CN104095963B - A kind of preparation method of rhodobryum roseum tablet and the application in suppression anus sarcoma cell S-180 cell proliferation thereof - Google Patents
A kind of preparation method of rhodobryum roseum tablet and the application in suppression anus sarcoma cell S-180 cell proliferation thereof 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 rhodobryum roseum tablet.The preparation method of rhodobryum roseum tablet of the present invention, be made up as crude drug of Ramulus Uncariae Cum Uncis 780g, Rhodobryum roseum Limpr. 910g, Radix Polygoni Multiflori 26g, Radix Astragali 260g, Radix Notoginseng 26g, Fructus Crataegi 650g, Radix Glycyrrhizae 260g, adopt supercritical extraction and microwave extracting to be prepared from, make ginsenoside Rg
1content improves a lot.Present invention also offers the application of rhodobryum roseum tablet in preparation suppression mice anus sarcoma cell S-180 cell proliferation.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method of rhodobryum roseum tablet and suppressing the application in mice anus sarcoma cell S-180 cell proliferation.
Background technology
Return heart recovering capsule standard No. WS-10737 (ZD-0737)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine and feel concerned about fascicle.Be made up as crude drug of Ramulus Uncariae Cum Uncis 780g, Rhodobryum roseum Limpr. 910g, Radix Polygoni Multiflori 26g, Radix Astragali 260g, Radix Notoginseng 26g, Fructus Crataegi 650g, Radix Glycyrrhizae 260g, there is benefiting QI for activating blood circulation, effect of calm suppressing the hyperactive liver.For blood stasis due to qi deficiency, the thoracic obstruction that excessive rising of liver-YANG causes, dizzy, disease sees the thoracic obstruction, uncomfortable in chest, cardiopalmus, dizzy grade for disease, coronary heart disease, hypertension belongs to above-mentioned patient.
In prior art, not yet have back heart recovering capsule extracting the report adopting supercritical and microwave technology in preparation, and powder directly beaten by Chinese medicine, 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.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of preparation method of rhodobryum roseum tablet.
Another object of the present invention is to provide a kind of rhodobryum roseum tablet to suppress the application in mice anus sarcoma cell S-180 cell proliferation in preparation.
The object of the invention is by following scheme realize:
A kind of preparation method of rhodobryum roseum tablet, be made up as crude drug of Ramulus Uncariae Cum Uncis 780g, Rhodobryum roseum Limpr. 910g, Radix Polygoni Multiflori 26g, Radix Astragali 260g, Radix Notoginseng 26g, Fructus Crataegi 650g, Radix Glycyrrhizae 260g, described preparation method is made up of the following step: get, and Radix Notoginseng, Radix Polygoni Multiflori, Rhodobryum roseum Limpr., 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 60% ethanol of 10L, 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, 60% 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 400, the heavy 0.3g of every sheet.
In the preparation method of above-mentioned rhodobryum roseum tablet, described CO
2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
In the preparation method of above-mentioned rhodobryum roseum tablet, described CO
2in supercritical extraction, extracting pressure is 25MPa.
In the preparation method of above-mentioned rhodobryum roseum tablet, described CO
2in supercritical extraction, extraction temperature is 40 DEG C.
In the preparation method of above-mentioned rhodobryum roseum tablet, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
In the preparation method of above-mentioned rhodobryum roseum tablet, described CO
2in supercritical extraction, extraction time is 200min.
In the preparation method of above-mentioned rhodobryum roseum tablet, described microwave extracting power is 700W.
In the preparation method of above-mentioned rhodobryum roseum tablet, each extraction time of described microwave extracting is 8 minutes.
Above-mentioned rhodobryum roseum tablet suppresses the application in mice anus sarcoma cell S-180 cell proliferation in preparation.
In prior art, return each 5 of heart recovering capsule, 3 times on the one.Return heart recovering capsule dosage large.The heavy 0.3g of the every sheet of the rhodobryum roseum tablet 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 Determination of Content of Ginsenoside Rg_1 in rhodobryum roseum tablet prepared by test one, distinct methods
L, instrument and reagent rhodobryum roseum tablet of the present invention: by the preparation of embodiment 1 method, use 3012g crude drug, makes 400 through extracting, the heavy 0.3g of every sheet.Former time heart recovering capsule, prepares according to WS-10737 (ZD-0737)-2002 standard method.Agilent1200 high performance liquid chromatograph; METTLERAE240 electronic analytical balance; Ginsenoside Rg1's reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VD).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; With acetonitrile-water (20:80) for mobile phase; Determined wavelength is 203nm.Number of theoretical plate calculates should be not less than 6000 by ginsenoside Rg1 1 peak.
The preparation of reference substance solution is taken at the 60 DEG C of drying under reduced pressure ginsenoside Rg1 of 2 hours 1 reference substances in right amount, accurately weighed, adds methanol and makes the solution of every 1ml containing 0.5mg, to obtain final product.
Rhodobryum roseum tablet of the present invention is got in the preparation of product need testing solution of the present invention, porphyrize, accurately weighed, porphyrize, get 1.2g, accurately weighed, put in apparatus,Soxhlet's, add diethyl ether appropriate, reflux 1 hour, discard ether solution, ether flung to by medicinal residues, put in apparatus,Soxhlet's, add methanol appropriate, heating and refluxing extraction is to colourless, methanol solution evaporate to dryness, residue adds water, and 20ml is warm makes dissolving, let cool, 4 times are extracted with water saturated n-butyl alcohol jolting, each 30ml, merge n-butanol extracting liquid, 2 times are extracted with 1% sodium hydroxide solution jolting, each 30ml, discard alkali liquor, the saturated water washing of n-butyl alcohol liquid n-butyl alcohol 2 times, each 30ml, discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 15ml, the warm dissolving of gradation, let cool, by processed good D101 type macroporous resin column (internal diameter 1.5cm, long 15cm), with water 50ml eluting, discard water liquid, use 20% ethanol 50ml eluting again, discard 20% ethanol elution, continue with 70% ethanol 100ml eluting, collect eluent, evaporate to dryness, residue adds methanol to be made dissolving and is quantitatively transferred in 10ml measuring bottle, add methanol to scale, shake up, obtain.
40 (Film coated tablets removing coatings) of this contrast are got in the preparation of reference product need testing solution, accurately weighed, porphyrize, get 3g, accurately weighed, put in apparatus,Soxhlet's, add diethyl ether appropriate, reflux 1 hour, discard ether solution, ether flung to by medicinal residues, put in apparatus,Soxhlet's, add methanol appropriate, heating and refluxing extraction is to colourless, methanol solution evaporate to dryness, residue adds water, and 20ml is warm makes dissolving, let cool, 4 times are extracted with water saturated n-butyl alcohol jolting, each 30ml, merge n-butanol extracting liquid, 2 times are extracted with 1% sodium hydroxide solution jolting, each 30ml, discard alkali liquor, the saturated water washing of n-butyl alcohol liquid n-butyl alcohol 2 times, each 30ml, discard water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 15ml, the warm dissolving of gradation, let cool, by processed good D101 type macroporous resin column (internal diameter 1.5cm, long 15cm), with water 50ml eluting, discard water liquid, use 20% ethanol 50ml eluting again, discard 20% ethanol elution, continue with 70% ethanol 100ml eluting, collect eluent, evaporate to dryness, residue adds methanol to be made dissolving and is quantitatively transferred in 10ml measuring bottle, add methanol to scale, shake up, obtain.
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 rhodobryum roseum tablet of the present invention, the content of ginsenoside Rg1 is 2-4mg/ sheet; And the content of ginsenoside Rg1 is 0.38mg/ bag in former time heart recovering capsule, the Determination of Content of Ginsenoside Rg_1 each serving consumption 2 is 2-4 times of former granule content, and when dose reduces, Determination of Content of Ginsenoside Rg_1 improves a lot.
Above-mentioned research shows, adopts rhodobryum roseum tablet prepared by preparation method of the present invention, and prepared by the method that active constituent content is recorded higher than WS-10737 (ZD-0737)-2002 standard far away returns heart recovering capsule.
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
Hook taking rattan 780g, Rhodobryum roseum Limpr. 910g, Radix Polygoni Multiflori 26g, Radix Astragali 260g, Radix Notoginseng 26g, Fructus Crataegi 650g, Radix Glycyrrhizae 260g, get Radix Notoginseng, Radix Polygoni Multiflori, Rhodobryum roseum Limpr. 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, 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 60% ethanol of 10L, 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, 60% 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 400, the heavy 0.3g of every sheet.
After testing, in finished product, the content of ginsenoside Rg1 is 3.89mg/ sheet.
Embodiment 2
Hook taking rattan 780g, Rhodobryum roseum Limpr. 910g, Radix Polygoni Multiflori 26g, Radix Astragali 260g, Radix Notoginseng 26g, Fructus Crataegi 650g, Radix Glycyrrhizae 260g, get Radix Notoginseng, Radix Polygoni Multiflori, Rhodobryum roseum Limpr. 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, for subsequent use; Get all the other Chinese medicines, pulverize, add 60% ethanol of 10L, 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, 60% 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 400, the heavy 0.3g of every sheet.
After testing, in finished product, the content of ginsenoside Rg1 is 2.59mg/ sheet.
Embodiment 3
Hook taking rattan 780g, Rhodobryum roseum Limpr. 910g, Radix Polygoni Multiflori 26g, Radix Astragali 260g, Radix Notoginseng 26g, Fructus Crataegi 650g, Radix Glycyrrhizae 260g, get Radix Notoginseng, Radix Polygoni Multiflori, Rhodobryum roseum Limpr. 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, 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 60% ethanol of 10L, 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, 60% 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 400, the heavy 0.3g of every sheet.
After testing, in finished product, the content of ginsenoside Rg1 is 1.98mg/ sheet.
Embodiment 4: rhodobryum roseum tablet suppresses the experimentation data of mice anus sarcoma cell S-180 cell proliferation
1. experiment material
1.1 experiment cell strains
Mice anus sarcoma cell S-180 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: rhodobryum roseum tablet of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: take 100mg rhodobryum roseum tablet, is dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and 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); NaHC0
3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration); Trypsin (AMRESCO company); EDTA (AMRESCO company); PenicillinGSodiumSalt (AMRESCO company 1); StreptomycinSulfate (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); 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/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) L5178Y 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 rhodobryum roseum tablet solution, continue to cultivate 24h.
4) add 20 μ lMTT 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 MicrosoftExcel2007 software and Studentt inspection, 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 L5178Y 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 rhodobryum roseum tablet is to L5178Y cell inhibitory effect influence research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 9.56±7.47 |
| 2 | 10 | 17.98±9.18* |
| 3 | 15 | 33.62±13.51** |
| 4 | 20 | 44.75±16.09** |
Note: compare with matched group, * P<O.01; * P<0.001.
5. experiment conclusion
Rhodobryum roseum tablet of the present invention can suppress L5178Y cell proliferation, and reduce the Growth of Cells number of L5178Y cell, this effect is dose dependent.
Claims (9)
1. the preparation method of a rhodobryum roseum tablet, be made up as crude drug of Ramulus Uncariae Cum Uncis 780g, Rhodobryum roseum Limpr. 910g, Radix Polygoni Multiflori 26g, Radix Astragali 260g, Radix Notoginseng 26g, Fructus Crataegi 650g, Radix Glycyrrhizae 260g, it is characterized in that, described preparation method is made up of the following step: get Radix Notoginseng, Radix Polygoni Multiflori, Rhodobryum roseum Limpr., 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 60% ethanol of 10L, 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, 60% 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 400, the heavy 0.3g of every sheet.
2. the preparation method of the rhodobryum roseum tablet according to claim l, is characterized in that, described CO
2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
3. the preparation method of the rhodobryum roseum tablet according to claim l, is characterized in that, described CO
2in supercritical extraction, extracting pressure is 25MPa.
4. the preparation method of the rhodobryum roseum tablet according to claim l, is characterized in that, described CO
2in supercritical extraction, extraction temperature is 40 DEG C.
5. the preparation method of the rhodobryum roseum tablet according to claim l, is characterized in that, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
6. the preparation method of the rhodobryum roseum tablet according to claim l, is characterized in that, described CO
2in supercritical extraction, extraction time is 200min.
7. the preparation method of the rhodobryum roseum tablet according to claim l, is characterized in that, described microwave extracting power is 700W.
8. the preparation method of the rhodobryum roseum tablet according to claim l, is characterized in that, each extraction time of described microwave extracting is 8 minutes.
9. the rhodobryum roseum tablet described in claim l suppresses the application in mice anus sarcoma cell S-180 cell proliferation in preparation.
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| Title |
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| 回心康片对大鼠实验性心肌缺血的保护作用;雷秀玲等;《中国民族民间医药杂志》;19991231(第03期);163-165,186 * |
| 回心康片抗心肌细胞缺氧损伤的作用及机制;蔡小军等;《中国医院药学杂志》;20121231;第32卷(第14期);第1118-1121页 * |
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