An extract of abnormal black melanin maturation agent, its preparation method and application

Abstract

The application provides an abnormal black bile mature agent extract and a preparation method and application thereof, and belongs to the technical field of biological medicines, and the preparation method comprises the following steps: mixing and degreasing abnormal black bile mature agent dry powder with petroleum ether, filtering and taking filter residues, mixing the filter residues with water, stirring and dissolving, filtering and taking filtrate, adding 80-90% ethanol solution into the filtrate, and precipitating and taking precipitates at 3-8 DEG C for 12-36 hours to obtain the abnormal black bile mature agent extract. The abnormal black bile mature agent raw material of the traditional Uyghur medicine is widely sourced, cheap and easy to obtain, and has no toxic and side effects on the human body. In the preparation method, the abnormal black bile mature agent is separated and extracted by using petroleum ether, water and ethanol solution, so that the active ingredients in the abnormal black bile mature agent can be effectively extracted, the abnormal black bile mature agent extract prepared by the application can effectively treat diabetes, and the application provides a new choice for clinical drugs for treating diabetes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an abnormal melanin cholesterin maturation agent extract, a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a metabolic disease characterized by chronic hyperglycemia. More than 90% of diabetes patients are type 2 diabetes patients. Type 2 diabetes is caused by insulin resistance, which causes a series of chronic metabolic disorders of protein, carbohydrate and fat, and gradually leads to damage to tissues and organs such as kidney, eye, heart and limbs. Severe cases can develop acute metabolic disorders, hyperosmolar hyperglycemic syndrome and ketoacidosis. Hypoglycemic drugs are an important means of treating type 2 diabetes. Although there are now a variety of effective hypoglycemic drugs available for clinicians to choose from, they still cannot meet the needs of the large number of diabetes patients, reducing the overall blood glucose target rate. Domestic and foreign scholars are looking for active substances that can assist in reducing blood sugar from natural products, hoping to provide more options for diabetes patients.

[0003] The abnormal melanin cholesterin maturation agent is a compound preparation composed of ten medicinal materials, which can regulate the abnormal melanin cholesterin type of body fluid, restore the balance of four body fluids such as melanin cholesterin, mucus, cholesterin and blood, and has the effect of preventing and treating difficult diseases such as abnormal melanin cholesterin type tumors, asthma, anxiety, cardiovascular diseases, psoriasis, aging, depression and mental illness. Therefore, it is of great significance to extract effective components from the abnormal melanin cholesterin maturation agent and detect their biological activity and medicinal value. SUMMARY

[0004] The present application aims to provide an abnormal melanin cholesterin maturation agent extract, a preparation method and application thereof. The abnormal melanin cholesterin maturation agent extract prepared by the present application can effectively treat diabetes.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a preparation method of an abnormal melanin cholesterin maturation agent extract, comprising the following steps:

[0007] The dry powder of the abnormal melanin cholesterin maturation agent is mixed with petroleum ether for degreasing, and the residue is obtained by filtration. The residue is mixed with water, stirred and dissolved, and the filtrate is obtained by filtration. 80% to 90% ethanol solution is added to the filtrate, and the precipitate is obtained by standing at 3 to 8℃ for 12 to 36 hours.

[0008] Preferably, the mass-volume ratio of the residue to water is 1:8 to 12g / mL, the stirring and dissolving temperature is 65 to 95℃, and the stirring and dissolving time is 10 to 40min.

[0009] Preferably, the volume ratio of the filtrate to the 80%-90% ethanol solution is 1:10-14.

[0010] Preferably, the mass-volume ratio of the dry powder of the abnormal melanin mature agent to petroleum ether is 1:4-6 g / mL.

[0011] Preferably, the defatting temperature is 55-65℃, and the defatting time is 1-2 h.

[0012] The application further provides an abnormal melanin mature agent extract prepared according to the preparation method.

[0013] The application further provides a medicine for treating diabetes, wherein the active ingredient of the medicine comprises the abnormal melanin mature agent extract.

[0014] Preferably, the excipient comprises one or more of a diluent, a wetting agent, a binder, a lubricant, an absorbent, and a preservative.

[0015] The application further provides the use of the abnormal melanin mature agent extract in the preparation of a medicine for treating diabetes.

[0016] Preferably, the diabetes is type 2 diabetes.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] The application provides an abnormal melanin mature agent extract, a preparation method and use thereof. DETAILED DESCRIPTION

[0019] The application provides a preparation method of an abnormal melanin mature agent extract, which comprises the following steps:

[0020] The dry powder of the abnormal melanin mature agent is mixed with petroleum ether for defatting, and the residue is obtained by filtration; the residue is mixed with water, stirred and dissolved, and the filtrate is obtained by filtration; 80%-90% ethanol solution is added to the filtrate, and the precipitate is obtained by standing at 3-8℃ for 12-36 h.

[0021] In the present application, the dry powder of abnormal black bile maturation agent is mixed with petroleum ether for degreasing, and the residue is filtered. In the present application, the raw material composition of the abnormal black bile maturation agent and its preparation method are disclosed in Chinese patent application No. 02130082.8. In the present application, the abnormal black bile maturation agent is composed of the following raw materials by weight: lavender 7 parts, fennel 7 parts, psilopegan fruit 15 parts, jujube 15 parts, roseroot 7 parts, lemon balm 7 parts, licorice 10 parts, adiantum capillus-veneris 7 parts, perfoliate knotweed 7 parts, and acanthopanax 15 parts. The preparation method of the abnormal black bile maturation agent is as follows: the above-mentioned abnormal black bile maturation agent except acanthopanax is cut into pieces, 12 times of water is added, heating is conducted for 20 minutes, then heating is stopped, soaking is conducted, and standing is conducted for 18 hours, then warm heating is conducted for 2 hours, filtering is conducted, the filtrate is heated and acanthopanax is added and stirred to dissolve, then filtering is conducted again, and the filtrate is diluted with distilled water to 5 times to obtain the abnormal black bile maturation agent. The abnormal black bile maturation agent is dried at 40-50℃ to obtain the dry powder of abnormal black bile maturation agent.

[0022] In the present application, the mass-volume ratio of the dry powder of abnormal black bile maturation agent to petroleum ether is preferably 1:4-6 g / mL, and further preferably 1:4.5-5.5 g / mL, the degreasing temperature is preferably 55-65℃, and further preferably 58-63℃, and the degreasing time is preferably 1-2 hours. In the present application, petroleum ether is used for degreasing the dry powder of abnormal black bile maturation agent, which can effectively remove the fat-soluble impurities in the dry powder of abnormal black bile maturation agent. The filtering method is not particularly limited in the present application, and the method known in the art can be used.

[0023] In the present application, the residue is mixed with water, stirred and dissolved, and the filtrate is obtained by filtering. The mass-volume ratio of the residue to water is preferably 1:8-12 g / mL, and further preferably 1:9-11 g / mL. The stirring and dissolving temperature is preferably 65-95℃, and further preferably 70-90℃, and the stirring and dissolving time is preferably 10-40 minutes, and further preferably 15-35 minutes. In the present application, the active ingredients in the abnormal black bile maturation agent can be extracted by water extraction.

[0024] In the present application, 80%-90% ethanol solution is added to the filtrate, and the precipitate is obtained by standing at 3-8℃ for 12-36 hours. The volume ratio of the filtrate to 80%-90% ethanol solution is preferably 1:10-14. The solvent of the ethanol solution is preferably water, and the concentration of the ethanol solution is volume percentage. After the precipitate is obtained, drying at 40-50℃ until there is no ethanol smell is further included in the present application. In the present application, the content of effective ingredients in the abnormal black bile maturation agent is significantly improved by water extraction and alcohol precipitation.

[0025] The present application also provides the abnormal melanin chondroitin maturation agent extract prepared by the above preparation method.

[0026] The present application also provides a medicine for treating diabetes, wherein the active ingredient comprises the abnormal melanin chondroitin maturation agent extract and the pharmaceutically acceptable adjuvant.

[0027] In the present application, the medicine can be in the form of powder, granule, tablet, capsule, drop pill, pill, powder, lyophilized powder injection or solution. The medicine of the present application can be administered in unit dosage form, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneal or rectal administration, etc. The medicine of the present application can also be administered by injection, including intravenous injection, intramuscular injection, subcutaneous injection and intradermal injection, etc. The adjuvant comprises one or more of diluent, humectant, binder, lubricant, absorbent and preservative. The diluent and absorbent can be one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; the humectant and binder can be one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; the lubricant can be one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc.; and the preservative can be one or more of sodium benzoate, nipagin, sorbic acid, etc.

[0028] The administration dose of the medicine of the present application depends on many factors, such as the nature and severity of the disease to be prevented or treated, the gender, age, weight, character and individual response of the patient or animal, the administration route, administration frequency, treatment purpose, etc. Therefore, the therapeutic dose of the present application can vary greatly. Generally, the use dose of the medicine in the present application is known to those skilled in the art. The actual amount of the medicine contained in the medicine preparation of the present application can be appropriately adjusted to achieve the therapeutically effective amount and achieve the prevention or treatment purpose of the present application. The daily use amount of the medicine of the present application is 0.001-150 mg / kg body weight, preferably 0.01-100 mg / kg body weight, more preferably 0.01-60 mg / kg body weight, and most preferably 0.1-10 mg / kg body weight.

[0029] The present application also provides the use of the abnormal melanin chondroitin maturation agent extract in the preparation of a medicine for treating diabetes.

[0030] In the present application, the diabetes is type 2 diabetes.

[0031] In the present application, all raw material components are commercially available products well known to those skilled in the art, unless otherwise specified.

[0032] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0033] Embodiment 1

[0034] A preparation method of an abnormal black bile maturation agent extract, steps as follows:

[0035] 100 g of dry powder of abnormal black bile maturation agent is mixed with 500 mL of petroleum ether, heated at 60℃ for 1.5 h for degreasing, the filter residue is obtained by filtration, the filter residue is mixed with water at a mass-volume ratio of 1:10 g / mL, stirred and dissolved at 80℃ for 30 min, the filtrate is obtained by filtration, 85% ethanol solution is added to the filtrate, mixed uniformly, the volume ratio of the filtrate to 85% ethanol solution is 1:12, precipitated at 5℃ for 24 h, the precipitate is dried at 45℃ until no ethanol smell, and the abnormal black bile maturation agent extract is obtained.

[0036] Embodiment 2

[0037] A preparation method of an abnormal black bile maturation agent extract, steps as follows:

[0038] 100 g of dry powder of abnormal black bile maturation agent is mixed with 400 mL of petroleum ether, heated at 55℃ for 2 h for degreasing, the filter residue is obtained by filtration, the filter residue is mixed with water at a mass-volume ratio of 1:12 g / mL, stirred and dissolved at 95℃ for 10 min, the filtrate is obtained by filtration, 80% ethanol solution is added to the filtrate, mixed uniformly, the volume ratio of the filtrate to 80% ethanol solution is 1:14, precipitated at 3℃ for 12 h, the precipitate is dried at 50℃ until no ethanol smell, and the abnormal black bile maturation agent extract is obtained.

[0039] Embodiment 3

[0040] A preparation method of an abnormal black bile maturation agent extract, steps as follows:

[0041] The 100 g dry powder of abnormal black melanin maturation agent was mixed with 600 mL petroleum ether, heated for 1 h at 65 °C for degreasing, and the residue was filtered. The residue was mixed with water at a mass-volume ratio of 1:8 g / mL, stirred and dissolved at 65 °C for 40 min, and the filtrate was filtered. 90% ethanol solution was added to the filtrate, mixed uniformly, and the volume ratio of the filtrate to 90% ethanol solution was 1:10. The precipitate was obtained by standing at 8 °C for 36 h and drying at 40 °C until no ethanol smell was detected. Thus, the extract of abnormal black melanin maturation agent was obtained.

[0042] Test Example 1

[0043] Treatment effect of the extract of abnormal black melanin maturation agent prepared in Example 1 on type 2 diabetic mice

[0044] Preparation of experimental reagents:

[0045] The extract of abnormal black melanin maturation agent prepared in Example 1 was dissolved in distilled water to prepare 30 mg / mL, 60 mg / mL and 120 mg / mL abnormal black melanin maturation agent extract solutions, respectively.

[0046] 1% streptozotocin (STZ) preparation:

[0047] 0.2 g of citric acid was added to 10 mL of ddH2O to obtain a citric acid solution, and 0.294 g of sodium citrate was added to 10 mL of ddH2O to obtain a sodium citrate solution. The citric acid solution and the sodium citrate solution were mixed at a ratio of 1:1, and the pH value of the buffer was adjusted to 4.4 to obtain a citric acid-sodium citrate buffer. STZ was weighed and mixed with the citric acid-sodium citrate buffer at a ratio of 1 g:100 mL to prepare a 1% STZ solution.

[0048] Animal grouping and administration:

[0049] Experimental animals

[0050] C57 / BL6J male mice (18-20 g, 3-5 weeks old) were purchased from Hunan Slike Jingda Experimental Animal Co., Ltd. The animal breeding temperature was 24±1 °C, the humidity was 50±10%, the light and dark were regularly alternated, and the animals were free to eat and drink. The animals were adaptively fed for 1 week.

[0051] Sixty male C57 / BL6J mice were randomly divided into six groups after one week of feeding: normal control group (NC), type 2 diabetes model group, low, medium and high concentration groups of abnormal black bile maturation agent extract, and metformin positive control group. Except for the normal control group, the other five groups were fed a high-fat model diet (Nantong Trofi Feed Technology Co., Ltd.). Mice were fasted overnight for 12 hours. Mice fed the high-fat diet were injected intraperitoneally with 50 mg / kg of pre-cooled 1% STZ solution for 3 consecutive days. Mice in the NC group were injected intraperitoneally with buffer for 3 consecutive days. After one week of stabilization, the fasting blood glucose level at the tail tip of the mice was measured to be >11.1 mmol / L to indicate successful modeling. Mice with successfully modeled type 2 diabetes were continuously fed a 60% high-fat model diet.

[0052] Six mice were administered the following treatments to mice that had successfully developed type 2 diabetes: a normal control group (NC), a type 2 diabetes model group, low-, medium-, and high-concentration groups of abnormal black bile maturation agent extract, and a metformin positive control group. The low-, medium-, and high-concentration groups of abnormal black bile maturation agent extract were administered via gavage at concentrations of 30 mg / mL, 60 mg / mL, and 120 mg / mL, respectively. The gavage dose was calculated at 0.2 mL per 20 g mouse. The normal control group and the type 2 diabetes model group were administered an equal volume of distilled water via gavage. The metformin positive control group was administered 300 mg / kg once daily for 60 days.

[0053] Indicator Testing:

[0054] Mouse weight level detection: Throughout the experiment, mouse weight was measured on days 10, 20, 30, 40, 50 and 60.

[0055] Fasting blood glucose levels in mice: Fasting blood glucose levels in mice were measured on days 10, 20, 30, 40, 50, and 60. All groups were fasted for 12 hours prior to the test, and blood glucose was rapidly measured from the tail tip using a glucometer.

[0056] Glucose tolerance test: Mice were fasted for 12 hours beforehand. On day 60, they were injected intraperitoneally with 20% glucose solution (2g / kg) while fasting. Blood glucose levels were measured at 0.5h, 1h, and 2h.

[0057] Table 1. Blood glucose variation trend in a diabetic mouse model (unit: nmol / L)

[0058]

[0059] As shown in Table 1, the mouse model of type 2 diabetes was successfully established.

[0060] Table 2. Blood glucose levels in mice from different groups after day 60 (unit: nmol / L)

[0061]

[0062] The results in Table 2 show that, compared with the type 2 diabetes model group, the fasting blood glucose levels of the high, medium, and low concentration groups of the abnormal black bile maturation agent extract and the metformin positive control group were significantly lower. Among them, the high concentration group of the abnormal black bile maturation agent extract was at a normal blood glucose level, and the medium concentration group of the abnormal black bile maturation agent extract was close to a normal blood glucose level.

[0063] Table 3. Changes in blood glucose levels in different groups of mice after day 60 (unit: nmol / L)

[0064]

[0065]

[0066] The results in Table 3 show that, with the extension of time, the blood glucose levels of the low, medium and high concentration groups of the abnormal black bile maturation agent extract and the metformin positive control group decreased, and the low, medium and high concentration groups of the abnormal black bile maturation agent extract after 2 hours were significantly different from those of the type 2 diabetes model group.

[0067] The abnormal black bile maturation agent extract prepared by the preparation method of the present invention was used to treat a mouse model of type 2 diabetes. The results showed that the abnormal black bile maturation agent extract of the present invention could significantly reduce the fasting blood glucose level of mice in each group and improve glucose tolerance. Therefore, the abnormal black bile maturation agent extract prepared by the preparation method of the present invention can effectively treat type 2 diabetes.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of an extract of an abnormal black bile maturation agent in the preparation of a drug for treating diabetes, characterized in that, The preparation method of the abnormal black bile maturation agent extract includes the following steps: The dry powder of the abnormal black bile maturation agent is mixed with petroleum ether for defatting, filtered to obtain the residue, the residue is mixed with water, stirred to dissolve, filtered to obtain the filtrate, 80% to 90% ethanol solution is added to the filtrate, and the mixture is allowed to stand at 3 to 8°C for 12 to 36 hours to precipitate and obtain the precipitate, which is the extract of the abnormal black bile maturation agent. The mass-to-volume ratio of the filter residue to water is 1:8-12 g / mL, the stirring and dissolving temperature is 65-95℃, and the stirring and dissolving time is 10-40 min; The volume ratio of the filtrate to the 80%–90% ethanol solution is 1:10–14; The mass-to-volume ratio of the dry powder of the abnormal black bile maturation agent to petroleum ether is 1:4-6 g / mL. The degreasing temperature is 55-65℃, and the degreasing time is 1-2 hours.

2. The application according to claim 1, characterized in that, The diabetes mentioned is type 2 diabetes.

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