AU2021100482A4 - An Pleurotus ostreatus Galactomannan and Its Preparation Method and Application - Google Patents
An Pleurotus ostreatus Galactomannan and Its Preparation Method and Application Download PDFInfo
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- 235000007685 Pleurotus columbinus Nutrition 0.000 title claims abstract description 93
- 240000001462 Pleurotus ostreatus Species 0.000 title claims abstract description 93
- 235000001603 Pleurotus ostreatus Nutrition 0.000 title claims abstract description 93
- OMDQUFIYNPYJFM-XKDAHURESA-N (2r,3r,4s,5r,6s)-2-(hydroxymethyl)-6-[[(2r,3s,4r,5s,6r)-4,5,6-trihydroxy-3-[(2s,3s,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]methoxy]oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@H](O)[C@H](O)O1 OMDQUFIYNPYJFM-XKDAHURESA-N 0.000 title claims abstract description 56
- 229920000926 Galactomannan Polymers 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 150000004676 glycans Chemical class 0.000 claims abstract description 41
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 41
- 239000005017 polysaccharide Substances 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 13
- 235000013402 health food Nutrition 0.000 claims abstract description 12
- 238000001125 extrusion Methods 0.000 claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 210000003098 myoblast Anatomy 0.000 claims abstract description 10
- 238000010828 elution Methods 0.000 claims abstract description 9
- 230000004792 oxidative damage Effects 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 4
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 4
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 42
- 239000000243 solution Substances 0.000 claims description 36
- 239000000843 powder Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 19
- 230000000694 effects Effects 0.000 claims description 18
- 239000011259 mixed solution Substances 0.000 claims description 17
- 239000002244 precipitate Substances 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 12
- OAABHEHWRQAHEJ-UHFFFAOYSA-N butan-1-ol;chloroform Chemical compound ClC(Cl)Cl.CCCCO OAABHEHWRQAHEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000008213 purified water Substances 0.000 claims description 10
- 239000011550 stock solution Substances 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 239000003963 antioxidant agent Substances 0.000 claims description 8
- 230000003078 antioxidant effect Effects 0.000 claims description 8
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 7
- 230000009089 cytolysis Effects 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 210000003494 hepatocyte Anatomy 0.000 claims description 6
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims description 5
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 5
- 229930091371 Fructose Natural products 0.000 claims description 5
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 5
- 239000005715 Fructose Substances 0.000 claims description 5
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-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
- 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 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
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 5
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-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
- 210000002421 cell wall Anatomy 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 229930182830 galactose Natural products 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000011068 loading method Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 230000003647 oxidation Effects 0.000 claims description 4
- 238000007254 oxidation reaction Methods 0.000 claims description 4
- 230000001590 oxidative effect Effects 0.000 claims description 4
- 208000027418 Wounds and injury Diseases 0.000 claims description 3
- 230000006378 damage Effects 0.000 claims description 3
- 208000014674 injury Diseases 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 238000004192 high performance gel permeation chromatography Methods 0.000 claims description 2
- 238000002329 infrared spectrum Methods 0.000 claims description 2
- 210000005229 liver cell Anatomy 0.000 claims description 2
- 150000002772 monosaccharides Chemical class 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims 2
- 229910021641 deionized water Inorganic materials 0.000 claims 2
- 238000004440 column chromatography Methods 0.000 claims 1
- 239000003826 tablet Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 10
- 241000222350 Pleurotus Species 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 241000233866 Fungi Species 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 230000005714 functional activity Effects 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 238000004587 chromatography analysis Methods 0.000 description 7
- 238000005457 optimization Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 3
- 101500000959 Bacillus anthracis Protective antigen PA-20 Proteins 0.000 description 3
- 238000004566 IR spectroscopy Methods 0.000 description 3
- 101150025129 POP1 gene Proteins 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- WQZGKKKJIJFFOK-UHFFFAOYSA-N hexopyranose Chemical compound OCC1OC(O)C(O)C(O)C1O WQZGKKKJIJFFOK-UHFFFAOYSA-N 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- NNZXDXMEXBYSRF-UHFFFAOYSA-N 2-methyl-4h-pyrazol-3-one Chemical compound CN1N=CCC1=O NNZXDXMEXBYSRF-UHFFFAOYSA-N 0.000 description 2
- 229920001491 Lentinan Polymers 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229940115286 lentinan Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- 238000003809 water extraction Methods 0.000 description 2
- 241001633942 Dais Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000002443 hepatoprotective effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000000874 microwave-assisted extraction Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 231100000862 numbness Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- 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
- 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
-
- 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
-
- 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
-
- 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
- C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Mycology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Animal Behavior & Ethology (AREA)
- Materials Engineering (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
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- Sustainable Development (AREA)
- Emergency Medicine (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to an Pleurotus ostreatus galactomannan its preparation method
and application, belonging to the field of preparation of effective components of edible
fungi. The Pleurotus ostreatus galactomannan is obtained by extracting dried fruiting body
of Pleurotus ostreatus bylysis method at normal temperature, and by combining extrusion
separation, protein removal and ion exchange chromatography. In this preparation method,
the extraction time is shortened, the reagent consumption is reduced, the energy is saved,
and the polysaccharide yield is improved. The extrusion filtration technology is suitable
for the separation of viscous solution. The obtained Pleurotus ostreatus galactomannan has
the functional activity of protecting myoblast from oxidative damage, which can be used
to prepare auxiliary medicines and health foods for protecting myoblast from oxidative
damage.
Allf PONlI
-NaCI 0.30
2.5
0.25
Ligh PO6
Value (j
(0.0
0.00
0 5 it0 15 20 25 30 35
Tube numbers
Figure 1.
The elution curve of Pleurotus ostreatusgalactomannan DEAL 52
Description
Allf PONlI -NaCI 0.30 2.5 0.25
Ligh PO6
Value (j
(0.0
0.00 0 5 it0 15 20 25 30 35
Tube numbers
Figure 1.
The elution curve of Pleurotusostreatusgalactomannan DEAL 52
PATENTS ACT 1990
An Pleurotus ostreatus Galactomannan and Its Preparation Method and Application
The invention is described in the following statement:-
An Pleurotus ostreatus Galactomannan and Its Preparation Method and Application
The invention relates to a Pleurotus ostreatus galactomannan and its preparation
method and application, belonging to the field of natural products.
Pleurotusostreatus is one of the top three edible fungi in China. Pleurotusostreatus
is rich in protein, polysaccharide, vitamins, trace elements and other nutrients. Traditional
Chinese medicine believes that it has the effect of treating lumbocrural pain, numbness of
limbs and relaxing muscles and activating collaterals. Polysaccharide is the main active
component of Pleurotus ostreatus. Limited by the preparation of Pleurotus ostreatus
polysaccharide and the related technology development of drugs or health food and other
products is not mature, especially the research on the efficacy of Pleurotus ostreatus
polysaccharide, such as the hepatoprotective activity and immunomodulatory activity of
Pleurotus ostreatus polysaccharide is still in the laboratory stage, so there is a lack of
related products on the market.
In recent years, the optimization of extraction conditions, structural analysis and
pharmacological activity screening of Pleurotus ostreatus polysaccharides have been
studied. At present, the extraction methods of Pleurotus ostreatus polysaccharide mainly
include hot water extraction, enzymatic hydrolysis, ultrasonic-assisted extraction,
microwave-assisted extraction and so on. Hot water extraction is the most commonly used
method to extract polysaccharide, which is simple, but it has some shortcomings, such as long extraction time, large amount of solvent used, and the structure of polysaccharides is easy to be destroyed under high temperature. Enzymatic hydrolysis has strong specificity, which can only hydrolyze specific glycosidic bonds, and the cost of enzyme preparation is high (Yong Yang, Shou Kunxiu, Li Lilang, et al. Optimization of extraction process of
Pleurotusostreatuspolysaccharideby compound enzyme method [J]. journal of mountain
agriculture and biology, 2019,38 (1): 29-34); Moreover, ultrasonic-assisted extraction is
noisy (Deng Bin. Optimization of extraction technology of Pleurotus ostreatus
polysaccharide by ultrasonic combined with enzymatic method [J]. Journal of
HeilongjiangBayi Agricultural University, 2019,31 (5): 66-72); In addition, microwave
assisted extraction is limited by equipment, and the extraction amount is small (ZhangXiao,
Zhu Caiping, Hong Deng, et al. Optimization of ultrasonic-assistedenzymatic extraction
of Pleurotus ostreatus polysaccharide [J]. Food and Machinery, 2016,32(9):166-171).
Therefore, none of the above-mentioned existing technologies can meet a large number of
demands for industrial development and production of such products.
The invention aims to provide an Pleurotusostreatus galactomannan its preparation
method and application aiming at the deficiency of existing research and technology.
The Pleurotus ostreatus galactomannan is extracted form Pleurotus ostreatus, an
alpha-pyranoglycan composed of fucose, arabinose, galactose, glucose and fructose, with
a molecular weight range of 2.75 x 10' Da to 8.70 x 10' Da.
The Pleurotus ostreatus galactomannan is obtained by extracting dried fruiting body of Pleurotusostreatus by lysis method at normal temperature, and by combining extrusion separation, protein removal and ion exchange chromatography.
And the preparation method comprises the following steps:
A. Crushing the dried fruiting bodies of Pleurotusostreatus, which is sieved with a
-mesh sieve to form dried fruiting body powder of Pleurotusostreatus, putting the dried
fruiting body powder of Pleurotusostreatusinto a cellwall lysis machine, adding deionized
water according to the ratio of material to liquid of 1: 10-1: 80, running the machine for 2
min, squeezing and filtering the mushroom to obtain a stock solution. Then concentrating
and extracting the stock solution at 70-90°C, while the volume of the concentrated solution
is 1 / 3-1 / 4 of the original solution. After cooling it to room temperature, anhydrous
ethanol of 3-4 times the volume of the concentrated solution is added to form mixture 1.
After standing the mixture at 4 °C for 12 h, the mixture 1 is centrifuged at 5000-10000 r
/ min for 5-10 min. Collecting the precipitate in the lower layer, evaporating residual ethanol,
and freeze-drying the precipitate, wherein the dried powder is the crude polysaccharide of
Pleurotusostreatus fruiting body.
B. Adding purified water to prepare a crude polysaccharide solution of Pleurotus
ostreatus fruiting body with the concentration of 5mg/mL, adding chloroform-n-butanol
mixed solution, magnetically stirring, centrifugally layering, collecting the upper layer
solution, repeatedly adding chloroform-n-butanol mixed solution once, and performing
centrifuging. Taking supernatant, which is concentrated under reduced pressure to 1/3-1/4
of the original volume, adding 3-4 times of absolute ethanol, which is stood at 4°C for 12
hours to form mixed solution 2. Centrifuging the mixed solution 2 at 5,000-10,000 r/min for 5-10 min, collecting the lower layer precipitate, volatilizing the residual ethanol, and freeze-drying, wherein the dried powder is Pleurotus ostreatus fruiting body polysaccharide.
C. Dissolving polysaccharide of Pleurotus ostreatus fruiting body with deionized
water, fixing the volume, loading the polysaccharide to DEAE 52 cellulose column
chromatography, which is eluted with purified water and 0.1mol/L NaCl solution in turn.
The flow rate is 5ml / 3min, and 5ml is collected in each tube. The sugar content in each
tube is determined by phenol sulfuric acid method. According to the sugar content in each
tube, collecting the elution peak of 0.1mol/1 NaCl solution, and freeze-drying the mixture
to prepare Pleurotusostreatus galactomannan.
The extrusion filter described in step A is a single screw extruder. The diameter of the
die hole of the single screw extruder is 8-16mm, the temperature of the extruder sleeve is
-90 °C, and the screw speed is 160-240r / min.
The application of Pleurotus ostreatus galactomannan in the preparation of health
food and medicine mentioned above.
A health food and pharmaceutical composition which can be applied for protecting
myoblast injury that the health food and pharmaceutical composition contains the
Pleurotusostreatus galactomannan component.
The dosage form of the health food and pharmaceutical composition can be tablet,
granule, capsule or solution.
Beneficial effects:
This is the first time that the extraction and separation method of wall breaking at
room temperature combined with extrusion filtration has been adopted in the preparation
process of Pleurotusostreatus galactomannan, which shortens the extraction time, reduces
the reagent consumption, saves energy and improves the polysaccharide yield, and the
extrusion filtration method is suitable for the separation of viscous solution. Meanwhile,
the galactomannan from Pleurotus ostreatus has the functional activity of protecting
myoblasts from oxidative damage, which can be used for preparing auxiliary medicines
and health foods for protecting muscle cells, as well as providing basic data for the
development of deep processing products of Pleurotus ostreatus with important economic
and market values.
The present invention will be further explained with reference to the accompanying
drawings and specific embodiments.
Fig. 1 is the elution curve of Pleurotus ostreatus galactomannan DEAE 52.
Fig. 2 is the high performance gel permeation chromatography of galactomannan from
Pleurotusostreatus.
Fig. 3 is the infrared spectrum of galactomannan from Pleurotusostreatus.
Fig. 4 is the analysis of monosaccharide composition of galactomannan in Pleurotus
ostreatus.
Fig. 5 shows the effect of galactomannan from Pleurotus ostreatus on the activity of
antioxidant enzyme SOD in liver cells damaged by oxidation.
Fig. 6 shows the effect of Pleurotus ostreatus galactomannan on the activity of
antioxidant enzyme GSH-Px in oxidative damaged hepatocytes.
Fig. 7 shows the effect of galactomannan from Pleurotus ostreatus on the amount of
MDA produced by oxidative damage of hepatocytes.
Fig. 8 shows the effect of Pleurotusostreatus galactomannan on ROS production of
oxidative damaged hepatocytes.
The following will further explain the present invention with specific embodiments,
but the claimed scope of the present invention is not limited to the following embodiments.
Example 1
1. Crushing the dried fruiting bodies of Pleurotus ostreatus, which is sieved with a
-mesh sieve to form dried fruiting body powder of Pleurotusostreatus, putting the dried
fruiting body powder of Pleurotusostreatusinto a cellwall lysis machine, adding deionized
water according to the ratio of material to liquid of 1: 40, running the machine for 10min,
squeezing and filtering the mushroom to obtain a stock solution. The extrusion filtration
adopts a single screw extruder with a die hole diameter of16mm, an extruder sleeve
temperature of 58°C and a screw rotation speed of 220r/min. Then concentrating and
extracting the stock solution at 70°C, while the volume of the concentrated solution is 1 /
3 of the original solution. After cooling it to room temperature, anhydrous ethanol of 3
times the volume of the concentrated solution is added to form mixture 1. After standing
the mixture at 4 °C for 12 h, the mixture is centrifuged at 10000 r / min for 5 min. Collecting the precipitate in the lower layer, evaporating residual ethanol, and freeze-drying the precipitate, wherein the dried powder is the crude polysaccharide of Pleurotus ostreatus fruiting body.
2. Adding purified water to prepare a crude polysaccharide solution of Pleurotus
ostreatus fruiting body with the concentration of 5mg/mL, adding chloroform-n-butanol
mixed solution(volume ratio 4:1), magnetically stirring, centrifugally layering, collecting
the upper layer solution, repeatedly adding chloroform-n-butanol mixed solution
once(volume ratio 4:1), and performing centrifuging. Taking supernatant, which is
concentrated under reduced pressure to 1/3 of the original volume, adding 3 times of
absolute ethanol, which is stood at 4°C for 12 hours. Centrifuging the mixed solution at
,000 r/min for 5 min, collecting the lower layer precipitate, volatilizing the residual
ethanol, and freeze-drying, wherein the dried powder is Pleurotus ostreatus fruiting body
polysaccharide.
3. Dissolving polysaccharide of Pleurotus ostreatus fruiting body with deionized
water, fixing the volume, loading the polysaccharide to DEAE 52 cellulose column
chromatography (4.0x30cm), which is eluted with purified water and 0.1mol/L NaCl
solution in turn. The flow rate is 5ml / 3min, and 5ml is collected in each tube. The sugar
content of each tube is determined by phenol-sulfuric acid method, and the elution peaks
are collected according to the sugar content of each tube, and the polysaccharide
components POP-1, POP-2 and POP-3 are obtained. The results are shown in Figure 1. Gel
permeation chromatography (TSK-gel G-3000PWXL column (7.8x300mm)) is carried out
on POP-2. chromatographic conditions: Dionex TM CarboPac TM PA20 column. Mobile
phase: A phase:ddH20; B phase: 200mMNaOH; C phase: 200mMNaOH/500mMNaAC.
The flow rate is 0.5mL/min. The molecular weight is calculated as gMw =-0.228t+7.831,
and the galactomannan of with an average molecular weight of 3.98x1O Dais obtained.
The results are shown in Fig. 2. The galactomannan of Pleurotus ostreatus is detected as
a-pyranose by infrared spectroscopy, and the results are shown in Figure 3. 2mg of
galactomannan from Pleurotusostreatus is hydrolyzed by methanol containing IM of HCl.
After hydrolyzed by 2MTFA, it is derivatized by1-phenyl-3-methyl-5-pyrazolone (PMP),
separated by Compass C18 column(250x4.6mm), analyzed by HPLC, and detected by
UV245nm. The results are shown in Figure 4. The results showed that galactomannan from
Pleurotusostreatus was composed of fucose, arabinose, galactose, glucose and fructose.
Example 2
1. Crushing the dried fruiting bodies of Pleurotus ostreatus, which is sieved with a
-mesh sieve to form dried fruiting body powder of Pleurotusostreatus, putting the dried
fruiting body powder of Pleurotusostreatusinto a cellwall lysis machine, adding deionized
water according to the ratio of material to liquid of 1: 10, running the machine for 20min,
squeezing and filtering the mushroom to obtain a stock solution. The extrusion filtration
adopts a single screw extruder with a die hole diameter of 8mm, an extruder sleeve
temperature of 90°C and a screw rotation speed of 240r/min. Then concentrating and
extracting the stock solution at 90°C, while the volume of the concentrated solution is 1 /
4 of the original solution. After cooling it to room temperature, anhydrous ethanol of 4
times the volume of the concentrated solution is added to form mixture. After standing the
mixture at 4 °C for 12 h, the mixture is centrifuged at 5000 r / min for 5 min. Collecting
the precipitate in the lower layer, evaporating residual ethanol, and freeze-drying the
precipitate, wherein the dried powder is the crude polysaccharide of Pleurotus ostreatus fruiting body.
2. Adding purified water to prepare a crude polysaccharide solution of Pleurotus
ostreatus fruiting body with the concentration of 5mg/mL, adding chloroform-n-butanol
mixed solution(volume ratio 4:1), magnetically stirring, centrifugally layering, collecting
the upper layer solution, repeatedly adding chloroform-n-butanol mixed solution
once(volume ratio 4:1), and performing centrifuging. Taking supernatant, which is
concentrated under reduced pressure to 1/3 of the original volume, adding 3 times of
absolute ethanol, which is stood at 4°C for 12 hours. Centrifuging the mixed solution at
,000 r/min for 5 min, collecting the lower layer precipitate, volatilizing the residual
ethanol, and freeze-drying, wherein the dried powder is Pleurotus ostreatus fruiting body
polysaccharide.
3. Dissolving polysaccharide of Pleurotus ostreatus fruiting body with deionized
water, fixing the volume, loading the polysaccharide to DEAE 52 cellulose column
chromatography (4.0x30cm), which is eluted with purified water and 0.1mol/L NaCl
solution in turn. The flow rate is 5ml / 3min, and 5ml is collected in each tube. The sugar
content of each tube is determined by phenol-sulfuric acid method, and the elution peaks
are collected according to the sugar content of each tube, and the polysaccharide
components POP-1, POP-2 and POP-3 are obtained. The results are shown in Figure 1. Gel
permeation chromatography (TSK-gel G-3000PWXL column (7.8x300mm)) is carried out
on POP-2. chromatographic conditions: Dionex TM CarboPac TM PA20 column. Mobile
phase: A phase: ddH20; B phase: 200mMNaOH; C phase: 200mMNaOH/500mMNaAC.
The flow rate is 0.5mL/min. The molecular weight is calculated as gMw =-0.228t+7.831,
and the galactomannan of Pleurotus ostreatus with an average molecular weight of
2.75x10' Da is obtained. The results are shown in Fig. 2. The galactomannan of Pleurotus
ostreatus is detected as a-pyranose by infrared spectroscopy, and the results are shown in
Figure 3. 2mg of galactomannan from Pleurotus ostreatus is hydrolyzed by methanol
containing IM of HCl. After hydrolyzed by 2MTFA, it is derivatized by 1-phenyl-3
methyl-5-pyrazolone (PMP), separated by Compass C18 column(250x4.6mm), analyzed
by HPLC, and detected by UV245nm. The results are shown in Figure 4. The results
showed that galactomannan from Pleurotus ostreatus is composed of fucose, arabinose,
galactose, glucose and fructose.
Example 3
1. Crushing the dried fruiting bodies of Pleurotus ostreatus, which is sieved with a
-mesh sieve to form dried fruiting body powder of Pleurotusostreatus, putting the dried
fruiting body powder of Pleurotusostreatusintoa cellwall lysis machine, adding deionized
water according to the ratio of material to liquid of 1: 80, running the machine for 2min,
squeezing and filtering the mushroom to obtain a stock solution. The extrusion filtration
adopts a single screw extruder with a die hole diameter of16mm, an extruder sleeve
temperature of 40°C and a screw rotation speed of 160r/min. Then concentrating and
extracting the stock solution at 80°C, while the volume of the concentrated solution is 1 /
3 of the original solution. After cooling it to room temperature, anhydrous ethanol of 3
times the volume of the concentrated solution is added to form mixture. After standing the
mixture at 4 °C for 12 h, the mixture is centrifuged at 7000 r / min for 8 min. Collecting
the precipitate in the lower layer, evaporating residual ethanol, and freeze-drying the
precipitate, wherein the dried powder is the crude polysaccharide of Pleurotus ostreatus
fruiting body.
2. Adding purified water to prepare a crude polysaccharide solution of Pleurotus
ostreatus fruiting body with the concentration of 5mg/mL, adding chloroform-n-butanol
mixed solution(volume ratio 4:1), magnetically stirring, centrifugally layering, collecting
the upper layer solution, repeatedly adding chloroform-n-butanol mixed solution
once(volume ratio 4:1), and performing centrifuging. Taking supernatant, which is
concentrated under reduced pressure to 1/3 of the original volume, adding 3 times of
absolute ethanol, which is stood at 4°C for 12 hours. Centrifuging the mixed solution at
7,000 r/min for 8 min, collecting the lower layer precipitate, volatilizing the residual
ethanol, and freeze-drying, wherein the dried powder is Pleurotus ostreatus fruiting body
polysaccharide.
3. Dissolving polysaccharide of Pleurotus ostreatus fruiting body with deionized
water, fixing the volume, loading the polysaccharide to DEAE 52 cellulose column
chromatography (7.8x300mm), which is eluted with purified water and 0.1mol/L NaCl
solution in turn. The flow rate is 5ml / 3min, and 5ml is collected in each tube. The sugar
content of each tube is determined by phenol-sulfuric acid method, and the elution peaks
are collected according to the sugar content of each tube, and the polysaccharide
components POP-1, POP-2 and POP-3 are obtained. The results are shown in Figure 1. Gel
permeation chromatography (TSK-gel G-3000PWXL column (7.8x300mm)) is carried out
on POP-2. chromatographic conditions: Dionex TM CarboPac TM PA20 column. Mobile
phase: A phase: ddH20; B phase: 200mMNaOH; C phase: 200mMNaOH/500mMNaAC.
The flow rate is 0.5mL/min. The molecular weight is calculated as gMw =-0.228t+7.831,
and the galactomannan of Pleurotus ostreatus with an average molecular weight of
8.70x10' Da is obtained. The results are shown in Fig. 2. The galactomannan of Pleurotus ostreatus is detected as a-pyranose by infrared spectroscopy, and the results are shown in
Figure 3. 2mg of galactomannan from Pleurotus ostreatus is hydrolyzed by methanol
containing IM of HCl. After hydrolyzed by 2MTFA, it is derivatized by 1-phenyl-3
methyl-5-pyrazolone (PMP), separated by Compass C18 column(250x4.6mm), analyzed
by HPLC, and detected by UV245nm. The results are shown in Figure 4. The results
showed that galactomannan from Pleurotus ostreatus is composed of fucose, arabinose,
galactose, glucose and fructose.
The following tests prove the application of Pleurotusostreatusgalactomannan which
can protect myoblasts from oxidative damage in medicines and health foods:
Test example 1:
The effect of galactomannan from Pleurotusostreatuson antioxidant enzyme activity
of myoblasts damaged by oxidation is detected by antioxidant kit. L6 cells in logarithmic
growth phase are inoculated into 96-well plates at a rate of 6x104 cells /mL, with 100L
per well. Grouping: 0 control group; © Positive group (lentinan 1mg/mL); Pleurotus
ostreatus galactomannan (2mg/mL, 1.5mg/mL, lmg/mL, 0.5mg/mL, 0.25mg/mL,
0.125mg/mL) groups, with 6 replicates in each group. After the cells grow for 24 hours,
they adhered well, so H202 (400M) is added and cultured for 4 hours. Collect culture
solution and resuspend cells, and operate according to the instructions of SOD, GSH-Px
and MDA kits, with 6 replicates in each group.
Test example 2:
The effect of galactomannan from Pleurotus ostreatus on the production of reactive
oxygen species (ROS) in myoblasts damaged by oxidation is detected by DCFH-DA fluorescent probe. L6 cells in logarithmic growth phase are inoculated into 96-well plates at a rate of 6x104 cells /mL, with 100OL per well. Grouping: 0 control group; © Positive group (lentinan lmg/mL); Pleurotus ostreatus galactomannan (2mg/mL, 1.5mg/mL, lmg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL) groups, with 6 replicates in each group.
After the cells grow for 24 hours, they adhered well, so H202 (400M) is added and
cultured for 4 hours. Serum-free DMEM medium containing 10 LDCFH-DA fluorescent
probe is added to each well, incubated in an incubator at 37°C for 30min, slowly washed
with PBS for three times, and placed in a multifunctional fluorescence microplate reader
to detect the fluorescence intensity and draw a bar chart (excitation wavelength is 488nm,
emission wavelength is 525nm).
As shown in Figure 5, Figure 6, Figure 7 and Figure 8, the results of the above
mentioned antioxidant enzyme activity detection and active oxygen generation
experiments show that when the concentration of galactomannan in Pleurotusostreatus is
0.25 ~ 1.5 mg/mL, it can enhance the antioxidant enzyme activity, reduce the active oxygen
generation, reduce the level of oxidative damage of cells, and protect the oxidative damage
of myoblasts.
This concludes the description of embodiments of the present invention.
Claims (7)
1. A Pleurotus ostreatus galactomannan is characterized in that it is extracted form
Pleurotus ostreatus, an alpha-pyranoglycan composed of fucose, arabinose, galactose,
glucose and fructose, with a molecular weight range of 2.75 x 105 Da to 8.70 x 105 Da.
2. The Pleurotus ostreatus galactomannan according to claim 1 is characterized in
that the Pleurotusostreatus galactomannan is obtained by extracting dried fruiting body of
Pleurotus ostreatus by lysis method at normal temperature, and by combining extrusion
separation, protein removal and ion exchange chromatography.
3. The preparation method of Pleurotusostreatus galactomannan according to claim
2 is characterized in the following steps:
A. Crushing the dried fruiting bodies of Pleurotusostreatus, which is sieved with a
-mesh sieve to form dried fruiting body powder of Pleurotusostreatus, putting the dried
fruiting body powder of Pleurotusostreatusinto a cellwall lysis machine, adding deionized
water according to the ratio of material to liquid of 1: 10-1: 80, running the machine for 2
min, squeezing and filtering the mushroom to obtain a stock solution. Then concentrating
and extracting the stock solution at 70-90°C, while the volume of the concentrated solution
is 1 / 3-1 / 4 of the original solution. After cooling it to room temperature, anhydrous
ethanol of 3-4 times the volume of the concentrated solution is added to form mixture 1.
After standing the mixture at 4 °C for 12 h, the mixture 1 is centrifuged at 5000-10000 r /
min for 5-10 min. Collecting the precipitate in the lower layer, evaporating residual ethanol,
and freeze-drying the precipitate, wherein the dried powder is the crude polysaccharide of
Pleurotusostreatus fruiting body.
B. Adding purified water to prepare a crude polysaccharide solution of Pleurotus
ostreatus fruiting body with the concentration of 5mg/mL, adding chloroform-n-butanol
mixed solution, magnetically stirring, centrifugally layering, collecting the upper layer
solution, repeatedly adding chloroform-n-butanol mixed solution once, and performing
centrifuging. Taking supernatant, which is concentrated under reduced pressure to 1/3-1/4
of the original volume, adding 3-4 times of absolute ethanol, which is stood at 4°C for 12
hours to form mixed solution 2. Centrifuging the mixed solution 2 at 5,000-10,000 r/min
for 5-10 min, collecting the lower layer precipitate, volatilizing the residual ethanol, and
freeze-drying, wherein the dried powder is Pleurotus ostreatus fruiting body
polysaccharide.
C. Dissolving polysaccharide of Pleurotus ostreatus fruiting body with deionized
water, fixing the volume, loading the polysaccharide to DEAE 52 cellulose column
chromatography, which is eluted with purified water and 0.1mol/L NaCl solution in turn.
The flow rate is 5ml / 3min, and 5ml is collected in each tube. The sugar content in each
tube is determined by phenol sulfuric acid method. According to the sugar content in each
tube, collecting the elution peak of 0.1mol/1 NaCl solution, and freeze-drying the mixture
to prepare Pleurotusostreatus galactomannan.
4. The preparation method of Pleurotus ostreatus galactomannan according to claim
3 is characterized in that the extrusion filter described in step A is a single screw extruder.
The diameter of the die hole of the single screw extruder is 8-16mm, the temperature of the
extruder sleeve is 40-90 °C, and the screw speed is 160-240r / min.
5. The application of Pleurotusostreatus galactomannan in the preparation of health food and medicine according to claim 1 to claim 3.
6. A health food and pharmaceutical composition which can be applied for protecting
myoblast injury is characterized in that the health food and pharmaceutical composition
contains the Pleurotusostreatus galactomannan component as claimed in claim 1 to claim
3.
7. The health food and pharmaceutical composition for protecting myoblast injury
according to claim 6, which is characterized in that the dosage form of the health food and
pharmaceutical composition can be tablet, granule, capsule or solution.
-1/8- 2021100482
Figure 1.
The elution curve of Pleurotus ostreatus galactomannan DEAE 52
-2/8- 2021100482
Figure 2.
The high performance gel permeation chromatography of galactomannan from
Pleurotus ostreatus
-3/8- 2021100482
Figure 3.
The infrared spectrum of galactomannan from Pleurotus ostreatus
-4/8- 2021100482
Figure 4.
The analysis of monosaccharide composition of galactomannan in Pleurotus
ostreatus
-5/8- 2021100482
Figure 5.
The effect of galactomannan from Pleurotus ostreatus on the activity of antioxidant
enzyme SOD in liver cells damaged by oxidation
-6/8- 2021100482
Figure 6.
The effect of Pleurotus ostreatus galactomannan on the activity of antioxidant
enzyme GSH-Px in oxidative damaged hepatocytes
-7/8- 2021100482
Figure 7.
The effect of galactomannan from Pleurotus ostreatus on the amount of MDA
produced by oxidative damage of hepatocytes
-8/8- 2021100482
Figure 8.
The effect of Pleurotus ostreatus galactomannan on ROS production of oxidative
damaged hepatocytes
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