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1
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
2
Dedication
To the spirits of my late father and my late mother,
To all my family with love
3
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.
4
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).
5
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
6
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.
7
ملخص األطروحة
إحصائية على استنادا مليون طن 4.98 للحمضيات ب السنوييقدر اإلنتاج
م أن 1996 بوفيل عام م أوضح 1997 العام يف) الفاو( ألزراعه و األغذيةأجرا منظمة
أجلانبيه من النواتج ينتج عنها كميات كبريةألفواكههعملية إنتاج العصري من هذه
اخلايلالبذور و لب الثمار ، تتكون من القشورواليت أجلانبيه النواتج حتتوى هذه)الثانوية(
جذبت اليت و احليوية ألفاعليه ذات الطبيعية على كميات كبرية من املواد ،العصريمن
الكتشاف الدراسة و عليه فقد متت هذه صحية الباحثني ملا تتميز به من خصائص انتباه
حيوية و مضادات حشرية هلا خواص كمبيدات بيهأجلان من هذه النواتج طبيعيةمواد
.آمنه
أظهر الكشف األوىل عن الدراسة هذه يف مت إجراؤها التجارب قدالعديد من
قشور -نقشورا لليمو، الدراسة هذه يف أملستخدمه ألنباتيه للمواد ألكيميائيهاملكونات
-الثالثي لتريبني ا- أن اإلستريولاليوسفي قشور القريب فروت و قشور - الربتقال
. أهم املكونات هلذه القشورهيالكمارين و الفالفونويد
8
قشور الربتقال و قشور القريب - من قشور الليمونألطياره الزيوت استخالصمت
و لقد GC/MS و من مث مت حتليلها كيميائيا بواسطةالبخاريفروت بواسطة التقطري
.الزيوت تكوين يفى نسبه ميثل أعلالليمونينيأوضحت النتائج أن مركب
الكيولكس ضد يرقات بعوضة حشرية كمبيدات الثالثة هذه الزيوت اختربت
و لقد دلت النتائج على أن مجيع هذه الزيوت هلا خاصية اإلباده كوينكويفاسياتص
أظهر أعلى فاعليه حيث أعطى أقل الذي خاصة زيت الربتقال أحلشره ضد هذه احلشرية
.االختبار من جمموع يرقات البعوض حتت 50 % تركيز يكفى لقتل
ال ينحصر فقط تأثري هذه الزيوت على بعوضة الكيولكس كما أوضحت النتائج أيضا أن
.للبعوضة أحلياتيه كل بقية األطوار إىلعلى الريقات بل ميتد أيضا
، البترويلاأليتر ، ألعضويه بواسطة عدد من احملاليل اليوسفي قشور استخالصمت
الكحول األثيلى و يفروفورم و األستون مث استخلص املستخلص القابل للذوبان الكلو
. و الكلوروفورمالكحويلاملستخلص غري القابل للذوبان فيه من مستخلصات اإليثر
ضد عدد من البكترييا موجبة اجلرام و حيوية هذه املستخلصات كمضادات اختربت
الكحول هو أكثر يفالقابل للذوبان أوضحت النتائج أن املستخلص ، سالبة اجلرام
.املستخلصات فاعليه حيث أعطى أقل تركيز يكفى لتثبيط البكتريا
9
يف املستخلص القابل للذوبان يف ألفعالهمت إجراء جتربه مبسطه لزيادة تركيز املادة
ببكترييا اإلستافيلوكوكس ) قبل فصلها عن الثمرة (اليوسفيالكحول و ذلك حبقن قشرة
املستخلص أظهرت النتائج أن . كما سبق ذكرها آنفا االستخالص إجراء عملية و من مث
من نظريه أكثر فاعليه القشور احملقونة بالبكترييا الكحول من يف للذوبان القابل
.املستخلص من القشور غري احملقونة بالبكترييا
قا من قبل جيابراكاش و مت التعرف عليها ساباليت الفالفون متعدد امليثايل و ،ألفعالهاملواد
HPLC بواسطة الدراسة هذه يف مت التعرف عليها NMR بواسطة 2000آخرون عام
التاجنرتني- 1: وهى
النوبيلتني-2
اتبعت اليت ألطريقه للبكترييا لعصري الليمون متبعني ذات املضادة ألفاعليه اختبارمت
استخدمت اليت التركيز أن كل أوضحت النتائج.اليوسفي حالة مستخلصات قشور يف
ضد كل من البكتريا موجبة اجلرام و البكترييا سالبة عالية ذات فعالية الليمون من عصري
.اجلرام
مليغراما من محض 58مبا أن املليلتلر من عصري الليمون الطازج حيتوى على
عصري منعديدة لتركيز للبكتريا املضادة ألفاعليه بني أملقارنهالستريك فقد جرت
للبكترييا لعصري املضادة ألفاعليه من حامض الستريك و لقد تبني أن اما يقابلهالليمون و
10
. حيدثها محض الستريك عند ذات التركيزاليت تركيز تساوى متاما تلك أيالليمون عند
. محض الستريكهي عصري الليمون يف للبكتريا املضادة املادةو عليه فقد ثبت أن
إستربتومايسني مع عصري الليمون أظهرت احليوياعلية املضاد عند مقارنة ف
تعطى ذات قطر اإلثباط الناتج عن احليويمل من هذا املضاد / ممليجرا 100النتائج أن
.أضعافعصري الليمون املركز أربعة
11
Table of Contents
Chapter 1
Introduction and Literature
Review…………………………………..1
1.1 Background about the citrus
fruits……………………………......1
1.1.1Botany of citrus
fruits……………………………………………..1
1.1.1. a The flavedo (exocarp)…………………………………….……..1
(i)Components of volatile
part…………………………….………1
(ii)Components of non-volatile
part…………….…………………2
(iii)Additional
components………………………………………..2
1.1.1. b The albedo
(mesocarp)…………………………………………..2
(i)Peptic
substances…………………………….………….………2
(ii)Additional
components…………………………………………2
1.1.1. c The membranous segments
(endocarp)………………………….2
(i)The
juice………………………………………………………...3
(ii)The
seeds…………………………………….…………………3
12
1.1.2 Phytochemicals in citrus
fruits…………………………………...3
1.1.2. a Nutrient
phytochemicals…………………………………………4
(i)Carbohydrates…………………………………………………...
4
(ii)Vitamins………………………………………………………..
4
(iii)Carotenoids…………………………………………………….
5
(iv)Folic
acid…………………………………….………………...7
(v)Potassium……………………………………………………….
7
1.1.2. b Nov-nutrient
phytochemicals……………………………………8
(i)Flavonoids………………………………………………………
8
(ii)Limonoids………………………………………………………
9
(iii)Coumarin……………………………………………………..1
0
1.1.3 Some analytical methods for citrus fruit
extracts……………..10
(i)Gas chromatography
(GC)…………………………………….10
(ii)Gas chromatography coupled with mass spectrometry
(GC/MS)………………………………………………………….1
1
13
(iii)Capillary electro-chromatography
(CEC)……………………11
(iv)High performance liquid chromatography
(HPLC)………….12
1.1.4 Folkloric uses of citrus
fruits……………………………………12
1.2 Background about the studied organisms……………………….13
1.2.1The mosquito….Culex
quinquefasciatus………………………….13
1.1.2 Bacteria…………………………………………………………...14
1.3 Background about insecticides and
antibiotics……………….....15
1.3.1
Insecticides……………………………………………………….15
1.3.1. a Some natural
insecticides……………………………….……...16
(i)Pyrethrum……………………………………………………...1
6
(ii)Nicotine…………………………………….…………………1
6
(iii)Rotenone……………………………………………………..1
6
(iv)Limonene……………………………………………………..1
6
(v)Neem……………………………………….…………………1
6
1.3.1. b Some natural
mosquitocides……………………………………17
14
1.3.2
Antibiotics………………………………………………………..18
1.4 The biological activities and health effect of citrus fruits…….....18
1.4.1Insecticidal
activity………………………………………....18
1.4.2Antimicrobial
activity……………………………………....20
1.4.3 Antiparasitic
activity……………………………………….22
1.4.4 Citrus fruits and cardiovascular
diseases…………………..23
1.4.5 Citrus fruits and eye
condition……………………………..25
1.4.6 Anticancer activity of citrus
fruits…………………………25
1.5 Research objectives……………………………………………….28
1.5.1The overall aim of the
study………………………………..28
1.5.2The specific aims of the study……………………………...28
Chapter 2
Materials and
Methods………………………………………………..30
2.1
Materials…………………………………………………………...30
2.1.1 Plants……………………………………………………………..30
2.1.2 Chemicals………………………………………………………...30
2.1.3Apparatus and
glasses……………………………………………..31
15
2.1.4 Laboratory animals and microorganisms………………………...32
(i)Insect: Culex quinquefasciatus………………………………..32
(ii) Microorganisms: four strains of
bacteria…………………….32
2.2 Methods…………………………………………………….……...32
2.2.1 Preliminary screening for non-nutrient phytochemicals from
peels of citrus
fruits……………………………………………………32
(i)Test for unsaturated sterols and
triterpenoids…………………32
(ii)Test for
flavonoids………………………….…………………32
(iii)Test for
coumarin…………………………………….………33
(iv)Test for
alkaloids……………………………………………..34
(v)Test for
tannins………………………………………………..34
(vi)Test for
saponin…………………………….………………...34
2.2.2 Larvicidal activity of citrus oils against Culex quinquefasciatus
larvae…………………………………………………………………...3
5
2.2.2. a Collection and maintenance of
mosquito……………………….35
2.2.2. b Extraction of citrus
oils………………………………………...36
(i) Cold
pressing………………………………………….………36
16
(ii)Steam
distillation……………………………………………...37
2.2.2. c Quantitative analysis of the extracted citrus oils by
GC/MS………………………………………………………….……….3
7
2.2.2. d Insect
bioassay…………………………….…………….……...39
(i)Toxicity on
larvae……………………….….………………….39
(ii)Fecundity of mosquito that survived sub-lethal
concentration……………………………………………………39
(a)When treated as
larvae………………………………………...39
(b)When treated as
adults………………………………………...40
(iii) Latent effects of citrus oils on
mosquito…………………….40
2.2.2. e Statistical
analysis………………………………………………40
2.2.3 Mandarin peels extracts as antibacterial
agent………………..41
2.2.3.1 Inoculums
preparation………………………………….……….42
2.2.3.2 Preparation of plant
materials………………………….………..42
2.2.3.3 Extraction of plant
materials…………………………………….42
17
2.2.3.4 In vitro antibacterial activity
tests………………………………43
(i) Agar diffusion
method…………………………………….43
(ii) Liquid dilution
method……………………………………44
2.2.3.5 Simple method for increasing concentration of the active
compounds………………………………………………………………4
4
2.2.3.6 Isolation and purification of the active
compounds……………..44
2.2.3.7 Antibacterial activity of the isolated
compounds……………….45
2.2.3.8 Chemical analysis of the isolated
compounds…………………..45
2.2.4 Antibacterial activity of lime
juice……………………………...45
2.2.4.1 Preparation of the
juice…………………………………..46
2.2.4.2 Preparation of concentrated
juice………………………...46
2.2.4.3 Antibacterial activity of natural and concentrated lime
juice………………………………………………………………4
6
2.2.4.4 Determination of the active compound in the lime
Juice………………………………………………………………4
6
18
2.2.4.5 Comparison between antibacterial activity of lime juice
and that of
streptomycin………………………………………………46
Chapter 3
Results
………………………………………………………………….47
3.1 Preliminary screening for non-nutrient phytochemicals from
peels of citrus
fruits……………………………………………………47
3.2 Larvicidal activity of citrus fruits’ oils against Culex
quinquefasciatus
larvae……………………………………….……….49
3.2.1 The extracted
oils………………………………………………….49
(i)The yields of the extracted
oils………………………………...49
(ii)The physical characters of the extracted
oils………………….49
(iii)The quantitative analysis of the extracted
oils…….…………49
3.2.2 The toxicity of citrus oils against Culex quinquefasciatus
larvae...54
3.2.3 Effects on fecundity of females that survived sub-lethal
concentration……………………………………………………………5
6
(i) When treated as
larvae…………………………………….56
19
(ii) When treated as
adults…………………………………….57
3.2.4 Latent effects of citrus oils on the developing stages…………….57
3.3 Antibacterial activity of mandarin peels
extracts……………….58
3.3.1 Susceptibility of bacteria to the different fractions from mandarin
peels……………………………………………………………………..5
8
3.3.2 Minimum inhibitory concentrations (MIC) for the different
mandarin peels’ fractions against
bacteria………………………………65
3.3.3 Increasing concentrations of the active
compounds………………66
3.3.4 Identification of the isolated
compounds………………………….67
3.3.5 Antibacterial activity of the isolated
compounds…………………68
3.4 Antibacterial activity of lime
juice………………………………..69
3.4.1 Antibacterial activity of natural and concentrated lime
juice……..69
3.4.2 Comparison between antibacterial activity of lime juice and that of
citric
acid………………………………………………………………..70
3.4.3 Comparison between antibacterial activity of lime juice and that of
streptomycin…………………………………………………………….7
2
Chapter 4
20
Discussion………………………………………………………………7
4
Conclusion………………………………………………………………7
8
Recommendations………………………………………………………7
8
References……………………………………………………………....7
9
21
List of Tables
Table 1: The typical GC/MS parameters………………………………38
Table 2: The non- nutrient phytochemicals in the peels of citrus
fruits..47
Table 3: Some physical characters of the extracted citrus
oils…………49
Table 4: Chemical composition of the extracted lime oil……………..51
Table 5: Chemical composition of the extracted grapefruit oil……….53
Table 6: Chemical composition of the extracted orange oil…………...54
Table 7: Toxicity of lime oil on different developing stages of Culex
quinquefasciatus………………………………………………………...5
5
Table 8: Toxicity of orange oil on different developing stages of Culex
quinquefasciatus………………………………………………………...5
5
Table 9: Toxicity of grapefruit oil on different developing stages of
Culex
quinquefasciatus…………………………………………………56
Table 10: Relative efficiency and sub-lethal concentrations values of
citrus oils against Culex quinquefasciatus
larvae……………………….56
Table 11: Fecundity of C. quinquefasciatus that survived LC50 of citrus
oils after treatment of larvae……………………………………………57
Table 12: Fecundity of C. quinquefasciatus that survive LC50 of citrus
oils after treatment of adult…………………………………………….57
22
Table 13: Latent effect on the developmental stages of
C.quinquefasciatus resulting from eggs lay by females treated as adults
with citrus oils (LC50)…………………………………………………58
Table 14: Latent effect on the developmental stages of
C.quinquefasciatus resulting from eggs lay by females treated as larvae
with citrus oils
(LC50)………………………………………………….58
Table 15: Minimum inhibitory concentrations (MIC) of mandarin peel
fractions against
bacteria………………………………………………..65
Table 16: Physical appearance, Spots under UV (365nm), Rf values,
retention time and wavelength of the isolated active compound from
mandarin
peels…………………………………………………………..67
Table 17: Diameters of inhibition zones caused by natural lime juice and
citric acid at concentration of
58mg/ml…………………………………70
Table 18: Diameters of inhibition zones caused by di-concentrated lime
juice and citric acid at concentration of 116mg/ml…………………….70
Table 19: Diameters of inhibition zones caused by four-concentrated
limejuice and citric acid at concentration of
232mg/ml………………...70
Table 20: Diameters of inhibition zones caused by four-times
concentrated lime juice and streptomycin at concentration of
100mg/ml………………………………………………………………..7
2
23
List of Figures
Fig. 1: a histogram showing the antibacterial activity of hexane extract
from mandarin peels, at different concentrations, against the studied
bacterial strains…………………………………………………………59
Fig. 2: a histogram showing the antibacterial activity of chloroform
extract from mandarin peels, at different concentrations, against the
studied bacterial
strains…………………………………………………60
Fig. 3: a histogram showing the antibacterial activity of acetone extract
from mandarin peels, at different concentrations, against the studied
bacterial strains…………………………………………………………61
Fig. 4: a histogram showing the antibacterial activity of ethanol-soluble
fraction from mandarin peels, at different concentrations, against the
studied bacterial
strains…………………………………………………62
Fig. 5: a histogram showing the antibacterial activity of ethanol-
insoluble fraction from mandarin peels, at different concentrations,
against the studied bacterial
strains…………………………………………………63
Fig. 6: A photographed plate showing the inhibition zones caused by the
different fractions from mandarin peels against Escherichia
coli………64
Fig. 7: A photographed plate showing the inhibition zones caused by the
ethanol-soluble fraction of inoculated and non-inoculated mandarin peels
against Staphylococcus
aureus………………………………………….66
24
Fig. 8: A photographed plate showing the inhibition zones caused by the
ethanol-soluble fraction and isolated active
compounds………………..68
Fig. 9: A histogram showing the antibacterial activity of lime juice at
different
concentrations…………………………………………………69
Fig. 10: A photographed plate showing the inhibition zones caused by
natural lime juice and citric acid at concentration of 58mg/ml against
Staphylococcus
aureus…………………………………………………..71
Fig. 11: A photographed plate showing the inhibition zones caused by
four-times concentrated lime juice and streptomycin at a concentration of
100 mg/ml against Escherichia
coli…………………………………….73
25
CHAPTER 1
Introduction and Literature Review
1.1 Background about the citrus fruits
1.1.1 Botany of citrus fruits
The citrus fruits are of Asiatic origin, then distributed
allover the world. They come from the genus Citrus,
subfamily Aurautiacea, family Rutacea and order Rulates.
The fruit is hesperidium, that is special berries with a juicy
pulp divided into segments, where the seeds are contained,
are the specific character of this fruit. By sectioning the citrus
fruit one can identify many fundamental parts:
1.1.1. a The flavedo (exocarp)
This is the colored outer peel. It is composed of few
cells layers that become progressively thicker in the internal
part. The epidermis layer is covered with wax and contains
small number of stoma which are closed when the fruit ripes.
When the fruit ripes the flavedo cells contain carotenoids
(mostly xantofils) inside chromatoplastides that, in previous
stadium contained chlorophyll. The flavedo internal part is
rich in multicultural bodies with spherical shape, the oil sacs
that are full of essential oils.
In general flavedo is formed by cellulose material and
contains mainly the essential oils and other components:
(i) Components of the volatile part
iTerpene
26
iAliphatic not terpenic compounds
iAromatic hydrocarbons
iEsters containing nitrogen
(ii) Components of the non-volatile part
iParaffin wax
iSteroids and triterpenoids
iFatty acid
iCoumarins, psoralens and flavones
(iii) Additional compounds
iPigments (carotenoids- chlorophyll- flavonoids)
iBitter principles (limonin)
iEnzymes (oxide-reducers, proteolytics, acetyl-esterase,
phosphatase, pectic enzyme
1.1.1. b The albedo (mesocarp)
This is the spongy internal part of the peel. It is white or pink.
It is composed of layers of cells generally big and less
compact. Albedo is constituted by:
(i) Pectic substances
iPectin
iPectic acid
iPectinic acid
(ii) Additional components
iBitter principle (limonin)
iEnzymes (oxide-reducers, proteolitics, acetil-esterase,
phosphatase, pectic enzymes)
iFlavonoids
27
1.1.1. c The membranous segments (endocarp)
Endocarp makes up the edible part. A thick radial film
divides it into segments, which contain a variable number of
monoembryonic seeds, of various sizes according to variety.
It contains:
(i) The juice
Found in the juice sacs. It contains
iCarbohydrates (mono and disaccharides)
iOrganic acids (citric acid and malic acid)
iNitrogenous components (protein, peptides, amino
acids)
iInorganic constituents (ashes)
iVitamins (vitamin C)
iLipids
iVolatile aromas (ethylic alcohol, acetone,
acetaldehyde, formic acid etc.)
iPigments (carotenoids, chlorophyll, flavonoids)
The juice yield depends, beside the species and variety, on
the ripe degree, the cultivation technique and extraction
method.
(ii) The seeds
Are constituted by cellulose material with presence of
iRaw protein
iOils
iBitter principles (limonoids)
1.1.2 Phytochemicals in citrus fruits
28
Citrus yield highly nutritional fruits (grapefruit, orange,
lemon, mandarin, tangerine, bergamot and lime). They are
consumed fresh or used in forms of candied fruit, beverages,
liqueurs and perfumes (citrus aromas are a favorite around
the world). Citrus fruits are fat free, sodium free and
cholesterol free. They contain carbohydrates, fibers, vitamin
C, potassium, folic acid, calcium, riboflavin, thiamine,
niacin, vitamin B6, copper, phosphorous, magnesium,
riboflavin, pantothenic acid and other varieties of
phytochemicals.
29
1.1.2. a Nutrient phytochemicals
(i) Carbohydrates
The main energy yielding in citrus is carbohydrates;
citrus contains the simple carbohydrates (sugars) fructose,
glucose and sucrose, as well as citric acid, which can also
provide a small amount of energy. Citrus fruit also contain
non- starch polysaccharides (NSP), commonly known as
dietary fiber, which is a complex carbohydrate with
important health benefits. The predominant fiber in citrus is
pectin, making up 65 to 70 percent of the total fiber.
The remaining fiber is in the form of cellulose, hemi-
cellulose and trace amounts of gums. Citrus also contains
lignin, a fiber-like component. In the body, NSP holds water-
soluble nutrients in a gel matrix which delays gastric
emptying and slows digestion and absorption. This tends to
promote satiety, and may reduce the rate of glucose up-
take following consumption of glycaemic (available)
carbohydrate, thus help in balancing of a sugar in blood
glucose levels. Improper regulation of blood glucose results
in either hyperglycaemia (high blood glucose) or
hypoglycaemia (low blood glucose). NSP can also interfere
with the re-absorption of bile acids, which may help in
lowering plasma cholesterol levels.
(ii) Vitamin C:
This water-soluble antioxidant vitamin plays an essential
role in collagen (a primary component of much of the
connective tissue in the body) formation, strengthen bones
30
and blood vessels, anchoring teeth in gums, absorbing
inorganic iron and zinc and helping in repair of tissues. It has
also been used in the treatment of anemia and stress. It is
necessary for prevention of the deficiency disease scurvy,
but in recent years has become of increasing interest in
relation to antioxidant capacity. As an antioxidant, it helps
prevent the cell damage done by ‘free radical’ molecules as
they oxidize protein, fatty acids and deoxyribonucleic (DNA)
in the body.
Free radical damage has been implicated in the
progression of several diverse and important disease states
including cancer, cardiovascular disease and cataract
formation. Being a good antioxidant if regularly consumed,
citrus can be an important part of a diet aimed at reducing
the risk of such chronic diseases.
Only 10 mg of vitamin C per day required preventing
vitamin C deficiency and the devastating disease scurvy.
However, for good health and sufficient body storage of
vitamin C, 30 to 100 mg/day is generally recommended,
although some recent studies have provided evidence that
more than 200mg/day may be optimal for the prevention of
chronic disease. Too much vitamin C (above 500mg/day)
may be dangerous, especially for those at a risk of iron
overload.
70mg and 56mg respectively can be obtained from
one orange or one grapefruit. A 225 ml glass of orange juice
contains approximately 125 mg of vitamin C.
31
(iii) Carotenoids
Citrus fruits have been recognized as sources of
pigment as well as biologically active compounds.
Carotenoids present in citrus fruits and vegetables are
important biological precursors of vitamin A and are widely
believed to keep human beings healthy. More than 600
carotenoids have been identified, but only a few are found in
measurable quantities in the human body: alpha-carotene,
beta-carotene, lycopene, lutein, zeaxanthin and
cryptoxanthin.
(1) Beta-carotene
It is the most well known carotene. Like many
carotenoids, beta-carotene is a powerful antioxidant (a
striking example being the protection it offers to the algae
from which it is commercially harvested against harmful
ultraviolet radiation from the sun). It may play a role in
slowing the progression of cancers and in population studies
has been identified as having a protective role against a
number of conditions such as lung and oral cancers. It may
play a role in immunity, cataracts and may slow the build-up
of plaque in arteries.
Beta-carotene also is a precursor of vitamin A (retinal,
and retinoic acid, which have been demonstrated to have
the ability to reduce differentiation of neoplastic and
preneoplastic cells. However, intervention trials in
populations at risk of skin, cervix, colon and lung cancer,
have failed to demonstrate any health benefits.
32
(2) Lycopene
It is mainly present in navel orange, pink grapefruit and
tomato. It is a very powerful antioxidant, which has been
associated with reduced risk of prostate cancer, risk of
macular degenerative disease, serum lipid oxidation and
cancers of the lung, bladder, cervix and skin. It has also
been linked to breast cancer prevention but studies are
sparse.
In the body lycopene is deposited in the liver, lung, prostate
gland, colon and skin. Its concentration in body tissues tends
to be higher than all other carotenoids.
(3) Lutein, Zeaxanthin and Cryptaxanthin
Lutein and zeaxanthin, which are present in citrus as
well as other vegetables and fruits, are carotenoids that are
linked to macular degeneration, as they are required for
proper pigmentation of the macular region of the eye .The
yellow pigments they form are believed to filter out harmful
blue light and protect against age macular degeneration,
the leading cause of blindness in those over 65 years. Lutein
and zeaxanthin have also been linked to reduce risk of lung
cancer.
Cryptoxanthin has been associated with reduced risk of
cervical cancer. It is abundant in orange fruits especially
orange, tangerines, mangoes, and papaya. Nishino et
al.(2004) reported that, cryptoxanthin showed a higher
anticancer activity in mice against both skin and large
intestine cancers compared to β-carotene.
33
(iv) Folic acid
The vitamin folic acid, from oranges and orange juice,
green leafy vegetables and the outer layers of many seeds
and grains, plays an important metabolic role in the synthesis
of DNA, and in situations requiring the transfer of a methyl
group to a biological acceptor molecule. It has thus been
investigated in relation to cancer prevention.
The recommended daily intake of folate is 180 mcg for
females and 200 mcg for males. A225 ml glass of orange
juice provides 75 mcg of folic acid .Methylation of DNA itself
appears to be an important mechanism for controlling the
expression of many genes, including those involved in cell
proliferation – abnormal methylation states of DNA (usually
low methylation) have been associated with a number of
neoplastic (formation of new tissues as in case of cancer)
and preneoplastic conditions.
Folate also plays a role in prevention of heart disease
through an effect on homocysteine. Homocysteine Is a
sulphur-containing amino acid derived from enzymic
transformations of the essential dietary amino acid
methionine.
(v) Potassium
Potassium is an essential mineral that works to maintain
the body’s water and acid balance. As an important
electrolyte, it plays a role in transmission nerve impulses to
muscles, in muscle contraction and in the maintenance of
normal blood pressure.
34
The daily requirement of potassium is approximately
2000mg. Deficiency of potassium (is some concern that a
high sodium-to-potassium intake ratio) may be a risk factor
for chronic disease. Increasing consumption of citrus fruit and
juices is a good means of increasing potassium intake. One
orange and one 225 ml glass of orange juice provide
approximately 235 and 500 mg of potassium, respectively.
400 mcg of potassium are associated with the prevention of
neural tube defects, severe birth defects.
1.1.2. b Non-nutrient phytochemicals
The phenolic compounds of citrus fruits such as
flavonoids (flavanones, flavones and flavonols), the
anthocyanins, the coumarins and the psoralens are
secondary metabolic products that are believed to be
produced as a result of plant’s interaction with the
environment and may play a major role in both plant and
animal health.
The non-phenolic compounds of citrus oils such as the
limonoids also have a vital role.
(i) Flavonoids
Are polyphenolic compounds found in the fruits and the
peels of citrus fruits. They constitute one of the most
characteristic classes of compounds widely distributed in
plant. Although they are considered to be non-nutritional
agent there is an increasing interest in these substances
because of their possible effects on human health.
35
Flavonoids have a variety of biological effects such as
GSE (glutathione S- transferase) inducers, phytoalexins, larval
growth inhibition activity, antitumor activity (Attawy 1994),
antiviral and antimicrobial activity, hypotensive properties
and antioxidant activity (Middleton and Kandaswoarli 1999).
The flavones in citrus are found in glycosylated and
aglycon states, the latter showing a greater variety of
compounds with their structure frequently multi-substituted
by hydroxyl and methoxyl groups. Among these, poly-
methoxylated flavones play an important role in plants,
acting as antioxidant and inhibitory of numerous enzymes
such as phenolases and pectinmethyltransferases (De Swardt
et al., 1967).
Moreover, because they show a characteristic
distribution pattern, they can be used for taxonomic
purposes (Ooghe et al., 1994).
Furthermore, they have numerous pharmacological
applications due to their anti-thrombogenic properties,
which regulate human blood erythrocyte concentration and
aggregation (Robbins, 1976), and cardioto- ic action
(Itoigawa et al., 1994). They have also been shown to have a
cytotoxic effect toward cancerous cell lines (Kupchan et al
1965) and to act as anti-mutagenics (Francis et al., 1989).
These compounds, together with the other compounds
of the essential oils, probably confer a certain degree of
36
resistance against microbial infections in citrus (Ben-Aziz,
1967; Huet, 1982).
(ii) Limonoids
Limonoids are highly oxygenated triterpenoids. They
are abundantly present in Rutaceae (citrus fruits) and
Meliaceae (neem) families of the order Rutales. Out of 36
limonoids aglycones and 17 limonoid glycosides have been
isolated from citrus and its hybrids (Hasegawa and
Miyake.1996).
Limonin is largely responsible for delayed bitterness in
citrus juice and processed citrus products (Ozaki et al.1991).
However, bitter limonoid aglycones turn to tasteless
glycosides with maturity of fruit.
Limonoids have attracted attention due to biological
functions such as antifeedant activity against termites
activity (Alford and Bently, 1986, Alford et al.1987, Serit, et al.
1991), antifeedant activity against insects (Alford et al., 1986;
Bently, et al., 1990; Klock and Kubo 1982), inducing
glutathione S- transferase (enzyme in heart and liver) activity
(Lam et al.,1989), anticarcinogenic activity (Huang et
al.,1994) cholesterol lowering activity in Hep G2 cells
(Elzbieta et al., 2000) and growth regulating activity
(Champagne et al. 1992).
More recently their anticarcingenic and
antitumorogenic activities attracted attention (Hasegawa et
al.1996). More than 20 epidemiological studies suggest an
inverse relation between consumption of citrus fruits
37
limonoids and many types of cancers. The ability to induce
detoxifying enzyme system, glutathione S- transferase may
be the possible mode of action of limonoids in cancer
chemoprevention.
Limonoids have shown to reduce the risk of many types
of cancers such as oral cavity (Miller, et al., 1989), larynx,
esophagus, stomach, pancreas, lung, colon and rectum.
Also they have property of inhibition of the proliferation of
human breast cancer cell (Miller, et al., 1989; Guthrie et al.,
1997).
(iii) Coumarin
They response to pathogens attacks on citrus (Nahrsdt,
1979). Psoralens (linear furocoumarins) are toxic to insects,
especially in the presence of UV light and have been
identified as phytoalexins in celery.
Evidence indicated that these compounds and others,
which are found in the flavedo, are conferring insect
resistance on their fruits. In particular the Mediterranean fruit
fly (Ceratitis capitata) does not survive in the lemon. These
two observations may also be related to a particular
hydroxylation pattern of a flavonoid compound the
importance of which has been demonstrated for larval
growth inhibition.
1.1.3 Some analytical methods for citrus extracts
(i) Gas chromatography (GC)
High resolution GC, conventional stationary phases, is
the technique which best helps in detecting cit adulteration.
38
The information obtained with the GC analysis of the volatile
fraction of oils can be sufficient to determine whether the
product is genuine or not, and sometimes when the prodict is
adulterated, the kind and the level of adulteration can be
detected.
Since essential oils are complex samples, the time
required for complete GC resolution of the components of
interest can be of the order of hours. In fact quality control
analysis of essential samples is usually carried out on very
long columns with 0.32 or 0.25 mm i.d. and slow temperature
program.
(ii) Gas chromatography coupled with mass spectrometry
(GC/MS)
Gas chromatography coupled with mass spectrometry
detection, led to a variety of analytical uses, which include
quantitative analysis of illicit drug samples and forensics
evidence (Ueki 1998), trace analysis of pesticides and other
toxic residues present in soil and ground water samples and
performing quality control analysis in both pharmaceutical
and food product industries (Vink et al, 1980; Chamblee et
al,. 1991).
Many of the essential oils belong to a family of
compounds known as terpenes and terpenoids. Terpenes are
small organic hydrocarbon molecules; they may be cyclic
or acyclic, saturated or unsaturated. Terpenoids are
oxygenated derivatives of terpenes, which may contain
hydroxyl groups or carbonyl groups. Regardless of their
39
structural diversity, terpenes and terpenoids share certain
structural similarities. This mixture is best analyzed
quantitatively and qualitatively with GC/MS.
(iii) Capillary electro-chromatography (CEC)
Capillary electrochromatography (CEC) is a relatively
new method. Several applications were performed by CEC in
different fields of analytical chemistry, and in different cases
it was demonstrated that electro-chromatographic methods
could offer some advantages over high performance liquid
chromatography (HPLC). Employing CEC can offer some
advantages such as reduction of time of analysis, higher
resolution, and enhancing peak efficiency .
(iv) High- performance liquid chromatography (HPLC)
It is a form of liquid chromatography, in which the
separation of components of mixture is achieved by forcing
the mixture over an immobilized chemical system in a
column by means of a flowing liquid solvent steam. HPLC
systems are used for determining the amount of organic
substances, at low concentrations, in environmental, food,
drug, or biological samples.
Rouseff and Ting, (1979) determined the major
polymethoxylated flavones (PMFs) in orange juice by this
quantitative high-performance liquid chromatography.
1.1.4 Folkloric uses of citrus fruits
In the folklore, the sour citrus fruits such as lemon and
lime are the most ones used mainly as flavoring in food and
drinks and in hot and spicy dishes. Traditionally these fruits
40
have many medicinal uses, they may be employed
externally to treat cough, cold, dengue fever and throat-
ache (Perry and Metzger 1980). It is also used for diabetes
(Mahabir and Gulliford 1997), fever in infants, period pain,
and rheumatism. The juices are taken as tonic to relieve
stomach ailments (Burkill 1935). The juice mixed with oil gives
as vermifuge. Poulticing with the sour juice may treats
gonorrhea. The sour juice also used with arsenic for treating
yaws (Burkill 1935).
In Fiji, the fresh fruit juice has been used for eye drops,
nasal bleeding, sinus and nausea (Singh 1986). While in India
the pickled lime is eaten to relieve digestion. In Haiti, the
fresh juice is used externally for wounds and sores and orally
for epilepsy, toothache, flu, cough, and urethritis and to
stimulate appetite (Wengier et al. 1986).
In Guatemala, sour juice is used topically for
conjunctivitis, eye irritation, wounds. Ulcers, bruises, sores,
infections of the skin and mucosa, ringworm, skin fungal
diseases, leucorrhoea and vaginitis (Giron et al.1988).
In Nicaragua, the lime has been used for child birth,
diarrhea, fever, infections, skin rashes, cold, cough, wounds,
intestinal parasites, malaria and as a digestive agent (Coee
and Anderson 1996).
In Nigeria, the sour citrus fruits are used as a fertilization
stimulant (Elujoba 1995); also the juice used for treatment of
irregular menstrual flow and prevents scurvy (Bhat et al.
1990).
41
In West Indies, the sour juice has been used for
dysmenorrhea, fever and worms (Ayensu 1978).
In Senegal and Sierra Leone, according to what was
stated in Quisumbing 1951, the juice is sometimes given as
vermifuge, mitigated by being mixed with oil. Externally the
fresh juice is used as a cleanser or stimulant of wound
surfaces. The cut sour lime roasted and applied to chronic
sores, yaws, etc. (Ouisumbing 1951)
In Belize, the fresh lime is used to help expel sputum. In
Peru, the sour juice of the fresh fruit is used as a
contraceptive given to both sexes, as well as treatment of
fever (Duke 1994).
1.2 Background about the studied organisms
1.2.1The mosquito--- Culex quinquefasciatus
Mosquitoes belong to the class Insecta, order Diptera
and family Culicidae. This family is divided into three
subfamilies: Anophelinae, Culicinae and Toxorhynchitinae.
There are about 2500 described species of mosquitoes
in the world, about 150 occur in temporate North America
and there are abundant species in the tropics. The species
Culex quinquefasciatus is widely spread all over the world. It
is largely responsible for the transmission of nocturnal
periodic form of Wucheraria bancrofti in Africa and Asia as
well as in transmission of bird malaria, heart worm of dogs
(Dirofilaria immitis), avian poxivirus and st. Louis encephalitis
in Eastern USA.
42
In Sudan however, it does not transmit any disease till
now, but it is a vigorous biter and causes nuisance to the
people especially in the cities including the capital
Khartoum.
As all other mosquitoes, it goes through four separate
and distinct stages of the life cycle: egg, larva, pupa and
adult. Culex quinquefasciatus lay their eggs on the surface of
fresh or stagnant water. They usually lay their eggs at night
over a period of time sticking them together to form a raft of
from 100 to 300 eggs. A raft of eggs looks like a speck of soot
floating on the water.
The larvae live in water from 4 to 14 days depending on
water temperature. They feed constantly on algae plankton,
fungi, bacteria and other microorganisms. They hang upside
down at the surface of the water with the breathing tube up.
During growth, the larva sheds its skin (molts) four times. Pupa
is comma shaped, lives in water from 1 to 4 days depending
on water temperature .It is lighter so it floats on the surface of
water and does not feed. The adult splits the pupal case and
emerges to the surface of the water where it rests until its
body becomes dry and hard.
1.2.2 Bacteria
Of the most common pathogenic bacterial strains are
the gram- positive and gram -negative groups. In this work
the gram- positive were represented by Staphylococcus
aureus and Bacillus subtilis while the gram- negative were
represented by Escherichia coli and Pseudomonas
43
aeruginosa. Staphylococcus areus is more spread in
newborn nurseries and postoperative wounds infections and
infection with positive strains lead to staphylococcal
poisoning.
Bacillus subtilis is a positive aerobic spore forming rod
shaped organism. It causes food poisoning.
Escherichia coli was found to be the causative agent
for a series of outbreak of diarrhoea in hospital new-born
nurseries. Pseudomonas aeruginosa is the causative agent of
the respiratory infection in the form of pneumonia.
1.3 Background about insecticides and antibiotics
1.3.1 Insecticides
Prior to the discovery of the organo-chlorine and
organo-phosphate insecticides botanical insecticides were
important products for pest management in industrialized
countries. The importation of plant materials or derivatives
used as insecticides represented a considerable enterprise:
for example over 6700 tons of Derris elliptica roots was
imported into the USA from Southeast Asia in 1947, but this
decreased to 1500 tons in 1963 (Wink, 1993). This reflects the
extent to which botanicals have been displaced by synthetic
insecticides. In 1990, an import of pyrethrum in the USA was
found to be just over 350 tons. Also, some botanical
insecticides that were used in North America and Western
Europe have lost their regulatory status as approved
products.
44
From the academic point of view plants represent a
vast storehouse of potentially useful natural products, and
indeed many laboratories all over the world have screened
thousands of species of higher plants not only for research of
pharmaceuticals, but also for pest control products . These
studies have pointed to numerous plant species possessing
potential pest controlling properties under laboratory
conditions, but the step from the laboratory to the field
eliminates many contenders.
1.3.1. a Some Natural Insecticides
(i)Pyrethrum
This is extracted from the flowers of a chrysanthemum
grown in Kenya and Ecuador. It is one of the oldest and
safest insecticides available. It causes immediate paralysis. It
contains a mixture of four compounds Pyrethrins I and ii and
Cinerins I and II. This insecticide has the same effect as the
synthetic Pyrethroids and DDT. They affect both the peripheral
and central nervous system of the insect.
(ii) Nicotine
It is an alkaloid extracted from tobacco. It is effective
against most types of insect pests, but it is used particularly
for aphids and soft bodied insects. It mimics acetylcholine at
the neuromuscular function in mammals and results in
twitching, convulsion and death, all in rapid order. In insects
the same action is observed but only in the central nervous
system ganglia.
(iii) Rotenone
45
Is produced in the root of two genera of the legume
family: Derris and Lonchocarpus (also called Cube). It is a
respiratory enzyme inhibitor resulting in failure of the
respiratory functions.
(iv)Limonene or d- Limonene
Is the latest addition to the natural insecticides.
Extracted from citrus peels, it is effective against all external
pests of pets, including fleas, lice, mites and ticks and is
virtually non-toxic to worm-blooded animals. Its mode of
action is similar to that of pyrethrum.
(v) Neem
Oil extract are squeezed from the seeds. They contain
the active ingredient azadirichtin, a triterpenoids belonging
to the limonoids. It has shown some rather sensational
insecticide, fungicide and bactericidal properties. It is
disrupts molting by inhibiting biosynthesis or metabolism of
ecdysone, the juvenile molting hormone.
1.3.1. b Some natural mosquitocides
Some plant products are very promising against
mosquitoes and can be used as insecticides and/or
repellents. They offer a safer alternative to synthetic
chemicals and can be obtained by individuals and
communities easily at a very low cost.
Neem oil and other derivatives of neem can be used
alone or in combination with other products for effective
protection against mosquitoes (Mulla ans Su1999;
Schmutterer 1990; Mittal et al. 1995; Dhar et al. 1996; Batra et
46
al. 1998; Nagpal et al. 1995; Sukumar et al. 1991; Sharma et
al. 1993; Rajnikant and Bhat 1994; Mishra et al. 1995; Sharma
et al. 1995; Sharma et al. 1996; Moore et al. 2002; Sharma et
al. 1993; Ansari and Razdan 1996).
Other herbal derivatives of Lantana camara (Sukumar
et al. 1991; Dua et al. 1996), Cymbopogon spp. (Sukumar et
al. 1991; Govere et al. 2000), Mentha piperita (Sukumar et al.
1991; Ansari et al. 1999; Ansari et al. 2000), Eucalyptus spp.
(Sukumar et al. 1991), Tagetes minuta (Green et al. 1991;
Perich et al. 1994; Pathak et al. 2000; Sukumar et al. 1991),
have also shown repellency effects against different
mosquito species and can be used for personal protection
against mosquitoes by individuals, thus minimizing the
dependency on synthetic chemicals.
Similarly, certain other plants derivatives obtained from
Citrus spp. (Al- Dakhil and Morsy 1999; Ezeonu et al. 2001;
Mwaiko 1992; Mwaiko and Savaeli 1994; Jayaprakasha et al.
1997), Solanum nigrum (Singh et al. 2002; Ahmed et al. 2001),
Ageratum conyzoides (Saxena et al. 1992), Annona
squamosa (Saxena et al. 1993), have also shown insecticidal
and /or growth inhibition activity against mosquitoes but their
potential for mosquito control under field conditions need to
be evaluated.
1.3.2 Antibiotics
Antibiotics are too often prescribed and taken. As a
result new bacterial strains are cropping up, improved and
resistant to one form of antibiotics or another. Over the last 30
47
years, doctors have had alarming confirmation of this. Our
future source of medicines, antibiotics in particular, is in
jeopardy and needs further exploration and
experimentation.
Even though, one in four medications has an active
ingredient derived from a plant, there are no plant-derived
antibiotics on the market. Plants have been a source of
disease treatment for thousands of years, beginning with the
earliest herbal and folk remedies. Of the 250,000 flowering
plant species, less than 1% has been analyzed for potential
human therapies.
Through biological assays with human pathogens,
scientists have tested the antibiotics properties of
representative genera obtained through literature searches.
They have shown through agar diffusion tests, how plants
may hold the key to our future well- being and provide us
with medicinal options. A potential antibiotics resource is
waiting to be tapped into revealing the medical and
economic value of plant life.
1.4 The biological activities and health effect of citrus fruits
A vast number of studies have been carried out to
demonstrate the biological activity of citrus fruits. These
studies included insecticidal activity, antimicrobial activity,
antiparasitic activity, anticancer activity and many other
activities.
1.4.1 Insecticidal activity
48
The insecticidal activity of citrus fruits extracts has been
the main issue in many scientific researches:
Al Dakhil and Morsy (1998), demonstrated the larvicidal
action of three ethanol extracts of peel oil of lemon,
grapefruit and novel orange. They tested against the early 4th
instars larvae of Culex pipiens and the resulting pupae. The
LC50 were 18.5, 20.3and 26.5 respectively.
Mwaiko (1992), made a susceptibility test in Culex
quinquefasciatus larvae using peel oil of bitter orange (Citrus
aurantium), sweet orange (Citrus sinensis) and lemon (Citrus
limon). Larvae mortalities were observed indicating that the
extracts may contain potentially useful insecticides.
Mwaiko and Savaeli (1994) demonstrated the mosquito
larvicidal activity of lemon peel oil extracts. The oil was found
to be toxic to the larvae, pupae and eggs of Culex
quinquefasciatus. The oil also fulfilled other required
specifications like suitable specific gravity, spreading
pressure and viscosity. It is also toxic at wide PH range,
stable to heat and light in terms of chemical change, which
could alter larvicidal action. However, it is volatile and did
not form a permanent film on water surface for long periods.
This also affected the larvicidal action.
Fan et al. (1995), made a preliminary study on
bioactivity of orange and tangerine peel extracts against
aphids and mites. They used residual film (topical method)
against Aphis semia and slide – dip (immersion method)
49
against mites. Test results showed that these extracts have
strong bioactivity against aphids and mites.
Jayaprakasha et al. (1997) investigated the molt
inhibiting activity of limonoids from Citrus reticulata in the 4th
instar larvae of Mosquito Culex quinquefasciatus. They
isolated three limonoids namely, limonin, nomilin, and
obacunnone from the seeds. The lethal concentration for
50% of the population (LC50) for inhibition of adult
emergence was 6.31, 26.61 and 59.57 ppm for obacunnone,
nomilin, and limonin respectively. The pattern of mortality at
around the lethal concentration for 50% of the population
(LC50) levels was indicative of molt inhibiting activity.
Shalaby et al. (1998) studied the insecticidal properties
of citrus oils against Culex pipiens and Musca domestica.
They used peel oils of lemon, grapefruit, and novel orange.
Their findings revealed that lemon peel oil is the most
effective against larvae and adults of Culex pipiens and
Musca domestica, while grapefruit oil is toxic to adults of
Musca domestica. On the other hand, the orange oil peel is
the least effective against larvae and adults of both species.
In addition Ezeonu et al. (2001) studied the insecticidal
properties of volatile extracts of orange peels. They studied
the volatile extracts of sweet orange (Citrus sinensis) and
lime (C.aurantifolia) against mosquito, cockroach and
housefly. The results indicated that the insecticidal activity is
better after 60 minutes than after30 minutes spraying of
rooms. Volatile extracts of sweet orange showed greater
50
insecticidal potency. The cockroach is the most susceptible
to the orange peel oil among the three studied insects.
1.4.2 Antimicrobial Activity
The antimicrobial activity of citrus fruits extracts had
been the main issue for many scientific researchers.
Jayaprakasha et al. (2000) investigated the
antibacterial activity of Citrus reticulata (Yousof Afandi) peel
extracts. The citrus peel was extracted successively with
hexane, chloroform and acetone using soxhlet extractor. The
hexane and chloroform extracts were fractionated into
alcohol soluble and alcohol insoluble fractions. These
fractions were tested against different gram- positive and
gram-negative bacteria .The ethanol-soluble fraction was
found to be the most effective.
On the other hand, de Castillo et al. (2000) have studied
the bactericidal activity of lemon juice (natural and
concentrated) and lemon derivatives (essential oils, fresh
and dehydrated peels) against Vibrio cholerae. The results
revealed that concentrated lemon juice and essential oils
inhibited Vibrio cholerae completely at all studied dilutions
and exposure times. Fresh and dehydrated lemon peel
partially inhibited growth of the bacteria.
At the same line, D’A quino and Teves (1994) studied
the natural biocidal activity of lime juice in order to explore
its possible use as a disinfectant and inhibitor of Vibrio
cholerae in drinking water for areas lacking water treatment.
The results showed that lime juice could actively prevent
51
survival of Vibrio cholerae but that such activity is reduced in
markedly alkaline water, so the degree of alkalinity of water
is determined by the minimum concentration of lime juice
required.
Moreover, Mata et al (1994) reported that millions of
Vibrio cholerae were rapidly eliminated with lime juice when
added to commercial ceviche prepared by marinating of
Mahi- Mahi fish, contaminated cabbage and lettuce.
Rodrigues et al (2000) did laboratory experiments to
elucidate the inhibitory effect of different concentrations of
lime juice on the survival of Vibrio cholerae in moals. The
results showed that Vibrio cholerae thrives in rice with peanut
sauce but lime juice inhibited its growth.
Whereas, Stange et al (1993), demonstrated the
antifungal compound produced by grapefruit (Citrus
paradisi) and Valencia orange (Citrus sinensis) after
wounding of the peels. A new compound was isolated from
the injured peel of orange and grapefruit showing a high
activity as fungicide.
While, Caccioni et al (1998), investigated the
relationship between volatile compounds of essential oils of
sweet orange (Citrus sinensis), sour orange (Citrus
aurantium), mandarin (Citrus delicosa), grapefruit (Citrus
paradisi) and lemon (Citrus limon) and antimicrobial action
on
Penicillium digitatum and P.italicum. Results showed a
positive correlation between monoterpenes (other than
52
limonine) and sequiterpene contents of the oils and the
pathogen fungi inhibition. The best result was shown by
lemon oil.
Vargas et al (1999) isolated the antimicrobial and
antioxidant compounds in the non-volatile portion of the
expressed orange essential oil. The results indicated that
these compounds exhibited antifungal activity against
phytopathogenic species and food contaminants. The
isolated hexa and hepta methoxyflavones exhibited
important fungicidal activity against Geotricum candidum,
which is not inhibited by the commercial broad-spectrum
fungicide, Benomyle.
Alderman and Marth (1976) found that orange and
lemon oil is inhibitory to mold growth and aflatoxin
production of Aspergillus parasiticus than d- limonene, the
main constituent of the two peel oils. After seven days at
28ºC 2000 ppm of lemon oil and 3000 ppm of orange oil in
grapefruit juice, as medium, afforded maximum suppression
of mold growth and toxin formation. When the glucose- yeast
extract medium was used 3000ppm of either oil are needed
to give the same results.
Kim et al (2001) isolated a new flavanone triglycoside
(naringenin, hespertin, hesperidin and narivutin). They
concluded that hesperetin 7-0-(2, 6-dialpha
rhamnopyranosyl)-beta-glucopyranoside is reported for the
first time from this plant .It inhibits the influenza virus.
53
Heggers et al., (2002), studied the mechanisms of
action and in vitro toxicity of grapefruit seed extract, they
concluded that the grapefruit seeds extract (GSE) is a broad-
spectrum antibiotic.
1.4.3 Antiparasitic Activity
Fujioka et al. (1989) tested the antimalarial activity of
thirty acradian alkaloids obtained from plants of genera
citrus. The antimalarial activity of these alkaloids showed
LC50 less than 10 µg/ml in vitro and vivo.
While Bhat and Suroha (2001), reported that the
aqueous and organic solvent extracts obtained from specific
parts of the plants Swertia chirata, Carica papaya and Citrus
sinensid were tested on malaria strain Plasmodium
falciparum FCK2 in vitro. Among the three plants, two had
significant inhibiting effect on the parasite.
Moreira et al (2002), on the other hand, made an
interview on housing conditions, epidemiological aspects,
prevention, standard clinical treatment and alternative
therapies for American tegumentary leishmaniasis in the
Amazon Region in the state of Maranhao, Brazil. Citrus limon
is the plant frequently used and 15.4% of the interviewees
used it as a powder spread on the wound
1.4.4 Citrus fruits and Cardiovascular Diseases
It is well accepted that a diet low in saturated fat and
cholesterol and rich in fruit and vegetables reduces the risk
of heart disease. Therefore, many researchers are looking for
potent antioxidants, which are able to inhibit the low density
54
lipoprotein (LDL) oxidation and thus lower the risk for
atherosclerosis.
Jeon et al (2001), made a comparison between the
antioxidant cholesterol lowering drugs lovastatin and
probucol and the citrus bioflavonoid (naringin) (respectively)
in twenty male rabbits fed with a high cholesterol diet or high
cholesterol diet supplement. They concluded that
thelovastatin and probucol were very potent in the
antioxidative defense system, whereas naringin exhibited a
comparable antioxidant capacity based on increasing the
gene expressions in the antioxidant enzymes, while also
increasing the hepatic superoxide dismultase (SOD) and
catalase (CAT) activities, sparing plasma vitamin E and
decreasing the hepatic mitochondria hydrogen peroxide.
Terpstra et al. (2002) found that citrus peels have a
cholesterol lowering activity on hamsters. Ginter et al (1979)
suggested that pectin and ascorbic acid are forming a
natural hypercholesterolemic agent. On the same line,
Baekey et al (1988) concluded that dietary grapefruit pectin
supplementation inhibits hypercholesterolemia and appears
to be proportionately protective against atherosclerosis.
While, Jeon et al. (2000), comparing the activity of citrus
bioflavonoid, naringin, and the cholesterol lowering drug,
lovastatin, in rabbits fed a high-cholesterol diet
supplemented with either naringin (0.5% cholesterol, 0.05%
naringin, w/w) or lovstatin (0.5% cholesterol, 0.03% lovastatin,
w/w). The results appear to indicate that, naringin plays an
55
important role in regulating antioxidative capacities by
increasing the superoxide dismultase (SOD) and catalase
(CAT) activities, up-regulating the gene expressions of SOD,
catalase, and glutathione peroxide (GSH-PX), and protecting
the plasma vitamin E. In contrast, lovastatin exhibited an
inhibitory effect on the plasma and hepatic lipid
peroxidation and increased the hepatic catalase activity in
high cholesterol fed rabbits.
A low dietary intake of folate (that present in folate-rich
food such as oranges and orange juice) contributes to the
decrease of plasma folate and the raising of plasma
homocysteine (a toxic agent for the vascular wall) levels. The
high level of homocysteine in plasma increases the risk of
cardiovascular disease.
Grassmann et al (2001), found that lemon oil and one of
its components, gamma-terpinene, are efficiently slowing
down the oxidation of LDL. .
Vinson and Jang (2001) indicated that, the combination
of citrus extract and vitamin C increased the lag time of
lipoprotein oxidation, compared with vitamin C alone, and
was a significantly better antioxidant than vitamin E. Kim et al. (1999) separated some polymethoxyflavone form the immature
peels of Citrus unshiu and they studied their antiallergic ability. The results indicated
that 3, 4, 5, 6, 7, 8-hexamethoxyflavone and 5-hydroxy-3, 4, 6, 7, 8-
pentamethoxyflavone inhibited dose-dependently histamine release from the rat
peritoneal mast cells activated by compound 48/80 or anti-dinitrophenyl
immunoglobulin E (anti-DNP IgE).
1.4.5 Citrus fruits and Eye conditions
56
Recent researches have shown a role for two
carotenoids, lutein and zeaxanthin, in protection from
macular degeneration, a major cause of blindness with
aging.
1.4.6 Anticancer Activity of citrus fruits
Cancer has received by far the most attention in the
epidemiological literature in relation to the potential effect of
fruits especially citrus fruits. Many researchers were
interested in this field ( Iwase et al., (2001); Murakami et al,
(1997); Chen et al., (1998); Mak et al (1996) ;Iwase et al.,
(2000); Einspahr et al., (2003); Proteggente et al., (2003); and
Silalahi (2002). They all proved that citrus phytochemicals are
promising anticancer agents.
In 1993, Sugiyama et al. isolated flavones from
methanol extract of Citrus reticulata peels. These flavones
showed differentiation including activity towards mouse
myeloid leukemia cells (MI), which resulted in the
phagocytic activity of the cells.
Hirano et al. (1995) reported that citrus flavone
tangeretin inhibits leukemic HL-60 growth partially with less
cytotoxicity on normal lymphocytes than the chemical
anticancer agents.
Moreover, Takemura et al. (1995) investigated the
inhibitory effect of Epstein Barr Virus (causative agent of
infectious mononucleosis) activation by using 25 alkaloids
from citrus plants. Some of these alkaloids showed
remarkable inhibitory effects.
57
The same results were reached by Iwase et al. (1999),
who investigated the inhibitory effect of-Epstein Barr Virus
(EBV) activation by citrus fruits, a cancer preventer. Iwase
reached this conclusion by doing extracts of fruits peels and
seeds of 78 species of the genus citrus and other closely
related species.
On the other hand, Manthey et al. (1999), isolated
polymethoxylated flavones from citrus. They found that this
flavonoid has an anti-inflammatory response through
suppression of cytokine expression by human monocytes.
Nevertheless, Crowell (1999) studied the possibility of
prevention and therapy of cancer by dietary monoterpenes.
The study revealed that d- limonene which comprises more
than 90% of orange peel oil has chemopreventive activity
against rodent mammary, skin, liver, lung and fore- stomach
cancers. Monoterpenes other than limonene also have
chemopreventive activity against different types of cancers
during the initial phase as reported by Crowell (1999). For
instance, perillyl alcohol has promotion phase
chemopreventive activity against rat liver cancer. Geraniol
has in vivo antitumor activity against maurine leukemia cells.
Miyake et al. (1999), isolated three coumarins from the
flavedo of the lemon peel using high performance liquid
chromatography (HPLC) method. The results suggested that
these coumarins are promising chemopreventive agents by
inhibiting free radical geneation.
58
Hakim et al. (2000) studied the relationship between
citrus peel consumption and human cancers. The results
showed that peel consumption, the major source of dietary
d- limonene, is not uncommon and may have a potential
preventive in relation to skin squamous cell (SCC).
On the same line, Hakim and Harris 2001 investigated
the relationships between citrus peel used and black tea
intake and squamous cell carcinoma of the skin.
Mak et al. (1996) investigated the in vitro effects of
Citrus reticulata peels extract on the growth and
differentiation of recently characterized murine myeloid
leukemic cell clone WEHI 3B JCR. They found that the citrus
peels extracts not only inhibited the proliferation of JCS cells
in a dose dependent manner, but also induced
differentiation of JCS cells into macrophages and
granulocytes, the thing that increases the phagocytic activity
of the cells.
Many researchers studied the cancer chemopreventive
effects of monoterpenes from citrus fruit (Crowell 1999;
Bardon et al. 1998). The results show that citrus monoterpenes
exhibited chemotherapeutic and chemopreventive actions.
They concluded that, monoterpenes are a new class of
therapeutic agent for breast cancer.
59
1.5 Research Objectives
1.5.1 The Overall Aim of the Study:
The use of synthetic chemical insecticides increased
and hence is the increasing awareness of the hazards
associated with this use (Morsey et al., 1998). Amr et al.
(1996), reported hepatitis B virus sermarkers among Egyptian
pesticides applicators. These facts have evoked a worldwide
interest in investigation for safe degradable and target
specific insecticides of plant origin (Jacobson 1958 and
Peterson 1989).
On the other hand, synthetic antibiotics are consistently
over prescribed and wrongly prescribed in the last few
decades, the thing that helps developing of strong strains of
bacteria. 13300 patients died in US hospitals from drug-
resistant infection in one year according to Time Magazine
(March 28th 1994). Half the annual production of antibiotics
is fed to cattle and poultry as a prophylactic and increase
bulk. Resistant bacteria developed in the animals are a
source of dangerous infection to human.
60
On the same time, over 75 million metric tons of citrus
fruits are produced annually throughout the world. Of which
some 60% is sold as fresh fruits, the remainder being taken up
by the citrus fruits processing industry, which produced a
large amount of wastes.
The peels of the citrus fruits are of the most part,
discarded as waste products. So, this work was carried out to
see to what extent such byproducts might be considered as
a potential source of save alternative insecticides and
antibiotics agents.
1.5.2 The specific aims of the study:
(1) To estimate the degree of toxicity of citrus oil (extracted
from the peels) on the different developmental stages of the
mosquito (Culex quinquefasciatus).
(2) To isolate and identify the active antibacterial agents
present in mandarin peel extracts.
(3) To detect the antibacterial activity of lime juice and to
identify its active compound.
61
CHAPTER 2
Materials and Methods
2.1 Materials
2.1.1 Plants
1 Lime peels
2 Orange peels
3 Grapefruit peels
4 Mandarin peels
5 Lime juice
62
2.1.2 Chemicals
Hexane
Chloroform, Riedel-de Haen-Germany
Acetone
Ethanol
Ethyl acetate
Petroleum ether (60-80ºC)
Acetic anhydride
Conc. Sulfuric acid, Merck-Germany
Methanol
Ferric chloride, BDH chemical Ltd. Poole, England
Aluminium chloride
Conc. Hydrochloric acid
Magnesium (turnings)
Lead acetate solution
Potassium hydroxide solution
Acetic acid
Ammonia solution
Gelatin
Normal saline
Sodium chloride
Nutrient agar
Nutrient broth
Silica gel for TLC
Streptomycin powder
Mayer’ reagent
63
2.1.3Apparatus and glasses
GC/MS system
HPLC system
Rotary evaporator
UV-source
Distillator
Autoclave
Oven
Pasteur pipette
Micropipette
Glass for TLC
Petri-dishes
Vials
Plastic trays
Glass jars
Aspirator
.1.4 Laboratory Animals and Microorganisms
(i) Insect: Mosquito (Culex quinquefasciatus)
(ii) Microorganisms: four strains of bacteria:
1 Escherichia coli ---------------------- ATCC 25922
2 Pseudomonas aeruginosa ---------- ATCC 27853
3 Staphylococcus aureus -------------- ATCC 25923
4 Bacillus subtilis ------------------------ NCTC 8236
64
2.2 Methods
2.2.1Preliminary screening for non-nutrient phytochemicals
from peels of citrus fruits
Citrus processing waste streams contain high amounts
of the secondary natural products that normally occur in
peels. Two of the main classes of natural products that have
attracted attention for their potential health promoting
properties include the phenolic compounds and the
triterpenoid limonoids.
(i) Test for unsaturated sterols and triterpenoids (Leibermann-
Buchard test)
1 g of powdered plant material was macerated with
20ml petroleum ether (60-80ºC) for six hours. The extract was
filtered and evaporated to dryness. The residue was
dissolved in acetic anhydride. 2ml was transferred to a test
tube, and concentrated sulfuric acid was added
continuously along the side of the tube. Possible presence of
sterols and/or triterpenenes is indicated by the immediate
appearance of violet color in case of triterpenes, which
changes to green on standing in case of sterols.
(ii) Test for flavonoids
6g of the powdered material were extracted with 100ml
methanol in a soxhlet extractor for three hours. The extract
was evaporated to dryness using rotary evaporator. The
residue was dissolved in 50ml of water and filtered. The
residue and the aqueous extracts were tested for the
possible presence of flavonoidas follow:
65
To a 2ml portion of the extract in a test tube, 5 drops of
2% ferric chloride solution in methanol were added.
Formation of green color may indicate the presence of
flavonoids compounds.
To a 2ml portion of the extract in a test tube, 1ml of 1%
ACL3 solution in methanol was added. Formation of yellow
color indicated the presence of flavonols, flavones, and or
/chalcones (flavonoid compounds).
To a 2ml portion of the extract in a test tube, 2ml of
concentrated hydrochloric acid and 0.2 g magnesium
(turnings) were added. Production of a definite color
changing to pink or red was taken as presumptive evidence
that flavonol or flavanone were present.
To a 2ml portion of the extract in a test tube 1ml of
strong lead acetate solution was added. Formation of yellow
precipitate indicates the presence of flavonoids.
To 2ml of the filtrate in a test tube, 1ml of 1% potassium
hydroxide solution was added. Formation of bright yellow
color indicated the presence of flavonoid compounds
flavones, flvanones, chalcone, and /or flavonols).
(iii) Test for coumarin
A 10ml of aliquot (PE) was extracted twice with
petroleum ether and then concentrated. Few drops were
spotted on a piece of filter paper, dried and examined under
UV light. The presence of coumarin compound is indicated
by the blue fluorescence, which changes to bluish green on
exposure to ammonia vapor.
66
(iv) Test for alkaloids
10g powdered material was macerated with 50ml of
10% acetic acid in 80% methanol for 24 hours. The extract
was filtered and concentrated.2-3 drops of alkaloid reagent
were added for one portion
The remaining portion of the extract was made alkaline with
strong ammonia solution, and allowed to stand for one hour
and filtered. The residue was air dried and extracted with
chloroform. The chloroform extract was evaporated to
dryness and the residue was dissolved in few ml of methanol.
The methanolic solution was acidified with diluted
hydrochloric acid and tested for the presence of alkaloids.
The test was recorded positive when turbidity or precipitate
obtained.
(v) Test for tannins
2ml of an aqueous extract was placed in a test tube
and 2-3 drops of ferric chloride solution were added.
Formation of blue or green color indicates the presence of
tannins.
To 2ml of the aqueous extraction in test tube 2-3 drops
of gelatin or gelatin salt solution (1%) were added. Formation
of white precipitate indicates the presence of tannins.
(vi) Test for saponin
67
5ml of an aqueous extract was placed in a test tube
and 5ml of water was added. The tube was crocked and
shaken vigorously. Formation of persistent foam which
remains stable for at least one hour indicate the possible
present of saponins.
5% of blood suspension in normal saline was prepared.
The aqueous extract was made isotonic with 5ml of sodium
chloride in test tube. A control test tube was carried out by
the addition of 5ml normal saline only to 5ml of the blood
suspension. All the tubes were shaken gently and observed
after 2 hours. In presence of saponin, the blood suspension
containing the aqueous extract shows a homogenous red
color and contains no residue at the bottom of the tube.
2.2.2 Larvicidal activity of citrus oils against Culex
quinquefasciatus larvae
Of the most dangerous pests are mosquitoes. Besides
being very annoying-insects, they transmit many serious
diseases as filariasis, malaria, yellow fever (acute febrile
illness), dengue fever (break-bone fever) and tularaemia
(deer- fly fever).
The common house mosquito Culex quinquefasciatus,
which is the vector of Wuchereria bancrofti in many countries
other than Sudan, had developed resistance to various types
of synthetic insecticides particularly chlorinated
hydrocarbons. In view of this resistance problem, many
research centers have been continuing trying to develop
new insecticides for effective control.
68
The present study reports the larvicidal potency of peel
oils of grapefruit (Citrus paradisi), orange (Citrus sinensis) and
lime (Citrus aurantifolia) on mosquito larvae. These citrus
peels (from which the oils are extracted) are either
discarded as waste or used as animal feed.
Ashbell et al. (1987), reported that peel is nutritionally
rich in protein and a large amount of lactate- assimilating
yeasts. Su et al. (1972a), reported efficiency of citrus oils as
protectans of black-eye peas against Cow weevils, the peel
has also found use as a mosquito repellant among people
living thatched houses near rivers. Anaso et al. (1990) have
demonstrated the potency of orange peel as a mosquito
fumigant. Ayedoun and Sossou, (1996), studied the volatile
constituents of the peel and leaf oils of some citrus species. .
2.2.2. a Collection and maintenance of mosquito
The larvae of Culex quinquefasciatus were collected
from channels at El Grief west using two methods:
I. The classical dipping method: using a tray (20X15X3cm) II
Using dip-net (small nylon gauze net mounted on a circular
frame (15 cm diameter) and attached to a wooden handle
(1m long)
Since the egg rafts and larvae of Culex quinqefasciatus
were floating on the water surface, only the classical dipping
method was used.
The larvae collected from the field were used for
exploratory tests to define the toxicity range of the citrus oils.
Some collected larvae were allowed to develop into pupae
69
and were strained off, washed with clean water, placed in
small bowls and put under cages for emergence to adults
(for colony maintenance).
The temperature of the colony was kept at 27± 3ºC
through out the study period. Emergent adults, were retained
in strictly labeled cages, each was 60X60X60 cm. Adult
mosquito were maintained on 10% aqueous sucrose solution
in a large test tube (cotton were impregnated with this
sucrose solution) and blood from a lived old pigeon
fledgling. The pigeon after having its back carefully and
neatly plucked was tightly in small topless wooden cage
placed snide the mosquito cage.
A petri dish containing water was kept inside the
mosquito cage to provide an egg-laying medium. Egg raft
was transferred into a wide specimen tube half filled with
dechlorinated tap water (as hatching medium) Hatched
larvae were transferred to rearing dishes using Pasteur
pipette. The actual bioassay was carried out on laboratory
bred mosquito larvae only.
2.2.2. b Extraction of Citrus Oils
(i) Cold Pressing
For the preliminary investigation crude oils were
extracted by modification of (Mwaiko, 1992) from the peels
of lime, grapefruit and orange using the presser.
(ii) Steam Distillation
70
For the actual investigation the crude oils were
extracted using the steam distillation method. Appropriate
citrus fruit were selected. The rind was freshly grated using
the finest texture of a common cheese grater. Only the
flavedo, the colored portion of the peel was grated and
avoids abrading the albedo, the white portion of the inner
peel. It is also essential to avoid abrading the pulp to avoid
excessive water contamination.
The fresh grated peels were put in the boiling bottle and
immerse with distilled water. After boiling, the condensed
steam was collected in the receiver. A few milligrams of
sodium anhydrous sulphate were added to the collected oils
to absorb any water mixed with the oil. A clean dry sample
tube was weighed before and after transferring the oil in it.
The oil was kept in a cool, dark place till used.
2.2.2. c Qualitative Analysis of the isolated Citrus Fruit Oils by
GC/MS
The crude citrus oil was diluted with 1.0 ml of
dichloromethane, and 0.25 µl of the resulting solution was
injected (split injection, 92:1) into the GC/MS. The time
interval between evaporation and injection was minimized in
order to prevent oxidation of the essential oils.
Using an interfaced Pentium I I computer running
Shimazu’s Class 5000 software, this referenced NIST libraries
of mass spectra. (NIST12.LIB and NIST62.LIB, V1.0, P/N 225-
01860-93). The capillary column used was a RESTEX XTI-5
capillary column (95% dimethyl and 5% diethyl
71
polysiloxane), 30m in length, 0.25-µm i.d., and 0.25µm - film
thickness. For exact instrumental conditions, refer to table (1.)
Table 1: The typical GC/MS Parameters
Oven
Injection temperature 200ºC
Detector interface temperature 260ºC
Initial temperature 50ºC for 3 min.
Ramp 30ºC per min.
Final temperature 250ºC for 1 min.
Column
Column length 30 m
Column diameter 0.25 mm
Carrier gas Helium, 99.9999% purity
Carrier gas pressure 28.7 Kpa
Column flow 0.7 ml per min.
Linear velocity 30.6 cm per min.
Split ratio 92
Total flow 66.1 ml per min.
Mass Spectrometer
M/Z range 20 to 350 amu
Scan interval 0.5 s
Threshold 1000
Scan speed 1000 amu per s
Solvent cut 3 min.
Detector 1.0 K V
72
2.2.2. d Insect Bioassay
(i) Toxicity on Larvae
Larvae used for the susceptibility tests were obtained
from the eggs produced by adult Culex quinquefasciatus
which were maintained under laboratory conditions
(Magayuka and White 1972).
The early 4th instar larvae of Culex quinquefasciatus
were used. A total of 25 larvae were exposed to water (in to
which ethanol was added to 2%) treated with different
concentrations of citrus oils, orange, grapefruit and lime (12,
24, 36, 48, 60 and 72ppm).
Exposure to extracts was for 24 hours in tap water
according to the (WHO 1970). After these 24 hours, each
group of the treated larvae were carefully washed and
transferred, using Pasteur pipette, to trays containing 250 ml
clean tap water. Food was given as usual.
Each test was repeated four times side by side with
control experiments (in which only water containing 2%
ethanol was used). Experiments were kept under close
observation.
73
Mortality were determined daily until pupation.
Surviving pupae were counted and transferred to jars
containing tap water (inside the cage) for further observation
of death and adult emergence. The mortality percentage
was submitted to probit analysis (Finney 1971).
(ii) Fecundity of mosquito that survived sublethal
concentrations
(a) When treated as larvae
4th instar larvae were exposed for 24 hours to water
treated with LC50 for the three citrus oils (49, 56 and 65 ppm
for orange, grapefruit and lime respectively).
The adult that succeeded to emerge (after exposing to
LC50) was kept and provide with sugar solution and blood
feeding. The eggs laid were counted and left until hatching
occurs. The larvae of the second generation were reared
and the eggs laid by the emerging adults also counted and
left until hatching occur.
(b) When treated as adults
Blood feeding females, were collected from inside
cages using the aspirator, and then exposed to LC50 (that
were calculated from regression line of mortality probit
against logarithm of concentrations) of the three oils in
plastic tubes lined with filter paper impregnated with citrus
oil. They were exposed for 2 hours side by side with the
control experiments.
74
After the 2- hour time the adults were placed in
screening cages and their eggs were counted. The eggs
watched until hatched.
In all cases the percentage of female fecundity was counted
using Crystal and Lachance formula (Crytal and Lachance
1963).
% Fecundity = (number of eggs per treated female / number
of eggs per untreated female) x 100
(iii) Latent Effects of Citrus Oils on Eggs and Developing
Larvae
A random sample of eggs lay by treated adults and
adult resulting from treated larvae were planted. The
numbers of larvae, which develop to pupae, were counted
and place in cages until the adult emergence. Mortality,
pupation and adult progeny were determined.
2.2.2. e Statistical Analysis
Data on toxicity were subjected to double
transformation probit regression analysis according to
Busvine (1957). The results of the analyzed data were
presented in tabular and graphical forms together with
relevant statistical data. In each case the equation of the
straight line: Y = a + b X was computed. In the equation, Y =
probit mortality; a = intercept of the regression line with the
vertical axis; b = the slope (the angle between the regression
line and the horizontal line).
Assessment of acute toxicity was done through the
calculation of the
75
Concentration of the test solution that kills 50 percent of the
population of the insect used (LC50), after 24 hours exposure.
These calculation were carried out to permit comparison as
which oil was more potent under the conditions of the tests.
The lowest the concentration, the more potent the extract
was.
2.2.3 Mandarin Peel Extracts as Antibacterial Agent
Mandarin peels, which are normally discarded as waste
products containing many bioactive compounds of which
flavones are the most group that attracted attention for their
biological activities.
The flavones in citrus are found in glycosylated and
aglycon states, the latter showing a greater variety of
compounds with their structure frequently multisubstituted by
hydroxy and/or methoxy groups. Among these poly
methoxylated flavones are sinensetin, tangeretin,
quercetogetin, and nobiletin.
Polymethoxylated flavones (PMF) are an interesting
group of bioactive compounds present in citrus fruits. Like
other flavonoids, they play an important role in plants, acting
as antioxidants and inhibitors of numerous enzymes such as
phenolases (Challice and Willins 1970) and
pectinmethyltransferases (De Swardt et al. 1967). Moreover,
because they show a characteristic distribution pattern, they
can be used for taxonomic purpose (Ooghe et al 1994).
Furthermore, they have numerous pharmacological
applications due to the antithrombogenic properties of
76
nobiletin and sinensetin, which regulate human blood
erythrocyte concentration and aggregation (Robbins 1974;
1976; Bracke et al. 1994) and cardiotonic action (Itoigawa et
al. 1994).
They have also been shown to have a cytotoxic effect
toward cancerous cell lines (Kupehan et al. 1965) where
nobiletin and tangeretin are more potent inhibitors of tumor
cell growth, due to better membrane uptake of these PMF
(Kandaswami et al. 1991; Francis et al. 1989).
These compounds together with the other components
of the essential oil probably confer a certain degree of
resistance against microbial infections in citrus (Ben-Aziz,
1967; Huet 1982). PMF are also showing anti-inflammatory
properties and they inhibit histamine release thereby
reducing allergic reactions (Middleton and Dzrewiecki 1982).
In the present study the PMF was identified and tested
against microorganisms to see whether such byproduct may
consider as a potential source of natural antibiotics.
2.2.3.1 Inoculums preparation (Jayaprakasha et al., 2000)
Strains of Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, and Bacillus subtilis, were obtained
from the stock culture collection of Microbiology Department
of The National Center For Researches. The bacterial cultures
were maintained at 4ºC on nutrient agar slants and sub
cultured at 15- day intervals. Prior to use, the cultures were
grown in nutrient broth at 37 º C for 24 hours. A preculture
was prepared by transferring 1 ml of this culture to 9 ml
77
nutrient broth and incubated for 48 hours at 37ºC. (One
hundred micro-liters is approximately 10³ cfu/ml).
2.2.3.2 Preparation of plant materials
Mandarin peels were obtained from fresh citrus fruits
bought from the local market at El Grief west, state of
Khartoum, Sudan. The peels were separated, washed
thoroughly in cold distilled water and dried. The dried peels
then finely powdered.
2.2.3.3 Extraction of Plant Materials
Powdered peels (100 g) were successively extracted
with hexane, chloroform and acetone using cold method
(tow days for each) and soxhlet apparatus. The extracts were
filtered and concentrated with Rotary evaporator and the
yield of each extract was calculated. One ml each of
hexane and chloroform extracts was mixed with 20ml of
ethanol. The precipitate formed (alcohol insoluble) was
filtered and the supernatant (alcohol soluble) was
concentrated using rotary evaporator. Then all fractions
(hexane, chloroform, acetone, alcohol soluble and alcohol
insoluble) were tested for antibacterial activity using disc
diffusion method and broth dilution method (to determine
MIC minimal inhibitory concentration).
2.2.3.4 In Vitro Antibacterial activity tests
(i) Agar Diffusion Method
500 ml of nutrient agar medium were distributed in 20
ml into vials and then sterilized in an autoclave. The molten
sterile medium inoculated with 200 µl of the tested organism
78
using micropipette (100 µl) and gently mixed to insure
uniform distribution of the organisms. The inoculated medium
then poured into sterile petri- dishes (95mm internal
diameter) and allowed to solidify at room temperature.
In each solidified medium number of holes (2-5) were
made using sterile crock borer (10mm in diameter). The
holes were filled with the extract (100 µl) of different
concentrations (200, 400, 800, 1600, and 3200µg/ml) and one
hole used as control filled with the solvent used for preparing
the concentration.
The plates were allowed to diffuse at room temperature
for two hours and then incubated in up right down position at
37 ºC for overnight. The diameters of inhibition zones were
measured by viewing the plates against the suitable
background using a ruler. The result was tabulated as
susceptible, intermediate and resistant.
(ii) Liquid Dilution Method (Naganawa et al .1996)
Minimum inhibitory concentrations (MIC) were
determined for the extract that showed high activity using
broth dilution technique .A serial of increasing
concentrations of the plant extracts was made in nutrient
broth inoculated with the tested organisms. The minimum
concentration that shows no any bacterial growth is the MIC.
79
2.2.3.5 Simple Method for Increasing Concentration of the
Active Compounds (New method)
Mandarins were selected and placed in a 37ºC
incubator over night to equilibrate. The next morning a 0.1ml
of Staphylococcus aureus culture was inoculated (using 1 ml
syringe) onto scar of warm mandarin that made using ml
pipette tip. The inoculated mandarin was allowed to stand
overnight at 37ºC in a covered glass container. The peel,
after removed with caution in a hood, was rolled in foil and
sterilized in an autoclave. The peel then dried, powdered
and extracted using the same solvents, hexane-chloroform
and acetone.
All the fractions (hexane-chloroform-alcohol-soluble-
alcohol-insoluble and acetone were tested against S. aureus.
2.2.3.6 Isolation and Purification of the Active Compounds
The ethanol soluble fraction was spotted on TLC and
developed using hexane: EtOAc (85:15 v/v). TLC plates were
sprayed with 10% sulfuric acid in methanol (v/v) and heated
at 110 ºC, for 10 min. The Rƒ (retardation factor) values of the
compounds were calculated. The bands on TLC were
scratched and dissolved with methanol, filtered and
crystallized. The isolated Compounds (1and 2) were
identified using HPLC.
80
2.2.3.7 Antibacterial activity of the isolated compound
Because of the weak yield of the two isolated
compounds, the activity test was done using the total two
compounds, using disk diffusion method and broth dilution
method.
2.2.3.8 Chemical Analysis of the isolated compounds by
HPLC
A quantitative high-performance liquid
chromatography (HPLC) procedure and mass spectrometry
(MS) were help in the determination of the two major
polymethoxylated flavones (PMFs) in mandarin peel extract
has been developed. In HPLC a unique ternary solvent
system with coupled UV-fluoresence detection was
employed.
2.2.4 Antibacterial activity Of Lime Juice
Lime is mostly valued for its juice, which contains sugars
and fruit acids, mainly citric acid. Lime juice displays a
unique, intensive acidity.
In folklore, lime juice is extensively used for treating many
diseases, such as scurvy (a deficiency disease caused by
lack of vitamin C), oral diseases, throat diseases, digestive
problem, fever, hemorrhage in internal organs, rheumatic
affections, obesity, cold, circulatory disorders, cholera, and
recently, there is an interest in the role of lime and lemon
juice as an AIDS protective for developing countries where
vaginal use of lemon juice has been linked to a lower
transmission rate of the HIV (human immunodeficiency virus).
81
A number of studies are currently underway in Australia to
assess whether citrus juice in general and especially lime
and lemon juice may have any unforeseen detrimental
effects used in this way and to see if it can indeed inactivate
the HIV virus in controlled trials. There is also interest in
assessing whether the low pH caused by the citrus juice
could exert a similar microbicidal effect on other bacteria.
2.2.4.1 Preparation of the Juice
The fresh lime fruits were bought from the local market
at El Grief West. The natural, fresh juice was prepared
manually by macerating the fruit with hand and filtered. Then
the juice was concentrated by evaporating by heat.
2.2.4.2 Preparation of Concentrated Juice
3 tubes each containing 40ml of fresh natural lime juice
was evaporated in water bath to 20, 10 and 5ml respectively.
2.2.4.3 Antibacterial Activity of Natural and Concentrated
Lime Juice
The fresh and concentrated juice was tested for
antibacterial activity using disc diffusion method (as
described previously).Both concentrated and natural juices
showed high antibacterial activity.
2.2.4.4 Determination of the Active compound In Lime Juice
As mentioned in the literature that 1 ml from lime juice
contains 58mg/ml citric so one ml of natural juice compared
with 58mg of citric acid dissolved in one ml of distilled water.
They gave the same diameter of inhibition zone. All other
82
concentrations of the juice (2-times, 4-times and 8-times)
gave the same diameter of the equivalent concentrations of
citric acids, 116,232 and 464 mg/ml respectively. This result
proved that the active ingredient is mainly the citric acid.
2.2.4.5 Comparison Between The antibacterial Activity of
Lime Juice and that of streptomycin
The different concentration of the lime juice was
compared with streptomycin (100mg/ml) using the same
procedure, disc diffusion method.
CHAPTER 3
Results
3.1 Preliminary Screening for Non-Nutrient phytochemicals
from peels of citrus fruits
Chemical screening of the non-nutrient phytochemicals
from the peels of citrus fruits (lime, grapefruit, orange and
mandarin) was showing in table (2) Table (2): The non-nutrient-phytochemicals in the peels of citrus fruits
Compoun
d
Test Lime peel Orange
peel
Grapefruit
peel
mandarin
peel
Triterpenoi
ds
Pet. ether
extract
dissolved in
acetic acid.
Appearanc
e of violet
color
(+)
Appearanc
e of violet
color
(+)
Appearanc
e of violet
color
(+)
Appearanc
e of violet
color
(+)
83
Conc.
Sulfuric acid
added
Unsaturat
ed sterol
The above
test
The violet
color
change to
green on
standing
(+)
The violet
color
change to
green on
standing
(+)
The violet
color
change to
green on
standing
(+)
The violet
color
change to
green on
standing
(+)
Flavonoid Pet. Ether
extract
dissolved in
water, 5
drops of
ferric
chloride was
added
Formation
of green
color
(+)
Formation
of green
color
(+)
Formation
of green
color
(+)
Formation
of green
color
(+)
To the
aqueous
extract
above, 1 ml
of 1% ALCL3
in methanol
was added
Formation
of yellow
color (+).
Flavone,
flavonol
and or
chalcone
were
present
Formation
of yellow
color (+).
Flavone,
flavonol
and or
chalcone
were
present
Formation
of yellow
color (+).
Flavone,
flavonol
and or
chalcone
were
present
Formation
of yellow
color (+).
Flavone,
flavonol
and or
chalcone
were
present
Coumarin Few drops
from pet-
ether
extract were
spotted on a
Slight blue
fluorescenc
e (trace)
Slight blue
fluorescenc
e (trace)
Slight blue
fluorescenc
e (trace
Slight blue
fluorescenc
e (trace)
84
piece of
filter paper,
dried and
examined
under UV
light
Alkaloids Plant
powder
material
was
macerated
with 10%
acetic acid
in 80%
methanol, 2-
3 drops of
alkaloid
reagent
(Mayer’s)
were added
No turbidity
or
precipitate
(-)
No turbidity
or
precipitate
(-)
No turbidity
or
precipitate
(-)
No turbidity
or
precipitate
(-)
Tannin 2-3 drops of
ferric
chloride
were added
to an
aqueous
extract
Formation
for blue or
green color
(trace)
Formation
for blue or
green color
(trace)
Formation
for blue or
green color
(trace)
Formation
for blue or
green color
(trace)
Saponin Powder
plant
material
was
Formation
of foam but
not existed
for long
Formation
of foam but
not existed
for long
Formation
of foam but
not existed
for long
Formation
of foam but
not existed
for long
85
vigorously
shaking in
water
time (trace) time (trace time (trace time (trace
3.2 Larvicidal Activity of Citrus Oils Against Culex
quinqiuefasciatus Larvae:
3.2.1The Extracted Oils:
(i) The Yields of the Extracted Oils:
The biological yields of the extracted oils are low, (1.3%,
3.5% and 2.3%) for lime, orange and grapefruit respectively,
but consider the fact that a great quantity of these peels is
generated by citrus juice producing industries.
(ii) The Physical Characters of the Extracted Citrus Oils:
Table (3) shows some of the physical characteristics of
the extracted citrus oils (which all cause eye irritation) Table (3): Some Physical Characters of the Extracted Citrus Oils
Citrus oil Color Odor Taste Acidity Water
solubility
Solubility in
absolute
alcohol
Lime Pale
yellow-
green
Fresh and
pleasant
lemon
smell
Bitter Acidic Insoluble Completely
soluble
Orange Pale
yellow-
orange
Aromatic
flavor of
the fresh
orange
Bitter Acidic Insoluble Completely
soluble
Grapefruit Pale-
yellow
Similar to
fresh
grapefruit
Bitter Acidic Insoluble Completely
soluble
86
(iii) The quantitative analysis of the extracted citrus oils
Tables (4, 5 and 6) show the chemical compositions of
the volatile parts, of the three citrus oils, lime, grapefruit and
orange, that were identified using GC/MS method, in which
one can easily knows that limonene is forming the bulk of the
oils.
87
Table (4) Chemical composition of the extracted lime oil
Peak R.T Compounds Formula % Of
total
1 14.42 1R-∝-Pinene
C10H16 1.73
2 17.35 Bicyclo [3.1.1] heptane, 6,6-dimethyl-
2-methylene-, (1S)-
C10H16 16.07
3 20.66 Limonene
C10H16 32.29
4 21.38 1,3,7-Octariene, 3,7-dimethyl-
C10H16 0.38
5 22.32 1,4-Cyclohexadiene, 1-methyl-4- (1-
methylethyl)-
C10H16 3.01
6 23.97 Cyclohexane, 1-methyl-4- (1-
methylethylidene)-
C10H16 0.32
7 25.11 1,6-Octadien-3-ol, 3,7-dimethyl-
C10H18O 1.47
8 30.84 3-Cyclohexene-1-ol, 4-methyl—1-(1-
methylethyl)-
C10H18O .37
9 31.97 3-Cyclohexene-1-methanol, ∝4- C10H18O 3.21
88
trimethyl-
10 32.31 Decanal
C10H20O 0.65
11 35.17 2,6-Octadienal, 3,7-dimethyl-, (Z)-
C10H16O 13.23
12 36.39 2,6-Octadien-1-ol, 3,7-dimethyl-, (Z)-
C10H18O 2.95
13 37.37 2,6-Octadienal, 3,7-dimethyl-
C10H16O 11.25
14 42.51 1,6-Octadien-3-ol, 3,7-dimethyl-,
acetate
C12H20O 0.47
15 43.85 2,6-Octadien-1-ol, 3,7-dimethyl-,
acetate, (Z)-
C12H20O 0.88
16 44.68 Cyclohexane, 1-ethenyl-1-methyl-2,
4-bis (1-methylethenyl)-, [1S-(1∝, 2∝,
4∝)]-
C15H24 0.55
17 46.76 Caryophyllene
C15H24 1.57
18 47.43 Bicyclo [3.1.1] hept-2-ene, 2,6-
dimethyl-6- (4-methyl-3-pentenyl)-
C15H24 1.83
19 50.51 1 H-Cyclopenta [1,3] cyclopropal [1,2]
benzene, octahydro-7-methyl-3-
methylene-4- (1-methylethyl)-, [3As-
(3∝β, 3b∝, 4∝, 7∝,
C15H24 0.56
89
20 52.13 Cyclohexene, 1-methyl-4- (5-methyl-
1-methylene-4-hexenyl)-, (S)-
C15H24 4.26
21 55.27 Ć-Elemene
C15H24 0.41
22 59.37 (-)-Spathulenol
C15H24O 0.40
Table (5) Chemical composition of the extracted grapefruit oil
Peak
R.T. Compounds Formula % of
total
1 14.46 1R-∝-Pinene
C10H16 0.79
2 16.68 ∝ –Phellandrene
C10H16 0.41
3 17.54 ∝ –Pinene
C10H16 3.19
4 20.45 Limonene
C10H16 92.46
5 21.21 1,3,6-Octatriene, 3,7-dimethyl-,(E)-
C10H16 0.20
6 22.98 2-Furanmethanol, 5-ethenyltetrahydro- C10H18O2 0.91
90
∝, β,5-trimethyl-,cis-
7 24.05 2-Furanmethanol, 5-ethenyltetrahydro-
∝,β5-trimethyl-,cis-
C10H18O2 0.40
8 30.65 3-Cyclohexen-1-ol, 4-methyl-1-(1-
methylethyl)-
C10H18O 0.14
9 31.69 3-Cyclohexene-1-methanol, ∝, β4-
trimethyl-
C10H18O 0.33
10 32.18 Decanal C10H20O 0.31
11 34.43 2,6-Octadienal,3,7-dimethyl-,(Z)- C10H16O 0.11
12 36.43 2,6-Octadienal, 3,7-dimethyl-
C10H18O 0.10
13 43.69 Copaene
C15H24 0.18
14 46.60 Caryophyllene
C15H24 0.26
15 52.60 Naphthalene,1,2,4a,5,8,8a-hexahydro-
4,7-dimethyl-1-(1-methylethyl)-,[1S-
(1∝,4a∝,,8a∝)]
C15H24 0.15
Table (6) Chemical composition of the extracted orange oil
Peak R.T. Compounds Formula % of
total
1 14.36 Bicyclo[3.1.1]hept-2-ene,2,6,6-trimethyl-
,(ñ)-
C10H16 0.64
2 16.60 ∝ –Phellandrene C10H16 0.40
3 17.45 ∝ –Myrcene C10H16 1.81
4 20.21 D-Limonene C10H16 97.15
91
3.2.2 The Toxicity of Citrus Oils Against Culex quinquefasciatus
Larvae
The susceptibility tests carried out using peel oil extracts
of sweet orange (Citrus sinensis), grapefruit (Citrus paradisi)
and lime (Citrus aurantifolia), against Culex quinquefasciatus
larvae, indicated that, these oils might contain potentially
insecticides (P>0.05)
Tables (7-10) summarize the potency of citrus oils on
Culex quinquefasciatus 4th instar larvae. Tables (7-9) showed
that the citrus oil toxicity applied to parental larvae was
extended to pupal and adult stages.
The relative potency indicated that orange and grapefruit
oils were (1.32) and (1.15) times more effective than lime oil
against Culex quinquefasciatus larvae.
Table (7): Toxicity of lime oil on different Developing stages of Culex
qyuinquefasciatus
Concentration (ppm) % Of larval mortality
12 6
24 15
92
36 30
48 37
60 45
72 60
Control 1
Table (8): Toxicity of orange oil on different developing stages of Culex
quinquefasciatus
Concentration (ppm) % Of larval mortality
12 13
24 26
36 38
48 48
60 59
72 82
Control 1
Table (9): Toxicity of grapefruit oil on different developing stages of
Culex quinquefasciatus
Concentration; (ppm) % Of larval mortality
12 8
24 20
36 35
93
48 43
60 54
72 76
Control 1
Table (10): Relative efficiency and sub-lethal concentrations (LC50)
values of Citrus Oils against Culex quinquefasciatus larvae
Citrus oils LC50 in ppm Relative efficiency
Grapefruit 49 1.32
Orange 56 1.15
Lime 65 1.00
3.2.3 Effects on Fecundity of Females That Survive Sub lethal
Concentration:
The results showed that fecundity of the females was
significantly reduced by citrus oils treatment (P > 0.05).
(i) When Treated as Larvae
Table (11) shows that, treated 4th instar larvae with citrus
oils at LC50 caused slight decrease in the number of
deposited eggs per female by (20.35% ,15.17% and 13.39%)
for orange, grapefruit and lime respectively.
Table (11): Fecundity of C. quinquefasciatus that survive LC50 of citrus
oils after treatment of larvae
Citrus oils Average eggs/F % Reduction in
oviposition
Fecundity
94
Orange 89 20.53 79.46
Grapefruit 95 15.18 84.82
Lime 97 13.39 86.60
Control 112 0 0
(ii) When Treated as Adults
Whereas, table (12) showed that the effect on fecundity
was more obvious on the treated adults. Orange, grapefruit
and lime reduced the number of eggs per female by
(54.90%, 31.37% and 24.50) respectively. Table (12): Fecundity of Culex quinquefasciatus that survive LC50 of
citrus oils after treatment of adult
Citrus oils Average eggs/F % Reduction in
oviposition
Fecundity
Orange 46 54.90 45.09
Grapefruit 70 31.37 68.62
Lime 77 24.50 75.49
Control 102 0 0
3.2.4 Latent effects on the developmental stages
Tables (13 and 14) show that larval and adult treatments
with citrus oils caused serious latent effect on the
developmental stages.
In both treated adults and larvae, the application of
citrus oils reduced the hatchability percentages. But, the
effects on hatchability of eggs laid by female resulting from
treated adults were lower than that lay by treated larvae
when the percentages of egg hatching were (74.85% -
95
68.14%), (78.00%–72.88%) and (80.20%- 77.10%) for orange,
grapefruit and lime respectively.
While table (14) shows that, in case of orange and
grapefruit oils treatments only 15.03% and 40.88% of the
larvae formed pupae respectively, whereas, in case of lime
oil 45.3% of the larvae pupated.
On the other hand, treatment of larvae with orange,
grapefruit and lime oils resulted in 9.35%, 20.20% and 25.0%
progeny respectively.
Table (13): Latent effect on the developmental stages of
C.quinquefasciatus resulting from eggs lay by females treated as adults
with citrus oils (LC50)
Citrus oils % Eggs hatched % Pupation % Emergence
Orange 74.85 45.00 41.29
Grapefruit 78.00 46.29 36.29
Lime 80.20 50.33 44.21
Control 95.00 92.00 90.00
Table (14): Latent effect on the developmental stages of
C.quinquefasciatus resulting from eggs lay by females treated as
larvae with citrus oils (LC50)
Citrus oils % Eggs hatched % Pupation % Emergence
Orange 68.14 15.03 9.35
Grapefruit 72.88 40.88 20.20
96
Lime 77.10 45.30 25.00
Control 95 92 92.00
3.3 Antibacterial Activity of Mandarin Peels Extracts
The yields of the different mandarin peels extract were
4.5 %, 1.4 % and 3.0% for hexane extract, chloroform extract
and acetone extract respectively.
3.3.1 Susceptibility of bacteria to the Different Fractions from
Mandarin Peel
The antibacterial activity of different fractions from
mandarin peel (as shown in fig 1-5) shows that all fractions
suppressed the growth of gram-positive bacteria,
Staphylococcus aureus and Bacillus saubtilis at
concentrations lower than that required for gram-negative
bacteria, Escherichia coli and Pseudomonas aeruginosa.
Fig. 6 reveals that ethanol-soluble fraction was the most
active extract against all the bacterial strains. The acetone
was found to be the least effective of the all tested fractions.
97
Fig (1) A histogram showing the antibacterial activity of hexane extract
of mandarin peels, at different concentrations, against the studied
bacterial strains
0
20
40
60
80
100
120
200 400 800 1600 2300
StaphBacEschPseu
98
0 = Resistant
50 = Intermediate
100 = Susceptible
Fig (2) A histogram showing the antibacterial activity of chloroform
extract of mandarin peels, at different concentrations, against the
studied bacterial strains
99
0
20
40
60
80
100
120
200 400 800 1600 2300
StaphBacEschPseu
0 = Resistant
50 = Intermediate
100 = Susceptible
100
Fig (3) A histogram showing the antibacterial activity of acetone
extract of mandarin peels, at different concentrations, against the
studied bacterial strains
0
20
40
60
80
100
120
200 400 800 1600 2300
StaphBacEschPseu
0 = Resistant
50 = Intermediate
100 = Susceptible
101
Fig (4) A histogram showing the antibacterial activity of ethanol-
soluble fraction of mandarin peels, at different concentrations, against
the studied bacterial strains
0
20
40
60
80
100
120
200 400 800 1600 2300
StaphBacEschPseu
0 = Resistant
50 = Intermediate
100 = Susceptible
102
Fig (5) A histogram showing the antibacterial activity of ethanol
insoluble-fraction of mandarin peels, at different concentrations,
against the studied bacterial strains
0
20
40
60
80
100
120
200 400 800 1600 2300
StaphBacEschPseu
0 = Resistant
50 = Intermediate
100 = Susceptible
103
Fig. (6): A photographed plate showing the Inhibition zones caused by
different mandarin fractions against bacteria
A= Hexane
B= Et-insoluble fraction
C= Chloroform
D= Et-soluble fraction
104
3.3.2 Minimum Inhibition Concentration (MIC) For the
Different Fractions from Mandarin Peel Extract
MIC (a complete inhibition of bacterial growth) for the
ethanol – soluble fraction against gram positive bacteria,
Staphylococcus aureus and Bacillus subtilis were observed at
the level of 360 ppm and 600 ppm respectively, while in case
of gram- negative bacteria, Escherichia coli and
Pseudomonas aeruginosa, they were observed at the level of
1440 ppm and 720 ppm respectively (Table 15)
Table (15): Minimum inhibitory concentration MIC (in µg/ml) of
mandarin peel fractions against bacteria
Gram +ve
bacteria
Hexane
extract
Chloroform
extract
Acetone
extract
Ethanol
soluble
fraction
Ethanol
insoluble
fraction
Staphylococcus
aureus
720 960 1200 360 960
Bacillus subtilis
720
840 960 600 960
Gram - ve
bacteria
105
Escherichia coli 1920 1920
2640 1440 2160
Pseudomonas
aeruginosa
1200 1440 1920 720 1200
3.3.3 Increasing concentrations of the active compounds
The minimum inhibitory concentrations of the un-
inoculated peel extracts were higher than that of inoculated
peels extract against Staphylococcus aureus. They were
found to be, 750-400ppm; 960-540ppm; 1200-1100ppm; 360-
200ppm and 960-900ppm, for hexane, chloroform, acetone
ethanol-soluble fraction and ethanol insoluble fraction for un-
inoculated and inoculated peels respectively. This result
proved that the concentration of the active compounds was
increased by inoculation of the peel with the microorganism.
106
Fig. (7): A photographed plate showing the Inhibition zones caused by
Et-soluble fraction of inoculated and un-inoculated mandarin peels
against Escherichia coli
A= Non-inoculated peel extract
B= Inoculated peel extract
107
3.3.4 Identification of the isolated compounds
As was shown in table 16, according to the physical
appearance, melting point, illumination at 365nm UV, Rf-
value, and the results obtained from MS analysis of the TLC
isolated bands, which were identified as pure substances by
HPLC, revealed that these compounds are: Compound1:
Tangeretin (5,6,7,8,4-pentamethoxyflavone)
Compound 2: Nobiletin (5, 6, 7, 3, 4-
hexamethoxyflavone)
Table (16): Physical appearance, Spots under UV (365nm), Rf-values,
retention time RT and Wavelength of the isolated compounds
Compou
nd
Physical
appearan
ce
Spots
at
365n
m
Rf
valu
e
RT λmax Identification
1 Colorless
needles,
mp 156-
Bright
blue
0.35 25.
5
240,271,3
23
Tangeretin
(5,6,7,8,4-
penta-
108
157ºC methoxyflavo
ne)
2 Pale
yellow
needles,
mp 138-
139ºC
Gray 0.30 16.
9
250,270,3
37
Nobletin
(5,6,7,8,3,4-
hexa-
methoxyflavo
ne)
3.3.5 Antibacterial Activity of the isolated compounds
Due to the low yields of the isolated compounds, the
antibacterial activity test was carried out for the whole
flavones (compound 1and 2)
The results revealed that, there was a clear difference
between the effects of the pure compounds and the other
fractions (figure 8). The Staphylococcus aureus was inhibited
very effectively by the isolated flavones. The inhibition was
stronger than that recorded by Mori et al (1987).
109
Fig. (8): A photographed plate showing the Inhibition zones caused by
Et-soluble fraction and Polymethoxylated flavones against
Staphylococcus aureus
110
A= Isolated compounds
B= Et-soluble fraction
3.4 Antibacterial Activity of Lime Juice
111
4.4.1Antibacterial Activity of Natural and Concentrated Lime
Juice
The antibacterial activity tests for the lime juice
indicated that lime juice exhibited strong antibacterial
activity against both gram-positive and gram-negative
bacteria at all concentrations used. As shown in fig. 11
112
Fig. (9): A histogram showing the antibacterial activity of lime juice at
different concentrations.
0102030405060708090
100
natural 2-conc 4-conc 8-c0n
S.aureusB.subtilisE.coliPS.aeuginosa
100 = susceptible
113
3.4.2 Comparison between Antibacterial Activity of Lime
Juice and Citric Acid
Tables (17-19) indicate that the active ingredient in the
lime juice is mainly the citric acid, since it gave the same
activity, in vitro, as that of corresponding concentrations of
lime juice. Fig.12 proved that natural lime juice and citric
acid at concentration of 58 mg/ml have the same inhibitory
effect in vitro Table (17): Diameters of inhibition zones (in mm) caused by natural
lime juice and citric acid at concentration of 58mg/ml
Bacteria Lime juice Citric acid
S.aureus 30 30
B. Subtilis 23 23
E.coli 22 22
Ps. Aeurginosa 25 25
Table (18): Diameters of inhibition zones (in mm) caused by double-
concentrated limejuice and citric acid at concentration of 116mg/ml
Bacteria Lime juice Citric acid
S.aureus 33 33
B. Subtilis 26 26
E.coli 25 25
Ps. aeurginosa 26 26
Table (19): Diameters of inhibition zones (in mm) caused by four-time
concentrated lime juice and citric acid at concentration of 232mg/ml
Bacteria Lime juice Citric acid
114
S.aureus 39 39
B. Subtilis 36 37
E.coli 36 36
Ps. aeurginosa 35 34
Fig. (10): A photographed plate showing Inhibition zones caused by
natural lime juice and citric acid at 58mg/ml against Staphylococcus
aureus
A= Natural lime juice
B= Citric acid at concentration of 58mg/ml
115
3.4.3 Comparison between Antibacterial Activity of Lime
Juice and that of Streptomycin
Table (20) and fig. (13) Revealed that streptomycin
100mg/ml has the same inhibitory effect (in vitro) with the
lime juice that concentrated four times. Table (20): Diameters of inhibition zones (in mm) for tri-concentrated
lime juice, streptomycin (100mg/ml)
Bacteria Streptomycin Lime juice
Staphylococcus.
aureus
38 38
Bacillus subtilis
36 35
Escherichia. Coli
35 36
Pseudomonas
aeurginosa
37 36
116
Fig (11): Inhibition zones caused by four-time concentrated lime juice
and streptomycin 100mg/ml against Escherichia coli
A= Streptomycin at 100mg/ml
B= Lime juice concentrated four times
117
CHAPTER 4
Discussion
Chemicals used as insecticides and antibiotics are
expensive and most of them retain serious side effects, thus
the present study is an attempt to investigate safer and less
expensive substitutes for both.
In this study the essential oils of particular citrus fruits,
lime, orange and grapefruit, were extracted by distillation
from the fresh peels, The yields of the oils were found to be
1.3%, 3.5% and 2.3% for lime, orange and grapefruit
respectively.
The chemical constituents for these oils were analyzed
using gas chromatography coupled with mass spectrometry
(GC/MS). The extraction procedure can be considered as
adequate since all the oils obtained do not contain P-
118
cimene, which is an indicator of oxidation of monoterpenes
in citrus oils. Limonene, a monoterpene compound, was
found to constitute the bulk of the three oils (orange 97.15%,
grapefruit 92.46% and lime 32.29%).
The insecticidal activity of these oils were tested against
mosquito, Culex quinquefasciatus 4th instar larvae. All the oils
showed insecticidal activity. The activity was found to be in
the same order of the limonene percentage i.e. Orange oil
showed the higher activity (with LC50= 49 ppm) followed by
grapefruit (LC50=56 ppm) and lastly lime oil (LC50= 65 ppm).
These results are in consistence with the findings of
Abbassy et al (1979) in Egypt, who studied the activity of
some citrus oils against immature and adult stages of
mosquito, Culex pipiens. The results revealed that, orange
exhibited the highest activity followed by grapefruit and
lastly lime oil.
Also, this result is in agreement with the result of Ezeonu
et al (2001), who found that , orange (Citrus sinensis ) has a
promising insecticidal activity against mosquitoes and
cockroaches.
On the other hand, the present result disagrees with the
result of al- Dakhil and Morsy (1998). They demonstrated the
larvicidal action of three ethanol extracts of peel oils of
lemon, grapefruit and orange. They tested these oils against
the 4th instar larvae of Culex pipiens and the resulting pupae.
The results revealed that lemon has the highest activity
followed by grapefruit and lastly orange oil.
119
This disagreement may be due to the different method
of oil extraction used by authors, the thing that may alter the
chemical composition of the oils.
It was found that the citrus oils used in this study, not
only affect the larval stage, but also, the other developing
stages. Hence affecting the oviposition and hatchability of
mosquito eggs. The effect on the oviposition was found to be
more obvious when the insect was treated as adult. Orange
oil, grapefruit oil and lime oil reduced the oviposition by
54.90%, 31.37% and 24.52% respectively (when the insect
was treated as adult). The reduction in number of eggs per
female was found to be 26.53%, 15.18% and 13.39% for
orange oil, grapefruit oil and lime oil respectively, when the
insect was treated as larvae.
The effect on egg hatchability is more obvious when the
insect was treated as larvae (opposite to effect on
oviposition). Orange oil, grapefruit oil and lime oil gave
hatchability percentages of 74.85%, 78.0% and 80.20%
respectively when the insect was treated as adult. But the
hatchability percentages were lower, 68.14% , 72.88% and
77.10% for orange oil, grapefruit oil and lime oil respectively,
when the insect was treated as larvae.
These results were similar to those reported by Shalaby
et al (1998) who found that the toxicity effect of citrus oils on
larvae extends to the other developmental stages, affecting
the oviposition and hatchability of eggs when they studied
120
the effect of citrus oils against the 4th instar larvae of Culex
pipiens.
When antibacterial activity of mandarin peels was
studied, the peels were dried, powdered and then extracted
successively by hexane, chloroform and acetone .Different
concentrations (200,400,800 1600 and 3200 µg/ml) were
prepared from these extracts and then were tested against
some gram-positive bacteria, Staphylococcus aureus –
Bacillus subtilis, and gram-negative bacteria, Escherichia
coli – Pseudomonas aeruginosa, using disc diffusion method
and broth dilution techniques.
The hexane and chloroform extract showed the highest
activity, whereas, acetone has a low activity. The active
extracts, hexane and chloroform, which may contain the
active compounds were further fractionated into ethanol-
soluble fraction and ethanol-insoluble fraction. Ethanol-
soluble fraction showed the highest antibacterial activity,
amongst all extracts. The activity of ethanol soluble fraction
showed lower minimum inhibition concentration (MIC)
against gram-positive bacteria, than that against gram-
negative bacteria. It showed MIC of 360 ppm, 600 ppm, 1440
pmm and 720 ppm for Staphylococcus aureus, Bacillus
subtilis, Escherichia coli and Pseudomonas aeruginosa
respectively.
Similar results were reported by Jayaprakasha et al
(2000), although the MIC in the present study showed
relatively higher values than those reported by them. This
121
may be due to the fact that, the chemical composition of the
fruit from different areas may vary. They also used the
method of Chen et al (1998), as antibacterial test, in which
the activity was estimated by calculating the number of the
developing bacterial colonies.
The active compounds were isolated from ethanol-
soluble fraction using thin layer chromatography (TLC) and
then identified as nobiletin and tangeretin
(polymethoxylated flavones) by high performance liquid
chromatography (HPLC). These pure compounds showed
higher activity than all other fractions. This may prove that
the antibacterial activity of mandarin peel extracts is mainly
due to the presence of these polymethoxylated flavones.
These results are in agreement with the results of
Jayaprakasha et al (2000) and Vargas et al (1999). The
latter, isolated the antimicrobial and antioxidant compounds
in the non-volatile portion of the expressed orange essential
oils. The results indicated that these compounds were
polymethoxylated flavones.
Inoculation of the peels with the bacteria gave more
active extract
Than un-inoculated peels. This may be due to fact that the
injected bacteria stimulate certain cells to increase the
active compounds or to release new antibacterial agents.
This result may be, to some extent, in consistence with
the findings of Stange et al (1993), who demonstrated the
antifungal compound produced by grapefruit (Citrus
122
paradisi) and Valencia orange (Citrus sinensis) after
wounding of the peel. A new compound was isolated from
the injured peel of grapefruit and orange showing a high
activity as fungicides.
Conclusion
In this study, we conclude that all citrus essential oils
have a potential insecticidal activity against the different
stages of the mosquito Culex quinquefasciatus, but orange
oil gave promising results as future natural insecticides, while
lime juice and flavones from citrus peels may be good
natural preservatives.
Recommendations
1. The insecticidal activity of pure limonene (the active
compound of the citrus fruit oils) should be investigated.
2. Production of antibiotics against specific pathogens by
injection of the citrus peel with that pathogen should take
more interest and more investigation chance.
123
3. Since lime juice is a common ingredient of salad, sauce
and many food types, its use should be further encouraged
to prevent transmission of food born bacteria in the
household during outbreaks
4. The promising anticancer activity of the citrus
phytochemicals should be intensively studied.
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Regression
Descriptive Statistics
49.8333 26.4077 631.0000 20.9284 6
MORTALITCONCENTR
Mean Std. Deviation N
143
Correlations
1.000 .984.984 1.000
. .000.000 .
6 66 6
MORTALITCONCENTRMORTALITCONCENTRMORTALITCONCENTR
Pearson Correlation
Sig. (1-tailed)
N
MORTALIT CONCENTR
Variables Entered/Removedb
CONCENTR
a . Enter
Model1
VariablesEntered
VariablesRemoved Method
All requested variables entered.a.
Dependent Variable: MORTALITb.
Model Summary
.984a .968 .960 5.2691 .968 121.593 1Model1
R R SquareAdjustedR Square
Std. Error ofthe Estimate
R SquareChange F Change df1
Change Statisti
Predictors: (Constant), CONCENTRa.
ANOVAb
3375.781 1 3375.781 121.593 .000a
111.052 4 27.7633486.833 5
RegressionResidualTotal
Model1
Sum ofSquares df Mean Square F Sig.
Predictors: (Constant), CONCENTRa.
Dependent Variable: MORTALITb.
Coefficientsa
11.345 4.100 2.767 .050 -.038 22.721.242 .113 .984 11.027 .000 .929 1.554
(Constant)CONCENTR
Model1
B Std. Error
UnstandardizedCoefficients
Beta
Standardized
Coefficients
t Sig. Lower Bound Upper Bound95% Confidence Interval for
Dependent Variable: MORTALITa.
144
Coefficient Correlationsa
1.0001.268E-02
CONCENTRCONCENTR
CorrelationsCovariances
Model1
CONCENTR
Dependent Variable: MORTALITa.
Collinearity Diagnosticsa
1.851 1.000 .07 .07.149 3.529 .93 .93
Dimension12
Model1
EigenvalueCondition
Index (Constant) CONCENTRVariance Proportions
Dependent Variable: MORTALITa.
Regression
Descriptive Statistics
44.3333 24.5167 642.0000 22.4499 6
MORTALITCONCENTR
Mean Std. Deviation N
Correlations
1.000 .990.990 1.000
. .000.000 .
6 66 6
MORTALITCONCENTRMORTALITCONCENTRMORTALITCONCENTR
Pearson Correlation
Sig. (1-tailed)
N
MORTALIT CONCENTR
145
Variables Entered/Removedb
CONCENTR
a . Enter
Model1
VariablesEntered
VariablesRemoved Method
All requested variables entered.a.
Dependent Variable: MORTALITb.
Model Summary
.990a .980 .975 3.8993 .980 193.657 1 4Model1
R R SquareAdjustedR Square
Std. Error ofthe Estimate
R SquareChange F Change df1 df2 Si
Change Statistics
Predictors: (Constant), CONCENTRa.
ANOVAb
2944.514 1 2944.514 193.657 .000a
60.819 4 15.2053005.333 5
RegressionResidualTotal
Model1
Sum ofSquares df Mean Square F Sig.
Predictors: (Constant), CONCENTRa.
Dependent Variable: MORTALITb.
Coefficientsa
-1.067 3.630 -.294 .783 -11.145 9.0121.081 .078 .990 13.916 .000 .865 1.297
(Constant)CONCENTR
Model1
B Std. Error
UnstandardizedCoefficients
Beta
Standardized
Coefficients
t Sig. Lower Bound Upper Bound95% Confidence Interval for
Dependent Variable: MORTALITa.
Coefficient Correlationsa
1.0006.034E-03
CONCENTRCONCENTR
CorrelationsCovariances
Model1
CONCENTR
Dependent Variable: MORTALITa.
146
Collinearity Diagnosticsa
1.899 1.000 .05 .05.101 4.330 .95 .95
Dimension12
Model1
EigenvalueCondition
Index (Constant) CONCENTRVariance Proportions
Dependent Variable: MORTALITa.
Regression
Descriptive Statistics
40.5000 24.9620 642.0000 22.4499 6
MORTALITCONCENTR
Mean Std. Deviation N
Correlations
1.000 .991.991 1.000
. .000.000 .
6 66 6
MORTALITCONCENTRMORTALITCONCENTRMORTALITCONCENTR
Pearson Correlation
Sig. (1-tailed)
N
MORTALIT CONCENTR
Variables Entered/Removedb
CONCENTR
a . Enter
Model1
VariablesEntered
VariablesRemoved Method
All requested variables entered.a.
Dependent Variable: MORTALITb.
Model Summary
.991a .983 .979 3.6430 .983 230.752 1 4Model1
R R SquareAdjustedR Square
Std. Error ofthe Estimate
R SquareChange F Change df1 df2 Si
Change Statistics
Predictors: (Constant), CONCENTRa.
147
ANOVAb
3062.414 1 3062.414 230.752 .000a
53.086 4 13.2713115.500 5
RegressionResidualTotal
Model1
Sum ofSquares df Mean Square F Sig.
Predictors: (Constant), CONCENTRa.
Dependent Variable: MORTALITb.
Coefficientsa
-5.800 3.391 -1.710 .162 -15.216 3.6161.102 .073 .991 15.191 .000 .901 1.304
(Constant)CONCENTR
Model1
B Std. Error
UnstandardizedCoefficients
Beta
Standardized
Coefficients
t Sig. Lower Bound Upper Bound95% Confidence Interval for
Dependent Variable: MORTALITa.
Coefficient Correlationsa
1.0005.266E-03
CONCENTRCONCENTR
CorrelationsCovariances
Model1
CONCENTR
Dependent Variable: MORTALITa.
Collinearity Diagnosticsa
1.899 1.000 .05 .05.101 4.330 .95 .95
Dimension12
Model1
EigenvalueCondition
Index (Constant) CONCENTRVariance Proportions
Dependent Variable: MORTALITa.
148