CN113072632A - Preparation method of plant functional protein and application of plant functional protein in protection of acute alcoholic liver injury - Google Patents
Preparation method of plant functional protein and application of plant functional protein in protection of acute alcoholic liver injury Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention discloses a preparation method of plant functional protein and application of the plant functional protein in protection of acute alcoholic liver injury, and belongs to the technical field of functional materials. The invention takes natural defatted wheat germ powder as a raw material, prepares the wheat germ globulin by salt extraction, can effectively reduce the rise of liver index, spleen index and kidney index caused by acute alcohol stimulation, reduces the levels of total cholesterol and triglyceride in serum and plays a role in protecting the liver; can improve the glutathione content and the superoxide dismutase activity of the liver, reduce the malondialdehyde content and improve the liver oxidative stress caused by alcohol; can also relieve the degree of hepatocyte necrosis, and protect acute alcoholic liver injury. Compared with the existing medicine for dispelling the effects of alcohol and protecting the liver, the wheat germ globulin is safer and more effective, has no toxic or side effect, and obviously improves physiological and biochemical abnormalities caused by alcohol. The wheat germ globulin has considerable development and application values in the aspect of improving acute alcoholic liver injury, and has wide market application prospects.
Description
Technical Field
The invention belongs to the technical field of functional materials, and particularly relates to a preparation method of plant functional protein and application of the plant functional protein in protection of acute alcoholic liver injury.
Background
Alcoholic liver injury (ALD) can not only damage the health of drinkers, but also harm families and cause social problems. The data shows that 2016 years of global over-drinking caused approximately 300 million deaths. Liver damage can be caused by excessive drinking once or by long-term intake of a large amount of alcohol, and is classified into chronic and acute liver damage according to different causes. Alcoholic liver injury is generally thought to be due to oxidative stress mediated by ethanol and metabolites, the body's immune response and liver cell damage.
Early stage of alcoholic liver injury manifests as fatty liver, mainly due to accumulation of fat (triglycerides, phospholipids, cholesterol esters, etc.) in hepatocytes. Early studies showed that alcohol consumption increased the NAPDH/NAD ratio in hepatocytes, disrupting the β -oxidized fatty acids in mitochondria, leading to steatosis. Alcohol intake enhances the lipid supply from the small intestine to the liver, increasing the absorption by the liver of fatty acids metabolized from adipose tissue. The long-term drinking can also cause nutrient malabsorption, so that the absorption of antioxidants in food is reduced, further the oxidation promoting substances in the organism are increased, the oxidation resisting substances are reduced, the oxidative stress is promoted, and finally, the liver cells are damaged and even necrotized.
There are many kinds of drugs for treating alcoholic liver injury. The treatment period of the chemical medicine is short, but adverse reactions are often caused, and the chemical medicine is not suitable for the old, the weak and the pregnant women to take and has certain limitations. The traditional Chinese medicine has small side effect but long treatment period, and except for partial Chinese patent medicines which are verified, other prescription medicines are different according to different people, thus being not beneficial to large-scale clinical popularization and application.
Along with the improvement of living standard and the increase of health consciousness, the homology of medicine and food causes wide attention of people. Based on the theory, compared with chemical medicines, the vegetable protein has the advantages of convenient absorption, wide sources, good safety and the like, can participate in but not interfere in human metabolism, has almost no adverse reaction, and is suitable for various crowds except renal insufficiency patients. Due to its high nutritive value and functional activity, it can repair liver injury and improve malnutrition and complications due to alcoholism to some extent. When the nutritional status of the patient is improved, the immune function of the patient is also obviously improved. More and more studies are beginning to intervene in alcoholic liver injury by using natural active substances extracted from food.
Disclosure of Invention
The invention aims to develop a plant functional protein which is safe, effective and free of side effect and can effectively protect acute liver injury caused by alcohol.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
a preparation method of plant functional protein comprises the following preparation steps: adding defatted wheat germ powder into NaCl solution at a mass-to-volume ratio of 1:10w/v, stirring uniformly, and shaking at constant temperature; after the shaking is finished, centrifuging and taking supernatant; the supernatant was passed through 0.22 μm and 0.1 μm microfiltration membranes in sequence using a buchner funnel with a vacuum pump; collecting the filtrate by a dialysis bag, and magnetically stirring to obtain the plant functional protein solution.
Further, the mass concentration of the NaCl solution is 0.9%.
Further, the constant temperature shaking time is 1.5h, and the temperature is 30 ℃.
Further, the centrifugal speed is 4000rpm, and the centrifugal temperature is 20 ℃; the centrifugation time was 10 min.
Further, the cut-off molecular weight of the dialysis bag was 3500.
Further, the time of magnetic stirring was 12 hours and the temperature was 4 ℃.
The plant functional protein can be applied to protecting liver injury caused by acute alcoholic intoxication.
Wheat germ globulin (WEG) has multiple physiological activities of resisting inflammation, resisting oxidation, enhancing immunity and the like, is a high-quality plant active protein, and has wide raw material sources, simple and convenient extraction method and good application prospect. At present, the in vitro anti-inflammatory activity and in vitro antioxidant activity of the wheat germ globulin are effectively verified, and the wheat germ globulin is subjected to KEGG metabolic pathway analysis based on proteomics, so that the wheat germ globulin can participate in an alcoholism metabolic pathway, and the wheat germ globulin is tried to be developed into an alcoholic liver injury protective agent on the basis.
Advantageous effects
According to the invention, natural defatted wheat germ powder is used as a raw material, salt solution and wheat germ are accurately proportioned, wheat germ globulin is prepared by salt extraction, and the obtained protein can effectively reduce the increase of liver index, spleen index and kidney index caused by acute alcohol stimulation, reduce TC and TG levels in serum and play a role in liver protection; can improve GSH content and SOD activity of liver, reduce MDA content, and improve liver oxidative stress caused by alcohol; meanwhile, the degree of hepatocyte necrosis can be relieved, and the protective effect on acute alcoholic liver injury is achieved. Compared with the existing medicine for dispelling the effects of alcohol and protecting the liver, the wheat germ globulin is safer and more effective, has no toxic or side effect, and obviously improves physiological and biochemical abnormalities caused by alcohol. The wheat germ globulin has considerable development and application values in the aspect of improving acute alcoholic liver injury, and has wide market application prospects.
Drawings
FIG. 1 shows the effect of the wheat blastoglobulin of the present invention on the blood lipid of mice with acute alcoholic liver injury;
(a) the effect of WEG on TC; (b) effect of WEG on TG;
FIG. 2 shows the effect of wheat germ globulin of the present invention on the pathological change of liver tissue of mice with acute alcoholic liver injury;
wherein NC is blank group, AC is experimental group, PC is positive control group, LG is WEG low dose group, MG is WEG middle dose group, HG is WEG high dose group, 1/2 is comparative example 1/2.
Detailed Description
The technical solution of the present invention is further described below with reference to specific embodiments, but is not limited thereto.
Example 1
A preparation method of plant functional protein comprises the following preparation steps: adding defatted wheat germ powder into NaCl solution at a mass-to-volume ratio of 1:10w/v, stirring uniformly, and shaking at constant temperature; after the shaking is finished, centrifuging and taking supernatant; the supernatant was passed through 0.22 μm and 0.1 μm microfiltration membranes in sequence using a buchner funnel with a vacuum pump; collecting the filtrate by a dialysis bag, and magnetically stirring to obtain the plant functional protein solution.
The mass concentration of the NaCl solution is 0.9%.
The constant temperature shaking time is 1.5h, and the temperature is 30 ℃.
The centrifugal speed is 4000rpm, and the centrifugal temperature is 20 ℃; the centrifugation time was 10 min.
The cut-off molecular weight of the dialysis bag is 3500.
The magnetic stirring time was 12h and the temperature was 4 ℃.
The plant functional protein can be applied to protecting liver injury caused by acute alcoholic intoxication.
Comparative example 1
A preparation method of plant functional protein comprises the following preparation steps: adding defatted wheat germ powder into NaCl solution at a mass-to-volume ratio of 1:5w/v, stirring uniformly, and shaking at constant temperature; after the shaking is finished, centrifuging and taking supernatant; the supernatant was passed through 0.22 μm and 0.1 μm microfiltration membranes in sequence using a buchner funnel with a vacuum pump; collecting the filtrate by a dialysis bag, and magnetically stirring to obtain the plant functional protein solution.
The mass concentration of the NaCl solution is 0.9%.
The constant temperature shaking time is 1.5h, and the temperature is 30 ℃.
The centrifugal speed is 4000rpm, and the centrifugal temperature is 20 ℃; the centrifugation time was 10 min.
The cut-off molecular weight of the dialysis bag is 3500.
The magnetic stirring time was 12h and the temperature was 4 ℃.
This comparative example was conducted in the same manner as in example 1 except that the ratio of the defatted wheat germ powder to the NaCl solution was 1:5 w/v.
Comparative example 2
A preparation method of plant functional protein comprises the following preparation steps: adding defatted wheat germ powder into NaCl solution at a mass-to-volume ratio of 1:20w/v, stirring uniformly, and shaking at constant temperature; after the shaking is finished, centrifuging and taking supernatant; the supernatant was passed through 0.22 μm and 0.1 μm microfiltration membranes in sequence using a buchner funnel with a vacuum pump; collecting the filtrate by a dialysis bag, and magnetically stirring to obtain the plant functional protein solution.
The mass concentration of the NaCl solution is 0.9%.
The constant temperature shaking time is 1.5h, and the temperature is 30 ℃.
The centrifugal speed is 4000rpm, and the centrifugal temperature is 20 ℃; the centrifugation time was 10 min.
The cut-off molecular weight of the dialysis bag is 3500.
The magnetic stirring time was 12h and the temperature was 4 ℃.
This comparative example was conducted in the same manner as in example 1 except that the ratio of the defatted wheat germ powder to the NaCl solution was 1:20 w/v.
Test examples
Mouse assay
Experimental animals:
KM mice, SPF grade, purchased at zhengzhou university laboratory animal center, certification No.: DW 2019100103.
Test materials and reagents:
defatted wheat germ flour, zhengzhou wandanxue ltd; silibinin, tianjin tianshi saint, gmbh; hematoxylin eosin staining kit, paraformaldehyde, DTNB, SDS, EDTA, Beijing Soilebao science and technology Co., Ltd; reduced glutathione, Shandong-Xiya chemical Co., Ltd; sulfosalicylic acid, shanghai source phyllo biotechnology limited; tetraethoxypropane, NBT, MET, Shanghai Michelin Biochemical Co., Ltd.; TBA, shanghai national chemicals chemical limited; BCA protein concentration assay kit, U.S. GlpBio; other reagents are all domestic analytical purifiers.
Test equipment and instruments:
model XW-80A vortex mixer, beijing xin tenda instruments ltd; model F570 ultra-low temperature refrigerator, Eppendorf, germany; 3k-18 type centrifuge, sigma company, germany; FA1004B model electronic balance, shanghai precision instruments ltd; SynergyHTX multifunctional microplate reader, BioTek, usa.
Experiment design:
blank control, commercial drug groups and comparative examples 1-2 are designed as controls, and compared and verified with the technical effects of the embodiments of the present invention, mice are randomly divided into 8 groups after being bred for 7d, namely, blank group (NC), experimental group (AC), positive control group (PC), WEG low dose group (LG), WEG medium dose group (MG), WEG high dose group (HG), comparative example 1 and comparative example 2, 10 mice per group, and 2 cages per group. The specific test design is shown in table 1:
TABLE 1 grouping and treatment of acute alcoholic liver injury mice
The experimental test method comprises the following steps:
fasted for 12h after the last gavage and body weight recorded. The mice were anesthetized with ether, and after the eyeballs of the mice were picked up with medical ophthalmic forceps, the venous blood of the eyeballs was immediately collected. Standing at room temperature for 2h, centrifuging at 4 deg.C and 3500r/min for 20min, collecting supernatant, namely serum, placing in EP tube, and storing in-80 deg.C ultra-low temperature refrigerator for testing. The mice were sacrificed by cutting off the neck immediately after blood collection, dissected and tissues such as liver, spleen, kidney, etc. were collected and weighed for recording. 0.3g of the same part of the right lobe of the liver was taken from each mouse, and 9 times of precooled physiological saline was added thereto, and the homogenate was ground on ice. Centrifuging at 4 deg.C and 3000r/min for 10min, collecting supernatant, one part for measuring malonaldehyde and reduced glutathione content, and the other part for measuring superoxide dismutase content, wherein the supernatant is obtained by adding saturated ammonium sulfate solution at a ratio of 3:1, centrifuging at 4 deg.C and 10000r/min to obtain crude enzyme solution. Storing at-80 deg.C for use.
Data processing:
all experimental data are expressed as mean ± Standard Deviation (SD). The experimental data were subjected to one-way analysis of variance (ANOVA) and LSD tests using SPSS19.0 software, and significant differences were considered when P < 0.05.
Example 3
Index of visceral organs
Organ coefficient (g)/mouse last body weight (g) × 100%.
The change of the organ index can preliminarily reflect the degree of the alcohol-mediated organism injury. The liver index, which is the main indicator in this change, is the proportion of the wet weight of the mouse liver to its body weight. Alcohol-mediated liver damage is mainly manifested by cell damage and lipid metabolism disorder, and liver is heavily fatted, and its specific gravity of wet weight relative to body weight is increased. As can be seen from the data in table 2, the liver coefficients of the model group were significantly higher than those of the blank group, whereas the liver coefficients of the WEG-treated group were significantly lower than those of the model group with no significant difference from the blank group (P < 0.05). The effect of the medium dose was significantly better than that of the high dose treatment group. Accordingly, spleen and kidney indices were significantly elevated in the model group mice compared to the blank group mice, and intervention of WEG significantly improved abnormalities in these visceral indices. Shows that WEG has positive effect on protecting acute alcoholic liver injury and has better effect of medium dosage. While the positive effect on the organ index is significantly reduced in comparative examples 1-2, in which the process parameters were changed. The ratio of the defatted wheat germ powder to the NaCl solution is 1:10w/v, which is the best process parameter.
TABLE 2 organ index of acute alcoholic liver injury mice
Note: there was a significant difference between the values of the different letter designations (P < 0.05).
Example 4
Blood fat
TC and TG levels in serum of acute ALD mice were measured using a full-biochemical automatic analyzer.
Lipid metabolism disorder is one of the manifestations of alcoholic liver injury. Alcohol promotes TC and TG synthesis, increasing liver lipid content by high density lipoprotein transfer from the liver to the periphery, leading to worsening of ALD. The severity of hepatic steatosis can be judged by detecting the content of TC and TG in serum. As shown in fig. 1, serum TC and TG levels of the model mice were significantly increased compared to the blank group, while blood lipid levels of the liver-injured mice were significantly decreased (P <0.05) compared to the model group under the protection of wheat germ globulin. Therefore, WEG can well protect liver fatty lesion mediated by alcohol.
Example 5
Determination of GSH content
Standard curve: taking 1mmol/L of GSH standard solution and physiological saline to prepare 0, 20, 40, 100, 200, 300 and 400 mu mol/L of GSH standard solution respectively, adding 4.5mL of DTNB into the solutions respectively, mixing the solutions uniformly and reacting for 10min in a dark place, measuring the absorbance at the wavelength of 420nm, and drawing a standard curve according to the result.
Tissue supernatant: 0.5mL of 10% homogenate +0.5mL of 4% sulfosalicylic acid, centrifuged at 3500rpm at room temperature for 10min, and the supernatant was collected.
And (3) determination: blank wells: 200 μ L of 4% sulfosalicylic acid +1.8mL DTNB;
measuring a tube: 200 μ L tissue supernatant +1.8mL DTNB;
after the plate was attached, the plate was left standing for 10min, and the absorbance was measured at a wavelength of 420 nm.
Determination of SOD Activity
Preparing crude enzyme solution, adding saturated (NH) into liver homogenate supernatant at ratio of 3:14)2SO4The solution was centrifuged at 10000rpm for 10min at 4 ℃ and the supernatant was collected for SOD content determination.
Blank group: 2mL of 0.1mol/L PBS (pH 8.0) +2mL of NBT reaction solution
Measurement group: 2mL of tissue crude enzyme solution +2mL of NBT reaction solution
After 4min of uniform illumination, the absorbance was measured at a wavelength of 560 nm.
Determination of the MDA content
Blank tube: 0.1mL 8.1% SDS +0.75mL 0.2M acetate buffer +0.75mL 0.8% TBA +
0.4mL of distilled water. (As A)
Measuring a tube: 0.1mL of 8.1% SDS +0.75mL of 0.2M acetate buffer +0.75mL of 0.8% TBA +0.3mL of distilled water +0.1mL of 10% tissue homogenate supernatant. (as B)
③ standard pipe: 0.1mL of 8.1% SDS +0.75mL of 0.2M acetate buffer +0.75mL of 0.8% TBA +0.3mL of distilled water +0.1mL of 40nmol/mL of tetrachloropropane. (as F)
Mixing, boiling in water bath for 1h in dark place, cooling, centrifuging for 5min, and measuring absorbance at 532nm wavelength.
Wherein: c: a tetrachloro propane concentration (40 nmol/ml); k: diluting the concentration; protein concentration: (nmol/mg protein).
The BCA method is adopted to determine the concentration of the liver homogenate protein, and the specific experimental steps refer to the kit description.
The test results are shown in table 3:
TABLE 3 influence of WEG on oxidative stress of liver in mice with acute alcoholic liver injury
Note: there was a significant difference between the values of the different letter designations (P < 0.05).
After alcohol treatment, the GSH and SOD were significantly reduced and the MDA content was significantly increased in the model group mice (P <0.05) compared to the blank group mice. Compared with the model group, the intervention of WEG obviously improves the SOD and GSH activity in the liver and reduces the MDA activity (P < 0.05). The SOD activity of the dosage group in WEG is not only remarkably higher than that of the model group, but also remarkably higher than that of the blank group. While the level of the comparative group was comparable to the positive control group. In conclusion, WEG can improve oxidative stress injury of liver caused by acute ethanol intake, and the effect is best in medium dosage.
Example 6
H & E staining of liver pathological tissue section
Tissue blocks with the volume of about 1.0 multiplied by 0.2(cm) are picked from the left leaves of the liver of the mouse, fixed in 10 percent paraformaldehyde fixing solution for 48 hours, and sequentially dehydrated in alcohol solutions with different concentrations in a gradient way. Adding a xylene-alcohol mixed solution (v: v ═ 1:1), adding xylene after 15min, and carrying out transparency for 30 min. The tissue is waxed 2 times at 65-70 deg.C for 30min each time. After being embedded by an embedding machine, the slices are cut into 5 mu m slices by a slicer, the slices are dyed according to the steps of the instruction, and the pathological changes of the liver are observed under an optical microscope.
The test results are shown in fig. 2:
the blank control group has normal hepatocyte structure and morphology, regular cell arrangement, uniform size, clear boundary, large and round nucleus and less necrosis. The liver cell structure of the model group mouse is obviously damaged, the phenomena of liver cell necrosis and cell nucleus shrinkage are generated, the model is successfully made, and the liver of the mouse is seriously damaged by acute alcohol stimulation. The liver injury is relieved to different degrees by the three treatment groups of wheat germ globulin, wherein the effect of the low-dose group is poor, and the protection effect of the medium-dose group and the high-dose group is basically consistent with that of the positive control group.
In conclusion, the invention adopts a method of oral triple-time excessive drinking to cause acute alcoholic liver injury of mice, and tests prove that:
the results of the liver antioxidant capacity show that the activity of GSH and SOD in the liver tissues of the model mice is obviously reduced (P <0.05), and the MDA content is obviously increased (P < 0.05). Compared with the model group mice, the intervention of WEG obviously improves the contents of SOD and GSH in liver tissues of the liver injury mice (P <0.05) and obviously reduces the content of MDA (P < 0.05).
The result of the liver pathological tissue section shows that the hepatocyte structure of the model group mouse is obviously damaged and arranged disorderly, the phenomena of hepatocyte necrosis and cell nucleus shrinkage are generated, and the intervention of WEG obviously relieves the phenomenon of hepatocyte apoptosis and necrosis caused by alcohol.
The WEG low dose group has poor effect in part of indexes, and the WEG medium and high doses do not show obvious dose dependence and play a good role in protection, so the WEG medium dose has the best protection effect.
By combining the experimental results, the wheat blastoglobulin prepared by the invention has good protection effect on acute alcoholic liver injury. Can be used for protecting acute liver injury caused by alcohol, and has the advantages of safety, effectiveness and no toxic or side effect.
It should be noted that the above-mentioned embodiments are only some of the preferred modes for implementing the invention, and not all of them. Obviously, all other embodiments obtained by persons of ordinary skill in the art based on the above-mentioned embodiments of the present invention without any creative effort shall fall within the protection scope of the present invention.
Claims (7)
1. The preparation method of the plant functional protein is characterized by comprising the following preparation steps: adding defatted wheat germ powder into a NaCl solution at a ratio of 1:10w/v, uniformly stirring, and vibrating at constant temperature; after the shaking is finished, centrifuging and taking supernatant; the supernatant was passed through 0.22 μm and 0.1 μm microfiltration membranes in sequence using a buchner funnel with a vacuum pump; collecting the filtrate by a dialysis bag, and magnetically stirring to obtain the plant functional protein solution.
2. The method for producing a plant functional protein according to claim 1, wherein the NaCl solution is contained at a concentration of 0.9% by mass.
3. The method for preparing functional plant protein according to claim 1, wherein the shaking time at constant temperature is 1.5h and the temperature is 30 ℃.
4. The method for preparing functional plant protein according to claim 1, wherein the centrifugation speed is 4000rpm, and the centrifugation temperature is 20 ℃; the centrifugation time was 10 min.
5. The method for preparing functional plant protein according to claim 1, wherein the dialysis bag has a molecular weight cut-off of 3500.
6. The method for preparing functional plant protein according to claim 1, wherein the magnetic stirring is carried out for 12 hours at a temperature of 4 ℃.
7. The use of the functional plant protein according to any one of claims 1 to 6 for the preparation of a medicament for protecting liver from acute alcoholic injury.
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| CN111513173A (en) * | 2020-04-15 | 2020-08-11 | 河南工业大学 | Application of wheat germ globulin in improving cognitive dysfunction caused by aging |
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