CN103690647A - Preparation method of Yikangbuyuan tablet and application of Yikangbuyuan tablet in medicines for inhibiting mouse leukaemia cell L6565 proliferation - Google Patents
Preparation method of Yikangbuyuan tablet and application of Yikangbuyuan tablet in medicines for inhibiting mouse leukaemia cell L6565 proliferation Download PDFInfo
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- YKRGDOXKVOZESV-WRJNSLSBSA-N Paeoniflorin Chemical compound C([C@]12[C@H]3O[C@]4(O)C[C@](O3)([C@]1(C[C@@H]42)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C)OC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-WRJNSLSBSA-N 0.000 description 11
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of traditional Chinese medicines, and particularly provides a preparation method and application of a Yikangbuyuan tablet. The Yikangbuyuan tablet is prepared from 280g of radix astragali, 80g of Michelia hedyosperma Lew, 190g of angelica, 220g of yerbadetajo, 190g of rhizoma atractylodis, 150g of safflower, 140g of red paeonia, 140g of peach kernel, 150g of radix achyranthis bidentatae, 120g of hemlock parsley, 70g of fructus aurantii and 100g of balloonflower by supercritical extraction and microwave extraction, so that the penoniflorin content is greatly enhanced. The invention also provides application of the Yikangbuyuan tablet in preparing medicines for inhibiting mouse mononuclear macrophage leukaemia cell L6565 proliferation.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method and application in suppressing mouse leukemia cell L6565 cell proliferation medicine thereof of Yikang complement sheet.
Background technology
Yikang complement granule standard No. WS-10983(ZD-0983)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine taste fascicle.By Radix Astragali 280g, fiber crops rare 80g, Radix Angelicae Sinensis 190g, Herba Ecliptae 220g, Rhizoma Atractylodis 190g, Flos Carthami 150g, Radix Paeoniae Rubra 140g, Semen Persicae 140g, Radix Achyranthis Bidentatae 150g, Rhizoma Chuanxiong 120g, Fructus Aurantii 70g, Radix Platycodonis 100g, as crude drug, made, there is benefiting QI for activating blood circulation, the effect of strengthening spleen, tonifying kidney.The spiritlessness and weakness causing for blood stasis due to qi deficiency, deficiency of spleen and stomach, short breath, insomnia, soreness of the waist and knees, the forgetful grade of lack of appetite disease.
In prior art, not yet have Yikang complement granule aspect extraction preparation, adopting the report of supercritical and microwave technology, and volatile oil extract, the method that decocting boils, technique is coarse, backward, and impurity is many, cause 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 Yikang complement sheet.
Another object of the present invention is to provide a kind of Yikang complement sheet to suppress the application in mouse leukemia cell L6565 cell proliferation medicine in preparation.
The object of the invention is to realize by following scheme:
A kind of preparation method of Yikang complement sheet, by Radix Astragali 280g, fiber crops rare 80g, Radix Angelicae Sinensis 190g, Herba Ecliptae 220g, Rhizoma Atractylodis 190g, Flos Carthami 150g, Radix Paeoniae Rubra 140g, Semen Persicae 140g, Radix Achyranthis Bidentatae 150g, Rhizoma Chuanxiong 120g, Fructus Aurantii 70g, Radix Platycodonis 100g, as crude drug, made, described preparation method is comprised of the following step: get that fiber crops are rare, Radix Angelicae Sinensis, Rhizoma Atractylodis, Semen Persicae, Rhizoma Chuanxiong, 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 ℃, CO
2flow l-3ml/g crude drug min, extraction time 180-220min, obtains supercritical extract, standby; 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 300, every heavy 0.3g.
In the preparation method of above-mentioned Yikang complement sheet, 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 Yikang complement sheet, described CO
2in supercritical extraction, extracting pressure is 25MPa.
In the preparation method of above-mentioned Yikang complement sheet, described CO
2in supercritical extraction, extraction temperature is 60 ℃.
In the preparation method of above-mentioned Yikang complement sheet, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
In the preparation method of above-mentioned Yikang complement sheet, described CO
2in supercritical extraction, extraction time is 200min.
In the preparation method of above-mentioned Yikang complement sheet, described microwave extracting power is 700W.
In the preparation method of above-mentioned Yikang complement sheet, the each extraction time of described microwave extracting is 8 minutes.
Above-mentioned Yikang complement sheet suppresses the application in mouse leukemia cell L6565 cell proliferation medicine in preparation.
In prior art, each 1 bag of Yikang complement granule, every bag of 7g, 3 times on the one.Yikang complement granule dosage is large, and granule preparation need to add a large amount of sugar, and diabetics is not suitable for, and the words taste of not sugaring is not good, and patient is not easy under clothes, and compliance is poor.The every heavy 0.3g of Yikang complement sheet that adopts the inventive method to be prepared into only needs 2 at every turn, within 1st, takes 3 times.Greatly reduced dose having under the condition of more active component.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 paeoniflorin content in Yikang complement sheet prepared by test one, distinct methods
L, instrument and reagent Yikang complement of the present invention sheet: press embodiment 1 method preparation, use 1510g crude drug, make 300 through extracting, every heavy 0.3g.Former Yikang complement granule, according to WS-10983(ZD-0983)-2002 standard method preparations.Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Peoniflorin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
According to high performance liquid chromatography (appendix V D of Chinese Pharmacopoeia version in 2010), measure.
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Acetonitrile-0.2% phosphate aqueous solution (15: 85) is mobile phase; Detection wavelength is 230nm.Number of theoretical plate calculates and should be not less than 3000 by peoniflorin peak.
The preparation of the reference substance solution phosphorus pentoxide drying under reduced pressure peoniflorin reference substance of 36 hours of learning from else's experience is appropriate, accurately weighed, adds methanol and makes every 1ml containing the solution of 0.1mg, obtains.
Yikang complement sheet of the present invention is got in the preparation of product need testing solution of the present invention, and porphyrize, gets 2.25g, accurately weighed, to put in tool plug conical flask, precision adds the close plug of methanol 25ml., weighed weight, supersound process (power 250w. frequency 33kHz) 1 hour, lets cool, weighed weight again, the weight of supplying less loss with methanol, shakes up, and filters, with microporous filter membrane (0.45 μ m), filter, obtain.。
The Yikang complement granule of this contrast is got in the preparation of reference product need testing solution, and porphyrize, gets 2.5g, accurately weighed, to put in tool plug conical flask, precision adds the close plug of methanol 25ml., weighed weight, supersound process (power 250w. frequency 33kHz) 1 hour, lets cool, weighed weight again, the weight of supplying less loss with methanol, shakes up, and filters, with microporous filter membrane (0.45 μ m), filter, obtain.
Algoscopy is accurate reference substance solution and each 10 μ L of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
3, result
Result shows, in Yikang complement sheet of the present invention, the content of peoniflorin is 5-8mg/ sheet; And the content of peoniflorin is 4.8mg/ bag in former Yikang complement granule, each serving the paeoniflorin content of 2 of consumptions be former granule content 2-4 doubly, in the situation that dose reduces, paeoniflorin content improves a lot.
Above-mentioned research shows, the Yikang complement sheet that adopts preparation method of the present invention to prepare, active constituent content is far away higher than WS-10983(ZD-0983) the Yikang complement granule prepared of the method recorded of-2002 standards.
The specific embodiment
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 Radix Astragali 280g, fiber crops rare 80g, Radix Angelicae Sinensis 190g, Herba Ecliptae 220g, Rhizoma Atractylodis 190g, Flos Carthami 150g, Radix Paeoniae Rubra 140g, Semen Persicae 140g, Radix Achyranthis Bidentatae 150g, Rhizoma Chuanxiong 120g, Fructus Aurantii 70g, Radix Platycodonis 100g, Jiang Mahan, Radix Angelicae Sinensis, Rhizoma Atractylodis, Semen Persicae, Rhizoma Chuanxiong 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 25MPa, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 200min, obtains supercritical extract, standby; 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 300, every heavy 0.3g.
After testing, in finished product, the content of peoniflorin is 7.8mg/ sheet.
Embodiment 2
Get Radix Astragali 280g, fiber crops rare 80g, Radix Angelicae Sinensis 190g, Herba Ecliptae 220g, Rhizoma Atractylodis 190g, Flos Carthami 150g, Radix Paeoniae Rubra 140g, Semen Persicae 140g, Radix Achyranthis Bidentatae 150g, Rhizoma Chuanxiong 120g, Fructus Aurantii 70g, Radix Platycodonis 100g, Jiang Mahan, Radix Angelicae Sinensis, Rhizoma Atractylodis, Semen Persicae, Rhizoma Chuanxiong 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 30MPa, temperature 50 C, CO
2flow 3ml/g crude drug min, extraction time 180min, obtains supercritical extract, standby; 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 300, every heavy 0.3g.
After testing, in finished product, the content of peoniflorin is 5.6mg/ sheet.
Embodiment 3
Get Radix Astragali 280g, fiber crops rare 80g, Radix Angelicae Sinensis 190g, Herba Ecliptae 220g, Rhizoma Atractylodis 190g, Flos Carthami 150g, Radix Paeoniae Rubra 140g, Semen Persicae 140g, Radix Achyranthis Bidentatae 150g, Rhizoma Chuanxiong 120g, Fructus Aurantii 70g, Radix Platycodonis 100g, Jiang Mahan, Radix Angelicae Sinensis, Rhizoma Atractylodis, Semen Persicae, Rhizoma Chuanxiong 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 15MPa, 30 ℃ of temperature, CO
2flow 1ml/g crude drug min, extraction time 220min, obtains supercritical extract, standby; 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 300, every heavy 0.3g.
After testing, in finished product, the content of peoniflorin is 6.2mg/ sheet.
Embodiment 4: Yikang complement sheet suppresses the experimentation data of mouse leukemia cell L6565 cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse leukemia cell L6565 cell, Shandong University's laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Yikang complement sheet of the present invention: press embodiment 1 method preparation.
Medicinal liquid liquid storage: take 100mg Yikang complement sheet, be dissolved in 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ ldoff 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
DMEM (Cat.No.12100-061Lot.No.758137 of GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); NaHC03 (Cat.No.11810-033Lot.No.1088387 of Shanghai Jiu Yi chemical reagent company limited); 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): the autogamy of PBS(laboratory);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DMIL); Visible-ultraviolet light microwell plate detector (U.S. MD company model: SPECTRAMAX190); C02 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. Sprlng company model: S/N020579); 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); 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) L6565 cell uses DMEM+10%FBS in 37 ℃, 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 3ml0.25% 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 concentration of cell suspension 3 * 10
4individual/ml.
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%C0
2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add Yikang complement 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 low-speed oscillation 10min on shaking table, as entered 200 μ l dimethyl sulfoxide, is put in every hole, 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 disposition
Adopt correlation analysis and Student t check in Microsoft Excel2007 software, data represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, with matched group comparison, when dosage reaches 5mg/ml, to L6565 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.
Table 1 Yikang complement sheet is on L6565 cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 7.64±8.57 |
| 2 | 10 | 19.86±9.78* |
| 3 | 15 | 32.98±15.22** |
| 4 | 20 | 40.07±16.92** |
Note: with matched group comparison, * P<O.01; * P<0.001.
5. experiment conclusion
Yikang complement sheet of the present invention can suppress L6565 cell proliferation, reduces the Growth of Cells number of L6565 cell, and this effect is dose dependent.
Claims (9)
1. the preparation method of a Yikang complement sheet, by Radix Astragali 280g, fiber crops rare 80g, Radix Angelicae Sinensis 190g, Herba Ecliptae 220g, Rhizoma Atractylodis 190g, Flos Carthami 150g, Radix Paeoniae Rubra 140g, Semen Persicae 140g, Radix Achyranthis Bidentatae 150g, Rhizoma Chuanxiong 120g, Fructus Aurantii 70g, Radix Platycodonis 100g, as crude drug, made, it is characterized in that, described preparation method is comprised of the following step: get that fiber crops are rare, Radix Angelicae Sinensis, Rhizoma Atractylodis, Semen Persicae, Rhizoma Chuanxiong, 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 ℃, CO
2flow l-3ml/g crude drug min, extraction time 180-220min, obtains supercritical extract, standby; 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 300, every heavy 0.3g.
2. according to the preparation method of the Yikang complement sheet 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 Yikang complement sheet described in claim l, it is characterized in that described CO
2in supercritical extraction, extracting pressure is 25MPa.
4. according to the preparation method of the Yikang complement sheet described in claim l, it is characterized in that described CO
2in supercritical extraction, extraction temperature is 60 ℃.
5. according to the preparation method of the Yikang complement sheet described in claim l, it is characterized in that described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
6. according to the preparation method of the Yikang complement sheet described in claim l, it is characterized in that described CO
2in supercritical extraction, extraction time is 200min.
7. according to the preparation method of the Yikang complement sheet described in claim l, it is characterized in that, described microwave extracting power is 700W.
8. according to the preparation method of the Yikang complement sheet described in claim l, it is characterized in that, the each extraction time of described microwave extracting is 8 minutes.
9. the Yikang complement sheet described in claim l suppresses the application in mouse leukemia cell L6565 cell proliferation medicine in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104398812A (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 breast tumor cell C127 |
| CN104398813A (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 leukemia cell L6565 |
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2013
- 2013-12-11 CN CN201310671539.5A patent/CN103690647B/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104398812A (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 breast tumor cell C127 |
| CN104398813A (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 leukemia cell L6565 |
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