Discovery of cyclooxygenase inhibitors from medicinal plants used to treat inflammation

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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% (IC50). Acetyl-11-keto-β-boswellic acid, β-boswellic acid, acetyl-α-boswellic acid, acetyl-β-boswellic acid, and betulinic acid were COX-1 selective inhibitors with IC50 values of approximately 10 μM. Senkyunolide O and cryptotanshinone were COX-2 selective inhibitors with IC50 values of 5 μM and 22 μM, respectively. Roburic acid and phenethyl-trans-ferulate inhibited COX-1 and COX-2 equally. COX inhibition and the IC50 values of most of these natural product ligands have not been reported previously.

Introduction

Rheumatoid arthritis and osteoarthritis are the most common forms of arthritis and are the major causes of morbidity, limitation of physical activity and health care utilization, especially in the elderly [1]. Although there is no cure, medications including steroids, non-steroidal anti-inflammatory drugs and opioids are commonly used for the treatment of arthritis. Since most of these drugs are associated with undesirable side effects such as gastrointestinal disturbances [2], new anti-inflammatory drugs are needed and complementary and alternative medicines are being sought [3].

An example of a botanical dietary supplement used to treat arthritis and related disorders is Huo-Lou-Xiao-Lin Dan (HLXL) [4]. HLXL contains 11 Chinese herbs including Ruxiang (Boswellia carterii Birdw., defatted gum resin); Qianghuo (Notopterygium incisum Ting ex H.T. Chang., root and rhizome); Danggui (Angelica sinensis (Oliv.) Diels., root); Baishao (Paeonia lactiflora Pall., root); Gancao (Glycyrrhiza uralensis Fisch., root); Yanhusuo (Corydalis yanhusuo W.T. Wang., root); Danshen (Salvia miltiorrhiza Bge., root); Chuanxiong (Ligusticum chuanxiong S.H. Qiu., root); Qinjiao (Gentiana macrophylla Pall., root); Guizhi (Cinnamomum cassia Presl., twigs); and Duhuo (Angelica pubescens Maxim., root) [2], [5]. To facilitate in vitro, in vivo and clinical investigations of safety and efficacy of this botanical dietary supplement [2], [5], we prepared a chemically standardized HLXL product employing an HPLC fingerprint method [2]. However, to be able to replicate the biological/pharmacological profiles in subsequent studies, it is essential to standardize HLXL both biologically and chemically [6], [7], [8]. Furthermore, mechanism of action studies would benefit from the identification of pharmacologically active compounds.

In support of biological and chemical standardization studies of HLXL employing bioassays relevant to arthritis of the knee, as well as to assist in determining potential mechanisms of action of the clinical preparation, extracts of the 11 plant components of HLXL, their isolated chemical compounds and the crude extract of HLXL were tested for anti-inflammatory activities. The isolated compounds included steroids, terpenes, alkaloids, flavonoids, glycolated compounds, and acids. This paper describes cyclooxygenase (COX)-1 and COX-2 screening studies of HLXL and its constituent plants and compounds using pulsed ultrafiltration liquid chromatography–mass spectrometry (LC–MS), the identification of COX ligands, and the determination of the concentration of each ligand that inhibits COX-1 or COX-2 by 50% (IC50).

Pulsed ultrafiltration LC–MS is an established approach for the screening of complex mixtures such as combinatorial libraries or natural product extracts for the discovery of ligands to macromolecular targets such as enzymes (see outline of the pulsed ultrafiltration LC–MS approach in Fig. 1) [9], [10], [11], [12], [13], [14], [15]. By applying this technique, we identified 17 ligands in extracts of HLXL and its constituent plants that bind to COX-2 and characterized additional ligands which will require further isolation and structure determination. Through the use of a COX functional assay based on LC–tandem mass spectrometry (LC–MS–MS), the inhibitory activities of these ligands towards COX-1 and COX-2 were determined. Selective and non-selective COX-1 and COX-2 inhibitors were identified with IC50 values as low as 5 μM. Together, the COX inhibitors in HLXL might be responsible, at least in part, for the anti-inflammatory activity of this traditional Chinese medicine.

Section snippets

Chemicals and reagents

All 11 medicinal plants were procured and authenticated by Dr. Chen Shilin who is a botanist and the Director of the Institute of Medicinal Plants Development at the Academy of Chinese Medicine in Beijing, China. The 11 individual plant components were extracted and formulated into HLXL under GMP conditions by Phytoway (Changsha, Hunan, China). Briefly, each of the 11 botanical powders was macerated separately in 70% aqueous acetone overnight at a ratio of 250 g of herb/L solvent. The

Results

Extracts HLXL, the 11 plant components of HLXL and mixtures of standard compounds were screened for ligands to COX-2 using pulsed ultrafiltration LC–MS. Enhancement of LC–MS peak heights for ultrafiltrates from incubations with active COX-2 compared with those from incubations using denatured COX-2 indicated the presence of ligands to COX-2. For example, phenethyl-trans-ferulate was detected in HLXL as a COX-2 ligand using pulsed ultrafiltration LC–MS (Fig. 2A). Based on the elemental

Discussion

The IC50 of celecoxib, the COX-2 selective positive control compound, was 50 nM for human COX-2 and 30 μM for ovine COX-1. These data are similar to those of Penning et al., who reported celecoxib IC50 values of 40 nM for COX-2 and 15 μM for COX-1 [26]. The 600-fold selectivity of celecoxib for COX-2 (COX-1 IC50/COX-2 IC50) measured in this functional assay is also consistent with other previously reported COX-2 selectivities of >300 for celecoxib [27], [28]. The natural product standard used in

Acknowledgments

This research was supported by grants P01 CA48112 from the National Cancer Institute (NCI) and P01 AT002605 and P50 AT00155 from the National Center for Complementary and Alternative Medicine (NCCAM), the Office of Dietary Supplements (ODS) and the Office for Research on Women's Health (ORWH) of the National Institutes of Health (NIH). The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NCI, NCCAM, ODS, ORWH, or the

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