Tung et al. BMC Complementary and Alternative Medicine (2015) 15:423 DOI /s

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1 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 DI /s RESEARCH ARTICLE Antioxidtive phytochemicls from Rhododendron oldhmii Mxim. lef extrcts reduce serum uric cid levels in potssium oxonte-induced hyperuricemic mice pen Access Yu-Tng Tung 1, Lei-Chen Lin 2, Y-Ling Liu 3, Shng-Tse Ho 3, Chi-Yng Lin 3, Hsio-Li Chung 4, Chien-Cho Chiu 5, Chi-Chng Hung 1* nd Jyh-Horng Wu 3* Abstrct Bckground: Some of the genus Rhododendron ws used in trditionl medicine for rthritis, cute nd chronic bronchitis, sthm, pin, inflmmtion, rheumtism, hypertension nd metbolic diseses nd mny species of the genus Rhododendron contin lrge number of phenolic compounds nd ntioxidnt properties tht could be developed into phrmceuticl products. Methods: In this study, the ntioxidtive phytochemicls of Rhododendron oldhmii Mxim. leves were detected by n online HPLC DPPH method. In ddition, the nti-hyperuricemic effect of the ctive phytochemicls from R. oldhmii lef extrcts ws investigted using potssium oxonte (P)-induced cute hyperuricemi. Results: Six phytochemicls, including (2R, 3R)-epictechin (1), (2R, 3R)-txifolin (2), (2R, 3R)-stilbin (3), hyposide (4), guijverin (5), nd quercitrin (6), were isolted using the developed screening method. f these, compounds 3, 4, 5, nd 6 were found to be mjor bioctive phytochemicls, nd their contents were determined to be ± 10.9, ± 8.5, ± 4.7, nd ± 4.0 mg per grm of EtAc frction, respectively. In ddition, the four mjor bioctive phytochemicls t the sme dosge (100 mmol/kg) were dministered to the bdominl cvity of potssium oxonte (P)-induced hyperuricemic mice, nd the serum uric cid level ws mesured fter 3 h of dministrtion. H&E stining showed tht P-induced kidney injury cused renl tubulr epithelium nucler condenstion in the cortex res or the ppernce of numerous hyline csts in the medull res; tretment with 100 mmol/kg of EtAc frction, (2R, 3R)-stilbin, hyposide, guijverin, nd quercitrin significntly reduced kidney injury. In ddition, the serum uric cid level ws significntly suppressed by 54.1, 35.1, 56.3, 56.3, nd 53.2 %, respectively, by the dministrtions of 100 mmol/kg EtAc frction nd the derived mjor phytochemicls, (2R, 3R)-stilbin, hyposide, guijverin, nd quercitrin, compred to the P group. The dministrtion of 10 mg/kg benzbromrone, well-known uricosuric gent, significntly reduced the serum uric cid level by 45.5 % compred to the P group. Conclusion: The in vivo decrese in uric cid ws consistent with free rdicl scvenging ctivity, indicting tht the mjor phytochemicls of R. oldhmii leve extrcts nd the derived phytochemicls possess potent hypouricemic effects, nd they could be potentil cndidtes for new hypouricemic gents. Keywords: Rhododendron oldhmii mxim, Lef, Phytochemicl, nline HPLC DPPH, Hyperuricemi * Correspondence: d @gmil.com; eric@nchu.edu.tw Equl contributors 1 Grdute Institute of Sports Science, Ntionl Tiwn Sport University, Toyun 333, Tiwn 3 Deprtment of Forestry, Ntionl Chung Hsing University, Tichung 402, Tiwn Full list of uthor informtion is vilble t the end of the rticle 2015 Tung et l. pen Access This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 Pge 2 of 10 Bckground The mjor function of xnthine oxidse (XD) is to ctlyze the oxidtion of hypoxnthine to xnthine nd to further ctlyze the oxidtion of xnthine to uric cid in purine metbolism [1]. Excess mounts of uric cid in the body result in the deposition of urte crystls in the joints nd kidneys, cusing inflmmtion s well s gouty rthritis nd uric cid nephrolithisis [2, 3]. Recent studies lso noted tht hyperuricemi is ssocited with risk of chronic nephritis, renl dysfunction, crdiovsculr diseses, hypertension, dibetes, nd metbolic syndrome [4, 5]. Thus, there hs been incresing interest in the serch of more effective or novel bioctive compounds in order to improve the insufficient uric cid excretion from wide vriety of trditionl herbl plnts [6 8]. The genus Rhododendron is widely distributed throughout most of the world, with the exception of Afric nd South Americ [9]. Some members of the genus Rhododendron were used in trditionl medicine for rthritis, cute nd chronic bronchitis, sthm, pin, inflmmtion, rheumtism, hypertension, nd metbolic diseses [10 12]. In ddition, Rhododendron sp. used in Unni system of medicine for tretment of gout [13, 14]. Recent studies hve shown tht mny species of the genus Rhododendron contin lrge number of phenolic compounds nd ntioxidnt properties tht could be developed into phrmceuticl products [15, 16]. In ddition, recent studies noted tht phenolics re strong XD-inhibitors [17] nd hve the potentil to lower the risk of hyperuricemi nd gout [18]. In previous studies, it ws found tht Rhododendron yedoense contins lrge mounts of flvonoids nd shows excellent XD-inhibitory ctivities [19]. Therefore, methnolic extrcts of R. oldhmii leves my be good cndidtes for further development s cliniclly relevnt ntihyperuricemic gents. However, to the best of our knowledge, there re no prior reports on R. oldhmii lef extrcts nd its derivtives possessing ntioxidnt ctivity in vitro nd hyperuricemic ctivity in vivo. Therefore, in this study, we investigted the hypouricemic effect of methnolic extrct nd its mjor phytochemicls from R. oldhmii leves in mice for the first time. Methods Chemicls Benzbromrone, potssium oxonte, 1,1 -diphenyl-2- picrylhydrzyl rdicl (DPPH. ), hypoxnthine, xnthine oxidse, nitroblue tetrzolium chloride (NBT), Folin- Cioclteu regent, potssium dihydrogen phosphte (KH 2 P 4 ), nd (+)-ctechin were purchsed from Sigm Chemicl Co. (St. Louis, M, USA). The other chemicls nd solvents used in this experiment were HPLC-grde. Plnt mterils The leves of Rhododendron oldhmii Mxim. from Lion Hed Mountin of Tipei county in Tiwn (Lt '16"N., Long '07"E.) were collected t the end of April The voucher specimen (voucher no. 6) ws deposited t the herbrium of the Deprtment of Forestry nd Nturl Resources, Ntionl Chiyi University (NCYU), Tiwn. The species were identified by Dr. Lei-Chen Lin (NCYU). The mterils were ir dried t mbient temperture (25 C) nd stored in refrigertor t 4 C prior to tretments. Extrction, frctiontion, nd isoltion Leves were soked in methnol t mbient temperture for 7 dys. The extrcts were decnted nd filtered through Whtmn No.1 filter pper, nd the filtrtes were concentrted in rotry evportor nd then lyophilized. Furthermore, the resulting methnolic crude extrcts of R. oldhmii were successively frctionted with n-hexne, ethyl cette (EtAc), n-butnol (Bu), nd wter to yield soluble frctions of hexne, EtAc, Bu, nd wter. The phytochemicls from the EtAc frction were seprted nd purified by semi-preprtive HPLC on Jsco PU-2080 instrument (Tokyo, Jpn) equipped with MD-2010 photo-diode rry detector (Jsco) nd 250 mm 10.0 mm i.d., 5-μm Supelco RPmide column (Bellefonte, PA, USA). The mobile phse ws solvent A, 100 % Me; nd solvent B, ultrpure wter. The elution conditions were 0 15 min of % A to B (liner grdient); min of % A to B (liner grdient); min of % A to B (liner grdient); nd 27 in of % A to B t flow rte of 4 ml/min. ESI-MS dt were collected by Finnign MAT-95S mss spectrometer, nd NMR spectr were recorded by Bruker Avnce 500 MHz FT- NMR spectrometer. The structures of compound 1, compound 2, compound 3, compound 4, compound 5, nd compound 6 were identified by ESI-MS nd NMR. Quntifiction The phytochemicls were quntified by LC-MS (Thermo, Dioxed UltiMte 3000 UHPLC; Bruker, mzon speed) with 250 mm 4.6 mm i.d., 2.6-μm C-18 column (Phenomenex, Torrnce, CA, USA). The mobile phse ws solvent A, 100 % Me; nd solvent B, ultrpure wter. The elution conditions were 0 15 min of % A to B (liner grdient); min of % A to B (liner grdient); min of % A to B (liner grdient); nd 27 in of % A to B t flow rte of 0.5 ml/min using detector. For the preprtion of the clibrtion curve, stndrd stock solutions of compounds were prepred in methnol, filtered through Millipore filters (0.45 μm), nd ppropritely diluted to obtin the desired concentrtions in the

3 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 Pge 3 of 10 quntifiction rnge. The clibrtion grphs were plotted fter the liner regression of the pek res versus concentrtions. DPPH rdicl-scvenging ctivity (DPPH ssy) ThescvengingctivityoftheDPPHfreerdiclbythe test smples ws determined ccording to the method reported by Ho et l. [20]. Ten μl ofthetestsmplesws mixed with 200 μl of 0.1 mm DPPH-ethnol solution nd 90 μl of 50 mm Tris HCl buffer (ph 7.4). Methnol (10 μl) lone ws used s the control in this experiment. After 30 min of incubtion t room temperture, the reduction in DPPH free rdicls ws mesured by reding the bsorbnce t 517 nm using Thermo Scientific Multiskn G microplte reder (Vnt, Finlnd). (+)-Ctechin ws used s the positive control. The inhibition rtio ws clculted using the following eqution: % inhibition = [(bsorbnce of control bsorbnce of test smple)/ bsorbnce of control] 100. Superoxide rdicl-scvenging ctivity (NBT ssy) The mesurement of superoxide rdicl-scvenging ctivity ws crried out using the method described by Tung et l. [21], nd (+)-ctechin ws used s the stndrd. First, 20 μl of15mmn 2 EDTA in buffer (50 mm KH 2 P 4 /K, ph 7.4), 50 μl of 0.6 mm NBT in buffer, 30 μl of 3 mm hypoxnthine in 50 mm K, 5 μl of the test smples in methnol, nd 145 μl of buffer were mixed in 96-well micropltes (Flcon, USA). The rection ws initited by dding 50 μl of xnthine oxidse solution in buffer (1 unit in 10 ml buffer) to the mixture. The rection mixture ws incubted t room temperture, nd the bsorbnce t 570 nm ws determined every 20 s up to 5 min using plte reder. The control ws 5 μl of methnol insted of the smple solution. (+)-Ctechin ws used s the positive control. The inhibition rtio ws clculted using the following eqution: % Inhibition = [(rte of control rte of smple - rection)/ rte of control] 100. Reducing power ssy This ssy ws determined ccording to the method reported by Lin et l. [16], with slight modifictions. Briefly, 1 ml of the rection mixture contining 500 μl the test extrcts or compounds in 500 μl phosphte buffer (0.2 M, ph 6.6) ws incubted with 500 μl potssium ferricynide (1 %, w/v) t 50 C for 20 min. The rection ws terminted by dding trichlorocetic cid (10 %, w/v), nd the mixture ws centrifuged t 3000 rpm for 10 min. The superntnt solution (500 μl) ws mixed with distilled wter (500 μl) nd 100 μl of ferric chloride (0.1 %, w/v) solution, nd the bsorbnce ws mesured t 700 nm. The reducing bility ws expressed s (+)-ctechin equivlents (CE) in milligrms per grm smple. Determintion of totl phenolics The totl phenolic content ws determined ccording to the Folin-Cioclteu method [16], using gllic cid s the stndrd. The test smple (5 mg) ws dissolved in 5 ml of methnol/wter (50:50, v/v). The extrct solution (500 μl) ws mixed with 500 μl of 50 % Folin-Cioclteu regent. The mixture ws kept for 5-min period, which ws followed by the ddition of 1.0 ml of 20 % N 2 C 3. After 10 min of incubtion t room temperture, the mixture ws centrifuged for 8 min (1200 g) nd the bsorbnce of the superntnt ws mesured t 730 nm. The totl phenolic content ws expressed s gllic cid equivlents (GAE) in milligrms per grm smple. n-line DPPH rdicl-scvenging nlysis The best ntioxidnt ctivity of the extrct (EA frction) from R. oldhmii leves ws further monitored by the on-line RP HPLC DPPH method. The EA frction (stock concentrtion = 20 mg/ml) ws monitored by nlytic HPLC on model PU-2080 instrument (Jsco, Jpn) with 250 mm 10.0 mm i.d., 5-μm Supelco RPmide column (Bellefonte, PA, USA). The mobile phse ws solvent A, 100 % Me; nd solvent B, ultrpure wter. The elution conditions were 0 15 min of % A to B (liner grdient); min of % A to B (liner grdient); min of % A to B (liner grdient); nd 27 in of % A to B t flow rte of 4 ml/min, using Jsco MD-2010 photo diode rry t 280 nm wvelength. For the on-line DPPH rdicl-scvenging nlysis, the flow of DPPH regent (300 mg/l in methnol) ws set to 4 ml/min, nd the induced bleching ws detected photometriclly s negtive pek t 517 nm. Animls Mle ICR mice with body weight of pproximtely 30 g (8 weeks old) were purchsed from BioLASC (A Chrles River Licensee Corp., Yi-Ln, Tiwn). Mice were given stndrd lbortory diet nd distilled wter d libitum, nd they were kept on 12-h light/drk cycle t 22 ± 2 C. All niml experimentl protocols were pproved by the Institutionl Animl Cre nd Use Committee (IACUC) of Ntionl Tiwn Sport University, nd the study conformed to the guidelines of the protocol IACUC pproved by the IACUC ethics committee. Potssium oxonte (P) induced hyperuricemi in mice For the hyperuricemi study, potssium oxonte (P), n uricse inhibitor, ws employed to induce cute hyperuricemi ccording to Tung et l. [22]. Sixty-four mice were rndomly ssigned to eight groups for tretment (n = 8 per group): (1) vehicle group; (2) P group; (3) P + BZM group; (4) P + EA group; (5) P + AS

4 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 Pge 4 of 10 group; (6) P + HP group; (7) P + GJ group; nd (8) P + QR group. Briefly, mice were intrperitonelly (i.p.) injected with PBS contining P (200 mg/kg) 1 h before the test smples were dministered to increse the serum uric cid level. For comprtive study, the sme dosge t 100 mmol/kg of the EtAc frction (EA, 45.0 mg/kg), (2R, 3R)-stilbin (AS, 45.0 mg/kg), hyposide (HP, 46.4 mg/kg), guijverin (GJ, 43.4 mg/kg), nd quercitrin (QR, 44.8 mg/kg) dissolved in DMS were delivered i.p. for 1 h post P dministrtion. In this study, 10.0 mg/kg benzbromrone (BEM), well-known uricosuric gent, ws used s the reference control. Pthologicl histology Kidney tissue ws fixed in 10 % buffered formldehyde nd exmined using hemtoxylin nd eosin (H&E) stining. Mesurement of serum BUN, cretinine, nd uric cid level Blood smples were collected by retro-orbitl bleeding fter 3 h P dministrtion. Blood smples were centrifuged t 1,400 g t 4 C for 15 min, nd the levels of BUN, cretinine nd uric cid in the serum superntnts were mesured using n utonlyzer (Hitchi 7060, Hitchi, Jpn). Sttisticl nlysis The dt for in vitro nd in vivo ssys re expressed s the men ± SD (n = 3) nd the men ± SEM (n = 8), respectively. The significnce of difference ws clculted by Scheffe s test, nd the results with P < 0.05 were considered to be sttisticlly significnt. Results nd discussion DPPH rdicl-scvenging ctivity of R. oldhmii lef extrct nd its derived soluble frctions In the present study, the free rdicl-scvenging ctivity of R. oldhmii lef extrct ws ssessed by DPPH ssy. Accordingly, s shown in Fig. 1, the DPPH rdicl-scvenging ctivity of the methnolic extrct nd its derived soluble frctions from leves of R. oldhmii, including the soluble frctions of n-hexne, EtAc, Bu, nd wter, were shown in dose-dependent mnner. f these, the EtAc frction showed the strongest ctivity. Menwhile, except for the n-hexne nd wter-soluble frctions, ll extrcts showed good inhibitory ctivity ginst the DPPH rdicls. The IC 50 vlues (the concentrtion required to inhibit rdicl formtion by 50 %) of the crude extrct, hexne, EtAc, Bu, nd wter frction were 7.5 ± 0.2, 77.6 ± 2.3, 5.0 ± 0.1, 6.1 ± 0.1 nd 30.0 ± 0.6 μg/ml, respectively. The IC 50 vlue of (+)-ctechin, well-known ntioxidnt compound used s the reference control in this study, Fig. 1 Antioxidnt ctivities of the methnolic extrct nd its derived soluble frctions from the leves of R. oldhmii. DPPH rdicl scvenging ctivity. b Superoxide rdicl scvenging ctivity. c Reducing power. d Totl phenolic contents. The results re presented s the men ± SD (n =3). The brs mrked by different letters re significntly different t the level of P < 0.05, ccording to Scheffe s test

5 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 Pge 5 of 10 ws 1.9 ± 0.0 μg/ml. Lin et l. [16] reported tht the IC 50 vlues of the crude extrcts of Rhododendron species incresed in the following order: R. pseudochrysnthum (7.5 μg/ml), R. oldhmii (7.5 μg/ml), R. knehiri (7.7 μg/ml), R. breviperultum (8.8 μg/ml), R. rubropilosum vr. tiwnlpinum (10.4 μg/ml), R. formosnum (10.7 μg/ml), R. simsii (11.8 μg/ml), R. rubropilosum vr. rubropilosum (12.1 μg/ml), R. ellipticum (14.2 μg/ml), nd R. mriesii (14.7 μg/ml). These results showed tht the crude extrcts of the Rhododendron species exhibited good DPPH rdicl scvenging ctivity. Superoxide rdicl-scvenging ctivity of R. oldhmii lef extrct nd its derived soluble frctions Superoxide rdicl ws generted by the hypoxnthinexnthine oxidse nd NBT systems in this ssy. Figure 1b shows the superoxide rdicl-scvenging ctivity of the methnolic extrct nd its derived frctions from R. (A) Injector Mixer RP-mide column Detector H 2 Me Pump Rhododendron (Lef extrct) Pump DPPH solution n-line RP HPLC DPPH chromtogrms Absorbnce (mau) nm 517 nm Retention time (min) (B) H 6 1' R 1 R 2 1: R 1 = H 2 ; R 2 = H 2: R 1 = ; R 2 = H 3: R 1 = ; R 2 = Rhm H R 1' 4: R = Gl 5: R = Ar 6: R = Rhm H H CH 3 Rhm = -L-Rhmnopyrnosyl CH 2 H Gl = -D-Glctopyrnosyl H Ar = -L-Arbinopyrnosyl Fig. 2 The online HPLC DPPH chromtogrms of the EtAc frction from R. oldhmii lef extrct. b Isolted nd identified phytochemicls: (2R, 3R)-epictechin (1), (2R, 3R)-txifolin (2), (2R, 3R)-stilbin (3), hyposide (4), guijverin (5), nd quercitrin (6)

6 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 Pge 6 of 10 oldhmii leves compred with (+)-ctechin. At the 10 μg/ml test concentrtion, the superoxide rdicl inhibition of R. oldhmii lef extrct nd its derived frctions decresed in the following order: Bu frction (49.6 %) > EtAc frction (42.6 %) > crude extrct (37.7 %) > wter frction (9.1 %) > n-hexne frction (4.7 %). The IC 50 vlues of (+)-ctechin, crude extrct, n-hexne frction, EtAc frction, Bu frction, nd wter frction were 15.1 ± 0.5, 18 ± 2.1, ± 9.0, 11.1 ± 0.4, 9.6 ± 0.6, nd 60.1 ± 4.8 μg/ml, respectively. The comprisons of these dt reveled tht the crude extrct, the EtAc frction, nd the Bu frction exmined in this study exhibited gret superoxide rdiclscvenging ctivity. The present study reveled tht the EtAc frction nd the Bu frction showed excellent performnces in superoxide rdicl inhibition nd their inhibitory ctivities were better thn tht of (+)-ctechin. Lin et l. [16] showed tht mong ll soluble frctions from R. pseudochrysnthum leves, both the EtAc frction nd the Bu frction exhibited the best superoxide rdicl-scvenging ctivity. This my be becuse the mjor components of the EtAc nd Bu frctions were flvonoids nd phenolic cids, which hve gret superoxide rdicl-scvenging effects. Reducing power of R. oldhmii lef extrct nd its derived soluble frctions The reducing power of the crude extrct nd its derived frctions ws clculted s (+)-ctechin equivlents (CE) in milligrms per grm smple. As shown in Fig. 1c, the reducing power of the EtAc frction (367.9 ± 6.3 mg/g) ws higher thn tht of the Bu frction (357.4 ± 9.3 mg/g), the crude extrct (285.2 ± 4.1 mg/g), the wter frction (89.3 ± 2.8 mg/g), nd the hexne frction (19.5 ± 0.5 %). These results reveled tht the EtAc frction possessed the highest ntioxidnt ctivity, which ws the sme s the DPPH rdicl-scvenging ctivity results. These results imply tht there is n bundnce of ntioxidtive phytochemicls present in the EtAc frction. Totl phenolic contents of R. oldhmii lef extrct nd its derived soluble frctions Phenolic compounds re commonly found in the plnt kingdom, nd they hve been reported to hve multiple biologicl effects [23, 24]. A correltion between the content of phenolic compounds nd ntioxidnt ctivities hs been described in mny studies [25 27]. The phenolic compounds re very importnt plnt constituents becuse of their bility to scvenge free rdicls. Figure 1d shows the content of totl phenolics in the crude extrct nd its derived frctions clculted s gllic cid equivlents (GAE) in milligrms per grm of smple. Apprently, the totl phenolic content of the EtAc frction (330.3 ± 3.1 mg/g) ws higher thn tht of the Bu frction (295.4 ± 2.9 mg/g), the crude extrct (217.6 ± 2.4 mg/g), the wter frction (66.4 ± 7.2 mg/g), nd the hexne frction (16.6 ± 0.6 mg/g). These results imply tht there were bundnt ntioxidtive phytochemicls present in the lef extrct of R. oldhmii, especilly in the EtAc frction. n-line RP HPLC DPPH method The on-line RP HPLC DPPH method cn be used for rpid ssessment of pure ntioxidnt compounds in complex mixtures, prticulrly plnt extrcts [28]. The more rpidly the bsorbnce decreses, the more potent the ntioxidnt ctivity of the compound will be in terms of hydrogen-donting bility [29]. The combined UV (positive signls) nd DPPH quenching (negtive signls) chromtogrms under grdient conditions of the EtAc frction from the lef extrct of R. oldhmii re presented in Fig. 2. Severl eluted phytocompounds in the EtAc frction were detected nd gve positive peks on the UV detector (280 nm). Among them, (2R, 3R)-epictechin (1), (2R, 3R)-txifolin (2), (2R, 3R)-stilbin (3), hyposide (4), guijverin (5), nd quercitrin (6) (Fig. 2b) showed hydrogen-donting cpcity (negtive pek) towrds the DPPH rdicl t the pplied concentrtion. The results reveled tht the method cn be pplied for quick screening of ntioxidnt compounds or to more precisely determine the rdiclscvenging ctivity of compounds. Thus, it is no longer necessry to isolte nd purify non-trget phytocompounds, leding to very significnt reductions in costs nd fster results. Quntifiction nd ntioxidnt ctivities of mjor ctive compounds in lef extrct of R. oldhmii According to the screening results from the on-line RP HPLC DPPH system, (2R, 3R)-epictechin (1), (2R, 3R)-txifolin (2), (2R, 3R)-stilbin (3), hyposide Tble 1 Antioxidnt ctivities nd contents of mjor phytochemicls of the EtAc frction from R. oldhmii leves Phytochemicls Content (mg/g of EtAc frction) IC 50 (μm) DPPH rdicl scvenging Superoxide rdicl scvenging (2R,3R)-Epictechin (1) 15.0 ± ± ± 1.3 (2R, 3R)-Txifolin (2) 27.0 ± ± 0.2 bc 17.9 ± 1.0 b (2R, 3R)-Astilbin (3) ± ± 0.5 b 33.2 ± 1.7 Hyperoside (4) ± ± 0.4 c 16.1 ± 0.3 bc Guijverin (5) ± ± 0.1 bc 15.8 ± 0.4 bc Quercitrin (6) ± ± 0.2 c 13.0 ± 0.9 c Different letters within column indicte significnt difference t the P < 0.05 level ccording to Scheffe s test

7 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 (4), guijverin (5) nd quercitrin (6) were found to be the mjor bioctive phytochemicls in the EtAc frction, nd their contents were determined to be 15.0 ± 0.3, 27.0 ± 0.4, ± 10.9, ± 8.5, ± 4.7, nd ± 4.0 mg per grm of EtAc frction, respectively (Tble 1). To determine the ntioxidnt ctivities of these mjor ctive compounds, DPPH nd NBT ssys were performed. As shown in Tble 1, the IC50 vlues for the DPPH rdicl-scvenging ctivity of these mjor phytochemicls were 8.4 ± 0.2, 7.6 ± 0.2, 8.2 ± 0.5, 6.8 ± 0.4, 7.4 ± 0.1, nd 6.9 ± 0.2 μm, respectively. However, for superoxide rdicl-scvenging ctivity, Vehicle Pge 7 of 10 the IC50 vlues of these compounds were 34.1 ± 1.3, 17.9 ± 1.0, 33.2 ± 1.7, 16.1 ± 0.3, 15.8 ± 0.4, nd 13.0 ± 0.9 μm, respectively. The DPPH nd NBT ssys showed tht compounds 4, 5 nd 6 hd better ctivities. These results demonstrted tht flvonoids contining the 2,3 double bond in the C ring hve better ntioxidnt ctivities. In contrst, flvns hve sturted heterocyclic C ring, nd the consequent lck of conjugtion between the A nd B rings mens tht electrons re less ble to be deloclized from the B ring to the A ring, thus lowering their ntioxidnt ctivities. P + AS P P + HP P + BZM P + GJ P + QR P + EA Fig. 3 The pthologicl histology of the EtAc frction nd its derived phytochemicls of R. oldhmii lef extrct in P-induced hyperuricemic mice. The H&E stining showed tht P-induced kidney injury cused renl tubulr epithelil nucler condenstion (kryopyknosis) in the cortex or the ppernce of numerous hyline csts in the medull res. The levels of renl tubulr epithelil nucler condenstion or numerous hyline csts were significntly reduced by benzbromrone, the EtAc frction, (2R, 3R)-stilbin, hyposide, guijverin, nd quercitrin tretments

8 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 Pge 8 of 10 Anti-hyperuricemic effect in hyperuricemic mice Benzbromrone nd llopurinol re widely used for the tretment of hyperuricemi. Allopurinol inhibits RS production by working s n X inhibitor but not s rdicl scvenger, wheres benzbromrone decreses RS production directly nd indirectly by working s both uric cid trnsporter 1 inhibitor nd rdicl scvenger [30]. In this study, the mjor compounds of R. oldhmii lef extrct were good rdicl scvengers. Thus, the nti-hyperuricemic effect of R. oldhmii lef extrct nd its phytochemicls on P-induced hyperuricemi in mice ws studied, nd benzbromrone ws the reference control used in this study. The hyperuricemic mice were generted by single intrperitonel injection of P (200 mg/kg). P, urte oxidse inhibitor, cn rise the serum uric cid concentrtion by inhibiting the decomposition of uric cid by uricse [31]. H&E stining showed tht P-induced kidney injury cused renl tubulr epithelium nucler condenstion (kryopyknosis) in the cortex or the ppernce of numerous hyline csts in the medull res (Fig. 3). The levels of renl tubulr epithelil nucler condenstion or numerous hyline csts were significntly reduced fter the dministrtion of 100 mmol/kg of EtAc frction, (2R, 3R)-stilbin, hyposide, guijverin, nd quercitrin. Xiong et l. [32] lso exhibited tht NF-κB plys role in the pthogenesis of chronic glomerulonephritis, nd rhododendron root my ttenute renl dmges by downregulting the ctivtion of NF-κB. The hyperuricemic nimls 3 h fter i.p. of P exhibited higher serum levels of BUN (40.2 ± 1.2 mg/dl), cretinine (0.44 ± 0.02 mg/dl), nd uric cid (2.9 ± 0.3 mg/dl) when compred to the vehicle control group (16.6 ± 0.7, 0.10 ± 0.01, nd 0.8 ± 0.1 mg/dl, respectively). Therefore, t 3 h post P injection, the serum uric cid level showed more thn 3-fold increse compred with the vehicle control (P < 0.05). The dministrtion of benzbromrone (10 mg/kg) significntly reduced the serum uric cid level by 45.5 % compred to the P group. At n equimolr dose (100 mmol/kg), nimls treted with the EtAc frction, (2R, 3R)-stilbin, hyposide, guijverin, nd quercitrin showed significnt reduction in uric cid by 54.1, 35.1, 56.3, 56.3, nd 53.2 %, respectively, compred to the P group (P < 0.05) (Fig. 4). In ddition, treting hyperuricemic mice with benzbromrone, the EtAc frction, (2R, 3R)-stilbin, hyposide, guijverin, nd quercitrin slightly reduced both cretinine nd uric cid serum levels. Wng et l. [33] reported tht cinnmldehyde hd good inhibitory ctivity on xnthine oxidse ctivity nd could significntly reduce the serum uric cid level by 60 % compred with the P group t dosge of 150 mg/kg. Tung et l. [22] reported tht the flvonoids of Acci confus hertwood, ( )-2,3-cis-3,4-cis- 3,3,4,4,7,8-hexhydroxyflvn, ( )-2,3-cis-3,4-cis-4 -methoxy- 3,3,4,7,8-penthydroxyflvn, melnoxetin, trnsilitin, (A) BUN (mg/dl) (B) Cretinine (mg/dl) (C) Uric cid (mg/dl) P Tretment b b c b bc BZM EA AS HP GJ nd oknin, showed good inhibitory ctivity on xnthine oxidse ctivity, nd significntly reduced the serum uric cid level by 66, 72, 75, 65 nd 69 %, respectively, compred with the P group. Zhu et l. [34] lso found tht dministrtion of the flvonoids quercetin nd rutin significntly reduced the serum uric cid level by 36 % nd 32 %, respectively, t dosges of 150 mg/kg. Comprisons of the forementioned results indicted tht the mjor flvonoids from the R. oldhmii lef extrct hve n bc b bc bc bc QR Fig. 4 The reductions in serum BUN, cretinine, nd the uric cid level by the EtAc frction nd its derived phytochemicls of R. oldhmii lef extrct in P-induced hyperuricemic mice. The results re presented s the men ± SEM of eight mice. Different letters re significntly different t the level of P < 0.05 ccording to the Scheffe s test

9 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 Pge 9 of 10 excellent effect on reducing urte levels. Among the flvonoid constituents exmined, hyposide, guijverin, nd quercitrin were comprble with benzbromrone nd showed n excellent effect on reducing urte levels. Conclusion It is well-known tht RS hve high correltion with severl diseses, such s geing, therosclerosis, inflmmtory injury, cncer, nd crdiovsculr disese. This study demonstrted, for the first time, tht mong the lef extrcts of R. oldhmii, the EtAc frction exhibited the highest ntioxidnt ctivity. Thus, the EtAc frction ws pplied to the online HPLC DPPH method, nd six specific nd excellent ntioxidnts were detected nd identified. In ddition, the study lso demonstrted tht the mjor constituents of R. oldhmii lef extrcts possessed potent in vivo hypouricemic effects in hyperuricemic mice pretreted with potssium oxonte. Thus, the dietry use of R. oldhmii lef extrcts nd their constituents my provide some options for the prevention nd/or tretment of hyperuricemi. Competing interests The uthors declre no competing of interests. Authors contributions Yu-Tng Tung, Chi-Chng Hung nd Jyh-Horng Wu designed the experiments. Lei-Chen Lin, Y-Ling Liu, Shng-Tse Ho, Chi-Yng Lin, Hsio-Li Chung, Chien-Cho Chiu, Chi-Chng Hung nd Jyh-Horng Wu crried out the lbortory experiments. Yu-Tng Tung, Chi-Chng Hung nd Jyh-Horng Wu nlyzed the dt, interpreted the results, prepred figures, nd wrote the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgement This work ws finncilly supported by reserch grnt from the Ntionl Chung Hsing University. Author detils 1 Grdute Institute of Sports Science, Ntionl Tiwn Sport University, Toyun 333, Tiwn. 2 Deprtment of Forestry nd Nturl Resources, Ntionl Chiyi University, Chiyi 600, Tiwn. 3 Deprtment of Forestry, Ntionl Chung Hsing University, Tichung 402, Tiwn. 4 Ntionl Lbortory Animl Center, Ntionl Applied Reserch Lbortories, Tipei 11529, Tiwn. 5 Division of Animl Resource, Animl Technology Lbortories, Agriculturl Technology Reserch Institute, Hsinchu City 30093, Tiwn. Received: 19 September 2015 Accepted: 26 November 2015 References 1. Hrris MD, Siegel LB, Allowy JA. Gout nd hyperuricemi. Am Fm Physicin. 1999;59: Krmer HM, Curhn G. The ssocition between gout nd nephrolithisis: the Ntionl Helth nd Nutrition Exmintion Survey III, Am J Kidney Dis. 2002;40: Tomit M, Mizuno S, Ymnk H, Hosod Y, Skum K, Mtuok Y, et l. Does hyperuricemi ffect mortlity? A prospective cohort study of Jpnese mle workers. J Epidemiol. 2000;10: Lee JM, Kim HC, Cho HM, h SM, Choi DP, Suh I. 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10 Tung et l. BMC Complementry nd Alterntive Medicine (2015) 15:423 Pge 10 of Wng SY, Yng CW, Lio JW, Zhen WW, Chu FH, Chng ST. Essentil oil from leves of Cinnmomum osmophloeum cts s xnthine oxidse inhibitor nd reduces the serum uric cid levels in oxonte-induced mice. Phytomedicine. 2008;15: Zhu JX, Wng Y, Kong LD, Yng C, Zhng X. Effects of Biot orientlis extrct nd its flvonoid constituents, quercetin nd rutin on serum uric cid levels in oxonte-induced mice nd xnthine dehydrogense nd xnthine oxidse ctivities in mouse liver. J Ethnophrmcol. 2004;93: Submit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-submission inquiries ur selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online submission Thorough peer review Inclusion in PubMed nd ll mjor indexing services Mximum visibility for your reserch Submit your mnuscript t