A macroalgal oligosaccharide sulfate and a preparation method and application thereof

Abstract

The application discloses a kind of giant kelp oligosaccharide sulfate and its preparation method and application, belong to marine sugar chemical technical field, the giant kelp oligosaccharide sulfate is prepared by using giant kelp as raw material, through extraction, directional cleavage, chemical modification, purification and the like process preparation obtains, preparation method is simple and easy to operate, easy to scale production, the giant kelp oligosaccharide sulfate prepared has double aldehyde structure;Verified by experiment, the giant kelp oligosaccharide sulfate provided by the application has the effect of efficient anticoagulant activity, good transdermal performance and increase the intersegmental vascular blood flow velocity of zebra fish, has good microcirculation improvement function, can be used in the field of cosmetics, such as whitening, spot lightening, dark circle lightening and other skin microcirculation improvement needs.

Description

Technical Field

[0001] This invention belongs to the field of marine glycochemistry technology, specifically relating to a giant kelp oligosaccharide sulfate ester, its preparation method, and its application. Background Technology

[0002] As a "nutrient delivery network" connecting the epidermis and dermis, the patency of the skin's microcirculation directly determines the skin's health and appearance, making it a crucial target in cosmetic development. From a physiological perspective, the microcirculation system delivers essential nutrients such as oxygen and amino acids to the basal cells of the epidermis via capillaries, while simultaneously removing metabolic waste (such as lactic acid and free radicals). When microcirculation is impaired, insufficient nutrient supply leads to a decreased cell renewal rate, abnormal accumulation of dead skin cells, and problems such as dullness, roughness, and lack of radiance. Furthermore, impaired microcirculation hinders collagen fiber synthesis in the dermis, accelerating signs of aging such as sagging and fine lines, and even causing sensitivity and redness due to the accumulation of metabolic waste.

[0003] For cosmetic development, improving microcirculation can achieve breakthroughs in multiple skincare benefits. In the whitening field, improved microcirculation can accelerate melanin metabolism and excretion, reducing pigmentation. In the anti-aging field, improved microcirculation can provide sufficient nutrient supply to activate fibroblast activity, promote collagen regeneration, and enhance skin elasticity. In sensitive skin care, a certain degree of improved microcirculation can regulate the skin's immune microenvironment, alleviating discomfort such as redness and burning. Furthermore, improved microcirculation can enhance the overall penetration rate of active ingredients in cosmetics. Smooth microcirculation optimizes the hydration state and barrier function of the stratum corneum, opening absorption channels for active ingredients and addressing the industry pain point of "effective ingredients but insufficient penetration." Therefore, whether for functional or basic skincare cosmetics, improving microcirculation is incorporated into the formulation design logic.

[0004] Giant kelp extract, rich in polysaccharides, amino acids, minerals, and other active ingredients, offers multiple skincare benefits including moisturizing, repairing, and anti-aging. Its gentle nature makes it suitable for most skin types, and it is an important raw material for alginate, medicinal herbs, and mannitol, making it a popular ingredient in the cosmetics industry. Giant kelp is rich in acidic polysaccharides, which possess some anticoagulant activity; however, they are not yet effective in improving skin microcirculation. Summary of the Invention

[0005] To address the shortcomings of the above-mentioned technologies, the present invention aims to provide a giant kelp oligosaccharide sulfate, its preparation method, and its application. The giant kelp oligosaccharide sulfate provided by the present invention is prepared from giant kelp through processes such as selective extraction of polysaccharides, directional cleavage, chemical modification, and purification. This extract exhibits superior efficacy in improving microcirculation.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention adopts the following technical solution:

[0007] This invention provides a method for preparing giant kelp oligosaccharide sulfate, the method comprising the following steps:

[0008] S1: Add giant kelp to sodium carbonate aqueous solution for extraction, add calcium chloride solution to the filtrate for precipitation, add hydrochloric acid solution to the precipitate for acidification, and collect the precipitate; add sodium hydroxide solution to the precipitate to dissolve and adjust the pH to 7.0~7.5 to obtain the solution;

[0009] S2: Add sodium periodate to the solution for directional cleavage, then add ethylene glycol to remove residual sodium periodate, and finally add ethanol for alcohol precipitation. Take the precipitate and dry it.

[0010] S3: The dried precipitate was added to a chlorosulfonic acid-formamide mixed solution for sulfation modification, and then ethanol was added for alcohol precipitation to obtain a paste-like precipitate.

[0011] S4: Dissolve the paste-like precipitate in water, then adjust the pH to 8-9 with sodium hydroxide solution, and then perform the first ultrafiltration fraction. Perform the second ultrafiltration fraction on the filtrate, retain the retentate obtained from the second ultrafiltration fraction, and dry it to prepare giant kelp oligosaccharide sulfate.

[0012] Furthermore, in step S1, the mass concentration of the sodium carbonate aqueous solution is 1% to 5%; the ratio of the giant kelp to the sodium carbonate aqueous solution is 1:30 to 1:35.

[0013] Furthermore, in step S1, the mass concentration of the hydrochloric acid solution is 5% to 10%.

[0014] Furthermore, in step S2, the sodium periodate has a mass concentration of 3% to 5% in the solution and is subjected to directional pyrolysis at room temperature for 5 to 7 hours.

[0015] Furthermore, in step S2 of the preparation method, sodium periodate is used to oxidize and ring-open the C2OH-C3OH (cis-diol) residues of some mannuronic acid and guluronic acid residues in the giant kelp oligosaccharide obtained in step S1 to form dialdehyde monosaccharide residues. The oxidative ring-opening reaction can make the polysaccharide chain more extended.

[0016] Furthermore, in step S2, the molar ratio of ethylene glycol to sodium periodate is 1:1.

[0017] Furthermore, in step S3, the mass ratio of precipitate:chlorosulfonic acid:formamide is 1:2.8~3.1:10, and the modification reaction is carried out at a temperature of 60~70℃ for 2~4 h.

[0018] Furthermore, in the chlorosulfonic acid-formamide mixed solution, the mass ratio of chlorosulfonic acid to formamide is 2.8~3.1:10. The partially oxidized and ring-opened giant kelp oligosaccharide is modified by sulfation using the chlorosulfonic acid-formamide method. By controlling the reaction conditions, the hydroxyl groups in the remaining unopened ring residues are replaced by sulfate groups.

[0019] Furthermore, in step S4, the molecular weight cutoff for the first ultrafiltration fraction is 30 kDa, and the molecular weight cutoff for the second fraction is 3.5 kDa.

[0020] Furthermore, the first ultrafiltration stage removes unreacted, excessively large molecular weight components, and the second ultrafiltration stage removes small molecule impurities, including components with excessively small molecular weights after the reaction and residual inorganic salts, thereby further narrowing the molecular weight distribution.

[0021] Furthermore, in step S4, the drying method includes spray drying or freeze drying.

[0022] The present invention also provides a giant kelp oligosaccharide sulfate obtained by the above preparation method, wherein the oligosaccharide sulfate has the following structural characteristics:

[0023]

[0024] The giant kelp oligosaccharide sulfate includes a modified oligosaccharide derivative composed of saturated mannuronic acid sulfate M-SO4, saturated guluronic acid sulfate G-SO4, ring-opening mannuronic acid M', and ring-opening guluronic acid G' linked by 1→4 glycosidic bonds; in the monosaccharide residues with R substituents, R is H or SO3. - And there is at least one SO3 residue on the same monosaccharide residue. - ;6≤n≤36.

[0025] Furthermore, the average molecular weight of the giant kelp oligosaccharide sulfate is 5 kDa to 30 kDa, and the molar percentage of monosaccharide residues containing a dialdehyde structure in the giant kelp oligosaccharide sulfate is 0.01% to 10%.

[0026] Preferably, the average molecular weight of the giant kelp oligosaccharide sulfate is 12.1 kDa to 18.3 kDa, and the molar percentage of monosaccharide residues containing a dialdehyde structure in the giant kelp oligosaccharide sulfate is 6.3% to 8.9%.

[0027] Furthermore, the sulfate group mass percentage of the giant kelp oligosaccharide sulfate ester ranges from 20% to 40%.

[0028] Furthermore, the sulfate group mass percentage of the giant kelp oligosaccharide sulfate ester ranges from 0.2% to 33.1%.

[0029] Preferably, the sulfate group mass percentage of the giant kelp oligosaccharide sulfate ester ranges from 26.7% to 30.4%.

[0030] The present invention also provides the application of the aforementioned giant kelp oligosaccharide sulfate in the preparation of cosmetics with the effect of improving skin microcirculation.

[0031] Furthermore, the mass concentration of giant kelp oligosaccharide sulfate contained in the cosmetic is 0.1~10 mg / mL.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. This invention focuses on improving skin microcirculation as the activity enhancement target and uses in vitro anticoagulant activity as the evaluation index. It uses giant kelp (Macrocystis Pryifera) as raw material and prepares an oligosaccharide sulfate ester through processes such as polysaccharide selective extraction, directional cleavage, chemical modification, and purification. Compared with traditional polysaccharide-modified sulfate esters, the obtained oligosaccharide sulfate ester has a partial dialdehyde active structure. This structure makes the sugar chain more flexible and easier to absorb through the skin, and has a superior effect on improving microcirculation. This provides an innovative path for developing a new generation of high-performance marine-derived functional cosmetic raw materials.

[0034] 2. The oligosaccharide sulfate provided by this invention can effectively prolong APTT, PT, and TT in vitro, reduce FIB formation, and exhibit good anticoagulant activity. Simultaneously, this oligosaccharide sulfate can significantly increase intersegmental blood flow velocity in zebrafish in vivo. This oligosaccharide sulfate has excellent potential to improve skin microcirculation, and coupled with its superior transdermal absorption, it can be used as an active ingredient in cosmetics to improve skin health. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0036] Figure 1 This is a monosaccharide composition diagram of the giant kelp oligosaccharide sulfate ester in Example 1;

[0037] Figure 2 The image shows the relative molecular mass spectrum of the giant kelp oligosaccharide sulfate ester in Example 1. Detailed Implementation

[0038] To make the application objectives, technical solutions, and beneficial technical effects of this invention clearer, the following detailed description of this invention is provided in conjunction with specific embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or manufacturer-suggested conditions are followed. Where the manufacturers of reagents or instruments are not specified, they are all commercially available conventional products.

[0039] The material-liquid ratio mentioned in this invention refers to the mass concentration, specifically the ratio of the mass of giant kelp (unit: kg) to the volume of sodium carbonate aqueous solution (unit: L).

[0040] The unit for the mass concentration of the sodium carbonate aqueous solution, sodium hydroxide aqueous solution, and hydrochloric acid solution is g / L; the unit for the mass concentration of the sodium periodate is g / L.

[0041] Example 1

[0042] This embodiment provides a method for preparing giant kelp oligosaccharide sulfate, which specifically includes the following steps:

[0043] 1. Take 1 kg of dried giant kelp (Macrocystis Pryifera), crush it, and pass it through a 2-mesh sieve. Add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v), stir and extract at 70℃ for 3 h, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to allow for sufficient precipitation, centrifuge and collect the precipitate. Add 5% hydrochloric acid solution to the precipitate for sufficient acidification, centrifuge and collect the precipitate. Add 1% sodium hydroxide aqueous solution to the precipitate to dissolve it, and adjust the pH of the solution to 7.0~7.5 to obtain the dissolved solution.

[0044] 2. Add sodium periodate to the solution until the final mass concentration of sodium periodate in the solution is 4%. React at room temperature in the dark for 6 h. Then add ethylene glycol in an equimolar amount of sodium periodate and react until the residual sodium periodate is removed. Then add 3 times the volume of 95% ethanol solution to the above solution for alcohol precipitation. Centrifuge to collect the precipitate and dry it at 60℃. The dried precipitate is an intermediate with a dialdehyde ring-opening structure.

[0045] 3. Add chlorosulfonic acid-formamide sulfation reagent to the intermediate with the dialdehyde ring-opening structure at a mass ratio of intermediate:chlorosulfonic acid:formamide of 1:2.9:10 (m / m / m), and react at 65℃ for 3 h. After the reaction is completed, add 3 times the volume of 95% ethanol to the reaction solution to precipitate, and take the paste precipitate.

[0046] 4. The obtained paste-like precipitate was dissolved in an appropriate amount of water and adjusted to pH 8-9 with sodium hydroxide solution. The resulting neutralized solution was filtered through microfiltration (MF) to remove impurities, and then subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The filtrate was collected. The filtrate was then subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa. The retentate was collected. The retentate was freeze-dried to obtain giant kelp oligosaccharide sulfate.

[0047] Example 2

[0048] This embodiment provides a method for preparing giant kelp oligosaccharide sulfate, which differs from the preparation method described in Example 1 mainly in the amount of chlorosulfonic acid-formamide sulfation reagent added and the reaction time. Specifically, it includes the following operations:

[0049] 1. Take 1 kg of dried giant kelp, crush it and pass it through a 2-mesh sieve; add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v), stir and extract at 70℃ for 3 h, filter and collect the filtrate; add excess calcium chloride solution to the filtrate to fully precipitate, centrifuge and collect the precipitate; add 5% hydrochloric acid solution to the precipitate to fully acidify, centrifuge and collect the precipitate; add 1% sodium hydroxide aqueous solution to the precipitate to dissolve it, and adjust the pH of the solution to 7.0~7.5 to obtain the solution.

[0050] 2. Add sodium periodate to the solution until the final mass concentration of sodium periodate in the solution is 4%, react at room temperature in the dark for 6 h, and then add ethylene glycol in an equimolar amount of sodium periodate to react until the residual sodium periodate is removed; add 3 times the volume of 95% ethanol solution to the above solution for alcohol precipitation, centrifuge to collect the precipitate and dry it at 60°C. The dried precipitate is an intermediate with a dialdehyde ring-opening structure.

[0051] 3. Add chlorosulfonic acid-formamide sulfation reagent to the intermediate with the dialdehyde ring-opening structure at a mass ratio of intermediate:chlorosulfonic acid:formamide of 1:3.1:10 (m / m / m), and react at 65℃ for 4 h. After the reaction is completed, add 3 times the volume of 95% ethanol to the reaction solution to precipitate, and take the paste precipitate.

[0052] 4. Dissolve the obtained paste-like precipitate in an appropriate amount of water and adjust the pH to 8-9 with sodium hydroxide solution; after removing impurities by microfiltration, the resulting neutralized solution is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the filtrate is collected; the filtrate is then subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa, and the retentate is collected; the retentate is then freeze-dried to obtain giant kelp oligosaccharide sulfate.

[0053] Example 3

[0054] This embodiment provides a method for preparing giant kelp oligosaccharide sulfate. The main difference between this embodiment and the preparation method described in Example 1 lies in the mass concentration of sodium periodate added and the reaction time. Specifically, the method includes the following steps:

[0055] 1. Take 1 kg of dried giant kelp, crush it and pass it through a 2-mesh sieve; add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v), stir and extract at 70℃ for 3 h, filter and collect the filtrate; add excess calcium chloride solution to the filtrate to fully precipitate, centrifuge and collect the precipitate; add 5% hydrochloric acid solution to the precipitate to fully acidify, centrifuge and collect the precipitate; add 1% sodium hydroxide aqueous solution to the precipitate to dissolve it, and adjust the pH of the solution to 7.0~7.5 to obtain the solution.

[0056] 2. Add sodium periodate to the solution until the final mass concentration of sodium periodate in the solution is 5%, react at room temperature in the dark for 7 h, then add ethylene glycol in an equimolar amount of sodium periodate and react until the residual sodium periodate is removed; add 3 times the volume of 95% ethanol solution to the above solution for alcohol precipitation, centrifuge to collect the precipitate and dry it at 60℃. The dried precipitate is an intermediate with a dialdehyde ring-opening structure.

[0057] 3. Add chlorosulfonic acid-formamide sulfation reagent to the intermediate with the dialdehyde ring-opening structure at a mass ratio of intermediate:chlorosulfonic acid:formamide of 1:2.9:10 (m / m / m), and react at 65℃ for 3 h. After the reaction is completed, add 3 times the volume of 95% ethanol to the reaction solution to precipitate, and take the paste precipitate.

[0058] 4. The obtained paste-like precipitate is dissolved in an appropriate amount of water and neutralized with sodium hydroxide solution to pH 8-9. After removing impurities by microfiltration, the resulting neutralized solution is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the filtrate is collected. The filtrate is then subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa, and the retentate is collected. The retentate is then freeze-dried to obtain giant kelp oligosaccharide sulfate.

[0059] Comparative Example 1

[0060] Comparative Example 1 provides a method for preparing giant kelp oligosaccharide sulfate, the main difference from Example 1 being the mass concentration of sodium periodate added and the difference in reaction time, specifically including the following operations:

[0061] 1. Take 1 kg of dried giant kelp, crush it and pass it through a 2-mesh sieve; add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v), stir and extract at 70℃ for 3 h, filter and collect the filtrate; add excess calcium chloride solution to the filtrate to fully precipitate, centrifuge and collect the precipitate; add 5% hydrochloric acid solution to the precipitate to fully acidify, centrifuge and collect the precipitate, add 1% sodium hydroxide aqueous solution to the precipitate to dissolve it, and adjust the pH of the solution to 7.0~7.5 to obtain the dissolved solution.

[0062] 2. Add sodium periodate to the above solution until the final mass concentration of sodium periodate in the solution is 8%, react at room temperature in the dark for 10 h, then add ethylene glycol in an equimolar amount of sodium periodate and react until the residual sodium periodate is removed; add 3 times the volume of 95% ethanol solution to the above solution for alcohol precipitation, centrifuge to collect the precipitate and dry it at 60℃. The dried precipitate is an intermediate with a dialdehyde ring-opening structure.

[0063] 3. Add chlorosulfonic acid-formamide sulfation reagent to the intermediate with the dialdehyde ring-opening structure at a mass ratio of intermediate:chlorosulfonic acid:formamide of 1:2.9:10 (m / m / m), and react at 65℃ for 3 h. After the reaction is completed, add 3 times the volume of 95% ethanol to the reaction solution to precipitate, and take the paste precipitate.

[0064] 4. Dissolve the obtained paste-like precipitate in an appropriate amount of water and adjust the pH to 8-9 with sodium hydroxide solution; after removing impurities by microfiltration, the resulting neutralized solution is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the filtrate is collected; the filtrate is then subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa, and the retentate is collected; the retentate is then freeze-dried to obtain giant kelp oligosaccharide sulfate.

[0065] Comparative Example 2

[0066] Comparative Example 2 provides a method for preparing giant kelp oligosaccharide sulfate, which differs from Example 1 mainly in the amount of chlorosulfonic acid-formamide sulfation reagent, sodium periodate, and reaction time. Specifically, it includes the following steps:

[0067] 1. Take 1 kg of dried giant kelp, crush it and pass it through a 2-mesh sieve; add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v), stir and extract at 70℃ for 3 h, filter and collect the filtrate; add excess calcium chloride solution to the filtrate to fully precipitate, centrifuge and collect the precipitate; add 5% hydrochloric acid solution to the precipitate to fully acidify, centrifuge and collect the precipitate, add 1% sodium hydroxide aqueous solution to the precipitate to dissolve it, and adjust the pH of the solution to 7.0~7.5 to obtain the dissolved solution.

[0068] 2. Add sodium periodate to the above solution until the final mass concentration of sodium periodate is 2%, react at room temperature in the dark for 4 h, then add ethylene glycol in an equimolar amount of sodium periodate and react until the residual sodium periodate is removed; add 3 times the volume of 95% ethanol solution to the above solution for alcohol precipitation, centrifuge to collect the precipitate and dry it at 60℃. The dried precipitate is an intermediate with a dialdehyde ring-opening structure.

[0069] 3. Add chlorosulfonic acid-formamide sulfation reagent to the intermediate with the dialdehyde ring-opening structure at a mass ratio of intermediate:chlorosulfonic acid:formamide of 1:3.2:10 (m / m / m), and react at 65℃ for 3 h. After the reaction is completed, add 3 times the volume of 95% ethanol to the reaction solution to precipitate, and take the paste precipitate.

[0070] 4. Dissolve the obtained paste-like precipitate in an appropriate amount of water and adjust the pH to 8-9 with sodium hydroxide solution; after removing impurities by microfiltration, the resulting neutralized solution is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the filtrate is collected; the filtrate is then subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa, and the retentate is collected; the retentate is then freeze-dried to obtain giant kelp oligosaccharide sulfate.

[0071] Comparative Example 3

[0072] Comparative Example 3 provides a method for preparing giant kelp oligosaccharide sulfate, the main difference from Example 1 being that sodium periodate was not added for directional cleavage, specifically including the following operations:

[0073] 1. Take 1 kg of dried giant kelp, crush it and pass it through a 2-mesh sieve; add it to a 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v), stir and extract at 70℃ for 3 h, filter and collect the filtrate; add excess calcium chloride solution to the filtrate to fully precipitate, centrifuge and collect the precipitate; add 5% hydrochloric acid solution to fully acidify the precipitate, centrifuge and collect the precipitate, add 1% sodium hydroxide aqueous solution to the precipitate to dissolve it, and neutralize the solution to pH 7.0~7.5 to obtain the dissolved solution.

[0074] 2. Add 3 times the volume of 95% ethanol solution to the above solution for alcohol precipitation, centrifuge to collect the precipitate and dry it at 60°C.

[0075] 3. Add chlorosulfonic acid-formamide sulfation reagent to the dried precipitate at a mass ratio of precipitate:chlorosulfonic acid:formamide of 1:2.9:10 (m / m / m), and react at 65℃ for 3 h. After the reaction is complete, add 3 times the volume of 95% ethanol to the reaction solution to precipitate, and take the paste-like precipitate.

[0076] 4. Dissolve the obtained paste-like precipitate in an appropriate amount of water and adjust the pH to 8-9 with sodium hydroxide solution; after removing impurities by microfiltration, the resulting neutralized solution is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the filtrate is collected; the filtrate is then subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa, and the retentate is collected; the retentate is then freeze-dried to obtain giant kelp oligosaccharide sulfate.

[0077] Comparative Example 4

[0078] Comparative Example 4 provides a method for preparing giant kelp oligosaccharide sulfate ester. The main difference from Example 1 is that chlorosulfonic acid-formamide sulfation reagent was not added for acidification modification. Specifically, the method includes the following steps:

[0079] 1. Take 1 kg of dried giant kelp, crush it and pass it through a 2-mesh sieve; add it to a 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v), stir and extract at 70℃ for 3 h, filter and collect the filtrate; add excess calcium chloride solution to the filtrate to fully precipitate, centrifuge and collect the precipitate; add 5% hydrochloric acid solution to the precipitate to fully acidify, centrifuge and collect the precipitate, add 1% sodium hydroxide aqueous solution to the precipitate to dissolve it, and adjust the pH of the solution to 7.0~7.5 to obtain the dissolved solution.

[0080] 2. Add sodium periodate to the above solution until the final mass concentration of sodium periodate in the solution is 4%, react at room temperature in the dark for 6 h, and then add ethylene glycol in an equimolar amount of sodium periodate to react until the residual sodium periodate is removed; add 3 times the volume of 95% ethanol solution to the above solution for alcohol precipitation, centrifuge to collect the precipitate and dry it at 60℃. The dried precipitate is an intermediate with a dialdehyde ring-opening structure.

[0081] 3. After removing impurities by microfiltration, the obtained solution is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the filtrate is collected. The filtrate is then subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa, and the retentate is collected. The retentate is then freeze-dried to obtain giant kelp oligosaccharide sulfate.

[0082] Example 4

[0083] This embodiment analyzes the components and functions of the giant kelp oligosaccharide sulfates prepared in Examples 1-3 and Comparative Examples 1-4.

[0084] 1. Component analysis of giant kelp oligosaccharide sulfate ester

[0085] (1) Monosaccharide composition analysis

[0086] The intermediate with a dialdehyde ring-opening structure in the preparation process of the giant kelp oligosaccharide sulfate was analyzed for monosaccharide composition using 1-phenyl-3-methyl-5-pyrazolone derivatization-high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Hypersil BDS C18 HPLC column (250 mm × 4.6 mm, 3 μm); mobile phase: acetonitrile: 0.1 mol / L PBS solution (pH 7.0) = 17:83 (v / v); flow rate: 0.8 mL / min; column temperature: 30 °C; injection volume: 10 μL; detector: UV detector (254 nm).

[0087] (2) Relative molecular weight analysis method

[0088] The relative molecular weight of the giant kelp oligosaccharide sulfate was analyzed by gel permeation chromatography (GPC), using dextran molecular weight standard as a reference. The chromatographic conditions were as follows: TSK-Gel3000PW column. XL Gel chromatography column (300 mm × 7.5 mm, 7 μm); mobile phase: 0.2 mol / L NaNO3 solution; flow rate: 0.5 mL / min; column temperature: 35℃; injection volume: 20 μL; detector: differential refractive index detector (35℃).

[0089] (3) Molar ratio of monosaccharide residues with dialdehyde structure

[0090] The aldehyde content of the dialdehyde ring-opening intermediate in the preparation of giant kelp oligosaccharide sulfate was determined by hydroxylamine hydrochloride titration. 0.1 g of the intermediate with the dialdehyde ring-opening structure was weighed and dissolved in 25 mL of ultrapure water. The pH of the intermediate solution was adjusted to 5.0 using hydrochloric acid aqueous solution and sodium hydroxide aqueous solution, and the pH of 20 mL of hydroxylamine hydrochloride solution was also adjusted to 5.0. The two solutions were mixed thoroughly and reacted in a water bath at 40 °C for 4 h with stirring. The pH of the mixed solution was then titrated to 5.0 using 0.1 mol / L NaOH standard solution. The volume of NaOH standard solution consumed in the titration was recorded. The aldehyde content of the giant kelp oligosaccharide sulfate intermediate was calculated using Formula 1, and the molar ratio of monosaccharide residues with the dialdehyde structure in the giant kelp oligosaccharide sulfate intermediate was calculated using Formula 2.

[0091] Formula 1:

[0092] In the formula, V NaOH The volume (mL) of NaOH standard solution consumed in the titration is C. NaOH denoted as , where is the concentration of the NaOH standard solution (0.1 mol / L), and m is the mass of the giant kelp oligosaccharide sulfate intermediate (0.1 g).

[0093] Formula 2: Molar ratio of monosaccharide residues with dialdehyde structure (%) = ,

[0094] In the formula, C CHO 198 represents the aldehyde content (mmol / g); 198 represents the average molar mass (g / mol) of mannuronic acid or guluronic acid residues in sodium-type giant kelp alginate polysaccharide; 2000 is the conversion factor.

[0095] (4) Sulfate content analysis

[0096] Construction of the standard curve for sulfate content: Accurately measure potassium sulfate standard solution, dilute it serially with purified water to prepare standard solutions of 0.5, 1, 5, 10, 20, 30, and 50 μg / mL, shake well, and filter through a 0.22 μm filter membrane. Plot the standard curve with the concentration of potassium sulfate standard solution as the x-axis and the peak area as the y-axis. Sample treatment: Add 60 mL of purified water and 1 mL of 30% hydrogen peroxide to a combustion flask; accurately weigh 20 mg of the sample, degrade it using the oxygen flask combustion method, seal the flask with a small amount of purified water, shake and let stand for 30 min to allow complete absorption of the fumes; then heat the combustion flask until bubbles appear to remove excess hydrogen peroxide, cool the solution and dilute to 100 mL. Chromatographic conditions: SH-AC-37 anion exchange column (250 mm × 4 mm); mobile phase: anion exchange eluent; flow rate: 1.0 mL / min; column temperature: 35℃; injection volume: 25 μL; acquisition time: 25 min. The peak area of ​​the test sample was substituted into the regression equation to calculate the sulfate concentration C of the test sample. 供试品 Calculate the mass percentage of sulfate groups in the test sample according to Formula 3.

[0097] Formula 3: The mass percentage of sulfate groups in the test sample (%) =

[0098] In the formula, C 供试品 V is the sulfate concentration of the test sample (μg / mL); V is the final volume of the test sample solution (mL); m is the weighed mass of the test sample solution (g); 10 -6 1.0105 is the unit conversion factor; 1.0105 is the mass conversion factor between sulfate and sulfate groups.

[0099] The monosaccharide composition, average relative molecular weight, molar percentage of dialdehyde structure, and sulfate group content of the *Kochia scoparia* oligosaccharide sulfate esters obtained in Examples 1-3 and Comparative Examples 1-4 are shown in Table 1. Figures 1 and 2 are, respectively, the monosaccharide composition spectrum and the relative molecular mass spectrum of the *Kochia scoparia* oligosaccharide sulfate ester obtained in Example 1.

[0100] The natural alginate in giant kelp is mainly composed of two sugar units: β-D-mannuronic acid (M) and α-L-guluronic acid (G). Taking Example 1 as an example, combined with the attached... Figure 1 The results of the detection spectrum and monosaccharide composition analysis showed that the obtained giant kelp oligosaccharide sulfate contained only M and G and no other monosaccharide components, which is consistent with the structural characteristics of alginate from giant kelp. This confirms that the giant kelp oligosaccharide sulfate prepared in this application completely retains the original saccharide unit skeleton of giant kelp alginate.

[0101] Furthermore, the structure of the giant kelp oligosaccharide sulfate ester prepared in this application is shown below:

[0102]

[0103] The aforementioned giant kelp oligosaccharide sulfates include modified oligosaccharide derivatives composed of saturated mannuronic acid sulfate (M-SO4), saturated guluronic acid sulfate (G-SO4), ring-opening mannuronic acid (M'), and ring-opening guluronic acid (G') linked by 1→4 glycosidic bonds. In the M-SO4 and G-SO4 monosaccharide residues with R substituents, R is H or SO3. - And there is at least one SO3 residue on the same monosaccharide residue. - n satisfies 6≤n≤36.

[0104] Furthermore, the physicochemical structural analysis results in Table 1 show that the average relative molecular weight of the giant kelp oligosaccharide sulfates prepared in Examples 1-3 and Comparative Examples 1-4 ranges from 8.3 kDa to 76.3 kDa, the molar percentage of dialdehyde structure in the giant kelp oligosaccharide sulfates ranges from 0.8% to 15.3%, and the sulfate group content in the giant kelp oligosaccharide sulfates ranges from 0.2% to 33.1%.

[0105] This invention prepares a series of giant kelp oligosaccharide sulfates with different molecular weights, dialdehyde content, and sulfate substitution degrees by controlling the directional pyrolysis intensity, sulfation modification feed ratio, and reaction time. As shown in Examples 1, 3, and Comparative Examples 1-3, the sodium periodate concentration and reaction time directly determine the degree of polysaccharide pyrolysis. Stronger pyrolysis (Comparative Example 1, 8% concentration, 10 h) results in lower molecular weight and a higher proportion of dialdehyde structure in the product; weaker pyrolysis (Comparative Example 2, 2% concentration, 4 h) or no pyrolysis (Comparative Example 3) results in higher molecular weight and fewer dialdehyde structures in the product. In particular, Comparative Example 3 did not involve sodium periodate oxidation during the preparation of giant kelp oligosaccharide sulfates, resulting in an aldehyde content of 0.08 mmol / g and a dialdehyde structure molar percentage of 0.8%. As can be seen from Examples 1 and 2 and Comparative Examples 2 and 4, the chlorosulfonic acid feed ratio and reaction time determine the degree of sulfate substitution. In Examples 2 and 2, the chlorosulfonic acid ratio was increased and the reaction time was extended, resulting in a significant increase in sulfate content. In Comparative Example 4, no sulfonation reaction was performed when preparing giant kelp oligosaccharide sulfate ester, so its sulfate content was 0.2%.

[0106] Meanwhile, the proportions of mannuronic acid and guluronic acid in the products of each embodiment and comparative example remained basically stable, all conforming to the intrinsic monomer ratio of natural giant kelp alginate. This indicates that the process of the present invention only controls the length of the sugar chain and the degree of ring-opening modification, without destroying the original skeleton structure of oligosaccharides, and the product structure is stable and highly controllable.

[0107] Table 1. Physicochemical structural analysis results of a series of oligosaccharide sulfate esters

[0108]

[0109] 2. Activity evaluation of giant kelp oligosaccharide sulfate ester

[0110] (1) In vitro anticoagulation four-item test

[0111] The four in vitro anticoagulation parameters of the giant kelp oligosaccharide sulfate in Examples 1-3 and Comparative Examples 1-4 were determined. The experiment included a blank control group, a positive drug group (enoxaparin sodium), and an experimental group (100 μg / mL).

[0112] Preparation of test solutions and blood samples: Accurately weigh 12.0 mg of the test sample and add 1 mL of physiological saline to prepare a 12 mg / mL test sample solution. Dilute the solution sequentially to prepare test sample solutions of 6, 3, 1.5, 0.6, 0.3, and 0.06 mg / mL. Take 295 μL of sheep plasma and add 5 μL of different concentrations of test sample solutions to make the sample concentrations in the sheep plasma 200, 100, 25, 10, 5, 1, and 0 μg / mL, respectively.

[0113] Activated Partial Thromboplastin Time (APTT) Measurement: Before the experiment, preheat the APTT working solution and 0.025 mol / L CaCl2 solution in a 37°C constant temperature bath for 15 min. Take 100 μL of the blood sample to be tested and place it in a coagulation bath, add 100 μL of APTT working solution, and incubate at 37°C for 180 s. Add 100 μL of 0.025 mol / L CaCl2 solution preheated to 37°C, mix immediately, start timing, and record the plasma coagulation time. The APTT measurement is a key test for evaluating the function of the intrinsic coagulation pathway and the common coagulation pathway. By simulating the coagulation process in vitro, it measures the time required for plasma to form a fibrin clot under the action of specific reagents; the longer the time, the stronger the anticoagulant effect.

[0114] Prothrombin Time (PT) Measurement: Before the experiment, prepare the PT working solution according to the instructions, and then preheat the PT working solution at 37℃ for about 15 min. Take 100 μL of the blood sample to be tested into the coagulation bath and incubate at 37℃ for 180 s. Then add 200 μL of the preheated PT working solution at 37℃, mix immediately, start timing, and record the plasma coagulation time. The PT measurement is a screening test to assess the function of the extrinsic coagulation pathway. By detecting the plasma coagulation time, it reflects the activity levels of coagulation factors II, V, VII, and X, as well as the fibrinogen content, and is used to monitor the effect of anticoagulant drug treatment; it mainly reflects the coagulation capacity of the extrinsic coagulation pathway, and a prolonged PT indicates that extrinsic coagulation is inhibited.

[0115] Thrombin Time (TT) Measurement: Before the experiment, prepare the TT working solution according to the instructions, and then preheat the TT working solution at 37℃ for about 15 min. Take 100 μL of the blood sample to be tested into the coagulation bath and incubate at 37℃ for 180 s. Then add 100 μL of the preheated TT working solution at 37℃, mix immediately, start timing, and record the plasma coagulation time. The TT measurement can reflect the process of fibrinogen converting into fibrin; a prolonged TT indicates that terminal coagulation is inhibited.

[0116] Plasma fibrinogen (FIB) content determination: For standard curve preparation, reconstituted plasma was diluted with coagulation analysis diluent at ratios of 1:5, 1:10, 1:15, 1:20, and 1:30. Then, 200 μL of each dilution was taken, preheated at 37°C for 3 minutes, and 100 μL of thrombin solution was added. The coagulation time was measured. A standard curve was constructed with the fibrinogen content (mg / dL) of different concentrations of plasma as the x-axis and the corresponding coagulation time as the y-axis. For test plasma, the test plasma was diluted 1:10 with coagulation analysis diluent. 200 μL of the diluted plasma was taken, preheated at 37°C for 3 minutes, and 100 μL of thrombin solution was added. The coagulation time was measured. The measurement time was substituted into the standard curve to calculate the fibrinogen content. All experiments were repeated three times. Data are expressed as mean ± standard deviation, and comparisons were performed using a t-test (two-tailed test). The FIB assay primarily detects major clotting substrates in the blood. The lower the concentration of these substrates, the less likely the blood is to clot, directly reflecting the level of clotting substrates.

[0117] The experimental results are shown in Tables 2-5. Compared with the blank control group, the positive control drug enoxaparin sodium showed the most significant effects in prolonging APTT, PT, and TT and downregulating FIB levels. However, all test samples in Examples 1-3 and Comparative Examples 1-4 of this application could prolong APTT, PT, and TT clotting time to varying degrees and reduce plasma FIB fibrinogen levels, confirming that the giant kelp oligosaccharide sulfate prepared in this application can act simultaneously on intrinsic, extrinsic, and common coagulation pathways and has a clear in vitro anticoagulant effect.

[0118] Comparative Examples 2 and 3 showed higher sulfate group content than other groups, and exhibited the best APTT, PT, and TT prolongation effects, the largest fibrinogen downregulation, and the best anticoagulant effect. Example 2 had a higher sulfate group content than Examples 1 and 3, and its anticoagulant activity was among the highest in the example groups. Comparative Example 1 had significantly lower sulfate group content than other groups, resulting in a substantial reduction in anticoagulant activity. Comparative Example 4 underwent almost no sulfate modification, and its kelp oligosaccharide sulfate ester showed similar effects to the blank group, with no significant differences except for TT, essentially losing its in vitro anticoagulant activity. This demonstrates that sulfate groups are essential active groups for the in vitro anticoagulant activity of kelp oligosaccharide sulfate ester; the higher the degree of sulfate modification, the stronger the anticoagulant activity.

[0119] Further analysis of the data in Table 1 revealed a clear positive correlation between average relative molecular weight and anticoagulant activity. Comparative Example 3, with a molecular weight of 76.3 kDa, had the highest anticoagulant activity among all groups; Comparative Example 2, with a molecular weight of 25.3 kDa, had the second highest anticoagulant activity. The molecular weights of the Example groups decreased sequentially, with the anticoagulant effect gradually weakening. Comparative Example 1, with a molecular weight of only 8.3 kDa, showed severe molecular chain breakage and a significant decrease in anticoagulant activity. This indicates that within a reasonable structural range, a larger molecular weight is more conducive to enhancing the anticoagulant effect.

[0120] Therefore, the giant kelp oligosaccharide sulfate in Comparative Examples 2, 3 and Example 1 significantly prolonged APTT, PT and TT and reduced FIB content. Thus, the structure with high molecular weight and high sulfate content is crucial for improving in vitro anticoagulant activity.

[0121] Furthermore, the molar percentage of dialdehyde residues in the dialdehyde structure reflects the degree of ring-opening oxidation of the polysaccharide. Comparative Example 3 had the lowest dialdehyde residue content, resulting in a complete and stable polysaccharide backbone structure, which is more conducive to maintaining the spatial conformation and exerting an anticoagulant effect. Comparative Example 1 had an excessively high dialdehyde residue content, leading to excessive cleavage of the polysaccharide backbone and damage to the overall structure. Even with a small amount of sulfate groups, it was difficult to effectively exert an anticoagulant effect. In contrast, the dialdehyde residues in each example group were in a moderate range, ensuring successful grafting of sulfate groups while preserving the integrity of the main polysaccharide structure, thus meeting the requirements for exerting anticoagulant activity.

[0122] Table 2. APTT test results of a series of giant kelp oligosaccharide sulfates (100 μg / mL)

[0123]

[0124] Table 3. PT test results of a series of giant kelp oligosaccharide sulfates (100 μg / mL)

[0125]

[0126] Table 4. TT test results of a series of giant kelp oligosaccharide sulfates (100 μg / mL)

[0127]

[0128] Table 5. Results of FIB experiments on a series of giant kelp oligosaccharide sulfates (100 μg / mL)

[0129]

[0130] (2) Transdermal absorption assay

[0131] Transdermal absorption assays were performed on the series of samples. The experimental steps are as follows:

[0132] Accurately weigh 100 mg of the sample to be tested, add 10 mL of physiological saline to prepare a 1% (w / w) sample solution, mix well and set aside. Take the treated Bama miniature pig skin, cut it to an appropriate size, and place it in the diffusion cell with the keratin layer facing upwards. Add 7 mL of physiological saline to the receiving chamber, remove air bubbles, and add the sample solution above the pig skin. Perform transdermal absorption at 37℃ and 300 r / min. After 24 h, aspirate 2 mL of the receiving liquid from the sampling port. Use high-performance liquid chromatography (HPLC) to determine the permeation of the sample in the receiving liquid.

[0133] Transdermal absorption capacity is key to improving skin microcirculation. Table 6 lists the transdermal permeability of giant kelp oligosaccharide sulfate in Examples 1-3 and Comparative Examples 1-4 over 24 hours. The results show that, combined with the 24-hour in vitro transdermal absorption rate results in Table 6, the transdermal permeability of the samples is significantly negatively correlated with molecular weight. Comparative Examples 3 and 2 have relatively large molecular weights and large spatial volumes, making it difficult to penetrate the skin's stratum corneum barrier; their transdermal absorption rates are only 1.8% and 2.1%, respectively, indicating extremely poor skin permeability. As the molecular weight gradually decreases, the overall transdermal absorption capacity of the samples shows an upward trend, with the low molecular weight Comparative Example 1 showing significantly better transdermal performance than the high molecular weight group.

[0134] The molar percentage of monosaccharide residues with a dialdehyde structure reflects the degree of polysaccharide oxidation and cleavage. The higher the degree of oxidation, the more complete the molecular chain breakage, the smaller the molecular particle size, and the more conducive it is to skin penetration. Comparative Example 1 has the highest content of dialdehyde residues, a large degree of molecular cleavage, and good transdermal penetration ability; Comparative Example 3 has an extremely low proportion of dialdehyde structure, with complete and long molecular chains, making it difficult to penetrate the skin barrier, and has the worst transdermal effect.

[0135] The percentage of sulfate groups also affects transdermal performance. Sulfate groups are highly polar, and an excessively high percentage increases molecular hydrophilicity, making it more difficult for the skin's stratum corneum to penetrate, thus reducing transdermal absorption. Comparative Examples 2 and 3 showed excessively high sulfate content and strong hydrophilicity, further weakening skin penetration; samples with a moderate sulfate content were more likely to achieve good transdermal results.

[0136] The results show that low molecular weight, high dialdehyde structure ratio and appropriate sulfate mass percentage are beneficial to improving transdermal permeability. In addition, although high molecular weight samples have good anticoagulant activity in vitro, they have low transdermal permeability and are difficult to exert anticoagulant activity in vivo.

[0137] Table 6 Comparison of 24-hour transdermal absorption rates of a series of giant kelp oligosaccharide sulfate esters

[0138]

[0139] (3) Experiment on blood flow velocity in zebrafish blood vessels

[0140] Experiments were conducted to investigate the effects of a series of giant kelp oligosaccharide sulfates on intersegmental blood flow velocity in zebrafish.

[0141] The experiment included a normal control group, a positive control group (500 μg / mL hemolysin), and an experimental group (500 μg / mL series of giant kelp oligosaccharide sulfates).

[0142] Two dpf zebrafish were randomly selected and placed in 6-well plates, 15 fish per well. Different concentrations of extract 5 aqueous solutions were prepared and added at 3 mL per well. The plates were incubated at 28℃ in the dark for 24 h. Ten zebrafish from each group were randomly selected and placed in a blood flow and heart rate analyzer for video recording and data acquisition. The intersegmental blood flow velocity (V) of the zebrafish was analyzed. The following formula was used to calculate and determine whether the sample had the effect of improving microcirculation: Microcirculation improvement effect (%) = [(V (sample group) - V (normal control group)) / (V (normal control group))] × 100%.

[0143] All experiments were repeated three times. Data are expressed as mean ± standard deviation. Comparisons were performed using t-tests and two-tailed tests.

[0144] The experimental results are shown in Table 7. Compared with the normal control group, both the experimental group and the positive control group increased the blood flow velocity in the intersegmental vessels of zebrafish to a certain extent, confirming that the giant kelp oligosaccharide sulfate ester prepared in this application has the effect of improving microcirculation. Analysis of the physicochemical structural data in Table 1 shows that the differences in blood flow-promoting activity of the samples are mainly related to three modified structural characteristics: average relative molecular weight, the proportion of monosaccharide residues in the dialdehyde structure, and the mass fraction of sulfate groups.

[0145] First, the pro-blood-flow activity showed a significant negative correlation with molecular weight, generally exhibiting a pattern where smaller average relative molecular weight resulted in better effects on improving vascular blood flow velocity. Comparative Example 3, with the highest molecular weight, had the weakest effect, with a blood flow-improving efficacy of only 5.95%. As the molecular weight gradually decreased, the pro-blood-flow effect steadily increased, indicating that smaller molecular structures are more easily penetrated and act on microcirculatory tissues, effectively improving blood flow. Second, higher dialdehyde residue content facilitated the improvement of blood flow. Comparative Example 1, with the highest proportion of dialdehyde residues, showed a better blood flow improvement effect than the high molecular weight group. Finally, sulfate group content showed a positive correlation with pro-microcirculatory blood flow activity; higher sulfate group content resulted in a more pronounced effect on improving vascular blood flow. Comparative Example 4, with its extremely low sulfate group content and weaker effect on improving blood flow velocity, further confirmed that sulfate groups are the key structural basis for this effect.

[0146] Overall, the molecular weight, dialdehyde structure ratio, and sulfate content of Example 1 were most balanced, resulting in higher intersegmental blood flow velocity and improved efficacy compared to the positive control group and other samples, demonstrating the best microcirculation improvement effect. Example 3 showed the second-best structural parameters, with blood flow-promoting activity close to the positive control. Example 2's overall effect was slightly lower than the first two groups. In summary, the giant kelp oligosaccharide sulfate ester in Examples 1-3 showed the best efficacy in accelerating intersegmental blood flow velocity in zebrafish. Its molecular weight, sulfate content, and molar ratio of dialdehyde structure were in a relatively ideal balance, and its ability to improve vascular blood flow velocity was significantly better than that of the comparative samples, thus demonstrating superior efficacy in improving microcirculation in vivo.

[0147] Table 7. Effects of a series of giant kelp oligosaccharide sulfates on intersegmental blood flow velocity in zebrafish.

[0148]

[0149] In summary, combining the physicochemical indicators in Table 1 and the test results in Tables 2-7, it can be seen that molecular weight, the proportion of dialdehyde residues, and the sulfate group content directly determine the comprehensive efficacy and application performance of giant kelp oligosaccharide sulfate ester. This application obtained the products of Examples 1-3 through precise control of oxidative pyrolysis and sulfation modification processes. These products have a balanced and reasonable molecular structure ratio, possessing excellent in vitro anticoagulant activity, good transdermal skin penetration, and can effectively improve vascular blood flow velocity and skin microcirculation. The comparative products generally suffer from insufficient efficacy, poor transdermal effect, or structural imbalance, and their overall performance is significantly inferior to the samples in the examples of this application.

[0150] In some embodiments of this application, the giant kelp oligosaccharide sulfate prepared in Examples 1-3 is also used in cosmetics that improve skin microcirculation, and the cosmetics also include at least one of moisturizers, thickeners, emulsifiers and preservatives.

[0151] In some embodiments of this application, the mass concentration of giant kelp oligosaccharide sulfate contained in the above-mentioned cosmetic is 0.1~10 mg / mL.

[0152] The above examples only represent the technical solutions of this invention and are not intended to limit the experiments. Although we have improved the experimental scheme, researchers in the same field can still further improve the experimental scheme described above or make scientifically equivalent substitutions for the experimental steps. These changes do not cause the substance of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed in this invention.

Claims

1. A method for preparing giant kelp oligosaccharide sulfate, characterized in that, The preparation method includes the following steps: S1: Add giant kelp to sodium carbonate aqueous solution for extraction, add calcium chloride solution to the filtrate for precipitation, add hydrochloric acid solution to the precipitate for acidification, and collect the precipitate; add sodium hydroxide solution to the precipitate to dissolve and adjust the pH to 7.0~7.5 to obtain the solution; S2: Add sodium periodate to the solution for directional cleavage, then add ethylene glycol to remove residual sodium periodate, and finally add ethanol for alcohol precipitation. Take the precipitate and dry it. S3: The dried precipitate was added to a chlorosulfonic acid-formamide mixed solution for sulfation modification, and then ethanol was added for alcohol precipitation to obtain a paste-like precipitate. S4: Dissolve the paste-like precipitate in water, then adjust the pH to 8-9 with sodium hydroxide solution, and then perform the first ultrafiltration fraction. Perform the second ultrafiltration fraction on the filtrate, retain the retentate obtained from the second ultrafiltration fraction, and dry it to prepare giant kelp oligosaccharide sulfate.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass concentration of the sodium carbonate aqueous solution is 1% to 5%; the ratio of the giant kelp to the sodium carbonate aqueous solution is 1:30 to 1:

35.

3. The preparation method according to claim 1, characterized in that, In step S2, the sodium periodate has a mass concentration of 3% to 5% in the solution and is subjected to directional pyrolysis at room temperature for 5 to 7 hours.

4. The preparation method according to claim 1, characterized in that, In step S2, the molar ratio of ethylene glycol to sodium periodate is 1:

1.

5. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of precipitate:chlorosulfonic acid:formamide is 1:2.8~3.1:10, and the modification reaction is carried out at a temperature of 60~70℃ for 2~4 h.

6. The preparation method according to claim 1, characterized in that, In step S4, the molecular weight cutoff for the first ultrafiltration fraction is 30 kDa, and the molecular weight cutoff for the second fraction is 3.5 kDa.

7. The giant kelp oligosaccharide sulfate obtained by the preparation method according to any one of claims 1-6, characterized in that, The structure of the giant kelp oligosaccharide sulfate ester is shown below: ， The giant kelp oligosaccharide sulfate comprises a modified oligosaccharide derivative consisting of saturated mannuronic acid sulfate M-SO4, saturated guluronic acid sulfate G-SO4, ring-opening mannuronic acid M', and ring-opening guluronic acid G' linked by 1→4 glycosidic bonds; in the monosaccharide residues with R substituents, R is H or SO3. - And there is at least one SO3 residue on the same monosaccharide residue. - ;6≤n≤36.

8. The giant kelp oligosaccharide sulfate ester according to claim 7, characterized in that, The average molecular weight of the giant kelp oligosaccharide sulfate is 5 kDa to 30 kDa, and the molar percentage of monosaccharide residues containing a dialdehyde ring-opening structure in the giant kelp oligosaccharide sulfate is 0.01% to 10%.

9. The giant kelp oligosaccharide sulfate according to claim 7, wherein the mass percentage of sulfate groups in the giant kelp oligosaccharide sulfate ranges from 20% to 40%.

10. The application of the giant kelp oligosaccharide sulfate ester according to claim 7 in the preparation of cosmetics with the effect of improving skin microcirculation, characterized in that, The cosmetic contains 0.1-10 mg / mL of giant kelp oligosaccharide sulfate.

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