CN101084979B - 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. The process for its preparation is also disclosed in the invention.
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, and it is formed by the Radix Codonopsis and the Radix Astragali prescription of equivalent, and the 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 accomplish 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 like application number 200310113944.1 one Chinese patent application; Method with alcohol reflux obtains the total saponins composition in the Radix Codonopsis and 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 that a kind of active constituent content is high, be fit to 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 through 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 gets total saponin extracts through the macroporous adsorptive resins purification;
(2) after the medicinal residues that obtain of step (1) are removed solvent, use water extraction, after extracting solution concentrated, the accent alcoholic degree precipitated to 50-80%; Deposition 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%, crude polysaccharides, with ultrafiltration behind the water dissolution; Concentrated solution further transfers alcoholic degree to precipitate to 70-90%, gets deposition, and drying and crushing gets total polysaccharide extractive.
(3) get (1) total saponin extracts of step and the total polysaccharide extractive of step (2), the ratio of the two weight is 0.1-8: 1, add suitable pharmaceutically acceptable carrier, and process 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 deposition, 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, deposition can be carried out according to the conventional perhaps known method in this area.
Described step (1) big pore resin can be any routine or known macroporous resin; Comprise nonpolar, low pole, Semi-polarity and strong polar macroporous resin, 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 for example commonly used
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 the conventional perhaps known method in this area.
The proteic method of removal of described step (2) can be the conventional or known method of art technology; Like 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 the conventional perhaps known method in this area.
It is that 3000 film carries out that the molecular weight that dams is preferably adopted in the ultrafiltration of described step (2), ultra-filtration process can be according to this area conventional or known method carry out.
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 this area perhaps known solvent such as water commonly used; Also can comprise osmotic pressure regulator, this regulator can be the commonly used or known regulator in this area, like glucose, sodium chloride, fructose, xylose etc.; The described pharmaceutically acceptable carrier of step (3) can also comprise and process 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 process 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.
The pharmaceutical preparation that is used for assistant treating cancer of preparation 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%; Wherein about the molecular weight of polysaccharide; Particularly preferably be: the weight average molecular weight Mw1 of polysaccharide should be 140000 ± 30000 in the said preparation; Wherein to account for the percentage ratio of total molecule be 100.0% to the molecule of Mw＞100000, and molecular weight distribution width Mw/Mn should be 1.02 ± 0.4; Mw2 should be 60000 ± 10000, and wherein to account for the percentage ratio of total molecule be 100.0% to the molecule of Mw＞20000, and molecular weight distribution width Mw/Mn should be 1.12 ± 0.4; Mw3 should be 4000 ± 1000, wherein the molecule of Mw＞2000 account for the percentage ratio of total molecule should be greater than 80.0%, molecular weight distribution width Mw/Mn should be 1.25 ± 0.4.
The present invention provides the method for preparing that above-mentioned preferred freeze-dried powder also is provided, and may further comprise 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) after the medicinal residues that obtain of step (1) were removed solvent, after use water extraction, extracting solution concentrated, the accent alcoholic degree precipitated to 50-80%; Deposition transfers after being dissolved in water alcoholic degree to precipitate to 30-40% again, gets supernatant, removes albumen with trichloroacetic acid method, further transfers alcoholic degree to precipitate to 70-90%; Crude polysaccharides, behind water dissolution, adopting the molecular weight that dams is that 3000 film carries out ultrafiltration; Concentrated solution further transfers alcoholic degree to precipitate to 70-90%, gets deposition, and drying and crushing gets total polysaccharide extractive.
(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, process freeze-dried powder.
The notion of the low unit alcohol here is the same, preferred alcohol.
The used low pole macroporous resin of described step (1) can be any routine or known low pole macroporous resin, D for example commonly used
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 the conventional perhaps known method in this area.
Method for preparing 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 5-10; Reflux, extract, 1-3 time each 1.5-3 hour, gets extracting solution and medicinal residues; Extracting solution is collected eluent and concentrate drying and is got Radix Codonopsis, Radix Astragali total saponins extract through concentrated, centrifugal and D101 type macroporous adsorptive resins purification;
(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%, after being dissolved in water, deposition transfer alcoholic degree to precipitate to 30-40% again, get supernatant; Remove albumen, 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%, gets deposition, 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, process 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 method for preparing of Chinese medicine composition of the present invention, its processing step and parameter are that the inventor confirms 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 25% (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 ability 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 following:
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 the astragaloside reference substance 60 ℃ of drying under reduced pressure to constant weights, processes the solution that every 1ml contains astragaloside 0.4mg with dehydrated alcohol, shakes up, and promptly gets.
These article content 350mg is got in the preparation of need testing solution, and accurate the title decides, and puts in the 25ml measuring bottle, and it is an amount of to add methanol, supersound process 20 minutes; Put coldly, add methanol and be diluted to scale, shake up, filter, precision is measured subsequent filtrate 5ml; Put water-bath steam in, residue dissolves with 50% methanol solution 20ml, solution with the speed of per minute 0.5ml through neutral alumina post (4g, 100～200 orders, column length 20cm; Internal diameter 1cm), and continue flushing with 50% methanol solution 40ml, merga pass liquid and washing liquid, water bath method, residue are used a small amount of anhydrous alcohol solution; Be transferred in the 10ml measuring bottle, be diluted to scale, shake up, as need testing solution with dehydrated alcohol.
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 (an appendix V of Chinese Pharmacopoeia version in 2000 A).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, the sighting target directrix curve prepare the method item under, from " adding 8% vanillin ... ", measure trap in accordance with the law.Calculate, promptly get.
2, Astragaloside content is measured
Measure according to HPLC (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler, 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 the astragaloside reference substance 60 ℃ of drying under reduced pressure to constant weights, adds dissolve with methanol, processes the solution that every 1ml contains 0.6mg, promptly gets.
The preparation precision of need testing solution takes by weighing the about 500mg of these article 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, and each 20ml; Merge n-butyl alcohol liquid, be transferred in the evaporating dish, water bath method is with dissolve with methanol and be transferred to the 5ml volumetric flask; Methanol is diluted to scale, shakes up, and the 0.45um membrane filtration promptly gets.
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 (mv.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, and promptly get.
3, polysaccharide molecular weight Determination of distribution
Measure according to HPLC (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test use gel to be filler, 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, processes 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 these article 160mg, puts in the 5ml measuring bottle, adds the mobile phase dissolving and is diluted to scale, shakes up, and promptly gets.
The accurate need testing solution 25 μ l that draw of algoscopy inject chromatograph of liquid, measure, and calculate, and promptly get.
4, total polysaccharides assay
Measure according to HPLC (an appendix VI of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test use gel to be filler, 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, and the mixing reference substance solution of 3.2mg/ml promptly gets.
The preparation precision of need testing solution takes by weighing these article 160mg, puts in the 5ml measuring bottle, adds the mobile phase dissolving and is diluted to scale, shakes up, and promptly gets.
The specific embodiment
Below in conjunction with embodiment the present invention is described further, 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 subsequent use), 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; Elder generation is with the distilled water washing resin post of 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.
(2) the above-mentioned medicinal residues of flinging to behind the solvent add 10 times of water gagings, in 60 ℃ of lixiviates 2 times, and 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, precipitate centrifugally, centrifugal gained deposition is added suitable quantity of water (water: crude drug in whole is 1: 1) dissolve; 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, gets supernatant, adds ethanol and reaches 80% to containing the alcohol amount; Hold over night filters, and gets crude polysaccharides, adds suitable quantity of water (water: primary crude drug is 1: 1) dissolving, with molecular cut off 3000 ultrafilter membrane ultrafiltration; Get concentrated solution, being evaporated to relative density is 1.20 ~ 1.30 (60 ℃), adds ethanol and reaches 80% to containing the alcohol amount, and hold over night filters; Get deposition, use absolute ethanol washing, vacuum drying is pulverized, and gets total polysaccharide extractive.
(3) getting weight ratio is 1: 3 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive, adds 2000ml water for injection and makes dissolving, adds 9.5% injection mannitol, 0.5% 30 POVIDONE K 30 BP/USP 30; And to use 10% sodium hydroxide solution adjust pH be 6.5～7.5, adds water for injection to 2500ml, filters with 0.22 μ m microporous filter membrane; In the fill 7ml cillin bottle, every bottle of 2.5ml, lyophilization; Gland seals, and promptly gets.
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.
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 subsequent use) 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; Elder generation is with the distilled water washing resin post of 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.
(2) the above-mentioned medicinal residues of flinging to behind the solvent add 6 times of water gagings, in 100 ℃ of lixiviates 2 times, and 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, precipitate centrifugally, centrifugal gained deposition is added suitable quantity of water (water: crude drug in whole is 1: 1) dissolve; 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, gets supernatant, adds ethanol and reaches 80% to containing the alcohol amount; Hold over night filters, and gets crude polysaccharides, adds suitable quantity of water (water: primary crude drug is 1: 1) dissolving, with molecular cut off 3000 ultrafilter membrane ultrafiltration; Get concentrated solution, being evaporated to relative density is 1.20 ~ 1.30 (60 ℃), adds ethanol and reaches 90% to containing the alcohol amount, and hold over night filters; Get deposition, use absolute ethanol washing, vacuum drying is pulverized, and gets total polysaccharide extractive.
(3) getting weight ratio is 1: 0.1 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive, adds 2000ml water for injection and makes dissolving, adds 9.5% injection mannitol, 0.5%Tween-80; And to use 10% sodium hydroxide solution adjust pH be 6.5～7.5, adds water for injection to 2500ml, filters with 0.22 μ m microporous filter membrane; In the fill 7ml cillin bottle, every bottle of 2.5ml, lyophilization; Gland seals, and promptly gets.
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 subsequent use) 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; Elder generation is with the distilled water washing resin post of 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.
(2) the above-mentioned medicinal residues of flinging to behind the solvent add 8 times of water gagings, in 80 ℃ of lixiviates 3 times, and 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, precipitate centrifugally, centrifugal gained deposition is added suitable quantity of water (water: crude drug in whole is 1: 1) dissolve; 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, gets supernatant, adds ethanol and reaches 70% to containing the alcohol amount; Hold over night filters, and gets crude polysaccharides, adds suitable quantity of water (water: primary crude drug is 1: 1) dissolving, with molecular cut off 6000 ultrafilter membrane ultrafiltration; Get concentrated solution, being evaporated to relative density is 1.20 ~ 1.30 (60 ℃), adds ethanol and reaches 70% to containing the alcohol amount, and hold over night filters; Get deposition, use absolute ethanol washing, vacuum drying is pulverized, and gets total polysaccharide extractive.
(3) getting weight ratio is 1: 8 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive, adds 2000ml water for injection and makes dissolving, adds 9.5% glucose; And to use 10% sodium hydroxide solution adjust pH be 6.5～7.5, adds water for injection to 2500ml, filters with 0.22 μ m microporous filter membrane; In the fill 7ml cillin bottle, every bottle of 2.5ml, lyophilization; Gland seals, and promptly gets.
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 subsequent use) decompression recycling ethanol also was concentrated into the thick paste that relative density is 1.15～1.25 (60 ℃), adds 5 times of water gagings and makes dissolving; Leave standstill, centrifugal, get supernatant and be added on the AB-8 type macroporous adsorptive resins of having handled well; Earlier with the distilled water washing resin post of 5 times of amounts, reuse 80% alcoholic acid eluting saponin component is collected 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, 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, and 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, precipitate centrifugally, centrifugal gained deposition is added suitable quantity of water (water: crude drug in whole is 1: 1) dissolve; 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, gets supernatant, adds ethanol and reaches 70% to containing the alcohol amount; Hold over night filters, and gets crude polysaccharides, adds suitable quantity of water (water: primary crude drug is 1: 1) dissolving, with molecular cut off 6000 ultrafilter membrane ultrafiltration; Get concentrated solution, being evaporated to relative density is 1.20 ~ 1.30 (60 ℃), adds ethanol and reaches 70% to containing the alcohol amount, and hold over night filters; Get deposition, use absolute ethanol washing, vacuum drying is pulverized, and gets total polysaccharide extractive.
(3) getting weight ratio is 1: 4 Radix Codonopsis, Radix Astragali total saponins and total polysaccharide extractive, adds 2000ml water for injection and makes dissolving, adds 9.5% glucose; And to use 10% sodium hydroxide solution adjust pH be 6.5～7.5, adds water for injection to 2500ml, filters with 0.22 μ m microporous filter membrane; In the fill 7ml cillin bottle, every bottle of 2.5ml, lyophilization; Gland seals, and promptly gets.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 one 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 is seen 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
Visible by table 1, 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 dose groups 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), explains that pharmaceutical preparation of the present invention can the anti-Mice Bearing Lewis Lung Cancer effect of enhanced electronic phosphamide.
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 is seen 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
Visible by table 2, 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 Combined application, anti-mice H
22The effect of hepatocarcinoma strengthens; Large, medium and small dose groups 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;, explain that pharmaceutical preparation of the present invention can strengthen the anti-mice H of radiotherapy
22The hepatocarcinoma effect.SHENQI FUZHENG ZHUSHEYE and radiotherapy Combined application, 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, explain that pharmaceutical preparation of the present invention strengthens the anti-mice H of radiotherapy
22The hepatocarcinoma effect is better than SHENQI FUZHENG ZHUSHEYE.
Claims (8)
1. pharmaceutical preparation that is used for assistant treating cancer, adopting the Radix Codonopsis and the Radix Astragali is raw material, makes through 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, add alcohol reflux, and get extracting solution and medicinal residues, extracting solution gets total saponin extracts through concentrated, centrifugal and low pole macroporous adsorptive resins purification;
(2) after the medicinal residues that obtain of step (1) are removed solvent, use water extraction, after extracting solution concentrated, the accent alcoholic degree precipitated to 50-80%; Deposition 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%, crude polysaccharides, using and adopting the molecular weight that dams behind the water dissolution is that 3000 film carries out ultrafiltration; Concentrated solution further transfers alcoholic degree to precipitate to 70-90%, gets deposition, and drying and crushing gets total polysaccharide extractive;
(3) total polysaccharide extractive of (1) total saponin extracts and step (2) of getting step is as active component, and the ratio of the two weight is 0.1-8: 1, add suitable pharmaceutically acceptable carrier, and process 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 method for preparing of the preparation of claim 5 may further comprise the steps:
(1) get Radix Codonopsis and Radix Astragali decoction pieces, add alcohol reflux, 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 concentrated, centrifugal and low pole macroporous adsorptive resins purification;
(2) after the medicinal residues that obtain of step (1) are removed solvent, use water extraction, after extracting solution concentrated, the accent alcoholic degree precipitated to 50-80%; Deposition transfers after being dissolved in water alcoholic degree to precipitate to 30-40% again, gets supernatant, removes albumen with trichloroacetic acid, further transfers 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%; Crude polysaccharides, behind water dissolution, adopting the molecular weight that dams is that 3000 film carries out ultrafiltration; Concentrated solution further transfers alcoholic degree to precipitate to 70-90%, gets deposition, 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, process 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, comprise the 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, gets extracting solution and medicinal residues; Extracting solution is collected eluent and concentrate drying and is got Radix Codonopsis, Radix Astragali total saponins extract through concentrated, centrifugal and D101 type macroporous adsorptive resins purification;
(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%, after being dissolved in water, deposition transfer alcoholic degree to precipitate to 30-40% again, get supernatant; Remove albumen, 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%, gets deposition, 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, process freeze-dried powder.
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| CN108635550A (en) * | 2018-08-20 | 2018-10-12 | 江苏省中医院 | A kind of pharmaceutical composition that treating gastric cancer and its application |
| CN111965315A (en) * | 2020-08-13 | 2020-11-20 | 山东大学 | Method for screening radix astragali total saponin extraction process based on zebra fish vascular injury model and application thereof |
| CN114949031A (en) * | 2021-02-26 | 2022-08-30 | 湖州新活医疗科技有限公司 | Traditional Chinese medicine composition for improving spleen and stomach functions and enhancing immunity and application |
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| CN1546152A (en) * | 2003-12-02 | 2004-11-17 | 张正生 | Freeze-dried 'Shenqifuzheng' powder for injection and its preparing process |
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