TW202247847A - Purpose for treating or preventing diabetes and facilitating islet regeneration and anti-inflammation using small molecule kefir exopolysaccharide extract capable of relieving or preventing diseases such as inflammation, inflammation-related diseases, hyperglycemia and related diseases, and diabetes and diabetic complications - Google Patents

Abstract

The invention discloses a purpose for treating or preventing diabetes and facilitating islet regeneration and anti-inflammation using small molecule kefir exopolysaccharide extract. Since the small molecule kefir exopolysaccharide extract has efficacies of inhibiting NF-kB inflammatory factors, controlling blood sugar, protecting islet cells, protecting kidney cells and the like, by delivering effective amount of the small molecule kefir exopolysaccharide extract disclosed in the invention or a composition containing the same, diseases, such as inflammation, inflammation-related diseases, hyperglycemia and related diseases, and diabetes and diabetic complications, can be relieved or prevented. The diabetic complications or hyperglycemia-related diseases include hyperphosphatemia, hyperlipidemia, diabetic nephropathy, etc.

Description

Application of small molecular kefir exopolysaccharide extract in treating or preventing diabetes, promoting islet regeneration and anti-inflammation

The present invention relates to the use of fermentation by-products, in particular to the use of a small molecular kefir exopolysaccharide extract for treating or preventing diabetes, promoting islet regeneration and anti-inflammation.

By the way, diabetes is currently one of the top ten most prevalent diseases in the world, and its mortality rate ranks seventh among the top ten causes of death in the world. Among the many types of diabetes, type 2 diabetes accounts for the largest number of people with diabetes, ranking No. The cause of type 2 diabetes is mainly caused by insulin resistance. The current drugs mainly reduce the symptoms caused by insulin resistance by increasing insulin secretion, reducing gluconeogenesis and promoting cell sugar uptake. However, most of these drugs have a strong Side effects and liver toxicity may bring a greater physical burden to patients.

Kefir fermented milk is obtained by fermenting milk, goat milk or horse milk with kefir grain composed of lactic acid bacteria and yeast. It is a fermented drink popular in North Caucasus, Eastern Europe and Russia. In recent years Many studies have confirmed that kefir fermented milk contains many small molecular peptides, which have the effect of improving obesity, gastrointestinal diseases, allergies and other diseases, and have antibacterial, wound healing, prevention of high blood pressure, anti-oxidation and other effects . Because kefir fermented milk is a very safe and functional food, long-term consumption will not cause adverse side effects on the human body, so kefir fermented milk is a high-quality nutritional supplement or raw material for functional food.

The main purpose of the present invention is to provide a second application of the small molecule kefir exopolysaccharide extract, because the small molecule kefir exopolysaccharide extract has multiple functions of anti-inflammation, regulating blood sugar and promoting islet regeneration Therefore, by administering an effective amount of small-molecule kefir exopolysaccharide extract or a composition containing it to an individual, the effect of treating or/and preventing inflammatory diseases, diabetes or its complications can be effectively achieved.

The reason is that, in order to achieve the above purpose, the present invention discloses a use of a small molecule kefir exopolysaccharide extract for preparing a functional composition, wherein the small molecule kefir exopolysaccharide extract is separated from Glucose-enriched mixture from kefir ferments with a molecular weight of 12 kDa.

Wherein, the functional composition can be a food, a nutritional supplement or a pharmaceutical composition, and can be prepared in different forms such as lozenges, powders, liquid beverages, etc. according to requirements.

In one embodiment of the present invention, the functional composition is an anti-inflammatory composition, which means that by administering an effective amount of small-molecule kefir exopolysaccharide extract to a body, it can effectively Inhibit or improve inflammatory response or its related diseases.

In one embodiment of the present invention, the anti-inflammatory composition can be used to inhibit the expression of pro-inflammatory factors in multiple organs, and these organs at least include any two of the following organs: spleen, lung, liver, pancreas, kidney, and The pro-inflammatory factor is NF-kB protein.

In another embodiment of the present invention, the functional composition is a composition for treating or/and preventing diabetes and related diseases, that is, by administering an effective amount of small molecule kefir exopolysaccharide The extract can effectively prevent or improve diseases such as diabetes, hyperlipidemia, hyperphosphatemia, diabetic nephropathy, diabetic liver injury or diabetic pancreatopathy when administered to an individual suffering from diabetes or hyperglycemia.

In one embodiment of the present invention, the composition for treating or/and preventing diabetes and its related diseases can be used to reduce symptoms of increased drinking water and increased urine output caused by diabetes.

In yet another embodiment of the present invention, the functional composition is a blood sugar regulating agent, specifically, the blood sugar regulating agent can increase the phosphorylation of PI3K protein and the expression of GLUT2 protein, so as to effectively delay The effect of exacerbation of diabetes or prevention of diabetes.

In the next embodiment of the present invention, the functional composition is a composition for promoting islet regeneration. Specifically, the composition for promoting islet regeneration can reduce the apoptosis of islet β cells and avoid the damage of islet β cells. In order to effectively achieve the effect of treating or improving diseases related to pancreatic islet damage.

The present invention discloses the use of a small molecule kefir exopolysaccharide extract for treating or preventing diabetes, promoting islet regeneration and anti-inflammation, and the small molecule kefir exopolysaccharide extract is a kefir fermented The secondary product, isolated from kefir powder, has a molecular weight of less than 12kDa and has the ability to inhibit NF-kB inflammatory factors, regulate blood sugar, maintain blood sugar stability, protect islet cells, and protect kidney cells. Therefore, by administering an effective amount of the present invention The small molecular kefir exopolysaccharide extract or the composition containing it can improve or treat inflammation, inflammation-related diseases, hyperglycemia and its related diseases, diabetes, diabetic complications, liver damage, or can effectively To prevent the occurrence of the above diseases, among them, diabetic complications or hyperglycemia-related diseases include hyperphosphatemia, hyperlipidemia, diabetic nephropathy, etc.

In the following, in order to illustrate the technical features of the present invention, some examples will be given together with diagrams for a more detailed description as follows.

The kefir powder used in the following examples is a commercial product (Sinochem Health Biotechnology, Taichung, Taiwan). Furthermore, kefir powder is obtained by fermenting the milk powder base with kefir grains as the fermentation bacteria at 20°C for 20 hours, and then drying and spraying. It contains small fractions of peptides and small molecule polysaccharides.

The KE extract used in the following examples is the fermented polysaccharide (Kefiran extract; KE) of a single lactic acid bacterium traditionally isolated from Kefir grains, which belongs to macromolecular polysaccharides (>805 kDa).

The cell lines used in the following examples, such as mouse macrophage cell line RAW264.7 (mouse macrophage cell line RAW264.7), are all commercial cell lines and do not need to be registered separately, and all cell tests are only to verify the present invention The functions of the disclosed technical features are not intended to limit the technical features and the scope of interpretation of the present invention.

The NF-κB-luciferase +/+ gene transfected mice (hereinafter referred to as NF-kB-Luc mice) used in the following examples are those carrying the luciferase gene driven by the NF-κB promoter Mice can reflect the activity of NF-κB by observing the activity of luciferase (the degree of luminescence).

The following animal experiments were carried out in accordance with the relevant guidelines for animal experiments, and were approved by the Animal Care and Utilization Committee of National Chung Hsing University.

Example 1: Preparation of Keffer Exopolysaccharide Extract

Dissolve a certain amount of kefir powder in 4 times the volume of distilled water, heat at 95-100°C for 4 hours, and then centrifuge at room temperature for 30 minutes (5000xg). After centrifugation, take the supernatant and add an equal volume of 20% Trichloroacetic acid solution (Trichloroacetic acid) was left to stand for 30 minutes, centrifuged at 4°C for 30 minutes (5000xg), the supernatant was taken, and the supernatant was filtered with a 0.45 µm filter, and the filtrate was added to three times the volume of 95 % alcohol, wait for the precipitation; centrifuge the precipitated liquid at 4°C for 30 minutes (5000xg), remove the supernatant and collect the precipitate, which is the Kevre exopolysaccharide extract disclosed by the present invention (hereinafter referred to as KEPS extract ), the yield was about 11.0 ± 0.4% (w/w). It was lyophilized for use in the following examples.

Example 2: Preparation of conventional kefran extract

Take Lactobacillus kefiranofaciens subsp. Kefiranofaciens (ATCC 43761) to ferment in MRS medium (Man-Rogosa-Sharpe broth medium, LAB 094, Heywood, United Kingdom) under anaerobic conditions at 30°C, collect the fermentation broth, add an equal volume The 20% trichloroacetic acid solution (Trichloroacetic acid) was centrifuged at 4°C for 30 minutes (5000xg) and filtered, the supernatant was taken, and three times the volume of 95% alcohol was added to the supernatant to wait for precipitation; the precipitated liquid was placed in Centrifuge at 4°C for 30 minutes (5000xg), and the product obtained is kefiran extract (kefiran, hereinafter referred to as KE extract), and the yield is about 0.08 ± 0.01% (w/v). It was lyophilized for use in the following examples.

Example 3: KEPS extract is a small molecule polysaccharide

The KEPS extract obtained in Example 1 and the KE extract obtained in Example 2 were redissolved in distilled water respectively, filtered (0.20 μm) and degassed, and then 20 μl of the solution was taken and used in HPSEC (high performance size-exclusion chromatography) column Analysis, the results are shown in Figure 1, where the standards (pullulan standards, Shodex, Kanagawa, Japan) were used as 6.2 kDa, 10 kDa, 21.7 kDa, 48.8 kDa, 113 kDa, 210 kDa, 366 kDa and 805 kDa as Points to detect the molecular weight of KEPS extract and KE extract.

In addition, KEPS extract and KE extract were analyzed by monosaccharide high performance liquid chromatography detection technology, and the results are shown in Table 1 below. In detail, 150mg of KEPS extract and KE extract were mixed with 2M trifluoroacetic acid (10ml) and heated, then evaporated and dried under vacuum, and then re-dissolved with an aliquot of 5ml deionized water to obtain KEPS extract respectively Extract and KE extract sample mixture. ABEE-derived monosaccharides (p-Aminobenzoic acid ethyl ester-derivatized monosaccharides) were prepared by a method disclosed in a previous study (Yasuno S et. al). Aliquots (30 μl) of each sample mixture and each standard solution were mixed with ABEE reagent solution (Seikagaku Co., Tokyo, Japan) and heated. After cooling, 600 μl of distilled water and 600 μl of chloroform were added, and centrifuged (4° C, 1800×g, 5 minutes), take out 5 μl of the upper aqueous layer for HPLC analysis, wherein, the HPLC analysis system uses a Gemini C 18 column (Phenomenex C18 250 mm × 4.6 mm), and the analysis conditions are: column (90:10 ) mobile phase is 0.04 M potassium borate buffer (pH 8.9, solvent A) and 100% acetonitrile (solvent B); 50°C; detection wave is 308nm; flow rate is 0.8ml/min; and, the 10 used Sugar standards were glucuronic acid, galacturonic acid, galactose, mannose, glucose, arabinose, ribose, xylose, fucose and rhamnose, respectively.

Table 1: Monosaccharide composition of KEPS extract and KE extract Monosaccharide KEPS extract (%) KE extract (%) Galactose 1.20±0.02 8.28±0.06 glucose 98.1±0.06 10.1±0.08 Mannose 0.74 ± 0.05 76.4±1.23

It can be known from the results in Figure 1 that the molecular weight of the KEPS extract disclosed by the present invention is only 12 kD, which is significantly smaller than that of the conventional KE extract (molecular weight > 805 kDa); and, from the results in Table 1, it can be seen that the KEPS extract and KE The composition of the extracts is different. Specifically, the main component of the KEPS extract is glucose, while the main component of the KE extract is mannose.

The results of Figure 1 and Table 1 clearly show that the KEPS extract disclosed by the present invention is a different composition from the conventional KE extract, which means that the KEPS extract disclosed by the present invention is different from the conventional Kepran, and It is a novel small molecule polysaccharide composition.

Example 3: Cytotoxicity Analysis

The mouse macrophage cell line RAW264.7 (1×10 ⁶ cells per well) was treated with different concentrations (0, 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 μg/ml) KEPS extract or KE extract, and cells treated with 1 μg/ml lipopolysaccharide as a positive control group were cultured for 24 hours and analyzed for cell viability (MTT analysis). The detection wavelength was 550nm, and the results are shown in Figure 2A and Figure 2B As shown, wherein, the culture conditions are 37°C, 5% carbon dioxide and 95% air humidity, using the addition of 10% fetal bovine serum, 4 mM L-glutamine, 100 μg/mL streptomycin and 100U/mL penicillin DMEM medium.

From the results of Figure 2A and Figure 2B, it can be seen that compared with cells treated with lipopolysaccharide, treating the cell line with KEPS extract or KE extract can improve the survival rate of cells, and when the concentration of KEPS extract or KE extract When the concentration is higher than 2 μg/ml, it has the activity of stimulating cell proliferation; in other words, the results of Figure 2A show that the KEPS extract disclosed by the present invention has no cytotoxicity.

Example 4: Anti-inflammatory test

Take the mouse macrophage cell line RAW264.7 (1×10 ⁶ cells per well), wherein the culture conditions are as described in Example 3. The mouse macrophage cell line RAW264.7 was divided into group A and group B. Among them, group A was the model of untreated lipopolysaccharide, and KEPS with different concentrations (0, 7.8, 31.3, 125 μg/ml) Cells were treated with extract or KE extract; group B is the mode of processing lipopolysaccharide (1μg/ml), which means that cells in each well are treated with lipopolysaccharide and different concentrations (0, 7.8, 31.3, 125 μg/ml) of KEPS at the same time Extract or KE extract; the cells in each well of group A and group B were treated according to the treatment conditions and cultured for 24 hours, and the cell culture supernatant was collected, and the cytokines were detected by ELISA kit (Abcam, Cambridge, MA, USA). Determination, the results are shown in Figure 3A and Figure 3B.

From the results in Figure 3A, it can be seen that under the condition that the cells were not treated with lipopolysaccharide, the administration of KEPS extract or KE extract had no effect on the amount of IL-6 secreted by the cells; but from the results in Figure 3B, it was known that when the cells were cultured After lipopolysaccharide is added to the environment, cells will be stimulated by lipopolysaccharide to secrete IL-6, and if KEPS extract or KE extract is administered at the same time, cells will be inhibited from secreting IL-6 stimulated by lipopolysaccharide, and when administered with KEPS extract When the concentration of the substance is 7.8 and 125 μg/ml, it is better to inhibit the cells from secreting IL-6 stimulated by lipopolysaccharide.

The results of Fig. 3A and Fig. 3B prove that the KEPS extract disclosed in the present invention can inhibit the secretion of inflammatory hormones in cells, thereby achieving the effect of preventing or slowing down the inflammatory response.

Example 5: Bioluminescent Image Analysis (1)

A plurality of 8-week-old male and female NF-kB-Luc mice were randomly divided into 5 groups, and the mice in each group were treated with the following conditions:

Control group: no drug or sample was administered;

LPS group: administered 12.5 mg/kg lipopolysaccharide;

KEPS/LPS group: administer 12.5 mg/kg lipopolysaccharide and 100 mg/kg KEPS extract disclosed in the present invention;

KE/LPS group: 12.5 mg/kg lipopolysaccharide and 100 mg/kg conventional KE extract were administered;

ASA/LPS group: Administer 12.5 mg/kg lipopolysaccharide and 12.5 mg/kg aspirin (acetylsalicylic acid);

Among them, KEPS/LPS group and KE/LPS group were given KEPS extract or KE extract for 7 days first, and then LPS was administered by intraperitoneal injection; ASA/LPS group was administered intraperitoneally before LPS 1 hour to cast aspirin.

Except for the mice in the control group, all other mice were irradiated with In Vivo Imaging System 24 hours after LPS administration. The in vivo luminescence imaging of mice in each group was analyzed and quantified. The results are shown in Figure 4A to Figure 4D and sacrifice the mice in each group, take the heart, spleen, lung, kidney, pancreas, intestine and other tissues, observe and analyze the luminescence imaging of each organ of the mice in each group with live luminescence imaging and quantify them, the results are shown in Figure 5A to Figure 5D.

From the results in Figure 4 and Figure 5, it can be seen that compared with the control group, the luminescence intensity of the whole body and internal organs of the mice in the LPS group was significantly increased, and the luminescence intensity of the intestinal tissue of the female NF-kB-Luc mice was the highest (eg Figure 4A and Figure 5A). For female NF-kB-Luc mice, the luminous intensity of the whole body and internal organs of KEPS/LPS group, KE/LPS group and ASA/LPS group were significantly lower than those of LPS group; while for male NF-kB- For Luc mice, administration of LPS could only stimulate the luminescent signal in the spleen and pancreas, but the intensity of luciferase in the KEPS/LPS group and/or KE/LP group was significantly lower than that in the LPS group.

The results shown in Figure 4 and Figure 5 show that when the individual is exposed to high oxidative stress environments, such as the oxidative stress induced by lipopolysaccharide, administering a certain amount of the KEPS extract disclosed by the present invention can protect the individual or its internal organs against Oxidative stress can effectively avoid or prevent diseases or organ damage caused by high oxidative stress.

Example 6: Analysis of the expression of inflammation-related cytokines (1)

The spleen, lung, and liver of each group of NF-kB-Luc mice in Example 5 were taken, and after protein extraction and electrophoresis colloid separation, the expression of inflammation-related cytokines in the organs of the mice in each group was detected by Western blot method, including There are NF-κB, MAPK, p-NF-κB or p-MAPK, and the results are shown in Fig. 6 to Fig. 8 .

From the results of Figure 6 to Figure 8, it can be seen that compared with the control group, the expression of p-NF-κB in the spleen, lung and liver of NF-kB-Luc mice in the LPS group was significantly increased, and the expression of p-NF-κB in female NF-kB-Luc mice was significantly increased. -The expression of p-MAPK in the spleen of Luc mice increased, indicating that the administration of lipopolysaccharide will promote the production of pro-inflammatory cytokines in individual organs, and then produce an inflammatory response. Compared with the LPS group, the expressions of p-NF-κB and p-MAPK in the organs of NF-kB-Luc mice in the KEPS/LPS group and the KE/LPS group were significantly decreased, indicating that administration of the KEPS extract disclosed in the present invention It can effectively inhibit the expression of pro-inflammatory factors, that is, it can regulate the expression of inflammation-related proteins: p-NF-κB and p-MAPK, and then achieve the effect of preventing or treating inflammation or related diseases.

Example 7: Bioluminescent Image Analysis (2)

With reference to the process described in Example 7, normal mice and NF-kB-Luc mice were taken, and NF-kB-Luc mice were randomly divided into groups, and the mice in each group were treated with the following conditions:

Normal control group: normal mice without any drugs or test samples;

LPS group: for NF-kB-Luc mice, intraperitoneal administration of 5 mg/kg lipopolysaccharide;

LPS/KEPSL group: NF-kB-Luc mice were first administered with the KEPS extract disclosed in the present invention for 7 days at a dose of 50 mg/kg, and then intraperitoneally administered with 5 mg/kg lipopolysaccharide;

LPS/KEPSH group: NF-kB-Luc mice were first administered with the KEPS extract disclosed in the present invention for 7 days at a dose of 100 mg/kg, and then intraperitoneally administered with 5 mg/kg lipopolysaccharide;

Among them, 24 hours after administration of lipopolysaccharide, the mice in each group were irradiated and analyzed with in vivo luminescent images, and the results are shown in FIG. 9A and FIG. 9B .

From the results in Figure 9A and Figure 9B, compared with the mice in the normal control group, the mice in the LPS group had a highly luminescent image, indicating that the administration of lipopolysaccharide can indeed induce a systemic inflammatory response in the mice, and in the kidney , intestines, pancreas, lungs, brain and other organs had obvious inflammatory reactions; compared with the mice in the normal control group, the luminescence intensity of the mice in the LPS/KEPSL group and the LPS/KEPSH group decreased, and with the As the dose of the KEPS extract of the present invention increased, the effect of inhibiting the intensity of luminescence was improved, showing that the KEPS extract disclosed by the present invention can indeed inhibit the systemic inflammatory response induced by lipopolysaccharide, and the inhibitory effect is with the administration of Increased with increasing dose.

Example 8: Analysis of the expression of inflammation-related cytokines (2)

Sacrifice the mice in each group that completed the experiment in Example 7, and take out their pancreas and kidneys respectively. After protein extraction and electrophoresis colloid separation, quantitative analysis of NF-kB and p-NF in each tissue was carried out by Western blot method. The expression of -κB, the results are shown in Figure 10A and Figure 10B.

The results of Figure 10A and Figure 10B show that the mice in the LPS group have a large amount of protein expression of NF-kB inflammatory factor in the pancreas and kidney, indicating that the administration of lipopolysaccharide will promote the inflammatory response of internal organs; compared with LPS For mice, the expression of NF-kB inflammatory factors in the pancreas and kidneys of mice in the LPS/KEPSL group and LPS/KEPSH group was significantly reduced, indicating that administration of the KEPS extract disclosed in the present invention can inhibit the NF-kB induced by lipopolysaccharide. The expression of kB inflammatory factor, and the effect of inhibiting inflammatory factor is improved with the increase of the administered dose.

Example 9: Animal Experiments

Eight-week-old male Sprague Dawley (SD) strain rats (purchased from Taiwan Lesco Biotechnology Co., Ltd.) were used for the test period of 8 weeks, and the rats in each group were sacrificed after the test period expired. During the test time, they were fed with 60% high-fat feed. At the 4th week of the test, after fasting for 16 hours, STZ (35 mg/kg/bw, dissolved in citrate buffer at pH 4.4) was injected intraperitoneally for diabetes induction, and three days later, the rats were fasted for 12 hours to detect the Mouse blood glucose, insulin content in serum, insulin index and β cell function index were obtained through HOMA-IR formula and HOMA-β cell function formula, and 2 g/kg/bw glucose solution was fed for glucose tolerance test (Oral glucose tolerance test (OGTT), and then record the blood glucose levels of the rats at 30, 60, 90, and 120 minutes respectively. The above test results are shown in Figures 11A to 11F.

From the results of Figure 11A to Figure 11D, it can be seen that the blood sugar level of the rats induced by STZ increased significantly, the HOMA-IR value was higher than the normal value (2.8), and the β-cell function was significantly decreased; it can be seen from the results of Figure 11E and Figure 11F The blood glucose of the rats induced by STZ exceeded the normal value by 200 mg/dl, and the area under the OGTT curve calculated by the PGAUC formula was 501.4 mg h/dl higher than the average value of the rats not induced by STZ. The results shown in Figure 11A to Figure 11F show that the induction of high-fat diet and STZ at the same time can indeed establish the type 2 diabetes model.

in:

HOMA-IR index = fasting blood glucose value (mg/dl) x serum insulin value (µIU/mL)/405

HOMA-β cell function index=20 x serum insulin value (µIU/mL)/(fasting blood glucose value (mg/dl)/18-3.5)

Rats whose fasting blood glucose was greater than or equal to 200 mg/dl and whose blood glucose was greater than or equal to 200 mg/dl 2 hours after feeding glucose solution (2 g/kg/bw) were divided into groups. The treatment conditions of each group of rats are described as follows:

The first group is the normal group, and the induction treatment program replaces STZ with citrate buffer as a placebo, and orally feeds distilled water;

The second group is the blank control group, injecting STZ to induce diabetes, and feeding distilled water orally during induction;

The third group is the KEPS high-dose group, which was injected with STZ to induce diabetes, and began to orally feed the KEPS extract disclosed by the present invention at the fifth week of the test, with a dose of 100 mg/kg/day;

The fourth group is the low-dose KEPS group. Diabetes was induced by STZ injection, and the KEPS extract disclosed in the present invention was orally fed at the fifth week of the test, with a dose of 50 mg/kg/day.

Example 10: Analyzing the physiological values of rats in each group

The rats in each group in Example 9 were tested weekly for body weight, urine output, food intake, water intake and blood sugar in each group during the test period, and the results are shown in Figures 12A to 12E.

From the results in Figure 12A and Figure 12B, it can be seen that the body weight of rats in groups 2 to 4 showed a steady increase under the induction of high-fat diet from week 0 to week 4 of the experiment, and because after injecting STZ at week 5, Body weight decreased; from the results in Figure 12C and Figure 12D, it can be seen that the drinking water and urine volume of the rats in the fourth group were significantly lower than those of the rats in the second group, and were close to the drinking water of the rats in the first group amount and urine output, it is shown that administering the KEPS extract disclosed by the present invention is helpful to improve polydipsia and polyuria symptoms caused by diabetes, and the effect is better at high doses; as can be seen from the results of Figure 12E, the second group When the rats in the fourth group were co-induced by high-fat diet and STZ, their blood sugar was significantly higher than that of the rats in the first group, and the fasting blood glucose values of all rats were higher than 200 mg/dl, just like the rats in the second group The average fasting blood sugar level was 353.71 mg/dl, and the blood sugar level of the rats in Group 3 and Group 4 decreased as the time of administering the KEPS extract disclosed by the present invention increased, and there was a relationship between the degree of blood sugar decrease and the administered dose. Positive correlation.

In other words, the results of Figure 12A to Figure 12E show that the KEPS extract disclosed in the present invention can effectively lower the blood sugar level of diabetic individuals and improve or relieve symptoms caused by diabetes, such as polydipsia or polyuria, and when the dose is administered When the amount increases, the effect of improving blood sugar and diabetes-related symptoms will increase, which means that the KEPS extract disclosed in the present invention has the ability to treat or prevent diabetes or its related diseases.

Example 11: Analysis of the effect of KEPS extract on the improvement of islet function in blood sugar regulation

Each group of rats in the example nine carried out the following tests after the end of the 8-week test:

Collect the blood of rats in each group, detect the blood glucose level and insulin level, and check the HOMA-IR index and HOMA-β cell function index. The results are shown in Figure 13A to Figure 13D; The obtained results are compared, and the results are shown in Figure 13G to Figure 13H;

Tube-fed 2 g/kg/bw glucose solution for glucose tolerance test, recorded the blood glucose levels of the rats at 30, 60, 90, and 120 minutes respectively, and compared the results with those in the fourth week of the test in Example 9 The results of the glucose tolerance test were compared and the results are shown in Figure 13E and Figure 13F.

From the results of Figure 13A to Figure 13H, it can be seen that the rats in the second group developed diabetes under the induction of high-fat diet and STZ, so that the blood sugar increased significantly and the glucose tolerance increased, and it can be seen from Figure 13C and D that the rats in the second group Insulin resistance increases and islet β-cell function decreases. Compared with the rats in the second group, the blood sugar levels of the rats in the third group and the fourth group were lowered, and the severity of the glucose tolerance reduction could be prevented, which means that the glucose tolerance of the individual could be improved, indicating that the present invention The disclosed KEPS extract can effectively regulate blood sugar, so that individual blood sugar can be maintained stable no matter on an empty stomach or after a meal; in addition, the insulin resistance of rats in groups 3 and 4 decreased significantly, and the islet β-cell function index increased ( 13B to 13D ), it shows that the KEPS extract disclosed in the present invention can indeed regenerate the islet β cells and improve the function of the islet β cells.

The results from Figure 13A to Figure 13F prove that administering a certain amount of the KEPS extract disclosed in the present invention to individuals suffering from diabetes or unstable blood sugar can effectively treat or prevent diabetes or its related diseases, and can enhance pancreatic beta cells function to reduce insulin resistance, and the effect of the KEPS extract disclosed in the present invention in treating or preventing diabetes and its related diseases is enhanced with the increase of the dosage.

Example 12: Analysis of the effect of KEPS extract on liver and kidney metabolism

In Example 9, after the 8-week test, the rats in each group took their blood respectively, and detected high-density lipoprotein (high density lipoprotein, HDL for short), low-density lipoprotein/very low-density lipoprotein (low-density lipoprotein) with a commercial detection kit. density lipoprotein/ very low density lipoprotein (LDL/ VLDL for short), triglyceride, phosphorus, blood urea nitrogen (BUN for short), the results are shown in Figure 14A to Figure 14E.

From the results of Figure 14A to Figure 14C, it can be seen that the content of low-density lipoprotein and triglyceride in the serum of the rats in the third group or the fourth group is higher than that in the serum of the rats in the second group. content is low. As is well known to those skilled in the art of the present invention, an increase in the content of low-density lipoprotein and triglyceride is a symptom of hyperlipidemia, and hyperlipidemia is the most common disease in diabetic individuals. Therefore, from FIG. 14A to FIG. The results of 14C can prove that administering a certain amount of the KEPS extract disclosed by the present invention to individuals suffering from diabetes or individuals with unstable blood sugar can effectively reduce the content of low-density lipoprotein and triglyceride in their serum. Furthermore, the effect of treating or preventing hyperlipidemia caused by diabetes and its related diseases can be achieved, and the effect of treatment or prevention is improved with the increase of the dosage.

Furthermore, from the results in Figure 14D and Figure 14E, compared with the rats in the first group, the levels of phosphorus and blood urea nitrogen in the serum of the rats in the second group were significantly increased, which means that the rats in the second group were suffering from diabetes or Hyperglycemia damages kidney function, resulting in hyperphosphatemia or diabetic nephropathy; compared with rats in group 2, the levels of phosphorus in serum and blood urea nitrogen in rats in group 3 or group 4 The content is relatively low, showing that the KEPS extract disclosed in the present invention can protect the kidneys from being damaged by hyperglycemia, and achieve the effect of reducing or preventing kidney damage in patients with diabetes or hyperglycemia. In other words, by administering an effective amount of the KEPS extract disclosed in the present invention, it is possible to delay or prevent kidney diseases caused by hyperglycemia or diabetes, such as hyperphosphatemia and diabetic nephropathy.

Example 13: Pancreas tissue section analysis

In Example 9, the rats in each group were sacrificed after the experiment, and their pancreas were taken for tissue sections, and hematoxylin and eosin staining (H&E) staining and immunohistochemical staining were performed respectively. The results are shown in Figure 15A Show.

Further analysis of the pancreatic tissue sections of rats in each group included scoring the degree of pancreatic lesions for the degree of β cell apoptosis, analyzing the number of islets in the islet sections and the proportion of insulin expression area in immunostaining, and the score of islet lesions was based on the severity The degree is divided into 5 grades, among which, grade 1 is the smallest lesion, the lesion area is less than 1%; grade 2 is mild lesion, the lesion area is less than 1-25%; It is a moderate/severe lesion with a lesion area of less than 51-75%; Grade 5 is a severe/higher lesion with a lesion area of less than 76-100%; the analysis results of pancreatic tissue sections are shown in Figure 15B to Figure 15D.

From the results of Figure 15A to Figure 15C, it can be seen that the islet cells of the rats in Group 1 are the most complete and their surrounding shapes are closer to arcs; the islets of Group 2 are relatively shrunken, and there are fine edges and corners around the islets , and the internal β cells showed a large number of apoptosis; in the H&E stained section of the pancreas of the rats in the third group, the islet atrophy phenomenon was improved compared with the second group, and the surrounding islets were as in the second group Fine edges and corners, according to the pathological interpretation report, the cell apoptosis (black arrow) was higher than that of group 1, but significantly lower than that of group 2 (Fig. 15B); the pancreas of rats in group 4 The shape and integrity of the islets in the H&E stained sections were closer to those of group 1, and the edges and corners around the islets were less than those in groups 2 and 3 and were closer to arcs, while in the islets, although Apoptosis still occurred, but the amount of apoptosis was significantly decreased compared with group 2 (Fig. 15B).

Furthermore, from the results in Figure 15A, it can be seen that the insulin distribution area of the rats in the first group is very dense and its color is darker, indicating a higher concentration; the insulin staining area of the rats in the second group is relatively fragmented, and the color is lighter; The insulin staining of rats in group 3 was similar to that of group 2, but the area density of insulin staining was higher than that of group 2; the shape of insulin staining area of rats in group 4 was nearly complete, which was not as good as in group 2. Insulin presents fragmented state. Also, as shown in Figure 15D, the insulin expression area of rats in group 2 was significantly lower than that of group 1 rats; although the insulin expression area of rats in group 3 was lower than that of group 1 rats, it was larger than that of group 2 rats Rats are still significantly improved; the insulin expression area of the 4th group rats not only presents the result of a significant increase compared with the 2nd group rats, and is almost significantly different from the 1st group rats, which is expressed in the high dose of the present invention. The administration of KEPS extract can regenerate islet cells.

The results from Figure 15A to Figure 15D show that the KEPS extract disclosed in the present invention can indeed effectively reduce the atrophy and apoptosis of islets, and the high dose of the KEPS extract disclosed in the present invention has the effect of promoting the regeneration of pancreatic islets Efficacy, it can be seen that administering an effective amount of the KEPS extract disclosed by the present invention to individuals suffering from diabetes or hyperglycemia can effectively prevent or treat diabetes and its related diseases, and can delay the deterioration of diabetes .

Example 14: Analysis of Kidney Tissue Sections

In Example 9, the rats in each group were sacrificed after the end of the experiment, and their kidneys were taken for histological sections, and stained with hematoxylin and eosin respectively. The results are shown in Figure 16A; The degree of convoluted tubule lesion is scored, and the ratio of kidney to body weight is calculated, and the disease score of renal proximal convoluted tubule is divided into 5 grades according to the severity, among which, grade 1 is the smallest lesion, and the lesion area is less than 1%; grade 2 is Mild lesions, less than 1-25% of the lesion area; Grade 3, moderate lesions, less than 26-50% of the lesion area; Grade 4, moderate/severe lesions, less than 51-75% of the lesion area; Grade 5, severe/higher lesions, The lesion area was less than 76-100%; the analysis results are shown in Figure 16B and Figure 16C.

From the results of Figure 16A and Figure 16B, it can be seen that in the kidney tissue of rats in group 2, a large number of proximal convoluted tubules have enlarged lumen, which means that the kidney tissue has been diseased and watery degeneration (black arrow); in group 3 Although multiple watery degenerations of the proximal convoluted tubules also appeared in the kidney tissues of the rats in the fourth group, compared with the rats in the second group, the kidney tissues of the rats in the third and fourth groups The watery degeneration of the proximal convoluted tubule has been improved, and the size of the vacuoles has been significantly improved in the renal tissue sections of the rats in the fourth group. From the results in Fig. 16C, it can be seen that kidney tissue lesions and watery degeneration will increase the kidney weight, and compared with the kidney weight of the rats in the second group, the kidney weights of the rats in the third and fourth groups are relatively decreased.

From the results of Fig. 16A to Fig. 16C, it can be confirmed that the KEPS extract disclosed in the present invention can protect kidney cells, so as to improve or slow down the kidney disease caused by hyperglycemia or diabetes. In other words, administering an effective amount of the KEPS extract disclosed in the present invention to individuals suffering from diabetes or hyperglycemia can effectively prevent or treat kidney disease caused by diabetes.

Example 15: Analysis of the effect of KEPS extract on the expression of proteins related to glucose metabolism

In Example 9, the rats in each group were sacrificed after the experiment, and their livers were taken, and the expression of PIK3, AKT and GLUT2 proteins in the liver were detected and quantified by Western blot method. The results are shown in Figure 17 to Figure 19 .

From the results of Figure 17A to Figure 17C, it can be seen that the expression levels of AKT protein in the livers of rats in each group are similar; in terms of the expression level of p-AKT protein, the rats in the 2nd to 4th groups are all higher than the rats in the 1st group ; and the phosphorylation expression of AKT protein was calculated according to the expression of AKT protein and p-AKT protein (Figure 17C), it can be seen that the phosphorylation expression of AKT protein in the liver of rats in groups 2 to 4 showed an increasing trend.

From the results of Figure 18A to Figure 18C, it can be seen that in terms of the expression level of PI3K protein, the expression level of rats in Group 2 to Group 4 was lower than that of rats in Group 1, but the rats in Group 4 were lower than those in Group 2. The expression level of the rats was significantly improved; in terms of the expression level of p-PI3K protein, the rats in the third group and the fourth group were significantly higher than the rats in the second group; further, the expression level of PI3K phosphorylation (Figure 18C) It can be seen that the phosphorylation expression of PI3K protein in the rats in the second group was significantly lower than that in the rats in the first group, and compared with the rats in the second group, the phosphorylation expression of PI3K protein in the liver of the rats in the third and fourth groups was not only Significantly increased, even surpassed the phosphorylation expression of PI3K protein in group 1 rats.

As can be seen from the results in Figure 19, in terms of the expression of GLUT2 protein, the expression level of the rats in the second group was significantly lower than that of the rats in the first group, and the expression level of the rats in the third group was almost the same as that of the rats in the first group The same, and the performance of the rats in the fourth group was even higher than that of the rats in the third group.

The results from Figure 17 to Figure 19 show that the KEPS extract disclosed in the present invention can stimulate the PI3K/AKT pathway by increasing the phosphorylation of PI3K protein, thereby increasing the expression of GLUT2 protein, so as to effectively reduce blood sugar or treat diabetes and related diseases. The effect of disease.

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Figure 1 is the result of analyzing the molecular weight of the KEPS extract disclosed in the present invention and the conventional KE extract. Figure 2A is the cell survival rate of the mouse macrophage cell line RAW264.7 treated with different concentrations of KEPS extract for 24 hours. Figure 2B shows the cell viability of the mouse macrophage cell line RAW264.7 treated with different concentrations of KE extract for 24 hours. Figure 3A shows the results of detecting the expression of IL-6 after the mouse macrophage cell line RAW264.7 was treated with different concentrations of KEPS extract or KE extract. Figure 3B shows the results of detecting the expression of IL-6 in each well after the mouse macrophage cell line RAW264.7 was simultaneously treated with lipopolysaccharide and different concentrations of KEPS extract or KE extract. Figure 4A is the result of irradiating individual female mice in each group with in vivo luminescent images. Fig. 4B is the result of statistical analysis of the bioluminescence of female mice in each group. Figure 4C is the result of irradiating individual male mice in each group with in vivo luminescent images. Fig. 4D is the result of statistical analysis of the bioluminescence of male mice in each group. Figure 5A shows the results of irradiating the hearts, spleens, lungs, kidneys, pancreas, and intestines of female mice in each group with in vivo luminescence images. Fig. 5B is the result of statistical analysis of the bioluminescence in the hearts, spleens, lungs, kidneys, pancreas, and intestines of female mice in each group. Figure 5C is the result of irradiating the hearts, spleens, lungs, kidneys, pancreas, and intestines of male mice in each group with in vivo cold light images. Fig. 5D is the result of statistical analysis of the bioluminescence in heart, spleen, lung, kidney, pancreas, and intestine of male mice in each group. Figure 6A shows the expression and quantitative results of NF-κB and p-NF-κB in the spleens of female mice in each group. Fig. 6B shows the expression and quantitative results of NF-κB and p-NF-κB in the lungs of female mice in each group. Fig. 6C shows the expression and quantitative results of NF-κB and p-NF-κB in the livers of female mice in each group. Figure 7A shows the expression and quantitative results of NF-κB and p-NF-κB in the spleens of male mice in each group. Figure 7B shows the expression and quantitative results of NF-κB and p-NF-κB in the lungs of male mice in each group. Fig. 7C shows the expression and quantitative results of NF-κB and p-NF-κB in the liver of male mice in each group. Figure 8A shows the expression of MAPK and p-MAPK in the spleens of female and male mice in each group. Figure 8B shows the expression of MAPK and p-MAPK in the lungs of female and male mice in each group. . Fig. 8C shows the expression and quantitative results of MAPK and p-MAPK in the livers of female and male mice in each group. Figure 9A is the result of irradiating individual mice of each group with in vivo luminescent images. FIG. 9B is the result of statistical analysis of the bioluminescence of mice in each group. Fig. 10 A shows the expression and quantitative results of NF-κB and p-NF-κB in pancreatic line of mice in each group. Figure 10B shows the expression and quantitative results of NF-κB and p-NF-κB in the kidneys of mice in each group. Figure 11A shows the results of detecting the blood glucose levels of normal rats and rats induced by STZ. Figure 11B shows the results of detecting the serum insulin levels of normal rats and STZ-induced rats. Fig. 11C is the HOMA-IR index of normal rats and rats induced by STZ. Fig. 11D is the index of β-cell function in normal rats and rats induced by STZ. Fig. 11E is a graph showing the glucose tolerance curves of normal rats and STZ-induced rats. Fig. 11F is the result of statistical analysis of the integral of the area under the glucose tolerance curve of normal rats and rats treated with STZ induction. Figure 12A shows the results of detecting the body weight of rats in each group from the 4th week to the 8th week of the test. Figure 12B is the result of analyzing the body weight changes of rats in each group from the 4th week to the 8th week of the experiment. Fig. 12C is the result of detecting the water intake of rats in each group from the 4th week to the 8th week of the experiment. Fig. 12D is the result of detecting the urine output of rats in each group from the 4th week to the 8th week of the experiment. Figure 12E shows the results of detecting the blood glucose of rats in each group from the 4th week to the 8th week of the experiment. Figure 13A shows the results of detecting the blood glucose levels of rats in each group after the test. Fig. 13B is the result of detecting the insulin content in the serum of rats in each group after the test. Fig. 13C shows the results of analyzing the HOMA-IR index of each group of rats after the test. Fig. 13D is the result of analyzing the β-cell function index of rats in each group after the test. Fig. 13E is the result of comparing the glucose tolerance of normal rats and rats in each group induced by STZ after the test. Fig. 13F is a comparison of the results of the area under the glucose tolerance curve of normal rats and rats in each group induced by STZ. Fig. 13G compares the results of HOMA-IR between normal rats and rats in each group induced by STZ after the test. Fig. 13H is a comparison of the islet β-cell function index results of normal rats and rats in each group induced by STZ. Fig. 14A is the result of detecting the content of high-density lipoprotein in the serum of rats in each group after the test. Fig. 14B is the result of detecting the content of low-density lipoprotein in the serum of each group of rats after the test. Fig. 14C is the result of detecting the content of triglyceride in the serum of rats in each group after the test. Figure 14D is the result of detecting the phosphorus content in the serum of rats in each group after the test. Figure 14E is the result of detecting the blood urea nitrogen content in the rats of each group after the test. Fig. 15A is the result of staining of pancreatic tissue sections of rats in each group. Fig. 15B is the result of scoring the degree of lesion according to the degree of apoptosis of β-cells in the pancreatic tissue sections of rats in each group. Fig. 15C is the result of statistical analysis of the number of islets in the pancreatic tissue sections of rats in each group. Figure 15D is the result of quantifying the insulin-stained area of the islets by IMAGE J after immunohistostaining of the pancreatic tissues of the rats in each group, and analyzing the ratio of the insulin expression area of the rats in each group. Fig. 16A is the result of staining of kidney tissue sections of rats in each group. Fig. 16B is the result of scoring according to the lesion degree of the proximal convoluted tubule in the renal tissues of rats in each group. Fig. 16C is the result of statistical analysis of the kidney weight ratio of rats in each group. Fig. 17A is the result of quantitative expression of AKT protein in liver of rats in each group. Fig. 17B is the result of quantitative expression of p-AKT protein in liver of rats in each group. Fig. 17C is the result of quantification of AKT protein phosphorylation in liver of rats in each group. Fig. 18A is the result of quantitative expression of PI3K protein in liver of rats in each group. Fig. 18B is the result of quantitative expression of p-PI3K protein in liver of rats in each group. Fig. 18C is the results of quantitative quantification of PI3K protein phosphorylation in liver of rats in each group. Figure 19 is the result of quantitative expression of GLUT2 protein in liver of rats in each group.

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Claims (10)

A use of small-molecule kefir exopolysaccharide extract for preparing an anti-inflammatory composition, wherein the small-molecule kefir exopolysaccharide extract is a glucose-rich mixture separated from kefir fermentation, Its molecular weight is 12kDa. As described in claim 1, the use of the small molecule kefir exopolysaccharide extract for the preparation of an anti-inflammatory composition, wherein the anti-inflammatory composition can inhibit the expression of pro-inflammatory factors in multiple organs, and these organs Contains at least two of the following organs: spleen, lung, liver, pancreas and kidney. As described in claim 1, the use of the small molecule kefir exopolysaccharide extract for the preparation of an anti-inflammatory composition, wherein the pro-inflammatory factor is NF-kB protein. A use of small-molecule kefir exopolysaccharide extract for preparing compositions for treating or/and preventing diabetes and related diseases, wherein the small-molecule kefir exopolysaccharide extract is isolated from kefir exopolysaccharide Fermented and glucose-rich mixture with a molecular weight of 12kDa. As described in claim 4, the use of small molecule kefir exopolysaccharide extracts for the preparation of compositions for treating or/and preventing diabetes and its related diseases, wherein the diabetes related diseases are hyperlipidemia, hyperemia Phosphorus, diabetic nephropathy, diabetic liver damage, or diabetic pancreatopathy. As described in claim 4, the use of the small molecule kefir exopolysaccharide extract for the preparation of a composition for the treatment or/and prevention of diabetes and its related diseases, wherein the treatment or/and prevention of diabetes and its related diseases The composition can reduce the symptoms of increased drinking water and increased urine output caused by diabetes. A use of a small-molecule kefir exopolysaccharide extract for the preparation of a blood sugar regulator, wherein the small-molecule kefir exopolysaccharide extract is a glucose-rich mixture separated from kefir fermentation products, which The molecular weight is 12kDa. As described in Claim 7, the small molecule kefir exopolysaccharide extract is used to prepare a blood sugar regulating agent, wherein the blood sugar regulating agent can increase the phosphorylation of PI3K protein and the expression of GLUT2 protein. A use of a small-molecule kefir exopolysaccharide extract for preparing a composition for promoting islet regeneration, wherein the small-molecule kefir exopolysaccharide extract is a glucose-rich mixture isolated from kefir fermentation , with a molecular weight of 12 kDa. As described in Claim 9, the small molecule kefir exopolysaccharide extract is used to prepare a composition for promoting islet regeneration, wherein the composition for promoting islet regeneration can reduce the apoptosis of pancreatic beta cells.

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