A kind of method and application thereof of extracting antitumor component from Cordyceps militaris (L.) Link.
Technical field
The present invention relates to biological product processing technique field, be specifically related to a kind of method of extracting antitumor component from Cordyceps militaris (L.) Link., and this active component is in the application of preparing in antitumor drug.
Background technology
Tumor is a kind of genopathy, but is not hereditary, it be phalangeal cell under carcinogenic factor effect, having there is change in gene, loses the normal regulation to its growth, causes monoclonicity paraplasm and the neoplasm that forms.2008, the data show that WHO issues, China has become second-biggest-in-the-world cancer state occurred frequently, and annual newly-increased 2,200,000 cancer patients of China, account for 20% of global cancer patient sum, and especially hepatocarcinoma, gastric cancer and pulmonary carcinoma become main Health Killer.How research is eliminated cancer, is allowed cancer patient can live ground better, it is the research emphasis in field of medicaments, therefore find efficient, low toxicity, the desirable new type antineoplastic medicine that has clinical value that selectivity is strong and be a long-term and difficult task, the important directions that seeking antitumor medicine has become antitumor drug exploitation from the nontoxic of medicine-food two-purpose and Chinese medicine that raw material is cheap and easy to get with wish place.
Cordyceps is that Cordyceps colonizes in the entomogenous fungi complex take it as carrier in Lepidoptera Hepialidae larva, contain rich in protein, fat, cordycepic acid, nucleosides material and several amino acids isoreactivity material, taste delicate fragrance, has lung nourishing and qi invigorating, increases smart the kidney invigorating, hemostasis and phlegm, inhibition tumor, cough-relieving calmness, wind dispelling, heat radiation, stays effect of Rong Meiyan, slow down aging, raising immunity of organisms.
Cordyceps militaris (L.) Link. (Cordycepsmilitaris) is attributed to Cordyceps with Cordyceps is same on taxonomy, is a kind of medicinal fungi of preciousness, and its chemical composition is similar to Cordyceps with pharmacological action, often substitutes clinically Cordyceps and is used as medicine.Because wild cordyceps militaris resource is very limited, can not meet the market demand day by day increasing, Artificial Cultivation of Cordyceps militaris Link becomes essential.At present, Artificial Cultivation of Cordyceps militaris Link is succeeded, and has realized large-scale production.
Cordyceps militaris (L.) Link. has the multiple active substance relevant to antitumor action, and some active substance has been studied more deeply as the antitumor action of cordycepin, Cordyceps polysaccharide etc.; Some active substance is as less in the research of anti-tumor aspect in carotenoid, protein etc.Cordyceps militaris (L.) Link. active substance fails farthest to be develop and useedd, so research has the new active substance of antitumor action, constantly new physiologically active and the pharmacological action of the existing active substance of developing are significant, can provide fundamental basis and industrialization background in the application aspect medical for further researching and developing Cordyceps militaris (L.) Link..
The extracting method of traditional Cordyceps militaris (L.) Link. antitumor component, in " in-vitro screening of Cordyceps militaris (L.) Link. antitumor and Immunoactive site " (the edible fungi journal .2011.18 (1): 41-45) delivering as Du Xiuju etc., a kind of militaris sporocarp extract preparation method is disclosed: Cordyceps militaris (L.) Link.sporophore powder is crossed after 60 mesh sieves, with chloroform extraction 2 hours, filter, filtering residue is air-dry rear with ethyl acetate extraction 2 hours, filter, air-dry rear use 95% ethanol extraction of filtering residue 2 hours, filter, 30 times of 100 ℃ of distilled water extraction of the air-dry rear use of filtering residue 2 hours, the centrifugal 20min postprecipitation of 400g extracts 2 hours with 15 times 100 ℃ 3% ammonium oxalate solutions, the centrifugal 20min postprecipitation of 400g extracts 2 hours with 10 times of 100 ℃ of 5%NaOH solution, after the centrifugal 20min of 400g, supernatant continues with 4 ℃ of neutralizations of 1mol/L hydrochloric acid, the centrifugal 20min of 400g, the filtrate that each step is obtained or supernatant vacuum concentration, lyophilization, obtain each extract components.The extraction separation method step of this Cordyceps militaris (L.) Link.sporophore effective ingredient is numerous and diverse, and the active component extracting is few.In addition, adopt this traditional solvent extraction to carry out the extraction of effective active component, between batch, do not there is repeatability, cannot guarantee that material composition and second batch that first extraction obtains extract the concordance between the material composition obtaining.
A kind of isolation and purification method of target compound is disclosed in separation and purification and the antitumor action of-adenosine " the N6-(2-ethoxy in Cordyceps militaris (L.) Link.sporophore) " (edible fungi journal .2013.20 (1): 62-65) that and for example Zhu Lina etc. delivers, it adds in distilled water 100 ℃ to extract 2 hours in Cordyceps militaris (L.) Link.sporophore powder, repeat to extract 2 times, merge extractive liquid,, then carries out the extraction of succeeding target composition with this extracting solution.The method belongs to the extracting method of traditional Effective Component of Chinese Medicine, carry out the extraction of effective ingredient with the supernatant of water extraction, and the precipitation of water extraction is not carried out to deep research, this likely causes the loss of a large amount of active components, thereby has hindered the progress of Cordyceps militaris (L.) Link. aspect antitumor drug exploitation.
Summary of the invention
Technical problem to be solved by this invention is for above the deficiencies in the prior art, break through the restriction that existing Chinese medicine active component extracts, a kind of method of extracting antitumor component from Cordyceps militaris (L.) Link. is provided, the method is precipitated as raw material with the Cordyceps militaris (L.) Link.sporophore water extraction that there is no anti-tumor activity on cellular level, after acetone extraction, isolates strong active antitumor component again by the active guiding of preparative liquid chromatography technology.
The technical solution adopted in the present invention is:
A method of extracting antitumor component from Cordyceps militaris (L.) Link., the method comprises the following steps:
1, pulverizing at ultralow temperature:
The Cordyceps militaris (L.) Link.sporophore of getting oven dry is raw material, puts into super micron mill and carries out micronizing, pulverizes the overall process water of 2~10 ℃ as micronized pulverization machine internal coolant simultaneously, and the micronizing time is 5-20min, obtains superfine powder.
Above-mentioned grinding time is preferably 12min.
2, solvent extraction:
2.1 decocting in water: add ultra-pure water in superfine powder, the addition of ultra-pure water is 8~10 times of superfine powder quality, 100 ℃ of heating are extracted for 3~10 hours, and centrifuging and taking precipitation, then repeats 1~3 time, and the precipitation obtaining is merged, dry, obtains crude product A.
This step is different from the maximum of at present traditional Chinese medicine active component or extracts active ingredients.In traditional Chinese medicine, the extracting method of active component or active component is all to carry out the extraction of effective ingredient take the supernatant of water extraction as intermediate raw material, in Chinese medicine, to drink be also like this to Chinese medicine, after decocting in water, getting supernatant is patients, because understand routinely, effective ingredient after boiling water decocts in medical material is just dissolved in liquid, therefore comprised required active component in supernatant.And the limitation of this understanding has exactly limited the exploitation of active component in current species or active component, make a lot of active components or effective ingredient be outwelled, further not be found and develop as waste residue.This is not also because most researchers has been ignored the probability that active component in waste residue or effective ingredient exist, but because waste residue may not have anti-tumor activity in vitro or activity is very weak, the crude product A obtaining as this step does not have anti-tumor activity after testing on cellular level, such testing result makes most researchers abandon it to be further analyzed and to study, thereby has caused the loss of a large amount of active components.The present invention is by applicant's further investigation, analysis and experiment, find not have the water extraction precipitation of anti-tumor activity to there is the probability of object active component on cellular level, thereby precipitate, from water extraction, by a large amount of experimental exploring and process optimization, finally summing up a set of is the extracting method of Cordyceps militaris (L.) Link. antitumor component of the present invention, finally successfully extracts object effective active component.
2.2 acetone precipitations: add acetone in crude product A, the addition of acetone is 2~4 times of crude product A quality, lixiviate 12~36 hours under room temperature, gets precipitation after centrifugal, then repeats 1~3 time, and the precipitation obtaining is merged, dry, obtains crude product B.
Above-mentioned centrifugal rotating speed is preferably 6000rpm.
Above-mentioned dry being preferably in 60 ℃ of air dry ovens carried out.
3, chromatographic isolation:
3.1 get crude product for B 50% ethyl acetate-hexane solution (being that ethyl acetate and normal hexane form take the volume ratio proportioning of 1:1) be dissolved to concentration as 50mg/ml, cross the organic microporous filter membrane of 0.45 μ m, then separate by preparative liquid chromatography, type of elution is 100% normal hexane isocratic elution, 25% ethyl acetate-hexane solution (being that ethyl acetate and normal hexane form with the volume ratio proportioning of 1:3) isocratic elution, 100% ethyl acetate isocratic elution, detection wavelength is 260nm, obtain each component after anti tumor activity in vitro screening, choose the strongest active component concentrated by rotary evaporation final vacuum dry, obtain component A.
Particularly, silica gel (Silica), size that the chromatographic column filler using is particle diameter 10 μ m are 150 × 250mm, type of elution is 100% normal hexane isocratic elution 10min, 25% ethyl acetate-hexane solution isocratic elution 25min, 100% ethyl acetate isocratic elution 15min, applied sample amount is 200ml, and flow velocity is 700ml/min.
The selection of this step solvent is extremely important, through overtesting, in 50% ethyl acetate-hexane solution, ethyl acetate can all be dissolved crude product B, normal hexane to crude product B for being partly dissolved, and select 50% ethyl acetate-hexane solution can accomplish to make crude product B to be dissolved to 50mg/ml, and when eluting, adopt the eluting order of 100% normal hexane, 25% ethyl acetate-hexane solution, 100% ethyl acetate, material composition in crude product B is separated as far as possible, be conducive to the extraction of object active component, improve extraction ratio.
3.2 to get component A 100% acetic acid ethyl dissolution to concentration be 50mg/ml, cross the organic microporous filter membrane of 0.45 μ m, then separate by preparative liquid chromatography, type of elution is 100% normal hexane isocratic elution 10min, 10% ethyl acetate-hexane solution eluting 50min, 15% ethyl acetate-hexane solution eluting 20min, 30% ethyl acetate-hexane solution eluting 15min, 60% ethyl acetate-hexane solution eluting 20min, 100% methanol isocratic elution 10min, type of elution is 100% normal hexane isocratic elution, 10% ethyl acetate-hexane solution isocratic elution, 15% ethyl acetate-hexane solution isocratic elution, 30% ethyl acetate-hexane solution isocratic elution, 60% ethyl acetate-hexane solution isocratic elution, 100% methanol isocratic elution, detection wavelength is 260nm, obtain each component after anti tumor activity in vitro screening, choose the strongest active component concentrated by rotary evaporation final vacuum dry, obtain B component.
Particularly, silica gel (Silica), size that the chromatographic column filler using is particle diameter 10 μ m are 150 × 250mm, type of elution is 100% normal hexane isocratic elution 10min, 10% ethyl acetate-hexane solution eluting 50min, 15% ethyl acetate-hexane solution eluting 20min, 30% ethyl acetate-hexane solution eluting 15min, 60% ethyl acetate-hexane solution eluting 20min, 100% methanol isocratic elution 10min, applied sample amount is 400ml, flow velocity is 700ml/min.
The eluant (100% normal hexane, 10% ethyl acetate-normal hexane, 15% ethyl acetate-normal hexane, 30% ethyl acetate-normal hexane, 60% ethyl acetate-normal hexane, 100% methanol) that this step eluting adopts is different from 3.1, by the difference of eluting rate, the component originally being eluted by a concentration is separated to eluting, be conducive to the acquisition of follow-up active component.
3.3 to get B component 100% dissolve with methanol to concentration be 50mg/ml, cross the organic microporous filter membrane of 0.45 μ m, then separate by preparative liquid chromatography, type of elution is 100% ethyl acetate~100% methanol gradient elution, detection wavelength is 260nm, obtain each component after anti tumor activity in vitro screening, choose the strongest active component concentrated by rotary evaporation final vacuum dry, obtain component C.
Particularly, silica gel (Silica), size that the chromatographic column filler of use is particle diameter 10 μ m are 150 × 250mm, and type of elution is 100% ethyl acetate~100% methanol gradient elution 30min, and applied sample amount is 400ml, and flow velocity is 700ml/min.
3.4 get component for C 20% dichloromethane-hexane solution to be dissolved to concentration be 50mg/ml, cross the organic microporous filter membrane of 0.45 μ m, then separate by preparative liquid chromatography, type of elution is 100% normal hexane isocratic elution, 20% dichloromethane-hexane solution isocratic elution, 25% dichloromethane-hexane solution isocratic elution, detection wavelength is 260nm, obtain each component after anti tumor activity in vitro screening, choose the strongest active component, concentrated by rotary evaporation final vacuum is dry, is the object of the invention antitumor component.
Particularly, silica gel (Silica), size that the chromatographic column filler using is particle diameter 10 μ m are 21.2 × 250mm, type of elution is 100% normal hexane isocratic elution 8min, 20% dichloromethane-hexane solution isocratic elution 37min, 25% dichloromethane-hexane solution isocratic elution 15min, applied sample amount is 2ml, and flow velocity is 15ml/min.
The key of said extracted method is chromatographic isolation, and the key of chromatographic isolation is mobile phase, by selection, the repeatedly selection of different mobile phase kinds and different proportionings between chromatographic isolation of different proportion of mobile phase in the selection of mobile phase kind, a same to chromatographic separation process, component in Cordyceps militaris (L.) Link. is fully separated, thereby make successfully from Cordyceps militaris (L.) Link., to extract object antitumor component, make to extract the antitumor component purity obtaining high.
In described step (3), anti tumor activity in vitro screening refers to and suppresses people's lung cancer A549 cell growth test and suppress the test of people's hepatocarcinoma Hep-G2 Growth of Cells, particularly, refer to the growing state that adopts real-time unmarked dynamic cellular analytical technology to detect in real time dosing descendant lung cancer A549 cell and people's hepatocarcinoma Hep-G2 cell, can obtain the cytological effect curve of medicine mediation simultaneously, judge the whether growth of anticancer of this medicine by this curve.Be with traditional method (MTT) comparative benefits, experimental implementation is simple, and step is few, and personal error is little.Adopt unmarked method, do not have labeling effciency problem, and to cell not damaged, cell detects approaching most under physiological status, and result precision is high.Experimental result is objective, reproducible, does not need to arrange repeating hole.The method can judge the anti-tumor activity of component to be measured well, because in experimenting, we find out that, each component to be measured is synchronous to the inhibition of these two kinds of cells substantially, do not exist No. 1 component to people's lung cancer A549 cell inhibitory action preferably and the 2nd component to the best situation of people's hepatocarcinoma Hep-G2 cyto-inhibition, substantially the object component of choosing or be all best to the inhibitory action of two kinds of cells, otherwise all poor to all component inhibitions of the best and another kind of cell of a kind of cyto-inhibition wherein.
The present invention also further provides the above-mentioned antitumor component obtaining of extracting from Cordyceps militaris (L.) Link. in the application of preparing in antitumor drug.This active component is made all kinds of pharmaceutical formulations with pharmaceutically acceptable any carrier according to a conventional method as effective ingredient.
Compared with prior art, the present invention has following remarkable advantage and beneficial effect:
1, the present invention adopts super micron mill to carry out superfine comminution at low temperature, to abolish the hard cell wall of Cordyceps militaris (L.) Link.sporophore, is conducive to the stripping of content, has effectively improved the extraction ratio of antitumor component in Cordyceps militaris (L.) Link.sporophore;
2, the present invention adopts preparative liquid chromatography to carry out the separation and purification of material composition, the technology of uses advanced is to being not have the water extraction precipitation of anti-tumor activity to carry out separation and purification on cellular level, select by the reagent to each separating step, the optimization of process conditions and technological parameter, and good synergism between each separating step, finally successfully extract antitumor component, with respect to traditional solvent extraction, not only good stability of the extracting method of this antitumor component, reproducible, between the material composition obtaining batch, concordance is high, and extraction ratio is high, the active component obtaining has powerful antitumor activity,
3, the present invention judges the extracorporeal anti-tumor ability of Cordyceps militaris (L.) Link. antitumor component by people's lung cancer A549 cell growth inhibition test and people's hepatocarcinoma Hep-G2 cell growth inhibition test, and establishes positive control, visual result, easily judgement;
4, the extracting method technique of Cordyceps militaris (L.) Link. antitumor component of the present invention is simple, easy to implement, have quick, efficient, reproducible, stablize controlled, separation and purification preparation amount large, be applicable to the features such as large-scale industrialization production, the Cordyceps militaris (L.) Link. antitumor component of gained can be used for research and development and prepares antitumor drug, and because this active component antitumor active constituent content is high, while using this active component to be prepared antitumor drug, active constituent content is high, can meet the demand of different preparations carrier forms to active constituent content;
5, the present invention has expanded the raw material sources of antitumor drug or health food, has expanded the range of application of Cordyceps militaris (L.) Link.sporophore, makes Cordyceps militaris (L.) Link.sporophore become the active component raw material of antitumor drug, has significantly improved the added value of Cordyceps militaris (L.) Link.sporophore.
Accompanying drawing explanation
Shown in Fig. 1 is embodiment of the present invention 1(3) chromatographic fractionation figure that (a) step obtains;
Shown in Fig. 2 is embodiment of the present invention 1(3) chromatographic fractionation figure that (b) step obtains;
Shown in Fig. 3 is embodiment of the present invention 1(3) chromatographic fractionation figure that (c) step obtains;
Shown in Fig. 4 is embodiment of the present invention 1(3) chromatographic fractionation figure that (d) step obtains;
Shown in Fig. 5 is that Cordyceps militaris (L.) Link. antitumor component prepared by the embodiment of the present invention 1 suppresses the cell detection figure in real time that people's lung cancer A549 cell is grown, and wherein C310-12-1-17 is object component (intermediate curve), and hlxa is cyclophosphamide (lower surface curve);
Shown in Fig. 6 is the cell detection figure in real time that the Cordyceps militaris (L.) Link. antitumor component prepared of the embodiment of the present invention 1 suppresses people's hepatocarcinoma Hep-G2 Growth of Cells, and wherein C310-12-1-17 is object component (intermediate curve), and hlxa is cyclophosphamide (lower surface curve);
Shown in Fig. 7 is the molecular structural formula of peroxy-ergosterol.
The specific embodiment
Below in conjunction with embodiment, the present invention is further described in detail.Be noted that following illustrating is all exemplary, be intended to the invention provides further instruction.Except as otherwise noted, all Science and Technology terms that the present invention uses have the identical meanings of conventionally understanding with the technical field of the invention personnel.
Embodiment 1: extract antitumor component from Cordyceps militaris (L.) Link.
1, raw material, material:
Cordyceps militaris (L.) Link.sporophore is this Laboratory Production.
2, reagent:
Ultra-pure water is produced by Mi Libo pure water instrument; Acetone is purchased from the north, Tianjin day medical chemistry chemical reagent work; Chromatographic grade ethyl acetate, normal hexane, dichloromethane, methanol are purchased from Honeywell company.
3, instrument and equipment:
Super micron mill is purchased from Sanqing Stainless Steel Apparatus Co., Ltd., Shandong; Tabletop refrigerated centrifuge is purchased from Thermo company; Rotary Evaporators is purchased from Shanghai Ya Rong biochemical instrument factory; Chromatographic column (10 μ msilica150 × 250mm) is purchased from Jiangsu Di Wote instrument and equipment Science and Technology Ltd.; Chromatographic column (10 μ msilica21.2 × 250mm) is purchased from Tianjin Bonaaijieer Technology Co.,Ltd.
The step of extracting antitumor component from Cordyceps militaris (L.) Link.sporophore is as follows:
(1) pulverizing at ultralow temperature:
The Cordyceps militaris (L.) Link.sporophore of getting oven dry is raw material, take the Cordyceps militaris (L.) Link.sporophore that 150g is dried, put into super micron mill and carry out micronizing, pulverize the overall process water of 2~10 ℃ as micronized pulverization machine internal coolant simultaneously, pulverize temperature to reduce, guarantee release and bioactive the stablizing of effective ingredient, the micronizing time is 5-20min, grope 12min for the most suitable through practice, obtain superfine powder.
(2) solvent extraction:
(a) decocting in water: add ultra-pure water in superfine powder, the addition of ultra-pure water is 8~10 times of superfine powder quality, 100 ℃ of heating are extracted for 3~10 hours, 6000rpm centrifuging and taking precipitation, then repeat twice, the precipitation obtaining for three times is merged, at 60 ℃ of air dry oven inner dryings, obtain crude product A.
(b) acetone precipitation: add acetone in crude product A, the addition of acetone is 3 times of crude product A quality, lixiviate 12~36 hours under room temperature, after 6000rpm is centrifugal, get precipitation, then repeat twice, the precipitation obtaining for three times is merged, at 60 ℃ of air dry oven inner dryings, obtain crude product B.
(3) chromatographic isolation:
(a) get crude product for B 50% ethyl acetate-hexane solution to be dissolved to concentration be 50mg/ml, cross the organic microporous filter membrane of 0.45 μ m, then separate by preparative liquid chromatography.Chromatographic column filler is that silica, the size of particle diameter 10 μ m is 150 × 250mm, type of elution is 100% normal hexane isocratic elution 10min, 25% ethyl acetate-hexane solution isocratic elution 25min, 100% ethyl acetate isocratic elution 15min, applied sample amount is 200ml, flow velocity is 700ml/min, detection wavelength is 260nm, and chromatogram as shown in Figure 1, obtains each component after anti tumor activity in vitro screening, choose the strongest active component concentrated by rotary evaporation final vacuum dry, obtain component C3-10.
(b) get component C3-10 100% ethyl acetate 50 and spend ultrasonicly to be just dissolved to concentration be 50mg/ml, cross the organic microporous filter membrane of 0.45 μ m, then separate by preparative liquid chromatography.Chromatographic column filler is the silica of particle diameter 10 μ m, size is 150 × 250mm, type of elution is 100% normal hexane isocratic elution 10min, 10% ethyl acetate-hexane solution eluting 50min, 15% ethyl acetate-hexane solution eluting 20min, 30% ethyl acetate-hexane solution eluting 15min, 60% ethyl acetate-hexane solution eluting 20min, 100% methanol isocratic elution 10min, applied sample amount is 400ml, flow velocity is 700ml/min, detection wavelength is 260nm, chromatogram as shown in Figure 2, obtain each component after anti tumor activity in vitro screening, choose the strongest active component concentrated by rotary evaporation final vacuum dry, obtain component C310-12.
(c) get component C310-12 100% methanol 50 and spend ultrasonicly to be just dissolved to concentration be 50mg/ml, cross the organic microporous filter membrane of 0.45 μ m, then separate by preparative liquid chromatography.Chromatographic column filler is that silica, the size of particle diameter 10 μ m is 150 × 250mm, type of elution is 100% ethyl acetate~100% methanol gradient elution 30min, applied sample amount is 400ml, flow velocity is 700ml/min, detection wavelength is 260nm, and chromatogram as shown in Figure 3, obtains each component after anti tumor activity in vitro screening, choose the strongest active component concentrated by rotary evaporation final vacuum dry, obtain component C310-12-1.
(d) get component for C310-12-1 20% dichloromethane-hexane solution 50 spend ultrasonicly to be just dissolved to concentration be 50mg/ml, cross the organic microporous filter membrane of 0.45 μ m, then separate by preparative liquid chromatography.Chromatographic column filler is the silica of particle diameter 10 μ m, size is 21.2 × 250mm, type of elution is 100% normal hexane isocratic elution 8min, 20% dichloromethane-hexane solution isocratic elution 37min, 25% dichloromethane-hexane solution isocratic elution 15min, applied sample amount is 2ml, flow velocity is 15ml/min, detection wavelength is 260nm, chromatogram as shown in Figure 4, obtain each component after anti tumor activity in vitro screening, choose the strongest active component, concentrated by rotary evaporation final vacuum is dry, obtain component C310-12-1-17, 0.3g altogether, be the object of the invention antitumor component.
Antitumor component C310-12-1-17 obtained above is through the further separation and purification of preparative hplc and NMR carbon spectrum hydrogen analysis of spectrum, and preliminary judgement mainly contains peroxy-ergosterol (molecular structural formula as shown in Figure 7) and other unknown materials.
Embodiment 2: suppress people's lung cancer A549 cell growth test
Main material and reagent:
People's lung cancer A549 cell is given by Acea Bio. (Hangzhou) Co., Ltd.; F-12K culture medium is given by Gibco company; Cyclophosphamide is purchased from Tianjin Jinshi Pharmaceutical Co., Ltd..
Blank hole: do not add medicine;
Positive control hole: add cyclophosphamide;
Experimental port: the antitumor component C310-12-1-17 that adds embodiment 1 gained.
Cell culture:
At 5%CO
2, 37 ℃, under saturated humidity, with F-12K(10%FBS+1%PS) culture medium culturing people lung cancer A549 cell, choose the cell of logarithmic (log) phase growth as experimental cell.After cell counting, be the about cell suspension of 5.7 ten thousand/mL with culture medium dilution.
Growth of Cells condition monitoring:
Cell real-time monitor is put into 5%CO
2, in 37 ℃ of saturated humidity incubators.Get 8 orifice plates, every hole adds 150 μ LF-12K culture medium, puts into cell real-time monitor and walks baseline, after covering baseline, take out octal plate, every hole adds the A549 cell suspension 345 μ L that diluted, to every porocyte number approximately 20,000, leave standstill 3min, under inverted microscope, whether observation of cell is even.It is required drug level (25 μ g/ml) that every hole adds medicine (C310-12-1-17) to the final concentration that 5 μ L have diluted, with the cyclophosphamide of same concentrations as positive controls, contain the culture medium of 0.1%DMSO as blank group, put into cell real-time monitor and detect, detect Taking Pictures recording when complete.As shown in Figure 5, result shows experimental result: the present invention extracts the Cordyceps militaris (L.) Link. antitumor component C310-12-1-17 obtaining and can reach in the time of 25 μ g/ml 100% inhibition human lung cancer cell A549's growth.
Embodiment 3: suppress the test of people's hepatocarcinoma Hep-G2 Growth of Cells
Main material and reagent:
People's hepatocarcinoma Hep-G2 cell is given by huge Technology Park; MEM culture medium is given by Gibco company; Cyclophosphamide is purchased from Tianjin Jinshi Pharmaceutical Co., Ltd..
Blank hole: do not add medicine;
Positive control hole: add cyclophosphamide;
Experimental port: the antitumor component C310-12-1-17 that adds embodiment 1 gained.
Cell culture:
At 5%CO
2, 37 ℃, under saturated humidity, with MEM(10%FBS, 1%PS) and culture medium people's pulmonary carcinoma Hep-G2 cell, choose cell that growth conditions is good as experimental cell.After cell counting, be the about cell suspension of 5.7 ten thousand/mL with culture medium dilution.
Growth of Cells condition monitoring:
Cell real-time monitor is put into 5%CO
2, in 37 ℃ of saturated humidity incubators.Get 8 orifice plates, every hole adds 150 μ LMEM culture medium, puts into cell real-time monitor and walks baseline, after covering baseline, take out octal plate, every hole adds the Hep-G2 cell suspension 345 μ L that diluted, to every porocyte number approximately 40,000, leave standstill 3min, under inverted microscope, whether observation of cell is even.It is required drug level (25 μ g/ml) that every hole adds medicine (C310-12-1-17) to the final concentration that 5 μ L have diluted, with the cyclophosphamide of same concentrations as positive controls, contain the culture medium of 0.1%DMSO as blank group, put into cell real-time monitor and detect, detect Taking Pictures recording when complete.As shown in Figure 6, result shows experimental result: the present invention extracts the Cordyceps militaris (L.) Link. antitumor component C310-12-1-17 obtaining can reach 100% inhibition human liver cancer cell Hep-G2 growth in the time of 25 μ g/ml.
Because Cordyceps militaris (L.) Link. itself is with regard to edible, so it has the features such as nontoxic, safe, can obtain effective antitumour new drug and/or anti-tumor health care product by Cordyceps militaris (L.) Link. antitumor component of the present invention, there is feature efficient, nontoxic and that the relative Cordyceps of raw material is cheap and easy to get.Because C310-12-1-17 mainly contains 2,6-dimethyl 3-hydroxyl decane, in the time that concentration is 25 μ g/ml, can reach the growth of 100% inhibition human lung cancer cell A549 and human liver cancer cell Hep-G2, this has shown the potentiality of C310-12-1-17 aspect further exploitation effective antitumour monomer medicine.Next we will carry out active component C310-12-1-17 to prepare on a large scale, strive for obtaining the strong activated monomer compound of antitumor, develop strong active antitumor monomer medicine, for the mankind's health is offered as a tribute our meagre strength.
Material, reagent and experimental facilities that the embodiment of the present invention relates to, if no special instructions, be the common commercially available prod that meets biological product processing.
The above, be only the preferred embodiments of the present invention, should be understood that; for those skilled in the art; not departing under the prerequisite of core technology of the present invention, can also make improvements and modifications, these improvements and modifications also should belong to scope of patent protection of the present invention.Any change in implication and the scope suitable with claims of the present invention, all should think to be included in the scope of claims.