CN103656493B - A kind of preparation method of blood stasis dispelling stomach reinforcing sheet and application - Google Patents
A kind of preparation method of blood stasis dispelling stomach reinforcing sheet and application Download PDFInfo
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Abstract
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method and application of blood stasis dispelling stomach reinforcing sheet.The preparation method of blood stasis dispelling stomach reinforcing sheet of the present invention, be made up as crude drug of Radix Astragali 198g, Radix Salviae Miltiorrhizae 198g, Cortex Phellodendri 198g, Rhizoma Paridis 119g, Radix Glycyrrhizae 119g, Rhizoma Curcumae 119g, Rhizoma Polygoni Cuspidati 198g, Radix Notoginseng 132g, Herba Hedyotidis Diffusae 198g, Endothelium Corneum Gigeriae Galli 119g, Rhizoma Atractylodis Macrocephalae 119g, Pseudobulbus Bletillae (Rhizoma Bletillae) 132g, adopt supercritical extraction and microwave extracting to be prepared from, emodin content is improved a lot.Present invention also offers the application of blood stasis dispelling stomach reinforcing sheet in preparation suppression mouse brain neuroma cell Neuro-2a cell proliferation.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method and application of blood stasis dispelling stomach reinforcing sheet.
Background technology
Blood stasis dispelling Yiwei Capsule standard No. WS-10823(ZD-0823)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine taste fascicle.Be made up as crude drug of Radix Astragali 198g, Radix Salviae Miltiorrhizae 198g, Cortex Phellodendri 198g, Rhizoma Paridis 119g, Radix Glycyrrhizae 119g, Rhizoma Curcumae 119g, Rhizoma Polygoni Cuspidati 198g, Radix Notoginseng 132g, Herba Hedyotidis Diffusae 198g, Endothelium Corneum Gigeriae Galli 119g, Rhizoma Atractylodis Macrocephalae 119g, Pseudobulbus Bletillae (Rhizoma Bletillae) 132g, there is invigorating the spleen and regulating the stomach, effect of blood-activating analgetic.For the acute and chronic gastritis of insufficiency of the spleen caused by energy stagnation and blood stasis, chronic atrophic gastritis.
In prior art, not yet have blood stasis dispelling stomach reinforcing sheet extracting the report adopting supercritical and microwave technology in preparation, and the method for powder, soak by water directly beaten by Chinese medicine, 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 applied clinically.
In prior art, blood stasis dispelling stomach reinforcing sheet is oral, one time 5,3 times on the one.Blood stasis dispelling stomach reinforcing sheet dosage is large.The heavy 0.5g of the every sheet of the blood stasis dispelling stomach reinforcing sheet adopting the inventive method to be prepared into, only needs 2 at every turn, within 1st, takes 3 times.Dose is greatly reduced under the condition with more active component.This conclusion can be proved by following test.
The comparison of emodin content in blood stasis dispelling stomach reinforcing sheet prepared by test one, distinct methods
L, instrument and reagent blood stasis dispelling stomach reinforcing of the present invention sheet: by the preparation of embodiment 1 method, use 1849g crude drug, makes 250 through extracting, the heavy 0.5g of every sheet.Former blood stasis dispelling stomach reinforcing sheet, according to WS-10823(ZD-0823)-2002 standard method preparations, in contrast.Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Emodin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex V D).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Methanol-water (77: 23) is mobile phase; Determined wavelength is 254nm.Number of theoretical plate calculates should be not less than 2000 by emodin peak.
The preparation precision of reference substance solution takes emodin reference substance in right amount, adds methanol and makes the solution of every 1ml containing 15 g, to obtain final product.
Blood stasis dispelling stomach reinforcing sheet of the present invention is got in the preparation of product need testing solution of the present invention, and porphyrize, gets 0.4g, accurately weighed, put in tool plug conical flask, precision adds methanol 20ml, close plug, weighed weight, supersound process (power 250W, frequency 30kHz) 60 minutes, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, centrifugal (rotating speed per minute 3500 turns), get supernatant to filter, get subsequent filtrate, to obtain final product.
The blood stasis dispelling Yiwei Capsule of contrast is got in the preparation of reference product need testing solution, and porphyrize, gets 1g, accurately weighed, put in tool plug conical flask, precision adds methanol 20ml, close plug, weighed weight, supersound process (power 250W, frequency 30kHz) 60 minutes, let cool, more weighed weight, supply the weight of less loss with methanol, shake up, centrifugal (rotating speed per minute 3500 turns), get supernatant to filter, get subsequent filtrate, to obtain final product.
Algoscopy is accurate respectively draws reference substance solution and each 10 l ~ 20 l of need testing solution, injection liquid chromatography, measures, to obtain final product.
3, result
Result shows, in blood stasis dispelling stomach reinforcing sheet of the present invention, the content of emodin is 500-800 g/ sheet; And the content of emodin is 72 g/ sheets in former blood stasis dispelling stomach reinforcing sheet, every sheet emodin content is equivalent to the 8-12 of original capsule content doubly, and when dose reduces, emodin content improves a lot.
Above-mentioned research shows, adopt blood stasis dispelling stomach reinforcing sheet prepared by preparation method of the present invention, active constituent content is far away higher than WS-10823(ZD-0823) the blood stasis dispelling stomach reinforcing sheet prepared of method recorded of-2002 standards.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of preparation method of blood stasis dispelling stomach reinforcing sheet.
Another object of the present invention is to provide a kind of blood stasis dispelling stomach reinforcing sheet to suppress the application in mouse brain neuroma cell Neuro-2a cell proliferation in preparation.
The object of the invention is by following scheme realize:
A kind of preparation method of blood stasis dispelling stomach reinforcing sheet, be made up as crude drug of Radix Astragali 198g, Radix Salviae Miltiorrhizae 198g, Cortex Phellodendri 198g, Rhizoma Paridis 119g, Radix Glycyrrhizae 119g, Rhizoma Curcumae 119g, Rhizoma Polygoni Cuspidati 198g, Radix Notoginseng 132g, Herba Hedyotidis Diffusae 198g, Endothelium Corneum Gigeriae Galli 119g, Rhizoma Atractylodis Macrocephalae 119g, Pseudobulbus Bletillae (Rhizoma Bletillae) 132g, it is characterized in that, described preparation method is made up of the following step: get Endothelium Corneum Gigeriae Galli and Radix Notoginseng, join CO
2in 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 DEG C, CO
2flow l-3ml/g crude drug min, extraction time 180-220min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 50% ethanol of 1.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extracts 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 250, the heavy 0.5g of every sheet.
In the preparation method of above-mentioned blood stasis dispelling stomach reinforcing sheet, described CO
2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
In the preparation method of above-mentioned blood stasis dispelling stomach reinforcing sheet, described CO
2in supercritical extraction, extracting pressure is 25 MPa.
In the preparation method of above-mentioned blood stasis dispelling stomach reinforcing sheet, described CO
2in supercritical extraction, extraction temperature is 60 DEG C.
In the preparation method of above-mentioned blood stasis dispelling stomach reinforcing sheet, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
In the preparation method of above-mentioned blood stasis dispelling stomach reinforcing sheet, described CO
2in supercritical extraction, extraction time is 200min.
In the preparation method of above-mentioned blood stasis dispelling stomach reinforcing sheet, described microwave extracting power is 700W.
In the preparation method of above-mentioned blood stasis dispelling stomach reinforcing sheet, each extraction time of described microwave extracting is 8 minutes.
Above-mentioned blood stasis dispelling stomach reinforcing sheet suppresses the application in mouse brain neuroma cell Neuro-2a cell proliferation in preparation.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art more understands the present invention, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Radix Astragali 198g, Radix Salviae Miltiorrhizae 198g, Cortex Phellodendri 198g, Rhizoma Paridis 119g, Radix Glycyrrhizae 119g, Rhizoma Curcumae 119g, Rhizoma Polygoni Cuspidati 198g, Radix Notoginseng 132g, Herba Hedyotidis Diffusae 198g, Endothelium Corneum Gigeriae Galli 119g, Rhizoma Atractylodis Macrocephalae 119g, Pseudobulbus Bletillae (Rhizoma Bletillae) 132g, Endothelium Corneum Gigeriae Galli and Radix Notoginseng are joined CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 25MPa, temperature 40 DEG C, CO
2flow 2ml/g crude drug min, extraction time 200min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 50% ethanol of 1.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 700W, extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 500, the heavy 0.5g of every sheet.
After testing, in finished product, the content of emodin is 780 g/ sheets.
Embodiment 2
Get Radix Astragali 198g, Radix Salviae Miltiorrhizae 198g, Cortex Phellodendri 198g, Rhizoma Paridis 119g, Radix Glycyrrhizae 119g, Rhizoma Curcumae 119g, Rhizoma Polygoni Cuspidati 198g, Radix Notoginseng 132g, Herba Hedyotidis Diffusae 198g, Endothelium Corneum Gigeriae Galli 119g, Rhizoma Atractylodis Macrocephalae 119g, Pseudobulbus Bletillae (Rhizoma Bletillae) 132g, Endothelium Corneum Gigeriae Galli and Radix Notoginseng are joined CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4%, extracting pressure 30MPa, temperature 50 C, CO
2flow 3ml/g crude drug min, extraction time 180min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 50% ethanol of 1.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extracts 2 times, each 5 minutes, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 500, the heavy 0.5g of every sheet.
After testing, in finished product, the content of emodin is 780 g/ sheets.
Embodiment 3
Get Radix Astragali 198g, Radix Salviae Miltiorrhizae 198g, Cortex Phellodendri 198g, Rhizoma Paridis 119g, Radix Glycyrrhizae 119g, Rhizoma Curcumae 119g, Rhizoma Polygoni Cuspidati 198g, Radix Notoginseng 132g, Herba Hedyotidis Diffusae 198g, Endothelium Corneum Gigeriae Galli 119g, Rhizoma Atractylodis Macrocephalae 119g, Pseudobulbus Bletillae (Rhizoma Bletillae) 132g, Endothelium Corneum Gigeriae Galli and Radix Notoginseng are joined CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 6%, extracting pressure 15MPa, temperature 30 DEG C, CO
2flow 1ml/g crude drug min, extraction time 220min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 50% ethanol of 1.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 800W, extracts 2 times, each 10 minutes, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 500, the heavy 0.5g of every sheet.
After testing, in finished product, the content of emodin is 540 g/ sheets.
Embodiment 4: blood stasis dispelling stomach reinforcing sheet suppresses the experimentation data of mouse brain neuroma cell Neuro-2a cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse brain neuroma cell Neuro-2a cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: blood stasis dispelling stomach reinforcing sheet of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: take 100mg blood stasis dispelling stomach reinforcing sheet, be dissolved in 5ml dehydrated alcohol, 0.2 m frit, 500 ldoff pipe subpackages ,-20 DEG C of storages, 0.2 m frit dehydrated alcohol is in order to the use of matched group simultaneously.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061 Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHC0
3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033 Lot.No. 1088387 of a specified duration); Trypsin(AMRESCO company); EDTA(AMRESCO company); Penicillin G Sodium Salt(AMRESCO company 1); Streptomycin Sulfate (AMRESCO); Dehydrated alcohol (Zibo Ya Dulan Trade Co., Ltd.); MTT (Biosharp lot number: 0793): PBS(laboratory autogamy);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DMIL); Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX 190); C0
2incubator (FORMA model: 3111); Super-clean bench (safe and sound Inc. of Su Jing group moulding number: SW-CJ-ZFD); Pure water instrument (Sprlng company of U.S. 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 company in Shanghai 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 m filter (MILLIPORE model: SLGP033RB); 1cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, pipet, Tips are some.
2. experimental technique
1) NEURO-2a cell DMEM+10%FBS is in 37 DEG C, 5%C0
2carry out cellar culture (10cm culture dish), when Growth of Cells is to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, add 5ml complete medium neutralization reaction wherein, proceeded in centrifuge tube after piping and druming cell, the centrifugal 5min of 1000rpm, adjustment concentration of cell suspension 3 × 10
4individual/ml.
2) enter in 96 well culture plates by cell kind, every hole adds cell suspension 180 l, culture plate put into cell culture incubator (37 DEG C, 5%C0
2) cellar culture.
3) according to cell growth status, generally grow to 50%-70%, add blood stasis dispelling stomach reinforcing sheet solution, continue to cultivate 24h.
4) add 20 l MTT 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, as entered 200 l dimethyl sulfoxide, is put low-speed oscillation 10min on shaking table, crystal is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) background (do not add cell, only add culture fluid) is set, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide) simultaneously, often organizes the multiple hole of setting 6.
7) result represents with the suppression ratio of medicine to cell: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value), control wells OD value × 100%.Experiment repetition 3 times.
3. statistical disposition
Adopt the correlation analysis in Microsoft Excel 2007 software and Student t to check, data represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to NEURO-2a cell inhibitory effect variant (P<0.05), dosage this difference when 10mg/ml has significance (P<0.01), has pole significant difference (P<0.001) when dosage reaches 15-20mg/ml.
Table 1 blood stasis dispelling stomach reinforcing sheet is to NEURO-2a cell inhibitory effect influence research (X ± SD)
Note: compare with matched group, * P<O.01; * P<0.001.
5. experiment conclusion
Blood stasis dispelling stomach reinforcing sheet can suppress NEURO-2a cell proliferation, and reduce the Growth of Cells number of NEURO-2a cell, this effect is dose dependent.
Claims (7)
1. the application of blood stasis dispelling stomach reinforcing sheet in preparation suppression mouse brain neuroma cell Neuro-2a cell proliferation, it is characterized in that, described blood stasis dispelling stomach reinforcing sheet is made up as crude drug of Radix Astragali 198g, Radix Salviae Miltiorrhizae 198g, Cortex Phellodendri 198g, Rhizoma Paridis 119g, Radix Glycyrrhizae 119g, Rhizoma Curcumae 119g, Rhizoma Polygoni Cuspidati 198g, Radix Notoginseng 132g, Herba Hedyotidis Diffusae 198g, Endothelium Corneum Gigeriae Galli 119g, Rhizoma Atractylodis Macrocephalae 119g, Pseudobulbus Bletillae (Rhizoma Bletillae) 132g, the preparation method of described blood stasis dispelling stomach reinforcing sheet is made up of the following step: get Endothelium Corneum Gigeriae Galli and Radix Notoginseng, join CO
2in 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 DEG C, CO
2flow l-3ml/g crude drug min, extraction time 180-220min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 50% ethanol of 1.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extracts 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 250, the heavy 0.5g of every sheet.
2. the blood stasis dispelling stomach reinforcing sheet according to claim l suppresses the application in mouse brain neuroma cell Neuro-2a cell proliferation in preparation, it is characterized in that, described CO
2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
3. the blood stasis dispelling stomach reinforcing sheet according to claim l suppresses the application in mouse brain neuroma cell Neuro-2a cell proliferation in preparation, it is characterized in that, described CO
2in supercritical extraction, extracting pressure is 25 MPa.
4. the blood stasis dispelling stomach reinforcing sheet according to claim l suppresses the application in mouse brain neuroma cell Neuro-2a cell proliferation in preparation, it is characterized in that, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
5. the blood stasis dispelling stomach reinforcing sheet according to claim l suppresses the application in mouse brain neuroma cell Neuro-2a cell proliferation in preparation, it is characterized in that, described CO
2in supercritical extraction, extraction time is 200min.
6. the blood stasis dispelling stomach reinforcing sheet according to claim l suppresses the application in mouse brain neuroma cell Neuro-2a cell proliferation in preparation, and it is characterized in that, described microwave extracting power is 700W.
7. the blood stasis dispelling stomach reinforcing sheet according to claim l suppresses the application in mouse brain neuroma cell Neuro-2a cell proliferation in preparation, and it is characterized in that, each extraction time of described microwave extracting is 8 minutes.
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| CN104398817A (en) * | 2014-11-13 | 2015-03-11 | 山东中大药业有限公司 | Preparation method of nine flavor swertia bimaculata tablet and application of nine flavor swertia bimaculata tablet in preparation of drugs for inhibition of cell proliferation of mastocytoma cell P815 |
| CN104306821A (en) * | 2014-11-13 | 2015-01-28 | 山东中大药业有限公司 | Preparation method of liver benefiting tablet containing nepal dock root and day lily and application of liver benefiting tablet to preparation of drug for restraining multiplication of mastocytoma cell P815 cell |
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Effective date of registration: 20150929 Address after: 9 first people's Hospital of Qingdao Development Zone, Huangpu Road, Huangdao District, Shandong, Qingdao 266555, China Applicant after: Chai Guifeng Applicant after: Wang Weike Address before: 250062, No. ten, No. 16369, Lixia District, Ji'nan City, Shandong Province Applicant before: SHANDONG ZHONGDA PHARMACEUTICAL CO., LTD. |
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