CN104597184A - Construction method of traditional Chinese medicine primary plant in vivo node metabolic network - Google Patents
Construction method of traditional Chinese medicine primary plant in vivo node metabolic network Download PDFInfo
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Abstract
The embodiment of the invention discloses construction of a metabolic network taking methyl-n-nonylketone of Houttuynia cordata as a node and explains a construction method of a traditional Chinese medicine primary plant in vivo node metabolic network. The construction method comprises the following steps: selecting an effective constituent of the traditional Chinese medicine primary plant in vivo metabolic network as the node, capturing a metabolic enzymes system corresponding to the node, anchoring each metabolic constituent of the node metabolic network by utilizing content change of metabolic constituent, that is, each network point of the node metabolic network, and calculating metabolic balance constant of each metabolic constituent of the node metabolic network. By utilizing the construction method, the entire traditional Chinese medicine primary plant metabolic network is split to a plurality of small networks, the entire metabolic network is constructed by constructing small networks, the construction method is applied to quality control of traditional Chinese medicine, a current point control mode is combined, and then crossing of a surface control mode of one measurement with multiple calculation, one measurement with multiple evaluation and one measurement with multiple control can be realized.
Description
Technical field
The present invention relates to the construction method of the metabolism network in the former plant of a kind of Chinese medicine, be specifically related to the construction method of a kind of Chinese medicine former plant interior nodes metabolism network.
Background technology
As everyone knows, traditional Chinese medicine quality is the factor that concerns affecting tcm clinical practice curative effect and security, is the motive power promoting TCM Modernization process.Therefore, in recent years, traditional Chinese medicine quality is paid close attention to extremely both at home and abroad.In order to ensure the application in TCM of high-quality in clinical, the quality control method of a kind of suitable " Chinese medicine " must be set up.Current traditional Chinese medicine quality control model continues to use Western medicine pattern mostly, namely detects the content of a certain or several composition of Quality Control Chinese medicine.When the content of tested composition reaches specified value, namely judge that this traditional Chinese medicine quality is qualified.This pattern belongs to " point " control model of one or two composition controlled in Chinese medicine, only have when tested composition be this Chinese medicine play a certain clinical efficacy material base and unique time, the index composition that could really control as this traditional Chinese medicine quality, this obviously and many Therapeutic Characteristics of the multicomponent Mutiple Targets comprehensive effect of Chinese medicine do not match.Therefore, existing " point " control model can not meet Chinese medicine system complicated and changeable.
Chinese medicine is different from Western medicine, and the former plant of Chinese medicine all passes through complicated metabolism in growth course, forms the plant metabolism network be made up of traditional Chinese medicine ingredients, metabolic enzyme, metabolic pathway.If Chinese medicine former plant metabolism network figure can be drawn out, and calculates the metabolic balance coefficient of each traditional Chinese medicine ingredients; Then this coefficient of balance is applied to traditional Chinese medicine quality control; Again in conjunction with current " point " control model, the leap of " face " control model of " survey calculate ", " survey comment " and " survey is multi-control " can be realized more more.
But the former plant metabolism network of Chinese medicine has the biosynthetic metabolism approach differing from and study in modern molecular biology and molecule pharmacognosy, and it is the network system built by metabolic balance constant between each composition by many traditional Chinese medicine ingredients.The subjects such as current plant physiology and molecule pharmacognosy have carried out large quantity research to medical raw plant metabolism network.Although by several metabolic pathways in the former plant metabolism network of Study of Traditional Chinese Medicine, traditional Chinese medicine ingredients is pressed textural classification, utilize the route of synthesis of each class Metabolite of technical research such as isotope labeling tracking, and study its key enzyme, plant modification bio-reactor or macroscopical gene regulation traditional Chinese medicine quality etc., achieve great successes.But also do not make a breakthrough about the research of the overall metabolism network of Chinese medicinal plant always.
Summary of the invention
In order to overcome the above problems, the invention provides the construction method of a kind of Chinese medicine former plant interior nodes metabolism network.
The former plant metabolism network of Chinese medicine has the biosynthetic metabolism approach differing from and study in modern molecular biology and molecule pharmacognosy, and it is the network system built by metabolic balance constant between each composition by many traditional Chinese medicine ingredients.Chinese medicine former plant metabolism network can dissect as being made up of many " node " metabolism networks.Should refer to centered by a certain composition by " node " metabolism network, this composition upper level metabolic response thing and next stage metabolism product are " site ", metabolic balance constant between composition is bridge, and with corresponding father and son's metabolic enzyme, (synzyme composes name " father's enzyme ", catabolic enzyme composes name " sub-enzyme ") cyberrelationship that forms for " media ", or claim metabolism " LAN (Local Area Network) ", " father and son's metabolism network ", " the periphery metabolism network " of this composition.So research first from " node " plant metabolism network of a certain composition of Chinese medicine, and then can extend to other compositions, the docking of " node " plant metabolism network will be owned, just can realize the structure of the former plant metabolism network of this Chinese medicine thus.
In order to realize above object, technical scheme provided by the invention is: the construction method of " node " metabolism network in the former plant of a kind of Chinese medicine, and concrete steps are as follows:
(1) choosing an effective constituent in Chinese medicine former plant intracellular metabolite network is " node ", catches the metabolic enzyme system corresponding with described " node ";
(2) Metabolite content is utilized, each Metabolite in " node " metabolism network described in grappling, each " site " namely in " node " metabolism network;
(3) the metabolic balance constant of each Metabolite in described " node " metabolism network is calculated.
In construction method of the present invention, described in step (1), the former plant of Chinese medicine comprises cordate houttuynia, rheum officinale, safflower, honeysuckle.
In construction method of the present invention, described in step (1), effective constituent comprises methylnonanone, archen, Kaempferol, chlorogenic acid.
In construction method of the present invention, each " site " described in step (2) comprises upper level metabolic response thing and the next stage metabolic product of methylnonanone metabolism.
Further, in construction method of the present invention, affinity chromatography is utilized to catch the metabolic enzyme system corresponding with methylnonanone.
Further, in construction method of the present invention, GC-MS is utilized to analyze fat-soluble Metabolite content.
Further, in construction method of the present invention, HPLC is utilized to analyze the change of water-soluble metabolic component content.
Relative to prior art, the present invention has following beneficial effect:
1, the present invention opens the former plant metabolism network of Chinese medicine and builds new approaches and new method, being split as several little networks, reaching the structure of whole metabolism network by building little network by whole Chinese medicine former plant metabolism network;
2, the structure of interior " node " metabolism network of the former plant of Chinese medicine, formed by the associated metabolic composition of Chinese medicine " node " composition, " node ", i.e. " node " metabolism network of forming of upper level metabolic response thing and next stage metabolism product and the metabolic enzyme system corresponding with " node ", metabolic balance constant;
3, by building interior " node " metabolism network of the former plant of Chinese medicine and then building whole metabolism network in the former plant of Chinese medicine, and traditional Chinese medicine quality control is applied to; In conjunction with current " point " control model, the leap of " face " control model of " survey calculate ", " survey comment " and " survey is multi-control " can be realized more more.
Accompanying drawing explanation
Fig. 1, with methylnonanone be " node " each composition of metabolism network between the mathematical model of metabolic balance.
Embodiment
Carry out clear, complete description below in conjunction with in the embodiment of the present invention to technical scheme of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment
The structure of " node " metabolism network being effective constituent with methylnonanone in cordate houttuynia.
(1) affinity chromatography is utilized to catch the associated metabolic enzyme system with methylnonanone:
(A) active gross protein in fresh cordate houttuynia is extracted:
A, configuration solution:
1. 2mol/L trishydroxymethylaminomethane (Tris): get 24.228g Tris, add deionized water dissolving, be settled to 1000mL;
2. 2mol/LHCl: measure 16.7mL concentrated hydrochloric acid, adds deionized water and is settled to 100mL;
3. 1mol/L (PH=7.5) Tris-HCl damping fluid: the Tris getting 100mL 2Mol/L, adds the HCl of 80.6mL 2mol/L; Add deionized water and be settled to 200mL, then dripping dense HCl, to be adjusted to PH be 7.5;
4. extracting solution of protein (PH=7.5): get polyvinylpyrrolidone (PVP) powder 2g, in beaker, add appropriate amount of deionized water dissolve, be poured in volumetric flask, measure 15mL 2mol/L Tris-HCl damping fluid, 25mL glycerine imports in volumetric flask, add deionized water and be settled to 100mL;
B, extract active total protein:
Precision takes fresh cordate houttuynia 6.1370g in mortar, adds 1 ~ 2g sand, gets extracting solution of protein 100mL; First add 10mL extracting solution of protein, grinding 90min, becomes pasty state; Be transferred in beaker, remaining 90mL extracting solution of protein joined in beaker, mixes; In 4 DEG C of standing 3h, centrifuge speed 12000r/min, centrifugal 20min; Discard precipitation, get supernatant solution, obtain the glycerite of active gross protein, be placed on-20 DEG C of freezen protective;
(B) methylnonanone media for affinity chromatography is prepared:
Get 1.7mL tetramethoxy-silicane (TMOS), 100 μ L methyl alcohol, the glycerine water solution of 300 μ L 50% (massfraction), 250 μ L water and 100 μ L 0.04mol/L HCl to mix, vortex oscillation 20min; In 4 DEG C of standing 10min; Add the phosphate buffered saline(PBS) (PBS damping fluid) that 2.45mL pH is 7.4; Add the PBS damping fluid 725 μ L containing 165 μ g methylnonanones immediately, slightly shake, gel generates rapidly, 4 DEG C of standing 20h, both obtains methylnonanone media for affinity chromatography;
(C) the metabolic enzyme system corresponding with methylnonanone is caught:
A, methylnonanone media for affinity chromatography wet method obtained in step (1) (B) is filled post, make suitable affinity chromatographic column; Wherein, glass pillar is selected in this experiment;
B, get glycerite (the being labeled as A) 0.2mL of the active gross protein in step (1) (A) .b, sample introduction is in populated glass pillar, treat the decline of sample liquid level and upper strata gel phase at ordinary times, add the PBS buffer solution elution that 8mL pH is 7.4; Collect efflux 7.5mL; Be concentrated into 0.4mL, the solution example after concentrated, is labeled as B;
C, blank group of glycerite (the being labeled as stoste) 0.2mL getting the active gross protein in step (1) (A) .b, sample introduction (does not add standard items methylnonanone to blank glass pillar, all the other operate same step (1) (C) .b, collect efflux sample, be labeled as blank group;
D, sample above-mentioned steps (1) (C) .b and (1) (C) .c obtained do protein electrophoresis, and what protein electrophorese figure result demonstrated metabolic enzyme system corresponding with methylnonanone catches situation;
(2) in metabolism environment, utilize metabolic enzyme system to the catalytic action of the metabolic response of corresponding composition, the feature of the change of described corresponding component amount can be caused, each Metabolite of " node " metabolism network described in grappling, each " site " namely in " node " metabolism network;
(A) adopt the fat-soluble total volatile oil in extraction by steam distillation cordate houttuynia, preserve in the refrigerator of-20 DEG C, for subsequent use;
(B) described fat-soluble total volatile oil is made GC-MS to analyze, obtain finger-print, it can thus be appreciated that the relative content of each constituent in described fat-soluble total volatile oil;
(C) the active protease system corresponding with methylnonanone metabolism that step (1) (C) .d catches is inserted the content observing each constituent of fat-soluble volatile oil in described fat-soluble volatile oil system, what obtain methylnonanone metabolism thus belongs to fat-soluble upper level metabolic response thing and next stage metabolic product;
(D) extract the water soluble ingredient in cordate houttuynia with decocting method, preserve in the refrigerator of-5 DEG C, for subsequent use;
(E) water soluble ingredient is made HPLC to analyze, obtain the relative content of each constituent in described water soluble ingredient;
(F) step (C) is repeated, insert catching the active protease system corresponding with methylnonanone metabolism obtained the content observing each composition in water soluble ingredient system, what obtain methylnonanone metabolism belongs to water miscible upper level metabolic response thing and next stage metabolic product;
(3) measuring and calculating take methylnonanone as the metabolic balance constant between each composition of metabolism network of " node ";
1. founding mathematical models:
For six kinds of Metabolite founding mathematical models in methylnonanone upper level metabolic response thing and next stage metabolic product, as shown in Figure 1; Wherein A, A ', B, B ', C, C ' are reactant or the product of the metabolic pathway of methylnonanone; K
a, K
a, K
b, K
b ', K
c, K
c 'be the methylnonanone metabolic response equilibrium constant; E
a, E
a ', E
b, E
b ', E
c, E
c 'be father and son's metabolic enzyme of methylnonanone, then:
For above-mentioned parameter, least square method is adopted to carry out parameter fitting;
Make K
i=K
i 0+ h
i, then:
Solve above-mentioned hexa-atomic linear function group and just can obtain one group of equilibrium constant, then one group of equilibrium constant can be tried to achieve by iterating;
Above-mentioned model is only applicable to linear dynamics system, if nonlinear, then solves after first converting linear relationship to parameter replacement;
2. setting up in cordate houttuynia take methylnonanone as " node " metabolism network of effective constituent:
Comprehensive above step (1), (2), (3), draw by Chinese medicine " node ", the upper level metabolic response thing of described node and next stage metabolic product, plant " node " metabolism network that the corresponding metabolic enzyme system of described node, metabolic balance constant are formed.
Comprehensive the above results is successfully " node " metabolism network of effective constituent with methylnonanone in structure cordate houttuynia.
Disclosed embodiment of this invention is only the section Example in the scope of the invention.Based on the technical scheme in the present invention and embodiment, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Claims (7)
1. a construction method for Chinese medicine former plant interior nodes metabolism network, is characterized in that, comprise the steps:
(1) choosing an effective constituent in Chinese medicine former plant intracellular metabolite network is node, catches the metabolic enzyme system corresponding with described node;
(2) Metabolite content is utilized, each Metabolite in node metabolism network described in grappling, each site namely in node metabolism network;
(3) the metabolic balance constant of each Metabolite in described node metabolism network is calculated.
2. construction method according to claim 1, is characterized in that, described in step (1), the former plant of Chinese medicine comprises cordate houttuynia, rheum officinale, safflower, honeysuckle.
3. construction method according to claim 1, is characterized in that, described in step (1), effective constituent comprises methylnonanone, archen, Kaempferol, chlorogenic acid.
4. construction method according to claim 1, is characterized in that, each Metabolite described in step (2) comprises upper level metabolic response thing and the next stage metabolic product of methylnonanone metabolism.
5. construction method according to claim 1, is characterized in that, utilizes affinity chromatography to catch the metabolic enzyme system corresponding with methylnonanone.
6. construction method according to claim 1, is characterized in that, utilizes GC-MS to analyze fat-soluble Metabolite content.
7. construction method according to claim 1, is characterized in that, utilizes HPLC to analyze the change of water-soluble metabolic component content.
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Citations (4)
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| CN101694485A (en) * | 2009-09-27 | 2010-04-14 | 上海交通大学 | Method for utilizing affinity column to analyze holoprotein |
| CN102622532A (en) * | 2012-02-27 | 2012-08-01 | 中国药科大学 | Method for building complex drug material group in vivo and vitro associated metabolic network |
| WO2013006278A2 (en) * | 2011-07-01 | 2013-01-10 | Metabolon, Inc. | Identity elucidation of unknown metabolites |
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Patent Citations (4)
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| JP2008040812A (en) * | 2006-08-07 | 2008-02-21 | Keio Gijuku | Metabolism network estimation method and metabolism network estimation device |
| CN101694485A (en) * | 2009-09-27 | 2010-04-14 | 上海交通大学 | Method for utilizing affinity column to analyze holoprotein |
| WO2013006278A2 (en) * | 2011-07-01 | 2013-01-10 | Metabolon, Inc. | Identity elucidation of unknown metabolites |
| CN102622532A (en) * | 2012-02-27 | 2012-08-01 | 中国药科大学 | Method for building complex drug material group in vivo and vitro associated metabolic network |
Non-Patent Citations (3)
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| P.A.N. PUNYASIRI ET AL: "Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways", 《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》, vol. 431, 11 September 2004 (2004-09-11), pages 22 - 30, XP 004581254, DOI: doi:10.1016/j.abb.2004.08.003 * |
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