CN102988691A - Preparation method and application of liver-strengthening tablet - Google Patents
Preparation method and application of liver-strengthening tablet Download PDFInfo
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- CN102988691A CN102988691A CN2012103897467A CN201210389746A CN102988691A CN 102988691 A CN102988691 A CN 102988691A CN 2012103897467 A CN2012103897467 A CN 2012103897467A CN 201210389746 A CN201210389746 A CN 201210389746A CN 102988691 A CN102988691 A CN 102988691A
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Abstract
The invention provides a preparation method of a liver-strengthening tablet. The liver-strengthening tablet is prepared by using 100g of daylily roots, 150g of isatis roots, 250g of oriental wormwoods, 150g of danshen roots and 100g of Chinese dates as bulk drugs and adopting supercritical extraction and microwave-assisted extraction modes. By adopting the method, the ketone content of the danshen root is greatly improved; and the invention also provides application of the liver-strengthening tablet in preparation of drugs for inhibiting cell proliferation of mice melanoma cells B16.
Description
Technical field
The present invention relates to the Chinese medicine preparation technical field, be specifically related to a kind of preparation method and application of strong liver sheet.
Background technology
Strong liver sheet is recorded in Ministry of Public Health standard WS3-B-0376-90, is made as crude drug by Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, can clearing away heat-damp and promoting diuresis.Be used for acute hepatitis.
In the prior art, not yet there is strong liver sheet to adopt the report of supercritical and microwave technology aspect the preparation extracting, and adopts the method that powder and decocting boil of beating, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, be inconvenient to take, had a strong impact on this product and used clinically.
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 the application of a kind of strong liver sheet in preparation inhibition B16 mouse melanoma cell line cell proliferation medicine.
Technical scheme: the objective of the invention is to realize by following scheme:
A kind of preparation method of strong liver sheet, made as crude drug by Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, described method is comprised of the following step: get Herba Artemisiae Scopariae, join in the 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 gets supercritical extract, and is for subsequent use; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in the 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 is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
The preparation method of above-mentioned a kind of strong liver sheet, described CO
2The percent by volume that the 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.
The application of above-mentioned strong liver sheet in preparation inhibition B16 cell proliferation medicine.
In the 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, takes 2 times in 1st, 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 the strong liver sheet of test one, distinct methods preparation
1, the strong liver sheet of instrument and reagent the present invention: press the preparation of embodiment 3 methods, use the 690g crude drug, make 1500 through extraction, every heavy 0.5g.Former strong liver sheet by the preparation of ministry standard method, uses the 690g crude drug, makes 1652 through extraction, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; The METTLERAE240 electronic analytical balance; TANSHINONES reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute).
2, method
Chromatographic condition and system suitability: be filler with octadecylsilane chemically bonded silica; Methanol-water-phosphoric acid (40:60:0.2) is mobile phase; The detection wavelength is 280nm.Number of theoretical plate calculates by the TANSHINONES peak, should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing at 4 hours TANSHINONES reference substance of 60 ℃ of drying under reduced pressure an amount of, adds methanol and makes the solution that every 1ml contains 18 μ g, and get final product.
The preparation of need testing solution: get the strong liver sheet of the present invention and former strong liver sheet, porphyrize, mixing is got 1g, and is accurately weighed, the accurate 70% ethanol 20ml that adds, close plug, supersound process 10 minutes, centrifugal, get supernatant, and get final product.
Algoscopy is accurate reference substance solution and each 20 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and be get final product.
3, result
The result shows that the content of TANSHINONES is the 1.43mg/ sheet in the strong liver sheet of the present invention; And the content of TANSHINONES is the 0.24mg/ sheet in the former strong liver sheet, and in the situation that dose reduces, TANSHINONES content improves a lot.
Above-mentioned studies show that, the strong liver sheet that adopts the present invention to prepare, 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 that the scope of the above-mentioned theme of the present invention only limits to following example, all technology that realizes 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, with Herba Artemisiae Scopariae, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in the 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 is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content of TANSHINONES is the 1.48mg/ sheet in the finished product.
Embodiment 2
Get Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, with Herba Artemisiae Scopariae, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in the 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 is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content of TANSHINONES is the 1.53mg/ sheet in the finished product.
Embodiment 3
Get Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, with Herba Artemisiae Scopariae, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in the 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 is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content of TANSHINONES is the 1.43mg/ sheet in the finished product.
Embodiment 4: strong liver sheet suppresses the experimentation data of B16 cell proliferation
1 experiment material
1.1 experiment cell strain
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 the liver sheet: press the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing the strong liver sheet of 100mg, be dissolved in the 5ml dehydrated alcohol, 0.2 μ m filter filters, and 500 μ l doff manage packing ,-20 ℃ of storages, and 0.2 μ m filter filters dehydrated alcohol in order to the usefulness of matched group simultaneously.
1.3 experiment reagent
The Cat.No.12100-061Lot.No.758137 of DMEM(GIBCO company); Hyclone (Hangzhoupro, sky, Zhejiang bio tech ltd Lot.No.100419); NaHCO3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration); 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); As seen-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 makes 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 (the accurate experimental facilities in Shanghai 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) the B16 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%CO2, when Growth of Cells during to logarithmic (log) phase, collecting cell discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% trypsin-0.04%EDTA, behind 37 ℃ of digestion 2min, to wherein adding 5ml complete medium neutralization reaction, behind the piping and druming cell it is changed in the centrifuge tube, the centrifugal 5min of 1000rpm adjusts 3 * 104/ml of concentration of cell suspension.
2) the 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 the Growth of Cells situation, generally grow to 50%-70%, add strong liver sheet solution, continue to cultivate 24h.
4) add 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) behind the 24h, continue to cultivate 4h.
5) the buckle method is removed supernatant behind the 4h, pats dry gently with absorbent paper, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on the shaking table, and crystal is fully dissolved.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 490nm place.
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) is set 6 multiple holes for every group.
7) result represents with the suppression ratio of medicine to cell:
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 the mtt assay experiment, compare with matched group, 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), and utmost point significant difference (P＜0.001) is arranged when dosage reaches 15-20mg/ml.
The strong liver sheet of table 1 is on B16 cell inhibitory effect impact research
Annotate: compare * P＜0.01 with matched group; * P＜0.001
5 experiment conclusion
Strong liver sheet can suppress the B16 cell proliferation, reduces the Growth of Cells number of B16 cell, and this effect is dose dependent.
Claims (5)
1. the preparation method of a strong liver sheet, made as crude drug by Radix Hemerocallis 100g, Radix Isatidis 150g, Herba Artemisiae Scopariae 250g, Radix Salviae Miltiorrhizae 150g, Fructus Jujubae 100g, it is characterized in that described method is comprised of the following step: get Herba Artemisiae Scopariae, join in the CO2 supercritical extraction device, ethanol is as entrainer, the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 ℃, CO2 flow 1-3m1/g crude drug min, extraction time 150-180min gets supercritical extract, and is for subsequent use; Get Radix Hemerocallis, Radix Isatidis, Radix Salviae Miltiorrhizae, Fructus Jujubae, pulverize, add 70% ethanol of 2L, drop in the 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 is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
2. the preparation method of described a kind of strong liver sheet according to claim 1 is characterized in that described CO
2The percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
3. the preparation method of described a kind of strong liver sheet according to claim 1 is characterized in that described microwave extracting power 500W, extracts 6 minutes at every turn.
4. the preparation method of described a kind of strong liver sheet according to claim 1 is characterized in that described CO
2The extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2Flow 2ml/g crude drug min, extraction time 160min.
5. the according to claim 1 application of described a kind of strong liver sheet in preparation inhibition B16 mouse melanoma cell line cell proliferation medicine.
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| CN201210389746.7A CN102988691B (en) | 2012-10-15 | 2012-10-15 | Preparation method and application of liver-strengthening tablet |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104435569A (en) * | 2014-11-13 | 2015-03-25 | 山东中大药业有限公司 | Preparation method of rumex nepalensis and hemerocallis tablet for benefiting liver and application of rumex nepalensis and hemerocallis tablet to preparation of drug for inhibiting proliferation of melanoma cell B16 |
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| CN1383880A (en) * | 2002-05-20 | 2002-12-11 | 张俊峰 | Tumor treating pill |
| CN1546103A (en) * | 2003-12-03 | 2004-11-17 | 贵州德祥制药有限责任公司 | Liver benefiting Huangxuan Yigan Powder and its preparing process |
| CN1679898A (en) * | 2005-01-13 | 2005-10-12 | 刘桂香 | Chinese medicine composition for treating hepatitis and cancers |
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2012
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| CN1383880A (en) * | 2002-05-20 | 2002-12-11 | 张俊峰 | Tumor treating pill |
| CN1546103A (en) * | 2003-12-03 | 2004-11-17 | 贵州德祥制药有限责任公司 | Liver benefiting Huangxuan Yigan Powder and its preparing process |
| CN1679898A (en) * | 2005-01-13 | 2005-10-12 | 刘桂香 | Chinese medicine composition for treating hepatitis and cancers |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104435569A (en) * | 2014-11-13 | 2015-03-25 | 山东中大药业有限公司 | Preparation method of rumex nepalensis and hemerocallis tablet for benefiting liver and application of rumex nepalensis and hemerocallis tablet to preparation of drug for inhibiting proliferation of melanoma cell B16 |
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| CN102988691B (en) | 2014-04-23 |
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