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. The gel elec opho esis s udy on
my osinase enzyme ac i i y showed highe ac i i y in A. us icana compa ed o
A. mac oca pa. In A. us icana, he old oo had highe my osinase ac i i y han
he young oo and lea , which had simila my osinase ac i i y. In A.
89
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