A preparation method of low molecular weight sulfated glycosaminoglycan and its skin care application

Abstract

The present invention relates to the field of cosmetic technology, and proposes a preparation method and skin care application of a low molecular weight sulfated glycosaminoglycan. The low molecular weight sulfated glycosaminoglycan is obtained by extracting and degrading heparin-like byproducts as raw materials. The low molecular weight sulfated glycosaminoglycan of the present invention is a biogenic material, has good skin affinity, and at the same time, the lower molecular weight can better enter the deep tissue of the skin, can inhibit the aggregation of zebrafish embryonic neutrophils, has a significant soothing effect, and can also promote the expression of zebrafish type I collagen gene, has the effect of promoting type I collagen regeneration, supports anti-wrinkle and firming efficacy claims, and has a broad prospect for development as anti-aging cosmetics. Through the above technical scheme, the problem that heparin-like byproducts in the related art cannot be utilized is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a preparation method and skin care application of low molecular weight sulfated glycosaminoglycans. Background Art

[0002] As consumers' awareness of scientific skin care continues to improve, skin problem-oriented functional skin care and precision skin care methods have become important development trends in cosmetics. Glycosaminoglycans are long-chain, linear polysaccharides composed of repeating disaccharide units. They are endogenously expressed at high levels in the skin and are commonly found in skin cells, cell surfaces, and extracellular matrix. They account for 0.1% to 0.3% of the total weight of the skin and are a recognized skin care target.

[0003] Depending on whether they are sulfated, glycosaminoglycans include non-sulfated hyaluronic acid and sulfated heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and keratan sulfate. As a non-sulfated glycosaminoglycan, hyaluronic acid has been widely studied and applied in the field of cosmetics. However, there are few studies on the application of sulfated heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and keratan sulfate in cosmetics.

[0004] Heparan sulfate, dermatan sulfate and chondroitin sulfate are all present in heparin extraction by-products and can be separated from heparin extraction by-products. Heparan sulfate and dermatan sulfate work synergistically and can be used as raw materials for sulodexide, which are used to prevent and treat thrombotic diseases and diabetic nephropathy; chondroitin sulfate has an anticoagulant effect and can prevent and treat coronary heart disease. After separating heparan sulfate, dermatan sulfate and chondroitin sulfate from heparin extraction by-products, the heparin-like by-products are usually discarded as scraps, resulting in a waste of resources. Summary of the invention

[0005] The present invention provides a preparation method and skin care application of a low molecular weight sulfated glycosaminoglycan, which solves the problem in the related art that the heparin-like byproducts after heparan sulfate, dermatan sulfate and chondroitin sulfate are separated from heparin extraction byproducts cannot be utilized.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing low molecular weight sulfated glycosaminoglycans comprises the following steps:

[0008] S1, dissolving the heparin-like by-product in an acetic acid solution to obtain an acetic acid solution of the heparin-like by-product;

[0009] S2, adding the sodium nitrite solution to the acetic acid solution of the heparin-like byproduct to react to obtain a first reaction solution;

[0010] S3, filtering the first reaction liquid to obtain a first filtrate, adjusting the pH of the first filtrate to alkaline, adding an oxidant, performing an oxidation reaction, and obtaining a second reaction liquid;

[0011] S4, filtering the second reaction liquid to obtain a second filtrate, adding an organic solvent to the second filtrate until the volume fraction of the organic solvent is 49% to 54%, and precipitating to obtain a precipitate and a supernatant;

[0012] S5, continue to add the organic solvent to the supernatant until the volume fraction of the organic solvent is ≥80%, stand and separate, collect the precipitate, and dry to obtain low molecular weight sulfated glycosaminoglycan;

[0013] The heparin-like by-product is the heparin-like by-product remaining after sodium heparin is separated from crude intestinal mucosal heparin by salt hydrolysis, thermal denaturation, ion exchange, oxidation, and organic solvent precipitation, and then heparan sulfate, dermatan sulfate, and chondroitin sulfate are extracted.

[0014] In the present invention, based on the volume fraction of the organic solvent in the entire liquid medicine after the organic solvent is added, when the volume fraction of the organic solvent after the organic solvent is added in step S4 is not in the range of 49%-54%, and the volume fraction of the organic solvent after the organic solvent is added in step S5 is less than 80%, low molecular weight sulfated glycosaminoglycans with a molecular weight of less than 6000 and a specific rotation of ≥﹢10° can hardly be obtained.

[0015] As a further technical solution, the volume concentration of the acetic acid is 4% to 10%, and the mass volume ratio of the heparin-like by-product to the acetic acid solution is 1 g: 10 to 25 mL.

[0016] As a further technical solution, the mass volume ratio of the heparin-like by-product to the acetic acid solution is 1 g: 10-25 mL.

[0017] As a further technical solution, the mass ratio of the sodium nitrite to the heparin-like by-product is 1-2.5:100.

[0018] As a further technical solution, the specific rotation of the heparin-like by-product is -10°~-40°. Specifically, when the specific rotation of the heparin-like by-product is -10°~-20°, the mass ratio of the sodium nitrite to the heparin-like by-product is 2~2.5:100;

[0019] When the specific rotation of the heparin-like by-product is -20° to -30°, the mass ratio of the sodium nitrite to the heparin-like by-product is 1.5 to 2:100;

[0020] When the specific rotation of the heparin-like by-product is -30° to -40°, the mass ratio of the sodium nitrite to the heparin-like by-product is 1 to 1.5:100.

[0021] In the present invention, different masses of sodium nitrite need to be added to heparin-like byproducts with different specific rotations to finally obtain low molecular weight sulfated glycosaminoglycans with a molecular weight of less than 6000 and a specific rotation of ≥+10°.

[0022] As a further technical solution, in step S3, the oxidant includes hydrogen peroxide and / or peracetic acid, and the amount of the oxidant added is 2% to 5% of the volume of the first filtrate.

[0023] As a further technical solution, in step S4 and step S5, the organic solvent includes one or more of methanol, ethanol, and acetone.

[0024] The present invention also provides a low molecular weight sulfated glycosaminoglycan, which is prepared according to the preparation method.

[0025] As a further technical solution, the molecular weight of the low molecular weight sulfated glycosaminoglycan is less than 6000, and the specific rotation is ≥﹢10°.

[0026] In the present invention, when the molecular weight of the low molecular weight sulfated glycosaminoglycan is less than 6000 and the specific rotation is ≥+10°, the low molecular weight sulfated glycosaminoglycan has a significant skin care effect, inhibits the aggregation of neutrophils, promotes collagen regeneration, promotes skin wrinkle removal and skin tightening. On the contrary, if the molecular weight and specific rotation are not within the above range, the skin care effect is not significant.

[0027] The present invention also proposes the use of the low molecular weight sulfated glycosaminoglycan in the preparation of skin care products.

[0028] As a further technical solution, the low molecular weight sulfated glycosaminoglycan is used in the preparation of skin care products with skin wrinkle removal and firming effects.

[0029] As a further technical solution, the low molecular weight sulfated glycosaminoglycan is used in the preparation of skin care products with soothing effects.

[0030] In the present invention, low molecular weight sulfated glycosaminoglycans are used to prepare skin care products, which can give full play to the soothing, wrinkle-removing and firming effects of low molecular weight sulfated glycosaminoglycans.

[0031] The working principle and beneficial effects of the present invention are:

[0032] 1. In the present invention, the heparin-like byproducts obtained by extracting heparin from crude heparin and then extracting heparan sulfate, dermatan sulfate and chondroitin sulfate are used as raw materials, and low molecular weight sulfated glycosaminoglycans are prepared by acidification, cracking, oxidation, graded precipitation and the like. This part of the heparin-like byproducts is usually discarded by various enterprises. The present invention redevelops and utilizes this part of the waste, solving the problem in the prior art that the heparin-like byproducts obtained by separating heparan sulfate, dermatan sulfate and chondroitin sulfate from heparin extraction byproducts cannot be utilized.

[0033] 2. In the present invention, the low molecular weight sulfated glycosaminoglycan product prepared by using heparin-like by-product as raw material has skin care effect. In the zebrafish model, the product can significantly inhibit the aggregation of neutrophils and has broad application potential in soothing skin care products; in addition, the product can also significantly upregulate the expression of type I collagen gene, promote collagen regeneration, and play an important role in skin wrinkle removal and firming. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The preferred implementation modes will be described below in a clear and understandable manner with reference to the accompanying drawings to further illustrate the above-mentioned characteristics, technical features, advantages and implementation methods of the present invention.

[0035] Figure 1 This is a soothing efficacy diagram of the model control group in Experimental Example 2 of the present invention;

[0036] Figure 2 This is a soothing effect diagram of Example 1 group in Experimental Example 2 of the present invention;

[0037] Figure 3 This is a soothing effect diagram of the comparative example 1 group in Experimental Example 2 of the present invention;

[0038] Figure 4 This is a soothing effect diagram of the comparative example 3 group in the experimental example 2 of the present invention;

[0039] Figure 5 This is a bar graph of the relative expression levels of zebrafish type I collagen gene in groups 1 to 5 in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings and other implementation methods can be obtained based on these drawings without creative work.

[0041] In addition, in the description of the present application, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0042] In the following embodiments, the heparin-like by-product is obtained by separating the heparin sodium from the crude intestinal mucosal heparin by salt hydrolysis, heat denaturation, ion exchange, oxidation, and organic solvent precipitation, and then extracting heparan sulfate, dermatan sulfate, and chondroitin sulfate to obtain the remaining heparin-like by-product.

[0043] Example 1

[0044] A method for preparing low molecular weight sulfated glycosaminoglycans comprises the following steps:

[0045] S1. Weigh 100 g of a heparinoid byproduct with a specific rotation of -17°, add 2000 mL of a 5% (V / V) acetic acid solution, and stir to dissolve to obtain an acetic acid solution of the heparinoid byproduct;

[0046] S2. Take 48 mL of 5% (W / V) sodium nitrite solution and slowly and uniformly add it to the acetic acid solution of the heparin-like byproduct for 3.5 hours. After the addition is completed, stir and react for 20 hours.

[0047] S3, filter, adjust the solution pH to 10.9 with sodium hydroxide, add 3.5% (V / V) hydrogen peroxide by volume of the filtrate, and react for 12 hours;

[0048] S4, filtering, adjusting the pH of the filtrate to 6.8, adding methanol under stirring until the volume fraction of methanol is 49% (calculated based on the volume fraction of methanol in the entire liquid after the addition of methanol), and letting it stand;

[0049] S5. Separate the precipitate and the supernatant, dry the precipitate and keep it for other uses, continue to add methanol to the supernatant until the volume fraction of methanol is 85% (calculated as the volume fraction of methanol in the entire solution after the addition of methanol), let it stand, remove the supernatant, collect the precipitate, and obtain 38 g of low molecular weight sulfated glycosaminoglycan.

[0050] Example 2

[0051] A method for preparing low molecular weight sulfated glycosaminoglycans comprises the following steps:

[0052] S1. Weigh 100 g of heparinoid byproduct with a specific rotation of -25°, add 1000 mL of 9% (V / V) acetic acid solution and stir to dissolve;

[0053] S2. Measure 38 mL of 5% (W / V) sodium nitrite solution, slowly and uniformly add it to the acetic acid solution of the heparin-like byproduct for 3 hours and 15 minutes. After the addition is completed, stir and react for 20 hours.

[0054] S3, filter, adjust the solution pH to 10.6 with sodium hydroxide, add 4.9% (V / V) hydrogen peroxide by volume of the filtrate, and react for 12 hours;

[0055] S4, filtering, adjusting the pH of the filtrate to 6.6, adding ethanol under stirring until the volume fraction of ethanol is 50% (calculated based on the volume fraction of ethanol in the entire liquid after the addition of ethanol), and letting it stand;

[0056] S5. Separate the precipitate and the supernatant, dry the precipitate and keep it for other uses, continue to add ethanol to the supernatant until the volume fraction of ethanol is 85% (calculated as the volume fraction of ethanol in the entire solution after the addition of ethanol), let it stand, remove the supernatant, collect the precipitate, and obtain 31g of low molecular weight sulfated glycosaminoglycan.

[0057] Example 3

[0058] A method for preparing low molecular weight sulfated glycosaminoglycans comprises the following steps:

[0059] S1. Weigh 100 g of heparinoid byproduct with a specific rotation of -32°, add 2500 mL of 4% (V / V) acetic acid solution and stir to dissolve;

[0060] S2. Weigh 22 mL of 5% (W / V) sodium nitrite solution, slowly and uniformly add it to the acetic acid solution of the heparin-like byproduct for 4 hours. After the addition is completed, stir and react for 20 hours.

[0061] S3, filter, adjust the solution pH to 10.8 with sodium hydroxide, add 2.0% (V / V) peracetic acid by volume of the filtrate, and react for 12 hours;

[0062] S4, filtering, adjusting the pH of the filtrate to 6.5, adding acetone under stirring until the volume fraction of acetone is 54% (based on the volume fraction of acetone in the entire solution after the addition of acetone), and letting it stand;

[0063] S5. Separate the precipitate and the supernatant, dry the precipitate and keep it for other uses, continue to add acetone to the supernatant until the volume fraction of acetone is 85% (calculated as the volume fraction of acetone in the entire solution after the addition of acetone), let it stand, remove the supernatant, collect the precipitate, and obtain 31 g of low molecular weight sulfated glycosaminoglycan.

[0064] Example 4

[0065] A method for preparing low molecular weight sulfated glycosaminoglycans comprises the following steps:

[0066] S1. Weigh 100 g of a heparinoid byproduct with a specific rotation of -12°, add 2000 mL of a 5% (V / V) acetic acid solution, and stir to dissolve to obtain an acetic acid solution of the heparinoid byproduct;

[0067] S2. Take 40 mL of 5% (W / V) sodium nitrite solution and slowly and uniformly add it to the acetic acid solution of the heparin-like byproduct for 3 hours and 20 minutes. After the addition is completed, stir and react for 20 hours.

[0068] S3, filter, adjust the solution pH to 11 with sodium hydroxide, add 3.5% (V / V) peracetic acid by volume of the filtrate, and react for 12 hours;

[0069] S4, filtering, adjusting the pH of the filtrate to 6.6, adding methanol under stirring until the volume fraction of methanol is 54% (calculated based on the volume fraction of methanol in the entire liquid after the addition of methanol), and letting it stand;

[0070] S5. Separate the precipitate and the supernatant, dry the precipitate and keep it for other uses, continue to add methanol to the supernatant until the volume fraction of methanol is 85% (calculated as the volume fraction of methanol in the entire solution after the addition of methanol), let it stand, remove the supernatant, collect the precipitate, and obtain 32 g of low molecular weight sulfated glycosaminoglycan.

[0071] Example 5

[0072] A method for preparing low molecular weight sulfated glycosaminoglycans comprises the following steps:

[0073] S1. Weigh 100 g of heparinoid byproduct with a specific rotation of -21°, add 1000 mL of 9% (V / V) acetic acid solution and stir to dissolve;

[0074] S2. Take 31 mL of 5% (W / V) sodium nitrite solution and slowly and uniformly add it to the acetic acid solution of the heparin-like byproduct for 3 hours and 45 minutes. After the addition is completed, stir and react for 20 hours;

[0075] S3, filter, adjust the solution pH to 10.9 with sodium hydroxide, add 4.9% (V / V) peracetic acid by volume of the filtrate, and react for 12 hours;

[0076] S4, filtering, adjusting the pH of the filtrate to 6.6, adding ethanol under stirring until the volume fraction of ethanol is 53% (based on the volume fraction of ethanol in the entire liquid after the addition of ethanol), and letting it stand;

[0077] S5. Separate the precipitate and the supernatant, dry the precipitate and keep it for other uses, continue to add ethanol to the supernatant until the volume fraction of ethanol is 85% (calculated as the volume fraction of ethanol in the entire solution after the addition of ethanol), let it stand, remove the supernatant, collect the precipitate, and obtain 33 g of low molecular weight sulfated glycosaminoglycan.

[0078] Example 6

[0079] A method for preparing low molecular weight sulfated glycosaminoglycans comprises the following steps:

[0080] S1. Weigh 100 g of heparinoid byproduct with a specific rotation of -37°, add 2500 mL of 4% (V / V) acetic acid solution and stir to dissolve;

[0081] S2. Weigh 28 mL of 5% (W / V) sodium nitrite solution, slowly and uniformly add it to the acetic acid solution of the heparin-like byproduct for 3 h 40 min. After the addition is completed, stir and react for 20 h.

[0082] S3, filter, adjust the solution pH to 10.1 with sodium hydroxide, add 2.0% (V / V) hydrogen peroxide by volume of the filtrate, and react for 12 hours;

[0083] S4, filtering, adjusting the pH of the filtrate to 6.5, adding acetone under stirring until the volume fraction of acetone is 49% (based on the volume fraction of acetone in the entire solution after the addition of acetone), and letting it stand;

[0084] S5. Separate the precipitate and the supernatant, dry the precipitate and keep it for other uses, continue to add acetone to the supernatant until the volume fraction of acetone is 85% (calculated as the volume fraction of acetone in the entire solution after the addition of acetone), let it stand, remove the supernatant, collect the precipitate, and obtain 36 g of low molecular weight sulfated glycosaminoglycan.

[0085] Example 7

[0086] The only difference between this embodiment and embodiment 1 is that in step S1, 3000 mL of 15% (V / V) acetic acid solution is added.

[0087] Finally, 36 g of low molecular weight sulfated glycosaminoglycan was obtained.

[0088] Example 8

[0089] The only difference between this embodiment and embodiment 1 is that in step S2, 58 mL of a sodium nitrite solution with a mass concentration of 5% is measured.

[0090] Finally, 34 g of low molecular weight sulfated glycosaminoglycan was obtained.

[0091] Comparative Example 1

[0092] A method for preparing low molecular weight sulfated glycosaminoglycans comprises the following steps:

[0093] S1. Weigh 100 g of heparinoid byproduct with a specific rotation of -17°, add 500 mL of 2% (V / V) acetic acid solution and stir to dissolve;

[0094] S2. Take 10 mL of 5% (W / V) sodium nitrite solution and slowly and uniformly add it to the acetic acid solution of the heparin-like byproduct for 4 hours. After the addition is completed, stir and react for 20 hours;

[0095] S3, filter, adjust the solution pH to 9.0 with sodium hydroxide, add 1.5% (V / V) peracetic acid by volume of the filtrate, and react for 12 hours;

[0096] S4, filtering, adjusting the pH of the filtrate to 6.9, adding methanol under stirring until the volume fraction of methanol is 40%, and letting it stand;

[0097] S5. Separate the precipitate and the supernatant, dry the precipitate and keep it for other use, continue to add methanol to the supernatant until the volume fraction of methanol is 85%, let it stand, remove the supernatant, collect the precipitate, and obtain 56 g of sulfated glycosaminoglycan.

[0098] Comparative Example 2

[0099] The only difference between this comparative example and Example 1 is that in step S1, 500 mL of 2% (V / V) acetic acid solution is added.

[0100] Finally, 43 g of sulfated glycosaminoglycan was obtained.

[0101] Comparative Example 3

[0102] The only difference between this comparative example and Example 1 is that in step S2, 35 mL of a sodium nitrite solution with a mass concentration of 5% is measured.

[0103] Finally, 37 g of sulfated glycosaminoglycan was obtained.

[0104] Comparative Example 4

[0105] The only difference between this comparative example and Example 1 is that in step S4, methanol is added under stirring until the volume fraction of methanol is 40%.

[0106] Finally, 58 g of sulfated glycosaminoglycan was obtained.

[0107] Comparative Example 5

[0108] The difference between this comparative example and Example 1 is that in step S4, methanol is added under stirring until the volume fraction of methanol is 60%.

[0109] Finally, 1 g of low molecular weight sulfated glycosaminoglycan was obtained.

[0110] Comparative Example 6

[0111] The only difference between this comparative example and Example 1 is that in step S5, methanol is added under stirring until the volume fraction of methanol is 75%.

[0112] Finally, 0 g of low molecular weight sulfated glycosaminoglycan was obtained.

[0113] Experimental Example 1 Molecular weight and specific rotation detection

[0114] The products prepared in Examples 1 to 8 and Comparative Examples 1 to 5 were tested for weight average molecular weight according to the European Pharmacopoeia 11.8 low molecular weight heparin detection method, and the specific rotation was tested according to the Chinese Pharmacopoeia 2020 specific rotation detection method.

[0115] The test results are shown in Table 1:

[0116] Table 1 Molecular weight and specific rotation test results

[0117]

[0118] As can be seen from Table 1, the molecular weights of the low molecular weight sulfated glycosaminoglycans of Examples 1 to 8 are all <6000, and the specific rotations are all ≥﹢10°, while the molecular weights of the sulfated glycosaminoglycans of Comparative Examples 1 to 4 are >6000, and the specific rotations are <﹢10°; although the molecular weight and specific rotation of the low molecular weight sulfated glycosaminoglycans obtained in Comparative Example 5 are within the standard range, the amount of low molecular weight sulfated glycosaminoglycans obtained in Comparative Example 5 is very small.

[0119] Experimental Example 2 Soothing Effect Test

[0120] Test method: copper sulfate was used to induce damage to the neuromast cells in the lateral line region of zebrafish embryos to cause neutrophil aggregation. 32 fish embryos were divided into 4 groups:

[0121] Group 1 was exposed to 10 µM anhydrous copper sulfate and served as the model control group;

[0122] The second group was exposed to 0.2 wt % of the low molecular weight sulfated glycosaminoglycan solution of Example 1, which is the Example 1 group;

[0123] The third group was exposed to 0.2 wt % of the low molecular weight sulfated glycosaminoglycan solution of Comparative Example 1, which is Comparative Example 1 group;

[0124] The fourth group was exposed to 0.2 wt % of the low molecular weight sulfated glycosaminoglycan solution of Comparative Example 3, which is Comparative Example 3 group;

[0125] After 40 min of exposure, the fish embryos were fixed and stained with Sudan black, and the number of neutrophils in the lateral line region was counted and statistically analyzed.

[0126] The analysis results are as follows Figures 1 to 4(corresponding to groups 1 to 4, respectively), the dark dots in the figure are stained neutrophils. It can be seen from the figure that the neutrophil aggregation inhibition rate in the model control group is 0%, with no soothing effect; the neutrophil aggregation inhibition rate in Example 1 group is 20%, with a significant soothing effect; the neutrophil aggregation inhibition rate in Comparative Example 1 group is only 5%, with an insignificant soothing effect; the neutrophil aggregation inhibition rate in Comparative Example 3 group is only 7%, with an insignificant soothing effect. This indicates that the low molecular weight sulfated glycosaminoglycan in Example 1 of the present invention has a high inhibition rate of 20% on the aggregation of zebrafish embryo neutrophils, and has broad application potential in soothing skin care products.

[0127] Experimental Example 3: Anti-wrinkle / tightening effect test

[0128] The distribution of type I collagen in zebrafish is identical to that in humans and shows a high degree of conservation with humans, with their innate expression dropping significantly at 6 days post fertilization and becoming very low between 10 and 12 days post fertilization.

[0129] Acetyl hexapeptide-8 is a recognized raw material with anti-wrinkle / firming effects, so acetyl hexapeptide-8 was used as a control.

[0130] Test method: 45 zebrafish 6 days after fertilization were divided into 5 groups:

[0131] Group 1 was exposed to fish embryo culture medium and served as a blank control group;

[0132] Group 2 was exposed to 0.08 wt% acetyl hexapeptide-8 and served as the positive control group (acetyl hexapeptide-8 group);

[0133] The third group was exposed to 0.2 wt % of the low molecular weight sulfated glycosaminoglycan solution of Example 1, which is the Example 1 group;

[0134] The fourth group was exposed to 0.2 wt % of the sulfated glycosaminoglycan solution of Comparative Example 1, which is Comparative Example 1 group;

[0135] The fifth group was exposed to 0.2 wt % of the sulfated glycosaminoglycan solution of Comparative Example 3, and was referred to as Comparative Example 3 group.

[0136] After 24 h of exposure, zebrafish RNA was extracted and cDNA synthesis and real-time PCR amplification were performed. β-actin As the housekeeping gene, Ct was used as the amplification result to calculate the type I collagen gene ( col1a1a , col1a1b and col1a2 ) and performed statistical analysis.

[0137] The analysis results are shown in Table 2 and Figure 5 shown.

[0138] Table 2 Wrinkle removal / tightening efficacy test

[0139]

[0140] From Table 2 and Figure 5 It can be seen from the figure that the low molecular weight sulfated glycosaminoglycans in Example 1 can significantly promote the expression of zebrafish type I collagen gene at a concentration of 0.2 wt%. col1a1a , col1a1b and col1a2 The promotion rates of gene expression were 11% (p=0.0085), 36% (p=0.037), and 53% (p=0.0011), respectively, where p is the significant difference between Example 1 and the blank control group. The sulfated glycosaminoglycans in Comparative Example 1 and Comparative Example 3 groups had a significant effect on zebrafish at a concentration of 0.2wt%. col1a1a , col1a1b and col1a2 There is no obvious promoting effect on gene expression, indicating that the low molecular weight sulfated glycosaminoglycans in Example 1 of the present invention can significantly upregulate the expression of type I collagen gene, promote collagen regeneration, and play an important role in skin wrinkle removal and firming.

[0141] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing low molecular weight sulfated glycosaminoglycans, characterized in that: The following steps are involved: S1, dissolving the heparin-like by-product in an acetic acid solution to obtain an acetic acid solution of the heparin-like by-product; S2, adding the sodium nitrite solution to the acetic acid solution of the heparin-like byproduct to react to obtain a first reaction solution; S3, filtering the first reaction liquid to obtain a first filtrate, adjusting the pH of the first filtrate to alkaline, adding an oxidant, performing an oxidation reaction, and obtaining a second reaction liquid; S4, filtering the second reaction liquid to obtain a second filtrate, adding an organic solvent to the second filtrate until the volume fraction of the organic solvent is 49% to 54%, and precipitating to obtain a precipitate and a supernatant; S5, continue to add the organic solvent to the supernatant until the volume fraction of the organic solvent is ≥80%, stand and separate, collect the precipitate, and dry to obtain low molecular weight sulfated glycosaminoglycan; The heparin-like byproduct is the heparin-like byproduct remaining after the crude intestinal mucosal heparin is subjected to salt hydrolysis, heat denaturation, ion exchange, oxidation, and organic solvent precipitation to separate heparin sodium, and then extracted with heparan sulfate, dermatan sulfate, and chondroitin sulfate; In step S1, the volume concentration of the acetic acid is 4% to 10%, and the mass volume ratio of the heparin-like byproduct to the acetic acid solution is 1 g: 10 to 25 mL; The specific rotation of the heparinoid byproduct is -12°~-17°, -21°~-25°, -32°~-37°; When the specific rotation of the heparin-like by-product is -12° to -17°, the mass ratio of the sodium nitrite to the heparin-like by-product is 2 to 2.4:100; When the specific rotation of the heparin-like by-product is -21° to -25°, the mass ratio of the sodium nitrite to the heparin-like by-product is 1.55 to 1.9:100; When the specific rotation of the heparin-like by-product is -32° to -37°, the mass ratio of the sodium nitrite to the heparin-like by-product is 1.1 to 1.4:100; The molecular weight of the low molecular weight sulfated glycosaminoglycan is 2205-5564, and the specific rotation is +25.7°-+42.4°.

2. The method for preparing a low molecular weight sulfated glycosaminoglycan according to claim 1, characterized in that: In step S3, the oxidant includes hydrogen peroxide and / or peracetic acid.

3. The method for preparing a low molecular weight sulfated glycosaminoglycan according to claim 1, characterized in that: In step S3, the amount of the oxidant added is 2% to 5% of the volume of the first filtrate.

4. A low molecular weight sulfated glycosaminoglycan, characterized in that: According to the preparation method according to any one of claims 1 to 3, the low molecular weight sulfated glycosaminoglycan has a molecular weight of 2205~5564 and a specific rotation of +25.7°~+42.4°.

5. Use of the low molecular weight sulfated glycosaminoglycan according to claim 4 in the preparation of skin care products.

6. The use according to claim 5, characterized in that: The low molecular weight sulfated glycosaminoglycan is used in the preparation of skin care products with skin wrinkle removal and firming effects.

7. The use according to claim 5, characterized in that: The low molecular weight sulfated glycosaminoglycan is used in the preparation of skin care products with soothing effects.

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