An ultra-low molecular weight heparinoid fusion molecule, a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine and cosmetics, and relates to a kind of ultra-low molecular weight heparin fusion molecules and its preparation method and application.The structure is as shown in formula I, wherein x is greater than or equal to 0, y is greater than or equal to 1;R1 and R2 are each independently selected from -SO3Na or -H;R3 is;Weight average molecular weight is 3-4kDa.The structure of the ultra-low molecular weight heparin fusion molecule provided by the application contains hydrophobic amide thiazole resorcinol structure and hydrophilic ultra-low molecular weight heparin structure at the same time, which are combined by covalent ester bond, has higher safety and excellent water solubility, and improves the problems of poor water solubility of amide thiazole resorcinol and safety risk of heparin products used in cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and cosmetic technology, and relates to an ultra-low molecular weight heparin-like fusion molecule, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Skin whitening efficacy is an important research direction in the cosmetics and pharmaceutical fields. Its core lies in inhibiting melanin production and deposition, and tyrosinase, as a key enzyme in melanin synthesis, is a focus of research and development for whitening active ingredients. Amide-thiazole-based resorcinol compounds are a new type of whitening molecule. With resorcinol as the core, through modification of the thiazole ring and amide group, they combine potent tyrosinase inhibition, chemical stability, and metabolic controllability to form a multi-target synergistic whitening effect. The resorcinol structure of these compounds can interact with the Cu²⁺ molecule at the active site of tyrosinase. + Chelating and competing for substrate sites, the thiazole ring enhances stability and transdermal permeability, while the amide group optimizes lipophilicity and reduces irritation, all working together to achieve highly effective skin whitening. For example, Thiamidol (isobutyramidothiazol-resorcinol) in this class of compounds has an IC50... 50 At a concentration of 0.12 μM, clinical trials showed that a formulation containing 0.2% Thiamidol reduced the facial pigmentation index (MASI) by 42.7% after 8 weeks of use, and inhibited melanin production by 78% in vitro, superior to the 62% reduction of hydroquinone at the same concentration. This class of compounds also overcomes the photosensitivity of traditional resorcinol derivatives, exhibiting high stability (activity retention >95%) and superior safety compared to hydroquinone, making it a model for next-generation skin-whitening ingredients. However, the low solubility of these compounds in water limits their application in cosmetics to some extent.

[0004] Heparin-based products are widely used in cosmetics due to their anti-inflammatory, anti-allergic, microcirculation-improving, and skin metabolism-promoting bioactivities. They not only soften and moisturize the skin, promote cell proliferation and accelerate wound healing, but also provide nutrients and promote the excretion of metabolic waste, making them particularly suitable for products such as eye creams, lotions, creams, masks, and serums. Ultra-low molecular weight heparin, as a new generation of glycosaminoglycan derivatives, has attracted much attention due to its lower molecular weight and higher bioactivity. Compared with traditional heparin, it has better safety, higher bioavailability, lower anticoagulant activity, and a longer half-life in vivo. It exhibits higher anticoagulant activity and lower antithrombin IIa activity, significantly reducing side effects such as bleeding and thrombocytopenia. In cosmetics, ultra-low molecular weight heparin exerts anti-inflammatory and repairing effects by inhibiting prostaglandins and the complement system, promoting the healing of damaged skin; its polysaccharide structure can absorb moisture, enhancing moisturizing ability and repairing the skin barrier; simultaneously, it can inhibit blood clotting, reduce redness and pigmentation, promote connective tissue regeneration, and improve skin smoothness and evenness. Furthermore, ultra-low molecular weight heparin is naturally derived, highly stable, and stable across a wide pH range. It is also resistant to high temperatures and light, making it suitable for various formulations. It also possesses multiple benefits, including antioxidant and anti-allergic properties, meeting the needs of different skin concerns. However, the use of heparin-based products in cosmetics presents safety risks and other issues. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an ultra-low molecular weight heparin fusion molecule, its preparation method, and its application. This ultra-low molecular weight heparin fusion molecule contains both a hydrophobic amide-thiazolyl resorcinol structure and a hydrophilic ultra-low molecular weight heparin structure, which are covalently bonded by ester bonds. This results in high safety and excellent water solubility, improving upon the poor water solubility of amide-thiazolyl resorcinol and the safety risks associated with using heparin-based products in cosmetics.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Firstly, an ultra-low molecular weight heparin-like fusion molecule, the structure of which is shown in Formula I.

[0007] Where x≥0, y≥1; R1 and R2 are each independently selected from -SO3Na or -H; R3 is The weight-average molecular weight is 3~4 kDa.

[0008] In a second aspect, a method for preparing an ultra-low molecular weight heparin fusion molecule as described in the second aspect of the present invention includes the step of esterifying the ultra-low molecular weight heparin of Formula II with the compound of Formula III to obtain the compound of Formula I.

[0009] Where n = x + y, x ≥ 0, y ≥ 1; R1 and R2 are each independently selected from -SO3Na or -H; R3 is .

[0010] Thirdly, a composition comprising an active ingredient and excipients, wherein the active ingredient is the ultra-low molecular weight heparin-like fusion molecule described in the first aspect of the present invention.

[0011] Fourthly, the use of an ultra-low molecular weight heparin fusion molecule as described in the first aspect of the present invention or a composition as described in the third aspect in the preparation of a skin whitening product.

[0012] The beneficial effects of this invention are as follows: 1. The ultra-low molecular weight heparin fusion molecule provided by the present invention fuses the hydrophobic amide-thiazole-based resorcinol structure with the hydrophilic ultra-low molecular weight heparin structure through covalent ester bonds. This structural design significantly improves the problem of poor water solubility of amide-thiazole-based resorcinol compounds, enabling them to be completely soluble in water, and greatly expanding their application in cosmetics and pharmaceutical preparations.

[0013] 2. The ultra-low molecular weight heparin-like fusion molecule provided by this invention retains the biological activity of heparin while reducing the safety risks that may arise when traditional heparin is used in cosmetics through molecular modification. Cytotoxicity and skin irritation experiments show that this fusion molecule has good biocompatibility, is non-toxic and non-irritating, and is suitable for long-term topical use.

[0014] 3. The ultra-low molecular weight heparin-like fusion molecule provided by this invention can release ultra-low molecular weight heparin and N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropionamide in vivo via esterase decomposition, achieving a synergistic dual effect of anti-inflammation, promoting microcirculation, and inhibiting melanin production. Zebrafish experiments show that this fusion molecule can significantly promote blood flow velocity in zebrafish and significantly inhibit melanin production in zebrafish embryos, with the effects of promoting blood circulation and inhibiting melanin production being significantly better than those of single components or physical mixtures.

[0015] 4. The ultra-low molecular weight heparin-like fusion molecule provided by this invention has excellent multiple effects, including reducing dark circles, moisturizing, anti-oxidation, anti-wrinkle, anti-aging, and skin repair. Human trial results show that continuous use of a formulation containing this fusion molecule for 8 weeks can significantly reduce the area of ​​brown spots and brighten skin tone, with the effect continuously increasing over time, verifying its high efficiency and stability in practical applications. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 The NMR spectrum of the ultra-low molecular weight heparin prepared in Example 1.

[0018] Figure 2 The NMR spectrum of the ultra-low molecular weight heparin-like fusion molecule prepared in Example 2.

[0019] Figure 3 The figure shows a comparison of the water solubility test results of the ultra-low molecular weight heparin-like fusion molecule (a) and N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropionamide (b) of the present invention.

[0020] Figure 4 This is a diagram showing the relative velocity measurement location in the zebrafish embryo blood flow velocity test in an embodiment of the present invention. a is a schematic diagram of the measurement location, and b is an actual image of the measurement location.

[0021] Figure 5 This is a bar chart showing the test results of blood flow velocity in zebrafish embryos in an embodiment of the present invention. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] Given the poor water solubility of amide-thiazolyl resorcinol and the poor safety of heparin-like products in cosmetics, which makes it difficult to use them in whitening products, this invention proposes an ultra-low molecular weight heparin-like fusion molecule, its preparation method, and its application in order to solve the above technical problems.

[0025] A typical embodiment of the present invention provides an ultra-low molecular weight heparin-like fusion molecule, the structure of which is shown in Formula I.

[0026] Where x≥0, y≥1; R1 and R2 are each independently selected from -SO3Na or -H; R3 is The weight-average molecular weight is 3~4 kDa.

[0027] In some embodiments, the degree of substitution of R3 in Formula I is 0.02-0.2.

[0028] In some embodiments, the degree of substitution of -SO3Na in the fused molecule is 2.0 to 3.0. The degree of substitution of -SO3Na in the fused molecule refers to the average amount of -SO3Na in each unit. Studies have shown that under these conditions, fused molecules, especially those with a degree of substitution of -SO3Na of 2.0 to 2.5, exhibit better performance.

[0029] In some embodiments, the weight-average molecular weight is 3.4~3.6 kDa.

[0030] Another embodiment of the present invention provides a method for preparing the above-mentioned ultra-low molecular weight heparin fusion molecule, comprising the step of esterifying the ultra-low molecular weight heparin of Formula II with the compound of Formula III to obtain the compound of Formula I.

[0031] Where n = x + y, x ≥ 0, y ≥ 1; R1 and R2 are each independently selected from -SO3Na or -H; R3 is .

[0032] In some embodiments, the method further includes the step of obtaining the ultra-low molecular weight heparin of Formula II via the following reaction route;

[0033] Where m > n, R4 and R5 are each independently selected from -SO3Na or -H.

[0034] Specifically, the steps are as follows: (1) Degradation of heparin: Heparin was dissolved in deionized water and the pH of the solution was adjusted to 4.5-5.5. Vitamin C and hydrogen peroxide were added for degradation. After degradation, sodium chloride was added and dissolved. The reaction solution was then added to ethanol. The solution was filtered and dried to obtain ultra-low molecular weight heparin. (2) Sulfonation of ultra-low molecular weight heparin: Sulfur trioxide N,N-dimethylformamide complex was added dropwise to N,N-dimethylformamide, followed by the addition of ultra-low molecular weight heparin solution. The temperature was controlled at 20~30℃ during the addition process, and the reaction was carried out for 3~5 hours. (3) Add triethylamine to the reaction system of step (2) to adjust the pH to 9-10, add N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropamide and condensing agent, and stir the reaction at 20-30℃ for 15-17 hours. Purification: Add the reaction solution to deionized water, filter it through a 0.45-micron microporous membrane, remove impurities through a nanofiltration membrane (molecular weight cutoff 500 Da), wash, and freeze dry to obtain the product.

[0035] A third embodiment of the present invention provides a composition comprising an active ingredient and excipients, wherein the active ingredient is the aforementioned ultra-low molecular weight heparin-like fusion molecule.

[0036] The compositions described in this invention are for oral or topical use. Those skilled in the art can select appropriate excipients according to conventional methods to prepare the compositions of this invention into suitable dosage forms or usage formats. In some embodiments, the excipients include, but are not limited to, thickeners, gelling agents, neutralizing agents, antioxidants, buffers, pH adjusters, fillers, emulsifiers, co-emulsifiers, emollients, solvents, stabilizers, solubilizers, hardening agents, suspending agents, binders, thickeners, penetration enhancers, preservatives, humectants, fragrances, etc.

[0037] In some embodiments, the composition is formulated as a solution, suspension, emulsion, cream, liniment, plaster, ointment, gel, foam, patch, medicated plaster, etc. For example, when the composition is formulated as a gel, the excipient is a gelling agent. Specifically, the gelling agent is a gel formed by crosslinking sodium alginate and calcium chloride. The preparation process is as follows: the ultra-low molecular weight heparin fusion molecule described in this invention is dissolved in water with sodium alginate, and then calcium chloride is added for crosslinking to form a gel.

[0038] A fourth embodiment of the present invention provides the application of the above-mentioned ultra-low molecular weight heparin fusion molecule or composition in the preparation of skin whitening products.

[0039] The product described in this invention can be a pharmaceutical or a cosmetic. When the product is a cosmetic, it can include, but is not limited to, toners, lotions, serums, gels, foundations, creams, and masks. The application scope of the cosmetic includes, but is not limited to, facial cleansing and care, body cleansing and care, and hair cleansing and care. The whitening product described in this invention is a whitening product with functions such as removing dark circles, moisturizing, replenishing skin nutrition, anti-inflammation, anti-oxidation, wrinkle removal, anti-aging, and / or skin repair.

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0041] The reaction route for preparing ultra-low molecular weight heparin-like fusion molecules is shown in the following examples:

[0042] Example 1: Preparation of ultra-low molecular weight heparin (Formula II) 1000g of heparin-like substances were dissolved in 20L of deionized water, and the pH of the solution was adjusted to 5. 70g of vitamin C and 500mL of 30% hydrogen peroxide were added, and the solution was degraded at 40℃ for 1 hour. Another 70g of vitamin C and 500mL of 30% hydrogen peroxide were added, and the degradation was completed after 1 hour. 1000g of sodium chloride was added to the reaction solution and dissolved. The reaction solution was then added to 60L of 95% ethanol, resulting in the precipitation of a yellow solid. The solid was filtered, and dried at 50℃ to obtain ultra-low molecular weight heparin-like substances with an average molecular weight of 3.5kDa. Its NMR spectrum is shown below. Figure 1 As shown.

[0043] Example 2 Preparation of ultra-low molecular weight heparin-like fusion molecule (Formula I) Sulfonation: 1000g of the ultra-low molecular weight heparin from Example 1 was dissolved in 5 times (w / v) N,N-dimethylformamide for later use. A sulfur trioxide N,N-dimethylformamide complex of 2 times (w / v) of the ultra-low molecular weight heparin was added dropwise to 3 times (w / v) N,N-dimethylformamide, followed by the addition of the ultra-low molecular weight heparin solution. The temperature was maintained at 25°C during the addition process, and the reaction was carried out for 4 hours. After the reaction was completed, triethylamine was added to adjust the pH to 9-10, and 180g of N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropionamide and 250g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added. The mixture was stirred at 25°C for 16 hours.

[0044] Purification: The reaction solution was added to 50 L of deionized water, filtered through a 0.45 μm microporous membrane, and then passed through a nanofiltration membrane (500 Da molecular weight cutoff) to remove impurities. After washing and lyophilization, ultra-low molecular weight heparin-like fusion molecules were obtained with an average molecular weight of 3.5 kDa and an R3 substitution degree of 0.09. Its NMR spectrum is shown below. Figure 2 As shown.

[0045] The ultra-low molecular weight heparin fusion molecules described in this invention have a weight-average molecular weight of 3-4 kDa, with an average of 3.5 kDa, and the degree of substitution of the R3 structure in the ultra-low molecular weight heparin fusion molecules is 0.02-0.2. Ultra-low molecular weight heparin fusion molecules within this range exhibit similar and stable properties. Changing the parameters in the preparation process shown in Examples 1 and 2 above can affect the molecular weight and degree of substitution of the product. Obtaining the desired molecular weight and degree of substitution by adjusting the above experimental process is easily achievable by those skilled in the art, and will not be elaborated further in this invention.

[0046] The performance of the ultra-low molecular weight heparin-like fusion molecule of the present invention will be further described below.

[0047] Example 3 Water solubility test Evaluation of solubility: The compounds of formula I prepared in Example 2 were dissolved in purified water at a concentration of 5 mg / ml. N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropamide was used as a control. It was confirmed that N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropamide is almost insoluble in water at the same concentration and is therefore opaque, while the compounds of formula I of the present invention are all completely soluble in water (see [link to original text]). Figure 2 It exhibits significantly improved water solubility.

[0048] Example 4: In vitro cytotoxicity and irritation experiments A purified aqueous solution of the compound of formula I prepared in Example 2 was prepared at a concentration of 5 mg / ml to obtain test sample 1. According to GB / T 16886.5-2017 / ISO 10993-5:2009 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity tests", in vitro cytotoxicity tests were performed on test sample 1 to verify biocompatibility. The MTT assay was used for evaluation. The cell line used was L929 cells. 10% DMSO solution was used as the positive control group, 3% fetal calf serum medium was used as the negative control group, and the experimental group consisted of 3% fetal calf serum medium extract of the test sample. The culture was carried out at 37°C with shaking for 24 h.

[0049] The specific experimental steps are as follows: L929 cells passaged for 48 hours were prepared into 1×10⁻⁶ cells using cell culture medium. 5 Prepare a cell suspension of 1 × 10⁶ cells / mL; 5Cell suspension of 100 μL / well was seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 h to form a semi-confluent monolayer. After 24 h, the original culture medium was discarded, and 100 μL of sample extract, positive control solution, and negative control solution were added to each well (5 wells per group). The plates were then incubated at 37°C with 5% CO2 for 24 h. 50 μL of LTT solution was added to each well, and the plates were incubated at 37°C with 5% CO2 for another 2 h. Discard the MTT solution, add 100 μL of DMSO solution to each well, shake the plate, place it on a microtiter plate spectrophotometer and measure the absorbance at 570 nm, calculate cell proliferation (RGR, %), and evaluate cytotoxicity according to the standards in the table below.

[0050] Table 1. In vitro cytotoxicity grading table

[0051] The experimental results are shown in Table 2. The in vitro cytotoxicity of test sample 1 is grade 1, indicating that it has good biocompatibility and will not produce toxicity or cause toxic residues.

[0052] Table 2 Results of in vitro cytotoxicity experiments

[0053] Fifteen healthy rabbits, weighing approximately 2 kg, were randomly divided into three groups of five. 24 hours before the experiment, the hair on both sides of the rabbits' backs was removed. 24 hours after the removal, the skin in the hair-removed area was checked for injury. Rabbits with injured skin were not suitable for skin irritation experiments. The test product was applied three times a day for seven consecutive days. The experimental results were observed and are shown in Table 3.

[0054] Table 3 Results of skin irritation test

[0055] Note: "+" indicates that the rabbit has red, swollen, inflamed and congested skin; "++" indicates that the rabbit has red, swollen, inflamed and congested skin and there is a tendency for the condition to worsen; "-" indicates that the rabbit has no red, swollen, inflamed and congested skin.

[0056] As can be seen from the experimental results in Table 3, the compound of formula I prepared in Example 2 is not irritating to the skin.

[0057] Example 5: Efficacy in reducing dark circles (test of blood flow velocity and melanin production inhibition rate in zebrafish embryos) This experiment tested the anti-inflammatory, anti-aging, and wrinkle-reducing effects of the test samples.

[0058] Experimental groups and test samples: Sample 1: Sodium alginate was dissolved in deionized water to prepare a solution with a concentration of 2 wt%; under mechanical stirring, sodium heparin was added to the sodium alginate solution at a concentration of 0.5 mg / ml; a calcium chloride solution with a concentration of 0.2 wt% was added, stirred for 20 min, and then allowed to stand for 24 h to obtain sodium heparin gel as Sample 1.

[0059] Sample 2: Sodium alginate was dissolved in deionized water to prepare a 2 wt% solution; under mechanical stirring, sodium heparin and N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropionamide were added to the sodium alginate solution at a concentration of 0.5 mg / ml; a 0.2 wt% calcium chloride solution was added, stirred for 20 min, and then allowed to stand for 24 h to obtain a gel as Sample 2.

[0060] Sample 3: Sodium alginate was dissolved in deionized water to prepare a solution with a concentration of 2 wt%; under mechanical stirring, the compound of formula I prepared in Example 2 was added to the sodium alginate solution at an addition amount of 0.5 mg / ml; a calcium chloride solution with a concentration of 0.2 wt% was added, stirred for 20 min, and then allowed to stand for 24 h to obtain an ultra-low molecular weight heparin-like fusion molecular gel as Sample 3.

[0061] Sample 4: Sodium alginate was dissolved in deionized water to prepare a 2 wt% solution; under mechanical stirring, N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropionamide was added to the sodium alginate solution at a concentration of 0.5 mg / ml; a 0.2 wt% calcium chloride solution was added, stirred for 20 min, and then allowed to stand for 24 h to obtain a gel, which was taken as Sample 4.

[0062] Sample 5: Sodium alginate was dissolved in deionized water to prepare a solution with a concentration of 2 wt%; under mechanical stirring, a calcium chloride solution with a concentration of 0.2 wt% was added, stirred for 20 min, and then allowed to stand for 24 h to obtain the gel, which was the blank sample 5.

[0063] Testing Principle: Dark circles under the eyes are a common symptom with many causes, including genetic factors, psychological factors, and excessive eye strain. They primarily result from venous congestion around the eyes, reduced oxygen levels, and hemoglobin buildup, manifesting as a dark color around the eyes. Excessive sun exposure and illness can also cause pigmentation around the eyes, leading to dark circles. Therefore, removing dark circles mainly involves improving microcirculation and whitening / lightening the skin. Skin microcirculation refers to blood circulation between arterioles and venules. Blood flow analyzers can be used to detect the blood flow velocity in zebrafish to evaluate the improvement in microcirculation. The main pathway for whitening and lightening melanin is through inhibiting tyrosinase activity, thereby reducing melanin production. The melanin regulation mechanism in zebrafish skin is highly similar to that in humans and can be clearly observed under a microscope. Image analysis can assess the degree of melanin inhibition in the samples.

[0064] Test methods Blood flow velocity: Three-day-old zebrafish embryos were exposed to samples 1-5, with three biological replicates per group and eight zebrafish embryos per replicate. 5 mL of the corresponding concentration working solution was added to each well. After two hours of exposure, the aorta of each zebrafish embryo was photographed under a microscope for 20 seconds. The blood flow velocity in the aorta of each embryo was then analyzed using software. The test locations are shown below. Figure 4 As shown, the average value was taken and statistical analysis was performed.

[0065] Melanin grayscale values: Three-day-old zebrafish embryos were exposed to samples 1-5, with three biological replicates per group and eight zebrafish embryos per replicate. 5 mL of working solution corresponding to each concentration group was added to each well. All test groups were incubated in a 28.5±0.5 ℃ incubator for 24 hours. The embryos were demembranes using 1 mg / mL streptomycin solution and fixed with 3% methylcellulose. They were then observed and photographed under a stereomicroscope. During photography, the zebrafish embryos should be positioned with their heads to the left, abdomens down, and bodies horizontal. All zebrafish embryo photographs must be taken under the same instrument and environmental conditions, and the embryo positions must be consistent. ImageJ software was used to analyze the melanin grayscale values ​​of each embryo, and a significance test was performed on the data from each group.

[0066] Table 4. Experimental results of the effect of zebrafish embryos on promoting blood circulation.

[0067] Table 5. Experimental results of the inhibitory effect of zebrafish embryonic blood melanin.

[0068] Through Table 4 and Figure 5It is known that both heparin sodium and N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropamide can promote aortic blood circulation, and their combined use can significantly promote blood circulation, but neither is as effective as the ultra-low molecular weight heparin fusion molecule of this invention. Table 5 shows that heparin sodium does not have the ability to inhibit melanin, while N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)-2-hydroxypropamide can significantly inhibit melanin, and the combined effect with heparin is even better, but neither is as effective as the ultra-low molecular weight heparin fusion molecule of this invention. The ultra-low molecular weight heparin fusion molecule of this invention can significantly promote blood flow velocity in zebrafish (p<0.05) and significantly inhibit melanin production in zebrafish embryos (p<0.05), thus having the effect of removing dark circles under the eyes.

[0069] Example 6: Examination of the Spot-Removing Effect Studies have shown that Asians and people with darker skin are more prone to pigmentation. This invention selected 30 Chinese female volunteers aged 25-55 with facial melasma and sensitive skin to study and evaluate the depigmentation effect of ultra-low molecular weight heparin fusion molecules. Pigmentation was assessed by measuring gray levels from 0 to 255 (grayscale levels: 0 = black, 255 = white, with increasing values ​​indicating enhanced depigmentation) spectrophotometrically, and variance analysis was performed using the Dunnett-t test to calculate p-values. Sample 3 of Example 5 was applied twice daily to designated areas of the volunteers' faces, with an interval of more than one hour between applications, for eight consecutive weeks. Skin images were acquired using Siascope to identify chromophores such as melanin. The grayscale (color) and surface area of ​​the designated points and their adjacent normal skin were analyzed. The results showed that Sample 3 reduced the area and color of melasma (see Table 6).

[0070] Table 6. Effects of ultra-low molecular weight heparin fusion molecules on skin color and brown spot surface area.

[0071] The results in Table 6 show that the ultra-low molecular weight heparin-like fusion molecules have significant effects on improving skin tone (brightening), reducing the area of ​​brown spots, and improving the color grade of the surrounding skin, and these effects are enhanced over time.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An ultra-low molecular weight heparin-like fusion molecule, characterized in that, Its structure is shown in Equation I. Where x≥0, y≥1; R1 and R2 are each independently selected from -SO3Na or -H; R3 is The weight-average molecular weight is 3~4 kDa.

2. The ultra-low molecular weight heparin-like fusion molecule as described in claim 1, characterized in that, The value of y / (x+y) is 0.02~0.

2.

3. The ultra-low molecular weight heparin-like fusion molecule as described in claim 1, characterized in that, - The degree of substitution of SO3Na in the fused molecule is 2.0~3.0; or 2.0~2.

5.

4. The ultra-low molecular weight heparin-like fusion molecule as described in claim 1, characterized in that, The weight-average molecular weight is 3.4~3.6kDa.

5. A method for preparing the ultra-low molecular weight heparin-like fusion molecule according to claim 1, characterized in that, The step includes the esterification reaction of the ultra-low molecular weight heparin of Formula II and the compound of Formula III to obtain the compound of Formula I. Where n = x + y, x ≥ 0, y ≥ 1; R1 and R2 are each independently selected from -SO3Na or -H; R3 is .

6. The preparation method according to claim 5, characterized in that, It also includes the step of obtaining the ultra-low molecular weight heparin shown in Formula II via the following reaction route; Where m > n, R4 and R5 are each independently selected from -SO3Na or -H.

7. A composition comprising an active ingredient and excipients, characterized in that, The active ingredient is any one of the ultra-low molecular weight heparin fusion molecules as described in claims 1 to 4.

8. The composition of claim 7, characterized in that, The excipients include one or more of the following: thickeners, gelling agents, neutralizing agents, antioxidants, buffers, pH adjusters, fillers, emulsifiers, co-emulsifiers, emollients, solvents, stabilizers, solubilizers, hardeners, suspending agents, adhesives, tackifiers, penetration enhancers, preservatives, humectants, and fragrances. Alternatively, the composition may be formulated as a solution, suspension, emulsion, cream, liniment, plaster, ointment, gel, foam, patch, or plaster.

9. The use of an ultra-low molecular weight heparin fusion molecule as described in any one of claims 1 to 4 or the composition as described in claim 7 or 8 in the preparation of a skin whitening product.

10. The application as described in claim 9, characterized in that, The product in question is a pharmaceutical or cosmetic product. Alternatively, the whitening product may be a whitening product with functions such as removing dark circles, moisturizing, replenishing skin nutrition, anti-inflammation, anti-oxidation, wrinkle removal, anti-aging and / or skin repair.

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