Discovery of cyclooxygenase inhibitors from medicinal plants used to treat inflammation

Abstract

Eleven authenticated botanicals used in the traditional Chinese medicine Huo-Luo-Xiao-Ling Dan were screened for ligands to cyclooxygenase (COX) using pulsed ultrafiltration liquid chromatography-mass spectrometry, and a mass spectrometry-based enzyme assay was used to determine the concentration of each of 17 ligands that inhibited COX-1 or COX-2 by 50% (IC(50)). Acetyl-11-keto-beta-boswellic acid, beta-boswellic acid, acetyl-alpha-boswellic acid, acetyl-beta-boswellic acid, and betulinic acid were COX-1 selective inhibitors with IC(50) values of approximately 10 microM. Senkyunolide O and cryptotanshinone were COX-2 selective inhibitors with IC(50) values of 5 microM and 22 microM, respectively. Roburic acid and phenethyl-trans-ferulate inhibited COX-1 and COX-2 equally. COX inhibition and the IC(50) values of most of these natural product ligands have not been reported previously.

Figure 1

Experimental design of pulsed ultrafiltration LC-MS screening of solutions for ligands to cyclooxygenases. 1. COX is incubated with a mixture of potential ligands; 2. COX-ligand complexes are separated from unbound compounds by using ultrafiltration; 3. COX ligands are released by denaturing the protein with organic solvent; 4. ligands are characterized using LC-MS/MS and identified by comparison to standards.

Figure 2

Pulsed ultrafiltration LC-MS screening of compounds that bind to COX-2. Enhancement of peak heights in the experiment using active COX-2 compared with the control containing denatured enzyme indicates specific binding to COX-2. (A) Pulsed ultrafiltration LC-MS screening of HLXL for COX-2 ligands. The computer-reconstructed mass chromatogram of m/z 297 shows the detection of the deprotonated molecule of the COX-2 ligand phenethyl trans-ferulate. (B) Total ion chromatogram of a mixture of COX-2 ligands (0.7 μg/mL each) including the HLXL compounds liquiritigenin, isoliquiritigenin and phenethyl trans-ferulate. Resveratrol and celecoxib were included in the assay as positive controls. The peaks eluting at approximately 12 and 28 min are impurities in the mobile phase or ion source, and the peak eluting at 25 min is p-hydroxyphenethyl anisate which not in HLXL but was included in this particular assay as another positive control.

Figure 3

Chemical structures COX-2 ligands from HLXL identified using pulsed ultrafiltration LC-MS with comparison to standards.

Figure 4

Identification of senkyunolide O and cryptotanshinone in HLXL using LC-MS-MS. In addition to demonstrating identical elemental compositions using high resolution mass spectrometry with accurate mass measurements and identical tandem mass spectra, co-chromatography was demonstrated using LC-MS-MS. Note that the signals for the SRM transitions corresponding to senkyunolide O and cryptotanshinone have been summed into one chromatogram.

Figure 5

Determination of IC₅₀ values of cryptotanshinone and senkyunolide O for the inhibition of COX-1 and COX-2. Both compounds showed selectivity for COX-2 inhibition.