3.1. biology of plant materials 3.1.1. taxonomy of acalypha...

3. MATERIALS AND METHODS 3.1. Biology of plant materials

3.1.1. Taxonomy of Acalypha indica L.

Kingdom : Plantae

Division : Angiospermae

Class : Dicotyledones

Sub class : Monochlamydeae

Series : Unisexuales

Family : Euphorbiaceae

Binomial : Acalypha indica L.

Tamil: Kuppaimeni. English: Indian Acalypha. The plant occurs

throughout the plains of India. An erect annual herb with 30-75 cm in

height, leaves are ovate and mosaic type in arrangement (Plate X-a).

Phytochemical constituents

Kaempferol, acalyphamide amides, quinine, sterols and cyanogenic

glycoside

Medicinal Properties

The plant is used as expectorant. It has a diuretic action. It is a

useful remedy for bronchitis, asthma and pneumonia; also for rheumatism.

The leaves are used for scabies. A decoction of the leaves is given for ear

ache. The plant is used in congestive headaches. The powder of the dry

leaves is used in bed sores and wounds caused by worms. The leaf of this

plant is said to be antiparasitic (Nadkarni, 1976; Kumar et al., 2007).

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3.1.2. Taxonomy of Annona squamosa L.

Kingdom : Plantae


Class : Dicotyledones

Sub class : Polypetalae

Series : Thalamiflorae

Order : Ranales

Family : Annonaceae

Binomial : Annona squamosa L.

Tamil Sitaaphalam, Atta, English: custard apple, sugar apple,

sweet-sop.

The plants are widely distributed in the tropical regions of the

world. Now it is cultivated throughout India. Native places are South

America and the West Indies. The plant is a small tree about 3 to 8 metres,

more or less an evergreen tree. Leaves are simple and the entire flower is

yellowish green perianth type. The fruit has a pleasant flavour (Plate X-b).

Phytochemical constituents

Higenamine, anonaine, roemerine, isocordine, glaucine, camphor,

squamamolane, etc.

Medicinal properties

Roots are used as purgative for mental depression and spinal

disorders. Leaves are used as insecticidal and for destroying lice. Fruit is

sweet, haematinic, cooling, sedative, stimulant, expectorant, good remedy

for anemia and burning sensation. Seed is aborti facient, insecticides, and

destroying lice in the hair (Nadkarni, 1976; Jaswanth et al., 2002).

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3.1.3. Taxonomy of Vitex negundo L.

Kingdom : Plantae


Class : Dicotyledonae

Sub class : Gamopetale

Series : Bicarpellatae

Order : Lamiales

Family : Verbenaceae

Binomial : Vitex negundo L.

Tamil: Nochchi, English: five leaved chaste, Indian privet.

Hindi : Nirgundi

The plant is almost entirely tropical or subtropical in distribution. It

is a large aromatic shrub, upto 4.5 metres in height. It is common in waste

place around villages, river banks, moist localities and deciduous forests.

Leaves are simple and they are 3-7 foliate quadrangular stems. Fruit

consists of four nutlets and when ripens, it is black is colour

(Plate X-c).

Phytochemical Constituents

Beta-sitosterol (stem), Casticin (leaf), Lerteolin, (stem),

Leucoanthocyanidins (stem), n-Hentriacontane, n-Nonacosane,

p-Hydoroxybenzoic acid, Tritriacontane, vanillic acid etc.

Medicinal properties

Leaves are antimalarial, parasiticide, plasmodicide, protisticide,

antibacterial, antimutagenic, cancer preventive, pesticide, etc. Stem is

aldose-reductase-inhibitor, anti-HIV, antiallergic, antibacterial,

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anticarcinomic, anticataract, antidermatic, antiherpetic, antitumor, etc.

Seed is anti-inflammatory cosmetic, diuretic, anti-mutagenic, pesticide

etc. (Kirtikar and Basu, 1991; Sivarajan and Balachandran, 2002).

3.2. Biology of mosquitoes

3.2.1. Systematic position of Aedes aegypti Liston

Phylum : Arthropoda

Class : Insecta

Subclass : Pterygota

Order : Diptera

Family : Culicidae

Genus : Aedes

Species : aegypti

Geographic distribution

Aedes aegypti is the most important vector in the tropics and sub

tropical regions such as Southern United States and Asia. Aedes aegypti is

the primary vector for dengue fever. It also transmits the chickengunya

and yellow fever viruses.

Egg laying

Eggs are laid singly directly on the damp soil that will be flooded

by water. Most eggs hatch into larvae within 48 h (Plate VI-a).

Life span

The life span of this mosquito is determined by the abiotic factors

particularly temperature.

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Breeding places

They breed in areas of stagnant water, such as flower vases,

uncovered barrels, buckets, and discarded tyres, but the most dangerous

areas are the wet shower floors and toilet tanks, as they allow the

mosquitoes to breed right in the residence.

Travel

Aedes mosquitoes are strong fliers and are known to fly many miles

from their breeding sources.

Biting activity

Aedes mosquitoes are painful and persistent biters, attacking during

daylight hours. They do not enter dwellings, but they prefer to bite

mammals like humans.

Preferred food

Male mosquitoes feed on plant juices and nectar, while females

feed only blood meal from human being. The mouth parts of male

mosquitoes are adapted to feed on plant juices while female mouth parts

are adapted to feed on blood from mammals.

Vector

They transmit viruses which cause dengue fever, chickengunya,

yellow fever and other diseases.

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Characteristic features

A pair of characteristic lyre shaped white marking present on meso

thorax. Tibia without white rings; Hind tarsi completely white mid femora

without preapical white spot.

Mating

Generally mating is initiated by males. Male mosquitoes are poly

gamous while females are eurygamous. The duration of the mating is

2-5 min.

Resting

It commonly rests outdoors (exophilia) at day time.

Biology

The life cycle of Aedes aegypti includes egg, larva, pupa and adult.

The duration of life cycle depends on abiotic and biotic factors.

Eggs

Aedes lay their eggs on damp soil that will be flooded by water.

Most eggs hatch into larvae within 48 h.

Larvae

The larvae live in water from 7 to 14 days depending on water

temperature. Larvae must come to the surface at frequent intervals to

obtain oxygen through a breathing tube called siphon. The larvae eat algae

and small organisms which live in water. The larvae is metamorphosed

into pupa (Plate VII-a).

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Pupa

The pupa lives in water from 1 to 4 days, depending upon the

temperature. The pupa floats at the surface of water. It takes oxygen

through two breathing tubes called “trumpets”. The pupa does not eat. The

adult mosquito splits the papal case and emerges to the surface of water

where it rests until its body gets dry and hard (Plate VIII-a).

Adult

The adult Aedes have a slender body. It is composed of head,

thorax and abdomen.

The head is black in colour and it contains two compound eyes and

proboscis. The head is 0.6 mm long and 0.5 mm broad. The silvery flat

scales are on tori and eyes. The proboscis is 2.2 mm long, straight

cylindrical and black in colour.

The thorax has one pair of wings and one pair of halters. The

specialized marking in the form of lyre is on thorax. The forewings are 3.8

mm long, 0.95 mm broad, unspotted only with dark scales, squama and

anular convex, transparent, fringe on hind margin. The halter is 0.18 mm

long, 0.14 mm road, yellow, rounded at tip. The hind leg is 8.5 mm long,

slender, longer than body. A narrow basal white band is on 1st, 2nd and 3rd

tarsal segment of fore and hind legs.

The abdomen is 3.7 mm long, 0.7 mm broad. The colour is

blackish brown but white patches are also found (Plate IX-a).

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3.2.2. Systematic position of Anopheles stephensi Liston.

Phylum : Arthropoda

Class : Insecta


Order : Diptera

Family : Culicidae

Genus : Anopheles

Species : stephensi


Anopheles stephensi is limited to tropical areas, most notoriously

the regions of sub-Saharan Africa. Anopheles that can transmit malaria are

found not only in malaria- endemic areas, but also in areas where malaria

has been eliminated. The latter areas are thus constantly at rise in the

re-introduction of disease.

Egg laying

Adult females lay 50-200 eggs per oviposition. Eggs are laid

singly directly on water and are unique in having floats on either side

(Plate VI-b).

Life span

The duration from egg to adult varies considerably among species

and is strongly influenced by ambient temperature. It can develop from

egg into adult in 5 days but usually takes 10-14 days in tropical

conditions. Their life span depends on temperature, humidity and also

their ability to successfully obtain blood meal while avoiding host

defences.

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Breeding Places

The breeding places of A. stephensi are fresh or salt marshes,

mangrove swamps, rice fields, grassy ditches, the edges of streams, rivers

and small temporary rain pools.

Travel

Mosquitoes are not strong flyers, making only 1-2 km/h.

Biting activity

They are puscular (active at dusk or dawn) or nocturnal (active at

night). They feed indoors (endophagic) and or feed outdoors (exophagic).

Preferred food

Male mosquito feed on plant juices, nectar, and other sources of

sugar while female feed only blood meal from human (anthrophilic) and

cattle (zoophilic). Mouth parts of a male mosquito are not developed for

piercing and they do not suck blood but females have well developed

piercing and sucking type of mouthparts which are helpful for the blood

sucking behaviour.

Vector

Mosquitoes are a vector agent that carries disease- carrying viruses

and parasites from person to person without catching the disease

themselves. Anopheles can transmit malarial parasite plasmodium.

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Anopheles mosquitoes can be distinguished from other mosquitoes

by the palps, which are long as the proboscis and by the presence of

discrete blocks of black and white scales on the wings. Adult anopheles

can also be identified by their typical resting position. Males and females

rest with their abdomens sticking up in the air rather than parallel to the

surface on which they are resting.

Mating

Adult mosquitoes usually mate within few days after emerging

from the pupal stage. In A. stephensi the males form large swarms, usually

around dusk, and the females fly into the swarms to mate.

Resting

It commonly rests indoors (endophilia) both before and after

feeding.

Biology

The life cycle of an Anopheles mosquito includes four stages

namely egg, larva, pupa and adult. The duration of the life cycle depends

on the abiotic factors.

Eggs

Adult females lay 50-200 eggs per ovi position. Eggs are laid

singly directly on water and are unique in having floats on either side.

Eggs are not resistant to drying and hatch within 2-3 weeks in colder

climates (Plate VI-b).

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Larvae

Anopheles larvae have a well-developed head with mouth brushes

used for feeding, a large thorax and a segmented abdomen. They lack a

respiratory siphon and for this reason they position themselves so that

their body is parallel to the surface of water (Plate VII-b).

Larvae breathe through spiracles. The larvae feed on algae, bacteria

and other microorganisms in the surface micro layer. Larvae

metamorphoses into pupae.

Pupae

The pupa is comma shaped. The head and thorax are merged into

cephalo thorax with a curved abdomen. After few days as a pupa, the

dorsal surface of the cephalo thorax splits and the adult mosquito emerges

(Plate VIII-b).

Adult

The adult Anopheles has slender bodies with the sections namely

head, thorax and abdomen. The head of the adult is specialized for

acquiring sensory information and for feeding. Three pairs of legs and a

pair of wings are attached to the thorax. The abdomen is specialized for

food digestion and egg development. These segmented body parts expand

considerably when a female sucks blood (Plate IX-b).

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3.2.3. Systematic position of Culex quinquefasciatus Say.

Phylum : Arthropoda

Class : Insecta


Order : Diptera

Family : Culicidae

Genus : Culex

Species : quinquefasciatus


Culex quinquefasciatus is probably the most abundant mosquito in

towns and cities of the tropical countries. This is an important vector of

Japanese encephalitis virus. Culex mosquitoes are found more or less

worldwide, but they are absent in the extreme northern parts of the

temperate zones.

Egg laying

Females lay single raft of 140-340 eggs on heavily polluted small

water collection after each blood meal. Eggs hatch in one to two days

(Plate VI-c).

Life span

The life cycle from egg to adult may be completed in 8-12 days in

tropical countries, but it may vary in temperate regions from 10 to 14

days.

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Breeding places

The breeding places of Cx. quinquefasciataus vary considerably.

All types of polluted water habitat like, ditches, sewage plants, septic tank

and cess pool act as potential breeding habitat for mosquitoes.

Travel

Mosquito can travel upto 3,600 feet in height.

Biting activity

In general, Culex bite in the evening or in the early part of the

night. It is an indoor biting insect.

Preferred food

The mouthparts of the males are not developed for piercing and

they do not suck blood. Their food consists of plant juices, nectar and

other liquids. Female mosquitoes feed only blood meal from human

beings (anthrophilic) and cattle (zoophilic).

Vector

It is the vector of St. Louis encephalitis virus (SLE), West Nile

virus (WN) and Easter equine encephalitis virus (EEE).


Medium-sized mosquito of brownish appearance; proboscis dark

but often with some pale scaling mid way on the underside; scutellum

with golden and bronzy narrow scales; wings are dark scaled; abdominal

tergites dark scaled with pale basal bands constricted laterally and not

merging with lateral patches except perhaps on terminal segments,

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sternites generally pale scaled but with a few to more dark scales scattered

medially.

Mating

Usually mating is initiated by males. Females are eurygamous and

mate only once and they store male gametes in spermatheca only once.

Male mosquitoes are polygamous and they mate more than once. The

duration of mating is 3-5 min.

Resting

It commonly rests indoors (endophilia) both before and after

feeding.

Biology

The life cycle of a mosquito includes four stages namely egg, larva,

pupa and adult. The duration of the life cycle depends on the abiotic factor

particularly temperature and biotic factors.

Eggs

The female lay eggs on stagnant water. The eggs are mainly laid on

polluted water. The eggs are brown, long, cylindrical and cigar shaped

tapering at one end. The eggs are laid at night time. The eggs are hatched

in 1-2 days and larva emerges from the lower end of each egg

(Plate VI-c).

Larva

The larvae are called “wrigglers” because of the wriggling

movement and they are microscopic at hatching time. There are four

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active larval instars and larval life lasting from 3 to 4 days, based on the

temperature. During this period, it moulds four times. The larva breathes

air through siphon and comes to the surface to take-in air while resting,

the larvae pierces the surface film of water by its siphon which projects

just above the surface and draws in air and hangs by the siphon with its

head downwards (Plate VII-c).

Bottom feeders, larvae float obliquely with the head lowermost.

There are no palmate hairs. A long respiratory siphon is present on the 8th

abdominal segment. In tropical countries the period from egg hatching to

pupation can be as short as 7-14 days, while in temperate areas the larval

period may last several weeks or months (Plate VIII-c).

Adult

The body is small, soft, slender and covered with brown scaling

scutum and accessory pale scaling on the lower surface of the proboscis.

Culex quinquefasciatus measures about 3-4 mm in length. It has a body

well built with stouter legs. At rest, the body lays parallel with surface;

wings not spotted; palpi short in female; scutellum is trilobed (Plate IX-c).

Frequently but not always the thorax, legs and wings, veins of the

adult are covered with sombre-coloured often brown scales. The abdomen

is often covered with brown or blackish scale but some whitish scales may

also occur on most segments. The tip of the abdomen of female is blunt.

The claws of all tarsi are simple and those of hind tarsi are very simple.

Examination under a microscope shows that all tarsi have a pair of small

fleshy pulvilli. In tropical countries adult female mosquitoes probably live

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an average of 1-2 weeks, whereas in temperate countries adult longevity is

likely to be 3-4 weeks.

3.3. Screening of mosquitoes

The mosquitoes were collected from ten resting places (old Bus

stand, Srinivasapuram, Medical college, Kodimarathu moolai, Keelavasal,

Karanthai, Nanjikkottai, Mela Vasthadachavedi, Tholkappiar square and

Mariyammankovil) in and around Thanjavur (Feb.2006 to Jan.2007),

Thanjavur district, Tamil Nadu, India (Plate I and II). Adult mosquitoes

were collected with the help of suction tube from different resting places

viz., cattle shed, human dwellings, mixed dwellings, etc. The collected

adult mosquitoes were killed with ether, packed in cellophane paper and

transported to the Research Laboratory of Zoology Department, A.V.V.M.

Sri Puhspam College (Autonomous), Poondi, Thanjavur, for further

studies. Mosquitoes were identified using the key of Christophers (1933),

Barraud (1934) and Catalog of Knight and Stone (1977) (Plate III and

XII).

3.3.1. Method of collection of larvae

The method adopted by Russel and Baisas (1935) was followed to

collect the larvae from cesspits, cesspools, drainages, septic tank, tree

holes, discarded tiers, containers and manmade or natural pond. To find

out whether the habitat contained the larvae or not, first the water was

collected with the help of white background spoon. If there is mosquito

larvae in the sample water, the standard dipper of 250 ml capacity with 3

feet handles were used. One side of the dipper has a wire mesh through

which water is filtered out and the larvae were retained inside. The

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collections were carried out twice a month. Three dips were made in each

spot (Plate IV).

The collected larvae were placed in plastic containers and the

nature of habitat, date and time of collection were labelled and identified.

Then the larvae were discarded.

3.4. Collection of plant materials

Leaves of Acalypha indica L., Annona squamosa L. and Vitex

negundo L. were collected from Botanical and Research Garden of

A.V.V.M. Sri Pushpam College, Poondi, Thanjavur district, Tamil Nadu,

India. Voucher specimen of each plant sample was drymounted,

photographed and preserved for future reference and deposited in the

herbarium of the Postgraduate and Research Department of Botany,

A.V.V.M. Sri Pushpam College, Poondi, Thanjavur district, Tamil Nadu.

3.5. Extraction of plant materials (Harborne, 1984)

Leaves of Acalypha indica, Annona squamosa, and Vitex negundo

were washed with dechlorinated water, dried in shade and powdered with

the help of an electric blender. The test materials (1.0 kg) were extracted

with different organic solvents viz., acetone, hexane, petroleum ether,

chloroform and ethanol in a soxhlet apparatus for 8 h and the extract was

concentrated in a rotary vacuum evaporator to yield crude extract, which

was used in bioassays (Plate XI-a, b).

3.6. Larvicidal Assay

Larvicidal bioassay was carried out as per the protocol of WHO

(1996). Beaker of 500 ml capacity containing 250 ml of water and 25

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numbers of early IV instar mosquito larvae of Ae. aegypti, An. stephensi

and Cx. quinquefasciatus were taken separately to treat in various

concentrations of plant extracts. Five different concentrations of each

plant extract were taken to carry out experiments, with six replicates.

Control was maintained by 1 ml of solvent in 249 ml of water and

mortality was recorded after 24 h separately. Larvicidal experiments were

carried out separately under controlled laboratory conditions (temperature

27 2°C) against laboratory reared Ae. aegypti, An. stephensi and

Cx. quinquefasciatus larvae.

3.7. Oviposition deterrent activity (Xue et al., 2001)

Adult Ae. aegypti, An. stephensi and Cx. quinquefasciatus were

collected from study areas and maintained at 27 2°C; 70-80% relative

humidity and a photoperiod of L:D 14:10. Larvae of each test mosquito

were fed with 3:1 mixture of dog biscuits and yeast powder. Adults were

provided with a 10% sucrose solution and were periodically blood fed on

restrained 5-7 week-old chicks. Six days old adult mosquitoes were used

for oviposition deterrent activity.

The oviposition deterrent test of hexane extract of Acalypha indica,

petroleum ether extract of Annona squamosa and ethanol extract of Vitex

negundo were performed using the method of Xue et al. (2001). Fifteen

gravid female Ae. aegypti, An. stephensi and Cx. quinquefasciatus were (6

days old, 4 days after blood feeding) transferred to each mosquito cage

(45x38x38 cm) separately covered with a plastic screen, with a glass top

and a muslin sleeve for access. A 10% sucrose solution was available at

all times. Serial dilutions of leaf extract of each test plant was made in

ethanol. Enamel bowls containing 100 ml of water treated with leaf

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extract to obtain test solutions of 0.01, 0.025, 0.050, 0.075 and 1.0%. Two

enamel bowls holding 100 ml of water were placed in the opposite corners

of each cage, one treated with the test material and the other with a solvent

control (1 ml). The positions of the bowls were alternated between the

different replicates so as to nullify any effect on oviposition. Five

replicates for each concentration were run, with cages placed side by side

for each bio-assay. All experiments were run at ambient temperature (27

2°C) with a relative humidity of 70-80%. After 24 h, the number of eggs

laid in treated and control bowls were recorded.

The per cent effective repellency for each plant leaf extract

concentration was calculated using the following formula:

ER(%) = NC- NT

100 (%)NC

where, ER = Per cent effective repellency

NC = Number of eggs in control

NT = Number of eggs in treatment

3.8. Skin repellent activity

The duration of protection period from all the three mosquito bites

provided by each test plant leaf extracts were determined separately using

the method of Fradin and Day (2002).

From the stock solution of hexane extract of Acalypha indica,

petroleum ether extract of Annona squamosa and ethanol extract of Vitex

negundo, each test plant leaf extract was diluted with ethanol to obtain test

solutions of 0.001, 0.005, 0.01, 0.015 and 0.02%. For each test solution,

10 disease free, laboratory reared, unfed adult mosquitoes of test species

that were between 8 and 14 days old were introduced into separate

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laboratory cages (45 38 38 cm) (Plate V). Before each test, the skin of

volunteers was washed with unscented soap, and the leaf extract of each

test plant being tested was applied from elbow to fingertips. After the

application, the arm was not rubbed, touched, or wetted. An arm treated

with ethanol was served as control. In each cage, one arm of the volunteer

was inserted into the cage for one test solution. Both test solution and

control were repeated five times in separate cages. In each replicate,

different volunteers were used to nullify any effect of skin differences on

repellency. Volunteers were asked to follow the testing protocol.

Volunteers conducted their test of each concentration by inserting their

treated arm and control arm into a separate cage for one minute at every

five minutes. If they were not bitten within 20 min, then the arms were

reinserted for one minute at every 15 min, until the first bite occurred.

3.8.1. Qualitative Analysis

Phytochemical analysis of the plant extracts was undertaken using

standard qualitative methods as described by various authors (Kapoor et

al., 1969; Odebiyi and Sofowora, 1990). The plant extracts were screened

for the presence of biologically active compounds such as alkaloids,

flavonoids, carbohydrates, phytosterols, proteins, phenolics, tannins and

saponins.

Preparation of plant extracts

The leaves were cleaned and dried in shade for 7 days, then ground

well to fine powder. About 500 g of dry powder was extracted with

methanol (80%) at 70°C by continuous hot percolation using soxhlet

apparatus. The extraction was continued for 24 h. The methanolic extract

was then filtered and kept in hot air oven at 40°C for 24 h to evaporate the

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methanol from it. A dark brown residue was obtained. The residue was

kept separately in air tight containers and stored in a deep freezer.

Alkaloids (Salehi Surmaghi et al., 1992)

Dragendroff’s test (Kraut reagent – Potassium bismuth iodide)

8 g of Bi (NO3)3 5 H2O was dissolved in 20 ml of HNO3 and 2.72 g

of potassium iodide in 50 ml of water. They were mixed and allowed to

stand till KNO3 got crystallized. The supernatant was decanted and made

upto 100 ml with distilled water. The alkaloids were regenerated from the

precipitate by treating with Na2CO3 followed by extraction of the liberated

base with ether. To 0.5 ml of plant extract 2 ml of HCl was added. Then 1

ml of reagent was added to this acidic medium. An orange red precipitate

was produced immediately, which indicated the presence of alkaloids.

Wagner’s reagent (Iodine-Potassium iodide solution)

1.2 g of iodine and 2.0 g of potassium iodide were dissolved in 5

ml of H2SO4 and the solution was diluted to 100 ml. 10 ml of plant extract

was acidified by adding 1.5% v/v HCl and a few drops of Wagner’s

reagent. The formation of a yellowish brown precipitate confirmed the

presence of alkaloids.

Meyer’s reagent (Potassium mercuric iodide)

1.36 g of mercuric chloride was dissolved in 60 ml of distilled

water and 5 g of potassium iodide in 10 ml of water. The two solutions

were mixed and diluted to 100 ml with distilled water.

A few drops of the reagent were added to 1 ml of the plant extract.

The formation of a pale precipitate showed the presence of alkaloids.

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Flavonoids (Somolenski et al., 1972)

In a test tube containing 0.5 ml of plant extract, 5-10 drops of

diluted HCl and a small piece of zinc or magnesium were added, and the

solution was boiled for a few minutes. In the presence of flavonoids, a

reddish pink or dirty brown colour was produced.

Carbohydrates

Fehling’s test (Kokate, 1994)

Solution A: 34.65 g of copper sulphate was dissolved in water and made

upto 500 ml.

Solution B: 125 g of potassium hydroxide and 173 g of Rochelle’s salt

(sodium potassium tasrtarate) were dissolved in water and made upto

500 ml.

The solutions ‘A’ and ‘B’ were added. The contents were boiled

for a few minutes. The formation of a red or brick red precipitate indicated

the presence of carbohydrates.

Benedict’s test

173 g of sodium citrate and 100 g of sodium carbonate were

dissolved in 500 ml of distilled water. 17.3 g of copper sulphate dissolved

in 100 ml of distilled water was added to the above solution.

To 0.5 ml of plant extract, 5 ml of Benedict’s reagent was added

and boiled for 5 min. The formation of a bluish green colour showed the

presence of carbohydrates.

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Proteins

Million’s test (Walsh and Farrel, 1961)

One part of mercury was digested with 2 parts of concentrated

HNO3 and the resulting solution was diluted with 2 volumes of water.

To a small quantity of plant extract, 5-6 drops of Millon’s reagent

was added. A white precipitate which turned red on heat indicated the

presence of proteins.

Phenols (Malick and Singh, 1980)

To 1 ml of plant extract, 2 ml of distilled water followed by a few

drops of 10 per cent aqueous FeCl3 solution were added. Formation of a

blue or green precipitate indicated the presence of phenols.

Lead acetate test

1 ml of plant extract was diluted to 5 ml with distilled water and

then a few drops of 1 per cent aqueous solution of lead acetate was added.

Appearance of yellow precipitate indicated the presence of phenols.

Libermann’s test

A small amount of plant extract was dissolved in 0.5 ml of 20 per

cent sulphuric acid solution followed by the addition of a few drops of

aqueous sodium nitrate solution. A red colour was obtained on dilution

and it turned blue when made alkaline with aqueous sodium hydroxide

solution, which indicated the presence of phenol.

Saponins (Malick and Singh, 1980)

In a test tube containing about 5 ml of plant extract, a drop of

sodium bicarbonate was added. The mixture was shaken vigorously and

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kept for 3 min. Formation of a honey comb like froth showed the presence

of saponins.

Tannins (Segelman et al., 1969)

Ferric chloride test

To 1-2 ml of plant extract, a few drops of 5 per cent aqueous FeCl3

solution were added. A bluish black colour was formed, which then

disappeared on addition of few ml of dilute H2SO4. This was followed by

the formation of yellowish brown precipitate.

Lead acetate test

In a test tube containing about 500 ml of plant extract, a few drops

of 1 per cent solution of lead acetate was added. Formation of yellow or

red precipitate indicated the presence of tannins.

Phytosterols (Malick and Singh, 1980)

About 0.5 ml of test solution was mixed with minimum quantity of

chloroform and the 3-4 drops of acetic acid and one drop of concentrated

H2SO4 were added. Formation of a deep blue or green colour showed the

presence of steroids.

3.8.2. Quantitative Analysis

Estimation of total carbohydrate (Hodege and Hofreiter, 1962)

Carbohydrates are the important components of storage and

structural materials in the plants. They exist as free sugars and

polysaccharides. Carbohydrates were quantitatively estimated following

the method of Hodege and Hofreiter (1962). Carbohydrates were first

hydrolyzed into simple sugars using dilute hydrochloric acid. In hot acid

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medium, glucose was dehydrated to hydroxyl methyl purpurate. This

compound formed a green colour product in addition to anthrone with an

absorption maximum at 630 nm.

100 mg of the sample was weighed and kept in a boiling tube. It

was hydrolysed by keeping in a boiling water bath for three hours with

5 ml of 2.5 N HCl and cooled to room temperature. It was neutralized

with solid sodium carbonate until the effervescence ceases. The volume

was made upto 100 ml and centrifuged. The supernatant was collected and

0.5, 1 ml aliquots were taken for analysis. The volume was made upto

1 ml in all the tubes by adding distilled water. Then 4 ml of anthrone

reagent was added to each tube and kept in a boiling water bath for 8 min.

The tubes were cooled rapidly and the green to dark green colour was read

at 630 nm. Standard graph was prepared by plotting concentration of the

standard on the X-axis versus absorbance on the Y-axis. From the graph,

the amount of carbohydrate present in the sample tube was calculated,

using the following formula:

Total carbohydrate =mg of glucose

100 volume of test sample

Estimation of Protein (Lowry et al., 1951)

The carbamyl group of protein molecules reacts with copper and

potassium of the Lowry’s reagent to give a blue coloured complex,

together with tyrosine and phenolic compounds present in the proteins

reduce the phosphomolybdic, phosphotungstate compounds in the Folin’s

coicalteau reagent.

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500 mg of each sample were homogenized thoroughly with 5 ml of

distilled water and centrifuged at 5,000 rpm for 10 min. The supernatant

was collected and cooled, to which 5 ml of 10 per cent TCA was added

and centrifuged at 3,000 rpm for 5 min. The precipitated pellet was

dissolved in 3 ml of 0.1 N NaOH and the total soluble protein content was

determined as follows.

0.2 ml of the extract was taken and made upto 1 ml with distilled

water. To this 5 ml of 0.1 per cent CuSO4 and 2.5 ml of 12.5 per cent

Na2CO3 was added and allowed to stand for 10 min. Then 0.5 ml of Folin-

Ciocalteau reagent (25%) was added. Similarly blank and standard were

prepared. The intensity of the blue colour developed was colorimetrically

read at 660 nm. Protein content was expressed as mg protein per gram

fresh weight of the sample. This was calculated by plotting OD values on

the graph using BSA (Bovine Serum Albumin) as standard.

Alkaloids (Harborne, 1973)

5 g of sample was weighed and taken into a 250 ml beaker. 200 ml

of 10 per cent acetic acid in ethanol was added, covered with aluminium

foil and allowed to stand for 4 h. This was filtered and the extract was

concentrated on a water bath to one quarter of the original volume.

Concentrated ammonium hydroxide was added dropwise to the extract

until the completion of precipitation. The whole solution was allowed to

settle and the precipitate was collected and washed with dilute ammonium

hydroxide and then filtered. The alkaloid residue was dried and weighed.

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Flavonoids (Boham and Kocipai-Abyazan, 1974)

10 g of plant sample was extracted repeatedly with 100 ml of 80

per cent aqueous methanol at room temperature. The whole solution was

filtered through Whatman No.42 filter paper. The filterate was later

transferred into a crucible and evaporated into dryness over a water bath

and weighed.

Tannins (Van-Burden and Robinson, 1981)

500 mg of the sample was weighed and transferred to a 100 ml

plastic bottle. 50 ml of distilled water was added and shaken for 1 hour in

a mechanical shaker. This was filtered into a 50 ml volumetric flask and

made upto the mark. Then 5 ml of the filterate was pipetted out into a test

tube and mixed with 2 ml of 0.1 M FeCl3 in 0.1 N HCl and 0.008 M

potassium ferrocyanide. The absorbance was measured at 120 nm within

10 min.

Phenols (Malick and Singh, 1980)

The powdered sample was boiled with 50 ml of ether for the

extraction of the phenolic components for 15 min 5 ml of the extract was

pipetted into a 50 ml flask, then 10 ml of distilled water was added. 2 ml

of ammonium hydroxide solution and 5 ml of concentrated amyl alcohol

were also added. The samples were made upto the volume (50 ml) and left

to react for 3 min for colour development. This was measured at 505 nm.

Saponins (Obadoni and Ochuko, 2001)

The samples were ground well and 20 g of each was put into a

conical flask and 100 cm3 of 20 per cent aqueous ethanol was added. The

samples were heated over a hot water bath for 4 h with continuous stirring

59

at about 55°C. The mixture was filtered and the residue was re-extracted

with another 200 ml ethanol. The combined extracts were reduced to 40

ml over water bath at about 90°C. The concentrate was transferred into a

250 ml separating funnel and 20 ml of diethylether was added and shaken

vigorously. The aqueous layer was recovered while the ether layer was

discarded. The purification process was repeated and 60 ml of n-butanol

was added. The combine n-butanol extracts were washed twice with 10 ml

of 5 per cent aqueous sodium chloride. The remaining solution was heated

in water bath. After evaporation the samples were dried in the hot air oven

to constant weight; the saponin content was calculated and represented in

terms of percentage.

Phytosterols (Malick and Singh, 1980)

0.5 g of the dried samples were hydrolyzed with 10 ml of

concentrated HCl. Then samples were extracted three times with 10 ml of

chloroform. The chloroform was evaporated to obtain the residue. The

residue was weighed and dissolved in glacial acetic acid to obtain a

solution of 10 mg/ml. To this 5 ml of acetic anhydride and concentrated

sulphuric acid was added and kept in dark for 20 min. The colour

developed was read on spectrophotometer at 540 nm. The values were

compared with the calibration curve of the standard cholesterol (Himedia).

3.9 Gas Chromatography – Mass Spectroscopy (GC-MS) analysis (Ivanova et al., 2002)

Sample preparation

The powdered sample (20 g) were soaked and dissolved in 75 ml of

hexane, petroleum ether and ethanol for 24 h. Then the filtrates were

collected by evaporated under liquid nitrogen.

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The GC-MS analysis was carried out using a Clarus 500 Perkin-

Elmer (Auto System XL) Gas Chromatograph equipped and coupled to a

mass detector Turbo mass gold – Perking Elmer Turbomas 5.2

spectrometer with an Elite-1 (100% Dimethyl ply siloxane), 300 m x 0.25

mm x 1 m df capillary column. The instrument was set to an initial

temperature of 110°C, and maintained at this temperature for 2 min. At

the end of this period, the oven temperature was raised upto 280°C, at the

rate of an increase of 5°C/min, and maintained for 9 min. Injection port

temperature was ensured as 250°C and Helium flow rate as 1 ml/min. The

ionization voltage was 70 eV. The samples were injected in split mode as

10:1. Mass Spectral scan range was set at 45-450 (mhz).

The chemical constituents were identified by GC-MS. The

fragmentation patterns of mass spectra were compared with those stored

in the spectrometer database using National Institute of Standards and

Technology Mass Spectral database (NIST-MS). The percentage of each

component was calculated from relative peak area of each component in

the chromatogram.

3.10. Statistical analysis

The larvicidal bio-assays and per cent control mortality were

calculated using Abbyy’s transformation (Abbott, 1925). LC50 and LC90

(lethal concentrations causing 50 and 90 per cent mortality) were

calculated using Probit analysis (Finney, 1971). Data from larval mortality

was subjected to an analysis of variance. Statistical software SPSS 11.5

was used for data analysis.

3.1. biology of plant materials 3.1.1. taxonomy of acalypha...

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