CN116058504B

CN116058504B - Synergistic hypoglycemic composition and hypoglycemic health-care beverage

Info

Publication number: CN116058504B
Application number: CN202310120523.9A
Authority: CN
Inventors: 李伟; 牟书才; 王海峰; 张秀春
Original assignee: Heilongjiang Beibikang Biotechnology R & D Co ltd
Current assignee: Heilongjiang Beibikang Biotechnology R & D Co ltd
Priority date: 2023-02-15
Filing date: 2023-02-15
Publication date: 2023-07-07
Anticipated expiration: 2043-02-15
Also published as: CN116058504A

Abstract

The invention provides a synergistic hypoglycemic composition and a hypoglycemic health-care drink, which belong to the technical field of research and development of health-care foods and functional foods. The synergistic hypoglycemic composition with the specific proportion provided by the invention can realize the optimal hypoglycemic effect, and the effect is superior to that of the independent components. The sensory score of the hypoglycemic drink prepared by the invention is 92.9, the quality is excellent, and the hypoglycemic drink is very suitable for industrial popularization. The active ingredients of the health-care drink of the invention are not lost due to the necessary product sterilization process, and the health-care drink can be put into mass production. The invention can promote the fine and deep processing of the lonicera caerulea fruits, improves the commercial value of the lonicera caerulea fruits and lays a foundation for local prop industry.

Description

Synergistic hypoglycemic composition and hypoglycemic health-care beverage

Technical Field

The invention relates to the technical field of research and development of health-care foods and functional foods, in particular to a synergistic hypoglycemic composition and a hypoglycemic health-care beverage.

Background

The incidence of type ii diabetes increases dramatically with aging population, increasing obesity rate, decreasing exercise amount, and changing eating habits, and is expected to continue to increase. The current therapeutic strategies for diabetes include diet adjustment, moderate exercise, oral medication, insulin injection, and the like. However, most of the medicines in the market have adverse effects on human bodies, such as hypoglycemia, fluid retention, osteoporosis, heart failure and the like, while treating diabetes. A great deal of researches show that the natural products and active ingredients have little toxicity and adverse effect on human bodies while resisting diabetes, and a possible hypoglycemic mechanism thereof is gradually becoming a research hot spot. Multiple natural products are used in combination, possibly further resulting in multi-component multi-target therapies that have proven to be more effective and less toxic than traditional single-target drugs (ESPINOZA-FONSECA L m. The peptides of the multi-target approach in drug design anddiscovery [ J ]. Bioorganic & Medicinal Chemistry,2006,14 (4): 896-897.).

The indigo fruit fresh food has sour and astringent taste, is not accepted by consumers, but is rich in active substances such as anthocyanin, flavonol, polyphenol acid and the like, and the anthocyanin content is 7-12 times of that of blueberries. As the lonicera caerulea has a functional health care function, the hot tide of developing the third-generation fruits with nutrition and health care double effects represented by berries such as the lonicera caerulea is raised.

At present, a synergistic health care preparation taking lonicera caerulea fruits as a raw material is not yet seen.

Disclosure of Invention

The invention aims to provide a synergistic hypoglycemic composition and a hypoglycemic health-care beverage, which solve the problem that a synergistic health-care preparation taking lonicera caerulea fruits as a raw material is lacked in the prior art.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a synergistic hypoglycemic composition which comprises lonicera caerulea fruit extract, rhizoma polygonati extract and a birch mushroom extract, wherein the volume ratio of the lonicera caerulea fruit extract to the rhizoma polygonati extract to the birch mushroom extract is 3-5:1:1.

Preferably, the concentration of the lonicera caerulea fruit extract is 2-3 g/mL, the concentration of the rhizoma polygonati extract is 1-3 g/mL, and the concentration of the birch mushroom extract is 1-3 g/mL.

Preferably, the indigo fruit extract, the rhizoma polygonati extract or the birch mushroom extract are respectively prepared from indigo fruit, rhizoma polygonati or birch mushroom serving as raw materials through water extraction treatment.

Preferably, the water extraction treatment comprises the following steps:

pulverizing the raw materials, sieving, mixing with water, performing first ultrasonic extraction, and centrifuging to obtain a first supernatant and a first filter residue;

mixing the first filter residue with water, performing second ultrasonic extraction, and performing second centrifugation after the extraction to obtain a second supernatant and a second filter residue;

mixing the first supernatant and the second supernatant, and concentrating to obtain extractive solution.

Preferably, the mesh number of the sieving is 30-50 mesh;

the weight ratio of the raw materials to the water is 1:5-15.

Preferably, the temperature of the first ultrasonic extraction and the second ultrasonic extraction is independently 35-45 ℃;

the time of the first ultrasonic extraction and the second ultrasonic extraction is independently 50-70 min.

Preferably, the concentration adopts rotary evaporation concentration, and the temperature of the rotary evaporation concentration is 40-50 ℃.

The invention also provides application of the synergistic hypoglycemic composition in preparing a medicament for treating diabetes.

The invention also provides a blood sugar reducing health-care drink, which contains the synergistic blood sugar reducing composition and auxiliary materials, wherein the auxiliary materials comprise sucralose, sodium citrate, gamma-cyclodextrin and essence.

Preferably, the adding amount of the sucralose is 0.02-0.03%;

the addition amount of the sodium citrate is 1-2%;

the addition amount of the gamma-cyclodextrin is 0.02-0.06%;

the addition amount of the essence is 0.02-0.06%.

The invention has the technical effects and advantages that:

the invention takes lonicera caerulea, rhizoma polygonati and the betulina as main raw materials, a novel hypoglycemic health-care product is successfully developed, the optimal hypoglycemic effect can be realized through the synergistic hypoglycemic composition with the specific proportion, the effect is better than that of the independent components, and the lonicera caerulea extract, the rhizoma polygonati extract and the betulina extract have synergistic effect in hypoglycemic. Experiments prove that the sensory score of the hypoglycemic drink prepared by the invention is 92.9, the quality is excellent, and the hypoglycemic drink is very suitable for industrial popularization. The active ingredients of the health-care drink are increased after pasteurization, the health-care drink is not lost due to the necessary product sterilization process, and the health-care drink can be put into mass production. The invention can promote the fine and deep processing of the lonicera caerulea fruits, improves the commercial value of the lonicera caerulea fruits and lays a foundation for local prop industry.

Further experiments prove that the hypoglycemic health-care drink provided by the invention has no toxicity to mice, and relieves the weight reduction of diabetic mice to a certain extent. After the diabetic mice are irrigated with the stomach LPI for 28 days, the symptoms of overeating and polydipsia of each dosing group can be effectively relieved, wherein the LPIM group has the best effect. The organ indexes of each administration group are relieved compared with the model group, and the lesions can be relieved to a certain extent. The LPI provided by the invention has remarkable antioxidant activity, and plays a role in protecting liver tissues by improving the redox state of liver tissues of a diabetic mouse through the synergistic induction of high-fat diet and STZ, thereby being beneficial to the recovery of the glycolipid metabolic functions of the liver tissues. LPI can control blood sugar by inhibiting the activity of immune factors, alleviate insulin resistance, has a relieving effect on mouse inflammation, and presumably can control blood sugar by promoting the activity of anti-inflammatory factors, and alleviate insulin resistance.

Drawings

FIG. 1 shows the inhibition results of indigo honeysuckle extract;

FIG. 2 shows the inhibition results of the extract of Inonotus obliquus;

FIG. 3 shows the inhibition results of Polygonatum sibiricum extract;

FIG. 4 shows the inhibition rate results of the hypoglycemic compositions in different ratios;

FIG. 5 is a graph showing the analysis results of the composition and content of anthocyanin in the hypoglycemic beverage before sterilization;

FIG. 6 is a graph showing the analysis results of the composition and content of anthocyanin in the sterilized hypoglycemic beverage;

FIG. 7 is a polysaccharide content calibration curve;

FIG. 8 is a particle size distribution of a hypoglycemic beverage before sterilization;

FIG. 9 is a particle size distribution of the sterilized hypoglycemic beverage;

FIG. 10 shows the change in body weight of mice in each group during gastric lavage;

FIG. 11 shows changes in diet of mice in each group during gastric lavage;

FIG. 12 shows the variation in water uptake during gastric lavage in groups of mice;

FIG. 13 shows the FBG changes of mice in each group during lavage;

FIG. 14 is an oral glucose tolerance curve for each group of mice during gastric lavage;

FIG. 15 is the AUC area of each group of mice during intragastric administration;

FIG. 16 is the effect of LPI on serum insulin in T2D mice;

FIG. 17 is the effect of LPI on T2D mouse serum HOMA-IR;

FIG. 18 is the effect of LPI on T2D mouse liver glycogen;

FIG. 19 is the effect of LPI on liver tissue morphology in T2D mice;

FIG. 20 is the effect of LPI on pancreatic tissue morphology in T2D mice.

Detailed Description

The invention provides a synergistic hypoglycemic composition, which comprises an indigo honeysuckle extract, a rhizoma polygonati extract and a birch mushroom extract, wherein the volume ratio of the indigo honeysuckle extract to the rhizoma polygonati extract to the birch mushroom extract is 3-5:1:1, preferably 3.5-4.5:1:1; in the invention, the lonicera caerulea extracting solution is preferably prepared by the following steps: cleaning, crushing and sieving the lonicera caerulea raw material, mixing with water, performing first ultrasonic extraction, and performing first centrifugation after the extraction to obtain a first supernatant and first filter residues; mixing the first filter residue with water, performing second ultrasonic extraction, and performing second centrifugation after the extraction to obtain a second supernatant and a second filter residue; combining the first supernatant and the second supernatant, and concentrating to obtain an indigo honeysuckle extract, wherein the mesh number of the screening is preferably 30-50 meshes, more preferably 35-45 meshes, and the weight ratio of the indigo honeysuckle raw material to water is preferably 1:5-15, more preferably 1:8-12; the temperature of the first ultrasonic extraction is preferably 35-45 ℃, more preferably 38-42 ℃, and the time of the first ultrasonic extraction is preferably 50-70 min, more preferably 55-65 min; the temperature of the second ultrasonic extraction is preferably 35-45 ℃, more preferably 38-42 ℃, and the time of the second ultrasonic extraction is preferably 50-70 min, more preferably 55-65 min; the concentration is preferably rotary evaporation concentration, and the temperature of the rotary evaporation concentration is preferably 40-50 ℃, and more preferably 43-47 ℃; in the invention, the rhizoma polygonati extract is preferably prepared by the following steps: cleaning rhizoma Polygonati, pulverizing, sieving, mixing with water, performing first ultrasonic extraction, and centrifuging to obtain first supernatant and first residue; mixing the first filter residue with water, performing second ultrasonic extraction, and performing second centrifugation after the extraction to obtain a second supernatant and a second filter residue; mixing the first supernatant and the second supernatant, and concentrating to obtain a rhizoma polygonati extract, wherein the mesh number of the sieve is preferably 30-50 meshes, more preferably 35-45 meshes, and the weight ratio of the rhizoma polygonati raw material to water is preferably 1:5-15, more preferably 1:8-12; the temperature of the first ultrasonic extraction is preferably 35-45 ℃, more preferably 38-42 ℃, and the time of the first ultrasonic extraction is preferably 50-70 min, more preferably 55-65 min; the temperature of the second ultrasonic extraction is preferably 35-45 ℃, more preferably 38-42 ℃, and the time of the second ultrasonic extraction is preferably 50-70 min, more preferably 55-65 min; the concentration is preferably rotary evaporation concentration, and the temperature of the rotary evaporation concentration is preferably 40-50 ℃, and more preferably 43-47 ℃; in the invention, the betulina extract is preferably prepared by the following steps: cleaning raw materials of the birch mushroom, crushing, sieving, mixing with water, performing first ultrasonic extraction, and performing first centrifugation after the extraction to obtain a first supernatant and first filter residues; mixing the first filter residue with water, performing second ultrasonic extraction, and performing second centrifugation after the extraction to obtain a second supernatant and a second filter residue; mixing the first supernatant and the second supernatant, and concentrating to obtain a birch mushroom extract, wherein the mesh number of the sieve is preferably 30-50 meshes, more preferably 35-45 meshes, and the weight ratio of the raw material of the birch mushroom to water is preferably 1:5-15, more preferably 1:8-12; the temperature of the first ultrasonic extraction is preferably 35-45 ℃, more preferably 38-42 ℃, and the time of the first ultrasonic extraction is preferably 50-70 min, more preferably 55-65 min; the temperature of the second ultrasonic extraction is preferably 35-45 ℃, more preferably 38-42 ℃, and the time of the second ultrasonic extraction is preferably 50-70 min, more preferably 55-65 min; the concentration is preferably rotary evaporation concentration, and the temperature of the rotary evaporation concentration is preferably 40-50 ℃, and more preferably 43-47 ℃; in the invention, after the preparation of the lonicera caerulea extract, the rhizoma polygonati extract and the birch mushroom extract is completed, the lonicera caerulea extract, the rhizoma polygonati extract and the birch mushroom extract are preferably stored at the temperature of 3-5 ℃ for later use.

The invention also provides application of the synergistic hypoglycemic composition in preparing a medicament or hypoglycemic health-care food for treating diabetes, wherein the medicament or the health-care food preferably takes the synergistic hypoglycemic composition as the only active ingredient; the medicine preparation is preferably powder, tablets, granules, capsules, solutions, emulsions, suspensions or injections and the like; the health food is preferably selected from honey paste, dew, soft capsule, powder, fresh juice, hard capsule, tablet, tea, oral liquid, medicated wine or granule; the medicine or health food of the invention preferably further comprises auxiliary materials, wherein the auxiliary materials can be fillers, sweeteners or other auxiliary materials for adjusting taste, disintegrants, lubricants, adhesives, coatings, colorants, preservatives and the like.

The invention also provides a blood sugar-reducing health-care drink, which contains the synergistic blood sugar-reducing composition and auxiliary materials, wherein the auxiliary materials comprise sucralose, sodium citrate, gamma-cyclodextrin and essence; the addition amount of the sucralose is preferably 0.02 to 0.03%, more preferably 0.023 to 0.027%; the addition amount of the sodium citrate is preferably 1 to 2%, more preferably 1.3 to 1.7%; the addition amount of the gamma-cyclodextrin is preferably 0.02 to 0.06%, more preferably 0.03 to 0.05%; the addition amount of the essence is preferably 0.02-0.06%, more preferably 0.03-0.05%, and the blood glucose-reducing health-care beverage with the addition amount of each component is optimal in color, flavor, tissue state and taste; the hypoglycemic health-care drink is preferably sterilized by a pasteurization process, wherein the temperature of the pasteurization process is preferably 80-90 ℃, and the time is preferably 10-20 min.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Washing and crushing lonicera caerulea fruits, sieving with a 40-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:10, carrying out ultrasonic-assisted extraction at 40 ℃ for 60min, extracting for 2 times, centrifuging to obtain supernatant, combining the two supernatant filtrates, carrying out rotary evaporation concentration at 45 ℃, and storing in a refrigerator at 4 ℃ for later use.

Cleaning and crushing rhizoma polygonati, sieving with a 40-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:10, carrying out ultrasonic-assisted extraction at 40 ℃ for 60min, extracting for 2 times, centrifuging to obtain supernatant, combining the two supernatant filtrates, carrying out rotary evaporation concentration at 45 ℃, and storing in a refrigerator at 4 ℃ for later use.

Cleaning and crushing the birch mushroom, sieving with a 40-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:10, carrying out ultrasonic-assisted extraction at 40 ℃ for 60min, extracting for 2 times, centrifuging to obtain supernatant, mixing the two supernatant filtrates, carrying out rotary evaporation concentration at 45 ℃, and storing in a refrigerator at 4 ℃ for later use.

Mixing indigo honeysuckle extract, rhizoma polygonati extract and birch mushroom extract in a volume ratio of 4:1:1 to obtain the synergistic hypoglycemic composition.

Example 2

Washing and crushing lonicera caerulea fruits, sieving with a 30-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:5, carrying out ultrasonic-assisted extraction at 40 ℃ for 50min, extracting for 2 times, centrifuging to obtain supernatant, combining the two supernatant filtrates, carrying out rotary evaporation concentration at 50 ℃, and storing in a refrigerator at 4 ℃ for later use.

Cleaning and crushing rhizoma polygonati, sieving with a 30-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:10, carrying out ultrasonic-assisted extraction at 35 ℃ for 70min, extracting for 2 times, centrifuging to obtain supernatant, combining the two supernatant filtrates, carrying out rotary evaporation concentration at 45 ℃, and storing in a refrigerator at 4 ℃ for later use.

Cleaning and crushing the birch mushroom, sieving with a 30-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:5, carrying out ultrasonic-assisted extraction at 45 ℃ for 60min, extracting for 2 times, centrifuging to obtain supernatant, mixing the two supernatant filtrates, carrying out rotary evaporation concentration at 40 ℃, and storing in a refrigerator at 4 ℃ for later use.

Mixing indigo honeysuckle extract, rhizoma polygonati extract and birch mushroom extract in a volume ratio of 3:1:1 to obtain the synergistic hypoglycemic composition.

Example 3

Washing and crushing lonicera caerulea fruits, sieving with a 50-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:15, carrying out ultrasonic-assisted extraction at 40 ℃ for 70min, extracting for 2 times, centrifuging to obtain supernatant, combining the two supernatant filtrates, carrying out rotary evaporation concentration at 45 ℃, and storing in a refrigerator at 4 ℃ for later use.

Cleaning and crushing rhizoma polygonati, sieving with a 50-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:15, carrying out ultrasonic-assisted extraction at 35 ℃ for 60min, extracting for 2 times, centrifuging to obtain supernatant, combining the two supernatant filtrates, carrying out rotary evaporation concentration at 50 ℃, and storing in a refrigerator at 4 ℃ for later use.

Cleaning and crushing the birch mushroom, sieving with a 50-mesh sieve, accurately weighing 10.00g, adding water according to a material-water ratio of 1:10, carrying out ultrasonic-assisted extraction at 45 ℃ for 50min, extracting for 2 times, centrifuging to obtain supernatant, combining the two supernatant filtrates, carrying out rotary evaporation concentration at 40 ℃, and storing in a refrigerator at 4 ℃ for later use.

Mixing indigo honeysuckle extract, rhizoma polygonati extract and birch mushroom extract in a volume ratio of 5:1:1 to obtain the synergistic hypoglycemic composition.

Comparative example 1

The difference from example 1 was only that the indigo honeysuckle extract, the rhizoma Polygonati extract and the birch mushroom extract were mixed in a volume ratio of 2:1:1 to obtain a hypoglycemic composition.

Comparative example 2

The difference from example 1 is only that the indigo honeysuckle extract, the rhizoma polygonati extract and the birch mushroom extract were mixed in a volume ratio of 6:1:1 to obtain a hypoglycemic composition.

Example 4

Taking the synergistic hypoglycemic composition of the example 1, adding 0.025% of sucralose, 1.50% of sodium citrate, 0.04% of gamma-cyclodextrin and 0.04% of essence, and sterilizing by a pasteurization process at 85 ℃ for 15min to obtain the hypoglycemic beverage.

Example 5

Taking the synergistic hypoglycemic composition of the example 2, adding 0.02% of sucralose, 1.20% of sodium citrate, 0.03% of gamma-cyclodextrin and 0.05% of essence, and sterilizing by a pasteurization process at 80 ℃ for 10min to obtain the hypoglycemic beverage.

Example 6

Taking the synergistic hypoglycemic composition of the example 3, adding 0.03% of sucralose, 1.80% of sodium citrate, 0.05% of gamma-cyclodextrin and 0.03% of essence, and sterilizing by a pasteurization process at 95 ℃ for 20min to obtain the hypoglycemic beverage.

Experimental example 1 synergistic Effect verification

Determination of the inhibition ratio of α -glucosidase: a substrate PNPG solution of 7.5mmol/L and an alpha-glucosidase solution of 0.2U/mL were prepared with phosphate buffer solution of pH6.9 and 0.1mol/L, respectively. Respectively transferring 100 μL of sample solution to be tested with different concentrations into 96-well plates by adopting a 96-well plate reaction system, respectively adding 50 μL of alpha-glucosidase solution, incubating at 37 ℃ for 10min, adding 50 μL of PNPG solution, reacting at 37 ℃ for 20min, and adding 100 μL of Na ₂ CO ₃ Measurement of absorbance A of the solution (1 mol/L) at 405nm wavelength ₁ . The solution to be measured is replaced by distilled water with equal volume, and the absorbance A is measured ₀ The method comprises the steps of carrying out a first treatment on the surface of the The absorbance A is measured by replacing alpha-glucosidase solution with equal volume of phosphate buffer solution ₂ . By the following methodThe formula calculates the alpha-glucosidase inhibition rate:

determination of the inhibition of alpha-amylase: 30 μl of alpha-amylase (2U/ml) and 40 μl of the extracts at different concentrations were mixed in 96 well plates. After incubation at 37℃for 10 minutes, 30. Mu.l of substrate-2% soluble starch solution prepared in 0.1M phosphate buffer (pH 6.9) was added and incubated for a further 15 minutes at 37 ℃. 100 μl of 3,5 dinitrosalicylic acid (DNS) was then added as a colorant to the mixture and boiled for about 10 minutes. Absorbance A1 was measured at 540nm by an enzyme-labeled instrument. The solution to be measured is replaced by distilled water with equal volume, and the absorbance A is measured ₀ The method comprises the steps of carrying out a first treatment on the surface of the The absorbance A is measured by replacing alpha-glucosidase solution with equal volume of phosphate buffer solution ₂ The alpha-amylase inhibition rate was calculated using the following formula:

IC of indigo fruit extract, rhizoma Polygonati extract and birch mushroom extract for alpha-glucosidase in the examples was calculated by SPSS analysis with the help of curve data ₅₀ Values, results show: indigo fruit extract alpha-glucosidase IC ₅₀ The value is 4.142mg/mL, the inhibition rate is shown in figure 1, the birch mushroom extract is 9.822mg/mL, the inhibition rate is shown in figure 2, the rhizoma Polygonati extract is 114.076mg/mL, and the inhibition rate is shown in figure 3, wherein the IC of rhizoma Polygonati is shown in figure 3 ₅₀ The value is the largest, and the inhibition effect is the worst among the three.

IC of the synergistic hypoglycemic composition of example 1, the hypoglycemic compositions of comparative examples 1 and 2 for alpha-glucosidase were calculated separately ₅₀ Values, results show:

IC of synergistic hypoglycemic composition in example 1 ₅₀ The value was 3.829mg/mL, and the hypoglycemic compositions IC in comparative example 1 and comparative example 2 ₅₀ The values were 4.639mg/mL and 6.002mg/mL, respectively, and the results are shown in FIG. 4. From the results, it can be seen that by the present inventionThe provided synergistic hypoglycemic composition with specific proportion can realize the optimal IC for alpha-glucosidase ₅₀ The value, effect is better than that of the single component.

The CI combination index was calculated by means of CompuSyn software, the specific principle being as follows:

wherein: d1, D2 and D3 are mass concentrations (mg/mL) when the combined action inhibition rate of the compound 1, the compound 2 and the compound 3 is 50%, and Dx1, dx2 and Dx3 are mass concentrations (mg/mL) when the independent action inhibition rate of the compound 1, the compound 2 and the compound 3 is 50%. When CI <1, the combined action effect of the compound 1, the compound 2 and the compound 3 has a synergistic effect; when ci=1, the combined action effect of compound 1, compound 2 and compound 3 has a superposition effect; when CI >1, compound 2 and compound 3 have antagonism in their combined effect.

The results show that CI combination index of the lonicera caerulea extract, the rhizoma polygonati extract and the birch mushroom extract is 0.63 (< 1), further illustrating that the three have synergistic effect.

Experimental example 2 sensory evaluation experiment

The hypoglycemic drink (denoted as LPI) obtained in example 4 was evaluated.

10 students were selected and subjected to sensory evaluation in terms of color, flavor, texture state, and mouthfeel, and the total score was 100 points, and the evaluation criteria are shown in table 1. All the people participating in the sensory evaluation are randomly selected, and the conditions of physical health, no anorexia and the like are required, so that the sensory evaluation capability is good. Three evaluations were performed under the same conditions to reduce experimental errors.

Table 1 organoleptic criteria for hypoglycemic drinks

The results show that the sensory score of the hypoglycemic drink in the example 4 is 92.9, the quality is excellent, and the hypoglycemic drink is very suitable for industrial popularization.

Experimental example 3 determination of active substance

1. Anthocyanin content determination

The anthocyanin content of the hypoglycemic beverage (LPI) before and after sterilization in example 4 was compared and analyzed by high performance liquid chromatography (Shimadzu LC-16 ultraviolet detector) at 530nm with reference to NY/T2640-2014. The chromatographic column was phenanthromen C18 (250 x 4.6 mm), column temperature 35 ℃, mobile phase A, B respectively 1% formic acid-water/acetonitrile, sample injection amount: 20 μl, flow rate: 0.8mL/min. Determining the anthocyanin content in the LPI before and after sterilization respectively, wherein the analysis results of the anthocyanin composition and the anthocyanin content in the LPI before sterilization are shown in figure 5; the analysis results of the composition and content of anthocyanin in the sterilized LPI are shown in figure 6.

The results show that: 2 anthocyanin monomers were detected before LPI sterilization, and the content was as follows from high to low: procyanidins 122.95mg/L, procyanidins and their glycosides have been indicated as promising candidates for dietary compounds with antidiabetic effects. The cyanidin-3-glucoside has high alpha-amylase inhibiting effect, and IC thereof ₅₀ Is 0.3mmoL/L. Paeoniflorin IC ₅₀ At 4.54mg/L, paeoniflorin produced a significant hypoglycemic effect in diabetic rats, and this hypoglycemic effect was also observed in normoglycemic rats. The total content of the LPI sterilized procyanidine is 127.49mg/L. After sterilization, 2 anthocyanin monomers are detected in total, and the content of the anthocyanin monomers is as follows: cornflower pigment 130.69mg/L and paeoniflorin 4.98mg/L. The total anthocyanin content after LPI sterilization is 135.67mg/L. Therefore, the anthocyanin content of the health-care beverage is increased after pasteurization, the anthocyanin is not lost due to the necessary product sterilization process, and the health-care beverage can be put into mass production.

2. Polysaccharide content determination

The polysaccharide content was determined by phenol sulfuric acid method with reference to SN/T4260-2015, and the polysaccharide content in LPI before and after sterilization was compared and analyzed, and the standard curve is shown in FIG. 7.

The results show that the polysaccharide content in LPI before sterilization is 158.52 +/-3.84 mg/mL; the content of polysaccharide in the sterilized LPI is 160.15+/-3.44 mg/mL, and the content of polysaccharide is increased after sterilization. The polysaccharide has better thermal stability, realizes polysaccharide functional release through the sterilization process, increases the activity of reducing blood sugar, and further illustrates that the sterilization process of the product can not cause the loss of active ingredients of the health-care beverage.

Experimental example 4 stability analysis

1. Particle size analysis

Particle size analysis is a necessary means to detect the stability of liquid formulations. The larger the particle size, the easier the aggregation, affecting the stability of the liquid formulation system. The smaller the particle size, the better the product stability, and the particle size distribution of LPI before and after pasteurization is shown in fig. 8 and 9.

2. Other physical index

Soluble Solids (TSS), pH, turbidity and centrifuge stability factor before and after LPI pasteurization were measured and TSS was measured according to GB/T12143-2008. The pH of the juice mixture sample was measured at ambient temperature (25.+ -. 1 ℃ C.) using a digital pH meter (FZ-600T; chengdu century ark technologies Co., ltd., china Chengdu); the centrifugal stability factor was measured by using a spectrophotometer (WFJ 7200; shanghai Union instruments Co., ltd.) with a detection wavelength of 720nm, measuring LPI diluted 100 times and centrifuging the solution at 4000r/min for 10min to obtain absorbance X ₁ X is X ₂ The stability factor W is X ₂ And X ₁ If W is more than or equal to 90 percent, the stability is better. The change in LPI stability before and after pasteurization was evaluated and the results are shown in table 2 below:

TABLE 2 comparison of basic physical Properties before and after pasteurization

Note that: the different superscript letters in the same column represent a significant difference between the two (p < 0.05)

From the results, the centrifugal stability factor of LPI after pasteurization was changed from 81.67.+ -. 1.79% to more than 90%, and the solid content of each component was suspended in LPI, indicating an increase in stability. The pH after pasteurization of LPI changed from 4.03.+ -. 0.02 to 4.01.+ -. 0.01, the difference was not significant (P > 0.05), but the TSS was lower (P < 0.05), the decrease in TSS being attributable to the inactivation of some soluble solids by high temperature treatment. The absorbance of the LPI measured turbidity after pasteurization was reduced from 0.195+ -0.002 to 0.175+ -0.007, the difference was significant (P < 0.05), and the reduction in turbidity was positive for the stability of the liquid formulation. The combination of the physical index test results shows that the pasteurization treatment can improve the stability of the LPI.

Experimental example 5 improving effect on physiological and biochemical index of type II diabetes mice

Establishing and grouping experimental animal models: healthy male clean grade C57BL/6J mice, purchased from Peking Vitre Liwa laboratory animal technologies Co., ltd., license number: SCXK (Beijing)

2016-0011,4-5 weeks old, adapted to 1 week of feeding, ambient temperature 22+ -2deg.C, humidity 60% + -5%, 12h light/dark cycle. After normal feed is fed for 4 weeks, 60 mice are randomly selected for high fat cultivation for 4 weeks; another 10 blank groups (NC) were taken and fed with normal feed. After 4 weeks, 50 mice were intraperitoneally injected with STZ diluted with 0.1mol/L sodium citrate buffer at a dose of 100mg/kg to establish a model of type II diabetes mice. And (3) after the STZ injection is carried out for 3 days, the fasting blood sugar of all mice is measured, the blood sugar value is more than or equal to 10mmol/L, the successful modeling of the type II diabetes mice is confirmed, and the rest blood sugar values do not reach the diabetes diagnosis standard, so that the experiment is eliminated.

42 mice successfully molded were divided into 6 groups: blank (NC), model (T2D), metformin Positive Control (PC), sample low-dose intervention (LPIL), medium-dose intervention (LPIM) and high-dose intervention (LPIH). Dosage groups were given l0, 20 and 30mL/kg doses of sample (health drink obtained in example 4) for gavage, positive control group metformin (100 mg/kg. Bw), and other groups were gavaged with equal volumes of distilled water, respectively, in accordance with the low, medium and high doses. The mice were fed for 28 days, the diet and water intake were recorded daily, and the body weight of the mice was measured every 4 days.

Collecting a blood sample: mice in each group were fasted overnight, and the next day was collected by early eye drop and blood was collected by pressing the heart rapidly at about 800. Mu.L. Standing at room temperature for 2h to separate out serum, centrifuging at 3500r/min for 20min, collecting supernatant, and storing at-20deg.C.

Leaving a tissue specimen: the mice are killed after blood is taken, hearts, spleens, livers, kidneys, pancreas and colon are dissected and taken out, viscera are rinsed in pre-cooled normal saline, the pancreas, the right leaf width of the livers, i cm and the colon (3 of each group) are put into a centrifuge tube filled with 4% paraformaldehyde fixing solution for temporary storage, marks are made for HE staining, and pathological morphological changes of tissues are observed. The rest tissue is quickly frozen in liquid nitrogen and quickly transferred to a refrigerator at-80 ℃ for freezing and storing for subsequent determination.

The method for measuring fasting blood glucose comprises the following steps: the fasting blood glucose of the mice was measured periodically every 4 days, tail vein blood was taken after 6 hours of fasting, and was dropped on the reaction end of the blood glucose test strip, and Fasting Blood Glucose (FBG) was measured and recorded with a blood glucose meter. Repeating the steps twice, wherein the results of the two steps are similar, namely the data are available, and the two times have large difference, so that the measurement is re-performed.

Method for measuring oral glucose tolerance: oral glucose tolerance test at day 22 of intragastric administration: the mice were fasted without water for 12 hours, initial blood glucose was measured, and then 2g/kg glucose solution was filled, and blood glucose values were measured at 30, 60, 90 and 120 min.

Effects on the morphology of the mouse organ tissue: the liver, colon, spleen and pancreas tissues are fixed by 4% paraformaldehyde fixing solution, taken out and placed in an embedding box for running water to be washed for 12 hours, dehydrated by gradient alcohol, embedded in paraffin after xylene is used for replacing alcohol to be transparent, hot water is used for ironing to avoid wrinkles after slicing, drying is carried out, xylene is dewaxed and alcohol is used for water, hematoxylin and eosin are used for dyeing, and after sealing, the slice is observed and photographed under a Philips EM400T type transmission electron microscope.

Determination of the sugar metabolism index: serum and liver tissues are used for testing biochemical indexes of mice in each group. The detection indexes are as follows: fasting glucose (FBG), oral glucose tolerance (OGTT), serum Insulin (INS), hexokinase activity (HK), pyruvate kinase activity (PK), succinate dehydrogenase activity (SDH), lactate dehydrogenase activity (LDH), glycated Serum Protein (GSP), liver glycogen, glucose-6-phosphate hydrolase (G6 Pase), glycogen Phosphorylase (GP), and the like, according to the kit instructions.

Measurement of lipid metabolism index: serum was used to determine lipid metabolism index for each group of mice. The detection indexes are as follows: total Cholesterol (TC), triglycerides (TG), low density lipoproteins (LDL-C), high density lipoproteins (HDL-C), leptin (Leptin), adiponectin (ADP). According to the kit instruction steps.

Measurement of antioxidant index: the liver was used to determine the antioxidant index for each group of mice. The detection indexes are as follows: superoxide dismutase (SOD), malondialdehyde activity (MDA), glutathione peroxidase activity (GSH-Px), and Catalase Activity (CAT). According to the kit instruction steps.

Measurement of inflammatory factors: the content of IL-6, IL-10, TNF-alpha and IL-1 beta inflammation related factors in the serum of the mice is measured by ELISA method, and the specific measuring method refers to the related kit instruction.

1. Impact on mouse body weight

The fur of the mice before molding has luster, good spirit, dark yellow hair of the mice after molding by combining high fat with STZ, listlessness, increased food intake, increased water intake, increased urination and weight loss. The body weight of the mice was measured every 2 days within 28 days of LPI intragastric administration, and the results are shown in FIG. 10. Of the initial average body weights of the mice in each group, the NC group was lower (P < 0.05), and the remaining groups were not significantly different. Mice in the blank group grew more rapidly with increasing days, tended to be 26g more rapidly, and the model group had a weight decrease and then a slow rise within 0-12 days.

Mice in the group given the drug had a decreased body weight due to pancreatic injury caused by STZ injection affecting appetite, and then showed some recovery due to the drug effect, and the body weight tended to rise slightly and smoothly. At the end of 28 days of gastric lavage, the average body weight of the PC group mice was not different from that of the NC group mice (P > 0.05), the average body weight of the LPIH group was highest in the LPI different dose groups, was 24.84g, and the average body weight of the T2D group mice was lowest, was 23.65g. The mice in each group can ingest and take water normally within 28 days of gastric lavage, have quick response to the outside, have no listlessness, have no toxicity under the dosage of LPI, and relieve the weight loss of the diabetic mice to a certain extent.

2. Effects on mice diet and Water intake

Diabetic mice commonly have symptoms of overeating and polydipsia, and the food intake and water intake of the T2D group are both obviously increased (P < 0.05), so that a model with the characteristics of diabetes is obviously established. The changes in food intake of mice in each group during gastric lavage are shown in FIG. 11 and changes in food intake are shown in FIG. 12. From fig. 11, it can be seen that the daily food intake of NC groups is substantially constant, and fluctuates occasionally, with little variation; but the T2D group was nearly 50% higher than the NC group. The feed intake was improved in each of the dosing groups compared to the T2D group, with the greatest reduction in feed intake in the LPIM group (P < 0.05). The daily food intake of each rat in NC group, T2D group and LPIM group was 3.34.+ -. 0.15g, 6.44.+ -. 0.34g and 5.35.+ -. 0.21g, respectively, and the average food intake in LPIM group was reduced by about 16.92% as compared with that in T2D group.

As shown in FIG. 12, the NC group had normal water intake, each mouse had an average of 4.57.+ -. 0.46mL per day, while the diabetic mice had a rapid increase in water intake, and the T2D group had an average of 15.14.+ -. 2.01mL, which was more than 3 times that of the NC group. The middle dose group of LPI group has the greatest alleviation of polydipsia symptoms, which is on average 10.54+ -2.37 mL, about 30% lower than that of T2D group.

From the results, the symptoms of overeating and polydipsia of each dosing group can be effectively relieved after the diabetic mice are subjected to gastric lavage for 28 days, wherein the LPIM group has the best effect.

Effect of lpi on fasting blood glucose in mice:

glucose concentration typically fluctuates within a narrow range and dysfunction in glucose utilization may impair glucose homeostasis, causing elevated blood glucose. The effect of LPI on fasting blood glucose in mice during gastric lavage is shown in fig. 13.

The metabolic homeostasis of the mice is destroyed by the high-fat feed, the blood sugar of the mice in the T2D group is continuously and dynamically increased to about 8mmol/L, the blood sugar is rapidly increased after the STZ injection for 3 days, and the mice are judged to be successfully modeled after the STZ injection is higher than 10 mmol/L. During gastric lavage, NC groups fluctuated at 3.70-5.80mmol/L, each dosed group was reduced, LPIL was not significantly changed (P > 0.05), and the final mean blood glucose of LPIM group was as low as 12.01mmol/L, but compared to LPIH group was more reduced, approaching PC group. Each group has a certain dose dependency relationship, and the effects of the LPIH group and the PC group are better, the LPIM group is inferior, and the fasting blood glucose value of the LPIL group is reduced to the minimum extent. After 28 days of gastric lavage, the fasting blood glucose levels in mice were significantly reduced (P < 0.05) compared to the LPIH group and T2D group, indicating that LPI has an anti-diabetic effect.

Effect of lpi on oral glucose tolerance in mice: the oral glucose tolerance test (oral glucose tolerance test, OGTT) reflects the ability of the body to regulate blood glucose concentration, a common and effective method for diagnosing early stage diabetes and metabolic disease, and fig. 14 and 15 are oral glucose tolerance curves and AUC area plots under the curves for mice.

As shown in FIG. 14, the blood glucose was reduced after reaching a peak value for 30min within 0min to 120 min. The concentration of blood sugar in NC group is 12.30+ -1.82 mmol/L at 30min, is reduced to 7.53+ -0.25 mmol/L at 60min, and is 5.30+ -0.95 mmol/L at 120min, and has no significant difference (P > 0.05) from that before the solution of glucose is infused into stomach. The blood glucose rise in the T2D group was 29.50.+ -. 0.30mmol/L at 30min, followed by a slight decrease but not yet restored to the 0min level, which may be related to a deterioration of insulin function due to destruction of its islets. The trend was approximately the same for each dosing group, the effect was not apparent (P > 0.05) for the LPIL group, the blood glucose rate was the fastest in the LPIM group, and the LPIH group was the next most. As can be seen from fig. 15, the PC group and the LPIM group significantly improved glucose tolerance (P < 0.01), and the LPIH group significantly improved glucose tolerance (P < 0.05).

Effect of lpi on mouse serum insulin and insulin resistance index: the activation of polyadenylation and nitric oxide release following STZ administration, resulting in beta cell dysfunction, decreased insulin secretion, and thus hyperglycemia, and feedback-driven insulinosis, is shown in figure 16. The insulin resistance index (HOMA-IR) has been widely used in clinical evaluation of insulin sensitivity in diabetics. The insulin resistance index of each group of mice is shown in fig. 17.

As shown in fig. 16, T2D mice had very significantly elevated insulin levels (P < 0.01) compared to NC group. The LPI was improved to a different extent in each dose group compared to the T2D group, the reduction was significant in the PC group, the LPIL group and the LPIH group (P < 0.05), and the LPIM group was extremely significantly reduced (P < 0.01). Insulin intuitively reflects the condition of blood glucose regulation in vivo, the rise of the T2D group indicates successful modeling and insulin resistance, and the decrease of insulin after administration of a test object also indicates that LPI can possibly increase tissue sensitivity, reduce insulin production and improve insulin resistance to regulate blood glucose. HOMA-IR of the T2D group is 7.23+ -0.06, which is far higher than 1.95+ -0.07 of the NC group and is 3.70 times of the NC group. Each treatment group had a significant improvement over the T2D group (P < 0.05), with the PC group having the best effect, and the LPIH group being inferior.

Effect of lpi on mouse liver glycogen: glycogen is a key to regulating blood glucose balance, and insulin-induced liver glycogen synthesis and deposition in the postprandial state is a direct response to elevated blood glucose levels, playing an important role in maintaining glucose homeostasis. The insulin-AKT-GSK 3 signaling axis is considered to be the most important pathway regulating glycogen synthesis and responsible for glucose clearance. The liver glycogen content of each group of mice is shown in FIG. 18.

As can be seen from fig. 18, the hepatic glycogen content of the T2D group was significantly reduced (P < 0.05), and the decrease in hepatic glycogen synthesis probably means an increase in the flux of glucose to fat synthesis and causes hepatic triglyceride accumulation and metabolic abnormality. Compared with the T2D group, the liver glycogen content of mice in the LPIM group and the PC group is obviously increased (P < 0.05), and the mice in the LPIM group and the PC group are not different from those in the NC group (P > 0.05).

Effect of lpi on organ index in mice

Organ weight may reflect whether an inflammatory response or a lesion and degree of development occurs in an animal organ. The repair of damage to important organs of glucose metabolism in mice by different doses of LPI was evaluated by cardiac, spleen, liver, kidney, pancreas index, and the results are shown in table 3 below:

TABLE 3 organ index of mice in each group

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Note that: NC: blank control group; T2D: a diabetes model group; PC: metformin positive control group; LPIL: a low dose sample set; LPIM: a medium dose sample set; LPIH: high dose sample groups. Different superscript letters in the same column represent a significant difference (p < 0.05) between the two.

From the statistics in the table above, the spleen and kidney indices of the T2D mice were significantly increased (P < 0.05), pancreas and liver were significantly increased (P < 0.01), and heart index was significantly decreased (P < 0.05) compared to NC. The organ index may be raised due to pathological enlargement of the organ caused by obesity of the body of the mice after high-fat feeding, and inflammatory reaction in the body of the mice after insulin resistance. Heart failure leads to heart atrophy. The organ indexes of each administration group are relieved compared with the model group, and the lesions can be relieved to a certain extent.

Influence of LPI on the morphology of organs and tissues of mice

The observation under an optical microscope at a magnification of 200 x after HE staining of liver and pancreatic tissue is shown in fig. 19 to 20.

Liver staining as shown in fig. 19, the liver cells of the normal group were orderly and densely arranged, the cell size and morphology were basically consistent, and the cell nucleus was clear. The shape of the liver cells of the mice in the model group is irregular, the limit is blurred, gaps are increased, even cavitation exists, the cell nuclei are unevenly arranged, even aggregation is achieved, and cytoplasm is fused. The liver tissue structure of mice in the administration group is recovered to a certain extent, wherein the LPIH group has the best effect, the cells are closely arranged, the LPIM and PC groups are secondarily distributed, the cells are unevenly distributed, and the LPIL group has an improving effect and still has vacuoles.

Pancreas staining as shown in fig. 20, nc mice had orderly arranged pancreatic cells, abundant cytoplasm, small voids, and uniform cell nucleus size. Compared with NC group, T2D group mice have disordered pancreatic cell structure, partially dissolved islet cells, and increased loose clearance of whole structure due to STZ damage to islet cells. The pancreatic tissue structure recovery degree of mice in each dosing group is obvious, the LPIH group has the best effect, the pancreatic cell structure recovery degree of the mice is better, the cell boundaries are clearer, and the gaps are smaller in order arrangement. LPIM group and PC group.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The synergistic hypoglycemic composition is characterized by comprising an indigo honeysuckle extract, a rhizoma polygonati extract and a birch mushroom extract, wherein the volume ratio of the indigo honeysuckle extract to the rhizoma polygonati extract to the birch mushroom extract is 3-5:1:1; the lonicera caerulea extract, the rhizoma polygonati extract or the birch mushroom extract are respectively prepared from lonicera caerulea, rhizoma polygonati or birch mushroom serving as raw materials through water extraction treatment.

2. The synergistic hypoglycemic composition according to claim 1, wherein the concentration of the lonicera caerulea fruit extract is 2-3 g/mL, the concentration of the rhizoma polygonati extract is 1-3 g/mL, and the concentration of the betulina extract is 1-3 g/mL.

3. The synergistic hypoglycemic composition as claimed in claim 1, wherein the water extraction treatment comprises the steps of:

4. A synergistic hypoglycemic composition as claimed in claim 3, wherein the screened mesh number is 30-50 mesh; the weight ratio of the raw materials to the water is 1:5-15.

5. The synergistic hypoglycemic composition as claimed in claim 4, wherein the temperature of the first ultrasonic extraction and the second ultrasonic extraction is independently 35-45 ℃;

6. The synergistic hypoglycemic composition as claimed in claim 5, wherein the concentration is by rotary evaporation concentration, and the temperature of the rotary evaporation concentration is 40-50 ℃.

7. Use of the synergistic hypoglycemic composition according to any one of claims 1 to 6 for the preparation of a medicament for the treatment of diabetes.

8. Use of the synergistic hypoglycemic composition according to any one of claims 1 to 6 for the preparation of hypoglycemic foods.

9. A hypoglycemic health-care drink, which is characterized by comprising the synergistic hypoglycemic composition and auxiliary materials according to any one of claims 1-6;

the auxiliary materials comprise sucralose, sodium citrate, gamma-cyclodextrin and essence.

10. The hypoglycemic health drink according to claim 9, wherein the adding amount of the sucralose is 0.02-0.03%;

the addition amount of the sodium citrate is 1-2%;

the addition amount of the gamma-cyclodextrin is 0.02-0.06%;

the addition amount of the essence is 0.02-0.06%.