Plackett-burman design and path analysis for improving the yield of polysaccharides from terminalia ferdinandiana bailey and the preparation method and application thereof

Abstract

The application belongs to the technical field of plant polysaccharides, and particularly relates to a Plukenetia volubilis shell polysaccharide with improved vascular function and a preparation method and application thereof. The Plukenetia volubilis shell polysaccharide is polymerized by galactose, arabinose, rhamnose and galacturonic acid, and has a structure as shown in formula (I); formula (I). The Plukenetia volubilis shell polysaccharide provided in the application can be used for improving vascular function and maintaining a healthy level of blood pressure. The Plukenetia volubilis shell polysaccharide with a specific fine structure has a purity of 95.04%, and greatly reduces the residues of impurities such as proteins and lipids, thereby solving the problems of difficult extraction and low purity of the Plukenetia volubilis shell polysaccharide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant polysaccharides, and particularly relates to a Plukenetia volubilis shell polysaccharide with improved vascular function, and a preparation method and application thereof. BACKGROUND

[0002] Vascular endothelial cells generally refer to a monolayer of flat epithelium lining the inner surface of the heart, blood vessels and lymphatic vessels, and are an important component of the blood vessel wall. Vascular endothelial cells have the functions of regulating vasomotion, phagocytizing harmful substances in blood vessels, immune regulation and anti-thrombosis. Dysfunction of vascular endothelial cells can cause an increase in the permeability of the blood vessel wall and an increase in the viscosity of the blood, thereby easily causing thrombosis or embolic diseases. In addition, vascular endothelial cells can secrete vasodilator nitric oxide (NO) and vasoconstrictor endothelin-1 (ET-1) to regulate vasodilation and vasoconstriction, thereby maintaining the stability of blood pressure in the body. However, the dysfunction of vascular endothelial cells can cause an imbalance in the secretion of vasodilators and vasoconstrictors in the body, leading to an increase in blood pressure in the human body, and thereby causing a series of complications such as renal dysfunction and myocardial infarction. The drugs currently used to treat vascular endothelial cell damage usually have large side effects and certain toxicity. Therefore, it is imperative to find a treatment method for endothelial cell damage with small side effects and low toxicity.

[0003] Polysaccharides refer to high-molecular-weight carbohydrates with more than ten monosaccharides connected by glycosidic bonds. They have attracted widespread attention in the food and biomedical industries due to their non-toxicity, good biological activity, biocompatibility and biodegradability. Polysaccharides are involved in various biological metabolisms in the human body and have various biological effects such as blood pressure reduction, liver protection, anti-depression and blood glucose reduction.

[0004] Plukenetia volubilis is a plant with rich nutritional value and multiple uses. Plukenetia volubilis can be used for beauty, treatment of muscle pain, rheumatoid arthritis and atopic dermatitis, etc. Plukenetia volubilis oil and Plukenetia volubilis protein are mainly used in the food industry. The water extract of Plukenetia volubilis shell is reported to have the effect of reducing blood pressure. Plukenetia volubilis shell polysaccharide has the effects of immune regulation and alleviating kidney damage. However, the purity and activity of the Plukenetia volubilis shell polysaccharide prepared at present are still not good, and the research on its application also needs to be further developed. SUMMARY

[0005] The present application provides a Plukenetia volubilis shell polysaccharide, a preparation method and application thereof, effectively solving the problems of difficult extraction and low extraction purity of Plukenetia volubilis shell polysaccharide.

[0006] The present application provides a Plukenetia volubilis shell polysaccharide, a preparation method and application thereof, effectively solving the problems of difficult extraction and low extraction purity of Plukenetia volubilis shell polysaccharide.

[0007] Formula (I).

[0008] The application further provides a preparation method of the Plukenetia volubilis shell polysaccharide.

[0009] S1, after the Plukenetia volubilis shell is pulverized, hot water extraction, freeze-drying, the Plukenetia volubilis shell water extract is subjected to fractional extraction with ethanol solution, and the obtained component is subjected to ultrafiltration treatment to obtain a solution one;

[0010] S2, after the solution one is subjected to protein removal treatment, the water phase is collected, dialysis is performed to obtain a solution two;

[0011] S3, the solution two is subjected to decolorization treatment, freeze-drying is performed to obtain Plukenetia volubilis shell crude polysaccharide powder;

[0012] S4, after the Plukenetia volubilis shell crude polysaccharide powder is subjected to purification with an anion exchange chromatography column, dialysis is performed, freeze-drying is performed, and the obtained powder is subjected to purification with a dextran gel column to obtain the Plukenetia volubilis shell polysaccharide.

[0013] Further, in step S1, the temperature of hot water extraction is 70-90 ℃, and the time of hot water extraction is 1-5 h;

[0014] In step S1, the molecular weight of the ultrafiltration membrane is 10-50 kDa;

[0015] In step S1, the fractional extraction with the ethanol solution specifically comprises the following steps: after the Plukenetia volubilis shell water extract is subjected to fractional extraction with anhydrous ethanol, an ethanol solution with a volume concentration of 75% and an ethanol solution with a volume concentration of 50%, ethanol is removed by rotary evaporation, and the component obtained after the ethanol solution with a volume concentration of 50% is collected.

[0016] Further, in step S2, the protein removal treatment specifically comprises the following steps: ammonium sulfate is added to the solution one, an equal volume of tert-butyl alcohol is added, constant-temperature oscillation is performed, standing is performed, and the water phase is collected.

[0017] Preferably, after the ammonium sulfate is added, the saturation degree of the ammonium sulfate is 10-30%.

[0018] Preferably, the equal volume of tert-butyl alcohol specifically refers to that the volume ratio of the tert-butyl alcohol to the added solution is 1:1.

[0019] Further, in step S3, the decolorization treatment specifically comprises the following steps: activated carbon is added to the solution two, constant-temperature oscillation is performed, centrifugation is performed, suction filtration is performed, and the supernatant is collected.

[0020] Preferably, in step S3, the mass ratio of the activated carbon to the solution two is 0.5%-3%. Preferably, the mass ratio of the activated carbon to the solution two is 2.5%.

[0021] Further, the purification by using the anion exchange chromatography column in step S4 specifically comprises: gradient elution by using membrane water, 0.1M and 0.3M NaCl solution in sequence, and collecting the polysaccharide component eluted by 0.3M NaCl solution.

[0022] Further, the purification by using the Sephadex column in step S4 specifically comprises: adding the powder purified by the anion exchange chromatography column into the Sephadex column, eluting by using water, and collecting the component with high response value.

[0023] Preferably, in the elution by using water in step S4, the loading amount is 2mL, the flow rate is 0.2mL / min, and the component collected is 150-255min.

[0024] The application further provides application of the above-mentioned Opuntia tuna husk polysaccharide or the Opuntia tuna husk polysaccharide prepared by any one of the above-mentioned preparation methods in preparation of a product for improving vascular function or a product for maintaining a healthy level of blood pressure.

[0025] Further, the product comprises a medicine, a health food or a food.

[0026] The application further provides application of the above-mentioned Opuntia tuna husk polysaccharide or the Opuntia tuna husk polysaccharide prepared by any one of the above-mentioned preparation methods in preparation of a medicine for improving vascular endothelial cell damage.

[0027] The application has the following advantages:

[0028] The Opuntia tuna husk polysaccharide and the preparation method thereof have the following advantages:

[0029] The Opuntia tuna husk polysaccharide can be used for improving vascular function and maintaining a healthy level of blood pressure, has the effect of treating vascular endothelial cell damage, promotes secretion of endothelial cell vasodilator NO and reduces expression of vasoconstrictor ET-1, and improves the antioxidant capacity of vascular endothelial cells, thereby providing support for application of the Opuntia tuna husk polysaccharide. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0031] Figure 1 The active carbon decoloring process optimization in the test example 1 of the application;

[0032] Figure 2 The anion exchange column elution curve in the test example 1 of the application;

[0033] Figure 3 The molecular weight and uniformity detection spectrum of the sericosphaera intermedia shell polysaccharide in the test example 2 of the application;

[0034] Figure 4 The full wavelength scanning graph of the sericosphaera intermedia shell polysaccharide in the test example 2 of the application;

[0035] Figure 5 The Fourier infrared spectrum of the sericosphaera intermedia shell polysaccharide in the test example 2 of the application;

[0036] Figure 6 The total ion flow graph and secondary proton chromatogram of the sericosphaera intermedia shell polysaccharide glycosidic bond type detection in the test example 2 of the application;

[0037] Figure 7 The nuclear magnetic spectrum of the sericosphaera intermedia shell polysaccharide in the test example 2 of the application;

[0038] Figure 8 The scanning electron microscope graph of the sericosphaera intermedia shell polysaccharide in the test example 2 of the application;

[0039] Figure 9 The sericosphaera intermedia shell polysaccharide influence graph on the endothelial cell survival rate, NO secretion and ET-1 secretion level in the test example 3 of the application; (average value ± standard deviation, **** p<0.0001 compared with the control group CON, #p<0.05; ##p<0.01; ###p<0.001 compared with the model group ANG);

[0040] Figure 10 The sericosphaera intermedia shell polysaccharide graph of improving the antioxidant capacity of endothelial cells in the test example 3 of the application; (average value ± standard deviation, *p<0.05; **** p<0.0001 compared with the control group CON, #p<0.05; ##p<0.01; ###p<0.001 compared with the model group ANG);

[0041] Figure 11 The sericosphaera intermedia shell polysaccharide graph of regulating the expression of endothelial cell function related genes in the test example 3 of the application; (average value ± standard deviation, **** p<0.0001 compared with the control group CON, ##p<0.01; ###p<0.001; ####p<0.0001 compared with the model group ANG);

[0042] Figure 12Figure for regulating the secretion of endothelial cell function-related protein by the Serjania polyssacharide in the test example 3 of the present application; (average value ± standard deviation, **p<0.01; ***p<0.001; ****p<0.0001 compared with the control group CON, #p<0.05; ##p<0.01; ###p<0.001 compared with the model group ANG). DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. In the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0044] In one aspect, the present application provides a Serjania polyssacharide, which is polymerized by galactose, arabinose, rhamnose and galacturonic acid, and has a structure as shown in formula (I).

[0045] Formula (I).

[0046] It should be pointed out that among the four kinds of monosaccharides provided in the embodiments of the present application, galactose is abbreviated as Gal, rhamnose is abbreviated as Rha, arabinose is abbreviated as Ara, and galacturonic acid is abbreviated as GalA.

[0047] The Serjania polyssacharide described in the embodiments of the present application is derived from natural plant raw materials, has high safety and small toxic and side effects. The Serjania polyssacharide is polymerized by galactose, arabinose, rhamnose and galacturonic acid, the main chain is composed of →6)-β-D-Galp-(1→, →3,6)-β-D-Galp-(1→, →2)-α-L-Rha-(1→ and →4)-α-D-GalAp-(1→, the side chain is composed of α-L-Araf-(1→, →2,5)-α-L-Araf-(1→ and β-D-Galp-(1→, the Serjania polyssacharide with specific fine structure has a purity of 95.04%. The molecular weight of the Serjania polyssacharide obtained in the embodiments of the present application is 57.51 kDa.

[0048] In a preferred embodiment of the present application, the Serjania polyssacharide is composed of 9 types of glycosidic bonds, which are Araf-(1→, Arap-(1→, →2)-Rhap-(1→, →5)-Araf-(1→, Galp-(1→, →4)-GalAp-(1→, →2,5)-Araf-(1→, →6)-Galp-(1→ and →3,6)-Galp-(1→.

[0049] In another aspect, the present application provides a preparation method of the polysaccharide from the shell of Plukenetia volubilis L., comprising the following steps:

[0050] S1, after the shell of Plukenetia volubilis L. is powdered, hot water extraction, freeze-drying, the water extract of the shell of Plukenetia volubilis L. is subjected to fractional extraction with ethanol solution, and the obtained component is subjected to ultrafiltration treatment to obtain a solution one;

[0051] S2, after the solution one is subjected to protein removal treatment, the water phase is collected, dialysis is performed to obtain a solution two;

[0052] S3, the solution two is subjected to decolorization treatment, freeze-drying to obtain a crude polysaccharide powder from the shell of Plukenetia volubilis L.;

[0053] S4, after the crude polysaccharide powder from the shell of Plukenetia volubilis L. is subjected to purification with an anion exchange chromatography column, dialysis is performed, freeze-drying, the obtained powder is subjected to purification with a dextran gel column to obtain the polysaccharide from the shell of Plukenetia volubilis L.

[0054] In an embodiment of the present application, in step S1, the temperature of hot water extraction is 70-90 ℃, and the time of hot water extraction is 1-5 h. Preferably, in step S1, the temperature of hot water extraction is 80 ℃, and the time of hot water extraction is 3 h.

[0055] In an embodiment of the present application, in step S1, the molecular weight of the ultrafiltration membrane is 10-50 kDa. Preferably, in step S1, the molecular weight of the ultrafiltration membrane is 30 kDa.

[0056] In an embodiment of the present application, in step S1, the fractional extraction with ethanol solution specifically comprises: after the water extract of the shell of Plukenetia volubilis L. is subjected to fractional extraction with anhydrous ethanol, an ethanol solution with a volume concentration of 75%, and an ethanol solution with a volume concentration of 50%, ethanol is removed by rotary evaporation, and the component obtained after the ethanol solution with a volume concentration of 50% is collected.

[0057] Preferably, in step S1, the temperature of rotary evaporation is 60 ℃.

[0058] In an embodiment of the present application, in step S2, the protein removal treatment specifically comprises: ammonium sulfate is added to the solution one, an equal volume of tert-butyl alcohol is added, constant temperature shaking is performed, standing is performed, and the water phase is collected. In the embodiment of the present application, the three-phase separation method is used for the protein removal treatment of the solution one.

[0059] In an embodiment of the present application, in step S2, the temperature of constant temperature shaking is 30-40 ℃, the time of constant temperature shaking is 0.5-1.5 h, and the time of standing is 10-30 min. Preferably, in step S2, the temperature of constant temperature shaking is 35 ℃, the time of constant temperature shaking is 1 h, and the time of standing is 20 min.

[0060] In an embodiment of the present application, in step S2, the tert-butyl alcohol is added in an equal volume, specifically, the volume ratio of the tert-butyl alcohol to the added solution is 1:1.

[0061] In an embodiment of the present application, in step S2, after the addition of the ammonium sulfate, the saturation of the ammonium sulfate is 10-30%. Preferably, after the addition of the ammonium sulfate, the saturation of the ammonium sulfate is 20%. It should be noted that the saturation of the ammonium sulfate refers to the saturation of the ammonium sulfate in the system.

[0062] In an embodiment of the present application, in step S2, during the dialysis, the molecular weight of the dialysis bag is 3500 Da.

[0063] In an embodiment of the present application, in step S3, the decoloring treatment specifically comprises: adding activated carbon into the solution two, constant temperature oscillation, centrifugation, suction filtration, and collection of the supernatant.

[0064] In an embodiment of the present application, in step S3, the mass ratio of the activated carbon to the solution two is 0.5%-3%. Preferably, the mass ratio of the activated carbon to the solution two is 2.5%.

[0065] In an embodiment of the present application, in step S3, the temperature of the constant temperature oscillation is 30-40 ℃, and the time of the constant temperature oscillation is 0.1-1.5 h. Preferably, in step S3, the temperature of the constant temperature oscillation is 35 ℃, and the time of the constant temperature oscillation is 10 min.

[0066] In an embodiment of the present application, in step S3, the centrifugation is specifically 3000-4000 rpm for 5-15 min. Preferably, in step S3, the centrifugation is specifically 3500 rpm for 10 min.

[0067] In an embodiment of the present application, in step S3, the suction filtration is performed by using a Buchner funnel.

[0068] In an embodiment of the present application, in step S4, the purification by using the anion exchange chromatography column specifically comprises: gradient elution by using membrane water, 0.1M, and 0.3M NaCl solution in sequence, and collection of the polysaccharide component eluted after the 0.3M NaCl solution.

[0069] Preferably, in step S4, during the gradient elution, the flow rate is 1 mL / min, and each tube is collected for 5 mL.

[0070] Preferably, in step S4, the filler used by the anion exchange chromatography column is DEAE FF, which is a weak anion exchange medium formed by bonding diethylaminoethyl (DEAE) on a fast-flowing agarose gel.

[0071] In an embodiment of the present application, in step S4, the purification by the Sephadex column comprises: adding the powder purified by the anion exchange column into the Sephadex column, eluting with water, and collecting components with high response values.

[0072] In an embodiment of the present application, in step S4, when eluting with water, the loading amount is 2 mL, the flow rate is 0.2 mL / min, and the components in 150-255 min are collected.

[0073] In an embodiment of the present application, in step S4, the dialysis is performed by using 3500 Da.

[0074] In another aspect, the present application also provides use of the any one of the above-mentioned Opuntia tuna husk polysaccharides or the Opuntia tuna husk polysaccharides prepared by the any one of the above-mentioned preparation methods in the preparation of a product for improving vascular function or a product for maintaining a healthy level of blood pressure. The product comprises a medicine, a health food or a food.

[0075] Preferably, the present application also provides use of the any one of the above-mentioned Opuntia tuna husk polysaccharides or the Opuntia tuna husk polysaccharides prepared by the any one of the above-mentioned preparation methods in the preparation of a medicine for improving vascular endothelial cell damage.

[0076] The Opuntia tuna husk polysaccharide provided by the present application has the effects of treating vascular endothelial cell damage, promoting the secretion of the endothelial cell vasodilator NO and reducing the expression of the vasoconstrictor ET-1, and improving the antioxidant capacity of the vascular endothelial cells, thereby providing support for the application of the Opuntia tuna husk polysaccharide.

[0077] Preferably, the Opuntia tuna husk polysaccharide is used in the preparation of a medicine for improving vascular endothelial cell damage in vitro. More preferably, the vascular endothelial cell damage is induced by angiotensin II (Ang II).

[0078] The present application will be described in detail below with reference to the accompanying drawings.

[0079] Example 1 A preparation method of an Opuntia tuna husk polysaccharide comprises the following steps:

[0080] (1) Opuntia tuna husk is powdered, sieved, extracted in a water bath at 80°C for 3 h, and then collected as a powder after rotary evaporation and freeze-drying, to obtain an Opuntia tuna husk water extract; the Opuntia tuna husk water extract is subjected to fractional extraction with anhydrous ethanol, 75% ethanol and 50% ethanol, and then the ethanol is removed by rotary evaporation at 60°C, to collect the 50% ethanol extraction sample; the 50% ethanol extraction sample is subjected to ultrafiltration by using a 30 kDa ultrafiltration membrane, to collect a component greater than 30 kDa, to obtain a solution one;

[0081] (2) The protein removal treatment is performed by using three-phase separation technology, specifically including adding the greater than 30 kDa component after ultrafiltration into ammonium sulfate to make the saturation degree 20%, then adding t-butyl alcohol according to 1:1 (V / V), constant temperature oscillation at 35 ℃ for 1 h, standing for 20 min, collecting the aqueous phase, dialysis for 72 h at 3500 Da, to obtain solution two;

[0082] (3) The decolorization treatment is performed by using activated carbon, specifically including adding 5 mL of the crude polysaccharide solution with a concentration of 2 mg / mL into activated carbon, the mass ratio of activated carbon to solution two is 2.5%, constant temperature oscillation at 35 ℃ for 10 min, that is, the decolorization time is 10 min, centrifugation, suction filtration, taking the supernatant, freeze-drying, to obtain the powder of the crude polysaccharide from the Syzygium cumini shell;

[0083] (4) The DEAE FF anion exchange chromatography column is selected for further purification, the DEAE FF filler is poured into the chromatography column, and then the membrane water balance of 2 BV is performed. The sample solution with a concentration of 50 mg / mL is prepared, filtered through a 0.22 μm filter, after the sample is added, the membrane water, 0.1 and 0.3 NaCl solutions are sequentially used for gradient elution, the flow rate is 1 mL / min, 5 mL is collected per tube, the 0.3 M NaCl elution component is collected, dialysis (3500 Da) is performed, and the sample is collected by freeze-drying;

[0084] (5) Sephadex G100 is used for further purification. First, the dextran gel G100 is poured along the wall of the chromatography column (1.6*80 cm), and then the membrane water is used for balance at a flow rate of 0.2 mL / min for 2 BV. The sample solution obtained in step (4) is prepared at a concentration of 50 mg / mL, the sample amount is 2 mL, the flow rate is 0.2 mL / min, the membrane water is used for elution, 3 mL is collected per tube, the phenol-sulfuric acid method is used to determine the absorbance A490 of each tube, the tube number is used as the abscissa, and the absorbance is used as the ordinate to plot a graph, and the part with a high response value is collected, that is, the 150-255 min component is collected, and freeze-drying is performed to obtain the Syzygium cumini shell polysaccharide sample.

[0085] Comparative Examples 1 to 4

[0086] The same as in Example 1, except that in step (2), the ammonium sulfate with saturation degrees of 0%, 40%, 60%, and 80% is respectively used instead of the ammonium sulfate with a saturation degree of 20% for the protein removal treatment.

[0087] Comparative Examples 5 to 10

[0088] The same as in Example 1, except that in step (3), the decolorization time is respectively replaced by 20 min, 30 min, 40 min, 50 min, 60 min, and 70 min instead of 10 min.

[0089] Comparative Examples 11 to 15

[0090] The same as example 1, except that in step (3), the mass ratio of activated carbon to solution two is replaced by 0.5%, 1%, 1.5%, 2%, and 3% instead of 2.5%, respectively.

[0091] Comparative Example 16

[0092] The same as example 1, except that in step (4), gradient elution is performed using membrane-passed water, 0.1M, 0.3M, and 0.5M NaCl solutions, and the fractions eluted by membrane-passed water, 0.1M, 0.3M, and 0.5M NaCl are collected, respectively.

[0093] Test Example 1 Process condition optimization of polysaccharide from Opuntia dillenii Haw. shell

[0094] (1) Optimization of deproteinization process

[0095] The protein and total sugar contents of the polysaccharide samples from Opuntia dillenii Haw. shell obtained in test example 1 and comparative examples 1-4 were tested.

[0096] The total sugar was determined by the phenol-sulfuric acid method, which specifically included the following steps: 10 mg of anhydrous glucose dried to constant weight at 105 ℃ was weighed into a 250 mL volumetric flask, water was added to the mark, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mL were respectively taken, each was supplemented with 2.0 mL of distilled water, then 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid were added, shaken well, cooled, and placed at room temperature for 20 min, then the absorbance was measured at 490 nm, 2.0 mL of water was used as a blank, the abscissa was the glucose concentration (mg / mL), and the ordinate was the absorbance A, a standard curve was obtained, the Opuntia dillenii Haw. shell polysaccharide sample was prepared to 1 mg / mL, 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid were added, shaken well, cooled, and placed at room temperature for 20 min, then the absorbance was measured at 490 nm. 2.0 mL of water was used as a blank, and the same color developing operation was performed, three samples were measured in parallel for each determination, and the total sugar content was calculated by substituting the standard curve.

[0097] Protein content was determined using Coomassie Brilliant Blue assay, specifically as follows: 10 mg of bovine serum albumin (BSA) was weighed and dissolved in 100 mL of physiological saline to obtain a 100 μg / mL protein standard solution. BSA solutions of 0, 20, 40, 60, 80, and 100 μg / mL were prepared. 1 mL of each concentration was accurately pipetted into separate 10 mL stoppered test tubes. 5 mL of Coomassie Brilliant Blue G250 reagent was added, and after mixing and incubation for 20 min, the absorbance was measured at 595 nm to plot a standard curve. 1 mL of a 1 mg / mL *Coccus mutans* shell polysaccharide sample solution was added to 5 mL of Coomassie Brilliant Blue G250 reagent, and after mixing and incubation for 20 min, the absorbance was measured at 595 nm. Triple replicates were performed. 1.0 mL of water was used as a blank. The protein content was then determined using the standard curve.

[0098] The results showed that the higher the ammonium sulfate saturation, the higher the protein removal rate. The total sugar content was significantly increased when the ammonium sulfate saturation was 20%, while the sugar content decreased significantly when the ammonium sulfate saturation was higher than 20%. Therefore, considering all factors, the effect was best when the ammonium sulfate saturation was 20%.

[0099] Table 1 Optimization of deproteinization process

[0100]

[0101] (2) Decolorization process optimization

[0102] The supernatant obtained in step (3) of Example 1, Comparative Examples 5-10, and Comparative Examples 11-15 was tested. The total sugar content and decolorization rate in the supernatant were determined. The total sugar content was determined using the same phenol-sulfuric acid method as described above. The decolorization rate was obtained by directly measuring the absorbance of the supernatant at 450 nm. A comprehensive score was calculated, with the total sugar content and decolorization rate each accounting for 50%. That is, the comprehensive score = A1 / A0*100*0.5 + B1 / B0*100*0.5, where A1 is the decolorization rate, A0 is the maximum detected decolorization rate, B1 is the polysaccharide retention rate, and B0 is the maximum detected polysaccharide retention rate. The results are as follows: Figure 1 As shown.

[0103] like Figure 1 A, Figure 1 As shown in Figure B, the decolorization rate increased continuously with the extension of decolorization time, gradually leveling off after 20 minutes. At 10 minutes, the sugar retention rate was 65%, which significantly decreased and reached equilibrium after 20 minutes. The combined score of polysaccharide retention rate and decolorization rate was highest at a decolorization time of 10 minutes; therefore, a decolorization time of 10 minutes was chosen for further experiments. Figure 1 C Figure 1As shown in D, the decolorization rate increases with increasing activated carbon content, while the sugar retention rate decreases. The highest overall score is achieved when the activated carbon content is 2.5%. Therefore, an activated carbon content of 2.5% and a decolorization time of 10 minutes yield the best results.

[0104] (3) Optimization of anion exchange column process

[0105] The total sugar content of the fractions eluted with membrane-passing water, 0.1 M NaCl solution, 0.3 M NaCl solution, and 0.5 M NaCl solution in step (4) of Example 1 and Comparative Example 16 was determined using the phenol-sulfuric acid method, as described above. The results are as follows. Figure 2 As shown.

[0106] Depend on Figure 2 It can be seen that the sugar content of the eluted components obtained by distilled water, 0.1 M NaCl solution and 0.5 M NaCl solution is relatively low, while the sugar content of the eluted components obtained by 0.3 M NaCl solution is relatively high. Therefore, 0.3 M NaCl solution has the best elution effect.

[0107] Test Example 2 Structural characterization of Sapote salsa polysaccharides

[0108] (1) Determination of molecular weight and purity of polysaccharides from the shell of *Sapindus mukorossi*

[0109] Accurately weigh 5 mg of the *Sapindus mukorossi* shell polysaccharide sample obtained in Example 1, dissolve it in 1 mL of mobile phase to prepare a 5 mg / mL sample solution, centrifuge at 12000 rpm for 10 min, and pass the supernatant through a 0.22 μm water-washed microporous membrane. Place the supernatant in a 1.8 mL sample vial for analysis. Weigh 5 mg of dextran standard, process it using the same method as the sample, and place it in a sample vial for later use. Determine the molecular weight of the polysaccharide by high-performance gel permeation chromatography (HPGPC). The mobile phase was 0.05 M NaCl solution, the column was a BRT105-103-101 tandem gel column (8 × 300 mm), the flow rate was 0.8 mL / min, the column temperature was 40 ºC, the injection volume was 25 μL, and the detector was a RID-20 differential detector. A standard curve was constructed based on the retention time and molecular weight of the standard. The molecular weight of the *Sapindus mukorossi* shell polysaccharide was calculated by substituting the retention time of the sample into the standard curve. The purity of *Sagittaria sagittifolia* shell polysaccharides was determined using the phenol-sulfuric acid method. A UV spectrophotometer was used for a full wavelength scan from 200 to 800 nm.

[0110] The molecular weight distribution of polysaccharides from the shell of *Sagittaria sagittifolia* was determined using HPGPC. Figure 3As shown, the GPC spectrum of *Sagittaria sagittifolia* shell polysaccharide shows only a single peak at 29.73 min, exhibiting narrow and symmetrical characteristics, indicating high purity and good homogeneity. Based on the standard curve, the molecular weight of *Sagittaria sagittifolia* shell polysaccharide is calculated to be 57.51 kDa. Figure 4 As shown, the polysaccharide from the shell of *Sapindus mukorossi* showed no absorption peaks at 260 nm and 280 nm, indicating that it does not contain nucleic acids or proteins. Furthermore, the sugar content of the polysaccharide was determined using the phenol-sulfuric acid method, revealing a purity of 95.04 ± 1.64%.

[0111] (2) Fourier transform infrared analysis of polysaccharides from the shell of *Sapindus mukorossi*

[0112] Weigh 1 mg of dried *Saussurea involucrata* shell polysaccharide obtained in Example 1, add 100 mg of KBr particles, grind into a homogeneous sample without obvious particle texture, compress into tablets using a tablet press, and press at a wavelength of 4000-500 cm⁻¹. -1 Infrared spectroscopy was performed, with KBr as a blank control.

[0113] Figure 5 Fourier transform infrared (FTIR) spectra of polysaccharides from the shell of *Citrus medica*. The polysaccharides from *Citrus medica* are observed at 3397, 2937, 1613, 1419, and 1070 cm⁻¹. -1 All showed absorption peaks, typical signal peaks for polysaccharides. The polysaccharide from the shell of *Sagittaria sagittifolia* showed an absorption peak at 3397 cm⁻¹. -1 The signal peak at 2937 cm⁻¹ is mainly generated by the stretching vibrations of OH molecules between and within monosaccharide molecules; -1 The signal peaks that appeared were absorption peaks of CH, including stretching and bending vibrations of CH, CH2, and CH3 in the shell polysaccharide of *Caulis Chamomile*; 1613 cm⁻¹ -1 The absorption peak at 1760 cm⁻¹ is caused by the asymmetric stretching vibration of C=O and carboxyl esterification in the polysaccharide of *Sapindus mukorossi* shell, but... -1 ~1740 cm -1 No absorption peak was observed within the range, indicating that there is no carboxyl esterification in the polysaccharide from the shell of *Sapindus mukorossi*; 1419 cm⁻¹ -1 The signal peak is generated by the CH stretching and bending vibrations of the sacha inca peel polysaccharide; 1070 cm⁻¹ -1 The stretching vibrations of COC and CO in the *Caulis Chamaecyparis* husk polysaccharide were attributed to these vibrations, indicating the presence of galactopyranose in the polysaccharide; 895 cm⁻¹ and 812 cm⁻¹. -1 The absorption peak at the surface indicates the presence of α- and β-glycosidic bonds in the polysaccharide from the shell of *Sapindus mukorossi*. Therefore, based on the infrared results, the infrared spectrum of *Sapindus mukorossi* polysaccharide is a typical polysaccharide spectrum, and both α- and β-glycosidic bonds are present in the polysaccharide.

[0114] (3) Methylation analysis of the polysaccharide from the shell of Plukenetia volubilis

[0115] A 2 mg sample of the freeze-dried polysaccharide from the shell of Plukenetia volubilis was weighed into a reaction bottle and 1 mL of anhydrous dimethyl sulfoxide was added. After rapid addition of an anhydrous base solution, the bottle was closed, ultrasonically dissolved, and then iodomethane solution was added. The mixture was stirred magnetically in a water bath at 30 °C for 60 min, and then 2 mL of ultrapure water was added to terminate the methylation reaction. The methylated polysaccharide product was taken and 1 mL of 2 M trifluoroacetic acid was added to hydrolyze it for 90 min. After evaporation under reduced pressure, 2 mL of ultrapure water and 60 mg of sodium borohydride were added, and the mixture was reacted for 8 h. After neutralization with glacial acetic acid, the mixture was concentrated under reduced pressure and dried in an oven at 101 °C. Acetylation was performed by adding 1 mL of acetic anhydride at 100 °C for 1 h. After cooling, 3 mL of toluene was added, and the mixture was concentrated under reduced pressure and dried. This process was repeated 4-5 times to remove excess acetic anhydride. The acetylated product was dissolved in 3 mL of dichloromethane and transferred to a separatory funnel. A small amount of distilled water was added and the mixture was shaken well. The upper aqueous solution was removed and the process was repeated 4 times. The dichloromethane layer was dried with an appropriate amount of anhydrous sodium sulfate and concentrated to 1 mL, and then placed in a liquid-phase vial. The acetylated product of the polysaccharide from the shell of Plukenetia volubilis was determined by gas chromatography-mass spectrometry. The chromatographic column was HP-INNOWAX (30 m x 0.32 mm x 0.25 μm), the initial temperature was 140 °C and the temperature was increased to 230 °C at a rate of 1 °C / min, the detector temperature and the injection port temperature were both 250 °C, helium was used as the carrier gas at a flow rate of 1 mL / min.

[0116] Figure 6 The main glycosidic bond connection mode of the polysaccharide from the shell of Plukenetia volubilis was determined. The GC-MS spectrum of the polysaccharide from the shell of Plukenetia volubilis showed 9 types of methylated sugar alcohol acetates, namely 2,3,5-Me3-Araf, 2,3,4-Me3-Arap, 3,4-Me2-Rhaf, 2,3-Me2-Araf, 2,3,4,6-Me4-Galp, 2,3,6-Me3-Galp, 3-Me1-Araf, 2,3,4-Me3-Galp, and 2,4-Me2-Galp, indicating that the polysaccharide from the shell of Plukenetia volubilis is composed of 9 types of glycosidic bonds, namely Araf-(1→, Arap-(1→, →2)-Rhap-(1→, →5)-Araf-(1→, Galp-(1→, →4)-GalAp-(1→, →2,5)-Araf-(1→, →6)-Galp-(1→, →3,6)-Galp-(1→.

[0117] (4) NMR analysis of the polysaccharide from the shell of Plukenetia volubilis

[0118] 50 mg of the sample was dissolved in 0.5 mL of heavy water and freeze-dried, re-dissolved in 0.5 mL of heavy water, and freeze-dried again. The above process was repeated to fully exchange the active hydrogen. Finally, the sample was dissolved in 0.5 mL of heavy water, and deuterated acetone was added as an internal standard. The 1D NMR, 13C NMR, DEPT-135 NMR, HSQC, HMBC, NOESY, and COSY of the sample were obtained using a 600 MHz NMR instrument.

[0119] Table 2 Carbon-hydrogen assignment of the polysaccharide from the shell of S. robusta H.B. et K.

[0120]

[0121] NMR technology is mainly used to determine the conformation and connection mode of the polysaccharide. Figure 7 Table 2 is the NMR result of the polysaccharide from the shell of S. robusta H.B. et K. The anomeric hydrogen signals are mainly concentrated in 3.0-5.5 ppm, the non-anomeric hydrogen signals are concentrated in 3.0-4.3 ppm, the anomeric hydrogen signals are distributed in 4.3-5.5 ppm, and the anomeric carbon chemical shifts of the polysaccharide from the shell of S. robusta H.B. et K. are concentrated in 90-110 ppm, i.e. 110.62 ppm, 109.25 ppm, 108.77 ppm, 108.60 ppm, 106.10 ppm, 105.14 ppm, 104.69 ppm, 100.38 ppm, and 99.88 ppm, which are typical polysaccharide signal spectra. According to the two-dimensional NMR spectra combined with the methylation result, the main chain of the polysaccharide from the shell of S. robusta H.B. et K. is composed of →6)-β-D-Galp-(1→, →3,6)-β-D-Galp-(1→, →2)-α-L-Rha-(1→, and →4)-α-D-GalAp-(1→, and the side chain is composed of α-L-Araf-(1→, →2,5)-α-L-Araf-(1→, and β-D-Galp-(1→. The specific molecular structure is shown in formula (I).

[0122] (5) Analysis of the morphological structure of the polysaccharide from the shell of S. robusta H.B. et K.

[0123] The polysaccharide powder sample was fixed on double-sided tape, and a gold layer was sprayed in a vacuum environment. The surface structure of the polysaccharide was observed under 100, 500, 2000, and 5000 times using SEM.

[0124] The surface morphology of the polysaccharide from the shell of S. robusta H.B. et K. was observed under 100, 500, 2000, and 5000 times. Figure 8 The surface morphology of the polysaccharide from the shell of S. robusta H.B. et K. was observed under 100, 500, 2000, and 5000 times. The polysaccharide mainly presents a block structure with irregular shape, loose structure, rough surface, and many honeycomb structures.

[0125] Test Example 3 Effect test of Opuntia dillenii Hull polysaccharide on endothelial cell injury

[0126] Experimental design (1) Vascular endothelial cell culture

[0127] The vascular endothelial cells HUVECs were cultured with ECM medium and placed in a 37 ℃, 5% CO2 incubator for culture.

[0128] (2) Cell treatment

[0129] HUVECs were divided into 5 groups for treatment, and the specific treatment was as follows:

[0130] a) The CON group was a blank control group, which was cultured with ECM medium.

[0131] b) The ANG group was a model group, which was treated with 1 μM Ang Ⅱ for 24 h.

[0132] c) The 100 μg / mL group was a low-dose group, which was treated with 100 μg / mL Opuntia dillenii Hull polysaccharide for 24 h, and then treated with 1 μM Ang Ⅱ for 24 h.

[0133] d) The 200 μg / mL group was a medium-dose group, which was treated with 200 μg / mL Opuntia dillenii Hull polysaccharide for 24 h, and then treated with 1 μM Ang Ⅱ for 24 h.

[0134] e) The 400 μg / mL group was a high-dose group, which was treated with 400 μg / mL Opuntia dillenii Hull polysaccharide for 24 h, and then treated with 1 μM Ang Ⅱ for 24 h.

[0135] (3) Index determination

[0136] NO, ET-1, malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) were determined according to the kit instructions.

[0137] (4) RT-qPCR analysis

[0138] Add 1 mL of Trizol reagent to the cells, mix well, and lyse in a centrifuge tube for 5 min. Add 200 μL of chloroform, shake vigorously for 15 s, and stand at room temperature for 3 min. Centrifuge at 12000 rpm and low temperature for 15 min. Take the supernatant to a centrifuge tube, add an equal volume of isopropanol, mix well, stand for 10 min, centrifuge at low temperature and 12000 rpm for 10 min, remove the supernatant, add 75% ethanol 1 mL to wash the precipitate, centrifuge at 4°C and 12000 rpm for 3 min, remove the supernatant, dry for 5-10 min, add 20-30 μL of sterile enzyme-free water to dissolve the precipitate, and measure the concentration at 260 nm and 280 nm using a UV spectrophotometer. Calculate the concentration and purity. Use total mRNA as a template to reverse transcribe cDNA. The reverse transcription product can be used for PCR and fluorescent quantitative PCR. The internal reference gene is β-actin, and the relative expression amount of mRNA is calculated by the 2-ΔΔCt method.

[0139] (5) Immunoblotting analysis

[0140] Wash the cells with pre-cooled PBS, add 200 μL of RIPA lysis buffer, and lyse at low temperature for 10 min. Collect the supernatant after centrifugation at 4°C for 15 min for subsequent experiments. Determine the protein content by BCA method. Add 5x loading buffer to 100 uL of protein supernatant and mix well, then boil in water for 5 min to prepare protein samples. Prepare 10% separating gel and 5% concentrated gel, add Marker and prepared protein samples to the lanes. The electrophoresis voltage is 75 V, and the time is 130 min. After electrophoresis, transfer the membrane, the transfer conditions are 300 mA constant current, wash the nitrocellulose (NC) membrane with PBST once, and block with 5% skim milk powder at room temperature for 90 min. Dilute the primary antibody, incubate the NC membrane with the primary antibody at 4°C overnight, then wash with PBST for 3 times, 10 min each time. Dilute the secondary antibody and incubate with the NC membrane at room temperature for 90 min, then wash with PBST for 3 times, 15 min each time. Use ECL chemiluminescence solution to incubate with the membrane for 1 min for color development reaction, and take a photo record using a gel imaging system.

[0141] Experimental results (1) Opuntia tuna shell reduces vascular endothelial cell damage

[0142] As Figure 9As shown, the concentration of M. oleifera husk polysaccharide was 0~400 mg / mL, which had no toxic effect on endothelial cells. High-dose M. oleifera husk polysaccharide could promote the proliferation of endothelial cells induced by Ang II. Ang II could significantly reduce the secretion of NO in endothelial cells, while M. oleifera husk polysaccharide at medium and high concentrations could significantly promote the secretion of NO; Ang II could significantly promote the secretion of ET-1, a vasoconstrictor, while M. oleifera husk polysaccharide at low, medium and high concentrations could significantly inhibit the secretion level of ET-1. Therefore, M. oleifera husk polysaccharide could promote the secretion of NO and reduce the expression of ET-1 to alleviate the endothelial dysfunction induced by Ang II.

[0143] (2) M. oleifera husk polysaccharide improves the antioxidant capacity of vascular endothelial cells

[0144] Oxidative stress is another important factor causing endothelial dysfunction. Under normal circumstances, the production and elimination of ROS in cells is in a balanced state, but when the level of ROS in cells exceeds the clearance capacity of the antioxidant system, the redox balance is destroyed, which eventually leads to cell dysfunction. For example, Figure 10 As shown, after Ang II treatment, the fluorescence intensity of ROS in HUVECs was significantly enhanced compared with the blank group, while the fluorescence intensity of HUVECs treated with M. oleifera husk polysaccharide was significantly decreased. Quantitative analysis of fluorescence intensity found that M. oleifera husk polysaccharide could significantly inhibit the production of ROS in endothelial cells induced by Ang II. MDA is one of the end products of lipid peroxidation reaction. When lipid molecules in cells are attacked by ROS, lipid peroxidation reaction occurs, leading to an increase in the content of MDA in cells. SOD is an important antioxidant enzyme in cells, which can effectively scavenge free radicals in cells. Determination of MDA content and SOD activity found that Ang II could significantly promote the production of MDA and reduce the enzyme activity of SOD, destroy the redox balance in HUVECs, while M. oleifera husk polysaccharide treatment significantly inhibited the production of MDA in endothelial cells induced by Ang II and increased the enzyme activity of SOD. Therefore, M. oleifera husk polysaccharide can significantly reduce the production of ROS and MDA in HUVECs, increase the activity of SOD, and alleviate the oxidative damage of endothelial cells induced by Ang II.

[0145] (3) M. oleifera husk polysaccharide regulates the expression of genes related to the function of vascular endothelial cells and protein analysis

[0146] RT-qPCR and WB experiments were used to further explore the mechanism of M. oleifera husk polysaccharide in alleviating endothelial cell damage induced by Ang II. AT1R is the receptor of Ang II, and Ang II binds to its receptor and further stimulates the downstream pathway, leading to a decrease in NO and an increase in ET-1. As shown, Figure 11 and 12As shown, Ang II induced the gene expression and protein secretion of AT1R in endothelial cells, and after the administration of different concentrations of Opuntia Milpa Alta polysaccharides, the expression and secretion levels were significantly decreased, indicating that Opuntia Milpa Alta polysaccharides can inhibit the gene expression of AT1R in endothelial cells induced by Ang II. eNOS can promote the generation of vasodilator NO, and Ang II can significantly reduce the gene expression of eNOS, and after the administration of Opuntia Milpa Alta polysaccharides, the expression level of eNOS in endothelial cells was significantly increased, indicating that Opuntia Milpa Alta polysaccharides can promote the gene expression and protein secretion of eNOS. In addition, the ACE2-Ang (1-7)-MasR pathway plays a negative feedback regulation role in Ang II-induced endothelial cell dysfunction, ACE2 can hydrolyze Ang II to Ang (1-7) to prevent Ang II from binding to its receptor AT1R, and the hydrolyzed Ang (1-7) can bind to its receptor Mas to activate the downstream pathway. The ACE2, Ang (1-7) and MasR in the Ang II group were significantly reduced, and the administration of Opuntia Milpa Alta polysaccharides can significantly increase the gene expression and protein secretion levels of ACE2, Ang (1-7) and MasR. In addition, Opuntia Milpa Alta polysaccharides can also promote the expression of AKT, the downstream gene of ACE2-Ang (1-7)-MasR pathway. Therefore, Opuntia Milpa Alta polysaccharides can inhibit the expression of Ang II receptor AT1R and increase the expression of eNOS, ACE2, Ang (1-7), MasR and AKT protein, thereby relieving the endothelial dysfunction induced by Ang II.

[0147] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Schinopsis gumei husk polysaccharide, characterized in that, It is polymerized by galactose, arabinose, rhamnose and galacturonic acid, and has a structure as shown in formula (I); Formula (I).

2. The method of preparing a Schinopsis gmelina husk polysaccharide according to claim 1, characterized in that, It comprises the following steps: S1, after the powdering of the Terminalia ferdinandiana shell, hot water extraction, freeze-drying, the obtained Terminalia ferdinandiana shell water extract is subjected to fractional extraction with ethanol solution, and the obtained component is subjected to ultrafiltration treatment to obtain solution one; S2, after the protein removal treatment of the above-mentioned solution one, the water phase is collected, dialyzed to obtain solution two; S3, the above-mentioned solution two is subjected to decolorization treatment, freeze-drying to obtain Terminalia ferdinandiana shell crude polysaccharide powder; S4, the above-mentioned Terminalia ferdinandiana shell crude polysaccharide powder is subjected to purification with an anion exchange chromatography column, dialysis, freeze-drying, and the obtained powder is subjected to purification with a dextran gel column to obtain Terminalia ferdinandiana shell polysaccharide.

3. The preparation method according to claim 2, wherein, in step S1, the temperature of hot water extraction is 70-90 ℃, and the time of hot water extraction is 1-5 h; in step S1, the molecular weight of the ultrafiltration membrane is 10-50 kDa; in step S1, the fractional extraction with ethanol solution specifically comprises: the Terminalia ferdinandiana shell water extract is subjected to fractional extraction with anhydrous ethanol, 75% volume concentration ethanol solution and 50% volume concentration ethanol solution, then ethanol is removed by rotary evaporation, and the component obtained after the 50% volume concentration ethanol solution extraction is collected.

4. The preparation method according to claim 2, wherein, in step S2, the protein removal treatment specifically comprises: ammonium sulfate is added to the above-mentioned solution one, then an equal volume of tert-butyl alcohol is added, constant temperature shaking, standing, and the water phase is collected.

5. The preparation method according to claim 4, wherein, after the addition of ammonium sulfate, the saturation of ammonium sulfate is 10-30%.

6. The preparation method according to claim 4, wherein, the equal volume addition of tert-butyl alcohol specifically refers to that the volume ratio of tert-butyl alcohol to the added solution is 1:

1.

7. The preparation method according to claim 2, wherein, in step S3, the decolorization treatment specifically comprises: activated carbon is added to solution two, constant temperature shaking, centrifugation, suction filtration, and the supernatant is collected.

8. The preparation method according to claim 7, wherein, in step S3, the mass ratio of activated carbon to solution two is 0.5%-3%.

9. The preparation method according to claim 8, wherein, the mass ratio of activated carbon to solution two is 2.5%.

10. The preparation method according to claim 2, wherein, in step S4, the purification with an anion exchange chromatography column specifically comprises: membrane water, 0.1M and 0.3M NaCl solution are sequentially used for gradient elution, and the polysaccharide component eluted after the 0.3M NaCl solution is collected.

11. The preparation method according to claim 2, wherein, in step S4, the purification with a dextran gel column specifically comprises: the powder after the anion exchange chromatography column purification is added to the dextran gel column, water elution is adopted, and the component with a high response value is collected.

12. The preparation method according to claim 11, wherein, In step S4, the water elution was performed with a sample loading amount of 2 mL and a flow rate of 0.2 mL / min, and fractions collected from 150 to 255 min.

13. Use of the Opuntia tuna husk polysaccharide of claim 1, or the Opuntia tuna husk polysaccharide prepared by the method of any one of claims 2-12, in the preparation of a medicament for improving vascular function, or in the preparation of a medicament for maintaining a healthy level of blood pressure.

14. Use of the Opuntia tuna husk polysaccharide of claim 1, or the Opuntia tuna husk polysaccharide prepared by the method of any one of claims 2-12, in the preparation of a medicament for improving vascular endothelial cell damage.

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