CN101560268A - Cs-4 fermentation mycelium polysaccharide and preparation method and applications thereof - Google Patents
Cs-4 fermentation mycelium polysaccharide and preparation method and applications thereof Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention adopts paecilomyces hepiali Chen (Cs-4) fermentation mycelium as a raw material and uses methods including water extracting, alcohol precipitation, decolorization, ultrafiltration and the like to obtain intracellular polysaccharide of Cs-4 fermentation mycelium of cultured cordyceps sinensis with molecular weight below 500,000. Pharmacological and pharmacodynamic tests show that the polysaccharide provided by the invention has better resistance to glomerular sclerosis and renal interstitial fibrosis and can be used for preparing drugs for resistance to glomerular sclerosis and renal interstitial fibrosis.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, being specifically related to a kind of is the Cs-4 fermentation mycelium polysaccharide and preparation method thereof and the purposes of feedstock production with peacilomyce hepiahi (Cs-4) mycelium.
Background technology
Cordyceps sinensis is that section ergot fungus cordyceps sinensis bacterium Cordyceps sinensis (Berk.) Sacc. colonizes in stroma and the larva cadaveric complex on the Hepialidae insect larvae, is rare traditional Chinese medicine (hereinafter to be referred as Chinese caterpillar fungus).Because the natural cs growing environment is special, and price is higher, can not satisfy the demand in market.Therefore, people utilize modern biofermentation technique to cultivate Cordyceps mycelium to replace natural cs.The Cordyceps sinensis sample that Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences gathered in nineteen eighty-two from the Tibetan autonomous prefecture, Deqen in Yunnan Province separates tens bacterial strains of acquisition, and 4 bacterial strains have wherein been carried out cultivating and morphologic research, through evaluation called after peacilomyce hepiahi (Paecilonyces hepialid chen ﹠amp; Dai Cs-4 is hereinafter to be referred as Cs-4; The physiological Study of Cordyceps mycelium. Shao Aijuan, Dai Ruqin etc., Chinese Chinese materia medica science and technology, 1994 the 1st volume the 5th phases [J] .).Institute of Materia Medica,Chinese Academy of Medical Sciences nineteen eighty-two separation from the fresh Cordyceps sinensis that Qinghai Hua Longxian gathers obtains the Cs-4 bacterial strain, also is peacilomyce hepiahi through identifying.Afterwards, be that the medicine that raw material is made successfully goes on the market with the mycelium of peacilomyce hepiahi cultivation and fermentation, nomenclature of drug is a paecilomyces hepiall chen.
Cordyceps polysaccharide has physiologically active widely.A large amount of medical experiments confirm, Cordyceps polysaccharide can improve the human immunological competence, improve respiratory system, suprarenal gland weight, blood plasma cortisol, aldosterone and suprarenal gland inner cholesterol content are increased, have to promote adrenal effect, and have antitumor, radioprotective, hypoglycemic and cough-relieving, reduce phlegm, moistening lung and pharmacological action such as delay senility.Along with the development of modern biotechnology, the artificial fermentation cultivates becomes the new way of obtaining Cordyceps polysaccharide.
It is the extracting method that main raw material prepares Cordyceps polysaccharide that Chinese patent CN200810120873.0 discloses with the Cs-4 fermented waste fluid, the content of Cordyceps polysaccharide is lower in the fermented waste fluid, impurity is many, unsuitable separation and purification, its polysaccharide is mainly exocellular polysaccharide, be not from intramycelial intracellular polyse, yet there are no the relevant report that is applied to field of medicaments from the intracellular polyse in the Cs-4 ferment cordyceps sinensis mycelium.
Glomerular sclerosis and kidney region fibrosis are the common pathologic basis that various chronic renal diseases are developed to whole latter stage, and the process that therefore effectively delays glomerular sclerosis, kidney region fibrosis is the progress of delaying chronic kidney depletion effectively.TGF-β 1 is most important glomerular sclerosis, the kidney region fibrosis factor of causing, TGF-β 1 is synthetic by stimulating inoblast, messangial cell etc. to increase extracellular matrix (ECM) composition, the activity that suppresses multiple ECM degrading enzyme, suppress the degraded of ECM, stimulate the tubule epithelial cell to change and be divided into myofibroblast, thereby cause the fibrosis of nephridial tissue.Therefore, TGF-β 1 is the target of the anti-renal fibrosis of ideal.
Summary of the invention
The inventor is a raw material with the Cs-4 fermentation mycelium, adopt that water is carried, methods such as alcohol precipitation, decolouring and ultrafiltration obtain molecular weight at the Cs-4 ferment cordyceps sinensis mycelium intracellular polyse (hereinafter to be referred as the Cs-4 polysaccharide) below 500,000, by the pharmacological effect test, find that polysaccharide of the present invention has the effect of good anti-glomerular sclerosis, anti-kidney region fibrosis.
Polysaccharide of the present invention also has the effect of good enhance immunity power.
Polysaccharide of the present invention is based on the intracellular polyse of Cs-4 fermentation mycelium, and molecular weight is between 10,000 to 500,000, and or rather, polysaccharide molecular weight of the present invention can have better pharmacological effect between 10,000 to 200,000.
Polysaccharide of the present invention can be used for preparation treatment and prevention glomerular sclerosis and kidney region fibrosis medicine or healthcare products.
But the intracellular polyse extract application medicaments of Cs-4 fermentation mycelium of the present invention goes up acceptable auxiliary, makes pharmaceutical dosage forms such as tablet, capsule, lozenge, soft capsule, freeze-drying lozenge, liquid capsule, injection liquid.
The present invention can be achieved through the following technical solutions:
The preparation of Cs-4 ferment cordyceps sinensis mycelium polysaccharides (hereinafter to be referred as the Cs-4 polysaccharide)
Get Cs-4 ferment cordyceps sinensis mycelium, pulverize, the water heating is extracted 3 times, each water that adds 6-12 times of weight, heating was extracted 1-3 hour, filter, the filtrate standing over night, high speed centrifugation (20000 rev/mins), it with molecular weight cut-off 10000 ultra-filtration membrane ultrafiltration and concentration, being concentrated into trapped fluid volume and Cordyceps mycelium weight ratio is 1: 2 (g/ml), add ethanol and make and contain alcohol amount and reach 20%, standing over night filters, get supernatant liquor, continue to add ethanol and make and contain alcohol amount and reach 60%-70%, leave standstill, filter, the precipitation water dissolution, use the ethanol alcohol precipitation more once, make to contain alcohol amount and reach 70%-80%, leave standstill, get the precipitation adding distil water and make dissolving, add 20% trichoroacetic acid(TCA) solution and make its concentration reach 1%, refrigeration is left standstill, high speed centrifugation (20000r/min), it is 2: 1 with crude drug amount ratio that centrifugate adds water to soup, with molecular weight cut-off is 500000 hollow fiber column ultrafilter, and ultrafiltration and concentration liquid is with 3 times of amount distilled water ultrafiltration washings, and the ratio that the filtrate that ultrafiltration goes out is concentrated to medicine liquid volume and Cordyceps mycelium body weight is 1: 2, adding ethanol makes and contains alcohol amount and reach 80%, leave standstill, suction filtration, precipitation is washed residual to the greatest extent trichoroacetic acid(TCA) with 80% ethanol, again for several times with washing with alcohol, 70 ℃, vacuum-drying under the condition of 6.6KPa, perhaps lyophilize, pulverize, get Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract (Cs-4 polysaccharide).The present invention is further illustrated below by experimental example.
One, Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract content Determination of Polysaccharide of the present invention
1, content Determination of Polysaccharide
Instrument and reagent
Ultraviolet-visible spectrophotometer (day island proper Tianjin UV-2450 type), digital display thermostat water bath HH-4 (state China Electrical Appliances Co., Ltd), glucose reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number 110833-200602), phenol, the vitriol oil (Beijing chemical reagents corporation is analytical pure)
The preparation of typical curve
It is an amount of that precision takes by weighing the glucose standard substance, is mixed with the standard solution of 0.1mg/ml with distilled water, accurate respectively glucose reference liquid 0.1,0.3,0.5,0.7,0.9, the 1.1ml of drawing, place tool plug test tube, it is 2.0ml that adding distil water makes volume, adds 6% phenol liquid 1.0ml again, shakes up, drip vitriol oil 5.0ml rapidly, shake up, placed 5 minutes, put on the boiling water bath and heated 15 minutes, take out, cold water is cooled to room temperature; With distilled water 2.0ml, as blank, utilize the spectrophotometric determination absorption value in addition in the 490nm place with above-mentioned operation.(μ g) is X-coordinate with the standard substance glucose amount, and absorption value is an ordinate zou, calculates regression equation: Y=0.0077X+0.0057, and R=0.9997, description standard product glucose amount is good linear relationship with absorption value in 0～110 μ g scope.
Samples contg is measured
It is an amount of that precision takes by weighing Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract (embodiment 3 gained polyoses extracts), be mixed with the need testing solution of 0.2mg/ml with distilled water, draw need testing solution 0.1ml, adding distil water is to 2.0ml, press the method preparation of typical curve preparation below respectively, measure absorption value, the content of trying to achieve Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract polysaccharide by equation of linear regression is 67.8%.
Two, the effect experiment example of Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract
1, material
1.1 laboratory animal
60 of healthy male Wistar rats, body weight 180-220g is provided by Military Medical Science Institute's animal center, and raises at this center Animal Lab..
1.2 medicine
Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract (provide by Jiangxi JINSHUIBAO medicine company, specifically make) by embodiment 3 technologies; NIAODUQING KELI (Kangchen Pharmaceutical Co., Ltd., Guangzhou); VITAMIN B4 (Chemical Reagent Co., Ltd., Sinopharm Group).
1.3 main agents
The mouse TGF-β of the rabbit Chinese People's Anti-Japanese Military and Political College 1 polyclonal antibody, two step method immunologic combined detection reagent kit (Beijing Zhong Shan Bioisystech Co., Ltd); The RT-PCR primer of TGF-β 1R I, TGF-β 1R II (Shanghai Bo Ya Bioisystech Co., Ltd), Trizol test kit (American I nvitrogen company) provides, agarose (U.S. Sigma company).
2 methods
2.1 modeling is got 12 rats at random and given distilled water filling stomach as normal group, all the other rats press 250mgkg for 2.5% VITAMIN B4 suspension
-1D
-1Irritate stomach, successive administration 21d.Get eyeground vein blood 0.5ml, survey serum creatinine (Scr), blood urea nitrogen (BUN), conclusive evidence modeling success.
2.2 grouping
The modeling rat is divided into 4 groups, 10 every group: pathological model group, NIAODUQING group, Cs-4 ferment cordyceps sinensis mycelium polysaccharides (hereinafter to be referred as Cordyceps polysaccharide) high dose group (160mg/kg), Cs-4 ferment cordyceps sinensis mycelium polysaccharides low dose group (80mg/kg).
2.3 after the administration modeling success, Cs-4 ferment cordyceps sinensis mycelium low dose group is pressed 80mgkg
-1D
-1Irritate stomach; High dose group is pressed 160mgkg
-1D
-1The NIAODUQING group is pressed 1.81gkg
-1D
-1Irritate stomach, normal group and pathological model group adopt 2mlkg
-1D
-1Distilled water is irritated stomach, treats 60d altogether.Rat ad lib, drinking-water in the treatment.
2.4 draw materials with vetanarcol anesthesia, put to death, cut open inspection after weighing with electronic scales, get fresh and alive nephridial tissue, observe the kidney form, claim weight in wet base.Nephridial tissue rip cutting, a part are put into 4% paraformaldehyde solution and are fixed, and another part is preserved in liquid nitrogen.
3 detect
3.1 the section of kidney pathological observation routine paraffin wax, H E, Masson dyeing.Light microscopic is observed matter between renal glomerulus, uriniferous tubules, kidney down, the kidney blood vessel structure changes.
3.2 immunohistochemical methods
One is anti-: rabbit anti-TGF-beta 1 polyclonal antibody; Two is anti-: the anti-rabbit I of biotinylated goat g G.Concrete steps: the paraffin section de-waxing dehydration fever is repaired antigen 1 0min, drips normal goats serum confining liquid, room temperature 10min, get rid of deblocking liquid, drip one anti-(1: 50) in 4 ℃ of refrigerator overnight, drip two anti-(1: 75), 20 ℃ of 20min, the DAB 10-30min that develops the color, Hematorylin is slightly redyed, and conventional dehydration is transparent, mounting, microscopically is observed.There is even matter fine particulate material positive in the cell, is distributed in cytolemma and the endochylema, based on cytolemma.5 visuals field are got in each section at random, measure positive stained area and positive cell number, represent the content of this material in this tissue with positive cell percentage.
3.3RT-PCR detect
The extraction of total tissue RNA is carried out according to the Trizol specification sheets.Configuration reverse transcription system: 5xcDNA Synthesis buffer4 μ l, 0.1M DTT 1 μ l, RNase OUT (40U/ μ l), DEPC-treated water 1 μ l, ThermoSctipt
TMRT (15U/ μ l), 1 μ l.Fully behind the mixing, draw in the reaction tubes after 8 μ l adding is hatched, put into the PCR instrument then, 50 ℃, 60min carries out the synthetic cDNA of reverse transcription, carries out pcr amplification as template.
Pcr amplification condition: 94 ℃, 5min; 94 ℃, 30s, (ACTIN:55 ℃, 1RII:54 ℃ of 1min of I:56 ℃ of TGF-β 1R, TGF-β, 72 ℃, 1min, 30 circulations; 72 ℃, 10min.Detect through 2% agarose gel electrophoresis, with the gel imaging system analysis and take pictures.Analyze each electrophoretic band gray integration with ImageTool 5.0, as standard correction, carry out semi-quantitative analysis with the gray integration of Actin.
4, image analysis
The interstitial fibrosis semi-quantitative analysis adopts colored multi-media semi-quantitative analysis system, get the Masson stained, every group is selected 10 visuals field at random, regulates by gray scale and distinguishes positive signal area in the visual field, calculate a positive general objective and a statistics total area ratio, get average and carry out statistical study.The immunohistochemical methods semi-quantitative analysis is by high-definition color pathology picture and text report analytical system, and every section 10 high power fields of picked at random (* 400) are measured nephridial tissue TGF-β 1, calculate its mean light absorbency value, and big more its expression amount of expression of absorbancy is many more.
5 statistical method experimental datas adopt the analysis of SPSS 10.0 statistical softwares, relatively use variance analysis between many groups, and the result is with (X ± S) expression.
6, test-results
6.1, respectively organize the variation of rat BUN, Scr
The perfusion VITAMIN B4 compared with normal group after 21 days, and each is organized BUN, Scr and significantly raises (P＜0.01), and the result shows that the modeling success has comparability between group.
Respectively organize relatively (X ± S) of BUN, Scr after table 1 modeling
Annotate: compare with normal group,
*P＜0.01
The test-results explanation, model modeling success.
6.2, gross morphology changes
The HE coloration result: normal group uriniferous tubules skin, medullary substance are demarcated clear, the no abnormal change of matter between kidney, and no proliferation of fibrous tissue, the tubule tube chamber does not have the crystallisate deposition; Pathological model group uriniferous tubules skin, medullary substance boundary are failed to understand, the uriniferous tubules atrophy, and epithelial cell sex change, necrosis, interstitial fibers is netted or the sheet hyperplasia, visible crystallisate deposition in the part tubule; The slight atrophy of Cordyceps polysaccharide high dose group renal cells, sex change, there is the minute quantity inflammatory cell in a matter, and proliferation of fibrous tissue is not obvious; Matter changes between Cordyceps polysaccharide high dose group and pathological model group between NIAODUQING group and Cordyceps polysaccharide low dose group uriniferous tubules.
The HE coloration result shows that Cs-4 ferment cordyceps sinensis mycelium polysaccharides high dose group has better action to renal fibrosis.
6.3, the interstitial fibrosis sxemiquantitative
The matter part is narrow between around the normal group nephridial tissue collagen staining tubule.The a large amount of inflammatory cell infiltrations of matter between pathological model group kidney, interstitial fibers hamartoplasia, area broadening, the atrophy of tubule part, obliteration or expansion.Treatment back ID becomes and alleviates, and fibrosis obviously improves (P＜0.01), and wherein the high dose group result of treatment is best, has compared significant difference (P＜0.01) with the NIAODUQING group.
Table 2 is respectively organized relatively (X ± S) of interstitial fibrosis half-quantitative detection result
Annotate: compare * * P＜0.01 with the pathological model group; Compare with the NIAODUQING group,
△ △P＜0.01
Test-results shows that Cs-4 ferment cordyceps sinensis mycelium polysaccharides high dose group has significant effect to the renal fibrosis due to the VITAMIN B4 group.
6.4 immunohistochemistry
The expression of TGF-β 1 in nephridial tissue mainly concentrates on renal cells, based on endochylema dyeing, and also visible a small amount of expression of renal glomerulus.Other are respectively organized expression ratio TGF-β 1 normal group and obviously strengthen (P＜0.01), and the treatment group obviously suppresses the expression of TGF-β 1, with model group significant difference (P＜0.01) are arranged relatively.Wherein the high dose group result of treatment is obvious, with the NIAODUQING group significant difference (P＜0.01) is arranged relatively, with low dose group significant difference (P＜0.05) is arranged relatively.
Respectively organize rat kidney TGF-β 1 after table 3 treatment and express mean level (ML) relatively
Annotate: compare with the pathological model group,
*P＜0.001; Compare with the NIAODUQING group,
△ △P＜0.01;
Test-results shows that Cs-4 ferment cordyceps sinensis mycelium polysaccharides has the effect of remarkable inhibition TGF-β 1.
Specific embodiment
Embodiment 1
Cs-4 ferment cordyceps sinensis mycelium polysaccharides tablet
The preparation of Cs-4 ferment cordyceps sinensis mycelium polysaccharides
Get Cs-4 ferment cordyceps sinensis mycelium 10kg, pulverize, the water heating is extracted 3 times, each water that adds 8 times of weight, heating was extracted 2 hours, filter, the filtrate standing over night, high speed centrifugation (20000 rev/mins), it with molecular weight cut-off 10000 ultra-filtration membrane ultrafiltration and concentration, concentrating under reduced pressure trapped fluid to trapped fluid volume and Cordyceps mycelium weight ratio is 1: 2 (g/ml) then, add ethanol and make and contain alcohol amount and reach 20%, standing over night filters, get supernatant liquor, continue to add ethanol and make and contain alcohol amount and reach 70%, leave standstill, filter, precipitation water low-grade fever (50 ℃) makes dissolving, put coldly, add ethanol again and make and contain alcohol amount and reach 80%, leave standstill, suction filtration, get the precipitation adding distil water and make dissolving, add 20% trichoroacetic acid(TCA) solution and make its concentration reach 1%, refrigeration is left standstill, high speed centrifugation (20000r/min), it is 2: 1 with crude drug amount ratio that centrifugate adds water to soup, is 500000 hollow fiber column ultrafilter with molecular weight cut-off, and ultrafiltration and concentration liquid is with 3 times of amount distilled water ultrafiltration washings, the ratio that the filtrate that ultrafiltration goes out is concentrated to medicine liquid volume and Cordyceps mycelium body weight is 1: 2, add ethanol and make and contain alcohol amount and reach 80%, leave standstill suction filtration, precipitation is washed residual to the greatest extent trichoroacetic acid(TCA) with 80% ethanol, again for several times with washing with alcohol, 70 ℃, vacuum-drying under the condition of 6.6KPa, pulverize, get Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract
By acceptable auxiliary on the pharmaceutics, compacting in flakes, the bag film-coat, get final product Cs-4 ferment cordyceps sinensis mycelium polysaccharides tablet of the present invention.
Embodiment 2
Cs-4 ferment cordyceps sinensis mycelium polysaccharides capsule
The preparation of Cs-4 ferment cordyceps sinensis mycelium polysaccharides
Get Cs-4 ferment cordyceps sinensis mycelium 5kg, pulverize, the water heating is extracted 3 times, each water that adds 10 times of weight, heating was extracted 1 hour, filter, the filtrate standing over night, high speed centrifugation (20000 rev/mins), with molecular weight cut-off is 10000 ultra-filtration membrane ultrafiltration and concentration, and concentrating under reduced pressure trapped fluid to trapped fluid volume and Cordyceps mycelium weight ratio is 1: 1 (g/ml) then, adds 1.5% gac, stir, it is that 1: 2 (g/ml) adds ethanol and make and contain the alcohol amount and reach 20%, standing over night that suction filtration, filtrate are concentrated into medicine liquid volume and Cordyceps mycelium weight ratio, filter, get supernatant liquor, continue to add ethanol and make and contain alcohol amount and reach 60%, leave standstill, filter, precipitation water low-grade fever (50 ℃) makes dissolving, puts coldly, adds ethanol again and makes and contain alcohol amount and reach 70%, leave standstill, suction filtration is got the precipitation adding distil water and is made dissolving, adds 20% trichoroacetic acid(TCA) solution and makes its concentration reach 1%, refrigeration is left standstill, it is 5: 1 with crude drug amount ratio that high speed centrifugation (20000r/min), centrifugate add water to soup, is 500000 hollow fiber column ultrafilter with molecular weight cut-off, the ratio that the filtrate that ultrafiltration goes out is concentrated to medicine liquid volume and Cordyceps mycelium body weight is 1: 2, add ethanol and make and contain alcohol amount and reach 80%, leave standstill suction filtration, precipitation is washed residual to the greatest extent trichoroacetic acid(TCA) with 80% ethanol, again for several times with washing with alcohol, 70 ℃, vacuum-drying under the condition of 6.6KPa, pulverize, get Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract.
By acceptable auxiliary on the pharmaceutics, compacting in flakes, the bag film-coat, get final product Cs-4 ferment cordyceps sinensis mycelium polysaccharides tablet of the present invention.
Embodiment 3
Cs-4 ferment cordyceps sinensis mycelium polysaccharides lozenge
The preparation of Cs-4 ferment cordyceps sinensis mycelium polysaccharides
Get Cs-4 ferment cordyceps sinensis mycelium 10kg, pulverize, the water heating is extracted 3 times, each water that adds 8 times of weight, heating was extracted 1.5 hours, filter, filtrate high speed centrifugation (20000 rev/mins), the centrifugate molecular weight cut-off is 10000 ultra-filtration membrane ultrafiltration and concentration, concentrating under reduced pressure trapped fluid to trapped fluid volume and Cordyceps mycelium weight ratio is 1: 1 (g/ml) then, adds 1.0% gac, heating, stir, suction filtration, filtrate are concentrated into medicine liquid volume and the Cordyceps mycelium weight ratio is 1: 2 (g/ml), adds ethanol and makes and contain alcohol amount and reach 20%, standing over night, filter, get supernatant liquor, continue to add ethanol and make and contain the alcohol amount and reach 60%, leave standstill, filter, precipitation water low-grade fever (50 ℃) makes dissolving, puts cold, adding ethanol again makes and contains alcohol amount and reach 80%, leave standstill, suction filtration is got the precipitation adding distil water and is made dissolving, adding 20% trichoroacetic acid(TCA) solution makes its concentration reach 1%, refrigeration is left standstill, high speed centrifugation (20000r/min), and it is 3: 1 with crude drug amount ratio that centrifugate adds water to soup, it with molecular weight cut-off 200000 hollow fiber column ultrafilter, the ratio that the filtrate that ultrafiltration goes out is concentrated to medicine liquid volume and Cordyceps mycelium body weight is 1: 2, adds ethanol and makes and contain the alcohol amount and reach 80%, leaves standstill, suction filtration, precipitation is washed residual to the greatest extent trichoroacetic acid(TCA) with 80% ethanol, more for several times with washing with alcohol, and lyophilize, pulverize, get Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract.
Get Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract, add sucrose, N.F,USP MANNITOL, low-substituted hydroxypropyl cellulose, a small amount of Magnesium Stearate, mixing, compressing tablet, promptly.
Embodiment 4
Cs-4 ferment cordyceps sinensis mycelium polysaccharides injection liquid
The preparation of Cs-4 ferment cordyceps sinensis mycelium polysaccharides
Get Cs-4 ferment cordyceps sinensis mycelium 10kg, pulverize, the water heating is extracted 3 times, each water that adds 8 times of weight, heating was extracted 1.5 hours, filter, filtrate high speed centrifugation (20000 rev/mins), the centrifugate molecular weight cut-off is 10000 ultra-filtration membrane ultrafiltration and concentration, concentrating under reduced pressure trapped fluid to trapped fluid volume and Cordyceps mycelium weight ratio is 1: 1 (g/ml) then, adds 1.0% gac, heating, stir, suction filtration, filtrate are concentrated into medicine liquid volume and the Cordyceps mycelium weight ratio is 1: 2 (g/ml), adds ethanol and makes and contain alcohol amount and reach 20%, standing over night, filter, get supernatant liquor, continue to add ethanol and make and contain the alcohol amount and reach 60%, leave standstill, filter, precipitation water low-grade fever (50 ℃) makes dissolving, puts cold, adding ethanol again makes and contains alcohol amount and reach 80%, leave standstill, suction filtration is got the precipitation adding distil water and is made dissolving, adding 20% trichoroacetic acid(TCA) solution makes its concentration reach 1%, refrigeration is left standstill, high speed centrifugation (20000r/min), and it is 3: 1 with crude drug amount ratio that centrifugate adds water to soup, it with molecular weight cut-off 200000 hollow fiber column ultrafilter, the ratio that the filtrate that ultrafiltration goes out is concentrated to medicine liquid volume and Cordyceps mycelium body weight is 1: 2, adds ethanol and makes and contain the alcohol amount and reach 80%, leaves standstill, suction filtration, precipitation is washed residual to the greatest extent trichoroacetic acid(TCA) with 80% ethanol, more for several times with washing with alcohol, and lyophilize, pulverize, get Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract.
Get Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract, add the injection water and make dissolving, make every 1ml and contain polysaccharide 10mg, add 0.2% medicine active carbon, keep little and boiled 15 minutes, filter while hot, filtrate filters with the millipore filtration of 0.22 μ m, the filtrate embedding, sterilization, promptly.
Claims (4)
1, a kind of Cordyceps mycelium polyoses extract is characterized in that it is that feedstock production forms with peacilomyce hepiahi (Cs-4) fermentation mycelium, and polysaccharide molecular weight is 1 * 10
4～5 * 10
5Between, by weight percentage, the content of its polysaccharide is not less than 50%.
2, Cordyceps mycelium polyoses extract according to claim 1, it is characterized in that: polysaccharide molecular weight is 1 * 10
4～2 * 10
5Between, by weight percentage, its polysaccharide content is not less than 50%.
3, the preparation method of the described polyoses extract of the arbitrary claim of claim 1 or claim 2 is characterized in that this polyoses extract can be prepared by following method:
Get Cs-4 ferment cordyceps sinensis mycelium, pulverize, the water heating is extracted 3 times, each water that adds 6-12 times of weight, heating was extracted 1-3 hour, filter, the filtrate standing over night, high speed centrifugation (20000 rev/mins), it with molecular weight cut-off 10000 ultra-filtration membrane ultrafiltration and concentration, being concentrated into trapped fluid volume and Cordyceps mycelium weight ratio is 1: 1 (g/ml), use activated carbon decolorizing, suction filtration, filtrate is concentrated into medicine liquid volume and the Cordyceps mycelium weight ratio is 1: 2 (g/ml), adds ethanol and make and contain alcohol amount and reach 20%, standing over night, filter, get supernatant liquor, continue to add ethanol and make and contain alcohol amount and reach 60%-70%, leave standstill, filter, precipitation use water dissolution, uses the ethanol alcohol precipitation more once, makes to contain alcohol and measure and to reach 70%-80%, leave standstill, get the precipitation adding distil water and make dissolving, add 20% trichoroacetic acid(TCA) solution and make its concentration reach 1%, refrigeration is left standstill, high speed centrifugation (20000r/min), it is 2: 1 with crude drug amount ratio that centrifugate adds water to soup, is 500000 hollow fiber column ultrafilter with molecular weight cut-off, and ultrafiltration and concentration liquid is with 3 times of amount distilled water ultrafiltration washings, the ratio that the filtrate that ultrafiltration goes out is concentrated to medicine liquid volume and Cordyceps mycelium body weight is 1: 2, add ethanol and make and contain alcohol amount and reach 80%, leave standstill suction filtration, precipitation is washed residual to the greatest extent trichoroacetic acid(TCA) with 80% ethanol, again for several times, 70 ℃ with washing with alcohol, 6.6KPa condition under vacuum-drying, perhaps lyophilize, pulverize, get Cs-4 ferment cordyceps sinensis mycelium polysaccharides extract (Cs-4 polysaccharide).
4, the application of peacilomyce hepiahi (Cs-4) fermentation mycelium polyoses extract in preparation treatment and prevention glomerular sclerosis, kidney region fibrosis medicine or healthcare products.
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