TWI487529B - A composition for reducing insulin resistance - Google Patents

Description

Composition for reducing insulin resistance

The present invention relates to the use of a meat and/or green algae hydrolyzed extract for the preparation of a composition for reducing insulin resistance, and to a method of preparing the composition.

Diabetes is a chronic hyperglycemic metabolic disease and one of the most serious chronic diseases recognized worldwide. According to the World Health Organization's 2011 statistics, there are currently about 220 million people worldwide suffering from diabetes (World Health Organization. 2011. Media centre-Diabetes-Fact sheets.). Diabetes can be mainly divided into type I, type 2 diabetes, gestational diabetes and other types of diabetes (American Diabetes Association. 2010. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care. 33: S63-S65), of which type 1 diabetes Because the pancreas can not normally secrete insulin, it needs to be treated with insulin for a lifetime. The second type of diabetes patients account for 90-95% of the diabetic patients. The pathogenesis is mainly due to the resistance of the surrounding tissues to insulin, which reduces the insulin utilization rate. , or with the relative lack of insulin, it leads to an increase in blood sugar. The drugs currently used to treat patients with type 2 diabetes are mainly divided into four categories: sulfonium urea (stimulating pancreatic insulin secretion), biguanides (inhibiting liver gluconeogenesis and enhancing liver and skeletal muscle sensitivity to insulin). , Thiazolidinedione (reducing lipolysis, increasing sensitivity of liver and skeletal muscle to insulin) and α-glucosidase inhibitor (reducing carbohydrate absorption in the intestine), however, the first three drugs are easy to have Serious side effects such as hypoglycemia, lactic acid, etc., have relatively small side effects of the relative α-glucosidase inhibitor. Among the α-glucosidase inhibitors, the currently widely used drug is Acarbose, which is a pseudo-tetrasaccharide structure that simultaneously inhibits α-glucosidase and α-amylase to delay blood sugar. The effect of ascending (Bischoff, H. 1995. The mechanism of alpha-glucosidase inhibition in the management of diabetes. Clinical & Investigative Medicine. 18: 303-11.).

In recent years, many diabetes drugs have been used to treat type 2 diabetes, which can be divided into (1) incretin analogs, such as glucagon-like peptide-1 (GLP-1). ), can stimulate insulin secretion, delay islet cell apoptosis, slow gastric emptying, suppress appetite and inhibit glycoside; (2) dipeptidyl peptidase-4 (DPP-4) inhibitor For example, sitagliptin inhibits DPP-4 in the blood and increases GLP-1 and glucose-dependent insulinotropic hormones (GIP), thereby increasing insulin secretion to lower blood glucose; (3) amyloid analogues (amylin analogues) ), can slow gastric emptying, inhibit digestive enzyme secretion, and synergize insulin to control glucose.

Chlorella is a green algae of the green algae plant. It is a single-celled microalgae. Chlorella is rich in nutrients, mainly containing protein, carbohydrates and lipids. It also contains minerals such as calcium, phosphorus, iron, potassium and magnesium. , sodium, zinc, vitamin A, C, E, B group, carotene, chlorophyll, green algae, folic acid and inositol and other nutrient sources (Spolaore, P. et al.2006.Commercial applications of microalgae.Journal of Bioscience and Bioengineering. 101: 87-96). However, green algae have cell walls, so it is necessary to physically or enzymatically hydrolyze lipids to release internal active substances. Regarding the application of green algae in addition to lowering blood pressure and blood lipids in living, it has also been reported that feeding streptococcal (STZ)-induced diabetic mice with green algae can increase liver and muscle utilization of glucose, and also reduce serum The concentration of esterified fatty acids, which in turn enhances insulin sensitivity, achieves hypoglycemic effects (Chemg, JY et al. 2006. Improving glycogenesis in Streptozocin (STZ) diabetic mice after administration of green algae Chlorella. Life Sciences. 78: 1181-1186 In addition, after feeding the diabetic rats with green algae for 6 weeks, the fasting glucose concentration of the rats given Chlorella was significantly decreased, while the normal rats did not cause hypoglycemia after 6 weeks of feeding (Jeong, H. et al . 2009.Hypoglycemic effect of Chlorella vulgaris intake in type 2 diabetic Goto-Kakizaki and normal Wistar rats Nutrition Research and Practice 3:.. 23-30); also reported that a hydrolyzate of algae for lysing α- glucosidase Enzymes and alpha-amylases have good inhibitory capacity.

Taiwanese is commonly known as Lazi, which is the main species of farmed alfalfa in Taiwan. Its yield is the third in the edible economic shellfish after oysters and clams. Its nutritional value is high. The general composition of loquat meat contains 85% of water and protein 5.3. %, fat 2.0%, sugar 7.0%, ash 0.7%, calcium 133 mg, phosphorus 92 mg, vitamin A 1900 IU, riboflavin 0.4 mg, and the like. Regarding the application of the meat extract, it has been reported that the β-alanyl-L-histidine contained in the meat extract has the ability to resist oxidation and scavenge free radicals (Reddy, VP et al. 2005. Carnosine). :a versatile antioxidant and antiglycating agent.Science of aging knowledge environment.18:pe12). In addition, the hydrolyzed extract of scorpion meat was found to have good inhibitory ability against angiotensin converting enzyme (ACE), and the isolated three peptides Val-Lys-Pro and Val-Lys-Lys can effectively inhibit ACE. After eight weeks of feeding the hypertensive rats with the hydrolyzed extract of medlar meat, systolic blood pressure and diastolic blood pressure were significantly reduced (Tsai, J Set al. 2006. The inhibitory effects of freshwater clam (Corbicula fluminea, Muller) muscle Protein hydrolysates on angiotensin I converting enzyme. Process Biochemistry. 41:2276-2281). On the other hand, it has also been reported that the extract of clam meat can reduce the content of cholesterol and triglyceride in the living body, and the hyperglycemic rats are fed with the hot water extract of the meat. After two weeks, the cholesterol content in the plasma can be effectively reduced and increased. Excretion of bile acid in feces; after feeding the Sprague-Dawley rats for four weeks, the plasma triglyceride and total cholesterol were reduced by 65.8% and 26.1%, respectively, in the liver, triglyceride and Total cholesterol decreased by 27.6% and 50.5% compared with the control group, total cholesterol output in feces was 2.1 times that of the control group, and bile acid content was 1.2 times higher than that of the control group. However, there is no literature showing the use of mash meat hydrolysate or in combination with sorghum hydrolysate and green algae hydrolysate to reduce insulin resistance.

The present invention unexpectedly finds that the hydrazine hydrolyzed extract or the combined hydrazine hydrolyzed extract and the green algae hydrolyzed extract have the effect of reducing insulin resistance. Therefore, the present invention provides a hydrolyzed extract of sorghum or combined with hydrazine hydrolysis. The extract and the green algae hydrolyzed extract are used for the preparation of a composition for reducing insulin resistance.

Accordingly, in one aspect, the present invention provides a composition for reducing insulin resistance which simultaneously has the effect of lowering blood glucose and treating or preventing diabetes, comprising an effective amount of a meat extract. Wherein the meat extract is prepared by the following steps, comprising reacting the meat with water at an extraction temperature for an extraction time, wherein the extraction temperature is 70 to 150 ° C, and the extraction time is 30 to 50 minutes; The meat, the protease is added, and reacted at a hydrolysis temperature for a hydrolysis time to obtain the meat extract, wherein the protease is 0.5 to 2% of the protein content of the meat, the hydrolysis temperature is 35 to 60 ° C, and the hydrolysis The time is 4 to 6 hours.

In one embodiment of the invention, the extraction temperature of the extracted meat is about 100 The extraction time was about 40 minutes at °C.

In one embodiment of the invention, the protease is about 1% of the protein content of the meat, the hydrolysis temperature is about 50 ° C, and the hydrolysis time is about 5 hours.

In one embodiment of the invention, the meat is from Corbicula fluminea .

In another aspect, the present invention provides a composition for lowering blood glucose, reducing insulin resistance, and treating or preventing diabetes, comprising an effective amount of a combination of a meat extract and a green algae extract, wherein the green algae extract and the The ratio of the meat extract is 1:1 to 3:1.

In a specific embodiment of the present invention, the green algae extract is prepared by the following steps, comprising reacting the green algae powder with a cellulase and a protease at a hydrolysis temperature for a hydrolysis time to obtain the green algae extract, wherein The cellulase is 4 to 6% by weight of the green algae powder, and the protease is 1 to 3% by weight of the green algae powder, the hydrolysis temperature is 40 to 60 ° C, and the hydrolysis time is 2 to 4 hours.

In one embodiment of the invention, the green algae powder is from Chlorella sorokiniana .

The compositions of the present invention can be formulated into a pharmaceutical composition, or a food composition, or a beverage composition.

The invention also provides a use of a meat extract for preparing a composition for reducing insulin resistance, and a combination of a meat extract and a green algae extract for preparing blood sugar lowering, reducing insulin resistance, and treating or preventing diabetes. The use of the composition.

Various specific examples of the invention are detailed below. Other features of the present invention will be apparent from the following detailed description, drawings and claims.

Figure 1 shows the synergistic effect of different ratios of lysine hydrolyzed extracts and cyanobacterial hydrolyzed extracts on inhibition of alpha-glucosidase.

Figure 2 shows the results obtained by filtration of colloidal green algae hydrolyzed extract by colloidal chromatography. According to its molecular weight, the partitions P ₁ (1710-1480 Da), P ₂ (860-740 Da), P ₃ (570- 490 Da), P ₄ (350-300 Da) and C ₁ (1250-1050 Da).

Figure 3 shows the results obtained by gel filtration chromatography of a cyanobacteria hydrolyzed extract on a BPG column.

Figure 4 shows the results of further separation of the fraction P ₄ obtained by colloidal filtration chromatography with a C ₁₈ semi-preparative column by reverse phase high performance liquid chromatography, and seven major peak species can be proposed.

Figure 5 shows the results of a C ₁₂ column analysis of the second peak material obtained by reverse phase high performance liquid chromatography of a C ₁₈ semi-preparative column, and four major peak species were proposed.

Figure 6 shows the results of further separation of the fraction P ₄ obtained by colloidal filtration chromatography by high performance liquid chromatography using a HILIC semi-preparative column, and two major peaks can be proposed within 30 minutes. H ₁ and H ₂ .

Figure 7 shows the results obtained by analyzing the peak H ₂ obtained by high performance liquid chromatography of HILIC semi-preparative column and then analyzing by C ₁₂ column.

Figure 8 shows the results of a glucose tolerance test after eight weeks of administration of a green algae hydrolyzed extract and a hydrolyzed extract of sorghum (CH, PX and CH/PX) in STZ diabetic rats.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. As used herein, in the event of a conflict, the present document, including definitions, will control.

The term "a" as used herein, unless otherwise specified, means that the grammatical acceptance of the article is one or more (ie, at least one). For example, "a component" means one component or more than one component.

In the present invention, it has been unexpectedly found that the hydrolyzed extract of the meat and/or the hydrolyzed extract of the green algae has the effect of reducing insulin resistance.

In the example of the present invention, the hydrolysis extract of the meat, the green algae hydrolysis extract and the combination thereof are reacted with α-glucosidase, α-amylase or DPP4, and the hydrolysis extract of the meat and the hydrolysis of the green algae are found. The extract and its combination have an inhibitory effect on the above enzymes. Further, when the hydrolyzed extract of the meat and the green algae hydrolyzed extract are combined in a ratio of 1:3 to 1:1, the α-glucosidase inhibitory effect is 1.1 to 1.7 times that of the individual hydrolyzed extract, indicating The composition has a synergistic effect.

In another embodiment of the present invention, the extract of the meat is hydrolyzed and the water with the green algae The combination of extract extracts was administered to STZ-induced diabetic rats, and it was found to be effective in reducing insulin and insulin resistance in plasma of rats, and at the same time reducing the fasting blood glucose and increasing glucose tolerance.

Accordingly, the present invention provides a composition for reducing insulin resistance comprising an effective amount of a meat extract or a combination thereof with a green algae extract.

The present invention also provides a composition for lowering blood sugar, lowering insulin resistance, and treating or preventing diabetes, comprising a synergistic amount of a combination of a meat extract and a green algae extract, wherein the green algae extract and the alfalfa extract The ratio of meat extract is 1:1 to 3:1.

As used herein, "effective amount" means that the amount of the drug or agent causes the desired pharmacological or physiological result, or the treatment, cure, prevention, or treatment of the disease, abnormality or side effect, as compared to the corresponding individual who does not receive the amount. Or improve, or reduce the occurrence of disease or abnormalities. The effective amount or effective dose to be administered may vary depending on the particular active ingredient employed, the mode of administration, age, size, and conditions of the individual to be treated. The precise dose of the drug is administered at the discretion of the physician and varies by individual.

The invention also provides a use of a meat extract or a combination thereof with a green algae extract for the preparation of a composition for reducing insulin resistance.

The composition of the present invention can be further formulated into a pharmaceutical composition, or a food composition, or a beverage composition, which has the effects of lowering insulin and insulin resistance while reducing fasting blood glucose and increasing glucose tolerance, thereby It can be used to treat and prevent diabetes.

According to the present invention, an oral pharmaceutical, food or drink can be prepared by using appropriate dietary materials as needed in accordance with the teachings of the present invention in a manner known to those of ordinary skill in the art.

The invention also includes a pharmaceutical composition for lowering blood glucose, reducing insulin resistance, or treating or preventing diabetes, comprising a synergistic amount of a combination of a meat extract and a green algae extract, and a pharmaceutically acceptable Carrier.

As used herein, the word "acceptable" means that the carrier must be compatible with the active ingredient of the composition (preferably to stabilize the active ingredient) and not deleterious to the subject being treated.

The carrier can act as a diluent, carrier, excipient or base for the active ingredient. Examples of some suitable excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, gum arabic, calcium phosphate, alginate, scutellaria, gelatin, calcium citrate, microcrystalline cellulose, poly Vinyl pyrrolidone, cellulose, sterilized water, syrup and methylcellulose. The composition can be additionally These include lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; and flavoring agents.

The pharmaceutical composition according to the present invention may be in the form of a tablet, a pill, a powder, a tablet, a pouch, a drug pack, a medicinal liquor, a suspension, an emulsion, a solution, a syrup, a soft gelatin capsule and a hard gelatin capsule, a suppository, a sterile injection. The form of the solution and the packaged powder.

The invention is further illustrated by the following examples, which are provided for illustrative purposes and are not intended to limit the invention.

Instance Materials and Methods material

Chlorella sorokiniana (green algae powder that has been processed to break the cell wall) was supplied by Taiwan Green Algae Industry Co., Ltd. Bic (Corbicula fluminea) was purchased from Renai Market in Keelung City, cultured in Kouhu Township, Yunlin County, and harvested in November 2010. Cellulase (Cellulase AP3) was purchased from Amano Pharmaceutical Ltd (Nagoya, JP). Protease N was purchased from Amano Pharmaceutical Ltd (Nagoya, JP); Protease (Protamex®) was purchased from Novo Nordisk A/S (Denmark). --glucosidase, dipeptidyl peptidase-4 (DPP-4), α-amylase, p-nitrophenyl-α-glucose (p-Nitrophenyl) -α-glucopyranoside, p-NPG) was purchased from Sigma (St. Louis, MO). Blood glucose analysis reagent (AD747GP), triglyceride analysis reagent (AD841T), and cholesterol analysis reagent (AD744CH) were purchased from Audit Diagnostics (Carrigtwohill, Cork, Ireland). Insulin assay reagents (10-1250-01) were purchased from Mercodia (Sylveniusgatan, Uppsala, Sweden). The leptin assay reagent (ADI-900-015A) was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY).

Preparation of Hydrolyzed Extract of Pork Meat

Laboratory preparation: The meat is boiled for 40 minutes. When the solid content is 3%, the filtrate is called the hot water extract of the meat. The remaining fat after the above hot water is taken out is made into a lyophilized powder. The broth powder was added to 10 times (w/w) distilled water, heat-sterilized in a boiling water bath for 10 minutes, and then hydrolyzed at 1 ° (w/w, protein content based substrate) protease (Protamex®) at 50 ° C. The reaction was allowed to proceed for 5 hours, and the hydrolysis reaction was terminated by heating in a boiling water bath for 10 minutes to inactivate the protease. The hydrolysis reaction product was centrifuged at 12000 x g for 20 minutes, followed by a No. 2 filter paper (Advantec No. 2 Toyo Roshi Kaisha, Ltd. Japan) Filtration, and finally the filtrate was freeze-dried by a vacuum freeze dryer (Freeze drying system, Labconco AST Instruments Corporation, U.S.A.) to prepare a powder.

Intermediate factory preparation: After the meat is boiled in 1 times water (w/w) for about 40 minutes, the remaining meat is taken out and chopped by a chopper (Cutter, Model blixer 5 plus, Robot Coupe, England). Double kettle, add 2 times (w / w) deionized water, heat sterilization at 98 ± 2 ° C for 10 min, after cooling to 50 ° C, add 1% (w / w, based on the protein content of the meat powder The protease (Protamex®) having a protein content of 62.5% of the meat of the oyster meat was subjected to a hydrolysis reaction in a double vessel at 50 ° C for 5 hours, and the hydrolysis reaction was terminated by heating in a boiling water bath for 10 minutes to inactivate the protease. The hydrolysis reaction product was filtered in a 140 mesh soymilk bag (3 m × 3 m), and the obtained filtrate was a hydrolyzed extract (PX), and the filtrate was freeze-dried to prepare a powder for subsequent use.

Preparation of Chlorella Hydrolyzed Extract

Laboratory preparation: the green algae powder was added to 6 times (w/w) deionized water, and heat-sterilized in a boiling water bath for 10 minutes. After cooling, 5% (w/w) cellulase was added to the algae matrix. At the same time, 2% (w/w) protease (Protease N) was added to the algae as a substrate, and hydrolysis reaction was carried out at 50 ° C for 3 hours, and the hydrolysis reaction was finally heated in a boiling water bath for 10 minutes to inactivate the cellulase and the protease. After cooling to room temperature, the hydrolyzate was centrifuged at 12000 × g for 20 minutes, and then filtered with No. 2 filter paper (Advantec No. 2 Toyo Roshi Kaisha, Ltd. Japan), and finally the filtrate was subjected to a Freeze drying system (Freeze drying system, Labconco AST Instruments Corporation, USA) freeze-dried to form a powder.

Intermediate plant preparation: Chlorella powder and 6 times (w/w) deionized water were added to a double kettle and boiled for 30 minutes for sterilization. After cooling to 50 ° C, 5% cellulase (based on algal substrate, w / w) and 2% protease (Protease N) (based on algal protein as matrix, w / w) for hydrolysis for 3 hours, The hydrolysis reaction was terminated by heating in a boiling water bath for 15 minutes to inactivate the cellulase and protease. The hydrolysis reaction product was filtered in a 140 mesh soybean milk bag (3 m × 3 m), and the obtained filtrate was a green algae hydrolysis extract (CH), and the filtrate was freeze-dried to prepare a powder for subsequent use.

Analysis of the properties of hydrolyzed extracts Soluble protein content

Take 0.1 g of the dried extract of the hydrolyzed extract and the ethanol precipitate to deionized water After constant volume to 10 ml, filter with 0.2 μm filter (MF-Millipore Membrane Filter, Millipore Co., USA), dilute appropriately, take 0.1 ml of diluent, add 0.5 ml of reagent A and 4 ml of reagent B ( Bio-Rad DC Protein Assay Kit, Bio-Rad Laboratories Inc., USA) mixed and shaken, after standing for 15 minutes, using a spectrophotometer (UV-160A UV-Visible Recording Spectrophotometer, Shimadzu Co., Japan) at a wavelength of 750 nm The absorbance is detected and the soluble protein content is expressed in mg/g from the standard calibration curve of the bovine serum albumin standard.

Peptide content

Determine the peptide content in the sample with o-Phthaldialdehyde solution, each taking 25 ml of 100 mM sodium tetraborate and 2.5 ml of 20% sodium dodecyl sulphate. SDS), 1 ml of OPA (40 mg of phthalaldehyde dissolved in 1 ml of methanol) and 100 μl of β-mercaptoethanol were added to the brown bottle and quantified to 50 with deionized water. Ml, well prepared is the o-phthalaldehyde solution.

Take 0.1 g of hydrolyzate to freeze the dry powder, dilute to 10 ml with deionized water, filter through 0.2 μm and 5000 Da filters and dilute appropriately, and mix 50 μl of sample with 2 ml of o-phthalaldehyde solution. After 20 seconds, after standing for 1 minute and 40 seconds, the absorbance was detected by a spectrophotometer at a wavelength of 340 nm, and the content of the peptide was calculated by the standard calibration curve obtained from the Leu-Gly standard substance, and the unit was mg/g.

Total sugar content determination

The total sugar content in the sample was determined by the Phenol-sulfuric acid method. After properly diluting the sample, take 1 ml of the liquid, mix it with 1 ml of 5% phenol solution, and quickly add 5 ml of concentrated sulfuric acid. After 20 minutes of reaction, absorb the light at a wavelength of 490 nm with a spectrophotometer. Value, and the total sugar content in terms of glucose as a standard calibration line, in mg/g.

Determination of glycosidic enzyme inhibitory activity by hydrolyzed extracts Determination of α-glucosidase inhibition ability

The α-glucosidase (5.7 U) was dissolved in 28.5 ml of a 100 Mm phosphate buffer solution (pH 6.9), and the α-glucosidase activity was 0.2 U/ml. Take another matrix of p-Nitrophenyl-α-D-glucopyranoside (p-NPG) 38 mg, and make up to 50 ml with a phosphate buffer solution (pH 6.9) at a concentration of 2.5 mM. . Take 300 μl of the sample dilution (diluted with 100 mM phosphate buffer (pH 6.9) and filtered through a 0.2 μm filter), 60 μl of α-glucosidase solution and 60 μl of the same phosphate buffer solution. After reacting for 10 minutes in a 37 ° C constant temperature water bath, 60 μl of p-NPG solution was sequentially added, and then reacted in a constant temperature water bath at 37 ° C for 20 minutes, and finally 240 μl of 1 M sodium carbonate (Na ₂ CO ₃ ) was added to terminate the reaction. The mixture after completion of the reaction was measured by a spectrophotometer (UV-160A UV-Visible Recording Spectrophotometer, Shimadzu Co., Japan) at a wavelength of 405 nm to determine the product o-nitrophenol (p-) isolated from the matrix p-NPG. The absorbance of Nitrophenol, p-NP), the percentage of inhibition of α-glucosidase activity calculated from the absorbance.

Determination of IC ₅₀ value of α-glucosidase

0.1 g of hydrolyzate was used to freeze the dried powder, and the volume was adjusted to 10 ml with deionized water. After filtration through a 0.2 μm filter and appropriate dilution, the logarithm of the different dilutions of the sample was plotted on the horizontal axis. The inhibition rate of glucosidase is plotted on the vertical axis, and the optimal regression curve is obtained. The regression equation is used to determine the concentration of the sample required to inhibit the activity of α-glucosidase 50%, which is the IC ₅₀ value.

Determination of α-amylase inhibitory activity

The α-amylase (25 Unit) was dissolved in 12.5 ml of a 0.02 M phosphate buffer solution (pH 6.9), and the α-amylase activity was 2 U/ml. Another substrate 0.5% starch solution was taken in the same phosphate buffer solution. Take 500 μl of the sample dilution (diluted with 20 mM phosphate buffer (pH 6.9) and filtered through a 0.2 μm filter) and mix with 500 μl of α-amylase solution, and place in a 37 ° C water bath for 10 minutes. Then, 500 μl of starch matrix solution was added, and the reaction was continued in a 37 ° C water bath for 10 minutes. Finally, 1 ml of DNS reagent was added to terminate the reaction, and then heated in boiling water for 5 minutes and then cooled to room temperature. After the reaction mixture was completed, the absorbance of the reducing sugar of the product separated from the starch matrix solution was measured by a spectrophotometer (UV-160A UV-Visible Recording Spectrophotometer, Shimadzu Co., Japan) at a wavelength of 540 nm. The absorbance value is calculated as a percentage of inhibition of alpha-amylase activity.

Determination of IC ₅₀ value of α-amylase

The experimental procedure is the same as the above determination of the IC ₅₀ value of α-glucosidase. After the reaction is completed, the logarithmic value of the sample dilution of the different concentrations is the horizontal axis, and the inhibition rate of the α-amylase is plotted on the vertical axis. The optimal regression curve was obtained, and the regression equation was used to determine the concentration of the sample required to inhibit the 50% activity of the α-amylase.

Determination of the inhibitory activity of hydrolyzed extracts on dipeptidyl peptidase 4 ( DPP4 ) Determination of the inhibitory activity of hydrolyzed extracts on DPP4

DPP4 (0.91 U) was dissolved in 9.1 ml of 0.1 M Tris-HCl buffer (pH 8.0), diluted 10 times with the same buffer, and DPP4 activity was 0.01 U/ml. Another substrate Gly-Pro-p-nitroanolide 13.152 mg was dissolved in 1 ml of 0.1 M Tris-HCl buffer and diluted 25-fold with the same buffer. Take 25 μl of the sample dilution (diluted with 0.1 M Tris-HCl buffer (pH 8.0) and filtered through a 0.2 μm filter) and 25 μl of Gly-Pro-p-nitroanolide solution at 37 ° C for 20 minutes. Then, 50 μl of DPP4 solution was added and mixed for 60 minutes. Finally, 100 μl of 1 M sodium acetate buffer (pH 4.0) was added to terminate the reaction. The mixture after the reaction was measured for absorbance at a wavelength of 385 nm by an enzyme immunoassay, and the percentage of inhibition of DPP4 activity was calculated from the absorbance.

Determination of IC ₅₀ value of DPP4

0.1 g of hydrolyzate was used to freeze the dried powder, and the volume was adjusted to 10 ml with Tris-HCl buffer. After filtration through a 0.2 μm filter and appropriate dilution, the logarithm of the sample dilutions at different concentrations was the horizontal axis. The inhibition rate of DPP4 is plotted on the vertical axis, and the optimal regression curve is determined. The regression equation is used to determine the concentration of the sample required to inhibit 50% of DPP4 activity, which is the IC ₅₀ value.

Colloidal filtration chromatography Division of small molecular weight substances 1. GPC pipe column

The colloidal chromatography conditions were eluted with deionized water at a flow rate of 0.5 ml/min (MINIPULS 3, Gilson Medical Electronic Inc., France). Take 0.1 g of green algae or broth hydrolysate to freeze the dry powder, dilute to 10 ml with deionized water, filter through 0.2 μm, molecular weight 3000 Da filter, take 2 ml sample for injection; C16/900 ( 16 x 900 mm, Amersham Pharmacia Biotech AB Uppsala, Sweden) is a filter column packed internally with SephadexTM G-25 (Pharmacia Biotech AB Uppsala, Sweden). The precipitate (5 ml/tube) was collected by a divided collector (Fc 203, Gilson Medical Electronic Inc., France), and the above-mentioned divided collection was spectrophotometer (UV-160A UV-Visible Recording Spectrophotometer, Shimadzu Co., Japan). The absorbance was measured at a wavelength of 280 nm, and the fraction was subjected to a phenol sulfuric acid colorimetric test, and the absorbance at a wavelength of 490 nm was measured. The molecular weight of the precipitate is converted by a standard calibration curve obtained from a standard product. The molecular weight of the standard is as follows: Insulin B chain: 3495.9 Da, Bacitracin: 1422 Da, Tryptophan: 204.2 Da.

2. BPG column

The colloidal chromatographic conditions were eluted with deionized water at a flow rate of 19.5 ml/min (ACCU Pumps, Blossom Biotechnologies, Inc., U.S.A.). Take 1.5 g of green algae or broth hydrolysate to freeze the dry powder, dilute to 50 ml with deionized water, filter through 0.2 μm, molecular weight 3000 Da filter, take 40 ml sample for injection; C100/950 ( 100 x 950 mm, General Electric Company Amersham Biosciences AB, Sweden) is a filter column packed internally with SephadexTM G-25 (Pharmacia Biotech AB Uppsala, Sweden). The precipitate (195 ml/tube) was collected, and the above-mentioned divided collection was measured by a spectrophotometer (UV-160A UV-Visible Recording Spectrophotometer, Shimadzu Co., Japan) at a wavelength of 280 nm, and the fraction was subjected to phenol. After the sulfuric acid colorimetric test, the absorbance at a wavelength of 490 nm was measured.

Determination of α-glucosidase inhibitory activity by dividing the collection

Each of the partitions after the colloid filtration was collected, and a tube of the precipitate was collected before and after, and a total of three tubes were collected as one tube. The freeze-dried powder was adjusted to a volume of 1 ml with a 100 mM phosphate buffer solution (pH 6.9), and the α-glucosidase inhibitory activity was measured by the above method, and the effective inhibition percentage was calculated.

Reverse phase high performance liquid chromatography 1. C18 pipe column

The C18 semi-preparative column (Synergi 4μ Hydro-RP 80A, 250 x 10 mm, Phenomenex, U.S.A.) was analyzed while using a protective column (Synergi 4μ Hydro-RP 80A, 50 x 10 mm, Phenomenex, U.S.A.). The chromatographic conditions were as follows: extract A (containing 0.1% trifluoroacetic acid in deionized water), extract B (containing 0.1% trifluoroacetic acid in cyanide solution) as the mobile phase, and extracting the extract B in 120 minutes. The concentration was increased from 0 to 100% for gradient elution at a flow rate of 1.5 ml/min (Pump L-7100, Hitachi, Ltd., Japan), the sample injection amount was 500 μl, and the detector was detected at a wavelength of 220 nm. The peak substances were collected to measure the inhibition rate of α-glucosidase, and the effective inhibition percentage was calculated.

Each collected peak material was analyzed by C12 analytical column (Synergi 4μ MAX-RP 80A, 250 × 4.6 mm, Phenomenex, USA), and a protective column (Synergi 4 μ MAX-RP, 4 × 3.0 mm, Phenomenex, USA). In 60 minutes The concentration of the extract B was increased from 0 to 100% for gradient elution at a flow rate of 1.5 ml/min (Pump L-7100, Hitachi, Ltd., Japan), the sample injection amount was 20 μl, and the detector was at the wavelength. The mixture was detected at 220 nm, and the collected material was confirmed to be a single peak, which was freeze-dried by a vacuum freeze dryer (Freeze drying system, Labconco AST Instruments Corporation, USA).

2. HILIC column

Analyzed by HILIC semi-preparative column (Inertsil 5μ HILIC 4.6 x 250 mm, GL Sciences, Japan), chromatographic conditions with extract A (containing 10% 15 mM ammonium acetate solution) as mobile phase, within 60 minutes The concentration of the extract A was 100%, the flow rate was 5 ml/min (Pump L-7100, Hitachi, Ltd., Japan), the sample injection amount was 500 μl, and the detector was at a wavelength of 280 nm. Detection, collection of each peak material to determine its inhibition rate of α-glucosidase, and calculate the effective inhibition percentage.

Each collected peak material was analyzed by C12 analytical column (Synergi 4μ MAX-RP 80A, 250 × 4.6 mm, Phenomenex, USA), and a protective column (Synergi 4 μ MAX-RP, 4 × 3.0 mm, Phenomenex, USA) was used. ). The concentration of the extract B was increased by 100% from 0 in 60 minutes, and the flow rate was 1.5 ml/min (Pump L-7100, Hitachi, Ltd., Japan), and the sample injection amount was 20 μl. The device was detected at a wavelength of 220 nm, and the collected material was confirmed to be a single peak, which was freeze-dried by a vacuum freeze dryer (Freeze drying system, Labconco AST Instruments Corporation, USA).

Long-term animal feeding test Animal grouping and induction

After 40 weeks of 6-week-old SD rats were domesticated for 2 weeks, they were divided into 5 groups according to body weight, 8 in each group. Group 1 was the normal control group, and the other 4 groups were treated with streptozotocin (STZ, Streptozotocin) plus Nicotinamide to induce type 2 diabetes and grouped according to fasting blood glucose. Group 2 was pathology of diabetic rats. In the control group, the third group was fed with a green algae hydrolysis extract group for diabetic rats. The fourth group was fed with the hydrolyzed extract of diabetic rats, and the fifth group was fed with the mixed group of green algae and alfalfa in diabetic rats, which were (1) normal control group and (2) pathological control group (DM) (3) ) Chlorella Hydrolyzed Extract (DM+CH) (4) Hydrolyzed Extract of Porphyrin (DM+PX) (5) Hydrolyzed Extract of Chlorella and Porphyrin (DM+CH/PX).

Experimental animal feeding conditions and experimental projects

6-week-old SD rats (40) were housed at 23 ± 1 ° C, relative humidity In the 55±5% animal room, the daily light and dark periods were controlled for 12 hours each. Before the end of domestication, the blood glucose was evaluated. After confirming that 32 of them had been induced as type 2 diabetic rats, they were randomly divided into 4 groups according to their fasting blood glucose, 8 rats in each group, free to feed and drink, and record feed and drink every week. Liquid intake and rat body weight. The experimental period was 8 weeks. A glucose tolerance test was performed before the fourth cycle of the experiment and before sacrifice. The glucose metabolism of each group of rats was observed. The experiment was completed at the end of the 8th cycle, and after 12 hours of fasting, the body weight was measured and blood was collected for biochemical analysis.

Animal plasma analysis

The blood obtained by drawing blood was mixed with heparin sodium (500 IU/ml) into a centrifuge tube, and immediately centrifuged at 3000 rpm (1570×g) for 20 minutes in a centrifuge (Hitachi, CF7D2, Tokyo, Japan) to collect plasma. Perform the following analysis.

Determination of glucose concentration

10 μl of plasma was uniformly mixed with 1 ml of Glucose Enzymatic Kit (Audit Diagnostics, Cork, Ireland) reagent, and the absorbance was detected at a wavelength of 500 nm after 5 minutes at 37 ° C, and the plasma glucose concentration was compared with the standard.

Determination of insulin concentration in plasma

25 μl of plasma was analyzed by Rat insulin ELISA Kit (cat.10-1124-10; Mercodia AB Inc., Uppsala, Sweden), TMB was used as a coloring agent, and the detector detected the absorbance at a wavelength of 450 nm. The plasma insulin concentration was converted to a cubic spline.

Determination of leptin concentration

100 μl of plasma was analyzed with a Rat leptin Enzyme immunometric Kit (cat. 900-015; Assay Design, Inc., Ann Arbor, MI, USA), TMB was used as a coloring agent, and the absorbance was detected at a wavelength of 450 nm. Standards are converted to leptin concentration.

Determination of total cholesterol concentration

10 μl of plasma was uniformly mixed with 1 ml of Cholesterol Enzymatic Kit (Audit Diagnostics, Cork, Ireland) reagent, and after 5 minutes at 37 ° C, the light value was detected at a wavelength of 500 nm, and the plasma cholesterol concentration was compared with the standard.

Determination of triglyceride concentration

10 μl of plasma was uniformly mixed with 1 ml of Triacylglycerol Enzymatic Kit (Audit Diagnostics, Cork, Ireland) reagent, and after 5 minutes at 37 ° C, wave The absorbance was measured at 505 nm and the concentration of triglyceride in plasma was compared to the standard.

Statistical Analysis

The data were statistically analyzed by the Statistical Analysis System (SAS 1988) statistical software for the analysis of variance (ANOVA), and the differences between the samples were investigated by Duncan's multiple range test.

Example 1: Composition of Chlorella and Pork Hydrolyzed Extracts

The composition of the cyanobacteria was hydrolyzed by adding 5% cellulase (Cellulase AP3) and 2% protease (Protease N) for 3 hours, and the components thereof are shown in Table 1. Soluble protein, peptide and carbohydrate content were 633.7 ± 2.8, 304.1 ± 1.5 and 250.4 ± 4.5 mg / g, respectively. The components of the glutinous meat powder were hydrolyzed with 1% protease Protamex® for 5 hours, and their compositions are shown in Table 1. The soluble protein, peptide and carbohydrate contents were 694.6 ± 1.3, 179.70 ± 0.90 and 365.3 ± 4.8 mg / g, respectively.

Example 2: Hydrolysis of Chlorella and Poria can inhibit glycosidase and DPP4 activity

The inhibitory effects of the cytotoxic green algae hydrolysis extract on α-glucosidase and α-amylase are shown in Table 2. The IC ₅₀ values of α-glucosidase and α-amylase were 72.46 and 33.52 mg/ml, respectively, and Acarbose was positive control group. The hydrazine hydrolyzed extract has an IC ₅₀ value of 32.08 mg/ml for α-glucosidase, and is non-inhibitory for α-amylase.

The results of inhibition of DPP4 by the hydrolyzed extract of cyanobacteria and the hydrolyzed extract of sorghum were as shown in Table 3. The IC ₅₀ values of the hydrolyzed extracts of the cyanobacteria and the hydrolyzed extracts of the cockroach were 5.43 and 8.24 mg/ml, respectively, and the Diprotin A was the positive control group.

Further analysis of the effect of green algae (CH) and meat extracts (PX) mixed in different proportions on α-glucosidase activity. As a result, it was found that the IC ₅₀ values of the compositions of PX and CH at 1:3, 1:1 and 3:1 for α-glucosidase were 20.27, 21.99 and 32.16 mg/ml, respectively (Table 4), indicating that PX/CH= The 1:3 and 1:1 compositions have a better inhibitory effect on α-glucosidase, and the inhibitory effects are 1.4 and 1.3 times, respectively, for the single substance PX, and 1.7 and 1.1 times for the single substance CH. Figure 1 also shows that when PX/PX+CH=25/100 and 50/100, the value of a single substance can be significantly increased, indicating that the PX/CH=1/3 and 1/1 compositions have synergistic effects. effect.

Example 3: Separation of active constituents from hydrolyzed extracts of green algae and hydrolyzed extracts of alfalfa 1. Separation of the components of the hydrolyzed extract of the meat by membrane partitioning

The inhibitory effect of the meat extract and its membrane fraction on α-glucosidase is shown in Table 5. The IC ₅₀ value of the hydrolyzed extract of oyster meat is 32.08 mg/ml, which shows that the small molecular partition of the hydrolyzed extract of 蚬 meat (less than 1000 Da) has better α-glucosidase inhibition ability, IC ₅₀ The value was 25.69 mg/ml, which increased the inhibition rate of α-glucosidase by 39.8% compared to the crude hydrolyzate.

The results of inhibition of DPP4 by the hydrolyzed extract of medlar meat are shown in Table 6. The IC ₅₀ value is 8.24 mg/ml, which is divided by the film to show that the small molecular fraction (less than 1000 Da) of the hydrolyzed extract of medlar meat has a higher content. Good DPP4 inhibition with an IC ₅₀ value of 8.54 mg/ml.

2. Separation of green algae hydrolyzed extract by colloidal chromatography

When the green algae is hydrolyzed by cellulase, the cell wall is decomposed into small molecular sugars such as reducing sugars and oligosaccharides, and the green algae protein is hydrolyzed into short-chain peptides or amino acids via proteases. The green algae hydrolysis extract was separated by colloidal filtration chromatography (analytical column, Sephadex G-25), and the results are shown in Fig. 2. According to the different absorbance values of peptide (280 nm) and total sugar (490 nm), the green algae hydrolysis extract can be divided into five regions, wherein the peptide substances are divided into P ₁ , P ₂ , P ₃ and P ₄ ; The sugar substance is classified as C ₁ . The molecular weight ranges are, respectively, P ₁ (1710-1480 Da), P ₂ (860-740 Da), P ₃ (570-490 Da), P ₄ (350-300 Da) and C ₁ (1250-1050). Da). In order to improve the efficiency of the collection of the partitions, the BPG column was used for gel filtration chromatography (filling Sephadex G-25) and the effective partitions were collected. The results are shown in Fig. 3. It is found that the BPG column can be used for the collection of the partitions. Increase collection efficiency by 75 times.

The inhibitory effects of different partitions on α-glucosidase activity were measured, and the results are shown in Table 7, which shows that the partitions P ₂ and P ₄ having molecular weights of 860-740 and 350-300 Da have better inhibition of α-glucosidase. capacity, an IC ₅₀ values of 1.42 and 3.62 mg / ml. Next, the purification treatment will be carried out for P ₄ .

In terms of DPP4 inhibition ability (Table 8), the partition P ₁ having a molecular weight of 1710 to 1480 Da had the best inhibitory ability, and its IC ₅₀ value was 3.80 mg/ml.

Example 4: Purification of active constituents of green algae hydrolysis extract Reverse phase high performance liquid chromatography C ₁₈ column

The green algae hydrolyzed extract, the fraction P ₄ obtained by colloidal filtration chromatography (Sephadex G-25), and the C ₁₈ semi-preparative column (Synergi 4μ Hydro-RP 80A, 250×10 mm, Phenomenex, USA) Separation by reverse phase high performance liquid chromatography. The results are shown in Figure 4. Seven major peaks can be extracted in 50% gradients within 60 minutes.

The respective peaks were collected and their inhibition rates for α-glucosidase were measured, and the effective inhibition percentage (IER) was calculated. The results are shown in Table 9. Among the 7 peaks, the second peak has a better inhibitory effect, and its inhibitory capacity is 14.4%. If the IER value is 1082.7%/mg/ml calculated from the peptide substance, the IER value of the sugar substance is 194.33. %/mg/ml, the results of Table 9 show the reaction of the peptide with the sugar substance.

Next, the second wave front was analyzed by a C ₁₂ column (Synergi 4 μ MAX-RP 80A, 250 × 4.6 mm, Phenomenex, USA), and the results obtained are shown in Fig. 5. Four major wave fronts were proposed with a 25% gradient and 15 minutes. It is speculated that when the C ₁₈ column is purified, the substances with similar polarities are not separated. Therefore, consider purifying with other columns.

2. HILIC column

The green algae hydrolyzed extract was separated by colloidal filtration chromatography (Sephadex G-25) P ₄ and separated by high performance liquid chromatography with HILIC semi-preparative column (Inertsil 5μ HILIC 4.6 x 250 mm). . The main peaks (H ₁ and H ₂ ) can be rushed within 30 minutes, as shown in Figure 6. The peak substances were collected and the inhibition rate of α-glucosidase was measured, and the effective inhibition percentage (IER) was calculated. The results are shown in Table 10.

Among the two peaks, H ₂ has a better inhibitory effect, and its inhibitory ability is 60.19%. If the peptide and the saccharide are calculated, the IER values are 1941.5 and 21496.6%/mg/ml, respectively. The H _{2 was} analyzed by a C ₁₂ column (Synergi 4 μ MAX-RP 80A, 250 × 4.6 mm, Phenomenex, USA), and the results obtained after the analysis are shown in Fig. 7. The main peak was clarified by a 25% gradient in 15 minutes, and H ₂ was determined to be composed of a single substance, and the active substance of the monosaccharide peptide was presumed to be analyzed according to its composition.

Example 5: Hydrolyzed extract of medlar meat and hydrolyzed extract of green algae can effectively inhibit STZ-induced diabetes

1. The hydrolyzed extract of medlar meat and the hydrolyzed extract of green algae reduce the fasting blood glucose of diabetic rats

The STZ-induced diabetic rats were measured for fasting blood glucose levels after four weeks and eight weeks of feeding the green algae hydrolyzed extract and the hydrolyzed extract of the meat. The results are shown in Table 11.

The fasting blood glucose levels of each group after feeding for four weeks were 149.68±6.32, 166.81±8.50, 154.41±7.45, 151.02±12.21 and 142.52±8.34 mg/dl, respectively, indicating that diabetic rats were fed CH, PX and CH/PX groups. Compared with the pathological control group, the fasting plasma glucose concentration decreased by 4.1, 9.5, and 14.56%, respectively (P < 0.05), and there was no significant difference in blood glucose values compared with normal rats. After 8 weeks of feeding, the fasting blood glucose levels of each group were 136.50±8.51, 167.00±3.91, 151.48±10.7, 158.67±4.48, and 148.80±5.65 mg/dl, respectively, indicating that the diabetes mellitus fed CH, PX, and CH/PX groups was large. Compared with the pathological control group, the fasting plasma glucose concentration of the white mice decreased by 8.1, 3.7, and 9.6% (P<0.05), respectively. It showed that the hydrolysis of the green algae extract and the hydrolyzed extract of the scorpion meat significantly improved the fasting plasma glucose concentration in diabetic rats. Effect.

2. Glucose tolerance test

Rats fed with STZ-induced diabetes were fed with a green algae hydrolyzed extract, a hydrolyzed extract of medlar meat and a combination thereof (CH, PX and CH/PX) and subjected to a glucose tolerance test after eight weeks to determine the plasma glucose concentration. The result is shown in Figure 8. The results showed that before glucose feeding, the blood glucose levels of rats fed CH, PX and CH/PX were 8.1, 3.7 and 9.6% lower than those in the diabetic pathological control group, respectively, and 60 minutes and 120 minutes after glucose administration. The blood glucose level of the CH/PX group was 11.6% and 10.5% lower than that of the pathological control group (P<0.05). It was shown that the green algae hydrolysis extract and the mash fermentation extracts (CH, PX and CH/PX) have the effect of delaying STZ-induced glucose uptake in diabetic rats.

3. Changes in plasma insulin levels

STZ-induced diabetic rats were given plasma insulin concentrations four weeks and eight weeks after feeding the green algae hydrolyzed extract and the hydrolyzed extract of the meat. The results are shown in Table 12.

The plasma insulin concentrations in normal rats and diabetic rats after feeding for four weeks were 0.62 ± 0.15, 1.35 ± 0.34, 0.63 ± 0.21, 0.67 ± 0.12, and 0.55 ± 0.16 μg / l, respectively, and fed CH, PX, and CH/PX diabetes. The plasma insulin concentration in rats was 53.33, 50.37 and 59.26% lower than that in the control group (P<0.05). The plasma insulin concentrations in normal rats and diabetic rats after feeding for 8 weeks were 1.39±0.37, 1.92±0.29, 1.88±0.53, 1.54±0.38, and 1.19±0.36μg/l, indicating that diabetic rats fed CH, PX and CH/PX had lower insulin concentrations, and the plasma insulin concentration in CH/PX group decreased by 38.0% (P<0.05), with significant difference.

4. HOMA-IR value change

The HOMA-IR value can be used to show the insulin resistance of diabetic patients. In addition, studies have shown that the decrease in HOMA-IR value can also improve cardiovascular disease. Table 13 shows that the HOMA-IR values of normal rats and diabetic rats after feeding the green algae hydrolyzed extract or the hydrolyzed extract of the scorpion meat were 5.51±1.39, 13.34±3.40, 5.75±1.99, 6.11±1.39, and 4.60±1.13, respectively. Gg. Mmol/L. L, the insulin concentrations of diabetic rats fed CH, PX and CH/PX were 56.89, 54.19 and 65.52% lower than those of the diabetic pathological control group, respectively (P<0.05). The HOMA-IR values of normal rats and diabetic rats were 11.06±2.94, 18.81±3.09, 16.81±4.66, 14.52±3.65 and 9.59±1.98 μg, respectively, after feeding the green algae hydrolyzed extract or the hydrolyzed extract of the meat. Mmol/L. L, showed that diabetic rats fed CH, PX and CH/PX had lower HOMA-IR values, of which CH/PX group had significant difference compared with diabetic pathological control group, and HOMA-IR value decreased by 51.95% (P <0.05).

From the above experimental results, it was found that STZ-induced diabetic rats given the hydrolyzed extract of green algae and the hydrolyzed extract of sorghum had no insulin resistance at the fourth week. The shape shows that the hydrolyzed extract of green algae and the hydrolyzed extract of alfalfa can delay the insulin resistance of STZ-induced diabetic rats. After eight weeks of administration of the green algae hydrolyzed extract and the hydrolyzed extract of medlar (CH/PX), the STZ-induced diabetic rats showed an effect of significantly improving their insulin concentration and HOMA-IR value, indicating that the green algae hydrolyzed extract and the cockroach meat The hydrolyzed extract can achieve the effect of regulating blood sugar by improving insulin resistance.

5. Plasma lipid changes

Diabetes patients, in addition to significantly higher blood glucose levels, can also cause abnormal lipid metabolism, such as: increase plasma cholesterol concentration, reduce HDL-C concentration and increase the concentration of VLDL-TG, resulting in hypertriglyceridemia, abnormal lipid metabolism is An important risk factor for cardiovascular disease.

The rats in which STZ-induced diabetes were fed were fed with the green algae hydrolyzed extract and the meat extracts (CH, PX and CH/PX) and the plasma total cholesterol concentrations were measured after four weeks and eight weeks. The results are shown in Table 14.

The total cholesterol in the blood of rats after feeding the caries or green algae hydrolyzed extracts was 45.04±5.90, 68.40±10.80, 51.82±7.91, 142.86±7.97 and 48.50±6.65 mg/dl, respectively, indicating that CH, PX and CH were fed. /PX significantly reduced total cholesterol by 24.2, 37.3, and 29.1%, respectively (P < 0.05). After feeding the carmine or green algae hydrolysis extract for eight weeks, the total blood of the rats Cholesterol was 49.70±6.90, 71.35±15.4, 55.84±10.9, 48.84±14.4, and 44.03±8.5 mg/dl, respectively. Compared with the diabetic pathological control group, the total cholesterol in plasma was significantly decreased. Feeding CH, PX and CH/PX was possible. They dropped by 21.7, 31.6 and 38.3% respectively.

The change of triglyceride is shown in Table 15. The normal and diabetic rats have a triglyceride value of 133.46±5.89, 152.61±10.22, and 144.46±8.16, respectively, after feeding the meat or green algae hydrolysis extract for four weeks. 133.33±6.83 and 139.16±6.45 mg/dl showed that the feeding of CH, PX and CH/PX decreased the glycerol ester values of 5.3, 12.6 and 8.8%, respectively (P<0.05). The triglyceride values of normal and diabetic rats were 125.20, 147.51, 128.84, 129.22, and 119.23 mg/dl after feeding the carmine or green algae hydrolyzed extract for eight weeks, respectively. Compared with the diabetic pathological control group, the CH was fed. The PX and CH/PX groups had the effect of lowering plasma triglyceride by 12.0, 12.4, and 19.2%, respectively (P < 0.05).

From the results of the above analysis, it was found that the effects of the total cholesterol and the triglyceride concentration in the diabetic rats were significantly reduced after the administration of the green algae hydrolyzed extract and the hydrolyzed extract of the alfalfa (CH, PX and CH/PX) for eight weeks.

6. Changes in plasma leptin

Plasma rat leptin concentrations were measured in normal rats and the cultured extracts of green algae and the hydrolysate STZ induced by STZ in diabetic rats (CH, PX and CH/PX) after the fourth and eighth weeks of the experiment. 16, the results show that between plasma leptin concentration in rats with diabetic and normal rats no significant difference statistically, which values were 2.81 ± 0.57,2.65 ± 0.59,3.12 ± 1.59,3.30 ± 0.08 and 2.67 ± 0.06 ng/ml.

In summary, it has been confirmed by in vitro and in vivo experiments that the hydrolyzed extract of medlar and green algae or a combination thereof has the effects of lowering blood sugar and blood lipids, can be used for regulating blood sugar and blood lipids, and treating diabetes.

The present invention may be applied to its broadest scope without further elaboration, based on the description of the present invention. Therefore, it is to be understood that the claims and claims are not intended to limit the scope of the invention.

Claims (8)

A use of a combination of a meat extract and a green algae extract for the preparation of a pharmaceutical composition for reducing insulin resistance, wherein the combination of the meat extract and the green algae extract has a synergistic effect on reducing insulin resistance Exist in the pharmaceutical composition; wherein the ratio of the green algae extract to the meat extract is 1:1. The use according to claim 1, wherein the meat extract is prepared by reacting a piece of meat in water at an extraction temperature for an extraction time, wherein the extraction temperature is 70 to 150 ° C. And the extraction time is 30 to 50 minutes; and adding a protease and reacting at a suitable hydrolysis temperature for a suitable hydrolysis time to obtain the meat extract, wherein the protease is 0.5 to 2% of the protein content of the meat, The hydrolysis temperature is 35 to 60 ° C, and the hydrolysis time is 4 to 6 hours. The use of claim 2, wherein the extraction temperature is about 100 ° C and the extraction time is about 40 minutes. The use of claim 2, wherein the protease is about 1% of the protein content of the meat. The use of claim 2, wherein the hydrolysis temperature is about 50 ° C and the hydrolysis time is about 5 hours. The use of claim 2, wherein the meat is from Corbicula fluminea . The use according to claim 1, wherein the green algae extract is prepared by reacting a green algae powder with a cellulase and a protease at a hydrolysis temperature to obtain a hydrolysis time. The green algae extract, wherein the cellulase is 4 to 6% by weight of the green algae powder, and the protease is 1 to 3% by weight of the green algae powder, and the hydrolysis temperature is 40 to 60. °C, and the hydrolysis time is 2 to 4 hours. The use of claim 1, wherein the green algae powder is from Chlorella sorokiniana .