CN116732125A - Extraction method for improving hypoglycemic activity of mulberry leaf peptide - Google Patents
Extraction method for improving hypoglycemic activity of mulberry leaf peptide Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
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Abstract
The application relates to an extraction method for improving hypoglycemic activity of mulberry leaf peptide, which comprises the steps of cleaning and mincing fresh mulberry leaves, adding water, adding alkaline protease, extracting, and inactivating enzyme after the extraction is finished to obtain Sang Shetai extract; filtering by a plate frame to obtain clear liquid and filter residues; the obtained clear liquid is fractionated by ultrafiltration and nanofiltration membrane to obtain components with different molecular weight ranges; concentrating the obtained components in different molecular weight ranges, sterilizing the filter element, and spray drying to obtain the product of the components in different molecular weight ranges. The application relates to a grading purification technology in the Sang Shetai extraction process, which is used for grading Sang Shetai products with different specifications and aiming at different hypoglycemic cases and scenes.
Description
Technical Field
The application belongs to the fields of medicine and biochemistry, and in particular relates to an extraction method for improving hypoglycemic activity of mulberry leaf peptide.
Background
Mulberry leaf, which is originally carried in Shennong Ben Cao Jing, has the effects of reducing blood sugar, blood pressure, blood fat, aging resistance, tumor resistance, inflammation diminishing, swelling eliminating and the like; is rich in flavone, alkaloid, mulberry leaf polysaccharide, gamma-aminobutyric acid and other active components. Mulberry leaves are recorded in the chinese pharmacopoeia 2015 edition as a substance homologous to drugs and foods, and their extracts are generally used for development of hypoglycemic agents. The mulberry leaves or the mulberry leaf extract are used as raw materials to produce more products related to reducing blood sugar, but products aiming at Sang Shetai as active ingredients are few.
The patent (201911374156.5) discloses a process for extracting high-activity mulberry leaf oligopeptide powder, which comprises the steps of firstly deconstructing cellulose wrapped outside protein in a directional enzymolysis mode before extracting the protein, and then carrying out enzymolysis on the protein by selecting proper protease and enzymolysis conditions, so that 84.14% of the obtained peptide fragments are oligopeptide with molecular weight not higher than 1000 and easy to be absorbed by human bodies, and the free radical removing performance of the product, the content of active ingredient total flavonoids and r-aminobutyric acid are also reserved to the greatest extent in the extraction process.
Disclosure of Invention
The application provides an extraction method for improving the hypoglycemic activity of mulberry leaf peptide, which adopts a grading purification technology in the Sang Shetai extraction process, and the graded Sang Shetai products with different specifications aim at different hypoglycemic cases and scenes.
In order to achieve the purpose, the application adopts the following technical scheme:
mulberry leaf pulping, extracting, separating, classifying, concentrating, sterilizing, drying, packing and obtaining the finished product.
An extraction method for improving hypoglycemic activity of mulberry leaf peptide, comprising the following steps:
step 1, cleaning and mincing the harvested fresh mulberry leaves, adding water, adding protease, extracting, and inactivating enzyme after the completion of the extraction to obtain Sang Shetai extract;
step 2, filtering the extract obtained in the step 1 through a plate frame to obtain clear liquid and filter residues;
step 3, grading the clear liquid obtained in the step 2 through ultrafiltration and nanofiltration membranes to obtain components with different molecular weight intervals;
step 4, concentrating the components with different molecular weight intervals obtained in the step 3 respectively;
step 5, sterilizing the concentrated solution obtained in the step 4 through a filter element;
step 6, spray drying the product concentrate obtained in the step 5 to obtain products with different molecular weight interval components; the process for improving the hypoglycemic activity of the mulberry leaf peptide is completed.
Preferably, in the step 1, 3-6 times of water is added, protease with 1% -3% of leaf weight is added at 50-60 ℃, ultrasonic extraction is performed with or without ultrasonic wave for 1-5 hours, and ultrasonic power is 80-300w.
Preferably, in the step 1, before the alkaline protease is added for enzymolysis, the pH is controlled to be in the range of 8-10, and the pulse light treatment condition is that: the pulse light intensity is 10-5 uW/cm 2 The pulse times are 1 time/s and 5-20s.
Further preferably, in the step 1, sodium hydroxide, calcium hydroxide, ammonia, hydrochloric acid, phosphoric acid or sulfuric acid may be used for adjusting the pH.
Preferably, in the step 2, the obtained clear liquid is subjected to freezing treatment, and after thawing, the clear liquid is subjected to step 3 treatment, wherein the freezing temperature is-18 ℃, and the freezing is carried out for 3-6 hours.
Preferably, in the step 3, the components in different molecular weight intervals obtained by ultrafiltration and nanofiltration membrane fractionation are respectively: less than 500D,500-2000D, 2000-5000D.
Preferably, in the step 5, the pore size of the filter element is 0.15-0.3 μm.
Preferably, in the step 6, in the spray drying, the air inlet is 180+/-20 ℃ and the air outlet is 90+/-5 ℃.
Preferably, sang Shetai is obtained by the process for increasing the hypoglycemic activity of Sang Shetai, which is obtained by Sang Shetai products of different molecular weight interval components:
a product of less than 500D: DNJ content is 5.5-7.5%, protein content is 50-65%, total sugar content is 25-35%, and gamma aminobutyric acid content is 13000-22000ppm;
500-2000D: DNJ content 1.1-1.9%, protein content 41-47%, total sugar content 23-29%, gamma aminobutyric acid content 2000-2600ppm;
2000-5000D product: DNJ content is 0.06-0.14%, protein content is 22-30%, total sugar content is 45-55%, and gamma aminobutyric acid content is 300-450ppm.
Preferably, sang Shetai is obtained by the process for increasing the hypoglycemic activity of Sang Shetai, which is obtained by Sang Shetai products of different molecular weight interval components:
the product less than 500D is used for preparing medicines or health care products or foods for patients with type II diabetes with fasting blood glucose more than 12 mmol/L;
the 500-2000D product is used for preparing medicines or health care products or foods for patients with type II diabetes with fasting blood glucose less than 12 mmol/L;
2000-5000D is used for preparing medicine or health product or food for slightly diabetic patients with fasting blood glucose less than 8mmol/L or weight-reducing product required by weight-controlling crowd.
The application has the following beneficial effects:
in the method, the inventor performs extraction and grading research on Sang Shetai extraction and improving the hypoglycemic activity, and after numerous experiments and researches, the method finds that after high pressure under alkaline conditions, the method adopts ultrasonic technology pretreatment to generate unexpected effects, and the main appearance is that: (1) The pH is controlled within the range of 8-10, and the dissolution of active ingredients in Sang Shetai can be greatly improved by adopting pulse light treatment, so that the enzymolysis efficiency is improved, and the use amount of an enzyme preparation is reduced; (2) In the enzymolysis process, 80-300w of ultrasonic wave is adopted to assist the enzymolysis process, so that the extraction rate can be increased, and the extraction time can be shortened; (3) Although the mechanism is not quite clear, after the primary filtrate is frozen, the primary filtrate is thawed, insoluble macromolecular impurities can be further removed, the activity of Sang Shetai can be obviously improved, and the content of active ingredients is increased by detection, wherein the freezing parameter is-18 ℃, and the freezing time is 3-6 hours; (4) The extract liquid with impurities removed by freezing is further subjected to membrane fractionation, so that different components obtained have unexpected effects on the distribution of certain active ingredients, and the activity is correspondingly improved and reduced, so that Sang Shetai can be used in different scenes. The classifying membranes are ultrafiltration membranes and nanofiltration membranes, and the pore diameters are 500D,2000D and 5000D respectively. (5) The feed-liquid ratio is properly controlled between 1:15 and 1:25, and too high feed-liquid ratio can increase the concentration cost and reduce the production efficiency. Further, in order to obtain higher extraction yield and Sang Shetai activity,
according to the application, membrane classification is carried out in the process, 3 products with different specifications are obtained, the content of relevant active ingredients is different, the blood sugar reducing effect is also different, and the method is suitable for different scenes. And separating 500D,2000D and 5000D by using membranes with different apertures to obtain products with the sizes smaller than three intervals of 500D,500-2000D and 2000-5000D. The components (DNJ content, protein content, total sugar content, gamma aminobutyric acid content) were compared, and the hypoglycemic effect was obtained. The product with the content of the alkaloid DNJ smaller than 500D has higher protein peptide content, relatively lower mulberry leaf polysaccharide content and higher hypoglycemic activity, can be used for developing hypoglycemic drugs and aims at patients with type II diabetes with higher fasting blood glucose (larger than 12 mmol/L); the product of 500-2000D has moderate content of alkaloid DNJ and protein peptide, and can be used for patients with type II diabetes with less serious illness state and fasting blood glucose less than 12 mmol/L; the product with the content of the alkaloid DNJ of 2000-5000D is low, the content of the protein peptide is high, and the product can be used for patients with mild diabetes (fasting blood glucose is less than 8), and has the requirement of controlling weight of people, so that the weight-losing product is developed.
The specific embodiment is as follows:
the application is further illustrated by the following specific examples.
In vitro blood glucose lowering and antioxidant activity (DPPH radical scavenging) detection method:
alpha-glucosidase inhibitory Activity assay:
0.1mL of 0.2U/mL alpha-glucosidase solution is taken and added with 2mL of 0.1mol/L phosphate buffer solution (pH 6.8), water bath is carried out for 15min at 37 ℃, and after 10min of reaction is carried out after the sample solution is added, 0.25mL of 25mmol/L substrate PNPG is added. After 30min in the water bath, 0.1mol/L2mLNa2CO3 was added to terminate the reaction, and absorbance (A2) was measured at 400 nm; 1mL of buffer solution is used for replacing the sample solution, and the absorbance value is (A0); the absorbance was measured by substituting 0.1mL of the buffer for the enzyme solution and was (A1). The assay was repeated 3 times and acarbose was used as a positive control. Alpha-glucosidase inhibition rate/% = [1- (A2-A1)/A0 ]. Times.100
Determination of free radical scavenging Activity on Sang Shetai by DPPH method:
the specific detection method comprises the following steps: 1.5mL of the sample was added to 1.5mL of 0.1mmol/L DPPH (95% ethanol), mixed well and incubated at 25℃for 30min. Absorbance was measured at 517 nm. The VC solution was used as a control. DPPH clearance w (%) was calculated as follows:
wherein A0 is an absorbance value of 1.5mL of distilled water and 1.5mL of 95% ethanol containing 0.1mmol/L DPPH, A1 is an absorbance value of 1.5mL of hydrolysate containing 0.1mmol/L DPPH, and A2 is an absorbance value of 1.5mL of hydrolysate and 1.5mL of 95% ethanol.
Sang Shetai extraction rate calculation:
sang Shetai the extraction rate is obtained by drying Sang Shetai solution obtained by a series of extraction, purification, classification and other processes, namely Sang Shetai, and the ratio of the Sang Shetai solution to the mulberry leaf raw material is Sang Shetai, and is calculated as follows:
W(％)＝m (Sang Shetai) /m (Mulberry leaf)
Example 1
An extraction method for improving hypoglycemic activity of mulberry leaf peptide, comprising the following steps:
step 1, cleaning and mincing the harvested fresh mulberry leaves, adding water, adding alkaline protease, extracting, and inactivating enzyme after the completion of the extraction to obtain Sang Shetai extract;
step 2, filtering the extract obtained in the step 1 through a plate frame to obtain clear liquid and filter residues;
step 3, carrying out ultrafiltration and nanofiltration membrane fractionation on the supernatant obtained in the step 2 to obtain components with different molecular weight intervals;
step 4, concentrating the components with different molecular weight intervals obtained in the step 3 respectively, wherein the concentration is 30%;
step 5, sterilizing the concentrated solution obtained in the step 4 through a filter element;
step 6, spray drying the product concentrate obtained in the step 5 to obtain products with different molecular weight interval components;
the process for improving the hypoglycemic activity of the mulberry leaf peptide is completed.
Preferably, in the step 1, 4 times of water is added, alkaline protease with 2% of leaf weight is added at 55 ℃, ultrasonic extraction is performed for 3 hours, and ultrasonic power is 80w.
Preferably, in the step 3, the components in different molecular weight intervals are respectively: less than 500D,500-2000D, 2000-5000D.
Preferably, in the step 5, the pore size of the filter element is 0.22 μm.
Preferably, in the step 6, in the spray drying, the air inlet is 180 ℃ and the air outlet is 90 ℃.
Example 2
An extraction method for improving hypoglycemic activity of mulberry leaf peptide, comprising the following steps:
step 1, cleaning and mincing the harvested fresh mulberry leaves, adding water, adding alkaline protease, extracting, and inactivating enzyme after the completion of the extraction to obtain Sang Shetai extract;
step 2, filtering the extract obtained in the step 1 through a plate frame to obtain clear liquid and filter residues;
step 3, carrying out ultrafiltration and nanofiltration membrane fractionation on the supernatant obtained in the step 2 to obtain components with different molecular weight intervals;
step 4, concentrating the components with different molecular weight intervals obtained in the step 3 respectively, wherein the concentration is 30%;
step 5, sterilizing the concentrated solution obtained in the step 4 through a filter element;
step 6, spray drying the product concentrate obtained in the step 5 to obtain products with different molecular weight interval components;
the process for improving the hypoglycemic activity of the mulberry leaf peptide is completed.
Preferably, in the step 1, 5 times of water is added, alkaline protease with 3% of leaf weight is added at 50 ℃, and ultrasonic extraction is performed for 1h, wherein the ultrasonic power is 300w.
Preferably, in the step 3, the components in different molecular weight intervals are respectively: less than 500D,500-2000D, 2000-5000D.
Preferably, in the step 5, the pore size of the filter element is 0.15 μm.
Preferably, in the step 6, in the spray drying, the air inlet is 190 ℃ and the air outlet is 95 ℃.
Example 3
An extraction method for improving hypoglycemic activity of mulberry leaf peptide, comprising the following steps:
step 1, cleaning and mincing the harvested fresh mulberry leaves, adding water, adding alkaline protease, extracting, and inactivating enzyme after the completion of the extraction to obtain Sang Shetai extract;
step 2, filtering the extract obtained in the step 1 through a plate frame to obtain clear liquid and filter residues;
step 3, carrying out ultrafiltration and nanofiltration membrane fractionation on the supernatant obtained in the step 2 to obtain components with different molecular weight intervals;
step 4, concentrating the components with different molecular weight intervals obtained in the step 3 respectively, wherein the concentration is 30%;
step 5, sterilizing the concentrated solution obtained in the step 4 through a filter element;
step 6, spray drying the product concentrate obtained in the step 5 to obtain products with different molecular weight interval components;
the process for improving the hypoglycemic activity of the mulberry leaf peptide is completed.
Preferably, in the step 1, 6 times of water is added, alkaline protease with a leaf weight of 1.5% is added at 60 ℃, and ultrasonic extraction is performed for 4 hours, wherein the ultrasonic power is 400w.
Preferably, in the step 3, the components in different molecular weight intervals are respectively: less than 500D,500-2000D, 2000-5000D.
Preferably, in the step 5, the pore size of the filter element is 0.25 μm.
Preferably, in the step 6, in the spray drying, the air is introduced at 170 ℃ and the air is discharged at 85 ℃.
The properties of the products obtained in the three examples are shown in tables 1 to 3 below.
Example 4
Based on example 3, in the step 1, 6 times of water is added, and before the enzymolysis by adding alkaline protease, the pH is controlled to 8, and the pulsed light treatment is performed under the conditions that (pulsed light intensity is 10A 4uW/cm 2 The number of pulses was 1/s, 15 s). Alkaline protease 1% of the leaf weight was added and extracted with ultrasound for 4h at a power of 400w.
Otherwise, the same as in example 3 was conducted.
Example 5
On the basis of example 3, the clear liquid obtained in the step 2 is subjected to freezing treatment, and after thawing, the clear liquid is subjected to the step 3 treatment, wherein the freezing temperature is-18 ℃, and the freezing time is 4 hours. Otherwise, the same as in example 3 was conducted.
Example 6
Based on example 3, in the step 1, before the alkaline protease is added for enzymolysis, the pH is controlled to be in the range of 8-10, and the pulsed light treatment is performed under the conditions that (pulsed light intensity is 10-4 uW/cm 2 The number of pulses was 1/s, 15 s). Otherwise, the same as in example 3 was conducted.
And (3) freezing the obtained clear liquid in the step (2), thawing, performing the step (3), and freezing for 4 hours at the freezing temperature of-18 ℃. Otherwise, the same as in example 3 was conducted.
Example 7
On the basis of example 3, in the above-mentioned step 1, ultrasonic extraction was not employed for 4 hours.
TABLE 1 index of products of the components in the molecular weight range of example 1, in vitro hypoglycemic and antioxidant activities
TABLE 2 index of products of example 2 molecular weight range composition and in vitro hypoglycemic and antioxidant Activity
TABLE 3 index of products of example 3 molecular weight Interval Components and in vitro hypoglycemic and antioxidant Activity
As shown in tables 1-3, after ultrafiltration and nanofiltration membrane fractionation treatment, the DNJ, protein, total sugar content and gamma aminobutyric acid content of the products with different molecular weight intervals are different, and the alpha-glucosidase inhibitory activity and DPPH clearance capability are greatly different. The gamma aminobutyric acid content, alpha-glucosidase inhibitory activity and DPPH clearance of the products smaller than 500D are higher than those of the products of 500-2000D and 2000-5000D, and different product application scenes can be opened according to the requirements.
TABLE 4 index of whether products treated with pulsed light were used and in vitro hypoglycemic and antioxidant activity
As is clear from Table 4, in example 4, the pH was controlled to be about 8, and compared with example 3, the gamma aminobutyric acid content was greatly improved, and the alpha-glucosidase inhibitory activity and DPPH scavenging ability of the product of example 4 were much higher than those of example 3, and in example 4, a relatively high extraction rate (7.7%) was achieved by using a relatively small enzyme addition amount (1%) as well, i.e., the dissolution of the active ingredient in Sang Shetai was greatly improved by using a pulsed light treatment, which was advantageous for improving the enzymolysis efficiency and reducing the use amount of the enzyme preparation.
TABLE 5 index of whether to select the products of freeze-dried impurities and in vitro hypoglycemic and antioxidant activity
As is clear from Table 5, in example 5, compared with example 3, the gamma aminobutyric acid content was greatly improved, and the alpha-glucosidase inhibitory activity and DPPH scavenging ability of the product of example 5 were much higher than those of example 3, namely, after the primary filtrate was frozen, the primary filtrate was thawed again, insoluble macromolecular impurities could be further removed, the activity of Sang Shetai stock solution could be remarkably improved, and the total content of the effective products for separating the components at a later stage could be improved.
TABLE 6 index of products of the components in the molecular weight range of example 6 and in vitro hypoglycemic and antioxidant activities
As can be seen from Table 6, compared with examples 3-5, the gamma aminobutyric acid content, alpha-glucosidase inhibitory activity and DPPH scavenging ability of the product of example 6 are far higher than those of examples 3-5, namely, the product is frozen by combining pulsed light with primary filtrate and then thawed, so that the dissolution of active ingredients in Sang Shetai can be greatly improved, insoluble macromolecular impurities can be removed, the activity of Sang Shetai stock solution can be obviously improved, and the total content of the active products for separating each component at a later stage can be improved.
TABLE 7 index of products of comparative example 1 molecular weight interval component and in vitro hypoglycemic and antioxidant activity
As is clear from Table 7, in comparative example 1, the stock solution of comparative example 1 was slightly lower in gamma aminobutyric acid content, alpha-glucosidase inhibitory activity, DPPH scavenging ability than in example 3 without ultrasonic treatment, and the extraction rate was much lower than in example 3. Namely, the ultrasonic wave is adopted to assist the enzymolysis process, so that the extraction rate can be increased, and the extraction time can be shortened.
Animal experiments were performed using the different interval fractions obtained in example 6
1, method:
1.2.1 Effect of mulberry leaf polypeptide on blood sugar of Normal mice
Healthy male Kunming mice are fasted for 3-5 hours, drink water freely, test fasting blood glucose, and divide 50 mice into 5 groups according to fasting blood glucose level, 10 each group is normal control group, mulberry leaf polypeptide less than 500D group, mulberry leaf polypeptide 500-2000D group, mulberry leaf polypeptide 2000-5000D group and health care product group. The test group was given different test sample solutions at doses of 2.01g/kg,2.01g/kg,1.05g/kg,2.01g/kg, and a dosing volume of 0.2ml/10g, and the control group was given the same volume of sterile distilled water, and after continuous dosing for 30 days, fasting was conducted for 16 hours, fasting blood glucose was measured, and fasting blood glucose values of the two groups of animals were compared.
1.2.2 influence of mulberry leaf polypeptide on blood glucose in a rat model of alloxan-induced insulin-resistant glucose/lipid metabolism disorder
Healthy SD male rats 105, normal maintenance feed, were fed for 3-5 days, fasted for 3-5 hours, tail blood was taken, and the blood glucose level before glucose administration (i.e., 0 hour) was measured, and the blood glucose level after 2.5g/kg BW glucose was 0.5, 2 hours, as the base value for the batch of animals. The blood glucose levels of 0 and 0.5 hours are divided into 7 groups which are respectively a blank control group and a model control group, the mulberry leaf polypeptide is smaller than 500D high-dose group, the mulberry leaf polypeptide is smaller than 500D medium-dose group, the mulberry leaf polypeptide is smaller than 500D low-dose group, the mulberry leaf polypeptide is 500-2000D group and the mulberry leaf polypeptide is 2000-5000D group, and 15 mulberry leaves are used in each group. The blank control group is not treated, 5 experimental groups are subjected to gastric lavage and are respectively provided with samples with different concentrations, the administration doses are respectively 2.01g/kg, 0.67g/kg, 0.335g/kg, 2.01g/kg and 1.05g/kg, and the model control group is provided with the solvent with the same volume for 33 continuous days. The model control group and 3 dose groups were fed with maintenance feed for 1 week and were fed with replacement hyperthermia feed, and after 3 weeks, the model control group and 3 doses were fasted for 24 hours (without water) and 105mg/kg of tetraoxapyrimidine was administered by intraperitoneal injection at an injection rate of 1ml/100g body weight. The high heat energy feed is fed for 3-5 days after injection. At the end of the test, animals of each group were fasted for 16 hours and tested for fasting blood glucose, glucose tolerance, serum insulin, cholesterol and triglyceride levels.
1.3 data processing and result determination
Generally, analysis of variance is adopted, but the variance alignment is firstly checked according to the program of the analysis of variance, the F value is calculated, the F value is less than F0.05, and the conclusion is that: the difference between the average numbers of the groups is not significant; f value is more than or equal to F0.05, P is less than or equal to 0.05, and statistics is carried out by a pairwise comparison method of average numbers between a plurality of experimental groups and a control group; proper variable conversion is carried out on the data with non-normal or variance, and statistics is carried out on the converted data after the normal or variance alignment requirement is met; if the normal or variance alignment purpose is not achieved after the variable conversion, the rank sum test is used for statistics.
1.3.1 index determination
1.3.1.1 blood glucose reducing test for normal animals
Blood glucose index: the dosage group of the sample for fasting blood glucose has no statistical significance compared with the control group, and the blood glucose of normal animals is judged to be not influenced.
1.3.1.2 hypoglycemic tests in hyperglycemic models
Fasting blood glucose index: on the premise that the model is established, the dose group of the sample to be tested is compared with the model control group, the fasting blood glucose is reduced or the blood glucose reduction percentage is increased, which has statistical significance, and the fasting blood glucose index result of the sample to be tested is judged to be positive.
Sugar tolerance index: on the premise that the model is established, comparing the tested sample dosage group with the model control group, and judging that the tested sample glucose tolerance index result is positive, wherein the blood glucose is reduced (or the blood glucose reduction percentage is increased) at any time point of 0.5 and 2 hours after glucose or medical starch is fed, or the area reduction under the blood glucose curve of 0, 0.5 and 2 hours is statistically significant.
Blood lipid index: on the premise that the model is established, the dose group of the tested sample is compared with the model control group, and the reduction of serum cholesterol or triglyceride has statistical significance, so that the positive blood lipid reduction index of the tested sample can be judged.
1.3.2 result determination
Scheme II: one of the two indexes of fasting blood glucose and glucose tolerance is positive, blood fat (total cholesterol and triglyceride) is not obviously increased, and the normal animal fasting blood glucose is not influenced, so that the tested sample can be judged to be positive in functional animal experiment results which are helpful for maintaining blood glucose health level.
2 results
2.1 fasting blood glucose in Normal mice
The weight and blood glucose values of normal mice before grouping are shown in Table 8, after 30 days of administration, the weight of each administration group is not obviously changed compared with that of a control group, the fasting blood glucose values of the mulberry leaf polypeptide are less than 500D, the mulberry leaf polypeptide is 500-2000D and the fasting blood glucose values of the mulberry leaf polypeptide is 2000-5000D are reduced compared with that of the control group, and the blood glucose values of the mulberry leaf polypeptide are reduced by less than 500D, so that the blood glucose values of the groups are statistically significant. The health product group has no obvious change compared with the control group.
The experimental result shows that the mulberry leaf polypeptide is less than 500D, so that the blood sugar level of normal animals can be reduced, and the mulberry leaf polypeptide 500-2000D, the mulberry leaf polypeptide 2000-5000D and the health care product group have no obvious influence on the blood sugar level of the normal animals. The results are shown in Table 9.
Table 8 pre-dose fasting glycemia in normal mice
P <0.05, < P <0.01 compared to normal group;
table 9 fasting blood glucose in normal mice 30 days after administration
P <0.05, < P <0.01 compared to normal group;
2.2 index of fasting blood sugar, glucose tolerance and blood lipid of rat
The blood glucose index before rat grouping is shown in Table 10, and there is no obvious difference in blood glucose index among rats in each group.
After the rats are dosed for 33 days, the blood sugar, sugar tolerance and blood lipid levels of the rats are shown in table 11, and as can be seen from the table, the blood sugar values of a normal control group, a model control group and each experimental group for 0h are all 3.2-6.3mmol/L, the blood sugar rise of the model group is obviously different from that of the normal group, and the blood sugar rise of each dosing group is not obviously changed from the model group; after glucose is given for 0.5h, the blood sugar of each group is more than 10.0mmol/L, and the blood sugar values of the low, medium and high dosage groups of mulberry leaf polypeptide less than 500D are lower than those of a model control group, but the difference is not obvious; the blood sugar values of the mulberry leaf polypeptide 500-2000D and the mulberry leaf polypeptide 2000-5000D are obviously reduced compared with the model control group, and the difference is obvious, so that the mulberry leaf polypeptide has statistical significance. After glucose is given for 2 hours, the blood sugar value of each group of rats is obviously reduced compared with 0.5 hour, and the blood sugar value of the model group is still more than 10.0mmol/L, compared with the normal control group, the area under the curve is obviously increased, and the difference has statistical significance, so that the model of the glucose metabolism disorder is successfully copied. The blood glucose value of each experimental group is obviously lower than that of the model control group, and the difference is obvious, so that the experimental groups have statistical significance. The results indicate that the mulberry leaf polypeptide is less than 500D, and the high, medium and low dosage groups, the mulberry leaf polypeptide 500-2000D and the mulberry leaf polypeptide 2000-5000D have obvious blood sugar reducing effect.
After 33 days of administration, the serum of the rats in the model group is obviously increased compared with the normal group, and the rats have statistical significance, and the TG model group is increased but has no statistical significance compared with the normal group. Compared with the model group, the administration groups have no obvious change in indexes of CHO and TG (the TG of the group of the mulberry leaf polypeptide 2000-5000D is obviously increased compared with the model group, and the overall average value and standard deviation increase caused by abnormal increase of TG of individual animals can be considered for elimination), and the result indicates that the mulberry leaf polypeptide is less than 500D, the mulberry leaf polypeptide is 500-2000D and the mulberry leaf polypeptide is 2000-5000D has no obvious effect on blood fat.
TABLE 10 blood glucose index @ before rat dosingn＝15)
P <0.05, < P <0.01 compared to normal group; compared to the model group, #p <0.05, #p <0.01;
table 11 blood sugar and blood lipid index @ 33 days after rat administrationn＝15)
P <0.05, < P <0.01 compared to normal group; compared to the model group, #p <0.05, #p <0.01;
conclusion 3
In the normal mouse hypoglycemic experiment, the fasting blood glucose of the mice in the mulberry leaf polypeptide 500-2000D, the mulberry leaf polypeptide 2000-5000D and the health care product group has no statistical significance compared with the fasting blood glucose of the mice in the control group, and the normal animal blood glucose is judged to be not influenced.
On the premise that the rat glucose metabolism disorder model is established, compared with a model control group, the tested sample group has obvious difference between the mulberry leaf polypeptide 500-2000D and the mulberry leaf polypeptide 2000-5000D blood sugar after glucose is given for 0.5h and has statistical significance; and the blood sugar of each experimental group is obviously different from that of the model group, so that the blood sugar of the tested sample is statistically significant, and the result of the sugar tolerance index of the tested sample is positive, and the mulberry leaf polypeptide is less than 500D, the mulberry leaf polypeptide is 500-2000D and the mulberry leaf polypeptide is 2000-5000D, so that the blood sugar reducing effect is achieved. In addition, as can be seen from the blood glucose indexes of the three experimental groups with the mulberry leaf polypeptide less than 500D, the blood glucose reducing effect of the mulberry leaf polypeptide less than 500D has certain dose dependency, and the higher the dose, the more obvious the blood glucose reducing effect after 2 hours.
According to the result judging method for maintaining the blood sugar balance, one index of the fasting blood sugar and the sugar tolerance is positive, the blood fat (total cholesterol and triglyceride) is not obviously increased, the normal animal fasting blood sugar is not influenced, and the tested sample can be judged to be positive in the functional animal experiment result which is helpful for maintaining the blood sugar health level. Therefore, the mulberry leaf polypeptide 500-2000D and the mulberry leaf polypeptide 2000-5000D are beneficial to maintaining blood sugar health level, and the mulberry leaf polypeptide less than 500D has a certain effect of reducing blood sugar of normal animals and is obvious in blood sugar reducing effect on hyperglycemic animals.
Aiming at the animal experiment results, we find that the hypoglycemic activity of the three components of the mulberry leaf polypeptide is as follows: less than 500D components > 500-2000D components > 2000-5000D components. According to the characteristic, the mulberry leaf polypeptide can be purified and graded, and the obtained product can be used for targeting the actual diseases.
Because the content of the product of 500D of alkaloid DNJ is higher, the protein peptide content is higher, the mulberry leaf polysaccharide content is relatively lower, the hypoglycemic activity is higher, and the product can be used for developing hypoglycemic drugs and aiming at patients with type II diabetes with higher fasting blood glucose (more than 12 mmol/L); the product of 500-2000D has moderate content of alkaloid DNJ and protein peptide, and can be used for patients with type II diabetes with less serious illness state and fasting blood glucose less than 12 mmol/L; the product with the content of the alkaloid DNJ of 2000-5000D is low, the content of the protein peptide is high, and the product can be used for patients with mild diabetes (fasting blood glucose is less than 8), and has the requirement of controlling weight of people, so that the weight-losing product is developed.
The above embodiments are merely preferred embodiments of the present application, and should not be construed as limiting the present application, and the embodiments and features of the embodiments of the present application may be arbitrarily combined with each other without collision. The protection scope of the present application is defined by the claims, and the protection scope includes equivalent alternatives to the technical features of the claims. I.e., equivalent replacement modifications within the scope of this application are also within the scope of the application.
Claims (8)
1. An extraction method for improving hypoglycemic activity of mulberry leaf peptide, which is characterized by comprising the following steps:
step 1, cleaning and mincing the harvested fresh mulberry leaves, adding water, adding alkaline protease, extracting, and inactivating enzyme after the completion of the extraction to obtain Sang Shetai extract;
step 2, filtering the extract obtained in the step 1 through a plate frame to obtain clear liquid and filter residues;
step 3, grading the clear liquid obtained in the step 2 through ultrafiltration and nanofiltration membranes to obtain components with different molecular weight intervals;
step 4, concentrating the components with different molecular weight intervals obtained in the step 3 respectively;
step 5, sterilizing the concentrated solution obtained in the step 4 through a filter element;
step 6, spray drying the product concentrate obtained in the step 5 to obtain products with different molecular weight interval components;
the technology for improving the hypoglycemic activity of the mulberry leaf peptide is completed;
in the step 1, water with the mass of 3-6 times is added, alkaline proteinase with the weight of 1% -3% of the leaf weight is added at 50-60 ℃, ultrasonic extraction is carried out for 1-5h, and the ultrasonic power is 80-300w;
in the step 2, the obtained clear liquid is frozen, thawed and then subjected to the step 3 treatment, wherein the freezing temperature is-18 ℃, and the freezing time is 3-6 hours;
in the step 3, the components in different molecular weight intervals obtained by ultrafiltration and nanofiltration membrane classification are respectively as follows: less than 500D,500-2000D, 2000-5000D.
2. The process for improving the hypoglycemic activity of Sang Shetai according to claim 1, wherein in the step 1, before the alkaline protease is added for enzymolysis, the pH is controlled to be in the range of 8-10 by adopting pulse light treatment, and the pulse light treatment conditions are as follows: the pulse light intensity is 10-4 uW/cm square, the pulse times are 1 time/s, and 5-20s.
3. The process for improving the hypoglycemic activity of Sang Shetai according to claim 2, wherein in the step 1, sodium hydroxide, calcium hydroxide, ammonia, hydrochloric acid, phosphoric acid or sulfuric acid is used for adjusting the pH.
4. The process of claim 1, wherein in step 5, the filter element has a pore size of 0.15 to 0.3 μm.
5. The process for improving the hypoglycemic activity of Sang Shetai according to claim 1, wherein in the spray drying in the step 6, the air intake is 180+ -20 ℃ and the air outlet is 90+ -5 ℃.
6. The process for improving the hypoglycemic activity of Sang Shetai according to any one of claims 1-5, wherein the process gives Sang Shetai products of different molecular weight interval components,
a product of less than 500D: DNJ content is 5.5-7.5%, protein content is 50-65%, total sugar content is 25-35%, and gamma aminobutyric acid content is 13000-22000ppm;
500-2000D: DNJ content 1.1-1.9%, protein content 41-47%, total sugar content 23-29%, gamma aminobutyric acid content 2000-2600ppm;
2000-5000D product: DNJ content is 0.06-0.14%, protein content is 22-30%, total sugar content is 45-55%, and gamma aminobutyric acid content is 300-450ppm.
7. The use of Sang Shetai in the manufacture of a medicament for the treatment of type II diabetes in accordance with claim 6, wherein said process results in a Sang Shetai product of different molecular weight range fractions,
the product less than 500D is used for preparing medicines or health care products or foods for patients with type II diabetes with fasting blood glucose more than 12 mmol/L;
the 500-2000D product is used for preparing medicines or health care products or foods for patients with type II diabetes with fasting blood glucose less than 12 mmol/L;
2000-5000D is used for preparing medicine or health product or food for slightly diabetic patients with fasting blood glucose less than 8mmol/L or weight-reducing product required by weight-controlling crowd.
8. The use of Sang Shetai in the manufacture of a health product for aiding in the reduction of blood glucose according to claim 6.
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