CN103494843A - Yellow mushroom standardized component manufacturing method and application of components to treatment of lung cancer - Google Patents
Yellow mushroom standardized component manufacturing method and application of components to treatment of lung cancer Download PDFInfo
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Abstract
The invention relates to a yellow mushroom standardized component manufacturing method. The method comprises the following steps: (1) drying and crushing yellow mushrooms, then performing water extraction, centrifugally collecting mushroom residues, extracting the mushroom residues with acetone, centrifugally collecting filtrate, and performing reduced pressure concentration on the filtrate to form paste, namely an acetone extract; (2) dissolving the acetone extract in an n-hexane-ethyl acetate mixed solution, then filtering to obtain a sample solution containing the acetone extract, and separating the sample solution containing the acetone extract on a preparation chromatogram to obtain 14 standardized component samples, namely Fr1, Fr2,..., Fr13 and Fr14; and (3) performing anti-lung cancer cell model screening on the 14 standardized component samples in vitro by an iCelligence real-time label-free cell analyzer, thus determining that the 7th, 8th and 14th standardized components have human lung cancer cell adherence and proliferation inhibition activity in vitro. The yellow mushroom standardized component manufacturing method provided by the invention has the advantages of simple process and easiness in implementation. Simultaneously, the obtained active components can be applied to treatment of lung cancer.
Description
Technical field
The present invention relates to pharmaceutical chemistry and biological medicine technology field, relate in particular to T. gambasum mushroom standardization component method for making and the application in lung cancer therapy thereof.
Background technology
Pulmonary carcinoma is modal lung primary malignant tumor, is also one of modal malignant tumor in the world today.According to World Health Organization (WHO)/IARC's statistics, pulmonary carcinoma accounts for 19% of whole malignant tumor, be positioned at first of various mortality of malignant tumors, the annual newly-increased pulmonary carcinoma case in the whole world surpasses 1,200,000, and the speed with 3% left and right increases, and pulmonary carcinoma has formed great threat to human survival and health.At present the research of pulmonary carcinoma method of early diagnosis, targeting drug delivery system and Chinese medicine medicine for preventing has been become the focus of global concern.
Natural drug has long history for the treatment of the difficult and complicated illness such as pulmonary carcinoma, and the active component in natural drug can be brought into play its of medical care effect by number of ways, a plurality of target spot biology.The particularly extract of originated from fungus and reactive compound, as ganoderan, pachyman, Cordyceps polysaccharide, vincristine, irreplaceable effect has been brought into play in the extensive use as a line medication such as paclitaxel in the treatment of pulmonary carcinoma and other disease.The edible and medicinal fungi abundant species, the metabolic pathway complexity is various, the metabolite structure type is various, it is through advantages such as mutagenic breeding, the industrialization that is easy to ferment in addition, and world's pharmaceuticals industry has the important sources of biologically active drug and finds one of main target of novel anti-lung-cancer medicament using its metabolite as screening.
The T. gambasum mushroom is the rare wild edible and medical fungi that grows in Qinghai-Tibet Platean.Limited qualitative investigation at present shows, contain more rich protein, aminoacid, saccharide, alkaloid and a small amount of organic acid, flavone, cardiac glycoside, steroidal triterpenes, glycoside, saponin, volatile oil, coumarin terpenoid, tannin, phenolic compound etc. in the Armillaria luteo-virens sporophore, this compounds has the biological activitys such as influenza, control neuritis, vitamin B1 deficiency, promotion childhood development, antioxidation and antitumor.
Preparative liquid chromatography is technology stable with the major diameter column separating purification, homogeneous components.Along with natural drug is modern, develop rapidly, preparative liquid chromatography is subject to the attention of the research fields such as natural product chemistry, medicine synthetic chemistry as a kind of isolation technics rapidly and efficiently.A lot of sample component can only be separated by the preparative liquid chromatography technology, the positive preparative high performance liquid chromatography is as one of method of applying more disintegrate-quality homogeneous, stable, reliable component in preparative hplc, become a kind of requisite separation and purification means at present, apply more and more extensive.
Chinese patent application CN 200910154901.5 " method of synthesizing betulic acid by carrying out biocatalysis on betulin " has related to Armillaria luteo-virens biotransformation synthesizing betulic acid; Chinese patent application CN200710069803.2 " a kind of yellow-green halimasch fibrinolytic enzyme and production method thereof " has related to the synthetic fibrinolysin of Armillaria luteo-virens metabolism; Chinese patent application CN200710066667.1 " a kind of method of microbial cell biotransformation p-Hydroxybenzylalcohol synthetic gastrodin " has related to Armillaria luteo-virens by the biotransformation synthetic gastrodin; Chinese patent application CN200610155189.7 " a kind of Armillaria luteo-virens and cultural method thereof and application ", in careful patent " a kind of anticancer Armillaria luteo-virens polysaccharide and extraction process ", " a kind of submerged fermentation of Armillaria luteo-virens and application thereof ", relate generally to Armillaria luteo-virens Mycelium culture and polysaccharide extracting process.Do not relate to T. gambasum mushroom chemical composition.Above-mentioned document, about the research of T. gambasum mushroom, mainly concentrates on biotransformation, to the research of T. gambasum mushroom chemical composition seldom.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of technique T. gambasum mushroom standardization component method for making simple, easy to implement.
Another technical problem to be solved by this invention is to provide the application of active component in lung cancer therapy of this T. gambasum mushroom standardization component method for making gained.
For addressing the above problem, T. gambasum mushroom standardization component method for making of the present invention comprises the following steps:
(1), after T. gambasum mushroom drying being pulverized, the solid-liquid ratio of pressing 1g:4mL ~ 20 mL extracts 3 times at 40 ~ 90 ℃ with analyzing pure water, each 1 ~ 10h, and centrifugal collection bacterium slag, this bacterium slag is pressed 1g:1mL ~ 20 mL solid-liquid ratio with acetone extracts 3 times at 10 ~ 80 ℃, each 5 ~ 72h, and centrifugal collection filtrate; Described filtrate, through being evaporated to paste, obtains acetone extract;
(2) in described acetone extract, add 1 ~ 6 times of normal hexane of its volume-ethyl acetate mixed liquor to be dissolved, cross organic filter membrane of 0.45 μ m after dissolving, obtain the sample solution containing acetone extract; The described sample solution containing acetone extract separates take on the preparative hplc that 10 μ m silica gel are isolation medium, obtain Fr1, Fr2 ..., Fr13, Fr14 totally 14 standardization component samples;
(3) adopt the real-time n cell analyser of iCelligence to carry out in the steps below In Vitro Anti lung carcinoma cell model discrimination in described 14 standardization component samples:
1. confirm iCelligence test macro exact connect ion, 37 ℃, 5%CO
2incubator is working properly;
2. add culture medium by 150 μ L/ holes in 8 porocyte plates, room temperature is placed to be placed on the iCelligence testboard in 15 minutes and is surveyed baseline; Described culture medium refers to the F-12k culture medium containing 10% hyclone;
3. each component sample in described 14 standardization component samples is mixed with to the sample solution that concentration is 5mg/mL with DMSO;
4. by the human lung cancer cell A549 of exponential phase, after digesting according to a conventional method with pancreatin, with the centrifugal 5min of the speed of 950rpm, cell is resuspended in described culture medium, and cell concentration is adjusted to 2.85 * 10
4individual/mL, obtain human lung cancer cell A549's suspension of exponential phase, and by the amount of 345 10000 cells in ,Mei hole, μ L/ hole, human lung cancer cell A549's suspension inoculation of described exponential phase entered in the respective aperture of described 8 porocyte plates, and room temperature is placed 30 min;
5. described 14 standardization component samples being added to the amount that sample solution, every hole that the described concentration of 5 μ L is 5mg/mL add human lung cancer cell A549's suspension of the described exponential phase of 345 μ L by every hole in two batches inoculates in the respective aperture of described 8 porocyte plates, and be placed in described iCelligence testboard, put into described incubator and start to detect; Obtain the activity detection figure of two the anti-pulmonary carcinoma standardization of T. gambasum mushroom components after 45 h; Wherein vertical coordinate is cell index (cell index), and abscissa is real-time cell proliferation dynamic effect (RTCA);
6. detect figure according to the activity of described two the anti-pulmonary carcinoma standardization of T. gambasum mushroom components, the trend that presents rising gently or do not rise when the cell proliferation curve, illustrate that cell division slows down or stops, be that cell can not divide normally, thereby determine that the 7th, the 8th and No. 14 standardization component has the adherent and proliferation activity of vitro inhibition human lung carcinoma cell.
The described step (1) condition of middle concentrating under reduced pressure refers to that vacuum is 0.03 ~ 0.1 MPa, and temperature is 30 ~ 80 ℃.
Described step (2) middle normal hexane-ethyl acetate mixed liquor refers to the solution that normal hexane mixes in the ratio of 1 mL:0.1 mL ~ 10 mL with ethyl acetate.
The described step condition that (2) middle preparative hplc is separated refers to that column size is 260 * 50 mm; Employing A is that the binary mobile phase system that normal hexane, B are ethyl acetate is separated: 0 ~ 15 min, 100%A ~ 100%A; 15 ~ 35 min, 60%A ~ 60%A; 35 ~ 50 min, 0%A ~ 0%A; Detecting wavelength is 260 nm; Applied sample amount is 1.0 g.
The application of active component in lung cancer therapy of T. gambasum mushroom standardization component method for making gained as above, it is characterized in that: this active component is made all kinds of pharmaceutical formulations with pharmaceutically acceptable any carrier according to a conventional method as effective ingredient.
The present invention compared with prior art has the following advantages:
1, the present invention adopts the conventional methods such as solvent extraction, centrifugal, preparing chromatograph in industry, separates the standardization component (referring to Fig. 1, Fig. 2) that has obtained having anti-lung cancer activity from the T. gambasum mushroom, and not only technique is simple, and easy to implement.
2, the present invention utilizes the iCELLigence system to detect, but Real-time and Dynamic Detection cell attachment and breeding provide the cytological effect collection of illustrative plates of high information quantity, and a large amount of, important dynamic response information is provided; Unmarked, AT detection of electrons can not grown and analyze by interference cell; But the quantitative measurement Growth of Cells of high precision/degree of accuracy, provide the dynamic range higher than 2 orders of magnitude; Whole experimentation automatic data collection, real-time analysis, improve the result of only analyzing at maximal end point greatly; Detect the cell quality in culture hole, reached the purpose (referring to Fig. 3, Fig. 4) of living cells quality control.
3, the present invention be take cell growth curve as the impact of basic index criterion component on cellular physiological activities such as adherent, breedings, visual result, easily judgement; The active component of gained can be used for the anti-lung cancer therapy medicine of research and development preparation.
4, the present invention has expanded the raw material sources of anti-lung-cancer medicament, has enlarged the purposes of T. gambasum mushroom, makes the T. gambasum mushroom become the active component raw material of anti-lung-cancer medicament, significantly improves the added value of T. gambasum mushroom.
The accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
The chromatographic fractionation figure A that Fig. 1 is T. gambasum mushroom standardization component of the present invention.
The chromatographic fractionation figure B that Fig. 2 is T. gambasum mushroom standardization component of the present invention.
The anti-lung cancer activity detection figure A that Fig. 3 is T. gambasum mushroom standardization component of the present invention.
The anti-lung cancer activity detection figure B that Fig. 4 is T. gambasum mushroom standardization component of the present invention.
The specific embodiment
embodiment 1t. gambasum mushroom standardization component method for making comprises the following steps:
(1), after T. gambasum mushroom drying being pulverized, the solid-liquid ratio of pressing 1g:4mL extracts 3 times at 40 ℃ with analyzing pure water, each 10h, and centrifugal collection bacterium slag, and this bacterium slag is pressed 1g:1mL solid-liquid ratio with acetone extracts 3 times at 10 ℃, each 72h, and centrifugal collection filtrate; Filtrate vacuum be under 0.03 MPa, the temperature condition that is 30 ℃ through being evaporated to paste, obtain acetone extract.
(2) in acetone extract, add 1 times of normal hexane of its volume-ethyl acetate mixed liquor to be dissolved, cross organic filter membrane of 0.45 μ m after dissolving, obtain the sample solution containing acetone extract; Separate take on the preparative hplc that 10 μ m silica gel are isolation medium containing the sample solution of acetone extract, obtain Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10, Fr11, Fr12, Fr13, Fr14 totally 14 standardization component samples.
Wherein:
The condition that preparative hplc is separated refers to that column size is 260 * 50 mm; Employing A is that the binary mobile phase system that normal hexane, B are ethyl acetate is separated: 0 ~ 15 min, 100%A ~ 100%A; 15 ~ 35 min, 60%A ~ 60%A; 35 ~ 50 min, 0%A ~ 0%A; Detecting wavelength is 260 nm; Applied sample amount is 1.0 g.Normal hexane-ethyl acetate mixed liquor refers to the solution that normal hexane mixes in the ratio of 1 mL:0.1 mL with ethyl acetate.
(3) adopt the real-time n cell analyser of iCelligence to carry out in the steps below In Vitro Anti lung carcinoma cell model discrimination in 14 standardization component samples:
1. confirm iCelligence test macro exact connect ion, 37 ℃, 5%CO
2incubator is working properly.
2. add culture medium by 150 μ L/ holes in 8 porocyte plates, room temperature is placed to be placed on the iCelligence testboard in 15 minutes and is surveyed baseline.
Wherein: culture medium refers to the F-12k culture medium containing 10% hyclone.
3. each component sample in 14 standardization component samples is mixed with to the sample solution that concentration is 5mg/mL (being to contain 5 mg component sample in every mLDMSO) with DMSO.
4. by the human lung cancer cell A549 of exponential phase, after digesting according to a conventional method with pancreatin, with the centrifugal 5min of the speed of 950rpm, cell is resuspended in culture medium, and cell concentration is adjusted to 2.85 * 10
4individual/mL, obtain human lung cancer cell A549's suspension of exponential phase, and by the amount of 345 10000 cells in ,Mei hole, μ L/ hole, human lung cancer cell A549's suspension inoculation of exponential phase entered in the respective aperture of 8 porocyte plates, and room temperature is placed 30 min.
5. 14 standardization component samples being added to the amount that sample solution, every hole that 5 μ L concentration are 5mg/mL add human lung cancer cell A549's suspension of 345 μ L exponential phases by every hole in two batches inoculates in the respective aperture of 8 porocyte plates, and be placed in the iCelligence testboard, put into incubator and start to detect; Obtain the activity detection figure of two the anti-pulmonary carcinoma standardization of T. gambasum mushroom components after 45 h; Wherein vertical coordinate is cell index (cell index), and abscissa is real-time cell proliferation dynamic effect (RTCA).
6. detect figure according to the activity of two the anti-pulmonary carcinoma standardization of T. gambasum mushroom components, the trend that presents rising gently or do not rise when the cell proliferation curve, illustrate that cell division slows down or stops, be that cell can not divide normally, thereby determine that the 7th, the 8th and No. 14 standardization component has the adherent and proliferation activity of vitro inhibition human lung carcinoma cell.
embodiment 2t. gambasum mushroom standardization component method for making comprises the following steps:
(1), after T. gambasum mushroom drying being pulverized, the solid-liquid ratio of pressing 1g:20 mL extracts 3 times at 90 ℃ with analyzing pure water, each 1h, and centrifugal collection bacterium slag, and this bacterium slag is pressed 1g:20 mL solid-liquid ratio with acetone extracts 3 times at 80 ℃, each 5h, and centrifugal collection filtrate; Filtrate vacuum be under 0.1 MPa, the temperature condition that is 80 ℃ through being evaporated to paste, obtain acetone extract.
(2) in acetone extract, add 6 times of normal hexane of its volume-ethyl acetate mixed liquor to be dissolved, cross organic filter membrane of 0.45 μ m after dissolving, obtain the sample solution containing acetone extract; Separate take on the preparative hplc that 10 μ m silica gel are isolation medium containing the sample solution of acetone extract, obtain Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10, Fr11, Fr12, Fr13, Fr14 totally 14 standardization component samples.
Wherein:
The condition that preparative hplc is separated is same
embodiment 1.Normal hexane-ethyl acetate mixed liquor refers to the solution that normal hexane mixes in the ratio of 1 mL:10 mL with ethyl acetate.
(3) adopt the real-time n cell analyser of iCelligence to press in 14 standardization component samples
embodiment 1described step is carried out In Vitro Anti lung carcinoma cell model discrimination, determines that the 7th, the 8th and No. 14 standardization component has the adherent and proliferation activity of vitro inhibition human lung carcinoma cell.
embodiment 3t. gambasum mushroom standardization component method for making comprises the following steps:
After T. gambasum mushroom drying is pulverized, the solid-liquid ratio of pressing 1g:12 mL extracts 3 times at 65 ℃ with analyzing pure water, each 5.5h, and centrifugal collection bacterium slag, and this bacterium slag is pressed 1g:10mL solid-liquid ratio with acetone extracts 3 times at 45 ℃, each 38h, and centrifugal collection filtrate; Filtrate vacuum be under 0.06 MPa, the temperature condition that is 55 ℃ through being evaporated to paste, obtain acetone extract.
(2) in acetone extract, add 3.5 times of normal hexane of its volume-ethyl acetate mixed liquor to be dissolved, cross organic filter membrane of 0.45 μ m after dissolving, obtain the sample solution containing acetone extract; Separate take on the preparative hplc that 10 μ m silica gel are isolation medium containing the sample solution of acetone extract, obtain Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10, Fr11, Fr12, Fr13, Fr14 totally 14 standardization component samples.
Wherein:
The condition that preparative hplc is separated is same
embodiment 1.Normal hexane-ethyl acetate mixed liquor refers to the solution that normal hexane mixes in the ratio of 1 mL:5 mL with ethyl acetate.
(3) adopt the real-time n cell analyser of iCelligence to press in 14 standardization component samples
embodiment 1described step is carried out In Vitro Anti lung carcinoma cell model discrimination, determines that the 7th, the 8th and No. 14 standardization component has the adherent and proliferation activity of vitro inhibition human lung carcinoma cell.
Above-mentioned
embodiment 1 ~ 3the active component of middle T. gambasum mushroom standardization component method for making gained is made all kinds of pharmaceutical formulations with pharmaceutically acceptable any carrier according to a conventional method as effective ingredient and is applied in lung cancer therapy.
Claims (5)
1. T. gambasum mushroom standardization component method for making comprises the following steps:
(1), after T. gambasum mushroom drying being pulverized, the solid-liquid ratio of pressing 1g:4mL ~ 20 mL extracts 3 times at 40 ~ 90 ℃ with analyzing pure water, each 1 ~ 10h, and centrifugal collection bacterium slag, this bacterium slag is pressed 1g:1mL ~ 20 mL solid-liquid ratio with acetone extracts 3 times at 10 ~ 80 ℃, each 5 ~ 72h, and centrifugal collection filtrate; Described filtrate, through being evaporated to paste, obtains acetone extract;
(2) in described acetone extract, add 1 ~ 6 times of normal hexane of its volume-ethyl acetate mixed liquor to be dissolved, cross organic filter membrane of 0.45 μ m after dissolving, obtain the sample solution containing acetone extract; The described sample solution containing acetone extract separates take on the preparative hplc that 10 μ m silica gel are isolation medium, obtain Fr1, Fr2 ..., Fr13, Fr14 totally 14 standardization component samples;
(3) adopt the real-time n cell analyser of iCelligence to carry out in the steps below In Vitro Anti lung carcinoma cell model discrimination in described 14 standardization component samples:
1. confirm iCelligence test macro exact connect ion, 37 ℃, 5%CO
2incubator is working properly;
2. add culture medium by 150 μ L/ holes in 8 porocyte plates, room temperature is placed to be placed on the iCelligence testboard in 15 minutes and is surveyed baseline; Described culture medium refers to the F-12k culture medium containing 10% hyclone;
3. each component sample in described 14 standardization component samples is mixed with to the sample solution that concentration is 5mg/mL with DMSO;
4. by the human lung cancer cell A549 of exponential phase, after digesting according to a conventional method with pancreatin, with the centrifugal 5min of the speed of 950rpm, cell is resuspended in described culture medium, and cell concentration is adjusted to 2.85 * 10
4individual/mL, obtain human lung cancer cell A549's suspension of exponential phase, and by the amount of 345 10000 cells in ,Mei hole, μ L/ hole, human lung cancer cell A549's suspension inoculation of described exponential phase entered in the respective aperture of described 8 porocyte plates, and room temperature is placed 30 min;
5. described 14 standardization component samples being added to the amount that sample solution, every hole that the described concentration of 5 μ L is 5mg/mL add human lung cancer cell A549's suspension of the described exponential phase of 345 μ L by every hole in two batches inoculates in the respective aperture of described 8 porocyte plates, and be placed in described iCelligence testboard, put into described incubator and start to detect; Obtain the activity detection figure of two the anti-pulmonary carcinoma standardization of T. gambasum mushroom components after 45 h; Wherein vertical coordinate is cell index, and abscissa is real-time cell proliferation dynamic effect;
6. detect figure according to the activity of described two the anti-pulmonary carcinoma standardization of T. gambasum mushroom components, the trend that presents rising gently or do not rise when the cell proliferation curve, illustrate that cell division slows down or stops, be that cell can not divide normally, thereby determine that the 7th, the 8th and No. 14 standardization component has the adherent and proliferation activity of vitro inhibition human lung carcinoma cell.
2. T. gambasum mushroom standardization component method for making as claimed in claim 1 is characterized in that: described step (1) in the condition of concentrating under reduced pressure refer to that vacuum is 0.03 ~ 0.1 MPa, temperature is 30 ~ 80 ℃.
3. T. gambasum mushroom standardization component method for making as claimed in claim 1 is characterized in that: described step (2) in normal hexane-ethyl acetate mixed liquor refer to the solution that normal hexane mixes in the ratio of 1 mL:0.1 mL ~ 10 mL with ethyl acetate.
4. T. gambasum mushroom standardization component method for making as claimed in claim 1 is characterized in that: described step (2) in the preparative hplc condition of separating refer to that column size is 260 * 50 mm; Employing A is that the binary mobile phase system that normal hexane, B are ethyl acetate is separated: 0 ~ 15 min, 100%A ~ 100%A; 15 ~ 35 min, 60%A ~ 60%A; 35 ~ 50 min, 0%A ~ 0%A; Detecting wavelength is 260 nm; Applied sample amount is 1.0 g.
5. the application of active component in lung cancer therapy of T. gambasum mushroom standardization component method for making gained as claimed in claim 1, it is characterized in that: this active component is made all kinds of pharmaceutical formulations with pharmaceutically acceptable any carrier according to a conventional method as effective ingredient.
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| CN105687255A (en) * | 2014-11-28 | 2016-06-22 | 天津耀宇生物技术有限公司 | A method of extracting volatile oil from armillaria luteo-virens |
| CN105687253A (en) * | 2014-11-28 | 2016-06-22 | 天津耀宇生物技术有限公司 | A method of extracting anti-liver-cancer active components from armillaria luteo-virens and applications of the active components |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105687254A (en) * | 2014-11-28 | 2016-06-22 | 天津耀宇生物技术有限公司 | A method of extracting anti-lung-cancer active components from armillaria luteo-virens and applications of the active components |
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