CN111748600A - Target activity screening and evaluating method applied to curcuma zedoaria - Google Patents

Target activity screening and evaluating method applied to curcuma zedoaria Download PDF

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CN111748600A
CN111748600A CN201910241783.5A CN201910241783A CN111748600A CN 111748600 A CN111748600 A CN 111748600A CN 201910241783 A CN201910241783 A CN 201910241783A CN 111748600 A CN111748600 A CN 111748600A
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curcuma zedoary
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梁鑫淼
王纪霞
王志伟
刘艳芳
周维佳
于广璞
薛珍珍
单彩龙
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Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
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Abstract

The invention relates to the field of target activity screening and evaluation of traditional Chinese medicines, in particular to a target activity screening and evaluation method applied to curcuma zedoary. The invention establishes a target activity screening and evaluating method of curcuma zedoary by using a GPCR stable transformation cell screening model based on a FLIPR Tetra high-flux real-time fluorescence detection analysis system. The activity screening and evaluating method provided by the invention has the advantages of high flux, strong reliability, convenience in operation and the like, can quickly find target active ingredients of the traditional Chinese medicine, provides a new idea for finding targets of complex traditional Chinese medicines, and has important significance for explaining molecular mechanisms of clinical application of the traditional Chinese medicines.

Description

Target activity screening and evaluating method applied to curcuma zedoaria
Technical Field
The invention relates to the field of target activity screening and evaluation of traditional Chinese medicines, in particular to a target activity screening and evaluation method applied to curcuma zedoary.
Background
The traditional Chinese medicine zedoary is the rhizome of a plant in the genus of curcuma in the family of zingiberaceae, is a common traditional Chinese medicine in China, and has the effects of promoting qi circulation, removing blood stasis, removing food retention and relieving pain. The chemical components of Curcumae rhizoma mainly include curcumin components and volatile oil components, wherein the volatile oil mainly contains curcumenol, beta-elemene, zedoary turmeric epoxy ketone, zedoary turmeric cyclodiene, curcumenol ketone, curcumenol, etc. Modern researches find that the medicinal material has the effects of resisting cancer, resisting blood coagulation, resisting oxidation and protecting liver, and is clinically used for treating early cervical cancer, malignant lymphoma, primary liver cancer, ischemic encephalopathy, skin ulcer, neurodermatitis, infantile autumn diarrhea, infantile acute respiratory infection, chronic bronchitis and other diseases. With the development and utilization of more and more medicinal values, the curcuma zedoary becomes a medicinal material with great development potential and broad market prospect. However, the total extract of zedoary is mainly used as a medicine at present, the pharmacodynamic substance is unclear, and the research on the action target is very little. Therefore, the screening and evaluation of the action target of the curcuma zedoary is of great significance to the subsequent drug development. At present, no report exists about a target activity screening and evaluating method of an FLIPR Tetra high-throughput real-time fluorescence detection analysis system applied to curcuma zedoary.
Disclosure of Invention
The invention aims to solve the problem that the acting target of curcuma zedoary is undefined, and establishes a target activity screening and evaluating method of curcuma zedoary by means of a FLIPR Tetra high-throughput real-time fluorescence detection analysis system and a GPCR (G protein coupled receptor) stable-transfer cell screening model. The activity screening and evaluating method provided by the invention has the advantages of high flux, strong reliability, convenience in operation and the like, can quickly find target active ingredients of the traditional Chinese medicine, provides a new idea for finding targets of complex traditional Chinese medicines, and has important significance for explaining molecular mechanisms of clinical application of the traditional Chinese medicines.
The technical scheme of the invention is as follows:
a target activity screening and evaluating method applied to curcuma zedoary is based on a FLIPR Tetra high-throughput real-time fluorescence detection analysis system, utilizes a muscarinic receptor M3 cell screening model, a dopamine receptor D2 cell screening model, an opioid receptor delta cell screening model and an opioid receptor kappa cell screening model, takes curcuma zedoary fraction as a screening object, and screens and evaluates activity action targets through agonistic analysis and antagonistic analysis.
Furthermore, the cell screening model is to inoculate HEK-293 cells of stable transfer receptors in a 96-well plate compatible with cells, and the inoculated cell density is 8 × 104The number of the cells per well is 100 muL per well, and the cell culture time after inoculation is 16-22 h.
Further, the curcuma zedoary fraction comprises curcuma zedoary water layer, ethyl acetate layer and n-heptane layer fractions, and the preparation method comprises the following steps:
firstly, dividing the total extract of the curcuma zedoaria into three fractions by solvent distribution, and specifically operating as follows: weighing X kg of rhizoma Curcumae tuber, slicing, extracting with 10 times of 50-90% ethanol solution under heating and refluxing for 1-3 hr for three times, mixing extractive solutions, vacuum filtering, and rotary evaporating at 60 deg.C under vacuum to concentrate volume to 4X-7X L. Sequentially eluting with n-heptane (12X-20X L) and ethyl acetate (25X-30X L), and rotary evaporating the concentrated solution to obtain Curcumae rhizoma water layer, ethyl acetate layer and n-heptane layer fractions.
Further, the screening and evaluation of the zedoary turmeric fraction on a muscarine receptor M3 cell model specifically comprises the following steps:
A. adding the zedoary turmeric fraction dissolved in DMSO (0.05-0.1%) at a concentration of 1-100 μ g/mL into a 96-well plate, and detecting the response signal of the fraction; continuously adding carbachol agonist with the concentration of 1-10 nM, and detecting the response signal of carbachol;
B. adding scopolamine antagonist into 96-well plate at concentration of 0.5-10 μ M, adding Curcumae rhizoma fraction dissolved in DMSO containing 0.05-0.1% at concentration of 1-100 μ g/mL, and detecting response signal of fraction;
further, the screening and evaluation of the zedoary turmeric fraction on a dopamine receptor D2 cell model specifically comprises the following steps: adding the fractions dissolved in the aqueous layer of Curcumae rhizoma, ethyl acetate layer and n-heptane layer containing 0.05-0.1% DMSO into 96-well plate at a concentration of 1-100 μ g/mL, and detecting the response signal of the fractions; and then continuously adding the dopamine agonist with the concentration of 1-20 nM to detect the response signal of the dopamine.
Further, the screening and evaluation of the zedoary turmeric fraction on an opioid receptor delta cell model specifically comprises the following steps: adding the fractions dissolved in the aqueous layer of Curcumae rhizoma, ethyl acetate layer and n-heptane layer containing 0.05-0.1% DMSO into 96-well plate at a concentration of 1-100 μ g/mL, and detecting the response signal of the fractions; and then continuously adding enkephalin with the concentration of 1-50 nM to detect the response signal of the enkephalin.
Further, the screening and evaluation of the zedoary turmeric fraction on an opioid receptor kappa cell model specifically comprises the following steps: adding the fractions dissolved in the aqueous layer of Curcumae rhizoma, ethyl acetate layer and n-heptane layer containing 0.05-0.1% DMSO into 96-well plate at a concentration of 1-100 μ g/mL, and detecting the response signal of the fractions; and then the dynorphin with the concentration of 500-1000 nM is added continuously to detect the response signal of the dynorphin.
The target activity screening and evaluating method provided by the invention has the advantages of high flux, strong reliability, convenience in operation and the like, can quickly find the action target of the curcuma zedoary, provides a new idea for finding the target of a complex traditional Chinese medicine, has important significance for explaining the molecular mechanism of clinical application of the traditional Chinese medicine, and simultaneously provides candidate active ingredients for treating related diseases of muscarinic receptor M3, dopamine receptor D2, opioid receptor delta and kappa such as chronic obstructive pulmonary disease, Alzheimer's disease, Parkinson's disease, schizophrenia and pain.
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FIG. 1, (A) the signals of the zedoary fraction in a 50 μ g/mL concentration on a HEK-293-M3 muscarinic receptor cell model; (B) the zedoary fraction responded to signals on a model HEK-293-M3 cell at a concentration of 50 μ g/mL after 1 μ M scopolamine pretreatment of HEK-293-M3 cells.
FIG. 2 (A) the response signals of the zedoary turmeric fraction at a concentration of 50 μ g/mL on a HEK-293-D2 dopamine receptor cell model; (B) response signals of dopamine at 10nM concentration on HEK-293-D2 cell models following pretreatment of HEK-293-D2 cells with zedoary turmeric fraction at a concentration of 50 μ g/mL.
FIG. 3 (A) the response signals of the zedoary turmeric fraction at a concentration of 50 μ g/mL on a HEK-293-delta opioid receptor cell model; (B) response signals of enkephalin at 20nM concentration on HEK-293-delta cell model after pretreatment of HEK-293-delta cells with zedoary fraction at 50. mu.g/mL.
FIG. 4 (A) the response signals of the zedoary turmeric fraction at a concentration of 50 μ g/mL on a HEK-293-kappa opioid receptor cell model; (B) response signals of dynorphin at a concentration of 800 nM on a model HEK-293-kappa cell after pretreatment of HEK-293-kappa cells with a 50. mu.g/mL zedoary fraction.
Detailed Description
The present invention will now be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.
Example 1: screening and evaluation of activity of zedoary turmeric fraction on muscarine receptor M3 cell model
Human embryonic kidney HEK-293-M3 cells were obtained from a laboratory self-constructed cell bank, purchased from OLYMPUS under an inverted microscope, and purchased from TCI and Aladdin, respectively, under carbachol and scopolamine. The cell culture plate is an optical biocompatible 96 micro-porous plate purchased from corning company, and the detection platform is a FLIPR Tetra high-flux real-time fluorescence detection analysis system of Molecular Devices.
Inoculating HEK-293-M3 cells in logarithmic growth phase into an optical biocompatible 96 micro-porous plate, wherein the culture medium is DMEM (C1199550 BT, GIBCO), the inoculation volume of each hole is 100 mu L, and the number of the cells inoculated in each hole is 8.0 × 104And (3) placing the inoculated cell plate in a cell culture box for culturing for 16-22 h until the cell fusion degree reaches 80-90%, and performing an activity experiment. Adding 10% Calcium-6 Hank's balanced salt solution (containing 20 mM HEPES) into a microplate, adding 100 μ L per well, incubating in a cell culture box for 1h, and collectingPutting the sample in a FLIPR Tetra high-flux real-time fluorescence detection analysis system; detecting a substrate signal between 800-1100, adding a blank control and scopolamine into a microplate, adding 50 mu L of the blank control and the scopolamine into each hole, wherein the scopolamine concentration is 1 mu M, paralleling for 2 times, monitoring an RFU signal for 5min in real time, adding carbachol and a zedoary turmeric fraction into the microplate, wherein the volume of each hole is 50 mu L, the zedoary turmeric fraction concentration is 50 mu g/mL, and the carbachol concentration is 5 nM. The RFU signal is monitored in real time for 3 min. The maximum fluorescence intensity of the cells under the action of the zedoary turmeric fraction and the carbachol in the blank and scopolamine respectively is detected, and the result is shown in figure 1. Compared with a blank control, the water layer of the curcuma zedoary has obvious agonistic activity, and the relative fluorescence intensity reaches 869; compared with carbachol, the aqueous layer of the curcuma zedoary is inhibited by scopolamine, and the relative fluorescence intensity reaches 105 and 290, which shows that the aqueous layer fraction of the curcuma zedoary has obvious M3 target point agonistic activity.
Example 2: activity screening and evaluation of zedoary fraction on dopamine receptor D2 cell model
Human embryonic kidney HEK-293-D2 cells were obtained from a laboratory self-constructed cell bank, purchased from OLYMPUS under an inverted microscope, and purchased from TCI and Aladdin, respectively, under carbachol and scopolamine. The cell culture plate is an optical biocompatible 96 micro-porous plate purchased from corning company, and the detection platform is a FLIPR Tetra high-flux real-time fluorescence detection analysis system of Molecular Devices.
Inoculating HEK-293-D2 cells in logarithmic growth phase into an optical biocompatible 96 micro-porous plate, wherein the culture medium is DMEM (C1199550 BT, GIBCO), the inoculation volume of each hole is 100 mu L, and the number of the cells inoculated in each hole is 8.0 × 104And (3) placing the inoculated cell plate in a cell culture box for culturing for 16-22 h until the cell fusion degree reaches 80-90%, and performing an activity experiment. Adding 10% Calcium-6 Hank's balanced salt solution (containing 20 mM HEPES) into a microplate, adding 100 mu L of the solution into each well, putting the well into a cell culture box, incubating for 1h, taking out, and putting the well into a FLIPR Tetra high-flux real-time fluorescence detection analyzer; detecting the substrate signal between 800-1100, adding blank control, haloperidol and zedoary turmeric fraction into the microplate, wherein the volume of each well is 50 muL, and the concentration of the zedoary turmeric fraction is 50Mu g/mL, the haloperidol concentration is 1 mu M, the parallel is carried out for 2 times, the RFU signal is monitored for 5min in real time, and then dopamine is added into the micropore plate, the volume of each hole is 50 mu L, and the concentration of the dopamine is 10 nM. The RFU signal is monitored in real time for 3 min. The maximum fluorescence intensity of the cell under the blank action of the zedoary turmeric fraction and the maximum fluorescence intensity of dopamine under the action of the zedoary turmeric fraction were detected, and the results are shown in FIG. 2. Studies show that none of the zedoary turmeric fractions has obvious agonistic activity compared with a blank control, and none of the zedoary turmeric fractions has dopamine-inhibiting activity compared with haloperidol, which indicates that the zedoary turmeric fractions have no activity on a D2 target point.
Example 3: activity screening and evaluation of zedoary fraction on opioid receptor Delta cell model
Human embryonic kidney HEK-293-delta cells were obtained from a laboratory self-constructed cell bank, purchased from OLYMPUS under an inverted microscope, and purchased from TCI and MCE respectively for enkephalin and naloxone. The cell culture plate is an optical biocompatible 96 micro-porous plate purchased from corning company, and the detection platform is a FLIPR Tetra high-flux real-time fluorescence detection analysis system of Molecular Devices.
Inoculating HEK-293-delta cells in logarithmic growth phase into an optical biocompatible 96-microplate, wherein the culture medium is DMEM (C1199550 BT, GIBCO), the inoculation volume of each well is 100 mu L, and the number of the cells inoculated in each well is 8.0 × 104And (3) placing the inoculated cell plate in a cell culture box for culturing for 16-22 h until the cell fusion degree reaches 80-90%, and performing an activity experiment. Adding 10% Calcium-6 Hank's balanced salt solution (containing 20 mM HEPES) into a microplate, adding 100 mu L of the solution into each well, putting the wells into a cell culture box, incubating for 1h, taking out, and putting the wells into a FLIPR Tetra high-flux real-time fluorescence detection and analysis system; detecting a substrate signal between 800-1100, adding a blank control, naloxone and zedoary fraction into a microporous plate, adding the volume of each hole into the microporous plate to be 50 mu L, adding the concentration of the zedoary fraction into the microporous plate to be 50 mu g/mL, and adding the naloxone into the microporous plate to be 1 mu M, paralleling for 2 times, monitoring an RFU signal for 5min in real time, adding enkephalin into the microporous plate, adding the volume of each hole into the microporous plate to be 50 mu L, and adding the concentration of the enkephalin into the microporous plate to be 20 nM. The RFU signal is monitored in real time for 3 min. Detecting the maximum fluorescence intensity of the cell under the blank action of the zedoary turmeric fraction and the enkephalin in the zedoary turmericThe maximum fluorescence intensity by the action of the fractions is shown in FIG. 3. Studies show that none of the zedoary turmeric fractions has obvious agonistic activity compared with a blank control, none of the zedoary turmeric fractions has inhibitory activity on enkephalin compared with naloxone, and the zedoary turmeric fractions have no activity on delta targets.
Example 4: screening and evaluation of activity of zedoary turmeric fraction on opioid receptor Kappa cell model
Human embryonic kidney HEK-293-kappa cells were obtained from a laboratory self-constructed cell bank, purchased from OLYMPUS under an inverted microscope, and purchased from TCI and MCE respectively for dynorphin and naloxone. The cell culture plate is an optical biocompatible 96 micro-porous plate purchased from corning company, and the detection platform is a FLIPR Tetra high-flux real-time fluorescence detection analysis system of Molecular Devices.
HEK-293-kappa cells in logarithmic growth phase were inoculated in an optically biocompatible 96-well plate using DMEM (C1199550 BT, GIBCO) as the medium, the inoculation volume per well was 100 μ L, and the number of cells inoculated per well was 8.0 × 104And (3) placing the inoculated cell plate in a cell culture box for culturing for 16-22 h until the cell fusion degree reaches 80-90%, and performing an activity experiment. Adding 10% Calcium-6 Hank's balanced salt solution (containing 20 mM HEPES) into a microplate, adding 100 mu L of the solution into each well, putting the wells into a cell culture box, incubating for 1h, taking out, and putting the wells into a FLIPR Tetra high-flux real-time fluorescence detection and analysis system; detecting a substrate signal between 800-1100, adding a blank control, naloxone and zedoary fraction into a microporous plate, adding the volume of each hole into the microporous plate to be 50 mu L, the concentration of the zedoary fraction to be 50 mu g/mL, the concentration of the naloxone to be 10 mu M, paralleling for 2 times, monitoring an RFU signal for 5min in real time, adding dynorphin into the microporous plate, adding the volume of each hole into the microporous plate to be 50 mu L, and the concentration of the dynorphin to be 800 nM. The RFU signal is monitored in real time for 3 min. The maximum fluorescence intensity of the cell fraction subjected to zedoary turmeric under the blank action and the maximum fluorescence intensity of dynorphin under the zedoary turmeric distillation action are detected, and the result is shown in figure 4. Studies show that none of the zedoary turmeric fractions has obvious agonistic activity compared with a blank control, and none of the zedoary turmeric fractions has inhibition of dynorphin activity compared with naloxone, which indicates that none of the zedoary turmeric fractions has activity on a kappa target.
The invention provides a method for screening and evaluating the target activity of curcuma zedoary, which has the advantages of high flux, strong reliability, convenient operation and the like, can quickly discover the target active ingredients of traditional Chinese medicines, provides a new idea for discovering the target of complex traditional Chinese medicines, has important significance for explaining the molecular mechanism of clinical application of the traditional Chinese medicines, and simultaneously provides candidate active ingredients for treating related diseases of muscarinic receptor M3, dopamine receptor D2, opioid receptor delta and kappa, such as chronic obstructive pulmonary disease, Alzheimer's disease, Parkinson's disease, schizophrenia and pain.

Claims (7)

1. A target activity screening and evaluating method applied to curcuma zedoary is characterized in that based on a FLIPR Tetra high-throughput real-time fluorescence detection analysis system, a muscarinic receptor M3 cell screening model, a dopamine receptor D2 cell screening model, an opioid receptor delta cell screening model and an opioid receptor kappa cell screening model are utilized, curcuma zedoary fraction is taken as a screening object, and activity action targets are screened and evaluated through agonistic analysis and antagonistic analysis.
2. The method as claimed in claim 1, wherein the cell screening model is HEK-293 cells inoculated with stable transgenic receptors in a 96-well cell-compatible plate, and the cell density of the inoculation is 8 × 104The number of the cells per well is 100 muL per well, and the cell culture time after inoculation is 16-22 h.
3. The method for screening and evaluating the activity of a target applied to curcuma zedoary according to any one of claims 1 or 2, wherein the curcuma zedoary fraction comprises a curcuma zedoary aqueous layer, an ethyl acetate layer and an n-heptane layer fraction, and the steps of obtaining are as follows:
firstly, dividing the total extract of the curcuma zedoaria into three fractions by solvent distribution, and specifically operating as follows: weighing X kg of rhizoma Curcumae tuber, slicing, extracting with 10 times of 50-90% ethanol solution under heating and refluxing for 1-3 hr for three times, mixing extractive solutions, vacuum filtering, and rotary evaporating at 60 deg.C under vacuum to concentrate volume to 4X-7X L;
sequentially eluting with n-heptane (12X-20X L) and ethyl acetate (25X-30X L), and rotary evaporating the concentrated solution to obtain Curcumae rhizoma water layer, ethyl acetate layer and n-heptane layer fractions.
4. The method for screening and evaluating the activity of a target applied to curcuma zedoary according to claim 3, wherein the screening and evaluation of the curcuma zedoary fraction on a muscarine receptor M3 cell model specifically comprises the following steps:
A. adding the zedoary turmeric fraction dissolved in DMSO (0.05-0.1%) at a concentration of 1-100 μ g/mL into a 96-well plate, and detecting the response signal of the fraction; continuously adding carbachol agonist with the concentration of 1-10 nM, and detecting the response signal of carbachol;
B. adding scopolamine antagonist into 96-well plate at concentration of 0.5-10 μ M, adding Curcumae rhizoma fraction dissolved in DMSO containing 0.05-0.1% at concentration of 1-100 μ g/mL, and detecting response signal of fraction.
5. The method for screening and evaluating the activity of a target applied to curcuma zedoary, according to claim 3, wherein the screening and evaluation of the curcuma zedoary fraction on a dopamine receptor D2 cell model specifically comprises the following steps: adding the fractions dissolved in the aqueous layer of Curcumae rhizoma, ethyl acetate layer and n-heptane layer containing 0.05-0.1% DMSO into 96-well plate at a concentration of 1-100 μ g/mL, and detecting the response signal of the fractions; and then continuously adding the dopamine agonist with the concentration of 1-20 nM to detect the response signal of the dopamine.
6. The method for screening and evaluating the activity of a target applied to curcuma zedoary according to claim 3, wherein the screening and evaluation of the curcuma zedoary fraction on the opioid receptor delta cell model specifically comprises the following steps: adding the fractions dissolved in the aqueous layer of Curcumae rhizoma, ethyl acetate layer and n-heptane layer containing 0.05-0.1% DMSO into 96-well plate at a concentration of 1-100 μ g/mL, and detecting the response signal of the fractions; and then continuously adding enkephalin with the concentration of 1-50 nM to detect the response signal of the enkephalin.
7. The method for screening and evaluating the activity of a target applied to curcuma zedoary according to claim 3, wherein the screening and evaluation of the curcuma zedoary fraction on an opioid receptor kappa cell model specifically comprises the following steps: adding the fractions dissolved in the aqueous layer of Curcumae rhizoma, ethyl acetate layer and n-heptane layer containing 0.05-0.1% DMSO into 96-well plate at a concentration of 1-100 μ g/mL, and detecting the response signal of the fractions; and then the dynorphin with the concentration of 500-1000 nM is added continuously to detect the response signal of the dynorphin.
CN201910241783.5A 2019-03-28 2019-03-28 Target activity screening and evaluating method applied to curcuma zedoaria Pending CN111748600A (en)

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060275835A1 (en) * 2003-07-11 2006-12-07 Jacobson Marlene A Methods for identifying cell surface receptor protein modulators
US7655396B1 (en) * 2006-09-29 2010-02-02 Allergan, Inc. Methods for detecting receptor modulator activity
US20110281278A1 (en) * 2010-05-13 2011-11-17 University Of South Carolina High Throughput Assay for Discovering New Inhibitors of the GIRK1/4 Channel
CN102818868A (en) * 2012-08-27 2012-12-12 浙江大学 Screening method of active ingredients in complex natural product and its application
CN103487531A (en) * 2013-01-28 2014-01-01 中国药科大学 Efficient preparation method for compound library applicable to natural product high-throughput screening
US20140057804A1 (en) * 2011-03-30 2014-02-27 Institute Of Pharmacology And Toxicology Academy Of Military Medical Sciences P.L.A. China Cell Model and Method for Screening c-Fms Kinase Inhibitors
CN105445245A (en) * 2015-11-20 2016-03-30 中国药科大学 Fluorescence resonance energy transfer technique based high-throughput screening method for dopamine 5 receptor inhibitor
CN105462930A (en) * 2014-06-24 2016-04-06 中国人民解放军军事医学科学院毒物药物研究所 Cell model for screening [kappa] opioid receptor agonist, and screening method thereof
CN105963352A (en) * 2016-06-15 2016-09-28 江苏康缘药业股份有限公司 Cinnamon oil and application of main ingredient cinnamaldehyde of cinnamon oil
CN108570450A (en) * 2017-03-11 2018-09-25 华中科技大学鄂州工业技术研究院 A kind of application of cell model and its construction and screening Serotonin receptor target active substance

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060275835A1 (en) * 2003-07-11 2006-12-07 Jacobson Marlene A Methods for identifying cell surface receptor protein modulators
US7655396B1 (en) * 2006-09-29 2010-02-02 Allergan, Inc. Methods for detecting receptor modulator activity
US20110281278A1 (en) * 2010-05-13 2011-11-17 University Of South Carolina High Throughput Assay for Discovering New Inhibitors of the GIRK1/4 Channel
US20140057804A1 (en) * 2011-03-30 2014-02-27 Institute Of Pharmacology And Toxicology Academy Of Military Medical Sciences P.L.A. China Cell Model and Method for Screening c-Fms Kinase Inhibitors
CN102818868A (en) * 2012-08-27 2012-12-12 浙江大学 Screening method of active ingredients in complex natural product and its application
CN103487531A (en) * 2013-01-28 2014-01-01 中国药科大学 Efficient preparation method for compound library applicable to natural product high-throughput screening
CN105462930A (en) * 2014-06-24 2016-04-06 中国人民解放军军事医学科学院毒物药物研究所 Cell model for screening [kappa] opioid receptor agonist, and screening method thereof
CN105445245A (en) * 2015-11-20 2016-03-30 中国药科大学 Fluorescence resonance energy transfer technique based high-throughput screening method for dopamine 5 receptor inhibitor
CN105963352A (en) * 2016-06-15 2016-09-28 江苏康缘药业股份有限公司 Cinnamon oil and application of main ingredient cinnamaldehyde of cinnamon oil
CN108570450A (en) * 2017-03-11 2018-09-25 华中科技大学鄂州工业技术研究院 A kind of application of cell model and its construction and screening Serotonin receptor target active substance

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
《中国中医药年鉴》编辑委员会编: "《中国中医药年鉴》", 上海辞书出版社, pages: 323 - 324 *
刘明等: "细胞分子生物学技术在靶向药物筛选中的应用", 生物技术通报, vol. 6, no. 6, pages 62 - 66 *
王晓明;等: "高通量活性筛选技术在中药研究中的应用", 山东医药, vol. 56, no. 22, pages 105 - 107 *

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