608 Synergistic effect of rhein in combintion with mpicillin or oxcillin ginst methicillin-resistnt Stphylococcus ureus DAE-KI JOUNG 1, HEE JOUNG 1, DA-WUN YANG 1, DONG-YEUL KWON 2, JANG-GI CHOI 2, SEO WOO 3, DONG-YOUNG SHIN 3, OH-HYUN KWEON 4, KEE-TAE KWEON 5 nd DONG-WON SHIN 1 1 Deprtment of Orientl Medicine Resources, College of Bio Industry Science, Sunchon Ntionl University, Sunchon, Jeonnm 540-742; 2 Deprtment of Orientl Phrmcy, College of Phrmcy, Wonkwng Orientl Medicines Reserch Institute, Wonkwng University, Iksn, Jeonbuk 570-749; 3 Deprtment of Development in Plnt Resources, College of Bio Industry Science, Sunchon Ntionl University, Sunchon, Jeonnm 540-742; 4 Deprtment of Physicl Therpy, Andong Science College, Seohu-Myeon, Andong-Si, Gyongbuk 760-820; 5 Ministry of Helth nd Welfre Office for Helthcre Policy Division of Trditionl Koren Medicine, Jongro-gu, Seoul 110-793, Republic of Kore Received October 22, 2011; Accepted December 28, 2011 DOI: 10.3892/etm.2012.459 Abstrct. Methicillin-resistnt Stphylococcus ureus (MRSA) is bcterium responsible for number of infections in humns tht re difficult to tret, nd s result, is substntil contributor to morbidity nd mortlity. In the present study, in serch of nturl products cpble of inhibiting this multidrug-resistnt bcterium, we investigted the ntimicrobil ctivity of rhein isolted from Rheum plmtum L. (Polygoncee) ginst 16 different strins of the bcterium. New ntimicrobil ctivity ws found using the pper disc diffusion method [miniml inhibitory concentrtions (MICs)], MTT test nd checkerbord dilution test. Aginst the 16 strins, the disc diffusion test ws in the rnge of 20-29 mm nd the MICs of rhein were in the rnge of 7.8-31.25 µg/ml. From these results we performed the checkerbord test to determine the synergism of rhein in combintion with mpicillin (AM) or oxcillin (OX) ginst ll strins. The combined ctivity of rhein nd the two ntimicrobil gents (AM nd OX) ginst ll strins resulted in frctionl inhibitory concentrtion index rnging from 0.28-1 nd from 0.18-1, respectively. The effect of rhein with AM nd OX ws found to be synergistic or prtilly synergistic. We found tht rhein reduced the MICs of AM nd OX. Rhein in combintion with AM or OX could led to the development of new combintions of ntibiotics ginst MRSA infection. Introduction Methicillin-resistnt Stphylococcus ureus (MRSA) is serious nd urgent clinicl problem worldwide. Few new drugs re vilble ginst MRSA, since it hs the bility to cquire resistnce to most ntibiotics. An effective method for treting MRSA my be to develop ntibiotics from nturl products which re likely to hve lower toxicity nd fewer side effects. Rheum plmtum, populrly known s Dhung, hs trditionlly been used s n orientl folk medicine. Rhein nturlly occurs in nthrquinone (1,3,8-trihydroxy-6-methyl nthrquinone) (Fig. 1), which is found in Rheum plmtum nd relted plnts such s rhubrb. Anthrquinone components include loe-emodin, rhein, emodin, chrysophnol nd physcion (1). Rhein is used for treting numerous complints, including constiption, jundice, gstrointestinl hemorrhge nd ulcers (2). It hs lso been shown to hve good ntitumor (3-6) nd nti-inflmmtory properties (7,8), nticncer (9), purgtive (10), nephric protection (11,12), liver protection (13), ntimicrobil nd hemosttic properties (14,15). As such, it hs been widely used for the tretment of gstrointestinl diseses, heptitis nd chronic renl filure. However, little is known bout its ntimicrobil effects on MRSA. Thus, we present the current study demonstrting the ntimicrobil ctivity of rhein ginst MRSA nd methicillin-sensitive S. ureus (MSSA) strins, s well s its synergistic effect. Mterils nd methods Correspondence to: Dr Dong-Won Shin, Deprtment of Orientl Medicine Resources, College of Bio Industry Science, Sunchon Ntionl University, Sunchon, Jeonnm 540-742, Republic of Kore E-mil: sdw@sunchon.c.kr Key words: ntibcteril, methicillin-resistnt Stphylococcus ureus, rhein, Rheum plmtum Test microorgnisms. Among the 16 S. ureus strins used in this study, 14 clinicl isoltes of MRSA were obtined from 14 different ptients t Wonkwng University Hospitl (Iksn, Republic of Kore). The other two strins used were S. ureus ATCC 33591 (methicillin-resistnt strin) nd S. ureus ATCC 25923 (methicillin-susceptible strin). ATCC 25923 (Americn Type Culture Collection, Mnsss, VA, USA) nd ATCC 33591 were commercilly purchsed. Before use, ll
JOUNG et l: METHICILLIN-RESISTANT Stphylococcus ureus 609 Tble I. The S. ureus strins used in the experiments. S. ureus Clss meca gene Antibiotic strin resistnce pttern Figure 1. Structures of importnt nthrquinone derivtives. Rhein: R 1 =COOH; R 2 =H. Aloe-emodin: R 1 =CH 2 OH; R 2 =H. Emodin: R 1 =CH 3 ; R 2 =OH. Chrysophnol: R 1 =CH 3 ; R 2 =H. Physcion: R 1 =CH 3 ; R 2 =OCH 3. bcteri were stored in 30% glycerol nd frozen t -70 C. The bcteri were cultured in Mueller-Hinton broth (MHB) nd Mueller-Hinton gr (MHA) (Difco Lbortories, Bltimore, MD, USA). Bcteri were suspended in MHB nd then incubted t 37 C for 24 h. Disc diffusion method. The disc diffusion method ws performed s described by the Clinicl nd Lbortory Stndrds Institute (CLSI) nd by using modified gr well diffusion method (CLSI, 2001) (16). Bcteril strins grown on MHA t 37 C for 18 h were suspended in MHB nd djusted to turbidity of 0.5 on the McFrlnd stndrd scle (pproximtely 1.5x10 8 CFU/ml). The MHA ws poured into Petri dishes nd inoculted with 100 µl of the suspension. Sterile pper discs (dimeter, 6 mm; Tokyo Roshi Kihs, Jpn) were punched into the gr nd ech well ws filled with 500 nd 250 µg of ech drug. The dissolution of the mpicillin (AM), oxcillin (OX) nd rhein ws fcilitted by the ddition of 50% (v/v) DMSO (50% DMSO ws not ctive ginst ll strins) (DMSO; Sigm, St. Louis, MO, USA). AM nd OX were used s the positive controls, nd the discs treted with DMSO were used s the negtive control. The pltes were plced in n incubtor t 37 C for 18 h in the drk. The inhibition zone dimeter round ech of the discs ws mesured nd recorded t the end of the incubtion period. Minimum inhibitory concentrtion. The minimum inhibitory concentrtion (MIC) ws determined using the broth microdilution method ccording to the CLSI guidelines, 2000 (17). Briefly, the inocultion of the microorgnisms ws crried out on 24 h broth cultures, nd the suspensions were djusted to 0.5 McFrlnd stndrd turbidity (pproximtely 1.5x10 8 CFU/ ml). Finl inoculum size ws djusted to 1.5x10 6 CFU/ml. These serilly diluted cultures were then incubted t 37 C for 18 h. MIC ws defined t the lowest concentrtion of AM, OX nd rhein. At the end of the incubtion period, the well pltes were visully exmined for turbidity. Cloudiness indicted tht bcteril growth hd not been inhibited by the concentrtion of the ntimicrobil gent contined in the medium. Checkerbord dilution test. The synergistic combintions were investigted in the preliminry checkerbord method performed using the MRSA nd MSSA strins, nd one clinicl isolte strin vi MIC determintion, ccording to ATCC 25923 MSSA - - ATCC 33591 MRSA + AM, OX DPS-1 MRSA + AM, OX DPS-2 MRSA + AM, OX DPS-3 MRSA + AM, OX DPS-4 MRSA + AM, OX DPS-5 MRSA + AM, OX DPS-6 MRSA + AM, OX DPS-7 MRSA + AM, OX DPS-8 MRSA + AM, OX DPS-9 MRSA + AM, OX DPS-10 MRSA + AM, OX DPS-11 MRSA + AM, OX DPS-12 MRSA + AM, OX DPS-13 MRSA + AM, OX DPS-14 MRSA + AM, OX Stphylococcus ureus strins from the Deprtment of Plstic Surgery, Wonkwng University Hospitl, Iksn, Kore. (+), positive; (-), negtive; AM, mpicillin; OX, oxcillin. the CLSI guidelines (18). The MIC ws defined s the lowest concentrtion of drug lone or in combintion tht inhibited the visible growth. The in vitro interction ws quntified by determining the frctionl inhibitory concentrtion (FIC). The FIC index ws clculted s follows: FIC = (MIC of drug A in combintion/mic of drug A lone) + (MIC of drug B in combintion/mic of drug B lone). FIC indices (FICIs) were interpreted s follows: <0.5, synergy; 0.5-0.75, prtil synergy; 0.76-1.0, dditive effect; >1.0-4.0, indifference; nd >4.0, ntgonism. Finlly, the vrying rtes of synergy between the two gents were determined. All experiments were independently repeted three times. Colorimetric ssy using MTT test. A colorimetric ssy bsed on MTT for the rpid detection of the presence of bcteri ws performed s previously described (19-21). Briefly, stock solution of 5 mg/ml MTT (Sigm) ws prepred in phosphtebuffered sline nd kept t -70 C. A finl concentrtion of 1 mg/ml of MTT ws used in the ssy. Following 24 h of incubtion 37 C, 20 µl of the yellow MTT ws dded to the 96-well microtiter plte (0.3 ml volume) nd incubted for n dditionl 20 min. The presence of blue color indicted the presence of bcteri. Results Tble I shows the S. ureus strins used in the experiments. Tble II shows the mens of inhibition zones produced ginst the tested bcteri rnged between 20 nd 29 mm. The growth of ll the tested strins of MRSA nd MSSA ws inhibited t 500 nd 250 µg/disc. As shown in Tble III, rhein demon-
610 Tble II. The ntimicrobil ctivity (s inhibitory zone dimeters) of rhein, AM nd OX ginst the S. ureus strin. Zone of inhibition (mm) -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Rhein (µg/ml) Ampicillin (µg/ml) Oxcillin (µg/ml) ------------------------------------------------- ------------------------------------------------ ------------------------------------------------- S. ureus strin 500 250 500 250 500 250 ATCC 25923 25 22 43 41 37 35 ATCC 33591 29 23 16 15 18 15 DPS-1 19 14 18 14 ND ND DPS-2 25 20 18 17 17 15 DPS-3 21 17 21 16 ND ND DPS-4 21 18 20 15 ND ND DPS-5 22 18 17 14 ND ND DPS-6 20 16 17 14 ND ND DPS-7 21 18 11 10 ND ND DPS-8 21 17 12 11 ND ND DPS-9 23 19 13 12 ND ND DPS-10 25 20 12 11 ND ND DPS-11 25 21 11 9 ND ND DPS-12 24 20 12 10 ND ND DPS-13 24 19 18 14 ND ND DPS-14 20 16 15 11 ND ND Stphylococcus ureus strins from the Deprtment of Plstic Surgery, Wonkwng University Hospitl, Iksn, Kore. ND, no detected ctivity t this concentrtion. AM, mpicillin; OX, oxcillin. Tble III. The MICs of rhein, AM nd OX ginst the S. ureus strin. ----------------------------------------------------------------------------- S. ureus strin Rhein Ampicillin Oxcillin ATCC25923 15.62 7.8 7.8 ATCC33591 15.62 1,000 250 DPS-1 15.62 31.25 500 DPS-2 15.62 1,000 500 DPS-3 15.62 31.25 500 DPS-4 31.25 31.25 500 DPS-5 7.8 31.25 500 DPS-6 7.8 31.25 250 DPS-7 31.25 250 500 DPS-8 31.25 250 500 DPS-9 31.25 125 500 DPS-10 31.25 250 500 DPS-11 31.25 250 500 DPS-12 31.25 250 500 DPS-13 7.8 31.25 1,000 DPS-14 7.8 250 500 Stphylococcus ureus strins from the Deprtment of Plstic Surgery, Wonkwng University Hospitl, Iksn, Kore. MIC, miniml inhibitory concentrtion; AM, mpicillin; OX, oxcillin. strted ntimicrobil ctivity ginst ll the tested strins of MRSA s well s the MSSA strin. The MICs of rhein ginst S. ureus rnged from 7.8-31.25 µg/ml. As shown in Tbles IV nd V, tests were performed to determine the ction of rhein lone s well s its synergistic ction with AM or OX ginst the MRSA clinicl isolte, the stndrd MRSA strin or the stndrd MSSA strin. Rhein lowered the MICs of AM nd OX ginst the MRSA strins. For the stndrd MSSA strin, rhein lowered the MICs of both AM nd OX. The combined ctivity of rhein nd the two ntimicrobil gents (AM nd OX) ginst ll strins resulted in FICI rnging from 0.28-1 nd from 0.18-1, respectively. In conclusion, the combintion effect of rhein with AM nd OX ws found to be synergistic or prtilly synergistic. Discussion In the present study, we investigted the ntimicrobil ctivity of rhein ginst clinicl isoltes of MRSA nd stndrd MSSA strin. We report tht the rhein gent lso shows synergistic ctivity with AM nd OX ginst MRSA. When combined, these ntibiotic effects were drmticlly incresed. While the results obtined in the present study cnnot currently be pplied in clinicl prctice, we consider tht the combintion tretment of rhein isolted with AM or OX will prove to be helpful in treting MRSA nd MSSA. Further medicinl, clinicl nd mechnism studies re required to verify the mechnisms by which rhein enhnces the ntibcteril ctivity. At present, the continued emergence of multi-drug resistnt bcteri nd the infectious diseses cused by them re serious globl problems. It is hoped tht it will be possible to reduce the use of existing ntibcteril drugs nd increse the use of nturl product drugs, such s rhein. At this point in time, the product is still under investigtion. In our study, rhein mrk-
JOUNG et l: METHICILLIN-RESISTANT Stphylococcus ureus 611 Tble IV. Result of the combined effect of rhein nd AM ginst S. ureus. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- S. ureus strin Rhein lone With AM AM lone With rhein FICI ATCC 25923 15.62 3.9 7.8 1.95 0.5 ATCC 33591 15.62 0.97 1,000 250 0.31 DPS-1 15.62 3.5 31.25 7.8 0.5 DPS-2 15.62 3.9 1,000 250 0.5 DPS-3 15.62 7.8 31.25 0.97 0.53 DPS-4 31.25 15.62 31.25 0.97 0.53 DPS-5 7.8 3.9 31.25 15.62 1 DPS-6 7.8 3.9 31.25 7.9 0.75 DPS-7 31.25 7.8 250 62.5 0.5 DPS-8 31.25 7.8 250 7.8 0.28 DPS-9 31.25 7.8 125 3.9 0.28 DPS-10 31.25 7.8 250 7.8 0.28 DPS-11 31.25 7.8 250 15.62 0.31 DPS-12 31.25 7.8 250 62.5 0.5 DPS-13 7.8 3.9 31.25 15.62 1 DPS-14 7.8 0.48 250 125 0.56 Stphylococcus ureus strins from the Deprtment of Plstic Surgery, Wonkwng University Hospitl, Iksn, Kore. ND, no detected ctivity t this concentrtion; MIC, miniml inhibitory concentrtion; AM, mpicillin; FICI, frctionl inhibitory concentrtion index. Tble V. Result of the combined effect of rhein nd OX ginst S. ureus. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- S. ureus strin Rhein lone With OX OX lone With rhein FICI ATCC 25923 15.62 1.95 7.8 1.95 0.37 ATCC 33591 15.62 1.95 250 15.62 0.18 DPS-1 15.62 7.8 500 31.25 0.53 DPS-2 15.62 3.9 500 125 0.5 DPS-3 15.62 7.8 500 15.62 0.53 DPS-4 31.25 7.8 500 125 0.5 DPS-5 7.8 1.95 500 125 0.5 DPS-6 7.8 1.95 250 7.8 0.53 DPS-7 31.25 7.8 500 125 0.5 DPS-8 31.25 7.8 500 62.5 0.37 DPS-9 31.25 7.8 500 125 0.5 DPS-10 31.25 7.8 500 62.5 0.37 DPS-11 31.25 7.8 500 125 0.5 DPS-12 31.25 7.8 500 250 0.75 DPS-13 7.9 0.48 1,000 500 0.56 DPS-14 7.9 3.9 500 250 1 Stphylococcus ureus strins from the Deprtment of Plstic Surgery, Wonkwng University Hospitl, Iksn, Kore. ND, no detected ctivity t this concentrtion; MIC, miniml inhibitory concentrtion; OX, oxcillin; FICI, frctionl inhibitory concentrtion index. edly lowered the MICs of AM nd OX ginst the two MRSA strins nd one MSSA strin. While the product is still under investigtion, the present results re promising nd my help to promote the use of nturl products rther thn ntibiotics. Acknowledgements This study ws supported by the Sunchon Ntionl University Reserch Fund (2011).
612 References 1. Yn D, M Y, Shi R, Xu D nd Zhng N: Phrmcokinetics of nthrquinones in Xiexin decoction nd in different combintions of its constituent herbs. Phytother Res 23: 317-323, 2009. 2. Chinese Phrmcopoei Commission: Phrmcopoei of the People's Republic of Chin. Chemicl Industry Press, Beijing, p17, 2005. 3. Dorsey JF nd Ko GD: Aloe(-emodin) for cncer? More thn just comforting slve. Cncer Biol Ther 6: 89-90, 2007. 4. Hung Q, Lu G, Shen HM, Chung MC nd Ong CN: Anti-cncer properties of nthrquinones from rhubrb. Med Res Rev 27: 609-630, 2007. 5. Lee HZ, Hsu SL, Liu MC nd Wu CH: Effects nd mechnisms of loe-emodin on cell deth in humn lung squmous cell crcinom. Eur J Phrmcol 431: 287-295, 2001. 6. Shi YQ, Fuki T, Skgmi H, Kurod J, Miyok R, Tmur M, Yoshid N nd Nomur T: Cytotoxic nd DNA dmge-inducing ctivities of low moleculr weight phenols from rhubrb. Anticncer Res 21: 2847-2853, 2001. 7. Cuellr MJ, Giner RM, Recio MC, Mnez S nd Rios JL: Topicl nti-inflmmtory ctivity of some Asin medicinl plnts used in dermtologicl disorders. Fitoterpi 72: 221-229, 2001. 8. Ding Y, Zho L, Mei H, Zhng SL, Hung ZH, Dun YY nd Ye P: Explortion of emodin to tret lph-nphthylisothiocynte-induced cholesttic heptitis vi nti-inflmmtory pthwy. Eur J Phrmcol 590: 377-386, 2008. 9. Ci J, Rzzk A, Hering J, Sed A, Bbcock TA, Helton S nd Espt NJ: Fesibility evlution of emodin (rhubrb extrct) s n inhibitor of pncretic cncer cell prolifertion in vitro. JPEN J Prenter Enterl Nutr 32: 190-196, 2008. 10. Hung KC: The Phrmcology of Chinese Herbs. CRC Press, Boc Rton, FL, pp233-234, 1993. 11. Yokozw T, Suzuki N, Okud I, Our H nd Nishiok I: Chnges in the urinry constituents in rts with chronic renl filure during orl dministrtion of rhubrb extrct. Chem Phrm Bull (Tokyo) 33: 4508-4514, 1985. 12. Wng J, Zho Y, Xio X, Li H, Zho H, Zhng P nd Jin C: Assessment of the renl protection nd heptotoxicity of rhubrb extrct in rts. J Ethnophrmcol 124: 18-25, 2009. 13. Zho YL, Wng JB, Zhou GD, Shn LM nd Xio XH: Investigtions of free nthrquinones from rhubrb ginst α-nphthylisothiocynte-induced cholesttic liver injury in rts. Bsic Clin Phrmcol Toxicol 104: 463-469, 2009. 14. Dictionry of Trditionl Chinese Medicine: New Medicl College of Jingsu. 1st edition. People's Publishing Co. of Shnghi, p102, 1997. 15. WHO: Monogrphs on Selected Medicinl Plnts. World Helth Orgniztion, Genev, pp231-240, 1999. 16. Clinicl nd Lbortory Stndrds Institute: Performnce stndrds for ntimicrobil disk susceptibility tests. Approved stndrds. CLSI document M2 A7. Wyne, PA, 2001. 17. Clinicl nd Lbortory Stndrds Institute: Methods for dilution ntimicrobil susceptibility tests for bcteri tht grow erobiclly. Approved stndrds. CLSI document M7-A5. Wyne, PA, 2000. 18. Mzumdor K, Dutt NK, Kumr KA nd Dstidr SG: In vitro nd in vivo synergism between tetrcycline nd the crdiovsculr gent oxyfedrine HCI ginst common bcteril strins. Biol Phrm Bull 28: 713-717, 2005. 19. Abte G, Mshn RN nd Miorner H: Evlution of colorimetric ssy bsed on 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyl tetrzolium bromide (MTT) for rpid detection of rifmpicin resistnce in Mycobcterium tuberculosis. Int J Tuberc Lung Dis 2: 1011-1016, 1988. 20. Scheuber PH, Scheuber PH, Mossmnn H, Beck G nd Hmmer DK: Direct skin test in highly sensitized guine pigs for rpid nd sensitive determintion of stphylococcl enterotoxin B. Appl Environ Microbiol 46: 1351-1356, 1983. 21. Shi YJ, Chen J nd Xu M: A new method for ntimicrobil susceptibility testing of in vitro-cultured bcteri by mens of resonnce light scttering technique. J Microbiol Biotechnol 18: 118-123, 2008.