A laminarin proteoglycan purified extract, its preparation method and use

Abstract

The application provides a laminarin proteoglycan purified extract and a preparation method and application thereof. In the preparation process of the purified extract, ultrasonic-assisted water extraction and anion exchange chromatography are used for purification, the extraction efficiency is high, the purification effect is good, the effective components of laminarin proteoglycan in laminaria can be effectively enriched, the obtained extract has high purity and strong activity, the high-value utilization of laminaria resources is realized, the laminarin proteoglycan is extracted and purified as a whole and activity research is conducted, and the synergistic effect is fully exerted. In the application, through the Caenorhabditis elegans model, the zebra fish model and the human kidney epithelial cell (HK-2) model, it is verified that the laminarin proteoglycan purified extract has a significant protection effect on the damage of the organism caused by microplastics, and a new candidate drug and scientific basis are provided for the development of a microplastic damage protection drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a purified extract of kelp proteoglycan, its preparation method, and its application. Background Technology

[0002] Microplastics, as a widespread pollutant in the environment, can enter organisms through various routes such as ingestion, inhalation, and skin contact, accumulating in multiple tissues and organs including the liver, kidneys, heart, and intestines, causing a series of bodily damages. The mechanisms of damage caused by microplastics mainly include inducing oxidative stress, triggering inflammatory responses, disrupting cell membrane integrity, and inducing apoptosis. They can also affect the growth and development of organisms and cardiovascular function. Currently, there are no specific drugs to prevent and treat damage caused by microplastics, making the development of safe and effective natural product-based protective drugs a research hotspot.

[0003] Kelp is the dried thallus of plants belonging to the Laminariaceae or Alginate families. It is cold in nature and salty in taste, and enters the liver, stomach, and kidney meridians. It has the effects of softening hard masses, resolving phlegm, and promoting diuresis. Kelp contains abundant proteoglycan bioactive components. Studies have shown that kelp proteoglycans have antioxidant, anti-inflammatory, and immunomodulatory effects. However, current extraction and purification methods for kelp proteoglycans are complex, resulting in products with low purity and poor activity. Furthermore, there are no reports on the application of kelp proteoglycans in the prevention and treatment of microplastic-induced bodily damage.

[0004] Existing methods for extracting kelp polysaccharides mostly involve single-component water or alcohol extraction, and purification often employs single-column separation, resulting in low extraction rates and poor purification effects. Furthermore, current research largely focuses on individual kelp polysaccharides or proteins, neglecting the extraction, purification, and activity studies of proteoglycans as a whole, leading to low utilization efficiency of kelp resources. Therefore, developing an efficient and simple method for preparing purified kelp proteoglycan extracts and exploring their preventative and therapeutic effects against microplastic-induced damage to the body has significant clinical value and practical implications. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing purified extracts of kelp proteoglycans based on existing technologies. This method combines ultrasonic-assisted water extraction and anion exchange chromatography for purification, resulting in high extraction efficiency and good purification effect. It can effectively enrich the effective components of kelp proteoglycans in kelp, remove small molecule impurities and ineffective components, and is simple to operate, low in cost, environmentally friendly, and suitable for large-scale production.

[0006] The second objective of this invention is to provide a purified extract of kelp proteoglycan obtained by the above preparation method, which has high purity and strong activity. The purified extract contains a polysaccharide content of not less than 2.64 mg / mL, a kelp protein content of not less than 0.06 mg / mL, and a fucose molar percentage of not less than 75% in the monosaccharides that make up the polysaccharides. The glycopeptide bonds are N-linked, and it has excellent antioxidant and anti-inflammatory activities.

[0007] The third objective of this invention is to provide the application of the above-mentioned purified kelp proteoglycan extract in the preparation of drugs for the prevention or treatment of microplastic-induced bodily damage, particularly showing significant improvement effects on reduced cell viability, cell membrane damage, apoptosis, growth retardation, abnormal heart rate, increased oxidative stress levels, and upregulation of inflammatory factor expression caused by polystyrene microplastics.

[0008] The present invention also provides the application of the above-mentioned purified kelp proteoglycan extract in the preparation of drugs that improve oxidative stress and inflammatory response caused by microplastics, and in the preparation of drugs that protect against liver and kidney damage, cardiovascular damage and developmental damage caused by microplastics.

[0009] The present invention also provides a pharmaceutical composition having the above-mentioned purified extract of kelp proteoglycan as the active ingredient or main active ingredient, supplemented by a pharmaceutically acceptable carrier.

[0010] The technical solution of the present invention is as follows:

[0011] A method for preparing a purified extract of kelp proteoglycans includes the following steps:

[0012] (1) Degreasing and decolorization treatment: Kelp is mixed with anhydrous ethanol and refluxed at 160-200℃ to carry out degreasing and decolorization treatment, then dried, pulverized and set aside;

[0013] (2) Ultrasonic-assisted water extraction: The pulverized kelp was mixed with ultrapure water and extracted by ultrasonication. The resulting extract was filtered, and then the filtrate was rotary evaporated, precipitated with alcohol, centrifuged, and freeze-dried to obtain the crude extract.

[0014] (3) Anion exchange chromatography column purification: The crude extract obtained in step (2) was dissolved in ultrapure water, and the resulting mixed solution was loaded onto a DEAE FF anion exchange chromatography column for purification. The purification conditions were as follows: gradient elution was performed using mobile phase A and mobile phase B, where mobile phase A was ultrapure water and mobile phase B was 2 M NaCl aqueous solution. Gradient elution was performed according to the concentrations of NaCl aqueous solution of 0 M, 0.05 M, 0.1 M, 0.25 M, 0.5 M, 1 M and 2 M. The fraction eluted by 0.5 M NaCl aqueous solution was collected, concentrated and desalted to obtain the purified extract.

[0015] In the preparation of the purified extract of kelp proteoglycans, this invention uses kelp as raw material. First, it is extracted by defatting and decolorization and ultrasonic-assisted water extraction to obtain a crude extract. Then, the crude extract is purified by anion exchange chromatography to obtain a purified extract. The purified extract contains a polysaccharide content of not less than 2.64 mg / mL, a kelp protein content of not less than 0.06 mg / mL, and a fucose molar percentage of not less than 75% among the monosaccharides that make up the polysaccharides.

[0016] In a preferred embodiment, the purified extract contains 2.64 mg / mL of polysaccharide, 0.06 mg / mL of laminarin, and 75.29% of the monosaccharides that make up the polysaccharide.

[0017] In another preferred embodiment, the purified extract contains 76.3% polysaccharide by mass, 1.73% kelp protein by mass, and 75.29% fucose by molar percentage among the monosaccharides that make up the polysaccharide.

[0018] In this invention, in step (1), kelp is mixed with anhydrous ethanol. The ratio of kelp to anhydrous ethanol is 1:5-15 g / mL, which can be, but is not limited to, 1:5 g / mL, 1:6 g / mL, 1:7 g / mL, 1:8 g / mL, 1:9 g / mL, 1:10 g / mL, 1:11 g / mL, 1:12 g / mL, 1:13 g / mL, 1:14 g / mL or 1:15 g / mL. Preferably, the ratio of kelp to anhydrous ethanol is 1:10 g / mL.

[0019] In step (1), kelp is mixed with anhydrous ethanol and refluxed at 160-200°C. The reflux temperature can be, but is not limited to, 160°C, 170°C, 175°C, 180°C, 185°C, 190°C or 160°C. Preferably, the reflux temperature is 175°C-185°C, more preferably lower, with a reflux temperature of 180°C.

[0020] In step (1), the reflow time is 2-6 hours, which can be, but is not limited to, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. Preferably, the reflow time is 3 hours.

[0021] In step (1), the operation is repeated 2-4 times before drying, preferably 3 times.

[0022] In step (1), the drying temperature is 50℃-70℃, which can be but is not limited to 50℃, 55℃, 60℃, 65℃ or 70℃, and preferably, the drying temperature is 60℃.

[0023] In a preferred embodiment, in step (1), kelp and anhydrous ethanol are mixed at a ratio of 1:10 (g / mL), and refluxed at 180°C for 3 hours. This process is repeated 3 times to fully remove fat-soluble impurities and pigments. The treated kelp is then dried in an oven at 60°C, pulverized after drying, and set aside for later use.

[0024] In this invention, in step (2), the pulverized kelp is mixed with ultrapure water, wherein the amount of ultrapure water added is based on the kelp that has not been degreased and decolorized in step (1). The ratio of kelp to ultrapure water in step (1) is 1:5-15 g / mL, and can be, but is not limited to, 1:5 g / mL, 1:6 g / mL, 1:7 g / mL, 1:8 g / mL, 1:9 g / mL, 1:10 g / mL, 1:11 g / mL, 1:12 g / mL, 1:13 g / mL, 1:14 g / mL, or 1:15 g / mL. Preferably, the ratio of kelp to ultrapure water in step (1) is 1:8 g / mL.

[0025] In step (2), the pulverized kelp is mixed with ultrapure water and ultrasonically extracted. The ultrasonic extraction power is 100W-200W, which can be but is not limited to 100W, 110W, 120W, 130W, 140W, 150W, 160W, 170W, 180W, 190W or 200W. Preferably, the ultrasonic extraction power is 150W. The ultrasonic extraction time is 20-40 min, which can be but is not limited to 20 min, 25 min, 30 min, 35 min or 40 min. Preferably, the ultrasonic extraction time is 30 min.

[0026] In step (2), the pulverized kelp is mixed with ultrapure water and extracted by ultrasonic extraction. The extraction is repeated 2-4 times, preferably 3 times.

[0027] In step (2), the pulverized kelp is mixed with ultrapure water and extracted by ultrasonication. The extraction is repeated 2-4 times. The resulting extract is filtered and the filtrates are combined. The total filtrate is then rotary evaporated. The rotary evaporation conditions are as follows: temperature is 40-60℃, preferably 50℃, and speed is 60 rpm-70 rpm, preferably 65 rpm.

[0028] In step (2), the total filtrate is rotary evaporated to 2.5%-3.5% of the original filtrate volume. The filtrate volume can be, but is not limited to, 2.5%, 2.55%, 2.6%, 2.65%, 2.7%, 2.75%, 2.8%, 2.85%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, or 3.5%. Preferably, the total filtrate is rotary evaporated to 2.75% of the original filtrate volume.

[0029] In step (2), the rotary evaporated liquid obtained after rotary evaporation is added to anhydrous ethanol for alcohol precipitation. During alcohol precipitation, the obtained rotary evaporated liquid is added to 2-6 times the amount of anhydrous ethanol. It is possible, but not limited to, adding 2 times, 3 times, 4 times, 5 times or 6 times the amount of anhydrous ethanol. Preferably, 4 times the amount of anhydrous ethanol is added for alcohol precipitation.

[0030] In step (2), the conditions for alcohol precipitation are as follows: temperature is 20-30℃, time is 8-16 hours, which can be but is not limited to 8 hours, 10 hours, 12 hours, 14 hours or 16 hours, preferably 12 hours.

[0031] In step (2), after alcohol precipitation, the resulting alcohol precipitate is centrifuged and freeze-dried to obtain a crude extract. The centrifugation conditions are as follows: the rotation speed is 2000 rpm-4000 rpm, preferably 3000 rpm, and the time is 4 min-6 min, preferably 5 min.

[0032] In a preferred embodiment, in step (2), the pulverized kelp is mixed with ultrapure water (the ratio of kelp to ultrapure water in step (1) is 1:8 g / mL), the ultrasonic extraction power is 150W, the ultrasonic extraction time is 30 min, and the extraction is repeated 3 times to improve the extraction rate of effective components. After the obtained extract is filtered, the filtrates are combined and rotary evaporated at 50℃ and 65 rpm to 2.5%-3.5% (preferably 2.75%) of the original filtrate volume. Then, 4 times the amount of anhydrous ethanol is added to the obtained rotary evaporated liquid, and alcohol precipitation is carried out at 20-30℃ for 12 hours. The obtained alcohol precipitation liquid is centrifuged at 3000 rpm for 5 min and the supernatant is discarded. After the obtained precipitate is freeze-dried, the crude extract is obtained.

[0033] For the purposes of this invention, in step (3), the anion exchange column is a DEAE FF anion exchange column. The specific operation is as follows: the crude extract obtained in step (2) is dissolved in ultrapure water, and the resulting mixed solution is loaded onto a DEAE FF anion exchange column and purified using an AKTA PURE protein purifier. The purification conditions are as follows: gradient elution is performed using mobile phase A and mobile phase B, with the flow rate controlled at 1.0 mL / min-2.0 mL / min, preferably 1.5 mL / min; the upper limit of pressure is 0.2 MPa-0.5 MPa, preferably 0.3 MPa; and the ultraviolet absorption wavelength is 270 nm-290 nm, preferably 280 nm. In this process, mobile phase A was ultrapure water, and mobile phase B was a 2 M NaCl aqueous solution. Gradient elution was performed with NaCl aqueous solution concentrations of 0 M, 0.05 M, 0.1 M, 0.25 M, 0.5 M, 1 M, and 2 M, corresponding to the proportions of phase B as 0%, 2.5%, 5%, 12.5%, 25%, 50%, and 100%. The fraction eluted with the 0.5 M NaCl aqueous solution was collected, concentrated, and desalted to obtain the purified extract.

[0034] The method for preparing purified extract of kelp proteoglycan provided by this invention includes the following more detailed steps:

[0035] (1) Degreasing and decolorization treatment: Kelp and anhydrous ethanol are mixed at a ratio of 1:10 (g / mL), refluxed at 180℃ for 3 hours, and the operation is repeated 3 times. The mixture is then dried at 60℃, pulverized, and set aside for later use.

[0036] (2) Ultrasonic-assisted water extraction: The pulverized kelp was mixed with ultrapure water (the ratio of kelp to ultrapure water in step (1) was 1:8 g / mL), and ultrasonic extraction was performed. The ultrasonic extraction power was 150W and the ultrasonic extraction time was 30min. The extraction was repeated 3 times. The resulting extract was filtered and the filtrates were combined. The filtrates were rotary evaporated at 50℃ and 65rpm to 2.75% of the original filtrate volume. The resulting rotary evaporated liquid was then added to 4 times the amount of anhydrous ethanol and precipitated at 20-30℃ for 12 hours. The precipitate was centrifuged at 3000rpm for 5min and the supernatant was discarded. The precipitate was freeze-dried to obtain the crude extract.

[0037] (3) Anion exchange chromatography column purification: The crude extract obtained in step (2) was dissolved in ultrapure water, and the resulting mixed solution was loaded onto a DEAE FF anion exchange chromatography column for purification. The purification conditions were as follows: gradient elution was performed using mobile phase A and mobile phase B, with the flow rate controlled at 1.0 mL / min-2.0 mL / min, preferably 1.5 mL / min; the upper limit of pressure was 0.2 MPa-0.5 MPa, preferably 0.3 MPa; and the ultraviolet absorption wavelength was 270 nm-290 nm, preferably 280 nm. Among them, mobile phase A was ultrapure water, and mobile phase B was 2 M NaCl aqueous solution. Gradient elution was performed according to the concentrations of NaCl aqueous solution of 0 M, 0.05 M, 0.1 M, 0.25 M, 0.5 M, 1 M and 2 M, corresponding to the proportions of phase B of 0, 2.5%, 5%, 12.5%, 25%, 50% and 100%; the fraction eluted by 0.5 M NaCl aqueous solution was collected, concentrated and desalted to obtain the purified extract.

[0038] The purified kelp proteoglycan extract provided by this invention can be used in the preparation of drugs to prevent or treat microplastic-induced damage to the body, wherein the microplastic can be polystyrene microplastics. In particular, it has a significant improving effect on reduced cell viability, cell membrane damage, apoptosis, growth retardation, abnormal heart rate, increased oxidative stress levels, and upregulation of inflammatory factor expression caused by polystyrene microplastics.

[0039] The purified extract of kelp proteoglycan provided by this invention can be used in the preparation of drugs that improve oxidative stress and inflammatory responses caused by microplastics, wherein the microplastics can be polystyrene microplastics.

[0040] The purified extract of kelp proteoglycan provided by this invention can be used in the preparation of drugs to protect against liver and kidney damage, cardiovascular damage, and developmental damage caused by microplastics, wherein the microplastics can be polystyrene microplastics.

[0041] This invention also provides a pharmaceutical composition using the above-mentioned purified extract of kelp proteoglycan as the active ingredient or main active ingredient, supplemented by a pharmaceutically acceptable carrier. This pharmaceutical composition can be a liquid, solid, or semi-solid dosage form, particularly an injection, tablet, granule, powder, capsule, or oral liquid. Pharmaceutically acceptable excipients play a crucial role in the development of pharmaceutical technology; excipients constitute a large portion of many formulations, thus their properties largely determine the properties of the formulation. Excellent excipients can enhance the stability of the active pharmaceutical ingredient (API), prolong its shelf life, regulate the release rate of the API in vivo and in vitro, and alter drug absorption in vivo, increasing bioavailability. No specific type of excipient is limited.

[0042] The advantages of using the technical solution of this invention are as follows:

[0043] The present invention provides a method for preparing purified kelp proteoglycan extract, which combines ultrasound-assisted water extraction and anion exchange chromatography for purification. This method achieves high extraction efficiency and good purification results, effectively enriching the effective components of kelp proteoglycan while removing small molecule impurities and ineffective components. Furthermore, the method is simple to operate, low in cost, and environmentally friendly, making it suitable for large-scale production. This invention realizes the high-value utilization of kelp resources by extracting, purifying, and studying the activity of kelp proteoglycan as a whole, fully leveraging its synergistic effects.

[0044] The kelp proteoglycan purified extract obtained by the preparation method of the present invention has high purity and strong activity. The purified extract contains a polysaccharide content of not less than 2.64 mg / mL, a kelp protein content of not less than 0.06 mg / mL, and a fucose molar ratio of not less than 75% in the monosaccharides that make up the polysaccharides. The glycopeptide bonds are N-linked, and it has excellent antioxidant and anti-inflammatory activities.

[0045] This invention validated the significant protective effect of purified kelp proteoglycan extract against microplastic-induced damage using Caenorhabditis elegans, zebrafish, and human kidney epithelial cell (HK-2) models. It can significantly improve cell viability after microplastic exposure, reduce cell membrane damage, inhibit apoptosis, improve symptoms such as growth retardation and abnormal heart rate, reduce oxidative stress levels, and alleviate inflammatory responses, providing new candidate drugs and scientific basis for the development of drugs to protect against microplastic damage. Attached Figure Description

[0046] Figure 1 This is a DEAE FF column chromatography elution curve of the crude kelp proteoglycan extract in this invention;

[0047] Figure 2 This is the Fourier transform infrared spectrum of the purified extract of kelp proteoglycan in this invention;

[0048] Figure 3 This is a 200-400 nm wavelength scan of the purified extract of kelp proteoglycan in this invention;

[0049] Figure 4 This is a graph showing the effect of the purified extract of kelp proteoglycan in this invention on the survival rate of microplastic-exposed Caenorhabditis elegans.

[0050] Figure 5 This is a graph showing the effect of the purified extract of kelp proteoglycan in this invention on the body wiggling frequency of microplastic-exposed Caenorhabditis elegans.

[0051] Figure 6The figure shows the effect of the purified extract of kelp proteoglycan in this invention on the levels of ROS and lipofuscin in microplastic-exposed Caenorhabditis elegans.

[0052] Figure 7 The figure shows the effect of the purified extract of kelp proteoglycan in this invention on the heart rate of zebrafish exposed to microplastics.

[0053] Figure 8 This is a graph showing the effect of the purified extract of kelp proteoglycan in this invention on the SOD enzyme activity of microplastic-treated zebrafish.

[0054] Figure 9 The figure shows the effect of the purified extract of kelp proteoglycan in this invention on the viability of microplastic-exposed HK-2 cells.

[0055] Figure 10 This is a graph showing the effect of the purified extract of kelp proteoglycan in this invention on the LDH level of HK-2 cells exposed to microplastics.

[0056] Figure 11 This figure shows the effect of the purified extract of kelp proteoglycan in this invention on the apoptosis rate of microplastic-exposed HK-2 cells. Detailed Implementation

[0057] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0059] The present invention will be further described below with reference to specific embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0060] Example 1: Preparation method of purified extract of kelp proteoglycan

[0061] (1) Degreasing and decolorizing treatment: Weigh 100 g of kelp, add 1000 mL of anhydrous ethanol and mix. Reflux at 180℃ for 3 hours. Repeat the operation 3 times. Then spread the degreased and decolorized kelp on tin foil, dry it in an oven at 60℃, crush it and set it aside.

[0062] (2) Ultrasonic-assisted water extraction: 800 mL of ultrapure water was added to the pulverized kelp and mixed. Ultrasonic extraction was performed with an ultrasonic extraction power of 150 W and an ultrasonic extraction time of 30 min. The extraction was repeated 3 times. The resulting extracts were filtered and combined to obtain 2400 mL of filtrate. The filtrate was rotary evaporated at 50℃ and 65 rpm to 66 mL (2.75% of the original filtrate volume) to obtain the rotary evaporated liquid. Four times the amount of anhydrous ethanol was added to the rotary evaporated liquid and ethanol precipitation was performed at 20-30℃ for 12 hours. The precipitate was centrifuged at 3000 rpm for 5 min and the supernatant was discarded. The precipitate was freeze-dried to obtain 8.462 g of crude extract freeze-dried powder.

[0063] (3) Anion exchange chromatography column purification: The lyophilized crude extract obtained in step (2) was dissolved in 40 mL of ultrapure water. The resulting mixed solution was loaded onto a DEAE FF anion exchange chromatography column and purified using an AKTA PURE protein purifier. The purification conditions were as follows: gradient elution was performed using mobile phase A and mobile phase B, with a flow rate of 1.5 mL / min; the upper limit of pressure was 0.3 MPa; and the ultraviolet absorption wavelength was 280 nm. Among them, mobile phase A was ultrapure water, and mobile phase B was 2 M NaCl aqueous solution. Gradient elution was performed according to the concentrations of NaCl aqueous solution of 0 M, 0.05 M, 0.1 M, 0.25 M, 0.5 M, 1 M and 2 M, corresponding to the proportions of phase B of 0, 2.5%, 5%, 12.5%, 25%, 50% and 100%; the fraction eluted by 0.5 M NaCl aqueous solution was collected, concentrated and desalted to obtain the purified extract of kelp proteoglycan ( Figure 1 ).

[0064] (4) Component analysis:

[0065] The polysaccharide content in the purified extract was determined to be 2.64 mg / mL, with a mass content of 76.3%, using the phenol-sulfuric acid method. The specific operating steps are as follows:

[0066] 1) 0-0.3 mg / mL standard curve: Pipettes 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, and 60 μL of 1 mg / mL glucose standard solution into 1.5 mL centrifuge tubes, adds water to a final volume of 200 μL, adds 100 μL of 6% phenol solution to each tube, shakes until homogeneous, quickly adds 500 μL of concentrated sulfuric acid, shakes until homogeneous, incubates for 10 min, then incubates in an 80°C water bath for 15 min. After cooling to room temperature, pipette 200 μL into a 96-well plate and determine A. 490 .

[0067] 2) Take 200 μL of kelp proteoglycan sample, dilute it 10 times, and determine A according to the above steps. 490 The concentration and content of polysaccharides were calculated based on the standard curve.

[0068] The Bradford method determined the kelp protein content to be 0.06 mg / mL, with a mass content of 1.73%. The specific steps are as follows: Prepare protein standard solutions of 0, 0.125, 0.25, 0.5, 0.75, 1, and 1.5 mg / mL, mixing thoroughly after each dilution. Add 5 μL of each protein standard solution to a 96-well plate; add 5 μL of kelp proteoglycan sample solution to a 96-well plate; add 250 μL of G250 staining solution to each well; and measure A using a microplate reader. 595 The protein concentration in the sample is calculated based on the standard curve.

[0069] The composition of monosaccharides in the polysaccharides was analyzed by ion chromatography as follows: fucose:rhamnose:galactose:glucose:xylose:mannose:glucuronic acid = 75.29:3.82:8.50:0.83:0.85:4.69:6.02, with fucose accounting for 75.29% of the total content. The specific operating steps were as follows: Standard solutions and sample solutions of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid were prepared. A Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA) was used to analyze the monosaccharide components using a pulsed amperometric (PAD) detector. A Dionex™ CarboPac™ PA20 (150 × 3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 mL / min; column temperature 30℃; elution gradient: 0 min A / B / C (95:5:0, V / V), 26 min A / B / C (85:5:10, V / V), 42 min A / B / C (85:5:10, V / V), 42.1 min A / B / C (60:0:40, V / V), 52 min A / B / C (60:40:0, V / V), 52.1 min A / B / C (95:5:0, V / V), 60 min A / B / C (95:5:0, V / V).

[0070] Fourier transform infrared spectroscopy revealed the presence of characteristic functional groups such as OH, NH, and CH. Figure 2 The specific operating steps are as follows: Take an appropriate amount of purified kelp proteoglycan into a dry mortar, add potassium bromide powder and grind until it becomes a fine powder like flour. Transfer the powder to a tablet press and compress it into a tablet. Place the sample film into a Fourier transform infrared spectroscopy scanner for scanning. The scanning temperature is 15-25℃ and the scanning range is 400-4000 cm⁻¹. -1 The number of scans was 64.

[0071] β-elimination reaction indicates that the glycopeptide bond is N-linked. Figure 3The specific operating steps are as follows: Weigh 10 mg of kelp proteoglycan sample into a 1.5 mL centrifuge tube, add 300 μL of 0.3 mol / L NaOH and 300 μL of 1 mol / L NaBH4 to dissolve, and incubate in a water bath at 45℃ for 2 h to obtain the solution after elimination. Similarly, weigh 10 mg of kelp proteoglycan sample, add 600 μL of water to dissolve, and obtain the solution before elimination. At the same time, measure the A of the above two solutions. 240nm .

[0072] Example 2: Detection of the protective effect of purified kelp proteoglycan extract against microplastic-exposed Caenorhabditis elegans.

[0073] 1. Experimental groups: A blank group, a PS-MPs (50nm polystyrene microplastics, 100 mg / L) exposure group, and a combined exposure group of low, medium and high concentrations (0.5 mg / mL, 1 mg / mL, 1.5 mg / mL) of purified kelp proteoglycan extract were set up, with 3 biological replicates for each group.

[0074] 2. Nematode culture and treatment: Caenorhabditis elegans synchronized to the L3 stage was placed in NGM medium of each experimental group and cultured at a constant temperature of 20±0.5℃. The nematodes were observed and fed daily. After 10 days of treatment, relevant indicators were detected.

[0075] 3. Detection indicators:

[0076] (1) Survival rate: The number of surviving nematodes was recorded daily, and the survival rate was calculated. The results showed that the survival rate of nematodes in the poisoned group was significantly reduced to 78%, while the survival rate of the combined poisoning groups of 0.5 mg / mL, 1 mg / mL, and 1.5 mg / mL recovered to 92%-94%, which was not significantly different from the control group. Figure 4 ).

[0077] (2) Body wiggling frequency: The number of times the nematode's body bent within 30 seconds was recorded under a stereomicroscope and converted into wiggling frequency per minute. The results showed that the wiggling frequency was significantly reduced in the poisoned group, while the wiggling frequency in the combined poisoning groups of 0.5 mg / mL and 1.5 mg / mL significantly increased, returning to the level of the blank group. Figure 5 ).

[0078] (3) ROS and lipofuscin levels: ROS was detected using the DCFH-DA fluorescent probe, and lipofuscin autofluorescence was detected using the DAPI channel. Results showed that ROS and lipofuscin levels were significantly increased in the poisoned group, while the combined poisoning group significantly reduced ROS and lipofuscin levels. The 0.5 mg / mL group showed the best ROS scavenging effect, and the 1 mg / mL group showed the best inhibitory effect on lipofuscin. Figure 6 ).

[0079] Example 3: Detection of the protective effect of purified kelp proteoglycan extract on microplastic-treated zebrafish.

[0080] 1. Experimental groups: A blank group, a PS-MPs (20 mg / L) treatment group, and a combined treatment group of purified kelp proteoglycan extract (50 mg / L, 100 mg / L, 200 mg / L) were set up. Each group was biologically replicated 3 times, with 30 zebrafish embryos per well.

[0081] 2. Zebrafish culture and treatment: AB wild-type zebrafish embryos with a growth rate of 6 hpf were selected and placed in the culture medium of each experimental group. They were cultured at a constant temperature of 28.5±0.5℃, with the medium changed daily. After reaching 120 hpf, relevant indicators were detected.

[0082] 3. Detection indicators:

[0083] (1) Heart rate: The number of ventricular beats in zebrafish over 30 seconds was counted under an inverted fluorescence microscope, and the heart rate per minute was calculated. The results showed that the heart rate in the poisoned group decreased significantly to about 100 bpm, while the heart rate in the combined poisoning groups of 100 mg / L and 200 mg / L increased back to 120 bpm and 130 bpm, respectively. Figure 7 ).

[0084] (2) Body length and interocular distance: The length from the snout to the end of the caudal fin and the interocular distance of zebrafish were measured under a microscope. The results showed that the body length and interocular distance of the poisoned group were significantly reduced, while the combined poisoned group could significantly recover. The 100 mg / L group had the best recovery effect on interocular distance, and the 200 mg / L group had the best recovery effect on body length.

[0085] (3) SOD enzyme activity: The SOD enzyme activity in zebrafish was detected using a kit; the results showed that the SOD activity in the poisoned group decreased significantly to 30%, while the SOD activity in the 50 mg / L and 100 mg / L combined poisoned groups recovered to 45% and 50%, respectively. Figure 8 ).

[0086] Example 4: Detection of the protective effect of purified kelp proteoglycan extract on microplastic-exposed HK-2 cells.

[0087] 1. Experimental groups: A blank group, a PS-MPs (2 mg / mL, 2.5 mg / mL, 4 mg / mL) exposure group, and a kelp proteoglycan purified extract (2 mg / mL, 6 mg / mL, 10 mg / mL) combined exposure group were set up.

[0088] 2. Cell culture and treatment: HK-2 cells were seeded in culture plates and cultured until they adhered. Then, culture medium for each experimental group was added and the cells were cultured in a 37°C, 5% CO2 incubator. After 24 h of exposure to the virus, the cells were administered the drug for 24 h, and relevant indicators were detected.

[0089] 3. Detection indicators:

[0090] (1) Cell viability: Cell viability was detected by CCK8 assay; the results showed that the cell viability of the 2 mg / mL treatment group decreased to 55%, while the cell viability of the 6 mg / mL and 10 mg / mL combined treatment groups recovered to 125% and 110%, respectively; the cell viability of the 4 mg / mL treatment group decreased to 5%, while the cell viability of the 6 mg / mL and 10 mg / mL combined treatment groups recovered to 40% and 45%, respectively. Figure 9 ).

[0091] (2) LDH level: The kit was used to detect LDH activity in the cell culture medium; the results showed that the LDH level in the poisoned group was significantly increased, while the LDH level in the combined poisoned group was significantly decreased, and cell membrane damage was alleviated. Figure 10 ).

[0092] (3) Apoptosis rate: Apoptosis rate was detected by Annexin V-FITC / PI double staining flow cytometry; the results showed that the apoptosis rate in the 2 mg / mL treatment group increased to 25%, while the apoptosis rate in the 6 mg / mL combined treatment group decreased to 16%; the apoptosis rate in the 2.5 mg / mL treatment group increased to 26%, while the apoptosis rate in the 6 mg / mL combined treatment group decreased to 18%. Figure 11 ).

[0093] This invention aims to provide a purified extract of kelp proteoglycans and its preparation method, realizing the high-value utilization of kelp resources. The preparation method is simple to operate, low in cost, and environmentally friendly. The obtained extract has high purity and strong activity. Furthermore, its significant protective effect against microplastic-induced damage to the body has been verified through nematodes, zebrafish, and cell models, providing a new direction and scientific basis for the development of drugs to protect against microplastic damage.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a purified extract of kelp proteoglycan, characterized in that, Includes the following steps: (1) Degreasing and decolorization treatment: Kelp is mixed with anhydrous ethanol and refluxed at 160-200℃ to carry out degreasing and decolorization treatment, then dried, pulverized and set aside; (2) Ultrasonic-assisted water extraction: The pulverized kelp was mixed with ultrapure water and extracted by ultrasonication. The resulting extract was filtered, and then the filtrate was rotary evaporated, precipitated with alcohol, centrifuged, and freeze-dried to obtain the crude extract. (3) Anion exchange chromatography column purification: The crude extract obtained in step (2) was dissolved in ultrapure water, and the resulting mixed solution was loaded onto a DEAE FF anion exchange chromatography column for purification. The purification conditions were as follows: gradient elution was performed using mobile phase A and mobile phase B, where mobile phase A was ultrapure water and mobile phase B was 2 M NaCl aqueous solution. Gradient elution was performed according to the concentrations of NaCl aqueous solution of 0 M, 0.05 M, 0.1 M, 0.25 M, 0.5 M, 1 M and 2 M. The fraction eluted by 0.5 M NaCl aqueous solution was collected, concentrated and desalted to obtain the purified extract. The purified extract contains a polysaccharide content of not less than 2.64 mg / mL, a laminarin content of not less than 0.06 mg / mL, and fucose molar percentage of the monosaccharides constituting the polysaccharides is not less than 75%. In step (1), the ratio of kelp to anhydrous ethanol is 1:5-15 g / mL; the reflux temperature is 175℃-185℃; the reflux time is 2-6 hours; the operation is repeated 2-4 times before drying; the drying temperature is 50℃-70℃. In step (2), the ratio of kelp to ultrapure water in step (1) is 1:5-15 g / mL; the ultrasonic extraction power is 100W-200W; the ultrasonic extraction time is 20-40 min; the extraction is repeated 2-4 times; the rotary evaporation conditions are as follows: temperature is 40-60℃, speed is 60 rpm-70 rpm; the filtrate is rotary evaporated to 2.5%-3.5% of the original filtrate volume; during alcohol precipitation, the obtained rotary evaporated liquid is added to 2-6 times the amount of anhydrous ethanol; the alcohol precipitation conditions are as follows: temperature is 20-30℃, time is 8-16 hours; the centrifugation conditions are as follows: speed is 2000 rpm-4000 rpm, time is 4 min-6 min; In step (3), the flow rate is 1.0 mL / min-2.0 mL / min; the upper limit of pressure is 0.2 Mpa-0.5 Mpa; and the ultraviolet absorption wavelength is 270 nm-290 nm.

2. The method for preparing the purified extract of kelp proteoglycan according to claim 1, characterized in that, The purified extract contained 2.64 mg / mL of polysaccharide and 0.06 mg / mL of laminarin. Fucose accounted for 75.29% of the monosaccharides that made up the polysaccharide.

3. The method for preparing the purified extract of kelp proteoglycan according to claim 1, characterized in that, The purified extract contained 76.3% polysaccharide by mass, 1.73% kelp protein by mass, and 75.29% fucose by molar percentage among the monosaccharides that make up the polysaccharide.

4. The method for preparing the purified extract of kelp proteoglycan according to claim 1, characterized in that, In step (1), the ratio of kelp to anhydrous ethanol is 1:10 g / mL; the reflux temperature is 180℃; the reflux time is 3 hours; the operation is repeated 3 times before drying; the drying temperature is 60℃. In step (2), the ratio of kelp to ultrapure water in step (1) is 1:8 g / mL; the ultrasonic extraction power is 150W; the ultrasonic extraction time is 30 min; the extraction is repeated 3 times; the rotary evaporation conditions are as follows: temperature is 50℃, speed is 65 rpm; the filtrate is rotary evaporated to 2.75% of the original filtrate volume; during alcohol precipitation, the obtained rotary evaporated liquid is added to 4 times the amount of anhydrous ethanol; the alcohol precipitation conditions are as follows: temperature is 20-30℃, time is 12 hours; the centrifugation conditions are as follows: speed is 3000 rpm, time is 5 min; In step (3), the flow rate is 1.5 mL / min; the upper limit of pressure is 0.3 MPa; and the ultraviolet absorption wavelength is 280 nm.

5. A purified extract of kelp proteoglycans, characterized in that, The purified extract is obtained by the preparation method according to any one of claims 1-4.

6. The use of the purified kelp proteoglycan extract according to claim 5 in the preparation of a medicament for the prevention or treatment of microplastic-induced bodily damage.

7. The application according to claim 6, characterized in that, The microplastic is a polystyrene microplastic; the damage to the body includes reduced cell viability, cell membrane damage, apoptosis, growth retardation, abnormal heart rate, increased oxidative stress, and upregulation of inflammatory factor expression.

8. The use of the purified kelp proteoglycan extract according to claim 5 in the preparation of a medicament for improving oxidative stress and inflammatory response caused by microplastics.

9. The use of the purified kelp proteoglycan extract according to claim 5 in the preparation of a medicament for protecting against liver and kidney damage, cardiovascular damage, and developmental damage caused by microplastics.

10. A pharmaceutical composition, characterized in that, It uses the purified extract of kelp proteoglycan as described in claim 5 as the active ingredient or main active ingredient, supplemented by a pharmaceutically acceptable carrier.

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