CHAPTER 1

INTRODUCTION

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 INTRODUCTION

According to world health organization (WHO) plants can provide variety of drugs
for medicinal purposes [1]. Medicinal plants accumulate different organic compounds
which are biologically active and used for the treatment of various human and animal
ailments. The medicines obtained from plants are used in pharmaceutical and cosmetic
industries [2]. Helminthiasis is the most important animal diseases amongst the tropical
people. The disease is highly prevalent in the third world countries [3]. The increasing
problems of helmintic infections have led to the proposal of screening medicinal plants for
their anthelmintic activity. The plants provide a source of botanical anthelmintics [4].
Azadirachta indica, commonly known as Neem, belongs to Family Meliaceae, is one of
the most versatile medicinal plants that has gained worldwide importance due to medicinal
and insecticide properties. There are several studies showing the effects of Azadirachta
indica in experimental and clinical models.[5]
If you are discovering Neem for the first time, here are some quick amazing Neem facts:
 the tree is known to cure more than ONE HUNDRED diseases.
 neem is both a pest control material and as well as a fertilizer, making it aninvaluable farm
companion
 .neem is an intelligent pest repeller - it affects only harmful pests and does not affect
useful species in the farm
 neem has a four-pronged repellant action and complex molecular structure which
ensures that pests don't become immune to it.
 it gives more oxygen than other trees, making it a champion pollution fighter.
 just sleeping under the shade of a neem tree is therapeutic.
 the tree is known to halt spreading deserts.
 It's leaf and it's extract are a natural substance that don't simply mask symptoms but
encourage your body to heal itself.
 Its completely safe and causes no harm or side-effects. It has been used as a health
and beauty aid in India for more than 5000 years!
 It was considered a divine tree by ancient Indians.
 A world conference is held every three years to discuss the research and discoveries
modern science is making, on this single tree. And there is lots to show and tell

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 1.1 Screening:

Screening is the process of separation and isolation of active principle from plant sources.

Screening is helpful:

1 To get lead for discovery of new therapeutic agent.

2 To find new sources for economic material.
3 To help expand chemotaxonomy.
4 To produce semi synthetic derivatives.

For this purpose, following three essential steps are prescribed-

1 Selection of plant.
2 Phytochemical screening.
3 Phytopharmacological evaluation.

1.2 Phytochemical:-

Phytochemical are non- nutritive plant chemicals that have protective or disease
Preventive properties. There are more than thousand known Phytochemical. It is well-
known that plant produces these chemicals to protect itself but recent research
demonstrates that they can protect humans against diseases. Some of the well known
Phytochemical are lycopene In tomatoes Is flavones in soy and flavonoids in fruits. They
are not essential nutrients and are not required by the human body for sustaining Life.

The different Phytochemical are as follows:-

1 Alkaloids
2 Saponins glycosides
3 Cardenolides and Bufadionolides
4 Flavonoids
5 Tannins and Polyphenolic compound
6 Anthraquinone
7 Cyanoganic glycosides
8 Carbohydrates ,Fixed oils, Fats and Volatile oils.

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 1.2.1 Alkaloids:

Alkaloids are naturally occurring chemical compound containing basis nitrogen

atoms. The name derives from the word alkaline and was used to describe any including
Bacteria, Fungi, Plants, and Animals and is part of the group of natural products (also
called secondary metabolites). Many alkaloids can be purified from crude extracts by Acid
–base extraction. Many alkaloids are toxic to other organisms. They often have
Pharmacological effects and are used as medications, as recreational drugs, or in
entheogenic rituals. Examples are the local anesthetic and stimulant cocaine, the stimulant
caffeine, nicotine, the analgesic morphine, or antimalarial drug quinine. Some alkaloids
have a bitter taste.

1.2.2 Saponin Glycosides:

Saponins are a class of chemical compounds, one of many secondary metabolites found
In natural sources, with Saponins found in particular abundance in various plant species.
Specifically, they are amphipathic glycosides grouped phenomenological by the soap-like
Foaming they produce when shaken in aqueous solutions, and structurally by their being
Composed of one or more hydrophilic glycoside moieties combined with a lipophilic
Triterpene derivative. In chemistry, glycosides are molecules in which a sugar is bound to
a non-carbohydrate Moiety, usually a small organic molecule. Glycosides play numerous
important roles in Living organism. Many plants store chemicals in the form of inactive
glycosides; which can be activated by enzyme hydrolysis. This causes the sugar part to be
broken off making the chemical available for use. Many such plant glycosides are used as
medication. In animals (including humans), poisons are often bound to sugar molecules As
part of their elimination from the body. Formally, a glycoside is any molecule in which a
suger group is bonded through it anomeric carbon to another group via a glycosidic bond.
Glycosides can be linked by An O- (an-glycoside), N-(a glycosylamine), S-(a-
thioglycoside) or C-(a-C-glycosyl) glycosidic bond. The given definition is the one used
by IUPAC. Many authors require in addition that the sugar be bonded to a non sugar
for the molecule to qualify as a glycoside, thus excluding polysaccharides. The sugar
group is then known as the glycone and the non sugar group as the aglycone or genin part
of the glycoside.

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 1.2.3 Flavonoids:-

Flavonoids (or bioflavooid), also collectivelly known as Vitamin P and Citrin, are
a class of plant secondary metabolites. According to the nomen clature, they can be
Classified into:

1. Flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1, 4-benzopyrone)

structure (example: quercetin,rutin).

2. Isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1, 4-benzopyroe)

Structure.

3. Neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1, 2-benzopyrone)

structure.

The three flavonoids classes above are all ketone-contaning compounds, and as such,
are flavonoids flavonols. This class was the first to be termed “bioflavonoid.” The terms
flavonoids and bioflavonoid have also been more loosely used to describe non-ketene
polyhydroxy polyphenol compound which are more specifically termed flavonoids, flavan
-3-ols, or catechins (although catechins are actually a subgroup of flavonoids). Flavonoids
(both flavonols and flavonols) are most commonly known for their Antioxidant
activity. Flavonoids (specifically flavonoids such as the catechins) are “the most common
group of polyphenolic compound in the human diet and are found ubiquitously in plants”.
Flavonols, the original bioflavonoid such as quercetion, are also found ubiquitously, but In
lesser quantities. Both sets of compounds have evidence of health-modulating effects in
animals which eat them.

1.2.4 Tannins and Polyphenolic compounds:-

Tannins are astringent, bitter plant polyphenol that either bind or Precipitate or shrink
Proteins. The astringency from the tannins is what causes the dry and pucker feeling in
The mouth following the consumption of unripened fruit or red wine Likewise, the
Destruction or modification of tannins with time plays an important role in the ripening Of
fruit and the aging of wine. The term tannin (from tanna, an Old High German Word for
oak or fir tree) refers to the Use of wood tannins from oak in tanning animal hides into
leather however, the terms is Widely applied to any large Polyphenolic compound
containing sufficient hydroxyls and Other suitable groups (such as carbonyls) to form

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 strong complexes with the proteins and Other macromolecules. The compound are widely
distributed in many species of plants, Where they play a role in Protection from predation
and perhaps also in growth regulation.

Tannins have molecular weights ranging from 500 to over 9,000. Tannins are
incompatible with alkalis, gelatin, heavy metals, iron, lime water, metallic salts, strong
Oxidizing agents and zinc sulfate.

1.2.4.1. Occurrence:-

Tannis are distributed in species throughout the plant kingdom. They are commonly
found in both gymnosperms as well as angiosperms. Histological tannins are mainly
physically located in the vacuoles or surface wax of plant. These strong sites keep tannins
active against plant predators, but also keep some tannin from affecting plant metabolism
while the plant tissue is alive; it is only after cell breakdown and death that the tannins are
active in metabolic effects.

Tannins are found in leaf, bud, seed, root, and stem tissue. An example of the location
of the tannins in stem tissue is that they are often found in the growth areas of trees, such
as the secondary phloem and xylem and the layer between the cortex and epidermis
tannins may help regulate the growth of these tissues.

They are also found in the heartwood of conifers and may play a role in
inhibiting microbial activity, thus resulting in the natural durability of the wood.

There may be a loss in the bioavailability of still other tannis in plants due to birds,
pests, and other pathogens.

The leaching of tannins from the decaying leaves of vegetation adjoining a stream may

Produce what is known as a black water river.

1.2.4.2. Hydrolysable Tannins:-

At the ceter of a hydrolysable tannin molecule, there is a carbohydrate (usually D-

glucose).The hydroxyl group of the carbohydrate is partially or totally esterifies with
phenol group such as eallic acid (in gallotanins) or ellagic acid (in elligitannins).
Hydrolysable tannins are hydrolyzed by weak acids or weak base to produce carbohydrate
and phenol acids.

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 Examples of gallotannins are the gallic acid esters of glucose in tannic acid (C76H52O46)

Found in the leaves and bark of many plant species.

1.2.4.3. Condensed tannis:-

Condensed tannins, also known as Proanthocyannidins, are polymers of 2 to 50 (or

more) flavonoids unit s that are joined by carbon-carbon bonds, which are not susceptible
to being cleared by hydrolysis. White hydrolysable tannins and most condensed tannins
are water soluble; some very large condenced tannins are insoluble.

Condensed tannins from Lithocarpus glaber leaves have been analyzed through acid-
catalyzed degradation in presence of cysteamin and have a potent free radical scavenging
activity.

1.2.5. Anthraquinone:-

Anthraquinone (9, 10-dioxoanthracene) is an aromatic organic compound. It is a

derivative of anthracene. It has the appearance of yellow or light gray to gray-green solid
crystalline powder.

Its other names are 9,10-anthracenedione, anthradione, 9,10-anthrachinon, anthracene

9,10-quinoe, 9,10-dihydro,9,10-dioxanthracene.

1.2.6. Cyanogenic Glycosides:-

In this case, the aglycone contains a cyanide group, and the glycoside can release the
poisonous hydrogen cyanide if acted upon by some enzyme. They are stored in the
vacuole but if the plant is attacked they are released and become activated by enzyme in
the cytoplasm. These remove the sugar part of the molecule and release toxic hydrogen
cyanide. Storing them in inactive forms in the cytoplasm prevents them from damaging
the plant under normal conditions. An example of these is amygdaline from almonds.
They can also be found in the fruit (and wilting leaves) of the rose family (including
cherries, apples, plums, almond, peaches, apricots, raspbrries, carbapples).

Cassava, an important food plant in Africa and South America, contains cyanogenic
glycoside and therefore has to be washed and ground under running water prior to
consumption. Sorghum (sorghum bicolor) expresses cyanogenic glycoside in its roots and
thus is resistant to pests such as rootworms (Diabrotica spp.) that plague its cousin maize

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 (Zea mays L.). Some cyanogenic glycoside may have anti cancer properties. A recent
study may also show that increasing CO2 levels, caused by anthropogenic emission,
may result in much higher levels of cyanogenic glycoside Production in sorghum
and Cassava plants, making them highely toxic and inconsumable. A doubling of co2
concentration was found to double the concentration of cyaogenic glycosides in the leaves.

1.2.7. Carbohydrates:-

Carbohydrateor saccharine are the most abundant of the four major classes of
bimolecular. They fill numerous role in living things, such as the storage and transport of
energy (e.g.,starch, glycogen) and structural components (e.g., cellulose in plants and
chitin in animals). In addition, carbohydrate and their derivatives play major roles in the
working process of the immune system, fertilization, pathogenesis, blood clotting, and
development.

Crbohydrates are simple organic compounds that are aldehydes or ketone with many
hydroxyl group added, usually one on each carbon atom that is not part of the aldehydes or
ketone functional group. The basic carbohydrate units are called monosaccharide example
are glucose, galactose, and fructose. The general stoichiometric formula of an unmodified
monosaccharide is (CH2O)n, where n is any number of three or greater; however, not all
carbohydrates conform to this preise stoichiometric deinition (e.g., uronic acids, deoxy-
sugar such as fructose),nor all chemicals that do conform to this definition automatically
classified as carbohydrates.

Monosaccharide can be linked together in to what are called polysaccharides (or

Oligosaccharides) in a large varity of ways. Many carbohydrates contain one or more
modified monosaccharide units that have had one or more groups replaced or removed for
example, deoxyribose, a componennt of DNA, is a modified version of ribose; chitin Is
composed of repeating units of N-acetyle glucosamine, a nitrogen-containing form of
Glucose.

While the scientific nomenclature of carbohydrates is complex, the names of

carbohydrates very often end in the suffix ose. Glycoinformatics is the specialized field of
study that deals with the specific and unique bioinformatics of carbohydrates.

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 1.2.8 Fats:-

Fats consist of a wide group of compound that is generally soluble in organic solvents
and largely insoluble in water. Chemically, fats are generally triesters of glycerol and fatty
acids. Fats may be either solid or liquid at normal room temperature, depending on their
structure and composition. Although the words “oils”, “fats”, and “lipids” are all used to
refers to fats, “oils” is usually used to refer to fats that are liquids at normal room
temperature, while “fats” is usually used to refer to fats that are solids at normal room
temperature, “lipids” is used to refer to both liquid and solid fats, along with other
related substances. The word “oil is used for any substance that does not mix with water
and has a greasy feel, such as petroleum (or crude oil) and heating oil, regardless of its
chemical structure.

Fats form a category of lipid, distinguished from other lipids by their chemical structure
and physical properties. This category of molecules is important for many forms of life,
serving both structural and metabolic functions. They are an important part of the diet of
most heterotrophs (including human). Fats or lipids are broken down in the body by
enzyme called lipases produced in the pancreas.

Example of edible anima l fats is lard (pig fat), fish oil, and butter or ghee. They are
obtained from fats in the milk, meat and under the skin of the animal. Example of edible
plant fats are Peanut, soya been, sesame, coconut, olive, and vegetable oils ,sunflower
margarine and vegetable shortening, which can be derived from the above oils, are used
mainly for baking. These examples of fats can be categorized into saturated fats and
unsaturated fats. [6]

1.2.9. Volatile oils:-

An essential oil is a concentrated, hydrophobic liquid containing volatile aroma

compound from plants. Essential oils are also known as volatile or ethereal oils, or simply
as the “oil of” the plant from which they were extracted, such as oil of clove. Oil is
“essential” in the sense that it carries a distinctive scent, or essence, of the plant
essential oils do not as a group needs to have any specific chemical properties in
common, beyond conveying characteristic fragrances. They are not to be confused with
essential fatty acids.

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 Essential oils are generally extracted by distillation. Other processes including
expression or solvent extraction. They are used in perfumes, cosmetic and products, for
flavoringfood and drink, and for scenting incense and household cleaning products.

Various essential oils have been used medicinally at different periods in history.
Medical application proposed by those who sell medicinal oils range from skin
treatments to remedies for cancer, and are often based on historical use of these oils for
these purposes. Such claims are now subject to regulation in the most countries, and have
grown vaguer to

Stay within these regulations.

Interest in essential oils has revived in recent decades with the popularity of
aromatherapy, a branch of alternative medicine which claims that the specific aromas
carried by essential oils have curative effects. Oils are volatilized or diluted in carrier oil
and used in massage, diffused in the air by a nebulizer or by heating over a candle flame or
burned as incense.

1.2.10. Proteins:-

Proteins (also known as polypeptides) are organic compound made of amino acids
arranged in a linear chain and folded into a globular form. The amino acids in a polymer
chain are joined together by the peptide bonds between the carboxyl and amino groups of
adjacent amino acid residues. The sequence of amino acids I a protein is defined by the
sequence of a gene which is encoded in the genetic code. In general, the genetic code
specifies 20 standard amino acids; however, in certain organism the genetic code
caninclude selenocysteine and in certain archaea pyrrolysine.

Shortly after or even during synthesis, the residue in a protein are often chemically
modified by post translational modification, which alter the physical and chemical
properties, folding, stability,activity and ultimately, the function of the proteins, proteis
can also work together to achieve a particular function, and they often associate to form
stable complexes.

Like other biological macromolecule such as polysaccharides and nucleic acids,

proteins are essential parts of organisms and participate in virtually every process within
cells. Many proteins are enzyme that catalyze biochemical reaction and are vital to

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 metabolism. Proteins also have structural or mechanical function, such as action and
myosin in muscle and the proteins in the cytoskeleton, which from a system of
scaffolding that maintains cell shape. Other proteins are important in cell signaling,
immune responses, cell adhesion, and the cell cycle. Proteins are also necessary in
animal’s diets, since animals cannot synthesize all the amino acids they need and must
obtain essential amino acids from food. Through the process of digestion, animals break
down ingested protein into free amino acids that are then used in metabolism.

Proteins were first described and named by the Swedish Chemist Jones Jacob Berzelius in

1838; however, the central role of proteins in living organisms was not fully appreciated
until 1926, when James B. Sumner showed that the enzyme urease was a proteins. The
first protein to be sequenced was insulin, by Frederick Sanger, who won the Nobel Prize
for this achievement in 1958. The first protein structures to be solved were hemoglobin
and myoglobin, by Max Perutz and Sir John Cowdery Kendrew, respectively, in 1958.

It is a process of tracing plant constituents. For example you want to found out if
certain lent contains alkaloids (a plant constituent) then, you will be performing a
Phytochemical screening procedures for alkaloids (in this case Mayer’s and Wagner’s
test).

There are general plant constituents that can be performed with a standard test. The
plant is a biosynthetic laboratory, not only for chemical compound such as carbohydrates
proteins and lipids that are food by man , but also for a multitude of compouds like
glycoside , alkaloids, volatile oils, tannins etc. that exert a physiological and therapeutic
effect. The compound that are responsible for medicinal property of the drug are
secondary metabolites.

A systematic study of crude drug embraces , through consideration of primary

and secondary metabolites derived as a result of plant metabolism. The plant material
is subject to dried to preliminary Phytochemical screening for the detection of various
plant constituents on following lines.

The extracts with different solvents can also be prepared successive macerating (cold
Extraction).

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 1.3. Phytochemical screening:

It is a process of tracing plant constituents. For example you want to found out if
certain lent contains alkaloids (a plant constituent) then, you will be performing a
Phytochemical screening procedures for alkaloids (in this case Mayer’s and Wagner’s
test).

There are general plant constituents that can be performed with a standard test. The
plant is a biosynthetic laboratory, not only for chemical compound such as carbohydrates
proteins and lipids that are food by man , but also for a multitude of compouds like
glycoside , alkaloids, volatile oils, tannins etc. that exert a physiological and therapeutic
effect. The compound that are responsible for medicinal property of the drug are
secondary metabolites.

A systematic study of crude drug embraces , through consideration of primary

and secondary metabolites derived as a result of plant metabolism. The plant material
is subject to dried to preliminary Phytochemical screening for the detection of various
plant constituents on following lines.

The extracts with different solvents can also be prepared successive macerating (cold
Extraction)

1.4. The Phytochemical Investigations Involve Four Different Stages:-

-The procurement of raw material and quality control.

-Extraction, purification and characterization of the constituents of

Pharmaceutical interest and In process quality control.

-Quantitative evolution.

1.4.1. The procurement of raw material and quality control:-

Quality control is an essential operation of the pharmaceuticle industry. Drugs must be

marketed as safe and therapeutically active formulation whose performance is consistent
and predictable. New and better medicinal agents are being produced at an accelerated
rate. At the same time more exacting and sophisticated analytical methods are being
developed for their evaluation.

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 WHO Guidelines for Quality Standardized Herbal formulation:-

Standardization and quality control parameters for herbal formulation are based on
following fundamental parameters:

- Quality control of crude drugs material, plant preparations and finished products.
- Stability assessment and shelf life.
- Safety assessment; documentation of safety based on experience or toxicological studies.
- Assessment of efficacy by ethnomedical iformation and biological activity evaluations.

Quality Control of Crude Material:-

According to pharmaceutical manufacturers association of U.S. “quality is the sum of

all the factors which contribute directly or indirectly to the safety, effectiveness and
acceptability of the product’. Standardization describes all measures taken during
manufacturing process and quality control leads to reproducible quality of particular
product. Growing need for standardization and quality control of herbal medicines is
recognized by WHO.

In policies and checklist on Traditional Medicine(TM) WHO has given emphasis on

development of national standards and technical guidelines and methodology for
evaluating safety, efficacy and quality of TM. As well as WHO also gives stress on
development of national pharmacopoeia and monographs of medicinal plants, cultivation
and conversion of medicinal plant to ensure their sustainable use are also prime
importance as botanicals are considered.

Standardization of botanicals offers many obstacles because synthetic drugs have well
defined structure and other analytical parameters as well as reference standard for
comparison also established assays and pharmacopoeias. Therefore, quality control is not
problematic for synthetic drug. There are several challenges as standardization of herbal
product is considered like controversial identify of various plant deliberated adulteration
of plant material, problems in storage and transport, which should be considered.

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 1.4.2. Extraction, Purification and characterization:

Extraction:-

The commonly employed technique for separation of active substance from crude drug
is called as ‘Extraction’ which involves the use of different solvents. The plant material
used for extraction should be properly authenticated or identified. The choice of the plant
material for extraction depends upon its nature and the components required being
isolated. The dried powdered plant material is commonly used for extraction. The solvent
used for extraction is called menstrum and the residue is known as marc.

Methods of extraction:-

There are various methods of extraction. Some of them are described below:

1.4.2.1 Maceration:

The word maceration means softening. It is the simplest method of crude drug
extraction and was official in I.p.1996. The process consists of keeping the crude drug in
intimate contact with whole menstrum in a closed vessel with occasional shaking for seven
days, straining, pressing the marc, mixing the liquids and finally clarifying by subsidence
or filtration. The process may take up to 14 days in some cases for complete extraction.
The drug: menstrum ratio should be 1:10.

1.4.2.2 Infusion:

Infusion is usually prepared form vegetable drugs containing water soluble and easily
extractable principles. The process consisted of moistening the drug with water,
macerating it with boiling water, straining and making up the volume.

1.4.2.3 Digestion:

This is a modified maceration process In which extraction is accomplished at a higher

temperature at which the active ingredients are not adversely affected . use of higher
temperature provides for enhanced solvent action of menstrum and constant mechanical
agitation of the system acceleratas establishment of equilibrium in a short time.

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 1.4.2.4 Decoction:

Decoction is also employed for extracting vegetable drugs containing water-soluble and
heat-soluble constitutes. The process consisted of boiling the drug with water, cooling,
expressig, straining liquid and finally makes up the volume.

1.4.2.5 Percolation:

Percolation is extraction process in which granulated or powdered drugs is deprived of

its constant by the descent of a suitable menstrum through it. In Greek, the word
‘percolate’ means ‘to pass through’. The process implies a slow passage of menstrum
under the

Influence of gravity through a column of the drug. During this movement, the menstrum
goes on extracting the drug particle layer wise, it being replaced by other layers above as

it moves downwards.

1.4.2.6 Ultrasonic extraction:

The speed of drug extraction is enhanced by application of ultrasonic vibrations. The

mixture of the drug and the menstrum is subjected to ultrasonic waves of 20 to 450
kilocycles\second followed by extraction in a soxhlet extractor. The treatment with
ultrasonic vibrations provides rapid and superior extraction.

1.4.2.7 Soxhlet extraction or hot continuous extraction: In this method, finely ground
sample is placed in a porous bag or “thimble” made from a strong filter paper or cellulose,
which is place, is in thimble chamber of the Soxhlet apparatus . Extraction solvents is
heated in the bottom flask, vaporizes into the sample thimble, condenses in the condenser
and drip back. When the liquid content reaches the siphon arm , the liquid contents
emptied into the bottom flask again and the process is continued.

1.5. Chromatography:

Among the various methods of separating plant constiuents the chromatographic

procedure originated by Tswett is one of the most commonly used techniques of general
applications.

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 In 1944, consider, Gordon and martin introduced a method of partition chromatography
using strip of filter paper for the analysis of amino acid mixture.

Chromatography represents a group of methods for separating molecular mixtures that

depend on the differential affinities of the solute between two immiscible phases. One of
the phases is a fixed bed of area which is called a stationary phase, while the other is a
fluid or gas which moves through over or over the surface, which is called the mobile
phase. The stationary phase may be a porous or finely divided solid or liquid that has been
coated as thin layer on inert support material. The mobile phase may be a pure liquid or a
mixture of solutions or it may be a gas or mixture of gasses.

For the separation some biocnstituents, it is necessary to use a two-dimensional

chromatography using two different solvent systems. The resolved components of original
mixture can be separately eluted from chromatogram by treating the cur-out spots wuth a
suitable solvent and then determined quantitatively by some suitable instrumental method
of analysis, for example fluorescence analysis, colorimetry, or ultraviolet absorption.

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 CHAPTER 2
LITERATURE REVIEW

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LITRATURE REVIEW
Since ancient times, neem has been associated with healing in the sub-continent of India. A
large number of medicinals, cosmetics, toiletries and pharmaceuticals are now based on neem
derivatives because of it's unique properties.
Bark: Neem bark is cool, bitter, astringent, acrid and refrigerant. It is useful in tiredness,
cough, fever, loss of appetite, worm infestation. It heals the wounds and is also used in
vomiting, skin diseases and excessive thirst.
Leaves: According to Ayurveda, Neem leaves help in the treatment of vatik disorders (neuro
muscular pains). Neem leaves are also reported to remove toxins, purify blood and prevent
damage caused by free radical in the body by neutralising them. Neem leaves are reported to
be beneficial in eye disorders and insect bite poisons. It treats Vatik Disorders (neuro and
muscular pains )
Fruits: Neem fruits are bitter, purgative, antihemorrhodial and anthelmintic in nature.
Flowers: Neem flowers are used in vitiated conditions of pitta ( balancing of the body heat)
and kapha ( cough formation ). They are astringent, anthelmintic and non-toxic.
Seeds: Neem seeds are also described as anthelminitic, antileprotic, antipoisonous and bitter
in taste.
Oil: Neem oil derived from crushing the seeds is antidermatonic, a powerful anthelmintic
and is bitter in taste. It has a wide spectrum of action and is highly medicinal in nature.
Mixture: Five parts of Neem tree i.e, Bark, Root, Fruit, Flower and Leaves together are used
in diseases of blood. It is also used in vitiated conditions of excess heat, itching, wound,
burning sensation in body and skin diseases.
 The plant is traditionally used and possesses antibacterial, antifungal, antiviral,
antihistamine, anthelmintic and antiseptic properties. The neem leaves, flowers,
seeds, roots, bark and fruits are utilized to treat inflammation, infections, skin
diseases etc.[6]

Hogade et.al.,,(2014) carried out evaluation of anthelmintic activity of aqueous and

ethanolic extracts of the bark of Azadirachta indica (Family–Meliaceae) and its different
extracts (viz. ethanol and aqueous) against Ascardi galli and Eudrilus eugeniae. Various
concentrations (50&100 mg/ml) of ethanolic and aqueous extracts and its various fractions
were evaluated in the bioassay involving determination of time of Paralysis (P) and time of

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Death (D) of both types of the worms. Piperazine citrate was used as standard anthelmintic
drug and distilled water was used as control. The results of the present study indicated that
the alcoholic ethanol and aqueous extracts of the bark significantly exhibited paralysis (P <
0.05) in worms in lower dose 50 mg/ml and also caused death of worms especially at higher
concentration of 100 mg/ ml, as compared to standard drug. [7]

Christiane et.al(2010)stated that The effectiveness of neem (Azadiracta indica) oil on

the growth, morphology, sporulation, viability of spores, aflatoxin B1 and B2 production by
A. flavus on Yeast Extract-Sucrose medium was determined. Neem oil inhibited the fungal
growth (i.e. mycelia dry weight, diameter of colony and growth rate) on solid media at
concentrations from 0.5 to 5.0% v/v, although it significantly increased sporulation in the
same conditions. Spores obtained from cultures grown without neem oil reduced germination
when incubated in a neem-oil supplemented medium. Colonies grown on solid media and in
submerged cultures in the presence of neem oil exhibited morphological alterations, including
granular cytoplasm, atypical hyphae branching pattern, abnormal and undifferentiated
conidiophores. High Performance Liquid Chromatography was used to measure aflatoxins. In
submerged cultures, neem oil at concentrations from 0.5 to 4.0% v/v caused approximately
95% inhibition in Aflatoxin B1 and B2. On other hand, these conditions failed to suppress
fungal growth. Current research emphasized that neem oil was not fungistatic or fungicidal,
but exhibited anti-aflatoxigenic activity[8]

Haque Rabiu et.al.,[2011] reported that The aqueous extract of Azadirachta Indica Leaves
was investigated for anthelmintic activity using earthworms (Pheretima posthuma),
tapeworms (Raillietina spiralis) and roundworms (Ascaridiagalli). Various concentrations
(10-70 mg/ml) of plant extract were tested in the bioassay. Piperazine citrate (10 mg/ml) was
used as reference standard drug whereas distilled water as control. Determination of paralysis
time and death time of the worms were recorded. Extract exhibited significant anthelmintic
activity at the concentration of 40 mg/ml. The result shows that aqueous extract possesses
vermicidal activity and found to be effective as an anthelmintic. Therefore, the anthelmintic
activity of the aqueous extract of Azadirachta Indica Leaves has been reported.[9]

Amir et.al., given some knowledge about,Neem (Azadirachta indica), is native of India and
naturalized in most of tropical and subtropical countries are of great medicinal value and
distributed widespread in the world. The chemical constituents contain many biologically
active compounds that can be extracted from neem, including alkaloids,

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lavonoids, triterpenoids, phenolic compounds, carotenoids, steroids and ketones, biologically
most active compound is azadirachtin A-G and azadirachtin E is more effective. Other
compounds that have a biologaical activity are salannin, volatile oils, meliantriol and
nimbin2. It is evergreen but under severe drought it may shed most or nearly all of its leaves.
For thousands of years, the beneficial properties of Neem have been recognized in the Indian
tradition. The importance of neem tree has been recognized by the US National Academy of
Sciences, which published a report in 1992 entitled ‘Neem – a tree for solving global
problems’. The advancement of Neem research has earlier been documented.[10]

Gavalapu Venkateswara Rao e.l at.[2017]The aim of the present study is to evaluate
anthelmintic activity of crude ethanolic extract of leaf rachis of Azadirachta indica linn.
(neem) against pheretima posthuma as test worm. Four concentrations (10 mg/mL, 15
mg/mL, 25 mg/ml, 50 mg/ml) of ethanolic extract were studied in the activity, which
involved the determination of the time taken for paralysis (P) and the time taken for death (D)
of the worms. Albendazole (10 mg/ml) was included as standard reference and saline water
as control. The results of present study indicated that crude ethanolic extract exhibited
significant anthelmintic activity at concentration 50 mg/ml compared to reference
Albendazole. The anthelmintic activity of ethanolic extract of Leaf Rachis of Azadirachta
Indica linn therefore being demonstrated for the first time and showed the best anthelmintic
activity. Key words: Azadirachta Indica linn (Neem), Pheretima posthuma, Anthelmintic
activity, Albendazole,[11]

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 CHAPTER 3
PLANT PROFILE

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PLANT PROFILE:
Common names (Synonyms)
Hindi – Neem
Bengali - Nim, Nimgachh
Konkani - Beva-rooku
Marathi – Kadunimb
Gujarati – Leemdo
Tamil - Vembu, Vempu
Punjabi – Nimb
Malayalam - Veppu, Aryaveppu, Aruveppu, Kaippan, Veppu, Vepa
Simhalee – Nimu
Oriya – Nimo
Telegu – Vepa
Kannada - Bevinmar, Kahibevu
English - Margosa, Neem, Indian Lilac
French - Azarirae d'lnde, Margousier
German - Indischer Zadrach
Persian - Azade Darakhte Hindi
Arabic - Azad Darkhtu Hind
Burmese - Tamabin, Kamakha
Malay - Dawoon Nambu, Baypay
Latin - Azadirachta indica A. Juss or Melia azadirachta Linn
Farsi - Azad darkht 1 hindi (Free tree of India)
Singapore - Kohumba, nimba
Indonesia – Mindi
Nigeria - Don goyaro
Spanish – Margosa
Nepal – Nim
Portuguese - Margosa, Nimbo
Taxonomical Classification
Kingdom Plantae

Subkingdom Tracheobionta

Superdivision Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Rosidae

Order Sapindales

Family Meliaceae

Genus Azadirachta .

Species A indica .

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 [Azadirachta indica,]

Plant Description:

Normally it thrives in areas with sub-arid to sub-humid conditions, with an annual

rainfall of 400–1,200 millimetres (16–47 in). It can grow in regions with an annual rainfall
below 400 mm, but in such cases it depends largely on ground water levels. Neem can grow
in many different types of soil, but it thrives best on well drained deep and sandy soils. It is a
typical tropical to subtropical tree and exists at annual mean temperatures of 21–32 °C (70–
90 °

Neem is a fast-growing tree that can reach a height of 15–20 metres (49–66 ft), and
rarely 35–40 metres (115–131 ft). It is evergreen, but in severe drought it may shed most of
its leaves or nearly all leaves. The branches are wide and spreading. The fairly dense crown is
roundish and may reach a diameter of 20–25 metres (66–82 ft). The neem tree is very similar
in appearance to its relative, the Chinaberry (Melia azedarach).

The opposite, pinnate leaves are 20–40 centimetres (7.9–15.7 in) long, with 20 to 31 medium
to dark green leaflets about 3–8 centimetres (1.2–3.1 in) long. The terminal leaflet often is
missing. The petioles are short.

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 The (white and fragrant) flowers are arranged in more-or-less
drooping axillary panicles which are up to 25 centimetres (9.8 in) long. The inflorescences,
which branch up to the third degree, bear from 250 to 300 flowers. An individual flower is 5–
6 millimetres (0.20–0.24 in) long and 8–11 millimetres (0.31–0.43 in) wide. Protandrous,
bisexual flowers and male flowers exist on the same individual tree.

The fruit is a smooth (glabrous), olive-like drupe which varies in shape from elongate oval to
nearly roundish, and when ripe is 1.4–2.8 centimetres (0.55–1.10 in) by 1.0–1.5 centimetres
(0.39–0.59 in). The fruit skin (exocarp) is thin and the bitter-sweet pulp (mesocarp) is
yellowish-white and very fibrous. The mesocarp is 0.3–0.5 centimetres (0.12–0.20 in) thick.
The white, hard inner shell (endocarp) of the fruit encloses one, rarely two, or three,
elongated seeds (kernels) having a brown seed coat.

The neem tree is often confused with a similar looking tree called bakain. Bakain also has
toothed leaflets and similar looking fruit. One difference is that neem leaves are pinnate but
bakain leaves are twice- and thrice-pinnate. Its fruit are shaped like miniature apples.

Chemical Components:
 The most important active constituent is azadirachtin and the others are nimbolinin,
nimbin, nimbidin, nimbidol, sodium nimbinate, gedunin, salannin, and quercetin.
 Leaves contain ingredients such as nimbin, nimbanene, 6-desacetylnimbinene,
nimbandiol, nimbolide, ascorbic acid, n-hexacosanol and amino acid, 7-desacetyl-7-
benzoylazadiradione, 7-desacetyl-7-benzoylgedunin, 17-hydroxyazadiradione, and
nimbiol [12–13].
 Quercetin and ß-sitosterol, polyphenolic flavonoids, were purified from neem fresh
leaves and were known to have antibacterial and antifungal properties [14] and
seeds hold valuable constituents including gedunin and azadirachtin.

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 CHAPTER -4
AIM AND OBJECTIVE

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AIM AND OBJECTIVE:

The aim of the present study was to evaluate the phytochemical and anthelmentic
activities of Azadirachta indica

PLAN OF WORK:

The plan of work for selected plant includes the following:

 Collection, identification and preparation of plant materials.

 Phytochemical study of plant extract (Alkaloid, Glycoside, Carbohydrate, Tannins,
Flavonoids Test)
 Extraction of plant material maceration method using ethanol.
 Screening for anthelmentic activity of plant extracts.

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 BIBLIOGRAPHY

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BIBLIOGRAPHY

1. R. N. S. Yadav and M. Agarwala, Phytochemical Analysis of Some Medicinal Plants,

Journal of Phytology, 3, 12 (2011).
2. L. M. Ndam, A. M. Mih, A. G. N. Fongod, A. S. Tening, R. K. Tonjock, J. E. Enang and
Y. Fujii, Phytochemical Screening of The Bioactive Compounds in Twenty (20)
Cameroonian Medicinal Plants, Int. J. Curr. Microbiol. App. Sci., 3, 12 (2014).
[3] Dhar DN, Sharma RL and Bansal GC. Gastrointestinal nematodes in sheep in Kashmir. Vet.
Parasitol, 1982; 11: 271-277.

[4] Satyavati GV, Raina MK and Sharma M. Medicinal plants of India. Vol.1

5.. Dubey S, Kashyap P. Azadirachta indica : A Plant With Versatile Potential, 2014; 39–46.
6.] Sondhi SM., Shahu R., Magnan Archana. Indian Drugs. 1994; 31(7): 317-320 .

7.Invitro Anthelmintic Activity of Bark of Azadirachta indica against Ascardi galli and Eudrilus
eugeniae Maheshwar G. Hogade1*, Jalalpure SS.2, Somnath D. Bhinge3, Sonali Kuthar4, Kosgi SS.4
journal of natural remadies 14(1):48-51.

8. Advances in Bioscience and Biotechnology, 2010, 1, 292-299 ABB doi:10.4236/abb.2010.14038

Published Online October 2010 (http://www.SciRP.org/journal/abb/). Published Online October

2010 in SciRes.

9. Haque Rabiu, Mondal Subhasish, “Investigation of in Vitro Anthelmintic activity of Azadirachta

Indica Leaves”, Int. J. Drug Dev. & Res., Oct-Dec 2011, 3(4): 94-100

10. THERAPEUTIC POTENTIAL OF “NEEM” (Azadirachta indica )

Amir Khan*, Arvind Kumar Yadav,pharma research library

11. PRELIMINARY PHYTOCHEMICAL SCREENING AND IN VITRO ANTHELMINTIC ACTIVITY OF

ETHANOLIC EXTRACT OF LEAF RACHIS OF AZADIRACHTA INDICA Linn. (NEEM), Gavalapu
Venkateswara Rao* Vol.3, Issue-1, 2017, 8-12

12. Ali A. Textbook of Pharmacognosy. New Delhi, India: Publication and Information Directorate;
1993.

13. Kokate C., Purohit A. P., Gokhale S. B. Pharmacognosy. Maharashtra, India: Nirali Prakashan;
2010.

14. Govindachari T. R., Suresh G., Gopalakrishnan G., Banumathy B., Masilamani S. Identification of
antifungal compounds from the seed oil of Azadirachta
indica . Phytoparasitica. 1998;26(2):109–116. doi: 10.1007/bf02980677.

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