CN101323640B - Enzymolysis products with inhibitory activity of α-glucosidase and application thereof - Google Patents

Abstract

The invention relates to an alpha-glucosidase inhibitor, in particular to an enzymolysis product with alpha-glucosidase activity, which can be obtained by enzymolysis of oyster soluble protein through pepsin. The invention applies modern enzyme catalysis technology to carry out enzymolysis on soluble protein of oyster, utilizes biomolecule separation technology to carry out preliminary separation on enzymolysis products, researches the inhibition mechanism of the component with alpha-glucosidase inhibitory activity, researches the alpha-glucosidase inhibitory activity and stability of the component in vivo and in vitro, and results show that the enzymolysis products have stronger hypoglycemic effect. Therefore, the active component and the derivative or the salt thereof can be used as long-term treatment medicines for diabetics or can be used as food additives to prepare health-care foods.

Description

Enzymolysis products with inhibitory activity of α-glucosidase and application thereof

technical field

The invention relates to an alpha-glucosidase inhibitor, in particular to an enzymatic hydrolysis product obtained by treating oyster soluble protein with pepsin and its application.

Background technique

Diabetes is a chronic disease that seriously threatens people's health and quality of life. According to the survey of the World Health Organization: At present, the number of diabetics in the world is as high as 180 million, and the diabetics in China account for 1/3 of the world. The disability and mortality caused by diabetes and its complications have become the "third killer" that threatens human health. If not treated in time, diabetics can cause numbness of limbs, body pain, blindness, renal failure, brain damage, etc. Hemorrhage, sudden death and other serious consequences. Type II diabetes accounts for about 90% of the total number of diabetic patients. Excessive postprandial blood glucose is the direct cause of complications of type 2 diabetes, and controlling postprandial blood glucose can slow down the progression of diabetes in patients with type 2 diabetes. α-glucosidase can degrade polysaccharides or oligosaccharides into monosaccharide glucose, and inhibiting the activity of α-glucosidase can effectively inhibit the rise of postprandial blood glucose. Therefore, research on α-glucosidase inhibitors is a hotspot in the development of drugs for the prevention of diabetic complications.

Existing synthetic drugs for the treatment of hyperglycemia such as: metformin, acarbose, vogboose, glimepiride and other α-glucosidase inhibitors all have many side effects, and α-glucose derived from food protein The glucosidase inhibitor has no toxic and side effects and has a good market prospect.

Contents of the invention

The invention provides a food-sourced, high-safety, cheap, industrializable enzymatic hydrolyzate with activity of inhibiting α-glucosidase and its application.

To achieve the above object, the technical solution adopted in the present invention is:

An enzymatic hydrolysis product with the activity of inhibiting α-glucosidase, which can be obtained by hydrolyzing oyster soluble protein with pepsin.

The operation process of the pepsin enzymolysis oyster soluble protein is as follows:

Oyster soluble protein is dissolved in the phosphate buffer solution of PH=2-4, and the mass volume concentration of protein in the buffer system is 1.5-5%, adds pepsin, and the mass ratio of enzyme and substrate oyster soluble protein is 1: 1-1000, enzymolysis at 25-40°C for 1-48 hours; heat the obtained enzymolysis solution to 80-100°C and keep it for 5-30 minutes to inactivate the protease; then adjust the pH of the hydrolysis solution to 6-8 , centrifuged, filtered by ultrafiltration membrane, the molecular weight cut-off MW of the ultrafiltration membrane is 8000-12000 Daltons, and the small molecular components passing through the ultrafiltration membrane are collected, which is the enzymatic hydrolysis agent with the activity of inhibiting α-glucosidase product. Vacuum freeze-drying, oyster oligopeptides, stored at -20°C;

Chromatographically separate the small molecular components that pass through the ultrafiltration membrane through a Sephadex LH-20 column, elute with a methanol solution with a volume concentration of 20-40%, the column temperature is room temperature, and the detection wavelength is 280 nanometers, and collected according to time; Fractions with strong α-glucosidase inhibitory activity were collected, and the hydrolyzed products were further purified. The concentration of salt in the phosphate buffer may be 0.01-0.2 mol/L.

The oyster soluble protein can be obtained as follows:

Shell the fresh oysters, remove the viscera, chop the remaining white muscles, gills and body fluids with a homogenizer at low temperature, add them to an equal volume of PBS buffer solution with a pH of 6-8, and stir thoroughly; After standing still for 2-10 hours at 0-8°C, centrifuge; take the supernatant, add ammonium sulfate, and make the final concentration reach 75-100%, and stand at 0-8°C for 2 -10 hours, centrifuged; the precipitate was dissolved in PBS buffer solution of PH=6-8, packed into a dialysis bag, the molecular weight cut-off MW of the dialysis bag was 8000-12000 Daltons, dialyzed with tap water for 12-36 hours, and then used The deionized water running water is dialyzed for 12-36 hours, and then the content in the dialysis bag is vacuum freeze-dried to obtain the oyster soluble protein.

The enzymatic hydrolysis products and their salts have alpha-glucosidase inhibitory activity and can be used in the preparation of medicines for treating and preventing hyperglycemia.

The medicine can be various forms of powders, granules, tablets, capsules, aqueous solutions, suspensions, emulsions, sprays or powders formed by enzymatic hydrolysis products and their salts and fillers. They have α-glucose Glucosidase inhibitory activity; through oral ingestion or in the form of injection; its use can take oral and non-oral input methods: non-oral input can be subcutaneous and intravenous injection, anorectal injection, etc.; injection For the production, physiological saline, glucose, stabilizers, preservatives, suspending agents, emulsifiers, etc. can be arbitrarily selected.

The medicine can be added to various foods as a health food for controlling blood sugar; the food can be in liquid form, such as refreshing drinks, lactic acid drinks, condiments, soups, solid cheese, ham, snacks and the like.

Said salts can be the salts (for example, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid) produced by enzymatic hydrolysis product and acid reaction; Formic acid, acetic acid, propionic acid, glycocholic acid, malic acid, citric acid, tartaric acid or succinic acid and other organic acids;), salts formed with metal ions (including sodium salts, potassium salts, calcium salts or ammonium salts), or salts formed with aminoethanol, triethylamine or dicyclic ethylamine Amine salts and the like are not particularly limited.

The usage amount of the α-glucosidase inhibitor related to the present invention is not particularly limited, but it should be appropriately determined according to the degree of hyperglycemia, the patient's age, body weight, physical condition, administration method and other factors.

The present invention uses modern enzymatic catalysis technology to enzymatically hydrolyze oyster soluble protein, utilizes biomolecular separation technology to preliminarily separate enzymolyzed products with α-glucosidase inhibitory activity, and isolate active components in vivo and in vitro The inhibitory activity of α-glucosidase has been studied, and the results show that this active component has a strong inhibitory activity of α-glucosidase in vitro; this active component can still be maintained after being treated under different temperature conditions of 4-100°C The original inhibitory activity on α-glucosidase; this active component can still maintain the original inhibitory activity on α-glucosidase after being treated with different acid-base conditions of pH=2-12; The original inhibitory activity on α-glucosidase can still be maintained after the treatment of intestinal conditions; this active component has a small molecular weight and is an oligopeptide small molecule substance, which is easy to absorb in the body and is not easy to produce immunogenicity; the isolated ileum The peristalsis test shows that the intake of this active ingredient will not affect the normal peristalsis of the ileum; this active ingredient is obtained from the degradation of natural food by pepsin, and does not produce the common toxic and side effects of synthetic drugs; the results of animal experiments show that this active ingredient is in the It has a strong hypoglycemic effect in the body. Therefore, the active ingredient and its derivatives or its salts can be used as long-term treatment drugs for diabetic patients, or used as food additives to make health food.

Description of drawings

Fig. 1 is the liquid chromatogram of Sephadex LH-20 column separation enzymatic hydrolysis product;

Fig. 2 is the relationship curve between the concentration of the active component obtained from the primary separation of the enzymatic hydrolysis product and the inhibitory activity of α-glucosidase.

Detailed ways

The present invention will be further elaborated below in conjunction with embodiment:

The preparation of embodiment 1 oyster soluble protein

Shell the fresh oysters, remove the viscera, chop the remaining white muscles, gills and body fluids with a homogenizer at low temperature, add them to an equal volume of PBS buffer solution with pH=6, and stir thoroughly; After standing still for 2 hours at ℃, centrifuge; take the supernatant, add ammonium sulfate, and make the final concentration reach 75%, and let stand for 2 hours at 0℃, centrifuge; dissolve the precipitate in pH = 6 in PBS buffer solution, put it into a dialysis bag, the molecular weight cut-off of the dialysis bag is 8000 Daltons, dialyze with running water for 12 hours, then dialyze with deionized water for 12 hours, and then vacuum freeze-dry the contents in the dialysis bag , to obtain oyster soluble protein.

The preparation of embodiment 2 oyster soluble protein

Shell the fresh oysters, remove the viscera, chop the remaining white muscles, gills and body fluids with a homogenizer at low temperature, add them to an equal volume of PBS buffer solution with a pH of 8, and stir thoroughly; After standing for 10 hours under the condition of ℃, centrifuge; take the supernatant, add ammonium sulfate, and make the final concentration reach 100%, and under the condition of 8℃, let stand for 10 hours, centrifuge; dissolve the precipitate in pH = 8 in PBS buffer solution, put it into a dialysis bag, the molecular weight cut-off of the dialysis bag is 12,000 Daltons, dialyze with running water for 36 hours, and then dialyze with deionized water for 36 hours, and then vacuum freeze-dry the contents in the dialysis bag , to obtain oyster soluble protein.

The enzymolysis of embodiment 3 oyster soluble protein

Dissolve oyster soluble protein in phosphate buffer solution with pH=2, protein concentration is 1%, add pepsin, the mass ratio of enzyme and substrate oyster soluble protein is 1:1 (mass ratio), at 25°C Enzymatic hydrolysis for 1 hour. The obtained enzymolysis solution was heated to 80°C and kept for 5 minutes to inactivate the protease. Regulate hydrolyzate pH=6 afterwards, centrifuge, filter through ultrafiltration membrane, the molecular weight cut-off of ultrafiltration membrane is 8000 Daltons, collect the small molecule component that passes through ultrafiltration membrane, and it promptly has inhibitory alpha-glucosidase Active enzymatic hydrolysis products.

The enzymolysis of embodiment 4 oyster soluble protein

Dissolve oyster soluble protein in phosphate buffer solution with pH=4, the mass volume concentration of protein in the buffer system is 5%, add pepsin, the mass ratio of enzyme and substrate oyster soluble protein is 1:1000, at 40 Enzymolysis under the condition of ℃ for 48 hours; the obtained enzymolysis solution was heated to 100℃ and kept for 30 minutes to inactivate the protease; then the hydrolysis solution was adjusted to pH=8, centrifuged, and filtered through ultrafiltration membrane, the molecular weight cut-off of ultrafiltration membrane was 12,000 Daltons, collect the small molecular components that pass through the ultrafiltration membrane, which is the enzymatic hydrolysis product that has the activity of inhibiting α-glucosidase.

The primary separation of embodiment 5 enzymolysis products

Carry out chromatographic separation (as shown in Figure 1) from 15ml of oyster soluble protein hydrolyzate through Sephadex LH-20 column, elute with 30% methanol solution, column temperature is room temperature, detection wavelength is 280 nanometers, collect according to the number of tubes , 20 minutes/tube, collect fractions with strong α-glucosidase inhibitory activity.

The determination of embodiment 6α-glucosidase inhibitory activity

The assay of α-glucosidase inhibitory activity was slightly modified according to Yong-Mu Kim's method (see Carbohydrate Research, Volume 339, 2004, Pages 715-717).

Materials and Instruments:

Preparation of pH 6.8 buffer solution: Weigh KH ₂ PO ₄ , 2.72 grams; KOH, 0.64 grams; MgCl, 2.6 grams; H ₂ O, 0.13 grams, dissolve the test drug, and dilute to 100 mL.

Preparation of experimental reagents: 0.1 unit/ml α-glucosidase and substrate (3 mmol/L p-nitrophenol pyranoside dissolved in buffer) were prepared with buffer.

Experimental method: Add 20 μL of α-glucosidase to 20 μL of experimental samples, incubate for 5 minutes in a 37°C constant temperature water bath, then add 20 μL of substrate solution, react for 30 minutes, add terminator 0.1mol/L Na ₂ CO ₃ 40 μL, and then measure the absorbance at a wavelength of 405 nm.

Specific experiment: the active components obtained from the separation of enzymatic hydrolysis products were prepared at different concentrations to detect the inhibitory activity of α-glucosidase

Sample concentration (mg/ml) 1 5 10 20 25 α-glucosidase inhibition rate (%) 2 29 58 80 85

As shown in Figure 2, the calculated final IC ₅₀ =8.8mg/ml

Claims (6)

1. one kind has the enzymolysis product that suppresses alpha-glucosidase activity, it is characterized in that: it obtains through stomach en-enzymolysis oyster soluble proteins;

Said oyster soluble proteins obtains by following process:

Internal organ are shelled, removed to fresh oyster, with white muscle, the gill and the body fluid of remainder with refiner after chopping low-temperature condition under, it is added in the PBS damping fluid of isopyknic PH=6-8 abundant stirring; Under 0-8 ℃ of condition, leave standstill 2-10 hour after, centrifugal; Get supernatant, add ammonium sulfate, and make its ultimate density reach 75-100%, and under 0-8 ℃ of condition, leave standstill 2-10 hour, centrifugal; Deposition is dissolved in the PBS damping fluid of PH=6-8; The dialysis tubing of packing into; The molecular weight cut-off MW of dialysis tubing is 8000-12000 dalton, and tap water flowing water dialysis 12-36 hour is again with deionized water flowing water dialysis 12-36 hour; With the content vacuum lyophilization in the dialysis tubing, obtain the oyster soluble protein then;

The operating process of said stomach en-enzymolysis oyster soluble proteins is following:

The oyster soluble proteins is dissolved in the phosphoric acid buffer of PH=2-4; Proteic mass and size concentration is 1-5% in the buffer system; Add stomach en-, the mass ratio of enzyme-to-substrate oyster soluble protein is 1: 1-1000, under 25-40 ℃ of condition enzymolysis 1-48 hour; The gained enzymolysis solution is heated to 80-100 ℃, and keeps 5-30 minute, makes the proteolytic enzyme inactivation; Regulate enzymolysis solution pH=6-8 afterwards, centrifugal, through ultrafiltration membrance filter, the molecular weight cut-off MW of ultra-filtration membrane is 8000-12000 dalton, collects the small molecule component that sees through ultra-filtration membrane, and it is has the enzymolysis product that suppresses alpha-glucosidase activity;

To carry out chromatographic separation through Sephadex LH-20 post through the small molecule component of ultra-filtration membrane, with the methanol solution wash-out of volumetric concentration 20-40%, column temperature is a room temperature, and detecting wavelength is 280 nanometers, collects by the time; Collection has strong alpha-glucosidase and suppresses active component, and enzymolysis product is further purified.

2. according to the said enzymolysis product of claim 1, it is characterized in that: the concentration of salt is 0.01-0.2mol/L in the said phosphoric acid buffer.

3. described enzymolysis product of claim 1 and its esters application in preparation treatment and prevention hyperglycemia medicine.

4. according to the application of described enzymolysis product of claim 3 and its esters; It is characterized in that: said medicine is enzymolysis product and its esters and the formed powder of filling agent, granule, tablet, capsule, the aqueous solution, suspension-s, emulsion, spray or pulvis, and they have alpha-glucosidase and suppress active.

5. according to the application of described enzymolysis product of claim 3 and its esters, it is characterized in that: said medicine adds in the middle of the various food, as the controlling blood sugar protective foods.

6. according to the application of described enzymolysis product of claim 3 and its esters, it is characterized in that: the salt that said salt is the salt that produces of enzymolysis product and acid-respons, form with metals ion or with monoethanolamine, the amine salt that forms of triethammonia or two ring second ammonia.

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