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Phytochemical Constituents from the Aerial Part of Ducrosia ismaelis Asch.
Phytochemical Constituents from the Aerial Part of Ducrosia ismaelis Asch.
Natural Product Sciences. 2015. Mar, 21(1): 6-13
Copyright © 2015, The Korean Society of Pharmacognosy
  • Received : September 05, 2014
  • Accepted : September 25, 2014
  • Published : March 31, 2015
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About the Authors
Abubaker M. A. Morgan
College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea
Jang Hoon Kim
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
Hyun Woo Lee
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
Sang-Hyun Lee
Department of Food and Nutrition, Hannam University, Daejeon 305-811, Korea
Chi-Hwan Lim
College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea
Hae-Dong Jang
Department of Food and Nutrition, Hannam University, Daejeon 305-811, Korea
haedong@hnu.kr
Young Ho Kim
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
yhk@cnu.ac.kr

Abstract
Phytochemical investigation of the aerial components of Ducrosia ismaelis Asch. led to the isolation of six known compounds, psoralen ( 1 ), isopsoralen ( 2 ), cnidioside A ( 3 ), (−)-syringaresinol- O - β -D-glucopyranoside ( 4 ), ( E )-plicatin B ( 5 ), trilinolein ( 6 ). The chemical structures of these compounds were elucidated from spectroscopic data and by comparison of these data with previously published results. The antioxidant, anti-osteoporotic and cardiovascular related activities of the isolated compounds were assessed using oxygen radical absorbance capacity (ORAC), reducing capacity, tartrate-resistant acid phosphatase (TRAP), and soluble epoxide hydrolase (sEH) inhibitory activity assays. Compounds ( 3-5 ) showed potent peroxyl radical-scavenging capacities with ORAC values of 11.06 ± 0.39, 7.98 ± 0.10, and 13.99 ± 0.06 Trolox equivalent (TE) at concentrations of 10 μM, respectively. Only compounds 4 and 5 was able to significantly reduce Cu 2+ ions, with a reduction value of 9.06 ± 0.32 and 4.61 ± 0.00 μM Trolox Equivalent (TE) at a concentration of 10 μM. Compound 5 at 10 μM exhibited a potent inhibitory effect on osteoclastic TRAP activity with a TRAP value of 86.05 ± 6.55% of the control. Compounds 1 , 3 and 5 potently inhibited sEH activity with IC 50 values of 41.6 4.9, 16.0 1.1, and 49.0 5.7 μM, respectively.
Keywords
Introduction
Oxidative stress arises when the production of reactive oxygen species (ROS) overwhelms the intrinsic anti-oxidant defences. Reactive oxygen species play important roles as second messengers in many intracellular signalling cascades aimed at maintaining the cell in homeostasis with its immediate environment. 1 Severe oxidative stress has been implicated in many chronic and degenerative diseases, including osteoporosis, cancer, ageing, and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. 2 Oxidative stress is enhanced in hypertension, atherosclerosis, and other forms of cardiovascular disease and participates in the mechanisms of vascular injury. 3 There are several methods that have been developed to estimate the antioxidant capacity of phytochemicals in foods. The most popular and most well characterised method is the peroxyl radical scavenging assay. 4 , 5 In addition, reducing capacity is also a popular assay, frequently used for measuring the electrondonating capacity of food extracts or antioxidants. 4 , 6
Osteoporosis, one of the most frequently encountered degenerative diseases in ageing communities, is characteristic by a decrease in bone mass and density, causing bones to become fragile and more prone to fracture. 2 Bone formation is related to osteoblastic proliferation, alkaline phosphatase (ALP) activity, and osteocalcin and collage synthesis. Bone resorption is associated with osteoclast formation and differentiation, and tartrateresistant acid phosphatase activity (TRAP). 7
In mammals, epoxides of arachidonic acids (called epoxyeicosatrienoic acids or EETs) and of other fatty acids are important lipid mediators that have key roles in the regulation of hypertension, inflammation, and other cardiovascular-related diseases as well as in modulating both inflammatory and neuropathic pain. 8 However, endogenous metabolism of these epoxy-fatty acids to their corresponding hydrated products by soluble epoxide hydrolase (sEH EC 3.3.2.10) generally reduces these biological activities. 9 , 10 Both in vitro and in vivo studies have demonstrated that the anti-hypertensive and cardio protective effects mediated by the EETs are inversely dependent on the extent of sEH hydrolysis of the EETs. 10 , 11 Previous report explained for the first time that EETs increase antioxidant enzyme expression and activity in cancer cells. This effect enables tumor cells to resist the oxidative damage and the subsequent apoptosis induced by arsenic trioxide (ATO). 12 Thus, maintaining the in vivo concentration of EETs through sEH inhibition is a promising therapeutic pathway to treat cardiovascular and other diseases. 8
Ducrosia ismaelis (commonly known as Haza or Geshea) belong to the Apiaceae family, is a perennial, herbaceous, and branched plant with a characteristic aromatic odor. 7 The whole herb, especially its aerial parts, has been used in traditional medicines to treat skin infections and to repel insects and reptiles. 13 Previous phytochemical investigation on D. ismaelis led to the isolation of flavonoids, lignans, and phenolic compounds from the methanol extract of the aerial parts. 7 Pharmacological studies on some of these compounds showed antiosteoporotic and antioxidant activities. 7 In a continuation of our phytochemical study on D. ismaelis to provide promising lead compounds for the treatment of osteoporosis and cardiovascular diseases, we inves-tigated the chemical constituents of the D. ismaelis plant and evaluated their biological activities. Compounds 1 - 6 were isolated from the aerial parts of D. ismaelis ( Fig. 1 ). This report details the isolation, structural determination, antioxidant, antiosteoporotic, and inhibitory activity on sEH of these compounds using ORAC, reducing capacity, TRAP and sEH inhibitory activity assays. Dried aerial parts of D. ismaelis were extracted with 100% methanol at room temperature. The crude extract was suspended in water and then successively partitioned with dichloromethane and ethyl acetate to give CH 2 Cl 2 , EtOAc, and water fractions, respectively. Using various types of column chromatography, psoralen ( 1 ), 14 isopsoralen ( 2 ), 15 cnidioside A ( 3 ), 16 (−)-syringaresinol- O - β -D-glucopyranoside ( 4 ), 17 ( E )-plicatin B ( 5 ), 18 trilinolein ( 6 ), 19 were isolated for the first time from D. ismaelis . Their chemical structures ( Fig. 1 ) were elucidated based on 1D and 2D NMR spectra, MS data, and by comparisons with previously published data acquired from similar compounds.
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Chemical structures of isolated compounds (1 - 6).
Experimental
Plant material − Dried aerial parts of D. aismaelis were purchased from a folk medicine market, in Wad-Medani, Gezira state, Central Sudan, in December 2012. The plant material was identified by Professor A. M. Hamdoun, Faculty of Agricultural Sciences, University of Gezira, Sudan. A voucher specimen (CNU12113) was deposited at the herbarium, College of Pharmacy, Chungnam National University.
Extraction and isolation − Dried aerial parts of D. ismaelis . (3.0 kg) were extracted with 100% methanol at room temperature three times. After removal of the solvent under reduced pressure, the crude extract (258.0 g) was dissolved in 1.0 L of H 2 O to form a suspension that was successively partitioned with dichloromethane (CH 2 Cl 2 ), and ethyl acetate (EtOAc), to give CH 2 Cl 2 (72.9 g), EtOAc (8.5 g), and aqueous extract (177.0 g), respectively. CH 2 Cl 2 extract was chromatographed over silica gel (70 - 230 mesh), using n-hexane/EtOAc/MeOH (20 : 1 : 0.1 - 1 : 1 : 0.1 v/v/v) gradually as eluents, to afford fractions (1a - 1f). Repeated silica gel column chromatography of fractions 1b, 1c, 1d, and 1e with n-hexane/EtOAc (5 : 1 - 1 : 1 v/v), to give 6 (70.0 mg), 2 (50.0 mg), 1 (20.0 mg), and 5 (30.0 mg), respectively. EtOAc extract was subjected to column chromatography using silica gel (70 - 230 mesh), eluting with CH 2 Cl 2 /MeOH/H 2 O (20 : 1 : 0.2 - 5 : 1 : 0.1v/v/v) to give five fractions (2a - 2e). Repeated silica gel column chromatography of fraction 2d with CH 2 Cl 2 /MeOH/H 2 O (8 : 1 : 0.1v/v/v), further by YMC column and eluting with MeOH/H 2 O (1 : 1), and (1.5 : 1 v/v) to obtain 3 (25.0mg). Finally aqueous extract was chromatographed on a column of highly porous polymer (Diaion HP-20) and eluted with H 2 O and MeOH, successively to give four fractions (3a-3d). Fraction 3c was chromatographed over silica gel eluting gradually with CHCl 3 /MeOH/H 2 O (12 : 1 : 0.1 - 1 : 1 : 0.1 v/v/v), further by YMC column and eluting with MeOH/Me 2 CO/H 2 O (0.3 : 0.3 : 1 v/v/v), to obtain 4 (15.0 mg).
Psoralen (1) −White amorphous powder, mp 158 - 161 ℃; UV λ max (MeOH) nm: 328.0, 290.0, 244.0; FT-IR (KBr): ν max 2925.6, 2359.4, 1722.8, 1575.7, 1284.4, 823.9, 748.9 cm −1 ; 1 H and 13 C NMR data were in accordance with previous reported data; 14 ESI-MS m/z 187 [M + H] + .
Isopsoralen (2) − Yellow amorphous powder, mp 137 - 140 ℃; UV λ max (MeOH) nm: 297.0, 246.0; FT-IR (KBr): ν max 2921.0, 2364.6, 1736.4, 1613.6, 1113.4, 829.5, 746.3 cm −1 ; 1 H and 13 C NMR data were in accordance with previous reported data; 15 ESI-MS m/z 187 [M + H] + .
Cnidioside A (3) − White amorphous powder,
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= −29.4 ( c 0.05, MeOH); UV λ max (MeOH) nm: 285.0, 244.0; FT-IR (KBr): ν max 3396.1, 2925.0, 1711.6, 1467.9, 1294.8, 1073.4 cm −1 ; 1 H and 13 C NMR data were in accordance with previous reported data; 16 ESI-MS m/z 369 [M + H] + .
(−)-Syringaresinol-O-β-D-glucopyranoside (4) − Colorless needles,
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= −20.0 ( c 0.1, MeOH); UV λ max (MeOH) nm: 269.0, 243.0; FT-IR (KBr): ν max 3448.4, 2924.3, 1560.5, 1119.1 cm −1 ; 1 H and 13 C NMR data were in accordance with previous reported data; 17 EI-MS m/z 603 [M + Na] + .
(E)-Plicatin B (5) −White amorphous powder; mp 83 - 88℃
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= +4.84 ( c 0.05 MeOH); FT-IR (KBr): ν max 3404.2, 2924.0, 1700.4, 1624.8, 1198.5, 831.5 cm −1 ; 1 H and 13 C NMR data were in accordance with previous reported data; 18 HR-ESI-Q-TOF MS m/z 269.1164 [M+Na] + (calcd for C 15 H 18 O 3 Na, 269.1148).
Trilinolein (6) − Yellow pale; mp 248 - 250 ℃; FT-IR (KBr): ν max 1747.5, 1461.7, 1377.8, 1159.1 cm −1 ; 1 H and 13 C NMR data were in accordance with previous reported data; 19 EI-MS m/z 879.8 [M] + .
Oxygen radical absorbance capacity (ORAC) assay − ORAC assay, was carried out using a Tecan GENios multifunctional plate reader (Salzburg, Austria) with fluorescent filters (excitation wavelength: 485 nm, emission filter: 535 nm). In the final assay mixture, fluorescein (40 nM) was used as a target of free radical attack with AAPH (20 mM) as a peroxyl radical generator in the peroxyl radical-scavenging capacity assay. The analyzer was programmed to record fluorescein fluorescence every 2 min after AAPH had been added. All fluorescence measurements were expressed relative to the initial reading. Final values were calculated based on the difference in the area under the fluorescence decay curve between the blank and test sample. All data are expressed as net protection area (net area). Trolox (1 μM) was used as the positive control to scavenge peroxyl radicals. 20
Reducing capacity assay − The electron-donating capacities of extracted compounds ( 1 - 6 ) to reduce Cu 2+ to Cu + were assessed according to the method of Aruoma et al. 2 Forty microliters of different concentrations of compounds dissolved in ethanol were mixed with 160 μL of a mixture containing 0.5 mM CuCl 2 and 0.75 mM neocuproine, a Cu + specific chelator, in10 mM phosphate buffer. Absorbance was measured using a microplate reader at 454 nm for 1 h. Increased absorbance of the reaction mixture indicated greater reducing power. 20
Tartrate-resistant acid phosphatase (TRAP) Assay − RAW 264.7 cells were seeded in 96-well plates (1 × 10 4 cells/well) containing DMEM medium plus 10% FBS, and the medium was replaced with test samples in differentiation medium containing 50 ng/mL RANKL. The differentiation medium was changed every 2 days. After 5 days, the medium was removed, and the cell monolayer was gently washed twice using ice-cold PBS. The cells were fixed in 3.5% formaldehyde for 10 min and ethanol-acetone (1 : 1) for 1 min. Subsequently, the dried cells were incubated in 50 mM citrate buffer (pH 4.5) containing 10 mM sodium tartrate and 6 mM PNPP. After 1 h incubation, the reaction mixtures were transferred to new well plates containing an equal volume of 0.1 N NaOH. Absorbance was measured at 405 nm using an enzyme-linked immunoassay reader, and TRAP activity was expressed as the percent of the untreated control. 20
sEH inhibitory activity assay − The sEH inhibitory activity was determined using a hydrolysis reaction of PHOME in the presence or absence of the Inhibitor. The final reaction volume was 200 μL, and contained 25 mM bis-Tris HCl buffer (including 0.1% BSA (bovine serum albumin), pH 7.0), 20 μL of various concentrations of samples, 50 μL sEH enzyme, and 50 μL of 40 μM PHOME, AUDA (IC 50 : 7.2 ± 2.7 nM) was used as the positive control. Reaction systems were incubated at 37 ℃ for 1 h, and fluorescence intensity was then monitored every 2 min (during 1 h) using a Genios microplate reader (Tecan, Mannedorf, Switzerland) at excitation and emission wavelengths of 330 and 465 nm, respectively. sEH inhibitory activity for each sample was calculated as follows:
  • Enzyme inhibition (%) = 100 − [(S−S0) / (C−C0)] × 100
where C is the fluorescence of the control (enzyme, buffer, MeOH, and substrate) after 60 min of incubation, C 0 is the fluorescence of the control at zero time, S is the fluorescence of the tested samples (enzyme, buffer, sample solution, and substrate) after incubation, and S 0 is the fluorescence of the tested samples at zero time. 21 , 22
sEH kinetic assay − To study the kinetics of sEH inhibition by isolated compounds 1 - 3 , various concentrations of compounds 1 - 3 were added in 96 well plate containing 80 μl of 25 m-M bis-Tris HCl buffer (including 0.1% BSA (bovine serum albumin), pH 7.0) with a series of substrate concentrations range of 10 - 80 μM, and fluorescence intensity was monitored at 37 ℃ e-very 2 mins for 20 mins. The Ki value was also derived by plotting slopes obtained from Lineweaver-Burk plots and Dixon plots using SigmaPlot (SPSs Inc., Chicago, IL). 22 , 23
Statistical analysis − All data are presented as means ± standard deviations (SD). Statistical analysis was carried out using the IBM SPSS statistical package (Version 21.0, IBM, New York, USA) program, and the significance of each group was verified with a one-way analysis of variance (ANOVA) followed by Duncan's test. A p value < 0.05 was considered significant.
Results and Discussion
A methanolic extract of the dried aerial parts of D. ismaelis was partitioned with dichloromethane (CH 2 Cl 2 ), and ethyl acetate (EtOAc), successively. Repeated column chromatography on silica gel, C-18, and Diaion HP-20 of the CH 2 Cl 2 , EtOAc, and aqueous soluble fractions led to the isolation of six compounds ( 1 - 6 ) ( Fig. 1 ). To determine the structures of compounds, the combined analyses with a series of 1D and 2D NMR, infrared, and mass spectra were accomplished. In addition, all physical and spectroscopic data obtained in this present study were compared with those of previously published manuscripts.
Structural elucidation of the isolated compounds − Compound 1 ; 1 H-NMR spectrum showed two proton doublets at δ H 6.35 ( J = 9.6 Hz) and δ H 7.78 ( J = 9.6 Hz) characteristic of α -pyrone protons assignable to H-3 and H-4 respectively, and a pair of doublets occurring at δ H 6.82 ( J = 2.0 Hz) and δ H 7.69 ( J = 2.0 Hz) typical of furanic protons assignable to H-3' and H-2' respectively. 24 The spectrum further showed two protons singlet at δ H 7.68 and 7.45 assignable to C-5 and C-8 of the furanocoumarin structure. The 13 C NMR spectrum exhibited 11 carbon resonances including six methines, one carbonyl and four quaternary carbons. This NMR data compared favourably with that published for psoralen ( 1 ). 14 Compound 2 ; Both of the 1 H- and 13 C-NMR spectrum of 2 were similar to those of 1 , except that the spectra of 2 showed a pair of doublets occurring at δ H 7.28 ( J = 8.5 Hz) and δ H 7.33 ( J = 8.5 Hz) assignable to a H-5 and H-6, typical the angular furanocoumarin structure. This NMR data compared favourably with that published for isopsoralen ( 2 ). 15 Compound 3 ; 1 H-NMR and 1 H- 1 H COSY spectrum showed signals due to an anomeric proton at δ H 4.86 (1H, d, J = 7.8 Hz), methylene protons at δ H 2.58 (2H, d, J = 7.3 Hz) and 3.00 (2H, d, J = 7.3 Hz) coupled with each other and four aromatic protons at δ H 6.61(lH, br s), 7.25 (1H, s), 7.28 (1H, br s) and 7.53 (IH, br s), suggesting that 3 is an aromatic glycoside. The 13 C NMR spectrum of 3 gave seventeen carbon signals caused by one β -glucopyranosyl unit with an ether linkage, one carbonyl at δ C 176.48, two methylenes at δ C 26.13 and 34.59, three oxygen-bearing aromatic carbons at δ C 144.61, 153.64 and l54.49, two aromatic carbons at δ C 121.91, 126.08 and three aromatic carbons bearing a proton at δ C 98.44, 105.82 and 120.85. By the comparison of its spectral data with those of literature values, the structure of 3 was identified as cnidioside A. 16 Compound 4 ; 1 H-NMR and 13 C NMR spectrum, indicated the presence of one glucose, two benzene rings, two propane units and four methoxyl groups in the molecule. On the basis of the above spectral evidence, compound 4 was concluded to be (−)-syringaresinol- O - β -D-glucopyranoside. The physiochemical and spectral data were good agreement with those published. 17 Compound 5 ; 1 H NMR spectrum showed an ortho-coupled aromatic protons at δ H 7.35 (1H, d, J = 8.40 Hz) and δ H 6.69 (1H, d, J = 8.40 Hz) and an isolated proton at δ H 7.48 (1H, s). The presence of a trans -methyl cinnamate moiety was shown by a pair of doublets of olefinic protons at δ H 6.76 (1H, d, J = 15.92 Hz) and 5.74 (1H, d, J = 15.92 Hz) and methoxyl group at δ H 3.65 (3H, s). In addition, the spectrum showed the presence of a prenyl group (two methyl group at δ H 1.71 (6H, s); one proton at δ H 5.25 (1H, m) and two protons at δ H 3.27 (2H, d, J = 7.8 Hz). The 13 C NMR spectrum determined in the DEPT mode, showed the presence of 15 carbon atoms with three methyl at δ C 17.96, 25.88 and 51.44, one methylene at δ C 29.78, six methines at δ C 115.44, 116.33, 121.63, 130.15, 132.66 and 143.97, four quaternary carbon atoms at δ C 126.60, 127.32, 134.92 and 156.67), and one carbonyl at δ C 167.27. By the comparison of its spectral data with those of literature values, the structure of 5 was identified as ( E )-plicatin B. 18 Compound 6 ; 1 H NMR spectrum showed methine protons resonated only between δ H 5.28 and 5.37, the methyl groups at δ H 0.9 and the rest of the peaks being methylene protons. The deshielded methylene protons at δ H 4.12 to 4.33 and methine proton at δ H 5.28 suggests protons attached to oxygen bearing carbons, imblecating a glycerol ester type of structure. The olefinic complex multiplet resonating at δ H 5.4 was assigned to the aliphatic double bonds (H-9/10 and 12/13), the doublet of doublets between δ H 4.12 and 4.33 to H-1' (2H) and H-3' (2H) and the deshielded multiplet at δ H 5.28 to the tertiary oxomethine (H-2', C-68.5 ppm). The 13 C NMR spectra showed six double bonds at δ C 127.80, 128.40, 128.93, 129.51, 129.83 and 130.45 and three carbonyl (two at δ C 173.07 and one at δ C 172.71). On the basis of the above spectral evidence, compound 6 was concluded to be trilinolein. The physio-chemical and spectral data were good agreement with those published. 19 , 25
In vitro Antioxidant activities − The antioxidant activities of compounds 1 - 6 were investigated in terms of their peroxyl radical-scavenging and reducing capacity. Based on the chemical reactions involved, major antioxidant capacity assays can be roughly divided into hydrogen atom transfer-based and single electron transfer-based assays. 26 ORAC assay is based on hydrogen atom transfer while reducing capacity assay depends upon single electron transfer. 26 The scavenging activities of compounds 1 - 6 in the presence of peroxyl radicals were shown in Fig. 2 , at dose-dependent manner at 1 - 10 μM. Among them, compounds ( 3 - 5 ) showed potent peroxyl radical-scavenging activities with 11.06 ± 0.39, 7.98 ± 0.10, and 13.99 ± 0.06 Trolox equivalent (TE) at concentrations of 10 μM, respectively. In particular, at a concentration of 10 μM, 5 showed the strongest peroxyl radical-scavenging activity against peroxyl radicals generated from (AAPH). Based on structural analysis of the active isolates ( 3 , 4 and 5 ), the hydroxyl groups on the structure seem to be responsible for peroxyl radical-scavenging activity by donating hydrogen atoms to peroxyl radicals. 2 The ability of the isolated compounds 1 - 6 to stimulate the reduction of copper ions (Cu 2+ to Cu + ) by donating electrons was investigated to determine whether their peroxyl radicalscavenging capacities, could be related to their reduction capacities. As shown in Fig. 3 , only compounds 4 and 5 was able to significantly reduce Cu 2+ ions, with a reduction value of 9.06 ± 0.32 and 4.61 ± 0.00 μM TE at a concentration of 10 μM. Present data suggest that the ability of compounds 4 and 5 to donate hydrogen atoms or electrons to peroxyl radicals and to convert them into relatively stable compounds may contribute to their peroxyl radicalscavenging capacity. Although compound 3 showed potent peroxyl radical-scavenging capacities, it appeared to have lower capacities for reducing Cu 2+ to Cu + compared to compounds 4 and 5 . These data suggest that the hydrogen donating ability of compounds 3 is strong, but that its single electron transferring activity is weak. This may be due to the absence of hydroxyl substituent on the aromatic ring, which results in unfavourable electron transfer to the Cu 2+ ion. This data agrees with the conclusions of previous studies. 2 , 20
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Peroxyl radical-scavenging activity (Trolox Equivalent, μM) of compounds (1 - 6) in ORAC assay.
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Reducing capacities of compounds 1 - 6. The results represent the mean SD of values obtained from three measurements.
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Tartrate-resistant acid phosphatase (% of control) of compounds (1 - 6). C: control, which was not treated; TC: treated control, which was treated with RANKL.
In vitro anti-osteoporotic activities − The anti-osteoporotic activities of compounds 1 - 6 were determined using TRAP assay. TRAP activity was expressed as the percent of the untreated control (mean ± one standard deviation, n = 3). RANKL treatment induced osteoclast formation from RAW 264.7 pre-osteoclast cells and dramatically enhanced TRAP activity up to 170.9%, as shown in Fig. 3 . Compounds 5 , 4 , and 2 suppressed osteoclast formation in a dose-dependent manner with TRAP values of 86.05 ± 6.55, 109.30 ± 9.37, and 110.12 ± 5.15 (% of control, daidzein used as a positive control) at concentrations of 10 μM, respectively. Compound 5 exhibited the highest anti-osteoporotic activity among tested compounds, as indicated by the relatively low TRAP value ( Fig. 2 ). This may be due to the high cell membrane permeability of 5 , resulting from the absence of a glucose unit and the presence of two hydrophobic side chains. 7 When compared both anti-osteoporotic and antioxidant activities of 1 - 6 , the potent TRAP, peroxyl radical scavenging and reducing capacity activities were observed in 4 and 5 among 6 isolated compounds. Therefore, hese results imply that compound 4 and 5 may augment antiosteoporotic activity by directly scavenging intracellular reactive oxygen species produced during RANKL-mediated osteoclast differentiation through donating hydrogen atom or electron. 20
In vitro sEH inhibitory activity − sEH inhibitory activity of isolated compounds 1 - 6 was evaluated in vitro using a fluorescent method based on hydrolysis of the specific substrate PHOME in the presence of sEH. AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid] one of the most effective sEH inhibitors, was used as a positive control (IC 50 : 7.2 ± 2.7 nM). The sEH inhibitory activity was assessed at doses of 50 and 100 μM. At a concentration of 50 μM, isolated compounds showed sEH Inhibition values ranging from 14.2% ( 6 ) to 77.4% ( 3 ) ( Fig. 5 ). Compounds 1 , 3 and 5 inhibited sEH more than 50% and were assessed for further investigation. Effects of those compounds were examined at concentrations from 3.1 to 100 μM, and the 50% inhibitory concentration (IC 50 ) was calculated using a dose-dependent response curve ( Fig. 6 ). Among the 6 isolated compounds, compounds 1 , 3 and 5 exhibited high sEH inhibitory activity; their IC 50 values were 41.6 ± 4.9, 16.0 ± 1.1, and 49.0 ± 5.7 μM, respectively. The enzyme kinetic was measured with a series of substrate concentrations and various inhibitor concentrations. The enzyme inhibition mechanisms of 1 , 3 and 5 were modeled using double-reciprocal plots (Lineweaver-Burk and Dixon plots). The Lineweaver-Burk plot (1/V vs 1/[S]) resulted in a series of straight lines passing through the same point on the x-axis ( Fig. 7 A-C). These ndings indicate that compounds 1 , 3 and 5 exhibit non-competitive inhibition characteristics. The Ki values were calculated from the Dixon plots ( Fig. 7 D-F), which are useful for determining the Ki value of the inhibitor. From these kinetic plots, the sEH inhibitors 1 , 3 and 5 , had inhibition constants ( Ki ) of 40.1 ± 1.8, 4.9 ± 0.2, and 21.2 ± 0.4 M, respectively ( Table 1 ). Also when compared antioxidant and sEH inhibitory activities of 1-6 , the potent peroxyl radical scavenging reducing capacity and sEH inhibitory activities were observed in 5 among 6 isolated compounds. Therefore, these results imply that potential activity of sEH inhibitors may be enhance the antioxidant defense mechanisms. 12 Further study using an in vivo animal model may be required to elucidate the correlation between antioxidant and sEH inhibitory activities of the phytochemicals from D. ismaelis .
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Inhibitory effect of the isolated compounds 1 - 6 on sEH activity determined using the uorometric method. The sEH activities expressed as the percentage of control activity. Values represent means ± SD from triplicate experiments. (n = 3). (AUDA (12.5 nM): positive control).
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Effects of compounds 1, 3 and 5 on the activity of sEH for hydroysis of substrate.
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Graphical determination of the inhibition type for isolated compounds 1, 3 and 5. (A -C) Lineweaver-Burk plots for the inhibition of compounds 1, 3, and 5 on sEH for the hydrolysis of substrate. (D - F) Dixon plots for the inhibition of compounds 1, 3, and 5 to determined ki value.
In vitrosEH inhibitory activity and kinetic study of compounds1,3and5
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aAll compounds were examined in a set of experiments three times bInhibition constant cPositive control
Acknowledgements
This study was supported by the Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093815), Republic of Korea.
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