Sophora flower bud and mulberry leaf composition beneficial to blood glucose metabolism health and preparation method and application thereof

Abstract

The invention relates to a sophora flower bud and mulberry leaf composition beneficial to blood glucose metabolism health as well as a preparation method and application of the sophora flower bud and mulberry leaf composition. The sophora flower bud and mulberry leaf composition comprises a sophora flower bud extract and a purified mulberry leaf extract, wherein the mass ratio of the sophora flower bud extract to the purified mulberry leaf extract is (1: 0.3)-(1: 0.5); moreover, according to the mass percent, the sophora flower bud extract contains 10%-20% of quercetin glucoside, 20%-30% of general flavone and 3%-8% of polysaccharide; the mulberry leaf purified extract contains 1%-3% of DNJ. Through research, the synergistic sugar control effect of the sophora flower bud extract and the folium mori purified extract with specific component contents under a specific mass ratio is found, and the optimal bioavailability and sugar control effect can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional foods and natural medicines, and particularly relates to a sophora japonica leaf composition beneficial to blood glucose metabolism health, a preparation method thereof, and an application thereof. Background Art

[0002] With the rapid growth of the population with diabetes and pre-diabetes, the demand for blood glucose control has become a core issue in the global health field. Although existing blood glucose control drugs (such as metformin, DPP4 inhibitors) are effective, they have side effects and long-term dependence. In traditional Chinese medicine, both sophora japonica and mulberry leaves are recorded to relieve "consumptive thirst" (symptoms of diabetes), but the bioavailability of single components is low and the action mechanism is single, making it difficult to meet the modern blood glucose control requirements.

[0003] In the prior art, the enzymatic hydrolysis technology of sophora japonica powder and the extraction process of DNJ (1-deoxynojirimycin) in mulberry leaves have not achieved high-efficiency synergistic effects. In addition, traditional formulas lack scientific verification and cannot clarify their multi-pathway blood glucose control mechanisms (such as inhibiting sugar absorption, promoting GLP-1 secretion, and repairing pancreatic islet function). Therefore, there is an urgent need to develop a blood glucose control composition based on natural ingredients, with multi-mechanism synergy and clinical verification. Summary of the Invention

[0004] Aiming at the deficiencies of the above-mentioned prior art, the present invention improves the extraction methods of sophora japonica and mulberry leaves, increases the content of related active ingredients therein, and combines them to achieve synergistic effects, providing a sophora japonica leaf composition beneficial to blood glucose metabolism health.

[0005] The specific technical solution of the present invention is as follows: In the first aspect of the present invention, there is provided a sophora japonica leaf composition beneficial to blood glucose metabolism health, which comprises a sophora japonica extract and a purified mulberry leaf extract, and the mass ratio of the sophora japonica extract to the purified mulberry leaf extract is (1:0.3)-(1:0.5); moreover, by mass percentage, the sophora japonica extract contains 10%-20% of quercetin glucoside, 20%-30% of total flavonoids, and 3%-8% of polysaccharides; the purified mulberry leaf extract contains 1%-3% of DNJ.

[0006] By selecting sophora japonica compositions and purified mulberry leaf extracts with specific component contents, and through scientific screening, it is found that the synergistic blood glucose control effect of the sophora japonica extract and the purified mulberry leaf extract at a specific mass ratio (1:0.3)-(1:0.5) achieves the best bioavailability and blood glucose control effect.

[0007] Preferably, the sophora japonica extract in the composition of the present invention is prepared by sequentially treating sophora japonica pulverizate with cellulase and α-1,4-galacturonase.

[0008] Flos Sophorae Immaturus contains various active ingredients such as flavonoids, saponins, and polysaccharides. By enzymatically hydrolyzing Flos Sophorae Immaturus with cellulase and α-1,4-galacturonase, the bioavailability of quercetin glucoside in Flos Sophorae Immaturus can be significantly improved.

[0009] Preferably, the dosage of the cellulase is 5%-10% of the weight of the crushed Flos Sophorae Immaturus, the enzymatic hydrolysis temperature is 45-65°C, the enzymatic hydrolysis pH is 4.5-5.5, and the enzymatic hydrolysis time is 1-2 hours; The dosage of the α-1,4-galacturonase is 5%-10% of the weight of the crushed Flos Sophorae Immaturus, the enzymatic hydrolysis temperature is 40°C-60°C, the enzymatic hydrolysis pH is 4.0-6.0, and the enzymatic hydrolysis time is 1-2 hours.

[0010] Preferably, the preparation method of the purified mulberry leaf extract in the composition of the present invention includes the steps: 1) The frosted mulberry leaves are dried in the shade and crushed to obtain frosted mulberry leaf powder, and then enzymatically hydrolyzed with cellulase, pectinase, and β-glucosidase, inactivated, and dried or freeze-dried to obtain a crude mulberry leaf extract; 2) The obtained crude mulberry leaf extract is extracted under hot water and ultrasonic conditions, the extraction solutions are filtered and combined, and concentrated and dried to obtain a mulberry leaf extract; 3) The purified mulberry leaf extract is dissolved in an acidic aqueous solution with a pH of 2.5-3.0 and dynamically adsorbed by a strongly acidic cation exchange resin after pretreatment, and the adsorption flow rate is 2-3 BV / h; 4) After the adsorption is completed, the resin is eluted in stages with hot water at 50°C with a pH of 3.0, hot water at 70°C with a pH of 4.5, and hot water at 85°C with a pH of 6.0, and finally the 85°C eluate is collected; 5) The eluate is concentrated to 1 / 10 of the original volume by a nanofiltration membrane and freeze-dried or spray-dried to obtain a purified mulberry leaf extract with a DNJ content of 1%-3%.

[0011] Frosted mulberry leaves are generally harvested between September and October after the frost. After being frosted, the content of their nutritional and medicinal components is relatively high. Cellulase can break down the cellulose in the mulberry leaf cell wall and release DNJ; pectinase is used to degrade pectin and improve the dissolution rate of cell contents; β-glucosidase can selectively hydrolyze glycosidic bonds and reduce polysaccharide interference. Sequentially using cellulase, pectinase, and β-glucosidase for enzymatic hydrolysis is more conducive to the release of the active ingredient DNJ. Then, resin adsorption and purification are adopted, which can extract the active ingredient DNJ more efficiently and quickly without destroying the structure of the active ingredient.

[0012] Preferably, in step 1), in the enzymatic hydrolysate, the weight-volume ratio of mulberry leaves to the solvent is 1:15 - 20, the dosage of cellulase is 5% - 10% of the weight of mulberry leaves, the dosage of pectinase is 2% - 5% of the weight of mulberry leaves, the dosage of β-glucosidase is 2% - 5% of the weight of mulberry leaves, the enzymatic hydrolysis temperature is 45 - 65 °C, the enzymatic hydrolysis pH is 4.5 - 5.5, and the enzymatic hydrolysis time is 1 - 2 hours.

[0013] Preferably, in step 2), the hot water temperature is 70 °C - 80 °C, the ultrasonic frequency is 200 - 400 W, the extraction times are 3 times, and the extraction time for each time is 15 - 30 min.

[0014] Preferably, in step 3), the strongly acidic cation exchange resin is of D001 type, and the pretreatment method is activation with 4% - 6% HCl for 2 - 4 hours, followed by washing with water until the pH is 5.0 - 6.0.

[0015] Preferably, in step 5), the molecular weight cut-off of the nanofiltration membrane is 200 - 500 Da.

[0016] Compared with the traditional water extraction or alcohol extraction of DNJ in mulberry leaf extract, when preparing the purified mulberry leaf extract of the present invention, enzyme treatment is first used, and then resin adsorption purification is adopted. The content of DNJ in the obtained purified mulberry leaf extract can be as high as 1% - 3%, which is increased by 10 - 30 times.

[0017] In the second aspect of the present invention, there is also provided the use of the said composition in the preparation of a functional food or drug for controlling blood sugar, improving insulin resistance or preventing diabetic complications. The said functional food includes baked foods (such as bread, biscuits, etc.), flour products, beverages (solid beverages, liquid beverages), tablet candies, oral liquids; the said drug includes oral preparations or enteric-coated preparations.

[0018] In the third aspect of the present invention, there is also provided a sugar-control solid beverage, which contains the said composition and food-grade acceptable excipients, wherein the addition amount of the composition is 0.5 - 1.5 g / 100 g.

[0019] The beneficial effects of the present invention are as follows: (1) Through research, the present invention discovers the synergistic blood sugar control effect of the sophora flower extract with specific component content and the purified mulberry leaf extract at a specific mass ratio. Specifically, sophora flower quercetin glucoside improves insulin sensitivity by activating the PPARγ pathway, and mulberry leaf DNJ inhibits α-glucosidase. The two work together to regulate the GLP-1 / insulin axis, and finally achieve the effects of inhibiting sugar absorption, promoting GLP-1 secretion, and repairing pancreatic islet function; this ratio is scientifically screened to achieve the best bioavailability and blood sugar control effect.

[0020] (2) By improving the preparation methods of the purified extracts of mulberry leaves and sophora japonica fruits, the present invention increases the content of related active ingredients therein. Specifically, by enzymatically hydrolyzing sophora japonica fruits with cellulase and α-1,4-galacturonase, the bioavailability of quercetin glucoside in sophora japonica fruits is significantly improved. By first treating with multiple enzymes and then using the method of resin adsorption and purification to prepare the purified extract of mulberry leaves, the content of DNJ in the extract can be as high as 1%-3%, which is 15-30 times higher than that of the traditional water extraction or alcohol extraction methods.

[0021] (3) The composition of the present invention has safe ingredients, all of which are natural ingredients, meet the standards of medicine and food homology, have no chemical additives, and have good tolerance in human trials. It can be used in functional foods or drugs. Detailed implementation manners

[0022] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0023] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0024] Example 1 This example provides a sophora japonica fruit and mulberry leaf composition beneficial to blood glucose metabolism health, including a sophora japonica fruit extract and a purified mulberry leaf extract; it is prepared by the following method: (1) Preparation of the sophora japonica fruit extract.

[0025] 100 g of sophora japonica fruits are selected, cleaned, dried, and pulverized through a 60-100 mesh sieve; The obtained sophora japonica fruit powder is dissolved in a buffer solution with a pH of 5.0 at 20 times (v / w), 10% of the cellulase based on the mass of the sophora japonica fruit powder is added, and enzymatic hydrolysis is carried out at 50 °C for 2 h, and the enzyme is inactivated by a 10-minute water bath at 90 °C; Then, 10% of α-1,4-galacturonase based on the mass of the sophora japonica fruit powder is added, and enzymatic hydrolysis is carried out at 50 °C and a pH of 5.0 for 2 h; the enzyme is inactivated by a 10-minute water bath at 90 °C; The supernatant is taken by centrifugation and spray-dried (inlet air temperature 180 °C) to obtain the sophora japonica fruit extract.

[0026] After detection, the mass percentage content of the active ingredients therein is: quercetin glucoside 16%, total flavonoids 25%, and polysaccharides 5%.

[0027] (2) Preparation of the purified mulberry leaf extract Raw materials: 50 g of frosted mulberry leaves are selected, cleaned, dried, and pulverized through a 40-60 mesh sieve.

[0028] 1) Enzyme treatment: Mulberry leaf powder was dissolved in a buffer solution with 20 times (v / w) pH 5.0, 10% of cellulase based on the mass of mulberry leaf powder was added, enzymolysis was carried out at 50 °C for 2 h, and the enzyme was inactivated; then 4% of pectinase based on the mass of mulberry leaf powder was added, enzymolysis was carried out at 50 °C for 1.5 h, and the enzyme was inactivated; finally, 3% of β-glucosidase based on the mass of mulberry leaf powder was added, enzymolysis was carried out at 50 °C for 1 h, and the enzyme was inactivated; after concentration, it was freeze-dried to obtain the crude mulberry leaf extract; 2) The obtained crude mulberry leaf extract was added with 10 times the volume (v / w) of hot water, and extracted at 70 °C and 400 W ultrasonic wave for 20 min; filtered, and 10 times the volume (v / w) of hot water was added to the filter residue again, and extracted at 70 °C and 400 W ultrasonic wave for 20 min; filtered, and 10 times the volume (v / w) of hot water was added to the filter residue, and extracted at 70 °C and 400 W ultrasonic wave for 20 min; filtered, the filtrates were combined and concentrated to obtain the mulberry leaf extract; 3) The mulberry leaf extract was dissolved in an acidic aqueous solution with 10 times the volume (v / w) of pH 2.5, and then dynamically adsorbed by a strongly acidic cation exchange resin after pretreatment and activation, and the adsorption flow rate was 2 BV / h; the strongly acidic cation exchange resin was of D001 type, and the pretreatment method was activation with 4% HCl for 4 hours and washed with water to pH 5.5.

[0029] 4) After the adsorption was completed, the resin was eluted in stages with hot water at 50 °C (pH 3.0), hot water at 70 °C (pH 4.5), and hot water at 85 °C (pH 6.0), and the 85 °C eluate was collected; 5) The eluate was concentrated to 1 / 10 of the original volume by a nanofiltration membrane with a cut-off molecular weight of 200 Da, and freeze-dried or spray-dried to obtain the purified mulberry leaf extract.

[0030] After detection, the mass percentage content of the active ingredient was: DNJ 2.8%.

[0031] (3) The sophora flower bud extract and the purified mulberry leaf extract obtained in steps (1) and (2) were mixed according to a mass ratio of 1:0.3; after mixing, they were passed through an 80-mesh sieve to prepare the composition.

[0032] Example 2 This example provides a sophora flower bud and mulberry leaf composition beneficial to blood glucose metabolism health, including sophora flower bud extract and purified mulberry leaf extract; it is prepared by the following method: (1) Preparation of sophora flower bud extract.

[0033] 100 g of sophora flower buds were selected, cleaned, dried, and pulverized through a 60-100 mesh sieve; The obtained sophora flower bud powder was dissolved in a buffer solution with 20 times (v / w) pH 5.5, 8% of cellulase based on the mass of sophora flower bud powder was added, enzymolysis was carried out at 55 °C for 1 h, and the enzyme was inactivated by a 90 °C water bath for 10 minutes; Then add α-1,4-galacturonase at 8% of the mass of sophora flower powder, and carry out enzymatic hydrolysis at 60 °C, pH 6.0 for 1 h; inactivate the enzyme in a water bath at 90 °C for 10 minutes; Centrifuge to obtain the supernatant, and spray-dry it (inlet air temperature 180 °C) to obtain sophora flower extract.

[0034] After detection, the mass percentage content of the active ingredients is as follows: quercetin glucoside 12%, total flavonoids 22%, and polysaccharides 4%.

[0035] (2) Preparation of purified mulberry leaf extract Raw materials: 50 g of frosted mulberry leaves, selected, dried, and pulverized through a 40-60 mesh sieve.

[0036] 1) Enzymatic treatment: Dissolve mulberry leaf powder in a buffer solution with a pH of 5.0 at 20 times (v / w), add cellulase at 10% of the mass of mulberry leaf powder, carry out enzymatic hydrolysis at 55 °C for 1 h, and inactivate the enzyme; then add pectinase at 5% of the mass of mulberry leaf powder, carry out enzymatic hydrolysis at 55 °C for 1 h, and inactivate the enzyme; finally add β-glucosidase at 5% of the mass of mulberry leaf powder, carry out enzymatic hydrolysis at 50 °C for 1 h, and inactivate the enzyme; concentrate and then freeze-dry to obtain crude mulberry leaf extract; 2) Add 10 times the volume (v / w) of hot water to the obtained crude mulberry leaf extract, and extract it at 80 °C and 400 W ultrasonic waves for 15 min; filter, add 10 times the volume (v / w) of hot water to the filter residue again, and extract it at 80 °C and 400 W ultrasonic waves for 15 min; filter, add 10 times the volume (v / w) of hot water to the filter residue, and extract it at 80 °C and 400 W ultrasonic waves for 10 min; filter, combine the filtrates, and concentrate to obtain mulberry leaf extract; 3) Dissolve the mulberry leaf extract in an acidic aqueous solution with a pH of 2.5 at 10 times the volume (v / w), and then dynamically adsorb it with a strongly acidic cation exchange resin after pretreatment and activation, with an adsorption flow rate of 3 BV / h; the strongly acidic cation exchange resin is of the D001 type, and the pretreatment method is to activate it with 4% HCl for 4 hours and wash it with water until the pH is 5.5.

[0037] 4) After the adsorption is completed, elute the resin in stages with hot water at 50 °C (pH 3.0), hot water at 70 °C (pH 4.5), and hot water at 85 °C (pH 6.0), and collect the eluate at 85 °C; 5) Concentrate the eluate to 1 / 10 of the original volume through a nanofiltration membrane with a cut-off molecular weight of 200 Da, and freeze-dry or spray-dry it to obtain purified mulberry leaf extract.

[0038] After detection, the mass percentage content of the active ingredient is: DNJ 2.2%.

[0039] (4) Mix the sophora japonica extract obtained in steps (1) and (2) with the purified mulberry leaf extract at a mass ratio of 1:0.5; after mixing, pass through an 80-mesh sieve to obtain the composition.

[0040] Comparative Example 1 This comparative example provides a sophora japonica mulberry leaf composition, including sophora japonica powder and purified mulberry leaf extract; it is prepared by the following method: (1) Preparation of sophora japonica powder.

[0041] 100 g of sophora japonica, selected, dried, pulverized and passed through a 60-100 mesh sieve; the obtained sophora japonica powder.

[0042] After detection, the mass percentage content of the active ingredients is: total flavonoids 20%, polysaccharides 0.1%. And since it is only simply pulverized, quercetin glucoside is not effectively extracted, and the detected content is 0%.

[0043] (2) Preparation of purified mulberry leaf extract, the same as in Example 1.

[0044] (3) Mix the sophora japonica powder obtained in steps (1) and (2) with the purified mulberry leaf extract at a mass ratio of 1:0.3; after mixing, pass through an 80-mesh sieve to obtain the composition.

[0045] Comparative Example 2 This comparative example provides a sophora japonica composition, including sophora japonica extract; it is prepared by the following method: (1) Preparation of sophora japonica extract, the same as in Example 1.

[0046] (2) Pass the sophora japonica extract obtained in step (1) through an 80-mesh sieve to obtain the composition.

[0047] Comparative Example 3 This comparative example provides a sophora japonica mulberry leaf composition, including sophora japonica extract and purified mulberry leaf extract; it is prepared by the following method: (1) Preparation of sophora japonica extract, the same as in Example 1.

[0048] (2) Preparation of purified mulberry leaf extract, the same as in Example 1.

[0049] (3) Mix the sophora japonica extract obtained in steps (1) and (2) with the purified mulberry leaf extract at a mass ratio of 1:0.2; after mixing, pass through an 80-mesh sieve to obtain the composition.

[0050] Comparative Example 4 This comparative example provides a sophora japonica mulberry leaf composition, including sophora japonica extract and mulberry leaf extract; it is prepared by the following method: (1) Preparation of sophora japonica extract, the same as in Example 1.

[0051] (2) Preparation of mulberry leaf extract; Raw materials: 50 g of frosted mulberry leaves, selected, dried, pulverized and sieved through a 40 - 60 mesh sieve.

[0052] Water extraction: Add 10 times the volume (v / w) of water to the mulberry leaf powder, extract at 85 °C for 30 min, filter, add 10 times the volume of water (v / w) to the filter residue, extract at 85 °C for 20 min, combine the filtrates, and concentrate to dryness to obtain the mulberry leaf extract.

[0053] After detection, the mass percentage content of the active ingredient is: DNJ 0.1%.

[0054] (3)Mix the sophora flower extract and the purified mulberry leaf extract obtained in steps (1) and (2) according to a mass ratio of 1:0.3; pass through an 80 - mesh sieve after mixing to obtain the composition.

[0055] Comparative Example 5 This comparative example provides a sophora flower - mulberry leaf composition, including sophora flower extract and mulberry leaf extract; it is prepared by the following method: (1)Preparation of sophora flower extract, the same as in Example 1.

[0056] (2) Preparation of mulberry leaf extract; Raw materials: 50 g of frosted mulberry leaves, selected, dried, pulverized and sieved through a 40 - 60 mesh sieve.

[0057] 1) Enzyme treatment: Dissolve the mulberry leaf powder in 20 times (v / w) of a buffer solution with pH 5.0, add 10% of the mass of the mulberry leaf powder of cellulase, enzymatically hydrolyze at 50 °C for 2 h, inactivate the enzyme; then add 4% of the mass of the mulberry leaf powder of pectinase, enzymatically hydrolyze at 50 °C for 1.5 h, inactivate the enzyme; finally add 3% of the mass of the mulberry leaf powder of β - glucosidase, enzymatically hydrolyze at 50 °C for 1 h, inactivate the enzyme; concentrate and freeze - dry to obtain the crude mulberry leaf extract; 2) Add 10 times the volume (v / w) of hot water to the obtained crude mulberry leaf extract, extract at 70 °C and 400 W ultrasonic wave for 20 min; filter, add 10 times the volume (v / w) of hot water to the filter residue again, extract at 70 °C and 400 W ultrasonic wave for 20 min; filter, add 10 times the volume (v / w) of hot water to the filter residue, extract at 70 °C and 400 W ultrasonic wave for 20 min; filter, combine the filtrates, and concentrate to obtain the mulberry leaf extract; After detection, the mass percentage content of the active ingredient is: DNJ 0.8%.

[0058] (3)Mix the sophora flower extract and the mulberry leaf extract obtained in steps (1) and (2) according to a mass ratio of 1:0.3; pass through an 80 - mesh sieve after mixing to obtain the composition.

[0059] Effect verification: (1)In vitro experiment (α-glucosidase inhibition rate) Experimental grouping and experimental methods Experimental grouping: All samples and reagents were prepared with PBS (0.1 mol / L, pH 6.9). It was divided into a blank group, a positive control group (acarbose 1 mg / mL), Example 1, and groups D1 - D5. The sample in the Example 1 group was a sample solution containing the composition of Example 1 at a concentration of 1 mg / mL, and D1 - D5 were sample solutions containing the compositions of Comparative Examples 1 - 5 at a concentration of 1 mg / mL respectively.

[0060] Experimental procedure: 1) Reaction system construction (96-well plate): Blank group: 50 μL PBS + 50 μL enzyme solution + 50 μL substrate; Experimental groups: 50 μL of sample (different groups) → pre-incubate at 37 °C for 10 min → add 50 μL of enzyme solution → react at 37 °C for 15 min → add 50 μL of substrate.

[0061] 2) Termination and detection: Add 100 μL of 0.1 M Na2CO3 to terminate the reaction.

[0062] 3) Measure the absorbance at 405 nm (microplate reader).

[0063] Calculation formula: Inhibition rate (%) = [ (A 空白 - A 样品 ) / A 空白 ] × 100% Other parameters: Enzyme activity unit: 0.5 U / mL (extracted from rat intestine); Substrate concentration: 5 mM PNPG; Reaction temperature: 37 ± 0.5 °C; Detection time window: The measurement should be completed within 20 min after termination.

[0064] Data verification design Replication setting: 6 replicates for each group; Statistical analysis: ANOVA was used for inter-group difference analysis; Dunnett's test was performed between Example 1 and the comparative example groups (p < 0.01).

[0065] Experimental results Table 1 α-Glucosidase inhibition rate and significant differences in each group

[0066] Note: ** indicates significant difference between groups D1 - D5 and Example 1 group (P < 0.01) α-glucosidase releases glucose by hydrolyzing glucosidic bonds, participates in blood glucose metabolism in the human body, thereby increasing blood glucose. Inhibiting its activity can delay the absorption of carbohydrates in the intestine, thus achieving the effect of lowering blood glucose. From the results in Table 1, it can be seen that each experimental group has an inhibitory effect on α-glucosidase. However, there are significant differences when comparing the data between Example 1 and each comparative group, indicating that the inhibitory effect of the compositions in each comparative group on α-glucosidase is far less than that of the composition in Example 1. That is to say, the composition of the present invention has a certain in vitro blood glucose lowering effect. And through the comparative examples, it is found that in the raw materials of the composition, the compound enzymolysis process significantly improves the bioavailability of the active ingredients in sophora japonica, and synergistically inhibits α-glucosidase with high-purity DNJ, and the effect is better than that of single components or traditional processes.

[0067] (2) Animal experiments An animal experiment was conducted using a diabetic mouse model induced by STZ (streptozotocin). Grouping: blank control group, model group, Example 1 group, D1-D5 groups (corresponding to Comparative Examples 1-5 respectively), and continuous intragastric administration for 4 weeks.

[0068] Establishment of diabetic mouse model: STZ injection protocol: - STZ (streptozotocin) was purchased from Sigma-Aldrich, batch number S0138, and stored at -20°C in the dark; - After fasting for 12 h, STZ (40 mg / kg, dissolved in 0.1 M citrate buffer, pH 4.5) was intraperitoneally injected; - Continuously injected for 5 days; - After 72 h, a fasting blood glucose > 11.0 mmol / L was regarded as successful model establishment.

[0069] Experimental process: 80 C57BL / 6 mice were randomly divided into 8 groups. According to the above grouping, except for the blank group, the other groups established diabetic mouse models. The blank control group was fed with normal feed and intragastrically administered an equal volume of normal saline every morning; the model group was fed with normal feed and intragastrically administered an equal volume of normal saline every morning; the other test groups were fed with normal feed and intragastrically administered the corresponding composition of 200 mg / kg every morning.

[0070] Detection indexes: At the end of the fourth week, after fasting for 6 h, 2 g / kg of glucose was intragastrically administered, and the mice were sacrificed after 120 min. Blood was taken from the orbital cavity, 1. Blood glucose 2 h after meal, measured by a portable blood glucose meter; 2. Serum GLP-1 level 2 h after meal, detected by ELISA kit (Jingkang Biology JK-E4011); 3. Survival rate of pancreatic β cells, HE staining + insulin immunohistochemistry, and finally ImageJ was used to calculate the survival rate.

[0071] result: Table 2 Blood sugar test results of the embodiments and comparative examples

[0072] Note: # indicates that there are significant differences between Example 1 and D1-D5 and the model group (p < 0.05); ## indicates that there are extremely significant differences (p < 0.01); ** indicates that there are extremely significant differences between Example 1 and D1-D5 (p < 0.01) 1. From the results in Table 2, it can be seen that the postprandial blood glucose of the model group mice is significantly higher than that of the blank control group, indicating that the postprandial blood glucose of the STZ model mice cannot be well controlled. The blood glucose of Example 1 is significantly lower than that of the model group, indicating that the composition of Example 1 has a significant effect of inhibiting the increase in blood glucose caused by STZ modeling in mice (p < 0.01). D1-D5 and the model group items also have a good and obvious effect of controlling blood glucose (p < 0.05), but comparing the data of Example 1 and D1-D5, the effect of D1-D5 in controlling blood glucose increase is significantly worse than that of Example 1 (P < 0.01), so it can be explained that the composition of the present invention is effective in inhibiting the uncontrolled increase of postprandial blood glucose in mice due to STZ modeling.

[0073] 2. From the results in Table 2, it can be seen that after STZ modeling, the postprandial GLP-1 level of mice was significantly lower than that of the blank control group. Compared with the model group in Example 1, the serum GLP-1 level was significantly improved (p < 0.01). Compared with the model group, D1-D5 showed different degrees of improvement in the data, but a closer look showed that D1 had no significant difference, D2 and D4 had significant differences (p < 0.05), and D3 and D5 had extremely significant differences (p < 0.01). Combined with the comparison results between Example 1 and D1-D5 groups, there were significant differences (p < 0.01), indicating that D1-D2 was far less effective than Example 1 in improving the decrease in GLP-1 levels in postprandial mice caused by STZ modeling.

[0074] 3. From the results in Table 2, it can be seen that after the mice were modeled by STZ, the β cells in the pancreatic islets of the mice were damaged and their survival rate was greatly reduced. After continuous intragastric administration of the composition of Example 1 or D1-D5, the survival rate of the β cells was improved to varying degrees. Compared with the model group, each experimental group had a significant effect of improving the survival rate of β cells (p < 0.05); compared with Example 1 and D1-D5 groups, the effect of Example 1 was significantly better than that of D1-D5 (p < 0.01).

[0075] In summary, the composition of Example 1 has the effect of improving the β-cell damage in the islets of STZ model mice, increasing the survival rate, thus improving the insufficient secretion of GLP-1, and ultimately achieving the effect of controlling the postprandial blood glucose elevation.

[0076] (3)Human experiment (randomized double-blind) Subjects: People with prediabetes (n = 80), who consumed a solid beverage containing 1 g of the composition daily for 12 weeks.

[0077] Inclusion criteria: Aged 25 - 60 years old, ADA prediabetes diagnostic criteria (fasting blood glucose 5.6 - 6.9 mmol / L, HbAlc 5.7 - 6.4%), BMI 23 - 30.

[0078] Exclusion criteria: Use of hypoglycemic drugs / insulin, history of severe gastrointestinal diseases, pregnancy / lactation.

[0079] Grouping and intervention: As shown in Table 3.

[0080] Table 3 Description of grouping and intervention in human experiment

[0081] Evaluation indicators and detections: 1. 2-hour postprandial blood glucose (portable blood glucose meter), measured at the end of 12 weeks.

[0082] 2. HbAlc (high-performance liquid chromatography), measured at the time of enrollment and at the end of 12 weeks.

[0083] 3. HOMA-IR: HOMA-IR = (fasting insulin × fasting blood glucose) / 22.5, measured at the time of enrollment and at the end of 12 weeks.

[0084] 4. Gastrointestinal tolerance questionnaire Test procedure: In the 1st week, enrollment and baseline detection.

[0085] In the 2nd - 12th weeks: Intervention period, a test meal of 500 kcal (carbohydrates accounting for about 60%) was provided uniformly every day, and the test drug was taken with meals.

[0086] In the 12th week: Final evaluation.

[0087] Others: During the intervention period, liver and kidney functions (ALT, Cr) were tracked and detected weekly.

[0088] Test results and data Table 4 Fasting and postprandial blood glucose levels (mmol / L)

[0089] Table 5 Changes in HbAlc and HOMA-IR values before enrollment and after the experiment

[0090] Table 6 Changes in each index

[0091] The test results are shown in Tables 4 - 6. During the 12-week intervention period, the subjects had no gastrointestinal discomfort, and there were no significant changes in liver and kidney function indicators (ALT, Cr), which proved the long-term safety of the composition of the present invention. The specific analysis is as follows: 1. Regarding the change in blood glucose 2 hours after a meal, combining the results of Tables 4 and 6, when taking the composition of the present invention with meals, the blood glucose 2 hours after a meal increased by 12.18%, while that of the control group increased by 43.28%, indicating that the composition of the present invention has the effect of controlling blood glucose elevation; 2. HbAlc is glycated hemoglobin, which is a standard for evaluating the long-term blood glucose control of diabetic patients. The normal range is 4% - 6%, pre-diabetes is 5.7 - 6.4%, and diabetes diagnosis is ≥6.5%. From the results of Tables 5 and 6, the HbAlc value of the experimental group at the time of enrollment was 6.32%. After 12 weeks of the experiment, the HbAlc value decreased to 5.87%, a decrease of 0.45%, indicating that the blood glucose control situation has improved. The change in the control group was not obvious.

[0092] 3. HOMA-IR is the homeostasis model assessment of insulin resistance, which is an index calculated based on fasting blood glucose and fasting insulin levels and is used to evaluate the degree of insulin resistance. <1 indicates good insulin sensitivity, 1 - 2.9 belongs to the normal range, 3 - 5 is early insulin resistance, and >5 is significant insulin resistance. From the results of Tables 5 and 6, the experimental group decreased from 3.51 at the time of enrollment to 2.71, a decrease of 22.79%; the control group increased from 3.49 at the time of enrollment to 3.54, an increase of 1.43%, with little change. It shows that after 12 weeks of the experiment, the insulin resistance of the experimental group decreased and the metabolic health improved.

[0093] In summary, clinical verification shows that the composition provided by the present invention can effectively control blood glucose fluctuations, improve insulin resistance, and has no gastrointestinal adverse reactions, with the potential for market transformation.

[0094] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A composition of Sophora japonica bud and mulberry leaves beneficial to blood sugar metabolism, characterized in that: The composition comprises a Sophora japonica extract and a purified mulberry leaf extract, wherein the mass ratio of the Sophora japonica extract to the purified mulberry leaf extract is (1:0.3)-(1:0.5); and, in terms of mass percentage, the Sophora japonica extract contains 10%-20% of quercetin glucoside, 20%-30% of total flavonoids and 3%-8% of polysaccharides; and the purified mulberry leaf extract contains 1%-3% of DNJ.

2. The composition of Sophora japonica bud and mulberry leaves beneficial to blood sugar metabolism health according to claim 1, characterized in that: The sophora japonica extract is prepared by treating sophora japonica crushed material with cellulase and α-1,4-galacturonic acid hydrolase in sequence.

3. The composition of Sophora japonica bud and mulberry leaves beneficial to blood sugar metabolism health according to claim 2, characterized in that: The cellulase dosage is 5%-10% of the weight of the crushed Sophora japonica seeds, the enzymolysis temperature is 45-65°C, the enzymolysis pH is 4.5-5.5, and the enzymolysis time is 1-2 hours; The dosage of the α-1,4-galacturonic acid hydrolase is 5%-10% of the weight of the crushed Sophora japonica seeds, the enzymolysis temperature is 40° C.-60° C., the enzymolysis pH is 4.0-6.0, and the enzymolysis time is 1-2 hours.

4. The composition of Sophora japonica bud and mulberry leaves beneficial to blood sugar metabolism health according to claim 1, characterized in that: The method for preparing the purified mulberry leaf extract comprises the following steps: 1) The frosted mulberry leaves are dried in the shade and crushed to obtain frosted mulberry leaf powder, and then enzymatically treated with cellulase, pectinase, and β-glucosidase to inactivate the enzymes, and then dried or freeze-dried to obtain a crude mulberry leaf extract; 2) extracting the obtained mulberry leaf crude extract under hot water and ultrasonic conditions, filtering and combining the extracts, concentrating and drying to obtain a mulberry leaf extract; 3) dissolving the mulberry leaf extract in an acidic aqueous solution with a pH of 2.5-3.0, and dynamically adsorbing the pretreated strong acidic cation exchange resin at an adsorption flow rate of 2-3 BV / h; 4) After adsorption is completed, the resin is eluted in stages with hot water at 50°C with pH=3.0, hot water at 70°C with pH=4.5, and hot water at 85°C with pH=6.0, and the 85°C eluate is collected at last; 5) The eluate is concentrated to 1 / 10 of its original volume through a nanofiltration membrane, and freeze-dried or spray-dried to obtain a purified mulberry leaf extract with a DNJ content of 1%-3%.

5. The Sophora japonica bud and mulberry leaf composition beneficial to blood sugar metabolism health according to claim 4, characterized in that: In the step 1), in the enzymatic hydrolysis solution, the weight volume ratio of mulberry leaves to solvent is 1:15-20, the amount of cellulase is 5%-10% of the weight of the mulberry leaves, the amount of pectinase is 2%-5% of the weight of the mulberry leaves, the amount of β-glucosidase is 2%-5% of the weight of the mulberry leaves, the enzymatic hydrolysis temperature is 45-65°C, the enzymatic hydrolysis pH is 4.5-5.5, and the enzymatic hydrolysis time is 1-2 hours.

6. The composition of Sophora japonica bud and mulberry leaves beneficial to blood sugar metabolism health according to claim 4, characterized in that: In the step 2), the hot water temperature is 70° C.-80° C., the ultrasonic frequency is 200-400W, the extraction times are 3 times, and each extraction time is 15-30 minutes.

7. The composition of Sophora japonica bud and mulberry leaves beneficial to blood sugar metabolism health according to claim 4, characterized in that: In the step 3), the strongly acidic cation exchange resin is of type D001, and the pretreatment method is activation with 4%-6% HCl for 2-4 hours and washing with water to a pH of 5.0-6.

0.

8. The composition of Sophora japonica bud and mulberry leaves beneficial to blood sugar metabolism health according to claim 4, characterized in that: In the step 5), the molecular weight cut-off of the nanofiltration membrane is 200-500 Da.

9. Use of the composition according to any one of claims 1 to 8 in the preparation of functional foods or drugs for controlling blood sugar, improving insulin resistance or preventing diabetic complications, wherein the functional foods include one or more of baked foods, flour products, beverages, compressed candies, and oral liquids; and the drugs include oral preparations or enteric-coated preparations.

10. A sugar-controlled solid beverage, comprising the composition according to any one of claims 1 to 8 and food-related acceptable auxiliary materials, wherein the added amount of the composition is 0.5-1.5 g / 100 g.

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