CN108896500A - A kind of screening and extracting method of white Chinese actinidia root anti-tumor active substance - Google Patents
A kind of screening and extracting method of white Chinese actinidia root anti-tumor active substance Download PDFInfo
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Abstract
The present invention is the screening and extracting method of a kind of white Chinese actinidia root anti-tumor active substance.The present invention includes：(1) pre-process：It is crushed after taking rattan pears root herb to dry, crosses medicine and sieve to obtain evengranular powder；(2) ligroin extraction is prepared：White Chinese actinidia root powder is taken, petroleum ether is measured and impregnates white Chinese actinidia root powder, separate the white Chinese actinidia root dregs of a decoction with extracting solution after ultrasonic extraction, extracting solution filtering and concentrating obtains ligroin extraction；(3) ethyl acetate extract is prepared：By step (2) in the drying of the white Chinese actinidia root dregs of a decoction be placed in ethyl acetate and impregnate, then with ethyl acetate be that solvent is extracted using percolation, the dregs of a decoction are stand-by, and percolate reduced pressure obtains ethyl acetate extract；(4) ethanol extract is prepared：By step (3) in the drying of the white Chinese actinidia root dregs of a decoction, it is solvent using percolation extraction with ethyl alcohol that ethyl alcohol, which is added, and impregnates again, and percolate filtering and concentrating obtains ethanol extract；(5) activity rating：Anticancer experiment in vitro detects influence of three kinds of extracts to activity of tumor cells.
Description
Technical field
The present invention relates to screening bioactive compounds in Chinese medicine and extractive technique field, especially a kind of white actinidia root antitumor
The screening and extracting method of active material.
Background technique
With society and expanding economy, production environment and living-pattern preservation, malignant tumour, which becomes, threatens the mankind strong
One of principal disease of health.Chinese Cancer Mortality and the death rate are in sustainable growth trend since the 1970s,
In liver cancer, esophagus, the malignant tumours such as stomach, lung, Chinese new cases and death toll occupy first place in the world.
A large amount of clinical and experimental datas showed that traditional Chinese medicine had its original in the therapeutic process of tumor patient in recent years
Effect, has become the important component of combined therapy of tumour.Chinese medicine is improving tumor patient clinical symptoms, and it is raw to improve patient
Quality is deposited, prevents tumor recurrence from shifting, and has good curative effect with Chemotherapy plus Synergy and attenuation etc..But Chinese medicine
Contained chemical component is sufficiently complex, both contains effective component, also there is invalid components, and daily decoction is taken also inconvenient.Chinese medicine mentions
It takes and exactly extracts wherein effective ingredient to greatest extent using some technologies, so that the inherent quality of Chinese materia medica preparation and clinical treatment effect
Fruit is improved, and the effect of Chinese medicine is enable to play to greatest extent.It extracts the effective component in Chinese medicine and is further separated, is pure
Change, obtaining effective monomer is an important content in current traditional Chinese medicine research field.Extracting from Chinese herbal medicine antitumor has
Imitate active constituent, it has also become the important sources of anti-tumor drug exploitation.
Actinidia eriantha (Actinidia eriantha Benth), i.e. our rattan pears for being commonly called as, are Actinidiaceae Mi
Monkey peach family plant seed is distributed widely in Zhejiang, Fujian, Jiangxi, Guangxi and Guangdong.White Chinese actinidia root is the dry root of actinidia eriantha,
Cold in nature, bitter and puckery flavor has heat-clearing, dampness removing, activating blood circulation and reducing swelling, removing toxic substances and other effects, is the conventional crude drugs of Yi people's folk therapy tumour, extensively
It is used for antitumor treatment generally.But the effective active position of white actinidia root antitumor and indefinite for a long time, lead to it
The extraction of effective active composition is also lack of pertinence.Existing extraction process generally uses water or ethyl alcohol to carry out reflux for solvent and mentions
It takes, obtains water extract or alcohol extract；Or extracted with 95% alcohol dipping, it is extracted, is closed with 75% alcohol dipping again after having extracted
And extracting solution, ethanol total extract is obtained, then extracted with petroleum ether, chloroform, the ethyl acetate solvent different with n-butanol isopolarity
It takes, finally obtains different effective component.Thermal extraction method is be easy to cause brokenly for the effective component of thermally labile in medicinal material
Bad or structure changes, and Aqueous extracts or alcohol extract difficult solvent recovery, not easy to maintain or transport.In addition, extraction process compares
Phenomena such as many and diverse, opposed polarity solvent extraction is not enough in extraction process, and methylene chloride extraction is also also easy to produce emulsification, operation
It is time-consuming, be not suitable for industrialized production.
Summary of the invention
The object of the present invention is to provide the effectively screenings and extracting method of activity substance in a kind of white Chinese actinidia root.
The technical solution adopted by the present invention is that complete in the following way：A kind of white Chinese actinidia root anti-tumor active substance
The screening technique of screening technique, the white Chinese actinidia root anti-tumor active substance includes the following steps：
(1) pre-process：Take a certain amount of rattan pears root herb, high speed disintegrator crushes after drying, cross medicine sieve to obtain particle it is equal
Even white Chinese actinidia root powder；
(2) ligroin extraction is prepared：Appropriate white Chinese actinidia root powder is weighed, the petroleum ether for measuring certain volume impregnates rattan
The white Chinese actinidia root dregs of a decoction are separated after ultrasonic extraction with extracting solution, after extracting solution is filtered with filter paper, use Rotary Evaporators by pears root powder
It is concentrated under reduced pressure, obtains ligroin extraction；
(3) ethyl acetate extract is prepared：The white Chinese actinidia root dregs of a decoction after step (2) middle ultrasonic extraction are dried in an oven,
Remaining solvent is volatilized, the dregs of a decoction after drying, which are placed in the ethyl acetate of certain volume, to be impregnated, the white Chinese actinidia root dregs of a decoction after immersion
It is solvent using percolation extraction with ethyl acetate, the dregs of a decoction are stand-by, and percolate depressurizes in Rotary Evaporators after being filtered with filter paper
Concentration, obtains ethyl acetate extract；
(4) ethanol extract is prepared：By the white Chinese actinidia root dregs of a decoction after step (3) middle seepage pressure effects through baking oven dry out solvent, add
The ethyl alcohol for entering certain volume impregnates, and the white Chinese actinidia root dregs of a decoction after immersion are solvent using percolation extraction with ethyl alcohol, and percolate is used
It is concentrated under reduced pressure in Rotary Evaporators after filter paper filtering, obtains ethanol extract；
(5) activity rating：Anticancer experiment in vitro is carried out using RTCA DP real-time cell analysis system, detects three of the above
Influence of the extract to activity of tumor cells filters out the optimal extract of antitumous effect according to experimental result.
In a kind of screening technique of white Chinese actinidia root anti-tumor active substance, acetic acid in three kinds of extracts
Contained anti-tumor active substance content highest in ethyl ester extract acts on inhibiting tumour cells obvious, ethyl acetate extract
Effect, IC are significantly inhibited to human hepatoma HepG2 cell50For 7.6 μ g/mL；There are inhibiting effect, IC to typeⅡ pneumocyte50
For 150 μ g/mL.
In a kind of screening technique of white Chinese actinidia root anti-tumor active substance, the antitumous effect is optimal to be mentioned
Taking object is ethyl acetate extract, and active constituent therein is triterpene compound.
In a kind of screening technique of white Chinese actinidia root anti-tumor active substance, the step (5) in select tumour
Cell is at least one of typeⅡ pneumocyte, human hepatoma HepG2 cell.
In a kind of screening technique of white Chinese actinidia root anti-tumor active substance, the concrete operations of the step (5)
For：The tumour cell of logarithmic growth phase, is made single cell suspension after pancreatin digestion, adjustment cell concentration is 5 × 104A/mL,
First hole every on E-Plate plate is put into 50 μ L culture mediums and surveys baseline, and 100 μ L cell suspensions are then added, are placed in 37 DEG C, 5%CO2
It cultivates for 24 hours, records cell index (CI) in incubator；If Normal group, 0.2%DMSO control group, three kinds of extract tests
Group, every group sets 3 multiple holes and certain density extract-treated cell is added in test group after culture for 24 hours, continues to cultivate 72h,
CI value is recorded, screening acts on preferable sample to inhibiting tumour cells and carries out 200,100,50,25,12.5,6.25 μ g/mL6
The CI value of concentration detects, and calculates IC50。
A kind of extracting method of white Chinese actinidia root anti-tumor active substance, the extraction side of the white Chinese actinidia root anti-tumor active substance
Method includes the following steps：
(1) pre-process：Take a certain amount of rattan pears root herb, high speed disintegrator crushes after drying, cross medicine sieve to obtain particle it is equal
Even white Chinese actinidia root powder；
(2) petroleum ether separates：Appropriate white Chinese actinidia root powder is weighed, the petroleum ether for measuring certain volume impregnates white Chinese actinidia root powder
End separates the white Chinese actinidia root dregs of a decoction with petroleum ether extract after ultrasonic extraction, by the ingredient of polarity very little contained in white Chinese actinidia root
It separates, to improve subsequent extracted purity；
(3) ethyl acetate extract is prepared：The white Chinese actinidia root dregs of a decoction after step (2) middle ultrasonic extraction are dried in an oven,
Remaining solvent is volatilized, the dregs of a decoction after drying, which are placed in the ethyl acetate of certain volume, to be impregnated, the white Chinese actinidia root dregs of a decoction after immersion
It is solvent using percolation extraction with ethyl acetate, percolate is concentrated under reduced pressure in Rotary Evaporators after being filtered with filter paper, obtains
Ethyl acetate extract；
In a kind of extracting method of white Chinese actinidia root anti-tumor active substance, (2) concrete operations are the step：
White Chinese actinidia root powder 10g~1000g is weighed, the petroleum ether that 5 times of amounts (solvent volume and medicinal material weight ratio) are added impregnates 30min
Afterwards, ultrasonic extraction 2 times, each 30min, combined extract isolate the white Chinese actinidia root dregs of a decoction.
In a kind of extracting method of white Chinese actinidia root anti-tumor active substance, (3) concrete operations are the step：
By step (2) in the white Chinese actinidia root dregs of a decoction dried through 65 DEG C of baking oven, volatilize solvent, 5 times of amounts (solvent volume and medicinal material weight be added
Than) ethyl acetate impregnate 12h, with the ethyl acetate seepage pressure effects of 10 times of amounts (solvent volume and medicinal material weight ratio), diacolation speed
Degree is 2ml/min, collects percolate, and filter paper filters after merging, is concentrated under reduced pressure in Rotary Evaporators, and the condition of reduced pressure is pressure
Power 150mbar, 39 DEG C of temperature, recycling design obtains ethyl acetate extract, and yield is 0.72%~1.08%.
It is surveyed using the content of total triterpene in the effective active position of ultraviolet spectrophotometry dialogue actinidia root antitumor
Fixed, using ursolic acid as standard items, 5% vanillic aldehyde-glacial acetic acid and perchloric acid develop the color, and absorbance is measured at 550nm, substitute into
The standard curve of ursolic acid measures its total triterpene contents.
Rattan pears are extracted from small to large by solvent polarity when anti-tumor active substance in screening white Chinese actinidia root in the present invention
Ingredient in root efficiently and rapidly extracts the small polar component of white Chinese actinidia root using petroleum ether ultrasonic extraction；Second is successively used again
Acetoacetic ester, ethanol percolation extract, and obtain maximum extracted yield as far as possible, play preferable separating effect.Extracorporeal anti-tumor
It is ethyl acetate extract that testing sieve, which selects the antitumor useful effect position of rattan pears root, specifies white actinidia root antitumor
Position where material base is triterpene compound in conjunction with the active material in the information analyses ethyl acetate such as finger-print.This
Inventive step is simple to operate, and suitable for the extraction of white Chinese actinidia root effective component, total triterpene extraction yield is relatively high, is suitable for
The extensive extraction of medicinal material, is conducive to industrialized popularization.
Detailed description of the invention
Fig. 1 is influence diagram of the RTCA DP real-time cell analysis system detection ligroin extraction to A549 cell activity.
Fig. 2 is influence diagram of the RTCA DP real-time cell analysis system detection ethyl acetate extract to A549 cell activity.
Fig. 3 is influence diagram of the RTCA DP real-time cell analysis system detection ethanol extract to A549 cell activity.
Fig. 4 is influence diagram of the RTCA DP real-time cell analysis system detection ligroin extraction to HepG2 cell activity.
Fig. 5 is influence of the RTCA DP real-time cell analysis system detection ethyl acetate extract to HepG2 cell activity
Figure.
Fig. 6 is influence diagram of the RTCA DP real-time cell analysis system detection ethanol extract to HepG2 cell activity.
Fig. 7 is the high-efficiency liquid-phase fingerprint of ethyl acetate extract.
Specific embodiment
The present invention will be further explained below with reference to examples.
A kind of screening technique of white Chinese actinidia root anti-tumor active substance, specifically includes following steps：
(1) pre-process：Appropriate rattan pears root herb is taken, 65 DEG C of baking oven is placed in and dries for 24 hours, high speed disintegrator crushes, and crosses 5
Number sieve (i.e. 80 meshes), obtain evengranular white Chinese actinidia root powder；
(2) ligroin extraction is prepared：White Chinese actinidia root powder 1000g is weighed, the petroleum ether for measuring 5000mL impregnates rattan pears
Root powder 30min, ultrasonic extraction 2 times, each 30min, the dregs of a decoction are for later use, combined extract, filter paper filtering, filtered
Extracting solution is concentrated under reduced pressure in BUCHI Rotary Evaporators, and the condition of reduced pressure is pressure 250mbar, 39 DEG C of temperature, recycles molten
Agent obtains ligroin extraction, yield 0.24%；
(3) ethyl acetate extract is prepared：The white Chinese actinidia root dregs of a decoction after step (2) middle ultrasonic extraction are dried in 65 DEG C of baking ovens
Dry 12h, volatilizes remaining solvent, and the dregs of a decoction after drying are placed in the ethyl acetate of 5 times of amounts (solvent volume and medicinal material weight ratio)
Soaked overnight, the ethyl acetate that the white Chinese actinidia root dregs of a decoction after immersion measure (solvent volume and medicinal material weight ratio) with 10 times are adopted for solvent
It is extracted with percolation, diacolation speed is 2mL/min, and the dregs of a decoction are stand-by, collects percolate, merges percolate, percolate filter paper mistake
It is concentrated under reduced pressure in BUCHI Rotary Evaporators after filter, the condition of reduced pressure is pressure 150mbar, 39 DEG C of temperature, is recycled molten
Agent obtains ethyl acetate extract, yield 0.89%；
(4) ethanol extract is prepared：By step (3) in the white Chinese actinidia root dregs of a decoction after seepage pressure effects through 65 DEG C of drying 12h of baking oven,
95% ethyl alcohol soaked overnight of 5 times of amounts (solvent volume and medicinal material weight ratio) is added, the white Chinese actinidia root dregs of a decoction after immersion are with 10 times
Amount (solvent volume and medicinal material weight ratio) ethyl alcohol is that solvent is extracted using percolation, and diacolation speed is 2mL/min, collects diacolation
Liquid merges percolate, and percolate is concentrated under reduced pressure in BUCHI Rotary Evaporators after being filtered with filter paper, and the condition of reduced pressure is
Pressure 90mbar, 39 DEG C of temperature, recycling design obtains ethanol extract, yield 5.47%；
(5) activity rating：By obtained white Chinese actinidia root ligroin extraction, ethyl acetate extract, ethanol extract difference
It is configured to 50 μ g/mL, 100 μ g/mL concentration；Anticancer experiment in vitro, cell are carried out using RTCA DP real-time cell analysis system
Cancer cell A549, HepG2 is respectively adopted, the cell of logarithmic growth phase is made single cell suspension after pancreatin digestion, adjusts cell
Concentration is 5 × 104A/mL, first hole every on E-Plate plate is put into 50 μ L culture mediums and surveys baseline, and it is outstanding that 100 μ L cells are then added
Liquid is placed in 37 DEG C, 5%CO2It cultivates for 24 hours, records cell index (CI) in incubator；After culture for 24 hours, if Normal group,
0.2%DMSO control group, ligroin extraction, ethyl acetate extract, ethanol extract group, respectively with sample concentration 100,50
μ g/mL handles cell, and every group sets 3 multiple holes, continues to cultivate 72h, records CI value；Screening acts on preferable sample to inhibition of cancer cell
The CI value detection for carrying out 200,100,50,25,12.5,6.25 μ g/mL6 concentration, calculates IC50；Experimental result is shown in attached drawings 1~attached
Fig. 6, it can be seen that white Chinese actinidia root ligroin extraction, ethyl acetate extract and ethanol extract to typeⅡ pneumocyte and
Human hepatoma HepG2 cell's proliferation activity has inhibiting effect, and compared with Normal group and 0.2%DMSO control group, three kinds are mentioned
Object is taken to have a good inhibiting effect to A549 cell and HepG2 cell in 200~6.25 μ g/mL of experimental concentration range, three kinds
Ethyl acetate extract antitumous effect is best in extract, IC of the different extracts to typeⅡ pneumocyte 48h50Respectively
2mg/mL, 0.15mg/mL and 3.8mg/mL, IC of the different extracts to human hepatoma HepG2 cell 48h50Respectively 75 μ g/mL,
7.6 μ g/ml and 71 μ g/mL.
(6) active material determines in ethyl acetate extract：Using the finger of efficient liquid phase method measurement ethyl acetate extract
Line map, wherein mobile phase be -0.1% phosphoric acid water gradient elution of acetonitrile, flow velocity 1.0mL/min, 20 DEG C of column temperature, obtained fingerprint
Map is as shown in Fig. 7, and active material is triterpene compound, grinds in conjunction with dialogue actinidia root antitumor activity in existing literature
Report is studied carefully it is found that the anti-tumor active substance in ethyl acetate extract is exactly triterpene compound.
A kind of extracting method of white Chinese actinidia root anti-tumor active substance, specifically includes following steps：
(1) pre-process：Appropriate rattan pears root herb is taken, 65 DEG C of baking oven is placed in and dries for 24 hours, high speed disintegrator crushes, and crosses 5
Number sieve (i.e. 80 meshes), obtain evengranular white Chinese actinidia root powder；
(2) petroleum ether separates：White Chinese actinidia root powder 1000g is weighed, the petroleum ether for measuring 5000mL impregnates white Chinese actinidia root powder
30min, ultrasonic extraction 2 times, each 30min separates the ingredient of polarity very little contained in white Chinese actinidia root, subsequent to improve
DNA purity, the dregs of a decoction isolated are used；
(3) ethyl acetate extract is prepared：The white Chinese actinidia root dregs of a decoction after step (2) middle ultrasonic extraction are dried in 65 DEG C of baking ovens
Dry 12h, volatilizes remaining solvent, and the dregs of a decoction after drying are placed in the ethyl acetate of 5 times of amounts (solvent volume and medicinal material weight ratio)
Soaked overnight, the ethyl acetate that the white Chinese actinidia root dregs of a decoction after immersion measure (solvent volume and medicinal material weight ratio) with 10 times are adopted for solvent
Extracted with percolation, diacolation speed be 2mL/min, collect percolate, merge percolate, percolate filtered with filter paper after
It is concentrated under reduced pressure in BUCHI Rotary Evaporators, the condition of reduced pressure is pressure 150mbar, and 39 DEG C of temperature, recycling design obtains
Ethyl acetate extract medicinal extract, yield 0.89%；
(4) concentration mensuration：
1. Specification Curve of Increasing：Ursolic acid is to be present in one of natural plants pentacyclic triterpenoid, the present invention
Taking ursolic acid is that standard sample draws standard curve, and precision weighs ursolic acid standard items 2.5mg in 10mL volumetric flask, adds acetic acid
Ethyl ester is diluted to scale, shakes up, and is configured to the reference substance solution of the 0.25mg/mL containing ursolic acid；Accurate measurement reference substance is molten respectively
Liquid 0.1,0.2,0.4,0.6,0.8,1.0mL are placed in 10mL volumetric flask, are dried with nitrogen, and are added the 5% of 0.5mL after removing solvent
The perchloric acid solution of vanillic aldehyde-glacial acetic acid and 1.2mL mix, close plug, 15min is heated in 70 DEG C of waters bath with thermostatic control, is taken out for developing the color
Cold water is cooled to room temperature, ethyl acetate constant volume, is shaken up, and makees blank control with corresponding reagent, light absorption value is measured in 550nm, to inhale
Luminosity A is ordinate, and concentration C is abscissa, draws standard curve, and the equation of linear regression for obtaining ursolic acid is A=59.478C-
0.0289 (r=0.9994), is presented good linear relationship within the scope of 0.0025~0.025mg/mL of concentration.
2. ethyl acetate extract sample configures：Precision weighs white Chinese actinidia root ethyl acetate extract powder 0.1g, is placed in
In 10mL volumetric flask, add ethyl acetate constant volume, be ultrasonically treated 10min, let cool, mend weight, shake up, filters to get sample mother liquor；It takes
1mL sample mother liquor is placed in 5mL volumetric flask to get sample solution, concentration 2mg/mL.
3. sample size measures：White Chinese actinidia root ethyl acetate extract powder 0.02g is weighed, it is accurately weighed, it is placed in 10mL appearance
Measuring bottle adds ethyl acetate constant volume, and being configured to solution concentration is 2mg/mL, takes 0.1mL in the measurement method by step 1., in
Absorbance is measured at 550nm wavelength, is 46.95% from the content for calculating total triterpene in ethyl acetate extract on standard curve,
RSD is 1.46%.
4. sample recovery rate is tested：The white Chinese actinidia root ethyl acetate extract powder about 0.02g of known total triterpene contents is weighed,
It is placed in 10mL volumetric flask, ethyl acetate is added to be diluted to graduation mark, accurate draw solution 0.1mL is placed in 10mL volumetric flask, adds
Enter ursolic acid reference substance (concentration 0.25mg/mL) 0.5mL, be dried with nitrogen solvent, it is my 5% vanillic aldehyde-ice second that 0.5mL, which is added,
Acid and 1.2mL perchloric acid solution mix, close plug, and 15min is heated in 70 DEG C of waters bath with thermostatic control, take out cold water and are cooled to room temperature, acetic acid second
Ester constant volume, shakes up, and absorbance is measured at 550nm wavelength, obtains the average of white Chinese actinidia root ethyl acetate extract total triterpene and returns
Yield is 97%, RSD 1.13%, shows that measure resulting result is closer to actual value, accuracy is higher, is loaded back
Yield is higher, and this method can satisfy measurement and require, and feasibility is strong.
Claims (8)
1. a kind of screening technique of white Chinese actinidia root anti-tumor active substance, it is characterised in that the white Chinese actinidia root anti-tumor active substance
Screening technique include the following steps：
(1) pre-process：A certain amount of rattan pears root herb is taken, high speed disintegrator crushes after drying, and medicine sieves to obtain evengranular excessively
White Chinese actinidia root powder；
(2) ligroin extraction is prepared：Appropriate white Chinese actinidia root powder is weighed, the petroleum ether for measuring certain volume impregnates white Chinese actinidia root
The white Chinese actinidia root dregs of a decoction are separated after ultrasonic extraction with extracting solution, after extracting solution is filtered with filter paper, are depressurized with Rotary Evaporators by powder
Concentration, obtains ligroin extraction；
(3) ethyl acetate extract is prepared：The white Chinese actinidia root dregs of a decoction after step (2) middle ultrasonic extraction are dried in an oven, are volatilized
Remaining solvent, the dregs of a decoction after drying, which are placed in the ethyl acetate of certain volume, to be impregnated, the white Chinese actinidia root dregs of a decoction second after immersion
Acetoacetic ester is that solvent is extracted using percolation, and the dregs of a decoction are stand-by, and percolate is concentrated under reduced pressure in Rotary Evaporators after being filtered with filter paper,
Obtain ethyl acetate extract；
(4) ethanol extract is prepared：By the white Chinese actinidia root dregs of a decoction after step (3) middle seepage pressure effects through baking oven dry out solvent, it is added one
The ethyl alcohol for determining volume impregnates, and the white Chinese actinidia root dregs of a decoction after immersion are solvent using percolation extraction, percolate filter paper with ethyl alcohol
It is concentrated under reduced pressure in Rotary Evaporators after filtering, obtains ethanol extract；
(5) activity rating：Anticancer experiment in vitro is carried out using RTCA DP real-time cell analysis system, detection three of the above is extracted
Influence of the object to activity of tumor cells filters out the optimal extract of antitumous effect according to experimental result.
2. a kind of screening technique of white Chinese actinidia root anti-tumor active substance according to claim 1, it is characterised in that described
Three kinds of extracts in contained anti-tumor active substance content highest in ethyl acetate extract, inhibiting tumour cells are acted on bright
Aobvious, ethyl acetate extract significantly inhibits effect, IC to human hepatoma HepG2 cell50For 7.6 μ g/mL；It is thin to human lung cancer A549
Born of the same parents have inhibiting effect, IC50For 150 μ g/mL.
3. a kind of screening technique of white Chinese actinidia root anti-tumor active substance according to claim 1 or 2, it is characterised in that institute
Stating the optimal extract of antitumous effect is ethyl acetate extract, and active constituent therein is triterpene compound.
4. a kind of screening technique of white Chinese actinidia root anti-tumor active substance according to claim 1, it is characterised in that described
(5) the middle tumour cell selected is at least one of typeⅡ pneumocyte, human hepatoma HepG2 cell to step.
5. a kind of screening technique of white Chinese actinidia root anti-tumor active substance according to claim 1, it is characterised in that described
The concrete operations of step (5) are：The tumour cell of logarithmic growth phase, is made single cell suspension after pancreatin digestion, adjustment cell is dense
Degree is 5 × 104A/mL, first hole every on E-Plate plate is put into 50 μ L culture mediums and surveys baseline, and it is outstanding that 100 μ L cells are then added
Liquid is placed in 37 DEG C, 5%CO2It cultivates for 24 hours, records cell index (CI) in incubator；If Normal group, 0.2%DMSO are compareed
Group, three kinds of extract test groups, every group sets 3 multiple holes, and after culture for 24 hours, three groups of extract test groups are extracted with a certain concentration respectively
Object handles cell, continues to cultivate 72h, records CI value, screening to inhibiting tumour cells act on preferable sample carry out 200,100,
50, the CI value detection of 25,12.5,6.25 μ g/mL6 concentration, calculates IC50。
6. a kind of extracting method of white Chinese actinidia root anti-tumor active substance, it is characterised in that the white Chinese actinidia root anti-tumor active substance
Extracting method include the following steps：
(1) pre-process：A certain amount of rattan pears root herb is taken, high speed disintegrator crushes after drying, and medicine sieves to obtain evengranular excessively
White Chinese actinidia root powder；
(2) petroleum ether separates：Appropriate white Chinese actinidia root powder is weighed, the petroleum ether for measuring certain volume impregnates white Chinese actinidia root powder, surpasses
Sound separates the white Chinese actinidia root dregs of a decoction with petroleum ether extract after extracting, and the ingredient of polarity very little contained in white Chinese actinidia root is separated
It opens, to improve subsequent extracted purity；
(3) ethyl acetate extract is prepared：The white Chinese actinidia root dregs of a decoction after step (2) middle ultrasonic extraction are dried in an oven, are volatilized
Remaining solvent, the dregs of a decoction after drying, which are placed in the ethyl acetate of certain volume, to be impregnated, the white Chinese actinidia root dregs of a decoction second after immersion
Acetoacetic ester is that solvent is extracted using percolation, and percolate is concentrated under reduced pressure in Rotary Evaporators after being filtered with filter paper, obtains acetic acid
Ethyl ester extract.
7. a kind of extracting method of white Chinese actinidia root anti-tumor active substance according to claim 6, it is characterised in that described
(2) concrete operations are step：White Chinese actinidia root powder 10g~1000g is weighed, 5 times of amounts (solvent volume and medicinal material weight ratio) are added
After petroleum ether impregnates 30min, ultrasonic extraction 2 times, each 30min, combined extract isolates the white Chinese actinidia root dregs of a decoction.
8. a kind of extracting method of white Chinese actinidia root anti-tumor active substance according to claim 6, it is characterised in that described
(3) concrete operations are step：By step (2) in the white Chinese actinidia root dregs of a decoction through 65 DEG C of baking oven dry, volatilize solvent, it is (molten that 5 times of amounts be added
Agent volume and medicinal material weight ratio) ethyl acetate impregnate 12h, with the ethyl acetate of 10 times of amounts (solvent volume and medicinal material weight ratio)
Seepage pressure effects, diacolation speed are 2mL/min, collect percolate, and filter paper filters after merging, is concentrated under reduced pressure, subtracts in Rotary Evaporators
The condition of pressure concentration is pressure 150mbar, and 39 DEG C of temperature, recycling design obtains ethyl acetate extract, yield is 0.72%~
1.08%.
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