CN112521522B - Preparation method and application of Siberian Nitraria polysaccharide effective part - Google Patents
Preparation method and application of Siberian Nitraria polysaccharide effective part Download PDFInfo
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- CN112521522B CN112521522B CN202011446283.4A CN202011446283A CN112521522B CN 112521522 B CN112521522 B CN 112521522B CN 202011446283 A CN202011446283 A CN 202011446283A CN 112521522 B CN112521522 B CN 112521522B
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- polysaccharide
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- nitraria
- siberian nitraria
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to a preparation method and application of Siberian Nitraria polysaccharide effective parts, which comprises the steps of drying Siberian Nitraria fruit, crushing, degreasing with petroleum ether, decoloring with chloroform-methanol, refluxing with ethanol, extracting with hot water, ultrasonic assisted extraction, cellulase extraction, pectinase + ultrasonic assisted extraction and the like, centrifuging the extract, purifying with macroporous adsorption resin, concentrating, precipitating with absolute ethanol, centrifuging, and freeze-drying the polysaccharide precipitate in vacuum to prepare the Siberian Nitraria polysaccharide effective parts. The effective part of the nitraria tangutorum bobr polysaccharide obtained by the method is verified to have stronger antioxidant activity and probiotic growth promoting activity. The research and development of natural polysaccharide antioxidants and intestinal flora regulators have important practical significance for improving the immune function of organisms. The invention can provide a basis for the application of the Siberian nitraria polysaccharide effective part in the fields of food, medicine and cosmetics, the creation of health food with specific functional activity and the realization of the industrialized application of technical achievements.
Description
Technical Field
The invention relates to a preparation method and application of Siberian white polysaccharide effective parts.
Background
Nitrariaceae (Nitrariaceae) shrubs, 11 species in total, are widely distributed in Asia, Europe, Africa, etc. Xinjiang, Qinghai, Gansu, Ningxia and inner Mongolia areas in northwest of China are distributed. Both fruits and leaves of Nitraria plant can be used as medicine. Siberian white thorn (Nitraria sibirica Pall.) is a plant of Zygophyllaceae, is distributed in various regions of Xinjiang in China, is a wild plant with homology of medicine and food, and has a fruit which is a rare wild berry in desert.
Siberian nitraria is used in traditional Chinese medicine for treating various diseases, including hypertension, weakness of spleen and stomach, menstrual disorder and the like. Meanwhile, modern pharmacological studies prove that the nitraria tangutorum bobr fruits have various effects of reducing blood pressure, reducing blood fat, expanding coronary artery, resisting inflammation, oxidation and fatigue, improving the immunity of the organism and the like. Siberian nitraria fruit is rich in various bioactive substances such as polysaccharide, protein, amino acid, alkaloid, flavone and phenols. Throughout the research reports at home and abroad, the chemical components of the Nitraria tangutorum bobr have more research on the Nitraria tangutorum bobr, but the research reports on the chemical components and the biological activity of the Nitraria sibirica are less, and the research is mainly focused on the compounds such as pigments, alkaloids, flavones and the like. At present, researches on extraction and separation, structural identification and related biological activity screening of Siberian Nitraria polysaccharide biomacromolecule compounds are in an initial stage. Therefore, the research on polysaccharide components with wide biological activity of Siberian nitraria has important significance, provides technical support for the comprehensive utilization of Siberian nitraria resources, and provides material basic reference for developing health care products for improving the body immunity function by taking Siberian nitraria as a raw material.
Polysaccharide compounds are natural high-molecular polymers having a complex structure in which a plurality of monosaccharide residues are bonded by glycosidic bonds, and are widely found in animals and plants, microorganisms, algae, and the like. Different plant polysaccharides show various pharmacological effects due to different structures. Most research reports show that the polysaccharide compound has various biological activities, including multiple functions of resisting oxidation, inhibiting bacteria, reducing blood sugar, resisting tumors, regulating immunity, improving intestinal flora and the like. Polysaccharides are widely used in the pharmaceutical industry due to their broad therapeutic action and extremely low cytotoxicity. Meanwhile, because of the good functional characteristics of the polysaccharide compound, the polysaccharide compound comprises water retention property, oil retention property, emulsibility, emulsion stability and the like, and is widely applied to the food industry to be used as an emulsifier, a food additive and the like. Besides, the polysaccharide compound has wide application prospect in the fields of cosmetics, environmental management and the like.
Antioxidant activity is one of the most widespread biological activities of polysaccharide compounds. Reactive oxygen species are by-products of normal oxygen metabolism and play an important role in cell signaling and maintaining normal functioning of the body. However, excessive active oxygen beyond the range of the body's scavenging ability seriously affects the body's health, and studies have shown that oxidative stress causes cell damage, leading to various problems such as aging, DNA mutation, etc., thereby causing serious health problems. Therefore, a scientifically reasonable supplementation with appropriate antioxidants is required. Antioxidants slow or prevent oxidation by themselves to remove free radicals, inhibiting further oxidation reactions from occurring. However, most of the antioxidants currently on the market are synthesized and may cause aging, liver damage, carcinogenesis, and the like. Therefore, it is of great practical significance to screen natural antioxidants from natural animals and plants in nature that can scavenge free radicals in the body to maintain the health of the body.
In recent years, people's health consciousness is continuously improved, and the relationship between health and diet is more and more emphasized, stimulating the demand of people for functional and healthy food. Prebiotics are nondigestible food ingredients that play a vital role in promoting host health by selectively stimulating the growth and metabolic activity of the gastrointestinal flora. The intestinal flora can inhibit the growth of foreign bacteria and form a biological barrier, thereby influencing the immune function of the organism. Studies have shown that carbohydrates may maintain or improve human health by regulating the host gut flora, polysaccharides are not degraded by enzymes in the gut and can be used as a carbon source by gut bacteria via anaerobic metabolism. Meanwhile, the finally generated short-chain fatty acids such as lactic acid enable the intestinal tract to be in an acid environment, and inhibit the growth of pathogens, so that the composition of intestinal flora is changed, and the health condition of a host is improved.
Disclosure of Invention
The invention aims to provide a preparation method and application of Siberian nitraria fruit polysaccharide effective part. The effective part of Siberian nitraria fruit polysaccharide obtained by the method is proved by verification to be: has stronger antioxidant activity and probiotic growth promoting activity; the research and development of polysaccharide natural antioxidant and intestinal flora regulator to improve the immunity of organism is of great practical significance. The application of the obtained Siberian Nitraria polysaccharide effective part in the fields of food, medicine and cosmetics, and creation of health food with specific functional activity, and provides a foundation for realizing industrial application of technical achievements.
The invention relates to a preparation method of Siberian Nitraria polysaccharide effective parts, which takes Siberian Nitraria fruit as a raw material, adopts hot water extraction, ultrasonic-assisted extraction, cellulase-assisted extraction, pectinase-assisted extraction or ultrasonic + pectinase extraction, and specifically comprises the following steps:
hot water extraction:
a. collecting mature Siberian nitraria fruits, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3-4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeatedly extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, carrying out reflux extraction on the degreased and decolored Siberian nitraria fruit powder at the temperature of 90 ℃, carrying out extraction for 2 hours at 2 times, combining the extracting solutions, carrying out centrifugation at 7000rpm for 10min, purifying the extract by using AB-8 type macroporous adsorption resin, and concentrating the extract to obtain polysaccharide concentrated solution;
e. d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part;
ultrasonic assisted extraction:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3-4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeatedly extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, extracting for 1 time at 7000rpm for 50min at 50 ℃, centrifuging for 10min, purifying by using AB-8 type macroporous resin, and concentrating to obtain polysaccharide concentrated solution;
e. d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part;
auxiliary extraction of cellulase:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3-4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeatedly extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, adding 0.5% by weight of cellulase, carrying out reflux extraction at the temperature of 50 ℃, extracting for 6 hours, raising the temperature to 95 ℃ after the extraction is finished, carrying out enzyme deactivation, keeping for 15min at 7000rpm, centrifuging for 10min, purifying by using AB-8 type macroporous resin, and concentrating to obtain a polysaccharide concentrated solution;
e. d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part;
and (3) auxiliary extraction of pectinase:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3-4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeatedly extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, adding 0.5% of pectinase according to the weight ratio, carrying out reflux extraction at the temperature of 50 ℃, extracting for 6 hours, raising the temperature to 95 ℃ after the extraction is finished, carrying out enzyme deactivation, keeping the temperature for 15min at 7000rpm, centrifuging for 10min, purifying by using AB-8 type macroporous resin, and concentrating to obtain polysaccharide concentrated solution;
e. d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part;
ultrasonic and pectinase extraction:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3-4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeatedly extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, adding 0.5% of cellulase with the ultrasonic power of 500W, extracting at 50 ℃ for 50min for 1 time, putting the mixture into a boiling water bath for enzyme deactivation after the extraction is finished, keeping the mixture for 15min at 7000rpm, centrifuging the mixture for 10min, purifying and concentrating the AB-8 type macroporous resin to obtain polysaccharide concentrated solution;
e. and d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part.
The Siberian nitraria polysaccharide effective part obtained by the method is used for preparing antioxidant drugs.
The application of the Siberian Nitraria polysaccharide effective part obtained by the method in preparing a probiotic growth medicament.
The Siberian nitraria polysaccharide effective part obtained by the method is used for preparing food or health care products for promoting the growth of probiotics.
The Siberian nitraria polysaccharide effective part obtained by the method is used for preparing antioxidant food or health care products.
The application of the Siberian Nitraria polysaccharide effective part obtained by the method in preparing antioxidant cosmetics.
The experiments show that the Siberian nitraria polysaccharide effective part obtained by the method of the invention has the following characteristics: has antioxidant activity and strong total reducing power, has certain capacity of eliminating 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical and hydroxyl free radical, and can promote the growth of bifidobacterium adolescentis, lactobacillus delbrueckii subsp bulgaricus and enterococcus faecalis. At present, the search for new antioxidants and probiotic growth promoters from natural plants is a new direction for the development of modern pharmaceutical and food industries. The research can provide a foundation for the application of the Siberian nitraria polysaccharide effective part in the fields of food, medicine and cosmetics, the creation of health food with specific functional activity and the realization of the industrial application of technical achievements.
Drawings
FIG. 1 is an infrared spectrum of the present invention;
FIG. 2 is a maximum absorption spectrum of a Congo red composite of the present invention;
FIG. 3 is a scanning electron microscope image of the present invention; wherein A is hot water extraction of polysaccharide;
FIG. 4 is a scanning electron microscope image of the present invention; wherein B is polysaccharide extracted by ultrasonic;
FIG. 5 is a scanning electron microscope image of the present invention; wherein C is cellulose extracted polysaccharide;
FIG. 6 is a scanning electron microscope image of the present invention; wherein D is polysaccharide extracted from pectinase;
FIG. 7 is a scanning electron microscope image of the present invention; wherein E is ultrasonic and pectase extracted polysaccharide;
FIG. 8 is a surface elemental analysis chart of Siberian Nitraria polysaccharide of the present invention;
FIG. 9 is a graph of the DPPH radical scavenging ability of Siberian Nitraria polysaccharide of the present invention;
FIG. 10 is a graph of hydroxyl radical scavenging ability of Siberian Nitraria polysaccharide of the present invention;
FIG. 11 is a graph of the total reduction capacity of Siberian Nitraria polysaccharide of the present invention.
Detailed Description
Example 1
Hot water extraction:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria fruit powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeating the steps until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, carrying out reflux extraction on the degreased and decolored Siberian nitraria fruit powder at the temperature of 90 ℃, carrying out extraction for 2 hours at 2 times, combining the extracting solutions, carrying out centrifugation at 7000rpm for 10min, purifying the extract by using AB-8 type macroporous adsorption resin, and concentrating the extract to obtain polysaccharide concentrated solution;
e. and d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part.
Example 2
Ultrasonic extraction:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr fruit powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, extracting until the color of the ethanol layer solution is lighter, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, extracting for 1 time at 7000rpm for 50min at 50 ℃, centrifuging for 10min, purifying by using AB-8 type macroporous resin, and concentrating to obtain polysaccharide concentrated solution;
e. and d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part.
Example 3
And (3) extracting cellulase:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr fruit powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, adding 0.5% by weight of cellulase, carrying out reflux extraction at the temperature of 50 ℃, extracting for 6 hours, raising the temperature to 95 ℃ after the extraction is finished, carrying out enzyme deactivation, keeping for 15min at 7000rpm, centrifuging for 10min, purifying by using AB-8 type macroporous resin, and concentrating to obtain a polysaccharide concentrated solution;
e. and d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part.
Example 4
And (3) extracting pectinase:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria fruit powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeating the steps until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, adding 0.5% of pectinase according to the weight ratio, carrying out reflux extraction at the temperature of 50 ℃, extracting for 6 hours, raising the temperature to 95 ℃ after the extraction is finished, carrying out enzyme deactivation, keeping the temperature for 15min at 7000rpm, centrifuging for 10min, purifying by using AB-8 type macroporous resin, and concentrating to obtain polysaccharide concentrated solution;
e. and d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part.
Example 5
Ultrasonic and pectinase extraction:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr fruit powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, adding 0.5% of cellulase with the ultrasonic power of 500W, extracting at 50 ℃ for 50min for 1 time, putting the mixture into a boiling water bath for enzyme deactivation after the extraction is finished, keeping the mixture for 15min at 7000rpm, centrifuging for 10min, purifying and concentrating AB-8 type macroporous resin to obtain polysaccharide concentrated solution;
e. and d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part.
Example 6
Infrared spectrum of siberian nitraria polysaccharide:
the infrared spectrogram analysis results of Siberian Nitraria polysaccharide effective part samples obtained in examples 1-5 (figure 1) show that the polysaccharide prepared by different methods is 3400cm-1The strong absorption peak is near, and the absorption peak is caused by stretching vibration of polysaccharide hydroxyl; at 2900cm-1The nearby absorption peak is caused by C-H stretching vibration; about 1600cm-1The nearby strong absorption peak is caused by the hydration vibration peak of the polysaccharide compound, and the Siberian Nitraria polysaccharide is 500cm at 4000--1Typical polysaccharide absorption peaks are shown in the range.
Example 7
Conformational analysis of siberian nitraria polysaccharide:
conformational analysis of the siberian nitraria polysaccharide effective part samples obtained in examples 1 to 5 was performed by congo red experiment, as shown in fig. 2, when the concentration of sodium hydroxide was in the range of 0 to 0.5mol/L, the maximum absorption wavelengths of siberian nitraria polysaccharide were all significantly red-shifted compared with the maximum absorption wavelength of the control group (congo red), which indicates that the siberian nitraria polysaccharide effective parts extracted by different methods all have triple-helical structures.
Example 8
Scanning electron microscope and energy spectrum analysis of Siberian Nitraria polysaccharide:
the scanning electron microscope-energy dispersive analysis (SEM-EDX) figures of the Siberian Nitraria polysaccharide effective parts obtained in examples 1-5 are shown in figures 3 and 4, compared with the hot water extraction method, the polysaccharide surface structure, particle shape, particle size and the like are changed after ultrasonic or enzymolysis treatment, and Siberian Nitraria polysaccharide contains main elements such as carbon, oxygen and the like, and also contains normal trace elements such as Na, Mg, P, S, K, Ca and the like which are beneficial to human bodies.
Example 9
The antioxidant activity test of the Siberian Nitraria polysaccharide effective part comprises the following steps:
(1) determination of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) radical scavenging Capacity:
putting 100 μ L of Siberian Nitraria polysaccharide effective part solution of example 1-5 with each concentration (0.1-2mg/mL) and 100 μ L of 0.2mmol/L DPPH-methanol solution in a 96-well plate, reacting at room temperature in a dark place for 30min, measuring absorbance at 517nm, and calculating clearance rate;
(2) measurement of hydroxyl radical scavenging ability:
50 mu L of Siberian Nitraria polysaccharide effective part solution obtained in example 1-5 with 50 mu L of ferrous sulfate solution of 6mmol/L, 50 mu L of salicylic acid-ethanol solution of 6mmol/L and 50 mu L of hydrogen peroxide solution of 0.1% are put in a 96-well plate, and reacted for 30min at room temperature in a dark place, and the absorbance is measured at 510nm to calculate the clearance rate;
(3) the total reducing capacity of the effective part of Siberian Nitraria polysaccharide is determined:
200 μ L of each concentration (0.1-2mg/mL) of the Siberian Nitraria polysaccharide effective fraction solution obtained in example 1-5 was mixed with 100 μ L of phosphoric acid buffer solution, 200 μ L of 1% potassium ferricyanide solution, and 100 μ L of 10% trichloroacetic acid solution, reacted at 50 ℃ for 20min, 400 μ L of distilled water and 100 μ L of ferric chloride solution were added, left at room temperature for 10min, and absorbance was measured at 700nm to calculate clearance.
The in vitro antioxidant activity analysis of the Siberian Nitraria polysaccharide effective part related by the invention shows that: the prepared Siberian Nitraria polysaccharide effective components have total reducing ability, and ability of eliminating DPPH and hydroxyl free radical, and the results are shown in figures 5-7, which indicate that the extraction method has great influence on the antioxidant activity of polysaccharide; the removal rate half-inhibition concentrations of polysaccharide effective parts obtained by hot water extraction, ultrasonic extraction, cellulase extraction, pectinase extraction and ultrasonic + pectinase extraction on DPPH free radicals are respectively 1.171,0.209,0.209,0.356 and 1.740mg/mL, wherein the removal capacity of the polysaccharide obtained by ultrasonic-assisted extraction and cellulase-assisted extraction on DPPH free radicals is obviously stronger than that of the polysaccharide obtained by the other three methods; however, the half-inhibitory concentrations of the polysaccharides prepared by the five methods for scavenging hydroxyl radicals are respectively 0.815,0.168,0.149,0.725 and 0.138mg/mL, wherein the scavenging ability of the polysaccharides obtained by ultrasonic and pectinase assisted extraction for scavenging hydroxyl radicals is obviously stronger than that of the polysaccharides obtained by the other four methods. Compared with a hot water extraction method, the ultrasonic-assisted extraction and the enzymolysis extraction have the advantages that the polysaccharide is extracted under a milder condition, and the better antioxidant activity is shown. Siberian nitraria polysaccharide can become a potential antioxidant and anti-aging agent due to the strong antioxidant activity of Siberian nitraria polysaccharide, and is widely applied to the fields of food, medicine, health-care products and the like.
Example 10
The probiotics growth promoting activity of Siberian Nitraria polysaccharide:
respectively inoculating lactobacillus delbrueckii subsp bulgaricus, bifidobacterium adolescentis and streptococcus faecalis liquid into corresponding culture media, respectively adding samples with different concentrations into a basic culture medium for culturing for 16-18 hours, detecting the number of bacteria by using a cell counter, and repeating each sample for 3 times;
the results of the in vitro probiotic growth activity promotion results of the Siberian Nitraria polysaccharide effective part provided by the invention show that: the prepared siberian nitraria polysaccharide effective parts have obvious growth promoting activity on bifidobacterium adolescentis, lactobacillus delbrueckii subsp bulgaricus and streptococcus faecalis, and the results are shown in tables 1-3:
TABLE 1
TABLE 2
TABLE 3
The research result shows that: different methods of hot water extraction, ultrasonic-assisted extraction, cellulase-assisted extraction, pectinase-assisted extraction or ultrasonic + pectinase extraction are adopted to greatly influence the growth activity of probiotics on the Siberian polysaccharide effective part, wherein the polysaccharide obtained by hot water extraction has the strongest growth promotion effect on bifidobacteria, the polysaccharide prepared by cellulase-assisted extraction has the strongest growth promotion activity on Lactobacillus delbrueckii subspecies bulgaricus, and the polysaccharide extracted by pectinase-assisted extraction has the strongest growth promotion effect on enterococcus faecalis. The polysaccharide has different biological activities due to the influence of different extraction methods on the polysaccharide content, monosaccharide composition, molecular weight, sugar chain structure and the like.
The verification of the effective part of the Siberian polysaccharide prepared by the invention shows that: has strong antioxidant activity and probiotic growth promoting activity. The research and development of polysaccharide natural antioxidants and intestinal flora regulators so as to improve the immune function of organisms have important practical significance, and provide a basis for the application of Siberian nitraria polysaccharide effective parts in the fields of foods, medicines and cosmetics, the creation of healthy foods with specific functional activity and the realization of industrial application of technical achievements.
Claims (1)
1. The application of the Siberian Nitraria polysaccharide effective part in preparing the medicines, foods or health products for promoting the growth of probiotics is disclosed, wherein the probiotics is bifidobacterium adolescentis, the preparation method of the Siberian Nitraria polysaccharide effective part adopts hot water extraction or ultrasonic auxiliary extraction, and the specific operation is carried out according to the following steps:
hot water extraction:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3-4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeatedly extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, carrying out reflux extraction on the degreased and decolored Siberian nitraria fruit powder at the temperature of 90 ℃, carrying out extraction for 2 hours at 2 times, combining the extracting solutions, carrying out centrifugation at 7000rpm for 10min, purifying the extract by using AB-8 type macroporous adsorption resin, and concentrating the extract to obtain polysaccharide concentrated solution;
e. d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part;
ultrasonic assisted extraction:
a. collecting mature Siberian nitraria fruit, drying at 40 ℃, mechanically crushing, and sieving by a 40-mesh sieve to obtain Siberian nitraria fruit powder;
b. b, adding petroleum ether into the powder obtained in the step a according to the material-liquid ratio of 1:8g/mL, stirring for 2 hours, extracting until a petroleum ether layer is colorless, performing suction filtration, and air-drying the powder until no petroleum ether solvent remains to obtain degreased Siberia nitraria fruit powder;
c. b, adding the degreased Siberian nitraria tangutorum bobr powder obtained in the step b into a chloroform-methanol mixed solution with the volume ratio of 4:1 according to the material-liquid ratio of 1:5g/mL, stirring for 2 hours at room temperature, extracting for 3-4 times, carrying out suction filtration and natural air drying, adding 90% ethanol with the material-liquid weight ratio of 1:10g/mL, refluxing and heating for 3 hours, repeatedly extracting until the color of the ethanol layer solution is light, carrying out suction filtration and natural air drying to obtain degreased and decolored Siberian nitraria tangutorum bobr fruit powder;
d. c, adding water into the degreased and decolored Siberian nitraria fruit powder obtained in the step c according to the material-liquid ratio of 1:20g/mL, extracting for 1 time at 7000rpm for 50min at 50 ℃, centrifuging for 10min, purifying by using AB-8 type macroporous resin, and concentrating to obtain polysaccharide concentrated solution;
e. and d, precipitating the polysaccharide concentrated solution obtained in the step d by using absolute ethyl alcohol according to the volume ratio of 1:5, standing at the temperature of 4 ℃ for 12h at 7000rpm, centrifuging for 10min, and carrying out vacuum freeze drying on the polysaccharide precipitate to obtain the Siberian nitraria polysaccharide effective part.
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