CN1939530A - Freeze-drying powder injection of Bozhi glycopeptide and its preparation - Google Patents
Freeze-drying powder injection of Bozhi glycopeptide and its preparation Download PDFInfo
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Abstract
A freeze-dried powder injection of thin tree ganoderma's glycopeptide extracted from the thin tree ganoderma is proportionally prepared from said glycopeptide and the freeze-drying excipient.
Description
Technical field
The present invention relates to the ganoderma's glycopeptide preparation, relate in particular to ganoderma's glycopeptide injection preparation and preparation method thereof.
Background technology
Ganoderma is stated from the Shennong's Herbal of Han dynasty as medicine head.The ancient medicine man thinks that all Ganoderma can treat multiple disease, is strengthening by means of tonics, the precious medicine of strengthening the body resistance.Ganoderma is commonly called as Ganoderma lucidum seu Japonicum, and among the people have legends such as " Ganoderma are controlled all kinds of diseases and ailments ".Ganoderma is a kind of medicinal fungi of preciousness, belongs to Basidiomycetes, Polyporaceae, Ganoderma.This genus has 100 kinds approximately, and common have Ganoderma lucidum (Leyss. Ex Fr.) Karst. (Ganoderma lucidum), Ganoderma (G.japonicum), a ganoderma capense (G.capense) etc.Be referred to as " Herba mesonae chinensis " ancient times.Ganoderma is a medicinal plants precious in motherland's pools of traditional Chinese medicine.Ganoderma is warm in nature, and is lightly seasoned, and having calms the nerves supports essence, strengthening by means of tonics, and the effect of relieving cough and asthma, the Chang Zuowei tonic comes across in the ancient book.Studies show that, the Ganoderma main component is ergosterol, fungal lysozyme, mannitol, polypeptide, aminoacid etc., has strengthening the body resistance, integrally-regulated, enhance immunity, inhibition tumor, brain-strengthening kidney tonifying, strengthening by means of tonics, detoxifies, protects the liver, many-sided biological activity such as heart tonifying, calmness, anti-hypoxia, convulsion, relieving cough and asthma; Be used for diseases such as angina pectoris, atherosclerosis, hyperlipidemia, acute viral hepatitis, neurasthenia, chronic bronchitis.
Ganoderma also has significant curative effect to diseases such as gastrointestinal, liver, kidney, leukemia, neurasthenia, chronic bronchitis, asthma, allergy except effective in cure to cancer, cerebral hemorrhage, the heart disease of the human three big causes of the death.In addition, Ganoderma also has strong essence, antiinflammatory, analgesia, antibiotic, detoxifcation, diuresis, multiple effect and effect such as blood only.Ganoderma not only has good health-care effect, and multiple person in middle and old age's chronic metabolic disease is had the auxiliary treatment effect.The anticancer research of Ganoderma also is subjected to attracting attention of Chinese scholars, and the Ganoderma anticancer drugs successfully go on the market, and have obtained excellent curative.Pharmacological research both domestic and external proves that all Ganoderma has the obvious suppression function of tumor, and tumour inhibiting rate is 50%～70%.Ganoderma can also significantly reduce the mortality rate of the radiation exposure animal or the injection chemotherapeutic animal of fatal dose.
China's ganoderan mainly extracts from Ganoderma sporophore at present.The artificial culture Ganoderma sporophore production cycle is long, and adopts the deep layer cultured method to obtain Ganoderma mycelium and ganoderan, can shorten the production cycle.The ganoderma's glycopeptide that Beijing match crude drug is already produced is to extract in the dry mycelium powder of Ganoderma ganoderma capense [Ganoderma capense (Lloyd) Teng] that liquid submerged fermentation makes with ganoderma capense (G.capense) bacterial strain to make, and its main component is polypeptide and polysaccharide.Ganoderma's glycopeptide has the effect of regulating body's immunity, and body nonspecific immunity, humoral immunization and cellular immunization etc. are all had facilitation; Have antioxidation, can remove oxygen-derived free radicals; In addition, effects such as the nucleic acid of promotion, protein biosynthesis are still arranged.Ganoderma's glycopeptide is applicable to progressive muscular dystrophy, myotonia atrophica, and vestibular dysfunction, and diseases such as the dizzy and autonomic nervous dysfunction that causes such as hypertension, epilepsy, insomnia.The auxiliary treatment that also can be used for tumor, hepatitis.
Lin Yongqi, horse Biao etc. studied boron aglucon affinity chromatograph technology purification ganoderma's glycopeptide method (biotechnology, 2004,14, No.z1,31-32).Method is: investigate condition of different pH with ammonium acetate buffer and add the influence of variable concentrations NaCl to the ganoderma's glycopeptide adsorption-desorption down, find out static optimal adsorption desorption condition; Obtaining maximum static adsorption capacity, absorption constant and dynamic adsorption sugar and proteic capacity according to the Chase model. the result shows: the adsorbance that adds to ganoderma's glycopeptide under the 0.15mol/L condition in pH8.2, NaCl concentration reaches maximum, is the optimal adsorption desorption condition therefore; Maximum static adsorption capacity qm=55.57mg/g wet basis with this understanding, absorption constant Kd=5.312g/L; Dynamic adsorption polysaccharide capacity is the 43.1mg/g wet basis, and the dynamic adsorption protein capacity is the 10.3mg/g wet basis.Affinity chromatograph that this system uses separation and purification ganoderma's glycopeptide effectively is described.
Through clinical application for many years, proved that ganoderma's glycopeptide has better curative effect.Have only aqueous injection through the intravenously administrable dosage form in the market, have only the ganoderma's glycopeptide injection through state approval production.This medicine in clinical practice for many years, curative effect is reliable, the market consumption is big, but has poor stability, clarity is bad, product yield is low, preserves, is subject in the transportation problems such as influence of temperature change.
Summary of the invention
At the above-mentioned technical problem that prior art exists, the inventor successfully develops the ganoderma's glycopeptide lyophilized injectable powder, and it has greatly improved stability of drug, stores all more stablely under low temperature and higher temperature, and has solved the bad problem of clarity; And improved product yield, transportation, easy to carry.
Therefore, one object of the present invention is to provide a kind of injection ganoderma's glycopeptide freeze-dried powder.
Another object of the present invention is to provide the preparation method of this freeze-dried powder.
According to an aspect of the present invention, the invention provides a kind of injection ganoderma's glycopeptide freeze-dried powder, its characteristics are:
Described lyophilized injectable powder is to be prepared by ganoderma's glycopeptide concentrated solution and freeze-dried excipient;
The weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.3～7;
Described ganoderma's glycopeptide concentrated solution is to extract to make from thin tree ganoderma, and every milliliter of ganoderma's glycopeptide concentrated solution contains polysaccharide and is not less than 2.5mg with glucose meter, and polypeptide is not less than 0.5mg.
Described freeze-dried excipient can be amino acids, as glycine, alanine, histidine, arginine, aspartic acid, agedoite etc.; Also can be mannitol, sorbitol, lactose, glucose, sucrose, dextran, polyhydric alcohol inositol, albumin, gelatin, Polyethylene Glycol etc.
Preferably, described freeze-dried excipient is to be selected from one or more of mannitol, dextran, glycine etc.
Preferred, described freeze-dried excipient is mannitol, dextran or glycine.
Preferably, the weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.3～1.
Most preferred, the weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.5.
Aqueous injection of the present invention is that ganoderma's glycopeptide adds the sterile water solution that suitable stabilizing agent is made, and described lyophilized injectable powder is that ganoderma's glycopeptide adds the lyophilized injectable powder that suitable adjuvant is made.
In better embodiment of the present invention, described lyophilized injectable powder contains polysaccharide with glucose (C
6H
12O
6) meter and polypeptide be not less than 90% of labelled amount usually.
Described ganoderma's glycopeptide concentrated solution is to extract in the dry mycelium powder of Ganoderma ganoderma capense [Ganoderma capense (Lloyd) Teng] that liquid submerged fermentation makes with ganoderma capense (G.capense) bacterial strain to make, and its main component is polypeptide and polysaccharide.
Use other kind of Ganoderma, as Ganoderma lucidum (Leyss. Ex Fr.) Karst., Ganoderma etc., also can prepare and comprise polysaccharide and polypeptide amount and the similar glycopeptide extracting solution of the used ganoderma's glycopeptide concentrated solution of the present invention, it will be understood by those skilled in the art that the injectable powder that is prepared by these glycopeptide extracting solution also should comprise within the scope of the present invention.
Injectable powder provided by the invention can give the patient with effective dose according to the conventional using method of injectable powder.
According to a further aspect in the invention, the invention provides a kind of preparation method of freeze-drying powder injection of Bozhi glycopeptide, comprise the steps:
A, extract from thin tree ganoderma and make the ganoderma's glycopeptide concentrated solution, every milliliter of ganoderma's glycopeptide concentrated solution contains polysaccharide and is not less than 2.5mg with glucose meter, and polypeptide is not less than 0.5mg;
B, be that 10: 0.3～7 ganoderma's glycopeptide concentrated solution mixes with freeze-dried excipient, divide the bottle of packing into, after the lyophilization, seal, promptly get freeze-drying powder injection of Bozhi glycopeptide weight ratio.
According to the common practise of this area, the ganoderma's glycopeptide concentrated solution that is used to prepare lyophilized injectable powder should be aseptic apyrogeneity, divides the bottle of packing into to carry out under aseptic condition.
When preparation ganoderma's glycopeptide lyophilized injectable powder, extracting the used thin tree ganoderma of ganoderma's glycopeptide concentrated solution usually should be for ease of the dry mycelium of industrial ganoderma capense.
Preferably, described freeze-dried excipient is to be selected from one or more of mannitol, dextran, glycine etc.
Preferred, described freeze-dried excipient is mannitol, dextran or glycine.
Preferably, the weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.3～1.
Most preferred, the weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.5.
In the present invention, described ganoderma's glycopeptide concentrated solution can adopt traditional isolation and purification method such as hot water or ethanol precipitation to extract from the dry mycelium powder of Ganoderma ganoderma capense to make; Also can prepare the ganoderma's glycopeptide crude extract earlier, adopt the affinitive layer purification technology to prepare then.
Therefore, in steps A, described ganoderma's glycopeptide concentrated solution can prepare by following step:
A1, the dry mycelium of ganoderma capense is pulverized, is sieved, mycelium powder;
A2, with mycelium powder with 92～94% alcohol reflux 3～6 times, filtered while hot, merging filtrate in being evaporated to small size below 70 ℃, adding water and is evaporated in the distillate till the no ethanol again, the ethanol immersion;
A3, above-mentioned ethanol immersion is moved in the separatory funnel, water layer liquid is collected in the extraction 8～12 times that adds diethyl ether, and ether layer liquid is added water washing, water lotion is incorporated in the water layer liquid, water layer liquid;
A4, water layer liquid put in the water-bath concentrate, drive ether, the ganoderma's glycopeptide concentrated solution.
In steps A, described ganoderma's glycopeptide concentrated solution also can prepare by following step:
B1, the dry mycelium of ganoderma capense is pulverized, is sieved, mycelium powder;
B2, employing second alcohol and water obtain the ganoderma's glycopeptide crude extract as extracting solvent from above-mentioned mycelium powder extraction separation;
B3, employing boron aglucon affinity chromatography obtain the ganoderma's glycopeptide concentrated solution from the separation and purification of above-mentioned ganoderma's glycopeptide crude extract.
Among the step b3 the concrete process conditions of used boron aglucon affinity chromatography can referring to disclosed pertinent literatures such as the strange forever, Ma Biao of woods (ganoderma's glycopeptide affinitive layer purification technology, biotechnology, 2004,14, No.z1,31-32).
In step B, described lyophilization is vacuum lyophilization, and carries out according to following steps:
Earlier with 0.1～10 ℃ of quick-freezing of per minute to-45 ℃, press freeze-drying curve and keep vacuum, under vacuum state, slowly be warming up to 35 ℃ with 0.5～10 ℃ speed per hour, stop to heat.
In a specific embodiment of the present invention, the preparation method of described freeze-drying powder injection of Bozhi glycopeptide is as follows:
1, preparation ganoderma's glycopeptide concentrated solution;
2, according to preparation specification, batch and prescription requirement, prepare aseptic pyrogen-free ganoderma's glycopeptide concentrated solution;
3, dissolve pharmaceutically acceptable auxiliaries with water for injection, the ultrafiltration depyrogenation;
4, at hundred grades of clean areas above-mentioned supplementary material is measured by the prescription requirement, place abundant mixing in the aseptic apyrogeneity liquid dispensing container;
5, through 0.22 μ m filter membrane aseptic filtration;
6, filtrate accurately is sub-packed in medicinal inner packing bottle, puts vacuum drying oven;
7, lyophilization: earlier with 0.1～10 ℃ of quick-freezing of per minute to-45 ℃, press freeze-drying curve and keep vacuum, under vacuum state, slowly be warming up to 35 ℃ with 0.5～10 ℃ speed per hour, stop to heat;
8, natural cooling takes out after reducing to room temperature, and gland promptly gets lyophilized injectable powder immediately.
Influence factor's result of the test shows, no significant change before and after the ganoderma's glycopeptide freeze-dried powder provided by the invention, and the character of liquid drugs injection and pH value have taken place than significant change, illustrate that powder pin stability is apparently higher than liquid drugs injection.
At present, the ganoderma's glycopeptide injection adopts control antibiotic bottle packing, bottle stopper contacts for a long time with injection, cause and when clarity test, check out many fibers and glass point, cause product yield lower, and freeze-dried powder provided by the invention has avoided medicine to contact with the long-term of bottle stopper, thereby has improved clarity, has improved product yield greatly.
The advantage of injection ganoderma's glycopeptide freeze-dried powder provided by the invention is:
1, freeze-drying powder injection of Bozhi glycopeptide waits other dosage form to compare with ganoderma's glycopeptide injection (liquid drugs injection) to have good stability, easily transportation, easily advantage such as preservation.
2, through stability test, influence factor's test, accelerated test and room temperature reserved sample observing, compare with liquid drugs injection, the freeze-dried powder steady quality is placed sample appearance, pH value, content for a long time and is had no significant change.
3, freeze-dried powder was preserved 10 days for 60 ℃, and catabolite is just arranged, and liquid drugs injection was preserved 2 days for 60 ℃, had catabolite to produce.This shows that freeze-dried powder stability improves, and anti-degradation capability is better than liquid drugs injection.
4, freeze-dried powder adopts cillin bottle, and steady quality is easy to keeping and transportation.
5, product yield improves.
6, solved the bad problem of ganoderma's glycopeptide aqueous injection clarity.
In order to understand essence of the present invention better,, describe in detail but do not limit the present invention below by description to better embodiment of the present invention.
The specific embodiment
The used test material of the present invention if no special instructions, is commercially available purchase product.
The preparation and the quality standard thereof of the dry mycelium powder of ganoderma capense
[embodiment 1]
Through the liquid fermentation and culture method, prepare Ganoderma ganoderma capense [GanodermaCapense (Lloyd) Teng] dry mycelium (providing) by the ganoderma capense bacterial strain by Beijing Saier Bio-Pharmaceutical Co., Ltd., pulverize through pulverizer, cross 20 mesh sieves, get the powdery mycelium.
According to the dry mycelial quality standard check of ganoderma capense, mycelium powder of the present invention meets corresponding quality standard.Quality standard is as follows:
1, character: this product is filbert and the yellowish-brown particulate powder.
2, loss on drying: get this product, be dried to constant weight, subtract weight loss and must not cross 8% at 105 ℃.
3, toxicity and anti-nicotinamide effect are checked: get dry mycelium 30 grams, with 90% alcohol reflux 2 hours, filtered while hot, continue with alcohol reflux 3 times, merge ethanol extract, in water-bath, be evaporated to small size, after add the water of about 1/3 volume again, continue to be evaporated to distillate and do not have till the ethanol.Add an amount of extraction of ether about 10 times again, water layer is concentrated in water-bath, drive ether, spissated extracting solution is made into the dry mycelial solution of suitable 1 gram with every ml of the dry mycelial solution of suitable 2 grams of every ml, do toxicity and anti-nicotinamide effect inspection respectively.
3.1 anti-nicotinamide effect: 20 of the mices of getting 18～22 gram body weight, two groups of five equilibriums, the dry mycelial solution of suitable 1 gram of every ml of one group of above-mentioned preparation of lumbar injection (dosage is by per kilogram of body weight 40 grams), the matched group injection is with the normal saline of volume, the nicotinamide that the equal intravenous injection concentration of every afterwards Mus is 0.2mg/ml (dosage is by per kilogram of body weight 1mg), the record mice is because of the tonic convulsion death toll.
Toxicity test is carried out in ※ P＜0.05, as repeating once at the edge, as death is arranged in the mice before not injecting nicotinamide, then can make after the suitably dilution repeated experiments again.
| The control group mice death toll | Medication group dead mouse number must not surpass |
| 10/10 | 5/10 |
| 9/10 | 4/10 |
| 8/10 | 3/10 |
3.2 toxicity test: with 10 of body weight 18～22 gram mices, the above-mentioned every ml of lumbar injection is equivalent to the dry mycelium solution of 2 grams (dosage is pressed per kilogram 40 grams), and in 24 hours, the dead mouse number must not be crossed 5.
The preparation of ganoderma's glycopeptide concentrated solution and quality standard thereof
The preparation of [embodiment 2] ganoderma's glycopeptide concentrated solution
Method one: the dry mycelium of Ganoderma ganoderma capense [GanodermaCapense (Lloyd) Teng] that the ganoderma capense bacterial strain makes through the liquid fermentation and culture method is pulverized through pulverizer, crossed 20 mesh sieves, get the powdery mycelium.Get the 30g mycelium powder, added 92～94% alcohol refluxs 2 hours, filtered while hot.Continue with alcohol reflux 3 times again, merge ethanol extract, after being evaporated to small size in the water-bath, add the water of about 1/3 volume again, being evaporated to distillate again, to measure proportion with the ethanol gravimeter be 0 only.Put coldly, add about about 10 times of an amount of extraction of ether, discard the intermediate layer, collect ether layer and water layer.Water layer is concentrated in water-bath, drive ether, be spissated thin sesame injection part, get concentrated solution and be made into 100% and 200% two kind of each 5ml of concentration, carry out biology inspection.
Method two: get the dry mycelium that is up to the standards and pulverize, cross 20 mesh sieves, get the powdery mycelium through pulverizer.With 92～94% alcohol reflux of 5 times of amounts, 4 times of amounts, 3 times of amounts four times, refluxed filtered while hot 2 hours at every turn.Merging filtrate, in being evaporated to small size below 70 ℃, add water again row be evaporated in the distillate till the no ethanol, must the ethanol immersion.Above-mentioned ethanol immersion is moved in the separatory funnel, add diethyl ether that it is more shallow to be extracted to ether layer color, collect water layer liquid, and ether layer liquid is added the low amounts of water washing, water lotion also is incorporated in the water layer liquid, water layer liquid.Water layer liquid put in the water-bath concentrate, drive ether, the ganoderma's glycopeptide concentrated solution, test by ganoderma's glycopeptide concentrated solution quality standard.
[embodiment 3] ganoderma's glycopeptide concentrated solution quality standard
According to the check of ganoderma's glycopeptide concentrated solution quality standard, ganoderma's glycopeptide concentrated solution of the present invention meets corresponding quality standard.Quality standard is as follows:
This strain ganoderma capense bacterial strain, the dry mycelium of Ganoderma ganoderma capense [GanodermaCapense (Lloyd) Teng] that makes through the liquid fermentation and culture method extracts the solution that makes, and every ml soln contains polysaccharide with glucose (C
6H
12O
6) must not count and be lower than 2.5mg, polypeptide must not be lower than 0.5mg.
1, character: this product is faint yellow to fallow clear liquid.
2, differentiate
(1) gets this product 1ml, add anthrone solution and (get anthrone 0.2g, add sulphuric acid 100ml and make dissolving; Face with newly joining) 5ml, to put water-bath and place, solution is aeruginous.
(2) get this product 2ml, add the about 10mg of 1,2,3-indantrione monohydrate, shake up, drip on filter paper, bluish violet should appear in hot blast drying.
(3) get this product 10ml, little fire heating is concentrated into about 2ml, be cooled to room temperature after, add 2 of bismuth potassium iodide test solutions, promptly produce the precipitation of orange red or rufous.
3, check
PH value: get this product, be diluted to water for injection and contain the about 2.5mg/ml of polysaccharide or contain polypeptide 0.5mg/ml, measure (two appendix VI of Chinese Pharmacopoeia version in 2000 H) in accordance with the law, should be 5.0～7.0.
Protein: get this product 2ml, add 20% sulfosalicylic acid solution 1ml, shake up, solution must not produce muddiness.
Tannin: get this product 1ml, add 2～3 of 4% gelatin solutions, shake up, solution must not produce muddiness.
The undue toxicity: get this product, be diluted to water for injection and contain the about 0.5mg/ml of polypeptide or contain polysaccharide 2.5mg/ml, check in accordance with the law and (two appendix XI of Chinese Pharmacopoeia version in 2000 C) press the administration of vein method, should be up to specification.
Bacterial endotoxin: get this product, check (two appendix XI of Chinese Pharmacopoeia version in 2000 E) in accordance with the law, the contained bacterial endotoxin of every 1mg polypeptide should be less than 10EU, and the contained bacterial endotoxin of every 1mg polysaccharide should be less than 2EU.
4, assay
Polypeptide: it is an amount of to measure this product, adds water and makes the solution that contains polypeptide 0.15mg among every 1ml, as need testing solution.Measure according to forint phenol algoscopy (seeing embodiment 4), from regression equation, obtain content of peptides.
Polysaccharide: the about 0.1g of anhydrous glucose that the preparation of reference solution is learnt from else's experience 105 ℃ and is dried to constant weight, accurate claim surely, put in the 100ml measuring bottle, be dissolved in water and be diluted to scale, shake up.
The preparation of need testing solution: precision is measured this product, and the every 1ml of thin up contains the solution of 0.0375mg approximately.
The preparation of standard curve: precision is measured reference solution 1.0ml, 2.0ml, 4.0ml, 6.0ml, 8.0ml, puts respectively in the 100ml measuring bottle, and thin up shakes up to scale.Accurate respectively each 1.0ml of reference solution of measuring above-mentioned 5 concentration, other 1.0ml that fetches water does blank, put respectively in the tool plug test tube, place ice-water bath, the accurate respectively anthrone solution 5.0ml that adds shakes up, put in the water-bath and reacted 10 minutes, take out, be cooled to room temperature, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 B) with cold water, wavelength place at 620nm measures trap, carries out regression equation calculation to record the trap concentration corresponding with it.Algoscopy: precision is measured need testing solution 1.0ml, puts in the tool plug test tube, rises from " placing ice-water bath, the accurate respectively anthrone solution 5.0ml that adds " under the sighting target directrix curve preparation, measures trap in accordance with the law, calculates the content of polysaccharide from regression equation.
5, classification: immunomodulator.
6, storage: lucifuge, cryopreservation.
[embodiment 4] forint phenol algoscopy
Reagent alkaline copper test solution is got sodium hydroxide 10g, and sodium carbonate 50g adds water 400ml and makes dissolving, as first liquid; Get Soluble tartar. 0.5g, add water 50ml and make its dissolving, other gets copper sulfate 0.25g, adds water 30ml its dissolving is mixed two liquid as second liquid.
Before facing usefulness, merge first, second two liquid, and add water to 500ml.
The bovine serum albumin reference substance is got in the preparation of maneuver contrast solution, adds water and makes the solution that contains 0.3mg among every 1ml.
Preparing of need testing solution according to the method preparation of regulation down of each kind item.
The preparation precision of standard curve is measured contrast solution 0.0ml, 0.1ml, 0.3ml, 0.5ml, 0.7ml, 0.9ml, put respectively in the tool plug test tube, respectively add water to 1.0ml, add alkaline copper test solution 1.0ml more respectively, shake up, each adds forint phenol test solution (getting the stock solution 1 → 16 in the forint test solution) 4.0ml, mixing immediately, put in 55 ℃ of water-baths accurate response 5 minutes, put psychrolusia 10 minutes, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 B), measure trap at the wavelength place of 650nm; Manage as blank with No. 0.Calculate regression equation with reference substance solution concentration and corresponding absorbance.
The algoscopy precision is measured need testing solution 1.0ml, and the method under the preparation of sighting target directrix curve from " adding the alkaline copper test solution ", is measured in accordance with the law, from the content of regression equation calculation polypeptide, and multiply by extension rate, promptly.
The preparation of freeze-drying powder injection of Bozhi glycopeptide and quality standard thereof
The preparation of [embodiment 5] freeze-drying powder injection of Bozhi glycopeptide
The detailed process and the step of preparation freeze-drying powder injection of Bozhi glycopeptide of the present invention are as follows:
1, the preparation of ganoderma's glycopeptide concentrated solution
With reference to the method for embodiment 2, and, prepare aseptic pyrogen-free ganoderma's glycopeptide concentrated solution according to preparation specification, batch and prescription requirement;
2, dosing
Prepare: various dosing articles for use on deck;
Measure liquid: measure the ganoderma's glycopeptide concentrated solution (pressing finished product calculates) of recipe quantity, two people check;
Adjuvant preparation: it is an amount of to take by weighing pharmaceutic adjuvant mannitol, and being dissolved to final concentration with water for injection is 10%, the ultrafiltration depyrogenation;
Aseptic filtration: at hundred grades of clean areas above-mentioned supplementary material and diluent are measured by the prescription requirement, place abundant mixing in the aseptic apyrogeneity liquid dispensing container; Cross the 0.22um filter membrane;
The medicinal liquid of aseptic medicine should be no more than 3 hours from the interval that is formulated into aseptic filtration, checks semi-finished product content, pH and bacterial endotoxin etc., clears out a gathering place.
3, fill
Test run: install machine, energized, check each working order, by low speed to high-speed cruising intact after, can use
Prepare: the bottling top plug, cillin bottle is sent into bottle arranging disk, plug is packed into manage in the plug device;
Transfer loading amount: the result determines loading amount by the semi-finished product assay, and rotation accent amount nut is transferred to definite loading amount.
Desirable loading amount 1 ± 0.2ml;
Fill: the reason dish turns clockwise, bottle is sent into reason bottle track, enter the calibration fluted disc, the calibration fluted disc is with 10-50 intermittently speed rotation of per minute, bottle is pushed filling position, filling mechanism carries out fill with the operation up and down at synchronous intermittence, and the cillin bottle after the fill goes to by the plug position, carries out by plug.
Plug is sent to by plug wheel place by the defeated plug of reason plug device track, and intermittently transposition is done in plug firm following by the plug wheel under pull of vacuum, makes plug aim at bottleneck, to push away on the bottle by the plug push rod, make plug aim at bottleneck, this moment, vacuum was closed automatically, plug is thrown off by the plug wheel, enters defeated bottle track with bottle.
With the medicine bottle sabot of fill false add plug, send into freeze dryer, clear out a gathering place.
Finish to be no more than 7 hours at interval from filtration to the fill concluding time.
4, lyophilization
4.1 pre-freeze: baffle temperature is controlled at-35 ℃ to-40 ℃, and products temperature reaches below-35 ℃, from the visible glaciation of form;
4.2 distillation: slowly be warming up to goods complete white (about 25 ℃) early stage, simultaneously the system vacuum degree remained on 6～8Pa, then, programming rate can be approaching to plate temperature and goods slightly soon.
4.3 dry again: 38 ℃～40 ℃ in goods kept 4～6 hours
3.4 clear out a gathering place.
5, roll lid, leak detection, lamp inspection after, draw samples send quality testing department check, promptly gets freeze-drying powder injection of Bozhi glycopeptide after the assay was approved.
The preparation of [embodiment 6] freeze-drying powder injection of Bozhi glycopeptide
It is an amount of to get the aseptic pyrogen-free ganoderma's glycopeptide concentrated solution that is up to the standards, and adds an amount of mannitol, and making mannitol ultimate density in solution is 5%, and the sterile filling lyophilizing promptly gets the injection ganoderma's glycopeptide.Concrete preparation method is with embodiment 4.
The preparation of [embodiment 7] freeze-drying powder injection of Bozhi glycopeptide
It is an amount of to get the aseptic pyrogen-free ganoderma's glycopeptide concentrated solution that is up to the standards, and adds an amount of dextran, and making dextran ultimate density in solution is 5%, and the sterile filling lyophilizing promptly gets the injection ganoderma's glycopeptide.Concrete preparation method is with embodiment 4.
The preparation of [embodiment 8] freeze-drying powder injection of Bozhi glycopeptide
It is an amount of to get the aseptic pyrogen-free ganoderma's glycopeptide concentrated solution that is up to the standards, and adds an amount of glycine, and making glycine ultimate density in solution is 5%, and the sterile filling lyophilizing promptly gets the injection ganoderma's glycopeptide.Concrete preparation method is with embodiment 4.
The quality standard of [embodiment 9] freeze-drying powder injection of Bozhi glycopeptide
According to the check of freeze-drying powder injection of Bozhi glycopeptide quality standard, freeze-drying powder injection of Bozhi glycopeptide of the present invention meets corresponding quality standard.Quality standard is as follows:
This product is the ganoderma's glycopeptide lyophilized injectable powder, is to use the ganoderma capense bacterial strain, and the dry mycelium extraction of Ganoderma ganoderma capense [Ganoderma Capense (Lloyd) Teng] that makes through the liquid fermentation and culture method makes, and contains polysaccharide with glucose (C
6H
12O
6) meter and polypeptide all must not be lower than 90.0% of labelled amount.
1, character: this product is that white is to faint yellow freeze-dried powder.
2, differentiate
(1) gets 1 of this product and add water 2ml, therefrom measure 1ml, add anthrone solution and (get anthrone 0.2g, add sulphuric acid 100ml and make dissolving; Face with newly joining) 5ml, to put water-bath and place, solution is aeruginous.
(2) get 1 of this product, add water 2ml, add the about 10mg of 1,2,3-indantrione monohydrate, drip on filter paper, bluish violet should appear in hot blast drying.
(3) get 3 of this product, add water 1ml dissolving altogether, add 2 of bismuth potassium iodide test solutions, promptly produce the precipitation of orange red or rufous.
3, check
PH value: get test sample, the 2ml water for injection of whenever drawing dissolving, (two appendix VIH of Chinese Pharmacopoeia version in 2000) check pH value in accordance with the law, should be 5.5～7.0.
Protein: get 1 of this product, add water 2ml, add 20% sulfosalicylic acid solution 1ml, shake up, solution must not produce muddiness.
Tannin: get 3 of this product, add water 2ml respectively, measure 1ml behind the mixing, add 2～3 of 4% aqueous gelatin solutions, shake up, solution must not produce muddiness.
The undue toxicity: get this product, check in accordance with the law and (two appendix XI of Chinese Pharmacopoeia version in 2000 D) press intravenous administration, should be up to specification.
Pyrogen: get every of test sample and check in accordance with the law (two appendix XID of Chinese Pharmacopoeia version in 2000) that with the dissolving of 2ml water for injection dosage is by the every 1kg injection of rabbit body weight 1ml.
The clarity of solution: get this product, every with 2ml water for injection dissolving, and solution should be clarified.
Loss on drying: get this product, check (Chinese Pharmacopoeia version appendix in 2000 VIIIL) in accordance with the law,, subtract weight loss and must not cross 5.0% 60 ℃ of drying under reduced pressure 4 hours.
Other: should meet every regulation relevant under the injection item (two appendix IB of Chinese Pharmacopoeia version in 2000).
4, assay
Polypeptide: get 3 of this product and add water and make the solution that contains polypeptide 0.15mg among every 1ml, as need testing solution.Measure according to forint phenol algoscopy, from regression equation, obtain content of peptides.
Polysaccharide:
The about 0.1g of anhydrous glucose that the preparation of reference solution is learnt from else's experience 105 ℃ and is dried to constant weight accurate claims surely, puts in the 100ml measuring bottle, is dissolved in water and is diluted to scale, shakes up.
The preparation of need testing solution: precision is measured this product 3ml, puts in the 200ml measuring bottle, and thin up is to scale.
The preparation of standard curve: precision is measured reference solution 1.0ml, 2.0ml, 4.0ml, 6.0ml, 8.0ml, puts respectively in the 100ml measuring bottle, and thin up shakes up to scale.Accurate respectively each 1.0ml of reference solution of measuring above-mentioned 5 concentration, make blank with water, put respectively in the tool plug test tube, place ice-water bath, the accurate respectively anthrone solution 5.0ml that adds, shake up, put in the water-bath accurate response 10 minutes, take out, be cooled to room temperature with cold water, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 B), measure absorbance at the wavelength place of 620nm, carry out regression equation calculation to record the trap concentration corresponding with it.
Algoscopy: precision is measured need testing solution 1.0ml, puts in the tool plug test tube, rises from " placing ice-water bath, the accurate respectively anthrone solution 5.0ml that adds " under the sighting target directrix curve preparation, measures trap in accordance with the law, calculates the content of polysaccharide from regression equation.
5, specification: 5mg (polysaccharide), 1mg (polypeptide).Shading, shady and cool place preserves.Tentative 2 years of effect duration.
The stable comparative test of thin sesame sugar acid anhydride lyophilized injectable powder and injection
[embodiment 10]
Influence factor's experimental condition and the results are shown in Table 1 and 2.As can be seen from the test results, no significant change before and after the influence factor of powder pin tests, and the character of liquid drugs injection and pH value have taken place than significant change, illustrate that powder pin stability is apparently higher than liquid drugs injection.
Table 1 freeze-drying powder injection of Bozhi glycopeptide influence factor result of the test (n=6)
| Condition | Outward appearance | The pH value | Clarity | Content (%) | ||
| Polysaccharide | Polypeptide | |||||
| 0 day | Faint yellow loose lyophilized powder opisthosoma | 6.40 | Qualified | 108.1 | 98.0 | |
| 60℃ | 5d | No change | 6.42 | Qualified | 109.8 | 100.4 |
| 10d | No change | 6.48 | Qualified | 109.0 | 99.7 | |
| 4500 lx | 5d | No change | 6.40 | Qualified | 109.0 | 99.5 |
| 10d | No change | 6.48 | Qualified | 107.8 | 100.2 | |
Table 2 ganoderma's glycopeptide injection influence factor's result of the test (n=6)
| Condition | Outward appearance | The pH value | Clarity | Content (%) | ||
| Polysaccharide | Polypeptide | |||||
| 0 day | Faint yellow clear liquid | 6.40 | Qualified | 106.2 | 97.3 | |
| 60℃ | 5d | The buff clear liquid | 6.1 | Qualified | 105.0 | 97.9 |
| 10d | Buff liquid | 5.7 | Qualified | 103.2 | 98.6 | |
| 4500 lx | 5d | The buff clear liquid | 6.3 | Qualified | 104.6 | 97.9 |
| 10d | Buff liquid | 6.0 | Qualified | 105.4 | 98.4 | |
More than the description of better embodiment of the present invention is not limited the present invention, those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (11)
1, a kind of freeze-drying powder injection of Bozhi glycopeptide is characterized in that,
Described lyophilized injectable powder is to be prepared by ganoderma's glycopeptide concentrated solution and freeze-dried excipient;
The weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.3～7;
Described ganoderma's glycopeptide concentrated solution is to extract to make from thin tree ganoderma, and every milliliter of ganoderma's glycopeptide concentrated solution contains polysaccharide and is not less than 2.5mg with glucose meter, and polypeptide is not less than 0.5mg.
2, lyophilized injectable powder according to claim 1 is characterized in that, described freeze-dried excipient is to be selected from one or more of mannitol, dextran, glycine.
3, lyophilized injectable powder according to claim 2 is characterized in that, described freeze-dried excipient is mannitol, dextran or glycine.
4, lyophilized injectable powder according to claim 1 is characterized in that, the weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.3～7.
5, lyophilized injectable powder according to claim 4 is characterized in that, the weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.3～1.
6, a kind of preparation method of freeze-drying powder injection of Bozhi glycopeptide comprises the steps:
A, extract from thin tree ganoderma and make the ganoderma's glycopeptide concentrated solution, every milliliter of ganoderma's glycopeptide concentrated solution contains polysaccharide and is not less than 2.5mg with glucose meter, and polypeptide is not less than 0.5mg;
B, be that 10: 0.3～7 ganoderma's glycopeptide concentrated solution mixes with freeze-dried excipient, divide the bottle of packing into, after the lyophilization, seal, promptly get freeze-drying powder injection of Bozhi glycopeptide weight ratio.
7, preparation method according to claim 6 is characterized in that, described freeze-dried excipient is to be selected from one or more of mannitol, dextran, glycine.
8, preparation method according to claim 7 is characterized in that, described freeze-dried excipient is mannitol, dextran or glycine.
9, preparation method according to claim 6 is characterized in that, the weight ratio of described ganoderma's glycopeptide concentrated solution and freeze-dried excipient is 10: 0.3～1.
10, preparation method according to claim 6 is characterized in that, in steps A, described ganoderma's glycopeptide concentrated solution is to prepare by following step:
A1, the dry mycelium of ganoderma capense is pulverized, is sieved, mycelium powder;
A2, with above-mentioned mycelium powder, with 92～94% alcohol reflux 3～6 times, filtered while hot, merging filtrate,, add water and be evaporated to again in the distillate till the no ethanol in being evaporated to small size below 70 ℃, the ethanol immersion;
A3, above-mentioned ethanol immersion is moved in the separatory funnel, water layer liquid is collected in the extraction 8～12 times that adds diethyl ether, and ether layer liquid is added water washing, water lotion is incorporated in the water layer liquid, water layer liquid;
A4, above-mentioned water layer liquid put in the water-bath concentrate, drive ether, the ganoderma's glycopeptide concentrated solution.
11, preparation method according to claim 6 is characterized in that, in steps A, described ganoderma's glycopeptide concentrated solution is to prepare by following step:
B1, the dry mycelium of ganoderma capense is pulverized, is sieved, mycelium powder;
B2, employing second alcohol and water are as extracting solvent, and extraction separation obtains the ganoderma's glycopeptide crude extract;
B3, employing boron aglucon affinity chromatography, separation and purification obtains the ganoderma's glycopeptide concentrated solution.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103027859A (en) * | 2011-09-29 | 2013-04-10 | 北京奥唯康健生物科技有限公司 | Cosmetic containing Ganoderma capense glycopeptides, and its application |
| CN103335959A (en) * | 2012-12-28 | 2013-10-02 | 通化吉通药业有限公司 | Method for determining peptide content in CZ-81 irpex cacteus extract |
| CN104634913A (en) * | 2015-01-30 | 2015-05-20 | 北京赛升药业股份有限公司 | Quality control method of bozhi glycopeptide injection liquid |
| CN104817646A (en) * | 2015-04-07 | 2015-08-05 | 广东药学院 | Polysaccharides in ganoderma capense, extraction method and applications thereof |
| CN105733956A (en) * | 2016-02-18 | 2016-07-06 | 天津泰创生物科技有限公司 | Ganoderma capense (Lloyd) Teng glycopeptide production fungi and application thereof |
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2005
- 2005-09-28 CN CNB2005100865318A patent/CN100427140C/en active Active
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103027859A (en) * | 2011-09-29 | 2013-04-10 | 北京奥唯康健生物科技有限公司 | Cosmetic containing Ganoderma capense glycopeptides, and its application |
| CN103027859B (en) * | 2011-09-29 | 2015-01-28 | 北京奥唯康健生物科技有限公司 | Cosmetic containing Ganoderma capense glycopeptides, and its application |
| CN103335959A (en) * | 2012-12-28 | 2013-10-02 | 通化吉通药业有限公司 | Method for determining peptide content in CZ-81 irpex cacteus extract |
| CN104634913A (en) * | 2015-01-30 | 2015-05-20 | 北京赛升药业股份有限公司 | Quality control method of bozhi glycopeptide injection liquid |
| CN104817646A (en) * | 2015-04-07 | 2015-08-05 | 广东药学院 | Polysaccharides in ganoderma capense, extraction method and applications thereof |
| CN104817646B (en) * | 2015-04-07 | 2017-06-06 | 广东药学院 | Polysaccharide and its extracting method and application in ganoderma capense |
| CN105733956A (en) * | 2016-02-18 | 2016-07-06 | 天津泰创生物科技有限公司 | Ganoderma capense (Lloyd) Teng glycopeptide production fungi and application thereof |
| CN105733956B (en) * | 2016-02-18 | 2018-10-26 | 天津泰创生物科技有限公司 | Ganoderma's glycopeptide produces bacterium and its application |
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|---|---|
| CN100427140C (en) | 2008-10-22 |
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Address after: 100176 No. 8 prosperous street, Beijing economic and Technological Development Zone Patentee after: Beijing Science Sun Pharmaceutical Co., Ltd. Address before: 100176 No. 8 prosperous street, Beijing economic and Technological Development Zone Patentee before: Beijing Saisheng Pharmaceutical Co., Ltd. |