CHAPTER 4 SCREENING OF THE PLANTS THAT …shodhganga.inflibnet.ac.in/bitstream/10603/19716/11/11_chapter 4.pdfCHAPTER – 4 SCREENING OF THE PLANTS THAT HAVE PEST CONTROL ABILITY 4.1

CHAPTER – 4 SCREENING OF THE PLANTS

THAT HAVE PEST CONTROL

ABILITY

4.1 INTRODUCTION

Recent research has focused on natural product alternatives for pest control in

developing countries (Keita et al., 2001). Screening for biological activity using simple and

fast bioassays has now been added to give a better indication of the usefulness of the plants.

Comparative phytochemical examinations of 127 species have been studied by Goh et al.

(1997). Further agriculturists and businessmen have to store the black gram, V.mungo for a

longer time. It is easily prone to the attack of the stored grain pest pulse beetle, C.maculatus.

Not much work is done on the control of this pest by using botanicals in Tirunelveli district.

In order to find out the potential hierarchy and the biocontrol ability of the plants, it is

imperative to study the preliminary phytochemistry of these plants.

In the present study the plant extracts were screened for the presence of biologically

active compounds like phenols, alkaloids and flavonoids. Functions of secondary metabolites

are: deterrence against predators and pathogens, attraction and deterrence against pollinators,

allelopathic action, attraction of symbionts, food for pollinators, symbionts, herbivores,

pathogens and decomposers and UV protectants (Morton, 1981).

The secondary metabolites produced by the plants have a wide range of mode of

actions. These compounds are deleterious to insects and other herbivores in multiple ways,

such as acute toxicity affecting insect behavior disrupting growth and development of insects

, acting as repellents, oviposition deterrents, ovicidal compounds, enzyme inhibitors, and

interfering with the consumption and (or) utilization of food (Wheeler et al., 2001; Nathan,

2006).

Plants produce a number of chemicals which act as a defensive material against

insects and protect the plants against any microbial invasion and insect infestation. Crude

extracts and biochemical products of approximately 3000 plant species have worked out for

insecticidal properties (Shankunthala Nair and Jionthomas, 2000; and Verma et al., 1981).

The plants have chemical resistance or antiherbivore mechanisms. These mechanisms are

qualitatively allelochemicals, phenolic acids, tannins, flavonoids, alkaloids and terpenoids

and quantitatively, carbohydrates, proteins, lipids and nitrogens. Antifeedant is defined as a

chemical that inhibits the physiological activity and which does not kill the insect directly.

The insect remains in the treated foliage and dies through starvation. Antifeedant will be of

great value in protecting crops from insect attack and pest.Flavonoid pigments are known to

act as feeding stimulants / deterrents in insects which contribute to cyanic colour (orange, red

and blue) as well as yellow and white (Harbornd, 1976).

The aim of the present study is designed to help the agriculturists and other people to

minimize economic loss and to store the grains for a longer time. The main objective is to

study the preliminary phytochemistry of the aerial parts of 10 locally available plant based

pesticides such as, Azadirachta indica, Calotropis gigantea, Catharanthus roseus, Cynodon

dactylon, Morinda pubescens, Ocimum tenuiflorum, Phyllanthus amarus, Sesbania

grandiflora, Tephrosia purpurea and Vitex negundo.

4.2. MATERIALS AND METHODS

Aerial parts of 10 selected plants were collected from Parvathiapuram, Kaduvetti,

Konarkulam and Kattabomman nagar of Tirunelveli district, Tamilnadu, India. They were

washed thrice with distilled water and once with tap water and were shade dried for two

weeks. Twenty g each of the aerial parts powder samples of Azadirachta indica, Calotropis

gigantea, Catharanthus roseus, Cynodon dactylon, Morinda pubescens,Ocimum

tenuiflorum,Phyllanthus amarus,Sesbania grandiflora, Tephrosia purpurea and Vitex

negundo separately were successively extracted with methanol and water in a soxhlet

apparatus. The extracts were tested for alkaloids, phenolic compounds and flavonoids.The

various phytochemical tests were performed following the methods of Brinda et al. (1981)

with slight modifications to find out the secondary metabolites.

4.2.1. I. DESCRIPTION OF SELECTED PLANTS

Plant 1. Azadirachta indica:

Scientific Classification:

Kingdom : Plantae

Division : Mangoliophyta

Order : Sapindales

Family : Meliaceae

Genus : Azadirachta

Species : Indica

Neem (Azadirachta indica) is a tree in the mahogany family Meliaceae. It is one of

the two species in the genus Azadirachta and is native to India, Myanmar, Bangladesh, Sri

Lanka, Malaysia and Pakistan growing in tropical and Semitropical regions. The branches are

wide spread.

Entomological Usages:

Neem is a source of environment – friendly biopesticide.

Plant 2. Calotropis gigantea


Kingdom : Plantae

Order : Gentianales

Family : Apocynaceae

Subfamily : Asclepiadoideae

Genus : Calotropis

Species : gigantea

This species is a large shrub or small tree, about 3 – 5(10) m tall. Its stems are erect,

up to 20 cm in diameter; 6.5– 10 cm x 3-5 cm.Calotropis gigantea is a common weed in open

waste ground, road sides and railway lines, as well as village surroundings. It grows

especially on littoral sandy soils and dry uncultivated land, with periodic dry periods.

Plant 3. Catharanthus roseus

Scientific classification

Kingdom : Plantae

Order : Gentianales

Family : Apocynaceae

Genus : Catharanthus

Species : roseus

Catharanthus roseus (Madagascar Periwinkle) is a species of Catharanthus native and

endemic to Madagascar. Synonyms include Vinca rosea (the basionym), Ammocallis rosea,

and Lochnera rosea; Other English names occasionally used include Cape Periwinkle, Rose

Periwinkle, Rosy Periwinkle, and “Old-main”. In the wild, it is an endangered plant; the main

cause of decline is habitat destruction by slash and burn agriculture. It is also however widely

cultivated and is naturalized in subtropical and tropical areas of the world.In traditional

Chinese medicines, extracts from it have been used to treat numerous diseases, including

diabetes, malaria, and Hodgkin’s disease.

Plant 4 . Cynodon dactylon


Kingdom : Plantae

Order : Poales

Family : Poaceae

Genus : Cynodon

Species : dactylon

Cynodon dactylon is widely cultivated in warm climates all over the world between

about 30o south and 30

o North latitude, and that get between 625 – 1,750 mm (24.6 – 68.9 m)

of rainfall a year (or less if irrigation is available). Cynodon dactylon has many medicinal

properties including antimicrobial and antiviral properties, as well as treatment of urinary

tract infections, prostatitis, syphilis, and dysentery. A fine to robust Stoloni ferrous peremial,

mostly with rhizomes can penetrate 40 – 50 cm in clay soil and 70 – 80 cm in sand.

Plant 5. Morinda pubescens


Kingdom : Plantae

Order : Gentianales

Family : Rubiaceae

Sub family : Rubioideae

Genus : Morinda

Species : pubescens

It comprises approximatively 80 species, distributed in all tropical regions of the

world. These species may be trees, shrubs, or vines. Morinda pubescens are trees that very

much resemble vines.

Plant 6. Ocimum tenuiflorum


Kingdom : Plantae

Order : Lamiales

Family : Lamiaceae

Genus : Ocimum

Species : tenuiflorum

Tulsi has been used for thousands of years in Ayurveda for its diverse healing

properties. It is mentioned by Charaka samhita, an ancient Ayurvedic text. Tulsi is considered

to be an adaptogen, balancing different processes in the body and helpful for adapting to

stress.

It is found throughout India ascending upto 1,800m in the Himalayas and in the

Andaman and Nicobar islands. At least two types of Ocimum tenuiflorum are encountered

with in cultivation. The green type (Sri Tulsi) is the most common; the second type (Krishna

tulsi) bears purple leaves.

Plant 7. Phyllanthus amarus


Kingdom : Plantae

Division : Angiospermae

Class : Dicotyledoneae

Order : Tubiflorae

Family : Euphorbiaceae

Genus : Phyllanthus

Species : amarus

It is considered as deobstruent, diuretic, astringer and cooling. It is prescribed as dry

powder or fresh juice for jaundice.

Traditional use:

The plant is bitter, astringer, cooling, diuretic, stomachic, febrifuge and antiseptic. It

is useful in dropsy, jaundice, diarrhoea, dysentery, intermittent fevers, and disease of urino-

genital system, scabies, ulcers and wounds. Phyllanthus amarus and Eclipta Alba were tested

for their invitro inactivation property of Hepatitis B surface antigen (HBs Ag).

Plant 8. Sesbania grandiflora


Kingdom : Plantae

Division : Mangoliophyta

Class : Mangoliopsida

Family : Fabaceae

Genus : Sesbania

Species : grandiflora

Sesbania grandiflora (also known as agati, syn Aeschynomene grandiflora) or

humming bird tree / scarlet wisteria is a small tree in the genus Sesbania. It is believed to

have originated either in India or South East Asia and grows primarily in hot and humid

tropical areas of the world. In India this plant is known as Agathi and both the leaves and the

flowers have culinary uses.

Plant 9. Tephrosia purpurea

Scientific classification:

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Fabales

Family : Fabaceae

Genus : Tephrosia

Species : purpurea

Tephrosia is grown as a common waste and weed. It is used traditionally as folk

medicine. In many parts it is cultivated as green manure crop. In India, Tephrosia purpurea

has been used as traditional medicine, for the treatment of various inflammatory disorders. It

is considered beneficial for liver, spleen and kidney disorders. Also it has the property to cure

all type of wounds.

Though Tephrosia had insecticidal activity against very limited pests, such

information was not available for any Lepidopteran pests. In this context, testing of

Tephrosia seed extracts for bio-etticacy and antifieedant effects against an economically

important Lepidopteron insect pest is of utmost importance.

Plant 10. Vitex negundo


Kingdom : Plantae

Order : Lamiales

Family : Lamiaceae

Genus : Vitex

Species : negundo

Grows abundantly in the wasteland up to 2000 metres in the Himalayas. The juice of

the leaves is used for removing foetid discharges and worms from ulcers; oil prepared with

the leaf juice is applied to sinuses and scrofulous sores. The plant is said to be a malarial

preventative and is also used in the treatment of bacterial dysentery. Extracts of the leaves

have shown bactericidal and antitumor activity. The leaves are used to repel insects in grain

stores. Extracts of the leaves have insecticidal activity. The fresh leaves are burnt with grass

as a fumigant. The leaves of V. negundo possess discutient properties and are applied to

rheumatic swellings of the joints and in sprains.

II. DESCRIPTION OF THE SELECTED STORED GRAIN PESTS

Pest.1. Pulse beetle: Callosobruchus maculatus


Kingdom : Animalia

Phylum : Arthropoda

Class : Insecta

Order : Coleoptera

Family : Chrysomelidae

Genus : Callosobruchus

Species : maculatus

Bean beetles, Callosobruchus maculatus (Coleoptera: Bruchidae), are tropical and

subtropical agricultural pest insects. This species is among the most tractable and robust

laboratory animal systems but is not widely used outside of research laboratories.

Callosobruchus maculatus is extremely easy to manipulate, maintain and has a very rapid life

cycle. Extensive past and present research on Callosobruchus maculatus (more than 150

journal articles in the past 10 years) provides opportunities for connections between

undergraduate laboratory studies and research in ecology, evolutionary biology and animal

behavior.

Callosobruchus maculatus is a cosmopolitan pest of stored legumes. Females

colonize seeds both in the field and in storage, cementing their eggs to the surface of the host

seeds. Approximately 4.5 days later (at 28oC), the eggs hatch and the first instar larvae

burrow into the seed, directly beneath the egg. Larval development and pupation are

completed entirely within a single seed. Emerging adults are well adapted to storage

conditions, requiring neither food nor water to reproduce (Edvardsson and Tregenza, 2005).

The adult (Callosobruchus maculatus) is a relatively small beetle, 3 - 4 mm in length,

somewhat teardrop or triangular in shape, and dull-colored with white, reddish, or black

markings. Adults are 1/8-inch long, reddish-brown slightly elongate beetles compared to the

typical rounded appearance of other members of this family (bruchids). Although weevil-like

they are not true weevils (Curculionidae) and do not have their prolonged anterior part into a

long “snout.” Wing covers (elytra) are marked with black and gray and there are two black

spots near the middle (Note: Ebeling says 2 red spots on elytra!). The elytra are short, leaving

the last segment of the abdomen exposed. This last abdominal segment also has two black

spots visible. The larva is whitish and somewhat C-shaped with a small head (Messina,

2004).

Life Cycle:

Adults may be found outdoors in flowers in early spring. Eggs laid by females hatch

in 5 to 20 days. Larvae typically feed inside the Cowpea, taking from 2 weeks to 6 months to

develop before pupating there. Six or seven generations may occur per year. The eggs are

glued to the bean or the pod. On hatching the larvae bores into the seed where it makes a

translucent 'window' in the seed before pupating. The larval and pupal stages are spent inside

the bean. The adult emerges through the 'window' leaving a neat round hole. Infestations can

begin in the field. Adults move to bean fields from trash beans left in sacks, harvesters,

planters, or feed areas (Savalli et al., 2000).

Habitat and Food Source:

Mouthparts are for chewing. They prefer dried cowpeas but will attack other beans

and peas in storage. Adults move about readily and can infest seeds in the field, but can also

breed continuously in stored dry cowpeas. Larvae typically develop inside the dried peas.

Larvae chew near the surface and leave a thin covering uneaten which appears as a window.

Later the adult emerges from the “Window.”

Damage

The larval stage of the pest tunnel and develop within the beans. They may consume

nearly the entire bean contents. Pupation occurs in the beans and adults emerge through a

round hole in the seed coat. Damage is a combination of the feeding and contamination.

Pest .2. Rice weevil: Sitophilus oryzae


Kingdom : Animalia

Phylum : Arthropoda

Class : Insecta

Order : Coleoptera

Family : Curculionidae

Genus : Sitophilus

Species : oryzae

The rice weevil (Sitophilus oryzae) is a serious stored product pest which attacks

several economically important crops, including wheat, rice, and maize.

Description

The adults are around 2 mm long with a long snout. The body color appears to be

brown/black, but on close examination, four orange/red spots are arranged in a cross on the

wing covers. It is easily confused with the similar looking maize weevil, but there are several

distinguishing features:

Biology

Adult rice weevils survive for up to 2 years. Females lay 2-6 eggs per day and up to

300 eggs over their lifetime. The female uses strong mandibles to chew a hole into a grain

kernel after which she deposits a single egg within the hole and seals the hole with secretions

from her ovipositor. The larva develops within the grain, hollowing it out while feeding. It

then pupates within the grain kernel and emerges 2–4 days after eclosion.

Male S. oryzae produce an aggresive pheromone ((4S, 5R)-5-Hydroxy-4-

methylheptan-3-one) to which males and females are drawn. A synthetic version is available

which attracts rice weevils, maize weevils and grain weevils. Females produce a pheromone

which attracts only males.

Sitophilus is a cosmopolitan genus of weevils found on rice, maize and tamarind. It

has also been found on Chickpea.Notable species, the Rice weevil, S. oryzae and the Maize

weevil (S. zeamais) both damage a variety of standing crops, and other stored cereals.

Species

Sitophilus granarius (wheat weevil or granary weevil)

Sitophilus linearis (tamarind weevil)

Sitophilus oryzae (rice weevil)

Sitophilus zeamais (maize weevil)

Appearance

The rice weevil is small, 1/10 inch (2 to 3 mm) and stout in appearance. It is very

similar in appearance to the granary weevil. However, the rice weevil is reddish-brown to

black in color with four light yellow or reddish spots on the corners of the elytra (the hard

protective forewings). The snout is long (1 mm), almost 1/3 of the total length. The head with

snout is as long as the prothorax or the elytra. The prothorax (the body region behind the

head) is strongly pitted and the elytra have rows of pits within longitudinal grooves. The larva

is legless and stays inside the hollowed grain kernel. It is fat with a cream colored body and

dark head capsule.

Habits

The rice weevil is one of the most serious stored grain pests worldwide. This pest of

whole grain originated in India and has been spread worldwide by commerce. It now has a

cosmopolitan distribution. It is a serious pest in the southern United States. Both the adults

and larvae feed on whole grains. They attack wheat, corn, oats, rye, barley, sorghum,

buckwheat, dried beans, cashew nuts, wild bird seed, and cereal products, especially

macaroni. The adult rice weevil can fly and is attracted to lights. When disturbed, adults pull

in their legs, fall to the ground, and feign death. The larval rice weevil must complete its

development inside a seed kernel or a man-made equivalent, like macaroni products. Larval

rice weevils have been known to develop in hard caked flour. The adult female makes a

cavity into a seed and then deposits a single egg in the cavity, sealing in the egg with

secretions from her ovipositor. The larva develops within the seed, hollowing it out while

feeding. The larva then pupates within the hollow husk of the grain kernel.

Biology

The adult female rice weevil lays an average of 4 eggs per day and may live for four

to five months. The full life cycle may take only 26 to 32 days during hot summer months,

but requires a much longer period during cooler weather. The eggs hatch in about 3 days. The

larvae feed inside the grain kernel for an average of 18 days. The pupa is naked and the pupal

stage lasts an average of 6 days. The new adult will remain in the seed for 3 to 4 days while it

hardens and matures.

Control

The most important aspect of control is location of the source of the infestation.

Sticky traps should be placed around the room to locate the infestation, if not initially or

easily located. Sticky traps with a higher density of rice weevils attached are probably closest

to the infestation site. All life stages can be killed by extreme heat (120°F for one hour) or

cold (0°F for a week). The best control measure is to store products likely to be infested in

pest-proof containers of plastic, glass, or metal. Seeds and nuts can be stored for a long term

by adding a 1 inch cube of dry ice (solid carbon dioxide) to a quart mason jar of seeds and

sealing the lid. The carbon dioxide atmosphere discourages all stored product pests. (Baloch,

1992)

Infestations in non-food areas can be treated with space sprays or crack and crevice

treatments with residual insecticides having rice weevils listed on the label. Infestations in

large quantities of grain are controlled by fumigation.

III. STORED GRAIN TAKEN FOR THE STUDY

Black gram (Vigna mungo)


Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Fabales

Family : Fafaceae

Subfamily : Faboideae

Tribe : Phascoleae

Genus : Vigna

Species : mungo

Urad (Vigna mungo) (in Tamil- Ulundu) is referred to as black gram or black lentil

(not to be confused with the much smaller true black lentil Lens culinaris). Vigna mungo is a

bean grown in southern Asia. It is largely used to make dhal from the whole or split,

dehusked seeds. It along with the mung bean was placed in Phaseolus but has been

transferred to Vigna. Black gram originated in India where it has been in cultivation from

ancient times and is one of the most highly prized pulses of India. It has also been introduced

to other tropical areas mainly by Indian immigrants.

Food Value of Black gram:

Blackgram is boiled and eaten whole or after splitting into dhal. It is extensively, used

in various culinary preparation like curries and Papad. The green pods are eaten as vegetables

and they are highly nutritious. The hulls or the outer covering of gram and straw are used as

cattle feed. (Bakr et al.,2004).

Food Value : Minerals and Vitamins

Moisture-10.9 % : Calcium – 154 mg

Protein – 24.0 % : Phosphorous – 385mg

Fat – 1.4 % : Iron – 9.1 mg

Fibre – 0.9 % : Small amount of Vitamin B complex

Minerals – 3.2% : Values per 100 gm’s edible portion.

Carbohydrates – 52.9 % : Calorific value – 34

Natural Benefits and curative properties of Black gram:

Black gram is demulcent or soothing and it is a cooling agent. It is an aphrodisiac and

nervine tonic. However, excessive use of black gram causes flatulence which can, however,

be prevented by adding little asafoetida, pepper and ginger in the culinary preparations. It

should not be taken by those who are easily predisposed to rheumatic disease and urinary

calculi as it contains oxalic acid in high concentrations.

4.2.2 TLC STUDY

4.2.2. 1.COLLECTION OF PLANT MATERIALS

The healthy leaves of selected plants were collected from Parvathiapuram, Kaduvetti,

Konarkulam and Kattabomman nagar, Tirunelveli District, Tamil Nadu, India. They were

collected in early morning and were washed in tap water and shade-dried for 10 days.

4.2.2.2. PREPARATION OF PLANT EXTRACTS

The shade dried plant material was powdered using kitchen blender and that powder

was subjected to Soxhlet extraction with methanol (60oC) and water (100

oC) for 24 h. Each

solvent extract was distilled and condensed at 40oC. The condensed extract was stored at

room temperature in air tight bottles and was used.

4.2.2.3. SEPARATION AND IDENTIFICATION OF PHYTOCHEMICALS OF

SELECTED BOTANICALS

The presence of bioactive Phytocompounds i.e the secondary metabolites from the

leaves of selected plants were qualitatively analysed by Thin layer chromatography.

4.2.2.4. TLC PLATE PREPARATION

The solid phase of silica gel was kept in hot air oven at 100oC for 20 minutes. Then

the silica powder was mixed with petroleum ether and the slurry was prepared. The 20 x 20

cm clean TLC glass plates were taken and were covered with that slurry and allowed to air

dried. After drying the plates were kept in hot air oven at 72oC for 1 h. After developing the

plates the condensed filtrate was spotted using capillary tube. The different spots were

separated using a different solvent mixture which acts as a mobile phase. The different steps

involved in the process are given below.

4.2.2.5. (a) TLC STUDY FOR ALKALOIDS

About one g powdered leaves of selected plants were wetted with a half diluted

NH4OH and lixiviated with EtOAc for 24h at Room Temperature. The organic phase is

separated from the acidified filtrate and basified with NH4OH (pH 11-12). It is extracted with

chloroform (3x), condensed by evaporation and used for chromatography. The alkaloid spots

were separated using the solvent mixture chloroform and methanol in the ratio of 1:5. The

color and Rf value of the separated alkaloids were recorded both under Ultra Violet (UV

254nm) and visible light after spraying with Dragendorff’s reagent.

4.2.2.5. (b) TLC STUDY FOR FLAVONOIDS

One g powdered leaves of selected plants were extracted with 10 ml methanol on

water bath (60oC/5min). The filtrate was condensed by evaporation and a mixture of water

and EtOAc at a ratio of 10:1 was added and mixed thoroughly. The EtOAc phase thus

retained is used for chromatography. The flavonoid spots were separated using chloroform

and methanol solvent mixture in the ratio of 19:1. The color and Rf value of these spots were

recorded under ultraviolet (UV254nm) light.

4.2.2.5. (C) TLC STUDY FOR PHENOLS

The powdered leaves of selected plants were lixiviated in methanol on rotary shaker (180

thaws/min) for 24h. The condensed filtrate was used for chromatography. The phenols were

separated using chloroform and methanol solvent mixture in the ratio of 1:5. The colour and

Rf values of these phenols spots were recorded under visible light after spraying the plates

with Folin-Ciocalteu’s reagents heating at 80oC/10min

4.2.3. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) STUDY

The analytical equipment consisted of a Perkin Elmer Series 200LC pump and Auto

sampler with 200 µl loop (Norwalk, CT, USA), a Perkin Elmer LC 235 C Diode Array

detection system (Norwalk, CT, USA), and a PE Nelson 600 Series link (Norwalk, CT,

USA). For the analysis of Plant extracts, the samples were analysed using an RP.18 analytical

column (Luna 3µ C18, 100x4.60 mm i.d. Phenomenex, USA) with a fitted security guard

cartridge (C18 ODS (Octadecyl) 3x4 mm, Phenomenex, USA). The mobile phase flow rate

was 1.0 ml/min for a total run time of 20 minutes, with UV detection at 255 nm. The mobile

phase consisted of Isopropanol: Chloroform (1:1). The gradient profile was developed using

the computer assisted optimization program. Identification of peaks was performed by

comparison with reference compound retention times and UV spectra recorded by photodiode

array detection.

4.3. RESULTS AND DISCUSSION

4.3.1. TLC PROFILE OF SELECTED PLANTS

The preliminary phytochemical investigation revealed the presence of alkaloids,

flavonoids and phenols as shown in Table 4. 3. The results showed that not all compounds are

present in all plants. Whereas one of the compounds is present in one plant is not in other

plant. For example, the presence of alkaloids is found out in the leaf of Azadirachta indica

and the absence of phenols is noted in the leaf of Phyllanthus amarus. Further it is

interesting to note that all the three tested insecticidal compounds viz.,alkaloids,phenols and

flavonoids are present in Azadirachta indica, Tephrosia purpurea, Calotropis gigantea,

Cynodon dactylon and Ocimum tenuiflorum. Some of the findings are not in agreement with

the previous findings. This might be due to climatic and other environmental factors.

The medicinal properties of plants are perhaps due to the presence of these secondary

metabolites such as alkaloids, flavonoids, glycosides, phenols, saponins, sterols etc. True

triterpenoids, steroids, saponins, and cardiac glycosides are the classes of terpenoids. Tannins

are considered as an important compound, which act as a barrier to herbivory

(Ananthakrishnan, 1992).

In the present study, it was recorded that methanol extract of Cynodon dactylon,

Morinda pubescens and Ocimum tenuiflorum showed the presence of flavonoids. Almost all

the plants in the plant kingdom possess flavones and flavonols. Other water insoluble

chemicals present in the plants are phenolic compounds (Harbone, 1984). Both tannin and

flavonoids are collectively called polyphenols. Results of the present study showed that 9

plants possess polyphenols. They mainly reduce the damage caused by insects through their

deterrent and or antifeedant effects (Echeverri et al., 1991; Pavela, 2006). Tannin and

flavonoids were reported in many plants. All the plants possess at least two or more than two

secondary metabolites. So they can be utilized as pesticidal plants. However the quantity

may be determined before selecting them for pest management purpose.

The TLC profile of secondary metabolites (Alkaloids, Flavonoids and Phenols) is

tabulated in the Table 4.4 and Figure 4.3.1.a – 4.3.1.c. Among the three groups of

phytochemicals determined from the leaves, Flavonoids were found to be the most abundant

one followed by Phenols and Alkaloids.

The data of secondary metabolites of Azadirachta indica revealed the presence of

pink to intense black colored (Plate 9) secondary metabolites with Rf values of 0.79, 0.73 and

0.66.The data of secondary metabolites of Tephrosia purpurea revealed the presence of pink

to intense black colored (Plate 8) secondary metabolites with Rf values of 0.74, 0.83, 0.63. In

C.gigantea, the Rf values are 0.86, 0.63, 0.72, and P.amarus are 0.75, 0.63, 0.68. The data of

secondary metabolites of Sesbania grandiflora revealed the presence of, pink to intense black

colored (Plate 2) secondary metabolites with Rf values of 0.84, 0.73 and 0.63. Morinda

pubescens revealed the presence of, pink to intense black colored secondary metabolites with

Rf values of 0.73, 0.68, 0.67. Similarly the leaf extract of Vitex negundo also revealed the

presence of, pink to intense black colored secondary metabolites with Rf values of 0.74, 0.65

and 0.63. This study helps to formulate a safe ecofriendly plant based chemical for pest

control practices.

The term retention factor Rf, is commonly used to describe the chromatographic

behavior of sample solutes. The Rf value for each substance is the distance it has moved

divided by the distance the solvent front has moved. Usually, the center of each spot is the

point taken for measurement. Comparison of Rf values makes it possible to research complex

mixtures qualitatively. The extent of the surface of the spot is a measure for the quantity of

the material present (Fritz and Schenk, 1987). Previous phytochemical studies of the genus

Tephrosia (Leguminosae; subfamily Papilinoideae; tribe Tephrosieae) had led to the isolation

and identification of numerous flavonoids and rotenoids, some of which possess insecticidal

and pesticidal properties (Gomez-Garibay et al., 2001).

Table 4.1.

Plants selected for extracting compounds

S.No. Common Name Botanical Name Family Parts used

1 Neem Azadirachta indica Meliaceae Leaves

2 Eruku Calotropis gigantea Apocynaceae Leaves

3 Nithya Kalyani Catharanthus roseus Apocynaceae Leaves

4 Arukkampillu Cynodon dactylon Poaceae Leaves

5 Manjanathi Morinda pubescens Rubiaceae Leaves

6 Tulsi Ocimum tenuiflorum Lamiaceae Leaves

7 Keelanelli Phyllanthus amarus linn. Euphorbiaceae Leaves

8 Akathikeerai Sesbania grandiflora Fabaceae Leaves

9 Kolinji Tephrosia purpurea Fabaceae Leaves

10 Nocchi Vitex negundo lamiaceae Leaves

Table 4.2

Preliminary phytochemical Screening Tests

S.No. Tests Observation Inference

1.

Test solution+minimum amount of CHCl3 + 3

drops of acetic anhydride + 2 drops of

concentrated H2SO4 (Liberman–Burchard

test).

Purple colour

changing to blue

or green

Presence of

steroids

2.

Test solution shaken with 2NHCl, Aqueous

layer formed, decanted and added one or two

drops of Mayer’s reagent

White turbidity

or precipitate

Presence of

alkaloids

3. Alcoholic solution of test solution + one drop

of Ferric chloride Intense colour

Presence of

phenolic

compounds.

4. Test solution + H2O and shaken well Foamy Lather Presence of

Saponins

5.

Test solution + Magnesium powder and

treated with concentrated HCl and heated. The

tube was cooled under running water

Orange Colour Presence of

Flavonoids

Table.4.3

Phytochemicals present in selected plants

S.No. Name of the plant Alkaloids Flavonoids Phenols

1 Azadirachta indica + + +

2 Calotropis gigantea + + +

3 Catharanthus roseus + - +

4 Cynodon dactylon + + +

5 Morinda pubescens - + +

6 Ocimum tenuiflorum + + +

7 Phyllanthus amarus + + +

8 Sesbania grandiflora - + +

9 Tephrosia purpurea + + +

10 Vitex negundo - + +

Table 4.4

TLC Profile of phytochemicals

S.No. Plant Name Colour of the spot Name of the Secondary

metabolites compound Rf value

1. Azadirachta

indica

Pink Alkaloids 0.79

Yellow Flavonoids 0.73

Blue Phenols 0.66

2. Calotropis

gigantea

Pink Alkaloids 0.86


Blue Phenols 0.72

3 Catharanthus

roseus

Pink Alkaloids 0.79


Blue Phenols 0.53

4 Cynodon

dactylon

Pink Alkaloids 0.78


Blue Phenols 0.69

5 Morinda

pubescens

Pink Alkaloids 0.73


Blue Phenols 0.67

6 Ocimum

tenuiflorum

Pink Alkaloids 0.78


Blue Phenols 0.69

7 Phyllanthus

amarus

Pink Alkaloids 0.75


Blue Phenols 0.68

8 Sesbania

grandiflora

Pink Alkaloids 0.84


Blue Phenols 0.63

9 Tephrosia

purpurea

Pink Alkaloids 0.74


Blue Phenols 0.63

10 Vitex negundo

Pink Alkaloids 0.74


Blue Phenols 0.63

4.3.2. HPLC PROFILE OF SELECTED PLANTS

The results are presented in Table 4.5.1 - 4.5.10 and Fig.4.3.2.a - 4.3.2.j Simultaneous

quantitative estimation of two biologically active flavonoidal compounds, quercetin and rutin

in Tephrosia purpurea leaves was performing high-performance thin-layer chromatography

(HPTLC) (Avijeet Jain et al., 2009). One of the most widely used herbs in ayurvedic

medicine is Phyllanthus amarus, which is predominantly grown in Indian subcontinent. Other

plants which are taken for the study are also available enormously in to the study area. An

investigation was undergone to quantify the phytochemicals present in these plants through

HPLC.

In Azadirachta indica, 1st peak - the area was 16.872, % of area was 26.5, height was

1.668, % of height was 39.0 % and retention time was 2.66.

21 3

1 -dethydrosalannol

In Azadirachta indica, 2nd

peak - the area was 46.438, % of area was 72.9, height

was 2.582, % of height was 60.3% and retention time was 2.857.

Nimbolide

In Azadirachta indica, 3rd

peak - the area was 0.431, % of area was 0.7, height was


Salannin

HPLC analysis of the extracts of the plant Azadirachta indica showed 3 peaks. The

corresponding compounds for these 3 peaks are 213

1- dethydrosalannol, Nimbolide and

Salannin. Crude extract of these compounds was found to be effective in reducing

oviposition, adult emergence and adult mortality in C.maculatus. The insecticidal activities of

these compounds were reported by earlier workers (Robert Irving Krieger, 2001).

In Calotropis gigantea, 1st peak - the area was 991.049, % of area was 95.9, height


n-Hexadecaonoic acid

In Calotropis gigantea, 2nd



Solvent peak- methanol

In Catharanthus roseus, 1st peak - the area was 6.565, % of area was 32.8, height

was 1.326, % of height was 42.3 and retention time and 1.923. .

Solvent peak- petroleum ether

In Catharanthus roseus, 2nd


was 1.046, % of height was 33.4 and retention time was 2.310.

Solvent peak-ethanol

In Catharanthus roseus, 3rd


0.418, % of height was 13.3 and retention time was 2.610.

Solvent peak- methanol

In Catharanthus roseus, 4th


was 0.343, % of height was 11.0 and retention time was 6.377.


In Cynodon dactylon, 1st peak - the area was 1123.266, % of area was 94.1, height

was 61.340, % of height was 92.8 % and retention time was 2.918.

2-Furancarboxaldehyde, 5-(hydroxymethyl)-

In Cynodon dactylon, 2nd


4.545, % of height was 6.9% and retention time was 4.218.

Solvent peak-ethanol

In Cynodon dactylon, 3rd



Solvent Peak-methanol

In Cynodon dactylon, 4th

peak-the area was 1.710, % of area was 0.1, height was

0.103, % of height was 0.2 %, and retention time was 5.592.

3H-Pyrazol-3-one, 2,4-dihydro-2,4,5-trimethyl

In the present study HPLC analysis of the extracts of the plant Cynodon dactylon

showed 4 peaks. The corresponding compounds for the 1st and 4

th peaks are 2

Furancaboxyaldehyde and 3 H- pyrazole-3-one, 2,4-dihydro-2,4,5- trimethyl. Among these 2

compounds, the compound 3-Hpyrazol-3-one had been reported by earlier workers that

exhibit insecticidal activity. In the present study the crude extract of C.dactylon was found to

inhibit the growth and development of stored grain pest C.maculatus. The compound 3 H-

pyrazol-3-one, 2-4 dihydro- 2, 4, 5-trimethyl might have inhibited the growth and

development of C.maculatus as reported by earlier worker (Pat O’Connor-maer, 2006).

In Morinda pubescens, 1st peak - the area was 1391. 652, % of area was 98.7, height


Deacety laspe rulos idic acid

In Morinda pubescens, 2nd




In Ocimum tenuiflorum, 1st peak – the area was 7.071, % of area was 33.4, height was


1-hydroxy-2-methoxy-4-allylbenzene

In Ocimum tenuiflorum, 2nd



Solvent Peak- methanol

In Ocimum tenuiflorum, 3rd



Isothymusin (6, 7-dimethoxy-5, 8, 4'-trihydroxyflavone)

HPLC analysis of the extracts of the plant Ocimum tenuiflorum showed 3 peaks. The

compounds for the 1st and 3

rd peaks are 1-hydroxy-2-methoxy-4 allyl benzene and

isothymusin (6,7 dimethoxy -5-8-4’-trihydroxy flavone) respectively. Among these two

compounds the insecticidal activity of isothymusin had been reported by earlier workers. In

the present study the crude extract of Ocimum tenuiflorum was found to inhibit the growth

and development of C.maculatus as reported by earlier workers (Hong-ago et al., 2007).

In Phyllanthus amarus, 1st peak-the area was 497.816, % of area was 93.2, height

was 30.346, % of height was 95.0 % and retention time was 2.880.

Phyllanthin

In Phyllanthus amarus, 2nd



Hypo Phyllanthin

In Phyllanthus amarus, 3rd



Solvent Peak-ethanol

In the present study, HPLC analysis of the extracts of the plant Phyllanthus amarus

showed three peaks. The compounds for the first two peaks were identified. They are

phyllanthin and hypophyllanthin. In the present study crude extract of P.amarus was found to

inhibit the growth and development of C.maculatus. This finding was supported by (Eiri

Board, 2006).

In Sesbania grandiflora, 1st peak - the area was 28.681, % of area was 100, height

was 2.972, % of height was 100% and retention time was 3.070.

Kaempferol

In Tephrosia purpurea, 1st peak - the area was 427.172, % of area was 26.7, height


Solvent peak-Ethanol

In Tephrosia purpurea, 2nd

peak – the area was 1173, % of area was 73.3, height was


Solvent peak- Petroleum ether

In Vitex negundo, 1st peak - the area was 260.242, % of area was 77.3, height was


Butane, 1, 1-diethoxy-3-methyl

In Vitex negundo, 2nd



2, 3-Dihydrothiophene 1, 1-dioxide

In Vitex negundo, 3rd

peak - the area was 29.414, % of area was 8.7, height was 2.878,

% of height was15.3 % and retention time was 3.740.

Solvent Peak-Methanol

In Vitex negundo, 4th


% of height was 8.1 % and retention time was 3.952.

Solvent peak- Ethanol




4, 9-Decadienoic acid, 2-nitro-, ethyl ester




10, 13-Octadecadiynoic acid, methyl ester




Azulene, 1, 4-dimethyl-7-(1-methylethyl)-

HPLC analysis of the extracts of the plant V.negundo showed seven peaks. The

corresponding compounds for the peaks except 3rd

and 4th

are Butane 1,1-diethoxy -3-methyl,

2,3-dihydrothiophene ,1,1-dioxide,4,9 Decadienoic acid, 2- nitro- ethyl ester, 10,13-

octadecadiynoic acid, methyl ester and Azulene 1,4-dimethyl-7- (1- methylethyl)-. Among

these 5 compounds, 4, 9 Decadienoic acid, 2 nitro ethyl ester was reported as an insecticidal

compound by previous worker (Rebecca Baldwin, 2008). Crude extract of V.negundo was

found to inhibit the growth and development of C.maculatus. The compound 4, 9,

Decadienoic acid, 2 nitro ethyl ester might be responsible for this deterrent activity.

4.5. HPLC profile of phytochemicals

Table 4.5.1.

HPLC profile of A.indica

S.No Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 2.665 16.872 1.668 26.5 39.0 0.13

2 2.857 46.438 2.582 72.9 60.3 0.26

3 4.627 0.431 0.031 0.7 0.7 0.18

Total 63.741 4.281 100.0 100.0 -

Table 4.5.2.

HPLC profile of C. gigantea

S.No Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 3.090 991.049 67.079 95.9 94.6 0.20

2 4.657 42.105 3.820 4.1 5.4 0.16

Total 1033.154 70.899 100.0 100.0 -

Table 4.5.3.

HPLC profile of C.roseus

S.No Reten.time

(Min)

Area

(mV-s)

Height

(mV)

Area

(%)

Height

(%)

W05

(Min)

1 1.923 6.565 1.326 32.8 42.3 0.08

2 2.310 6.411 1.046 32.1 33.4 0.09

3 2.610 4.150 0.418 20.8 13.3 0.12

4 6.377 2.859 0.343 14.3 11.0 0.13

Total 19.985 3.133 100 100

Table 4.5.4.

HPLC profile of C.dactylon

Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 2.918 1123.266 61.340 94.1 92.8 0.25

2 4.218 68.012 4.545 5.7 6.9 0.20

3 5.437 0.823 0.081 0.1 0.1 0.20

4 5.592 1.710 0.103 0.1 0.2 0.27

Total 1193.811 66.069 100.0 100.0

Table 4.5.5.

HPLC profile of M.pubescens

Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 2.963 1391.652 56.400 98.7 99.2 0.35

2 5.285 18.553 0.435 1.3 0.8 0.90

Total 1410.206 56.835 100.0 100.0

Table 4.5.6.

HPLC profile of O.tenuiflorum

Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 1.763 7.071 1.672 33.4 49.7 0.07

2 2.207 8.816 1.102 41.7 32.7 0.12

3 2.583 5.274 0.593 24.9 17.6 012

Total 21.161 3.367 100.0 100.0

Table 4.5.7.

HPLC profile of P.amarus

Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 2.880 497.816 30.346 93.2 95.0 0.24

2 3.552 25.057 1.065 4.7 3.3 0.29

3 4.423 11.344 0.524 2.1 1.6 0.26

Total 534.218 31.935 100.0 100.0

Table 4.5.8.

HPLC profile of S.grandiflora

Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 3.070 28.681 2.972 100.0 100.0 0.15

Total 28.681 2.972 100.0 100.0

Table 4.5.9.

HPLC profile of T.purpurea

Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 2.815 427.172 59.210 26.7 46.4 0.10

2 2.928 1173.020 68.414 73.3 53.6 0.25

Total 1600.192 127.625 100.0 100.0

Table 4.5.10.

HPLC profile of V.negundo

Reten.time

(min)

Area

(mVs)

Height

(Mv)

Area

(%)

Height

(%)

W05

(min)

1 2.905 260.242 11.293 77.3 60.1 0.31

2 3.635 16.777 2.476 5.0 13.2 0.11

3 3.740 29.414 2.878 8.7 15.3 0.19

4 3.952 19.382 1.517 5.8 8.1 0.20

5 4.310 5.016 0.332 1.5 1.8 0.24

6 4.732 1.970 0.144 0.6 0.8 0.26

7 5.010 3.711 0.143 1.1 0.8 0.29

Total 336.512 18.782 100.0 100.0

Fig. 4.3.2 a HPLC profile of A.indica

Fig. 4.3.2 b HPLC profile of C.gigantea

Fig. 4.3.2 c HPLC profile of C. roseus

Fig. 4.3.2 d HPLC profile of C. dactylon

Fig. 4.3.2e HPLC profile of M. pubescens

Fig. 4.3.2 f HPLC profile of O.tenuiflorum

Fig. 4.3.2 g HPLC profile of P.amarus

Fig. 4.3.2 h HPLC profile of S.grandiflora

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CHAPTER 4 SCREENING OF THE PLANTS THAT …shodhganga.inflibnet.ac.in/bitstream/10603/19716/11/11_chapter 4.pdfCHAPTER – 4 SCREENING OF THE PLANTS THAT HAVE PEST CONTROL ABILITY 4.1