CN103977044B - Hooker winghead root n-butanol portion extract and its production and use - Google Patents
Hooker winghead root n-butanol portion extract and its production and use Download PDFInfo
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- CN103977044B CN103977044B CN201410217723.7A CN201410217723A CN103977044B CN 103977044 B CN103977044 B CN 103977044B CN 201410217723 A CN201410217723 A CN 201410217723A CN 103977044 B CN103977044 B CN 103977044B
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Abstract
The present invention relates to a kind of hooker winghead root n-butanol portion extract and its production and use.The main component of the extract is iridoid glycoside, and the preparation method of the extract is：Take hooker winghead root medicinal material, alcohol extracting under heated reflux condition, alcohol is recovered under reduced pressure in extract solution, is concentrated to give one-level extract；Add water to be suspended one-level extract again, extracted with lipophylic organic solvents, to remove oil-soluble impurities；Then extracted again with n-butanol, hooker winghead root n-butanol portion extract is obtained after recycling design.The invention also discloses purposes of the said extracted thing in antineoplastic is prepared, the extract has the effect of softening and resolving hard mass, and the performance common to malignant tumour is such as：Stagnation of blood stasis, healthy tendency virtual loss, substantive enclosed mass etc. have the effect of notable.
Description
Technical field
The invention belongs to the field of Chinese medicines, more particularly to a kind of hooker winghead root n-butanol portion extract, its preparation method and
Purposes in antineoplastic is prepared.
Background technology
Malignant tumour is the major disease for seriously endangering human life and health, and its death rate occupies human diseases death people
Several first.GLOBOCAN (2008) report point out, global new cancer cases 12,700,000 in 2008, cancer mortality case
7600000, it is contemplated that the year two thousand thirty whole world cancer new cases will be added to 22,200,000.At present, chemotherapy is still that modern clinical tumor is controlled
One of Main Means for the treatment of, but chemotherapy lacks the selectivity to normal cell and tumour cell, in the same of killing tumor cell
When, also there is great damage to normal cell, not only damage the function of the vitals such as the heart, liver, lung, kidney, marrow, and destroy
Immune system, self-protection barrier of the body to tumour is caused to be lost.The toxic side effect of chemotherapy and the drug resistance of tumour make mostly
The treatment of number tumor patient is hard to carry on, limits the treatment intensity and frequency of use of chemotherapeutics.Therefore, it is raising intra-tumor
The therapeutic effect of section, it is always the struggle mesh that researcher seek assiduously to find antineoplastic safe and effective, that toxic side effect is small
Mark.
Hooker winghead root system Dipsacaceae wing head flower plants, whole world wing head flower plants there are about 25 kinds, and China only has spoon leaf
Hooker winghead root Pterocephalus hookeri (C.B.Clarke) Hoeck and decomposite leaf hooker winghead root Pterocephalus
Two kinds of bretschneideri (Batal.) Pretz, is distributed mainly on Tibet various regions, Qinghai, western Sichuan and the north, Yun Nanxi
The ground such as the north, with root or all herbal medicine, it is Tibetan medicine's medicinal herbs most in use, is widely used in Tibetan medicinal preparation, such as 12 taste wings are first is dissipated, 20
Five tastes donkey blood ball, 20 Six-element emblic balls, nine taste green grass or young crops rocs dissipate, pomegranate Pu'an dissipates etc..Tibetan medicine thinks that hooker winghead root is cold in nature, bitter,
There is clearing heat and detoxicating, wind-damp dispelling, analgesic, available for diseases such as treatment pest poison, bi Zheng, arthritis.To the medicine chemistry with
Pharmacological research is in the ascendant.
The content of the invention
It is an object of the invention to provide a kind of n-butanol portion extract of hooker winghead root.
Another object of the present invention is to provide the preparation method of above-mentioned hooker winghead root n-butanol portion extract.
It is still another object of the present invention to provide above-mentioned hooker winghead root n-butanol portion extract in antineoplastic is prepared
Purposes.
According to the above-mentioned purpose of the present invention, the invention provides a kind of hooker winghead root n-butanol portion extract, the extract
Main component be iridoid glycoside.
The present invention also provides the preparation method of above-mentioned hooker winghead root n-butanol portion extract, and the method includes the steps of：
(1) hooker winghead root medicinal material is taken, alcohol extracting is carried out under heated reflux condition, alcohol is recovered under reduced pressure in extract solution, is concentrated to give
One-level extract；
(2) add water to be suspended above-mentioned one-level extract, extracted with lipophylic organic solvents, to remove oil-soluble impurities；So
Extracted again with n-butanol afterwards, the hooker winghead root n-butanol portion extract is obtained after recycling design.
Wherein, the Extraction solvent used in the alcohol extracting in above-mentioned steps (1) be methanol, ethanol, volumetric concentration be more than 50%
Methanol aqueous solution or volumetric concentration be more than 50% ethanol water.The dosage of Extraction solvent used in alcohol extracting is hooker winghead root
2~10 times of quality of medicinal material.During alcohol extracting, the temperature being heated to reflux is 50~100 DEG C, and the number of heating and refluxing extraction is 1~5
It is secondary, it is heated to reflux every time 0.5~2 hour.
Lipophylic organic solvents described in above-mentioned steps (2) are petroleum ether, hexamethylene, n-hexane, benzene, carbon tetrachloride,
Chloroform or dichloromethane.Lipophylic organic solvents extraction times are 1~5 time, and the solvent load extracted every time is to be extracted object
Long-pending 1~5 times.
When being extracted in step (2) with n-butanol, extraction time is 1~5 time, and the dosage for adding n-butanol every time is to be extracted
1~5 times of object product.
The present invention also provide the hooker winghead root n-butanol portion extract that is prepared according to the method described above prepare it is antitumor
Purposes in medicine.Specifically, i.e., using hooker winghead root n-butanol portion extract provided by the present invention as active ingredient, add
Pharmaceutically acceptable carrier, with the method for Chinese medicinal of routine, various form of Chinese drug are prepared, for preventing or treating evil
Property tumor disease.
Hooker winghead root n-butanol portion extract provided by the present invention, its raw material hooker winghead root crude drug source is extensive, price is low
It is honest and clean, and the method kind for preparing the n-butanol portion extract is simple and easy.The present invention uses for reference clinical according to traditional Chinese medicine theory
Medication experience, using traditional Tibetan medicine material hooker winghead root, its n-butanol active component is extracted, the active component can significantly inhibit a variety of
The growth of tumour cell, induces liver cancer cells Hep3B apoptosis, and this result is also confirmed in nude mice experiment in vivo.Therefore, originally
The involved hooker winghead root n-butanol effective part extract of invention, available for the medicine that treatment malignant tumour is made, has softening hard masses
The effect of dissipating bind, the performance common to malignant tumour is such as：Stagnation of blood stasis, healthy tendency virtual loss, substantive enclosed mass etc. have significant treat
Effect, exploitation is expected to be a kind of curative for effect, safe ready, toxic side effect is small, cheap antineoplastic.
Brief description of the drawings
Fig. 1 is the HPLC collection of illustrative plates of iridoid glycoside composition in hooker winghead root n-butanol portion extract；
Fig. 2 shows the inhibitory action that hooker winghead root n-butanol portion extract grows to various tumor cell strains：
Wherein, Fig. 2-a are that extract is extract to Eca-109 cells to Hep3B cell line inhibitory action figures, Fig. 2-b
Strain inhibitory action figure, Fig. 2-c are that extract is extract to A549 cell lines to Hep G2 cell line inhibitory action figures, Fig. 2-d
Inhibitory action figure；
Fig. 3 shows half inhibiting rate IC50 of the hooker winghead root n-butanol portion extract to various tumor cell lines；
Fig. 4 shows the influence that hooker winghead root n-butanol portion extract is bred to normal hepatocytes embryonic cell BNL-CL2；
Fig. 5 shows that the Hep3B cells of fluorescence microscope hooker winghead root n-butanol portion extract induction in vitro culture wither
The morphological feature (× 400 times) died；
Fig. 6 shows transmission electron microscope observing Hep3B cell ultra micros metamorphosis (× 8200 times)；
Fig. 7 shows hooker winghead root n-butanol portion extract inducing cell apoptosis and the pass of PTEN/PI3K/AKT signal paths
System；Wherein, 1 control group is represented, 2 represent hooker winghead root n-butanol portion extract group (the μ g/ml of concentration 200), and 3 represent LY294002
(PI3K inhibitor) group, 4 represent the extract low concentration group treated through LY294002, and 5 represent what is treated through LY294002
Concentration group in extract, 6 represent the extract high concentration group treated through LY294002,；
Fig. 8 shows shadow of the double dye detection hooker winghead root n-butanol portion extracts of Annexin-V/PI to Hep3B Apoptosis
Ring；
Fig. 9 shows hooker winghead root n-butanol portion to nude mice index and spleen index and tumor weight, the influence of nude mice body weight；Wherein,
Fig. 9-a are nude mice index and spleen index figure, and Fig. 9-b are nude mouse multigraph, and Fig. 9-c are nude mice knurl weight figure, and Fig. 9-d are tumor control rate
Scheme, the abscissa in each figure represents concentration group, extract high concentration in CMCNa groups, extract low concentration group, extract successively
Group, positive drug fluorouracil group.
Embodiment
N-butanol portion extraction is obtained from hooker winghead root bulk drug below by way of the specific embodiment explanation present invention for preparing
The method of thing, but they are not limitation of the invention.
Embodiment 1：
1.1 Med Mat Appreciation
Spoon leaf hooker winghead root Pterocephalus hookeri (C.B.Clarke) Hoeck are collected in China in April, 2012
Qinghai Province of people's republic.Identification is awarded through Chinese medicine institute of Shanghai Univ. of Traditional Chinese Medicine Zhao Zhi the Confucian or feudal ethical codes.It is big that sample is stored in Shanghai Chinese
Learn Chinese pharmacology school's medicine laboratory (sample number：YSC-20120415).
It is prepared by 1.2 medicinal material extracts
Hooker winghead root medicinal material 2000g, the ethanol 7L for adding volumetric concentration to be 95%, heats refluxing extraction 3 times at 80 DEG C, every time
2h, merge extract solution, be concentrated under reduced pressure into 1/5 volume, add isometric water, evaporate into no alcohol taste, successively with 2 times of volume oil
Ether, dichloromethane extract 3 times, then with 2 times of volume extracting n-butyl alcohols 3 times, merge n-butanol, recycling design obtains the positive fourth of hooker winghead root
Alcohol extractive part.
Embodiment 2：
2.1 Med Mat Appreciation：With embodiment 1
It is prepared by 2.2 medicinal material extracts
Hooker winghead root medicinal material 2000g, adds absolute ethyl alcohol 7L, heats refluxing extraction 3 times at 80 DEG C, each 2h, merges extraction
Liquid, 1/5 volume is concentrated under reduced pressure into, adds isometric water, evaporate into no alcohol taste, successively with 2 times of volume hexamethylenes, chloroform extraction 3
It is secondary, then with 2 times of volume extracting n-butyl alcohols 3 times, merge n-butanol, recycling design obtains hooker winghead root n-butanol portion extract.
Embodiment 3
3.1 Med Mat Appreciation：With embodiment 1
It is prepared by 3.2 medicinal material extracts
Hooker winghead root medicinal material 200g, adds methanol 900mL, heats refluxing extraction 3 times at 80 DEG C, each 2h, merges extract solution,
1/5 volume is concentrated under reduced pressure into, adds isometric water, evaporates into no alcohol taste, successively with 2 times of volume hexamethylenes, dichloromethane extraction 3
It is secondary, then with 2 times of volume extracting n-butyl alcohols 3 times, merge n-butanol, recycling design obtains hooker winghead root n-butanol portion extract.
Embodiment 4
4.1 Med Mat Appreciation：With embodiment 1
It is prepared by 4.2 medicinal material extracts
Hooker winghead root medicinal material 1000g, the ethanol 4L for adding volumetric concentration to be 75%, heats refluxing extraction 2 times at 50 DEG C, every time
0.5h, merge extract solution, be concentrated under reduced pressure into 1/5 volume, add isometric water, evaporate into no alcohol taste, successively with 1 times of volume just oneself
Alkane, carbon tetrachloride extraction 3 times, then with 3 times of volume extracting n-butyl alcohols 4 times, merge n-butanol, recycling design obtains the positive fourth of hooker winghead root
Alcohol extractive part.
Embodiment 5
5.1 Med Mat Appreciation：With embodiment 1
It is prepared by 5.2 medicinal material extracts
Hooker winghead root medicinal material 1000g, the methanol 3.5L for adding volumetric concentration to be 80%, heats refluxing extraction 5 times at 100 DEG C,
Each 1h, merge extract solution, be concentrated under reduced pressure into 1/5 volume, add isometric water, evaporate into no alcohol taste, successively with 5 times of volumes
Benzene, dichloromethane extract 3 times, then with 5 times of volume extracting n-butyl alcohols 2 times, merge n-butanol, recycling design obtains the positive fourth of hooker winghead root
Alcohol extractive part.
It is first the wing of the present invention is expanded on further by test example (by taking extract prepared by embodiment 1 as an example) again below
The beneficial effect of careless n-butanol portion extract, these test examples include cytotoxicity experiment and animal pharmacodynamic experiment.
Embodiment 6：The inhibitory action that hooker winghead root n-butanol portion extract grows to various tumor cell strains
6.1 experiment material
6.1.1 cell line：
Lung cancer A549 cell strain, esophageal carcinoma Eca-109 cell line strain, HepG2 cell, liver cancer Hep3B cell lines,
Purchased from OEG cell institute of the Chinese Academy of Sciences, preserved by Shanghai Univ. of Traditional Chinese Medicine's prescription laboratory passage.
6.1.2 reagent：
DMEM high sugared nutrient solution (Hyclone, Thermo companies of the U.S., lot number：SH30243.01), hyclone
(Gibco, Invitrogen company, lot number：100099-141), 0.25% trypsase (Gibco, Invitrogen company, is criticized
Number：25200-072)
6.1.3 instrument：
Electronic balance (Shanghai Ao Haosi Instrument Ltd., model：AR124CN) CO2gas incubator (the U.S.
Thermo Fisher companies, model：3111), superclean bench (Shanghai Li Shen Scientific Instruments Corporations Co., Ltd, model：
HFsafe-1500), all-wave length ELIASA (gene gene companies of the U.S., model：Synergy HT), centrifuge (Sigma companies,
Model：2-16PK).
6.2 experimental method：
(1) inoculating cell：Cell is made into the high sugared nutrient solution of RPMI1640 nutrient solutions and DMEM containing 10% hyclone
Suspension, 96 orifice plates, every group of 4-6 multiple holes, per the μ l of pore volume 100 are inoculated into every 3000-5000 cell in hole.If Experimental agents
Group, positive controls and blank control group；It is put into 37 DEG C, 5%CO2Cultivated in incubator；
(2) dosing：After cell attachment, original fluid is drawn, adds the new nutrient solution (medicine group of the medicine containing various concentrations
Each concentration is closed as 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml；It is positive
Comparison medicine vincristine concentration is 0.5 μ g/ml) per the μ l of hole 100, continue to cultivate 48h；
(3) colour generation：After cultivating 48h, 5mg/ml MTT solution 10ul are added per hole, continues to be incubated 4h, terminates culture, inhale training
Hole supernatant is supported, DMSO150 μ l are added per hole, 10min is shaken, crystal is fully melted；
(4) colorimetric：490nm wavelength is selected, each hole absorbance is determined on enzyme mark detector；
(5) inhibiting rate is calculated：
6.3 experimental result
As a result as shown in Figure 2, hooker winghead root n-butanol portion extract can substantially suppress human esophagus cancer cell Eca109, people
Liver cancer cells Hep3B, HepG2, the activity of human lung cancer cell A549's growth.As shown in Figure 3, half inhibiting rate (IC50) respectively
For Hep3B90.92 μ g/ml, HepG2126.18 μ g/ml, A549110.4 μ g/ml, Eca109112.0 μ g/ml, illustrate the extraction
Thing can suppress kinds of tumor cells growth concentration dependent.
The inhibitory action that the hooker winghead root n-butanol portion extract of embodiment 7 is bred to normal liver cell line BNL-CL2
7.1 experiment materials are the same as embodiment 6
7.2 experimental methods are the same as embodiment 6
7.3 experimental result
As shown in Figure 4, the inhibitory action that hooker winghead root n-butanol portion extract grows to normal liver cell line BNL-CL2
Unobvious, inhibiting rate are only 22.16% when maximum concentration reaches 500 μ g/ml.
The form of the fluorescence microscope hooker winghead root n-butanol portion extract inductor Hep3B Apoptosis of embodiment 8
Feature
8.1 experiment material：
PBS (Hyclone, Thermo company, lot number：), SH30256.01B Hoechst33258 dye liquors (Sigma
Products, lot number：861405), it is kept in dark place.4% paraformaldehyde (Chemical Reagent Co., Ltd., Sinopharm Group, lot number：
20120514).Fluorescence microscope (Japanese OLYMPUS companies, model：IX71)
8.2 experimental method：
A) learnt from else's experience various concentrations hooker winghead root n-butanol portion extract effect 48h cell, add 4% paraformaldehyde to consolidate
Determine 30min；
B) after PBS washs cell, add Hoechst33258 dye liquors (per μ l of hole 30), act on 10min；
C) PBS is washed 3 times, each 5min, adds anti-quencher mounting；
D) micro- Microscopic observation, accompanying drawing 5. is as a result seen
8.3 experimental result：
Cell after Hoechst dyeing, the distribution of cellular control unit nuclear dna is relatively uniform, and core is in circle or oval, without solid
Contracting, is in faint blueness in the visual field；It is solid in fine and close dense dye, core and medication group part apoptosis, nucleus color depth
Contracting, deformation.Compared with control group, the middle and high concentration group apoptotic cells increased of extract.
The form of the transmission electron microscope observing hooker winghead root n-butanol portion extract inductor Hep3B Apoptosis of embodiment 9 is special
Sign
9.1 experiment material：
Transmission electron microscope (Dutch PHILIPS Co., model：Philips Tecnai-12 types), Hep3B cells, glutaraldehyde (on
Marine medical pharmaceutical university experimental center for scientific technology Electron Microscopy Room provides)
9.2 experimental method：
A) various concentrations hooker winghead root n-butanol portion extract of learning from else's experience acts on 48h cell, adds penta 2 after digestion, centrifugation
Aldehyde is fixed；
B) Shanghai Univ. of Traditional Chinese Medicine's experimental center for scientific technology Electron Microscopy Room embedding, section are sent
C) machine is observed on, as a result such as accompanying drawing 6
9.3 experimental result：
Control group Hep3B cell volumes are larger, and cell membrane is complete, and chromatin enriches in core, and kernel is obvious, and organelle is complete
It is whole, see Fig. 6 a；And after hooker winghead root n-butanol portion extract-treated 48h, it is seen that cell volume reduces, and core has pyconsis,
Chromatic agglutination in core, chromatin are unevenly assembled along under nuclear membrane, and reticulum dilatation, vacuolization increases, organelle swelling
Occur Deng Apoptosis feature (see Fig. 6 b, 6c).
The hooker winghead root n-butanol portion extract inducing cell apoptosis of embodiment 10 and the pass of PTEN/PI3K/AKT signal paths
System
10.1 experiment materials：
Electrophoresis apparatus (BIO-RAD companies of the U.S., type 552BR102298), transferring film instrument (BIO-RAD companies of the U.S., model：
153BR81259), multi-functional decolorization swinging table (Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd., model：ZHWY-344), constant temperature gold
Category bath (Hangzhou BIOER Technology Co., Ltd, model：CHB-100), gel imaging system (BIO-RAD companies of the U.S., model：
GelDoc XR), ECL luminescent solutions (Thermo companies, lot number：32106), PTEN, PI3K, AKT and p-PTEN, p-PI3K, p-
AKT antibody (Rabbit mAb) is Cell Signaling Tehnology Products, the albumen Maker (U.S.
Fermentas products, lot number：SM1811), other experiment reagents are purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
10.2 experimental methods：
The Hep3B cells through various concentrations hooker winghead root n-butanol portion extract-treated 48h are collected respectively, and nutrient solution is inhaled
Abandon, with the PBS 2 times of precooling, add 200 μ L cell pyrolysis liquid RIPA, ice bath cracking 20min, 14000r/min, 4 DEG C from
Heart 20min, takes supernatant, carries out protein quantification with BCA methods, supernatant is placed in -20 DEG C of preservations after packing.Take equal protein (30 μ
G) SDS-PAGE electrophoretic separation is carried out, first in the constant pressure 100V electrophoresis subsequent 150V electrophoresis 60min of 20min, then in 250mA bar
Electric transferring film 90min under part, by protein delivery to pvdf membrane, 5% skimmed milk power room temperature closing 1h, it is separately added into corresponding primary antibody
(internal reference GAPDH dilution factor is 1:5000, the dilution factor of primary antibody is 1:1000) stay overnight for 4 DEG C；Add corresponding HRP marks
Secondary antibody (1:5000 dilutions) colour developing of room temperature 2h, ECL luminescence method, test and be repeated 3 times under the same conditions.
10.3 experimental results are shown in accompanying drawing 7
7 display can be seen that with reference to the accompanying drawings：Compared with control group, PTEN and p-PTEN expression increases, and it is dense with medicine
Degree increases and ascendant trend is presented；Akt, p-Akt, PI3K, p-PI3K expression have declined, gradual with drug concentration increase expression
Decline.Illustrate that the Hep3B cells through the effect of hooker winghead root n-butanol portion extract are presented with difference on protein level, pass through
PTEN/PI3K/Akt signal paths work.
The double dye methods of the flow cytometer Annexin-V/PI of embodiment 11 detect hooker winghead root n-butanol portion extract to Hep3B
The influence of Apoptosis
11.1 experiment materials：
Hep3B cells DMEM high sugared nutrient solution (Hyclone, Thermo companies of the U.S., lot number：SH30243.01) tire ox blood
(Gibco, Invitrogen company, lot number clearly：100099-141) 0.25% trypsase, without EDTA, (Chinese medicines group chemistry
Reagent Co., Ltd, lot number：20130103) 6 well culture plates (Corning companies of the U.S., model：3516), 15ml centrifuge tubes are (equal
For U.S.'s Corning Products), AnnexiV/Fitc-PI kits (U.S. company BD, lot number:), flow cytometer is (beautiful
BD companies of state, model：FACSCalibur), streaming pipe (U.S. company BD, model：352052)
11.2 experimental methods：
1) plate (6 orifice plates, Hep3B cells 2*10 are planted5/ hole, bed board next day dosing, action time 48h)
2) cell is collected：Medium in 6 orifice plates is sucked in corresponding 15ml centrifuge tubes respectively；It is separately added into
1mlPBS, gently swing and wash cell；Vitellophag；Centrifugation：1000rpm,5min；Operated according to kit specification, add 100 μ
L/ pipe binding buffer, cell is resuspended, is separately added into 5 μ l Annexin and PI, lucifuge is incubated 30min, adds 400 μ l/
Upper machine testing in pipe binding buffer, 1h.
11.3 experimental results：See accompanying drawing 8
8 display can be seen that with reference to the accompanying drawings：As shown：Compared with control group, apoptosis-induced related PTEN and p-
PTEN expression increases, and increase with drug concentration and ascendant trend is presented；Suppress apoptotic proteins Akt, p-Akt, PI3K, p-PI3K
Expression has declined, and is gradually reduced with drug concentration increase expression.Illustrate through the effect of hooker winghead root n-butanol portion extract
Hep3B cells are presented with difference on protein level, are worked by PTEN/PI3K/Akt signal paths.
The hooker winghead root n-butanol portion extract Anticancer effect in vivo of embodiment 12 is studied
12.1 experiment materials:
12.1.1 instrument, reagent：Mouse stomach pin, 1ml syringes, electronic balance (Industrial Co., Ltd. of upper Nereid section, YP
Type), slide measure.(Chinese medicines group chemical reagent is limited by sodium carboxymethylcellulose carboxymethylcellulose sodium
Company, lot number：20130113) Fluorouracil Injection (Shanghai Xudong Hipu Medicine Co., Ltd, lot number：FA1209012)
12.1.2 animal：BALB/c-nu nude mices, body weight 16-18g, 5 week old, male, had by Shanghai Si Laike experimental animals
Limit responsible company to provide, experimental animal production licence：SCXK (Shanghai) 2007-0005；Raise in Shanghai Univ. of Traditional Chinese Medicine animal
Experimental center no-special pathogen (SPF) environment.Temperature：20-25 DEG C, humidity 40-70%.
12.1.3 knurl kind：Hep3B cell lines
12.2 experimental methods:
12.2.1 establish liver cancer Hep3B and subcutaneously suppress knurl model：
12.2.1.1 tumour culture cell collects 5min after the Hep3B cells in exponential phase are digested with pancreatin, uses
PBS is washed 2 times, and adjustment concentration of cell suspension is 1 × 107Individual/mL.
12.2.1.2 the inoculating cell of tumour culture cell is counted as 1 × 107Individual/mL, the right armpit notch graft of every nude mice
Kind 2 × 106Individual cell (0.2mL/ is only).After 2-3 days, the formational situation of transplanted tumor in nude mice is observed, measures tumor size (tumour body
Product TV calculation formula be:TV=1/2*a*b2, wherein a, b represent the length and width of tumour respectively), from being inoculated with, successful transplantable tumor is naked
It is more consistent that tumor size is selected in mouse, gross tumor volume is up to 60~70mm3The 30 of left and right are served only for experimental study.
12.2.2 experiment packet is grouped at random by gross tumor volume size, every group 6, totally 30.
Blank control group:0.5% CMC (CMC-Na) solution is given by 0.1mL/10g body weight, gavage, is connected
Continuous 21d.
Each dosage group of extract：By 0.1mL/10g dosages, gavage, continuous 21d.
Positive drug 5-FU groups ((fluorouracil group):20mg/kg times, 5-FU0.2mL/d, intraperitoneal injection, every other day one
It is secondary, 21d is used in conjunction.
Each dosage group of extract --- according to each dosage group of oral liquid (μ g/ of low dosage 100 obtained by preparation method of the present invention
Ml, the μ g/ml of middle dosage 300, the μ g/ml of high dose 1000), last dose next day weighs, and puts to death animal, strips tumour, spleen simultaneously
Weigh.
12.2.3 observation index：1) ordinary circumstance and changes of weight of mouse are observed daily, and slide measure was used every 1 day
Measure 1 nude mice knurl volume.The calculation formula of gross tumor volume (T V) is:T V=1/2*a*b2, wherein a, b represent tumour respectively
Length and width.2) calculate each group knurl weight and calculate tumour inhibiting rate：Tumor-like hyperplasia (%)=(1-T/C) × 100%, wherein C are control
The average knurl weight of group, T are the average knurl weight of each test group.3) index and spleen index：SI=spleen weights (mg)/10g body weight * 100%.
12.3 experimental results are shown in accompanying drawing 9.
9 display can be seen that with reference to the accompanying drawings：Compared with 0.5%CMC-Na (hydroxymethyl cellulose) control group, extract
Each dosage group can reduce the knurl weight of nude mice；Each group the weight of animals is also statistically significant；In terms of immunologic mechanism is adjusted, extract
Influence to index and spleen index (SI) is smaller, not statistically significant；And positive control drug fluorouracil influences to have on index and spleen index
Statistical significance, therefore think that the toxic side effect of the extract is small compared with chemotherapeutic 5-FU (fluorouracil) groups.
It will be understood by those skilled in the art that the respective embodiments described above are to realize the specific embodiment of the present invention,
And in actual applications, can to it, various changes can be made in the form and details, without departing from the spirit and scope of the present invention.
Claims (6)
1. purposes of the hooker winghead root n-butanol portion extract in antineoplastic is prepared, it is characterised in that the extract
Main component is iridoid glycoside；
Wherein, the preparation method of the extract comprises the steps of：
(1) hooker winghead root medicinal material is taken, alcohol extracting is carried out under heated reflux condition, alcohol is recovered under reduced pressure in extract solution, is concentrated to give one-level
Extract；
(2) add water to be suspended above-mentioned one-level extract, extracted with lipophylic organic solvents, to remove oil-soluble impurities；Then again
Extracted with n-butanol, the hooker winghead root n-butanol portion extract is obtained after recycling design；
Wherein, the temperature being heated to reflux in step (1) is 50~100 DEG C, and the number of heating and refluxing extraction is 1~5 time, is added every time
Heat backflow 0.5~2 hour.
2. purposes according to claim 1, it is characterised in that the Extraction solvent used in alcohol extracting in step (1) is methanol,
The ethanol water that the methanol aqueous solution or volumetric concentration that ethanol, volumetric concentration are more than 50% are more than 50%.
3. purposes according to claim 1, it is characterised in that the dosage of the Extraction solvent used in alcohol extracting in step (1)
For 2~10 times of hooker winghead root quality of medicinal material.
4. purposes according to claim 1, it is characterised in that the lipophylic organic solvents described in step (2) are oil
Ether, hexamethylene, n-hexane, benzene, carbon tetrachloride, chloroform or dichloromethane.
5. purposes according to claim 1, it is characterised in that the number that lipophylic organic solvents extract in step (2) is 1
~5 times, the solvent load extracted every time is 1~5 times of extract volume.
6. purposes according to claim 1, it is characterised in that the number that n-butanol extracts in step (2) is 1~5 time, often
The dosage of secondary addition n-butanol is 1~5 times of extract volume.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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