CN105796825A - Application of yiganle tablets in preparing drugs for inhibiting proliferation of gastric adenocarcinoma cell SGC-7901 - Google Patents

Application of yiganle tablets in preparing drugs for inhibiting proliferation of gastric adenocarcinoma cell SGC-7901 Download PDF

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CN105796825A
CN105796825A CN201610187708.1A CN201610187708A CN105796825A CN 105796825 A CN105796825 A CN 105796825A CN 201610187708 A CN201610187708 A CN 201610187708A CN 105796825 A CN105796825 A CN 105796825A
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herba
diisopropylamine dichloroacetate
yiganle
tablets
supercritical
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不公告发明人
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Jinan Xinshidai Medicine Science and Technology Co Ltd
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Jinan Xinshidai Medicine Science and Technology Co Ltd
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    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction

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Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to the application of yiganle tablets in preparing drugs for inhibiting proliferation of the gastric adenocarcinoma cell SGC-7901 and a preparing method of the yiganle tablets.The yiganle tablets are prepared from 200 g of Chinese fevervine herb, 160 g of rhizoma polygoni cuspidate, 200 g of herba verbenae, 200 g of junesnow, 200 g of Chinese wedelia herb, 200 g of Japanese raspberry herb, 170 g of pink reineckea herb, 100 g of Chinese mahonia stem and 70 g of clear weed.The yiganle tablets are prepared through supercritical extraction, so that the content of emodin is increased greatly.

Description

Preferably diisopropylamine dichloroacetate sheet is in preparation suppression gastric adenocarcinoma cells SGC-7901 cell proliferation Application
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preferably diisopropylamine dichloroacetate sheet at preparation suppression gastric adenocarcinoma cells SGC- Application in 7901 cell proliferation and the preparation method of suitable diisopropylamine dichloroacetate sheet.
Background technology
Preferably Ganle Granules standard No. WS-10148(ZD-0148)-2002, it is recorded in country standard for traditional Chinese medicines compilation internal medicine liver Gallbladder fascicle.By fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g make as crude drug, have heat-clearing and toxic substances removing, effect of promoting the function of the gallbladder to alleviate jaundice.For liver and gall Damp and hot caused acute or chronic hepatitis B.
In prior art, not yet there is suitable diisopropylamine dichloroacetate sheet in preparation suppression human gastric adenocarcinoma SGC-7901 cell proliferation In the report of application, also there are no suitable diisopropylamine dichloroacetate sheet and extract preparation aspect and use the report of supercritical extraction, and traditional water decoction is boiled, The method of alcohol reflux, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, it has not been convenient to take, have a strong impact on This product is applied clinically.
Summary of the invention
Goal of the invention: it is an object of the invention to provide a kind of preferably diisopropylamine dichloroacetate sheet at preparation suppression gastric adenocarcinoma cells SGC-7901 Application in cell proliferation.
Further object is that the preparation method that a kind of suitable diisopropylamine dichloroacetate sheet is provided.
It is an object of the invention to by following scheme realization:
Preferably diisopropylamine dichloroacetate sheet application in preparation suppression gastric adenocarcinoma cells SGC-7901 cell proliferation, described suitable diisopropylamine dichloroacetate sheet is by chicken Vow rattan 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g make as crude drug, the preparation method of described suitable diisopropylamine dichloroacetate sheet comprise the steps: to take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, cold water Flower 70g, joins CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for the percent by volume of total extractant and is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 700-800min, Obtain supercritical extract, supercritical extract is added starch, 70% ethanol granule, it is dried, tabletting, makes 200, every Weight 0.30g.
Preferably, above-mentioned suitable diisopropylamine dichloroacetate sheet answering in preparation suppression gastric adenocarcinoma cells SGC-7901 cell proliferation With, the preparation method of described suitable diisopropylamine dichloroacetate sheet comprises the steps: to take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2Supercritical extraction In device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, is added by supercritical extract and forms sediment Powder, 70% ethanol granule, it is dried, tabletting, makes 200, every tablet weight 0.30g.
In prior art, suitable Ganle Granules is administered orally, a 10g, 3-4 time on the one.Preferably Ganle Granules dosage is big.Use this Suitable every tablet weight 0.30g of diisopropylamine dichloroacetate sheet that inventive method is prepared as, the most only needs 2, within 1st, takes 3-4 time.There is more activity Dose is greatly reduced under conditions of composition.This conclusion can be proved by following test.
The comparison of emodin content in suitable diisopropylamine dichloroacetate sheet prepared by test one, distinct methods
L, instrument and the reagent present invention suitable diisopropylamine dichloroacetate sheet: prepare by embodiment 1 method, use 1500g crude drug, extracted make 200, every tablet weight 0.30g.Former suitable Ganle Granules, according to WS-10148(ZD-0148) prepared by-2002 standard methods. Agilent1200 high performance liquid chromatograph；METTLERAE240 electronic analytical balance；(China's medicine is biological for rheum emodin reference substance Goods examine and determine institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia the 4th note on the use 15 of version in 2015).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；Methanol-0.1% phosphoric acid Solution (80: 20) is flowing phase；Detection wavelength is 437nm.Number of theoretical plate is calculated by rheum emodin peak should be not less than 4000.
It is appropriate that the preparation precision of reference substance solution weighs rheum emodin reference substance, adds methanol and makes molten containing 50 g of every 1ml Liquid, to obtain final product.
The preparation of the need testing solution of the suitable diisopropylamine dichloroacetate sheet of the present invention takes the suitable diisopropylamine dichloroacetate sheet 5 of the present invention, finely ground, takes 0.12g, accurately weighed, to put in tool plug conical flask, add dehydrated alcohol 20ml, supersound process (power 250W, frequency 25kHZ) 1 is little Time, let cool, filter, medicinal residues absolute ethanol washing 4 times, each 10ml, merging filtrate and cleaning mixture, it is evaporated, residue adds dilute salt Acid 10ml makes dissolving, adds chloroform 20ml, is heated to reflux 30 minutes, lets cool, move in separatory funnel, holds with a small amount of chloroform Device, is incorporated in separatory funnel；Dividing and take chloroform solution, acid solution adds chloroform 2 times, each 10ml, and combined chloroform liquid is evaporated, residue Accurate addition methanol 10ml, weighed weight, slight fever makes dissolving, lets cool, more weighed weight, adds methanol and supplies the weight of less loss, shakes Even, filter, take subsequent filtrate, to obtain final product.
The preparation of reference product need testing solution takes the suitable Ganle Granules 20g of this product comparison, finely ground, takes 4g, accurate title Fixed, put in tool plug conical flask, add dehydrated alcohol 20ml, supersound process (power 250W, frequency 25kHZ) 1 hour, let cool, filter, Medicinal residues absolute ethanol washing 4 times, each 10ml, merging filtrate and cleaning mixture, it is evaporated, residue adds dilute hydrochloric acid 10ml makes dissolving, Add chloroform 20ml, be heated to reflux 30 minutes, let cool, move in separatory funnel, with a small amount of chloroform container, be incorporated to separatory funnel In；Dividing and take chloroform solution, acid solution adds chloroform 2 times, each 10ml, and combined chloroform liquid is evaporated, and residue precision adds methanol 10ml, weighed weight, slight fever makes dissolving, lets cool, more weighed weight, adds methanol and supplies the weight of less loss, shakes up, and filters, takes continuous Filtrate, to obtain final product.
Algoscopy precision respectively draws reference substance solution and each 5 l of need testing solution, injects chromatograph of liquid, measures, to obtain final product.
3, result
Result shows, in the present invention suitable diisopropylamine dichloroacetate sheet, the content of rheum emodin is 0.95-1.86mg/ sheet；And it is big in former suitable Ganle Granules The content of flavin is 0.91mg/ bag (every bag of 10g Han granule), and the emodin content each serving consumption 2 is former granule 10g 2-4 times of content, in the case of dose reduces, emodin content improves a lot.
The studies above shows, uses suitable diisopropylamine dichloroacetate sheet prepared by preparation method of the present invention, and active constituent content is significantly larger than WS- 10148(ZD-0148) suitable Ganle Granules prepared by the method that-2002 standards are recorded.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, in order to those skilled in the art knows more about The present invention, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below, all above-mentioned based on the present invention The technology that content is realized belongs to the scope of the present invention.
Embodiment 1
Take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for total extraction The percent by volume taking solvent is 5%, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, is dried, tabletting, makes 200 Sheet, every tablet weight 0.30g.
After testing, in finished product, the content of rheum emodin is 1.85mg/ sheet.
Embodiment 2
Take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for total extraction The percent by volume taking solvent is 4%, extracting pressure 15MPa, temperature 30 DEG C, CO2Flow 1ml/g crude drug min, extraction time 900min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, is dried, tabletting, makes 200 Sheet, every tablet weight 0.30g.
After testing, in finished product, the content of rheum emodin is 1.21mg/ sheet.
Embodiment 3
Take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for total extraction The percent by volume taking solvent is 6%, extracting pressure 30MPa, temperature 60 C, CO2Flow 3ml/g crude drug min, extraction time 800min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, is dried, tabletting, makes 200 Sheet, every tablet weight 0.30g.
After testing, in finished product, the content of rheum emodin is 0.95mg/ sheet.
Embodiment 4: the preferably experimentation data of diisopropylamine dichloroacetate sheet suppression human gastric adenocarcinoma SGC-7901 cell proliferation
1. experiment material
1.1 experiment cell strains
Human gastric adenocarcinoma SGC-7901 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention suitable diisopropylamine dichloroacetate sheet: prepare by embodiment 1 method.
Medicinal liquid liquid storage: weigh 100mg suitable diisopropylamine dichloroacetate sheet, be dissolved in 5ml dehydrated alcohol, 0.2 m filter filters, 500 ldoff Pipe subpackage ,-20 DEG C of storages, 0.2 m filter filters dehydrated alcohol in case matched group is used simultaneously.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061Lot.No.758137)；(Hangzhoupro, sky, Zhejiang biotechnology has hyclone Limit company Lot.No.100419)；NaHC03(the long hundred million chemical reagent company limited Cat.No.11810-in Shanghai 033Lot.No.1088387)；Trypsin(AMRESCO company)；EDTA(AMRESCO company)； PenicillinGSodiumSalt(AMRESCO company 1)；StreptomycinSulfate(AMRESCO)；Dehydrated alcohol is (black Bo Yadulan Trade Co., Ltd.)；MTT (Biosharp lot number: 0793): PBS(laboratory autogamy)；
1.4 experiment equipment
Lycra inverted microscope (Germany Leica model: DMIL)；Visible-ultraviolet light microwell plate detector (MD company type of the U.S. Number: SPECTRAMAX190)；C02Incubator (FORMA model: 3111)；Super-clean bench (safe and sound company of Su Jing group manufacture model: SW-CJ-ZFD)；Pure water instrument (Sprlng company of U.S. model: S/N020579)；Accurate pipettor (Gilson Inc of France type Number: P2)；Electronic balance (Sai Duolisi company limited of Germany model: BT323S)；(Japan SANYO is public for full-automatic high-pressure autoclave Department model: MLS-3020)；Table electrothermal air dry oven (Shanghai precision experimental facilities company model: DHG9123A)；Refrigerator (Siemens Company's model: KG18V21TI)；Liquid nitrogen container (CBS model: 2001)；Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai Model: KA-1000)；0.2 m filter (MILLIPORE model: SLGP033RB)；1cm culture dish (NEST company), 96 holes are cultivated Plate (NEST company)；Cell counting count board；Centrifuge tube, pipet, Tips are some.
2. experimental technique
1) SGC-7901 cell DMEM+10%FBS is in 37 DEG C, 5%C02Carry out cellar culture (10cm culture dish), when cell is raw When length is to logarithmic (log) phase, collecting cell, discard culture fluid, PBS rinses 3 times, addition 3ml0.25% trypsin-0.04%EDTA, and 37 After DEG C digestion 2min, it is added thereto in 5ml complete medium and react, blowing and beating after cell and proceeded in centrifuge tube, 1000rpm is centrifuged 5min, adjusts concentration of cell suspension 3 × 104Individual/ml.
2) entering in 96 well culture plates by cell kind, every hole adds cell suspension 180 l, and culture plate is put in cell culture incubator (37 DEG C, 5%C02) cellar culture.
3) according to cell growth status, general long to 50%-70%, add suitable diisopropylamine dichloroacetate sheet solution, continue to cultivate 24h.
4) add 20 lMTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.
5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole adds 200 l dimethyl sulfoxide, puts shaking table Upper low-speed oscillation 10min, makes crystal fully dissolve.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm.
6) arranging background (being not added with cell, only add culture fluid), (cell, the medicine dissolution of same concentrations are situated between control wells simultaneously Matter, culture fluid, MTT, dimethyl sulfoxide), often group sets 6 multiple holes.
7) suppression ratio of cell is represented by result with medicine: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD Value), control wells OD value × 100%.Experiment is repeated 3 times.
3. statistical disposition
Using the correlation analysis in MicrosoftExcel2007 software and Studentt inspection, data are with mean ± S.D. table Show.
4. experimental result
Statistical result showed after mtt assay experiment, compares with matched group, when dosage reaches 5mg/ml, increases SGC-7901 cell Growing suppression variant (P < 0.05), dosage this difference when 10mg/ml has significance (P < 0.01), when dosage reaches 15- Pole significant difference (P < 0.001) is had during 20mg/ml.
The suitable diisopropylamine dichloroacetate sheet of table 1 is to SGC-7901 cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 14.16±5.03
2 10 27.35±6.98*
3 15 39.25±10.16**
4 20 48.88±13.54**
Note: compare with matched group, * P < 0.01；**P<0.001.
5. experiment conclusion
The suitable diisopropylamine dichloroacetate sheet of the present invention can suppress SGC-7901 cell proliferation, reduces the cell growing number of SGC-7901 cell, This effect is dose dependent.

Claims (2)

1. preferably diisopropylamine dichloroacetate sheet application in preparation suppression gastric adenocarcinoma cells SGC-7901 cell proliferation, described suitable diisopropylamine dichloroacetate sheet by Fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, contribution Wood 100g, Rhizoma Belamcandae 70g make as crude drug, it is characterised in that the preparation method of described suitable diisopropylamine dichloroacetate sheet comprises the steps: Take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, merit Labor wood 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for total extractant Percent by volume be 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction Time 700-800min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, is dried, tabletting, Make 200, every tablet weight 0.30g.
2. according to the suitable diisopropylamine dichloroacetate sheet described in claim l in preparation suppression gastric adenocarcinoma cells SGC-7901 cell proliferation Application, it is characterised in that the preparation method of described suitable diisopropylamine dichloroacetate sheet comprises the steps: to take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, horsewhip Grass 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, is extracted by supercritical Take thing and add starch, 70% ethanol granule, be dried, tabletting, make 200, every tablet weight 0.30g.
CN201610187708.1A 2016-03-29 2016-03-29 Application of yiganle tablets in preparing drugs for inhibiting proliferation of gastric adenocarcinoma cell SGC-7901 Withdrawn CN105796825A (en)

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