CN101084981B - Medicinal preparation for cancer auxiliary treatment and its preparation method - Google Patents
Medicinal preparation for cancer auxiliary treatment and its preparation method Download PDFInfo
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Abstract
The invention provides a medicinal preparation for the subsidiary treatment of cancer, which has high content of active ingredients, high effectiveness and low toxicity. The preparation is suitable for intravenous injection.
Description
Technical field
The present invention relates to a kind of cancer adjuvant therapy medicament preparation and preparation method thereof that is used for, particularly a kind of is the cancer adjuvant therapy medicament preparation and preparation method thereof 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.SHENQI FUZHENG ZHUSHEYE is a water preparation, has transportation inconvenience, and therefore problems such as quality stability difference have researcher to improve its technology, and it is prepared into freeze-dried powder.It as application number 200310113944.1 Chinese patent application, with the method acquisition Radix Codonopsis of alcohol reflux and the total saponins composition in the Radix Astragali, the method of medicinal residues reuse water extract-alcohol precipitation obtains the total polysaccharides composition in the Radix Codonopsis and the Radix Astragali, total saponins and total polysaccharide extractive are mixed, add the adjuvant lyophilization and get freeze-dried powder.The advantage of this method is to have kept the total saponins total polysaccharides effective constituents in the crude drug simultaneously, yet, owing to after the extraction of total saponins, there is not further purge process, be not suitable for being prepared into the powder injection formulation that suitable vein is used in practice.
Summary of the invention
The purpose of this invention is to provide the pharmaceutical preparation that is used for assistant treating cancer of a kind of active constituent content height, suitable intravenous injection use, high-efficiency low-toxicity.
Another object of the present invention is to provide a kind of method for preparing the said medicine preparation.
The pharmaceutical preparation that is used for assistant treating cancer provided by the invention is that the employing Radix Codonopsis and the Radix Astragali are raw material, makes by the method that may further comprise the steps:
(1) getting weight ratio is 1: the Radix Codonopsis of 0.5-2 and Radix Astragali decoction pieces, and add low first alcohol reflux and extract, get extracting solution and medicinal residues, extracting solution is further used alkaline purification by macroporous resin behind concentrated, centrifugal and macroporous adsorptive resins purification, get total saponin extracts;
(2) behind the medicinal residues removal solvent that step (1) obtains, use water extraction, after extracting solution concentrates, transfer alcoholic degree to precipitate to 50-80%, precipitation transfers after being dissolved in water alcoholic degree to precipitate to 30-40% again, gets supernatant, removes albumen, further transfer alcoholic degree to precipitate to 70-90%, get crude polysaccharides, adopting the molecular weight that dams with order behind the water dissolution is 10,000,10,000 and 3,000 ultrafilter membrane ultrafiltration, concentrated solution further transfer alcoholic degree to precipitate to 70-90%, get precipitation, drying and crushing can get total polysaccharide extractive A, B and C respectively.
(3) get total polysaccharide extractive A, the B of (1) total saponin extracts of step and step (2), one or both or three kinds among the C as active component, the ratio of the two weight is 0.1-8: 1, add suitable pharmaceutically acceptable carrier, make injection, infusion solution or freeze-dried powder.
Used low first alcohol is meant the alcohol of C1-C4 among the present invention, be that this area is generally used for extracting the Chinese crude drug effective ingredient, or the alcohol of the conventional or known C1-C4 of effective ingredient or impurity in the precipitation, purification of aqueous solutions, for example methanol, ethanol, isopropyl alcohol, propylene glycol, n-butyl alcohol and/or isobutanol, preferred alcohol.Processes such as described extraction, purification, precipitation can be carried out according to this area routine or known method.
Described step (1) first step big pore resin can be a macroporous resin any routine or known, comprise nonpolar, low pole, Semi-polarity and strong polar macroporous resin, for example Chang Yong ADS-21 type macroporous resin, ADS-17 type macroporous resin, ADS-16 type macroporous resin or ADS-F8 type macroporous resin, DM-130, D-101 and D-4020, 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.Described (1) second step of step big pore resin is that the alkalescence macroporous adsorbent resin can be an alkalescence macroporous resin any routine or known, preferred D941, D301 type macroporous adsorbent resin, also can be any routine or known alkalescence macroporous resin strong basicity macroporous resin, preferred D941, D301 type macroporous adsorbent resin.
The proteic method of removal of described step (2) can be the method art technology routine or known, as Sevage method, trichloroacetic acid method (TCA method) and tannic acid method (TA method), preferred trichloroacetic acid method, purge process can be carried out according to this area routine or known method.
The described pharmaceutically acceptable carrier of step of the present invention (3), comprise and be prepared into the needed solvent of injection, this solvent can be that this area is used always or known solvent such as water, also can comprise osmotic pressure regulator, this regulator can be the commonly used or known regulator in this area, as glucose, sodium chloride, fructose, xylose etc.; The described pharmaceutically acceptable carrier of step (3) can also comprise and make the needed excipient of lyophilized injectable powder, for example mannitol, lactose, glycine etc.Described injection, infusion solution and lyophilized injectable powder can according to the routine of this area or known method carry out; Described pharmaceutically acceptable carrier can also comprise as required and make needed buffer agent of lyophilized injectable powder and/or cosolvent etc.
The preferred 0.5-5 of ratio of the weight of described step (3) total saponin extracts and total polysaccharide extractive: 1, most preferably 3: 1.
Zhi Bei the pharmaceutical preparation that is used for assistant treating cancer according to the method described above, preferred freeze-dried powder, this freeze-dried powder is preferably: the weight percentage of total saponins is 10-32.5% in the active component, the weight percentage of total polysaccharides is 25-87%, and the weight percentage of astragaloside is 2.5-7%; More preferably: the weight percentage of total saponins is 12.5-32.5% in the active component, and the weight percentage of total polysaccharides is 35-87%, and the weight percentage of astragaloside is 4.3-7%.
The invention provides the preparation method that above-mentioned preferred freeze-dried powder also is provided, may further comprise the steps:
(1) getting weight ratio is 1: the Radix Codonopsis of 0.5-2 and Radix Astragali decoction pieces, and add low first alcohol reflux and extract, get extracting solution and medicinal residues, extracting solution is further used alkaline purification by macroporous resin behind concentrated, centrifugal and macroporous adsorptive resins purification, get total saponin extracts;
(2) behind the medicinal residues removal solvent that step (1) obtains, use water extraction, after extracting solution concentrates, transfer alcoholic degree to precipitate to 50-80%, precipitation transfers after being dissolved in water alcoholic degree to precipitate to 30-40% again, gets supernatant, removes albumen, further transfer alcoholic degree to precipitate to 70-90%, get crude polysaccharides, adopting the molecular weight that dams with order behind the water dissolution is 10,000,10,000 and 3,000 ultrafilter membrane ultrafiltration, concentrated solution further transfer alcoholic degree to precipitate to 70-90%, get precipitation, drying and crushing can get total polysaccharide extractive A, B and C respectively.
(3) get total polysaccharide extractive A, the B of (1) total saponin extracts of step and step (2), one or both or three kinds among the C as active component, the ratio of the two weight is 0.1-8: 1, add suitable pharmaceutically acceptable carrier, make injection, infusion solution or freeze-dried powder.
The notion of low unit alcohol herein is the same, preferred alcohol.
Described step (1) first step big pore resin can be a macroporous resin any routine or known, comprise nonpolar, low pole, Semi-polarity and strong polar macroporous resin, for example Chang Yong ADS-21 type macroporous resin, ADS-17 type macroporous resin, ADS-16 type macroporous resin or ADS-F8 type macroporous resin, DM-130, D-101 and D-4020, 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.Described (1) second step of step big pore resin is that the alkalescence macroporous adsorbent resin can be an alkalescence macroporous resin any routine or known, preferred D941, D301 type macroporous adsorbent resin, also can be any routine or known alkalescence macroporous resin strong basicity macroporous resin, preferred D941, D301 type macroporous adsorbent resin.
Preparation method provided by the invention preferably may further comprise the steps:
(4) get Radix Codonopsis and Radix Astragali decoction pieces, adding low first alcohol reflux extracts, extracting solution and medicinal residues, extracting solution is through concentrated, centrifugal, low pole macroporous adsorptive resins purification and alkalescence macroporous adsorptive resins purification, collection eluent and concentrate drying get Radix Codonopsis, Radix Astragali total saponins extract;
(5) behind the medicinal residues removal solvent that step (1) obtains, use water extraction, after extracting solution concentrates, transfer alcoholic degree to precipitate to 50-80%, precipitation transfers after being dissolved in water alcoholic degree to precipitate to 30-40% again, gets supernatant, removes albumen with trichloroacetic acid, further transfer alcoholic degree to precipitate to 70-90%, crude polysaccharides, with ultrafiltration behind the water dissolution, concentrated solution further transfers alcoholic degree to precipitate to 70-90%, get crude polysaccharides, behind water dissolution, adopting the molecular weight that dams is that 3000 film carries out ultrafiltration, and concentrated solution further transfers alcoholic degree to precipitate to 70-90%, get precipitation, drying and crushing gets Radix Codonopsis, the Radix Astragali Mongolici total polysaccharide extract.
The Radix Codonopsis of combining step (1) and step (2), Radix Astragali total saponins extract and Radix Codonopsis, Radix Astragali Mongolici total polysaccharide extract add suitable pharmaceutically acceptable carrier, make freeze-dried powder.
Freeze-dried powder of the present invention before use, can be dissolved in freeze-dried powder in water for injection or other injection, also can be dissolved in the infusion solution, as be used for the isotonicity medium that venous tree is annotated, for example in normal saline solution or 5% glucose solution.
According to disease, therapeutic process and patient's concrete condition, the consumption that uses pharmaceutical preparation of the present invention generally can every day 1~2, and every is equivalent to crude drug 5-15g.
The preparation method of Chinese medicine composition of the present invention, its processing step and parameter are that the inventor determines that on a large amount of experiment basis the inventor has paid a large amount of performing creative labours for this reason, the Radix Astragali total saponins extract of method preparation of the present invention,
Always Saponin content is more than 80% (W/W), and the rate of transform is more than 40%; In the total polysaccharide extractive, the content of total polysaccharides is 80% More than, the rate of transform is more than 40%.
Pharmaceutical preparation of the present invention has active constituent content height, good effect, characteristics that toxicity is low.Prove through pharmacodynamics, though pharmaceutical preparation of the present invention itself does not have obvious antineoplastic, but potentiation and Attenuation are arranged with radiotherapy or chemotherapy drugs in combination application, while energy enhancing body's immunological function and anti-stress ability, its effect is equivalent to or is better than the SHENQI FUZHENG ZHUSHEYE of present clinical use.Pharmaceutical preparation of the present invention does not find that through acute toxicity test, long term toxicity test, safety testing and experimental studies such as allergy, stimulation it has tangible toxicity.
The used assay method of the present invention is as follows:
1, Radix Astragali total saponins assay
Measure according to spectrophotography (" appendix VA of Chinese pharmacopoeia version in 2000).
The preparation precision of reference substance solution takes by weighing at the astragaloside reference substance of 60 ℃ of drying under reduced pressure to constant weight, makes the solution that every 1ml contains astragaloside 0.4mg with dehydrated alcohol, shakes up, promptly.
This product content 350mg is got in the preparation of need testing solution, and accurate the title decides, and puts in the 25ml measuring bottle, it is an amount of to add methanol, and supersound process 20 minutes is put cold, add methanol and be diluted to scale, shake up, filter, precision is measured subsequent filtrate 5ml, puts water bath method, and residue dissolves with 50% methanol solution 20ml, solution passes through neutral alumina post (4g with the speed of per minute 0.5ml, 100~200 orders, column length 20cm, internal diameter 1cm), and continue to wash with 50% methanol solution 40ml, merga pass liquid and washing liquid, a small amount of anhydrous alcohol solution of water bath method, residue, be transferred in the 10ml measuring bottle, be diluted to scale with dehydrated alcohol, shake up, as need testing solution.
Accurate reference substance solution 0,100,200,300,400,500, the 600ul of drawing of the preparation of standard curve places 10ml tool plug scale test tube respectively, adds dehydrated alcohol respectively to 0.5ml.Name adds 8% vanillin dehydrated alcohol reagent 0.5ml, shakes up.Put and slowly add 72% sulphuric acid 5ml in the ice-water bath, shake up 62 ℃ of water bath heat preservation 20min.Taking-up is put ice-water bath immediately and is cooled to room temperature, measures trap at the 538nm place according to spectrophotography (appendix VA of Chinese Pharmacopoeia version in 2000).With reference substance concentration is that abscissa, trap value A are ordinate, the drawing standard curve.
The accurate need testing solution 0.5ml that draws of algoscopy puts in the 10ml tool plug scale test tube, and the method under the preparation of sighting target directrix curve rose from " adding 8% vanillin ... ", measured trap in accordance with the law.Calculate, promptly.
2, Astragaloside content is measured
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica, and acetonitrile-water (37: 63) is a mobile phase, detects wavelength 203nm.Number of theoretical plate should be not less than 3000 by astragaloside.
The preparation precision of reference substance solution takes by weighing at the astragaloside reference substance of 60 ℃ of drying under reduced pressure to constant weight, adds dissolve with methanol, makes the solution that every 1ml contains 0.6mg, promptly.
The preparation precision of need testing solution takes by weighing the about 500mg of this product content, adds water 10ml dissolving, is transferred in the separatory funnel, with water saturated n-butanol extraction 4 times, each 20ml merges n-butyl alcohol liquid, with ammonia solution washing 3 times, each 20ml merges n-butyl alcohol liquid, be transferred in the evaporating dish, water bath method, with dissolve with methanol and be transferred to the 5ml volumetric flask, methanol is diluted to scale, shake up, the 0.45um membrane filtration promptly.
Accurate respectively above-mentioned reference substance solution 5 μ l, 10 μ l, 15 μ l, 20 μ l, the 30 μ l of drawing of the preparation of standard curve, inject chromatograph of liquid respectively, measure the astragaloside peak area by above-mentioned chromatographic condition, with reference substance sample size logX (μ g) is that abscissa, peak area value logY (m v.s) are ordinate, the drawing standard curve
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
3, polysaccharide molecular weight Determination of distribution
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test are filler with gel, and 0.1M sodium chloride is mobile phase, differential refraction detector, and sensitivity is 32.GPC software.
The drafting of standard curve is got molecular weight and is respectively 1270,11600, and 23800,48600,80900,147600 glucosan reference substance is an amount of, makes the solution that contains 2mg among every 1mL approximately with the mobile phase dissolving respectively, injects chromatograph of liquid respectively, the record chromatogram.Logarithm logM with standard dextran molecule amount
WBe vertical coordinate, with retention time t
RBe abscissa, carry out regression treatment, the drawing standard curve.
The preparation precision of need testing solution takes by weighing this product 160mg, puts in the 5ml measuring bottle, adds the mobile phase dissolving and is diluted to scale, shakes up, promptly.
The accurate need testing solution 25 μ l that draw of algoscopy inject chromatograph of liquid, measure, and calculate, promptly.
4, total polysaccharides assay
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test are filler with gel, and 0.1M sodium chloride is mobile phase, differential refraction detector, and sensitivity is 32.GPC software.
The preparation precision of reference substance solution takes by weighing molecular weight Mw and is respectively 147600,48600, and 1270 glucosan is a reference substance, is configured to concentration difference 0.2,0.1, the mixing reference substance solution of 3.2mg/ml, promptly.
The preparation precision of need testing solution takes by weighing this product 160mg, puts in the 5ml measuring bottle, adds the mobile phase dissolving and is diluted to scale, shakes up, promptly.
The specific embodiment
The invention will be further described below in conjunction with embodiment, and embodiment does not limit the present invention in any way:
Embodiment 1
(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, eluent is crossed D941 type macroporous adsorbent resin, and a small amount of 80% ethanol elution of reuse is collected effluent and eluent, 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, incline and supernatant, precipitate centrifugally, 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%, standing over night to containing the alcohol amount, filter, get crude polysaccharides, add suitable quantity of water (water: primary crude drug is 1: 1) dissolving, with molecular cut off 100000 ultrafilter membrane ultrafiltration, get concentrated solution, 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 A; Permeate is got concentrated solution with 10000 ultrafilter membrane ultrafiltration, and being evaporated to relative density is 1.20~1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, and standing over night filters, and gets precipitation, uses absolute ethanol washing, and vacuum drying is pulverized, and gets total polysaccharide extractive B; Concentrated solution is got in the 3000 ultrafilter membrane ultrafiltration of permeate reuse, and being evaporated to relative density is 1.20~1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, standing over night filters, and gets precipitation, uses absolute ethanol washing, vacuum drying is pulverized, and gets total polysaccharide extractive C;
(3) getting weight ratio is 1: 3 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive (comprising A, B and C), 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.
Embodiment 2
Method according to embodiment 1 prepares pharmaceutical preparation of the present invention, just gets Radix Codonopsis 3500g in (1) step, and Radix Astragali 6500g, used macroporous adsorbent resin are respectively AB-8 type macroporous adsorbent resin and D
301The type macroporous resin, it is n-butyl alcohol that (2) step was extracted used low first alcohol, precipitating used low first alcohol is isopropyl alcohol; The total polysaccharide extractive in (3) step is A and B.
Embodiment 3
Method according to embodiment 1 prepares pharmaceutical preparation of the present invention, just gets Radix Codonopsis 6500g in (1) step, Radix Astragali 3500g, and the used macroporous adsorbent resin of step (1) is an ADS-17 type macroporous adsorbent resin, the proteic method of removal of step (2) is the Sevage method.
Embodiment 4
(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, earlier with the distilled water washing resin post of 10 times of amounts, reuse 80% alcoholic acid eluting saponin component is collected 3 times of ethanol elution of 80% to the resin column volume, and eluent is crossed D
201The type macroporous resin, a small amount of 80% ethanol elution of reuse is collected effluent and eluent, and 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, incline and supernatant, precipitate centrifugally, 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%, standing over night to containing the alcohol amount, filter, get crude polysaccharides, add suitable quantity of water (water: primary crude drug is 1: 1) dissolving, with molecular cut off 100000 ultrafilter membrane ultrafiltration, get concentrated solution, 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 A; Permeate is got concentrated solution with 10000 ultrafilter membrane ultrafiltration, and being evaporated to relative density is 1.20~1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, and standing over night filters, and gets precipitation, uses absolute ethanol washing, and vacuum drying is pulverized, and gets total polysaccharide extractive B; Concentrated solution is got in the 3000 ultrafilter membrane ultrafiltration of permeate reuse, and being evaporated to relative density is 1.20~1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, standing over night filters, and gets precipitation, uses absolute ethanol washing, vacuum drying is pulverized, and gets total polysaccharide extractive C;
(3) getting weight ratio is 1: 0.1 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive (comprising total polysaccharides B and C), add 2000ml water for injection and make dissolving, add 9.5% injection mannitol, 0.5%Tween-80, 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.
Embodiment 5
(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, earlier with the distilled water washing resin post of 5 times of amounts, reuse 80% alcoholic acid eluting saponin component is collected 2 times of ethanol elution of 80% to the resin column volume, and eluent is crossed D
301The type macroporous resin, a small amount of 80% ethanol elution of reuse is collected effluent and eluent, and 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 100000 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 A; Permeate is got concentrated solution with 10000 ultrafilter membrane ultrafiltration, and being evaporated to relative density is 1.20~1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, and standing over night filters, and gets precipitation, uses absolute ethanol washing, and vacuum drying is pulverized, and gets total polysaccharide extractive B; Concentrated solution is got in the 3000 ultrafilter membrane ultrafiltration of permeate reuse, and being evaporated to relative density is 1.20~1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, standing over night filters, and gets precipitation, uses absolute ethanol washing, vacuum drying is pulverized, and gets total polysaccharide extractive C;
(3) getting weight ratio is 1: 8 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive (comprising extract A, B and C), 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.
Embodiment 6
(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, reuse 80% alcoholic acid eluting saponin component with 5 times of amounts, collect 3 times of ethanol elution of 70% to the resin column volume, eluent is crossed D
301The type macroporous resin, a small amount of 80% ethanol elution of reuse is collected effluent and eluent, and 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 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, incline and supernatant, precipitate centrifugally, 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%, standing over night to containing the alcohol amount, filter, get crude polysaccharides, add suitable quantity of water (water: primary crude drug is 1: 1) dissolving, with molecular cut off 100000 ultrafilter membrane ultrafiltration, get concentrated solution, 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 A; Permeate is got concentrated solution with 10000 ultrafilter membrane ultrafiltration, and being evaporated to relative density is 1.20~1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, and standing over night filters, and gets precipitation, uses absolute ethanol washing, and vacuum drying is pulverized, and gets total polysaccharide extractive B; Concentrated solution is got in the 3000 ultrafilter membrane ultrafiltration of permeate reuse, and being evaporated to relative density is 1.20~1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, standing over night filters, and gets precipitation, uses absolute ethanol washing, vacuum drying is pulverized, and gets total polysaccharide extractive C;
(3) getting weight ratio is 1: 4 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive (comprising extract A and C), 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.
Embodiment 7 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 1 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 7.044%, 43.65% and 38.86%, 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 2 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 57.35%, 47.65% and 25.54%, compares with independent application combination radiotherapy group (tumour inhibiting rate is 24.76), 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 36.54%, 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 anti-mice H of radiotherapy
22The hepatocarcinoma effect is better than SHENQI FUZHENG ZHUSHEYE.
Claims (10)
1. pharmaceutical preparation that is used for assistant treating cancer, adopting the Radix Codonopsis and the Radix Astragali is raw material, makes by following steps:
(1) getting weight ratio is 1: the Radix Codonopsis of 0.5-2 and Radix Astragali decoction pieces, and add low first alcohol reflux and extract, get extracting solution and medicinal residues, extracting solution is further used alkaline purification by macroporous resin behind concentrated, centrifugal and macroporous adsorptive resins purification, get total saponin extracts;
(2) behind the medicinal residues removal solvent that step (1) obtains, use water extraction, after extracting solution concentrates, transfer alcoholic degree to precipitate to 50-80%, precipitation transfers after being dissolved in water alcoholic degree to precipitate to 30-40% again, gets supernatant, removes albumen, further transfer alcoholic degree to precipitate to 70-90%, get crude polysaccharides, adopting the molecular weight that dams with order behind the water dissolution is 10,000,10,000 and 3,000 ultrafilter membrane ultrafiltration, concentrated solution further transfer alcoholic degree to precipitate to 70-90%, get precipitation, drying and crushing can get total polysaccharide extractive A, B and C respectively;
(3) get total polysaccharide extractive A, the B of (1) total saponin extracts of step and step (2), one or both or three kinds among the C as active component, the ratio of the two weight is 0.1-8: 1, add suitable pharmaceutically acceptable carrier, make injection, infusion solution or freeze-dried powder.
2. preparation according to claim 1, wherein the ratio of the weight of the total saponin extracts of step (3) and total polysaccharide extractive is 0.5-5: 1.
3. preparation according to claim 2, wherein the ratio of the weight of the total saponin extracts of step (3) and total polysaccharide extractive is 3: 1.
4. preparation according to claim 1, the freeze-dried powder of step (3) wherein, the weight percentage of total saponins is 10-32.5% in the active component, and the weight percentage of total polysaccharides is 25-87%, and the weight percentage of astragaloside is 2.5-7%.
5. preparation according to claim 4, the freeze-dried powder of step (3) wherein, the weight percentage of total saponins is 12.5-32.5% in the active component, and the weight percentage of total polysaccharides is 35-87%, and the weight percentage of astragaloside is 4.3-7%.
6. the preparation method of the preparation of claim 5, form by following steps:
(1) gets Radix Codonopsis and Radix Astragali decoction pieces, adding low first alcohol reflux extracts, extracting solution and medicinal residues, extracting solution is through concentrated, centrifugal, low pole macroporous adsorptive resins purification and alkalescence macroporous adsorptive resins purification, collection eluent and concentrate drying get Radix Codonopsis, Radix Astragali total saponins extract;
(2) behind the medicinal residues removal solvent that step (1) obtains, use water extraction, after extracting solution concentrates, transfer alcoholic degree to precipitate to 50-80%, precipitation transfers after being dissolved in water alcoholic degree to precipitate to 30-40% again, gets supernatant, removes albumen with trichloroacetic acid, further transfer alcoholic degree to precipitate to 70-90%, crude polysaccharides, with ultrafiltration behind the water dissolution, concentrated solution further transfers alcoholic degree to precipitate to 70-90%, get crude polysaccharides, behind water dissolution, adopting the molecular weight that dams is that 3000 film carries out ultrafiltration, and concentrated solution further transfers alcoholic degree to precipitate to 70-90%, get precipitation, drying and crushing gets Radix Codonopsis, the 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.
7. according to the method for claim 6, wherein said low pole macroporous resin is D101 type macroporous resin or AB-8 type macroporous resin.
8. according to the method for claim 7, wherein said alkalescence macroporous resin is D941, D301 or D201 type macroporous resin.
9. method according to Claim 8, wherein said low first alcohol is ethanol.
10. according to the method for claim 9, form by following steps:
(1) gets the Radix Codonopsis and the Radix Astragali decoction pieces of equivalent, add the 60-80% ethanol of doubly measuring with 5-10, reflux, extract, 1-3 time, each 1.5-3 hour, get extracting solution and medicinal residues, extracting solution is through concentrated, centrifugal, D101 type macroporous adsorptive resins purification and D941 type purification by macroporous resin, and collection eluent and concentrate drying get Radix Codonopsis, Radix Astragali total saponins extract;
(2) behind the medicinal residues removal solvent that step (1) obtains, add the water that 6-12 doubly measures, in 50-100 ℃, lixiviate 1-3 time, each 1-2.5 hour, transfer alcoholic degree to precipitate to 60-80%, precipitation transfers after being dissolved in water alcoholic degree to precipitate to 30-40% again, gets supernatant, removes albumen, further transfer alcoholic degree to precipitate to 70-90%, get crude polysaccharides, adopting the molecular weight that dams with order behind the water dissolution is 10,000,10,000 and 3,000 ultrafilter membrane ultrafiltration, concentrated solution further transfer alcoholic degree to precipitate to 70-90%, get precipitation, drying and crushing gets Radix Codonopsis, Radix Astragali Mongolici total polysaccharide extract;
(3) get total polysaccharide extractive A, the B of (1) total saponin extracts of step and step (2), one or both or three kinds among the C as active component, the ratio of the two weight is 0.1-8: 1, add suitable pharmaceutically acceptable carrier, make freeze-dried powder.
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