Low molecular weight enteromorpha polysaccharide with antioxidant activity, and preparation method and application thereof

Abstract

The application discloses a low-molecular-weight Enteromorpha polysaccharide with antioxidant activity and a preparation method and application thereof. c The method for preparing the low-molecular-weight Enteromorpha polysaccharide by degrading the prepared Enteromorpha polysaccharide by microwave-assisted H2O2 / V c The method for preparing the low-molecular-weight Enteromorpha polysaccharide by degrading the prepared Enteromorpha polysaccharide by microwave-assisted H2O2 / V c The method for preparing the low-molecular-weight Enteromorpha polysaccharide by degrading the prepared Enteromorpha polysaccharide by microwave-assisted H2O2 / V c The total antioxidant capacity of the low-molecular-weight Enteromorpha polysaccharide LEP IIIa obtained by separation and purification is increased from 315.2 U / mL before degradation to 661.8 U / mL, and the iron ion reduction capacity is increased from 51.3% before degradation to 83.4%; and the prepared Enteromorpha polysaccharide LEP IIIa has a good effect of relieving exercise-induced fatigue.

Description

TECHNICAL FIELD

[0001] The present application relates to the processing application field of marine green algae, and particularly relates to low-molecular-weight Enteromorpha polysaccharide with antioxidant activity, a preparation method and application thereof. BACKGROUND

[0002] In recent years, with the in-depth study of the structure-activity relationship of polysaccharide macromolecular compounds, various biological activities of polysaccharides have been reported, such as antiviral, antioxidant, hypoglycemic, hypolipidemic, anti-fatigue and the like. Nowadays, the research on polysaccharides has been extended from terrestrial organisms to marine algae. Marine polysaccharides have become an important source of bioactive polysaccharides in the fields of biological pharmaceuticals, health food development and the like due to their strong biological activities.

[0003] Enteromorpha prolifera, commonly known as moss strips, belongs to the Enteromorpha prolifera of the Chlorophyta Enteromorpha, and is widely grown in the eastern coastal areas of Shandong Peninsula, Jiangsu, Zhejiang and the like. In recent years, the reproduction of Enteromorpha prolifera has also been found in the sea area of Yingkou, Liaoning in the northeast, and is an important edible and medicinal economic seaweed. It is recorded in the herbal books that it has the effects of clearing heat and detoxifying, promoting water and reducing swelling, and the like. It is rich in carbohydrates, minerals, crude fiber and a small amount of protein and fat, and Enteromorpha polysaccharide is the main active ingredient of its cell wall. In recent years, Enteromorpha polysaccharide has been widely reported to have anticancer, antioxidant, hypolipidemic, hypoglycemic and immunomodulatory activities, and the like.

[0004] The antioxidant activity of polysaccharide is greatly influenced by the molecular weight, branching structure and spatial structure. At present, low-molecular-weight Enteromorpha polysaccharide can be obtained through enzymatic hydrolysis, acid hydrolysis and oxidative degradation and the like. Enzymatic hydrolysis has high specificity for the cleavage of glycosidic bonds in polysaccharide chains, and cannot be used for industrial development. Acid hydrolysis and oxidative degradation need high concentration or long reaction time, which leads to the change of sugar unit structure and the destruction of necessary life activity groups. At present, the microwave-assisted extraction technology has been successfully used for the extraction of bioactive compounds of seaweed polysaccharides.

[0005] Chinese patent application number CN201510040162.2 discloses a method for microwave extraction of Enteromorpha polysaccharide, (1) fresh Enteromorpha is rinsed with clean water to remove impurities; (2) the Enteromorpha is thinly laid in a blast drying oven and dried at 30-40℃ to a moisture content of 6-10% or is naturally air-dried to a moisture content of 6-10%; (3) the Enteromorpha is crushed and sieved; the crushed Enteromorpha has a particle size of 40-60 mesh; (4) 5g of the Enteromorpha powder is weighed, deionized water is added at a liquid-to-material ratio of 1:30, and under the conditions of a power of 500W and a temperature of 90℃, the Enteromorpha is extracted for 15min, a total of 2 times, and the extraction solutions of the two times are combined; (5) the extraction solution is centrifuged to remove the precipitate, and the supernatant is obtained; (6) the supernatant is concentrated, anhydrous ethanol is added to a concentration of 80% by mass, and the mixture is placed at 4℃ for 24h, centrifuged at 4000rpm for 15min, and the precipitate is taken, and after freeze-drying, Enteromorpha polysaccharide is obtained. The method mainly improves the extraction efficiency of Enteromorpha polysaccharide, but cannot reduce the molecular weight of the Enteromorpha polysaccharide.

[0006] Chinese patent application number CN201510955842.7 discloses a method for improving the biological activity of Enteromorpha polysaccharide, crude Enteromorpha polysaccharide (EP) is degraded by H2O2 / V c combined method to obtain degraded Enteromorpha polysaccharide (DEP); carboxymethylation of the degraded Enteromorpha polysaccharide is carried out with chloroacetic acid as a carboxymethylation reagent to prepare carboxymethylated Enteromorpha polysaccharide (CDEP); in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), CDEP is coupled with hydroxylamine to obtain hydroxamic acidized degraded Enteromorpha polysaccharide (HCDEP). The prepared HCDEP has a low molecular weight (30-13kDa), an iron chelating capacity of 0.2-1.2mmol / g, high in-vitro antioxidant activity and antibacterial activity. Although the method can reduce the molecular weight of the Enteromorpha polysaccharide and improve the antioxidant capacity, the preparation process involves a large amount of organic reagents, is complicated, requires a long time, and the obtained degraded Enteromorpha polysaccharide components are not separated and their structures are not analyzed. Therefore, there is an urgent need to develop an efficient and environmentally friendly low-molecular-weight Enteromorpha polysaccharide with antioxidant activity, a preparation method and application thereof. SUMMARY

[0007] To solve the above technical problems, the present application provides a low-molecular-weight Enteromorpha polysaccharide with antioxidant activity, a preparation method and application thereof.

[0008] To achieve the above-mentioned purposes, the present application is implemented according to the following technical solutions:

[0009] The first object of the present application is to provide a preparation method of a low-molecular-weight Enteromorpha polysaccharide with antioxidant activity, comprising the following steps:

[0010] S1, collect fresh Enteromorpha prolifera, wash with water, dry at 60°C to constant weight, crush and pass through an 80-100 mesh sieve to obtain Enteromorpha prolifera powder;

[0011] S2, take the Enteromorpha prolifera powder, add an excess of anhydrous ethanol, reflux extract at 75°C for 1-2h, cool, filter and dry to obtain defatted and impurity-removed Enteromorpha prolifera powder;

[0012] S3, take the defatted and impurity-removed Enteromorpha prolifera powder, add 50 times the volume of deionized water, and place in a microwave reactor closed container for auxiliary extraction for 10-20min to obtain an extract;

[0013] S4, centrifuge the extract of step S3, concentrate under reduced pressure to 1 / 5 of the original volume, add 4 times the volume of 95% ethanol, and stand at 4°C overnight to obtain an alcohol precipitation solution;

[0014] S5, centrifuge the alcohol precipitation solution of step S4, dissolve the precipitate in deionized water, add papain with a concentration of 2-4%(W / V) to hydrolyze for 1h to obtain an enzymatic hydrolysate, precipitate the enzymatic hydrolysate with 4 times the volume of 95% ethanol, collect the precipitate after centrifugation to obtain Enteromorpha prolifera crude polysaccharide, dissolve the Enteromorpha prolifera crude polysaccharide in deionized water again to obtain an Enteromorpha prolifera crude polysaccharide solution, further remove protein from the Enteromorpha prolifera crude polysaccharide solution using the Sevag method, and concentrate and freeze-dry the aqueous solution to obtain white powder Enteromorpha prolifera crude polysaccharide EP;

[0015] S6, dissolve the Enteromorpha prolifera crude polysaccharide EP obtained in step S5 in 200mL deionized water, add 0.1-0.3mL of 30% H2O2 and 1-3mL of 1.0mol / L V c solution, mix, place in a microwave reactor closed container, use a magnetic stirrer to stir, and assist in degradation for 10-20min, dialyze the obtained solution in deionized water overnight, and freeze-dry to obtain low molecular weight Enteromorpha prolifera polysaccharide LEP;

[0016] S7, use a DEAE-52 chromatography column to chromatographically separate the low molecular weight Enteromorpha prolifera polysaccharide LEP: use deionized water, 0.1mol / L and 0.5mol / L NaCl as eluents to elute, a total of 3 elution peaks appear, numbered LEP I, LEP II and LEP III, collect and concentrate each sugar-containing peak component, and record as polysaccharide components LEP I, LEP II and LEP III, respectively;

[0017] S8, chromatographically separate polysaccharide component LEP III on a Sephadex G-100 gel column using deionized water as an eluent to separate two peaks, LEP IIIa and LEP IIIb, combine the eluent of the main peak of LEP IIIa, concentrate and freeze-dry to obtain white powder purified low molecular weight Enteromorpha prolifera polysaccharide LEP IIIa.

[0018] Further, in the step S2, the volume ratio of the Enteromorpha powder to the anhydrous ethanol is 1:15-25.

[0019] Further, in the step S3, the power of the microwave reactor is 500-800 W, and the temperature is 70-90℃.

[0020] Further, in the step S5, the pH is 6-8, and the temperature is 40-60℃ when the papain is hydrolyzed.

[0021] Preferably, in the step S6, the power of the microwave reactor is 400-600 W, and the temperature is 50-60℃.

[0022] The second object of the present application is to provide a low molecular weight Enteromorpha polysaccharide with antioxidant activity prepared by the above method.

[0023] The third object of the present application is to provide an application of the low molecular weight Enteromorpha polysaccharide with antioxidant activity in the preparation of a functional food or health product with antioxidant, lactic acid decomposition accelerating, lactic dehydrogenase activity improving, and exercise-induced fatigue relieving functions.

[0024] Compared with the prior art, the present application prepares a low molecular weight Enteromorpha sulfated polysaccharide by using microwave-assisted H2O2 / V c The method for preparing a low molecular weight Enteromorpha sulfated polysaccharide by degrading the prepared Enteromorpha crude polysaccharide using microwave-assisted H2O2 / V BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1Elution profile of DEAE-52 column chromatography of LEP.

[0026] Figure 2 Acetate cellulose electrophoresis profile of different components of LEP.

[0027] Figure 3 Sephadex G-100 gel column chromatography profile of LEP III.

[0028] Figure 4 Effect of microwave-assisted H2O2 / V c Effect of degradation on total antioxidant capacity of EP.

[0029] Figure 5 Effect of microwave-assisted H2O2 / V c Effect of degradation on ferrous ion reducing capacity of EP.

[0030] Figure 6 Infrared spectrum scanning profile of LEP IIIa.

[0031] Figure 7 HPLC chromatogram of monosaccharide standard (A) and LEP IIIa (B).

[0032] Figure 8 Effect of LEP IIIa on pole climbing time of mice.

[0033] Figure 9 Effect of LEP IIIa on exhaustive swimming time of mice under load

[0034] Figure 10 Effect of LEP IIIa on urea nitrogen concentration (A) and lactate dehydrogenase activity (B) in serum of mice.

[0035] Figure 11 Effect of LEP IIIa on liver glycogen (A) and muscle glycogen (B) content of mice. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with embodiments. The specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0037] The Enteromorpha prolifera sample used in the following embodiments is the Enteromorpha prolifera green tide in the coastal area of Yingkou Laoheqian, Liaoning.

[0038] Embodiment 1

[0039] (1) The Enteromorpha prolifera was washed with tap water, dried at 60°C to constant weight, and ground by a high-speed multifunctional grinder, and then sieved through a 100-mesh sieve to obtain Enteromorpha prolifera powder. A certain amount of the ground and sieved Enteromorpha prolifera powder was added with 20 times the volume of anhydrous ethanol, and refluxed at 75°C for 2 hours to remove alcohol-soluble impurities such as lipids, flavonoids, and polyphenols. After cooling, the mixture was filtered and dried to obtain defatted and impurity-removed Enteromorpha prolifera powder. 10 g of the defatted Enteromorpha prolifera powder was added with 500 mL of deionized water, and the mixture was thoroughly mixed and placed in a microwave reactor closed container for auxiliary extraction at 500 W and 80°C for 15 min.

[0040] (2) The extraction liquid in step (1) was centrifuged, concentrated under reduced pressure to 100 mL, and then added with 400 mL of 95% ethanol and placed at 4°C overnight. The mixture was centrifuged at 4000 r / min for 10 min, and the precipitate was dissolved in 200 mL of deionized water. 3% (W / V) papain was added, and the pH was adjusted to 6.5. The mixture was hydrolyzed at 50°C for 1 hour to remove protein impurities. The enzymatic hydrolysate was precipitated with 4 times the volume of 95% ethanol, and then centrifuged at 4000 r / min for 10 min. The precipitate was dissolved in 100 mL of deionized water again, and 20 mL of chloroform and 4 mL of n-butanol were added to further remove free proteins. The water phase solution was concentrated and freeze-dried to obtain white powder Enteromorpha prolifera crude polysaccharide EP.

[0041] (3) The EP extracted in step (2) was dissolved in 200 mL of deionized water, and then added with 0.2 mL of 30% H2O2 and 2 mL of 1 mol / L V c solution, respectively. After mixing, the mixture was placed in a microwave reactor closed container, stirred with a magnetic stirrer, and subjected to auxiliary degradation at 500 W and 55°C for 15 min. The obtained solution was dialyzed against deionized water overnight and freeze-dried to obtain low molecular weight Enteromorpha prolifera polysaccharide LEP with a molecular weight of 23.8 kDa.

[0042] Example 2

[0043] (1) The Enteromorpha prolifera was washed with tap water, dried at 60°C to constant weight, and ground by a high-speed multifunctional grinder, and then sieved through a 100-mesh sieve to obtain Enteromorpha prolifera powder. A certain amount of the ground and sieved Enteromorpha prolifera powder was added with 20 times the volume of anhydrous ethanol, and refluxed at 75°C for 2 hours to remove alcohol-soluble impurities such as lipids, flavonoids, and polyphenols. After cooling, the mixture was filtered and dried to obtain defatted and impurity-removed Enteromorpha prolifera powder. 10 g of the defatted Enteromorpha prolifera powder was added with 500 mL of deionized water, and the mixture was thoroughly mixed and placed in a microwave reactor closed container for auxiliary extraction at 500 W and 80°C for 15 min.

[0044] (2) The extraction solution in step (1) was centrifuged, concentrated to 100 mL under reduced pressure, 400 mL of 95% ethanol was added, and it was placed at 4°C overnight; centrifuged at 4000 r / min for 10 min, the precipitate was dissolved in 200 mL of deionized water, 2% (W / V) papain was added, the pH was adjusted to 6, and it was hydrolyzed at 60°C for 1 h to remove protein impurities, the enzymatic solution was precipitated with 4 times the volume of 95% ethanol, centrifuged at 4000 r / min for 10 min, the precipitate was dissolved in 100 mL of deionized water again, 15 mL of chloroform and 3 mL of n-butanol were added to further remove free proteins, the water phase solution was concentrated and freeze-dried to obtain white powder-like Enteromorpha prolifera crude polysaccharide EP.

[0045] (3) The EP extracted in step (2) was dissolved in 200 mL of deionized water, 0.1 mL of 30% H2O2 and 1 mL of 1 mol / L V c solution were added respectively, after mixing, it was placed in a microwave reactor closed container, stirred with a magnetic stirrer, auxiliary degradation at 600 W power for 10 min at 50°C, the obtained solution was dialyzed with deionized water overnight, and freeze-dried to obtain low molecular weight Enteromorpha prolifera polysaccharide LEP with a molecular weight of 23.4 kDa.

[0046] Example 3

[0047] (1) The Enteromorpha prolifera was washed with tap water, dried at 60°C to constant weight, and crushed with a high-speed multifunctional crusher, and then sieved through a 90-mesh sieve to obtain Enteromorpha prolifera powder; a certain amount of the crushed and sieved Enteromorpha prolifera powder was added with 25 times the volume of anhydrous ethanol, and refluxed at 75°C for 1 h to remove alcohol-soluble impurities such as lipids, flavones, and polyphenols, and then filtered and dried after cooling to obtain defatted and impurity-removed Enteromorpha prolifera powder. 10 g of the defatted Enteromorpha prolifera powder was added with 500 mL of deionized water, mixed thoroughly, and placed in a microwave reactor closed container, and auxiliary extraction was performed at 600 W power for 20 min at 90°C.

[0048] (2) The extraction solution in step (1) was centrifuged, concentrated to 100 mL under reduced pressure, 400 mL of 95% ethanol was added, and it was placed at 4°C overnight; centrifuged at 4000 r / min for 10 min, the precipitate was dissolved in 200 mL of deionized water, 2% (W / V) papain was added, the pH was adjusted to 6, and it was hydrolyzed at 60°C for 1 h to remove protein impurities, the enzymatic solution was precipitated with 4 times the volume of 95% ethanol, centrifuged at 4000 r / min for 10 min, the precipitate was dissolved in 100 mL of deionized water again, 15 mL of chloroform and 3 mL of n-butanol were added to further remove free proteins, the water phase solution was concentrated and freeze-dried to obtain white powder-like Enteromorpha prolifera crude polysaccharide EP.

[0049] (3) The EP extracted in step (2) was dissolved in 200 mL of deionized water, 0.3 mL of 30% H2O2 and 3 mL of 1 mol / L V cThe solution was mixed and then placed in a microwave reactor sealed container, stirred using a magnetic stirrer, 400W power, 60°C auxiliary degradation for 20min, the obtained solution was dialyzed overnight with deionized water, and freeze-dried to obtain low molecular weight Enteromorpha polysaccharide LEP with a molecular weight of 24.2kDa.

[0050] Taking Example 1 as an example, the yield of low molecular weight Enteromorpha polysaccharide LEP was calculated, and the polysaccharide yield (X) calculation formula was as follows:

[0051]

[0052] After calculation, it was found that after the pretreatment of removing impurities, polysaccharide extraction, enzyme combined Sevag method for removing protein, microwave assisted H2O2 / V c Degradation, dialysis, concentration and freeze-drying, the texture was relatively fluffy, which was convenient for subsequent dissolution operation. The obtained white low molecular weight Enteromorpha polysaccharide was 1.17±0.05g, the polysaccharide yield was 11.7±0.5%, and the protein content was 0.65±0.3%.

[0053] Example 4

[0054] (1) DEAE-52 chromatography column was used for chromatographic separation of low molecular weight Enteromorpha polysaccharide LEP prepared in Example 1. 1g of low molecular weight Enteromorpha polysaccharide LEP was dissolved in 20mL of deionized water and injected into a DEAE-52 chromatography column equilibrated with deionized water. After loading, the column was eluted with deionized water at a flow rate of 6mL / 5min / tube, and then eluted with 0.1, 0.5, 1.0mol / L NaCl solution respectively, and the polysaccharide components were collected.

[0055] (2) Phenol-sulfuric acid method combined with cellulose acetate membrane electrophoresis was used for detection, and the non-single component was concentrated, and further separated by Sephadex G-100 gel chromatography with deionized water elution, and the obtained single components were freeze-dried.

[0056] The elution curve of LEP by DEAE-52 column chromatography is shown in Figure 1 It can be seen from Figure 1 that after elution of LEP with different ionic strength solutions, three elution peaks appeared, which were numbered as LEP I, LEP II and LEP III, corresponding to deionized water, 0.1mol / L and 0.5mol / L NaCl respectively. A peak with relatively high polysaccharide content (LEP III) appeared in the 0.5mol / L NaCl elution stage. The components in each sugar-containing peak were collected and concentrated, and the polysaccharide content was determined to be (0.095±0.005, 0.304±0.003, 0.598±0.028)g / g LEP. Figure 1 It shows that the separation effect of LEP III polysaccharide component is poor. LEP III was analyzed by cellulose acetate membrane electrophoresis (Figure 2 It was found that it was not a single component.

[0057] Furthermore, the LEPⅢ polysaccharide fraction was further purified by Sephadex G-100 gel column chromatography, and the results are as follows: Figure 3 As shown. From Figure 3 As can be seen, LEPⅢ, after being separated by Sephadex G-100 gel column chromatography and eluted with deionized water, yielded two peaks, LEPⅢa and LEPⅢb, with polysaccharide contents of (0.542±0.022) g / g LEP, respectively. Since LEPⅢb had a lower sugar content, it was not studied further. The eluent from the main peak of LEPⅢa was combined, concentrated, and lyophilized. The resulting low molecular weight *Ulva prolifera* polysaccharide LEPⅢa was a white powder, with a polysaccharide yield of 6.3±0.3%.

[0058] Example 5

[0059] The LEP used in this embodiment is the polysaccharide component isolated and purified in Example 4.

[0060] (1) Take 0.1g, 0.2g, 0.4g, 0.8g and 1.0g of LEP samples with different components and undegraded EP samples, add them to 100mL of deionized water, mix well and obtain sample solutions of different concentrations (1mg / mL, 2mg / mL, 4mg / mL, 8mg / mL and 10mg / mL).

[0061] (2) With the corresponding concentration of V c This serves as a positive control. The total antioxidant capacity of each sample solution was determined using the T-AOC kit.

[0062] The measurement results show that microwave-assisted H2O2 / V c The total antioxidant capacity of each component of LEP prepared by degradation method is as follows: Figure 4 As shown in the figure, at the same polysaccharide concentration, the total antioxidant capacity of each polysaccharide component after EP degradation was significantly enhanced compared with that before degradation (P<0.05). Meanwhile, the LEPⅢa component showed relatively high antioxidant capacity, which was significantly different from that of LEPⅠ and LEPⅡ components (P<0.05). For the same polysaccharide component, the total antioxidant capacity increased with increasing concentration, and tended to stabilize after a concentration of 8 mg / mL. At this point, the total antioxidant capacity of LEPⅢa increased from (315.2±10.3) U / mL before degradation to (661.8±18.3) U / mL, which was higher than the total antioxidant capacity of (624.5±17.3) U / mL of Vc at 2 mg / mL.

[0063] Example 6

[0064] The LEP used in this embodiment is the polysaccharide component isolated and purified in Example 4.

[0065] The specific steps for determining the iron ion reducing power of LEP are as follows:

[0066] (1) Take 1 mL of sample solution of different components and different concentrations in the total antioxidant capacity determination, add the same volume of 0.2 mol / L pH 6.6 phosphate buffer and 1% potassium hexacyanate ferrate, react at 50℃ for 20 min, then add the same volume of 10% trichloroacetic acid to terminate the reaction, take 2 mL of reaction solution, add 2 mL of distilled water and 0.4 mL of 0.1% FeCl3 solution, react at room temperature for 10 min, and measure the absorbance at 700 nm.

[0067] (2) With the corresponding concentration of V c This serves as a positive control. Results are expressed as the ratio of the sample to the corresponding concentration V. c The absorbance percentage is expressed as a percentage.

[0068] The measurement results show that microwave-assisted H2O2 / V c The iron ion reducing power of each component of LEP prepared by degradation method is as follows: Figure 5 As shown in the figure, at the same polysaccharide concentration, the iron ion reducing capacity of each polysaccharide component after EP degradation was significantly enhanced compared with that before degradation (P<0.05). For the same polysaccharide component, the iron ion reducing capacity increased with increasing concentration, and tended to stabilize after the concentration reached 8 mg / mL. At this point, the iron ion reducing capacity increased from (51.3±1.0)% before degradation to (83.4±2.2)%. Meanwhile, at the same concentration, the iron ion reducing capacities of LEPⅠ and LEPⅡ components were (74.2±1.6)% and (70.2±1.3)%, respectively. The iron ion reducing capacity of LEP showed the same trend as the total antioxidant capacity.

[0069] Example 7

[0070] The LEP used in this embodiment is the polysaccharide component LEPⅢa obtained by separation and purification in Example 4.

[0071] The structural characterization of LEPⅢa is performed using the following steps:

[0072] (1) Mix 2-5 mg of dried LEPⅢa sample with 200-500 mg of dried KBr, compress into tablets, and determine the LEPⅢa concentration at wavenumbers of 4000-400 cm⁻¹ using an infrared spectrometer. -1 Infrared absorption spectrum in the range.

[0073] (2) 2-5 mg of LEPⅢa sample was added to 2-5 mL of trifluoroacetic acid and hydrolyzed in a sealed container at 121 °C for 1-2 h. The hydrolysate was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), and its monosaccharide composition was analyzed by reversed-phase high-performance liquid chromatography. Mobile phase: 0.1 mol / L pH 6.9 phosphate buffer / acetonitrile (20:80, V / V), lactose as internal standard, differential refractive index detector, column temperature 25 °C, injection volume 5 μL. The molar ratio of each monosaccharide component was calculated using peak area. Monosaccharide standards mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), and xylose (Xyl) were analyzed using the same method.

[0074] The LEPⅢa infrared spectral characterization results are as follows: Figure 6 As shown. LEPⅢa is at approximately 3411 cm. -1 There is a strong and broad absorption peak at 2964 cm⁻¹, which is the characteristic stretching vibration peak of O-H. -1 2830cm -1 The absorption peak at 2715 cm⁻¹ is due to the C-H stretching vibration in the —CH₃ and —CH₂— groups. -1 A weak C-H stretching vibration peak of the aldehyde group is present at 2345 cm⁻¹. -1 The weak absorption peak at 1600 cm⁻¹ is related to the stretching vibration of CO₂ adsorbed in the sample. -1 The relatively broad absorption peak nearby corresponds to the stretching vibration of the carbonyl C=O group, and also indicates the presence of bound water, 1363 cm⁻¹. -1 The absorption peak at 1137 cm⁻¹ is due to the stretching vibration of a carboxyl group (C-O). These two absorption peaks indicate that LEPⅢa contains a uronic acid component. -1 The strong absorption peak at 844 cm⁻¹ is caused by the symmetric stretching vibration of S=O. -1 620cm -1 The characteristic absorption at this location likely corresponds to the bending vibration of the sulfate group in its axial position, indicating that LEPⅢa is a sulfated polysaccharide. 775cm -1 The peak at 900-1000 cm⁻¹ represents the stretching vibration of pyranoside. -1 The weak peaks in the region are due to stretching vibrations of C-O or glycosidic bonds, and bending vibrations of C-OH bonds. Infrared spectroscopy analysis shows that LEPⅢa has characteristic absorption peaks of polysaccharides, indicating that it is a sulfated polysaccharide containing pyranoside.

[0075] The results of LEPⅢa high-performance liquid chromatography analysis are as follows: Figure 7The 7 monosaccharide markers appeared at different retention times. The LEP IIIa hydrolysate appeared at 8.43, 11.26, 12.31, and 17.73 min, respectively. Compared with the retention times of the monosaccharide markers, it was indicated that the LEP IIIa was mainly composed of D-(+)-glucose, and also contained a small amount of L-(+)-rhamnose, D-glucuronic acid, and a trace amount of D-(+)-mannose. The molar ratio of each monosaccharide in the molecular composition was determined according to the peak area, which was 0.97:4.43:3.13:24.8.

[0076] Example 8

[0077] The anti-fatigue effect of LEP IIIa on mice was detected according to the method of Example 4, and the specific steps were as follows:

[0078] (1) The experimental animals were SPF healthy male ICR mice (6-8 weeks, 18-22 g) purchased from Liaoning Changsheng Biotechnology Co., Ltd. After being adaptively fed in a barrier environment for one week, 80 mice were randomly divided into 6 groups, 15 in each group (5 in the rest blank control group A), and were fed in separate cages, which were marked as the rest blank control group A, the blank control group B, the rhodiolos positive control group C, the LEP IIIa low-dose group D, the LEP IIIa medium-dose group E, and the LEP IIIa high-dose group F. Among them, the A and B blank groups were intragastrically administered with normal saline at a dose of 100 mg / kg / d, the rhodiolos positive control group C was intragastrically administered with rhodiolos at a dose of 100 mg / kg / d, and the groups D-F were intragastrically administered with LEP IIIa at doses of 50 mg / kg / d, 100 mg / kg / d, and 200 mg / kg / d, respectively.

[0079] (2) The mice were intragastrically administered according to the above dose grouping at a unified time every day, and the administration was continuously performed for 28 days. During the experiment, the mice in each group were normally fed and watered, and all the mice were fed according to the Guidelines for Laboratory Animal Feeding Management and Use.

[0080] (3) On the 28th day of the experiment, 30 min after intragastrical administration, 5 mice in each of the groups B-F were randomly selected, and the pole climbing experiment was performed according to the following steps.

[0081] (4) The mice were placed on the organic glass rod of the rotating rod fatigue instrument, the rotating speed was set to 40 rpm to make the muscles in a tense state, and the time for the mice to fall off the organic glass rod due to muscle fatigue was recorded. The experiment was terminated at the 3rd falling, and the average time of the cumulative 3 times was taken as the pole climbing time.

[0082] The results are shown in Table 1. Figure 8The pole-climbing time of mice in the low-dose LEP IIIa group was prolonged from 354 ± 5s of the blank control group to 486 ± 7s, and the medium-dose and high-dose LEP IIIa groups showed the same efficacy as the salidroside positive control group. The pole-climbing time of mice in each dose group of LEP IIIa was significantly prolonged compared with the blank control group B (P < 0.05), and there was a dose-dependent effect, showing the efficacy of relieving physical fatigue.

[0083] (5) On the 28th day of the experiment, 5 mice in groups B to F were randomly selected 30 minutes after intragastrical administration, and the exhaustive swimming test under load was performed according to the following steps.

[0084] (6) The body weight of the selected 25 mice was measured, and a weight of 5% of the body weight was hung on the tail of each mouse. The mice were placed in a water tank to swim under load, and the water temperature was 25 ± 2°C. The height of the water surface from the bottom of the pool was 50 cm to ensure that the mice could completely enter the water and could not touch the bottom. The timing started when the mice swam into the water, and the mice were considered exhausted when they sank into the water and could not return to the water surface within 8s. The timing was stopped immediately, and the exhaustive swimming time of the mice was recorded.

[0085] The results are shown in Figure 9 . The exhaustive swimming time of mice in each dose group of LEP IIIa was significantly prolonged compared with the blank control group B (P < 0.05), and there was a dose-dependent effect. The high-dose group showed the same efficacy as the salidroside positive control group, and the exhaustive swimming time was prolonged from 437 ± 10s of the blank control group to 643 ± 20s, showing strong efficacy in relieving physical fatigue.

[0086] (7) The remaining 5 mice in groups B to F were taken for 1h swimming exercise without load, and the 5 mice in the rest blank control group A were taken as the no-swimming blank control group. Thirty minutes after the swimming exercise, the mice were enucleated to collect blood, and the collected blood was loaded into a disposable negative pressure vacuum blood collection tube to collect serum (4000 rpm centrifugation for 10 min). According to the instructions of the kit, the serum urea nitrogen (BUN) and lactate dehydrogenase (LDH) activity were determined. Then the mice were sacrificed, dissected, and the liver and skeletal muscle were taken and immediately frozen in liquid nitrogen, and stored at -80°C for later use in determination of liver glycogen and muscle glycogen content.

[0087] The results of serum biochemical index detection are shown in Figure 10The concentration of urea nitrogen (BUN) in serum of the blank control group mice was significantly increased (P < 0.05) and the activity of lactate dehydrogenase (LDH) was significantly increased (P < 0.05) after swimming, indicating that the mice were in a state of fatigue. The LEP Ⅲa by gavage can significantly reduce (P < 0.05) the accumulation of urea nitrogen (BUN) in serum and the increase of lactate dehydrogenase (LDH) activity caused by fatigue, and there is a dose-dependent effect, which shows the effect of relieving physical fatigue. The detection values of the two biochemical indicators in serum of the mice by gavage of high-dose LEP Ⅲa are almost the same as those of the Rhodiolin positive control group.

[0088] The detection results of the glycogen content of the mouse body are shown in Table 2. Figure 11 The liver glycogen and muscle glycogen levels of the mice after swimming for 1 hour were significantly decreased (P < 0.05), indicating that the body was extremely fatigued after swimming. The muscle glycogen of the mice by gavage of medium and high doses of LEP Ⅲa was up-regulated to the normal level, and the liver glycogen level of the mice by gavage of medium and high doses of LEP Ⅲa was equivalent to that of the Rhodiolin positive control group. Therefore, medium and high doses of LEP Ⅲa can up-regulate the glycogen level of the body and regulate the energy metabolism during exercise, thereby playing an anti-fatigue role.

[0089] In summary, the low molecular weight Enteromorpha polysaccharide prepared by the present application has stronger antioxidant capacity. The total antioxidant capacity of a low molecular weight Enteromorpha polysaccharide LEP Ⅲa obtained by separation and purification is increased from 315.2 U / mL before degradation to 661.8 U / mL, and the iron ion reducing capacity is increased from 51.3% before degradation to 83.4%. Animal experiments show that the prepared Enteromorpha polysaccharide LEP Ⅲa shows good anti-fatigue effect. The pole climbing time and weight-bearing swimming time of the mice in the low, medium and high dose groups of Enteromorpha polysaccharide LEP Ⅲa are significantly prolonged (P < 0.05), the serum urea nitrogen (BUN) content is significantly reduced (P < 0.05), the serum lactate dehydrogenase (LDH) activity of the mice in the medium and high dose groups of Enteromorpha polysaccharide LEP Ⅲa is significantly reduced (P < 0.05), after weight-bearing swimming, the liver glycogen content of the mice in the medium and high dose groups of Enteromorpha polysaccharide LEP Ⅲa is equivalent to that of the Rhodiolin positive control group C, at the same time, the muscle glycogen content of the medium and high dose groups is maintained at a normal level, which is significantly improved (P < 0.05) compared with the control weight-bearing swimming mice, indicating that a low molecular weight Enteromorpha polysaccharide LEP Ⅲa has good effect of relieving exercise-induced fatigue.

[0090] The technical scheme of the present application is not limited to the above specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. The application of a low molecular weight *Ulva prolifera* polysaccharide with antioxidant activity in the preparation of food or health products with the function of relieving exercise-induced fatigue, characterized in that, A method for preparing low molecular weight *Ulva prolifera* polysaccharides with antioxidant activity includes the following steps: S1. Collect fresh seaweed, wash it with water, dry it at 60℃ to constant weight, pulverize it and pass it through an 80-100 mesh sieve to obtain seaweed powder; S2. Take the seaweed powder, add excess anhydrous ethanol, reflux at 75°C for 1-2 hours, cool, filter and dry to obtain defatted and impurity-removed seaweed powder. S3. Take the defatted and impurity-removed seaweed powder, add 50 times the volume of deionized water, place it in a sealed container of a microwave reactor for 10-20 minutes to assist extraction, and obtain the extract. S4. Centrifuge the extract from step S3, concentrate it under reduced pressure to 1 / 5 of the original volume, add 4 times the volume of 95% ethanol, let it stand overnight at 4°C to obtain the alcohol precipitate. S5. After centrifuging the alcohol precipitate from step S4, dissolve the precipitate in deionized water, add 2-4% (w / v) papain to hydrolyze for 1 hour to obtain the enzymatic hydrolysate. The pH of the papain hydrolysate is 6-8 and the temperature is 40-60℃. The enzymatic hydrolysate is precipitated with 4 times the volume of 95% ethanol, and the precipitate is collected after centrifugation. The crude polysaccharide of Ulva prolifera is then dissolved in deionized water to obtain a crude polysaccharide solution. The crude polysaccharide solution is further deproteinized using the Sevag method. The aqueous phase solution is concentrated and freeze-dried to obtain a white powder of crude polysaccharide EP. S6. Dissolve the crude polysaccharide EP obtained in step S5 in 200 mL of deionized water, and add 0.1-0.3 mL of 30% H2O2 and 1-3 mL of 1.0 mol / L V2O2. c After mixing the solution, place it in a sealed container of a microwave reactor and stir with a magnetic stirrer for 10-20 minutes to assist in degradation. Dialyze the resulting solution with deionized water overnight and freeze-dry to obtain low molecular weight Ulva prolifera polysaccharide LEP. S7. Low molecular weight Ulva prolifera polysaccharide LEP was separated by chromatography using a DEAE-52 column: deionized water, 0.1 mol / L, and 0.5 mol / L NaCl were used as eluents, and three elution peaks were observed, which were numbered LEPⅠ, LEPⅡ, and LEPⅢ, respectively. The fractions containing sugar peaks were collected and concentrated, and were denoted as polysaccharide fractions LEPⅠ, LEPⅡ, and LEPⅢ, respectively. S8. The polysaccharide component LEPⅢ was subjected to Sephadex G-100 gel column chromatography and eluted with deionized water to separate two peaks, namely LEPⅢa and LEPⅢb. The eluent of the main peak of LEPⅢa was combined, concentrated and lyophilized to obtain a white powder of purified low molecular weight Ulva prolifera polysaccharide LEPⅢa.

2. The application of the low molecular weight *Ulva prolifera* polysaccharide with antioxidant activity according to claim 1 in the preparation of food or health products with the function of relieving exercise-induced fatigue, characterized in that, In step S2, the volume ratio of seaweed powder to anhydrous ethanol is 1:15-25.

3. The application of the low molecular weight *Ulva prolifera* polysaccharide with antioxidant activity according to claim 1 in the preparation of food or health products with the function of relieving exercise-induced fatigue, characterized in that, In step S3, the power of the microwave reactor is 500-800W and the temperature is 70-90℃.

4. The application of the low molecular weight *Ulva prolifera* polysaccharide with antioxidant activity according to claim 1 in the preparation of food or health products with the function of relieving exercise-induced fatigue, characterized in that, In step S6, the power of the microwave reactor is 400-600W and the temperature is 50-60℃.

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