µ¦ r ¦³ ° ´·Í ° ª£µ¡ µ ° Á ¥Á «Ã ¥Ä µ ¸É¤¸¸o ÊÎ· · 2012-07-02 · ´Ê (R2 = 0.9835) ®¤ Î´µ®¦ ¨ µ¦ª·Á ¦µ³® ¸ ´Ê´r Ã ¥ µ¦ª

2553

2553

EXTRACTION OF BIOACTIVE COMPONENTS FROM CINNAMON

(CINNAMOMUM ZEYLANICUM) BY SUBCRITICAL WATER

By

Nucha Sayputikasikorn

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

Department of Food Technology

Graduate School

SILPAKORN UNIVERSITY

2010

“

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……...........................................................

( . )

.......... .................... . ...........

1. .

2. .

3. .

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( . )

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( . ) ( . )

............/......................../.............. ............/......................../..............

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( . ) ( . )

............/......................../.............. ............/......................../..............

51403207 :

: / /

:

. : . . , . .

. . . 74 .

(semi-continuous)

60 3 / 100, 150

200

protocatechuic acid, ferulic acid,

catechin, p-coumaric acid, vanillic acid caffeic acid

50

200

catechin

two-site kinetic

2,2-diphenyl-

1-picrylhydrazyl (DPPH) 200

10

50 30

DPPH

(R2 = 0.9835)

(log P)

(hydrophilic)

2553

........................................

1. ....................... 2. ................... 3. .......................

51403207 : MAJOR : FOOD TECHNOLOGY KEY WORDS : PHENOLIC COMPOUNDS/ANTIOXIDANT/BIOACTIVE COMPOUND

NUCHA SAYPUTIKASIKORN : EXTRACTION OF BIOACTIVE COMPONENTS FROM CINNAMON (CINNAMOMUM ZEYLANICUM) BY SUBCRITICAL WATER. THESIS ADVISORS : ASST.PROF.PRAMOTE KHUWIJITJARU, Ph.D., ASST.PROF.SUCHED SAMUHASANEETOO, Ph.D., AND ASST.PROF.PRASONG SIRIWONGWILAICHAT, Ph.D. 74 pp.

The aim of this research was to study the subcritical water extraction (SWE) of bioactive components from cinnamon (Cinnamomum zeylanicum) bark using a semi-continuous system at a constant pressure of 60 bar and water flow rate of 3 ml/min. Effects of extraction temperature (100, 150 and 200°C) were determined on the amount of cinnamaldehyde, coumarin, cinnamic acid and cinnamyl alcohol compared to those obtained by ultrasonic-assisted extraction (UAE) using methanol as a solvent and the amount of phenolic compounds (protocatechuic acid, ferulic acid, catechin, p-coumaric acid, vanillic acid and caffeic acid) and the total phenolic content compared to those obtained by UAE using 50% methanol as a solvent. The amount of cinnamaldehyde obtained by UAE was higher than SWE, whereas the amount of coumarin and cinnamic acid in the extracts obtained by SWE and UAE were not significantly different. However, the amount of cinnamyl alcohol obtained by SWE was higher than UAE. The amount of phenolic compounds and the total phenolic content increased with an increasing in subcritical water temperature. At 200°C, these values from SWE were higher than UAE. However, catechin was not found from SWE. The extraction data were fitted with two-site kinetic model and the results indicated that the extraction rates in the first fraction for cinnamaldehyde, coumarin, cinnamic acid and cinnamyl alcohol were higher than those for phenolic compounds. In addition, antioxidant capacity of the extract that measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay from the first collected fraction of SWE at 200°C was comparable to that obtained by UAE using 50% methanol as a solvent. And the antioxidant capacity was linearly related to the total phenolic content (R2 = 0.9835). The polarity of the extracts was assessed by determining the octanol-water partition coefficient (log P). The extracts obtained from both SWE and UAE were found to be hydrophilic.

Department of Food Technology Graduate School, Silpakorn University Academic Year 2010 Student's signature ........................................Thesis Advisors' signature 1. ....................... 2. ....................... 3. .......................

.

. .

. .

“

2”

......................................................................................................

..................................................................................................

........................................................................................................

..............................................................................................................

.................................................................................................................

1 .............................................................................................................. 1

.............................................................. 1

........................................................................... 2

.............................................................................. 2

................................................................................. 2

2 .......................................................................... 3

..................................................................................................... 3

- (partition coefficient) ........... 10

.............................................................................................. 12

................................... 15

3 ............................................................................................. 17

.................................................................................................... 17

................................................................................................... 17

................................................................................ 18

........................................................................................... 20

................................................................. 20

(UAE) .............................................. 21

(Partition coefficient) ............. 21

................................................................................. 22

.......................................................... 23

.............................. 23

4 ............................................................ 24

...................................................................... 24

....... 26

..................................................................... 31

.......................................................... 39

....................................... 42

...................................................................... 43

(partition) ...................................... 44

5 ......................................................................................... 47

......................................................................................................... 48

............................................................................................................. 55

................................................................................................. 56

................................................................................................. 58

................................................................................................. 67

.......................................................................................................... 74

1

............................................................................ 5

2

.................................................................................................. 6

3 ...................................................... 7

4 ...... 9

5 F, k1 k2

two-site kinetic................................................................................ 29

6

(SWE) 60

(UAE) 30

................................................................................................. 31

7

........................................................................................... 32

8 F, k1 k2

two-site kinetic ....................... 37

9

(SWE) 60

(UAE) 50

30 .................................................................. 38

10 F, k1 k2

two-site kinetic ................... 40

11

(SWE) 60

(UAE) 50

30 .................................................................. 41

12 (log P)

DPPH ............................................ 45

13

.................................................................... 68

14

........................................................................ 68

15

.................................................................... 69

16

....................................................... 69

17 protocatechuic acid

50 ....................................... 69

18 ferulic acid

50 .................................................... 70

19 catechin

50 ........................................................ 70

20 p-coumaric acid

50 ............................................. 70

21 vanillic acid

50 .................................................... 71

22 caffeic acid

50 .................................................... 71

23

50 ............................... 71

24 DPPH

- 50

30 .................................................................... 72

25 (log P)

50 ....................................... 72

26 DPPH

100

- ...... 72

27 DPPH

150

- ...... 73

28 DPPH

200

- ...... 73

1 (a) (b) (c)

(d) .............................................................................. 4

2 ................................................................................... 7

3 ................................................................................ 12

4 ( )

(log (KW)) 15 MPa........................................ 13

5 ....................................................... 15

6 (semi-continuous

system)........................................................................................... 18

7 (semi-

continuous system) (a) (b)

............................................ 19

8 100,

150 200

(UAE)....................................................... 25

9

(a) 250 (b)

280 ................................................................................. 26

10 (a)

(b) (c) (d)

100°C ( ), 150°C ( ) 200°C ( )

two-site kinetic 100°C ( ),

150°C ( ) 200°C ( ) ..................................................... 27

11

200 10 (a)

250 (b) 280 (c)

320 ......................................................................... 33

12 (a)

protocatechuic acid (b) ferulic acid (c) p-coumaric acid (d)

vanillic acid (e) caffeic acid

100°C ( ), 150°C ( ) 200°C ( )

two-site kinetic

100°C ( ), 150°C ( ) 200°C ( ) ............................... 36

13

100°C ( ), 150°C ( ) 200°C ( )

two-site kinetic 100°C

( ), 150°C ( ) 200°C ( ) .......................................... 39

14 DPPH

(SWE)

(UAE) 50 30 .. 43

15

DPPH ....................... 44

16 ........ 57

17 (a) protocatechuic acid, (b) catechin, (c)

vanillic acid, (d) caffeic acid, (e) p-coumaric acid (f) ferulic

acid ................................................................................................ 63

18

............................................................................................ 64

19

DPPH............................................................................ 65

1

1

1.1

(Adisakwattana,

2007)

(Subash Babu ,

2007)

(Chang

, 2001) (

, 2551)

( , 2549)

100

374

(Smith, 2002)

(Herrero , 2006)

(polarity)

(Ozel , 2003) (Khuwijitjaru , 2008)

2

1.2

1.

2. DPPH

3. -

1.3

1.

DPPH

-

2.

1.4

100 - 200

(semi-continuous)

3

2

2.1

Lauraceae

30 – 40

4 – 5 ( , 2527) (Cinnamomum) 250

(Jayaprakasha , 2003)

(Cinnamomum zeylanicum)

Ceylon Cinnamon ( ) True Cinnamon ( )

3

(cinnamaldehyde)

(cinnamic aldehyde) 60 - 75 ( , 2549)

4

OH O O

2.1.1

(cinnamaldehyde)

(cinnamic acid) (cinnamyl alcohol) (coumarin)

( 1)

(Woehrlin , 2010) 1

1 (a) (b) (c)

(d)

5 - 20

45 streptozotocin (STZ)

20

glycosylated hemoglobin (HbA1C)

high-density lipoprotein (HDL-cholesterol) (Subash

Babu , 2007)

OH

O

H

O (a) (b)

(c) (d)

5

1

(mg/kg) (mg/kg)

(mg/kg)

(mg/kg)

cinnamon powder

mean value 11100 64 252 183

median 11500 <LOD 235 <LOD

range 2080 - 24800 <LOD - 297 88 - 436 <LOD - 509

cinnamon sticks

mean value 16700 185 231 1210

median 17400 160 208 434

range 3930 - 28200 <LOD - 486 62 - 522 <LOD - 8140

<LOD are considered as 0

: Woehrlin (2010)

Singh (2007)

97.7 50.0

Chang (2001)

(2551)

30 4,

6, 8 10 (% yield)

4 10

6

2

2

DPPH radical-scavenging Extraction

time

(hour)

Yieldns

(%)

(dry basis) EC50

( g DM/ g DPPH)

ARP

(1/EC50)

Total phenolic

contents (mg of

GAE/g DM)

4 26.46±0.24 5.74±0.08 a 0.18±0.01 a 281.51±4.85 ab

6 24.80±0.74 5.80±0.42 a 0.17±0.01 a 278.34±0.68 a

8 27.70±0.88 5.04±0.57 ab 0.20±0.03 ab 283.28±4.65 b

10 24.76±0.90 4.15±0.17 b 0.24±0.01 b 300.67±3.20 c a, b, c Means with different letters within the column are significantly different (p 0.05)

: (2551)

(secondary metabolite)

, (Pentose phosphate, Shikimate

Phenylpropanoid pathway) (Balasundram , 2006)

(-OH) 1

2

(Vermerris Nicholson, 2006)

7

2

: Vermerris Nicholson (2006)

(Naczk Shahidi, 2004)

3 (phenolic acids), (flavonoids)

(tannins) (Balasundram ,

2006)

3

: Balasundram (2006)

8

(Phenolic acids) 2

(Hydroxybenzoic acid) 1 (C6-C1)

(hydroxycinnamic acids)

3 C6-C3 (Balasundram , 2006)

(Flavonoids)

8,000 15

C6-C3-C6 A B

3

(heterocyclic ring) C C

(flavonols), (flavones),

(flavanones), (flavanols catechins), (isoflavones),

(flavanonols) (anthocyanidins) (Balasundram , 2006)

(Tannins)

( ,

2549) Condensed tannins Hydrolysable tannins

4

9

4

rutin

quercetin

kaempferol

isorhamentin

catechin

0.896 ±0.028

0.550 ±0.095

0.492 ±0.134

0.113 ±0.015

2.30 ±0.049

mg/100g

(fresh wt)

Al-Numair

(2007)

quercetrin

quercetin

kaempferol

0.122±0.005

0.267±0.001

0.005±0.000

mg/100g

(dry wt)

Prasad

(2009)

catechin

caffeic acid

454.4

24.2

mg/100g

(dry wt)

Shan

(2005)

p-coumaric

acid

10.3 ±0.03 mg/100g

(dry wt)

Wojdylo

(2007)

vanillic acid

caffeic acid

ferulic acid

- - Muchuweti

(2007)

(free radical)

10

ROO• + PPH ROOH + PP•

RO• + PPH ROH + PP•

ROO•, RO• PPH

2 (Shahidi Naczk, 2004)

ROO• + PP• ROOPP

RO• + PP• ROPP

3

( , 2549)

1. (chelating agent)

2. (chain breaking antioxidant)

3. (regenerate)

2.2 - (partition coefficient)

(antioxidants)

(Maisuthisakul , 2006)

(polarity)

, (surfactants), - (pH),

(hydrophilic antioxidants)

11

(lipophilic antioxidants)

polar paradox

(Shahidi Zhong, 2011)

(oil-

in-water)

-

(Huang , 1997)

(partition coefficient, P) 2

(immiscible) (Cheng , 2010)

(1-octanol)

2

(Noubigh , 2009)

Partition coefficient (P) = aqueous

octanol

C

C

Coctanol

Caqueous

25

12

2.3

( 3) (subcritical water superheated

water) ( 100

374 )

3

: (2549)

2.3.1

(dielectric constant) (ion product) 4

(relative dielectric constant, )

79.0 25 35.5

200 20.7 300

( 32 25

) ( 21 25 -

)

13

(KW) 15 MPa Log(KW) -14

25 -11.3 200-250

(H3O+) (OH- ) 1×10-7 mol/L

2.2 ×10-6 mol/L 20

(acid-base catalyst)

( , 2549)

4 ( ) (log

(KW)) 15 MPa

: (2549)

2.3.2 (Subcritical water extraction)

100

374

(Smith, 2002)

(Herrero

, 2006)

Thymbra spicata (Ozel , 2003)

(Centella asiatica) (Kim , 2009)

14

(Rangsriwong , 2009) shikimic acid (Illicium verum Hook. f.)

(Ohira , 2009) quercetin (Ko , 2011)

(Anekpankul , 2007)

1.

(Coriandrum

sativum L.) 100 125 linalool

150 175 linalool

(Eikani , 2007)

(Terminalia chebula ) 120 180

gallic acid ellagic acid 220

corilagin 120

220 corilagin (Rangsriwong

, 2009)

2. Budrat Shotipruk (2008)

Anekpankul (2007) (Momordica

charantia)

3.

(Petroselinum crispum)

asiatic acid asiaticoside (Centella asiatic) (Gámiz-Gracia Luque de

Castro, 2000, Luthria, 2008 Kim , 2009)

4. (

) 40

glycyrrhizin ephedrine (Eng , 2007)

5.

(Luthria, 2008 Ohira

, 2009)

15

6. (solid-to-solvent ratio)

100 250 91.3

500 78.3 78.8 750 1,000

(Luthria, 2008)

2.4

4 ( 5) (1)

(2)

(3)

(4)

(Khajenoori

, 2009)

solute-solid interaction (adsorption),

solute-fluid interaction (solubility) mass transfer

(upscale) (Sovová, 2005)

5

: Ong (2006)

16

two-site kinetic

2

(F) k1

(1-F) k2 Two-site kinetic (Kubátová

, 2002)

tktkt eFFeSS 21

0

11

St = t (mg/g)

S0 = (mg/g)

0

SS

t = t

F =

1-F =

k1 = (min-1)

k2 = (min-1)

savory (Kubátová , 2002)

Zataria multiflora Boiss (Khajenoori , 2009) damnacanthal

(Anekpankul ,2008) (Kim

Mazza, 2006)

17

3

3.1

(Cinnamomum zeylanicum)

500 4

8.17%

Karl Fisher Titration

3.2

3.2.1 Methanol (Merck, Germany)

3.2.2 Acetonitrile (Burdick & Jackson Reagent Plus, Korea)

3.2.3 Glacial acetic acid (Mallinckrodt Chemicals, USA)

3.2.4 DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, Germany)

3.2.5 Folin-ciocalteu (Merck, Germany)

3.2.6 Sodium carbonate anhydrous (Na2CO3) (Ajax Finechem, Australia)

3.2.7 Ascorbic acid (Poch, Poland)

3.2.8 Glass wool (Ajax Finechem, Australia)

3.2.9 Silicon dioxide acid washed (Riedel-de Haën, Germany)

3.2.10 protocatechuic acid , gallic acid monohydrate, trans-

cinnamaldehyde, cinnamyl alcohol coumarin (Sigma-

Aldrich,Germany), trans-cinnamic acid , vanillic acid , caffeic acid ,

catechin, p-coumaric acid ferulic acid (Fluka, UK)

3.2.11 HPLC grade (Fisher Scientific, UK)

3.2.12 1-octanol (Panreac, Spain)

3.2.13

18

3.3

3.3.1 ( 6 7)

- 10 ( 30 108 mm Thar Instruments

Inc. 5794-13, USA)

- (Shimadzu LC-20AD, Japan)

- (back pressure regulator valve) (Upchurch

Scientific P-880, USA)

- (stainless steel tubing : i.d. 1/16 in.)

- (Temperature controller) (oven)

( )

- (Fluk 52 II, China)

- type-K

6 (semi-continuous system)

distilled

water

pump

cooling bath

oven

vial

extraction vessel

back pressure regulator

thermocouple T

pressure gauge

19

7 (semi-continuous

system) (a) (b)

3.3.2 (HPLC)

HPLC (Dionex P680A LPG-4, Germany), Photodiode array detector (Dionex PDA-

100, USA) Inertsil ODS-3 ( 4.6 250 GL Science, Japan)

3.3.3 (Thermo Spectronic Genesys 10uv, USA)

oven pump

temperature

controller

cooling

bath

back pressure regulator

Distilled

water

(a)

extraction vessel

heating coil

(b)

20

3.3.4 Karl Fisher Titration (KF Titrino 787 KF Titrino,

703 Ti Stand Metrohm, Switzerland)

3.3.5 4 (Sartorius AG ED224S, Germany)

3.3.6 (Binder FD 115, USA)

3.3.7 (Crest Ultra Sonics 275DAE, Malaysia)

3.3.8 (Büchi, Japan)

3.3.9

3.3.10 (Germany)

3.3.11 (Hettich Zentrifugen, Universal 16/16R, Germany)

3.3.12 (ALC PK 121R, Italy)

3.3.13 (water bath)

3.3.14

3.3.15

3.4

(UAE)

50

3.4.1

3.4.1.1 1.0 silicon dioxide 7.0

(glass wool)

3.4.1.2 (

0.45

)

3.4.1.3 (pre-heating coil) 5

(temperature controller)

21

( 100 9 –

11 150 10 – 12 200

12 – 15 )

10

10 -18

3

3.4.2 (UAE)

3.4.2.1 1.0

( )

50 ( ) 50

( 27 – 35 )

30

3.4.2.2 945.8 g 15

3.4.2.3 whatman 1

3.4.2.4 50

-18 3

3.4.3 (Partition coefficient)

Maisuthisakul (2006)

3.4.3.1 100, 150 200

60

50 30

(freeze dry)

3.4.3.2 3,000 ppm HPLC

grade (Fisher Scientific, UK)

3.4.3.3 2.0

16 100

22

3.4.3.4 1-octanol 2.0

3.4.3.5 (vortex) 3 20 20

3.4.3.6 2,879 g 40 20

3.4.3.7

-18

3.4.3.8

DPPH

3.4.3.9 (Partition coefficient)

Partition coefficient (P) = aqueous

octanol

C

C

Coctanol

–

Caqueous

Partition coefficient (P) = after

afterbefore

C

CC

Cbefore Cafter

3.4.4

-

(HPLC) 2

He (2005) ( )

-

(HPLC) 2

Prasad (2009) ( )

23

- Folin-Ciocalteu

3 Jayaprakasha (2007)

(mg GAE/g

sample) ( )

- DPPH radical scavenging

3 Brand-Williams (1995)

(mg

VCEAC/g sample) ( )

3.4.4

two-site kinetic non-linear

regression Microsoft Excel solver F,

k1 k2

3.4.5

Completely Randomized Design (CRD)

3 (ANOVA, Analysis of

Variance)

LSD

95 ( = 0.05)

24

4

4.1

100, 150 200

3 60

10 60

30

(He , 2005)

50

(Zhao Hall, 2008) 20

( 8)

100

150 200

(4.65%) (59.55%)

(Peter, 2001)

(Caramelization)

(Maillard Browning Reaction) (Hata , 2008 Plaza

, 2010)

25

150 200

10 20

(mucilage) (Lapointe Solomon, 2006)

8 100, 150

200

(UAE)

100°C

150°C

200°C

0-10 21-30 41-50 11-20 31-40 51-60

50%MeOH

UAE

100%MeOH

UAE

26

4.2

HPLC

9 250

280

9

(a) 250 (b) 280

0.0 10.0 20.0 30.0 40.0 55.0-100

500

1,000

1,500

2,000 mAU

min

coum

arin

cinn

amic

aci

d

cinn

amal

dehy

de

WVL:280 nm

0.0 10.0 20.0 30.0 40.0 55.0-100

500

1,000

1,500

2,000 mAU

min

cinn

amyl

alc

ohol

WVL:250 nm

(a)

(b)

27

10

10

0

20

40

60

80

100

0 10 20 30 40 50 60 ( )

0

20

40

60

80

100

0 10 20 30 40 50 60

( )

0

20

40

60

80

100

0 10 20 30 40 50 60

( )

0

20

40

60

80

100

0 10 20 30 40 50 60

( )

10 (a)

(b) (c) (d)

100°C ( ), 150°C ( )

200°C ( ) two-site kinetic

100°C ( ), 150°C ( ) 200°C ( )

(c) (d)

(b) (a)

28

two-site kinetic

(F) k1

(1-F) k2

F, k1 k2

Microsoft Excel solver 5

two-site

kinetic 10

k1 k2 5 k1

k2

k1 k1

100 200

k1 150 100

200 150 k1 100,

150 200

5 k1 100

10

100

29

5

F, k

1 k

2

two-

site

kinet

ic

F

k 1

k 2

10

0°C

150°

C 20

0°C

10

0°C

150°

C 20

0°C

10

0°C

150°

C 20

0°C

4.

6410

-1

5.48

10-1

5.

5910

-1

2.

72

7.77

10-2

2.

4810

-1

8.

5210

-3

0.00

0.

00

4.

0110

-1

8.52

10-1

7.

6710

-1

6.

9010

-1

5.35

10-2

2.

1710

-1

1.

8210

-2

0.00

1.

0310

-4

8.

3110

-1

7.77

10-1

9.

1310

-1

3.

9410

-1

2.38

10-2

3.

4110

-1

2.

4310

-3

2.32

10-2

8.

3410

-4

5.

0910

-1

9.31

10-1

8.

1810

-1

3.

27

6.02

10-2

2.

3710

-1

3.

1310

-5

6.02

10-2

7.

1810

-4

30

60

30

6

38.78 Woehrlin

(2010)

(cinnamon powder) 7

2.08 – 24.80 (cinnamon sticks)

15 3.93 – 28.20

(p 0.05)

100 150 200 28.16

21.76 22.80

Friedman

(2000)

(benzaldehyde)

60

(benzoic acid) (Chen , 2009) Woehrlin

(2010)

(p > 0.05)

31

150

200

(p > 0.05)

100

6 (SWE)

60

(UAE) 30

( )*

(ºC)

UAE 38.78±0.78a 4.44±0.10 0.68±0.01 0.27±0.00a

SWE 100 28.16±4.02b 3.89±1.32 0.60±0.03 0.14±0.01b

150 21.76±1.30c 3.68±0.16 0.65±0.03 0.25±0.06a

200 22.80±2.11c 3.40±0.13 0.64±0.04 0.22±0.02a

a-c

(p 0.05)

* ± (n = 3)

4.3

HPLC retention time 7

protocatechuic acid, ferulic acid,

catechin, p-coumaric acid, vanillic acid caffeic acid

11 protocatechuic acid vanillic acid

250 catechin 280

caffeic acid, p-coumaric acid ferulic acid 320

( , 2549)

32

protocatechuic acid

(OH) 2 (Cai , 2006)

(Tanaka , 2011)

7

protocatechuic acid / quercetin nd

catechin / rutin nd

p-coumaric acid / rosmarinic acid nd

vanillic acid / tannic acid nd

caffeic acid / ellagic acid nd

ferulic acid / syringic acid nd

gallic acid nd gentisic acid nd

chlorogenic acid nd 4-hydroxybenzoic acid nd

/ nd

protocatechuic acid,

catechin p-coumaric acid

protocatechuic acid, ferulic acid, p-coumaric acid, vanillic acid caffeic acid

catechin

12

20 vanillic

acid

33

11

200 10 (a) 250 (b)

280 (c) 320

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 44.0-10

20

40

60

80

100 mAU

min

caffe

ic a

cid

p-co

umar

ic a

cid

feru

lic a

cid

WVL:320 nm

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 44.0-10

20

40

60

80

100 mAU

min

WVL:280 nm

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 44.0-10

20

40

60

80

100 mAU

min

prot

ocat

echu

ic a

cid

vani

llic

acid

WVL:250 nm

(a)

(b)

(c)

34

100 200

protocatechuic acid, ferulic acid, p-coumaric acid,

vanillic acid caffeic acid (p 0.05) 9

(solute-matrix

interaction)

(Ong , 2006)

Ju (2011) 120

180 p-coumaric acid,

ferulic acid vanillic acid

F, k1 k2 two-site kinetic

8

two-site

kinetic 12

k1 k2

5 k1 k2

8 k1

k1


protocatechuic acid

8 k1 k2 0 100

protocatechuic acid

protocatechuic acid

100

35

Clifford (1999)

k1 150 200

150 200

k1 k2

200 150

vanillic acid 200 k1

k2

12

50

30

protocatechuic acid 200

(p 0.05) p-coumaric acid

(p > 0.05)

200

100 150 (p 0.05)

catechin 13.28 100

catechin

36

0

20

40

60

80

100

0 10 20 30 40 50 60 ( )

0

20

40

60

80

100

0 10 20 30 40 50 60 ( )

0

20

40

60

80

100

0 10 20 30 40 50 60 ( )

0

20

40

60

80

100

0 10 20 30 40 50 60

( )

0

20

40

60

80

100

0 10 20 30 40 50 60 ( )

12 (a)

protocatechuic acid (b) ferulic acid (c) p-coumaric acid (d) vanillic acid

(e) caffeic acid 100°C

( ), 150°C ( ) 200°C ( )

two-site kinetic 100°C ( ), 150°C ( ) 200°C ( )

(c) (d)

(e)

(a) (b)

37

8

F

, k1

k2

two-

site

kinet

ic

F

k 1

k 2

10

0°C

150°

C 20

0°C

10

0°C

150°

C 20

0°C

10

0°C

150°

C 20

0°C

prot

ocat

echu

ic

acid

2.

6010

-1

3.81

10-1

6.05

10-1

2.

41

5.24

10-3

7.

2710

-2

0.

00

5.24

10-3

7.

2710

-2

feru

lic a

cid

- 9.

8410

-1

6.06

10-1

-

1.00

10-2

6.

0110

-2

-

1.00

10-2

6.

0110

-2

cate

chin

-

- -

-

- -

-

- -

p-co

umar

ic

acid

-

3.94

10-1

5.86

10-1

-

1.72

10-2

7.

7610

-2

-

1.72

10-2

7.

7610

-2

vani

llic a

cid

- 3.

7910

-10.

00

-

1.42

10-3

2.

7110

-4

-

1.42

10-3

2.56

10-2

caffe

ic a

cid

- 3.

8410

-15.

9510

-1

-

7.28

10-3

7.

9710

-2

-

7.28

10-3

7.

9710

-2

-

38

9

(S

WE)

6

0

(UAE

) 5

0

30

( 1

00

)*

(ºC)

prot

ocat

echu

ic a

cid

feru

lic a

cid

cate

chin

p-

coum

aric

aci

d va

nillic

aci

d ca

ffeic

aci

d

UAE

5.

23±0

.42b

nd

13.2

8±0.

88a

3.65

±0.2

2a nd

nd

SWE

100

6.66

±7.1

4b nd

nd

nd

nd

nd

15

0 6.

20±0

.50b

5.30

±0.5

5b nd

2.

74±0

.29b

0.28

±0.2

4b 0.

76±0

.03b

20

0 25

.65±

3.88

a 13

.03±

0.57

a nd

4.

19±0

.65a

3.52

±0.9

0a 2.

19±0

.64a

a-c

(p

0

.05)

* ±

(n

= 3

)

nd

39

4.4

50 Folin-Ciocalteu

13

100, 150 200

10

F, k1 k2

10 k1 k2 k1 k2

0

20

40

60

80

100

0 10 20 30 40 50 60

( )

13

100°C ( ), 150°C ( ) 200°C ( )

two-site kinetic 100°C ( ), 150°C ( )

200°C ( )

40

10 F, k1 k2

two-site kinetic

100°C 150°C 200°C

F 4.74 10-1 5.02 10-1 2.60 10-1

k1 2.69 9.68 10-2 2.75

K2 5.70 10-3 7.59 10-3 7.54 10-2

60

50 30

11 100

( )

(Teo ,

2010) 200

(Kw)

(Wahyudiono

, 2008)

200

(p 0.05)

41

11

(SWE) 60

(UAE) 50 30

(ºC)

(mg GAE/g dw)*

UAE 139.75±6.20b

SWE 100 116.70±10.43c

150 125.47±6.59bc

200 185.64±12.91a

a-c

(p 0.05)

* ± (n = 3)

Folin-Ciocalteu HPLC

Burns (2000) Gheldof (2002)

HPLC tannins, phenolic diterpenes ,

phenolic volatile Gu (2004)

dimeric , trimeric oligomeric

proanthocyandins

Folin-Ciocalteu

Folin-Ciocalteu (Stevanato , 2004)

42

4.5

DPPH radical

scavenging

(mg Vitamin C Equivalent Antioxidant Capacity (VCEAC)/g dw) 14

100, 150 200 10, 20, 30, 40, 50

60 50

30 0-10

10

50

200 0 -10 (167.42 mg VCEAC/g dw)

(179.70 mg VCEAC/g dw)

(p > 0.05) 100

150 0-10

129.83 78.97 mg VCEAC/g dw ( 14)

13

0-10

200, 100 150 -

30

200 10

43

b

fg g g g g

a

c

ef de

fg g g

a

d

efg g g g0

50

100

150

200

UAE 0-10 11-20 21-30 31-40 41-50 51-60 ( )

(mg

VCEA

C/g

dw

)

SWE 100ºCSWE 150ºCSWE 200ºC

14 DPPH

(SWE)

(UAE)

50 30

4.6

100, 150 200 -

10, 20, 30, 40, 50 60

50

15

44

R2 = 0.9835

0

50

100

150

200

0 20 40 60 80 100 120 140 160

(mg GAE/g dw)

DPP

H

(mg

VCEA

C/g

dw

)100°C150°C200°CUAE

15

DPPH

DPPH

(R2) 0.9835 (Cai , 2004; Wojdylo ,

2007 Jayaprakasha , 2008)

(Amarowicz , 2004)

4.7 (partition)

(log Poctanol/water)

100,150 200

50

DPPH 12

45

12 (log P)

DPPH

DPPH

(mg VCEAC/g dry extract)

(°C) log P

UAE -1.54±0.07 506.09±21.08a 492.42±11.60a

SWE 100 -1.61±0.13 367.70±35.88b 321.12±36.05b

150 -1.72±0.24 204.30±10.83c 187.47±4.98c

200 -1.72±0.29 130.00±30.35d 131.61±24.03d

a-d

(p 0.05)

* ± (n = 3)

(log P)

0

50

(p > 0.05)

50

DPPH

(p 0.05)

100, 150 200

46

( 4.4)

-

(protein-polyphenol complexes) (Staszewski , 2011)

DPPH

DPPH (p > 0.05)

DPPH

47

5

protocatechuic acid, ferulic acid, p-coumaric acid, vanillic acid, caffeic acid

(volatile compounds)

two-site kinetic

48

. 2535. 2. . .

71-80.

. 2549. (subcritical water)

. Food & Health 14: 51-53.

. 2535. . . . 161

.

. 2551. .

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57

1.

100, 150 200

16

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80

( )

()

100ºC

150ºC

200ºC

16

59

1.

(HPLC)

He (2005)

1.1

1.1.1 Inertsil ODS-3 C18 ( GL Sciences, Japan

4.6×250 )

1.1.2 mobile phase :

0.04% 25 : 75 (v/v) 0.45

30

1.1.3 mobile phase 1.0

1.1.4

280 250

1.2

1.2.1

1.006-201.200, 0.016-162.000, 0.011-109.000

0.043-432.000

1.2.2 syringe filter 0.45

20 HPLC

1.2.3 ( )

60

y = 93.122x - 0.5217, R2 = 0.9999

y = 139.30x + 1.0854, R2 = 0.9998

y = 160.29x + 0.3344, R2 = 0.9999

y = 168.46x + 3.2907, R2 = 0.9998

y = (mAU*min)

x = ( )

1.3


20 HPLC

1.3.2 retention time

retention time 23, 27, 37 48

2.

(HPLC) Prasad (2009)

2.1

2.1.1 Inertsil ODS-3 C18 ( GL Sciences, Japan

4.6×250 )

2.1.2 mobile phase A 2%(v/v)

mobile phase B : (10:15 , v/v) 0.45

30

2.1.3 mobile phase 1.0

2.1.4 protocatechuic acid vanillic acid

250 catechin 280

caffeic acid, p-coumaric acid ferulic acid 320

gradient

61

( ) (mL/min) A B

0 1.00 90% 10%

10 1.00 80% 20%

15 1.00 70% 30%

25 1.00 60% 40%

30 1.00 50% 50%

40 1.00 50% 50%

42 1.00 75% 25%

44 1.00 90% 10%

2.2

2.2.1 protocatechuic acid ,

catechin , vanillic acid , caffeic acid, p-coumaric acid ferulic acid

0.510-61.200 , 0.480-48.000 , 0.052-52.000, 0.052-52.000 , 0.022-22.000 0.021-

20.800


20 HPLC

2.2.3 ( )

protocatechuic acid y = 49.44x + 0.1299, R2 = 0.9997

catechin y = 12.365x - 0.216, R2 = 0.9970

vanillic acid y = 106.82x + 0.0185, R2 = 0.9998

caffeic acid y = 114.6x + 0.117, R2 = 1.0000

p-coumaric acid y = 139.41x - 0.044, R2 = 1.0000

ferulic acid y = 107.78x + 0.1323, R2 = 0.9999

y = (mAU*min)

x = ( )

62

2.3

2.3.1 10

(Rotary evaporator) 5


20 HPLC

2.3.3 retention time ( 17)

protocatechuic acid, catechin, vanillic acid, caffeic acid, p-coumaric

acid ferulic acid retention time 9, 12, 15,16, 20 22

0

200

400

600

250 270 290 310 330 350

Wavelength (nm)

Valu

e

0

10

20

30

40

250 270 290 310 330 350

Wavelength (nm)

Valu

e

0

200

400

600

250 270 290 310 330 350

Wavelength (nm)

Valu

e (m

*AU

)

0

200

400

600

250 270 290 310 330 350

Wavelength (nm)

Valu

e (m

*AU

)

(a) (b)

(c) (d)

63

0

200

400

600

250 270 290 310 330 350

Wavelength (nm)

Valu

e (m

*AU

)

0

200

400

600

800

250 270 290 310 330 350

Wavelength (nm)

Valu

e (m

*AU

)

17 (a) protocatechuic acid, (b) catechin, (c) vanillic

acid, (d) caffeic acid, (e) p-coumaric acid (f) ferulic acid

3. Folin-Ciocalteu

Jayaprakasha (2007)

3.1

3.1.1 0.025±0.001 ( )

3.1.2 25

100

3.1.3 20, 40,

60 80

3.1.4 400

Folin-ciocalteu 10 2.0

3

3.1.5 (Na2CO3) 7.5

1.6

3.1.6 120

3.1.7 765

3.1.8 blank 400 Folin-ciocalteu

10 2.0 Na2CO3 7.5

1.6

(e) (f)

64

3.1.9 (

)

y = 0.0108x + 0.0339

R2 = 0.9991

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 20 40 60 80 100 120

( )

18

3.2

3.2.1

3.2.2 3.1.4 – 3.1.8

3.2.3

(mg gallic acid equivalent; mg

GAE) 1 (dw)

4. DPPH radical

scavenging Brand-Williams (1995)

4.1

4.1.1 0.025±0.001 ( )

3.4.2 25

1.0 stock solution

4.1.3 stock solution 0.1, 0.3, 0.5

0.7

65

4.1.4 50

4.1.5 DPPH 0.1 mM 5

30

4.1.6 517

blank

4.1.7 % Radical scavenging activity

% Radical scavenging activity = 100controlOD

sampleODcontrolOD

control OD = DPPH

sample OD = DPPH

4.1.8 % Radical scavenging activity

( )

y = 118.92x + 1.7285

R2 = 0.9983

0

20

40

60

80

100

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

( )

% R

adic

al s

cave

ngin

g ac

tivity

19

DPPH

66

4.2

4.2.1

4.2.2 3.3.1.4 – 3.3.1.7

4.2.3

vitamin C equivalent antioxidant capacity

(VCEAC) vitamin C equivalents 1 (dw)

68

(ANOVA, Analysis of

Variance) SPSS

LSD 95

( =0.05)

13

Source DF Sum of Squares Mean Square F Value Pr>F

Model 3 546.3748596 182.1249532 31.83 <.0001

Error 8 45.7718293 5.7214787

Corrected Total 11 592.1466889

14


Model 3 1.74957400 0.58319133 1.30 0.3396

Error 8 3.59006067 0.44875758


69

15


Model 3 0.01071300 0.00357100 4.02 0.0512

Error 8 0.00709867 0.00088733


16


Model 3 0.02319092 0.00773031 6.47 0.0157

Error 8 0.00956400 0.00119550



50


Model 3 869.072550 289.690850 17.45 0.0007

Error 8 132.792517 16.599065


70

18 ferulic acid

50


Model 3 341.7433182 113.9144394 731.55 <.0001

Error 8 1.2457287 0.1557161


19 catechin

50


Model 3 396.6669722 132.22232414 760.16 <.0001

Error 8 1.3915287 0.1739411


20 p-coumaric acid

50


Model 3 30.70470025 10.23490008 73.46 <.0001

Error 8 1.11462067 0.13932758


71

21 vanillic acid

50


Model 3 26.58780300 8.86260100 41.08 <.0001

Error 8 1.72587800 0.21573475


22 caffeic acid

50


Model 3 9.55969200 3.18656400 30.67 <.0001

Error 8 0.83108200 0.10388525


23

50


Model 3 8469.535172 2823.178391 31.61 <.0001

Error 8 714.407709 89.300964


72

24 DPPH

-

50 30


Model 18 182362.8854 10131.2714 136.77 <.0001

Error 38 2814.9389 74.0773


25 (log P)

50


Model 3 0.06944395 0.02314798 0.56 0.6574

Error 8 0.33190844 0.04148855


26 DPPH

100

-


Model 1 3253.380204 3253.380204 2.51 0.1880

Error 4 5174.435281 1293.608820


73

27 DPPH

150

-


Model 1 424.9743360 424.9743360 5.98 0.0708

Error 4 284.2691680 71.0672920


28 DPPH

200

-


Model 1 3.859224 3.859224 0.01 0.9462

Error 4 2997.300335 749.325084


74

- ( )

( ) MISS NUCHA SAYPUTIKASIKORN

27 73000

. . 2550

. . 2550

2554 “

” 4

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