Development of an Enzyme-Based Thin-Layer Chromatographic Assay for the Detection of Cyclooxygenase-2 Inhibitors

Abstract

The search for new anti-inflammatory drugs with less side effects requires simple, fast and reliable screening methods. In this context, we have developed a sensitive thin-layer chromatographic (TLC) assay on silica gel plates to detect cyclooxygenase-2 (COX-2) inhibition. COX-2 catalyzes two sequential enzymatic reactions: a first oxygenation step that converts arachidonic acid into prostaglandin G₂, and a subsequent reduction of prostaglandin G₂ into prostaglandin H₂. Our test is based on the co-oxidation during this peroxidation step of a co-substrate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), leading to a blue-grey product. As a consequence, COX-2 inhibitors appear on the TLC plate after revelation as clear spots against the colored background. Parameters such as concentrations of enzyme, substrate, and chromogenic reagent have been optimized. The limit of detection was found to be below the microgram for standard COX-2 inhibitors such as celecoxib or ibuprofen. The developed TLC assay was also conclusive when applied to 60 various natural pure compounds and some complex natural extracts. Results demonstrated a COX-2 inhibitory activity mostly for triterpene and sterol derivatives. This COX-2 TLC assay appears as a suitable low-cost and reliable strategy for the screening of natural extracts to discover new anti-inflammatory compounds.

1. Introduction

Inflammation is an innate defense mechanism of the immune system, triggered by external aggression such as infection or injury. Although the acute inflammatory process is beneficial to the organism, it also leads to unpleasant sensations, such as redness and swelling of the skin at the site of injury, as well as pain or tenderness, and a feeling of heat. Non-steroidal anti-inflammatory drugs such as ibuprofen, diclofenac or celecoxib are widely used to reduce inflammation. However, the search for effective anti-inflammatory compounds with less side effects is an ongoing and relevant effort [1], which requires methods suitable for large screenings. Currently, most of the assays are cell-based models (measuring the expression of pro-inflammatory cytokines such as TNF-α, NF-κB, etc.), ex vivo assays (measuring levels and activity of cytokines), or even in vivo assays using the carrageenan-induced rat hind-paw edema method [2]. All these bioassays require specific skills, sophisticated equipment, regular and careful maintenance of the cells, or animal testing regulations and authorization. Some commercial screening kits are also available to detect inhibitors of cyclooxygenase (COX)-catalyzed prostaglandin biosynthesis. These kits are generally based on enzyme immunoassay (ELISA) or fluorometric reactions. Other methods have been developed to detect COX inhibitors with the measurement of prostaglandins generated from arachidonic acid, using immunoassays, radiometric techniques, LC-MS or UV detection [3,4]. Although these bioassays are sensitive and reliable, they are generally too tedious or expensive to allow large-scale screening or bioguided purification and are not suitable for complex mixtures such as plant extracts that sometimes contain minor active compounds or molecules with antagonist effects.

For these reasons, we have developed a novel assay that is cheap, quick to perform, and informative, based on thin-layer chromatography. Cyclooxygenases (COX, EC 1.14.99.1) play an essential role in the inflammatory response by converting arachidonic acid into prostaglandin PGH₂. While COX-1 is constitutively expressed in most cell types, COX-2 is only expressed in response to various pro-inflammatory stimuli and represents therefore a target of choice in the treatment of acute inflammation. We have therefore developed a thin-layer chromatographic (TLC) assay for the detection of COX-2 inhibitors, based on the oxidation of TMPD, a chromogenic co-substrate.

2. Materials and Methods

2.1. Chemicals and Reagents

Recombinant human cyclooxygenase-2 (COX-2) was obtained from Sigma-Aldrich (Saint Louis, MO, USA), as well as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD or Wurster’s blue). Arachidonic acid, hematin porcine, celecoxib, ibuprofen, diclofenac, eugenol, salicylic acid, ascorbic acid, caffeine, quercetin, vanillin, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were also purchased from Sigma-Aldrich. Sitosterol and betulinic acid were obtained from Extrasynthese (Genay, France) and acetylsalicylic acid from Cooper (Melun, France). Tris Base HCl was purchased from Millipore (Burlington, MA, USA). The suppliers of the 60 standard compounds tested for the specificity assay are not mentioned here for readability. Ethanol, DMSO and methyl tert-butyl ether were obtained from Fisher Scientific (Illkirch, France), tetrahydrofurane from VWR (Radnor, PA, USA), and cyclohexane from Sigma-Aldrich.

2.2. Preparation of Solutions

A stock solution of COX-2 was prepared in DMSO. For TLC assays, this solution was dissolved in 100 mM Tris-HCl buffer pH 8.0 containing 1 µM of hematin to give a final solution of 1 U/mL. Stock solutions of arachidonic acid (1 mM) and TMPD (2 mM) were prepared in ethanol and DMSO, respectively. Arachidonic acid solution was diluted with Tris-HCl buffer pH 8.0 and combined with TMPD to obtain the final reaction mixture of arachidonic acid (5 µM) and TMPD (1 mM). All standard compounds were dissolved in ethanol in suitable concentrations for TLC application. A 0.1% (m/v) DPPH solution was prepared in methanol, and vanillin sulfuric reagent was prepared by dissolving 0.5 g of vanillin in 100 mL of sulfuric acid/ethanol (5:95 v/v).

2.3. Thin-Layer Chromatography

Experiments were performed on pre-washed (MeOH) 20 × 20 cm aluminum TLC or HPTLC silica gel plates 60 F₂₅₄ Merck (Darmstadt, Germany). Pure compounds were applied with appropriate volumes with a 100 μL syringe (Hamilton, Bonaduz, Switzerland) onto TLC plates as bands of 2 mm in length, with a CAMAG Linomat 5 (Muttenz, Switzerland) under a stream of nitrogen. Ethyl acetate extracts of clove (Syzygium aromaticum), Fomitopsis pinicola and Hypholoma fasciculare [5] were deposited (50 µg) in the same conditions as bands of 8 mm in length and then eluted with the solvent system methyl tert-butyl ether/tetrahydrofurane/cyclohexane (5:1:4 v/v/v) previously developed [5].

2.4. Cyclooxygenase-2 Inhibition Assay

After complete removal of solvents, the TLC plates were sprayed with 2 mL of the COX-2 solution using a Camag Derivatizer (Muttenz, Switzerland), and placed on a stand in a Petri dish lined with moist filter paper. This Petri dish was then incubated at 37 °C for 10 min in a KB 23 incubator Binder (Tuttlingen, Germany) to activate the enzyme. Immediately after, the chromogenic reaction was initiated by spraying 2 mL of the solution of arachidonic acid/TMPD with the Derivatizer. After 5 min, images of the TLC plates were acquired with CAMAG Reprostar 3 (Muttenz, Switzerland) under visible light, where COX-2 inhibitors appear as clear spots against the lavender blue background.

2.5. Sensitivity and Linearity of the TLC Assay

The visual limit of detection (LOD) was evaluated by applying on the TLC plate decreasing amounts of several COX-2 inhibitors: celecoxib, ibuprofen, diclofenac, and aspirin (from 10 to 1 µg with interval of 1 µg, then from 0.9 to 0.1 with interval of 0.1 µg, then from 0.09 to 0.05 µg with interval of 0.01 µg). The assays were performed in triplicate without any chromatographic elution. For determination of linearity, inhibition was performed between 5 and 100 µg. Images were then converted to 8-bit greyscale and the area of inhibitions zones was measured in mm² using ImageJ^® 1.53k software (Rasband WS, NIH, Bethesda, MD, USA).

2.6. Specificity of the Reaction

Sixty natural compounds and three complex natural extracts from Fomitopsis pinicola, Hypholoma fasciculare, and Syzygium aromaticum—reported to have or not to have COX-2 inhibitory effects—were evaluated with this COX-2-based TLC assay (at 20 µg for pure compounds and 50 µg for the extracts). In order to check that clear spots were not false positive reactions due to antioxidant compounds, a parallel revelation with DPPH was performed.

3. Results and Discussion

Several assays to detect enzyme inhibitors have been already developed on TLC plates, to detect glucosidase, acetylcholinesterase, or tyrosinase inhibitors, for example [6,7]. In any case, the visualization of an enzymatic activity on TLC plates requires the use of a reagent that will generate only after enzymatic reaction a coloration, or another form of detection, such as fluorescence for instance. This compound can be a direct substrate of the enzyme or a co-product of successive reaction steps. Cyclooxygenases catalyze two consecutive reactions, an oxygenation of arachidonic acid to prostaglandin G₂ followed by a reduction of PGG₂ to PGH₂, via a peroxidase activity. During this second step, oxidation of a peroxidase co-substrate can occur and be measured [8,9]. Among these compounds, TMPD was found to be a suitable co-subtrate to detect peroxidase activity, generating quickly a grey-blue coloration [10,11].

3.1. Preliminary Assays

The initial concentrations of enzyme and arachidonic acid to be tested were adjusted from conditions reported in the literature for assays in 96-well plates. The concentration of the COX-2 solution was generally comprised between 2 and 20 units/mL, most of the time around 5 U/mL; the working concentration of arachidonic acid was reported between 5 and 50 µM, in accordance with the apparent Michaelis constant (Km). Publications dealing with TMPD are scarce, but one mentioned in microtiter plates 200 units of enzyme for 100 µM arachidonic acid and 100 µM TMPD, without indicating the working volume [12]. Another one used 100 µM arachidonic acid and 170 µM TMPD, but the concentration of COX-2 was not indicated [13]. In a more recent study, the mixture reaction contained 133 µM of arachidonic acid and TMPD, but again there was no information about COX-2 concentration [14]. Furthermore, when transposing a colorimetric microwell-plate method to a TLC plate, we have to consider that there will be only a very thin layer of reaction solution onto the surface of the TLC plate, and so concentrations have to be increased in order to observe a coloration visible to the eye.

Based on these data, we have chosen to set for the preliminary assay a concentration of COX-2 at 8 U/mL, and to use 1 mM arachidonic acid and 2 mM TMPD. The incubation conditions were similar to those reported in the literature: 10 min at 37 °C in a moist atmosphere. In order to check that the coloration occurs on TLC plates only in the presence of the enzyme, substrate and co-substrate, four clean TLC plates have been prepared without any inhibitors. One has been sprayed with TMPD only, the second one with arachidonic acid and TMPD, the third one with COX-2 and TMPD, and the last one with COX-2, arachidonic acid, and TMPD. All plates have been incubated for 10 min at 37 °C. After 30 min, only the TLC plate containing all the different reagents developed a blue coloration (Figure A1). This proves that the coloration appears only after reaction of the enzyme with arachidonic acid and subsequent reaction with TMPD, and that autoxidation of TMPD is imperceptible in these conditions, and so insignificant.

After this, a first assay with a COX inhibitor was carried out by applying 100 µg of ibuprofen on a TLC plate, followed by subsequent reaction with COX-2 and a mixture of arachidonic acid and TMPD; after incubation, ibuprofen appeared as a clear spot on the TLC plate, confirming the feasibility of this enzyme-based TLC assay.

3.2. Optimization of Reagent Concentrations

TLC bioassays are considered easy to perform and generally inexpensive. However, when reagents are expensive—which is often the case with enzymes—it makes sense to reduce the concentration of reagents to decrease the cost of experiments. For this reason, the concentrations of COX-2 and substrates have been adjusted so as to reduce them as much as possible while maintaining a contrast allowing inhibition zones to be observed. The concentration of COX-2 was first lowered and finally set at 1 U/mL to keep a grey-blue coloration. Then various combinations were evaluated, ranging from 1 µM to 10 mM of arachidonic acid, and from 10 µM to 2 mM of TMPD; various ratios of arachidonic acid/TMPD were also tested with these different concentrations (5:1, 1:1, 1:10, 1:100, 1:200).

The mixture of arachidonic acid/TMPD with respective concentrations of 5 µM and 1 mM, and 1:200 ratio was found to give the most intense coloration on TLC plates, allowing a better contrast for the detection of inhibitors. Lower concentrations, especially of TMPD, gave poor contrast. TMPD is known to be rather unstable and undergoes fairly rapid autoxidation, even if the literature mentions that the absorbance of autoxidized TMPD remains below its absorbance in the presence of active enzymes, and even below its absorbance in the presence of a COX-2 inhibitor such as celecoxib [14]. During our assays, we observed that the coloration of the plate can be more or less intense according to the time elapsed between the preparation of the TMPD solution and the TLC assay. It is therefore very important to prepare the reagents solutions extemporaneously. We also observed that the higher contrast was obtained 5 to 10 min after chromogenic agent application, and that the blue coloration vanished from the plate after approximately one hour.

Several compounds have been selected to develop this TLC assay, including four common anti-inflammatory drugs: ibuprofen and diclofenac, non-selective reversible inhibitors, aspirin, a non-selective irreversible inhibitor, and celecoxib, a selective COX-2 inhibitor. In addition, eugenol and salicylic acid were both evaluated. Eugenol is known to inhibit the expression of COX-2, but docking studies have suggested that it should also inhibit COX activity [15], and salicylic acid has been reported as a very weak COX inhibitor in vitro [16]. Under the conditions set out and tested at 50 µg, all these compounds led to pale inhibition spots, more or less diffused (Figure 1).

3.3. Limits of Detection and Linearity

Once all parameters were set, the sensitivity of the TLC assay was estimated for the COX inhibitors celecoxib, ibuprofen, diclofenac, and aspirin, by assessing the visual limit of detection for each compound. The LOD values were comprised between 0.1 µg and 0.6 µg, i.e., between 0.5 and 2.0 nanomole depending on the inhibitor (

Table 1. Minimum quantity required to observe a clear inhibition spot on TLC (LOD).

Compound	LOD (µg)
celecoxib	0.4
diclofenac	0.6
ibuprofen	0.2
aspirin	0.1

).

As shown in Figure A2, the intensity of the inhibition zone was quite well correlated to the amount of inhibitor on the plate; however, the measurement of the linearity of the response was not very conclusive due the diffusion of the inhibition zone for high quantities (above 10 µg), leading to blurred edges of inhibition spots. Regression equations correlating peak area to inhibitor quantity were obtained for diclofenac and celecoxib, but with not-outstanding coefficients of determination (y = 0.7331x + 1.6945, R² = 0.962 for diclofenac; y = 0.3808x + 3.775, R² = 0.920 for celecoxib). Despite poor linearity, the sensitivity of the developed COX-2 TLC assay allows the detection of inhibitors at quantities below 1 µg and is therefore suitable for screenings.

3.4. Specificity of the COX-2 TLC Assay

In order to evaluate the selectivity of the assay, several pure compounds were evaluated with the developed COX-2 assay: caffeine, coumarin, ascorbic acid, quercetin, sitosterol, and betulinic acid. Sitosterol and betulinic acid have been previously reported to have COX-2 inhibitory activity and both gave a clear spot characteristic of an enzyme inhibition (Figure 2). Caffeine, known to have no COX-2 activity, did not induce any clear spot, similar to coumarin. Quercetin, known to suppress COX-2 expression, seemed to give at first glance an inhibition spot, but it presented a yellow-green coloration indicating that a blue coloration developed on the yellow spot of quercetin, and so there is no COX-2 inhibition. On the contrary, ascorbic acid, a strong antioxidant, induced a large and intense inhibition area. Based on the specific reaction that occur during the second step of the enzymatic reaction—an oxidation of TMPD—it could be likely that antioxidants prevent the oxidation of TMPD, whether the enzyme is active or not, leading to false positive reactions.

To check this hypothesis, 60 natural products were spotted on two TLC plates, one revealed with the COX-2 reaction, the other one with TMPD (

Table 2. COX-2 inhibition and antioxidant activity for some standard natural products (tested at 20 µg). Celecoxib was used as positive control for COX-2 inhibition assay.

Compound	COX-2 Inhibition a	Antioxidant Activity b	Compound	COX-2 Inhibition a	Antioxidant Activity b
betulinic acid	+	−	menthol	−	−
madecassic acid	+	−	farnesene	−	−
α-hederin	+	−	α-humulene	−	−
ginsenoside Rd	+	−	trans-caryophyllene	−	−
sabinene	+	−	santonin	−	−
parthenolide	+	−	ginkgolide A	−	−
campesterol	+	−	betulin	−	−
β-sitosterol	+	−	epicatechin gallate	−	+
quinine bromhydrate	+	−	quercetin	−	+
stigmasterol	±	−	quercetol	−	+
lupeol	±	−	kampferol	−	+
uvaol	±	−	myricetin	−	+
piperine	±	−	rutin	−	+
aloin A	±	−	taxifolin	−	+
psoralen	±	−	protocatechuic acid	−	+
ascorbic acid	+	+	arbutin	−	+
capsaicin	+	+	rosmarinic acid	−	+
ononin	−	−	resveratrol	−	+
tomatin	−	−	procyanidin A2	−	+
senecionin	−	−	procyanidin B1	−	+
lobeline sulfate	−	−	procyanidin B2	−	+
taurine	−	−	scopoletin	−	+
L-carnitine	−	−	carvacrol	−	+
glucobrassicin	−	−	α-tocopherol	−	+
sinigrin	−	−	naringin	−	±
astaxanthin	−	−	vitexin	−	±
caffeine	−	−	liliroside	−	±
anethol	−	−	tyrosol	−	±
borneol	−	−	lawsone	−	±
eucalyptol	−	−	cyanidin	nd c	nd c

^a inhibition detected as a clear spot on TLC plate after revelation with COX-2/arachidonic acid/TMPD, ^b inhibition detected as a clear spot on TLC plate after revelation with DPPH, ^c could not be determined due to the very dark color of the spot.

). These compounds were selected to represent the chemical diversity of natural products, including 12 flavonoids, 12 other phenolics, 24 terpene derivatives (mono-, sesqui-, di-, triterpenes, and sterols), as well as 12 other compounds including alkaloids, glucosinolates, vitamin or amino acids.

Twenty compounds did not induce any activity in both assays, notably nitrogen and sulfur compounds, as well as monoterpenes (Table 2). As expected, antioxidant activity was observed for most phenolic compounds, but none of them inhibited COX-2 in the TLC assay. A dozen compounds were found to induce an intense inhibition spot against COX-2 without any effect on DPPH, mostly triterpene and sterol derivatives. The triterpene acid derivatives betulinic acid, madecassic acid, and α-hederin, were the three compounds exhibiting the most intense inhibition spot. Finally, only two compounds, ascorbic acid and capsaicin, were found to be active in both assays, confirming the non-interference of antioxidant compounds with the detection of COX-2 inhibition in the developed assay.

This study also showed that strongly colored compounds could make it difficult to evaluate clear inhibition spots. However, pre- and post-development photos showed that yellow compounds exhibited a greenish color after TMPD oxidation, confirming the oxidation of TMPD into a blue derivative and so the absence of COX-2 inhibition.

3.5. Application of the COX-2 TLC Assay on Complex Natural Extracts

In the previous assay with pure natural products, we observed that tested phenolic compounds were devoid of COX-2 inhibitory activity contrarily to triterpene derivatives that often exhibited an intense inhibition spot. Plants contain ubiquitous phenolic compounds, but on the contrary, many mushrooms are devoid of phenolic compounds and contain mainly triterpenoids. We have therefore submitted to the developed TLC assay plant and fungal extracts. First, we have evaluated an extract from Fomitopsis pinicola (Fomitopsidaceae), a common decay fungus growing on dead trees. This mushroom does not contain any phenolic compound but is rich in terpene derivatives. Furthermore, it was reported to contain lanostane triterpenoids strongly inhibiting COX-2 activity [17]. The TLC enzymatic assay with TMPD confirmed the presence of COX-2 inhibitors in this mushroom, as many clear inhibition zones could be observed (Figure 3A). Revelation with sulfuric vanillin gave purple or blue colorations, suggesting that these active compounds could be terpene derivatives. Revelation with DPPH confirmed that these active zones were not correlated with radical scavenging or antioxidant activity. Similar results were obtained on HPTLC plates (Figure A3).

Hypholoma fasciculare (Strophariaceae) was also evaluated in this bioassay. As F. pinicola, this mushroom contains mainly terpene derivatives. No COX inhibitory activity has been reported up to now for this species, but an anti-inflammatory effect due to a decrease of COX-2 expression was mentioned for a closely-related species, H. lateritium [18]. After enzyme reaction, four inhibition zones could be clearly distinguished on the elution profile of H. fasciculare (Figure 3B). Actually, a fifth thin distinct pale band was also visible at Rf 0.05 before diffusion of the zone at the bottom of the plate. As for F. pinicola, this activity seemed to be due to terpene derivatives and not correlated to an antioxidant activity.

A crude plant extract from clove flower buds was also tested. Clove was previously reported to inhibit COX-2 activity [19] and eugenol was found to inhibit COX-2 activity in our preliminary assay. In the clove crude extract, eugenol gave an intense antioxidant spot on the plate revealed with DPPH but was barely visible with enzyme reaction (Figure 4). Yet, two well-defined COX-2 inhibition zones could be detected from the clove extract. These two bands were colored in blue with vanillin-sulfuric acid reagent and could be triterpene derivatives. This would be in accordance with results obtained with natural pure compounds and with mushroom extracts, suggesting a high potential of triterpene derivatives as COX-2 inhibitors.

4. Conclusions

A sensitive TLC effect-directed analysis has been developed on silica gel for the rapid identification of cyclooxygenase-2 inhibitors in complex mixtures. Reagent concentrations have been optimized to assure a low-cost assay while maintaining a good sensitivity below the microgram. The procedure is easy to perform and reliable, either on TLC or HPTLC plates. This novel enzyme-based assay represents a good alternative to tedious cell-based assays or expensive commercial kits and will allow large-scale screening of complex mixtures and bioguided fractionation for the identification of new anti-inflammatory compounds.