Potentilla anserine composition with anti-hypoxia function and preparation method thereof

Abstract

The invention discloses a silverweed cinquefoil root composition with an anti-hypoxia function and a preparation method thereof, and belongs to the technical field of medical compositions.The silverweed cinquefoil root composition comprises silverweed cinquefoil root polysaccharide, silverweed cinquefoil root polyphenol and silverweed cinquefoil root glycoside compounds according to the mass ratio of (0.6-0.8): (0.2-0.4): (0-0.05), and the silverweed cinquefoil root glycoside compounds comprise roxburgh rose glycoside and multiflora rose glycoside according to the mass ratio of (0.05-0.45): 1. The compounding of the potentilla anserine polysaccharide and the potentilla anserine polyphenol can provide a comprehensive free radical scavenging effect, maintain the dynamic balance of generation and scavenging of oxygen free radicals in an organism, coordinate cell energy metabolism and help the organism to better utilize limited oxygen resources. The silverweed cinquefoil root glycoside compound is used as an additional antioxidant, oxidative stress reaction and side reaction can be reduced, and the obtained silverweed cinquefoil root composition can well improve the anti-hypoxia capacity of an organism.

Description

Technical Field

[0001] The invention belongs to the technical field of medical compositions, and in particular relates to a Potentilla anserine composition with anti-hypoxia function and a preparation method thereof. Background Art

[0002] Oxygen is an important component of life activities. For oxygen, a basic substance necessary for life activities, cells have evolved complex regulatory mechanisms to support the oxygen supply required for survival. However, in some high-altitude areas, the harsh climate environment of low pressure, low oxygen, and high radiation will result in low oxygen supply or low body utilization. The body will produce excessive oxidative stress due to lack of oxygen, causing inflammatory reactions, cell apoptosis, and energy metabolism disorders, etc., which will lead to damage to various tissues and organs, making it difficult for people entering the plateau to adapt.

[0003] At present, the commonly used anti-hypoxia drugs in clinical practice are generally Western medicines such as nimodipine and dexamethasone, which have a certain effect on alleviating hypoxia symptoms, but their side effects are large, and their application is therefore limited. Potentilla anserina is a plant used as both medicine and food in plateau areas. It mainly has the effects of anti-oxidation, anti-hypoxia, protecting myocardial cells, enhancing immunity, protecting the liver, and replenishing blood. The use of Potentilla anserina to protect the body's tolerance to hypoxia has few side effects. However, the bioavailability of Potentilla anserina to the human body is relatively low, making it difficult to achieve the expected effect, and long-term or high-dose use still has certain adverse reactions. Summary of the invention

[0004] The purpose of the present invention is to provide a bracken ansemen composition with anti-hypoxia function and a preparation method thereof, so as to solve the problem of various body injuries caused by human body hypoxia under a hypoxic environment, and the effect is fast and the side effects are small.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] In a first aspect, the present invention provides a bracken anserine composition having an anti-hypoxia function, wherein the bracken anserine composition comprises bracken anserine polysaccharides, bracken anserine polyphenols and bracken anserine glycoside compounds in a mass ratio of (0.6-0.8): (0.2-0.4): (0-0.05).

[0007] Preferably, the terpene glycoside compound comprises roxburghii glycoside and rosa roxburghii glycoside in a mass ratio of (0.05-0.45):1.

[0008] Preferably, the bracken ansemen composition of the present invention can have a certain anti-hypoxia function even without containing bracken ansemen glycoside compounds.

[0009] In a low-pressure, low-oxygen environment, the dynamic balance of oxygen free radical production and removal in the body will be destroyed, triggering oxidative stress reaction, increasing the free radical content in the body, and reducing the ability to remove free radicals, causing a large amount of free radicals to accumulate, thereby causing cell membrane lipid peroxidation damage. And the available oxygen content in the mitochondrial electron transport chain is reduced, resulting in a large amount of reactive oxygen species, causing brain damage. By adopting the above technical scheme, Potentilla polysaccharide has the activity of removing free radicals, and also has a good scavenging effect on superoxide anions. Under hypoxic conditions, Potentilla polysaccharide can improve the activity of superoxide dismutase in the body and myocardial cells, thereby improving the body's ability to remove free radicals, reducing the occurrence of oxidative stress reactions in the body, protecting the body's cells from damage, and can also regulate the cell metabolism capacity under hypoxic stress, improve the utilization rate of oxygen by cells, and help the body to maintain normal functions in hypoxia. Potentilla polyphenols can improve the body's ability to remove oxygen free radicals, prevent lipid peroxidation, improve mitochondrial function after hypoxia, and can also promote vasodilation and increase blood flow, thereby improving the oxygen supply of tissues under hypoxia.

[0010] The potholes composition of the present invention is compounded with potholes polysaccharide and potholes polyphenol. Under a certain ratio, the two can show good synergistic effect. Specifically, potholes polysaccharide can remove free radicals through non-enzymatic pathways, and potholes polyphenol can directly remove free radicals and chelate metal ions to achieve the removal of oxygen free radicals. The combination of the two can provide a more comprehensive free radical scavenging effect, and maintain the dynamic balance of oxygen free radical generation and removal in the body. At the same time, the vasodilator effect of potholes polyphenol can help improve blood circulation and increase oxygen delivery, and potholes polysaccharide can regulate the energy metabolism of cells and tissues in this process, helping the body to better utilize limited oxygen resources, thereby improving the body's ability to resist hypoxia.

[0011] And the anti-hypoxia function of the composition can be improved by adding a glycoside compound to the bracken anserine composition of the present invention. The bracken anserine glycoside compound is a mixture of roxburghii glycoside and wild rose glycoside. Roxburghii glycoside and wild rose glycoside are one of the potential quality marker components extracted from bracken anserine, and have good anti-oxidative stress effect. As an additional antioxidant, the bracken anserine glycoside compound can improve the free radical scavenging ability of the body under a hypoxic environment together with bracken anserine polysaccharide and bracken anserine polyphenol, and reduce the damage of oxidative stress to cells. In addition, the present invention finds that during the process of bracken anserine polyphenol dilating blood vessels, the bracken anserine glycoside compound can protect the dilated blood vessels, effectively reduce the generation of side effects, further improve blood circulation, and increase the delivery of oxygen in the body, thereby improving the body's anti-hypoxia ability.

[0012] In a second aspect, the present invention provides a method for preparing a Potentilla anserina composition having an anti-hypoxia function, comprising the following process steps:

[0013] S1. Pretreating Potentilla anserina to obtain Potentilla anserina concentrate;

[0014] S2. Extracting and purifying polysaccharides, polyphenols and glycosides from the concentrated solution of Potentilla anserina respectively;

[0015] S3. Mix Potentilla anserine polysaccharides, Potentilla anserine polyphenols and Potentilla anserine glycoside compounds in corresponding proportions and dissolve them in ethanol, concentrate and freeze-dry the mixture to prepare a Potentilla anserine composition powder, which is a Potentilla anserine composition with anti-hypoxia function.

[0016] Preferably, the pretreatment comprises the following steps:

[0017] S101. The bracken hemp was washed, dusted and dried, and then crushed and sieved to obtain bracken hemp powder;

[0018] S102. Reflux and defatten the bracken powder in a solvent for 2 to 3 times;

[0019] S103. After evaporating the solvent, add the Potentilla anserine powder into distilled water, extract at 80-90° C., and concentrate the extract by centrifugation and reduced pressure to obtain Potentilla anserine concentrate.

[0020] More preferably, the average particle size of the Potentilla anserina powder is 100 mesh.

[0021] More preferably, the solvent is one of anhydrous ethanol and petroleum ether.

[0022] More preferably, the solid-liquid ratio of the extraction is 1 g: (15-20) mL.

[0023] Preferably, the extraction and purification of Potentilla anserina polyphenols comprises the following steps: extracting Potentilla anserina concentrate with n-butanol, separating and purifying the final eluted fractions through a macroporous resin after chromatographic separation and gradient elution of the obtained extract, and finally obtaining Potentilla anserina polyphenols through water washing and alcohol washing.

[0024] Preferably, the purification of the glycoside compounds of the bracken angustifolia comprises the following steps: preparing a solvent of n-hexane-ethyl acetate-methanol-water in a volume ratio of 1:5:2:5, and ultrasonically degassing the solvent for standby use; mixing the bracken angustifolia concentrate with water, eluting the mixture through adsorption by a macroporous resin, and then concentrating the mixture by reduced pressure evaporation, adding the mixture to the standby solvent, and separating and purifying the bracken angustifolia glycoside compounds by high-speed countercurrent chromatography.

[0025] By adopting the above technical scheme, the bracken ansemen is extracted by heating to obtain the bracken ansemen extract. At a certain temperature, the mass transfer rate of the active substances in the bracken ansemen after repeated defatting to the extract is increased, which is beneficial to the subsequent extraction and purification of each active ingredient. The purified active ingredients are then combined to obtain a bracken ansemen composition with anti-hypoxia function.

[0026] Preferably, the extraction and purification of Potentilla anserina polysaccharide comprises the following steps:

[0027] S201. Adding amylase to the concentrated solution of Pteridium anserine, heating to 50-60°C, stirring for enzymolysis, and obtaining a de-starched concentrated solution of Pteridium anserine;

[0028] S202. The destarch-free Pteridium anserine concentrate was added to anhydrous ethanol for alcohol precipitation, and then centrifuged to obtain a crude product of Pteridium anserine polysaccharide;

[0029] S203. The crude product of Potentilla anserina polysaccharide is prepared into an aqueous solution, a protease, a phenolic antioxidant and a polyethylene glycol-conjugated compound are added, the pH value of the solution is adjusted to 7 to 8, the temperature is 35 to 45° C., the solution is stirred for enzymatic hydrolysis and then centrifuged to remove the precipitate, the obtained liquid phase is mixed with a Sevage reagent, the mixture is evenly mixed and then allowed to stand for stratification, and the supernatant is retained;

[0030] S204. Add hydrogen peroxide to the obtained supernatant, adjust the solution pH to 7.5-8, the temperature to 65-75°C, stir to decolorize, centrifuge to remove the precipitate, and obtain Potentilla anserina polysaccharide after alcohol precipitation.

[0031] Preferably, the amount of amylase added is 0.05-0.1% of the Potentilla anserina concentrate.

[0032] Preferably, the mass fraction of the aqueous solution of the crude product of Potentilla anserina polysaccharide is 1-2%.

[0033] Preferably, the phenolic antioxidant comprises a combination of one or more of catechins, gallic acid and anthocyanins; the added amount of the phenolic antioxidant is 0.01-0.2% of the mass of the crude product of Potentilla anserina polysaccharide.

[0034] Preferably, the raw materials of the polyethylene glycol-conjugated compound include epoxidized polyethylene glycol, iminooxalic acid and copper salt in a mass ratio of 1:(0.6-0.7):(0.5-0.6).

[0035] Preferably, the copper salt includes a combination of one or more of copper sulfate, copper nitrate, copper carbonate, copper acetate and copper chloride.

[0036] Preferably, the amount of polyethylene glycol-conjugated compound added is 2-5% of the mass of the crude product of Potentilla anserina polysaccharide.

[0037] Preferably, the polyethylene glycol-conjugated compound is prepared according to the following method:

[0038] Preparation of epoxidized polyethylene glycol: Add polyethylene glycol to a solvent, stir to dissolve, then add epichlorohydrin and 40-50% by mass of boron trifluoride-ether solution, react for 12-24 hours, then add sodium hydroxide, stir to react for 5-6 hours, then filter and dry to obtain epoxidized polyethylene glycol.

[0039] Preparation of polyethylene glycol-conjugated compound: add iminodiacetic acid to sodium carbonate solution and stir to dissolve, add epoxidized polyethylene glycol, increase the temperature to 60-70°C, stir to react for 12-15 hours, and finally extract with chloroform, dehydrate and rotary evaporate to obtain a pre-reactant; dissolve copper salt in water, adjust the pH value of the solution to 4-4.5, add the pre-reactant, stir to react for 5-6 hours, and then extract with chloroform, dehydrate and rotary evaporate to obtain a polyethylene glycol-conjugated compound.

[0040] More preferably, the molecular weight of polyethylene glycol is 3500-4500.

[0041] More preferably, the solvent includes one of benzene, ethyl acetate, acetone and N,N-dimethylformamide.

[0042] More preferably, the amount of epichlorohydrin added is 4 to 5% of the amount of polyethylene glycol added.

[0043] More preferably, the amount of sodium hydroxide added is 0.8 to 1% of the amount of polyethylene glycol added.

[0044] Preferably, the protease is one of trypsin, neutral protease and papain; the added amount of the protease is 0.05-0.1% of the mass of the crude product of Potentilla anserina polysaccharide.

[0045] Preferably, the Sevage reagent is a mixed solution of chloroform and n-butanol in a volume ratio of (4-6):1; the liquid phase to the Sevage reagent solid-liquid ratio is (1-3):1.

[0046] Preferably, the amount of hydrogen peroxide added is 15-20% of the mass of the supernatant.

[0047] By adopting the above technical scheme, in the purification process of Potentilla anserina polysaccharide, protein is its main impurity. Its presence increases the hygroscopicity of polysaccharide, and the charge it carries will also adsorb a large number of other impurities, increasing the difficulty of removing other impurities. At the same time, protein will also adsorb polysaccharide, which brings difficulties to the separation of polysaccharide. At the same time, in the compounding process of Potentilla anserina polysaccharide and Potentilla anserina polyphenol, if the protein impurities in Potentilla anserina polysaccharide are not cleaned up, the residual protein impurities will interact with the polyphenol non-covalently, and the formed complex will reduce the solubility of Potentilla anserina polyphenol and change the bioavailability and activity of Potentilla anserina polyphenol, thereby resulting in a decrease in the anti-hypoxia ability of the obtained Potentilla anserina composition.

[0048] Therefore, in order to improve the anti-hypoxia ability of the composition, it is necessary to optimize the purification process of Potentilla anserina polysaccharide and improve the removal rate of protein. In the purification process of Potentilla anserina polysaccharide of the present invention, phenolic antioxidants and polyethylene glycol-conjugated compounds are added together with protease. The addition of phenolic antioxidants can protect the structure of Potentilla anserina polysaccharide from being destroyed during the enzymatic hydrolysis process, help inhibit the activity of residual enzymes, and prevent them from acting on Potentilla anserina polysaccharide, resulting in polysaccharide decomposition, thereby improving the yield and biological activity of Potentilla anserina polysaccharide and enhancing the anti-hypoxia ability of Potentilla anserina composition; compared with other antioxidants, phenolic antioxidants will not affect the activity of subsequent Potentilla anserina polyphenols, but can protect Potentilla anserina polyphenols from inactivation.

[0049] The addition of polyethylene glycol-conjugated compounds can utilize conjugated chelated metal compounds to selectively bind to proteins during enzymatic hydrolysis, and metal ions can coordinate with specific amino acid residues on the surface of proteins, thereby enhancing the binding force between proteins and polyethylene glycol-conjugated compounds, which is beneficial to the separation of proteins from Potentilla anserina polysaccharides. At the same time, the steric hindrance effect of polyethylene glycol-conjugated compounds can greatly reduce the non-specific interaction with Potentilla anserina polysaccharides on the one hand, and can also help reduce the effective volume between protein molecules on the other hand, which is beneficial to the aggregation and precipitation of proteins, thereby protecting the structure and activity of Potentilla anserina polysaccharides while improving the removal rate of proteins, which is beneficial to the further separation of proteins from Potentilla anserina polysaccharides, thereby improving the yield and purity of Potentilla anserina polysaccharides, and improving the activity and anti-hypoxia ability of the Potentilla anserina composition.

[0050] Beneficial effects of the present invention:

[0051] 1. The bracken anserine composition with anti-hypoxia function of the present invention is a composition of bracken anserine polysaccharide, bracken anserine polyphenol and bracken anserine glycoside compounds. The compound of bracken anserine polysaccharide and bracken anserine polyphenol can provide a comprehensive free radical scavenging effect and maintain the dynamic balance of oxygen free radical generation and scavenging in the body; bracken anserine polyphenol can improve blood circulation, and bracken anserine polysaccharide can regulate energy metabolism. The two can work together to help the body better utilize limited oxygen resources. As an additional antioxidant, bracken anserine glycoside compounds can help improve the free radical scavenging hydrogen energy under hypoxic stress, reduce oxidative stress reactions, protect dilated blood vessels, and reduce the generation of side reactions. The bracken anserine composition obtained can well improve the body's anti-hypoxia ability.

[0052] 2. In the purification process of the Potentilla anserine polysaccharide in the Potentilla anserine composition of the present invention, phenolic antioxidants and polyethylene glycol-conjugated compounds are also added. The phenolic antioxidants can protect the Potentilla anserine polysaccharide from being destroyed during the enzymatic hydrolysis process, and the polyethylene glycol-conjugated compounds can help aggregate and precipitate proteins, thereby improving the protein separation rate, thereby improving the activity and yield of the obtained Potentilla anserine polysaccharide, reducing impurities in the polysaccharide, and improving the activity and anti-hypoxia ability of the Potentilla anserine composition. DETAILED DESCRIPTION

[0053] 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. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Preparation Example

[0055] Preparation Example 1: A polyethylene glycol-conjugated compound was prepared according to the following method:

[0056] Add 50 g of polyethylene glycol 4000 to 250 mL of benzene, stir to dissolve, add 2.5 g of epichlorohydrin and 50% by mass of boron trifluoride-ether solution, react for 24 h, prepare 0.5 g of sodium hydroxide into a 40% by mass sodium hydroxide aqueous solution, add to the above reaction solution, stir to react for 5 h, then filter and dry to obtain epoxidized polyethylene glycol;

[0057] Add 6.5 g of iminodiacetic acid to 100 mL of 2 mol / L sodium carbonate solution and stir to dissolve, add 10 g of the above-obtained epoxidized polyethylene glycol, raise the temperature to 70° C., stir to react for 15 h, and finally extract with chloroform, dehydrate, and remove chloroform by rotary evaporation to obtain a pre-reactant; dissolve 5.5 g of copper sulfate in 100 mL of water, adjust the solution pH to 4, add the pre-reactant, stir to react for 6 h, and then extract with chloroform, dehydrate, and remove chloroform by rotary evaporation to obtain a polyethylene glycol-conjugated compound.

[0058] Preparation Example 2, a polyethylene glycol-conjugated compound, is different from Preparation Example 1 only in that the amount of iminodiacetic acid added is 6 g; the amount of copper sulfate added is 5 g.

[0059] Preparation Example 3, a polyethylene glycol-conjugated compound, is different from Preparation Example 1 only in that the amount of iminodiacetic acid added is 7 g; the amount of copper sulfate added is 6 g.

[0060] Preparation Example 4, a polyethylene glycol-conjugated compound, is different from Preparation Example 1 only in that the amount of iminodiacetic acid added is 5 g; the amount of copper sulfate added is 4 g.

[0061] Preparation Example 5, a polyethylene glycol-conjugated compound, is different from Preparation Example 1 only in that the amount of iminodiacetic acid added is 8 g; the amount of copper sulfate added is 7 g.

[0062] Example

[0063] Example 1, a bracken ansemen composition with anti-hypoxia function, prepared by the following process steps:

[0064] S1. Pretreating Potentilla anserina to obtain Potentilla anserina concentrate;

[0065] S101. The fern was washed, dusted and dried and crushed, and passed through a 100-mesh sieve to obtain fern powder;

[0066] S102. The powder of Pteris anserina was degreased by reflux in petroleum ether for 3 times, each degreasing time was 2 h;

[0067] S103. After evaporating the petroleum ether, add the bracken powder to distilled water with a solid-liquid ratio of 1 g:20 mL, extract at 85°C for 1 h, and obtain the extract by centrifugation and vacuum concentration to obtain the bracken concentrate.

[0068] S2. Extracting and purifying polysaccharides, polyphenols and glycosides from the concentrated solution of Potentilla anserina respectively;

[0069] The extraction and purification of Potentilla anserine polyphenols includes the following steps: extracting Potentilla anserine concentrate with n-butanol, with the solid-liquid ratio of 1 g:15 mL; subjecting the obtained extract to chromatographic separation and gradient elution, separating and purifying the final eluted fraction through a macroporous resin (model: Diaion HP-20 resin); and finally obtaining Potentilla anserine polyphenols through water washing and alcohol washing.

[0070] The purification of the glycoside compounds of the bracken angustifolia comprises the following steps: preparing a solvent of n-hexane-ethyl acetate-methanol-water in a volume ratio of 1:5:2:5, and setting it aside after ultrasonic degassing; mixing the bracken angustifolia concentrate with water, and then eluting it through adsorption by a macroporous resin (model: HPD-100 resin), and then concentrating it through reduced pressure evaporation, adding it to the stand-by solvent, and separating and purifying it through high-speed countercurrent chromatography to obtain the bracken angustifolia glycoside compounds.

[0071] The extraction and purification of Potentilla anserina polysaccharide includes the following steps:

[0072] S201. Adding α-amylase to the concentrated solution of Potentilla anserina, wherein the amount of α-amylase added is 0.05% of the concentrated solution of Potentilla anserina, heating to 55 ° C, stirring for enzymolysis, and obtaining a destarched concentrated solution of Potentilla anserina;

[0073] S202. The destarch-free Pteridium anserine concentrate was added to anhydrous ethanol for alcohol precipitation, and then centrifuged to obtain a crude product of Pteridium anserine polysaccharide;

[0074] S203. The crude product of Potentilla anserina polysaccharide is added to water to prepare a 2% aqueous solution of the crude product of Potentilla anserina polysaccharide by mass, and then 0.08% trypsin, 0.1% catechin and 3% of the polyethylene glycol-conjugated compound prepared in Preparation Example 1 are added, the pH value of the solution is adjusted to 7.5 and the temperature is 40° C. After stirring for enzymatic hydrolysis, the precipitate is removed by centrifugation, and the obtained liquid phase is mixed with Sevage reagent (a mixed solution of chloroform and n-butanol in a volume ratio of 5:1), the liquid phase and the Sevage reagent have a solid-liquid ratio of 2:1, and after mixing evenly, the mixture is allowed to stand for stratification, and the supernatant is retained;

[0075] S204. Add 20% hydrogen peroxide to the obtained supernatant, adjust the solution pH to 8, the temperature to 70°C, stir to decolorize, centrifuge to remove the precipitate, and obtain Potentilla anserina polysaccharide after alcohol precipitation.

[0076] S3. Mix Potato polysaccharides, Potato polyphenols and Potato glycosides in a mass ratio of 0.7:0.3:0.02 and dissolve them in ethanol, wherein the Potato glycosides are a mixture of rosin and rhododendron in a mass ratio of 0.25:1. Concentrate and freeze-dry the mixed solution to prepare a Potato composition powder, which is a Potato composition with anti-hypoxia function.

[0077] Example 2, a Potentilla anserine composition with anti-hypoxia function, which is different from Example 1 only in that the mass ratio of Potentilla anserine polysaccharide, Potentilla anserine polyphenol and Potentilla anserine glycoside compound is 0.6:0.4:0.02, wherein the Potentilla anserine glycoside compound is a mixture of roxburghii glycoside and wild rose glycoside in a mass ratio of 0.45:1.

[0078] Example 3, a Potentilla anserine composition with anti-hypoxia function, which is different from Example 1 only in that the mass ratio of Potentilla anserine polysaccharide, Potentilla anserine polyphenol and Potentilla anserine glycoside compound is 0.8:0.2:0.02, wherein the Potentilla anserine glycoside compound is a mixture of roxburghii glycoside and wild rose glycoside in a mass ratio of 0.05:1.

[0079] Example 4, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that no Potentilla anserine glycoside compound is added for mixing.

[0080] Example 5, a Potentilla anserine composition with anti-hypoxia function, which is different from Example 1 only in that the mass ratio of Potentilla anserine polysaccharide, Potentilla anserine polyphenol and Potentilla anserine glycoside compounds is 0.6:0.4:0.05.

[0081] Example 6, a Potentilla anserine composition with anti-hypoxia function, which differs from Example 1 only in that the step S203 of extracting and purifying Potentilla anserine polysaccharide is as follows: add the crude Potentilla anserine polysaccharide product to water to prepare an aqueous solution of the crude Potentilla anserine polysaccharide product with a mass fraction of 2%, then add 0.1% trypsin, 0.2% catechin and 5% of the polyethylene glycol-conjugated compound prepared in Preparation Example 2, adjust the solution pH to 8 and the temperature to 40°C, stir for enzymatic hydrolysis and then centrifuge to remove the precipitate, mix the obtained liquid phase with Sevage reagent (a mixed solution of chloroform and n-butanol in a volume ratio of 4:1), the liquid phase and the Sevage reagent have a solid-liquid ratio of 1:1, mix evenly and let stand for stratification, and retain the supernatant.

[0082] Example 7, a Potentilla anserine composition with anti-hypoxia function, which differs from Example 1 only in that the step S203 of extracting and purifying Potentilla anserine polysaccharide is as follows: add the crude Potentilla anserine polysaccharide product to water to prepare a 2% aqueous solution of the crude Potentilla anserine polysaccharide product by mass, then add 0.05% trypsin, 0.02% catechin and 2% of the polyethylene glycol-conjugated compound prepared in Preparation Example 3, adjust the solution pH to 7 and the temperature to 40°C, stir for enzymatic hydrolysis and then centrifuge to remove the precipitate, mix the obtained liquid phase with Sevage reagent (a mixed solution of chloroform and n-butanol in a volume ratio of 6:1), the liquid phase and the Sevage reagent have a solid-liquid ratio of 3:1, mix evenly and let stand for stratification, and retain the supernatant.

[0083] Example 8, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that the polyethylene glycol-conjugated compound prepared in Preparation Example 1 is replaced by an equal amount of the polyethylene glycol-conjugated compound prepared in Preparation Example 4.

[0084] Example 9, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that the polyethylene glycol-conjugated compound prepared in Preparation Example 1 is replaced by an equal amount of the polyethylene glycol-conjugated compound prepared in Preparation Example 5.

[0085] Example 10, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that the added amount of the polyethylene glycol-conjugated compound prepared in Preparation Example 1 is 1% of the mass of the crude product of Potentilla anserine polysaccharide.

[0086] Example 11, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that the added amount of the polyethylene glycol-conjugated compound prepared in Preparation Example 1 is 6% of the mass of the crude product of Potentilla anserine polysaccharide.

[0087] Example 12, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that an equal amount of polyethylene glycol 4000 is used to replace the polyethylene glycol-conjugated compound prepared in Preparation Example 1.

[0088] Example 13, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that the polyethylene glycol-conjugated compound prepared in Preparation Example 1 is not added.

[0089] Example 14, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that catechin is replaced by an equal amount of ascorbic acid.

[0090] Example 15, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that catechins are not added.

[0091] Example 16, a Potentilla anserine composition with anti-hypoxia function, which is different from Example 1 only in that catechins and the polyethylene glycol-conjugated compound prepared in Preparation Example 1 are not added.

[0092] Example 17, a Potentilla anserine composition with anti-hypoxia function, which is different from Example 1 only in that the Potentilla anserine glycoside compound is Rosa roxburghii glycoside.

[0093] Example 18, a Potentilla anserine composition with anti-hypoxia function, which is different from Example 1 only in that the Potentilla anserine glycoside compound is rosa glycoside.

[0094] Comparative Example

[0095] Comparative Example 1, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that the mass ratio of Potentilla anserine polysaccharide, Potentilla anserine polyphenol and Potentilla anserine glycoside compounds is 0.9:0.1:0.02.

[0096] Comparative Example 2, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that the mass ratio of Potentilla anserine polysaccharide, Potentilla anserine polyphenol and Potentilla anserine glycoside compound is 0.5:0.5:0.02.

[0097] Comparative Example 3, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that the mass ratio of Potentilla anserine polysaccharide, Potentilla anserine polyphenol and Potentilla anserine glycoside compound is 0.7:0.3:0.1.

[0098] Comparative Example 4, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that no Potentilla anserine polyphenol is added.

[0099] Comparative Example 5, a Potentilla anserine composition with anti-hypoxia function, is different from Example 1 only in that no Potentilla anserine polysaccharide is added.

[0100] Comparative Example 6, a Potentilla anserine composition with anti-hypoxia function, is different from Example 4 only in that no Potentilla anserine polyphenol is added.

[0101] Comparative Example 7, a Potentilla anserine composition with anti-hypoxia function, is different from Example 4 only in that no Potentilla anserine polysaccharide is added.

[0102] Performance testing:

[0103] 1. ROS level detection:

[0104] (1) Sample preparation: The mouse aorta was cleaned and adipose tissue was separated to prepare 2 mm × 2 mm artery slices, which were cultured in an incubator at 37°C and 5% carbon dioxide. The cultured cells were then digested with trypsin to obtain a cell suspension, and the cell number was adjusted to 10 with PBS. 6 / mL, divided into test tubes, and divided into negative group, cultured at room temperature, untreated and unstained cell group; normal group, cultured at room temperature, untreated stained cells; control group, the cell suspension was placed in an oxygen-free chamber, and a 95% N2, 5% CO2 mixed gas was passed at 37°C, and the anoxic culture was carried out for 6 hours; experimental group, 0.2 mg / ml of the fern hemp composition obtained in the embodiment and the comparative example was added to the cell suspension, and then placed in an oxygen-free chamber, and a 95% N2, 5% CO2 mixed gas was passed at 37°C, and the anoxic culture was carried out for 6 hours.

[0105] (2) Intracellular reactive oxygen species detection: Flow cytometry was used to detect the reactive oxygen species content of the above samples.

[0106] The above test results are shown in Table 1.

[0107] 2. Detection of myocardial cell metabolic activity:

[0108] (1) Sample preparation: Mouse ventricular myocardium was cut into tissue blocks of about 1 mm × 1 mm × 1 mm, and then myocardial cells were isolated for primary culture. Myocardial cells cultured for 4 days were taken, and serum-free DMEM / F-12 medium was used for continuous culture for 24 hours. The normal culture medium was replaced with D-Hanks solution (pre-saturated with 95% N2, 5% CO2 mixed gas for 30 minutes) and randomly divided into a normal group, a control group and an experimental group. The control group was cultured in anoxic culture for 6 hours, and the experimental group was added with 0.2 mg / mL of the bracken hemp composition obtained in the embodiment and the comparative example. Then the cultured cells were quickly moved into a mixed gas incubator (95% N2, 5% CO2, O2 concentration <1%) and cultured in anoxic culture at 37°C for 6 hours.

[0109] (2) Detection of cardiomyocyte metabolic activity: The MTT method was used to determine the metabolic activity of cardiomyocytes. Specifically, after 6 hours of hypoxia, 20 μL MTT was added to each well and incubated for 4 hours in a 37°C, 5% CO2 incubator. The culture was terminated. 150 μL of dimethyl sulfoxide was added and shaken for 15 minutes to fully dissolve the crystals. The absorbance (OD) of each well was measured at a measurement wavelength of 570 nm and a reference wavelength of 630 nm.

[0110] The above test results are shown in Table 2.

[0111] Table 1 ROS level detection test results

[0112]

[0113] Table 2 Results of the cardiomyocyte metabolic activity test

[0114]

[0115] According to Table 1 and Table 2, in combination with Example 1, the normal group and the control group, it can be seen that the reactive oxygen level and the metabolic activity of the cardiomyocytes in Example 1 are close to those in the normal group, and are significantly better than those in the control group, indicating that the bracken composition obtained in Example 1 can maintain the balance between the generation and removal of oxygen free radicals in a hypoxic environment, will not cause an explosive increase in the reactive oxygen content, and also improves the metabolic activity of the cardiomyocytes that decreases under hypoxic stress to maintain normal vital signs.

[0116] In combination with Example 1, Example 2 to Example 7, it can be seen that the fluctuations in the active oxygen levels and myocardial cell metabolic activity of Example 2 to Example 7 compared with Example 1 are not obvious, among which the change in Example 4 compared with Example 1 is relatively the most obvious one, indicating that the anti-hypoxia ability of the bracken hemp composition obtained in Example 2 to Example 7 is not much different from that in Example 1. The reason may be that the bracken hemp composition of Example 4 does not add bracken hemp glycoside compounds for compounding, which can achieve a certain anti-hypoxia effect, but due to the lack of additional antioxidants, the anti-hypoxia performance is still reduced compared with Example 1. Compared with Example 1, Example 2, Example 3, Example 5 to Example 7 only changed the ratio of raw materials within the required range, and the anti-hypoxia performance of the bracken hemp composition obtained fluctuated within the allowable range.

[0117] Combining Example 1, Example 8, Example 9 and Example 12, it can be seen that the active oxygen levels of Examples 8, Example 9 and Example 12 are improved compared with Example 1, and the metabolic activity of cardiomyocytes is reduced, among which the change in Example 12 is more obvious, indicating that the anti-hypoxia ability of Examples 8, Example 9 and Example 12 is reduced compared with Example 1. The reason may be that, in the purification process of bracken polysaccharide in Example 8, the metal ion content of the added polyethylene glycol-conjugated compound complex is low, and the ability to attract and aggregate proteins is reduced. Therefore, the effect of removing, settling and separating proteins is reduced, resulting in an increase in the impurity content of bracken polysaccharide, which will not only affect the activity of the polysaccharide but also the activity of the compounded bracken polyphenols, resulting in a decrease in the overall anti-hypoxia performance of the obtained bracken composition. In Example 12, polyethylene glycol is not compounded with conjugated compounds, and the effect of metal ions is lacking, which has a greater impact on the removal ability of bracken polysaccharide protein impurities. In Example 9, the content of metal ion complex is increased, which will cause excess metal ions to attack bracken polysaccharide, which is not conducive to the maintenance of the polysaccharide structure and the enhancement of activity, thereby resulting in a decrease in the overall anti-hypoxia performance of the bracken composition.

[0118] Combining Example 1, Example 10, Example 11 and Example 13, it can be seen that the active oxygen levels of Example 10, Example 11 and Example 13 are improved compared with Example 1, and the metabolic activity of myocardial cells is decreased, among which the change in Example 13 is more obvious, indicating that the anti-hypoxia ability of Example 10, Example 11 and Example 13 is decreased compared with Example 1. The reason may be that in Example 10, during the purification process of Potentilla anserina polysaccharide, the amount of polyethylene glycol-conjugated compound added was reduced, resulting in a decrease in the aggregation of proteins and the protective effect of polysaccharides. In Example 13, polyethylene glycol-conjugated compounds were not added, and the performance degradation was more obvious; in Example 11, the amount of polyethylene glycol-conjugated compounds added was increased, which would lead to the mutual combination of polyethylene glycol and polysaccharides, and introduce new impurities in the purification of Potentilla anserina polysaccharides, which is not conducive to the enhancement of the activity of Potentilla anserina polysaccharides.

[0119] Combining Example 1, Example 14 and Example 15, it can be seen that the active oxygen levels of Example 14 and Example 15 are improved compared with Example 1, and the metabolic activity of cardiomyocytes is decreased, indicating that the anti-hypoxia ability of Example 14 and Example 15 is decreased compared with Example 1. The reason may be that in Example 14, a general antioxidant is used to replace the phenolic antioxidant. Although it can also play a certain role in protecting Potentilla polysaccharides during the enzymatic hydrolysis process, it will affect the subsequent compounding process of Potentilla polyphenols and affect the activity of Potentilla polyphenols, resulting in a decrease in the anti-hypoxia ability of the Potentilla composition; in Example 15, no antioxidant is added, and in the enzymatic hydrolysis process, the polysaccharide structure is also easily decomposed and destroyed by the action of residual enzymes, and the activity decreases, which leads to a decrease in the overall anti-hypoxia ability.

[0120] Combining Example 1, Example 17 and Example 18, it can be seen that the active oxygen levels in Example 17 and Example 18 are improved compared with Example 1, and the metabolic activity of myocardial cells is reduced, indicating that the anti-hypoxia ability of Example 17 and Example 18 is reduced compared with Example 1. The reason may be that the terpenoid glycosides in Example 17 and Example 18 are single glycoside compound components, and compared with the compound effect of roxburghii glycoside and wild rose glycoside, the anti-hypoxia ability of a single component is reduced.

[0121] Combining Example 1, Example 4, and Comparative Examples 1 to 7, it can be seen that the active oxygen levels of Comparative Examples 1 to 7 are significantly improved compared to Example 1, and the metabolic activity of myocardial cells is significantly reduced, indicating that the anti-hypoxia ability of Comparative Examples 1 to 7 is significantly reduced compared to Example 1. The reason may be that the proportion of Potentilla polyphenols is reduced in Comparative Example 1, and the scavenging ability of oxygen free radicals is reduced, and the proportion of Potentilla polysaccharides is reduced in Comparative Example 2, and the scavenging ability of oxygen free radicals is also reduced, so the anti-hypoxia ability of Comparative Examples 1 and 2 is reduced. In Comparative Example 3, the addition amount of Potentilla glycoside compounds is increased, which will affect the composite synergistic effect of Potentilla polysaccharides and Potentilla polyphenols, resulting in a decrease in performance. Compared with Example 1, Comparative Examples 4 and 5, and Comparative Examples 6 and 7 compared with Example 4, respectively, did not add Potentilla polyphenols and Potentilla polysaccharides. The antioxidant capacity of the single components was significantly reduced, and the antioxidant compound effect and metabolic capacity enhancing effect of Potentilla polysaccharides and Potentilla polyphenols were significantly enhanced compared with the single components in terms of their ability to resist hypoxia.

[0122] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0123] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Potentilla anserine composition having an anti-hypoxia function, characterized in that: The bracken anserine composition comprises bracken anserine polysaccharides, bracken anserine polyphenols and bracken anserine glycoside compounds in a mass ratio of (0.6-0.8):(0.2-0.4):(0-0.05).

2. The Potentilla anserine composition with anti-hypoxia function according to claim 1, characterized in that: The terpene glycoside compound comprises roxburghii glycoside and rosa roxburghii glycoside in a mass ratio of (0.05-0.45):

1.

3. A method for preparing a Potentilla anserine composition having an anti-hypoxia function according to any one of claims 1 to 2, characterized in that: The process steps include: S1. Pretreating Potentilla anserina to obtain Potentilla anserina concentrate; S2. Extracting and purifying polysaccharides, polyphenols and glycosides from the concentrated solution of Potentilla anserina respectively; S3. Mix Potentilla anserine polysaccharides, Potentilla anserine polyphenols and Potentilla anserine glycoside compounds in corresponding proportions and dissolve them in ethanol, concentrate and freeze-dry the mixture to prepare a Potentilla anserine composition powder, which is a Potentilla anserine composition with anti-hypoxia function.

4. The method for preparing the Potentilla anserina composition having anti-hypoxia function according to claim 3, characterized in that: The pre-processing comprises the following steps: S101. The bracken hemp was washed, dusted and dried, and then crushed and sieved to obtain bracken hemp powder; S102. Reflux and defatten the bracken powder in a solvent for 2 to 3 times; S103. After evaporating the solvent, add the Potentilla anserine powder into distilled water, extract at 80-90° C., and concentrate the extract by centrifugation and reduced pressure to obtain Potentilla anserine concentrate.

5. The method for preparing the Potentilla anserina composition having anti-hypoxia function according to claim 3, characterized in that: The extraction and purification of Potentilla anserina polysaccharide comprises the following steps: S201. Adding amylase to the concentrated solution of Potentilla anserina, heating to 50 to 60 ° C, stirring for enzymolysis, and obtaining a de-starched concentrated solution of Potentilla anserina; S202. The destarch-free Pteridium anserine concentrate was added to anhydrous ethanol for alcohol precipitation, and then centrifuged to obtain a crude product of Pteridium anserine polysaccharide; S203. The crude product of Potentilla anserina polysaccharide is prepared into an aqueous solution, a protease, a phenolic antioxidant and a polyethylene glycol-conjugated compound are added, the pH value of the solution is adjusted to 7 to 8, the temperature is 35 to 45° C., the solution is stirred for enzymatic hydrolysis and then centrifuged to remove the precipitate, the obtained liquid phase is mixed with a Sevage reagent, the mixture is evenly mixed and then allowed to stand for stratification, and the supernatant is retained; S204. Add hydrogen peroxide to the obtained supernatant, stir to decolorize, centrifuge to remove the precipitate, and obtain Potentilla anserina polysaccharide after alcohol precipitation.

6. The method for preparing the Potentilla anserina composition having anti-hypoxia function according to claim 5, characterized in that: The phenolic antioxidant comprises a combination of one or more of catechins, gallic acid and anthocyanins; the added amount of the phenolic antioxidant is 0.01-0.2% of the mass of the crude product of Potentilla anserina polysaccharide.

7. The method for preparing the Potentilla anserina composition having anti-hypoxia function according to claim 5, characterized in that: The raw materials of the polyethylene glycol-conjugated compound include epoxidized polyethylene glycol, iminooxalic acid and copper salt in a mass ratio of 1:(0.6-0.7):(0.5-0.6).

8. The method for preparing the Potentilla anserina composition having anti-hypoxia function according to claim 5, characterized in that: The added amount of the polyethylene glycol-conjugated compound is 2-5% of the mass of the crude product of Potentilla anserina polysaccharide.

9. The method for preparing the Potentilla anserina composition having anti-hypoxia function according to claim 5, characterized in that: The protease is one of trypsin, neutral protease and papain; the added amount of the protease is 0.05-0.1% of the mass of the crude product of Potentilla anserina polysaccharide.

10. The method for preparing the Potentilla anserine composition having anti-hypoxia function according to claim 5, characterized in that: The Sevage reagent is a mixed solution of chloroform and n-butanol in a volume ratio of (4-6):1; the liquid phase and the Sevage reagent have a solid-liquid ratio of (1-3):1.

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