CN110790848A - Preparation method and application of total seabuckthorn polysaccharide - Google Patents

Abstract

The invention relates to a preparation method and application of total polysaccharide of sea buckthorn. In the method, the dried and ripe fruits of sea buckthorn are mechanically pulverized, sieved to separate impurities, degreased with petroleum ether, naturally air-dried and then continuously extracted with a solvent, centrifuged, concentrated, and dehydrated with ethanol. Precipitate, centrifuge again, and dry the polysaccharide precipitate at 40°C to prepare a water-soluble crude polysaccharide. The crude polysaccharide was purified, deproteinized, separated and purified by ion exchange resin and dextran gel to obtain neutral polysaccharide and acid polysaccharide. The extraction rate of the total crude polysaccharide obtained by this method reached 10.61%. The polysaccharide site has been verified to have antioxidant activity and free radical scavenging ability. Finding new antioxidants from natural plants is a new direction for the development of modern medicine and food industries. It is of great practical significance to study the antioxidant effects of polysaccharides and their derivatives and develop new natural antioxidants. This study can provide a reference for the application and development of seabuckthorn polysaccharides in antioxidant and anti-fatigue functional foods.

Description

Preparation method and application of total seabuckthorn polysaccharide

technical field

The invention relates to a preparation method and application of total seabuckthorn polysaccharide.

Background technique

Sea buckthorn (Hippophae rhamnoides), also known as vinegar willow, sour thorn, is a shrub or small tree of the Elaeaceae family. The fruit is orange-yellow when ripe, the pulp and seed surface are oily and smooth, and the seeds are oval dark brown with a sour taste. Sea buckthorn is widely distributed in the temperate regions of Europe and Asia, and is cultivated in China, the United Kingdom, Mongolia, etc. China is a country with many species of sea buckthorn and a relatively wide distribution area in the world. There are 4 species and 5 subspecies of sea buckthorn. In 13 provinces including Xinjiang, Shanxi, Shaanxi, Liaoning and Gansu.

In 1977, the Chinese Pharmacopoeia included sea buckthorn for the first time, and it was later identified as a medicinal and edible plant. Sea buckthorn has high economic benefits. The annual output of sea buckthorn in Burqin County, Altay, can reach 5 million tons, with a total output value of 30 million. In 2015, the General Administration of Quality Supervision, Inspection and Quarantine of China approved the large fruit sea buckthorn grown in Altay, Xinjiang, for the protection of geographical indication products. In addition, sea buckthorn can retain the water content in the soil, resist wind erosion and sand burial, and can play a certain role in improving the ecological environment. At this stage, a large number of sea buckthorn are planted in some areas. First, it can strengthen the construction of ecological projects, and second, it can help farmers get rid of poverty and become rich. Sea buckthorn not only has broad development in medicine, health care, food, etc., but also has certain application prospects in the field of feed. In recent years, researchers have extracted polysaccharides and flavonoids from sea buckthorn, and there have been many studies on their purification. There are few reports on the effects of polysaccharides on antioxidant and anti-fatigue functions at home and abroad.

Polysaccharide is a natural macromolecular polymer in which aldose or ketose are linked by glycosidic bonds, which are common in the cell membranes of animals, plants and microorganisms. Polysaccharides have a variety of biological activities, and are safe, non-toxic, and non-drug-resistant. They have become a research hotspot in medicine, agriculture, and food. Most foreign pharmaceutical companies are engaged in the research and development of carbohydrate drugs. Studies have found that polysaccharides have various functions such as anti-tumor, anti-coagulation and immune regulation. Polysaccharides are one of the most important active components of medicinal plants. Plant polysaccharides have broad application prospects in the fields of medicine, food, cosmetics and environmental management due to their extensive therapeutic effects and extremely low cytotoxicity. Therefore, in recent years, the research on polysaccharides has attracted more and more attention and has become a "hot spot" of today's research. Some polysaccharides extracted from natural resources have attracted great attention in the fields of biochemistry and pharmacology, especially traditional Chinese medicine polysaccharides, which have received more attention due to the unique effects of traditional Chinese medicines and in-depth research on the mechanism of action of traditional Chinese medicines. my country has made major breakthroughs in the separation, purification, molecular structure determination and dose-response relationship control of active polysaccharides, reaching the world's leading level.

The human body is carrying out redox reactions all the time. If it is in a pathological or stressful state, it can cause an imbalance between the oxidation and antioxidant status of the body, and excess free radicals will be produced, and these excess free radicals will cause damage to the body. The human body has formed its own antioxidant system, including the antioxidant zymogen system, endogenous antioxidants and essential nutrients with antioxidant activity. The above antioxidant systems help the body to remove endogenous active oxidative free radicals, but the elimination of oxidative free radicals will decline with the aging of the body. When the cells and tissues of the body are under oxidative stress by reactive oxygen species and free radicals, macromolecular substances such as lipids, proteins, and carbohydrates in the cells and tissues will undergo various oxidative reactions, which will lead to the destruction of cell structure and function. Causes injuries and lesions, leading to some diseases. A large number of studies have shown that polysaccharides have immune regulation, anti-tumor, hypoglycemic and anti-aging effects.

Fatigue is a subjective feeling of fatigue and weakness, accompanied by adverse reactions such as dizziness, headache, dizziness, and rapid heart rate. If the fatigue produced by the human body cannot be eliminated in time, it will gradually accumulate and lead to poor mood, thereby aggravating the feeling of fatigue, and even leading to "death from overwork" in severe cases. At this stage, traditional anti-fatigue drugs are mostly banned because they contain stimulants, which are harmful to human health. At present, the search for natural, non-toxic and side-effect anti-fatigue health foods and medicines has become a hot spot of social concern. Studies have shown that polysaccharides have anti-fatigue effects, but there are few studies on the anti-fatigue effects of seabuckthorn polysaccharides.

With the development of society and the improvement of the quality of life, the consumption of medicines has gradually changed from chemical medicines to botanical medicines that use natural resources as raw materials. Traditional Chinese medicine has unique advantages in treatment, rehabilitation and health care and improving human immunity. Become an important part of the world's pharmaceutical industry.

There are many kinds of polysaccharides, and the extraction and separation methods of different polysaccharides are different. The extraction of polysaccharides mostly uses water, weak acid and dilute alkali solutions at different temperatures as extraction solvents. The extraction methods of polysaccharides are roughly divided into the following categories.

(1) Solvent extraction method

The solvent generally used in the solvent extraction method is water, weak acid, dilute alkali, etc. The latter two extraction methods have higher extraction rates, but they are easy to destroy the structure and activity of polysaccharides. The hot water extraction method, that is, the traditional extraction method, takes a long time and has a low extraction rate, but it has high safety and is not easy to destroy the physicochemical properties of polysaccharides.

(2) Enzymatic extraction

Enzymes are highly specific, and enzymatic extraction takes advantage of this feature. The enzymatic method decomposes the raw material tissue through an enzymatic reaction, accelerates the release and extraction of active ingredients, selects appropriate conditions to decompose and remove impurities that affect the extraction, and promotes the conversion of some low-polarity fat-soluble components into sugars. At the same time, enzymatic extraction can also selectively degrade the impurities contained in the raw material components. Protease or compound enzymes can be used to degrade and remove most of the proteins, so that the extraction and separation are more thorough and the extraction rate is higher. Due to the advantages of mild conditions, easy removal of impurities, high recovery rate and energy saving, the application prospect of enzymatic extraction is very broad.

(3) Ultrasonic extraction

The ultrasonic-assisted extraction method uses the unique cavitation effect of ultrasonic waves to generate great pressure, which makes the cell wall of the raw material cells easily broken and the entire organism is broken. extraction rate. The experimental environment of ultrasonic extraction is mild, and the extraction temperature is low, which can avoid the destruction of the biological activity of water-soluble polysaccharides due to high temperature, and has many advantages such as low energy consumption and resource saving.

(4) Reflux extraction

The reflux extraction method is that in a reflux device, the solvent and the raw materials are mixed in a round-bottomed flask, and heated to dissolve the water-soluble polysaccharide components. The solvent distillate is liquefied in a cold state, and then returned to the round-bottomed flask to repeatedly extract the raw materials until the active ingredients are completely extracted. This method extracts polysaccharides, and the loss of the extract is low, but the extraction time is long, and some polysaccharides are easily degraded under long-term high temperature conditions.

(5) Supercritical fluid extraction method

In the supercritical state, the supercritical fluid is in contact with the target, so that it sequentially extracts components with different polarities, boiling points and molecular weights. When the pressure and temperature return to normal, the components dissolved in the fluid will immediately dissolve The liquid state of the absorption liquid is separated from the gaseous fluid, so as to achieve the purpose of extraction. Supercritical extraction technology has strong extraction capacity, high extraction efficiency, short production cycle, easy discovery of new active ingredients, minimal loss of volatile components or destruction of physiologically active substances, no solvent residue, and high product quality.

(6) Ultra-high pressure extraction method

The ultra-high pressure extraction method refers to the use of static pressure in the ultra-high pressure range of 100-1000 mpa to act on the raw material liquid under normal temperature conditions. The active ingredients can also reach a sufficient dissolution balance, and the pressure is quickly relieved at the time of equilibrium, and the active ingredients in the cells will be flushed out of the cells due to the sudden increase of the osmotic pressure difference between inside and outside the cells, passing through the heavy obstacles of the cells and transferring to the cells outside the cells. In the extract, so as to achieve the purpose of extracting active ingredients. The ultra-high pressure extraction method is carried out at room temperature to avoid the structural change and physiological activity of the active ingredients due to thermal effects. At the same time, ultra-high pressure extraction is generally operated in a closed environment, and the solvent volatilization is very small, which greatly reduces the pollution to the environment. Green" environmental protection technical requirements.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a total polysaccharide of sea buckthorn and its preparation method and application. The seabuckthorn total polysaccharide of the present invention comprises one neutral polysaccharide and two acidic polysaccharides, preferably, the neutral polysaccharide is a neutral polysaccharide eluted by water, and the acidic polysaccharide is an acidic polysaccharide eluted by NaCl. The preparation method of the total polysaccharide of sea buckthorn of the present invention comprises mechanically pulverizing the dried and ripe fruits of sea buckthorn (sea buckthorn belongs to the genus Sea buckthorn, a deciduous shrub. The origin of the material used in the present invention is the Altay area of Xinjiang Province), and sieving and separating Impurities, degreased with petroleum ether, naturally air-dried and then used solvent for continuous extraction, centrifugation, concentration, anhydrous ethanol precipitation, centrifugation again, and drying the polysaccharide precipitate at 40°C to prepare water-soluble crude polysaccharide. The crude polysaccharide was purified, deproteinized, separated and purified by ion exchange resin and dextran gel to obtain neutral polysaccharide and acid polysaccharide. The extraction rate of the total crude polysaccharide obtained by this method reached 10.61%. This polysaccharide site has been verified to have antioxidant activity and scavenging ability to free radicals. Finding new antioxidants from natural plants is a new direction for the development of modern medicine and food industries. It is of great practical significance to study the antioxidant effects of polysaccharides and their derivatives and develop new natural antioxidants. This study can provide a reference for the application and development of seabuckthorn polysaccharides in antioxidant and anti-fatigue functional foods.

The preparation method of sea buckthorn total polysaccharide according to the present invention, the method takes the dried and ripe fruits of sea buckthorn as raw material, uses distilled water as a solvent for extraction, precipitation with absolute ethanol, deproteinization, ion exchange resin, glucan gel, etc. The neutral polysaccharide and the acidic polysaccharide are obtained by separation and purification, and the specific operation is carried out according to the following steps:

a. Mechanically pulverize the dried and ripe fruits of sea buckthorn, the pulverization time is 80-120 seconds, pass through a 60-mesh sieve, and separate sundries to obtain powder;

b, the powder obtained in step a is 1:5 by weight ratio of material to liquid, adding petroleum ether, extracting in a water bath at room temperature for 1-2 hours, repeatedly extracting 2-3 times until no oil is extracted, filtering, and naturally air-dried to no Until there is petroleum ether solvent residue, get degreasing sea buckthorn powder;

c, three kinds of polysaccharide concentrates are prepared respectively by the defatted sea buckthorn powder obtained in step b:

According to the weight ratio of 1:20, water was added to the obtained defatted sea buckthorn powder, and the temperature was 70 °C for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate for subsequent use;

The filtered residue was collected, air-dried naturally, and the weight ratio of the material to liquid was 1:20. Water was added to the air-dried filter residue, and the temperature was 70° C. for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate. ;

d. Add the two polysaccharide concentrates obtained in step c to 25% volume of Sevage reagent (V chloroform:V n-butanol=4:1) and mix at 2000r/min for 20min in a vortex mixer. After mixing, centrifuge at room temperature for 20 min at a speed of 4000 r/min, and take the supernatant and repeat deproteinization 5 times.

The deproteinized polysaccharide concentrate was added to anhydrous ethanol for precipitation, the temperature was 4°C overnight, suction filtration, the polysaccharide precipitate was washed, and the polysaccharide precipitate was washed with anhydrous ethanol, acetone, and ether in sequence, suction filtered, and the precipitate was left. Dry in an oven at 40° C. until crystals, to obtain yellow water-soluble sea buckthorn crude polysaccharide, wherein the volume ratio of polysaccharide concentrate to absolute ethanol is 1:3.

e. Purify the crude polysaccharide obtained in step d with ion exchange resin DEAE-52 and Sephadex G-150 to obtain a neutral sugar eluted with water and an acidic polysaccharide washed with two different concentrations of NaCL, Finally, three total polysaccharides of sea buckthorn were obtained.

The yield of total seabuckthorn polysaccharide obtained by the above method was 10.61%; the yield of neutral polysaccharide was 41 mg/g, the yield of acid polysaccharide 1 was 86 mg/g, and the yield of acid polysaccharide 2 was 110 mg/g.

The use of the seabuckthorn total polysaccharide of the present invention in the preparation of antioxidative and antifatigue medicines and/or foods and/or agents.

The high-efficiency preparation method of seabuckthorn total polysaccharide of the present invention has the advantages of high yield of polysaccharide, easy to scale up in pilot scale, and on the basis of retaining the original physical and chemical properties and nutritional components of polysaccharide, by using low temperature or vacuum Or freeze-drying technology, which avoids the disadvantage of traditional drying or vacuum filtration and some compounds lose their activity. The total polysaccharide of sea buckthorn obtained by this method has been verified to have antioxidant activity, scavenging ability to DPPH free radicals and hydroxyl free radicals, and anti-fatigue activity.

Description of drawings

Fig. 1 is the flow chart of the present invention, wherein a is the sea buckthorn raw material pretreatment figure, b is the extraction flow chart of the sea buckthorn crude polysaccharide, and c is the sea buckthorn crude polysaccharide separation and purification figure;

Fig. 2 is an infrared (IR) spectrum of the present invention, wherein a is a neutral polysaccharide, b is an acid 1 polysaccharide, and c is an acid 2 polysaccharide;

Figure 3 is a graph showing the scavenging ability of crude polysaccharide to DPPH free radicals;

Fig. 4 is the scavenging ability diagram of crude polysaccharide to hydroxyl radical scavenging ability;

Fig. 5 is a graph showing the scavenging ability of crude polysaccharide to superoxide anion free radicals;

Figure 6 is a graph showing the effect of crude polysaccharide on the weight-bearing swimming time of mice; in which: "*" indicates that compared with the blank group, there is significant (P<0.05), and "**" indicates that compared with the blank group, there is a very significant sex (P<0.01), the same below.

Figure 7 is a graph showing the effect of crude polysaccharide on liver glycogen in fatigued mice;

Figure 8 is a graph showing the effect of crude polysaccharide on BUN in the serum of fatigued mice;

Figure 9 shows the effect of crude polysaccharide on LA in the serum of fatigued mice

Detailed ways

Example 1

a. Mechanically pulverize the dried and ripe fruits of sea buckthorn, the pulverization time is 80 seconds, pass through a 60-mesh sieve, and separate impurities to obtain powder;

b, the powder obtained in the step a is 1:5 by weight ratio of material to liquid, adding sherwood oil, extracting in a water bath at room temperature for 1 hour, repeatedly extracting 2 times until no oil is extracted, filtering, and naturally air-dried until there is no petroleum ether solvent Until the residue is left, the defatted sea buckthorn powder is obtained;

c, three kinds of polysaccharide concentrates are prepared respectively by the defatted sea buckthorn powder obtained in step b:

According to the weight ratio of 1:20, water was added to the obtained defatted sea buckthorn powder, and the temperature was 70 °C for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate for subsequent use;

The filtered residue was collected, air-dried naturally, and the weight ratio of the material to liquid was 1:20. Water was added to the air-dried filter residue, and the temperature was 70° C. for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate. ;

d. Add the two polysaccharide concentrates obtained in step c to 25% volume of Sevage reagent (V chloroform:V n-butanol=4:1) and mix at 2000r/min for 20min in a vortex mixer. After mixing, centrifuge at room temperature for 20 min at a speed of 4000 r/min, and take the supernatant and repeat deproteinization 5 times.

The deproteinized polysaccharide concentrate was added to anhydrous ethanol for precipitation, the temperature was 4°C overnight, suction filtration, the polysaccharide precipitate was washed, and the polysaccharide precipitate was washed with anhydrous ethanol, acetone, and ether in sequence, suction filtered, and the precipitate was left. Dry in an oven at 40° C. until crystals, to obtain yellow water-soluble sea buckthorn crude polysaccharide, wherein the volume ratio of polysaccharide concentrate to absolute ethanol is 1:3.

e. Purify the crude polysaccharide obtained in step d with ion exchange resin DEAE-52 and Sephadex G-150 to obtain a neutral sugar eluted with water and an acidic polysaccharide washed with two different concentrations of NaCL, Finally, three total polysaccharides of sea buckthorn were obtained.

Example 2

a. Mechanically pulverize the dried and ripe fruits of sea buckthorn, the pulverization time is 100 seconds, pass through a 60-mesh sieve, and separate impurities to obtain powder;

b, the powder obtained in the step a is 1:5 by weight ratio of material to liquid, adding sherwood oil, extracting in a water bath at room temperature for 2 hours, repeatedly extracting 2 times until no oil is extracted, filtering, and naturally air-dried until there is no sherwood oil solvent Until the residue is left, the defatted sea buckthorn powder is obtained;

c, three kinds of polysaccharide concentrates are prepared respectively by the defatted sea buckthorn powder obtained in step b:

According to the weight ratio of 1:20, water was added to the obtained defatted sea buckthorn powder, and the temperature was 70 °C for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate for subsequent use;

The filtered residue was collected, air-dried naturally, and the weight ratio of the material to liquid was 1:20. Water was added to the air-dried filter residue, and the temperature was 70° C. for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate. ;

d. Add the two polysaccharide concentrates obtained in step c to 25% volume of Sevage reagent (V chloroform:V n-butanol=4:1) and mix at 2000r/min for 20min in a vortex mixer. After mixing, centrifuge at room temperature for 20 min at a speed of 4000 r/min, and take the supernatant and repeat deproteinization 5 times.

The deproteinized polysaccharide concentrate was added to anhydrous ethanol for precipitation, the temperature was 4°C overnight, suction filtration, the polysaccharide precipitate was washed, and the polysaccharide precipitate was washed with anhydrous ethanol, acetone, and ether in sequence, suction filtered, and the precipitate was left. Dry in an oven at 40° C. until crystals, to obtain yellow water-soluble sea buckthorn crude polysaccharide, wherein the volume ratio of polysaccharide concentrate to absolute ethanol is 1:3.

e. Purify the crude polysaccharide obtained in step d with ion exchange resin DEAE-52 and Sephadex G-150 to obtain a neutral sugar eluted with water and an acidic polysaccharide washed with two different concentrations of NaCL, Finally, three total polysaccharides of sea buckthorn were obtained.

Example 3

a. Mechanically pulverize the dried and ripe fruits of sea buckthorn, the pulverization time is 120 seconds, pass through a 60-mesh sieve, and separate impurities to obtain powder;

b, the powder obtained in the step a is 1:5 by weight ratio of material to liquid, adding sherwood oil, extracting in a water bath at room temperature for 2 hours, repeatedly extracting 3 times until there is no oil and fat to be extracted, filter, and naturally air-dry until there is no petroleum ether solvent Until the residue is left, the defatted sea buckthorn powder is obtained;

c, three kinds of polysaccharide concentrates are prepared respectively by the defatted sea buckthorn powder obtained in step b:

According to the weight ratio of 1:20, water was added to the obtained defatted sea buckthorn powder, and the temperature was 70 °C for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate for subsequent use;

The filtered residue was collected, air-dried naturally, and the weight ratio of the material to liquid was 1:20. Water was added to the air-dried filter residue, and the temperature was 70° C. for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate. ;

d. Add the two polysaccharide concentrates obtained in step c to 25% volume of Sevage reagent (V chloroform:V n-butanol=4:1) and mix at 2000r/min for 20min in a vortex mixer. After mixing, centrifuge at room temperature for 20 min at a speed of 4000 r/min, and take the supernatant and repeat deproteinization 5 times.

The deproteinized polysaccharide concentrate was added to anhydrous ethanol for precipitation, the temperature was 4°C overnight, suction filtration, the polysaccharide precipitate was washed, and the polysaccharide precipitate was washed with anhydrous ethanol, acetone, and ether in sequence, suction filtered, and the precipitate was left. Dry in an oven at 40° C. until crystals, to obtain yellow water-soluble sea buckthorn crude polysaccharide, wherein the volume ratio of polysaccharide concentrate to absolute ethanol is 1:3.

e. Purify the crude polysaccharide obtained in step d with ion exchange resin DEAE-52 and Sephadex G-150 to obtain a neutral sugar eluted with water and an acidic polysaccharide washed with two different concentrations of NaCL, Finally, three total polysaccharides of sea buckthorn were obtained.

Example 4

The results of infrared spectrum analysis of the total polysaccharide samples of sea buckthorn obtained in Examples 1-3 respectively showed that typical polysaccharide absorption peaks appeared for the three components in the range of 4000-400 cm ^-1 . The broad peak around 3420cm ^-1 is the stretching vibration peak of hydroxyl OH; the absorption peak around 2920cm ^-1 is the CH stretching vibration peak; the absorption peak around 1620cm ^-1 is the polysaccharide hydration vibration peak; around 1400cm ^-1 is the CH in-plane bending The vibration signal peaks; the three weak absorption peaks between 1000-1200cm ^-1 are the bending vibration peaks of CO bond stretching vibration absorption peak COH; the absorption peak near 835cm ^-1 is the characteristic peak of α-glycosides; the absorption near 880cm ^-1 The peaks are characteristic of β-glycosides; these data indicate that the extract is indeed characteristic of polysaccharides.

Example 5

Antioxidative activity test of sea buckthorn polysaccharide

The sea buckthorn water-extracted crude polysaccharide samples were prepared into solutions of different concentrations, and the antioxidant activities of DPPH (diphenylpicryl phenylhydrazine) free radical scavenging ability, hydroxyl radical scavenging ability and superoxide anion free radical scavenging ability were carried out. test.

DPPH (diphenylpicryl phenylhydrazine) free radical scavenging ability: add 1mL 1, 2, 3, 4, 5mg/mL seabuckthorn polysaccharide solution (the seabuckthorn crude polysaccharide obtained in Example 2) into a 10mL colorimetric tube respectively. 2 mL of 0.2 mmol/LDPPH ethanol solution was added respectively, and the volume was adjusted to the mark with anhydrous ethanol. Shake well, react in the dark at room temperature for 30min, take absolute ethanol as blank control, VC (vitamin C, purchased from Sinopharm Chemical Reagent Co., Ltd., use water as solvent to configure vitamin C at 1, 2, 3, 4, 5 mg/mL) C solution) is the positive control, absorbance is measured at 517nm, A1: sample to be tested (1, 2, 3, 4, 5 mg/mL of seabuckthorn polysaccharide solution) + DPPH ethanol solution; A2: sample to be tested (respectively 1 , 2, 3, 4, and 5 mg/mL of sea buckthorn polysaccharide solution) + anhydrous ethanol; A0: anhydrous ethanol + DPPH ethanol solution; calculate its clearance rate according to formula (1).

Clearance rate (%)=[A0-(A1-A2)]/A×100% (1)

Para-hydroxyl radical scavenging ability: Take 1 mL of seabuckthorn polysaccharide solution with concentrations of 0.1, 0.5, 1, 1.5, 2.0 mg/mL in test tubes, and add 800 μL of ferrous sulfate (6 mmol/L) and 800 μL of salicylic acid to each tube. (6mmol/L) after shaking, add 50 μL of 0.1% H ₂ O ₂ to each tube and mix well, take a water bath at 37°C for 30 minutes, take distilled water as blank control, take VC (vitamin C) as positive control, measure the absorbance of each reaction solution at 510 nm Values A0: 1 mL H ₂ O + 800 μL FeSO ₄ + 50 μL H ₂ O ₂ ; A1: 1 mL sea buckthorn polysaccharide solution (5 concentrations of polysaccharide solutions of 0.1, 0.5, 1, 1.5, 2.0 mg/mL, respectively) + 800 μL FeSO ₄ + 50 μL H ₂ O ₂ ; A2: 1 mL sea buckthorn polysaccharide solution (5 concentration polysaccharide solutions of 0.1, 0.5, 1, 1.5, 2.0 mg/mL respectively)+800 μL FeSO ₄ +50 μL H ₂ O. The clearance rate was calculated according to formula (2).

Clearance rate (%)=[A0-(A1-A2)]/A0×100% (2)

For superoxide anion free radical scavenging ability: take 4.0mL of 0.05mol/L Tris-HCl buffer (pH=8.2) into a test tube, water bath at 25°C for 20min, add 1mL of 1, 2, 4, 6, 8 mg/mL respectively. The seabuckthorn polysaccharide solution and 20 μL of 80 mmol/L pyrogallol were thoroughly mixed. After mixing, the reaction was performed in a water bath at 25°C for 5 min. After the reaction, 200 μL of 10 mol/L hydrochloric acid solution was added to terminate the reaction. VC (vitamin C) was used as a positive control. Measure the absorbance at 325nm, A1: 4mL Tris-HCl+20μL pyrogallol+1mL seabuckthorn polysaccharide solution (5 concentrations of 1, 2, 4, 6, 8mg/mL polysaccharide solutions respectively); A2: 4mL Tris-HCl+20μL distilled water +1mL seabuckthorn polysaccharide solution (1, 2, 4, 6, 8mg/mL five concentrations of polysaccharide solution); A3: 4mL Tris-HCl + 20μL pyrogallol + 1mLl distilled water. Calculate its clearance rate according to formula (3).

Clearance rate (%)=[1-(A1-A2)/A3]×100% (3)

The oxidative metabolism process of life activities constantly produces various reactive oxygen radicals, such as DPPH radicals, hydroxyl radicals, superoxide anion radicals, etc. In addition to participating in the physiological and metabolic processes of cells, these reactive oxygen species can also cause damage to cell membranes and DNA, resulting in lipid peroxidation and reduced permeability of biological membranes, leading to various diseases such as cancer, atherosclerosis, Alzheimer's disease Haimer's disease, etc. Although the body has oxidative defense and repair mechanisms, it cannot completely prevent the body damage caused by excessive free radicals, so it is very necessary to supplement exogenous antioxidants. However, many synthetic antioxidants have many safety problems, so the research and development of natural antioxidants have attracted extensive attention. A large number of studies have shown that polysaccharides have immunomodulatory, anti-tumor, anti-inflammatory, anti-viral, hypoglycemic and anti-aging effects. These effects are inseparable from the antioxidant activity of polysaccharides. Antioxidation is one of the most important biological activities of polysaccharides. In recent years, domestic and foreign scholars have done a lot of research work on the antioxidant activity of polysaccharides.

The in vitro antioxidant activity analysis of the seabuckthorn polysaccharide involved in the invention shows that the water-soluble crude polysaccharide has a certain scavenging ability to DPPH free radicals, OH free radicals and O ₂ ^- free radicals within a certain concentration range,

1. When the concentration of seabuckthorn polysaccharide gradually increases, the scavenging rate of DPPH free radicals increases. The scavenging ability of DPPH free radicals increases rapidly when the concentration is 1-3 mg/mL and 4-5 mg/mL, and the scavenging capacity for DPPH free radicals increases rapidly at 3-4 mg. The scavenging ability of DPPH free radical increased slowly. The scavenging effect of seabuckthorn polysaccharide on DPPH free radical was lower than that of _VC when the concentration of seabuckthorn polysaccharide was 1-4 mg/mL. When the concentration was 5 mg/mL, the scavenging rate of seabuckthorn polysaccharide and _VC on DPPH free radical were 93.69% and 95.28%, respectively. The rate was similar, indicating that the antioxidative ability of seabuckthorn polysaccharide was similar to that of _VC when the concentration of seabuckthorn polysaccharide was 5 mg/mL.

2. When the concentration of seabuckthorn polysaccharide increases gradually, the scavenging rate of OH free radicals increases. The scavenging ability of seabuckthorn polysaccharides for OH free radicals is lower than that of _VC within the concentration of 0.1-2 mg/mL. · The _IC50 value of free radical scavenging ability is 0.97 mg/mL.

3. When the concentration of seabuckthorn polysaccharide increases gradually, the scavenging rate of O ₂ ^- · free radicals increases accordingly. When the concentration is 1-6 mg/mL, the scavenging rate for O ₂ ^- · free radicals increases slowly, and the concentration is 6-8 mg. /mL, the scavenging rate of O ₂ ^- · free radicals increased rapidly. When the concentration was 1～8mg/mL, the scavenging ability of seabuckthorn polysaccharide to O ₂ ^- · was lower than that of _VC . The calculated IC ₅₀ value of seabuckthorn polysaccharide on O ₂ ^- ·radical scavenging ability was 7.95 mg/mL.

It can be seen that the seabuckthorn polysaccharide has relatively strong scavenging ability to DPPH free radicals and OH · free radicals, but weak scavenging ability to O ₂ ^- · free radicals. Therefore, the polysaccharide part can be used as a potential antioxidant and has broad application prospects in the fields of food, medicine and health care products. This study can provide a reference for the application and development of seabuckthorn polysaccharide in antioxidant and anti-aging functional foods.

Example 6

Anti-fatigue activity test of sea buckthorn polysaccharide

40 mice with swimming ability were selected and divided into blank group (normal saline), low-dose group (seabuckthorn polysaccharide 100mg/kg), middle-dose group (seabuckthorn polysaccharide 200mg/kg,), high-dose group (Seabuckthorn polysaccharide 400mg/kg) was administered into groups for 15 days, with 10 animals in each group, during which they ate normal food and water. The anti-fatigue effect of seabuckthorn polysaccharide was evaluated by the swimming time of mice and the determination of LA (lactic acid) and BUN (urea) content in serum and glycogen content in liver.

It can be seen from Figure 6 that the weight-bearing swimming time of the low, medium and high dose groups of sea buckthorn polysaccharide was prolonged by 29.87%, 56.21% and 37.64% respectively compared with the blank group, indicating that sea buckthorn polysaccharide can relieve the fatigue state of mice to a certain extent, making the mice Weight-bearing swimming time increased. The weight-bearing swimming time in the middle-dose group of seabuckthorn polysaccharide increased significantly (P<0.01), and the weight-bearing swimming time in the high-dose group increased significantly (P<0.05). It shows that the anti-fatigue effect of different doses of seabuckthorn polysaccharide on mice is also different, and the middle dose has better effect.

During exercise, glucose and glycogen are the main energy substances, and increasing the glycogen reserve in the liver is beneficial to improve exercise endurance and reduce fatigue. It can be seen from Figure 7 that the liver glycogen content in the low (100mg/kg), medium (200mg/kg) and high (400mg/kg) dose groups of seabuckthorn polysaccharide increased by 12.41%, 30.75% and 15.35% respectively compared with the blank group. The results indicated that the middle dose of seabuckthorn polysaccharide had the best effect on increasing the hepatic glycogen reserve and reducing fatigue.

The change of BUN content can evaluate the fatigue state of mice, and the decrease of BUN content indicates that the body can effectively relieve exercise fatigue. As can be seen from Figure 8, compared with the blank group, the BUN content of the low-dose, medium-dose and high-dose seabuckthorn polysaccharide groups decreased by 22.07%, 25.89% and 23.72%, respectively. Among them, the content of BUN in the middle and high dose groups of seabuckthorn polysaccharide was significantly decreased (P<0.01), and the content of BUN in the low dose group of seabuckthorn polysaccharide decreased significantly (P<0.05), indicating that seabuckthorn polysaccharide can reduce the content of BUN and relieve the fatigue of swimming. .

It can be seen from Figure 9 that the serum LA in the low, medium and high dose groups of seabuckthorn polysaccharide was 4.40%, 21.90%, and 22.42% lower than those in the blank group, respectively. Compared with the blank group, the LA content of the low-dose was less different. The results showed that the anti-fatigue effect of the low-dose seabuckthorn polysaccharide was weaker than that of the middle-dose and high-dose.

Fatigue refers to the complex physiological and biochemical processes of the body that the body's physiological process cannot sustain its function at a specific level, or that each organ cannot maintain its predetermined exercise intensity. Polysaccharides can improve the body's anti-fatigue ability by promoting glycogen synthesis, reducing exercise metabolites, increasing hypoxia tolerance, etc., and play an important role in preventing and eliminating exercise-induced fatigue.

The analysis of the anti-fatigue results of the sea buckthorn polysaccharide involved in the present invention shows that, compared with the blank group, each dosage group of sea buckthorn polysaccharide can prolong the swimming time of mice under load, increase the content of liver glycogen, and reduce the content of LA and BUN in the serum. Has anti-fatigue effect.

Claims (5)

1. A total polysaccharide of sea buckthorn, comprising one neutral polysaccharide and two acidic polysaccharides. 2 . The total seabuckthorn polysaccharide according to claim 1 , wherein the neutral polysaccharide is a water-eluting neutral polysaccharide, and the acidic polysaccharide is a NaCl-eluting acid polysaccharide. 3 . 3. the preparation method of seabuckthorn total polysaccharide described in any one of claim 1-2, it is characterized in that this method takes seabuckthorn dry ripe fruit as raw material, uses distilled water as solvent to carry out extraction, through absolute ethanol precipitation, deproteinization, ionization Neutral polysaccharides and acidic polysaccharides are obtained by separation and purification of exchange resin, glucan gel, etc. The specific operations are carried out according to the following steps: a. Mechanically pulverize the dried and ripe fruits of sea buckthorn, the pulverization time is 60-180 seconds, pass through a 60-mesh sieve, and separate sundries to obtain powder; b, the powder obtained in the step a is 1:5 by the weight ratio of material to liquid, adding petroleum ether, stirring at room temperature for 1-2 hours, repeatedly extracting 3-4 times until no oil is extracted, filtering, and air-drying until no more Until the petroleum ether solvent remains, the degreasing sea buckthorn powder is obtained; c, three kinds of polysaccharide concentrates are prepared respectively by the defatted sea buckthorn powder obtained in step b: According to the weight ratio of 1:20, water was added to the obtained defatted sea buckthorn powder, and the temperature was 70 °C for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate for subsequent use; The filtered residue was collected, air-dried naturally, and the weight ratio of the material to liquid was 1:20. Water was added to the air-dried filter residue, and the temperature was 70° C. for ultrasonic-assisted extraction for 22 minutes, filtered, and the extracts were combined and concentrated to obtain a polysaccharide concentrate. ; d. Add the two polysaccharide concentrates obtained in step c to 25% volume of Sevage reagent (V chloroform:V n-butanol=4:1) and mix at 2000r/min for 20min in a vortex mixer. After mixing, centrifuge at room temperature for 20 min at a speed of 4000 r/min, and take the supernatant and repeat deproteinization 5 times. The deproteinized polysaccharide concentrate was added to anhydrous ethanol for precipitation, the temperature was 4°C overnight, suction filtration, the polysaccharide precipitate was obtained, and the polysaccharide precipitate was washed with anhydrous ethanol, acetone, and ether in sequence, suction filtered, and the precipitate was left. Dry in an oven at 40° C. until crystals, to obtain yellow water-soluble sea buckthorn crude polysaccharide, wherein the volume ratio of polysaccharide concentrate to absolute ethanol is 1:3. e. Purify the crude polysaccharide obtained in step d with ion exchange resin DEAE-52 and Sephadex G-150 to obtain a neutral sugar eluted with water and an acidic polysaccharide washed with two different concentrations of NaCL, Finally, three total polysaccharides of sea buckthorn were obtained. 4. Use of the total polysaccharide of sea buckthorn according to any one of claims 1-2 in the preparation of antioxidative biologically active medicines and/or foods and/or agents. 5. Use of the seabuckthorn total polysaccharide according to any one of claims 1-2 in the preparation of anti-fatigue medicines and/or foods and/or agents.

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