Abstract
In this study 160 plant samples representing more than 30 plant families collected from the Malaysian forests were assessed for their ability to inhibit lipoxygenase activity. The lipoxygenase inhibiting activity was measured using the 96-well microplate-based ferric oxidation of xylenol orange (FOX) assay. The screening parameters, including z’ factor, indicated that the assay method adopted was robust and suitable for high-throughput screening. In the preliminary screen, four plant extracts displayed inhibitory activity of 70% or higher. The active plant extracts were isolated from Agelaea borneensis Merr. (Connaraceae) (bark) (IC50, 1.6 μg/mL), Chisocheton polyandrus Merr. (Meliaceae) (bark) (3 μg/mL), Garcinia cuspidata King (Guttiferae) (bark) (28.3 μg/mL) and Timonius flavescens Baker (Rubiaceae) (leaf) (8.9 μg/mL).
Introduction
Inflammation is a complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells or irritants. The cell damage associated with inflammation causes cell membranes to release arachidonic acid (CitationLevick et al., 2007). Arachidonic acid undergoes two metabolic pathways: the cyclooxygenase (COX) pathway involving cyclooxgenase-1 (COX-1) and cyclooxgenase-2 (COX-2) to produce the prostaglandins and thromboxanes (CitationRainsford, 2007); and the lipoxygenase (LOX) pathway, involving 5-lipoxygenase (5-LOX), 12-lipoxygenase (12-LOX) and 15-lipoxygenase (15-LOX), to produce the leukotrienes and hydroperoxy fatty acids (CitationSamuelsson et al., 1987; CitationYedgar et al., 2007).
The products of the COX and LOX pathways are involved in the induction of numerous pathologies, especially inflammatory diseases. These include arthritis (CitationHonda et al., 2006), fever, chronic pain (CitationMabuchi et al., 2004), sepsis, burn injury (CitationHahn & Gamelli, 2000), inflammatory bowel disease (CitationSubbaramaiah et al., 2004), and carcinogenic processes in colorectal cancer (CitationBacklund et al., 2005). In the treatment of inflammation, non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used, and are effective in the management of pain, fever, redness and edema arising as a consequence of inflammatory mediator release (CitationFerreira, 2002). NSAIDs achieve these effects by inhibiting the activity of COX-1 and COX-2. COX-1 inhibitors are associated with a number of side effects including gastrointestinal erosions and renal and hepatic insufficiency (CitationBurdan et al., 2004). The newer drugs, which are COX-2 inhibitors, are more effective against inflammation and are claimed to have fewer gastrointestinal side effects. However, one of the COX-2 inhibitors, Vioxx• was withdrawn in 2004 due to serious cardiovascular events (CitationRainsford, 2007).
It has now been suggested that the therapeutic effects of agents inhibiting only COX-1 and COX-2 may be limited by preferential conversion of arachidonic acid to leukotrienes through the LOX pathway since leukotriene B4 (LTB4) is the main leukotriene that plays a major role in the inflammatory response (CitationHudson et al., 1993). Therefore, medications that inhibit both COX-1/2 and 5-LOX pathways are believed to be superior to conventional NSAIDs since they could produce a synergistic effect and achieve optimal anti-inflammatory activity by blocking the production of both leukotrienes and prostaglandins (CitationCelotti & Laufer, 2001; CitationMartel-Pelletier et al., 2003). This is supported by a significant reduction of collagen-induced arthritis in animal models upon concomitant administration of NSAID and leukotriene synthesis inhibitors (CitationMartel-Pelletier et al., 2003; CitationNickerson-Nutter & Medvedeff, 1996).
5-LOX is the first and the key enzyme involved in the arachidonic acid pathway to produce leukotrienes (CitationZhang et al., 2002). The 5-LOX pathway has been associated with various diseases including asthma (CitationMcMillan, 2001), inflammatory bowel diseases (CitationJupp et al., 2007), cancers (prostate, pancreatic and breast) (CitationAvis et al, 2001; Ghosh & Myers, 1998; CitationRomano et al., 2001) and cardiovascular diseases including atherosclerosis, heart attack and stroke (CitationFunk, 2005; Mehrabian & Allayee, 2003). Among the leukotrienes produced by 5-LOX activity, LTB4 is the most significant in the inflammatory response. For example, elevated levels of LTB4 have been found in patients with rheumatoid arthritis (CitationChen et al., 2006) and inflammatory bowel disease (CitationSchmidt et al., 1995).
The isozymes 12-LOX and 15-LOX are associated with carcinogenesis, bronchial asthma and inflammatory vascular diseases, which include artherosclerosis, diabetes and hypertension (CitationKuhn & O’Donnell, 2006; CitationNatarajan & Nadler, 2004). Tissue levels of two 15-LOX products, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE), are elevated during inflammation. For example, increased 15-HETE levels were found in human asthmatic bronchitis (CitationProfita et al., 2000) and human proctocolitis (CitationZijlstra et al., 1991). Therefore, LOX inhibitors are considered promising agents for the treatment of inflammatory diseases on the basis of the important roles of LOX pathways (CitationNaveau, 2005).
Previously, we have reported G-protein coupled receptor binding and antimicrobial activities of some Malaysian plants and isolated compounds in our ongoing screening programme (CitationChung et al., 2004, Citation2005a, Citation2005b, Citation2006, Citation2008). In the current study we evaluated LOX inhibiting activity of Malaysian plants using a microplate-based ferric oxidation of xylenol orange (FOX) assay. The aim was to qualitatively identify plants that exhibit significant LOX inhibiting activity for further bioassay-guided isolation of the active constituents. Soy LOX containing mainly LOX-1 was used as an in vitro biochemical model, since it resembles human LOX in its substrate specificity and inhibition characteristics (CitationBoyington et al., 1993; CitationMahesha et al., 2007; CitationPrigge et al., 1997).
Materials and methods
Materials
Soybean lipoxygenase (LOX) (type V), linoleic acid, xylenol orange (XO), indomethacin and phenidone were purchased from Sigma Chemicals (St. Louis, MO). 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (Sc-560) was obtained from Cayman Chemicals (Ann Arbor, MI). Ferrous sulfate, hydrogen peroxide and potassium permanganate were from BDH (Poole, Dorset). Dimethyl sulfoxide (DMSO), absolute ethanol, methanol and sulfuric acid were supplied by Fisher Scientific (Loughborough, Leicestershire).
Plant samples and extracts
A total of 160 plant samples were collected from the state of Sabah, Malaysia. The voucher specimens were kept at the Sandakan (SAN) Herbarium, Forest Research Center, Sepilok, Sandakan, Sabah, Malaysia. The identifiers for the voucher specimens are with the prefix “SAN” follows by the voucher numbers in . The plant samples were dried, ground, and macerated (100-200 g) with sufficient methanol in conical flasks for 7 days with sonication (2 x 30 min). Methanol extracts were collected and filtered at 48, 96, and 168 h, and the conical flasks with plant materials were replaced with fresh methanol to continue extraction. The pooled extracts were evaporated at 50°C in vacuo and the residues freeze-dried and stored in glass vials at -20°C until use.
Plant extracts were made up as 50 mg/mL stock solutions in DMSO, and serially diluted in 50 mM Tris HCl buffer, pH 7.4. A final DMSO concentration of 0.2% v/v was achieved by adding 20 μL of the diluted plant extracts (final concentration 100 μg/mL) to each well of the 96-well microplate. For the determination of IC50 values, the plant extracts were tested at 2-128 μg/mL (final concentration). Control and blank wells contained an equivalent concentration of DMSO.
Lipoxygenase assay
The 96-well microplate based-FOX assay was carried out according to the method described by CitationWaslidge and Hayes (1995), with minor modifications. An aliquot of 50 μL LOX in 50 mM Tris HCl buffer, pH 7.4 (final concentration, 100 ng protein/mL), was pre-incubated with 20 μL test sample (plant extracts or standard inhibitor) in each well of the 96-well microplate at 25°C for 5 min. For the control, 50 μL of LOX solution and 20 μL of buffer containing 0.2% v/v DMSO (final concentration) were pipetted into the wells. Blanks (background) contained the enzyme LOX during incubation, but the substrate (linoleic acid) was added after the FOX reagent.
The reaction was initiated by the addition of 50 μL linoleic acid (final concentration, 140 μM) in 50 mM Tris HCl buffer, pH 7.4, and the reaction mixture was incubated at 25°C for 20 min in the dark. The final concentrations of LOX and linoleic acid were based on a total volume of 120 μL for the reaction mixture. The assay was terminated by the addition of 100 μL freshly prepared FOX reagent: sulfuric acid (30 mM), xylenol orange (100 μM), iron (II) sulfate (100 μM), methanol/water (9:1). After termination, the Fe3+–dye complex was allowed to develop for 30 min at 25°C before being measured at 560 nm on a microplate reader (Anthos Labtec HT3, Lagerhausstrassee, Salzburg, Austria).
Data analysis
All data points were measured in triplicate and the results are presented as mean ± SD. z’ factor analysis, originally described by CitationZhang et al. (1999) to evaluate the quality of screening assays, was performed on replicate control and blank wells. To calculate the percentage of lipoxygenase inhibition in the presence of the test extracts, a standard data reduction algorithm was used as shown below:
where Abscontrol = absorbance of control well, Absbackground = absorbance of background (blank) well, and Abssample = absorbance of sample well.
Results and discussion
The assay used in this study measures the conversion of linoleic acid to linoleic hydroperoxide in the presence of LOX. The linoleic hydroperoxide then oxidizes Fe2+ in the FOX reagent to Fe3+ ions, which then interact with the acidified xylenol orange to give a colored Fe3+–dye complex that absorbs light at 560 nm. Any extracts containing inhibitors of LOX will reduce the formation of the Fe3+–dye complexes, providing a rapid, colorimetric assay for high-throughput screening of plant samples.
The sensitivity of FOX reagent towards linoleic hydroperoxide generated from linoleic acid by the action of LOX was evaluated by replacing the substrate linoleic acid with hydrogen peroxide. The results indicated the reaction was linear between 0–20 μM hydrogen peroxide (r2 = 0.990), after which it became saturated, where this was in agreement with the findings of CitationJiang et al. (1992) and CitationSödergren et al. (1998).
The Michaelis-Menten constant (Km) of the reaction of LOX with various concentrations of linoleic acid was estimated to be 41 μM at 25°C, which was higher than that reported by CitationAxelrod et al. (1981) and CitationHuang et al. (2006) (12 μM) for soy LOX-1. The reaction of 140 μM linoleic acid in the presence of LOX (100 ng protein/mL) gave a linear response up to 20 min. Thus, 100 ng protein/mL LOX and 20 min of incubation time for LOX/linoleic acid with or without plant extracts were selected to give an optimal signal-to-background ratio. The IC50 for phenidone (positive control) was 4.3 μM, which was similar to the values reported by CitationHlasta et al. (1991) (0.48 μM) and CitationKingston (1981) (15 μM). Both indomethacin and Sc-560 (COX inhibitors; negative control) gave less than 2.4% inhibition over the 0-20 μM concentration range.
Intraplate variations of signal and background were determined from 48 data points each, the values were 0.70 ± 0.015 (mean ± SD) (%CV, 2.1) and 0.07 ± 0.004 (%CV, 5.4), respectively, to give a signal-to-background ratio of 9.5. Interplate variations of signal and background were analyzed from 6 data points per microplate of a total of 8 microplates. The %CV of the signal and background were 4% and 5.4%, respectively. The results clearly show intraplate and interplate variations that are minimal and acceptable for the purpose of high-throughput screening (HTS), and this is further supported by the z’ factor analysis. Assays with a z’ factor between 0.5 and 1 are considered to be reliable, robust, and suitable for HTS. The z’ factor of 0.91 indicates the assay adopted is suitable for HTS purposes (CitationZhang et al., 1999).
In the preliminary screen, the lipoxygenase inhibiting effects of 160 plant extracts were tested at 100 μg/mL, and the results are as depicted in . Of these plant extracts, 2.5% (four plant extracts) were classified as highly active against lipoxygenase (more than 70% inhibition), 15.6% gave moderate activity (41-70% inhibition), and 81.9% exhibited low or insignificant activity (0-40% inhibition). This hit rate is similar to our earlier report on central nervous system receptor binding activities of some Malaysian plants, but it is higher than the hit rate of 0.1-0.5% from a typical HTS screening program (Chung et al., Citation2005a, Citation2006). The higher hit rate is probably due to the phylogenetic approach adopted in the collection of the plant samples.
Table 1. Percentage inhibition of lipoxygenase activity by plant extracts (100 μg/mL).
The highly active extracts (more than 70% inhibition) were tested between 0-128 μg/mL in the confirmation assay, and they showed a concentration-dependent inhibition of lipoxygenase activity. The active plants are Agelaea borneensis Merr. (Connaraceae) (bark) (IC50, 1.6 μg/mL), Chisocheton polyandrus Merr. (Meliaceae) (bark) (3 μg/mL), Garcinia cuspidata King (Guttiferae) (bark) (28.3 μg/mL), and Timonius flavescens Baker (Rubiaceae) (leaf) (8.9 μg/mL). These IC50 values are similar to those of other LOX active crude extracts in the range of 1-100 μg/mL (CitationSchneider & Bucar, 2005).
Our preliminary chemical studies and the literature indicate that these plants or closely related species contain coumarins, flavonoids and terpenoids, and that some have been reported to show anti-inflammatory activity. For example, both anti-inflammatory flavones (tricin) and coumarins (dicoumarol, 4-hydroxycoumarin) have been found in other Agelaea species (CitationHidenori et al., 2003; CitationLiu et al., 1998; CitationRoos et al., 1997; CitationVickery & Vickery, 1980). Therefore, similar compounds might be present in A. borneensis. Although there are no phytochemical or biological investigations reported on C. polyandrus, chemical compounds have been isolated from other Chisocheton species. These include limonoids (CitationGunning et al., 1994; CitationInada et al., 1993; CitationYadav et al., 1999), meliacins (CitationBordoloi et al., 1993; CitationSaikia et al., 1978), tetranotriterpenoids (CitationChatterjee et al., 1989; CitationConnolly, et al., 1979), and triterpenoids (CitationInada et al., 1993). Many of these triterpenes have been reported to exert anti- inflammatory effects (CitationPatočka, 2003; CitationScholz et al., 2004).
Garcinia species have been reported to contain phloroglucinols (garcinielliptones A, B, C, D, F, H, I, K, L & M), terpenoids (garcinielliptone E, G, J, N & O), xanthones (mangostins), and their derivatives, and some have been shown to possess anti-inflammatory effects (CitationGopalakrishnan et al., 1980; CitationShankaranarayan et al., 1979; Weng et al., Citation2003a, Citation2003b, Citation2004). The pericarp of G. mangostana containing high levels of xanthones has been shown to be highly effective in scavenging free radicals and suppressing the production of pro- inflammatory cytokines (CitationChomnawang et al., 2007). Thus, it is conceivable that the inhibition on LOX exhibited by G. cuspidata is due to the presence of these compounds. Although phytochemical and biological investigations on T. flavescens have not been reported, numerous chemical compounds have been isolated from other Timonius species. These include triterpenes and alkaloids (CitationErdelmeier et al., 1994; CitationJohns & Lamberton, 1970; CitationKhan et al., 1993).
In summary, the modified FOX assay to determine LOX inhibiting activity on 160 plant extracts covering over 30 families of the Malaysian flora has successfully identified four active plant extracts. These active plants are A. borneensis Merr. (bark), C. polyandrus Merr. (bark), G. cuspidata King (bark), and T. flavescens Baker (leaf). The LOX inhibiting effects of these plant extracts were concentration-dependent, giving IC50 values of 1.6-28.3 μg/mL. These active plants have now been selected for further testing and bioassay-guided fractionation to identify active constituents.
Acknowledgements
This project was partially supported by the Biotechnology Directorate, Ministry of Science, Technology and Innovation, Malaysia (IRPA 26-02-06-0127).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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