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International Journal of Molecular Sciences Article Anti-Osteoclastic Activity of Artemisia capillaris Thunb. Extract Depends upon Attenuation of Osteoclast Differentiation and Bone Resorption-Associated Acidification Due to Chlorogenic Acid, Hyperoside, and Scoparone Sang-Hyun Lee, Jung-Yun Lee, Young-In Kwon and Hae-Dong Jang * Department of Food and Nutrition, Hannam University, Daejeon 34430, Korea; [email protected] (S.-H.L.); [email protected] (J.-Y.L.); [email protected] (Y.-I.K.) * Correspondence: [email protected]; Tel.: +82-426-298-795 Academic Editor: Maurizio Battino Received: 14 December 2016; Accepted: 26 January 2017; Published: 4 February 2017 Abstract: The present study attempts to elucidate the anti-osteoporotic activity of Artemisia capillaris Thunb. in the form of anti-osteoclastic effect and responsible bioactive compounds. The contents of chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenic acid A, and scoparone in Artemisia capillaris hydroethanolic extract (ACHE) were 38.53, 0.52, 4.07, 3.03, 13.90, and 6.59 mg/g, respectively. ACHE diminished osteoclast differentiation and bone resorption due to chlorogenic acid, hyperoside, and scoparone. In addition, ACHE attenuated acidification as well as reducing tumor necrosis factor receptor-associated factor 6 (TRAF6) expression and its association with vacuolar H+ -adenosine triphosphatase (V-ATPase). Furthermore, chlorogenic acid, hyperoside, and scoparone from A. capillaris abrogated the association of V-ATPase with TRAF6, suggesting that the blockage of bone resorption by A. capillaris was partially mediated by reducing acidification through down-regulating interaction of V-ATPase with TRAF6 due to scoparone as well as chlorogenic acid and hyperoside. These results imply that the anti-osteoclastic effect of A. capillaris through down-regulating osteoclast differentiation and bone resorption may contribute to its anti-osteoporotic effect. Keywords: Artemisia capillaris; osteoclast differentiation; bone resorption; acidification 1. Introduction The bone remodeling cycle is an elaborately controlled process of bone metabolism, in which osteoclasts resorb the mineralized matrix and osteoblasts form new bone matrix [1]. Accordingly, bone mass depends upon an orchestrated balance between osteoclastic bone resorption and osteoblastic bone formation. Osteoclast differentiation from precursors and tartrate-resistant acid phosphatase (TRAP) activity belong to essential factors for bone resorption by osteoclasts. Osteoblast differentiation and proliferation, alkaline phosphatase activity, and type I collagen synthesis are required for bone formation by osteoblasts [2]. However, the imbalance between bone resorption and bone formation can cause metabolic bone diseases such as osteoporosis and osteopetrosis [3]. Osteoclasts, bone-resorbing multinucleated cells, are differentiated by the fusion of their mononuclear precursors, monocytes and macrophages, which is triggered by the receptor activator of nuclear factor-κB ligand (RANKL) produced by osteoblasts [4–6]. Bone resorption requires that osteoclasts have the ability to produce protons since an acidic pH is required for the solubilization of the alkaline salts of bone matrix and the digestion of bone matrix by acid enzymes secreted by Int. J. Mol. Sci. 2017, 18, 322; doi:10.3390/ijms18020322 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 322 Int. J. Mol. Sci. 2017, 18, 322 2 of 14 2 of 14 + -adenosine localized on[7]. theThis ruffled border of osteoclasts, which H hydrolyzes ATP to produce protons and extrudes osteoclasts acidification is due to vacuolar triphosphatase (V-ATPase) localized them into extracellular It was reported that the interaction of V-ATPase necrosis on the ruffled border ofspace. osteoclasts, which hydrolyzes ATP to produce protonswith andtumor extrudes them factor receptor-associated factor 6 (TRAF6) is required for its activation [8]. into extracellular space. It was reported that the interaction of V-ATPase with tumor necrosis factor Artemisia capillaris Thunb. belongs the genus Artemisia and[8]. has been widely used as an edible receptor-associated factor 6 (TRAF6) is to required for its activation herbalArtemisia medicine in Asian countries. Many have reported that A. widely capillaris extracts or its capillaris Thunb. belongs to thestudies genus Artemisia and has been used as an edible bioactive compounds such as caffeoylquinic acids, flavonoids, andA.coumarins [9–12] or have various herbal medicine in Asian countries. Many studies have reported that capillaris extracts its bioactive biological functions including antioxidant activity [13,14], anti-inflammatory anticompounds such as caffeoylquinic acids, flavonoids, and coumarins [9–12] haveactivity various[15], biological microbiological activity [16], anti-tumor activity [17], cytoprotective effects [14], hepatoprotective functions including antioxidant activity [13,14], anti-inflammatory activity [15], anti-microbiological effects [18,19], and anti-osteoporosis [20]. The recent demonstrated thateffects the water extract activity [16], anti-tumor activity [17],effects cytoprotective effectsstudy [14], hepatoprotective [18,19], and of A. capillaris suppressed RANKL-induced osteoclast differentiation anti-osteoporosis effects [20]. The recent study demonstrated that the water from extractbone of A. marrow capillaris macrophages and bone resorption by the attenuated expression of biomarkers including cathepsin K suppressed RANKL-induced osteoclast differentiation from bone marrow macrophages and bone and ATPasebyV(0) domain (ATPv0d2) Unfortunately, therecathepsin is no systematic study on antiresorption the attenuated expression[20]. of biomarkers including K and ATPase V(0)the domain osteoclastic effect of bioactivethere compounds of A. study capillaris the down-regulation of (ATPv0d2) [20]. Unfortunately, is no systematic on thethrough anti-osteoclastic effect of bioactive acidification. compounds of A. capillaris through the down-regulation of acidification. The Theobjective objectiveof ofthe the present present study study was was to to investigate investigate the the anti-osteoclastic anti-osteoclasticeffects effectsof ofA. A. capillaris capillaris and responsible bioactive compounds using an osteoclastic cells model for developing functional and bioactive compounds using an osteoclastic cells model for developing functional food food the treatment of bone diseases caused by bone in theintreatment of bone diseases caused by bone loss. loss. Results 2.2.Results 2.1.The TheContent ContentofofSix SixMarker MarkerCompounds Compoundsin inArtemisia Artemisiacapillaris capillaris Hydroethanolic Hydroethanolic Extract Extract (ACHE) (ACHE) 2.1. Chlorogenicacid, acid,caffeic caffeicacid, acid, hyperoside, hyperoside,isoquercitrin, isoquercitrin,isochlorogenic isochlorogenicacid acidA, A,and and scoparone scoparone Chlorogenic demonstratedin inFigure Figure11have havebeen beenreported reportedas assix siximportant importantconstituents constituentsof ofA. A.capillaris capillaris[11]. [11].Thus, Thus, demonstrated they were identified by the retention time and ultraviolet (UV) spectrum data of standard substances they were identified by the retention time and ultraviolet (UV) spectrum data of standard substances andtheir theircontents contents were were determined determined by and by straight straight calibration calibrationwith withaastandard standardcurve. curve.As Asshown shownininFigure Figure2, the retention time of chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenic acid and 2, the retention time of chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenicA,acid scoparone were 13.78, 14.78, 22.52, 22.88, 26.61, and 27.39 min, respectively. The content of chlorogenic A, and scoparone were 13.78, 14.78, 22.52, 22.88, 26.61, and 27.39 min, respectively. The content of acid, caffeicacid, acid,caffeic hyperoside, isoquercitrin, isochlorogenic acid A, and acid scoparone ACHE were chlorogenic acid, hyperoside, isoquercitrin, isochlorogenic A, andinscoparone in 38.53, 0.52, 4.07, 3.03, 13.90, and 6.59 mg/g, respectively (Table 1), indicating that chlorogenic acidthat and ACHE were 38.53, 0.52, 4.07, 3.03, 13.90, and 6.59 mg/g, respectively (Table 1), indicating isochlorogenic were the major components ACHE. This resultin appear toThis be consistent with chlorogenic acidacid andAisochlorogenic acid A were theinmajor components ACHE. result appear previous study in which the content acid Aofwas 43.14 mg/g followed by 43.14 chlorogenic to be consistent with previous studyofinisochlorogenic which the content isochlorogenic acid A was mg/g acid (21.06 mg/g), hyperoside (8.44 mg/g), and scoparone (5.56 mg/g) [11]. followed by chlorogenic acid (21.06 mg/g), hyperoside (8.44 mg/g), and scoparone (5.56 mg/g) [11]. Figure1.1.Chemical Chemicalstructures structuresof ofsix sixcompounds. compounds. Figure Int. J. Mol. Sci. 2017, 18, 322 3 of 14 Int. J. Mol. Sci. 2017, 18, 322 3 of 14 Figure 2. High performance liquid chromatography (HPLC) chromatograms of ethanol extract of Figure 2. High performance liquid chromatography (HPLC) chromatograms of ethanol extract of Artemisia capillaris. The detection wavelength was set at 255 nm for hyperoside and isoquercitrin, at Artemisia capillaris. The detection wavelength was set at 255 nm for hyperoside and isoquercitrin, at 320 nm for chlorogenic acid, caffeic acid, and isochlorogenic acid, and at 340 nm for scoparone. 320 nm for chlorogenic acid, caffeic acid, and isochlorogenic acid, and at 340 nm for scoparone. Table 1. The content of six marker compounds of hydroethanolic extract of A. capillaris. (n = 3). Table 1. The content of six marker compounds of hydroethanolic extract of A. capillaris. (n = 3). Compounds Compoundsacid Chlorogenic Chlorogenic acid Caffeic acid Caffeic acid Hyperoside Hyperoside Isoquercitrin Isoquercitrin Isochlorogenic acid Isochlorogenic acid Scoparone Scoparone (1) Content (1) (mg/g) (1) (mg/g) Content 38.526 ± 0.927 38.526 0.927 0.515 ±±0.048 0.515 ± 0.048 4.072 ± 0.220 4.072 ± 0.220 3.031 ± 0.148 3.031 ± 0.148 13.898 ± 0.667 13.898 ± 0.667 6.589 ± 0.193 6.589 ± 0.193 (1) Each Each value is expressed deviation triplicate experiments. value is expressedas asmean mean ± ± standard standard deviation inin triplicate experiments. 2.2. Suppressive Suppressive Effect Effect of of ACHE ACHE and and Its Its Six Six Marker Marker Compounds Compounds on on Osteoclast Osteoclast Differentiation Differentiation 2.2. Tostudy studythethe anti-osteoporosis of ACHE through the attenuated bone resorption by To anti-osteoporosis effecteffect of ACHE through the attenuated bone resorption by osteoclasts, osteoclasts, the suppressive effects of ACHE on RANKL-induced osteoclast differentiation were the suppressive effects of ACHE on RANKL-induced osteoclast differentiation were analyzed. RANKL, analyzed. RANKL, is required of formonocytes/macrophages the differentiation of monocytes/macrophages which is required forwhich the differentiation into osteoclasts, was usedinto as osteoclasts, was used as an inducer of osteoclast differentiation from RAW 264.7 [18]. induced RANKL an inducer of osteoclast differentiation from RAW 264.7 cells [18]. RANKL (50 cells ng/mL) (50 ng/mL) induced TRAP-positive formation from cells, which was TRAP-positive osteoclast formation osteoclast from pre-osteoclastic cells,pre-osteoclastic which was visualized by light visualized by light(Figure microphotography ACHE at 1–20 attenuated μg/mL dosemicrophotography 3A). However, (Figure ACHE at3A). 1–20However, µg/mL dose-dependently the dependently attenuated the number of osteoclasts (Figure 3B). In addition, ACHE at μg/mL number of osteoclasts (Figure 3B). In addition, ACHE at 1–20 µg/mL dose-dependently1–20 diminished dose-dependently TRAP of osteoclasts was up significantly (p RANKL < 0.001) the TRAP activity ofdiminished osteoclasts the which wasactivity significantly (p < 0.001)which enhanced to 172.1% by enhanced up to 172.1% by RANKL treatment compared to the control group (Figure 3C). To exclude treatment compared to the control group (Figure 3C). To exclude the possibility that the inhibitory the possibility that the inhibitory effect was due to the decreased viability and/or the proliferation of effect was due to the decreased viability and/or the proliferation of the pre-osteoclastic cells, cell the pre-osteoclastic cells, cell was that checked. The showed that ACHE diddoses not affect cytotoxicity was checked. Thecytotoxicity results showed ACHE didresults not affect cell viability at the that cell viability at the doses that effectively suppressed osteoclast differentiation (Figure 2D). effectively suppressed osteoclast differentiation (Figure 2D). To determine determine which which of of the the six six marker marker compounds compounds of of ACE ACE contributed contributed to to inhibitory inhibitory activity activity of of To ACHE ininRANKL-induced RANKL-induced osteoclast differentiation, TRAP was activity was atmeasured at the ACHE osteoclast differentiation, TRAP activity measured the concentration concentration of 10 μM at which scoparone moderately inhibited RANKL-induced osteoclast of 10 µM at which scoparone moderately inhibited RANKL-induced osteoclast differentiation in our differentiation ourthe previous study [21]; the compounds cell cytotoxicity of six compounds wasThe not potent found previous study in [21]; cell cytotoxicity of six was not found (Figure 3D). (Figure 3D). The potent inhibitory effect on TRAP activity was observed in chlorogenic acid, inhibitory effect on TRAP activity was observed in chlorogenic acid, hyperoside, and scoparone as hyperoside, and scoparone as compared with caffeic acid, isoquercitrin, and isochlorogenic acid at 10 μM (Figure 3E). On the other hand, isoquercitrin displayed the pro-osteoclastic effect in this Int. J. Mol. Sci. 2017, 18, 322 4 of 14 Int. J. Mol. Sci. 2017, 18, 322 4 of 14 compared with caffeic acid, isoquercitrin, and isochlorogenic acid at 10 µM (Figure 3E). On the other experiment. Thus, displayed this resultthe suggests that the effect inhibitory of ACHE onthis RANKL-induced hand, isoquercitrin pro-osteoclastic in thiseffect experiment. Thus, result suggests osteoclast differentiation may be attributed to three compounds such as chlorogenic acid, hyperoside, that the inhibitory effect of ACHE on RANKL-induced osteoclast differentiation may be attributed to and scoparone. three compounds such as chlorogenic acid, hyperoside, and scoparone. Figure 3. Cont. Int. J. Mol. Sci. 2017, 18, 322 Int. J. Mol. Sci. 2017, 18, 322 5 of 14 5 of 14 Figure Inhibitoryeffect effectofofhydroethanolic hydroethanolicextract extractof ofA. A.capillaris capillaris and and its six marker Figure 3. 3. Inhibitory marker compounds compounds on (A) The Thevisualized visualizedtartrate-resistant tartrate-resistantacid acid phosphatase (TRAP)-positive onosteoclast osteoclast differentiation. (A) phosphatase (TRAP)-positive multinucleated osteoclasts with capillaris hydroethanolic extract (ACHE) treatment; (B,C) multinucleated osteoclasts with A. A. capillaris hydroethanolic extract (ACHE) treatment; (B,C) TheThe counted number and TRAP of TRAP-positive multinucleated ACHE counted number and TRAP activityactivity of TRAP-positive multinucleated osteoclasts osteoclasts with ACHE with treatment; (D) The cytotoxicity of A.and capillaris its six compounds; marker compounds; The TRAP activity (D)treatment; The cytotoxicity of A. capillaris its sixand marker (E) The(E) TRAP activity of multinucleated osteoclasts withwith six marker compounds treatment. RAW 264.7 cells were exposed of multinucleated osteoclasts six marker compounds treatment. RAW 264.7 cells were exposed to receptor activator of nuclear factor-κB ligand (RANKL; 50 50 ng/mL) forfor 5 days in in thethe absence and to receptor activator of nuclear factor-κB ligand (RANKL; ng/mL) 5 days absence and presence of ACHE. After 5 days in culture, thethe cells were fixed andand stained using a leukocyte acid presence of ACHE. After 5 days in culture, cells were fixed stained using a leukocyte acid phosphatase kit.kit. TRAP-positive multinucleated osteoclasts were visualized at 200-fold magnification phosphatase TRAP-positive multinucleated osteoclasts were visualized at 200-fold magnification under light microscopy. TRAP-positive multi-nucleated osteoclasts were counted and TRAP activity under light microscopy. TRAP-positive multi-nucleated osteoclasts were counted and TRAP activity was measured at λat=λ405 nm.nm. Data areare expressed as as percentages of of thethe value of of cells treated with was measured = 405 Data expressed percentages value cells treated with RANKL (means ± standard deviations, SD, n =n 3). Data areare expressed as as percentages of of thethe values of of RANKL (means ± standard deviations, SD, = 3). Data expressed percentages values untreated cellscells (means ± standard deviations, n = 3). Different letters indicate significant untreated (means ± standard deviations, n = 3). corresponding Different corresponding letters indicate ## p < 0.01, ##### p < 0.001 ### differences at p < 0.05 by Duncan’s test. vs. C. C: control, which was not was significant differences at p < 0.05 by Duncan’s test. p < 0.01, p < 0.001 vs. C. C: control, which treated; TC: treated control,control, which was treated with RANKL; CHA: chlorogenic acid; CAA: not treated; TC: treated which was treated with RANKL; CHA: chlorogenic acid;caffeic CAA:acid; caffeic HY:acid; hyperoside; IQ: isoquercitrin; ICA: isochlorogenic acid; SC:acid; scoparone; NS: not significant. HY: hyperoside; IQ: isoquercitrin; ICA: isochlorogenic SC: scoparone; NS: not significant. Inhibitory Effect of ACHE Three Bioactive Compounds Bone Resorption of Osteoclast 2.3.2.3. Inhibitory Effect of ACHE andand Three Bioactive Compounds on on Bone Resorption of Osteoclast The inhibitory effect ACHE bone resorption was determined RANKL-differentiated The inhibitory effect of of ACHE onon bone resorption was determined in in RANKL-differentiated osteoclasts cultured 5 days onplates well coated plates coated with fluoresceinamine-labeled chondroitin osteoclasts cultured for 5for days on well with fluoresceinamine-labeled chondroitin sulfate sulfate (FACS) andphosphate calcium phosphate (CaP) using a bone resorption assay kitBio, (CosMo Bio, Tokyo, (FACS) and calcium (CaP) using a bone resorption assay kit (CosMo Tokyo, Japan). Japan). FACS bound CaP was released from the CaP into by theosteoclastic medium byresorption. osteoclastic The FACS The bound to CaP wasto released from the CaP layer into thelayer medium resorption. Therefore, bone resorption activity istoproportional fluorescence intensity FACS in Therefore, bone resorption activity is proportional fluorescencetointensity of FACS in the of medium. the medium. Theon resorbed area onvisualized the plates was visualized 200-fold under magnification under light The resorbed area the plates was under 200-foldunder magnification light microscopy. microscopy. RANKL treatment markedly increased thesize number and size of bone resorption areas RANKL treatment markedly increased the number and of bone resorption areas (Figure 4A). (Figure 4A). theactivity bone resorption activity significantly (p <RANKL 0.001) (Figure enhanced Consistently, theConsistently, bone resorption was significantly (p was < 0.001) enhanced by 4B).by 4B). In contrast, ACHE at 1–20 μg/mL dose-dependently alleviated bonests. resorption In RANKL contrast,(Figure ACHE at 1–20 µg/mL dose-dependently alleviated bone resorption of osteocla of osteocla sts. Int. J. Mol. Sci. 2017, 18, 322 Int. J. Mol. Sci. 2017, 18, 322 6 of 14 6 of 14 Figure 4. Inhibitory Inhibitory effect effectofofhydroethanolic hydroethanolic extract capillaris its bioactive compounds on Figure 4. extract of of A. A. capillaris andand its bioactive compounds on bone bone resorption. (A) The visualized absorbed area with ACHE treatment; (B) The fluorescence resorption. (A) The visualized absorbed area with ACHE treatment; (B) The fluorescence intensity of the intensity thewith absorbed area with with ACHE treatment ACHE and (C)compounds. three bioactive compounds. A assay bone absorbedof area treatment andwith (C) three bioactive A bone resorption resorption assayusing was aperformed using commercial assay RAW 264.7tocells were(100 exposed to was performed commercial assaya kit. RAW 264.7 cellskit. were exposed RANKL ng/mL) RANKL ng/mL) forand 5 days in the absenceorand of ACHE or threeareas compounds. The for 5 days(100 in the absence presence of ACHE threepresence compounds. The absorbed on each plate absorbed areas at on200-fold each plate were visualized at 200-fold magnification under lightwas microscopy. were visualized magnification under light microscopy. Fluorescence intensity measured Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a fluorometric plate wavelength nm using a fluorometric plate reader. Data arecells expressed asstandard percentages of the reader. Data of are535 expressed as percentages of the values of untreated (means ± deviations, values untreated cells (means ±letters standard deviations, n = 3).differences Different corresponding indicate n = 3). ofDifferent corresponding indicate significant at p < 0.05 byletters Duncan’s test. ### p < 0.001 ### p < 0.001 vs. C. C: control, which was not treated; significant differences p < 0.05which by Duncan’s test. vs. C. C:atcontrol, was not treated; TC: treated control, which was treated with TC: treated which wasHP: treated with RANKL; CA: chlorogenic acid; HP: hyperoside; SP: RANKL; CA:control, chlorogenic acid; hyperoside; SP: scoparone. scoparone. Bone resorption can be achieved by multi-nucleated osteoclasts after the differentiation process. Bone resorption can be achieved by multi-nucleated osteoclasts after the differentiation process. Because the strong inhibitory effect on osteoclastic differentiation was found in three compounds such Because the strong inhibitory effect on osteoclastic differentiation was found in three compounds as chlorogenic acid, hyperoside, and scoparone, these three bioactive compounds were used for bone such as chlorogenic acid, hyperoside, and scoparone, these three bioactive compounds were used for bone resorption assay. To determine which of the three compounds of ACHE with potential antiosteoclastic activity could be related with the inhibitory activity of ACHE on bone resorption of Int. J. Mol. Sci. 2017, 18, 322 7 of 14 Int. J. Mol.assay. Sci. 2017, 322 7 of 14 resorption To18,determine which of the three compounds of ACHE with potential anti-osteoclastic activity could be related with the inhibitory activity of ACHE on bone resorption of osteoclasts, the osteoclasts, the bone resorption assay was carried out using a bone resorption assay kit. Though all bone resorption assay was carried out using a bone resorption assay kit. Though all of three compounds of three compounds showed the inhibition effect on bone resorption (Figure 4C), the strongest showed the inhibition effect on bone resorption (Figure 4C), the strongest inhibition effect was observed inhibition effect was observed in scoparone. These data imply that scoparone as well as chlogenic in scoparone. These data imply that scoparone as well as chlogenic acid and hyperoside may be major acid and hyperoside may be major components of ACHE, contributing to its inhibitory activity on components of ACHE, contributing to its inhibitory activity on bone resorption of osteoclasts. bone resorption of osteoclasts. 2.4. Suppressive Effect of ACHE and Three Bioactive Compounds on the Acidification by Osteoclasts 2.4. Suppressive Effect of ACHE and Three Bioactive Compounds on the Acidification by Osteoclasts The effect of ACE on acidification was examined by measuring pH since the acidic pH is essential The effect of ACE on acidification was examined by measuring pH since the acidic pH is for essential bone resorption ostaoclasts. RANKL significantly (p < 0.001) the pHthe of pH a culture for bone by resorption by ostaoclasts. RANKL significantly (p <diminished 0.001) diminished of a medium as compared to the control group, but ACHE at 1–20 µg/mL reversed this decrease and culture medium as compared to the control group, but ACHE at 1–20 μg/mL reversed this decrease further augmented pH in pH a dose-dependent manner (Figure 5A).5A). In addition, the enhancing and further augmented in a dose-dependent manner (Figure In addition, the enhancingeffect effectof three compounds on pH is clearly shown in Figure 5B. Furthermore, scoparone markedly increased pH of three compounds on pH is clearly shown in Figure 5B. Furthermore, scoparone markedly increased as compared to chlorogenic acid and hyperoside, which is in line with the result of their suppressive pH as compared to chlorogenic acid and hyperoside, which is in line with the result of their effects on bone effects resorption. suppressive on bone resorption. Figure 5. (A) Inhibitory effect of hydroethanolic extract of A. capillaris and (B) three bioactive Figure 5. (A) Inhibitory effect of hydroethanolic extract of A. capillaris and (B) three bioactive compounds on acidification. RAW 264.7 cells were exposed to RANKL (50 ng/mL) for 5 days in the compounds on acidification. RAW 264.7 cells were exposed to RANKL (50 ng/mL) for 5 days in absence and presence of ACHE. After 5 days, the pH of the differentiated medium was measured the absence and presence of ACHE. After 5 days, the pH of the differentiated medium was measured using a pH meter. Data are expressed as percentages of the values of untreated cells (means ± standard using a pH meter. Data are expressed as percentages of the values of untreated cells (means ± standard deviations, n = 3). Different corresponding letters indicate significant differences at p < 0.05 by deviations, n = 3).##Different ### corresponding letters indicate significant differences at p < 0.05 by Duncan’s Duncan’s test. p < 0.01, p < 0.01 vs. C. C: control, which was not treated; TC: treated control, which ## ### test. p < 0.01, p < 0.01 vs. C. C: control, which was not treated; TC: treated control, which was was treated with RANKL; CA: chlorogenic acid; HP: hyperoside; SP: scoparone. treated with RANKL; CA: chlorogenic acid; HP: hyperoside; SP: scoparone. Int. J. Mol. Sci. 2017, 18, 322 Int. J. Mol. Sci. 2017, 18, 322 8 of 14 8 of 14 Down-Regulating Effect Effect of of ACHE ACHE and andThree ThreeBioactive BioactiveCompounds Compoundson onTRAF6 TRAF6Expression Expressionand andItsItsBinding 2.5. Down-Regulating to V-ATPase Binding to V-ATPase RANKL binding to RANK localized on the membrane of precursor cells may recruit TRAF6 to induce osteoclast differentiation and to enhance enhance bone bone resorption. resorption. For efficient bone resorption by osteoclasts, acidification is required and the primary mechanism responsible for this acidification is V-ATPase interaction of V-ATPase V-ATPase which which transports transports H H++ to extracellular resorption lacunae [7]. The interaction with ofof nuclear factor-κB (RANK) results in its [8]. with TRAF6 TRAF6recruited recruitedby bythe thereceptor receptoractivator activator nuclear factor-κB (RANK) results in activation its activation Therefore, the the effect of ACHE onon expression ofof TRAF6 [8]. Therefore, effect of ACHE expression TRAF6and andV-ATPase V-ATPaseand andtheir their interaction interaction was investigated using western blot analysis and immunoprecipitation. immunoprecipitation. RANKL significantly (p < 0.001) enhanced the expression of both TRAF6 and V-ATPase V-ATPase (Figure (Figure 6A). 6A). However, However, ACHE ACHE at at20 20µg/mL μg/mL significantly significantly (p (p < 0.001) diminished the TRAF6 expression enhanced by RANKL, but showed no effect on V-ATPase V-ATPase expression expression increased increased by by RANKL. RANKL. Furthermore, to determine whether V-ATPase binds with TRAF6 for its activation, the association of TRAF6 withwith V-ATPase was investigated. In anti-TRAF6 with TRAF6 for its activation, the association of TRAF6 V-ATPase was investigated. In antiimmunoprecipitates, RANKLRANKL significantly (p < 0.001) augmented V-ATPase bound to TRAF6, and TRAF6 immunoprecipitates, significantly (p < 0.001) augmented V-ATPase bound to TRAF6, ACHE at 20at µg/mL significantly (p < (p 0.001) abrogated this this increased V-ATPase (Figure 6B). 6B). and ACHE 20 μg/mL significantly < 0.001) abrogated increased V-ATPase (Figure Which of of the thethree threecompounds compoundscontributed contributed inhibitory effect on expression of TRAF6 toto thethe inhibitory effect on expression of TRAF6 and and its interaction V-ATPase was examined. According to Figure 6C, the significant difference its interaction withwith V-ATPase was examined. According to Figure 6C, the significant difference in the in the protein level of TRAF6 and V-ATPase not found among three compounds (Figure protein level of TRAF6 and V-ATPase was was not found among three compounds (Figure 6C).6C). In In addition, anti-V-ATPaseimmunoprecipitates, immunoprecipitates,RANKL RANKLsignificantly significantly(p(p << 0.001) 0.001) induced induced the addition, in in anti-V-ATPase interaction V-ATPase with with TRAF6. TRAF6. The most potent inhibition effect on interaction V-ATPase with TRAF6 followed byby hyperoside andand chlorogenic acidacid (Figure 6D). 6D). This data TRAF6 was wasobserved observedininscoparone, scoparone, followed hyperoside chlorogenic (Figure This is consistent with the result the inhibitory effect of three from A. capillaris data is consistent with theofresult of the inhibitory effectcompounds of three compounds from on A. acidification capillaris on by osteoclasts. these results these strongly suggest that suggest scoparone well as chlorogenic acidification byConsequently, osteoclasts. Consequently, results strongly thatasscoparone as well as acid and hyperoside responsible ACE for attenuating association V-ATPase chlorogenic acid andare hyperoside arecompounds responsible of compounds of ACE forthe attenuating theofassociation with TRAF6. with TRAF6. of V-ATPase Figure 6. Cont. Int. J. Mol. Sci. 2017, 18, 322 9 of 14 Int. J. Mol. Sci. 2017, 18, 322 9 of 14 Figure 6. 6. Suppressive Suppressiveeffect effectofofhydroethanolic hydroethanolicextract extract capillaris and three bioactive compounds Figure ofof A.A. capillaris and three bioactive compounds on +-adenosine on tumor necrosis factor receptor-associated factor 6 (TRAF6) and vacuolar H + tumor necrosis factor receptor-associated factor 6 (TRAF6) and vacuolar H -adenosine triphosphatase triphosphatase (V-ATPase) expression and (A) their The hydroethanolic effect of A. capillaris (V-ATPase) expression and their interaction. Theinteraction. effect of A. (A) capillaris extract hydroethanolic extract (ACHE) on TRAF6 and V-ATPase expression and (B) their interaction. (C) The (ACHE) on TRAF6 and V-ATPase expression and (B) their interaction. (C) The effect of three bioactive effect of three compounds onexpression TRAF6 and V-ATPase andRAW (D) their compounds onbioactive TRAF6 and V-ATPase and (D) theirexpression interaction. 264.7interaction. cells were RAW 264.7 cells were exposed to RANKL (50 ng/mL) for 5 days in the absence and presence exposed to RANKL (50 ng/mL) for 5 days in the absence and presence of ACHE (20 µg/mL)oforACHE three (20 μg/mL) or three (10 bioactive compounds μM). were lysed and cell lysates were bioactive compounds µM). The cells were(10 lysed andThe cellcells lysates were incubated with TRAF6 or incubated with TRAF6 or V-ATPase antibody and protein A beads. After washing the beads, V-ATPase antibody and protein A beads. After washing the beads, precipitated proteins and cell precipitated proteins and cell lysates weresulfate-polyacrylamide separated by sodium gel dodecyl sulfate-polyacrylamide gel lysates were separated by sodium dodecyl electrophoresis (SDS-PAGE) and electrophoresis for (SDS-PAGE) andproteins immunoblotted for theantibodies. indicated proteins specific antibodies. immunoblotted the indicated using specific Data are using expressed as percentages Data expressed as percentages of the of untreated of theare values of untreated cells (means ± values standard deviations,cells n = (means 3). ## p ±< standard 0.01, ### pdeviations, < 0.001 vs.nC;= ## p < 0.01, ### p < 0.001 vs. C; *** p < 0.001 vs. TC. C: control, which was not treated; TC: treated 3). *** p < 0.001 vs. TC. C: control, which was not treated; TC: treated control, which was treated with control, which was treated with RANKL; CA: chlorogenic acid; HP: RANKL; CA: chlorogenic acid; HP: hyperoside; SP: scoparone; NS: nothyperoside; significant. SP: scoparone; NS: not significant. 3. Discussion 3. Discussion The osteoclast responsible for bone resorption is a multinucleated cell differentiated from The osteoclast responsible for monocytes bone resorption is a multinucleated from mononuclear precursors including and macrophages [3]. Thecell lowdifferentiated level of nontoxic mononuclear precursors monocytes and growth macrophages low level of nontoxic amount of reactive oxygen including species (ROS) with several factors [3]. and The cytokines is induced by the amount of reactive oxygen species (ROS) with several growth factors and cytokines is induced by the RANKL binding to the receptors [5]. This increase in ROS may play an important role as a secondary RANKL binding to the receptors [5]. This increase in ROS may play an important role as a secondary messenger in RANKL-mediated signaling pathways for osteoclast differentiation [6]. Previous reports messenger in RANKL-mediated signaling pathwaysameliorating for osteoclast differentiation [6]. Previous have demonstrated that potent flavonoid antioxidants osteoclastic differentiation have reports have demonstrated that potent flavonoid antioxidants ameliorating osteoclastic been also found in genistein [22], luteolin [23], baicalein [24], epigallocatechin-3-gallate [25], and differentiation been study also found in genistein [22], luteolin [23], baicalein [24], epigallocatechin-3fisetin [4]. Our have previous confirmed that scopoletin with strong cellular antioxidant capacity gallate [25], and fisetin [4]. Our previous study confirmed that scopoletin with strong cellular reduces ROS production as superoxide anions to suppress osteoclastic differentiation from RAW 264.7 antioxidant capacity reduces ROS production as superoxide anions to suppress osteoclastic cells [26]. In addition, the potent antioxidant activity of A. capillaris was recently reported [13,27]. differentiation fromhave RAW [26]. In the addition, the potent antioxidant of A. capillaris Thus, these reports led264.7 us tocells investigate inhibitory effect of A. capillarisactivity on RANKL-induced was recently reported [13,27]. Thus, these reports have led us to investigate the inhibitory effect of A. osteoclast differentiation. capillaris on RANKL-induced osteoclast differentiation. In the present study, the anti-osteoclastic effect of A. capillaris was confirmed by measuring In the present study, the anti-osteoclastic of is A.consistent capillaris was measuring the the number and TRAP activity of osteoclasts,effect which withconfirmed the recentby study reporting number and TRAP activity of osteoclasts, which is consistent with the recent study reporting the the suppressive effect of A. capillaris on osteoclast differentiation [20]. This anti-osteoclastic effect of suppressive effect A. capillaris osteoclast differentiation [20]. This anti-osteoclastic effect of A. A. capillaris may beofattributed to on three bioactive compounds such as chlorogenic acid, hyperoside, capillaris may be attributed to three bioactive compounds such as differentiation chlorogenic acid, and and scoparone because the potent suppressive effect on osteoclast washyperoside, found in them scoparone because the potent suppressive effect on osteoclast differentiation was found in them compared to caffeic acid, isoquercitrin, and isochlorogenic acid. Recently, it has been reported that Int. J. Mol. Sci. 2017, 18, 322 10 of 14 compared to caffeic acid, isoquercitrin, and isochlorogenic acid. Recently, it has been reported that scoprone can attenuate RANKL-induced osteoclastic differentiation through suppressing ROS production and scavenging [21] and chlorogenic acid can inhibit RANKL-induced osteoclast differentiation [28], implying that the anti-osteoclastic effect of A. capillaris may be attributed to the antioxidant activity of three bioactive compounds including chlorogenic acid, hyperoside, and scoparone. Excessive bone resorption by osteoclasts can result in osteoporosis in older post-menopausal women. The inhibitory effects of natural polyphenols including silibinin and phloretin on bone resorption have been also reported [29,30]. To check the possibility of A. capillaris in the treatment of osteoporosis through regulating bone resorption, the suppressive effect of A. capillaris on bone resorption by osteoclasts was observed in the current study. For efficient bone resorption, the solubilization of bone mineral and the hydrolysis of organic bone matrix by enzymes are required, which depends on the pH of the environment. The result that A. capillaris significantly augmented pH lowered by RANKL treatment appears to be associated with decreased bone resorption. According to Figure 4 and 5, the inhibitory effect of scoparone on bone resorption and acidification was more potent than that of chlorogenic acid and hyperoside. In addition, the inhibitory effect of chlorogenic acid on bone resorption was reported in the previous study [28]. Therefore, these data support that scoparone as well as chlorogenic acid and hyperoside may be major active components of A. capillaris, attenuating bone resorption of osteoclast through controlling acidification. The primary cellular protein of the acidification by osteoclasts is known as V-ATPase which hydrolyzes ATP to produce protons [7]. In addition, the interaction of V-ATPase with TRAF6 is essential for its activation [8]. The present study revealed that A. capillaris markedly alleviated TRAF6 expression and the association of TRAF6 with V-ATPase compared to RANKL treatment. In addition, although three bioactive compounds including chlorogenic acid, hyperoside, and scoparone displayed the potent inhibitory effect on bone resorption in comparison with caffeic acid, isoquercitrin, and isochlorogenic acid, they did not significantly affect the expression of either TRAF6 or V-ATPase. In contrast, they vigorously interrupted the association of V-ATPase with TRAF6 compared to RANKL treatment even though the underlying mechanism remains unclear. The possible explanation for the reduced bone resorption of A. capillaris due to suppresssed acidification may be the reduced osteoclast differentiation and/or the attenuated interaction of V-ATPase with TRAF6. Accordingly, this finding indicates that scoparone, as well as chlorogenic acid and hyperoside, may be partially responsible for the inhibitory effect of A. capillaris on bone resorption through disrupting the association of V-ATPase with TRAF6. 4. Materials and Methods 4.1. Reagents and Cell Culture Materials Neocuproine, Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium alpha medium (α-MEM), fetal bovine serum (FBS), 2,5-diphenyltetrazolium (MTT), Triton X-100, RANKL, sodium tartrate, p-nitro-phenylphosphate (PNPP), fluoresceinamine-labeled chondroitin sulfate (FACS), phosphate-buffered saline (PBS, pH 7.4), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), dimethylsulfoxide (DMSO), chlorogenic acid, caffeic acid, isoquercitrin, and isochlorogenic acid A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hyeroside was purchased from Hwz Analytic GmbH (Ruelzheim, Germany). Scoparone was obtained from Phytolab GmbH&Co (Vestenbergreuth, Germany). Antibodies and protein A-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), including anti-TRAF6 (sc-7221), V-ATPase (sc-20946), and β-actin (sc-130656). A bone resorption assay kit was purchased from CosMo Bio Co., Ltd. (Tokyo, Japan). MC3T3-E1 subclone 4 and RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Int. J. Mol. Sci. 2017, 18, 322 11 of 14 4.2. Preparation of A. capillaris Hydroethanolic Extract (ACHE) The aerial parts of A. capillaris Thunb. were purchased at Kyoungdong market, an herbal medicine market in Seoul, and taxonomically identified by Young-Ho Kim who is a professor in the College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea. A voucher specimen (CNU-10103) was deposited at the Herbarium of the College of Pharmacy, Chungnam National University. The plants were cut in 1-cm lengths and were extracted with a 6-fold volume of 50% ethanol at 60 ◦ C for 6 h. Then, a second extraction was done with a 5-fold volume of 50% ethanol at 50 ◦ C for 16 h. After the pooled extraction solution was concentrated under a vacuum and spray-dried, it was kept at −20 ◦ C until used. 4.3. High Performance Liquid Chromatography (HPLC) Analysis The HPLC analysis was carried out using a HP Agilient HPLC system (Santa Clara, CA, USA) with a solvent delivery unit, an online degasser, a column oven, an autosampler and a photodiode array (PDA) detector. The analytical column was C18 (21.5 × 300 mm, particle size 10 µM, Tosoh, Tokyo, Japan) maintained at 40 ◦ C. The mobile phases were distilled water (A) and phosphoric acid in 60% acetonitrile (pH 2.4, B). The gradient flow rate was composed of 0%–10% B for 0–5 min, 10%–50% B for 5–30 min, and 50%–100% B for 30–35 min. The flow rates and injection volumes were 1.0 mL/min and 10 µL, respectively. The detection wavelength was 255 nm for hyperoside and isoquercitrin, 320 nm for chlorogenic acid, caffeic acid, and isochlorogenic acid A, and 340 nm for scoparone. Six pure compounds such as chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenic acid A, and scoparone, were used as chemical markers. The identification of the compounds was performed by comparison of the retention time and UV spectrum. 4.4. Cell Cytotoxicity by MTT Assay RAW 264.7 cells were plated in 24-well plates at a density of 2 × 104 cells/mL in triplicate. Cells were treated with RANKL (50 ng/mL) and increasing concentrations of ACHE (1–50 µg/mL), or six compounds (1–20 µM) were added to DMEM supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. After 5 days, MTT reagent was added to each well. The plate was incubated at 37 ◦ C for 1 h. After removing the medium, the plate was washed twice with PBS. DMSO was then added to dissolve the intracellular insoluble formazan. The absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Salzburg, Austria), and the percentage proliferation was calculated. 4.5. TRAP Staining and Activity RAW 264.7 cells were plated in 96-well plates at a density of 1 × 104 cells/well. The cells were treated with RANKL (50 ng/mL) and ACHE (1–20 µg/mL), or six compounds (10 µM) were added to DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. The medium was changed every 2 days. After 5 days, the cells were fixed in 3.5% formalin for 10 min and stained with an acid phosphatase kit. The multinucleated osteoclast cells which were stanined with TRAP, were observed at 200-fold magnification with a light microscopy. For measuring TRAP activity, the cells was washed twice with ice-cold PBS and fixed in 3.5% formaldehyde and ethanol/acetone (1:1) for 10 and 1 min, respectively. The dried cells were then incubated in 50 mM citrate buffer (pH 4.5) supplemented with 10 mM sodium tartrate and 6 mM PNPP for 40 min. After the mixtures were added to the new wells with an equal volume of 0.1 N NaOH, the absorbance was determined at 405 nm using an ELISA reader. 4.6. Bone Resorption Assay A bone resorption assay kit (CosMo Bio, Tokyo, Japan) was used for bone resorption assay. In this assay, phenol red-free DMEM was used to avoid the interruption of fluorescence measurement due to Int. J. Mol. Sci. 2017, 18, 322 12 of 14 phenol red. RAW 264.7 cells were seeded in FACS and CaP-coated 24-well plates (1 × 104 cells/well) containing phenol red-free DMEM plus 10% FBS, and the medium was replaced with test sample (1 to 20 µg/mL ACHE or 10 µM six compounds) in DMEM containing 50 ng/mL RANKL. After 5 days, 100 µL of the medium and 50 µL resorption assay buffer (catalogue number CSR-BRA-B1, CosMo Bio Co. Ltd) were transferred into the wells of a 96-well plate and mixed for 3 min under dark conditions. A fluorometric plate reader (Tecan GENios, Salzburg, Austria) was used to measure the fluorescence at an excitation of 485 nm and an emission of 535 nm. After washing the cells with 5% sodium hypochlorite, the resorbed areas on the plate were visualized at 200-fold magnification under light microscopy. 4.7. pH Measurement RAW 264.7 cells were seeded in 60 mm dishes (1 × 104 cells/dish) containing DMEM plus 10% FBS, and the medium was replaced with test sample (1–20 µg/mL ACHE or 10 µM of six compounds) in differentiation medium. The differentiation medium was changed every 2 days. After 5 days, the pH of the differentiated media was measured using a pH meter (Thermo Scientific, Hudson, NH, USA). 4.8. Immunoprecipitation and Immunoblotting Immunoprecipitation was performed according to the method of Cho et al. with a slight modification [31]. RAW 264.7 cells were plated in 6-well plates a density of 1 × 104 cells/well. The cells were treated with RANKL (50 ng/mL) and ACHE (1–20 µg/mL) or six compounds (10 µM) were added to DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. The medium was changed every 2 days. After 5 days, the cell were harvested using a cell scraper and centrifuged at 7500× g for 20 min. The cell pellets were lysed in NaCl EDTA Tris-nonyl phenoxypolyethoxylethanol (NP)-40 lysis (NET-NL) buffer (1 M Tris at pH 7.5, 0.5 M EDTA, 1 M NaCl, 1 M DTT, 10% NP-40, 0.1 M PMSF, 10 mg/mL bovine serum albumin (BSA), and protease inhibitor cocktail). The cell lysates were immunoprecipitated with 2 µg of TRAF6 and V-ATPase antibodies, and protein A–agarose beads by overnight incubation. The immune complexes were then washed three times with NaCl EDTA Tris-NP-40 washing (NET-NW) buffer (1 M Tris at pH 7.5, 0.5 M EDTA, 1 M NaCl, 1 M DTT, 10% NP-40, and 0.1 M PMSF) and centrifuged at 570× g for 30 s. The immunoprecipitated proteins were mixed with sample loading buffer, resolved by 8%–10% SDS-PAGE, and immunoblotted using anti-TRAF6, anti-V-ATPase, and anti-β-actin antibodies. The proteins on nirocellulose membranes were detected with a chemiluminescence kit (Intron Biotechnology, Seoul, Korea) and analyzed using a LAS4000 chemiluminescent image analyser (Fuji, Tokyo, Japan). 4.9. Statistical Analysis All data are presented as mean ± standard deviation. Statistical analysis were done by the statistical package for the social sciences (SPSS, Chicago, IL, USA) program. Student’s t-test was used the parameters between two groups. One-way analysis of variance (ANOVA) and Duncan’s test were used to compare the parameters among more than three groups and a p < 0.05 was considered statistically significant. 5. Conclusions The current report demonstrates that ACHE, an A. capillaris hydroethanolic extract, attenuated osteoclastic effect including RANKL-induced osteoclast differentiation and bone resorption activity; the responsible compounds for this effect were chlorogenic acid, hyperoside, and scoparone. The blockage of bone resorption by A. capillaris was in part mediated by reducing acidification through down-regulating the interaction of V-ATPase with TRAF6 due to scoparone as well as chlorogenic acid and hyperoside. For these reasons, A. capillaris extract may have a high potential as an important bioactive resource of functional food for the prevention or treatment of bone diseases caused by bone loss. However, additional studies using in vivo models and clinical trials are required. Int. J. Mol. Sci. 2017, 18, 322 13 of 14 Acknowledgments: This work was supported by a research grant from Hannam University, Dajeon, Korea in 2016. 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