CN113663014A - Preparation method of toad feeding particles for lung cancer - Google Patents

Preparation method of toad feeding particles for lung cancer Download PDF

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CN113663014A
CN113663014A CN202111090363.5A CN202111090363A CN113663014A CN 113663014 A CN113663014 A CN 113663014A CN 202111090363 A CN202111090363 A CN 202111090363A CN 113663014 A CN113663014 A CN 113663014A
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solvent
sunflower
lung cancer
ethanol
water
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赵荣华
谢春祥
蒋震
谢永鑫
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Heilongjiang Chanbao Biotechnology Development Co ltd
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Heilongjiang Chanbao Biotechnology Development Co ltd
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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
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/65—Amphibians, e.g. toads, frogs, salamanders or newts
    • 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
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/076—Poria
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/284—Atractylodes
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/481—Astragalus (milkvetch)
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0215—Solid material in other stationary receptacles
    • B01D11/0253—Fluidised bed of solid materials
    • B01D11/0257—Fluidised bed of solid materials using mixing mechanisms, e.g. stirrers, jets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/028—Flow sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0288—Applications, solvents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass

Abstract

The invention discloses a preparation method of toad feeding particles for lung cancer, which comprises the following steps: weighing Ganoderma, nidus Vespae, Bombyx Batryticatus, greedy worm, radix astragali, parched Atractylodis rhizoma, Poria and medulla Junci, mixing, pulverizing, adding the pulverized medicinal materials into an extractor, adding solvent multiple times by pressure pump, reflux extracting, filtering under reduced pressure, concentrating the filtrate, and drying to obtain Bufonis worm granule. The obtained toad feeding particles and vinorelbine can inhibit the growth, proliferation and transfer of non-small cell lung cancer cells in various ways, and the inhibition effect is improved; the side effect is low, and the side effects of constipation and the like caused by singly using the vinorelbine are effectively improved; the extractor of the invention can be used to fully extract the used medicinal materials, has high extraction rate and simple and convenient operation.

Description

Preparation method of toad feeding particles for lung cancer
Technical Field
The invention relates to the field of toad larva processing, in particular to a preparation method of toad larva granules for lung cancer.
Background
Primary bronchogenic carcinoma, lung cancer for short, is one of common malignant tumors and occupies the first cause of cancer death in the world. The onset of lung cancer is mainly influenced by the deterioration of environmental pollution, the increase of smoking population, genetic effect, radiation, specific occupation and other factors, and shows a great trend of increasing year by year. Lung cancer usually occurs in bronchi or mucosal epithelial cells of bronchi, and accounts for more than nine folds of lung parenchymal tumors. Non-small cell lung cancer (NSCLC) accounts for over 85% of the total lung cancer, and in recent decades, poor prognosis of NSCLC has resulted in patient survival rates of less than 5 years, despite significant improvements in diagnostic and therapeutic techniques. The final treatment prognosis of lung cancer patients is still not ideal due to cytotoxicity or drug resistance and other adverse reactions generated after non-small cell lung cancer is treated by western medicine. Therefore, new therapeutic strategies or drugs are important to improve the survival rate of lung cancer patients.
Traditional Chinese medicine treatment is one of the treatment strategies, however, due to the different polarities of active ingredients of the medicines in a plurality of Chinese herbal medicines, the traditional Chinese medicine extraction is often incomplete, and the waste of medicinal material resources is caused.
Disclosure of Invention
Aiming at the situation, the invention aims to provide a preparation method of toad eating insect granules related to lung cancer, wherein the toad eating insect granules are prepared by mixing medicinal herbs such as ganoderma lucidum, nidus vespae, white muscardine silkworm, greedy eating insect, raw astragalus, fried bighead atractylodes rhizome, poria cocos and corduroy, and changing the polarity of a solvent by utilizing pressure difference to fully release active ingredients in the medicinal herbs, and the prepared toad eating insect granules are combined with a western medicine vinorelbine to treat non-small cell lung cancer, and have remarkable effect and low side effect.
The technical scheme for solving the problem is as follows:
a preparation method of toad feeding worm particles related to lung cancer comprises the following steps: s1, weighing Ganoderma, nidus Vespae, Bombyx Batryticatus, greedy worm, radix astragali, parched Atractylodis rhizoma, Poria, and medulla Junci at a certain proportion; s2, mixing and crushing the raw materials in the step S1, adding the mixture into an extractor, opening a pressure pump, adding a solvent, and soaking for 1-2 hours to obtain a medicinal material soaking solution; s3, refluxing and extracting the medicinal material soak solution, and filtering under reduced pressure to obtain filtrate and filter residue; s4, pressing solvent, reflux extracting, filtering under reduced pressure to obtain filtrate and residue, and releasing active ingredients with different polarities from the herbal medicine in S1 by using the polarity of pressure difference solvent; s5, collecting the filtrate obtained in the steps S3 and S4, concentrating, spray drying, and sieving with a 80-100 mesh sieve to obtain medicine extraction powder; s6, adding dextrin into the medicine extraction powder obtained in the step S5, uniformly mixing, granulating, dripping an ethanol wetting agent, uniformly mixing to obtain wet granules, and drying to obtain the toad feeding particles.
Further, in the step S1, the ratio of the lucid ganoderma, the nidus vespae, the white muscardine silkworm, the toad larva, the sunflower polysaccharide, the raw astragalus, the fried bighead atractylodes rhizome, the poria cocos and the juncus effuses is as follows: 6-10 parts of 9-12 parts of 23-35 parts of 0.05-0.1 part of 15-25 parts of 10-15 parts of 10-23 parts of 8-13 parts of C.
Further, the specific operations of pressing solvent extraction in steps S2 and S3 are: the amount of the pressed solvent is 6-10 times of the total mass of the raw materials, and the reflux extraction time is 60-90min each time.
Further, sunflower polysaccharide is added when the concentration is half the volume in step S5.
Further, in step S6, the mass ratio of the drug extract powder to the dextrin is 1:2, and the ethanol concentration in the ethanol wetting agent is 60%.
Further, the sunflower polysaccharide is extracted by the following method: (1) drying sunflower stem core at 45 deg.C to remove water, pulverizing, and sieving with 80 mesh sieve to obtain sunflower powder; (2) adding 20 times of 95% ethanol, reflux-extracting for 3-4 times, reflux-extracting with 80% ethanol for 3-4 times, and removing residual ethanol and water to obtain sunflower residue; (3) adding 20 times of ultrapure water, leaching twice at 90 ℃, leaching for 4h each time, filtering, collecting filtrate, concentrating to 100mL, adding 400mL of 80% ethanol, standing for 24h, centrifuging at 5000rpm for 20min, drying the precipitate to remove water to obtain crude sunflower polysaccharide; (4) adding 15 times of ultrapure water to dissolve crude sunflower polysaccharide, adding 20% Sevag reagent (n-butanol: chloroform = 1: 4), shaking at constant speed for 30min, standing for 5min, collecting supernatant, and repeating for several times until no milky denatured protein is precipitated to obtain sunflower polysaccharide.
Further, the toad feeding insects are obtained by the following method: (1) cleaning living toads with clear water, then putting the toads into the mixed solution, taking out after 72h, cleaning again, beating to cause dizziness, finally pulping the toads uniformly, putting the toads into the chrysomyia megacephala to be produced to produce maggots, collecting the maggots, and cleaning to obtain fresh and alive toads eating; (2) heating Bufonis venenum to 45 deg.C, drying at 50-60 deg.C for 10-14 hr to remove water to obtain Bufonis venenum; the preparation method of the mixed solution comprises the following steps: 100 g of magnolia officinalis, 100 g of immature bitter orange, 100 g of mirabilite and 100 g of rhubarb are taken, mixed and crushed, decocted in water for 30min and filtered, 25-30 kg of clean water is added into the filtrate, and the water temperature is kept at 26-28 ℃.
Further, the extractor comprises a material barrel, a feed inlet is arranged at the top of the material barrel, a reflux device covers the top of the material barrel, a stirring device and a temperature control heating device are arranged at the bottom of the material barrel, a liquid collecting tank is arranged below the bottom of the material barrel, the material barrel is connected with the liquid collecting tank through a solid-liquid filter, a check valve is arranged on the solid-liquid filter, a liquid outlet is arranged at the bottom of the liquid collecting tank, an air outlet is arranged at the top of the liquid collecting tank and connected with a pressure reducing device, a guide pipe is arranged at the feed inlet of the material barrel, one end of the guide pipe extends into the feed inlet, the other end of the guide pipe is connected with a solvent tank, a pressure pump is arranged on the guide pipe, a first solvent tank and a second solvent tank are arranged in the solvent tank, the heights of the first solvent tank and the second solvent tank are the same, the volume of the second solvent tank is 2 times that of the first solvent tank, the guide pipe extends into the lower part of the first solvent tank, the top of the first solvent tank is communicated with the bottom of the second solvent tank, the top of the second solvent box is provided with a liquid inlet.
Further, before the extractor works, the solvent in the first solvent tank is deionized water, and the solvent in the second solvent tank is ethanol.
Furthermore, the toad feeding particle and the vinorelbine are used together to achieve a synergistic effect on resisting the non-small cell lung cancer cells.
The toad is pungent in taste, cool in nature and low in toxicity, can counteract toxicity and kill parasites, relieve pain, eliminate edema and break abdominal mass, and has an effect of inhibiting cancer cell proliferation; the maggot is salty and cold in taste, enters spleen and stomach channels, clears away heat and toxic materials, removes food retention, has slight toxicity, and the toad eating maggot obtained by breeding the maggot by taking the toad as food has the double effects of the toad and the maggot and can reduce the toxic and side effects caused by the toad.
The lucid ganoderma is sweet and bitter in taste and cool in nature, and has the effects of clearing away heat and toxic materials, regulating qi, relieving pain, relieving cough and asthma; the white muscardine silkworm is pungent and salty in flavor and neutral in nature, enters liver and lung channels, and can dispel wind, relieve spasm, extinguish wind, relieve pain, resolve phlegm and dissipate stagnation; the nidus vespae is neutral in nature and sweet in flavor, enters stomach channel, and has the effects of killing parasites, counteracting toxic substances, relieving swelling and alleviating pain; the raw astragalus root is sweet and warm in nature, enters lung and spleen channels, and has the effects of tonifying qi, consolidating superficial resistance, promoting urination, expelling pus, healing sore and promoting tissue regeneration; the bighead atractylodes rhizome is sweet, pungent and warm in flavor, enters the channels of the heart, spleen, stomach, kidney and triple energizer, and can remove dampness, promote digestion, tonify qi, strengthen yin and benefit qi of the waist and navel; poria has sweet and mild taste, and has effects of promoting diuresis, eliminating dampness, invigorating spleen, and calming heart, and medulla Junci can clear heart fire, induce diuresis and treat stranguria. According to the application, lucid ganoderma, white muscardine silkworm, nidus vespae and toad insect eating are combined to tonify qi and nourish yin, spleen is acquired, qi and blood are generated, lung qi is generated by spleen soil, lung qi is abundant, whether the function of transportation and transformation of food essence of spleen is normal or not depends on, and a series of spleen and lung strains caused by lung cancer can be effectively relieved by tonifying lung and spleen with astragalus, bighead atractylodes rhizome, poria cocos and rush.
According to the modern traditional Chinese medicine theory, the yield mode of A549 is influenced by adopting lucid ganoderma, white muscardine silkworm, nidus vespae and toad insect to interfere the gene of key enzyme and protein in the glycolysis reaction process and the activity of protein expression, so that the proliferation of the A549 is hindered, the apoptosis of cells is promoted, and the growth of lung cancer tumor bodies is improved; the sunflower contains flavonoid compounds, terpenoid compounds, phenolic compound organic compounds, polysaccharide and other components, and plays an anti-inflammatory role by inhibiting the content of TNF-alpha, thereby indirectly inhibiting the proliferation of lung cancer tumor cells and simultaneously reducing the migration of the lung cancer tumor cells; meanwhile, the immunity function is enhanced and the anti-tumor activity is improved by using the astragalus, the bighead atractylodes rhizome, the poria cocos and the rush. The effect of the combination of the vinorelbine on the treatment of the non-small cell lung cancer is greatly improved compared with the effect of the combination of the vinorelbine and the vinorelbine, the combination of the vinorelbine and the combination of the vinorelbine can inhibit the growth, the proliferation and the transfer of the non-small cell lung cancer cells from various ways, and can effectively reduce the side effects of constipation and the like caused by the single use of the vinorelbine.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages:
1. according to the preparation method of the toad larva eating particles related to lung cancer, the polarity of the mixed solvent of ethanol and water is changed by utilizing the pressure difference, so that active ingredients with different polarities in the medicinal herbs of ganoderma lucidum, nidus vespae, white muscardine silkworm, greedy larva eating, raw astragalus, fried bighead atractylodes rhizome, poria cocos and wick are fully released, the extraction efficiency is good, and the operation is simple and convenient;
2. the toad eating particle prepared by the preparation method of the toad eating particle related to the lung cancer is combined with vinorelbine to inhibit the growth, proliferation and transfer of the non-small cell lung cancer cells from various ways, so that the inhibition effect is improved;
3. the toad eating particle prepared by the preparation method of the toad eating particle related to the lung cancer is combined with the vinorelbine, so that the overall side effect is low, and the side effects of constipation and the like caused by singly using the vinorelbine are effectively improved.
Drawings
FIG. 1 is a diagram of an extractor according to the present invention.
Detailed Description
The foregoing and other technical and scientific aspects, features and utilities of the present invention will be apparent from the following detailed description of the embodiments, which is to be read in connection with the accompanying drawings of fig. 1. The structural contents mentioned in the following embodiments are all referred to the attached drawings of the specification.
Example 1
A preparation method of toad feeding worm particles related to lung cancer comprises the following steps: s1, weighing Ganoderma, nidus Vespae, Bombyx Batryticatus, greedy worm, radix astragali, parched Atractylodis rhizoma, Poria, and medulla Junci at a certain proportion; s2, mixing and crushing the raw materials in S1, adding the mixture into a material barrel of an extractor, opening a pressure pump, adding a solvent, and soaking for 1 hour to obtain a medicinal material soaking solution; s3, refluxing and extracting the medicinal material soak solution, and filtering under reduced pressure to obtain filtrate and filter residue; s4, pressing solvent, reflux extracting, filtering under reduced pressure to obtain filtrate and residue, and fully releasing active ingredients with different polarities in the herbal medicine of S1 by using the polarity of pressure difference solvent for 3 times; s5, collecting the filtrate obtained in the steps S3 and S4, concentrating, spray drying, and sieving with a 80-mesh sieve to obtain medicine extraction powder; s6, adding dextrin into the medicine extraction powder obtained in the step S5, uniformly mixing, granulating, dripping an ethanol wetting agent, uniformly mixing to obtain wet granules, and drying to obtain the toad feeding granule.
6 parts of lucid ganoderma, 9 parts of nidus vespae, 8 parts of white muscardine silkworm, 23 parts of toad eating insects, 0.05 part of sunflower polysaccharide, 15 parts of raw astragalus membranaceus, 10 parts of fried bighead atractylodes rhizome, 10 parts of poria cocos and 8 parts of juncus effuses in the S1.
The specific operations of pressing solvent extraction in steps S2 and S3 are: the amount of the pressed solvent is 6 times of the total mass of the raw materials, and the reflux extraction time is 60min each time.
Sunflower polysaccharide was added when the concentration was half volume in step S5.
In step S6, the mass ratio of the drug extract powder to dextrin is 1:2, and the ethanol concentration in the ethanol wetting agent is 60%.
The sunflower polysaccharide is extracted by the following method: (1) taking 1kg of sunflower stem core, drying at 45 ℃ to remove water, crushing, and sieving with a 80-mesh sieve to obtain sunflower powder; (2) adding 20 times of 95% ethanol, extracting under reflux for 3 times, extracting with 80% ethanol under reflux for 3 times, and removing residual ethanol and water to obtain sunflower residue; (3) adding 20 times of ultrapure water, leaching twice at 90 ℃, leaching for 4h each time, filtering, collecting filtrate, concentrating to 100mL, adding 400mL of 80% ethanol, standing for 24h, centrifuging at 5000rpm for 20min, drying the precipitate to remove water to obtain crude sunflower polysaccharide; (4) adding 15 times of ultrapure water to dissolve crude sunflower polysaccharide, adding 20% Sevag reagent (n-butanol: chloroform = 1: 4), shaking at constant speed for 30min, standing for 5min, collecting supernatant, and repeating for several times until no milky denatured protein is precipitated to obtain sunflower polysaccharide.
The toad feeding insect is obtained by the following method: (1) cleaning 1kg of living toads with clear water, then putting the toads into 10 times of mixed liquor, taking out after 72h, cleaning again, beating the toads to cause dizziness, finally pulping the toads uniformly, putting the toads into chrysomyia megacephala to be produced to produce maggots, collecting the maggots, and cleaning to obtain fresh and alive toads eating; (2) heating Bufonis venenum to 45 deg.C, drying at 50 deg.C for 10 hr to remove water to obtain Bufonis venenum; the preparation method of the mixed solution comprises the following steps: 100 g of magnolia officinalis, 100 g of immature bitter orange, 100 g of mirabilite and 100 g of rhubarb are taken, mixed and crushed, decocted in water for 30min and filtered, 25 kg of clear water is added into the filtrate, and the water temperature is kept at 26 ℃.
As shown in fig. 1, the extractor comprises a material barrel 1, a feed inlet 3 is arranged at the top of the material barrel 1, a reflux device 2 is covered at the top of the material barrel 1, a stirring device 4 and a temperature control heating device 5 are arranged at the bottom of the material barrel 1, a liquid collecting tank 7 is arranged below the bottom of the material barrel 1, the material barrel 1 is connected with the liquid collecting tank 7 through a solid-liquid filter 4, a stop valve 6 is arranged on the solid-liquid filter 4, a liquid outlet 9 is arranged at the bottom of the liquid collecting tank 7, an air outlet 8 is arranged at the top of the liquid collecting tank 7, the air outlet 8 is connected with a pressure reducing device, a conduit 10 is arranged at the feed inlet 3 of the material barrel 1, one end of the conduit 10 extends into the feed inlet 3, the other end of the conduit 10 is connected with a solvent tank, a pressure pump 11 is arranged on the conduit, a first solvent tank 12 and a second solvent tank 13 are arranged in the solvent tank, the height of the first solvent tank 12 is the same as that of the second solvent tank 13, and the volume of the second solvent tank is 2 times that of the first solvent tank 12, the conduit 10 extends into the lower part of the first solvent tank 12, the top of the first solvent tank 12 is communicated with the bottom of the second solvent tank 13, and the top of the second solvent tank 12 is provided with a liquid inlet 14.
Before the extractor works, the solvent in the first solvent tank 12 is deionized water, and the solvent in the second solvent tank 13 is ethanol.
Example 2
A preparation method of toad feeding worm particles related to lung cancer comprises the following steps: s1, weighing Ganoderma, nidus Vespae, Bombyx Batryticatus, greedy worm, radix astragali, parched Atractylodis rhizoma, Poria, and medulla Junci at a certain proportion; s2, mixing and crushing the raw materials in S1, adding the mixture into a material barrel of an extractor, opening a pressure pump, adding a solvent, and soaking for 1.5 hours to obtain a medicinal material soaking solution; s3, refluxing and extracting the medicinal material soak solution, and filtering under reduced pressure to obtain filtrate and filter residue; s4, pressing solvent, reflux extracting, filtering under reduced pressure to obtain filtrate and residue, and fully releasing active ingredients with different polarities in the herbal medicines in S1 by using the polarity of pressure difference solvent for 4 times; s5, collecting the filtrate obtained in the steps S3 and S4, concentrating, spray drying, and sieving with a 80-mesh sieve to obtain medicine extraction powder; s6, adding dextrin into the medicine extraction powder obtained in the step S5, uniformly mixing, granulating, dripping an ethanol wetting agent, uniformly mixing to obtain wet granules, and drying to obtain the toad feeding granule.
8 parts of lucid ganoderma, 10 parts of nidus vespae, 9 parts of white muscardine silkworm, 24 parts of toad eating insects, 0.08 part of sunflower polysaccharide, 20 parts of raw astragalus membranaceus, 12 parts of fried bighead atractylodes rhizome, 16 parts of poria cocos and 10 parts of juncus effuses in the S1.
The specific operations of pressing solvent extraction in steps S2 and S3 are: the amount of the pressed solvent is 8 times of the total mass of the raw materials, and the reflux extraction time is 75min each time.
Sunflower polysaccharide was added when the concentration was half volume in step S5.
In step S6, the mass ratio of the drug extract powder to dextrin is 1:2, and the ethanol concentration in the ethanol wetting agent is 60%.
The sunflower polysaccharide is extracted by the following method: (1) taking 1.2kg of sunflower stem core, drying at 45 ℃ to remove water, crushing, and sieving with a 80-mesh sieve to obtain sunflower powder; (2) adding 20 times of 95% ethanol, extracting under reflux for 3 times, extracting with 80% ethanol under reflux for 3 times, and removing residual ethanol and water to obtain sunflower residue; (3) adding 20 times of ultrapure water, leaching twice at 90 ℃, leaching for 4h each time, filtering, collecting filtrate, concentrating to 100mL, adding 400mL of 80% ethanol, standing for 24h, centrifuging at 5000rpm for 20min, drying the precipitate to remove water to obtain crude sunflower polysaccharide; (4) adding 15 times of ultrapure water to dissolve crude sunflower polysaccharide, adding 20% Sevag reagent (n-butanol: chloroform = 1: 4), shaking at constant speed for 30min, standing for 5min, collecting supernatant, and repeating for several times until no milky denatured protein is precipitated to obtain sunflower polysaccharide.
The toad feeding insect is obtained by the following method: (1) cleaning 1.2kg of living toads with clear water, then putting the toads into 10 times of mixed liquor, taking out after 72h, cleaning again, beating the toads to cause dizziness, finally pulping the toads uniformly, putting the toads into chrysomyia megacephala to be produced to produce maggots, collecting the maggots, and cleaning to obtain fresh and alive toad eating insects; (2) heating Bufonis venenum to 45 deg.C, drying at 55 deg.C for 12 hr to remove water to obtain Bufonis venenum; the preparation method of the mixed solution comprises the following steps: mixing cortex Magnolia officinalis 100 g, fructus Aurantii Immaturus 100 g, Natrii sulfas 100 g, and radix et rhizoma Rhei 100 g, pulverizing, decocting in water for 30min, filtering, adding clear water 28 kg into the filtrate, keeping the water temperature at 27 deg.C
Before the extractor works, the solvent in the first solvent tank is deionized water, and the solvent in the second solvent tank is ethanol.
Example 3
A preparation method of toad feeding worm particles related to lung cancer comprises the following steps: s1, weighing Ganoderma, nidus Vespae, Bombyx Batryticatus, greedy worm, radix astragali, parched Atractylodis rhizoma, Poria, and medulla Junci at a certain proportion; s2, mixing and crushing the raw materials in S1, adding the mixture into a material barrel of an extractor, opening a pressure pump, adding a solvent, and soaking for 2 hours to obtain a medicinal material soaking solution; s3, refluxing and extracting the medicinal material soak solution, and filtering under reduced pressure to obtain filtrate and filter residue; s4, pressing solvent, reflux extracting, filtering under reduced pressure to obtain filtrate and residue, and releasing active ingredients with different polarities from the herbs in S1 by using the polarity of pressure difference solvent for 5 times; s5, collecting the filtrate obtained in the steps S3 and S4, concentrating, spray drying, and sieving with a 100-mesh sieve to obtain medicine extraction powder; s6, adding dextrin into the medicine extraction powder obtained in the step S5, uniformly mixing, granulating, dripping an ethanol wetting agent, uniformly mixing to obtain wet granules, and drying to obtain the toad feeding granule.
10 parts of lucid ganoderma, 12 parts of nidus vespae, 10 parts of white muscardine silkworm, 35 parts of toad eating insect, 0.1 part of sunflower polysaccharide, 25 parts of raw astragalus, 15 parts of fried bighead atractylodes rhizome, 23 parts of poria cocos and 13 parts of juncus effuses in the S1.
The specific operations of pressing solvent extraction in steps S2 and S3 are: the amount of the pressed solvent is 10 times of the total mass of the raw materials, and the reflux extraction time is 90min each time.
Sunflower polysaccharide was added when the concentration was half volume in step S5.
In step S6, the mass ratio of the drug extract powder to dextrin is 1:2, and the ethanol concentration in the ethanol wetting agent is 60%.
The sunflower polysaccharide is extracted by the following method: (1) taking 1.3kg of sunflower stem core, drying at 45 ℃ to remove water, crushing, and sieving with a 80-mesh sieve to obtain sunflower powder; (2) adding 20 times of 95% ethanol, reflux-extracting for 4 times, reflux-extracting with 80% ethanol for 4 times, and removing residual ethanol and water to obtain sunflower residue; (3) adding 20 times of ultrapure water, leaching twice at 90 ℃, leaching for 4h each time, filtering, collecting filtrate, concentrating to 100mL, adding 400mL of 80% ethanol, standing for 24h, centrifuging at 5000rpm for 20min, drying the precipitate to remove water to obtain crude sunflower polysaccharide; (4) adding 15 times of ultrapure water to dissolve crude sunflower polysaccharide, adding 20% Sevag reagent (n-butanol: chloroform = 1: 4), shaking at constant speed for 30min, standing for 5min, collecting supernatant, and repeating for several times until no milky denatured protein is precipitated to obtain sunflower polysaccharide.
The toad feeding insect is obtained by the following method: (1) cleaning 1.3kg of living toads with clear water, then putting the toads into 10 times of mixed liquor, taking out after 72 hours, cleaning again, beating the toads to cause dizziness, finally pulping the toads uniformly, putting the toads into chrysomyia megacephala to be produced to produce maggots, collecting the maggots, and cleaning to obtain fresh and alive toad eating insects; (2) heating Bufonis venenum to 45 deg.C, drying at 60 deg.C for 14 hr to remove water to obtain Bufonis venenum; the preparation method of the mixed solution comprises the following steps: 100 g of magnolia officinalis, 100 g of immature bitter orange, 100 g of mirabilite and 100 g of rhubarb are taken, mixed and crushed, decocted in water for 30min and filtered, 30 kg of clear water is added into the filtrate, and the water temperature is kept at 28 ℃.
Before the extractor works, the solvent in the first solvent tank is deionized water, and the solvent in the second solvent tank is ethanol.
Comparative example 1
S1, weighing 30 parts of lucid ganoderma, 30 parts of dried toad skin, 15 parts of nidus vespae and 15 parts of white muscardine silkworm for later use; s2, mixing and crushing ganoderma lucidum, nidus vespae, white muscardine silkworm and dried toad skin, adding into an extractor, opening a pressure pump, adding a solvent, and soaking for 1h to obtain a medicinal material soaking solution; s3, refluxing and extracting the medicinal material soak solution, and filtering under reduced pressure to obtain filtrate and filter residue; s4, performing reflux extraction and reduced pressure filtration according to the steps of pressing a solvent in, and obtaining filtrate and filter residue for 3 times; s5, collecting the filtrate obtained in the steps S3 and S4, concentrating, spray drying, and sieving with a 80-mesh sieve to obtain medicine extraction powder; s6, adding dextrin into the medicine extraction powder obtained in the step S5, uniformly mixing, granulating, dripping an ethanol wetting agent, uniformly mixing to obtain wet granules, and drying to obtain the traditional Chinese medicine granules.
Comparative example 2
The solvent in the second solvent tank of the extractor in example 1 was replaced by ethanol to de-ionized water, and the rest of the procedure was unchanged to obtain the traditional Chinese medicine particles.
Comparative example 3
The operation of adding sunflower polysaccharide in the preparation process of the toad feeding granule described in example 1 was removed to obtain a traditional Chinese medicine granule.
Test example 1
Proliferation inhibitory effect on a549 cell line:
(1) culture and treatment of human lung cancer cells (a 549): human lung cancer cells (A549) were cultured in DMEM medium containing 10% fetal bovine serum and 1% diabody (100U/mL penicillin, 100mg/L streptomycin) at 37 deg.C and 5% CO2Subculturing in an incubator. Adherent cells were digested with 0.25% trypsin 2 passages per week and all cells used in the experiment were cells in logarithmic growth phase. (2) The MTT method detects the influence of different drugs on the proliferation of A549 cells: taking human lung cancer cell (A549) in logarithmic growth phase, and adjusting cell number to 3-5 × 10 with DMEM culture solution of 10% fetal calf serum5And (4) inoculating the cells per mL into a 96-well cell culture plate, wherein each well is 200 mu L, and when 70-80% of the cells adhere to the wall, the old culture solution is discarded. Adding 200 μ L culture solution into each well of blank group and control group, adding 6mg/mL Bufonis insect granule culture solution 200 μ L prepared in example 1 and 6mg/mL Chinese medicinal granule culture solution 200 μ L and 6mg/mL prepared in comparative example 1 into each well of drug group200. mu.L of the culture solution of Chinese medicinal granules prepared in comparative example 2, 200. mu.L of the culture solution of Chinese medicinal granules prepared in comparative example 3 at 6mg/mL, 200. mu.L of the culture solution of Bufonis venenum granules prepared in example 1 at 4mg/mL and 200. mu.L of the culture solution of vinorelbine at 2mg/mL, each set of 5 replicates. After 48 hours of incubation in the same manner, 20. mu.L of MTT (5mg/mL) was added to each well, incubation was continued for 4 hours, the old culture solution was aspirated, 150. mu.L of DMSO was added to each well, the mixture was shaken at 37 ℃ for 15 minutes at a low speed, and the absorbance (A value) was measured at 490 nm. The cell growth inhibition rate was calculated as follows: the inhibition rate was 1- (drug OD value-blank OD value)/(control OD value-blank OD value) × 100%. The results are shown in Table 1:
Figure 550727DEST_PATH_IMAGE001
as can be seen from the experimental results in table 1, compared with the traditional Chinese medicine granule in comparative example 1, the toad feeding granule prepared by the method has a significant inhibition effect on the in vitro proliferation of the human lung cancer a549 cells, and as can be seen from comparative example 2, the medicinal material extracted by the extractor of the method has a significantly enhanced proliferation inhibition effect; the addition of sunflower polysaccharide helps to improve the inhibition rate of A549 cell proliferation; the toad feeding particles and the vinorelbine are used together to achieve the synergistic inhibition effect, and the inhibition rate is as high as 95%.
Test example 2
Effect on inhibition of growth of a549 cell nude mouse transplanted tumor:
inoculation of a549 cell nude mouse graft tumor: the BALB/c nude mice are bred, the mice are 5-6 weeks old, and the male mice are about 20g in weight. The subcutaneous inoculation transplantation method of cultured cells is adopted. Taking A549 cells in logarithmic growth phase, washing a culture bottle with PBS (phosphate buffer solution) for 2 times, adding 0.25% pancreatin for digestion, centrifuging, discarding supernatant, adding 10mL of DMEM culture solution without fetal calf serum, blowing and uniformly mixing by using a suction pipe, removing supernatant, repeating for 1 time, adding serum-free DMEM culture solution, resuspending cells, adjusting the number of the cells to be about 3 x 107/mL, and inoculating 200 mu L of the cells per nude mouse to the right forelimb oxter subcutaneous tissue. The drug has the following effects of inhibiting the lung cancer transplantation tumor of a nude mouse: after 2 weeks of inoculation, the tumor grows to 100-150mm3Thereafter, the nude mice were randomly divided into 4 groups, eachGroup 10 only: model control group (1 physiological saline injection every 2 days), example 1 group (1 tail vein administration every 2 days, 4mg/kg toad feeding granule of example 1), vinorelbine group (tail vein administration every 2 days)
Figure 533727DEST_PATH_IMAGE002
Vein administration is carried out for 1 time, 4mg/kg of the traditional Chinese medicine granules in the comparative example 1), and a combined drug group (tail vein administration is carried out for 1 time every 2 days, 2mg/kg of the toad feeding granules in the example 1 and 2mg/kg of vinorelbine) are carried out for 6 times. The excretion of the mice before and after administration was observed, and the longest diameter a and the shortest diameter b of the transplanted tumor were measured with a vernier caliper before each administration. At the end of the experiment, all nude mice were sacrificed by dislocation, and the longest diameter a and the shortest diameter b of the tumor were measured with a vernier caliper and the tumor was weighed. Wherein the tumor volume V is 1/2 × a × b2(ii) a The tumor inhibition rate is (tumor weight of model control group-tumor weight of drug use group)/tumor weight of model control group x 100%, and the experimental results are shown in table 2:
as can be seen from the experimental results in table 2, the graft tumor of the model control group increased significantly with time, while the graft tumor of the drug group increased relatively slowly. After 12d administration, the mean tumor volume of the model control group was (1238.5. + -. 114.6) mm3Example 1 set was (624.3. + -. 77.4) mm3The vinorelbine group is (585.4 + -54.2) mm3In contrast, the combination was only (364.1. + -. 38.6) mm3. In addition, the tumor inhibition rates of the group 1, the vinorelbine group and the combined drug group are respectively 51.9%, 40.3% and 69.3%, and it can be seen that the tumor inhibition rate of the combined drug group is significantly higher than that of the single drug group, which shows that the combined drug of the buformin-feeding particles and the vinorelbine has a synergistic effect in inhibiting the proliferation of the human lung cancer A549 cells, and in addition, the constipation of the combined drug group is improved compared with that of the single drug group of the vinorelbine.
While the invention has been described in further detail with reference to specific embodiments thereof, it is not intended that the invention be limited to the specific embodiments thereof; for those skilled in the art to which the present invention pertains and related technologies, the extension, operation method and data replacement should fall within the protection scope of the present invention based on the technical solution of the present invention.

Claims (8)

1. A preparation method of toad feeding worm particles related to lung cancer is characterized by comprising the following steps: s1, weighing Ganoderma, nidus Vespae, Bombyx Batryticatus, greedy worm, radix astragali, parched Atractylodis rhizoma, Poria, and medulla Junci at a certain proportion; s2, mixing and crushing the raw materials in the step S1, adding the mixture into an extractor, opening a pressure pump, adding a solvent, and soaking for 1-2 hours to obtain a medicinal material soaking solution; s3, refluxing and extracting the medicinal material soak solution, and filtering under reduced pressure to obtain filtrate and filter residue; s4, pressing solvent, reflux extracting, filtering under reduced pressure to obtain filtrate and residue, and releasing active ingredients with different polarities from the herbal medicine in S1 by using the polarity of pressure difference solvent; s5, collecting the filtrate obtained in the steps S3 and S4, concentrating, spray drying, and sieving with a 80-100 mesh sieve to obtain medicine extraction powder; s6, adding dextrin into the medicine extraction powder obtained in the step S5, uniformly mixing, granulating, dripping an ethanol wetting agent, uniformly mixing to obtain wet granules, and drying to obtain the toad feeding particles.
2. The method for preparing the buformin granules relating to lung cancer according to claim 1, wherein the steps of S2 and S3 are carried out by pressing a solvent for extraction: the amount of the pressed solvent is 6-10 times of the total mass of the raw materials, and the reflux extraction time is 60-90min each time.
3. The method of claim 1, wherein the sunflower polysaccharide is added when the concentration is half of the volume in step S5.
4. The method of claim 1, wherein the mass ratio of the drug extract powder to the dextrin in step S6 is 1:2, and the ethanol concentration in the ethanol humectant is 60%.
5. The method for preparing the buformin granules relating to lung cancer according to claim 1, wherein the sunflower polysaccharides are extracted by the following method: (1) drying sunflower stem core at 45 deg.C to remove water, pulverizing, and sieving with 80 mesh sieve to obtain sunflower powder; (2) adding 20 times of 95% ethanol, reflux-extracting for 3-4 times, reflux-extracting with 80% ethanol for 3-4 times, and removing residual ethanol and water to obtain sunflower residue; (3) adding 20 times of ultrapure water, leaching twice at 90 ℃, leaching for 4h each time, filtering, collecting filtrate, concentrating to 100mL, adding 400mL of 80% ethanol, standing for 24h, centrifuging at 5000rpm for 20min, drying the precipitate to remove water to obtain crude sunflower polysaccharide; (4) adding 15 times of ultrapure water to dissolve crude sunflower polysaccharide, adding 20% Sevag reagent (n-butanol: chloroform = 1: 4), shaking at constant speed for 30min, standing for 5min, collecting supernatant, and repeating for several times until no milky denatured protein is precipitated to obtain sunflower polysaccharide.
6. The preparation method of the buforms granule for treating lung cancer according to claim 1, wherein the buforms is obtained by the following steps: (1) cleaning living toads with clear water, then putting the toads into the mixed solution, taking out after 72h, cleaning again, beating to cause dizziness, finally pulping the toads uniformly, putting the toads into the chrysomyia megacephala to be produced to produce maggots, collecting the maggots, and cleaning to obtain fresh and alive toads eating; (2) heating Bufonis venenum to 45 deg.C, drying at 50 deg.C for 14 hr to remove water to obtain Bufonis venenum; the preparation method of the mixed solution comprises the following steps: 100 g of magnolia officinalis, 100 g of immature bitter orange, 100 g of mirabilite and 100 g of rhubarb are taken, mixed and crushed, decocted in water for 30min and filtered, 25-30 kg of clean water is added into the filtrate, and the water temperature is kept at 26-28 ℃.
7. The method for preparing the toad feeding particles related to the lung cancer according to claim 1, wherein the extractor comprises a material barrel, a feed inlet is arranged at the top of the material barrel, a reflux device is covered at the top of the material barrel, a stirring device and a temperature control heating device are arranged at the bottom of the material barrel, a liquid collecting tank is arranged below the bottom of the material barrel, the material barrel is connected with the liquid collecting tank through a solid-liquid filter, a check valve is arranged on the solid-liquid filter, a liquid outlet is arranged at the bottom of the liquid collecting tank, an air outlet is arranged at the top of the liquid collecting tank and connected with a pressure reducing device, a guide pipe is arranged at the feed inlet of the material barrel, one end of the guide pipe extends into the feed inlet, the other end of the guide pipe is connected with a solvent tank, a pressure pump is arranged on the guide pipe, a first solvent tank and a second solvent tank are arranged in the solvent tank, the first solvent tank and the second solvent tank are the same in height, and the volume of the second solvent tank is 2 times that of the first solvent tank, the guide pipe stretches into the lower part of the first solvent box, the top of the first solvent box is communicated with the bottom of the second solvent box, and the top of the second solvent box is provided with a liquid inlet.
8. The method for preparing the buformin granules as claimed in claim 7, wherein the solvent in the first solvent tank is deionized water and the solvent in the second solvent tank is ethanol before the extractor is operated.
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