Inhibitory Effects of Acetylmelodorinol, Chrysin and Polycarpol from Mitrella kentii on Prostaglandin E2 and Thromboxane B2 Production and Platelet Activating Factor Receptor Binding

Acetylmelodorinol, chrysin and polycarpol, together with benzoic acid, benzoquinone and stigmasterol were isolated from the leaves of Mitrella kentii (Bl.) Miq. The compounds were evaluated for their ability to inhibit prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) production in human whole blood using a radioimmunoassay technique. Their inhibitory effect on platelet activating factor (PAF) receptor binding to rabbit platelet was determined using 3H-PAF as a ligand. Among the compounds tested, chrysin showed a strong dose-dependent inhibitory activity on PGE2 production (IC50 value of 25.5 µM), which might be due to direct inhibition of cyclooxygenase-2 (COX-2) enzymatic activity. Polycarpol, acetylmelodorinol and stigmasterol exhibited significant and concentration-dependent inhibitory effects on TXB2 production with IC50 values of 15.6, 19.1 and 19.4 µM, respectively, suggesting that they strongly inhibited COX-1 activity. Polycarpol and acetylmelodorinol showed strong dose-dependent inhibitory effects on PAF receptor binding with IC50 values of 24.3 and 24.5 µM, respectively.


Introduction
The hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs), is catalyzed by phospholipase A 2 (PLA 2 ). Prostaglandin E 2 (PGE 2 ), a metabolite of AA through the cyclooxygenase-2 (COX-2) pathway, has received great attention because of its role and contribution to inflammation. It is also involved in a variety of other functions, such as vasodilation, febrile responses, and altered microvascular permeability [1]. Thromboxane A 2 (TXA 2 ) is an oxidation product derived from AA in cyclooxygenase and thromboxane synthase dependent reactions. TXA 2 is rather unstable and is rapidly hydrolyzed into the almost inactive, stable and more measurable metabolite thromboxane B 2 (TXB 2 ) [2]. The determination of serum TXB 2 production by platelets following blood coagulation is a specific and most common method for evaluation of COX-1 activity in human and other species [3]. TXA 2 is a vasoconstrictor and a promoter of platelet aggregation and plays an important role in the maintenance of vascular homeostasis. TXA 2 has been postulated to be a mediator contributing to the pathophysiology of several of disease processes such as thrombosis, atheroscleoris and myocardial ischemia [4]. The radioimmunoassay technique (RIA) is the most common way to quantify PGE 2 and TXB 2 in plasma as it is able to detect and quantify the antigen-antibody interaction [5].
PAF is a potent glycerophospholipid mediator, involved in several pathophysiological conditions such as inflammation [6], allergy [7], asthma [8] and thrombosis [9]. Specific receptors for PAF have been reported in a variety of cell membranes including those from platelets [10]. Therefore, compounds which inhibit the specific binding between PAF and receptors may be used as leads in the development of therapeutic agents in a variety of inflammation, respiratory, immunological and cardiovascular disorders [11]. It has been reported that the synthesis of both PAF and prostanoids shares common metabolic pathways, but the relationships between them have not been defined extensively [12]. Some authors have suggested that TXA 2 can be the mediator of the PAF effects in some pathophysiologic conditions [13]. In addition, PAF induce an increased PGE 2 synthesis in cultured rat mesangial cells. Moreover, they suggested an intermediate PAF role in the prostanoids secondary production [14].
Mitrella kentii (Bl.) Miq. is a tree-climbing liana belonging to the custard apple family called Annonaceae. Its synonyms are Melodorum pisocarpum and Me. elegans, while the common (Indonesian) name is 'kiawi'. The plant is found in the tropics, especially in the Asia-Pacific regions. It is used traditionally in Malaysia as a drink in the form of a root decoction to treat fever [15]. Earlier phytochemical investigation on the bark of the plant had reported the isolation of four alkaloids, namely liriodenine, anonaine, and asimilobinem and aequaline [16]. Terpenylated dihydrochalcones: (−)-neolinderatin, (−)-linderatin and 2',6'-dihydroxy-4' methoxydihydrochalcone, and (+)-catechin have also been identified in the stem bark of M. kentii [17].
In our screening study to identify compounds from tropical plants as potential anti-inflammatory agents, we observed that the methanol extract of the leaves of M. kentii showed strong inhibitory effects on PGE 2 and TXB 2 production in human whole blood (>50.0% inhibition) and PAF receptor binding to rabbit platelets (>60% inhibition). In this paper, we report on the isolation of acetylmelodorinol, chrysin and polycarpol, together with benzoic acid, benzoquinone and stigmasterol, from this plant and their ability to inhibit COX-1 and COX-2 activities through inhibition of the production of TXB 2 and PGE 2 in human whole blood and displacement of 3 H-PAF-specific binding in washed rabbit platelets.

Isolation and Identification of Compounds
In this study, six compounds have been isolated from the leaves of Mitrella kentii by chromatographic techniques. The compounds were identified as benzoic acid, acetylmelodorinol [18], chrysin [19], benzoquinone [20], stigmasterol [21] and polycarpol [18] by comparing their physicochemical and spectroscopic properties with literature values, in addition to their 2D NMR spectra and ESI-MS spectra (See Figure 1 for the structures of acetylmelodorinol, chrysin and polycarpol). Alkaloids and terpenylated dihydrochalcones reportedly identified in the bark of the plant in previous studies were not found in the leaves of the plant [16,17].

Inhibition of Production of PGE 2 and TXB 2
The cell viability test carried out to evaluate the cytotoxicity of the compounds on the blood cells at 1.25 and 10.0 µg/mL indicated that the blood cells were viable (>95%) after 24 h incubation. The compounds were investigated for their inhibitory effects on production of PGE 2 and TXB 2 in whole blood at 10 µg/mL. Compounds which showed strong inhibitory activity (>50% inhibition) were subsequently tested at serial concentrations for determination of their IC 50 values. Amongst the compounds tested, chrysin exhibited the strongest inhibitory effect on the production of PGE 2 (63.6% inhibition) induced by lipopolysaccharide (LPS) in whole blood (Table 1). Chrysin was further investigated at various concentrations and it showed dose-dependent response, i.e., as the concentration of the compound increased, the percentage inhibition increased (Table 2). However, the IC 50 value of chrysin (25.5 µM) was higher than that of indomethacin (9.5 µM), a potent cyclooxygenase inhibitor [22].
The inhibition of PGE 2 production might be due to direct inhibition of cyclooxygenase-2 (COX-2) enzymatic activity, possibly by the anti-oxidant effects of the compound or related to its capacity to bind COX-2 active site. However, phospholipase A 2 (PLA 2 ) may also be as a potential target for the compound. Table 1. Percentage inhibition (%) of compounds isolated from Mitrella kentii at 10 µg/mL on PGE 2 production in human whole blood induced by LPS. Values are presented as mean ± SD (n = 3); * P < 0.05 as compared with control. Values are presented as mean ± SD (n = 3); IC 50 values in µM are presented in parentheses.

Compound % Inhibition
In the TXB 2 assay, acetylmelodorinol displayed the highest percentage inhibition (61.5%), followed by polycarpol (60.6%), stigmasterol (54.3%) and crysin (51.2%) ( Table 3). The percentage inhibition of the compounds at different concentrations is shown in Table 4. The results showed that the compounds inhibited TXB 2 production in human whole blood in a dose-dependent manner, i.e., as the concentrations of the compounds increased the percentages of inhibition increased ( Table 4). The IC 50 values for polycarpol, acetylmelodorinol, stigmasterol and crysin were 15.6, 19.2, 19.4 and 39.3 µM, respectively. The IC 50 value of polycarpol was comparable to that of indomethacin (12.8 µM). Since the measurement of serum TXB 2 production by the platelets following blood coagulation is a specific test for assessment of COX-1 activity, the results suggest that the inhibitory effect of the compounds could be due to direct inhibition of COX-1 enzymatic activity.
Chrysin, a flavone devoid of carboxylic acid funtional group, which showed strong inhibitory effects on PGE 2 and TXB 2 production in human whole blood could be used as a lead compound in the development of COX inhibitors. Chrysin has been reported to have several biological activities including antioxidant, anti-allergic, anti-inflammatory, anticancer, antiestrogenic, hypoglycemic and anxiolytic activities [23]. Of all the compounds tested, polycarpol showed the strongest inhibitory activity on TXB 2 production, reflecting its specific inhibition of COX-1 activity. The butenolide, acetylmelodorinol, which was the major compound in M. kentii, also showed significant inhibitory activity on TXB 2 production in human whole blood. Stigmasterol which showed moderate effect on TXB 2 production has been reported to possess strong anti-inflammatory activity [24]. In a study conducted by Gomez et al. [25], stigmasterol was found to be more effective as topical anti-inflammatory agent in acute than in chronic processes.

Effect of the Compounds on 3 H-PAF Receptor Binding
The inhibitory effect of the compounds on PAF receptor binding to rabbit platelets at the concentration of 18.2 µg/mL is shown in Figure 2. Among the compounds studied, acetylmelodorinol and polycarpol showed significant inhibitory effects on the PAF receptor binding with percentages of inhibition of 70.3 and 57.3%, respectively. The percentage inhibition of acetylmelodorinol was comparable to that of the positive control, cedrol (78.8%), a known PAF antagonist from natural sources [26]. The inhibitory effects of the compounds were then evaluated at various concentrations and the IC 50 values of acetylmelodorinol and polycarpol were determined by probit analysis as 24.5 and 24.3 µM, respectively (Table 5). Acetylmelodorinol and polycarpol showed relatively strong PAF antagonistic activity and their dose-dependent responses are shown in Figure 3. The compounds inhibited the specific binding between PAF and receptors and thus they may be used as leads in the development of therapeutic agents in a variety of inflammation, respiratory, immunological and cardiovascular disorders [27]. Polycarpol has been shown to exhibit interesting antitrypanosomal activity but its anti-inflammatory effect has not been reported [28]. Acetylmelodorinol has been reported to exhibit general antiproliferative activity toward tumor cells [18].

Plant Material
Fresh leaves of Mitrella kentii were collected from the Angsi mountain forest in Negeri Sembilan, Malaysia in July 2008 and identified by Dr Kamarudin Mat Salleh, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM). A voucher specimen (AZ 69) was deposited at the Herbarium of Faculty of Science and Technology, UKM.

Extraction and Isolation of Compounds
Dried ground leaves of M. kentii (1,000 g) were extracted successively with hexane (3 × 2.5 L, 24 h each), ethyl acetate (3 × 2.5 L, 24 h each) and methanol (3 × 2.5 L, 24 h each) using maceration technique. The solvents were then evaporated using a rotatory evaporator to yield hexane (14.9 g, 1.5%), ethyl acetate (29.5 g, 3.0%) and methanol (67.8 g, 6.8%) extracts, respectively. The extracts were subjected to column chromatography (CC) on silica gel 60 (230-400 mesh), eluted with a gradient system of n-hexane, ethyl acetate and methanol to afford six known compounds. The hexane extract yielded stigmasterol (a white amorphous solid, 52.0 mg) and polycarpol (a white amorphous solid, 43.0 mg), the ethyl acetate extract yielded benzoic acid (6.1 mg), acetylmelodorinol (colorless needles, 1.0 g), chrysin (a light yellow powder, 500.0 mg) and benzoquinone (colorless crystals, 2.0 mg), while acetylmelodorinol (54.0 mg) and chrysin (35.2 mg) were isolated from the methanol extract. The compounds were identified by comparison of their physicochemical and spectroscopic properties with literature values. Purity of the compounds was >95%, based on their physicochemical properties, NMR and ESI-MS data.

Cell Viability
Cell viability was determined by the standard trypan blue exclusion method. The blood cells (1 × 10 6 /mL) were incubated with 1.25 and 10.0 µg/mL of compounds, each in triplicate at room temperature for 24 h. The blue dye uptake was an indication of cell death. The percentage viability was calculated from the total cell counts.

Radioimmunoassay for Prostaglandin E 2 (PGE 2 ) and Thromboxane B 2 (TXB 2 )
Radioimmunoassay was carried out to determine the levels of PGE 2 and TXB 2 productions by blood cells following incubation with compounds and coagulation according to the modified method of Patrignani et al. [29]. The use of human blood was approved by the Ethics Committee of Universiti Kebangsaan Malaysia (UKM) (approval no. FF-168-2007). Radioimmunoassay procedures were carried out in triplicate for each compound.

Preparation of Standards
A series of concentrations of PGE 2 and TXB 2 standards were prepared, ranging from 2.45-240 and 2.05-500 pg/0.1 mL, respectively. One hundred µL of PGE 2 standard solution was added to 100 µL of anti-PGE 2 and 100 µL of [ 3 H]-PGE 2 . Meanwhile, 100 µL of TXB 2 standard solution was added to 100 µL of anti-TXB 2 and 100 µL of [ 3 H] -TXB 2 . The mixtures were incubated at 4 °C for 18-24 h. After incubation, the mixtures were added with 200 µL of dextran charcoal and were incubated again for 10 min. After centrifugation at 2000× g for 15 min at 4 °C, 3 mL of liquid scintillation cocktail was added to 300 µL of supernatant. The radioactivity was measured by a liquid scintillation counter.

Prostaglandin E 2 Radioimmunoassay
Briefly, venous blood was obtained in polypropylene tube containing 10% (v/v) of 2% EDTA by aseptic vein puncture from healthy human volunteers who fulfilled the following inclusion criteria: non-smoker, fasted overnight and did not take any medicine or supplements within the last two weeks. One mL of blood was incubated at 37 °C for 24 h with 10 µL of LPS and 10 µL of serial dilutions of each compound in DMSO and ethanol (1:1 ratio) (1.25-10 µg/mL) or control. DMSO and ethanol (1:1 ratio) was used as a negative control and indomethacin, a known cyclooxygenase inhibitor was used as a positive control. After incubation, the blood was centrifuged at 2,000 × g for 10 min at 4 °C to separate the plasma. The reaction mixtures consisted of 100 µL of plasma, 100 µL of anti-PGE 2 and 100 µL of [ 3 H]-PGE 2 were incubated at 4 °C for 18-24 h. After incubation, the mixtures were added with 200 µL of dextran charcoal and were incubated again for 10 min. The final concentrations of the samples in the mixture were 10.0, 5.0, 2.5 and 1.25 µg/mL. After centrifugation at 3,000 × g for 15 min at 4 °C, 3 mL of liquid scintillation cocktail was added to 300 µL of supernatant. The radioactivity was measured by a liquid scintillation counter.

Thromboxane B 2 Radioimmunoassay
Thromboxane B 2 assay was carried out similarly to the PGE 2 assay. In this assay, 1 mL of blood mixed with 10 µL of serial dilutions of each compound in DMSO and ethanol (1:1 ratio) (1.25-10 µg/mL) or control was allowed to clot for 60 min at 37 °C. DMSO and ethanol (1:1 ratio) was used as a negative control and indomethacin, a known cyclooxygenase inhibitor was used as a positive control. The blood was centrifuged at 2,000 × g for 10 min at 4 °C to separate the serum as supernatant. The reaction mixtures consisted of 100 µL of serum, 100 µL of anti-TXB 2 and 100 µL of [ 3 H]-TXB 2 were incubated at 4 °C for 18-24 h. After incubation, the mixtures were added with 200 µL of dextran charcoal and were incubated again for 10 min. The final concentrations of the samples in the mixture were 10.0, 5.0, 2.5 and 1.25 µg/mL. After centrifugation at 3,000 × g for 15 min at 4 °C, 3 mL of liquid scintillation cocktail was added to 300 µL of supernatant. The radioactivity was measured by liquid scintillation counter.

Calculation of PGE 2 and TXB 2 Concentrations
The readings obtained for each set of triplicate were averaged. The net counts for all standards and samples were calculated by substracting the value of the antibody binding to the antigen in the sample (Bx) with non specific binding (Nc). The normalized percentage bound (% B/Bo) for each standard and sample (Bx) were calculated as follows: The % B/Bo for each standard versus the corresponding picogram (pg) concentration of PGE 2 and TXB 2 were plotted using semi-logarithmic graph. The concentrations of PGE 2 and TXB 2 in each sample were determined by interpolation from the standard curve. Percentage inhibition of samples was obtained as follows: % 100 2 2 2 2 100

PAF Receptor Binding Inhibitory Assay (PAF Assay)
The assay was carried out according to the method as described previously by Jantan et al. [30]. 3 µg/mL. Cedrol, a known PAF antagonist was used as a positive control 0.1% DMSO in saline was used as a control. The final concentration of DMSO in the reaction mixture was fixed at 0.1% to avoid interference with the receptor binding studies. The reaction mixture was incubated at room temperature for 1 h. The free and bound ligands were separated by a filtration technique using Whatman GF/C glass fiber filters. The radioactivity was measured by a scintillation counter. The difference between total amounts of bound 3 H-PAF in the absence and the presence of excess unlabeled PAF is defined as specific binding of the radiolabeled ligand. The IC 50 values of the compounds were obtained from at least three independent determinations. Percentage inhibition of the sample was obtained by the following equation:

% 100
Tc = Total binding of control; Ts = Total binding of sample; Nc = Nonspecific binding of control; Ns = Nonspecific binding of sample

Statistical Analysis
All the data were analysed using the Statistical Package for Social Sciences (SPSS) software. Each sample was measured in triplicate and the data are presented as means ± standard deviation (SD). Probit programme was used to determine the IC 50 values for the active pure compounds. The values were obtained from at least three determinations. Data were analysed using one way ANOVA. P < 0.05 was considered to be statistically significant.

Conclusions
Among the compounds isolated from the leaves of Mitrella kentii, chrysin showed a potent dose-dependent inhibitory activity on PGE 2 production in human blood induced by LPS, indicating that it might directly inhibit COX-2 enzymatic activity. The results of this study indicate that the strong inhibitory activity of M. kentii on TXB 2 production was due to polycarpol, acetylmelodorinol and stigmasterol which exhibited significant and concentration-dependent inhibitory effect on COX-1 activity. The strong PAF antagonistic effect of the plant was contributed by polycarpol and acetylmelodorinol which showed strong and dose-dependent inhibitory effects on PAF receptor binding. Acetylmelodorinol, chrysin and polycarpol have the potential to be used as lead structures for the development of potent anti-inflammatory agents. However, further studies need to be carried out to study their mechanisms of anti-inflammatory actions such as the cell free assays for COX enzyme activity and to find derivatives with maximum inhibitory effects.