CN103566168B - Preparation method and application of tranquilizing tablets - Google Patents
Preparation method and application of tranquilizing tablets Download PDFInfo
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- CN103566168B CN103566168B CN201310528851.9A CN201310528851A CN103566168B CN 103566168 B CN103566168 B CN 103566168B CN 201310528851 A CN201310528851 A CN 201310528851A CN 103566168 B CN103566168 B CN 103566168B
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a preparation method and application of tranquilizing tablets. The tranquilizing tablets are prepared by adopting 40g of fried spina date seed, 47g of ligusticum wallichii, 112g of rhizoma anemarrhenae, 92g of radix ophiopogonis, 32g of prepared polygonum multiflorum, 97g of schisandra chinensis, 130g of salvia miltiorrhiza, 97g of poria as crude drugs and adopting supercritical extraction and microwave extraction, so that the content of schizandrin is greatly increased. The invention also provides application of the tranquilizing tablets in preparation of a medicament for inhibiting cell proliferation of myeloma cell SP2/0 of mice.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method and application of mind tranquilizing tablet.
Background technology
ANSHEN JIAONANG is recorded in one the 717th page of Chinese Pharmacopoeia version in 2010, by Semen Ziziphi Spinosae (parched) 40g, Rhizoma Chuanxiong 47g, Rhizoma Anemarrhenae 112g, Radix Ophiopogonis, 92g, Radix Polygoni Multiflori Preparata 32g, Fructus Schisandrae Chinensis 97g, Radix Salviae Miltiorrhizae 130g, Poria 97g make as crude drug, there is the YIN nourishing of enriching blood, effect of tranquilizing by nourishing the heart.For deficiency of YIN-blood, insomnia and dreamful sleep, palpitation and uneasiness, dysphoria with feverish sensation in the chest palms and soles, night sweat tinnitus.
In prior art, not yet there is ANSHEN JIAONANG at the report that adopts supercritical and microwave technology aspect extraction preparation, and medicine is directly played powder, dipping diafiltration, the method that decocting boils, 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.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of preparation method of mind tranquilizing tablet.
Another object of the present invention is to provide a kind of mind tranquilizing tablet to suppress the application in murine myeloma cell SP2/0 cell proliferation medicine in preparation.
The object of the invention is to realize by following scheme:
A kind of preparation method of mind tranquilizing tablet, by Semen Ziziphi Spinosae (parched) 40g, Rhizoma Chuanxiong 47g, Rhizoma Anemarrhenae 112g, Radix Ophiopogonis, 92g, Radix Polygoni Multiflori Preparata 32g, Fructus Schisandrae Chinensis 97g, Radix Salviae Miltiorrhizae 130g, Poria 97g make as crude drug, and described preparation method is made up of the following step: Semen Ziziphi Spinosae (parched), Fructus Schisandrae Chinensis are joined to 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, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 500, every heavy 0.3g.
In the preparation method of above-mentioned mind tranquilizing tablet, described CO
2in supercritical extraction, to account for the percent by volume of total extractant be 5% to entrainer.
In the preparation method of above-mentioned mind tranquilizing tablet, described CO
2in supercritical extraction, extracting pressure is 25 MPa.
In the preparation method of above-mentioned mind tranquilizing tablet, described CO
2in supercritical extraction, extraction temperature is 60 DEG C.
In the preparation method of above-mentioned mind tranquilizing tablet, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
In the preparation method of above-mentioned mind tranquilizing tablet, described CO
2in supercritical extraction, extraction time is 200min.
In the preparation method of above-mentioned mind tranquilizing tablet, described microwave extracting power is 700W.
In the preparation method of above-mentioned mind tranquilizing tablet, the each extraction time of described microwave extracting is 8 minutes.
Above-mentioned mind tranquilizing tablet suppresses the application in murine myeloma cell SP2/0 cell proliferation medicine in preparation.
In prior art, each 4 of ANSHEN JIAONANG, 3 times on the one.The every 0.3g of mind tranquilizing tablet that adopts the inventive method to be prepared into only needs 2 at every turn, within 1st, takes 3 times.Under the condition with more active component, greatly reduce dose.This conclusion can be by following evidence.And be prepared into tablet, patient swallows with water, dosage is little and without bitterness, patient is easy to accept.
The comparison of schisandrin content in mind tranquilizing tablet prepared by test one, distinct methods
L, instrument and reagent mind tranquilizing tablet of the present invention: press embodiment 1 method preparation, use 647g crude drug, make 500 through extracting, every heavy 0.3g.Former ANSHEN JIAONANG, by 2010 editions standards of pharmacopoeia method preparations, is used 647g crude drug, makes 50 bags through extracting, every bag heavy 10g.Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Schisandrin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (annex V D).
Chromatographic condition and system suitability are taking octadecylsilane chemically bonded silica as filler; Taking methanol-water (58:42) as mobile phase; Detection wavelength is 250nm.Number of theoretical plate calculates and should be not less than 3000 by schisandrin peak.
It is appropriate that schisandrin reference substance is got in the preparation of reference substance solution, accurately weighed, add methanol and make every 1ml containing the solution of 30 μ g, to obtain final product.
Mind tranquilizing tablet of the present invention is got in the preparation of product need testing solution of the present invention, and porphyrize is got about 0.6g, accurately weighed, to put in tool plug conical flask, precision adds chloroform-methanol (2:1) mixed solution 25ml, close plug, weighed weight, supersound process (power 250W, frequency 25kHz) 40 minutes, let cool, more weighed weight, the weight of supplying less loss with chloroform-methanol (2:1) mixed solution, shakes up, and filters, get subsequent filtrate, to obtain final product.
This contrast ANSHEN JIAONANG content is got in the preparation of reference product need testing solution, and porphyrize is got about 1g, accurately weighed, to put in tool plug conical flask, precision adds chloroform-methanol (2:1) mixed solution 25ml, close plug, weighed weight, supersound process (power 250W, frequency 25kHz) 40 minutes, let cool, more weighed weight, the weight of supplying less loss with chloroform-methanol (2:1) mixed solution, shakes up, and filters, get subsequent filtrate, to obtain final product.
Algoscopy is accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, and note people chromatograph of liquid, measures, and to obtain final product.Every of this product contains Fructus Schisandrae Chinensis with schisandrin (C
24h
32o
7) meter, must not be less than 0.20mg.
3, result
Result shows, in mind tranquilizing tablet of the present invention, the content of schisandrin is 0.80-1.0mg/ sheet; And the content of schisandrin is 0.24mg/ sheet in former ANSHEN JIAONANG, in the situation that dose reduces, schisandrin content improves a lot.
Above-mentioned research shows, the mind tranquilizing tablet that adopts preparation method of the present invention to prepare, the ANSHEN JIAONANG that active constituent content is prepared higher than the method for recording in 2010 editions standards of pharmacopoeia far away.
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 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 Semen Ziziphi Spinosae (parched) 40g, Rhizoma Chuanxiong 47g, Rhizoma Anemarrhenae 112g, Radix Ophiopogonis 92g, Radix Polygoni Multiflori Preparata 32g, Fructus Schisandrae Chinensis 97g, Radix Salviae Miltiorrhizae 130g, Poria 97g, Semen Ziziphi Spinosae (parched), Fructus Schisandrae Chinensis are joined to 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, 40 DEG C of temperature, 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, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 500, every heavy 0.3g.
After testing, in finished product, the content of schisandrin is 0.98mg/ sheet.
Embodiment 2
Get Semen Ziziphi Spinosae (parched) 40g, Rhizoma Chuanxiong 47g, Rhizoma Anemarrhenae 112g, Radix Ophiopogonis 92g, Radix Polygoni Multiflori Preparata 32g, Fructus Schisandrae Chinensis 97g, Radix Salviae Miltiorrhizae 130g, Poria 97g, Semen Ziziphi Spinosae (parched), Fructus Schisandrae Chinensis are joined to 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, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 500, every heavy 0.3g.
After testing, in finished product, the content of schisandrin is 0.88mg/ sheet.
Embodiment 3
Get Semen Ziziphi Spinosae (parched) 40g, Rhizoma Chuanxiong 47g, Rhizoma Anemarrhenae 112g, Radix Ophiopogonis 92g, Radix Polygoni Multiflori Preparata 32g, Fructus Schisandrae Chinensis 97g, Radix Salviae Miltiorrhizae 130g, Poria 97g, Semen Ziziphi Spinosae (parched), Fructus Schisandrae Chinensis are joined to 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, 30 DEG C of temperature, 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, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 500, every heavy 0.3g.
After testing, in finished product, the content of schisandrin is 0.82mg/ sheet.
Embodiment 4: mind tranquilizing tablet suppresses the experimentation data of murine myeloma cell SP2/0 cell proliferation
1. experiment material
1.1 experiment cell strains
Murine myeloma cell SP2/0 cell, Shandong Academy of Medical Sciences's laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: mind tranquilizing tablet of the present invention: press embodiment 1 method preparation.
Medicinal liquid liquid storage: take 100mg mind tranquilizing tablet, be dissolved in 5ml dehydrated alcohol, 0.2 m filter filters, 500 ldoff pipe subpackages ,-20 DEG C of storages, 0.2 m filter filters the use of dehydrated alcohol in order to matched group simultaneously.
1.3 experiment reagent
DMEM (the Cat.No.12100-061 Lot.No.758137 of GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); NaHC0
3(the Shanghai hundred million Cat.No.11810-033 Lot.No. of chemical reagent company limited 1088387 of a specified duration); Trypsin(AMRESCO company 1); EDTA(AMRESCO company 1); Penicillin G Sodium Salt(AMRESCO company 1); Streptomycin Sulfate (AMRESCO); Dehydrated alcohol (Nanjing Chemistry Reagent 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 company of Su Jing group manufactures model: 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 (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 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) SP2/0 cell uses DMEM+10%FBS in 37 DEG C, 5%C0
2carry out cellar culture (10cm culture dish), 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 DEG C 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 concentration of cell suspension 3 × 10
4individual/ml.
(2) cell kind is entered in 96 well culture plates, 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 Growth of Cells situation, generally grow to 50%-70%, add mind tranquilizing tablet solution, continue to cultivate 24h.
(4) after 24h, add 20 l MTT solution (5mg/ml, i.e. 0.5%MTT), continue to cultivate 4h.
(5) after 4h, buckle method is removed supernatant, pats dry gently with absorbent paper, and low-speed oscillation 10min on shaking table, as entered 200 l dimethyl sulfoxide, is put in every hole, 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), 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 disposition
Adopt correlation analysis and Student t inspection in Microsoft Excel 2007 softwares, data represent with mean ± S.D..The results are shown in Table 1.
Table 1 mind tranquilizing tablet is on SP2/0 cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 18.7±15.32 |
| 2 | 10 | 28.6±13.96* |
| 3 | 15 | 36.6±12.98** |
| 4 | 20 | 44.1±15.27** |
Note: with matched group comparison, * P<0.01; * P<0.001.
4. experimental result
Statistical result showed after mtt assay experiment, with matched group comparison, in the time that dosage reaches 5mg/ml, to SP2/0 cell inhibitory effect variant (P<0.05), dosage this difference in the time of 10mg/ml has significance (P<0.01), has utmost point significant difference (P<0.001) in the time that dosage reaches 15-20mg/ml.
5. experiment conclusion
Mind tranquilizing tablet can suppress SP2/0 cell proliferation, reduces the Growth of Cells number of SP2/0 cell, and this effect is dose dependent.
Claims (8)
1. the preparation method of a mind tranquilizing tablet, by Semen Ziziphi Spinosae (parched) 40g, Rhizoma Chuanxiong 47g, Rhizoma Anemarrhenae 112g, Radix Ophiopogonis, 92g, Radix Polygoni Multiflori Preparata 32g, Fructus Schisandrae Chinensis 97g, Radix Salviae Miltiorrhizae 130g, Poria 97g make as crude drug, it is characterized in that, described preparation method is made up of the following step: Semen Ziziphi Spinosae (parched), Fructus Schisandrae Chinensis are joined to 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, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 500, every heavy 0.3g.
2. according to the preparation method of the mind tranquilizing tablet described in claim l, it is characterized in that described CO
2in supercritical extraction, to account for the percent by volume of total extractant be 5% to entrainer.
3. according to the preparation method of the mind tranquilizing tablet described in claim l, it is characterized in that described CO
2in supercritical extraction, extracting pressure is 25 MPa.
4. according to the preparation method of the mind tranquilizing tablet described in claim l, it is characterized in that described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
5. according to the preparation method of the mind tranquilizing tablet described in claim l, it is characterized in that described CO
2in supercritical extraction, extraction time is 200min.
6. according to the preparation method of the mind tranquilizing tablet described in claim l, it is characterized in that, described microwave extracting power is 700W.
7. according to the preparation method of the mind tranquilizing tablet described in claim l, it is characterized in that, the each extraction time of described microwave extracting is 8 minutes.
8. the mind tranquilizing tablet described in claim l suppresses the application in murine myeloma cell SP2/0 cell proliferation medicine in preparation.
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Application publication date: 20140212 Assignee: Ji'nan Ding Long Chemical Technology Co., Ltd. Assignor: Yao Ye Contract record no.: 2014370000196 Denomination of invention: Preparation method and application of tranquilizing tablets Granted publication date: 20140806 License type: Exclusive License Record date: 20141218 |
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