Total Phenol content assay

8/19/2019 Total Phenol content assay

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16.2.16 no vortexing before shaking and sonication

80%ethanol (0.1%HCl to water)H was nearl! 1

"tandard c#rve ($bs. &s ' "td. (in ')*allic "td. (in +Conc (+,)

! - 0.008x 2/ 0.02/ 0.08 8.

/0 0.0/ 0.16666 166.66666666/ 0.0/ 0.2/ 2/0

100 0.1 0. .

12/ 0.12/ 0.1666 16.66666666

skin shaking 1 3./30113

2 100.82/86

skin sonication 1 10./621831

2 10/./1213

seed shaking 1 21.3108

2 21.01132/

seed sonication 1 21.21312 218.8/0/1

skin 5rst dil#tion is 10 100 ' sa+le dil#ted to 000 '4 so

seed 5rst dil#tion is 10 100 ' sa+le dil#ted to 000 '4 so

Conc (',)*allic

7ote9 :il#tion factor



7:il fac

',ol&g*al $bs *allicacid60n+

0.6

1./62.1

2.36

.612

283.1 28.3108 0.82 skin shaking

010./ 0.1082/3 0.8

1068.3 1.06836//12 0.301 skin sonication

16//.1 1.6//1218 0.318

61.3 6.1310/ 1.86 seed shaking

6/10./ 6/.1082/3 1.888

63/1.2 63./1213 2.016 seed sonication6/6//.1 6/.6//1218 1.30

dil#tion is 0

dil#tion is

+,ol&g*allicacid e;#ivalent



/%ethanol4(0.1%HCl to water)H was

18.2.16 vortexing before shaking and sonicatio

stock sol#tion-10+, of trolox

"tandard c#rve ($bs. &s ' of ascor

! - 0.008x 2/ 0.02//0 0.0/

/ 0.0/

100 0.1

12/ 0.12/

"$,<="

skin shaking 1 0.1

2 0.1

skin sonication 1 0.1

2 0.1

seed shaking 1 0.12 0.1

seed sonication 1 0.1

2 0.1

skin e +ade to 20 +4 so 5rst dil#tion is 10

seed e +ade to 10 +4 so 5rst dil#tion is 10

"td. (in')*allicacid

"td. (in+)*allic acid

d.w(+ake #tototal +l)


too high readiwere dil#ted



"tandard c#rve ($bs. &s ' of ascor "td. (in ')"td. (in +

! - 0.008x 2/ 0.02/

/0 0.0/

/ 0.0/

100 0.112/ 0.12/

"$,<="

skin shaking 1 0.1

2 0.1

skin sonication 1 0.1

2 0.1

seed shaking 1 0.1

2 0.1

seed sonication 1 0.1

2 0.1

skin is 600 le +ade to 20 +4 so 5rst dil#tion is 1

seed is 1000 +ade to 10 +4 so 5rst dil#tion is 10

t4>est9 >wo4"a+le $ss#+ing ?ne;#al ariances

skin Variable 1 Variable 2

,ean 21.12/ 2.1

ariance 0.02/1 0.2812/

@bservations 2 2

H!othesiAed ,ean :iBerence 0

df 1

d.w(+ake #tototal +l)




t "tat 4./3/

<(>-t) one4tail 0.08/2

t Critical one4tail 6.1/2

<(>-t) two4tail 0.1/08

t Critical two4tail 12.062

this res#lt is in accordance to the reference aer DEfects o extracting conditions on phenolic comp"a+avardhana et al. wh.ere no signi5cant diBerence be



nearl! 1. since ref.aer had /% eth.

n 7:il fac

0.08 8.0.166666666 166.666666666

0.2/ 2/0

0. .

0.16666666 16.666666666

106.62/ 138./ 1.38/

103.12/ 2./ 2./

112.62/ 8./ .8/

11./ 2/ .2/

22./ 63/0 63./26 0800 0.8

2/2 /600 /.6

28 100 1.

100 ' sa+le dil#ted to 000 '4 so dil#tion is 0 th#s total dil#tio


0+in 0+in

shaking sonic. <4val#e

skin

1.38/ .8/ 0.120362/8

2./ .2/ 0.020/2300

seed

63./ /.6 0.31816

0.8 1.

skin

/.2138/

.3013608

seed

.803/281

0.8061/

Conc (+,)*allicacid Conc (',)*allic acid

',ol&g*allic acide;#ivalent


% increase in henol insonication+ethod

ngs of abs. so the sa+les



Conc (+,)*allic aci ',ol&g*allic acid e;#i

0.08 8.

0.166666666 166.666666666

0.2/ 2/0

0. .0.16666666 16.666666666

6./ 2182/ 21.82/

6 21600 21.6

3.12/ 2/ 2./

.8/ 222/ 22.2/

8./ 8/00 8./

8./ 8/00 8./

30./ 30/0 30./

83./ 83/0 83./

/0 ' sa+le dil#ted to 000 '4 so dil#tion is 60 th#s total dil#tio


0+in 0+in

shaking sonic.

skin

21.82/ 2./

21.622.2/

seed

8./ 30./

8./ 83./

skin

.028/336

.3/03/03/

seed

./8126216

/.83/8212

t4>est9 >wo4"a+le $ss#+ing ?ne;#al

seed Variable 1

,ean 86

ariance ./

@bservations 2

H!othesiAed ,ean :i 0

df 1

Conc (',)*allic acid+,ol&g*allicacid e;#ivalent

% increase in henolin sonication+ethod



t "tat 42.68360111

<(>-t) one4tail 0.111618

t Critical one4tail 6.1/1/16

<(>-t) two4tail 0.226226


unds and antioxidant activity rom diferent grape processing byproducts'tween shaking and sonicn extractn +ethodsE altho#gh slighl! higher val#es b! sonica



0.61./6

2.1

2.36

.612

0.8/ skin shaking

0.8

0.301 skin sonication

0.318

1.86 seed shaking1.888

2.016 seed sonication

1.30

0

$bs*allicacid60n+

0 /0

0

0./

1

1./

2

2./

./

f(x) - 0.008

F - 0.331

diBerence is not signi5cant 9shaking better



0.6

1./6

2.1

2.36.612

0.231 skin shaking

0.288

0.1 skin sonication

0.0

0. seed shaking

0.66

0.26 seed sonication

0.18

0

<4val#e

0.138/61

0.18300613

G4>est >wo4"a+le for ariances

riances

shaking sonic.

Variable 2 ,ean 21.12/ 2.1

30.2/ ariance 0.02/1 0.2812/

0./ @bservations 2 2

2 df 1 1

G 0.03

<(G-f) one4tail 0.18//

$bs*allicacid60n+

diBerence is not signi5cant 9shaking referable



G Critical one4tail 0.00613

ion +et.




Variable 1 Variable 2,ean 2.62/ .1062/

ariance 0.2812/ 0.202012/

@bservations 2 2

H!othesiAed ,ean :i 0

df 2

t "tat 4./01826

<(>-t) one4tail 0.0/62681

t Critical one4tail 2.31338//801

<(>-t) two4tail 0.012/236

t Critical two4tail .026/223/

100 1/0 200 2/0 00 /0 00 /0

232x

1623

Col#+n

inear (Col#



0 08. 0.6

166.666 1./6

2/0 2.1

. 2.36

16.666 .612

Conc (',)

$bs*allicacid

60n+

+n )



:?>@ /%ethanol4since ref.aer had this

18.2.16 vortexing before shaking and sonication

"tandard c#rve ($b"td. (in ')*allic acid "td. (in +Conc (+,)*allic

! - 0.008x 2/ 0.02/ 0.08

/0 0.0/ 0.166666666

/ 0.0/ 0.2/100 0.1 0.

12/ 0.12/ 0.16666666

"@C$>= +in

skin 1/

0

/

60

/

seed s 1/0

/

60

/

skin is 00 le +ade to 10 +4 so 5rst dil#tion is 10 100 ' sa

seed is 600 +le +ade to 10 +4 so 5rst dil#tion is 20 100 ' s

skin

seed

skin

seed





diBt.ti+es of inc#bation

7:il fac

',ol&g*allic acid e;#ivalent

8.

166.666666666

2/0.

16.666666666

18.18/ /6.2/ ./62/

1/ 100 .1

16/.12/ 3/3./ 3./3/

10./ /1112./ /1.112/

18.3/ /681.2/ /.6812/

10.62/ 621/ 62.1/11.12/ 68/ 68./

12.12/ / ./

12.062/ 32./ 3.2/

10.18/ 8112./ 8.112/

+le dil#ted to 000 '4 so dil#tion is 0 th#s total dil#tion fa

+le dil#ted to 000 '4 so dil#tion is 0 th#s total dil#tion

sonic. sonic. <4val#e1/+in /+in

./62/ /.6812/ 0.26/86318

62.1/ 8.112/ not signi5cant

1.181213

26.08111286

Conc (',)*allic acid+,ol&g*allicacid e;#ivalent

% increase in henol in /&s1/ +in



$bs *allicacid60n+

0.6

1./6

2.12.36

.612

1 2 avg. +in

1.1 1.13 1.18// 1/

1.22/ 1.28 1.2/6 0

1.1 1.28 1.22/ /

1. 1.83 1.6 60

1.2 1. 1.1/ /

0.81 0.81 0.823 1/0.831 0.3/ 0.31 0

0.368 1.018 0.33 /

1.0 1.063 1.0/6/ 60

1.038 1.1/ 1.121/ /

0



2.2.16 oti+isation of

>otal <henol

abs

"kin 80%,e@H 80%,I$

1 0.2/ 0.8/

2 0.2 0.11

"eed1 0.61/ 1.101

2 0.6 0.30

>otal Glavonoids

abs

"kin 80%,e@H 80%,I$

1 0.063 0.122

2 0.0/ 0.12/

"eed1 0.2 0.21

2 0.22 0.23

>otal anthoc!anins

b#Ber H-1

"==: /20n+ 00n+ /20400n+

80%,e@H1 0.216 0.016 0.2

80%,e@H2 0.226 0.02 0.206

80%,I$1 0.26 0.02 0.226

80%,I$2 0.26/ 0.02/ 0.2

80%=t@H1 0.2 0.018 0.212

80%=t@H2 0.28 0.02 0.22/

80%=I$1 0.2/1 0.02 0.228

80%=I$2 0.23 0.021 0.228

"J /20n+ 00n+ /20400n+

80%,e@H1 0.62 0.021 0.6/1



80%,e@H2 0.6/ 0.02 0.6//

80%,I$1 0.8 0.013 0.68

80%,I$2 0.63/ 0.018 0.6

80%=t@H1 0.82 0.06 0.

80%=t@H2 0.88 0.03 0.3

80%=I$1 0.831 0.0/ 0.8/6

80%=I$2 0.386 0.0 0.32



abs

80%=t@H 80%=I$ "kin 1 2

0.286 0.232 80%,e@H 0.2/ 0.2

0.13 0.26 80%,I$ 0.8/ 0.11

80%=t@H 0.286 0.130.23 0.3 80%=I$ 0.232 0.26

0. 0./

"tandard =icatechin9stock9100

80%=t@H 80%=I$ (') +

0.0/ 0.08 0

0.03 0.031 2/ 0.02/

/0 0.0/0.2/ 0.2 / 0.0/

0.2 0.23 100 0.1

/0 0./

/00 0./

b#Ber H-./

"==: /20n+ 00n+ /20400n+ $ (H14H./)

80%,e@H1 0.01 0.003 0.008 0.132

80%,e@H2 0.01 0.003 0.008 0.138

80%,I$1 0.023 0.016 0.01 0.21

80%,I$2 0.022 0.003 0.01 0.22

80%=t@H1 0.01 0.018 0.01 0.133

80%=t@H2 0.02 0.00 0.01 0.212

80%=I$1 0.028 0.012 0.016 0.212

80%=I$2 0.0 0.01/ 0.013 0.203

"J /20n+ 00n+ /20400n+ $ (H14H./)

80%,e@H1 0.0/2 0.00 0.0/ 0.606



80%,e@H2 0.0/ 0.00/ 0.08 0.60

80%,I$1 0.0 0.00 0.06 0.01

80%,I$2 0.06 0.00/ 0.062 0.61/

80%=t@H1 0.086 0.02 0.06 0.11

80%=t@H2 0.08 0.026 0.0/ 0.632

80%=I$1 0.06 0.018 0.0/8 0.38

80%=I$2 0.1 0.01 0.063 0.8



7:G

Conc (',)*allic acid Conc',&g*allic acid

1b 2b 1b 2b "eed

2.12/ 0.8/ 132/ 18/2/ 80%,e@H

8.12/ /1./ 28875 30825 80%,I$

/./ 3.8/ 21/0 232/ 80%=t@H6./ 21300 13800 80%=I$

+

:K('l) total('l) conc.+ conc.b /10n+400n+

0 0 0

2/ 2/00 1 1000 0.023

2/0 2/00 2 2000 0.03222/ 2/00 000 0.1

200 2/00 000 0.136

21/0 2/00 1 1000 0.//3

2000 2/00 20 20000 0.3

000

000

+g&

$7,K7:G71000&=<"=@7

86.36/3 skin is 2

8.016 seed is 266.6

3!"02#8

#00#!3"0#

8.823

3/.130306

3/.130306

321.3/02

$7,K7:G71000&=<"=@7

2.080/

7ote9 :il#tion fa



21.32/2

166./368

28.0626

211.11

12/.88/1

3"0$!7057

3$3!$38



7:G

abs Conc (',)*allic acid Conc',&g*allic acid

1 2 1b 2b 1b 2b

0.61/ 0.6 6.8/ 80./ 1//0 160/0

1.101 0.30 1.62/ 11 275250 22"000

0.23 0. /.62/ // 102/0 1100000.3 0./ /.8/ /3./ 103/0 118/0

2. 100

1 gra+ sa+le +ade to 10 +4 so 5rst dil#tion is 10 1/0 ' sa+le dil#ted to 100 '4

0./ gra+ sa+le +ade to 10 +4 so 5rst dil#tion is 20 00 ' sa+le dil#te

1.

266.666666666

ctor

0 / 10 1/ 20 2/

0

0.1

0.2

0.

0.

0./

0.6

0.

0.8

0.3

1

f(x) - 0.06821086x

F - 0.33/00308

Col#+n

inear (



skin is 600 1 gra+ sa+le +ade to 10 +4 so 5rst dil#tion is 10 /0 ' sa+le dil#

seed is 2000 0./ gra+ sa+le +ade to 10 +4 so 5rst dil#tion is 20 0 ' sa+le di

skin is 2/0 1 gra+ sa+le +ade to 10 +4 s

seed 0./ gra+ sa+le +ade to 10 +4

abs conc.(+)"kin 1 2 1b

80%,e@H 0.063 0.0/ 1.606/12

80%,I$ 0.122 0.12/ 2.8203

80%=t@H 0.0/ 0.03 1.186

80%=I$ 0.08 0.031 2.022//8

al! dil.factor

abs conc.

"kin 1 2 1b80%,e@H 0.063 0.0/ 01.1623

80%,I$ 0.122 0.12/ 70!302

80%=t@H 0.0/ 0.03 6.06/1

80%=I$ 0.08 0.031 /0/.813/

o dil#tion is 2. th#s total dil#tion factor is 2



<

ol#+n <)



ed to 000

l#ted to 0

5rst dil#tion is 10 100 ' sa+le

so 5rst dil#tion is 20 100 ' sa+l

abs conc.2b "eed 1 2 1b 2b

1.186 0.2 0.22 /.882 /.3/88

2.30636 0.21 0.23 .6/116 6.8203

1.8203 0.2/ 0.2 /.636 /.6186

2.116231 0.2 0.23 /./813/ /.3063

abs conc.

2b "eed 1 2 1b 2b6.06/1 0.2 0.22

72"!7$$ 0.21 0.23

/3.02 0.2/ 0.2

/23.063 0.2 0.23



>@>$ <H=@"

"kin 80%,e@H 80%,I$ 80%=t@H 80%=I$

conc. n skin 132/ 28875 21/0 21300

18/2/ 30825 232/ 13800


Variable 1 Variable 2

,ean 18300 2268./

ariance 2812/0 062812./

@bservations 2 2

H!othesiAed ,ean :iBer 0

df 1

t "tat 42.3230632

<(>-t) one4tail 0.1020/


<(>-t) two4tail 0.2036031t Critical two4tail 12.0620



,ean 238/0 208/0

ariance 13012/0 220/000

@bservations 2 2


df 2t "tat 6.28108011

<(>-t) one4tail 0.01221122

t Critical one4tail 2.31338//801

<(>-t) two4tail 0.02228/

t Critical two4tail .026/223/

"eed 80%,e@H 80%,I$ 80%=t@H 80%=I$

1b 1//0 275250 102/0 103/0

2b 160/0 22"000 110000 118/0


%eed Variable 1 Variable 2

,ean 1/2/0 10862/

ariance 2/00000 812/0

@bservations 2 2


nosigni5cant

diB.between80%+ethand eth.

signi5cantdiB.

between80%+eth

and eth Iacid

signi5cantdiB.

between



df 1

t "tat 12.303/3

<(>-t) one4tail 0.02/6/168


<(>-t) two4tail 0.031/06




,ean 2/062/ 112/0

ariance 1212812/0 0/00000

@bservations 2 2


df 1

t "tat /.8/<(>-t) one4tail 0.0/8/21


<(>-t) two4tail 0.11//0


80%+ethand eth

nosigni5cant

diB.between80%+eth

and ethIacid



$nova9 "ingle Gactor

"?,,$L

Groups Count Sum Average Variance

Col#+n 1 2 800 18300 2812/0

Col#+n 2 2 /300 238/0 13012/0

Col#+n 2 // 2268./ 062812

Col#+n 2 100 208/0 220/000

$@$

Source of Variation SS df MS F !valueMetween *ro#s 1.=I008 /62128 2.36/ 0!00$#0$#

Kithin *ro#s /01 1862/8

>otal 1.=I008

$nova9 "ingle Gactor

"?,,$L

Groups Count Sum Average Variance

Col#+n 1 2 1/00 1/2/0 2/00000

Col#+n 2 2 /012/0 2/062/ 1.2=I003



Col#+n 2 212/0 10862/ 812/0

Col#+n 2 228/00 112/0 0/00000

$@$

Source of Variation SS df MS F !value

Metween *ro#s 2.6=I010 8.6=I003 26.3081 0!00$##3035Kithin *ro#s 1.=I003 .2=I008

>otal 2.=I010



F crit 6./3182



F crit

6./3182



0/.0.2016 0./%HCl 4since extra added to water assa!

onl! skin >otal <henol /0'lI23/0'l :K

"kin :il#tion Gactor

1st 1 0.8 60

2 0.6

0.82nd1 0.

2 0./

0.0

rd 1 0.31

2 0.36

0./8

<ooled1 0.

2 0.8

0.02

>otal Glavonoids

"kin /10n+400n+ :il#tion Gactor

1st 1 0.06/ /0

2 0.06

0.068

2nd1 0.082

2 0.066

0.0

rd 1 0.063

2 0.0/3

0.01

<ooled1 0.06

2 0.06

0.06

>otal anthoc!anins

b#Ber H-1

"kin /20n+ 00n+

1st 1 0.13 0.008

2 0.1/8 0.00

0.1/ 0.006

2nd1 0.12 0.001

2 0.1 0.001

0.12 0.00

rd 1 0.18 0.002

1ho#r

shaking

2ho#rshaking

ho#rshaking

all ooled

/0'lI2000'l :K

1ho#rshaking

2ho#rshaking



2 0.1 0.00

0.18 0.00

<ooled1 0.1 0.001

2 0.1 0.001

0.12 40.001

>otal anthoc!anins

b#Ber H-./

"kin /20n+ 00n+

1st 1 0.0/3 0.006

2 0.01 0.008

0.0 0.00

2nd1 0.0/3 0.00

2 0.0/ 0.008

0.06 0.00rd 1 0.0/1 0.008

2 0.06 0.008

0.01 0.01

<ooled1 0.0 0.008

2 0.0 0.003

0.06 0.00

ho#rshaking

all ooled



skin is 600 de to 10 +4 so 5rst dil#tion is 10 /0 ' sa+le dil#ted to 000

skin is /00 de to 10 +4 so 5rst dil#tion is 10 /0 ' sa+le dil#ted to 2/00

+g& total anth

/20400n+ $ (H14H./) $7,K7:G71000&=<"=@7 avg.

0.11 0.08 /2.36/ 0./

0.1/ 0.031 11.06232/

0.163 0.033 .2002/

0.11 0.083 02.028// 12./68

0.1 0.06 .0/

0.163 0.103 32.20/

0.16 0.10 6/.26302/ $3"!""





0.16 0.068 0.163

0.18 0.113 /./82/

0.1 0.10 63.862 1.0

0.16 0.08/ 8.3/38/

0.1 0.086 88.0/

+g&

/20400n+ $ (H14H./) $7,K7:G71000&=<"=@7

0.0/

0.06

0.0

0.0/2

0.06

0.060.0

0.068

0.061

0.063

0.061

0.0/



'4 so dil#tion is 60 th#s total di

'4 so dil#tion is /0 th#s total di

:il#tion Gactor

/

skin is 2 +le dil#ted to 100 '4 so dil#tion is 2. th#s total dil#tion factor is 2




03.0.2016 0./%HCl 4sinec extra added to w

>otal <henol /0'lI23/0'l :K

"kin :il#tion Gactor

1st 1 0.28 60

2 0.232

0.66

2nd1 0./

2 0.23

0.2

rd 1 0.1/1

2 0.11

0.11

<ooled1 0.28

2 0.26/

0.21

>otal Glavonoids

"kin 0n+400n+ :il#tion Gactor

1st 1 0.06 66.6666

2

0.0 1

2nd1 0.063

2 0.061

0.03rd 1 0.028

2 0.013

0.022

<ooled1 0.0/2

2 0.13

:il#tion Gactor >otal anthoc!anins

/

b#Ber H-1

"kin /20n+ 00n+ 20400n+

1st 1 0.8 0.11 0.66/

2 0.32 0.12 0.668

0.8/ 0.1 0.0

2nd1 0.82 0.0/ 0.

2 0.13 0.026 0.23

0.26 0.016 0.22

1stextract

2ndextract

rdextract

allooled

0

/0



rd 1 0.038 0.01/ 0.08

2 0.062 0.01 0.0/2

0.0/ 0.008 0.06

<ooled1 0.62 0.01 0.21

2 0.6/ 0.06 0.13

0./ 0.0 0.238

>otal anthoc!anins

b#Ber H-./

"kin /20n+ 00n+ 20400n+

1st 1 0.11 0.086 0.22/

2 0.2 0.086 0.21

0. 0.081 0.2/3

2nd1 0.16 0.026 0.18

2 0.13 0.02 0.126 0.103 0.016 0.03

rd 1 0.02 0.01 0.02

2 0.02 0.00 0.0/

0.06 0.00 0.03

<ooled1 0.16 0.01 0.1

2 0.162 0.02 0.1

0.1/3 0.02 0.12



ater

:il#tion Gactor //0'lI3/0'l :K

8.181818

+g&

$ (H14H $7,K7:G71000&=<"=@7

0.

0.2

0.8

0.203

0.16

0.12

lI130'l :K

lI13/0'l :K



0.0/1

0.01

0.028

0.188

0.183

0.11

+g&

$ (H14H $7,K7:G71000&=<"=@7



a

b

Total Phenol content assay

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