CN101615222A - A kind of Chinese prescription designing technique based on the Chinese medicine effective component group - Google Patents
A kind of Chinese prescription designing technique based on the Chinese medicine effective component group Download PDFInfo
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Abstract
The present invention is to be research object with the huanglian jiedu decoction, comprehensive also integrated information integrated (foundation of chemicobiology information database), data mining (knowledge base of existing pharmacological action of prescription and molecular mechanism is set up), system modelling is (based on pathogenetic molecular pathology model of pyemia and cell, the pharmacophore model of integral level makes up), step analysis is (based on traditional Chinese medical science compatibility theory, modern molecular biology and pharmacological effective constituent heap sort and based on the pyemia molecule, cell, the symptom classification of integral level pharmacophore model), effectively identification (area of computer aided drug design technology and the checking of inside and outside pharmacophore model), optimal design (based on pathogenetic pathological model of pyemia and area of computer aided drug design The Application of Technology), the overall evaluation multinomial technology such as (many indexs pyemia whole animal model and many index comprehensives index assessment methods), be used for the prescription design of huanglian jiedu decoction Chinese medicine effective component group, finally determine huanglian jiedu decoction Chinese medicine effective component group prescription.The Chinese prescription technology of the Chinese medicine effective component group that the present invention sets up have demonstration, efficient, fast, economy, novel features.Use this invention and can produce Chinese medicine molecule new compound, for Chinese medicine compound prescription modernization, Chinese medicine compound prescription innovation provide a feasibility thinking based on the Chinese medicine effective component group.
Description
Technical field:
The present invention is directed to the critical bottleneck problem of Chinese medicinal formulae modern study, by information integrated, data mining, system modelling, step analysis, effectively identification, optimal design, the overall evaluation, foundation is suitable for the Chinese prescription designing technique of traditional Chinese medicine mass action characteristics, be Chinese medicine and the modern research of compound thereof, simultaneously also provide technical support for the secondary development of Chinese medicine and the research and development of original new drug.
Background technology:
Chinese medicine and prescription thereof (compound) are the principal modes of tcm clinical practice medication.At present, the research of Chinese medicine has two kinds of basic ideas: a kind of is as plant or natural drug with Chinese medicine, method by the traditional classical formula is studied, extract its active site, the separation chemistry composition, fixed structure, and even synthetic, carry out pharmacodynamics, pharmacokinetics, toxicologic research then, carry out the transition to clinical testing at last.This research thinking still accounts for suitable status at present, and they mainly study single medicinal material, therefrom find monomer then, are applied to clinical.Another kind of thinking is progressively to form over nearly more than 30 years, it promptly is the viewpoint of traditional Chinese medicine, exactly Chinese medicine and compound thereof are studied by the viewpoint of the combination of Chinese tradiational and Western medicine, the characteristics of its research are to follow traditional Chinese medical theory, in conjunction with the tcm clinical practice curative effect, the research means of utilization modern science is carried out the research of Chinese medicine and compound closely, and its fundamental purpose is to illustrate traditional Chinese medical theory, illustrate prescription rule and developing new drug, development new drug, promote the modernization of Chinese medicine.
Basic ideas of the present invention are: with the traditional Chinese medicine compatibility theory is guidance, with clinical effective prescription is research object, based on existing Chinese medicine active principle basis and Study on mechanism achievement, with Chemoinformatics, bioinformatics, molecular biology, molecular pharmacology, mathematical statistics is theoretical the support, serve as theme with system modelling and optimal design, set up Chinese prescription designing technique based on the Chinese medicine effective component group.That is: select a kind of clinical common and pathogenesis relatively clearly disease plant for research is sick, the more deep clinical effective prescription of preferred effective substance research is a research object, set up molecule, cell, whole three levels not only additional mutually but also reflect the system's medicine efficacy screening and the verification model of card mutually, create monarch, minister, assistant, make the identification of Chinese medicine effective component group and the association study method thereof of four levels, integrated information is learned, modern biology, computer science, modern pharmacology, five major techniques such as mathematics pharmacology, critical bottleneck problem at the Chinese medicinal formulae modern study, by information integrated, data mining, system modelling, step analysis, effectively identification, optimal design, the overall evaluation, foundation is suitable for the Chinese prescription designing technique of traditional Chinese medicine mass action characteristics, be the modern research of Chinese medicine compound prescription, simultaneously also provide technical support for the secondary development of Chinese medicine and the research and development of original new drug.
Summary of the invention:
The present invention is under the guidance of traditional Chinese medical science compatibility theory, appliance computer ancillary drug designing technique, modern biology technology and mathematical statistics method, by system modelling and optimisation technique, the relation of the clear and definite Chinese medicine effective component group and the compatibility of medicines in a prescription, foundation is based on the Chinese prescription designing technique of effective component group, for the secondary development of Chinese medicine compound prescription and the research and development of original new drug provide technical support.At present, the research of Chinese medicinal formulae mainly concentrates on based on medicinal material or medicine materical crude slice (macroscopic aspect) with based on the research of active component (middle sight aspect), lack research, restricted the secondary development of Chinese medicine compound prescription and the research and development of original new drug based on effective component group (microcosmic point).The useful trial that the Chinese prescription designing technique based on the Chinese medicine effective component group that the present invention proposes addresses the above problem just has positive meaning to the modernization that advances Chinese medicine compound prescription,
The present invention is research object with the huanglian jiedu decoction, comprehensive also integrated information integrated (chemistry, the biological information database is set up), data mining (knowledge base of existing pharmacological action of prescription and molecular mechanism is set up), system modelling is (based on pathogenetic molecular pathology model of pyemia and cell, the pharmacophore model of integral level makes up), step analysis is (based on traditional Chinese medical science compatibility theory, modern molecular biology and pharmacological effective constituent heap sort and based on the pyemia molecule, cell, the symptom classification of integral level pharmacophore model), effectively identification (area of computer aided drug design technology and the checking of inside and outside pharmacophore model), optimal design (based on pathogenetic pathological model of pyemia and area of computer aided drug design The Application of Technology), the overall evaluation multinomial technology such as (many indexs pyemia whole animal model and many index comprehensives index assessment methods), be used for the design of huanglian jiedu decoction Chinese medicine effective component group prescription, finally determine huanglian jiedu decoction Chinese medicine effective component group prescription.Chinese medicine effective component group compound or Chinese medicine molecule compound with compare with the Chinese medicine compound prescription of medicine materical crude slice or active component compatibility, it is clear to have chemical constitution, and pharmacological action is clear and definite, molecular mechanism is clear, and quality standard is controlled, plurality of advantages such as pharmaceutical preparation is simple, and clinical practice is convenient.Huanglian jiedu decoction Chinese medicine effective component group composition design technique has typical case and exemplary role, also can be used for that other chemical constitution is clear relatively, pharmacological action is clear and definite relatively, the prescription flavour of a drug are formed less relatively Chinese medicine compound prescription effective component group prescription design.
Concrete steps are:
1. select the pyemic effective prescription huanglian jiedu decoction of clinical treatment (coptis, the root of large-flowered skullcap, golden cypress, cape jasmine) as research object, with traditional Chinese medical science compatibility theory monarch is guidance, prescription is carried out monarch function effective components group's research according to pyemia pathogenesis molecular mechanism.
2. applied chemistry information science and correlation technique thereof by methods such as literature survey, database accesss, are set up the chemical constitution information database of each simple of huanglian jiedu decoction.Comprising compound title, molecular formula, molecular weight, structural formula (two dimension, three-dimensional), physico-chemical property, pharmacological action, molecular mechanism, medicine generation, toxicity, clinical practice etc.
3. by methods such as literature survey, database accesss, set up huanglian jiedu decoction compound database.Comprising composition, dosage, function and cure mainly, chemistry, pharmacological action, molecular mechanism, medicine generation, toxicity, clinical practice etc.
4. on the basis of literature survey, database access, data mining, at the main and clear and definite pharmacological action of prescription, appliance computer ancillary drug designing technique, system are set up its molecule pharmacological model.
5. according to molecule pharmacological model The selection result, to the effective constituent cluster and just arrange with score right.
6. according to area of computer aided drug design functional classification method, determine its monarch effect, and to its cluster.
7. according to area of computer aided drug design molecule pharmacological model The selection result, in the vivo and vitro pharmacological model of correspondence, verify respectively.
8. according to screening of molecule pharmacological model and monarch cluster result, obtain complexity, from every class model, choose several molecular composition compounds in conjunction with vivo and vitro pharmacological model pharmacological action intensity, the effect of many target spots and compound.
9. in the whole animal model that embodies the Syndrome in TCM marquis, prescription is verified.Obtain the Chinese prescription based on effective part group of reflection compound mass action characteristics.
10. by optimal design, systematic study and checking repeatedly, finally set up the Chinese prescription designing technique that is suitable for traditional Chinese medicine mass action characteristics based on effective component group.
Every flavor medicine monarch of living in the Chinese medicine compound prescription, minister, help, make the status difference, but they Each performs its own functions, the complementation of helping each other, mutual restriction, coordinate mutually, keeping perfect combination, performance integral body or colony's effect.The present invention at first adopts area of computer aided drug design technology, and system sets up pyemia table disease molecule pharmacological model.According to molecule pharmacological model The selection result, to the effective constituent cluster and just arrange with score right, according to area of computer aided drug design functional classification method, determine its monarch effect, and to its cluster, be divided into monarch, minister equally, help, make the group containing effective constituent in the compound according to The selection result, inquire into the mass action between the medicine of different effective parts then.Therefore, its pharmacodynamics test, pharmacological toxicology are learned and are tested and should carry out drug effect, the pharmacological toxicology test of each " effective part group " respectively with such integral body for being tried material.Reflect the drug action and the toxicity situation of this colony with animal experiment, for clinical research provides safety, validity basis.The present invention fully tests by whole animal, Chemoinformatics, bioinformatics, the computer virtual triage techniques, carry out the mensuration of compound activity in the effective part group, the research in the mutual relationship between the effective part group and each stage of prescription, with the modern plants chemistry, Chemoinformatics combines with mathematics pharmacology is close, use in conjunction, at last by this research tool of pharmacology, be beneficial to we from microcosmic to objective, from the part to integral body, curative effect material base from the comprehensive research tradition medicine of each aspect, obtain more information, for clinical research provides necessary foundation.
The Chinese medicine composition technology of the Chinese medicine effective component group that the present invention sets up has efficient quick, economic novel features.This invents the generation of direct corresponding Chinese medicine molecule novel compound.The chemical constitution molecule that the foregoing invention step screens can directly be ordered mostly, and the composition that minority can not be bought also can extract from medicinal material, and it is convenient feasible to draw materials.This molecule new compound has been inherited wherein a kind of pharmacological action of former side from former Chinese medicine compound prescription, has more specific aim, but abandoned the shortcoming that Chinese medicinal formulae is unfavorable for the preparation poor compliance, by after the drug effect toxicological experiment checking, but just preparation Cheng Fang declares new drug.This invention leading portion experimental design is finished under the computer virtual state, need not to use and expend chemical agent, economic security, and the pharmacological evaluation in later stage is verified the actual drug effect of this method, makes this invention have more rationality.The present invention provides a feasibility thinking for Chinese medicine compound prescription modernization, Chinese medicine compound prescription innovation.
Description of drawings
Fig. 1 is a huanglian jiedu decoction chemical constitution library of molecules (part).
Fig. 2 is a pyemia pathogenesis molecular mechanism system modelling.
Fig. 3 is a target behind the pyemia pathogenesis system modelling.
Fig. 4 effective constituent molecule prescription (compatibility), overall evaluation technology path.
Embodiment:
Below in conjunction with embodiment, the present invention is described further, but the present invention is not limited to following embodiment.
Determining of embodiment 1 research object
The sick kind and the selection of prescription: choose the pyemia of clinical common refractory and chemical constitution is clear, main pharmacodynamics is clear and definite, clinical efficacy is sure treatment pyemia prescription " huanglian jiedu decoction " and be research object.
The foundation of embodiment 2 compound chemical composition databases
Applied chemistry information science of the present invention and correlation technique thereof, by methods such as literature survey, database accesss, set up in the chemical constitution of " huanglian jiedu decoction (coptis, the root of large-flowered skullcap, golden cypress, cape jasmine) " each simple and the compound owing to decoct the chemical constitution information database of new generation such as the effect of grade.Specialty chemical database and these two compound databases of Dr.Duke ' sPhytochemical and Ethnobotanical Databases based on Shanghai organic chemistry research institute of the Chinese Academy of Sciences, collect the chemical constitution of two each simple of side by the database access mode, and, the new component that forms because of effects such as decoctions in the compound is carried out addendum in conjunction with literature survey.The information content of compound chemical composition database comprises compound Chinese and English title, molecular formula, molecular weight, structural formula (two dimension, three-dimensional), physico-chemical property, pharmacological action, molecular mechanism, medicine generation, toxicity, clinical practice, pertinent literature etc.The database management function that mainly utilizes the molecular structure of ISIS/Draw to draw function and ISIS/Base is created " huanglian jiedu decoction ".The present invention has made up four chemical constitution word banks of huanglian jiedu decoction four flavor prescription medicinal materials, and amounting to the molecule number is 222.(Fig. 1)
With the pyemia is example, sets up corresponding pathomechanism system model, sees accompanying drawing 2 pyemia pathogenesis molecular mechanism bio-networks figure.
In pyemic pathomechanism system model, inflammation, bacterial infection, heating are the principal contradictions in the disease, and occupy the target spot of upstream in these pathology approach and the target spot that plays a decisive role is crucial target.See accompanying drawing 3 " monarch " effective constituent molecular group and interpretation of the cause, onset and process of an illness target mapping figure.
The function of embodiment 5 effective constituents is hived off
With traditional Chinese medical science compatibility theory " monarch " is guidance, based on the system model of setting up according to pyemia pathogenesis molecular mechanism, " huanglian jiedu decoction " prescription is carried out " monarch, minister, help, make " function effective components hive off.
1. " monarch drug in a prescription " molecular group: according to theory of traditional Chinese medical science, monarch drug in a prescription is the medicine that plays main therapeutic action in the compound prescription at disease and disease, promptly is drug ingedient at main interpretation of the cause, onset and process of an illness target with pharmacological modern languages explaination.In pyemic pharmacological mechanism system model, inflammation is the principal contradiction in the disease, and some crucial targets that distributing in these pathology approach.The screening of monarch drug in a prescription molecular group is promptly carried out at these crucial targets.Method for sieving integrated use modern computer drug design and triage techniques and modern molecular biology triage techniques.The material sieving technology route is initial operating module with computing machine drug design and triage techniques, a large amount of compound chemical composition molecules is tentatively sieved, preliminary screening result enters molecular biology screening passage again and verifies and screening once more, saves cost and manpower and materials with this.I. computing machine drug design and triage techniques: A. molecular docking: the target for can find three-dimensional crystalline structure in PDB protein molecular storehouse, directly adopt the technology of molecular docking to study.The molecular docking technology of the target molecules three-dimensional structure that forms based on true experimental data can guarantee the precision of screening.The docking technique link adopts the patented technology Induced-fit in the up-to-date Schrodinger software package, the active binding pocket of target is really realized handling flexibly, and the gentle change of the flexible and shape of processing target pocket volume, target molecules and compound chemical composition molecule realize that real " induced-fit " formula docks, and have strengthened active precision of prediction greatly.B. the homology mould is built: the target proteins for still there not being at present the three-dimensional crystalline structure report, utilize the homology mould to build the technique construction target proteins, and build at this mould and to carry out molecular docking on the protein-base.C. pharmacophore search: for not only not having three-dimensional crystalline structure, but also can't carrying out the target proteins that the homology mould is built, then utilize its ligand structure to make up active relevant pharmacophore, the Pharmacophore Model of utilizing structure to finish is searched for the compound chemical composition library of molecules as the enquirement structure, and the compound that hits promptly is the chemical constitution that possible have this target organisms activity.Simultaneously, this technology is used with above two kinds of technology simultaneously, strengthens the screening precision.II. molecular biology screening:, enter molecular biology screening passage and verify and regrading by the compound that above computing machine drug design and triage techniques sieve out.Final definite monarch drug in a prescription molecular group.
2. " ministerial drug " molecular group: ministerial drug is the medicine that auxiliary monarch drug in a prescription plays therapeutic action in the prescription in theory of traditional Chinese medical science, so with the language explaination of modern pharmacology exactly at the compound chemical composition molecule of less important or the interpretation of the cause, onset and process of an illness target that helps out.The screening of ministerial drug molecular group is the same with the screening flow process of monarch drug in a prescription molecular group, and different is at different targets.
3. " adjutant " molecular group: according to theory of traditional Chinese medical science, adjutant is an assistant principal drug assistance ministerial drug in the prescription, or with can the oppose each other and yet also complement each other medicine of therapeutic action of monarch drug in a prescription ministerial drug.The modern pharmacology explaination of adjutant effect may be relevant with some non-therapeutic drug targets, as P-glycoprotein, MRP etc.P-glycoprotein is maximum in ATP combination and a transport vehicle albumen subbreed, it has distribution at many tissues, it is a kind of ATP dependence film transporter, as drug transporter, its effect is similar to extraction pump, medicine can be effluxed in the cell and make that drug concentration reduces in the born of the same parents, thereby reduce drug effect.Therefore, the interaction energy between P-glycoprotein and substrate and adjusting influences absorption, distribution, metabolism, the drainage of medicine.Suppress the drug action that P-glycoprotein can effectively strengthen main pharmacodynamics composition " the monarch and his subjects " medicine.Therefore, Chinese medicine compound prescription " adjutant " molecular group helps effect to be likely to realize by this class non-therapeutic drug target generation effect to P-glycoprotein to the assistant of monarch-minister drug.The above theoretical analysis of screening foundation to the adjutant molecular group adopts the area of computer aided drug screening technology, serves as the screening template with non-therapeutic target spots such as P-glycoprotein, the adjutant molecule in the screening compound chemical composition library of molecules.
4. the molecular group that " makes medicine ": " making medicine " in theory of traditional Chinese medical science is that all medicines of guiding reach ill internal organs, relevant channels and collaterals, sick position in the prescription.Discover that some compound molecules can impel the medicament contg that enters cell to increase by increasing the water glycerol channel albumen of being responsible for medicament transport, the content of organic yin, yang ion transport body; On the other hand, some compound molecules can be by changing the interior environment of other drug molecular action, as increasing the permeability of cell membrane, and reach the effect of " tying-in ", can change permeability of cell membrane as the borneol in the borneol, promote blood cerebrospinal fluid barrier open, priming is gone into brain." making medicine " effect is all included in these effects in, and the compound chemical composition molecule screening with these effects enters " making medicine " molecular group.Make the screening of medicine molecular group at first utilize computing machine QSAR (D-M (Determiner-Measure) construction-activity relationship) pharmaceutically active forecasting techniques in conjunction with artificial intelligence technology such as artificial neural network and support vector machine technology, with the known compound with above effect is training set, training produces and makes the medicine model of cognition, chemical constitution molecule in the compound is carried out Classification and Identification, and screening may have the chemical constitution of " making medicine " function.The compound that screening is come out adopts correlation techniques such as cell biology, molecular biology that it is verified and regrading again, by measuring these chemical constitution molecules to checking such as the content of water glycerol channel albumen etc., the change degree of permeability of cell membrane and estimate finally " making medicine " molecular group of screening.
The computing machine drug design and the triage techniques-molecular docking of embodiment 6 monarch drug in a prescription effective component groups
Molecular docking: the target for can find three-dimensional crystalline structure in PDB protein molecular storehouse, directly adopt the molecular docking technology to study.The three-dimensional structure of going into to record molecule in the selection database is that the molecular docking technical research is carried out on the basis as target molecules, has to screen precision preferably.The docking technique link adopts the patented technology Induced-fit in the up-to-date Schrodinger software package, the active binding pocket of target is really realized handling flexibly, and the gentle change of the flexible and shape of processing target pocket volume, target molecules and compound chemical composition molecule realize that real " induced-fit " formula docks, and strengthen active precision of prediction greatly.222 molecules of huanglian jiedu decoction library of molecules dock result's marking and see Table 1 with COX-2, and by the ordering of marking height.Statistics finds that giving a mark has 25 greater than the molecule more than 5.0, illustrates to exist effective COX-2 to suppress active chemical components in the huanglian jiedu decoction, and suppress the active forulic acid of reporting as existing COX-2, the marking of forulic acid in the present invention is 5.56, the ordering top ten.
Table 1 COX-2 molecular docking marking table
| ??Compound?Name | ??Score |
| ??10-acetyl?geniposide | ??6.82 |
| ??5-hydroxy-7，8-dimethoxyflavone | ??6.42 |
| ??geranyl?acetone | ??6.10 |
| ??5，5’-dimethylfurfural?ether | ??5.84 |
| ??herculin | ??5.83 |
| ??5，2’，5’-trihydroxy-6，7，8-trimethoxyflavanone | ??5.82 |
| ??methylnonyl?ketone | ??5.69 |
| ??salidroside | ??5.64 |
| ??cordycepic?acid | ??5.61 |
| ??ferulic?acid | ??5.56 |
| ??alpha-cubebene | ??5.55 |
| ??isoeugenol | ??5.55 |
| ??7-methoxybaicalein | ??5.49 |
| ??methyldihydrojasmonate | ??5.45 |
| ??phthalic?acid-dioctyl-ester | ??5.38 |
| ??baicalein | ??5.29 |
| ??moslosooflavone | ??5.25 |
| ??neocnidilide | ??5.22 |
| ??4-phenyl-but-cis-3-en-2-one | ??5.21 |
| ??genipin | ??5.16 |
| ??rivularin | ??5.15 |
| ??5，2’，7’-trihydroxy-8-methoxyflavanone | ??5.13 |
| ??n-butylphthalide | ??5.08 |
| ??panicolin | ??5.02 |
| ??skullcapflavoneI | ??5.02 |
| ??scuteamoenin | ??4.93 |
The computing machine drug design and the triage techniques-homology mould of embodiment 7 monarch drug in a prescription effective component groups are built
The homology mould is built: the target proteins for still there not being at present the three-dimensional crystalline structure report, utilize the homology mould to build the technique construction target proteins, and build at this mould and to carry out molecular docking on the protein-base.
Adopt this method at the screening of IKK-2 among the present invention.Technology path is: 1) sequence alignment: utilize Prime module comparison IKK-2 in the Schrodinger software package and the homologous protein in the protein pool, find suitable homology mould to build the albumen template.2) the albumen mould is built: with the homologous protein that finds is template, carries out the kinase whose albumen mould of IKK-2 and builds, and particularly builds very key for the mould of binding pocket, adopts the method for building with known ligand associating mould, obtains accurate albumen model.3) molecular docking: the IKK-2 albumen of building with the homology mould is the butt joint that template is carried out the compound library of molecules.GALAHAD obtains 20 of Pharmacophore Model, in conjunction with the analysis that IKK-β homology mould is built the body avtive spot, considers the marking of Pharmacophore Model energy simultaneously, the selected Pharmacophore Model that obtains.This Pharmacophore Model is inserted the homology mould of structure such as Andrew Baxter and built in the active binding cavity of IKK-2, find that the critical sites of this model and IKK-2 binding pocket is very identical.Carry out the compound search of Unity for " enquirement template " with this Pharmacophore Model.Obtain hitting 40 of compounds.Listed all 40 of searching in the table 2 and hit compound, QFIT represents that compound and Pharmacophore Model hit the marking of degree
The IKK-2 that table 2 Unity searches suppresses active pharmacophore and hits compound
| ??Compound | ??QFIT |
| ??wogonin-5-beta-D-glucoside | ??28.21 |
| ??5，7，2’，6’-tetrahydroxyflavone | ??12.36 |
| ??10-acetyl?geniposide | ??17.42 |
| ??oroxylin-7-O-glucuronide-methyl-ester | ??12.48 |
| ??(2R，3R)-2’，3，5，7-tetrahydroxyflavanone | ??30.41 |
| ??chrysin-6-C-beta-glucoside | ??32.62 |
| ??5，2’，5’-trihydroxy-6，7，8-trimethoxyflavanone | ??11.4 |
| ??viscidulinII | ??12.36 |
| ??5，2’，5’-trihydroxy-7，8-dimethoxyflavone | ??12.36 |
| ??dihydrooroxylin?A | ??20.71 |
| ??baicalein-7-O--D-glucopyranoside | ??25.77 |
| ??oroxylin?A | ??21.07 |
| ??oroxylin | ??21.07 |
| ??geniposidie?acid | ??35.77 |
| ??piscidinol?A | ??10.89 |
| ??5，2’，6’trihydroxy-7，8-dimethoxyflavone | ??12.36 |
| ??jasminoside | ??35.89 |
| ??viscidulinI | ??43.74 |
| ??wogonoside | ??12.43 |
| ??gardoside | ??33.42 |
| ??chrysin-7-o-beta-glucoside | ??12.16 |
| ??(2R，3R)-3，5，7-trihydroxyflavanone | ??30.41 |
| ??(2S)-7，2’，6’-trihydroxy-5-methoxyflavano | ??11.4 |
| ??scutellarein | ??21.07 |
| ??baicalein | ??21.07 |
| ??chlorogenic?acid | ??18.11 |
| ??5’-caffeoylquinic?acid | ??18.11 |
| ??tenaxinII | ??21.07 |
| ??5，7，4’-trihydroxy-6-methoxyflavanone | ??21.07 |
| ??wogonin-7-beta-D-gluconide-methyl-ester | ??12.41 |
| ??oroxylin-A-7-O-glucuronide | ??12.49 |
| ??salidroside | ??35.17 |
| ??(2S)-2’，5，6’，7-tetrahydroxyflavanone | ??11.4 |
| ??hispiol?B | ??15.83 |
| ??picrocrocinic?acid | ??44.21 |
| ??scuteamoenin | ??11.4 |
| ??5，5’-dimethoxylariciresinol | ??5.14 |
| ??3，5，7，2’，6’-pentahydroxyflavanone | ??30.41 |
| ??chrysin-8-C-beta-D-glucoside | ??30.66 |
| ??geniposide | ??29.14 |
The virtual screening of the anti-inflammatory target spot PDE-4 of embodiment 8 monarch drug in a prescription effective component groups
Huanglian jiedu decoction chemical constitution molecule and PDE-4's docks result such as table 3, and according to the ordering of butt joint marking height.Because active pocket and the misclosure binding cavity of PDE-4, the collision situation of butt joint molecule and binding cavity residue is less, so whole butt joint marking there is no too many low the branch or negative the branch, suppresses active compound but the high molecule of butt joint marking still should be considered as having PDE-4.Statistics shows that butt joint marking has 23 greater than 5.0 molecule, has natural PDE-4 inhibitor in this explanation huanglian jiedu decoction and exists.
Table 3 PDE-4 molecular docking marking table
| ??Compound?Name | ??Score |
| ??rutin | ??6.93 |
| ??3，4-di-O-caffeoylquinic?acid | ??6.17 |
| ??6”-p-coumaroyl-genipingentiobioside | ??6.1 |
| ??jatrorrhizine | ??5.91 |
| ??isoscutellarein | ??5.83 |
| ??eriodictyol | ??5.67 |
| ??isomartynoside | ??5.62 |
| ??10-acetyl?geniposide | ??5.59 |
| ??2，6，2’，4’-tetrahydroxy-6’-methoxychalcone | ??5.59 |
| ??2’，5，8-trihydroxy-6，7-dimethoxyflavone | ??5.4 |
| ??worenine | ??5.36 |
| ??gardoside | ??5.26 |
| ??baicalein-7-O-D-glucopyranoside | ??5.18 |
| ??5，7，2’-trihydroxyflavone | ??5.16 |
| ??verbascoside | ??5.15 |
| ??amurensin | ??5.13 |
| ??chlorogenic?acid | ??5.13 |
| ??melianone | ??5.1 |
| ??methyldihydrojasmonate | ??5.07 |
| ??ligustilide | ??5.03 |
| ??gardenin | ??5.02 |
| ??5，2’，5’-trihydroxy-6，7，8-trimethoxyflavone | ??5.02 |
| ??isoscutellarein-8-o-glucoside | ??5.01 |
| ??berberrubine | ??4.97 |
| ??epiberberine | ??4.94 |
| ??palmatine | ??4.94 |
| ??5，5’-dimethyl?furfural?ether | ??4.92 |
| ??tetrahydrojatrorrhizine | ??4.92 |
| ??coptisine | ??4.91 |
| ??baicalin | ??4.9 |
The comprehensive evaluation of chemical constitution in the embodiment 9 monarch drug in a prescription effective component groups
Molecule with COX-2 and PDE-4 molecular docking marking ordering preceding 20% suppresses bioactive molecule as having COX-2 and PDE-4, the molecule of IKK-2 pharmacophore search hit suppresses bioactive molecule as having IKK-2, analyzes many targets guide effect of huanglian jiedu decoction chemical constitution.Table 4 has been listed the molecule that may suppress 2 and 2 above target enzymes simultaneously, amounts to 28, and wherein 2 molecules all present inhibiting effect to three targets.
The comprehensive evaluation at many targets of table 4 huanglian jiedu decoction chemical constitution
| ??Compound?Name | ??COX-2 | ??PDE-4 | ??IKK-2 |
| ??(2S)-2’，5，6’，7-tetrahydroxyflavanone | ??+ | ??- | ??+ |
| ??10-acetyl?geniposide | ??+ | ??+ | ??+ |
| ??2’，5，8-trihydroxy-6，7-dimethoxyflavone | ??+ | ??+ | ??- |
| ??2，6，2’，4’-tetrahydroxy-6’-methoxychalcone | ??+ | ??+ | ??- |
| ??5，2’，5’-trihydroxy-6，7，8-trimethoxyflavanone | ?+ | ?- | ?+ |
| ??5，2’dihyrdoxy-6，7，8-trimethoxyflavone | ?+ | ?+ | ?- |
| ??5，5’-dimethylfurfural?ether | ?+ | ?+ | ?- |
| ??5，7，4’-trihydroxy?6-methoxyflavanone | ?+ | ?- | ?+ |
| ??5，8-dihydroxy-6，7-dimethoxyflavone | ?+ | ?+ | ?- |
| ??baicalein | ?- | ?+ | ?+ |
| ??chlorogenic?acid | ?- | ?+ | ?+ |
| ??chrysin-6-C-beta-glucoside | ?- | ?+ | ?+ |
| ??gardoside | ?- | ?+ | ?+ |
| ??geniposide | ?- | ?+ | ?+ |
| ??herculin | ?+ | ?+ | ?- |
| ??isoscutel?larein-8-O-glucoside | ?+ | ?+ | ?- |
| ??ligustilide | ?+ | ?+ | ?- |
| ??linalool | ?+ | ?+ | ?- |
| ??methyldihydrojasmonate | ?+ | ?+ | ?- |
| ??oroxylin-A-7-O-glucuronide | ?- | ?+ | ?+ |
| ??phthalic?acid-dioctyl-ester | ?+ | ?+ | ?- |
| ??rivularine | ?+ | ?+ | ?- |
| ??salidroside | ?+ | ?+ | ?+ |
| ??scuteamoenin | ?+ | ?- | ?+ |
| ??viscidulinII | ?+ | ?- | ?+ |
| ??wogonin-5-beta-D-glucoside | ?- | ?+ | ?+ |
| ??wogonin-7-beta-D-gluconide-methyl-ester | ?- | ?+ | ?+ |
| ??wogonoside | ?- | ?+ | ?+ |
Choose clear and definite inflammation target COX-2, IKK-2, PDE-4 at present among the present invention, with serve as the screening target spot huanglian jiedu decoction chemical constitution library of molecules is carried out virtual screening, and carry out analysis-by-synthesis on this basis, find to exist in the huanglian jiedu decoction molecule that has the guide effect of many targets in a large number, can be simultaneously to two or more target spot generation effects, the activity that suppresses them, many targets therapeutic action of this explanation Chinese medicine compound prescription has material base, and many target spots chemical constitution of screening under this basis is carried out the research of inside and outside pharmacologically active.
Molecular biology screening-COX-2 enzyme inhibition activity the test of embodiment 10 monarch drug in a prescription effective component groups
COX-2 enzyme inhibition activity test: use vitro enzyme reaction experiment system, adopt the EIA method to observe the influence of test-compound to the pure enzymatic activity of Cycloxygenase-2 (COX-2).Reference reagent box instructions, 96 hole ELISA Plate are established reagent blank control wells, S1-S8 standard control hole, TA hole, NSB hole, BO hole, COX-2 blank hole, COX-2 100% enzymatic activity hole, sample well etc., and two multiple holes are established in every hole.Add 950 μ L reaction buffers [0.1M Tris-HCl (pH8.0) contains 5mMEDTA and2I1M phenol], 10uL heme, 10 μ L in the sample well.The test-compound solution of the variable concentrations of COX-2,20 μ L doubling dilutions; 100% enzymatic activity hole is except that not adding the test-compound all the other same sample wells: COX-2 blank hole adds the COX 2 10 μ L of 970 μ L reaction buffers, 10 μ L hemes and deactivation.After 37 ℃ in each hole is incubated 10 minutes in advance, add behind arachidonic acid (the 100 μ mol/L) mixing 37 ℃, water-bath added 50 μ L1MHC1 and stops enzyme reaction after 2 minutes.Take out each pipe of back and add 100 μ LSnC
12The solution mixing is put 5 fens kinds of room temperature lucifuge reaction.The standard control control wells adds the PGE of the variable concentrations of multiple proportions dilution
2Detect prostate PGE with enzyme immunoassay (EIA method)
2 α, with PGE
2 αGrowing amount calculate the activity of enzyme.Calculate IC50, analyze its inhibition strength and selectivity enzymatic activity.
Molecular biology screening-the IKK-2 of embodiment 11 monarch drug in a prescription effective component groups suppresses active testing
IKK-2 suppresses active testing: experimental technique: the solution of IkB-α substrate and ATP is added on to cultivate in advance with test-compound began reaction in 5 minutes in the polypropylene board that contains the IKK-2 enzyme.Then reaction mixture was cultivated 1 hour in 25 ℃, placed on ice to stop reaction by adding 150 μ l, 10% trichloroacetic acid and 5% disodium pyrophosphate.After the mixing, the whole reaction mixture that stops to react is moved into the Packard Unifilter filter of prewetting, suction and with 250 μ LddH
2O washes 6 times, uses PackardFiltermate Harvester.Then that filter is air-dry, replenish 40 μ L Microscint, 20 scintillation solutions, use the PackardTopCount scintillation counter quantitative
33The reaction product of P-mark can obtain different Compound I KK-2 and suppress active.
Molecular biology screening-the PDE-4 of embodiment 12 monarch drug in a prescription effective component groups suppresses active testing
PDE-4 suppresses active testing: experiment material: 96 hole microtiter plates (MTS); Potpourri (20mM Tris (pH7.4), 5mM MgCl
2, 0.5 μ M cAMP, [
3H] cAMP (about 30,000cpm/ measure), test compound mainly contains people's neutrophil cell cytosol of PDE4 active matter); PDE-3 specific inhibitor Motapizone (1 μ M); DMSO; 5 '-nucleosidase (rattle snake venom (Crotalus atrox snake venom); QAE Sephadex A-25 post; Ammonium formate (pH6.0).
Experimental technique: 37 ℃ of precincubation added substrate (cAMP) and start reaction after 5 minutes, measured thing 15 minutes in 37 ℃ of further incubations.Add 50 μ l 0.2N HCL cessation reactions, place on ice and measure about 10 minutes of thing.To measure thing upper prop to QAESephadex A-25 post (1ml bed volume) with 25 μ g 5 ' nucleosidases (rattle snake venom (Crotalus atrox snake venom)) incubation after 10 minutes at 37 ℃.Post is measured radioactivity with 2ml 30mM ammonium formate (pH6.0) wash-out, counting eluate, draws different compounds then PDE-4 is suppressed active.
Molecular biology screening-the HMGB-1 of embodiment 13 monarch drug in a prescription effective component groups suppresses active testing
HMGB-1 suppresses active testing: experiment material: the animal model preparation: cleaning level male Wistar rat, body weight 220～250g.Rat adaptability is raised 1 week back row CLP, duplicates serious abdominal cavity infection and causes the pyemia model.Before the art rat is weighed, numbers, and fasting 12h, freely drink water.Mix by 8: 3 volume ratios with Ketamine Hydrochloride Inj. and the new II parenteral solution of speed dormancy, fixing behind the intramuscular anesthesia rat, routine disinfection, shop aseptic hole-towel.Make an otch that is about 1.5cm along the abdomen median line, expose caecum and, avoid ligation ileum and mesocecum blood vessel, connect 3 formation of puncture caecums intestinal fistula with No. 16 puncture needles, and keep somewhere the rubber tissue of a wide 2.0mm in case the pin hole closure in the root ligation.Caecum is also received the abdominal cavity, the layer-by-layer suture incision of abdominal wall, art finishes the anti-shock of hypodermic injection physiological saline immediately, and postoperative is freely drunk water.Animal grouping and administration: rat is divided into normal control group, sham operated rats, model group and treatment by the table of random number method organizes 4 groups.The treatment group respectively at CLP after 0.5,12,24,36,48 and 60h inject test sample through vena dorsalis penis, be divided into behind the CLP 2,8,24,48 and the 72h time point according to observing time point, 8 animals of each time point; Model group is according to dividing into groups and parallel injection equivalent physiological saline with method.Collection of specimens and processing: by corresponding time point with Animal Anesthesia after, the abdominal aorta sterile blood sampling is left and taken 3ml blood in massfraction is 3.8% sodium citrate (1: 9) anti-freezing test tube, 4 ℃ of centrifugal 5min down, separated plasma ,-80 ℃ of preservations are to be measured.Detect index: blood plasma HMGB-1 content: with HMGB-1 enzyme linked immunosorbent assay (ELISA) kit measurement blood plasma HMGB-1 content, detect the strict by specification of step and carry out,, calculate sample HMGB-1 concentration with sample absorbance (A) value substitution typical curve.Test-compound adopts above method to detect to the inhibiting effect of HMGB-1.
Biology screening-the endotoxin of embodiment 14 monarch drug in a prescription effective component groups suppresses active experiment
Endotoxin suppresses active Bioexperiment: external tachypleus amebocyte lysate qualitative method: test is established and is tried group of molecules, dexamethasone positive controls, endotoxin group, solvent control group, negative control group.Candidate group of molecules, dexamethasone are a series of desired concns with the solvent dilution, endotoxin are dissolved (10IU/ml) with agent dissolves liquid. add the 0.1ml soup at the 0.01ml endotoxin, insulation is 1 hour or 4 hours in 37 ℃ of waters bath with thermostatic control.Other group similarity condition temperature is down bathed.After temperature is bathed, other gets, and a cover test tube adds tachypleus amebocyte lysate 0.1ml respectively and temperature is bathed the reactant liquor 0.1ml mixing that reacts the back, puts in 37 ℃ of waters bath with thermostatic control again and is incubated 1 hour, and temperature is taken out test tube rack after bathing gently for the second time, slowly, observe and judge test findings 180 ° of test tube rack reversings.Be (+) to be solid gel person in managing, be not gel or be gel but can not the person of being kept perfectly be (-).Tried the external anti-endotoxin effect of molecule and provided foundation in vivo studies dosage design by this experimental observation.Antiendotoxin activity experiment in the body: get Kunming mouse, random packet, every group of 12 mouse, normal group is irritated stomach tap water 0.5mL every day, 4d continuously; Be subjected to the examination group to irritate huanglian jiedu decoction 4 d of stomach various dose, be subjected to examination group, normal group, Dexamethasone group lumbar injection D-GALN 150mg/kg after the last administration, the 24h posterior orbit is got blood system from serum, puts-30 ℃ of refrigerators, IL-2 to be measured.
Biology screening-the bacteriostatic experiment of embodiment 15 monarch drug in a prescription effective component groups
Bacteriostatic experiment: be primarily aimed at the common source of infection Gram-negative bacteria of pyemia, also comprise the part gram-positive bacteria, specific as follows: streptococcus pneumonia (31002), staphylococcus aureus (26003), Pseudomonas aeruginosa (10104), Escherichia coli (44102).Activated, the growth period bacterium liquid streptococcus pneumonia that experimentizes of taking the logarithm after identifying is cultivated 18h for 37 ℃, and outside streptococcus pneumonia was cultivated with serum broth and blood agar culture-medium, other 3 kinds of bacterium were all used meat extract soup and plain agar medium culture.1) agar diffusion method (mensuration of inhibition zone diameter size): cut-off footpath 12cm plate, impouring nutrient culture media (blood agar culture-medium or plain agar nutrient culture media) 15mL, dip in to get with the cotton swab of sterilization after the condensation and be uniformly coated on media surface, place certain hour under the room temperature for examination bacterium bacterium liquid.Card punch with diameter 6mm punches on agar plate, and every hole adds a certain amount of soup, and every kind of soup is established 3 holes.Take out behind 37 ℃ of cultivation 24h, survey inhibition zone diameter, and obtain the mean value of 3 hole inhibition zone diameters of every kind of soup.2) tube dilution method (mensuration of MIC, MBC) is got some of sterile test tube, after doubly diluting medicine with fluid nutrient medium (serum broth or meat extract soup) etc., each pipe adds 0.1mL for examination bacterium liquid, establish nutrient culture media control tube and bacterium liquid control tube simultaneously, take out observations behind 37 ℃ of cultivation 24h, under the satisfactory situation of control tube, observe each developmental tube one by one, with can inhibition test the high dilution of medicine of bacteria growing be minimum inhibitory concentration (MIC), and the culture in the test tube of each asepsis growth is forwarded on the agar plate of corresponding nutrient culture media.37 ℃ are taken out observations after cultivating 24h, are minimum bactericidal concentration (MBC) with the high dilution of the medicine that kills 99.9% test organisms thalline.
Biology screening-the refrigeration function of embodiment 16 monarch drug in a prescription effective component groups
The zoopery of refrigeration function: get 50 of Wistar kind rats, body weight 220-250g, male and female half and half are divided into three groups of the high, normal, basic dosage of test-compound, aspirin positive controls group and physiological saline blank group.All the other respectively organize rat to screening the target compound that draws with reference dose except that the physiological saline group, and the 2h administration of injection back is surveyed 1 anus temperature every 1h. continuous 8h.The body temperature of the body temperature and the back different time of taking medicine changed before record was taken medicine.The cooling-down effect of test-compound and control group relatively and carry out statistical analysis judge that the refrigeration function of test-compound also provides the optimal dose reference.
The research of embodiment 17 other effective constituent molecular groups
Each effective part group all adopts above-mentioned research method to carry out screening system, analyzes and researches one by one at different effectively groups with pathological model at different target spots, draws effective constituent molecule crucial in each effective part group.
Embodiment 18 effective constituent molecule prescriptions (compatibility), overall evaluation principle and method
The cardinal rule that prescription (compatibility) is followed: through screening system and " monarch " effective constituent molecular group that checking obtains, the compatibility design that also must carry out effective constituent obtains the new prescription of identical with former side's pharmacological action (or greater than former side's pharmacological action).The research of Chinese medicine effective component group compatibility its essence is the dynamic (dynamical) research of drug interaction.In the middle of during pharmaceutically active ingredient flock mating 5, dissimilar influencing each other takes place between middle pharmaceutically active ingredient, general performance is the enhancing of drug effect or weakens etc., by analyzing the quantitative rule between a plurality of effective constituent various dose and effect, finally realizes the optimization of Chinese medicine effective component group compatibility.The cardinal rule of Chinese medicine effective component group prescription is: reasonable, favourable, certificate arranged.Reasonablely be exactly: on theory of traditional Chinese medical science and pharmacological mechanism, prescription or compatibility have rational document or experimental basis.Favourablely be exactly: synergy is arranged on the drug effect and toxicity does not increase; Toxicity reduces or the toxicity organ disperses, and drug effect does not subtract; Onset or complementation action time; The drug effect range expansion acts on multiple symptom.Have according to being exactly: the component medicine is selected rationally; Dosage ratio is suitable, near best proportioning; Dosage is appropriate, and too small drug effect reduces, and is excessive and unnecessary.Secondly, science, reasonable, objective Chinese medicine effective component group compound must be selected desirable overall evaluation method.The desirable evaluation method of Chinese medicine effective component group prescription is: can stand on the mathematics.Mathematical model is rigorous; Feasible on the specialty.Having acology is worth; Credible on the statistics.Testing index is reasonable; Reasonable in logic.According to above principle,, many indexs should be carried out simplification and handle, be i.e. analysis-by-synthesis because Chinese medicine effective component group compound is multicomponent, multiple dose and many ratios compatibility.Many index comprehensives index method is estimated so be more suitable for Chinese medicine effective component group prescription owing to can carry out analysis-by-synthesis to many indexs.By research process such as above information integrated, data mining, system modelling, step analysis, effectively identification, optimal design, the overall evaluations, set up huanglian jiedu decoction Chinese medicine effective component group prescription technology.Final definite huanglian jiedu decoction Chinese medicine effective component group prescription.
Embodiment 19 effective constituent molecule prescriptions (compatibility), overall evaluation technology path
Following traditional Chinese medical science compatibility theory and Chinese medicine effective component group prescription principle reasonable, favourable, that certificate is arranged carries out.According to the principle of uniform Design, be the investigation factor with " monarch " four effective molecular groups, the molecule number is the investigation level, derives new compatibility prescription.Each molecule is with the optimal dose compatibility side of going into.Prescription and overall evaluation technology path are seen Fig. 4.
Embodiment 20 Chinese medicine effective component group molecule prescription inclusion criterias
1. the strong molecule of drug action preferentially is selected in the similar Chinese medicine effective component group molecule; When drug action was identical or close in the 2. similar Chinese medicine effective component group molecule, reference computers screening, in-vitro screening and bibliographical information result carried out comprehensive evaluation, select the superior.3. above-mentioned 1. when 2. identical or close in the similar Chinese medicine effective component group molecule, many persons are preferential for action target spot.4. in the similar Chinese medicine effective component group molecule when above-mentioned 1., 2. with 3. identical or close, easily winner's (in Chinese medicine active constituent content height, preparation method simple) is preferential.5. when above-mentioned 1., 2., 3. with 4. identical or close, the person is preferential for dosage little (consumption is few) in the similar Chinese medicine effective component group molecule.
Embodiment 21 selected effective constituent molecule prescription posteriority confirmatory tests
Set up experimental animal model, rat is divided into normal control group, sham operated rats, model group, positive controls and treatment group by the table of random number method at different pathological models.The treatment group is according to the clinical administration administration.Model group is according to the also parallel equivalent physiological saline that gives that divides into groups with method, and positive controls gives the huanglian jiedu decoction decoction respectively, and observation is obtained a result.
The pharmacological evaluation drug effect checking of the pre-side of antiinflammatory action molecule in embodiment 22 effective constituents
Set up the inflammation pharmacological model of mice caused by dimethylbenzene xylene auricle edema, each composition in the pre-side of the huanglian jiedu decoction antiinflammatory action molecule that embodiment 17 is obtained: jamaicin, forulic acid, 7-methoxyl baicalein, Geniposide carry out anti-inflammatory agent effect card respectively.
1. material:
1.1 animal used as test: this research institute breeds Kunming mouse voluntarily, male and female half and half, body weight: 20.0 ± 2.0g.
1.2 medicine: jamaicin, forulic acid, 7-methoxyl baicalein, Geniposide are all purchased the company in sigma.Standby with the dissolved in distilled water wiring solution-forming.The positive control medicine boils concentrate (0.8g crude drug/ml) for the huanglian jiedu decoction decocting.Dimethylbenzene (analyzing pure) is purchased the Ke Wei company in University Of Tianjin.
1.3 instrument: precise electronic balance, Japan, ShimadzuAUY220.
2. experimental technique:
Get 60 mouse and be divided into 6 groups at random, 10 every group, each group is gastric infusion respectively, and the blank group is irritated with distilled water, is subjected to the examination group to give 4 kinds respectively and is subjected to the reagent thing, and all according to the 150mg/kg administration, positive controls is pressed the 25ml/kg administration, continuously 7d.All mouse, 12h after art time administration, auris dextra is coated with dimethylbenzene 0.05ml, behind the 2h, takes off vertebra and puts to death mouse, and clip mouse two ears are got its auricle with the card punch of diameter 0.9cm, weigh.The mice auricle swelling degree calculates: swelling degree=auris dextra weight-left ear weight.Inhibitory rate of intumesce calculates: inhibitory rate of intumesce=(blank group swelling degree-administration group swelling degree)/blank group swelling degree * 100%.
3. experimental result:
Table 5 anti-inflammatory The pharmacological results
Annotate: compare with the blank group,
*P＜0.01,
*P＜0.001
Experimental result (table 5) shows that 5 kinds of medicines all have certain antiphlogistic effects, can be used for the prescription element of " huanglian jiedu decoction " antiinflammatory action molecule new compound.
The dosage ratio and the experimental design of embodiment 23 antiinflammatory action molecule new compounds
Utilization uniform Design software UD2.2,4 molecules that obtain with embodiment 18 are factor of influence, and the factor is got 7 levels, and each factor level is all got 200,100,50,25,12.5,6.25,0 (unit: mg/kg).Software generates uniform designs table U7, arrange each factor and level according to the design table, obtain the experimental program of table 2, wherein one classify a kind of medicine as, delegation is exactly a kind of new prescription, gets corresponding medicine mixing by line number value in the table and promptly gets this prescription, totally 7 kinds, wherein the 7th group is the blank group, establishes huanglian jiedu decoction water cooking liquid positive controls simultaneously.
The anti-inflammatory pharmacological evaluation of embodiment 24 antiinflammatory action molecule new compounds
1. material
1.1 animal used as test: this research institute breeds Kunming mouse voluntarily, male and female half and half, body weight: 20.0 ± 2.0g.
1.2 medicine: jamaicin, forulic acid, 7-methoxyl baicalein, Geniposide are all purchased the company in sigma.Standby with the dissolved in distilled water wiring solution-forming.The positive control medicine boils concentrate (0.8g crude drug/ml) for the huanglian jiedu decoction decocting.Dimethylbenzene (analyzing pure) is purchased the Ke Wei company in University Of Tianjin.
1.3 instrument: precise electronic balance, Japan, ShimadzuAUY220.
2. experimental technique:
Get 80 mouse and be divided into 8 groups at random, 10 every group, mouse is irritated stomach by table 1 respectively and is subjected to the reagent thing, and positive controls is pressed the 25ml/kg administration, continuously 7d.All mouse, 12h after the last administration, auris dextra are coated with dimethylbenzene 0.05ml, behind the 2h, take off vertebra and put to death mouse, and clip mouse two ears are got its auricle with the card punch of diameter 0.9cm, weigh.The mice auricle swelling degree calculates: swelling degree=auris dextra weight-left ear weight.Inhibitory rate of intumesce calculates: inhibitory rate of intumesce=(blank group swelling degree-administration group swelling degree)/blank group swelling degree * 100%.
3. experimental result:
The results are shown in Table 6.Huanglian jiedu decoction decocting liquid positive controls inhibitory rate of intumesce is 71.25%.Intuitive analysis, various prescriptions all have good inflammation inhibition effect, especially first kind of prescription inflammation inhibiting rate even have surpassed huanglian jiedu decoction decocting liquid positive control.
Regression equation by the software generation: y=16.3+0.24*X (1)+0.128*X (2)+0.0802*X (3)+0.246*X (4)
X (1) represents the jamaicin dosage; X (2) represents the forulic acid dosage
X (3) represents 7-methoxyl baicalein dosage; X (4) represents the Geniposide dosage
Y represents inhibitory rate of intumesce.
Because regression coefficient represented in a way its to dependent variable to the contribution of inflammation inhibiting rate as a result, compared 0.24: 0.128: 0.0802 by regression coefficient: 0.246 ≈ 6: 3: 2: 6, rough infer that this huanglian jiedu decoction antiinflammatory action molecule new compound is with jamaicin: forulic acid: 7-methoxyl baicalein: Geniposide=6: 3: 2: 6 the proportioning side of one-tenth inflammation suppresses effect may be better, verify through pharmacological evaluation, its inhibitory rate of intumesce is 71.02%, therefore keeps first kind of prescription 32: 16: 8: 1 dosage ratio.
Table 6 prescription uniform designs table and experimental result
Annotate: compare with the blank group,
*P＜0.01,
*P＜0.001
Claims (7)
1, the invention provides a kind of Chinese prescription designing technique based on effective component group, it is characterized in that this technology integrated information integration (chemistry, the biological information database is set up), data mining (knowledge base of existing pharmacological action of prescription and molecular mechanism is set up), system modelling is (based on pathogenetic molecular pathology model and cell, the pharmacophore model of integral level makes up), step analysis is (based on traditional Chinese medical science compatibility theory, modern molecular biology and pharmacological effective constituent heap sort and based on the illness molecule, cell, the symptom classification of integral level pharmacophore model), effectively identification (area of computer aided drug design technology and the checking of inside and outside pharmacophore model), optimal design (based on pathogenetic pathological model and area of computer aided drug design The Application of Technology), the overall evaluation multinomial technology such as (many indexs whole animal model and many index comprehensives index assessment methods) is used for the design of Chinese medicine effective component group prescription.Mainly may further comprise the steps:
1) select clinical effective prescription huanglian jiedu decoction as research object.
2) applied chemistry information science and correlation technique thereof by methods such as literature survey, database accesss, are set up the chemical constitution information database of each simple of prescription huanglian jiedu decoction.Comprising compound title, molecular formula, molecular weight, structural formula (two dimension, three-dimensional), physico-chemical property, pharmacological action, molecular mechanism, medicine generation, toxicity, clinical practice etc.
3) by methods such as literature survey, database accesss, set up huanglian jiedu decoction compound database.Comprising composition, dosage, function and cure mainly, chemistry, pharmacological action, molecular mechanism, medicine generation, toxicity, clinical practice etc.
4) on the basis of literature survey, database access, data mining, at the main and clear and definite pharmacological action of prescription, system sets up its pyemia molecule pharmacological model.
5) be guidance with traditional Chinese medical science compatibility theory monarch, prescription carried out monarch function effective components group's research according to pyemia pathogenesis molecular mechanism.
6) appliance computer ancillary drug designing technique, and binding molecule pharmacological model The selection result, to the effective constituent cluster and just arrange with score right.
7) according to area of computer aided drug design molecule pharmacological model The selection result, in the vivo and vitro pharmacological model of correspondence, verify respectively.
8) according to screening of molecule pharmacological model and monarch cluster result, obtain complexity, from every class model, choose several molecular composition molecule compounds in conjunction with vivo and vitro pharmacological model pharmacological action intensity, the effect of many target spots and compound.
9) in the whole animal model that embodies the Syndrome in TCM marquis, prescription is verified.Obtain the Chinese prescription based on effective part group of reflection compound mass action characteristics.
10) by optimal design, systematic study and checking repeatedly, finally set up the Chinese prescription designing technique that is suitable for traditional Chinese medicine mass action characteristics, obtain new Chinese medicine molecule compound based on effective component group.
2, a kind of Chinese prescription designing technique according to claim 1 based on effective component group, it is characterized in that this method not only is applied to the huanglian jiedu decoction of being mentioned in (1) in the claim 1, can be applied to also simultaneously that clinical treatment is effective, clear and definite other any prescriptions of study of pathogenesis.
3, according to claim 1 and the described a kind of Chinese prescription designing technique of claim 2 based on effective component group, it is characterized in that this method not only is applied to the pyemia pathological model of being mentioned in (1) in the claim 1, also can be applied to the research of other clear and definite any pathological models of study of pathogenesis simultaneously.
4, a kind of Chinese prescription designing technique according to claim 1 based on effective component group, it is not only cited as embodiment to it is characterized in that (4) in this method relate to software and the method used when the molecule pharmacological model uses a computer the ancillary drug design in setting up, and effect technology and method also can replace mutually on an equal basis.
5, a kind of Chinese prescription designing technique according to claim 1 based on effective component group, it is characterized in that (5) relate to effective constituent similarity cluster in this method, with the compound molecular amounts is index, control cluster similarity, in the molecular group of structural similarity, select the forward molecule of quasi-medicated property scoring and enter butt joint, to accelerate butt joint speed, increase work efficiency.
6, a kind of Chinese prescription designing technique according to claim 1 based on effective component group, it is characterized in that (8) are according to screening of molecule pharmacological model and monarch cluster result in this method, obtain complexity in conjunction with vivo and vitro pharmacological model pharmacological action intensity, the effect of many target spots and compound, select candidate molecules to form recruit's compound, wherein the number of candidate molecules does not have strict restriction, draws reasonable number according to each experimental result analysis-by-synthesis.
7, a kind of Chinese prescription designing technique according to claim 1 based on effective component group, it is characterized in that single medicinal material effective substance, the mechanism of action and effective substance that this method is equally applicable to complicated component are that pharmacological screening and verification model are set up, research object in step (1), (2), (3) replaces with single medicinal material, and other steps according to claim 1.
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