CN100384982C - Antrodia camphorata mycelium fermented extract and application thereof - Google Patents
Antrodia camphorata mycelium fermented extract and application thereof Download PDFInfo
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- CN100384982C CN100384982C CNB2005100448080A CN200510044808A CN100384982C CN 100384982 C CN100384982 C CN 100384982C CN B2005100448080 A CNB2005100448080 A CN B2005100448080A CN 200510044808 A CN200510044808 A CN 200510044808A CN 100384982 C CN100384982 C CN 100384982C
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Abstract
The present invention discloses an Antrodia camphorate mycelium fermentation extract which is prepared by alcohol extract and dryness. Bacterial strains are Antrodia camphorata Ac001 which is preserved in 'Chinese microorganism culture collection collegium common microorganism center ' on 21st September, 2005, and the preservation number is CGMCC No. 1460. The Antrodia camphorata mycelium fermentation extract has the wide application to the preparation of medicines for inhibiting HBV DNA and the secretion of antigen HBsAg and e-antigen HBeAg and the preparation of cancer resisting medicines, particularly liver cancer resisting medicines.
Description
Technical field
The present invention relates to a kind of Antrodia camphorata mycelium fermented extract and application thereof.
Background technology
The camphor tree sesame (Antrodia camphorata) claim the Cinnamomum kanahirai hay mushroom again.Generally be grown on the hollow heartwood inwall of leaflet sassafras mitriform; Thalamium is orange, bitter.Camphor tree sesame new variety mycelium is orange, the digested tankage look after cultivating.Microscopic examination, mycelia is elongated, have every, have clamp connexion, and a large amount of conidiums is arranged, conidium is orange, digested tankage look or yellow because of the culture condition difference.
Recently some progress have been obtained, Chinese patent 01123613.2 " solid culture method of camphor tree sesame, gained solid culture and products thereof and purposes " about the crude extract of Antrodia Camphorata mycelium culture or the pharmacology of its polysaccharide or bioactivity research; 200410008046.4 " from the mycelial new blend of Antrodia camphorata and compound and uses thereof " and 200410011142.4 " basidiomycetous polysaccharide body of Antrodia camphorata and uses thereof " all related to effect and the purposes of camphor tree sesame, but, in described patent, but do not see to have to carry via satellite and improve the breed, to improve the report of its pharmacological effect.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides a kind of Antrodia camphorata mycelium fermented extract and application thereof.
The Antrodia camphorata mycelium fermented extract that the present invention relates to is made by following method:
(1) gets Antrodia camphorata mycelium fermented full liquid,, make its volume be concentrated to 1/2～1/5 of original volume its concentrating under reduced pressure in a usual manner;
(2) be that 60～95% ethanol extracts above-mentioned spissated fermented liquid with volume percent, wherein, add the alcoholic acid amount and be 2～5 times of concentrated solution volume, can make that alcohol concn reaches 60～90% in the extracting solution;
Under (3) 50～70 ℃ of conditions, heated 1～2 hour;
(4) separate with ordinary method, and remove impurity, separate obtaining ethanol extract by cascade filtration;
(5) with above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5～1/10 of original volume;
(6) be ethanol 95%, 4～8 times of ethanol concentrated solution volume amounts with volume percent, above-mentioned concentrated ethanol extract is carried out 12～24 hours precipitation process;
(7) isolate throw out with ordinary method;
(8) with throw out vacuum-drying or frozen drying in a usual manner, Antrodia camphorata mycelium fermented extract;
Wherein, described Antrodia Camphorata mycelium is CGMCCNo.1460, this bacterial strain is called camphor tree sesame (Antrodia camphorata) Ac001, has been deposited on September 21st, 2005 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number are CGMCC No.1460.
Above-mentioned camphor tree sesame (Antrodia camphorata) Ac001 CGMCC No.1460 is the camphor tree sesame bacterial classification that is separated to by camphor tree sesame sporophore, send national space flight and aviation portion Dongfanghong aerospace center, it has been carried out the satellite lift-launch, the condition of carrying is: the satellite quality is 2100 kilograms, satellite temperature is 5～28 ℃, microgravity magnitude 10
-3～10
-5, 200～210 kilometers of perigee altitudes, 300～400 kilometers of altitude of the apogees, the orbital period is 90 minutes, space travel 18 days; After satellite carries mutagenesis, after activation culture repeatedly, obtain.
The fermentation culture method of above-mentioned camphor tree sesame (Antrodia camphorata) Ac001 CGMCC No.1460 is, be inoculated into camphor tree sesame bacterial classification in the Erlenmeyer flask that liquid nutrient medium is housed with ordinary method, with 20～35 ℃ of temperature, shaking bottle rotating speed is 80～280r/min, under pH 3～8 conditions, vibrations were cultivated 7～15 days; In the cultivation when the pH value drops to 2.5～4, the seed that shakes in the bottle is inoculated in the nutrient solution of 50L fermentor tank, with 20～35 ℃ of temperature, fermentor tank pressure 0.1～0.2 kg/cm, pH 3～8, air flow 0.5～1.1vvm, the condition that stirring velocity is 100～280 rev/mins, cultivated 7～15 days, and can utilize Antrodia camphorata mycelium fermented full liquid preparation to get thing.
Wherein, in the fermentation culture method of described camphor tree sesame (Antrodia camphorata) Ac001 CGMCC No.1460, culture temperature is the suitableeest to be 28～30 ℃, and cultivating pH the suitableeest is 6.
Wherein, above-mentioned Antrodia camphorata mycelium fermented full liquid is meant the filtrate of mycelium and fermented liquid.
Wherein, above-mentioned seed or fermentative medium formula in gram are for/100 milliliters:
W-Gum 1% glucose 1%
Peptone 0.2% yeast extract paste 0.2%
Sal epsom 0.1% potassium primary phosphate 0.05%
Antrodia camphorata mycelium fermented extract of the present invention is preparing the application in hepatitis B surface antigen(HBsAg) HBsAg, e antigen HBeAg and the inhibiting medicine of HBV DNA excretory.
Utilize Antrodia camphorata mycelium fermented extract of the present invention to study the effect of its anti-hepatitis B virus; The Bel7402 2.2.15 of hepatitis B virogene transfection is adopted in experiment, studies the toxicity of its pair cell and hepatitis B surface antigen(HBsAg) HBsAg, e antigen HBeAg and HBV DNA excretory are suppressed effect.
Experimental result shows: 3 times of Antrodia camphorata mycelium fermented extract dilutions added cell cultures 8 days, pair cell median toxic concentration TC
50Be 0.153mg/ml.Maximal non-toxic concentration TCO is 0.039mg/ml.Camphor tree sesame fermentation broth extract maximal non-toxic concentration 0.039mg/ml is 59.0% to the inhibiting rate of HBVDNA, its half-inhibition concentration IC
50Being 0.0086mg/ml, is 36.1% to the inhibiting rate of hepatitis B surface antigen(HBsAg) HBsAg, its half-inhibition concentration IC
50Be 0.026mg/ml, and be 49.8%, its half-inhibition concentration IC the inhibiting rate of e antigen HBeAg
50Be 0.022mg/ml.
The application of Antrodia camphorata mycelium fermented extract of the present invention in the preparation cancer therapy drug.
Antrodia camphorata mycelium fermented extract of the present invention is the application in the preparation medicines resistant to liver cancer especially.
Utilize Antrodia camphorata mycelium fermented extract of the present invention to carry out antitumor animal experiment, the result shows: the Antrodia camphorata mycelium fermented extract of each metering group all has certain restraining effect to the growth of S180 murine sarcoma and H22 rat liver cancer cell, and antitumor activity is generally between 30～45%.Wherein, the inhibiting rate of S180 murine sarcoma is reached as high as 43.37% (1000mg/kg body weight), can reach 48.88% (160mg/kg body weight) the highest inhibiting rate of H22 rat liver cancer cell.Analyze by experiment, such antitumor activity that is subjected to the reagent thing may be by immune conciliation is produced tumor-inhibiting action.
Description of drawings
Camphor tree sesame of the present invention (Antrodia camphorata) Ac001, be deposited on September 21st, 2005 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; address: in the Microbe Inst., Chinese Academy of Sciences of Zhongguancun, Beijing City; postcode is 100080, deposit number is CGMCC No.1460.
Fig. 1 camphor tree sesame Ac001 bacterial strain satellite carries back electron microscopic observation figure
Fig. 2 camphor tree sesame Ac001 starting strain electron microscopic observation figure
Fig. 3 camphor tree sesame different strain mycelium dry weight relatively
Fig. 4 camphor tree sesame different varieties liquid fermenting polysaccharide content relatively
Fig. 5 camphor tree sesame different strain esterase (EST) isozymogram
Fig. 6 Antrodia camphorata mycelium fermented extract polysaccharide high performance liquid chromatography
Fig. 7 Antrodia camphorata mycelium fermented extract sugar compositional analysis
Embodiment
Embodiment 1:
The camphor tree sesame bacterial classification that camphor tree sesame (Antrodia camphorata) is separated to by camphor tree sesame sporophore, send national space flight and aviation portion Dongfanghong aerospace center, it is carried out satellite carry, the condition of lift-launch is: the satellite quality is 2100 kilograms, satellite temperature is 5～28 ℃, microgravity magnitude 10
-3～10
-5, 200～210 kilometers of perigee altitudes, 300～400 kilometers of altitude of the apogees, the orbital period is 90 minutes, space travel 18 days is carried out satellite and is carried mutagenesis.
Camphor tree sesame bacterial classification repeatedly after the activation culture, is observed form, relatively the difference of bacterial strain before and after the mutagenesis to it with ordinary method after satellite carries mutagenesis.
1. mycelium morphology is observed
Antrodia Camphorata mycelium is observed by light microscopic and environmental scanning electronic microscope after flat board is cultivated, and can observe primary hyphae and secondary hyphae and exist, and the primary hyphae wall is thin, and is transparent, born of the same parents footpath 2.7 μ m～3.5 μ m; Secondary hyphae thin-walled, transparent little Huang, the tool clamp connexion has branch, born of the same parents footpath 3.1 μ m～3.9 μ m; And starting strain mycelium width only is 2.4～2.7um.
2. conidium and conidial fructification morphologic observation
Observe discovery by light microscopic and ESEM: Antrodia Camphorata mycelium can produce conidium at dull and stereotyped cultivation stage.
Sprout in the conidium that the Antrodia Camphorata mycelium cultivation stage produces belongs to and grow phialospore (phialospore) in the type (blastic), this class spore is to produce from a special ampuliform conidiogenous cell, and this chalice cell is called bottle stalk (phialide).The outer wall of conidiogenous cell does not form conidial wall.The conidium light red orange, ellipse, outer wall is smooth, born of the same parents footpath 2.7～3.5 μ m * 5.7～7.7 μ m; And starting strain spore size only is 4.0～5.2um (seeing Fig. 1, Fig. 2).
As seen starting strain has all produced bigger variation from the mycelia form to the spore size after satellite carries.
Satellite is carried camphor tree sesame bacterial strain called after camphor tree sesame (Antrodia camphorata) Ac001 after the mutagenesis, send that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " preservation, deposit number is CGMCC No.1460.
Above-mentioned camphor tree sesame Ac001 bacterial strain mycelium is cultivated
The dull and stereotyped cultivation: camphor tree sesame Ac001 bacterial strain mycelium is inoculated on the flat board, puts 28 ℃ and cultivated 10 days.
Shake-flask culture: 5～10 (1cm of Antrodia Camphorata mycelium that the plate of making even is cultivated
2/ piece) move in the 500ml triangular flask, the liquid nutrient medium with following culture medium prescription making wherein is housed, under 28 ℃, the condition of pH 6, placed on the rotary shaker rotating and culturing 7 days, rotating speed is 110 rev/mins.
Described culture medium prescription in gram is for/100 milliliters:
W-Gum 1% glucose 1%
Peptone 0.2% yeast extract paste 0.2%
Sal epsom 0.1% potassium primary phosphate 0.05%
Fermentor cultivation:
Substratum is the same.Above-mentioned triangular flask is cultivated resulting bacterial classification inoculation in the substratum of 50L fermentor tank, at 28 ℃, pH6, fermentor tank pressure 0.15 kg/cm, air flow is with 1: under the condition that the speed of 1.0vvw, stirring velocity are 200 rev/mins, cultivated 7 days, promptly obtain Antrodia camphorata mycelium fermented full liquid, comprising mycelium and filtered liquid.
Result: can get 30～35 liters of fermentation liquids after per 50 liters of ferment tanks finish.Wherein mycelium dry weight is 0.3 kilogram.The proportion of fermentation liquid is 1.02.Utilize Antrodia camphorata mycelium fermented full liquid can prepare its extract.
Embodiment 2: the strain culturing characteristic that the camphor tree sesame is carried after preceding and the lift-launch compares
(1) temperature comparison test
Analytical test is the result show: the mycelial sprouting temperature range of camphor tree sesame Ac001 is 6-36 ℃, enlarges than starting strain (10-32 ℃) temperature range; The optimal temperature scope of camphor tree sesame Ac001 mycelial growth is between 24 ℃-32 ℃ (bacterium colony is orange), and starting strain suitable culture temperature is 28-30 ℃ (bacterium colony is orange).
(2) pH comparison test
Find through test; When initial pH was in pH3～pH12 scope, camphor tree sesame Ac001 mycelium can both be grown; The pH scope of suitable antrodia camphorata mycelium bulk-growth is pH5～7; When pH=7, the cultivation proterties is good.
And the initial pH of starting strain mycelial growth when pH4 and pH10 is very weak, and the pH scope of suitable antrodia camphorata mycelium bulk-growth is about pH6.
(3) mycelium dry weight relatively
In the camphor tree sesame different varieties liquid culture process, every day, its mycelium dry weight was surveyed in sampling, and the result shows that camphor tree sesame Ac001 bacterial strain mycelium day growth amount reaches the highest apparently higher than camphor tree sesame original species at the mycelium dry weight of cultivating the 6th, seven day two bacterial strain; The average dry weight of Ac001 bacterial strain exceeds 22.8mg/100ml than camphor tree sesame original species.(see figure 3)
(4) polysaccharide content relatively
In the camphor tree sesame different varieties liquid culture process, every day, its polysaccharide content was surveyed in sampling, and the result shows that the Ac001 bacterial strain reaches the climax at the 8th day polysaccharide content of cultivation, and just peaks at the tenth day camphor tree sesame original species; It seems relatively that from polysaccharide content the Ac001 bacterial strain can reach 28.4mg/ml, camphor tree sesame original species are the highest to have only 21.7mg/ml.(see figure 4)
(5) esterase isozyme relatively
Adopt camphor tree sesame different varieties mycelium to carry out the esterase isozyme analysis, the result shows that two kinds all have four identical enzyme bands, and during 0.25,0.55,0.60,0.75, the Ac001 bacterial strain has a new enzyme band to the Rf value at Rf value 0.375 place respectively.(see figure 5)
Embodiment 3: the preparation of Antrodia camphorata mycelium fermented extract
(1) gets Antrodia camphorata mycelium fermented full liquid (filtrate of mycelium and fermented liquid),, make its volume be concentrated to 1/4 of original volume its concentrating under reduced pressure in a usual manner;
(2) be that 70% ethanol extracts above-mentioned spissated fermented liquid with volume percent, wherein, add the alcoholic acid amount and be 4 times of concentrated solution volume;
Under (3) 70 ℃ of conditions, heated 1 hour;
(4) separate with ordinary method, and remove impurity, separate obtaining ethanol extract by cascade filtration;
(5) with above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5 of original volume;
(6) be ethanol 95%, 5 times of ethanol concentrated solution volume amounts with volume percent, above-mentioned concentrated ethanol extract is carried out 20 hours precipitation process;
(7) isolate throw out with ordinary method;
(8) with throw out vacuum-drying or frozen drying in a usual manner, Antrodia camphorata mycelium fermented extract.
Embodiment 4: the preparation of Antrodia camphorata mycelium fermented extract
(1) gets Antrodia camphorata mycelium fermented full liquid (filtrate of mycelium and fermented liquid),, make its volume be concentrated to 1/2 of original volume its concentrating under reduced pressure in a usual manner;
(2) be that 75% ethanol extracts above-mentioned spissated fermented liquid with volume percent, wherein, add the alcoholic acid amount and be 5 times of concentrated solution volume;
Under (3) 60 ℃ of conditions, heated 2 hours;
(4) separate with ordinary method, and remove impurity, separate obtaining ethanol extract by cascade filtration;
(5) with above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5 of original volume;
(6) be ethanol 95%, 7 times of ethanol concentrated solution volume amounts with volume percent, above-mentioned concentrated ethanol extract is carried out 16 hours precipitation process;
(7) isolate throw out with ordinary method;
(8) with throw out vacuum-drying or frozen drying in a usual manner, Antrodia camphorata mycelium fermented extract.
Embodiment 5: the preparation of Antrodia camphorata mycelium fermented extract
Get camphor tree sesame fermentation liquid 30L, be concentrated into 1/3 of original volume in concentration tank, 80% the ethanol of then taking advantage of heat to add 4 times of amounts adds extraction heat, and temperature control meter is built in about 50 ℃, and vacuum tightness is 0.02 kg/cm.
Behind the alcohol extraction, then adopt vacuum pump that extraction liquid is filtered through 180 purpose strainers, with mycelia in the original fermented solution and liquid separation, wash mycelium 3 times repeatedly with pure water after, merging filtrate then reclaims the ethanol in the extraction liquid in thickener.Vacuum degree control is in 0.02 kg/cm at this moment, and temperature is controlled at about 80 ℃.
Ethanol reclaims the remaining camphor tree sesame fermentation liquid extraction liquid that is in back, then it is carried out concentrating under reduced pressure, and the concentration tank temperature is 50 ℃, and vacuum tightness is 0.01 kg/cm.Extraction liquid is concentrated into 1/10 of original volume, and specific gravity control is at 1.06.
When concentrated solution is cooled to below 25 ℃, it is emitted, measure its volume and place in the stainless cylinder bucket of 100L, and add 95% ethanol, add-on is 6 times of concentrated solution volume, leaves standstill under the room temperature 24 hours.Shift out the ethanol supernatant liquor with transfer pipet then, with obtaining camphor tree sesame fermentation liquid ethanol extraction after the solid-liquid separation, put into the vacuum drying oven drying immediately, temperature is controlled at below 50 ℃, and vacuum tightness is below 0.02 kg/cm.
Dried extract is pulverized, and crosses 200 mesh sieves, puts cryodrying place and preserves standby.
Extraction of active ingredients is separating obtained after extracting from fermentation liquid with separating behind the above-mentioned Antrodia Camphorata mycelium liquid fermenting.
Embodiment 6: Antrodia camphorata mycelium fermented extract polysaccharide molecular weight and sugar component test
Testing method:
(1) purity and molecular weight determination:
(2) sugared composition measuring:
Sample thief 10mg is dissolved in the 2mol/1 trifluoroacetic acid; tube sealing; 100 ℃ of hydrolysis 2h under nitrogen oxygen protection, hydrolyzed solution add the methyl alcohol final vacuum and are concentrated into driedly, add as above repeatedly twice again of method of methyl alcohol again; the less water dissolving; divide two partly, the inspection of a thin plate chromatography has not uronic acid existence, and another part is used sodium borohydride reduction; acetylize then, gas chromatography.
Tester: Shanghai Pharmaceutical Inst., Chinese Academy of Sciences polysaccharide laboratory
Test result:
Sugar compositional analysis (see figure 7):
The Antrodia camphorata mycelium fermented extract sample contains pectinose, wood sugar, semi-lactosi and glucose sugar; Its mol ratio is 1: 1.5: 0.4: 10.5.
Sample purity detects and molecular weight:
At least contain five above components according to this sample of HPLC result's (condition determination is seen Fig. 6);
Wherein: RT is that 30.38 molecular weight is 510,000
RT is that 34.05 molecular weight is 7.7 ten thousand
RT is that 38.20 molecular weight is 1.7 ten thousand
RT is that 38.70 molecular weight is 1.5 ten thousand
RT is that 42.07 molecular weight is 0.5 ten thousand
Embodiment 7: Antrodia camphorata mycelium fermented extract total triterpene contents, Determination of Total Alkaloid
Tester: Shanghai Pharmaceutical Inst., Chinese Academy of Sciences polysaccharide laboratory
Measuring method: ordinary method (summary)
Test result:
Total triterpene contents (%) total alkaloid content
Antrodia camphorata mycelium fermented extract 0.32% 0.22mg/ml
Embodiment 8: the Antrodia camphorata mycelium fermented extract determined amino acid
Tester: Laiyang centralab of institute of man
Sample treatment: hydrochloric acid hydrolysis
Analytical instrument: the 835-50 of Hitachi type high-speed amino acid analyzer
Test result:
ASP aspartic acid 2.13%; ILE Isoleucine 1.54%; THR Threonine 1.15%;
LEU leucine 1.72%; SER Serine 1.27%; TYR tyrosine 0.79%;
GLU L-glutamic acid 2.84%; PHE phenylalanine 0.97%; GLY glycine 1.66%;
LYS Methionin 1.08%; ALA L-Ala 1.53%; NH
3Ammonia (disregarding) 0.59%;
CYS Gelucystine 0.32%; HIS Histidine 0.40%; VAL Xie Ansuan 1.20%;
ARG arginine 1.26%; MET methionine(Met) 0.61%; PRO proline(Pro) 1.27%.
Amino acid summation 21.74.
Embodiment 9: use the Antrodia camphorata mycelium fermented extract sample in the human liver cancer cell 2.2.15 of hepatitis B virus transfection clone is cultivated, detect its influence to hepatitis B virus surface antigen (HBsAg) e antigen (HBeAg) secretion and HBV DNA.
Measuring unit: Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences
Material and method:
One. medicine: camphor tree sesame fermentation broth extract is the liquid preparation that Laiyang Agricultural College medicinal fungi institute provides, the light brown suspension.4 ℃ of preservations of mother liquor are made into desired concn with the 2.2.15 cell culture fluid during use during experiment.
Two .2.2.15 cells: the 2.2.15 clone of hepatitis B virus (HBV) dna clone transfection human liver cancer cell (HepG2), U.S. Mount Sinai medical center makes up, the cultivation of going down to posterity voluntarily after introduce this chamber.Cell is with containing foetal calf serum 10%, 3% glutamine 1%, G418380pg/ml, and Eagle ' the sMEM nutrient solution of kantlex 50U/ml is at 37 ℃, 5%CO
2Cultivate in the incubator, an about week goes down to posterity once.
Three. reagent and instrument: HBsAg, HBeAg solid phase radioimmunoassay box, available from Beifang Inst. of Immune Reagents, Chinese Isotopes Co.: radio isotope α 32P dCTP is an inferior brightness biomedical engineering company, specific activity: 111TBq/nmol; The random primer test kit that probe mark is used is available from Promerga company.
Microplate reader: BIO-RAO 3550 types; The Y-calculating instrument is a U.S. DPC company product.
Four. experimental technique:
1. medicine pair cell toxicity test
The mother liquor of medicine camphor tree sesame fermentation broth extract, be made into beginning concentration with 10 times of dilutions of 2.2.15 cell culture fluid, then with nutrient solution from beginning 3 times of dilutions totally 8 concentration, add 96 porocyte culture plates, same concentration liquid was changed in per 4 days in every concentration 4 holes, established no drug cell control group. and with the observation of cell pathology is index, 8 days microscopically observation of cell pathologies, completely destroy is 4; 75% is 3; 50% is 2; 25% is 1; Anosisly become 0.Calculate every concentration liquid average cell lesion degree and suppress %.Press Reed ﹠amp; The Muench method is calculated the poisonous concentration of half (TC50), maximal non-toxic concentration (TC0)
2. medicine is to HBeAg, HBsAg inhibition test
Every milliliter of 100,000 2.2.15 cell inoculations, 96 porocyte culture plates, every hole 200ul, 37 ℃ of 5%CO
2Cultivated 24 hours, and added the following 3 times of dilution test soups of non-toxic concn, 4 extent of dilution are respectively 3 concentration of maximal non-toxic concentration and following 3 times of dilutions thereof, and no drug cell control group is established in every concentration 4 holes, 37 ℃ of 5%CO
2Cultivate, changed the cultivation of original content soup in per 4 days, results nutrient solution in the time of the 8th day ,-20 ℃ are frozen.HBsAg and HBeAg are measured in a collection of experiment simultaneously.HBsAg, the HBeAg positive and negative control and cell contrast are established in experiment.Measure with HBsAg and HBeAg solid phase radioimmunoassay box, method is seen specification sheets, measures every hole cpm value with γ-calculating instrument, calculates inhibiting rate after 4 hole parallel holes are got average.Press Reed ﹠amp; The Muench method is calculated the poisonous concentration of half (IC50).
A=log＞50% drug level B=log＜50% drug level C=log extension rate
3. medicine is to the inhibition test of HBV DNA
2.2.15 cell conditioned medium night each concentration group soup and each control group and cell calculate inhibiting rate after extracting the A value of its HBV DNA, each sample dot hybridization, radioautograph, each hybridization point of measurement by molecular cloning experimental technique method.Press Reed﹠amp; The Muench method is calculated the poisonous concentration of half (IC50).
A=log＞50% drug level B=log＜50% drug level C=log extension rate
4. selectivity index (SI): SI=TC
50/ IC
50
The result:
(1) cytotoxicity of camphor tree sesame broth extraction matter sample in the 2.2.15 cell cultures
For observing the toxicity of camphor tree sesame broth extraction matter sample to people's liver cancer 2.2.15 cell of hepatitis B virogene transfection, behind inoculation 2.2.15 cell, added 3 times of dilute liquid medicines in 24 hours, establish the normal cell contrast simultaneously.Changed soup in 4 days one time, kept 8 days, use the microscope observing cell pathology, by formula calculate the poisonous concentration of half (TC50), maximal non-toxic concentration (TC0) is done a collection of experimental result and is seen Table 1.
The toxicity of table 1. camphor tree sesame broth extraction matter sample in the 2.2.15 cell cultures
Annotate: the cytopathy completely destroy is 4; 75% is 3; 50% is 2; 25% is 1; Anosisly become 0
(2) sample in the 2.2.15 cell cultures to HBeAg and HBsAg excretory restraining effect
3 concentration of sample maximal non-toxic concentration and following 3 times of dilutions thereof add in the 2.2.15 cell and cultivate, and the 8th day inhibition effect to HBsAg and HBeAg sees Table 2 and table 3.
Table 2. camphor tree sesame broth extraction matter sample the 8th day restraining effect in the 2.2.15 cell to HBsAg
Table 3. camphor tree sesame broth extraction matter sample the 8th day restraining effect in the 2.2.15 cell to HBeAg
(3) camphor tree sesame broth extraction matter sample in the 2.2.15 cell cultures to the restraining effect of HBV DNA
3 concentration of camphor tree sesame broth extraction matter sample maximal non-toxic concentration and following 3 times of dilutions thereof add in the 2.2.15 cell and cultivated 8 days, and camphor tree sesame broth extraction matter sample the results are shown in Table 4 to HBV DNA effect.
Table 4. camphor tree sesame broth extraction matter sample the 8th day restraining effect in the 2.2.15 cell to HBV DNA
Conclusion:
Camphor tree sesame broth extraction matter sample the results are shown in Table 5 to the 2.2.15 cytotoxicity with in the 2.2.15 cell cultures to the inhibiting of HBV DNA.The lamivudine (3TC) of positive drug 1 μ M inhibiting rate to HBV DNA in the 2.2.15 cell cultures is 46.4％。Camphor tree sesame broth extraction matter sample sees Table 6,7 to HBeAg and HBsAg excretory restraining effect in the 2.2.15 cell cultures.
Table 5. camphor tree sesame broth extraction matter sample is to the restraining effect conclusive table of 2.2.15 cytotoxicity and HBV DNA
Table 6. camphor tree sesame broth extraction matter sample is to the 2.2.15 cytotoxicity with to the restraining effect conclusive table of HBsAg
Table 7. camphor tree sesame broth extraction matter sample is to the 2.2.15 cytotoxicity with to the restraining effect conclusive table of HBeAg
Embodiment 10: use the Antrodia camphorata mycelium fermented extract sample and carry out the active detection of anti-tumor in vivo:
Measuring unit: pharmaceutical college of Shandong University new drug institute of pharmacology
1. material
1.1 given the test agent:
Antrodia camphorata mycelium fermented extract is the brown powder shape, and institute provides by the Laiyang Agricultural College medicinal fungi, kept dry.Make suspension with distilled water during experiment.
Tegafur (T207): Qilu Pharmaceutical Factory's product, lot number: 0306007.
1.2 tumor cell line: the S180 murine sarcoma, the H22 rat liver cancer derives from medical courses in general institute institute of materia medica, Shandong Province.The ascites preservation of going down to posterity.
1.3 animal: Kunming mouse, body weight 19～22g, the male and female dual-purpose is provided production licence by Shandong University's Experimental Animal Center: production permit (Shandong) 20030004.
2. method:
5～7 days mouse tumor ascites after aseptic extraction is gone down to posterity is through dilution, adjustment cell count to 2～5 * 10, counting back
6(H22 is 1～2 * 10 to/ml
7/ ml), every right side of mice subcutaneous abdomen inoculation 0.2ml.The mouse random packet, is respectively big metering group (1000mg/kg), middle metering group (400mg/kg), small dose group (160mg/kg), positive controls (FT207 120ml/kg), several 14～18 of control animals by 10 every group.Administration next day after kind of the knurl is only irritated the long-pending 0.8ml/ of body of stomach, and control group is given and equal volume distilled water.Administration in morning every day, continuous 12～15 days, put to death mouse behind the last administration 24h, get knurl and weigh, calculate tumour inhibiting rate.
3. result:
With camphor tree sesame extract, respectively S180 murine sarcoma and H22 rat liver cancer are carried out different batches inhibition test, summary result.
4. presentation of results:
(1) continuous irrigation stomach camphor tree sesame bacterial strain extract is 12～15 days.Each metering group to the general situation of animal and body weight gain all significantly better than positive controls.
(2) the camphor tree sesame bacterial strain extract of each metering group all has certain restraining effect to the growth of S180 murine sarcoma and H22 rat liver cancer cell, and antitumor activity is generally between 30～45%.Wherein, the inhibiting rate of S180 murine sarcoma is reached as high as 43.37% (1000mg/kg body weight), can reach 48.88% (160mg/kg body weight) the highest inhibiting rate of H22 rat liver cancer cell.Analyze by experiment, such antitumor activity that is subjected to the reagent thing may be by immune conciliation is produced tumor-inhibiting action.
Claims (7)
1. Antrodia camphorata mycelium fermented extract is made by following method:
(1) gets Antrodia camphorata mycelium fermented full liquid,, make its volume be concentrated to 1/2～1/5 of original volume its concentrating under reduced pressure in a usual manner;
(2) be that 60～95% ethanol extracts above-mentioned spissated fermented liquid with volume percent, wherein, add the alcoholic acid amount and be 2～5 times of concentrated solution volume, can make that alcohol concn reaches 60～90% in the extracting solution;
Under (3) 50～70 ℃ of conditions, heated 1～2 hour;
(4) separate with ordinary method, and remove impurity, separate obtaining ethanol extract by cascade filtration;
(5) with above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5～1/10 of original volume;
(6) be ethanol 95%, 4～8 times of ethanol concentrated solution volume amounts with volume percent, above-mentioned concentrated ethanol extract is carried out 12～24 hours precipitation process;
(7) isolate throw out with ordinary method;
(8) with throw out vacuum-drying or frozen drying in a usual manner, Antrodia camphorata mycelium fermented extract;
Wherein, described Antrodia Camphorata mycelium is CGMCCNo.1460, this bacterial strain is called camphor tree sesame (Antrodia camphorata) Ac001, has been deposited on September 21st, 2005 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number are CGMCC No.1460.
2. Antrodia camphorata mycelium fermented extract as claimed in claim 1, it is characterized in that, the fermentation culture method of described camphor tree sesame (Antrodiacamphorata) Ac001 CGMCC No.1460 is, be inoculated into camphor tree sesame bacterial classification in the Erlenmeyer flask that liquid nutrient medium is housed with ordinary method, with 20～35 ℃ of temperature, shaking bottle rotating speed is 80～280r/min, and under pH 3～8 conditions, vibrations were cultivated 7～15 days; In the cultivation when the pH value drops to 2.5～4, the seed that shakes in the bottle is inoculated in the nutrient solution of 50L fermentor tank, with 20～35 ℃ of temperature, fermentor tank pressure 0.1～0.2 kg/cm, pH 3～8, air flow 0.5～1.1vvm, the condition that stirring velocity is 100～280 rev/mins, cultivated 7～15 days, and can utilize Antrodia camphorata mycelium fermented full liquid preparation to get thing.
3. Antrodia camphorata mycelium fermented extract as claimed in claim 2 is characterized in that, in the fermentation culture method of described camphor tree sesame (Antrodiacamphorata) Ac001 CGMCC No.1460, culture temperature is 28～30 ℃, and cultivating pH is 6.
4. as claim 1,2 or 3 described Antrodia camphorata mycelium fermented extracts, it is characterized in that described Antrodia camphorata mycelium fermented full liquid is meant the filtrate of mycelium and fermented liquid.
5. as claim 2 or 3 described Antrodia camphorata mycelium fermented extracts, it is characterized in that described seed or fermentative medium formula in gram are for/100 milliliters:
W-Gum 1% glucose 1%
Peptone 0.2% yeast extract paste 0.2%
Sal epsom 0.1% potassium primary phosphate 0.05%
6. the described Antrodia camphorata mycelium fermented extract of claim 1 is preparing the application in hepatitis B surface antigen(HBsAg) HBsAg, e antigen HBeAg and the inhibiting medicine of HBV DNA excretory.
7. the application of the described Antrodia camphorata mycelium fermented extract of claim 1 in the preparation cancer therapy drug.
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| CN100394927C (en) * | 2005-09-28 | 2008-06-18 | 莱阳农学院 | Liver cancer resistant Antrodia camphorata and preparation method thererof |
| CN101225066B (en) * | 2007-01-18 | 2010-09-22 | 国鼎生物科技股份有限公司 | Cyclohexenone extract of antrodia camphorata |
| TWI394575B (en) * | 2007-07-09 | 2013-05-01 | Golden Biotechnology Corp | Application of Cynanchum auranthone Cyclohexenone Compounds in the Preparation of Drugs for the Suppression of Hepatitis B |
| CN101417934B (en) * | 2007-10-24 | 2012-04-18 | 国鼎生物科技股份有限公司 | New compounds separated from Antrodia camphorate extract |
| CN104211627A (en) * | 2013-05-29 | 2014-12-17 | 曾卉菱 | Antrodia camphorate compound, extract, and application thereof |
| CN103860605A (en) * | 2013-09-09 | 2014-06-18 | 青岛农业大学 | Preparation method of antrodia-camphorate-fermented full-liquid alcohol extract with avian influenza virus resistance |
| CN103819569B (en) * | 2013-09-09 | 2016-04-13 | 青岛农业大学 | A kind of preparation method of camphor tree sesame fermentation mycelium Crude polysaccharides of anti-avian influenza virus |
| CN104212856A (en) * | 2014-09-17 | 2014-12-17 | 中山安荞生物科技有限公司 | Method for extracting adenosine from antrodia camphorate bacteria |
| CN104293841A (en) * | 2014-10-20 | 2015-01-21 | 中山安荞生物科技有限公司 | Efficient extraction method of antrodia camphorata oil |
| CN104371931A (en) * | 2014-10-20 | 2015-02-25 | 中山安荞生物科技有限公司 | Taiwanofungus camphorates culture medium and culturing method |
| CN105708869B (en) * | 2014-12-05 | 2020-10-13 | 葡萄王生技股份有限公司 | Antrodia camphorata mycelium active substance and composition for protecting nerve cells |
| TWI552755B (en) * | 2015-07-09 | 2016-10-11 | Grape King Bio Ltd | A mycelia of mycelia, a method for preparing the same, a pharmaceutical composition comprising the same, and a use thereof |
| CN105580642B (en) * | 2016-01-28 | 2018-06-01 | 厦门北化生物产业研究院有限公司 | A kind of method using culture of continuous cultivation production Antrodia camphorata thalline |
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