CN101559083A - Fruiting body extract of Ganoderma Lucidum with antitumor activity and preparation method - Google Patents
Fruiting body extract of Ganoderma Lucidum with antitumor activity and preparation method Download PDFInfo
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Abstract
The invention discloses a fruiting body extract of Ganoderma Lucidum with antitumor activity and a preparation method, wherein the content of triterpenes components in the fruiting body extract of Ganoderma lucidum is 30-70wt%; meanwhile, the extract comprises active components represented by the following method: the extract is characterized by the condition 1, which is composed of components with chromatographic peak retention time being 80-128min; the extract is characterized by the condition 2, which is composed of five components with chromatographic peak retention times being 14.89-15.67min, 30.74-31.35min, 34.36-34.98min and 37.03-37.64min, 39.47-40.01min. The invention overcomes the problem that only triterpenic acid components in Ganoderma Lucidum are used by the prior art and the other components with antitumor activity are neglected, can take full advantage of Ganoderma Lucidum medicinal materials better, and greatly reduces the environmental pollution generated by organic solvent, thereby protecting environment.
Description
Technical field:
The present invention relates to the Chinese medicine Ganoderma sporophore extract, specifically relate to a kind of Ganoderma sporophore extract with anti-tumor activity, and this preparation method with Ganoderma sporophore extract of anti-tumor activity.
Background technology:
Ganoderma is the Polyporaceae fungus, and medicinal history in several thousand is arranged, and records this agrostology monograph Shennong's Herbal the earliest in China.2,000 for many years, and people prevent, treat tumor with Ganoderma always; Modern scientific research shows that also Ganoderma has stronger antitumor action.
The Ganoderma triterpenoids constituents is acknowledged as one of Ganoderma antineoplastic effective composition.About the extraction of Ganoderma triterpenoids constituents, alcohol heating reflux commonly used extracts, and it exists, and organic solvent residual is many, extraction temperature height, the more high relatively shortcoming of cost.Supercritical CO
2Fluid technique is a state-of-the-art technology of extracting the Ganoderma triterpenoids constituents, has overcome above shortcoming.Chinese patent 200510047263.9 discloses CO
2-SFE is the application on the triterpene substance in extracting Ganoderma sporophore.
Though at present about the Ganoderma sporophore supercritical CO
2The Study on extraction of extraction triterpenes component is more, but less relatively to the purification research of triterpenes component, and is clinical many with supercritical CO
2The extract direct drug injection, or the Ganoderma triterpenoids acids composition behind the purification is used for the treatment of.
Li Baoming (referring to: Li Baoming, Liu Chao, Wang Hongqing, Deng. the research [J] of Ganoderma total triterpenes acid content assay method. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2007,32 (12) 1234-1236.) report Ganoderma sporophore is extracted with alcohol heating reflux, the chloroform dissolving, saturated sodium bicarbonate solution alkali is carried, hcl acidifying, and the method for chloroform extraction obtains Ganoderma triterpenoids acids composition; Chinese patent 200310117112.7 discloses a kind of extracting method of ganoderma lucidum triterpene antitumor component, it with Ganoderma sporophore with methanol or the lixiviate of ethanol ultrasonic circulating, carry with saturated sodium bicarbonate solution alkali then, hcl acidifying, the triterpenic acid constituents of reuse chloroform extraction after with acidify, evaporated under reduced pressure obtains triterpene acids component.More than report has a something in common, and the chloroform layer after promptly all saturated sodium bicarbonate solution alkali being carried discards, and only keeps the triterpenic acid constituents in the saturated sodium bicarbonate solution alkali extract, with it acidify and extract.
Summary of the invention
Purpose of the present invention provides a kind of Ganoderma sporophore extract with anti-tumor activity, and this is a kind of from the Ganoderma sporophore supercritical CO
2The extract that obtains in the extract with anti-tumor activity.The present invention also provides this preparation method with Ganoderma sporophore extract of anti-tumor activity.The present invention will overcome and generally use triterpenic acid constituents in the Ganoderma triterpenoids in the past, have the problem that anti-tumor active ingredient utilizes and ignore other that wherein contain.
The scheme of finishing the 1st invention task of the application is, a kind of Ganoderma sporophore extract with anti-tumor activity is characterized in that, wherein the content of triterpenes components is 30～70wt%; Simultaneously, the effective ingredient that contains in this extract also characterizes in order to following method: characterize extract with high-efficient liquid phase chromatogram condition 1, be grouped into (Fig. 1) by the one-tenth of chromatographic peak retention time between 80～128min; Characterize extract with high-efficient liquid phase chromatogram condition 2, mainly comprising the chromatographic peak retention time is 5 compositions (Fig. 2) of 14.89～15.67min, 30.74～31.35min, 34.36～34.98min, 37.03～37.64min, 39.47～40.01min.
Described high-efficient liquid phase chromatogram condition 1 is: ZORBAX SB-C18 post (4.6mm * 250mm, 5 μ m); Mobile phase: A: acetonitrile, B:0.05% phosphate aqueous solution, linear gradient elution, 0min:27%A; 40min:27%A; 60min:35%A; 80min:45%A; 90min:65%A; 100min:75%A; 110min:85%A; 120min:100%A; 128min:100%A; Flow velocity: 1.0mLmin-1; Detect wavelength: 254nm; Column temperature: 30 ℃.
With this understanding, Ganoderma sporophore supercritical CO
2The crude extract of extraction is formed (Fig. 3) by all the components of chromatographic peak retention time between 0～128min; The sodium bicarbonate extract layer of this crude extract (i.e. the Ganoderma triterpenoids acids composition that in the past generally used) is formed (Fig. 4) by all the components of chromatographic peak retention time between 0～80min, and all the components of chromatographic peak retention time between 80～128min formed this extract.
High-efficient liquid phase chromatogram condition 2 is: ZORBAX SB-C18 post (4.6mm * 250mm, 5 μ m); Mobile phase: A: acetonitrile, B:0.05% phosphate aqueous solution, linear gradient elution, 0min:60%A; 25min:90%A; 40min:100%A; 45min:100%A; Flow velocity: 1.0mLmin-1; Detect wavelength: 240nm; Column temperature: 30 ℃.
The triterpenes components content assaying method: using spectrophotography, is reference substance with the ursolic acid, is to measure under the 543nm detecting wavelength, and the spectral scan figure of test sample and blank sample sees Fig. 5, Fig. 6, Fig. 7.
The applicant discovers the Ganoderma sporophore supercritical CO
2The crude extract that abstraction technique extracts, the chloroform layer after saturated sodium bicarbonate solution alkali is carried, the material that discards in the promptly above report also has very strong anti-tumor activity.So the present invention has set up a kind of Ganoderma triterpenoids acid method of extract in addition of extracting from Ganoderma sporophore, obtain a kind of Ganoderma sporophore extract with anti-tumor activity.
Say that more specifically and more optimally the Ganoderma sporophore extract with anti-tumor activity of the present invention is meant the extract that adopts following method to prepare:
(1) Ganoderma sporophore is ground into coarse powder, sieves;
(2) with ethanol the entrainer supercritical CO with the Ganoderma sporophore coarse powder
2Abstraction technique extracts, and obtains Ganoderma sporophore crude extract alcoholic solution, and decompression and solvent recovery obtains the Ganoderma sporophore crude extract;
(3) the Ganoderma sporophore crude extract is dissolved with ethyl acetate, the saturated sodium bicarbonate solution extraction obtains saturated sodium bicarbonate layer and ethyl acetate layer respectively;
(4) get ethyl acetate layer decompression and solvent recovery, vacuum drying gets the Ganoderma sporophore antineoplastic extract.
Another object of the present invention provides the preparation method of said extracted thing, it is characterized in that, step is as follows:
(1) Ganoderma sporophore is ground into coarse powder, sieves;
(2) with ethanol the entrainer supercritical CO with the Ganoderma sporophore coarse powder
2Abstraction technique extracts, and obtains Ganoderma sporophore crude extract alcoholic solution, and decompression and solvent recovery obtains the Ganoderma sporophore crude extract;
(3) the Ganoderma sporophore crude extract is dissolved with ethyl acetate, the saturated sodium bicarbonate solution extraction obtains saturated sodium bicarbonate layer and ethyl acetate layer respectively;
(4) get ethyl acetate layer decompression and solvent recovery, vacuum drying gets the Ganoderma sporophore antineoplastic extract.
In above-mentioned steps (1), when Ganoderma sporophore is pulverized, cross 10～50 mesh sieves.In preferred embodiment of the present invention, the Ganoderma sporophore coarse powder is crossed 12 mesh sieves.
In above-mentioned steps (2), supercritical CO
2Extraction conditions is: extracting pressure is 25MPa, and extraction temperature is 45 ℃, and the extraction time is 1.5h, and entrainer ethanol consumption is 3mLg
-1Crude drug.
In above-mentioned steps (2), when Ganoderma sporophore crude extract alcoholic solution reclaimed ethanol, temperature was controlled at 30～60 ℃, and Optimal Temperature Control is at 50 ℃.
In above-mentioned steps (3), when dissolving with ethyl acetate, the ratio of Ganoderma sporophore crude extract and ethyl acetate is a Ganoderma sporophore crude extract quality (g): ethyl acetate (mL)=1: 10～1: 100, be preferably 1: 40～1: 80, and most preferably be 1: 50.
In above-mentioned steps (3), with saturated sodium bicarbonate solution extraction ethyl acetate solution, extraction times is 1～8 time, and preferred extraction times is 2～6 times, and most preferably extraction times is 4 times.
In above-mentioned steps (3), during with saturated sodium bicarbonate solution extraction ethyl acetate solution, ethyl acetate and saturated sodium bicarbonate solution volume ratio are 1: 3～3: 1, are preferably 1: 2～2: 1, most preferably are 1: 1.
In above-mentioned steps (4), when reclaiming ethyl acetate solvent and vacuum drying extract, temperature is controlled at 30～60 ℃, is preferably 50 ℃.
The pitchy solid that obtains in above-mentioned steps (4) is the Ganoderma sporophore antineoplastic extract.
The pharmaceutical usage of Ganoderma sporophore extract of the present invention be with described Ganoderma sporophore extract as anti-tumor active ingredient, add acceptable accessories, the medicine of any dosage form of making.
Beneficial effect of the present invention
1. the present invention uses supercritical CO
2The active component of fluid technique extracting ganoderma sporophore can reduce the environmental pollution that organic solvent produces significantly, the protection environment.
2. antineoplastic extract of the present invention has better antitumous effect more in the past than the Ganoderma triterpenoids acids composition that generally uses, and making full use of extract of the present invention can be better, this flavor valuable ingredient of Chinese medicine of more abundant reasonable use Ganoderma.
Description of drawings
Fig. 1 is an extractive HPLC collection of illustrative plates of the present invention (chromatographic condition 1);
Fig. 2 is an extractive HPLC collection of illustrative plates of the present invention (chromatographic condition 2);
Fig. 3 is the Ganoderma sporophore supercritical CO
2Crude extract HPLC collection of illustrative plates (chromatographic condition 1);
Fig. 4 is the Ganoderma sporophore supercritical CO
2The HPLC collection of illustrative plates of the sodium bicarbonate extract layer of crude extract (chromatographic condition 1);
Fig. 5 is the spectral scan figure of ursolic acid reference substance;
Fig. 6 is the spectral scan figure of test sample;
Fig. 7 is the spectral scan figure of blank sample.
Fig. 8 is for obtaining a kind of preparation method of extract flow chart with anti-tumor activity from Ganoderma sporophore.
The specific embodiment
Below in conjunction with example the present invention is described in further detail, but scope of the present invention is not subjected to the restriction of these examples.
Embodiment 1
Ganoderma sporophore is pulverized, crossed 12 mesh sieves, get the 1L extraction kettle that 100g places supercritical extraction unit, setting the extraction kettle temperature is 45 ℃, and the separation reactor I temperature is 50 ℃, and separation reactor I I temperature is 36 ℃, opens CO
2Steel cylinder (needing to keep outlet pressure more than 4MPa), regulating extraction kettle pressure simultaneously is that 25MPa, separation reactor I pressure are that 7MPa, separation reactor I I pressure are 4MPa, regulates CO
2Flow is 30L/h, extraction time 1.5h, and entrainer ethanol consumption is 300mL.Make supercritical CO
2Fluid carries out extraction cycle under the pressure of setting, temperature, after the extraction time that reaches the test setting, receive extract from separation reactor I and separation reactor I I discharging opening; With the extracting solution that obtains, 50 ℃ of decompression and solvent recoveries obtain the Ganoderma sporophore crude extract.
With above-mentioned resulting Ganoderma sporophore crude extract, with 50 times of ethyl acetate dissolvings; Reuse saturated sodium bicarbonate solution extraction 4 times, saturated sodium bicarbonate solution and ethyl acetate volume ratio are 1: 1 during extraction, obtain sodium bicarbonate solution layer and ethyl acetate layer; 50 ℃ of reclaim under reduced pressure ethyl acetate solvents, and, get pitchy pressed powder 1.06g in 50 ℃ of vacuum dryings.
Triterpenes components assay in the extract of the present invention:
Get Ganoderma sporophore antineoplastic extract 50mg, to 50mL, measure the content of triterpenes components with visible spectrophotometry with the dehydrated alcohol standardize solution.
The preparation of reference substance solution: precision takes by weighing ursolic acid reference substance 2.21mg, places the 10mL measuring bottle, adds dehydrated alcohol and makes dissolving in right amount, and standardize solution shakes up, promptly.
Coloration method and detection wavelength determination: get above-mentioned ursolic acid reference substance solution 0.5mL, put in the 10mL tool plug test tube, volatilize solvent in the boiling water bath, add the 5% vanillin-glacial acetic acid solution 0.5mL of new preparation successively, perchloric acid 0.8mL, close plug, in 60 ℃ of water-baths, heat 15min, take out, cool off 5min in the ice-water bath, add glacial acetic acid 5mL, shake up.Carrying out full wavelength scanner with the visible spectrophotometer of UV-2802 type, at the 543nm place maximum absorption band is arranged, is 543nm so select to detect wavelength.
The preparation of standard curve: the accurate respectively ursolic acid reference substance solution 0.2,0.3,0.4 of drawing, 0.5,0.6,0.7,0.8mL, measuring absorbance at the 543nm place by above-mentioned condition and step, is vertical coordinate (Y) with the absorbance, and ursolic acid reference substance content (μ g) is abscissa (X), the drawing standard curve, linear equation is: Y=0.0059X+0.0035, and r=0.9995, the result shows that ursolic acid presents good linear relationship at 44.2～154.7 μ g.
Assay: get the Ganoderma sporophore antineoplastic extract alcoholic solution 60 μ L of above-mentioned preparation, measure absorbance down, calculate the content of Ganoderma triterpenoids according to standard curve by " coloration method " item.Result: see Table 2.
The assay of solid content: precision is measured above-mentioned Ganoderma sporophore antineoplastic extract alcoholic solution 10mL, puts in the evaporating dish of dry constant weight, and water-bath volatilizes solvent, puts constant weight in 105 ℃ of baking ovens, and it is fixed to claim, calculates the content of solid content, the results are shown in Table 1.
The triterpene content of table 1 extract of the present invention, the mensuration of solid content
Embodiment 2
Ganoderma sporophore is pulverized, crossed 10 mesh sieves, get the 1L extraction kettle that 100g places supercritical extraction unit, setting the extraction kettle temperature is 45 ℃, and the separation reactor I temperature is 50 ℃, and separation reactor I I temperature is 36 ℃, opens CO
2Steel cylinder (needing to keep outlet pressure more than 4MPa), regulating extraction kettle pressure simultaneously is that 25MPa, separation reactor I pressure are that 7MPa, separation reactor I I pressure are 4MPa, regulates CO
2Flow is 30L/h, extraction time 1.5h, and entrainer ethanol consumption is 300mL.Make supercritical CO
2Fluid carries out extraction cycle under the pressure of setting, temperature, after the extraction time that reaches the test setting, receive extract from separation reactor I and separation reactor I I discharging opening; With the extracting solution that obtains, 30 ℃ of decompression and solvent recoveries obtain the Ganoderma sporophore crude extract.
With above-mentioned resulting Ganoderma sporophore crude extract, with 100 times of ethyl acetate dissolvings; Reuse saturated sodium bicarbonate solution extraction 6 times, saturated sodium bicarbonate solution and ethyl acetate volume ratio are 2: 1 during extraction, obtain sodium bicarbonate solution layer and ethyl acetate layer; 40 ℃ of reclaim under reduced pressure ethyl acetate solvents, and, obtain pitchy pressed powder 0.95g in 40 ℃ of vacuum dryings.
Embodiment 3
Ganoderma sporophore is pulverized, crossed 50 mesh sieves, get the 1L extraction kettle that 100g places supercritical extraction unit, setting the extraction kettle temperature is 45 ℃, and the separation reactor I temperature is 50 ℃, and separation reactor I I temperature is 36 ℃, opens CO
2Steel cylinder (needing to keep outlet pressure more than 4MPa), regulating extraction kettle pressure simultaneously is that 25MPa, separation reactor I pressure are that 7MPa, separation reactor I I pressure are 4MPa, regulates CO
2Flow is 30L/h, extraction time 1.5h, and entrainer ethanol consumption is 300mL.Make supercritical CO
2Fluid carries out extraction cycle under the pressure of setting, temperature, after the extraction time that reaches the test setting, receive extract from separation reactor I and separation reactor I I discharging opening; With the extracting solution that obtains, 50 ℃ of decompression and solvent recoveries obtain the Ganoderma sporophore crude extract.
With above-mentioned resulting Ganoderma sporophore crude extract, with 10 times of ethyl acetate dissolvings; Reuse saturated sodium bicarbonate solution extraction 2 times, saturated sodium bicarbonate solution and ethyl acetate volume ratio are 1: 3 during extraction, obtain saturated sodium bicarbonate solution solution layer and ethyl acetate layer; 30 ℃ of reclaim under reduced pressure ethyl acetates, 30 ℃ of vacuum dryings obtain pitchy pressed powder 1.31g.
Embodiment 4
Ganoderma sporophore is pulverized, crossed 12 mesh sieves, get the 1L extraction kettle that 100g places supercritical extraction unit, setting the extraction kettle temperature is 45 ℃, and the separation reactor I temperature is 50 ℃, and separation reactor I I temperature is 36 ℃, opens CO
2Steel cylinder (needing to keep outlet pressure more than 4MPa), regulating extraction kettle pressure simultaneously is that 25MPa, separation reactor I pressure are that 7MPa, separation reactor I I pressure are 4MPa, regulates CO
2Flow is 30L/h, extraction time 1.5h, and entrainer ethanol consumption is 300mL.Make supercritical CO
2Fluid carries out extraction cycle under the pressure of setting, temperature, after the extraction time that reaches the test setting, receive extract from separation reactor I and separation reactor I I discharging opening; With the extracting solution that obtains, 60 ℃ of decompression and solvent recoveries obtain the Ganoderma sporophore crude extract.
With above-mentioned resulting Ganoderma sporophore crude extract, with 80 times of ethyl acetate dissolvings; Reuse saturated sodium bicarbonate solution extraction 5 times, saturated sodium bicarbonate solution and ethyl acetate volume ratio are 3: 1 during extraction, obtain saturated sodium bicarbonate solution and ethyl acetate layer; 60 ℃ of reclaim under reduced pressure ethyl acetates, 60 ℃ of vacuum dryings obtain pitchy pressed powder 0.81g.
Embodiment 5
Ganoderma sporophore is pulverized, crossed 12 mesh sieves, get the 1L extraction kettle that 100g places supercritical extraction unit, setting the extraction kettle temperature is 45 ℃, and the separation reactor I temperature is 50 ℃, and separation reactor I I temperature is 36 ℃, opens CO
2Steel cylinder (needing to keep outlet pressure more than 4MPa), regulating extraction kettle pressure simultaneously is that 25MPa, separation reactor I pressure are that 7MPa, separation reactor I I pressure are 4MPa, regulates CO
2Flow is 30L/h, extraction time 1.5h, and entrainer ethanol consumption is 300mL.Make supercritical CO
2Fluid carries out extraction cycle under the pressure of setting, temperature, after the extraction time that reaches the test setting, receive extract from separation reactor I and separation reactor I I discharging opening; With the extracting solution that obtains, 50 ℃ of decompression and solvent recoveries obtain the Ganoderma sporophore crude extract.
With above-mentioned resulting Ganoderma sporophore crude extract, with 40 times of ethyl acetate dissolvings; Reuse saturated sodium bicarbonate solution extraction 4 times, saturated sodium bicarbonate solution and ethyl acetate volume ratio are 1: 2 during extraction, obtain saturated sodium bicarbonate solution layer and ethyl acetate layer; 40 ℃ of reclaim under reduced pressure ethyl acetate solvents, 40 ℃ of vacuum dryings obtain pitchy pressed powder 1.19g.
Embodiment 6
Ganoderma sporophore is pulverized, crossed 12 mesh sieves, get the 1L extraction kettle that 100g places supercritical extraction unit, setting the extraction kettle temperature is 45 ℃, and the separation reactor I temperature is 50 ℃, and separation reactor I I temperature is 36 ℃, opens CO
2Steel cylinder (needing to keep outlet pressure more than 4MPa), regulating extraction kettle pressure simultaneously is that 25MPa, separation reactor I pressure are that 7MPa, separation reactor I I pressure are 4MPa, regulates CO
2Flow is 30L/h, extraction time 1.5h, and entrainer ethanol consumption is 300mL.Make supercritical CO
2Fluid carries out extraction cycle under the pressure of setting, temperature, after the extraction time that reaches the test setting, receive extract from separation reactor I and separation reactor I I discharging opening; With the extracting solution that obtains, 50 ℃ of decompression and solvent recoveries obtain the Ganoderma sporophore crude extract.
With above-mentioned resulting Ganoderma sporophore crude extract, with 60 times of ethyl acetate dissolvings; Reuse saturated sodium bicarbonate solution extraction 5 times, saturated sodium bicarbonate solution and ethyl acetate volume ratio are 1: 1 during extraction, obtain saturated sodium bicarbonate solution layer and ethyl acetate layer; 60 ℃ of reclaim under reduced pressure ethyl acetate solvents, 60 ℃ of vacuum dryings obtain pitchy pressed powder 0.90g.
Embodiment 7
Ganoderma sporophore is pulverized, crossed 12 mesh sieves, get the 1L extraction kettle that 100g places supercritical extraction unit, setting the extraction kettle temperature is 45 ℃, and the separation reactor I temperature is 50 ℃, and separation reactor I I temperature is 36 ℃, opens CO
2Steel cylinder (needing to keep outlet pressure more than 4MPa), regulating extraction kettle pressure simultaneously is that 25MPa, separation reactor I pressure are that 7MPa, separation reactor I I pressure are 4MPa, regulates CO
2Flow is 30L/h, extraction time 1.5h, and entrainer ethanol consumption is 300mL.Make supercritical CO
2Fluid carries out extraction cycle under the pressure of setting, temperature, after the extraction time that reaches the test setting, receive extract from separation reactor I and separation reactor I I discharging opening; With the extracting solution that obtains, 50 ℃ of decompression and solvent recoveries obtain the Ganoderma sporophore crude extract.
With above-mentioned resulting Ganoderma sporophore crude extract, with 80 times of ethyl acetate dissolvings; Reuse saturated sodium bicarbonate solution extraction 8 times, saturated sodium bicarbonate solution and ethyl acetate volume ratio are 1: 1 during extraction, obtain saturated sodium bicarbonate solution layer and ethyl acetate layer; 50 ℃ of reclaim under reduced pressure ethyl acetates, 50 ℃ of vacuum dryings obtain pitchy pressed powder 0.76g.
Embodiment 8
Ganoderma sporophore is pulverized, crossed 12 mesh sieves, get the 1L extraction kettle that 100g places supercritical extraction unit, setting the extraction kettle temperature is 45 ℃, and the separation reactor I temperature is 50 ℃, and separation reactor I I temperature is 36 ℃, opens CO
2Steel cylinder (needing to keep outlet pressure more than 4MPa), regulating extraction kettle pressure simultaneously is that 25MPa, separation reactor I pressure are that 7MPa, separation reactor I I pressure are 4MPa, regulates CO
2Flow is 30L/h, extraction time 1.5h, and entrainer ethanol consumption is 300mL.Make supercritical CO
2Fluid carries out extraction cycle under the pressure of setting, temperature, after the extraction time that reaches the test setting, receive extract from separation reactor I and separation reactor I I discharging opening; With the extracting solution that obtains, 60 ℃ of decompression and solvent recoveries obtain the Ganoderma sporophore crude extract.
With above-mentioned resulting Ganoderma sporophore crude extract, with 70 times of ethyl acetate dissolvings; Reuse saturated sodium bicarbonate solution extraction 1 time, saturated sodium bicarbonate solution and ethyl acetate volume ratio are 1: 1 during extraction, obtain saturated sodium bicarbonate solution layer and ethyl acetate layer; 40 ℃ of reclaim under reduced pressure ethyl acetates, 40 ℃ of vacuum dryings obtain pitchy pressed powder 1.79g.
Embodiment 9
The anticancer experiment in vitro of extract of the present invention:
Human hepatocellular carcinoma BEL-7402 cell's strain
The trophophase human hepatocellular carcinoma BEL-7402 cell (available from Shanghai RESEARCH ON CELL-BIOLOGY institute of Chinese Academy of Sciences cell bank) that takes the logarithm uses trypsinization, is mixed with certain density cell suspension with containing calf serum RPMI 1640 culture medium, and the concentration of cell is 5 * 10
4Individual/mL, be inoculated in 96 orifice plates then, every hole adds 100 μ L, puts 37 ℃, 5%CO
2Cultivate 24h under incubator and the saturated humidity condition, discard the culture medium that contains calf serum then, every hole adds 90 μ L serum-free mediums, and the medicine of 10 μ L variable concentrations, and the positive drug group gives 60 μ gmL
-1Cisplatin solution, blank group adds isopyknic PBS buffer salt solution, establishes two these bottom outlets (promptly do not contain tumor cell, only contain corresponding culture medium and medicine) for every group in addition, to remove the influence of culture medium and medicinal liquid color.Be subjected to reagent to be respectively extract of the present invention (being the ethyl acetate layer) and Ganoderma sporophore triterpenic acid extract (being the saturated sodium bicarbonate layer), all establish 5 dosage groups, being diluted to concentration with PBS respectively is 20,5,1.25,0.3125,0.0781 μ g extract mL-1, establishes 3 parallel holes for every group.After 37 ℃, 5%CO2 incubator were cultivated 48h, every hole added MTT solution (5mgmL
-1) 10 μ L, continue to cultivate 4h, stop cultivating, inhale and abandon supernatant in the hole, every then hole adds 100 μ L DMSO dissolving MTT first a ceremonial jade-ladle, used in libation granule, behind microoscillator vibration 10min, measure optical density value (OD) in enzyme-linked immunosorbent assay instrument 570nm wavelength, experiment repeats 3 times, averages, calculate the suppression ratio of medicine, the results are shown in Table 2 tumor cell.
Medicine suppression ratio=[OD
Matched group-OD
The medicine group]/OD
Matched group* 100%
Table 2 extract of the present invention and Ganoderma sporophore triterpenic acid extract
To human hepatocellular carcinoma BEL-7402 cell's inhibited proliferation relatively (X ± s, n=3)
Annotate: positive control cisplatin group inhibitory rate of cell growth is respectively: (70.47 ± 0.65) %
"---" represent that suppression ratio all less than 50%, can not calculate IC
50
The result shows that extract of the present invention has the activity of stronger inhibition human hepatocellular carcinoma BEL-7402 cell propagation; And under same concentration,, has the activity of better inhibition people hepatocarcinoma BEL-7402 tumor cell proliferation than Ganoderma sporophore triterpenic acid extract suppression ratio height.
People's pulmonary carcinoma SPC-A-1 cell strain
The trophophase people pulmonary carcinoma of taking the logarithm SPC-A-1 cell (available from Shanghai RESEARCH ON CELL-BIOLOGY institute of Chinese Academy of Sciences cell bank) is used trypsinization, makes it take off wall, and with the resuspended one-tenth single cell suspension of 10% hyclone RPMI1640 culture fluid, the concentration of cell is 5 * 10
4Individual/mL, be inoculated in 96 porocyte culture plates then, every hole adds 100 μ L.Be subjected to reagent to be respectively extract of the present invention (being the ethyl acetate layer) and Ganoderma sporophore triterpenic acid extract (being the saturated sodium bicarbonate layer), all establish 5 dosage groups, being diluted to concentration with PBS respectively is 20,10,5,1.25,0.3125 μ g extract mL-1, establishes 3 parallel holes for every group.All the other operations are identical with the human hepatocellular carcinoma BEL-7402 cell's, calculate the suppression ratio of medicine to tumor cell, the results are shown in Table 3.
Table 3 extract of the present invention and Ganoderma sporophore triterpenic acid extract
To the inhibited proliferation of people's pulmonary carcinoma SPC-A-1 cell relatively (X ± s, n=3)
Annotate: positive control cisplatin group inhibitory rate of cell growth is respectively: (74.08 ± 0.59) %
"---" represent that suppression ratio all less than 50%, can not calculate IC
50
The gained result shows that extract of the present invention has the proliferation activity of stronger inhibition people pulmonary carcinoma SPC-A-1; And under same concentration,, has the activity of better inhibition SPC-A-1 tumor cell proliferation than Ganoderma sporophore triterpenic acid extract suppression ratio height.
The anti-tumor in vivo experiment of extract of the present invention:
Select inoculation 7d, well-grown lotus Heps liver cancer mouse, the cervical vertebra dislocation is put to death, and aseptic extraction ascites becomes 2 * 10 with physiological saline solution by 1: 4 dilution proportion
7The tumor cell suspension of individual/mL, it is subcutaneous to be inoculated in the healthy mice right fore, every 0.2mL.Inoculate next day, be divided into 6 groups at random, be respectively: normal saline blank group by body weight; The cyclophosphamide positive controls; The high low dose group of extract of the present invention (140mg extract kg-1d-1,70mg extract kg-1d-1); The high low dose group of Ganoderma sporophore triterpenic acid extract (140mg extract kg-1d-1,70mg extract kg-1d-1).The cyclophosphamide positive controls is lumbar injection (20mgkg
-1D
-1), other group is for gastric infusion, is every day 1 time, continuously 7d.During this time, observe the situation such as general activity, fur, feces of mice every day.Put to death mice with cervical vertebra dislocation behind the 24h after the last administration and strip tumor tissue, cut open simultaneously and get mouse spleen, thymus, weigh, calculate tumour inhibiting rate, spleen index, thymus index, the results are shown in Table 4, table 5.
Tumour inhibiting rate (%)=(it is heavy that the average tumor of average tumor weight/matched group is organized in the 1-treatment) * 100%;
Heavy (the mg)/mice body weight (g) of spleen index=spleen;
Heavy (the mg)/mice body weight (g) of thymus index=thymus.
Table 4 extract of the present invention and Ganoderma sporophore triterpenic acid extract
To the inhibitory action of Heps transplanted tumor (X ± s) relatively
Annotate: compare with negative control group
*P＜0.05
The result shows that extract of the present invention has hepatocarcinoma Heps growth of tumor effect in the stronger inhibition mice body, and its high and low dose group all has significance to reduce (P＜0.05) with the tumor heavy phase ratio of matched group; And under same concentrations,, has the activity of hepatocarcinoma Heps growth of tumor in the better inhibition mice body than Ganoderma sporophore triterpenic acid extract tumour inhibiting rate height.
Table 5 extract of the present invention and Ganoderma sporophore triterpenic acid extract
To the influence of tumor-bearing mice immune organ (X ± s) relatively
Annotate: compare with negative control group
*P＜0.05
Experimental result shows that the spleen index of extract group of the present invention and thymus index and blank group be there was no significant difference relatively, shows that promptly Ganoderma sporophore extract has no side effect to the immune organ thing of tumor-bearing mice, can keep the immunocompetence of body.
Claims (9)
1, a kind of Ganoderma sporophore extract with anti-tumor activity is characterized in that, wherein the content of triterpenes components is 30～70wt%; Simultaneously, the effective ingredient in this extract also characterizes in order to following method: characterize extract with high-efficient liquid phase chromatogram condition 1, be grouped into by the one-tenth of chromatographic peak retention time between 80～128min; Characterize extract with high-efficient liquid phase chromatogram condition 2, comprising the chromatographic peak retention time is 5 compositions of 14.89～15.67min, 30.74～31.35min, 34.36～34.98min, 37.03～37.64min, 39.47～40.01min;
Described high-efficient liquid phase chromatogram condition 1 is: ZORBAX SB-C18 post; Mobile phase: A: acetonitrile, B:0.05% phosphate aqueous solution, linear gradient elution, 0min:27%A; 40min:27%A; 60min:35%A; 80min:45%A; 90min:65%A; 100min:75%A; 110min:85%A; 120min:100%A; 128min:100%A; Flow velocity: 1.0mLmin-1; Detect wavelength: 254nm; Column temperature: 30 ℃;
Described high-efficient liquid phase chromatogram condition 2 is: ZORBAX SB-C18 post (4.6mm * 250mm, 5 μ m); Mobile phase: A: acetonitrile, B:0.05% phosphate aqueous solution, linear gradient elution, 0min:60%A; 25min:90%A; 40min:100%A; 45min:100%A; Flow velocity: 1.0mLmin-1; Detect wavelength: 240nm; Column temperature: 30 ℃.
2, the Ganoderma sporophore extract with anti-tumor activity according to claim 1 is characterized in that, this Ganoderma sporophore extract with anti-tumor activity is meant the extract that adopts following method to prepare:
(1) Ganoderma sporophore is ground into coarse powder, sieves;
(2) with ethanol the entrainer supercritical CO with the Ganoderma sporophore coarse powder
2Abstraction technique extracts, and obtains Ganoderma sporophore crude extract alcoholic solution, and decompression and solvent recovery obtains the Ganoderma sporophore crude extract;
(3) the Ganoderma sporophore crude extract is dissolved with ethyl acetate, the saturated sodium bicarbonate solution extraction obtains saturated sodium bicarbonate layer and ethyl acetate layer respectively;
(4) get ethyl acetate layer decompression and solvent recovery, vacuum drying gets the Ganoderma sporophore antineoplastic extract.
3, the described preparation method with Ganoderma sporophore extract of anti-tumor activity of a kind of claim 1 is characterized in that step is as follows:
(1) Ganoderma sporophore is ground into coarse powder, sieves;
(2) with ethanol the entrainer supercritical CO with the Ganoderma sporophore coarse powder
2Abstraction technique extracts, and obtains Ganoderma sporophore crude extract alcoholic solution, and decompression and solvent recovery obtains the Ganoderma sporophore crude extract;
(3) the Ganoderma sporophore crude extract is dissolved with ethyl acetate, the saturated sodium bicarbonate solution extraction obtains saturated sodium bicarbonate layer and ethyl acetate layer respectively;
(4) get ethyl acetate layer decompression and solvent recovery, vacuum drying gets the Ganoderma sporophore antineoplastic extract.
4, the preparation method with Ganoderma sporophore extract of anti-tumor activity according to claim 3 is characterized in that,
In described step (1), when Ganoderma sporophore is pulverized, cross 10～50 mesh sieves;
In described step (2), the supercritical CO 2 extraction conditions is: extracting pressure is 25MPa, and extraction temperature is 45 ℃, and the extraction time is 1.5h, and entrainer ethanol consumption is 3mLg
-1Crude drug; When Ganoderma sporophore crude extract alcoholic solution reclaimed ethanol, temperature was controlled at 30～60 ℃;
In described step (3), when dissolving with ethyl acetate, the ratio of Ganoderma sporophore crude extract and ethyl acetate is a Ganoderma sporophore crude extract quality (g): ethyl acetate (mL)=1: 10～1: 100;
In described step (3), during with saturated sodium bicarbonate solution extraction ethyl acetate, extraction times is 1～8 time;
In described step (3), during with saturated sodium bicarbonate solution extraction ethyl acetate, ethyl acetate and saturated sodium bicarbonate solution volume ratio are ethyl acetate: saturated sodium bicarbonate solution=1: 3～3: 1;
In described step (3), during with saturated sodium bicarbonate solution extraction ethyl acetate, extraction times is 1～8 time.
5, the preparation method with Ganoderma sporophore extract of anti-tumor activity according to claim 4 is characterized in that,
In described step (1), the Ganoderma sporophore coarse powder is crossed 12 mesh sieves;
In described step (2), when Ganoderma sporophore crude extract alcoholic solution reclaimed ethanol, temperature was controlled at 50 ℃;
In described step (3), when dissolving with ethyl acetate, the ratio of Ganoderma sporophore crude extract and ethyl acetate is Ganoderma sporophore crude extract quality g: ethyl acetate volume mL is 1: 40～1: 80;
In described step (3), during with saturated sodium bicarbonate solution extraction ethyl acetate, extraction times is 2～6 times.
6, the preparation method with Ganoderma sporophore extract of anti-tumor activity according to claim 5 is characterized in that,
In described step (3), when dissolving with ethyl acetate, the ratio of Ganoderma sporophore crude extract and ethyl acetate is Ganoderma sporophore crude extract quality g: ethyl acetate volume mL is 1: 50.
In described step (3), during with saturated sodium bicarbonate solution extraction ethyl acetate, extraction times is 4 times.
7, according to the described preparation method of one of claim 4～6, it is characterized in that with Ganoderma sporophore extract of anti-tumor activity,
In described step (3), during with saturated sodium bicarbonate solution extraction ethyl acetate, ethyl acetate and saturated sodium bicarbonate solution volume ratio 1: 3～3: 1;
In described step (4), when reclaiming ethyl acetate solvent and vacuum drying extract, temperature is controlled at 30～60 ℃.
8, the preparation method with Ganoderma sporophore extract of anti-tumor activity according to claim 7 is characterized in that,
In described step (3), during with saturated sodium bicarbonate solution extraction ethyl acetate, ethyl acetate and saturated sodium bicarbonate solution volume ratio are 1: 2～2: 1.
In described step (4), when reclaiming ethyl acetate solvent and vacuum drying extract, temperature is controlled at 50 ℃.
9, the preparation method with Ganoderma sporophore extract of anti-tumor activity according to claim 8 is characterized in that,
In described step (3), during with saturated sodium bicarbonate solution extraction ethyl acetate, ethyl acetate and saturated sodium bicarbonate solution volume ratio are 1: 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106474164A (en) * | 2015-08-25 | 2017-03-08 | 苏庆华 | Ganoderma extract works in coordination with the anticancer medical composition of Amphotericin B |
| CN109168964A (en) * | 2018-11-28 | 2019-01-11 | 山东农业大学 | A kind of method of ganodenic acid content in raising ganoderma lucidum fruitbody |
| CN110156862A (en) * | 2019-03-14 | 2019-08-23 | 延安大学 | A kind of method that separation prepares antineoplastic component ganoderic acid |
-
2009
- 2009-05-25 CN CN2009100272306A patent/CN101559083B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106474164A (en) * | 2015-08-25 | 2017-03-08 | 苏庆华 | Ganoderma extract works in coordination with the anticancer medical composition of Amphotericin B |
| CN109168964A (en) * | 2018-11-28 | 2019-01-11 | 山东农业大学 | A kind of method of ganodenic acid content in raising ganoderma lucidum fruitbody |
| CN110156862A (en) * | 2019-03-14 | 2019-08-23 | 延安大学 | A kind of method that separation prepares antineoplastic component ganoderic acid |
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