Antimicrobial activity of natural photosensitizing anthraquinones

Transcription

1 Antimicroil ctivity of nturl photosensitizing nthrquinones S.C. Núñez Montoy, #, L.R. Comini, # nd J.L. Crer. Phrmcognosy, Deprtment of Phrmcy, School of Chemicl Sciences, Ntionl Ntionl University of Córdo, IMBIV-CNICET, Ciudd Universitri 5 Córdo, Argentine. # S.C.N.M. nd L.R.C. contriuted eqully to this puliction It ws demonstrted tht nthrquinone-rich extrcts, otined from the phototoxic vegetl species eterophylle pustult ook f. (Ruicee), exhiited cteriosttic ctivity on Micrococcus luteus ATCC 934, selectively inhiiting oth oxcillin-sensitive nd resistnt Stphylococcus ureus, nd ntifungl ctivity ginst importnt opportunist microorgnisms nd ginst those involved in superficil mycosis, ll from nosocomil origin. The cute in vitro cytotoxicity evlution of ech nthrquinone (AQ) isolted from these ioctive extrcts, on mmmlin eukryotic cell line (Vero cells), llowed us to estlish the non-cytotoxic concentrtion rnge, which ws used to evlute the ntimicroil effect. Four from nine AQs tested, sornjidiol, ruidin, dmncnthl nd (S)-5,5'-isornjidiol, showed in vitro cteriosttic/ctericide ctivity ginst S. ureus. The ction mechnism seems to involve n increse in the levels of superoxide nion nd/or singlet oxygen moleculr. Moreover, the effect of ctinic irrdition s oosting gent for the production of oth rective oxygen species s well s its influence on nticteril effect ws ssessed. Keywords nthrquinones; ntimicroil ctivity. Introduction Throughout history, nturl products hve een rich source of compounds tht hve found mny pplictions in the field of medicine. In microiology, prticulrly, severl plnt-derived compounds hve een studied with this im, including lkloids, flvonoids, tnnins, quinones, essentil oils nd other secondry metolites []. Among them, nthrquinone derivtives (AQ) hve roused specil interest since they hve demonstrted potentil therpeutic uses s nticteril, ntivirl, ntifungl gents nd other iologicl ctivities [-5]. Within this fmily of compounds, severl AQs hve een thoroughly studied in reltion to their photosensitizing properties in photodynmic rections [, 7]. n the sis of this, some of them show good nticteril nd ntivirl effects, y producing rective oxygen species (RS) such s superoxide nion ( ), hydroxyl rdicl (.) nd singlet moleculr oxygen (), in the presence of light, with susequent oxidtive dmge [8, 9]. ypericin, photosensitizing AQ found in certin memers of the genus ypericum, is cler cse showing significnt ntimicroil ctivity y mens of photodynmic photosensitiztion, cting minly through the genertion of RS, prticulrly [-]. It is widely ccepted tht sustnces with photosensitizing chrcteristics hve ecome prticulrly relevnt due to their potentil pplictions in photodynmic ntimicroil chemotherpy (PACT), which involves photosensitizers nd visile or ultrviolet light. This therpeutic (PACT) hs een proposed in the tretment of locl infections, especilly those from cries, periodontl diseses, orl cndidisis s well s wounds [9, 3]. In this context, ering in mind the mny potentil pplictions of photosensitizers nd the need for new chemicl structures with this prticulr feture, we strted series of chemicl, physicl nd iologicl studies on photosensitizing AQs isolted from phototoxic plnt species, eterophylle pustult ook f. (Ruicee). This vegetl species grows in the Anden mountin rnge in the northwest of Argentin [4] nd the nimls tht ingest the eril prts of this plnt experience typicl primry photosensitiztion rection, cliniclly reveled y dermtitis nd lindness in severe cses [5, ]. The chemicl investigtion of this plnt reveled the presence of severl constituents (AQs, flvonoids nd iridoids) with n evident predominnce of glicone-9,-aqs. From leves nd stems, nine AQs nmely, sornjidiol, sornjidiol -methyl ether, ruidin, ruidin -methyl ether, dmncnthl, dmncnthol, heterophylline, pustuline nd (S)-5,5 -isornjidiol (Fig. ), were isolted nd purified y using repeted comintion of severl chromtogrphic techniques. The identifiction of ech metolite ws mde y pplying different spectroscopic/spectrometric techniques (UV-V, IR, -RMN, 3C-RMN, RMS) [7, 8]. In previous studies we demonstrted tht these AQs lso exhiit photosensitizing properties y genertion of (Type I mechnism) nd/or (Type II mechnism) [9, ], which re directly involved in the phototoxic effects tht. pustult produces on cttle []. Following previous studies, we hve focused our ttention on studying the in vitro nticteril nd ntifungl ctivity of enriched AQ extrcts otined from. pustult. To nlyze further the results otined, we evluted the nticteril ctivity of ech isolted AQ, centering our interest on determining whether this effect ws solely due to photodynmic process or to concurrent comintion of light-driven nd drk processes. FRMATEX 3

2 8 R5 7 R 9 8 A C R4 4 5 R Anthrquinone nucleous AQs sornjidiol sornjidiol -methyl ether ruidin ruidin -methyl ether dmncnthl dmncnthol -hydroxi-3-methyl AQ heterophylline pustuline 5 R3 (S)-5,5'-isornjidiol R C3 C3 C3 C3 R C3 C3 C3 C3 C C- C3 R3 C3 C3 R4 R5 C3 C3 Fig. Anthrquinones structures used in this study.. In vitro nticteril nd ntifungl ctivity of enriched AQ extrcts otined from eterophylle pustult. Since the AQ-rich extrcts otined from severl plnt species hve shown significnt ntimicroil ctivity [], we decided to evlute the in vitro nticteril nd ntifungl properties of enriched AQ extrcts otined from. pustult [7]. In order to prepre extrcts, the ir-dried plnt mteril ws seprted into stems nd leves. Both were ground seprtely nd extrcted with enzene in Soxhlet pprtus. Phytochemicl studies hve shown tht enzenic stem extrct hs dmncnthl, dmncnthol, ruidin, sornjidiol nd ruidin -methyl ether. Benzenic lef extrct hs similr chemicl composition except for the sence of dmncnthl nd dmncnthol, nd the presence of sornjidiol -methyl ether, heterophylline, pustuline nd 5,5'-isornjidiol. A screening with the enzenic stem nd lef extrcts ws performed in reltion to their in vitro nticteril ctivity ginst the reference strin Micrococcus luteus ATCC 934, oxcillin-sensitive nd resistnt Stphylococcus ureus, cogulse-negtive (c.n.) S. sprophyticus, Escherichi coli (two different strins), Proteus mirilis, Pseudomons eruginos (two different strins) nd the fungl species Cndid licns, C. glrt, C. krusei, C. prpsilosis, C. tropiclis, Cryptococcus neoformns, Aspergillus fumigtus, A. flvus, Trichophyton mentgrophytes, T. rurum, nd T. floccosum [7]. The clinicl importnce of these pthogenic species (oxcillin-resistnt nd sensile S. ureus; the lter, Bet-lctmse producer) should e stressed, since they prticipte in diverse infections such s scesses, endocrditis, pneumoni, meningitis nd osteomyelitis. In ddition, Cndid is the fourth germ isolted from hospitl hemocultures, ssocited with 38% of mortlity. C. licns prticiptes in cutneous nd deep cndidisis. C. krusei offers nturl resistnce to fluconzole, n ntiiotic commonly dministered in numerous tretments. C. prpsilosis represents the min cuse of cndidemi in children nd ctheter-ering ptients. Cryptococcosis nd spergillosis produce mortlity rtes higher thn 95% nd re trnsmitted y the inhltory route, frequently ffecting AIDS ptients. T. mentgrophytes is ssocited with superficil skin, nil nd hir mycosis [7]. The ntimicroil ctivity of enzenic stem nd lef extrcts ws determined using the gr-well diffusion method modified ccording to the following experimentl conditions, using nutrient gr for cteri or Sourud gr for fungi [7]. Except for M. luteus, ll cteril microorgnisms were pthogenic strins isolted from ptients with different infections. All fungl microorgnisms were isolted from different lesions of ptients from the ospitl de Clínics (Buenos Aires), s descried y Pérez et l. [3]. Six-millimeter-dimeter wells were punched into the gr nd filled with different extrct dilutions of stock solution ( mg/ml in Et) nd solvent lnks or stndrd ntiiotic solutions, ccording to the sensitivity of the microorgnism tested [4]. The ntimicroil ctivity ws evluted y mesuring the inhiition-zone dimeter oserved. In ddition, the miniml ctericidl concentrtion (MBC) ws determined [7]. Cylindricl pieces (4 mm dimeter) were extrcted from the inhiition-zones of S. ureus produced y the highest concentrtion of the different. pustult extrcts. The pieces were trnsferred to sterile tues contining tryptose roth. The tues were incuted t ºC for 4 h. A ml liquot of this roth ws spred over Petri pltes contining sterile nutrient gr. This ws 4 FRMATEX

3 incuted t 3 ºC for 4 h nd the development of microorgnisms ws checked. The miniml extrct concentrtion tht reduces the vile cteri to : or less is considered MBC. The enzenic stem extrct shows selective ctivity on Micrococcus luteus ATCC 934, oxcillin-sensitive nd resistnt Stphylococcus ureus, without cting ginst other Grm + s S. sprophyticus c.n. neither Grm s Escherichi coli (two different strins), Proteus mirilis nor Pseudomons eruginos (two different strins). In this spect the lef extrct showed similr ctivity to tht of the stem [7]. A cteriosttic effect of the different. pustult extrcts ginst oxcillin-sensile nd resistnt S. ureus is implied y the MBC/MIC (minimum inhiitory concentrtion) rtio, whose results re clerly higher thn [7]. Moreover, the ntimicroil spectrum of the enzenic stem extrct proves interesting ecuse of its ctivity ginst fungl species from nosocomil origin. Indeed, it ws useful ginst fungl cultures isolted from different corporl lesions such s severl strins of C. licns, C. krusei, C. prpsilosis, C. tropiclis, C. neoformns, A. fumigtus, A. flvus nd T. mentgrophytes (Tle ). Tle Antifungl ctivity of eterophylle pustult stem extrct (mg/ml) Microorgnism pportunistic mycosis Cndid licns I Cndid licns II Cndid licns III Cndid licns IV Cndid licns V Cndid licns VI Cndid licns VII Cndid glrt I Source mteril Inhiition Dimeter (mm) Extrct Mz I.A. (u) Cndid glrt II Cndid glrt III Cndid krusei I Cndid krusei II Cndid prpsilosis I hemoculture urineculture MC hemoculture 373 hemoculture 374 hemoculture 554 hemoculture 55 hemoculture 999 penis sw (cndidemi y C. glrt) 3534 hemoculture 35 hemoculture 393 lryngel prothesis urineculture hemoculture Cndid prpsilosis II Cndid tropiclis Cryptococcus neoformns Aspergillus fumigtus Aspergillus flvus hemoculture 57 hemoculture 89 CSF (IV ptient) 77 nsl polyps (fngl sinusitis) nsl sw (in leukemic ptient) Superficil mycosis Trychophyton rurum Trychophyton mentgrophytes Epidermophyton floccosum Tine pedis (mle ptient) toe nil Tine pedis ( yer evolution in IV ptient) Control: miconzole (MZ) ( mg/ml). I.A.: inoculum sornce t 58 nm. Inoculum dilution in the culture medium ws /8 v/v. Vlues re mens of to 4 dt tht differ in less thn % from the men. - = no ctivity detected n the other hnd, cute toxicity studies were performed y dministrtion schedules using unique doses on femle mice of CF strin, 3-month old nd weight rnging etween 3 nd g [7]. Becuse of the low soluility chrcteristics of the enzenic stem nd lef extrct, mixture of dimethylsulfoxide (DMS)/ (3:) (v/v) ws selected mong severl solvents. The extrct ws dministered sucutneously in doses rnging from to 4, mg/kg. The intrvenous dministrtion ws performed through the til veins with doses within 7 to 8 mg/kg rnge. In oth cses, the control mice were injected with the corresponding vehicle. The toxicity signs, including deth, were oserved during 8 dys following the extrct dministrtion. The LD5 ws estimted through the up-nd-down method for smll smples y Dixon [5]. FRMATEX 5

4 Upon sucutneous dministrtion, the mice tolerted doses of up to 4, mg/kg ody wt. without signs of toxicity, indicting LD5 equl or higher thn tht level. With the im of determining LD5 vlues nd tking into ccount the difficulty of otining the extrct, cute toxicity following intrvenous dministrtion ws studied. We estlished LD5 vlue of 3 mg/kg ody wt. without higher signs of toxicity in the surviving mice. In oth cses, the enzenic extrct of stem nd lef ppers to exhiit similr ptterns [7]. In conclusion, the AQ-rich extrcts hve proved nticteril nd ntifungl effect, without mnifest toxicity in experimentl nimls t the concentrtion ssyed. Some of these experiments were performed in the Deprtment of Phrmcology, School of Dentistry, University of Buenos Aires, under the direction of Dr. Cristin Pérez. 3. Evlution in vitro of iologicl effects of ech AQ isolted from. pustult. 3. In vitro nticteril ctivity of ech AQ Considering the ove results of the ntimicroil ctivity of AQ-rich extrcts, otined from. pustult, we evluted the in vitro nticteril ctivity of ech AQ ginst Grm (+) cterium with the im of estlishing the metolites responsile for the effect shown y the extrcts. Routine susceptiility tests, including the determintion of MIC, re n estimte of cteriosttic ction. In this cse, mesurement of the MIC for ech AQ ws crried ccording to interntionl stndrds from the Clinicl nd Lortory Stndrds Institute (CLSI) y mens of roth mcrodilution method in Mueller-inton medium (M, Britni) []. The strin used for this purpose ws S. ureus ATCC 93 (x5 colony-forming units (cfu)/ml). The working solutions of ech AQ were prepred y mens of serited dilutions from 5 g/ml up to.5 g/ml. MIC determintion ws crried out fter 4 h of incution t 37 ºC y oserving turidity. ur studies led to the conclusion tht only sornjidiol, ruidin, dmncnthl nd 5,5'-isornjidiol showed susceptiility to MICs within 3-4 g/ml rnge. Bering in mind tht for mny sustnces hving nticteril ctivity there re mechnisms tht elicit physiologicl response in cteri y producing rective oxygen species (RS) [7-9], our ojective is demonstrted if the nticteril effect shown y sornjidiol, ruidin, dmncnthl nd 5,5'-isornjidiol (through the determintion of the MIC) is directly linked to the increse of nd/or levels. Nevertheless, it should e noted tht some photosensitizers lso generte cteril photoinctivtion minly y mens of the genertion of RS, prticulrly singlet moleculr oxygen (), ut through photodynmic photosensitiztion [3]. Therefore, s the AQs studied re photosensitizing gents, we lso ssessed whether the irrdition cuses photosensitiztion y these AQs, which consequently increses the production of RS nd its nticteril effect. In order to rech this conclusion, the experimentl model ws crried out ccording to the following guidelines: the AQs were dissolved in phosphte uffer solution (PBS) nd the concentrtions used throughout were elow the miniml inhiitory concentrtion (MIC) (3-4 g/ml), specificlly ws selected the sutoxic doses of g/ml ecuse higher concentrtions proved to e toxic for norml mmml cells (Africn green monkey kidney cells Vero) (see results elow) [3]. The four AQs tht showed susceptiility to S ureus (sornjidiol, ruidin, dmncnthl nd 5,5'-isornjidiol) were exmined to scertin whether this ctivity ws ssocited with n incresed genertion of with respect to the sl production. The determintion ws sed on the NBT ssy []. Two ssys were simultneously performed in drkness. ne ws crried out in the sence of AQs to mesure the sl level of the production in the cteri (control). The second preformed y dding AQs to the culture medium, in order to estlish whether these compounds induce n increse in the genertion through physiologicl response. Both ssys were independently repeted under ctinic irrdition to evlute the photosensitized response. The strin used ws S. ureus ATCC 93 (9 cfu/ml). In the NBT ssy, the cteril suspension ws incuted with ech AQ solution nd NBT t 37 ºC in drkness. Agin, the control experiments were crried out under the sme conditions ut without dding the AQ. Triplicte sets were incuted under ctinic irrdition. All these experiments were independently mesured s function of time []. Moreover, the sme set of AQs used in the preceding prgrph ws exmined to evlute n increse of genertion with respect to the sl production. The production in the rection medium ws followed y spectrophotometricl determintion of the consumption of methionine (MET) t 3 nm s function of time [3]. This compound rects chemiclly with the generted with rte constnt (kr) of. x 7 L mol-s- [3]. ere gin, two sets of experiments were done; one in drkness nd the other under ctinic irrdition. For ech set, the procedure is descried elow: i) In order to determine the sl production generted y cteri (control), we incuted cteril suspension (9 cfu/ml), MET nd PBS. FRMATEX

5 ii) The production of in the presence of AQs ws mesured incuting cteril suspension (9 cfu/ml), MET, ml of AQ nd PBS. By using sodium zide (NN3) s physicl quencher (kt = 5,8 8 L mol-s-) we cn confirm whether the consumption of MET is direct consequence of the genertion in the medium [33]. Due to its lrge quenching constnt, NN3 ws dded in concentrtion high enough to suppress the consumption of y MET s descried elow: iii) The cteril suspension (9 cfu/ml) ws incuted with MET, ml of AQ, NN3 nd PBS. Fig. shows the increse in percentge of in S. ureus with respect to sl sitution when this strin ws treted with ech AQ in drkness nd under ctinic rdition t, nd min. As oserved, there is no cler indiction of t min, neither for drkness nor for irrdition. At nd min, ll the AQs induce n increse in production with respect to the sl sitution in drkness. Except for dmncnthl, this effect is more noticele t longer times. Under ctinic irrdition, there is further increse in production with respect to sl sitution with the sme exception shown previously. Increse in percentge of superóxide nion production with respect to sl sitution 8 AQ-Drkness 7 7,7 AQ-Irrdition, 4,5 5,3 5,5 5, 38 3,4 9,8 3 7, 5,3, 3,9,5 35, 3 Time (min) - Dmncnthl Ruidin Sornjidiol 5,5'-isornjidiol Results re given s men SD, n=3. p.5 mesured with respect to drkness p.5 mesured with respect to min time Fig. NBT ssy. Increse in percentge of in S. ureus ATCC 93 with respect to sl sitution for every AQ nd time mesured. Fig. 3 shows the MET consumption cused y the generted in S. ureus under the conditions outlined in i, ii, iii (evlution of the genertion), oth for drkness nd under ctinic irrdition for 5,5'-isornjidiol. As cn e oserved, in the sence of AQ, smll mounts of re produced due to the norml rething process. MET consumption ws higher when 5,5'-isornjidiol ws present, which mens tht the production incresed. This phenomenon is oserved under oth working conditions (drkness nd ctinic irrdition). The generted ws countercted y ddition of NN3. This physicl quencher efficiently competes with MET for the dectivtion of nd consequently, the trend line is similr to control experiments. These results confirm tht MET consumption is direct consequence of the genertion. The preceding discussion is referred only to 5,5'-isornjidiol. The other AQs tested lso show similr ehvior, though with smller increse in genertion. FRMATEX 7

6 Drkness Control of 5,5'-isornjidiol of NN Control of 5,5'-isornjidiol of NN3 Actinic Irrdition Time (s) Time (s) Fig. 3 Consumption of MET cused y in S. ureus ATCC 93 treted with 5,5'-isornjidiol ( µg/ml), in drkness nd under irrdition. Averge of three experiments. Ruidin Ruidin drk + drk light + light Fig. 4 shows the MET consumption cused y the genertion in cteri treted with ech AQ (see ii: evlution of the genertion), in drkness vs. irrdition. Its nlysis (y comprison of trend lines) shows tht the production is higher in the presence of ctinic rdition with respect to drkness ecuse MET consumption incresed, except for dmncnthl. 5 drk + drk light+ light Dmncnthl Dmncnthl 5 drk + drk light + light 5 5,5'-isornjidiol 5,5'-isornjidiol TimeTime (s) (s) TimeTime (s) (s) Sornjidiol Sornjidiol drk + drk light + light TimeTime (s) (s) TimeTime (s) (s) Fig. 4 Consumption of MET cused y in S. ureus ATCC 93 treted with ech AQ ( µg/ml), y compring the process occurring in drkness nd under ctinic irrdition. Averge of three experiments. Lstly, ering in mind tht, while those compounds tht cn reduce minimum of 3 cfu/ml (3. Log) re considered ctericide gents, those elow tht rnge (which merely inhiit growth) re considered cteriosttic gents [7, 34, 35], we crried out ssys with the purpose of estlishing whether the AQs show ny of such chrcteristics. With this ojective, S. ureus ATCC 93 (8 cfu/ml) ws incuted in triplicte with ech AQ (dmncnthl, 8 FRMATEX

7 Log cfu/ml Log cfu/ml ruidin, sornjidiol nd 5,5'-isornjidiol) t 37 ºC independently in drkness nd under irrdition, replicting lso the whole set to e mesured t, nd 9 min []. After incution time, cteri were serilly -fold diluted with PBS nd ech dilution ws plced on plte count gr M nd incuted for 4 h t 37 ºC. Bctericidl ctivity ws determined y mens of plte recount of colony-forming units per milliliter (cfu/ml). A prticulr experiment ws crried out to prove whether the suppression of production y chemicl quencher chnges the reduction in the numer of cfu/ml. A cteril suspension (8 cfu/ml) with 5,5'-isornjidiol under irrdition ws treted with MET, fter min of incution, llowing the system to proceed normlly nd mesuring the cfu/ml t 9 min. Fig. 5 shows Log chnge in cfu/ml of S. ureus treted with ech AQ in drkness, including the control. As noted, decrese, more pronounced for 5,5'-isornjidiol, is seen fter n initil increse. This reduction could relte to the cpility of producing physiologicl response in cteri tht generte (Fig. ) s well s (Fig. 3) in the sence of light. 5,5'-isornjidiol ws the only AQ tht showed ctericide effect on this microorgnism with reduction of 3. Log of cfu/ml t 9 min. Ruidin nd sornjidiol produce reduction of. nd. Log of cfu/ml, respectively. The increse in production for ruidin nd sorinjidiol is similr to tht of 5,5'-isornjidiol (Fig. ). Nevertheless, the increse in genertion is lower thn the inthrquinone. For dmncnthl, only n inhiitory effect without producing deth is noticed. No AQ Dmncnthl Ruidin Sornjidiol 5,5'-isornjidiol NoAQ Dmncnthl Ruidin Sornjidiol 5,5'-isornjidiol 5,5'-isornjidiol + MET ( min.) Time (min.) Time (min) Error rs represent SD of the men. Fig. 5 Log chnge in colony-forming units per milliliter (cfu/ml) of S. ureus ATCC 93 treted with ech AQ ( µg/ml) in drkness. Fig. Log chnge in colony-forming units per milliliter (cfu/ml) of S. ureus ATCC 93 treted with ech AQ ( µg/ml) under irrdition. n the other hnd, the ctinic rdition nd the consequent nd increse y mens of photosensitiztion phenomenon (Figs. nd 4), further incresed the reduction of cfu/ml for ll AQs, execept dmncnthl (Fig. ). These results clerly demonstrte tht 5,5'-isornjidiol shows higher nticteril ctivity thn tht of ruidin nd sornjidiol, lthough the three increse production t out the sme level s function of time (Fig. ) regrdless tht they re cting in drkness or under irrdition. In contrst, the production for 5,5'-isornjidiol is high irrespective of conditions (drkness or irrdition) []. This would suggest tht, mong the different species tht comprise RS, is the one minly involved in the ctericidl effect, s lredy reported y Becerr et l. [3] using precisely S. ureus. This ecomes evident since, fter min of incution, the ddition of MET to smple hving 5,5'-isornjidiol shows tht even under irrdition, the cfu/ml reduction is completely suppressed t 9 min (open squres, Fig. ). In conclusion, these ssys llowed us to identify the AQs: ruidin, sornjidiol, dmncnthl nd 5,5'-isornjidiol s the compounds responsile for the nticteril effects shown y AQ-rich extrcts otined from the phototoxic vegetl species,. pustult. In ddition, our results suggest tht the nticteril effect on S. ureus, found for these AQs, is closely linked to the increse in nd/or levels. This increse could result from the interction etween cteri nd the AQs without needing light, i.e., without photosensitizing process to produce physiologicl response eliciting nd. Actinic irrdition, on the other hnd, genertes photosensitiztion for ruidin, sornjidiol nd 5,5'-isornjidiol, consequently incresing their nticteril effects (prticulrly ctericide). This effect is, in turn, suppressed when specific quencher of is dded, thus suggesting tht the ctericidl ctivity derives minly from tht prticulr RS s lredy proposed in other reports [3]. Most importntly, the prticulr ctericidl ctivity shown under irrdition for ruidin, sornjidiol nd 5,5'isornjidiol t the concentrtion used, which does not ffect norml mmml cells (sutoxic concentrtion - see results ove), led us to consider these AQs s potentil gents for photodynmic nticteril chemotherpy tretments. FRMATEX 9

8 Some of these experiments were performed interdisciplinrily with Dr. Inés Ales (Lortory of Phrmceuticl Microiology, Deprtment of Phrmcy) nd Dr. Gustvo A. Argüello (Deprtment of Physicl Chemistry), School of Chemicl Sciences, Ntionl University of Cordo. 3. Cytotoxic evlution in vitro of ech AQ Prior to the nticteril tests of ech AQ, it ws necessry to evlute the cytotoxicity of these compounds on mmmlin eukryotic cell line (Vero cells) [37], with the im of estlishing the non-cytotoxic concentrtion rnge for evluting the ntimicroil effect of ech AQ. For this purpose, Africn green monkey kidney cells (Cercopithecus ethiops, Vero 7 ATCC CRL-587) were used. The cytopthic effect produced y ech AQ on the morphology of Vero cells ws oserved y opticl microscopy [38]. From stock solution of ech AQ ( mg/ml in PBS with % DMS s co-solvent), 5 consecutive dilutions were prepred with PBS, within rnge of to 3-5 µg/ml ccording to AQ soluility. Ech dilution ws inoculted in duplicte on confluent cell monolyer (.5 ±. x 5 cells/ml, 48 h incution). Cell controls (CC), contining only mintennce medium (MM: Egle s minimum essentil medium (MEM) with % Fetl clf serum (FCS), % Lglutmine, gentmicine (5 µg/ml) nd % DMS), were included. The cells were incuted t 37 C during 7 h, nd the development of cellulr ltertions such s rounding, memrne retrction, cell detchment nd the presence of grnules in the cytoplsm ws dily oserved [38]. The cellulr viility (CV) depending on the concentrtion of ech AQ ws mesured y mens of the uptke Neutrl Red (NR) ssy [39]. Ech dilution ws inoculted in triplicte on confluent monolyer of cells (.5 ±. x 5 cells/ml). The sornce of the NR extrcted fter 48 h of incution t 37 C ws mesured t 5 nm on microplte reder (BioTek ELx8). The percentge of cellulr viility (CV%) ws clculted y comprison with CC (% viility). The concentrtion of compound tht reduces the vile cells to 5% (CC5) ws determined y regression (R>.9) from the plot of cellulr viility percentge vs. AQ concentrtion [39]. Mximum Non-Cytotoxic Concentrtion (MNCC) ws defined s the mximl smple concentrtion showing more thn 9% vile cells nd exerting no cytotoxic effect s detected y microscopic monitoring []. ur results showed tht, wheres ruidin -methyl ether, dmncnthol nd pustuline stnd out y showing MNCC vlues within nd µg/ml rnge, this concentrtion ws not higher thn μg/ml for the remining AQs (Tle ). Thus, the MNCC for sornjidiol -methyl ether, heterophylline nd 5,5'-isornjidiol ws ner µg/ml, nd pproximtely µg/ml for sornjidiol, ruidin nd dmncnthl (Tle ). Tle Cytotoxic Concentrtion to 5% (CC5), Mximum Non-Cytotoxic Concentrtion (MNCC) nd Sutoxic Concentrtion for ech AQ tested on Vero cells. AQs ruidin -methyl ether dmncnthol pustuline sornjidiol -methyl ether heterophylline (S)- 5,5'-isornjidiol dmncnthl sornjidiol ruidin CC5 ( g/ml) 34.4 ± ±. nc 7. ±. 3.9 ±.4.7 ±.. ±. 7.5 ±. 4.9 ±. MCNC ( g/ml). ±.4 9. ±.. ±.3.5 ± ±. 9.5 ±..7 ±. c.3 ±. c 5.3 ±.3 c Sutoxic concentrtion ( g/ml).8 ±. 3.9 ±..3 ±. 8.4 ±. 5.4 ± ±.. ±. 9.9 ±. 8. ±. nc: not clculted p <.5 clculted with respect to p <.5 clculted with respect to c Similr results were otined y nlyzing the CC5 determined for ech AQ (Tle ), which estlished tht ruidin -methyl ether ws less cytotoxic, followed, in incresing order of cytotoxicity, y dmncnthol, pustuline, sornjidiol -methyl ether, heterophylline, 5,5'-isornjidiol, dmncnthl, sornjidiol nd ruidin. The CC5 of pustuline ws not estimted ecuse concentrtions higher thn 3 g/ml could not e tested due to soluility prolems; however, its MNCC ws estlished. In ddition, sutoxic concentrtion ws determined for ll AQs tested (Tle ), defined s the concentrtion tht cuses - % cellulr deth [4] nd produces slight morphologic chnges oserved y microscopy. In ddition, in previous studies we demonstrted tht some of the AQs tested hd the ility to increse the production in humn leukocytes [9, ]. Considering tht this effect could generte the cytotoxic ctivity of AQs on Vero cells, the ility of ech AQ to produce this RS nd its reltion to the cytotoxic effect were evluted. To this im, the NBT reduction iossy ws performed nd ech AQ ws evluted t µg/ml, which represents noncytotoxic or sutoxic concentrtion depending on the compound studied [4, 43]. FRMATEX

9 Thus, ruidin -methyl ether, dmncnthol nd pustuline did not increse the production of t g/ml (Fig. 7) since it is non-cytotoxic concentrtion for these AQs (pproximtely 95% CV) without evidencing ny cytopthic effect [3]. For the other AQs, this mount corresponds to sutoxic vlue, incresing the genertion of (Fig. 7). It ws noted tht those compounds producing lrge increse in the genertion t g/ml (sornjidiol nd dmncnthl, Fig. 7) exhiit low CV% (out 8%) with incresed cell dmge [3]. owever, those AQs tht t the sme concentrtion showed smll increse in the production (sornjidiol -methyl ether, heterophylline, 5,5'isornjidiol nd ruidin), revel high CV% (etween 8 nd 9%), except for ruidin [3]. In generl, we might conclude tht n incresed production of cuses n s importnt cytopthic effect s oserved y microscopy, with concomitnt decrese in cellulr viility. Ruidin is excepted from this ehvior, producing significnt cytopthic effect t g/ml, which results in significnt decrese in CV (3%), despite hving low production of (Fig 7) [3]. We my therefore ssume the presence of nother mechnism in the cytotoxicity of this compound. In ddition, when ech AQ ws tested t their CC5, the increse in production ws not the sme in ll AQs. Therefore, the intrcellulr increse of this RS would not e the sole cuse for the loss of cellulr viility t CC5. Fig. 7 NBT ssy. Increse in percentge of in Vero cells with respect to sl sitution, produced y two different concentrtions of every AQ. Finlly we crried out n AQ incorportion ssy in Vero cells [9], which constitutes spectrophotometric determintion of intrcellulr content of AQs needed to stimulte production nd other iologic effects. Sornjidiol ws chosen to study the incorportion of AQs in Vero cells ecuse this AQ is the predominnt compound in the eril prts of. pustult. Thus, y mens of this ssy, we hve estlished tht sornjidiol enters Vero cells, 9 ± 3% with respect to the initil concentrtion fter 3 min incution. Although only single AQ ws tested, no significnt differences re expected in the rte of incorportion for the other AQs since they ll hve similr prtition coefficients [44]. In conclusion, this work llowed us to estlish the concentrtion rnge where ech AQ exhiits low or no cytotoxic effect nd therefore, these concentrtions my e used in order to test their potentil ntimicroil effects. From the nine AQs tested, we were le to identify three derivtives: ruidin -methyl ether, dmncnthol nd pustuline, with low or no cytotoxicity (95 ± 5% VC) in concentrtion rnge limited y the MNCC (Tle ). The estimtion of the sutoxic concentrtion for the other AQs (sornjidiol, sornjidiol -methyl ether, ruidin, dmncnthl, heterophylline nd 5,5'-isornjidiol) llowed us to consider tht concentrtion of out g/ml could e used to test different iologicl ctivities, since this concentrtion ensured in our experiments more thn 8% CV (Tle ). Acknowledgements The support y Agenci Ncionl de Promoción Científic y Tecnológic (FNCYT), Consejo Ncionl de Investigciones Científics y Técnics (CNICET), Ministerio de Cienci y Tecnologí de l Provinci de Córdo (Argentin), nd Secretrí de Cienci y Tecnologí de l Universidd Ncionl de Córdo (SeCyT-UNC) is grtefully cknowledged. References [] Cown MM. Plnt Products s ntimicroil gents. Clin. Microiol. Rev. 999;(4): [] Wijnsm R, Verpoorte R. Anthrquinones in the Ruicee. In: ill RA, Kres Ch, Verpoorte R, Wijnsm R, erz W, Grisech, Kiry GW, Tmm Ch, eds. Progress in the Chemistry of rgnic Nturl Products. Wien: Springer-Verlg; 98,49: [3] Rth G, Ndonzo M, ostettmnn K. Antifungl nthrquinones from Morind lucid. Int. J. Phrmcognosy. 995;33():74. FRMATEX

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