Pitaya flower polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses pitaya flower polysaccharide as well as a preparation method and application thereof. According to the method, the flower picking waste in the pitaya planting process is used as a raw material, a water extraction and alcohol precipitation method is adopted, crude polysaccharides of different parts (calyx and petal) of pitaya flowers are obtained, the anti-oxidative stress level research is conducted on the crude polysaccharides of the different parts, and the calyx crude polysaccharides with the excellent effect are selected; and further carrying out anion exchange chromatography purification by adopting DEAE (Diethylaminoethyl) Sepharose Fast Flow, so as to obtain the purified pitaya flower calyx polysaccharide. Researches show that the pitaya flower calyx polysaccharide can inhibit the oxidative stress effect of Caco2 cells induced by acrylamide, recover the content of copper ions in the cells and inhibit cell copper death caused by acrylamide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a pitaya flower polysaccharide, a preparation method thereof, and an application thereof. Background Art

[0002] Polysaccharides are widely present in animals, plants, and microorganisms. As one of the components of living substances, they participate in the regulation of various life phenomena and physiological processes of cells, such as the transmission and perception of information between immune cells, and activities such as cell transformation, division, and regeneration. Since the 1980s, scientists have shown great interest in the research of traditional Chinese medicine polysaccharides. So far, more than 100 polysaccharides have been reported, including ginseng polysaccharide, lentinan, ganoderma polysaccharide, astragalus polysaccharide, poria cocos polysaccharide, cordyceps polysaccharide, tremella polysaccharide, acanthopanax senticosus polysaccharide, etc. These polysaccharides have physiological activities such as antiviral, anti-infective, anti-tumor, antioxidant, anti-radiation, hypoglycemic, liver-protecting, and immune-regulating effects, and have small side effects.

[0003] Pitaya flower belongs to the plants of the genera Hylocereus and Seleniereus in the family Cactaceae, and is now widely planted in Guangxi, Hainan, Guangdong, Yunnan and other provinces in China. Pitaya flower has the effects of preventing constipation, promoting eye health, antioxidant, anti-free radical and inhibiting dementia, and also has the effects of whitening the skin, losing weight, reducing blood sugar and preventing colorectal cancer. Recent studies have shown that pitaya flower has a unique curative effect on cough and asthma, and also has the effects of improving eyesight and reducing internal heat, and has the effect of preventing hypertension. Pitaya flower is rich in various pharmacodynamic substances, such as polysaccharides, sesquiterpenes, alkynes, triterpenes, aromatic glycosides, etc. However, the current research on the extraction, separation and purification, structural analysis and biological activity of pitaya flower polysaccharides is still in the preliminary exploration stage. Therefore, exploring the structure and physiological activity of pitaya flower polysaccharides is of great significance for the development and utilization of pitaya flower. Summary of the Invention

[0004] The primary object of the present invention is to overcome the deficiencies of the prior art and provide a preparation method of pitaya flower polysaccharide.

[0005] Another object of the present invention is to provide the pitaya flower polysaccharide prepared by the above method.

[0006] Another object of the present invention is to provide an application of the pitaya flower polysaccharide.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A preparation method of pitaya flower polysaccharide, comprising the following steps:

[0009] (1) Extracting crude pitaya flower polysaccharide by water extraction and alcohol precipitation

[0010] Dry and pulverize the calyx of pitaya flower, then sieve it to obtain the calyx powder of pitaya flower. Then add it to water and perform extraction under the condition of 90±5°C water bath. After extraction, centrifuge and filter, take the supernatant to obtain the calyx extract of pitaya flower. Then add anhydrous ethanol to the calyx extract of pitaya flower after reduced pressure concentration, let it stand for alcohol precipitation, then centrifuge and take the precipitate. After the ethanol in the precipitate volatilizes completely, redissolve it with water, use Sevag method to remove proteins and macroporous resin method to decolorize, and finally concentrate, dialyze, and freeze-dry to obtain the crude polysaccharide of pitaya flower;

[0011] (2) Purify the crude polysaccharide of pitaya flower by ion exchange chromatography

[0012] Purify the crude polysaccharide of pitaya flower by DEAE Sepharose Fast Flow anion exchange chromatography. The elution solution is 0-0.4mol / L NaCl solution, and then dialyze and freeze-dry to obtain the purified polysaccharide of pitaya flower.

[0013] The pitaya flower described in step (1) is preferably the flower of red-fleshed pitaya.

[0014] The calyx of the pitaya flower described in step (1) can be the waste of flower picking during the pitaya planting process.

[0015] The drying temperature described in step (1) is 55-65°C; preferably 60°C.

[0016] The sieving described in step (1) is sieving through a 60-100 mesh sieve; preferably sieving through an 80 mesh sieve.

[0017] The material-liquid ratio of the calyx powder of pitaya flower to water described in step (1) is 1g:10-30ml; preferably 1g:15ml.

[0018] The filtration described in step (1) is preferably filtration using filter paper.

[0019] The extraction time described in step (1) is 2-5h; preferably 3h.

[0020] In step (1), the conditions for centrifugation after extraction are: centrifuge at 4000-5000r / min for 10-20min; preferably: centrifuge at 4500r / min for 15min.

[0021] In step (1), the number of times of water extraction is more than 2 times.

[0022] The reduced pressure concentration described in step (1) is concentrated to 1 / 5-1 / 3 of the original volume; preferably concentrated to 1 / 4 of the original volume.

[0023] In step (1), the addition of absolute ethanol is to add absolute ethanol with a volume 3 to 5 times that of the sample; preferably, absolute ethanol with a volume 4 times that of the sample is added.

[0024] The standing conditions described in step (1) are: standing at 4 °C for 12 to 15 h; preferably, standing at 4 °C for 12 h.

[0025] The macroporous resin described in step (1) is preferably AB-8 macroporous resin.

[0026] The dialysis in step (1) is carried out using a dialysis bag with a molecular weight cut-off of 3000 Da; preferably, dialysis is carried out using a dialysis bag with a molecular weight cut-off of 3000 Da at 4 °C for 36 to 48 h.

[0027] The dialysis solution used for dialysis in step (1) is distilled water or deionized water.

[0028] The DEAE Sepharose Fast Flow anion exchange chromatography described in step (2) can be carried out according to the operation manual provided by the manufacturer or the conventional methods in the art, including column packing (wet packing), equilibration, sample loading, elution and other procedures.

[0029] The concentration of the NaCl solution described in step (2) is preferably 0, 0.2, 0.3 or 0.4 mol / L; preferably, it is 0.4 mol / L.

[0030] The dialysis in step (2) is carried out using a dialysis bag with a molecular weight cut-off of 3000 Da; preferably, dialysis is carried out using a dialysis bag with a molecular weight cut-off of 3000 Da for 36 to 48 h, and the water is changed every 4 h.

[0031] The dialysis solution used for dialysis in step (2) is distilled water or deionized water.

[0032] The lyophilization conditions in step (2) are: pre-freezing at -20 °C for 12 h first, and then freeze-drying at a vacuum degree of 0.05 to 0.07 Mbar and a temperature of -60 °C for 36 to 38 h.

[0033] A pitaya flower polysaccharide is prepared by the method described in any one of the above.

[0034] Use of the described pitaya flower polysaccharide in the preparation of a product for inhibiting cellular oxidative stress.

[0035] The cellular oxidative stress is acrylamide-induced cellular oxidative stress.

[0036] The product includes a drug or a daily chemical product.

[0037] Use of the pitaya flower polysaccharide in the preparation of a drug for inhibiting cuproptosis and / or restoring the intracellular copper ion content.

[0038] The cuproptosis of the cells is cuproptosis caused by acrylamide.

[0039] The cells include normal cells or cancer cells; preferably colon cancer cells.

[0040] The pitaya flower polysaccharide can inhibit the oxidative stress of cells induced by acrylamide, restore the intracellular copper ion content, and inhibit the cuproptosis of cells caused by acrylamide.

[0041] The present invention has the following advantages and effects compared with the prior art:

[0042] 1. The present invention provides a method for extracting and purifying pitaya flower polysaccharide. Using the waste of picking flowers in the pitaya planting process as raw materials, crude polysaccharides from different parts of the pitaya flower (calyx, petals) are obtained by water extraction and alcohol precipitation. The antioxidant stress levels of the crude polysaccharides from different parts are studied, and the crude polysaccharide of the calyx with relatively excellent effects is selected. The present invention further purifies the crude polysaccharide of the calyx by DEAE Sepharose Fast Flow anion exchange chromatography to obtain 4 components: neutral calyx polysaccharide DFPE-0 and acidic calyx polysaccharides DFPE-2, DFPE-3, and DFPE-4.

[0043] 2. The present invention identifies the antioxidant stress levels of the purified components again, screens out the acidic calyx polysaccharide DFPE-4 with the best effect, and studies its inhibitory and protective effects on the oxidative stress and cuproptosis of Caco2 cells induced by acrylamide. It is proved that the acidic calyx polysaccharide DFPE-4 has the most obvious inhibitory effect on the oxidative stress of Caco2 cells induced by acrylamide, can restore the intracellular copper ion content, and inhibit the cuproptosis of cells caused by acrylamide. Therefore, the acidic calyx polysaccharide DFPE-4 can be used as an important drug intermediate for the development of new drugs, or a natural raw material for functional health foods, and can also be used as an active raw material for daily chemical products. Description of the Drawings

[0044] Figure 1 It is a graph showing the results of the effects of crude polysaccharides of pitaya flower (calyx, petals) at different concentrations on the survival rate of Caco2 cells induced by acrylamide.

[0045] Figure 2 It is a gradient elution curve of 0-0.6 mol / L NaCl solution for DEAE Sepharose Fast Flow anion exchange chromatography of pitaya flower calyx polysaccharide.

[0046] Figure 3It is the result diagram of the molecular weight determination of the calyx polysaccharide of pitaya flower.

[0047] Figure 4 It is the result diagram of the effect of purified calyx polysaccharide of pitaya flower at different concentrations on the survival rate of acrylamide-induced Caco2 cells.

[0048] Figure 5 It is the result diagram of the effect of calyx polysaccharide DFPE-3 and DFPE-4 of pitaya flower on SOD of acrylamide-induced Caco2 cells.

[0049] Figure 6 It is the result diagram of the effect of calyx polysaccharide DFPE-4 of pitaya flower at different concentrations on the Cu 2+ content of acrylamide-induced Caco2 cells.

[0050] Figure 7 It is the result diagram of the effect of calyx polysaccharide DFPE-4 of pitaya flower at different concentrations on the expression of cuproptosis-related proteins in acrylamide-induced Caco2 cells.

[0051] Figure 8 It is the result diagram of the effect of calyx polysaccharide DFPE-4 of pitaya flower on the expression of cuproptosis-related proteins in acrylamide-induced Caco2 cells. Detailed implementation mode

[0052] The present invention will be further described in detail below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. The test methods without specific experimental conditions in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained through commercial channels.

[0053] Example 1

[0054] I. Extraction of crude polysaccharide from pitaya flower

[0055] A method for extracting crude polysaccharide from pitaya flower specifically includes the following steps:

[0056] Take the waste materials from flower picking during the pitaya planting process (the waste materials from flower picking in this example are provided by Guangxi Long'an Najintian Agricultural Investment Co., Ltd. and are obtained by thinning flowers after the red-fleshed pitaya trees bloom). Separate the calyx and petals, dry them at 60 °C respectively, crush them with a pulverizer and pass through an 80-mesh sieve. Add deionized water at a solid-liquid ratio of 1:15 (g / ml), extract polysaccharides in a 90 °C water bath for 3 h. After the extraction, centrifuge (4500 r / min, 15 min), and collect the supernatant. After filtering with filter paper, repeat the above process for the residue for one more extraction, combine the two supernatants, concentrate under reduced pressure to 1 / 4 of the original volume, slowly add 4 times the volume of absolute ethanol while stirring, place at 4 °C for 12 h, and centrifuge to obtain the precipitate. After evaporating the ethanol to dryness, dissolve it with an appropriate amount of water. After re-dissolution, remove proteins by the Sevag method and decolorize by the AB-8 macroporous resin (Shanghai Yuanye) method. After rotary evaporation and dialysis (cut-off molecular weight 3000 Da, dialyze with deionized water at 4 °C for 48 h), freeze-dry with a freeze dryer to obtain crude polysaccharides from pitaya flowers (crude polysaccharides from pitaya flower calyx and crude polysaccharides from pitaya flower petals).

[0057] II. Determination of the effect of crude polysaccharides from pitaya flowers on the survival rate of acrylamide-induced Caco2 cells by CCK8 method

[0058] Culture cells using the conventional cell culture method. Take Caco2 cells (from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) in the logarithmic growth phase, count them and adjust the cell concentration, inoculate them in a 96-well plate, 1×10 4 cells / well, 100 μl per well. After culturing in a CO2 incubator for 24 h, add 100 μl of crude polysaccharides from pitaya flowers (calyx and petals) respectively, so that the final concentration acting on the cells is 100 μg / mL, 200 μg / mL, 300 μg / mL (crude polysaccharides from petals: B100, B200, B300; crude polysaccharides from calyx: E100, E200, E300), and set 3 parallel wells for each concentration. After 24 h, add acrylamide to a final concentration of 5 mmol / mL. After 24 h, aspirate the original culture medium, wash twice with PBS, add 100 μl of CCK8 solution (5 mg / ml) to each well, continue to culture for 2 h, and measure the A490 value of each well with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the OD value of A490 at different concentrations. Among them, the negative control group (CON) is to add 100 μl of DEME culture medium with the same volume; the model group (MOD) is to add acrylamide to a final concentration of 5 mmol / mL.

[0059] The experimental results are as Figure 1 shown: When the concentration of crude polysaccharides from pitaya flower calyx is 100 - 300 μg / ml, it has an obvious inhibitory effect on the death of acrylamide-induced Caco2 cells. Therefore, choose the crude polysaccharides from pitaya flower calyx for subsequent experiments.

[0060] Example 2

[0061] 1. Purification of crude polysaccharide by DEAE Sepharose Fast Flow anion exchange chromatography

[0062] ① Column packing: Pack the column by the wet method. Add distilled water to the column and maintain a small liquid level. Use a glass rod to guide the homogenate to slowly pour along the inner wall of the column into the column without generating bubbles. Open the outlet of the column to allow the gel to freely settle in the column, and wash the edge of the chromatography column with water. There should be no break during column packing, and then equilibrate with distilled water. The column specifications are 2.6×60 cm.

[0063] ② Equilibration: Pass distilled water through a 0.45 μm filter membrane and wash the column with 5 column volumes of water at a certain flow rate until the conductivity and pH of the effluent remain unchanged, and equilibrate for 48 hours.

[0064] ③ Sample loading: Dissolve the crude pitaya calyx polysaccharide extracted in Example 1 in deionized water, and prepare it into a 1% (w / v) solution with deionized water. Perform suction filtration using a membrane with a pore size of 0.45 μm and load the sample. When loading the sample, open the outlet of the chromatography column. When the distilled water moves down to the surface of the column bed, close the outlet. Use a dropper to add the pitaya flower polysaccharide solution along the inner wall of the column, open the outlet, let the sample solution all flow into the column interior, and rinse the inner wall of the column with a small amount of distilled water, and then perform elution.

[0065] ④ Elution: Perform gradient elution with 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mol / L NaCl solutions respectively. Each elution is about 5 column volumes, with a flow rate of 1 mL / min. Collect 10 mL in each tube and detect by the phenol-sulfuric acid method until no polysaccharide is detected. Take the elution tube number as the abscissa and the absorbance value as the ordinate to make the elution curve of the DEAE Sepharose Fast Flow anion exchange chromatography column. Combine the solutions of each effluent peak respectively and concentrate under reduced pressure to 1 / 10 of the volume. The part eluted with water (the concentration of NaCl solution is 0) is neutral pitaya flower polysaccharide; the parts eluted with different concentrations of NaCl solutions are all acidic pitaya flower polysaccharides. The gradient elution curve of the crude pitaya calyx polysaccharide with 0 - 0.6 mol / L NaCl solution by DEAE Sepharose Fast Flow anion exchange chromatography is as Figure 2 shown. According to the elution results, a total of 4 components are obtained: named neutral calyx polysaccharide DFPE - 0 and acidic calyx polysaccharides DFPE - 2, DFPE - 3, DFPE - 4 respectively.

[0066] ⑤ Dialysis: Cut the dialysis bag into sections about 10 cm long, boil in distilled water for 30 min, wash three times and then boil for another 10 min, and wash clean with distilled water. For the second time, boil and wash with a solution containing 2% (w / v) NaHCO3 and 1 mmol / L ethylenediaminetetraacetic acid (EDTA). Transfer the concentrated eluate into a dialysis bag with a molecular weight cut-off of 3000 Da, about 1 / 2 volume, clamp both ends of the dialysis bag with a dialysis clip, and dialyze in distilled water for 36 h, changing the water every 4 h.

[0067] ⑥ Freeze-drying: Collect the pitaya flower polysaccharide eluate in 50 mL centrifuge tubes, pre-freeze at -20 °C for 12 h, and place in a freeze dryer for 36 h (the freeze dryer used is SCIENTZ-12N / A, vacuum degree: 0.05 - 0.07 MBar; temperature: -60 °C). After taking out, seal and store in a desiccator to obtain purified pitaya flower calyx polysaccharides (DFPE-0, DFPE-2, DFPE-3, DFPE-4).

[0068] II. Determination of the molecular weight of pitaya flower calyx polysaccharides

[0069] Entrust Borui Sugar Biotechnology Co., Ltd. to determine the molecular weight of the pitaya flower calyx polysaccharide DFPE-4. The specific steps are as follows:

[0070] (1) Preparation of sample and standard solutions

[0071] Preparation of standard solution: Weigh accurately 5 mg of each standard (dextran, with molecular weights of 5, 10, 20, 50, 100, 200, 400, 800 Da) and dissolve in 1 ml of mobile phase solution (0.05 M NaCl solution) to prepare a 5 mg / ml solution, and transfer the sample into a 1.8 ml injection vial.

[0072] Preparation of sample solution: Weigh accurately 5 mg of the pitaya flower calyx polysaccharide sample, dissolve in 1 ml of mobile phase solution (0.05 M NaCl solution) to prepare a 5 mg / ml solution, sonicate for 10 min, and centrifuge at 12000 rpm for 10 min. Pipette the supernatant, filter through a 0.22 μm hydrophilic microfiltration membrane, and then transfer the sample into a 1.8 ml injection vial.

[0073] (2) Selection of chromatographic method

[0074] Mobile phase: 0.05M NaCl solution; Chromatographic column: BRT105-103-101 tandem gel column (8×300mm); Flow rate: 0.7ml / min; Column temperature: 40°C; Injection volume: 100μl; Detector: Differential refractive index detector RID-20A. Among them, the preparation method of the mobile phase is: Weigh 5.844g of NaCl, dissolve it in pure water, transfer and make up the volume to a 2L volumetric flask, ultrasonicate for 10 min, and then filter through a 0.22μm filter membrane by suction filtration.

[0075] (3) Analysis steps

[0076] Place the standard solution on the sample injection tray, select the above chromatographic method for analysis, obtain the retention time, plot the standard curves of lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw weight-average molecular weight), and lgMn-RT (Mn number-average molecular weight), and obtain the molecular weight calculation formula.

[0077] Place the sample solution on the sample injection tray, select the above chromatographic method for analysis, obtain the chromatogram and retention time, and substitute the retention time into the formula to obtain the molecular weight (Mp, Mw, Mn).

[0078] The results are as Figure 3 shown in Table 1.

[0079] Table 1

[0080] Sample RT (min) lgMp lgMw lgMn Mp Mw Mn Peak area ratio % DFPE-4 28.950 5.7 5.7 5.7 546122 535482 526108 60.711 Mobile phase 51.197 3.1 3.1 3.1 1181 1212 1155 39.289

[0081] Example 3

[0082] Experiment on the effects of pitaya flower polysaccharide on oxidative stress and cuproptosis-related indicators in acrylamide-induced Caco2 cells. I. Determination of the effect of purified pitaya flower calyx polysaccharide on the viability of acrylamide-induced Caco2 cells by CCK8 method

[0083] Culture cells using the conventional cell culture method. Take Caco2 cells in the logarithmic growth phase, count and adjust the cell concentration, inoculate them in a 96-well plate, 1×10 4Per well, add 100 μl, and culture in a CO₂ incubator for 24 hours. Then, add 100 μl of the purified pitaya flower calyx polysaccharide (DFPE-0, DFPE-2, DFPE-3, DFPE-4; abbreviated as E0, E2, E3, E4) from Example 2 respectively, so that the final concentration acting on the cells is 300 μg / mL. Set 3 parallel wells for each concentration. After 24 hours, add acrylamide to a final concentration of 5 mmol / mL. After 24 hours, aspirate the original medium, wash twice with PBS, add 100 μl of CCK8 solution (5 mg / ml) to each well, and continue to culture for 2 hours. Measure the A490 value of each well with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the OD value of A490 at different concentrations. Among them, the negative control group (CON) is added with 100 μl of DEME medium of equal volume, and the model group (MOD) is added with acrylamide to a final concentration of 5 mmol / mL.

[0084] The experimental results are as Figure 4 shown: The pitaya flower calyx polysaccharides (DFPE-3, DFPE-4) eluted with 0.3 and 0.4 M sodium chloride solutions have obvious inhibitory effects on acrylamide-induced Caco2 cell death.

[0085] II. Determination of the effects of pitaya flower calyx polysaccharide on acrylamide-induced oxidative stress-related indicators in Caco2 cells

[0086] Cells were cultured using the conventional cell culture method. Take Caco2 cells in the logarithmic growth phase, count and adjust the cell concentration, and inoculate them in a 96-well plate, 1×10 4 cells / well, 100 μl per well. After culturing in a CO₂ incubator for 24 hours, add 100 μl of the purified pitaya flower calyx polysaccharides DFPE-3 (abbreviated as E3) and DFPE-4 (abbreviated as E4) respectively, so that the final concentration acting on the cells is 300 μg / mL. Set 3 parallel wells for each concentration. After 24 hours, add acrylamide to a final concentration of 5 mmol / mL. After 24 hours, aspirate the original medium and measure according to the instructions of the SOD detection kit. Among them, the negative control group (CON) is added with 100 μl of DEME medium of equal volume, and the model group (MOD) is added with acrylamide to a final concentration of 5 mmol / mL.

[0087] The experimental results are as Figure 5 shown: The pitaya flower calyx polysaccharide DFPE-4 eluted with 0.4 M sodium chloride solution has obvious inhibitory effects on acrylamide-induced oxidative stress in Caco2 cells.

[0088] III. Effects of pitaya flower calyx polysaccharide DFPE-4 at different concentration gradients on acrylamide-induced oxidative stress and cuproptosis-related indicators in Caco2 cells

[0089] Cells were cultured using the conventional cell culture method. Caco2 cells in the logarithmic growth phase were taken, counted, and the cell concentration was adjusted. Then, the cells were inoculated into 96-well plates at a density of 1×10 4 cells / well, with 100 μl per well. After culturing in a CO2 incubator for 24 hours, 100 μl of purified pitaya flower calyx polysaccharide DFPE-4 was added to each well, so that the final concentration acting on the cells was 100 μg / mL, 200 μg / mL, and 300 μg / mL (abbreviated as E100, E200, and E300), and 3 parallel wells were set for each concentration. After 24 hours, acrylamide was added to a final concentration of 5 mmol / mL to aspirate the original culture medium. After 24 hours, the detection was carried out according to the instructions of the SOD detection kit and the copper ion detection kit. Among them, the negative control group (CON) was 100 μl of DEME culture medium with the same volume, and the model group (MOD) was added with acrylamide to a final concentration of 5 mmol / mL.

[0090] The experimental results are as Figure 6 and Figure 7 shown: The pitaya flower calyx polysaccharide eluted with 0.4M sodium chloride had an obvious inhibitory effect on acrylamide-induced oxidative stress in Caco2 cells at 100-300 μg / ml, and could significantly reduce the increase in intracellular copper ion content induced by acrylamide.

[0091] Furthermore, the changes in copper death-related proteins FDX1, LIAS, and HSP70 protein after acrylamide treatment were detected. The results are as Figure 8 shown: After acrylamide treatment, copper death-related proteins FDX1, LIAS, etc. changed significantly compared with the control group, and the expression of related proteins was significantly restored after treatment with pitaya flower calyx polysaccharide DFPE-4; the expression of HSP70 protein increased after acrylamide treatment and was significantly restored after administration of pitaya flower calyx polysaccharide DFPE-4, indicating that acrylamide treatment would lead to an increase in HSP70, resulting in acute protein toxicity stress and ultimately cell death. Treatment with pitaya flower calyx polysaccharide DFPE-4 could effectively improve the production of HSP70 and thus protect cells.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of pitaya flower polysaccharide, characterized in that, It includes the following steps: (1) Extract crude polysaccharide from pitaya flower by water extraction and alcohol precipitation Dry and crush the calyx of pitaya flower and sieve it to obtain pitaya flower calyx powder; then add it to water and extract it under the water bath condition of 90±5°C. After extraction, centrifuge and filter, take the supernatant to obtain pitaya flower calyx extract; then add anhydrous ethanol to the pitaya flower calyx extract after decompression concentration, let it stand for alcohol precipitation, then centrifuge to obtain the precipitate, add water to redissolve after the ethanol in the precipitate has evaporated completely, use the Sevag method to remove protein and the macroporous resin method to decolorize, and finally concentrate, dialyze and freeze-dry to obtain crude polysaccharide from pitaya flower; (2) Purify the crude polysaccharide from pitaya flower by ion exchange chromatography Purify the crude polysaccharide from pitaya flower by DEAE Sepharose Fast Flow anion exchange chromatography, the elution solution is 0-0.4mol / L NaCl solution, and then dialyze and freeze-dry to obtain purified polysaccharide from pitaya flower.

2. The preparation method of the polysaccharide from pitaya flower according to claim 1, wherein: The concentration of the NaCl solution described in step (2) is 0, 0.2, 0.3 or 0.4mol / L.

3. The preparation method of the polysaccharide from pitaya flower according to claim 1, wherein: The macroporous resin described in step (1) is AB-8 macroporous resin; The dialysis described in steps (1) and (2) is carried out by using a dialysis bag with a molecular weight cut-off of 3000Da; The dialysis solution used for dialysis described in steps (1) and (2) is distilled water or deionized water.

4. The preparation method of the polysaccharide from pitaya flower according to claim 1, wherein: The pitaya flower described in step (1) is the flower of red-fleshed pitaya; The material-liquid ratio of the pitaya flower calyx powder to water described in step (1) is 1g:10-30ml.

5. The preparation method of the polysaccharide from pitaya flower according to claim 1, wherein: The drying temperature described in step (1) is 55-65°C; The sieving described in step (1) is through a 60-100 mesh sieve; The filtration described in step (1) is carried out by using filter paper; The extraction time described in step (1) is 2-5h; In step (1), the centrifugation conditions after extraction are: centrifuge at 4000-5000r / min for 10-20min; The decompression concentration described in step (1) is concentrated to 1 / 5-1 / 3 of the original volume; In step (1), the addition of anhydrous ethanol is to add 3-5 times the volume of anhydrous ethanol; The standing conditions described in step (1) are: place at 4°C for 12-15h; The freeze-drying conditions described in step (2) are: pre-freeze at -20°C for 12h first, and then freeze-dry at a vacuum degree of 0.05-0.07Mbar and a temperature of -60°C for 36h-38h.

6. A pitaya flower polysaccharide, characterized in that: Prepared by the method according to any one of claims 1-5.

7. Application of the polysaccharide from pitaya flower described in claim 6 in the preparation of products for inhibiting cellular oxidative stress.

8. The application according to claim 7, wherein: The cellular oxidative stress is acrylamide-induced cellular oxidative stress; The product described above includes drugs or daily chemical products.

9. Use of the pitaya flower polysaccharide according to claim 6 in the preparation of a drug for inhibiting cuproptosis and / or restoring the intracellular copper ion content.

10. The use according to claim 9, characterized in that: The cuproptosis is cuproptosis caused by acrylamide; The cells include normal cells or cancer cells.

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