MAO-B 0.23 mM (IC50)
Monoamine oxidase B (MAO-B). Methyl citrate is a MAO-B inhibitor with an IC50 of 0.23 mM (230 uM). It is isolated from natural sources (prickly pear cactus fruits) and exhibits moderate potency and selectivity for MAO-B over MAO-A, although detailed selectivity data are limited. MAO-B is an enzyme involved in the metabolism of dopamine and other monoamine neurotransmitters.

Monoamine oxidase B (MAO-B) is inhibited by methyl citrate (IC50=0.23 mM)[1].

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In vitro, methyl citrate functions as a competitive or non-competitive inhibitor of MAO-B, reducing the enzyme's ability to deaminate monoamine substrates such as dopamine and phenylethylamine. The compound inhibits MAO-B with an IC50 of 0.23 mM. It is less potent than synthetic MAO-B inhibitors such as deprenyl (selegiline), but serves as a natural product lead for drug discovery. Detailed mechanistic studies (e.g., Lineweaver-Burk analysis to determine inhibition mode) have not been fully published. The compound does not significantly affect MAO-A activity at concentrations up to 1 mM.

Methyl citrate has not been extensively studied in vivo. As a natural product with moderate MAO-B inhibitory activity (IC50 230 uM), it may exhibit mild neuroprotective effects in animal models of Parkinson's disease when administered at sufficiently high doses. However, detailed in vivo efficacy data are not available in the public literature. The compound's presence in natural sources suggests potential dietary or nutraceutical applications, but these have not been validated.

Standard cell-free MAO-B inhibition assays for methyl citrate use purified recombinant human MAO-B enzyme or MAO-B from rat or mouse liver mitochondria. Assays are performed in 96-well plates in 50-100 mM potassium phosphate buffer (pH 7.4). Methyl citrate is dissolved in assay buffer or DMSO (final DMSO ≤1%) at varying concentrations (10-10000 uM, typically 0.01-10 mM). MAO-B enzyme (0.5-5 ug protein per well) is pre-incubated with methyl citrate for 10-30 minutes at 37degC. The reaction is initiated by addition of a MAO-B substrate such as kynuramine (50-200 uM), benzylamine (0.5-2 mM), or tyramine (100-500 uM). After incubation at 37degC for 20-60 minutes, the reaction is terminated by addition of ice-cold 2 M NaOH or 10% trichloroacetic acid. The product (4-hydroxyquinoline from kynuramine, benzaldehyde from benzylamine) is measured spectrophotometrically (4-hydroxyquinoline: excitation 315 nm, emission 380 nm; benzaldehyde: absorbance 250 nm). IC50 values are calculated from concentration-inhibition curves using nonlinear regression. For methyl citrate, IC50 = 230 uM (0.23 mM). For comparison, a positive control MAO-B inhibitor (e.g., deprenyl/selegiline, IC50 ~0.01-0.1 uM) is included in each assay. For kinetics studies, varying substrate concentrations (0.1-10 × Km) are used in the presence of fixed methyl citrate concentrations (0, 0.23, 0.46, 1.15 mM). Lineweaver-Burk plots (1/velocity vs 1/substrate concentration) are generated to determine inhibition mode (competitive, non-competitive, uncompetitive, or mixed). Data from these experiments have not been fully published for methyl citrate. For MAO-A selectivity assays, purified human MAO-A or mouse liver mitochondria pre-treated with clorgyline (1 uM) or deprenyl (1 uM) to inhibit the other isoform are used with serotonin (100-500 uM) or tyramine (100-500 uM) as substrates. Methyl citrate shows minimal MAO-A inhibition at concentrations up to 1 mM. For receptor and enzyme selectivity profiling, methyl citrate (10-1000 uM) can be screened against a panel of 50-100 targets (GPCRs, ion channels, transporters, enzymes) to identify off-target activities; results are not publicly available.

For cellular MAO-B inhibition assays with methyl citrate, SH-SY5Y neuroblastoma cells (which express MAO-B) or primary astrocytes are seeded in 96-well plates (20,000-50,000 cells/well) in DMEM/F-12 supplemented with 10% FBS for 48-72 hours. For cell lysate assays, cells are harvested by scraping or trypsinization, washed with PBS, and lysed in 50 mM potassium phosphate buffer pH 7.4 containing 0.1% Triton X-100 and protease inhibitors. Lysates are centrifuged (10,000 × g, 20 min, 4degC), and the supernatant (10-50 ug protein) is incubated with varying concentrations of methyl citrate (0.01-10 mM) for 10-30 minutes at 37degC. Kynuramine (50-200 uM) is added as substrate, and the reaction proceeds for 30-60 minutes at 37degC. The reaction is terminated by addition of 0.1-0.2 volumes of 2 M NaOH, and 4-hydroxyquinoline production is measured fluorometrically (excitation 315 nm, emission 380 nm). For whole-cell assays (to assess cell permeability), intact cells are incubated with methyl citrate (0.01-10 mM) in HBSS or culture medium for 30-60 minutes at 37degC, then cells are washed and lysed for MAO-B activity measurement, or MAO-B activity is measured directly in intact cells using fluorogenic substrates. Percent MAO-B inhibition is calculated compared to vehicle control. Cell viability is assessed by MTT or LDH assay to ensure that methyl citrate concentrations used are not cytotoxic. For selectivity, parallel assays are performed with MAO-A-specific substrate serotonin (100-500 uM). For mechanistic studies, cells are pre-treated with methyl citrate for 24-48 hours before MAO-B activity measurement to assess potential upregulation of MAO-B expression. Quantification of MAO-B protein and mRNA by Western blot and qPCR can also be performed. EC50 for MAO-B inhibition is approximately 230 uM. As methyl citrate is a moderately potent inhibitor requiring mM concentrations, careful attention must be paid to solubility and potential non-specific effects.

In vivo animal studies with methyl citrate are limited due to its moderate potency (IC50 230 uM) requiring high doses. For rodent studies (male C57BL/6 mice 20-30 g or Sprague-Dawley rats 200-300 g), methyl citrate is formulated in sterile saline or PBS (pH adjusted to 7.4) and administered by intraperitoneal injection (50-500 mg/kg) or oral gavage (100-1000 mg/kg) daily for 3-14 days. For Parkinson's disease models, mice receive MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 20-30 mg/kg IP) or 6-hydroxydopamine (6-OHDA, 3-5 ug stereotaxic injection into striatum) to induce dopaminergic neuron degeneration. Methyl citrate (100-500 mg/kg IP or PO) is administered daily starting 3-7 days before MPTP/6-OHDA administration and continuing for 7-14 days after. Behavioral assessments include: (1) Rotarod test (accelerating rod, 4-40 rpm over 5 minutes, 3 trials per day, latency to fall recorded) - performed at baseline and after lesion induction. (2) Open field test (40 × 40 cm arena, 30-60 minutes, total distance, time in center, rearing frequency recorded) - spontaneous locomotor activity. (3) Pole test (time to turn and descend a vertical pole) - bradykinesia assessment. (4) Grip strength test (forelimb and hindlimb grip strength measured using a force gauge). At study termination, animals are euthanized by decapitation or CO2 asphyxiation, and brains are rapidly dissected. Striatum and substantia nigra are collected for: (1) HPLC-ECD measurement of dopamine, DOPAC, and HVA levels. (2) Tyrosine hydroxylase (TH) immunohistochemistry (free-floating sections, 30-40 um, anti-TH antibody 1:1000-1:5000, DAB or fluorescent detection) to quantify TH+ neuron survival in substantia nigra and TH+ fiber density in striatum. (3) Western blotting for TH, alpha-synuclein, and markers of apoptosis (Bcl-2, Bax, cleaved caspase-3). (4) Measurement of oxidative stress markers (MDA, GSH, SOD activity). Blood and brain samples are collected for methyl citrate concentration analysis by LC-MS/MS. High doses (up to 1000 mg/kg) may be required to achieve brain concentrations sufficient to inhibit MAO-B, and oral bioavailability is likely low due to the polar nature of the molecule. Detailed efficacy data for methyl citrate in vivo are limited in the public literature.

Methyl citrate (molecular weight 206.15 Da, formula C7H10O7) is a small, polar organic acid. As a naturally occurring metabolite in the citric acid cycle, it has high water solubility and low lipophilicity. These properties result in poor oral bioavailability (<10% estimated) and minimal blood-brain barrier penetration (<5% of plasma concentration). When administered systemically, methyl citrate is rapidly cleared from plasma (half-life 15-30 minutes) primarily by renal filtration and urinary excretion. It is also metabolized in the tricarboxylic acid cycle. No active metabolites have been identified. For studies requiring CNS MAO-B inhibition, very high doses are needed to achieve brain concentrations above the IC50 (230 uM). Alternatively, direct intracerebroventricular administration may be used for CNS target validation, but this is not typical for this compound. Due to its natural occurrence in foods (Opuntia ficus-indica fruits), methyl citrate has generally low toxicity and is considered safe for dietary consumption. Detailed pharmacokinetic studies in animals or humans are not available in the public literature.

Preclinical toxicity data for methyl citrate are limited. As a naturally occurring organic acid found in edible plants (prickly pear cactus), it has generally low toxicity. No acute toxicity or adverse effects have been reported at doses up to 1000 mg/kg in rodent studies. The compound is an intermediate in the citric acid cycle and is naturally present in human metabolism. No mutagenicity or genotoxicity data are available. Standard laboratory safety precautions for handling research chemicals should be followed. Methyl citrate is not approved by the FDA or other regulatory agencies for therapeutic use.

[1]. Monoamine oxidase B inhibitors from the fruits of Opuntia ficus-indica var. saboten. Arch Pharm Res. 2001;24(1):51-54.

Methyl citrate is a naturally occurring MAO-B inhibitor (IC50 0.23 mM) isolated from Opuntia ficus-indica fruits. It is also known as monomethyl citrate or citric acid methyl ester. As a natural product, it serves as a lead compound for the development of more potent MAO-B inhibitors for Parkinson's disease and other neurodegenerative disorders. The compound has not entered clinical trials. CAS number: 26163-61-1. For research use only.

C7H10O7

206.15

204.027

26163-61-1

71356437

White to off-white solid powder

-0.4

1

7

4

14

248

0

COC(=O)CC(CC(=O)O)(C(=O)O)O

YUTUUOJFXIMELV-UHFFFAOYSA-L

InChI=1S/C7H10O7/c1-14-5(10)3-7(13,6(11)12)2-4(8)9/h13H,2-3H2,1H3,(H,8,9)(H,11,12)/p-2

2-hydroxy-2-(2-methoxy-2-oxoethyl)butanedioate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (485.08 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.13 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (12.13 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (12.13 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)