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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142

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