Plukenetia volubilis linneo shell polysaccharide with blood vessel improving function as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of plant polysaccharides, and particularly relates to a plukenetia volubilis linneo shell polysaccharide with a function of improving blood vessels as well as a preparation method and application thereof. The plukenetia volubilis linneo shell polysaccharide is formed by polymerizing galactose, arabinose, rhamnose and galacturonic acid, and has a structure as shown in a formula (I), # imgabs0 # formula (I): (1). The plukenetia volubilis linneo shell polysaccharide provided by the invention can be used for improving the blood vessel function and maintaining the blood pressure health level. The purity of the plukenetia volubilis linneo shell polysaccharide with a specific fine structure can reach 95.04%, residues of impurities such as protein and lipid are greatly reduced, and the problems that the plukenetia volubilis linneo shell polysaccharide is difficult to extract and low in purity are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant polysaccharides, and in particular relates to a sargassum chinensis shell polysaccharide capable of improving vascular function, and a preparation method and application thereof. Background Art

[0002] Vascular endothelial cells usually refer to the single layer of squamous epithelium lining the inner surface of the heart, blood vessels and lymphatic vessels, and are an important component of the vascular wall. Vascular endothelial cells have the functions of regulating vasoconstriction, phagocytizing harmful substances in blood vessels, immunomodulation and anti-thrombosis. Dysfunction of vascular endothelial cells may lead to increased permeability of vascular walls and increased blood viscosity, which can easily cause thrombotic or embolic diseases. In addition, vascular endothelial cells can secrete vasodilator factor nitric oxide (NO) and vasoconstrictor factor endothelin-1 (ET-1) to regulate vascular contraction and relaxation, thereby maintaining blood pressure stability in the body. However, vascular endothelial cell dysfunction can lead to an imbalance in the secretion of vasodilation and contraction factors in the body, leading to increased blood pressure in the human body, and then leading to a series of complications, such as renal dysfunction, myocardial infarction and other diseases. At present, drugs for the treatment of vascular endothelial cell damage usually have large side effects and certain toxicity. Therefore, it is imperative to find a method for treating 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 extensive attention in the food and biopharmaceutical industries because of their non-toxicity, good biological activity, biocompatibility and biodegradability. Polysaccharides participate in a variety of biological metabolism in the human body and have a variety of biological effects such as lowering blood pressure, protecting the liver, anti-depression, and lowering blood sugar.

[0004] Sacha Inchi is a plant with rich nutritional value and multiple uses. Sacha Inchi can be used for beauty, treatment of muscle pain, rheumatoid arthritis and atopic dermatitis. Sacha Inchi oil and Sacha Inchi protein are mostly used in the food field. Sacha Inchi shell water extract is reported to have the effect of lowering blood pressure. Sacha Inchi shell polysaccharide has the effects of immunomodulation and alleviating kidney damage. However, the purity and activity of the Sacha Inchi shell polysaccharide prepared at present are still poor, and research on its application needs to be further developed. Summary of the invention The invention provides a polysaccharide of the fruit shell of the safflower oil, and a preparation method and application thereof, which effectively solve the problems of difficult extraction and low extraction purity of the polysaccharide of the fruit shell of the safflower oil.

[0005] The present invention provides a polysaccharide of the fruit shell of the pineapple vine, which is polymerized by galactose, arabinose, rhamnose and galacturonic acid and has a structure as shown in formula (I); Formula (I).

[0006] The present invention also provides a method for preparing the above-mentioned citrus chinensis shell polysaccharide, comprising the following steps: S1, after the husk of the vine is pulverized, it is extracted with hot water and freeze-dried. The obtained husk water extract is further graded and extracted with an ethanol solution, and the obtained components are subjected to ultrafiltration to obtain solution 1; S2, after removing protein from the above solution 1, collecting the aqueous phase and dialyzing to obtain solution 2; S3, decolorizing the above solution 2, freeze-drying, and obtaining crude polysaccharide powder of the fruit shell of the citrus vine; S4. Purify the above-mentioned crude polysaccharide powder of the fruit shell of the pine needle by an anion exchange chromatography column, dialyze, and freeze-dry. The obtained powder is purified by a dextran gel column to obtain the polysaccharide of the fruit shell of the pine needle.

[0007] Furthermore, in step S1, the temperature of hot water extraction is 70-90°C, 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 graded extraction with ethanol solution specifically includes: gradedly extracting the water extract of the fruit shell of the citrus indica with anhydrous ethanol, an ethanol solution with a volume concentration of 75%, and an ethanol solution with a volume concentration of 50%, removing the ethanol by rotary evaporation, and collecting the components obtained after extraction with the ethanol solution with a volume concentration of 50%.

[0008] Furthermore, in step S2, the protein removal treatment specifically includes: adding ammonium sulfate to the above solution 1, and then adding tert-butanol in equal volume, shaking at a constant temperature, standing, and collecting the aqueous phase; Preferably, after adding ammonium sulfate, the saturation of ammonium sulfate is 10-30%; Preferably, the equal volume of tert-butanol is added, specifically the volume ratio of tert-butanol to the added solution is 1:1.

[0009] Furthermore, in step S3, the decolorization treatment specifically includes: adding activated carbon to solution 2, shaking at a constant temperature, centrifuging, filtering, and collecting the supernatant; Preferably, in step S3, the mass ratio of activated carbon to solution 2 is 0.5% to 3%. Preferably, the mass ratio of activated carbon to solution 2 is 2.5%.

[0010] Furthermore, in step S4, purification using an anion exchange chromatography column specifically includes: gradient elution using membrane water, 0.1 M, and 0.3 M NaCl solutions in sequence, and collecting the polysaccharide component eluted with the 0.3 M NaCl solution.

[0011] Furthermore, in step S4, purification using a dextran gel column specifically includes: adding the powder purified by the anion exchange chromatography column to the dextran gel column, eluting with water, and collecting components with high response values; Preferably, in step S4, when water elution is used, the sample volume is 2 mL, the flow rate is 0.2 mL / min, and the 150-255 min fraction is collected. The present invention also proposes the use of the above-mentioned citrus chinensis shell polysaccharide, or the citrus chinensis shell polysaccharide prepared by any of the above-mentioned preparation methods, in the preparation of products for improving vascular function, or in the preparation of products for maintaining healthy blood pressure levels. Furthermore, the product includes medicine, health food or food.

[0012] The present invention also proposes the use of the above-mentioned Safflower Chitosan husk polysaccharide, or the Safflower Chitosan husk polysaccharide prepared by any of the above-mentioned preparation methods, in the preparation of a drug for improving vascular endothelial cell damage.

[0013] The present invention has the following advantages: The present invention proposes a polysaccharide of the fruit shell of the Chinese ginseng and a preparation method thereof. By screening specific preparation conditions, the polysaccharide of the fruit shell of the Chinese ginseng with a specific fine structure, which is polymerized by galactose, arabinose, rhamnose and galacturonic acid, is discovered for the first time. The purity of the polysaccharide can reach 95.04%, which greatly reduces the residual impurities such as protein and lipid, and fills the problem of difficult extraction and low purity of the polysaccharide of the fruit shell of the Chinese ginseng.

[0014] The polysaccharide of the fruit shell of the present invention can be used to improve vascular function and maintain a healthy blood pressure level. It has the effect of treating vascular endothelial cell damage, promoting the secretion of endothelial cell vasodilator factor NO and reducing the expression of vasoconstrictor factor ET-1, and improving the antioxidant capacity of vascular endothelial cells. It provides support for the application of polysaccharide of the fruit shell of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1 The activated carbon decolorization process in Test Example 1 of the present invention is optimized; Figure 2 is the elution curve of the anion exchange column in Test Example 1 of the present invention; Figure 3 This is the molecular weight and uniformity detection spectrum of the polysaccharide of the carpel shell of the Chinese vine in Experimental Example 2 of the present invention; Figure 4 This is a full wavelength scanning image of the polysaccharide of the Chinese vine fruit shell in Experimental Example 2 of the present invention; Figure 5This is the Fourier infrared spectrum of the chitosan polysaccharide in Experimental Example 2 of the present invention; Figure 6 The total ion flow diagram and secondary proton chromatogram of the glycosidic bond type detection of the Sinomeria japonica shell polysaccharide in Test Example 2 of the present invention; Figure 7 This is the NMR spectrum of the chitosan polysaccharide of the Chinese vine fruit in Experimental Example 2 of the present invention; Figure 8 This is a scanning electron microscope image of the polysaccharide of the fruit shell of the Chinese vine in Experimental Example 2 of the present invention; Fig. 9 This is a graph showing the effect of the polysaccharide from the fruit shell of the Chinese vine on the survival rate of endothelial cells, NO secretion and ET-1 secretion level in Experimental Example 3 of the present invention; (mean ± 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); Fig.10 This is a graph showing the improvement of the antioxidant capacity of endothelial cells by polysaccharide from the fruit shell of the Chinese vine in Experimental Example 3 of the present invention; (mean ± 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); Fig.11 This is the gene expression diagram related to the regulation of endothelial cell function by polysaccharide from the fruit shell of the Chinese vine in Experimental Example 3 of the present invention; (mean ± 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); Fig.12 This is a graph showing the secretion of proteins related to the regulation of endothelial cell function by polysaccharide from the fruit of the Chinese vine in Experimental Example 3 of the present invention; (mean ± 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

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0018] On the one hand, the embodiment of the present invention provides a polysaccharide of the fruit shell of the fruit ... Formula (I).

[0019] It should be noted that the four monosaccharides proposed in the embodiments of the present invention include galactose, abbreviated as Gal; rhamnose, abbreviated as Rha; arabinose, abbreviated as Ara; and galacturonic acid, abbreviated as GalA.

[0020] The saitang fruit shell polysaccharide described in the embodiment of the present invention is derived from natural plant raw materials, has high safety, and has little toxic side effects. It is polymerized from galactose, arabinose, rhamnose and galacturonic acid, and 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→, and the side chain is composed of α-L-Araf-(1→, →2,5)-α-L-Araf-(1→ and β-D-Galp-(1→). The saitang fruit shell polysaccharide with a specific fine structure has a purity of up to 95.04%. The molecular weight of the saitang fruit shell polysaccharide obtained in the embodiment of the present invention is 57.51 kDa.

[0021] In a preferred embodiment of the present invention, the polysaccharide of the fruit shell 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→.

[0022] On the other hand, the present invention provides a method for preparing a polysaccharide from the fruit shell of the fruit ... S1, after the husk of the vine is pulverized, it is extracted with hot water and freeze-dried. The obtained husk water extract is further graded and extracted with an ethanol solution, and the obtained components are subjected to ultrafiltration to obtain solution 1; S2, after removing protein from the above solution 1, collecting the aqueous phase and dialyzing to obtain solution 2; S3, decolorizing the above solution 2, freeze-drying, and obtaining crude polysaccharide powder of the fruit shell of the citrus vine; S4. Purify the above-mentioned crude polysaccharide powder of the fruit shell of the pine needle by an anion exchange chromatography column, dialyze, and freeze-dry. The obtained powder is purified by a dextran gel column to obtain the polysaccharide of the fruit shell of the pine needle.

[0023] In one embodiment of the present invention, in step S1, the temperature of hot water extraction is 70-90°C, and the time of hot water extraction is 1-5 hours. Preferably, in step S1, the temperature of hot water extraction is 80°C, and the time of hot water extraction is 3 hours.

[0024] In one embodiment of the present invention, 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. In one embodiment of the present invention, in step S1, the graded extraction using ethanol solution specifically includes: gradedly extracting the water extract of the fruit shell of the citrus indica with anhydrous ethanol, an ethanol solution with a volume concentration of 75%, and an ethanol solution with a volume concentration of 50%, removing the ethanol by rotary evaporation, and collecting the components obtained after extraction with the ethanol solution with a volume concentration of 50%.

[0025] Preferably, in step S1, the temperature of rotary evaporation is 60°C. In one embodiment of the present invention, in step S2, the protein removal treatment specifically includes: adding ammonium sulfate to the above solution 1, then adding an equal volume of tert-butyl alcohol, shaking at a constant temperature, standing, and collecting the aqueous phase. In the embodiment of the present invention, the protein removal treatment of solution 1 is carried out by a three-phase separation method. In one embodiment of the present invention, in step S2, the temperature of the constant temperature oscillation is 30-40°C, the time of the constant temperature oscillation is 0.5-1.5 h, and the standing time is 10-30 min. Preferably, in step S2, the temperature of the constant temperature oscillation is 35°C, the time of the constant temperature oscillation is 1 h, and the standing time is 20 min. In one embodiment of the present invention, in step S2, adding tert-butanol in equal volumes specifically means that the volume ratio of tert-butanol to the added solution is 1:1.

[0026] In one embodiment of the present invention, in step S2, after adding ammonium sulfate, the saturation of ammonium sulfate is 10-30%. Preferably, after adding ammonium sulfate, the saturation of ammonium sulfate is 20%. It should be noted that the saturation of ammonium sulfate refers to the saturation of ammonium sulfate in the system.

[0027] In one embodiment of the present invention, in step S2, during dialysis, the molecular weight of the dialysis bag is 3500 Da. In one embodiment of the present invention, in step S3, the decolorization treatment specifically includes: adding activated carbon to solution 2, shaking at a constant temperature, centrifuging, filtering, and collecting the supernatant.

[0028] In one embodiment of the present invention, in step S3, the mass ratio of activated carbon to solution 2 is 0.5% to 3%. Preferably, the mass ratio of activated carbon to solution 2 is 2.5%.

[0029] In one embodiment of the present invention, in step S3, the temperature of the constant temperature oscillation is 30-40°C, 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°C, and the time of the constant temperature oscillation is 10 min. In one embodiment of the present invention, in step S3, the centrifugation is specifically performed at 3000-4000 rpm for 5-15 min. Preferably, in step S3, the centrifugation is specifically performed at 3500 rpm for 10 min.

[0030] In one embodiment of the present invention, in step S3, a Buchner funnel is used for suction filtration.

[0031] In one embodiment of the present invention, in step S4, purification using an anion exchange chromatography column specifically includes: gradient elution using membrane water, 0.1 M, and 0.3 M NaCl solutions in sequence, and collecting the polysaccharide component eluted with the 0.3 M NaCl solution.

[0032] Preferably, in step S4, during gradient elution, the flow rate is 1 mL / min, and 5 mL is collected in each tube.

[0033] Preferably, in step S4, the filler used in the anion exchange chromatography column is DEAE FF, which is a weak anion exchange medium formed by diethylaminoethyl (DEAE) bonded to a fast-flow agarose gel.

[0034] In one embodiment of the present invention, in step S4, purification using a dextran gel column specifically includes: adding the powder purified by the anion exchange chromatography column to the dextran gel column, eluting with water, and collecting components with high response values. In one embodiment of the present invention, in step S4, when water elution is used, the sample volume is 2 mL, the flow rate is 0.2 mL / min, and the 150-255 min fraction is collected.

[0035] In one embodiment of the present invention, in step S4, the dialysis is performed using 3500 Da.

[0036] In another aspect, the present invention also provides the use of any of the above-mentioned sargassum chinensis polysaccharides, or the sargassum chinensis polysaccharides prepared by any of the above-mentioned preparation methods, in the preparation of products for improving vascular function, or in the preparation of products for maintaining a healthy level of blood pressure. The products include medicines, health foods or foods.

[0037] Preferably, the embodiments of the present invention further provide the use of any of the above-mentioned Safflower Chitosan husk polysaccharides, or Safflower Chitosan husk polysaccharides prepared by any of the above-mentioned preparation methods, in the preparation of a drug for improving vascular endothelial cell damage.

[0038] The savoy vine shell polysaccharide proposed in the embodiment of the present invention has the effect of treating vascular endothelial cell damage, promoting the secretion of endothelial cell vasodilator factor NO and reducing the expression of vasoconstrictor factor ET-1, improving the antioxidant capacity of vascular endothelial cells, and providing support for the application of savoy vine shell polysaccharide.

[0039] Preferably, the polysaccharide from the fruit shell of Safflower is used in the preparation of a drug for improving vascular endothelial cell damage in vitro. More preferably, the vascular endothelial cell damage is induced by angiotensin II (Ang II). Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

[0040] Example 1 A method for preparing safflower shell polysaccharide comprises the following steps: (1) The husk of the fruit of the Chinese tomato is powdered, sieved, extracted in a water bath at 80°C for 3 h, and the powder is collected after rotary evaporation and freeze-drying, i.e., the aqueous extract of the fruit of the Chinese tomato; the aqueous extract of the fruit of the Chinese tomato is graded and extracted with anhydrous ethanol, 75% ethanol, and 50% ethanol, and then the ethanol is removed by rotary evaporation at 60°C, and the 50% ethanol extract sample is collected, and ultrafiltration is performed using an ultrafiltration membrane with a molecular weight of 30 kDa to collect the components larger than 30 kDa to obtain solution 1; (2) The protein was removed by three-phase separation technology, which specifically included adding ammonium sulfate to the ultrafiltration component larger than 30 kDa to make its saturation reach 20%, then adding tert-butanol at a ratio of 1:1 (V / V), shaking at 35 °C for 1 h, standing for 20 min, collecting the aqueous phase, and dialyzing at 3500 Da for 72 h to obtain Solution 2; (3) Using activated carbon for decolorization, specifically, adding activated carbon to 5 mL of crude polysaccharide solution with a concentration of 2 mg / mL, with the mass ratio of activated carbon to solution 2 being 2.5%, shaking at a constant temperature of 35°C for 10 min, that is, the decolorization time is 10 min, centrifuging, filtering, taking the supernatant, and freeze-drying to obtain the crude polysaccharide powder of the fruit shell of the fruit ... (4) Select anion exchange chromatography column DEAE FF for further purification. After pouring DEAE FF filler into the chromatography column, balance 2 BV with membrane water. Prepare a sample solution with a concentration of 50 mg / mL, pass it through a 0.22 μm filter membrane, and after the sample is loaded, use membrane water, 0.1, and 0.3 NaCl solutions for gradient elution in sequence at a flow rate of 1 mL / min. Collect 5 mL in each tube, collect the 0.3 M NaCl elution fraction, dialyze (3500 Da), and freeze-dry the collected sample; (5) Further purification using Sephadex G100. First, pour the Sephadex G100 along the wall of the chromatography column (1.6×80cm). After natural sedimentation, use water to equilibrate 2 BV at a flow rate of 0.2 mL / min. Prepare a 50 mg / mL sample solution obtained in step (4), load 2 mL, flow rate 0.2 mL / min, elute with water, collect 3 mL in each tube, use the phenol-sulfuric acid method to determine the absorbance value A490 in each tube solution, plot with the tube number as the horizontal axis and the absorbance value as the vertical axis, collect the part with the highest response value, that is, collect the 150~255 min component, freeze-dry, and obtain the safflower shell polysaccharide sample.

[0041] Comparative Examples 1 to 4 The same as Example 1, except that in step (2), ammonium sulfate with saturation of 0%, 40%, 60% and 80% is used to replace the saturation of 20% for deproteinization.

[0042] Comparative Examples 5-10 The same as Example 1, except that in step (3), the decolorization time is 20 min, 30 min, 40 min, 50 min, 60 min, and 70 min respectively instead of 10 min.

[0043] Comparative Examples 11-15 The same as Example 1, except that in step (3), the mass ratio of activated carbon to solution 2 is 0.5%, 1%, 1.5%, 2%, and 3% respectively instead of 2.5%.

[0044] Comparative Example 16 The same as Example 1, except that in step (4), gradient elution is performed using membrane water, 0.1 M, 0.3 M, and 0.5 M NaCl solutions, and the components eluted with membrane water, 0.1 M, 0.3 M, and 0.5 M NaCl are collected respectively.

[0045] Test Example 1 Optimization of Processing Conditions for Polysaccharide from the Shell of the Fruit ... (1) Optimization of deproteinization process The protein and total sugar contents in the polysaccharide samples of the nutcracker shell obtained in Example 1 and Comparative Examples 1 to 4 were tested.

[0046] Total sugar was determined by the phenol-sulfuric acid method, which specifically includes: weighing 10 mg of anhydrous glucose dried to constant weight at 105 °C into a 250 mL volumetric flask, adding water to the scale, and taking 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mL respectively, and each was made up to 2.0 mL with distilled water, and then 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid were added, shaken and cooled, and the absorbance was measured at 490 nm after leaving at room temperature for 20 min. 2.0 mL of water was used as a blank according to the same color development operation. The horizontal axis was the glucose concentration (mg / mL), and the vertical axis was the absorbance A. The standard curve was obtained. The sample of Miteng fruit shell polysaccharide was prepared into 1 mg / mL, and 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid were added. After shaking and cooling at room temperature for 20 minutes, the absorbance was measured at 490 nm. Use 2.0 mL of water as blank and perform the same color development operation. Make three parallel samples for each determination and substitute them into the standard curve to calculate the total sugar content.

[0047] Protein was determined by Coomassie Brilliant Blue, specifically including: weighing 10 mg of bovine serum albumin and dissolving it in 100 mL of physiological saline to obtain a 100 μg / mL protein standard solution; taking the protein standard solution to prepare 0, 20, 40, 60, 80, and 100 μg / mL bovine serum albumin solutions, accurately taking 1 mL of each of the above concentration solutions and placing them in 10 mL stoppered test tubes, adding 5 mL of Coomassie Brilliant Blue G250 reagent, mixing and placing for 20 min, and then colorimetrically measuring the absorbance at 595 nm to draw a standard curve; taking 1 mL of a 1 mg / mL samovar polysaccharide sample solution, adding 5 mL of Coomassie Brilliant Blue G250 reagent, mixing and placing for 20 min, and then colorimetrically measuring the absorbance at 595 nm, and repeating 3 times. Use 1.0 mL of water as a blank by the same operation. Substitute the standard curve to determine the protein content. The results showed that the higher the ammonium sulfate saturation, the higher the protein removal rate, and the total sugar content increased significantly when the ammonium sulfate saturation was 20%, and when the ammonium sulfate saturation was higher than 20%, the sugar content decreased significantly. Therefore, considering all factors, the best effect was achieved when the ammonium sulfate saturation was 20%.

[0048] Table 1 Optimization of deproteinization process

[0049] (2) Decolorization process optimization The supernatant obtained in step (3) of test example 1, comparative examples 5-10, and comparative examples 11-15 was tested to determine the total sugar content and decolorization rate in the supernatant. The total sugar was determined by the same phenol-sulfuric acid method as described above, and the decolorization rate was obtained by directly measuring the absorbance of the supernatant at 450 nm, and a comprehensive score was performed. The comprehensive score was calculated by selecting the total sugar content and the decolorization rate as 50%, that is, the comprehensive score = A1 / A0*100*0.5+B1 / B0*100*0.5, wherein A1 is the decolorization rate, A0 is the maximum decolorization rate value detected, B1 is the polysaccharide retention rate, and B0 is the maximum polysaccharide retention rate detected. The results are as follows Figure 1 shown.

[0050] like Figure 1 A. Figure 1 As shown in B, as the decolorization time increases, the decolorization rate increases continuously. After 20 min, the decolorization rate gradually rises. At 10 min, the sugar retention rate is 65%, which decreases significantly and reaches equilibrium after 20 min. When the decolorization time is 10 min, the comprehensive score of the polysaccharide retention rate and decolorization rate is the highest. Therefore, the decolorization time of 10 min was selected for further experiments. Figure 1 C. Figure 1 D shows that with the increase of activated carbon addition, the decolorization rate continues to increase, while the sugar retention rate continues to decrease. When the activated carbon addition is 2.5%, the comprehensive score is the highest. Therefore, the best effect is achieved when the activated carbon addition is 2.5% and the decolorization time is 10 min.

[0051] (3) Anion exchange column process optimization The total sugar content of the components eluted by the membrane 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 detected. The specific method was the same as the above-mentioned total sugar determination using the phenol-sulfuric acid method. The results are as follows: Figure 2 shown.

[0052] Depend on Figure 2 It can be seen that the sugar content detected in the components eluted with distilled water, 0.1 M NaCl solution and 0.5 M NaCl solution is low, while the sugar content detected in the component obtained with 0.3 M NaCl solution is high. Therefore, the elution effect of 0.3 M NaCl solution is the best.

[0053] Test Example 2 Structural Characterization of Polysaccharide from the Shell of the Fruit ... (1) Determination of molecular weight and purity of polysaccharide from the fruit shell of Miteng Weigh 5 mg of the polysaccharide sample of the fruit shell of the fruit of the vine obtained in Example 1 accurately, dissolve it in 1 mL of mobile phase, prepare a sample solution of 5 mg / mL, centrifuge at 12000 rpm for 10 min, take the supernatant and pass it through a 0.22 μm water-washed microporous filter membrane, and put it into a 1.8 mL injection bottle for testing. Weigh 5 mg of the dextran standard, treat it in the same way as the sample, put it into the injection bottle for standby use. The molecular weight of the polysaccharide is determined by high performance gel permeation chromatography (HPGPC). The mobile phase is 0.05 M NaCl solution, the chromatographic column is BRT105-103-101 tandem gel column (8× 300 mm), the flow rate is 0.8 mL / min, the column temperature is 40 ºC, the injection volume is 25 μL, and the detector is a differential detector RID-20. A standard curve is made according to the retention time and molecular weight of the standard, and the retention time of the sample is brought into the standard curve to calculate the molecular weight of the polysaccharide of the fruit shell of the fruit of the vine. The purity of the polysaccharide from the fruit shell of the fruit ... HPGPC was used to detect the molecular weight distribution of polysaccharides from the fruit shell of Miteng Figure 3 As shown in the figure, the GPC spectrum of the polysaccharide from the fruit shell of the pineapple tree has only a single peak at 29.73 min, and it is narrow and symmetrical, indicating that the polysaccharide from the fruit shell of the pineapple tree has a high purity and good uniformity. According to the standard curve, the molecular weight of the polysaccharide from the fruit shell of the pineapple tree is 57.51 kDa. Figure 4 As shown in the figure, the polysaccharide of the fruit shell of the pineapple has no absorption peaks at 260 nm and 280 nm, which can be judged that the polysaccharide of the fruit shell of the pineapple does not contain nucleic acids and proteins. At the same time, the sugar content in the polysaccharide of the fruit shell of the pineapple is determined by the phenol-sulfuric acid method, and the purity of the polysaccharide of the fruit shell of the pineapple is 95.04±1.64%.

[0054] (2) Fourier infrared analysis of polysaccharide from the shell of Miteng Fruit 1 mg of the dried polysaccharide of the fruit shell obtained in Example 1 was weighed, and 100 mg of KBr particles were added, and the mixture was ground into a uniform sample without obvious granularity. The sample was pressed by a tablet press and the wavelength was 4000-500 cm -1 Infrared spectroscopy was performed under the conditions of 40 °C and KBr was used as blank control. Figure 5 This is the Fourier infrared spectrum of the polysaccharide of the fruit shell of the pine cone. The polysaccharide of the fruit shell of the pine cone has the following peaks at 3397, 2937, 1613, 1419, and 1070 cm -1 All of them have absorption peaks, which are typical signal peaks of polysaccharides. -1 The signal peak at 2937 cm is mainly caused by the stretching vibration of OH between and within monosaccharide molecules; -1The signal peaks that appeared were the absorption peaks of CH, including the stretching and bending vibrations of CH, CH2, and CH3 in the polysaccharide of the fruit shell; 1613 cm -1 The absorption peak at 1760 cm -1 ~1740 cm -1 There is no absorption peak in the range of 1419 cm -1 The signal peak is generated by the CH stretching vibration and bending of the polysaccharide of the fruit shell of the fruit ... -1 The stretching vibrations of COC and CO of the husk polysaccharide of the pine vine indicate the presence of galactopyranose in the husk polysaccharide of the pine vine; 895 cm-1 and 812 cm -1 The absorption peak at 10 indicates that there are α and β glycosidic bonds in the polysaccharide of the fruit shell of the pine cone. Therefore, according to the infrared results, the infrared spectrum of the polysaccharide of the fruit shell of the pine cone is a typical polysaccharide result spectrum, and there are both α and β glycosidic bonds in the polysaccharide of the fruit shell of the pine cone.

[0055] (3) Methylation analysis of polysaccharide from the fruit shell of Miteng Weigh 2 mg of freeze-dried safflower shell polysaccharide sample and place it in a reaction bottle and add 1 mL of anhydrous dimethyl sulfoxide. Quickly add anhydrous alkaline solution and seal it, dissolve it by ultrasonication, then add iodomethane solution, stir it magnetically in a water bath at 30 °C for 60 min, and add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction. Take the methylated safflower shell polysaccharide product, add 1 mL of 2 M trifluoroacetic acid to hydrolyze it for 90 min, concentrate it under reduced pressure and evaporate it to dryness, then add 2 mL of ultrapure water and 60 mg of sodium borohydride, react for 8 h, add glacial acetic acid to neutralize it, concentrate it under reduced pressure and dry it in an oven at 101 °C, add 1 mL of acetic anhydride to acetylate it at 100 °C for 1 h, cool it, add 3 mL of toluene, concentrate it under reduced pressure and evaporate it to dryness, repeat 4 to 5 times to remove excess acetic anhydride. The acetylated product is dissolved with 3 mL of dichloromethane and transferred to a separatory funnel, add a small amount of distilled water to fully shake it, remove the upper aqueous solution, and repeat this 4 times. The dichloromethane layer was dried with an appropriate amount of anhydrous sodium sulfate and concentrated to 1 mL, and placed in a liquid phase vial. The acetylation products of the polysaccharide from the shell of the fruit ...

[0056] Figure 6This is the main glycosidic bond connection mode of the polysaccharide from the fruit shell of the fruit of the pine tree. Nine methylated sugar alcohol acetates were characterized in the GC-MS spectrum of the polysaccharide from the fruit of the Chinese ginseng tree, 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 fruit of the Chinese ginseng tree was composed of nine 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→).

[0057] (4) NMR analysis of polysaccharides from the fruit shell of Mitochon Weigh 50 mg of the polysaccharide sample of the fruit of the vine, dissolve it in 0.5 mL of heavy water and freeze-dry it, redissolve it in 0.5 mL of heavy water, continue to freeze-dry it, and repeat the above process 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 one-dimensional spectra 1H NMR, 13C NMR, DEPT-135 NMR and two-dimensional spectra HSQC, HMBC, NOESY, COSY of the polysaccharide of the fruit of the vine were obtained using a 600 MHz nuclear magnetic resonance instrument.

[0058] Table 2 Carbon-hydrogen assignments of polysaccharides from the shell of the fruit of the pine tree

[0059] Nuclear magnetic resonance technology is mainly used to determine the conformation and connection mode of polysaccharides. Figure 7Table 2 shows the NMR results of the polysaccharide from the fruit shell of Miteng. The anomeric hydrogen signals of the polysaccharide are mainly concentrated in 3.0-5.5 ppm, the non-anomeric hydrogen signals are concentrated in 3.0-4.3 ppm, and the anomeric hydrogen signals are distributed in 4.3-5.5 ppm. The anomeric carbon chemical shifts of the polysaccharide from Miteng are concentrated in 90-110 ppm, which are 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, respectively, which are typical polysaccharide signal spectra. According to the two-dimensional NMR spectrum combined with the methylation results, the main chain of the polysaccharide of the fruit of Miteng 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→. Its specific molecular structure is shown in formula (I).

[0060] (5) Morphological and structural analysis of polysaccharide from the fruit shell of Miteng The powder sample of the polysaccharide from the fruit shell of the vine was fixed on a double-sided adhesive tape and a gold layer was sprayed under a vacuum environment. The surface structure of the polysaccharide from the fruit shell of the vine was observed by SEM at 100, 500, 2000, and 5000 times.

[0061] SEM was used to further analyze the surface morphology of the polysaccharide from the fruit shell of the fruit ... Figure 8 The surface morphology of the polysaccharide of the fruit shell of the vine was observed under a microscope of 100, 500, 2000 and 5000 times. The polysaccharide of the fruit shell of the vine mainly presents a block structure, irregular shape, loose structure, rough surface, and more honeycomb structures.

[0062] Test Example 3 Experimental study on the effect of polysaccharide from the shell of citrus vine in treating endothelial cell injury Experimental design (1) Vascular endothelial cell culture HUVECs were cultured in ECM medium at 37 °C and 5% CO2 incubator.

[0063] (2) Cell treatment HUVECs were divided into 5 groups and treated as follows: a) CON group was a blank control group and cultured with ECM medium.

[0064] b) ANG group was the model group and was treated with 1 μM Ang Ⅱ for 24 h.

[0065] c) The 100 μg / mL group was a low-dose group, that is, after being treated with 100 μg / mL of S. caenorhabditis elegans polysaccharide for 24 h, it was then treated with 1 μM Ang Ⅱ for 24 h.

[0066] d) The 200 μg / mL group was the medium-dose group, that is, the cells were treated with 200 μg / mL of P. caenese polysaccharide for 24 h, and then with 1 μM Ang Ⅱ for 24 h.

[0067] e) The 400 μg / mL group was a high-dose group, that is, after being treated with 400 μg / mL of S. caesalpinia quinata polysaccharide for 24 h, it was then treated with 1 μM Ang Ⅱ for 24 h.

[0068] (3) Index determination NO, ET-1, malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) were determined according to the kit instructions.

[0069] (4) RT-qPCR analysis Add 1 mL of Trizol reagent to the cells, mix thoroughly and lyse in a centrifuge tube for 5 min, add 200 μL of chloroform and shake vigorously for 15 s and stand at room temperature for 3 min, centrifuge at 12000 rpm for 15 min, take the upper liquid phase to the RNase-Free centrifuge tube, add an equal volume of isopropanol, mix and stand for 10 min, centrifuge at 12000 rpm for 10 min, remove the supernatant, add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 4 ℃, 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, measure the concentration at 260 nm and 280 nm using an ultraviolet spectrophotometer, and calculate the concentration and purity. Reverse transcribe cDNA using total mRNA as a template. The reverse transcription product can be used for PCR reaction and fluorescent quantitative PCR reaction. The internal reference gene was β-actin, and the relative expression of mRNA was calculated using the 2-ΔΔCt method.

[0070] (5) Immunoblotting analysis The cells were washed with pre-cooled PBS and lysed at low temperature for 10 min with 200 μL of RIPA lysis buffer. The supernatant was collected after centrifugation at 4 °C for 15 min for subsequent experiments. The protein content was determined by BCA method. 5× loading buffer was added to 100 uL of protein supernatant and mixed well. The protein sample was prepared by boiling water for 5 min. A 10% separation gel and a 5% concentration gel were prepared, and the marker and the prepared protein sample were added to the lane. The electrophoresis voltage was 75 V and the time was 130 min. After the electrophoresis, the membrane was transferred at a constant current of 300 mA. The nitrocellulose (NC) membrane was washed once with PBST and blocked with 5% skim milk powder at room temperature for 90 min. After the primary antibody was diluted, the NC membrane was incubated with the primary antibody at 4 °C overnight. After the incubation, it was washed 3 times with PBST for 10 min each time. The diluted secondary antibody was incubated with the NC membrane at room temperature for 90 min. After the incubation, it was washed 3 times with PBST for 10 min each time. The membrane was incubated with ECL chemiluminescent solution for 1 min for color development, and the gel imaging system was used to take pictures and record.

[0071] Experimental results (1) The husk of the fruit of the vine reduces the damage of vascular endothelial cells like Fig. 9 As shown in the results, the polysaccharide of the fruit shell of Safflower has no toxic effect on endothelial cells at a concentration of 0-400 mg / mL. High doses of Safflower shell polysaccharide can promote the proliferation of endothelial damaged cells induced by Ang II. Ang II can significantly reduce the secretion of NO in endothelial cells, while Safflower shell polysaccharide can significantly promote the secretion of NO at medium and high concentrations; Ang II can significantly promote the secretion of vasoconstrictor factor ET-1, while Safflower shell polysaccharide can significantly inhibit the secretion level of ET-1 at low, medium and high concentrations. Therefore, Safflower shell polysaccharide can promote the secretion of NO and reduce the expression of ET-1 to alleviate Ang II-induced endothelial cell dysfunction.

[0072] (2) Mitochondrial shell polysaccharide improves the antioxidant capacity of vascular endothelial cells Oxidative stress is another important factor causing endothelial cell dysfunction. Under normal circumstances, the production and elimination of ROS in cells are in a balanced state, but when the intracellular ROS level exceeds the clearance capacity of the antioxidant system, the redox balance is destroyed, which can eventually lead to cell dysfunction. Fig.10As shown in the figure, 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 was significantly decreased after treatment with safflower shell polysaccharide. Quantitative analysis of the fluorescence intensity showed that safflower shell 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 will occur, resulting in an increase in the content of MDA in cells. SOD is an important antioxidant enzyme in cells, which can effectively remove free radicals in cells. The determination of MDA content and SOD activity showed that Ang II could significantly promote the production of MDA and reduce the enzyme activity of SOD, destroying the balance of redox reaction in HUVECs cells, while safflower shell polysaccharide treatment significantly inhibited the production of MDA in endothelial cells induced by Ang II and increased the enzyme activity of SOD. Therefore, safflower shell 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.

[0073] (3) Analysis of gene expression and protein related to regulation of vascular endothelial cell function by polysaccharide from the fruit shell of Safflower RT-qPCR and WB experiments were used to further explore the mechanism by which the polysaccharide from the fruit shell of the fruit ... Fig.11 and 12As shown in the results, Ang Ⅱ induced the gene expression and protein secretion of endothelial cell AT1R, while after administration of different concentrations of sainfoin, its expression and secretion levels decreased significantly, indicating that sainfoin can inhibit the gene expression of AT1R in endothelial cells induced by Ang Ⅱ. eNOS can promote the production of vasodilator factor NO, while Ang Ⅱ can significantly reduce the gene expression of eNOS. After administration of sainfoin, the expression level of eNOS in endothelial cells increased significantly, indicating that sainfoin can promote the gene expression and protein secretion of eNOS. In addition, the ACE2-Ang (1-7)-MasR pathway plays a negative feedback regulatory role in Ang Ⅱ-induced endothelial cell dysfunction. ACE2 can hydrolyze Ang Ⅱ into Ang (1-7), preventing Ang Ⅱ from binding to its receptor AT1R, while the hydrolyzed Ang (1-7) can bind to its receptor Mas and activate the downstream pathway. In the Ang Ⅱ group, the levels of ACE2, Ang (1-7) and MasR in the cells were significantly reduced, while the administration of saitenghus polysaccharide significantly increased the gene expression and protein secretion levels of ACE2, Ang (1-7) and MasR. In addition, saitenghus polysaccharide can also promote the expression of AKT, a downstream gene of the ACE2-Ang (1-7)-MasR pathway. Therefore, saitenghus polysaccharide can inhibit the expression of Ang Ⅱ receptor AT1R and increase the expression of eNOS, ACE2, Ang (1-7), MasR and AKT proteins, thereby alleviating Ang Ⅱ-induced endothelial dysfunction.

[0074] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A polysaccharide of the fruit shell of Mitsugu, characterized in that It is polymerized from galactose, arabinose, rhamnose and galacturonic acid and has a structure as shown in formula (I); Formula (I).

2. The method for preparing the polysaccharide of the fruit shell of the fruit ... The steps include: S1, after the husk of the vine is pulverized, it is extracted with hot water and freeze-dried. The obtained husk water extract is further graded and extracted with an ethanol solution, and the obtained components are subjected to ultrafiltration to obtain solution 1; S2, after removing protein from the above solution 1, collecting the aqueous phase and dialyzing to obtain solution 2; S3, decolorizing the above solution 2, freeze-drying, and obtaining crude polysaccharide powder of the fruit shell of the citrus vine; S4. Purify the above-mentioned crude polysaccharide powder of the fruit shell of the pine needle by an anion exchange chromatography column, dialyze, and freeze-dry. The obtained powder is purified by a dextran gel column to obtain the polysaccharide of the fruit shell of the pine needle.

3. The preparation method according to claim 2, characterized in that: In step S1, the temperature of hot water extraction is 70-90°C, and the time of hot water extraction is 1-5 hours; In step S1, the molecular weight of the ultrafiltration membrane is 10-50 kDa; In step S1, the graded extraction with ethanol solution specifically includes: gradedly extracting the water extract of the fruit shell of the citrus indica with anhydrous ethanol, an ethanol solution with a volume concentration of 75%, and an ethanol solution with a volume concentration of 50%, removing the ethanol by rotary evaporation, and collecting the components obtained after extraction with the ethanol solution with a volume concentration of 50%.

4. The preparation method according to claim 2, characterized in that: In step S2, the protein removal treatment specifically includes: adding ammonium sulfate to the above solution 1, then adding tert-butyl alcohol in equal volume, shaking at a constant temperature, standing, and collecting the aqueous phase; Preferably, after adding ammonium sulfate, the saturation of ammonium sulfate is 10-30%; Preferably, the equal volume of tert-butanol is added, specifically the volume ratio of tert-butanol to the added solution is 1:

1.

5. The preparation method according to claim 2, characterized in that: In step S3, the decolorization treatment specifically includes: adding activated carbon to solution 2, shaking at a constant temperature, centrifuging, filtering, and collecting the supernatant; Preferably, in step S3, the mass ratio of activated carbon to solution 2 is 0.5% to 3%. Preferably, the mass ratio of activated carbon to solution 2 is 2.5%.

6. The preparation method according to claim 2, characterized in that: In step S4, purification using an anion exchange chromatography column specifically includes: gradient elution using membrane water, 0.1 M, and 0.3 M NaCl solutions in sequence, and collecting the polysaccharide component eluted with the 0.3 M NaCl solution.

7. The preparation method according to claim 2, characterized in that: In step S4, purification using a dextran gel column specifically includes: adding the powder purified by the anion exchange chromatography column to the dextran gel column, eluting with water, and collecting components with high response values; Preferably, in step S4, when water elution is used, the sample volume is 2 mL, the flow rate is 0.2 mL / min, and the 150-255 min fraction is collected.

8. Use of the sargassum chinensis polysaccharide according to claim 1, or the sargassum chinensis polysaccharide prepared by the preparation method according to any one of claims 2 to 7 in preparing a product for improving vascular function, or in preparing a product for maintaining a healthy level of blood pressure.

9. The use according to claim 8, characterized in that: The products include medicines, health foods or foods.

10. Use of the polysaccharide of the fruit shell of the saussurea chinensis according to claim 1, or the polysaccharide of the fruit shell of the saussurea chinensis obtained by the preparation method according to any one of claims 2 to 7 in the preparation of a drug for improving vascular endothelial cell damage.

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