CN111329871A - Preparation method and application of product of cordyceps militaris for preventing and treating liver cancer - Google Patents
Preparation method and application of product of cordyceps militaris for preventing and treating liver cancer Download PDFInfo
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- CN111329871A CN111329871A CN202010175547.0A CN202010175547A CN111329871A CN 111329871 A CN111329871 A CN 111329871A CN 202010175547 A CN202010175547 A CN 202010175547A CN 111329871 A CN111329871 A CN 111329871A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
- A61K36/066—Clavicipitaceae
- A61K36/068—Cordyceps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
Abstract
The invention establishes a preparation method and application of a product for preventing and treating liver cancer by cordyceps militaris, and belongs to the technical field of development of cordyceps militaris products. Cordyceps militaris is used as a raw material, active ingredients of the anti-liver cancer drug are extracted and separated, and the product is prepared according to a certain formula process. The experimental results of preliminary toxicity, drug effect and the like show that the product is nontoxic and has the efficacy of treating liver cancer. Because the active ingredients and the auxiliary materials are extracted from the cordyceps militaris, the product has no toxic or side effect on human bodies, and can be safely used for preventing and treating liver cancer by the public.
Description
Technical Field
The invention relates to the technical field of cordyceps militaris development, and particularly relates to development and application of products for preventing and treating liver cancer by cordyceps militaris.
Background
According to the latest statistics, China has become a genuine big liver cancer country, and the youngest patients are only 19 years old. In 2019, more than half of liver cancer cases all occur in China all over the world, 80% of the cases are late-stage, and the prevention of liver cancer is the key point for solving the problems.
The cordyceps sinensis is an entomomycete complex with cordyceps sinensis parasitizing in lepidoptera hepialidae larvae and taking the cordyceps sinensis as a carrier, contains rich active substances such as protein, fat, cordycepic acid, nucleoside substances, various amino acids and the like, has faint scent taste, and has the record of good effects of keeping lung, tonifying kidney, replenishing essence and marrow, stopping bleeding and reducing phlegm, and treating diaphragm diseases as early as the 'Bencao Chun Xin (New materia Medica'). The traditional Chinese medicine considers that the cordyceps sinensis enters the lung and kidney two channels, can tonify lung yin and kidney yang, is mainly used for treating kidney deficiency, impotence and spermatorrhea, soreness and pain of waist and knees, weakness after illness, chronic cough and weakness, cough with phlegm and blood, spontaneous perspiration and night sweat and the like, and is the only traditional Chinese medicine capable of balancing and adjusting yin and yang simultaneously. The research at present proves that the cordyceps militaris has the effects of inhibiting tumors, relieving cough and calming, dispelling wind, dissipating heat, retaining beauty and keeping young, delaying senility, improving the immunity of the organism, calming and hypnotizing and the like.
Cordyceps militaris (Cordyceppsmilitaris) belongs to new resource food (No. 3 of 2009, bulletin of Ministry of health), belongs to the genus of Cordyceps together with Cordyceps in taxonomy, and is a precious medicinal fungus, and the laboratory researches prove that the chemical components of Cordyceps militaris and Cordyceps are similar to more than 70%, the pharmacological action is similar to that of Cordyceps, and the Cordyceps militaris can be clinically used as a medicament instead of Cordyceps. Because the requirements of the cordyceps sinensis on the growth environment are strict, the cordyceps sinensis resource is extremely limited and cannot meet the increasing market demand. At present, the artificial cultivation of cordyceps militaris is successful, the large-scale production is realized, and the artificial cultivation of cordyceps militaris can be used for replacing cordyceps militaris to be used as a medicine.
Cordyceps militaris is selected as a raw material, an anti-insomnia effective component ergosterol is obtained through solvent extraction and chromatographic separation, and cordycepin and a cordyceps militaris extract are matched to develop a series of green and healthy foods or medicines with the effects of preventing and treating liver cancer, so that the health problem of the public who prevent liver cancer and live affected by liver cancer is solved.
Disclosure of Invention
The invention establishes a preparation method and application of a product for preventing and treating liver cancer by cordyceps militaris, the product has no toxic or side effect on a human body, can be safely used for preventing and treating liver cancer by the human body, and comprises the following steps:
(1) preparation of ergosterol (C310-6-2), cordycepin, C3-12
(a) Pulverizing oven-dried fruiting body of Cordyceps militaris at ultralow temperature. Adding a certain amount of ultrapure water into the superfine powder, heating and extracting for 3 times, each time for 3h, mixing the extractive solutions, concentrating for use, dispersing the concentrate with a proper amount of water, adding 95% ethanol until the final concentration of ethanol is 80%, mixing well, precipitating in a refrigerator at 4 deg.C overnight, centrifuging to obtain supernatant and precipitate, concentrating, and drying for use;
(b) taking the supernatant obtained in the step (a), adding 15% methanol-water to dissolve until the concentration is 200mg/ml, filtering by using a microporous membrane, separating by using high performance liquid chromatography, wherein a chromatographic column is DAC150(150 x 250mm C1810 mu m), the elution mode is 15% methanol-water isocratic elution for 70min, the ultraviolet detection wavelength is 260nm, collecting 35-50min fractions, concentrating and drying to obtain a cordycepin sample;
(c) taking the precipitate obtained in the step (a), adding acetone for extraction, centrifuging, taking supernatant, separating by using a high performance liquid chromatography, eluting for 45min by using a DAC150(150 x 250mm Silica 10 mu m) chromatographic column which sequentially comprises 100% of n-hexane for 10min, 25% of ethyl acetate-n-hexane for 25min and 100% of ethyl acetate for 10min, detecting the ultraviolet wavelength to be 260nm, collecting 18-25min fraction, drying for later use, collecting 35-45min fraction, concentrating and drying to obtain a C3-12 sample;
(d) collecting 18-25min fraction from (C), dissolving in dichloromethane to concentration of 200mg/ml, filtering with microporous membrane, separating with high performance liquid chromatography (DAC 150(150 x 250mm Silica 10 μm), eluting for 40min, ultraviolet detecting wavelength of 260nm, collecting 18-30min fraction, concentrating, and drying to obtain ergosterol (C310-6-2) sample;
(2) product manufacture
The product is prepared by mixing ergosterol (C310-6-2), cordycepin and C3-12 as raw materials, wherein the ergosterol (C310-6-2) accounts for 30% by mass, the cordycepin accounts for 30% by mass and the C3-12 accounts for 40% by mass;
according to different requirements of later-stage dosage forms, ergosterol (C310-6-2), cordycepin and C3-12 can be further processed into series products such as tablets, capsules, granules and the like by matching with other dosage form auxiliary materials according to any proportion.
Preferably, the ultrafine grinding treatment in the step (1) (a) is to perform coarse grinding treatment on the cordyceps militaris raw material after drying treatment by using a grinder with a 50-80-mesh screen, and perform grinding treatment (300-500 mesh) on the cordyceps militaris raw material after coarse grinding treatment by using an ultrafine grinder to obtain the cordyceps militaris ultrafine powder.
Preferably, the extraction in step (1) (a) is performed by stirring once every 0.5h for 3h, combining the extracts, and concentrating.
Preferably, the mass of the extraction solvent added in the step (1) (a) is 1.5 to 2.5 times of the mass of the precipitate.
Preferably, the centrifugation treatment in the step (1) (a) refers to placing the concentrated extract solution after temperature reduction treatment in a centrifuge bottle, wherein the centrifugation temperature is 8-12 ℃, the centrifugation time is 0.5-1.5h, and the rotation speed is 3000-6000 rpm/min.
Preferably, the chromatographic column packing used for preparing the liquid phase in the step (1) (b) is C18 with the diameter of 10 μm, the size is 150mm, × 250mm, the elution mode is that the C18 is eluted with 15% methanol-water isocratic for 70min, the ultraviolet detection wavelength is 260nm, the sample loading amount is 200mL, and the flow rate is 600 mL/min.
Preferably, the chromatographic column filler used for preparing the liquid phase in the step (1) (c) is Silica with the diameter of 10 microns, the size is 150mm × 250mm, the elution mode is that the chromatographic column filler is eluted for 45min, and the chromatographic column filler is 100% n-hexane for 10min, 25% ethyl acetate-n-hexane for 25min and 100% ethyl acetate for 10min in sequence, the ultraviolet detection wavelength is 260nm, 18-25min fractions are collected, the sample loading amount is 200mL, and the flow rate is 600 mL/min.
Preferably, the temperature of the concentration and drying treatment in the step (1) (c) is 60-70 ℃.
Preferably, the chromatographic column packing used for preparing the liquid phase in the step (1) (d) is Silica with the diameter of 10 μm and the size of 150mm × 250 mm.
Preferably, the product in the step (2) is prepared by mixing ergosterol (C310-6-2), cordycepin and C3-12 as raw materials, wherein the mass ratio of the ergosterol (C310-6-2) is 30%, the mass ratio of the cordycepin is 30% and the mass ratio of the C3-12 is 40%.
Preferably, the ergosterol (C310-6-2), cordycepin and C3-12 can be further processed into a series of products such as tablets, capsules, granules and the like according to different requirements of later-stage dosage forms by matching with other dosage form auxiliary materials in any proportion.
The invention establishes a preparation method and application of a product for preventing and treating liver cancer by cordyceps militaris, and belongs to the technical field of development of cordyceps militaris products. Cordyceps militaris is used as a raw material, ergosterol which is an anti-liver cancer medicinal effective component is obtained through solvent extraction and chromatographic separation, and cordycepin and a cordyceps militaris extract are matched to develop a series of green and healthy foods or medicines with the effects of preventing and treating liver cancer, so that the health problem of people who prevent liver cancer and live under the influence of liver cancer is solved.
The following description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood and to be implemented according to the content of the description, the preferred embodiments of the present invention are described below.
Drawings
FIG. 1 is a flow chart showing the preparation of natural products for preventing and treating liver cancer according to examples 1 and 2 of the present invention in FIG. 1;
FIG. 2 shows acute toxicity test of products (five kinds of mixture) of Cordyceps militaris for preventing and treating liver cancer. a. The average weight change result of each group of mice, and b, the food intake change result of each group of mice;
FIG. 3 shows the death status of animals in each group of acute toxicity test of the product (five kinds of mixture) for preventing and treating liver cancer by cordyceps militaris. a. A five-mixture low-dose group (1g/kg), a b.five-mixture medium-dose group (2g/kg), and a c.five-mixture high-dose group (4 g/kg);
FIG. 4 shows the acute toxicity test of the product (five mixtures) for preventing and treating liver cancer of Cordyceps militaris, the influence of the five mixtures on each main organ (heart, liver, lung, kidney, brain) of mouse;
FIG. 5 shows the average tumor weight (g) of mice in H22 tumor resistant experiment of the product (five kinds of mixture) of Cordyceps militaris for preventing and treating liver cancer;
FIG. 6 shows the average liver weight (g) of mice in H22 tumor resistant experiment of the product (five kinds of mixture) for preventing and treating liver cancer of Cordyceps militaris;
FIG. 7 shows the average change (g) of net weight of mice in H22 tumor resistant experiment of the product (five kinds of mixture) for preventing and treating liver cancer of Cordyceps militaris;
FIG. 8 shows the tumor tissues of H22-resistant experimental mice of Cordyceps militaris products (mixture of five types) for preventing and treating liver cancer;
FIG. 9 shows H22 tumor-resistant H22 mouse liver tissue of the product (five kinds of mixture) for preventing and treating liver cancer of Cordyceps militaris.
Detailed Description
The present invention will be described in further detail with reference to examples. It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention provides a preparation method of a product for preventing and treating liver cancer by cordyceps militaris, which specifically comprises the following steps:
(1) preparation of ergosterol (C310-6-2), cordycepin, C3-12
(a) Pulverizing oven-dried fruiting body of Cordyceps militaris at ultralow temperature. Adding a certain amount of ultrapure water into the superfine powder, heating and extracting for 3 times, each time for 3h, mixing the extractive solutions, concentrating for use, dispersing the concentrate with a proper amount of water, adding 95% ethanol until the final concentration of ethanol is 80%, mixing well, precipitating in a refrigerator at 4 deg.C overnight, centrifuging to obtain supernatant and precipitate, concentrating, and drying for use;
(b) taking the supernatant obtained in the step (a), adding 15% methanol-water to dissolve until the concentration is 200mg/ml, filtering by using a microporous membrane, separating by using high performance liquid chromatography, wherein a chromatographic column is DAC150(150 x 250mm C1810 mu m), the elution mode is 15% methanol-water isocratic elution for 70min, the ultraviolet detection wavelength is 260nm, collecting 35-50min fractions, concentrating and drying to obtain a cordycepin sample;
(c) taking the precipitate obtained in the step (a), adding acetone for extraction, centrifuging, taking supernatant, separating by using a high performance liquid chromatography, eluting for 45min by using a DAC150(150 x 250mm Silica 10 mu m) chromatographic column which sequentially comprises 100% of n-hexane for 10min, 25% of ethyl acetate-n-hexane for 25min and 100% of ethyl acetate for 10min, detecting the ultraviolet wavelength to be 260nm, collecting 18-25min fraction, drying for later use, collecting 35-45min fraction, concentrating and drying to obtain a C3-12 sample;
(d) collecting 18-25min fraction from (C), dissolving in dichloromethane to 200mg/ml, filtering with microporous membrane, separating with high performance liquid chromatography (DAC 150(150 x 250mm Silica 10 μm), eluting for 40min, detecting ultraviolet wavelength at 260nm, collecting 18-30min fraction, concentrating, and drying to obtain ergosterol (C310-6-2) sample;
(2) product manufacture
The product is prepared by mixing ergosterol (C310-6-2), cordycepin and C3-12 as raw materials, wherein the ergosterol (C310-6-2) accounts for 30% by mass, the cordycepin accounts for 30% by mass and the C3-12 accounts for 40% by mass;
according to different requirements of later-stage dosage forms, ergosterol (C310-6-2), cordycepin and C3-12 can be further processed into series products such as tablets, capsules, granules and the like by matching with other dosage form auxiliary materials according to any proportion.
Example 1 was carried out: acute toxicity test of the product (mixture of five types) on mice
Examination of acute toxicity of five classes of mixtures
[ drugs and reagents ]
And (3) testing a sample: five kinds of mixture sample (batch: 20130930, purity: 100%, preservation method: refrigeration at 4 deg.C, preparation method: grinding and mixing with 2% Tween 80)
Additive: tween 80 (split package by beijing bornurite technologies ltd).
[ Experimental animals ]
KM mice (18-22 g, half male and female) (animal license number: SCXK (Jing) 2012-0001, certificate number: 11400700020779), animal source (SCXK (Jun) 2012-0004, Experimental animal center of military medical academy of China, SCXK (Jun) 2012-0004), feed source (SCXK (Jun) 2007-001), breeding environment: normal grade, room temperature 21 ℃, and conventional feeding.
[ Experimental methods ]
1. Grouping and administration method
The 40 experimental mice are divided into 4 groups, 10 mice in each group, half of each male and female, ① mixture high dose group (4g/kg), ② mixture medium dose group (2g/kg), ③ mixture low dose group (1g/kg) and ④ blank group (2% Tween 80 normal saline), and the groups are subjected to intragastric administration once according to the administration dose of each group after 12 hours of fasting.
2. Observation index
The mice of each group were weighed daily, diet was calculated, and the mice were observed for death and toxic reactions according to the attached table (standard reference table), recorded, and observed dissected on day 14.
[ Experimental results ]
From the results shown in Table 1, it can be preliminarily presumed that the five types of mixed drugs have no effect on the body weight and diet of mice when administered orally in a dose range of 1g/kg to 4g/kg (see FIG. 2 in particular). The survival rate can reach 80% at a high dose (4g/kg), and the death time is 1 day after administration (see fig. 3 specifically, except that the self-dissolving phenomenon of organs with longer death time is not easy to observe, the caecum of observable mouse organs is larger, and the death is probably related to the excessive diet). In the adverse reactions, various degrees of toxic reactions were observed in the administered dose range of 1g/kg to 4g/kg, including piloerection, perioral hair wetness, drowsiness, limp stools, and lacrimation, involving the autonomic nervous system and the central sleep system (see Table 2 for details).
The surviving mice were dissected 14 days after administration and observed for major organs of the body, and no significant organ changes were observed (see in particular fig. 4).
According to the experimental result, five kinds of mixed drugs LD are calculated by using Bliss method50As a result, LD50 was 57.94 g/kg.
TABLE 1 acute toxicity test results for five kinds of mixtures
General observations of the acute toxicity test with attached tables and tissues, organs, systems that may be involved
Example 2 was carried out: the product (five kinds of mixture) can be used for resisting H22 tumor
Five kinds of mixture anti-liver cancer tumor effects are examined by adopting H22 tumor-bearing mouse model
[ drugs and reagents ]
H22 hepatoma strain (generation 2), five mixture samples (20130930), cyclophosphamide tablet (20130101 from Tianjin Jinshi pharmaceutical Co., Ltd.), and Tween 80 (subpackaged by Beijing Borun Lett technologies Co., Ltd.).
[ test animals ]
KM mouse (18-22 g) (animal license number SCXK (Jing) 2012-0001, certificate number 11400700020779)
[ instruments and apparatus ]
Syringe, aseptic plate, centrifuging tube, surgical instruments, superclean bench, tissue homogenizer.
[ procedure ]
1. Establishment of mouse H22 liver cancer tumor-bearing model
Taking a tumor-bearing dislocation with good inoculated tumor state for 8-13 days for killing, sterilizing body surface alcohol, peeling the tumor of a mouse in a sterile (super clean bench) environment to a sterile flat dish containing normal saline, shearing to small blocks, putting the small blocks of tumor into a tissue homogenizer, adding the normal saline into the tissue homogenizer according to the volume ratio of about 1:3 for homogenizing, and pouring the homogenate into a 50ml sterile centrifuge tube for later use. 0.2ml of the tumor cell suspension was prepared under sterile conditions (in a clean bench) by injecting 0.2ml of the suspension into the right underarm of KM mice using a 1ml syringe. The tumor was completed within 60 minutes from removal to completion of tumor inoculation.
2. Process for the preparation of a medicament
Five kinds of mixture intragastric administration agents: weighing five types of mixture samples with certain weight into a mortar, adding Tween-80 with the volume of 2% of the required solvent, fully grinding, adding physiological saline with the required volume, and uniformly mixing for later use.
Cyclophosphamide intragastric agent: removing the film coat of the cyclophosphamide tablet, weighing, adding a certain weight into a mortar, adding physiological saline with required volume, fully grinding, and mixing uniformly for later use.
3. Grouping and administration method
50 test mice were divided into 5 groups of 10 mice each, each half of the mice were divided into ① mixture low dose (67mg/kg), ② mixture medium dose (200mg/kg), ③ mixture high dose (600mg/kg), ④ cyclophosphamide and ⑤ model groups, and the groups were administered once daily, 0.2ml each, for 10 days.
4. Observation index
4.1 general State
The body weight and the food intake of the mice are weighed and recorded every day, and the net weight of the mice after tumor removal is calculated for the last time.
4.2 tumor weight and tumor inhibition Rate
The right axilla of the mice were observed daily, and the tumor volume (mm) was determined and recorded when the growth of tumor mass was clearly discernible by naked eye3) 0.5 × major diameter × minor diameter2. After the last administration for 24h, dissecting and taking out tumor tissue, weighing the tumor weight, and calculating the tumor growth inhibition rate according to the following formula.
4.3 liver tissue weight and cancer liver metastasis Rate
After the last administration for 24h, the liver tissue is dissected and taken out, the weight of the liver tissue is weighed, whether the liver tissue changes or not is observed, and the number of mice with liver cancer metastasis is counted.
5. Data statistics
The corresponding data of each group of mice are shown in the specification
Statistics are shown using SPSS software, and one-way ANOVA tests are used for each group.
[ results ] A method for producing a compound
The experimental results of table 2 and fig. 5 to 9 show that the five kinds of mixtures have significant difference (P <0.05) when the administration dose is 600mg/kg and the administration days are 10 days compared with H22 liver cancer tumor model mice, and the liver cancer tumor has higher inhibition rate when the dose is 600 mg/kg; the cyclophosphamide group has very significant difference (P <0.05) compared with the model group, and the tumor inhibition rate has no significant difference when the dosage is 600mg/kg compared with the cyclophosphamide group.
No cancer cell liver metastasis phenomenon appears in each group of mice.
In conclusion, the five mixtures are presumed to have certain curative effects on mouse H22 liver cancer tumor models when the administration dose is 600mg/kg and the administration dose is 10 days.
TABLE 2 results of antitumor tests
Note: p <0.01, p <0.05 compared to model groups.
Claims (10)
1. The preparation method of the product for preventing and treating liver cancer by cordyceps militaris is characterized by comprising the following steps:
(1) preparation of ergosterol (C310-6-2), cordycepin, C3-12
(a) Pulverizing oven-dried fruiting body of Cordyceps militaris at ultralow temperature, adding a certain amount of ultrapure water into the superfine powder, heating and extracting for 3 times, each for 3 hr, mixing extractive solutions, and concentrating. Dispersing the concentrate with appropriate amount of water, adding 95% ethanol until the final concentration of ethanol is 80%, mixing, precipitating in a refrigerator at 4 deg.C overnight, centrifuging to obtain supernatant and precipitate, concentrating, and drying;
(b) taking the supernatant obtained in the step (a), adding 15% methanol-water to dissolve until the concentration is 200mg/ml, filtering by using a microporous membrane, separating by using high performance liquid chromatography, wherein a chromatographic column is DAC150(150 x 250mm C1810 mu m), the elution mode is 15% methanol-water isocratic elution for 70min, the ultraviolet detection wavelength is 260nm, collecting 35-50min fractions, concentrating and drying to obtain a cordycepin sample;
(c) taking the precipitate obtained in the step (a), adding acetone for extraction, centrifuging, taking supernatant, separating by using a high performance liquid chromatography, eluting for 45min by using a DAC150(150 x 250mm Silica 10 mu m) chromatographic column which sequentially comprises 100% of n-hexane for 10min, 25% of ethyl acetate-n-hexane for 25min and 100% of ethyl acetate for 10min, detecting the ultraviolet wavelength to be 260nm, collecting 18-25min fraction, drying for later use, collecting 35-45min fraction, concentrating and drying to obtain a C3-12 sample;
(d) collecting 18-25min fraction from (C), dissolving in dichloromethane to 200mg/ml, filtering with microporous membrane, separating with high performance liquid chromatography (DAC 150(150 x 250mm Silica 10 μm), eluting for 40min, detecting ultraviolet wavelength at 260nm, collecting 18-30min fraction, concentrating, and drying to obtain ergosterol (C310-6-2) sample;
(2) product manufacture
The product is prepared by mixing ergosterol (C310-6-2), cordycepin and C3-12 as raw materials, wherein the ergosterol (C310-6-2) accounts for 30% by mass, the cordycepin accounts for 30% by mass and the C3-12 accounts for 40% by mass;
according to different requirements of later-stage dosage forms, ergosterol (C310-6-2), cordycepin and C3-12 can be further processed into series products such as tablets, capsules, granules and the like by matching with other dosage form auxiliary materials according to any proportion.
2. The method for extracting and separating the effective components of the anti-liver cancer medicament from the cordyceps militaris as the raw material as claimed in claim 1, is characterized in that: the ultramicro-pulverization treatment in the step (1) (a) is to perform coarse pulverization treatment on the dried cordyceps militaris raw material by using a pulverizer with a 50-80-mesh screen, and perform pulverization treatment (300-500 mesh) on the cordyceps militaris raw material subjected to coarse pulverization treatment by using an ultramicro pulverizer to obtain the cordyceps militaris ultramicro powder.
3. The method for extracting and separating the effective components of the anti-liver cancer medicament from the cordyceps militaris as the raw material as claimed in claim 1, is characterized in that: the extraction operation in the step (1) (a) is that the mixture is stirred once every 0.5h, the extraction time is 3h every time, and the extracting solutions are combined and concentrated.
4. The method for extracting and separating the effective components of the anti-liver cancer medicament from the cordyceps militaris as the raw material as claimed in claim 1, is characterized in that: the mass of the extraction solvent added in the step (1) (a) is 1.5-2.5 times of the mass of the precipitate.
5. The method for extracting and separating the effective components of the anti-liver cancer medicament from the cordyceps militaris as the raw material as claimed in claim 1, is characterized in that: the centrifugation treatment in the step (1) (a) refers to placing the concentrated extracting solution after temperature reduction treatment in a centrifuge bottle, wherein the centrifugation temperature is 8-12 ℃, the centrifugation time is 0.5-1.5h, and the rotating speed is 3000-.
6. The method for extracting and separating the effective components of the anti-liver cancer drug from the cordyceps militaris as the raw material according to claim 1, wherein the filler of the chromatographic column used in the liquid phase prepared in the step (1) (b) is C18 with the diameter of 10 μm and the size of 150mm × 250mm, the elution mode is that the elution is carried out in 15% methanol-water isocratic elution for 70min, the ultraviolet detection wavelength is 260nm, the sample loading amount is 200mL, and the flow rate is 600 mL/min.
7. The method for extracting and separating effective components of an anti-liver cancer drug from Cordyceps militaris as a raw material according to claim 1, wherein the chromatographic column filler used in the step (1) (c) is Silica with a diameter of 10 μm and a size of 150mm × 250mm, the elution mode is that the chromatographic column filler is eluted for 45min, and the chromatographic column filler, the elution mode is sequentially 100% n-hexane for 10min, 25% ethyl acetate-n-hexane for 25min and 100% ethyl acetate for 10min, the ultraviolet detection wavelength is 260nm, 18-25min fractions are collected, the sample loading amount is 200mL, and the flow rate is 600 mL/min.
8. The method for extracting and separating the effective components of the anti-liver cancer drug from the cordyceps militaris as the raw material as claimed in claim 1, wherein the chromatographic column filler used in the liquid phase preparation in the step (1) (d) is Silica with the diameter of 10 μm and the size of 150mm × 250 mm.
9. The method for preparing a product for preventing and treating liver cancer according to claim 1, wherein the product comprises: the ergosterol (C310-6-2) is prepared by mixing 30% by mass, the cordycepin 30% by mass and C3-12% by mass.
10. The method for preparing a product for preventing and treating liver cancer according to claim 1, wherein the product comprises: the product takes ergosterol (C310-6-2), cordycepin and C3-12 as raw materials, and the ergosterol (C310-6-2), cordycepin and C3-12 can be further processed into a series of products such as tablets, capsules, granules and the like according to different requirements of later-stage dosage forms by matching with other auxiliary materials of the dosage forms in any proportion.
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| CN111956672A (en) * | 2020-07-23 | 2020-11-20 | 贵州贵安精准医学研究院股份有限公司 | Extraction and application of cordyceps militaris antibacterial active substance |
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