identification of polyphenols in tobacco leaf and their antioxidant and antimicrobial...

7/21/2019 Identification of polyphenols in tobacco leaf and their antioxidant and antimicrobial activities.docx

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Identification of polyphenols in tobacco leaf

and their antioxidant and antimicrobial

activities HaiyanWanga,c,

MoumingZhaoa, , ,

BaoYangb,

YuemingJiangb,

GuohuaRaoa

Show more

Abstract

Crude polyphenols were e!rac!ed "rom !obacco lea" by #$% e!hanol solu!ion wi!h ul!rasonic

!rea!men! and !hen puri"ied by a macroporous resin& 'he polyphenols "rom !obacco lea" ()'*+

were subec!ed !o analyses by re-erse.phase high.per"ormance li/uid chroma!ography (R).

H)*C+ and elec!rospray ioni0a!ion mass spec!rome!ry (1S2.MS+& 'he dominan! polyphenols in

!obacco lea" were iden!i"ied as chlorogenic acid and ru!in& 3ur!hermore, !he an!ioidan! ac!i-i!ies

o" )'* were in-es!iga!ed, including sca-enging ac!i-i!ies o" 4,4.diphenyl.5.picrylhydra0yl

(6))H+ radicals (7&$5 8g9ml 2C7$-alue+, hydroyl radicals (:;&< 8g9ml 2C7$ -alue+ and

superoide anion radicals (::&$ 8g9ml 2C7$-alue+, inhibi!ion ac!i-i!y o" lipid peroida!ion

(4=5 8g9ml 2C7$-alue+ and reducing power& 'he proli"era!ion inhibi!ion ac!i-i!ies onEscherichia

coli, Staphylococcus aureusandBacillus subtiliswere also measured "or e-alua!ing !he

an!imicrobial ac!i-i!y o" )'*& 'he diame!ers o" inhibi!ion 0ones were 5$&5= > $&:5, 4?& $ $&5; mm, respec!i-ely& 'he resul!s showed !ha! )'* had grea! po!en!ial asan!ioidan! and an!imicrobial agen!&

Keywords

'obacco@
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)olyphenol@

An!ioidan! ac!i-i!y@

An!imicrobial ac!i-i!y@

H)*CMS

1. Introduction

'obacco is a -ery impor!an! economic crop& 2n 5$$


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2.2. Chemicals

Fi!ro blue !e!ra0olium (FB'+, phena0ineme!hosulpha!e ()MS+, nico!inamide adenine

dinucleo!ide (FA6H+, 6))H, .!ocopherol and !hiobarbi!uric acid ('BA+ were purchased "rom

Sigma Chemical Co& (S!& *ouis, M, ISA+& S# macroporous resin was purchased "rom !he

Chemical Company o" FanDai Ini-ersi!y ('ianin, China+& All o!her chemicals and reagen!s used

were o" analy!ical grade&

2.3. xtraction and purification

'he e!rac!ion o" )'* was done according !o !he me!hod o" *ee and WicDer (4;;4+, wi!h some

modi"ica!ions& 'obacco lea" powder (5$ g+ was weighed and pu! in!o a 57$ ml conical "lasD& 'wo

hundred millili!res o" #$% e!hanol solu!ion were added& 'he conical "lasD was placed in anul!rasonic cleaner (:$ DH0, K.=$$61, Sonica!or Company, unshan, China+ "or 47 min a!

room !empera!ure& 'he e!rac! was cen!ri"uged a! 4$ #7:gin a ubo!a


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Analy!ical R).H)*C analysis was conduc!ed on a 62F1P summi! li/uid chroma!ograph

(6ione Corpora!ion, Sunny-ale, ISA+, "i!!ed wi!h a C4# re-erse.phase column (57$ :&< mm,

6iamonsil, 6iDma 'echnologies, Beiing, China+, a 6ione )6A.4$$ pho!odiode array de!ec!or

and 6ione )


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whereA$indica!es !he absorbance o" blanD@A4is !he absorbance o" !he mi!ure in !he presence o"

sample@A5is !he absorbance o" !he mi!ure in !he absence o" sample& 'he plo! o" sca-enging

ac!i-i!y on hydroyl radical was done and 2C7$-alue (concen!ra!ion o" sample !o sca-enge 7$%

o" !he hydroyl radicals+ was calcula!ed&

2.,.2. +uperoxide anion radical&scaven-in- activity

'he superoide anion radical.sca-enging ac!i-i!y was assessed by !he me!hod o" Yu e! al& (5$$


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Sca-enging ac!i-i!y(%+(A74?o" con!rol.A74?o"

sample+9A74?o" con!rolT4$$&

'he plo! o" sca-enging ac!i-i!y on 6))H radical was done and 2C7$-alue (concen!ra!ion o"

sample !o sca-enge 7$% o" !he 6))H radicals+ was calcula!ed&

2.,.!. $educin- power

'he reducing power o" )'* was de!ermined according !o !he me!hod o" Ahmadi, adi-ar, and

Shahedi (5$$?+& Sample solu!ion ($7$ 8g9ml, 5 ml+, phospha!e bu""er (5 ml, $&5 M, pH


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de!ermined by using !he disc di""usion me!hod ( Bauer, irby, Sheriss, 'urcD, 4; 5&= mg

CA19g&

H)*C, wi!h re-erse phase column !echnology, is !he analy!ical !echni/ue !ha! has domina!ed !he

separa!ion and charac!eri0a!ion o" phenolic compounds (Robbins, 5$$=+& 2n !he presen! s!udy, !he

maor componen!s in )'* were iden!i"ied by R).H)*C and MS analyses& 'he chroma!ogram o"

)'* was recorded a! =:7 nm and is shown in3ig& 4& 'he R).H)*C pro"ile o" )'* indica!ed !ha!

!he R).H)*C sys!em could separa!e easily !he dominan! polyphenols& By comparing wi!h !he

commercial s!andards, !wo main peaDs a! 4=&:? and 4;&=7 min were iden!i"ied as chlorogenic

acid and ru!in, respec!i-ely& 'heir IO-isible scanning spec!ra, "rom !he diode array de!ec!or

o-er !he range 5$$:$$ nm, were also de!ermined (3ig& 4+& Chlorogenic acid had !hree

absorbance peaDs a! 54#&;, 5::&4 and =5#&? nm, respec!i-ely, while ru!in also had !hree

absorbance peaDs a! 5$7&4, 57? and =7


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dihydroycinnamoyl group& M.HT m9z


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3ig& 5&

Mass spec!ra o" chlorogenic acid and ru!in&

3igure op!ions

3.2. Antioxidant activity

3.2.1. 'ydroxyl radical&scaven-in- activity

Hydroyl radical is !he mos! reac!i-e "ree radical and i! can be "ormed "rom superoide anion

and hydrogen peroide in !he presence o" me!al ions, such as copper or iron& Hydroyl radicals

reac! wi!h lipid, polypep!ides, pro!eins and 6FA, especially !hiamine and guanosine& 'he

resul!ing radical can undergo "ur!her reac!ions, such as reac!ing wi!h oygen !o gi-e

peroylradicals, or decomposing !o phenoyl.!ype radicals by wa!er elimina!ion (i!ada,

2garashi, Hirose, i!agawa, 4;?;+& 'he sca-enging ac!i-i!ies o" )'* on hydroyl radicals are

shown in 3ig& =& 'hese were dose.dependen!& Moreo-er, when !he !es!ed concen!ra!ion was

abo-e :$ 8g9ml, )'* showed higher sca-enging ac!i-i!y !han did -i!amin C, which is considered

!o be a po!en! hydroyl radical.sca-enger&

3ig& =&

Sca-enging ac!i-i!ies o" )'* and -i!amin C on hydroyl radicals&V, -i!amin C@ , )'*&

3igure op!ions

3.2.2. +uperoxide anion radical&scaven-in- activity
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Superoide anion is a reduced "orm o" molecular oygen, by recei-ing an elec!ron& 2! is also an

ini!ial "ree radical "ormed "rom mi!ochondrial elec!ron !ranspor! sys!ems& Mi!ochondria genera!e

energy using "our elec!ron chain reac!ions, reducing oygen !o wa!er& Some o" !he elec!rons

escaping "rom !he chain reac!ion o" mi!ochondria direc!ly reac! wi!h oygen and "orm superoide

anion& 2! plays an impor!an! role in !he "orma!ion o" o!her reac!i-e oygen species, such as

hydrogen peroide, hydroyl radical, or single! oygen in li-ing sys!ems (ulisic, Radonic,

a!yalinic, 5$$7+& 'he e""ec!s o" )'* on superoide anion radicals were de!ermined and !he

resul!s are shown in 3ig& :& )'* had a signi"ican! sca-enging ac!i-i!y on !he superoide anion

radicals in a dose.dependen! manner& Compared wi!h -i!amin C, )'* showed insigni"ican!

di""erence (PX $&$7+ wi!h regard !o superoide anion radical.sca-enging ac!i-i!y&

3ig& :&

Sca-enging ac!i-i!ies o" )'* and -i!amin C on superoide anion radicals&V, -i!amin C@ ,

)'*&

3igure op!ions

3.2.3. %%' radical&scaven-in- activity

6))H radical is commonly used as a subs!ra!e !o e-alua!e an!ioidan! ac!i-i!y@ i! is a s!able "ree

radical !ha! can accep! an elec!ron or hydrogen radical !o become a s!able molecule& 'he

reduc!ion o" 6))H radical was de!ermined by !he decrease in i!s absorbance a! 74? nm inducedby an!ioidan!s& 3ig& 7shows !he sca-enging e""ec! o" )'* on 6))H "ree radicals& )'* ehibi!ed

a s!rong abili!y !o /uench 6))H radicals& 'he sca-enging e""ec! increased wi!h increasing

concen!ra!ions used in !he !es!& 'he 6))H radical.sca-enging ac!i-i!y o" )'* a! low

concen!ra!ion was signi"ican!ly higher (PU $&$7+ !han !ha! o" -i!amin C, a commercial

an!ioidan! used in !he "ood indus!ry& 'his indica!ed !ha! )'* was a good an!ioidan! wi!h s!rong

6))H radical.sca-enging ac!i-i!y&
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3ig& 7&

Sca-enging ac!i-i!ies o" )'* and -i!amin C on 6))H radicals&V, -i!amin C@ , )'*&

3igure op!ions

3.2.!. $educin- power

Reducing power is o"!en used as an indica!or o" elec!ron.dona!ing ac!i-i!y, which is an impor!an!

mechanism "or !es!ing an!ioida!i-e ac!ion o" phenolics (Yildirim, Ma-i, ara, 5$$4+& 'he

reducing power has also been used as an impor!an! !es! o" an!ioidan! ac!i-i!y o" medicinal herbs

(6uh and Yen, 4;;?and 6uh e! al&, 4;;;+& A good correla!ion be!ween an!ioidan! ac!i-i!y and

reducing power in some plan! e!rac!s has been es!ablished (Yen, Chen, )eng, 5$$$+&

'here"ore, reducing power may be used as an indica!or o" po!en!ial an!ioidan! ac!i-i!y& 3ig&


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3ig&


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which were signi"ican!ly lower (PU $&$7+ !han !hose o" !he con!rol, -i!amin C& 'he 2C7$-alue o"

)'* (4=5 8g9ml+ "or lipid peroida!ion was sligh!ly higher !han !ha! o" !he con!rol, -i!amin 1,

possibly due !o !he weaDer liposolubili!y o" )'* !han -i!amin 1& According !o !he resul!s in 'able

4, a conclusion could be drawn !ha! )'* possessed s!rong an!ioidan! ac!i-i!y&

'able 4&

Comparison o" 2C7$-alue o" )'* and s!andard an!ioidan!s

+amples

'ydroxyl radical&

scaven-in- activity

-4ml5

+uperoxide anion

radical&scaven-in-

activity -4ml5

%%' radical&

scaven-in- activity

-4ml5

(ipid

peroxidation

-4ml5

)'* :;&< ::&$ 7&$5 4=5

Oi!amin C 7=&= :


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!. Conclusions

'he main polyphenols in )'* were iden!i"ied as chlorogenic acid and ru!in by analyses o" R).

H)*C and MS& S!rong sca-enging ac!i-i!ies on hydroyl radicals, superoide anion radicals and

6))H radicals were "ound "or )'*& 'he analyses o" lipid peroida!ion inhibi!ion ac!i-i!y andreducing power also indica!ed !ha! )'* possessed good an!ioidan! ac!i-i!y& Meanwhile, !he

proli"era!ion inhibi!ion ac!i-i!y o" )'* onE. coli, S. aureusandB. subtiliswere in-es!iga!ed in

!his s!udy& 'he resul!s con"irmed !ha! i! had good an!imicrobial ac!i-i!y& 3rom !he abo-e resul!s,

i! appears impor!an! !o de-elop na!ural an!ioidan!s and bac!erial inhibi!ors "rom !obacco lea",

and !his may be a good way "or e!ensi-ely u!ili0ing !he !obacco resource&

identification of polyphenols in tobacco leaf and their antioxidant and antimicrobial...

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