CN110179831A - The pharmaceutical applications of sulphur fungus extract - Google Patents
The pharmaceutical applications of sulphur fungus extract Download PDFInfo
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- CN110179831A CN110179831A CN201910376194.8A CN201910376194A CN110179831A CN 110179831 A CN110179831 A CN 110179831A CN 201910376194 A CN201910376194 A CN 201910376194A CN 110179831 A CN110179831 A CN 110179831A
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Abstract
The present invention provides a kind of application of sulphur fungus extract on the drug for preparing anti-hepatitis virus.Sulphur fungus extract is vulcanized ester fructification or the extract that mycelium alcoholic solvent flows back.The alcoholic solvent includes ethyl alcohol, and the concentration of ethyl alcohol is 95% or more.The vulcanized ester and its ingredient that the present invention obtains are hepatitis B infected for preventing, and treat hepatitis B infected property hepatitis, prevent liver cancer, treat HBV-associated hepatocellular carcinoma, prevent from recurring.Lesion overall process has positive effect.To solving, clinical treatment is hepatitis B infected, especially solves the problems, such as that HBV-associated hepatocellular carcinoma technology is of great significance.
Description
Technical field
The present invention relates to a kind of sulphur fungus extract and the pharmaceutical applications of main component, and in particular to sulphur fungus extract and
Application of the main component on the drug for preparing anti-hepatitis virus.
Background technique
Human liver cancer HepG2/2.2.15 cell strain is derived by liver cancer cell lines HepG2, incorporates two end to end
Connected HBV full genome is in HepG2 genome, which can stablize and continuous expression has infective HBV particle, and
HBV GAP-associated protein GAP HBsAg, HBeAg and HBcAg etc. are expressed, a large amount of virus replication intermediates are generated.The mRNAs of hepatitis B
It is separately encoded core antigen (HBcAg), envelope protein antigen (HBsAgs), polymerase (pol) and X protein (HBx).HBV has
Double casing, hepatitis B surface antigen (HBsAg) are the memebrane proteins of HBV shell.HbsAg coating enters interior after hepatitis B virus duplication
In matter net, later by being secreted in the cell of virus infection, thus it is clinically that HBsAg expression in patients serum is positive as
Infect the mark of HBV.We are indirect by the HBsAg concentration being secreted into culture medium in HepG2/2.2.15 cell cultivation process
Reflect the content of HBV.
HBV belongs to liver DNA virus family, and HBV gene group encodes tetra- genes of S/preS, C/preC, P and X.Hepatitis B
Viral X protein (HBx) is a kind of soluble protein, molecular weight 17kDa, by 4 open reading frames of HBV gene group
(ORF) the smallest gene X gene coding in, has played key effect in HBV biology and liver cancer development.HBx is one
Kind of multifunctional protein, it is existing research shows that the HBV of HBx gene defect virus replication amount will appear and significantly reduce in vivo and in vitro,
Prompt HBx has played important function for the lasting duplication of HBV and the continuous infection of human virus.By in measurement cell
The expression of HBx can reflect the content of HBV in cell.
China is hepatitis type B virus (HBV), the district occurred frequently Hepatitis C Virus (HCV).Virus causes China's liver cancer
Etiological is at present HBV-associated hepatocellular carcinoma there are about 85% patient.Liver cancer is the 5th common malignant tumour, but its disease
Dead rate but occupies second in cancer case fatality rate.The new hair and dead liver cancer patient that the whole world has 50% or more are in China.More
Fearful is that 5 years survival rates of Chinese liver cancer are only 12.5%.Postoperative 5 years recurrence rates are but up to 70%.The onset of liver cancer is more hidden
It hides, early stage typically no any symptom seriously affects China's liver cancer treatment effect.Drug therapy is still the important of liver cancer prevention and treatment
Therapeutic modality.Either classic chemotherapy or targeted therapy, curative effect is because drug resistance is by serious influence.As current liver cancer is controlled
The line molecular targeted agents Sorafenib treated to the life span of extension advanced liver cancer patient, or even obtains long term survival
Etc. have the unexistent income of the past drug therapy liver cancer.However, the appearance of drug resistance significantly affects curative effect.Its drug resistance
Mechanism is complicated.Therefore, even targeted drug treatment liver cancer also still difficulties, so the diagnosis and treatment method of liver cancer is clinical needs
One of great critical issue to be solved.
The difficulty that liver cancer treatment faces is largely because the characteristic that virus affects liver cancer: Hepatitis B correlation liver
The treatment of cancer becomes body, tumour, virus, drug by body, tumour, drug triadic relation in conventional oncotherapy and closes
System.Virus is to influence liver cancer recurrence, transfer and drug resistant major reason.Cause tradition using apoptosis as the chemotherapeutical medicine curative effect of target
It is bad.Therefore the relationship of virus with tumour is fully considered.Liver cancer is prevented and treated using New Measure, is the positive of raising liver cancer treatment effect
Outlet.
Virus is that the major reason for influencing curative effect of medication has: 1, virus influences drug resistance: HBX gene passes through in HBV gene group
It is integrated into liver cell, plays a significant role in the occurrence and development and drug resistance of liver cancer.Suo Feilani drug resistance and HBX pass through up-regulation
TGF-β/II signal path of T β R- is related.2, virus influences liver cancer recurrence and transfer: HBX can be logical by signals such as COX-2/PEG2
Road promotes liver cancer tissue Angiogenesis, promotes the transfer of liver cancer.HBX can activate IGF-1R, promote canceration of hepatic cell, and to liver
Key effect is played when cancerous invasion and transfer.Therefore, targeting IGF-1R treatment can inhibit growth of cancer cells, prevent liver cancer recurrence and
A kind of transfer, it has also become new direction of liver cancer biological therapy.But cellular uptake is low, lacks specific target sequence and causes non-specific toxicities
Reaction etc. limits its application.3, virus influences tumour cell characteristic: the Ras of HBX and host cell, c-Jun, NF- κ B, c-
The transcription factors such as Fos, FAK formed protein-protein complex, activation or change transcription factor activity, influence Apoptosis and
DNA is repaired, and can be promoted canceration of hepatic cell or be improved cancer cell invasion and transfer ability.HBX also passes through trans-activation, raises a system
The cell and viral gene of column.Therefore, carry out hepatitis B while the country advocates HBV associated hepatocellular carcinoma antineoplaston at present
Treatment, reduce postoperative recurrence and transfer.Even if can not be good with the treatment for carrying out hepatitis B while antineoplaston
The problems such as solving adverse drug reaction and curative effect well, less can solve virus infection and asks to the blocking developed into liver cancer overall process
Topic.Develop high-efficiency low-toxicity can antiviral but also anti-tumor drug as necessity.
Vulcanized ester [Laetiporus sulphureus (Fr.) Murrill] alias Huang sesame, Jin Zhi, Shennong's Herbal,
" Mingyi Bielu ", Compendium of Material Medica are on the books.Vulcanized ester is also the common qi-restoratives medicine of Tujia, has effects that complement is strong.
Food and medicament dual-purpose, securely and reliably.Vulcanized ester has anti-inflammatory effect according to reports, and treatment colitis, rheumatism and rheumatoid arthritis are existing
For a long time history.Vulcanized ester main component is clear, and eburicoic acid, 3- hydrogenation pine Siberian cocklebur acid B are two kinds of Triterpenoid principal components.Eburicoic acid is
The ingredient of generally acknowledged specific anti-inflammatory effect, and there is anticancer, liver protection, anti-ulcer effect etc..
Summary of the invention
In view of the above technical problems, the present invention provides a kind of drug of anti-hepatitis virus, i.e. sulphur fungus extract.
Vulcanized ester (Laetiporus sulphureus (Fr.) Murr.) genus polyporus Cordycepps, sulphur Pseudomonas, alias sulphur are more
Pore fungi, Polyporus sulphureus, Huang Zhi, which are that a kind of food medicine dual-purpose macro fungi is often edible, can promote health.
The sulphur fungus extract is vulcanized ester fructification or the extract that mycelium alcoholic solvent flows back.
The alcoholic solvent includes ethyl alcohol, and the concentration of ethyl alcohol is 95% or more.(used methanol, ethyl alcohol, acetic acid second
Ester, water, petroleum ether extract.Methanol, ethyl alcohol, ethyl acetate are ok, but ethyl alcohol is easy to get safely, and effective component contains in extract
Highest is measured, so 70-100% is ok with ethyl alcohol, but concentration is preferably 90-95%.)
Three hydrogenation pine Siberian cocklebur acid B answering on the drug for preparing anti-hepatitis virus in the vulcanized ester ethanol extract
With.
Application of the eburicoic acid on the drug for preparing anti-hepatitis virus in the vulcanized ester ethanol extract.
The technical solution of the present invention loose Siberian cockleburs of three hydrogenations that sulphur fungus extract, sulphur fungus extract separate it has been confirmed by experiments that
Sour B (Trametenolic acid B), eburicoic acid (Eburicoic acid) have anti-HBV effect.
Technical solution of the present invention also provides a kind of drug of anti-hepatitis virus, and active constituent is that claim 1-5 is any
Sulphur fungus extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid described in；The drug includes with the extraction of active constituent vulcanized ester
Object or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid are primary formulation, and auxiliary material is tablet, capsule or ointment made of auxiliary agent
Agent.
The tablet is that auxiliary material and active constituent sulphur fungus extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid are mixed pressure
Piece forms；It further include the magnesium stearate that mass fraction is 0.5-1.5% in the auxiliary material, the auxiliary material includes microcrystalline cellulose
Element, modified starch, ethyl cellulose, methylcellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose
One of sodium is a variety of.
The capsule is by auxiliary material and active constituent sulphur fungus extract or three hydrogenation pine Siberian cocklebur acid B or perforation acid-mixed
It closes, granulation, whole grain is encapsulated to form, and the auxiliary material includes microcrystalline cellulose, modified starch, ethyl cellulose, Methyl cellulose
One of element, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose are a variety of.
The ointment is to mention adjunct ingredient lanolin, vaseline, single stearic acid glycerine lipoprotein and active constituent vulcanized ester
Object or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid are taken to mix；Wherein lanolin, vaseline, single stearic acid glycerine lipoprotein by etc. matter
Amount is than mixing.
In drug of the invention, pine is hydrogenated comprising vulcanized ester alcohol extract 0.1-0.5g or eburicoic acid three in every 100g auxiliary material
Siberian cocklebur acid B10-100mg or eburicoic acid 10-100mg.
The vulcanized ester and its ingredient that the present invention obtains are hepatitis B infected for preventing, and treat hepatitis B infected property hepatitis, prevent liver
Cancer treats HBV-associated hepatocellular carcinoma, prevents from recurring.Lesion overall process has positive effect.To solving, clinical treatment is hepatitis B infected,
Especially solve the problems, such as that HBV-associated hepatocellular carcinoma technology is of great significance.Being embodied in vulcanized ester and its ingredient can prevent
Hepatitis B sense or as disinfection medication.It carries out disinfection to by hepatitis B pollutant.Hepatitis B infected property hepatitis is treated, prevents hepatitis to liver cancer
Progress.HBV-associated hepatocellular carcinoma is treated, prevents from recurring.It can also enhance drug effect with existing drug combination, prevent drug resistance, resist
Virus reduces recurrence.
Detailed description of the invention
Fig. 1 is the silicagel column elution object high-efficient liquid phase analysis figure of vulcanized ester alcohol extract.
Fig. 2 is ELISA kit standard curve.
Fig. 3 is that 3- hydrogenates the pine Siberian cocklebur acid B effect influence to HepG2/2.2.15 cell HBsAg (ng/mL) secretion for 24 hours,
Middle * P < 0.05, * * P < 0.01.
The influence that Fig. 4 3- hydrogenation pine Siberian cocklebur acid B effect 48h secretes HepG2/2.2.15 cell HBsAg (ng/mL),
Middle * * P < 0.01.
Fig. 5 eburicoic acid effect influence to HepG2/2.2.15 cell HBsAg (ng/mL) secretion for 24 hours.
The influence that Fig. 6 eburicoic acid effect 48h secretes HepG2/2.2.15 cell HBsAg (ng/mL), wherein * P <
0.05。
Fig. 7 vulcanized ester alcohol extract acts on the influence to HepG2/2.2.15 cell HBsAg (ng/mL) secretion for 24 hours, wherein * *
P < 0.01.
Fig. 8 vulcanized ester alcohol extract acts on the influence that 48h secretes HepG2/2.2.15 cell HBsAg (ng/mL), wherein * *
P < 0.01.
Expression of HBx under the action of 3- hydrogenates pine Siberian cocklebur acid B, wherein * P < 0.05, * * P < in Fig. 9 liver cancer cells
0.01。
Expression of HBx under the action of eburicoic acid in Figure 10 liver cancer cells, wherein * * P < 0.01.
Expression of HBx under the action of vulcanized ester alcohol extract in Figure 11 liver cancer cells, wherein * P < 0.05, * * P <
0.01。
Specific embodiment
Embodiment 1
The preparation of sulphur fungus extract, three hydrogenation pine Siberian cocklebur acid B and eburicoic acid
Experimental material used in the present invention
Vulcanized ester (Laetiporus sulphureus (Fr.) Muttill) is by Three Gorges Tourism Vocationl Technical College Wang Shaobai
Professor identifies and provides fructification and strain.
Agents useful for same of the present invention
95% medicinal alcohol, distilled water, chromatographic grade acetonitrile, petroleum ether, acetone etc..
Laboratory apparatus used in the present invention
KQ-500DB type ultrasonic washing instrument, Ai Lang Rotary Evaporators (Germany), ten a ten thousandth electronics day of ME215S type
Flat (German Sartorios company), ultraviolet specrophotometer (UV -2201, Japanese Shimadzu Corporation), high performance liquid chromatograph
(DIONEX Ultimate 3000, Dai An liquid chromatogram Co., Ltd, the U.S.), Nuclear Magnetic Resonance (UltrashiedTM
400MHZ Plus, bruker Brooker company), freeze drier (FREEZ0NE PLUS 6, LABCONCO).
The preparation of vulcanized ester alcohol extract
95% alcohol reflux of 100g vulcanized ester fructification is extracted three times, each 1.5h is filtered while hot and merged three
Secondary filtrate, and is removed under reduced pressure ethyl alcohol on Rotary Evaporators, obtains liquid extract 50g, then extract is dried in 50 DEG C, obtains alcohol extract,
Total 25g.
By above-mentioned vulcanized ester alcohol extract petroleum ether: acetone=3:1 makees eluant, eluent through silica gel post separation, and gained eluate accounts for
The 24% of alcohol extracting thing, then eluate is analyzed into its ingredient through HPLC, condition is that distilled water (A)-acetonitrile (B) eluent system is washed
It takes off, B/A=95%, there are mainly two types of materials compounds 1 and compounds 2 in eluate (see Fig. 1).
60mg eluate accurately is weighed, makees mobile phase with B/A=95%, through high performance liquid chromatography separation, obtains 18mg chemical combination
Object 1 and 40mg compound 2.Prepare compound 1 and compound 2 by the same method, for experiment.
It is identified through nuclear-magnetism, compound 1:13C-NMR(DMSO-d6,500MHz):177.1,134.4,133.5,131.2,
124.0,76.9,50.2,49.2,47.6,46.7,43.9,38.7,36.7,35.4,32.3,30.2,28.5,28.2,27.7,
26.6,26.1,25.7,25.6,24.2,20.4,19.1,18.0,17.5,15.9,15.7；1H-NMR(DMSO-d6,
500MHz):0.68(s,3H),0.69(s,3H),0.81(s,3H),0.90(s,3H),0.90(s,3H),1.52(s,3H),
1.62 (s, 3H), 4.25 (d, J=3.5Hz, 1H), 5.06 (s, 1H), 11.96 (s, 1H)
It is compareed with document, meets the structure of three hydrogenation pine Siberian cocklebur acid B.
Compound 2:13C-NMR(Pyr-d5, 125MHz) and δ: 37.4,28.7,78.0,39.6,50.9,18.8,26.8,
134.4,135.3,37.4,19.4,29.4,44.9,49.9,30.9,27.5,47.8,16.4,21.3,49.2,178.7,
31.8,32.8,155.9,34.2,21.9,21.3,28.7,24.5,16.4,107.0；1H-NMR(Pyr-d5,400MHz)δH:
3.46(1H,t),1.61(1H,m),2.31,2.67(2H,m),1.98,2.30(2H,m),1.04(3H,s),1.10(3H,s),
1.03 (3H, d, J=5.5Hz), 1.02 (3H, d, J=5.5Hz), 1.18 (3H, s), 1.20 (3H, s), 1.30 (3H, s),
4.95,4.91(2H,s).
It is compareed with document, meets the structure of eburicoic acid.
Embodiment 2
Sulphur fungus extract, three hydrogenation pine Siberian cocklebur acid B and eburicoic acid are as the application on anti-hepatic-B virus medicine
Drug and cell strain
Vulcanized ester alcohol extract, eburicoic acid, 3- hydrogenate loose Siberian cocklebur acid B, are provided by this laboratory.When cell experiment, DMSO is used:
Dehydrated alcohol=1: 9 solution dissolution drug is finally made 10mg/mL initial mass by 0.22 μm of millipore water membrane filtration
The drug of concentration, then cause required concentration to carry out cell experiment with culture medium dilution.
Human liver cancer HepG2/2.2.15 cell strain is purchased from biological Co., Ltd, Nanjing section one hundred.
Main agents used in present invention process
Human Hepatitis B Surface Antigen (HBsAg) ELISA detection kit, Beijing Clinical Diagnosis Reagent Co., Ltd.
Product；
Q-PCR kit, Sangon Biotech's product；
Reverse Transcriptase kit, RNA isolater Total RNAExtraction Reagent (lysate), Nanjing promise
Wei Zan Biotechnology Co., Ltd product；
MEM dehydrated medium, U.S.'s Gibco Products；
Sodium Pyruvate (NaP), Non Wssential Amino Acids (NEAA), Shanghai Mike's woods biochemistry section
Skill Co., Ltd product；
Chloroform, isopropanol (cold), PBS, DEPC water.
Key instrument used in present invention process
CFX96Touch quantitative fluorescent PCR is bought in Bio Rad Laboratories；
NanoQuant microplate reader, the purchase of Yu Ruishi Tecan company；
One two-sided superclean bench is bought in Purifying Equipment Co., Ltd., Suzhou；
Water-jacket typ CO2 incubator, Yu Shanghai tri- rise Instrument Ltd.'s purchase；
Medical low-temperature refrigerator, the purchase of Yu Zhongke Dou Ling commercial appliance limited liability company；
- 20 DEG C of refrigerators, the purchase of Yu Haier company；
5415R small desk high speed freezing centrifuge, the purchase of Yu Deguo Eppendorf AG；
Vortex-Genie2 turbula shaker, the purchase of Yu Meiguo Scientific Industries company；
The liquid-transfering gun of various ranges is bought in general Ube Industries, Ltd, Germany.
Embodiment 3
ELISA kit detects hepatitis B surface antigen secretion
The HepG2/2.2.15 cell of logarithmic growth phase is selected, pancreatin is digested to single cell suspension, and 2 × 104mL-1Concentration
Inoculating cell, 100 holes μ L/ are inoculated in 96 orifice plates, set 37 DEG C, 5%CO212h is cultivated in incubator, is then respectively adding containing 0.0
The MEM culture medium of each drug of the μ g/mL of (control), 2.5,5,10.0,15.0 and 20.0, each concentration set 3 multiple holes.Cultivate 24 Hes
After 48h, the cell supernatant of 24 and 48h of culture is collected respectively, is saved in -20 DEG C.According to the specification of ELISA kit, survey
Determine the content of HBsAg in supernatant.Wavelength 450nm is selected, each hole OD value is read with microplate reader reading, takes mean value, calculate and inhibit
Percentage.
After collection part supernatant, the MTT that 20 μ L concentration are 5g/L is added in every hole, continues to cultivate 4h in 37 DEG C of incubators,
Liquid is discarded supernatant, every hole is added after 150 μ L dimethyl sulfoxides sufficiently vibrate mixing and is read respectively with microplate reader (Detection wavelength 490nm)
Hole absorbance (A) value records result.Cell proliferation inhibition rate=(control wells A value-dosing holes A value)/control wells A value ×
100%.
Embodiment 4
The expression of qRT-PCR method detection liver cancer cells HBx
The extraction of total serum IgE
(1) with 6 orifice plates culture cell, dosing for 24 hours is cultivated, dosing difference action time is 48h, 72h for 24 hours.Discard culture
Base is washed 2 times with PBS.
(2) 1mL lysate is added in every hole cell, is allowed to sufficiently cover cell surface, is blown and beaten cell with liquid-transfering gun
Into lysate.
(3) lysate is transferred in the centrifuge tube of 1.5mL, blows and beats lysate repeatedly with pipettor, when visually observing nothing
In the presence of obvious granular, 5min is waited in placement quietly on ice.
(4) 200 μ L chloroforms are added in Xiang Shangshu lysate, acutely shakes 15S and forms emulsion.4 DEG C of standing 5min.
(5) 4 DEG C of centrifugation 5min of 12000g.
(6) upper strata aqueous phase is carefully sucked into a new centrifuge tube.Draw 400 μ L.
(7) isopropanol of 400 μ L pre-cooling is added into centrifuge tube, is mixed by turning upside down.4 DEG C of standing 10min.
(8) 4 DEG C of centrifugation 10min of 12000g, usually can see that white precipitate.
(9) supernatant is abandoned, 75% ethyl alcohol of 1mL is added.Abundant sluicing pipe lid and tube wall, make precipitating suspend, and stand 3-5min.
(10) 12000g is centrifuged 5min, abandons supernatant.
(11) drying precipitated 2-5min is added 20 μ L DEPC water dissolution precipitating, is stored in -80 DEG C.
(12) utilize reverse transcription reagent box by the RNA reverse transcription extracted at c DNA
The removal of genomic DNA
Mixing, 42 DEG C of 2min are blown and beaten with liquid-transfering gun.
Reverse transcription reaction system
It is mixed with liquid-transfering gun, reverse transcription system: 50 DEG C of 15min, 85 DEG C of 5sec.
RT-PCR reaction detection HBx gene expression dose
The cDNA of reverse transcription is diluted one times
Q-PCR reaction condition
All experimental datas are with mean ± standard deviationIt indicates, statistical analysis uses spss21 software, comparison among groups
It is analyzed with One-Way ANOVA, P < 0.05 is with significant difference, and P < 0.01 illustrates that difference has extremely significant property.
Embodiment 5
The influence that ELISA kit detection drug secretes hepatitis B surface antigen
ELISA kit standard curve is measured to be illustrated in fig. 2 shown below.
ELISA kit detects the influence that three kinds of drugs secrete HBsAg
ELISA kit test analysis, three kinds of drugs show the HBsAg secretion of HepG2/2.2.15 cell certain
20,15,10,5,2.5 μ g/mL of concentration is arranged in the inhibiting effect of degree, three kinds of drugs.Such as Fig. 3-8, we are can be found that with medicine
The growth of object action time, various drugs also enhance the inhibiting effect that HBsAg secretes therewith, but the concentration difference opposite sex is unknown
It is aobvious.And compare between three kinds of drugs, to the inhibiting rate of the HBsAg secretion of HepG2/2.2.15 cell: vulcanized ester alcohol extract >
3- hydrogenates loose Siberian cocklebur acid B > eburicoic acid.
QRT-PCR method detects expression of the liver cancer cells HBx under three kinds of drug effects
Hepatitis B virus x protein (HBx) is in three kinds of drug effects in qRT-PCR method detection hepatocellular carcinoma H22/2.2.15
Under expression, drug concentration is respectively 20 μ g/mL, 15 μ g/mL, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL.It acts on for 24 hours,
A degree of raising is presented in the expression of HBx, and as the reduction elevated-levels of concentration are more obvious, but vulcanized ester alcohol extracting
The expression no significant difference compared to the blank group of object HBx in higher concentrations；After acting on 48h and 72h, HepG2/2.2.15 is thin
The expression of HBx is lowered in born of the same parents, and there are time and dose dependents for downward degree.Three groups of drugs compare, to HepG2/
2.2.15 in cell HBx express inhibiting rate: vulcanized ester alcohol extract > 3- hydrogenate pine Siberian cocklebur acid B > eburicoic acid (result is shown in Fig. 9-
11)。
Embodiment 6
The preparation of vulcanized ester mycelia
Culture medium is prepared
Make potato culture medium: potato (peeling) 200g, glucose 20g, agar 20g, water 1000ml.Potato is cut
It is put into pot at fritter, adds water 1000ml, boiled 20 minutes, with filtered through gauze, moisturizing to 1000ml is put into agar and grape
Sugar is adjusted PH6.0 (potassium dihydrogen phosphate, dipotassium hydrogen phosphate solution) after heating dissolution, culture medium is dispensed in conical flask, every bottle
It is packed into 1/3 capacity culture medium, 0.1-0.15MPa steam high-voltage sterilizing 30min.
Actication of culture
Vulcanized ester strain is taken out from strain library uses shaking table shaken cultivation in being inoculated on aseptic operating platform.Revolving speed
150r/min, is cultivated 5-7 days by 25 DEG C of temperature.
Inoculation and culture
Make culture solution: glucose 20g, gypsum 2g, soybean powder 10g, malt flour 2g, 20% potato juice 1000ml are prepared
PH6.0 (potassium dihydrogen phosphate, dipotassium hydrogen phosphate solution) is adjusted afterwards, is sub-packed in the triangular flask of 500ml, per bottled 150ml, high pressure is gone out
Bacterium.In the strain 5-10ml for being inoculated with activation on aseptic operating platform, shaking table shaken cultivation is used.Revolving speed 120r/min, 25 DEG C of temperature,
Culture 5-20 days.Collect mycelia, 60-100 DEG C of drying.
Eburicoic acid, three hydrogenate loose Siberian cocklebur acid B content measurement in vulcanized ester and mycelium
With efficient liquid phase measurement vulcanized ester in and mycelium in eburicoic acid content.Eburicoic acid accurately is weighed, uses chromatographic grade
Methanol is made into the solution of various concentration, and 0.22 μm of membrane filtration is as standard items.It takes vulcanized ester, each 1.5g of mycelium through 95%
80 DEG C of ultrasonic extractions of ethyl alcohol, filter while hot, take filtrate, and evaporation drying is made into isometric solution, 0.22 μm of filter with hplc grade methanol
Film filtering is used as sample to be tested.
Draw standard curve, analysis condition: chromatographic column Diamonsil C18 (250mm*4.6mm.id., 5um), column temperature 25
DEG C, mobile phase: acetonitrile 10-100%, ultrapure water 90%~0%, elution time 30min；100% acetonitrile, 10min；It is total
40min.Detection eburicoic acid wavelength is set as 203nm, and the titer of sample introduction various concentration calculates standard curve: obtaining eburicoic acid acid
Calibration curve equation Y=19.07x+3.38, R2=0.999；Three hydrogenate loose Siberian cocklebur acid B standard curvilinear equation Y=110.7X+
1.499 coefficient R 2=0.999.Standard curve is linearly preferable.Sample introduction 10uL analyte sample fluid, is repeated 5 times, and calculates RSD,
The repeatability of detection method, RSD=1.46%, method repeatability are good.In addition a Duplicate Samples are taken, every 0h, 4h, 7h, 10h,
The accuracy of detection method, RSD=2.47%, internal stability is preferable for 24 hours for this method.Sample introduction measures according to the above method, real by son
Body and mycelium dry weight calculate content.
The content of eburicoic acid, the loose Siberian cocklebur acid B of three hydrogenations in vulcanized ester fructification and mycelium
Vulcanized ester fructification and mycelium alcohol extract yield, eburicoic acid, the content of the loose Siberian cocklebur acid B of three hydrogenations are almost the same.Alcohol
Eburicoic acid, three total contents for hydrogenating loose Siberian cocklebur acid two kinds of main components of B are more than 50% in extract.Therefore, eburicoic acid, three hydrogenate loose Siberian cocklebur
Sour B is vulcanized ester principle active component.
Embodiment 7
The drug preparation process of sulphur fungus extract, three hydrogenation pine Siberian cocklebur acid B and eburicoic acid is as follows:
The preparation of medicinal tablet of the present invention
Vulcanized ester alcohol extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid are taken, starch and 1% magnesium stearate, tabletting is added, is made every
The piece extract 0.1-1.0g of mykol containing sulphur, or eburicoic acid 10-100mg or eburicoic acid 10-100mg.(auxiliary material used can be micro-
Crystalline cellulose, modified starch, ethyl cellulose, methylcellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl
One or more of common auxiliary material of the tablets such as sodium cellulosate).
The preparation of medicine capsule of the present invention
Vulcanized ester alcohol extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid are taken, starch is added, granulation, whole grain is encapsulated, and every
The extract of mykol containing sulphur 0.1-0.5g in capsule, or the loose Siberian cocklebur acid B 10-100mg or eburicoic acid 10-100mg of three hydrogenations.It is (used
It is fine that auxiliary material can be microcrystalline cellulose, modified starch, ethyl cellulose, methylcellulose, hydroxyethyl cellulose, hydroxypropyl methyl
Tie up one or more of common auxiliary materials of capsules such as element, sodium carboxymethylcellulose).
The preparation of drug ointment agent of the present invention
Vulcanized ester alcohol extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid are taken, lanolin, vaseline, glycerol monostearate is added
The auxiliary materials such as rouge are fabricated to ointment, and the extract 0.1-0.5g of mykol containing sulphur or eburicoic acid three hydrogenate loose Siberian cocklebur acid in every 100g ointment
B10-100mg eburicoic acid 10-100mg.
Claims (10)
1. a kind of application of sulphur fungus extract on the drug for preparing anti-hepatitis virus.
2. application according to claim 1, which is characterized in that sulphur fungus extract is that vulcanized ester fructification or mycelium are used
The extract of alcoholic solvent reflux.
3. application according to claim 2, which is characterized in that the alcoholic solvent includes ethyl alcohol, and the concentration of ethyl alcohol is 95%
Or more.
4. application according to claim 3, which is characterized in that three hydrogenation pine Siberian cocklebur acid B in vulcanized ester ethanol extract exist
Prepare the application on the drug of anti-hepatitis virus.
5. application according to claim 3, which is characterized in that the eburicoic acid in vulcanized ester ethanol extract is preparing anti-second
Application on the drug of hepatovirus.
6. a kind of drug of anti-hepatitis virus, which is characterized in that active constituent is the described in any item vulcanized esters of claim 1-5
Extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid；The drug includes hydrogenating loose Siberian cocklebur with active constituent sulphur fungus extract or three
Sour B or eburicoic acid are primary formulation, and auxiliary material is tablet, capsule or ointment made of auxiliary agent.
7. the drug of anti-hepatitis virus according to claim 6, which is characterized in that the tablet be by auxiliary material and activity at
Sulphur fungus extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid mixed pressuring plate are divided to form；It further include quality point in the auxiliary material
Number is the magnesium stearate of 0.5-1.5%, and the auxiliary material includes microcrystalline cellulose, modified starch, ethyl cellulose, Methyl cellulose
One of element, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose are a variety of.
8. the drug of anti-hepatitis virus according to claim 6, which is characterized in that the capsule is by auxiliary material and activity
Ingredient sulphur fungus extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid mixing, granulation, whole grain is encapsulated to form, the auxiliary material
Including microcrystalline cellulose, modified starch, ethyl cellulose, methylcellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose,
One of sodium carboxymethylcellulose is a variety of.
9. the drug of anti-hepatitis virus according to claim 6, which is characterized in that the ointment is by adjunct ingredient sheep
Hair rouge, vaseline, single stearic acid glycerine lipoprotein and active constituent sulphur fungus extract or three hydrogenation pine Siberian cocklebur acid B or eburicoic acid mixing
It forms.
10. the drug of anti-hepatitis virus according to claim 6, which is characterized in that include vulcanized ester in every 100g auxiliary material
Alcohol extract 0.1-0.5g or eburicoic acid three hydrogenate loose Siberian cocklebur acid B 10-100mg or eburicoic acid 10-100mg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910376194.8A CN110179831A (en) | 2019-05-07 | 2019-05-07 | The pharmaceutical applications of sulphur fungus extract |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910376194.8A CN110179831A (en) | 2019-05-07 | 2019-05-07 | The pharmaceutical applications of sulphur fungus extract |
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Family
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667608A (en) * | 2021-03-24 | 2021-11-19 | 孙长春 | Sulfur cinnabarinus and application thereof in treating digestive tract ulcer and colitis |
| CN113717859A (en) * | 2021-03-17 | 2021-11-30 | 孙长春 | Sulfur red mushroom and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103961379A (en) * | 2014-05-15 | 2014-08-06 | 神农架天润生物科技有限责任公司 | Laetiporus sulphureus extract and application thereof |
-
2019
- 2019-05-07 CN CN201910376194.8A patent/CN110179831A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103961379A (en) * | 2014-05-15 | 2014-08-06 | 神农架天润生物科技有限责任公司 | Laetiporus sulphureus extract and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| LUNG M Y等: "Antioxidant properties of polysaccharides from Laetiporus sulphureus in submerged cultures", 《AFRICAN JOURNAL OF BIOTECHNOLOGY》 * |
| 胡亚平等: "硫磺菌多糖的分离纯化及体外抗氧化、抗肿瘤活性初探", 《中国现代中药》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113717859A (en) * | 2021-03-17 | 2021-11-30 | 孙长春 | Sulfur red mushroom and application thereof |
| CN113717859B (en) * | 2021-03-17 | 2023-08-22 | 孙长春 | Armillariella tabescens and application thereof |
| CN113667608A (en) * | 2021-03-24 | 2021-11-19 | 孙长春 | Sulfur cinnabarinus and application thereof in treating digestive tract ulcer and colitis |
| CN113667608B (en) * | 2021-03-24 | 2023-08-22 | 孙长春 | Thiobacillus cinnabarinus and application thereof in treatment of digestive tract ulcer and colonitis |
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