CN104888186A - Application of auricularia polytricha protein extract in preparation of product for relieving alcoholic hepatocyte damage - Google Patents
Application of auricularia polytricha protein extract in preparation of product for relieving alcoholic hepatocyte damage Download PDFInfo
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Abstract
The invention discloses an application of auricularia polytricha protein extract in preparation of a product for relieving the alcoholic hepatocyte damage. The auricularia polytricha protein extract prepared with a method does not have the cytotoxic effect on L-O2 cells, can increase the survival rate of alcoholic damaged L-O2 cells, has the liver protection effect, and can be used for relieving the alcoholic hepatocyte damage. When the concentration of protein in the auricularia polytricha protein extract in a culture solution is 30 mu g/mL, the survival rate of the L-O2 cells in a treatment group is the highest, can be up to 87.86% and is higher than that of cells in a damage model group by 42.85%, and the survival rate of the cells in the damage model group is 45.01%. The auricularia polytricha protein extract can effectively reduce the content of TG (triglyceride) and ROS (reactive oxygen species) in the cells and can form statistic significant difference (P is smaller than 0.05) with the model group. Meanwhile, the auricularia polytricha protein extract can reduce cell damage, reduce release of endoenzyme ALT (alanine aminotransferase) and AST (aspartate aminotransferase) and protect cell integrity, and has the liver protection effect.
Description
Technical field
The present invention relates to Auricularia polytricha protein extract and prepare the application in alleviation of alcohol hepatocyte injury product.
Background technology
Liver is an organ based on metabolic function in health, to ex vivo with external many non-nutritive substances as some metabolite in various medicine, poisonous substance and body, there is biotransformation, by metabolism, they thoroughly decomposed or excrete with original shape.Enter ethanol 90% in body at liver metabolism, ethanol has direct detrimental effect to liver, and its micromechanism of damage is caused at hepatocyte intracellular metabolite by ethanol.If long-term excessive drinking, can make hepatocyte that steatosis, necrosis occur repeatedly, finally develop into alcoholic hepatitis and alcoholic cirrhosis, severe patient threat to life safety.For prevention and therapy alcoholic liver injury, except alleviating alcohol addiction, also answer polyphagia to be rich in high protein, vitamin and antioxidative food with some, increase the protection to liver.
Auricularia polytricha (Mout) Sacc., formal name used at school (Auricularia Polytricha), also known as white dorsal body setae Auricularia, belongs to the Auricularia polytricha (Mout) Sacc. kind in Basidiomycota, Hymenomycetes, Auriculariale, Auriculariaceae, Auricularia.Auricularia polytricha (Mout) Sacc. sporophore colloid is sliding tough, and early stage cup-shaped, fades to ear, lobate or irregularly shaped, the upper surface of sporophore layer be purplish grey to black, lower surface long wool hair, Chang Chengqing brown, shallow dark brown are to dark grey color.Analyzing according to Chinese Academy of Sciences's Chengdu biology, in Auricularia polytricha (Mout) Sacc. dry product, amino acid whose total amount is 41.68%, has essential amino acid 7 kinds, accounts for 42.31% of amino acid contained total amount, has 9 kinds, other aminoacid, accounts for 57.69% of amino acid contained total amount.Auricularia polytricha (Mout) Sacc. is a kind of fungus of Medicine Food Homology, has the prospect of potential Application and Development.
Summary of the invention
Technical problem to be solved by this invention is how alleviation of alcohol hepatic injury.
For solving the problems of the technologies described above, the invention provides the application of following A or B:
A, Auricularia polytricha protein extract are preparing the application in alleviation of alcohol hepatocyte injury product;
B, Auricularia polytricha protein extract are preparing the application in alleviation of alcohol hepatic injury product;
Described Auricularia polytricha protein extract is prepared by following method:
1) Auricularia polytricha (Mout) Sacc. sporophore is pulverized rear flooding, collect water-soluble substances and obtain Auricularia polytricha (Mout) Sacc. water solubility extract;
2) ammonium sulfate precipitation that saturation is 80% is carried out to described Auricularia polytricha (Mout) Sacc. water solubility extract, collecting precipitation, after described precipitate with deionized water dialysis, obtain described Auricularia polytricha protein extract.
In above-mentioned application, described flooding to can be at 2-6 DEG C with flooding 10-14 hour, specifically can be at 4 DEG C by flooding 12 hours.Described water can be deionized water.
In above-mentioned application, described Auricularia polytricha (Mout) Sacc. sporophore can be crushed to 50-100 order, as 100 orders.
In above-mentioned application, the mass ratio of described Auricularia polytricha (Mout) Sacc. sporophore and water can be 1:40-60, and as 1:40, the quality of described Auricularia polytricha (Mout) Sacc. sporophore is dry weight.
In above-mentioned application, described Auricularia polytricha (Mout) Sacc. sporophore can be fresh sporophore and also can be dry sporophore.
In above-mentioned application, the sporophore of described drying obtains dry under room temperature (as 20-25 DEG C) for fresh Auricularia polytricha (Mout) Sacc. sporophore.
In above-mentioned application, water-soluble substances described in collected by centrifugation can be adopted.The centrifugal force of described centrifugal employing can be 6000-15000g (as 12000g), and centrifugation time can be 10-20 minute (as 15 minutes).
In above-mentioned application, described saturation is ammonium sulfate the saturation of 4 DEG C.
In above-mentioned application, can adopt described in collected by centrifugation and precipitate; The centrifugal force of described centrifugal employing can be 6000-15000g (as 12000g), and centrifugation time can be 10-20 minute (as 15 minutes).
In above-mentioned application, the molecular cut off of the semipermeable membrane that described dialysis adopts is 3kDa; Described dialysis can be at 2-6 DEG C of dialysis 10-14 hour, specifically can be 4 DEG C of dialysis 12 hours.
Above-mentioned application, also comprise by dialysis after semipermeable membrane in liquid at 7000-14000g (as 8500g), centrifugal 10-20 minute (as 15 minutes), collect supernatant, this supernatant is carried out lyophilization, is prepared into the step of Auricularia polytricha protein extract dry powder.
In above-mentioned application, described alleviation of alcohol hepatocyte injury is following 1)-5) at least one:
1) hepatocellular survival rate is improved;
2) content of triglyceride in hepatocyte is reduced;
3) active o content in hepatocyte is reduced;
4) hepatocyte is reduced to the burst size of glutamate pyruvate transaminase;
5) hepatocyte is reduced to the burst size of glutamic oxaloacetic transaminase, GOT;
Described alleviation of alcohol hepatic injury is following 1)-5) at least one:
1) hepatocellular survival rate is improved;
2) content of triglyceride in hepatocyte is reduced;
3) active o content in hepatocyte is reduced;
4) hepatocyte is reduced to the burst size of glutamate pyruvate transaminase;
5) hepatocyte is reduced to the burst size of glutamic oxaloacetic transaminase, GOT.
Above, described alcoholic liver cell injury can be specifically people's alcoholic liver cell injury, and described alcoholic liver injury can be specifically people's alcoholic liver injury.Described hepatocyte specifically can be L-O2 cell.
Experiment proves, the Auricularia polytricha protein extract adopting method of the present invention to prepare, to L-O2 cell no cytotoxicity, can promote the survival rate of Alcoholic damage L-O2 cell, have the effect protected the liver, can be used for alleviation of alcohol hepatocyte injury.When protein concentration in Auricularia polytricha protein extract in culture fluid is 30 μ g/mL, treatment group L-O2 cell survival rate is the highest, can 87.86% be reached, improve 42.85% than the cell survival rate of damage model group (cell survival rate is 45.01%).Auricularia polytricha protein extract can reduce the content of TG and ROS in cell effectively, enables treatment group and damage model group form statistically significant difference (P<0.05).Meanwhile, Auricularia polytricha protein extract can also reduce L-O2 cell injury, reduces the release of endocellular enzyme ALT and AST, and the integrity of protection L-O2 cell, has the effect protected the liver.The intracellular TG content for the treatment of group (817 ± 33 μm of ol/mg protein) L-O2 reduces 68.90% than damage model group (2627 ± 11 μm of ol/mg protein); The average fluorescent strength (4898 ± 406) of the intracellular ROS for the treatment of group L-O2 reduces 53.56% than damage model group (10548 ± 319); The ALT content (8096 ± 194U/mg protein) for the treatment of group L-O2 cell release reduces 51.06% than damage model group (16544 ± 468U/mg protein); The AST content (6979 ± 221U/mg protein) for the treatment of group L-O2 cell release reduces 53.70% than damage model group (15072 ± 579U/mg protein).
Accompanying drawing explanation
Fig. 1 is the impact of different concentration ethanol on L-02 cytoactive.
Fig. 2 is that the cytotoxicity of Auricularia polytricha protein extract detects.
Fig. 3 is the design sketch of Auricularia polytricha protein extract protection Alcoholic damage L-O2 cell.In figure, damage group is damage model group, and 10,20,30,40,50 are respectively the treatment group that No. 1-5 contains the culture fluid of ethanol and Auricularia polytricha protein extract.
Wherein, the protein concentration in Fig. 2 is the final concentration of protein in culture fluid in Auricularia polytricha protein extract.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
No. 3, Auricularia polytricha (Mout) Sacc. (Auricularia polytricha (Mont.) Sacc.) in following embodiment (Zhao Shuan etc. Auricularia polytricha (Mout) Sacc. mycelium produces the optimization of polysaccharide fermentation condition. food science and technology .2012 the 37th volume the 4th phase) public can obtain this strain from Beijing City Agriculture and Forestry Institute, to repeat the application's experiment.
The product that L-O2 cell strain (human liver cell) in following embodiment is Chinese Academy of Medical Sciences's tumor cell storehouse.
RPMI RPMI-1640 in following embodiment is Invitrogen Products, and catalog number is 11875-093.
BCA protein quantification test kit in following embodiment is Beijing Bo Maide Bioisystech Co., Ltd product, and catalog number is PP0103.
It is Beijing Puli's lema gene Technology Co., Ltd. product that tissue triglycerides in following embodiment measures test kit, and catalog number is E1013.
In tissue in following embodiment, quantitation active oxygen test kit is Beijing Puli's lema gene Technology Co., Ltd. product, and catalog number is C1300.
In serum in following embodiment, glutamate-pyruvate transaminase determination reagent kit is that Science and Technology Ltd.'s product is built up in Nanjing, and catalog number is C009-2.
In serum in following embodiment, glutamic oxaloacetic transaminase, GOT measures test kit is that Science and Technology Ltd.'s product is built up in Nanjing, and catalog number is C010-2.
The preparation of embodiment 1, Auricularia polytricha protein extract
One, Auricularia polytricha (Mout) Sacc. sporophore is prepared
Auricularia polytricha (Mout) Sacc. (Auricularia polytricha (Mont.) Sacc.) No. 3 slant strains are inoculated in one-level kind culture medium and activate, 25 DEG C of constant temperature culture, covering with after test tube until mycelia is inoculated in second-generation culture medivm, 25 DEG C of constant temperature culture rooms are cultured to mycelia and cover with, carry out under secondary kind being inoculated into the condition of in the cultivating bag that culture medium for cultivating is housed 25 DEG C sending out bacterium, carry out mycelium stimulation after strain covers with cultivating bag and move into warmhouse booth, Cultivation condition keeps humidity more than 90%, temperature is about 20-25 DEG C, collect the first damp sporophore, obtain Auricularia polytricha (Mout) Sacc. (Auricularia polytricha (Mont.) Sacc.) 3 work song entities.
Wherein, the culture medium that this experiment is used is as follows:
One-level kind culture medium: 200g Rhizoma Solani tuber osi, 20g glucose, 20g agar powder, 3g KH
2pO
4, 10mg vitamin B1,5g peptone, 1.5gMgSO
4, 1000mL distilled water, through 121 DEG C, 30min autoclaving.
Second-generation culture medivm: cotton seed hulls 80%, wheat bran 18%, Gypsum Fibrosum 1%, sugar 1%, material-water ratio is 1:1, through 121 DEG C, 30min autoclaving.
Culture medium for cultivating: wood flour 50%, cotton seed hulls 28%, wheat bran 20%, sugar 1%, Gypsum Fibrosum 1%, material-water ratio is 1:1-1.2.Through 121 DEG C, 30min autoclaving.
Two, Auricularia polytricha (Mout) Sacc. water solubility extract is prepared
1, collect Auricularia polytricha (Mout) Sacc. (Auricularia polytricha (Mont.) Sacc.) 3 work song entities, after 20-25 DEG C of dry in the sun obtains dry product, pulverize grinding and make the homogeneous Auricularia polytricha (Mout) Sacc. powder of 100 object;
2, soak the Auricularia polytricha (Mout) Sacc. powder of 1 mass parts at 4 DEG C with the deionized water of 40 mass parts, soak 12 hours, the centrifugal 15min of 12000g, collect supernatant, this supernatant is Auricularia polytricha (Mout) Sacc. water solubility extract.
Three, Auricularia polytricha protein extract is prepared
At 4 DEG C, the ammonium sulfate precipitation (namely adding ammonium sulfate to the saturation of ammonium sulfate in this supernatant is 80%) that saturation is 80% is carried out to the Auricularia polytricha (Mout) Sacc. water solubility extract of step 2, after precipitation 4h, the centrifugal 15min of 12000g, collecting precipitation, dialyses to this precipitation.Wherein, the molecular cut off of the semipermeable membrane that dialysis adopts is 3kDa, and precipitation is 4 DEG C of dialysis 12h in deionized water.By the liquid in the semipermeable membrane after dialysis at the centrifugal 15min of 8500g, collect supernatant, after this supernatant is adopted liquid nitrogen freezing, carry out lyophilization, be prepared into Auricularia polytricha protein extract dry powder.
Embodiment 2, Auricularia polytricha protein extract alleviation of alcohol hepatocyte injury are tested
One, the foundation of human liver cell Alcoholic damage model
1, cell culture: add penicillin, streptomycin mixed liquor and hyclone (FBS) in RPMI RPMI-1640, penicillin and the streptomycin mixed liquor final volume percentage composition in culture fluid is made to be 1%, the final volume degree of FBS in culture fluid is 10%, is namely mixed with the RPMI RPMI-1640 containing 10%FBS.Adopt the trypsin of 0.25% to carry out digesting in the L-O2 cell of exponential phase and use the RPMI1640 culture fluid collection re-suspended cell containing 10%FBS, being inoculated in 96 well culture plates that (every hole 100 μ L, cell density is 8 × 10
3individual/hole), being placed in temperature is 37 DEG C, and relative humidity is 95%, CO
2concentration is cultivate 24h in the incubator of 5%.
2, medicine ordinance: add dehydrated alcohol to containing in the RPMI1640 culture fluid of 10%FBS, be configured to the culture fluid that ethanol contend percentage composition is respectively 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.5% and 3%.
3, drug incubation: after cell culture terminates, absorb the cell culture fluid in culture hole, add the culture fluid (experimental group) of the different volumes percentage composition ethanol in 200 μ L steps 2 respectively, matched group adds isopyknic RPMI1640 culture fluid (volumn concentration of ethanol is 0) containing 10%FBS, being placed in temperature is 37 DEG C, relative humidity is 95%, CO
2concentration is that after continuing to cultivate 48h in the incubator of 5%, drug incubation terminates.The multiple hole of Setup Experiments three.
4, MTT detects: after drug incubation terminates, absorbed by the culture fluid containing medicine, every hole adds 200 μ L MTT diluents (MTT concentration is 1mg/mL), and be placed in 37 DEG C, relative humidity is 95%, CO
2concentration is hatch 4h in the incubator of 5%.After hatching end, absorb the liquid in every hole, every hole adds 200 μ L DMSO solution, and mix homogeneously, measures the OD in every hole in the long microplate reader of fluorescence all-wave
562value.
Cell survival rate formula: the OD of cell survival rate (%)=experimental group
562the OD of value/matched group
562value × 100%.
The cell survival rate of matched group is set as 100%, the ethanol of different volumes percentage composition for the impact of L-O2 cytoactive in table 1, volumn concentration be 1% and 1.2% ethanol on cytoactive impact less, cell survival rate is respectively 96.15% and 94.62%.Along with the increase of ethanol contend percentage composition, L-O2 cell survival rate obviously declines (table 1 and Fig. 1), when ethanol contend percentage composition is 3%, cell survival rate is reduced to 45.38%, basis of microscopic observation finds that cell floats in a large number, as the hepatocellular experimental concentration of damage when being 3% using ethanol contend percentage composition, be that L-O2 cell after the culture fluid effect of 3% is as human liver cell Alcoholic damage model using ethanol contend percentage composition.
The ethanol of table 1, different volumes percentage composition is on the impact of L-02 cytoactive
Two, the experiment of alleviation of alcohol human liver cell damage
1, the preparation of alleviation of alcohol human liver cell damage medicine
Dissolve Auricularia polytricha protein extract dry powder prepared by embodiment 1 by phosphate-buffered saline (PBS), obtaining protein concentration is the medicine that the Auricularia polytricha protein extract aqueous solution of 2mg/mL damages as alleviation of alcohol human liver cell.
Wherein, in Auricularia polytricha protein extract aqueous solution, the mensuration of protein content adopts BCA protein quantification test kit to carry out, and description is shown in concrete operations.
2, the Cytotoxic detection of Auricularia polytricha protein extract
1) cell culture: operate same step one.
2) Pharmaceutical formulations: add the Auricularia polytricha protein extract aqueous solution that protein concentration is 2mg/mL to containing in the RPMI1640 culture fluid of 10%FBS, is configured to the culture fluid containing Auricularia polytricha protein extract that protein concentration is respectively 20,50,100,200 and 500 μ g/mL.
3) drug incubation: after cell culture terminates, absorb the cell culture fluid in culture hole, add 200 μ L steps 2 respectively) in different proteins concentration containing the culture fluid (experimental group) of Auricularia polytricha protein extract, matched group adds isopyknic RPMI RPMI-1640 (protein concentration is 0) containing 10%FBS, being placed in temperature is 37 DEG C, relative humidity is 95%, CO
2concentration is that after continuing to cultivate 24h in the incubator of 5%, drug incubation terminates.The multiple hole of Setup Experiments three.
4) MTT detects: operate same step one.
Cell survival rate formula: the OD of cell survival rate (%)=experimental group
562the OD of value/matched group
562value × 100%.
The cell survival rate of matched group is set as 100%, result as shown in Figure 2, the culture fluid containing Auricularia polytricha protein extract of above-mentioned different proteins concentration does not have significant difference for the impact of L-O2 cytoactive, and cell survival rate is all more than 90%, illustrate that Auricularia polytricha protein extract does not have cytotoxicity to L-O2 cell, can be used for liver-protecting function research.
3, the cell survival rate experiment of Auricularia polytricha protein extract alleviation of alcohol human liver cell damage
1) cell culture: operate same step one.
2) Pharmaceutical formulations: to containing adding the Auricularia polytricha protein extract aqueous solution that dehydrated alcohol and protein concentration are 2mg/mL in the RPMI1640 culture fluid of 10%FBS, be configured to following No. 1-5 culture fluid containing ethanol and Auricularia polytricha protein extract: No. 1 (ethanol contend percentage composition is 3%, protein concentration is 10 μ g/mL), No. 2 (ethanol contend percentage composition is 3%, protein concentration is 20 μ g/mL), No. 3 (ethanol contend percentage composition is 3%, protein concentration is 30 μ g/mL), No. 4 (ethanol contend percentage composition is 3%, protein concentration is 40 μ g/mL) and No. 5 (ethanol contend percentage composition is 3%, protein concentration is 50 μ g/mL).
3) drug incubation: after cell culture terminates, absorb the cell culture fluid in culture hole, add 200 μ L steps 2 respectively) in No. 1-5 culture fluid (treatment group) containing ethanol and Auricularia polytricha protein extract, matched group adds isopyknic RPMI1640 culture fluid containing 10%FBS, and (ethanol contend percentage composition is 0, protein concentration is 0 μ g/mL), damage model group adds isopyknic culture fluid containing 3% (volumn concentration) ethanol, being placed in temperature is 37 DEG C, relative humidity is 95%, CO
2concentration is that after continuing to cultivate 48h in the incubator of 5%, drug incubation terminates.The multiple hole of Setup Experiments three.
4) MTT detects: operate same step one.
Cell survival rate formula: the OD of cell survival rate (%)=experimental group
562the OD of value/matched group
562value × 100%.
The cell survival rate of matched group is set as 100%; result as shown in Figure 3; the L-O2 cell survival rate for the treatment of group is all higher than the survival rate of the L-O2 cell of damage model group, and Auricularia polytricha protein extract has protective effect in various degree to L-O2 cell, can promote the survival rate of cell.When protein concentration in Auricularia polytricha protein extract in culture fluid is 30 μ g/mL, treatment group L-O2 cell survival rate is the highest, can 87.86% be reached, improve 42.85% than the cell survival rate of damage model group (cell survival rate is 45.01%), therefore this concentration is set as that the best protects the liver dosage.
4, the characteristics index of Auricularia polytricha protein extract alleviation of alcohol human liver cell damage detects
Adopt the trypsin of 0.25% to carry out digesting in the L-O2 cell of exponential phase and use the RPMI1640 culture fluid collection re-suspended cell containing 10%FBS, being inoculated in 6 well culture plates that (every hole 3mL, cell density is 3 × 10
5individual/hole), being placed in temperature is 37 DEG C, and relative humidity is 95%, CO
2concentration is cultivate 24h in the incubator of 5%.
After cell culture terminates, absorb the cell culture fluid in culture hole, treatment group adds the culture fluid that 3mL contains 3% (volumn concentration) ethanol and the 30 μ g/mL concentration of protein (in the Auricularia polytricha protein extract) protein; Matched group adds isopyknic RPMI1640 culture fluid containing 10%FBS, and (ethanol contend percentage composition is 0, protein concentration is 0 μ g/mL), damage model group adds isopyknic culture fluid containing 3% (volumn concentration) ethanol, and to be placed in temperature be 37 DEG C, relative humidity is 95%, CO
2concentration is that after continuing to cultivate 48h in the incubator of 5%, drug incubation terminates.The multiple hole of Setup Experiments three.
1) detection of intracellular triglyceride (TG) content
After drug incubation terminates, collect cell adherent in culture dish after absorbing culture fluid, adopt the detection organizing triglyceride mensuration test kit to carry out TG content, adopt the protein content in BCA protein quantification kit measurement sample, concrete operations are see description simultaneously.In cell, the content of TG is with a μm ol/mg Representation of Proteins.
Result is as shown in table 2, the intracellular TG content for the treatment of group L-O2 is starkly lower than damage model group, the intracellular TG content for the treatment of group (817 ± 33 μm of ol/mg protein) L-O2 reduces 68.90% than damage model group (2627 ± 11 μm of ol/mg protein), Auricularia polytricha protein extract effectively reduces the intracellular TG content of L-O2, decreases the damage of L-O2 cell.Treatment group and damage model group can form statistically significant difference (P < 0.05).
2) detection of reactive oxygen species (ROS) content
After drug incubation terminates, absorb the culture fluid in culture hole, collect cell adherent in culture dish, add fluorescent probe, adopt active oxygen detection kit in tissue to carry out the detection of ROS content, concrete operations are see description.
Result is as shown in table 2, the intracellular ROS content for the treatment of group L-O2 is starkly lower than damage model group, the average fluorescent strength (4898 ± 406) of the intracellular ROS for the treatment of group L-O2 reduces 53.56% than damage model group (10548 ± 319), Auricularia polytricha protein extract effectively reduces the intracellular ROS content of L-O2, decreases the damage of L-O2 cell.Treatment group and damage model group can form statistically significant difference (P < 0.05).
3) detection of the outer glutamate pyruvate transaminase (ALT) of born of the same parents and glutamic oxaloacetic transaminase, GOT (AST) content
After drug incubation terminates, collect the culture fluid in every hole, glutamic oxaloacetic transaminase, GOT in glutamate-pyruvate transaminase determination reagent kit and serum is adopted in serum to measure the content that test kit measures outer ALT and AST of born of the same parents respectively, adopt the protein content in BCA protein quantification kit measurement sample, concrete operations are see description simultaneously.The content of ALT and AST is all with U/mg Representation of Proteins.
Result is as shown in table 2, the content of ALT and AST for the treatment of group L-O2 cell release is starkly lower than damage model group, the ALT content (8096 ± 194U/mg protein) for the treatment of group L-O2 cell release reduces 51.06% than damage model group (16544 ± 468U/mg protein), the AST content (6979 ± 221U/mg protein) for the treatment of group L-O2 cell release reduces 53.70% than damage model group (15072 ± 579U/mg protein), Auricularia polytricha protein extract effectively reduces the content of L-O2 cell release ALT and AST, protect the integrity of L-O2 cell.
Table 2, Auricularia polytricha protein extract protection L-O2 characteristic indices of cell value
| Index name | Matched group | Treatment group | Damage model group |
| TG (μm ol/mg protein) | 309±41 | 817±33 | 2627±11 |
| ROS(F 525nm) | 3318±232 | 4898±406 | 10548±319 |
| ALT (U/mg protein) | 4014±279 | 8096±194 | 16544±468 |
| AST (U/mg protein) | 3018±627 | 6979±221 | 15072±579 |
Note: F 525nm is the fluorescent value at 525nm place.
Claims (8)
1. Auricularia polytricha protein extract is preparing the application in alleviation of alcohol hepatocyte injury product, and described Auricularia polytricha protein extract is prepared by following method:
1) Auricularia polytricha (Mout) Sacc. sporophore is pulverized rear flooding, collect water-soluble substances and obtain Auricularia polytricha (Mout) Sacc. water solubility extract;
2) ammonium sulfate precipitation that saturation is 80% is carried out to described Auricularia polytricha (Mout) Sacc. water solubility extract, collecting precipitation, after described precipitate with deionized water dialysis, obtain described Auricularia polytricha protein extract.
2. application according to claim 1, is characterized in that: described step 2) in, at 4 DEG C, the ammonium sulfate precipitation that saturation is 80% is carried out to described Auricularia polytricha (Mout) Sacc. water solubility extract.
3. application according to claim 1 and 2, is characterized in that: described deionized water dialysis adopts molecular cut off to be that 3kDa semipermeable membrane carries out.
4., according to described application arbitrary in claim 1-3, it is characterized in that: described alleviation of alcohol hepatocyte injury is following 1)-5) at least one:
1) hepatocellular survival rate is improved;
2) content of triglyceride in hepatocyte is reduced;
3) active o content in hepatocyte is reduced;
4) hepatocyte is reduced to the burst size of glutamate pyruvate transaminase;
5) hepatocyte is reduced to the burst size of glutamic oxaloacetic transaminase, GOT.
5. Auricularia polytricha protein extract is preparing the application in alleviation of alcohol hepatic injury product; Described Auricularia polytricha protein extract is prepared by following method:
1) Auricularia polytricha (Mout) Sacc. sporophore is pulverized rear flooding, collect water-soluble substances and obtain Auricularia polytricha (Mout) Sacc. water solubility extract;
2) ammonium sulfate precipitation that saturation is 80% is carried out to described Auricularia polytricha (Mout) Sacc. water solubility extract, collecting precipitation, after described precipitate with deionized water dialysis, obtain described Auricularia polytricha protein extract.
6. application according to claim 5, is characterized in that: described step 2) in, at 4 DEG C, the ammonium sulfate precipitation that saturation is 80% is carried out to described Auricularia polytricha (Mout) Sacc. water solubility extract.
7. the application according to claim 5 or 6, is characterized in that: described deionized water dialysis adopts molecular cut off to be that 3kDa semipermeable membrane carries out.
8., according to described application arbitrary in claim 5-7, it is characterized in that: described alleviation of alcohol hepatic injury is following 1)-5) at least one:
1) hepatocellular survival rate is improved;
2) content of triglyceride in hepatocyte is reduced;
3) active o content in hepatocyte is reduced;
4) hepatocyte is reduced to the burst size of glutamate pyruvate transaminase;
5) hepatocyte is reduced to the burst size of glutamic oxaloacetic transaminase, GOT.
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