Gynostemma pentaphyllum, Trigonella foenum-graecum seeds, Polygala tenuifolia, Ginkgo biloba leaves, Astragalus membranaceus, Radix paeoniae alba and Suanzaorenhehuan were purchased from Harbin Xinheng Traditional Chinese Medicine Materials Ltd. (Harbin, China) and authenticated by Professor Lianjie Su, Heilongjiang University of Chinese Medicine. HP-20 macroporous resin and Sephadex LH-20 were obtained from Soledad Technology Co., Ltd. (Beijing, China). Clorgiline, pargilin, fluoxetine (Flu) were purchased from Sigma (St. Louis, MO, USA). Casein, vanilla acid, 4-aminoantipyrine and horseradish peroxidase were purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China).
Clorgiline
Manufactured by Merck Group
Sourced in United States
Clorgiline is a laboratory equipment product manufactured by Merck Group. It is a selective and irreversible inhibitor of monoamine oxidase type B (MAO-B), an enzyme involved in the breakdown of certain neurotransmitters in the brain. The core function of Clorgiline is to facilitate the study of MAO-B and its role in various biological processes.
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Lab products found in correlation
Selegiline, by Merck Group (2 mentions)
Fluoxetine flu, by Merck Group (1 mentions)
Quercetin 3 o glucoside, by Extrasynthese (1 mentions)
Quercetin 3 o rutinoside, by Extrasynthese (1 mentions)
Dntb 5 5 dithiobis 2 nitrobenzoic acid, by Thermo Fisher Scientific (1 mentions)
Monoamine oxidase a mao a, by Merck Group (1 mentions)
Apigenin 6 c glucoside, by Extrasynthese (1 mentions)
Acetylcholinesterase ache, by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Pyrogallol, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Juglone, by Thermo Fisher Scientific (1 mentions)
Peonidin 3 o glucoside, by Extrasynthese (1 mentions)
Folin ciocalteu reagent, by Chem-Lab (1 mentions)
Mao a, by Merck Group (1 mentions)
Mao b, by Merck Group (1 mentions)
Deprenyl, by Merck Group (1 mentions)
Peroxidase from horseradish, by Merck Group (1 mentions)
6 protocols using clorgiline
1
Multi-herb Extract Preparation and Evaluation
2
Phytochemical and Enzymatic Analysis
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Acetonitrile (99.9%) was of HPLC grade from Fisher Scientific (Lisbon, Portugal). Phenolic compound standards (apigenin-6-C-glucoside, quercetin-3-O-glucoside, quercetin-3-O-rutinoside, peonidin-3-O-glucoside) were from Extrasynthèse (Genay, France). Formic acid, DPPH· (2,2-diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), ATCI (acetylthiocholine iodide), acetylcholinesterase (AChE), monoamine oxidase A (MAO A), clorgiline, tris–HCl and pyrogallol were purchased from Sigma–Aldrich (St. Louis, MO, USA). DNTB (5,5′-dithiobis (2-nitrobenzoic acid)) and juglone (5-hydroxy-1,4-naphthoquinone) were from Alfa Aesar (Ward Hill, MA, USA), Folin-Ciocalteu reagent was purchased from Chem-lab (Zeldelgem, Belgium). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).
3
Inhibition of MAO-B for Parkinson's Disease
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EXAMPLE 12
Characterization of inhibition of MAO B (Monoamine oxidases) as a potential drug for treating Parkinson's Disease.
Selective inhibitors MAO-B increase dopamine levels in the CNS affected in Parkinson's disease without increasing levels of the other neurotransmisors (epinephrine, norepinephrine or serotonine), in contrast to no selective MAO inhibitors (MAO-A and MAO B). The MAO-B inhibitors can be used also to treat depressions.
Protocol: Human recombinant monoamine oxidase proteins MAO-A and MAO-B were purchased from Sigma Aldrich (Reference M7316 and M7441 respectively). In order to monitor the MAO enzymatic activities and their inhibition rate a fluorescence based assay was used The substrate for the assay, kynuramine, is non-fluorescent until undergoing oxidative domination by MAOs resulting in the fluorescent product 4-hydroxyquinoline. Kynuramine is a substrate for both MAO-A and -B (non-specific substrate). Clorgiline and Deprenyl (Sigma Aldrich) were used as controls for specific inhibition of MAO-A and MAO-B respectively.
Results snow that 5-(4-(2-(5-(1-hydroxyethyl)pyridine-2-yl)ethoxy)benzyl)thiazolidine-2,4-dione inhibits MAO B with a IC50 of 70.5 nM. In contrast, this compound did not inhibit MAO A protein.
4
Selective MAO Inhibition Protocols
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For pharmacological tests, selegiline and clorgiline were chosen for selective MAO-A and MAO-B inhibition and purchased from Sigma-Aldrich (St. Louis, MO). The reversible MAO-B inhibitor KDS2010 was provided by KIST. For in vivo FSCV and M-CSWV, selegiline (10 mg kg−1; i.p.), clorgiline (10 mg kg−1; i.p.) or KDS2010 (10 mg kg−1; i.p.) was dissolved in DI water and injected as a single dose of 500 µL. For ex vivo DA imaging, each compound (100 nM) was applied to the bath. For ex vivo patch-clamp experiments, acute brain slices were pre-incubated in each compound (100 nM).
5
Fluorescence-Based Inhibition Assay for MAO-A and MAO-B
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Ampliflu™ Red (10-Acetyl-3,7-dihydroxyphenoxazine), hMAO-A, hMAO-B, peroxidase from horseradish, tyramine hydrochloride, H2O2, clorgiline and selegiline were acquired from Sigma-Aldrich (Steinheim, Germany) and retained under the proposed conditions by supplier. A Biotek Precision XS robotic system (USA) was used for all pipetting operations. Measurements were performed with the use of BioTek-Synergy H1 microplate reader (USA) based upon the fluorescence generated (excitation, 535 nm, emission, 587 nm) over a 30 min period, in which the fluorescence increased linearly.
Enzymatic assay was performed according to recent method pronounced by our research group17 ,20–22 (link). Control, blank and all concentrations of obtained compounds were tested in quadruplicate and inhibition percent was calculated with following equation:
FCt2: Fluorescence of a control well measured at t2 time, FCt1: Fluorescence of a control well measured at t2 time, FIt2: Fluorescence of an inhibitor well measured at t2 time, FIt1: Fluorescence of an inhibitor well measured at t1 time,
The IC50 values were calculated using a dose-response curve achieved by plotting the percentage inhibition versus the log concentration using GraphPad ‘PRISM’ software (version 5.0). The results were showed as mean ± SD.
Enzymatic assay was performed according to recent method pronounced by our research group17 ,20–22 (link). Control, blank and all concentrations of obtained compounds were tested in quadruplicate and inhibition percent was calculated with following equation:
FCt2: Fluorescence of a control well measured at t2 time, FCt1: Fluorescence of a control well measured at t2 time, FIt2: Fluorescence of an inhibitor well measured at t2 time, FIt1: Fluorescence of an inhibitor well measured at t1 time,
The IC50 values were calculated using a dose-response curve achieved by plotting the percentage inhibition versus the log concentration using GraphPad ‘PRISM’ software (version 5.0). The results were showed as mean ± SD.
6
Photosensitive DAAQ Compound Analysis
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Benzaldehyde, benzylamine, and glacial acetic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan). Sodium dihydrogenphosphate dihydrate (NaH 2 PO 4 :2H 2 O) and disodium hydrogen phosphate (Na 2 HPO 4 ) were obtained from Nacalai Tesque (Kyoto, Japan). DAAQ and clorgiline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol (HPLC grade) were obtained from Kanto Chemical Company (Tokyo, Japan). The water used was purified by a Simpli Lab UV (Millipore, Bedford, MA, USA). Phosphate buffer solution (PBS, 0.1 M, pH 7.8) was prepared by dissolving 0.13 g of Na 2 HPO 4 and 0.14 g of NaH 2 PO 4 :2H 2 O in 100 mL distilled water and a Horiba F22 pH-meter was used to check the pH of the buffer. Stock solution of Benzaldehyde (10 mM) was prepared in acetonitrile and stock solutions of clorgiline (0.2 mM) and benzylamine (5.6 mM) were prepared in PBS. 8.0 mM and 0.8 mM solutions of DAAQ were prepared in glacial acetic acid. Because of the expected photosensitivity of DAAQ, its solution was kept in amber-colored glass bottles.
It was found to be stable for at least 2 weeks when kept at 4 °C in the refrigerator.
It was found to be stable for at least 2 weeks when kept at 4 °C in the refrigerator.
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