阅读说明：本技术 一种增强免疫调节和抗氧化活性的硒化金樱子多糖及其制备方法与应用 (Selenized cherokee rose fruit polysaccharide for enhancing immunoregulation and antioxidant activity, and preparation method and application thereof ) 是由 吴晖 詹麒平 董洲 赖富饶 贺萍 王倩 于 2021-08-10 设计创作，主要内容包括：本发明公开了一种增强免疫调节和抗氧化活性的硒化金樱子多糖及其制备方法与应用。该方法为：取金樱子果肉为原料,经过粉碎、脱脂、水提取、脱色、Sevag法除蛋白、醇沉、透析和干燥后即得金樱子粗多糖,经阴离子交换层析0-0.5M梯度NaCl洗脱后和凝胶渗透层析分离出金樱子酸性多糖,经HNO-(3)-Na-(2)SeO-(3)法,调节pH、离心、透析、冻干后即得；当浓度200μg/mL时,与空白对照相比,RAW264.7细胞分泌TNF-α、IL-6和NO能力分别显著提高；并且显著提高其抗氧化能力,在其浓度为3mg/mL时红细胞溶血抑制率可达94.25％,显著抑制AAPH诱导的氧化损伤,保护红细胞维持正常的细胞形态。(The invention discloses a selenized cherokee rose fruit polysaccharide for enhancing immunoregulation and antioxidant activity and a preparation method thereofA preparation method and application. The method comprises the following steps: collecting fructus Rosae Laevigatae pulp as raw material, pulverizing, defatting, extracting with water, decolorizing, removing protein by Sevag method, precipitating with ethanol, dialyzing, and drying to obtain fructus Rosae Laevigatae crude polysaccharide, eluting with 0-0.5M gradient NaCl by anion exchange chromatography, separating with gel permeation chromatography to obtain fructus Rosae Laevigatae acidic polysaccharide, and HNO 3 ‑Na 2 SeO 3 The method comprises the steps of adjusting pH, centrifuging, dialyzing and freeze-drying; when the concentration is 200 mug/mL, compared with a blank control, the TNF-alpha, IL-6 and NO secretion capacities of RAW264.7 cells are respectively and obviously improved; and the oxidation resistance of the red blood cell is obviously improved, the hemolysis inhibition rate of the red blood cell can reach 94.25% when the concentration of the red blood cell is 3mg/mL, the oxidation damage induced by AAPH is obviously inhibited, and the red blood cell is protected to maintain normal cell morphology.)

1. A method for preparing selenized fructus Rosae Laevigatae polysaccharide for enhancing immunoregulation and antioxidant activity is characterized in that fructus Rosae Laevigatae pulp is taken as a raw material, subjected to crushing, degreasing, 95 ℃ water extraction, decoloring, Sevage protein removal, alcohol precipitation, dialysis and drying, subjected to DEAE-Sepharose anion exchange chromatography and Sephadex G100 gel permeation chromatography to separate and purify fructus Rosae Laevigatae acidic polysaccharide, and subjected to HNO₃-Na₂SeO₃The preparation method comprises dialyzing, lyophilizing to obtain the final product with immunoregulatory, antioxidant, and anti-erythrocyte hemolysis effectsActive selenized acidic fructus Rosae Laevigatae polysaccharide.

2. The method of claim 1, comprising the steps of:

(1) extracting the cherokee rose fruit crude polysaccharide: adding water into the sieved and degreased fructus Rosae Laevigatae pulp crushed material, stirring at 95 ℃, centrifuging, collecting supernatant, concentrating, adopting D101 macroporous resin, putting into a shaking table for decoloring, adding Sevage reagent for deproteinization after decoloring, collecting supernatant, adding absolute ethyl alcohol, standing, centrifuging, adding a proper amount of distilled water into obtained precipitate for redissolving, dialyzing, and freeze-drying to obtain fructus Rosae Laevigatae crude polysaccharide;

(2) separation and purification: dissolving the fructus rosae laevigatae crude polysaccharide obtained in the step (1) by using water, adding the fructus rosae laevigatae crude polysaccharide into a column filled with DEAE-Sepharose anion filler, eluting by using ultrapure water, discarding an ultrapure water elution component, performing continuous gradient elution by using a 0-0.5M NaCl eluent, dialyzing and freeze-drying an eluent which is traced by a phenol-sulfuric acid method and has an absorption peak at 490nm wavelength, adding water into a component which is eluted by using 0-0.5M continuous gradient NaCl, adding the component into a chromatographic column filled with Sephadex G100 filler, eluting by using ultrapure water, sharing 1 absorption peak at 490nm wavelength, collecting and freeze-drying, wherein the component is fructus rosae laevigatae acidic polysaccharide;

(3)HNO₃-Na₂SeO₃selenium enrichment: using 50mL of HNO with the mass fraction of 0.4-0.6% to 500mg of the cherokee rose fruit acidic polysaccharide obtained in the step (2)₃Dissolving, stirring for 30min at room temperature, and adding 300-500 mgNa₂SeO₃Stirring in 70 deg.C water bath for 6-8h under dark condition and oxygen isolation, cooling to room temperature, and adding Na₂CO₃Adjusting pH to 7-8, centrifuging, collecting supernatant, dialyzing with 1000Da dialysis bag until the conductivity of dialysate is lower than 2ppm, collecting, and lyophilizing to obtain selenized fructus Rosae Laevigatae acidic polysaccharide product.

3. The preparation method of selenized rosa laevigata polysaccharide for enhancing immunoregulation and antioxidant activity according to claim 2, wherein in the step (1), the mass-to-volume ratio of the defatted rosa laevigata crushed material to water is 1g:10 mL-1 g:30 mL;

the stirring time is 1-3 h; the centrifugation is carried out at room temperature of 4000-6000 r/min for 10-20 min.

4. The method for preparing selenized rosa laevigata polysaccharide for enhancing immunoregulation and antioxidant activity as claimed in claim 2, wherein in the step (1), the volume ratio of the D101 macroporous resin to the supernatant is 1: 2-1: 3, the rotation speed of the shaking table is 160-.

5. The method for preparing selenized cherokee rose-hip polysaccharide for enhancing immunoregulation and antioxidant activity according to claim 2, wherein in the step (1), the obtained decolored supernatant is deproteinized by Sevage reagent with 3-5 times volume, and the supernatant is collected and repeated for 3-5 times;

collecting supernate, concentrating, adding 5-10 times of anhydrous ethanol, and standing for 8-12 h at 4-8 ℃.

6. The preparation method of the selenized fructus rosae laevigatae polysaccharide for enhancing the immunoregulation and antioxidant activity according to claim 2, wherein in the step (2), the fructus rosae laevigatae crude polysaccharide is dissolved by water according to the proportion of 5g:1ml to 10g:1 ml; eluting with ultrapure water at the flow rate of 1.0-2 mL/min for 10 min/tube, collecting 30 tubes, discarding, and continuously gradient-eluting with 0-0.5M NaCl solution at the flow rate of 1.0-2 mL/min for 10 min/tube, and collecting 50 tubes.

7. The preparation method of the selenized cherokee rose-hip polysaccharide for enhancing immunoregulation and antioxidant activity as claimed in claim 2, wherein in the step (2), the component eluted by 0-0.5M NaCl is added with water according to the proportion of 5G:1 mL-10G: 1mL, and then added with a chromatographic column filled with Sephadex G100 filler, the sample volume is 1-2 mL, and then eluted by ultrapure water, the elution flow rate is 0.5-1.0 mL/min, 5 min/tube, and 20 tubes are collected.

8. The preparation method of the selenized fructus rosae laevigatae polysaccharide for enhancing the immunoregulation and antioxidant activity according to claim 2, wherein the centrifugation in the step (3) is 4000-6000 r/min at room temperature for 10-20 min.

9. The selenized cherokee rose-hip polysaccharide prepared by the preparation method of any one of claims 1-8, which can enhance the immunoregulation and antioxidant activity.

10. The use of selenized acidic polysaccharides of cherokee rose-hip as claimed in claim 9, wherein: the selenized cherokee rose fruit acidic polysaccharide can be used as an antioxidant, an immunopotentiator and a selenium supplement food to be used in functional foods and food additives.

Background

Cherokee rose-hip is widely distributed in Shaanxi, Anhui, Jiangxi, Jiangsu, Zhejiang, Hubei, Hunan, Guangdong, Guangxi, Taiwan, Fujian, Sichuan, Yunnan, Guizhou and other places in China. Fructus Rosae Laevigatae can be used as diuretic and antitussive, and can also be used for treating skin tumor, burn, scald, neurasthenia, hypertension, nervous headache, and chronic nephritis. Wherein the main functions are cherokee rose fruit flavone, alkaloids and polysaccharides. At present, researches on cherokee rose fruit mainly focus on small molecular substances in cherokee rose fruit, and polysaccharides, which are one of the main components, are rarely reported.

Polysaccharides extracted from natural plants are receiving more and more attention due to their better biological activities, such as anti-infection, anti-tumor, immunity enhancement, blood sugar reduction, antivirus, etc., and especially their immune regulation and anti-oxidation activities make them widely used in food and medicine. Selenium, as an important micronutrient for maintaining human health, cannot be synthesized in the human body and can only be obtained from food. The daily selenium intake of adults recommended by the Chinese academy of nutrition is 60-250ug, but the daily selenium intake of two thirds of the population in China is lower than the minimum recommended intake, and the serious selenium deficiency phenomenon is particularly prominent in China. When the intake of selenium in the diet is insufficient, various selenium deficiency-related diseases such as keshan disease, diabetes, tumor, immunodeficiency and cardiovascular and cerebrovascular diseases can be caused. Thus, increasing selenium intake can effectively reduce the risk of developing diseases associated with selenium deficiency. Selenium mainly exists in the forms of inorganic selenium (selenious acid, sodium selenite, etc.) and organic selenium (seleno-amino acid, seleno-polysaccharide, etc.). Inorganic selenium severely limits the application of the selenium due to high toxicity, while organic selenium has the advantages of high bioavailability, low toxicity and the like, and the industrial application prospect of the selenium is greatly expanded.

The selenium polysaccharide has the physiological activity of both selenium and polysaccharide, and is easy to be absorbed and utilized by human body. A large number of researches prove that compared with inorganic selenium, polysaccharide or a mixture of the inorganic selenium and the polysaccharide, the selenized polysaccharide has better capacities of resisting oxidation, regulating immunity, resisting tumors, reducing blood sugar and removing free radicals, thereby having wider application prospects in foods and medicines. Plant selenium polysaccharide is a main source for human body to absorb selenium, but due to environmental and regional factors, natural selenium polysaccharide in plants is few, and selenium polysaccharide can be obtained after the polysaccharide is combined with a selenium common rail, so that the synthesis of selenium polysaccharide by a chemical method becomes a hot problem for researchers to study. The invention provides a preparation method of selenized cherokee rose fruit acidic polysaccharide with the functions of enhancing immunoregulation and antioxidation activity on the basis.

Disclosure of Invention

The invention aims to provide an economical and efficient selenized cherokee rose fruit acidic polysaccharide with immunoregulation and antioxidation (anti-erythrocyte hemolysis) activity and a preparation method thereof, and develops the application of the selenized cherokee rose fruit acidic polysaccharide as an antioxidant, an immunomodulator, a selenium supplement food and the like; when the concentration of the selenized cherokee rose-hip polysaccharide is 200 mug/mL, compared with a blank control, the capabilities of the RAW264.7 cell for secreting TNF-alpha, IL-6 and NO are respectively and obviously improved by 449.19%, 703.38% and 485.01%; and the oxidation resistance of the red blood cell is obviously improved, the hemolysis inhibition rate of the red blood cell can reach 94.25% when the concentration of the red blood cell is 3mg/mL, the oxidation damage induced by AAPH is obviously inhibited, and the red blood cell is protected to maintain normal cell morphology.

The purpose of the invention is realized by the following technical scheme.

A method for preparing selenized fructus Rosae Laevigatae acidic polysaccharide with immunoregulation and antioxidant activity comprises taking fructus Rosae Laevigatae pulp as raw material, pulverizing, degreasing, extracting with 95 deg.C water, decolorizing, removing protein by Sevag method, precipitating with ethanol, dialyzing, drying, eluting with DEAE-Sepharose anion exchange chromatography 0-0.5M gradient NaCl, separating and purifying with SephadexG100 gel permeation chromatography to obtain fructus Rosae Laevigatae acidic polysaccharide, and purifying with HNO₃-Na₂SeO₃The selenized cherokee rose fruit acidic polysaccharide with immunoregulation and antioxidant activity is obtained after dialysis and freeze-drying.

Preferably, the method comprises the steps of:

(1) extracting cherokee rose fruit polysaccharide: adding water into the sieved defatted cherokee rose fruit pulp crushed material, stirring at 95 ℃, centrifuging, collecting supernatant, concentrating, decoloring by adopting D101 macroporous resin, adding Sevage reagent for protein removal after decoloring, collecting supernatant, adding absolute ethyl alcohol, standing, centrifuging, adding a proper amount of distilled water into obtained precipitate for redissolving, dialyzing, freeze-drying, and obtaining the cherokee rose fruit crude polysaccharide.

(2) Separation and purification: dissolving the fructus Rosae Laevigatae crude polysaccharide obtained in step (1) with water, adding into a column filled with DEAE-Sepharose anion filler, performing gradient elution with eluent, dialyzing and freeze-drying the eluent with absorption peak at 490nm by phenol-sulfuric acid method, and continuously eluting with 0-0.5M NaCl to obtain high and sharp absorption peak, so that the component can be continuously separated and purified. Adding water into the fraction eluted with 0.5M NaCl, adding into chromatographic column filled with Sephadex G100 filler, eluting with ultrapure water, collecting and lyophilizing to obtain fructus Rosae Laevigatae acidic polysaccharide product with total absorption peaks at 490nm wavelength.

(3)HNO₃-Na₂SeO₃Selenizing the acidic polysaccharide of the cherokee rose fruit by a method:

diluting the acidic polysaccharide of fructus Rosae Laevigatae obtained in step (2) with diluted HNO₃Stirring at room temperature for 0.5h to dissolve, adding Na₂SeO₃Stirring in water bath at 70 deg.C in the absence of light and oxygen, cooling to room temperature, adding Na₂CO₃Adjusting pH to 7-8, centrifuging, intercepting and dialyzing the supernatant with a 1000Da dialysis bag until the conductivity of the dialysate is lower than 2ppm, collecting and freeze-drying to obtain the finished product of the selenized fructus Rosae Laevigatae acidic polysaccharide.

Adopt infrared absorption spectrum to compare the structure of fructus Rosae Laevigatae polysaccharide and selenizing fructus Rosae Laevigatae polysaccharide to carry out the comparison to activity between them, obtain the selenizing fructus Rosae Laevigatae polysaccharide with stronger activity.

Furthermore, the mass-volume ratio of the degreased fructus rosae laevigatae crushed material in the step (1) to water is 1g:10 mL-1 g:30 mL.

Further, the stirring time in the step (1) is 1-3 h; the centrifugation is carried out at room temperature of 4000-6000 r/min for 10-20 min.

Further, in the step (1), the volume ratio of the D101 macroporous resin to the supernatant is 1: 2-1: 3, any type of shaking table can be used as the shaking table, the rotation speed of the shaking table is 160-.

Further, in the step (1), collecting the supernatant, concentrating, and adding 5-10 times of volume of absolute ethyl alcohol.

Further, the standing in the step (1) is carried out for 8-12 h at 4-8 ℃.

Further, in the step (1), collecting supernatant of the obtained precipitate by using Sevage reagent with the volume of 3-5 times, and repeating for 3-5 times.

Further, in the step (2), the cherokee rose fruit crude polysaccharide is dissolved by water according to the proportion of 5g:1 mL-10 g:1 mL; and performing gradient elution by using a 0-0.5M NaCl solution, wherein the elution flow rate is 1.0-2 mL/min and the elution flow rate is 10 min/tube.

Further, in the step (2), water is added into the component eluted by 0.5M NaCl according to the proportion of 5G:1 mL-10G: 1mL, a chromatographic column filled with Sephadex G100 filler is added, the sample adding volume is 1-2 mL, and the elution is carried out by using ultrapure water, wherein the elution flow rate is 0.5-1.0 mL/min and 5 min/tube.

Further, in the step (3), the fructus Rosae Laevigatae acidic polysaccharide is 500mg, and HNO₃Is 50mL, the mass fraction is 0.4-0.6 percent, and Na₂SeO₃300-500 mg, and the stirring time is 6-8 h.

Further, in the step (3), the centrifugation is carried out at the room temperature of 4000-6000 r/min for 10-20 min.

In the above method, the activity evaluation in step (3): including immune activity analysis (macrophage RAW264.7 secretion of TNF-alpha, IL-6 and NO) and antioxidant (against erythrocyte hemolysis) ability analysis.

Further, in the above method, the water used for preparing the solution may be distilled water or ultrapure water, except that the ultrapure water is specified water.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the method adopts the D101 macroporous resin to decolor the cherokee rose fruit crude polysaccharide, the decoloring effect is higher than 90 percent, the polysaccharide loss rate is low, the polysaccharide purity is improved to more than 85 percent from 53 percent, and the D101 macroporous resin is cheap, so that the production cost is greatly saved.

(2) The method is simple to operate, low in production cost and suitable for industrial production, and compared with the traditional organic product obtained by biological selenium conversion or direct ethanol precipitation after selenylation, the method is short in production time effect, and effectively avoids potential safety hazards caused by inorganic selenium residues in the product.

(3) Compared with the original polysaccharide, the selenated cherokee rose fruit acidic polysaccharide prepared by the invention obviously enhances the immune and antioxidant activity of the original polysaccharide, can be added into health-care medicines or food additives with the functions of enhancing immune regulation, resisting oxidation (resisting red blood cell hemolysis) and supplementing selenium, is more favorable for absorption, has good product stability, and can be widely applied to the industries of foods and medicines.

(4) The invention promotes the development and utilization of the cherokee rose fruit resources, improves the medical and edible value of the cherokee rose fruit, provides a new way for preparing novel selenized cherokee rose fruit acidic polysaccharide with the functions of enhancing immunoregulation and antioxidation activity, and promotes the application and development of the selenized cherokee rose fruit acidic polysaccharide in food and medicine.

Drawings

FIG. 1 is a process flow diagram provided by the present invention.

FIG. 2 is an infrared spectrum of selenized fructus Rosae Laevigatae acidic polysaccharide and fructus Rosae Laevigatae acidic polysaccharide.

FIG. 3 is a graph of the effect of selenizing the acidic polysaccharides of cherokee rose fruit and its stimulation of TNF- α, IL-6 and NO secretion from RAW264.7 cells.

FIG. 4 is a scanning electron microscope image of the effect of selenizing the acidic polysaccharides of Rosa laevigata and the acidic polysaccharides of Rosa laevigata on AAPH-induced erythrolysis.

Detailed Description

For better understanding of the present invention, the present invention is further specifically described in detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and for process parameters not specifically noted, it can be performed with reference to the conventional art.

The process flow diagram of the invention is shown in figure 1.

Example 1

Preparation and structural characterization of selenized cherokee rose fruit polysaccharide:

(1) preparing a cherokee rose fruit polysaccharide crude product: cleaning fructus Rosae Laevigatae pulp, sun drying, pulverizing, sieving with 100 mesh sieve to obtain fructus Rosae Laevigatae pulp pulverized material, and placing in dry place; condensing and refluxing the crushed materials by using 95% ethanol at the reflux temperature of 70 ℃ for 2 hours, removing impurities and small molecular substances, carrying out suction filtration, removing supernatant, and naturally airing filter residues for later use; adding water into filter residue at a ratio of 1:30(g/mL), stirring at 95 deg.C for 1h, 4000r/min, centrifuging at room temperature for 10min, collecting supernatant, concentrating, adding D101 macroporous resin at a volume ratio of 2:1, decolorizing for 2h at 180 r/min; collecting supernatant with Sevage reagent of 3 times volume, repeating for 3 times, concentrating, adding 4 times volume of anhydrous ethanol, standing at 4 deg.C for 8 hr at 4000r/min, and centrifuging at room temperature for 10 min; redissolving the obtained precipitate with distilled water, dialyzing, and freeze-drying to obtain fructus Rosae Laevigatae crude polysaccharide, wherein the yield of polysaccharide is 4.47%;

(2) isolated pure of cherokee rose fruit acidic polysaccharideAnd (3) conversion and selenization: dissolving the crude cherokee rose fruit polysaccharide in the step (1) in water to prepare a solution of 5mg/mL, and filtering the solution by using a filter membrane of 0.22 mu m. 5mL of the filtrate was passed through a DEAE Sepharose Fast Flow anion column, and the column was washed with ultrapure water and a continuous gradient solution of 0 to 0.5mol/L NaCl at a rate of 1.0mL/min and collected by an automatic collector (10 min/tube). Obtaining 0.5mol/L NaCl solution elution component, dialyzing the collected component through a 1000Da dialysis bag for desalination, and dissolving the component with water after freeze drying. The components are prepared into a solution with the concentration of 5mg/mL by water, and after the solution is filtered by a filter membrane with the thickness of 0.22 mu m, 1mL of the solution is added into a Sephadex G-100 gel column for chromatographic separation. The flow rate is 0.5mL/min, samples are collected at 5 min/tube, the absorbance of the elution components is measured at 490nm, and an elution curve is drawn. Collecting the eluate after passing through the gel column, and freeze-drying. 500mg of fructus Rosae Laevigatae acidic polysaccharide is added with 50mL of 0.5% HNO₃Stirring at room temperature for 0.5h to dissolve, adding 400mg Na₂SeO₃Stirring in 70 deg.C water bath for 8 hr in the absence of light and oxygen, cooling to room temperature, adding Na₂CO₃Adjusting pH to 7-8, 6000r/min, centrifuging at room temperature for 10min, intercepting and dialyzing the supernatant with a 1000Da dialysis bag until the conductivity of the dialysate is lower than 2ppm, collecting and freeze-drying to obtain a finished product of the selenized fructus Rosae Laevigatae acidic polysaccharide, and measuring the selenized fructus Rosae Laevigatae acidic polysaccharide by a hydride atomic fluorescence method to obtain the selenized fructus Rosae Laevigatae acidic polysaccharide with the selenium content of 547-862 μ g/g after wet digestion.

(3) Structure contrast of selenized fructus Rosae Laevigatae acidic polysaccharide and fructus Rosae Laevigatae acidic polysaccharide

Taking 2mg of dried cherokee rose fruit polysaccharide and selenized cherokee rose fruit acidic polysaccharide samples, respectively adding 150mg of dried KBr, grinding uniformly by using a mortar, pressing into slices by a tablet press, and then adopting an iS5N type Fourier infrared spectrometer to process 4000-400 cm-doped cherokee rose fruit acidic polysaccharide^-1Scanning the wave bands to respectively obtain the infrared spectrograms of the acidic polysaccharide of the cherokee rose fruit and the polysaccharide of the selenized cherokee rose fruit (figure 2). 763.78cm newly appeared in the selenized cherokee rose fruit acidic polysaccharide infrared spectrogram by comparing with the original cherokee rose fruit acidic polysaccharide infrared spectrogram^-1Corresponding to Se ═ O stretching vibration absorption peak, 915.19cm^-1Corresponding to the C-O-Se stretching vibration absorption peak, the success of selenizing the cherokee rose fruit acidic polysaccharide is shown.

Example 2

Evaluation of immune and anti-erythrocyte hemolytic activity of selenized fructus rosae laevigatae acidic polysaccharide:

(1) the prepared selenized cherokee rose fruit acidic polysaccharide of the embodiment of the invention is subjected to immunological activity determination: dissolving the selenized polysaccharide in a DMEM culture medium to prepare a mother solution of 1000 mug/mL, and diluting the mother solution into three dosage groups of 200 mug/mL, 100 mug/mL and 50 mug/mL in an isocratic manner; dissolving the orthogenic polysaccharide by a DMEM culture medium to prepare a solution of 200 mu g/mL; then unfreezing the mouse macrophage RAW264.7, quickly transferring the unfrozen cells to a 15mL sterile centrifuge tube, adding 12mL DMEM medium, uniformly mixing, and then centrifuging at low speed (1000rpm/min, 5 min). The medium was removed, fresh medium was added, and the cells were suspended and transferred to a cell culture flask. Is placed in CO₂Cultured in an incubator (37 ℃, 5 vol% CO)₂) Changing the culture medium every two days, adding 100 mu L of selenized fructus Rosae Laevigatae acidic polysaccharide solution and original fructus Rosae Laevigatae acidic polysaccharide solution with different concentrations, adding 100 mu L of culture medium containing lipopolysaccharide (20 mu g/mL) into the positive control group, adding 100 mu L of DMEM culture medium into the zero adjustment group and the negative control group respectively, and setting 3 parallel holes in each group. The cells were put back into CO₂After 24 hours of culture in an incubator, the expression level of each cytokine (TNF-alpha, IL-6 and NO) in cell supernatant is determined by using an Elisa kit and a nitric oxide detection kit, and the result is shown in figure 3, wherein different letters represent that different groups have significant difference, and p is<0.05. When the selenized polysaccharide reaches 200 mu g/mL, the capacity of promoting RAW264.7 cells to secrete TNF-alpha, IL-6 and NO is respectively improved by 16.28%, 14.85% and 24.02% compared with the original polysaccharide; the improvement compared with the blank control is 449.19%, 703.38% and 485.01% respectively.

(2) The selenized cherokee rose fruit acidic polysaccharide prepared by the embodiment of the invention is subjected to the determination of the antioxidant (anti-erythrolysis) activity: meanwhile, the change of the anti-erythrocytic hemolytic activity of the Cherokee rose fruit acidic polysaccharide before and after selenization was compared by using a system of selenizing Cherokee rose fruit acidic polysaccharide and Cherokee rose fruit acidic polysaccharide solutions (0.75, 1.5 and 3mg/mL, PBS dilution) with different concentrations and 0.4mL AAPH solution of 0.2mL of erythrocyte suspension, 0.2mL of selenization, and the result is shown in FIG. 4. When the concentration of the selenized fructus rosae laevigatae acidic polysaccharide is 3mg/mL, the erythrocyte hemolysis inhibition rate can reach 94.25%. When the concentration of the selenized fructus rosae laevigatae acidic polysaccharide is 1.5mg/mL, the hemolysis inhibition rate of erythrocytes is 78.48 percent, which is obviously increased by 17.17 percent compared with the original fructus rosae laevigatae acidic polysaccharide and is obviously increased by 154.39 percent compared with a positive group, so that erythrocytes are prevented from being oxidized and damaged by AAPH, and the normal basic morphology of erythrocytes is maintained.

Example 3

Preparation method of selenized cherokee rose fruit acidic polysaccharide

(1) Preparing a cherokee rose fruit polysaccharide crude product: cleaning fructus Rosae Laevigatae pulp, sun drying, pulverizing, sieving with 100 mesh sieve to obtain fructus Rosae Laevigatae pulp pulverized material, and placing in dry place; condensing and refluxing the crushed materials by using 95% ethanol at the reflux temperature of 70 ℃ for 2 hours, removing impurities and small molecular substances, carrying out suction filtration, removing supernatant, and naturally airing filter residues for later use; adding water into filter residue at a ratio of 1:30(g/mL), stirring at 95 deg.C for 1h, 4000r/min, centrifuging at room temperature for 10min, collecting supernatant, concentrating, adding D101 macroporous resin at a volume ratio of 3:1, 160r/min, and decolorizing for 1 h; collecting supernatant with Sevage reagent of 3 times volume, repeating for 3 times, concentrating, adding 4 times volume of anhydrous ethanol, standing at 4 deg.C for 8 hr at 4000r/min, and centrifuging at room temperature for 10 min; redissolving the obtained precipitate with distilled water, dialyzing, and freeze-drying to obtain fructus Rosae Laevigatae crude polysaccharide, wherein the yield of polysaccharide is 6.18%;

(2) separating and purifying the cherokee rose fruit acidic polysaccharide and selenizing: dissolving the crude cherokee rose fruit polysaccharide in the step (1) in water to prepare a solution of 5mg/mL, and filtering the solution by using a filter membrane of 0.22 mu m. 5mL of the filtrate was passed through a DEAE Sepharose Fast Flow anion column, and the column was washed with ultrapure water and a continuous gradient solution of 0 to 0.5mol/L NaCl at a rate of 1.0mL/min and collected by an automatic collector (10 min/tube). Obtaining 0.5mol/L NaCl solution elution component, dialyzing the collected component through a 1000Da dialysis bag for desalination, and dissolving the component with water after freeze drying. The components are prepared into a solution with the concentration of 5mg/mL by water, and after the solution is filtered by a filter membrane with the thickness of 0.22 mu m, 1mL of the solution is added into a Sephadex G-100 gel column for chromatographic separation. The flow rate is 0.5mL/min, samples are collected at 5 min/tube, the absorbance of the elution components is measured at 490nm, and an elution curve is drawn. Collecting the eluate after passing through the gel column, and freeze-drying. 500mg of fructus Rosae Laevigatae acidic polysaccharide is added with 50mL of 0.4% HNO₃Stirring at room temperature for 0.5h to dissolve, adding 300mg Na₂SeO₃Stirring in 70 deg.C water bath for 6 hr in the absence of light and oxygen, cooling to room temperature, adding Na₂CO₃Adjusting the pH value to 7-8, 6000r/min, centrifuging for 10min at room temperature, intercepting and dialyzing the supernatant by a 1000Da dialysis bag until the conductivity of the dialysate is lower than 2ppm, collecting and freeze-drying to obtain a finished product of the selenized cherokee rose acidic polysaccharide, and measuring the selenized cherokee rose acidic polysaccharide by a hydride atomic fluorescence method to obtain the selenized cherokee rose acidic polysaccharide with the selenium content of about 329-containing acid 553 mug/g through wet digestion.

Example 4

Preparation method of selenized cherokee rose fruit acidic polysaccharide

(1) Preparing a cherokee rose fruit polysaccharide crude product: cleaning fructus Rosae Laevigatae pulp, sun drying, pulverizing, sieving with 100 mesh sieve to obtain fructus Rosae Laevigatae pulp pulverized material, and placing in dry place; condensing and refluxing the crushed materials by using 95% ethanol at the reflux temperature of 70 ℃ for 2 hours, removing impurities and small molecular substances, carrying out suction filtration, removing supernatant, and naturally airing filter residues for later use; adding water into filter residue at a ratio of 1:10(g/mL), stirring at 95 deg.C for 1h, 4000r/min, centrifuging at room temperature for 10min, collecting supernatant, concentrating, adding D101 macroporous resin at a volume ratio of 2:1, decolorizing for 2h at 180 r/min; collecting supernatant with Sevage reagent of 3 times volume, repeating for 3 times, concentrating, adding 4 times volume of anhydrous ethanol, standing at 4 deg.C for 8 hr at 4000r/min, and centrifuging at room temperature for 10 min; redissolving the obtained precipitate with distilled water, dialyzing, and freeze-drying to obtain fructus Rosae Laevigatae crude polysaccharide, wherein the yield of polysaccharide is 3.18%;

(2) separating and purifying the cherokee rose fruit acidic polysaccharide and selenizing: dissolving the crude cherokee rose fruit polysaccharide in the step (1) in water to prepare a solution of 5mg/mL, and filtering the solution by using a filter membrane of 0.22 mu m. 5mL of the filtrate was passed through a DEAE Sepharose Fast Flow anion column, and the column was washed with ultrapure water and a continuous gradient solution of 0 to 0.5mol/L NaCl at a rate of 1.0mL/min and collected by an automatic collector (10 min/tube). Obtaining 0.5mol/L NaCl solution elution component, dialyzing the collected component through a 1000Da dialysis bag for desalination, and dissolving the component with water after freeze drying. Preparing the above components into 5mg/mL solution with water, filtering with 0.22 μm filter membrane, adding 1mL into Sephadex G-100 gelCarrying out chromatographic separation in the column. The flow rate is 0.5mL/min, samples are collected at 5 min/tube, the absorbance of the elution components is measured at 490nm, and an elution curve is drawn. Collecting the eluate after passing through the gel column, and freeze-drying. 500mg of fructus Rosae Laevigatae acidic polysaccharide is added with 50mL of 0.6% HNO₃Stirring at room temperature for 0.5h to dissolve, adding 500mg Na₂SeO₃Stirring in 70 deg.C water bath for 8 hr in the absence of light and oxygen, cooling to room temperature, adding Na₂CO₃Adjusting the pH value to 7-8, 6000r/min, centrifuging for 10min at room temperature, intercepting and dialyzing the supernatant by a 1000Da dialysis bag until the conductivity of the dialysate is lower than 2ppm, collecting and freeze-drying to obtain a finished product of the selenized cherokee rose fruit acidic polysaccharide, and measuring the selenized cherokee rose fruit acidic polysaccharide by a hydride atomic fluorescence method to obtain the selenized cherokee rose fruit acidic polysaccharide with the selenium content of about 631-.

In conclusion, compared with the original fructus rosae laevigatae acidic polysaccharide, the selenized fructus rosae laevigatae acidic polysaccharide prepared by the embodiment of the invention can obviously improve the immunity and the antioxidant activity, and the selenized fructus rosae laevigatae acidic polysaccharide is a bioactive substance with higher application value and a selenium supplement.

The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.