A macroalgal oligosaccharide extract, and a method of preparing and using the same

Abstract

The application discloses a kind of giant kelp oligosaccharide extract and its preparation method and application, belong to marine sugar chemistry technical field.The giant kelp oligosaccharide extract is prepared by using giant kelp as raw material, and is prepared by selective extraction, two-step directional cleavage, separation and purification process, and the obtained giant kelp oligosaccharide extract has dialdehyde structure.It is proved that the giant kelp oligosaccharide extract provided by the application can significantly inhibit the excessive release of NO in LPS-induced RAW264.7 cells, down-regulate the excessive expression of TNF-alpha, IL-1beta mRNA, reduce the excessive release of ROS in HSF cells caused by UVA damage, has good anti-inflammatory and antioxidant biological activity, and can be used for the development of cosmetic products with soothing effect.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic raw material development technology, specifically relating to a giant kelp oligosaccharide extract, its preparation method, and its application. Background Technology

[0002] As consumers become more aware of skin health and the proportion of people with "problem skin" increases, functional skincare products are gradually replacing traditional daily skincare products and becoming the mainstream in the market. Unlike traditional skincare products, which primarily focus on basic cleansing, hydration, and moisturizing, functional skincare products are designed to address specific skin problems, such as anti-inflammation, soothing, barrier repair, oil control and acne treatment, and whitening and fading dark spots. Their core advantages lie in: higher concentrations of active ingredients, more clearly defined targets, simpler formulas, and proven safety.

[0003] Among numerous efficacy claims, "anti-inflammatory" and "soothing" have become core areas of research and development in skincare. Modern lifestyles (such as environmental pollution, UV exposure, stress, and improper skincare) easily induce skin barrier damage, activating inflammatory signaling pathways such as NF-κB and MAPK, leading to the release of pro-inflammatory factors (such as NO, TNF-α, and IL-1β), causing discomfort symptoms such as erythema, burning, stinging, and itching. Therefore, developing efficacy ingredients with clear anti-inflammatory, antioxidant, and other skin-soothing effects can not only alleviate acute inflammatory responses but also prevent "inflammatory aging" from the source and maintain skin homeostasis. Against this backdrop, the development of naturally derived, highly biocompatible, and low-irritant anti-inflammatory and soothing cosmetic raw materials has attracted much attention.

[0004] Giant kelp (Macrocystis pyrifera), also known as giant seaweed or giant kelp algae, is a large marine brown alga belonging to the genus Macrocystis in the phylum Phaeophceae. It is mainly distributed in the cold-water areas along the North Pacific coast (such as California, Chile, New Zealand, and southern Australia), and is rich in polysaccharides (such as alginic acid and fucoidan), minerals, amino acids, phenolic compounds (such as algal polyphenols), and vitamins. Giant kelp extract (INCI name: MACROCYSTIS PYRIFERA (KELP) EXTRACT) has been widely used in cosmetics and other personal care products. However, most commercially available giant kelp extracts are currently crude extracts or simple hydrolysates, only functioning in basic ways such as moisturizing and barrier protection. High-performance raw materials prepared through selective extraction and targeted modification to enhance efficacy remain very scarce. Summary of the Invention

[0005] The main objective of this invention is to provide a giant kelp oligosaccharide extract, its preparation method, and its applications. The extract provided by this invention is prepared from giant kelp through selective extraction, two-step directional lysis, separation, and purification processes. This extract exhibits excellent anti-inflammatory and antioxidant properties.

[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 a giant kelp oligosaccharide extract, the method comprising the following steps:

[0008] (1) Add sodium carbonate solution to extract giant kelp and collect the filtrate; add calcium chloride solution to the obtained filtrate to precipitate and collect the precipitate;

[0009] (2) The precipitate is acidified by adding hydrochloric acid solution, and the acidified precipitate is dissolved and neutralized by adding sodium hydroxide solution to obtain a neutralized solution;

[0010] (3) Add hydrogen peroxide to the solution to perform directional cleavage of glycosidic bonds to obtain a polysaccharide lysis solution; add catalase to the obtained polysaccharide lysis solution to remove residual hydrogen peroxide to obtain an oligosaccharide solution;

[0011] (4) Add sodium periodate to the oligosaccharide solution to perform cis-diol bond directional cleavage to obtain oligosaccharide cleavage solution; add ethylene glycol to the obtained oligosaccharide cleavage solution to remove residual sodium periodate to obtain dialdehyde oligosaccharide solution.

[0012] (5) The dialdehyde oligosaccharide solution is subjected to ultrafiltration fractionation, and the filtrate is subjected to nanofiltration fractionation. The nanofiltration retentate is taken; the nanofiltration retentate is dried to obtain the giant kelp oligosaccharide extract.

[0013] Furthermore, the hydrogen peroxide has a mass concentration of 2% to 5% in the solution and is subjected to directional decomposition at 70 to 90°C for 4 to 8 hours.

[0014] Furthermore, the final mass concentration of sodium periodate in the lysis solution is 4%~6%, and it is directionally pyrolyzed for 6~15 h under room temperature and light-protected conditions.

[0015] Furthermore, the molecular weight cutoff for the ultrafiltration fraction is 5000 Da, and the molecular weight cutoff for the nanofiltration fraction is 300 Da.

[0016] Furthermore, the ratio of the giant kelp to the sodium carbonate solution is 1:25 to 1:35, and the mass concentration of the sodium carbonate solution is 1 to 5%.

[0017] Furthermore, the hydrochloric acid solution has a mass concentration of 5% to 10%, and the sodium hydroxide solution has a mass concentration of 2% to 5%.

[0018] In the above preparation method, the present invention first utilizes the physicochemical property that alginate (prepared by mixing giant kelp with sodium carbonate solution) forms a precipitate with calcium ions, while other giant kelp polysaccharides (including fucoidan and β-glucan) do not form a precipitate with calcium ions, thereby achieving selective extraction of alginate polysaccharides. Furthermore, the present invention utilizes hydrogen peroxide to achieve free radical directional cleavage of glycosidic bonds, and utilizes the oxidizing property of hydrogen peroxide to oxidize the reducing end hemiacetal of the polysaccharide into a diacid structure, further enhancing the anionic properties of the product. By controlling the degradation conditions, the molecular weight of the product is controlled, thus achieving the first step of directional cleavage. Further, sodium periodate is used to oxidize and ring-open the C2OH-C3OH cis-diol in some mannouronic acid and guluronic acid residues to form a dialdehyde structure, further enhancing the reducing property of the product, thus achieving the second step of directional cleavage. Finally, ultrafiltration is used to remove residual large molecular enzymes and unreacted, excessively large molecular weight components, followed by nanofiltration to remove residual inorganic salts and other small molecular weight impurities, as well as components with excessively small molecular weights after the reaction, thereby further narrowing the molecular weight distribution.

[0019] The present invention also provides a giant kelp oligosaccharide extract prepared by the above preparation method, wherein the giant kelp oligosaccharide extract has the following structural characteristics:

[0020]

[0021] The giant kelp oligosaccharide extract comprises an oligosaccharide backbone formed by 1→4 glycosidic bonds of saturated mannuronic acid, saturated guluronic acid, open-ring mannuronic acid, and open-ring guluronic acid, where 1≤n≤6; the giant kelp oligosaccharide extract also comprises guluronic acid or mannuronic acid diacid with C1-position oxidized ring-opening attached to the end of the oligosaccharide backbone; the molar percentage of sugar units containing dialdehyde structures in the giant kelp oligosaccharide extract ranges from 0.01% to 20%.

[0022] Furthermore, the average relative molecular weight of the giant kelp oligosaccharide extract is 500 Da to 5000 Da.

[0023] Furthermore, the average relative molecular weight of the giant kelp oligosaccharide extract ranges from 890 Da to 4130 Da, and the molar percentage of sugar units with dialdehyde structures in the giant kelp oligosaccharide extract ranges from 9.28% to 15.33%.

[0024] Furthermore, in the guluronic acid or mannuronic acid monosaccharide residues connected to the end of the oligosaccharide backbone, the C1 position of the guluronic acid or mannuronic acid monosaccharide residue is oxidized and ring-opened to form a diacid structure.

[0025] The present invention also provides the application of the giant kelp oligosaccharide extract in the preparation of cosmetics with anti-inflammatory and / or antioxidant soothing effects.

[0026] Furthermore, the giant kelp oligosaccharide extract can significantly reduce the excessive release of NO in LPS-induced RAW264.7 cell inflammation, downregulate the mRNA expression levels of IL-1β and TNF-α, and reduce the excessive release of ROS in HSF cells caused by UVA damage.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The giant kelp oligosaccharide extract provided by the present invention is prepared by selective extraction, two-step directional lysis, separation and purification of giant kelp as raw material, and has a novel structure.

[0029] 2. The giant kelp oligosaccharide extract provided by this invention is designed with the enhancement of skin-soothing effects such as anti-inflammatory and antioxidant properties in mind. It has dual advantages in anti-inflammatory and antioxidant effects, which provides an innovative path for developing a new generation of high-performance marine-derived cosmetic raw materials. Attached Figure Description

[0030] 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.

[0031] Figure 1 This is a monosaccharide composition diagram of the giant kelp oligosaccharide extract in Example 1;

[0032] Figure 2 The image shows the relative molecular mass spectrum of the giant kelp oligosaccharide extract from Example 1. Detailed Implementation

[0033] 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. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0034] The material-liquid ratio mentioned in this invention refers to the mass concentration, specifically the ratio of the mass (unit: kg) of giant kelp (Macrocystis pyrifera) to the volume (unit: L) of sodium carbonate aqueous solution.

[0035] The units for the mass concentrations of sodium carbonate aqueous solution, hydrochloric acid solution, hydrogen peroxide, sodium periodate, and sodium hydroxide solution described in this invention are all g / L.

[0036] Example 1

[0037] This embodiment provides a method for preparing giant kelp oligosaccharide extract, specifically including the following steps:

[0038] 1. Weigh 1 kg of dried giant kelp and add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v). Extract at 70℃ with stirring for 3 h. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0039] 2. Add a 5% hydrochloric acid solution to the above precipitate to submerge it, stir thoroughly to acidify, and filter to collect the precipitate. Add a 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and adjust the pH of the solution to 7.0~7.5 to obtain the solution.

[0040] 3. Add an appropriate amount of hydrogen peroxide to the solution until the mass concentration of hydrogen peroxide in the solution is 4%. Degrade at 80℃ for 5 h. Then add an appropriate amount of catalase to remove the residual hydrogen peroxide and obtain oligosaccharide solution.

[0041] 4. Add sodium periodate to the oligosaccharide solution until the sodium periodate concentration in the oligosaccharide solution is 5%, react at room temperature in the dark for 8 h, and then add ethylene glycol in an equimolar amount of sodium periodate to react until the residual sodium periodate is removed, to obtain the dialdehyde oligosaccharide solution.

[0042] 5. After filtering the obtained dialdehyde oligosaccharide solution to remove insoluble impurities, ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The filtrate was then fractionated using a nanofiltration membrane with a molecular weight cutoff of 300 Da, and the nanofiltration retentate was collected. The obtained nanofiltration retentate was spray-dried to obtain the giant kelp oligosaccharide extract.

[0043] Example 2

[0044] This embodiment provides a method for preparing giant kelp oligosaccharide extract. The main difference from Example 1 lies in the reaction time of the initial directional cleavage and the secondary directional cleavage. The specific steps include:

[0045] 1. Weigh 1 kg of dried giant kelp and 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. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0046] 2. Add 5% hydrochloric acid solution to the above precipitate to submerge it, stir thoroughly to acidify, filter and collect the precipitate; add 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and adjust the pH of the solution to 7.0~7.5 to obtain the solution.

[0047] 3. Add an appropriate amount of hydrogen peroxide to the solution until the mass concentration of hydrogen peroxide in the solution is 4%, degrade at 80°C for 4 h, and then add an appropriate amount of catalase to remove residual hydrogen peroxide to obtain oligosaccharide solution.

[0048] 4. Add sodium periodate to the above oligosaccharide solution until the mass concentration of sodium periodate in the oligosaccharide solution is 5%, react at room temperature in the dark for 9 h, and then add ethylene glycol in an equimolar amount of sodium periodate to react until the residual sodium periodate is removed, to obtain dialdehyde oligosaccharide solution.

[0049] 5. After filtering the obtained dialdehyde oligosaccharide solution to remove insoluble impurities, ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The filtrate was then fractionated using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration retentate was collected and spray-dried to obtain the giant kelp oligosaccharide extract.

[0050] Example 3

[0051] This embodiment provides a method for preparing giant kelp oligosaccharide extract. The main difference from Example 1 is the reaction time for the first-stage directional pyrolysis and the second-stage directional pyrolysis. Specifically, it includes the following steps:

[0052] 1. Weigh 1 kg of dried giant kelp and add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v). Extract at 70℃ with stirring for 3 h. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0053] 2. Add a 5% hydrochloric acid solution to the precipitate to submerge it, stir thoroughly to acidify, and filter to collect the precipitate. Add a 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and adjust the pH of the solution to 7.0-7.5 to obtain the dissolved solution.

[0054] 3. Add an appropriate amount of hydrogen peroxide to the solution until the mass concentration of hydrogen peroxide in the solution is 4%, degrade at 80°C for 6 h, and then add an appropriate amount of catalase to remove residual hydrogen peroxide to obtain oligosaccharide solution.

[0055] 4. Add sodium periodate to the oligosaccharide solution until the sodium periodate concentration in the oligosaccharide solution is 5%, 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, to obtain the dialdehyde oligosaccharide solution.

[0056] 5. After filtering the obtained solution to remove insoluble impurities, ultrafiltration fractionation is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The obtained filtrate is then fractionated by nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration retentate is collected and spray-dried to obtain giant kelp oligosaccharide extract.

[0057] Comparative Example 1

[0058] Comparative Example 1 provides a method for preparing giant kelp oligosaccharide extract. The main difference between Comparative Example 1 and Example 1 lies in the mass concentration of hydrogen peroxide and the initial directional cleavage time. Specifically, it includes the following steps:

[0059] 1. Weigh 1 kg of dried giant kelp and 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. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0060] 2. Add a 5% hydrochloric acid solution to the precipitate to submerge it, stir thoroughly to acidify, and filter to collect the precipitate. Add a 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and neutralize the solution to a pH of 7.0-7.5 to obtain the dissolved solution.

[0061] 3. Add an appropriate amount of hydrogen peroxide to the solution until the mass concentration of hydrogen peroxide in the solution is 2%, degrade at 80°C for 3 h, and then add an appropriate amount of catalase to remove residual hydrogen peroxide to obtain oligosaccharide solution.

[0062] 4. Add sodium periodate to the above oligosaccharide solution until the mass concentration of sodium periodate in the oligosaccharide solution is 5%, react at room temperature in the dark for 8 h, and then add ethylene glycol in an equimolar amount of sodium periodate to react until the residual sodium periodate is removed, to obtain dialdehyde oligosaccharide solution.

[0063] 5. After filtering the obtained dialdehyde oligosaccharide solution to remove insoluble impurities, ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The filtrate was then fractionated using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration retentate was collected and spray-dried to obtain the giant kelp oligosaccharide extract.

[0064] Comparative Example 2

[0065] Comparative Example 2 provides a method for preparing giant kelp oligosaccharide extract. The main difference from Example 1 lies in the mass concentration of hydrogen peroxide and the time of initial directional cleavage. Specifically, it includes the following steps:

[0066] 1. Weigh 1 kg of dried giant kelp and add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v). Extract at 70℃ with stirring for 3 h. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0067] 2. Add a 5% hydrochloric acid solution to the above precipitate to submerge it, stir thoroughly to acidify, and filter to collect the precipitate. Add a 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and neutralize the solution to a pH of 7.0-7.5 to obtain the dissolved solution.

[0068] 3. Add an appropriate amount of hydrogen peroxide to the solution until the mass concentration of hydrogen peroxide in the solution is 6%, degrade at 80°C for 8 hours, and then add an appropriate amount of catalase to remove residual hydrogen peroxide to obtain oligosaccharide solution.

[0069] 4. Add sodium periodate of 5% to the oligosaccharide solution and react at room temperature in the dark for 8 hours. Then add ethylene glycol of equimolarity to the sodium periodate solution and react until the residual sodium periodate is removed to obtain the dialdehyde oligosaccharide solution.

[0070] 5. After filtering the obtained dialdehyde oligosaccharide solution to remove insoluble impurities, ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The filtrate was then fractionated using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration retentate was collected and spray-dried to obtain the giant kelp oligosaccharide extract.

[0071] Comparative Example 3

[0072] Comparative Example 3 provides a method for preparing giant kelp oligosaccharide extract, the main difference from Example 1 being the timing of the secondary directional lysis, specifically including the following steps:

[0073] 1. Weigh 1 kg of dried giant kelp and 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. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0074] 2. Add a 5% hydrochloric acid solution to the precipitate to submerge it, stir thoroughly to acidify, and filter to collect the precipitate. Add a 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and neutralize the solution to a pH of 7.0-7.5 to obtain the dissolved solution.

[0075] 3. Add an appropriate amount of hydrogen peroxide to the solution until the mass concentration of hydrogen peroxide in the solution is 4%, degrade at 80°C for 5 h, and then add an appropriate amount of catalase to remove residual hydrogen peroxide to obtain oligosaccharide solution.

[0076] 4. Add sodium periodate to the above oligosaccharide solution until the sodium periodate concentration in the oligosaccharide solution is 5%, react at room temperature in the dark for 15 h, and then add ethylene glycol in an equimolar amount of sodium periodate to react until the residual sodium periodate is removed, to obtain dialdehyde oligosaccharide solution.

[0077] 5. After filtering the obtained dialdehyde oligosaccharide solution to remove insoluble impurities, ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The filtrate was then fractionated using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration retentate was collected and spray-dried to obtain the giant kelp oligosaccharide extract.

[0078] Comparative Example 4

[0079] Comparative Example 4 provides a method for preparing giant kelp oligosaccharide extract. The main difference from Example 1 is the order of solvents used in the two directional cleavage processes. Specifically, it includes the following steps:

[0080] 1. Weigh 1 kg of dried giant kelp and 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. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0081] 2. Add a 5% hydrochloric acid solution to the above precipitate to submerge it, stir thoroughly to acidify, and filter to collect the precipitate. Add a 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and neutralize the solution to pH 7.0~7.5 to obtain the solution.

[0082] 3. Add an appropriate amount of sodium periodate to the solution until the sodium periodate concentration in the solution is 5%, react at room temperature in the dark for 8 hours, and then add ethylene glycol in an equimolar amount of sodium periodate to react until the residual sodium periodate is removed, thus obtaining a preliminary directional lysis solution.

[0083] 4. Add an appropriate amount of hydrogen peroxide to the obtained preliminary directional lysis solution until the mass concentration of hydrogen peroxide in the oligosaccharide solution is 4%. Degrade at 80℃ for 5 hours. Then add an appropriate amount of catalase to remove residual hydrogen peroxide and obtain the secondary directional lysis solution.

[0084] 5. After removing insoluble impurities from the obtained secondary directional lysis solution, ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The obtained filtrate was then fractionated using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration retentate was collected and spray-dried to obtain the giant kelp oligosaccharide extract.

[0085] Comparative Example 5

[0086] Comparative Example 5 provides a method for preparing giant kelp oligosaccharide extract. The main difference from Example 1 is that secondary directional cleavage with sodium periodate was not performed. The method specifically includes the following steps:

[0087] 1. Weigh 1 kg of dried giant kelp and add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v). Extract at 70℃ with stirring for 3 h. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0088] 2. Add a 5% hydrochloric acid solution to the above precipitate to submerge it, stir thoroughly to acidify, and filter to collect the precipitate. Add a 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and neutralize the solution to a pH of 7.0-7.5 to obtain the dissolved solution.

[0089] 3. Add an appropriate amount of hydrogen peroxide to the solution until the mass concentration of hydrogen peroxide in the solution is 4%, degrade at 80°C for 5 h, and then add an appropriate amount of catalase to remove residual hydrogen peroxide to obtain oligosaccharide solution.

[0090] 4. After filtering the obtained oligosaccharide solution to remove insoluble impurities, ultrafiltration fractionation is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The obtained filtrate is then fractionated by nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration retentate is collected and spray-dried to obtain the giant kelp oligosaccharide extract.

[0091] Comparative Example 6

[0092] Comparative Example 6 provides a method for preparing giant kelp oligosaccharide extract, the main difference from Example 1 being that hydrogen peroxide-directed pyrolysis was not performed, and specifically includes the following steps:

[0093] 1. Weigh 1 kg of dried giant kelp and add it to 30 L of 1% sodium carbonate aqueous solution at a material-to-liquid ratio of 1:30 (m / v). Extract at 70℃ with stirring for 3 h. After extraction, filter and collect the filtrate. Add excess calcium chloride solution to the filtrate to fully calcify and precipitate. Filter and collect the precipitate.

[0094] 2. Add a 5% hydrochloric acid solution to the above precipitate to submerge it, stir thoroughly to acidify, and filter to collect the precipitate. Add a 2% sodium hydroxide aqueous solution to the obtained precipitate to dissolve it, and neutralize the solution to pH 7.0~7.5 to obtain the solution.

[0095] 3. Add sodium periodate to the solution until the mass concentration of sodium periodate in the solution is 5%, react at room temperature in the dark for 8 hours, and then add ethylene glycol in an equimolar amount of sodium periodate to react until the residual sodium periodate is removed, thereby obtaining the directional lysis solution.

[0096] 4. After filtering the obtained directional lysis solution to remove insoluble impurities, ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The obtained filtrate was then fractionated using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration retentate was collected and spray-dried to obtain giant kelp oligosaccharide extract.

[0097] Example 4

[0098] This embodiment combines the physicochemical properties of the giant kelp oligosaccharide extracts prepared in Examples 1-3 and Comparative Examples 1-6.

[0099] 1. Monosaccharide composition analysis

[0100] The monosaccharide composition of the above nine giant kelp oligosaccharide extracts was analyzed by 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).

[0101] 2. Relative molecular mass analysis

[0102] Relative molecular weight analysis was performed using gel permeation chromatography (GPC), with dextran molecular weight standard as a reference. The chromatographic conditions were as follows: TSK-Gel 3000PW 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℃).

[0103] 3. Molar ratio of monosaccharide residues with dialdehyde structure

[0104] The aldehyde content in the giant kelp oligosaccharide extract was determined by hydroxylamine hydrochloride titration. 0.1 g of the prepared giant kelp oligosaccharide extract was weighed and dissolved in 25 mL of ultrapure water. The pH of the giant kelp oligosaccharide extract 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. Then, the pH of the mixed solution was 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 in the giant kelp oligosaccharide extract was calculated using Formula 1, and the molar ratio of monosaccharide residues with dialdehyde structures in the giant kelp oligosaccharide extract was calculated using Formula 2.

[0105] Formula 1: ,

[0106] 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 extract (0.1 g).

[0107] Formula 2: Molar ratio of monosaccharide residues with dialdehyde structure (%) = ,

[0108] In the formula, C CHO 198 represents the aldehyde content (mmol / g) of the giant kelp oligosaccharide extract; 198 represents the average molar mass (g / mol) of mannuronic acid or guluronic acid residues; 2000 is the conversion factor.

[0109] The monosaccharide composition, average relative molecular weight, and molar percentage of dialdehyde structures of the oligosaccharide extracts from *Kelpia macrophylla* in Examples 1-3 and Comparative Examples 1-6 are shown in Table 1. Figure 1 Appendix Figure 2 The images shown are, in order, the monosaccharide composition and relative molecular mass spectra of the giant kelp oligosaccharide extract from Example 1.

[0110] 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 detection chromatogram and monosaccharide composition results show that the obtained giant kelp oligosaccharide extract contains 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 extract prepared by this invention completely retains the original saccharide unit skeleton of giant kelp alginate.

[0111] Furthermore, the structure of the giant kelp oligosaccharide extract prepared by this invention is shown below:

[0112]

[0113] The above-mentioned giant kelp oligosaccharide extract is mainly composed of M and G. Specifically, the giant kelp oligosaccharide extract includes an oligosaccharide backbone composed of saturated mannuronic acid (M), saturated guluronic acid (G), open-ring mannuronic acid (M'), and open-ring guluronic acid (G') linked by 1→4 glycosidic bonds, with 1≤n≤6 and oxidized open-ring guluronic acid or mannuronic acid diacid structure at C1 position. The molar percentage of sugar units containing dialdehyde structures (i.e., M' and G') in the giant kelp oligosaccharide extract ranges from 0.01% to 20%. Among them, the dialdehyde structure of sugar unit M' is formed by the oxidation and ring-opening of the C2OH-C3OH cis-diol structure (C2 and C3 positions cis-vicial diol structure) of the saturated mannuronic acid sugar ring. Similarly, the dialdehyde structure of sugar unit G' is formed by the oxidation and ring-opening of the C2OH-C3OH cis-diol structure (C2 and C3 positions cis-vicial diol structure) of the saturated guluronic acid sugar ring.

[0114] The present invention prepares the giant kelp oligosaccharide extract as shown in Table 1 through selective extraction, two-step directional cleavage, separation and purification steps. The average relative molecular weight ranges from 890 Da to 4130 Da, and the molar percentage of sugar units with dialdehyde structure in the giant kelp oligosaccharide extract ranges from 2.27% to 23.21%.

[0115] This invention prepares a series of giant kelp oligosaccharide extracts with different molecular weights and molar proportions of dialdehyde structures, as shown in Table 1, by controlling the directional pyrolysis intensity of hydrogen peroxide, the duration of the sodium periodate oxidation ring-opening reaction, and the process sequence. Examples 1-3 and Comparative Examples 1, 2, and 6 show that the concentration of hydrogen peroxide degradation and the reaction time directly determine the degree of polysaccharide chain pyrolysis. Higher pyrolysis intensity (e.g., Comparative Example 2, high concentration, long degradation time) results in a lower average relative molecular weight of the product; weaker pyrolysis (Comparative Example 1, low concentration, short degradation time) or no pre-pyrolysis (Comparative Example 6, no hydrogen peroxide degradation) results in insufficient sugar chain breakage and a significantly higher molecular weight of the product. Comparative Example 6, which did not undergo directional pyrolysis with hydrogen peroxide, yielded a product with an average relative molecular weight as high as 4130 Da, indicating insufficient sugar chain degradation and the inability to form a uniform oligosaccharide structure.

[0116] As can be seen from Examples 1-3 and Comparative Examples 3 and 5, the reaction time of sodium periodate oxidation directly determines the amount of dialdehyde structure generated in the product. When the oxidation time is extended (Comparative Example 3), the degree of ring opening of the vicinal diol of the sugar ring is significantly increased, and the molar proportion of the dialdehyde structure increases significantly, resulting in over-oxidation. Without sodium periodate oxidation (Comparative Example 5), the sugar ring hardly undergoes a ring-opening reaction, and the molar proportion of the dialdehyde structure is only 2.27%, with the target ring-opening structure being severely lacking.

[0117] As can be seen from Example 1 and Comparative Example 4, the process reaction sequence has a significant impact on the structural characteristics of the product. The process sequence of first oxidizing and opening the ring and then degrading (Comparative Example 4) results in a decrease in oxidation reaction efficiency due to the steric hindrance of long-chain polysaccharides. Compared with the process of first directional cleavage and then oxidizing and opening the ring in this invention, the amount of dialdehyde structure generated is significantly reduced.

[0118] 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.

[0119] Table 1 Physicochemical properties and structural analysis of the oligosaccharide extracts from giant kelp in the examples and comparative examples.

[0120]

[0121] Example 5

[0122] This embodiment investigates the inhibitory effect of giant kelp oligosaccharide extract on lipopolysaccharide (LPS)-induced inflammatory response in mouse monocytes RAW264.7.

[0123] Experimental Methods: A mouse monocyte / macrophage RAW264.7 cell inflammation model induced by LPS was constructed. The experiment included a blank control group (no LPS, no giant kelp oligosaccharide extract), a model control group (LPS only), and an experimental group (LPS and giant kelp oligosaccharide extract). RAW264.7 cells were cultured at 8 × 10⁸ cells per well. 5 Cells were seeded at a density of 1 mL per well in 6-well plates and incubated overnight at 37°C with 5% CO2. After cell attachment, the supernatant was discarded in a biosafety cabinet. Equal volumes of fresh complete culture medium were added to the blank and positive control groups, while the experimental groups were added to fresh complete culture medium containing 100 μg / mL of giant kelp oligosaccharide extract. The plates were then pre-incubated at 37°C with 5% CO2 for 6 h, followed by the addition of LPS (final concentration 1 μg / mL) to each well for an additional 18 h of co-incubation. After centrifugation, the supernatant and cells at the bottom of the plate were collected separately.

[0124] The NO content in cell supernatant was determined using a NO detection kit: After bringing the NO detection kit to room temperature, 1 M NaNO2 standard solution was diluted with complete culture medium to prepare 0, 1, 2, 5, 10, 20, 40, 60, and 100 μM standard solutions. A new 96-well plate was prepared, and 50 μL of cell supernatant or standard solution was added to each well. Then, 50 μL of reagent I was added to each well. At this point, the red color of the culture medium faded. Next, 50 μL of reagent II was added to each well. The solution color turned purple-red. The absorbance was measured at 540 nm using a microplate reader. A standard curve was plotted, and the NO concentration in the sample was calculated based on the standard curve.

[0125] RNA extraction from RAW264.7 cells was performed using the kit as follows: (1) Collect and lyse the cells. Carefully aspirate the culture medium and wash with 1 mL of PBS. Aspirate the PBS and add 350 μL of lysis buffer to a 6-well plate. After 10 min, add all the cell lysis buffer to the DNA removal column (place the removal column in the collection tube). (2) Immediately centrifuge at 13000 rpm for 60 seconds and retain the filtrate (RNA is in the filtrate). Ensure that all liquid is filtered out after centrifugation and that there is no residue on the membrane. (3) Add 350 μL of 70% ethanol, mix by pipetting, and add to the adsorption column RA (place the adsorption column in the collection tube). Centrifuge at 13000 rpm for 30 s and discard the waste liquid. (4) Add 700 μL of protein removal solution RW1, incubate at room temperature for 1 min, centrifuge at 12000 rpm for 30 s, and discard the waste liquid. (5) Add 500 μL of wash buffer RW, centrifuge at 12000 rpm for 30 s, discard the waste liquid, add another 500 μL of wash buffer RW, and repeat once. (6) Put the adsorption column RA back into the empty collection tube, centrifuge at 13000 rpm for 2 min, and try to remove the wash buffer to avoid residual ethanol in the wash buffer inhibiting the downstream reaction. (7) Take out the adsorption column RA and put it into an RNase-free centrifuge tube. Add 30~50 μL of RNase-free water to the middle of the adsorption membrane according to the expected RNA yield, place at room temperature for 1 min, and centrifuge at 12000 rpm for 1 min. (8) Collect the liquid after centrifugation. At this time, the RNA is in the collected liquid. Place it on a cooled plate and use Nanodrop to detect the RNA concentration and purity.

[0126] Perform the genomic DNA removal reaction and reverse transcription to synthesize DNA as follows: Add 4 μL of 4×g DNA wiper Mix and an appropriate amount of RNA solution to each well, based on an RNA concentration of 1 μg per sample. Finally, add ultrapure water to bring the volume to 16 μL. Use a Bio-Rad T100 gradient PCR instrument, incubate at 42 ℃ for 2 min, and then maintain at 4 ℃. Store the resulting cDNA at -80 ℃.

[0127] Perform the RT-qPCR reaction as follows: Prepare the designed primers into a solution. The reaction system consists of 5 μL SYBR Mix, 0.5 μL Forward Primer, 0.5 μL Reverse Primer, and 4 μL of cDNA at an appropriate concentration, for a total reaction volume of 10 μL. The amplification conditions for TNF-α, IL-1β, and 18S are shown in Table 2.

[0128] Table 2 Reaction Procedure

[0129]

[0130] Based on the circulation threshold (Ct) of the target gene and the Ct value of the 18S internal reference gene, according to 2 -△△Ct The relative expression level of the target gene mRNA in the sample was calculated using this method.

[0131] The blank group was designated as 1, and the relative contents of TNF-α and IL-1β mRNA in each group were calculated.

[0132] Primer sequences are shown in Table 3:

[0133] Table 3 Primer Sequences

[0134]

[0135] All experiments were repeated three times. Data are expressed as mean ± standard deviation and t-tests and two-tailed tests were used.

[0136] This invention prepared a series of giant kelp oligosaccharide extracts with different molecular weights and molar proportions of dialdehyde structures by controlling the directional cleavage intensity of hydrogen peroxide, the duration of the sodium periodate oxidation ring-opening reaction, and the process sequence. Using an LPS-induced RAW264.7 inflammatory cell model, the inhibitory activity of different structural products on the overexpression of inflammatory mediators NO, TNF-α, and IL-1β was investigated, clarifying the structure-activity relationship between product structure and anti-inflammatory efficacy. The experimental results are shown in Tables 4-6. Compared with the blank control group, the NO content and TNF-α and IL-1β mRNA expression levels in the LPS model group were significantly increased, indicating intracellular inflammation and proving successful model establishment. Compared with the positive model group, all the giant kelp oligosaccharide extracts prepared in the examples and comparative examples could, to some extent, reduce the NO content produced by LPS-induced RAW264.7 cells and downregulate the mRNA expression levels of TNF-α and IL-1β.

[0137] As demonstrated in Examples 1-3 and the comparative examples, the giant kelp oligosaccharide extract, possessing both a suitable molecular weight and a moderate content of dialdehyde structures, exhibits excellent anti-inflammatory activity. The products of Examples 1-3, with molecular weights ranging from 1380 to 2160 Da and a dialdehyde structure molar percentage within the optimal range of 9.28% to 15.33%, showed inhibition rates of 49.91% to 51.29% on the release of the inflammatory factor NO, 34.25% to 38.96% on TNF-α mRNA overexpression, and a high inhibition rate of 59.04% to 61.64% on IL-1β mRNA overexpression. Overall, their anti-inflammatory effects were significantly superior to those of the comparative samples.

[0138] As can be seen from Examples 1 and 3 and Comparative Examples 1, 2 and 6, the degree of polysaccharide cleavage directly affects the anti-inflammatory activity of the product. Insufficient cleavage leads to an excessively high molecular weight (Comparative Example 6, 4130 Da), excessively long sugar chains, and reduced bioavailability, resulting in a significant decrease in the inhibitory effects on NO, TNF-α, and IL-1β. Excessive cleavage leads to an excessively low molecular weight (Comparative Example 2, 890 Da), impaired sugar chain structure integrity, and inability to effectively exert anti-inflammatory effects, with the inhibitory activity against inflammatory factors being significantly lower than that of the samples in the examples of this invention.

[0139] As can be seen from Example 1 and Comparative Examples 3 and 5, the content of the dialdehyde structure is significantly positively correlated with the anti-inflammatory activity. In Comparative Example 5, which did not undergo sodium periodate oxidation and ring-opening, the molar proportion of the dialdehyde structure was only 2.27%, the target active structure was missing, and the inhibitory ability against various inflammatory mediators was greatly weakened. In Comparative Example 3, which underwent excessive oxidation with sodium periodate, the proportion of the dialdehyde structure was too high and the sugar chain structure was destroyed, so the anti-inflammatory activity could not be further improved and was even lower than that of the moderately oxidized example sample. This proves that the appropriate content of the dialdehyde structure is the core condition for ensuring excellent anti-inflammatory activity.

[0140] As can be seen from Example 1 and Comparative Example 4, the order of the preparation process has a significant impact on the anti-inflammatory activity of the product. The reverse process of first oxidizing and opening the ring and then directionally cleaving (Comparative Example 4) resulted in insufficient formation of the dialdehyde structure and poor structural uniformity. The inhibition rates of NO, TNF-α, and IL-1β were only 23.15%, 18.29%, and 21.39%, respectively, which were the lowest among all samples. This further verifies that the process order of first directional cleaving and then oxidizing and opening the ring is the key to preparing highly active giant kelp oligosaccharide extract.

[0141] Therefore, it is fully demonstrated that the moderate molecular weight and moderately open-ring structure of the dialdehyde obtained by the present invention through precise control of pyrolysis intensity and oxidation degree is the core structural basis for the excellent anti-inflammatory activity of giant kelp oligosaccharide extract. The process is highly controllable and the product has outstanding activity advantages.

[0142] Table 4. Inhibitory effect of giant kelp oligosaccharide extract (100 μg / mL) on LPS-induced NO over-release in LAW264.7 cells in the examples and comparative examples.

[0143]

[0144] Table 5. Inhibitory effect of giant kelp oligosaccharide extract (100 μg / mL) on LPS-induced TNF-α mRNA overexpression in LAS264.7 cells in the examples and comparative examples.

[0145]

[0146] Table 6. Inhibitory effect of giant kelp oligosaccharide extract (100 μg / mL) on LPS-induced IL-1β mRNA overexpression in LAW264.7 cells in the examples and comparative examples.

[0147] Example 6

[0148] This embodiment investigates the inhibitory effect of giant kelp oligosaccharide extract on long-wave ultraviolet (UVA)-induced oxidative stress damage in human skin fibroblasts (HSF).

[0149] Experimental methods: Using human skin fibroblasts (HSF) as the research object, a UVA-induced human HSF damage model was constructed.

[0150] The experiment included a blank control group (no UVA irradiation and giant kelp oligosaccharide extract), a damage group (UVA irradiation only), a ROS positive group (positive control in the ROS kit), and an experimental group (UVA irradiation and giant kelp oligosaccharide extract).

[0151] HSF cells were spaced at 1 × 10⁶ cells per well. 4Cells were seeded at a density of 100 μL per well in 96-well plates (3 replicates per group). The plates were incubated at 37°C with 5% CO2 for 24 h. The supernatant was discarded in a biosafety cabinet, and the cells were washed twice with PBS. Equal volumes of fresh complete culture medium were added to the blank control group, damage control group, and ROS-positive group. The experimental groups were added to fresh complete culture medium containing 100 μg / mL *Gnaphalium affine* oligosaccharide extract. The cells were incubated at 37°C with 5% CO2 for 24 h. The supernatant was discarded, and the cells were washed twice with PBS. 50 μL of PBS was added to each well, and the cells were irradiated under a UV lamp for 8 min. The PBS was discarded, and equal volumes of fresh complete culture medium were added to the blank control group, damage control group, and ROS-positive group. The experimental groups were added to fresh complete culture medium containing 100 μg / mL *Gnaphalium affine* oligosaccharide extract. Cells were incubated for another 24 h. A ROS detection kit was used to load the cells with fluorescent probes, and fluorescence detection was performed using a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Calculate the ROS content for each group.

[0152] All experiments were repeated three times. Data are expressed as mean ± standard deviation. Comparisons were performed using t-tests and two-tailed tests.

[0153] This embodiment further investigates the inhibitory effect of giant kelp oligosaccharide extracts on UVA-induced oxidative stress damage in HSF cells. The structure-activity relationship between the product structure and its antioxidant and anti-photodamage activities was verified by detecting the level of excessive ROS release in cells. The experimental results are shown in Table 7. Compared with the blank control group, the ROS level in the damaged group was significantly increased, indicating successful model establishment. Compared with the damaged group, the giant kelp oligosaccharide extracts prepared in this invention can effectively reduce the excessive release of ROS in UVA-induced HSF cells, demonstrating significant superiority and a good ability to resist oxidative damage and alleviate skin inflammation caused by oxidative damage.

[0154] Among them, the giant kelp oligosaccharide extracts prepared in Examples 1-3 still exhibited the best antioxidant activity, with an inhibition rate of 24.40%-27.70% against UVA-induced excessive ROS release in HSF cells, significantly reducing the relative release of cellular ROS and restoring the oxidative stress level to near the blank control group. Compared with the example groups, the ROS inhibitory activity of each comparative sample was significantly reduced. The ROS inhibition rates of comparative examples 1-3, 5, and 6 were only 11.94%-15.32%, while comparative example 4, with the reversed process order, showed the worst activity, with a ROS inhibition rate of only 7.36%.

[0155] Table 7. Inhibitory effect of giant kelp oligosaccharide extract on UVA-induced excessive ROS release in HSF in the examples and comparative examples.

[0156]

[0157] Example 7

[0158] This embodiment explores the dose-response relationship of anti-inflammatory and antioxidant effects of the giant kelp oligosaccharide extract prepared in Example 1.

[0159] As demonstrated in Examples 5 and 6, the giant kelp oligosaccharide extract from Example 1 exhibited significant bioactivity in multiple models: it not only effectively inhibited LPS-induced excessive NO release and TNF-α and IL-1β mRNA overexpression in RAW264.7 cells, but also significantly reduced UVA-induced excessive ROS accumulation in HSF cells. This further indicates that the M / G ratio and the proportion of dialdehyde structures are key structural factors determining the anti-inflammatory and antioxidant activities of this extract. Therefore, subsequent studies will use the giant kelp oligosaccharide extract from Example 1 to further explore the dose-response relationship of its anti-inflammatory and antioxidant effects.

[0160] Experimental Methods: Following the experimental model established in Examples 5-6, three concentrations of the giant kelp oligosaccharide extract from Example 1 were set up for the experimental group: 20 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. The experimental results are shown in Tables 8-11. Compared with the model group, the giant kelp oligosaccharide extract obtained in Example 1 exhibited dose-dependent anti-inflammatory and antioxidant effects, demonstrating its potential as a functional ingredient in soothing cosmetics.

[0161] Regarding anti-inflammatory activity, as the sample concentration increased from 20 μg / mL to 200 μg / mL, the product's inhibition rate on the excessive release of the inflammatory factor NO increased from 39.80% to 59.75%, the inhibition rate on the overexpression of the inflammatory factor TNF-α mRNA increased from 24.26% to 45.33%, and the inhibition rate on the overexpression of the inflammatory factor IL-1β mRNA increased from 50.76% to 67.60%. It can gradient-inhibit the secretion of inflammatory mediators and the expression of inflammatory genes, effectively alleviating cellular inflammatory responses. In terms of anti-photooxidative damage activity, the dose-response relationship was also significant. As the sample concentration increased, the product's ability to inhibit the excessive release of ROS in UVA-induced HSF cells continuously strengthened. At a concentration of 20 μg / mL, the ROS inhibition rate was 15.97%, at 100 μg / mL it increased to 26.59%, and at a high concentration of 200 μg / mL it reached 34.26%. It can gradient-inhibit intracellular oxidative stress products and reduce oxidative damage to skin cells caused by ultraviolet radiation.

[0162] Table 8. Dose-response relationship of the giant kelp oligosaccharide extract in Example 1 in inhibiting LPS-induced NO over-release in RAW264.7 cells.

[0163]

[0164] Table 9. Dose-response relationship of the giant kelp oligosaccharide extract in Example 1 in inhibiting LPS-induced TNF-α mRNA overexpression in RAW264.7 cells.

[0165]

[0166] Table 10. Dose-response relationship of the giant kelp oligosaccharide extract in Example 1 in inhibiting LPS-induced IL-1β mRNA overexpression in RAW264.7 cells.

[0167]

[0168] Table 11. Dose-response relationship of the inhibitory effect of giant kelp oligosaccharide extract in Example 1 on UVA-induced excessive ROS release in HSF cells.

[0169]

[0170] In summary, this invention, through precise control of the cleavage and oxidation processes, yields a highly active giant kelp oligosaccharide extract with a suitable molecular weight and appropriate proportion of dialdehyde structures. Process parameters can precisely control the molecular structure and bioactivity of the product; imbalances in sugar chain cleavage, improper oxidation levels, and disordered process sequence all significantly reduce the product's anti-inflammatory and antioxidant activities. The extract prepared by this invention exhibits good concentration-dependent bioactivity, maintaining stable efficacy even at low concentrations, making it suitable for conventional cosmetic additive systems. It can effectively block skin inflammatory responses, scavenge UV-induced free radicals, and achieve skin soothing, repair, anti-oxidation, and anti-photoaging effects, demonstrating excellent prospects for industrial application.

[0171] In some embodiments of the present invention, the giant kelp oligosaccharide extract prepared by the present invention can be applied to skin care cosmetics such as toners, repair lotions, serums, masks, and creams, and is compatible with conventional water-based formulation systems. Combined with cell activity dose-effect experiments, the effective addition concentration of this extract in cosmetics is not less than 0.002% (20 μg / mL). Under these minimum addition concentrations and above, it can stably achieve the core effects of anti-inflammatory and / or antioxidant soothing. By scavenging skin free radicals and improving UVA-induced skin oxidative stress and inflammatory damage, it can be effectively used to prepare cosmetics with anti-inflammatory, antioxidant, and soothing effects.

[0172] 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 a giant kelp oligosaccharide extract, characterized in that, The preparation method includes the following steps: (1) Add giant kelp to sodium carbonate solution for extraction, and collect the filtrate; add calcium chloride solution to the obtained filtrate for precipitation, and collect the precipitate; (2) The precipitate is acidified by adding hydrochloric acid solution, and the acidified precipitate is dissolved and neutralized by adding sodium hydroxide solution to obtain a neutralized solution; (3) Add hydrogen peroxide to the solution to perform directional cleavage of glycosidic bonds to obtain a polysaccharide lysis solution; add catalase to the obtained polysaccharide lysis solution to remove residual hydrogen peroxide to obtain an oligosaccharide solution; (4) Add sodium periodate to the oligosaccharide solution to perform cis-diol bond directional cleavage to obtain oligosaccharide cleavage solution; add ethylene glycol to the obtained oligosaccharide cleavage solution to remove residual sodium periodate to obtain dialdehyde oligosaccharide solution. (5) The dialdehyde oligosaccharide solution is subjected to ultrafiltration fractionation, and the filtrate is subjected to nanofiltration fractionation. The nanofiltration retentate is taken; the nanofiltration retentate is dried to obtain the giant kelp oligosaccharide extract.

2. The preparation method according to claim 1, characterized in that, The hydrogen peroxide has a mass concentration of 2% to 5% in the solution and is subjected to directional decomposition at 70 to 90°C for 4 to 8 hours.

3. The preparation method according to claim 1, characterized in that, The sodium periodate has a mass concentration of 4% to 6% in the oligosaccharide solution and is directionally pyrolyzed at room temperature in the dark for 6 to 15 hours.

4. The preparation method according to claim 1, characterized in that, The molecular weight cutoff for the ultrafiltration fraction is 5000 Da, and the molecular weight cutoff for the nanofiltration fraction is 300 Da.

5. The preparation method according to claim 1, characterized in that, The ratio of giant kelp to sodium carbonate solution is 1:25 to 1:35, and the mass concentration of sodium carbonate solution is 1% to 5%.

6. The preparation method according to claim 1, characterized in that, The hydrochloric acid solution has a mass concentration of 5% to 10%, and the sodium hydroxide solution has a mass concentration of 2% to 5%.

7. The giant kelp oligosaccharide extract prepared by the preparation method according to any one of claims 1-6, characterized in that, The structure of the giant kelp oligosaccharide extract is shown below: The giant kelp oligosaccharide extract comprises an oligosaccharide backbone consisting of saturated mannuronic acid, saturated guluronic acid, open-ring mannuronic acid, and open-ring guluronic acid linked by 1→4 glycosidic bonds, where 1≤n≤6; the giant kelp oligosaccharide extract also includes guluronic acid or mannuronic acid diacid with C1-position oxidized ring-opening attached to the end of the oligosaccharide backbone; the monosaccharide residues containing dialdehyde structures account for a molar percentage of 0.01%~20% of the giant kelp oligosaccharide extract.

8. The giant kelp oligosaccharide extract according to claim 7, characterized in that, The average relative molecular weight of the giant kelp oligosaccharide extract is 500 Da to 5000 Da.

9. The use of the giant kelp oligosaccharide extract according to claim 7 in the preparation of cosmetics with anti-inflammatory and / or antioxidant soothing effects.

10. The application according to claim 9, characterized in that, The giant kelp oligosaccharide extract can significantly reduce the excessive release of NO in LPS-induced inflammation of RAW264.7 cells, downregulate the mRNA expression levels of IL-1β and TNF-α, and reduce the excessive release of ROS in HSF cells caused by UVA damage.

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