CN105255986B - A kind of preparation method of Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound - Google Patents
A kind of preparation method of Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound, invented technology is simple, the rate of recovery is high, repeated preferable, stable and controllable for quality, and influence of the invention using cell growth curve as basic index criterion component and sample to cellular physiological activities such as adherent, breedings, visual result, easily judgement；Obtained Armillaria luteo-virens fructification anti-liver cancer and anti-sterols active sample (Fr8-1 sample and Fr8-4 sample) is through NMR hydrogen spectrum, carbon spectrum analysis, it is determined as 3 β-hydroxy- (22E respectively, 24R)-ergosta-5,8,22-trien-7-one and (3 α, 5 α), (8 β, 11 β)-diepidioxy-ergost-22E-en-12-one.
Description
Technical field
The present invention relates to pharmaceutical chemistry and biomedicine technical field more particularly to a kind of anti-livers of Armillaria luteo-virens fructification
The preparation method and applications of cancer reactive sterol class compound.
Background technique
Liver cancer cells (hepatocellular carcinoma, HCC, hereinafter referred to as liver cancer) be a kind of grade malignancy it is high,
The dangerous tumour of prognosis, survival rate only has 7% or so within 5 years, is the global 5th most common malignant tumour, in whole world tumour
Third position is in the lethality of patient.China's onset of liver cancer number accounts for about more than half of whole world, and age of onset tends to be young
Change.Liver cancer annual death rate in China's is 20/,100,000 or so at present, occupies the 2nd of the various mortality of malignant tumors in China, and close
It is in rising trend always over 10 years.Liver cancer has become the big killer for seriously threatening the health of our people and life, dangerous
Property can not be ignored.
Studies have shown that some Chinese medicine compound prescriptions or single the effective elements of the medicine can induce apoptosis of tumor cells, anticancer therapeutic
It is related with herb induction apoptosis of tumor cells.Armillaria luteo-virens fructification is the rare wild edible medicinal for being grown on Qinghai-Tibet Platean
Bacterium.At present it is limited studies have shown that in Armillaria luteo-virens fructification contain more rich protein, amino acid, carbohydrate, alkaloid
With a small amount of organic acid, flavones, cardiac glycoside, steroidal triterpenes, glycoside, saponin(e, volatile oil, cumarin terpene, tannin, phenols chemical combination
Object etc., such compound have anti influenza, prevention and treatment neuritis, athlete's foot, promote child development, the anti-oxidant and biologies such as antitumor
Activity.
Chinese patent application CN 103494843A " yellow mushroom standardizes component preparation method and its application in lung cancer therapy "
The preparation method of Armillaria luteo-virens fructification standardization component is related to CN 103494843A and its in lung cancer and liver cancer
Using, and the not yet explicitly specific chemical composition in component.It is same to the research of Armillaria luteo-virens fructification chemical component in document
There is a serious shortage in the supply.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of simple processes, a kind of Armillaria luteo-virens easy to implement
The preparation method of entity resisting liver cancer activity sterols component.
Another technical problem to be solved by this invention is to provide the Armillaria luteo-virens sterols reactive compound in liver
Application in cancer treatment.
To solve the above problems, a kind of Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound of the present invention
Preparation method, comprising the following steps:
(1) 1~6 times of 10%~50% n-hexane-ethyl acetate mixing of its volume is added in Armillaria luteo-virens acetone extract
Liquid is dissolved, and 0.45 μm of organic filter membrane is crossed after dissolution, obtains the sample solution containing acetone extract；It is described to contain acetone extraction
The sample solution of object separate and be dried under reduced pressure in the preparation chromatography using 10 μm of silica gel as isolation medium to get Fr1, Fr2, Fr3,
Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10, Fr12, Fr13, Fr14 totally 14 standardization component samples；
(2) 14 standardization component samples are pressed into following steps using the real-time n cell analyzer of iCelligence
It is rapid to carry out anti-liver cancer cell model discrimination:
1. confirmation iCelligence test macro correctly connects, 37 DEG C, 5%CO2Incubator is working properly；
2. culture medium is added by 150 holes μ L/ in 8 porocyte plates, it is placed at room temperature for 15 minutes and is placed on iCelligence test
Baseline is surveyed on platform；The culture medium refers to the low sugar DMEM culture medium containing 10% fetal calf serum；
3. dividing sample to be configured to concentration with DMSO each of described 14 standardization component sample is 5mg/mL's
Sample solution；
4. by the human liver cancer cell HepG2 of logarithmic growth phase, after being digested according to a conventional method with pancreatin, with the rate of 950rpm
It is centrifuged 5min, cell is resuspended in the culture medium, and cell concentration is adjusted to 2.85 × 104A/mL, obtains logarithmic growth
The human liver cancer cell HepG2 suspension of phase, and by 345 holes μ L/, 10000 cells in every hole amount by the people of the logarithmic growth phase
Hepatocellular carcinoma H22 suspension is inoculated in the corresponding aperture of 8 porocyte plates, is placed at room temperature for 30min；
Add the sample that concentration described in 5 μ L is 5mg/mL molten by every hole in two batches 5. standardizing component sample for described 14
Liquid, every hole add the amount of the human liver cancer cell HepG2 suspension of logarithmic growth phase described in 345 μ L to be inoculated with the phase into 8 porocyte plates
Ying Kongzhong is placed in the iCelligence testboard, is put into the incubator and starts to detect；Obtained after 45h two it is yellowish green
The Activity determination figure of the sub- anti-liver cancer and anti-standardization component of halimasch；Wherein ordinate is cell index, and abscissa is real-time cell increasing
Grow dynamic effect；
6. according to the Activity determination figure of the sub- anti-liver cancer and anti-standardization component of two Armillaria luteo-virens, when cell Proliferation curve
It presenting and rises trend that is gentle or not rising, then illustrate that cell division is slowed or shut off, i.e., cell not can be carried out normal division,
So that it is determined that the 7th, the 8th, the 9th, the 10th, the 11st, the 13rd and No. 14 standardization component has external inhibition human liver cancer cell
Adherent and proliferation activity.
(3) 2~5 times of 5%~20% n-hexane-alcohol mixeding liquids that its volume is added in No. 8 standardization component are carried out molten
Solution crosses 0.45 μm of organic filter membrane after dissolution, obtain the sample solution containing No. 8 standardization component；It is described to contain No. 8 standardization
The sample solution of component separate and be dried under reduced pressure in the preparation chromatography using 10 μm of XAmide as isolation medium to get Fr8-1,
Fr8-2, Fr8-3, Fr8-4 totally 4 samples；
(4) 4 samples are carried out in vitro in the steps below using the real-time n cell analyzer of iCelligence
The screening of anti-liver cancer and anti-cell model:
1. confirmation iCelligence test macro correctly connects, 37 DEG C, 5%CO2Incubator is working properly；
2. culture medium is added by 150 holes μ L/ in 8 porocyte plates, it is placed at room temperature for 15 minutes and is placed on iCelligence test
Baseline is surveyed on platform；The culture medium refers to the low sugar DMEM culture medium containing 10% fetal calf serum；
3. each sample in 4 samples is configured to the sample solution that concentration is 5mg/mL with DMSO；
4. by the human liver cancer cell HepG2 of logarithmic growth phase, after being digested according to a conventional method with pancreatin, with the rate of 950rpm
It is centrifuged 5min, cell is resuspended in the culture medium, and cell concentration is adjusted to 2.85 × 104A/mL, obtains logarithmic growth
The human liver cancer cell HepG2 suspension of phase, and by 345 holes μ L/, 10000 cells in every hole amount by the people of the logarithmic growth phase
Hepatocellular carcinoma H22 suspension is inoculated in the corresponding aperture of 8 porocyte plates, is placed at room temperature for 30min；
5. adding concentration described in 5 μ L to add 345 μ for the sample solution of 5mg/mL, every hole by every hole in two batches in 4 samples
The amount of the human liver cancer cell HepG2 suspension of logarithmic growth phase described in L is inoculated in the corresponding aperture of 8 porocyte plates, is placed in
The iCelligence testboard, is put into the incubator and starts to detect；Two anti-livers of Armillaria luteo-virens are obtained after 45h
The Activity determination figure of cancer standardization component；Wherein ordinate is cell index, and abscissa is real-time cell proliferation dynamics effect；
6. according to the Activity determination figure of the sub- liver cancer standardization component of two Armillaria luteo-virens, when cell Proliferation curve is in
Now rising trend that is gentle or not rising, then illustrates that cell division is slowed or shut off, i.e., cell not can be carried out normal division, from
And determining Fr8-1 sample and Fr8-4 sample has external inhibition human liver cancer cell adherent and proliferation activity.
(1) (3) the middle condition being concentrated under reduced pressure refers to that vacuum degree is 0.06~0.09MPa, temperature to the step with the step
It is 50~70 DEG C.
(1) the middle condition for preparing chromatographic isolation refers to that column size is 260 × 50mm to the step；Oneself is positive using A
Alkane, the binary-mobile phase system that B is ethyl acetate are separated: 0~15min, 100%A~100%A；15~35min, 60%
A~60%A；35~50min, 0%A~0%A；Detection wavelength is 260nm；Applied sample amount is 1.0g.
(3) condition that the step prepares chromatographic isolation refers to that column size is 250 × 20mm；Use A for n-hexane, B
It is separated for the binary-mobile phase system of ethyl alcohol: 0~40min, 97%A~92%A；Detection wavelength is 260nm；Applied sample amount is
0.15 or 0.3g.
The resulting activity of preparation method of Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound as described above
Sample is it is characterized in that application in liver cancer treatment.
The resulting activity of preparation method of Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound as described above
Sample is it is characterized by: the active sample is made respectively with pharmaceutically acceptable any carrier according to a conventional method as effective component
Class pharmaceutical formulation.
Compared with the prior art, the present invention has the following advantages:
1, the present invention is isolated from Armillaria luteo-virens fructification to have anti-liver cancer and anti-using preparation chromatography two-step purifying
Active sample (referring to Fig. 1, Fig. 3), not only simple process, and also it is easy to implement.
2, the present invention utilizes iCELLigence system detection, can Real-time and Dynamic Detection cell is adherent and breeding, provide
The cytological effect map of high information quantity provides a large amount of, important dynamic response information；Unmarked, hurtless measure detection of electrons is not
Cell can be interfered to grow and analyze；Can the growth of high precision/accuracy quantitative measurement cell, provide dynamic higher than 2 orders of magnitude
State range；Whole experiment process automatic data collection is analyzed in real time, is substantially improved only in the result of final point analysis；It is training
It supports in hole and detects cell quality, achieved the purpose that living cells quality control (referring to fig. 2, Fig. 4).
3, the present invention is basic index criterion component and sample to cells such as adherent, breedings using cell growth curve
The influence of physiological activity, visual result, easily judgement；Resulting sterols active sample can be used for researching and developing preparation anti-liver cancer and anti-medicine
Object.
4, the present invention has expanded the raw material sources of medicines resistant to liver cancer, expands the purposes of Armillaria luteo-virens fructification, makes Huang
Green halimasch fructification becomes effective sample material of medicines resistant to liver cancer, significantly improves the added value of Armillaria luteo-virens fructification.
5, present invention process is simple, the rate of recovery is high, repeated preferable, stable and controllable for quality, obtained Armillaria luteo-virens
Fructification anti-liver cancer and anti-sterols active sample (Fr8-1 sample and Fr8-4 sample) is divided through NMR hydrogen spectrum, carbon spectrum analysis
It is not determined as 3 β-hydroxy- (22E, 24R)-ergosta-5,8,22-trien-7-one and (3 α, 5 α), (8 β, 11 β)-
Diepidioxy-ergost-22E-en-12-one (referring to Fig. 5).
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 prepares chromatogram for Armillaria luteo-virens fructification of the present invention standardization component.
Fig. 2 is the resisting liver cancer activity detection figure that Armillaria luteo-virens fructification of the present invention standardizes component.
Fig. 3 is of the present invention Armillaria luteo-virens fructification the 8th and standardizes component and prepare chromatogram.
Fig. 4 is that the resisting liver cancer activity of Armillaria luteo-virens fructification Fr8-1~Fr8-4 sample of the present invention detects figure.
The structural formula of compound of Fig. 5 Armillaria luteo-virens fructification Fr8-1 and Fr8-4 sample of the present invention.
Specific embodiment
A kind of preparation method of the Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound of embodiment 1, including it is following
Step:
(1) 1 times of 50% n-hexane-ethyl acetate mixed liquor that its volume is added in Armillaria luteo-virens acetone extract carries out molten
Solution crosses 0.45 μm of organic filter membrane, obtains the sample solution containing acetone extract after dissolution；The sample containing acetone extract
Solution separate and be dried under reduced pressure in the preparation chromatography using 10 μm of silica gel as isolation medium to get Fr1, Fr2, Fr3, Fr4, Fr5,
Fr6, Fr7, Fr8, Fr9, Fr10, Fr12, Fr13, Fr14 totally 14 standardization component samples；
Wherein:
The condition of reduced pressure refers to that vacuum degree is 0.06MPa, and temperature is 70 DEG C.
The condition of preparation chromatographic isolation refers to that column size is 260 × 50mm；Using A for n-hexane, B is ethyl acetate
Binary-mobile phase system separated: 0~15min, 100%A~100%A；15~35min, 60%A~60%A；35~
50min, 0%A~0%A；Detection wavelength is 260nm；Applied sample amount is 1.0g.
(2) 14 standardization component samples are pressed into following steps using the real-time n cell analyzer of iCelligence
It is rapid to carry out anti-liver cancer cell model discrimination:
1. confirmation iCelligence test macro correctly connects, 37 DEG C, 5%CO2Incubator is working properly；
2. culture medium is added by 150 holes μ L/ in 8 porocyte plates, it is placed at room temperature for 15 minutes and is placed on iCelligence test
Baseline is surveyed on platform；The culture medium refers to the low sugar DMEM culture medium containing 10% fetal calf serum；
3. dividing sample to be configured to concentration with DMSO each of described 14 standardization component sample is 5mg/mL's
Sample solution；
4. by the human liver cancer cell HepG2 of logarithmic growth phase, after being digested according to a conventional method with pancreatin, with the rate of 950rpm
It is centrifuged 5min, cell is resuspended in the culture medium, and cell concentration is adjusted to 2.85 × 104A/mL, obtains logarithmic growth
The human liver cancer cell HepG2 suspension of phase, and by 345 holes μ L/, 10000 cells in every hole amount by the people of the logarithmic growth phase
Hepatocellular carcinoma H22 suspension is inoculated in the corresponding aperture of 8 porocyte plates, is placed at room temperature for 30min；
Add the sample that concentration described in 5 μ L is 5mg/mL molten by every hole in two batches 5. standardizing component sample for described 14
Liquid, every hole add the amount of the human liver cancer cell HepG2 suspension of logarithmic growth phase described in 345 μ L to be inoculated with the phase into 8 porocyte plates
Ying Kongzhong is placed in the iCelligence testboard, is put into the incubator and starts to detect；Obtained after 45h two it is yellowish green
The Activity determination figure of the sub- anti-liver cancer and anti-standardization component of halimasch；Wherein ordinate is cell index, and abscissa is real-time cell increasing
Grow dynamic effect；
6. according to the Activity determination figure of the sub- anti-liver cancer and anti-standardization component of two Armillaria luteo-virens, when cell Proliferation curve
It presenting and rises trend that is gentle or not rising, then illustrate that cell division is slowed or shut off, i.e., cell not can be carried out normal division,
So that it is determined that the 7th, the 8th, the 9th, the 10th, the 11st, the 13rd and No. 14 standardization component has external inhibition human liver cancer cell
Adherent and proliferation activity.
(3) 2 times of 20% n-hexane-alcohol mixeding liquids that its volume is added in No. 8 standardization component are dissolved, dissolved
The organic filter membrane for crossing 0.45 μm afterwards obtains the sample solution containing No. 8 standardization component；It is described to contain No. 8 standardization component
Sample solution separate and be dried under reduced pressure in the preparation chromatography using 10 μm of XAmide as isolation medium to get Fr8-1, Fr8-2,
Fr8-3, Fr8-4 totally 4 samples；
Wherein:
The condition of reduced pressure refers to that vacuum degree is 0.06MPa, and temperature is 70 DEG C；
The condition of preparation chromatographic isolation refers to that column size is 250 × 20mm；Use A for n-hexane, B are ethyl alcohol two
First flow visualizing is separated: 0~40min, 97%A~92%A；Detection wavelength is 260nm；Applied sample amount be 0.15 or
0.3g。
(4) 4 samples are carried out in vitro in the steps below using the real-time n cell analyzer of iCelligence
The screening of anti-liver cancer and anti-cell model:
1. confirmation iCelligence test macro correctly connects, 37 DEG C, 5%CO2Incubator is working properly；
2. culture medium is added by 150 holes μ L/ in 8 porocyte plates, it is placed at room temperature for 15 minutes and is placed on iCelligence test
Baseline is surveyed on platform；The culture medium refers to the low sugar DMEM culture medium containing 10% fetal calf serum；
3. each sample in 4 samples is configured to the sample solution that concentration is 5mg/mL with DMSO；
4. by the human liver cancer cell HepG2 of logarithmic growth phase, after being digested according to a conventional method with pancreatin, with the rate of 950rpm
It is centrifuged 5min, cell is resuspended in the culture medium, and cell concentration is adjusted to 2.85 × 104A/mL, obtains logarithmic growth
The human liver cancer cell HepG2 suspension of phase, and by 345 holes μ L/, 10000 cells in every hole amount by the people of the logarithmic growth phase
Hepatocellular carcinoma H22 suspension is inoculated in the corresponding aperture of 8 porocyte plates, is placed at room temperature for 30min；
5. adding concentration described in 5 μ L to add 345 μ for the sample solution of 5mg/mL, every hole by every hole in two batches in 4 samples
The amount of the human liver cancer cell HepG2 suspension of logarithmic growth phase described in L is inoculated in the corresponding aperture of 8 porocyte plates, is placed in
The iCelligence testboard, is put into the incubator and starts to detect；Two anti-livers of Armillaria luteo-virens are obtained after 45h
The Activity determination figure of cancer standardization component；Wherein ordinate is cell index, and abscissa is real-time cell proliferation dynamics effect；
6. according to the Activity determination figure of the sub- anti-liver cancer and anti-standardization component of two Armillaria luteo-virens, when cell Proliferation curve
It presenting and rises trend that is gentle or not rising, then illustrate that cell division is slowed or shut off, i.e., cell not can be carried out normal division,
So that it is determined that Fr8-1 sample and Fr8-4 sample have external inhibition human liver cancer cell adherent and proliferation activity.
Obtained Armillaria luteo-virens fructification anti-liver cancer and anti-sterols active sample (Fr8-1 sample and No. Fr8-4
Sample) through NMR hydrogen spectrum, carbon spectrum analysis, it is determined as 3 β-hydroxy- (22E, 24R)-ergosta-5 respectively, 8,22-trien-
7-one and (3 α, 5 α), (8 β, 11 β)-diepidioxy-ergost-22E-en-12-one (referring to Fig. 5).
A kind of preparation method of the Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound of embodiment 2, including it is following
Step:
(1) 6 times of 10% n-hexane-ethyl acetate mixed liquor that its volume is added in Armillaria luteo-virens acetone extract carries out molten
Solution crosses 0.45 μm of organic filter membrane, obtains the sample solution containing acetone extract after dissolution；The sample containing acetone extract
Solution separate and be dried under reduced pressure in the preparation chromatography using 10 μm of silica gel as isolation medium to get Fr1, Fr2 ..., Fr13,
Fr14 totally 14 standardization component samples；
Wherein:
The condition of reduced pressure refers to that vacuum degree is 0.09MPa, and temperature is 50 DEG C.
Prepare chromatographic isolation condition with 1 step of embodiment (1).
By 5 component samples using the real-time n cell analyzer of iCelligence by step described in embodiment 1 into
Row anti-liver cancer cell model discrimination determines the 7th, the 8th, the 9th, the 10th, the 11st, the 13rd and No. 14 standardization component tool
There is external inhibition human liver cancer cell adherent and proliferation activity.
(3) 5 times of 5% n-hexane-alcohol mixeding liquids that its volume is added in No. 8 standardization component are dissolved, after dissolution
The organic filter membrane for crossing 0.45 μm obtains the sample solution containing No. 8 standardization component；The sample for containing No. 8 standardization component
Product solution separate and be dried under reduced pressure in the preparation chromatography using 10 μm of XAmide as isolation medium to get Fr8-1, Fr8-2,
Fr8-3, Fr8-4 totally 4 samples；
Wherein:
The condition of reduced pressure refers to that vacuum degree is 0.09MPa, and temperature is 50 DEG C；
Prepare chromatographic isolation condition with 1 step of embodiment (2).
(4) 4 samples are subjected to body by step described in embodiment 1 using the real-time n cell analyzer of iCelligence
Outer anti-liver cancer and anti-cell model screening determines that Fr8-1 and Fr8-4 sample have external inhibition human liver cancer cell adherent and increase
Grow activity.
A kind of preparation method of the Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound of embodiment 3, including it is following
Step:
(1) 3 times of 30% n-hexane-ethyl acetate mixed liquor that its volume is added in Armillaria luteo-virens acetone extract carries out molten
Solution crosses 0.45 μm of organic filter membrane, obtains the sample solution containing acetone extract after dissolution；The sample containing acetone extract
Solution separate and be dried under reduced pressure in the preparation chromatography using 10 μm of silica gel as isolation medium to get Fr1, Fr2 ..., Fr13,
Fr14 totally 14 standardization component samples；
Wherein:
The condition of reduced pressure refers to that vacuum degree is 0.08MPa, and temperature is 60 DEG C.
Prepare chromatographic isolation condition with 1 step of embodiment (1).
By 5 component samples using the real-time n cell analyzer of iCelligence by step described in embodiment 1 into
Row anti-liver cancer cell model discrimination determines the 7th, the 8th, the 9th, the 10th, the 11st, the 13rd and No. 14 standardization component tool
There is external inhibition human liver cancer cell adherent and proliferation activity.
(3) 3 times of 10% n-hexane-alcohol mixeding liquids that its volume is added in No. 8 standardization component are dissolved, dissolved
The organic filter membrane for crossing 0.45 μm afterwards obtains the sample solution containing No. 8 standardization component；It is described to contain No. 8 standardization component
Sample solution separate and be dried under reduced pressure in the preparation chromatography using 10 μm of XAmide as isolation medium to get Fr8-1, Fr8-2,
Fr8-3, Fr8-4 totally 4 samples；
Wherein:
The condition of reduced pressure refers to that vacuum degree is 0.08MPa, and temperature is 60 DEG C；
Prepare chromatographic isolation condition with 1 step of embodiment (2).
(4) 4 samples are subjected to body by step described in embodiment 1 using the real-time n cell analyzer of iCelligence
Outer anti-liver cancer and anti-cell model screening determines that Fr8-1 and Fr8-4 sample have external inhibition human liver cancer cell adherent and increase
Grow activity.
In above-described embodiment 1~3 obtained by the preparation method of Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound
Reactive compound all kinds of pharmaceutical formulations be made with pharmaceutically acceptable any carrier according to a conventional method as effective component answer
For in liver cancer treatment.
Claims (1)
1. a kind of preparation method of Armillaria luteo-virens fructification resisting liver cancer activity phytosterin compound, which is characterized in that including with
Lower step:
Armillaria luteo-virens acetone extract be added 1~6 times of 10%~50% n-hexane-ethyl acetate mixed liquor of its volume into
Row dissolution crosses 0.45 μm of organic filter membrane, obtains the sample solution containing acetone extract after dissolution；It is described containing acetone extract
Sample solution separate and be dried under reduced pressure in the preparation chromatography using 10 μm of silica gel as isolation medium to get Fr1, Fr2, Fr3, Fr4,
Fr5, Fr6, Fr7, Fr8, Fr9, Fr10, Fr12, Fr13, Fr14 totally 14 standardization component samples, 14 component samples
Separation shown in Figure 1 prepare chromatogram；
By 14 standardization component samples using the real-time n cell analyzer of iCelligence in the steps below into
Row anti-liver cancer cell model discrimination:
1. confirmation iCelligence test macro correctly connects, 37 DEG C, 5%CO2Incubator is working properly；
2. culture medium is added by 150 holes μ L/ in 8 porocyte plates, it is placed at room temperature for 15 minutes and is placed on iCelligence testboard
Survey baseline；The culture medium refers to the low sugar DMEM culture medium containing 10% fetal calf serum；
3. dividing sample to be configured to the sample that concentration is 5mg/mL with DMSO each of described 14 standardization component samples
Solution；
4. the human liver cancer cell HepG2 of logarithmic growth phase after being digested according to a conventional method with pancreatin, is centrifuged with the rate of 950rpm
Cell is resuspended in the culture medium, and cell concentration is adjusted to 2.85 × 10 by 5min4A/mL, obtains logarithmic growth phase
Human liver cancer cell HepG2 suspension, and by 345 holes μ L/, 10000 cells in every hole amount by the human liver cancer of the logarithmic growth phase
Cell HepG2 suspension is inoculated in the corresponding aperture of 8 porocyte plates, is placed at room temperature for 30min；
5. standardizing component samples for described 14 adds sample solution that concentration described in 5 μ L is 5mg/mL, every by every hole in two batches
Hole adds the amount of the human liver cancer cell HepG2 suspension of logarithmic growth phase described in 345 μ L to be inoculated with the corresponding aperture into 8 porocyte plates
In, it is placed in the iCelligence testboard, is put into the incubator and starts to detect；Two yellowish green sweet rings are obtained after 45h
The Activity determination figure of mushroom anti-liver cancer and anti-standardization component；Wherein ordinate is cell index, and abscissa is that real-time cell proliferation is dynamic
State effect；
6. according to the Activity determination figure of the sub- anti-liver cancer and anti-standardization component of two Armillaria luteo-virens, when cell Proliferation curve is presented
Rising trend that is gentle or not rising, then illustrates that cell division is slowed or shut off, i.e., cell not can be carried out normal division, thus
Determine that the 7th, the 8th, the 9th, the 10th, the 11st, the 13rd and No. 14 standardization component has external inhibition human liver cancer cell adherent
And proliferation activity.
(3) 2~5 times of 5%~20% n-hexane-alcohol mixeding liquids that its volume is added in No. 8 standardization component are dissolved,
Organic filter membrane that 0.45 μm is crossed after dissolution obtains the sample solution containing No. 8 standardization component；It is described to contain No. 8 standardization group
Point sample solution separate and be dried under reduced pressure in the preparation chromatography using 10 μm of XAmide as isolation medium to get Fr8-1,
Fr8-2, Fr8-3, Fr8-4 totally 4 samples, the separation of 4 samples it is shown in Figure 3 prepare chromatogram；
(4) 4 samples are subjected to external anti-liver using the real-time n cell analyzer of iCelligence in the steps below
Cancer cell model discrimination:
1. confirmation iCelligence test macro correctly connects, 37 DEG C, 5%CO2Incubator is working properly；
2. culture medium is added by 150 holes μ L/ in 8 porocyte plates, it is placed at room temperature for 15 minutes and is placed on iCelligence testboard
Survey baseline；The culture medium refers to the low sugar DMEM culture medium containing 10% fetal calf serum；
3. each sample in 4 samples is configured to the sample solution that concentration is 5mg/mL with DMSO；
4. the human liver cancer cell HepG2 of logarithmic growth phase after being digested according to a conventional method with pancreatin, is centrifuged with the rate of 950rpm
Cell is resuspended in the culture medium, and cell concentration is adjusted to 2.85 × 10 by 5min4A/mL, obtains logarithmic growth phase
Human liver cancer cell HepG2 suspension, and by 345 holes μ L/, 10000 cells in every hole amount by the human liver cancer of the logarithmic growth phase
Cell HepG2 suspension is inoculated in the corresponding aperture of 8 porocyte plates, is placed at room temperature for 30min；
5. adding concentration described in 5 μ L to add 345 μ L institutes for the sample solution of 5mg/mL, every hole by every hole in two batches in 4 samples
The amount for stating the human liver cancer cell HepG2 suspension of logarithmic growth phase is inoculated in the corresponding aperture of 8 porocyte plates, is placed in described
ICelligence testboard is put into the incubator and starts to detect；The sub- anti-liver cancer and anti-mark of two Armillaria luteo-virens is obtained after 45h
The Activity determination figure of standardization component；Wherein ordinate is cell index, and abscissa is real-time cell proliferation dynamics effect；
6. according to the Activity determination figure of the sub- anti-liver cancer and anti-standardization component of two Armillaria luteo-virens, when cell Proliferation curve is presented
Rising trend that is gentle or not rising, then illustrates that cell division is slowed or shut off, i.e., cell not can be carried out normal division, thus
Determine that Fr8-1 sample and Fr8-4 sample have external inhibition human liver cancer cell adherent and proliferation activity；
Wherein, (1) (3) the middle condition being concentrated under reduced pressure refers to that vacuum degree is 0.06~0.09MPa, temperature to the step with the step
It is 50~70 DEG C；(1) the middle condition for preparing chromatographic isolation refers to that column size is 260 × 50mm to the step；It is positive using A
Hexane, the binary-mobile phase system that B is ethyl acetate are separated: 0~15min, 100%A~100%A；15~35min,
60%A~60%A；35~50min, 0%A~0%A；Detection wavelength is 260nm；Applied sample amount is 1.0g；In the step (3)
The condition of preparation chromatographic isolation refers to that column size is 250 × 20mm；Using A is the binary mobile phase of ethyl alcohol for n-hexane, B
System is separated: 0~40min, 97%A~92%A；Detection wavelength is 260nm；Applied sample amount is 0.15 or 0.3g.
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