A method for extracting small molecule active peptides from animal eyeballs and its application

Abstract

The present application relates to a method for extracting small molecule active peptides from animal eyeballs and their application. The method comprises the following steps: S1, crushing the animal eyeballs and mixing them with water to obtain a mixed solution, and then adjusting the pH value of the mixed solution to alkaline to obtain a pretreatment solution; S2, using pancreatic enzyme and neutral protease to enzymolyze the pretreatment solution in turn to obtain an enzymolyzed solution; S3, concentrating and alcohol precipitating the enzymolyzed solution to obtain a concentrated solution; S4, cold precipitating the concentrated solution, removing the precipitate, and obtaining a decontaminated concentrated solution; S5, decolorizing and filtering the decontaminated concentrated solution to obtain a small molecule active peptide extract. The molecular weight of the small molecule active peptides extracted by the method is less than 3KDa, and has excellent anti-cataract function, and has better efficacy in preventing and treating early cataract diseases. It is also found that small molecule active peptides with isoelectric points near 11 and 7 have good antioxidant and anti-apoptotic abilities.

Description

Technical Field

[0001] The present application relates to the field of polypeptide and biomedicine technology, and in particular to a method for extracting small molecule active peptides from animal eyeballs and their application. Background Art

[0002] Cataract is the leading cause of blindness in the world. According to statistics from the Ophthalmology Branch of the Chinese Medical Association, the incidence of cataract in people aged 60 to 89 in my country is about 80%. With the acceleration of the aging of the population in my country, the number of people suffering from cataracts is increasing year by year. According to the World Health Organization, there are about 20 million people blinded by cataracts in the world, and the trend continues to rise, and may reach 50 million by 2050. Cataracts can be divided into two categories according to their causes: congenital and acquired. The latter is further divided into metabolic cataracts, senile cataracts, radiation cataracts, drug-induced cataracts, traumatic cataracts, post- or secondary cataracts, and concurrent cataracts, among which congenital, metabolic, and senile cataracts account for the vast majority. The pathogenesis of cataracts is relatively complex and is related to multiple factors such as nutrition, metabolism, environment, and genetics. It is the result of the long-term combined effects of various factors inside and outside the body on the lens. At present, the only effective way to treat cataracts is surgery. However, early prevention and treatment of cataracts are also very important, and the use of anti-cataract drugs is the main method at this stage.

[0003] Biochemical preparations extracted from the whole eyeball of animals by enzymatic hydrolysis can improve blood circulation and metabolism in the eyes, promote the absorption of vitreous opacities, prevent the development of cataracts, improve visual function, and are clinically used for early senile cataracts. At present, there is a large domestic market demand for such products, but the extraction effect is not good, and the composition of the extracted products is complex. Although it has been clinically proven to be effective, the specific effective ingredients and mechanism of action are still unclear, which is not conducive to further optimization of prescriptions and prediction of the efficacy of cataracts under different pathological conditions. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the present application provides a method for extracting small molecule active peptides from animal eyeballs. The small molecule active peptides extracted by the method have a molecular weight of less than 3KDa and have anti-cataract function, especially the above-mentioned small molecule active peptides with isoelectric points near 11 and 7, and can therefore be better used in the prevention and early treatment of cataract diseases.

[0005] To this end, the first aspect of the present application provides a method for extracting small molecule active peptides from animal eyeballs, the method comprising the following steps:

[0006] S1, mincing the animal eyeball and mixing it with water to obtain a mixed solution, and then adjusting the pH value of the mixed solution to alkaline to obtain a pretreatment solution;

[0007] S2, enzymolyzing the pretreatment solution with trypsin and neutral protease in sequence to obtain an enzymolysis solution;

[0008] S3, concentrating and precipitating the enzymatic hydrolysate to obtain a concentrated solution;

[0009] S4, cold precipitating the concentrated solution, removing the precipitate, and obtaining a decontaminated concentrated solution;

[0010] S5, decolorizing and filtering the impurity-removed concentrated solution to obtain a small molecule active peptide extract.

[0011] In the present application, the animal eyeball is selected from the eyeball of a mammal, such as a pig eyeball.

[0012] The method described in the present application obtains a small molecule active peptide extract by enzymatic hydrolysis, concentrated alcohol precipitation, cold precipitation, decolorization and filtration of the pretreated animal eyeball, and the small molecule active peptide extract has a good effect on the early treatment and prevention of cataracts. Subsequent separation of peptides with different isoelectric points and verification of peptide functions found that the peptides with PIs near 7 and 11 have better antioxidant and anti-apoptotic abilities.

[0013] In some embodiments, in step S1, the mass ratio of the animal eyeball to water is 1: (1-3), preferably 1: 2. The present application controls the mass ratio of the animal eyeball to water to 1: (1-3), especially 1: 2, so that the performance of the small molecule active peptide extract obtained can be the best.

[0014] In some embodiments, in step S1, the pH value of the mixed solution is adjusted to 8.5-9.5, preferably 9.0. By adjusting the pH value of the mixed solution to 8.5-9.5, especially 9.0, the present application can better exert the effects of pancreatic enzyme and neutral protease, thereby fully degrading the protein in the animal eyeball into small molecule active peptides, and improving the performance of the obtained small molecule active peptide extract.

[0015] The present application does not clearly limit the pH adjusting agent used for pH adjustment, which can be a conventional choice in the art. In some specific embodiments, a 10 wt % NaOH solution can be used for pH adjustment.

[0016] In some embodiments, the enzymatic hydrolysis conditions of pancreatic enzyme are as follows: adding 60 to 70 active units of pancreatic enzyme per gram of animal eyeball, the enzymatic hydrolysis temperature is 40 to 45° C., and the enzymatic hydrolysis time is 6 to 8 hours.

[0017] In some preferred embodiments, the enzymatic hydrolysis conditions of pancreatic enzyme are as follows: 64 active units of pancreatic enzyme are added to each gram of animal eyeball, the enzymatic hydrolysis temperature is 43° C., and the enzymatic hydrolysis time is 6 hours.

[0018] In some embodiments, the enzymatic hydrolysis conditions of the neutral protease are: adding 100 to 160 activity units of neutral protease per gram of animal eyeball, the enzymatic hydrolysis temperature is 45 to 48° C., and the enzymatic hydrolysis time is 4 to 6 hours.

[0019] In some preferred embodiments, the enzymatic hydrolysis conditions of the neutral protease are: adding 128 activity units of neutral protease per gram of animal eyeball, the enzymatic hydrolysis temperature is 45° C., and the enzymatic hydrolysis time is 4 hours.

[0020] In some more preferred embodiments, the neutral protease is fungal protease.

[0021] In some embodiments, the pancreatic enzyme is added in three times, specifically: 40-50% of the added amount of pancreatic enzyme is added in the first time, and enzymatic hydrolysis is carried out for 2-2.5 hours; 20-30% of the added amount of pancreatic enzyme is added in the second time, and enzymatic hydrolysis is carried out for 2-2.5 hours; and the remaining pancreatic enzyme is added in the third time, and enzymatic hydrolysis is carried out for 2-2.5 hours.

[0022] In some preferred embodiments, the pancreatic enzyme is added in three times, wherein the first time 50% of the added amount of pancreatic enzyme is added and enzymolysis is performed for 2 hours; the second time 25% of the added amount of pancreatic enzyme is added and enzymolysis is performed for 2 hours; the third time 25% of the added amount of pancreatic enzyme is added and enzymolysis is performed for 2 hours.

[0023] In some embodiments, the neutral protease is added in two times, specifically: 50-55% of the added amount of neutral protease is added in the first time, and enzymolysis is performed for 2-2.5 hours; the remaining neutral protease is added in the second time, and enzymolysis is performed for 2-2.5 hours.

[0024] In some preferred embodiments, the neutral protease is added twice, specifically: 50% of the added amount of neutral protease is added for the first time, and enzymolysis is performed for 2 hours; and 50% of the added amount of neutral protease is added for the second time, and enzymolysis is performed for 2 hours.

[0025] In the present application, after the pancreatic enzymatic hydrolysis is completed, the solution needs to be left to stand for 12 to 18 hours before adding neutral protease for enzymatic hydrolysis.

[0026] By controlling the types, enzymatic hydrolysis conditions and enzymatic hydrolysis methods of pancreatic enzymes and neutral proteases within the above ranges, the present application can fully release the small molecule active peptides in the animal eyeball and degrade them into appropriate sizes, thereby ultimately improving the performance of the obtained small molecule active peptide extract.

[0027] In some embodiments, in step S3, the enzymatic hydrolysate is firstly subjected to impurity removal before being concentrated and precipitated with alcohol; the impurity removal method is: adjusting the pH value of the enzymatic hydrolysate to 4.5-5.0, heating and boiling, and then letting it stand to collect the supernatant.

[0028] In the present application, the enzymatic hydrolysis can be terminated by heating and boiling, and the non-enzymatically hydrolyzed protein can be denatured and precipitated; and the pH value of the enzymatic hydrolyzate is adjusted to 4.5-5.0 in order to further enhance the precipitation effect.

[0029] In some embodiments, in step S3, the concentration is performed under vacuum, the concentration temperature is 50-65° C., and the volume after concentration is 5-10% of the volume before concentration.

[0030] In some embodiments, in step S3, the alcohol precipitation uses an ethanol solution with a volume concentration of more than 85%, and the amount of ethanol solution added is such that the final volume concentration of ethanol in the solution is 70% to 75%; after adding the ethanol solution, let it stand for 12 to 24 hours, take the supernatant and concentrate it again to obtain a concentrated solution.

[0031] In the present application, the conditions for the second concentration are the same as those for the first concentration, that is, the second concentration is carried out under vacuum, the concentration temperature is 50-65°C, and the volume after concentration is 15-20% of the volume before concentration.

[0032] In some embodiments, in step S4, the temperature of the cold precipitation is -5 to 0°C and the time is 72 to 84 hours.

[0033] The present application can further remove impurities such as macromolecular proteins in the small molecule active peptide extract by cold precipitation, thereby improving the performance of the small molecule active peptide extract.

[0034] In some embodiments, in step S5, the decolorization operation method is: adjusting the pH value of the impurity-removing concentrated solution to 5.0-7.0, adding activated carbon and heating the impurity-removing concentrated solution to boiling for decolorization; the amount of activated carbon added is such that the concentration of activated carbon in the impurity-removing concentrated solution is 0.4-0.5wt%.

[0035] In other embodiments, the filtering operation method is: cooling the temperature of the decolorized impurity-removed concentrated solution to less than 50° C., and then filtering through a titanium rod filter and a 0.45 μm cartridge filter respectively to obtain a small molecule active peptide extract.

[0036] The present application further removes impurities in the extract by decolorization and filtration, thereby improving the purity of the small molecule active peptides in the extract, and further improving the performance of the extracted small molecule active peptide extract.

[0037] In some embodiments, the molecular weight of the small molecule active peptide is less than 3 KDa.

[0038] In the present application, the molecular weight of the small molecule active peptide in the extract can be detected by SDS-PAGE and LC-MS / MS methods. Through SDS-PAGE detection, it was found that there were no large molecular proteins and polypeptides (>10KDa) in the small molecule active peptide extract described in the present application. Further, through LC-MS / MS detection, it was shown that the molecular weight of the small molecule active peptide was less than 3KDa, specifically 328 to 2968Da.

[0039] The second aspect of the present application provides a use of a small molecule active peptide extracted by the method described in the first aspect of the present application in the preparation of a drug for preventing and treating cataract disease.

[0040] In some preferred embodiments, the isoelectric point of the small molecule active peptide is 6.8-7.2 and / or 10.8-11.2.

[0041] The present application separates polypeptides with different isoelectric points from the small molecule active peptides by precipitation at different pH values, and then verifies the efficacy of the polypeptides with different isoelectric points. It is found that polypeptides with isoelectric points near 11 and 7 have good antioxidant and anti-apoptotic abilities; especially polypeptides with isoelectric points near 11 (10.8-11.2) have the best antioxidant and anti-apoptotic abilities.

[0042] In the present application, the pH regulator used to separate polypeptides with different isoelectric points by adjusting the pH value is preferably a weak acid and a weak base, such as acetic acid and ammonia water, because the use of strong acids (such as concentrated hydrochloric acid) and strong bases (such as caustic soda) to adjust the pH value may accelerate the hydrolysis of peptide bonds, thereby reducing the amount of precipitation (separated polypeptides).

[0043] In some preferred embodiments, the cataract disease is early cataract disease.

[0044] Through efficacy verification, the present application found that the small molecule active peptide extract extracted in the present application has good efficacy in the early treatment and prevention of cataracts, and therefore can be well used in the prevention and treatment of early cataracts.

[0045] In this application, it is necessary to construct a cell model of cataract disease when verifying the efficacy of the drug. Specifically, different concentrations of H 2 O 2 The cell culture medium (400 μM to 1000 μM) was co-cultured with cataract-derived cells to construct a cell model of cataract disease. 2 O 2 The concentrations selected were 400 μM, 800 μM and 1000 μM, corresponding to the early stage, middle stage and late stage of cataract pathological development, respectively.

[0046] In the present application, the above-mentioned cataract-derived cells can be human lens epithelial cell line SRA01 / 04; the cell culture medium can be DMEM high-glucose culture medium with 10% fetal bovine serum, wherein the culture medium can also contain 100 μg / ml penicillin and 100 μg / ml streptomycin as antibacterial agents; the co-culture time can be 1 to 24 hours, preferably 1 hour.

[0047] In the present application, when performing efficacy verification, the reconstituted solution of the small molecule active peptide extract or the polypeptide precipitated at different pH values ​​is mixed with H 2 O 2 The order of addition can be: (1) first add H 2 O 2 Then add the small molecule active peptide extract or the reconstituted solution of the peptide precipitated at different pH (therapeutic efficacy verification); (2) first add the small molecule active peptide extract or the reconstituted solution of the peptide precipitated at different pH and then add H 2 O 2 (Preventive efficacy verification); (3) Reconstitution of small molecule active peptide extracts or peptides precipitated at different pH with H 2 O 2 Also added (preventive efficacy verification).

[0048] The beneficial technical effect of the present application is: the present application provides a method for extracting small molecule active peptides from animal eyeballs, the molecular weight of the small molecule active peptides extracted by the method is less than 3KDa, and has excellent anti-cataract function. In addition, through functional verification, it is further clarified that the small molecule active peptides have better efficacy in preventing and treating early cataract diseases. At the same time, by comparing the efficacy of peptides with different isoelectric point ranges, it is found that small molecule active peptides with isoelectric points near 11 and 7 have good antioxidant and anti-apoptotic abilities, providing new development ideas for the development of efficient cataract prevention and treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the SDS-PAGE gel electrophoresis result of the small molecule active peptide extract. DETAILED DESCRIPTION

[0050] In order to make the present application easier to understand, the present application will be further described in detail below in conjunction with the examples, which are merely illustrative and are not intended to limit the scope of application of the present application. The raw materials or components used in the present application can be obtained through commercial routes or conventional methods unless otherwise specified.

[0051] Example 1: Extraction of small molecule active peptides from pig eyeballs

[0052] 5kg of pig eyeballs were minced and put into a 20L extraction tank containing 10kg of purified water, stirred evenly, and the pH value of the liquid was adjusted to 9 with a 10wt% sodium hydroxide solution. The liquid was heated to 43°C, and 1 / 2 of the prescribed amount of pancreatic enzyme (the prescribed amount is 80g, calculated at 4000μ / g, the same below) was added in sequence to hydrolyze for 2 hours; 1 / 4 of the prescribed amount of pancreatic enzyme, hydrolyzed for 2 hours; 1 / 4 of the prescribed amount of pancreatic enzyme, hydrolyzed for 2 hours; and left to stand overnight. The temperature of the hydrolyzate was adjusted and controlled at 45°C, and 1 / 2 of the prescribed amount of mycoproteinase (the prescribed amount is 160g, calculated at 4000μ / g, the same below) was added in sequence to hydrolyze for 2 hours; 1 / 2 of the prescribed amount of mycoproteinase, hydrolyzed for 2 hours. The pH value of the liquid was adjusted to 4.5 with a dilute hydrochloric acid solution, heated to boil, cooled and left to stand for 45 minutes, and the supernatant (about 8-10L) was taken. The supernatant was vacuum concentrated to 1.60L at 50°C, and an ethanol solution with a volume concentration of 85% was added while stirring (20°C) to make the final volume concentration of ethanol reach 75%. It was left to stand at room temperature for 12 hours, and the supernatant was concentrated twice in the same way to obtain a concentrated solution. After the concentrated solution was cooled, it was placed in a cold storage for cold precipitation at -5°C for 72 hours, and then taken out of the cold storage for thawing, filtering and impurity removal to obtain a concentrated solution after impurity removal. The concentrated solution after impurity removal was adjusted to pH 5.0 by adding sodium hydroxide, and activated carbon (for injection, the concentration of activated carbon in the concentrated solution after impurity removal was 0.4wt%) was added, stirred, heated to boiling for about 45 minutes, and cooled after 30 minutes. The feed solution cooled to <50°C was filtered through a titanium rod filter and a 0.45μm cartridge filter respectively to obtain a small molecule active peptide extract.

[0053] Example 2: Extraction of small molecule active peptides from pig eyeballs

[0054] The extraction method is basically the same as that of Example 1, except that the pH value of the feed solution is adjusted to 8.5 using 10 wt % sodium hydroxide solution.

[0055] Example 3: Extraction of small molecular active peptides from pig eyeballs The extraction method is basically the same as that of Example 1, except that the pH value of the feed solution is adjusted to 9.5 using 10 wt % sodium hydroxide solution.

[0056] Example 4: Extraction of small molecule active peptides from pig eyeballs

[0057] The extraction method is basically the same as in Example 1, except that the temperature of the hydrolyzate is adjusted and controlled at 45° C., the prescribed amount of mycoproteinase is added at one time, and the hydrolysis is carried out for 4 hours.

[0058] Example 5: Extraction of small molecule active peptides from pig eyeballs

[0059] The extraction method is basically the same as that of Example 1, except that the concentrate is not subjected to cold precipitation, but directly subjected to subsequent decolorization and filtration.

[0060] Example 6: Molecular weight detection of small molecule active peptides

[0061] 1. SDS-PAGE gel electrophoresis experiment

[0062] Electrode buffer solution preparation: Tris (trishydroxymethylaminomethane) 4.53g, glycine 28.2g, SDS 1.5g, mix well and add water to 1.5L. Prepare and use immediately, dissolve in water and then ultrasonicate, and store in plastic wrap at room temperature.

[0063] Preparation of staining solution: 80 mg of Coomassie Brilliant Blue, 25 mL of methanol, 8 mL of acetic acid, mix well and add water to 100 mL.

[0064] Preparation of decolorizing solution: Mix 50 mL of methanol and 16 mL of acetic acid evenly and add water to 200 mL.

[0065] Sample configuration: ① The small molecule active peptide extract obtained in Example 1 at the original concentration ② The small molecule active peptide extract obtained in Example 1 diluted 1 times ③ The small molecule active peptide extract obtained in Example 1 diluted 5 times ④ The small molecule active peptide extract obtained in Example 1 diluted 10 times ⑤ Loading buffer. After the sample is diluted (sample dilution is performed with purified water), mix it with the loading buffer at a ratio of 1:1.

[0066] Preparation before loading: boil each sample in boiling water for 5 minutes, and use Bio-Tech SDS-PAGE precast gel (Tris-Gly, 10%, 12 wells).

[0067] Experimental operation: Install the electrophoresis chamber, add the sample buffer to the outer chamber first and then to the inner chamber, use a dropper to remove bubbles, add the sample, and add the sample at 10 μl intervals, and mark the first grid. Electrophoresis conditions: 80V electrophoresis for 20 minutes, then 120V electrophoresis for about 60-90 minutes.

[0068] Staining: After the electrophoresis is completed, take out the plate, disassemble the plate and stain it with the above-mentioned Coomassie Brilliant Blue staining solution for about 4 hours.

[0069] Decolorization: Take out the stained gel and decolorize it twice with the above decolorizing solution, 100 mL each time, with an interval of 3 hours.

[0070] The results are as follows Figure 1 As shown. Figure 1 It can be seen that there are no large molecular weight proteins and polypeptides (>10KDa) in the small molecular weight active peptide extract extracted in Example 1.

[0071] 2. LC-MS / MS analysis of peptide composition of small molecule active peptide extract

[0072] Sample pretreatment: Add dithiothreitol (DTT) solution to an appropriate amount of the small molecule active peptide extract obtained in Example 1 to a final concentration of 10 mmol / L, and reduce in a 56°C water bath for 1 h. Add iodoacetamide (IAA) solution to a final concentration of 50 mmol / L, and react in the dark for 40 min. Desalt using a self-filled desalting column, and evaporate the solvent in a 45°C vacuum centrifugal concentrator.

[0073] LC-MS / MS detection:

[0074] Capillary liquid chromatography conditions:

[0075] Pre-column: 300μm id×5mm, packed with Acclaim PepMap RPLC C18, 5μm,

[0076] Analytical column: 150μm id×150mm, packed with Acclaim PepMap RPLC C18, 1.9μm,

[0077] Mobile phase A: 0.1% formic acid;

[0078] Mobile phase B: 0.1% formic acid, 80% ACN (acetonitrile);

[0079] Flow rate: 600nL / min;

[0080] Analysis time for each component: 66min;

[0081] During the analysis, the content of mobile phase B in the mobile phase is shown in Table 1.

[0082] Table 1

[0083] time Content of mobile phase B 0 4％ 2 8％ 45 28％ 55 40％ 56 95％ 66 95％

[0084] Mass spectrometry conditions:

[0085] Primary mass spectrometry parameters:

[0086] Resolution: 70,000

[0087] AGC target: 3e6

[0088] Maximum IT: 40ms

[0089] Scan range: 300~1800m / z

[0090] Secondary mass spectrometry parameters:

[0091] Resolution: 17,500

[0092] AGC target: 1e5

[0093] Maximum IT: 60ms

[0094] Top N: 20

[0095] NCE / steppedNCE:27

[0096] The results of the database search showed that the molecular weight of the polypeptide in amiodine eye drops ranged from 328 to 2968 Da.

[0097] Example 7: Separation of peptides with different isoelectric points from small molecule active peptides

[0098] Take 10 mL (3 portions) of the small molecule active peptide extract obtained in Example 1, concentrate 5 times to 2 mL by rotary evaporation at 50°C, and then adjust the pH to 3.0, 7.0 and 11.0 with acetic acid or ammonia water. Then centrifuge at 4°C, 10000 r / min, twice at an interval of 20 minutes each time. Collect the precipitate to obtain polypeptides with isoelectric points of 3.0, 7.0 and 11.0, respectively. Add 10 mL of sterile water to the polypeptides, redissolve them to the original volume, and obtain a resolubilized solution of the polypeptides precipitated at pH 3.0, 7.0 and 11.0, respectively.

[0099] Example 8: Pharmacodynamics Study

[0100] Human lens epithelial cell line SRA01 / 04 was used as cataract-derived cells to construct a cell model of cataract disease. The small molecule active peptide extracts used in the following experiments were all small molecule active peptide extracts obtained in Example 1.

[0101] 1. Cell Culture

[0102] Human lens epithelial cell line SRA01 / 04 was cultured in DMEM high glucose medium containing 10% fetal bovine serum, 100 μg / mL penicillin and 100 μg / mL streptomycin in a T25 flask at 37°C and 5% CO. 2 The cells were cultured in an incubator with sufficient saturated humidity, and the medium was changed every 2-3 days. After the cells grew to confluence, they were digested with 0.25% trypsin solution and centrifuged to prepare a single cell suspension, which was passaged at a ratio of 1:2.

[0103] 2. MTT experiment of cataract cell model group

[0104] SRA01 / 04 cells in the logarithmic growth phase were cultured at 5×10 3 The cells were seeded in 96-well plates at a density of 100 μL per well and incubated in CO 2Incubator (37°C, 5% CO 2 ) for 24 h. After incubation, remove the old culture medium and add H 2 O 2 . Divided into 5 hours 2 O 2 Treatment group (H 2 O 2 concentrations of 100 μM, 200 μM, 400 μM, 800 μM and 1000 μM) and a 2 O 2 The control group was treated with culture medium 2 O 2 Dilute to the corresponding concentration and add 100 μL of the above-mentioned solution with corresponding H 2 O 2 The blank group was filled with 100 μL PBS buffer and placed in a CO 2 Incubator (37°C, 5% CO 2 ) for 1h and 24h, respectively, and then 10μL MTT (5mg / mL) was added to each well. The culture was continued at 37℃ for 4h, the old medium was removed, and 150μL DMSO was added to each well for fixation. The absorbance (OD value) of each well was measured at a wavelength of 490nm on a microplate reader. The cell survival rate was calculated by substituting the following formula, and the cell viability at different concentrations of H was calculated. 2 O 2 The cell survival rates under the treatments are shown in Tables 2 and 3, respectively.

[0105] Cell viability (%) = [OD(treated) - OD(blank)] / [OD(control) - OD(blank)] × 100%; wherein, OD(sample): OD value of the treated group; OD(blank): OD value of the blank group; OD(control): OD value of the control group.

[0106] Table 2: Different H 2 O 2 The cell viability after 1 hour of coexistence of the concentration

[0107]

[0108]

[0109] Table 3: Different H 2 O 2 The cell survival rate after 24 hours of coexistence of the concentration and cells

[0110]

[0111] From Table 2 and Table 3, we can see that as H 2 O 2With the increase of H concentration, the cell survival rate gradually decreased, and it was related to the exposure to different concentrations of H 2 O 2 Compared with 1h, exposure to different concentrations of H 2 O 2 The cell survival rate at 24 h was lower, indicating that 2 O 2 It can inhibit the activity of human lens epithelial cells, and the higher the concentration and the longer the time, the greater the damage. Therefore, it is feasible to establish a method for cataract disease using hydrogen peroxide oxidation. At the same time, according to the IC50 value, 400uM, 800uM and 1000uM H 2 O 2 The amount added corresponds to the early stage, middle stage and late stage of cataract pathological development.

[0112] 3. MTT experiment of small molecule active peptide extract group

[0113] (1) Co-culture for 1 h

[0114] SRA01 / 04 cells in the logarithmic growth phase were cultured at 5×10 3 The cells were seeded in 96-well plates at a density of 100 μL per well and incubated in CO 2 Incubator (37°C, 5% CO 2 ) and incubate for 24 hours. After the incubation, the old culture medium was removed and small molecule active peptide extracts were added according to the experimental groups. The cells were divided into 9 small molecule active peptide extract sample groups (the concentrations of the small molecule active peptide extracts were diluted 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 300 times, 500 times, and 1000 times, respectively) and a control group without adding small molecule active peptide extracts. The small molecule active peptide extracts were diluted to the corresponding concentrations with culture medium, and 100 μL of the small molecule active peptide extracts diluted to the corresponding concentrations were added to each well. The cell-free wells with 100 μL of PBS buffer were used as blank groups and placed in CO 2 Incubator (37°C, 5% CO 2 ) were incubated for 1 hour, 10 μL MTT (5 mg / mL) was added to each well, and the culture was continued at 37°C for 4 hours. The old culture medium was removed, 150 μL DMSO was added to each well for fixation, and the absorbance (OD value) of each well was measured at a wavelength of 490 nm on an ELISA reader. The cell survival rate was calculated by substituting the following formula, and the cell survival rate under different concentrations of small molecule active peptide extracts was calculated. The results are shown in Table 4.

[0115] Cell viability (%) = [OD (sample) - OD (blank)] / [OD (control) - OD (blank)] × 100% wherein OD (sample): OD value of the sample group; OD (blank): OD value of the blank group; OD (control): OD value of the control group.

[0116] Table 4: Cell survival rate after co-existence of different small molecule active peptide extract concentrations with cells for 1 hour

[0117]

[0118] As can be seen from Table 4, as the concentration of the small molecule active peptide extract decreases, the cell survival rate shows a trend of first increasing and then decreasing, and finally remains basically unchanged after being diluted 200 times. When the small molecule active peptide extract is at a high concentration (diluted 2 times), it inhibits the growth of SRA01 / 04 cells. When the dilution multiple increases and the concentration of the small molecule active peptide decreases, the inhibition of SRA01 / 04 cell growth is significantly reduced. When the small molecule active peptide extract stock solution is diluted 100 times, the cell survival rate is the highest, and it has a certain promoting effect on the growth of SRA01 / 04 cells.

[0119] (2) Co-culture for 24 hours

[0120] SRA01 / 04 cells in the logarithmic growth phase were cultured at 5×10 3 The cells were seeded in 96-well plates at a density of 100 μL per well and incubated in CO 2 Incubator (37°C, 5% CO 2 ) and incubate for 24 hours. After incubation, remove the old culture medium and add small molecule active peptide extract according to the experimental groups. Divide into 5 small molecule active peptide extract sample groups (the concentrations of small molecule active peptide extract were diluted 5 times, 10 times, 50 times, 100 times, and 500 times, respectively) and a control group without adding small molecule active peptide extract. Dilute the small molecule active peptide extract to the corresponding concentration with culture medium, add 100 μL of the small molecule active peptide extract diluted to the corresponding concentration to each well, add 100 μL of PBS buffer to the cell-free well as the blank group, and place it in CO 2 Incubator (37°C, 5% CO 2 ) for 24 hours, 10 μL MTT (5 mg / mL) was added to each well, and the cells were cultured for another 4 hours at 37°C. The old medium was removed, and 150 μL DMSO was added to each well for fixation. The absorbance (OD value) of each well was measured at a wavelength of 490 nm on a microplate reader. The cell survival rate was calculated by substituting the following formula, and the cell survival rate at different concentrations of small molecule active peptide extracts was calculated. The results are shown in Table 5.

[0121] Cell viability (%) = [OD (sample) - OD (blank)] / [OD (control) - OD (blank)] × 100% wherein OD (sample): OD value of the sample group; OD (blank): OD value of the blank group; OD (control): OD value of the control group.

[0122] Table 5: Cell survival rate after 24 hours of co-culture with cells at the same concentration of small molecule active peptide extract

[0123]

[0124]

[0125] As shown in Table 5, after the in vitro cultured human lens epithelial cell line SRA01 / 04 was exposed to different concentrations of small molecule active peptide extracts (diluted 5, 10, 50, 100 and 500 times) for 24 hours, the MTT method was used to detect the effect of different concentrations of small molecule active peptide extracts on cell survival rate. The results showed that with the decrease of the concentration of small molecule active peptide extracts, the cell survival rate first increased and then decreased, and finally reached a peak value when diluted 100 times. The change pattern was similar to the result of co-culture for 1 hour. After that, the experiment selected the small molecule active peptide extract diluted 100 times as the uniform sample concentration, and the co-culture time was set to 1 hour.

[0126] 4. Small molecule active peptide extracts for MTT experiments in oxidative stress models

[0127] (1) First H 2 O 2 Small molecule active peptide extract (therapeutic efficacy verification group): SRA01 / 04 cells in the logarithmic growth period were cultured at 5×10 3 The cells were seeded in 96-well plates at a density of 100 μL per well and incubated in CO 2 Incubator (37°C, 5% CO 2 ) for 24 h. After incubation, remove the old culture medium and add H 2 O 2 .H 2 O 2 Treatment group (H 2 O 2 The concentrations were 400 μM, 800 μM and 1000 μM, respectively. 2 O 2 A control group (medium dilution) and a blank group without cells (PBS buffer dilution) were added with 100 μL per well and placed in CO 2 Incubator (37°C, 5% CO 2 ) and incubate for 1 hour. Remove the old culture medium and add 100 μL of the experimental groups (① 100-fold diluted solution of peptides precipitated at pH 3.0, ② 100-fold diluted solution of peptides precipitated at pH 7.0, ③ 100-fold diluted solution of peptides precipitated at pH 11.0, ④ 100-fold diluted small molecule active peptide extract, ⑤ PBS) to each well. Add equal amounts of complete culture medium and PBS buffer to the control group and blank group, respectively. Place in CO 2 Incubator (37°C, 5% CO 2) for 1 hour. 10 μL MTT (5 mg / mL) was added to each well, and the culture was continued at 37°C for 4 hours. The old culture medium was removed, and 150 μL DMSO was added to each well for fixation. The absorbance (OD value) of each well was measured at a wavelength of 490 nm on an ELISA reader. The results are shown in Table 6.

[0128] (2) First, extract the small molecule active peptides and then H 2 O 2 (Preventive efficacy verification group 1): SRA01 / 04 cells in the logarithmic growth phase were cultured at 5×10 3 The cells were seeded in 96-well plates at a density of 100 μL per well and incubated in CO 2 Incubator (37°C, 5% CO 2 ) for 24 h. After incubation, remove the old culture medium, add 100 μL of the experimental group (① 100-fold diluted solution of peptide precipitated at pH 3.0, ② 100-fold diluted solution of peptide precipitated at pH 7.0, ③ 100-fold diluted solution of peptide precipitated at pH 11.0, ④ 100-fold diluted small molecule active peptide extract, ⑤ PBS) to each well; add equal amounts of complete culture medium and PBS buffer to the control group and blank group respectively; place in CO 2 Incubator (37°C, 5% CO 2 ) for 1 h. The old culture medium was removed and H 2 O 2 (Concentrations were 400μM, 800μM and 1000μM, respectively), and the control group and blank group were added with equal volume of complete culture medium and PBS buffer, respectively. 2 Incubator (37°C, 5% CO 2 ) for 1 hour. 10 μL MTT (5 mg / mL) was added to each well, and the culture was continued at 37°C for 4 hours. The old culture medium was removed, and 150 μL DMSO was added to each well for fixation. The absorbance (OD value) of each well was measured at a wavelength of 490 nm on an ELISA reader. The results are shown in Table 6.

[0129] (3) Small molecule active peptide extract and H 2 O 2 At the same time (preventive efficacy verification group 1): SRA01 / 04 cells in the logarithmic growth phase were cultured at 5×10 3 The cells were seeded in 96-well plates at a density of 100 μL per well and incubated in CO 2 Incubator (37°C, 5% CO 2 ) for 24 h. After incubation, remove the old culture medium and add H 2 O 2 . Divided into 3 H 2 O 2 Treatment group (H2 O 2 The concentrations were 400 μM, 800 μM and 1000 μM, respectively. 2 O 2 100 μL of the experimental group (① 100-fold diluted solution of peptide precipitated at pH 3.0, ② 100-fold diluted solution of peptide precipitated at pH 7.0, ③ 100-fold diluted solution of peptide precipitated at pH 11.0, ④ 100-fold diluted small molecule active peptide extract, ⑤ PBS) was added to the treatment group. In addition, the control group and the blank group were added with equal amounts of complete culture medium and PBS buffer. 2 Incubator (37°C, 5% CO 2 ) for 1 hour, then the old medium was removed. 10 μL MTT (5 mg / mL) was added to each well, and the cells were cultured for another 4 hours at 37°C. The old medium was removed, and 150 μL DMSO was added to each well for fixation. The absorbance (OD value) of each well was measured at a wavelength of 490 nm on an ELISA reader. The results are shown in Table 6.

[0130] Table 6: Results of MTT experiments using small molecule active peptide extracts in oxidative stress models

[0131]

[0132]

[0133] As shown in Table 6, comparing experimental group ④ with experimental group ⑤, the addition of small molecule active peptide extract can significantly increase the OD value of the solution in the corresponding well, indicating that the small molecule active peptide extract can significantly increase the cell survival rate and improve its growth environment. Therefore, the small molecule active peptide extract can be used as an effective ingredient for anti-cataract biological preparations.

[0134] By comparing the MTT data of experimental groups ①~④ (① 100-fold diluted re-solution of the polypeptide precipitated at pH 3.0, ② 100-fold diluted re-solution of the polypeptide precipitated at pH 7.0, ③ 100-fold diluted re-solution of the polypeptide precipitated at pH 11.0, ④ 100-fold diluted small molecule active peptide extract), it was found that the MTT data of experimental groups ①~③ were all smaller than those of experimental group ④, indicating that in the process of anti-cataract treatment, polypeptides with different isoelectric points (PI 3~11) in the small molecule active peptide extract of pig eyes play a synergistic therapeutic effect. Further comparison of experimental groups ① to ③ (① 100-fold dilution of the resolution of the peptide precipitated at pH 3.0, ② 100-fold dilution of the resolution of the peptide precipitated at pH 7.0, ③ 100-fold dilution of the resolution of the peptide precipitated at pH 11.0) revealed that the therapeutic effects of experimental groups ① to ③ were: resolution of the peptide precipitated at pH 11.0 (100-fold dilution) > resolution of the peptide precipitated at pH 7.0 (100-fold dilution) > resolution of the peptide precipitated at pH 3.0 (100-fold dilution). This indicates that peptides with PIs around 7 and 11 have better antioxidant and anti-apoptotic abilities.

[0135] In addition, for each experimental group and H 2 O 2 The order of addition was investigated. MTT results showed that: 2 O 2 Adding it to each experimental group at the same time has the least effect on cell growth. 2 O 2 The experimental group came first, followed by the H 2 O 2 Worst. Possible reasons: (1) Experimental group first, then H 2 O 2 , the cells may first absorb and utilize the amino acids or peptides in each experimental group, resulting in extracellular H 2 O 2 / polypeptide (reducing) concentration ratio increases, affecting the absorbance. This indicates that the small molecule active peptide extract has no significant long-term preventive function (2) 2 O 2 In the latter experimental group, cells were first exposed to H 2 O 2 , which would damage cell viability, and then add each experimental group to cause extracellular H 2 O 2 / peptide (reducing) concentration ratio decreased, affecting the absorbance. This indicates that the small molecule active peptide extract has a certain therapeutic ability for cataracts (3) H 2 O 2 The experimental group was added at the same time, and the two may have a direct effect. 2 O 2 The concentration of extracellular H 2 O2 The ratio of H / peptide (reducing) concentration remained unchanged. This indicates that the peptide (reducing) in each experimental group has a certain inhibitory effect on oxidative stress, but H 2 O 2 Oxidative damage is still the main influencing factor. The small molecule active peptide extract showed the effect of cataract prevention.

[0136] Finally, we compared the effects of each experimental group on different stages of cataract (400uM H 2 O 2 Treatment, 800uM H 2 O 2 Treatment, 1000uM H 2 O 2 treatment), it was found that with the increase of H 2 O 2 With the increase of concentration, the therapeutic effect of small molecule active peptide extract and its PI separated polypeptide components was significantly reduced, indicating that small molecule active peptide extract and its PI separated polypeptide components have good therapeutic effect on early cataract.

[0137] In summary, the small molecule active peptide extract from pig eyes has a good effect on the early treatment and prevention of cataracts. Its main active ingredients are small molecule peptides (<3KDa), among which peptides with PIs around 7 and 11 have better antioxidant and cell apoptosis abilities.

[0138] Example 9: Pharmacological Efficacy Verification of the Small Molecular Active Peptide Extracts Obtained in Examples 2-5 The small molecular active peptide extracts obtained in Example 8 were used in the MTT experiment in the oxidative stress model. 2 O 2 The small molecule active peptide extract (therapeutic efficacy verification group) was used to verify the efficacy of the small molecule active peptide extract obtained in Example 2-5. The specific steps are as follows:

[0139] SRA01 / 04 cells in the logarithmic growth phase were cultured at 5×10 3 The cells were seeded in 96-well plates at a density of 100 μL per well and incubated in CO 2 Incubator (37°C, 5% CO 2 ) for 24 h. After incubation, remove the old culture medium and add H 2 O 2 .H 2 O 2 Treatment group (H 2 O 2 The concentrations were 400 μM, 800 μM and 1000 μM, respectively. 100 μL was added to each well and placed in CO 2 Incubator (37°C, 5% CO 2) and incubate for 1 hour. Remove the old culture medium and add 100 μL of the experimental groups (① the small molecule active peptide extract obtained in Example 2 diluted 100 times, ② the small molecule active peptide extract obtained in Example 3 diluted 100 times, ③ the small molecule active peptide extract obtained in Example 4 diluted 100 times, and ④ the small molecule active peptide extract obtained in Example 5 diluted 100 times) to each well. Place in CO 2 Incubator (37°C, 5% CO 2 ) for 1 hour. 10 μL MTT (5 mg / mL) was added to each well, and the culture was continued at 37°C for 4 hours. The old culture medium was removed, and 150 μL DMSO was added to each well for fixation. The absorbance (OD value) of each well was measured at a wavelength of 490 nm on an ELISA reader. The results are shown in Table 7.

[0140] Table 7: Results of MTT experiments using the small molecule active peptide extracts obtained in Examples 2-5 in oxidative stress models

[0141]

[0142] As can be seen from Table 7, the MTT data of the small molecule active peptide extracts obtained in Examples 2-5 are all smaller than the MTT data of the small molecule active peptide extract obtained in Example 1. This indicates that the small molecule active peptide extract obtained in Example 1 has better therapeutic effect.

[0143] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. A method for extracting small molecule active peptides from animal eyeballs, characterized in that: The method comprises the following steps: S1, mincing the animal eyeball and mixing it with water to obtain a mixed solution, and then adjusting the pH value of the mixed solution to alkaline to obtain a pretreatment solution; S2, enzymolyzing the pretreatment solution with trypsin and neutral protease in sequence to obtain an enzymolysis solution; S3, concentrating and precipitating the enzymatic hydrolysate to obtain a concentrated solution; S4, cold precipitating the concentrated solution, removing the precipitate, and obtaining a decontaminated concentrated solution; S5, decolorizing and filtering the impurity-removed concentrated solution to obtain a small molecule active peptide extract; The enzymatic hydrolysis conditions of the pancreatic enzyme are as follows: adding 60-70 active units of pancreatic enzyme per gram of animal eyeball, the enzymatic hydrolysis temperature is 40-45° C., and the enzymatic hydrolysis time is 6-8 hours; The enzymatic hydrolysis conditions of the neutral protease are as follows: adding 100-160 activity units of neutral protease per gram of animal eyeball, the enzymatic hydrolysis temperature is 45-48° C., and the enzymatic hydrolysis time is 4-6 hours; The pancreatic enzyme is added in three times, specifically: 40-50% of the added amount of pancreatic enzyme is added in the first time, and the enzymatic hydrolysis is carried out for 2-2.5 hours; 20-30% of the added amount of pancreatic enzyme is added in the second time, and the enzymatic hydrolysis is carried out for 2-2.5 hours; the remaining pancreatic enzyme is added in the third time, and the enzymatic hydrolysis is carried out for 2-2.5 hours; The neutral protease is added in two times, specifically: 50-55% of the added amount of neutral protease is added in the first time, and enzymolysis is performed for 2-2.5 hours; the remaining neutral protease is added in the second time, and enzymolysis is performed for 2-2.5 hours; The decolorization operation method is: adjusting the pH value of the impurity removal concentrate to 5.0-7.0, adding activated carbon and heating the impurity removal concentrate to boiling for decolorization; the amount of activated carbon added is such that the concentration of activated carbon in the impurity removal concentrate is 0.4-0.5wt%; The filtering operation method is: cooling the temperature of the decolorized impurity-removing concentrated solution to less than 50° C., and then filtering through a titanium rod filter and a 0.45 μm cartridge filter respectively to obtain a small molecule active peptide extract.

2. The method according to claim 1, characterized in that In step S1, the mass ratio of the animal eyeball to water is 1:(1-3); and / or In step S1, the pH value of the mixed solution is adjusted to 8.5-9.

5.

3. The method according to claim 2, characterized in that In step S1, the pH value of the mixed solution is adjusted to 9.

0.

4. The method according to claim 1 or 2, characterized in that: In step S3, the enzymatic hydrolysate is firstly removed from impurities before being concentrated and precipitated with alcohol; the method of removing impurities is: adjusting the pH value of the enzymatic hydrolysate to 4.5-5.0, heating and boiling, and then letting it stand to collect the supernatant.

5. The method according to claim 1 or 2, characterized in that: In step S3, the concentration is carried out under vacuum, the concentration temperature is 50-65° C., and the volume after concentration is 15-20% of the volume before concentration; and / or In step S3, the alcohol precipitation uses an ethanol solution with a volume concentration of more than 85%, and the amount of the ethanol solution added is such that the final volume concentration of ethanol in the solution is 70% to 75%; after adding the ethanol solution, the solution is allowed to stand for 12 to 24 hours, and the supernatant is taken and concentrated again to obtain a concentrated solution.

6. The method according to claim 1 or 2, characterized in that: In step S4, the temperature of the cold precipitation is -5 to 0°C, and the time is 72 to 84 hours.

7. The method according to claim 1 or 2, characterized in that: The molecular weight of the small molecule active peptide is less than 3KDa.

8. Use of a small molecule active peptide extracted by the method as claimed in any one of claims 1 to 7 in the preparation of a drug for preventing and treating cataract disease.

9. The use according to claim 8, characterized in that: The isoelectric point of the small molecule active peptide is 6.8-7.2 and / or 10.8-11.

2.

10. The use according to claim 8, characterized in that: The cataract disease is an early stage cataract disease.

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