CN104398810A - 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 neoplastic lymphoid cell P388D1 - Google Patents

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 neoplastic lymphoid cell P388D1 Download PDF

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Publication number
CN104398810A
CN104398810A CN201410638687.1A CN201410638687A CN104398810A CN 104398810 A CN104398810 A CN 104398810A CN 201410638687 A CN201410638687 A CN 201410638687A CN 104398810 A CN104398810 A CN 104398810A
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preparation
extraction
herba swertiae
swertiae bimaculatae
radix
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权栋栋
王洪涛
刘艳辉
权威宇
胡乃合
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Shandong Zhongda Pharmaceutical Co Ltd
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Shandong Zhongda Pharmaceutical Co Ltd
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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 a nine flavor swertia bimaculata tablet. The nine flavor swertia bimaculata tablet is prepared from 53.6g of swertia bimaculata, 17.9g of chrysosplenium, 35.7g of herba aconiti, 35.7g of radix aucklandiae, 35.7g of lagotis brevituba maxim, 42.9g of indian lettuce, 35.7g of hypecoum erectum, 28.6g of cortex berberidis and 14.2g of herpetospermum seed as raw material medicines by supercritical fluid extraction, microwave extraction and macroporous resin adsorption, and the content of berberine hydrochloride is greatly improved. The invention also provides application of the nine flavor swertia bimaculata tablet in preparation of drugs for inhibition of cell proliferation of neoplastic lymphoid cell P388D1 in mice.

Description

The preparation method of nine taste Herba Swertiae bimaculatae sheets and the application in preparation suppression lymph sample oncocyte P388D1 cell proliferation thereof
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method of nine taste Herba Swertiae bimaculatae sheets and suppress the application in mouse lymph sample oncocyte P388D1 cell proliferation in preparation.
Background technology
Nine taste Herba Swertiae bimaculatae capsule standard WS-11182(ZD-1182)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine liver and gall fascicle.Be made up as crude drug of Herba Swertiae bimaculatae 53.6g, Chrysosplenium nudicaule Bunge 17.9g, list loud, high-pitched sound 35.7g, Radix Aucklandiae 35.7g, Radix Pecteilis susannae 35.7g, Herba Ixeritis Denticulatae 42.9g, Radix Hypecol Erecti 35.7g, Radix Berberidis Amurensis 28.6g, Herpetospermum caudigerum Wall. 14.2g, there is heat clearing away, antiinflammatory, effect of pain relieving.For cholecystitis, acute icterohepatitis.
In prior art, nine taste Herba Swertiae bimaculatae capsules are not yet had to adopt CO in extraction preparation 2the report that supercritical extraction, microwave technology and macroporous adsorption resin technology extract, and the method that powder is got directly beaten by Chinese medicine, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, is inconvenient to take, and has had a strong impact on this product and has applied clinically.
In prior art, nine taste Herba Swertiae bimaculatae capsule orals, a 4-5 grain, 2-3 time on the one.Nine taste Herba Swertiae bimaculatae capsule dosages are large.The heavy 0.3g of the every sheet of nine taste Herba Swertiae bimaculatae sheets adopting the inventive method to be prepared into, only needs 2 at every turn, within 1st, takes 2-3 time.Dose is greatly reduced under the condition with more active component.This conclusion can be proved by following test.
The comparison of content of berberine hydrochloride in nine taste Herba Swertiae bimaculatae sheets prepared by test one, distinct methods
L, instrument and reagent the present invention nine taste Herba Swertiae bimaculatae sheet: by the preparation of embodiment 1 method, use 300g crude drug, make 200 through extracting, the heavy 0.3g of every sheet.Former nine taste Herba Swertiae bimaculatae capsules, according to WS-11182(ZD-1182)-2002 standard method preparations, in contrast.Agilent1200 high performance liquid chromatograph; AE240 electronic analytical balance; Berberine hydrochloride reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Acetonitrile-0.05mol/L sodium dihydrogen phosphate (by phosphoric acid adjust ph to 3) (22:78) is mobile phase; Determined wavelength is 347nm.Number of theoretical plate calculates should be not less than 5000 by berberine hydrochloride peak.
The preparation precision of reference substance solution takes berberine hydrochloride reference substance in right amount, adds the solution that methanol makes every 1ml hydrochloric berberine 28 μ g, to obtain final product.
Nine taste Herba Swertiae bimaculatae sheets of the present invention are got in the preparation of product need testing solution of the present invention, and porphyrize, gets 0.1g, accurately weighed, put in tool plug conical flask, precision adds methanol 50ml, weighed weight, supersound process 50 minutes, lets cool, weighed weight again, supplies the weight of less loss, shakes up with methanol, get supernatant, filter with microporous filter membrane (0.45 μm), get subsequent filtrate, to obtain final product.
Nine taste Herba Swertiae bimaculatae capsules of this contrast are got in the preparation of reference substance need testing solution, and porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, precision adds methanol 50ml, weighed weight, supersound process 50 minutes, lets cool, weighed weight again, supplies the weight of less loss, shakes up with methanol, get supernatant, filter with microporous filter membrane (0.45 μm), get subsequent filtrate, to obtain final product.
Algoscopy is accurate respectively draws each 5 μ l of reference substance solution, product need testing solution of the present invention and reference substance need testing solution, injection liquid chromatography, measures, to obtain final product.
3, result
Result shows, in the present invention nine taste Herba Swertiae bimaculatae sheet, the content of berberine hydrochloride is 3.6-7mg/ sheet; And the content of berberine hydrochloride is 0.71mg/ grain in former nine taste Herba Swertiae bimaculatae capsules, every sheet content of berberine hydrochloride is equivalent to the 5-10 of original capsule content doubly, and when dose reduces, content of berberine hydrochloride improves a lot.
Above-mentioned research shows, the nine taste Herba Swertiae bimaculatae sheets adopting preparation method of the present invention to prepare, and active constituent content is far away higher than WS-11182(ZD-1182) the nine taste Herba Swertiae bimaculatae capsules 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 nine taste Herba Swertiae bimaculatae sheets.
Another object of the present invention is to provide a kind of nine taste Herba Swertiae bimaculatae sheets to suppress the application in mouse lymph sample oncocyte P388D1 cell proliferation in preparation.
The object of the invention is by following scheme realize:
A kind of preparation method of nine taste Herba Swertiae bimaculatae sheets, be made up as crude drug of Herba Swertiae bimaculatae 53.6g, Chrysosplenium nudicaule Bunge 17.9g, list loud, high-pitched sound 35.7g, Radix Aucklandiae 35.7g, Radix Pecteilis susannae 35.7g, Herba Ixeritis Denticulatae 42.9g, Radix Hypecol Erecti 35.7g, Radix Berberidis Amurensis 28.6g, Herpetospermum caudigerum Wall. 14.2g, described preparation method is made up of the following step: get the Radix Aucklandiae, Radix Hypecol Erecti, be ground into fine powder, 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 20-30MPa, temperature 30-50 DEG C, CO 2flow l-3ml/g crude drug min, extraction time 130-170min, obtains supercritical extract, for subsequent use, the Radix Aucklandiae after extraction, Radix Hypecol Erecti residue and other seven flavor medicine materials, pulverize, add 60% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extract 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 60% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, by supercritical extract, the mixing of microwave extraction thing adds starch, 60% ethanol granule, dry, tabletting, make 200, the heavy 0.3g of every sheet.
In the preparation method of nine above-mentioned taste Herba Swertiae bimaculatae sheets, described CO 2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
In the preparation method of nine above-mentioned taste Herba Swertiae bimaculatae sheets, described CO 2in supercritical extraction, extracting pressure is 25 MPa.
In the preparation method of nine above-mentioned taste Herba Swertiae bimaculatae sheets, described CO 2in supercritical extraction, extraction temperature is 60 DEG C.
In the preparation method of nine above-mentioned taste Herba Swertiae bimaculatae sheets, described CO 2cO in supercritical extraction 2flow 2ml/g crude drug min.
In the preparation method of nine above-mentioned taste Herba Swertiae bimaculatae sheets, described CO 2in supercritical extraction, extraction time is 150min.
In the preparation method of nine above-mentioned taste Herba Swertiae bimaculatae sheets, described microwave extracting power is 700W.
In the preparation method of nine above-mentioned taste Herba Swertiae bimaculatae sheets, each extraction time of described microwave extracting is 8 minutes.
Nine above-mentioned taste Herba Swertiae bimaculatae sheets suppress the application in mouse lymph sample oncocyte P388D1 cell proliferation in preparation.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art more understands the present invention, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Herba Swertiae bimaculatae 53.6g, Chrysosplenium nudicaule Bunge 17.9g, list loud, high-pitched sound 35.7g, Radix Aucklandiae 35.7g, Radix Pecteilis susannae 35.7g, Herba Ixeritis Denticulatae 42.9g, Radix Hypecol Erecti 35.7g, Radix Berberidis Amurensis 28.6g, Herpetospermum caudigerum Wall. 14.2g: get the Radix Aucklandiae, Radix Hypecol Erecti, be ground into fine powder, 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 150min, obtains supercritical extract, for subsequent use; The Radix Aucklandiae after extraction, Radix Hypecol Erecti residue and other seven flavor medicine materials, pulverize, add 60% ethanol of 2L, 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 Diaion HP-10 macroporous adsorptive resins, 60% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, supercritical extract, the mixing of microwave extraction thing are added starch, 60% ethanol granule, dry, tabletting, make 200, the heavy 0.3g of every sheet.
After testing, in finished product, the content of berberine hydrochloride is 7.05mg/ sheet.
Embodiment 2
Get Herba Swertiae bimaculatae 53.6g, Chrysosplenium nudicaule Bunge 17.9g, list loud, high-pitched sound 35.7g, Radix Aucklandiae 35.7g, Radix Pecteilis susannae 35.7g, Herba Ixeritis Denticulatae 42.9g, Radix Hypecol Erecti 35.7g, Radix Berberidis Amurensis 28.6g, Herpetospermum caudigerum Wall. 14.2g: get the Radix Aucklandiae, Radix Hypecol Erecti, be ground into fine powder, 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 130min, obtains supercritical extract, for subsequent use; The Radix Aucklandiae after extraction, Radix Hypecol Erecti residue and other seven flavor medicine materials, pulverize, add 60% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 60% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, supercritical extract, the mixing of microwave extraction thing are added starch, 60% ethanol granule, dry, tabletting, make 200, the heavy 0.3g of every sheet.
After testing, in finished product, the content of berberine hydrochloride is 3.60mg/ sheet.
Embodiment 3
Get Herba Swertiae bimaculatae 53.6g, Chrysosplenium nudicaule Bunge 17.9g, list loud, high-pitched sound 35.7g, Radix Aucklandiae 35.7g, Radix Pecteilis susannae 35.7g, Herba Ixeritis Denticulatae 42.9g, Radix Hypecol Erecti 35.7g, Radix Berberidis Amurensis 28.6g, Herpetospermum caudigerum Wall. 14.2g: get the Radix Aucklandiae, Radix Hypecol Erecti, be ground into fine powder, 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 20MPa, temperature 30 DEG C, CO 2flow 1ml/g crude drug min, extraction time 170min, obtains supercritical extract, for subsequent use; The Radix Aucklandiae after extraction, Radix Hypecol Erecti residue and other seven flavor medicine materials, pulverize, add 60% ethanol of 2L, 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 Diaion HP-10 macroporous adsorptive resins, 60% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, supercritical extract, the mixing of microwave extraction thing are added starch, 60% ethanol granule, dry, tabletting, make 200, the heavy 0.3g of every sheet.
After testing, in finished product, the content of berberine hydrochloride is 4.88mg/ sheet.
Embodiment 4: nine taste Herba Swertiae bimaculatae sheet suppresses the experimentation data of mouse lymph sample oncocyte P388D1 cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse lymph sample oncocyte P388D1 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention nine taste Herba Swertiae bimaculatae sheet: prepare by embodiment 1 method.
Medicinal liquid liquid storage: take 100mg nine taste Herba Swertiae bimaculatae 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.12100-061 Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHC0 3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033 Lot.No. 1088387 of a specified duration); Trypsin(AMRESCO company); EDTA(AMRESCO company); Penicillin G Sodium Salt(AMRESCO company 1); Streptomycin Sulfate (AMRESCO); Dehydrated alcohol (Zibo Ya Dulan Trade Co., Ltd.); MTT (Biosharp lot number: 0793): PBS(laboratory autogamy);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DMIL); Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX 190); C0 2incubator (FORMA model: 3111); Super-clean bench (safe and sound Inc. of Su Jing group moulding number: SW-CJ-ZFD); Pure water instrument (Sprlng company of U.S. model: S/N 020579); Accurate pipettor (French Gilson Inc model: P2); Electronic balance (German Sai Duolisi company limited model: BT323S); Full-automatic high-pressure autoclave (Japanese SANYO company model: MLS-3020); Table electrothermal air dry oven (the accurate experimental facilities company in Shanghai model: DHG9123A); Refrigerator (Siemens Company's model: KG18V21TI); Liquid nitrogen container (CBS model: 2001); Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai's model: KA-1000); 0.2 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) P388D1 cell DMEM+10%FBS is in 37 DEG C, 5%C0 2carry out cellar culture (10cm culture dish), when Growth of Cells is to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, add 5ml complete medium neutralization reaction wherein, proceeded in centrifuge tube after piping and druming cell, the centrifugal 5min of 1000rpm, adjustment concentration of cell suspension 3 × 10 4individual/ml.
2) enter in 96 well culture plates by cell kind, every hole adds cell suspension 180 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 nine taste Herba Swertiae bimaculatae 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 Excel 2007 software and Student t to check, data represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to P388D1 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 nine taste Herba Swertiae bimaculatae sheet is to P388D1 cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 14.26±7.44
2 10 28.06±10.24*
3 15 40.08±12.98**
4 20 50.04±17.99**
Note: compare with matched group, * P<O.01; * P<0.001.
5. experiment conclusion
Nine taste Herba Swertiae bimaculatae sheets can suppress P388D1 cell proliferation, and reduce the Growth of Cells number of P388D1 cell, this effect is dose dependent.

Claims (9)

1. the preparation method of a taste Herba Swertiae bimaculatae sheet, be made up as crude drug of Herba Swertiae bimaculatae 53.6g, Chrysosplenium nudicaule Bunge 17.9g, list loud, high-pitched sound 35.7g, Radix Aucklandiae 35.7g, Radix Pecteilis susannae 35.7g, Herba Ixeritis Denticulatae 42.9g, Radix Hypecol Erecti 35.7g, Radix Berberidis Amurensis 28.6g, Herpetospermum caudigerum Wall. 14.2g, it is characterized in that, described preparation method is made up of the following step: get the Radix Aucklandiae, Radix Hypecol Erecti, be ground into fine powder, 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 20-30MPa, temperature 30-50 DEG C, CO 2flow l-3ml/g crude drug min, extraction time 130-170min, obtains supercritical extract, for subsequent use, the Radix Aucklandiae after extraction, Radix Hypecol Erecti residue and other seven flavor medicine materials, pulverize, add 60% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extract 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 60% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, by supercritical extract, the mixing of microwave extraction thing adds starch, 60% ethanol granule, dry, tabletting, make 200, the heavy 0.3g of every sheet.
2. the preparation method of nine taste Herba Swertiae bimaculatae sheets 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 nine taste Herba Swertiae bimaculatae sheets according to claim l, is characterized in that, described CO 2in supercritical extraction, extracting pressure is 25MPa.
4. the preparation method of nine taste Herba Swertiae bimaculatae sheets according to claim l, is characterized in that, described CO 2in supercritical extraction, extraction temperature is 60 DEG C.
5. the preparation method of nine taste Herba Swertiae bimaculatae sheets according to claim l, is characterized in that, described CO 2cO in supercritical extraction 2flow 2ml/g crude drug min.
6., according to the preparation method of nine taste Herba Swertiae bimaculatae sheets described in claim l, it is characterized in that, described CO 2in supercritical extraction, extraction time is 150min.
7. the preparation method of nine taste Herba Swertiae bimaculatae sheets according to claim l, it is characterized in that, described microwave extracting power is 700W.
8. the preparation method of nine taste Herba Swertiae bimaculatae sheets according to claim l, it is characterized in that, each extraction time of described microwave extracting is 8 minutes.
9. nine taste Herba Swertiae bimaculatae sheets described in claim l suppress the application in mouse lymph sample oncocyte P388D1 cell proliferation in preparation.
CN201410638687.1A 2014-11-13 2014-11-13 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 neoplastic lymphoid cell P388D1 Pending CN104398810A (en)

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