CN103479758A - S.officinalis L.-containing anti-influenza virus medical composition - Google Patents
S.officinalis L.-containing anti-influenza virus medical composition Download PDFInfo
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Abstract
The present invention relates to an anti-influenza virus medical composition, which comprises a therapeutically effective amount of S.officinalis L. extract or an active compound isolated from the S.officinalis L. extract, a hydrolyzate thereof or a pharmaceutically acceptable salt. The present invention further relates to an application of the S.officinalis L. extract or the active compound thereof in anti-influenza virus drug preparation.
Description
Technical field
The present invention system, about a kind of medical composition of resisiting influenza virus, particularly includes the medical composition of Radix Sanguisorbae extract or its reactive compound.The present invention also about Radix Sanguisorbae extract or its reactive compound as the application for preparing anti-influenza virus medicament.
Background technology
Influenza virus (being called for short influenza virus) is one of virus of most threatening property in the human life, from just occurring in 20th century, four influenzas are very popular, respectively 1918～1919 years (spanish influenzas, H1N1), nineteen fifty-seven (Asia influenza, H2N2), nineteen sixty-eight (Mao flu, H3N2) and 1977 (Russian influenza, H1N1).Wherein, the most serious with spanish influenza in 1918 again, its H1N1 virus strain is to be formed by bird flu and human influenza virus sudden change, causes 2～4,000 ten thousand people's death in the whole world.And novel H1N1 Influenza epidemic situation is broken out in Mexico in 2009, suspected and had more than 100 ten thousand people to infect, cause getting killed of people more than 10,000, (Neumann, G. in also continuing at present to spread; Noda, T.; Kawaoka, Y.Emergence and Pandemic Potential of Swine-Origin H1N1 Influenza Virus.Nature.459:931-939,2009).In addition, propose to pay special attention to the invasion and attack of seasonal A type H3N2 influenza in national influenza prevention and control meeting in 2010, this Strain also causes many injures and deaths in the past.
Influenza virus is that a kind of mankind of causing and animal suffer from grippal minus strand single-stranded RNA virus, to belong to Orthomyxoviridae family (Orthomyxoviridae), according to the difference of virus nucleoprotein, hereditary material, stromatin antigenic characteristic, can be divided into A, B, tri-serotypes of C.Wherein, A type influenza virus can cause the influenza between different hosts, two kinds of antigen hemagglutinin (hemagglutinin because of its viral appearance, HA) and neural amino acid enzyme (neuraminidase, NA) be pleiomorphism, found at present 16 kinds of HA(H1～16) and 9 kinds of NA(N1～N9), the heritability antigenic variation can occur; Less of Type B influenza antigen variation can cause that provincialism infects, and C type influenza virus is mainly to take pig as the host, more rare to the infection of human body, and therefore above-mentioned said worldwide large influenza is exactly the epidemic situation caused by A type influenza virus.
A type influenza virus structure from outer to inner can be divided into adventitia, stromatin and three parts of core.The have an appointment projection of 500 radial outside arrangements of adventitia is above-mentioned two kinds of antigenic types: columnar protrusions (HA) and mushroom projection (NA).HA can cause coagulation with the erythrocytic surface receptor absorption of many animals, after cracking, can be divided into heavy chain and light chain, makes the virus and host cell mutually merge; NA mainly has the sialic activity of hydrolysis, cuts off the last contact of virus and host cell, and virus is come off from the erythrocyte of absorption, prevents viral gathering, promotes the movement in mucus.Stromatin is comprised of M1, M2, the protection virus core is arranged and maintain viral structure.Core is comprised of 8 minus strand single-stranded RNA fragments in addition, and it combines and be wound in ribosome with nucleoprotein (NP) and RNA polymerase (PB1, PB2 and PA).And influenza virus why can to cause people's fear be mainly can produce new hypotype and let us is caught unprepared due to its variation mode, its sudden change mode has two kinds, one is antigenic drift (antigenic drift), and it refers to that the point mutation of antigen (NA) the amino acid sequence of influenza virus sub-strain produces little variation; Another is antigenic shift (antigenic shift), it is the influenza virus modal type that makes a variation, a large variation of antigenicity will occur every 10 years, and the reason caused is to infect because the host is subject to two kinds of different virus strains simultaneously, and viral RNA carries out gene recombinaton and produces new Strain, very large (the Chen Hongshan of impact, Zhang Xingquan, antiviral drugs and research method thereof, Chemical Industry Press, 328-331,2006.).
Influenza occurs in autumn and winter, early spring mostly, and the target of its invasion and attack is the respiratory mucosa epithelial cell, and breeds in host cell, causes that mucosa is congested, edema and cytopathy, the pathological changes such as come off.Common 1 to 3 day of incubation period; start to occur having a fever, feel cold, the symptoms such as headache, nasal obstruction, systemic pain; when spreading to lower respiratory tract; may cause bronchitis and interstitial pneumonia; because influenza virus can reduce the ability that the respiratory mucosa epithelial cell is removed and sticked foreign body; therefore often causing secondary pneumonia to infect, is that it mainly causes one of 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, mostly take community isolation and supportive Drug therapy for epidemic prevention and the treatment of influenza, and use the Prevention mode to be divided into vaccine and antiviral drugs.But for the virus increasingly made a variation, vaccine protection or risky property; And antiviral drugs mainly contains three kinds at present, the first is M2 inhibitor (M2 protein inhibitor), act on viral membrane-spanning protein M2 ion channels, hinder H+ and enter virus inside, make outer virionic membrane to merge with endosome, virus just can't discharge RNA, and this class medicine is Adamantan derivant (amantadine, rimantadine); The second is NA inhibitor (neuraminidase inhibitors), act on neural amino acid enzyme (NA), make virus can't be hydrolyzed sialic acid, and then can't the leaving from host cell, stop the virus diffusion, this class medicine has oseltamivir, zanamivir, peramivir and cyclopentane or pyrrolidine derivatives; The third is the RNApolymerase inhibitor, mainly suppresses RNA polymerase(PB1, PB2, PA) path of synthetic virus protein, medicine has 2 '-deoxy-2 ' fluoroguanosine(FdG), T-705; Also have in addition and utilize interferon and SiRNA(small interfering RNAs) etc. therapeutic modality prevent viral infection (Clercq, E.D.Antiviral Agents Active against Influenza A Viruses.Nat.Rev.Drug.Discov.5:1015-1025,2006).
Yet above-mentioned medicine all can't reach the various types of influenza virus of comprehensive inhibition, and the disappearance of Drug resistance and drug side effect is arranged.The antigenic variability of A type influenza virus is large, and infectiousness is strong, and the annual whole world approximately has 500,000,000 people to infect influenza, often causes very high mortality rate, for society, produces huge burden and economic loss, so the new Tamiflu of active development is an important topic in fact.
Summary of the invention
The present invention system proposes to prepare the medical composition of resisiting influenza virus with Radix Sanguisorbae (S.officinalis L.) extract first.
Therefore, one aspect of the present invention provides a kind of medical composition of resisiting influenza virus, and it comprises the Radix Sanguisorbae extract of effective dose and pharmaceutically acceptable supporting agent.
The present invention also proposes Radix Sanguisorbae (S.officinalis L.) extract as the application for preparing anti-influenza virus medicament.
In a specific embodiment, Radix Sanguisorbae extraction system is obtained by the preparation of alcohol extraction Radix Sanguisorbae.
On the other hand, the invention provides a kind of medical composition of resisiting influenza virus, it comprises the salt that can accept on formula I compound, its hydrolysate or the medicine of effective dose, and pharmaceutically acceptable supporting agent:
The present invention also provides the salt that can accept on formula I compound, its hydrolysate or medicine as the application for preparing anti-influenza virus medicament.
Other features of the present invention will be known and present via following detailed description, each instantiation and claim.
The accompanying drawing explanation
Foregoing invention content and the following specific embodiment and appended graphic reading in the lump will more increase understanding.For reaching the purpose of illustrating the present invention, graphic shown in instantiation be current preferably.Yet, should be appreciated that particular arrangement and the structure of the present invention shown in being not limited to.
Fig. 1 is the extraction procedure figure of Radix Sanguisorbae extract of the present invention.
Fig. 2 is the flow chart of the gained dichloromethane extract purifies and separates reactive compound according to the present invention.
Fig. 3 is the flow chart of the gained n-butyl alcohol extract purifies and separates reactive compound according to the present invention.
The specification specified of each instantiation of the present invention is as rear.The present invention's further feature will be via the detailed description in following each instantiation and claim and clear presenting.
Need not further set forth, the salty persond having ordinary knowledge in the technical field of the present invention of believing can utilize the present invention to the widest degree based on above stated specification.Therefore, be appreciated that the following description is only as illustrating, but not limit the scope of the invention by any way.
The specific embodiment
Unless otherwise, the technical and scientific term of all uses herein has the meaning of understanding as the common operator in skill under the present invention.
" one " second word that this paper is used, as do not specialize, mean the quantity of at least one (one or more).
The abbreviation that described it " influenza virus " is influenza virus herein, be that a kind of mankind of causing and animal suffer from grippal minus strand single-stranded RNA virus, to belong to Orthomyxoviridae family (Orthomyxoviridae), difference according to virus nucleoprotein, hereditary material, stromatin antigenic characteristic, can be divided into A type influenza virus, Type B influenza virus and C type influenza virus, then be divided into different hypotypes according to the antigenicity of hemagglutinin and neural amino acid enzyme.Comprise 6 key elements according to the name of World Health Organization (WHO) (WHO) strains of influenza viruses: type/host/separation area/strain sequence number/separation time (HnNn), wherein for human influenza virus, omit host's information, for B-mode and influenza virus C, omit hypotype information.In the present invention, alleged influenza virus comprises A type influenza virus, Type B influenza virus and C type influenza virus.In a specific embodiment, influenza virus is in particular the variant virus strain of H1N1, H3N2 or its tool Drug resistance, espespecially to the variant virus strain of Tamiflu (Tami flu) tool Drug resistance.
Described it " treatment effective dose " means that the salt that Radix Sanguisorbae extract of the present invention or formula I compound, its hydrolysate or medicine can be accepted can produce the required content of therapeutic effect to individuality herein.Haveing the knack of the effective dose that the technology person will understand an active component or compositions will play by ear, including, but not limited to the kind of for example this medicine and body weight, age and the health status of dosage form and this organism.
Described it " extract " pointer is extracted the product of gained to a material herein, normally by by the material of wish extraction soak or be mixed in solvent and the extract layer obtained.The preparation system of general extract from fresh plant or through grind or the plant sample of drying with the known various extracting process in this field, include but not limited to dipping, diafiltration, diafiltration again, clear up, counter-current extraction, turbine extraction, extruding/extrusion/squeeze or supercritical fluid carbon dioxide extracts it.Suitable solvent includes but not limited to ethanol, normal hexane, methanol, dichloromethane, water, n-butyl alcohol or other solvents.Optionally the concentration of the kind of selective reagent or allotment solvent, extracted to reach suitable polarity.The extract of different phase can merge mutually, also can for example, evaporate in the follow-up concentration step that carries out again, or purification or separating step, for example, filtration, centrifugal and chromatographic analysis.In one example, by all or part of (optionally through chopping or grind) of fresh plant or dry plant sample and suitable solvent or be soaked in wherein and stir and reach one section time enough, carry out or heat and carry out in room temperature, remove solid residue (filtering residue) via filtration, then collect the juice (extract) obtained; Optionally repeat to soak or blend step, merge gained juice, further concentrated, purification or separation.
Described it " Radix Sanguisorbae " has another name called beautiful drum, reddish brown, the red silk ball of acid herein, dry root for rosaceous plant Radix Sanguisorbae Sanguisorba officinalis L., Radix Sanguisorbae S.officinalisL.var.longifolia (Bert.) the Yu et Li that comes into leaves, system is said by TAO Hong-Jing: " its leaf is grown like elm; nascent step " and obtain its name, begin to be stated from " herbal classic ", classify middle product as.Cold nature, bitter in the mouth acid, nontoxic, return large intestine, liver, stomach, kidney channel, has the effects such as cooling blood for hemostasis, removing toxic substances sore.Radix Sanguisorbae is herbaceos perennial.Root is spindle more, and surperficial sepia or puce have vertical wrinkle and transverse crack.Stem is upright, and rib is arranged, and without hair or base portion, sparse glandular hair is arranged.Basal leaf is winglike compound leaf, lobule 4-6 couple; Petiole is without hair.Oval, the cylindrical or ovoid of spike, upright, long 1-3 centimetre, wide 0.5-1 centimetre, purple is to mulberry, opening from the picture of inflorescence top; Bract 2, lanceolar, there is pubescence at the back side and edge; Sepal 4, oval to width egg shape, aubergine; Stamen 4, filigree is thread, stigma tip dish type.Achene contains in calyx tube, the shape of falling ovum Long Circle.The florescence 7-10 month, the fruit phase 9-11 month.Be born in grassland, the patana of height above sea level 30-3000 meter, mainly be distributed in the ground such as northeast, East China, southwest and Henan, Hubei, Guangxi.Usually the Radix Sanguisorbae root of all can gathering in spring, autumn, its character is cylindrical, slightly distorted shape bending, long 18-22cm, diameter 0.5-2cm, adnation supporting root as seen sometimes.The matter heavily fortified point, slightly crisp, fracture is smooth, slightly the tool opaque.The transverse section cambium ring is obvious, and skin zone is faint yellow, and woody part brown color or band pink, be remarkable radial arrangement.Feeble QI, mildly bitter flavor puckery (Song Liren, China's book on Chinese herbal medicine, the 4:281-286 of Shanghai science tech publishing house, 1999).In one of the present invention specific embodiment, be to use Radix Sanguisorbae Sanguisorba officinalis L., it uses base portion is root.
Described solvent it " percentage ratio " or " % " mean the percent by volume that solvent is water-soluble herein, and for example, 95% ethanol refers to the aqueous solution that contains 95 percent by volume ethanol or for example, 80% methanol refers to the aqueous solution that contains 80 percent by volume methanol.
According to the present invention, can not expectedly find that Radix Sanguisorbae (S.officinalis L.) extract has the effect of resisiting influenza virus, includes but not limited to H1N1 and H3N2 influenza virus.Therefore a kind of medical composition of resisiting influenza virus is provided, and it comprises the Radix Sanguisorbae extract for the treatment of effective dose and pharmaceutically acceptable supporting agent.
Embodiment according to the present invention, this Radix Sanguisorbae extraction system is obtained by the preparation of alcohol extraction Radix Sanguisorbae, and it also can comprise further quintessence.For example, this Radix Sanguisorbae extract can be obtained by the method preparation of the following step of tool:
(1) with the alcohol extraction Radix Sanguisorbae, to obtain ethanolic extract; And
(2) extract this ethanolic extract with methanol and normal hexane (1:1), to obtain the methanol extraction thing.
Can further with dichloromethane and water (1:1), extract this methanol extraction thing again, to obtain dichloromethane extract.
Another embodiment according to the present invention, this Radix Sanguisorbae extraction system is by aforementioned methanol extraction thing with dichloromethane and water (1:1) extraction, and the water intaking extract extracts this water extract with n-butyl alcohol and water (1:1) again, to obtain n-butyl alcohol extract.
In aforementioned preparation method, the ethanol used is 50% to 99% ethanol, is preferably 95% ethanol.
In aforementioned preparation method, the methanol used is 50% to 99% methanol, is preferably 90% methanol.
According to the present invention, go out active component by the further separation and purification of this Radix Sanguisorbae extract, and confirm that through biotic experiment it has splendid anti-influenza virus activity, even there is the activity of the strains of influenza viruses of antagonism tool Drug resistance.
Therefore, the present invention provides a kind of medical composition of resisiting influenza virus on the other hand, and it comprises the salt that can accept on formula I compound, its hydrolysate or the medicine for the treatment of effective dose, and pharmaceutically acceptable supporting agent:
According to the present invention, formula (I) compound can further be hydrolyzed and its hydrolysate Mycophenolic Acid (Mycophenolic acid), formula (I) compound and hydrolysate thereof have it through the biotic experiment checking and have splendid anti-influenza virus activity, even have the activity of the strains of influenza viruses of antagonism tool Drug resistance.The salt that can accept on its medicine of salty letter also has anti-influenza virus activity certainly.
This paper is used it " salt that can accept on medicine " to mean for the mankind or mammal and takes the safe and effective salt compounds of tool, has its required biological activity.The salt that can accept on medicine includes but not limited to acidity or the alkaline salt of formula of the present invention (I) compound, for example the basic salt synthetic with hydrochloric acid, hydrobromic acid, iodic acid, nitric acid, sulphuric acid, sodium bisulfate, phosphoric acid, phosphate ester, acetic acid, lactic acid, salicylic acid, citric acid, tartaric acid, pantothenic acid, liquor epinephrinae bitartratis ophthalmicus, ascorbic acid, succinic acid, maleic acid, fumaric acid, gluconic acid, formic acid, benzoic acid, glutamic acid, pyrovinic acid, p-methyl benzenesulfonic acid; Or the basic salt synthetic with aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and diethanolamine salt.
According to the present invention, this medical composition can include but not limited to parenteral or oral administration medicine supplying.Its form of the medical composition of parenteral dispensing comprises solution, suspension, emulsion, and can carve before use dissolving or be suspended in the solid Injectable composition in solvent.Can prepare this injection in diluent by dissolving, suspension or emulsifying one or many active component.The example of aforementioned diluent is distilled water, normal saline, vegetable oil, alcohols and the combination thereof for injection.Again, this injection can contain tranquilizer, cosolvent, suspending agent, emulsifying agent, smooth agent, buffer, preservative agent etc.These injection tie up in final preparation step sterilizing or prepare with sterile procedure.The present invention's medical composition also can be formulated to the sterile solid preparation, for example, by lyophilization, and can carve before use sterilizing or be dissolved in sterile injectable water or other sterile diluents.But this medical composition is oral administration also, wherein said composition can be solid or liquid form.Solid composite comprises lozenge, pill, capsule, dispersibility powder, granule and analog thereof.Orally administered composition also comprises mouth-wash and sublingual tablet.Capsule comprises hard capsule and soft capsule.In this type of Orally-administered solid composition, one or many reactive compounds can mix voluntarily, or with diluent, bonding agent, loosely separate agent, lubricant, tranquilizer, cosolvent and mix, then with the prior art method preparation, become preparation.When needed, these preparations can smears coating, or can two or the coating of multiple coating layer.On the other hand, liquid oral compositions comprises pharmaceutically acceptable liquid solution, suspension, emulsion, syrup, medicated wine, and analog.In such composition, one or many reactive compounds can be dissolved, suspend or be emulsified in all-purpose diluent (as mixture of purified water, ethanol or its etc. etc.).Except this type of diluent, foregoing also can contain wetting agent, suspending agent, emulsifying agent, sweeting agent, flavoring agent, spice, preservative agent and buffer and analog thereof.
Need not further set forth, the salty persond having ordinary knowledge in the technical field of the present invention of believing can utilize the present invention to the widest degree based on above stated specification.Following embodiment is only as the use illustrated, but not limits by any way remaining disclosure.
The specification specified of each instantiation of the present invention is as rear.The present invention's technical characterictic will be via the detailed description in following each instantiation and claim and clearer presenting.
Example
Real Examination material
Solvent and reagent: except indicating especially, the component separating agents useful for same of embodiment, solvent, all purchased from the bright chemical industrial company of scape and friend and Trading Co., Ltd, and be the sterling of reagent level or liquid chromatography (LC) level.
Chromatographic material: except indicating especially, the chromatographic material of embodiment is all purchased from Taiwan Merck (Merck Taiwan) company and friend and Trading Co., Ltd.
(1) adsorptivity chromatography silica gel: Silica gel 60,70-230mesh, 230-400mesh, Diaion HP-20 ion exchange resin.
(2) thin layer chromatography sheet (TLC plate): DC Kieselgel 60 F254(positive thin layer chromatograph chromatography sheets), DC Kieselgel RP-18 F254S(anti-phase thin layer chromatograph chromatography sheet).
(3) low pressure preparation formula carbon 18 type reverse-phase chromatography posts: Lichroprep RP-18,40-63 μ m.
(4) carbon 18 type chromatographic columns for the preparation formula high-performance liquid chromatograph: the Cosmosil-pack of Nacalai Teaque company product, Prep-C18,10 μ m, 20mm * 250mm.
Equipment and instrument
Low pressure liquid chromatography side Pu: be the RP-SY-ICSC type low pressure side Pu that adopts FMI company product, be used in anti-phase low pressure chromatography.
High-performance liquid chromatograph:
1. system controller: Shimadzu SCL-8A;
2. side 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-480?Waterbath；
2.Buchi?R-114?Rotavapor；
3.Buchi?V-800?Vacuum?controller。
TLC spots detects:
1.UV(wavelength 360nm, shortwave 254nm);
2.p-Anisaldehyde being color reagent detects.
Nuclear magnetic resonance spectrometer: except the Varian GEMINI-300(300MHz that uses the national defence medical college), and entrust Univ Nat Taiwan's expensive instrument center on behalf of measuring (use instrument: Bruker Avance 500MHz FT-NMR).The hydrogen spectrum of mensuration and carbon light spectrum mean chemical shift with δ-value, and unit is ppm, and with TMS(tetramethylsilane) work as internal standard.S means unimodal (singlet); D means doublet (doublet); T means triplet (triplet); Br means broad peak (broad); Dd means two groups of doublets (doublet doublet).
Melting point apparatus: melting point compound is measured the melting point apparatus (not proofreading and correct) that system adopts U.S. Fischer-Johns company.
Infrared spectrometer: Shimadzu FT-IR8700 type.Polyethylene (polystyrene) tuning wavelength, except special instruction, all beat the ingot mode with potassium bromide (KBr) and measure; And measure in the fourier-transform infrared linear light spectrometer of Academia Sinica's genosome center Theremo Nicolet 380 types.
Ultraviolet spectrogram (Ultraviolet spectra, UV) is measured with Shimadzu UV-160spectrophotometer.
Mass spectrograph: entrust university of communications, Tsing-Hua University and Chung Hsing University's expensive instrument center on behalf of mensuration, its mass spectrometer is respectively Micromas TRIO-2000 GC-MS, MAT-95XL High Resolution Mass Spectrometer, High Resolution Mass Spectrometer.
Cell pharmacology experiment material
Cell strain (Cell line): MDCK:Madin-Darby canine kidney cells(Madin-DarbyShi Testis et Pentis Canis tubule cell strain).
Cell culture fluid:
1.10％?Fetal?bovine?serum(FBS）；
2.TPCK culture fluid (tosylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin);
3.DMEM（Dulbecco’s?modified?Eagle?medium）。
Cell physiological buffer: Phosphate-buffered saline(PBS).
Strain: Influenza A(H1N1/H3N2) (source: the Yao Zhen culture and education is awarded, pathology section of armed forces general hospital).
ELISA reading analyzer: BIOTEK CERES 900 EIR READER.
The preparation of embodiment 1. extracts
As shown in Figure 1, the Radix Sanguisorbae sample is ground to totally 10 kilograms of rear weighings, after extracting with 95% ethanol merceration under room temperature, concentrated with the concentrating under reduced pressure machine, obtain ethanolic extract and be total to 3400 g of gross weights.Ethanolic extract to be extracted with the equal-volume ratio with normal hexane after 90% dissolve with methanol, obtains N-hexane extract (SAOF-H) 134.20g and methanol extraction thing again.
The methanol extraction thing is extracted with equal-volume dichloromethane and water, obtained dichloromethane extract (SAOF-D) 159.50g.Wherein, water layer is extracted with isopyknic n-butyl alcohol again, because both can't separate, obtains n-butyl alcohol extract (SAOF-B) 3100g after merging.
The antiviral activity test of embodiment 2. extracts
The antiviral activity test is the degree of host cell infected virus of assessing with the survival rate of virus host cell, and the survival rate of host cell is the method with cell survival rate analysis (MTT assay).Principle is 3-(4, 5-dimethylthiazole-2-yl)-2, (3-(4 for 5-diphenyl tetrazole bromide, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, MTT) yellow aqueous solution solid can be by dehydrogenation ferment (dehydrogenase) metabolism in the Mitochondria of living cells (mitochondria), diazonium ring (tetrazolium ring) reduction is to purple infusible precipitate crystal formazan(3-(4, 5-dimethylthiazole-2-yl)-2, 5-bis-Ben Ji formazan 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-formazan) be deposited in cell, because the grain line body ferment of living cells 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 output to assess the survival rate of cell.
Specifically describe, the present embodiment selects the Strain of H1N1 and H3N2 to carry out the activity test of resisiting influenza virus, and its details is as follows:
One, cell culture
After cell thawing, cultivate in 37 ℃, 5% CO2 gas incubator, when to cell, covering with eighty per cant left and right, clean cell with phosphate buffer (PBS), add Trypsin Enzyme (trypsin) to react 5 minutes, make to be attached to the cell detachment of culture dish, add in the DMEM cell culture medium and the reaction of trypsin, centrifugal (1200rpm, 5 minutes), absorb supernatant, mix cell with a small amount of culture medium, after counting cells, add the DMEM cell culture fluid to be diluted to the required cell concentration of experiment, in order to carry out the anti-influenza virus activity screening.
Two, experimental procedure
1. cell dilution to prescribed concentration (2 * 104cells/well) is implanted in 96 hole culture dishs (96-well plate), cultivated 20～24 hours in 37 ℃, 5% CO2 gas incubator (incubator);
2. the cell of each well cleans twice with the cell physiological buffer (PBS) of 100 μ l, adds for the last time the TPCK culture fluid of 100 μ g/well, inserts incubator, administration again after drug dilution to be measured completes;
3. administration condition is divided into D+V, D, V, matched group (Mock) and blank group of (Blank) administration, in 37 ℃, 5% CO2 gas incubator (incubator), cultivates 48 hours;
4. two days later with microscope observing cell apoptosis situation; Then carry out the MTT detection, add MTT reagent 20 μ l (5mg/ml) respectively in D+V, D, V, matched group (Mock) and blank group (Blank), wait for 5 hours;
5. suck culture fluid, in D+V, D, V, matched group (Mock) and blank group (Blank), add 25 μ l glycine (glycine) buffer and 100 μ l dimethyl sulfoxide (dimethyl sulfoxide, DMSO).
6. read dish, measured wavelength 540nm light absorption value.
Three, administration condition
1.D+V: give 50 μ l variable concentrations medicines to be measured and 50 μ l influenza virus (0.01MOI) simultaneously;
2.D: give 50 μ l variable concentrations and treat side medicine and 50 μ l TPCK culture fluid;
3.V: give 50 μ l influenza virus (0.01MOI) and 50 μ l TPCK culture fluid;
4.Mock: containing mdck cell and 100 μ l TPCK culture fluid;
5.Blank: not containing mdck cell and TPCK culture fluid.
Four, influenza virus (H1N1/H3N2) infective dose
Impose on 0.01MOI(viral infection amount multiplicity of infection) strains of influenza viruses.
Five, drug level to be measured
1. extract: 100 μ g/ml, 50 μ g/ml, tri-kinds of concentration of 25 μ g/ml;
2. purified: 75 μ g/ml, 25 μ g/ml, tri-kinds of concentration of 12.5 μ g/ml.
Six, positive controls
Antiviral drugs Ribavirin.
Seven, MTT Inspection Measuring Knot Guo Read gets
1. Fine born of the same parents' survival rate:
2.0-25% cell survival rate (cell survival) record is +/-;
25-50% cell survival rate record for+;
The record of 50-75% cell survival rate is ++;
The record of 75-100% cell survival rate is +++;
100% cell survival rate record be ++ ++.
Wherein, medicine to be measured causes phenomena of apoptosis that return action person is arranged for virus, +++above be active drug, and active drug must have twice or above identical result.
In one embodiment, get N-hexane extract (SAOF-H), dichloromethane extract (SAOF-D) and three kinds of extracts of n-butyl alcohol extract (SAOF-B), carry out the screening active ingredients of resisiting influenza virus, experimental result refers to table 1 and table 2.
Table 1, extract resisiting influenza virus (H1N1) active testing result
Table 2, extract resisiting influenza virus (H3N2) Huo Measuring Try Knot fruit
As above shown in table 1 and table 2, n-butyl alcohol extract is at high dose (100 μ g/ml) and low dosage (25 μ g/ml) during simultaneously until side medicine and H1N1 or H3N2 influenza virus, all can make the mdck cell survival rate reach 75%, dichloromethane extract also can reach 75% survival under low dosage (25 μ g/ml).In addition, because dichloromethane extract and n-butyl alcohol extract are all taken from the methanol extraction thing, so the skill personage can understand the methanol extraction thing and also ought to have the effect of resisiting influenza virus.
The Radix Sanguisorbae extract is further selected to dichloromethane extract (SAOF-D) according to anti-influenza virus activity and n-butyl alcohol extract (SAOF-B) carries out the purification isolation active component, separation process as shown in Figures 2 and 3.
Get dichloromethane extract (SAOF-D) with the silica gel tube column chromatography, normal hexane: dichloromethane=1:1 is mobile phase, the mode polarity that the gradient punching is carried is cumulative to methanol, be divided into five layerings (SAOF-D-1～5), get the SAOF-D-3 that wherein has a large amount of mass crystallizations to separate out, separated by the silica gel tube column chromatography, take dichloromethane as mobile phase, it is cumulative to methanol that mode polarity is put forward in the gradient punching, gets five parts (SAOF-D-3-1～5).Get wherein SAOF-D-3-3 and separated by the silica gel tube column chromatography, movement be mutually the cumulative polarity of normal hexane: ethyl acetate=9:1 to ethyl acetate, continue and be divided into eight parts (SAOF-D-3-3-1～8).Getting wherein SAOF-D-3-3-4 take methanol/water via the reverse property of low pressure layer post and is separated as mobile, obtain six parts (SAOF-D-3-3-4-1～6), get wherein SAOF-D-3-3-4-4 through preparative high pressure liquid chromatography (HPLC) post with methanol/water separation and purification repeatedly, obtain compound S AOF-K09: methyl Mycophenolate Mofetil (methyl mycophenolate, 7mg).
The evaluation of embodiment 4. formula I compounds
By Radix Sanguisorbae extract of the present invention, be further purified and the active component SAOF-K09 that separates has splendid resisiting influenza virus effect, through being accredited as the following formula of tool (I) compound:
Chemical name methyl Mycophenolate mofeil (methyl mycophenolate), be white crystal, chemical formula: C18H22O6.Through identifying that its materialization data are:
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 compound (AnWen2150) of embodiment 5. formula I compounds
Be further purified and separate active component SAOF-K09 (formula (I) compound with splendid resisiting influenza virus effect by Radix Sanguisorbae extract of the present invention, 6mg) for starting material, be placed in the 5-mL test tube, added 1ml 0.1N NaOH (aq) jolting 1 as a child, after adding 1.5ml 0.1N HCl (aq) acidify, with ethyl acetate 5ml extraction 3 times, after combining extraction liquid, dewater, filter, concentrate post crystallization with anhydrous sodium sulfate 5g and obtain hydrolysis compound (AnWen2150,4.2mg), be accredited as Mycophenolic Acid (Mycophenolic acid), its materialization 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 embodiment 6. formula I compounds
Also can prepare according to following method by formula I compound S AOF-K09:
Mycophenolic Acid in ice bath (mycophenolic acid) 3.2g (10mmol) is dissolved in dichloromethane (dichloromethane) solution of 30ml, add 2 of oxalyl chloride (oxalyl chloride) 1.5ml (17.5mmol) and dimethyl formamides (dimethylformamide), at room temperature stir and should mix liquid 3 hours.Under vacuum, evaporation should mix liquid and obtain the 11a compound.The 11a compound is dissolved in methanol and at room temperature stirs and should mix liquid 10 minutes, under vacuum, evaporation should mix liquid and obtain crude product 11b compound (being formula (I) compound), this crude product from hexane (hexane) and dichloromethane (dichloromethane) (2:1) recrystallize and pure compound (2.3g, 67.3%).The preparation feedback flow process is as follows:
The antiviral activity test of embodiment 7. purifieds
As shown in table 3, by dichloromethane extract and purified that n-butyl alcohol extract separated in the activity test of anti-H1N1 influenza virus, SAOF-K09 (formula I compound) has preferably anti-influenza virus activity under low concentration (3.125 μ g/ml), and to cell without drug toxicity.
Table 3, purified resisiting influenza virus (H1N1) active testing result
As shown in table 4 again, SAOF-K09 has preferably anti-influenza virus activity under low concentration (3.125 μ g/ml), and to cell without drug toxicity.
Table 4, purified resisiting influenza virus (H3N2) active testing result
Minimal inhibitory concentration and the Drug toxicity trails of embodiment 8. purifieds
According to aforementioned resisiting influenza virus (H1N1/H3N2) active testing result, SAOF-K09 has anti-influenza virus activity, and carries out minimal inhibitory concentration and toxicity trial.Wherein, the experimental detail of minimal inhibitory concentration and toxicity trial system is same as the activity test of antiviral, and drug level to be measured is respectively 100,75,50,25,12.5,6.25,3.125,1.56,0.78,0.39,0.195,0.098 μ g/ml.
As shown in table 5, SAOF-K09 all at 0.098 μ g/ml, and has cytotoxicity for the minimal inhibitory concentration of anti-H1N1/H3N2 influenza virus activity more than 75 μ g/ml.
Table 5, SAOF-K09 resisiting influenza virus minimal inhibitory concentration and Drug toxicity trails result
According to the result of table 5, confirm, SAOF-K09 has the effect of resisiting influenza virus.
The minimal inhibitory concentration of embodiment 9. antagonistic drug sexually transmitted disease (STD) poison
According to preceding method, detecting is formula (I) compound and the following compounds minimal inhibitory concentration (MIC) for the drug resistance mutation of influenza virus:
Formula (I) compound;
AnWen2150: the hydrolysate (Mycophenolic acid) of formula (I) compound.
Tested strains of influenza viruses comprises:
(1) H1N1 T.R. – H1N1 Tamiflu (Tamiflu) resistance Strain: A type strains of influenza viruses (H1N1) is through mutation, and this Strain is similar to Influenza A/Taiwan/937/2009 after sequencing
(2) H3N2 – A type strains of influenza viruses (H3N2): be similar to strain Influenza A/New York/469/2004 after sequencing
(3) WSN – Influenza A/WSN/33 (H1N1): be similar to Influenza A/Hong Kong/470/97 after sequencing
(4) Influenza B – Type B strains of influenza viruses
(5) H1N1 – A type strains of influenza viruses (H1N1)
Table 8, to the minimal inhibitory concentration minimal inhibitory concentration (MIC) of tool drug resistance effect strains of influenza viruses
μg/ml?H1N1?T.R.H3N2?WSNInf?B?H1N1
The hydrolysate (AnWen2150) 0.39 1.56 0.195 of formula (I) compound
Formula (I) compound 0.098 0.098 0.097 0.078
According to the result of table 8, confirm, formula (I) compound has the strains of influenza viruses optimal efficacy of antagonism tool Drug resistance.
Although the present invention discloses as above with preferred embodiment; so it is not in order to limit the present invention, anyly has the knack of the technology of the present invention person, when can be without departing from the spirit and scope of the invention; do a little change and retouching, should belong to the protection domain that the present patent application the scope of the claims is defined.
Claims (9)
1. Radix Sanguisorbae (S.officinalis L.) extract is as the application for preparing anti-influenza virus medicament.
2. application as claimed in claim 1, wherein this Radix Sanguisorbae extract is to be obtained by the preparation of alcohol extraction Radix Sanguisorbae.
3. application as claimed in claim 2, wherein this Radix Sanguisorbae extract is to be obtained by the method preparation of the following step of tool:
(1) with the alcohol extraction Radix Sanguisorbae, to obtain ethanolic extract; And
(2) extract this ethanolic extract with methanol and normal hexane (1:1), to obtain the methanol extraction thing.
4. application as claimed in claim 2, wherein this Radix Sanguisorbae extract is to be obtained by the method preparation of the following step of tool:
(1) with the alcohol extraction Radix Sanguisorbae, to obtain ethanolic extract;
(2) extract this ethanolic extract with normal hexane and methanol (1:1), to obtain the methanol extraction thing; And
(3) extract this methanol extraction thing with dichloromethane and water (1:1), to obtain dichloromethane extract.
5. application as claimed in claim 2, wherein this Radix Sanguisorbae extract is to be obtained by the method preparation of the following step of tool:
(1) with the alcohol extraction Radix Sanguisorbae, to obtain ethanolic extract;
(2) extract this ethanolic extract with normal hexane and methanol (1:1), to obtain the methanol extraction thing; And
(3) extract this methanol extraction thing with dichloromethane and water (1:1), to obtain water extract; And
(4) extract this water extract with n-butyl alcohol and water (1:1),, to obtain n-butyl alcohol extract.
6. the salt that formula I compound, its hydrolysate or medicine can be accepted is as the application for preparing anti-influenza virus medicament:
7. application as claimed in claim 6, the hydrolysis compound that wherein said reactive compound is the formula I compound.
8. application as claimed in claim 7, wherein said hydrolysis compound is Mycophenolic Acid (Mycophenolic acid).
9. application as described as claim 1 or 6, the variant virus strain that wherein this influenza virus is H1N1, H3N2 or its tool Drug resistance.
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| WO2022215827A1 (en) * | 2021-04-08 | 2022-10-13 | 주식회사 큐제네틱스 | Sanguisorba officinalis linne extract composition inhibiting 3cl protease and rdrp activity of sars-cov-2 |
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