CN103977044A - Pterocephalus hookeri heck n-butyl alcohol extract, as well as preparation method and application thereof - Google Patents
Pterocephalus hookeri heck n-butyl alcohol extract, as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a pterocephalus hookeri heck n-butyl alcohol extract, as well as a preparation method and application thereof. Iridoid glycoside is the main component of the extract. The preparation method of the extract comprises the following steps: weighing the pterocephalus hookeri heck medicinal material; performing ethanol extraction under a heating refluxing condition; recycling ethanol from the extract in reduced pressure, and concentrating to obtain a primary extract; suspending the primary extract with water, and extracting with an amphipathic organic solvent to remove liposoluble impurities; extracting with n-butyl alcohol, recycling the solvent to obtain the pterocephalus hookeri heck n-butyl alcohol extract. The invention also discloses application of the extract in preparing anti-tumor medicaments. The extract has the effect of softening hardness to dissipate stagnation, and has remarkable curative effects on common syndromes of malignant tumors, such as blood stasis stagnation, vital energy deficiency, substantial mass and the like.
Description
Technical field
The invention belongs to the field of Chinese medicines, particularly a kind of Herba pterocephali n-butanol portion extract, its preparation method and in the purposes of preparing in antitumor drug.
Background technology
Malignant tumor is serious harm human life and healthy major disease, and its mortality rate occupies first of human diseases death toll.GLOBOCAN (2008) report is pointed out, global new cases of cancer 1,270 ten thousand in 2008, and cancer mortality case 7,600,000, expectation the year two thousand thirty whole world cancer new cases will be increased to 2,220 ten thousand.At present; chemotherapy remains one of Main Means of modern clinical therapy of tumor; but chemotherapy lacks the selectivity to normal cell and tumor cell; in killing tumor cell; normal cell is also had to great damage; not only damage the function of the vitals such as the heart, liver, lung, kidney, bone marrow, and destroy immune system, cause body to be lost the self-protection barrier of tumor.The toxic and side effects of chemotherapy and the drug resistance of tumor make the treatment of most of tumor patients hard to carry on, have limited treatment intensity and the frequency of utilization of chemotherapeutics.Therefore,, for improving the therapeutic effect of Internal Medicine-Oncology, find the antitumor drug safe and effective, toxic and side effects is little is the objective of the struggle that researcher is seek assiduously always.
Herba pterocephali is the first flower plant of the Dipsacaceae wing, the wing first flower plant in the whole world approximately has 25 kinds, only there are two kinds of spoon leaf Herba pterocephali Pterocephalus hookeri (C.B.Clarke) Hoeck and decomposite leaf Herba pterocephali Pterocephalus bretschneideri (Batal.) Pretz in China, mainly be distributed in various places, Tibet, Qinghai, western Sichuan and the north, the ground such as northwestern Yunnan Province, with root or all herbal medicine, for Tibetan medicine's medicinal herbs most in use, be widely used in Tibetan medicinal preparation, as first in the 12 taste wings loose, Ershiwuwei Luxue pellets, 20 Six-element Fructus Phyllanthi balls, the blue or green roc of nine tastes is loose, Punica granatum L. Pu'an is loose etc.Tibetan medicine thinks that Herba pterocephali is cold in nature, bitter in the mouth, has the effect of heat-clearing and toxic substances removing, wind-damp dispelling, pain relieving, can be used for treating the diseases such as pestilent toxicity, arthromyodynia, arthritis.Chemistry and pharmacological research to this medicine are in the ascendant.
Summary of the invention
The object of the present invention is to provide a kind of n-butanol portion extract of Herba pterocephali.
Another object of the present invention is to provide the preparation method of above-mentioned Herba pterocephali n-butanol portion extract.
A further object of the present invention is to provide above-mentioned Herba pterocephali n-butanol portion extract in the purposes of preparing in antitumor drug.
According to above-mentioned purpose of the present invention, the invention provides a kind of Herba pterocephali n-butanol portion extract, the main component of this extract is iridoid glycoside.
The present invention also provides the preparation method of above-mentioned Herba pterocephali n-butanol portion extract, and the method includes the steps of:
(1) get Herba pterocephali medical material, under reflux condition, carry out alcohol extraction, by extracting solution reclaim under reduced pressure alcohol, the concentrated one-level extract that obtains;
(2) above-mentioned one-level extract is added water suspendible, uses lipotropy organic solvent extraction, to remove oil-soluble impurities; And then with n-butanol extraction, after recovery solvent, obtain described Herba pterocephali n-butanol portion extract.
Wherein, the alcohol extraction extraction solvent used in above-mentioned steps (1) is that methanol, ethanol, volumetric concentration are that more than 50% methanol aqueous solution or volumetric concentration is more than 50% ethanol water.The consumption of alcohol extraction extraction solvent used is 2～10 times of Herba pterocephali quality of medicinal material.When alcohol extraction, the temperature of reflux is 50～100 DEG C, and the number of times of heating and refluxing extraction is 1～5 time, each reflux 0.5～2 hour.
Lipotropy organic solvent described in above-mentioned steps (2) is petroleum ether, cyclohexane extraction, normal hexane, benzene, carbon tetrachloride, chloroform or dichloromethane.Lipotropy organic solvent extraction number of times is 1～5 time, and the solvent load of each extraction is 1～5 times of extract volume.
In step (2), during with n-butanol extraction, extraction time is 1～5 time, and the consumption that at every turn adds n-butyl alcohol is 1～5 times of extract volume.
The present invention also provides the Herba pterocephali n-butanol portion extract preparing according to the method described above in the purposes of preparing in antitumor drug.Specifically,, taking Herba pterocephali n-butanol portion extract provided by the present invention as effective ingredient, add pharmaceutically acceptable carrier, with conventional method of Chinese medicinal, prepare various form of Chinese drug, for prevention or treatment malignancy disease.
Herba pterocephali n-butanol portion extract provided by the present invention, its raw material Herba pterocephali crude drug source is extensive, cheap, and it is simple to prepare the method kind of this n-butanol portion extract.The present invention is according to traditional Chinese medicine theory, use for reference clinical application experience, adopt traditional Tibetan medicine material Herba pterocephali, extract its n-butyl alcohol effective site, this effective site can significantly suppress the growth of kinds of tumor cells, induction hepatoma carcinoma cell Hep3B apoptosis, this result is tested and has also been obtained confirmation in nude mouse.Therefore, Herba pterocephali n-butyl alcohol effective part extract involved in the present invention, can be used for making the medicine for the treatment of malignant tumor, there is effect of hard masses softening and resolving, to the common performance of malignant tumor as: stagnation of blood stasis, healthy energy virtual loss, substantive enclosed mass etc. have significant curative effect, are expected to be developed as a kind of determined curative effect, safe ready, toxic and side effects is little, cheap antitumor drug.
Brief description of the drawings
Fig. 1 is the HPLC collection of illustrative plates of iridoid glycoside composition in Herba pterocephali n-butanol portion extract;
Fig. 2 shows the inhibitory action of Herba pterocephali n-butanol portion extract to various tumor cell strains growth:
Wherein, Fig. 2-a be extract to Hep3B cell strain inhibitory action figure, Fig. 2-b be extract to Eca-109 cell strain inhibitory action figure, Fig. 2-c be extract to Hep G2 cell strain inhibitory action figure, Fig. 2-d is that extract is to A549 cell strain inhibitory action figure;
Fig. 3 shows the half suppression ratio IC50 of Herba pterocephali n-butanol portion extract to various tumor cell lines;
Fig. 4 shows the impact of Herba pterocephali n-butanol portion extract on normal hepatocytes embryonic cell BNL-CL2 propagation;
Fig. 5 shows the apoptotic morphological characteristic of Hep3B (× 400 times) of fluorescence microscope Herba pterocephali n-butanol portion extract induction In vitro culture;
Fig. 6 shows transmission electron microscope observing Hep3B cell ultra micro metamorphosis (× 8200 times);
Fig. 7 shows the relation of Herba pterocephali n-butanol portion extract cell death inducing and PTEN/PI3K/AKT signal path; Wherein, 1 represents matched group, 2 represent Herba pterocephali n-butanol portion extract group (concentration 200 μ g/ml), 3 represent LY294002 (PI3K inhibitor) group, 4 represent the extract low concentration group of processing through LY294002,5 represent concentration group in the extract of processing through LY294002, and 6 represent the extract high concentration group of processing through LY294002;
Fig. 8 shows that the two detection Herba pterocephali n-butanol portion extracts that dye of Annexin-V/PI are on the apoptotic impact of Hep3B;
Fig. 9 shows the impact of Herba pterocephali n-butanol portion on nude mice index and spleen index and tumor weight, nude mice body weight; Wherein, Fig. 9-a is nude mice index and spleen index figure, Fig. 9-b is nude mouse multigraph, Fig. 9-c is nude mice tumor multigraph, Fig. 9-d is tumor control rate figure, and the abscissa in each figure represents concentration group in CMCNa group, extract low concentration group, extract, extract high concentration group, positive drug fluorouracil group successively.
Detailed description of the invention
From Herba pterocephali crude drug, obtain below the method for n-butanol portion extract by concrete Preparation Example explanation the present invention, but they are not limitation of the invention.
Embodiment 1:
1.1 medical material qualifications
Spoon leaf Herba pterocephali Pterocephalus hookeri (C.B.Clarke) Hoeck is collected in Qinghai Province of the People's Republic of China (PRC) in April, 2012.Through Shanghai Univ. of Traditional Chinese Medicine, the Zhao Zhi of Chinese medicine institute the Confucian or feudal ethical codes is awarded qualification.Specimen is stored in Chinese materia medica school of Shanghai Univ. of Traditional Chinese Medicine medicine laboratory (mark this shop: YSC-20120415).
1.2 medicinal material extract preparations
Herba pterocephali medical material 2000g, add volumetric concentration and be 95% ethanol 7L, heating reflux, extract, 3 times at 80 DEG C, each 2h, merge extractive liquid,, be evaporated to 1/5 volume, add isopyknic water, evaporate into without alcohol taste, use successively 2 times of volume petroleum ether, dichloromethane extraction 3 times, then use 2 times of volume n-butanol extractions 3 times, merge n-butyl alcohol, reclaim solvent and obtain Herba pterocephali n-butanol portion extract.
Embodiment 2:
2.1 medical material qualifications: with embodiment 1
2.2 medicinal material extract preparations
Herba pterocephali medical material 2000g, add dehydrated alcohol 7L, heating reflux, extract, 3 times at 80 DEG C, each 2h, merge extractive liquid,, be evaporated to 1/5 volume, add isopyknic water, evaporate into without alcohol taste, use successively 2 times of volume cyclohexane extraction, chloroform extraction 3 times, then use 2 times of volume n-butanol extractions 3 times, merge n-butyl alcohol, reclaim solvent and obtain Herba pterocephali n-butanol portion extract.
Embodiment 3
3.1 medical material qualifications: with embodiment 1
3.2 medicinal material extract preparations
Herba pterocephali medical material 200g, add methanol 900mL, heating reflux, extract, 3 times at 80 DEG C, each 2h, merge extractive liquid,, be evaporated to 1/5 volume, add isopyknic water, evaporate into without alcohol taste, use successively 2 times of volume cyclohexane extraction, dichloromethane extraction 3 times, then use 2 times of volume n-butanol extractions 3 times, merge n-butyl alcohol, reclaim solvent and obtain Herba pterocephali n-butanol portion extract.
Embodiment 4
4.1 medical material qualifications: with embodiment 1
4.2 medicinal material extract preparations
Herba pterocephali medical material 1000g, add volumetric concentration and be 75% ethanol 4L, heating reflux, extract, 2 times at 50 DEG C, each 0.5h, merge extractive liquid,, be evaporated to 1/5 volume, add isopyknic water, evaporate into without alcohol taste, use successively 1 times of volume normal hexane, carbon tetrachloride extraction 3 times, then use 3 times of volume n-butanol extractions 4 times, merge n-butyl alcohol, reclaim solvent and obtain Herba pterocephali n-butanol portion extract.
Embodiment 5
5.1 medical material qualifications: with embodiment 1
5.2 medicinal material extract preparations
Herba pterocephali medical material 1000g, add volumetric concentration and be 80% methanol 3.5L, heating reflux, extract, 5 times at 100 DEG C, each 1h, merge extractive liquid,, be evaporated to 1/5 volume, add isopyknic water, evaporate into without alcohol taste, use successively 5 times of volume benzene, dichloromethane extraction 3 times, then use 5 times of volume n-butanol extractions 2 times, merge n-butyl alcohol, reclaim solvent and obtain Herba pterocephali n-butanol portion extract.
The beneficial effect of below further setting forth again Herba pterocephali n-butanol portion extract of the present invention by test example (extract of preparing taking embodiment 1 is example), these test examples have comprised cytotoxicity experiment and animal pharmacodynamic experiment.
Embodiment 6: the inhibitory action of Herba pterocephali n-butanol portion extract to various tumor cell strains growth
6.1 experiment material
6.1.1 cell strain:
Lung cancer A549 cell strain, esophageal carcinoma Eca-109 cell line strain, HepG2 cell, hepatocarcinoma Hep3B cell strain, all purchased from biochemical cell institute of the Chinese Academy of Sciences, by the preservation of going down to posterity of Shanghai Univ. of Traditional Chinese Medicine side's medicine laboratory.
6.1.2 reagent:
The high sugared culture fluid (Hyclone of DMEM, Thermo company of the U.S., lot number: SH30243.01), hyclone (Gibco, Invitrogen company, lot number: 100099-141), 0.25% trypsin Gibco, Invitrogen company, lot number: 25200-072)
6.1.3 instrument:
Electronic balance (Shanghai Ao Haosi Instrument Ltd., model: AR124CN) CO2 gas incubator (Thermo Fisher company of the U.S., model: 3111), superclean bench (Shanghai company limited of Li Shen scientific instrument company, model: HFsafe-1500), the long microplate reader of all-wave (gene gene company of the U.S., model: Synergy HT), centrifuge (Sigma company, model: 2-16PK).
6.2 experimental techniques:
(1) inoculating cell: use containing RPMI1640 culture fluid and the high sugared culture fluid of DMEM of 10% hyclone and be made into cell suspension, be inoculated into 96 orifice plates with every hole 3000-5000 cell, every group of 4-6 multiple hole, every pore volume 100 μ l.If Experimental agents group, positive controls and blank group; Put into 37 DEG C, 5%CO
2in incubator, cultivate;
(2) dosing: after cell attachment, draw original fluid, (the each concentration of drug regimen is 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 to add new culture fluid containing variable concentrations medicine; Positive control drug vincristine concentration is 0.5 μ g/ml) every hole 100 μ l, continue to cultivate 48h;
(3) colour generation: cultivate after 48h, every hole adds 5mg/ml MTT solution 10ul, continues to hatch 4h, stops cultivating, and inhales culture hole supernatant, and every hole adds DMSO150 μ l, and concussion 10min, fully melts crystal;
(4) colorimetric: select 490nm wavelength, measure each hole absorbance on enzyme mark detector;
(5) calculate suppression ratio:
6.3 experimental result
As shown in Figure 2, Herba pterocephali n-butanol portion extract can obviously suppress the activity of human esophagus cancer cell Eca109, human liver cancer cell Hep3B, HepG2, human lung cancer cell A549's growth to result.As shown in Figure 3, half suppression ratio (IC
50) be respectively Hep3B90.92 μ g/ml, HepG2126.18 μ g/ml, A549110.4 μ g/ml, Eca109112.0 μ g/ml, illustrates that this extract can suppress kinds of tumor cells growth concentration dependent.
The inhibitory action of embodiment 7 Herba pterocephali n-butanol portion extracts to normal liver cell line BNL-CL2 propagation
7.1 experiment materials are with embodiment 6
7.2 experimental techniques are with embodiment 6
7.3 experimental result
As shown in Figure 4, Herba pterocephali n-butanol portion extract is not obvious to the inhibitory action of normal liver cell line BNL-CL2 growth, and suppression ratio is only 22.16% in the time that maximum concentration reaches 500 μ g/ml.
The apoptotic morphological characteristic of embodiment 8 fluorescence microscope Herba pterocephali n-butanol portion extract inductor Hep3B
8.1 experiment materials:
PBS buffer (Hyclone, Thermo company, lot number: SH30256.01B), Hoechst33258 dye liquor (Sigma company product, lot number: 861405), keep in Dark Place.4% paraformaldehyde (Chemical Reagent Co., Ltd., Sinopharm Group, lot number: 20120514).Fluorescence microscope (Japanese OLYMPUS company, model: IX71)
8.2 experimental techniques:
A) the learnt from else's experience cell of Herba pterocephali n-butanol portion extract effect 48h of variable concentrations, adds the fixing 30min of 4% paraformaldehyde;
B), after PBS washed cell, (every hole 30 μ l), act on 10min to add Hoechst33258 dye liquor;
C) PBS washing 3 times, each 5min, adds anti-quencher mounting;
D) micro-Microscopic observation, the results are shown in accompanying drawing 5.
8.3 experimental results:
Cell after Hoechst dyeing, cellular control unit nuclear dna distributes relatively even, and core is circle or oval, without pyknosis, is faint blueness in the visual field; And medication group part cell generation apoptosis, nucleus color depth, is fine and close dense dying, karyopycnosis, distortion.Compared with matched group, the middle and high concentration group of extract apoptotic cells increased.
The apoptotic morphological characteristic of embodiment 9 transmission electron microscope observing Herba pterocephali n-butanol portion extract inductor Hep3B
9.1 experiment materials:
Transmission electron microscope (Dutch PHILIPS Co., model: Philips Tecnai-12 type), Hep3B cell, glutaraldehyde (Shanghai Univ. of Traditional Chinese Medicine's experimental center for scientific technology Electron Microscopy Room provides)
9.2 experimental techniques:
A) the learnt from else's experience cell of variable concentrations Herba pterocephali n-butanol portion extract effect 48h, digestion, adds glutaraldehyde after centrifugal and fixes;
B) send Shanghai Univ. of Traditional Chinese Medicine's experimental center for scientific technology Electron Microscopy Room embedding, section
C) upper machine is observed, and result is as accompanying drawing 6
9.3 experimental results:
Matched group Hep3B cell volume is larger, and cell membrane is complete, and in core, chromatin is abundant, and kernel is obvious, and organelle is complete, sees Fig. 6 a; And after Herba pterocephali n-butanol portion extract-treated 48h, visible cell volume-diminished, core has pyconsis, chromatic agglutination in core, chromatin is unevenly along assembling under nuclear membrane, reticulum dilatation, cavity formation increases, and there is (seeing Fig. 6 b, 6c) in the apoptosis features such as organelle swelling.
The relation of embodiment 10 Herba pterocephali n-butanol portion extract cell death inducings and PTEN/PI3K/AKT signal path
10.1 experiment material:
Electrophresis apparatus (BIO-RAD company of the U.S., type 552BR102298), transferring film instrument (BIO-RAD company of the U.S., model: 153BR81259), multi-functional decolorization swinging table (Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd., model: ZHWY-344), constant-temperature metal bath (Hangzhou BIOER Technology Co., Ltd, model: CHB-100), gel imaging system (BIO-RAD company of the U.S., model: GelDoc XR), ECL luminescent solution (Thermo company, lot number: 32106), PTEN, PI3K, AKT and p-PTEN, p-PI3K, p-AKT antibody (Rabbit mAb) is Cell Signaling Tehnology company product, albumen Maker (U.S. Fermentas product, lot number: SM1811), other experiment reagents are all purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
10.2 experimental technique:
Collect respectively the Hep3B cell through variable concentrations Herba pterocephali n-butanol portion extract-treated 48h, culture fluid is inhaled and abandoned, clean 2 times with the PBS of pre-cooling, add 200 μ L cell pyrolysis liquid RIPA, ice bath cracking 20min, 14000r/min, 4 DEG C of centrifugal 20min, get supernatant, carry out protein quantification by BCA method, supernatant is placed in-20 DEG C of preservations after packing.(30 μ g) carry out SDS-PAGE electrophoretic separation to get equal protein, first at constant voltage 100V electrophoresis 20min 150V electrophoresis 60min subsequently, then electric transferring film 90min under the condition of 250mA, by protein delivery to pvdf membrane, 5% defatted milk powder room temperature sealing 1h, add respectively 4 DEG C of corresponding primary antibodies (dilution factor of internal reference GAPDH is 1:5000, and the dilution factor of primary antibodie is 1:1000) to spend the night; Add two anti-(1:5000 dilution) room temperature 2h of corresponding HRP labelling, the colour developing of ECL luminescence method, experiment repeats 3 times under the same conditions.
10.3 experimental results are shown in accompanying drawing 7
7 demonstration can be found out with reference to the accompanying drawings: with matched group comparison, PTEN and p-PTEN express to be increased, and increases and present ascendant trend with drug level; Akt, p-Akt, PI3K, p-PI3K expresses to some extent and declines, and increases to express gradually to decline with drug level.Illustrate on protein level, to show variantly through the Hep3B cell of Herba pterocephali n-butanol portion extract effect, work by PTEN/PI3K/Akt signal path.
The two methods of dying of embodiment 11 flow cytometer Annexin-V/PI detect Herba pterocephali n-butanol portion extract to the apoptotic impact of Hep3B
11.1 experiment material:
The high sugared culture fluid (Hyclone of Hep3B cell DMEM, Thermo company of the U.S., lot number: SH30243.01) hyclone (Gibco, Invitrogen company, lot number: 100099-141) 0.25% trypsin, not containing EDTA, (Chemical Reagent Co., Ltd., Sinopharm Group, lot number: 20130103) 6 well culture plates (Corning company of the U.S., model: 3516), 15ml centrifuge tube (being Corning company of U.S. product), AnnexiV/Fitc-PI test kit (U.S. company BD, lot number :), flow cytometer (U.S. company BD, model: FACSCalibur), streaming pipe (U.S. company BD, model: 352052)
11.2 experimental technique:
1) plant plate (6 orifice plates, Hep3B cell 2*10
5/ hole, bed board dosing next day, action time 48h)
2) collecting cell: the Medium in 6 orifice plates is sucked respectively in corresponding 15ml centrifuge tube; Add respectively 1mlPBS, swing gently and wash cell; Peptic cell; Centrifugal: 1000rpm, 5min; According to the operation of test kit description, add 100 μ l/ pipe binding buffer, re-suspended cell, adds respectively 5 μ l Annexin and PI, and lucifuge is hatched 30min, adds 400 μ l/ pipe binding buffer, upper machine testing in 1h.
11.3 experimental results: see accompanying drawing 8
8 demonstration can be found out with reference to the accompanying drawings: as figure shows: with matched group comparison, apoptosis-induced relevant PTEN and p-PTEN express to be increased, and increases and present ascendant trend with drug level; Apoptosis inhibit protein A kt, p-Akt, PI3K, p-PI3K expresses to some extent and declines, and increases to express gradually to decline with drug level.Illustrate on protein level, to show variantly through the Hep3B cell of Herba pterocephali n-butanol portion extract effect, work by PTEN/PI3K/Akt signal path.
Embodiment 12 Herba pterocephali n-butanol portion extract anti-tumor in vivo effect researchs
12.1 experiment material:
12.1.1 instrument, reagent: mouse stomach pin, 1ml syringe, electronic balance (above Industrial Co., Ltd. of Nereid section, YP type), slide gauge.Sodium carboxymethyl cellulose carboxymethylcellulose sodium (Chemical Reagent Co., Ltd., Sinopharm Group, lot number: 20130113) Fluorouracil Injection (Shanghai Xudong Hipu Medicine Co., Ltd, lot number: FA1209012)
12.1.2 animal: BALB/c-nu nude mice, body weight 16-18g, 5 week age, male, laboratory animal production licence: SCXK (Shanghai) 2007-0005 is provided by Shanghai Slac Experimental Animal Co., Ltd.; Raise in Shanghai Univ. of Traditional Chinese Medicine's zoopery center no-special pathogen (SPF) environment.Temperature: 20-25 DEG C, humidity 40-70%.
12.1.3 tumor kind: Hep3B cell strain
12.2 experimental technique:
12.2.1 set up the subcutaneous inhibition tumor of hepatocarcinoma Hep3B model:
12.2.1.1 tumor cultured cell is collected the 5min after trypsinization for cell in the Hep3B of exponential phase, washes 2 times with PBS, and adjusting concentration of cell suspension is 1 × 10
7individual/mL.
12.2.1.2 the inoculating cell of tumor cultured cell is counted as 1 × 10
7individual/mL, every right axillary fossa subcutaneous vaccination 2 × 10 of nude mice
6individual cell (0.2mL/ only).After 2-3 days, observe the formational situation of transplanted tumor in nude mice, (computing formula of gross tumor volume TV is: TV=1/2*a*b to measure tumor size
2, wherein a, b represent respectively the length and width of tumor), from inoculate successful transplanted tumor nude mice, select tumor size more consistent, gross tumor volume reaches 60～70mm
330 of left and right for experimentation.
12.2.2 gross tumor volume size random packet, 6 every group, totally 30 are pressed in experiment grouping.
Blank group: give 0.5% Sodium Tvlose (CMC-Na) solution by 0.1mL/10g body weight, gavage, continuously 21d.
The each dosage group of extract: press 0.1mL/10g dosage, gavage, continuously 21d.
Positive drug 5-FU group ((fluorouracil group): 20mg/kg time, 5-FU0.2mL/d, lumbar injection, every other day once, is used in conjunction 21d.
The each dosage group of extract---according to the each dosage group of preparation method gained oral liquid of the present invention (low dosage 100 μ g/ml, middle dosage 300 μ g/ml, high dose 1000 μ g/ml), last administration is weighed next day, put to death animal, strip tumor, spleen and weigh.
12.2.3 observation index: 1) observe ordinary circumstance and the body weight change of mice every day, used 1 nude mice tumor volume of vernier caliper measurement every 1 day.The computing formula of gross tumor volume (T V) is: T V=1/2*a*b
2, wherein a, b represent respectively the length and width of tumor.2) calculate each group of tumor and weigh and calculate tumour inhibiting rate: the heavy suppression ratio of tumor (%)=(1-T/C) × 100%, wherein C is the average tumor weight of matched group, T is the average tumor weight of each test group.3) index and spleen index: SI=spleen weight (mg)/10g body weight * 100%.
12.3 experimental results are shown in accompanying drawing 9.
9 demonstration can be found out with reference to the accompanying drawings: with the comparison of 0.5%CMC-Na (hydroxy methocel) matched group, the each dosage group of extract can reduce the tumor weight of nude mice; Each treated animal body weight also has statistical significance; Aspect adjusting immunologic mechanism, extract is less on the impact of index and spleen index (SI), not statistically significant; And positive control drug fluorouracil has statistical significance to index and spleen index impact, therefore think that the toxic and side effects of this extract organizes little compared with chemotherapeutic 5-FU (fluorouracil).
Persons of ordinary skill in the art may appreciate that the respective embodiments described above are to realize specific embodiments of the invention, and in actual applications, can do various changes to it in the form and details, and without departing from the spirit and scope of the present invention.
Claims (9)
1. a Herba pterocephali n-butanol portion extract, is characterized in that, the main component of described extract is iridoid glycoside.
2. the preparation method of the Herba pterocephali n-butanol portion extract of claim 1, is characterized in that comprising following steps:
(1) get Herba pterocephali medical material, under reflux condition, carry out alcohol extraction, by extracting solution reclaim under reduced pressure alcohol, the concentrated one-level extract that obtains;
(2) above-mentioned one-level extract is added water suspendible, uses lipotropy organic solvent extraction, to remove oil-soluble impurities; And then with n-butanol extraction, after recovery solvent, obtain described Herba pterocephali n-butanol portion extract.
3. preparation method according to claim 2, is characterized in that, the alcohol extraction extraction solvent used in step (1) is that methanol, ethanol, volumetric concentration are that more than 50% methanol aqueous solution or volumetric concentration is more than 50% ethanol water.
4. preparation method according to claim 2, is characterized in that, the consumption of alcohol extraction in step (1) extraction solvent used is 2～10 times of Herba pterocephali quality of medicinal material.
5. preparation method according to claim 2, is characterized in that, in step (1), the temperature of reflux is 50～100 DEG C, and the number of times of heating and refluxing extraction is 1～5 time, each reflux 0.5～2 hour.
6. preparation method according to claim 2, is characterized in that, the lipotropy organic solvent described in step (2) is petroleum ether, cyclohexane extraction, normal hexane, benzene, carbon tetrachloride, chloroform or dichloromethane.
7. preparation method according to claim 2, is characterized in that, in step (2), the number of times of lipotropy organic solvent extraction is 1～5 time, and the solvent load of each extraction is 1～5 times of extract volume.
8. preparation method according to claim 2, is characterized in that, in step (2), the number of times of n-butanol extraction is 1～5 time, and the consumption that at every turn adds n-butyl alcohol is 1～5 times of extract volume.
9. the Herba pterocephali n-butanol portion extract of claim 1 is in the purposes of preparing in antitumor drug.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410217723.7A CN103977044B (en) | 2014-05-20 | 2014-05-20 | Hooker winghead root n-butanol portion extract and its production and use |
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| CN201410217723.7A CN103977044B (en) | 2014-05-20 | 2014-05-20 | Hooker winghead root n-butanol portion extract and its production and use |
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| CN103977044B CN103977044B (en) | 2017-11-14 |
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| CN201410217723.7A Expired - Fee Related CN103977044B (en) | 2014-05-20 | 2014-05-20 | Hooker winghead root n-butanol portion extract and its production and use |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885385A (en) * | 2019-11-19 | 2020-03-17 | 西南大学 | Pterocephalus hookeri toxin A, application thereof and preparation method of pterocephalus hookeri extract with low liver injury toxicity |
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| JP2004155677A (en) * | 2002-11-05 | 2004-06-03 | Noevir Co Ltd | Moisture-retaining skin care preparation for external use and chapped skin-improving skin care preparation for external use |
| CN102526141A (en) * | 2011-03-09 | 2012-07-04 | 成都中医药大学 | Pterocephalus hookeri heck total iridoid glycoside extract and preparation method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004155677A (en) * | 2002-11-05 | 2004-06-03 | Noevir Co Ltd | Moisture-retaining skin care preparation for external use and chapped skin-improving skin care preparation for external use |
| CN102526141A (en) * | 2011-03-09 | 2012-07-04 | 成都中医药大学 | Pterocephalus hookeri heck total iridoid glycoside extract and preparation method and application thereof |
Non-Patent Citations (2)
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| 董娟娥等: "植物中环烯醚萜类化合物研究进展", 《西北林学院学报》 * |
| 雷旭东等: "翼首草总皂苷对体外培养的肿瘤细胞增殖的影响", 《时珍国医国药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885385A (en) * | 2019-11-19 | 2020-03-17 | 西南大学 | Pterocephalus hookeri toxin A, application thereof and preparation method of pterocephalus hookeri extract with low liver injury toxicity |
| CN110885385B (en) * | 2019-11-19 | 2020-09-25 | 西南大学 | Pterocephalus hookeri toxin A, application thereof and preparation method of pterocephalus hookeri extract with low liver injury toxicity |
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| CN103977044B (en) | 2017-11-14 |
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