Summary of the invention
Goal of the invention: in order to address the above problem, the object of the present invention is to provide a kind of preparation method of strong liver sheet.
Another object of the present invention is to provide a kind of strong liver sheet to suppress the application in B16 mouse melanoma cell line cell proliferation medicine in preparation.
Technical scheme: the object of the invention is to realize by following scheme:
A kind of preparation method of strong liver sheet, by Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, as crude drug, made, described method is comprised of the following step: get Herba Artemisiae Scopariae, join in CO2 supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 ℃, CO
2flow 1-3m1/g crude drug min, extraction time 150-180min, obtains supercritical extract, standby; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 400-600W, extracts 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on polyamide chinlon 66 chromatographic columns, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
The preparation method of above-mentioned a kind of strong liver sheet, described CO
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
The preparation method of above-mentioned a kind of strong liver sheet, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of above-mentioned a kind of strong liver sheet, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned strong liver sheet suppresses the application in B16 cell proliferation medicine in preparation.
In prior art, strong every 0.55g of liver sheet, each 8-10 sheet, 2 times on the one, the every 0.5g of strong liver sheet that adopts the present invention to be prepared into only needs 3 at every turn, within 1st, takes 2 times, has greatly reduced dose having under the condition of more active component.This conclusion can be by following evidence.
The comparison of TANSHINONES content in strong liver sheet prepared by test one, distinct methods
1, the strong liver sheet of instrument and reagent the present invention: press embodiment 3 method preparations, use 690g crude drug, make 1500 through extracting, every heavy 0.5g.Former strong liver sheet, by the preparation of ministry standard method, is used 690g crude drug, through extracting, makes 1652, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; METTLERAE240 electronic analytical balance; TANSHINONES reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica, be filler; Methanol-water-phosphoric acid (40:60:0.2) is mobile phase; Detection wavelength is 280nm.Number of theoretical plate calculates by TANSHINONES peak, should be not less than 3000.
The preparation of reference substance solution: precision takes at 60 ℃ of drying under reduced pressure TANSHINONES reference substance of 4 hours appropriate, adds methanol and makes every 1ml containing the solution of 18 μ g, obtains.
The preparation of need testing solution: get the strong liver sheet of the present invention and former strong liver sheet, porphyrize, mixes, and gets 1g, accurately weighed, and precision adds 70% ethanol 20ml, close plug, supersound process 10 minutes, centrifugal, get supernatant, obtain.
Algoscopy is accurate reference substance solution and each 20 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
3, result
Result shows, in the strong liver sheet of the present invention, the content of TANSHINONES is 1.43mg/ sheet; And the content of TANSHINONES is 0.24mg/ sheet in former strong liver sheet, in the situation that dose reduces, TANSHINONES content improves a lot.
Above-mentioned research shows, the strong liver sheet that adopts the present invention to prepare, and active constituent content is higher than the standby strong liver sheet of ministry standard legal system.
The specific embodiment
Form by the following examples, foregoing of the present invention is described in further detail again, but this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example, all technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, by Herba Artemisiae Scopariae, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4%, extracting pressure 15MPa, 30 ℃ of temperature, CO
2flow 1m1/g crude drug min, extraction time 150min, obtains supercritical extract, standby; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 400W, extracts 2 times, each 4 minutes, combining extraction liquid, concentrated, be added on polyamide chinlon 66 chromatographic columns, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of TANSHINONES is 1.48mg/ sheet.
Embodiment 2
Get Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, by Herba Artemisiae Scopariae, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 6%, extracting pressure 30MPa, temperature 50 C, CO
2flow 3m1/g crude drug min, extraction time 180min, obtains supercritical extract, standby; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on polyamide chinlon 66 chromatographic columns, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of TANSHINONES is 1.53mg/ sheet.
Embodiment 3
Get Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, by Herba Artemisiae Scopariae, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 20MPa, 40 ℃ of temperature, CO
2flow 2m1/g crude drug min, extraction time 160min, obtains supercritical extract, standby; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 500W, extracts 2 times, each 6 minutes, combining extraction liquid, concentrated, be added on polyamide chinlon 66 chromatographic columns, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of TANSHINONES is 1.43mg/ sheet.
Embodiment 4: strong liver sheet suppresses the experimentation data of B16 cell proliferation
1 experiment material
1.1 experiment cell strains
Mouse melanin tumor cell (B16), Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention is good for liver sheet: press embodiment 3 method preparations.
Medicinal liquid liquid storage: take the strong liver sheet of 100mg, be dissolved in 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ l doff pipe subpackages ,-20 ℃ of storages, 0.2 μ m filter filters dehydrated alcohol in order to the use of matched group simultaneously.
1.3 experiment reagent
The Cat.No.12100-061Lot.No.758137 of DMEM(GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); The NaHCO3(Shanghai Jiu Yi chemical reagent Cat.No.11810-033Lot.No.1088387 of company limited); Trypsin(AMRESCO company lot number: 2010/04); EDTA(AMRESCO company lot number: 2009/10); Penicillin G Sodium Salt(AMRESCO company lot number: 2010242); Streptomycin Sulfate(AMRESCO company lot number: 2010382); Dehydrated alcohol (Nanjing Chemistry Reagent Co., Ltd.'s lot number: 080310182); MTT (Biosharp lot number: 0793); The autogamy of PBS(laboratory);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DM1L); Visible-ultraviolet light microwell plate detector (U.S. MD company model: SPECTRAMAX 190); CO2 incubator (FORMA model: 3111); (safe and sound company of Su Jing group manufactures model to super-clean bench: SW-CJ-ZFD); Pure water instrument (U.S. Spring company model: S/N 020579); Accurate pipettor (French Gilson Inc model: P2); Electronic balance (German Sai Duolisi company limited model: BT323S); Full-automatic high-pressure autoclave (Japanese SANYO company model: MLS-3020); Table electrothermal air dry oven (Shanghai accurate 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's model: KA-1000); 0.2 μ m filter (MILLIPORE model: SLGP033RB); 10cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, pipet, Tips are some.
2 experimental techniques
1) B16 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%CO2, when Growth of Cells is during to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% trypsin-0.04%EDTA, after 37 ℃ of digestion 2min, add wherein 5ml complete medium neutralization reaction, after piping and druming cell, proceeded in centrifuge tube, the centrifugal 5min of 1000rpm, adjusts 3 * 104/ml of concentration of cell suspension.
2) cell kind is entered in 96 well culture plates, every hole adds cell suspension 180 μ l, culture plate put into cell culture incubator (37 ℃, 5%CO2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add strong liver sheet solution, continue to cultivate 24h.
4) after 24h, add 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT), continue to cultivate 4h.
5) after 4h, buckle method is removed supernatant, with absorbent paper, pats dry gently, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, and crystal is fully dissolved.At enzyme-linked immunosorbent assay instrument 490nm place, measure the light absorption value in each hole.
6) background (do not add cell, only add culture fluid) is set simultaneously, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide), sets 6 multiple holes for every group.
7) result represents the suppression ratio of cell with medicine:
Cell increment suppression ratio (%)=(control wells OD value-dosing holes OD value)/control wells OD value * 100%.Experiment repeats 3 times.
3 statistical dispositions
Adopt correlation analysis and Student t check in Microsoft Excel 2003 softwares, data represent with mean ± S.D..
4 experimental results
Statistical result showed after mtt assay experiment, with matched group comparison, when dosage reaches 5mg/ml, to B16 cell inhibitory effect variant (P<0.05), dosage this difference when 10mg/ml has significance (P<0.01), has utmost point significant difference (P<0.001) when dosage reaches 15-20mg/ml.
The strong liver sheet of table 1 is on B16 cell inhibitory effect impact research
Note: with matched group comparison, * P<0.01; * P<0.001
5 experiment conclusion
Strong liver sheet can suppress B16 cell proliferation, reduces the Growth of Cells number of B16 cell, and this effect is dose dependent.