The purposes of the preparation antiviral drugs of plain boiled pork Ganoderma extract and antiviral drugs
Technical field
The present invention relates to the new application of a kind of plain boiled pork Ganoderma extract, be specifically related to the disease-resistant of a kind of plain boiled pork Ganoderma extract
Poison purposes.
Background technology
Ganoderma (Ganoderma) in state-owned long medicinal history, be commonly used by people for tracheitis, hepatitis, high blood for a long time
The auxiliary treatment of the diseases such as pressure, tumor, immunity are disorderly.Modern pharmacology and clinical study results also indicate that Ganoderma contains suppression
Tumor and the active component of regulation immunity.Since DONK (1948) sets up Ganodermataceae (Ganodermataceae), the most full generation
The Ganodermataceae macro fungi that boundary has reported more than 200 is planted totally, and China Ganoderma on the books has 103 kinds, and wherein 14 kinds by people institute
Utilize.The most widely used kind includes Ganoderma lucidum (Leyss. Ex Fr.) Karst. (Ganoderma lucidum), Ganoderma (Ganoderma at present
Sinense) etc., other kind of the many of Ganoderma also has similar effect, is worth research further.
Herpes simplex virus (Herpes simple Virus, HSV) is a sickness rate height, global widely distributed people
Herpeslike virus, the mankind are unique natural hosts.Can cause after infection the positions such as skin, oral cavity and genitals primary/
, there are the phenomenons such as skin ulcer, pimple, inflammation in recurrent herpes.Simplexvirus α-herpes in herpetoviridae
Chordopoxvirinae, has two serotypes of HSV-1 and HSV-2.HSV virion is made up of kernel, capsid, tunicle and cyst membrane.HSV-1
The main infection body first half, classical symptom is that herpes of mouth, herpetic keratitis come off and herpes simplex encephalitis.Immunodeficiency person
Or the infected meeting of neonate causes serious disease such as central nervous system disease.Can if the encephalitis that HSV-1 causes is treated not in time
Can entail dangers to life.The development that anti-HSV medicine experiences more than 50 year, anti-HSV medicine in early days with idoxuridine as representative, its selectivity
Low, big to normal cytotoxicity.The medicine being now used clinically for treating HSV is often acyclovir (ACV, acyclovir), but
Because its antiviral spectrum is narrow, being easily generated drug resistance and expensive, serious limits clinical practice.
Summary of the invention
On the one hand, the present invention provide a kind of plain boiled pork Ganoderma extract for preparing antiviral drugs purposes.
Preferably, said extracted thing is extractive with organic solvent.
Preferably, said extracted thing is alcohol extraction thing, more preferably ethanol extraction.
Preferably, above-mentioned virus is herpesvirus, more preferably HSV virus, such as HSV-1 virus.
Preferably, above-mentioned plain boiled pork Ganoderma is plain boiled pork Ganoderma Ganoderma leucocontextum HMGIM-Z110122,
Deposit number is CCTCC NO:M 2016087.
The above-mentioned plain boiled pork Ganoderma of the present invention is picked up from the broad-leaf forest of Linzhi Area of Tibet Bowo County Zha Xi Gang Xiang, is identified as
Plain boiled pork Ganoderma Varieties By Uv Induced, and separate tissue acquisition original strain, named plain boiled pork Ganoderma Ganoderma
Leucocontextum HMGIM-Z110122, is preserved in China typical culture collection center (letter on March 7th, 2016
Claiming CCTCC, address is: Bayi Road Luo Jia Shan, Wuchang District, Wuhan City, Hubei Province), deposit number is CCTCC NO:M
2016087)。
The cultural method of above-mentioned plain boiled pork Ganoderma CCTCC M 2016087 novel bacterial, including making mother after separate tissue strain
Kind, making original seed, make to produce and plant, cultivation is cultivated and goes out sesame management.
In a preferred embodiment, the culture medium making mother's kind in the cultural method of above-mentioned plain boiled pork Ganoderma Varieties By Uv Induced is
Comprehensive PDA culture medium, the formula on described comprehensive PDA inclined-plane is Rhizoma Solani tuber osi 18-20%, glucose 1.5-by mass percentage
2%, agar 2-2.5%, potassium dihydrogen phosphate 0.15-0.3%, magnesium sulfate 0.05-0.15%, vitamin B1 trace, remaining is water.
In a preferred embodiment, the cultural method of above-mentioned plain boiled pork Ganoderma Varieties By Uv Induced makes the original seed material of original seed
Raw material composition is Sorghum vulgare Pers. 75-79%, Semen setariae 16-20%, CaCO by mass percentage31-2%, water content 55-60%.
In a preferred embodiment, the cultural method of above-mentioned plain boiled pork Ganoderma Varieties By Uv Induced makes the production kind of production kind
The raw material composition of material is cotton seed hulls 87-89%, wheat bran 10-12%, CaCO by mass percentage31-2% or cotton seed hulls 48-
52%, wood flour 25-30%, wheat bran 18-20%, calcium carbonate 1-2%, calcium sulfate 1-2%, water content 55-60%.
In a preferred embodiment, in the cultural method of above-mentioned plain boiled pork Ganoderma Varieties By Uv Induced, the raw material composition of planting material is pressed
Mass percent is calculated as wood flour 75-78%, wheat bran 20-22%, CaCO31-2%, water content 60-65%
The present invention provides the cultural method of a kind of preferred plain boiled pork Ganoderma CCTCC M 2016087 novel bacterial, including following
Step:
(1) make mother to plant: gather the strain plain boiled pork Ganoderma of separate tissue, be connected to comprehensive PDA inclined-plane by aseptic for strain, be placed in
Constant temperature light culture in 20-25 DEG C of incubator, until mycelia is covered with inclined-plane and i.e. obtains production mother's kind, wherein said comprehensive PDA inclined-plane
Formula is Rhizoma Solani tuber osi 18-20%, glucose 1.5-2%, agar 2-2.5%, potassium dihydrogen phosphate 0.15-by mass percentage
0.3%, magnesium sulfate 0.05-0.15%, vitamin B1 trace, remaining is water;
(2) original seed is made: mother will be produced and plant in aseptic access original seed bag, and be placed in constant temperature light culture in 25 DEG C of incubators, extremely
Mycelia i.e. obtains original seed after eating full material, wherein the raw material composition of original seed material is Sorghum vulgare Pers. 75-79%, Semen setariae 16-by mass percentage
20%, CaCO31-2%, water content 55-60%, load described original seed material in strain bag, prepare original seed bag;
(3) production kind is made: aseptic for original seed access produced in kind of bag, be placed in constant temperature light culture in 25 DEG C of incubators, extremely
Mycelia i.e. obtains production kind after eating full material, the raw material composition wherein producing kind of material is cotton seed hulls 87-89%, bran by mass percentage
Skin 10-12%, CaCO31-2% or cotton seed hulls 48-52%, wood flour 25-30%, wheat bran 18-20%, calcium carbonate 1-2%, sulphuric acid
Calcium 1-2%, water content 55-60%, by described, production kind material is loaded in strain bag, prepare and produce kind of a bag;
(4) inoculation cultivation: by the aseptic access cultivation material bag of production kind, lucifuge is cultivated in 25 DEG C ± 1 DEG C culturing room, treats
After mycelia in cultivating bag covers with planting material, continue shading After-mature cultivation and complete latter stage of ripening to mycelia, the wherein raw material of planting material
Composition is wood flour 75-78%, wheat bran 20-22%, CaCO by mass percentage31-2%, water content 60-65%, by described cultivation
Training material loads in strain bag, prepares cultivating bag;
(5) going out sesame management: regulation temperature is 20-21 DEG C, humidity 85-90%, illumination 100-200lux, stuffiness keeps
CO2Content at 2000ppm up to grow former base, adjust temperature be 26-28 DEG C, relative air humidity 85-90%, illumination about
200-300lux, stuffiness, continues to keep CO2Content, at more than 2000ppm, treats that stem terminates and enter cap growth elongating stage
During the phase, ventilation, adjust temperature and be 26-28 DEG C, humidity 85-95%, illumination 300-500lux, ventilation, keep CO2Content is less than
1200ppm, when the white edge to cap turns yellow completely, plucks.
Preferably, the relative air humidity of the culturing room in above-mentioned steps (4) is 60-70%.
Plain boiled pork Ganoderma CCTCC M 2016087 sporophore of the present invention has following morphological characteristic and an external appearance characteristic:
Cap 10-20*5-10cm, thickness 2-3cm at nearly handle, fan-shaped or semicircle fan-shaped, major part is red to bronzing, and children is tender
Time and ripe time edge white to the most light yellow, gradual change is yellow to bronzing, after maturation close to stem part be chocolate, dark violet reddish brown
Color or the most dark red brown, tool paint sample gloss, have concentricity, often have weak radial wrinkle.
When vent surface is fresh, white is to cream-colored, and injury band brown is to brown.Aperture subcircular, every millimeter 4-6.
Tube is up to 4-6mm, not stratified, reddish brown to ficelle or taupe.
Bacterial context thickness reaches 2.2cm, white, dry after cream-colored to near-white or micro-strip in vain, soft suberin to suberin, nearly epidermis
There is thin band brown close bed (cot).
Stem 5-8*3.5-5.5cm, cylindrical to the most flat, side life is to wilfully, and nearly stockless sometimes, chocolate is to dark violet brown
Color, has gloss, inner white.
Spore oval, top is truncate, light brown, double wall, and inwall tool spinule, non-starchy, including outermost spore layer
Time be 9.5-12.5*7-9 μm, do not include during outermost spore layer being 8.0-9.0*5-6.5 μm.
Another aspect of the present invention also provides for a kind of antiviral drugs, including above-mentioned plain boiled pork Ganoderma extract and pharmaceutically
Acceptable carrier.
The invention has the beneficial effects as follows: the present invention provides the new application of a kind of plain boiled pork Ganoderma, can effectively suppress herpesvirus,
For preparing antiviral drugs, especially for preparing anti-herpesvirus medicament, there is good medical value.
Accompanying drawing explanation
Fig. 1 is the subobject graph that the cultivation of the plain boiled pork Ganoderma of embodiment 1 obtains.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described.
Embodiment 1:
The artificial cultivation method of plain boiled pork Ganoderma (CCTCC M 2016087), comprises the following steps:
1) it is Rhizoma Solani tuber osi 20%, glucose 2%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate by mass percentage
0.15% and mcg vitamin B1, remaining is that the raw material composition routine of water makes comprehensive PDA inclined-plane, 0.11MPa atmospheric pressure, 121
DEG C High Temperature High Pressure moist heat sterilization 30min;
2) it is Sorghum vulgare Pers. 78%, Semen setariae 20%, CaCO by mass percentage3The raw material composition of 2%, water content 60%, system
Make original seed material, original seed material is loaded in the thermostable transparent polypropylene strain bag of 13cm × 25cm, beat in Bag Material with small wood
Hole, at the bottom of hole is deep to bag, then plastic hoop on bag mouth set, and buckle supporting lid, and preparing original seed bag, equivalent every original seed is packed dry
Material 300g;
3) it is cotton seed hulls 89%, wheat bran 10%, CaCO by mass percentage3The raw material composition of 1%, water content 55%,
Make and produce kind of a material, production kind material is loaded in the thermostable transparent polypropylene strain bag of 15cm × 30cm, with small wood at bag
Material burrows, at the bottom of hole is deep to bag, then plastic hoop on bag mouth set, and buckles supporting lid, prepare and produce kind of a bag, equivalent often give birth to
Produce kind of a packed siccative 350g;
4) it is wood flour 78%, wheat bran 20%, CaCO by mass percentage3The raw material composition of 2%, water content 60%, system
Make planting material, planting material is loaded in the thermostable transparent polypropylene strain bag of 17cm × 35cm, beat in Bag Material with small wood
Hole, at the bottom of hole is deep to bag, then plastic hoop on bag mouth set, and buckle supporting lid, prepare cultivating bag, equivalent every packed siccative
500g;
5) make mother to plant: gather the strain plain boiled pork Ganoderma CCTCC M 2016087 of separate tissue, be connected to combine by aseptic for strain
Close PDA inclined-plane, be placed in constant temperature light culture in 25 DEG C of incubators, until mycelia is covered with inclined-plane (about about 7 days) and i.e. obtains production mother's kind;
6) make original seed: female kind in aseptic access original seed bag will be produced, during inoculation, guarantee that female material block of planting is imbedded in original seed material,
Being subsequently placed in constant temperature light culture in 25 DEG C of incubators, after eating full material to mycelia, (about about 25 days) i.e. obtain original seed;
7) production kind is made: aseptic for original seed access produced in kind of bag, be placed in constant temperature light culture in 25 DEG C of incubators, to bacterium
Silk i.e. obtains production kind after eating full material;
8) inoculation cultivation: by the aseptic access cultivation material bag of production kind, at 25 DEG C ± 1 DEG C, relative air humidity 60-70%
Culturing room in lucifuge cultivate, after the mycelia in cultivating bag covers with planting material (about 30-35d), continue shading After-mature cultivation
15d completes latter stage of ripening to mycelia, cuts the collar and always plants block;
9) going out sesame management: regulation temperature is 20-21 DEG C, humidity 85-90%, illumination 100-200lux, stuffiness keeps
CO2Content at 2000ppm up to grow former base, regulation temperature is 26-28 DEG C, relative air humidity 85-90%, illumination about
200-300lux, stuffiness, continues to keep CO2Content, at more than 2000ppm, treats that stem terminates and enter cap growth elongating stage
During the phase, ventilation, adjust temperature and be 26-28 DEG C, humidity 85-95%, illumination 300-500lux, ventilation, keep CO2Content is less than
1200ppm, when the white edge to cap turns yellow completely, plucks.
The plain boiled pork Ganoderma that the present embodiment cultivation obtains is as shown in Figure 1.
Embodiment 2: antivirus test
1, reagent and instrument
African green monkey kidney cell (African green monkeykidney, Vero) (ATCC CCL-81TM) derives from
American Type Culture Collecti (ATCC);
HSV-1 virus is given in COHEN and EISENBERG (University of Pennsylvania), in being stored in
Academy of science of state Guangzhou biological medicine and health research institute laboratory;
Dulbecco ' s Modified Eagle Media (DMEM) culture fluid, pancreatin and ethylenediaminetetraacetic acid (EDTA);
Tissue culturing plate;Hyclone (FBS);Tetrazolium bromide (MTT) dimethyl sulfoxide (DMSO) is and is purchased;
Olympus IX71 microscope;Synergy HT multi-functional microwell plate detector.
2, test method
The acquisition of 2.1 ethanol extractions: after sample to be extracted is pulverized with pulverizer, carry 80 DEG C of backflows with 95% ethanol
Take 3 hours, 2 hours and 1 hour, merge three rotated evaporation and concentration of filtrate to paste, less than 60 DEG C drying, obtain to be extracted
The ethanol extraction of sample.
2.2 African green monkey kidney cell cells (vero cell) are cultivated: the FBS to DMEM adding 10% mass percent cultivates
In liquid, in inoculation African green monkey kidney cell to culture fluid, inoculum density is every milliliter 1.5 × 105Individual, by the cell suspension of inoculation
It is inoculated in 96 hole tissue culturing plates, every hole 100 μ L, is placed in 37 DEG C, 5%CO2Incubator for tissue culture in cultivate 24h;
2.3 viral suppression assay methods:
The ethanol extraction DMEM culture fluid doubling dilution treating side sample is become 5 concentration (respectively 5000ug/ml,
2500ug/ml, 1250ug/ml, 625ug/ml and 312.5ug/ml), obtain the sample liquid diluted, positive control ACV is
The antiviral drugs acyclovir of 12.5ug/ml concentration), negative control is the culture fluid (blank solvent) being added without extract.
Take the logarithm trophophase and the good Vero cell of growth conditions with 1.5 × 104The density in individual/hole inoculates 96 porocytes
Culture plate, incubated overnight, to growing up to cell monolayer.
Virus HSV-1 DMEM culture fluid is diluted to 0.5MOI/50ul solution.Divide in cell monolayer obtained above
The sample liquid that Jia Ru not dilute or comparison, every hole adds 50ul, immediately adds the virus liquid diluted, every hole 50ul.
Shaken well gently.37 DEG C, 5%CO2Incubator carries out the detection of β-Gal glycosidase activity after cultivating 24h.
β-Gal glycosidase activity detects: inhales and abandons mixed liquor, and every hole adds 100ul 1%NP40 cell pyrolysis liquid, places 5-
10min, cross vibrates three times halfway.Every hole is drawn 50ul lysate and is added on 96 hole ELISA Plate or 96 porocyte culture plates, with
β-Gal Activity determination the substrate buffer solution of rear addition 50ul, vibrates mixing gently.570nm dynamic optical is carried out in super microplate reader
The mensuration absorbed.Condition determination is 37 DEG C, surveys 570nm absorbance value, and once, continuous 25 readings, midway shakes every 2min reading
Swing.Result unit is milli-optical density units per min (mOD/min).Calculate each dense according to formula
The degree sample ethanol extraction suppression ratio to virus:
Suppression ratio=(negative control bore measurements-sample well measured value)/negative control bore measurements × 100%
The probit regression utilizing SPSS19 calculates the half suppression to HSV-1 virus of the sample ethanol extraction
Concentration IC50.
3, suppression Viral Assay:
The sample of the present embodiment is respectively as follows:
Sample 1: embodiment 1 cultivates the plain boiled pork Ganoderma Ganoderma leucocontextum obtained;
Sample 2: the Ganoderma Ganoderma lingzhi (Guangdong, Guangdong is micro-) being purchased;
Sample 3: the Ganoderma lucidum (Leyss. Ex Fr.) Karst. Ganoderma lucidum that artificial culture obtains
Sample 4: the heavy umbrella Ganoderma Ganoderma multipileum that artificial culture obtains
Sample 5: the coppery Ganoderma Ganoderma cupreum that artificial culture obtains.
The method using the ethanol extraction of 2.1 carries out the extraction of ethanol extraction respectively respectively to sample 1 to sample 5,
Obtain the ethanol extraction of sample 1 to sample 5.
Vero cell is cultivated by the method using 2.2, then by HSV-1 virus and the Vero co-culture of cells of cultivation
After 72h, collect cell in-80 DEG C of preservations.
The method using 2.3 measures viral suppression, is calculated the half-inhibition concentration of each sample ethanol extraction
IC50, result is as shown in table 1.
The IC50 result of the ethanol extraction of 1:5 sample of table
Table 1 is it is found that the plain boiled pork Ganoderma ethanol extraction of the present invention is substantially less than spirit to the half-inhibition concentration of HSV-1
Sesame and the ethanol extraction of Ganoderma lucidum (Leyss. Ex Fr.) Karst. and the ethanol extraction of other heavy umbrella Ganoderma and coppery Ganoderma etc., with other four sample phases
Ratio, the plain boiled pork Ganoderma ethanol extraction of the present invention all demonstrates the higher rejection to HSV-1, and this shows, plain boiled pork Ganoderma
The activity of ethanol extract anti-HSV-1 virus is much larger than Ganoderma lucidum (Leyss. Ex Fr.) Karst., Ganoderma, weighs the anti-of the ethanol extraction such as umbrella Ganoderma and coppery Ganoderma
The activity of HSV-1 virus.
The above, the only preferably specific embodiment of the present invention, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope that the invention discloses, according to technical scheme and
Conceive in addition equivalent or change, all should contain within the scope of the present invention.