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
Scientific Classification:
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
Scientific classification
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
Scientific Classification:
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
Scientific classification
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
Scientific Classification:
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
Scientific classification
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
Scientific classification:
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
Scientific Classification:
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
Scientific classification:
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)
Scientific classification:
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
Yellow Flavonoids 0.63
Blue Phenols 0.72
3 Catharanthus
roseus
Pink Alkaloids 0.79
Yellow Flavonoids 0.70
Blue Phenols 0.53
4 Cynodon
dactylon
Pink Alkaloids 0.78
Yellow Flavonoids 0.63
Blue Phenols 0.69
5 Morinda
pubescens
Pink Alkaloids 0.73
Yellow Flavonoids 0.68
Blue Phenols 0.67
6 Ocimum
tenuiflorum
Pink Alkaloids 0.78
Yellow Flavonoids 0.73
Blue Phenols 0.69
7 Phyllanthus
amarus
Pink Alkaloids 0.75
Yellow Flavonoids 0.63
Blue Phenols 0.68
8 Sesbania
grandiflora
Pink Alkaloids 0.84
Yellow Flavonoids 0.73
Blue Phenols 0.63
9 Tephrosia
purpurea
Pink Alkaloids 0.74
Yellow Flavonoids 0.83
Blue Phenols 0.63
10 Vitex negundo
Pink Alkaloids 0.74
Yellow Flavonoids 0.65
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
0.031, % of height was 0.7 % and retention time was 4.627.
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
was 67.079, % of height was 94.6% and retention time was 3.090.
n-Hexadecaonoic acid
In Calotropis gigantea, 2nd
peak - the area was 42.105, % of area was 4.1, height
was 3.820, % of height was 5.4% and retention time was 4.657.
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
peak - the area was 6.411, % of area was 32.1, height
was 1.046, % of height was 33.4 and retention time was 2.310.
Solvent peak-ethanol
In Catharanthus roseus, 3rd
peak - the area was 4.150, % of area was 20.8, height was
0.418, % of height was 13.3 and retention time was 2.610.
Solvent peak- methanol
In Catharanthus roseus, 4th
peak - the area was 2.859, % of area was 14.3, height
was 0.343, % of height was 11.0 and retention time was 6.377.
Solvent peak- petroleum ether
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
peak - the area was 68.012, % of area was 5.7, height was
4.545, % of height was 6.9% and retention time was 4.218.
Solvent peak-ethanol
In Cynodon dactylon, 3rd
peak - the area was 0.823, % of area was 0.1, height was
0.081, % of height was 0.1 % and retention time was 5.437.
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
was 56.400, % of height was 99.2% and retention time was 2.963.
Deacety laspe rulos idic acid
In Morinda pubescens, 2nd
peak - the area was 18.553, % of area was 1.3, height was
0.435, % of height was 0.8% and retention time was 5.285.
Solvent peak- petroleum ether
In Ocimum tenuiflorum, 1st peak – the area was 7.071, % of area was 33.4, height was
1.672, % of height was 49.7 % and retention time was 1.763.
1-hydroxy-2-methoxy-4-allylbenzene
In Ocimum tenuiflorum, 2nd
peak - the area was 8.816, % of area was 41.7, height was
1.102, % of height was 32.7% and retention time was 2.207.
Solvent Peak- methanol
In Ocimum tenuiflorum, 3rd
peak - the area was 5.274, % of area was 24.9, height was
0.593, % of height was 17.6 % and retention time was 2.583.
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
peak - the area was 25.057, % of area was 4.7, height was
1.065, % of height was 3.3 % and retention time was 3.552.
Hypo Phyllanthin
In Phyllanthus amarus, 3rd
peak - the area was 11.344, % of area was 2.1, height was
0.524, % of height was 1.6 % and retention time was 4.423.
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
was 59.210, % of height was 46.4% and retention time was 2.815.
Solvent peak-Ethanol
In Tephrosia purpurea, 2nd
peak – the area was 1173, % of area was 73.3, height was
68.414, % of height was 53.6% and retention time was 2.928.
Solvent peak- Petroleum ether
In Vitex negundo, 1st peak - the area was 260.242, % of area was 77.3, height was
11.293, % of height was 60.1 % and retention time was 2.905.
Butane, 1, 1-diethoxy-3-methyl
In Vitex negundo, 2nd
peak - the area was 16.777, % of area was 5.0, height was
2.476, % of height was 13.2 % and retention time was 3.635.
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
peak - the area was 19.382, % of area was 5.8, height was 1.517,
% of height was 8.1 % and retention time was 3.952.
Solvent peak- Ethanol
In Vitex negundo, 5th
peak - the area was 5.016, % of area was 1.5, height was 0.332,
% of height was 1.8 % and retention time was 4.310.
4, 9-Decadienoic acid, 2-nitro-, ethyl ester
In Vitex negundo, 6th
peak - the area was 1.970, % of area was 0.6, height was 0.144,
% of height was 0.8 % and retention time was 4.732.
10, 13-Octadecadiynoic acid, methyl ester
In Vitex negundo, 7th
peak - the area was 3.711, % of area was 1.1, height was 0.143,
% of height was 0.8 % and retention time was 5.010.
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