CN103156921A - Application of schisandra chinensis and extracts of schisandra chinensis in resisting severe acute respiratory syndrome (SARS) coronavirus infection - Google Patents
Application of schisandra chinensis and extracts of schisandra chinensis in resisting severe acute respiratory syndrome (SARS) coronavirus infection Download PDFInfo
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Abstract
The invention provides the application of schisandra chinensis and extracts of the schisandra chinensis in prevention or treatment of a severe acute respiratory syndrome (SARS) coronavirus infection. When the final concentration of the schisandra chinensis is 500 micrograms per milliliter, the inhibition ratio of isolated SARS coronavirus main protease by 95% ethanol extracts of the schisandra chinensis can reach 92%, and the inhibition ratio of the SARS coronavirus main protease by ethyl acetate extracts of the 95% ethanol extracts of the schisandra chinensis is 89.1%.
Description
Technical field
The present invention relates to pharmaceutical field, in particular to the application of Fructus Schisandrae Chinensis and extract thereof.
Background technology
Severe acute respiratory syndrome (severe acute respiratory syndrome, SARS) claim again severe acute respiratory syndrome, is that a kind of serious acute respiration systematic infection is sick.The SARS pathogen be coronavirus genus a kind of virus (Peiris J et al.Lancet, 2003,361:1319-1325).Coronavirus genus is under the jurisdiction of coronaviridae, coronavirus is positive chain RNA virus, and at present known positive chain RNA virus, their genome is maximum (Siddell, S.G et al.Coronaviruses, toroviruses and arteriviruses.in Topley ﹠amp; Wilson ' s Microbiology and Microbia Infections, 10
thEdition, Vol.Virology (eds.Mahy, B.W.J.﹠amp; Ter Meulen, V.) 823-856 (Hodder Arnold.London, 2005)).This genus contains 26 kinds of having an appointment; According to their natural reservoir (of bird flu viruses), gene order and serotype relation, this genus can be divided into again three cohorts: wherein the first cohort has transmissible gastro-enteritis virus (Porcine Transmissible Gastroenteritis Virus, TGEV) etc.; The second cohort has sars coronavirus etc.; The 3rd cohort has avian infectious bronchitis virus (Avian Infectious Bronchitis Virus, (the Spaan such as AIBV), W.J.M.and Cavanagh, D.Coronaviridae.in Virus taxonomy, VIIIth Report of the ICTV.945-62 (Elsevier-Academic Press., London, 2004).
The genome encoding of sars coronavirus 2/3 to 3/4 two replicase polyprotein (replicase polyproteins) ppla and pplab (Ziebuhr, Snijder et al.2000; Thiel, Herold et al.2001; Anand, Yang et al.2005; Ziebuhr 2005), they only just can make virus complete normal transcription, copy function (Ziebuhr, Snijder et al.2000 after the proteolytic cleavage of encoding viral is slit into independent subunit; Anand, Yang et al.2005; Bartlam, Yang et al.2005), sars coronavirus major protein enzyme (being called for short main protease, main protease) plays a major role in this process.If can suppress the hydrolysis of sars coronavirus main protease, will effectively resist sars coronavirus infecting human body so.Therefore, the main protease of sars coronavirus is a main target of anti-SARS drug screening.
Natural product claims again secondary metabolite, has structure diversity and biological activity multiformity.Natural product and derivant thereof have been brought into play promising effect in disease treatment in the past, also one of resource (Newman DJ, Gragg GM, the Snader KM.Nat Prod Rep of tool potentiality in current medicament research and development process, 2000,17 (3): 215-234; Lee K-H.J Nat Prod.2004,67 (2): 273-283).Now in the ascendant to the Natural products research in the conventional medicaments such as Chinese medicine, marine organisms and microbial metabolism, annual all can have the compound of a large amount of novel structures to be found, these compounds are the medicine that can't realize of synthetic method and the important source of lead compound, play an important role in the discovery of new drug and lead compound.Especially the research that derives from the natural product of Chinese medicine etc. for natural product is necessary, because the history that Chinese medicine was used in China in existing thousands of years, be a huge treasure-house of Medicine small molecule compound, the screening of therefore therefrom carrying out important virus or the relevant target protein inhibitor of important diseases in medicines natural products is necessary.
Chinese medicine Schisandra chinensis is the dry mature fruit of magnoliaceae schisandra (Schisandra chinensis (Turcz.) Baill.).Nature and flavor: sour, sweet, temperature.Return lung, the heart, kidney channel.Effect is astringent or styptic treatment for spontaneous sweating for convergence, supplementing QI for promoting the production of body fluid, kidney calming.Main chemical compositions is schizandrin (schizandrin), deoxidation schizandrin (de-oxyschizandrin), newly son element (neoschizandrin), schisandrol (schizan-drol), schizantherin (schisantherin, gomisin) A, B, C, D, E, F, G, H, J, K1, K2, K3, L1, L2, M1, M2, N2, O, R etc. simply.
Chen Jiancao etc. disclose in CN1539426A and have utilized plant polyphenol for the preparation of medicine and health product anti-SARS virus and treat and/or prevent disease due to SARS virus; The application of curcumin in the medicine of preparation treatment SARS disclosed in CN1568945A; The application of flavonol polyphenol in the medicine of preparation treatment SARS disclosed in CN1568968A.Shao Ningsheng etc. disclose in CN1443770A and have suppressed small RNA molecular and the action target spot thereof that SARS virus infects.The former Chinese medicine that discloses a kind for the treatment of and prevention SARS disease in CN1453030A of Zhao, it is made by the raw material of following weight proportioning, Radix Cynanchi Paniculati 3-10 part, Bombyx Batryticatus 3-10 part, Periostracum Cicadae 3-10 part, Rhizoma Dysosmae Versipellis 3-10 part, the blue or green 3-10 part of female, Rhizoma Osmundae 10-15 part, Semen Arecae 6-15 part, Cortex Magnoliae Officinalis 3-10 part, Fructus Tsaoko 3-6 part, Fructus Ligustri Lucidi 6-15 part, Radix Et Rhizoma Rhei 3-12 part.Described Chinese medicine can be made into oral drugs or the injection of water preparation, electuary, red ball, capsule, the patient that the SARS disease has been suffered from treatment.Wang Yifei etc. disclose the application of cepharanthine in the preparation SARS resisting medicine in CN1452967A.Gao Ping discloses heat clearing away, the moistening lung medicine of a kind of SARS for the treatment of in CN1552387A, be comprised of Herba Pogostemonis, thatch art, Talcum, Realgar, Radix Peucedani, Moschus, Cortex Magnoliae Officinalis, the Rhizoma Pinelliae, Radix Glycyrrhizae, Pericarpium Citri Reticulatae Viride, Fructus Trichosanthis, Bulbus Fritillariae Uninbracteatae; Heat clearing away, the antidote of a kind of SARS for the treatment of are disclosed in CN1552388A, by bulky, Cortex Magnoliae Officinalis, Fructus Tsaoko, Radix Glycyrrhizae, Radix Scutellariae, Flos Lonicerae, understand that vitriol, Fructus Arctii, the Rhizoma Anemarrhenae, Radix Bupleuri, Pericarpium Citri Reticulatae Viride form; Disclose the lung heat clearing medicine of a kind of SARS for the treatment of in CN1552390A, formed by the Rhizoma Anemarrhenae, Radix Glycyrrhizae, Radix Angelicae Sinensis, Radix Scutellariae, Pericarpium Citri Reticulatae, Radix Ophiopogonis, Cortex Lycii, Bulbus Lilii, Gypsum Fibrosum, Radix Bupleuri, the Rhizoma Pinelliae; Disclose antiinflammatory, the antipyretic of a kind of SARS for the treatment of in CN1552391A, formed by Pericarpium Citri Reticulatae, Flos Lonicerae, Radix Scutellariae, Cortex Phellodendri, Fructus Forsythiae, Radix Glycyrrhizae, Radix Aconiti Lateralis Preparata, Radix Bupleuri, Bulbus Fritillariae Cirrhosae, pollen, Radix Gentianae; A kind of Chinese medicine preparation that prevents SARS virus is disclosed in CN1552395A, wear into fine powder by Fructus Forsythiae, Radix Scutellariae, Radix Paeoniae Rubra, Radix Angelicae Sinensis, Flos Lonicerae, Herba Ephedrae, Radix Saposhnikoviae, Radix Et Rhizoma Rhei, Fructus Arctii, Folium Isatidis, pollen, Spina Gleditsiae, Herba Eupatorii, make Chinese powder medicine, this invention is on the basis of ancient secret recipe treatment pestilence in ancient times, the side of experience of continuous modification and perfection in the process of determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs.Xiao Wei etc. disclose the application of a kind of Chinese medicine composition in the medicine of preparation anti-SARS virus in CN1488380A, this kind Chinese medicine composition is made by raw materials such as Cornu Saigae Tataricae, Bulbus Fritillariae Ussuriensis, Radix Et Rhizoma Rhei, Radix Scutellariae, Lapis Chloriti, Gypsum Fibrosum, artificial Calculus Bovis and Radix Glycyrrhizaes.Yu Lingen discloses anti-SARS virus, has treated the Chinese medicine of cardiovascular and cerebrovascular vessel thromboembolism in CN1449817A, it consists of Rhizoma Typhonium Gigantei (Rhizoma Typhonii), Radix Aconiti Lateralis Preparata, Rhizoma Corydalis.Ding Runquan discloses a kind of Chinese medicine for the treatment of SARS virus in CN1454665A, mainly comprise Radix Scutellariae, Flos Lonicerae, Fructus Forsythiae, Folium Pyrrosiae, the Radix Astragali, Ganoderma, Radix Salviae Miltiorrhizae, Radix Paeoniae Rubra, Herba Eupatorii.Li Jianqing etc. disclose the purposes of Abiduoer in preparation prevention and treatment SARS virus medicine in CN1552321A, prove that by In vitro cell experiment Abiduoer has significant inhibitory action to SARS virus.Lou Shirong discloses the Chinese medicine for the treatment of and prevention SARS virus in CN1451434A, comprising the medicine that the raw material of Herba Solidaginis makes has fairly obvious specific aim to kill and inhibition to SARS virus and coronavirus.Luo Weisheng etc. disclose a kind of effect that the rhubarb anthraquinone compound of separation and Extraction has powerful inhibition SARS virus from Chinese herb rhubarb in CN1721387A, experiment in vitro shows, sars coronavirus 3C-likeproteinase (3C-protease) drug dose suppression ratio when the 100 μ g/mL is reached more than 85%.But relevant Fructus Schisandrae Chinensis there is not yet report in the research aspect the inhibition SARS virus.
Summary of the invention
In the process that Chinese medicine Schisandra chinensis is studied, we find that Fructus Schisandrae Chinensis has the effect that suppresses sars coronavirus main protease activity, it can be prepared into the sars coronavirus main proteinase inhibitor, thereby prevention or treatment sars coronavirus infect.
In one aspect, the invention provides the application of Fructus Schisandrae Chinensis in the medicine of preparation prevention or the infection for the treatment of sars coronavirus.
In above-mentioned application, described Fructus Schisandrae Chinensis refers to the dry mature fruit of magnoliaceae schisandra (Schisandra chinensis (Turcz.) Baill.).
In above-mentioned application, described Fructus Schisandrae Chinensis is through solvent extraction.Wherein, described solvent is selected from water, alcohol or its mixture.Described alcohol includes but not limited to methanol, ethanol, propanol, isopropyl alcohol, n-butyl alcohol, the tert-butyl alcohol, ethylene glycol etc., particular methanol or ethanol.In a specific embodiment, Fructus Schisandrae Chinensis obtains 95% ethanol extraction of Fructus Schisandrae Chinensis through 95% ethanol extraction.
In above-mentioned application, the Fructus Schisandrae Chinensis after extracting is further carried out organic solvent extraction.Wherein said organic solvent is selected from one or more in petroleum ether, chloroform, ethyl acetate or n-butyl alcohol.In a specific embodiment, the Fructus Schisandrae Chinensis after 95% ethanol extraction is obtained petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract with petroleum ether, ethyl acetate and n-butanol extraction successively.
In above-mentioned application, described Fructus Schisandrae Chinensis and extract thereof can be for the preparation of the sars coronavirus main proteinase inhibitors.
The method that the present invention adopts isolated activity to measure, Fructus Schisandrae Chinensis is carried out determination of activity, result shows, Fructus Schisandrae Chinensis and extract thereof have the effect of good inhibition sars coronavirus main protease activity, illustrate that Fructus Schisandrae Chinensis and extract thereof can become the potential drug of prevention or the infection for the treatment of sars coronavirus, perhaps become pharmaceutical composition with pharmaceutically acceptable suitable carrier well known in the art or vehicle group, be used for prevention or the infection for the treatment of sars coronavirus.
Description of drawings
Fig. 1 is that schisandra chinensis ethyl hydrate extract is to the activity inhibition curve chart of sars coronavirus main protease;
Fig. 2 is that each extract of schisandra chinensis ethyl hydrate extract is to the activity inhibition curve chart of sars coronavirus main protease;
Fig. 3 is the activity inhibition curve chart of each component to the sars coronavirus main protease after the segmentation of Fructus Schisandrae Chinensis ethyl acetate;
Fig. 4 separates 4 compounds obtaining the activity of sars coronavirus main protease is suppressed curve chart from Fructus Schisandrae Chinensis ethyl acetate extraction layer;
Fig. 5 is that deoxyschizandrin is to the active IC of the inhibition of sars coronavirus main protease
50Curve chart; And
Fig. 6 is that schisandrin B is to the active IC of the inhibition of sars coronavirus main protease
50Curve chart.
The specific embodiment
For the present invention is described better, below in detail the specific embodiment of the present invention will be described in detail.
First the ethanol extraction of Chinese medicine Schisandra chinensis carried out the screening of sars coronavirus main protease external activity, find that it has activity.Then its ethanol extraction is extracted segmentation, according to active guideline, active site is carried out separation and purification further, be intended to obtain to the inhibited particular compound of sars coronavirus main protease.
Chemical extraction separation and purification part
1) preparation of Fructus Schisandrae Chinensis extractum
Get a certain amount of schisandra chinensis medicinal material and carry out reflux, extract, with alcohol, water or its mixture, material medicine ratio is 2: 1～20: 1 (L/Kg), extracts 1～8 hour at every turn, extracts altogether 3～5 times.With extracting solution after filtering evaporated under reduced pressure get the extractum Fructus Schisandrae Chinensis extrat.
2) preparation of each extract of Fructus Schisandrae Chinensis
Each extract of Fructus Schisandrae Chinensis comprises petroleum ether extract, acetic acid ethyl ester extract and the n-butyl alcohol extract of schisandra chinensis ethyl hydrate extract.Their preparation process of brief description.
Get schisandra chinensis ethyl hydrate extract, after with 1～5 times of amount distilled water, it being disperseed, be poured in separatory funnel, use successively petroleum ether, ethyl acetate and n-butanol extraction, shake well, standing, isolate organic facies and water, every kind of solvent extraction 3 times merges the organic facies of every kind of solvent, and concentrate with Rotary Evaporators, obtain respectively Fructus Schisandrae Chinensis petroleum ether, ethyl acetate and the n-butyl alcohol extract of dried paste.
2) separation and purification of Fructus Schisandrae Chinensis acetic acid ethyl ester extract.
Under instructing, activity determines that the Fructus Schisandrae Chinensis acetic acid ethyl ester extract is the position that micromolecular inhibitor is mainly concentrated, we separate by various chromatographys such as silica gel column chromatography, Sephadex LH-20 gel filtration chromatography method, preparative HPLC repeatedly Fructus Schisandrae Chinensis ethyl acetate extraction layer, obtain a series of cyclohexyl biphenyl octene type lignan compounds.For brevity, the concrete method for separating and preparing of these compounds will provide in specific embodiment.
The pharmacologically active part of detecting
1.SARS the expression of coronavirus proteolytic, purification
According to document (Yang H et al.Proc Natl Acad Sci.2003 Nov; 100 (23): the expression and the purification that 13190-5) carry out the sars coronavirus main protease.Concrete grammar is as follows:
(1-1) structure of the expression vector of sars coronavirus main protease, concrete steps comprise:
A. utilize the cDNA library of the SARS virus strain that is numbered BJ01 that the large gene center of Beijing China provides, carry out amplification in vitro with round pcr;
Forward primer: 5 '-CGGGATCCAGTGGTTTTAGGAAAATG-3 '
Reverse primer: 5 '-CCGCTCGAGTCATTGGAAGGTAACACCAGA-3 '
B. through the genetic fragment of pcr amplification after BamHI and the two enzyme enzyme action of XhoI, reclaiming size with agarose gel electrophoresis is fragment about 1kb;
C. will reclaim fragment and be connected with the T carrier, and then transform colibacillus (Escherichia coli) DH5 α competent cell with connecting product, and be coated on LB flat board (containing the 100mg/L ampicillin) overnight incubation;
D. a plurality of monoclonals of picking from the flat board are inoculated in respectively and are equipped with approximately in the test tube of the LB of 5mL (add penbritin in this LB solution, making its final concentration is 100mg/L), overnight incubation.Then use plasmid extraction kit (vast Imtech Type B plasmid is the rapid extraction test kit in a small amount) to extract plasmid, and with BamHI and XhoI enzyme action, then reclaim with agarose gel the target gene fragment that size is about the 1kb left and right;
E. with destination carrier pGEX-4T-1 (available from Pharmacia company) BamHI and XhoI enzyme action, then reclaim the fragment of enzyme action with agarose gel;
F. the fragment that d is connected with e connects (mixes target gene segment and destination carrier fragment after the enzyme action recovery according to the molal quantity ratio of 3: 1～6: 1, requirement according to Takara DNA Ligation was reacted 30 minutes～18 hours at 16 ℃), transform colibacillus Escherichia coli DH5 α competent cell, be coated in the upper overnight incubation of LB flat board (containing the 100mg/L ampicillin).With the positive colony that screens, for the identification of and the order-checking.Sequencing result shows, the encoding gene of the main protease of sars coronavirus correctly is cloned in the pGEX-4T-1 carrier.
(1-2) expression and purification of sars coronavirus main protease, concrete steps comprise:
A. with the bacterial strain of the pGEX-4T-1 carrier Transformed E scherichia coli BL21 (DE3) that contains encoding SARS coronavirus proteolytic gene that obtains in above-mentioned steps (1-1), and with dull and stereotyped (the containing the 100mg/L ampicillin) screening positive clone of LB;
B. at picking positive colony on the LB flat board described in a (containing the monoclonal that grows out on the LB flat board of ampicillin), then overnight incubation changes the LB culture medium (containing the 100mg/L ampicillin) of 1L over to, works as OD
600When reaching 0.6-0.8, add the IPTG of 1mM left and right, cultivated about 12 hours at 16 ℃;
C.5000 the centrifugal 10～15min of～8000rpm, collecting cell, the then broken bacterium 20～30min of ice-bath ultrasonic; Collect supernatant after the broken centrifugal 20～40min of bacterium liquid 13000rpm～15000rpm;
D. supernatant is added in the GST affinity column (GE company) of PBS pre-equilibration, remove foreign protein with 20～30 bed volumes of PBS drip washing.The human rhinovirus's HRV 3CP that adds at last about 2ml 0.1mg/ml 4 ℃ of enzyme action 12～20 hours, is collected the sars coronavirus main protease afterwards;
E. use again Mono Q (GE company) anion-exchange chromatography to carry out purification the sars coronavirus main protease that obtains in step (1-2d), just can obtain the higher sars coronavirus main protease of purity.
2.SARS the screening technique of coronavirus proteolytic inhibitor
The method of screening sars coronavirus main proteinase inhibitor of the present invention is Rao Zi and disclosed screening technique in CN101418334A such as grade, and concrete grammar is as follows:
The determination of activity of sars coronavirus main protease is to use fluorogenic substrate MCA-AVLQSGFR-Lys (Dnp)-Lys-NH2 (purity greater than 95%, the biochemical company limited of Shanghai gill) to complete.The aminoacid sequence of this fluorogenic substrate derives from the N end of sars coronavirus main protease from shearing sequence.
The instrument that is used for fluorescent strength determining is Fluoraskan Ascent luminoscope (ThermoLabsystems, Helsinki, Finland), and exciting light and radiative wavelength are respectively 320nm and 405nm.
At buffer solution (50mM Tris-HCl (pH 7.3), 1mM EDTA (containing or do not contain DTT)) add sars coronavirus main protease (final concentration 0.5 μ M) in, add the DMSO solute of alternative sample (to make its final concentration be: 500 μ g/mL, concentration of substrate is 20 μ M, 298K placed after 10 minutes, add rapidly fluorescent labeling substrate (MCA-AVLQSGFRL (DNP) L-NH2, final concentration 20 μ M).Set contrast: do not add alternative sample, all the other conditions are identical.Excitation wavelength and emission wavelength are respectively 320nm and 405nm, and temperature keeps 298K, and record the first order fluorescence reading every 2 seconds, measures altogether 10 points.Take the time as X-axis, fluorescent value is that the Y-axis mapping just can obtain the movable mechanics curve of enzyme.The initial velocity V that just can obtain reacting by the numerical computations slope of scheming upper the first two point is defined as V with the initial velocity of reaction of negative control
0, add the initial velocity of reaction of alternative sample to be defined as V
iThereby, calculate the residual activity (V that adds protease after corresponding alternative sample
i/ V
0), the suppression ratio of corresponding alternative sample is (1-V
i/ V
0).Residual activity is carried out further separation and purification less than the alternative sample of 20% (or suppression ratio is greater than 80%), until be separated to pure compound.
3. compound suppresses active IC to the sars coronavirus main protease
50The mensuration of value
Each compound that solvent evaporates is done is dissolved into respectively the solution of 50mg/mL with 95% DMSO.The mentioned solution of getting 10 μ l carries out the gradient dilution of 2 times with 95% DMSO, be diluted to altogether the sample solution of 12 left and right.Residual activity with enzyme under the different chemical combination concentration of above-mentioned assay method test.Divided by the molecular weight of respective compound and get the denary logarithm value as X-axis, corresponding residual activity value is Y-axis, maps and calculates corresponding IC with GraphPad Prism 5 (GraphPad software company) with the compound concentration of dilution
50Value.
The following example is only used for explaining the present invention, and not should be understood to limit the scope of the invention by any way.
The preparation of embodiment 1 Fructus Schisandrae Chinensis 95% ethanol extraction
The schisandra chinensis medicinal material (the emerging Chinese crude drug company limited of Anhui Province's Bozhou City) of getting 2 kilograms carries out reflux, extract, three times with 8 liters of 95% ethanol, the each extraction 2 hours, extracting solution is merged after filtering, with EYELAN1001 type Rotary Evaporators (Japanese physics and chemistry company) concentrating under reduced pressure, after evaporate to dryness extractum 220 grams.
The preparation of petroleum ether, ethyl acetate and the n-butyl alcohol extract of embodiment 2 Fructus Schisandrae Chinensis 95% ethanol extractions
the schisandra chinensis ethyl hydrate extract of embodiment 1 gained is taken out 30g, suspend with 300 milliliters of distilled waters and disperseed in ultrasonic 2 hours, then be poured in 1 liter of separatory funnel, use successively petroleum ether according to organic solvent polarity, ethyl acetate, n-butyl alcohol extracts, every kind of solvent extraction 3 times, each with 500 milliliters of organic solvents, shake well, standing 3 hours, isolate the organic solvent phase and merge by the organic solvent kind, various organic solvent extraction liquid are concentrated with EYELAN1001 type Rotary Evaporators, obtain the Fructus Schisandrae Chinensis petroleum ether of dried paste, ethyl acetate, n-butyl alcohol extract, wherein obtain petroleum ether extract 4 grams, acetic acid ethyl ester extract 9 grams, n-butyl alcohol extract 6 grams.
The preparation of embodiment 3 deoxyschizandrins, schisandrin B, schisantherin A and schisandrin
After 9 gram acetic acid ethyl ester extract dissolvings, admix 8 gram silica gel (column chromatography 200-300 order), use silica gel column chromatography post (100 gram column chromatography silica gel 200-300 order) to separate, adopt petroleum ether-acetone solvent system as mobile phase, carry out eluting at 1: 1 with petroleum ether-acetone 30: 1, petroleum ether-acetone 20: 1, petroleum ether-acetone 10: 1, petroleum ether-acetone 5: 1, petroleum ether-acetone 2: 1, petroleum ether-acetone respectively, each gradient elution 3-5 column volume.Each fraction is carried out thin layer chromatography detect analysis, merge and form close component.Obtain 6 components, represent with E1-E6 respectively.According to the determination of activity result, determine that E2 component and E4 component are the position that micromolecular inhibitor is mainly concentrated.
E2 is carried out thin-layer chromatographic analysis, continuation separates it with silica gel column chromatography, adopt petroleum ether-acetone solvent system as eluant, use respectively petroleum ether-acetone 25: 1, 20: 1, 15: 1, 10: 1, carry out eluting at 5: 1, each gradient elution 4-5 column volume, merge according to the eluant gradient, the component 1 (being the concentrated part that obtains of eluant at 25: 1 with petroleum ether-acetone) that merging obtains is carried out adopting silica gel column chromatography to separate after thin-layer chromatographic analysis, use petroleum ether-ethyl acetate as eluant to carry out eluting at 10: 1, obtain respectively compound 1 (12mg) and compound 2 (15mg).The component 2 that merging is obtained (being at 20: 1 the concentrated part that obtains of eluant with petroleum ether-acetone) carries out adopting silica gel column chromatography to separate after thin-layer chromatographic analysis, use petroleum ether-acetone system 5: 1 repeatedly eluting obtain compound 4 (10mg).
Partly adopt methanol to carry out recrystallization E4, the adularescent insoluble matter carries out sucking filtration, then the sucking filtration thing is continued recrystallization with methanol, obtains white crystalline compound 3 (9mg).
These four compounds are identified:
Compound 1:C
24H
32O
6, white unformed powder
1H?NMR(CDCl
3，400MHz，δ，ppm)6.56(2H，d，J＝3.2Hz，H-4，H-11)，3.91(12H，m，H-1，3，12，14-OCH
3)，3.61(6H，d，J＝1.6Hz，H-2，13-OCH
3)，2.61(2H，m，H-6)，2.59(2H，m，H-6)，2.31(1H，dd，J＝13.2Hz，3.6Hz，H-9αH)，2.08(1H，d，J＝12.8Hz，H-9βH)，1.93(1H，m，8-H)，1.83(1H，m，7-H)，1.02(3H，d，J＝6.8Hz，8-Me)，0.76(3H，d，J＝6.8Hz，7-Me)；
13C?NMR(CDCl
3，150MHz，δ，ppm)151.5(C-1)，140.1(C-2)，152.9(C-3)，107.2(C-4)，139.2(C-5)，35.6(C-6)，40.8(C-7)，33.8(C-8)，39.1(C-9)，133.9(C-10)，110.5(C-11)，15.5(C-12)，139.7(C-13)，151.6(C-14)，123.4(C-15)，122.3(C-16)，12.7(C-17)，21.8(C-18)，60.9×3，60.5，55.9，55.8(-OCH
3)。
Compound 1
1H NMR,
13Deoxyschizandrin data consistent (the ZHAO Jing-feng of the people such as C NMR data and Zhao report, LI Liang, et al.Studies on the Chemical Constituents of Schisandra (II), Yunnan University's journal, 1999,314-316), be defined as deoxyschizandrin.
Compound 2:C
23H
28O
6, white unformed powder
1H?NMR(CDCl
3，400MHz，δ，ppm)6.57(1H，s，H-4)，6.50(1H，s，H-11)，5.96(12H，s，1，12，13，14-OCH
3)，3.92(3H，s)，3.90(3H，s)，3.85(3H，s)，3.57(3H，s)，0.99(3H，d，J＝7.2Hz，H-9)，0.75(3H，d，J＝6.8Hz，H-10)；
13C?NMR(CDCl
3，150MHz，δ，ppm)151.6(C-5)，151.5(C-1)，148.7(C-10)，141.1(C-14)，140.1(C-16)，137.8(C-3)，134.6(C-15)，134.1(C-12)，123.3(C-13)，121.4(C-2)，110.6(C-4)，102.9(C-11)，100.7(C-2，3-O-)，61.0，60.5，59.6，55.9(C-1，12，13，14-OCH
3)，40.7(C-7)，39.1(C-9)，35.5(C-6)，33.6(C-8)，21.5(C-8-CH
3)，12.8(C-7-CH
3)。
Compound 2
1H NMR,
13The schisandrin B data consistent of the report such as C NMR data and Shi Lin (Shi Lin, He Xiaoxia, Pan Ying etc., the research of Fructus Schisandrae Chinensis rattan chemical composition, Chinese herbal medicine, 2009,1707-1710), be defined as schisandrin B.
Compound 3:C
30H
32O
9, white crystals
1H?NMR(CDCl
3，400MHz)7.57-7.35(5H，m，OBen-H)，6.93(1H，s，H-4)，6.61(1H，s，H-11)，5.79，5.71(2H，d，1.3Hz，H-OCH
2O-)，5.78(1H，s，H-6)，3.95，3.85，3.47，3.29(12H，s，4-OCH
3)，2.60(1H，dd，14Hz，9.6Hz，H-9βH)，2.18(1H，d，13.6Hz，H-9α)，2.08(1H，m，H-8)，1.36(3H，s，Me-17)，1.20(3H，d，7.2Hz，H-Me-18)；
13C?NMR(CDCl
3，150MHz，δ，ppm)164.9，151.5，151.4，149.0，135.8，134.2，132.9，131.9，129.3，129.1，127.4，122.0，121.2，111.5，102.0，100.4，85.3，78.1，71.9，59.8，59.7，57.6，55.1，47.0，42.8，35.9，27.7，17.8。
Compound 3
1H NMR,
13The schisantherin A data consistent that C NMR data and Xu Xiumei etc. deliver (Xu Xiumei, Li Lei etc., the research of hair leaf fructus schizandrae chemical composition, Chinese crude drug, 2009,1399-1401), be defined as schisantherin A.
Compound 4:C
24H
32O
7, white crystals
1H?NMR(CDCl
3，400MHz，δ，ppm)6.61(1H，s)，6.54(1H，s)，3.91(3H，s)，3.90(3H，s)，3.89(3H，s)，3.88(3H，s)，3.59(3H，s)，3.58(3H，s)，2.65(2H，m)，2.37(2H，m)，1.88(2H，m)，1.56(1H，s)，1.26(3H，s)，0.99(3H，d，J＝7.2Hz)；
13CNMR(CDCl3，100MHz，δ，ppm)152.4，152.0，151.8，151.5，140.8，140.2，133.8，131.8，124.2，122.8，110.4，110.0，61.0，60.64，60.60，59.6，56.0，55.9，41.9，40.9，34.3，29.8，15.9。MS-ES
+：455(M+Na)；MS-ES
-：431(M-1)
Compound 4
1The schisandrin data consistent that H NMR data and Chen Wansheng etc. deliver (Chen Wansheng, Fan Wei etc., the research of Radix Polygoni Multiflori Preparata chemical composition, the The 2nd Army Medical College journal, 1999,20 (7), 438-440) be defined as schisandrin.
Embodiment 4 Fructus Schisandrae Chinensis extrats suppress active mensuration to the sars coronavirus main protease
With schisandra chinensis ethyl hydrate extract, petroleum ether extract, acetic acid ethyl ester extract and the n-butyl alcohol extract that obtains in embodiment 1 and 2, be dissolved into respectively the solution of 50mg/mL with 95% DMSO.
According to the screening technique in pharmacologically active part of detecting part 2, schisandra chinensis ethyl hydrate extract and each extract are successively carried out active testing, obtain each extract to the activity inhibition curve of SARS main protease, respectively as depicted in figs. 1 and 2.Calculate according to Fig. 1: under 500 μ g/mL concentration, schisandra chinensis ethyl hydrate extract is 92.0% to the inhibition activity of sars coronavirus main protease, calculate according to Fig. 2: under 500 μ g/mL concentration, Fructus Schisandrae Chinensis petroleum ether, Fructus Schisandrae Chinensis ethyl acetate and Fructus Schisandrae Chinensis n-butyl alcohol extract are respectively 47.0%, 89.1% and 20.5% to the suppression ratio of sars coronavirus main protease, hence one can see that, and the Fructus Schisandrae Chinensis acetic acid ethyl ester extract is best to the inhibition activity of sars coronavirus main protease, can carry out further separation and purification to it.
E1-E6 component in embodiment 5 Fructus Schisandrae Chinensis acetic acid ethyl ester extracts suppresses active mensuration to the sars coronavirus main protease.
The employing screening technique identical with embodiment 4 carries out active testing to the component E1-E6 that separates in acetic acid ethyl ester extract, acquisition suppresses curve as shown in Figure 3 to the activity of SARS main protease, obtain following result according to curve calculation: under 500 μ g/mL concentration, the suppression ratio of E1-E6 is respectively 54.7%, 95.2%, 42.4%, 87.1%, 38.6% and 28.0%, has inhibiting micromolecular compound mainly to be present in component E2 and component E4 to the sars coronavirus main protease in hence one can see that Fructus Schisandrae Chinensis acetic acid ethyl ester extract.
Embodiment 6 deoxyschizandrins, schisandrin B, schisantherin A and schisandrin suppress active mensuration to the sars coronavirus main protease
Adopt the screening technique identical with embodiment 4 to carry out primary dcreening operation to four compounds that are separated in embodiment 1, obtain these four compounds the activity of SARS main protease is suppressed curve as shown in Figure 4, calculate according to curve, obtain following result: under 500 μ g/mL concentration, deoxyschizandrin, schisandrin B, schisantherin A and schisandrin are respectively 92.9%, 96.7%, 57.2% and 39.7% to the suppression ratio of the coronavirus proteolytic of SARS, further deoxyschizandrin and schisandrin B are carried out IC
50PH-value determination pH.
The deoxyschizandrin and the schisandrin B that obtain in embodiment 4 are dissolved into respectively the solution of 50mg/mL with 95% DMSO.The mentioned solution of getting 10 μ l carries out the gradient dilution of 2 times with 95% DMSO, be diluted to altogether the sample solution of 12 left and right concentration, concentration is respectively: 50mg/mL, 25mg/mL, 12.5mg/mL, 6.25mg/mL, 3.125mg/mL, 1.5625mg/mL, 0.7812mg/mL, 0.3906mg/mL, 0.1953mg/mL, 0.09765mg/mL, 0.04882mg/mL and 0.02441mg/mL.The residual activity value that adopts the method identical with top described screening technique to test the protease under each compound variable concentrations.Divided by the molecular weight of respective compound and get the denary logarithm value as X-axis, corresponding residual activity value is mapped and calculates corresponding IC with GraphPad Prism 5 as Y-axis with the compound concentration of dilution
50Be worth, obtain the IC of deoxyschizandrin and schisandrin B
50Value is respectively 260.6 μ M and 61.8 μ M, the active IC of the inhibition of these two compounds
50Curve chart is seen respectively Fig. 5 and Fig. 6.
Can find out according to above-described embodiment, the E2 in schisandra chinensis ethyl hydrate extract, its acetic acid ethyl ester extract, acetic acid ethyl ester extract and E4 component and Fructus Schisandrae Chinensis deoxyschizandrin and schisandrin B have good inhibition activity to the sars coronavirus main protease.Show that Fructus Schisandrae Chinensis and extract thereof and corresponding extract can for the preparation of the sars coronavirus main proteinase inhibitor, infect thereby can prevent or treat sars coronavirus.
Although illustrated and described embodiments of the invention, for the ordinary skill in the art, be to be understood that without departing from the principles and spirit of the present invention and can carry out multiple variation, modification, replacement and modification to these embodiment, therefore, top description is intended to for explanation rather than is used for restriction.So scope of the present invention should not determine with reference to above-mentioned description, and should determine with reference to the determined four corner of doctrine of equivalents that following claims and these claim are enjoyed.
Claims (9)
1. the application of Fructus Schisandrae Chinensis in the medicine of preparation prevention or the infection for the treatment of sars coronavirus.
2. application according to claim 1, wherein, described Fructus Schisandrae Chinensis is Chinese medicine Schisandra chinensis (Schisandra chinensis (Turcz.) Baill.).
3. application according to claim 1, wherein, described Fructus Schisandrae Chinensis is through solvent extraction.
4. application according to claim 3, wherein, described solvent is selected from water, alcohol or its mixture.
5. application according to claim 4, wherein, described alcohol is methanol or ethanol.
6. application according to claim 3, wherein, described solvent is 95% ethanol.
7. application according to claim 3 wherein, is further carried out organic solvent extraction to the Fructus Schisandrae Chinensis after solvent extraction.
8. application according to claim 7, wherein, described organic solvent is selected from one or more in petroleum ether, chloroform, ethyl acetate or n-butyl alcohol.
9. according to claim 1-8 described application of any one, wherein, described Fructus Schisandrae Chinensis is for the preparation of the sars coronavirus main proteinase inhibitor.
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