CN106474103A - Application of the pentahydroxyflavone class compound in the medicine with HRV 3CP activity inhibition is prepared - Google Patents

Application of the pentahydroxyflavone class compound in the medicine with HRV 3CP activity inhibition is prepared Download PDF

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Publication number
CN106474103A
CN106474103A CN201610894199.6A CN201610894199A CN106474103A CN 106474103 A CN106474103 A CN 106474103A CN 201610894199 A CN201610894199 A CN 201610894199A CN 106474103 A CN106474103 A CN 106474103A
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China
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hrv
pentahydroxyflavone
medicine
class compound
compound
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CN201610894199.6A
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Inventor
魏艳红
尧晨光
奚彩丽
胡康洪
朱祥
纪梦莹
刘媛
孙鸽
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Hubei University of Technology
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Hubei University of Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention belongs to biomedicine field, is related to pentahydroxyflavone class compound and is preparing the application in the medicine with HRV 3CP activity inhibition as unique pharmacodynamic raw materials, the pentahydroxyflavone class compound structure formula is shown in formula I.By can effectively suppress HRV 3CP activity to pentahydroxyflavone class Compound ira vitro, and experiment proves that pentahydroxyflavone class compound or derivative can effectively suppress duplication of the EV71 in RD cell, it is the antiviral drugs design with HRV 3CP as target spot and research and development offer guidance.

Description

Pentahydroxyflavone class compound is preparing the medicine with HRV 3CP activity inhibition Application in thing
Technical field
The invention belongs to biomedicine field, and in particular to pentahydroxyflavone class compound is being prepared with HRV 3CP work Application in the medicine of property inhibitory action.
Background technology
HRV 3CP is the key protein enzyme for being catalyzed non-structural protein cracking in picornavirus precursor protein, with height Digestion specificity, virus life cycle in play very important effect, suppress its catalysis effectively suppress disease The cutting of malicious precursor protein, blocking virus are replicated, and are one of target spots of picornavirus study medication.The knowledge of HRV 3CP Other site is Leu Glu Val Leu Phe Gln ↓ GlyPro, and cleavage site is between Gln G1y, referred to as Q-G site. HRV 3CP has typical cysteine proteinase feature.Motif Gly-X-Cys-Gly, will be with catalysis by hydrogen bond network Amino acid Cys, His, Asp or Glu of activity connects to form active catalytic group；While the catalytic group is according to Cys-His- Glu/Asp order plays catalysis enzymolysis function successively.
HRV 3CP gene also has the conservative of height, and therefore HRV 3CP can be used as a preferable medicine effect Target spot.Compound AG7088 synthesized as shot design with HRV 3CP and the like, is the effective of rhinovirus HRV 3CP Inhibitor, and the compound of most promising Antipicornaviral so far.
Natural flavonoid compound is present in higher plant and the food with plant as raw material, is that a class is wide in plant kingdom The polyphenolic substance of general distribution, with numerous biologically actives and very high medical value.Pentahydroxyflavone class compound is derivative Thing Quercetin (Quecertin) is one of active highest compound in numerous flavone compounds.
Content of the invention
In order to solve problems of the prior art, the present invention provides a kind of pentahydroxyflavone with following structural formula Application in the medicine of class compound or derivative as unique pharmacodynamic raw materials in preparation with HRV 3CP activity inhibition,
R in formula1Can be oxo base, C1-6Alkyl amino, phenyl, and the hetero atom 5 containing N, S or O or 6 circle heterocycles Base, described phenyl and heterocyclic radical each arbitrarily can be replaced by hydroxyl, amino and other alkyl；R2Selected from hydrogen, hydroxyl, ammonia Base, sulfydryl, nitro, C1-6Alkyl amino.
Further, R in the compound of the formula (I) that the present invention is provided1For hydroxyl, oxo base, hydroxyl replace containing aerobic Heterocyclic radical；R2Select hydrogen, hydroxyl, amino.
Further, the compound of the formula (I) that the present invention is provided is Quercetin (Quecertin), quercitin And rutin (Rutin) (Quercitrin).
It is a further object of the present invention to provide the pentahydroxyflavone class compound shown in Formulas I or derivative are being prepared by suppression HRV 3CP activity processed come treat EV71 virus infection medicine in application.
The present invention can effectively suppress EV71 HRV 3CP to live by pentahydroxyflavone class compound or derivative in vitro Property, and experiment proves that pentahydroxyflavone class compound or derivative can effectively suppress duplication of the EV71 in RD cell, therefore In-depth study and exploitation can be carried out as the potential drug of prevention and/or treatment hand-foot-and-mouth disease.
Present invention firstly provides pentahydroxyflavone class compound or derivative are preparing prevention as HRV 3CP inhibitor And/or the application in the medicine of the hand-foot-and-mouth disease for causing is infected in treatment by EV71 and CVB3.
Description of the drawings
Toxicity of the Quercetin of Fig. 1 variable concentrations to RD cell；
The activity of the anti-EV71 of Quercetin of Fig. 2 variable concentrations；
The Quercetin suppression EV71 HRV 3CP activity of Fig. 3 variable concentrations；
Specific embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The enforcement for being provided Example is only the explanation to the inventive method, and limits remaining content of present invention announcement never in any form.
, mainly by taking Quercetin as an example, used main material and reagent are as follows for the following example：
Quercetin (Quecertin) is purchased from Calbiochem company, HRV 3CP fluorogenic substrate Dabcyl- RTATVQGPSLDFE-Edans is synthesized by Shanghai Kai Jing bio tech ltd, EV71 strain EV71/wuhan/3018/ 2010 are preserved by this laboratory.People's rhabdomyoma cell (RD), bacillus coli DH 5 alpha, BL21 are by this Laboratories Accession.Carrier PET-28a is given by Central China Normal University Life Science College professor Liu Deli.150i carbon dioxide cell is trained Foster case is purchased from BioTek purchased from Thermo Scientific, ELX800 ELIASA, and 96 orifice plates are purchased from Roche, dimethyl sulfoxide (DMSO) (DMSO) Gibco is purchased from purchased from BD, DMEM (Dulbecco modified Eagle mediuM), other are conventional use of inorganic Salt and organic solvent are all purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
【Embodiment 1】The anti-EV71 activity research of Quercetin
Experimental program：Cultured RD cell is accessed 96 orifice plates, when cell density reaches 80%-90%, antiviral Group adds 7X105EV71 infection cell, discards virus liquid after 1.5h, be simultaneously introduced the DMEM preparation with 2%FBS with medicine poison group Quercetin, concentration be respectively 0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM (due to Quercetin dissolve Property limit, highest drug toxicity and antiviral activity all only accomplish 200 μM), act on the survival shape of microscopic visual measurement cell after 48h Condition.Cell survival rate is surveyed using mtt assay, 96 orifice plate nutrient solutions are removed, per hole using 100 μ l PBS washed cell 2 times；Add per hole Enter the MTT of 30 μ l 5mg/ml, be placed in 37 DEG C of incubator incubation 2-4h；Remove MTT as clean as possible, add 40 μ l DMSO dissolving Mix；Light absorption value is read at ELIASA 490nm.
Experimental result：Fig. 1 shows that Quercetin has faint toxicity to RD cell in the range of 200 μM, and cell survival rate is equal More than 80%, do not possess concentration dependent, CC50 is more than 200 μM.Fig. 2 shows that Quercetin can significantly inhibit EV71 in RD Propagation in cell, improves cell survival rate, and with concentration dependent, EC50 is 15 μM.Therapeutic index SI (CC50/ EC50) it is more than 13.3.
【Embodiment 2】Quercetin suppresses EV71 HRV 3CP activity in vitro
The present invention utilizes FRET principle, first synthesizes one section of fluorescent polypeptide Dabcyl- RTATVQGPSLDFE-Edans as HRV 3CP substrate, whereinQGIt is HRV 3CP cleavage site.In 100 μ l reaction systems In, 50mM Tris-HCl, 200mM NaCl, 2mM DTT, 1 μM of HRV 3CP, compound of certain gradient concentration Quercetin, concentration gradient of the present invention are 200 μM, 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, after incubated at room 4-6h, plus Fluoroscopic examination is carried out immediately in multi-function microplate reader after the fluorogenic substrate mixing for entering 50 μM.If compound can suppress 3C egg in theory The activity of white enzyme, then the speed produced in the starting stage fluorescence of reaction after adding compound can reduce, and react the starting stage As at first order reaction, which plays that fluorescence intensity is linear in the range of certain time, according to the compound treatment of variable concentrations The speed that fluorescence is produced afterwards is that slope can calculate inhibiting rate, and the inhibiting rate further according to variable concentrations compound can try to achieve the chemical combination The IC50 of thing.
2 method therefor of the embodiment of the present invention can be divided into following steps：
1st, design of primers
EV71/wuhan/3018/2010 strain HRV 3CP gene order is inquired about by ncbi database, using Primer HRV 3CP primer of the 5 Software for Design N-terminals with his label, sequence are as follows：F 5 ,- CGCCCATGGGGCCCAGCTTAGACTTCG-3,；R 5 ,-CCCAAGCTTT TATTG CTCGCTGGC AAAATAAC-3, enzyme Enzyme site is Nco I and Hind III.Primer is synthesized by Huada gene company.
2nd, construction of recombinant plasmid
After EV71 infection RD cell 48h, extracting total serum IgE carries out reverse transcription, harvests cDNA as pcr template.To purpose piece Section enters performing PCR amplification, and the length according to primer TM value and purpose fragment determines that PCR reaction condition is：95 DEG C of denaturations 5min, 95 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 30 circulations, 72 DEG C of extension 5min.PCR primer and carrier pET- , respectively after Nco I and Hind III37 DEG C digestion 4h, the 4 DEG C of connections of T4 ligase are overnight for 28a.Connection product converts DH5 α, applies It is distributed in and receives the LB flat board of penicillin containing card, picking positive colony enrichment culture, identify through PCR and digestion after extracting plasmid, be sent to Huada gene company is sequenced.
3rd, HRV 3CP prokaryotic expression and purifying
Recombinant plasmid pET28a-3C is converted BL21, is coated and receives the LB flat board of penicillin containing card, picking single bacterium colony is in LB 37 DEG C of fluid nutrient medium, 220rpm concussion and cultivate, when bacterium solution OD600 reaches 0.8, the IPTG of final concentration 0.5mM is added in 18 DEG C overnight induction.Collects thalline, adds the lysate ultrasonic degradation 30min of 0.3 times of volume, power 30%, work 5s interval 5s. Cellular lysate liquid 13000rmp, 4 DEG C of centrifugation 20min, collect supernatant and are crude enzyme liquid.
Install Ni-NTA post, 2ml fresh resin is added, allow alcohol in post to drain off naturally under gravity, be subsequently adding 3 The lysis buffer balance pillar of times column volume.Crude enzyme liquid is injected after column equilibration, and coutroi velocity is 1mL/min or so, collects stream Go out liquid, add the dcq buffer liquid containing finite concentration imidazoles to rinse foreign protein after end of the sample, be eventually adding washing for 1 times of column volume Destination protein is eluted from pillar by de- liquid, collects the filtrate that filters every time.It is dense that Coomassie Brilliant Blue surveys albumen after purification Spend and carry out polyacrylamide gel electrophoresis.
4th, Quercetin is to HRV 3CP inhibitory action
Reaction system：50mM Tris-HCl, 200mM NaCl, 2mM DTT, 1 μM of HRV 3CP, 3Cization of certain gradient After compound incubated at room 4-6h, 50 μM of fluorescent peptide substrates are added, reactant liquor is prepared in ELISA Plate by above reaction system, shake Detected on multi-function microplate reader after mixing immediately, testing conditions are：Excitation wavelength is 340nm, and launch wavelength is 500nm, instead 37 DEG C of temperature being answered, a numerical value being read every 30s, total time is 20min.Using DMSO as negative control, Rutin is used as the positive Control, the data for obtaining is made the scatter diagram of fluorescence intensity and time and obtains slope, and inhibiting rate=(negative control group is oblique Rate-compound group slope)/negative control group slope * 100%.Compound half-inhibition concentration is obtained by SPSS software.
Experimental result：Fig. 3 shows that Quercetin can suppress EV71 HRV 3CP activity in vitro, and half-inhibition concentration reaches 18μM.
In sum, the pentahydroxyflavone class compound with Quercetin as representative have preferably anti-EV71 activity and The activity of suppression HRV 3CP, is the anti-EV71 drug design with HRV 3CP as target spot and research and development offer guidance.
SEQUENCE LISTING
<110>Hubei University Of Technology
<120>Application of the pentahydroxyflavone class compound in the medicine with HRV 3CP activity inhibition is prepared
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 27
<212> DNA
<213>HRV 3CP upstream primer
<400> 1
cgcccatggg gcccagctta gacttcg 27
<210> 2
<211> 32
<212> DNA
<213>HRV 3CP downstream primer
<400> 2
cccaagcttt tattgctcgc tggcaaaata ac 32

Claims (4)

1. a kind of pentahydroxyflavone class compound with following structural formula or derivative are preparing tool as unique pharmacodynamic raw materials The application having in the medicine of HRV 3CP activity inhibition,
Wherein, R1Selected from oxo base, C1-6Alkyl amino, phenyl, and the hetero atom 5 containing N, S or O or 6 circle heterocycles bases, institute The phenyl that states and heterocyclic radical each arbitrarily can be replaced by hydroxyl, amino and other alkyl；
R2Selected from hydrogen, hydroxyl, amino, sulfydryl, nitro, C1-6Alkyl amino.
2. application according to claim 1, R in the compound of formula (I)1For hydroxyl, oxo base, hydroxyl replace containing oxa- ring Base；R2Select hydrogen, hydroxyl, amino.
3. application according to claim 1, the compound of formula (I) is Quercetin, quercitin and rutin.
4. application according to claim 1, it is characterised in that described medicine is the medicine for treating EV71 virus infection.
CN201610894199.6A 2016-10-12 2016-10-12 Application of the pentahydroxyflavone class compound in the medicine with HRV 3CP activity inhibition is prepared Pending CN106474103A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113768920A (en) * 2021-09-06 2021-12-10 武汉市金银潭医院(武汉市传染病医院) Application of quercetin and/or medicinal derivatives thereof in preparation of anti-enterovirus medicines
CN115819526A (en) * 2022-12-02 2023-03-21 海雅美生物技术(珠海)有限公司 Recombinant botulinum neurotoxin and preparation method and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105255995A (en) * 2015-11-23 2016-01-20 江苏康缘药业股份有限公司 Hand-foot-and-mouth disease resistant drug activity detection method and kit

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105255995A (en) * 2015-11-23 2016-01-20 江苏康缘药业股份有限公司 Hand-foot-and-mouth disease resistant drug activity detection method and kit

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TSAI FUU-JEN等: "Kaempferol inhibits enterovirus 71 replication and internal ribosome entry site (IRES) activity through FUBP and HNRP proteins", 《FOOD CHEMISTRY》 *
WANG CHING-YING: "Antiviral Ability of Kalanchoe gracilis Leaf Extract against Enterovirus 71 and Coxsackievirus A16", 《EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113768920A (en) * 2021-09-06 2021-12-10 武汉市金银潭医院(武汉市传染病医院) Application of quercetin and/or medicinal derivatives thereof in preparation of anti-enterovirus medicines
CN115819526A (en) * 2022-12-02 2023-03-21 海雅美生物技术(珠海)有限公司 Recombinant botulinum neurotoxin and preparation method and application thereof

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Application publication date: 20170308