Butyrylcholinesterase (BChE): Pteryxin showed IC₅₀ = 12.96 ± 0.70 μg/ml (at 100 μg/ml, inhibition 91.62 ± 1.53%) [1].
Acetylcholinesterase (AChE): Pteryxin showed 9.30 ± 1.86% inhibition at 100 μg/ml [1].

Pteryxin at 100 μg/ml inhibited AChE by 9.30 ± 1.86% and BChE by 91.62 ± 1.53%. The IC₅₀ for BChE was 12.96 ± 0.70 μg/ml, which is more potent than the reference drug galanthamine (IC₅₀ = 22.16 ± 0.91 μg/ml, 81.93 ± 2.52% inhibition at 100 μg/ml) [1].
Molecular docking experiments revealed that pteryxin forms two hydrogen bonds with catalytic residues S198 and H438 of BChE, and a strong π-π stacking interaction with W231. No significant hydrogen bonding interactions with AChE catalytic residues (S203 and H447) were observed, consistent with low AChE inhibition [1].
In 3T3-L1 adipocytes, pteryxin (10, 15, 20 μg/ml) dose-dependently suppressed triglyceride (TG) content by 52.7%, 53.8%, and 57.4%, respectively (P < 0.05). When administered during early differentiation (Day 0-2), lipid accumulation was blocked by 50.6% (P < 0.05). Pteryxin (20 μg/ml) downregulated SREBP1c (18.0% reduction, P < 0.05), ACC1 (38.2% reduction, P < 0.05), PDK4, and MEST (42.8% reduction, P < 0.05); upregulated HSL (15.1% increase, P < 0.05), UCP2 (77.5% increase, P < 0.05), RORC, and FABP4 (202.0% increase); and showed a non-significant 36.1% suppression of FASN. PPARγ expression was lower than HP treatment but higher than control. GLUT4 was suppressed by 43.1% (P < 0.05) and IRS-1 by 36.6% (P > 0.05). No cytotoxicity was observed [2].
In HepG2 hepatocytes, pteryxin (10, 15, 20 μg/ml) inhibited TG content by 25.2%, 34.1%, and 27.4%, respectively (P < 0.05). Gene expression analysis showed suppression of hSREBP1 (72.3%, P < 0.05), hFASN (62.9%, P < 0.05), hSCD (44.5%, P < 0.05), and hACC1 (50.3%, P < 0.05), and upregulation of hPPARα (P < 0.05). No cytotoxicity was observed [2].

Cholinesterase inhibition assay (Ellman method): The inhibitory activity of pteryxin against AChE (from electric eel, Type-VI-S) and BChE (from horse serum) was measured using a slightly modified spectrophotometric method. Acetylthiocholine iodide and butyrylthiocholine chloride were used as substrates, and 5,5’-dithio-bis(2-nitrobenzoic) acid (DTNB) was used for measurement. In a 96-well microplate, 140 µL of sodium phosphate buffer (pH 8.0), 20 µL of DTNB, 20 µL of test solution, and 20 µL of enzyme solution were added and incubated for 15 min at 25°C. The reaction was initiated by adding 10 µL of substrate. Hydrolysis was monitored by formation of the yellow 5-thio-2-nitrobenzoate anion at 412 nm using a microplate reader. Percentage inhibition was calculated by comparing reaction rates of samples relative to blank (ethanol in phosphate buffer, pH 8). Galanthamine was used as reference. Experiments were run in six parallel sets [1].
Molecular docking experiment: The protein structure of BChE (PDB code: 4tpk) was downloaded from RCSB. Native ligand, non-protein atoms, and crystallographic waters were removed. Polar hydrogen atoms were added, and side chain amides and imidazoles were protonated assuming physiological pH using H++ server. AutoDock version 4.2.1 and Vina 1.1.2 were used for docking. AutoDock 4.2 employed Lamarckian genetic algorithm with 100 dockings, population size 150, random starting position, translation step ranges 2.0 Å, rotation step ranges 35°, elitism 1, mutation rate 0.02, crossover rate 0.8, local search rate 0.06, and 10 million energy evaluations. Grid was centered on the bound inhibitor with size 40×40×40 ų and spacing 0.375 Å. Results were clustered using MGL tools with a cutoff of 0.5 Å. The lowest energy conformation from each cluster was analyzed for protein-ligand interactions [1].

3T3-L1 preadipocyte differentiation and TG content assay: 3T3-L1 cells were seeded in 24-well plates at 1×10⁴ cells/well. Confluent fibroblasts were maintained for 2 days (Day 0). Differentiation was induced with 0.5 mM 3-isobutyl-1-methylxanthine, 0.25 μM dexamethasone, 10 μg/mL insulin, and 10% fetal bovine serum for 48 h (Day 0-2). From Day 2-6, cells were treated with pteryxin (10, 15, or 20 μg/mL) or controls. Medium was replaced every 2 days. For time-course study, pteryxin (20 μg/mL) was added at different intervals. On Day 6, cells were washed with phosphate-buffered saline, harvested into 10% Triton X-100 solution, and lysed by sonication. TG content was quantified using an enzymatic kit and expressed as mg triglyceride per mg cellular protein. Protein content was determined using a protein assay kit. Cell viability was assessed using a tetrazolium-based method. No cytotoxicity was observed [2].
HepG2 cell culture and TG content assay: HepG2 cells were maintained in DMEM with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO₂. Cells were seeded in 24-well plates at 5×10⁴ cells/well, grown to 70% confluence, then kept in serum-free DMEM containing 1% BSA overnight. Cells were treated with insulin (1 μM), or pteryxin (10, 15, or 20 μg/mL) for 24 h. TG content was measured as described above. No cytotoxicity was observed [2].
RNA extraction and quantitative real-time PCR: 3T3-L1 cells (3×10⁴ cells/3.0 cm dish) and HepG2 cells (15×10⁴ cells/dish) were treated as described. Total RNA was extracted using an RNA extraction kit. First-strand cDNA was generated from 2 μg total RNA using a reverse transcription kit. qPCR was performed on a real-time PCR system using SYBR Green master mix with the following protocol: one cycle at 95°C for 20 s, 40 cycles at 95°C for 3 s and 60°C for 30 s, then melting curve analysis starting at 95°C for 15 s, 60°C for 60 s, increasing by 0.3°C every 15 s. mRNA levels were normalized to β-actin (for 3T3-L1) or hGAPDH (for HepG2). Primers for mouse PPARγ, MEST, SREBP1c, FASN, ACC1, PDK4, RORC, C/EBPα, LPL, FABP4, UCP2, UCP3, HSL, FXRα, AdipoQ, GLUT4, IRS-1, PGC1α, and human SREBP1, FASN, SCD, ACC1, FXRα, PPARα were used [2].

Pteryxin at concentrations up to 20 μg/mL showed no detectable cytotoxicity in 3T3-L1 adipocytes as determined by a tetrazolium-based cell viability assay [2].
Pteryxin at concentrations up to 20 μg/mL showed no cytotoxicity in HepG2 hepatocytes [2].

[1]. Pteryxin - A promising butyrylcholinesterase-inhibiting coumarin derivative from Mutellina purpurea. Food Chem Toxicol. 2017 Nov;109(Pt 2):970-974.

[2]. Pteryxin: a coumarin in Peucedanum japonicum Thunb leaves exerts antiobesity activity through modulation of adipogenic gene network. Nutrition. 2014 Oct;30(10):1177-84.

Pteropterin is a coumarin compound. It has been reported to exist in Aster tataricus, Pseudomonas japonicus, and other organisms with relevant data.

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Pteryxin is a dihydropyranocoumarin derivative of the angular-type pyranocoumarins. The acyloxy groups are mentioned to play an important role in the bioactivity of dihydropyranocoumarins. Only a few studies on ChE inhibition of pyranocoumarins confirm that they display quite lower AChE inhibition, consistent with the data on pteryxin. No previous report on the effect of pteryxin towards the central nervous system (CNS) existed prior to this study [1].
In the anti-obesity study, during purification, Fr3 (which contained pteryxin) showed potent activity over the hexane phase, suggesting that other compounds in the hexane phase might counteract the anti-obesity activity of Fr3. Chlorogenic acid (CGA), a main constituent in coffee and previously identified in PJT, showed no TG suppression in hepatocytes [2].
The authors declared no conflicts of interest for both studies [1][2].

C21H22O7

386.3952

386.136

C, 65.28; H, 5.74; O, 28.98

13161-75-6

5281425

White to off-white solid powder

1.3±0.1 g/cm3

486.8±45.0 °C at 760 mmHg

81℃

211.5±28.8 °C

0.0±1.2 mmHg at 25°C

1.574

4.3

0

7

5

28

720

2

O1C2C([H])=C([H])C3C([H])=C([H])C(=O)OC=3C=2[C@]([H])([C@]([H])(C1(C([H])([H])[H])C([H])([H])[H])OC(C([H])([H])[H])=O)OC(/C(=C(/[H])\C([H])([H])[H])/C([H])([H])[H])=O

LYUZYPKZQDYMEE-YRCPKEQFSA-N

InChI=1S/C21H22O7/c1-6-11(2)20(24)27-18-16-14(28-21(4,5)19(18)25-12(3)22)9-7-13-8-10-15(23)26-17(13)16/h6-10,18-19H,1-5H3/b11-6-/t18-,19-/m1/s1

[(9R,10R)-9-acetyloxy-8,8-dimethyl-2-oxo-9,10-dihydropyrano[2,3-f]chromen-10-yl] (Z)-2-methylbut-2-enoate

Pteryxin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (~258.80 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.47 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 (6.47 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (6.47 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.)