NL2027683B1 - Auricularia heimuer Oligosaccharide for Protecting Hepatocytes from Oxidative Damage, Preparation Method Thereof and Use Thereof - Google Patents
Auricularia heimuer Oligosaccharide for Protecting Hepatocytes from Oxidative Damage, Preparation Method Thereof and Use Thereof Download PDFInfo
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- NL2027683B1 NL2027683B1 NL2027683A NL2027683A NL2027683B1 NL 2027683 B1 NL2027683 B1 NL 2027683B1 NL 2027683 A NL2027683 A NL 2027683A NL 2027683 A NL2027683 A NL 2027683A NL 2027683 B1 NL2027683 B1 NL 2027683B1
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- auricularia
- heimuer
- oligosaccharide
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- 241001057326 Auricularia heimuer Species 0.000 title claims abstract description 120
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 60
- 150000002482 oligosaccharides Chemical class 0.000 title claims abstract description 60
- 210000003494 hepatocyte Anatomy 0.000 title claims abstract description 24
- 230000004792 oxidative damage Effects 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
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- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 5
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 4
- 238000000265 homogenisation Methods 0.000 claims abstract description 3
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- 239000000243 solution Substances 0.000 claims description 34
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- 229920001282 polysaccharide Polymers 0.000 claims description 29
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 239000000843 powder Substances 0.000 claims description 25
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- 229910021641 deionized water Inorganic materials 0.000 claims description 15
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- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 10
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 10
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 10
- OAABHEHWRQAHEJ-UHFFFAOYSA-N butan-1-ol;chloroform Chemical compound ClC(Cl)Cl.CCCCO OAABHEHWRQAHEJ-UHFFFAOYSA-N 0.000 claims description 10
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- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 5
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
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- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 2
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- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
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- HMUNWXXNJPVALC-UHFFFAOYSA-N 1-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C(CN1CC2=C(CC1)NN=N2)=O HMUNWXXNJPVALC-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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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
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- 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
- 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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- 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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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01015—Polygalacturonase (3.2.1.15)
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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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Abstract
The present invention relates to an Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage, a preparation method thereof 5 and a use thereof, and belongs to the field of preparation of effective components of edible fungi. The Auricularia heimueroligosaccharide is obtained from a dried Auricularia heimuer fruit body by rapid homogenization combined with enzymatic extraction, extrusion filtration, protein removal, and ion exchange chromatography. The preparation method shortens the extraction time, reduces 10 the amount of reagents, saves energy and improves solubility; and the extrusion filtration step is suitable for the separation of viscous solutions with high gum content. Moreover, the Auricularia heimuer oligosaccharide has the function of protecting hepatocytes from oxidative damage, and can be used for the preparation of liver-protective auxiliary drugs and health food, which provides 15 basic data for the development of deep processing products of Auricularia heimuer and reveals important economic and market value.
Description
Auricularia heimuer Oligosaccharide for Protecting
Hepatocytes from Oxidative Damage, Preparation Method
Thereof and Use Thereof
The present invention relates to an Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage, a preparation method thereof and a use thereof, and belongs to the field of natural products.
The liver is the largest metabolic organ in the human abdominal cavity and plays a role in regulating immune tolerance, synthesizing proteins and maintaining the balance of the internal environment. More than 400,000 patients die from liver diseases in China every year, which is a physiological state common to many liver diseases. Staying up late, fatigue, drunkenness, viruses, drugs and genetics are all causes of liver injury, but liver injury induced by various incentives is accompanied by oxidative stress, lipid peroxide accumulation and other peroxidative states. Auricularia heimuer is the main variety of edible fungi in China, with annual production and consumption ranking among the best in edible fungi. The Auricularia heimuer polysaccharide is an important active component of Auricularia heimuer, which has many biological activities capable of lowering blood sugar, lowering blood lipid, enhancing immunity and anticoagulation. It has also been found that the Auricularia heimuer polysaccharide can prevent BCG-induced immune liver injury, but its structural characteristics and action pathway are not clear.
In addition, the hot water extraction is the simplest and most common method chosen to extract and prepare Auricularia heimuer polysaccharide, despite some disadvantages such as long extraction time, large amount of solvent used, and damage-prone polysaccharide structure at high temperature (Wei Hong. Auricularia heimuer Polysaccharide Extraction Process [J]. Food
Research and Development, 2010, 05: 117-120.). Moreover, most of Auricularia heimuer polysaccharides are colloidal components, and the high viscosity of water extract is also called "non-Newtonian fluid". So, acids, bases and bioenzymes are often added during the extraction process to improve the fluidity of Auricularia heimuer. However, acids and bases may destroy the structure of polysaccharide, the addition of bioenzymes also limits the extraction conditions to the optimum temperature and pH range of the enzyme, and the subsequent separation and purification process requires inactivation and removal of the enzyme, which makes the enzyme preparation more expensive (Li Xiao.
Optimization of Enzymatic Extraction of Active Polysaccharides from Auricularia heimuer by Response Surface Methodology [J]. Anhui Agricultural Science, 2010, 33: 475-478.). The ultrasonic-assisted extraction is noisy (Xu Xiuhui.
Ultrasonic Extraction of Polysaccharide from Auricularia heimuer [J].
Pharmaceutical and Clinical Research, 2011, 2 (19): 189 - 190.); whereas, the microwave-assisted extraction has a small extraction volume limited by the equipment (Zhu Lei. Optimization of Microwave-assisted Extraction of
Polysaccharide from Auricularia heimuer by Response Surface Methodology [J].
Journal of Chinese Institute of Food Science and Technology, 2009, 02: 58-65.).
None of the prior art mentioned above can meet a large number of demands for industrial development and production of such products.
The present invention provides an Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage, a preparation method thereof and a use thereof, aiming at solving the deficiencies and shortcomings of existing research and technology.
The present invention provides an Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage, wherein the Auricularia heimuer oligosaccharide is a B-type pyranose and consists of fucose, arabinose, galactose, glucose, xylose and mannose, with a molecular weight range of 6.882x108 to 22.684x103 Da.
The present invention provides a preparation method of an Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage, wherein the Auricularia heimuer oligosaccharide is obtained from a dried
Auricularia heimuer fruit body by rapid homogenization combined with enzymatic extraction, extrusion filtration, protein removal, and ion exchange chromatography.
The specific steps of the preparation method are as follows: a) crushing and allowing the dried Auricularia heimuer fruit body to pass through a 40-mesh sieve to obtain powder thereof, loading the powder into a homogenizer, and homogenizing for 5 — 20 min with the addition of deionized water according to a material-liquid ratio of 1:30 - 1:100; preparing an Auricularia heimuer enzyme extracting solution with addition of 10 mg/mL of pectinase solution, in which the mass of pectinase is 0.4% of the powder; extruding and filtering the Auricularia heimuer enzyme extracting solution to obtain an
Auricularia heimuerr filtrate; concentrating the Auricularia heimuer filtrate to 1/2 to 1/3 of the original volume at 80 — 100 °C, cooling to room temperature, adding anhydrous ethanol 3-4 times as much as the volume of concentrated Auricularia heimuer filtrate, and forming a mixed solution 1; standing at 4 °C for 12 h, centrifuging at 6000-10000 r/min for 5-10 min, volatilizing residual ethanol after centrifugation, and freeze-drying to obtain dried powder as crude polysaccharide of Auricularia heimuer fruit body; b) preparing the crude polysaccharide of Auricularia heimuer fruit body into 5 mg/mL of solution with addition of purified water, adding chloroform n-butanol mixed solution while magnetically stirring to allow centrifugal stratification, and extracting the upper solution; repeatedly adding chloroform n-butanol mixed solution once, centrifugalizing, and concentrating the supernatant to 1/2~1/3 of the original volume at a reduced pressure; adding anhydrous ethanol 3 - 4 times as much as the concentrated volume, standing at 4 °C for 12 h, centrifuging at 6,000-10,000 r/min for 5-10 min, volatilizing residual ethanol, and freeze-drying to obtain dried powder as an Auricularia heimuer fruit body polysaccharide; and c) dissolving the Auricularia heimuer fruit body polysaccharide in deionized water at constant volume, loading sample onto DEAE 52 cellulose column for chromatography, eluting with deionized water at the flow rate of 5mL/3min, collecting 5mL per tube, determining the sugar content of each tube, collecting the elution peak according to the sugar content of each tube, and obtaining an
Auricularia heimuer oligosaccharide.
The present invention provides a preparation method of an Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage, wherein the extrusion filtration step is completed by a single-screw extruder, with a die hole diameter of 8 - 16 mm, sleeve temperature of 60 - 100 °C and a screw speed of 160 - 260 r/min.
The present invention provides a use of an Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage in the preparation of health food and pharmaceuticals.
The present invention provides a health food and pharmaceutical composition for protecting hepatocytes from oxidative damage, wherein the health food and pharmaceutical composition contains the Auricularia heimuer oligosaccharide composition as claimed in claims 1 to 3.
Preferably, the dosage forms of the health food and pharmaceutical composition can be tablets, granules, capsules or solutions.
The advantageous effect of the present invention is as follow: it is the first time to obtain an Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage, a preparation method thereof, a use thereof in the preparation of health food and pharmaceutical products. In the preparation process of Auricularia heimuer oligosaccharide, a dried Auricularia heimuer fruit body is rapidly homogenized combined with enzymatic extraction, extrusion filtration, protein removal, and ion exchange chromatography, which shortens the extraction time, reduces the amount of reagents, saves energy and improves solubility. The extrusion filtration step is suitable for the separation of viscous solutions with high gum content. Moreover, the Auricularia heimuer oligosaccharide can protect functional activity of hepatocytes against oxidative damage by regulating the PI3K-akt signaling pathway, and can be used for the preparation of liver-protective auxiliary drugs and health food, which provides basic data for the development of deep processing products of Auricularia heimuer and reveals important economic and market value.
The present invention is further described below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is an elution curve of the Auricularia heimuer oligosaccharide DEAE 52 of the present invention;
Fig. 2 is an elution curve of high performance gel permeation chromatography of the Auricularia heimuer oligosaccharide of the present invention;
Fig. 3 shows the monosaccharide composition of the Auricularia heimuer oligosaccharide of the present invention;
Fig. 4 is an infrared spectrum of the Auricularia heimuer oligosaccharide of the present invention; 5 Fig. 5 shows the protective effect of the Auricularia heimuer oligosaccharide of the present invention on oxidatively damaged HepG2 cells;
Fig. 6 shows the effect of the Auricularia heimuer oligosaccharide of the present invention on the morphology of oxidatively damaged HepG2 cells;
Fig. 7 shows the effect of the Auricularia heimuer oligosaccharide of the present invention on superoxide dismutase activity of HepG2 cells;
Fig. 8 shows the effect of the Auricularia heimuer oligosaccharide of the present invention on peroxidase activity of HepG2 cells;
Fig. 9 shows the effect of the Auricularia heimuer oligosaccharide of the present invention on glutathione peroxidase activity of HepG2 cells;
Fig. 10 shows the effect of the Auricularia heimuer oligosaccharide of the present invention on the expression of apoptosis-related proteins in HepG2 cells.
The present invention is further described hereinafter with reference to preferred embodiments. However, the embodiments should not be construed as limited to the protection scope set forth herein.
Example 1 1. Crushing and allowing a dried Auricularia heimuer fruit body to pass through a 40-mesh sieve to obtain powder thereof, loading the powder into a homogenizer, and homogenizing for 10 min with the addition of deionized water according to a material-liquid ratio of 1:80; adding 10 mg/mL of pectinase solution, in which the mass of pectinase is 0.4% of the powder; heating in a water bath kettle at a constant temperature of 50 °C and reacting for 1 h, then placing the beaker in a 90 °C water bath for 1 h to inactivate the pectinase, extruding and filtering; in which the extrusion filtration step is completed by a single-screw extruder, with a die hole diameter of 16 mm, sleeve temperature of 60 °C and a screw speed of 240 r/min; concentrating the Auricularia heimuer filtrate to 1/3 of the original volume at 90 °C, cooling to room temperature, adding anhydrous ethanol 3 times as much as the volume of concentrated Auricularia heimuer filtrate, standing at 4 °C for 12 h, centrifuging at 10000 r/min for 5 min, volatilizing residual ethanol after centrifugation, and freeze-drying to obtain dried powder as crude polysaccharide of Auricularia heimuer fruit body; 2. preparing the crude polysaccharide of Auricularia heimuer fruit body into 5 mg/mL of solution, adding chloroform n-butanol mixed solution while magnetically stirring to allow centrifugal stratification, and extracting the upper solution; repeatedly adding chloroform n-butanol mixed solution once, centrifugalizing, and concentrating the supernatant to 1/2 of the original volume at a reduced pressure; adding anhydrous ethanol 4 times as much as the concentrated volume, standing at 4 °C for 12 h, centrifuging at 10,000 r/min for 5 min, volatilizing residual ethanol, and freeze-drying to obtain dried powder as an
Auricularia heimuer fruit body polysaccharide; 3. dissolving the Auricularia heimuer fruit body polysaccharide in deionized water at constant volume, loading sample onto DEAE 52 cellulose column for chromatography (4.0 x 30cm), eluting with deionized water at the flow rate of 5mL/3min, collecting 5mL per tube; determining the sugar content of each tube, collecting the elution peak according to the sugar content of each tube, and obtaining an Auricularia heimuer oligosaccharide, as shown in Fig. 1; and continuing the gel permeation chromatography analysis of the Auricularia heimuer oligosaccharide (TSK-gelG-3000PWXL chromatographic column (7.8 x 300mm), chromatographic conditions: chromatographic column:
DionexTMCarboPacTMPA20; mobile phase: A phase: DDH20; B phase: 200mMNaOH; C phase: 200mMNaOH/500mMNaAC; flow rate: 0.5mL/min).
Based on the molecular weight IgMw=7.831-0.228t+, an average molecular weight of the Auricularia heimuer oligosaccharide was 16.190kDa, as shown in
Fig. 2. The Auricularia heimuer oligosaccharide was B-pyranose as detected by infrared spectroscopy. The specific steps were as follows: hydrolyzing 2mg of
Auricularia heimuer oligosaccharide into a product by methanol containing 1MHCI, and hydrolyzing the product into another product by 2MTFA, which was derivatized by 1-phenyl-3-methyl-5-pyrazolone (PMP); separating the derivatized product by a CompassC18column (250x4.6mm) column and performing HPLC analysis, and detecting the eluted product at UV245 nm, as shown in Fig. 3. The results show that the Auricularia heimuer oligosaccharide consists mainly of glucose, galactose and mannose, and a small amount of rockulose, arabinose and xylose.
Example 2 1. Crushing and allowing a dried Auricularia heimuer fruit body to pass through a 40-mesh sieve to obtain powder thereof, loading the powder into a homogenizer, and homogenizing for 5 min with the addition of deionized water according to a material-liquid ratio of 1:100; adding 10 mg/mL of pectinase solution, in which the mass of pectinase is 0.4% of the powder; heating in a water bath kettle at a constant temperature of 50 °C and reacting for 1 h, then placing the beaker in a 100 °C water bath for 1 h to inactivate the pectinase, extruding and filtering; in which the extrusion filtration step is completed by a single-screw extruder, with a die hole diameter of 8 mm, sleeve temperature of 80 °C and a screw speed of 160 r/min; concentrating the Auricularia heimuer filtrate to 1/2 of the original volume at 100 °C, cooling to room temperature, adding anhydrous ethanol 3 times as much as the volume of concentrated
Auricularia heimuer filtrate, standing at 4 °C for 12 h, centrifuging at 6000 r/min for 10 min, volatilizing residual ethanol after centrifugation, and freeze-drying to obtain dried powder as crude polysaccharide of Auricularia heimuer fruit body; 2. preparing the crude polysaccharide of Auricularia heimuer fruit body into 5 mg/mL of solution, adding chloroform n-butanol mixed solution (volume ratio: 4:1) while magnetically stirring to allow centrifugal stratification, and extracting the upper solution; repeatedly adding chloroform n-butanol mixed solution (volume ratio: 4:1) once, centrifugalizing, and concentrating the supernatant to 1/2 of the original volume at a reduced pressure; adding anhydrous ethanol 3 times as much as the concentrated volume, standing at 4 °C for 12 h, centrifuging at 6000 r/min for 10 min, volatilizing residual ethanol, and freeze-drying to obtain dried powder as an Auricularia heimuer fruit body polysaccharide; 3. dissolving the Auricularia heimuer fruit body polysaccharide in deionized water at constant volume, loading sample onto DEAE 52 cellulose column for chromatography (4.0 x 30cm), eluting with deionized water at the flow rate of 5mL/3min, collecting 5 mL per tube; determining the sugar content of each tube, collecting the elution peak according to the sugar content of each tube, and obtaining an Auricularia heimuer oligosaccharide, as shown in Fig. 1. and continuing the gel permeation chromatography analysis of the Auricularia heimuer oligosaccharide (TSK-gelG-3000PWXL chromatographic column (7.8x300mm), chromatographic conditions: chromatographic column:
DionexTMCarboPacTMPA20; mobile phase: A phase: DDH20; B phase: 200mMNaOH; C phase: 200mMNaOH/500mMNaAC; flow rate: 0.5mL/min).
Based on the molecular weight IgMw=7.831-0.228t+, an average molecular weight of the Auricularia heimuer oligosaccharide was 22.684x10 3 Da, as shown in Fig. 2. The Auricularia heimuer oligosaccharide was B-pyranose as detected by infrared spectroscopy. The specific steps were as follows: hydrolyzing 2 mg of Auricularia heimuer oligosaccharide into a product by methanol containing 1MHCI, and hydrolyzing the product into another product by 2MTFA, which was derivatized by 1-phenyl-3-methyl-5-pyrazolone (PMP); separating the derivatized product by a CompassC18column (250x4.6mm) column, performing HPLC analysis, and detecting the eluted product at UV245 nm, as shown in Fig. 3. The results show that the Auricularia heimuer oligosaccharide consists mainly of glucose, galactose and mannose, and a small amount of rockulose, arabinose and xylose.
Example 3 1. Crushing and allowing a dried Auricularia heimuer fruit body to pass through a 40-mesh sieve to obtain powder thereof, loading the powder into a homogenizer, and homogenizing for 20 min with the addition of deionized water according to a material-liquid ratio of 1:30; adding 10 mg/mL of pectinase solution, in which the mass of pectinase is 0.4% of the powder; heating in a water bath kettle at a constant temperature of 50 °C and reacting for 1 h, then placing the beaker in a 80 °C water bath for 1 h to inactivate the pectinase, extruding and filtering; in which the extrusion filtration step is completed by a single-screw extruder, with a die hole diameter of 16 mm, sleeve temperature of 100 °C and a screw speed of 260 r/min; concentrating the Auricularia heimuer filtrate to 1/3 of the original volume at 80 °C, cooling to room temperature, adding anhydrous ethanol 4 times as much as the volume of concentrated Auricularia heimuer filtrate, standing at 4 °C for 12 h, centrifuging at 8000 r/min for 8 min, volatilizing residual ethanol after centrifugation, and freeze-drying to obtain dried powder as crude polysaccharide of Auricularia heimuer fruit body; 2. preparing the crude polysaccharide of Auricularia heimuer fruit body into mg/mL of solution, adding chloroform n-butanol mixed solution (volume ratio: 4:1) while magnetically stirring to allow centrifugal stratification, and extracting the upper solution; repeatedly adding chloroform n-butanol mixed solution (volume ratio: 4:1) once, centrifugalizing, and concentrating the supernatant to 5 1/3 of the original volume at a reduced pressure; adding anhydrous ethanol 4 times as much as the concentrated volume, standing at 4 °C for 12 h, centrifuging at 8000 r/min for 8 min, volatilizing residual ethanol, and freeze-drying to obtain dried powder as an Auricularia heimuer fruit body polysaccharide; 3. dissolving the Auricularia heimuer fruit body polysaccharide in deionized water at constant volume, loading sample onto DEAE 52 cellulose column for chromatography (4.0 x 30cm), eluting with deionized water at the flow rate of 5mL/3min, collecting 5 mL per tube; determining the sugar content of each tube, collecting the elution peak according to the sugar content of each tube, and obtaining an Auricularia heimuer oligosaccharide, as shown in Fig. 1. and continuing the gel permeation chromatography analysis of the Auricularia heimuer oligosaccharide (TSK-gelG-3000PWXL chromatographic column (7.8x300mm), chromatographic conditions: chromatographic column:
DionexTMCarboPacTMPA20; mobile phase: A phase: DDH20; B phase: 200mMNaOH; C phase: 200mMNaOH/500mMNaAC; flow rate: 0.5mL/min).
Based on the molecular weight IgMw=7.831-0.228t+, an average molecular weight of the Auricularia heimuer oligosaccharide was 6.882x10 3, as shown in
Fig. 2. The Auricularia heimuer oligosaccharide was B-pyranose as detected by infrared spectroscopy (AHP-0). The specific steps were as follows: hydrolyzing 2 mg of Auricularia heimuer oligosaccharide into a product by methanol containing 1MHCI, and hydrolyzing the product into another product by 2MTFA, which was derivatized by 1-phenyl-3-methyl-5-pyrazolone (PMP); separating the derivatized product by a CompassC18column (250x4.6mm) column, performing
HPLC analysis, and detecting the eluted product at UV245 nm, as shown in Fig. 3. The results show that the Auricularia heimuer oligosaccharide consists mainly of glucose, galactose and mannose, and a small amount of rockulose, arabinose and xylose.
The Auricularia heimuer oligosaccharide for protecting hepatocytes from oxidative damage, a preparation method thereof and a use thereof are demonstrated by the following tests:
Test example 1
Digesting the cells in logarithmic growth phase into cell suspension, adjusting to a concentration of 4x10% cells/mL, adding into a 96-well plate with 100 pL per well, incubating in a 5% CO: incubator for 24 h. Absorbing the medium and adding 100 uL of the corresponding culture solution according to the experimental groups. The experiment was carried out in six groups: 125, 250, 500 and 1000 pg/mL of Auricularia heimuer oligosaccharide and 0.7 mmol/L of
H2O2 complete culture solution were added to the experimental group; 0.7mmol/L of H202 complete culture medium was added to the model control group; and 6 replicates were set up for each group. Experimental steps are as follows: incubating in a CO2 incubator at the concentration of 5% for 48 h, discarding the drug-containing culture solution, adding 100 pL of fresh serum-free medium containing 10% CCK-8, and incubating for 1 h. The absorbance of each well was measured at 490 nm and the ICsowas calculated for each group.
Test example 2
Selecting HepG2 cells at logarithmic growth stage, adjusting to a concentration of 1x108 /mL, inoculating in a 6-well plate and incubating in the incubator for 24 h. Treating HepG2 cells with 125, 250, 500 and 1000 pg/mL of
Auricularia heimuer oligosaccharide and 0.7 mmol/L of H2O2 for 24 h, respectively. Discarding the medium and washing with DPBS for 3 times. Adding 70% ice ethanol and stabilizing at 4 °C for 15 min. Washing with DPBS for 3 times, staining with 1% crystal violet solution at room temperature for 10 min and then washing with DPBS for 3 times again. Performing microscopic examination at appropriate magnification and taking photos.
Test example 3 inoculating HepG2 cells in the logarithmic growth phase into a 6-well plate at 2x10 8 cells/mL and incubating for 24 h. The treatment conditions were the same as in Test Example 1: collecting culture solution, re-suspending cells and measuring the levels of antioxidant enzymes in the cell supernatant separately by commercially available kits (T-SOD, CAT, GSH-Px assay kits) according to the experimental protocol provided by the manufacturer. The experimental results were expressed with the values measured by ELISA at different absorbance. Each data set includes 3 replicate control wells.
Test example 4
HepG2 cells in logarithmic growth phase were passaged to 10 cm sterile culture dishes and incubated against the wall for 24 h. The cells were digested with trypsin and collected centrifugally. The experiment was performed in 6 groups, i.e. experimental control group, injury model group (0.7 mmol/L hydrogen peroxide) and administration group with different concentrations of
Auricularia heimuer oligosaccharide (125, 250, 500 and 1000 pg/mL) according to the following steps: incubating for 24 h with the addition of the corresponding culture solution according to the groups, discarding the culture medium, pre-cooling, and washing with DPBS twice; digesting the cells with trypsin, and lysing on ice for 30 min with the addition of 1 mL of lysis solution containing
PMSF, during which the centrifuge tube was shaken from time to time for complete lysis; then, centrifuging at 12,000 rpm/min and at 4 °C for 10 min, and determining the protein content of supernatant; in order to determine the protein content by the BCA kit, mixing the samples with Lodaing Buffer and heating at 95 °C for 5 min to denature the proteins; filling the denatured proteins and storing at -20 °C; selecting prefabricated gels with different densities according to the molecular weight of different proteins, and preparing the electrophoresis solution from ultrapure before use; taking above denatured protein samples for
SDS-PAGE electrophoresis at constant pressure of 110V for 75 min until the bromophenol blue indicator stain was about 1 cm from the low end of the gel; and then, transferring the membrane; after the membrane transfer, rinsing PVDF membrane in TBST buffer for 1 — 2 min to wash off the residual liquid; placing the PVDF membrane in 5% BSA, with the shaking table being at 10rpm/min, and closing for 60 min at 4 °C; immersing the closed PVDF membrane completely in the primary antibody dilution (diluted at a ratio of 1:1000), and incubating overnight at 4 °C while the shaking table was kept at 2 rpm/min; removing the
PVDF membrane with forceps and rinsing with TBST buffer for 4 times at 5 min/time; then, placing the PVDF membrane in secondary antibody horseradish peroxidase (HRP) and incubating for 60 min at 4 °C; rinsing with TBST for 4 times, 5 min each time, and keeping the shaking table at 4rpm/min; upon the completion of the secondary antibody incubation, removing the PVDF membrane and washing with TBST solution at 5 min/time for 3 times; mixing solutions A and B at a volume ratio of 1:1 and protecting from light according to the instructions of chemiluminescence kit; placing the PVDF membrane inside the ChemiDocXRS (Bio-Rad) instrument, adding the mixed luminescence reagent dropwise to the PVDF membrane evenly, and allowing the PVDF membrane to react for 2 minutes at room temperature in the dark. The gel imager automatically exposed to the picture in a signal accumulation mode.
Figs. 5 to 10 show the results of the above antioxidant enzyme activity assay and reactive oxygen species generation experiments. The Auricularia heimuer oligosaccharide at a concentration of 0.25-1.0 mg/mL inhibited the death of oxidatively damaged hepatocytes and increased the antioxidant enzyme activity of oxidatively damaged hepatocytes by regulating the PI3k-akt cell signaling pathway, which had a protective effect on oxidative damage of hepatocytes.
This concludes the description of the embodiments of the present invention.
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