Insecticidal and Antibacterial Activity of Citrus Fruits' Peels and Juices

148 Pages·2005·0.93 MB·English

Insecticidal and Antibacterial Activity of Citrus
Fruits’ Peels and Juices

By:
Hadia Mohammed Abdelatif
(M.Sc.)
Zoology

A thesis submitted to The Department of Zoology, Faculty of Science University of
Khartoum, in fulfillment of the requirements for the Degree of Doctor of Philosophy
(PhD).

Under the supervision of:
Professor Salah Ahmed Mohammed Ahmed

Co-supervisor:
Dr. Fathi M.A. El Rabaa

2004

1
Dedication

To the spirits of my late father and my late mother,

To all my family with love

2
Acknowledgements

I would like to express my deepest gratitude to my supervisor,
Professor Salah Ahmed, and co-supervisor, Dr. Fathi M.A. El Rabaa,
associate professor, for their guidance, patience and encouragement. I
am gratefully thankful to Dr. Idris Babikir, associate professor, and Dr.
Sania A. Shadad, Head Department of Pharmacology, for their
continuous encouragement.
I like to express my deepest gratitude to The Department of
Botany, Faculty of Science, University Of Khartoum, for allowing me to
do part of my lab work in their laboratories.
My thanks to The Department of Microbiology Of The National
Centre For Research, for providing me with the studied microorganisms.
I am also thankful to The Petroleum Laboratories Centre for helping me
in the chemical analysis.
I am strongly grateful for those who helped me in collection of
mosquito larvae. My due thanks to The University of Kordofan for the
financial support and for full release to complete this study.

3
Abstract

The annual world products of citrus fruits were estimated to be
98.4% million metric tons (FAO, 1997) and approximately 34% of the
fruits are processed into juice. As the juice yield is about half of the fruit
weight, processing of citrus fruits into juice results in large amounts of
byproducts (Bovill 1996). These byproducts which are mainly composed
of peels - seeds and macerated pulps, contain high amounts of secondary
natural bioactive compounds. These compounds attracted attention of
researchers for their potential health promoting properties. So this study
was done to investigate new natural and safe insecticidal and
antibacterial agents from the lime juice and peels of four types of citrus
fruits.
In this study, many experiments were carried out. Preliminary
phytochemical investigations for the studied citrus peels revealed that,
the non-volatile components of these peels are sterol, triterpenoids,
coumarins, and flavonoids.
Extraction by steam distillation and then chemical analysis by gas
chromatography coupled with mass spectrometry (GC/MS) for peel oils
of different citrus fruits, grapefruit (Citrus paradisi), sweet orange
(C.sinensis) and lime (C.aurantifolia) revealed that limonene, a terpene
compound, constitutes the bulk of the three oils (97,15%, 92.46% and
32.29% for orange, grapefruit and lime respectively). These oils were
then used against immature and adult stages of mosquito, Culex
quinquefasciatus. The results showed significant larvicidal activity for
all these oils specially the orange 0il, which showed the lowest
concentration for killing 50% (LC50) of the population under study (49
ppm)). These oils are not affecting only the larval stage but also the other
developmental stages (pupation and emergence of progeny).
4
The antibacterial activity of mandarin (Citrus reticulata) peel
extracts was studied. Firstly, the dried powdered mandarin peels were
successively extracted with hexane, chloroform and acetone using the
cold method (two days for each solvent). The yields from 1 kilogram of
dried peels were 5.0 g, 1.5 g, and 3.6 g respectively. The three extracts
were tested against gram positive bacteria Bacillus subtilis and
S6taphylococcus aureus, and gram negative bacteria Escherichia coli
and Pseudomonas aeruginosa using the disc diffusion method. Hexane
and chloroform extracts, that found to be the most active extracts, were
fractionated into alcohol-soluble and alcohol- insoluble fractions. All
fractions were tested against all bacterial strains using the disc diffusion
methods and broth dilution technique. The diameters of inhibition zone
were measured in millimeters (mm) and the result was tabulated as
susceptible, intermediate and resistant. The result revealed that alcohol-
soluble fraction was the most active fraction, with minimum inhibitory
concentration (MIC), (360µg/ml, 600µg/ml, 1440µg/ml and 720µg/ml
against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and
Pseudomonas aeuriginosa respectively) lower than that of all other
fractions.
A simple method was done to increase the concentration of the
active compounds in the extracts. The peel of mandarin was inoculated
with the Staphylococcus aureus before removing it from the fruit and
then incubated for 24 hours, removed, dried and powdered the peels.
The powdered peel then extracted using the same solvents. The
antibacterial activity test showed that these inoculated peels gave lower
MIC than the not inoculated (200µg/ml and 360µg/ml for inoculated
peels and non-inoculated peels respectively). This proved that
inoculation of the citrus peels with a microorganism increases the
5
concentration of the active compound against that particular
microorganism.
The active compounds that were previously identified by
Jayaprakasha et al. (2000) as polymethoxylated flavones (PMF) were
isolated from alcohol-soluble fraction using Thin Layer chromatography
(TLC), and identified using high performance liquid chromatography
(HPLC).
These isolated polymethoxylated flavones were found to be:
1 Tangeretin (penta-methoxyflavone)
2 Nobiletin (hexa-methoxyflavone)
The antibacterial activity of the lime juice against gram- positive
bacteria, Bacillus subtilis and Staphylococcus aureus and gram-negative
bacteria, Escherichia coli and Pseudomonas aeruginosa was studied,
using the same procedure that was repeated in case of mandarin peel
extracts. The result revealed that lime juice, natural and concentrated,
showed high antibacterial activity against both gram-positive and gram-
negative bacteria at all concentrations used (natural, 2-times
concentration, 4-times concentration and 8-times concentration).
When comparing the activity of lime juice (1 ml of lime juice
containing 58 mg citric acid) and equivalent concentration of citric acid,
they matched in their inhibitory activity, in vitro, in all concentrations
used. Thus the active compound in the lime juice was supposed to be
citric acid.
When comparing streptomycin with lime juice, it was found that
antibacterial activity attributed to 100mg/ml of streptomycin is equal to
that produced by four times the concentration of lime juice.

6
ﺔﺣﻭﺮﻃﻷﺍ ﺺﺨﻠﻣ

98.4
ﺔﻴﺋﺎﺼﺣﺇ ﻰﻠﻋ ﺍﺩﺎﻨﺘﺳﺍ ﻦﻃ ﻥﻮﻴﻠﻣ ﺏ ﺕﺎﻴﻀﻤﺤﻠﻟ ﻱﻮﻨﺴﻟﺍ ﺝﺎﺘﻧﻹﺍ ﺭﺪﻘﻳ
1996 1997
ﻥﺃ ﻡ ﻡﺎﻋ ﻞﻴﻓﻮﺑ ﺢﺿﻭﺃ ﻡ ﻡﺎﻌﻟﺍ ﰲ (ﻭﺎﻔﻟﺍ) ﻪﻋﺍﺭﺰﻟﺃ ﻭ ﺔﻳﺬﻏﻷﺍ ﺔﻤﻈﻨﻣ ﺎﺮﺟﺃ
ﻪﻴﺒﻧﺎﳉﺃ ﺞﺗﺍﻮﻨﻟﺍ ﻦﻣ ﺓﲑﺒﻛ ﺕﺎﻴﻤﻛ ﺎﻬﻨﻋ ﺞﺘﻨﻳ ﻪﻬﻛﺍﻮﻔﻟﺃ ﻩﺬﻫ ﻦﻣ ﲑﺼﻌﻟﺍ ﺝﺎﺘﻧﺇ ﺔﻴﻠﻤﻋ
ﱄﺎﳋﺍ ﺭﺎﻤﺜﻟﺍ ﺐﻟ ﻭ ﺭﻭﺬﺒﻟﺍ ،ﺭﻮﺸﻘﻟﺍ ﻦﻣ ﻥﻮﻜﺘﺗ ﱵﻟﺍﻭ ﻪﻴﺒﻧﺎﳉﺃ ﺞﺗﺍﻮﻨﻟﺍ ﻩﺬﻫ ﻯﻮﺘﲢ (ﺔﻳﻮﻧﺎﺜﻟﺍ)
ﺖﺑﺬﺟ ﱵﻟﺍ ﻭ ﺔﻳﻮﻴﳊﺍ ﻪﻴﻠﻋﺎﻔﻟﺃ ﺕﺍﺫ ﺔﻴﻌﻴﺒﻄﻟﺍ ﺩﺍﻮﳌﺍ ﻦﻣ ﺓﲑﺒﻛ ﺕﺎﻴﻤﻛ ﻰﻠﻋ ،ﲑﺼﻌﻟﺍ ﻦﻣ
ﻑﺎﺸﺘﻛﻻ ﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﺖﲤ ﺪﻘﻓ ﻪﻴﻠﻋ ﻭ ﺔﻴﺤﺻ ﺺﺋﺎﺼﺧ ﻦﻣ ﻪﺑ ﺰﻴﻤﺘﺗ ﺎﳌ ﲔﺜﺣﺎﺒﻟﺍ ﻩﺎﺒﺘﻧﺍ
ﺔﻳﻮﻴﺣ ﺕﺍﺩﺎﻀﻣ ﻭ ﺔﻳﺮﺸﺣ ﺕﺍﺪﻴﺒﻤﻛ ﺹﺍﻮﺧ ﺎﳍ ﻪﻴﺒﻧﺎﳉﺃ ﺞﺗﺍﻮﻨﻟﺍ ﻩﺬﻫ ﻦﻣ ﺔﻴﻌﻴﺒﻃ ﺩﺍﻮﻣ
.
ﻪﻨﻣﺁ
ﻦﻋ ﱃﻭﻷﺍ ﻒﺸﻜﻟﺍ ﺮﻬﻇﺃ ﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﰲ ﺎﻫﺅﺍﺮﺟﺇ ﰎ ﺪﻗ ﺏﺭﺎﺠﺘﻟﺍ ﻦﻣ ﺪﻳﺪﻌﻟﺍ
ﺭﻮﺸﻗ -ﻥﻮﻤﻴﻠﻟ ﺍﺭﻮﺸﻗ ،ﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﰲ ﻪﻣﺪﺨﺘﺴﳌﺃ ﻪﻴﺗﺎﺒﻨﻟﺃ ﺩﺍﻮﻤﻠﻟ ﻪﻴﺋﺎﻴﻤﻴﻜﻟﺃ ﺕﺎﻧﻮﻜﳌﺍ
-ﻲﺛﻼﺜﻟ ﺍ ﲔﺑﲑﺘﻟﺍ -ﻝﻭﲑﺘﺳﻹﺍ ﻥﺃ ﻲﻔﺳﻮﻴﻟﺍ ﺭﻮﺸﻗ ﻭ ﺕﻭﺮﻓ ﺐﻳﺮﻘﻟﺍ ﺭﻮﺸﻗ -ﻝﺎﻘﺗﱪﻟﺍ
.
ﺭﻮﺸﻘﻟﺍ ﻩﺬﳍ ﺕﺎﻧﻮﻜﳌﺍ ﻢﻫﺃ ﻲﻫ ﺪﻳﻮﻧﻮﻓﻼﻔﻟﺍ ﻭ ﻦﻳﺭﺎﻤﻜﻟﺍ
7
ﺐﻳﺮﻘﻟﺍ ﺭﻮﺸﻗ ﻭ ﻝﺎﻘﺗﱪﻟﺍ ﺭﻮﺸﻗ -ﻥﻮﻤﻴﻠﻟﺍ ﺭﻮﺸﻗ ﻦﻣ ﻩﺭﺎﻴﻄﻟﺃ ﺕﻮﻳﺰﻟﺍ ﺹﻼﺨﺘﺳﺍ ﰎ
GC/MS
ﺪﻘﻟ ﻭ ﺔﻄﺳﺍﻮﺑ ﺎﻴﺋﺎﻴﻤﻴﻛ ﺎﻬﻠﻴﻠﲢ ﰎ ﰒ ﻦﻣ ﻭ ﻱﺭﺎﺨﺒﻟﺍ ﲑﻄﻘﺘﻟﺍ ﺔﻄﺳﺍﻮﺑ ﺕﻭﺮﻓ

.ﺕﻮﻳﺰﻟﺍ ﻦﻳﻮﻜﺗ ﰲ ﻪﺒﺴﻧ ﻰﻠﻋﺃ ﻞﺜﳝ ﲔﻴﻧﻮﻤﻴﻠﻟﺍ ﺐﻛﺮﻣ ﻥﺃ ﺞﺋﺎﺘﻨﻟﺍ ﺖﺤﺿﻭﺃ

ﺲﻜﻟﻮﻴﻜﻟﺍ ﺔﺿﻮﻌﺑ ﺕﺎﻗﺮﻳ ﺪﺿ ﺔﻳﺮﺸﺣ ﺕﺍﺪﻴﺒﻤﻛ ﺔﺛﻼﺜﻟﺍ ﺕﻮﻳﺰﻟﺍ ﻩﺬﻫ ﺕﱪﺘﺧﺍ
ﻩﺩﺎﺑﻹﺍ ﺔﻴﺻﺎﺧ ﺎﳍ ﺕﻮﻳﺰﻟﺍ ﻩﺬﻫ ﻊﻴﲨ ﻥﺃ ﻰﻠﻋ ﺞﺋﺎﺘﻨﻟﺍ ﺖﻟﺩ ﺪﻘﻟ ﻭ ﺺﺗﺎﻴﺳﺎﻔﻳﻮﻜﻨﻳﻮﻛ
ﻞﻗﺃ ﻰﻄﻋﺃ ﺚﻴﺣ ﻪﻴﻠﻋﺎﻓ ﻰﻠﻋﺃ ﺮﻬﻇﺃ ﻱﺬﻟﺍ ﻝﺎﻘﺗﱪﻟﺍ ﺖﻳﺯ ﺔﺻﺎﺧ ﻩﺮﺸﳊﺃ ﻩﺬﻫ ﺪﺿ ﺔﻳﺮﺸﳊﺍ
. 50 %
ﺭﺎﺒﺘﺧﻻﺍ ﺖﲢ ﺽﻮﻌﺒﻟﺍ ﺕﺎﻗﺮﻳ ﻉﻮﻤﳎ ﻦﻣ ﻞﺘﻘﻟ ﻰﻔﻜﻳ ﺰﻴﻛﺮﺗ
ﻂﻘﻓ ﺮﺼﺤﻨﻳ ﻻ ﺲﻜﻟﻮﻴﻜﻟﺍ ﺔﺿﻮﻌﺑ ﻰﻠﻋ ﺕﻮﻳﺰﻟﺍ ﻩﺬﻫ ﲑﺛﺄﺗ ﻥﺃ ﺎﻀﻳﺃ ﺞﺋﺎﺘﻨﻟﺍ ﺖﺤﺿﻭﺃ ﺎﻤﻛ
.
ﺔﺿﻮﻌﺒﻠﻟ ﻪﻴﺗﺎﻴﳊﺃ ﺭﺍﻮﻃﻷﺍ ﺔﻴﻘﺑ ﻞﻛ ﱃﺇ ﺎﻀﻳﺃ ﺪﺘﳝ ﻞﺑ ﺕﺎﻗﲑﻟﺍ ﻰﻠﻋ
،ﱄﻭﺮﺘﺒﻟﺍ ﺮﺘﻳﻷﺍ ،ﻪﻳﻮﻀﻌﻟﺃ ﻞﻴﻟﺎﶈﺍ ﻦﻣ ﺩﺪﻋ ﺔﻄﺳﺍﻮﺑ ﻲﻔﺳﻮﻴﻟﺍ ﺭﻮﺸﻗ ﺹﻼﺨﺘﺳﺍ ﰎ
ﻭ ﻰﻠﻴﺛﻷﺍ ﻝﻮﺤﻜﻟﺍ ﰲ ﻥﺎﺑﻭﺬﻠﻟ ﻞﺑﺎﻘﻟﺍ ﺺﻠﺨﺘﺴﳌﺍ ﺺﻠﺨﺘﺳﺍ ﰒ ﻥﻮﺘﺳﻷﺍ ﻭ ﻡﺭﻮﻓﻭﺭﻮﻠﻜﻟﺍ
.
ﻡﺭﻮﻓﻭﺭﻮﻠﻜﻟﺍ ﻭ ﱄﻮﺤﻜﻟﺍ ﺮﺜﻳﻹﺍ ﺕﺎﺼﻠﺨﺘﺴﻣ ﻦﻣ ﻪﻴﻓ ﻥﺎﺑﻭﺬﻠﻟ ﻞﺑﺎﻘﻟﺍ ﲑﻏ ﺺﻠﺨﺘﺴﳌﺍ
ﻭ ﻡﺍﺮﳉﺍ ﺔﺒﺟﻮﻣ ﺎﻳﲑﺘﻜﺒﻟﺍ ﻦﻣ ﺩﺪﻋ ﺪﺿ ﺔﻳﻮﻴﺣ ﺕﺍﺩﺎﻀﻤﻛ ﺕﺎﺼﻠﺨﺘﺴﳌﺍ ﻩﺬﻫ ﺕﱪﺘﺧﺍ
ﺮﺜﻛﺃ ﻮﻫ ﻝﻮﺤﻜﻟﺍ ﰲ ﻥﺎﺑﻭﺬﻠﻟ ﻞﺑﺎﻘﻟﺍ ﺺﻠﺨﺘﺴﳌﺍ ﻥﺃ ﺞﺋﺎﺘﻨﻟﺍ ﺖﺤﺿﻭﺃ ،ﻡﺍﺮﳉﺍ ﺔﺒﻟﺎﺳ
.
ﺎﻳﺮﺘﻜﺒﻟﺍ ﻂﻴﺒﺜﺘﻟ ﻰﻔﻜﻳ ﺰﻴﻛﺮﺗ ﻞﻗﺃ ﻰﻄﻋﺃ ﺚﻴﺣ ﻪﻴﻠﻋﺎﻓ ﺕﺎﺼﻠﺨﺘﺴﳌﺍ
8
ﰲ ﻥﺎﺑﻭﺬﻠﻟ ﻞﺑﺎﻘﻟﺍ ﺺﻠﺨﺘﺴﳌﺍ ﰲ ﻪﻟﺎﻌﻔﻟﺃ ﺓﺩﺎﳌﺍ ﺰﻴﻛﺮﺗ ﺓﺩﺎﻳﺰﻟ ﻪﻄﺴﺒﻣ ﻪﺑﺮﲡ ﺀﺍﺮﺟﺇ ﰎ
ﺲﻛﻮﻛﻮﻠﻴﻓﺎﺘﺳﻹﺍ ﺎﻳﲑﺘﻜﺒﺑ (ﺓﺮﻤﺜﻟﺍ ﻦﻋ ﺎﻬﻠﺼﻓ ﻞﺒﻗ) ﻲﻔﺳﻮﻴﻟﺍ ﺓﺮﺸﻗ ﻦﻘﲝ ﻚﻟﺫ ﻭ ﻝﻮﺤﻜﻟﺍ
.
ﺺﻠﺨﺘﺴﳌﺍ ﻥﺃ ﺞﺋﺎﺘﻨﻟﺍ ﺕﺮﻬﻇﺃ ﺎﻔﻧﺁ ﺎﻫﺮﻛﺫ ﻖﺒﺳ ﺎﻤﻛ ﺹﻼﺨﺘﺳﻻﺍ ﺔﻴﻠﻤﻋ ﺀﺍﺮﺟﺇ ﰒ ﻦﻣ ﻭ
ﻩﲑﻈﻧ ﻦﻣ ﻪﻴﻠﻋﺎﻓ ﺮﺜﻛﺃ ﺎﻳﲑﺘﻜﺒﻟﺎﺑ ﺔﻧﻮﻘﶈﺍ ﺭﻮﺸﻘﻟﺍ ﻦﻣ ﻝﻮﺤﻜﻟﺍ ﰲ ﻥﺎﺑﻭﺬﻠﻟ ﻞﺑﺎﻘﻟﺍ
.
ﺎﻳﲑﺘﻜﺒﻟﺎﺑ ﺔﻧﻮﻘﶈﺍ ﲑﻏ ﺭﻮﺸﻘﻟﺍ ﻦﻣ ﺺﻠﺨﺘﺴﳌﺍ
ﻭ ﺵﺎﻛﺍﺮﺑﺎﻴﺟ ﻞﺒﻗ ﻦﻣ ﺎﻘﺑﺎﺳ ﺎﻬﻴﻠﻋ ﻑﺮﻌﺘﻟﺍ ﰎ ﱵﻟﺍ ﻭ ﻞﻳﺎﺜﻴﳌﺍ ﺩﺪﻌﺘﻣ ﻥﻮﻓﻼﻔﻟﺍ ،ﻪﻟﺎﻌﻔﻟﺃ ﺩﺍﻮﳌﺍ
HPLC NMR 2000
ﺔﻄﺳﺍﻮﺑ ﺔﺳﺍﺭﺪﻟﺍ ﻩﺬﻫ ﰲ ﺎﻬﻴﻠﻋ ﻑﺮﻌﺘﻟﺍ ﰎ ﺔﻄﺳﺍﻮﺑ ﻡﺎﻋ ﻥﻭﺮﺧﺁ
1
ﲔﺗﺮﳒﺎﺘﻟﺍ - :ﻰﻫﻭ
2
ﲔﺘﻠﻴﺑﻮﻨﻟﺍ -
ﺖﻌﺒﺗﺍ ﱵﻟﺍ ﻪﻘﻳﺮﻄﻟﺃ ﺕﺍﺫ ﲔﻌﺒﺘﻣ ﻥﻮﻤﻴﻠﻟﺍ ﲑﺼﻌﻟ ﺎﻳﲑﺘﻜﺒﻠﻟ ﺓﺩﺎﻀﳌﺍ ﻪﻴﻠﻋﺎﻔﻟﺃ ﺭﺎﺒﺘﺧﺍ ﰎ
.
ﺖﻣﺪﺨﺘﺳﺍ ﱵﻟﺍ ﺰﻴﻛﺮﺘﻟﺍ ﻞﻛ ﻥﺃ ﺞﺋﺎﺘﻨﻟﺍ ﺖﺤﺿﻭﺃ ﻲﻔﺳﻮﻴﻟﺍ ﺭﻮﺸﻗ ﺕﺎﺼﻠﺨﺘﺴﻣ ﺔﻟﺎﺣ ﰲ
ﺔﺒﻟﺎﺳ ﺎﻳﲑﺘﻜﺒﻟﺍ ﻭ ﻡﺍﺮﳉﺍ ﺔﺒﺟﻮﻣ ﺎﻳﺮﺘﻜﺒﻟﺍ ﻦﻣ ﻞﻛ ﺪﺿ ﺔﻴﻟﺎﻋ ﺔﻴﻟﺎﻌﻓ ﺕﺍﺫ ﻥﻮﻤﻴﻠﻟﺍ ﲑﺼﻋ ﻦﻣ
.
ﻡﺍﺮﳉﺍ
58
ﺾﲪ ﻦﻣ ﺎﻣﺍﺮﻐﻴﻠﻣ ﻰﻠﻋ ﻯﻮﺘﳛ ﺝﺯﺎﻄﻟﺍ ﻥﻮﻤﻴﻠﻟﺍ ﲑﺼﻋ ﻦﻣ ﺮﻠﺘﻠﻴﻠﳌﺍ ﻥﺃ ﺎﲟ
ﲑﺼﻋ ﻦﻣ ﺓﺪﻳﺪﻋ ﺰﻴﻛﺮﺘﻟ ﺎﻳﺮﺘﻜﺒﻠﻟ ﺓﺩﺎﻀﳌﺍ ﻪﻴﻠﻋﺎﻔﻟﺃ ﲔﺑ ﻪﻧﺭﺎﻘﳌﺃ ﺕﺮﺟ ﺪﻘﻓ ﻚﻳﺮﺘﺴﻟﺍ
ﲑﺼﻌﻟ ﺎﻳﲑﺘﻜﺒﻠﻟ ﺓﺩﺎﻀﳌﺍ ﻪﻴﻠﻋﺎﻔﻟﺃ ﻥﺃ ﲔﺒﺗ ﺪﻘﻟ ﻭ ﻚﻳﺮﺘﺴﻟﺍ ﺾﻣﺎﺣ ﻦﻣ ﺎﻬﻠﺑﺎﻘﻳ ﺎﻣ ﻭ ﻥﻮﻤﻴﻠﻟﺍ
9
.
ﺰﻴﻛﺮﺘﻟﺍ ﺕﺍﺫ ﺪﻨﻋ ﻚﻳﺮﺘﺴﻟﺍ ﺾﲪ ﺎﻬﺛﺪﳛ ﱵﻟﺍ ﻚﻠﺗ ﺎﻣﺎﲤ ﻯﻭﺎﺴﺗ ﺰﻴﻛﺮﺗ ﻱﺃ ﺪﻨﻋ ﻥﻮﻤﻴﻠﻟﺍ
.
ﻚﻳﺮﺘﺴﻟﺍ ﺾﲪ ﻲﻫ ﻥﻮﻤﻴﻠﻟﺍ ﲑﺼﻋ ﰲ ﺎﻳﺮﺘﻜﺒﻠﻟ ﺓﺩﺎﻀﳌﺍ ﺓﺩﺎﳌﺍ ﻥﺃ ﺖﺒﺛ ﺪﻘﻓ ﻪﻴﻠﻋ ﻭ
ﺕﺮﻬﻇﺃ ﻥﻮﻤﻴﻠﻟﺍ ﲑﺼﻋ ﻊﻣ ﲔﺴﻳﺎﻣﻮﺘﺑﺮﺘﺳﺇ ﻱﻮﻴﳊﺍ ﺩﺎﻀﳌﺍ ﺔﻴﻠﻋﺎﻓ ﺔﻧﺭﺎﻘﻣ ﺪﻨﻋ
100
ﻦﻋ ﺞﺗﺎﻨﻟﺍ ﻁﺎﺒﺛﻹﺍ ﺮﻄﻗ ﺕﺍﺫ ﻰﻄﻌﺗ ﻱﻮﻴﳊﺍ ﺩﺎﻀﳌﺍ ﺍﺬﻫ ﻦﻣ ﻞﻣ /ﻡﺍﺮﺠﻴﻠﻣ ﻥﺃ ﺞﺋﺎﺘﻨﻟﺍ

.ﻑﺎﻌﺿﺃ ﺔﻌﺑﺭﺃ ﺰﻛﺮﳌﺍ ﻥﻮﻤﻴﻠﻟﺍ ﲑﺼﻋ

10