Sapwood of Carob Tree (Ceratonia siliqua L.) as a Potential Source of Bioactive Compounds

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1 SHORT REPORT Re. Nt. Prod. 7:3 (2013) Spwood of Cro Tree (Certoni siliqu L.) s Potentil Soure of Biotive Compounds Luís Custódio 1*, An Luís Esp 1, João Ptrr 1, Ros Aligué 2, Fernndo Aleríio 3,4,5,6, Nuno Ros Neng 7, José Mnuel Florênio Nogueir 7 nd Anel Romno 1 1 Institute for Biotehnology nd Bioengineering, Centre of Genomis nd Biotehnology (IBB/CGB), Fulty of Sienes nd Tehnology, Ed. 8, Cmpus of Gmels, , Fro, Portugl 2 University of Brelon, Shool of Mediine, Deprtment of Cell Biology, Csnov 143, E-08036, Brelon, Spin 3 Institute for Reserh in Biomediine, Brelon Siene Prk, Bldiri Reix 10, 08028, Brelon, Spin 4 CIBER-BBN, Networking Centre on Bioengineering, Biomterils nd Nnomediine, Brelon Siene Prk, Bldiri Reix 10, Brelon, Spin 5 University of Brelon, Deprtment of Orgni Chemistry, Mrtí i Frnqués 1-11, Brelon, Spin 6 Shool of Chemistry, University of KwZulu-Ntl, 4001-Durn, South Afri 7 University of Lison, Fulty of Sienes, Chemistry nd Biohemistry Deprtment nd Centre of Chemistry nd Biohemistry, Cmpo Grnde Ed. C8, Lison, Portugl (Reeived Novemer 7, 2011; Revised April 1, 2012; Aepted My 17, 2013) Astrt: Methnol (ME) nd hot wter extrts (WE) of ro tree spwood (Certoni siliqu L.) exhiited high ntioxidnt tivity nd were rih in phenoli ompounds, with the min ompounds identified y HPLC/DAD s gentisi id nd (-)-epitehin. The ME displyed high in vitro ntitumor tivity ginst humn tumourl ell lines nd redued intrellulr ROS prodution y HeL ells fter tretment with H 2 O 2. (-)-Epitehin ws shown to ontriute to the ytotoxi tivity of the ME. This is the first report on the iologil tivity of ro tree spwood. Keywords: Antioxidnt tivity; ytotoxi; gentisi id; phenoli ompounds; ROS. 1. Plnt Soure Cro tree (Certoni siliqu L.) is n evergreen plnt from the Mediterrnen re. Cro fruits re minly used for loust en gum (LBG, E410) extrtion from the seeds, widely used in food industry s thikening gent nd stilizer. Spwood smples, whih were identified y J. Grç, were otined from mture femle trees of ultivr Mult in August of 2005, ultivr olletion field, Ministry of Agriulture nd Rurl Development nd Fisheries (Direção Regionl de Agriultur do Algrve, Tvir, Portugl). * Corresponding uthor: E-Mil: lustodio@ulg.pt (L. Custódio); Phone x7381. The rtile ws pulished y Ademy of Chemistry of Gloe Pulitions Pulished 5/28/2013 EISSN:

2 Spwood of ro tree (Certoni siliqu L.) Previous Studies Fruits nd leves of ro tree ontin proteins nd phenoli ompounds, nd exhiit ntioxidnt, ntiprolifertive nd ntimiroil tivities [1-16]. In previous work Bln [17] used ommon spetrosopi (Folin Ciolteu nd Rhodnin ssys) nd hromtogrphi (GC-MS) methods to determine the hemil omposition of methnol / wter extrts of hertwood nd spwood of ro tree from Turkey. Those extrts were frtionted y orgni solvents nd hydrolysed with hydrohlori id to determine ound hydrolysle tnnins. Before hydrolysis the min phenolis of diethyl ether nd diethyl ether / methnol frtions of spwood were glli id (GA) nd hlone (4 OH), wheres fter hydrolystion, the diethyl ether / methnol frtion ws minly omposed y methyl inositol, GA nd methyl GA. To our est knowledge, there re no reports on the iologil tivities of ro tree spwood. 3. Present Study Smples were dried t 40ºC for 2 dys, milled nd stored in drk t -20ºC. ME ws prepred y Soxhlet extrtion [8] nd WE ws otined y oiling smples (1 g) in 100 ml of distilled wter for 10 min, whih ws ooled nd filtered (Whtmnn nº 4). For the ell experiments, methnol ws removed under redued vuum t 40 C nd the dry extrt resuspended in the ulture medium. Stok solutions of the phenoli ompounds were prepred in phosphte-uffered sline (PBS, ph 7.4), nd diluted with ulture medium immeditely efore use in the ell ssys to the onentrtions orresponding to the mounts quntified in the ME. Totl ontent of phenoli ompounds (TPC), ondensed tnnins (TTC) nd flvonoids (TFC) were evluted s desried previously [8]. The HPLC nlysis nd identifition of the min phenoli ompounds in the extrts were onduted ording to [9-11]. The ntioxidnt tivities of the extrts t different onentrtions (125 to 1000 µg/ml) were evluted y their rdil-svenging tivity (RSA) on the DPPH [18, 19] nd ABTS [20, 21] rdils nd reduing power [22, 23]. Butylted hydroxytoluene (BHT, E321) ws used s positive ontrol. Four humn tumour ell lines (ervil: HeL, prostte: DU-145, rest: MDA-MB-231 nd olon: HCT-116 ells) were used to determine ytotoxiity of ME, s it exhiited the highest ntioxidnt tivity. Extrts, dissolved in ulture medium, were pplied to ells for 72h t different onentrtions ( µg/ml), nd the ell viility ws determined y WST-1 ssy in HeL ells [16] nd MTS method [17, 18] in other ell lines. Phenoli ompounds were pplied to HeL ells for 72h in onentrtions orresponding to the mounts quntified in the ME, nd ell viility ws determined y the MTT ssy [9, 11]. The pity of the ME to svenge intrellulr ROS genertion s result of n oxidtive stress indution y H 2 O 2 tretment of HeL ells ws evluted y 2',7'-dihlorodihydrofluoresin diette (DCFH-DA) method [9, 10]. 4. Results nd Disussion ME hd the highest level of TPC, lmost 5 times higher thn the level present in the WE (4.8 mg GAE/g, Tle 1). In previous study [17], it ws reported tht n queous extrt of spwood from ro tree hd TPC of 4.2 mg GAE/g dried mteril, s determined y F-C method, whih is similr to the vlue otined in this work for WE. The TTC ws 2.5 mg CE/g for the ME nd 1.2 mg CE/g for the WE, while flvonoids were present in tre mounts in oth smples (Tle 1). Our results indited tht the spwood of ro tree hs higher TPC thn leves, pulps nd germ flour, ut lower onentrtions of TTC nd TFC [8, 18]. Avllone et l. [9] lso oserved different mounts of phenoli ompounds in different orgns (pods, germ nd seeds) of femle ro trees. Gentisi id ws identified s mjor ompound, followed y (-)-epitehin, GA, (+)- tehin nd hlorogeni id (Tle 1, supporting informtion). Minor mounts of syringi id, vnnilin, rutin nd kempferol were lso oserved (Tle 1, supporting informtion). Bln [17] studied the phenoli omposition of queous / methnoli extrts of hertwood nd spwood from ro tree, fter frtioning using orgni solvents, efore nd fter hydrolysis with hydrohlori id.

3 227 Custódio et l., Re. Nt. Prod. (2013) 7: Before hydrolysis, the min ompounds identified on diethyl ether nd diethyl ether / methnol frtions of spwood were GA nd hlone (4 OH), while fter hydrolysis, the diethyl ether / methnol frtion ws minly omposed y methyl inositol, GA nd methyl GA. Our results differ from tht erlier reported, sine gentisi id ws identified s the min ompound in ro tree spwood. Tle 1. Phytohemil evlution nd rdil svenging tivity (RSA) of ro tree spwood extrtives. Phenolis ontent RSA (IC 50, µg/ml) TPC TTC TFC DPPH ABTS Smple Methnol extrt 23.8 ± ± ± ± ± 0.0 Hot wter extrt 4.8 ± ± 0.0 t ± ± 0.1 BHT * ± ± 0.0 TPC: Totl phenoli ontent, mg GAE/g extrt DW; TTC: totl tnnin ontent, mg CE/g extrt, DW; TFC: totl flvonoid ontent, mg,, RE/g extrt, DW; GAE: glli id equivlents; CE: tehin equivlents; RE: rutin equivlents; t: tre mounts; (-) not tested. Different letters in the sme olumn indites signifint differenes y Dunn s New Multiple Rnge Test t p < 0.05; *Referene ompound, 1 mg/ml. The extrts showed similr RSA on DPPH, nd even higher on ABTS rdils thn BHT (Tle 1), nd higher pity of reduing iron (Fig. 1, supporting informtion). Antioxidnt tivity of ro tree spwood ws found to e higher thn the reported erlier for lef, pulps nd germ flour extrts of the sme speies [16, 18], whih ould e due to its higher ontent of phenoli ompounds [24]. Tretment with ME resulted in the derese in ell viility, for ll ell lines tested (Fig. 1). However, only in HeL ells the pplition of inresing onentrtions of the extrt ws ompnied y the orresponding derese of ellulr viility, following ler onentrtiondependent pttern (Fig. 1). The IC 50 vlues otined for different ell lines were s follows: HeL, 41.3 µg/ml; MDA-MB-231, 21.8 µg/ml nd HCT-116, µg/ml. Cell viility (% of ontrol) d d Conentrtion (µg/ml) HeL MDA-MB-231 HCT-116 DU-145 Cell lines Figure 1. Effet of tretment with the ME from ro tree spwood on the ell viility of humn ner ell lines fter 72h of inution. Different letters in the sme ell line indites signifint differenes etween onentrtions y Dunn s New Multiple Rnge Test. The ME hd higher in vitro ntitumorl tivity ginst rest nd olon ell lines thn lef extrt of the sme speies [9], possily due to the higher ontent in phenoli ompounds. Phenolis disply in vitro ntiprolifertive nd ytotoxi tivities through different mehnisms, suh s poptosis nd ell yle rrest [25]. There re lso evidenes tht their ytotoxiity n e due to their oxidtive tivity, sine phenolis n e either ntioxidnts or pro-oxidnts [25]. In ft, their effet on the in vitro viility nd prolifertion of tumour ells is highly dependent on their struturl hrteristis (reviewed in [25]). Gentisi id hd protumourl tivity on HeL ells (ell viility: 125.9%, Fig. 2, supporting informtion). Gentisi id is n tive metolite of sliyli id degrdtion nd displys nlgesi, nti-inflmmtory, ntirheumti, ntirthriti, ytostti nd ntioxidnt tivities

4 Spwood of ro tree (Certoni siliqu L.) 228 [21]. However, our results indited tht under the experimentl onditions of this study, gentisi id ws not the responsile for the ytotoxi tivity of the rude extrt, sine it used signifint inrese in ell viility. Besides the struturl hrteristis of phenoli ompounds, other fetures, suh s dose, trget moleule, nd environment n modify their in vitro ntitumorl tivity [26]. (-)- Epitehin exhiited the strongest ytotoxi tivity, reduing ell viility to 59.4% (Fig. 2, supporting informtion). Epitehin, whih is one of the min omponents of green te, is flvonoid [27] nd together with epigllotehin gllte, epigllotehin, nd epitehin gllte, is responsile for the ntitumorl tivity of green te [27]. A signifint redution in ell viility ws oserved fter tretment with ll the remining phenoli ompounds, exept for hlorogeni id, whih hd no ytotoxi effet (Figure 2, supporting informtion). However, sine we tested the tivity of the totl extrt, we nnot exlude tht other ompounds might ontriute to the inhiition of ell viility. The iologil tivity of rude extrt nnot e ttriuted to single ompound ut lso to other omponents present in the extrt, often resulting from their synergisti effets. The pplition of the ME t the onentrtion of 400 µg/ml resulted in signifint derese of ROS prodution ompred with ontrol ells (Fig. 3, supporting informtion). ROS re the produts of norml ellulr metolism, nd re extremely retive nd potentilly dmging trnsient hemil speies to severl mromoleules, suh s DNA. The humn ody is equipped with enzymti nd nonenzymti ntioxidnt systems to protet ells ginst ROS indued dmge. Nevertheless, those systems my not e enough in ses involving severe or ontinued oxidtive stress, nd ertin mounts of exogenous ntioxidnts re onstntly required for the mintenne of dequte level of ntioxidnts in order to lne the ROS in humn ody [28]. As onlusion, ro tree spwood n e soure of ntioxidnt nd ytotoxi ompounds, nd (-)-epitehin n e one of its tive onstituents. Aknowledgments This work ws prtly supported y CICYT (CTQ /BQU), Instituto de Slud Crlos III (CB nd PI060624), the Generlitt de Ctluny (2005SGR 00662), the Institute for Reserh in Biomediine, nd the Brelon Siene Prk. L. Custódio thnks to the Portuguese Foundtion for Siene nd Tehnology (FCT) for post-dotorl grnt (grnt SFRH/BPD/20736/2004). Supporting Informtion Supporting Informtion ompnies this pper on Referenes [1] R. Avllone, M. Plessi, M. Brldi nd A. Monzni (1997). Determintion of hemil omposition of ro (Certoni siliqu): protein, ft, rohydrtes, nd tnnins, J. Food Comp. Anl. 10, [2] L. Corsi, R. Avllone, F. Cosenz, F. Frin, C. Brldi nd M. Brldi (2002). Antiprolifertive effets of Certoni siliqu L. on mouse heptoellulr rinom ell line, Fitoterpi 73, [3] S. Kumzw, M. Tniguhi, Y. Susuki, M. Shimur, M-S. Kwon nd T. Nkym (2002). Antioxidnt tivity of polyphenols in ro pods, J. Agri. Food Chem. 50, [4] R. Owen, R. Huner, W. Hull, G. Eren, B. Spiegelhlder, H. Brtsh nd B. Her (2003). Isoltion nd struture eluidtion of the mjor individul polyphenols in ro fire, Food Chem. Tox. 41, [5] M. Ppginnopoulos, H. Wollseifen, A. Mellenthin, B. Her nd R. Glens (2004). Identifition nd quntifition of polyphenols in ro fruits (Certoni siliqu L.) nd derived produts y HPLC-UV- ESI/MS, J. Agri. Food Chem. 52, [6] P. Dki, B. Wthelet nd M. Pquot (2007). Isoltion nd hemil evlution of ro (Certoni siliqu L.) seed germ, Food Chem. 102, [7] C. Bengoehe, A. Romero, A. Villnuev, G. Moreno, M. Aliz, F. Millán, A. Guerrero nd M. C. Puppo (2008). Composition nd struture of ro (Certoni siliqu L.) germ proteins, Food Chem. 107, [8] L. Custódio, E. Fernndes, A. L. Esp, R. Aligué, F. Aleríio nd A. Romno (2009). Antioxidnt tivity nd in vitro inhiition of tumor ell growth y lef extrts from the ro tree (Certoni siliqu L.), Phrm. Biol. 47,

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