CN103690647B - A kind of preparation method of Yikang complement sheet and the application in suppression mouse leukemia cell L6565 cell proliferation thereof - Google Patents
A kind of preparation method of Yikang complement sheet and the application in suppression mouse leukemia cell L6565 cell proliferation thereof 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 abstract description 12
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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 Yikang complement sheet.The preparation method of Yikang complement sheet of the present invention, be made up as crude drug of 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, adopt supercritical extraction and microwave extracting to be prepared from, paeoniflorin content is improved a lot.Present invention also offers the application of Yikang complement sheet in preparation suppression mouse leukemia cell L6565 cell proliferation.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method of Yikang complement sheet and suppressing the application in mouse leukemia cell L6565 cell proliferation.
Background technology
Yikang complement particulate level WS-10983(ZD-0983)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine taste fascicle.Be made up as crude drug of 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, there is benefiting QI for activating blood circulation, effect of strengthening spleen, tonifying kidney.For the spiritlessness and weakness that blood stasis due to qi deficiency, deficiency of spleen and stomach cause, short breath, insomnia, soreness of the waist and knees, lack of appetite is forgetful waits disease.
In prior art, Yikang complement granule is not yet had to extract the report adopting supercritical and microwave technology in preparation, and volatile oil is drawn, the method of soak by water, 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.
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 needs to add a large amount of sugar, and diabetics is not suitable for, and the words taste of not sugaring is not good, and under patient is not easy clothes, compliance is poor.The heavy 0.3g of the every sheet of the Yikang complement 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.And being prepared into tablet, patient swallows with water, and 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: by the preparation of embodiment 1 method, use 1510g crude drug, makes 300 through extracting, the heavy 0.3g of every sheet.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
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; Acetonitrile-0.2% phosphate aqueous solution (15: 85) is mobile phase; Determined wavelength is 230nm.Number of theoretical plate calculates should be not less than 3000 by peoniflorin peak.
The preparation of reference substance solution learns from else's experience the phosphorus pentoxide drying under reduced pressure peoniflorin reference substance of 36 hours in right amount, accurately weighed, adds methanol and makes the solution of every 1ml containing 0.1mg, to obtain final product.
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, 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, supply the weight of less loss with methanol, shake up, filter, filter with microporous filter membrane (0.45 μm), to obtain final product.。
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, 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, supply the weight of less loss with methanol, shake up, filter, filter with microporous filter membrane (0.45 μm), to obtain final product.
Algoscopy is accurate respectively draws reference substance solution and each 10 μ L of need testing solution, injection liquid chromatography, measures, to obtain final product.
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, the paeoniflorin content each serving consumption 2 is 2-4 times of former granule content, and when dose reduces, paeoniflorin content improves a lot.
Above-mentioned research shows, adopt Yikang complement sheet prepared by preparation method of the present invention, active constituent content is far away higher than WS-10983(ZD-0983) the Yikang complement granule 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 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 in preparation.
The object of the invention is by following scheme realize:
A kind of preparation method of Yikang complement sheet, be made up as crude drug of 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, described preparation method is made up of the following step: get numb 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 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 300, the heavy 0.3g of every sheet.
In the preparation method of above-mentioned Yikang complement 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 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 DEG C.
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, 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 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
Seldom 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 to get Radix Astragali 280g, fiber crops
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 300, the heavy 0.3g of every sheet.
After testing, in finished product, the content of peoniflorin is 7.8mg/ sheet.
Embodiment 2
Seldom 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 to get Radix Astragali 280g, fiber crops
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 300, the heavy 0.3g of every sheet.
After testing, in finished product, the content of peoniflorin is 5.6mg/ sheet.
Embodiment 3
Seldom 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 to get Radix Astragali 280g, fiber crops
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 300, the heavy 0.3g of every sheet.
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 storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Yikang complement sheet of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: take 100mg Yikang complement sheet, be dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, simultaneously 0.2 μm of frit dehydrated alcohol is in order to the use of matched group.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHC03 (Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.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: SPECTRAMAX190); C02 incubator (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/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 (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 μm of 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 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 3ml0.25% 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 μ 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 Yikang complement sheet solution, continue to cultivate 24h.
4) add 20 μ 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 μ 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 Excel2007 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 L6565 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 Yikang complement sheet is to L6565 cell inhibitory effect influence 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: compare with matched group, * P<O.01; * P<0.001.
5. experiment conclusion
Yikang complement sheet of the present invention can suppress L6565 cell proliferation, and reduce the Growth of Cells number of L6565 cell, this effect is dose dependent.
Claims (7)
1. the application of Yikang complement sheet in preparation suppression mouse leukemia cell L6565 cell proliferation, it is characterized in that, described Yikang complement sheet is made up as crude drug of 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, described Yikang complement piece preparation method is made up of the following step: get numb 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 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 300, the heavy 0.3g of every sheet.
2. the Yikang complement sheet according to claim l suppresses the application in mouse leukemia cell L6565 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 Yikang complement sheet according to claim l suppresses the application in mouse leukemia cell L6565 cell proliferation in preparation, it is characterized in that, described CO
2in supercritical extraction, extracting pressure is 25 MPa.
4. the Yikang complement sheet according to claim l suppresses the application in mouse leukemia cell L6565 cell proliferation in preparation, it is characterized in that, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
5. the Yikang complement sheet according to claim l suppresses the application in mouse leukemia cell L6565 cell proliferation in preparation, it is characterized in that, described CO
2in supercritical extraction, extraction time is 200min.
6. the Yikang complement sheet according to claim l suppresses the application in mouse leukemia cell L6565 cell proliferation in preparation, and it is characterized in that, described microwave extracting power is 700W.
7. the Yikang complement sheet according to claim l suppresses the application in mouse leukemia cell L6565 cell proliferation in preparation, and it is characterized in that, each extraction time of described microwave extracting is 8 minutes.
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