RU2826807C1 - Use of mulberry extract for preparing drug for reducing body weight in animals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the use of mulberry extract for preparing a drug for reducing body weight in animals. Use of the mulberry extract for the preparation of a drug for reducing body weight in animals, wherein said mulberry extract contains an alkaloid, a polysaccharide, an amino acid and a flavonoid, and wherein the weight content of each component, based on the weight of the mulberry extract, is as follows: alkaloid from 3% to 99%, polysaccharide from 0.2% to 70%, flavonoid from 0% to 10%, amino acid from 0% to 50% and other components from 0% to 25%.

EFFECT: said invention provides an effective agent for reducing body weight in animals.

12 cl, 6 dwg, 6 tbl

Description

AREA OF TECHNOLOGY

The present invention relates to the use of a mulberry extract for the production of a medicinal product for weight loss in animals.

LEVEL OF TECHNOLOGY

As the quality of life improves, the problem of obesity and overweight is increasing at an alarming rate worldwide. Epidemiological studies have shown that obesity and overweight are major risk factors for diabetes, cardiovascular diseases, cancer and premature death. The causes of obesity include an increase in the size and number of adipocytes, with the increase in the number of adipocytes caused by the differentiation of preadipocytes into mature adipocytes, and the differentiation of preadipocytes into mature adipocytes occurs due to the activation and expression of several transcription factors. Data show that the number of people suffering from diabetes is the largest in the world, among them, the proportion of overweight people is 41.0%, and the proportion of obese people is 24.3%. Compared with patients with simple obesity, patients with type 2 diabetes with obesity have difficulty losing weight and maintaining body weight (Experts' Consensus in China on Integrated Management of Type 2 Diabeteswith Obesity, China J Endocrinol Metab, 2016, 32(08): 623-627).

Due to the high nutritional and medicinal value of mulberry plants, they have been considered a valuable raw material for both medicinal and edible use since ancient times. According to the classics of traditional Chinese medicine and recipes from different years, there are records that mulberry branches, mulberry bark, mulberry leaves and mulberry berries were used to treat diseases. For example, the Materia Medica collection already mentions that “a decoction of mulberry leaves with juice can quench thirst instead of tea” and “drinking after cooking and decoction can quench thirst instead of tea”. The chemical components in mulberry plants mainly include flavonoid compounds, polysaccharide compounds, alkaloids and the like, and are currently widely used to prepare hypoglycemic, hypolipidemic, antiviral and immunomodulatory drugs.

There are a number of references in the prior art on the effect of mulberry extract on reducing body weight and reducing lipid levels. For example, patent application CN 1631246 A discloses the effect of mulberry twig extract on reducing body weight and reducing lipid levels, and specifically shows that an alcoholic extract of mulberry twigs can reduce body weight in NIH mice and also reduce blood triglyceride and cholesterol levels, but neither the administration route nor the dose-effect relationship are clear; another patent application, CN 102370708 A, discloses the use of an aqueous extract or an alcoholic extract of a Chinese herbal medicine from mulberry leaves for producing drugs for reducing body weight and reducing lipid levels, and also discloses the use of mulberry extract for reducing body weight and reducing lipid levels.

KKAy mice are a kind of animal model of type 2 diabetes. They are formed by transferring the mutant gene (ay) to KK mice; the reason for this is the environmental factor superimposed on genetic susceptibility. This model is very similar to the characteristics of type 2 diabetes in humans; in addition, due to small individual differences and good experimental repeatability, it is a relatively ideal animal model of spontaneous type 2 diabetes. KKAy mice have clinical signs of polydipsia, polyphagia and polyuria with obesity, and are characterized by hyperglycemia, high insulin resistance, islet cell deficiency, liver disease, kidney disease, etc. Studies show that the body weight as well as liver fat mass and epididymal fat mass of KKAy mice are significantly higher than those of normal mice. At present, there is no report on the study of the effect of mulberry extract with transparent composition on the weight of KKAy mice.

Brief description of the invention

The author of the present invention conducted research and found that the mulberry extract according to the present invention has a significant effect on reducing body weight in animals. Based on this, the present invention proposes the use of the mulberry extract for producing a drug for reducing body weight in animals.

In one embodiment of the present invention, the mulberry extract comprises an alkaloid, a polysaccharide, an amino acid and a flavone. Preferably, the weight content of each component, based on the weight of the mulberry extract, is as follows:

alkaloid from 3% to 99%,

polysaccharide from 0.2% to 70%,

flavone from 0% to 10%,

amino acid from 0% to 50%, and

other components from 0% to 25%;

more preferably, the mass content of each component, based on the mass of the mulberry extract, is as follows:

alkaloid from 50% to 99%,

polysaccharide from 0.2% to 35%,

flavone from 0% to 2%,

amino acid from 0% to 30%, and

other components from 0% to 20%; and

even more preferably, the mass content of each component, based on the mass of the mulberry extract, is as follows:

alkaloid from 50% to 99%,

polysaccharide from 0.2% to 25%,

flavone from 0% to 1%,

amino acid from 0% to 20%, and

other components from 0% to 20%,

even more preferably, the mass content of each component, based on the mass of the mulberry extract, is as follows:

alkaloid from 30% to 99%,

polysaccharide from 0.2% to 35%,

flavone from 0% to 2%,

amino acid from 0% to 30%, and

other components from 0% to 20%,

even more preferably, the mass content of each component, based on the mass of the mulberry extract, is as follows:

alkaloid from 40% to 99%,

polysaccharide from 0.2% to 35%,

flavone from 0% to 2%,

amino acid from 0% to 30%, and

other components from 0% to 20%.

In one embodiment, obtaining a mulberry extract comprises the following steps: preparing a crude extraction solution; optionally, separating with a cationic resin and/or an anionic resin; optionally subjecting the resin effluent to alcohol precipitation; and, optionally, concentrating and drying. Preferably, obtaining a mulberry extract comprises the following steps: step 1): preparing a crude extraction solution; step 2): separating with a cationic resin and/or, optionally, an anionic resin; optional step 3): subjecting the resin effluent obtained in step 2) to alcohol precipitation; and optional step 4): concentrating and drying.

In one embodiment, the mulberry extract was obtained according to the following steps: crushing mulberry twigs, mulberry leaves or mulberry bark; heating under reflux to extract with water and/or an alcohol solution or acidic water, the amount of solvent in which is 3-20 times greater than the amount of the original medicinal materials; repeating the extraction 1-3 times; combining the extraction solution and concentrating; loading a cation exchange resin; optionally, washing out unadsorbed impurities with water (preferably distilled) and eluting with 0.2-3 N ammonium hydroxide; concentrating with an eluent and loading an anion exchange resin; collecting the unadsorbed portion, adding ethanol, precipitation to remove impurities, centrifuging and concentrating (preferably, concentration under reduced pressure) with a pure solution or drying (preferably, spray drying or freeze drying) to obtain an extract.

In one embodiment, the mulberry extract was obtained according to the following steps: crushing mulberry twigs, mulberry leaves or mulberry bark; heating under reflux to extract with water and/or an alcohol solution or acidic water, the amount of solvent in which is 3-20 times greater than the amount of the original medicinal materials; repeating the extraction 1-3 times; combining the extraction solution and concentrating; loading a cation exchange resin; optionally, washing out unadsorbed impurities with water (preferably distilled) and eluting with 0.2-3 N ammonium hydroxide; concentrating with an eluent and loading an anion exchange resin; collecting the unadsorbed portion and concentrating (preferably concentrating under reduced pressure) or drying (preferably spray drying or freeze drying) to obtain an extract.

In one embodiment, the mulberry extract was obtained according to the following steps: crushing mulberry twigs, mulberry leaves or mulberry bark; heating under reflux to extract with water and/or an alcohol solution or acidic water, the amount of solvent in which is 3-20 times greater than the amount of the original medicinal materials; repeating the extraction 1-3 times; combining the extraction solution and concentrating; loading a cation exchange resin; optionally, washing out unadsorbed impurities with water (preferably distilled) and eluting with 0.2-3 N ammonium hydroxide and concentrating (preferably concentrating under reduced pressure) using an eluent or drying (preferably spray drying or freeze drying) to obtain an extract.

The kind of animal referred to by the term "animal" in the present invention is not particularly limited. Animals may be any animals with intrial organs, preferably mammals, more preferably rats, mice and humans, and most preferably humans.

In one embodiment of the present invention, the animals suffer from diabetes. Preferably, the diabetic animals have a body mass index BMI≥28.

Preferably the animals are healthy.

In one embodiment of the present invention, the medicament reduces body weight in animals by reducing fat accumulation in cells. Preferably, the cells are liver cells and/or epididymal adipocytes. More preferably, the cells are liver cells.

Preferably, the medicinal product further comprises a pharmaceutically acceptable carrier. The carrier may be an inactive component that is suitable for the routes or modes of administration and non-toxic to the human body. The carrier may be a solid or liquid excipient. Solid excipients include, for example, microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, polyvinylpolypyrrolidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium dodecyl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch and talc; and liquid excipients include, for example, water, ethanol, syrup and glycerin.

Preferably, the medicinal product has a dosage form for oral administration; more preferably, said medicinal product is a tablet, a capsule, an oral solution, an oral emulsion, a pill, a granule, a syrup and a powder.

The mulberry extract of the present invention can control the increase in body weight in mice in a spontaneous diabetes model with obesity; furthermore, due to the clear ingredients, controlled quality, no side effects, high safety and low cost, this extract provides a new opportunity for obese people with diabetes, as well as new opportunities for the development and screening of drugs for weight loss.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows the effects of long-term administration of the mulberry extract according to embodiment 9 on the body weight of KKAy mice: (A) the weight of each group before and after administration, and (B) the changes in weight before and after administration. **P<0.01, ***P<0.001 relative to the DM group.

Fig. 2 shows individual changes in body weight after 8 weeks of clinical administration in embodiment 11.

Fig. 3 shows individual changes in body weight after 24 weeks of clinical administration in embodiment 11.

Fig. 4 shows the changes in body weight of high-fat C57 mice in embodiment 13 after intragastric administration.

Fig. 5 shows the changes in body weight of high-fat C57 mice in embodiment 13 after injection.

Fig. 6 shows the effects of different doses of SZ-A on the accumulation of hepatic triglyceride and cholesterol induced by palmitic acid. SZ-A means the SZ-A mulberry twig extract treatment group, MET means the metformin positive control group, PA means the palmitic acid-stimulated insulin resistance model group, BSA means the normal control HepG2 cells (BSA0.25% treated) group. **P<0.01 relative to the normal control group.

Implementation options

The present invention will be further described in detail with reference to the drawings and embodiments of the invention. Thanks to these illustrative descriptions, the features and advantages of the present invention will become more clear and definite.

The specific term "exemplified" as used herein means "used as an example or embodiment, or being illustrative." Any embodiment described herein as "illustrative" is not necessarily to be construed as superior or better than others.

Furthermore, the technical features involved in the various embodiments of the present invention described below may be combined with each other if they do not contradict each other.

The content of the components used in the present invention was determined according to known methods (see the methods disclosed in patents with publication numbers CN 11077247 A and CN 110393738 A).

Implementation option 1: Obtaining 1 mulberry extract

1000 kg of fresh mulberry (Moms serrata Roxb-Yuesang No.11) twigs were collected and crushed, then 4000 L of water was added and extracted by heating under reflux for 2 h; the extraction solution was combined and filtered to remove insoluble matters, and thus a crude extraction solution was obtained. The crude extraction solution was concentrated by heating until the solid content percentage was 4% and kept at 50°C, and used as a loading solution for a cationic resin column.

150 kg of macroporous and weakly acidic cationic phenylpropene resin D113 type was used for packing the column, and 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; 1 mol/L sodium hydroxide solution was used for washing until the pH of the eluent was 8.5; 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; and deionized water whose volume was 5 times the column volume was used for washing, and thus the activation was completed. The concentrated extraction solution was loaded and then eluted using 1000 L of 2.5 mol/L ammonia water at an elution rate of 6 bed volumes/h; The eluent was collected when the effluent from the cation column was found to have a pH>7; the collection was stopped when the collected solution was up to 900 L, and the collected solution was purified directly through the anion column.

62.5 kg of macroporous and strongly alkaline anion resin type D218 was used to load the column, and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0; 1.5 mol/L hydrochloric acid solution was used to wash until the pH of the eluent was 3.5; and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0, and thus the activation was completed. The collected cationic eluent was loaded onto the anionic resin, and the effluent was collected until the effluent reached 870 L.

The collected solution was centrifuged to remove impurities, and then concentrated by a reverse osmosis membrane; the specific gravity of the concentrated liquid was 1.25; the concentrated liquid was transferred to an alcohol precipitation tank, and 25 L of anhydrous ethanol was added while the stirring stirrer was rotating at 500 rpm. After the addition of ethanol was completed, the stirring was stopped, and alcohol precipitation was carried out for 24 hours; the supernatant was collected and concentrated under reduced pressure, thereby obtaining an extract extract.

The effluent was concentrated under reduced pressure to obtain a mulberry twig extract extract, wherein the alkaloid accounted for 52%, the polysaccharide accounted for 22%, the flavone accounted for 0.8%, and the amino acid accounted for 20%.

Implementation option 2: Obtaining 2 mulberry extract

10 kg of fresh mulberry twigs (Sangteyou No.2) were collected and crushed, then 150 L of water was added twice, and extracted by boiling for 3 h each time; the extraction solution was combined and filtered to remove insoluble matter. The extraction solution was concentrated by heating until the solid content was 8%, then transferred to an alcohol precipitation tank, and 2367.9 g (3 L) of anhydrous ethanol was added under stirring at 300 rpm. After the addition of ethanol was completed, stirring was stopped, and alcohol precipitation was carried out for 24 h; the supernatant was taken as the loading solution for the cation resin column. 5 kg of a styrene-based strong acid cationic resin of type 002SC was used to pack the column, and the cationic resin was activated according to the method described in embodiment 1. The extraction solution was loaded after concentration and precipitation with alcohol, and then eluted with 100 L of 5 mol/L potassium chloride at an elution rate of 5 bed volumes/h; the effluent was detected with 20% silicotungstic acid, and collection was started after the formation of a white precipitate; collection was stopped until the collected solution was up to 25 L; and the collected solution was purified directly through the anion resin column.

10 kg of a strongly alkaline anion resin based on styrene type 711 was used to pack the column, and the anion resin was activated according to the method described in embodiment 3. The collected cationic eluent was loaded onto the anion resin, and the effluent was collected until the effluent reached 15 L. The collected solution was reloaded onto the cationic resin and separated twice using the cationic resin and the anion resin sequentially according to the above-mentioned method.

The collected solution obtained after three column separations was centrifuged to remove impurities, and then concentrated through a reverse osmosis membrane, and the specific gravity of the concentrated liquid was 1.25; it was transferred to an alcohol precipitation tank, and 125 g of anhydrous ethanol was added under a stirring speed of 1000 rpm. After the addition of ethanol was completed, stirring was stopped, and alcohol precipitation was carried out for 24 hours; the supernatant was collected and concentrated under reduced pressure, thereby obtaining an extract extract. In addition, fresh mulberry bark and mulberry leaves (Sangteyou No. 2) were again taken and extracted, and the extraction method and parameters were the same as those described above.

In the obtained mulberry twig extract, the alkaloid was 98%, the polysaccharide was 0.2%, the flavone was 0.05%, and the amino acid was 0.

In the obtained mulberry bark extract, alkaloid accounted for 95%, polysaccharide accounted for 2%, flavone accounted for 0.1%, and amino acid accounted for 1%.

In the obtained mulberry leaf extract, alkaloid accounted for 90%, polysaccharide accounted for 4%, flavone accounted for 0.1%, and amino acid accounted for 3%.

Implementation option 3: Preparation of 3 mulberry extract

1000 kg of fresh mulberry (Moms atropurpurea Roxb) twigs were collected and crushed, then 11500 L of water was added and extracted by heating under reflux for 2 h; the extraction solution was combined and filtered to remove insoluble matter, and thus a crude extraction solution was obtained. The crude extraction solution was centrifuged to remove impurities, and then concentrated by reverse osmosis membrane until the solid content percentage was 1%, and it served as a loading solution for the cationic resin column.

300 kg of macroporous and strongly acidic styrene-based cationic resin type D001 was used to pack the column, and the cationic resin was activated according to the method described in embodiment 1. The crude extraction solution was loaded after concentration, and then eluted with 5000 L of 0.04 mol/L ammonium nitrate at an elution rate of 5 bed volumes/h; the effluent was detected with 20% tungstic silicic acid, and collection was started after the formation of a white precipitate; collection was stopped until the collected solution reached 1000 L.

The collected solution obtained after separation on the cationic column was concentrated by nanofiltration and then concentrated under reduced pressure to obtain an extract extract.

In the obtained mulberry twig extract, the alkaloid was 15%, the polysaccharide was 20%, the flavone was 7%, and the amino acid was 45.

Implementation option 4: Obtaining 4 mulberry extract

333 kg of dried mulberry twigs (Yuesang No.11) were collected and crushed, then 4000 L of water was added and extracted twice by heating under reflux, 1 h for each reflux; the extraction solution was combined and filtered, and concentrated until the amount of crude drug reached 1 kg/L.

150 kg of macroporous and weakly acidic cationic phenylpropene resin D113 type was used for packing the column, and 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; 1 mol/L sodium hydroxide solution was used for washing until the pH of the eluent was 8.5; 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; and deionized water whose volume was 5 times the column volume was used for washing, and thus the activation was completed. The concentrated extraction solution was loaded and then eluted using 1000 L of 2.5 mol/L ammonia water at an elution rate of 6 bed volumes/h; The eluent was collected when the effluent from the cation column was found to have a pH>7; the collection was stopped when the collected solution was up to 900 L, and the collected solution was purified directly through the anion column.

125 kg of macroporous and strongly alkaline anion resin D218 was used to load the column, and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0; 1.5 mol/L hydrochloric acid solution was used to wash until the pH of the eluent was 3.5; and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0, and thus the activation was completed. The collected cationic eluent was loaded onto the anion resin, and the effluent whose pH was more than 8 was collected until the effluent reached 870 L.

The collected solution obtained after separation on the anion column was filtered by a microfiltration membrane, centrifuged to remove impurities, and then concentrated through a reverse osmosis membrane, and the specific gravity of the concentrated liquid was 1.1; it was transferred to an alcohol precipitation tank, and 15 g of anhydrous ethanol was added under a stirring speed of 400 rpm. After the addition of ethanol was completed, stirring was stopped and alcohol precipitation was carried out for 24 hours; the supernatant was collected and concentrated under reduced pressure, whereby mulberry twig extract extract was obtained. The content in the sample was: alkaloid was 80%, polysaccharide was 5%, flavone was 0.1%, amino acid was 4%.

Implementation option 5: Obtaining 5 mulberry extract

400 kg of dried mulberry twigs (Yuesang No.11) were collected and crushed, then 4000 L of water was added and extracted twice by heating under reflux, 1 hour for each reflux; the extraction solution was combined and filtered, and concentrated until the amount of crude drug reached 1 kg/L.

62.5 kg of macroporous and strongly alkaline anion resin type D218 was used to load the column, and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0; 1.5 mol/L hydrochloric acid solution was used to wash until the pH of the eluent was 3.5; and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0, and thus the activation was completed. The collected extract was concentrated and loaded into the anion resin, and the effluent was collected.

The collected solution obtained after separation by anion column was filtered by microfiltration membrane to remove impurities, then concentrated by reverse osmosis membrane, and further concentrated under reduced pressure and dried to obtain mulberry twig extract extract. The content in the sample was: alkaloid was 3%, polysaccharide was 70%, flavone was 10%, amino acid was 10%.

Implementation option 6: Obtaining 6 mulberry extract

1500 kg of fresh mulberry (Moms serrata Roxb-Yuesang No.11) twigs were collected and crushed, then 6000 L of water was added and extracted by heating under reflux for 2 h; the extraction solution was combined and filtered to remove insoluble matters, and thus a crude extraction solution was obtained. The crude extraction solution was concentrated by heating until the solid content percentage was 4% and kept at 50°C, and used as a loading solution for a cationic resin column.

100 kg of macroporous and weakly acidic cationic phenylpropene resin D113 type was used for packing the column, and 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; 1 mol/L sodium hydroxide solution was used for washing until the pH of the eluent was 8.5; 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; and deionized water whose volume was 5 times the column volume was used for washing, and thus the activation was completed. The concentrated extraction solution was loaded and then eluted using 1000 L of 2.5 mol/L ammonia water at an elution rate of 6 bed volumes/h; The eluent was collected when the effluent from the cation column was found to have a pH>7; the collection was stopped when the collected solution was up to 900 L, and the collected solution was purified directly through the anion column.

62.5 kg of macroporous and strongly alkaline anion resin type D218 was used to load the column, and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0; 1.5 mol/L hydrochloric acid solution was used to wash until the pH of the eluent was 3.5; and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0, and thus the activation was completed. The collected cationic eluent was loaded onto the anionic resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain a mulberry twig extract extract, wherein the alkaloid accounted for 30%, the polysaccharide accounted for 35%, the flavone accounted for 2%, and the amino acid accounted for 25%.

Implementation option 7: Obtaining 7 mulberry extract

1000 kg of fresh mulberry (Moms serrata Roxb-Yuesang No.11) twigs were collected and crushed, then 4000 L of water was added and extracted by heating under reflux for 2 h; the extraction solution was combined and filtered to remove insoluble matters, and thus a crude extraction solution was obtained. The crude extraction solution was concentrated by heating until the solid content percentage was 4% and kept at 50°C, and used as a loading solution for a cationic resin column.

100 kg of macroporous and weakly acidic cationic phenylpropene resin D113 type was used for packing the column, and 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; 1 mol/L sodium hydroxide solution was used for washing until the pH of the eluent was 8.5; 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; and deionized water whose volume was 5 times the column volume was used for washing, and thus the activation was completed. The concentrated extraction solution was loaded and then eluted using 1000 L of 2.5 mol/L ammonia water at an elution rate of 6 bed volumes/h; The eluent was collected when the effluent from the cation column was found to have a pH>7; the collection was stopped when the collected solution was up to 900 L, and the collected solution was purified directly through the anion column.

62.5 kg of macroporous and strongly alkaline anion resin D218 was used to load the column, and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0; 1.5 mol/L hydrochloric acid solution was used to wash until the pH of the eluent was 3.5; and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0, and thus the activation was completed. The collected cationic eluent was loaded onto the anionic resin, and the effluent was collected until the effluent reached 870 L. The effluent was concentrated under reduced pressure to obtain a mulberry twig extract extract, wherein the alkaloid accounted for 40%, the polysaccharide accounted for 25%, the flavone accounted for 0.5%, and the amino acid accounted for 25%.

Implementation option 8: Obtaining 8 mulberry extract

333 kg of dried mulberry twigs (Yuesang No.11) were collected and crushed, then 4000 L of water was added and extracted twice by heating under reflux, 1 h for each reflux; the extraction solution was combined and filtered, and concentrated until the amount of crude drug reached 1 kg/L.

150 kg of macroporous and weakly acidic cationic phenylpropene resin D113 type was used for packing the column, and 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; 1 mol/L sodium hydroxide solution was used for washing until the pH of the eluent was 8.5; 2 mol/L hydrochloric acid solution was used for washing until the pH of the eluent was 4.5; and deionized water whose volume was 5 times the column volume was used for washing, and thus the activation was completed. The concentrated extraction solution was loaded and then eluted using 1000 L of 2.5 mol/L ammonia water at an elution rate of 6 bed volumes/h; The eluent was collected when the effluent from the cation column was found to have a pH>7; the collection was stopped when the collected solution was up to 900 L, and the collected solution was purified directly through the anion column.

62.5 kg of macroporous and strongly alkaline anion resin type D218 was used to load the column, and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0; 1.5 mol/L hydrochloric acid solution was used to wash until the pH of the eluent was 3.5; and 1.5 mol/L sodium hydroxide solution was used to wash until the pH of the eluent was 9.0, and thus the activation was completed. The collected cationic eluent was loaded onto the anionic resin, and the effluent was collected until the effluent reached 870 L.

The collected solution obtained after separation on the anion column was filtered by a microfiltration membrane, centrifuged to remove impurities, and then concentrated through a reverse osmosis membrane, and the specific gravity of the concentrated liquid was 1.1; it was transferred to an alcohol precipitation tank, and 15 g of anhydrous ethanol was added under a stirring speed of 400 rpm. After the addition of ethanol was completed, stirring was stopped and alcohol precipitation was carried out for 24 hours; the supernatant was collected and concentrated under reduced pressure, whereby mulberry twig extract extract was obtained. The content in the sample was: alkaloid was 63%, polysaccharide was 23%, flavone was 1%, amino acid was 5%.

Implementation option 9: Effect of mulberry extract on body weight of KKAy mice with spontaneous type 2 diabetes

Twelve-week-old female KKAy mice were selected; after feeding with high-fat food for three weeks, they were equally divided into 3 groups (DM group, SZ-A 160 (mg mulberry extract/kg), SZ-A 320 (mg mulberry extract/kg)) according to the random blood glucose level, fasting blood glucose level, body weight and other indices, 8 mice in each group, and given the mulberry extract according to embodiment 8 once a day by intragastric administration; the drug was continuously administered for about 6 weeks, and the changes in the weight of the mice before and after the administration were recorded, as shown in Table 1, Table 2 and Fig. 1.

Before the administration of mulberry extract, the average body weight of the mice in each group was approximately 43 g. About 6 weeks after the administration of mulberry extract, the body weight of the mice in the two mulberry extract dosage groups was significantly reduced, namely, the body weight of the mice in the SZ-A160 dosage group was lower than that of the DM group by 2.0 g, the body weight loss rate was 4.2%, and the body weight of the mice in the SZ-A320 dosage group was lower than that of the DM group by 4.1 g, the body weight loss rate was 8.6%. During the administration, the body weight gain rate of mice in the two SZ-A dosage groups also decreased significantly, wherein: the average body weight gain of mice in the DM group was 4.4 g, the average body weight gain of mice in the SZ-A160 dosage group was 2.6 g, and the average gain in the SZ-A320 dosage group was 0.1 g, indicating that mulberry extract can remarkably control body weight gain in KKAy mice.

Table 2 Changes in body weight in KKAy mice with spontaneous type 2 diabetes

Implementation option 10: Clinical experiment 1

The mulberry twig extract according to embodiment 1 was taken and an appropriate amount of auxiliary material was added; after uniformly mixing and adding water, a soft material was obtained, and then granulated and dried; magnesium stearate was added and uniformly mixed, and tablets were obtained by pressing, thereby obtaining the mulberry twig extract, and each tablet contained a total of 50 mg of alkaloids.

Clinical cases and treatment:

(1) A 43-year-old Chinese male (84.2 kg, 170 cm, type 2 diabetes mellitus for 3 months) orally took tablets containing mulberry extract according to embodiment 1 (three times a day and two tablets each time orally for 24 weeks) and lost 10.2 kg of body weight.

(2) A 65-year-old male (68.3 kg, 173 cm, type 2 diabetes mellitus for 7 months with hyperlipidemia) orally took tablets containing mulberry extract according to embodiment 1 (three times a day and two tablets each time continuously for 24 weeks) and lost 6.3 kg of body weight.

(3) A 64-year-old male (75.0 kg, 168 cm, type 2 diabetes for 12 months) took acarbose for two months and then stopped; 8 months later, he orally took tablets containing mulberry extract according to embodiment 1 (three times a day and two tablets each time continuously for 24 weeks) and lost 7 kg of body weight.

(4) A 48-year-old woman (81.0 kg, 164 cm, type 2 diabetes mellitus for 8 months with hyperlipidemia) orally took tablets containing mulberry extract according to embodiment 1 (three times a day and two tablets each time continuously for 24 weeks) and lost 7 kg of body weight.

Implementation option 11: Clinical experiment 2

1. Task

Effect of mulberry twig extract on body weight loss in diabetic patients.

2. Experimental method

2.1 Grouping

The study design was randomized, double-blind, double-modeled, parallel-controlled, acarbose tablets, multicenter, non-inferiority. A total of 600 patients with type 2 diabetes mellitus (aged 18 to 70 years, 19 kg/ ^m2 ≤BMI≤30 kg/ ^m2 , 7%≤HbA1C≤10%) and poor blood glucose control were included and randomly assigned to receive alkaloid tablets (SZ-A, N=360) or acarbose (Glucobay®) (CON, N=240). All provided written informed consent.

The main exclusion criteria were as follows: 1) allergy or intolerance to alpha-glucosidase inhibitors; 2) the change in FBS levels between the first and second follow-up was greater than 2.5 mmol/L (>45 mg/dL); 3) a history of severe diabetic complications (proliferative stage diabetic retinopathy, diabetic nephropathy stage V, diabetic ketoacidosis, diabetic hypertensive coma, diabetic lactic acidosis); 4) combination drug therapy affecting glucose metabolism, such as antidiabetic Chinese medicine or glucocorticoids; 5) hyperlipidemia accompanied by a history of irregular use of lipid-lowering drugs; 6) chronic gastrointestinal dysfunction, obvious disturbances of digestion and absorption, or endocrine disorders such as hyperthyroidism, hypercortisolism, and acromegaly; 7) severe cardiac disease, myocardial infarction, unstable angina, chronic heart failure, or poor blood pressure control; 8) impaired liver or kidney function; and 9) pregnancy. 2.2 Route of Administration

The experimental groups were orally administered tablets (the tablets were prepared as follows: the extract according to embodiment 1 was collected and an appropriate amount of auxiliary substances were added; after uniformly mixing and adding water, a soft material was obtained, and then granulated and dried; magnesium stearate was added and uniformly mixed, and tablets were obtained by pressing, thereby obtaining the mulberry twig extract, each tablet containing 50 mg of total alkaloids) with an initial dose of one tablet each time and three times a day; four weeks later, the dose was successively increased to two tablets each time and three times a day; the control group was orally administered acarbose, one tablet each time and three times a day.

3. Detection and processing of experimental data

3.1 Detection Index

(1) From the 1st to the 24th week of administration, the height, body weight and body weight gain in each group were observed and recorded every day.

3.2 Statistical analysis

SPSS 20.0 software was used to process the data, and differences between groups were tested using the T-test.

4. Results

After 8 weeks and 24 weeks of administration, experimental data were obtained for different BMI groups of patients with type 2 diabetes mellitus in each group, as shown in Tables 3 and 4 and Figs. 2 and 3.

After 8 weeks of administration, as in patients with BMI≥28 kg/ ^m2 , the body weight in the experimental group was clearly reduced, compared with the control group, by an average of -0.503 kg and 0.210 kg, respectively (P=0.016); after 24 weeks of administration, as in patients with BMI≥28 kg/ ^m2 , the body weight in the experimental group was clearly reduced, compared with the control group, by an average of -1.228 kg and -0.182 kg, respectively (P=0.022); for other BMI groups, the changes in body weight were not statistically significant.

It has been shown that the use of mulberry twig extract in diabetic patients with BMI≥28 kg/ ^m2 can contribute to the treatment of type 2 diabetes mellitus, as well as weight loss.

Implementation Variant 12: Clinical Experimental Example 3

1. Task

Effect of mulberry twig extract on weight loss in healthy subjects

2. Method of experiment

2.1 Experimental equipment and materials

Scales

2.2 Experimental design

In the non-diabetic group, a total of 17 people, including 11 men and 6 women, were healthy people; they received normal nutrition during the drug administration period; the drug administration period was 0.7 to 8 months, the dose was 50 or 100 mg/time, and the drug administration frequency was 2 to 3 times/day.

2.3 Detection Index

Before and at the end of the experiments, the following body parameters were measured: weight, height.

The criteria for assessing weight loss were: (1) excellent, with the percentage of weight loss being greater than or equal to 10%; (2) good, with the percentage of weight loss being greater than or equal to 5% and less than 10%; (3) effective, with the percentage of weight loss being greater than or equal to 3% and less than 5%; and (4) ineffective, with the percentage of weight loss being less than 3%.

3. Results

There were 2 “excellent” cases, 9 “good” cases, 5 “effective” cases, and 1 “ineffective” case, and the overall effective rate was 94%. In addition, all subjects did not experience any adverse reactions after taking the drug, indicating that the drug has a significant effect on weight loss in non-diabetic patients. The information and experimental results for the subjects are shown in Table 5 below.

Overall Effectiveness Rate = (Excellent Case + Obvious Cases + Effective Cases)/Cases*100%.

TS: the medicine is taken three times a day BID: the medicine is taken twice a day.

Implementation option 13: Experiment on body weight loss in C57 mice fed a high-fat diet

1. Intragastric administration

Thirty healthy 6-week-old male C57 mice were randomly divided into a normal group, a model group and a SZ-A group, with 10 mice in each group, wherein the mice in the normal group were fed with the basal diet, and the mice in the model group and the SZ-A group were fed with the high-fat diet. After 14 weeks of feeding, the mice in each group were administered the corresponding drug by intragastric administration every day for 6 consecutive weeks, wherein the SZ-A group was administered intragastric administration at a dose of 400 mg/kg/day according to the total alkaloid amount of mulberry twig extract, and the normal group and the model group were administered intragastric administration of a corresponding dose of solvent. The weight of the mice was monitored during the drug treatment. After the end of drug administration, all the mice were weighed on an empty stomach for 12 hours.

As shown in Fig. 4, the average body weight of the mice in the model group was 50.3 g, and that of the mice in the administration group was 43.7 g, indicating that SZ-A could significantly reduce the body weight of mice fed with high-fat diet.

2. Injection

(1) Animal model: Thirty 6-week-old C57 mice were randomly divided into three groups, where the first group was fed with basal chow, the second and third groups were fed with high-fat chow, and feeding was continued for 14 weeks.

(2) Administration: Based on the body weight, the third group was given intraperitoneal injection of 200 mg/kg total alkaloids of mulberry twig extract, and the injection period was 6 weeks. The first and second groups were individually given physiological saline solution once a day according to the body weight.

(3) Measurement of body weight: After administration, the body weight of mice was measured using a scale and recorded.

As shown in Fig. 5, the average weight of the model group was 50.6 g and that of the administration group was 42.8 g, indicating that SZ-A injection could significantly reduce obesity induced by high-fat feed.

Implementation option 14: In vitro cell experiment

1. After splitting HepG2 cells at logarithmic growth stage, the cell density was adjusted to 2× ¹⁰⁵ cells/mL using complete culture medium DMEM (high glucose) containing 10% FBS, and the cells were transferred into a 6-well plate at 2 mL/well.

2. After culturing for 24 hours, the model cells were randomly divided into an SZ-A group (the mulberry twig extract SZ-A in Embodiment 1 at 50 μg/mL, 25 μg/mL, and 12.5 μg/mL), a metformin group (200 μmol/L MET), and a palmitic acid (PA)-stimulated model group; meanwhile, HepG2 cells served as a normal control group (0.25% BSA). In addition to the normal control group (0.25% BSA) and the model group (PA), the corresponding drugs and palmitic acid were added to the other groups separately, the palmitic acid stimulation model was replicated, and three replicates were provided for each group.

3. After culturing for 24 hours, pre-warmed PBS was added once for washing, and then 400 μl of Triton x-100 lysate containing 1% PMSF was added to each well of the 6-well plate. The 6-well plate was tapped on ice or at 4°C for 1 hour with gentle blowing with a pipette and shaking so that the lysate and cells were in full contact. The lysed liquid was not centrifuged, and 10 μl of triglyceride and 10 μl of total cholesterol were directly measured after shaking.

As shown in Fig. 6, excessive accumulation of fat in the liver can easily cause fatty liver. The formation of fatty liver tissue can be modeled by stimulating HepG2 liver cells with palmitic acid PA. By adding different doses of SZ-A, accumulation of triglycerides and cholesterol in the liver caused by inhibition of palmitic acid by SZ-A can be observed. The effect of SZ-A is similar to that of MET.

Referring to the above-mentioned method, the model cells were treated with the mulberry extract (dose was 25 μg/mL) obtained in embodiments 2 to 7, and the triglyceride level and total cholesterol in the cells were measured, respectively. According to the results, compared with the RA group, the accumulation of triglyceride and cholesterol in the liver caused by palmitic acid was reduced to a certain extent after treatment with the mulberry extract. The specific results are shown in Table 6 below.

The above embodiments show that mulberry extract plays a good role in controlling body weight. Long-term administration of mulberry extract can suppress body weight growth in mice and humans, and suppress body weight increase in type 2 diabetic mice and diabetic patients; in addition, it can suppress fat accumulation in organs.

With reference to preferred embodiments, the present invention is explained as above. However, these embodiments of the invention are merely exemplary and illustrative. Thus, various substitutions and improvements can be made to the present invention, and all these substitutions and improvements fall within the scope of protection of the present invention.

Claims (37)

1. The use of a mulberry extract for obtaining a medicinal product for reducing body weight in animals, wherein said mulberry extract contains an alkaloid, a polysaccharide, an amino acid and a flavonoid, and wherein the mass content of each component, based on the mass of the mulberry extract, is as follows: alkaloid from 3% to 99%, polysaccharide from 0.2% to 70%, flavonoid from 0% to 10%, amino acid from 0% to 50%, and other components from 0% to 25%. 2. The use according to paragraph 1, characterized in that the mass content of each component, calculated on the mass of the mulberry extract, is as follows: alkaloid from 50% to 99%, polysaccharide from 0.2% to 35%, flavonoid from 0% to 2%, amino acid from 0% to 30%, and other components from 0% to 20%. 3. The use according to paragraph 1, characterized in that the mass content of each component, calculated on the mass of the mulberry extract, is as follows: alkaloid from 50% to 99%, polysaccharide from 0.2% to 25%, flavonoid from 0% to 1%, amino acid from 0% to 20%, and other components from 0% to 20%. 4. The use according to paragraph 1, characterized in that the mass content of each component, calculated on the mass of the mulberry extract, is as follows: alkaloid from 30% to 99%, polysaccharide from 0.2% to 35%, flavonoid from 0% to 2%, amino acid from 0% to 30%, and other components from 0% to 20%. 5. The use according to paragraph 1, characterized in that the mass content of each component, calculated on the mass of the mulberry extract, is as follows: alkaloid from 40% to 99%, polysaccharide from 0.2% to 35%, flavonoid from 0% to 2%, amino acid from 0% to 30%, and other components from 0% to 20%. 6. Use according to any of paragraphs 1-5, characterized in that said animals are mammals, including humans and rodents. 7. Use according to any of paragraphs 1-6, characterized in that said animals suffer from diabetes. 8. Use according to any of paragraphs 1-6, characterized in that the said animals are healthy. 9. Use according to any of paragraphs 1-8, characterized in that said medicinal product additionally contains a pharmaceutically acceptable carrier. 10. Use according to any of paragraphs 1-9, characterized in that the said medicinal product has a dosage form for oral administration. 11. Use according to paragraph 10, characterized in that said medicinal product is a tablet, capsule, oral solution, oral emulsion, pill, granule, syrup, and powder. 12. Use according to paragraph 7, characterized in that said animals suffering from diabetes have a BMI≥28.