Application of benzyl isothiocyanate-loaded fish skin gelatin and pectin composite emulsion in preparation of hypoglycemic products

Abstract

The invention discloses application of a benzyl isothiocyanate loaded fish skin gelatin and pectin composite emulsion in preparation of a hypoglycemic product, and belongs to the technical field of functional carrier application. Benzyl isothiocyanate is embedded to prepare the benzyl isothiocyanate-loaded fish skin gelatin and pectin composite emulsion, and the specific method comprises the following steps: mixing a fish skin gelatin solution, a pectin solution and a certain amount of water to prepare a water phase; benzyl isothiocyanate is dissolved in edible oil, and an oil phase is prepared; and mixing the prepared water phase and oil phase, carrying out high-speed dispersion treatment, and then homogenizing to prepare the emulsion taking the fish skin gelatin and pectin as the compound emulsifier. The emulsion prepared by the invention has the capability of inhibiting the reduction of the activity of benzyl isothiocyanate, so that the emulsion can better improve the symptoms of diabetes mellitus and effectively reduce hyperglycemia.

Description

Technical Field

[0001] The invention relates to application of a fish skin gelatin and pectin composite emulsion loaded with benzyl isothiocyanate in the preparation of a blood sugar lowering product, belonging to the technical field of functional carrier application. Background Art

[0002] With the advancement of science and technology and the development of society, people's pace of life is accelerating, and eating habits are also quietly changing. Daily diets gradually tend to choose high-calorie, high-fat and high-sugar foods, but long-term intake can lead to problems such as obesity and insulin resistance. The convenience of modern life has also led to a decrease in people's physical activity. Long-term sitting and lack of exercise can lead to reduced muscle sensitivity to insulin. In addition, a high-pressure, fast-paced lifestyle can also easily lead to mental stress, which in turn affects the body's endocrine system. These factors can cause symptoms such as hyperlipidemia, hypertension, and hyperglycemia, and eventually develop into diabetes mellitus (DM).

[0003] Diabetes is usually a disease caused by insufficient insulin secretion or impaired insulin function. According to the cause, it can be divided into three categories: type 1 diabetes (T1DM), type 2 diabetes (T2DM) and gestational diabetes (GDM). Type 2 diabetes is the most common type of diabetes, accounting for more than 90% of all diabetes cases. The basic cause of its formation is insulin resistance (IR) and abnormal pancreatic β-cell function. Insulin resistance is mainly manifested in the reduced ability of insulin to take up and clear glucose in peripheral tissues, which in turn causes glucose metabolism disorders. In the initial stage of insulin resistance, in order to maintain blood sugar stability, insulin cells will react compensatorily and secrete too much insulin, but this resistance will weaken the ability of tissues such as the liver and fat to absorb glucose, while the liver's glucose output will increase. With the continuous aggravation of the body's insulin resistance symptoms, excessive insulin secretion cannot control the normal level of blood sugar. At the same time, the elevated insulin level increases the burden on the pancreas, followed by dysfunction of pancreatic β-cells, and then the function of insulin secretion is impaired, forming a vicious cycle.

[0004] Discovering active ingredients with high efficiency, low toxicity and less side effects from natural extracts to alleviate the impact of hyperglycemia symptoms has become a top priority in current research. Benzyl isothiocyanate (BITC) is a natural fat-soluble active ingredient that mainly exists in cruciferous vegetables and belongs to isothiocyanate compounds (ITCs). Studies have shown that BITC has the functional properties of regulating blood sugar. BITC can improve obesity and hepatic steatosis caused by a high-fat diet (HFD) and reduce insulin resistance in mice. However, BITC has the disadvantages of being volatile, having low water solubility and poor stability. When passing through the stomach phase, it will decompose due to its instability, resulting in its inability to function efficiently in the intestines. In addition, the release of BITC in cruciferous vegetables is affected by chewing and cooking, and the bioavailability is only 14-50%, which seriously limits its scope of application. Summary of the invention

[0005] [Technical issues]

[0006] The technical problem to be solved by the present invention is that BITC is easily volatile, has low water solubility, poor stability and low bioavailability, which limits its application in regulating blood sugar.

[0007] [Technical solution]

[0008] In order to solve the above problems, the present invention provides the use of a composite emulsion of fish skin gelatin (FSG) and pectin (PEC) loaded with BITC in the preparation of a blood sugar lowering product, wherein the composite emulsion is used to embed BITC, effectively improving its shortcomings such as high volatility, low water solubility and poor stability, so that BITC can be stable in the stomach and play a blood sugar regulating function after entering the intestine. The BITC can be widely used in the food, medicine and other industries, expanding its inherent application range.

[0009] The invention provides application of a BITC-loaded FSG and PEC composite emulsion in preparing a blood sugar-lowering product.

[0010] As an embodiment of the present invention, the product is food, medicine or health care product.

[0011] As an embodiment of the present invention, the method for preparing the BITC-loaded FSG and PEC composite emulsion comprises the following steps:

[0012] (1) Preparation of aqueous phase: FSG solution, PEC solution and a certain amount of water are mixed to prepare an aqueous phase; wherein the mass ratio of FSG to PEC in the aqueous phase is 16:1 to 1:6;

[0013] (2) Preparation of oil phase: dissolving BITC in edible oil to prepare an oil phase; the concentration of BITC in the oil phase is 2 to 10 mg / mL;

[0014] (3) Preparation of composite emulsion: The water phase prepared in step (1) is mixed with the oil phase prepared in step (2) at a volume ratio of 1:4 to 1:15, and high-speed dispersion treatment is performed, followed by homogenization to obtain an emulsion containing FSG and PEC as composite emulsifiers.

[0015] As an embodiment of the present invention, in step (1), the mass concentration of FSG in the aqueous phase is 1-2%.

[0016] As an embodiment of the present invention, in step (1), the mass concentration of PEC in the aqueous phase is 0.5-1%.

[0017] As an embodiment of the present invention, in step (1), the mass ratio of FSG to PEC in the aqueous phase is 2:1.

[0018] As an embodiment of the present invention, step (1) further comprises adjusting the pH of the aqueous phase to 7.

[0019] As an embodiment of the present invention, in step (2), the edible oil is corn oil.

[0020] As an embodiment of the present invention, in step (3), the volume ratio of the oil phase to the water phase is 1:9.

[0021] As an embodiment of the present invention, in step (3), high-speed dispersion: 8000-15000 r / min, high-speed dispersion 1-3 min.

[0022] As an embodiment of the present invention, in step (3), homogenization: the homogenization conditions are 7000-14000 psi, and high pressure homogenization is performed 4-8 times.

[0023] [Beneficial Effects]

[0024] BITC is a fat-soluble active ingredient with the function of regulating blood sugar, but its application in food is limited by its low bioavailability, chemical instability and poor water solubility. Using emulsion to deliver BITC is a low-cost method that can slow down the volatilization of BITC, enhance stability and improve its bioavailability, and has good application prospects in the food industry. Compared with pure BITC emulsion or emulsion using a single emulsifier to embed BITC, the composite emulsion encapsulating BITC prepared by the present invention can make diabetic mice drink more and eat more and improve their weight maintenance. It can also reduce the insulin (INS) level, fasting blood glucose (FBG) and insulin resistance index (HOMA-IR) of diabetic mice, and significantly improve the oral glucose tolerance (OGTT) of mice; reduce serum total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels, and improve high It can also reduce the content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CRE) in diabetic mice; increase the activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver, reduce the content of malondialdehyde (MDA) in the liver, reduce the damage of oxidative stress to the liver, improve the antioxidant capacity of the liver, and significantly improve the liver tissue damage and pancreatic islet damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The graph is a diagram of the change in the amount of food consumed by each group of mice every day after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively; ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively;

[0026] Figure 2 The water drinking amount of each group of mice after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4 was changed every day. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively, and ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0027] Figure 3 The figure is a graph showing the weight changes of mice in each group every day after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4;

[0028] Figure 4 The FBG changes of mice in each group every day after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4;

[0029] Figure 5INS graph after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4, * and # represent significant differences compared with the normal group and the model group (P<0.05), ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01);

[0030] Figure 6 The HOMA-IR graphs were obtained after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0031] Figure 7 This is a graph showing the determination of OGTT after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4;

[0032] Figure 8 The AUT graphs after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4, * and # represent significant differences compared with the normal group and the model group (P<0.05), ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01);

[0033] Fig. 9 The TC determination graph in serum after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4, * and # represent significant differences compared with the normal group and the model group (P<0.05), ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01);

[0034] Fig.10 The TG determination graph in serum after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4, * and # represent significant differences compared with the normal group and the model group (P<0.05), ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01);

[0035] Fig.11 This is a graph showing the determination of HDL-C in serum after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0036] Fig.12 The figure is the measurement of LDL-C in serum after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0037] Fig.13The figure is the measurement graph of ALT, an index of liver function in serum, after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0038] Fig.14 The AST test results of serum liver function index after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4, * and # respectively represent significant differences compared with the normal group and the model group (P<0.05), ** and ## respectively represent extremely significant differences compared with the normal group and the model group (P<0.01);

[0039] Fig.15 The graphs of renal function index CRE in serum after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4, * and # respectively represent significant differences compared with the normal group and the model group (P<0.05), ** and ## respectively represent extremely significant differences compared with the normal group and the model group (P<0.01);

[0040] Fig.16 The figure is the measurement graph of the liver function index BUN in serum after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4, * and # represent significant differences compared with the normal group and the model group (P<0.05), ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01);

[0041] Fig.17 The CAT activity in the liver was measured after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0042] Fig.18 The graph is a graph showing the determination of MDA content in the liver after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0043] Fig.19 The graph is a measurement of SOD content in the liver after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0044] Fig. 20The figure is the determination of GSH-Px content in the liver after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4, * and # represent significant differences compared with the normal group and the model group (P<0.05), ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01);

[0045] Fig.21 This is a cross-sectional view of liver tissue after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4;

[0046] Fig. 22 It is a cross-section of pancreatic tissue after intragastric administration using Examples 1 to 2 and Comparative Examples 1 to 4;

[0047] Fig.23 The figure is a graph showing the determination of TNF-α content in liver tissue homogenate after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0048] Fig.24 The IL-1β content in the liver tissue homogenate was measured after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively.

[0049] Fig.25 It is a graph showing the determination of IL-6 content in liver tissue homogenate after intragastric administration of Examples 1 to 2 and Comparative Examples 1 to 4. * and # represent significant differences compared with the normal group and the model group (P<0.05), respectively. ** and ## represent extremely significant differences compared with the normal group and the model group (P<0.01), respectively. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Source of raw materials

[0052] C57BL / 6 male mice (6–8 weeks old) fed with ordinary diet and high-fat and high-sugar diet (basic diet + 10% lard + 20% sucrose + 2.5% cholesterol + 1% cholesterol) were purchased from Liaoning Changsheng Biotechnology Co., Ltd.;

[0053] Streptozotocin (biochemical reagent) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0054] Citric acid (analytical grade) and sodium citrate (analytical grade) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0055] Metformin hydrochloride (biochemical reagent) was purchased from Beijing Solebow Technology Co., Ltd.;

[0056] Glucose (analytical grade) was purchased from Tianjin Damao Chemical Reagent Factory;

[0057] HDL-C assay kit, LDL-C assay kit, TC / TCH assay kit, TG assay kit, ALT test kit, AST test kit, CRE assay kit, BUN test kit, CAT assay kit, MDA assay kit, SOD assay kit, GSH-Px assay kit were purchased from Nanjing Jiancheng Bioengineering Institute;

[0058] INS kit, mouse tumor necrosis factor α (TNF-α) kit, mouse interleukin 1β (IL-1β) kit, and mouse interleukin 6 (IL-6) kit were purchased from Shanghai ELISA Biologicals Co., Ltd.

[0059] Example 1: Preparation of a BITC-loaded FSG and PEC composite emulsion

[0060] This embodiment provides a BITC-loaded FSG and PEC composite emulsion, which specifically includes the following steps:

[0061] S1. Preparation of aqueous phase: FSG solution, PEC solution and a certain amount of water are mixed to prepare an aqueous phase, so that the final concentration of FSG is 1wt%, the final concentration of PEC is 0.5wt%, and the pH of the solution is adjusted to 7;

[0062] S2. Preparation of oil phase: dissolving BITC in corn oil and thoroughly shaking and mixing to obtain a solution with a final concentration of 10 mg / mL of BITC in the oil phase;

[0063] S3. Preparation of a composite emulsion for regulating blood sugar: The water phase prepared in step S1 is mixed with the oil phase prepared in step S2, with the volume ratio of the oil phase to the water phase being 1:9, and a high-speed dispersion treatment is performed at 10,000 rpm for 2 minutes, followed by homogenization at a homogenization pressure of 10,000 psi, and the mixture is cycled 6 times to obtain an emulsion containing FSG and PEC as a composite emulsifier.

[0064] Example 2: Preparation of a BITC-loaded FSG and PEC composite emulsion

[0065] This embodiment provides a BITC-loaded FSG and PEC composite emulsion, which specifically includes the following steps:

[0066] S1. Preparation of aqueous phase: FSG solution, PEC solution and a certain amount of water are mixed to prepare an aqueous phase, so that the final concentration of FSG is 1wt%, the final concentration of PEC is 0.5wt%, and the pH of the solution is adjusted to 7;

[0067] S2. Preparation of oil phase: dissolving BITC in corn oil and thoroughly shaking and mixing to obtain a solution with a final concentration of 5 mg / mL of BITC in the oil phase;

[0068] S3. Preparation of a composite emulsion for regulating blood sugar: The water phase prepared in step S1 is mixed with the oil phase prepared in step S2, with the volume ratio of the oil phase to the water phase being 1:9, and a high-speed dispersion treatment is performed at 10,000 rpm for 2 minutes, followed by homogenization at a homogenization pressure of 10,000 psi, and the mixture is cycled 6 times to obtain an emulsion containing FSG and PEC as a composite emulsifier.

[0069] Comparative Example 1

[0070] The preparation method is consistent with that in Example 1, except that no PEC is added in step S1.

[0071] The details are as follows:

[0072] S1. Preparation of aqueous phase: prepare a solution with a FSG concentration of 1% and adjust the solution pH to 7.

[0073] Comparative Example 2

[0074] The preparation method is consistent with that in Example 1, except that no FSG is added in step S1.

[0075] The details are as follows:

[0076] S1. Preparation of aqueous phase: prepare a solution with a PEC concentration of 0.5% and adjust the solution pH to 7.

[0077] Comparative Example 3

[0078] The preparation method is the same as that of Example 1, except that BITC is not added in step S2.

[0079] The details are as follows:

[0080] S2. Preparation of oil phase: prepare a corn oil solution with a volume ratio of 1:9 to the water phase.

[0081] Comparative Example 4

[0082] The preparation method is consistent with that in Example 1, except that FSG and PEC are not added in step S1.

[0083] The details are as follows:

[0084] S1. Preparation of water phase: Take an aqueous solution with a volume ratio of 9:1 to the oil phase.

[0085] Example 3: Application of BITC-loaded FSG and PEC composite emulsion in regulating blood sugar

[0086] Test method:

[0087] Diabetic mouse modeling: After one week of adaptive feeding of basal diet and 5 weeks of high-fat and high-sugar diet, C57BL / 6 male mice were intraperitoneally injected with streptozotocin (STZ) solution at a dose of 55 mg / kg for 4 consecutive days according to their body weight, and their fasting blood glucose levels were measured using a blood glucose meter. The model was successfully established for mice with FBG values ​​≥11.1 mmol / L for two consecutive times, and they could be used as T2DM model mice.

[0088] Grouping and administration: The mice with successful modeling were randomly divided into a model group, a positive drug group, an example 1 group, an example 2 group, a comparative example 1 group, a comparative example 2 group, a comparative example 3 group, and a comparative example 4 group, with 10 mice in each group. The examples 1 to 2 groups and the comparative examples 1 to 4 groups were given 1 mL of the corresponding prepared emulsion, and the model group was given the same dose of normal saline. In addition, 10 normal mice were selected as the normal group and given the same amount of normal saline. The positive drug group was given 1 mL of metformin at 200 mg / kg of mouse body weight, gavage once a day for 30 days.

[0089] FBG measurement: Before each measurement, mice were fasted for 6 hours without food or water, and then their tails were cut to collect blood sugar. The blood sugar level was measured by a blood glucose meter. The fasting blood sugar was measured before intervention, which was the blood sugar level on the 0th day of intervention. After intervention, the fasting blood sugar was measured once every 5 days to investigate the effects of different gavage groups on the blood sugar level of diabetic mice.

[0090] OGTT determination: After 30 days of administration, mice in each group were fasted for 12 h but not water, and glucose solution (2 g / kg) was given by oral gavage. The blood glucose of mice was measured at 0, 30, 60, 90, and 120 min after gavage, and the OGTT curve was drawn, and the area under the blood glucose curve (AUC) was calculated.

[0091] Serum biochemical index detection: HDL-C, LDL-C, TC, TG and INS in serum were determined by enzyme-linked immunosorbent assay according to the kit method.

[0092] HOMA-IR=[FBG(mmol / L)×INS(mIU / L)]÷22.5

[0093] Where: 22.5 - Correction factor

[0094] Serum liver function and renal function determination: The liver function index ALT and trough AST, CRE and BUN in serum were determined according to the kit method.

[0095] Determination of related indicators in the liver: Oxidative stress indicators: The contents of CAT, MDA, SOD and GSH-Px in the liver were determined according to the kit method.

[0096] Levels of inflammatory factors: Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 in liver tissue homogenate, and all procedures were determined according to the kit method.

[0097] Liver and pancreas slice observation:

[0098] The liver and pancreas were separated and rinsed, fixed in 4% paraformaldehyde solution for 48 hours, dehydrated with alcohol, embedded in paraffin, and sliced ​​after solidification, stained with hematoxylin and eosin, and finally dehydrated and sealed. The pathological morphological changes of the liver and pancreas sections stained with hematoxylin-eosin (HE) were observed under an optical microscope.

[0099] Result analysis:

[0100] The present invention uses FSG and PEC to construct a composite emulsion and simultaneously embeds BITC to regulate blood sugar. The study found that:

[0101] Water intake and food intake of mice: Eating and drinking more is one of the characteristics of diabetes. Because glucose cannot be fully utilized, the sugar absorbed in the blood can only be excreted in the form of urine, and the body will be in a semi-starved state for a long time, which leads to eating and drinking more. Figure 1 As shown, the average daily food intake of mice in the model group was 1.46±0.05% times that of the normal group, showing obvious symptoms of polyphagia. The average daily food intake of the positive drug group, Examples 1-2, and Comparative Examples 1-4 was 0.81±0.04%, 0.84±0.01%, 0.89±0.01%, 0.85±0.04%, 0.88±0.02%, 0.91±0.02% and 0.90±0.05% times that of the model group, respectively. Figure 2 It can be seen that the amount of water consumed by the mice in the model group was significantly higher than that in the mice in the normal group, indicating that the diabetic mice had polydipsia. The average daily water consumption of the positive drug group, Example 1-2 groups, and Comparative Example 1-4 groups was 0.54±0.02%, 0.63±0.04%, 0.73±0.04%, 0.68±0.06%, 0.71±0.01%, 0.85±0.09% and 0.79±0.03% times that of the model group, respectively. The above results show that the positive group and Example 1 group significantly alleviated the occurrence of polyphagia and polydipsia in diabetic mice, and the comparative example 3 group had little effect on relieving the polyphagia and polydipsia in diabetic mice.

[0102] Weight changes in mice: Diabetes can hinder the interaction between insulin and its receptors, affecting the normal entry of glucose into cells, and then causing the body to break down its own fat and protein when it lacks energy, resulting in weight loss. Figure 3 As shown, at day 0, there was little difference in body weight between different groups of T2DM mice, and the body weight of mice in the model group showed a continuous downward trend over time. After 30 days, the body weight of the model group was 0.87±0.07% times its initial body weight, while the normal group, positive drug group, Example 1 group, and Comparative Examples 1 to 2 were 1.24±0.10%, 1.12±0.09%, 1.09±0.07%, 1.07±0.07% and 1.04±0.06% times their initial body weight, respectively. Although the body weight of Comparative Example 3 group increased, the degree was small. The results show that, except for the positive group, the emulsions of Example 1 to 2 groups and Comparative Example 1 to 2 groups improve the weight loss symptoms of diabetes by increasing the body weight of T2DM mice.

[0103] FBG: It is the most commonly used indicator to reflect abnormal glucose metabolism and normal function of pancreatic β cells. Figure 4 It can be seen that there was no significant difference in the fasting blood glucose values ​​of all groups of T2DM mice on the 0th day (P>0.05). After 30 days of intragastric administration, there was no significant change in the blood glucose value of the normal group, and the model group increased by 37.93±0.20% compared with its initial blood glucose value. The blood glucose of the positive drug group, the embodiment 1 group, the comparative example 1 group and the comparative example 2 group decreased by 41.37±1.12%, 26.13±5.98%, 20.00±3.64% and 14.95±0.64% respectively compared with their initial blood glucose values, and the blood glucose of the comparative example 3 group and the comparative example 4 group decreased by 4.83±1.71% and 6.37±2.08% respectively compared with their initial blood glucose values. Except for the positive group, the ability to regulate blood glucose was the strongest after long-term intragastric administration of the embodiment 1 group, and this ability was proportional to the BITC content. The higher the BITC content, the stronger its ability to regulate blood glucose; the ability to regulate blood glucose was the weakest after long-term intragastric administration of the comparative example 3 group, indicating that the regulation of blood glucose by the wall material alone was negligible.

[0104] INS and HOMA-IR: INS is a hormone secreted by pancreatic β cells in the pancreas that regulates blood sugar and plays an important role in regulating blood sugar in the body. INS regulates blood sugar by promoting the uptake and utilization of glucose by tissues and cells in the body and inhibiting glycogenolysis and gluconeogenesis. HOMA-IR means that insulin cannot effectively promote the uptake of glucose by peripheral tissues or inhibit the output of glucose by the liver, thus causing hyperglycemia. Figure 5It can be seen that compared with the normal group, the insulin level of mice in the model group was 2.87±0.72% times (P<0.01). The insulin levels of the positive drug group, Example 1-2 groups, and Comparative Example 1-4 groups were 0.43±0.06%, 0.46±0.9%, 0.70±0.06%, 0.51±0.05%, 0.62±0.03%, 0.91±0.02% and 0.80±0.09% times that of the model group, respectively. Among them, Example 1 group reduced the serum insulin level of mice closer to that of the positive group, and the reduction effect of Comparative Example 3 group was not significantly different from that of the model group. Figure 6 It can be seen that compared with the model group, the Example 1 group and the positive drug group significantly reduced the insulin resistance index of mice (P<0.01).

[0105] OGTT and AUC: OGTT is a method to evaluate the body's blood sugar regulation and pancreatic beta cell function, and is often used as a representative indicator for the diagnosis of diabetes. Figure 7 The OGTT changes of mice in different groups are shown. 30 minutes after gavage, the blood glucose of all groups of mice reached the highest value. The blood glucose values ​​of mice in the normal group tended to be normal within 90 minutes. The model mice had poor glucose tolerance. After oral administration of glucose solution, the blood glucose value rose rapidly to 33.3mmol / L, and remained above the initial value after 120 minutes. The change trend of Example 1 group was similar to that of the model group. After 120 minutes, the blood glucose value decreased significantly and was closer to its initial value. In order to better understand its change trend, the AUC ( Figure 8 ), compared with the model group, the AUC of the positive group, Example 1-2 groups, and Comparison 1-4 groups were reduced by 48.28±3.66%, 37.41±3.67%, 14.93±2.13%, 30.90±3.39%, 23.65±3.79%, 5.97±1.79% and 9.67±4.00%, respectively. Among them, only the comparative example 3 group had no significant difference with the model group. In summary, the emulsion in which BITC was embedded with an emulsifier significantly improved the OGTT and AUC of T2DM mice, while the wall material alone could not improve the OGTT and AUC of T2DM mice.

[0106] Serum biochemical indicators: Dyslipidemia is the core feature of metabolic syndrome and an important risk factor for cardiovascular and cerebrovascular diseases such as atherosclerosis, stroke and diabetes. Excessive levels of TC, TG and LDL-C are closely related to the formation of atherosclerosis and are the main factors that increase the risk of diabetes. HDL-C is regarded as a lipoprotein with anti-atherosclerotic effects. It transports cholesterol to the liver for metabolic clearance and is a protective factor for blood sugar health. In order to evaluate the lipid metabolism regulatory effect of BITC embedded in FSG-PEC emulsion on T2DM mice, the levels of four blood lipids in their serum were measured.

[0107] like Figures 9 to 12 The distribution of TC, TG, HDL-C and LDL-C in the serum of each group of mice can be known. The levels of TC, TG and LDL-C in the model group mice were significantly higher than those in the normal group (P<0.01), which were 1.37±0.12%, 2.16±0.21% and 1.71±0.04% times that of the normal group, respectively, while the HDL-C level of the model group mice was 0.66±0.01% times that of the normal group, which indicated that after the combined induction of diet and STZ, the model group mice showed obvious dyslipidemia, which are typical symptoms of diabetes. After 30 days of intragastric intervention, in the positive drug group, Example 1 group, and Comparative Examples 1-2 groups, TC was 0.75±0.05%, 0.77±0.13%, 0.82±0.05% and 0.84±0.09% times that of the model group, respectively; TG was 0.61±0.03%, 0.69±0.06%, 0.75±0.08% and 0.82±0.07% times that of the model group, respectively; LDL-C was 0.64±0.05%, 0.69±0.13%, 0.73±0.09% and 0.79±0.11% times that of the model group, respectively; HDL-C was 1.46±0.15%, 1.41±0.08%, 1.32±0.06% and 1.21±0.15% times that of the model group, respectively. There was no significant difference in TC, TG, LDL-C and HDL-C between the Comparative Examples 3-4 groups and the model group. In summary, the emulsion with higher BITC content has stronger ability to improve lipid metabolism disorder in T2DM mice, and the effect of Example 1 group is the most obvious except for the positive drug group; the gavage group using wall material and BITC embedded in no emulsifier has no improvement effect.

[0108] Serum liver function and kidney function: Liver function is an important indicator for measuring diabetes. Abnormal liver function will cause the release of ALT and AST in cells and then enter the blood circulation, leading to increased ALT and AST activity in serum. Therefore, testing the activity of ALT and AST in serum can assess the degree of liver damage and thus judge the degree of relief of diabetes. Figures 13-14As shown, the ALT and AST activities of the model group mice were 25.34±2.13U / L and 34.71±3.32U / L, respectively, which were 170.10±20.53% and 149.92±16.04% (P<0.01) of the ALT and AST activities of the normal group mice, respectively, indicating that the liver of the T2DM mice had severe liver damage. After 30 days of oral gavage treatment, the ALT activity of the positive drug group and the Example 1 group decreased by 54.17±3.41% and 43.69±11.67% compared with the model group, and the AST activity decreased by 51.51±6.77% and 44.72±7.81%. Among them, the embedded BITC content in all emulsion oral gavage groups showed a dose-effect relationship, and the Example 1 group had a better ability to inhibit the ALT and AST activities of T2DM mice; the comparative example 3 group had no significant difference in its ability to inhibit the ALT and AST activities compared with the model group. Long-term high blood sugar levels may increase the burden on the kidneys, thereby damaging the kidneys and leading to renal dysfunction. BUN and CRE in serum are important indicators for detecting the severity of renal damage. The BUN content is determined by the balance between urea production and renal excretion. When renal function is impaired, the BUN content in serum increases significantly. CRE is mainly excreted from the body through the kidneys, and when renal function is impaired, its content will increase. Fig.15 and 16 The BUN and CRE concentrations in the serum of mice were 164.96±37.81% and 54.95±13.87% higher than those in the normal group, respectively, indicating that diabetic mice had severe renal damage. After 30 days of intragastric administration, the BUN values ​​of the positive drug group and the Example 1 group decreased by 39.53±1.24% and 25.17±13.17%, respectively, and the CRE values ​​decreased by 32.84±7.55% and 28.51±0.11%, respectively. The BUN and CRE values ​​of the comparative example 3 group were not significantly different from those of the model group. This shows that the composite emulsion encapsulating BITC can partially alleviate the renal damage phenomenon in T2DM mice, the positive drug group and the Example 1 group can alleviate the liver damage and kidney damage caused by diabetes, and the comparative example 3 group cannot alleviate the liver damage and kidney damage caused by diabetes.

[0109] Oxidative stress: When the body is in a state of high blood sugar for a long time, excessive free radicals will be produced, which will cause oxidative stress and weaken the body's antioxidant function. MDA is a substance generated by the peroxidation reaction of free radicals and lipids. Its content can be used to indicate the degree of attack of oxygen free radicals on the body. Antioxidant enzymes such as CAT, SOD and GSH-Px have the ability to remove free radicals in the body. Their high content helps to reduce the damage caused by oxidative stress to cells and tissues. Figures 17-20As shown, the activities of CAT, SOD and GSH-Px in the model group were 0.56±0.11%, 0.69±0.01% and 0.52±0.06% times that of the normal group, while the MDA content was 2.02±0.31% times that of the normal group. This phenomenon indicates that diabetes causes oxidative stress in the body, which in turn causes oxidative damage. After 30 days of intragastric administration, compared with the model group, in the positive drug group, Example 1 group and Comparative Example 1 group, the CAT contents were 1.46±0.22%, 1.41±0.18% and 1.29±0.21% times, respectively, the SOD activities were 1.33±0.13%, 1.25±0.09% and 1.20±0.04% times, respectively, the GSH-Px activities were 1.81±0.25%, 1.54±0.12% and 1.45±0.11% times, respectively, and the MDA contents were 0.61±0.13%, 0.67±0.08% and 0.72±0.08% times, respectively, of the model group. There were no significant differences in the MDA contents, CAT, SOD and GSH-Px activities of the Comparative Examples 3 to 4 groups compared with the model group. The composite emulsion with high BITC content (Example 1 group) and the positive drug group have a regulating effect on the oxidative stress level of the liver of T2DM mice, while the composite emulsion with low BITC embedding rate (Example 2 group) has a relatively low effect on the oxidative stress of the liver of T2DM mice. In summary, the use of composite emulsifiers to embed BITC can enhance the antioxidant capacity of the body of T2DM mice, reduce free radicals in the body, and improve the antioxidant capacity of the body. However, the BITC with a single wall material and without emulsifier embedding has no effect on the antioxidant capacity of mice.

[0110] Liver and pancreas sections: HE staining was used to evaluate the effects of different intragastric administration solutions on the liver and pancreas of diabetic mice. Fig.21 As shown: In normal mice, the liver tissue structure is normal, the cell nucleus is not atrophic, the nucleus is evenly distributed, and the liver cells are arranged in an orderly and regular manner. The liver cell morphology of the model group mice deteriorated seriously, the cell wall became thinner, fusion and fragmentation occurred, the liver nucleus was irregular in shape, and inflammatory cells infiltrated. After 30 days of intervention in the positive drug group and Example 1 group, the liver tissue damage of the mice was significantly improved.

[0111] Pancreas HE staining Fig. 22As shown, the pancreatic islets in the normal group were normal in morphology, uniform in size, intact in structure, and clearly visible. Compared with the normal group, the pancreatic islets in the model group mice were severely damaged, shrunken in size, irregular in morphology, blurred in boundaries, and the number of cells in the pancreatic islets decreased. After intervention in the positive drug group, the pancreatic islet cells were significantly enlarged, intact in structure, and with clear boundaries. After 30 days of intragastric administration of the composite emulsion embedded with BITC, the number, morphology, and size of the pancreatic islet cells were improved, and the composite emulsion embedded with BITC had a dose-dependent effect on the repair of the pancreatic islets. Among them, the pancreatic islet tissue boundaries of the Example 1 group with a higher BITC content were clearer, and the repair was more thorough, and the effect was comparable to that of the positive drug group.

[0112] Inflammatory factors: Since long-term high-fat diet may cause liver inflammation, in order to explore the effect of BITC in the emulsion on liver inflammation, the liver inflammation indicators of mice were measured. This experiment evaluated the effect of BITC on the inflammation level of T2DM mice by measuring the content of inflammatory factors (TNF-α, IL-1β and IL-6) in the serum of each group of mice. Figures 23 to 25 The levels of TNF-α, IL-1β and IL-6 in the model group mice were 111.73±4.18 ng / L, 25.71±2.34 ng / L and 18.46±0.63 ng / L, respectively, which were significantly higher than those in the normal group (P<0.01), indicating that the levels of inflammatory factors in T2DM mice were higher than those in healthy mice. Compared with the model group, the TNF-α content of the mice in the positive drug group and the Example 1 group decreased by 20.84±6.92% and 15.66±4.17%, IL-1β decreased by 30.42±6.28% and 25.82±14.20%, and IL-6 decreased by 39.23±7.48% and 28.03±8.56%, respectively, which were significantly lower than the model group (P<0.05). The TNF-α content of the Example 2 group and the Comparative Examples 1 to 4 groups was not significantly different from that of the model group, and the IL-1β and IL-6 of the Comparative Examples 3 to 4 were not significantly different from those of the model group. From the above results, it can be seen that the composite emulsion using an emulsifier to embed BITC can effectively inhibit the increase of TNF-α, IL-1β and IL-6 content, thereby reducing the inflammatory response of T2DM mice caused by diabetes and improving their liver damage.

[0113] The above results indicate that a T2DM mouse model was established by inducing C57BL / 6 mice with high fat and STZ, and the in vivo blood glucose-alleviating effect of the composite emulsion delivery system of FSG-PEC-encapsulated BITC was evaluated. Compared with the BITC emulsions with a single emulsifier or without emulsifier embedding (Comparative Examples 1, 2, and 4), the composite emulsions with BITC embedded by emulsifier (Examples 1 to 2) significantly improved the polydipsia and polyphagia of diabetic mice, while the group using a single wall material for gavage (Comparative Example 3) did not significantly improve the polydipsia and polyphagia of diabetic mice; Example 1 and Example 2 groups could reduce INS, FBG and HOMA-IR of diabetic mice, and significantly improve OGTT of mice; reduce serum TC, TG and LDL-C levels, and increase HDL-C content; reduce ALT, AST content and BUN, CRE content of diabetic mice; increase CAT activity, SOD and GSH-Px content in the liver, reduce MDA content in the liver, reduce oxidative stress damage to the liver, improve liver antioxidant capacity, significantly improve liver tissue damage and pancreatic islet damage, and relieve inflammation: Comparative Examples 1 to 2 groups also improved the above indicators, but the effect was poor; Comparative Example 4 group had little effect on the above indicators; Comparative Example 3 group had basically no improvement effect on the above indicators. In summary, the emulsion using FSG and PEC to encapsulate BITC is optimal for improving diabetes.

[0114] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of fish skin gelatin and pectin composite emulsion loaded with benzyl isothiocyanate in the preparation of hypoglycemic products.

2. The use according to claim 1, characterized in that: The product is food, medicine or health product.

3. The use according to claim 1, characterized in that: The method for preparing the fish skin gelatin and pectin composite emulsion loaded with benzyl isothiocyanate comprises the following steps: (1) Preparation of an aqueous phase: mixing a fish skin gelatin solution, a pectin solution and a certain amount of water to prepare an aqueous phase; wherein the mass ratio of the fish skin gelatin to the pectin in the aqueous phase is 16:1 to 1:6; (2) Preparation of oil phase: dissolving benzyl isothiocyanate in edible oil to prepare an oil phase; the concentration of benzyl isothiocyanate in the oil phase is 2 to 10 mg / mL; (3) Preparation of composite emulsion: The aqueous phase prepared in step (1) is mixed with the oil phase prepared in step (2) at a volume ratio of the oil phase to the aqueous phase of 1:4 to 1:15, and a high-speed dispersion treatment is performed, followed by homogenization to obtain a composite emulsion of fish skin gelatin and pectin loaded with benzyl isothiocyanate.

4. The use according to claim 3, characterized in that: In step (1), the mass concentration of fish skin gelatin in the aqueous phase is 1-2%.

5. The use according to claim 3, characterized in that: In step (1), the mass concentration of pectin in the aqueous phase is 0.5-1%.

6. The use according to claim 3, characterized in that: In step (1), the mass ratio of fish skin gelatin to pectin in the aqueous phase is 2:

1.

7. The use according to claim 3, characterized in that: In step (1), the pH of the aqueous phase is also adjusted to 7.

8. The use according to claim 3, characterized in that: In step (2), the edible oil is corn oil.

9. The use according to claim 3, characterized in that: In step (3), the volume ratio of the oil phase to the water phase is 1:

9.

10. The use according to claim 3, characterized in that: In step (3), the conditions for high-speed dispersion are: 8000-15000 r / min, dispersion for 1-3 min; the conditions for homogenization are: 7000-14000 psi, homogenization for 4-8 times.

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