Article pubs.acs.org/jnp
Scopoletin and Scopolin Isolated from Artemisia iwayomogi Suppress Differentiation of Osteoclastic Macrophage RAW 264.7 Cells by Scavenging Reactive Oxygen Species Sang-Hyun Lee,† Yan Ding,‡ Xi Tao Yan,‡ Young-Ho Kim,*,‡ and Hae-Dong Jang*,† †
Department of Food and Nutrition, Hannam University, Daejeon 305-811, Republic of Korea College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
‡
ABSTRACT: Artemisia iwayomogi has been used as a folk medicine for treating various diseases including inflammatory and immune-related diseases. Scopoletin (1) and scopolin (2) were isolated from this species. Scopoletin (1) showed more potent peroxyl radical-scavenging capacity, reducing capacity, and cellular antioxidant capacity compared to scopolin (2). The inhibitory effect of 1 on the receptor activator of nuclear factor κB ligand-induced osteoclastic differentiation of RAW 264.7 macrophage cells was also more potent than that of 2. The production of general reactive oxygen species (ROS) and superoxide anions during differentiation of preosteoclastic RAW 264.7 cells into osteoclasts was attenuated by compounds 1 and 2. These findings indicate that the suppressive effects of 1 and 2 on the differentiation of preosteoclastic RAW 264.7 cells is partially due to their intracellular antioxidant capacity, as they can scavenge ROS and play an important signaling role in the differentiation process. among five fractions, and chlorogenic acid was identified as its major antioxidant. A polysaccharide fraction (AIP 1) from A. iwayomogi Kitam. was found to suppress pulmonary eosinophilia and Th2-type cytokine production in an ovalbumininduced allergic asthma model8 and to exhibit antitumor and immunomodulating activities.9 A methanol extract of A. iwayomogi Kitam. exhibited anti-inflammatory activity for lipopolysaccharide-activated macrophages.10 Scopoletin (1) is an active compound responsible for the anti-inflammatory effects of A. iwayomogi Kitam.,11 and the acetylcholinesterase inhibitory activities of scopoletin (1) and scopolin (2) have been reported.12 Additionally, phenolic compounds from A. iwayomogi Kitam., including 1 and 2, stimulate the differentiation of osteoblastic MC3T3-E1 cells.13 In the present study, scopoletin (1) and scopolin (2) were isolated from A. iwayomogi Kitam. collected in Jeju Island, Korea, and were investigated for their antioxidant activity and suppressive effects on differentiation of RAW 264.7 macrophages into osteoclasts via scavenging ROS.
T
he resorption of mineralized bone by osteoclasts is followed by osteoblastic bone formation during bone remodeling, so resorbed lacunae are filled to the original level by osteoblasts.1 Bone formation is related to osteoblastic proliferation, alkaline phosphatase activity, collagen synthesis, and mineralization, whereas bone resorption is associated with osteoclast formation and tartrate-resistant acid phosphatase (TRAP) activity.2 Excessive osteoclastic bone resorption relative to osteoblastic bone formation is often associated with osteopenic diseases including osteoporosis and rheumatoid arthritis.3 Osteoclasts are formed by multiple cellular fusions from their mononuclear precursors as monocytes and macrophages.4 Differentiation of precursors into osteoclasts can be induced by receptor activator of nuclear factor-κB ligand (RANKL) produced by osteoblasts.5 Binding of RANKL to its receptor induces small nontoxic amounts of reactive oxygen species (ROS) as various growth factors and cytokines including tumor necrosis factor-α.6 The low level ROS increase may play a crucial role as a secondary messenger in RANKL-induced signaling pathways for osteoclast differentiation.6 Artemisia iwayomogi Kitam. is a widely distributed perennial herb in Korea that belongs to the family Compositae and has been used as a folk medicine for treating various diseases including inflammatory and immune-related diseases. A methanol extract of A. iwayomogi Kitam. showed peroxynitrite (ONOO−)-scavenging activity, which is a potent cytotoxic oxidant formed by the reaction between nitric oxide (NO) and superoxide radical (O2−).7 In the same study, an ethyl acetate (EtOAc) fraction of the A. iwayomogi Kitam. MeOH extract displayed the most potent peroxyl radical-scavenging activity © 2013 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Scopoletin (1) Showed Potent Antioxidant Activity in Vitro. The antioxidant activities of scopoletin (1) and scopolin (2) were investigated in terms of their peroxyl radicalscavenging and reducing capacity. Figure 1A shows that the scavenging activities of 1 and 2 in the presence of peroxyl radicals were dose-dependent at 1−10 μM. At a concentration Received: November 24, 2012 Published: March 19, 2013 615
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convert them into relatively stable compounds may contribute to its peroxyl radical-scavenging capacity. Scopoletin (1) and Scopolin (2) Demonstrated Potential Cellular Antioxidant Capacity. The intracellular antioxidant capacities of 1 and 2 were investigated using a cellular antioxidant activity assay. HepG2 cells were preincubated with 1−10 μM 1 or 2 for 30 min and exposed to 60 μM AAPH or 1 mM H2O2 for 30 min. The cells were then treated with DCFH-DA, which is a fluorescent probe that detects ROS, for 30 min to measure intracellular oxidative stress induced by AAPH or H2O2. Intracellular oxidative stress in HepG2 cells increased 176.9% and 167.8% following treatment with AAPH and H2O2, respectively, as compared to the control group (Figure 2A and B). Trolox (10 μM), used as a positive control,
Figure 1. Peroxyl radical-scavenging capacity (A) and reducing capacity (B) of scopoletin (1) and scopolin (2). Data are expressed as means ± standard deviations of three individual experiments. Different corresponding letters indicate significant differences at p < 0.05 by Duncan’s test.
of 1 μM, 1 showed 3.3 Trolox equivalents (TE) against peroxyl radicals generated from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). In contrast, as 2 is devoid of a hydroxy group, its peroxyl radical-scavenging activity was very weak when compared to that of 1 at 1 μM (0.4 TE). This is in agreement with the conclusions of previous work in which rutin carrying rutinose at C-3 showed weaker peroxyl radicalscavenging capacity than that of quercetin.14 In order to confirm whether antioxidant activities of 1 and 2 resulted from a capacity to donate electrons, a reduction of Cu2+ to Cu+ was determined. As shown in Figure 1B, the reducing capacity of 1 increased from 2.1 μM to 16.8 μM with increasing concentrations of 1−10 μM, but that of 2 was not observed. At a concentration of 1 μM, 1 demonstrated a more potent reducing capacity as compared to that of Trolox, a water-soluble α-tocopherol analogue, used as a positive control (1.5 μM). Present data suggest that the ability of scopoletin (1) to donate hydrogens or electrons to peroxyl radicals and to
Figure 2. Cellular antioxidant capacity of scopoletin (1) and scopolin (2) against oxidative stress induced by AAPH (A) and H2O2 (B). Data are expressed as percentages of the value of untreated cells (mean ± standard deviation, n = 3) (###p < 0.001 vs control; *p < 0.05, **p < 0.01, ***p < 0.001 vs AAPH or H2O2).
decreased the intracellular oxidative stress caused by AAPH and H2O2 to 144.7% and 138.6%, respectively. The intracellular oxidative stress induced by AAPH and H2O2 was dosedependently alleviated by 1 at 1−10 μM, whereas only intracellular oxidative stress induced by H2O2 was reduced by 2. A more potent cellular antioxidant activity of 1 against oxidative stress induced by AAPH and H2O2 than 2 was observed at 10 μM. These results are different from in vitro 616
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Figure 3. Inhibitory effects of scopoletin (1) and scopolin (2) on differentiation (A, B) and tartrate-resistant acid phosphatase (TRAP) activity (C) of osteoclastic RAW 264.7 cells. RAW 264.7 cells were exposed to receptor activator of nuclear factor kappa-B ligand (RANKL) (50 ng/mL) for 5 days in the absence and presence of 1 and 2. After 5 days in culture, cells were fixed and stained using a leukocyte acid phosphatase kit. TRAPpositive multinucleated osteoclasts were visualized under light microphotography (A). TRAP-positive multinucleated osteoclasts were counted (B). Data are expressed as percentages of the value of cells treated with RANKL (means ± standard deviations [SD], n = 3). TRAP activity was measured at λ = 405 nm (C). Data are expressed as percentages of the values of untreated cells (means ± standard deviations, n = 3) (###p < 0.001 vs C; *p < 0.05, **p < 0.01, ***p < 0.001 vs TC. C: control, which was not treated; TC: treated control, which was treated with RANKL).
Figure 3B and C. Treatment with 1 and 2 reduced the number and TRAP activity of multinucleated TRAP-positive cells in a dose-dependent manner (Figure 3B and C). Additionally, substantial differences in suppressive effects on the differentiation of osteoclastic RAW 264.7 cells were observed between 1 and 2. This may be due to the higher cell membrane permeability of 1 than that of 2 resulting from the absence of a glucose unit in 1, as flavonoid aglycones more readily penetrate through cell membranes as compared to glycosides.16 The suppressive effects of natural flavonoids and stilbenoids on differentiation of osteoclast precursors by controlling ROS generation have also been confirmed in luteolin,17 baicalein,18 resveratrol,19 and epigallocatechin-3-gallate,20 which are wellknown potent antioxidants. Scopoletin (1) and Scopolin (2) Attenuated General ROS and Superoxide Anion Production. Previous studies have reported that the intracellular ROS level increases during RANKL-induced osteoclast differentiation and that this ROS generation can be attenuated by several antioxidants.21 Thus, to investigate whether 1 and 2 can inhibit ROS generation during osteoclast differentiation from preosteoclastic cells, intracellular ROS levels were analyzed with the cell-permeable, fluorescent probe DCFH-DA. First, the time-course of ROS generation was measured for 5 days in RANKL-stimulated RAW 264.7 cells. The intracellular ROS level increased to its largest value within 2 days and then declined (Figure 4A), which was in good
antioxidant activity such as peroxyl radical-scavenging capacity and reducing capacity because a very weak antioxidant activity was observed in 2 when compared to 1. Taken together, although a significant difference between 1 and 2 in intracellular antioxidant activity exists, both compounds may permeate the cell membrane to suppress intracellular oxidative stress induced by peroxyl radicals and H2O2. Scopoletin (1) and Scopolin (2) Suppressed Osteoclastic Differentiation. Osteoclast cells are responsible for bone resorption in the bone remodeling process and are coupled tightly with osteoblasts forming the new bone matrix. Excessive bone resorption by osteoclasts can result in bone loss, causing diseases such as osteoporosis and rheumatoid arthritis.13 In the present study, the inhibitory effects of 1 and 2 on osteoclast differentiation were investigated to establish their antiosteoporotic activity through suppressing excessive bone resorption by osteoclasts. Osteoclast differentiation from murine macrophage RAW 264.7 cells was induced by RANKL, which is essential for terminal differentiation of monocytes/ macrophages into osteoclasts.15 Compounds 1 and 2 at 1−10 μM were not toxic to RAW264.7 macrophage cells during a period of five days of differentiation (data not shown). TRAPpositive multinucleated osteoclasts are visualized by light microphotography (Figure 3A). RANKL treatment dramatically induced osteoclast formation from RAW 264.7 preosteoclast cells and enhanced TRAP activity up to 182.82%, as shown in 617
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Figure 4. Reactive oxygen species (ROS) production (A) and the suppressive effects of scopoletin (1) and scopolin (2) on general ROS (B), superoxide anion (C), and hydrogen peroxide (D) during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. RAW 264.7 cells were treated with RANKL (50 ng/mL), and general ROS production was monitored for 5 days. The suppressive effect was measured after 2 days’ incubation in the absence and presence of 1 and 2. Data are expressed as percentages of the value of untreated cells (means ± standard deviations, n = 3). Different corresponding letters indicate significant differences at p < 0.05 by Duncan’s test (###p < 0.001 vs C; *p < 0.05, **p < 0.01, ***p < 0.001 vs TC. C, control, which was not treated; TC, treated control, which was treated with RANKL; NS, not significant).
control following RANKL stimulation and suppressed by 10 μM 1 and 2 to 157.8% and 148.6%, respectively (Figure 4C). On the other hand, hydrogen peroxide level measured by DHR was not changed by treatment with RANKL or 1 and 2 as compared to that in the control (Figure 4D). This indicates that when the superoxide anions generated by RANKL treatment are transformed by superoxide dismutase into hydrogen peroxides in RAW 264.7 cells, they may be immediately broken down by catalase to produce water and oxygen. Therefore, the constant hydrogen peroxide level was maintained during treating with RANKL in RAW 264.7 cells. This finding can be supported by a previous report in which the hydrogen peroxide level increased by RANKL in bone marrowderived monocytes returned to the level before RANKL treatment within half an hour.6 These results imply that the suppressive effect of 1 on general ROS and superoxide anion
agreement with a previous report showing the largest ROS production after 2 days’ incubation in bone marrow-derived monocytes.22 It has been suggested that ROS-mediated RANKL-induced differentiation of RAW 264.7 cells into osteoclasts occurs through the electron transport chain in mitochondria23 and activation of NADPH oxidase homologues.4 After superoxide anions are produced, they are transformed into different ROS such as hydrogen peroxide and peroxynitrite. To assess the suppressive effects of 1 and 2 on the production of different ROS during 2 days’ incubation, DCFH-DA, DHE, and DHR fluorescent probes were used. The general ROS level determined by DCFH-DA was increased by RANKL treatment to 143.7% as compared to that of the control and was decreased by 10 μM 1 and 2 to 132.4% and 126.7%, respectively (Figure 4B). Superoxide anion level was also augmented by 176.7% when compared to that in the 618
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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.27 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. Reducing Capacity. The electron-donating capacities of 1 and 2 to reduce Cu2+ to Cu+ were assessed according to the method of Aruoma et al.28 Forty microliters of different concentrations of 1 and 2 dissolved in ethanol were mixed with 160 μL of a mixture containing 0.5 mM CuCl2 and 0.75 mM neocuproine, a Cu+ specific chelator, in 10 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. Cellular Antioxidant Capacity. Cellular oxidative stress due to ROS generated by AAPH or H2O2 was measured spectrofluorometrically using the DCFH-DA method.28 DCFH-DA diffuses through the cell membrane and is enzymatically hydrolyzed by intracellular esterase to nonfluorescent DCFH, which is rapidly oxidized to highly fluorescent DCF in the presence of ROS. HepG2 cells were first cultured in 96-well plates (5 × 105/mL) with DMEM for 24 h. After the cells were incubated with different concentrations of 1 and 2 dissolved in DMSO for 30 min, the medium was discarded, and the wells were gently washed twice with PBS. HBSS, which is fluorescently stable, was then added to each well instead of normal medium, and AAPH or H2O2 was used as an oxidative stress inducer. After the cells were treated with 60 μM AAPH or 1 mM H2O2 for 30 min, DCFHDA was added to the culture plates at a final concentration of 40 μM and incubated for 30 min at 37 °C in the dark. Trolox (10 μM) was used as the positive control. After the incubation, the cells were washed with HBSS, and DCF fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a Tecan GENios fluorometric plate reader. Cell Cytotoxicity by MTT Assay. RAW 264.7 cells were cultured in 24-well plates (2 × 104 cells/mL) containing DMEM supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics in a humidified atmosphere of 5% CO2 at 37 °C for 5 days, washed with PBS, and pretreated with different concentrations (1−10 μM) of samples to be tested. After 5 days’ incubation, MTT reagent was added to each well, and the plate was incubated at 37 °C for 1 h. The medium was removed, and the plate was washed twice with PBS. The intracellular insoluble formazan was dissolved in DMSO. The absorbance of each cell was recorded in DMSO. The absorbance of each cell was then measured at 570 nm using an ELISA reader, and the percentage proliferation was calculated. TRAP Staining. RAW 264.7 cells were seeded in 12-well plates (3 × 104 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 PBS. The cells were fixed in 3.5% formaldehyde for 10 min and washed with distilled water. Cells were incubated at 37 °C in a humid and light-protected incubator for 1 h in the reaction mixture of the leukocyte acid phosphatase assay kit (Sigma-Aldrich, cat. no. 387) as directed by the manufacturer. Cells were washed three times with distilled water, and TRAP-positive multinucleated cells containing three or more nuclei were counted under a light microscope. TRAP Activity. After differentiating the RAW 264.7 cells into osteoclasts for 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.
level is more potent than that of 2, which is similar to their inhibitory effects on RANKL-induced differentiation of RAW 264.7 cells into osteoclasts. Moreover, both 1 and 2 pronouncedly suppressed the differentiation of preosteoclatic cells into osteoclasts compared with their inhibitory effects on general ROS and superoxide anion production, indicating that ROS-sensitive signal pathways such as NF-κB, mitogenactivated protein kinases (MAPK), p38-MAPK, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase during osteoclastic differentiation may be involved in their suppressive effects besides scavenging ROS or ameliorating ROS production. Taken together, the suppression of preosteoclastic RAW 264.7 cell differentiation into osteoclasts by both 1 and 2 was partially due to their intracellular antioxidant activity of scavenging ROS such as peroxyl radicals and superoxide anions. In conclusion, it was found that 1 and 2 demonstrated intracellular antioxidant capacity and suppressed RANKLtriggered osteoclast differentiation through regulating intracellular ROS generation. Although further investigations are required, this study elucidates the mechanism by which scopoletin (1) and scopolin (2) act as inhibitors of RANKLinduced osteoclast differentiation via the control of ROS production. Furthermore, these natural products may be suggested as potential therapeutic agents for the control of bone diseases such as osteoporosis and rheumatoid arthritis by suppressing osteoclast differentiation.
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EXPERIMENTAL SECTION
Reagents and Cell Culture Materials. 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH), neocuproine, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), β-glycerophosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, Hank’s balanced salt solution (HBSS), 2′,7′dichlorofluorescein-diacetate (DCFH-DA), RANKL, leukocyte acid phosphatase assay kit, sodium tartrate, p-nitrophenylphosphate (PNPP), phosphate-buffered saline (PBS, pH 7.4), dihydroethidium (DHE), dihydrorhodamine (DHR), and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Plant Materials. Aerial parts of A. iwayomogi Kitam. were collected on Jeju Island in June 2007 and taxonomically identified by Y.H.K. A voucher specimen (CNU07105) has been deposited at the herbarium of the College of Pharmacy, Chungnam National University, Daejeon, Korea. Extraction and Isolation. The plants (3 kg) were extracted with 70% MeOH at room temperature for 1 day (10 L × 3 times). The 70% MeOH extract (294 g) was concentrated under vacuum to give a gummy residue, which was then suspended in H2O (3:l ratio). This solution was extracted with EtOAc (3 L × 3 times) to give 45 g of an EtOAc-soluble fraction and 220 g of a H2O-soluble fraction. The H2Osoluble fraction (215 g) was subjected to column chromatography over a highly porous synthetic resin using a stepwise gradient of MeOH− H2O (0%, 25%, 50%, 75%, and 100% MeOH) to yield five fractions (1−5). Fraction 3 was chromatographed on a silica gel column eluted with a stepwise gradient of CHCl3−MeOH−H2O (20:1:0.1− 10:1:0.1−5:1:0.1), to yield six subfractions (3A−3F). Compound 1 (100 mg) (mp 204−205 °C) and compound 2 (600 mg) (mp 218− 219 °C) were obtained from fractions 3B (0.25 g) and 3C (1.3 g), respectively, using a reverse-phase C18 column eluted with MeOH− H2O (2:3). These compounds exhibited spectroscopic values (1H NMR, 13C NMR, ESIMS) consistent with literature values.24,25 The percentage purity of scopoletin (1) and scopolin (2) was 98% and 97%, respectively, as determined by HPLC. Oxygen Radical Absorbance Capacity (ORAC) Assay. The ORAC assay, which has been employed extensively in previous antioxidant studies,26 was carried out using a Tecan GENios 619
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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. Detection of Intracellular General ROS, O2−, and H2O2. During differentiation of RAW 264.7 cells into osteoclasts for 5 days, ROS were measured using DCFH-DA. Scavenging capacity of 1 and 2 against general ROS, O2−, and H2O2 was investigated using fluorescent probes such as DCFH-DA (excitation [Ex]/emission [Em] = 485 nm/ 535 nm), DHE (Ex/Em = 518 nm/605 nm),29 and DHR (Ex/Em = 488 nm/543 nm),30 which react specifically with ROS. RAW 264.7 cells were seeded in 96-well plates (3 × 104 cells/well) containing DMEM medium plus 10% FBS and incubated for 24 h. The medium was then replaced with a differentiation medium containing 50 ng/mL RANKL, and the samples were tested. After 2 days’ incubation, the medium was discarded, and the wells were gently washed twice with PBS. HBSS, which is fluorescently stable, was then added to each well instead of normal medium. DCFH-DA, DHE, or DHR was added to the culture plates at a final concentration of 40, 50, and 20 μM, respectively, and incubated for 30 min at 37 °C in the dark. After the cells were washed twice with HBSS, the fluorescence intensities of DCF, DHE, and DHR were measured using a Tecan GENios fluorometric plate reader. Statistical Analysis. All data are presented as mean ± standard deviation. Statistical analyses were carried out using the SPSS statistical package (SPSS, Chicago, IL, USA) program, and the significance of each group was verified with one-way analysis of variance (ANOVA) followed by Duncan’s test or Student’s t-test. A p value < 0.05 was considered significant.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +82-42-629-8795 or +82-42-821-5933. Fax: +82-42-6298805 or +82-42-823-6566. E-mail:
[email protected] or yhk@ cnu.ac.kr. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was financially supported by the Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea, through the High Value-Added Food Technology Development Program.
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