Toxicity of the Aqueous and Ethanol Extracts of Ginger

1

Toxicity of the Aqueous and Ethanol Extracts of Ginger

(Zingiber officinale L.) Rhizome and Henna (Lawsonia inermis

L.) Leaves on Mosquitoes (Anopheles and Culex) Larvae

Ahmed Eldokhri Ahmed Adam

B.Sc. (Hons.) in Zoology, Faculty of Science, University of

Dalanj, (2013)

A Dissertation

Submitted to the University of Gezira in Partial Fulfillment of

the Requirements for the Award of the Degree of Master of

Science

in

Biosciences and Biotechnology (Biosciences)

Center of Biosciences and Biotechnology

Faculty of Engineering and Technology

October, 2015

2

Toxicity of the Aqueous and Ethanol Extracts of Ginger (Zingiber officinale L.)

Rhizome and Henna (Lawsonia inermis L.) Leaves on Mosquitoes (Anopheles and

Culex) Larvae


Supervision Committee:

Name Position Signature

Dr.: Mutaman Ali Abdalgadir Kehail Main Supervisor …………………….

Dr.: Abdalla Ibrahim Abdalla Co-Supervisor ……………………

Date: September, 2015

3


Rhizome and Henna (Lawsonia inermis L.) Leaves on Mosquitoes (Anopheles

and Culex) Larvae


Examination Committee:

Name Position Signature

Dr. Mutaman Ali Abdalgadir Kehail Chairperson …………………..

Prof. Elamin Mohamed Elamin External Examiner …………………..

Dr. Faiza Elgily Internal Examiner ……………………..

Date of Examination: /September/2015

4

Dedication In the name of Allah the merciful, the compassionate

With thank to my god I dedicate this work to:

To my beloved father and Mother

To my decent Brothers and Sisters

To my beloved colleagues To my sincere friend

To my family

5

ACKNOWLEDGEMENTS

First, I render Plentiful thanks and praise to Allah the Most

Merciful, for having given me patience, strength and health to

accomplish this work.

Specific thanks to my main Supervisor Dr. Mutaman Ali

Abdelgadir Kehail, for his keen supervision, sound advises; also

plentiful thanks are due to my Co- Supervisor Abdalla Ibrahim Abdalla

for his remarkable support and continuous help.

Thanks are due to my mother and Father. I am also indepted to my

brathers for their continuous encouragement, creation of most suitable

condition, invariable assistance and moral support which encouraged me

for accomplishing this study.

I am also thanks to all my colleagues in Centre of Biosciences and

Biotechnology, Faculty of Engineering and Technology,

University of Gezira.

6


Rhizome and Henna (Lawsonia inermis L.) Leaves on Mosquitoes (Anopheles

and Culex) Larvae


M.Sc. in Biosciences and Biotechnology (Biosciences), October 2015

Center of Biosciences and Biotechnology

Faculty of Engineering and Technology

University of Gezira

Abstract

Natural products are widely used in the fields of pharmaceutical, cosmetics and food

industries, while some of them exerted repellent or toxic effects against insect pests.

Ginger rhizomes and Henna leaves are of the popular materials used in the Sudanese

Homes. This study aimed at determining the toxicity (in term of LC50 and LC95) of the

aqueous and ethanol extracts of Ginger (Zingiber officinale) rhizome and Henna

Leaves (Lawsonia inermis) extracts on the larval stage of Anopheles and Culex

mosquitoes. The Ginger rhizome was brought from the Central market, Wad Medani

City, while Henna leaves were collected (fresh) from Hantoub east of Wad Medani

City also. The mosquito larvae were brought from the Insectory of the Blue Nile

Institute, University of Gezira and were used immediately in the toxicity tests. The

principles of testing the potentiality of these products (toxicity) on mosquito larvae

recommended by the World Health Organization (WHO) were carefully followed.

The submission period was 24 and 48 hours. Each test was triplicates. The

calculations of LC50 and LC95 for all extracts were run using the probit analysis

statistics. The results showed that, Culex larvae were more susceptible than Anopheles

larvae towards all extracts (except the ethanol extract of Ginger). The ethanol extract

was more potent than the aqueous extract against mosquito’s larvae. Respectively, for

Anopheles and Culex larvae, the LC50’s were 199.33 mg/L and 148 mg/L (for the

aqueous extract of Ginger), 171.73 mg/L and 143.67 mg/L (for the ethanol extract of

Ginger), 618.45 mg/L and 538.77 mg/L (for the aqueous extract of Henna leaves), and

173.15 mg/L and 141.11 mg/L (for the ethanol extract of Henna leaves). The

recommendation of this study was to continue investigation on natural products to

determine and add any products potential as mosquito larvicides

7

سمية المستخلص المائي والإيثانولي لرايزومات الجنزبيل وأوراق الحناء علي يرقات بعوض الأنوفلس والكيولكس أحمد آدمأحمد الدخري

2015ماجستير العلوم في العلوم والتقنية البيولوجية )العلوم البيولوجية( أكتوبر مركز العلوم والتقنية البيولوجية

كلية الهندسة والتكنولوجيا جامعة الجزيرة

الملخص العربي

تستخدم المنتجات الطبيعية بصورة كبيرة في مجالات الصناعات الدوائية, مستحضرات التجميل

والغذائية, بينما بعض منها قد أظهر تأثيرات طاردة أو سامة ضد بعض الآفات الحشرية. ريزومات الجنزبيل

منازل السودانية. هدف هذا البحث لتحديد السمية )بدلالة التركيز وأوراق الحناء من المواد الشائعة الإستخدام في ال

(( للمستخلصات المائية والإيثانولية للجنزبيل وأوراق الحناء علي 95LC) %95( و 50LC) %50الكافي لقتل

الأطوار اليرقية لبعوض الأنوفلس والكيولكس. تم إحضار عينات رايزومات الزنجبيل من السوق المركزي, مدينة

مدينة ود مدني أيضاً. تم إحضار يرقات حنتوب شرقود مدني, بينما أحضرت أوراق الحناء )طازجة( من

البعوض من بيت الحشرات لمعهد النيل الأزرق, جامعة الجزيرة والتي إستخدمت مباشرة في إختبارات السمية. تم

هذه المركبات )السمية( علي يرقات إتباع الأساسيات الموصي بها من منظمة الصحة العالمية لإختبار فاعلية

و 50LCساعة. كررت كل تجربة ثلاثة مرات. أجريت حسابات 48و 24البعوض بعناية. كان زمن التعريض

95LC لكل من المستخلص المائي والإيثانولي لكل المستخلصات بإستخدام تحليل بروبيت الإحصائي. أظهرت

بعوض الأنوفلس تجاه كل المستخلصات )عدا المستخلص النتائج أن بعوض الكيولكس أكثر حساسية من

الإيثانولي للجنزبيل(. المستخلصات الإيثانولية أظهرت سمية أعلي نسبياً من المستخلصات المائية ضد يرقات

148ملغ/لتر و 199.33كانت 50LCالبعوض. علي التوالي, بالنسبة ليرقات الأنوفلس والكيولكس, قيم

ملغ/لتر )للمستخلص الإيثانولي 143.67ملغ/لتر و 171.73المائي للجنزبيل(, ملغ/لتر )للمستخلص

ملغ/لتر و 173.15ملغ/لتر )للمستخلص المائي لأوراق الحناء(, 538.77ملغ/لتر و 618.45للجنزبيل(,

تجات ملغ/لتر )للمستخلص الإيثانولي لأوراق الحناء(. توصي هذه الدراسة بمواصلة البحوث علي المن 141.11

ضافة أي منتجات لها القدرة علي قتل يرقات البعوض. الطبيعية لتحديد وا

8

Table of Contents

Subject Page

Dedication iii

Acknowledgements iv

Abstract v

Arabic Abstract vi

Table of Contents vii

List of Tables ix

List of Figure x

CHAPTER ONE: INTRODUCTION 1

CHAPTER TWO: LITERATURE REVIEW 3

2.1 Mosquitoes: 3

2.1.1. Description and biology: 3

2.1.2. Identification: 4

2.1.3. Medical importance: 4

2.1.4. Larvae: 5

2.2. Ginger: Zingiber officinalis Roscoe 6

2.2.1 Morphology 6

2.2.2 Traditional uses 6

2.2.3 Chemistry 7

2.2.4 Nutritional importance 7

2.3. Henna Lawsonia inermis 8

2.3.1 Description 8

2.3.2 Cultivation 8

2.3.3 Uses as a medicine 9

2.3.4 Health effects 9

2.3.5 Chemical composition 9

CHAPTER THREE: MATERIALS AND METHODS 11

3.1. The study Area: 11

3.2. Collection and maintenance of larvae 11

3.3 Plants samples: 11

9

3.3.1 Preparation of extracts: 12

3.4 Bioassay of the test: 12

3.5 Statistical analysis: 12

CHAPTER FOUR: RESULTS AND DISCUSSION 13

4.1. Effect of Z. officinal rhizome on Anopheles and Culex larvae 13

4.1.1 Aqueous extract 13

.4.1 2 Ethanol extract 15

4.2. Effect of Henna L. inermis leaves on Anopheles and Culex larvae 17

.4 2. .1 Aqueous extract 17

.4 2.2. Ethanol extract 19

Chapter Five: Conclusions and Recommendations 23

5.1. Conclusions 23

5.2 Recommendations 23

References 24

10

List of Table and Figure

Table

No.

Title Page

4.1 Effect of aqueous extract of ginger Z. officinal rhizome on

Anopheles and Culex larvae after 24 and 48 hours submission

periods

14

4.2 Effect of ethanol extract of ginger Z. officinal rhizome on

Anopheles and Culex larvae after 24 and 48 hours submission

periods

16

4.3 Effect of aqueous extract of Henna L. inermis leaves on Anopheles

and Culex larvae after 24 and 48 hours submission periods

18

4.4 Effect of ethanol extract of Henna L. inermis leaves on Anopheles


20

4.1 22

11

CHAPTER ONE

INTRODUCTION

Mosquitoes are responsible for transmitting various infectious diseases;

hence, mosquito has been declared as ‘Public Enemy Number One’ (WHO, 1992).

Mosquitoes belonging to the genera Anopheles, Culex and Aedes are the vectors for

the pathogens of different diseases such as malaria, filariasis, Japanese encephalitis,

dengue and dengue haemorrhagic fever, epidemic poly arthritis.

In recent years, mosquito control programs had a setback because of the ever-

increasing insecticide resistance (WHO,1992; Liu et al., 2005). Besides insecticidal

resistance in arthropod vectors, the increased cost of insecticides and increased public

concern over environmental contamination have demanded a continued search for

alternative vector control strategies which would be environmentally safer and

specific in their action (Coats, 1994; Khan and Selman, 1996; Peng et al., 1998).

Extracts from leaves, flowers and roots of plants and oils were found to have

mosquito larvicidal activity (Sharma et al., 1998; Sosan et al., 2001).

Insecticides and fungicides should reduce insect and disease problems; be

target-specific (kill the pest, but not other organisms); break down quickly; have low

mammalian toxicity; and have minimal impact on the environment. Although

synthetic products (e.g., pyrethroids and neonicotinoids) have long been an important

part of pest management, there are risks to using them. As a result of this, as well as

the increased interest in organic gardening, many people are seeking less hazardous

alternatives to conventional pesticides. Lists of products that are acceptable in organic

plant production can be found at the Organic Materials Review Institute (Moreau et

al., 2006)

Zingiber officinalis Roscoe, commonly known as ginger belongs to family

Zingiberaceae is cultivated commercially in India, China, South East Asia, West

Indies, Mexico and other parts of the world. It is consumed worldwide as a spice and

flavoring agent and is attributed to have many medicinal properties. The British

Herbal Compendium reported its action as carminative, anti-emetic, spasmolytic,

peripheral circulatory stimulant and anti- inflammatory (Bradley, 1992).

The principal colouring matter of henna is lawsone, 2-hydroxy-1:4

napthaquinone (C10H6O3) besides lawsone other constituents present are gallic acid,

12

glucose, mannitol, fats, resin (2 %), mucilage and traces of an alkaloid. Leaves yield

hennatannic acid and an olive oil green resin, soluble in ether and alcohol. Flowers

yield an essential oil (0.01-0.02 %) with brown or dark brown colour, strong fragrance

and consist mainly of α-and β-ionones; a nitrogenous compound and resin. Seeds

contain proteins (5.0%), carbohydrates (33.62 %), fibers (33.5 %), fatty oils (10-11

%) composed of behenic acid, arachidic acid, stearic acid, palmitic acid, oleic acid

and linoleic acid. The unsaponified matter contains waxes and coloring matter. The

root contains a red coloring matter (Chaudhary et al., 2010).

Objectives:

To test the toxicity of the aqueous and ethanol extracts of Ginger (Zingiber

officinale L.) rhizome and Henna (Lawsonia inermis L.) leaves on mosquitoes (Anopheles

and Culex) larvae

.

13

CHAPTER TWO

LITERATURE REVIEW

2.1 Mosquitoes:

Mosquitoes belong to the order Diptera, a group of insects that only have one

pair of wing, located on the mesothorax. The hind pair is reduced to small, drumstick-

like organs, the halters. Diptera can only take fluid food, sometimes in the form of

blood. All species go through a complete metamorphosis in the life cycle (Nicholas

and Cowan, 1993).

In the more primitive suborder Nematocera, only females take blood, while

both sexes take sugar, usually from the nectar of flowers. The sugar is necessary for

activity of daily life, while the protein of blood is utilized for egg production (White,

1991).

2.1.1. Description and biology:

Approximately 3200 species of mosquitoes occur worldwide, and classified

into three subfamilies within the family culicidae. Most species of medical importance

are within the subfamily anophelinae (of which the most important genus is

Anopheles), and culicinae (Aedes, Culex and Mansonia) (Nicholas and Cowan, 1993).

Mosquitoes are small insects (about 3-6 mm long) with long legs, a globular

head, laterally compressed thorax and long cylindrical abdomen. The wings are rather

long and narrow and are carried flat over the back in repose. The mouth parts are used

for piercing and sucking. The piercing elements are more or less vestigial in the

males. The antennae are somewhat hairy in the female and bushy in the male. Each

blood meal generally provides enough nutrition for the female mosquito to produce a

batch of eggs, (30-150 in number); female anopheline needs at least two blood meals

before the first batch of eggs can be laid.

Male and female well live for several weeks, feeding repeatedly under natural

conditions. Breeding places of mosquitoes are always in water. Eggs may be

deposited on damp soil or vegetation, in moist tree-holes and sometimes directly on

the water. Larval development takes place about a week for most tropical mosquitoes,

but many temperate species over winter as larvae (White, 1991).

14

2.1.2. Identification:

Most medically important species of mosquitoes have close similarities to

other species which do not bite man. Specific identification may depend on minute

features of the male, the larvae or even the egg, often making it unreliable to identify

individual female specimens. For some species of mosquitoes it may be necessary to

look at their chromosomal characteristics protein or behavior, in order to distinguish

between species which are morphologically identical, or nearly so. The significance

of species complex is that some of the species are pests and vectors of disease, while

others are not (White, 1991).

The genus Anopheles includes over 400 species. A. gambiae Giles complex

are among the major vectors of malaria and filarial in Africa (White, 1974). The

relative significance of the six members of the complex in malaria transmission varies

considerable both with species and location (Colluzzi et al., 1979).

2.1.3. Medical importance:

Mosquitoes may serve as vectors of enzootic (i.e. restricted to animals or

birds) and some zoonotic diseases due to pathogens transmitted from animals to man,

e.g. yellow fever and subperiodic Burgian filariasis (White, 1989).

More than 400 viruses have been isolated and characterized as being arthropod

borne virus. They are all zoonotic, and almost 100 arboviruses have been recorded as

causing clinical symptoms in man, most are transmitted by vector mosquito or ticks

(Varma, 1989).

When Female mosquitoes of the genus Anopheles feed on the blood of an

infected person, any malarial parasites in the blood so taken being a new stage in their

life cycle within the mosquito. If the mosquito now bites a healthy person, the

parasites enter to his blood, destroying large numbers of red blood cells and producing

malaria (Jean, 1982).

Malaria is still the most important cause of fever and morbidity in the tropical

world. It is estimated that over 80% of the world clinical cases of malaria (250-450

million cases annually) and a very high proportion of the world's 1-2 million death

annually from malaria occur in tropical Africa (WHO, 1993). The measures used for

malaria control include these against the parasite through the widespread use of

antimalarial drugs for chemotherapy and chemosuppression and these against the

vectors. It is technically feasible to develop vaccines to protect humans from the

15

several encephalitis viruses, since the basic infection cycles will continue, however,

each new generation will require immunization (Reeves and Hardy, 1985).

2.1.4. Larvae:

The immature stages are always associated with free water, which may occur

in a wide range of locations. The mosquito egg hatches into a minute, worm-like larva

which feeds on microorganisms in water or on the water surface using paired mouth

brushes on the head. Vision is rudimentary but larva reacts rapidly to changes in light

intensity, moving actively with a wriggling or darting motion through water.

The bulky, thoracic part of the larva often has long bristles or hairs, which

assist in achieving balance. The abdomen has ten segments; the tenth segment is at an

angle to the ventral surface and has anal papillae. On the dorsal surface of the ninth

segment are the paired spiracles, sometime on the end of an extended siphon, through

which the larva obtains oxygen from the air/water interface. The larva passes through

four stages or instars molting to the pupal stage (Nicholas and Cowan, 1993).

Larvae will emerge from eggs within 2-3 days when environmental conditions

are ideal. All mosquito larvae go through four developmental stages called instars.

The first instars are barely noticeable to human eye. Last larval instars of some

species can be approximately 1/2 inch (12.7 mm) long. Larvae move through the

water in a serpentine motion when they sense a shadow or movement in their habitat,

larvae will quickly dive to the bottom to avoid the source of disturbance. Larvae are

well formed and lack legs. Upon careful inspection one can distinguish the wider

thorax from the long and slender abdomen. A tube-like structure, called a siphon, is

located at the tip of the abdomen. Larvae use the siphon to breathe air from water

surface. Larvae possessing a siphon (Culicine) hold their body roughly at a 45 degree

angle from the surface. Species of mosquitoes lacking a respiratory siphon

(Anopheline) hold their body horizontal to the water surface. They obtain air through

opening located on the dorsal surface of the abdomen. A few species will bore their

siphon into stems of aquatic plants to obtain oxygen.

Mosquito larvae can be found in a wide variety of habitats, including

temporary floodwater and snowmelt pools; more permanent water habitats like

marshes, swamps, lagoons, and ponds; stagnant waters; and natural and artificial

containers. Shallow water is ideal for larval survival because there is less turbulence

and wave action.

16

Upper water movement interferes with the surface feeding of some mosquito

species, and in most species, it hinders the larvae and pupae from obtaining oxygen at

the air-water interface. A deep-water environment presents bottom-feeding water

from reaching food that has accumulated at the lower levels of the water column.

Water quality in larval habitats can vary from fresh to saline to high in organic wastes.

Different species can tolerate and thrive in water with varying degrees of organic

contents.

Mosquito larvae eat a variety of living organisms, including detritus, algae,

bacteria, and fungi. Some mosquito species are predaceous and feed on other

mosquito larvae and small invertebrates. Depending on water temperature, crowding

and food availability, the larval stage is typically completed in 5-6 days (Renee and

Lourd, 2008).

2.2. Ginger: Zingiber officinalis Roscoe

2.2.1 Morphology

The ginger plant is an erect perennial growing from one to three feet in height. The

stem is surrounded by the sheathing bases of the two-ranked leaves. A club-like spike

of yellowish, purple-lipped flowers have show greenish yellow bracts beneath.

Unfortunately, ginger rarely flowers in cultivation. The ginger of commerce consists

of the thick scaly rhizomes (underground stems) of the plant. They branch with thick

thumb-like protrusions, thus individual divisions of the rhizome are known as "hands"

(Awang, 1992; Bisset and Wichtl, 1994). Rhizomes are 7-15 cm long and 1-1.5 cm

broad and laterally compressed. The branches arise obliquely from the rhizome are

about 1-3 cm long and terminate in depress scars or in undeveloped buds. The outer

surface is buff colored and longitudinally striated or fibrous (Evans, 2002). Fractured

surface shows a narrow cortex, a well marked endodermis and a wide stele (Ali,

1998).

2.2.2 Traditional uses

Ginger is extensively used around the world in foods as a spice. For centuries,

it has been an important ingredient in Chinese, Ayurvedic and Tibb-Unani herbal

medicines. In India the fresh and dried roots were measured distinct medicinal

products. Fresh ginger has been used for cold-induced disease, nausea, asthma, cough,

colic, heart palpitation, swellings, dyspepsia, loss of appetite, and rheumatism. In

short, it is used for the same purposes as in ancient China. In nineteenth century India,

17

one English writer observed that a popular preparation for cough and asthma

consisted of the juice of fresh ginger with a little juice of fresh garlic, mixed with

honey. A glue of powdered dried ginger was applied to the temples to mitigate

headache. To dispel nausea, fresh ginger was mixed with a little honey, topped off

with a nip of burnt peacock feathers. One modern government health guide in India

suggests 1-2 teaspoons of ginger juice with honey as a cough suppressant. Ginger is

as popular a home remedy in India today, as it was 2,000 years ago (Awang, 1992;

Bisset and Wichtl, 1994). The rhizomes of ginger are used as spice in food and

beverages and in traditional medicine as carminative, antipyrexia and treatment of

waist pain rheumatism and bronchitis. It is used for the treatment of gastrointestinal

disorders and piles (Banerjee, et al., 2011).

2.2.3 Chemistry

The pungency of ginger is due to gingerol, an oily liquid consisting of

homologous phenols. It is formed in the plant from phenylalanine, malonate and

hexonate (Evans, 2002). In the fresh ginger rhizome, the gingerols were identified as

the major active components and gingerol [5-hydroxy-1-(4-hydroxy-3-methoxy

phenyl) decan-3-one] is the most abundant constituent in the gingerol series. The

powdered rhizome contains 3-6% fatty oil, 9% protein, 60-70% carbohydrates, 3-8%

crude fiber, about 8% ash, 9-12% water and 2-3% volatile oil. The volatile oil consists

of mainly mono and sesquiterpenes; camphene, betaphellandrene, curcumene, cineole,

geranyl (Yoganarasimhan, 1996).

2.2.4 Nutritional importance

Fresh ginger contains 80.9% moisture, 2.3% protein, 0.9% fat, 1.2% minerals,

2.4% fiber and 12.3% Carbohydrates. The minerals present in ginger are iron, calcium

and phosphorous. It also contains vitamins such as thiamine, riboflavin, niacin and

vitamin C. The composition varies with the type, variety, agronomic conditions,

curing methods, drying and storage conditions (Govindarajan, 1982). Ginger

(Zingiber officinale) has been used as a spice for over 2000 years. Its roots and the

obtained extracts contain polyphenol compounds (gingerol and its derivatives), which

have a high antioxidant activity. Although the digestion stimulating effect of this

spice became known a long time ago, the stimulating effect on peptic juices, such as

gastric juice, bile, pancreatic and intestinal juices, was discovered later. Bile acids

play a major role in the uptake of fats and each upset in the metabolism of fats would

18

impede food digestion as a whole, because the fatty particles cover the other food

elements and make them inaccessible for the action of the digestive enzymes. Lipase

is the other key factor which plays a vital role in fat digestion. When ginger was

included in animal diets, it was found that there was a considerable increase in the

pancreatic and intestine lipase (Platel and Srinivasan, 2000).

2.3. Henna Lawsonia inermis

Also known as hina, the henna tree, the mignonette tree, and the Egyptian

privet is a flowering plant and the sole species of the Lawsonia genus (Bailey and

Bailey, 1976). The name henna also refers to the dye prepared from the plant and the

art of temporary based on those dyes. Henna has been used since antiquity to dye

skin, hair and fingernails, as well as fabrics including silk, wool and leather. The

name is used in other skin and hair dyes, such as black henna and neutral henna,

neither of which is derived from the henna plant (Dennis, 2013)

2.3.1 Description

Henna is a tall shrub or small tree, standing 1.8 to 7.6 m tall (6 to 25 ft). It

is multi-branched, with spine-tipped branchlets. The leaves grow opposite each other

on the stem. They are long and wider in the middle; average dimensions are 1.5–

5.0 cm x 0.5–2 cm or 0.6–2 in x 0.2–0.8 in (Bailey and Bailey, 1976). Henna flowers

have four sepals and a 2 mm (0.079 in) calyx tube, with 3 mm (0.12 in) spread lobes.

Its petals are obvate, with white or red stamens found in pairs on the rim of the calyx

tube. The ovary is four-celled, 5 mm (0.20 in) long, and erect. Henna fruits are small,

brownish capsules, 4–8 mm (0.16–0.31 in) in diameter, with 32–49 seeds per fruit,

and open irregularly into four splits (Kumar et al., 2005).

2.3.2 Cultivation

The henna plant is native to northern Africa, western and southern Asia, and

northern Australasia, in semi-arid zones and tropical areas (Dennis, 2013). It produces

the most dye when grown in temperatures between 35 and 45 °C (95 and 113 °F)

(Bechtold, 2009). During the onset of precipitation intervals, the plant grows rapidly,

putting out new shoots. Growth subsequently slows. The leaves gradually yellow and

fall during prolonged dry or cool intervals. It does not thrive where minimum

temperatures are below 11 °C (52 °F). Temperatures below 5 °C (41 °F) will kill the

henna plant.

http://en.wikipedia.org/wiki/Flowering_plant

http://en.wikipedia.org/wiki/Dye

http://en.wikipedia.org/wiki/Silk

http://en.wikipedia.org/wiki/Wool

http://en.wikipedia.org/wiki/Leather

http://en.wikipedia.org/wiki/Shrub

http://en.wikipedia.org/wiki/Sepal

http://en.wikipedia.org/wiki/Calyx_(botany)

http://en.wikipedia.org/wiki/Stamens

http://en.wikipedia.org/wiki/Ovary

http://en.wikipedia.org/wiki/South_Asia

http://en.wikipedia.org/wiki/Australasia

19

2.3.3 Uses as a medicine

Henna is known as a traditional medicine. It shows various health benefits such

as hypoglycaemic and hypolipidemic activities, inhibits the tuberculosis bacteria, and

useful in skin diseases. Moreover, henna extract prevents the liver damage occurred

from exposure of carbon tetrachloride (Kang and Lee, 2006). According to Ayurveda

for All by Murli Manohar, boiled aqueous extract of henna is effective remedy for the

urinary stones (Manohar, 2011).

2.3.4 Health effects

Henna is known to be dangerous to people with glucose-6-phosphate

dehydrogenase deficiency which is more common in males than females. Infants and

children of particular ethnic groups are especially vulnerable (Bechtold, 2009).

Though user accounts cite few other negative effects of natural henna paste, save for

occasional allergic reactions, pre-mixed henna body art pastes may have ingredients

added to darken stain, or to alter stain color. The health risks involved in pre-mixed

paste can be significant. The United States Food and Drug Administration (FDA)

does consider these risks to be adulterants and therefore illegal for use on skin

(Manohar, 2011). Some pastes have been noted to include: silver nitrate, carmine,

pyrogallol, disperse orange dye, and chromium (Kang and Lee, 2006). These have

been found to cause allergic reactions, chronic inflammatory reactions or late-onset

allergic reactions to hair-dressing products and textile dyes (Dron et al., 2007).

2.3.5 Chemical composition

The plant is reported to contain carbohydrates, proteins, flavonoids, tannins

and phenolic compounds, alkaloids, terpenoids, quinones, coumarins, xanthones and

fatty acids. The plant has been reported to have analgesic, hypoglycemic,

hepatoprotective, immunostimulant, anti-inflammatory, antibacterial, antimicrobial,

antifungal, antiviral, antiparasitic, antitrypanosomal, antidermatophytic, antioxidant,

antifertility, tuberculostatic and anticancer properties. It is now considered as a

valuable source of unique natural products for development of medicines against

various diseases and also for the development of industrial products. The principal

colouring matter of henna is lawsone, 2-hydroxy-1:4 napthaquinone (C10H6O3)

besides lawsone other constituents present are gallic acid, glucose, mannitol, fats,

resin (2 %), mucilage and traces of an alkaloid. Leaves yield hennatannic acid and an

olive oil green resin, soluble in ether and alcohol. Flowers yield an essential oil (0.01-

20

0.02 %) with brown or dark brown colour, strong fragrance and consist mainly of α-

and β- ionones; a nitrogenous compound and resin. Seeds contain proteins (5.0%),

carbohydrates (33.62 %), fibers (33.5 %), fatty oils (10-11 %) composed of behenic

acid, arachidic acid, stearic acid, palmitic acid, oleic acid and linoleic acid. The

unsaponified matter contains waxes and coloring matter. The root contains a red

colouring matter (Chaudhary et al., 2010).

21

CHAPTER THREE

MATERIALS AND METHODS

3.1. The study Area:

Wad Medani is located in the central part of Gezira state, Sudan and hence it

the mean status of habitat and susceptibilities of mosquitoes in the Gezira. Different

localities within Wad Medani city were selected for sampling mosquito larvae. A few

bushes and grasses were noticed to be scattered in these areas and no other

distinguishing features were observed.

3.2. Collection and maintenance of larvae:

A white enameled dipper was preferred, because this allows to see the larvae

most easily. According to WHO (1992) the dipper was used as follows:-

1. The dipper was lowered gently into the water at an angle of about 45o until

one side is just below the surface.

2. Care was taken during dipping, not to disturb the larvae and thus cause

them to swim down wards.

3. The dipper was moved along the breeding site, skimming the surface of the

water.

4. The dipper was left out of the water, so as not to spill the water containing

the larvae.

Mosquito’s larvae, pupae and other aquatic organisms were collected during

dipping and were put in large plastic container. Then larvae of each species

(Anopheles and Culex) were separated and transferred to the beakers of the toxicity

tests by means of a plastic dropper.

3.3 Plants samples:

The samples of Ginger (Zingiber officinal) rhizome was brought (dry) from

Wad Medani local market, Gezira State, whereas Henna (Lawsonia inermis) leaves

were collected (fresh) from Hantoub east of Wad Medani City at early morning and

the collected samples were immediately cleaned manually, dried in shade under room

temperature away from the direct sunlight. After being dried, the plant samples

(ginger rhizome and henna leaves) were grounded and kept in plastic containers for

the further steps.

22

3.3.1 Preparation of extracts:

Each dried sample was grounded to fine powder by using an electrical blender.

1.0 g of each powder was soaked into 10 ml of distilled water or ethanol in 10 ml

conical flasks for 24 hours and then filtered by using a filter paper. The dry weight of

the non-dissolved powder (filtrate) of each plant product into each solvent were

measured as same as the volume of each resulted extract, so as to calculate the

original concentrations (stock concentrations) in term of w/v (mg/ml).

3.4 Bioassay of the test:

In series (normally, 5 concentrations x 3 replicates for each extract) of beaker

(each of 250 ml volume), each of which was filled with a total volume of 250 ml tap

water, provided with 20 Anopheles or Culex larvae (of the third or early forth instars),

and applied with volumes of 0.5, 0.75, 1.00, 1.50 and 2.00 ml from the stock

concentration of each extract, in addition to one beaker as control, the toxicity tests

were run, following the instructions of WHO (1981). The submission periods were 24

and 48 hours. The signs of death to record the mortality data follow Busvine (1971).

3.5 Statistical analysis:

The concentrations used were transformed to the corresponding logarithms,

whereas, the mean (of three replicates) corrected mortalities were transformed to

probit (Finney, 1936). Simple regression analysis was run for log’s (X variable) and

probits (Y variable) so as to obtain the intercept (a) and the regression coefficient (b;

the slope), in addition to R2; the homogeneity factor. By using the regression equation

(Y = a + bX), the lethal concentrations; LC50 and LC95 (in mg/L) were calculated by

substituting Y (in each regression equation) by 5 and 6.64, respectively, according to

Abbott (1925). The least the LC value, the best will be the extract.

23

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1. Effect of Z. officinal rhizome on Anopheles and Culex larvae

4.1.1 Aqueous extract

The stock concentration of the aqueous extract of ginger rhizome was 200

mg/5 ml solvent (40 mg/L for each 1 ml). Volumes of 0.5, 0.75, 1, 1.5 and 2 ml from

this stock concentration were added separately to 250 ml water and the resulted tested

concentrations were 80, 120, 160, 240 and 320 mg/L. Table (4.1) showed that, the

corresponding corrected mortalities were 8, 18, 30,63 and 80%, respectively, in

Anopheles larvae, and 13, 15, 38, 83 and 93%, following the same order in Culex

larvae, just after 24 hours, with LC50 and LC95 of 199.53 and 547.14 mg/L,

respectively, for Anopheles larvae and 164.48 and 366.07 mg/L, followed the same

order for Culex larvae.

After 48 hours, the resulted mortalities did not differed than that resulted after

24 hours by more than 13% in most cases in Anopheles larvae, and by 8% in most

cases in Culex larvae. The R2 ranged between 0.93 and 0.99 in Anopheles and Culex

larvae during the two test periods, and seemed to reflect great homogeneity between

concentrations used and the corresponding mortalities.

The LC50’s values revealed that, Culex larvae (LC50 = 164.48 mg/L) were

more susceptible to ginger rhizome aqueous extract than Anopheles larvae (LC50 =

199.53 mg/L), i.e. the aqueous extract of ginger rhizome was relatively more toxic to

Culex larvae than Anopheles larvae.

Isman (2000), reported that Bio-efficacy of the essential oils of Mentha piperita,

Z. officinalis, Emblica officinalis, and Curcuma longa were evaluated for their

larvicidal activity against Spodoptera litoralis, M. domestica, Leptinotarsa

decemlineata, and Bovicola ocellatus. Essential oil of some plants including and Z.

officinale produced significant larval mortality against two mosquito species.

However, the highest larvicidal activity was observed in the essential oil from Z.

officinale against Cx. tritaeniorhynchus and An. subpictus (Marimuthu, 2011).

Rhizomes are externally effective as insect repellent against houseflies (Asolkar et

al., 1992).

24

Table (4.1) Effect of aqueous extract of ginger Z. officinal rhizome on Anopheles


48 hours 24 hours

Concentration Transformed

Probit value

Mortality Transformed

Probit value

Mortality

Culex An. Culex An. Culex An. Culex An. Log mg/L

4.08 4.16 18 20 3.87 3.59 13 8 1.90 80

4.26 4.69 23 38 3.96 4.08 15 18 2.08 120

5.25 4.75 60 40 4.69 4.48 38 30 2.20 160

5.95 5.67 83 75 5.95 5.33 83 63 2.38 240

7.05 6.48 98 93 6.48 5.84 93 80 2.51 320

Regression analysis

-5.65 -3.04 -5.46 -3.71 Intercept

4.95 3.70 4.72 3.78 Slope

0.94 0.94 0.93 0.99 R2

141.75 148.00 164.48 199.53 LC50

303.97 413.25 366.07 547.14 LC95

25

Products isolated/derived from Curcuma longa (turmeric) and Z. officinale (ginger)

have also been found effective as insect antifeedant and insect growth regulators

(Agarwal and Walia, 2003).

The active toxic material in ginger are arylalcanones such as gingerols and

gingerol analogues (Jiang et al., 2005) and most activities of ginger were attributed to

gingerol (Ahui et al., 2008). Rhizome of ginger contains tannins and flavonoids, some

of which have been identified as kaempferide, galangin and alpinin (Sastry, 1961)

.4.1 2 Ethanol extract

The stock concentration of the ethanol extract of ginger rhizome was 190 mg/

4 ml solvent (47.5 mg/L for each 1 ml). Volumes of 0.5, 0.75, 1, 1.5 and 2 ml from

this stock concentration were added separately to 250 ml water and the resulted tested

concentrations were 95, 143, 190, 285 and 360 mg/L as was showed in Table (4.2). So

the same table showed, the corresponding corrected mortalities were 15, 30, 43, 85

and 98%, respectively, in Anopheles larvae, and 8, 13, 67, 90 and 100%, following

the same order in Culex larvae, just after 24 hours, with LC50 and LC95 of 171.73 and

351.12 mg/L, respectively, for Anopheles larvae and 178.49 and 329.5 mg/L,

followed the same order for Culex larvae.






The LC50’s values revealed that, Culex larvae (LC50 = 178.49mg/L) were less

susceptible to ginger rhizome ethanol extract than Anopheles larvae (LC50 = 171.73

mg/L), i.e. the ethanol extract of ginger rhizome was relatively less toxic to Culex

larvae than Anopheles larvae.

26

Table (4.2) Effect of ethanol extract of ginger Z. officinal rhizome on Anopheles


48 hours 24 hours


Probit value

Mortality

Transformed

Probit value

Mortality


4.08 4.42 18 28 3.59 3.96 8 15 1.98 95

4.33 4.95 25 48 3.87 4.48 13 30 2.16 143

5.84 5.13 80 55 5.44 4.82 67 43 2.28 190

7.05 6.64 98 95 6.28 6.04 90 85 2.45 285

- - 100 100 - 7.05 100 98 2.56 360

Regression analysis

-9.49 -4.70 -8.87 -6.80 Intercept

6.68 4.50 6.16 5.28 Slope

0.91 0.88 0.91 0.94 R2

147.63 143.67 178.49 171.73 LC50

259.82 331.13 1000 351.12 LC95

27

4.2. Effect of Henna L. inermis leaves on Anopheles and Culex larvae

.4 2. .1 Aqueous extract

The stock concentration of the aqueous extract of Henna leaves was 200 mg/2

ml solvent (100 mg/L for each 1 ml). Volumes of 0.5, 0.75, 1, 1.5 and 2 ml from this

stock concentration were added separately to 250 ml water and the resulted tested

concentrations were 200, 300, 400, 600 and 800 mg/L. (Table, 4.3) showed that, the

corresponding corrected mortalities were 15, 23, 28, 45 and 65%, respectively, in

Anopheles larvae, and 15, 15, 40, 73 and 93%, following the same order in Culex

larvae, just after 24 hours, with LC50 and LC95 of 618.45 and 3194.1 mg/L,

respectively, for Anopheles larvae and 423.08 and 1000 mg/L, followed the same

order for Culex larvae.






The LC50’s values revealed that, Anopheles larvae (LC50 = 618.45mg/L) were

less susceptible to ginger rhizome aqueous extract than Culex larvae (LC50 = 423.08

mg/L), i.e. the aqueous extract of henna leaves was relatively less toxic to Anopheles

larvae than Culex larvae.

Bakhshi, (2014) recorded that the highest toxic effect of L. inermis was found at

4000 ppm and the lowest at 4 ppm against larval stages -1 and -2 of Anopheles

stephensi. Govindarajan and Sivakumar (2011), reported that tannin and tannic acid of

L. inermis cause the damage on epithelial membrane of gut of mosquito larvae. So

this compound is responsible for mortality of mosquito larvae. In fact, extract of L.

inermis is comprised of tannin and tannic acid and agent to damage the epithelial

membrane and suppress the immunity system of mosquito larvae.

28

Table (4.3) Effect of aqueous extract of Henna L. inermis leaves on Anopheles and

Culex larvae after 24 and 48 hours submission periods

48 hours 24 hours


Probit value

Mortality

Transformed

Probit value

Mortality


4.08 4.08 18 18 3.96 3.96 15 15 2.30 200

4.16 4.33 20 25 3.96 4.26 15 23 2.48 300

5.18 4.61 57 35 4.75 4.42 40 28 2.60 400

5.77 4.95 78 48 5.61 4.87 73 45 2.78 600

7.05 5.61 98 73 6.48 5.39 93 65 2.90 800

Regression analysis

-7.64 -1.61 -6.53 -1.42 Intercept

4.93 2.42 4.39 2.30 Slope

0.91 0.93 0.91 0.95 R2

584.43 538.77 423.08 618.45 LC50

788.05 2565.62 1000 3194.1 LC95

29

.4 2.2. Ethanol extract

The stock concentration of the ethanol extract of Henna leaves was 200 mg/5

ml solvent (40 mg/L for each 1 ml). Volumes of 0.5, 0.75, 1, 1.5 and 2 ml from this

stock concentration were added separately to 250 ml water and the resulted tested

concentrations were 80, 120, 160, 240 and 320 mg/L, as was showed in Table (4.4),

which showed that, the corresponding corrected mortalities were 18, 33, 35, 65 and

83%, respectively, in Anopheles larvae, and 23, 28, 38, 95 and 100%, following the

same order in Culex larvae, just after 24 hours, with LC50 and LC95 of 173.15 and

604.58 mg/L, respectively, for Anopheles larvae and 138.57 and 304.32 mg/L,

followed the same order for Culex larvae.






The LC50’s values revealed that, Culex larvae (LC50 = 138.57 mg/L) were

more susceptible to ginger rhizome aqueous extract than Anopheles larvae (LC50 =

173.15 mg/L), i.e. the aqueous extract of henna leaves was relatively more toxic to

Culex larvae than Anopheles larvae.

30

Table (4.4) Effect of ethanol extract of Henna L. inermis leaves on Anopheles and

Culex larvae after 24 and 48 hours submission periods

48 hours 24 hours


Probit value

Mortality

Transformed

Probit value

Mortality


4.95 4.33 48 25 4.26 4.08 23 18 1.90 80

5.13 4.69 55 38 4.42 4.56 28 33 2.08 120

5.25 5.00 60 50 4.69 4.61 38 35 2.20 160

7.05 5.61 98 73 6.64 5.39 95 65 2.38 240

- 6.48 100 93 - 5.95 100 83 2.51 320

Regression analysis

-3.34 -2.33 -5.28 -1.76 Intercept

4.18 3.41 4.80 3.02 Slope

0.74 0.94 0.77 0.95 R2

98.90 141.11 138.57 173.15 LC50

244.10 427.07 304.32 604.58 LC95

31

48 h-Cu 24 h-Cu 48 h-An 24h-An Product

141.75 164.48 148.00 199.53 Aq-z

147.63 178.49 143.67 171.73 Eth-z

584.43 423.08 538.77 618.45 Aq-hen

98.90 138.57 141.11 173.15 Eth-hen

32

33

CHAPTER FIVE

CONCLUSIONS AND RECOMMENDATIONS

5.1. Conclusions

1- The aqueous extract of ginger rhizome was relatively more toxic to Culex larvae

than Anopheles larvae.

2- The ethanol extract of ginger rhizome was relatively more toxic to Culex larvae


3- The aqueous extract of henna leaves was relatively more toxic to Culex larvae than

Anopheles larvae.

4- The ethanol extract of ginger rhizome was relatively more toxic to Culex larvae


5.2 Recommendations

1- To continue investigation on natural products to determine and add any products

potential as mosquito larvicides.

2- Other plant products should also be tested against mosquito larvae.

3- The tested plant extract can be used as an alternative larvicidal compound for

the IPM programs.

34

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