1. בירור הרכב החומרים מונעי החמצון בחלקים שונים של
Migal, Kiryat Shmona
בירור הרכב החומרים מונעי החמצון בחלקים שונים של
פרי הרימון ע"י סריקה של 29 זני רימונים שונים
יום עיון למגדלי רימונים, מכון וולקני, פברואר 2008
מוקדש לזכרו של משה זמירי ז"ל

2. Atherosclerosis lesion
development
Thrombus formation
Carotid Intima-Media Thickness
Percent of mortality
In western countries
OTHERS 25%
CHD 54%
CANCER 21%

3. pomegranate juice has been shown to:
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 juice
can lead to reduction of the
atherosclerosis lesion
Prof. Miki Aviram

4. * * Pomegranate Juice Consumption by Healthy
Volunteers Reduces LDL Oxidation A. 30 * * 20
(nmol TBARS/ mg LDL protein)
Copper Ion-Induced LDL Oxidation 10 1 2
Time (weeks)

5. Carotid IMT (mm) A. Control B. Pomegranate Juice Baseline After 1 year
Pomegranate Juice Consumption by Patients with Carotid Artery
Stenosis Reduces Carotid Intima-Media Thickness (IMT) 1 2 3 4
A. Control
B. Pomegranate Juice
Baseline
After 1 year
Carotid IMT (mm)

6. These activities are attributed to the Pomegranate high levels of antioxidant activity and its high total polyphenol content
Total polyphenols content of several fruit juices

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

8. To compare the levels of antioxidants,
Our main objectives:
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.
P.G
P.S. 21
PG128-29
C13

9. 2006 - PG 100-1 PG 101-2 PG 102-3 PG 103-4 PG 104-5 PG 105-6 PG 106-7
A17
C13
C14
C133
ERS
PS21
PS23
XBS
Camel
2006

10. 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

11. Antioxidant activity, total polyphenols, total anthocyanins,
*10^3 (mg/L)
capacity 1 2
Antioxidant activity
Antioxidant activity,
total polyphenols,
total anthocyanins,
and the level of
hydrolysable tannins
in aril juices
Polyphenols
*10^3 (mg/L)
Total
R2 = 0.76**
Total polyphenols 1 2
R2 = 0.46**
Total anthocyanins
anthocyanins
*10^2 (mg/L)
Total 1 2 3
R2 = 0.03
Punicalagin
*10^2 (mg/L)
m/z 1083
0.7
1.4
Punicalin
m/z 781
(mg/L) 2 4 6 8
R2 = 0.00
Gallagic acid
*10 (mg/L)
m/z 601 1 3
R2 = 0.01
Ellagic
acid PG PG PG PG PG
PG 104-5
PG 101-2
PG 105-6
PG 100-1
PG 106-7 PG PG PG PG PG PG
PG 102-3 PG PG
PG 108-9 PG
PG 103-4
Eve
Orange PG PG PG PG PG
** significant by Pirson test, α=0.01

12. organic acids in the aril juices
No positive correlation was found between the acid level and the content of
organic acids in the aril juices
Acid level 1 2
acid level
(%)
R2 = 0.08 2 3 4
pH level
R2 = 0.07
Citric acid level 1 2
Citric acid
level (%)
R2 = 0.05
Obtained by HPLC
Ascorbic acid level
Ascorbic acid
level (mg/L)
R2 = 0.00 4 8 12 16 20
Acetic acid level
0.2
0.4
0.6
0.8
Acetic acid
level (%)
R2 = 0.48** PG
130 - 31
127 28
116 17
109 10
128 29
104 5
101 2
105 6
100 1
106 7
121 22
ERS
Camel
118 19
114 15
112 13
102 3
123 24
120 21
108 9
119 20
103 4 C
133 A PS 23 14
XBS
** significant by Pirson test, α=0.01

13. Positive correlation was found between the TSS and the content of
Glucose and fructose in the aril juices
Antioxidant
capacity
(mM) 2 4 6 8 10
Antioxidant contents
%TSS 14 15 16 17 18
TSS (%)
R2 = 0.68** 13
Glucose level 4 5 6
Glucose
level (%)
R2 = 0.58**
Obtained by HPLC
Fructose level 4 5 6
Fructose
level (%)
R2 = 0.58** PG
130 - 31
127 28
116 17
109 10
128 29
104 5
101 2
105 6
100 1
106 7
121 22
ERS
Camel
118 19
114 15
112 13
102 3
123 24
120 21
108 9
119 20
103 4 C
133 A PS 23 14
XBS
** significant by Pirson test, α=0.01

14. ~ Antioxidant activity, total polyphenols, total anthocyanins,
and the inhibitory effect on
LDL oxidation in juices
prepared by juice extractor
Antioxidant
*10^4 (mg/L)
capacity
Polyphenols
*10^3 (mg/L)
Total
LDL Oxidation
Inhibition
*10 (%) 2 4 6 8
LDL oxidation inhibition
Lipid peroxides, R2 = 0.71**
TBARS, R2 = 0.69**
Total polyphenol
R2 = 0.61**
0.5 1
Antioxidant activity
Total anthocyanin 3
Anthocyanin
*10^2 (mg/L)
Pigment
R2 = 0.00
Gallagic acid
m/z 601
(mg/L)
R2 = 0.64** 4 8 36 40
*10^2 (mg/L)
Punicalin
m/z 781
R2 = 0.58** 2 6
Punicalagin
m/z 1083
R2 = 0.63** 18 20 5 10 15
Ellagic Acid
R2 = 0.68**
m/z 301 ~
The antioxidant was increased
by 5 fold compare to juice
prepared only from arils PG PG PG PG PG PG
PG 106-7
PG 100-1
PG 103-4 PG PG PG
Orange PG PG PG PG PG
PG 102-3
PG 108-9 PG PG
Eve
** significant by Pirson test, α=0.01

15. and the inhibitory effect on LDL oxidation in homogenates
Antioxidant
*10^4 (mg/L)
capacity
Orange
Polyphenols
Total
Anthocyanin
*10^2 (mg/L)
Pigment 1 2 3 4 5 6
Antioxidant activity
Total polyphenol
R2 = 0.90**
0.5
Total Anthocyanin
R2 = 0.05
Antioxidant activity,
total polyphenols,
total anthocyanins,
and the inhibitory effect on
LDL oxidation in homogenates
prepared from the whole fruit
Punicalagin 1 2 3
*10^4 (mg/L)
m/z 1083
Ellagic Acid 4
*10^2 (mg/L)
m/z 301
Punicalin
0.6
1.2
m/z 781
Gallagic acid
*10^3 (mg/L)
m/z 601 6 8
R2 = 0.79**
R2 = 0.44**
R2 = 0.54**
R2 = 0.55**
The antioxidant was increased
by 20 fold compare to juice
prepared only from arils PG PG
PG 104-5 PG PG
PG 102-3 PG PG PG PG
PG 100-1 PG
PG 106-7 PG
PG 101-2
PG 108-9 PG
PG 103-4 PG PG PG PG
PG 105-6 PG PG PG
Eve PG PJ
** significant by Pirson test, α=0.01

16. and the inhibitory effect on LDL oxidation in homogenates
Antioxidant
*10^5 (mg/L)
capacity
Polyphenols
*10^4 (mg/L)
Total
Anthocyanin
*10^2 (mg/L)
Pigment
Total anthocyanin
R2 = 0.01
0.5 1
Antioxidant activity
Total Polyphenol
R2 = 0.91**
1.5 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
Punicalagin
*10^5 (mg/L)
m/z 1083
Ellagic Acid 2 4 6 8
*10^2 (mg/L)
m/z 301
Punicalin 1
*10^4 (mg/L)
m/z 781
Gallagic acid
*10^3 (mg/L)
m/z 601 3
R2 = 0.39**
R2 = 0.72**
R2 = 0.49**
R2 = 0.89**
PG 106-7 PG PG
PG 102-3 PG PG
PG 101-2 PG
PG 100-1 PG PG PG PG PG
PG 108-9 PG
PG 105-6
Orange PG PG PG
PG 104-5
PG 103-4 PG PG PG
Eve PG PG
** significant by Pirson test, α=0.01

17. The antioxidant level is not correlated to the % of the peels from the fruits
Antioxidant contents 1 2 3 4
Antioxidant
*10^2 (mM)
capacity
R2 = 0.01
Fruit weight 2 4 6
*10^2 (gr)
R2 = 0.01
Peel weight 1 2 3
*10^2 (gr)
R2 = 0.06
Aril weight 1 2 3 4
*10^2 (gr) PG
106 - 7
114 15
123 24
102 3 PS 21
121 22
101 2
128 29
100 1
127 28
109 10
116 17
120
119 20
108 9
XBS
105 6 A
ERS
130 31
112 13
104 5
103 4 23 C PG
Eve
118 19 PG

18. Summary
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.

19. 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

20. Students Migal, Kiryat Shmona
Revital Zulker
Ira Glazer
Elinor Shwarzt
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

21. תודה על ההקשבה

22. רמת האנטיאוקסידנטיות במיצים מסחריים
Antioxidant capacity (mM) 10 20 30 40 50 60 70 80 NF D 2 PJ 4 U 71
spring H M
Total Polyphenols (mM)
רמת הפוליפנולים במיצים מסחריים 6 8
NF – נטוראפוד
מיץ אורגני מסחרי
D2 מיץ רימונים נסחט במסחטה
PJ מיץ מיקי מהול לשתייה
M- תרכיז רימונים ממולי
71- מיץ טרי מגרגירים בלבד
H- כנ"ל
יין רימונים- יקב דלתון
מיץ טבעי- מעמוס

24. Multiple Reaction Monitoring (MRM)
The identity of the hydrolysable-tannins obtained by using
Multiple Reaction Monitoring (MRM)
method of LC-MS-MS, according to their mother and daughter ions.
Multiple Reaction Monitoring (MRM)
The Mother ion was fragmented by
argon using different collision energies
to daughter ions. The level of the typical
daughter ion was then measured. Q1
Single Ion m/z Q2
Collision Q3
Single daughter Ion m/z