CN102038650B - G.japonicum mycelium polysaccharide refined substance freeze-dried powder injection and preparation method thereof - Google Patents
G.japonicum mycelium polysaccharide refined substance freeze-dried powder injection and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a G.japonicum mycelium polysaccharide refined freeze-dried powder injection, which is characterized by consisting of 5 to 25 weight percent of G.japonicum mycelium refined substance and 75 to 95 weight percent of freeze-dried excipient. The G.japonicum mycelium polysaccharide refined freeze-dried powder injection is prepared by the G.japonicum mycelium polysaccharide refined substance which contains more polysaccharide and has small molecular weight range, so that freeze-dried powder preparation has better tumor activity resistance, low response dosage and smaller toxicity; furthermore, the polysaccharide refined substance does not contain protein and other impurities, so, compared with the G.japonicum mycelium polysaccharide refined freeze-dried powder injection prepared by the prior art, the probability of causing adverse reaction by using the freeze-dried powder preparation is greatly reduced.
Description
Technical field
The present invention relates to field of traditional Chinese, be specifically related to refining composition freeze-dried powder injection of a kind of Ganoderma mycelium polysaccharides and preparation method thereof.
Background technology
Edible and medicinal fungi has the multiple efficacies such as anti-tumor virus, blood fat reducing, slow down aging, protecting liver and expelling toxin, promotion nucleic acid and protein biosynthesis, in state-owned long use history.Large quantity research shows both at home and abroad, and fungus polysaccharide separates and obtains from fungus sporophore, mycelium, fermentation liquid, is the active component of fungal drug, is to control cell division differentiation, regulates a class active polysaccharide of Growth of Cells aging.
Ganoderma (Ganoderma Sinense Zhao, Xu et Zhang) be the one of Ganoderma, less to Ganoderma research at present, Ganoderma extract (take polysaccharide as main) has the effect of antitumor, radioprotective, leukocyte increasing, and research shows that Ganoderma polysaccharide is for the main position of its drug effect.
Chinese patent 200710045369.4 " a kind of Ganoderma fermentation process and the Ganoderma mycelium making " discloses a kind of method of Ganoderma liquid submerged fermentation aerial mycelium, this mycelium extracts the crude polysaccharides content obtaining 39～75% with decoction and alcohol sedimentation technique, in its body, in pharmacological evaluation, oral dose has obvious anti-tumor activity while being 40mg/kg/day.Chinese patent 200710045368.x " intra-polysaccharides from mycelia of ganoderma sinensis and its preparation method and application " discloses a kind of mycelium producing from Ganoderma liquid submerged fermentation and has extracted the method for preparing crude polysaccharides.Relate to decoction and alcohol sedimentation technique extraction and obtained crude polysaccharides content 39～75%.Its pharmacological evaluation proves that it has obvious antitumor action (to the suppression ratio of mice H22 hepatocarcinoma and Lewis lung cancer respectively 45～55%, and 40～50%), and dosage is also 40mg/kg/day (administering mode: oral medicine feed).Yang Guohong, Yang Yifang, Jin Juandi (the antitumor active site research [J] of Ganoderma liquid submerged fermentation. Chinese herbal medicine, 2008,39 (6): 877-880) the anti-tumor activity polysaccharide of Ganoderma liquid submerged fermentation is studied, from Ganoderma mycelium, extract and obtain intracellular polysaccharide, pharmacological evaluation proves that its anti-tumor activity is suitable with sunrecome.
Polysaccharide, because its molecular weight is large, is difficult for seeing through biomembrane, therefore affect its absorbance.Ganoderma mycelium polysaccharides is prepared into lyophilized injectable powder can directly see through biomembrane, enters blood circulation, improves bioavailability.
The formula of polysaccharide lyophilized injectable powder, how containing dextran, has found that there is clinically its anaphylactic shock and has caused dead case a lot of.
In addition, traditional Ganoderma mycelium polysaccharides extracting method generally uses decoction and alcohol sedimentation technique, and the crude polysaccharides content that this method obtains is low, polysaccharide component complexity, and contain the macromolecular components such as protein pigment simultaneously, also easily cause infusion reaction.
In view of the situation of prior art, need to find prepare that polyoses content is high, molecular weight ranges is little, containing the impurity such as protein, there is obvious anti-tumor activity and onset dosage low, the Ganoderma mycelium polysaccharides that toxicity is less is refined the method for thing, thereby the lyophilized injectable powder of the refinishing polyose thing that can not produce untoward reaction is provided.
Summary of the invention
The applicant obtains mycelium by Ganoderma (Ganoderma Sinense Zhao, Xu et Zhang) through liquid submerged fermentation and extracts separation and purification, obtains the refining thing of Ganoderma mycelium polysaccharides.The polyoses content of this refinishing polyose thing reaches more than 90%, not containing protein.This polysaccharide proves to have obvious antitumor action through pharmacological evaluation, and onset dosage is low, and toxicity is very micro-.
The technical problem to be solved in the present invention is to utilize the anti-polysaccharide mycelium polysaccharides of Ganoderma to refine thing to be raw material, to provide the Ganoderma mycelium polysaccharides that a kind of anti-tumor activity is better, onset dosage is low, toxicity is less and adverse reaction rate is lower refining thing freeze-dried powder.
Therefore, first object of the present invention is to provide the refining composition freeze-dried powder injection of a kind of Ganoderma mycelium polysaccharides, it is characterized in that, the freeze-dried excipient of the 75-95wt% of the refining thing of its Ganoderma mycelium by 5-25wt% forms.
As present invention further optimization, the refining composition freeze-dried powder injection of described Ganoderma mycelium polysaccharides is made up of the refining thing of Ganoderma mycelium of 8-20wt% and the freeze-dried excipient of 80-92wt%.
More electedly, the refining composition freeze-dried powder injection of Ganoderma mycelium polysaccharides of the present invention is made up of the refining thing of Ganoderma mycelium of 15wt% and the freeze-dried excipient of 85wt%.
Further, described freeze-dried excipient is selected from one or more in mannitol, Polyethylene Glycol, hydroxypropyl beta cyclodextrin, sodium dihydrogen phosphate or sodium citrate.Preferred excipient is mannitol and/or sodium citrate.
Further, the pH of described injectable powder is 7.44-7.47.
Second object of the present invention is to provide the preparation method of the refining composition freeze-dried powder injection of above-mentioned Ganoderma mycelium polysaccharides, and described method is included in raw mixture and adds injection active carbon also to use the step of filtering with microporous membrane.
The preparation method of the refining thing of raw material Ganoderma mycelium polysaccharides of lyophilized injectable powder of the present invention comprises the steps: that Ganoderma mycelium obtains crude polysaccharides through water extract-alcohol precipitation, and crude polysaccharides, through ultrafiltration, concentrated and precipitating, obtains the refining thing of Ganoderma mycelium polysaccharides.
Concrete grammar is as follows:
Crude polysaccharides extracts:
Adopt Shandong Ganoderma strain (Ganoderma sinense) to carry out liquid fermentation, obtain fermentation liquid 1300L, obtain mycelium and wash with water, filter press.Get 3 times of water extraction heating extraction of mycelium 2 times, each 3h, extracting temperature is 90 ℃.Water extraction liquid merges and is concentrated into the mycelia scale of construction and amount of liquid medicine ratio is 1: 2, after add 4 times of 95% precipitate with ethanol and spend the night, precipitation is taken out and is dried, and obtains crude polysaccharides 119.22g.
Crude polysaccharides is refining:
Get 20 times of water dissolutioies for above-mentioned crude polysaccharides, the ultrafilter membrane that is 30,000 with molecular weight separates crude polysaccharides liquid, gets trapped fluid NF membrane and concentrates.
Get dried polysaccharide position 1g, be dissolved in 100mL deionized water, centrifugalize, gets supernatant and adds 2% trimethyl cetyl ammonium bromide, mix, centrifugalize, gets supernatant and adds 1% boric acid 50mL, adjust pH to 11 with 1MNaOH solution, continue to adjust pH to 7, centrifugalize with acetic acid.Get supernatant acetic acid and adjust pH to 4.4, add 1.5 times of ethanol precipitate with ethanol and spend the night, precipitation is got in centrifugalize, with dialysing with bag filter flowing water after water dissolution, takes out after the interior liquid of bag filter, and concentrated, lyophilization, obtains the refining thing of Ganoderma mycelium polysaccharides.
The yield of this refinishing polyose thing is 64.5%, and polyoses content is 92.5%, and contents of monosaccharides is 1.7%, and proteins react is negative.
The method of being prepared the refining composition freeze-dried powder injection of Ganoderma mycelium polysaccharides of the present invention by the refining thing of above-mentioned Ganoderma mycelium polysaccharides comprises the steps:
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add recipe quantity excipient, add injection active carbon, filtering with microporous membrane, is divided in cillin bottle, lyophilization, and sealing, produces injection.
The refining composition freeze-dried powder injection of Ganoderma mycelium polysaccharides of the present invention is owing to having adopted the refining thing preparation of Ganoderma mycelium polysaccharides, the latter's polyoses content is high, molecular weight ranges is little, and therefore this freeze-dried powder preparation has better anti-tumor activity and onset dosage is low, toxicity is less; On the other hand, because refinishing polyose thing is containing the impurity such as protein, therefore use probability that this freeze-dried powder preparation produces untoward reaction Ganoderma mycelium polysaccharides lyophilized injectable powder compared to prior art greatly to reduce.
The specific embodiment
In order to understand better the present invention, illustrate in order to understand better the present invention by following examples, but the present invention is not limited:
The concrete steps that phenol sulfuric acid process is measured polysaccharide (and monosaccharide) content are as follows:
Mark product preparation: accurately weighed dried glucose 25mg, be dissolved to 100ml, be made into 250ug/ml mark product solution, get respectively 0.1,0.2,0.4,0.5,0.7,0.80.ml is in different test tubes, add water and supply 1ml, add respectively 5% phenol 0.5ml, fully shake up, add fast concentrated sulphuric acid 3.5ml, put 40 ℃ of water-baths, react 30 minutes, then cold water placement 5min, mark product liquid made.
Blank preparation: get 2ml deionized water, add 5% phenol 1ml, concentrated sulphuric acid 7ml, heating in water bath, cooling, make blank solution.
Specification Curve of Increasing: liquid to be measured standard and blank solution are surveyed to its absorption value in 490nm place with ultraviolet spectrophotometry, and do standard curve.
Sample preparation: get the accurately weighed 20mg of crude polysaccharides sample, add in the deionized water of 10ml and prepare 2mg/ml solution, get 0.2ml in test tube, add water and supply 1ml, add successively phenol, concentrated sulphuric acid by mark product liquid, heating in water bath, cooling, make sample liquid.
Detect: testing sample ultraviolet spectrophotometry, in its absorption value of 490nm place survey, is surveyed to its polyoses content with standard curve.
As follows by the method for the serial molecular weight of demarcating the refining thing of Ganoderma mycelium polysaccharides of the present invention of dextran standard Dextron: (also please supplement concrete operation step and computational methods)
Contents of monosaccharides is measured
3,5-edlefsen's reagent: precision takes 1.000g 3,5-dinitrosalicylic acid, in 100mL beaker, first adds 20mL2mol/L sodium hydroxide solution, then adds 30g sodium potassium tartrate tetrahydrate, adds water and fully dissolves and be settled to 500mL
Glucose solution preparation: precision takes dried glucose 200mg, be dissolved to 100ml and be mixed with 2mg/ml solution, get respectively 0,0.4,0.8,1.2,1.6,2.0ml in 25ml volumetric flask, add water respectively and supply 2ml, respectively add 3,5-dinitrosalicylic acid 2ml, boiling water boiling 5min, cooling immediately, be diluted to scale with deionized water, shake up, survey its ultraviolet absorption value at 550nm place, do standard curve.
Sample detection: sample thief 12mg, in 10ml volumetric flask, adds deionized water 2ml and makes it to dissolve, then add 3,5-dinitrosalicylic acid 2ml, boiling water boiling 5min is cooling immediately, is diluted with water to scale.Survey its uv absorption at 550 places, bring standard curve into, calculate contents of monosaccharides.
Adopt efficient gel method to measure purity and molecular weight
Chromatographic condition: TSK-GEL GMPWx1 chromatographic column; Mobile phase is water; Flow velocity: 0.3mL/min; 35 ℃ of column temperatures; Detector is: differential detector.
Mark product Dextron, polysaccharide sample are used respectively water dissolution, sample introduction.
Measure glucosan Dextron series and sample retention time, do standard curve with the molecular weight logarithm value of retention time-Dextron series of Dextron, bring the retention time of polysaccharide sample into standard curve, can know the molecular weight at each polysaccharide peak by inference.
The concrete steps of protein negative reaction are as follows:
Ninhydrin solution compound method: 0.2g 1,2,3-indantrione monohydrate is dissolved in 100mL95% ethanol, concentration is 0.2% ninhydrin reaction (protein discriminating): sample solution is dripped on white plaque (evaporating dish), add a 1,2,3-indantrione monohydrate liquid, whether qualitative detection has protein.
Embodiment 1:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add mannitol and sodium citrate, stir 20 minutes, add injection active carbon 0.8mg, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.45.
Embodiment 2:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add mannitol and sodium citrate, stir 20 minutes, add 0.5% injection active carbon, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.47.
Embodiment 3:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add mannitol and sodium citrate, stir 20 minutes, add injection active carbon 0.8mg, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.46.
Embodiment 4:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add mannitol and sodium citrate, stir 20 minutes, add injection active carbon 1.6mg, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.46.
Embodiment 5:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add Polyethylene Glycol, stir 20 minutes, add injection active carbon 0.96mg, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.44.
Embodiment 6:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add hydroxypropyl beta cyclodextrin and sodium citrate, stir 20 minutes, add injection active carbon 0.8mg, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.46.
Embodiment 7:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add Polyethylene Glycol and sodium dihydrogen phosphate, stir 20 minutes, add injection active carbon 0.96mg, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.46.
Embodiment 8:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add mannitol and sodium dihydrogen phosphate, stir 20 minutes, add injection active carbon 0.8mg, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.46.
Embodiment 9:
Prescription composition
Get the refining thing of Ganoderma mycelium polysaccharides, with appropriate water for injection dissolving, add Polyethylene Glycol and sodium citrate, stir 20 minutes, add injection active carbon 0.96mg, in 80 ℃ heating 30 minutes, cooling rear with aperture be 0.22 μ m filtering with microporous membrane, be divided in cillin bottle, every bottle of 2mL, to freeze drying box lyophilization, sealing, produces injection.Gained injectable powder pH is 7.47.Embodiment 10: the made Ganoderma mycelium polysaccharides of the embodiment of the present invention 1,3,4,5 and 8 is refined composition freeze-dried powder injection to rat liver cancer H22, the effectiveness study of Mice Bearing Lewis Lung Cancer
1. animal
Strain: kunming mice
Production licence number: SCXK (Shanghai) 2003-0003
Occupancy permit number: SYXK (Shanghai) 2004-0015
Body weight: 18-20g
Sex: male and female have concurrently
Each treated animal number: 10,15 of matched groups (♀,
half and half)
2. transplanted tumor
Rat liver cancer H22, Mice Bearing Lewis Lung Cancer.
3. test method
Get well-grown rat liver cancer H22 ascites or Lewis lung cancer tumor piece, with normal saline dilution (about 1-2 × 10 of cell concentration
7individual/ml), every the right axil subcutaneous vaccination of mice 0.2ml, random packet, after inoculation, rise with the water for injection wiring solution-forming administration of the refining composition freeze-dried powder injection of the made Ganoderma mycelium polysaccharides of the embodiment of the present invention 1,3,4,5 and 8 next day; Positive control medicine is sunrecome (purchased from Xi'an Green Valley Pharmaceutical Co., Ltd., lot number is 070105), with the administration of water for injection wiring solution-forming; Blank is water for injection, and administration volume is 0.5ml/20g body weight, the continuous lumbar injection of rat liver cancer H22 7 days, the continuous lumbar injection of Lewis lung cancer 10 days.Inoculate latter 10 days and dissect H22, within 14th, dissect Lewis lung cancer, get tumor piece, claim tumor weight, result is judged according to following formula:
4. result of the test
The results are shown in Table 1 and 2.
The made Ganoderma mycelium polysaccharides of table 1. embodiment of the present invention 1,3,4,5 and 8 is refined the tumor-inhibiting action of composition freeze-dried powder injection to rat liver cancer H22
The made Ganoderma mycelium polysaccharides of table 2. embodiment of the present invention 1,3,4,5 and 8 is refined the tumor-inhibiting action of composition freeze-dried powder injection to Mice Bearing Lewis Lung Cancer
From the data of table 1 and 2, to compare with Chinese medicine compound preparation sunrecome, the dosage of the refining composition freeze-dried powder injection of Ganoderma mycelium polysaccharides of the present invention is very little, but tumour inhibiting rate is but very high.
Claims (6)
1. the refining composition freeze-dried powder injection of Ganoderma mycelium polysaccharides, it is characterized in that, the freeze-dried excipient of the refining thing of its Ganoderma mycelium by 5-25wt% and 75-95wt% forms, and described Ganoderma mycelium polysaccharides is refined the polyoses content of thing for being greater than 90%, and containing protein; And the preparation method of the refining thing of described Ganoderma mycelium polysaccharides comprises the steps: that Ganoderma mycelium obtains crude polysaccharides through water extract-alcohol precipitation, crude polysaccharides, through ultrafiltration, concentrated and precipitating, obtains the refining thing of Ganoderma mattress mycelium polysaccharide.
2. the refining composition freeze-dried powder injection of Ganoderma mycelium polysaccharides according to claim 1, is characterized in that, the freeze-dried excipient of the refining thing of its Ganoderma mycelium by 8-20wt% and 80-92wt% forms.
3. the refining composition freeze-dried powder injection of Ganoderma mycelium polysaccharides according to claim 1 and 2, is characterized in that, described freeze-dried excipient is selected from one or more in mannitol, Polyethylene Glycol, hydroxypropyl beta cyclodextrin, sodium dihydrogen phosphate or sodium citrate.
4. the refining composition freeze-dried powder injection of Ganoderma mycelium polysaccharides according to claim 3, is characterized in that, described excipient is mannitol and/or sodium citrate.
5. according to the refining composition freeze-dried powder injection of the Ganoderma mycelium polysaccharides described in the arbitrary claim of claim 1-4, it is characterized in that, the pH of described injectable powder is 7.44-7.47.
6. the preparation method of the refining composition freeze-dried powder injection of the arbitrary described Ganoderma mycelium polysaccharides of claim 1-5, described method is included in raw mixture and adds injection active carbon also to use the step of filtering with microporous membrane.
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| CN1288729A (en) * | 2000-07-19 | 2001-03-28 | 南京振中生物工程有限公司 | Freeze dried lentinan holoside powder injecta and its preparation |
| CN1579414A (en) * | 2004-03-04 | 2005-02-16 | 江卫世 | Glossy-ganoderma-spore polysaccharide for injection and its preparation process |
| CN101172125A (en) * | 2006-11-03 | 2008-05-07 | 四川省中医药研究院 | Ganoderma lucidum liquid fermentate, composition containing the fermentate , preparation method and application of the same |
| CN101376904A (en) * | 2007-08-29 | 2009-03-04 | 上海医药工业研究院 | Intra-polysaccharides from mycelia of ganoderma sinensis, and preparation and use thereof |
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|---|---|---|---|---|
| CN1288729A (en) * | 2000-07-19 | 2001-03-28 | 南京振中生物工程有限公司 | Freeze dried lentinan holoside powder injecta and its preparation |
| CN1579414A (en) * | 2004-03-04 | 2005-02-16 | 江卫世 | Glossy-ganoderma-spore polysaccharide for injection and its preparation process |
| CN101172125A (en) * | 2006-11-03 | 2008-05-07 | 四川省中医药研究院 | Ganoderma lucidum liquid fermentate, composition containing the fermentate , preparation method and application of the same |
| CN101376904A (en) * | 2007-08-29 | 2009-03-04 | 上海医药工业研究院 | Intra-polysaccharides from mycelia of ganoderma sinensis, and preparation and use thereof |
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