CN105456307B - Pinus massoniana twig bark extract for preventing and treating middle and late stage lung adenocarcinoma as well as preparation method and composition thereof - Google Patents
Pinus massoniana twig bark extract for preventing and treating middle and late stage lung adenocarcinoma as well as preparation method and composition thereof Download PDFInfo
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Abstract
The invention relates to a pinus massoniana twig bark extract for preventing and treating middle and late stage lung adenocarcinoma, a preparation method thereof and a composition thereof. The preparation method of the extract comprises the following steps: 1) pulverizing pinus massoniana twig bark to 800 meshes to obtain pinus massoniana twig bark powder, wherein the pinus massoniana twig bark is obtained by picking the twig of a pinus massoniana branch head branch in the same year in spring and summer; 2) adding water into the pinus massoniana twig bark powder at the temperature of 4-25 ℃, and soaking for 1-10 hours to prepare a pinus massoniana twig bark powder water soaking solution; 3) ultrasonically extracting a masson pine twig bark powder water soaking solution for 5min-2h, and filtering to obtain a masson pine twig bark water extracting solution; 4) drying the water extract of the tender branch bark of the masson pine. The masson pine twig bark extract is mainly used for preventing and treating middle and late stage lung adenocarcinoma and metastasis or complications thereof at lymph nodes, clavicles, ribs, thoracic vertebrae, lumbar vertebrae, pelvises and other parts, and has the advantages of high tumor inhibition rate of 93.66-99.58%, small toxic and side effects, high safety and the like.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine extracts, in particular to a Chinese red pine twig bark extract for preventing and treating middle and late stage lung adenocarcinoma, a preparation method and a composition thereof.
Background
The threat of cancer to human beings is increasing, and the cancer is becoming a first killer endangering human health and life. Overcoming cancer is an urgent task for all people, and all countries take it as an important subject and invest huge resources to overcome the problem. Because the surgical treatment, the radiotherapy and the chemical treatment of cancer have certain limitations and the toxic and side effects of the chemical treatment of cancer are continuously appeared, the international scientific community turns the attention to safer natural plant extracts and becomes a research hotspot for treating cancer.
In the 50 s of 20 th century, Masquerier Phragmites France firstly extracted a flavonoid natural antioxidant mixture, procyanidin, from bark of Pinus massoniana with antioxidant activity of VE50 times of Vc20 times of the total effective amount of the active oxygen, can remove excessive active oxygen free radicals in vivo, reduce lipid peroxidation degree of organism, and has effects of resisting oxidation, delaying aging, and enhancing immunity. In the later 50 s, the French maritime pine bark extract is registered as Pycnogenol drug and used for improving the resistance of blood vessels, reducing the fragility and permeability of capillary vessels, preventing the damage of vascular endothelium and the like. Huynh et al (Huynh et al, Anticancer Res, 1999) 1999 discovered that the extract from French maritime pine bark inhibits the activation of the cancer active metabolite NNK and prevents smoking-induced lung cancer.
Although the research reports about the biological activity of the extracts of the bark of Pinus maritima and Pinus radiata in New Zealand exist abroad, the living environment of the pine in China is unique, and the variety, the content and the property of the secondary metabolites in the pine body are different from those of the pine in China. Wujie et al have studied the difference of active ingredients in different kinds of pine bark in Pinaceae of China, and the results show that the content difference of active substances in different kinds of pine bark is significant, wherein the content of total polyphenol, total flavone, procyanidine and water-soluble polysaccharide in the masson pine bark extract is the highest. In addition, the research on masson pine bark extract mostly focuses on the extraction of active substances such as procyanidine from masson pine bark, and the extraction processes are different.
The extraction process comprises screening, oven drying and pulverizing Pinus massoniana Linne bark, extracting with pure water under heating, coarse-filtering, centrifuging, micro-filtering, removing suspended substances and macromolecular impurities, ultrafiltering with hollow fiber membrane to obtain filtrate containing bioactive components such as proanthocyanidins, nanofiltering, concentrating, removing impurities such as saccharides and inorganic salts and water, and drying. However, the extraction process adopts hot water extraction, the extraction temperature is high, volatile substances contained in the pine bark are lost, the activity of the extract is influenced, and the integrity of the active ingredients of the masson pine bark extract cannot be maintained due to the filtering and concentrating conditions and the removal of saccharides and other substances of the single procyanidin serving as a target substance in the purification process.
Ultrasound extracting Pinus massoniana lamb bark with thin glume method to obtain polyphenol material, which comprises cutting Pinus massoniana lamb bark, drying and pulverizing to obtain pine bark powder, taking 70% ethanol as solvent, performing ultrasound extraction at 30 deg.C with material-liquid ratio of 20:1 (ml: g) for 40min, centrifuging the extractive solution, filtering, and determining polyphenol yield. The method adopts ethanol as extraction solvent, the extraction temperature is slightly higher, and compared with low-temperature extraction with water as extraction solvent, the components of the extract have larger difference.
CN1923830A extracting procyanidin from Pinus massoniana bark with water solvent, specifically comprises taking cortex Pini as raw material, adding 5-10 times of water, boiling and refluxing for 1-2 hr, filtering with 300 mesh pipeline, discharging leaching solution, repeating once, and mixing leaching solutions; centrifuging, passing through column, concentrating, and drying to obtain powder. The method adopts boiling reflux to recover extract, and results in loss of effective components.
CN101468121A discloses mixing alfalfa, raspberry, areca catechu and masson pine bark according to a ratio of 1:1:1:1, adding 3 times of pure water by weight, soaking for 48h, decocting at a temperature of more than or equal to 100 ℃ for 5h, then primarily filtering, finely filtering and microporous bacteria-resistant finely filtering, concentrating, and spray-drying to obtain powder. The method for extracting the raw materials comprises alfalfa, raspberry and areca catechu besides the masson pine bark, so that the mixed components of the extracts are greatly different from the single masson pine bark extract, and a method of high-temperature decoction and spray drying is adopted, so that part of volatile matters of the effective components cannot be locked and lost.
The rhododendron and the like research different extraction methods and different extraction conditions such as reflux, Soxhlet extraction, ultrasonic extraction and the like, and the efficiency of extracting the procyanidine from the masson pine bark through the water solvent shows that the different extraction methods and the different extraction conditions can influence the yield of the procyanidine. And the optimum extraction temperature is 50 ℃ when the procyanidin is extracted by using the ultrasonic method, and the adverse teaching that the low-temperature condition can influence the extraction efficiency of the procyanidin is given.
CN101701013A discloses extracting procyanidin from pine bark with water as solvent, using pine bark as raw material and water as extraction solvent, and performing ultrasonic enhanced extraction to obtain procyanidin leaching solution; then carrying out membrane separation technology of microfiltration impurity removal, ultrafiltration purification and nanofiltration concentration to obtain concentrated solution; and then vacuum drying or freeze drying is carried out to obtain the procyanidin product. The method records that the extraction process is operated under the low temperature condition, but the specific implementation mode selects ultrasonic extraction at 45 ℃ and 50 ℃, the extracted procyanidin is still used as a main target, the molecular weight cut-off which is adaptive to the procyanidin is selected in the purification process, the content of the procyanidin in the obtained freeze-dried product can reach 67-68 wt%, and the total phenol content can reach 85-87 wt%.
In summary, the masson pine bark extract contains a compound formed by combining various components such as phenols, saccharides and the like, and different extraction solvents, extraction processes, extraction conditions, separation and purification operations and the like all cause differences of components and component proportions in the final extract. In addition, the researches on the masson pine bark all adopt the trunk aged bark of the masson pine as an extraction raw material, and no relevant report that the tender branch bark of the masson pine is specifically selected as the extraction raw material is found. The plant tissues of the tender branch bark and the main trunk old bark are in different growth stages, so that the contained bioactive substances are different, and the main trunk old bark grows for a longer time and is greatly influenced by environmental factors, so that the difference of the active ingredients of the extracts of the tender branch bark and the old bark is caused. The extract of masson pine bark is used for inhibiting lung adenocarcinoma, and procyanidine is extracted from the masson pine bark for inhibiting the proliferation activity of a lung adenocarcinoma cell strain Glc-82; the pine bark extract of Liwei et al is used for promoting apoptosis of human lung adenocarcinoma cell A549, but the active ingredient for inhibiting lung adenocarcinoma cell line is procyanidine.
Disclosure of Invention
The invention aims to provide a preparation method of a pinus massoniana twig bark extract for preventing and treating lung adenocarcinoma, which comprises the following steps: 1) pulverizing pinus massoniana twig bark to 800 meshes to obtain pinus massoniana twig bark powder, wherein the pinus massoniana twig bark is obtained by picking twigs of pinus massoniana twig head branches in spring and summer in the current year; 2) adding water into the pinus massoniana twig bark powder at the temperature of 4-25 ℃, and soaking for 1-10 hours, wherein the mass volume ratio of the pinus massoniana twig bark powder to the water is 1:2-1:15, preparing a pinus massoniana tender branch bark powder water soaking solution; 3) ultrasonically extracting a masson pine twig bark powder water soaking solution for 5min-2h, and filtering to obtain a masson pine twig bark water extracting solution; 4) and drying the water extract of the tender branch bark of the pinus massoniana to obtain the pinus massoniana.
In the preferred technical scheme of the invention, the pinus massoniana tender shoot skin is subjected to screening, impurity removal and drying treatment before being crushed, wherein the drying temperature is preferably 30-80 ℃, more preferably 40-70 ℃, and most preferably 50-60 ℃.
In the preferred technical scheme of the invention, the pinus massoniana twig bark is crushed to 250-700 meshes, preferably 300-600 meshes, and more preferably 350-500 meshes.
In the preferred technical scheme of the invention, the picking time of the pinus massoniana tender branch skin is 3-6 months, preferably 4-5 months.
In the preferred technical scheme of the invention, the extraction temperature is 8-20 ℃, and preferably 10-15 ℃.
In the preferred technical scheme of the invention, the soaking time is 2-8h, preferably 3-6h, and more preferably 4-5 h.
In the preferred technical scheme of the invention, the mass volume ratio of the pinus massoniana twig bark powder to the extraction water is 1:4-1:12, preferably 1:6-1: 10.
In a preferred embodiment of the present invention, the water is selected from any one of soft water, distilled water, and deionized water, or a combination thereof.
In the preferred technical scheme of the invention, the ultrasonic extraction times are 1-6 times, preferably 2-5 times, and more preferably 3-4 times.
In a preferred technical scheme of the invention, the ultrasonic extraction condition is ultrasonic for 10-100min, preferably ultrasonic for 15-80min, more preferably ultrasonic for 20-60min, and most preferably ultrasonic for 25-40 min.
In the preferred technical scheme of the invention, the ultrasonic extraction condition is that the ultrasonic power is 200-1000W, preferably 350-900W, and more preferably 500-750W.
In the preferable technical scheme of the invention, the ultrasonic extraction conditions are ultrasonic for 1-100s and interval for 1-100s, preferably ultrasonic for 5-80s and interval for 5-80s, more preferably ultrasonic for 10-70s and interval for 10-70s, and most preferably ultrasonic for 15-60s and interval for 15-60 s.
In the preferred technical scheme of the invention, the filtration comprises primary filtration, fine filtration and membrane-resistant bacteria filtration in sequence, preferably, the primary filtration is filtered by a 100-mesh screen, the fine filtration is repeatedly absorbed and filtered for 4-6 times by using raw white cloth, and the aperture of the membrane-resistant bacteria filtration is 0.22-0.45 mu m.
In a preferred embodiment of the present invention, the drying is selected from any one or a combination of vacuum concentration drying, freeze drying and spray drying.
In a preferred technical scheme of the invention, the effective components in the extract comprise polyphenol, total alkali and polysaccharide, preferably, the effective components contain 2-5% of polyphenol, 15-25% of total alkali and 60-80% of polysaccharide, and more preferably contain 2.39% of polyphenol, 18.07% of total alkali and 78.6% of polysaccharide.
The invention aims to provide a pinus massoniana twig bark extract for preventing and treating lung adenocarcinoma, which is prepared by the preparation method.
In a preferred technical scheme of the invention, the effective components in the extract comprise polyphenol, total alkali and polysaccharide, preferably, the effective components contain 2-5% of polyphenol, 15-25% of total alkali and 60-80% of polysaccharide, and more preferably contain 2.39% of polyphenol, 18.07% of total alkali and 78.6% of polysaccharide.
In a preferred technical scheme of the invention, the extract is powder, and preferably the powder is 100% full water-soluble light brown semitransparent crystals.
The invention aims to provide a pharmaceutical composition for preventing and treating lung adenocarcinoma, which consists of the pinus massoniana twig bark extract prepared by the preparation method and a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention can be prepared by using conventional preparation techniques in the art.
In a preferred technical scheme of the invention, the pharmaceutical composition is selected from any one of oral preparations, external preparations and enteric oral preparations.
In a preferred technical scheme of the invention, the oral preparation is selected from any one of tablets, capsules, granules, powder, oral liquid, suspension, pills, powder, dripping pills, syrup, mixture, distillate, effervescent, paste, emulsion and tea or enteric-coated preparations thereof.
In a preferred technical scheme of the invention, the enteric oral preparation is selected from any one of enteric tablets, enteric capsules, enteric granules, enteric powder, enteric oral liquid, enteric suspension, enteric pills, enteric powder, enteric dropping pills, enteric syrup, enteric mixture, enteric dew, enteric effervescent agents, enteric paste, enteric emulsion and enteric tea.
In a preferred technical scheme of the invention, the external preparation is any one selected from gels, emplastrums, creams, ointments, liniments, lotions, suppositories, smearing agents and ointments.
The pharmaceutically acceptable carrier according to the present invention is a commonly used excipient or adjuvant well known in the art for the preparation of the above-mentioned formulations. The excipients or adjuvants commonly used in oral preparations or external preparations include, but are not limited to, fillers (also called diluents), lubricants (also called glidants or antiadherents), dispersants, wetting agents, binders, regulators, solubilizers, antioxidants, bacteriostats, emulsifiers, flavors, or perfuming agents. Binders, such as syrup, acacia, gelatin, sorbitol, tragacanth, cellulose and its derivatives, gelatin syrup, starch syrup or polyvinylpyrrolidone, preferred cellulose derivatives being microcrystalline cellulose, sodium carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose; fillers, for example lactose, sugar powder, dextrin, starch and derivatives thereof, cellulose and derivatives thereof, inorganic calcium salts, sorbitol or glycine, preferably calcium sulfate, calcium phosphate, calcium hydrogen phosphate or precipitated calcium carbonate; lubricants, such as aerosil, magnesium stearate, talc, aluminium hydroxide, boric acid, hydrogenated vegetable oils or polyethylene glycols; disintegrants, for example starch and its derivatives, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone or microcrystalline cellulose, preferred starch derivatives being sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch or corn starch; wetting agents such as sodium lauryl sulfate, water or alcohols, and the like.
In the preferred technical scheme of the invention, the active ingredient and the pharmaceutically acceptable sustained-release carrier are mixed according to the preparation requirements, and then the mixture is prepared into pellets, such as sustained-release pellets or controlled-release pellets, according to the preparation method of the sustained-release preparation well known in the field, such as adding retardant coating or microencapsulating the active ingredient; the sustained and controlled release carrier comprises but is not limited to an oil-fat doping agent, a hydrophilic colloid, a coating retarder and the like, wherein the oil-fat doping agent is selected from any one or the combination of glyceryl monostearate, hydrogenated castor oil, mineral oil, polysiloxane or dimethyl siloxane; the hydrophilic colloid is selected from any one or combination of sodium carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, PVP, acacia, tragacanth or carbopol; the coating retarder is selected from any one of Ethyl Cellulose (EC), hydroxypropyl methyl cellulose (HMPC), polyvinylpyrrolidone (PVP), Cellulose Acetate Phthalate (CAP), acrylic resin or their combination.
In a preferred technical scheme of the invention, the pharmaceutical composition is a sustained-release preparation or a controlled-release preparation thereof, preferably, the sustained-release preparation is a sustained-release pellet, and the controlled-release preparation is a controlled-release pellet.
In a preferred technical scheme of the invention, the pharmaceutical composition is an enteric-coated tablet, preferably, a pharmaceutical excipient of the enteric-coated tablet is microcrystalline cellulose, and more preferably, the film coating material is hydroxypropyl methylcellulose (HPMC).
In the preferable technical scheme of the invention, the masson pine twig skin enteric-coated tablet contains 30-70 parts of masson pine twig skin extract, 1-10 parts of microcrystalline cellulose, preferably 40-50 parts of masson pine twig skin extract and 3-6 parts of microcrystalline cellulose.
In the preferable technical scheme of the invention, the masson pine twig skin extract enteric-coated tablet with the unit dose contains 15mg of microcrystalline cellulose and 250mg of masson pine twig skin extract.
In the preferable technical scheme of the invention, the masson pine twig skin extract enteric-coated tablet with the unit dosage contains 30mg of microcrystalline cellulose and 500mg of masson pine twig skin extract.
The invention also aims to provide application of the masson pine twig bark extract or the pharmaceutical composition thereof in preparing a medicament for treating lung adenocarcinoma.
In a preferred embodiment of the present invention, the lung adenocarcinoma is selected from middle and advanced lung adenocarcinoma or metastasis thereof at any site of lymph node, clavicle, rib, thoracic vertebra, lumbar vertebra, pelvis, or metastatic complications thereof.
In the preferred technical scheme of the invention, the dosage of the patient is related to factors such as age, sex, constitution, disease condition and the like. The dosage of the adult patient is preferably 500 mg/granule/time, 6 granules each time, and three times daily, more preferably, the dosage of the immature and children patients is half of that of the adult patient, or is properly adjusted according to the age, sex, constitution, disease condition, etc. The medicine is taken with warm water on an empty stomach.
Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.
Compared with the prior art, the invention has the following beneficial technical effects:
1. based on the comparative research on the tender branch bark and the old bark, the inventor selects the tender branch bark of masson pine as a pine bark extraction raw material, and adopts low-temperature ultrasonic water extraction and optimizes the extraction conditions, such as extraction at 4-25 ℃, so that the loss of volatile active ingredients, impurities and heat-sensitive substances are avoided, the purity and the damage of the active ingredients can be kept, the activity of the extract can be kept and the damage of the activity of the extract can be avoided, and the extract with special functional activity is obtained, wherein the extract contains 2-5% of polyphenol, 15-25% of total alkali and 60-80% of polysaccharide.
2. Researches show that other active ingredients except procyanidine in the pinus massoniana twig bark extract provided by the invention can obviously inhibit the proliferation of a lung adenocarcinoma cell line A549, the extract is used for preventing and treating middle and late stage lung adenocarcinoma and complications caused by metastasis of the lung adenocarcinoma in lymph nodes, clavicles, ribs, thoracic vertebrae, lumbar vertebrae and pelvis, the tumor inhibition rate is as high as 93.66-99.58%, and the pinus massoniana twig bark extract has the advantages of small toxic and side effects, high safety and the like.
3. The traditional extraction process adopts high-temperature operations such as heating extraction, heating concentration and the like, so that more than 70 percent of effective components in the extract are invalid, and if the traditional extraction (heating extraction and concentration) can not completely keep the original ecology and pure nature of the medicine; the activity of the traditional extraction is lost by heating (color depth); the traditional extraction heating (color depth and blackening) activity is damaged; the most effective component volatile matters can not be locked and lost when the traditional extraction heating temperature exceeds 100-110 ℃.
4. The masson pine twig bark extract is prepared by adopting a low-temperature ultrasonic water extraction method, so that the solvent pollution is reduced, the discharge of three wastes (waste water, waste residues and waste gases) and the pollution to the environment are avoided, and the safety of the production process is ensured; meanwhile, the method uses the dregs extracted from the tender branch skin of the pinus massoniana to prepare the biological fertilizer or the biomass energy, is beneficial to cyclic utilization of resources, and obviously improves the resource utilization rate.
5. The invention adopts the branch head branch 'twig bark' of the masson pine picked in spring and summer as the medicinal raw material, which is beneficial to protecting forest vegetation and can pull large-area planting in poor mountainous areas to create income and remove poverty to become rich.
Detailed Description
The present invention will be described in detail with reference to examples, which are provided only for illustrating the technical solutions of the present invention and are not intended to limit the spirit of the present invention.
Example 1Preparation of masson pine twig bark extract
The preparation method of the pinus massoniana twig skin extract comprises the following steps:
1) picking the tender branch skin of branches and heads of the pinus massoniana tree which are sent out in 3 months in the current year, screening the tender branch skin which has no visible foreign matters, no rot and no mildew and has the water content of less than or equal to 10 percent, removing impurities by air separation, and drying at 50 ℃ to obtain a pinus massoniana tender branch skin dry product;
2) crushing 1.5Kg of masson pine twig bark dry product to 400 meshes to prepare masson pine twig bark powder;
3) adding soft water into the pinus massoniana twig bark powder at the temperature of 15 ℃, and performing ultrasonic water extraction, wherein the mass volume ratio of the pinus massoniana twig bark powder to the soft water is 1:2, performing ultrasonic extraction under the conditions that the ultrasonic power is 550W, the ultrasonic is performed for 6s, the gap is 6s, and the ultrasonic is performed for 10min to prepare an ultrasonic water extract of the pinus massoniana twig bark;
4) filtering with 100 mesh filter screen, adsorbing and filtering with white cloth for 4 times, filtering the filtrate with 0.22 μm filter membrane for inhibiting bacteria, and freeze drying the filtrate to obtain Pinus massoniana twig bark extract 220 g.
5) The dregs of a decoction extracted from the tender branches and barks of the pinus massoniana are used for preparing the biological fertilizer, so that the resource utilization rate is improved.
Example 2Preparation of masson pine twig bark extract
The preparation method of the pinus massoniana twig skin extract comprises the following steps:
1) picking the tender branch skin of branches and heads of the pinus massoniana tree which are sent out in 4 months in the current year, screening the tender branch skin which has no visible foreign matters, no rot and no mildew and has the water content of less than or equal to 10 percent, removing impurities by air separation, and drying at 80 ℃ to obtain a pinus massoniana tender branch skin dry product;
2) crushing 2.0Kg of masson pine twig bark dry product to 500 meshes to prepare masson pine twig bark powder;
3) adding soft water into the pinus massoniana twig bark powder at the temperature of 25 ℃, and performing ultrasonic water extraction, wherein the mass volume ratio of the pinus massoniana twig bark powder to the soft water is 1: 8, performing ultrasonic extraction under the conditions that the ultrasonic power is 550W, the ultrasonic is performed for 8s, the gap is 8s, and the ultrasonic is performed for 12min to prepare an ultrasonic water extract of the pinus massoniana twig bark;
4) filtering with 100 mesh filter screen, repeatedly adsorbing and filtering with white cloth for 5 times, filtering the filtrate with 0.22 μm filter membrane for inhibiting bacteria, and spray drying to obtain 282g Pinus massoniana twig skin extract.
5) The dregs of a decoction extracted from the tender branches and barks of the pinus massoniana are used for preparing the biological fertilizer, so that the resource utilization rate is improved.
Example 3Preparation of masson pine twig bark extract
The preparation method of the pinus massoniana twig skin extract comprises the following steps:
1) picking the tender branch skin of branch and head branches of the pinus massoniana in the current 5 months, screening the tender branch skin without visible foreign matters, rot and mildew, and with the water content less than or equal to 10%, removing impurities by air separation, and drying at 60 ℃ to obtain a pinus massoniana tender branch skin dry product;
2) crushing 2.5Kg of masson pine twig bark dry product to 450 meshes to prepare masson pine twig bark powder;
3) adding soft water into the pinus massoniana twig bark powder at the temperature of 20 ℃, and performing ultrasonic water extraction, wherein the mass volume ratio of the pinus massoniana twig bark powder to the soft water is 1: 5, performing ultrasonic extraction under the conditions that the ultrasonic power is 600W, the ultrasonic time is 10s, the gap is 10s, and the ultrasonic time is 10min to prepare an ultrasonic water extract of the pinus massoniana twig bark;
4) filtering with 100 mesh filter screen, repeatedly adsorbing and filtering with white cloth for 6 times, filtering the filtrate with 0.45 μm filter membrane for inhibiting bacteria, and spray drying to obtain 356g Pinus massoniana twig bark extract.
5) The dregs of a decoction extracted from the tender branches and barks of the pinus massoniana are used for preparing the biological fertilizer, so that the resource utilization rate is improved.
Example 4Preparation of masson pine twig bark extract
The preparation method of the pinus massoniana twig skin extract comprises the following steps:
1) picking the tender branch skin of branches and heads of the pinus massoniana tree which are sent out in 6 months in the current year, screening the tender branch skin which has no visible foreign matters, no rot and no mildew and has the water content of less than or equal to 10 percent, removing impurities by air separation, and drying at 55 ℃ to obtain a pinus massoniana tender branch skin dry product;
2) crushing 3.0Kg of masson pine twig bark dry product to 200 meshes to prepare masson pine twig bark powder;
3) adding soft water into the pinus massoniana twig bark powder at the temperature of 18 ℃, and performing ultrasonic water extraction, wherein the mass volume ratio of the pinus massoniana twig bark powder to the soft water is 1:10, performing ultrasonic extraction under the conditions that the ultrasonic power is 700W, the ultrasonic time is 15s, the gap is 15s, and the ultrasonic time is 12min to prepare an ultrasonic water extract of the pinus massoniana twig bark;
4) filtering with 100 mesh filter screen, repeatedly adsorbing and filtering with white cloth for 5 times, filtering the filtrate with 0.22 μm filter membrane for inhibiting bacteria, and drying the filtrate under reduced pressure to obtain 396g Pinus massoniana twig skin extract.
5) The dregs of a decoction extracted from the tender branches and barks of the pinus massoniana are used for preparing the biological fertilizer, so that the resource utilization rate is improved.
Example 5Preparation of masson pine twig bark extract
The preparation method of the pinus massoniana twig skin extract comprises the following steps:
1) picking the tender branch skin of branches and heads of the pinus massoniana tree which are sent out in 4 months in the current year, screening the tender branch skin which has no visible foreign matters, no rot and no mildew and has the water content of less than or equal to 10 percent, removing impurities by air separation, and drying at 60 ℃ to obtain a pinus massoniana tender branch skin dry product;
2) crushing 4Kg of masson pine twig bark dry product to 400 meshes to prepare masson pine twig bark powder;
3) adding soft water into the pinus massoniana twig bark powder at the temperature of 4 ℃, and performing ultrasonic water extraction, wherein the mass volume ratio of the pinus massoniana twig bark powder to the soft water is 1:15, performing ultrasonic extraction under the conditions that the ultrasonic power is 700W, the ultrasonic treatment is performed for 20s, the interval is 20s, and the ultrasonic treatment is performed for 10min to prepare an ultrasonic water extract of the pinus massoniana twig bark;
4) filtering with 100 mesh filter screen, repeatedly adsorbing and filtering with white cloth for 5 times, filtering the filtrate with 0.22 μm filter membrane for inhibiting bacteria, and spray drying the filtrate to obtain Pinus massoniana twig bark extract 520 g.
5) The dregs of a decoction extracted from the tender branches and barks of the pinus massoniana are used for preparing the biological fertilizer, so that the resource utilization rate is improved.
Example 6Preparation of masson pine twig bark extract
The preparation method of the pinus massoniana twig skin extract comprises the following steps:
1) picking the tender branch skin of branch and head branches of the pinus massoniana in the current 5 months, screening the tender branch skin without visible foreign matters, rot and mildew, and with the water content less than or equal to 10%, removing impurities by air separation, and drying at 55 ℃ to obtain a pinus massoniana tender branch skin dry product;
2) crushing 5Kg of masson pine twig bark dry product to 450 meshes to prepare masson pine twig bark powder;
3) adding soft water into the pinus massoniana twig bark powder at the temperature of 10 ℃, and performing ultrasonic water extraction, wherein the mass volume ratio of the pinus massoniana twig bark powder to the soft water is 1:10, under the ultrasonic extraction conditions that the ultrasonic power is 700W, the ultrasonic is carried out for 25s, the gap is 25s, and the ultrasonic is carried out for 8min, so as to prepare an ultrasonic water extract of the pinus massoniana twig bark;
4) filtering with 100 mesh filter screen, repeatedly adsorbing and filtering with white cloth for 5 times, filtering the filtrate with 0.22 μm filter membrane for inhibiting bacteria, and spray drying to obtain cortex Pinus massoniana Radicis extract 603 g.
5) The dregs of a decoction extracted from the tender branches and barks of the pinus massoniana are used for preparing the biological fertilizer, so that the resource utilization rate is improved.
Comparative example 1Preparation of masson pine twig bark extract
The preparation method of the pinus massoniana twig skin extract comprises the following steps:
1) picking the tender branch skin of branch and head branches of the pinus massoniana in the current 5 months, screening the tender branch skin without visible foreign matters, rot and mildew, and with the water content less than or equal to 10%, removing impurities by air separation, and drying at 55 ℃ to obtain a pinus massoniana tender branch skin dry product;
2) crushing 5Kg of masson pine twig bark dry product to 100 meshes to prepare masson pine twig bark powder;
3) adding soft water into the pinus massoniana twig bark powder at the temperature of 30 ℃, and performing ultrasonic water extraction, wherein the mass volume ratio of the pinus massoniana twig bark powder to the soft water is 1:10, under the ultrasonic extraction conditions that the ultrasonic power is 700W, the ultrasonic is carried out for 25s, the gap is 25s, and the ultrasonic is carried out for 8min, so as to prepare an ultrasonic water extract of the pinus massoniana twig bark;
4) performing primary filtration by using a 100-mesh filter screen, repeatedly adsorbing and filtering for 5 times by using a raw white cloth, taking filtrate, performing bacteria-resistant filtration by using a filter membrane of 0.22 mu m, and performing spray drying on the filtrate to obtain 256g of masson pine twig bark extract.
5) The dregs of a decoction extracted from the tender branches and barks of the pinus massoniana are used for preparing the biological fertilizer, so that the resource utilization rate is improved.
Comparative example 2Preparation method of Pinus massoniana bark extract
The preparation method of the masson pine bark extract comprises the following steps:
1) screening masson pine barks without visible foreign matters, rottenness and mildew and with the water content less than or equal to 10 percent, removing impurities by winnowing, and drying at 55 ℃ to obtain a masson pine bark dry product;
2) crushing 5Kg of masson pine bark dry product to 450 meshes to prepare masson pine bark powder;
3) adding soft water into masson pine bark powder at the temperature of 16 ℃, and performing ultrasonic water extraction, wherein the mass volume ratio of the masson pine bark powder to the soft water is 1:10, performing ultrasonic extraction under the conditions that the ultrasonic power is 700W, the ultrasonic time is 25s, the interval is 25s, and the ultrasonic time is 8min to prepare masson pine bark ultrasonic water extract;
4) filtering with 100 mesh filter screen, adsorbing and filtering with white cloth for 5 times, filtering the filtrate with 0.22 μm filter membrane for inhibiting bacteria, and spray drying to obtain cortex Pteris Multifidae extract 258 g.
5) The masson pine bark extraction medicine dregs are used for preparing the biological fertilizer, so that the resource utilization rate is improved.
Example 7Detection of pinus massoniana twig bark extract
The pinus massoniana tender shoot bark extracts prepared in the examples 1-6 are light brown, bitter in smell and taste, sweet and bitter in taste, uniform in tissue form, free of visible foreign matter powder (sensory index), and 100% soluble in water; the extract of the comparative example 1 has light color, sweet taste and slight bitter taste, uniform tissue shape, no visible foreign powder, and 100 percent of water solubility; the extract of comparative example 2 was brownish red in color, bitter in flavor, bitter and pungent in taste, uniform in tissue morphology, free of visible foreign matter powder, and 100% soluble in water.
The contents of polyphenol, total alkali and polysaccharide in the masson pine bark extracts obtained in examples 1 to 6 and comparative examples 1 to 2 were measured by HPLC and phenol-sulfuric acid method, and the results are shown in Table 1.
TABLE 1
| Example numbering | Polyphenol (g/100g) | Total alkali (g/100g) | Polysaccharide (g/100g) |
| Example 1 | 4.12 | 20.23 | 73.12 |
| Example 2 | 2.39 | 18.07 | 78.6 |
| Example 3 | 3.47 | 21.72 | 73.20 |
| Example 4 | 4.52 | 24.35 | 65.33 |
| Example 5 | 2.63 | 16.59 | 70.4 |
| Example 6 | 3.12 | 23.24 | 62.56 |
| Comparative example 1 | 0.91 | 12.32 | 63.47 |
| Comparative example 2 | 1.53 | 15.64 | 74.25 |
As can be seen from Table 1, the pulverization degree and extraction conditions of the tender bark of Pinus massoniana, such as extraction temperature, have a great influence on the content of the effective components of the extract. The crushing particles are large, the extraction efficiency of the effective components can be influenced by high extraction temperature, and the loss rate of substances such as polyphenol, total alkali and the like can be increased by high extraction and purification temperature. The extraction results of the masson pine twig bark extract and the old bark extract under the same conditions show that the extraction rates of the masson pine twig bark extract and the old bark extract are greatly different, the effective components of the extracts are obviously different, and the contents of polyphenol and total alkali in the pine bark extract are lower than those of the twig bark extract.
Example 8Growth inhibition effect of pinus massoniana twig skin extract on human lung adenocarcinoma cells
5-fluorouracil is used as a positive drug, and the growth inhibition effect of the pinus massoniana twig skin extract prepared in the examples 1-6 on human lung adenocarcinoma cells A549 and human embryonic lung fibroblast MRC-5 (normal cells) is determined by adopting an MTT method.
The experimental cells (A549, MRC-5) were placed in a medium containing 5% CO2Culturing in a 37 ℃ incubator, digesting with 0.25% pancreatin EDTA after growing over 70-90% of the area, collecting cells, centrifuging for 5min at 1000r/min, pouring out supernatant, uniformly stirring cell precipitates with RM P I1640 culture solution containing 10% calf serum, inoculating into a 25mL culture bottle, and carrying out passage for 2-3 times per week in 5m L each bottle.
The test drug was dissolved in cell culture medium to give a stock solution of 10 mg/ml. Gradient dilutions were made in medium to give concentrations of 64, 32, 16, 8 and 4, 2, 1, 0.5. mu.g/ml drug. The human lung cancer cell A549 cell is paved in a 96-well plate in 10000/hole and 5000/hole for human embryonic lung fibroblast MRC-5 cell, and a culture medium with corresponding concentration is replaced after 48 hours, wherein each concentration is 6 multiple holes; and meanwhile, setting the wells only containing the culture medium as negative controls, sucking the culture medium in each well after incubation for 24h, washing the wells for 1-2 times by using PBS (phosphate buffer solution), replacing the culture medium containing 15% MTT (methyl thiazolyl tetrazolium), and continuing incubation for 4 h. The medium was aspirated off, 150ml DMSO was added, shaking was performed at low speed for 10min, and absorbance at 490nm was measured with a microplate reader at a reference wavelength of 630 nm. The inhibition rate was calculated using the following formula:
the pinus massoniana twig bark extract prepared in the example 1 has a strong growth inhibition effect on human lung adenocarcinoma cells A549, the IC50 value is 4.9 mu g/ml, and the pinus massoniana twig bark extract has no obvious toxic or side effect on normal cells MRC-5 under the concentration of 5 mg/ml. The IC50 value of 5-fluorouracil for human lung adenocarcinoma cells A549 was 2.2. mu.g/ml. The results are shown in Table 2.
TABLE 2 growth inhibition of A549 cells by Pinus massoniana twig bark extract
The growth inhibition effect of the pinus massoniana twig bark extract in example 2-6 on A549 cells is determined by the same method, and the IC50 is 3-7 mu g/ml, and has no obvious toxic or side effect on normal cells. The extracts of comparative examples 1-2 had poor inhibition of A549 cells, and the IC50 values were all higher than those of the shoot bark extracts described in examples 1-6.
The experimental result shows that the pinus massoniana twig bark extract has obvious growth inhibition effect on lung adenocarcinoma cells, and has no obvious toxic or side effect on normal cells. In addition, the Pinus massoniana twig skin extract has an obvious dose-effect relationship on the growth inhibition effect of lung adenocarcinoma cells, namely, the growth inhibition effect on the cells is gradually improved along with the increase of the concentration of the extract.
EXAMPLE 9 examination of anticancer active ingredients in shoot bark extract
The masson pine bark extract solution of example 2 was further treated to remove total alkali and polysaccharides therefrom to obtain a polyphenol extract having a concentration comparable to that of polyphenol in the bark extract. The results of determining the difference of the inhibitory activity of the two extracts on lung adenocarcinoma cell strain A549 are shown as follows:
table 3 comparison of the activity differences of the extracts of the invention and the polyphenol extracts
| Twig bark extract | Polyphenol extract of shoot bark | |
| Polyphenol | 2.39 | 2.3％ |
| Total alkali | 18.07 | 0 |
| Polysaccharides | 78.6 | 0 |
| IC50 of A549 | 3.8μg/ml | 12.3μg/ml |
As can be seen from the table 3, the masson pine twig skin extract is prepared by adopting a low-temperature ultrasonic water extraction method for the first time, the extract contains other components with the activity of inhibiting the lung adenocarcinoma, such as total alkali, polysaccharide and the like except procyanidine, and the effective components show a synergistic effect.
Example 10Preparation of masson pine twig and skin extract enteric-coated tablet
(1) The composition of the masson pine twig skin extract enteric-coated tablet core is as follows:
500g of masson pine twig bark extract
Microcrystalline cellulose 50g
Making into 1000 tablets.
(2) The composition of the enteric coating liquid is as follows:
about 20g of hydroxypropyl methylcellulose (HPMC) of the coating powder is prepared into 225g of coating solution with the concentration of 9 percent by using 95 percent ethanol as a solvent (225 g of coating solution is required for every 500g of medicine).
(3) The preparation method of the masson pine twig skin extract enteric-coated tablet comprises the following steps:
1) weighing 500g of the pinus massoniana twig bark extract prepared in the embodiment 2, adding 50g of microcrystalline cellulose, uniformly mixing, and preparing 1000 tablet cores according to a powder direct compression method;
2) adjusting the air inlet temperature to 85 ℃ and the air outlet temperature to 50 ℃; pouring the tablet core into a coating pan, starting the coating pan at the rotation speed of 1.5r/min, and slightly preheating until the tablet core has temperature; starting the spray liquid, taking attention to the condition of tablets in the coating pan, and properly regulating the speed to 2r/min-3 r/min; when the film coat is coated on the slices, stopping spraying liquid, drying for 3min, turning off hot air, cooling, and taking out.
Example 11Research on effect of pinus massoniana twig skin extract in prolonging life cycle of middle-stage and late-stage lung adenocarcinoma patients
The pinus massoniana twig skin extract prepared in the example 10 is orally taken enteric coated tablets and is used for treating volunteers with middle and advanced lung adenocarcinoma.
Twenty adult patients of volunteers diagnosed with middle and advanced lung adenocarcinoma were selected, of which three advanced patients had found bone metastasis of cancer cells, four advanced patients had found lymph node metastasis of cancer cells, and two advanced patients had found thoracic vertebra metastasis of cancer cells. The patients were dosed as follows: 500 mg/granule/time, six granules per time, three times a day, and is taken with warm boiled water.
The results show that the survival time of twenty patients is prolonged by 2-5 years on average, and the quality of life is obviously improved.
In summary, the masson pine twig skin extract is prepared by adopting a low-temperature ultrasonic water extraction method for the first time, the extract contains other components such as total alkali, polysaccharide and the like which have the activity of inhibiting the lung adenocarcinoma besides procyanidine, and the extract shows a synergistic interaction effect. The masson pine twig bark extract is mainly used for treating the lung adenocarcinoma at the middle and late stages and complications caused by the metastasis of lymph nodes, clavicles, ribs, thoracic vertebrae, lumbar vertebrae and pelvis, has the tumor inhibition rate as high as 93.66-99.58%, and has the advantages of small toxic and side effects, high safety and the like.
Claims (37)
1. A pinus massoniana twig bark extract for preventing and treating lung adenocarcinoma is disclosed, wherein the active ingredients in the extract comprise polyphenol, total alkali and polysaccharide, the active ingredients comprise 2-5% of polyphenol, 15-25% of total alkali and 60-80% of polysaccharide, and the preparation method of the extract comprises the following steps:
1) pulverizing pinus massoniana twig bark to 800 meshes at 200 meshes to obtain pinus massoniana twig bark powder, wherein the pinus massoniana twig bark is obtained by picking the twig of a pinus massoniana branch head branch in the current year in spring and summer, and the picking time of the pinus massoniana twig bark is 4-5 months;
2) adding water into the pinus massoniana twig bark powder at the temperature of 10-15 ℃, and soaking for 1-10h, wherein the mass volume ratio of the pinus massoniana twig bark powder to the water is 1:2-1:15, so as to prepare a pinus massoniana twig bark powder water soaking solution;
3) ultrasonically extracting a masson pine twig bark powder water soaking solution for 5min-2h, and filtering to prepare a masson pine twig bark water soaking solution, wherein the ultrasonic extraction conditions are that the ultrasonic power is 200-1000W, the ultrasonic power is 1-100s, the gap is 1-100s, and the ultrasonic extraction times are 1-6 times;
4) drying the water extract of the tender branch bark of the masson pine to obtain the masson pine extract.
2. The masson pine extract of claim 1, wherein the extract comprises 2.39% polyphenols, 18.07% total alkali and 78.6% polysaccharides.
3. A method for preparing the masson pine tender shoot bark extract for preventing and treating lung adenocarcinoma as claimed in claims 1-2, comprising the steps of:
1) pulverizing pinus massoniana twig bark to 800 meshes at 200 meshes to obtain pinus massoniana twig bark powder, wherein the pinus massoniana twig bark is obtained by picking the twig of a pinus massoniana branch head branch in the current year in spring and summer, and the picking time of the pinus massoniana twig bark is 4-5 months;
2) adding water into the pinus massoniana twig bark powder at the temperature of 10-15 ℃, and soaking for 1-10h, wherein the mass volume ratio of the pinus massoniana twig bark powder to the water is 1:2-1:15, so as to prepare a pinus massoniana twig bark powder water soaking solution;
3) ultrasonically extracting a masson pine twig bark powder water soaking solution for 5min-2h, and filtering to prepare a masson pine twig bark water soaking solution, wherein the ultrasonic extraction conditions are that the ultrasonic power is 200-1000W, the ultrasonic power is 1-100s, the gap is 1-100s, and the ultrasonic extraction times are 1-6 times;
4) drying the water extract of the tender branch bark of the masson pine to obtain the masson pine extract.
4. The preparation method according to claim 3, wherein the pinus massoniana tender shoot skin is subjected to screening, impurity removal and drying treatment before being crushed, and the drying temperature is 30-80 ℃.
5. The method according to claim 4, wherein the drying temperature is 40-70 ℃.
6. The method according to claim 5, wherein the drying temperature is 50-60 ℃.
7. The method as claimed in claim 3, wherein the bark of Pinus massoniana is crushed to a size of 250-700 mesh.
8. The method as claimed in claim 7, wherein the bark of Pinus massoniana is crushed to 300-600 mesh.
9. The method as claimed in claim 8, wherein the bark of Pinus massoniana is crushed to 350-500 mesh.
10. The method of claim 3, wherein the soaking is carried out for 2-8 h.
11. The method of claim 10, wherein the soaking is performed for 3-6 hours.
12. The method of claim 11, wherein the soaking is carried out for 4-5 hours.
13. The method according to claim 3, wherein the mass-to-volume ratio of the pinus massoniana shoot bark powder to the extraction water is 1:4 to 1: 12.
14. The method according to claim 13, wherein the mass-to-volume ratio of the pinus massoniana shoot bark powder to the extraction water is 1:6 to 1: 10.
15. The method of claim 3, wherein the water is selected from any one of soft water, distilled water, deionized water, or a combination thereof.
16. The method according to claim 3, wherein the ultrasonic extraction is performed for 2 to 5 times.
17. The method according to claim 16, wherein the ultrasonic extraction is performed for 3 to 4 times.
18. The preparation method according to claim 3, wherein the ultrasonic extraction is performed for 10-100min
19. The preparation method according to claim 18, wherein the ultrasonic extraction condition is ultrasonic for 15-80 min.
20. The preparation method according to claim 19, wherein the ultrasonic extraction condition is ultrasonic for 20-60 min.
21. The preparation method according to claim 20, wherein the ultrasonic extraction condition is ultrasonic for 25-40 min.
22. The preparation method according to claim 3, wherein the ultrasonic extraction condition is that the ultrasonic power is 350-900W.
23. The method as claimed in claim 22, wherein the ultrasonic extraction condition is that the ultrasonic power is 500-750W.
24. The preparation method according to claim 3, wherein the ultrasonic extraction conditions are 5-80s ultrasonic extraction and 5-80s intermittent extraction.
25. The method of claim 24, wherein the ultrasonic extraction conditions are ultrasonic for 10-70s and interval for 10-70 s.
26. The method of claim 25, wherein the ultrasonic extraction conditions are 15-60s ultrasonic and 15-60s interstitial.
27. The preparation method according to claim 3, wherein the filtration comprises primary filtration, fine filtration and membrane-resistant bacteria filtration in sequence, the primary filtration is carried out by using a 100-mesh screen, the fine filtration is carried out by using raw white cloth for repeated adsorption filtration for 4-6 times, and the aperture of the membrane-resistant bacteria filtration is 0.22-0.45 μm.
28. The method of claim 3, wherein the drying is selected from any one or a combination of vacuum concentration drying, freeze drying or spray drying.
29. A pharmaceutical composition for preventing and treating lung adenocarcinoma, which consists of the pinus massoniana twig bark extract as defined in claims 1-2 or the pinus massoniana twig bark extract prepared by the preparation method as defined in any one of claims 2-28 and a pharmaceutically acceptable carrier.
30. The pharmaceutical composition according to claim 29, wherein the pharmaceutical composition is selected from any one of oral preparation, external preparation and enteric oral preparation, the oral preparation is selected from any one of tablets, capsules, granules, powder, oral liquid, suspension, pills, powder, dripping pills, syrup, mixture, distillate, effervescent, paste, emulsion and tea, the enteric oral preparation is selected from any one of enteric tablets, enteric capsules, enteric granules, enteric powder, enteric oral liquid, enteric suspension, enteric pills, enteric powder, enteric dripping pills, enteric syrup, enteric mixture, enteric dew, enteric effervescent, enteric paste, enteric emulsion and enteric tea, the external preparation is selected from any one of gel, emplastrum, cream, ointment, liniment, lotion, suppository, smearing preparation and ointment.
31. The pharmaceutical composition according to any one of claims 29-30, which is a sustained-release formulation or a controlled-release formulation thereof, wherein the sustained-release formulation is sustained-release pellets and the controlled-release formulation is controlled-release pellets.
32. The pharmaceutical composition according to any one of claims 29 to 30, wherein the pharmaceutical excipient of the enteric-coated tablet is microcrystalline cellulose and the film coating material of the enteric-coated tablet is hydroxypropyl methylcellulose.
33. The pharmaceutical composition of claim 32, wherein the enteric-coated tablet comprises 30-70 parts of pinus massoniana twig bark extract and 1-10 parts of microcrystalline cellulose.
34. The pharmaceutical composition of claim 33, wherein the enteric-coated tablet comprises 40-50 parts of pinus massoniana twig bark extract and 3-6 parts of microcrystalline cellulose.
35. The pharmaceutical composition of claim 34, wherein the enteric tablet unit dose contains 15mg microcrystalline cellulose and 250mg pinus massoniana twig bark extract; or contains 30mg of microcrystalline cellulose and 500mg of Pinus massoniana twig bark extract.
36. Use of the masson pine twig bark extract as defined in claims 1-2 or the masson pine twig bark extract prepared by the preparation method as defined in any one of claims 3-28 or the pharmaceutical composition as defined in any one of claims 29-35 for the preparation of a medicament for the prevention and treatment of lung adenocarcinoma.
37. The use according to claim 36, wherein the lung adenocarcinoma is selected from middle and advanced lung adenocarcinoma or metastasis or metastatic complications thereof at any site of lymph node, clavicle, rib, thoracic vertebra, lumbar vertebra, pelvis.
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