CN106420932B

CN106420932B - Composition for increasing insulin sensitivity and application thereof

Info

Publication number: CN106420932B
Application number: CN201610793976.8A
Authority: CN
Inventors: 步世忠; 罗春; 袁燕燕; 邹欣蓉; 刘冰洋
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2016-08-31
Filing date: 2016-08-31
Publication date: 2020-02-18
Anticipated expiration: 2036-08-31
Also published as: CN106420932A

Abstract

The invention discloses a composition for increasing insulin sensitivity, which is characterized by being prepared by mixing and extracting 10-20% of silkworm excrement, 50-55% of white mulberry root-bark and 25-40% of mulberry leaves. The composition of the present invention shows a good effect on increasing insulin sensitivity.

Description

Composition for increasing insulin sensitivity and application thereof

Technical Field

The invention relates to a composition for increasing insulin sensitivity, which is prepared by mixing and extracting 10-20% of silkworm excrement, 50-55% of white mulberry root-bark and 25-40% of mulberry leaves.

Background

The mulberry is a perennial Morus plant of Moraceae, is widely distributed in tropical, subtropical and temperate zones of Asia, Europe, North America, south America, Africa and India, is planted in silkworm breeding bases of Zhejiang and Jiangsu provinces in southeast China, and contains abundant flavonoids. The cortex Mori extract is prepared from dried root bark of Morus genus plant of Moraceae family by picking in winter, cleaning, scraping off brown cork while fresh, separating bark from wood core, stripping off cortex Mori, and sun drying. Several studies have confirmed that cortex mori extracts (mori cortix extract) have various medicinal effects including anti-inflammatory and anti-hepatoma effects, such as inducing apoptosis of cancer cells by inhibiting microtubule assembly and eliminating lung inflammation including bronchitis. Cortex Mori extract has antidepressant effect on hippocampus by bidirectional phosphorylation of glucocorticoid receptor. The mulberry flavone G extracted from cortex Mori can also be used as antibacterial agent of oral cariogenic bacteria.

The main chemical components of cortex Mori include flavonoids, coumarins, polysaccharides, and other compounds. The flavonoids mainly comprise morin, cyclosporine, mulberrin, cyclosporine, morin, hydroxyl dihydro mulberrin, morinone A-V, mulberrin hydroperoxide, mulberrin-4-glucose, cyclosporine, flavanone mulberrin compound A, mulberrin A-D, mulberrin, 5, 7-dihydroxy chromone, Mochalcone A and the like. The coumarin includes scopoletin, 5, 7-hydroxycoumarin, scopoletin (6-methoxy-7-hydroxy-coumarin), and umbelliferone. The polysaccharides include chitosan, chitin, mucin, and mulberry polysaccharide. Other compounds also include mulberry aglycone A, 1-deoxynojirimycin, stilbene glycoside compounds, morin (A, B, C, D, F, G), butanol, glycoside derivatives, tannin, sitosterol, betulinic acid, volatile oil, daucosterol, oleanolic acid, ursolic acid, 3-methoxy-4-hydroxy-benzaldehyde, etc.

The study reports that the ethanol extract of the white mulberry root-bark can inhibit the activity of sucrase of intestinal epithelium and delay the absorption of monosaccharide, thereby reducing the rapid rise of postprandial blood sugar; hikino and other researches find that the white mulberry root-bark has hypoglycemic activity, and the glycoprotein of the moran A as the other component can obviously reduce the blood sugar level of normal mice and diabetes mice induced by alloxan; zhongfeng and the like establish a diabetic rat model by using high-fat diet and intraperitoneal injection of STZ, discuss the blood sugar reducing effect of the white mulberry root-bark total flavonoids, and find that the white mulberry root-bark total flavonoids can reduce the blood sugar and triglyceride level of a type 2 diabetic rat, and have the effects of resisting diabetes and regulating blood fat; the STZ-induced diabetic rats such as Turkey, etc. are subjected to intragastric administration of a white mulberry root-bark water extract to study the influence of the extract on the glycolipid metabolism of the diabetic rats, and the extract is found to have the hypoglycemic effect on experimental diabetic rats but has no influence on blood fat; wangning et al found that the aqueous extract of cortex Mori promoted HepG without the action of insulin₂Depletion of glucose by hepatocytes, low dose insulin-induced HepG₂The sugar uptake of cells has the function of auxiliary enhancement. The masson waves and the like are used for gastric perfusion of diabetes rats induced by alloxan, after a period of administration, the result shows that the gastric lavage of the mulberry bark extract has obvious improvement effect on the neuropathy of the diabetes rats, can increase the area of the cross section around the nerve myelin sheath, relieve edema, reduce the MDA content, increase the SOD activity and relieve the early lesion of the peripheral nerve of the diabetes rats; meanwhile, the cortex mori extract can increase the content of sciatic nerve synapsin, improve or increase the sensory and motor conduction speed of sciatic nerve of rats and shorten the latent period; in addition, the cortex Mori extract can improve the activity of sodium potassium pump enzyme, increase the content of cGMP and cAMP in sciatic nerve, and has certain effect of preventing and treating diabetic neuropathy.

The silkworm excrement is rich in chlorophyll, pectin, flavonoids, crude protein, trace elements necessary for a part of a human body and other effective components, researches on chemical components of silkworm excrement are continuously and deeply recorded in Chinese herbal medicine, the silkworm excrement contains chlorophyll derivatives including pheophytin a and B, 13-hydroxypheophytin a and B, 10-hydroxypheophytin a and the like, Chinese medicine dictionary, the silkworm excrement contains organic matter 83.77-90.44%, ash content 9.56-16.23%, total nitrogen content is 1.91-3.60%, phytol 0.25-0.29%, chlorophyll, free amino acid, unsaponifiable components β -sitosterol, cholesterol, sterol and tetracosanol, lupeol, β -sitosterol- β -glucoside, copper elements, a large amount of carotene and vitamin B, and phytohormone-spertisone, the silkworm excrement contains crude protein 70, crude fat, crude fiber, nitrogen-free protein, a crude protein, a trace element such as phytohormone, a vitamin E, a vitamin B, a vitamin E, a vitamin E, a vitamin E, a.

Since ancient times, the traditional Chinese medicine applies the mulberry leaves as the traditional Chinese medicine for treating diabetes (diabetes in modern medicine) to clinic, and Japanese ancient books (tea eating health preserving records) also record the function of the mulberry leaves for improving the 'drinking water disease' (diabetes in modern medicine). The research data at home and abroad prove that the alkaloid and the polysaccharide are the main blood sugar reducing components in the mulberry leaves.

The hypoglycemic effect of mulberry leaves is realized through two ways, namely, alkaloid DNJ (1-deoxynojirimycin) has an inhibitory effect on the activity of disaccharide lyase so as to inhibit the absorption of disaccharide by small intestine and reduce the high peak value of blood sugar after eating, and mulberry leaf alkaloid fagomine and mulberry leaf polysaccharide promote β cell insulin must, and insulin can promote the utilization of cells to sugar, the synthesis of liver glycogen and the improvement of sugar metabolism, thereby finally achieving the hypoglycemic effect.

In summary, silkworm excrement, cortex mori radicis and folium mori all have the effect of reducing blood sugar, but because the components are complex, it is not clear which component exerts the blood sugar reducing effect through which mechanism. In the course of treating diabetes, then, hypoglycemia is often the complication of treating diabetes, which is harmful to the human body, especially to elderly patients, rather than to hyperglycemia. The major hazards of hypoglycemia are: 1. when the blood sugar is low, in vivo glycemic hormones such as epinephrine, glucocorticoid, glucagon and growth hormone are increased, so that reactive sexual blood sugar (hematoxylin effect) is caused, blood sugar fluctuation is caused, and the disease condition is aggravated; 2. repeated and severe hypoglycemic attacks over a long period of time can cause irreversible damage to the central nervous system, causing personality changes, mental disorders, dementia and the like in patients; 3. hypoglycemia can also stimulate the cardiovascular system and promote arrhythmia, myocardial infarction, cerebral apoplexy, etc.; 4. a hypoglycemic coma may not be detected for a long time and may cause death.

In addition, different extraction methods have great difference on the types of substances extracted from silkworm excrement, cortex mori radicis and folium mori, and although different reports mostly show that the extracts of silkworm excrement, cortex mori radicis and folium mori have a certain blood sugar reducing effect, no report is made on the extracts and extraction methods of the three mixed extracts.

Disclosure of Invention

The present inventors have conducted creative experiments and diligent efforts to find that a novel extract has a significant effect on increasing insulin sensitivity and lowering blood glucose. The invention aims to provide a novel composition for increasing insulin sensitivity, which is prepared by mixing 10-20% of silkworm excrement, 50-55% of white mulberry root-bark and 25-40% of mulberry leaves and then extracting.

The silkworm excrement of the invention refers to silkworm excrement, and mulberry leaves, molt or other impurities mixed in the silkworm excrement during the excrement removal are separated. The cortex mori radicis the dry root bark of Morus alba L. of Moraceae, and can also be the dry substance of the root of a mulberry. The mulberry leaves of the invention are also from leaves of Morus alba L. of Moraceae, collected and dried in the sun for standby.

The above raw materials can be purchased from farmhouse or prepared by oneself, and the above raw materials are dried, pulverized into dry powder. The dry powder is mixed by 10-20% of silkworm excrement, 50-55% of white mulberry root-bark and 25-40% of mulberry leaf.

A method for extracting a composition for increasing insulin sensitivity, comprising the steps of:

a crushing step, namely drying the silkworm excrement, the white mulberry root-bark and the mulberry leaves respectively, and crushing into dry powder;

mixing, namely stirring and uniformly mixing 10-20% of silkworm excrement, 50-55% of white mulberry root-bark and 25-40% of mulberry leaves to obtain a mixture;

leaching, mixing the mixture with an extraction solution, wherein the extraction solution is 1, 3-butanediol with the water content of 40-60%, and placing the mixed solution in a water bath for stirring and extracting, wherein the temperature of the water bath is 80-95 ℃;

a filtering step, filtering the leached mixed solution to obtain a filtrate, and adjusting the pH value to 10 by using NaOH;

a concentration step, concentrating the filtrate under reduced pressure to 20-50% of the original concentration;

ion exchange resin treatment, namely, passing the concentrated solution through membrane treatment or ion exchange resin to obtain a composition stock solution;

and a drying step, drying the composition mixed solution to obtain brown powder.

Drawings

FIG. 1 comparison of total cholesterol TC in serum of rats of various groups.

FIG. 2 comparison of triglyceride TG in serum of rats of each group.

FIG. 3OGTT test blood glucose at 0, 30, 60, 120 min.

FIG. 4 statistics of area under the curve AUC of the OGTT test.

Detailed Description

Example 1

Silkworm excrement, white mulberry root-bark and mulberry leaf are from silkworm farmer in northern area of Zhejiang province, and are washed with deionized water, dried in a 30 deg.c drying box for over night to obtain dry product, and ground into powder or fine grains in a mortar or a grinder. Then 200g of silkworm excrement, 500g of white mulberry root-bark and 300g of mulberry leaf are respectively weighed and uniformly mixed, and the mixture is added into a large flask filled with 5L of extraction solution, wherein the extraction solution is 1, 3-butanediol with the water content of 50%.

The temperature of the water bath was set to 85 ℃ and the mixture was extracted with stirring for 48 hours and then filtered through filter paper (whatman corporation) to obtain 3.72L of a filtrate. The filtrate was adjusted to pH 10 with 1M NaOH solution. The filtrate was concentrated to 800ml at room temperature under reduced pressure using a rotary evaporator. Adsorbing the obtained concentrated solution by using an HPD400A macroporous resin column, wherein the filling amount of the resin column is 5Kg, and the diameter-height ratio is 1: and 4, repeatedly loading the sample for 3 times, and statically adsorbing for 60 minutes.

Eluting the resin column by deionized water with 4 times of column volume; then eluting with 8 times of column volume of 20% alkaline ethanol (NaOH, pH 10); then eluting with 20% ethanol with 3 times of column volume to neutrality; the adsorbed extract on the resin was eluted with 6 column volumes of 60% ethanol.

The eluate was also distilled under reduced pressure to recover ethanol, then vacuum dried, and the dried solid was pulverized and sieved through a 100 mesh sieve to give 102.1g of extract, which was designated as composition a.

Example 2

The extraction was carried out by the method of example 1 using 1,000g of silkworm excrement, mulberry bark and mulberry leaf, to obtain 78.4g of composition B, 104.3g of composition C and 112.8g of composition D, respectively.

EXAMPLE 3 determination of the Effect of the composition in increasing insulin sensitivity

Type II diabetes model production

Rats fed with high-fat feed are fasting for 12 hours, 1% streptozotocin (dissolved by 0.1mol/L sodium citrate buffer solution with pH of 4.2-4.4 and prepared before use) is intraperitoneally injected at one time with the dosage of 30mg/kg, and the standard diet group is synchronized with the sodium citrate buffer solution with the same dosage of intraperitoneal injection. And (3) after 72h, collecting blood from the tail-cutting tip vein of the rat injected with STZ, evaluating with fasting plasma glucose (FBG, mmol/L), and measuring the FBG with a one-touch glucometer, wherein the blood glucose value is more than or equal to 13.8mmol/L as the standard for successful model making of the rat.

2. Composition therapy

The composition A, B, C, D is applied to the diabetes treatment group in the treatment period of 12-20 weeks. The intragastric dose of the extract is 10g/kg d. For control, the remaining rats were also administered an equal volume of saline by gavage.

3. Blood glucose determination

FBG is measured once a week in the same time in each group of animals during the experiment period, rats are fasted for 12 hours without water prohibition before FBG measurement, blood is taken from tail tip veins of the rats the next day, blood glucose concentration is measured by a one-touch glucometer, and relevant records are made.

4. Oral glucose tolerance test

The Oral Glucose Tolerance Test (OGTT) was performed at the end of the treatment period. The blood glucose value of the rat tail tip vein blood sampling with the fasting state of 12h is recorded as the blood glucose value of 0 h. Then, the stomach is irrigated with 50% sugar solution according to the dose of 2.0g/kg, and the tail tip venous blood is taken for measuring the blood sugar at 30min, 60min and 120min respectively. After the experiment was completed, OGTT curves were drawn for 5 groups of rats with time (min) on the abscissa and blood glucose concentration (mmol/L) on the ordinate. And calculating the area under the curve (AUC) to judge the glucose tolerance state.

AUC (h.mmol/L) 1/4 × 0h +1/2 × 0.5h +3/4 × 1h +2 h. (formula 1)

Procedure of experiment

30 male SD rats were fed free diet water in the animal house to adapt to the new environment for 1 week. The test pieces were randomly divided into 6 experimental groups of N (normal group), M (diabetic group), M + A (diabetic treatment group to which the extract A of the present invention was added), M + B (diabetic treatment group to which the extract B of the present invention was added), M + C (diabetic treatment group to which the extract C of the present invention was added), and M + D (diabetic treatment group to which the extract D of the present invention was added), and 5 of them were each group. N groups were fed with a general diet, and M (diabetic group), M + a (diabetic group), M + B (diabetic group), M + C (diabetic group), and M + D (diabetic group) were continuously fed with a high fat phase for 12 weeks, and then 1% STZ (30mg/kg) was intraperitoneally injected to N groups of rats, and an equal amount of citric acid buffer was injected in the same manner to eliminate the influence of variables. And (3) after 72h, collecting blood from the tail-cutting tip vein of the rat injected with STZ, evaluating with fasting plasma glucose (FBG, mmol/L), and measuring the FBG with a one-touch glucometer, wherein the blood glucose value is more than or equal to 13.8mmol/L as the standard for successful model making of the rat. After the molding is successful, the treatment group enters the treatment stage, and the gastric lavage is respectively carried out by using the aqueous solution (10 g/kg. d) of the extract A, B, C, D composition, and the gastric lavage is carried out for 8 weeks. The OGTT test was performed at the end of the experiment at week 20; then, the rat is sacrificed, relevant biochemical detection is carried out on the plasma, the structural morphology condition of the liver is analyzed pathologically, and the gene related to fatty acid synthesis is detected by a Western blot method.

Results of the experiment

1. The extract A has good effect in reducing blood lipid TC (total cholesterol) and TG (triglyceride) levels

As shown in fig. 1 and 2, the TC and TG of the A, B, C, D-treated group were decreased compared to the diabetic rats, but the a-treated group was significantly decreased (P <0.05) to almost the level of normal mice, and thus the a-treated group had a better effect of reducing blood lipids than the B, C, D-treated group.

2. Extract A showed better effect on increasing insulin sensitivity

To assess the glucose tolerance status of rats after the administration of the extract of the invention, an OGTT test was performed at the end of the experiment, as shown in figure 3, the blood glucose profile of the OGTT test at 0, 30, 60, 120 min. The AUC of rats in the diabetic group was significantly higher than that of normal rats (P <0.05), as shown in fig. 4, the AUC of the treated group of the extract A, B, C, D of the present invention was significantly reduced compared to the diabetic group, and the extract a of the present invention had better effect on increasing insulin sensitivity compared to the extract B, C, D.

Claims

1. A method for extracting a composition for increasing insulin sensitivity, comprising the steps of:

a mixing step, namely stirring and uniformly mixing 10-20% of crushed silkworm excrement, 50-55% of white mulberry root-bark and 25-40% of mulberry leaf dry powder to obtain a mixture;

a leaching step, mixing the mixture with an extraction solution, wherein the extraction solution is 1, 3-butanediol with the water content of 40-60% to obtain a mixed solution, and placing the mixed solution in a water bath for stirring and extraction, wherein the water bath temperature is 80-95 ℃, and the leaching time is not less than 24 hours;

a filtering step, filtering the leached mixed solution to obtain a filtrate, and adjusting the pH value of the filtrate to 10 by using NaOH;

an ion exchange resin treatment step, namely treating the concentrated solution by using ion exchange resin to obtain a composition stock solution;

and a drying step, namely freeze-drying the composition stock solution to obtain brown powder.

2. The method of claim 1, wherein the extraction solution is 1, 3-butanediol having a water content of 50%.

3. The method of claim 1, wherein the water bath temperature is 85 ℃.