CN113143997A

CN113143997A - Application of mulberry extract in preparation of medicine for reducing animal weight

Info

Publication number: CN113143997A
Application number: CN202110272018.7A
Authority: CN
Inventors: 刘玉玲; 申竹芳; 刘率男; 曹慧; 刘志华; 刘泉; 李彩娜; 雷蕾; 陈艳敏; 朱向阳; 邹媛媛
Original assignee: Guangxi Wuhe Boao Pharmaceutical Co ltd; Beijing Wuhebao Pharmaceutical Co ltd
Current assignee: Guangxi Wuhe Boao Pharmaceutical Co ltd; Beijing Wuhebao Pharmaceutical Co ltd
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2021-07-23

Abstract

The invention relates to application of a mulberry extract in preparing a medicament for reducing animal weight, which is characterized in that the mulberry extract contains alkaloid, polysaccharide, amino acid and flavone, preferably, the content of each component is 3-99% of alkaloid, 0.2-70% of polysaccharide, 0-10% of flavone, 0-50% of amino acid and 0-25% of other components by weight based on the mulberry extract. The mulberry extract can control the weight increase accompanied by type 2 diabetes, and has good treatment effect on type 2 diabetes combined obesity.

Description

Application of mulberry extract in preparation of medicine for reducing animal weight

Technical Field

The invention relates to application of mulberry extract in preparing a medicament for reducing animal weight.

Background

With the increasing standard of living, the phenomena of obesity and overweight are growing at an alarming rate worldwide. Epidemiological studies have shown that obesity and overweight are important risk factors for diabetes, cardiovascular disease, cancer and premature death. The causes of obesity include an increase in the volume of adipocytes and an increase in the number of adipocytes, which are caused by the differentiation of precursor adipocytes into mature adipocytes, which are accomplished under the activation and expression of a series of transcription factors. The data show that the number of diabetics is the first global, wherein the proportion of overweight people is 41.0 percent, and the proportion of obese people is 24.3 percent. Compared with the simple obese patients, the type 2 diabetes mellitus combined obese patients have more difficulty in losing weight and maintaining the weight (Chinese type 2 diabetes mellitus combined obesity comprehensive management experts consensus, China journal of endocrine metabolism, 2016,32(08): 623-.

Moraceae plants are regarded as valuable materials for both medicine and food since ancient times due to higher nutritional and medicinal values, and there are records on traditional Chinese medicine books and prescriptions about diseases treated by mulberry twig, mulberry bark, mulberry leaf, mulberry fruit and the like, for example, as early as in Bencao gang mu, there are records on "mulberry leaf juice decocted for tea, capable of relieving thirst, and" roasted and decocted for drink, and quenched tea ". The chemical components of the Moraceae plants mainly comprise flavonoid compounds, polysaccharide compounds, alkaloids and the like, and are widely used for preparing medicines for reducing blood sugar, reducing blood fat, resisting viruses, regulating immunity and the like at present.

Some reports related to weight and lipid reduction of mulberry extract exist in the prior art, for example, patent application CN1631246A discloses the weight and lipid reduction effect of mulberry twig extract, which is specifically shown in the fact that the mulberry twig ethanol extract can reduce the weight of an NIH mouse and reduce the levels of triglyceride and cholesterol in blood, but the administration method and dose-effect relationship are not clear; patent application CN102370708A discloses the application of Chinese herbal medicine mulberry leaf water extract or alcohol extract in preparing weight-losing and lipid-lowering medicines, and records the application of mulberry leaf extract in weight-losing and lipid-lowering.

The KKAy mouse is an animal model mouse of type 2 diabetes mellitus, which is formed by transferring a mutant gene (ay) on the basis of a KK mouse, the morbidity of the mouse is induced by adding environmental factors on the basis of genetic susceptibility, the mouse is very similar to the expression of human type 2 diabetes mellitus, the individual difference of the mouse is small, the experimental repeatability is good, and the mouse is an ideal animal model of spontaneous type 2 diabetes mellitus. The KKAy mice are clinically manifested by polydipsia, polyphagia and polyuria with obesity and have the characteristics of hyperglycemia, high insulin resistance, insufficient islet function, liver lesion, kidney lesion and the like. Research shows that the weight, the liver fat and the epididymis fat weight of the KKAy mouse are obviously higher than those of a normal mouse. At present, no research report on the influence of the mulberry-related extract with definite ingredients on the body weight of KKAY mice exists.

Disclosure of Invention

The inventor researches to find that the mulberry extract has a remarkable effect on the weight loss of KKAy mice, and based on the research, the invention provides the application of the mulberry extract in preparing a medicament for reducing the weight of animals.

In one embodiment of the present invention, the mulberry extract contains alkaloids, polysaccharides, amino acids and flavones. Preferably, the weight content of each component based on the mulberry extract is

More preferably, the weight content of each component based on the mulberry extract is

Further preferably, the weight content of each component based on the mulberry extract is

In one embodiment, the preparation of the mulberry extract comprises the steps of: preparing a crude extract; optionally, separation on a cationic resin and/or an anionic resin; optionally, subjecting the resin effluent to an alcohol precipitation treatment; and optionally, concentrating and drying. Preferably, the preparation of the mulberry extract comprises the steps of: step 1): preparing a crude extract; step 2): separation on a cationic resin and/or optionally an anionic resin; optional 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, the mulberry extract is prepared by the following steps: pulverizing ramulus Mori, folium Mori or cortex Mori, extracting with water and/or alcohol solution or acid water under reflux, the solvent amount is 3-20 times of the original medicinal materials, extracting repeatedly for 1-3 times, mixing extractive solutions, concentrating, eluting with cation exchange resin, washing with distilled water to remove unadsorbed impurities, eluting with 0.2-3N ammonia water, concentrating the eluate, eluting with anion exchange resin, collecting unadsorbed part, adding ethanol, precipitating to remove impurities, centrifuging, and concentrating the clear solution under reduced pressure or spray drying or freeze drying to obtain extract.

In one embodiment, the mulberry extract is prepared by the following steps: pulverizing ramulus Mori, folium Mori or cortex Mori, extracting with water and/or alcohol solution or acid water under reflux, repeatedly extracting for 1-3 times, mixing extractive solutions, concentrating, eluting with cation exchange resin, washing with distilled water to remove unadsorbed impurities, eluting with 0.2-3N ammonia water, concentrating eluate, eluting with anion exchange resin, collecting unadsorbed part, and concentrating under reduced pressure or spray drying or freeze drying to obtain extract.

In one embodiment, the mulberry extract is prepared by the following steps: pulverizing ramulus Mori, folium Mori or cortex Mori, extracting with water and/or alcohol solution or acid water under reflux for 1-3 times, mixing extractive solutions, concentrating, adding cation exchange resin, washing with distilled water to remove unadsorbed impurities, eluting with 0.2-3N ammonia water, and concentrating the eluate under reduced pressure or spray drying or freeze drying to obtain extract.

The animal species of the "animal" of the present invention is not particularly limited, and may be any animal having an intestinal tract, preferably a mammal, more preferably a rat, a mouse and a human, and most preferably a human.

In one embodiment of the invention, the animal is a diabetic animal.

In one embodiment of the invention, the medicament reduces body weight in the animal by reducing fat accumulation in the cells. Preferably, the cell is a liver cell and/or an epididymal adipocyte. More preferably, the cell is a liver cell.

Preferably, 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, including microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropylcellulose, crospovidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, 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.

Preferably, the medicament is in an oral administration form; further preferably, the medicament is a tablet, a capsule, an oral solution, an oral emulsion, a pill, a granule, a syrup, and a powder.

The mulberry extract disclosed by the invention can control the weight increase of a spontaneous diabetes obesity model mouse, is definite in components, controllable in quality, free of adverse reaction, high in safety and low in cost, provides a new choice for diabetes obesity people, and provides a new variety for development and screening of weight-reducing drugs.

Drawings

Figure 1 is the effect of long-term administration of mulberry extract on the body weight of KKAy mice: (A) body weights of each group before and after administration, (B) body weight change before and after administration. P <0.01, P <0.001, compared to DM group.

FIG. 2 isA graph showing the effect of different doses of SZ-A on the accumulation of hepatic triglycerides and cholesterol by palmitic acid. SZ-A representsA mulberry twig extract SZ-A treatment group, MET isA metformin positive control group, PA representsA palmitic acid-stimulated insulin resistance model group, and BSA representsA HepG2 cell normal control group (0.25% BSA treatment). P <0.01, compared to normal control group.

Detailed Description

The invention is explained in more detail below with reference to the figures and examples. The features and advantages of the present invention will become more apparent from the exemplary descriptions.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

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).

Example 1 preparation of mulberry extract 1

Pulverizing fresh ramulus Mori (ramulus Mori 11 # with serrate Morus), adding 4000L water, extracting under reflux for 2 hr, 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 keeping the temperature at 50 ℃ to be used as a sample loading solution of the 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 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 until the effluent reaches 870L.

And centrifuging the collected liquid to remove impurities, concentrating the collected liquid by a counter ion permeable membrane, transferring the concentrated liquid with the specific gravity of 1.25 into an alcohol precipitation tank, and adding 25L of absolute ethyl alcohol into the alcohol precipitation tank at the speed of 500rpm 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.

Concentrating the effluent under reduced pressure to obtain ramulus Mori extract, wherein the alkaloid content is 52%, polysaccharide content is 22%, flavone content is 0.8%, and amino acid content is 20%.

Example 2 preparation of Mulberry extract 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 1, 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 to collect the effluent when white precipitate is generated, stopping collecting the effluent when the volume of the collected liquid reaches 25L, and directly purifying the collected liquid 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. And (4) loading the collected cation resin eluent to anion resin, and collecting the effluent until the effluent reaches 15L. 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 by the same method and parameters as above.

The obtained ramulus Mori extract contains alkaloid 98%, polysaccharide 0.2%, flavone 0.05%, and amino acids 0.

The obtained cortex Mori extract contains alkaloid 95%, polysaccharide 2%, flavone 0.1%, and amino acids 1%.

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%.

Example 3 preparation of Mulberry extract 3

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 subjected to centrifugation to remove impurities, and then is concentrated by a counter ion permeable membrane until the solid content reaches 1 percent, and the concentrated extract is used as a sample loading solution of a cation resin column.

The cation resin was activated by the method of preparation example 1 using 300kg of D001 type macroporous strongly acidic styrene cation resin packed in a column. 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 (4) concentrating the collected liquid obtained after the separation of the cation column by using a nanofiltration membrane, and concentrating under reduced pressure to obtain extract.

The obtained ramulus Mori extract contains alkaloid 15%, polysaccharide 20%, flavone 7%, and amino acids 45%.

Example 4 preparation of Mulberry extract 4

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 125kg 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 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 80%, the content of polysaccharide is 5%, the content of flavone is 0.1%, and the content of amino acid is 4%.

Example 5 preparation of Mulberry extract 5

Taking 400kg 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. Loading the collected concentrated extractive solution onto anion resin, and collecting effluent.

And (3) filtering the collected liquid obtained after the anion column separation by using a microfiltration membrane to remove impurities, concentrating by using a reverse ion permeable membrane, and further concentrating and drying under reduced pressure to obtain the mulberry twig extract. Sample content: the content of alkaloid is 3%, the content of polysaccharide is 70%, the content of flavone is 10% and the content of amino acid is 10%.

Example 6 preparation of Mulberry extract 6

Pulverizing fresh ramulus Mori (ramulus Mori 11), adding 6000L water, extracting under reflux for 2 hr, 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 keeping the temperature at 50 ℃ to be used as a sample loading solution of the cation resin column.

Loading 100kg 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 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 until the effluent reaches 870L. Concentrating the effluent under reduced pressure to obtain ramulus Mori extract, wherein the alkaloid content is 30%, polysaccharide content is 35%, flavone content is 2%, and amino acid content is 25%.

Example 7 preparation of Mulberry extract 7

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 until the effluent reaches 870L. Concentrating the effluent under reduced pressure to obtain ramulus Mori extract, wherein the alkaloid content is 40%, polysaccharide content is 25%, flavone content is 0.5%, and amino acid content is 25%.

Example 8 preparation of Mulberry extract 8

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%.

Example 9 Effect of Mulberry extract on body weight of idiopathic type 2 diabetic KKAy mice

Female KKAy mice of 12 weeks were selected, fed with high-fat diet for 3 weeks, and then divided into 3 groups (DM group, SZ-a 160(mg mulberry extract/kg), SZ-a 320(mg mulberry extract/kg)) on average according to indices such as random blood glucose, fasting blood glucose, and body weight, and 8 mice were each administered with the mulberry extract of example 8 by gavage once a day for about 6 weeks, and the change in body weight of the mice before and after administration was recorded.

TABLE 1 spontaneous type 2 diabetes KKAY mouse cohort data

The average body weight of each group of mice was about 43g before administration of the mulberry extract. After about 6 weeks of administration of the mulberry extract, the body weight of the mice of the two dose groups of the mulberry extract was significantly reduced, the body weight of the mice of the SZ-A100 dose group was reduced by 2.0g as compared with the DM group, and the body weight reduction rate was 4.2%; the mice in the SZ-A200 dose group lost 4.1g of weight and the weight loss rate was 8.6% compared with the DM group. During the administration period, the weight growth rate of mice in two dosage groups of SZ-A is also obviously reduced, the weight of mice inA DM group is averagely increased by 4.4g, the weight of mice inA SZ-A100 dosage group is averagely increased by 2.6g, and the weight of mice inA SZ-A200 dosage group is averagely increased by 0.1g, which indicates that the mulberry extract has an obvious effect of controlling the weight growth of KKAY mice.

TABLE 2 spontaneous type 2 diabetic KKAY mice weight changes

Data are expressed as ` X. + -. SD, n ═ 8, compared to DM; DM isA KKAy model control group, and SZ-A isA dosing group. P <0.01, P <0.001 compared to DM group

EXAMPLE 10 clinical trial

Taking the ramulus mori extract of the example 1, adding a proper amount of auxiliary materials, mixing uniformly, adding water to prepare a soft material, granulating, drying, adding magnesium stearate, mixing uniformly, and tabletting to obtain a ramulus mori extract preparation, wherein each tablet contains 50mg of total alkaloids.

Clinical cases and treatments:

(1) a Chinese male, 43 years old, 84.2 kg, 170cm in height, with type 2 diabetes for 3 months, orally administered with the tablet containing the mulberry extract of example 8 three times a day, two tablets at a time, for 24 weeks, and lost 10.2 kg in weight.

(2) A male 65 years old, 68.3 kg, 173cm in height, with type 2 diabetes for 7 months, combined with hyperlipidemia, orally administered the tablet of example 8 three times a day, two tablets at a time, for 24 weeks, losing 6.3 kg of body weight.

(3) A male aged 64 years old and 75.0 kg, 168cm tall, with type 2 diabetes mellitus for 12 months, was discontinued after two months of acarbose administration, and 8 months later, orally administered with the tablets of example 8 three times a day, two tablets at a time, for 24 weeks, losing 7 kg of body weight.

(4) A female aged 48 years old, 81.0 kg, 164cm in height, with type 2 diabetes for 8 months, combined with hyperlipidemia, orally administered the tablet of example 8 three times a day, two tablets at a time, for 24 weeks, losing 7 kg of body weight.

Example 11 in vitro cell assay

1. After digestion of HepG2 cells in logarithmic growth phase, the cell density was adjusted to 2X10 with DMEM (high-sugar) complete medium containing 10% FBS⁵Transfer to 6-well plates at 2 ml/well.

2. After 24h of culture, the model cells were randomly divided into SZ-A group (50ug/ml, 25ug/ml, 12.5ug/ml of the mulberry twig extract SZ-A of example 1), metformin group (200umol/l MET) and palmitic acid-stimulated insulin resistance model group (PA), while HepG2 cells were set asA normal control group (0.25% BSA). Except for the normal control group (0.25% BSA) and the model group (PA), the other groups were added with the corresponding drugs and palmitic acid, respectively, to replicate the insulin resistance model, and each group was provided with 3 replicate wells.

3. After 24h of culture, the cells were washed once with pre-warmed PBS and 400ul of 1% PMSF Triton x-100 lysate was added to each well of the 6-well plate. Placing on ice or cracking at 4 ℃ for 1h, and gently blowing and beating by a pipettor to ensure that the lysate is fully contacted with the cells. The lysed liquid was centrifuged and 10ul triglyceride and 10ul total cholesterol were measured directly after vortexing.

The test results are shown in FIG. 2, and fatty liver is easily formed due to excessive fat accumulation in liver. Stimulation of HepG2 liver cells with palmitic acid PA can mimic fatty liver formation. With the addition of different doses of SZ-A, it can be seen that SZ-A inhibits the accumulation of hepatic triglycerides and cholesterol caused by palmitic acid. SZ-A has the same effect as MET.

With reference to the above method, model cells were treated with the mulberry extract (dose of 25ug/ml) prepared in examples 2-7, and triglyceride and total cholesterol in the cells were measured, respectively. The results showed that the accumulation of hepatic triglycerides and cholesterol caused by palmitic acid was somewhat reduced after treatment with mulberry extract compared to the PA group. The specific results are shown in Table 3 below.

TABLE 3 Effect of Mulberry extract prepared from examples 1 to 7 on triglyceride and cholesterol levels

The above examples show that mulberry extract has a good weight control effect on diabetes combined with obesity. Long-term administration of mulberry extract can inhibit weight gain of type 2 diabetic mice and diabetic patients; and has inhibitory effect on visceral fat accumulation.

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. Use of a mulberry extract for the manufacture of a medicament for reducing the weight of an animal, wherein the mulberry extract comprises alkaloids, polysaccharides, amino acids and flavonoids in the respective weight fractions based on the mulberry extract

2. The use of claim 1, wherein the animal is a mammal, including humans and rodents.

3. Use according to claim 1 or 2, wherein the animal is a diabetic animal.

4. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.

5. The use of claim 1, wherein the medicament is in an oral dosage form; preferably, the medicament is a tablet, capsule, oral solution, oral emulsion, pill, granule, syrup, and powder.