CN103479758B - Resisiting influenza virus medical composition containing garden burnet - Google Patents

Abstract

The present disclosure generally relates to a kind of medical composition of resisiting influenza virus, this medical composition includes garden burnet (S.officinalis L.) extract of therapeutically effective amount, or the salt being subjected on reactive compound, its hydrolysate or the medicine isolated by garden burnet extract.The present invention also relates to garden burnet extract or its reactive compound as the application for preparing anti-influenza virus medicament.

Description

Resisiting influenza virus medical composition containing garden burnet

Technical field

The present disclosure generally relates to a kind of medical composition of resisiting influenza virus, particularly includes garden burnet extract or its activity The medical composition of compound.The present invention also relates to garden burnet extract or its reactive compound is used as and prepares anti-influenza virus medicament Application.

Background technology

Influenza virus (abbreviation influenza virus) is one of virus of most threatening property in human life, from 20th century It is very popular with regard to four influenzas occur, is 1918~1919 years (spanish influenza, H1N1), nineteen fifty-seven (Asia stream respectively Sense, H2N2), nineteen sixty-eight (Mao flu, H3N2) and 1977 (Russian influenza, H1N1).Wherein, and with Spain in 1918 Influenza is the most serious, and its H1N1 Strain is formed by bird flu and human influenza virus mutation, and 2~4 are caused in the whole world The death of million people.And new H1N1 Influenza epidemic situations are broken out in Mexico in 2009, having suspected has more than 100 ten thousand people infection, causes People's more than 10000 gets killed, (Neumann, G. in also persistently spreading at present；Noda,T.；Kawaoka,Y.Emergence and Pandemic Potential of Swine-Origin H1N1Influenza Virus.Nature.459:931-939, 2009).In addition, propose to pay special attention to the invasion and attack of seasonal A types H3N2 influenzas, this disease in national influenza prevention and control meeting in 2010 Strain is also causing many injures and deaths in the past.

Influenza virus is a kind of minus strand single-stranded RNA virus for causing the mankind to suffer from influenza with animal, is to belong to just glutinous Liquid Viraceae (Orthomyxoviridae), can according to virus nucleoprotein, inhereditary material, the difference of stromatin antigenic characteristic It is divided into tri- serotypes of A, B, C.Wherein, influenza A can cause the influenza between different hosts, because of its viral appearance Two kinds of antigen hemagglutinin (hemagglutinin, HA) and neural amino acid enzyme (neuraminidase, NA) be polymorphism, at present 16 kinds of HA (H1~16) and 9 kinds of NA (N1~N9) are had found, heredity antigenic variation can occur；Type B influenza antigen makes a variation Less can cause provincialism to infect, and for c-type influenza virus mainly using pig as host, the infection to human body is more rare, therefore on It is exactly the epidemic situation caused by influenza A to state described worldwide big influenza.

Influenza A structure can be divided into three outer membrane, stromatin and core parts from outer to inner.Outer membrane has about 500 The individual radial projection outwards arranged, it is above two antigenic type：Columnar protrusions (HA) and mushroom projection (NA).HA can be with The surface receptor absorption of many animals red blood cell causes aggegation, heavy chain and light chain can be divided into after cracking so that virus and host Cell mutually merges；NA mainly has an activity of hydrolysis sialic acid, cut-out virus with host cell is last contacts, make it is viral from The red blood cell of absorption comes off, and prevents the aggregation of virus, promotes the movement in mucus.Stromatin is made up of M1, M2, is had Protection virus core and the structure for maintaining virus.Other core is made up of 8 minus strand single-stranded RNA fragments, itself and nucleoprotein (NP) and RNA polymerase (PB1, PB2 and PA) is combined and is wound in ribosome.And why influenza virus can cause people Fear can produce new hypotype mainly due to its variation mode and let us is caught unprepared, its mutational formats has two Kind, one is antigenic drift (antigenic drift), and it refers to that the point of antigen (NA) Amino acid sequence of influenza virus sub-strain is dashed forward Small variation produced by change；Another is antigenic shift (antigenic shift), is the most common type of influenza virus variation, The reason for antigenic Big mutation rate will occur once every 10 years, cause is due to host simultaneously by two kinds of different virus strain senses Dye, viral RNA carry out genetic recombination and produce new Strain, influence it is very big (Chen Hongshan, Zhang Xingquan, antiviral drugs and its Research method, Chemical Industry Press, 328-331,2006.).

Influenza mostly occurs in autumn and winter, early spring, and its target attacked is respiratory mucosa epithelial cell, and in place Breed in chief cell, the lesion such as cause congested mucous membrane, oedema and cell degeneration, come off.In usual 1 to 3 day of incubation period, start to occur Have a fever, feel cold, having a headache, having a stuffy nose, the symptom such as Muscular stiffness, when spreading to lower respiratory tract, then may cause bronchitis and interstitial Property pneumonia, remove and stick the ability of foreign matter because influenza virus can reduce respiratory mucosa epithelial cell, therefore often result in Secondary pneumonia infects, and is that it mainly causes one of the influenza disease cause of death (Morens, D.M.；Taubenberger, J.K.；Fauci,A.S.Predominant Role of Bacterial Pneumonia as a Cause of Death in Pandemic Influenza:Implications for Pandemic Influenza Preparedness.J.Infect.Dis.198:962–970,2008)。

At present, the epidemic prevention for influenza takes cell isolate and supportive drug therapy mostly with treating, and uses and treat Precautionary approach is divided into vaccine and antiviral drugs.But the virus for making a variation increasingly, vaccine protection or risky property；And mesh Preceding antiviral drugs mainly has three kinds, and the first is M2 inhibitor (M2protein inhibitor), acts on virus and wears film egg White M2 ion channels, hinder H+ to enter viral internal so that outer virionic membrane can not merge with endosome, and virus just can not discharge RNA, this kind of medicine are Adamantan derivatives (amantadine, rimantadine)；Second is NA inhibitor (neuraminidase inhibitors), neural amino acid enzyme (NA) is acted on, virus can not be hydrolyzed sialic acid, and then can not Leave host cell, prevent virus diffusion, this kind of medicine have oseltamivir, zanamivir, peramivir and Cyclopentane or pyrrolidine derivatives；The third is RNA polymerase inhibitor, main to suppress RNA polymerase (PB1, PB2, PA) synthesize the path of virus protein, and medicine has 2'-deoxy-2'- fluoroguanosine(FdG)、T-705；Also have in addition and utilize interferon and SiRNA (small interfering RNAs) Infection (Clercq, E.D.Antiviral Agents the Active against of virus are prevented Deng therapeutic modality Influenza A Viruses.Nat.Rev.Drug.Discov.5:1015-1025,2006)。

However, above-mentioned medicine, which is all unable to reach, suppresses various types of influenza viruses comprehensively, and there are the resistance to the action of a drug and medicine The missing of side effect.The antigenic variability of influenza A is big, and infectiousness is strong, and the annual whole world there are about 500,000,000 people and infect influenza, often The very high death rate is caused, huge burden and economic loss, therefore the Tamiflu that active development is new are produced for society An actually important topic.

The content of the invention

Present invention system proposes prepare resisiting influenza virus with garden burnet (S.officinalis L.) extract medical group first Compound.

Therefore, one aspect of the present invention provides a kind of medical composition of resisiting influenza virus, and it includes the garden burnet extraction of effective dose Take thing and pharmaceutically acceptable supporting agent.

The present invention also proposes garden burnet (S.officinalis L.) extract as the application for preparing anti-influenza virus medicament.

In a specific embodiment, garden burnet extract is prepared by ethanol extraction garden burnet.

On the other hand, the present invention provides a kind of medical composition of resisiting influenza virus, and it includes the formula of effective dose (I) chemical combination The salt being subjected on thing, its hydrolysate or medicine, and pharmaceutically acceptable supporting agent：

It is susceptible as anti-current is prepared that the present invention also provides the salt being subjected on formula (I) compound, its hydrolysate or medicine The application of cytotoxic drug.

Other features of the present invention will be in clearly via described further below, each instantiation and claim It is existing.

Brief description of the drawings

Foregoing invention content and detailed description below are read in the lump with institute's accompanying drawings will more increase understanding.For up to illustrating this The purpose of invention, instantiation shown in schema are presently the preferred person.It should be appreciated, however, that the present invention be not limited to shown in it is specific Arrangement and structure.

Fig. 1 systems are the extraction procedure figure of garden burnet extract of the present invention.

Fig. 2 systems are the flow chart of the dichloromethane extract purifies and separates reactive compound according to obtained by the present invention.

Fig. 3 systems are the flow chart of the n-butyl alcohol extract purifies and separates reactive compound according to obtained by the present invention.

The specification specified of each instantiation of the present invention is as after.The further feature of the present invention will be via following each tool Detailed description and claim in body example and clearly appear from.

Need not be further elucidated above, it is believed that persond having ordinary knowledge in the technical field of the present invention is based on preceding description I.e. using the present invention to most wide degree.Accordingly, it is to be understood that the following description is merely possible to illustrate, rather than to appoint Where formula limits the scope of the invention.

Embodiment

Unless otherwise specified, all technical and scientific terms used here have the skill as belonging to the present invention In the meaning that is typically understood of usual operator.

" one " second word used herein, do not specialize such as, mean the quantity of at least one (one or more).

Its " influenza virus " system specifically described herein is influenza virus abbreviation, is to cause the mankind and animal for one kind Suffer from the minus strand single-stranded RNA virus of influenza, be to belong to Orthomyxoviridae family (Orthomyxoviridae), according to viral core Albumen, inhereditary material, the difference of stromatin antigenic characteristic, influenza A, Type B influenza virus and c-type influenza disease can be divided into Poison, it is divided into different hypotypes further according to the antigenicity of hemagglutinin and neural amino acid enzyme.According to the World Health Organization (WHO) influenza disease The name of strain includes 6 key elements：Type/host/separation area/strain sequence number/separation time (HnNn), wherein for the mankind Influenza virus, hosted information is omitted, hypotype information is omitted for B-mode and influenza virus C.It is alleged and his like susceptible in the present invention Poison includes influenza A, Type B influenza virus and c-type influenza virus.In a specific embodiment, influenza virus specifically for H1N1, H3N2 or its tool resistance to the action of a drug variant virus strain, espespecially have the variant virus strain of the resistance to the action of a drug to Tamiflu (Tamiflu).

Its " therapeutically effective amount " specifically described herein mean garden burnet extract of the present invention or formula (I) compound, its hydrolysate or The acceptable salt of medicine can produce the content needed for therapeutic effect to individual.It is familiar with the technology person and will be appreciated that an active component Or the effective dose of composition will play by ear, the including but not limited to species of such as medicine and formulation and the organism Body weight, age and health status.

Its " extract " specifically described herein refers to the product obtained by being extracted for a material, typically by will be intended to extract The material taken soaks or is mixed in the extract layer obtained in solvent.The preparation of general extract is from fresh plant or ground Or the plant sample of drying is including but not limited to impregnated with various extracting process known to this field, is percolated, is percolated, clears up again, be inverse Stream extraction, turbo-extraction, extruding/extrusion/squeezing or supercritical fluid carbon dioxide extract it.Appropriate solvent includes but unlimited In ethanol, n-hexane, methanol, dichloromethane, water, n-butanol or other solvents.Can optionally selective reagent species or tune Concentration with solvent, to be extracted up to appropriate polarity.The extract of different phase can mutually merge, also can be in subsequently entering again Row concentration step, for example, evaporation, or purifying or separating step, for example, filtering, centrifugation and chromatographic analysis.In one example, will Fresh plant or the plant sample of drying it is it is all or part of mixed (optionally through shredding or grinding) with appropriate solvent or It is soaked in wherein and stirs up to one section of time enough, carries out or heat to carry out in room temperature, solid residue is removed via filtering (filter residue), then collect obtained juice (extract)；Optionally repeat is soaked or blend step, merges gained juice, enters One step is concentrated, purified or separated.

Its " garden burnet " specifically described herein also known as beautiful drum, reddish brown, the red silk ball of acid, are rosaceous plant garden burnet Sanguisorba Officinalis L., garden burnet S.officinalisL.var.longifolia (Bert.) the Yu et Li that come into leaves dry root, System is said by Tao Hongjing：" its leaf is grown like elm, nascent step " and obtain its name, first recorded in " this warp ", it is classified as middle product.Cold nature, taste It is picric acid, nontoxic, return large intestine, liver, stomach, kidney channel, there is cooling blood and hemostasis, removing toxic substances sore and other effects.Garden burnet is herbaceos perennial. Root is in spindle, surface sepia or puce more, there is vertical wrinkle and transverse crack.Stem is upright, there is rib, and hairless or base portion has sparse Glandular hairs.Basal leaf is winglike compound leaf, 4-6 pairs of leaflet；Petiole is hairless.Spike ellipse, cylinder or ovoid, uprightly, Long 1-3 centimetres, wide 0.5-1 centimetres, purple to mulberry are open under the picture of inflorescence top；Bract 2, lanceolar, the back side and edge There is pubescence；Sepal 4, ellipse to width egg shape, aubergine；Stamen 4, filigree is thread, column cap tip dish type.Achene is contained in calyx tube It is interior, the Long Circle of falling ovate.The month at florescence 7-10, the fruiting period 9-11 months.Grassland, the patana of height above sea level 30-3000 meters are born in, mainly It is distributed in the ground such as northeast, East China, southwest and Henan, Hubei, Guangxi.Garden burnet root can be generally harvested in spring, autumn, its character For cylinder, sigmoid, long 18-22cm, diameter 0.5-2cm, the supporting root of viewable side life sometimes are slightly distorted.Matter is hard, slightly crisp, plane of rupture It is smooth, somewhat silty.Cross section cambium ring is obvious, and skin zone is faint yellow, woody part brown color or with pink, in significantly radial Arrangement.Gas is micro-, mildly bitter flavor puckery (Song Liren, China's book on Chinese herbal medicine, Shanghai science tech publishing house 4:281-286,1999).In the present invention One of in specific embodiment, be using garden burnet Sanguisorba officinalis L., it is root using base portion.

Solvent specifically described herein its " percentage " or " % " mean that solvent is dissolved in the percent by volume of water, for example, 95% second Alcohol refers to the aqueous solution containing 95 percent by volume ethanol or for example, 80% methanol refers to the water containing 80 percent by volume methanol Solution.

According to the present invention, have found unexpectedly that garden burnet (S.officinalis L.) extract have resisiting influenza virus it Effect, including but not limited to H1N1 and H3N2 influenza viruses.Therefore a kind of medical composition of resisiting influenza virus is provided, it includes The garden burnet extract and pharmaceutically acceptable supporting agent of therapeutically effective amount.

According to embodiments of the present invention, the garden burnet extract is prepared by ethanol extraction garden burnet, and it can also be included into The quintessence of one step.For example, the garden burnet extract can be prepared by the method for tool following step：

(1) garden burnet is extracted with ethanol, to obtain ethanolic extract；And

(2) with methanol and n-hexane (1:1) ethanolic extract is extracted, to obtain methanol extraction thing.

Again can be further with dichloromethane and water (1:1) the methanol extraction thing is extracted, to obtain dichloromethane extract.

According to another embodiment of the present invention, the garden burnet extract is by aforementioned methanol extract with dichloromethane and water (1:1) extract, water intaking extract is again with n-butanol and water (1:1) water extract is extracted, to obtain n-butyl alcohol extract.

In aforementioned preparation process, the ethanol system used is 50% to 99% ethanol, preferably 95% ethanol.

In aforementioned preparation process, the alkaline methanol used is 50% to 99% methanol, preferably 90% methanol.

According to the present invention, active component is further isolated and purified out by the garden burnet extract, and it is confirmed through Bioexperiment With splendid anti-influenza virus activity, or even the activity of the strains of influenza viruses with the confrontation tool resistance to the action of a drug.

Therefore, another aspect of the present invention provides a kind of medical composition of resisiting influenza virus, it include therapeutically effective amount it The salt being subjected on formula (I) compound, its hydrolysate or medicine, and pharmaceutically acceptable supporting agent：

According to the present invention, formula (I) compound can further hydrolyze and obtain its hydrolysate Mycophenolic Acid (Mycophenolic Acid), formula (I) compound and its hydrolysate are verified with it with splendid anti-influenza virus activity through Bioexperiment, even The activity of strains of influenza viruses with the confrontation tool resistance to the action of a drug.It is believed that the salt being subjected on its medicine also has anti-current susceptible certainly Cytotoxic activity.

Its " salt being subjected on medicine " used herein, which is meant, to be taken tool safety for the mankind or mammal and has The salt compounds of effect, there is the bioactivity needed for it.The salt being subjected on medicine includes but is not limited to formula (I) of the present invention The acidity or alkaline salt of compound, for example, with hydrochloric acid, hydrobromic acid, acid iodide, nitric acid, sulfuric acid, niter cake, phosphoric acid, phosphate, Acetic acid, lactic acid, salicylic acid, citric acid, tartaric acid, pantothenic acid, liquor epinephrinae bitartratis ophthalmicus, ascorbic acid, succinic acid, maleic acid, fumaric acid, Portugal Grape saccharic acid, formic acid, benzoic acid, glutamic acid, pyrovinic acid, the basic salt of p-methyl benzenesulfonic acid synthesis；Or with aluminium, calcium, lithium, magnesium, potassium, The basic salt of sodium, zinc and diethanolamine salt synthesis.

According to the present invention, the medical composition can include but is not limited to parenteral or oral administration medicine supplying.Parenteral is thrown Its form of the medical composition of medicine includes solution, suspension, emulsion, and can dissolve or be suspended in consolidating in solvent using preceding carve Body Injectable composition.The parenteral solution can be prepared in diluent by dissolving, suspending or emulsifying one or more active components.Before The example for stating diluent is for the distilled water of injection, physiological saline, vegetable oil, alcohols and combinations thereof.Also, the parenteral solution can contain There are stabilization agent, cosolvent, suspending agent, emulsifying agent, smooth agent, buffer solution, preservative agent etc..Such parenteral solution ties up to final preparation step Sterilize or prepared with sterile procedure in rapid.The medical composition of the present invention can also be formulated to sterile solid preparation, for example, by By being freeze-dried, and it can sterilize or be dissolved in sterile injectable water or other sterile diluents using preceding carve.The medicinal combination Thing also can oral administration, wherein said composition can be solid or liquid form.Solid composite include lozenge, pill, capsule, point Dissipate property pulvis, particle and the like.Orally administered composition also includes mouth-wash and sublingual tablets.Capsule includes hard shell capsules and flexible glue Capsule.In such Orally-administered solid composition, one or more reactive compounds can be mixed voluntarily, or with diluent, bonding agent, dissipate solution Agent, lubricant, stabilization agent, cosolvent mixing, then with prior art method preparation into preparation.When needed, these preparations can With coating agent, or can be coated with two or a variety of coating layers.On the other hand, liquid oral compositions include pharmaceutically acceptable Liquid solution, suspension, emulsion, syrup, medicinal liquor, and the like.In such composition, one or more reactive compounds can quilt Dissolve, suspend or be emulsified in all-purpose diluent (such as purified water, ethanol or its mixture etc.).Except such dilution Agent, foregoing also can contain wetting agent, suspending agent, emulsifying agent, sweetener, flavor enhancement, spices, preservative agent and buffer solution and Its analog.

Need not be further elucidated above, it is believed that persond having ordinary knowledge in the technical field of the present invention is based on preceding description I.e. using the present invention to most wide degree.Following embodiments are merely possible to illustrate and are used, rather than limit in any way Make remaining disclosure.

The specification specified of each instantiation of the present invention is as after.The technical characteristic of the present invention will be via following each tool Detailed description and claim in body example and become apparent from presenting.

Example

Real Examination materials

Solvent and reagent：In addition to especially indicating, composition separation agents useful for same, the solvent of embodiment, the bright chemistry of scape is purchased from Industrial group and friend and Trading Co., Ltd, and be the sterling of reagent level or liquid chromatography level.

Chromatographic material：In addition to especially indicating, the chromatographic material of embodiment is purchased from Taiwan Merck (Merck Taiwan) company and friend and Trading Co., Ltd.

(1) adsorptivity chromatographic silica gel：Silica gel 60,70-230mesh, 230-400mesh, Diaion HP-20 from Sub-exchange resin.

(2) thin-layer chromatography piece (TLC plate)：DC Kieselgel 60F254 (positive TLC chromatography sheet), DC Kieselgel RP-18F254S (anti-phase TLC chromatography sheet).

(3) the type reverse-phase chromatography post of low pressure formula carbon 18：Lichroprep RP-18,40-63 μm.

(4) the formula high-performance liquid chromatograph type chromatographic column of carbon 18：Nacalai Teaque companies produce it Cosmosil-pack, Prep-C18,10 μm, 20mm × 250mm.

Equipment and instrument

Low pressure liquid chromatography helps Pu：It is low to be used in anti-phase using the RP-SY-ICSC types low pressure side Pu of FMI companies product for system Laminate layer is analysed.

High-performance liquid chromatograph：

1. system controller：Shimadzu SCL-8A；

2. help Pu：Shimadzu LC-8A；

3. detector：Shimadzu SPD-10A UV spectrophotometric detector；

4. fraction collector：Shimadzu FCV-100B fraction collector；

5. register：Shimadzu C-R7A.

Thickener

1.Buchi B-480Waterbath；

2.Buchi R-114Rotavapor；

3.Buchi V-800Vacuum controller。

TLC spots detection：

1.UV (wavelength 360nm, shortwave 254nm)；

2.p-Anisaldehyde color reagents detect.

Nuclear magnetic resonance spectrometer：In addition to using the Varian GEMINI-300 (300MHz) of National Defense Medical Center, and entrust state Vertical Taiwan Univ.'s expensive instrument center (uses instrument on behalf of measure：Bruker Avance 500MHz FT-NMR).The hydrogen of measure Spectrum represents chemical shift, unit ppm with carbon spectrum with δ values, and works as internal standard with TMS (tetramethylsilane). S represents unimodal (singlet)；D represents doublet (doublet)；T represents triplet (triplet)；Br represents broad peak (broad)；Dd represents two groups of doublets (doublet doublet).

Melting point apparatus：Melting point compound measure system uses melting point apparatus (the non-school of Fischer-Johns companies of the U.S. Just).

Infrared spectrometer：Shimadzu FT-IR8700 types.Polyethylene (polystyrene) tuning wavelength, except special Illustrate, determined in a manner of KBr (KBr) plays ingot；And in Academia Sinica's genosome center Theremo Nicolet 380 The fourier-transform infrared linear light spectrometer measure of type.

Ultraviolet spectrogram (Ultraviolet spectra, UV) is with Shimadzu UV-160spectrophotometer Measure.

Mass spectrograph：University of communications, Tsing-Hua University and Chung Hsing University's expensive instrument center are entrusted on behalf of measure, its mass spectrograph Device be respectively Micromas TRIO-2000GC-MS, MAT-95XL High Resolution Mass Spectrometer, High Resolution Mass Spectrometer。

Cell pharmacology experiment material

Cell line (Cell line)：MDCK：Madin-Darby canine kidney cells (Madin-DarbyShi Dog renal tubular cell strain).

Cell culture fluid：

1.10%Fetal bovine serum (FBS)；

2.TPCK nutrient solutions (tosylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin)；

3.DMEM(Dulbecco’s modified Eagle medium)。

Cell physiological buffer solution：Phosphate-buffered saline(PBS).

Strain：Influenza A (H1N1/H3N2) (source：Yao Zhen culture and education is awarded, pathology portion of armed forces general hospital).

ELISA reading analyzers：BIOTEK CERES 900EIR READER.

The preparation of the extract of embodiment 1.

As shown in figure 1, garden burnet sample is ground into totally 10 kilograms of rear weighing, after being extracted at room temperature with 95% ethanol cold soaking, With the machine concentration that is concentrated under reduced pressure, ethanolic extract 3400 g of gross weight altogether is obtained.After ethanolic extract is dissolved with 90% methanol again and just Hexane is extracted with isometric ratio, obtains N-hexane extract (SAOF-H) 134.20g and methanol extraction thing.

Methanol extraction thing is extracted with isometric dichloromethane and water, obtains dichloromethane extract (SAOF-D) 159.50g.Wherein, water layer is extracted with isometric n-butanol again, and because both can not separate, n-butanol extraction is obtained after merging Take thing (SAOF-B) 3100g.

The antiviral activity experiment of the extract of embodiment 2.

Antiviral activity experiment is that the degree of host cell infected virus is assessed with the survival rate of virus host cells, and The survival rate of host cell is the method that (MTT assay) is analyzed with cell survival rate.Principle be 3- (4,5- dimethylthiazoles- 2- yls) -2,5- diphenyltetrazolium bromides (3- (4,5-dimethylthiazol-2-yl) -2,5- Diphenyltetrazolium bromide, MTT) yellow aqueous solution solid can be by the Mitochondria of living cells (mitochondria) dehydrogenation ferment (dehydrogenase) metabolism in, diazonium ring (tetrazolium ring) is reduced In purple infusible precipitate crystal formazan (3- (4,5- dimethylthiazole -2- bases) -2,5- Er Ben Ji formazan 3- (4,5- Dimethylthiazol-2-yl) -2,5-diphenyl-formazan) it is deposited in cell, because of the grain wire body ferment of living cells Element just has catalytic activity, therefore measured light absorption value can be proportional with living cells quantity, therefore the present embodiment system utilizes The number of formazan yield assesses the survival rate of cell.

Specifically describing, the present embodiment carries out the activity test of resisiting influenza virus from H1N1 and H3N2 Strain, its Details is as follows：

First, cell culture

Cell thaw after culture in 37 DEG C, 5% CO2gas incubator, wait to cell cover with eighty per cant or so when, with phosphorus Acid buffer (PBS) cleans cell, adds tryptose Enzyme (trypsin) and reacts 5 minutes, takes off the cell for being attached to culture dish Fall, add in DMEM cell culture mediums and trypsin reaction, centrifugation (1200rpm, 5 minutes), supernatant is absorbed, with a small amount of Culture medium mixes cell, and addition DMEM cell culture fluids are diluted to the required cell concentration of experiment after counting cell, to carry out Anti-influenza virus activity screens.

2nd, experimental procedure

1. cell is diluted into prescribed concentration (2 × 104cells/well) is implanted into 96 hole culture dishes (96-well plate) In, cultivated 20~24 hours in 37 DEG C, 5% CO2gas incubator (incubator)；

With 100 μ l cell physiological buffer solution (PBS) cleaning twice, 2. last time adds 100 μ to each well cell G/well TPCK nutrient solutions, incubator is inserted, is administered again after the completion of drug dilution to be measured；

3. administration condition is divided into D+V, D, V, control group (Mock) and blank group (Blank) administration, in 37 DEG C, 5% dioxy Change carbon incubator (incubator) to cultivate 48 hours；

4. two days later with micro- sem observation Apoptosis situation；Then MTT detections are carried out, respectively in D+V, D, V, control group (Mock) the μ l (5mg/ml) of MTT reagents 20 and in blank group (Blank) are added, are waited 5 hours；

5. sucking nutrient solution, 25 μ l Glys are added in D+V, D, V, control group (Mock) and blank group (Blank) (glycine) buffer solution and 100 μ l dimethyl sulfoxide (DMSO)s (dimethyl sulfoxide, DMSO).

6. carrying out disk-read, wavelength 540nm light absorption values are measured.

3rd, condition is administered

1.D+V：Give 50 μ l various concentrations medicine to be measured and 50 μ l influenza viruses (0.01MOI) simultaneously；

2.D：Give 50 μ l various concentrations and treat side medicine and 50 μ l TPCK nutrient solutions；

3.V：Give 50 μ l influenza viruses (0.01MOI) and 50 μ l TPCK nutrient solutions；

4.Mock：Containing mdck cell and 100 μ l TPCK nutrient solutions；

5.Blank：Without mdck cell and TPCK nutrient solutions.

4th, influenza virus (H1N1/H3N2) infective dose

Impose on 0.01MOI (viral infective dose multiplicity of infection) strains of influenza viruses.

5th, drug concentration to be measured

1. extract：100 μ g/ml, 50 μ g/ml, 25 μ g/ml, tri- kinds of concentration；

2. purified：75 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, tri- kinds of concentration.

6th, positive controls

Antiviral drugs Ribavirin.

7th, MTT Inspection Measuring Knot Guo Read take

1. Fine born of the same parents' survival rate：

2.0-25% cell survival rates (cell survival) record is +/-；

25-50% cell survival rates record for+；

50-75% cell survival rates are noted down ++；

75-100% cell survival rates are noted down +++；

>100% cell survival rate is noted down ++++.

Wherein, medicine to be measured causes phenomena of apoptosis to have return action person for virus, +++ it is above active drug, And active drug must have identical result twice or more.

In one embodiment, N-hexane extract (SAOF-H), dichloromethane extract (SAOF-D) and positive fourth are taken Three kinds of extracts of alcohol extract (SAOF-B), carry out the screening active ingredients of resisiting influenza virus, and experimental result refers to table 1 and table 2.

Table 1, extract resisiting influenza virus (H1N1) active testing result

As shown in upper table 1 and table 2, n-butyl alcohol extract in high dose (100 μ g/ml) and low dosage (25 μ g/ml) simultaneously Give when side medicine and during H1N1 or H3N2 influenza viruses, can make mdck cell survival rate up to 75%, dichloromethane extract Also up to 75% survival under low dosage (25 μ g/ml).Further, since dichloromethane extract and n-butyl alcohol extract are all derived from Methanol extraction thing, therefore those skilled in the art are appreciated that methanol extraction thing ought to also have the effect of resisiting influenza virus.

Embodiment 3. is purified with separating

Garden burnet extract is further selected to dichloromethane extract (SAOF-D) and n-butanol according to anti-influenza virus activity Extract (SAOF-B) is purified and separate active ingredients, and separation process is as shown in Figures 2 and 3.

Dichloromethane extract (SAOF-D) is taken with column chromatography on silica gel, n-hexane：Dichloromethane=1：1 is movement Phase, the mode polarity that gradient purges with is cumulative to methanol, is divided into five layerings (SAOF-D-1~5), taking wherein has a large amount of knots The SAOF-D-3 that partial crystallization goes out, is separated by column chromatography on silica gel, and using dichloromethane as mobile phase, it is cumulative that gradient purges with mode polarity To methanol, get five parts (SAOF-D-3-1~5).Wherein SAOF-D-3-3 is taken to be separated by column chromatography on silica gel, it is mobile It is mutually n-hexane：Ethyl acetate=9：1 cumulative polarity is continuous to be divided into eight parts (SAOF-D-3-3-1~8) to ethyl acetate.Take Wherein SAOF-D-3-3-4 is separated via the reverse property layer post of low pressure using methanol/water to be mobile, obtains six part (SAOF-D-3- 3-4-1~6), take wherein SAOF-D-3-3-4-4 to be isolated and purified repeatedly with methanol/water through preparative high pressure liquid chromatography (HPLC) post, obtain To compound SAOF-K09:Methyl Mycophenolate Mofetil (methyl mycophenolate, 7mg).

The identification of the formula of embodiment 4. (I) compound

It is further purified by garden burnet extract of the present invention and the active component SAOF-K09 of separation has splendid anti influenza Antiviral potency, it is identified as having following formula (I) compound：

Chemical name methyl Mycophenolate mofeil (methyl mycophenolate), it is for white crystal, chemical formula： C18H22O6.Its identified physicochemical data is：

1.Mol.Wt.:334.3637

2.Rf:0.3(EtOAc:Hexane=1:3)

3.mp:102-103℃

4.UV(MeOH):304(3.65),248(3.95),223(4.23)

5.IR(KBr)cm-1:3425,2993,2955,2918,1732,1622

6. 1H-NMR(DMSO-d6,300MHz),δ(ppm):

1.72 (3H, s, CH3C), 2.07 (3H, s, CH3Ar), 2.17 (2H, t, J=7.7Hz, CH2C), 2.34 (2H, t, J =7.8Hz, CH2CO), 3.28 (2H, d, J=6.9Hz, CH2Ar), 3.50 (3H, s, CH3OCO), 3.68 (3H, s, CH3OAr), 5.12 (1H, t, J=6.3Hz, CHC), 5.23 (2H, s, CH2O), 9.34 (1H, s, OH)

7. 13C-NMR(DMSO-d6,75MHz),δ(ppm):

10.80,15.63,22.20,32.06,33.91,50.88,60.47,68.52,106.88,115.94,122.25, 123.02,133.27,145.72,152.76,162.71,170.23,172.89

The Hydrolysis of compound (AnWen2150) of the formula of embodiment 5. (I) compound

The active component with splendid resisiting influenza virus effect is further purified and separates by garden burnet extract of the present invention SAOF-K09 (formula (I) compound, 6mg) is that starting material is placed in 5-mL test tubes, and it is small to add 1ml 0.1N NaOH (aq) shakings 1 When, after adding 1.5ml 0.1N HCl (aq) acidifyings, extracted 3 times with ethyl acetate 5ml, after combining extraction liquid, with anhydrous sulphur Hydrolysis of compound (AnWen2150,4.2mg) is crystallized to obtain after sour sodium 5g water removals, filtering, concentration, is accredited as Mycophenolic Acid (Mycophenolic acid), its physicochemical data are：

1.Mol.Wt.:320.3371

2.Rf:0.1(EtOAc:Hexane=1:3)

3.mp:139-141℃

4.UV(MeOH):304(3.85),251(3.97),225(4.50)

5.IR(KBr)cm-1:3450,2997,2959,2923,1735,1627

6. 1H-NMR(DMSO-d6,300MHz),δ(ppm):

1.72 (3H, s, CH3C), 2.07 (3H, s, CH3Ar), 2.15 (2H, t, J=8.1Hz, CH2C), 2.24 (2H, t, J =8.1Hz, CH2CO), 3.26 (2H, d, J=6.9Hz, CH2Ar), 3.68 (3H, s, CH3OAr), 5.12 (1H, t, J= 6.9Hz, C=CHC), 5.23 (2H, s, CH2O), 9.33 (1H, s, OH)

7. 13C-NMR(DMSO-d6,75MHz),δ(ppm):

10.80,15.63,22.19,31.96,33.87,60.47,68.52,106.88,115.94,122.25, 123.02,133.27,145.72,152.76,162.71,170.21,172.83.

The preparation of the formula of embodiment 6. (I) compound

Formula (I) compound SAOF-K09 can also be prepared according to following methods：

Mycophenolic Acid (mycophenolic acid) 3.2g (10mmol) in ice bath is dissolved in 30ml dichloromethane (dichloromethane) in solution, oxalyl chloride (oxalyl chloride) 1.5ml (17.5mmol) and dimethyl methyl are added Acid amides (dimethylformamide) 2 drips, and the mixed liquor is stirred at room temperature 3 hours.The mixed liquor is evaporated under vacuum and is obtained 11a compounds.11a compounds are dissolved in methanol and the mixed liquor are stirred at room temperature 10 minutes, the mixing is evaporated under vacuum Liquid and crude product 11b compounds (i.e. formula (I) compound), the crude product is from hexane (hexane) and dichloromethane (dichloromethane)(2:1) recrystallize and obtain pure compound (2.3g, 67.3%).It is as follows to prepare reaction process：

The antiviral activity experiment of the purified of embodiment 7.

As shown in table 3, by the separated purified of dichloromethane extract and n-butyl alcohol extract in anti-H1N1 influenzas disease In cytotoxic activity experiment, SAOF-K09 (formula (I) compound) has preferable resisiting influenza virus under low concentration (3.125 μ g/ml) Activity, and to cell without drug toxicity.

Table 3, purified resisiting influenza virus (H1N1) active testing result

Ribavirin systems are control group.

Again as shown in table 4, SAOF-K09 has preferable anti-influenza virus activity under low concentration (3.125 μ g/ml), and And to cell without drug toxicity.

Table 4, purified resisiting influenza virus (H3N2) active testing result

Ribavirin systems are control group.

The MIC and Drug toxicity trails of the purified of embodiment 8.

According to foregoing resisiting influenza virus (H1N1/H3N2) active testing result, SAOF-K09 has anti-influenza virus activity, And carry out MIC and toxicity trial.Wherein, the experimental detail system of MIC and toxicity trial Be same as antiviral activity test, and drug concentration to be measured is respectively 100,75,50,25,12.5,6.25,3.125,1.56, 0.78、0.39、0.195、0.098μg/ml。

As shown in table 5, SAOF-K09 for the MIC of anti-H1N1/H3N2 influenza activities all in 0.098 μ G/ml, and there is cytotoxicity more than 75 μ g/ml.

Table 5, SAOF-K09 resisiting influenza virus MIC and Drug toxicity trails result

According to table 5 as a result, it was confirmed that SAOF-K09 has the effect of resisiting influenza virus.

The MIC of the antagonistic drug venereal disease poison of embodiment 9.

It is that formula (I) compound and following compounds press down for the minimum of the anti-medicine mutation of influenza virus according to preceding method detection Concentration (MIC) processed：

Formula (I) compound；

AnWen2150:The hydrolysate (Mycophenolic acid) of formula (I) compound.

Tested strains of influenza viruses includes:

(1) H1N1T.R.-H1N1 Tamiflus (Tamiflu) resistance viral strain：Influenza A strain (H1N1) through mutation, This Strain is similar to Influenza A/Taiwan/937/2009 after sequencing

(2) H3N2-influenza A strain (H3N2)：It is similar to a plant Influenza A/New York/469/ after sequencing 2004

(3)WSN–Influenza A/WSN/33(H1N1)：It is similar to Influenza A/Hong Kong/ after sequencing 470/97

(4) Influenza B-Type B strains of influenza viruses

(5) H1N1-influenza A strain (H1N1)

Table 6, the MIC to having anti-drug effect strains of influenza viruses

According to table 6 as a result, it was confirmed that formula (I) compound has the strains of influenza viruses optimal efficacy of the confrontation tool resistance to the action of a drug.

Although the present invention is disclosed above with preferred embodiment, so it is not limited to the present invention, any to be familiar with the present invention Operator, when can without departing from the spirit and scope of the invention, make a little change with retouching, then it is special should to belong to the present patent application The protection domain that sharp scope defines.

Claims (3)

1. The acceptable salt of formula 1. (I) compound, its hydrolysate or medicine has the medicine of drug-fast influenza virus preparing confrontation Application in thing：

   Wherein the influenza virus is the H1N1 or H3N2 drug-fast variant virus strain of tool.
2. 2. application as claimed in claim 1, wherein the hydrolysate is Mycophenolic Acid.
3. 3. application as claimed in claim 1 or 2, the wherein drug-fast variant virus strain of the tool are to have the resistance to the action of a drug to Tamiflu.