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

Abstract

The invention discloses low-molecular-weight enteromorpha polysaccharide with antioxidant activity and a preparation method of the low-molecular-weight enteromorpha polysaccharide, and belongs to the technical field of polysaccharide preparation. The preparation method comprises the following steps: taking an enteromorpha crude polysaccharide solution, adding H2O2 to enable the concentration of the enteromorpha crude polysaccharide solution to be 0.04-0.08 M, and degrading for 90-120 minutes under ultraviolet rays to obtain the low-molecular-weight enteromorpha polysaccharide, the low-molecular-weight enteromorpha polysaccharide is composed of monosaccharides of rhamnose, glucose, galactose, xylose, mannose and arabinose according to the ratio of 1.08: 1.00: 0.83: 0.58: 0.69: 0.06, the scavenging capacity and the iron reducing capacity of DPPH, ABTS and hydroxyl free radicals are improved, and the low-molecular-weight enteromorpha polysaccharide is obtained. The compound has excellent antioxidant activity and can be used as or used for preparing an antioxidant active preparation. The preparation method disclosed by the invention is efficient and stable, can be used for preparing the low-molecular-weight enteromorpha polysaccharide with lighter color, lower molecular weight and higher antioxidant activity, and has higher potential application value.

Description

Technical Field

[0001] The present invention relates to a low-molecular-weight Enteromorpha polysaccharide with antioxidant activity and a preparation method thereof, belonging to the technical field of polysaccharide preparation. Background Art

[0002] Enteromorpha prolifera is a large marine green alga of the genus Ulva, rich in nutrients such as carbohydrates and proteins, and has the characteristics of high protein and low fat. At present, Enteromorpha prolifera is only used to produce low-value-added products, and how to promote its research and development has become a hot topic.

[0003] It has been reported in the prior art that Enteromorpha polysaccharide mainly composed of sulfated rhamnose has many biological activities such as antioxidant, anti-inflammatory, and anti-tumor, and is expected to be used as a natural active substance in the biomedical and functional food industries. As an important activity, antioxidant can play benefits such as scavenging free radicals and delaying aging. The antioxidant activity of Enteromorpha polysaccharide is restricted by factors such as molecular weight, polysaccharide structure, and extraction process. Polysaccharides with large molecular weights are often difficult to digest and absorb, while the lower the molecular weight, the easier it is to absorb and the better the activity.

[0004] At present, the auxiliary extraction methods of polysaccharides usually include enzyme-assisted method, ultrasonic-assisted method, microwave-assisted method, etc., among which the ultrasonic-assisted method also has the effect of auxiliary degradation. And lower-molecular-weight Enteromorpha polysaccharides are often obtained by methods such as enzymatic hydrolysis and acid hydrolysis, which often have defects such as instability and high pollution. The free radical degradation method has been successfully used for the degradation of natural active polysaccharides.

[0005] Chinese invention patent CN108239176A discloses a low-molecular-weight Enteromorpha polysaccharide and its preparation method, a sulfated low-molecular-weight Enteromorpha polysaccharide and its preparation method and application. After mixing the purified Enteromorpha polysaccharide with the Enteromorpha polysaccharide degrading enzyme prepared by fermenting a specific Bacillus alimentarius, an enzymatic hydrolysis reaction is carried out, and then enzyme inactivation and centrifugation are carried out in sequence to obtain the supernatant. The supernatant is subjected to alcohol precipitation and then centrifuged to obtain a low-molecular-weight Enteromorpha polysaccharide solution. The low-molecular-weight Enteromorpha polysaccharide solution is concentrated, dialyzed and freeze-dried to obtain a low-molecular-weight Enteromorpha polysaccharide. Although this method can reduce the molecular weight of Enteromorpha polysaccharide and improve its activity, too many reagents and steps greatly increase the processing cost. Summary of the Invention

[0006] In view of the above prior art, the present invention provides a low-molecular-weight Enteromorpha polysaccharide with antioxidant activity and a preparation method thereof.

[0007] The present invention is achieved by the following technical solutions: A preparation method of a low-molecular-weight Enteromorpha polysaccharide with antioxidant activity is as follows: Take Enteromorpha crude polysaccharide (UPP) solution, add H 2 O2 Adjust its concentration to 0.04 - 0.08 M, and degrade it under ultraviolet light for 90 - 120 minutes to obtain low molecular weight Enteromorpha polysaccharide (DUPP).

[0008] Furthermore, the concentration of the Enteromorpha crude polysaccharide solution is 1 - 10 mg / mL, preferably 5 mg / mL.

[0009] Furthermore, add H 2 O 2 to make its concentration 0.04 M, 0.06 M or 0.08 M.

[0010] Furthermore, the wavelength of the ultraviolet light is 200 - 280 nm, preferably 254 nm.

[0011] Furthermore, the degradation time is 90 minutes, 120 minutes or 150 minutes.

[0012] Furthermore, after degradation, add manganese dioxide to the degradation solution, shake it on a shaker, filter it by suction to obtain an Enteromorpha polysaccharide solution; concentrate it, add an ethanol solution, let it stand, centrifuge it, redissolve the precipitate in pure water, and freeze-dry it to obtain low molecular weight Enteromorpha polysaccharide.

[0013] Furthermore, the Enteromorpha crude polysaccharide is prepared by the following method: (1) Pretreatment of Enteromorpha: Take fresh Enteromorpha, dry it and crush it to obtain Enteromorpha powder; extract the Enteromorpha powder with ethanol, centrifuge it, and dry it to obtain defatted and decolorized Enteromorpha powder; (2) Extraction of Enteromorpha crude polysaccharide: Dissolve the defatted and decolorized Enteromorpha powder in water, extract it by ultrasonic wave, centrifuge it, and the supernatant is the Enteromorpha crude polysaccharide solution.

[0014] Furthermore, the specific method of extracting the Enteromorpha powder with ethanol is: Mix 20 g of Enteromorpha powder with 400 mL of 95% ethanol solution, stir it at room temperature and 500 rpm for 2 h.

[0015] Furthermore, the specific method of ultrasonic extraction is: The ultrasonic power is 648 W, the time is ultrasonic for 2 s and stop for 2 s, lasting for 200 min, and the extraction temperature does not exceed 45°C.

[0016] The low-molecular-weight Enteromorpha polysaccharide prepared by the above method has a weight-average molecular weight of 24.60 - 36.60 kDa and is composed of monosaccharides with a molar ratio of rhamnose:glucose:galactose:xylose:mannose:arabinose = 1.08:1.00:0.83:0.58:0.69:0.06 as detected. Experiments show that the low-molecular-weight Enteromorpha polysaccharide has improved scavenging abilities against DPPH, ABTS, hydroxyl radicals and iron-reducing ability, and is overall superior to commercially available Ulva lactuca polysaccharide within the concentration range of 0 - 8 mg / mL, with excellent antioxidant activity, and can be used as or for the preparation of antioxidant activity preparations.

[0017] The preparation method of the low-molecular-weight Enteromorpha polysaccharide of the present invention is efficient and stable, and the obtained waste is easy to handle. It can be degraded to prepare a low-molecular-weight Enteromorpha polysaccharide with a lighter color, lower molecular weight and higher antioxidant activity, and has high potential application value.

[0018] All the terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. Description of the Drawings

[0019] Figure 1 : Photo of UPP.

[0020] Figure 2 : Photo of DUPP.

[0021] Figure 3 : SEM image of UPP.

[0022] Figure 4 : SEM image of DUPP.

[0023] Figure 5 : Schematic diagram of the determination results of the monosaccharide composition of the standard product.

[0024] Figure 6 : Schematic diagram of the determination results of the monosaccharide composition of UPP.

[0025] Figure 7 : Schematic diagram of the determination results of the monosaccharide composition of DUPP.

[0026] Figure 8 : Schematic diagram of the Fourier transform infrared spectrum of UPP.

[0027] Figure 9 : Schematic diagram of the Fourier transform infrared spectrum of DUPP.

[0028] Figure 10 : Schematic diagram of the effects of UPP, DUPP and ULP on the scavenging rate of DPPH radicals.

[0029] Figure 11 : Schematic diagram of the effects of UPP, DUPP and ULP on the scavenging rate of ABTS radicals.

[0030] Figure 12 : Schematic diagram of the effects of UPP, DUPP, and ULP on the scavenging rate of hydroxyl radicals.

[0031] Figure 13 : Schematic diagram of the effects of UPP, DUPP, and ULP on the iron reducing power. Specific implementation manners

[0032] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0033] The instruments, reagents, and materials involved in the following embodiments are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels without special instructions. The experimental methods, detection methods, etc. involved in the following embodiments are all conventional experimental methods and detection methods existing in the prior art without special instructions.

[0034] Example 1 Preparation of low-molecular-weight Enteromorpha polysaccharide with antioxidant activity The steps are as follows: (1) Pretreatment of Enteromorpha: Take fresh Enteromorpha (collected from Qingdao, Shandong Province in 2023), wash it, drain the water, dry it in an oven at 45°C, crush it with a pulverizer, and pass through an 80-mesh sieve to obtain Enteromorpha powder; mix 20 g of Enteromorpha powder with 400 mL of 95% ethanol solution (solid-liquid ratio 1:20), place it on a magnetic stirrer, stir at room temperature and 500 rpm for 2 h; centrifuge (4000 rpm, 10 min) to obtain a precipitate, and dry it at 45°C to obtain defatted and decolorized Enteromorpha powder.

[0035] (2) Extraction of Enteromorpha crude polysaccharide: Mix 1 g of defatted and decolorized Enteromorpha powder with 75 mL of pure water (solid-liquid ratio 1:75), place it in an ultrasonic cell disruptor for ultrasonic extraction, set the ultrasonic power to 648 W, ultrasonic for 2 s and stop for 2 s, and continue for 200 min. During this period, control the extraction temperature not to exceed 45°C with an ice-water bath to obtain an ultrasonic extraction solution; centrifuge (4000 rpm, 10 min), and the supernatant is the Enteromorpha crude polysaccharide solution, which is rotary evaporated and concentrated to 5 mg / mL for standby.

[0036] (3) Preparation of low-molecular-weight Enteromorpha polysaccharide: Take 50 mL of 5 mg / mL Enteromorpha crude polysaccharide solution, place it in a 150 mm petri dish, and add 306 μL of 30% H 2 O 2 solution (mass fraction) to make H 2 O 2The concentration of [substance] is 0.06 M; place the petri dish in a UV crosslinker and degrade it for 120 min at a wavelength of 254 nm to obtain a degradation solution; (4) Add 1 g of manganese dioxide to the degradation solution, place it on a horizontal shaker, shake at 100 rpm for 2 h, and perform suction filtration (to remove H 2 O 2 and manganese dioxide) to obtain an Enteromorpha polysaccharide solution; concentrate it by rotary evaporation at 45 °C to 1 / 4 of the original volume, add 4 times the volume of 95% ethanol solution, let it stand in a refrigerator at 4 °C for 12 h, centrifuge (4000 rpm, 10 min), take the precipitate, redissolve it in 10 mL of pure water, pre-freeze it at -40 °C for 6 h, transfer it to a freeze dryer, and freeze-dry it for 48 h to obtain low-molecular-weight Enteromorpha polysaccharide. After testing, the weight-average molecular weight is 26.72 kDa.

[0037] Example 2 Preparation of Low-Molecular-Weight Enteromorpha Polysaccharide with Antioxidant Activity The steps are as follows: (1) Pretreatment of Enteromorpha: The same as in Example 1.

[0038] (2) Extraction of Enteromorpha crude polysaccharide: The same as in Example 1.

[0039] (3) Preparation of low-molecular-weight Enteromorpha polysaccharide: Take 50 mL of 5 mg / mL Enteromorpha crude polysaccharide solution, place it in a 150 mm petri dish, add 204 μL of 30% H 2 O 2 solution to make the concentration of H 2 O 2 be 0.04 M; place the petri dish in a UV crosslinker and degrade it for 90 min at a wavelength of 254 nm to obtain a degradation solution; (4) The same as in Example 1 to obtain low-molecular-weight Enteromorpha polysaccharide. After testing, the weight-average molecular weight is 36.60 kDa.

[0040] Example 3 Preparation of Low-Molecular-Weight Enteromorpha Polysaccharide with Antioxidant Activity The steps are as follows: (1) Pretreatment of Enteromorpha: The same as in Example 1.

[0041] (2) Extraction of Enteromorpha crude polysaccharide: The same as in Example 1.

[0042] (3) Preparation of low-molecular-weight Enteromorpha polysaccharide: Take 50 mL of 5 mg / mL Enteromorpha crude polysaccharide solution, place it in a 150 mm petri dish, add 408 μL of 30% H 2 O 2 solution to make the concentration of H 2 O 2The concentration was 0.08 M; the culture dish was placed in a UV cross-linker and degraded at a wavelength of 254 nm for 150 min to obtain a degradation solution; (4) The same as in Example 1, low-molecular-weight Enteromorpha polysaccharide was obtained. After detection, the weight-average molecular weight was 24.60 kDa.

[0043] Experiment 1 Characterization of the structure of Enteromorpha polysaccharide The structures of Enteromorpha crude polysaccharide (UPP) and low-molecular-weight Enteromorpha polysaccharide (DUPP) were characterized. Among them, DUPP was the low-molecular-weight Enteromorpha polysaccharide prepared in Example 1, and UPP was obtained by rotary evaporation, alcohol precipitation, and freeze-drying of the Enteromorpha crude polysaccharide solution prepared in Example 1 (the Enteromorpha crude polysaccharide solution was rotary-evaporated and concentrated to 1 / 4 of the original volume at 45 °C, 4 times the volume of 95% ethanol solution was added, and it was left to stand in a refrigerator at 4 °C for 12 h, centrifuged at 4000 rpm for 10 min, the precipitate was redissolved in 10 mL of pure water, pre-frozen at -40 °C for 6 h, transferred to a freeze-dryer, and freeze-dried for 48 h to obtain UPP).

[0044] (1) UPP and DUPP were directly observed and photographed. The photos of UPP and DUPP are as Figure 1 、 Figure 2 shown. The results showed that at the same magnification, the low-molecular-weight Enteromorpha polysaccharide was lighter in color and more delicate and uniform in particles than the Enteromorpha crude polysaccharide. In the redissolution process, the Enteromorpha crude polysaccharide usually needed to be dissolved by heating or ultrasonic assistance, and there were still some insoluble substances remaining; the same mass of low-molecular-weight Enteromorpha polysaccharide could be almost completely dissolved in water only by shaking at room temperature.

[0045] (2) Scanning electron microscope photos were taken: 5-10 mg of sample powder was evenly spread on the sample stage with conductive adhesive, the loose sample was blown off with an ear bulb, and it was vacuum-sprayed with gold for 30 s in a sputter coater; after taking out, it was put into the vacuum sample chamber of a Zeiss tungsten filament lamp scanning electron microscope, and photos were taken at a magnification of 200X. The SEM images of UPP and DUPP are as Figure 3 、 Figure 4 shown.

[0046] (3) HPLC determination of monosaccharide composition The steps are as follows: ① Weigh 2 mg of polysaccharide sample, add 1 mL of 2 M TFA acid solution, heat at 121 °C for 2 h; pass nitrogen and dry it; add 99.99% methanol for cleaning and then dry it again, repeat the methanol cleaning 3 times; add sterile water to dissolve, dilute 15 times, and transfer it to a chromatographic vial for determination.

[0047] ② Use a Dionex™ CarboPac™ PA20 (150*3.0 mm, 10μm) liquid chromatography column; the injection volume is 5 μL; mobile phase A (H 2Mobile phase A (0.1 M H₂SO₄), 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 °C; gradient elution; use software Chromeleon to process chromatographic data, determine substances by peak time, and quantify by peak area.

[0048] ③ Standard samples: Take quantitative different monosaccharide standard samples to prepare a standard mixed solution and measure according to ②.

[0049] The measurement results are as Figure 5 、 Figure 6 、 Figure 7 shown. The results show that the monosaccharide composition of Enteromorpha prolifera crude polysaccharide is: rhamnose:glucose:galactose:xylose:mannose = 1.26:1.00:0.56:0.26:0.22 (molar ratio); the monosaccharide composition of low molecular weight Enteromorpha prolifera polysaccharide is: rhamnose:glucose:galactose:xylose:mannose:arabinose = 1.08:1.00:0.83:0.58:0.69:0.06 (molar ratio). By comparison, it can be seen that the proportion of rhamnose decreases slightly after degradation, while the proportions of xylose, galactose and mannose increase, and a small amount of arabinose not contained in the crude polysaccharide is detected. Such results indicate that the degradation process has an impact on the monosaccharide composition of Enteromorpha prolifera polysaccharide, and rhamnose may be converted into other monosaccharides during the degradation process.

[0050] (4) Fourier transform infrared spectroscopy characterization Spectral pure potassium bromide is placed in an oven at 105 °C and dried for 6 h. Under an infrared lamp, 100 mg of potassium bromide and 1 mg of UPP powder or DUPP powder are mixed and ground according to a mass ratio of 100:1, and pressed into a thin film and scanned in the range of 400 - 4000 cm -1 with Fourier transform infrared spectroscopy.

[0051] The results are as Figure 8 、 Figure 9 shown. The infrared spectroscopy results show that the strong absorption peak of low molecular weight Enteromorpha prolifera polysaccharide at 3421 cm -1 represents O-H stretching vibration, and the peak at 2934 cm -1 is considered to be C-H stretching vibration. The absorption peak at 1642 cm -1 corresponds to the C=O vibration of the carbonyl group, indicating the presence of bound water. The peak at 1415 cm -1 is affected by the C-H bending vibration of carbohydrates. The peak at 1255 cm -1 corresponds to the stretching vibration of sulfate groups, and the characteristic peaks at 844 cm -1 both confirm the presence of sulfate groups, and the characteristic peak at 844 cm -1 also indicates that the low molecular weight Enteromorpha prolifera polysaccharide has an α configuration. In addition, the peak at 1052 cm -1The peak at [specific location] is the C-O stretching vibration in C-O-H, which also indicates that the sugar is pyranose. Therefore, the low-molecular-weight Enteromorpha polysaccharide is a sulfated pyranose with an α configuration. Compared with the crude Enteromorpha polysaccharide, the low-molecular-weight Enteromorpha polysaccharide shows no obvious absorption peak of S=O stretching vibration of sulfate groups representing axial coordination at 844 cm -1 There is no obvious absorption peak of S=O stretching vibration of sulfate groups representing axial coordination in the attachment, indicating that the content of sulfate groups in UPP may be lower than that in DUPP. The determination results by the barium chloride-gelatin method show that after the degradation of Enteromorpha polysaccharide, the content of sulfate groups increases from 11.86 ± 0.57% to 21.30 ± 0.98%, which confirms the above conclusion. Existing studies have shown that the higher the content of sulfate groups, the stronger the antioxidant activity. The above results indicate that the method of the present invention has a certain modifying effect on the structure of Enteromorpha polysaccharide and has the effect of enhancing antioxidant activity.

[0052] (5) Determination of polysaccharide molecular weight The SEC-Malls method was used, and the specific steps are as follows: ① Take 2 mg of the low-molecular-weight Enteromorpha polysaccharide sample, dissolve it in ultrapure water to prepare a 2 mg / mL sample solution, and filter it through a 0.22 μm filter membrane.

[0053] ② The mobile phase is 0.1 M Na 2 SO 4 , the chromatographic column model is Shodex Ohpak LB-806M, the column temperature is 30 °C, the injection volume is 100 μL, and the flow rate is 0.4 mL / min.

[0054] ③ The obtained results are processed by the ASTRA system, and the peak area is automatically calculated to generate the molecular weight value.

[0055] The results show that the weight-average molecular weight of the crude Enteromorpha polysaccharide is 168.87 kDa, and the weight-average molecular weight of the low-molecular-weight Enteromorpha polysaccharide is 26.72 kDa, indicating a significant reduction in molecular weight.

[0056] Experiment 2 Determination of antioxidant activities of UPP and DUPP (I) The scavenging rate of DPPH free radicals is as follows: (1) Prepare polysaccharide sample solutions with concentrations of 0, 0.25, 0.5, 1, 2, 4, and 8 mg / mL respectively using distilled water. Additionally, take 0.0025 g of DPPH and dissolve it in 25 mL of methanol to obtain the DPPH working solution.

[0057] (2) Take a 2 mL brown centrifuge tube, add 200 μL of the polysaccharide sample at the corresponding concentration, and then add 200 μL of the DPPH working solution. React at room temperature in the dark for 30 min, and measure the absorbance at 515 nm with an enzyme-labeling instrument and record it as A. The group with methanol replacing DPPH is denoted as A0, the group with methanol replacing the sample is denoted as A1, and the absorbance of methanol is denoted as A2. Use commercially available ulva lactuca polysaccharide (ULP) (purchased from Yuanye) as the control sample.

[0058] (3) Calculate the DPPH scavenging rate according to the following formula: DPPH scavenging rate (%) = (1 - ) * 100.

[0059] The test results are as Figure 10 shown. The results show that at low concentrations, the scavenging effects of UPP, DUPP, and ULP on DPPH radicals are poor. When the concentration is 0.5 - 8 mg / mL, the scavenging rate increases with the increase of the polysaccharide concentration, and the trend of DUPP is more obvious. Thus, it can be seen that within this concentration range, the scavenging effect of DUPP on DPPH radicals is better than that of UPP and ULP, indicating that the degradation process of the present invention has a great improvement in antioxidant activity and is better than the commercially available ulva lactuca polysaccharide of the same kind.

[0060] (2) Scavenging rate of ABTS radicals, the steps are as follows: (1) Prepare polysaccharide sample solutions with concentrations of 0, 0.25, 0.5, 1, 2, 4, and 8 mg / mL respectively with distilled water; take another 0.0066 g of K 2 S 2 O 8 , add 20 mL of deionized water to dissolve it, take 4 mL and add 0.0077 g of ABTS, react at room temperature in the dark for 14 h, and dilute it 25 times before use as the ABTS working solution.

[0061] (2) Take a 2 mL brown centrifuge tube, add 200 μL of the polysaccharide sample at the corresponding concentration, and then add 800 μL of the ABTS working solution, react at room temperature in the dark for 10 min, and measure the absorbance at 734 nm. The group with distilled water replacing ABTS is denoted as A0, the group with distilled water replacing the sample is denoted as A1, and the absorbance of distilled water is denoted as A2. Use commercially available ulva lactuca polysaccharide as the control sample.

[0062] (3) Calculate the ABTS scavenging rate according to the following formula: ABTS scavenging rate (%) = (1 - ) * 100.

[0063] The test results are as Figure 11As shown in the figure, the results showed that in the range of 0 - 8 mg / mL, the scavenging effect of DUPP on ABTS free radicals was dose-dependent, and reached 98.54% ± 0.30% at 8 mg / mL, showing good antioxidant ABTS free radical scavenging ability.

[0064] (III)Scavenging rate of hydroxyl free radicals, the steps are as follows: (1)Prepare polysaccharide sample solutions with concentrations of 0, 0.25, 0.5, 1, 2, 4, and 8 mg / mL respectively with distilled water; prepare 9 mmol / L FeSO 4 solution, 9 mmol / L salicylic acid solution and 8.8 mmol / L H 2 O 2 solution.

[0065] (2)Take 2 mL brown centrifuge tubes, and successively add 200 μL FeSO 4 solution, 200 μL salicylic acid solution, 200 μL sample solution and 200 μL H 2 O 2 solution, vortex and mix well, react in a water bath at 37 °C for 30 min, then cool with normal temperature water, and measure the absorbance A at 510 nm. The group with distilled water instead of H 2 O 2 solution is recorded as A1, and the group with distilled water instead of the sample is recorded as A0.

[0066] (3)Calculate the hydroxyl free radical scavenging rate according to the following formula: Hydroxyl free radical scavenging rate = (1 - ).

[0067] The test results are as Figure 12 shown. The results showed that the scavenging rates of UPP and DUPP on hydroxyl free radicals increased rapidly within the concentration range of 0 - 0.25 mg / mL and then tended to be stable. At a concentration of 1 mg / mL, the scavenging rate of DUPP on hydroxyl free radicals reached 50%, while UPP needed to exceed 4 mg / mL to achieve the same effect. In addition, the scavenging ability of ULP on hydroxyl free radicals was much lower than that of UPP in the mass concentration range of 0 - 4 mg / mL, while the degraded polysaccharide DUPP was significantly better than UPP, indicating the advantages of the degradation process of the present invention.

[0068] (IV)Effect on iron reducing power, the steps are as follows: (1)Prepare polysaccharide sample solutions with concentrations of 0, 0.25, 0.5, 1, 2, 4, and 8 mg / mL respectively with distilled water; prepare 0.1 mg / mL Trolox as a standard, and another acetic acid buffer solution with pH 3.6, 20 mM FeCl 3The solution and 10 mM TPTZ hydrochloride solution were mixed at a ratio of 10:1:1 and heated at 37 °C to obtain the FRAP working solution.

[0069] (2) Take a 2 mL brown centrifuge tube, add 100 μL of samples with different concentrations and 900 μL of the FRAP working solution, mix well, react at 37 °C in the dark for 15 min, and measure the absorbance A at 593 nm. Prepare a Trolox solution with a concentration gradient as a sample for synchronous detection, and draw a standard curve based on the absorbance.

[0070] (3) Calculate the Trolox equivalent of the sample according to the standard curve to represent the iron reduction ability.

[0071] The detection results are as Figure 13 shown. The results show that within the range of 0 - 8 mg / mL, the higher the polysaccharide concentration, the stronger the iron reduction ability. However, in terms of the growth rate, the upward trend of DUPP is much higher than that of UPP and ULP, and the iron reduction ability of UPP is slightly lower than that of ULP. 8 mg / mL of DUPP is equivalent to 55.39 μg of Trolox, while UPP and ULP are only equivalent to 22.78 μg and 24.72 μg of Trolox, highlighting the development and utilization potential of the low-molecular-weight Enteromorpha polysaccharide prepared by the present invention in terms of antioxidant.

[0072] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for preparing low molecular weight Enteromorpha polysaccharide with antioxidant activity, characterized in that: Take a crude Enteromorpha polysaccharide solution, add H2O2 to make its concentration 0.04-0.08 M, and degrade it under ultraviolet light for 90-120 minutes to obtain a low molecular weight Enteromorpha polysaccharide.

2. The method for preparing the low molecular weight Enteromorpha polysaccharide with antioxidant activity according to claim 1, characterized in that: The concentration of the Enteromorpha crude polysaccharide solution is 1-10 mg / mL.

3. The method for preparing the low molecular weight Enteromorpha polysaccharide with antioxidant activity according to claim 1, characterized in that: H2O2 was added to a concentration of 0.04 M, 0.06 M, or 0.08 M.

4. The method for preparing low molecular weight Enteromorpha polysaccharide with antioxidant activity according to claim 1, characterized in that: The wavelength of the ultraviolet rays is 254 nm, and the degradation time is 90 minutes, 120 minutes or 150 minutes.

5. The method for preparing low molecular weight Enteromorpha polysaccharide with antioxidant activity according to claim 1, characterized in that: After the degradation, manganese dioxide is added to the degradation solution, shaken on a shaker, and filtered to obtain a Enteromorpha polysaccharide solution; concentrated, ethanol solution is added, allowed to stand, centrifuged, the precipitate is redissolved in pure water, and freeze-dried to obtain a low molecular weight Enteromorpha polysaccharide.

6. The method for preparing low molecular weight Enteromorpha polysaccharide with antioxidant activity according to claim 1, characterized in that: The Enteromorpha crude polysaccharide is prepared by the following method: (1) Pretreatment of Enteromorpha: fresh Enteromorpha is taken, dried, and crushed to obtain Enteromorpha powder; the Enteromorpha powder is extracted with ethanol, centrifuged, and dried to obtain defatted and decolorized Enteromorpha powder; (2) Extraction of crude Enteromorpha polysaccharides: Dissolve the defatted and decolorized Enteromorpha powder in water, perform ultrasonic extraction, and centrifuge. The supernatant is the crude Enteromorpha polysaccharide solution.

7. The method for preparing low molecular weight Enteromorpha polysaccharide with antioxidant activity according to claim 6, characterized in that: The specific method of extracting the Enteromorpha powder with ethanol is as follows: 20 g of Enteromorpha powder is mixed with 400 mL of 95% ethanol solution, and stirred at room temperature and 500 rpm for 2 h.

8. The method for preparing low molecular weight Enteromorpha polysaccharide with antioxidant activity according to claim 6, characterized in that: The specific method of the ultrasonic extraction is: the ultrasonic power is 648 W, the ultrasonic time is 2 s on, 2 s off, and lasts for 200 minutes, and the extraction temperature does not exceed 45°C. 9 . The low molecular weight Enteromorpha polysaccharide prepared by the method for preparing the low molecular weight Enteromorpha polysaccharide with antioxidant activity according to any one of claims 1 to 8 .

10. Use of the low molecular weight Enteromorpha polysaccharide according to claim 9 in the preparation of an antioxidant active preparation.

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