CN109596749A - Based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method - Google Patents
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Abstract
The invention discloses a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method: the small-molecule mixture in Chinese medical extract or other sources being dissolved in buffer solution, enzyme is added and is incubated for altogether；The solution is filtered using ultracentrifugation concentration tube, it is repeated multiple times, merge concentrate, with buffer solution constant volume；The variation of chromatographic peak after being incubated for altogether using liquid chromatography-mass spectrometry analysis with enzyme.The present invention, which has been tested, screens Chinese medicine hypoglycemic activity ingredient using ultrafiltration-UPLC-MS, the experimental results showed that this method can carry out preliminary quick screening to active small molecule compound natural in complicated ingredient.The present invention has the features such as easy to operate, favorable reproducibility is tested in rapid batch screening, and experimental data is intuitive and reliable, provides new idea and method for the research of the effective substance of Chinese medicine or compound.
Description
Technical field
The invention belongs to the analysis of Chinese medicine active skull cap components and pharmaceutical synthesis fields, are related to a kind of based on ultrafiltration
(Ultrafiltration) method of-HPLC/UPLC-MS technology quick screening drugs active constituent.
Background technique
Diabetes are a kind of common chronic diseases of modern society, and main clinical manifestation is hyperglycemia and sugar, protein, rouge
The symptoms such as fat, water and electrolyte disturbance, clinical manifestation are more drinks, more foods, diuresis, out of strength, thin etc..According to theory of traditional Chinese medical science,
The disease largely belongs to " quenching one's thirst ", and diabetes then can be divided into I type and II type by modern medicine.The pathogenesis of diabetes is multiple
It is miscellaneous, and Chinese medicine is mainly reflected in multiple target effect in the advantage of hypoglycemic aspect, while hypoglycemic, take into account lipid-loweringing, anti-oxidant
Deng.Therefore, there is a need other than being controlled according to differential diagnosis in tcm opinion according to the modern times in terms for the treatment of the common chronic diseases such as diabetes
The research of pharmacology of Chinese materia medica finds new medicine activity component.
Modern Chinese herbal medicine pharmaceutical research shows that many Chinese medicine material medical instruments have good hypoglycemic effect, can reduce high blood
The blood glucose level of sugared animal model, clinical application is also very extensive, including the coptis, pueraria lobata, glutinous rehmannia, Radix Astragali, Radix Ophiopogonis, Stephania tetrandra etc.
Deng.However more Chinese medicine hypoglycemic effective substance is not clear, the mechanism and action target spot of hypoglycemic still need to further grind
Study carefully.
At the same time, the structure activity study of modern pharmacology then needs the action target spot of clear active material.For example, with
In the alpha-glucosidase inhibitor of type-2 diabetes mellitus treatment, by inhibiting the alpha-glucosidase of small intestinal mucosa brush border, thus
Delay the absorption of sugar, reduces postprandial hyperglycemia.The characteristics of such drug is steady hypoglycemic, highly-safe, is that minority can intervene sugar
Impaired one of the oral hypoglycemic agents of tolerance.Clinically used alpha-glucosidase inhibitor medicine has acarbose, Fu Gelie
Wave sugar etc..
The effective substance research of Chinese medicine is the key problem during the modernization of Chinese medicine.By " the active matter in Chinese medicine
Matter " is mapped with " action target spot ", specifies structure-activity relationship, even more one of the critical issue of the modernization of Chinese medicine.But it is traditional
There are heavy workload, low separation efficiency, active component screening are slow for the research mode of " ingredient separation-Structural Identification-screening active ingredients "
Deficiency；Meanwhile complicated component and the relatively low feature of effective substance content increase the research difficulty of this mode.
Ultrafiltration-UPLC-MS technology is now generally used for the detection of free state object in sample, not yet
It sees and carries out the report that active pharmaceutical ingredient is quickly screened using it.
Summary of the invention
The purpose of the present invention is to provide a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent
Method.
In order to achieve the above objectives, the invention adopts the following technical scheme:
1) it is divided into identical two parts after dissolving multicomponent sample with buffer solution, is to combine preceding sample with a copy of it,
To enzyme corresponding to quasi- screening drug target is added in another, obtain in conjunction with sample；
2) by being incubated for so that in conjunction with can be with the compound of the enzyme stable bond partly or entirely by dissociating in sample
State is changed into reference state, obtains Incubating Solution；By Incubating Solution ultrafiltration and filtrate is collected, by filtrate with the buffer solution constant volume, score
Analyse sample；
3) it will be handled according to incubation identical with step 2), ultrafiltration and constant volume process (i.e. referring to knot in conjunction with preceding sample
Sample is closed equally to handle), control sample is obtained, analysis sample and control sample are respectively adopted the liquid chromatogram-of the same terms
Mass spectrometric hyphenated technique is analyzed；
4) in the LC-MS analysis result of comparative analysis sample and control sample chromatographic peak variation, will occur
The drug candidate active constituent corresponding with the target spot that compound corresponding to the chromatographic peak of variation is obtained as preliminary screening
Or lead compound.
Preferably, the multicomponent sample is selected from Chinese medical extract (for example, herbal raw material medicament extract), Chinese materia medica preparation
The mixture of (for example, compound preparation), the compound obtained through separation means or compound；The sample is dissolved with buffer solution
When institute's expense >=0.01mg.
Preferably, the enzyme is selected from the enzyme of the diabetes target spots such as alpha-glucosidase, amylase or PTP1B.
Preferably, the condition of the incubation are as follows: the time is 5~60min, and temperature is 25~40 DEG C.
Preferably, the ultrafiltration using ultra-filtration centrifuge tube and combines centrifuge separation to realize the filtering to Incubating Solution, if repeatedly
Progress is ultrasonic after centrifugation, then merging filtrate, also, the preceding insoluble matter being once centrifugated mixs with buffer solution and standing at
Reason, is then centrifuged next time again.
Preferably, the ultra-filtration centrifuge tube uses the ultrafiltration membrane of low-protein Percentage bound, the molecule retention of ultra-filtration centrifuge tube
(MWCO) >=3000Da is measured, centrifugal force >=8000g of use is centrifugated.
Preferably, the buffer solution is selected from the volatility salt buffer system that pH is 4~10, wherein volatility salt
Including but not limited to ammonium formate, ammonium acetate or trifluoroacetic acid ammonium, pH adjust use reagent include but is not limited to formic acid, acetic acid,
Trifluoroacetic acid, ammonium hydroxide or triethylamine, the volatility salt buffer system is for example, by using ammonium formate/formic acid mixed liquor (i.e. formic acid
Ammonium buffer solution), ammonium acetate/acetic acid mixture (i.e. ammonium acetate buffer solution), trifluoroacetic acid ammonium/trifluoroacetic acid mixed liquor (i.e.
Trifluoroacetic acid buffer solution).Using the buffer system of above-mentioned pH, the activity of enzyme is kept in incubation, and effectively identification can be steady
The fixed compound (ensuring its presence in the form of reference state) in conjunction with enzyme.
Preferably, the volume of the constant volume is 0.1~10mL.
Preferably, the liquid chromatography-mass spectrometry is using high performance liquid chromatography or ultra performance liquid chromatography and matter
The combination equipment (for example, UPLC-MS) of spectrum (for example, the high resolution mass spectrums such as QTOF, Orbirap or string level four bars mass spectrum).
Preferably, the variation of the chromatographic peak include chromatographic peak component variation and/or identical retention time chromatographic peak
Strength Changes (for example, the reduction of chromatographic peak component or reduction of intensity).
Preferably, active (for example, hypoglycemic activity) ingredient of the drug candidate or lead compound are selected from natural
Small molecule compound or the small molecule compound of synthesis.
The beneficial effects of the present invention are embodied in:
The present invention is by being incubated for the identification realized in sample with the small molecule compound of target protein (enzyme) stable bond, in turn
The characteristics of being retained using ultrafiltration membrane molecular weight, " free small molecule " and " small molecule/target protein conjugate " are separated；Then
Secondary separation is carried out to " free small molecule " by liquid chromatogram (for example, UPLC), and mass spectrum (MS) the technology analysis of combination should be " small
The structure (qualitative) of molecule " and the variation (quantitative) for combining forward and backward content, so that analysis, which obtains having in sample, determines structure
The targeting feature of small molecule filters out potential active pharmaceutical ingredient in sample accordingly.The present invention have it is easy to operate, quickly batch
The features such as amount screening, tests favorable reproducibility, and experimental data is intuitive and reliable, especially suitable for complicated ingredient (for example, traditional Chinese medicine extraction
Object) pharmaceutical active ingredient screening.The present invention is Chinese medicine or the effective substance research of compound, targeting active Chinese drug component group
Research is divided to provide new idea and method.
Further, the MRM method in MS/MS, sample small molecular chemical combination can be used in quantitative analysis method of the invention
The detection sensitivity of object is very high, very good for micro or even trace constituent quantitative analysis, contains in itself suitable for Chinese medicine
Amount is few, but the object Quality Research that activity is high.Diagnostic cast relative to traditional " ingredient separation-Structural Identification-screening active ingredients "
The loss of formula, active constituent is considerably less, reduces the time loss and cost of drug research.
Further, the present invention carries out that high resolution mass spec can be used in qualitative using MS, it is hereby achieved that accurately
Mass number, and then molecular formula is calculated, it is provided conveniently for qualitative analysis.
Further, the present invention specifically uses Ultrafiltration-UPLC-MS technology to Chinese medicine hypoglycemic activity ingredient
Realize effective screening.
Detailed description of the invention
(b) is through the separating obtained filter of 10K super filter tube after Fig. 1 is (a) before Astragalus Root P.E is incubated for altogether with PTP1B enzyme, is incubated for altogether
The TIC map of the UPLC-QTOF of liquid.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and examples.
The characteristics of present invention is retained using ultrafiltration membrane molecular weight, for active small molecular compound in Chinese medicine (for example, molecule
Measure below 1000) with the power of specific protein combination stability, while be aided with UPLC-MS separation and qualitative, quantitative hand
Section is quickly screened the active small molecular compound for being directed to specific protein target spot in Chinese medicine, is mentioned for the basic research of Chinese medicine pharmaceutical substances
For new thinking.
(1) Chinese medicine hypoglycemic activity ingredient is quickly screened based on Ultrafiltration-UPLC-MS
The quick screening of PTP1B inhibitor in 1 Astragalus Root P.E of example
Radix Astragali clinical application is extensive, is the main ingredient in the hypoglycemics prescriptions such as JINQI JIANGTANG PIAN, 61 soup of Radix Astragali, and furthermore Radix Astragali is infused
Liquid is penetrated also to have a better effect the treatment of diabetes and diabetic nephropathy.
(1) the total incubation of Astragalus Root P.E and PTP1B enzyme
Astragalus Root P.E (75% ethanol extract, Xi'an biotech firm, Tianrui, 500 μ g/mL) 20 μ L, PTP1B enzyme
(hGST-PTP1 50nmol/L) 20 μ L and 100 μ L of ammonium acetate buffer solution (pH=5.2) are mixed in EP pipe, are obtained in conjunction with sample
Then the EP pipe is placed in 37 DEG C of water-baths by product, vibration is incubated for 30min.Be transferred to after incubation super filter tube (PALL Nanosep from
Heart concentration tube 10K) and 10min is centrifuged in 10000g.Upper layer insoluble matter continuously adds 100 μ L ammonium acetate buffer solution (pH=
5.2), ultrasonic 1min, standing 5min (free small molecule and combination small molecule need an equilibration time), 10000g is centrifuged afterwards
10min is repeated 2 times.It will be centrifuged gained lower layer's filtrate every time to merge, and be settled to ammonium acetate buffer solution (pH=5.2)
0.5mL carries out LC-MS analysis, compares in conjunction with preceding sample and combines sample to be incubated for, sample after ultrafiltration and final constant volume
The variation (being that PTP1B enzyme is not added in conjunction with preceding sample, other conditions are identical as sample is combined) of the total ion current of product.
(2) UPLC-QTOF is analyzed
Using Bruker MaxPlus Q-TOF analysis with PTP1B enzyme be incubated for altogether it is forward and backward (respectively correspond in conjunction with preceding sample,
In conjunction with sample, similarly hereinafter) chromatographic peak of corresponding constant volume sample, analysis condition are as follows；
Chromatographic condition:
Chromatographic column is Waters C18 chromatographic column (50mm*2.1mm*1.7 μm).Automatic sampling, 5 μ L of volume；Column temperature box temperature
Degree is 40 DEG C；Flow velocity 0.2mL/min, mobile phase use gradient elution, A liquid :+5% acetonitrile of 95% water (contains 0.1% formic acid)；B
Liquid: acetonitrile (contains 0.1% formic acid)；Gradient program: 0~1min, 0%B liquid；1~10min, 0%~90%B liquid；10~20min,
90%~99%B liquid；20~25min, 99%B liquid；25~28min, 99%~0%B liquid；28~30min, 0%B liquid.
Mass Spectrometry Conditions:
Electric spray ion source (ESI)；150 DEG C of ion source temperature；Analytical model is cation；Scanning range m/z be 100~
2000；Ion source spray voltage 4kV；Nitrogen flow rate 8L/min；Collision energy 10ev.
(3) data are analyzed
DataAnalysis2.1 carries out data collection and analysis, and correcting fluid is sodium formate, HTC correction mode；Software is automatic
It integrates and deconvolutes.
It is compareed by interpretation of mass spectra and document, the chromatographic peak (figure forward and backward to the total incubation of Astragalus Root P.E and PTP1B enzyme
1) it is studied, thus it is speculated that go out 32, possible PTP1B inhibitor, wherein flavonoids 4, Coumarins 6, alkaloids 10
It is a, triterpene saponin 8, other classes 4.The lead compound that this 32 compounds can be used as PTP1B inhibitor is further
Research is Astragaloside IV (M+H=785.4680) after wherein the chromatographic peak of 15.45min is compareed with document；And the compound is (yellow
Stilbene first glycosides) there is certain PTP1B inhibitor activity.
The quantitative experiment of Puerarin and alpha-glucosidase in example 2, pueraria lobata
Puerarin (puerarin) is also known as puerarin, is the isoflavone compound separated from Chinese medicament kudzu-vine root,
It is present in the root of leguminous plant Pueraria lobota and elegant jessamine etc., reporting, which has, brings down a fever, is calm and keep coronary blood flow increased
Effect.Nearest pharmaceutical research, which shows Puerarin also, to be had the function of reducing blood glucose, and action target spot may be phlorose
Glycosides enzyme.
(1) the total incubation of kudzu root extract and alpha-glucosidase
(Xi'an Ai Wo Biotechnology Co., Ltd, 1mg/mL, puerarin content are greater than 6%) 20 μ L, α-to kudzu root extract
20 μ L of glucuroide (0.2u/mL) and 100 μ L of ammonium acetate buffer solution (pH=5.2) are mixed in EP pipe, are obtained in conjunction with sample
Then the EP pipe is placed in 37 DEG C of water-baths by product, vibration is incubated for 30min.Be transferred to after incubation super filter tube (PALL Nanosep from
Heart concentration tube 30K) and 10min is centrifuged in 18000g.Upper layer insoluble matter continuously adds 50 μ L ammonium acetate buffer solution (pH=
5.2), ultrasonic 0.5min, 18000g is centrifuged 10min after standing 5min, is repeated 3 times.Gained lower layer's filtrate will be centrifuged every time to merge,
And be settled to 0.5mL with ammonium acetate buffer solution (pH=5.2), carry out LC-MS analysis, compare in conjunction with preceding sample and
It is incubated in conjunction with sample, the changes of contents of Puerarin (is that α-grape is not added in conjunction with preceding sample in sample after ultrafiltration and final constant volume
Glycosidase, other conditions are identical as sample is combined).
(2) UPLC-MS/MS analyzes puerarin content
It is incubated for the chromatographic peak of forward and backward corresponding constant volume sample altogether using Waters Xevo TQD analysis, analysis condition is as follows；
Chromatographic condition:
Chromatographic column is Waters C18 chromatographic column (50mm*2.1mm*1.7um).Automatic sampling, 5 μ L of volume；Column temperature box temperature
Degree is 40 DEG C；Flow velocity 0.2mL/min, mobile phase use gradient elution, A liquid :+5% acetonitrile of 95% water (contains 0.1% formic acid)；B
Liquid: acetonitrile (contains 0.1% formic acid)；Gradient program: 0~1min, 0%B liquid；1~3min, 0%~90%B liquid；3~4min,
90%~99%B liquid；4~6min, 99%B liquid；6~7min, 99%~0%B liquid；7~8min, 0%B liquid.
Mass Spectrometry Conditions:
Electric spray ion source (ESI)；150 DEG C of ion source temperature；Capillary voltage 3KV；Polyion reaction monitoring scans mould
Formula (MRM): parent ion and daughter ion are respectively 433.1 and 270.1；Orifice potential is 48V；Collision energy is 28eV；When resident
Between be 0.161s；N2The temperature and flow velocity of (dry gas) are 350 DEG C and 800L/h.
(3) data are analyzed
Mass Lynx software (Version 4.1) carries out data collection and analysis, Target LynxTM
Software and external standard method calculate the content of Puerarin.The concentration of Puerarin is dropped by 4 μ g/mL before total incubation under this experiment condition
150ng/mL after to total incubation；Puerarin has apparent alpha-glucosaccharase enzyme inhibition, can be used as hypoglycemic guide chemical combination
Object further investigation.
(2) precision, reproducibility and stability experiment
1. Precision Experiment: take after the total incubation prepared 1 part of sample, METHOD FOR CONTINUOUS DETERMINATION 7 times according to the method described above, to peak into
The RSD of row analysis, chemical shift and relative peak area between 0-0.38% and 0.1%-0.55%, illustrates this method respectively
Precision is good.
2. reproducibility is tested: taking after the total incubation of different batches 7 parts of sample, measure according to the method described above.Peak is divided
The RSD of analysis, chemical shift and relative peak area between 0-0.53% and 0.6%-2.68%, illustrates that this method is reappeared respectively
Property is good.
3. stability experiment: take after total incubation 1 part of sample, respectively 0,2,8,16,24,48h measurement, chemical shift and
The RSD of relative peak area is respectively less than 1.5%, and it is stable for illustrating that this method measures in 48h.Meet the verifying mark of methodology
It is quasi-.
Above embodiments are not the whole embodiments that the present invention can be implemented, drug effect (activity) object in Chinese medicine or compound
Matter component content is low, but action target spot is complicated.The pharmacotoxicological effect of Chinese medicine is likely to be one pack system-multiple target point, even
Multicomponent-multiple target point collective effect.The present invention targeted is that have characteristic target target active one in Chinese medicine or compound
Kind or a kind of compound screening, according to the combination of itself and target protein power, and the characteristics of retained using ultrafiltration membrane molecular weight into
Row separation；Qualitative and quantitative analysis is carried out in combination with UPLC-MS.Different types of chemical combination in different Chinese medicine or Same Chinese Herbal Medicine
The experiment condition of object is all different, and obtains the condition and range in the present invention accordingly.
The present invention selects Ultrafiltration-UPLC-MS to detect the sugar reducing substance ingredient in Chinese medicine or compound
For, it is that various Chinese medicines are mentioned in order to solve the problems, such as that Chinese medicine complicated ingredient is difficult to qualitative and quantitative detection for particular target
Take the small of natural origin small molecule compound in or mixtures thereof object, initial gross separation (such as through chromatography etc.) compound or synthesis
Molecular compound can be analyzed quickly.And with easy to operate, rapid batch realization, experiment favorable reproducibility, experimental data is straight
The features such as reliable is seen, is the medicine in Chinese medicine or its compound especially suitable for micro in Chinese medicine or even trace active constituent analysis
The further research or the research of targeting active Chinese drug component component of effect material composition provide new approaches and new method.
The methods of in addition, since analysis sample preparation is simple, be concentrated after analysis by albumen precipitation, refrigerated centrifuge,
Sample can very easily be recycled, for precious sample (for example, or mixtures thereof the compound obtained through separation means,
Especially natural origin small molecule compound or the small molecule compound of synthesis) further using provide convenience.
Claims (10)
1. a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method, it is characterised in that: including with
Lower step:
1) it is divided into identical two parts after dissolving multicomponent sample with buffer solution, is to combine preceding sample with a copy of it, to another
Enzyme corresponding to quasi- screening drug target is added in portion, obtains in conjunction with sample；
2) by being incubated for so that can partly or entirely be turned by free state with the compound of the enzyme stable bond in conjunction in sample
Become reference state, obtains Incubating Solution；By Incubating Solution ultrafiltration and filtrate is collected, by the filtrate buffer solution constant volume, obtains analysis sample
Product；
3) it will be handled in conjunction with preceding sample according to incubation identical with step 2), ultrafiltration and constant volume process, and obtain control sample,
The liquid chromatography-mass spectrometry that the same terms are respectively adopted to analysis sample and control sample is analyzed；
4) in the LC-MS analysis result of comparative analysis sample and control sample chromatographic peak variation, will change
Chromatographic peak corresponding to the drug candidate active constituent corresponding with the target spot that is obtained as preliminary screening of compound or elder generation
Lead compound.
2. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 1,
It is characterized by: the multicomponent sample is selected from Chinese medical extract, Chinese materia medica preparation, the compound or chemical combination obtained through separation means
The mixture of object；Sample institute's expense >=0.01mg when being dissolved with buffer solution.
3. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 1,
It is characterized by: the enzyme is selected from the enzyme of diabetes target spot.
4. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 1,
It is characterized by: the condition of the incubation are as follows: the time is 5~60min, and temperature is 25~40 DEG C.
5. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 1,
It is characterized by: the ultrafiltration is using ultra-filtration centrifuge tube and centrifuge separation is combined to realize the filtering to Incubating Solution, if repeatedly centrifugation,
Then merging filtrate, also, ultrasound and stewing process are first carried out before carrying out centrifugation next time.
6. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 5,
It is characterized by: the ultra-filtration centrifuge tube uses the ultrafiltration membrane of low-protein Percentage bound, the molecule interception of ultra-filtration centrifuge tube >=
3000Da is centrifugated centrifugal force >=8000g of use.
7. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 1,
It is characterized by: the buffer solution is selected from the volatility salt buffer system that pH is 4~10.
8. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 1,
It is characterized by: the volume of the constant volume is 0.1~10mL.
9. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 1,
It is characterized by: the liquid chromatography-mass spectrometry using high performance liquid chromatography or ultra performance liquid chromatography with it is mass spectrographic
It is combined equipment.
10. it is a kind of based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method according to claim 1,
It is characterized by: the variation of the chromatographic peak includes the variation of chromatographic peak component and/or the intensity of identical retention time chromatographic peak
Variation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111812252A (en) * | 2020-08-25 | 2020-10-23 | 山东省分析测试中心 | Screening and separating method for hypoglycemic functional compounds in plants |
| CN111983058A (en) * | 2020-07-30 | 2020-11-24 | 云南中医药大学 | Method for screening active substance of traditional Chinese medicine for resisting non-alcoholic fatty liver disease |
| CN114384180A (en) * | 2022-01-01 | 2022-04-22 | 中国科学院武汉植物园 | Method for rapidly screening anti-parasitic active compound |
| CN114384167A (en) * | 2021-12-14 | 2022-04-22 | 中国科学院武汉植物园 | Method for screening lotus leaf active compound for reducing blood sugar and blood fat |
| CN114594177A (en) * | 2022-01-25 | 2022-06-07 | 中国科学院武汉植物园 | Method for screening anti-aging active ligand |
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| CN114384180A (en) * | 2022-01-01 | 2022-04-22 | 中国科学院武汉植物园 | Method for rapidly screening anti-parasitic active compound |
| CN114384180B (en) * | 2022-01-01 | 2023-06-20 | 中国科学院武汉植物园 | Method for rapidly screening antiparasitic active compounds |
| CN114594177A (en) * | 2022-01-25 | 2022-06-07 | 中国科学院武汉植物园 | Method for screening anti-aging active ligand |
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