CN1830449A - Medical use of flavine-3-beta galactoside and its preparation - Google Patents

Abstract

An medical application of pentahydroxy flavoe-3-beta-galactoside, which can suppress the 3CL proteinase of SARS virus and HcoV-229E antigen-type cold virus, in preparing the medicine for preventing and treating SARS and cold is disclosed. Said medicine is also disclosed.

Description

The medical usage and the preparation thereof of Tricetin-3-beta galactose glycosides

Technical field

The present invention relates to the medical usage of Tricetin-3-beta galactose glycosides, be specifically related to the 3CL protease economic benefits and social benefits inhibitor of this chemical compound, the application in the medicine of SARS that preparation control coronavirus causes and cold disease as SARS virus and HCoV-229E antigenic type cold virus.The invention still further relates to this chemical compound is the pharmaceutical preparation of active component.

Background technology

Tricetin-3-beta galactose glycosides (English name is quercetin-3-d-galactoside or Hyperin or Hyperoside) is commonly called as hyperin or hyperin or hyperin or quercetin-3-galactoside, and its molecular formula is C ₂₁H ₂₀O ₁₂, molecular weight is 464, the CAS registration number is 482-36-0; Structural formula is as follows:

This chemical compound can extract from natural plants such as Radix Hyperici Monogyni (Herba Hyperici Monogyni), mossberry (cranberry), Flos abelmoschi manihot, Herba pyrolae japonicae and Fructus Crataegi and obtain, and has free radical resisting, antioxidation, and recent findings also has the analgesia function.

HCoV-229E antigenic type coronavirus is a kind of of common cold virus, and it is the cause of disease that causes human upper respiratory tract infection, often causes people's common cold, and can infect each age group.Human about 30% flu is caused by this virus, mainly occurs in winter and early spring.

SARS virus is a kind of new coronavirus, is the single strand RNA virus of normal chain, duplicates the intermediate without DNA, uses standard cipher.This virus be infectious atypical pneumonia (claim severe acute respiratory syndrome again, SevereAcute Respiratory Syndrome, pathogen SARS), by World Health Organization (WHO) in definite designation on April 16 in 2003.

Discover, all exist 3CL protease in SARS virus and the HCoV-229E antigenic type cold virus, this protease plays crucial regulating and controlling effect in the whole life cycle of virus, have only after the polyprotein of expressing viral is cut systematic function albumen by 3CL protease, virus just can be finished self transcribe, copy function (K.Anand, J.Ziebuhr etc., Science, 2003,300:1763-1767).Therefore this protease is an ideal drug design target spot, and the research of 3CL protease is also just become one of main direction of research and development treatment common cold and SARS medicine.If can effectively suppress this protease activities, just can blocking virus duplicating in vivo, thereby reach the purpose of the disease that human flu of treatment and SARS etc. cause by coronavirus.Up to now, flu and SARS still do not have specific treatment and preventive means.Particularly sars coronavirus is infected, still do not have the specificity antivirus therapy, and the antiviral therapy method and the medicine of bibliographical information coronavirus infection are seldom arranged.

Summary of the invention

The new medical usage that the purpose of this invention is to provide chemical compound Tricetin-3-beta galactose glycosides, promptly this chemical compound is preparing the application that prevents and treats in common cold and the SARS medicine as the 3CL protease economic benefits and social benefits inhibitor of SARS virus and cold virus.

It is the pharmaceutical preparation of main active that another object of the present invention is to provide with Tricetin-3-beta galactose glycosides.

Tricetin-3-beta galactose glycosides can extract preparation from natural plants such as Radix Hyperici Monogyni (Herba Hyperici Monogyni), mossberry (cranberry), Flos abelmoschi manihot, Herba pyrolae japonicae and Fructus Crataegi: material plant through 70～95% alcohol reflux, sucking filtration, concentrate after, obtain the ethyl acetate part with ethyl acetate extraction, the ethyl acetate part is through polyamide column chromatography (using the alcohol-water gradient elution), merge the ethanol elution part, get yellow solid after the drying.Through silica gel column chromatography (using the chloroform-methanol gradient elution), obtain the Tricetin-3-beta galactose glycosides of yellow powder shape again.Concrete operation method reference literature (X.Yan, B.Murphy, G.B.Hammond etc., Antioxidant Activities and Antitumor Screening of Extracts from Cranberry Fruit (Vaccinium macrocarpon), J.Agric.Food Chem., 2002,50:5844-5849).

The present invention is by computer simulation SARS and 3CL protease of HCoV-229E antigenic type cold virus and discovering of Tricetin-3-beta galactose glycosides molecular docking, this chemical compound can act on strong combination by hydrogen bond and hydrophobic etc. with the 3CL protease in above-mentioned two kinds of viruses, thereby can suppress 3CL protease activities in these two kinds of viruses.Simultaneously, result of calculation shows that Tricetin-3-beta galactose glycosides is very approaching to the inhibition constant of the 3CL protease in these two kinds of viruses, the inhibition ability is relatively more balanced.Therefore Tricetin-3-beta galactose glycosides can play simultaneously and suppress the effect that SARS virus and HCoV-229E antigenic type cold virus are duplicated, can be used as the 3CL protease economic benefits and social benefits inhibitor of these two kinds of viruses, and then can be used to prepare the medicine of anti-simultaneously SARS of treatment and flu.

Below by molecular biophysics experiment further specify Tricetin-3-beta galactose glycosides to the inhibition of 3CL protease in SARS virus and the HCoV-229E antigenic type cold virus active with combine activity:

Experiment one, Tricetin-3-beta galactose glycosides to the inhibition of the 3CL protease of SARS virus active with combine determination of activity

1, experimental principle:

The fluorogenic substrate of 3CL protease according to the specificity of 3CL protease substrate cutting (core sequence be Leu-Gln-↓-Ser) design is synthetic.The aminoacid sequence of substrate is KNSTLQSGLRKE.On the lysine (K) at dodecapeptide two ends and glutamic acid (E) side chain residue, be connected respectively fluorophor 5 '-(2 '-aminoethyl amino naphthalenes-1-sulfonic acid) (EDANS) and quenching group 4 '-(4 '-dimethylamino phenylazide) benzoic acid (Dabcyl).EDANS and Dabcyl are right for fluorescence-quencher molecule commonly used, and the suitableeest excitation wavelength is 340nm, and the suitableeest emission wavelength is 490nm.Dabcyl to the cancellation efficient of EDANS greater than 95%, very low with fluorescent method detection background value.When not adding 3CL protease, the distance between two fluorophors is very short, about 10-100 .Under the optical excitation state, the energy of EDANS is by the Dabcyl cancellation, and this quantum appearance is called resonance energy and shifts.After the cutting of protease specificity, the peptide substrate fracture, fluorophor separates with quenching group, recover the whole fluorescence of itself, so the peptide substrate of a low fluorescence cuts the material that becomes high fluorescence after the reaction through enzyme action, and the degree of the increase of fluorescence intensity and polypeptide hydrolysis is linear correlation.

2, experiment material and instrument

Fluorogenic substrate Dabcyl-KNSTLQSGLRKE-Edans: give birth to worker's biotechnology Services Co., Ltd by Shanghai and synthesize;

The fluorogenic substrate mother solution: with autoclaved buffer (20mM Tris-HCl, pH7.4,120mM NaCl) preparation, final concentration is 100 μ M.Be distributed into aliquot, frozen standby in-20 ℃;

Tricetin-3-beta galactose glycosides mother solution: with the 100%DMSO preparation, final concentration is 100mM, and 4 ℃ of preservations are standby;

HBS-EP working buffer liquid (10mM Hepes, 150mM NaCl, 3mM EDTA and 0.005% (v/v) surfactant P20, pH7.4);

Other chemical reagent: analytical pure, available from Sigma company;

U-2010 ultraviolet spectrophotometer (HITACHI company);

TECAN GENios microplate reader (Invitrogen company);

Biacore 3000 (utilizing the biosensor of surface plasma resonance technology) (Biacore AB company, Uppsala, Sweden);

The CM5 chip (BiacoreAB company, Uppsala, Sweden)

3, experimental technique:

3.1, SARS virus 3CL protease expression and purification

According to literature method (Haifang Sun, Haibin Luo, Changying Yu, Tao Sun etc, Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like Proteinase ofSARS coronavirus, Protein Expression and Purification, 2003,302-308) with pQE30 carrier (Qiagen company) construction expression plasmid pQE30-3CL, be converted among the escherichia coli M15 (Qiagen company) and express, the method purification by the NTA-Ni column chromatography obtains sars coronavirus 3CL protease.The protease assay of prepared fresh is at the absorbance value at 280nm wavelength place and be used for the IC of inhibitor after calculating protein concentration ₅₀PH-value determination pH.

3.2, Tricetin-3-beta galactose glycosides suppresses the IC of SARS virus 3CL protease ₅₀Measure

With Tricetin-3-beta galactose glycosides mother solution dilution, obtain Tricetin-3-beta galactose glycosides solution that concentration is followed successively by 100mM, 20mM, 10mM, 2mM, 1mM, 200 μ M, 100 μ M, 20 μ M, get each concentration Tricetin-3-beta galactose glycosides solution 1.2 μ l, 2 μ M 3CL protease with 120 μ l fully mix (final concentration of DMSO is 0.5%) respectively, hatch 2 hours at 4 ℃.Getting each concentration sample 100 μ l then is added in 96 orifice plates, the fluorogenic substrate solution that adds 100 μ l, 20 μ M more respectively begins reaction, this moment, the final concentration of 3CL protease was 1 μ M, the final concentration of fluorogenic substrate is 10 μ M, and the final concentration of chemical compound is respectively 500 μ M, 100 μ M, 50 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M.With the optical excitation of 340nm wavelength, the detection emission wavelength is that the fluorescent value under the 488nm changes, and reaction METHOD FOR CONTINUOUS DETERMINATION 1 hour is finished Tricetin-3-beta galactose glycosides to the inhibiting mensuration of SARS virus 3CL protease.Measure Compound I C ₅₀Set blank during value, promptly do not add chemical compound, but add the 3CL protease sample of same concentrations DMSO and the sample of independent fluorogenic substrate solution.Each sample is established multiple hole, and measured value is averaged.Under variable concentrations chemical compound existence condition, the response speed of SARS virus 3CL protease enzyme action fluorogenic substrate, compare with the response speed that does not add Tricetin-3-beta galactose glycosides but add 0.5%DMSO, calculate suppression ratio under each concentration (with the enzymatic activity that contains under the 0.5%DMSO condition is 100%, relative activity * 100% of the enzyme under the different inhibitor concentration of suppression ratio=100%-).Carry out nonlinear fitting according to the Logistic formula with origin software and calculate the IC that Tricetin-3-beta galactose glycosides suppresses the 3CL proteinase activity ₅₀Value.Formula is as follows: $A\left(I\right)/{\mathrm{<figure-callout\; id="0"\; label="A"\; filenames="A20051002434900112.png"\; state="\{\{state\}\}">A</figure-callout>}}_0=1-\frac{1}{1+{(I/{\mathrm{IC}}_{50})}^p}$ In the above-mentioned formula, A ₀Enzymatic activity when referring to add 0.5%DMSO, A (I) refers to the enzymatic activity under different Tricetins-3-beta galactose glycosides concentration, and I refers to the concentration of Tricetin-3-beta galactose glycosides, and p refers to the μ factor.

3.3, Tricetin-3-beta galactose glycosides combines active mensuration with SARS virus 3CL protease

Based on surface plasma resonance (SPR) principle, research SARS virus 3CL protease combines character with the enzyme kinetics of Tricetin-3-beta galactose glycosides.

3.3.1, the coupling of SARS virus 3CL protease

Biacore 3000 is steady to baseline with HBS-EP working buffer liquid balance.SARS virus 3CL protease is coupled on the Fc4 passage of CM5 chip.Set coupling method (standard amino coupled method) and link coupled aim parameter (4000RU) with the New Application Wizard in the Biacore software, carry out the coupling of 3CL protease.Protease is coupled at 25 ℃ to carry out.0.2M N-ethyl-N '-dimethyl aminopropyl carbodiimide (N-ethyl-N '-dimethylaminopropylcarbodiimide) and 50mM N-hydroxy-succinamide (EDC/NHS) mixed with 1: 1 ratio, 5 μ l/min sample introductions 7 minutes are with the activation chip surface.It is 25 μ g/ml that SARS virus 3CL protease is diluted to final concentration with 10mM sodium acetate (pH4.3), with 5 μ l/min flow velocity sample introductions, to the coupling amount be about 4000RU.At last, with 1M diethanolamine hydrochloride (pH8.5),, carry out the sealing of chip surface with 5 μ l/min flow velocity sample introductions 7 minutes, to final coupling amount be 4000RU.

3.3.2, kinetic determination

After the coupling of SARS virus 3CL protease was finished, equilibrate overnight was steady to baseline, carried out kinetic determination then.Working buffer liquid is with HBS-EP (containing 0.4%DMSO), Tricetin-3-beta galactose glycosides is made into different Concentraton gradient (80 μ M, 56 μ M, 39.2 μ M, 27.4 μ M, 19.2 μ M, 13.4 μ M and 9.4 μ M), with 30 μ l/min sample introduction 2min, the 4min that dissociates stablizes 4min with same buffer then.After obtaining Tricetin-3-beta galactose glycosides and the interactional sensing figure of SARS virus 3CL protease (Fig. 2), the steady-state model in the reuse Biacore analysis software (1: 1 Langmuir model) is analyzed the equilibrium dissociation constant (K of Tricetin-3-beta galactose glycosides and SARS virus 3CL protease _D).

4, experimental result

Measurement result is carried out computational analysis, and Tricetin-3-beta galactose glycosides is to the active IC of the inhibition of the 3CL protease of SARS virus ₅₀Value is 42.8 μ M, equilibrium dissociation constant K _DFor with 38.4 μ M.Suppress active measurement result and see Fig. 1.

Experiment two, Tricetin-3-beta galactose glycosides to the inhibition of the 3CL protease of HCoV-229E antigenic type cold virus active with combine determination of activity

1, experimental principle and experiment material, instrument are with experiment one.

2, experimental technique

2.1, the 3CL protease of HCoV-229E antigenic type cold virus expression and purification

According to literature method (Lili Chen, Chunshan Gui, Xiaomin Luo, Qingang Yang, etc.The old drugcinanserin is an inhibitor of the 3CL proteinase of SARS coronavirus and strongly reduces virusreplication in vitro.Journal of Virology, 2004, (accepted)) with carrier pGEX-4T-1 (Amersham company) construction expression plasmid pGEX-4T-1-3CL, be converted among the escherichia coli BL 21 (Promega company) and express, obtain the 3CL protease of HCoV-229E virus by the method purification of glutathion-sepharose 4B (Amersham-Pharmacia company) column chromatography.The protease assay of prepared fresh is at the absorbance value at 280nm wavelength place and be used for the screening and the IC of inhibitor after calculating protein concentration ₅₀PH-value determination pH.

2.2, Tricetin-3-beta galactose glycosides suppresses the IC of HCoV-229E antigenic type cold virus 3CL protease ₅₀Assay method and Tricetin-3-beta galactose glycosides combines active assay method with experiment one with HCoV-229E antigenic type cold virus 3CL protease, but when being coupled on the Biacore 3000CM5 chip HCoV-229E antigenic type cold virus 3CL protease to be diluted to final concentration be 10 μ g/ml.

3, experimental result

Measurement result is carried out computational analysis, and Tricetin-3-beta galactose glycosides is to the active IC of the inhibition of the 3CL protease of HCoV-229E antigenic type cold virus ₅₀Value is 52.1 μ M, equilibrium dissociation constant K _DFor with 10.8 μ M.Suppress active measurement result and see Fig. 3, the interactional sensing figure of 3CL protease of chemical compound and HCoV-229E sees Fig. 4.

Experiment one and experiment two result show, Tricetin-3-beta galactose glycosides to SARS virus 3CL protease and and the enzyme of the 3CL protease of HCoV-229E antigenic type cold virus live and suppress constant IC ₅₀The value and with these two bonded dissociation constant K of 3CL protease _dAll reach 10 ^-5The order of magnitude, visible Tricetin-3-beta galactose glycosides is the better inhibitor of the 3CL protease of SARS virus and HCoV-229E antigenic type cold virus.

As active component, be mixed with different pharmaceutical dosage forms with pharmaceutically acceptable excipient, carrier or diluent with Tricetin-3-beta galactose glycosides according to a conventional method.As injection or oral formulations.

Among the present invention, the effective content of Tricetin in the injection-3-beta galactose glycosides is 100mg/ml; The effective content of Tricetin in the oral formulations-3-beta galactose glycosides is 250mg/10ml.

Oral formulations can be made by pharmaceutical carrier and excipient commonly used in Tricetin-3-beta galactose glycosides and vitamin C and the oral formulations.Wherein pharmaceutical carrier and excipient comprise lactose; Starch and derivatives class thereof such as corn starch, carboxymethyl starch etc.; Cellulose derivative such as methyl and ethyl cellulose; Gelatin; Mineral-type chemical compound such as light magnesium oxide, Pulvis Talci etc.; Oil: as vegetable oil, Semen Sesami wet goods; Comprise agent such as beta-schardinger dextrin-; And di-alcohols such as Polyethylene Glycol.Oral formulations can be made tablet, capsule, Emulsion, solution etc., also can be applicable to solid dispersion sheet, Liposomal formulation, targeting preparation and controlled release preparation etc.In tablet, can make effervescent tablet with the disintegrating agent that tartaric acid or the acid of inflexible rubber mix composition with containing effective dose Tricetin-3-beta galactose glycosides, when meeting water, produce carbon dioxide and make the rapid disintegrate of tablet.

The canonic form of ejection preparation is injectable powder, suspension type injection etc., and the preparation medicine acceptable diluent that injection adopted can adopt isotonic saline solution, 5% D/W, water for injection, Oleum Camelliae, soybean oil, Oleum Sesami, ethanol, glycerol, propylene glycol, Polyethylene Glycol, benzyl benzoate, polyoxyethylene castor oil; Tween 80, lecithin, NaTDC, egg yolk lecithin; Acetic acid, sodium acetate, lactic acid, citric acid, tartaric acid and sodium salt, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate; Gelatin, sodium carboxymethyl cellulose, pectin, sorbitol solution, benzyl alcohol, butyl p-hydroxybenzoate and ethyl ester, chlorobutanol; Lignocaine; Cephalin, soybean phospholipid etc.

Beneficial effect

1, the invention provides the new medical usage of known compound Tricetin-3-beta galactose glycosides, excavated the purposes of this chemical compound, for the antiviral drugs of development control common cold and SARS has been opened up road as the 3CL protease economic benefits and social benefits inhibitor of SARS virus and cold virus.

2, Tricetin-3-beta galactose glycosides is present in the plants such as edible fruit, has reliable safety, and no obvious toxic-side effects utilizes this chemical compound can prepare little control common cold of side effect and the antiviral drugs of SARS.

Description of drawings

Fig. 1 is that Tricetin-3-beta galactose glycosides is to the active diagram of the inhibition of SARS virus 3CL protease.

Fig. 2 is Tricetin-3-beta galactose glycosides and the active sensing figure that combines of SARS virus 3CL protease, and the concentration of Tricetin-3-beta galactose glycosides is followed successively by 80 μ M, 56 μ M, 39.2 μ M, 27.4 μ M, 19.2 μ M, 13.4 μ M and 9.4 μ M from top to bottom.

Fig. 3 is that Tricetin-3-beta galactose glycosides is to the active diagram of the inhibition of the 3CL protease of cold virus HCoV-229E.

Fig. 4 is Tricetin-3-beta galactose glycosides and the active sensing figure that combines of the 3CL protease of cold virus HCoV-229E, and the concentration of Tricetin-3-beta galactose glycosides is followed successively by 80 μ M, 56 μ M, 39.2 μ M, 27.4 μ M, 19.2 μ M, 13.4 μ M and 9.4 μ M from top to bottom.

The specific embodiment:

The present invention is further elaborated below in conjunction with specific embodiment, but do not limit the present invention.

Embodiment 1: the preparation of Tricetin-3-beta galactose glycosides oral tablet

Tricetin-3-beta galactose glycosides 125 grams

Vitamin C 100 grams

Micropowder silica gel 40 grams

Pregelatinized Starch 10 grams

Magnesium stearate is an amount of

With additives mix homogeneously such as Tricetin-3-beta galactose glycosides and vitamin C, micropowder silica gels, with 10% pregelatinized Starch slurry as adhesive, wet granulation, vacuum drying.Add an amount of magnesium stearate and mix, be pressed into 500 in tablet, every contains Tricetin-3-beta galactose glycosides effective ingredient 0.25 gram.

Embodiment 2: the preparation of Tricetin-3-beta galactose glucoside oral liquid

Tricetin-3-beta galactose glycosides 25 grams

Vitamin C 50 grams

Sucrose 100 grams

Sodium carboxymethyl cellulose 5 grams

Tween is an amount of

Distilled water is an amount of

An amount of and above-mentioned additives heating mix homogeneously fully mixes with support one's family C and Tricetin-3-beta galactose glycosides after the cooling again, is made into 1000 milliliters of oral liquids.Every 10ml contains Tricetin-3-beta galactose glycosides effective ingredient 0.25 gram.

Embodiment 3: the preparation of Tricetin-3-beta galactose glycosides capsule

Tricetin-3--galactoside 125 grams

Vitamin C 50 grams

Low-substituted hydroxypropyl cellulose 10 grams

Micropowder silica gel 40 grams

Microcrystalline Cellulose is an amount of

70% ethanol is an amount of

Magnesium stearate is an amount of

With Tricetin-3-beta galactose glycosides and additives mix homogeneously such as vitamin C, micropowder silica gel and low-substituted hydroxypropyl cellulose, make wetting agent with 70% ethanol, wet granulation, vacuum drying.Add an amount of magnesium stearate and mix, the 1000 pieces of hard capsules of packing into, every piece of hard capsule contains Tricetin-3-beta galactose glycosides effective ingredient 0.125 gram.

Embodiment 4: the preparation of Tricetin-3-beta galactose glycosides injection

Tricetin-3-beta galactose glycosides 300 grams

45% ethanol 1350ml

Polyoxyethylene castor oil 1500 grams

With Tricetin-3-beta galactose glycosides and 45% ethanol and polyoxyethylene castor oil mix homogeneously, cold preservation, filtration, fill become 1000 bottles, and every bottle contains Tricetin-3-beta galactose glycosides effective ingredient 0.3 gram.Dilute intravenous drip before using with normal saline 250ml.

Embodiment 5: the preparation of the effervescent tablet of Tricetin-3-beta galactose glycosides

The granulates part

Tricetin-3-beta galactose glycosides 100 grams

Citric acid 450 grams

Lactose 50 grams

The alkali particulate fraction

NaHCO ₃390 grams

Lactose 40 grams

Aspartame 10 grams

Fructus Citri tangerinae flavor essence 4 grams

Behind granulates part and alkali particulate fraction difference mixing granulation, again these two parts are fully mixed, be pressed into 250.Every nearly weighs 4 grams, contains Tricetin-3-beta galactose glycosides effective ingredient 0.25 gram.

Claims (5)

1, Tricetin-3-beta galactose glycoside compound is as the application of the economic benefits and social benefits inhibitor of the 3CL protease of SARS virus and cold virus.

2, the application of Tricetin-3-beta galactose glycoside compound in the medicine of preparation control SARS.

3, the application of Tricetin-3-beta galactose glycoside compound in the anti-medicine that cures cold of preparation.

4, according to claim 2 or 3 described application, it is characterized in that with Tricetin-3-beta galactose glycosides be active component, make pharmaceutical preparation.

5, pharmaceutical preparation according to claim 4 is characterized in that said preparation is oral formulations or injection.

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