CN106432400B - Application and its preparation of the peroxy-ergosterol in the medicine that resisiting influenza virus infects - Google Patents
Application and its preparation of the peroxy-ergosterol in the medicine that resisiting influenza virus infects Download PDFInfo
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- CN106432400B CN106432400B CN201610806692.8A CN201610806692A CN106432400B CN 106432400 B CN106432400 B CN 106432400B CN 201610806692 A CN201610806692 A CN 201610806692A CN 106432400 B CN106432400 B CN 106432400B
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- 210000004969 inflammatory cell Anatomy 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- DBTMGCOVALSLOR-VPNXCSTESA-N laminarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1O[C@@H]1[C@@H](O)C(O[C@H]2[C@@H]([C@@H](CO)OC(O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-VPNXCSTESA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- PGZUMBJQJWIWGJ-ONAKXNSWSA-N oseltamivir phosphate Chemical compound OP(O)(O)=O.CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 PGZUMBJQJWIWGJ-ONAKXNSWSA-N 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000003909 pattern recognition Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000003726 plant lectin Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
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- 210000002345 respiratory system Anatomy 0.000 description 1
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- 239000000377 silicon dioxide Substances 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 229940061367 tamiflu Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/19—Acanthaceae (Acanthus family)
- A61K36/195—Strobilanthes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Steroid Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The present invention provides peroxy-ergosterol and is preparing the application in being used to prevent or treat the medicine of influenza infection, while also provides a kind of method that peroxy-ergosterol is separated from Radix Isatidis.The present invention provides application of the micromolecular compound peroxy-ergosterol in the medicine for preparing prevention or treatment influenza infection, so that for clinically the prevention and treatment of influenza provide a kind of effective medicine now.Present inventor has found that peroxy-ergosterol can effectively inhibit the pattern recognition receptors RIG I that influenza virus inducing host cell is overexpressed cause of disease identification；So as to the abnormal immune inflammation of suppression mode identification receptor downstream signal transduction mediation and the effect of mediating apoptosis.
Description
Technical field
The invention belongs to pharmaceutical technology field, more particularly to peroxy-ergosterol is preparing treatment or prevention influenza virus sense
Application in the medicine of dye, the method for further relating to separate peroxy-ergosterol from Radix Isatidis.
Background technology
Influenza virus (Influenza Virus) is enveloped RNA virus, belongs to orthomyxoviridae family
(Orthomyxoviridae), it is divided into tri- type of A, B, C, also referred to as first, second, the third three types.The long 13.6kb of influenza virus full genome, by
8 differ in size sub-thread strand RNA composition, be separately encoded 10 kinds of structural proteins (PB2, PB1, PA, HA, NP, NA, M1, M2,
PB1-F2 and NS2/NEP) and non-structural protein (NS1).The glycoprotein phytohemagglutin phytolectin (HA) of influenza surface and host are thin
Cellular surface sialic acid receptor mediates the endocytosis of influenza particles with reference to after and establishes influenza infection.The glycoprotein plant of avian influenza virus
Hemagglutinin (HA) is combined with sialic acid (sialic Acid, SA) a2-3Ga1 acceptors；Human influenza virus and SAa2-6Ga1 acceptor knots
Close.Bird flu causes avian influenza virus to be difficult to travel in crowd with the otherness that human influenza combines not isoacceptor.By influenza
Acute upper respiratory tract communicable disease caused by virus, has the characteristics that infectiousness is strong, spread speed is rapid, easily infects.
Since 20th century, influenza virus causes to be very popular several times in the world, such as：1918 " spanish influenza "；1957
Year " Asia influenza "；Nineteen sixty-eight " Mao flu ", each big influenza cause huge loss in the scope in the whole world.Exist recently
The popular bird flu H7N9 in Deposits in Eastern Coastal China area, is through counting 658 infection and death 221, the death rate
33.5%.As a kind of viral infectious, not only serious threat public health to influenza, but also to country and society
Bring serious financial burden.Since the antigenic variation of influenza virus is very competent and quite frequent, the development and life of vaccine
Production relatively lags behind, not high for the protective rate of Susceptible population.Current American Food and Drug Administration (FDA) approval is used to prevent
The medicine of influenza has M2 inhibitors of ion channels：More adamantane (Amantadine), Rimantadine (Rimantadine) and nerve
Propylhomoserin enzyme inhibitor：Oseltamivir (Oseltamivir), zanamivir (Zanamivir), but exist cause influenza respectively
The produced problem of viral persister.Therefore, new flu outbreak is likely to break out at any time.In this case, find
Tamiflu with preventive and therapeutic action be just particularly important with it is urgent.
At present, more research shows that the infection of influenza virus causes host immune system excessive activation, causes excessive inflammation
It is one of its main mechanism of causing a disease that the expression of the factor, which causes tissue damage,.Such as：The big influenza of Spain of 1918 is caused a disease with height
Higher incidence caused by H 5 N 1 avian influenza and case fatality rate cause then to occur " scorching with early stage recruitment to pulmonary inflammatory cell
Disease storm " is directly related.Infect 09H1N1 or HPAI (High Pathogenic AI) virus H5N1, H7N9 case produces severe viral lung
It is scorching.High expression inflammatory factor and chemotactic factor (CF) in 09H1N1 cases of infection：IP-10, MCP-1 and MIP-1.According to existing report,
Influenza causes " inflammatory factor storm " to produce and cause immunity of organism to damage, and therefore, control host body properly effectively removes disease
Former immune response, prevent too drastic immune this link of caused immunologic mjury, searches out effective modulate host inflammation
Medicine is very necessary.Influenza infection cause injury of lungs histopathology and Clinical symptoms show alveolar epithelial cells apoptosis,
Necrosis, alveolar septum collapses and serious hypoxemia, this is similar to the feature of ARDS patient.Research report recently, acute
Intrapulmonary expressing promoting apoptosis ligand FasL and TRAIL are the masters for inducing lung pathology damage in the progress of Respiratory Distress Syndrome(RDS) (ARDS)
Want factor.Antiapoptotic factors TRAIL, which is mainly derived from, to be raised to the monocyte and macrophage population of lung.TRAIL is overexpressed
In addition to mediation influenza causes injury of lungs, it can also be conducive to cause subsequent Secondary bacterial infections.Clinically apply now
Glucocorticoid has the function that to suppress immune response, anti-inflammatory etc..It is applied to the treatment of ARDS patient, can reduce inflammation and face
The symptom of bed.In addition, glucocorticoid prolonged application also has preferable effect in chronic inflammation COPD and asthma.Sugared cortical hormone
Element is also applied to acute inflammation display caused by influenza H1N1 infection and reduces injury of lungs and multiple organ failure.But
2009H1N1 influenza pandemic application glucocorticoid treatments but improve the case fatality rate of patient.Therefore, carried out using glucocorticoid
The effect of causing the treatment of immune inflammation to influenza still has to be evaluated.
Radix Isatidis is divided into south, north Radix Isatidis, is respectively Acanthaceae and Cruciferae, bitter cold in nature, has clearing heat and detoxicating, cool
The effect of blood relieving sore-throat, be clinical very common traditional resisiting influenza virus Chinese medicine.But effective the anti influenza composition and medicine of Radix Isatidis
The reason mode of action is still unclear.We separate the steroid obtained from rhizoma et Radix Baphicacanthis Cusiae first：Peroxy-ergosterol.Arrive
So far, the effect on peroxy-ergosterol in terms of the inflammatory reaction of influenza A virus mediation is suppressed is not found
Report.Pharmacological action according to existing report peroxy-ergosterol includes having antitumor, anti-inflammatory isoreactivity.The present invention first from
Separation obtains peroxy-ergosterol and is first applied to the peroxy-ergosterol separated in rhizoma et Radix Baphicacanthis Cusiae in rhizoma et Radix Baphicacanthis Cusiae
The treatment of influenza infection disease.
The content of the invention
The present invention provides peroxy-ergosterol and is preparing the application in being used to prevent or treat the medicine of influenza infection,
So as to for clinically the prevention and treatment of influenza provide a kind of effective medicine now.
Further, peroxy-ergosterol should in the medicine for preparing the inflammatory reaction that can suppress influenza A virus mediation
With.
Peroxy-ergosterol has as follows with regard to structural formula：
Further, the peroxy-ergosterol can be bought by commercial channel and be obtained, can also be by being produced from natural
Extraction separation is made in thing.As a kind of embodiment, peroxy-ergosterol can be separated from Radix Isatidis and obtained, preferably
, the Radix Isatidis is rhizoma et Radix Baphicacanthis Cusiae.
The present invention also provides a kind of method that peroxy-ergosterol is separated from Radix Isatidis, include the following steps：
S1：By the ethanol water heating and refluxing extraction that chromatogram of Radix Isatidis fragment volumetric concentration is 60~90%, extraction
Number can be one or many, and extracting solution is condensed into medicinal extract；
S2：Medicinal extract is dissolved into mixed liquor with water, water layer and chloroform layer is obtained with chloroform extraction mixed liquor, chloroform layer is depressurized
It is concentrated to give chloroform layer medicinal extract；Extraction times can be 1 time or repeatedly；
S3：Chloroform layer medicinal extract is added in silica gel, carrying out gradient elution collection with ethyl acetate-light petrol contains peroxide wheat
The cut of angle sterol simultaneously concentrates removal solvent.
Further including step S4, the step S4 is：Products therefrom in step S3 is dissolved with water, is added to anti-phase C18 columns
In, gradient elution is carried out with methanol-water, collects the cut containing peroxy-ergosterol.
As a kind of embodiment, when step S3, S4 is eluted, qualitative point is carried out to eluent with TLC
Analysis, required cut is collected according to TLC analysis results.
As a kind of embodiment, the Radix Isatidis is rhizoma et Radix Baphicacanthis Cusiae.
As a preferred embodiment, the silica gel is purification on normal-phase silica gel.
Preferably, in step S3, ethyl acetate-light petrol carries out gradient elution according to 9: 1~5: 5 volume ratio；More
Preferably, ethyl acetate-light petrol is eluted according to 9: 1,8: 2,7: 3,6: 4,5: 5 five gradients of volume ratio.
Preferably, in step S4, methanol-water carries out gradient elution according to 1: 9~6: 4 volume ratio；It is more highly preferred to, first
Alcohol-water is eluted according to volume ratio 1: 9,2: 8,3: 7,4: 6,5: 5,6: 4 six gradients.
The present invention also provides a kind of medicine for being used to preventing or treating influenza infection, the active ingredient of the medicine includes
Peroxy-ergosterol.Peroxy-ergosterol can be used as sole active agent, can also be combined with the material of other medicinal licenses.
As a kind of embodiment, pharmaceutically acceptable any formulation can be made in the formulation of the medicine, be
Formulation needed for preparing, the medicine can add the auxiliary material or carrier pharmaceutically allowed.Formulation is, for example, tablet, hard shell capsules, soft
The formulations such as capsule, parenteral solution, granule, dripping pill.Required pharmaceutically acceptable carrier or auxiliary material, it is specific as pharmaceutically commonly used
Diluent and absorbent, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, carbon
Sour calcium, white bole, microcrystalline cellulose, alumina silicate etc.；Pharmaceutically common wetting agent and adhesive, such as water, glycerine, poly- second two
Alcohol, ethanol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, carboxymethyl cellulose
Sodium, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.；Pharmaceutically common disintegrant, such as dry starch, sea
Alginates, agar powder, laminaran, sodium acid carbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, 12
Sodium alkyl sulfonate, methylcellulose, ethyl cellulose etc.；Disintegration inhibitor, for example, sucrose, glyceryl tristearate, cocoa butter,
Hydrogenated oil and fat etc.；Lubricant, such as talcum powder, silica, cornstarch, stearate, boric acid, atoleine, polyethylene glycol
Deng.No longer enumerate on pharmaceutically acceptable carrier or auxiliary material, those of ordinary skill in the art can be according to being grasped
Common knowledge carries out specifically chosen.
As a kind of embodiment, the peroxy-ergosterol in medicine obtains to be separated from Radix Isatidis, it is preferred that
The Radix Isatidis is rhizoma et Radix Baphicacanthis Cusiae.Peroxy-ergosterol in medicine can be bought by commercial channel and be obtained, or can be according to
Method as discussed above is made.
The present invention provides micromolecular compound peroxy-ergosterol to prepare the medicine of prevention or treatment influenza infection
Application in thing, so that for clinically the prevention and treatment of influenza provide a kind of effective medicine now.Present inventor adopts
Confirmed with fluorescence real-time quantitative PCR, peroxy-ergosterol can substantially suppress Influenza virus H1N1 infection A549 inductions pair
The overexpression of the pattern recognition receptors RIG-I of cause of disease identification；Peroxy-ergosterol can substantially suppress Influenza virus H1N1
The overexpression of the inflammatory factor (IL-6, IP-10, IL-8, IFN-lambda, IFN-β, TNF-a) of A549 cells induction is infected, is in
Dose-dependence；Peroxy-ergosterol can substantially suppress the Influenza virus H1N1 infection apoptosis-induced factor of A549 cells
The unconventionality expression of TRAIL.
Compared with existing medicine, prevention or treatment influenza infection medicine are prepared at least with as follows using peroxy-ergosterol
Outstanding advantage：1st, peroxy-ergosterol can effectively inhibit the pattern knowledge that influenza virus inducing host cell is overexpressed cause of disease identification
Other acceptor RIG-I；So as to the abnormal immune inflammation and mediating apoptosis of the mediation of suppression mode identification receptor downstream signal transduction
Effect.2nd, traditional treatment influenza Radix Isatidis is come from preferred solution, peroxy-ergosterol separation, it is secondary without obvious poison
Effect.
Brief description of the drawings
Fig. 1 peroxy-ergosterols suppress influenza induction A549 cells and are overexpressed pattern recognition receptors RIG-I；
Fig. 2 peroxy-ergosterols suppress the unconventionality expression of influenza induction A549 cellular inflammation genes；
Fig. 3 peroxy-ergosterols suppress influenza infection induction A549 cell expression antiapoptotic factors TRAIL.
Embodiment
Content for a better understanding of the present invention, below in conjunction with the accompanying drawings with specific embodiment to technical scheme
It is described further, but the protection content of the present invention is not limited to following embodiments.
Peroxy-ergosterol in following embodiments can be obtained by commercial channel, can also pass through following examples one
Method be made.
Embodiment one prepares rhizoma et Radix Baphicacanthis Cusiae peroxy-ergosterol compound
Step S1：Extraction
3Kg rhizoma et Radix Baphicacanthis Cusiae medicinal materials are cut into pieces, are heated to reflux extracting three respectively with the ethanol water of 80% (volume)
Secondary, the volume of each ethanol is 30L, 24L, 18L, obtains ethanol extract, merges ethanol extract, and ethanol is recovered under reduced pressure in heating
Obtain total extract medicinal extract.
Step S2：Extraction
Gained medicinal extract is mixed with water, and heating stirring, using the chloroform extraction gained mixed liquor of 1L obtain for 3 times water layer and
Chloroform layer, then extracts gained water layer 3 times using 1L ethyl acetate, obtains ethyl acetate layer and water layer.Respectively merge chloroform layer and
Ethyl acetate layer, and solvent is recovered under reduced pressure with heating and obtains chloroform layer medicinal extract and ethyl acetate layer medicinal extract.
Step S3：Purification on normal-phase silica gel processing
The chloroform layer medicinal extract of gained is added in silica gel, is carried out using the ethyl acetate-light petrol of volume ratio 9: 1~5: 5
Gradient elution, has carried out five gradients in the present embodiment, has been followed successively by 9: 1,8: 2,7: 3,6: 4,5: 5 altogether, each elution ladder
The elution amount of degree is 2L.By TLC analysis shows that determining to be divided into 13 cuts, and solvent is recovered under reduced pressure with heating and obtains 13 admittedly
Body residue.
Step S4：Anti-phase C18 processing
(1.8g) is dissolved with water and added in anti-phase C18 columns after No. 5 cuts are mixed with No. 6 cuts, is pressed using methanol-water
Gradient elution is carried out according to volume ratio 1: 9~6: 4, the present embodiment shares six gradients, is followed successively by 1: 9,2: 8,3: 7,4: 6,5
: 5,6: 4, the elution amount of each gradient is 1L, is shown by TLC, determines the cut corresponding to peroxy-ergosterol, this is evaporated
Divide and concentrated, is dry, obtain white powder (1.0mg).After testing, its purity is 95%.
The white powder of gained is detected, its structure elucidation is as follows：
1H-NMR spectrums show proton δ 3.97 (1H, M, H-3), δ 1.00 (3H, D, J=6.6Hz, H-21), δ 0.91 (D, J
=7.2Hz, 3H, H-28), δ 0.89 (S, 3H, H-19), δ 0.83 (D, J=6.6Hz, 3H, H-26), δ 0.82 (D, J=7.2Hz,
3H, H-26), and δ 0.82 (S, 3H, H-18), this shows that the compound should be sterol.
Simultaneous two double bond characteristic signals in 1H-NMR spectrums：δ 5.14 (1H, dd, J=15.6,7.8Hz, H-
22), δ 5.23 (1H, dd, J=15.6,7.8H, H-23), δ 6.51 (1H, d, J=9.0Hz, H-6), δ 6.25 (1H, d, J=
9.0Hz, H-7), in HMBC with carbon δ 132.3 (C-23), δ 135.2 (C-22), δ 135.39 (C-6), δ 130.68 (C-7) phase
Close.Two methene proton signals are in δ 1.94 (1H, m, H-4a) and 2.11 (1H, m, H-4b) and two carbon in H M B C
The significant correlation of signal δ 82.2 (C-5) and 135.4 (C-6) prompt the compound that there are 5 α, 8 α-epidioxy systems.At the same time
It is δ 82.2 (C-5) remote related that methylene signals δ 0.89 (s, 3H, H-19) is moved to carbon potential.Proton δ 1.94 (1H, m, H-4a)
Have correlation with 2.11 (1H, m, H-4b) and with carbon δ 79.4 (C-8) non-correlation.
In addition, olefinic protons δ 6.25 (1H, D, J=9.0Hz, H-7) and the carbon δ 51.1 in 5 α, 8 α-epidioxy systems
(C-9) it is related to 51.7 (C-14), and another olefinic protons δ 6.51 (1H, d, J=9.0Hz, H-6) and carbon δ 37.0 (C-4)
It is related to 36.9 (C-10).
After testing and structure elucidation, determine that all data of products therefrom are consistent with peroxy-ergosterol, its structure
It is as follows：
The influence of the infection induced host expresses pattern recognition receptors RIG-I of embodiment diperoxy ergosterol infected by influenza
1. experiment material：
1.1 medicine given the test agent
Peroxy-ergosterol (prepared by embodiment one), its purity is detected as 95% through HPLC.
1.2 cells and strain
A549 cells are purchased from ATCC, are stored in this laboratory；PR8 plants of H1N1virus (A/PR/8/34,
H1N1), it is stored in this laboratory
1.3 reagent
Reverse transcription reaction kit (is purchased from Takara companies；Article No.：RR036A)；(the purchase of real-time quantitative PCR reaction kit
From Takara companies；Article No.：RR390A)
2. experimental method
Include the following steps：
1) by A549 cells with 5X106Cell density kind to 6 orifice plates, be positioned over 37 DEG C of temperature, 5%CO2Incubator
In；
2) after being incubated overnight cell attachment, former culture medium is discarded, PBS is added and washs 2 times.Then add containing influenza virus A/
DMEM/DF12 (1: 1) culture medium adherent cell 2h of PR8/3/4 (H1N1) serum-free；Then, cell is washed into removing with PBS
Unadsorbed virus；
3) experimental group setting is carried out, including：Normal group, viral group, pharmaceutical intervention group and positive drug Oseltamivir compare
Group.Influenza virus A/PR8/3/4 (H1N1) (MOI=0.1) is diluted with DMEM/DF12 (1: 1) culture medium of serum-free and is added to
Adherent cell 2h is carried out in cell；Then, cell is washed with PBS and removes unadsorbed virus.Wherein pharmaceutical intervention group uses
Mode of drug, pharmaceutical intervention group addition various concentrations peroxy-ergosterol (PR8+ peroxy-ergosterols (150 μ g/ml),
PR8+ peroxy-ergosterols (300 μ g/ml))；Viral group is not added with medicine and is intervened；Positive drug Oseltamivir medicine compares
Group, adds the Oseltamivir of 300 μ g/ml.
4) continue culture 24 it is small when after, discard in 6 orifice plates after cells and supernatant, cold PBS is washed 2 times, adds 1ml
Trizol (Life technologies companies) is simultaneously incubated at room temperature 5min；It is (optional：Or go in 1.5mlEP pipes, in-
Preserved in 80 ° of refrigerators；Or directly extracted)；
5) subsequently carry out in accordance with the following steps：
1. adding 200ul chloroforms, 15sec is shaken, 2-3min is incubated in room temperature；It is placed in 4 DEG C of centrifuges and is centrifuged：
13000rpm x 15min；
2. centrifugation is finished, supernatant is transferred to new 1.5EP manages, and adds 500ul isopropanols；10min is incubated at room temperature；It is placed in
Centrifuged in 4 DEG C of centrifuges：12000rpm x 10min；
3. centrifugation is finished, add 75% ethanol of 1ml and washed once, be placed in 4 DEG C of centrifuges and centrifuged：7500rpm x
10min；
4. supernatant discarding, room temperature places 5-10min, adds no RNase water dissolving RNA, carries out reverse transcription at once into cDNA.
5. the concentration (ng/ μ l) of total serum IgE is measured in spectrophotometer and calculates the volume of total serum IgE：It is anti-to intend addition reverse transcription
The amount for answering total serum IgE in system is 1000ng, then the volume V=1000/RNA concentration of total serum IgE needed for calculating；
6. mRNA reverse transcriptions configure the following (kit of reverse transcription reaction system into cDNA：Takara RR036A)：
| Reagent | Volume |
| 5xPrimeScript RT Master Mix | 4ul |
| Total serum IgE | V |
| DH2O | 16-V |
| System cumulative volume | 20ul |
7. carrying out reverse transcription reaction, reaction condition is as follows：
| 37℃ | 15min |
| 85℃ | 5Sec |
| 4℃ | ∞ |
Sample is collected to carry out subsequent quantitation PCR experiment or be stored in -20 DEG C.
8. real-time quantitative PCR reaction kit (article No.：Takara RR390A)
It is as follows to configure reaction system：
Carry out Real time PCR reaction conditions (ABI7500)
Primer sequence and probe sequence are as follows：
Experimental result：Experimental result is referring to Fig. 1, and as seen from the figure, peroxy-ergosterol can substantially suppress Flu-A disease
Overexpression of the malicious H1N1 infection A549 inductions to the pattern recognition receptors RIG-I of cause of disease identification.
The influence of the infection induced host expresses inflammation gene expression of three peroxy-ergosterol infected by influenza of embodiment
1. experiment material：
1.1 medicine given the test agent
Peroxy-ergosterol (prepared by embodiment one).
1.2 cells and strain
A549 cells are purchased from ATCC, are stored in this laboratory；PR8 plants of H1N1virus (A/PR/8/34,
H1N1), it is stored in this laboratory.
1.3 reagent
Reverse transcription reaction kit (is purchased from Takara companies；Article No.：RR036A)；(the purchase of real-time quantitative PCR reaction kit
From Takara companies；Article No.：RR390A)
2. experimental method,
Include the following steps：
1) by A549 cells with 5X106Cell density kind to 6 orifice plates, be positioned over 37 DEG C of temperature, 5%CO2Incubator
In；
2) after being incubated overnight cell attachment, former culture medium is discarded, PBS is added and washs 2 times.Then add containing influenza virus A/
DMEM/DF12 (1: 1) culture medium adherent cell 2h of PR8/3/4 (H1N1) serum-free；Then, cell is washed into removing with PBS
Unadsorbed virus；
3) experimental group setting is carried out, including：Normal group, viral group, pharmaceutical intervention group and positive drug Oseltamivir compare
Group.Added to carefully with DMEM/DF12 (1: 1) culture medium dilution influenza virus A/PR8/3/4 (H1N1) (MOI=0.1) of serum-free
Adherent cell 2h is carried out in born of the same parents；Then, cell is washed with PBS and removes unadsorbed virus.Wherein pharmaceutical intervention group uses medicine
Thing treatment mode, pharmaceutical intervention group add various concentrations peroxy-ergosterol (PR8+ peroxy-ergosterols (150 μ g/ml), PR8+
Peroxy-ergosterol (300 μ g/ml))；Viral group is not added with medicine and is intervened.Positive drug Oseltamivir drug control group, adds
Enter the Oseltamivir of 300 μ g/ml.
4) continue culture 24 it is small when after, discard in 6 orifice plates after cells and supernatant, cold PBS is washed 2 times, adds 1ml
Trizol (Life technologies companies) is simultaneously incubated at room temperature 5min；It is (optional：Or go in 1.5mlEP pipes, in-
Preserved in 80 ° of refrigerators；Or directly extracted)；
The cell total rna subsequently carried out extracts, mRNA reverse transcriptions are implemented into eDNA and real-time quantitative PCR reaction reference
The corresponding steps of example two carry out, and repeat no more；Wherein, the present embodiment primer sequence and probe sequence in real-time quantitative PCR reaction
Row are as follows：
3. experimental result
As a result such as Fig. 2 is shown, influenza A/PR8/3/4 (H1N1) infection A549 cells carry out medicine peroxy-ergosterol intervention
Afterwards, hence it is evident that suppress the gene of inflammatory factor IL-6, IL-8, IP-10, IFN-beta, IFN-lambdal, TNF-a of influenza induction
Horizontal expression.
The expression of the infection induced host's apoptogene TRAIL of example IV peroxy-ergosterol infected by influenza influences
1. experiment material：
1.1 medicine given the test agent
Peroxy-ergosterol (prepared by embodiment one)
1.2 cells and strain
A549 cells are purchased from ATCC, are stored in this laboratory；PR8 plants of H1N1virus (A/PR/8/34,
H1N1), it is stored in this laboratory.
1.3 reagent
Reverse transcription reaction kit (is purchased from Takara companies；Article No.：RR036A)；(the purchase of real-time quantitative PCR reaction kit
From Takara companies；Article No.：RR390A)
2. experimental method
In accordance with the following steps：
1) by A549 cells with 5X106Cell density kind to 6 orifice plates, be positioned over 37 DEG C of temperature, 5%CO2Incubator
In；
2) after being incubated overnight cell attachment, former culture medium is discarded, PBS is added and washs 2 times.Then add containing influenza virus A/
DMEM/DF12 (1: 1) culture medium adherent cell 2h of PR8/3/4 (H1N1) serum-free；Then, cell is washed into removing with PBS
Unadsorbed virus；
3) experimental group setting is carried out, including：Normal group, viral group, pharmaceutical intervention group and positive drug Oseltamivir compare
Group.Added to carefully with DMEM/DF12 (1: 1) culture medium dilution influenza virus A/PR8/3/4 (H1N1) (MOI=0.1) of serum-free
Adherent cell 2h is carried out in born of the same parents；Then, cell is washed with PBS and removes unadsorbed virus.Wherein pharmaceutical intervention group uses medicine
Thing treatment mode, medicine group intervention group addition various concentrations peroxy-ergosterol (PR8+ peroxy-ergosterols (150 μ g/ml),
PR8+ peroxy-ergosterols (300 μ g/ml))；Viral group is not added with medicine and is intervened.Positive drug Oseltamivir medicine compares
Group, adds the Oseltamivir of 300 μ g/ml.
4) continue culture 24 it is small when after, discard in 6 orifice plates after cells and supernatant, cold PBS is washed 2 times, adds 1ml
Trizol (Life technologies companies) is simultaneously incubated at room temperature 5min；It is (optional：Or go in 1.5mlEP pipes, in-
Preserved in 80 ° of refrigerators；Or directly extracted)；
Follow-up cell total rna extracts, mRNA reverse transcriptions are equal with reference to embodiment two into cDNA and real-time quantitative PCR reaction
Corresponding steps carry out, repeat no more；Wherein, the present embodiment antiapoptotic factors TRAIL used in real-time quantitative PCR reaction draws
Thing sequence and probe sequence are as follows：
3. experimental result
The results are shown in Figure 3, rhizoma et Radix Baphicacanthis Cusiae peroxy-ergosterol after intervening the obvious apoptosis for suppressing influenza virus induction because
The gene expression dose of sub- TRAIL, and be in dose-dependence.
Above experimental result surface, peroxy-ergosterol can substantially suppress Influenza virus H1N1 infection A549 inductions
To the overexpression of the pattern recognition receptors RIG-I of cause of disease identification；Peroxy-ergosterol can substantially suppress influenza A virus
The inflammatory factor (IL-6, IP-10, IL-8, IFN-lambda, IFN-β, TNF-a) of H1N1 infection A549 cell inductions crosses table
Reach, in dose-dependence；Peroxy-ergosterol can substantially suppress Influenza virus H1N1 infection A549 cell inductions and wither
Die the unconventionality expression of factor TRAIL.Peroxy-ergosterol can be applied to the medicine for preparing prevention or treatment influenza infection, from
And for clinically the prevention and treatment of influenza provide a kind of effective medicine now.
Compared with existing medicine, prevention or treatment influenza infection medicine are prepared at least with as follows using peroxy-ergosterol
Outstanding advantage：Peroxy-ergosterol can effectively inhibit the pattern-recognition that influenza virus inducing host cell is overexpressed cause of disease identification
Acceptor RIG-I；So as to the abnormal immune inflammation and mediating apoptosis of the mediation of suppression mode identification receptor downstream signal transduction
Effect.
Common knowledge or routine techniques hand not specified in text, that to be those of ordinary skill in the art grasp according to it
Section can be appreciated or know, this is no longer repeated one by one.
The above described is only a preferred embodiment of the present invention, limitation in any form is not done to the present invention, therefore
All contents without departing from technical solution of the present invention, the technical spirit according to the present invention any are simply repaiied to made for any of the above embodiments
Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.
Claims (2)
1. the method for peroxy-ergosterol is separated from Radix Isatidis, it is characterised in that include the following steps：
S1：By the ethanol water heating and refluxing extraction that chromatogram of Radix Isatidis volumetric concentration is 60~90%, extracting solution is concentrated
Into medicinal extract；
S2：Medicinal extract is dissolved into mixed liquor with water, water layer and chloroform layer is obtained with chloroform extraction mixed liquor, chloroform layer is concentrated under reduced pressure
Obtain chloroform layer medicinal extract；
S3：Chloroform layer medicinal extract is added in silica gel, carries out gradient elution with ethyl acetate-light petrol, collection contains peroxide ergot
The cut of sterol simultaneously concentrates removal solvent；
S4：Products therefrom in step S3 is dissolved with water, is added in anti-phase C18 columns, gradient elution is carried out with methanol-water, is received
Collect the cut containing peroxy-ergosterol.
2. according to the method described in claim 1, it is characterized in that, the Radix Isatidis is rhizoma et Radix Baphicacanthis Cusiae；In step S3, the silicon
Glue is purification on normal-phase silica gel；In step S3, ethyl acetate-light petrol is according to 9:1~5:5 volume ratio carries out gradient elution；Step S4
In, methanol-water is according to 1:9~6:4 volume ratio carries out gradient elution.
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Address after: No. 151 Yanjiang Road, Yuexiu District, Guangzhou, Guangdong Co-patentee after: Guangzhou Respiratory Health Research Institute Patentee after: the First Affiliated Hospital of Guangzhou Medical University Co-patentee after: Macao University of Science and Technology Address before: 510120 151 Yanjiang Road, Yuexiu District, Guangzhou, Guangdong Co-patentee before: Guangzhou Institute of Respiratory Disease Patentee before: the First Affiliated Hospital of Guangzhou Medical University Co-patentee before: Macao University of Science and Technology |