A sophora japonica leaf and mulberry leaf composition for promoting blood sugar metabolism health, and a preparation method and application thereof

Abstract

The present application relates to a kind of sophora japonica leaf composition and its preparation method and application beneficial to the health of blood glucose metabolism.The sophora japonica leaf composition includes sophora japonica extract and mulberry leaf purified extract, and the mass ratio of sophora japonica extract and mulberry leaf purified extract is (1:0.3)-(1:0.5).Moreover, according to mass percentage, sophora japonica extract contains 10%-20% quercetin glucoside, 20%-30% total flavone and 3%-8% polysaccharide;Mulberry leaf purified extract contains 1%-3% DNJ.The present application finds that the synergistic sugar control effect of sophora japonica extract and mulberry leaf purified extract with specific ingredient content under specific mass ratio can achieve the best bioavailability and sugar control effect through research.

Description

Technical Field

[0001] This invention belongs to the field of functional food and natural medicine technology, specifically relating to a Sophora japonica and mulberry leaf composition that is beneficial to blood sugar metabolism, its preparation method and application. Background Technology

[0002] With the rapid increase in the number of people with diabetes and prediabetes, blood sugar control has become a core issue in the global health field. While existing blood sugar control drugs (such as metformin and DPP4 inhibitors) are effective, they have side effects and can lead to long-term dependence. In traditional Chinese medicine, both Sophora japonica buds and mulberry leaves are recorded as relieving "Xiao Ke Zheng" (symptoms of diabetes), but the bioavailability of these single components is low and their mechanisms of action are limited, making it difficult to meet modern blood sugar control needs.

[0003] In existing technologies, the enzymatic hydrolysis of Sophora japonica flower powder and the extraction process of DNJ (1-deoxynojirimycin) from mulberry leaves have not yet achieved highly efficient synergistic effects. Furthermore, traditional formulations lack scientific validation, making it impossible to clearly define their multi-pathway blood sugar control mechanisms (such as inhibiting sugar absorption, promoting GLP-1 secretion, and repairing pancreatic function). Therefore, there is an urgent need to develop a blood sugar control composition based on natural ingredients, with synergistic effects across multiple mechanisms and clinical validation. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention improves the extraction methods of Sophora japonica buds and mulberry leaves, increases the content of relevant active ingredients, and combines them synergistically to provide a Sophora japonica bud and mulberry leaf composition that is beneficial to blood sugar metabolism.

[0005] The specific technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a Sophora japonica and mulberry leaf composition beneficial to blood glucose metabolism, comprising 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) to (1:0.5); and, by mass percentage, the Sophora japonica extract contains 10%-20% quercetin glucoside, 20%-30% total flavonoids and 3%-8% polysaccharides; and the purified mulberry leaf extract contains 1%-3% DNJ.

[0007] By selecting a combination of Sophora japonica buds and purified mulberry leaf extract with specific component contents, and through scientific screening, it was found that the Sophora japonica bud extract and the purified mulberry leaf extract have a synergistic effect on blood sugar control at a specific mass ratio of (1:0.3) to (1:0.5), achieving the best bioavailability and blood sugar control effect.

[0008] Preferably, the Sophora japonica extract in the composition of the present invention is obtained by sequentially treating Sophora japonica powder with cellulase and α-1,4-galacturonic acid hydrolase.

[0009] Sophora japonica buds contain a variety of effective components such as flavonoids, saponins, and polysaccharides. By selectively hydrolyzing Sophora japonica buds with cellulase and α-1,4-galacturonase, the bioavailability of quercetin glucoside in Sophora japonica buds can be significantly improved.

[0010] Preferably, the amount of cellulase used is 5%-10% of the weight of the crushed Sophora japonica flower buds, the enzymatic hydrolysis temperature is 45-65℃, the enzymatic hydrolysis pH is 4.5-5.5, and the enzymatic hydrolysis time is 1-2 hours;

[0011] The amount of α-1,4-galacturonic acid hydrolase used is 5%-10% of the weight of the Sophora japonica bud powder, the enzymatic hydrolysis temperature is 40℃-60℃, the enzymatic hydrolysis pH is 4.0-6.0, and the enzymatic hydrolysis time is 1-2 hours.

[0012] Preferably, the method for preparing the purified mulberry leaf extract in the composition of the present invention includes the following steps:

[0013] 1) After drying and pulverizing the frost-covered mulberry leaves, frost-covered mulberry leaf powder is obtained. Then, it is enzymatically hydrolyzed with cellulase, pectinase, and β-glucosidase to inactivate the enzymes. After drying or freeze-drying, crude mulberry leaf extract is obtained.

[0014] 2) The crude mulberry leaf extract obtained was extracted under hot water and ultrasonic conditions, the extracts were filtered and combined, concentrated and dried to obtain mulberry leaf extract;

[0015] 3) The purified mulberry leaf extract was dissolved in an acidic aqueous solution with a pH of 2.5-3.0 and then dynamically adsorbed by a pretreated strongly acidic cation exchange resin at an adsorption flow rate of 2-3 BV / h.

[0016] 4) After adsorption is complete, the resin is eluted in stages with hot water at 50℃ (pH=3.0), hot water at 70℃ (pH=4.5), and hot water at 85℃ (pH=6.0). Finally, the eluent at 85℃ is collected.

[0017] 5) The eluent is concentrated to 1 / 10 of its original volume by nanofiltration, and then freeze-dried or spray-dried to obtain a purified mulberry leaf extract with a DNJ content of 1%-3%.

[0018] Mulberry leaves are generally harvested between September and October after the first frost. Mulberry leaves that have been exposed to frost have higher levels of nutrients and medicinal components. Cellulase can break down cellulose in the cell walls of mulberry leaves, releasing DNJ; pectinase degrades pectin, increasing the dissolution rate of cell contents; β-glucosidase selectively hydrolyzes glycosidic bonds, reducing polysaccharide interference. The sequential use of cellulase, pectinase, and β-glucosidase for enzymatic hydrolysis further facilitates the release of the active ingredient DNJ. Resin adsorption purification allows for more efficient and rapid extraction of DNJ without damaging its structure.

[0019] Preferably, in step 1), the weight-to-volume ratio of mulberry leaves to solvent in the enzymatic hydrolysate is 1:15-20, the amount of cellulase is 5%-10% of the weight of mulberry leaves, the amount of pectinase is 2%-5% of the weight of mulberry leaves, the amount of β-glucosidase is 2%-5% of the weight of mulberry leaves, the enzymatic hydrolysis temperature is 45-65℃, the enzymatic hydrolysis pH is 4.5-5.5, and the enzymatic hydrolysis time is 1-2 hours.

[0020] Preferably, in step 2), the hot water temperature is 70℃-80℃, the ultrasonic frequency is 200-400W, the extraction is performed 3 times, and the extraction time for each extraction is 15-30 minutes.

[0021] Preferably, in step 3), the strongly acidic cation exchange resin is type D001, and the pretreatment method is activation with 4%-6% HCl for 2-4 hours, followed by washing with water until the pH reaches 5.0-6.0.

[0022] Preferably, in step 5), the nanofiltration membrane has a molecular weight cutoff of 200-500 Da.

[0023] Compared to traditional water or alcohol extraction of mulberry leaf extracts containing DNJ, the mulberry leaf purified extract of this invention is prepared by first treating with enzymes and then purifying with resin adsorption, resulting in a DNJ content as high as 1%-3%, which is 10-30 times higher.

[0024] A second aspect of the invention also provides the use of the composition in the preparation of functional foods or pharmaceuticals for controlling blood sugar, improving insulin resistance, or preventing diabetic complications, wherein the functional foods include baked goods (bread, biscuits, etc.), flour products, beverages (solid beverages, liquid beverages), compressed candies, and oral liquids; and the pharmaceuticals include oral preparations or enteric-coated preparations.

[0025] In a third aspect, the present invention also provides a sugar-controlled solid beverage comprising the above-described composition and food-grade acceptable excipients, wherein the composition is added in an amount of 0.5-1.5 g / 100 g.

[0026] The beneficial effects of this invention are:

[0027] (1) Through research, this invention has discovered the synergistic blood sugar control effect of Sophora japonica extract and mulberry leaf purified extract with specific component contents at a specific mass ratio. Specifically, Sophora japonica quercetin glucoside improves insulin sensitivity by activating the PPARγ pathway, while mulberry leaf DNJ inhibits α-glucosidase. The two work together to regulate the GLP-1 / insulin axis, ultimately achieving the effects of inhibiting sugar absorption, promoting GLP-1 secretion, and repairing pancreatic function. This ratio has been scientifically screened to achieve the best bioavailability and blood sugar control effect.

[0028] (2) This invention improves the preparation methods of mulberry leaf purified extract and sophora japonica flower extract, thereby increasing the content of relevant active ingredients. Specifically, by using cellulase and α-1,4-galacturonidase to hydrolyze sophora japonica flowers, the bioavailability of quercetin glucoside in sophora japonica flowers is significantly improved. By first treating with multiple enzymes and then purifying with resin adsorption, the DNJ content in the mulberry leaf purified extract can reach as high as 1%-3%, which is 15-30 times higher than that of traditional water extraction or alcohol extraction methods.

[0029] (3) The composition of the present invention is safe, all of which are natural ingredients, meet the standards for food and medicine homology, contain no chemical additives, and have good tolerability in human trials. It can be used in functional foods or drugs. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] Example 1

[0033] This embodiment provides a Sophora japonica and mulberry leaf composition beneficial to blood glucose metabolism, comprising Sophora japonica extract and purified mulberry leaf extract; it is prepared by the following method:

[0034] (1) Preparation of Sophora japonica extract.

[0035] 100g of Sophora japonica buds, cleaned, dried, and pulverized through a 60-100 mesh sieve;

[0036] The obtained Sophora japonica powder was dissolved in 20 times (v / w) pH 5.0 buffer solution, and 10% (by weight) of cellulase of Sophora japonica powder was added. The mixture was enzymatically hydrolyzed at 50°C for 2 hours and then inactivated by water bath at 90°C for 10 minutes.

[0037] Then add 10% (by weight) of α-1,4-galacturonic acid hydrolase to the sophora japonica powder, and hydrolyze for 2 hours at 50°C and pH 5.0; then inactivate the enzyme by bathing in a 90°C water bath for 10 minutes.

[0038] Centrifuge to collect the supernatant, then spray dry (inlet air temperature 180℃) to obtain Sophora japonica extract.

[0039] The test results showed that the active ingredients contained the following percentages by mass: quercetin glucoside 16%, total flavonoids 25%, and polysaccharides 5%.

[0040] (2) Preparation of purified mulberry leaf extract

[0041] Raw materials: 50g of mulberry leaves, cleaned, dried, and pulverized through a 40-60 mesh sieve.

[0042] 1) Enzyme treatment: Mulberry leaf powder was dissolved in 20 times (v / w) pH 5.0 buffer solution. 10% (by weight) of cellulase was added to the mulberry leaf powder and enzymatically hydrolyzed at 50℃ for 2 hours to inactivate the enzyme. Then, 4% (by weight) of pectinase was added to the mulberry leaf powder and enzymatically hydrolyzed at 50℃ for 1.5 hours to inactivate the enzyme. Finally, 3% (by weight) of β-glucosidase was added to the mulberry leaf powder and enzymatically hydrolyzed at 50℃ for 1 hour to inactivate the enzyme. After concentration, the extract was freeze-dried to obtain crude mulberry leaf extract.

[0043] 2) The obtained crude mulberry leaf extract was added to 10 times its volume (v / w) of hot water and extracted at 70℃ and 400W ultrasound for 20 min; filtered, and the residue was added to the filter residue again to extract at 70℃ and 400W ultrasound for 20 min; filtered, and the residue was added to the filter residue to extract at 70℃ and 400W ultrasound for 20 min; filtered, the filtrates were combined and concentrated to obtain the mulberry leaf extract;

[0044] 3) Dissolve mulberry leaf extract in 10 times its volume (v / w) of an acidic aqueous solution with pH 2.5, and then dynamically adsorb it through a pretreated and activated strong acid cation exchange resin at an adsorption flow rate of 2 BV / h. The strong acid cation exchange resin is type D001, 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.

[0045] 4) After adsorption is complete, the resin is eluted in stages with hot water at 50℃ (pH 3.0), 70℃ (pH 4.5), and 85℃ (pH 6.0), and the eluent at 85℃ is collected.

[0046] 5) The eluent is concentrated to 1 / 10 of its original volume using a nanofiltration membrane with a molecular weight cutoff of 200 Da, and then freeze-dried or spray-dried to obtain a purified mulberry leaf extract.

[0047] The test results showed that the content of the active ingredient was DNJ 2.8% by mass.

[0048] (3) The Sophora japonica extract obtained in steps (1) and (2) is mixed with the purified mulberry leaf extract at a mass ratio of 1:0.3 and then passed through an 80-mesh sieve to obtain the composition.

[0049] Example 2

[0050] This embodiment provides a Sophora japonica and mulberry leaf composition beneficial to blood glucose metabolism, comprising Sophora japonica extract and purified mulberry leaf extract; it is prepared by the following method:

[0051] (1) Preparation of Sophora japonica extract.

[0052] 100g of Sophora japonica buds, cleaned, dried, and pulverized through a 60-100 mesh sieve;

[0053] The obtained Sophora japonica powder was dissolved in 20 times (v / w) pH 5.5 buffer solution, and 8% (by weight) of cellulase of Sophora japonica powder was added. The mixture was enzymatically hydrolyzed at 55℃ for 1 hour and then inactivated by water bath at 90℃ for 10 minutes.

[0054] Then add 8% (by weight of) α-1,4-galacturonic acid hydrolase of Sophora japonica powder, and hydrolyze for 1 hour at 60℃ and pH 6.0; then inactivate the enzyme by water bath at 90℃ for 10 minutes.

[0055] Centrifuge to collect the supernatant, then spray dry (inlet air temperature 180℃) to obtain Sophora japonica extract.

[0056] The test results showed that the active ingredients contained the following percentages by mass: quercetin glucoside 12%, total flavonoids 22%, and polysaccharides 4%.

[0057] (2) Preparation of purified mulberry leaf extract

[0058] Raw materials: 50g of mulberry leaves, cleaned, dried, and pulverized through a 40-60 mesh sieve.

[0059] 1) Enzyme treatment: Mulberry leaf powder was dissolved in 20 times (v / w) pH 5.0 buffer solution. 10% (by weight) of cellulase was added to the mulberry leaf powder and enzymatically hydrolyzed at 55℃ for 1 hour to inactivate the enzyme. Then, 5% (by weight) of pectinase was added to the mulberry leaf powder and enzymatically hydrolyzed at 55℃ for 1 hour to inactivate the enzyme. Finally, 5% (by weight) of β-glucosidase was added to the mulberry leaf powder and enzymatically hydrolyzed at 50℃ for 1 hour to inactivate the enzyme. After concentration, the extract was freeze-dried to obtain crude mulberry leaf extract.

[0060] 2) Add 10 times the volume (v / w) of hot water to the obtained crude mulberry leaf extract and extract for 15 min at 80℃ and 400W ultrasonication; filter, add 10 times the volume (v / w) of hot water to the residue again and extract for 15 min at 80℃ and 400W ultrasonication; filter, add 10 times the volume (v / w) of hot water to the residue and extract for 10 min at 80℃ and 400W ultrasonication; filter, combine the filtrates, and concentrate to obtain mulberry leaf extract;

[0061] 3) Dissolve mulberry leaf extract in 10 times its volume (v / w) of an acidic aqueous solution with pH 2.5, and then dynamically adsorb it through a pretreated and activated strong acid cation exchange resin at an adsorption flow rate of 3 BV / h. The strong acid cation exchange resin is type D001, 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.

[0062] 4) After adsorption is complete, the resin is eluted in stages with hot water at 50℃ (pH 3.0), 70℃ (pH 4.5), and 85℃ (pH 6.0), and the eluent at 85℃ is collected.

[0063] 5) The eluent is concentrated to 1 / 10 of its original volume using a nanofiltration membrane with a molecular weight cutoff of 200 Da, and then freeze-dried or spray-dried to obtain a purified mulberry leaf extract.

[0064] The test results showed that the content of the active ingredient was DNJ 2.2% by mass.

[0065] (4) The Sophora japonica extract obtained in steps (1) and (2) is mixed with the purified mulberry leaf extract at a mass ratio of 1:0.5 and then passed through an 80-mesh sieve to obtain the composition.

[0066] Comparative Example 1

[0067] This comparative example provides a Sophora japonica and mulberry leaf composition, comprising Sophora japonica powder and purified mulberry leaf extract; it is prepared by the following method:

[0068] (1) Preparation of Sophora japonica powder.

[0069] 100g of Sophora japonica buds are cleaned, dried, and pulverized through a 60-100 mesh sieve to obtain Sophora japonica bud powder.

[0070] The test results showed that the active ingredients contained the following percentages by mass: total flavonoids 20% and polysaccharides 0.1%. However, due to the simple crushing process, quercetin glucoside was not effectively extracted, and its content was 0%.

[0071] (2) Preparation of purified mulberry leaf extract, same as in Example 1.

[0072] (3) The Sophora japonica powder obtained in steps (1) and (2) is mixed with the purified mulberry leaf extract at a mass ratio of 1:0.3 and then passed through an 80-mesh sieve to obtain the composition.

[0073] Comparative Example 2

[0074] This comparative example provides a Sophora japonica flower composition, including Sophora japonica flower extract; it is prepared by the following method:

[0075] (1) The preparation of Sophora japonica extract is the same as in Example 1.

[0076] (2) The Sophora japonica extract obtained in step (1) is passed through an 80-mesh sieve to obtain the composition.

[0077] Comparative Example 3

[0078] This comparative example provides a Sophora japonica and mulberry leaf composition, comprising Sophora japonica extract and purified mulberry leaf extract; it is prepared by the following method:

[0079] (1) The preparation of Sophora japonica extract is the same as in Example 1.

[0080] (2) Preparation of mulberry leaf purified extract, same as in Example 1.

[0081] (3) The Sophora japonica extract obtained in steps (1) and (2) is mixed with the purified mulberry leaf extract at a mass ratio of 1:0.2 and then passed through an 80-mesh sieve to obtain the composition.

[0082] Comparative Example 4

[0083] This comparative example provides a Sophora japonica and mulberry leaf composition, comprising Sophora japonica extract and mulberry leaf extract; it is prepared by the following method:

[0084] (1) Preparation of Sophora japonica extract, as in Example 1.

[0085] (2) Preparation of mulberry leaf extract;

[0086] Raw materials: 50g of mulberry leaves, cleaned, dried, and pulverized through a 40-60 mesh sieve.

[0087] Water extraction: Add 10 times the volume (v / w) of water to mulberry leaf powder and extract at 85℃ for 30 min. Filter, add 10 times the volume (v / w) of water to the residue and extract at 85℃ for 20 min. Combine the filtrates and concentrate to dryness to obtain mulberry leaf extract.

[0088] The test results showed that the content of the active ingredient was 0.1% by mass.

[0089] (3) The Sophora japonica extract obtained in steps (1) and (2) is mixed with the purified mulberry leaf extract at a mass ratio of 1:0.3 and then passed through an 80-mesh sieve to obtain the composition.

[0090] Comparative Example 5

[0091] This comparative example provides a Sophora japonica and mulberry leaf composition, comprising Sophora japonica extract and mulberry leaf extract; it is prepared by the following method:

[0092] (1) The preparation of Sophora japonica extract is the same as in Example 1.

[0093] (2) Preparation of mulberry leaf extract;

[0094] Raw materials: 50g of mulberry leaves, cleaned, dried, and pulverized through a 40-60 mesh sieve.

[0095] 1) Enzyme treatment: Mulberry leaf powder was dissolved in 20 times (v / w) pH 5.0 buffer solution. 10% (by weight) of cellulase was added to the mulberry leaf powder and enzymatically hydrolyzed at 50℃ for 2 hours to inactivate the enzyme. Then, 4% (by weight) of pectinase was added to the mulberry leaf powder and enzymatically hydrolyzed at 50℃ for 1.5 hours to inactivate the enzyme. Finally, 3% (by weight) of β-glucosidase was added to the mulberry leaf powder and enzymatically hydrolyzed at 50℃ for 1 hour to inactivate the enzyme. After concentration, the extract was freeze-dried to obtain crude mulberry leaf extract.

[0096] 2) The obtained crude mulberry leaf extract was added to 10 times its volume (v / w) of hot water and extracted at 70℃ and 400W ultrasound for 20 min; filtered, and the residue was added to the filter residue again to extract at 70℃ and 400W ultrasound for 20 min; filtered, and the residue was added to the filter residue to extract at 70℃ and 400W ultrasound for 20 min; filtered, the filtrates were combined and concentrated to obtain the mulberry leaf extract;

[0097] The test results showed that the content of the active ingredient by mass percentage was 0.8% for DNJ.

[0098] (3) The Sophora japonica extract obtained in steps (1) and (2) is mixed with the mulberry leaf extract at a mass ratio of 1:0.3 and then passed through an 80-mesh sieve to obtain the composition.

[0099] Effect verification:

[0100] (1) In vitro experiment (α-glucosidase inhibition rate)

[0101] Experimental grouping and experimental methods

[0102] Experimental Groups: All samples and reagents were prepared using PBS (0.1 mol / L, pH 6.9). The experiments were divided into a blank group, a positive control group (acarbose 1 mg / mL), Example 1, and groups D1-D5. Example 1 consisted of a sample solution containing the composition of Example 1 at a concentration of 1 mg / mL. Groups D1-D5 consisted of sample solutions containing the compositions of Comparative Examples 1-5 at a concentration of 1 mg / mL, respectively.

[0103] Experimental procedure:

[0104] 1) Construction of the reaction system (96-well plate):

[0105] Control group: 50 μL PBS + 50 μL enzyme solution + 50 μL substrate;

[0106] Experimental group: 50 μL sample (different groups) → pre-incubate at 37℃ for 10 min → add 50 μL enzyme solution → react at 37℃ for 15 min → add 50 μL substrate.

[0107] 2) Termination and detection: Add 100 μL of 0.1 M Na2CO3 to terminate the reaction.

[0108] 3) Measure absorbance at 405 nm (ELISA reader).

[0109] Calculation formula: Inhibition rate (%) = [(A) 空白 -A 样品 ) / A 空白 ]× 100%

[0110] Other parameters:

[0111] Enzyme activity unit: 0.5 U / mL (derived from rat intestine extract);

[0112] Substrate concentration: 5 mM PNPG;

[0113] Reaction temperature: 37±0.5℃;

[0114] Detection time window: The measurement should be completed within 20 minutes after termination.

[0115] Data validation design

[0116] Repeat setting: 6 replicates per group;

[0117] Statistical analysis: ANOVA was used to analyze differences between groups; Dunnett's test was performed between Example 1 and the control group (p<0.01).

[0118] Experimental results

[0119] Table 1. Inhibition rate of α-glucosidase in each group and significant differences

[0120]

[0121] Note: ** indicates a significant difference between groups D1-D5 and group 1 of Example 1 (P < 0.01).

[0122] α-Glucosidase releases glucose by hydrolyzing glucosinolate bonds, participating in blood glucose metabolism and thus raising blood sugar levels. Inhibiting its activity can delay the absorption of carbohydrates in the intestine, thereby lowering blood sugar. Table 1 shows that all experimental groups inhibited α-glucosidase. However, significant differences were observed between the data from Example 1 and the comparative groups, indicating that the inhibitory effect of the comparative compositions on α-glucosidase was far less than that of the composition in Example 1. This demonstrates that the composition of the present invention has a certain in vitro blood glucose lowering effect. Furthermore, the comparative examples show that the compound enzymatic hydrolysis process significantly improves the bioavailability of the active ingredients in Sophora japonica, synergistically inhibiting α-glucosidase with high-purity DNJ, which is superior to single-component or traditional processes.

[0123] (2) Animal experiments

[0124] A diabetic mouse model induced by STZ (streptozotocin) was used for animal experiments. Groups included: blank control group, model group, Example 1 group, and D1-D5 groups (corresponding to ratios 1-5), administered by gavage for 4 consecutive weeks.

[0125] Establishment of a diabetic mouse model:

[0126] STZ injection regimen:

[0127] - STZ (streptozotocin) was purchased from Sigma-Aldrich, lot number S0138, and should be stored at -20°C protected from light.

[0128] - After fasting for 12 hours, administer STZ intraperitoneally (40 mg / kg, dissolved in 0.1 M citrate buffer, pH 4.5).

[0129] - Inject for 5 consecutive days;

[0130] - A fasting blood glucose level >11.0 mmol / L after 72 hours is considered a successful model establishment.

[0131] Experimental procedure:

[0132] Eighty C57BL / 6 mice were randomly divided into eight groups. According to the above grouping, except for the blank group, the other groups were used to establish a diabetic mouse model. The blank control group was fed a normal diet and was given an equal volume of physiological saline by gavage every morning; the model group was fed a normal diet and was given an equal volume of physiological saline by gavage every morning; the other experimental groups were fed a normal diet and were given 200 mg / kg of the corresponding composition by gavage every morning.

[0133] Testing indicators:

[0134] At the end of the fourth week, the animal was fasted for 6 hours, then given a gavage of 2g / kg of glucose. It was euthanized 120 minutes later, and blood was drawn from its orbital cavity.

[0135] 1. Postprandial 2-hour blood glucose level, using a portable blood glucose meter;

[0136] 2. Serum GLP-1 level 2 hours after meal, detected by ELISA kit (Jingkang Bio JK-E4011).

[0137] 3. Pancreatic β-cell survival rate: HE staining + insulin immunohistochemistry, and finally ImageJ calculation of survival rate.

[0138] result:

[0139] Table 2. Blood glucose test results of the examples and comparative examples.

[0140]

[0141] Note: # indicates a significant difference (p < 0.05) between Example 1 and D1-D5 and the model group; ## indicates a highly significant difference (p < 0.01); ** indicates a highly significant difference (p < 0.01) between Example 1 and D1-D5.

[0142] 1. As shown in Table 2, the postprandial blood glucose level in the model group mice was significantly higher than that in the blank control group, indicating that the postprandial blood glucose level in the STZ model mice could not be well controlled. The blood glucose level in Example 1 was significantly lower than that in the model group, indicating that the composition of Example 1 had a significant inhibitory effect on the increase in blood glucose caused by STZ modeling in mice (p < 0.01). The results for D1-D5 also showed a good and significant effect on controlling blood glucose levels compared to the model group (p < 0.05), but comparing the data of Example 1 and D1-D5, the effect of controlling blood glucose increase in D1-D5 was significantly less than that of Example 1 (P < 0.01). Therefore, this demonstrates the effectiveness of the composition of the present invention in inhibiting the uncontrolled increase in postprandial blood glucose in mice caused by STZ modeling.

[0143] 2. As shown in Table 2, the postprandial GLP-1 level in mice after STZ modeling was significantly lower than that in the blank control group. Compared with the model group, the serum GLP-1 level in Example 1 was significantly increased (p < 0.01). While there were varying degrees of increase in the levels of the D1-D5 groups compared to the model group, a closer look revealed no significant difference on D1, significant differences on D2 and D4 (p < 0.05), and highly significant differences on D3 and D5 (p < 0.01). Combining the results of Example 1 with the D1-D5 groups, all showed significant differences (p < 0.01), indicating that the effects of D1-D2 in improving the postprandial GLP-1 level decrease caused by STZ modeling in mice were far less effective than those of Example 1.

[0144] 3. As shown in Table 2, STZ modeling in mice damages β cells in the pancreas, significantly reducing their survival rate. Continuous gavage administration of the combination from Example 1 or D1-D5 improved β cell survival rates to varying degrees. Compared to the model group, each experimental group significantly improved β cell survival (p < 0.05); compared to the D1-D5 groups, Example 1 showed significantly better results (p < 0.01).

[0145] In summary, the composition of Example 1 has the effect of improving the survival rate of β-cell damage in the pancreatic islets of STZ model mice, thereby improving the insufficient secretion of GLP-1 and ultimately achieving the effect of controlling postprandial hyperglycemia.

[0146] (3) Human trials (randomized double-blind)

[0147] Subjects: Individuals with prediabetes (n=80) who consumed a solid beverage containing 1g of the composition daily for 12 weeks.

[0148] Inclusion criteria: age 25-60 years, ADA diagnostic criteria for prediabetes (fasting blood glucose 5.6-6.9 mmol / L, HbA1c 5.7-6.4%), BMI 23-30.

[0149] Exclusion criteria: use of hypoglycemic drugs / insulin, history of severe gastrointestinal disease, pregnancy / lactation.

[0150] Grouping and intervention: as shown in Table 3.

[0151] Table 3. Explanation of Human Trial Grouping Intervention

[0152]

[0153] Evaluation indicators and testing:

[0154] 1. Postprandial 2-hour blood glucose (portable blood glucose meter), measured at the end of 12 weeks.

[0155] 2. HbAlc (high-performance liquid chromatography), measured at enrollment and at the end of week 12.

[0156] 3. HOMA-IR: HOMA-IR = (fasting insulin × fasting blood glucose) / 22.5, measured at enrollment and at the end of week 12.

[0157] 4. Gastrointestinal Tolerance Questionnaire

[0158] Test procedure:

[0159] Week 1: Enrollment and baseline testing.

[0160] Weeks 2-12: Intervention period. A test meal of 500 kcal (approximately 60% carbohydrates) was provided daily, and the experimental drug was taken with the meal.

[0161] Week 12: Final Assessment.

[0162] Other: During the intervention period, liver and kidney function (ALT, Cr) were monitored weekly.

[0163] Experimental Results and Data

[0164] Table 4. Pre- and post-meal blood glucose levels (mmol / L)

[0165]

[0166] Table 5. Changes in HbA1c and HOMA-IR values ​​before and after the experiment.

[0167]

[0168] Table 6: Changes in Each Indicator

[0169]

[0170] The test results, as shown in Tables 4-6, indicate that during the 12-week intervention period, the subjects experienced no gastrointestinal discomfort, and there were no significant changes in liver and kidney function indicators (ALT, Cr), demonstrating the long-term safety of the composition of this invention. Specific analysis is as follows:

[0171] 1. Regarding the changes in blood glucose 2 hours after a meal, based on the results in Tables 4 and 6, the blood glucose level 2 hours after taking the composition of the present invention with a meal increased by 12.18%, while that in the control group increased by 43.28%, indicating that the composition of the present invention has the effect of controlling blood glucose rise.

[0172] 2. HbA1c, or glycosylated hemoglobin, is a standard for assessing long-term glycemic control in diabetic patients. The normal range is 4%-6%, 5.7%-6.4% for prediabetes, and ≥6.5% for a diagnosis of diabetes. As shown in Tables 5 and 6, the experimental group had an HbA1c value of 6.32% at enrollment. After 12 weeks, the HbA1c value decreased to 5.87%, a decrease of 0.45%, indicating improved glycemic control. The control group showed no significant change.

[0173] 3. HOMA-IR is a homeostasis model for insulin resistance assessment, calculated based on fasting blood glucose and fasting insulin levels, used to assess the degree of insulin resistance. <1 indicates good insulin sensitivity, 1-2.9 is within the normal range, 3-5 indicates early insulin resistance, and >5 indicates significant insulin resistance. From the results in Tables 5 and 6, the experimental group's insulin level decreased from 3.51 at enrollment to 2.71, a decrease of 22.79%; while the control group's level increased from 3.49 at enrollment to 3.54, an increase of 1.43%, showing little change. This indicates that after 12 weeks of experimentation, the experimental group experienced reduced insulin resistance and improved metabolic health.

[0174] In summary, clinical trials have shown that the composition provided by this invention can effectively control blood glucose fluctuations, improve insulin resistance, and has no gastrointestinal adverse reactions, thus possessing market commercialization potential.

[0175] The technical features of the embodiments described above can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. Use of a flos sophorae mume leaf and mulberry leaf composition in the manufacture of a food or drug for the improvement of blood sugar metabolism, characterized in that, The Sophora japonica and mulberry leaf composition comprises Sophora japonica extract and purified mulberry leaf extract, wherein the mass ratio of the Sophora japonica extract to the purified mulberry leaf extract is (1:0.3) to (1:0.5); and, by mass percentage, the Sophora japonica extract contains 10%-20% quercetin glucoside, 20%-30% total flavonoids and 3%-8% polysaccharides; the purified mulberry leaf extract contains 1%-3% DNJ. The Sophora japonica extract was prepared by sequentially treating Sophora japonica powder with cellulase and α-1,4-galacturonic acid hydrolase. The method for preparing the purified mulberry leaf extract includes the following steps: 1) After drying and pulverizing the frost-covered mulberry leaves, frost-covered mulberry leaf powder is obtained. Then, it is enzymatically hydrolyzed with cellulase, pectinase, and β-glucosidase to inactivate the enzymes. After drying or freeze-drying, crude mulberry leaf extract is obtained. 2) The crude mulberry leaf extract obtained was extracted under hot water and ultrasonic conditions, the extracts were filtered and combined, concentrated and dried to obtain mulberry leaf extract; 3) Dissolve mulberry leaf extract in an acidic aqueous solution with pH 2.5-3.0, and then dynamically adsorb it onto a pretreated strong acidic cation exchange resin at an adsorption flow rate of 2-3 BV / h. 4) After adsorption is complete, the resin is eluted in stages with hot water at 50℃ and pH=3.0, hot water at 70℃ and pH=4.5, and hot water at 85℃ and pH=6.0 respectively. Finally, the eluent at 85℃ is collected. 5) The eluent is concentrated to 1 / 10 of its original volume by nanofiltration, and then freeze-dried or spray-dried to obtain a purified mulberry leaf extract with a DNJ content of 1%-3%. The amount of cellulase used is 5%-10% of the weight of the crushed Sophora japonica flower buds, the enzymatic hydrolysis temperature is 45-65℃, the enzymatic hydrolysis pH is 4.5-5.5, and the enzymatic hydrolysis time is 1-2 hours. The amount of α-1,4-galacturonic acid hydrolase used is 5%-10% of the weight of the Sophora japonica bud powder, the enzymatic hydrolysis temperature is 40℃-60℃, the enzymatic hydrolysis pH is 4.0-6.0, and the enzymatic hydrolysis time is 1-2 hours; In step 1), the weight-to-volume ratio of mulberry leaves to solvent in the enzymatic hydrolysate is 1:15-20, the amount of cellulase used is 5%-10% of the weight of mulberry leaves, the amount of pectinase used is 2%-5% of the weight of mulberry leaves, the amount of β-glucosidase used is 2%-5% of the weight of mulberry leaves, the enzymatic hydrolysis temperature is 45-65℃, the enzymatic hydrolysis pH is 4.5-5.5, and the enzymatic hydrolysis time is 1-2 hours. In step 2), the hot water temperature is 70℃-80℃, the ultrasonic power is 200-400W, the extraction is performed 3 times, and the extraction time for each extraction is 15-30 minutes. The strongly acidic cation exchange resin is of type D001. The pretreatment method is to activate it with 4%-6% HCl for 2-4 hours and wash it with water until the pH reaches 5.0-6.

0. In step 5), the nanofiltration membrane has a molecular weight cutoff of 200-500 Da.

2. Use according to claim 1, characterized in that, The functional foods include one or more of beverages, compressed candies, and oral liquids; the drugs include oral preparations or enteric-coated preparations.

3. A sugar-controlled solid beverage comprising the Sophora japonica and mulberry leaf composition according to any one of claims 1-2 and functional food-grade acceptable excipients, wherein the amount of Sophora japonica and mulberry leaf composition added is 0.5-1.5g / 100g.

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