CN1799560A - Liver cancer resistant Antrodia camphorata and preparation method thererof - Google Patents
Liver cancer resistant Antrodia camphorata and preparation method thererof Download PDFInfo
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- CN1799560A CN1799560A CN 200510044806 CN200510044806A CN1799560A CN 1799560 A CN1799560 A CN 1799560A CN 200510044806 CN200510044806 CN 200510044806 CN 200510044806 A CN200510044806 A CN 200510044806A CN 1799560 A CN1799560 A CN 1799560A
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Abstract
The invention discloses an Antrodia camphorata capsule for resisting hepatic carcinoma, which comprises the following constituents (by weight portions): Antrodia camphorate mycelium fermentation extract 20-100, protein-free maize starch or medicinal starch gum 200-480. The Antrodia camphorate mycelium fermentation extract is abstracted by ethanol and dried. The Antrodia camphorata bacterial strain Ac001 was preserved in the China General Microbiological Culture Collection Center with a docket number of CGMCC No.1460. The Antrodia camphorate mycelium fermentation extract in the hepatic carcinoma resisting Antrodia camphorate capsule has appreciable actions in inhibiting hepatitis B virus HbsAg, e antigen HbeAg and HBV DNA secretion and resisting cancers especially liver cancer.
Description
Technical field
The present invention relates to a kind of capsule and preparation method thereof, relate in particular to Antrodia camphorata capsule of a kind of anti-hepatocarcinoma and preparation method thereof.
Background technology
Antrodia camphorata (Antrodia camphorata) claim Antrodia camphorata again.Generally be grown on the hollow heartwood inwall of lobule sassafras mitriform; Hymenium is salmon pink, bitter in the mouth.Antrodia camphorata new varieties mycelium is salmon pink, the meat powder color after cultivating.Microscopic examination, mycelia is elongated, have every, have clamp connection, and a large amount of conidium is arranged, conidium is salmon pink, meat powder color or yellow because of the condition of culture difference.
Recently some progress have been obtained, Chinese patent 01123613.2 " solid culture method of Antrodia camphorata, 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 chemical compound and uses thereof " and 200410011142.4 " basidiomycetous polysaccharide body of Antrodia Camphorata and uses thereof " all related to the effect and the purposes of Antrodia camphorata, 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, do not see yet and utilize the Antrodia camphorata preparation extract of improving the breed, be used to make the capsular report of Antrodia camphorata of anti-hepatocarcinoma.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides Antrodia camphorata capsule of a kind of anti-hepatocarcinoma and preparation method thereof.
The Antrodia camphorata capsule of the anti-hepatocarcinoma that the present invention relates to, form by following component and weight portion:
Antrodia camphorata mycelium fermented extract 20～100; Do not contain protein cornstarch or medicinal dextrin 200～480.
The Antrodia camphorata capsule of the anti-hepatocarcinoma that the present invention relates to, preferably form by following component and weight portion:
Antrodia camphorata mycelium fermented extract 50～100; Do not contain protein cornstarch or medicinal dextrin 200～450.
The Antrodia camphorata capsule of the anti-hepatocarcinoma that the present invention relates to, most preferably form by following component and weight portion:
Antrodia camphorata mycelium fermented extract 50; Do not contain protein cornstarch or medicinal dextrin 450.
In the Antrodia camphorata capsule of anti-hepatocarcinoma of the present invention, 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 fermentation liquid with percent by volume, wherein, add alcoholic acid amount and be 2～5 times of concentrated solution volume, can make that concentration of alcohol reaches 60～90% in the extracting solution;
Under (3) 50～70 ℃ of conditions, heated 1～2 hour;
(4) separate with conventional 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 percent by volume, above-mentioned concentrated ethanol extract is carried out 12～24 hours precipitation process;
(7) isolate precipitate with conventional method;
(8) with precipitate 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 Antrodia camphorata (Antrodia camphorata) Ac001, is deposited on the 21st in JIUYUE in 2005 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number are CGMCC No.1460.
Above-mentioned Antrodia camphorata (Antrodia camphorata) Ac001 CGMCC No.1460 is the Antrodia camphorata strain that is separated to by the Antrodia camphorata 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 mutation, after activation culture repeatedly, obtain.
The fermentation culture method of above-mentioned Antrodia camphorata (Antrodia camphorata) Ac001 CGMCC No.1460 is, be inoculated into the Antrodia camphorata strain in the conical flask that fluid medium is housed with conventional 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 pH value drops to 2.5～4, the seed that shakes in the bottle is inoculated in the culture fluid of 50L fermentation tank, with 20～35 ℃ of temperature, fermentation tank sterilization pressure 0.1～0.2 kg/cm, pH 3～8, ventilation 0.5～1.1vvm, the condition that mixing speed 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 Antrodia camphorata (Antrodia camphorata) Ac001 CGMCC No.1460, cultivation 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 fermentation liquid.
Wherein, above-mentioned seed or fermentative medium formula in gram are for/100 milliliters:
Corn starch 1% glucose 1%
Peptone 0.2% yeast extract 0.2%
Magnesium sulfate 0.1% potassium dihydrogen phosphate 0.05%
The capsular preparation method of the Antrodia camphorata of anti-hepatocarcinoma of the present invention is to take by weighing Antrodia camphorata mycelium fermented extract respectively, do not contain protein cornstarch or medicinal dextrin with described weight portion; All grinding is less than 200 purpose fine powders, abundant mixing under the drying condition; Adorn the amount capsule of packing into No. 0 of 0.5 gram with every capsules.
Wherein, the water content of described corn starch or medicinal dextrin requires in mass percent below 9%.
Antrodia camphorata mycelium fermented extract of the present invention is preparing the application in hepatitis B virus HBsAg, e antigen HBeAg and the excretory inhibiting medicine of HBV DNA.
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 to hepatitis B virus HBsAg, e antigen HBeAg and the excretory inhibition effect of HBV DNA.
Experimental result shows: 3 times of Antrodia camphorata mycelium fermented extract dilutions added cell culture 8 days, pair cell median toxic concentration TC
50Be 0.153mg/ml.Maximal non-toxic concentration TCO is 0.039mg/ml.Antrodia camphorata fermentation broth extract maximal non-toxic concentration 0.039mg/ml is 59.0% to the suppression ratio of HBVDNA, its half-inhibition concentration IC
50Being 0.0086mg/ml, is 36.1% to the suppression ratio of hepatitis B virus HBsAg, its half-inhibition concentration IC
50Be 0.026mg/ml, and be 49.8%, its half-inhibition concentration IC the suppression ratio 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 the antitumor animal experiment, the result shows: the Antrodia camphorata mycelium fermented extract of each metering group all has certain inhibitory action to the growth of S180 murine sarcoma and H22 rat liver cancer cell, and antitumor activity is generally between 30～45%.Wherein, the suppression ratio 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 suppression ratio 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
Antrodia camphorata of the present invention (Antrodia camphorata) Ac001, be deposited on the 21st in JIUYUE in 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 Antrodia camphorata Ac001 bacterial strain satellite carries back electron microscopic observation figure
Fig. 2 Antrodia camphorata Ac001 starting strain electron microscopic observation figure
Fig. 3 Antrodia camphorata different strain mycelium dry weight relatively
Fig. 4 Antrodia camphorata different cultivars liquid fermentation polyoses content relatively
Fig. 5 Antrodia camphorata 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 composition analysis
The specific embodiment
Embodiment 1:
The Antrodia camphorata strain that Antrodia camphorata (Antrodia camphorata) is separated to by the Antrodia camphorata 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 mutation.
The Antrodia camphorata strain repeatedly after the activation culture, is observed form, relatively the difference of bacterial strain before and after the mutation to it with conventional method after satellite carries mutation.
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 connection 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 flask 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 Antrodia camphorata bacterial strain called after Antrodia camphorata (Antrodia camphorata) Ac001 after the mutation, send that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " preservation, deposit number is CGMCC No.1460.
Above-mentioned Antrodia camphorata Ac001 bacterial strain mycelium is cultivated
The dull and stereotyped cultivation: Antrodia camphorata 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 fluid medium with following culture medium prescription making wherein is housed, under 28 ℃, the condition of pH6, 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:
Peptone 0.2% yeast extract 0.2%
Magnesium sulfate 0.1% potassium dihydrogen phosphate 0.05%
Fermentor cultivation:
Culture medium is the same.Above-mentioned triangular flask is cultivated resulting bacterial classification inoculation in the culture medium of 50L fermentation tank, 28 ℃, pH6, fermentation tank sterilization pressure 0.15 kg/cm, ventilation is with 1: under the condition that the speed of 1.0vvw, mixing speed are 200 rev/mins, cultivated 7 days, promptly obtain Antrodia camphorata mycelium fermented full liquid, comprising mycelium and filtrate.
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 Antrodia camphorata carries after preceding and the lift-launch compares
(1) temperature comparative test
Analytical test is the result show: the mycelial sprouting temperature range of Antrodia camphorata Ac001 is 6-36 ℃, enlarges than starting strain (10-32 ℃) temperature range; The preference temperature scope of Antrodia camphorata Ac001 mycelial growth is between 24 ℃-32 ℃ (bacterium colony is Chinese red), and starting strain suitable culture temperature is 28-30 ℃ (bacterium colony is Chinese red).
(2) pH comparative test
Find through test; When initial pH was in pH3～pH12 scope, Antrodia camphorata Ac001 mycelium can both be grown; The pH scope of suitable antrodia camphorata mycelium bulk-growth is pH5～7; When pH=7, the cultivation character 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 Antrodia camphorata different cultivars liquid culture process, every day, its mycelium dry weight was surveyed in sampling, and the result shows that Antrodia camphorata Ac001 bacterial strain mycelium day growth amount reaches the highest apparently higher than the Antrodia camphorata 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 Antrodia camphorata original species.(see figure 3)
(4) polyoses content relatively
In the Antrodia camphorata different cultivars liquid culture process, every day, its polyoses content was surveyed in sampling, and the result shows that the Ac001 bacterial strain reaches summit at the 8th day polyoses content of cultivation, and just peaks at the tenth day Antrodia camphorata original species; It seems relatively that from polyoses content the Ac001 bacterial strain can reach 28.4mg/ml, the Antrodia camphorata original species are the highest to have only 21.7mg/ml.(see figure 4)
(5) esterase isozyme relatively
Adopt Antrodia camphorata different cultivars 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 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 fermentation 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 fermentation liquid with percent by volume, wherein, add alcoholic acid amount and be 4 times of concentrated solution volume;
Under (3) 70 ℃ of conditions, heated 1 hour;
(4) separate with conventional 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 percent by volume, above-mentioned concentrated ethanol extract is carried out 20 hours precipitation process;
(7) isolate precipitate with conventional method;
(8) with precipitate 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 fermentation 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 fermentation liquid with percent by volume, wherein, add alcoholic acid amount and be 5 times of concentrated solution volume;
Under (3) 60 ℃ of conditions, heated 2 hours;
(4) separate with conventional 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 percent by volume, above-mentioned concentrated ethanol extract is carried out 16 hours precipitation process;
(7) isolate precipitate with conventional method;
(8) with precipitate 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 Antrodia camphorata 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 is 0.02 kg/cm.
Behind the alcohol extraction, then adopt vacuum pump that extract is filtered through 180 purpose filters, with mycelia in the original fermented solution and fluid separation applications, wash mycelium 3 times repeatedly with pure water after, merging filtrate then reclaims the ethanol in the extract in concentrator.Vacuum degree control is in 0.02 kg/cm at this moment, and temperature is controlled at about 80 ℃.
Ethanol reclaims the remaining Antrodia camphorata fermentation liquid extract that is in back, then it is carried out concentrating under reduced pressure, and the concentration tank temperature is 50 ℃, and vacuum is 0.01 kg/cm.Extract 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, addition is 6 times of concentrated solution volume, leaves standstill under the room temperature 24 hours.Shift out the ethanol supernatant with pipet then, with obtaining Antrodia camphorata 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 is below 0.02 kg/cm.
Dried extract is pulverized, and crosses 200 mesh sieves, puts cold drying 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 fermentation.
Embodiment 6: Antrodia camphorata mycelium fermented extract polysaccharide molecular weight and saccharic composition test
Method of testing:
(1) purity and molecular weight determination:
(2) sugared composition measuring:
Sample thief 10mg is dissolved in the 2mol/l trifluoroacetic acid; tube sealing; 100 ℃ of hydrolysis 2h under nitrogen oxygen protection, hydrolyzed solution add the methanol final vacuum and are concentrated into driedly, add as above repeatedly twice again of method of methanol again; the low amounts of water dissolving; divide two partly, the inspection of a thin plate chromatography has not alduronic acid existence, and another part is used sodium borohydride reduction; acetylation then, gas chromatography.
Tester: Shanghai Pharmaceutical Inst., Chinese Academy of Sciences's polysaccharide laboratory
Test result:
Sugar composition analysis (see figure 7):
The Antrodia camphorata mycelium fermented extract sample contains arabinose, xylose, galactose 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's polysaccharide laboratory
Assay method: conventional 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 Agricultural College central laboratory
Sample treatment: hydrochloric acid hydrolysis
Analytical tool: 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 glutamic acid 2.84%; PHE phenylalanine 0.97%; GLY glycine 1.66%;
LYS lysine 1.08%; ALA alanine 1.53%; NH
3Ammonia (disregarding) 0.59%;
CYS cystine 0.32%; HIS histidine 0.40%; VAL valine 1.20%;
ARG arginine 1.26%; MET methionine 0.61%; PRO proline 1.27%.
Aminoacid summation 21.74.
Embodiment 9: the capsular making of Antrodia camphorata
The capsular raw material of Antrodia camphorata is an Antrodia camphorata mycelium fermented extract, and adjuvant is a corn starch.
Take by weighing 50 kilograms of Antrodia camphorata mycelium fermented extracts (the no moisture absorption does not have caking); Do not contain 450 kilograms of protein cornstarch (water content is below 9% for food company's purchase, pure white).All grinding is less than 200 purpose fine powders, abundant mixing under the drying condition; Adorn the amount capsule of packing into No. 0 of 0.5 gram with every capsules.
Component is pressed Antrodia camphorata mycelium fermented extract 50mg/ grain in the capsule, and the prescription of corn starch 450mg/ grain is prepared, and uses the fill of fully-automatic capsule can packing machine behind the ball mill mix homogeneously.The polishing back is the Antrodia camphorata capsule finished product with fully-automatic capsule racking machine coating-dividing sealing (30 every bottle) after labelling.
Embodiment 10: the capsular making of Antrodia camphorata
The capsular raw material of Antrodia camphorata is an Antrodia camphorata mycelium fermented extract, and adjuvant is a medicinal dextrin.
Take by weighing 100 kilograms of Antrodia camphorata mycelium fermented extracts (the no moisture absorption does not have caking); 400 kilograms of medicinal dextrins.All grinding is less than 200 purpose fine powders, abundant mixing under the drying condition; Adorn the amount capsule of packing into No. 0 of 0.5 gram with every capsules.
Component is pressed Antrodia camphorata mycelium fermented extract 100mg/ grain in the capsule, and the prescription of corn starch 400mg/ grain is prepared, and uses the fill of fully-automatic capsule can packing machine behind the ball mill mix homogeneously.The polishing back is the Antrodia camphorata capsule finished product with fully-automatic capsule racking machine coating-dividing sealing (30 every bottle) after labelling.
Embodiment 11: use the Antrodia camphorata mycelium fermented extract sample in the human liver cancer cell 2.2.15 of hepatitis B virus transfection cell line 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: the Antrodia camphorata fermentation broth extract is the liquid preparation that Laiyang Agricultural College medicinal fungi institute provides, the light brown suspension.4 ℃ of preservations of mother solution 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 cell line 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 hyclone 10%, 3% glutamine 1%, G418 380pg/ml, and Eagle ' the sMEM culture fluid of kanamycin 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 is available from Beifang Inst. of Immune Reagents, Chinese Isotopes Co.; Radiosiotope 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 solution of medicine Antrodia camphorata fermentation broth extract, be made into beginning concentration with 10 times of dilutions of 2.2.15 cell culture fluid, then with culture fluid 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 matched group. and with the observation of cell pathological changes is index, 8 days microscopically observation of cell pathological changes, destroying fully 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 (TCO)
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%CO2 cultivated 24 hours, add the following 3 times of dilution test medicinal liquids of non-toxic concn, 4 dilution factors are respectively 3 concentration of maximal non-toxic concentration and following 3 times of dilutions thereof, no drug cell matched group is established in every concentration 4 holes, 37 ℃ of 5%CO
2Cultivate, changed the cultivation of original content medicinal liquid in per 4 days, results culture fluid 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 description, measures every hole cpm value with γ-calculating instrument, calculates suppression ratio 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 medicinal liquid and each matched group and cell calculate suppression ratio after extracting the A value of its HBV DNA, each sample dot blot hybridization, autoradiography, 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. selection index (SI): SI=TC
50/ IC
50
The result:
(1) cytotoxicity of Antrodia camphorata broth extraction matter sample in the 2.2.15 cell culture
For observing the toxicity of Antrodia camphorata broth extraction matter sample to people's hepatocarcinoma 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 medicinal liquid in 4 days one time, kept 8 days, use the microscope observing cell pathological changes, 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. Antrodia camphorata broth extraction matter sample in the 2.2.15 cell culture
| Medicine | Initial concentration | TC 50 | TC 0 |
| Barrier sesame fermentation broth extract | 3.2mg/ml | 0.153mg/ml | 0.039mg/ml |
Annotate: it is 4 that cytopathy is destroyed fully; 75% is 3; 50% is 2; 25% is 1; Anosisly become 0
(2) sample to 3 concentration of HBeAg and the excretory inhibitory action sample of HBsAg maximal non-toxic concentration and following 3 times of dilutions thereof, adds in the 2.2.15 cell and cultivates in the 2.2.15 cell culture, and the 8th day inhibition effect to HBsAg and HBeAg sees Table 2 and table 3.
Table 2. Antrodia camphorata broth extraction matter sample is the 8th day inhibitory action to HBsAg in the 2.2.15 cell
| Medicine | Drug level (mg/ml) | cpm(X±SD) | Suppress (%) | HBsAg IC 50(mg/ml) |
| The Antrodia camphorata fermentation broth extract | 0.0393 0.0131 0.0044 0.0015 | 6752 7364 6945 10959 | 49.8 34.1 24.5 - | 0.022 |
| The cell contrast | 10570 | |||
| Positive control | 62678 | |||
| Negative control | 392 |
Table 3. Antrodia camphorata broth extraction matter sample is the 8th day inhibitory action to HBeAg in the 2.2.15 cell
| Medicine | Drug level (mg/ml) | cpm(X±SD) | Suppress (%) | HBeAg IC 50(mg/ml) |
| The Antrodia camphorata fermentation broth extract | 0.0393 0.0131 0.0044 0.0015 | 2136 2804 3214 5226 | 49.8 34.1 24.5 - | 0.022 |
| The cell contrast | 4255 | |||
| Positive control | 55246 | |||
| Negative control | 477 |
(3) Antrodia camphorata broth extraction matter sample in the 2.2.15 cell culture to the inhibitory action of HBV DNA
3 concentration of Antrodia camphorata 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 Antrodia camphorata broth extraction matter sample the results are shown in Table 4 to HBV DNA effect.
Table 4. Antrodia camphorata broth extraction matter sample is the 8th day inhibitory action to HBV DNA in the 2.2.15 cell
| Medicine | Drug level (mg/ml) | The OD value | Suppress (%) | HBV DNA IC 50(mg/ml) |
| The Antrodia camphorata fermentation broth extract | 0.0393 0.0131 0.0044 0.0015 | 0.211 0.279 0.254 0.335 | 58.9 45.7 50.6 34.8 | 0.0086 |
| The contrast of | 0.238 0.514 | 46.4 |
Conclusion:
Antrodia camphorata broth extraction matter sample the results are shown in Table 5 to the 2.2.15 cytotoxicity with in the 2.2.15 cell culture to the inhibiting of HBV DNA.The lamivudine (3TC) of positive drug 1 μ M suppression ratio to HBV DNA in the 2.2.15 cell culture is 46.4％。Antrodia camphorata broth extraction matter sample sees Table 6,7 to HBeAg and the excretory inhibitory action of HBsAg in the 2.2.15 cell culture.
Table 5. Antrodia camphorata broth extraction matter sample is to the inhibitory action conclusive table of 2.2.15 cytotoxicity and HBV DNA
| Medicine | Cytotoxicity | HBV DNA | ||
| TC50 | TC0 | IC50 | SI | |
| The Antrodia camphorata fermentation broth extract | 0.153mg/ml | 0.039mg/ml | 0.0086mg/ml | 17.8 |
Table 6. Antrodia camphorata broth extraction matter sample is to the 2.2.15 cytotoxicity with to the inhibitory action conclusive table of HBsAg
| Medicine | Cytotoxicity | HBsAg | ||
| TC50 | TC0 | IC50 | SI | |
| The Antrodia camphorata fermentation broth extract | 0.153mg/ml | 0.039mg/ml | 0.026mg/ml | 5.9 |
Table 7. Antrodia camphorata broth extraction matter sample is to the 2.2.15 cytotoxicity with to the inhibitory action conclusive table of HBeAg
| Medicine | Cytotoxicity | HBsAg | ||
| TC50 | TC0 | IC50 | SI | |
| The Antrodia camphorata fermentation broth extract | 0.153mg/ml | 0.039mg/ml | 0.022mg/ml | 7.0 |
Embodiment 12: 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, provides kept dry by Laiyang Agricultural College medicinal fungi institute.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 number 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 mice 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 tumor is only irritated the long-pending 0.8ml/ of body of stomach, and matched group is given and the equal volume distilled water.Administration in morning every day, continuous 12～15 days, put to death mice behind the last administration 24h, get tumor and weigh, calculate tumour inhibiting rate.
3. result:
With the Antrodia camphorata 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 Antrodia camphorata 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 Antrodia camphorata bacterial strain extract of each metering group all has certain inhibitory action to the growth of S180 murine sarcoma and H22 rat liver cancer cell, and antitumor activity is generally between 30～45%.Wherein, the suppression ratio 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 suppression ratio 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 (9)
1. the Antrodia camphorata capsule of an anti-hepatocarcinoma is characterized in that, is made up of following component and weight portion:
Antrodia camphorata mycelium fermented extract 20～100; Do not contain protein cornstarch or medicinal dextrin 200～480.
2. the Antrodia camphorata capsule of anti-hepatocarcinoma as claimed in claim 1 is characterized in that, is made up of following component and weight portion:
Antrodia camphorata mycelium fermented extract 50～100; Do not contain protein cornstarch or medicinal dextrin 200～450.
3. the Antrodia camphorata capsule of anti-hepatocarcinoma as claimed in claim 2 is characterized in that, is made up of following component and weight portion:
Antrodia camphorata mycelium fermented extract 50; Do not contain protein cornstarch or medicinal dextrin 450.
4. as the Antrodia camphorata capsule of claim 1,2 or 3 described anti-hepatocarcinoma, it is characterized in that described 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 fermentation liquid with percent by volume, wherein, add alcoholic acid amount and be 2～5 times of concentrated solution volume, concentration of alcohol reaches 60～90% in the extracting solution making;
Under (3) 50～70 ℃ of conditions, heated 1～2 hour;
(4) separate with conventional 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 percent by volume, above-mentioned concentrated ethanol extract is carried out 12～24 hours precipitation process;
(7) isolate precipitate with conventional method;
(8) with precipitate vacuum drying or frozen drying in a usual manner, Antrodia camphorata mycelium fermented extract;
Wherein, described Antrodia camphorata mycelium fermented full liquid is meant the filtrate of mycelium and fermentation liquid; Described Antrodia Camphorata mycelium is CGMCCNo.1460, this bacterial strain is called Antrodia camphorata (Antrodia camphorata) Ac001, protected a surname on the 21st in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number are CGMCCNo.1460 in JIUYUE in 2005.
5. the Antrodia camphorata capsule of anti-hepatocarcinoma as claimed in claim 4, it is characterized in that, the fermentation culture method of described Antrodia camphorata (Antrodiacamphorata) Ac001 CGMCC No.1460 is, be inoculated into the Antrodia camphorata strain in the conical flask that fluid medium is housed with conventional 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 pH value drops to 2.5～4, the seed that shakes in the bottle is inoculated in the culture fluid of 50L fermentation tank, with 20～35 ℃ of temperature, fermentation tank sterilization pressure 0.1～0.2 kg/cm, pH 3～8, ventilation 0.5～1.1vvm, the condition that mixing speed is 100～280 rev/mins, cultivated 7～15 days, and can utilize Antrodia camphorata mycelium fermented full liquid preparation to get thing.
6. the Antrodia camphorata capsule of anti-hepatocarcinoma as claimed in claim 5 is characterized in that, described seed or fermentative medium formula in gram are for/100 milliliters:
Corn starch 1% glucose 1%
Peptone 0.2% yeast extract 0.2%
Magnesium sulfate 0.1% potassium dihydrogen phosphate 0.05%.
7. the Antrodia camphorata capsule of anti-hepatocarcinoma as claimed in claim 5 is characterized in that, in the fermentation culture method of described Antrodia camphorata (Antrodiacamphorata) Ac001 CGMCC No.1460, cultivation temperature is 28～30 ℃, and cultivating pH is 6.
8. the capsular preparation method of Antrodia camphorata of claim 1,2 or 3 described anti-hepatocarcinoma is characterized in that, takes by weighing Antrodia camphorata mycelium fermented extract respectively, does not contain protein cornstarch or medicinal dextrin with described weight portion; All grinding is less than 200 purpose fine powders, abundant mixing under the drying condition; Adorn the amount capsule of packing into No. 0 of 0.5 gram with every capsules.
9. as the capsular preparation method of Antrodia camphorata of anti-hepatocarcinoma as described in the claim 9, it is characterized in that the water content of described corn starch or medicinal dextrin requires in mass percent below 9%.
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