Study On Horseradish (Armoracia Rusticana) Essential Oil And Comparison With The Related Species – Debreceni Horseradish (Armoracia Macrocarpa).

S udy On Ho se adish (A mo acia Rus icana) Essen ial Oil And Compa ison Wi h The Rela ed Species – Deb eceni Ho se adish (A mo acia Mac oca pa). Dok o i (PhD) é ekezés Nguyen Minh Nha Téma eze ő: D . Vasas Gábo DEBRECENI EGYETEM Te mésze udományi Dok o i Tanács Juhász-Nagy Pál Dok o i Iskola Deb ecen, 2016. 1 2 Nyila koza ok 3 Ezen é ekezés a Deb eceni Egye em Te mésze udományi Dok o i Tanács Juhász-Nagy Pál Dok o i Iskola Biológia P og amja ke e ében készí e em a Deb eceni Egye em e mésze udományi dok o i (Ph.D) okoza ának elnye ése céljából. Deb ecen, 2016.10.17 ………………………... Nguyen Minh Nha Tanusí om, hogy Nguyen Minh Nha dok o jelöl 20011-2016 közö a en megne eze Dok o i Iskola biológia p og amjának ke e ében i ányí ásommal égez e munkájá . Az é ekezésben oglal e edményekhez a jelöl önálló alko ó e ékenységé el megha á ozóan hozzájá ul . Az é ekezés el ogadásá ja asolom. Deb ecen, 2016.10.17 …….……………………... D . Vasas Gábo 4 S udy On Ho se adish (A mo acia Rus icana) Essen ial Oil And Compa ison Wi h The Rela ed Species – Deb eceni Ho se adish (A mo acia Mac oca pa). É ekezés a dok o i (Ph.D) okoza megsze zése é dekében a biológia udományágban Í a: Nguyen Minh Nha , pha macis Készül a Deb eceni Egye em Juhász-Nagy Pál Dok o i Iskola (biológia dok o i p og amja) ke e ében Téma eze ő: D . Vasas Gábo A dok o i szigo la i bizo ság: elnök: D . ………………………… ………………... agok: D . ………………………… ……..…………. D . ………………………… ………………… A dok o i szigo la időpon ja: 2016…………………. Az é ekezés bí álói: D . ………………………… ………………… D . ……………………….... ………………… D . ………………………… ………………… 5 6 A bí áló bizo ság: elnök: D . …………………………. ………………… agok: D . …………………………. ………………… D . …………………………. ………………… D . …………………………. ………………… D . …………………………. ………………… Az é ekezés édésének időpon ja: 7 Table o con en s Page In oduc ion 14 Chap e I: Li e a u e Re iew 1. The plan 2. The o igin 3. The ela i e species 4. The c op 5. Possible uses 6. Possible pha macological s udies 6.1. ITCs inhibi ca cinogen-ac i a ing enzymes and induce ca cinogen-de oxi ying enzymes 6.2. ITCs igge apop osis pa hways 6.3. ITCs inhibi cell cycle p og ession 6.4. ITCs inhibi p o-in lamma o y and p o-ca cinogen signaling ac o 6.5. ITCs possess an imic obial and an ioxidan ac i i ies 6.6. ITCs a e conside ed o be oxidan s hemsel es 16 16 18 18 21 21 22 22 23 24 25 25 26 Chap e II: Essen ial Oil Dis illa ion Technology 1. Small-scaled dis illa ion 1.1. Ma e ials and sample p epa a ion 1.2. Me hods 1.3. Resul s and discussion 2. Medium-scaled dis illa ion 2.1 Ma e ials and echnology 2.2. Resul s and discussions 27 27 27 28 31 33 34 36 8 Chap e III: Analy ical S udies 1. Backg ound in o ma ion 1.1. Glucosinola es (GLSs) 1.2. GLSs hyd olysis eac ion 1.3. Iso hiocyana es (ITCs) 2. Gas ch oma og aphy and mass spec ome y s udy on ho se adish essen ial oil 2.1. Sample p epa a ion and me hod 2.2. Resul and discussions 3. Capilla ies elec opho esis (CE) s udy 3.1. Ma e ial p epa a ion and ins umen 3.2. Me hod 3.3. Resul and discussion 38 38 38 39 40 42 43 44 46 47 48 55 Chap e IV: Compa a i e Analysis o A. us icana and A. mac oca pa 1. Backg ound in o ma ion 1.1. My osinase 1.2. Pe oxidase 2. Liquid ch oma og aphy and mass spec ome y s udies on glucosinola es 2.1. Ma e ials and me hod 2.2. Resul s and discussion 3. Ana omical s udies 3.1. Ma e ial and me hods 3.2. Resul s and discussion 4. Gel elec opho esis s udies 4.1. Ma e ials and me hod 4.2. Resul s and discussions 71 71 71 72 74 74 75 78 79 79 83 84 84 9 Chap e I Li e a u e Re iew 1. THE PLANT A mo acia us icana, Cochlea ia a mo acia, and A mo acia lapa hi olia a e scien i ic names ha e e o a pe ennial plan o Mus a d amily (gene a-g oup Ca damineae o B assicaceae) commonly known as ho se adish (Mohlenb ock 1980).The plan can each he heigh o 120cm. I has a ha dy glab ous s em, om which wa y ma gin lea es a ise di ec ly (cauline lea ) ollowing a ci cula a angemen pa e n (basal ose e). Ho se adish lea is desc ibed o ha e a leng h o 30–100cm, a co da e base, long pe iole, and he shape sligh ly a ying om he lowe o he uppe mos lea . Whe eas a sho e pe iole and a lobe shape wi h en i e o se a e ma gin a e cha ac e is ics o lowe lea es, uppe lea es ha e a na ow base, ob use apex, oblong o lanceola e shape wi h c ena e o se a e ma gin. The ma gin is linea o almos en i e in he case o uppe mos lea es (Mohlenb ock 1980).Ho se adish has whi e, e ame ous lowe s a anged in acemes and a smoo h, b own angus isep a e ui —a ui la ened a a igh angle o he sep um, which usually con ains e y ew (≤ 6) o no seeds. In addi ion, he lack o e idence ha ho se adish g ows om seeds sugges s s e ili y (Sampline and Mille 2009). 16 Figu e 1. A mo acia us icana. Adap ed om h p://plan illus a ions.o g. 17 2. THE ORIGIN Ho se adish is belie ed o be na i e o Eas e n Eu opean coun ies (such as Romania o Uk aine) e en hough i can now be ound h oughou Eu ope. Ho se adish occu ence was ound only associa ed in a eas whe e he e a e people. No in o ma ion abou he wild popula ion o he plan has been ound. I is sugges ed ha he wild popula ion o ho se adish may ha e become ex inc o i may ha e been de i ed om ela ed species such as A mo acia mac oca pa and A mo acia sisymb oides (DC.) N.Busch ex Ganesh. I s abili y o sp ead by hizome could mean ha ho se adish is a p oblema ic weed, hough i is also possible ha i s wild popula ion is jus ye o be ound (Sampline and Mille 2009). 3. THE RELATIVE SPECIES A mo acia mac oca pa is na i e o Eas e n Eu ope, speci ically o he ma shes o he Cen al Danube Basin (e.g., in Hunga y, Czech Republic, Romania, and Bulga ia), whe eas Sibe ia is he na i e land o A mo acia sisymb oides. A mo acia mac oca pa g ows in eeds (Sci po-Ph agmi e um aus o-o ien ale), we meadows (Phala ide um a undinaceae) and sal meadows (Ag os i (o)- Alopecu e um p a ensis). I a o s alkaline soils, sal ole an , we o wa e co e ed adhe en loam, clay, sal soils, ich in nu ien s and alkaline mine als. In Hunga y, A mo acia mac oca pa can be ound in he No h Hunga ian Moun ains (Má a ma gins), G ea Hunga ian Plain (Danube egion, Danube- Tisza köze egion and Tiszán úl egion) and Sou h T ansdanubia. I has a agmen ed dis ibu ion, especially in no heas e n pa s o he coun y. Due o 18 i s habi a specializa ion and a con inuing decline in ex en and quali y o i s habi a , A mo acia mac oca pa is classi ied as a e (Hunga y, Romania) o e y a e (Se bia). A. mac oca pa and A. us icana a e nea ly iden ical. The lowe s and ui s o A. mac oca pa a e la ge han hose o A. us icana; u he , A. mac oca pa ui s con ain nume ous seeds, while he ui s o A. us icana a ely con ain seeds. A mo acia mac oca pa seems o ha e much la ge in lo escences han A. us icana .(Sampline and Mille 2009). 19 Figu e 2. A mo acia mac oca pa. Adap ed om h p://plan illus a ions.o g. 20 A mo cia sisymb ioides is dis inguishable om o he ela i e species by i s whi ish, glaucous lea es. Mo eo e , i s cauline lea es a e au icula e. Nei he o A mo acia us icana and A. mac oca pa ha e hese ea u es (Sampline and Mille 2009). The e is li le o no in o ma ion on hese wo species o A mo acia genus, no on he ela ion be ween hese species and ho se adish. Resea ch in his ield could help imp o e cu en ho se adish c ops, ia adi ional b eeding o bio echnological echniques. 4. THE CROP Fo p opaga ion, he oo s a e used exclusi ely. Ho se adish oo is whi e, cylind ical o ape ing, which can each a leng h o 60 cm in loose soil and consis o se e al la e al oo s. Deep, sil y loam soils wi h good d ainage and empe a e clima es a e a o able condi ions o bo h annual and pe ennial ho se adish c ops. Fo comme cial p oduc ion, ho se adish is usually cul i a ed as an annual c op, which usually s a s wi h plan a ion in ea ly sp ing because he oo s need he highe empe a u es o he summe (15–27oC) and he lowe empe a u es o he end o summe and all (11–22oC) o de elop. Usually he whole oo is ha es ed. In he case o pe ennial p oduc ion, he unde g ound shoo g own om o iginal oo is collec ed, and he o iginal oo is le in he ield o egene a ion. Majo ho se adish-g owing coun ies a e he Uni ed S a es and Hunga y (Sheha a e al. 2009). 5. POSSIBLE USES Due o he cha ac e is ic pungen , in ensi e lach yma o y odo and as e o he 21 oo , ho se adish is cul i a ed mos ly o condimen p oduc ion. I is also used as a ype o ood and o adi ional medical pu poses. Fo example, ho se adish has been used o ease pain such as low back pain and pain associa ed wi h scia ica and heuma ism. I is also adi ionally used as a u ina y, gas oin es inal, and espi a o y aid, o oo hache, and as aph odisiac. Combina ion o ho se adish oo and honey in wa m wa e is made o ea ing in luenza. Howe e , he unde lying mechanism o possible medicinal bene i s o ho se adish emains unclea (Sampline and Mille 2009). 6. POSSIBLE PHARMACOLOGICAL STUDIES Iso hiocyana es (ITCs) a e he p oduc s o hyd olysis eac ion o glucosinola es (GLSs), which occu s when he plan issues a e damages. ITCs a e po en ial an i-ca cinogenic agen s ha no only inhibi he de elopmen o cance cells bu also elimina e es ablished cance cells (Zhang 2004). The mechanism unde lying an i-ca cinogenic e ec o ITC is unclea e en hough he e ha e been many sugges ed hypo heses based on obse a ions in animal and human cell s udies. The e a e ye no clinical ials on ITCs o hei p ecu so s, GLSs (Valgimigli and Io i 2009). 6.1. ITCs inhibi ca cinogen-ac i a ing enzymes and induce ca cinogen- de oxi ying enzymes. The unde lying pha macology mechanisms o ITCs on cance cells could be due o he possibili ies o inhibi ing ca cinogen-ac i a ing enzymes and inducing ca cinogen-de oxi ying enzymes. Cellula enzymes such as hose belonging o he cy och ome P450 (CYP) amily a e known as ca cinogen- 22 ac i a ing enzymes due o hei abili y o ans o m p o-ca cinogens in o ca cinogens, he ac i e o m ha can be ha m ul o cells. ITCs showed he abili y o inhibi o down- egula e hese enzymes. In a s udy whe e animals we e ea ed wi h ca cinogenic agen (ni osamine), ITCs, especially a ylalkyl ITCs such as PEITC, inhibi ed CYP enzymes ha a e necessa y o ni osamine ac i a ion (Hech 2000). Phase II enzymes such as quinone educ ase 1 (QR1), glu a hione S- ans e ase (GST), and heme oxygensase 1 (HO-1) play impo an oles in cellula de ense mechanism agains oxidan s and ca cinogens and a e known as ca cinogen- de oxi ying enzymes. Inducing gene ansc ip ions o hose enzymes is hough o be one o he an i-ca cinogenic s a egies used by ITC. Modula ion o phase II enzymes is he consequence o he in e ac ion be ween ITCs and he complex consis ing o a nuclea ansc ip ion ac o , N 2 (NF-E2– ela ed ac o 2), and a p o ein ancho ed o he ac in cy oskele on known as Keap1 (Kelch-like ECH- associa ed [e y h oid cell-de i ed p o ein wi h CNC homology] p o ein 1). Binding o ITC o he complex in ol es he eac ion o ITC and p o ein sul hyd yl esidues o Keap1, leading o he dissocia ion o he complex in o N 2 and Keap1, as i is obse ed ha N 2 concen a ion is signi ican ly inc eased when ea ing human hepa oma HepG2 cells wi h 25 μM AITC (Jeong e al. 2005). F ee N 2 hen ansloca es in o he nucleus whe e oge he wi h o he ansc ip ion ac o s, i in e ac s wi h an ioxidan esponse elemen (ARE), esul ing in he ac i a ion o gene ansc ip ion o ca cinogen- de oxi ying enzymes (Dinko a-Kos o a e al. 2002). 6.2. ITCs igge apop osis pa hways. 23 Apop osis o p og ammed cell dea h esul s om clea age o speci ic cellula subs a es caused by he ac i i y o aspa a e-speci ic cys eine p o ease known as caspase. Main signaling pa hways leading o caspase ac i a ion a e ia dea h ecep o s such as umo nec osis ac o (TNF) ecep o s and ia mi ochond ia. AITC (10 μM) ac i a ed caspase 9 (mi ochond ia pa hway), caspase 8 (dea h ecep o pa hway), and caspase 12 (es ogen ecep o [ER] pa hway) in conjuga ion wi h caspase 3 ac i a ion in human leukemia HL60 cells (R. Yu e al. 1998). Mi ochond ial pa hway is egula ed la gely by membe s o Bcl2 amily— an iapop o ic membe s (Bcl-2, Bcl-x L), p oapop o ic membe s (Bax, Bak, Bok), and BH3-only p o ein (Bid, Bad, Bin). Changes in he egula o s o mi ochond ia pa hway we e accompanied wi h ITCs ea men in se e al s udies. Apa om he e ec on egula o s, ITCs a e sugges ed o exe di ec e ec on mi ochond ia i sel , causing he elease o cy och ome c. By binding o a heme g oup, cy och ome c becomes holocy och ome c, which has he abili y o ac i a e caspases (S i as a a e al. 2003; Singh e al. 2004; Xiao e al. 2003; Fimogna i e al. 2002; Chen e al. 1998; Xu and Tho nalley 2001). 6.3. ITCs inhibi cell cycle p og ession. The key egula o molecules o cell p oli e a ion h ough he cell cycle a e cyclins – he egula o y p o eins ha ac i a e a speci ic class o enzymes known as cyclin-dependen kinase (cdk). Toge he , hey ac as an ac i a ed complex ha pushes he cell h ough ce ain s ages o he cycle. Each s age o he cell cycle is ma ked wi h he ac i i y o ce ain ype o cyclin and kinases. AITC caused cell cycle a es in he G1 phase and in he G2/M phase (Zhang, Tang, 24 and Gonzalez 2003; Tang and Zhang 2004; Smi h e al. 2004). Cell cycle a es a e AITC ea men was accompanied wi h down- egula ion o cyclin B, cdk1, cdc25B, cdc25C, and ubulin dis up ion, sugges ing ha he inhibi o y e ec o ITCs is a complex p ocess. Cell ypes, dose and ime o exposu e may in luence he inhibi o y e ec o ITCs (Cla ke, Dashwood, and Ho 2008; Hwang and Lee 2006; Chiao e al. 2002). 6.4. ITCs inhibi p o-in lamma o y and p o-ca cinogen signaling ac o . Cance can be he consequence o ch onic in lamma ion p ocess in which p o- in lamma o y and p o-ca cinogen signaling ac o s sec e ed by cells play impo an oles. These ac o s a e cell-de i ed media o s (e.g., ni ic oxide (NO), p os aglandin E2 (PGE2), and umo nec osis ac o (TNF)). Inhibi ing he p oduc ion o sec e ion o hose molecules is possibly one o he an ica cinogenic mechanisms by which ITCs ac (Ge häuse e al. 2003; Ippoushi e al. 2002). 6.5. ITCs possess an imic obial and an ioxidan ac i i ies. ITCs possess an ibac e ial e ec agains se e al bac e ia, which is ela ed o an ica cinogenic e ec s, e.g., agains Helicoba e pylo i, one possible cause o s omach cance (Zsolnai 1971). Bac e icidal e ec agains Helicobac e pylo i, Esche ichia coli, Salmonella yphimu ium, S aphylococcus au eus, S ep ococcus mu ans, Penicillium no a um, Bacillus ce eus, and Vib io pa ahaemoly icus was epo ed a AITC concen a ion o 3.8–16.7 μM, wi h an ac i i y ha was 7.8–20.5 imes less han ha o PEITC (Shin, Masuda, and Naohide 2004; Luciano and Holley 2009; Tunc e al. 2007). The e a e se e al 25 using di ec hea ing mode. Rega dless o di e en amoun o added wa e , he plan ma e ials nea he bo om o he s ill cha ed, a ec ing he yield, quali y and odo o essen ial oil. A emp o dec ease hea ing powe oge he wi h ex end he dis illa ion du a ion led o he decomposi ion o essen ial oil's componen s due o he p olonged in e ac ion wi h ho wa e . Mo eo e , he insu icien a e o s eam p oduc ion due o he low hea ing powe (<90oC) causes he e lux o oil back o he sample con aining lask, leading o decomposi ion eac ion and poo oil quali y. The same phenomenon was obse ed in appa a us using s eam hea ing, plus he con ac su ace a ea o he plan ma e ials and s eam is limi ed, i.e. plan ma e ial ended o agg ega e, p e en ing s eam om hea ing he uppe , inne laye s. In ac ional dis illa ion, longe dis ance and low back o essen ial oil d ople s a e p obably he explaina ion o disc epancy in essen ial oil amoun . In he imp o ised hyd o-dis illa ion using wa e ba h hea ing, p oblems wi h bu ning and cha o plan ma e ials we e sol ed. The lask was hea ed a cons an empe a u e o boiling wa e (app oxima ely 100oC). No ma e ial cha was obse ed. The hea ing su ace a ea was inc eased as he lask was almos en i ely imme sed in o he boiling wa e . P oduced s eam was su icien o he p ocess. The yield o 0.071% (166.66 ± 27.54 μl) essen ial oil was ex ac ed om esh ho se adish main oo s (233 ± 38.74 g). Fo esh la e al oo s (151.67 ± 1.52 g), he yield o essen ial oil is 0.12% (187 ± 9.6 μl). The amoun o wa e added ollowing he a io o 1:3 olume / ma e ial weigh , esul s in he highes yield compa ed o o he a io in case o la e al oo dis illa ion. Fo he main oo s dis illa ion, less wa e was added (1:6 olume / ma e ial mass) because o hei highe wa e con en . The du a ion o 1.5 h was su icien o comple e ex ac ion o essen ial oil. In addi ion o p o ide he s able yield o 32 essen ial oil, his ex ac ing me hod comes wi h simplici y and he ease o use, i.e. equi ed minimum a en ion du ing he ope a ion. Figu e 7. Compa ing cha o ho se adish essen ial oil yields om di e en dis illa ion me hods. Cha was made by Lib eO ice Calc 5.2.1. 2. MEDIUM SCALED DISTILLATION The medium-scaled dis illa ion echnology was buil based on he expe iences om he labo a o y-scaled ex ac ing me hods. As he amoun o plan ma e ial is much highe in his case (app oxima ely 15 kg), di e en adjus men s on in luencing ac o s (e.g. du a ion o he p ocess, amoun o added wa e , hea ing empe a u e…) we e c ucial in o de o each he simila yield (0.07%) o labo a o y-scaled expe imen s. 33 0.00% 0.02% 0.04% 0.06% 0.08% 0.10% 0.12% 0.14% Yield (%) 2.1 Ma e ials and echnology The esh leshy ho se adish oo s we e cul i a ed in Újlé a and supplied by KELET PRODUCTION Z ., Hunga y. The plan ma e ials (≈15.5 kg) we e chopped, g ind by comme cial indus ial g inde and ans e ed immedia ely in o he s ill wi h g ea ca e due o he pungency o high ma e ial amoun . The s ill and i s componen s a e made o s ainless s eel o a oid co osi eness om essen ial oil. The plan ma e ial was mixed du ing dis illa ion by a o a ing ame wi h diagonal ba s, which is in oduced inside he s ill. Hea ing pla es a he bo om (n=3) and hea ing ings on he wall (n=3) o he s ill ensu e he su iciency o hea ing su ace a ea as well as hea ing powe . The o a ion ( pm) o mixing ame and powe o hea ing pla es as well as hei ac i a ing o de a e accessible h ough a con olling panel loca ed sepa a ely om he s ill. The spi al condense was used o su icien ly p ocess la ge amoun o s eam. 34 Figu e 8. Diag am o mid-scaled dis illa ion ins umen . Diag am was made by INKSCAPE, e sion 0.91. Legends – 1: he ank, 2: hea ing ings, 3: hea ing pla es, 4: o a ing mo o , 5: lid o he ank, 6,7: mixing ame wi h diagonal ba s, 8: he mos a , 9: al e, 10: neck connec o , 11: connec ing ube, 12: condense , 13: con ol panel. The impo an es ed ac o s o ex ac ing echnology includes: amoun o wa e added o he sys em, op imum empe a u e and hea ing mode. Di e en amoun s o added wa e (0, 250, 500, 750 mL) we e es ed and we e combined wi h a ious hea ing p og am (90, 92.5, 95, 97.5oC). Hea ing p og ams we e designed based on he ac i a ing o de o wall and bo om pla es and he desi ed end- empe a u e o he sys em (90÷97.5oC). The ex ac ed essen ial oil was sepa a ed om he wa e y dis illa e by using massi e cen i uga ion (Beckman A an i J-25) a 13000 pm o 10min. 35 2.2. Resul s and discussions A he same hea ing p og am (95oC), expe imen s wi h di e en added wa e amoun s showed ha sligh ly be e yield (0.03% yield) o essen ial oil was achie ed when no wa e was added compa ed o 0.02% essen ial oil yield in he o he cases (i.e. 0.25, 0.5, 0.75 L). This can be explained based on he la ge amoun o wa e comes om he esh oo s. I is impo an o no e ha adding mo e wa e in o he sys em would esul in high amoun o wa e y ex ac . The hea ing p og am was designed as ollowing: hea ing ings on he wall o he ank is ac i a ed in o de o he empe a u e in he s ill o each 70oC ( o dec ease he hea ing du a ion o subs ance agglome a ed a he bo om o he ank) and hea ing pla es on he bo om is ac i a ed o each desi ed end empe a u e. Low hea ing empe a u e (<95oC) esul s in p olonged du a ion o he dis illa ion while high empe a u e (>97oC) mo e likely esul s in sample cha . Good esul was achie ed when he se ing empe a u e alls a ound 95oC, i.e. be e yield (0.05%) compa ed o o he empe a u e se ings (i.e. 90oC – 0.02%, 92.5oC – 0.03% and 97.5oC – 0.03%). No sample bu ning was eco ded bu agglome a ions o plan ma e ial we e ound a he bo om o he s ill by he end o he p ocess, sugges ing ha he dis illa ion may no each i highes yield. Howe e , his is an ine i able echnical p oblem. The ex ac ing p ocess is conside ed o be comple ed in 2 h. Dis illa ion o a e age 15 kg o esh ho se adish main oo s using he op imal se ing esul ed in 12 mL essen ial oil (a e age yield 0.08%). On he o he hands, dis illa ion o esh la e al oo s (a e age yield o 0.01%) ailed o achie e he expec ed yield ega dless o di e en amoun o added wa e . Va ious amoun s oil we e ex ac ed om ho se adish oo s incuba ed in di e en pe iod. In some cases, e.g., dis illa ion 36 o oo s s o ed o 21 days in cold oom (4oC), be e yield was achie ed (i.e. 0.14%). Howe e , due o he disc epancy o he esul s, di e en incuba ing pe iods and s o ing condi ions a e no a eliable me hod compa ed o he dis illa ion using esh oo s. The newly de eloped ex ac ing echnology sa is ied he equi emen s. I p o ides a s able yield o essen ial oil and simila o ha om he labo a o y-scaled expe imen s. I is easy o handling, ope a ing and equi es easonable ime o a en ion. The p oposed echnology is sui able o ex ac ing high amoun o essen ial oil om esh ho se adish oo s. The in es iga ion o he e iciency o he new me hod is discussed in he ollowing chap e . 37 Chap e III Analy ical S udies 1. BACKGROUND INFORMATION 1.1. Glucosinola es (GLSs) A. us icana is ich in glucosinola es, he seconda y p oduc s ha play an impo an ole in he plan s de ensi e sys em. GLS is an o ganic anion ha is s able and soluble in wa e (Oe lemans e al. 2006). GLS’ s s uc u e consis s o β- hioglucoside N-hyd oxysul a es wi h a side chain (R) and a sul u -linked β- D-glucopy anose moie y (Figu e 9). Based on he s uc u e a ie y o he side chain (R), GLS can be classi ied in o di e en g oups, he mos common a e alipha ic, ω-me hyl hioalkyl, a oma ic, and he e ocyclic (indole) GLS (Fahey, Zalcmann, and Talalay 2001). Figu e 9. Gene al s uc u e o glucosinola es. Adap ed om www.wikipedia.o g. 38 Acco ding o p e ious s udies, eigh di e en GLS ha e been ound in ho se adish (Figu e 12b), in which sinig in (2-p openyl o allyl glucosinola e) and gluconas u iin (phene hylglucosinola e) we e ound in highe quan i y compa ed wi h he o he s. Sinig in is he main GLS ound in ho se adish and accoun s o 74% o he o al GLS in he plan ma e ial (Li and Kushad 2005). The quan i y and quali y o plan GLS con en a e signi ican ly a ec ed by he plan ’s age and en i onmen al ac o s (e.g. soil e ili y, wound, pa hogen challenge, e c…) (Bjö kman e al. 2011). 1.2. GLSs hyd olysis eac ion GLSs hyd olysis is igge ed when damage is done o he plan . This eac ion equi es an impo an enzyme called my osinase, which is sepa a ed unde no mal condi ions o a oid con ac wi h GLS. Once hyd olysis is igge ed, my osinase is eleased and eac s wi h GLS, esul ing in an uns able aglucone ( hiohyd oxima e O sul ona e), which hen gi es ise o di e en p oduc s depending on eac ion condi ions and pa icipa ion o o he ac o s. Fo example a pH 7, 37–45 ◦C, and unde he e ec o my osinase, he p oduc s o GLS hyd olysis eac ion a e mainly iso hiocyana es (ITCs). Ni ile and epi hioni ile a e inal p oduc s i he eac ion akes place a pH 3 and 6, espec i ely, in he p esence o Fe2+ ions and an epi hiospeci ie p o ein. The pa icipa ion o hiocyana e- o ming ac o in he eac ion esul s in he o ma ion o hiocyana e. ITC, hiocyana e, ni ile, epi hioni ile, and oxazolidine hione a e possible p oduc s o GLS hyd olysis, o which ITC has ecen ly a ac ed esea ch, as a po en ial an ica cinogenic agen (Zhang 2010; Li and Kushad 2005). 39 Figu e 10. Glucosinola es b eak-down pa hways. Figu e was made by Ma in JS e sion 16.9.12. 1.3. Iso hiocyana es (ITCs) S uc u e and p ope ies – The mos common p oduc s yielded om hyd olysis o GLSs a e ITCs ha sha e a common s uc u e consis ing o –NCS g oup and side chain –R ( igu e 11). Because o he –NCS g oup, speci ically he C a om o he g oup, ITCs possess elec ophilic cha ac e is ics, i.e. hey a o eac ions wi h nucleophilic molecules (e.g. cellula pep ides and amino acids), hus leading o possible pha macological e ec s. Elec ophilici y is in luenced by he side chain –R because o he s e ic hind ance e ec on he elec ophilic C a om. In addi ion o elec ophilici y, he side chain also in luences he lipophilici y o he molecule (Zhang 2004). ITCs a e mo e s able in acidic condi ions han in neu al o alkaline condi ions due o hei eac ions wi h wa e molecule’s OH− ions (Oh a, Taka ani, and Kawakishi 1995). O ganic sol en s (e.g., hexane, ace one, and e hyl ace a e) a e mo e a o able o s o age han aqueous solu ions, in which decomposi ion o ITCs is empe a u e 40 dependen . The decomposi ion a e is as a empe a u e 37oC, dec easing and s opping as he empe a u e dec eases o −5oC. The abili y o eac eadily wi h e hanol makes ITCs uns able in alcoholic solu ion. Addi ion o ci ic acid, suga es e s, o ege able oil may s abilize a solu ion o ITCs (Ina e al. 1981). Figu e 11. Gene al s uc u e o Iso hiocyana e. Adap ed om www.wikipedia.o g. Ho se adish ITCs – Allyl ITC (AITC) and 2-phenyle hyl ITC (PEITC) a e he mos common ITC componen s ound in ho se adish oo . Whe eas AITC is also ound o be p esen in bo h ho se adish and wasabi (Wasabi japonica), PEITC is only ound in ho se adish, which may a leas pa ly con ibu e o he di e ence in as e be ween he wo species (A. Dep ee, M. Howa d, and P. Sa age 1998). AITC cons i u es 78% o o al ho se adish ITCs. AITC is mos likely esponsible o he pungen , lach yma o y odo and as e o ho se adish oo . I is he inal p oduc om hyd olysis o sinig in. Also known by he common name “mus a d oil,” a oom empe a u e AITC appea s as a colo less liquid wi h a boiling poin o 150oC and mel ing poin o −80oC (Zhang 2010; E. Y. Yu e al. 2001). PEITC, also e e ed o as “phene hyl mus a d oil”, is he colo less o ligh yellow inal p oduc o he hyd olysis o gluconas u iin. 41 imme sed in boiling wa e o 10 minu es (wa e c ess) and 30 minu es (o he ege ables) o comple ely inac i a e he my osinase. 10 mL o MeOH was added o he cooked plan ma e ial, ollowed by ho ough homogeniza ion, and cen i uga ion a 13000 pm o 3 minu es. The supe na an was e apo a ed o d yness. P io o analysis, he d ied samples we e esuspended in wa e , cen i uged and subjec ed o analysis by bo h CE and LC/MS - a e dilu ion wi h wa e , i necessa y. These ex ac s a e e e ed o as “me hanolic ex ac s”. The ins umen – Me hod de elopmen was ca ied ou on a P inCE-C 700 capilla y elec opho esis ins umen . A 60 cm used silica capilla y wi h 50 µm (i.d.) was used. Fo my osinase ac i i y s udy, e ec i e leng h was 7.2 cm (sho -end injec ion). Fo de e mina ion o di e en glucosinola es o allyl iso hiocyana e quan i ica ion om concen a ed eal ma ices, e ec i e leng h was 52.8 cm (long-end injec ion). Capilla y p econdi ioning and pos - condi ioning we e p e iously desc ibed (Gonda e al. 2013), Sample injec ion was hyd odynamic (100 mba × 0.25 min.). Sinig in was quan i ied a 230, gluconas u iin a 210, ITC de i a es a 275 nm. The so wa es – ChemAxon Ma inSke ch was used o d awing chemical s uc u es and eac ions, while Calcula o Plugins we e used o s uc u e p ope y p edic ion and calcula ion. Ve sion 6.2.3_b915, 2014 was used. Figu es we e gene a ed using sc ip s in R 3.1.1. (R De elopmen Co e Team, 2009) using ggplo 0.9.3.1. 3.2. Me hod 3.2.1. Op imiza ion o sepa a ion 48 The basic o he new me hod is o be able o sepa a e GLSs and ITCs om he plan ma ix. Solu ions o pu e sinig in (SIN), gluconas u iin (GNT) and me hanol ex ac o ho se adish oo s we e used. The s a ing backg ound elec oly e (BGE) solu ion con ained N-T is(hyd oxyme hyl)me hyl-3- aminop opanesul onic acid (TAPS, 20mM), sodium deoxychola e (250 mM), sodium e abo a e (15 mM), pH was 8.50. In luencing pa ame e s (i.e., pH, concen a ion o elec oly es and su ac an s, addi ion o o ganic sol en s and pola i y (sho -end injec ion mode)) we e aken in o conside a ion in o de o imp o e he speed o he ope a ion wi h simila sensi i i y and s abili y. 3.2.2. De i a iza ion s udy Figu e 14. De i a iza ion scheme o iso hiocyana es gene a ed in- ial by my osinase media ed decomposi ion o glucosinola es. Figu e was made by Ma in JS e sion 16.9.12. Di hioca bama e is he p oduc o in- ial de i a iza ion eac ion o ITCs wi h me cap oace ic acid (MAA) ( igu e 14). MAA is he sui able de i a izing agen o ITCs because i is inexpensi e, miscible wi h wa e a any pH, and he p oduc (di hioca bama e) ca ies cha ges a he pH o BGE (pH = 9.0). The 49 s udy o op imal concen a ion o asco bic acid (enzyme ac i a o ) and MAA was designed as ollowing: 350 µg/mL AITC was de i a ized in phospha e bu e (10 mM, pH 7.5) wi h he di e en concen a ions o MAA (1, 5, 10 mM), and asco bic acid (0, 1, 5, 10 mM). The wo king pH ange o he eac ion was in es iga ed based on he quan i ica ion (using p oposed CE-MEKC me hod) o he gene a ed de i a ized p oduc s in di e en pH – 350 µg/mL AITC was de i a ized wi h MAA (5 mM), asco bic acid (1 mM) in a pH se ies o 5.5-9.5 (ace a e, phospha e o bo a e bu e s in 10 mM end-concen a ion). The cha ac e iza ion o de i a iza ion p oduc s by LC-MS was un on a The mo Accela HPLC a ached o a The mo LTQ XL Linea Ion T ap MS (column: Hype sil Gold 50 mm × 2.1 mm × 1.9µm). ESI ioniza ion pa ame e s we e as ollows: hea e empe a u e, 300 °C; shea h gas, N2; low a e, 20 a bi a y uni s (a b); aux gas low a e, 8 a b; sp ay ol age, 4 kV; capilla y empe a u e, 275 °C; capilla y ol age, -28.00 V, nega i e ion mode. G adien componen s we e A, wa e wi h 0.1% ( / ) o mic acid; B, MeCN wi h 0.1% ( / ) o mic acid. The ime p og am was 10% B: 0 – 2 min, 10 – 90% B: 2 – 7 min, 90%B: 7-13 min, 90 – 10% B: 13 – 13.1 min, 10% B: 13.1 – 15 min. Flow a e was 300 µL/min. 1 µL o a de i a ized sample (ace a e bu e , pH 5.0, me cap oace ic acid (5 mM), asco bic acid (1 mM)) con aining 1 µg/mL o allyl iso hiocyana e and phene hyl iso hiocyana e was injec ed. 50 Figu e 15. Cha ge o iso hiocyana es and hei me cap oace ic acid di hioca bama e p oduc s in he pH ange usually used o capilla y elec opho esis. Calcula ions we e done by ChemAxon Ma inSke ch 6.2.3., using de aul me hod pa ame e s. Abb e ia ions – AITC: allyl iso hiocyana e; PEITC: phene hyl iso hiocyana e; AITCp allyl iso hiocyana e di hioca bama e p oduc ; PEITCp: phene hyl iso hiocyana e di hioca bama e p oduc . 3.2.3. Valida ion The alida ion o he new CE me hod was pe o med using sho -end injec ion mode. Fo AITC, he 5-poin s calib a ion cu e (4.5, 9, 45, 90, 450 µg/ml) was p epa ed and measu ed as ollowing: 25 mg/ml AITC s ock solu ion was p epa ed wi h MeCN, which hen dilu ed wi h wa e o each he desi ed concen a ion. These solu ions we e mixed wi h de i a iza ion solu ion (NaH2PO4 (100 mM), me cap oace ic acid (50 mM), asco bic acid (10 mM), pH 7.50, ollowing 9:1 a io). In case o sinig in and gluconas u iin , 7-poin 51 calib a ion cu e (5, 10, 50, 100, 500, 1000, 5000 µg/ml) was p epa ed by dilu ion o hese glucohyd ola es wi h wa e . F om he calib a ion cu es, limi o de ec ion (LOD), limi o quan i a ion (LOQ), coe icien o de e mina ion (R2) and eg ession equa ions we e calcula ed. Rep oducibili y s udies we e designed as ollowing: i e injec ions o iso hiocyana e de i a ized solu ion (100 µg/ml), and 100µg/ml glucosinola e solu ion we e in oduced and measu ed pe day in 3 days. The ela i e s anda d de i a ion (RSD) be ween he a ea unde cu e (AUC) and e en ion ime was calcula ed by DAx 8.1. so wa e. Fo accu acy s udy, sample o 10 µl inac i a ed cold bu e ho se adish ex ac /100 µl olume was sepa a ely spiked wi h 1000 µg/ml s anda ds and he eco e ies we e calcula ed. The absence o esidual glucosinola es in hese plan ex ac s was con i med by injec ing samples wi hou adding glucosinola e s anda ds. 3.2.4. Quan i ica ion o GLSs and AITC om eal plan ma ix B ussels sp ou s, ho se adish, adish and wa e c ess me hanol ex ac we e measu ed by capilla y elec opho esis (long end injec ion mode) o glucosinola e p o ile and LC-ESI-MS o sinig in and gluconas u iin compa a i e s udy. Glucosinola e de e mina ion by LC-ESI-MS was done on a The mo Accela HPLC a ached o a The mo LTQ XL Linea Ion T ap MS, column: Kine ex XB-C18 (100 × 2.10 mm, 2.6 µm, Phenomenex). Fi e-poin calib a ion cu es o sinig in and gluconas u iin in wa e anging om 0.5 o 40 µg/mL we e used as calib a ion cu es. G adien componen s we e A, wa e wi h 0.1% ( / ) o mic acid; B, MeCN wi h 0.1% ( / ) o mic acid. The ime p og am was 5% B: 0 – 1 min, 5 – 25% B: 1 – 4 min, 25 – 60% B: 4-5 min, 60 – 5% B: 5 – 6 min, 5% B: 6-8 min. Flow a e was 250µL/min. 1µL o he 52 dilu ed me hanol ex ac was injec ed, ypically 5-100- old dilu ions wi h wa e we e app op ia e. The ins umen was uned au oma ically o sinig in o ob ain he op imal ESI pa ame e s. ESI ioniza ion pa ame e s we e as ollows: capilla y empe a u e, 275 °C; sou ce hea e empe a u e, 300 °C; shea h gas, N2; shea h gas low, 30 a bi a y uni s (a b); aux gas low, 5 a b; sou ce ol age, 3 kV; capilla y ol age, -1.00 V, nega i e ion mode. The new CE me hod is applied on he s udy o AITC con en o ood p oduc s (i.e., mus a d sauce, wo ypes o ho se adish sauces and wasabi c eam). The samples we e composed o 100 mg o ood p oduc dilu ed wi h 100µl bu e ed deodo iza ion solu ion ( NaH2PO4 (100 mM), asco bic acid (10 mM), me cap oace ic acid (50 mM), pH 7.5) and 800 µl wa e . A e mixing and cen i uga ion, he supe na an was in oduced di ec ly o CE (long-end injec ion mode) o measu emen . pH eadjus men ( o 7.5) is necessa y i he p oduc s con ain signi ican amoun o inega . 3.2.5. My osinase ac i i y de e mina ion and AITC elease s udy The my osinase ac i i y s udy was designed as ollowing: The my osinase- inac i a ed plan ex ac ob ained by bu e ex ac ion o boiled plan , se ed as nega i e con ols. The esh plan ex ac s by cold bu e we e dilu ed wi h bu e solu ion ( 100 mM NaH2PO4, 10 mM asco bic acid, pH 6.50) in 9:1 and wa e . 5 µL GLS s ock (10 mg/ml) was added o 190 µL o p e ious mix u e. Addi ion o he subs a e was he eac ion s a poin , nega i e con ols we e ob ained by using plan ex ac s ha we e p e iously boiled o inac i a e he my osinase. The eac ion was un a 25 °C o 5 minu es, and hen e mina ed by hea ing he es ubes o 100 °C o 5 minu es, ollowed by he sinig in 53 concen a ion de e mina ion by CE a e cen i uga ion a 13000 pm o 1 min. Subs a e concen a ion dec ease was kep below 10%. The plan ex ac s we e also checked o he p esence o esidual subs a es (no sinig in added). P o ein de e mina ion om hese ex ac s was done using B ad o d's eagen wi h bo ine se um albumine as s anda d. Unde he same condi ions, a se ies o di e en ini ial sinig in concen a ions we e es ed o ob ain he Km cons an o my osinase. Tes ed ini ial concen a ions we e 20, 35, 50, 65, 100, 150, 225, 300µg/ml, he de e mina ion was un in h ee eplica es. The added my osinase con aining 50- old dilu ed ho se adish ex ac was allowed o decompose sinig in o 5, 8.75, 12.5, 16.25, 25, 37.5, 56.25, 75 minu es, espec i ely, keeping decomposed subs a e below 10%, allowing he es ima ion o he ini ial eac ion a e ( 0). The Km alue was calcula ed by i ing he Michaelis Men en equa ion ( 0 = max[S] / Km+[S]) o he ob ained da a. Fo non-linea cu e- i ing, he nls package in R was used (n=3). The same ege able ex ac s we e also assayed o my osinase ac i i y by he widely used pH s a assay (Pieka ska e al., 2013). The eac ion mix u e was he same as ha o CE, excep ha i was no bu e ed: o 7.66 mL o wa e 80 µL o asco bic acid solu ion (100 mM, pH adjus ed o 6.50 wi h NaOH), 80 µL o plan ex ac (dilu ed i necessa y) was added. A e he pH d i s opped a e a ew minu es, he eac ion was ini ialized by addi ion o he subs a e ( inal concen a ion: 250 µg/mL). The ea e , eshly p epa ed 1 µM NaOH was added unde slow cons an s i ing o keep he pH a 6.50. The amoun o NaOH consumed by he eleased H+ du ing glucosinola e decomposi ion was egis e ed o 5 minu es. Ex ac s o he ou ege ables (B ussels sp ou s, ho se adish, adish, wa e c ess) we e compa ed o he sinig in aglycon – allyl iso hiocyana e con e sion a e. The eac ion mix u e was: 10 µL o bu e ed 54 de i a iza ion solu ion (100mM NaH2PO4, 10 mM asco bic acid, 50 mM me cap oace ic acid, pH 7.5), 60 µL bidis illed wa e , 10 µL enzyme con aining ex ac (no dilu ed) and 20 µL o sinig in s ock solu ion (10 mM). The expe imen was designed o esul in 2 mM allyl iso hiocyana e i he con e sion a io is 100%. 3.3. Resul and discussion 3.3.1.Op imiza ion o sepa a ion A good esolu ion o GLSs (sinig in / gluconas u iin) was obse ed in ho se adish me hanol ex ac using he s a ing BGE and long end injec ion mode. Howe e , he me hod could be u he de eloped in he aspec s o dec easing measu ing ime and inc easing sensi i i y. Comple ely emo ing o dec easing he concen a ion o BGE 's componen s can sho en he measu ing ime due o he possibili y o inc ease he ol age. Comple ely emo ing o bo a e and dec easing sodium deoxychola e concen a ion ( om 250 o 175 mM) showed no e ec on he esolu ion be ween GLSs. Fu he dec ease o analysis ime is also achie ed by inc easing pH o 9.0, i.e., gi ing he abili y o inc ease elec oosmo ic low (EOF). A pH 9.0, CHES (N-Cyclohexyl-2- aminoe hanesul onic acid) was used as a bu e ing agen (signi ican bu e ing capaci y, low UV abso p ion). O he s a egies such as addi ion o o ganic sol en s, employing di e en su ac an s (also as mix u es) esul ed in loss o esolu ion and/o sensi i i y o some analy es o in e es . 55 Figu e 16. Elec ophe og ams o he di e en me hods used du ing op imiza ion on P inCE-C 700 capilla y elec opho esis. a) Backg ound elec oly e was 20 mM TAPS, 15 mM Bo a e, 250 mM SDC, pH 8.5. b) Backg ound elec oly e was 20 mM TAPS, 250 mM SDC, pH 8.5. c) Backg ound elec oly e was 20 mM TAPS, 175 mM SDC, pH 8.5. 56 The mos e ec i e BGE con ains CHES (20 mM), sodium deoxychola e (175 mM), pH 9.0, applied ol age 20 kV. I showed good esolu ion o sinig in / gluconas u iin, and no majo in e e ing peak in he me hanol ex ac o ho se adish. Al hough AITC was sepa a ed om GLSs, because o i s low speci ic abso bance, he limi o de ec ion is so high which will be he p oblem o u he s udy. Inc easing he sensi i i y o he me hod o AITC was he nex s ep o me hod imp o emen . Figu e 17. Elec ophe og am o a my osinase inac i a ed (cooked) ho se adish oo ex ac , spiked wi h 500 ppm allyl iso hiocyana e wi hou de i a iza ion, using he p oposed CE-MEKC p ocedu e in long-end injec ion, wi h 52 cm e ec i e leng h. Backg ound elec oly e was 20 mM CHES, 175 mM SDC, a pH 9.0. As de ec ion wa eleng h, 230 nm was used. No e good esolu ion be ween GLSs, and low sensi i i y and esolu ion o he unde i a ized AITC. Abb e ia ions: AITC, allyl iso hiocyana e; G, gluconas u iin (phene hyl glucosinola e); S, sinig in; uG, uniden i ied glucosinola e. 3.3.2. De i a iza ion s udy 57 Table 4. Glucosinola e Con en o Fou Tes ed Vege ables, as Measu ed by he P oposed CE Me hod, o by LC-ESI-MS. Vege able CE LC-ESI-MS CE LC-ESI-MS Sinig in (µg/g (FW)) Gluconas u iin (µg g-1 (FW)) Radish n.d n.d n.d n.d B ussels sp ou 161 148.2 n.d n.d Wa e c ess n.d n.d 162.9 153.7 Ho se adish 2291.8 2784.4 248.5 244.3 Abb e ia ions: CE, capilla y elec opho esis; FW, esh weigh . The applica ion o p oposed me hod showed he es ed comme cial condimen s con ain 369 – 418 µg/g AITC. The me hod p o ided a good esolu ion wi h simple sample p epa a ion p ocedu e, as plo ed in igu e 22. The p esen ed long end injec ion me hod sepa a es he analy es o in e es wi hin 15 minu es om eal ma ices. This alls in he ange o he as es HPLC me hods a ailable o de e mina ion o sinig in and allyl iso hiocyana e (Tsao e al. 2002). Typical me hods las 20-25 minu es o mo e, consume 1mL/min sol en and equi e he emo al o p o ein and a be o e analysis (Budnowski e al. 2013; He zallah and Holley 2012). Di ec , simul aneous quan i ica ions om complex ma ices can be e en longe (Song e al. 2005). Iso hiocyana e de e mina ion – e en when de i a ized – can also be ime demanding. The p esen ed me hod is simila in speed o he as es a ailable CE me hods o glucosinola es, an analysis ime o 15-25 minu es is ypical (Ka che and El Rassi 1999). Howe e , he sepa a ion o iso hiocyana e adduc s usually equi es mo e ime (Bje gegaa d e al. 1999). Wi h he new me hod, sepa a ion o allyl iso hiocyana e di hioca bama e was also done wi hin 15 minu es. The CE 64 sc eening is also as e as compa ed o GC-MS me hods. The usual ime o a GC-MS o di e en iso hiocyana es measu emen is 30-35 minu e (Zhao, Tang, and Ding 2007). Figu e 22. De ec ion o allyl iso hiocyana e om ood p oduc s in long-end injec ion mode. a., mus a d (condimen ); b., ho se adish sauce wi h wasabi. Elec ophe og ams ob ained using he p oposed capilla y elec opho esis – micella elec okine ic ch oma og aphy (CE-MEKC) me hod showing possible applica ions. Backg ound elec oly e: CHES (20 mM), sodium deoxychola e (175 mM), pH 9.0. Iso hiocyana es a e p esen as di hioca bama es du ing sepa a ion. Sample ma ices: phospha e (10 mM, pH 7.5), asco bic acid (1 mM), me cap oace ic acid (5 mM). 3.3.5. S udy o my osinase ac i i y and allyl iso hiocyana e elease o ege able ex ac s The a e age Km alues ob ained by non-linea eg ession analysis o eac ion eloci y e sus sinig in concen a ion (a pH 6.50, 25 °C, 0.1 mM asco bic acid) ell in he ange o 0.129 ± 0.025µM. This is he same o de o magni ude ound o sinig in in se e al p e ious s udies o di e en my osinases, hus i can be s a ed ha he p esen ed ac i i y is uly ha o my osinase (Li and Kushad 65 2005; Nehmé e al. 2014). The ac i i ies (exp essed in µmol sinig in decomposed pe minu e (U)) o es ed ege ables a e shown in he able below. Ac i i y o sinig in decomposi ion anged om 4.42 U/g esh weigh (wa e c ess) o 208.26 U/g esh weigh (ho se adish) in 10 mM phospha e, 1 mM asco bic acid, pH 6.50, 25 °C, ini ial subs a e concen a ion: 250µg/ml. The me hod was shown o be sui able o measu e my osinase ac i i y om low ac i i y mix u es wi hou majo in e e ences. The ob ained my osinase ac i i ies wi h sinig in as he subs a e we e compa ed o hose ound in he widely used pH-s a assay. Unde he same condi ions (1 mM asco bic acid, pH 6.50, a 25 °C, ini ial subs a e concen a ion: 250 µg/mL), he my osinase con aining ex ac s o he ege ables had e y simila ac i i y (93.7% – 116.9%, a e age: 107.1%, able 5). Table 5. My osinase Ac i i ies o Fou Tes ed Vege ables Using Sinig in o Gluconas u iin as Subs a e, Measu ed By he P oposed CE Me hod and pH S a Assay Vege able Sinig in, CE (U/g FW) Sinig in, CE (U/mg p o ein) Sinig in, pH s a (U/g FW) Sinig in, pH s a (U/mg p o ein) Radish 10.31±1.31 3.41±0.43 11.00±0.94 3.64±0.31 B ussels sp ou 7.72±0.6 0.96±0.07 6.83±0.24 0.85±0.03 66 Wa e c ess 4.42±0.5 0.76±0.09 4.22±0.94 0.73±0.16 Ho se adish 208.26±42.94 27.69±5.71 178.13±4.42 23.69±0.59 Vege able Gluconas u ii n, CE (U/g FW) gluconas u ii n, CE (U/mg p o ein) S/G AR* ITC elease (%) Radish 5.35±1.02 1.77±0.34 1.93 92±4.39% B ussels sp ou 3.71±0.09 0.46±0.01 2.08 73.13±0.27% Wa e c ess 6.01±0.12 1.04±0.02 0.73 102.13±0.94% Ho se adish 197.94±33.98 26.32±4.52 1.05 98.25±3.02% One uni (U) o ac i i y is de ined as 1µmol pe minu e. Abb e ia ions: CE, capilla y elec opho esis; FW, esh weigh ; S/G AR: Ra io o my osinase ac i i y wi h sinig in as he subs a e / gluconas u iin as he subs a e. The p esen ed alues a e mean ± SD o h ee measu emen s. Compa ing he ac i i y a io o he same ex ac s wi h sinig in and gluconas u iin as he subs a e, some le el o speci ici y can be ound: B ussels sp ou s ha con ains sinig in bu no gluconas u iin (Table 5) had 2.08- old ac i i y agains sinig in as compa ed o gluconas u iin. Wa e c ess con aining gluconas u iin bu no sinig in (Table 5) also showed some speci ici y owa ds i s own glucosinola e. Ho se adish, which con ains bo h glucosinola es (Table 5), decomposed bo h wi h simila e icacy. Though he measu ed speci ici y is no ha s iking as i was p e ious desc ibed o C ambe abyssinica, i sugges s ha using sinig in as he sole subs a e o my osinase ac i i y de e mina ion may some imes esul in se ious unde - o o e es ima ion o he biologically 67 ele an my osinase ac i i y o plan ex ac s. The e o e, i possible, me hods capable o using subs a es o he han sinig in should be in eg a ed in o my osinase ac i i y es s. The phenomenon clea ly equi es mo e in-dep h s udy, o which he cu en me hod can be used. 3.3.6. Discussion The e a e popula me hods o my osinase measu emen , mos o hem a e based on spec opho ome y ins ead o ch oma og aphic sepa a ion. These include he indi ec measu emen o he glucose eleased (Wilkinson, Rhodes, and Fenwick 1984) o measu ing he b eakdown kine ics o he subs a e (dec ease o abso bance maximum). I is also possible o use he eleased H+ o quan i ica ion in a pH-s a assay (Pieka ska e al. 2013). As glucosinola e abso bance maxima a e usually a ound 210-230 nm, many compounds can in e e e wi h quan i ica ion in UV-Vis, especially when using mo e concen a ed aw ex ac s. Highe speci ici y can only be achie ed by subjec ing he eac ion mix u e o ch oma og aphic sepa a ion. Fo his pu pose, he CE me hods can be used. They ope a e wi h a minimal amoun o sample and a e able o s udy decomposi ion o di e en glucosinola es, bu nei he can gi e in o ma ion on he ITC elease a e ha is o p imal biological signi icance. HPLC me hods ha e excellen ep oducibili y and sensi i i y, bu a e equen ly ime consuming and equi e much mo e eagen s han CE measu emen s. The eac ion mix u es used in his s udy a e simple enough o use sho -end injec ion, which esul s in less, bu su icien esolu ion, and less analysis ime. In his case, he p esen ed me hod is capable o sepa a ing a glucosinola e – iso hiocyana e pai om he eagen s in 2.5 minu es. Wi h a capilla y econdi ioning applied a e e e y six h injec ion, a numbe o abou 68 wel e injec ions pe hou can be eached. This is compa able o he widely used assays' ime demand, ye , ch oma og aphic sepa a ion akes place, which gi es he leas in e e ences om o example asco bic acid. Fo many ege ables, con e sion o sinig in o allyl iso hiocyana e was no ound o be 100% in he li e a u e. The iso hiocyana e yield om he glucosinola e aglycon can ange om a ew pe cen o nea 100% (Pieka ska e al. 2013). This is usually a ibu ed o he p esence o speci ie p o eins ha cause he glucosinola e aglycon o ea ange in o di e en ola ile p oduc s such as ni iles, epi hioni iles, hiocyana es among o he s. In he cu en s udy, ou ege able ex ac s (B ussels sp ou s, ho se adish, adish, wa e c ess) we e success ully compa ed o ITC con e sion a e, wi h minimal amoun o eagen s. The p ocedu e also does no equi e labo ious sample p epa a ion (e.g. liquid-liquid ex ac ion) needed o s udy he ITC con en by GC-MS as in p e ious s udies. The amoun o allyl iso hiocyana e gene a ed om he same amoun o added sinig in (1mM) signi ican ly di e ed among he ege able ex ac s (p<0.05, n=3, ANOVA). Con e sion a e was ound o ange be ween 73.13±0.27% and 102.13±0.94%. The HPLC me hod success ully sepa a es allyl iso hiocyana e and sinig in om model ma ices wi h ac i e my osinase wi hin 6 minu es, and was success ully used o measu e ITC elease (Vas enhou e al. 2014). Howe e , i was no es ed o he abili y o measu e my osinase ac i i y o ITC elease om eal ma ices, and ope a es wi h a 1 mL/min sol en low. Tes ing o eal ma ices would also equi e he emo al o p o ein and a as sample p epa a ion s eps. The ad an ages o he p oposed CE me hod include sa ing ime and sol en s as compa ed o many HPLC me hods. Hence, i is sui able as a sc eening me hod o glucosinola es and allyl iso hiocyana e. The me hod was used as a highe speci ici y my osinase assay 69 ha also allows quan i ica ion o on-line gene a ed iso hiocyana es, he main bioac i e p oduc s. Only 25-50 µg glucosinola e pe sample is su icien o a my osinase s udy, which is especially impo an in he case o glucosinola es o he han sinig in. Analysis o ac o s a ec ing he glucosinola e – iso hiocyana e con e sion a e is also possible. 70 Chap e IV Compa a i e Analysis O A mo acia us icana And A mo acia mac oca pa 1. BACKGROUND INFORMATION 1.1. My osinase My osinase' s s uc u e and p ope ies – As men ioned ea lie , my osinase plays a c ucial ole in he hyd olysis eac ion o GLS. Ho se adish my osinase (β- hioglucoside glucohyd olase) is an S-glucosidase enzyme whose (β/α) 8- ba el s uc u e consis s o wo simila subuni s wi h a molecula weigh o 65 kDa linked by a zinc a om (Zhang 2010). To make con ac wi h GLS, ho se adish my osinase equi es a hyd oxyl g oup on C2 o he glucose moie y o GLS and a nucleophilic glu ama e o ca aly ic ac i i y (Fahey, Zalcmann, and Talalay 2001). In B assicaceae, my osinases a e gene ally classi ied based on he place whe e hey can be ound in he plan . The e a e h ee subg oups in his enzyme amily, my osinase A, my osinase B, and my osinase C (MA, MB, and MC, espec i ely). MA and MC can be ound only in he seed issue, whe eas MB can be ound in mos issues o he plan . Conside ing his poin , ho se adish my osinase should belong in he MB subg oup bu classi ica ion ge s mo e complica ed because ho se adish my osinase possesses a wa e -soluble p ope y, whe eas MB my osinase does no . This leads o he hypo hesis ha ho se adish my osinase may belong o an uniden i ied subg oup o he my osinase amily. My osinase is no subs a e speci ic. Ho se adish my osinase no only ca alyzes he hyd olysis o GLSs ound in ho se adish such 71 as sinig in, bu can also ca alyze hyd olysis o GLSs ound in di e en o he species (e.g. in b occoli), wi h a es depending on he subs a e (Li and Kushad 2005). My osinase' s ac i i ies – The op imum condi ions o ac i i y o my osinase a e when he empe a u e o he eac ion alls in he ange o 37–45oC and pH in he ange o 5–8. Ho se adish my osinase ac i i y was obse ed o inc ease a 23oC, emaining high (i.e. >80% o maximum) om 37oC, eaching i s maximum a 45oC, dec easing a empe a u es o e 50oC, and he enzyme becomes inac i e a empe a u es abo e 70oC. Wi hin he pH ange, inc easing my osinase ac i i y was no iced om a pH o 3–4, eaching maximum a pH 5.7 and he ac i i y emains high (i.e. >80% o maximum) a pH ange 5–8. Addi ion o 0.5mM asco bic acid can signi ican ly s imula e ho se adish my osinase, possibly due o a con o ma ion change in he enzyme leading o inc eased subs a e a ini y o ho se adish my osinase and eac ion eloci y (Bones and Rossi e 2006). Loss in endogenous asco bic acid was also sugges ed o cause he dec ease o my osinase ac i i y (A. Dep ee, M. Howa d, and P. Sa age 1998). In ano he s udy on my osinase isola ed om ho se adish g own in China, he bes condi ion o my osinase ac i i y is a empe a u e o 65oC and pH 4.0, wi h asco bic acid (2 mg/g powde ed oo ) added and an incuba ion pe iod o 120 min (Zi-Tao Jiang 2006). The inconsis ency on he de ails equi ed o op imal ac i i y be ween hese wo s udies may be ela ed o di e ences in geog aphic egions whe e he ho se adishes we e g own. Howe e , i can be concluded ha empe a u e, pH, and asco bic acid con en a e he main ac o s a ec ing he ac i i y o he enzyme. 1.2. Pe oxidase 72 S uc u e and p ope ies – Apa om my osinase, ho se adish con ains ano he enzyme ha has gained in e es : ho se adish pe oxidase (HP) – a heme- con aining enzyme ha u ili ies hyd ogen pe oxide o oxidize a wide a ie y o o ganic and ino ganic compounds, due o i s la ges-scaled comme cial uses, o example as a eagen o o ganic syn hesis and bio- ans o ma ion, as in coupled enzyme assays , chemiluminescen assays, immunoassay and he ea men o was e wa e . Fi een HP isoenzymes ha e been iden i ied om ho se adish oo . Based on hei isoelec ic poin alues, hese HPs a e e e ed o by codes as A1-3 (acidic), B1-3 and C1-C2 (neu al basic) and E1-E6 (basic). Among hose, he C isoenzyme is he mos abundan . Ho se adish pe oxidase isoenzyme C (HRPC) comp ises a single polypep ide o 308 amino acid esidues. The s uc u e o he enzyme is la gely α-helical and small egion o β-shee . HRPC con ain he heme g oup (i on (III) p o opo phy in IX), loca ed be ween he dis al and p oximal calcium binding domains. These me al cen e s a e c ucial o he s uc u al and unc ional in eg i y o he enzyme. Al hough so li le is known abou he unc ion o HP in plan , i is belie ed o be in ol ed in he con e sion o hyd ogen pe oxide o wa e and used by plan s o egula e le el o in acellula hyd ogen pe oxide. The adical p oduc s om HRP-calalysed eac ions possibly in ol e c oss-linking eac ions (e.g. he o ma ion o di e ula e linkages om polyme -a ached e ula e g oups o polysaccha ides o pec ins, he o ma ion o di y osine linkages, e c.), which may be exp essed in esponse o ex e nal ac o s such as he sounding o plan issue. Wa e loss and in asion by pa hogens can he e o e be limi ed by he o ma ion o a p o ec i e polyme ic ba ie such as sube in (Vei ch 2004). 73 i.e. he na ow co e o p ima y xylem is su ounded by an ex ensi e bu mainly pa enchyma ous seconda y xylem in which a ew achea y elemen s occu ( igu e 23). The well-de ined ascula cambium also p oduces cen i ugally a la gely pa enchyma ous seconda y phloem. IKI s ained c oss sec ions e ealed s a ch p esen in c oss sec ions based on he eac ion o iodine and he cen e o he helical s a ch molecules, gi ing he da k blue colo (longe molecules) o mo e ed colo (sho e molecules; igu e 24). The oil con en in he c oss sec ions o A. us icana and A. mac oca pa we e in es iga ed by exploi ing he a -soluble p ope y o Sudan III s ain, which is physically a ac ed o hyd ophobic s uc u es ( igu e 25, 26). These c oss-sec ions showed he simila i y in s uc u e o esh oo s om bo h species – A. us icana and i s ela i e, A. mac oca pa. 80 Figu e 23. C oss sec ions o he leshy oo s o A mo acia us icana and A mo acia mac oca pa s ained wi h oluidine blue solu ion. The images we e iewed wi h Olympus P o is AX70/A mic oscope. Figu e 24. C oss sec ions o he leshy oo s o A mo acia us icana and A mo acia mac oca pa s ained wi h IKI solu ion. The images we e iewed wi h Olympus P o is AX70/A mic oscope. 81 Figu e 25. C oss sec ion o he leshy oo s o A mo acia us icana s ained wi h Sudan III solu ion. The images we e iewed wi h Olympus P o is AX70/A mic oscope. 82 Figu e 26. C oss sec ion o he leshy oo s o A mo acia mac oca pa s ained wi h Sudan III solu ion. The images we e iewed wi h Olympus P o is AX70/A mic oscope. 4. THE GEL ELECTROPHORESIS STUDIES Gel elec opho esis is a lexible me hod o sepa a ion and analysis o p o eins, nucleic acids and o he cha ged molecules. In elec opho esis, cha ged molecules a e a eled h ough a po ous gel by an applied elec ic ield gene a ed in a bu e which pe mea es he gel, and a e sepa a ed based on hei di e en elec opho e ic mobili ies. Va ia ions in he gel and bu e make i possible o sepa a e molecules no only based on hei cha ges, bu also on hei molecula weigh , isoelec ic poin and bio-speci ic a ini y. The echnique is as , con enien , and inexpensi e, and is used bo h as an analy ical me hod and as a p epa a i e p ocedu e in he inal s ages o pu i ica ion. 83 4.1. Ma e ials and me hod Roo s and lea es o A. us icana and A. mac oca pa we e g ind by comme cial elec ic mixe . The ma e ial (app ox. 1 g) was hen ans e ed o 2 ml Eppendo ubes, ollowed by he addi ion o 1mL bu e (NaH2PO4/Na2HPO4 20 mM, 4oC, pH 6.55, VWR In e na ional L d.). The mix u es we e agg essi ely mixed and cen i uged a 13000 pm o 30 min using He aeus Bio uge in o de o ob ain he supe na an . The p o ein con en o he supe na an was assayed by he me hod o B ad o d. 40 μg p o ein was loaded in o each well o na i e 75% polyac ylamide gels. Elec opho esis was pe o med a 4oC. Fo my osinase ac i i y s udy, he gel was washed wi h dis illed wa e and s ained wi h solu ion (pH 8) con aining 0.25 mL 20 mM KH2PO4/K2HPO4, 0.05 ml (169 mg/10 ml) Asco bic acid, 0.5 mL 0.1% dye solu ion (me hyl ed), 1.25 (10 mg/mL) sinig in and 2.95 mL wa e o 1-2 min. The my osinase isoenzymes' band in ensi y we e e alua ed by ImageJ® and CP A las e sion 1.01 so wa e. The da a was g aphically p esen ed by Sigma plo 11.0 and Lib eo ice Calc so wa e. The ac i i y o pe oxidase was in es iga ed using spec opho ome y (SHIMADZU, UV-1601). Each sample o spec ome y con ains 970 μL 50mM KH2PO4/K2HPO4, 5 μL 3%H2O2, 20 μL 1 M pi ogallol and 5 μL pe oxidase enzyme om he supe na an . 4.2. Resul s and discussions Pe oxidase ac i i y – Da a ob ained om spec ome y measu emen ( igu e 27) compa ed he pe oxidase ac i i y in lea es, young oo and old oo om A. us icana and A. mac oca pa collec ed in july. In A. mac oca pa, he highes ac i i y was obse ed in he old oo (801.05 ± 31.04 ΔOD/min/mg p o ein), 84 ollowed by he young oo (412.6 ± 42.4 ΔOD/min/mg p o ein) and he leas ac i i y was in he lea e (55.75 ± 1.43 ΔOD/min/mg p o ein). In A. us icana, he same o de o pe oxidase ac i i ies we e measu ed, i.e. he highes ac i i y was eco ed in he old oo (671.25 ± 31.5 ΔOD/min/mg p o ein), ollowed by he young oo (206.95 ± 1.2 ΔOD/min/mg p o ein) and he leas ac i i ies was measu ed in he lea e (90.6 ± 2.4 ΔOD/min/mg p o ein). Acco ding o spec ome y esul s, he pe oxidase ac i i y in A. mac oca pa was highe compa ed o ha in A. us icana. Di e en esul s we e eco ded in o he measu emen s wi h oo s and lea es o bo h species collec ed in oc o be , in which pe oxidase ac i i y was highe in A. us icana (352.8 ± 1.4 ΔOD/min/mg p o ein, 16.4 ± 0.01ΔOD/min/mg p o ein in oo and lea , espec i ely) compa ed o ha o A. mac oca pa (232.9 ± 0.57 ΔOD/min/mg p o ein, 27.72 ± 0.18 ΔOD/min/mg p o ein in oo and lea , espec i ely). The di e en esul s in hese s udies could be ela ed o age and ime o ha es ing o oo s. Figu e 27. a) Pe oxidase ac i i ies measu ed by spec opho ome y. (A.m: A mo acia mac oca pa, A. : A mo acia us icana, L: Lea , j.R: young oo , o.R: old oo ). b) Pe oxidase (E.C. 1.11.1.7) ac i i y was isible due o da k ed-colo ed pu pu ogallin bands showed on he gel. Pic u e was aken by 85 a) b) a) b) a) Olympus 4040 came a. My osinase ac i i y – The da a on my osinase ac i i y was calcula ed based on he band in ensi ies on he PEG gel (pixel pe a ea – ppa). In A. us icana, my osinase ac i i ies in he young oo and lea e we e simila (1378.67 ± 113.2 ppa and 1342 ± 49.66ppa). The highes ac i i y was measu ed in he old oo (1881 ± 110ppa). In case o A. mac oca pa, he enzyma ic ac i i y was highe in he oo s (1628 ± 31.32ppa), in which he old oo has he highe ac i i y, simila ly o he case o A. us icana. Compa ing 2 species, my osinase ac i i y was o e all highe in A. us icana compa ed o i s ela i e. The highes my osinase ac i i y measu ed in A. mac oca pa old oo s was simila o he ac i i y measu ed in lea e and young oo s o A. us icana ( igu e 28). Figu e 28. a) Gel elec opho esis s udy on he my osinase ac i i y in A. us icana and i s ela i e, A. mac oca pa. (1: A. us icana young oo , 2: A. us icana old oo , 3: A. us icana lea , 4: A. mac oca pa young oo , 5: A. 86 b) a) mac oca pa old oo , 6: A. mac oca pa lea ). b) My osinase ac i i y measu ed by band in ensi ies on gel. (A. : A mo acia us icana, A.m: A mo acia mac oca pa, L: lea , y.R: young oo , o.R: old oo ). Pic u e was aken by Olympus 4040 came a. 87 Chap e V Summa y The i s pa o his s udy came up wi h a new echnology o ex ac ing high yield essen ial oil om esh ho se adish oo s. Wi h his me hod, o 15 kg o esh ho se adish oo , 12ml o essen ial oil can be ex ac ed ( he yield o 0.08%). Toge he wi h he s able and high yield, he ease o use and i s simplici y make su e ha he new me hod is sui able o he ho se adish essen ial oil p oduc ion. In es iga ion on he quali y o ho se adish essen ial oil by gas ch oma og aphy and mass spec ome y showed he p esences in high amoun o 2 main subs ances: allyl iso hiocyana e and 2-phene hyl iso hiocyana e. The o he h ee iden i ied iso hiocyana es a e sec-bu yl-, 3- bu enyl- and 4-pen enyl iso hiocyana e. In es iga ion on he comple ion o he ex ac ion (no glucosinola e / iso hiocyana e ound in he pos -dis illed mush) and he “ apped” con en o iso hiocyana e (5 ppm) in wa e y ex ac by gas ch oma og aphy and mass spec ome y con i med ha he new me hod is ecommended o la ge scaled ho se adish oil p oduc ion. The second pa o he s udy ocuses on he de elopmen o new capilla y elec opho esis (CE-MECK) me hod o simul aneous quan i ica ion o glucosinola es and iso hiocyana es. The assay in sho -end injec ion mode enables my osinase quan i ica ion as well as glucosinola e' aglycon o iso hiocyana e con e sion a e es ima ion. The me hod uses sinig in o gluconas u iin as subs a e, he main p oduc s o in e es a e de i a ized o a mo e sensi i ely de ec able di hioca bama e p oduc . I can also be a good al e na i e o es ablished me hods o quan i y my osinase ac i i y om aw plan ma e ials and simila ma ices, as well as cha ac e iza ion o soluble 88 my osinase enzymes, wi h espec o, o example, subs a e speci ici y and pH op ima. The me hod combines many ad an ages o equen ly used me hods: he speci ici y o ch oma og aphic sepa a ions and he simplici y, low cos and ime demand ha is he p ope y o he spec opho ome ic assays. The inal pa o his s udy compa es he ana omical s uc u e, glucosinola e p o ile and he enzyma ic ac i i ies in bo h A. us icana and A. mac oca pa. S udy on c oss sec ions s ained wi h oluidine blue solu ion, IKI solu ion and Sudan III om A. us icana and A. mac oca pa showed he simila i y in ana omical s uc u es o he oo o bo h species. S udy on glucosinola e p o ile in bo h species by liquid ch oma og aphy and mass spec ome y showed ha he e a e 6 iden i ied glucosinola es in A. us icana and 16 glucosinola es we e en a i ely iden i ied in A. mac oca pa. Gluconas u iin, glucob assicin, glucocochlea in, glucocon ingianin and glucoiba in a e he i e glucosinola es ound in bo h species. The absence o sinig in (pa en glucosinola e o allyl iso hiocyana e) can be he possible explana ion o he di e ence in he as e and smell o hese plan s. The ac i i ies o pe oxidase enzyme we e eco ded highe in A. us icana compa ed o A. mac oca pa s udied by spec opho ome y. Howe e , he opposi e esul s we e also eco ded, sugges ing ha pe oxidase ac i i y o bo h species hea ily depends on he age and he ime o ha es ing o he oo s. The old oo had he highes ac i i y compa ed o he young oo and he lea , whe e he lowes pe oxidase ac i i y was measu ed. 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