Abnormal melanin and chitin maturation agent extract against tumors, and preparation method and application thereof

Abstract

The application provides an anti-tumor abnormal melanochrous mature agent extract and a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: mixing and degreasing abnormal melanochrous mature agent dry powder with petroleum ether, filtering to obtain filter residue, adding 30-50% ethanol solution to the filter residue for extraction, filtering to obtain filtrate, and drying to obtain the abnormal melanochrous mature agent extract. The application uses traditional Chinese medicine abnormal melanochrous mature agent raw materials, which are widely sourced, cheap and easy to obtain, and have good safety and no toxic and side effects on human bodies. In the preparation method, the abnormal melanochrous mature agent is separated and extracted by using petroleum ether and ethanol solution, so that the active ingredients of the anti-tumor abnormal melanochrous mature agent extract can be effectively extracted. The abnormal melanochrous mature agent extract prepared by the application can effectively treat tumors, and provides a new choice for natural extraction drugs for treating tumors in clinical treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-tumor abnormal black bile maturation agent extract, its preparation method, and its application. Background Technology

[0002] Cancer, especially malignant cancer, is a leading cause of death and a major threat to human health, posing a formidable challenge to modern medicine. Traditional treatments for malignant cancer often involve Western medicine, radiotherapy, chemotherapy, or surgery, which are expensive and have significant side effects. Research indicates that extracts from many natural plants possess excellent anti-tumor and anti-inflammatory activities; therefore, exploring traditional Chinese medicine for drugs has become an effective approach for anti-inflammatory and anti-tumor drug research.

[0003] The Abnormal Black Bile Maturation Agent is a compound preparation composed of ten medicinal herbs. It can regulate the body fluid constitution of the Uyghur abnormal black bile type, restoring the balance of the four humoral constitutions: black bile, phlegm, choleric, and hemoglobin. It is effective in the prevention and treatment of intractable diseases such as abnormal black bile type tumors, asthma, anxiety, cardiovascular diseases, psoriasis, aging, depression, and mental illness. Therefore, the extraction of effective components from the Abnormal Black Bile Maturation Agent and the detection of their bioactivity and medicinal value are of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide an extract of abnormal black bile maturation agent for anti-tumor purposes, its preparation method, and its application. The abnormal black bile maturation agent extract prepared by this invention can effectively treat tumors.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing an extract of abnormal black bile maturation agent with anti-tumor properties, comprising the following steps:

[0007] The dry powder of the abnormal black bile ripening agent is mixed with petroleum ether for defatting, filtered to obtain the residue, and 30% to 50% ethanol solution is added to the residue for extraction. The filtrate is then filtered and dried to obtain the final product.

[0008] Preferably, the mass-to-volume ratio of the dry powder of the abnormal black bile ripening agent to petroleum ether is 1:4-6 g / mL.

[0009] Preferably, the degreasing temperature is 55-65°C, and the degreasing time is 1-2 hours.

[0010] Preferably, the extraction temperature is 45-55℃, the extraction is performed 1-3 times, and the extraction time for each extraction is 2-4 hours.

[0011] Preferably, the mass-to-volume ratio of the filter residue to the 30%–50% ethanol solution is 1:14–16 g / mL.

[0012] The present invention also provides an extract of abnormal black bile maturation agent prepared by the above preparation method.

[0013] The present invention also provides a medicament for treating antitumor diseases, the medicament comprising an active ingredient and pharmaceutically acceptable excipients, the active ingredient including the above-mentioned abnormal black bile maturation agent extract.

[0014] Preferably, the excipients include one or more of the following: diluent, flavoring agent, humectant, binder, lubricant, and preservative.

[0015] This invention also provides the application of an abnormal black bile maturation agent extract in the preparation of a tumor treatment drug.

[0016] Preferably, the tumor includes one or more of melanoma cells, breast cancer cells, and cervical cancer cells.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention provides an extract of abnormal black bile maturation agent for anti-tumor treatment and its preparation method and application. The invention uses abnormal black bile maturation agent from Uyghur medicine, which is widely available, inexpensive, and safe, with no toxic side effects on the human body. The preparation method of this invention is simple, using petroleum ether and ethanol solutions to separate and extract the abnormal black bile maturation agent, effectively extracting the anti-tumor active ingredient. The abnormal black bile maturation agent extract prepared by this invention can effectively treat tumors, providing a new option for naturally extracted drugs in clinical tumor treatment. Detailed Implementation

[0019] This invention provides a method for preparing an extract of abnormal black bile maturation agent with anti-tumor properties, comprising the following steps:

[0020] The dry powder of the abnormal black bile ripening agent is mixed with petroleum ether for defatting, filtered to obtain the residue, and 30% to 50% ethanol solution is added to the residue for extraction. The filtrate is then filtered and dried to obtain the final product.

[0021] In this invention, the raw material composition and preparation method of the abnormal black bile maturation agent are described in Chinese Invention Patent Application No. 02130082.8. In this invention, the abnormal black bile maturation agent is composed of the following raw materials in parts by weight: 7 parts lavender, 7 parts fennel, 15 parts *Clerodendrum trichotomum* fruit, 15 parts jujube, 7 parts *Hypericum perforatum*, 7 parts *Lemongrass*, 10 parts licorice, 7 parts *Adiantum capillus-veneris*, 7 parts *Euphorbia humifusa*, and 15 parts *Salix matsudana*. The preparation method of the abnormal black bile maturation agent involves chopping the herbs except for *Salix matsudana*, adding 12 times the amount of water, heating and decocting for 20 minutes, stopping the heating, soaking, letting it stand for 18 hours, then simmering again for 2 hours, filtering, adding *Salix matsudana* to the filtrate while hot and stirring to dissolve, filtering again, and diluting the filtrate with distilled water to 5 times to obtain the abnormal black bile maturation agent. The abnormal black bile maturation agent is then dried at 40-50°C to obtain the dry powder of the abnormal black bile maturation agent.

[0022] In this invention, the dry powder of the abnormal black bile maturation agent is mixed with petroleum ether for degreasing, and the residue is collected by filtration. To remove impurities in the abnormal black bile maturation agent that interfere with its antitumor activity, this invention uses petroleum ether to degrease the dry powder of the abnormal black bile maturation agent. The preferred mass-to-volume ratio of the dry powder of the abnormal black bile maturation agent to petroleum ether is 1:4–6 g / mL, more preferably 1:4.5–5.5 g / mL. The preferred degreasing temperature is 55–65°C, more preferably 58–63°C, and the preferred degreasing time is 1–2 hours. This invention does not specifically limit the filtration method; any method known in the art can be used.

[0023] In this invention, a 30%–50% ethanol solution is added to the filter residue for extraction, followed by filtration and drying of the filtrate. To increase the quantity and content of antitumor active ingredients in the filter residue, the extraction temperature is preferably 45–55°C, more preferably 48–52°C. This extraction temperature improves the extraction rate while ensuring the activity of the active ingredients in the extract. The number of extractions is preferably 1–3 times, and the extraction time for each extraction is preferably 2–4 hours. The mass-to-volume ratio of the filter residue to the 30%–50% ethanol solution is preferably 1:14–16 g / mL. The solvent for the ethanol solution is preferably water, and the ethanol concentration percentage is a volume percentage. This invention does not have specific limitations on the preparation of the ethanol solution; any preparation method known in the art can be used. This invention utilizes the above extraction method to increase the content of active ingredients in the abnormal black bile maturation agent.

[0024] The present invention also provides an extract of abnormal black bile maturation agent prepared by the above preparation method.

[0025] The present invention also provides a medicament for treating antitumor diseases, the medicament comprising an active ingredient and pharmaceutically acceptable excipients, the active ingredient including the above-mentioned abnormal black bile maturation agent extract.

[0026] This invention also provides the application of an abnormal black bile maturation agent extract in the preparation of a tumor treatment drug.

[0027] The dosage form of the drug can be powder, granules, tablets, capsules, drop pills, pills, powder, lyophilized powder for injection, or solution. The drug of the present invention can be administered in unit dose form, such as orally, intramuscularly, subcutaneously, nasally, through the oral mucosa, skin, peritoneum, or rectum. The drug of the present invention can also be administered by injection, including intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection. The excipients include one or more of the following: diluents, flavoring agents, humectants, binders, lubricants, and preservatives. The diluent may be one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, etc.; the humectant and binder may be one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; the lubricant may be one or more of talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc.; the preservative may be one or more of sodium benzoate, parabens, sorbic acid, etc. The flavoring agent includes one or more of sodium alginate, gum arabic, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, etc.

[0028] The dosage of the drug of this invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the frequency of administration, and the therapeutic purpose. Therefore, the therapeutic dosage of this invention can vary widely. Generally speaking, the dosage of the drug used in this invention is well known to those skilled in the art. Appropriate adjustments can be made to the actual amount of drug contained in the pharmaceutical preparation according to this invention to achieve the required therapeutic dose and fulfill the preventive or therapeutic purpose of this invention. The daily dosage of the drug of this invention 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] In this invention, the tumor includes one or more of melanoma cells, breast cancer cells, and cervical cancer cells.

[0030] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0031] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] Example 1

[0033] A method for preparing an extract of abnormal black bile maturation agent with anti-tumor properties, comprising the following steps:

[0034] Weigh 100g of the dry powder of abnormal black bile maturation agent and mix it with 500mL of petroleum ether. Heat at 60℃ for 1.5h to defatted the mixture. Filter and collect residue 1. Add 40% ethanol solution to residue 1 and mix evenly. The mass-volume ratio of the residue to the 40% ethanol solution is 1:15g / mL. Extract at 50℃ for 3h. Filter and collect filtrate a and residue 2. Mix residue 2 with 40% ethanol solution at a mass-volume ratio of 1:15g / mL. Extract at 50℃ for 2h. Filter and collect filtrate b. Combine filtrate a and filtrate b, concentrate under reduced pressure, and dry at 45℃ until no alcohol odor is detected. This yields the anti-tumor abnormal black bile maturation agent extract.

[0035] Example 2

[0036] A method for preparing an extract of abnormal black bile maturation agent with anti-tumor properties, comprising the following steps:

[0037] Weigh 100g of the dry powder of abnormal black bile maturation agent and mix it with 400mL of petroleum ether. Heat at 55℃ for 2 hours to defatted the mixture. Filter the mixture and collect the residue. Add 50% ethanol solution to the residue and mix well. The mass-volume ratio of the residue to the 50% ethanol solution is 1:16g / mL. Extract the mixture at 45℃ for 4 hours. Filter the mixture and collect filtrate a. Concentrate the filtrate under reduced pressure and dry it at 40℃ until there is no alcohol odor. This yields the anti-tumor abnormal black bile maturation agent extract.

[0038] Example 3

[0039] A method for preparing an extract of abnormal black bile maturation agent with anti-tumor properties, comprising the following steps:

[0040] Weigh 100g of the dry powder of abnormal black bile maturation agent and mix it with 600mL of petroleum ether. Heat at 65℃ for 1h to defatted the mixture. Filter and collect residue 1. Add 30% ethanol solution to residue 1 and mix evenly. The mass-volume ratio of the residue to the 30% ethanol solution is 1:14g / mL. Extract at 55℃ for 2h. Filter and collect filtrate a and residue 2. Mix residue 2 with 30% ethanol solution at a mass-volume ratio of 1:14g / mL and extract at 55℃ for 2h. Filter and collect filtrate b and residue 3. Mix residue 3 with 30% ethanol solution at a mass-volume ratio of 1:14g / mL and extract at 55℃ for 2h. Filter and collect filtrate c. Combine filtrate a, filtrate b, and filtrate c, concentrate under reduced pressure, and dry at 50℃ until no alcohol odor remains. This yields the anti-tumor abnormal black bile maturation agent extract.

[0041] Experimental Example 1

[0042] This study evaluated the antitumor activity of the abnormal black bile maturation agent extract prepared in Example 1 using breast cancer cell line MCF-7, melanoma B16, and HeLa cells.

[0043] Experimental grouping and drug administration:

[0044] Abnormal black bile maturation agent extract was weighed and prepared into abnormal black bile maturation agent extract solutions of 25, 50, 75 and 100 μg / mL using DMEM medium.

[0045] The blank control group was DMEM culture medium, and the sample treatment groups were 25, 50, 75, and 100 μg / mL of abnormal black bile maturation agent extract solution.

[0046] Test method:

[0047] MCF-7, B16, and HeLa cells were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2. Cells were passaged when they exhibited logarithmic growth. When cells reached 80%–90% confluence, they were seeded in 96-well plates (density: 4 × 10⁶ cells / well). 3Cells / well), 100 μL / well, incubated at 37℃, 5% CO2 for 24 h. After incubation, the supernatant was discarded, and 90 μL / well of DMEM complete medium containing 10% FBS was added. 10 μL / well of abnormal black bile maturation agent extract solution (25, 50, 75, 100 μg / mL) was added to each well, and 10 μL / well was added to the blank control group. Each experiment was performed in triplicate, and incubated for 72 h. The supernatant was discarded, and 20 μL of 5 mg / mL MTT solution was added to each well. Incubation continued for 4 h. The supernatant was carefully removed by pipette, and 150 μL of LDMSO was added to each well. After shaking for 15 min, the absorbance of each well was measured using a microplate reader (490 nm wavelength). The tumor inhibition rate was calculated as follows: Inhibition rate (%) = (OD2 / 2) * (OD2 / 2) * (D ... 空白对照组 -OD 样品处理组 )÷OD 空白对照组 ×100%, the results are shown in Table 1.

[0048] Table 1. Results of antitumor activity of extracts from different concentrations of abnormal black bile maturation agents.

[0049]

[0050] As shown in Table 1, the abnormal black bile maturation agent extract prepared in this invention can significantly inhibit the growth of breast cancer, melanoma and cervical cancer cells, and has significant anti-tumor activity.

[0051] 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 tumor-treating drug, characterized in that, The tumor is one or both of melanoma cells and breast cancer cells; 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, and 30% to 50% ethanol solution is added to the residue for extraction. The filtrate is then filtered and dried to obtain the final product. The extraction temperature is 45–55°C.

2. The application according to claim 1, characterized in that, The dry powder of the abnormal black bile maturation agent has a mass-to-volume ratio of 1:4 to 6 g / mL to petroleum ether.

3. The application according to claim 1, characterized in that, The degreasing temperature is 55-65℃, and the degreasing time is 1-2 hours.

4. The application according to claim 1, characterized in that, The extraction is performed 1 to 3 times, with each extraction lasting 2 to 4 hours.

5. The application according to claim 1, characterized in that, The mass-to-volume ratio of the filter residue to the 30%–50% ethanol solution is 1:14–16 g / mL.

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