Migal, Kiryat Shmona

בירור הרכב החומרים מונעי החמצון בחלקים שונים של זני רימונים שונים29פרי הרימון ע"י סריקה של

2008יום עיון למגדלי רימונים, מכון וולקני, פברואר מוקדש לזכרו של משה זמירי ז"ל

Thrombus formation

Carotid Intima-Media Thickness

Atherosclerosis lesiondevelopment

CANCER 21%

OTHERS 25%CHD 54%

Percent of mortalityIn western countries

Recent biological studies have been proven that certain compounds in the pomegranate juice has been shown to:

Reduce blood pressure; Reduce LDL oxidation; Reduce the atherosclerosis formation

Moreover: consumption of pomegranate juicecan lead to reduction of theatherosclerosis lesion

Prof. Miki Aviram

0

10

20

30

A.

**

0 1 2

Time (weeks)

Co

pp

er I

on

-In

du

ced

LD

L O

xid

atio

n

)n

mo

l T

BA

RS

/ m

g L

DL

pro

tein

(

Pomegranate Juice Consumption by Healthy

Volunteers Reduces LDL Oxidation

0

1

2

3

0

2

4

A. Control

B. Pomegranate Juice

Baseline After 1 year

Car

oti

d IM

T (

mm

)Pomegranate Juice Consumption by Patients with Carotid Artery

Stenosis Reduces Carotid Intima-Media Thickness (IMT)

These activities are attributed to the Pomegranate high levels of antioxidant activity and its high total polyphenol content

Po

lyp

hen

ols

(m

M)

0

1

2

3

4

5

6

7

Total Polyphenols content of several Juices

Pomeg

ranat

e

Juic

eRed

Win

e

Cranber

ry

Juic

eBlu

eber

ry

Juic

e

Green

Tea

Orange

Juic

e

Total polyphenols content of several fruit juices

Most of the data derived from one pomegranate cultivar called ‘wonderful’

P.G. 112-13

P.S. 21 PG128-29

C13

Our main objectives:

1. To compare the levels of antioxidants, total polyphenols, anthocyanins and four major hydrolysable tannins in 29 pomegranate cultivars.

2. To verify where the bioactive compounds are localize in the fruits (arils, lamellas, seeds, peels), and examine their chemical nature and their antioxidant activity.

PG 100-1 PG 101-2 PG 102-3 PG 103-4 PG 104-5

PG 105-6 PG 106-7 PG 108-9 PG 109-10 PG 112-13

PG 114-15 --PG 116-17 PG 118-19 PG 119-20

PG 121-22 PG 130-31PG 123-24

A17 C13 C14 C133

Camel

ERS

PS21 PS23 XBS

PG 120-21

PG 128-29PG 127-28

2006

Two different juices and two different homogenates were pretreated from each cultivar:

1. Arils juice

2. Juice prepare by Juice extractor

3. Homogenate prepared from the whole fruit

4. Homogenate prepared from the peels

0

R2 = 0.03

Pu

nic

alag

in

m/z

108

3*

10^2

) m

g/L

(

Punicalagin

0

0.7

1.4

Pu

nic

alin

m

/z 7

81

)m

g/L

(

Punicalin

2

4

6

8

R2 = 0.00

Gal

lag

ic a

cid

m

/z 6

01*

10)

mg

/L(

Gallagic acid

1

2

3

R2 = 0.01

PunicalaginPunicalagin

Ellagic acidEllagic acid

Gallagic acidGallagic acidPG 1

30-3

1

PG 127

-28

PG 116

-17

PG 109

-10

PG 128

-29

PG 104

-5

PG 101

-2

PG 105

-6

PG 100

-1

PG 106

-7

PG 121

-22

PG 201

-212

PG 206

-217

PG 118

-19

PG 114

-15

PG 112

-13

PG 102

-3

PG 123

-24

PG 120

-21

PG 108

-9

PG 119

-20

PG 103

-4

Eve

Orang

e

PG 204

-215

PG 200

-211

PG 205

-216

PG 203

-214

PG 202

-213

R2 = 0.46**

Total anthocyanins

To

tal

an

tho

cyan

ins *

10^2

) m

g/L

(

1

2

3

To

tal

P

oly

ph

eno

ls*

10^3

) m

g/L

(

R2 = 0.76**

Total polyphenols

0

1

2

An

tio

xid

ant

ca

pac

ity

*10

^3)

mg

/L(

1

2 Antioxidant activity

0

Antioxidant activity ,total polyphenols ,total anthocyanins ,

and the level of hydrolysable tannins

in aril juices

** significant by Pirson test, α=0.01

PG

130-3

1

PG

127-2

8

PG

116-1

7

PG

109-1

0

PG

128-2

9

PG

104-5

PG

101-2

PG

105-6

PG

100-1

PG

106-7

PG

121-2

2ERS

Camel

PG

118-1

9

PG

114-1

5

PG

112-1

3

PG

102-3

PG

123-2

4

PG

120-2

1

PG

108-9

PG

119-2

0

PG

103-4

C133 A1

7C1

3PS2

1PS2

3C1

4XBS

Acid level

0

1

2

acid

leve

l

(%) R2 = 0.08

2

3

4

pH level

pH

leve

l R2 = 0.07

Citric acid level

0

1

2

Cit

ric

acid

le

vel

(%)

R2 = 0.05

Ascorbic acid level

Asc

orb

ic a

cid

le

vel (

mg

/L)

R2 = 0.00

04

8

1216

20

Acetic acid level

0

0.2

0.4

0.6

0.8

Ace

tic

acid

le

vel

(%)

R2 = 0.48**

No positive correlation was found between the acid level and the content of organic acids in the aril juices

** significant by Pirson test, α=0.01

Obtained by HPLC

An

tio

xid

ant

ca

pac

ity

) m

M(

2

4

6

8

10Antioxidant contents

0%TSS

1415161718

TS

S (

%)

R2 = 0.68**

13Glucose level

4

5

6

Glu

cose

le

vel (

%)

R2 = 0.58**

Fructose level

4

5

6

Fru

cto

se

leve

l (%

)

R2 = 0.58**

PG

130-3

1

PG

127-2

8

PG

116-1

7

PG

109-1

0

PG

128-2

9

PG

104-5

PG

101-2

PG

105-6

PG

100-1

PG

106-7

PG

121-2

2ERS

Camel

PG

118-1

9

PG

114-1

5

PG

112-1

3

PG

102-3

PG

123-2

4

PG

120-2

1

PG

108-9

PG

119-2

0

PG

103-4

C133 A1

7C1

3PS2

1PS2

3C1

4XBS

** significant by Pirson test, α=0.01

Positive correlation was found between the TSS and the content of Glucose and fructose in the aril juices

Obtained by HPLC

** significant by Pirson test, α=0.01PG 1

20-2

1

PG 121

-22

PG 109

-10

PG 202

-213

PG 127

-28

PG 201

-212

PG 106

-7

PG 100

-1

PG 103

-4

PG 118

-19

PG 204

-215

PG 114

-15

Orang

e

PG 1

12-1

3

PG 128

-29

PG 130

-31

PG 203

-214

PG 123

-24

PG 102

-3

PG 108

-9

PG 200

-211

PG 205

-216

Eve

Gal

lag

ic a

cid

m/z

60

1 )m

g/L

(

Gallagic acidR2 = 0.64**

0

4

8

3640

Pu

nic

alin

m

/z 7

81

*

10

^2)

mg

/L(

PunicalinR2 = 0.58**

0

2

4

6

Pu

nic

ala

gin

m

/z 1

08

3*

10

^2)

mg

/L(

PunicalaginR2 = 0.63**6

18

024

20

0

5

10

15 Ellagic AcidR2 = 0.68**

Ella

gic

Ac

id

m/z

30

1

)m

g/L

(

~~

~~

Antioxidant activity ,total polyphenols ,total anthocyanins ,

and the inhibitory effect on LDL oxidation in juices

prepared by juice extractor

An

tio

xid

an

t

ca

pa

cit

y*

10

^4)

mg

/L(

To

tal

P

oly

ph

eno

ls * 1

0^3

) m

g/L

(

LD

L O

xid

ati

on

In

hib

itio

n

*1

0(%

)

0

2

4

6

8 LDL oxidation inhibitionLipid peroxides, R2 = 0.71** TBARS, R2 = 0.69**

Total polyphenolR2 = 0.61**

0

2

4

60

0.5

1 Antioxidant activity

Total anthocyanin

0

1

2

3

4

An

tho

cya

nin

Pig

me

nt

*1

0^2

) m

g/L

(

R2 = 0.00

The antioxidant was increased by 5 fold compare to juice prepared only from arils

PG 203

-214

PG 120

-21

PG 104

-5

PG 119

-20

PG 114

-15

PG 1

02-3

PG 123

-24

PG 201

-212

PG 202

-213

PG 200

-211

PG 100

-1

PG 121

-22

PG 106

-7

PG 109

-10

PG 1

01-2

PG 108

-9

PG 127

-28

PG 1

03-4

PG 116

-17

PG 128

-29

PG 204

-215

PG 112

-13

PG 1

05-6

PG 130

-31

PG 205

-216

PG 206

-217Eve

PG 118

-19

PJ

Punicalagin

0

1

2

3

Pu

nic

alag

in

m/z

108

3*

10^4

) m

g/L

(

Ellagic Acid

0

1

2

3

4

Ella

gic

Aci

d

m/z

301

*

10^2

) m

g/L

(

Punicalin

0

0.6

1.2

Pu

nic

alin

m

/z 7

81

*10

^4)

mg

/L(

Gal

lag

ic a

cid

m

/z 6

01*

10^3

) m

g/L

(

Gallagic acid

0

2

4

6

8

R2 = 0.79**

R2 = 0.44**

R2 = 0.54**

R2 = 0.55**

** significant by Pirson test, α=0.01

An

tio

xid

ant

ca

pac

ity

*10

^4)

mg

/L(

Orang

e

To

tal

P

oly

ph

eno

ls*

10^4

) m

g/L

(

An

tho

cyan

in

Pig

men

t

*10

^2)

mg

/L (

0

1

2

3

4

5

6

Antioxidant activity

Total polyphenolR2 = 0.90**

0

1

0.5

Total Anthocyanin

R2 = 0.05

0

1

2

3

4

Antioxidant activity ,total polyphenols ,total anthocyanins ,

and the inhibitory effect on LDL oxidation in homogenates

prepared from the whole fruit

The antioxidant was increased by 20 fold compare to juice prepared only from arils

PG 106

-7

PG 114

-15

PG 123

-24

PG 102

-3

PG 2

00-2

11

PG 121

-22

PG 101

-2

PG 128

-29

PG 100

-1

PG 127

-28

PG 109

-10

PG 116

-17

PG 120

-21

PG 119

-20

PG 1

08-9

PG 2

02-2

13

PG 105

-6

Ora

nge

PG 2

01-2

12

PG 130

-31

PG 112

-13

PG 104

-5

PG 103

-4

PG 2

05-2

16

PG 204

-215

PG 203

-214 Eve

PG 118

-19

PG 206

-217

0

Pu

nic

alag

in

m/z

108

3*

10^5

) m

g/L

(

Ellagic Acid

0

2

4

6

8

Ella

gic

Aci

d

m/z

301

*

10^2

) m

g/L

(

Punicalin

0

1

Pu

nic

alin

m

/z 7

81

*10

^4)

mg

/L(

Gallagic acid

2

4

6

8

Gal

lag

ic a

cid

m

/z 6

01*

10^3

) m

g/L

(

Punicalagin

0

1

2

3

4

R2 = 0.39**

R2 = 0.72**

R2 = 0.49**

R2 = 0.89**

An

tio

xid

ant

ca

pac

ity

*10

^5)

mg

/L(

To

tal

P

oly

ph

eno

ls

*10

^4)

mg

/L (

An

tho

cyan

in

Pig

men

t

*10

^2)

mg

/L(

Total anthocyaninR2 = 0.01

0

0.5

1 Antioxidant activity

0

Total PolyphenolR2 = 0.91**

0.5

1

1.5

0

2

4

6

8

Antioxidant activity ,total polyphenols ,total anthocyanins ,

and the inhibitory effect on LDL oxidation in homogenates

prepared from the peels

The antioxidant was increased by 40 fold compare to juice prepared only from arils

** significant by Pirson test, α=0.01

Antioxidant contents

0

1

2

3

4

An

tio

xid

ant

ca

pac

ity

*10

^2)

mM

(

PG

106-7

PG

114-1

5

PG

123-24

PG

102-3PS2

1

PG

121-22

PG

101-2

PG

128-2

9

PG

100-1

PG

127-28

PG

109-10

PG

116-17

PG

120-2

1

PG

119-20

PG

108-9

XBS

PG

105-6

A17

ERS

PG

130-31

PG

112-13

PG

104-5

PG

103-4PS2

3C1

3C

PG 203-

214

CEve

PG

118-19

PG 206

-207

R2 = 0.01

Fruit weight

0

2

4

6

Fru

it w

eig

ht

*

10^2

) g

r(

R2 = 0.01

Peel weight

0

1

2

3

Pee

l wei

gh

t

*10

^2)

gr

(

R2 = 0.06

Aril weight

0

1

2

3

4

Ari

l wei

gh

t

*10

^2)

gr

(The antioxidant level is not correlated to the % of the peels from the fruits

Summary

1. In arils juice- the antioxidant activity appear to be related to polyphenols and anthocyanins. This is accordance with other data demonstrated that in colored figs, apple, peaches and grapes the antioxidant level is significantly higher then in green fruits.

2. In homogenate prepared from the whole fruit, the level of the antioxidant increased by 20-fold suggested that the peels contributed significantly to antioxidant activity.

3. The predominant type of polyphenolic compounds extracted from the peels are the hydrosalbe tannins, composed mainly from punicalagin isomers, punicalin, gallagic acid and ellagic acid.

The study enlarge our knowledge about the natural variation of the antioxidant content and the compounds that give this activity.

It also identify the location and concentration of some phytochemical that have antioxidant activity in pomegranate fruits.

In the future, such information will enable breeders to select and breed genotypes having higher levels of health beneficial compounds and also provide useful information for addressing consumer choices for healthier products

Collaborators:

Dr. Doron Holland, Newe Ya’ar Research Center

Prof. Miki Aviram, Technion, Faculty of Medicine, Haifa

Dr. Hamutal Borochov, Mop Arava, Eilat

Dr. Igal Bar-Ilan, Migal, Kiryat Shmona

Prof. Ruth Ben-Arie, The fruit storage laboratory, Kiryat Shmona

Revital Zulker

Ira Glazer

Elinor Shwarzt

Students Migal, Kiryat Shmona

תודה על ההקשבה

רמת האנטיאוקסידנטיות במיצים מסחריים

Ant

ioxi

dant

cap

acit

y (m

M)

01020304050607080

NF D2 PJ 4U 71

sprin

g DHM

Tot

al P

olyp

heno

ls (

mM

)

רמת הפוליפנולים במיצים מסחריים

0

2

4

6

8

10

D2 NF 71 PJ 4U

sprin

g DM H

NFנטוראפוד – מיץ אורגני מסחרי

D2מיץ רימונים נסחט במסחטה PJמיץ מיקי מהול לשתייה Mתרכיז רימונים ממולי -

- מיץ טרי מגרגירים בלבד 71Hכנ"ל -

יין רימונים- יקב דלתוןמיץ טבעי- מעמוס

Q1Q1

Single Single Ion m/zIon m/z

Q2Q2

CollisionCollision

Q3Q3

Single daughter Single daughter Ion m/zIon m/z

Multiple Reaction Multiple Reaction Monitoring (MRM) Monitoring (MRM)

The identity of the hydrolysable-tannins obtained by using Multiple Reaction Monitoring (MRM)Multiple Reaction Monitoring (MRM)method of LC-MS-MS, according to their method of LC-MS-MS, according to their mother and daughter ions. mother and daughter ions.

The Mother ion was fragmented by argon using different collision energies to daughter ions. The level of the typicaldaughter ion was then measured.