Research Article Bioaccessibility In Vitro of Nutraceuticals from Bark of Selected Salix Species

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1 e Sientii World Journl, Artile ID , 10 pges Reserh Artile Bioessiility In Vitro o Nutreutils rom Brk o Seleted Slix Speies Urszul Gwlik-Dziki, 1 Dnut Sugier, 2 Driusz Dziki, 3 nd Piotr Sugier 4 1 Deprtment o Biohemistry nd Food Chemistry, University o Lie Sienes, Skromn Street 8, Lulin, Polnd 2 DeprtmentoIndustrilndMediinlPlnts,UniversityoLieSienesinLulin,Akdemik15,20-950Lulin,Polnd 3 Deprtment o Therml Tehnology, University o Lie Sienes, Doświdzln 44, Lulin, Polnd 4 Deprtment o Eology, Fulty o Biology nd Biotehnology, Mri Curie-Skłodowsk University, Akdemik 19, Lulin, Polnd Correspondene should e ddressed to Urszul Gwlik-Dziki; urszul.gwlik@up.lulin.pl Reeived 31 August 2013; Aepted 29 Deemer 2013; Pulished 17 Ferury 2014 Ademi Editors: P. Cos nd W. Gelderlom Copyright 2014 Urszul Gwlik-Dziki et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. The im o this study ws to investigte nd to ompre the extrtility, ioessiility, nd iovilility in vitro o ntioxidtive ompounds rom rk o seleted Slix speies: S. l(sa), S. dphnoides(sd), S. purpure(sp), nd S. dphnoides x purpure (SDP) hyrid willow lones originting rom their nturl hitts nd ultivted on the sndy soil. The highest mount o phenoli glyosides ws ound in the rk o SDP nd SD.TheestsoureophenoliswsrkoSDP. The highest ontent o lvonoids were ound in SD rk smples, wheres the highest onentrtion o ioessile nd ioville phenoli ids ws determined in SDP rk. Brk o ll tested Slix speies showed signiint ntirdil tivity. This properties re strongly dependent on extrtion system nd geneti tors. Regrdless o Slix genotypes, the lowest helting power ws ound or hemillyextrtle ompounds. Brk o ll Slix speies ontined ethnol-extrtle ompounds with reduing ility. Besides this, high ioessiility nd iovilility in vitro o Slix rk phytohemils were ound. Otined results indite tht extrts rom rk tested Slix genotypes n provide helth promoting eneits to the onsumers; however, this prolem requires urther study. 1. Introdution The willow rk is onstituent o mny herl drugs nd lso dietry supplements suh s n nlgesi, ntipyreti, ntiphlogisti nd weight loss enhnement remedies. The term Sliis Cortex [SC] is deined s whole or rgmented dried rk o young rnhes or whole dried piees o urrent yer twigs o vrious speies o the genus Slix [1]. SC is stndrdised sed on the ontent o sliin, ompound with nlgesi nd ntiphlogisti properties. However, linil trils suggest tht other ompounds lso present in Sliis Cortex n ontriute to the phrmologil eets. The results o linil trils suggest tht, esides sliyli derivtives, other sustnes like polyphenols (lvonoids, lvn-3- ols) nd simple phenols (phenoli ids) n ontriute to the therpeuti eets o SC [2, 3]. Phenoli ompounds possess strong ntioxidnt tivity; thus, this eet proly results rom synergisti intertions etween them nd sliin. Antioxidnt tivity is the undmentl property o phenoli mediinl plnt ompounds importnt or their helth proteting, inluding ntimutgeni, ntirinogeni, nd ntiging tivity. Retive oxygen speies (ROS) svenging preserves the genomi stility o ells through elimintion o rinogens nd intererene with DNA dduts ormtion [4]. Free rdils re onstntly genertedintheodysresultooxidtivemetolism.the retion o ROS lso is onneted with lipoxygense (LOX) nd xnthine oxidse (XO) tivity. Susequently, oxidtive stress ours i the ntioxidnt deense in the orgnism is not dequte. The ondition o in vivo oxidtive stress is deined s elevted levels o ree rdils or other ROS whih n eliit either diret or indiret dmge to the ody [5].

2 2 The Sientii World Journl Plnt extrts ontining seondry metolites hve served s ntioxidnts in phytotherpeuti mediines to protet ginst vrious diseses or enturies. Nturl ntioxidnts exhiit wide rnge o phrmologil tivities nd hve een shown to hve ntiner, nti-inlmmtory nd ntiging properties [6]. Aordingly, phytohemils n diretly interere with signling systems involved in the regultion o inlmmtory proesses, ngiogenesis nd ner invsion in mnner dependent on their ntioxidtive tivity nd onomitnt inhiitory eet on the enzymes involved with pthologil ondition [7, 8]. S. purpure L., S.dphnoidesVill., nd S. l L. re very populr herl speies irmed in the nturl hitts nd ield-ultivted in Polnd [9, 10]. The phenoli glyosides ontined in willow rk o this speies re known or their nti-inlmmtory, nlgesi, nd ever-reduing eets ndhveeenshowntorelieverheumtidisturnes, inetions, nd hedhe [3, 11]. Additionlly, rk o this speies ontins p-hydroxyenzoi, vnilli, innmi, p- oumri, eruli, nd ei ids phenoli ids nd nringenin [1]. However little is s yet known out their synergi eets. A prtiulrly importnt issue on the eetiveness o dietry supplements is iovilility nd iovilility o tive ompounds. Thus the im o this study ws to investigte nd to ompre the extrtility, ioessiility, nd iovilility in vitro o ntioxidtive ompounds rom rk o seleted Slix speies. 2. Mterils nd Methods 2.1. Plnt Mteril. Seleted S. l (SA), S. dphnoides (SD), S. purpure (SP), nd S. dphnoides x purpure (SDP) hyrid willow lones with the highest phenoli glyoside ontents [9, 10] originting rom their nturl hitts were ultivted on the sndy soil (hevy lomy snd). The study ws rried out in 2009 nd 2010 on 5-6 yer-old plnttion. The experiment ws ompletely rndomized lok design with three replites onduted in the re o experimentl ields ttheuniversityoliesienesinlulin(51 33 N; E). The plnttion ws estlished t the sping o m. Eh plot ws 16 m 2. The sndy soil ws hrterized y n verge ontent o humus (1.41%), very low phosphorus (17.3 mg kg 1 ), very low potssium (33.2 mg kg 1 ), very low mgnesium (15.0 mg kg 1 ), nd strong id retion (ph KCl 4.1). Minerl ertiliztion ws pplied in spring eore the eginning o vegettion in oth soil types: N 20 kg; P 13.1 kg; K 49.8 kg lulted per 1 h. WillowshootswerehrvestedinNovemereveryyer ( ) in three replites. The plnt mteril ws olletedintheormo60nnulshoots(20entireshoots per plot) rom every txon. The shoots were wshed with deionized wter. Brk ws seprted rom the wood y peeling nd susmpled or hemil nlysis. The rk mteril smpled or sliylte nlysis ws dried t room temperture nd intensively mixed nd homogenized. Ater drying, the phenoli glyosides ontent lulted on sliin ws determined y mens o the HPLC tehnique [12] in the Lortory o Lorm in Strogrd Gdński nd expressed s mg/mg dry mss (DM) Chemils. Ferrozine (3-(2-pyridyl)-5,6-is-(4- phenyl-suloni id)-1,2,4-trizine), ABTS (2,2 -zino-is (3-ethylenzothizoline-6-sulphoni id)) α-mylse, pnretin, pepsin, ile extrt, Folin-Ciolteu regent, linolei id, mmonium thioynte, nd hemogloin were purhsed rom Sigm-Aldrih Compny (Poznn, Polnd). All others hemils were o nlytil grde Extrts Preprtion Chemil Extrt (CE). Two grms o plnt mteril ws illed with 100 ml o 70% ethnol nd let in drkness y 2 weeks Buer Extrt (BE). Powdered smples o willow rk (1 g) were extrted or 1 h with 20 ml o PBS uer (phosphte uered sline, ph 7.4). The extrts were seprted y denttion nd the residues were extrted gin with 20 ml o PBS uer. Extrts were omined nd stored in drkness t 20 C Digestion In Vitro (DE). In vitro digestion nd sorption were perormed ording to Gwlik-Dziki [13]. The rk smples (1 g) were homogenized in stomher lortory lender or 1 min to simulte mstition with the presene o 15 ml o simulted slivry luid (prepred y dissolving 2.38 g N 2 HPO 4,0.19gKH 2 PO 4,nd8gNCl, 100 mg o muin in 1 liter o distilled wter. The solution ws djusted to ph = 6.75 nd α-mylse (E.C ) ws dded to otin 200 U per ml o enzyme tivity. For the gstri digestion 300 U/mL o pepsin (rom porine stomh muos, pepsin A, EC ) in 0.03 mol/l NCl, ph = 1.2 ws prepred. Further, simulted intestinl juie ws prepred y dissolving 0.05 g o pnretin (tivity equivlent 4 x USP) nd 0.3 g o ile extrt in 35 ml 0.1 mol/l NHCO 3 ), nd, susequently, the smples were shken or 10 min t 37 C. The smples were djusted to ph = 1.2 using 5mol/L HCl, nd, susequently, 15mL o simulted gstri luid ws dded. The smples were shken or 60 min t 37 C. Ater digestion with the gstri luid, the smples were djusted to ph = 6 with 0.1 mol/l o NHCO 3 nd then 15 ml o mixture o ile extrt nd pnretin ws dded. The extrts were djusted to ph = 7 with 1 mol/l NOH nd inlly 5 ml o 120 mmol/l NCl nd 5 ml o mmol/l KCl were dded to eh smple. The prepred smples were sumitted or in vitro digestion or 120 minutes, t 37 C in the drkness. Ater tht, smples were entriuged nd superntnts were used or urther nlysis Asorption In Vitro (AE). Considering tht ntioxidnts sorption tkes ple minly t the intestinl digestion stge, the resulting mixture (luids otined ter in vitro digestion) ws trnserred to the dilysis sks (D FT, Sigm-Aldrih), pled in n Erlenmeyer lsk ontining 50 ml o PBS uer nd inuted in rotry shker (2 times

3 The Sientii World Journl 3 per 2 h, 37 C). The PBS uer, together with the ompounds tht pssed through the memrne (dilyste) ws treted s n equivlent o the rw mteril sored in the intestine ter digestion Determintion o Totl Phenolis Content (TPC). Totl phenols were estimted ording to the Folin-Ciolteu method [14]. A 0.5mL smple o the extrt ws mixed with 0.5 ml o H 2 O,2mLoFolinregent(1:5H 2 O), nd ter 3 min with 10 ml o 10% N 2 CO 3.Ater30min,the sorne o mixed smples ws mesured t wvelength o 725 nm. The mount o totl phenolis ws expressed s glli id equivlents (GAE) per grm o dry mss (DM) Determintion o Totl Flvonoids (TFC). Totl lvonoids were estimted ording to the method desried y Bhorun et l. [15]. One milliliter o smple ws mixed with 1 ml 2 g/100 ml, AlCl 3 6H 2 O. Ater 10 min sorne t 430 nm ws mesured. The totl lvonoids ontent ws expresses s queretin equivlent (QE) in milligrms per grm o DM Determintion o Totl Phenoli Aids (TPA). Totl phenoli ids ontent ws determined ording to the Arnov method [16]. One milliliter o smple ws mixed with 5 ml o distilled wter, 1 ml 0.5 mol/l HCl, 1 ml o Arnov regent (10 g sodium molydte nd 10 g sodium nitrite dissolved in 100 ml o distilled wter) nd 1 ml 1 mol/l NOH nd omplete to 10 ml with distilled wter. Asorne ws mesured t 490 nm. The totl phenoli ids ontent ws expressed s ei id equivlent (CAE) in mirogrms per grm o DM Free Rdil Svenging Assy (AA). The experiments were perormed using n improved ABTS deoloriztion ssy [17]. ABTS + ws generted y the oxidtion o ABTS with potssium persulte. The ABTS rdil tion (ABTS + ) ws produed y reting 7 mmol/l stok solution o ABTS with 2.45 mmol/l potssium persulphte (inl onentrtion). The ABTS + solution ws diluted (with distilled wter) to n sorne o 0.7±0.05 t 734 nm. Then, 40 μlosmple ws dded to 1.8 ml o ABTS + solution nd the sorne wsmesuredttheendtimeo5min.theilityothe extrts to quenh the ABTS ree rdil ws determined using the ollowing eqution: svenging % =[ (A C A A ) A C ] 100, (1) where A C : sorne o ontrol nd A A :sorneo smple. Antioxidnt tivity ws expressed s IC 50 extrt onentrtion provided 50% o tivity sed on dose-dependent mode o tion Metl Chelting Ativity (CHEL). Chelting power ws determined y the method o Guo et l. [18]. The extrt smples (5 ml) were dded to 0.1 ml o 2 mm FeCl 2 solution nd 0.2 ml 5 mm errozine nd the mixture ws shken vigorously nd let stnding t room temperture or 10 min. Asorne o the solution ws then mesured spetrophotometrilly t 562 nm. The perentge o inhiition o errozine-fe 2+ omplex ormtion ws given elow ormul: %inhiition=[1 ( A P A C )] 100, (2) where A C : sorne o the ontrol nd A P :sornein thepreseneothesmple. Antioxidnt tivity ws expressed s IC 50 extrt onentrtion provided 50% o tivity sed on dose-dependent mode o tion Ferri Reduing Power (FRAP). Reduing power ws determined using the method desried y Oyizu [19]. Extrts (2.5 ml) were mixed with phosphte uer (2.5 ml, 200 mmol/l, ph 6.6) nd 2.5 ml o 1 g/100 ml queous solution o potssium erriynide K 3 [Fe(CN 6 )]. The mixture ws inuted t 50 C or 20 min. A portion (0.5 ml) o 10 g/100 ml trihloroeti id ws dded to the mixture, whih ws then entriuged t 25 g or10min.the upper lyer o solution (2.5 ml) ws mixed with distilled wter (2.5 ml) nd 0.5 ml o 0.1 g/100 ml FeCl 3,ndthe sorne ws mesured t 700 nm. IC 50 vlue (mg/ml) is the eetive onentrtion t whih the sorne ws 0.5 or reduing power nd ws otined y interpoltion rom liner regression nlysis Inhiition o Lipoxygense (LOXI). Lipoxygense tivity ws determined spetrophotometrilly t temperture o 25 C y mesuring the inrese o sorne t 234 nm over 2 min period [20]. The retion mixture ontined 2.45 ml 1/15 mol/l phosphte uer, 0.02 ml o lipoxygense solution (167 U/mL), nd 0.05 ml o inhiitor (vegetle extrt) solution. Ater preinution o the mixture t 30 C or 10 min, the retion ws initited y dding 0.08 ml 2.5 mmol/l linolei id. One unit o LOX tivity ws deined s n inrese in sorne o per minute t 234 nm. Antioxidnt tivity ws expressed s IC 50 extrt onentrtion provided 50% o tivity sed on dose-dependent mode o tion Inhiition o Xnthine Oxidse (XOI). The XOI tivities with xnthine s sustrte were mesured spetrophotometrilly [21], with the ollowing modiition: the ssy mixture onsisted o 0.5 ml o test solution, 1.3 ml o 1/15 mol/l phosphte uer (ph 7.5), nd 0.2 ml o enzyme solution (0.01 U/mL in M/15 phosphte uer). Ater preinution o the mixture t 30 Cor10min,theretion ws initited y dding 1.5mL o 0.15mmol/L xnthine solution.thessymixturewsinutedt30 Cndthe sorne (295 nm) ws mesured every minute or 10 min. XO inhiitory tivity ws expressed s the perentge

4 4 The Sientii World Journl inhiition o XO in the ove ssy mixture system nd ws lulted s ollows: %inhiition = (1 ΔA/min test ) 100, (3) ΔA/min lnk where ΔA/min test isthelinerhngeinsorneper minute o test mteril nd ΔA/min lnk is the liner hnge in sorne per minute o lnk. Antioxidnt tivity ws expressed s IC 50 extrt onentrtion provided 50% o tivity sed on dose-dependent mode o tion Theoretil Approh. The ollowing tors were determined to etter evlute the extrtility o phenoli ompounds: (i) the mstition eiieny tor (MEF) whih is n indition extrtility o the phytohemils during simulted mstition MEF = C BE C CE, (4) (ii) the digestion eiieny tor (DEF) whih is n indition extrtility o the phytohemils during simulted digestion DEF = C GE C CE, (5) (iii) the sorption eiieny tor (AEF) whih is n indition extrtility o the phytohemils during simulted sorption AEF = C AE, (6) C CE where C BE is the onentrtion o phenolis in rw extrt (BE), C GE is the onentrtion o phenolis in extrt ter simulted gstrointestinl digestion (GE), nd C AE is the onentrtion o phenolis in extrt ter simulted intestinl sorption (AE). The ollowing tors were determined to etter understnd the reltionships etween iologilly tive ompounds in the light o their ioessiility, iovilility, nd ioeiieny: (i) the ntioxidnt ioessiility index (BAC), whih is n indition o the ioessiility o ntioxidtive ompounds: BAC = A BE A GE, (7) (ii) the ntioxidnt iovilility index (BAV) whih is n indition o the iovilility o ntioxidtive ompounds: BAV = A GE A AE, (8) Sliin ontent (mg/g DM) S. l S. dphnoides S. purpure S. d x p Figure 1: Comprison o phenoli glyosides ontent in Slix rk. Brs (mens) ollowed y the dierent letters dier signiintly (Tukey-test, P < 0.05). (iii) the ntioxidnt ioeiieny index (BEF), whih is n indition o the iotivity o ioville ntioxidnt ompounds: BEF = A BE A AE, (9) where A BE is EC 50 o rw extrt (BE), A GE is IC 50 o extrt ter simulted gstrointestinl digestion (GE), nd AAE is IC 50 o extrt ter simulted intestinl sorption (AE) Sttistil Anlysis. All experimentl results re displyed s ± S.D. o three prllel experiments (n = 9)nd dt were evluted y nlysis o vrine (one-wy Anov). The sttistil dierenes etween the groups were estimted usingthetukeytest,α = Results nd Disussion 3.1. Comprison o Phytohemils Content. Slix rk smples were hrterized y diverse ontent o phenoli glyosides. The highest mounts were ound in the rk o SDP nd SD, wheres the lowest in the rk o SA (Figure 1). These results were in ordne with those otined y Sugier et l. [10]. Severl studies hve oused on phytohemil investigtion o Slix speies used or prepring the inl willow rk produts, nd, or exmple, S. dphnoides, S. pentndr, S. purpure, S. l, nds. rgilis hve een investigted or their ontent o phenoli glyosides. Sliyltes (lulted s sliin) re ound in ll memers o Slix speies ut S. dphnoides, S. rgilis, nd S. purpure ontin the gretest yield [22]. These onstituents re lso reported to possess ntirheumti, ntipyreti, hyperglyemi/hypoglyemi, uriosuri/ntiuriosuri tivities, inreses prothromin time, nd plsm-lumin inding [23]. In ddition to sliyltes, lvonoids nd ondensed tnnins onstitute the mjor groups o seondry metolites in Slix speies, nd these ompounds re elieved to ontriute

5 The Sientii World Journl 5 TPC TFC TPA Tle 1: Comprison o ontent nd extrtility tors o phenoli ompounds. Plnt mteril Extrts Extrtility tors Chemil Buer Digested Asored MEF DEF AEF S. l ± da ± 6.11 db ± 2.55 dc ± 2.12 dd S. dphnoides ± A ± 5.98 B ± 4.14 C ± 5.54 C S. purpure ± A ± 5.12 B ± 3.29 C ± 4.38 D S. d x p ± A ± 7.21 B ± 9.22 C ± 5.42 D S. l ± 0.99 da ± 0.55 db ± 0.77 dc 7.51 ± 0.33 dd S. dphnoides ± 9.58 A ± 2.69 B ± 1.23 B ± 1.25 C S. purpure ± 3.21 A ± 1.41 B ± 1.13 C ± 0.81 D S. d x p ± 7.89 A ± 2.54 B ± 2.25 B ± 0.84 C S. l ± 6.88 da ± 9.25 B ± 6.55 dc ± 3.45 D S. dphnoides ± A ± 9.13 A ± B ± 9.18 C S. purpure ± 8.45 A ± 7.77 B ± 9.23 C ± 7.13 dd S. d x p ± 9.48 A ± B ± C ± D The vlues designted y the dierent smll letters in the olumns o the tle (within individul extrt nd rtion) re signiintly dierent (α = 0.05). The vlues designted y the dierent smll letters in the lines o the tle re signiintly dierent (α = 0.05). to the nlgesi nd nti-inlmmtory eet o willow rk [24]. AsitneseenromTle 1, regrdless o kind o extrt, the est soure o phenoli ompounds (expressed s glli id equivlent) ws rk o SDP. However, the highest ontents o lvonoids were ound in smples otined rom SD rk. Tking into ount lohol nd uer extrts, the highest ontent o phenoli ids ws determined in smples otined rom SD rk; however the highest their onentrtion ws determined in extrts otined ter simulteddigestionndsorptionosdp rk. Tking into ount dt presented in Tle 1 itneonludedthtthe lestvlulesoureophenoliompoundswssa rk. Tking into ount extrtility o phenoli ompounds rtions, the highest extrtility o totl phenolis in ll smples hs een rehed in the simulted mstition stge, wheres simulted digestion eiieny ws signiintly lower. Importntly, eiieny o simulted sorption (with the exeption o SA rk) ws omprle to tht determined or the simulted digestion system. These results my indite high iovilility o phenoli ompounds rom Slix rk. Tking into ount lvonoids nd phenoli ids, the highest eiieny ws ound or simulted digestion system (exept SA phenoli ids), whih my indite high ioessiility o these ompounds. For lvonoids, eetiveness o simulted sorption system ws the lowest, whih my indite low iovilility o these ompounds, wheres or phenoli ids, AEF vlues were generlly omprle to DEF vlues, whih my indite high iovilility o phenoli ids rom tested soures (Tle 1). Results onerning totl phenolis ontent otined in our lortory were signiintly higher thn those onerning S. egypti [25]. The totl phenol ontent o 212 ± 4 mg GAE/g o DM ws otined in the ethnoli extrt o S. egypti rk while the lowest totl phenol ontent o 4± 1 mg GAE/g o dried smple ws otined in ylohexne extrt o rk. However, in omprison with our study, ethnoli extrt o S. egypti rk ontined higher mount o lvonoids 479 ± 63 mg CE/g o DM ws oserved in the ethnoli extrt o rk. This dierene my e prtilly explined y the t tht in our study, totl lvonoids ontent ws expressed y queretin equivlent. Aording to our reserh, rk o nlysed Slix genotypes ontined signiint mount o phenoli ids. The presene o p- hydroxyenzoi, vnilli, innmi, p-oumri, eruli nd ei ids ws reported in the genus Slix [1, 25]. Aording to work o Podłok-Oleh et l. [1] the orms o glyosides or ester derivtives re wider spred thn ree phenoli ids. Furthermore, the reveled presene o pyrotehol in the rk o some willow speies rises question regrding the sety o the pplition o this herl mediine, minly the twigs [1].Ontheotherhnd,Freishmidtetl.[26]onluded (rom the in vitro dt) tht not only lvonoids nd sliin derivtives, ut lso tehol n proly ontriute to the nti-inlmmtory tivity o willow rk extrts Extrtility o Antioxidnts. Severl phenoli ids, inluding sliyli nd ei ids, possess nti-inlmmtory nd nlgesi tivity whih hs een ssoited with their ntioxidnt tivity. Svenging oxygen ree rdils deides the nti-inlmmtory tivity o glli nd prototehui ids [27]. Antioxidnt tivity ws lso shown or ei, eruli nd hlorogeni ids [1]. Brk o ll tested Slix speies showed signiint ntirdil tivity (Figure 2). These properties strongly dependent o extrtion system nd geneti tors. Tking into ount extrtion proedure, etter results were otined when using EtOH thn PBS uer, with the exeption o SDP rk. Digestion in vitro in ll ses (exept SDP rk) relesed ompounds le to quenh ABTS rdils. Antirdil ompounds rom SA nd SP were esily ioville in vitro,wheres potentil iovilility o ntirdil ompounds relesed during in vitro digestion rom rk o SD nd SDP ws diiult. Regrdless o this, the highest ntirdil tivity ws ound or extrts otined rom SDP (Figure 2).

6 6 The Sientii World Journl IC 50 (mg DM/mL) d de d d e j h i gh g S. l S. dphnoides S. purpure S. d x p Chemil extrt Buer extrt Digested Asored Figure 2: Eet o extrtion system on ntirdil tivity o phytohemils rom rk o dierent Slix genotype. Brs (mens) ollowed y the dierent letters dier signiintly (Tukey-test, P< 0.05). IC50 (mg DM/mL) e d d d d S. l S. dphnoides S. purpure S. d x p Chemil extrt Buer extrt Digested Asored Figure 3: Eet o extrtion system on helting power o phytohemils rom rk o dierent Slix genotype. Brs (mens) ollowed y the dierent letters dier signiintly (Tukey-test, P< 0.05). e Tking into ount the helting power o nlyzed smples the similr reltionships hve een reeived or ll Slix vrieties (Figure 3). Regrdless o plnt mteril, the lowest tivity ws ound or hemilly extrtle ompounds. Signiintly higher tivity ws oserved in the ses o potentilly mstition-extrtle ompounds (uer extrts). Digestion in vitro relesed ompounds le to metl ions helte rom ll smples exept SDP rk. Most importntly, tive ompounds were highly ioville in vitro, wheres signiint dierenes etween smples were not ound (Figure 3). Brk o ll Slix speies ontined ethnol-extrtle ompounds with omprle reduing ility. Signiint dierenes were onerning potentilly mstition-extrtle phytohemils the highest tivity ws oserved in the se o SD nd SDP rk, wheres the lowest or rk o SP. Digestionin vitro, unexpetedly, did not relese tive ompounds-tivity o these extrts nd ws the lowest in ll ses. Most importntly, ompounds with reduing ility esily permeted dilysis memrne, whih my indite their iovilility. In ll ses tivity o extrts ter simulted sorption ws the highest (Figure 4). Antioxidnt tivity o Slix rk ws widely studied [24, 26, 27], ut omprtive nlysis o results ws diiult due to dierent wys o its mesure nd expression. And so, ntirdil tivity o rk o S. egypti (depending on the extrtion system) rnged rom 10 to 105 mg queretin equivlent/d DM [26]. The results rom in vitro studies showed tht methnoli extrt rom S. nigr rk is n eetive svenger o oth superoxide nd hydrogen peroxide rdils. This my e due to the phenoli ontent, prtiulrly lvonoids present in S. nigr extrt, whih re elieved to e potent superoxide rdil svengers. Moreover, presene o suh ompounds in the extrt my help neutrlize hydrogen peroxide into wter through eletron dontion. The ntioxidtive properties o methnoli extrt rom S. nigr on svenging hydrogen IC50 (mg DM/mL) de de d d h S. l S. dphnoides S. purpure S. d x p Chemil extrt Buer extrt Digested Asored Figure 4: Eet o extrtion system on reduing power o phytohemils rom rk o dierent Slix genotype. Brs (mens) ollowed y the dierent letters dier signiintly (Tukey-test, P< 0.05). peroxide rdils were ound to e superior (IC 50 vlue ws μg/ml) [23] while the ility to inhiit superoxide rdils ws lower thn tht reported or S. pre lower extrt [28]. This tivity o Slix rk is ttriuted to the high ntioxidnt ontents whih ould ret with ree rdils to stilize nd terminte rdil hin retions [29]. Oxidtive stress nd inlmmtion hve een reported to e losely ssoited with tumor promotion stge o rinogenesis. The intivtion o enzymes y ree rdils nd the umultion o oxidized proteins my ply ritil role in the ltertion o ellulr untion nd ell deth. Some essentil growth regultory proteins lose their untion when dmged y ree rdils [30]. In the light o dt given in Figure 5.itmyeonluded thtompoundsletoinhiitloxwereesilyethnolextrtle. Ativity o ll extrts ws omprle nd very high in omprison with other smples otined rom the sme plnt mteril. Signiintly lower eiieny ws g i

7 The Sientii World Journl 7 IC50 (mg DM/mL) d e e e d S. l S. dphnoides S. purpure S. d x p Chemil extrt Buer extrt Digested Asored Figure5:EetoextrtionsystemonLOX-inhiitorytivityo phytohemils rom rk o dierent Slix genotype. Brs (mens) ollowed y the dierent letters dier signiintly (Tukey-test, P< 0.05). d IC50 (mg DM/mL) e S. l S. dphnoides S. purpure S. d x p Chemil extrt Buer extrt d g Digested Asored Figure 6: Eet o extrtion system on XO-inhiitory tivity o phytohemils rom rk o dierent Slix genotype. Brs (mens) ollowed y the dierent letters dier signiintly (Tukey-test, P< 0.05). g oserved ter using PBS uer. Extrts ontining potentilly mstition extrtle phytohemils were hrterizedyomprletivity.signiintdiereneswere oserved or extrts otined ter simulted digestion. Digestion in vitro relesed tive ompounds rom ll smples exept S. l rk. Only in this se, tivity o simulted digest ws lower thn tivity o uer extrt. Unortuntely, ompounds le to inhiit LOX with diiulty permeted dilysis memrne. Ativity o extrts ter simulted sorption ws the lowest in ll ses exept SA smple (Figure 5). Most o the tumor promoters hve een reported to inrese the tivity o XO thus inresing superoxide rdil genertion nd enhning hydrogen peroxide genertion [30]. Tking into ount tivity o XO inhiitors, it my e onluded tht the est results were otined ter using ethnol or extrtion. Extrtility in uer system ws signiintly lower. Espeilly signiint dierenes were oserved or SA nd SD tive ompounds, wheres the lowest or phytohemils rom SDP. Chnges ourring during simulted digestion used derese o tivity in ll smples exept SD. This tendeny is strongly visile in SDP nd SP rk. All the more interesting is the t tht XO inhiitors esily permeted dilysis memrne (Figure6). In ll ses, the highest tivity ws ound or extrts ter simulted sorption. Results presented in this study onirm the results otined y Sultn nd Sleem [30]. Cited investigtors stted tht the pretretment o S. pre ws oserved to reverse the hemil indution in XO tivity nd hydrogen peroxide ontent. This suggests the ntitumor promoting potentil o S. pre. High ntioxidnt tivity under in vitro onditions o methnol extrtrom S. nigr ws lso onirmed. It hs gret potentil to meliorte the progression o ollgen-indued rthritis y ontrolling inlmmtory proteins, nitri oxide, nd ntioxidnt enzymes [23]. On the other hnd, is interesting to note tht wter extrt rom SD rk used s eliitor signiintly inresed LOX nd XO-inhiitory tivity in rooli sprouts. Tretment with 1% SD wter extrt lso signiintly improved SODlike tivity nd inrese o OH rdils svenging ility, proly y stimulting phenolis overprodution in rooli sprouts [31, 32] Studies o Potentil Bioessiility nd Biovilility. The gstrointestinl trt my e onsidered s n extrtor where oth the mehnil tion during mstition nd the hemil tion during the digestive phse ontriute to the extrtion o phenoli ompounds [33]. It should e tkenintoonsidertionththighontentndtivityo phytohemils determined in hemil system not lwys go hnd in hnd in high tivity in vivo. For this reson, the investigtions o ioessiility nd iovilility o iotive ompounds should e rried out. Models sed on humn physiology hve een developed s simple, hep, nd reproduile or investigting the ood omponents In vitro digestion models re widely used or studying struturl hnges, digestiility, nd relese o ood omponents under simulted gstrointestinl onditions [34]. Dt given in Tle 2 indite tht the est soure o ioessile ntirdil ompounds ws rk o SP, while the lowest BAC vlue ws otined or rk o SDP;however highly ioville in vitro were phytohemils le to quenh ree rdils relesed during in vitro digestion rom rk o SP nd SA. The highest ioeiieny ws ound or ntirdil rom SP. Tking into ount potentil ioessiility o ompounds le to metl ions helte smples my e ordered s ollows: SA > SD SP > SDP. Unexpetedly the highest iovilility nd ioeiieny in vitro were ound or SDP phytohemils. Similr reltionship ws oserved ompring iovilility nd ioeiieny in vitro o redutive ompounds the highest BAV nd BEF vlues were determined or SDP rk smples while their potentil ioessiility ws reltively low. Though reltively low ioessiility in vitro (BAC vlues not exeed 1), LOX inhiitors relesed rom tested Slix rk were strongly

8 8 The Sientii World Journl Tle 2: Comprison o ntioxidnts ioessiility (BAC), iovilility (BAV), nd ioeiieny (BEF) tors. Ativity Antirdil tivity Chelting power Reduing power LOX inhiitory tivity XO inhiitory tivity Plnt mteril Ftors BAC BAV BEF S. l S. dphnoides S. purpure S. d x p S. l S. dphnoides S. purpure S. d x p S. l S. dphnoides S. purpure S. d x p S. l S. dphnoides S. purpure S. d x p S. l S. dphnoides S. purpure S. d x p ioville nd ioeiient in vitro. The highest vlues o these prmeters were otined or S. l phytohemils, wheres the lowest or SDP ompounds. Brk o SA ontined lso ioville nd ioeiient ompounds le to inhiit XO. Although the highest BAC vlue ws determined in the se o SDP phytohemils, potentil iovilility o these ompounds ws reltively low. The highest ioeiieny ws determined or XO inhiitors derived rom the rk o SDP nd SA. While white willow is the willow speies most ommonly used or mediinl purposes, purple willow (S. purpure), nd violet willow (S. dphnoides) re ll sliin-rih nd my e soldunderthelelowillowrk.themjormetolites o sliin re gentisi id, sliyli id, nd sliyluri id, with sliyli id eing the mjor omponent in the serum. Ater orl ingestion o willow rk, pek levels o sliyli id were ound in less thn 2 hours. A sliin ontent o 240 mg orresponds to pproximtely 87 mg o etylsliylte, whih is more rdioprotetive thn nlgesi. It is lso ound tht the iovilility o the sliin in the ormultion eing evluted ws greter thn tht ound in other studies. This suggests tht dierent ormultions o willow rk extrt result in dierent iovililities, onept tht my e pplile to ll herl meditions [35]. onneted to physiologil proesses. This ide hs ound n pplition in phrmology during investigtions on omintions o ew metolites in multidiretionl therpy [36]. It hs een proposed tht lthough willow extrts hve een trditionlly used s nti-inlmmtory ompounds or their sliin ontent, the presene o high mounts o phenoli ompounds n ontriute to the eneiil eets [2, 3]. Our investigtions support this thesis. In the light o presenteddt,itmyeonluded,thttheestsoureo potentilly mstition-extrtle ompounds with multidiretoriliologiltivitywssdndsdprk.other tors, oten overlooked in studies o the iologil tivity o phytohemils, re their ioessiility nd iovilility. Our study shows high ioessiility nd iovilility in vitro o Slix rk phytohemils, whih my indite tht extrts rom this speies o plnts my provide helth promoting eneits to the onsumers; however, this issue requires urther study. Conlit o Interests The uthors delre tht there is no onlit o interests regrding the pulition o this pper. 4. Conlusion Unlike in the se o syntheti phrmeutils sed on n tivity o single (hemil) tive ompounds, numerous phytohemil ompounds t in eneiil mnner y n dditive o synergisti tivity in one or numerous trget sites Reerenes [1] L. Połok-Oleh, M. Kruze-Brnowsk, D. Głód, A. Kwik, nd E. Łojkowsk, Chromtogrphi nlysis o simple phenols in some speies rom the genus slix, Phytohemil Anlysis,vol.21,no.5,pp ,2010.

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