CN117903246A - Leech active peptide for improving nonalcoholic fatty liver and preparation method and application thereof - Google Patents
Leech active peptide for improving nonalcoholic fatty liver and preparation method and application thereof Download PDFInfo
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- CN117903246A CN117903246A CN202311686509.1A CN202311686509A CN117903246A CN 117903246 A CN117903246 A CN 117903246A CN 202311686509 A CN202311686509 A CN 202311686509A CN 117903246 A CN117903246 A CN 117903246A
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of bioactive peptides, and in particular relates to a leech bioactive peptide for improving non-alcoholic fatty liver, and a preparation method and application thereof. The peptide sequence of the active peptide is GFAGDDAPRA. The leech active peptide provided by the invention can effectively improve nonalcoholic fatty liver diseases, and has the effects of reducing liver lipid accumulation, oxidative stress and inflammatory reaction.
Description
Technical Field
The invention belongs to the field of bioactive peptides, and in particular relates to a leech bioactive peptide for improving non-alcoholic fatty liver, and a preparation method and application thereof.
Background
Non-alcoholic fatty liver (NAFLD) refers to a clinical pathological syndrome which is mainly characterized by diffuse liver cell bullous steatosis and is caused by removing alcohol and other definite liver damage factors, and comprises simple fatty liver and fatty hepatitis, liver cirrhosis and other diseases which are evolved from the simple fatty liver. The prevalence of NAFLD is statistically as high as 25% worldwide, and is still continuously rising with changes in dietary structure, however no drug to date has been approved by the FDA for treatment of NAFLD. Therefore, the timely intervention and treatment of NAFLD has practical significance for improving the health level of residents and relieving medical pressure.
Leech, first in Shennong Ben Cao Jing, is a dried whole body of leech WHITMANIA PIGRA WHITMAN, leech Hirudo nipponica Whitman or leech WHITMANIA ACRANULATA WHITMAN which is a leech family animal, enters liver meridian, has flat property, salty and bitter taste, has small toxicity, and has the effects of breaking blood, relieving dysmenorrhea, removing blood stasis and eliminating mass. The Hirudo contains a large amount of proteins, accounting for more than 70% of total insect population. However, macromolecular proteins cannot be directly absorbed through gastrointestinal tracts, can only be degraded into small molecular polypeptides or amino acids under the action of gastrointestinal digestive enzymes, and then absorbed into blood to exert the efficacy of the small molecular polypeptides or amino acids, but the efficiency of in vivo enzymolysis of proteins into small molecular peptides is extremely low, most of the proteins are directly removed from the body, so that a great deal of medicinal materials are wasted, and the curative effect is greatly reduced.
Disclosure of Invention
In order to solve the technical problems, the invention utilizes a bionic enzymolysis technology to separate and purify leech active peptide GFAGDDAPRA (GA-10) from leeches, researches the action mechanism of GA-10 for preventing NAFLD, provides theoretical and data support for high-value development and utilization of animal traditional Chinese medicine active peptide, and also provides candidate components for human development and treatment of NAFLD drugs or functional products.
The invention aims to solve the technical problem of providing a leech active peptide, a preparation method and application thereof aiming at the current state of the art, wherein the leech active peptide can be used for preparing related products such as medicines for preventing and treating nonalcoholic fatty liver diseases.
The technical scheme adopted for solving the technical problems is as follows:
In a first aspect of the invention, the active peptide derived from leech is characterized in that the peptide sequence of the active peptide has a structural sequence GFAGDDAPRA, shown in SEQ ID NO. 1, and has a molecular weight of 975.44Da, which is named GA-10.
In a second aspect of the present invention, there is provided a process for the preparation of the above-mentioned hirudin active peptide GA-10, comprising the steps of:
1) Adding 8-10 times of deionized water into Hirudo powder, and decocting for 15-20 min to obtain Hirudo decoction;
2) Adding HCl solution into Hirudo decoction, adjusting pH to 1.5-2.0, adding pepsin, and performing enzymolysis at 36-38deg.C for 1-1.5 hr; then NaOH solution is added to adjust the pH of the solution to 7.8-8.5, trypsin is added, and enzymolysis is continued for 2.5-3h at 36-38 ℃; obtaining leech enzymolysis liquid;
3) And (3) carrying out enzyme deactivation treatment on the leech enzymolysis liquid in the step (2), cooling, and centrifuging to obtain supernatant.
4) Desalting the supernatant obtained in the step 3), performing ultrafiltration with a 3000Da ultrafiltration membrane, concentrating the filtrate, and freeze-drying to obtain Hirudo polypeptide lyophilized powder;
5) And separating and purifying the leech polypeptide freeze-dried powder to obtain the leech active peptide GFAGDDAPRA.
Optionally, in step 1), the raw material of the leech active peptide is leech and/or Hirudo nipponica.
Optionally, in step 2), the concentration of the HCl solution and the NaOH solution is 0.3M-0.8M, and the enzyme bottom ratio of pepsin and trypsin is 0.5% -1.5%.
Preferably, in step 2), the concentration of the HCl solution and the NaOH solution is 0.5M, and the enzyme bottom ratio of pepsin to trypsin is 1.0%.
Optionally, in the step 3), the enzyme deactivation treatment mode is heating, and the temperature is 80-100 ℃.
Preferably, in step 3), the enzyme deactivation treatment is performed by heating at 90 ℃.
Optionally, in step 3), the centrifugation conditions are: centrifuging at 3000-4000rmp for 10-20min.
Preferably, in step 3), the centrifugation conditions are 3500rmp for 15min.
Optionally, in step 4), the desalination means is electrodialysis and/or dialysis bag desalination.
Preferably, in step 4), the desalination means is electrodialysis desalination.
In a third aspect of the invention, there is provided the use of the active peptide GA-10 in a medicament for the treatment of non-alcoholic fatty liver disease.
A medicament comprising the leech-derived active peptide GA-10.
The invention has the beneficial effects that: the leech active peptide GA-10 provided by the invention can effectively improve nonalcoholic fatty liver diseases, and has the effects of reducing liver lipid accumulation, oxidative stress and inflammatory reaction. The concrete steps are as follows: in NAFLD rat model experiments, GA-10 can reduce liver lipid drops and blood lipid levels TC, TG and the like of NAFLD rats; reducing liver injury index AST and ALT; decreasing MDA and increasing SOD oxidative stress levels, and decreasing inflammatory response by decreasing pro-inflammatory factors TNF- α, IL-6, IL-8. The GA-10 peptide is expected to become a candidate component for NAFLD drug development.
Drawings
FIG. 1 is a block diagram of GA-10;
FIG. 2 is a circular dichroism spectrum of GA-10;
FIG. 3 is an infrared spectrum of GA-10;
FIG. 4 is a graph showing changes in NAFLD rat body weight and liver volume ratio;
FIG. 5 is a comparison of NAFLD rat livers;
FIG. 6 is a graph showing the results of HE staining for liver injury in NAFLD rats;
FIG. 7 is a graph of NAFLD rat liver lipid droplet accumulation oil red O staining;
FIG. 8 is a statistical plot of NAFLD rat serum TC, TG, LDL-C, HDL-C changes;
FIG. 9 is a statistical plot of NAFLD rat serum AST and ALT changes;
FIG. 10 is a graph showing the statistics of changes in liver MDA and SOD of NAFLD rats;
FIG. 11 is a statistical plot of changes in the serum ELISA indicators TNF- α, IL-6 and IL-8 of NAFLD rats.
Detailed Description
In order to more clearly illustrate the invention, the invention will be described in further detail below with reference to the drawings and the preferred embodiments.
Example 1 preparation of the Hirudo active peptide GA-10 (I)
1) 50G of leech medicinal material powder is taken, 10 times of deionized water is added, and the leech medicinal material powder is decocted for 15min to obtain leech decoction.
2) Adding 0.5M HCl solution into leech decoction to adjust pH to 2.0, adding 1% pepsin (enzyme activity 1:15000), and performing enzymolysis at 37deg.C for 1 hr; then adding 0.5M NaOH solution to adjust the pH of the solution to 8.0, adding trypsin (enzyme activity 1:2500), and continuing to carry out enzymolysis for 3 hours at 37 ℃; and (5) obtaining leech enzymolysis liquid.
3) And (3) heating and inactivating enzyme of the leech enzymolysis liquid in the step (2) at 90 ℃, cooling, centrifuging 3500rmp for 15min, and taking supernatant.
4) And (3) carrying out electrodialysis desalination on the supernatant obtained in the step (3), then carrying out ultrafiltration with a 3000Da ultrafiltration membrane, concentrating and freeze-drying the obtained filtrate to obtain the leech polypeptide freeze-dried powder.
5) After the leech polypeptide freeze-dried powder is separated and purified, the active peptide GFAGDDAPRA with the strongest activity is obtained by the determination of an activated partial thromboplastin time assay, and the structure of the active peptide GFAGDDAPRA is shown in figure 1.
EXAMPLE 2 preparation of the hirudin GA-10 (II)
The preparation method of example 2 is the same as that of example 1, except that the leech source is leech.
Example 3 preparation of the Hirudo active peptide GA-10 (III)
The preparation method of example 3 is the same as that of example 1, except that the leech source is Hirudo japonica.
Example 4 round dichromatic detection of the Hirudo active peptide GA-10
1Mg of the hirudin GA-10 sample was weighed and dissolved in 10mL of deionized water. The sample solution was filtered through a 0.45 μm microporous filter membrane and subjected to round dichroism analysis. Ambient temperature
The optical path of the quartz cuvette is 0.1cm at 20 ℃, each spectrum record is an average value of 8 scans, the scanning range is 180-260nm, the scanning speed is 50nm/min, the data interval is 0.2nm, the bandwidth is 2nm, the sensitivity is 20mdeg, and the response time is 0.5s. Deionized water was used as a blank to correct for solvent effects. As shown in FIG. 2, GA-10 showed positive peaks at 189nm and 220nm, and was found to have an alpha-helix ratio of 16.8%, a beta-sheet ratio of 57.1%, a beta-turn ratio of 21.0% and a random coil ratio of 42.5% by CDNN software analysis and calculation. In summary, GA-10 is predominantly beta-sheet and random coil.
Example 5 Infrared Spectroscopy detection of the Hirudo active peptide GA-10
Mixing Hirudo active peptide 5.0mg with 200mg potassium bromide, grinding, tabletting under 15.0MPa for 3.0min. And then the sample piece is placed into an optical path of an infrared spectrometer for measurement, and the background of potassium bromide and air is subtracted in real time. The scanning range of the wave number of the spectrogram is 4 000-400cm -1, the resolution is 4cm -1, the scanning times are 10, the horizontal coordinate and the vertical coordinate of the infrared spectrogram are respectively the wave number (cm -1) and the light transmittance (T/%) and the obtained infrared spectrogram is subjected to automatic baseline correction and smoothing treatment by adopting OMNIC software. As shown in FIG. 3, the broad peak at 3300.04cm -1 is the telescopic vibration absorption peak of the associated hydroxyl group O-H; the strong peak at 2930.45cm -1 is the telescopic vibration absorption peak of methylene C-H; the broad peak at 16534.38cm -1 is the telescopic vibration absorption peak of amide c=o; an N-H flexural vibration absorption peak of amide at 1527.51cm -1; an O-H in-plane flexural vibration absorption peak of carboxylic acid at 1454.86cm -1; C-N flexural vibration absorption of amides at 1200.32cm -1.
EXAMPLE 6 pharmacodynamic experiments of Hirudo active peptide GA-10 in the treatment of NAFLD
1. Model construction and group drug administration intervention
NAFLD model was constructed using high-fat diet (HFD) induced SD rats. Prior to grouping, rats were fed adaptively for one week, and after observing the status of the rats, they were randomly divided into 5 groups of 10 animals each: normal control group (Con), normal diet; model group (Mod), high fat diet; model group + GA-10 low dose Group (GAL), high fat diet and 3mg/kg GA-10 treatment; model group + GA-10 high dose Group (GAH), high fat diet and 6mg/kg GA-10 treatment. All groups were normally fed for 16 weeks (treatment groups were fed with high fat diet for 10 weeks and were then intervened). The body weight change of the rats was recorded once a week.
2. Biological sample collection and index measurement
After the last dose, the rats of each group were fasted but not water. Rats were anesthetized with 1.0% sodium pentobarbital solution, then blood was taken from the abdominal aorta, left at room temperature for half an hour, centrifuged at 3000rpm for 15min, and the serum levels TC, TG, LDL-C, HDL-C, ALT and AST and liver levels ALT, AST, SOD and MDA were examined. Meanwhile, the livers of rats are picked up, the liver index is measured, and the liver lobules are fixed in a neutral 4% paraformaldehyde solution for H & E staining and oil red staining.
(1) H & E staining
Sequentially placing the liver slices into an environment-friendly dewaxing liquid I for 20min, an environment-friendly dewaxing liquid II for 20min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min and 75% alcohol for 5min, and washing with tap water. Then hematoxylin staining is carried out, the sections are stained with hematoxylin staining solution for 3-5min, tap water washing, differentiation solution differentiation, tap water washing, blue returning solution and running water washing are carried out. Then, eosin staining is carried out, the sections are dehydrated in gradient alcohol of 85% and 95% for 5min respectively, and then the sections are stained in eosin staining solution for 5min. Finally, sequentially placing the slices into absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-absolute ethyl alcohol III 5 min-dimethyl I5 min-dimethyl II 5min for transparency, and sealing the slices with neutral resin.
(2) Oil red dyeing
Taking out the frozen liver slices from the refrigerator at the temperature of minus 20 ℃ to return to room temperature, fixing the frozen liver slices with tissue fixing liquid for 15min, washing with tap water, airing, conducting oil red dyeing, fully and uniformly mixing 6 parts of saturated oil red O dye liquid with 4 parts of distilled water, standing at the temperature of 4 ℃ overnight, filtering once by qualitative filter paper the next day, and standing at the temperature of 4 ℃ for 24h for the second time, thus obtaining the oil red O working liquid. The slices are immersed in oil red dye liquor for 8-10min (covered and protected from light). The slices were taken out and left to stand for 3s, and then immersed in two cylinders of 60% isopropanol in sequence for differentiation for 3s and 5s respectively. The slices were immersed in 2 cylinders of pure water sequentially for 10s each. Then, the sections were taken out, left for 3 seconds, immersed in hematoxylin counterstain for 3-5min, immersed in 3-jar pure water for 5 seconds, 10 seconds and 30 seconds, respectively. Differentiation of the differentiation liquid is carried out for 2-8s, distilled water in 2 cylinders is used for washing for 10s respectively, blue returning liquid is used for returning blue for 1s, the slices are lightly immersed in tap water in 2 cylinders for immersion washing, and the microscopic examination and dyeing effects are carried out for 5s and 10s respectively. Finally, glycerol gelatin tablet sealing and tablet sealing.
3. Experimental results
3.1 Influence of GA-10 on body weight and liver ratio in NAFLD rats
As shown in fig. 4, the Mod group rats had significantly increased body weight and liver mass ratio (P < 0.01) compared to the Con group, while the GAL group rats had significantly decreased body weight and liver mass ratio (P < 0.01) compared to the Mod group rats. The results demonstrate that GA-10 can reduce the weight and liver mass ratio increase of rats induced by high fat diet, indicating its effect in treating fatty liver.
3.2 Effect of GA-10 on liver morphology and lipid accumulation in NAFLD rats
As shown in fig. 5, 6 and 7, from the apparent morphology of liver, HE staining and oil red O staining results, mod rats showed significant liver steatosis, lobular inflammation, severe vacuolation of hepatocytes and accumulation of lipid droplets compared with Con rats; compared with Mod group, after GA-10 administration, liver steatosis and cavitation degree of rat are reduced, and lipid drop accumulation is obviously reduced. The results indicate that GA-10 can improve liver steatosis and lipid accumulation in NAFLD rats caused by high fat diet.
3.3 Effect of GA-10 on NAFLD rat serum blood lipid factor
As shown in fig. 8, mod group rats had significantly increased serum TG, TC, LDL-C content (P < 0.01) and significantly decreased HDL-C content (P < 0.01) compared to con group; GA-10 treatment significantly reduced NAFLD rat serum TC, TG, LDL-C content (P < 0.01) and HDL-C content significantly increased (P < 0.01) compared to Mod group.
3.4 Effect of GA-10 on liver function in NAFLD rats
As shown in fig. 9, liver functions AST, ALT were significantly elevated in Mod group rats (P < 0.01) compared to Con group; compared with Mod group, GAH group mice AST and ALT are obviously reduced (P < 0.05), while GAL group ALT is not obviously changed, which shows that GA-10 has liver protection effect.
3.5 Influence of GA-10 on hepatic oxidative stress in NAFLD rats
SOD and MDA are important components of the body's oxidative stress defense mechanism. As shown in fig. 10, the Mod group rats had decreased SOD levels (P < 0.01) and increased MDA levels (P < 0.01) compared to the Con group, and an oxidative stress reaction occurred; GA-10 dosing significantly increased SOD levels and decreased MDA levels (P < 0.01) compared to the model group, indicating that GA-10 can prevent NAFLD by reducing oxidative stress.
3.6 Effect of GA-10 on NAFLD rat inflammatory factor
As shown in fig. 11, mod group rats had significantly increased secretion of TNF- α, IL-6, IL-8 (P < 0.01) compared to Con group; compared with Mod group, the serum TNF-alpha, IL-6 and IL-8 content of rats after GA-10 administration is significantly reduced (P < 0.01), and the dosage is dependent.
4. Conclusion of the experiment
The test adopts high-fat diet feed to induce SD rats to construct NAFLD model, and researches the therapeutic effect of the leech active peptide GA-10 on NAFLD. The result shows that GA-10 can reduce the weight, liver weight, serum ALT and AST indexes of NAFLD rats and improve liver injury; reducing liver lipid accumulation by lowering TC, TG, LDL-C, increasing HDL-C; reducing MDA levels by increasing SOD levels inhibits oxidative stress; in addition, GA-10 can also reduce secretion of inflammatory factors to reduce inflammatory responses. In conclusion, the GA-10 can effectively improve the non-alcoholic fatty liver induced by high-fat diet rats, and is expected to be a medicament for treating the non-alcoholic fatty liver.
Claims (9)
1. The active peptide derived from leech is characterized in that the peptide sequence of the active peptide is GFAGDDAPRA, and the structural sequence is shown as SEQ ID NO. 1.
2. The method for preparing the leech bioactive peptide according to claim 1, comprising the following steps:
1) Adding 8-10 times of deionized water into Hirudo powder, and decocting for 15-20min to obtain Hirudo decoction;
2) Adding HCl solution into Hirudo decoction, adjusting pH to 1.5-2.0, adding pepsin, and performing enzymolysis at 36-38deg.C for 1-1.5 hr; then NaOH solution is added to adjust the pH of the solution to 7.8-8.5, trypsin is added, and enzymolysis is continued for 2.5-3h at 36-38 ℃; obtaining leech enzymolysis liquid;
3) Inactivating enzyme of the leech enzymolysis liquid in the step 2), cooling, centrifuging and taking supernatant;
4) Desalting the supernatant obtained in the step 3), performing ultrafiltration with a 3000Da ultrafiltration membrane, concentrating the filtrate, and freeze-drying to obtain Hirudo polypeptide lyophilized powder;
5) And separating and purifying the leech polypeptide freeze-dried powder to obtain the leech active peptide GFAGDDAPRA.
3. The method for preparing a leech bioactive peptide according to claim 2, wherein the leech bioactive peptide is prepared from leech and/or Hirudo salicifolium and/or Hirudo japonica.
4. The method for preparing a leech bioactive peptide according to claim 2, wherein in step 2), the concentration of HCl solution and NaOH solution is 0.3M-0.8M, and the enzyme-to-enzyme ratio of pepsin and trypsin is 0.5% -1.5%.
5. The method for preparing a leech bioactive peptide as claimed in claim 2, wherein in step 3), the enzyme-inactivating treatment is performed by heating at 80-100deg.C.
6. The process for preparing a leech bioactive peptide as claimed in claim 2, wherein in step 3), the centrifugation conditions are: centrifuging at 3000-4000rmp for 10-20min.
7. The process for preparing a leech bioactive peptide as claimed in claim 2, wherein in step 4), the desalting means is electrodialysis and/or dialysis bag desalting.
8. Use of the active peptide of claim 1 in a medicament for the treatment of non-alcoholic fatty liver disease.
9. A medicament comprising the leech-derived active peptide as claimed in claim 1.
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