Preparation method of lotus seed pot polysaccharide and novel application of lotus seed pot polysaccharide in myocardial protection

Abstract

The invention discloses a lotus seed pot polysaccharide in the technical field of plant polysaccharide extraction, and the lotus seed pot polysaccharide mainly comprises rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose in a molar ratio of (1-2): (1-2): (1.5-5): (1-4): (1-2.5). The sugar is obtained by a water extraction and alcohol precipitation method and separation and purification of an anion exchange column and a gel column. The pectic polysaccharide regulates Nrf2 to inhibit apoptosis in a range of 0.25 mg / mL-1 mg / mL so as to play a myocardial protection role of an AC16 myocardial cell injury model induced by oxygen deficit reoxygenation (H / R), the protection role is increased in a concentration-dependent manner, and compared with a model group, the anti-myocardial cell injury effect of the pectic polysaccharide is 50%-80%.

Description

Technical Field

[0001] The invention relates to the technical field of plant polysaccharide extraction, in particular to a preparation method of lotus seed pod polysaccharide and a new application thereof in myocardial protection. Background Art

[0002] Cardiovascular disease remains the leading cause of death in industrialized countries, and its morbidity and mortality are also increasing rapidly worldwide. Many factors can affect the development of cardiovascular disease, such as age, gender, smoking, lack of exercise, alcoholism, unhealthy diet, obesity, genetic susceptibility, etc. Some Western medicines are known to have a protective effect on the heart, such as calcium channel blockers, angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists. However, natural compounds have a relatively low toxicity and side effects, and people are paying more and more attention to their role in the treatment and prevention of cardiovascular disease. Well, a large number of studies have shown that Chinese herbal polysaccharides show a profound effect in preventing or protecting cardiovascular diseases, such as astragalus polysaccharides, ginseng polysaccharides, wolfberry polysaccharides, etc.

[0003] Lotus pod is the receptacle of Nelumbinis Flos., a plant of the genus Nelumbinis of the family Nymphaeaceae. Its main function is to dissipate blood stasis and stop bleeding, and it mainly treats metrorrhagia, menorrhagia, blood in the stool, hematuria, etc. In the medicinal value of the entire lotus plant, lotus pods are mostly discarded as waste, resulting in a waste of resources. The main active ingredient of the lotus pod in current research is proanthocyanidins, which have multiple effects such as anti-oxidation, anti-liver oxidative damage, anti-myocardial ischemia and lipid-lowering; it is also found that lotus pod flavonoid extracts have anti-inflammatory and antioxidant effects in vivo and in vitro. There are few studies on lotus pod polysaccharides in the prior art, and it is only found that it is an acidic heteropolysaccharide, mainly containing galacturonic acid, but its structure has not been analyzed, and there is no related report on the protection of lotus pod polysaccharides on myocardial cells. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention designs a process and method for extracting, separating and purifying a lotus pod polysaccharide LS1 from a lotus pod plant.

[0005] One of the purposes of the present invention is to provide a lotus pod polysaccharide, which is mainly composed of rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose, and the molar ratios thereof are 1-2:1-2:1.5-5:1-4:1-2.5, respectively. The weight average molecular weight of the lotus pod polysaccharide is 10-50 kDa, and the dispersion coefficient D is 1-2.5.

[0006] Furthermore, the number average molecular weight (Mn) is 3 to 10 kDa.

[0007] Furthermore, the sugar residues of the polysaccharide have the following connection modes: terminal-linked arabinose, terminal-linked rhamnose, 1,5-linked arabinose, 1,2-linked rhamnose, terminal-linked glucuronic acid, terminal-linked galactose, 1,3,5-linked arabinose, 1,2,4-linked rhamnose, 1,4-linked galactose, 1,3-linked galactose, 1,6-linked galactose, 1,3,4-linked galacturonic acid, 1,3,6-linked galactose and 1,4-linked galacturonic acid, and the molar ratios thereof are 4-8: 1-3: 2-4: 1-2: 1-2: 3-6: 2-4: 2-4; 1-3: 2-4: 2-4: 1-2: 4-8: 16-25.

[0008] Further, the polysaccharide has the following structure:

[0009]

[0010] Among them, 1,4-α-D-galactopyranosyl uronic acid and 1,2-α-L-rhamnose pyranose are the main chains, and the C-4 position of 1,2-α-L-rhamnose pyranosyl uronic acid is composed of 1,4-β-D-galactopyranose, 1,5-α-L-arabinofuranose, 1,3,5-α-L-arabinofuranose or 1,3,6-β-D-galactopyranose, 1,3-β-D-galactopyranose, 1,6-D-galactopyranose residues; and the C-3 position of 1,4-α-D-galactopyranosyl uronic acid is composed of α-L-pyranohexenuronic acid or β-D-rhamnose or β-D-pyranoglucuronic acid.

[0011] The second object of the present invention is to provide a method for preparing lotus pod polysaccharide, comprising the following steps: 1) crushing: crushing the lotus pod with a grinder, and screening 100-300 mesh lotus pod powder for standby use;

[0012] 2) Soaking: Immerse the lotus pod powder in water and soak it at room temperature for 12 to 24 hours;

[0013] 3) Grinding: Grind 2 to 4 times using a colloid mill homogenizer;

[0014] 4) Decoction: heat to maintain slight boiling and decoct for 2 to 4 hours / time, decoct 2 to 4 times in total, and combine the filtrate;

[0015] 5) Dialysis: The filtrate is concentrated to 1 / 10-1 / 20 of the original volume, cooled naturally to room temperature, and the concentrate is dialyzed against running water through cellophane for 2-3 days;

[0016] 6) Alcohol precipitation: The dialysate is concentrated to 1 / 5-1 / 10 of the original volume, cooled naturally to room temperature, centrifuged at 4000-8000 rpm for 10-30 min, and the supernatant is taken and 3-6 times the volume of the supernatant is added with 95% ethanol while stirring, and allowed to stand for 12-24 hours;

[0017] 7) Freeze drying: centrifuge the alcohol precipitate at 4000-8000 rpm for 10-30 min, add water to the precipitate and heat to evaporate the remaining ethanol, freeze it and freeze dry it in a freeze dryer to obtain crude polysaccharide;

[0018] 8) Separation: The crude polysaccharide was separated by DEAE anion exchange and eluted with 0.10 M NaCl to obtain lotus seed pod polysaccharide.

[0019] Furthermore, the liquid-to-water ratio of water to lotus pod is 1:10 to 1:30.

[0020] Furthermore, the lotus pod is made of dried or fresh receptacle.

[0021] Furthermore, the crude polysaccharide in step 8) is separated by anion exchange column DEAE FAST FLOW with a sample load ranging from 30 to 50 g and a concentration of 40 to 80 mg / mL.

[0022] The third object of the present invention is to provide the use of lotus seed pod polysaccharide in preparing drugs for protecting myocardial cells or assisting in the treatment of myocardial injury.

[0023] Specifically, the administration concentration of lotus seed pod polysaccharide is 0.25 mg / mL to 1 mg / mL.

[0024] The fourth object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned lotus pod polysaccharide and pharmaceutically acceptable excipients.

[0025] The working principle and beneficial effects of the present invention are as follows: the present invention separates and purifies a lotus pod polysaccharide LS1 from the lotus pod, and the monosaccharide composition analysis by the sugar alcohol acetate derivatization method shows that the lotus pod polysaccharide LS1 is a pectin polysaccharide, which is composed of rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose, and the molar ratio thereof is 1-2:1-2:1.5-5:1-4:1-2.5.

[0026] According to research, 0.25mg / mL to 1mg / mL lotus seed pod polysaccharide LS1 has a significant inhibitory effect on AC16 cardiomyocyte injury induced by hypoxia / reoxygenation, with an inhibition rate of 50% to 80%. Pharmacological experiments show that the polysaccharide can inhibit hypoxia / reoxygenation-induced AC16 cardiomyocyte injury in a dose-dependent manner. Therefore, the polysaccharide LS1 is expected to be developed into a polysaccharide drug that protects cardiomyocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1This is a schematic diagram of the results of HPLC purity of lotus seed pod polysaccharide LS1;

[0028] Figure 2 This is a schematic diagram of the monosaccharide composition analysis of lotus seed pod polysaccharide LS1;

[0029] Figure 3 Lotus seed polysaccharide LS1 1 H NMR (A) and 13 C NMR (B) spectrum;

[0030] Figure 4 Schematic diagram of the effect of different concentrations of LS1 on the survival rate of cardiomyocytes;

[0031] Figure 5 Schematic diagram of the effect of different concentrations of LS1 on LDH levels in cardiomyocytes;

[0032] Figure 6 Schematic diagram of the effect of different concentrations of LS1 on the gene levels of ANP and BNP in cardiomyocytes;

[0033] Figure 7 Schematic diagram of the effect of compound LS1 on the expression of related proteins. DETAILED DESCRIPTION

[0034] The following is further described in detail through specific implementation methods:

[0035] Example 1. Polysaccharide extraction

[0036] 1. Soaking: Soak dried / fresh lotus pod receptacle with water at a ratio of 1:10-1:30 (g / mL) at room temperature for 12-24 hours;

[0037] 2. Decoction: decoct with slight boiling for 2 to 4 hours / time, decoct 2 to 4 times;

[0038] 3. Dialysis: The decoction is concentrated to 1 / 10-1 / 20 of the original volume, cooled naturally to room temperature, and dialyzed against running water through cellophane for 2-3 days to remove pigments and small molecules. After the dialysis is completed, it is filtered through gauze;

[0039] 4. Alcohol precipitation: The dialysate is concentrated to 1 / 5-1 / 10 of the original volume, cooled naturally to room temperature, centrifuged at 4000-8000rpm for 10-30min, and the supernatant is taken and 3-6 times the volume of the supernatant is added with 95% ethanol while stirring, and allowed to stand overnight;

[0040] 5. Freeze drying: centrifuge the alcohol precipitate at 4000-8000rpm for 10-30min, add 0.5-3L water to the precipitate, mix well, heat to evaporate the remaining ethanol, freeze it and freeze dry it in a freeze dryer to obtain the crude polysaccharide LS extracted from lotus humus;

[0041] 6. Separation: The crude polysaccharide LS extracted from lotus pod water was separated by DEAE anion exchange and eluted with 0.10 M NaCl to obtain lotus pod polysaccharide LS1.

[0042] Example 2. Polysaccharide purity, monosaccharide composition analysis and sugar residue connection mode

[0043] 1. Determination of polysaccharide purity:

[0044] Accurately weigh 2-6 mg of polysaccharide sample, dissolve in 300-600 μL of 0.1 M sodium nitrate, oscillate to fully dissolve the sample, centrifuge at 4000-8000 rpm for 5-10 min, and filter the supernatant through a 0.22 μM aqueous filter membrane for purity determination.

[0045] Polysaccharide-specific gel chromatography columns in series: Waters UltrahydrogelTM 2000 and UltrahydrogelTM 500 in series. Chromatographic conditions: mobile phase 0.1M sodium nitrate, flow rate 0.5mL / min, injection volume 10-20μL, column temperature 35℃, UV absorption wavelength 280nm, differential detector temperature 35℃, 40-60min / sample.

[0046] 2. Use sugar alcohol acetate derivatization method to analyze the monosaccharide composition of polysaccharides:

[0047] 1) Complete acid hydrolysis: Weigh 2-4 mg of sample, dissolve in 2 mL of distilled water, vortex and oscillate to dissolve as much as possible (heating or ultrasound can be used), add the sample solution to a heart-shaped bottle (specification 50 / 19), then add 2 mL of 4M trifluoroacetic acid (TFA) and mix well, add a hollow stopper to the heart-shaped bottle, and seal the stopper and the contact port of the heart-shaped bottle with medical rubber paste, and heat and hydrolyze at 110°C for 2-5 hours. After hydrolysis, cool, add methanol and evaporate under reduced pressure for several times to remove TFA. Add 200 μL of distilled water to dissolve the hydrolyzate.

[0048] 2) Reduction and acetylation: Dissolve the completely acid-hydrolyzed sample in 50uL of distilled water and add 1mL of 0.5M NaBH 4 DMSO solution (prepared and used immediately) was added, mixed and placed in a 40°C water bath for reduction for 1 hour and 30 minutes. After the reaction was completed, 100 μL of glacial acetic acid was added dropwise, and mixed until no bubbles were generated. Then 200 μL of 1-methylimidazole and 1 mL of acetic anhydride were added, vortexed and mixed, and acetylated in a 40°C water bath for 10 minutes. After the acetylation was completed, 2 mL of distilled water was added to terminate the reaction.

[0049] 3) Preparation of standard solution: Prepare 9 mg / mL of each monosaccharide with deionized water, take 100 μL of each and mix them, then there are 7 kinds of monosaccharide standard solutions, each with a concentration of 1 mg / mL. During derivatization, take out 50 μL of the standard mixture for reduction and acetylation.

[0050] 4) Extraction: Dissolve the acetylated product in 2 mL of chloroform, then add an equal volume of distilled water for extraction, and wash the chloroform layer three times with distilled water. Dry the chloroform layer with anhydrous sodium sulfate, concentrate it to an appropriate volume, filter it with a 0.22 μL microporous membrane, and perform GC-MS analysis.

[0051] 5) Gas phase analysis (GC):

[0052] The monosaccharide standard and polysaccharide samples were derivatized according to the above-mentioned sugar alcohol acetate derivatization method, and then subjected to gas chromatography-mass spectrometry. Chromatographic analysis conditions: gas chromatograph (Agilent), TR-5MS (Thermo) chromatographic column (60m×0.25mm×0.25μm), programmed temperature conditions: starting temperature 140℃, inlet sample temperature 250℃, carrier gas helium (He), flow rate 1mL / min.

[0053] Example 3: Analysis of the connection mode of sugar residues in polysaccharides

[0054] Take the lotus pod polysaccharide component LS1 for methylation analysis. Weigh 5-12 mg of the sample in advance into a 50 mL chicken heart bottle, add 2 mL of deionized water to dissolve, freeze-dry and place in a drying cabinet overnight (the reaction is guaranteed to be anhydrous), and completely dissolve in 2 ml of anhydrous DMSO the next day. After the polysaccharide sample is completely dissolved (if the sample is not well soluble in DMSO, it can be heated in a 70 ° C oil bath and stirred overnight to dissolve), add 50 mg of ground dry sodium hydroxide powder, and stir the reaction at room temperature for 7 minutes. In an ice water bath, slowly add 1 mL of iodomethane (about 35 minutes), remove the ice bath, and react at room temperature for 30 minutes away from light. After the reaction is completed, add 1 ml of deionized water to the reaction system to quench the reaction. The solution is concentrated under reduced pressure to remove excess unreacted iodomethane, dialyze against deionized water for 24 to 72 hours, and freeze-dry the solution. Repeat 4 to 5 times again. The sample after the reaction was completely acid hydrolyzed, 4 ml of 2M TFA was added and reacted at 110°C for 4 h. After cooling to room temperature, methanol was added several times to remove excess acid until there was no sour taste. After the reaction, 2 ml of water and 50 mg of sodium borohydride were added for reduction. After sealing, the reaction was carried out at room temperature for 3 h. Sodium borohydride was neutralized with 25% acetic acid solution to terminate the reaction. Methanol was repeatedly added to remove excess acid, and the sample was placed in a 100°C oven for drying for 15 min. 3 ml of acetic anhydride was added and placed in a 100°C oven for acetylation. The reaction was carried out for 1.5 h. Toluene was repeatedly added to remove excess acetic anhydride. Finally, 15 mL of chloroform and 15 mL of deionized water (v / v was 1:1) were added to the reaction bottle, extracted, and the aqueous phase was discarded. The organic phase was washed with deionized water 5 times, dried over anhydrous sodium sulfate, and the chloroform was concentrated under reduced pressure to 200 μL, filtered through a 0.22 μm organic phase filter membrane, and placed in a liquid phase vial. The connection mode of polysaccharides was detected and analyzed by GC-MS (Thermo Fisher ISQ7000).

[0055] like Figure 1 , 2 As shown, Figure 1 , LS1 differential detection diagram; Figure 2 , A: The peak order of the monosaccharide composition of 7 monosaccharide standards measured by sugar alcohol acetate derivatization and the LS1 monosaccharide composition analysis (Rha: rhamnose; Fuc: fucose; Ara: arabinose; Xyl: xylose; Man: mannose; Glc: glucose; Gal: galactose).

[0056] HPGPC purity analysis showed that the weight average molecular weight (Mw) of LS1 was 10-50 kDa, the number average molecular weight (Mn) was 3-10 kDa, and the dispersion coefficient D was 1-2.5.

[0057] Comparison of the monosaccharide composition of LS1 derivatized with sugar alcohol acetate before and after reduction showed that LS1 was an acidic polysaccharide containing rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose in a molar ratio of 1-2:1-2:1.5-5:1-4:1-2.5.

[0058] The analysis of the sugar residue connection patterns of LS1 by GC-MS showed that the following connection patterns were found in LS1: terminal-linked arabinose, terminal-linked rhamnose, 1,5-linked arabinose, 1,2-linked rhamnose, terminal-linked glucuronic acid, terminal-linked galactose, 1,3,5-linked arabinose, 1,2,4-linked rhamnose, 1,4-linked galactose, 1,3-linked galactose, 1,6-linked galactose, 1,3,4-linked galacturonic acid, 1,3,6-linked galactose and 1,4-linked galacturonic acid, and the molar ratios were 4-8: 1-3: 2-4: 1-2: 1-2: 3-6: 2-4: 2-4; 1-3: 2-4: 2-4: 1-2: 4-8: 16-25.

[0059] Application example: Determination of polysaccharide anti-cardiomyocyte injury

[0060] (1) Adjust the cell density of AC16 cells in the logarithmic growth phase to 5×10 4 cells / mL, inoculated into a 96-well microplate at 100 μL / well and placed in CO 2 Incubate in an incubator for 12 hours;

[0061] (2) Prepare each polysaccharide compound (3 mg / ml stock solution) with DMEM medium, and then dilute it with DMEM medium to 1, 0.5, and 0.25 mg / mL concentrations and administer it to cells for 24 hours. Then, replace the medium of the H / R group and each drug group with low-glucose medium and place it in an anoxic incubator for hypoxia induction treatment for 20 hours. After taking it out, replace it with high-glucose medium and place it in a normal incubator for reoxygenation treatment for 2 hours. The blank control group does not undergo H / R treatment. Each concentration has 3 replicates. After the cell treatment, add 5 mg / ml MTT solution, 20 μL / well; continue to culture for 3 hours, and then add 120 μL / well DMSO to each culture plate to dissolve the reaction product so that the optical density value (OD value) can be measured under an ELISA instrument.

[0062] The experimental observation index is: using an enzyme-labeled instrument to detect the OD value of each well of the 96-well plate at a wavelength of 490 nm to calculate the cell viability.

[0063] The results showed that the effect of lotus seed pod polysaccharide LS1 on cardiomyocyte activity ( Figure 4), treated the cells with different concentrations of LS1 (0.25, 0.5, 1 mg / mL) for 24 h, and then induced cardiomyocyte injury. The cell survival rate was calculated by CCK-8 and compared with the blank control group ( Figure 4 ), the cell survival rate of myocardial cells was significantly reduced to about 50% after injury (P<0.001). Then, LS1, 0.5 and 1 mg / mL of LS1, was given to inhibit myocardial cell death to varying degrees (P<0.05, P<0.001).

[0064] The results showed that the effect of lotus seed pod polysaccharide LS1 on LDH level in cardiomyocytes Figure 5 ), treated the cells with different concentrations of LS1 (0.25, 0.5, 1 mg / mL) for 24 h, then induced myocardial cell injury, collected the cells to detect LDH levels, and detected by the kit ( Figure 5 ), compared with the normal group cells, the LDH level in the model group was significantly increased after cell injury (P<0.01). Then, the administration of lotus seed pod polysaccharide LS1 could inhibit the LDH level in cardiomyocytes to varying degrees (P<0.05, P<0.01).

[0065] The results showed that the effect of lotus seed pod polysaccharide LS1 on the expression level of myocardial injury markers ( Figure 6 ), treated the cells with different concentrations of LS1 and LS1 (0.25, 0.5, 1 mg / mL) for 24 h, then induced myocardial cell injury, and collected the cells to detect the gene expression levels of ANP and BNP. Figure 6 The results showed that the expression levels of related genes ANP and BNP in the cells after myocardial cell injury were significantly higher than those in the blank control group (P < 0.001); 0.5 and 1 mg / mL of lotus pod polysaccharide LS1 could significantly inhibit the gene expression level of ANP (P < 0.05, P < 0.01), in addition, 1 mg / mL of lotus pod polysaccharide LS1 could also significantly inhibit the gene expression level of BNP (P < 0.05).

[0066] The results showed that the effect of lotus seed pod polysaccharide LS1 on the expression level of some proteins Figure 7 ), Western Blot is a method that can detect protein expression. By detecting the protein expression levels in different groups of cells, the mechanism of action of the compound against myocardial injury can be inferred. Figure 4As shown, 1 mg / mL of lotus pod polysaccharide LS1 can significantly increase the ratio of Bcl-2 / Bax (*P<0.05), and 0.25, 0.5 and 1 mg / mL of lotus pod polysaccharide LS1 can promote the protein expression levels of Nrf2 and phosphorylated AKT to varying degrees (P<0.05, P<0.01, P<0.001). Based on this result, it is speculated that the compound lotus pod polysaccharide LS1 can inhibit the nuclear translocation of Nrf2 due to cell damage, and inhibit cell apoptosis by regulating the ratio of Bcl-2 / Bax.

[0067] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A lotus seed pod polysaccharide, Features: It is mainly composed of rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose, and the molar ratios thereof are 1-2:1-2:1.5-5:1-4:1-2.

5. The weight average molecular weight of the lotus pod polysaccharide is 10-50 kDa, and the dispersion coefficient D is 1-2.

5.

2. The lotus pod polysaccharide according to claim 1, Features: The sugar residues of the polysaccharide have the following connection modes: terminal-connected arabinose, terminal-connected rhamnose, 1,5-connected arabinose, 1,2-connected rhamnose, terminal-connected glucuronic acid, terminal-connected galactose, 1,3,5-connected arabinose, 1,2,4-connected rhamnose, 1,4-connected galactose, 1,3-connected galactose, 1,6-connected galactose, 1,3,4-connected galacturonic acid, 1,3,6-connected galactose and 1,4-connected galacturonic acid, and the molar ratios thereof are 4-8:1-3:2-4:1-2:1-2:3-6:2-4:2-4; 1-3:2-4:2-4:1-2:4-8:16-25.

3. The lotus pod polysaccharide according to claim 1, Features: The polysaccharide has the following structure: Among them, 1,4-α-D-galactopyranosyl uronic acid and 1,2-α-L-rhamnose pyranose are the main chains, and the C-4 position of 1,2-α-L-rhamnose pyranosyl uronic acid is composed of 1,4-β-D-galactopyranose, 1,5-α-L-arabinofuranose, 1,3,5-α-L-arabinofuranose or 1,3,6-β-D-galactopyranose, 1,3-β-D-galactopyranose, 1,6-D-galactopyranose residues; and the C-3 position of 1,4-α-D-galactopyranosyl uronic acid is composed of α-L-pyranohexenuronic acid or β-D-rhamnose or β-D-pyranoglucuronic acid.

4. The method for preparing lotus pod polysaccharide according to any one of claims 1 to 3, Features: The following steps are involved: 1) Crushing: crush the lotus pods with a crusher, and select 100-300 mesh lotus pod powder for later use; 2) Soaking: Immerse the lotus pod powder in water and soak it at room temperature for 12 to 24 hours; 3) Grinding: Grind 2 to 4 times using a colloid mill homogenizer; 4) Decoction: heat to maintain slight boiling and decoct for 2 to 4 hours / time, decoct 2 to 4 times in total, and combine the filtrate; 5) Dialysis: The filtrate is concentrated to 1 / 10-1 / 20 of the original volume, cooled naturally to room temperature, and the concentrate is dialyzed against running water through cellophane for 2-3 days; 6) Alcohol precipitation: The dialysate is concentrated to 1 / 5-1 / 10 of the original volume, cooled naturally to room temperature, centrifuged at 4000-8000 rpm for 10-30 min, and the supernatant is taken and 3-6 times the volume of the supernatant is added with 95% ethanol while stirring, and allowed to stand for 12-24 hours; 7) Freeze drying: centrifuge the alcohol precipitate at 4000-8000 rpm for 10-30 min, add water to the precipitate and heat to evaporate the remaining ethanol, freeze it and freeze dry it in a freeze dryer to obtain crude polysaccharide; 8) Separation: The crude polysaccharide was separated by DEAE anion exchange and eluted with 0.10 M NaCl to obtain lotus seed pod polysaccharide.

5. The preparation method according to claim 4, Features: The liquid ratio of water to lotus pod is 1:10 to 1:

30.

6. The preparation method according to claim 5, Features: The crude polysaccharide in step 8) is separated by anion exchange column DEAEFAST FLOW with a sample loading range of 30 to 50 g and a concentration of 40 to 80 mg / mL.

7. Use of the lotus seed pod polysaccharide according to any one of claims 1 to 3 in the preparation of a drug for protecting myocardial cells or a drug for assisting the treatment of myocardial injury.

8. The use according to claim 7, Features: The dosage concentration of lotus pod polysaccharide is 0.25mg / mL to 1mg / mL.

9. A pharmaceutical composition comprising the lotus pod polysaccharide according to any one of claims 1 to 3, and pharmaceutically acceptable excipients.

Images