An extraction method to enhance the hypoglycemic activity of mulberry leaf peptides

Abstract

This invention relates to an extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides. Freshly harvested mulberry leaves are washed, crushed, and then water and alkaline protease are added for extraction. After extraction, the enzyme is inactivated to obtain the mulberry leaf peptide extract. The extract is then filtered through a plate and frame filter to obtain a clear liquid and a residue. The clear liquid is further fractionated using ultrafiltration and nanofiltration membranes to obtain components with different molecular weight ranges. These components are then concentrated, sterilized using filter cartridges, and spray-dried to obtain products with different molecular weight ranges. This invention utilizes a fractionation and purification technology in the mulberry leaf peptide extraction process, resulting in different grades of mulberry leaf peptide products suitable for various hypoglycemic cases and scenarios.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and biochemistry, specifically a method for extracting mulberry leaf peptides to enhance their hypoglycemic activity. Background Technology

[0002] Mulberry leaves, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), possess various medicinal properties, including lowering blood sugar, blood pressure, and blood lipids; anti-aging; anti-tumor; and anti-inflammatory effects. They are rich in flavonoids, alkaloids, mulberry leaf polysaccharides, and γ-aminobutyric acid (GABA), among other active ingredients. As a substance with both medicinal and edible uses, mulberry leaves are included in the 2015 edition of the *Chinese Pharmacopoeia*, and their extracts are commonly used in the development of hypoglycemic drugs. While many hypoglycemic products are manufactured using mulberry leaves or their extracts, products targeting mulberry leaf peptides as active ingredients are relatively rare.

[0003] The patent "An Extraction Process for Highly Active Mulberry Leaf Oligopeptide Powder (201911374156.5)" discloses an extraction process for highly active mulberry leaf oligopeptide powder. Before extracting the protein, the cellulose coating on the protein is deconstructed by targeted enzymatic hydrolysis. Then, appropriate proteases and enzymatic hydrolysis conditions are selected to hydrolyze the protein, so that 84.14% of the obtained peptides are oligopeptides with a molecular weight of no more than 1000 that are easily absorbed by the human body. Moreover, the product's free radical scavenging performance, the content of active ingredients total flavonoids and γ-aminobutyric acid are also preserved to the greatest extent during the extraction process. Summary of the Invention

[0004] This invention proposes an extraction method to enhance the hypoglycemic activity of mulberry leaf peptides. The method involves a graded purification technique during the extraction process to produce mulberry leaf peptide products of different grades, which are suitable for different hypoglycemic cases and scenarios.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Mulberry leaves – pulping – extraction – separation – grading – concentration – sterilization – drying – packaging – finished product.

[0007] An extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides, the method comprising the following steps:

[0008] Step 1: After the fresh mulberry leaves are harvested, they are washed, minced, and then water and protease are added for extraction. After extraction, the enzyme is inactivated to obtain mulberry leaf peptide extract.

[0009] Step 2: Filter the extract from Step 1 through a plate and frame filter to obtain a clear liquid and filter residue;

[0010] Step 3: The clear liquid obtained in Step 2 is fractionated by ultrafiltration and nanofiltration membranes to obtain components with different molecular weight ranges;

[0011] Step 4: Concentrate the components with different molecular weight ranges obtained in Step 3 respectively;

[0012] Step 5: Sterilize the concentrated solution obtained in Step 4 by passing it through a filter cartridge;

[0013] Step 6: Spray dry the product concentrate obtained in Step 5 to obtain products with different molecular weight ranges; thus completing the process of improving the hypoglycemic activity of mulberry leaf peptides.

[0014] Preferably, in step 1, 3-6 times the weight of water is added, and at 50-60°C, 1%-3% of the leaf weight of protease is added. Extraction is performed for 1-5 hours with or without ultrasound, and the ultrasound power is 80-300w.

[0015] Preferably, in step 1, before adding alkaline protease for enzymatic hydrolysis, pulsed light treatment is performed while controlling the pH within the range of 8-10. The pulsed light treatment conditions are: pulsed light intensity of 10^4-10^5 μW / cm. 2 The pulse count is 1 pulse / second, lasting 5-20 seconds.

[0016] More preferably, in step 1, the pH can be adjusted using sodium hydroxide, calcium hydroxide, ammonia, hydrochloric acid, phosphoric acid, or sulfuric acid.

[0017] Preferably, in step 2, the obtained clear liquid is frozen, thawed, and then processed in step 3. The freezing temperature is -18°C, and the freezing time is 3-6 hours.

[0018] Preferably, in step 3, the different molecular weight range components obtained by fractionation through ultrafiltration and nanofiltration membranes are: less than 500D, 500-2000D, and 2000-5000D.

[0019] Preferably, in step 5, the filter element pore size is 0.15-0.3 micrometers.

[0020] Preferably, in step 6, during spray drying, the inlet air temperature is 180±20℃ and the outlet air temperature is 90±5℃.

[0021] Preferably, the mulberry leaf peptides obtained by the process for enhancing the hypoglycemic activity of mulberry leaf peptides, and the mulberry leaf peptide products with different molecular weight ranges obtained by the process:

[0022] Products with less than 500D: DNJ content 5.5-7.5%, protein content 50-65%, total sugar content 25-35%, Gamma-aminobutyric acid content 13000-22000ppm;

[0023] Products with 500-2000D: DNJ content 1.1-1.9%, protein content 41-47%, total sugar content 23-29%, Gamma-aminobutyric acid content 2000-2600ppm;

[0024] Products with a D2 of 2000-5000D: DNJ content 0.06-0.14%, protein content 22-30%, total sugar content 45-55%, and GABA content 300-450ppm.

[0025] Preferably, the mulberry leaf peptides obtained by the process for enhancing the hypoglycemic activity of mulberry leaf peptides, and the mulberry leaf peptide products with different molecular weight ranges obtained by the process:

[0026] Products with a value less than 500D are used in the preparation of medicines, health products, or foods for patients with type 2 diabetes whose fasting blood glucose is greater than 12 mmol / L.

[0027] Products with a value of 500-2000D are used in the preparation of medicines, health products, or foods for patients with type 2 diabetes whose fasting blood glucose is less than 12 mmol / L.

[0028] Products with a value of 2000-5000D are used in the preparation of medicines, health products, or foods for patients with mild diabetes whose fasting blood glucose is less than 8 mmol / L, or for weight loss products for people who need to control their weight.

[0029] The present invention has the following beneficial effects:

[0030] In the method of this invention, the inventors conducted extraction and grading studies on mulberry leaf peptides and improved their hypoglycemic activity. After numerous experiments and studies, they found that under alkaline conditions and high pressure, pretreatment with ultrasonic technology produced unexpected effects, mainly in the following aspects: (1) Controlling the pH within the range of 8-10 and using pulsed light treatment can greatly improve the dissolution of effective components in mulberry leaf peptides, which is conducive to improving the enzymatic hydrolysis efficiency and reducing the amount of enzyme preparation used; (2) During the enzymatic hydrolysis process, using 80-300w ultrasonic waves to assist the enzymatic hydrolysis process can increase the efficiency of enzyme preparation. (3) Although the mechanism is not very clear, freezing the initial filtrate and then thawing it can further remove insoluble macromolecular impurities and significantly improve the activity of mulberry leaf peptides. The content of active ingredients has been increased. The freezing parameters are -18℃ and freezing time is 3-6h. (4) The extract after freezing and removing impurities is further subjected to membrane fractionation. Different components are obtained, and the distribution of certain active ingredients shows unexpected effects. The activity is also increased or decreased accordingly, so that mulberry leaf peptides can be used in different scenarios. The fractionation membranes are ultrafiltration and nanofiltration membranes with pore sizes of 500D, 2000D, and 5000D, respectively. (5) The material-liquid ratio is more suitable when controlled between 1:15 and 1:25. Too high a material-liquid ratio will increase the concentration cost and reduce the production efficiency. Furthermore, in order to obtain a higher extraction rate and mulberry leaf peptide activity,

[0031] This invention employs membrane fractionation during the manufacturing process, resulting in three different product specifications with varying contents of relevant active ingredients and different blood sugar-lowering effects, suitable for different scenarios. By separating products using membranes of different pore sizes (500D, 2000D, and 5000D), products in three ranges—less than 500D, 500-2000D, and 2000-5000D—were obtained. The blood sugar-lowering effects were compared based on the components (DNJ content, protein content, total sugar content, and GABA content). Products with a concentration less than 500D have higher levels of the alkaloid DNJ and protein peptides, but relatively lower levels of mulberry leaf polysaccharides. They exhibit high hypoglycemic activity and can be used for the development of hypoglycemic drugs, as well as for type 2 diabetes patients with high fasting blood glucose (greater than 12 mmol / L). Products with a concentration of 500-2000D have moderate levels of DNJ and protein peptides and can be used for type 2 diabetes patients with less severe conditions and fasting blood glucose less than 12 mmol / L. Products with a concentration of 2000-5000D have lower levels of DNJ and higher levels of protein peptides and can be used for patients with mild diabetes (fasting blood glucose less than 8 mmol / L) and for individuals who need to control their weight, thus enabling the development of weight loss products. Detailed implementation method:

[0032] The present invention will be further illustrated by the following specific embodiments.

[0033] In vitro methods for detecting hypoglycemic and antioxidant activity (DPPH free radical scavenging):

[0034] α-glucosidase inhibitory activity assay:

[0035] 0.1 mL of a 0.2 U / mL α-glucosidase solution was added to 2 mL of 0.1 mol / L phosphate buffer (pH 6.8), and the mixture was incubated at 37°C for 15 min. The sample solution was added, and the reaction was allowed to proceed for 10 min. Then, 0.25 mL of 25 mmol / L substrate PNPG was added. After incubating for 30 min, 2 mL of 0.1 mol / L Na₂CO₃ was added to terminate the reaction. The absorbance was measured at 400 nm (A₂). The absorbance was measured again when 1 mL of buffer was used instead of the sample solution (A₀). The absorbance was measured again when 0.1 mL of buffer was used instead of the enzyme solution (A₁). The measurements were repeated three times, with acarbose as a positive control. α-glucosidase inhibition rate / % = [1 - (A₂ - A₁) / A₀] * 100

[0036] The free radical scavenging activity of mulberry leaf peptides was determined using the DPPH method.

[0037] The specific detection method is as follows: Add 1.5 mL of sample to 1.5 mL of 0.1 mmol / L DPPH· (95% ethanol), mix well, and incubate at 25 °C for 30 min. Measure the absorbance at 517 nm. VC solution is used as a control. The DPPH· scavenging capacity w (%) is calculated as follows:

[0038]

[0039] Where A0 is the absorbance of 1.5 mL of distilled water and 1.5 mL of 95% ethanol containing 0.1 mmol / L DPPH, A1 is the absorbance of 1.5 mL of hydrolysis product containing 0.1 mmol / L DPPH, and A2 is the absorbance of 1.5 mL of hydrolysis product and 1.5 mL of 95% ethanol.

[0040] Calculation of mulberry leaf peptide extraction rate:

[0041] The extraction rate of mulberry leaf peptides refers to the ratio of the mulberry leaf peptide solution obtained after a series of extraction, purification, and grading processes to the raw mulberry leaves. This product, obtained through drying, is mulberry leaf peptides. The extraction rate is calculated as follows:

[0042] W(%)=m (桑叶肽) / m (桑叶)

[0043] Example 1

[0044] An extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides, the method comprising the following steps:

[0045] Step 1: After the fresh mulberry leaves are harvested, they are washed, minced, and then water and alkaline protease are added for extraction. After extraction, the enzyme is inactivated to obtain mulberry leaf peptide extract.

[0046] Step 2: Filter the extract from Step 1 through a plate and frame filter to obtain a clear liquid and filter residue;

[0047] Step 3: The supernatant obtained in Step 2 is fractionated by ultrafiltration and nanofiltration membranes to obtain components with different molecular weight ranges;

[0048] Step 4: Concentrate the different molecular weight range components obtained in Step 3 to a concentration of 30%;

[0049] Step 5: Sterilize the concentrated solution obtained in Step 4 by passing it through a filter cartridge;

[0050] Step 6: Spray dry the product concentrate obtained in Step 5 to obtain products with different molecular weight range components.

[0051] Complete the process to enhance the hypoglycemic activity of mulberry leaf peptides.

[0052] Preferably, in step 1, 4 times the mass of water is added, and at 55°C, 2% of the leaf weight of alkaline protease is added, followed by ultrasonic extraction for 3 hours at an ultrasonic power of 80W.

[0053] Preferably, in step 3, the components with different molecular weight ranges are: less than 500D, 500-2000D, and 2000-5000D.

[0054] Preferably, in step 5, the filter element pore size is 0.22 micrometers.

[0055] Preferably, in step 6, during spray drying, the air inlet temperature is 180°C and the air outlet temperature is 90°C.

[0056] Example 2

[0057] An extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides, the method comprising the following steps:

[0058] Step 1: After the fresh mulberry leaves are harvested, they are washed, minced, and then water and alkaline protease are added for extraction. After extraction, the enzyme is inactivated to obtain mulberry leaf peptide extract.

[0059] Step 2: Filter the extract from Step 1 through a plate and frame filter to obtain a clear liquid and filter residue;

[0060] Step 3: The supernatant obtained in Step 2 is fractionated by ultrafiltration and nanofiltration membranes to obtain components with different molecular weight ranges;

[0061] Step 4: Concentrate the different molecular weight range components obtained in Step 3 to a concentration of 30%;

[0062] Step 5: Sterilize the concentrated solution obtained in Step 4 by passing it through a filter cartridge;

[0063] Step 6: Spray dry the product concentrate obtained in Step 5 to obtain products with different molecular weight range components.

[0064] Complete the process to enhance the hypoglycemic activity of mulberry leaf peptides.

[0065] Preferably, in step 1, 5 times the mass of water is added, and at 50°C, 3% of the leaf weight of alkaline protease is added, followed by ultrasonic extraction for 1 hour at an ultrasonic power of 300W.

[0066] Preferably, in step 3, the components with different molecular weight ranges are: less than 500D, 500-2000D, and 2000-5000D.

[0067] Preferably, in step 5, the filter element pore size is 0.15 micrometers.

[0068] Preferably, in step 6, during spray drying, the inlet air temperature is 190°C and the outlet air temperature is 95°C.

[0069] Example 3

[0070] An extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides, the method comprising the following steps:

[0071] Step 1: After the fresh mulberry leaves are harvested, they are washed, minced, and then water and alkaline protease are added for extraction. After extraction, the enzyme is inactivated to obtain mulberry leaf peptide extract.

[0072] Step 2: Filter the extract from Step 1 through a plate and frame filter to obtain a clear liquid and filter residue;

[0073] Step 3: The supernatant obtained in Step 2 is fractionated by ultrafiltration and nanofiltration membranes to obtain components with different molecular weight ranges;

[0074] Step 4: Concentrate the different molecular weight range components obtained in Step 3 to a concentration of 30%;

[0075] Step 5: Sterilize the concentrated solution obtained in Step 4 by passing it through a filter cartridge;

[0076] Step 6: Spray dry the product concentrate obtained in Step 5 to obtain products with different molecular weight range components.

[0077] Complete the process to enhance the hypoglycemic activity of mulberry leaf peptides.

[0078] Preferably, in step 1, 6 times the mass of water is added, and at 60°C, 1.5% of the leaf weight of alkaline protease is added, followed by ultrasonic extraction for 4 hours at an ultrasonic power of 400W.

[0079] Preferably, in step 3, the components with different molecular weight ranges are: less than 500D, 500-2000D, and 2000-5000D.

[0080] Preferably, in step 5, the filter element pore size is 0.25 micrometers.

[0081] Preferably, in step 6, during spray drying, the inlet air temperature is 170°C and the outlet air temperature is 85°C.

[0082] The product performance obtained from the above three embodiments is shown in Tables 1-3 below.

[0083] Example 4

[0084] Based on Example 3, in step 1, 6 times the mass of water was added, and before adding alkaline protease for enzymatic hydrolysis at 60°C, the pH was controlled at 8, and pulsed light treatment was performed. The pulsed light treatment conditions were (pulse light intensity of 10^4 uW / cm). 2 Pulse frequency: 1 pulse / s, 15 s. Add 1% alkaline protease by weight of leaf and extract using ultrasound for 4 hours at a power of 400 W.

[0085] Everything else is the same as in Example 3.

[0086] Example 5

[0087] Based on Example 3, in step 2, the obtained clear liquid is frozen, thawed, and then subjected to step 3. The freezing temperature is -18°C, and the freezing time is 4 hours. Other steps are the same as in Example 3.

[0088] Example 6

[0089] Based on Example 3, in step 1, before adding alkaline protease for enzymatic hydrolysis, pulsed light treatment is performed while controlling the pH within the range of 8-10. The pulsed light treatment conditions are: pulsed light intensity of 10^4 uW / cm. 2 The number of pulses is 1 pulse / s, for 15 seconds. Other details are the same as in Example 3.

[0090] In step 2, the obtained clear liquid is frozen, thawed, and then processed in step 3. The freezing temperature is -18°C, and the freezing time is 4 hours. Other procedures are the same as in Example 3.

[0091] Example 7

[0092] Based on Example 3, in step 1, ultrasonic extraction for 4 hours is not used.

[0093] Table 1. Indicators and in vitro hypoglycemic and antioxidant activities of the product in the molecular weight range of Example 1.

[0094]

[0095] Table 2. Product indicators and in vitro hypoglycemic and antioxidant activities of the molecular weight range components in Example 2.

[0096]

[0097] Table 3. Indicators and in vitro hypoglycemic and antioxidant activities of the product components in Example 3 (molecular weight range).

[0098]

[0099] The above-mentioned stock solution is the clarified solution obtained in step 2. As shown in Tables 1-3, after fractionation treatment using ultrafiltration and nanofiltration membranes, the contents of DNJ, protein, total sugar, and gamma-aminobutyric acid (GABA) in products with different molecular weight ranges are different. At the same time, the α-glucosidase inhibitory activity and DPPH· scavenging ability vary significantly. Products with a molecular weight of less than 500D have higher GABA content, α-glucosidase inhibitory activity, and DPPH· scavenging ability than products with a molecular weight of 500-2000D and 2000-5000D, allowing for the development of different product application scenarios as needed.

[0100] Table 4. Indicators of products treated with pulsed light and their in vitro hypoglycemic and antioxidant activities.

[0101]

[0102]

[0103] As shown in Table 4, by controlling the pH to around 8, the content of gamma-aminobutyric acid in Example 4 is significantly higher than that in Example 3. Furthermore, the α-glucosidase inhibitory activity and DPPH· scavenging ability of the product in Example 4 are much higher than those in Example 3. Example 4 achieves a high extraction rate (7.7%) with a relatively small amount of enzyme added (1%). This means that pulsed light treatment can greatly improve the dissolution of effective components in mulberry leaf peptides, which is beneficial to improving the enzymatic hydrolysis efficiency and reducing the amount of enzyme preparation used.

[0104] Table 5. Indicators for whether cryopreservation was selected and their in vitro hypoglycemic and antioxidant activities.

[0105]

[0106] As shown in Table 5, compared with Example 3, the content of gamma-aminobutyric acid in Example 5 is significantly increased. Furthermore, the α-glucosidase inhibitory activity and DPPH scavenging ability of the product in Example 5 are much higher than those in Example 3. That is, freezing the initial filtrate and then thawing it can further remove insoluble macromolecular impurities, significantly improve the activity of the mulberry leaf peptide stock solution, and increase the total content of effective products of each component in the later separation.

[0107] Table 6. Indicators and in vitro hypoglycemic and antioxidant activities of the product components in Example 6 (molecular weight range).

[0108]

[0109] As shown in Table 6, compared with Examples 3-5, the gamma-aminobutyric acid content, α-glucosidase inhibitory activity, and DPPH scavenging ability of the product in Example 6 are much higher than those in Examples 3-5. That is, by using pulsed light combined with freezing and then thawing of the initial filtrate, the dissolution of the effective components in mulberry leaf peptides can be greatly improved, insoluble macromolecular impurities can be removed, the activity of the mulberry leaf peptide stock solution can be significantly improved, and the total content of the effective products of each component can be increased in the later separation.

[0110] Table 7. Comparative Example 1: Indicators and in vitro hypoglycemic and antioxidant activities of the products within the molecular weight range.

[0111]

[0112] As shown in Table 7, compared to Example 3, without ultrasonic treatment, the gamma-aminobutyric acid content, α-glucosidase inhibitory activity, and DPPH· scavenging ability of the stock solution in Comparative Example 1 were significantly lower than those in Example 3, and the extraction rate was also significantly lower. This indicates that using ultrasound-assisted enzymatic hydrolysis can increase the extraction rate and shorten the extraction time.

[0113] Animal experiments were conducted using components from different regions obtained in Example 6.

[0114] Method 1:

[0115] 1.2.1 Effects of mulberry leaf polypeptides on blood glucose in normal mice

[0116] Healthy male Kunming mice were fasted for 3-5 hours and given free access to water. Fasting blood glucose levels were measured. Fifty mice were randomly divided into five groups of ten mice each: a normal control group, a mulberry leaf polypeptide group (less than 500 D), a mulberry leaf polypeptide group (500-2000 D), a mulberry leaf polypeptide group (2000-5000 D), and a health supplement group. The experimental groups were given different test sample solutions at doses of 2.01 g / kg, 2.01 g / kg, 1.05 g / kg, and 2.01 g / kg, respectively, with a dosage of 0.2 ml / 10 g. The control group received the same volume of sterile distilled water. After 30 days of continuous administration, the mice were fasted for 16 hours, and fasting blood glucose levels were measured. The fasting blood glucose values ​​of the two groups were compared.

[0117] 1.2.2 Effects of mulberry leaf polypeptides on blood glucose in a rat model of alloxan-induced insulin resistance and glucose / lipid metabolism disorder

[0118] One hundred and five healthy male SD rats were acclimatized to a standard maintenance diet for 3-5 days, then fasted for 3-5 hours. Tail blood was collected, and blood glucose levels were measured before glucose administration (0 hours) and at 0.5 and 2 hours after administration of 2.5 g / kg BW glucose. These values ​​were used as the baseline values ​​for this batch of animals. The rats were divided into seven groups based on their 0 and 0.5 hour blood glucose levels: a blank control group, a model control group, a high-dose group (less than 500 D) of mulberry leaf polypeptide, a medium-dose group (less than 500 D) of mulberry leaf polypeptide, a low-dose group (less than 500 D) of mulberry leaf polypeptide, a 500-2000 D group of mulberry leaf polypeptide, and a 2000-5000 D group of mulberry leaf polypeptide, with 15 rats in each group. The blank control group received no treatment. The five experimental groups were administered different concentrations of the test sample by gavage at doses of 2.01 g / kg, 0.67 g / kg, 0.335 g / kg, 2.01 g / kg, and 1.05 g / kg, respectively. The model control group received the same volume of solvent. This treatment was continued for 33 consecutive days. After one week of maintenance feeding, the model control group and the three dosage groups were switched to a high-energy diet and fed for three weeks. The model control group and the three dosage groups were then fasted for 24 hours (water was permitted), and alloxan 105 mg / kg was administered intraperitoneally at a dose of 1 ml / 100g body weight. Following the injection, the animals were fed the high-energy diet for 3–5 days. At the end of the experiment, all animals were fasted for 16 hours, and fasting blood glucose, glucose tolerance, serum insulin, cholesterol, and triglyceride levels were measured.

[0119] 1.3 Data Processing and Result Determination

[0120] Generally, analysis of variance is adopted. However, homogeneity of variance test should be carried out first according to the procedure of analysis of variance. If the variances are homogeneous, calculate the F value. If the F value < F0.05, the conclusion is that there is no significant difference among the means of each group; if the F value ≥ F0.05 and P ≤ 0.05, statistical analysis is carried out using the pairwise comparison method for the means between multiple experimental groups and a control group; for non-normal or heterogeneous variance data, appropriate variable transformation is carried out. After meeting the requirements of normality or homogeneity of variance, statistical analysis is carried out using the transformed data; if the purpose of normality or homogeneity of variance is still not achieved after variable transformation, rank sum test is used for statistical analysis.

[0121] 1.3.1 Index determination

[0122] 1.3.1.1 Hypoglycemic test on normal animals

[0123] Blood glucose index: There is no statistical significance in the comparison of fasting blood glucose between the test sample dose group and the control group. It is determined that the test sample has no effect on the blood glucose of normal animals.

[0124] 1.3.1.2 Hypoglycemic test on hyperglycemic model

[0125] Fasting blood glucose index: On the premise that the model is established, when comparing the test sample dose group with the model control group, if there is statistical significance in the decrease of fasting blood glucose or the increase of the percentage of blood glucose decrease, it is determined that the result of the fasting blood glucose index of the test sample is positive.

[0126] Glucose tolerance index: On the premise that the model is established, when comparing the test sample dose group with the model control group, if there is statistical significance in the decrease of blood glucose (or the increase of the percentage of blood glucose decrease) at any time point of 0.5 and 2 hours after giving glucose or medical starch, or if there is statistical significance in the decrease of the area under the blood glucose curve at 0, 0.5, and 2 hours, it is determined that the result of the glucose tolerance index of the test sample is positive.

[0127] Blood lipid index: On the premise that the model is established, when comparing the test sample dose group with the model control group, if there is statistical significance in the decrease of serum cholesterol or triglyceride, it can be determined that the test sample has a positive hypolipidemic index.

[0128] 1.3.2 Result determination

[0129] Scheme 2: If one of the two indexes of fasting blood glucose and glucose tolerance is positive, and there is no obvious increase in blood lipids (total cholesterol, triglyceride), and it has no effect on the fasting blood glucose of normal animals, it can be determined that the result of the animal experiment on the function of the test sample to help maintain a healthy blood glucose level is positive.

[0130] 2 Results

[0131] 2.1 Fasting blood glucose of normal mice

[0132] Table 8 shows the pre-group body weight and blood glucose levels of normal mice. After 30 days of drug administration, the body weight of mice in each treatment group showed no significant change compared to the control group. However, the fasting blood glucose levels of mice in the mulberry leaf polypeptide groups (<500D), mulberry leaf polypeptide groups (500-2000D), and mulberry leaf polypeptide groups (2000-5000D) decreased compared to the control group. The decrease in blood glucose level in the mulberry leaf polypeptide group (<500D) was statistically significant. The health supplement group showed no significant change compared to the control group.

[0133] The results of this experiment suggest that mulberry leaf polypeptides smaller than 500D can reduce blood glucose levels in normal animals, while mulberry leaf polypeptides of 500-2000D, mulberry leaf polypeptides of 2000-5000D, and the health supplement group have no significant effect on blood glucose levels in normal animals. The results are shown in Table 9.

[0134] Table 8 Fasting blood glucose levels in normal mice before drug administration

[0135]

[0136] Compared with the normal group, *P<0.05, **P<0.01;

[0137] Table 9 Fasting blood glucose levels in normal mice after 30 days of drug administration.

[0138]

[0139] Compared with the normal group, *P<0.05, **P<0.01;

[0140] 2.2 Fasting blood glucose, glucose tolerance and lipid levels in rats

[0141] The blood glucose levels of the rats before grouping are shown in Table 10. There were no significant differences in blood glucose levels among the groups.

[0142] Table 11 shows the blood glucose, glucose tolerance, and blood lipid levels of rats 33 days after administration. The table indicates that the blood glucose levels at 0 h in the normal control group, model control group, and all experimental groups ranged from 3.2 to 6.3 mmol / L. The model group showed a significant difference in blood glucose levels compared to the normal group, while there were no significant changes between the administered groups and the model group. After 0.5 h of glucose administration, blood glucose levels in all groups were above 10.0 mmol / L. The blood glucose levels in the low, medium, and high dose groups (less than 500D of mulberry leaf polypeptide) were lower than those in the model control group, but the differences were not significant. The blood glucose levels in the 500-2000D and 2000-5000D mulberry leaf polypeptide groups were significantly lower than those in the model control group, with statistically significant differences. Two hours after glucose administration, blood glucose levels in all groups decreased significantly compared to 0.5 h, while the blood glucose level in the model group remained above 10.0 mmol / L. Compared to the normal control group, the area under the curve (AUC) was significantly increased, indicating a statistically significant difference and successful replication of the glucose metabolism disorder model. The 2-hour blood glucose levels in each experimental group were significantly lower than those in the model control group, and the differences were statistically significant. The results suggest that the high, medium, and low doses of mulberry leaf polypeptide (less than 500D), as well as the 500-2000D and 2000-5000D groups, all have a significant blood glucose-lowering effect.

[0143] After 33 days of administration, serum CHO levels in the model group rats were significantly higher than those in the normal group, which was statistically significant. TG levels in the model group were slightly higher than those in the normal group, but not statistically significant. Compared with the model group, there were no significant changes in CHO and TG levels in any of the administration groups (the TG level in the 2000-5000D mulberry leaf polypeptide group was significantly higher than that in the model group; this was due to abnormally high TG levels in individual animals, leading to an overall increase in the mean and standard deviation, and should be considered for exclusion). These results suggest that mulberry leaf polypeptide levels below 500D, 500-2000D, and 2000-5000D have no significant effect on blood lipids.

[0144] Table 10 Blood glucose levels in rats before drug administration ( n=15)

[0145]

[0146] Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01;

[0147] Table 11 Blood glucose and lipid levels in rats after 33 days of drug administration ( n=15)

[0148]

[0149] Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01;

[0150] 3. Conclusion

[0151] In a hypoglycemic experiment in normal mice, the fasting blood glucose levels of mice in the mulberry leaf polypeptide 500-2000D, mulberry leaf polypeptide 2000-5000D, and health supplement groups were not statistically different from those in the control group, indicating that they had no effect on blood glucose in normal animals.

[0152] Under the premise of establishing a rat model of glucose metabolism disorder, compared with the model control group, the blood glucose levels of the tested sample group and the model control group were significantly different at 0.5 h after glucose administration compared with the model group, which was statistically significant. Furthermore, the blood glucose levels of each experimental group at 2 h were significantly different from those of the model group, which was also statistically significant. Therefore, the glucose tolerance index of the tested samples was positive, and mulberry leaf peptides less than 500D, mulberry leaf peptides 500-2000D, and mulberry leaf peptides 2000-5000D all had a certain hypoglycemic effect. In addition, the blood glucose levels of the three experimental groups (low, medium, and high) of mulberry leaf peptide less than 500D showed that the hypoglycemic effect of mulberry leaf peptide less than 500D was dose-dependent; the higher the dose, the more significant the hypoglycemic effect at 2 h.

[0153] According to the method for determining the effect of maintaining blood glucose balance, if either fasting blood glucose or glucose tolerance is positive, and blood lipids (total cholesterol and triglycerides) are not significantly elevated, and there is no effect on fasting blood glucose in normal animals, then the test sample can be judged to have a positive result in functional animal experiments that help maintain healthy blood glucose levels. Therefore, both mulberry leaf polypeptide 500-2000D and mulberry leaf polypeptide 2000-5000D can help maintain healthy blood glucose levels. Mulberry leaf polypeptide less than 500D has a certain blood glucose lowering effect in normal animals, and its blood glucose lowering effect is also more obvious in hyperglycemic animals.

[0154] Based on the above animal experiment results, we found that the hypoglycemic activities of the three components of mulberry leaf peptides are in the following order: less than 500D component > 500-2000D component > 2000-5000D component. Based on this characteristic, we can purify and grade the mulberry leaf peptides, and the resulting products can be used for targeted treatment of specific diseases.

[0155] Because the 500D product has a high content of DNJ alkaloids and protein peptides, but a relatively low content of mulberry leaf polysaccharides, it has high hypoglycemic activity and can be used for the development of hypoglycemic drugs, as well as for type 2 diabetic patients with high fasting blood glucose (greater than 12 mmol / L); the 500-2000D product has moderate content of DNJ alkaloids and protein peptides, and can be used for type 2 diabetic patients with less severe conditions and fasting blood glucose less than 12 mmol / L; the 2000-5000D product has a lower content of DNJ alkaloids and a higher content of protein peptides, and can be used for patients with mild diabetes (fasting blood glucose less than 8), as well as for people who need to control their weight, and for the development of weight loss products.

[0156] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The embodiments and features described in these embodiments can be arbitrarily combined without conflict. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. An extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides, characterized in that, The method includes the following steps: Step 1: After harvesting, fresh mulberry leaves are washed, shredded, and then water and alkaline protease are added for extraction. The enzyme is then inactivated to obtain mulberry leaf peptide extract. Before adding the alkaline protease for enzymatic hydrolysis, the pH is controlled within the range of 8-10, and pulsed light treatment is performed. The pulsed light treatment conditions are: pulsed light intensity of 10... 4 μW / cm², pulse frequency 1 time / s, 5-20s, adjust pH using sodium hydroxide, calcium hydroxide, ammonia, hydrochloric acid, phosphoric acid or sulfuric acid; Step 2: Filter the extract from Step 1 through a plate and frame filter to obtain a clear liquid and filter residue; Step 3: The clear liquid obtained in Step 2 is fractionated by ultrafiltration and nanofiltration membranes to obtain components with different molecular weight ranges; Step 4: Concentrate the components with different molecular weight ranges obtained in Step 3 respectively; Step 5: Sterilize the concentrated solution obtained in Step 4 by passing it through a filter cartridge; Step 6: Spray dry the product concentrate obtained in Step 5 to obtain products with different molecular weight range components. Complete the process to enhance the hypoglycemic activity of mulberry leaf peptides; In step 1, add 3-6 times the weight of water, add 1%-3% of alkaline protease by weight of the leaves at 50-60℃, and extract using ultrasound for 1-5 hours with an ultrasound power of 80-300w. In step 2, the obtained clear liquid is frozen and then thawed before step 3. The freezing temperature is -18℃ and the freezing time is 3-6 hours. In step 3, the different molecular weight range components obtained by ultrafiltration and nanofiltration membrane fractionation are: less than 500D, 500-2000D, and 2000-5000D.

2. The extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides according to claim 1, characterized in that, In step 5, the filter element pore size is 0.15-0.3 micrometers.

3. The extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides according to claim 1, characterized in that, In step 6, during spray drying, the air inlet temperature is 180±20℃ and the air outlet temperature is 90±5℃.

4. Mulberry leaf peptides obtained by the extraction method for enhancing the hypoglycemic activity of mulberry leaf peptides according to any one of claims 1-3.

5. The use of the mulberry leaf peptide according to claim 4 in the preparation of a medicament for treating patients with type 2 diabetes.

6. The use of the mulberry leaf peptide according to claim 4 in the preparation of health products that help lower blood sugar.

Images