CN101088520B - Medicine composition for auxiliary treatment of cancer and its prepn and quality control method - Google Patents
Medicine composition for auxiliary treatment of cancer and its prepn and quality control method Download PDFInfo
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Abstract
The present invention provides one kind of medicine composition for auxiliary treatment of cancer and its preparation and quality control method. The medicine composition for auxiliary treatment of cancer is prepared with Dangshen and astragalus root as material. It features that the high performance liquid phase fingerprint has at least 6 absorption peaks, including 3 with single peak area over 10 % of the total peak area.
Description
Technical field
The present invention relates to a kind of cancer adjuvant therapy medicament compositions, its preparation and method of quality control of being used for, particularly a kind of is cancer adjuvant therapy medicament compositions, its preparation and the method for quality control of raw material with the Chinese herbal medicine.
Background technology
Radix Astragali source leguminous plant Radix Astagali A.membranaceus (Fisch.) Bge.var.mongholicus Bge.Hsiao dry root, the Qi-tonifying drug for traditional has invigorating QI to consolidate the body surface resistance, diuresis evacuation of pus, the effect of expelling pus and promoting granulation.Tradition is used to improve effects such as body immunity, also is used to QI invigorating simultaneously, such as fatigue, lacks symptoms such as energy.The chemical constitution study report of the Radix Astragali is a lot, therefrom isolates polysaccharide, saponin, flavone, aminoacid, trace element etc.Modern age, pharmacological research proved, astragalus polysaccharides (polysaccharides) and Radix Astragali saponin (total Astragloside) are main component wherein.Wherein, (Astragaluspolysaccharids APS) has effects such as the immunologic function of promotion, raising macrophage activity, inhibition EAS, two-ways regulation blood glucose to astragalus polysaccharides.Radix Codonopsis is the dry root of campanulaceae plant Radix Codonopsis Codonopsis pilosula (Franch.) Nannf., has invigorating the spleen and replenishing QI, the effect that spleen invigorating is promoted the production of body fluid.Radix Codonopsis contains polysaccharide, saponin, volatilization wet goods.
With the Radix Codonopsis and the Radix Astragali is that the SHENQI FUZHENG ZHUSHEYE of feedstock production is the wider Chinese medicine preparation of present clinical practice; it is formed by the Radix Codonopsis and the Radix Astragali prescription of equivalent; late tumor patient to qi-deficiency type has good curative effect clinically; effect with strengthening the body resistance, QI invigorating tonify deficiency; can help the cancer patient to finish radiotherapy, chemotherapy smoothly; reduce the toxic and side effects of chemotherapeutics; the hemopoietic function of protection body; patient's hemogram is raise; improve immunologic function and symptom of digestive tract etc.; improve patient's life quality, and can treat diseases such as the heart, cerebrovascular.
Chinese medicine fingerprint is meant chromatograph or spectrographic collection of illustrative plates common, that have distinctive certain class or number constituents in certain Chinese crude drug or the Chinese patent medicine.Do not have under the clear and definite situation in the present stage Effective Components of Chinese Herb overwhelming majority, Chinese medicine fingerprint has great importance for the quality of effective control Chinese crude drug or Chinese patent medicine.The Japan main manufacturing enterprise of Chinese prescription medicine just adopts the high-efficiency liquid-phase fingerprint control of quality in enterprises in the eighties in 20th century.Germany, France find that the medical function of Folium Ginkgo extract is extract gained material group's mass action result in the process that Folium Ginkgo extract is developed jointly, and to the quality control of such integral body, also adopt the high-efficiency liquid-phase fingerprint method.In the plant medical herbs guide of formulating U.S. FDA recent years clearly the method for quality control (FDA.Guidance of Industry:Botanical Drug (Draft) .2000August) of finger printing as the compounding substances group.Along with going deep into of research, it is found that, as the product of putting into practice of theory of Chinese medical science, Chinese medicine, especially herbal mixture, wherein contained arbitrary composition all can not be represented its whole curative effect.People recognize that gradually the existing quality standard with reference to Western medicine (synthetic drug) quality control pattern can not reflect the intrinsic quality of Chinese medicine rightly.From development trend, from existing quality control pattern to a kind of comprehensive, macroscopic, quantifiable discriminating combines with main active constituent content measuring is the trend that develops.
The working standard of medicine quality evaluated is to utilize spectrum or the discriminating of chromatograph means and measure a certain or several effective ingredient, active component or index components, and the routine examination project of pharmacopeia regulation.Record 602 kinds of medical materials and patent medicine kind altogether as Chinese Pharmacopoeia 2000 version [an one].Wherein have 992 thin layer chromatographys to differentiate that 308 kinds have assay (volumetric method, spectrographic method, liquid chromatography, gas chromatography and TLC scanning method), most of kinds have general inspection item.Obviously. the setting of these quality standards is the patterns of having imitated chemical drugs.The German medical herbs monograph that other country edits as the HANYAO in Britain, India, U.S.'s medical herbs allusion quotation, the Pharmacopeia of Japan and German CommissionE etc. has also adopted essentially identical content.For chemical drugs, its active ingredient is the unification compound of clear in structure, and structure activity relationship is clear and definite, and its content and purity are directly expressed it and effectively reached safety.Yet the characteristics of middle medical drugs are compound compatibilities, and any single content height effective or active component all can not be expressed its whole curative effect.For example, the contained astragaloside (aastraga losideIV) of the Radix Astragali is the current discriminating of quality standard and the most common target of assay of being selected as, but not according to clearly getting in touch that the function that proves the astragaloside and the Radix Astragali cures mainly.Equally, Rhizoma Coptidis, Cortex Phellodendri, Radix Berberidis all contain girder alkali, and be general all with its target as detection, completely different but three's function cures mainly.The situation of compound preparation is just complicated more.The traditional Chinese medical science is this not to be that man-to-man nonlinear theory and practice explanation Chinese medicine quality should adopt certain macroscopic comprehensive quality evaluation means.
SHENQI FUZHENG ZHUSHEYE is that primary raw material is made by Radix Codonopsis, the Radix Astragali, and its therapeutic effect has obtained clinical checking, and whether can guarantee that the quality of medicine is the basis of its curative effect of decision.If with one, two kind of active component illustrate its inherent quality, has certain one-sidedness, said nothing of the index components of no drug effect.Control its effect, only at one, two chemical constituents characterize and control is not enough, must be controlled its material group integral body.So, except " micro analysis ", also should characterize Chinese medicine quality on the whole effectively with certain " macroscopic analysis " method.Finger printing is become a consensus of the international community at present as Chinese herbal medicine and extraction of substance amount control method thereof.Now, more to the assay method of active component in the SHENQI FUZHENG ZHUSHEYE, but how can the macroscopic quality control method that it carries out quality control not appeared in the newspapers as yet from more macroscopic angle.
Summary of the invention
The purpose of this invention is to provide a kind of cancer adjuvant therapy medicament compositions, its preparation and method of quality control of being used for.
The pharmaceutical composition that is used for assistant treating cancer of the present invention, with the Radix Codonopsis and the Radix Astragali is that raw material is made, it is characterized in that having 6 absworption peaks at least in the high-efficiency liquid-phase fingerprint of described compositions, the absworption peak that wherein unimodal area surpasses total peak area 10% has 3, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%.
Pharmaceutical composition of the present invention preferably, has 6 absworption peaks at least in the finger printing of compositions, the absworption peak that wherein unimodal area surpasses total peak area 5% has 5, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 2 peaks, average retention time RT is 19.439min, and RSD is 0%, and peak area is 358.374, and RSD is 1.08%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%;
No. 6 peaks, average retention time RT is 48.72min, and RSD is 0%, and peak area is 538.678, and RSD is 5.42%.
Pharmaceutical composition of the present invention more preferably, has 6 absworption peaks at least in the finger printing, the absworption peak that wherein unimodal area surpasses total peak area 2% has 6, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 2 peaks, average retention time RT is 19.439min, and RSD is 0%, and peak area is 358.374, and RSD is 1.08%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 4 peaks, average retention time RT is 31.238min, and RSD is 0%, and peak area is 145.988, and RSD is 0.13%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%;
No. 6 peaks, average retention time RT is 48.72min, and RSD is 0%, and peak area is 538.678, and RSD is 5.42%.
Pharmaceutical composition of the present invention can be as required, add or do not add pharmaceutically acceptable carrier, make any type of pharmaceutical preparation, as tablet, capsule, powder, pill, oral liquid, injection, infusion solution or freeze-dried powder etc., preferred injection, infusion solution or freeze-dried powder.
The detection of the compositions of medicine of the present invention or the finger printing of preparation adopts following method to carry out:
According to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 D), measure in conjunction with the requirement of finger printing.
Chromatographic condition and system suitability test Diamonsil C
18Post (Di Ma company), granularity 5 μ m, volume 250 * 4.6mm; Mobile phase of acetonitrile and water linear gradient elution (seeing the following form); 30 ℃ of column temperatures; Flow velocity 1ml/min; The detection wavelength is 270nm; Writing time: 55 minutes.Number of theoretical plate calculates by maximum peak should be not less than 3000.
The linear gradient elution proportion of mobile phase changes program
The preparation of need testing solution
Get 1 bottle of this product, add water 5ml and make dissolving, shake up, microporous filter membrane (0.45 μ m) filters, and filtrate is as need testing solution.
The accurate need testing solution 20 μ l that draw of algoscopy inject chromatograph of liquid, measure.
The test sample finger printing compares with finger printing with contrast, and machine simulation similarity software for calculation " chromatographic fingerprints of Chinese materia medica similarity evaluation system " (Chinese Pharmacopoeia can be published, version 2 004A or B) calculates as calculated, and similarity should be between 0.90～1.00.
The method that comprises the steps that adopts of pharmaceutical composition of the present invention and preparation thereof prepares:
(1) get the Radix Codonopsis and the Radix Astragali decoction pieces of equivalent, add low first alcohol reflux and extract, extracting solution and medicinal residues, extracting solution is collected eluent and concentrate drying and is got Radix Codonopsis, Radix Astragali total saponins extract through low pole macroporous adsorptive resins purification;
(2) behind the medicinal residues removal solvent that step (1) obtains, through water carry, the low unit alcohol precipitation of 50-80%, the be dissolved in water low unit alcohol precipitation of back reuse 30-40% of precipitation is got supernatant, remove albumen with trichloroacetic acid method, further, get crude polysaccharides, behind water dissolution with the low unit alcohol precipitation of 70-90%, the employing molecular weight that dams is that 3000 film carries out ultrafiltration, concentrated solution further with the low unit alcohol precipitation of 70-90%, is got precipitation, and drying and crushing gets Radix Codonopsis, Radix Astragali Mongolici total polysaccharide extract.
(3) Radix Codonopsis, Radix Astragali total saponins extract and Radix Codonopsis, the Radix Astragali Mongolici total polysaccharide extract of combining step (1) and step (2) add suitable pharmaceutically acceptable carrier, make freeze-dried powder.
The notion of low unit alcohol herein is the same, preferred alcohol.
The used low pole macroporous resin of described step (1) can be low pole macroporous resin any routine or known, for example Chang Yong D
101Type macroporous adsorbent resin, AB-8 type macroporous adsorbent resin, ZTC-1 type macroporous adsorbent resin and DidaionHP20 type macroporous adsorbent resin etc., preferred AB-8 type macroporous adsorbent resin and D
101Type macroporous adsorbent resin, more preferably D
101The type macroporous adsorbent resin adopts the purge process of resin to carry out according to this area routine or known method.
Preparation method provided by the invention preferably may further comprise the steps:
(1) gets Radix Codonopsis and Radix Astragali decoction pieces, add the 60-80% ethanol of doubly measuring with 6-10, reflux, extract, 1-3 time, each 1.5-3 hour, get extracting solution and medicinal residues, extracting solution is collected eluent and concentrate drying and is got Radix Codonopsis, Radix Astragali total saponins extract through D101 type macroporous adsorptive resins purification;
(2) behind the medicinal residues removal solvent that step (1) obtains, add the water that 6-10 doubly measures, in 60-100 ℃, lixiviate 1-3 time, each 1.5-3 hour, the ethanol precipitation of 60-80%, the ethanol precipitation of reuse 30-40% was got supernatant after precipitation was dissolved in water, and removed albumen, further use the ethanol precipitation of 70-90%, get crude polysaccharides, with ultrafiltration behind the water dissolution, concentrated solution is further used the ethanol precipitation of 70-90%, get precipitation, drying and crushing gets Radix Codonopsis, Radix Astragali Mongolici total polysaccharide extract.
(3) Radix Codonopsis, Radix Astragali total saponins extract and Radix Codonopsis, the Radix Astragali Mongolici total polysaccharide extract of combining step (1) and step (2) add suitable pharmaceutically acceptable carrier, make freeze-dried powder.
The present invention also provides the method for quality control of described pharmaceutical composition and preparation thereof, comprises the steps:
(a) foundation of reference fingerprint
Get described pharmaceutical composition or preparation control sample, be dissolved in water, shake up, filter with 0.45 μ m microporous filter membrane, filtrate is product solution in contrast;
(b) mensuration of reference fingerprint:
Draw above-mentioned control sample solution and inject chromatograph of liquid, use high performance liquid chromatography to measure, obtain reference fingerprint, chromatographic condition: chromatographic column is filler with the octadecylsilane chemically bonded silica; Mobile phase is: the acetonitrile-water linear gradient elution, and the detection wavelength is 250-280nm;
(c) mensuration of product fingerprint collection of illustrative plates to be measured:
Get product to be measured, measure the finger printing of this product to be measured according to Step By Condition described in above-mentioned (a);
(d) described product fingerprint collection of illustrative plates to be measured and described reference fingerprint are compared, discern the quantity of the two common absworption peak that has, to determine whether product quality is qualified.
Preferred chromatographic condition is in described (a) step: the flow velocity 0.5～1.5ml/min of mobile phase, detect wavelength 265～275nm, 20～40 ℃ of column temperatures.
Following method is adopted in the foundation of preferred codonopsis pilosula finger printing:
(a) foundation of reference fingerprint
Get described pharmaceutical composition or preparation control sample, be dissolved in water, shake up, filter with 0.45 μ m microporous filter membrane, filtrate is product solution in contrast;
(b) mensuration of reference fingerprint:
Draw above-mentioned control sample solution and inject chromatograph of liquid, use high performance liquid chromatography to measure, obtain reference fingerprint, chromatographic condition: Diamonsil C
18Post (Di Ma company), granularity 5 μ m, volume 250 * 4.6mm; Mobile phase of acetonitrile and water linear gradient elution (seeing the following form); 30 ℃ of column temperatures; Flow velocity 1ml/min; The detection wavelength is 270nm; Writing time: 55 minutes.Number of theoretical plate calculates by maximum peak should be not less than 3000.
The linear gradient elution proportion of mobile phase changes program
(c) mensuration of product fingerprint collection of illustrative plates to be measured:
Get product to be measured, measure the finger printing of this product to be measured according to Step By Condition described in above-mentioned (a);
(d) described product fingerprint collection of illustrative plates to be measured and described reference fingerprint are compared, discern the quantity of the two common absworption peak that has, to determine whether product quality is qualified.
In the foundation of reference fingerprint, in the reference fingerprint of the use codonopsis pilosula that high effective liquid chromatography for measuring obtained 6 absworption peaks are arranged, the absworption peak that wherein unimodal area surpasses total peak area 10% has 3, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%.
In the foundation of reference fingerprint, in the reference fingerprint of the use codonopsis pilosula that high effective liquid chromatography for measuring obtained 6 absworption peaks are arranged, the absworption peak that wherein unimodal area surpasses total peak area 5% has 5, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 2 peaks, average retention time RT is 19.439min, and RSD is 0%, and peak area is 358.374, and RSD is 1.08%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%;
No. 6 peaks, average retention time RT is 48.72min, and RSD is 0%, and peak area is 538.678, and RSD is 5.42%.
In the foundation of reference fingerprint, in the reference fingerprint of the use codonopsis pilosula that high effective liquid chromatography for measuring obtained 6 absworption peaks are arranged, the absworption peak that wherein unimodal area surpasses total peak area 2% has 6, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 2 peaks, average retention time RT is 19.439min, and RSD is 0%, and peak area is 358.374, and RSD is 1.08%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 4 peaks, average retention time RT is 31.238min, and RSD is 0%, and peak area is 145.988, and RSD is 0.13%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%;
No. 6 peaks, average retention time RT is 48.72min, and RSD is 0%, and peak area is 538.678, and RSD is 5.42%.
The present invention adopts the quality of following method control pharmaceutical composition and preparation, get testing combination or preparation, adopt with the identical method of preparation method, chromatographic condition, assay method of control sample and measure, obtain finger printing, with compositions or preparation product fingerprint collection of illustrative plates to be measured and reference fingerprint comparison, when the two finger printing has absworption peak identical more than 3 or 3, think that just product to be measured is qualified products.
Preferably, codonopsis pilosula product fingerprint collection of illustrative plates to be measured and described codonopsis pilosula reference fingerprint relatively when the two finger printing has absworption peak identical more than 4 or 4, are thought the up-to-standard of described codonopsis pilosula product to be measured.
Comparatively preferably, codonopsis pilosula product fingerprint collection of illustrative plates to be measured and described codonopsis pilosula reference fingerprint relatively when the two finger printing has absworption peak identical more than 5 or 5, are thought the up-to-standard of described codonopsis pilosula product to be measured.
The most preferably, tested codonopsis pilosula product fingerprint collection of illustrative plates and contrast codonopsis pilosula finger printing relatively when the two finger printing has 6 identical absworption peaks, are thought the up-to-standard of described codonopsis pilosula product to be measured.
The present invention does the various composition fingerprint graphs in pharmaceutical composition or the preparation as a wholely to treat, pay attention to each front and back that constitute fingerprint characteristic peak order and mutual relation, pay attention to whole facial feature, avoided judging the one-sidedness of its total quality because of only measuring one, two chemical constituent, for complete, accurately estimate its quality new reference standard be provided.
The present invention has that method is easy, stable, precision is high, favorable reproducibility, the characteristics that are easy to grasp.
Stability test (S1～S5 represents same batch 5 parts in sample respectively)
Precision test (S1～S5 represents same batch 5 parts in sample respectively)
Repeatability test (S1～S5 represents same batch 5 parts in sample respectively)
To those skilled in the art, technology contents disclosed according to the present invention, those skilled in the art will very clear other embodiment of the present invention, and the embodiment of the invention is only as example.Under the situation of not violating purport of the present invention and scope, can carry out various changes and improvements to the present invention.For example, use the different measurement result that detecting instrument obtained possibilities different, but as long as use method of quality control of the present invention, all within protection domain of the present invention.
Description of drawings
The finger printing of the pharmaceutical preparation of embodiment 1.
The specific embodiment
Enumerate embodiment below and further describe the present invention, this embodiment only is used to illustrate the present invention, and the present invention is not construed as limiting.
Embodiment 1
The preparation of pharmaceutical composition and preparation:
(1) gets Radix Codonopsis, each 5000g of Radix Astragali decoction pieces, add 6 times of amount 70% ethanol, reflux, extract, 3 times, each 1.5 hours, merge extractive liquid, (medicinal residues fling to solvent after standby), decompression recycling ethanol also is concentrated into the thick paste that relative density is 1.15～1.25 (60 ℃), add 4 times of water gagings and make dissolving, leave standstill, centrifugal, get supernatant and be added on the D that has handled well
101On the type macroporous adsorptive resins, first distilled water washing resin post with 2 times of amounts, reuse 80% alcoholic acid eluting saponin component, collect 4 times of ethanol elution of 80% to the resin column volume, decompression recycling ethanol also is concentrated into thick paste, and concentrate is at 60 ℃ of dry down Radix Codonopsis, Radix Astragali total saponins extracts of getting.
(2) the above-mentioned medicinal residues of flinging to behind the solvent add 10 times of water gagings, in 60 ℃ of lixiviates 2 times, each 1.5 hours, merge the water extract, be concentrated into 1: 1 (every milliliter contains primary crude drug 1 gram), add ethanol, make determining alcohol reach 70%, leave standstill 24h, inclining supernatant, precipitates centrifugally, and centrifugal gained precipitation is added suitable quantity of water (water: crude drug in whole is 1: 1) dissolving, add ethanol again, make determining alcohol reach 35%, leave standstill 24h, centrifugal, get supernatant, reclaim ethanol to the greatest extent, adding suitable quantity of water adjustment volume to relative density is 1.00 ~ 1.05, adds 5% trichloroacetic acid solution with 1: 1 equal-volume, leaves standstill 4 hours, centrifugal, get supernatant, add ethanol and reach 80% to containing the alcohol amount, standing over night filters, and gets crude polysaccharides, add suitable quantity of water (water: primary crude drug is 1: 1) dissolving,, get concentrated solution with molecular cut off 3000 ultrafilter membrane ultrafiltration, being evaporated to relative density is 1.20 ~ 1.30 (60 ℃), adds ethanol and reaches 80%, standing over night to containing the alcohol amount, filter, get precipitation, use absolute ethanol washing, vacuum drying, pulverize, get total polysaccharide extractive.
(3) getting weight ratio is 1: 3 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive, add 2000ml water for injection and make dissolving, add 9.5% injection mannitol, 0.5% 30 POVIDONE K 30 BP/USP 30, and be 6.5～7.5 with 10% sodium hydroxide solution adjust pH, add water for injection to 2500ml, filter with 0.22 μ m microporous filter membrane, in the fill 7ml cillin bottle, every bottle of 2.5ml, lyophilization, gland seals, promptly.
Finger printing detects:
" finger printing " photograph high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 D), measure in conjunction with the requirement of finger printing.
Chromatographic condition and system suitability test Diamonsil C
18Post (Di Ma company), granularity 5 μ m, volume 250 * 4.6mm; Mobile phase of acetonitrile and water linear gradient elution (seeing the following form); 30 ℃ of column temperatures; Flow velocity 1ml/min; The detection wavelength is 270nm; Writing time: 55 minutes.Number of theoretical plate calculates by maximum peak should be not less than 3000.
The linear gradient elution proportion of mobile phase changes program
The preparation of need testing solution
Get 1 bottle of the product of present embodiment preparation, add water 5ml and make dissolving, shake up, microporous filter membrane (0.45 μ m) filters, and filtrate is as need testing solution.
The accurate need testing solution 20 μ l that draw of algoscopy inject chromatograph of liquid, measure.
Test sample finger printing (seeing accompanying drawing) compares with finger printing with contrast, and machine simulation similarity software for calculation " chromatographic fingerprints of Chinese materia medica similarity evaluation system " (Chinese Pharmacopoeia can be published, version 2 004A or B) calculates as calculated, and similarity is 0.99.
Embodiment 2
Method according to embodiment 1 prepares pharmaceutical preparation of the present invention, just get Radix Codonopsis 3500g in (1) step, Radix Astragali 6500g, used macroporous adsorbent resin are AB-8 type macroporous adsorbent resins, it is n-butyl alcohol that (2) step was extracted used low first alcohol, and precipitating used low first alcohol is isopropyl alcohol.
The test sample finger printing compares (pressing the method for embodiment 1) with contrast with finger printing, and as calculated machine simulation similarity software for calculation " chromatographic fingerprints of Chinese materia medica similarity evaluation system " (Chinese Pharmacopoeia can be published, version 2 004A or B) to calculate, similarity is 0.98.
Embodiment 3
(1) gets Radix Codonopsis, each 5000g of Radix Astragali decoction pieces, add 10 times of amount 60% ethanol, reflux, extract, 1 time, 3 hours, extracting solution (medicinal residues fling to solvent after standby) decompression recycling ethanol also is concentrated into the thick paste that relative density is 1.15～1.25 (60 ℃), adds 3 times of water gagings and makes dissolving, leaves standstill, centrifugal, get supernatant and be added on the D that has handled well
101On the type macroporous adsorptive resins, first distilled water washing resin post with 10 times of amounts, reuse 80% alcoholic acid eluting saponin component, collect 3 times of ethanol elution of 80% to the resin column volume, decompression recycling ethanol also is concentrated into thick paste, and concentrate is at 50 ℃ of dry down Radix Codonopsis, Radix Astragali total saponins extracts of getting.
(2) the above-mentioned medicinal residues of flinging to behind the solvent add 6 times of water gagings, in 100 ℃ of lixiviates 2 times, each 3 hours, merge the water extract, be concentrated into 1: 1 (every milliliter contains primary crude drug 1 gram), add ethanol, make determining alcohol reach 60%, leave standstill 24h, inclining supernatant, precipitates centrifugally, and centrifugal gained precipitation is added suitable quantity of water (water: crude drug in whole is 1: 1) dissolving, add ethanol again, make determining alcohol reach 35%, leave standstill 24h, centrifugal, get supernatant, reclaim ethanol to the greatest extent, adding suitable quantity of water adjustment volume to relative density is 1.00 ~ 1.05, adds 5% trichloroacetic acid solution with 1: 1 equal-volume, leaves standstill 4 hours, centrifugal, get supernatant, add ethanol and reach 80% to containing the alcohol amount, standing over night filters, and gets crude polysaccharides, add suitable quantity of water (water: primary crude drug is 1: 1) dissolving,, get concentrated solution with molecular cut off 3000 ultrafilter membrane ultrafiltration, being evaporated to relative density is 1.20 ~ 1.30 (60 ℃), adds ethanol and reaches 90%, standing over night to containing the alcohol amount, filter, get precipitation, use absolute ethanol washing, vacuum drying, pulverize, get total polysaccharide extractive.
(3) getting weight ratio is 1: 0.1 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive, add 2000ml water for injection and make dissolving, add 9.5% injection mannitol, 0.5%Tween-80, and be 6.5～7.5, add water for injection to 2500ml with 10% sodium hydroxide solution adjust pH, filter with 0.22 μ m microporous filter membrane, in the fill 7ml cillin bottle, every bottle of 2.5ml, gland, seal, promptly.
The test sample finger printing compares (pressing the method for embodiment 1) with contrast with finger printing, and as calculated machine simulation similarity software for calculation " chromatographic fingerprints of Chinese materia medica similarity evaluation system " (Chinese Pharmacopoeia can be published, version 2 004A or B) to calculate, similarity is 0.99.
Embodiment 4
(1) gets Radix Codonopsis, each 5000g of Radix Astragali decoction pieces, add 8 times of amount 60% ethanol, reflux, extract, 2 times, each 2 hours, extracting solution (medicinal residues fling to solvent after standby) decompression recycling ethanol also is concentrated into the thick paste that relative density is 1.15～1.25 (60 ℃), adds 5 times of water gagings and makes dissolving, leaves standstill, centrifugal, get supernatant and be added on the D that has handled well
101On the type macroporous adsorptive resins, first distilled water washing resin post with 5 times of amounts, reuse 80% alcoholic acid eluting saponin component, collect 2 times of ethanol elution of 80% to the resin column volume, decompression recycling ethanol also is concentrated into thick paste, and concentrate is at 60 ℃ of dry down Radix Codonopsis, Radix Astragali total saponins extracts of getting.
(2) the above-mentioned medicinal residues of flinging to behind the solvent add 8 times of water gagings, in 80 ℃ of lixiviates 3 times, each 2 hours, merge the water extract, be concentrated into 1: 1 (every milliliter contains primary crude drug 1 gram), add ethanol, make determining alcohol reach 80%, leave standstill 24h, inclining supernatant, precipitates centrifugally, and centrifugal gained precipitation is added suitable quantity of water (water: crude drug in whole is 1: 1) dissolving, add ethanol again, make determining alcohol reach 40%, leave standstill 24h, centrifugal, get supernatant, reclaim ethanol to the greatest extent, adding suitable quantity of water adjustment volume to relative density is 1.00 ~ 1.05, adds 5% trichloroacetic acid solution with 1: 1 equal-volume, leaves standstill 4 hours, centrifugal, get supernatant, add ethanol and reach 70% to containing the alcohol amount, standing over night filters, and gets crude polysaccharides, add suitable quantity of water (water: primary crude drug is 1: 1) dissolving,, get concentrated solution with molecular cut off 6000 ultrafilter membrane ultrafiltration, being evaporated to relative density is 1.20 ~ 1.30 (60 ℃), adds ethanol and reaches 70%, standing over night to containing the alcohol amount, filter, get precipitation, use absolute ethanol washing, vacuum drying, pulverize, get total polysaccharide extractive.
(3) getting weight ratio is 1: 8 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive, add 2000ml water for injection and make dissolving, add 9.5% glucose, and be 6.5～7.5, add water for injection to 2500ml with 10% sodium hydroxide solution adjust pH, filter with 0.22 μ m microporous filter membrane, in the fill 7ml cillin bottle, every bottle of 2.5ml, gland, seal, promptly.
The test sample finger printing compares (pressing the method for embodiment 1) with contrast with finger printing, and as calculated machine simulation similarity software for calculation " chromatographic fingerprints of Chinese materia medica similarity evaluation system " (Chinese Pharmacopoeia can be published, version 2 004A or B) to calculate, similarity is 0.97.
Embodiment 5
(1) gets Radix Codonopsis, each 5000g of Radix Astragali decoction pieces, add 9 times of amount 60% ethanol, reflux, extract, 2 times, each 1.5 hours, extracting solution (medicinal residues fling to solvent after standby) decompression recycling ethanol also is concentrated into the thick paste that relative density is 1.15～1.25 (60 ℃), add 5 times of water gagings and make dissolving, leave standstill, centrifugal, get supernatant and be added on the AB-8 type macroporous adsorptive resins of having handled well, first distilled water washing resin post with 5 times of amounts, reuse 80% alcoholic acid eluting saponin component, collect 3 times of ethanol elution of 70% to the resin column volume, decompression recycling ethanol also is concentrated into thick paste, and concentrate is at 60 ℃ of dry down Radix Codonopsis, the Radix Astragali total saponins extract.
(2) the above-mentioned medicinal residues of flinging to behind the solvent add 8 times of water gagings, in 70 ℃ of lixiviates 3 times, each 3 hours, merge the water extract, be concentrated into 1: 1 (every milliliter contains primary crude drug 1 gram), add ethanol, make determining alcohol reach 70%, leave standstill 24h, inclining supernatant, precipitates centrifugally, and centrifugal gained precipitation is added suitable quantity of water (water: crude drug in whole is 1: 1) dissolving, add ethanol again, make determining alcohol reach 350%, leave standstill 24h, centrifugal, get supernatant, reclaim ethanol to the greatest extent, adding suitable quantity of water adjustment volume to relative density is 1.00 ~ 1.05, adds 5% trichloroacetic acid solution with 1: 1 equal-volume, leaves standstill 4 hours, centrifugal, get supernatant, add ethanol and reach 70% to containing the alcohol amount, standing over night filters, and gets crude polysaccharides, add suitable quantity of water (water: primary crude drug is 1: 1) dissolving,, get concentrated solution with molecular cut off 6000 ultrafilter membrane ultrafiltration, being evaporated to relative density is 1.20 ~ 1.30 (60 ℃), adds ethanol and reaches 70%, standing over night to containing the alcohol amount, filter, get precipitation, use absolute ethanol washing, vacuum drying, pulverize, get total polysaccharide extractive.
(3) getting weight ratio is 1: 4 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive, add 2000ml water for injection and make dissolving, add 9.5% glucose, and be 6.5～7.5 with 10% sodium hydroxide solution adjust pH, add water for injection to 2500ml, filter with 0.22 μ m microporous filter membrane, in the fill 7ml cillin bottle, every bottle of 2.5ml, lyophilization, gland seals, promptly.
The test sample finger printing compares (pressing the method for embodiment 1) with contrast with finger printing, and as calculated machine simulation similarity software for calculation " chromatographic fingerprints of Chinese materia medica similarity evaluation system " (Chinese Pharmacopoeia can be published, version 2 004A or B) to calculate, similarity is 0.96.
Embodiment 6 antitumor synergism tests
Synergism test to the effect of cyclophosphamide anti-Lewis pulmonary carcinoma
Under aseptic condition, win the tumor piece of lotus Lewis lung cancer mice, remove slough, use glass homogenizer homogenate, dilute with physiological saline solution by 1: 4 times, then every mice (C under the aseptic condition
57The BL/6 kind) right fore axillary fossa subcutaneous injection 0.2ml claims the weight of animals and grouping behind the 24h.First group is lotus tumor matched group, gives the isometric(al) normal saline; Second group is the chemotherapeutics group, gives Neosar 30mg/kg; Third and fourth, five groups be pharmaceutical preparation of the present invention+chemotherapeutics group, give pharmaceutical preparation of the present invention (method of press embodiment 1 prepares) 3g crude drug/kg, 6g crude drug/kg, 12g crude drug/kg and Neosar 30mg/kg simultaneously.Second day beginning intraperitoneal injection behind the inoculated tumour, 0.2ml/10g, every day 1 time, continuous 10 days; Second day and the 6th day difference intraperitoneal injection of cyclophosphamide injection behind the inoculated tumour.24h claims the weight of animals after the administration in the 10th day, puts to death mice, peels off the tumor piece and weighs, and tumour inhibiting rate calculates the same.Result of the test sees Table 1.
Table 1 pharmaceutical preparation of the present invention is to the influence of cyclophosphamide anti-Lewis pulmonary carcinoma effect (x ± s)
Compare #P＜0.05, ##P＜0.01 with lotus tumor matched group; Compare * P＜0.05, * * P＜0.01 with the cyclophosphamide group
By table 12 as seen, it is heavy that this cyclophosphamide of testing used dosage can alleviate the tumor of Lewis lung cancer mice, with lotus tumor matched group significant difference (P＜0.05 or P＜0.01) arranged relatively.Pharmaceutical preparation of the present invention and cyclophosphamide combined are used, the effect of anti-Mice Bearing Lewis Lung Cancer strengthens, large, medium and small dosage group tumour inhibiting rate is followed successively by 69.77%, 44.65% and 36.74%, compare with independent application cyclophosphamide (tumour inhibiting rate is 35.81), heavy dose of group has significant difference (P＜0.05 or P＜0.01), illustrates that pharmaceutical preparation of the present invention can strengthen the anti-Mice Bearing Lewis Lung Cancer effect of cyclophosphamide.
To the anti-H of radiotherapy
22The synergism test of hepatocarcinoma effect
Extract lotus H down in aseptic condition
228 days mouse ascites of hepatocarcinoma dilutes with physiological saline solution by 1: 4 times, and every mice (ICR kind) lumbar injection 0.2ml under the aseptic condition claims the weight of animals and grouping behind the 24h then.First group for being lotus tumor matched group, to the isometric(al) normal saline; Second group is combination radiotherapy group, gives low dose of roentgen radiation x; The 3rd group is the SHENQI FUZHENG ZHUSHEYE group, gives SHENQI FUZHENG ZHUSHEYE 6g crude drug/kg; Fourth, fifth, six groups is pharmaceutical preparation of the present invention and low dose of roentgen radiation x, gives pharmaceutical preparation of the present invention (pressing the method preparation of embodiment one) 3g crude drug/kg, 6g crude drug/kg, 12g crude drug/kg and low dose of roentgen radiation x simultaneously.Second day begins intraperitoneal injection behind the inoculated tumour, 0.2ml/10g, every day 1 time, continuous 10 days; Gave disposable low dose of roentgen radiation x (whole body dose is 12.5mGy/min, and accumulated dose is 75mGy) behind the inoculated tumour on the 5th day.24h claims the weight of animals after the administration in the 10th day, puts to death mice, peels off the tumor piece and weighs, and tumour inhibiting rate calculates the same.Result of the test sees Table 13.
Table 2 pharmaceutical preparation of the present invention is to the anti-H of radiotherapy
22The influence of hepatocarcinoma effect (x ± s)
Compare ##P＜0.01 with lotus tumor matched group; Compare * * P＜0.01 with combination radiotherapy group
By table 13 as seen, this radiotherapy of testing used dosage can alleviate H
22The tumor of liver cancer mouse is heavy, compares with lotus tumor matched group, and significant difference (P＜0.01) is arranged.Pharmaceutical preparation of the present invention and radiotherapy use in conjunction, anti-mice H
22The effect of hepatocarcinoma strengthens, large, medium and small dosage group tumour inhibiting rate is followed successively by 56.38%, 47.87% and 25.00%, compares with independent application combination radiotherapy group (tumour inhibiting rate is 24.47), and significant difference (P＜0.01) is arranged,, illustrate that pharmaceutical preparation of the present invention can strengthen the anti-mice H of radiotherapy
22The hepatocarcinoma effect.SHENQI FUZHENG ZHUSHEYE and radiotherapy use in conjunction, anti-mice H
22The effect of hepatocarcinoma slightly strengthens, and tumour inhibiting rate is 36.28%, but compares there was no significant difference (P＞0.05) with independent application combination radiotherapy group; With with dosage pharmaceutical preparation of the present invention relatively, significant difference (P＜0.01) is arranged, illustrate that pharmaceutical preparation of the present invention strengthens the effect of the anti-mice H22 of radiotherapy hepatocarcinoma and is better than SHENQI FUZHENG ZHUSHEYE.
Claims (10)
1. pharmaceutical composition that is used for assistant treating cancer, with the Radix Codonopsis and the Radix Astragali is that raw material is made, it is characterized in that having 6 absworption peaks at least in the high-efficiency liquid-phase fingerprint of described compositions, the absworption peak that wherein unimodal area surpasses total peak area 10% has 3, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%
Pharmaceutical composition and preparation thereof adopt the method preparation that comprises the steps:
(1) get the Radix Codonopsis and the Radix Astragali decoction pieces of equivalent, add low first alcohol reflux and extract, extracting solution and medicinal residues, extracting solution is collected eluent and concentrate drying and is got Radix Codonopsis, Radix Astragali total saponins extract through low pole macroporous adsorptive resins purification;
(2) behind the medicinal residues removal solvent that step (1) obtains, through water carry, the low unit alcohol precipitation of 50-80%, the be dissolved in water low unit alcohol precipitation of back reuse 30-40% of precipitation is got supernatant, remove albumen with trichloroacetic acid method, further, get crude polysaccharides, behind water dissolution with the low unit alcohol precipitation of 70-90%, the employing molecular weight that dams is that 3000 film carries out ultrafiltration, concentrated solution further with the low unit alcohol precipitation of 70-90%, is got precipitation, and drying and crushing gets Radix Codonopsis, Radix Astragali Mongolici total polysaccharide extract;
(3) Radix Codonopsis, Radix Astragali total saponins extract and Radix Codonopsis, the Radix Astragali Mongolici total polysaccharide extract of combining step (1) and step (2) add suitable pharmaceutically acceptable carrier, make freeze-dried powder.
2. pharmaceutical composition as claimed in claim 1 is characterized in that having 6 absworption peaks at least in the finger printing of described compositions, and the absworption peak that wherein unimodal area surpasses total peak area 5% has 5, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 2 peaks, average retention time RT is 19.439min, and RSD is 0%, and peak area is 358.374, and RSD is 1.08%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%;
No. 6 peaks, average retention time RT is 48.72min, and RSD is 0%, and peak area is 538.678, and RSD is 5.42%.
3. pharmaceutical composition as claimed in claim 2 is characterized in that having 6 absworption peaks at least in the finger printing of described compositions, and the absworption peak that wherein unimodal area surpasses total peak area 2% has 6, is respectively:
No. 1 peak, average retention time RT is 12.807min, and RSD is 0%, and peak area is 1158.519, and RSD is 4.5%;
No. 2 peaks, average retention time RT is 19.439min, and RSD is 0%, and peak area is 358.374, and RSD is 1.08%;
No. 3 peaks, average retention time RT is 28.032min, and RSD is 0%, and peak area is 1555.306, and RSD is 2.46%;
No. 4 peaks, average retention time RT is 31.238min, and RSD is 0%, and peak area is 145.988, and RSD is 0.13%;
No. 5 peaks, average retention time RT is 43.481min, and RSD is 0%, and peak area is 2578.959, and RSD is 1.52%;
No. 6 peaks, average retention time RT is 48.72min, and RSD is 0%, and peak area is 538.678, and RSD is 5.42%.
4. contain the preparation of the pharmaceutical composition of right requirement 1,2 or 3, said preparation is injection, infusion solution or freeze-dried powder.
5. the detection method of the pharmaceutical composition of claim 4 comprises the steps:
(a) foundation of reference fingerprint
The weighting profit requires 4 pharmaceutical preparation control sample, is dissolved in water, and shakes up, and filters with 0.45 μ m microporous filter membrane, and filtrate is product solution in contrast;
(b) mensuration of reference fingerprint:
Draw above-mentioned control sample solution and inject chromatograph of liquid, use high performance liquid chromatography to measure, obtain reference fingerprint, chromatographic condition: chromatographic column is filler with the octadecylsilane chemically bonded silica; Mobile phase is: the acetonitrile-water linear gradient elution, and the detection wavelength is 250-280nm;
(c) mensuration of product fingerprint collection of illustrative plates to be measured:
Get product to be measured, measure the finger printing of this product to be measured according to Step By Condition described in above-mentioned (a);
(d) described product fingerprint collection of illustrative plates to be measured and described reference fingerprint are compared, discern the quantity of the two common absworption peak that has, to determine whether product quality is qualified.
6. detection method as claimed in claim 5 is characterized in that: the flow velocity 0.5～1.5ml/min of mobile phase in described (a) step, detect wavelength 265～275nm, 20～40 ℃ of column temperatures.
7. detection method as claimed in claim 6 is characterized in that, described product fingerprint collection of illustrative plates to be measured and described reference fingerprint relatively when the two finger printing has absworption peak identical more than 3 or 3, are thought the up-to-standard of described product to be measured.
8. detection method as claimed in claim 7 is characterized in that, described product fingerprint collection of illustrative plates to be measured and described reference fingerprint relatively when the two finger printing has absworption peak identical more than 4 or 4, are thought the up-to-standard of described product to be measured.
9. detection method as claimed in claim 8 is characterized in that, described product fingerprint collection of illustrative plates to be measured and described reference fingerprint relatively when the two finger printing has absworption peak identical more than 5 or 5, are thought the up-to-standard of described product to be measured.
10. detection method as claimed in claim 9 is characterized in that, described product fingerprint collection of illustrative plates to be measured and described reference fingerprint relatively when the two finger printing has 6 identical absworption peaks, are thought the up-to-standard of described product to be measured.
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