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In tea (Camellia sinensis L.) plant, polyphenols and alkaloids have been considered as

the main active components in many pharmacological studies [1-5. Lost o! 

 potential activities in plant e"tracts a!ter prolong e"traction and heating #ere usually

reported and that #ould be barrier to !urther study the !unctionality and potential o! these

e"tracts. $ence, a good e"traction method must be able to e"tract compounds o! interestcompletely and simultaneously avoid chemical modi!ication [%. $ot #ater (&'1''*) #as

!re+uently used in tea e"traction in many studies [1, , -. $o#ever, the purpose o! these

studies !ocused more on the sensory evaluation o! tea +uality by colour, !lavour and taste [&,

 provided little +uantitative in!ormation about the actual polyphenols and alkaloids present in

the solid samples. /any attempts have been made to investigate the !actors attributed to

e"traction yields and great progress has been made. 0ield o! polyphenols and alkaloids in

di!!erent e"traction parameters such as heating temperatures, e"traction solvents, p$ and

!ilter membranes have been analyed [, 2-1. $o#ever, comparison o! di!!erence e"traction

methods !or the e"traction yields o! tea lea! polyphenol and alkaloid contents !rom di!!erent

maturation stages has not been reported. In the present paper, the changes in polyphenol and

alkaloid components in tea leaves (lea! buds, young leaves and old leaves) and in the

commercial 3abah 4lack ea (34) in t#o di!!erent e"traction methods are e"amined.

/678I6L3 69: /7$;:3

*hemicals< 6uthentic (=)-catechin hydrate (*), ()-epicatechin (7*), ()-epicatechin gallate

(7*>), ()-epigallocatechin (7>*), ()-epigallocatechin gallate (7>*>), gallic acid (>6)

and ca!!eine (*a!) #ere bought !rom 3igma *hemical *o. (3t. Louis, /;). 6cetonitril

(6*9) and methanol (/e;$) #ere $?L* grade. :ichloromethane (:*/) and n-he"ane

($e") #ere >* grade. ;rtho-phosphoric acid-25@ ($ ?; ) #as reagent grade chemical.

$igh-puri!ied #ater #as !rom /illipore (4illerica, /6, A36).

3amples ?reparation< Bresh tea leaves o! the variety assamica #ere harvested !rom the !ield

o! 3abah ea ?lantation (3?). hey #ere categoried into three main groups, consisting o! 

tea lea! buds (L4), young leaves #ith light green in colour (0L) and old leaves #ith dark 

green in colour (;L). *ommercial 34 #as !rom the !actory o! 3?. hey #ere trans!erred

to Laboratory in the same day and cleaned be!ore being dried in a ventilated oven at '* !or 

2 hours. :ried plant samples #ere pulveried into po#ders and #ere used throughout in this

study

Identi!ication and Cuanti!ication o! ea ?olyphenols and 6lkaloids

6nalytical *ondition o! $igh ?er!ormance Li+uid *hromatography< 6n 6gilent echnologies

1'' series high per!ormance li+uid chromatography ($?L*) system #as used. he column

used #as an 7clipse D:4-*12 reversed phase (5 Em, 5' " . mm) #ith a *12 (5 Em, %' "

. mm) guard column. 6nalytical conditions #ere optimied based on the methods o! 0ao et

al. [1% and ;#uor and ;banda [1. he mobile phase composition started at F@ solvent 6

('.1@ a+ueou $ ?; ) and 2@ solvent 4 (1''@ 6*9) and linearly increased to 12@ solvent

4 in 5 minutes, !ollo#ing by a post-run o! % minutes be!ore the ne"t analysis cycle. *olumn

temperature #as set at %5* and the e!!luent #as monitored at 2' nm. 6ll standards and

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samples #ere !iltered through '.' Em ?G:B membrane !ilters and ali+uot o! ' EL #as

inHected into the $?L* system.

7stablishment o! *alibration *urves< Cuanti!ication o! catechins (*, 7*, 7*>, 7>* and

7>*>), ca!!eine (*a!) and gallic acid (>6) in sample e"tracts #ere carried out using $?L*

#ith e"ternal standard. 3tock standard solutions o! 1 mg.mL o! catechins, >6 and *a! #ere

 prepared. 6 1' di!!erent concentration o! each authentic standard ('.'5-5'.' Eg.mL ) #as

generated by plotting linear regression o! peak area in relation to kno#n concentration

(Eg.mL ) inHected into the $?L* system. he correlation coe!!icient (8 ) o! linear regression

curve !or all o! the standards #as then calculated using /icroso!t 7"cel. 6uthentic standards

o! minor catechin [(-)-catechin gallate (*>) and minor alkaloids [theobromine (b) and

theophylline (p) #ere not available in this study, the concentration o! *> #as +uanti!ied as

the e+uivalent o! its epimer 7*>, #hile concentration o! b and p #ere +uanti!ied as the

e+uivalent o! *a!.

7"traction /ethods

3o"hlet 7"traction (37)< 7ach plant samples (5' g) #ere separately !illed in !our sets o! 

so"hlet apparatus, each containing %5' mL o! $e" as the !irst e"traction solvent. he

temperature #as brought to boiling point (F*) and percolation #as carried out !or h. ;n

the ne"t day, $e" e"tract #as discharged and all plant materials #ere allo#ed to dry be!ore

the second solvent :*/ (%5' mL) #as re!illed in and percolation #as continued !or another 

h. he same process #as repeated !or /e;$ (%5' mL). 6t the end o! e"traction period,

:*/ and /e;$ e"tracts o! L4, 0L, ;L and 34 #ere individually distilled o!! at '*

under reduced pressure in rotary evaporator ;nce concentrated to a small volume, eache"tract #as trans!erred to pre-#eighed beaker and #as allo#ed to dry completely in a bench

top oven (%'*) overnight. 6 !inal concentration o! 1 mg.mL o! each :*/ and /e;$

e"tracts o! L4, 0L, ;L and 34 #ere prepared in '@ a+ueous 6*9 and !iltered prior to

$?L* analysis. Binal concentration o! each identi!ied compound #as e"pressed in mg.g :

(milligram per gram o! dried #eight).

:irect 7"traction (:7)< :irect e"traction o! plant samples (L4, 0L, ;L and 34) #ere

carried out #ith '@ a+ueous 6*9 under continuous stirring (1' rpm) !or h in the dark.

en grams o! each plant sample #ere placed in '' mL capped bottles, each o! #hich #as

e"tracted #ith 1'' mL " % o! '@ a+ueous 6*9. In the !ourth day, all '@ a+ueous 6*9e"tracts o! L4, 0L, ;L and 34 #ere !iltered prior to $?L* analysis. he mean

concentration #as taken !rom % subse+uent e"tractions o! each sample in % replications to

indicate the !inal concentration (mg.g : ) o! individual compound.

:ata 6nalysis and 3tatistics

3tatistical 6nalyses< 3tatistical ?ackage !or 3ocial 3cience so!t#are (3?33) 1.' Gersion !or 

indo#s #as chosen in this study. he means and standard deviation o! all the studied

 parameters #ithin the samples #ere calculated #ith /icroso!t 7"cel. 3tatistical signi!icance

di!!erences o! the e"traction yields bet#een 37 and :7 #ere analyed #ith one-#ay 69;G6

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(analysis o! variance) and the di!!erence o! means bet#een groups #as analyed #ith post

hoc test ukey $3:. 6 p-value o! '.'5 or less #as considered statistically signi!icant.

873AL3 69: :I3*A33I;9

6nalytical *onditions< *omplete baseline separation o! tea polyphenols (*, 7*, *>, 7*>,7>*, 7>*> and >6) and alkaloids (*a!, b and p) #as achieved in 5 min 6ll calibration

curves obtained #ere linear over the concentration ranges indicated in 3ection .%., #ith 8 

ranged !rom '.FF&& to 1.'''' (results not sho#ed). he $?L* chromatogram o! a standard

(*, 7*, 7*>, 7>*, 7>*>, >6 and *a!J % Eg per standard) and a typical chromatogram o! 

L4 are sho#n in Big. 1a and 1b, respectively. he maHor polyphenols and alkaloids present

are labeled in Big. 1a and 1b. he additional % peaks #ere unkno#n but they are most

 probably the minor alkaloid theobromine (b), theophylline (p) and minor catechins (-)-

catechin gallate (*>) [1%, 15, 1

0ields o! Individual ?olyphenol !rom 3o"hlet and :irect 7"traction /ethods< he 37-:*/

37-/e;$ and '@ 6*9 a+ueous :7 e"tractions !rom all samples (L4, 0L, ;L and 34)

#ere subHected to $?L* to determine their concentration o! polyphenol and alkaloid contents

and #ere sho#n in Big. %. he largest portion o! total polyphenols content #as 7>*, #ith L4

(:7, %.% mg.g J 37-/e;$, 1'.2 mg.g ) and 0L (:7, %5.2 mg.g , 37-/e;$, 1.2 mg.g )

 being the highest. he second largest portion o! total polyphenols is 7>*>, #ith L4 and 0L

contained the highest (:7, .%-%&.F mg.g J 37, 1.2-.2 mg.g ). /aHor catechin 7*> made

up the third largest portion in the total polyphenols contentJ #ith L4 contained the highest in

concentration (:7, .' mg.g J 37-/e;$, 1.F mg.g ), !ollo#ed by 0L (:7, 15.' mg.g J 37-

/e;$, &.F mg.g ), 34 (:7, 11.2 mg.g J 37-/e;$, 1.1 mg.g ) and ;L (:7, F. mg.g J37-/e;$, . mg.g ). 7* #as the !ourth highest tea maHor catechins o! total polyphenols

content and !ollo#s the same trend #ith L4 being the highest, !ollo#ed by 0L, 34 and ;L.

0ields o! Individual 6lkaloid !rom 3o"hlet and :irect 7"traction /ethods< 0ield o! total *a! 

!rom both 37 and :7 methods remains in the range o! 5.F-52.2 mg.g in L4, %.-%&. mg.g

in 0L and 1F.F-. mg.g in ;LJ #ith highest content obtained 1 !rom :7 method. In

contrast, *a! content obtained !rom 34 #as higher in the method o! 37 (.5 mg.g ) in

comparison to :7 (%'.5 mg.g ). Bor the minor alkaloids b and p, their present #as in much

lo#er +uantities than *a!. he b contents in lea! samples #ere !ound to be appro"imately

1'-!old lo#er than maHor alkaloid *a! #ith a relative ratio o! 11.' in L4, 11. in 0L, 2.5 in;L and F.5 in 34, #hereas p #as !ound to decrease !rom L4 K 0L K 34 K ;L. ;verall,

total *a! content in all o! the samples #ere mainly constant irrespective to maturity stages.

*ontents o! ?olyphenol and 6lkaloid in 3ample 7"tracts< Big. % sho#s the total concentration

o! all types o! polyphenols and alkaloids obtained. ea leaves #ere high in both polyphenols

and alkaloids in general. /ethod o! :7 consistently yields higher and greater polyphenols

and alkaloids in comparison to 37. ?olyphenols content in L4, 0L and ;L obtained !rom :7

method #ere 1.-1&.1 mg.g , but decreased by 5%.-2.1@ #hen 37 method #as used. he

impact o! e"traction methods on 34 #as small but yet signi!icant, #ith a 5.@ and .'@

decrease in polyphenol and alkaloid contents respectively. otal alkaloid contents in L4, 0L

and ;L #ere varied bet#een .-&. mg.g in :7 and '.2-52.1 mg.g in 37. Interestingly,

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the percentage variation o! alkaloid contents !rom :7 method to 37 method in 0L and ;L

#ere .%@ and .@, respectively. ?rolong percolation o! L4 at high temperature (b.p.

/e;$ 5*) in the 37 method yielded polyphenols content that is closed to the content

obtained in 34, 5F.' and 51.5 mg.g respectively.

*;9*LA3I;93

his study has demonstrated that direct e"traction can e"tract polyphenol and alkaloid #hich

its content is as similar to green tea, but #hen it is using so"hlet e"traction, the polyphenol

content is as similar to black tea. he yields o! both polyphenol and alkaloid #ere

signi!icantly higher in the e"tract obtained by direct e"traction method using '@ a+ueous

acetonitril than to the so"hlet e"traction method using dichloromethane and methanol

solvents. his indicated that total polyphenol and alkaloid contents in tea e"tracts are a!!ected

 by the e"traction methods used in sample preparation as #ell as maturity stages o! the tea

leaves used, #ith their contents decreased in the order o! L4 K 0L K 34 K ;L !or 

 polyphenol, #hile alkaloid !ollo#s the order o! L4 K 34 K 0L K ;L.

6*9;L7:>7/793

he authors #ould like to thank Aniversiti /alaysi 3abah (>rant number< B8>''5-51-

1M'') and the /inistry o! 3cience, echnology and Innovation (3cholarship<

/M'FM'1M''M4I;) !or !inancial support and to the 3abah ea ?lantation !or providing tea

samples that support this study.