CN111568948A

CN111568948A - Application of mulberry extract in preparing medicine for improving pancreatic islet function

Info

Publication number: CN111568948A
Application number: CN202010492849.0A
Authority: CN
Inventors: 刘玉玲; 申竹芳; 金毅群; 刘率男; 刘志华; 朱向阳; 刘泉; 李彩娜
Original assignee: Guangxi Wuhe Boao Pharmaceutical Co ltd; Beijing Wehand Bio Pharmaceutical Co ltd
Current assignee: Guangxi Wuhe Boao Pharmaceutical Co ltd; Beijing Wehand Bio Pharmaceutical Co ltd
Priority date: 2020-06-03
Filing date: 2020-06-03
Publication date: 2020-08-25

Abstract

The invention relates to application of a mulberry extract in preparing a medicament for improving pancreatic islet function, which is characterized in that the mulberry extract is prepared according to the following steps: step 1): preparing a mulberry crude extract; step 2): separation on a cationic resin and/or optionally an anionic resin; step 3): carrying out alcohol precipitation treatment on the resin effluent liquid in the step 2); optional step 4) concentration and drying treatment. The improvement of the pancreatic islet function comprises the regulation of the expression ratio of insulin to glucagon and the promotion of GLP-1 secretion.

Description

Application of mulberry extract in preparing medicine for improving pancreatic islet function

Technical Field

The invention relates to application of mulberry extract in preparing a medicament for improving pancreatic islet function.

Background

The function of the pancreatic islets refers to the function of the pancreatic islets to secrete hormones such as insulin and glucagon. Insulin is the only hormone in the body which reduces blood sugar, and is also the only hormone which promotes the synthesis of glycogen, fat and protein, and is secreted by pancreatic islet beta cells. Glucagon is secreted by islet alpha cells, and has strong effects of promoting glycogenolysis and gluconeogenesis, and can raise blood sugar obviously.

Studies show that the abnormal function of islet alpha cells persists in the occurrence and progression of type 2 diabetes, and the secretory function of islet beta cells declines progressively with the progression of the disease course. In addition, the islet cells of normal mice are arranged regularly, insulin is uniformly distributed in the islets, and glucagon is mainly distributed at the edges of the islets; and the pancreatic islet cell arrangement of the KKAy diabetic mouse is disordered, the cells are degenerated, the distribution of insulin and glucagon is disordered, the secretion ratio is unbalanced, and the pancreatic islet function is seriously damaged.

Due to the large side effects of chemical drugs, more and more research is being undertaken to develop insulinotropic agents from natural plants. Liudidi and the like prove that the pine nut dilute alkali extract of the Korean pine can obviously improve the oral glucose tolerance, the serum insulin level and the insulin secretion index of a Streptozotocin (STZ) -induced type 2 diabetes mouse model, thereby improving the pancreatic islet function and achieving better anti-diabetes effect (Liudidi and the like; pine nut extract of the Korean pine nut has hypoglycemic activity on diabetes mice [ J ] Chinese food academy of China.2019); fengxinhuan finds that the red date water extract can obviously improve hyperglycemia symptoms of a diabetes mouse model induced by STZ, promote serum insulin secretion and improve pancreatic islet function (the improvement effect of the Xinfenghuan red date water extract on the blood sugar of the diabetes mouse [ J ] food technology.2019); the humifuse euphorbia water extract can obviously improve the damage degree of islet cells of a diabetes mouse induced by alloxan, promote insulin secretion and reduce blood sugar (the effect of the humifuse euphorbia water extract on reducing blood sugar of the diabetes mouse [ J ] Nanchang university report 2014).

Glucagon-like peptide-1 (GLP-1) 1 is one of the secretes of secretin from L cells of the intestinal tract. After eating, nutrients (such as glucose, amino acids, fatty acids, etc.) can promote the expression and secretion of GLP-1, so that the GLP-1 can be combined with receptors on pancreatic islet beta cells, and glucose-dependent insulin secretion is promoted, thereby playing a role in regulating blood sugar balance. In addition, GLP-1 can induce the regeneration and differentiation of beta cells, inhibit the apoptosis of the beta cells and protect the function of pancreatic islets from being damaged. Studies have shown that continuous subcutaneous administration of GLP-1 to type 2 diabetic patients not only reduces blood glucose levels, but also improves both first and second phase insulin secretion. GLP-1 plays a role in protecting the function of pancreatic islets mainly from the following aspects: (1) after being combined with a specific receptor on an islet beta cell membrane, the insulin release is promoted, and the transcription of a proinsulin gene is induced to promote the synthesis of insulin; (2) inhibiting alpha cell from secreting glucagon, and making the ratio of insulin to glucagon accord with physiological state; (3) inducing the regeneration and differentiation of beta cells and inhibiting the apoptosis of the beta cells; (4) stimulates the endothelial cells of the pancreatic ducts to be transformed into insulin-like cells and plays a positive role in regulating the number and volume of islet cells and the like.

Because natural medicines have incomparable safety and effectiveness compared with chemical medicines, researches on promoting GLP-1 secretion of traditional Chinese medicines or compositions thereof exist at present, for example, the researches show that the composition of coptis chinensis extract and probiotics can effectively promote GLP-1 secretion of cells (Huimen capsule. medicine for stimulating glucagon-like peptide-1 secretion. ZL 201110054219.6); a Chinese medicine composition prepared from scutellaria root, pueraria root and coptis root in a certain proportion can promote synthesis of intestinal tract L cells and secretion of GLP-1 by activating bitter receptors and downstream channels of the intestinal tract L cells (Chinese medicine composition for promoting secretion of glucagon-like peptide-1 and preparation method thereof, CN 201610430442.9).

In conclusion, insulin and GLP-1 are important for maintaining the normal function of pancreatic islets. Although there have been reports on studies on natural active ingredients for treating diabetes and complications thereof by promoting the secretion of insulin and GLP-1, respectively, there are few studies on this aspect as a whole, especially few drugs capable of improving pancreatic islet function by simultaneously regulating the secretion of multiple hormones, and there is still a need to develop safer and more effective drugs for improving the impaired pancreatic islet function of diabetic patients.

Disclosure of Invention

In order to solve the problems, the invention provides application of a mulberry extract in preparing a medicament for improving pancreatic islet function. Specifically, the invention provides application of a mulberry extract in improving pancreatic islet function, which is characterized in that the mulberry extract is prepared according to the following steps: step 1): preparing a mulberry crude extract; step 2): separation on a cationic resin and/or optionally an anionic resin; step 3): carrying out alcohol precipitation treatment on the resin effluent liquid in the step 2); optional step 4) concentration and drying treatment.

In one embodiment of the present invention, the mulberry extract comprises alkaloids, polysaccharides, flavones and amino acids. Preferably, the alkaloids comprise DNJ (1-deoxynojirimycin ) and FAG (Fagomine).

In one embodiment of the present invention, the mulberry extract improves pancreatic islet function by regulating the rate and location of hormone expression in pancreatic islets. Further, the mulberry extract improves pancreatic islet function by promoting insulin secretion in pancreatic islets and inhibiting glucagon secretion. Further, the mulberry extract improves pancreatic islet function by increasing one-phase insulin secretion ability. Research shows that islet cells of a normal mouse are arranged regularly, insulin is uniformly distributed in islets, glucagon is mainly distributed at the edges of the islets, and the secretion levels of the insulin and the glucagon are in a balanced state; and the pancreatic islet cell arrangement of the KKAy diabetic mouse is disordered, the cells are degenerated, the distribution of insulin and glucagon is disordered, the expression level of insulin is reduced, the expression of glucagon is increased, and the function of pancreatic islets is seriously damaged. The mulberry extract can restore the ratio of insulin to glucagon of a diabetic mouse to a normal physiological state, thereby improving the function of pancreatic islets.

In one embodiment of the invention, the mulberry extract improves pancreatic islet function by promoting intestinal GLP-1 secretion. GLP-1 mainly realizes the regulation effect on sugar metabolism by promoting insulin secretion, promoting beta cell proliferation and inhibiting apoptosis. The mulberry extract can be used as GLP-1 secretagogue, and plays roles of inducing insulin secretion, protecting the structure and function of islet cells, improving glycolipid metabolism and the like.

In one embodiment of the present invention, the mulberry extract acts on animals, which are mammals, including humans and rodents. Preferably, the animal glycolipid metabolism is abnormal. More preferably, the animal is a human or a mouse.

In one embodiment of the present invention, the medicament further comprises a pharmaceutically acceptable carrier. The carrier is an inactive component which has no toxic action on human body and accords with the medication route or the administration mode. The carrier may be a solid or liquid vehicle. Solid excipients, for example, include microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, crospovidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, soybean oil, medium chain oil, cholesterol, soy lecithin, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch, and talc; liquid excipients include, for example, water, ethanol, syrup, and glycerol.

In one embodiment of the invention, the medicament is administered orally. Preferably, the pharmaceutical is in a dosage form selected from the group consisting of capsules, tablets, oral solutions, oral emulsions, pills, granules, syrups and powders.

Drawings

FIG. 1 isA graph showing the effect of mulberry extract SZ-A on the level of blood insulin secretion in the high glucose clamp of KKAY mice after long-term administration. DM, a model group; SZ-A400, 400mg/kg SZ-A treatment group.

FIG. 2 isA graph showing the effect of dual markers of insulin (red) and glucagon (green) immunity on pancreatic tissue sections from KKAY mice after chronic administration of mulberry extract SZ-A. WT, wild type control group; DM, a model group; SZ-A400, 400mg/kg SZ-A treatment group.

FIG. 3 is the effect of mulberry extract SZ-A on glucose-stimulated insulin secretion function of islet cell lines (A) the effect of SZ-A on Min6 cell basis and on glucose-stimulated insulin secretion (B) the effect of different concentrations of SZ-A on high-glucose-stimulated insulin secretion by NIT-1 cells (C) the effect of the mulberry extract SZ-A and its major components on high-glucose-stimulated insulin secretion by NIT-1 cells (P # P <0.01, compared to the Vehicle group at 2.8 mM), P <0.05, P <0.01, P <0.001, compared to the Vehicle group. Vehicle, blank control group; SZ-A50/100/200, 50/100/200 μ g/mL SZ-A treatment group; DNJ-40, 40. mu.g/mL DNJ treated group; FAG-20, 20. mu.g/mL FAG treated group; DAB-10, 10. mu.g/mL DAB treatment group.

FIG. 4 isA graph of the effect of mulberry extract SZ-A on fasting and post-oral glucose-stimulated (A) blood glucose, (B) serum insulin, and (C) active GLP-1 levels in KKAY mice 5 weeks after administration. DM, control group; SZ-A200/400, 200 or 400mg/kgSZ-A treatment group.

Detailed Description

The following examples and test examples are illustrative only and are not to be construed as limiting the invention. In addition, the technical features related to the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The contents of the components involved in the present invention were measured according to a known method (see methods described in patent publication Nos. CN111077247A and CN 110393738A).

Preparation example 1

Pulverizing fresh ramulus Mori (11 # Yue Mulberry), adding 4000L water, extracting for 2 hr under reflux, mixing extractive solutions, and filtering to remove insoluble substances to obtain crude extractive solution. And (3) carrying out heat concentration on the crude extract until the solid content reaches 4%, and using the crude extract as a sample loading solution of a cation resin column.

Loading 150kg of D113 type macroporous weakly acidic styrene series cationic resin into a column, and washing with 2mol/L hydrochloric acid solution until the pH of eluate is 4.5; washing with 1mol/L sodium hydroxide solution until the pH of the eluate is 8.5; washing with 2mol/L hydrochloric acid solution until the pH of an eluate is 4.5; and then washed with 5 column volumes of deionized water to complete the activation. And (3) loading the concentrated extracting solution, eluting by using 1000L of 2.5mol/L ammonia water at the elution speed of 6BV/h, collecting the eluent when the pH of the effluent of the cation column is detected to be more than 7, stopping collecting when the collected liquid reaches 900L, and directly purifying the collected liquid by using an anion column.

Loading 400kg of D218 macroporous strongly basic acrylic acid series anionic resin into a column, and washing by using 1.5mol/L sodium hydroxide solution until the pH of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; and (4) completing activation. And loading the collected cation resin eluent to anion resin, and collecting the effluent until the effluent reaches 870L.

And (3) filtering the collected liquid obtained after the anion column separation by using a microfiltration membrane to remove impurities, concentrating by using a counter ion permeable membrane, transferring the concentrated liquid into an alcohol precipitation tank, wherein the specific gravity of the concentrated liquid is 1.1, and adding 15kg of absolute ethyl alcohol into the alcohol precipitation tank at the speed of 400rpm of a stirring paddle. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, collecting supernatant, and concentrating under reduced pressure to obtain ramulus Mori extract. In addition, fresh cortex Mori and folium Mori (Yue Mulberry No. 11) are extracted by the same method and parameters as above.

In the obtained ramulus Mori extract, alkaloid content is 75%, polysaccharide content is 15%, flavone content is 0.7%, and amino acid content is 5%; the alkaloid contains DNJ (1-deoxynojirimycin ), FAG (Fagomine) and DAB (1, 4-dideoxy-1,4-imino-D-arabinitol, 1,4-dideoxy-1, 4-imino-D-arabinitol), wherein the content of DNJ is 72%.

In the obtained extract of cortex Mori extract, alkaloid content is 67%, polysaccharide content is 20%, flavone content is 0.8%, and amino acid content is 6%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 70%.

In the obtained mulberry leaf extract, the content of alkaloid is 50%, the content of polysaccharide is 27%, the content of flavone is 3% and the content of amino acid is 16%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 66%.

Preparation example 2

Pulverizing fresh ramulus Mori (Morus alba L. Ex Fr.2) 10kg, adding 150L water, adding into the pulverized ramulus Mori 2L water, decocting for 3 hr each time, mixing extractive solutions, and filtering to remove insoluble substances. The extract was concentrated by heating until the solid content reached 8%, and transferred to an alcohol precipitation tank, where 2367.9g of absolute ethanol (3L) was added under 300rpm of a stirring paddle. And stopping stirring after the ethanol is added, precipitating with ethanol for 24h, and taking the supernatant as a sample loading solution of the cationic resin column. The cationic resin was activated in the same manner as in example 3 using 002SC type strongly acidic styrene type cationic resin 5kg packed in a column. Sampling the extract subjected to concentration and alcohol precipitation, eluting with 100L of 5mol/L potassium chloride at the elution speed of 5BV/h, detecting the effluent with 20% silicotungstic acid, starting collection when white precipitate is generated, stopping collection when the volume of the collected liquid reaches 25L, and purifying the collected liquid directly through an anion column.

The anion resin was activated in the same manner as in example 3 by using 10kg of type 711 strongly basic styrene anion resin packed in a column. Loading the collected cation resin eluent to anion resin, and collecting the effluent until the effluent reaches

15L ends. The collected solution was re-loaded onto the cationic resin and re-separated twice with the cationic resin and the anionic resin in this order as described above.

And (3) centrifuging the collected liquid obtained after the three-time column separation to remove impurities, concentrating the collected liquid by a counter ion permeable membrane, transferring the concentrated liquid into an alcohol precipitation tank, and adding 125g of absolute ethyl alcohol into the alcohol precipitation tank at the speed of 1000rpm of a stirring paddle. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, collecting supernatant, and concentrating under reduced pressure to obtain extract. In addition, fresh cortex Mori and folium Mori (SANTOU No. 2) are extracted, and the extraction method and parameters are the same as above.

In the obtained ramulus Mori extract, alkaloid content is 98%, polysaccharide content is 0.2%, and flavone content is 0.05%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 99 percent.

In the obtained cortex Mori extract, alkaloid content is 95%, polysaccharide content is 2%, flavone content is 0.1%, and amino acid content is 1%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 96 percent.

In the obtained mulberry leaf extract, the content of alkaloid is 90%, the content of polysaccharide is 4%, the content of flavone is 0.1%, and the content of amino acid is 3%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 91%.

Preparation example 3

Pulverizing fresh Mori fructus (white mulberry fruit) 100g, adding 300ml alcohol water, adding for 2 times, and extracting under reflux each time

And (3) combining the extracting solutions, and filtering to remove insoluble substances to obtain a crude extracting solution. The crude extract is thermally concentrated until the solid content reaches 2%, and is used as a sample loading liquid of a cation resin column at the temperature of 25 ℃.

Column packing with 732 type strongly acidic styrene cation resin 5g, washing with 2.5mol/L hydrochloric acid solution

The pH of the effluent is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 8.0; washing with 2.5mol/L hydrochloric acid solution until the pH of an eluate is 3.5; then, the column was washed with 3 column volumes of deionized water to complete the activation. And (3) loading the concentrated extracting solution, eluting with 3L of 0.1mol/L ammonia water at the elution speed of 10BV/h, collecting the eluent when the pH of the effluent of the cation column is detected to be more than 7, stopping collecting when the collected liquid reaches 1L, and directly purifying the collected liquid through an anion column.

Loading 1.25g of D218 macroporous strongly basic acrylic acid series anion resin into a column, and washing by using 1.5mol/L sodium hydroxide solution until the pH value of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; and (4) completing activation. And (4) loading the collected cation resin eluent to anion resin, and collecting the effluent until the effluent reaches 1L.

And (3) centrifuging the collected liquid obtained after the anion column separation to remove impurities, concentrating the collected liquid through a counter ion permeable membrane, transferring the concentrated liquid into an alcohol precipitation tank, and adding 25g of absolute ethyl alcohol into the alcohol precipitation tank at the speed of 100rpm of a stirring paddle. Ethanol

Stopping stirring after the addition, precipitating with ethanol for 24 hr, collecting supernatant, and vacuum drying to obtain extract. Extracting fresh ramulus Mori, cortex Mori, and folium Mori (white mulberry), with the same extraction method and parameters as above.

In the obtained mulberry extract, the content of alkaloid is 45%, the content of polysaccharide is 28%, the content of flavone is 5%, and the content of amino acid is 20%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 60 percent.

In the obtained ramulus Mori extract, alkaloid content is 48%, polysaccharide content is 25%, flavone content is 4%, and amino acid content is 17%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 62%.

In the obtained cortex Mori extract, alkaloid content is 45%, polysaccharide content is 27%, flavone content is 6%, and amino acid content is 18%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 61%.

In the obtained mulberry leaf extract, the content of alkaloid is 30%, the content of polysaccharide is 34%, the content of flavone is 7% and the content of amino acid is 30%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 55 percent.

Preparation example 4

Pulverizing fresh ramulus Mori (ramulus Mori in Guangdong), adding 11500L water, heating and reflux-extracting for 2 hr, mixing extractive solutions, and filtering to remove insoluble substances to obtain crude extractive solution. The crude extract is centrifuged to remove impurities and then concentrated by a counter ion permeable membrane to solid

The content reaches 1 percent, and the product is used as the sample loading solution of the cation resin column.

150kg of D001 type macroporous strongly acidic styrene cationic resin was packed in a column, and the column was treated in the same manner as in preparation example 1

The cationic resin is activated. And (3) loading the concentrated crude extract, eluting with 5000L of 0.04mol/L ammonium nitrate at an elution speed of 5BV/h, detecting the effluent with 20% silicotungstic acid, starting to collect when a white precipitate is generated, and stopping collecting when the collected liquid reaches 1000L.

And (3) concentrating the collected liquid obtained after the separation of the cation column by using a nanofiltration membrane, transferring the concentrated liquid with the specific weight of 1.3 into an alcohol precipitation tank, and adding 1.7kg of absolute ethyl alcohol into the alcohol precipitation tank under the stirring speed of 600 rpm. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, collecting supernatant, and concentrating under reduced pressure to obtain extract. In addition, fresh cortex Mori and folium Mori (Guangdong mulberry) are extracted by the same method and parameters as above.

In the obtained ramulus Mori extract, alkaloid content is 15%, polysaccharide content is 40%, flavone content is 0.7%, and amino acid content is 40%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 55 percent.

In the obtained cortex Mori extract, alkaloid content is 10%, polysaccharide content is 42%, flavone content is 0.8%, and amino acid content is 41%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 50%.

In the obtained mulberry leaf extract, the content of alkaloid is 8%, the content of polysaccharide is 45%, the content of flavone is 0.6%, and the content of amino acid is 43%; DNJ, FAG and DAB are contained in the alkaloid, wherein the content of DNJ is 49 percent.

Preparation example 5

Taking 333kg of dry ramulus Mori (Yue Mulberry No. 11), pulverizing, adding 4000L of water, extracting by heating reflux method twice, refluxing for 1 hr each time, mixing extractive solutions, filtering, and concentrating the extractive solution to 1kg crude drug amount/L.

Loading 62.5kg of D218 macroporous strongly basic acrylic acid series anion resin into a column, and washing by using 1.5mol/L sodium hydroxide solution until the pH value of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; and (4) completing activation. And loading the collected cation resin eluent to anion resin, and collecting the effluent with the pH value of more than 8 until the effluent reaches 870L.

And (3) filtering the collected liquid obtained after the anion column separation by using a microfiltration membrane to remove impurities, concentrating by using a counter ion permeable membrane, transferring the concentrated liquid into an alcohol precipitation tank, wherein the specific gravity of the concentrated liquid is 1.1, and adding 15kg of absolute ethyl alcohol into the alcohol precipitation tank at the speed of 400rpm of a stirring paddle. Stopping stirring after the ethanol is added, precipitating with ethanol for 24h, collecting supernatant, and concentrating under reduced pressure to obtain ramulus Mori extract. Sample content: the content of alkaloid is 63%, the content of polysaccharide is 23%, the content of flavone is 1%, and the content of amino acid is 5%; the alkaloid contains DNJ, FAG and DAB, wherein the content of DNJ is 39%, the content of FAG is 10.46% and the content of DAB is 7%.

Test example 1

30 female KKAy mice, 8 weeks old, were selected and fed a high calorie diet (Research diet 12451). When the weight of the mice increased above 40 g, the mice were randomized into 3 groups (n = 10/group) with reference to fasting blood glucose and body weight: wild-type KKAy mice (WT) givenA normal diet, KKAy mouse model group (DM) givenA high fat diet, high fat diet and 400mg/kg of the mulberry twig extract SZ-A of preparation example 5. Gavage was performed 2 times a day, and the same dose of distilled water was administered to the WT group and the DM group for 8 weeks. After the administration, the effect of SZ-A400 mg/kg dose group on the glucose-stimulated insulin secretion function of KKAy mice was examined by using the high glucose clamp technique. After the experiment is finished, treating animals, fixing pancreatic tail tissues, embedding the pancreatic tail tissues, then carrying out an insulin and glucagon immunofluorescence labeling experiment, and investigating the influence of an SZ-A400 mg/kg dose group on the pancreatic hormone expression and the pancreatic islet function of a KKAY mouse.

As shown in fig. 1, the high glucose clamp test indicates that the extract of the preparation example after long-term administration can significantly increase the one-phase insulin secretion capacity of KKAy mice and relieve the two-phase compensatory secretion pressure.

As shown in fig. 2, pancreatic immunofluorescence results showed increased and irregular distribution of glucagon expression and decreased insulin expression in diabetic model mice; after the extract of the preparation example is administrated for a long time, the expression amount of insulin is increased and is uniformly distributed in the pancreatic islets; while glucagon expression is reduced and mainly accumulates at the islet borders.

The above results show that the extract of the invention can improve the insulin secretion dysfunction state of KKAy mice, regulate the hormone expression ratio in pancreatic islets and improve the pancreatic islet function.

Test example 2

Min6 cells were selected as test cells, cultured in 2.8 mM and 16.8 mM glucose environments, and added with 100. mu.g/mL SZ-A, respectively, to examine the effect of SZ-A on the insulin secretion level of Min6 cells under basal and high glucose stimulation conditions.

NIT-1 cells were selected as test cells and cultured inA 16.8 mM glucose environment, and SZ-A was added at 50, 100 and 200. mu.g/mL, respectively, to examine the effect of different concentrations of SZ-A on the insulin secretion level of NIT-1 cells under high glucose stimulation.

As shown in FIG. 3, the concentration of SZ-A (100. mu.g/mL) can directly stimulate the insulin secretion level in Min6 and NIT-1 cells under high glucose condition; in NIT-1 cells, the effect of stimulating insulin secretion of SZ-A at the concentration of 50-200 mug/mL has good dose-effect relationship; DNJ and FAG in SZ-A are the main substances that SZ-A stimulates the insulin secretion function of NIT-1 cells.

Test example 3

30 female KKAy mice, 8 weeks old, were selected and fed a high calorie diet (Research diet 12451). When the weight of the mice increased above 40 g, the mice were randomized into 3 groups (n = 10/group) with reference to fasting blood glucose and body weight: KKAY mouse model group (DM) given high fat diet, high fat diet and 200mg/kg of ramulus mori extract SZ-A, high fat diet and 400mg/kg of ramulus mori extract SZ-A. Gavage was performed 2 times daily, and the same dose of distilled water was administered to DM group for 5 weeks.

After 5 weeks of administration, KKAy mice were fasted overnight, fasting blood samples were taken to determine their fasting blood glucose, serum insulin and serum active GLP-1 levels, a mixture of glucose (2 g/kg) and drug solution was administered orally by gavage, and blood samples were taken 15 min thereafter to determine the above-mentioned indices and examine the glucose-stimulated insulin and GLP-1 secretion function.

As shown in FIG. 4, the extract of the preparation example after long-term administration was effective in reducing the blood glucose rise 15 min after oral administration of glucose to KKAY mice, and increasing the levels of serum insulin and blood active GLP-1 after fasting and glucose challenge. This demonstrates that the extract of the present invention can improve pancreatic islet function by promoting the mechanism of insulin and GLP-1 secretion.

The present invention has been described above in connection with preferred embodiments, but these embodiments are merely exemplary and merely illustrative. On the basis of the above, the invention can be subjected to various substitutions and modifications, and the substitutions and the modifications are all within the protection scope of the invention.

Claims

1. The application of the mulberry extract in preparing the medicine for improving the pancreatic islet function is characterized in that the mulberry extract is prepared according to the following steps:

step 1), preparing a crude extract;

step 2), separation on a cationic resin and/or an optional anionic resin;

step 3), carrying out alcohol precipitation treatment on the resin effluent liquid in the step 2);

optional step 4), concentrating and drying.

2. Use according to claim 1, wherein the mulberry extract comprises alkaloids, polysaccharides, flavones and amino acids, preferably wherein the alkaloids comprise DNJ and FAG.

3. The use according to claim 1, wherein the mulberry extract improves pancreatic islet function by regulating the hormone expression ratio in pancreatic islets.

4. The use of claim 3, wherein the mulberry extract improves pancreatic islet function by promoting insulin secretion from pancreatic islets and inhibiting glucagon secretion.

5. The use of claim 4, wherein the mulberry extract improves pancreatic islet function by increasing one-phase insulin secretion.

6. The use according to claim 1, wherein the mulberry extract improves pancreatic islet function by promoting intestinal GLP-1 secretion.

7. The use according to claim 1, wherein the mulberry extract acts on animals.

8. The use of claim 7, wherein the animal is a mammal, including humans and rodents.

9. Use according to claim 7, wherein the animal glycolipid metabolism is abnormal.

10. The use of claim 7, wherein the animal is a human or a mouse.

11. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.

12. Use according to claim 1, wherein the medicament is administered orally, preferably in a form selected from the group consisting of capsules, tablets, oral solutions, oral emulsions, pills, granules, syrups and powders.