thereof

No. 1 mouthwash: deionized water is used for preparing a low molecular weight chondroitin sulfate (molecular weight 3000) water solution with the mass percentage concentration of 0.5 percent, and the solution is used at present.

Mouthwash No. 2: deionized water is used for preparing a low molecular weight chondroitin sulfate (molecular weight 800) water solution with the mass percentage concentration of 0.5 percent, and the solution is used at present.

No. 3 mouthwash: deionized water is used for preparing a low molecular weight chondroitin sulfate (molecular weight 15000) water solution with the mass percentage concentration of 0.5 percent, and the solution is used as it is.

No. 4 mouthwash: the chondroitin sulfate (with the molecular weight of 20000) water solution with the mass percent concentration of 0.5 percent is prepared by deionized water and is used as the preparation.

Evaluation population (per experimental group): 5 women; 5 men.

Age: between 20 and 50 years old.

Ulcer condition: recurrent aphthous stomatitis, which is generated at the inner side of lip, buccal mucosa, vestibular groove, soft palate and other parts, has white affected part with diameter of 0.2-0.5cm, obvious pain and burning pain, 1-2 days of ulcer generation, healthy body and accordance with the volunteer selection standard of a subject.

The using method comprises the following steps: the test solution was repeatedly used for 3 times without swallowing, 3 times daily, and 2 days.

TABLE 4 evaluation of the efficacy of mouthwashes to alleviate canker sores