EVALUATION OF ANTIBACTERIAL ACTIVITY ON

LEAF EXTRACT OF PITHECELLOBIUM DULCE

A dissertation submitted to
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY, HYDERABAD
IN PARTIAL FULFILMENT FOR THE AWARD OF THE DEGREE IN
BACHELOR OF PHARMACY

Submitted by
MANDHAVADHA SAI VENNELA SRI - (20DE1R0051)
MANIKIREDDY SRAVANI - (20DE1R0052)
MEHATAJ AMZUM- (20DE1R0053)
MENDE PRASHANTH- (20DE1R0054)
MERIGA SAI ASHREETHA – (20DE1R0055)
Under the supervision of

Dr. K. CHAITANYA SRAVANTHI, M.Pharm, Ph.D.

ASSOCIATE PROFESSOR

DEPARTMENT OF PHARMACOGNOSY

ST. MARY’S COLLEGE OF PHARMACY

(Approved by AICTE, PCI and affiliated to JNTUH)
H. No 9/1/248, St. Francis Street Secunderabad-500025, Secunderabad,
Telangana.
ST. MARY’S COLLEGE OF PHARMACY
(Approved by AICTE, PCI and affiliated to
JNTUH)
H. No 9/1/248, St. Francis Street Secunderabad-
500025, Secunderabad, Telangana.
 CERTIFICATE BY THE PRINCIPAL

This is to certify that the dissertation entitled “EVALUATION OF ANTI-

BACTERIAL ACTIVITY ON LEAF EXTRACT OF PITHECELLOBIUM
DULCE” being submitted to JAWAHARLAL NEHRU TECHNOLOGICAL
UNIVERSITY, Hyderabad, in partial fulfillment for the award of the degree in
“Bachelor of Pharmacy” has been carried out by MANDHAVADHA SAI
VENNELA SRI (20DE1R0051) , MANIKIREDDY SRAVANI-(20DE1R0052),
MEHATAJ AMZUM-(20DE1R0053), MENDE PRASHANTH–
(20DE1R0054), MERIGA SAI ASHREETHA (20DE1R0055) during the
academic year 2023-2024 under the guidance of Dr. K. CHAITANYA
SRAVANTHI, Department of Pharmacognosy, St. Mary’s College of Pharmacy,
Secunderabad, and the work carried out is original and not submitted earlier to any
other university or college.

Date:
Place: Secunderabad

Dr. MURALIDHAR RAO M.Pharma., Ph.D.

Principal
St. Mary’s College of Pharmacy

External Examiner

ST. MARY’S COLLEGE OF PHARMACY

(Approved by AICTE, PCI and affiliated to
JNTUH)
H. No 9/1/248, St. Francis Street Secunderabad-
500025, Secunderabad, Telangana
CERTIFICATE BY THE HEAD OF THE DEPARTMENT

This is to certify that the dissertation entitled “EVALUATION OF ANTI-

BACTERIAL ACTIVITY ON LEAF EXTRACT OF PITHECELLOBIUM
DULCE” being submitted to JAWAHARLAL NEHRU TECHNOLOGICAL
UNIVERSITY, Hyderabad, in partial fulfillment for the award of the degree in
“Bachelor of Pharmacy” has been carried out by MANDHAVADHA SAI
VENNELA-(20DE1R0051), MANIKIREDDY SRAVANI-(20DE1R0052),
MEHATAJ AMZUM-(20DE1R0053), MENDE PRASHANTH(20DE1R0054),
MERIGA SAI ASHREETHA-(20DE1R0055) during the academic year 2023-
2024 under the guidance of Dr. K. CHAITANYA SRAVANTHI, Department of
Pharmacognosy, St. Mary’s College of Pharmacy, Secunderabad, and the work
carried out is original and not submitted earlier to any other university or college.

Date:
Place: Secunderabad
Dr. K. CHAITANYA SRAVANTHI, M.Pharm,Ph.D

Head of the department

St. Mary’s College of Pharmacy

External Examiner

ST. MARY’S COLLEGE OF PHARMACY

(Approved by AICTE, PCI and affiliated to
JNTUH)

H. No 9/1/248, St. Francis Street Secunderabad-

500025, Secunderabad, Telangana

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “EVALUATION OF ANTI-

 BACTERIAL ACTIVITY ON LEAF EXTRACT OF PITHECELLOBIUM
DULCE” that is being submitted to JAWAHARLAL NEHRU
TECHNOLOGICAL UNIVERSITY, Hyderabad, in partial fulfillment for the
award of the degree in “Bachelor of Pharmacy” has been successfully carried
out by MANDHAVADHA SAI VENNELA SRI- (20DE1R0051),
MANIKIREDDY SRAVANI-(20DE1R0052), MEHATAJ AMZUM-
(20DE1R0053), MENDE PRASHANTH-(20DE1R0054), MERIGA SAI
ASHREETHA-(20DE1R0055) during the academic year 2023-2024 under my
guidance.

Date:
Place: Secunderabad

Dr. K. CHAITANYA SRAVANTHI

Associate Professor
Department of Pharmacognosy

External Examiner

DECLARATION BY THE CANDIDATE

We hereby declare that the research work embodied in the dissertation is

original and carried out by us under the guidance of “EVALUATION OF
ANTIBACTERIAL ACTIVITY ON LEAF EXTRACT OF
PITHECELLOBIUM DULCE” Dr. K. CHAITANYA SRAVANTHI for
the award of degree in “Bachelor of Pharmacy”. This work has not been
submitted earlier to any university either in part or in full for the award of
any degree.
 Date:
Place: Secunderabad

MANDHAVADHA SAI VENNELA SRI - (20DE1R0051)

MANIKIREDDY SRAVANI - (20DE1R0052)
MEHATAJ AMZUM - (20DE1R0053)
MENDE PRASHANTH - (20DE1R0054)
MERIGA SAI ASHREETHA - (20DE1R0055)
 ACKNOWLEDGEMENT

It gives us immense pleasure to acknowledge with gratitude, the help and

guidance rendered to us by a host of people, to whom we owe a substantial
measure for the successful completion of this project work.

In the accomplishment of this project successfully, primarily, we would like to

express my special thanks of gratitude to our project guide “Dr. K.
CHAITANYA SRAVANTHI”, Associate Professor, Department of
Pharmacognosy for their guidance and support in completing our project.
Moreover, we would also like to extend our gratitude to our honorable Principal
Sir, “Dr. MURALIDHAR RAO” M.Pharm. Ph.D., St. Mary’s College of
Pharmacy, and to all the respected faculty and lab technicians for providing us
with all the facilities that were required.

Last but not the least, we are especially thankful to our parents and some other
friends for their unstinting support in doing this dissertation work, which shall
be remembered forever.

Date:
Place: Secunderabad

MANDHAVADHA SAI VENNELA SRI - (20DE1R0051)

MANIKIREDDY SRAVANI - (20DE1R0052)
MEHATAJ AMZUM - (20DE1R0053)
MENDE PRASHANTH - (20DE1R0054)
MERIGA SAI ASHREETHA - (20DE1R0055)

CONTENTS PAGE.NO
 I. ABSTRACT 1

II. INTRODUCTION 2-16

III. LITERATURE REVIEW 17-21

IV. AIM AND OBJECTIVES 22

V. MARERIALS AND METHODS 23-31

VI. RESULTS AND DISCUSSION 32-35

VII. CONCLUSION 36

VIII. REFERENCES 37-39

LIST OF FIGURES:

S.NO NAME PAGE.NO

1. PITHECELLOBIUM DULCE 3
2. WORLD WIDE DISTRIBUTION OF 4
PITHECELLOBIUM DULCE
3. PITHECELLOIUM DULCE IN 5
DIFFERENT CLIMATES
4. VARIETIES OF PITHECELLOBIUM 6
DULCE
5. VEGETATION PROPAGATION OF
6
PITHECELOBIUM DULCE
6. MULCHING
7
7. FRUIT OF PITHECELLOIUM DULCE
10
8. LEAVES OF PITHECELLOIUM DULCE 10
9. FLOWER OF PITHECELLOIUM DULCE 11
10. SEEDS OF PITHECELLOIUM DULCE 11
11. SOXHLET APPARATUS 23
12. SIMPLE DISTILLATION 24
13. INOCULUM OF BACTERIA 31
14. ANTI-BACTERIAL ACTIVITY OF 34
PITHECELLOBIUM DULCE PLANT-
SAMPLE 34
LIST OF TABLES:

S.N0 NAME PAGE NO

1. CLASSIFICATION OF 3
PITHECELLOBIUM DULCE

2. NUTRITIONAL VALUES OF 14
PITHECELLOBIUM DULCE

3. PRELIMINARY PHYTOCHEMICAL 32
ANALYSIS OF LEAF EXTRACTOF
PITHECELLOBIUM DULCE

4. CONSTITUENTS PRESENT IN 33
EXTRACT

5. ZONE OF INHIBITION 33

ABSTRACT:
Plants are the major source of natural drugs. Pithecellobium dulce is one of the plants
with many therapeutic activities belonging to the family Leguminosae.
Pithecellobium dulce is a tree with a spiny trunk and bipinnate leaves. Each pinna has
ovate-oblong leaflets, and its fragrant, sessile flowers reach 12 cm in length. The pod
produces a pink or white, edible pulp with black shiny seeds. The pollens are stitched-
together grains, and the tree can reach heights of 10 to 15 meters. The pod turns pink
when ripe and opens to expose the seed arils. Soxhlet extraction is used to extract the
drug. For 25 grams of shade-dried leaf powder, we got 12 grams of concentrated drug
extract. The preliminary phytochemical analysis of crude drug revealed primary
metabolites like carbohydrates, fats, and proteins. Secondary metabolites like
alkaloids, cardio glycosides, amino acids, flavonoids, steroids, and saponins. The disc
diffusion method is a culture-based microbiology assay used in diagnostic and drug
discovery laboratories to determine the susceptibility of bacteria to clinically
approved antibiotics. The evaluation of anti- bacterial activity was done by using agar
culture media and nutrient media. Anti-bacterial was done by disc diffusion method.
The microbes which were selected for determining the anti-bacterial activity are two-
gram negative microorganism (Escherichia coli, Pseudomonas aeruginosa) and two-
gram positive microorganism (Staphylococcus aureus, Bacillus subtillis). Ethanol was
taken as a negative control and streptomycin of concentration 1mg/ml was taken as
positive control of anti-microbial of plant extract i.e., sample was evaluated for anti-
bacterial activity. The plant extract of 100µl was tested against four bacteria i.e.,
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus
subtillis. The extract shows about 75% of anti-bacterial activity against Escherichia
coli, 83% of anti-bacterial activity against Pseudomonas aeruginosa ,40% of anti-
bacterial activity against Staphylococcus aureus and 65% of anti-bacterial activity
against Bacillus subtillis when compare to standard Streptomycin activity.
INTRODUCTION

1. INTRODUCTION:
Plants are the major source of natural drugs. Pithecellobium dulce is one of the plants
with many therapeutic activities belonging to the family Leguminosae. Along with
200 other American species, such as other woody legumes from the genera Gliricidia,
Leucaena, Prosopis, and Samanea, pithecellobium dulce was brought to the
Philippines from Mexico on board a Spanish government galleon that sailed between
Acapulco and Manila between 1521 and 1815 (Merrill, 1912).

Pithecellobium dulce is endemic to tropical America, but it was originally identified

and named in 1798 by Roxburgh from material gathered in India, where it was
supposedly transported from the Philippines. This suggests that it was introduced to
Asia before 1800. Additional evidence of P. dulce's early historical cultivation and
vast dispersal throughout the tropics comes from its extremely wide range as observed
today, its high degree of naturalization, and the plethora of regional colloquial names.

It is especially common and frequently weedy in the Caribbean, such as in Puerto

Rico where Prosopis pallida grows along mangrove edges, in Hawaii's pastures, and
in the drier, warmer climates of south India and the Gangetic plains (Troup and Joshi,
1983). It's a widespread shade and decorative tree elsewhere. In the mid-1980s,
Pithecellobium dulce seeds were part of a package of widely dispersed dry zone
species from Central America that were tested in a series of species elimination
studies (Hughes and Styles, 1984; Stewart and Dunsdon, 1994). Based on herbarium
records, Lock (1989), Lock and Simpson (1991), and Lock and Heald (1994)
summarize the distribution of Pithecellobium dulce today.(1)

P. dulce was categorized as a highly invasive plant by Binggeli et al. (1998).

According to Space et al. (2000a), it is one of the possibly invasive plants that was
brought to Rota in the Northern Mariana Islands. After discovering that it had become
naturalized, the authors advised against planting it any more. It has also been brought
to other parts of the Pacific, including Chuuk (Space et al., 2000b), Micronesia (Space
and Falanruw, 1999), New Caledonia, Fiji, and French Polynesia (Space and Flynn,
2001), where it is described as cultivated, common, or weedy. It has recently been
rerecorded as an invasive plant on Pohnpei Island, despite not having previously been
considered a concern species in Micronesia (PIER, 2016).(1)

P. dulce was introduced in about 1870 as a shade tree in dry lowlands (Little and
Skolmen, 2003). It is now found in dry pastures and wastelands on almost all of the
Hawaiian Islands (Motooka et al., 2003). In Hawaii, where it is known as "opiuma"
due to the resemblance of its seeds to opium seeds (Neal, 1965). Major Hawaiian
infestations were noted by Smith (1998) in 1998 in areas of Mokuleia, O'ahu,
Moloka'i, Lahaina, Maui, and South Kona. According to Brewbaker (1992), the
invasion of grass pasture in Hawaii mostly happened in areas with low nitrogen
levels, while in other places, the species' propensity to spread was influenced by
things like management and grazing pressure.

Pithecellobium dulce is reported to have naturalized and been rapidly spreading

throughout the Caribbean, including Puerto Rico (Francis and Liogier, 1991). At the
time of reporting in 1991, it was reported to have occupied less than 1000 hectares of
coast on the north and south of the island; additional information on its invasive status
in the Caribbean is required, as it was also reported to have naturalized in Florida,
USA, Jamaica, Cuba, and St. Croix (US Virgin Islands) (Parrotta, 1991). Despite
being classified as an invasive species in Florida by Csurhes and Edwards (1998), the
plant is not listed on the Florida Exotic Pest Plant Council list. Additionally,
Pithecellobium dulce has been found to be invasive (Csurhes and Edwards, 1998) or
naturalized (Parrotta, 1991) in eastern Africa and India.(1)

Products from Pithecellobium dulce plant:

Food: Pods, containing sweet and acidic pulp, are used in sweet drinks like lemonade
and roasted or fresh. They are also used in curries in India, Mexico, Cuba, and
Thailand, where they are harvested and sold on roadside stands.

Fodder: Hedge clippings' pods and leaves are consumed by livestock like horses,
goats, camels, cattle, and sheep, while seed oil extraction press cake residue can be
used as stock feed.

Apiculture: Bees visit flowers and produce high-quality honey.

Fuel: Pithecellobium dulce wood, despite its fast-growing and coppicing nature, is not
highly quality due to its smokiness and low calorific value, primarily used in India for
brick kiln fuel.

Timber: Pithecellobium dulce's wood is strong, durable, and soft, with a moderately
hard, straight-grained texture. It weighs around 590 kg/m³ and is easy to saw. In south
India, it's used for drums, while in China, it's used for matches. It's also used in
construction and posts, but its short spines and irregular growth make it less appealing
for wood use.

Gum or resin: The wounded bark emits a mucilaginous reddish-brown gum,

resembling gum Arabic.

Tannin or dyestuff: Tannin, a dyestuff extracted from bark, seeds, and leaves, is used
to soften leather and dye fishnets a yellow colour.

Lipids: Seeds contain 20% greenish oil, which can be used for food, soap production,
and as a substitute for kapok and ground nut seed oils after refining and bleaching.

Pithecellobium dulce is an evergreen tree belonging to the Leguminosae family that is

widely dispersed throughout Southeast Asia and a significant chunk of India. Among
them, Pithecellobium dulce is one of the more well-known species because of its sour
taste, which is akin to tamarind. Manila tamarind is another name for it. Regarding the
edible pulp of the pod, the species' Latin name, Pithecellobium dulce delicious, refers
to it.
The Greek words "Pithekos," which means an ape, and "lobos," which denotes a pod,
are the sources of the generic term "lobos."

The Leguminosae family is one of the largest groups of flowering plants, with about
650 genera and 18,000 species. Generally, subfamilies like Mimosoideae,
Caesalpiniodeae, and Papilionoideae are distinguished from the main family.(2)

Fig:1.1 Pithecellobium dulce

TAXONOMICAL CLASSIFICATION OF Pithecellobium dulce.

Domain Eukaryota
Kingdom Plantae
Phylum Spermatophyta
Subphylum Angiospermae
Class Dicotyledonae
Order Fabales
Family Leguminosae
Genus Pithecellobium
Species P. dulce
Binominal name Pithecellobium dulce
Table: 1.1 classification of Pithecellobium dulce
Classified into over 600 genera, the Leguminosae is one of the largest families of
flowering plants, with 12,000 species. Three subfamilies—Papilionoideae,
Caesalpinioideae, and Mimosoideae—typically make up the family. Within the
Mimosoideae subfamily, Pithecellobium is a genus comprising 100–200 species that
are distributed throughout the tropics, primarily in Asia and America. Of these, about
10 species are found in India: Pithecellobium globosum, Pithecellobium dulce,
Pithecellobium monadelphum, Pithecellobium arboreum, Pithecellobium flexicaule,
Pithecellobium jiringa, Pithecellobium parviflorum, and Pithecellobium mart, among
others.is one of the well-known species among them; because of its tamarind-like sour
flavor, it is sometimes known as manila tamarind. The genus name "Pithekos" comes
from the Greek word "lobos," which means "pod," and "ape." and the Latin name for
the species, dulce, refers to sweetness in reference to the pod's edible pulp. The plant
is also known as jungli jalebi due to the fruits' similarity to the Indian sweet jalebi.(2)

1.1 SYNONYM:

The local Tamil term for Manila tamarind is Jungle Jilebi, also known as Mitiambli,
Madras thorn, and Korkalikka. Other names for it include Ape's earring and bread-
and-cheese tree. Blackbead, Camachile, Thai Sweet Tamarind, Monkey Pod, Seema
Chintakayalu, and Kona Puliyankai (Twisted Tamarind).

1.2 BIOLOGICAL SOURCE:

The bark, leaves, flowers and fruits of Pithecellobium dulce belonging to the family
“Leguminosae”

1.3 HABITAT:

It is indigenous to Central America, South America, and Mexico. Portuguese and

Spanish settlers introduced it to Indonesia and the Philippines, respectively. In
Thailand, Malaysia, and India, it is typical. P. Bubalinum (Jack) in Malaysia and P.
Jiringa (Jack) Prain in Myanmar and western Malaysia are related species that are
farmed for their fruit. (1979) stated that it is not planted by humans, but rather is
imported as an invasive agricultural species to Hawaii.

Fig : 1.2 World Wide distribution of Pithecellobium dulce

1.4 AREA OF CULTIVATION:

The sweet tamarind is a woodland species with a huge but unreported area. It is
widely found in tropical and subtropical regions as a wayside and fence plant.
Systematic plantation as an orchard has not yet been tried since it has never been
counted under fruit species. The majority of the fruits that are sold and used for
different reasons are gathered from plantations on waste ground and by the side of the
road.

The Pithecellobium species is found in South East Asia, Latin America, Mexico, the
East Indies, and tropical Africa, especially coastal Africa. The main nations where
manila tamarind, also known as sweet tamarind, is typically grown are Guyana,
Venezuela, Brazil, Peru, Jamaica, Puerto Rico, Cuba, St. Croix, Philippines, and
India. It is found all over India; nevertheless, plantations have been established as
food trees on Andaman Island and as forest species in the northern states (Pareek and
Nath, 2006). Mahapatra (2008) has also noted that it exists in the jungles of Orissa.(2)

1.5 CLIMATE AND SOIL:

Within its natural range, the climate is tropical and subtropical, with mean rainfall
falling between 500 and 1000 mm. It is vulnerable to severe cold, yet it can withstand
shadow and dry conditions. It has been grown successfully in regions with maximum
dry seasons of 4–5 months and mean annual rainfall as low as 400 mm. In a semiarid
region of India, where the average monthly temperature varies from 7 to 8°C in
January to 40 to 42°C in May and June, sweet tamarind is said to grow well. Grown
on waste ground, sweet tamarind is a drought-tolerant plant. It may grow in a broad
variety of soil types, such as nutrient-poor sand, rocky limestone soils, clays, and soils
with a high, brackish water table. The tree is said to flourish successfully in highly
degraded Montana wastelands and saline environments in India.

Fig: 1.3 Pithecellobium dulce in different climate

1.6 VARIETY:

Mainly two types of sweet tamarind i.e. Red aril and creamy aril types are common in
most parts of the country.
 Fig : 1.4 varieties of Pithecellobium dulce

1.7 PROPAGATION BY SEEDS:

Usually, it is spread via seed. Seed is sown in a polythene bag that has equal amounts
of sand, clay, and FYM (farm yard manure). Scarification or other treatments are not
necessary for the germination of seeds. While dried seeds require thirty to thirty-five
days to germinate, freshly picked seeds do so readily in one to two days. In storage,
seeds can be kept alive for around six months. After four to six months, seedlings
cultivated in nurseries are used for new plantings. After six months, seedlings may be
poked out of the germination beds and placed in transplant beds or polythene bags.
Young plants require protection from hot, dry winds.(2)

1.8 PROPAGATION BY VEGETATIVE METHOD:

According to Pareek and Vishal Nath (2006), hardwood cuttings can be used to grow
it. July through August is the ideal time to take cuttings and treat with 1000 ppm. IBA
makes rooting better. In little quantities, budding, grafting, and layering also work
well.
 Fig: 1.5 Vegetation propagation of Pithecellobium dulce
1.9 CULTIVATION:
Planting:
This kind of tree is multipurpose. The way it is planted and cared for varies depending
on its use. In order to create an impenetrable hedge, seeds are spread in two to three
rows at a distance of 15 cm. Regular training and trimming will help the hedge grow.
Seedlings are transplanted around the orchard at a spacing of 3½4 m in order to form
a shelter belt. Inga seedlings are planted in a square system with an 8 x 8 m spacing in
order to produce fruit. Plants with vegetative multiplicity are spaced six by six meters
apart. The ideal months to plant are July and August, when the seedlings are placed in
60 x 60 x 60 cm well-prepared and filled trenches. Pit sizes in troublesome soil can be
increased as necessary.(2)
Irrigation:
This tree is resilient and thrives without irrigation. Irrigation is needed in the
beginning to help the new plant establish itself. It is not necessary to use irrigation
once fruit has been grown. Summertime irrigation increases fruit output and size.

Orchard management:

To improve soil management and prevent weeds, intercultural operations can be

introduced early on. As needed, one or two weeding’s can be completed.

Mulching:

It is the process of covering the top soil with plant material, such as grass, leaves,
twigs, crop leftovers, etc. This is also referred to as culturing. The activity of
earthworms and other soil organisms is increased by a mulch cover.
 Fig 1.6: Mulching
Manilla or sweet Although tamarind trees can withstand drought, other plants, such as
dry banana leaves or paddy straw, can be used as mulch beneath trees. Mulch made of
black polythene works wonders at retaining soil moisture.
1.10 Mineral nutrition:

Since the majority of the current plantations are located in shelter belts and roadside
plantations, where nutrients are typically not treated, there is no systematic research
available on the nutritional requirements of manila tamarind.
On the other hand, applying 50 kilograms FYM during the monsoon enhances a
bearing tree's fruit set, fruit size, and yield.
It has been discovered that applying 40–50 kilograms of FYM and 500grams of
phosphatic fertilizer per tree is advantageous. Fertilizers should be applied in the
months of February through March and July through August. Following fertilizer
application, mild irrigation should be conducted.(2)

1.11 Insect pests:

In general, illnesses and pests don't affect Manila tamarind. On the other hand, several
defoliating and burrowing insect pests have been documented. Damage is caused by
shoot hole borer, which can be stopped by filling the holes in the trunk with cotton
swabs dipped in kerosene or gasoline.
It is thorn's preferred host. Additionally, it has been mentioned that lac insects host on
it. Insects on pithecellobium were mentioned by Browne (1968). These insects
include Coleopterus Kelosterna scabrator, Sternoceras termicornis; Hemipterus
Kerria lacca, Nipaecoccus vastator; and Lepidopterus Cryptophlebia illeptida,
Eucosma stereoma, Euproctis scintillans, and Hypanartia hecabe.

1.12 Diseases:

Diseases called leaf spot severely harm plants. On manila tamarinds, fungi have been
identified as Bitzea ingae, Catacauma ingae, Fusarium semitechim var. majus,
Perisporium truncatum, Peziotrichum sacchardinum, Phyllostica ingae¬edulis,
Ravenelia ingae, Rhizoctonia spp., Colletrotrichum spp., Uredo ingae, and Corticum
salmonicolor has been identified.
Fungicide spraying is one way to control them. The mosaic virus also affects trees,
according to Witches Broom. The main diseases that affect this crop are twig blig,
leaf spot (Phyllostica pithecolubis) in Texas and Puerto Rico, Physalospora fusca and
Physalospora rhodina in Florida, and wood rot (Polyporus gilvus) in Hawaii.

1.13HARVESTING, YIELD, POSTHARVEST MANAGEMENT AND

STORAGE:

When the colour of the peel changes from green to pink or the pulp takes on a pinkish
hue, ripe fruits are hand plucked. However, because of the tree's prickly branches and
stem, climbing on it is dangerous. Thin and long bamboo poles with a sharp pruning
knife fastened at the top are used to pick the fruits from a tall (10½–15 m) tree.
Harvested pods are packaged in bamboo and wooden baskets for marketing after
being cut free of the twigs. Fruit kept at room temperature will keep for a few days.
Peel and seeds are removed from the pods to release the pulp. One consumes fresh
fruits. The fruits need to be consumed right away because they don't keep well.(2)

1.14 ECONOMICS OF CULTIVATION, TRADE AND MARKETING:

There is no commercial sweet tamarind or manila plantations. It is frequently gathered

from roadside and forest plantations and sold to ensure a living. The fruits are often
sold in country markets in Mexico and India for their sweet, acidic, and edible arils,
which can be eaten raw, roasted, or blended into a drink similar to lemonade.

1.15 FUTURE RESEARCH THRUST:

Sweet tamarind, also known as manila, is a resilient fruit crop that is not currently
being used for commercial purposes. Because of the trees many uses, high nutritional
value, and antioxidant characteristics, this crop species need additional study. The
species' maximum potential can only be realized following:

1. The development of a dwarf-statured, highly productive, and likely thorn-free

cultivar with excellent fruit quality and palatability.

2. Creating a commercial process to produce planting materials that are high-

quality and true to type.

3. Standardization of all agronomic procedures for fruit production across

different ecological zones.

4. The creation of diverse value-added products and the expansion of their current
application spectrum.

5. The creation of diverse value-added products and the expansion of their current
usage patterns.

6. This fruit's transformation from an underappreciated fruit to a wholesome staple

in the community. (2)
1.16 MORPHOLOGICAL CHARACTERISTICS:
FRUIT:

Fig: 1.7 Fruit of Pithecellobium dulce

COLOUR: Creamy and red colour.

TASTE: Sweet and tangy
ODOUR: Odourless.
SIZE: 5- 7cm in diameter.
SHAPE: Spirally coiled into a circle.
Each pod has 5- 10 shiny black coloured seeds, which are surrounded by thick spongy
dry pulp.(3)

LEAVES:
Fig: 1.8 Leaves of Pithecellobium dulce

COLOUR: Dark green colour.

SHAPE: Bipinnate.
Each pinna has a single pair of ovate- oblong leaflet that are 2- 4 cms long.
Spines are present in pairs at the base of the leaf.(4)

FLOWERS:

Fig: 1.9 Flower of Pithecellobium dulce

COLOUR: Creamy- white.

SPECIFORM PANICLES: 10- 20 cm in length, 1- 1.5 cm in diameter.
SIZE: 0.3- 0.5 cm with hairy corolla and calyx.
Each branch has round 15- 20 white flowers in round heads.

SEEDS:

Fig: 1.10 Seeds of Pithecellobium dulce

COLOUR: Glossy black

SHAPE: Oval- elliptical
SIZE: 0.9- 1.2 cm long and 0.7 cm wide.
It is covered with freshly white or pinkish edible aril, covering the lower half of the
seed.(5)
1.17 CHEMICAL CONSTITUENTS:
 The fruit has a 50% pulp content and a distinct flavor and taste. The leaves and
seed have a high protein content and can be used medicinally. Triterpene
saponins found in the seeds have anti-inflammatory properties. The seed's
fatty acid profile showed that roughly 26.5% of it contained linoleic acid
(18:2), and its lipid content was 30%.

 The predominant sugars found in arils are glucose and sterol glycoside. The
fruit is rich in nutrients, protein, polyphenols, anthocyanin, and distinctive
scent.

 The seed has been shown to include lipids, phospholipids, glycosides,

glycolipids, and polysaccharides in addition to a steroid saponin.
9 saturated and 17 unsaturated fatty acids were found in the seed extract
during fatty acid analysis.

 The seeds had the highest total protein content (50.3–67.1%), followed by the
stems, roots, leaves, fruits, and flowers.

 The fruit extract was discovered to contain flavonoids, including rutin,

quercitrin, kaempferol, naringin, and daidzein, and it was also shown to be
rich in phenolic compounds.

 The fruit pulp's glycoside-A steroid is a combination of beta-sit sterol, which

lowers cholesterol, camp sterol, and stigma sterol (anti-diabetic).

 The leaves include 29% protein and fiber, 1.14% calcium, and 0.35%
phosphorus.

 The seed contains 13.5% moisture, 17.6% protein, 17.1% crude fiber, and
41.4% starch.(6)
1.17.1 Chemical Constituents present in leaves of
Pithecellobium dulce:

Alkaloids:
1. Juliprosopine: This alkaloid has been isolated from the leaves of
Pithecellobium dulce and has been studied for its potential
pharmacological activities, including antimicrobial properties and effects
on blood pressure regulation.
Flavonoids:

1. Kaempferol: Another common flavonoid found in many plants, kaempferol

has antioxidant and anti-inflammatory properties and is associated with
potential benefits for heart health.
2. Rutin: Also known as quercetin-3-O-rutinoside, rutin is a flavonoid
glycoside that has antioxidant and anti-inflammatory effects and is often used
in traditional medicine.
3. Luteolin: This flavonoid is known for its antioxidant and anti-inflammatory
properties and has been studied for its potential role in various health
conditions.
4. Apigenin: Found in many plants, including certain parts of Pithecellobium
dulce, apigenin is known for its antioxidant and anti-inflammatory activities.
Tannins:
1. Hydrolysable Tannins: These tannins are esters of Gallic acid or Ellagic acid
with glucose or other polyols. Hydrolysable tannins are typically found in
fruits, seeds, and leaves of certain plant species.
2. Condensed Tannins (Proanthocyanidins): These tannins are polymers of
flavan-3-ol units (such as catechin and epicatechin). Condensed tannins are
commonly found in fruits, seeds, bark, and leaves.
1.18 NUTRITIONAL BENEFITS:

 The fruit known as jungle jalebi is rich in minerals like calcium, potassium,
phosphorus, and iron as well as a wide range of substances like dietary fiber,
carbs, lipids, and proteins.
 It also contains phytochemicals like phenols, flavonoids, and saponins.
 The nutritional composition of this fruit is given in the table below. (7)
Nutritional components Value per 100g
Carbohydrates 76.87 g
Protein 12.4 g
Dietary fibre 1.3 g
Fats 0.24 g
Potassium 0.2 g
Phosphorus 0.04 g
Calcium 0.01 g
Iron 0.005 g

Table no: 1.2 Nutritional values of Pithecellobium dulce

 Essential vitamins like vitamin C (antioxidant activity), vitamin E (delays

aging), thiamine, and riboflavin (contributes to skin and hair health) are found
in the fruits and seeds of Pithecellobium dulce.

 Essential amino acids, including valine, lysine, phenylalanine, and tryptophan;

Minerals, including calcium, sodium, potassium, and phosphorus, which
support healthy bones and teeth; and iron, which reduces fatigue.

 This plant's leaf extract contains substances including dulcitol, kaempferol,

afzelin, and quercetin.

 Saponins, flavonoids, and phenols are present in its fruit.

 The hydroxyl functional group of flavonoids and phenolic compounds has the
capacity to scavenge radicals and avert oxidative damage.

 Leaf content is high in fiber, and seed content is high in protein. (8,9,10)

1.19 HEALTH BENEFITS:

 1. Weight loss: The amounts of dietary fiber in jungle jalebi aid in appetite
regulation and hunger reduction. Having a glass of lemon juice that has been
infused with extracts from jungle jalebi pods is a great way to lose weight and
keep it off.

2. Treats Digestive Issues: Quercetin and flavonoids, two potent antioxidants,

are abundant in jungle jalebi pods. These work well to scavenge toxic
substances and dangerous free radicals from the stomach and intestines, which
treats diarrhea and dysentery (bloody diarrhea).

3. Controls Symptoms of Diabetes: The juice from extracts of jungle jalebi

pods has been shown to have anti-hyperglycemic properties. This suggests that
it can effectively lower blood sugar levels in people with type 2 diabetes.
Therefore, diabetics can frequently consume this tart juice to help control their
blood sugar levels.

4. Strengthens Bones and Muscles: The abundant calcium and phosphorous

found in jungle jalebi pods are essential nutrients for strong bones. Moreover,
these pods' exceptional protein content contributes to the growth of strong,
healthy muscles.

5. Boosts Immune Function: Drinking a glass of jungle jalebi juice provides

large amounts of vitamin C, an advantageous antioxidant that enhances
immunity. In addition to being a potent defense against infections and
illnesses, vitamin C improves the texture of the skin, minimizing dark spots
and acne while also giving the face a healthy, glowing appearance.

6. Relieves anxiety and depression: Alkaloid, flavonoid, and tannin-rich jungle

jalebi fruits relieve anxiety and depression. These phytonutrients have potent
bioactive properties that improve memory, cognition, and brain function in
addition to reducing anxiety and depressive symptoms and elevating mood.
(11,12,13)

SIDE EFFECTS:
Reflux and decreased renal function.

INTERACTIONS:
 Pithecellobium dulce has the potential to worsen bleeding risks and interact with
some drugs, including blood thinners.

CONTRAINDICATION:
Allergic Reactions:
Pithecellobium dulce allergies can occur in people who are allergic to other members
of the Fabaceae family of plants, which includes peanut allergies.

Pregnancy and Breastfeeding:

The safety of Pithecellobium dulce during pregnancy and lactation is not well
understood. Because of the possible hazards, it is advised that women who are
pregnant or nursing refrain from using it.

Digestive Issues:
In certain people, consuming too many Pithecellobium dulce seeds or pods might
result in digestive distress, such as diarrhoea or pain in the abdomen. (14)
LITERATURE
REVIEW
 2. Literature Reviews:

1. Antidiabetic and antihyperlipidemic activity:

Dnyaneshwar Madhukar nagamoti, et al, studied the seeds extract in streptozotocin-
induced diabetic rats. The study Investigated the attenuating effect of methanolic
extract of Pithecellobium dulce seeds on hyperglycaemia, hyperlipidaemia in
streptozotocin induced diabetic rats. Diabetes was induced by intraperitoneal
administration of streptozotocin-induced (50mg/kg) in male Wistar rats. Diabetic rates
were treated with oral doses of PDM for 21 days. Blood was withdrawn to measure
glycosylated haemoglobin, insulin, total proteins. The glycogen content of liver was
estimated. Histopathological examination of liver, kidney and pancreatic tissue was
also examined. Oral administration of PDM caused decrease in fasting blood glucose
and significant increase in body weight, serum, insulin, total protein and liver
glycogen levels in treated diabetic rats. It presents indicates that PDM possessed
significant antihyperglycemic, antihyperlipidemic, antioxidant potential which prove
beneficial in treatment of diabetes. (15)

ANTIDIABETIC ACTIVITY OF EXTRACTS OF PITHECELLOBIUM

DULCE BENTH LEAVES IN ALLOXAN INDUCED DIABETIC RATS:
Mule V.S. ,Naikwase N.S, Magdum C.S, Jagtap V.A had performed the antidiabetic
activity of extracts of Pithecellobium dulce leaves. The most prevalent kind of
diabetes is called diabetes mellitus (DM), which is brought on by an increase in
insulin resistance or a lack of the hormone insulin, both of which lead to
hyperglycemia. Insulin resistance and malfunctioning β cells in the pancreas are the
primary causes of diabetes, which is the most common and deadly metabolic illness in
the world. Diabetes mellitus (DM) is a disorder that is dependent on the degree of
hyperglycemia, leading to issues related to both macro and microvascular health. Oral
glucose tolerance test: The acclimated animals were split into seven groups of five
rats and allowed to fast for a full day at room temperature. The first groups were
control groups and were given distilled water. The second group controls diabetes.
Rats in groups 4–7 were given aqueous and ethanolic extracts at doses of 200 and 400
mg/kg, respectively, and the third group was given pioglitazone at an oral dose of 20
mg/kg. Following the withdrawal of the initial (0 hours) blood samples, the rats in all
groups were then given 2 g/kg of glucose orally. After glucose loading, blood glucose
levels were measured using glucose strips at intervals of 30, 60, 90, and 120 minutes
from the tail vein.
Effect of aqueous and ethanolic extract of P. dulce leaves on mean fasting blood
glucose level in alloxan induced diabetic rats. (16)
 2. Antibacterial activity:
Muneer Ahmad Bhat, et al, (2018) studied the extract of root was evaluated for
antibacterial activity against one gram positive (S. aureus) & three gram negative
(Acetobacter aceti, Klebsiella pneumoniae strains). Extract prepared in polar solvents
(Ethanol) of root was found to be most active against S. aureus, Acetobacter aceti &
Klebsiella pneumoniae strains.
The zone of inhibition of ethanolic root extract against S. aureus, Acetobacter aceti
& Klebsiella pneumoniae strains was 15.4, 11 & 19mm at 100mg conc.
The benzene extract showed some activity against Acetobacter aceti with the zone of
inhibition 10mm. The antibacterial activity of root, hexane extract was found to be
negligible against all 4 strains of bacteria. R.Kalavani Evaluation of anti-
inflammatory and antibacterial activity of pithecellobium dulce extract. For this
streptococcus progenes, staphylococcus aureus, Escherichia coli have been studied
for pithecellobium dulce extract.
This shows the presence of many difference secondary metabolites such as alkaloids,
flavonoids, glycosides, phenols, terpenoids, saponins, tannins , have been detected.
The Antibacterial response showed a +ve when compared with the standard drug
gentamycin at lower concentration. Hence ethanolic extract of pithecellobium dulce
can be used as potential agents as anti-inflammatory and Antibacterial activity. (17)

3. Antioxidant activity:
Shankar D. Katekhaye, et al, (2012). Studied the invitro anti-oxidant in the plant
Pithecellobium dulce. Methanol & 70% acetone extract of wood bark & leaves of P.
dulce were evaluated for their total phenolic & flavonoid content and anti- oxidant
activity. Total antioxidant activity in DPPH (2,2-diphenyl-1-picrylhydrazyl) assay by
methanol extract on bark is 150.23 ± 2.8µg/ml, acetone extract of bark 16.83
±0.38µg/ml, methanol extract of leaf 250.23 ± 4.8µg/ml and acetone extract of leaf
18.3 ± 0.43 µg/ml. The evidence shows that methanolic & 70% acetone extracts of P.
dulce wood bark and leaves are potential source of natural antioxidants.(18)

4. Antifungal activity:
Suman Kumari, (2016) evaluated anti- fungal activity, a protein of an apparent
molecular mass of 14.4kDa with antifungal activity was isolated from the seeds of P.
dulce using extraction with 100mM Tris-HCl buffer(pH=8), ppt with 80% ammonium
sulfate and bioassay purification via Anion exchange chromatography and Super Dex
200 Gel filtration chromatography. This plant lysozyme expressed antifungal activity
with a rather high thermal stability of up to 80°c for 15min at pH=8. It extracted an
antifungal action towards Macrophomina phaseolina but no antifungal activity
towards Phymatotrichopsis omnivore and Fusarium avenaceum.(19)

Narumon Sawasdipuksa et.al. performed a lysosome with antifungal activity from

pithecellobium dulce leaves. The P. dulce seed extracts were found to have inhibitory
effect against papain, chymotrypsin, and trypsin. P. dumosum seeds yielded a Kunitz-
type protease inhibitor that was discovered to have insecticidal qualities, while P.
dulce seeds yielded a protease inhibitor of the Kunitz family that was isolated and
described. This paper describes a lysozyme-like protein that has antifungal action
found in P. dulce seeds. Potato dextrose agar (PDA) on sterile Petri plates was used to
test the antifungal activity of the material. Sterile paper disks with a diameter of 0.625
cm were positioned 0.5 cm away from the mycelial colony's rim once it had begun to
spread outward from the application zone. Before mycelial growth from the central
region enveloped the peripheral disks containing the control and formed crescents of
inhibition (positive) or enveloped (negative) the disks with test samples for antifungal
activity, an aliquot (40 ml) of the fraction under investigation was added to the disk.
The plates were then incubated at 28 °C for 72 hours. The three fungi utilized in this
assay were Fusarium avenaceum, Phymatotrichopsis omnivora (cotton rot), and
Macrophomina phaseolina (charcoal rot), which were all received from the Plant
Biology Division of The Samuel Roberts Noble Foundation in Ardmore, Oklahoma,
USA. The antifungal growth activity (fungistatic and/or fungicidal) of the crude
protein extract from P. dulce seeds, which was produced following the precipitation
with 80% ammonium sulfate and dialysis, was assessed against three phytopathogenic
fungi. Using tandem mass spectrometry and chromatography techniques along with
Mascot database searching, a lysozyme has been isolated, purified, and identified
from Pithecellobium dulce seeds. With a molecular mass of 14.4 kDa, this plant
lysozyme is somewhat similar to the 14.3 kDa molecular mass of chicken egg white
lysozyme, with which it also exhibits a significant degree of partial amino acid
sequence similarity. Furthermore, Macrophomina phaseolina is susceptible to the
antifungal effects of plant lysozyme, which has a rather high degree of thermal
stability up to 80 °C for 15 min (at pH=8.0).(20)

5. Antimicrobial activity:
Mukesh Kumar, et al, (2012) evaluated of Pithecellobium dulce seed extract against
pathogenic bacteria in silio and in vitro methods.Extract from Pithecellobium dulce
seed and its ability to inhibit the growth of clinically relevant multi drug resistant
bacteria. It shows that Pithecellobium dulce extract is a rich source of D-Turanos
(55.82%), hexadecenoic acid (11.56%), indole-1-acetic acid (11.42%), inositol
(5.78%), and octadecanoic acid (4.36%). The extract shows Antibacterial activity
towards tested clinical bacterial strains with MIC values ranging from 233mg/ml for
Acinetobacter Baumanni to 300mg/ml for S. aureus and Escherichia coli. Possible
use of Pithecellobium dulce extract as a valuable source for drug development against
pathogenic drug resistant bacteria. Using the Agar well diffusion and broth
microdilution procedures, the antibacterial activity of Pithecellobium dulce root was
assessed against five different microorganisms in this study. Ethanol and water were
alternately macerated with P. dulce roots constantly. The outcomes demonstrated the
ethanolic extract’s Staphylococcus activity.
The results indicated that Bacillus subtilis had the greatest inhibitory zone
(11.67±0.58 mm) against Staphylococcus aureus, the lowest MBC (500 μg/ml)
against Bacillus subtilis (Turbidity method), and the lowest MIC (250 μg/ml) against
both bacteria (Diffusion). The water extract shown no activity against Saccharomyces
cerevisiae or Escherichia coli, however it did display the greatest inhibitory zone
(8.00±0.00 mm) and lowest MIC (62.5 μg/ml) against Staphylococcus aureus, as well
as the lowest MBC and MFC.
 >2000 μg/ml against Candida albicans, Bacillus subtilis, and Staphylococcus aureus.
The study's findings indicate that P. dulce extracts have significant antibacterial
activity due to the presence of bioactive chemicals. (21)

6. Anticancer activity:

Abdullah S. Alhamed. Analysed phytochemical of Pithecellobium dulce seed extract

in colorectal cancer cells.
The findings of this study indicated a significant cytotoxic effect of seeds of this plant
in colorecta cancer Characterized by induction of apoptosis, fell cycle arrest, and
reduction of migration. Collections of seeds and extraction is done with the distilled
water to remove any contaminates and dried in dark area. 426g of dried seeds were
milled into coarse powder using electrical grinding mill. This powder was extracted
three times by maceration.
The phytochemical analysis of extract was detected using high resolution liquid
chromatography- mass spectrometry to determine the constituents with known
anticancer properties. The analysis defines 14phythochemicals in +ve electrospray
ionization modes and 21 phytochemical in -ve electrospray ionization modes. Five of
these I dictates the phytochemical have known anticancer properties.(22)

7. Nanoparticles:
G. Madhumitha et.al. had performed the green synthesis, characterization and
antifungal activity of Pithecellobium dulce peel-mediated ZnO nanoparticles. Because
of their stability, broad surface area, and strong electron-hole binding energy (60
meV), zinc oxide (ZnO)NPs have been widely documented to degrade dyes. Thanks
to their catalytic activity and quantum efficiency, ZnO NPs are regarded as an
extremely essential photocatalyst in photocatalytic dye degradation, similar to TiO2
nanomaterials. The US Food and Drug Administration similarly considers them to be
safe. To breakdown dyes, large-surface-area, nanoscale zinc oxide nanoparticles (ZnO
NPs) are synthesised more readily when phytochemicals such phenolic compounds
are present. In the production of ZnO NPs, the phytochemicals function as capping
agents, modifying the NPs' surfaces while simultaneously regulating NP aggregation.
This inspired us to create ZnO NPs using dried peel extract from Pithecellobium
dulce.
It has been demonstrated that the highly bioactive phytochemicals found in the aerial
sections of P. dulce have antidiabetic, antivenomic, antioxidant, anti-inflammatory,
antibacterial, and antiulcer properties. P. dulce's larvicidal and antibacterial properties
have led to its usage in the synthesis of NPs. Iron NPs, silver NPs, and ZnO NPs have
primarily been synthesized using P. dulce leaf extracts. There isn't a single publication
that we could find on using the peel to make ZnO NPs for photocatalytic dye
degradation. Because of its antibacterial properties and ability to breakdown the dye
methylene blue (MB), we decided to produce ZnO NPs using P. dulce peel. In the
Vellore area of Tamil Nadu, India's Puttuthakku Forest is where the P. dulcepeel was
collected. AVRA Chemicals, Hyderabad, India provided the zinc acetate and MB, and
distilled water was used throughout the entire experiment. ZnO NPs were synthesised
in a single step utilizing a green technique, employing P. dulce peel extract as a
capping and stabilizing agent. Consequently, ZnO NPs produced with P. dulce may be
applied to more environmental science and nanotechnology research.
A one step eco-friendly phytosynthesis of ZnO nanoparticles is proposed.
The first report on peel of Pithecellobium dulce used for nanoparticles synthesis.
Performance of ZnO nanoparticles on methyelene blue degradation achieved. (23)
 3. AIM AND OBJECTIVES:

Aim:

Evaluation of the antibacterial activity of Pithecellobium dulce of leaf extract.

Objectives:

 To collect the plant material.

 To authentify the plant material.

 Preparation of extract.

 Preliminary phytochemical analysis.

MATERIALS
AND
METHODS
4. MATERIALS AND METHODS:
4.1 Collection of plant Material:

Fresh leaves of the plant, Pithecellobium dulce as shown in figure 1was collected
locally during the month of February to April from the local areas of Ranga Reddy
district of Telangana. The plant material was washed thoroughly, initially with tap
water and then with distilled water to remove any debris or dust particles and was
shad dried. The dried plant material was ground to a fine powder and stored at room
temperature in airtight containers until used further.

4.2 Preparation of leaf extract:

Leaves were washed with water and 50 grams of fresh leaves are shade dried (kept at
25°C for 5 days in the absence of sunlight). To prepare the leaf extract Pithecellobium
dulce of Soxhlet method is used.

Soxhlet extraction:

This process is otherwise known as continuous hot extraction. The apparatus is called
Soxhlet extractor made up of glass. It consists of a round bottom flask, extraction
chamber, siphon tube, and condenser at the top.

A dried, grinded, and finely powdered plant material of Pithecellobium dulce is

placed inside muslin bag made up of a clean cloth or strong filter paper and tightly
closed. The extraction solvent is poured into the bottom flask, followed by the thimble
into the extraction chamber. The solvent is then heated from the bottom flask,
evaporates, and passes through the condenser where it condenses and flow down to
the extraction chamber and extracts the drug by coming in contact. Consequently,
when the level of solvent in the extraction chamber reaches the top of the siphon, the
solvent and the extracted plant material flow back to the flask. (24)

Fig: 4.1 Soxhlet apparatus

The entire process continues repeatedly until the drug is completely extracted, a point
when a solvent flowing from extraction chamber does not leave any residue behind.
This method is suitable for plant material that is partially soluble in the chosen solvent
and for plant materials with insoluble impurities. However, it is not a suitable method
for thermolabile plant materials.

Advantages: Large amount of drug can be extracted with smaller amount of solvent.
It is also applicable to plant materials that are heat stable. No filtration is required, and
high amount of heat could be applied.

Disadvantages: Regular shaking is not possible, and the method is not suitable for
thermolabile materials.

The soxhlet method of extraction is followed by simple distillation method.

Distillation is a technique used by chemists to separate components of a liquid
mixture with different boiling points.

Fig: 4.2 Simple distillation

To do so, the liquid mixture is heated to only boil one component, which separates
from the mixture as vapour. This vapour then passes through a cold tube, condensing
it back into a liquid and flowing into a separate vessel. (25)

4.3 PHYTOCHEMICAL ANALYSIS:

Preliminary phytochemical analysis of the extract:

To assess the chemical composition of the various extracts qualitatively, a preliminary

phytochemical analysis was conducted according to the standard methods.
The presence of several phytochemicals like sterols, proteins, sugars, alkaloids,
flavonoids, saponins, amino acids and cardiac glycosides was evaluated. (26)

Chemical test for Carbohydrates:

1. Molisch test: To 2- 3 ml of test solution, add few drops of molish reagent solution
and shake. Then add concentrated sulphuric acid from the sides of the test tube. Violet
ring is formed at the junction of two liquids.

Benedict test: To 5 ml of benedict solution, add 1 ml of test solution and shake it

well. Place the test tube in a boiling water bath and heat it for 3 minutes. Remove the
test tube from the heat and allow then to cool. Formation of green-red or yellow
precipitate is observed.

Chemical test for Alkaloids:

1. Dragendorff’s test: Add 3 ml filtrate, add few drops of Dragendroff reagent

(potassium bismuth iodide solution). Formation of orange- brown-coloured
precipitate shows presence of alkaloids.
2. Mayer’s test: Add few drops of Mayer’s reagent (potassium mercuric iodide
solution) in 3 ml of filtrate. Formation of cream coloured precipitate, indicates
the presence of alkaloids.

3. Hager’s test: Add small quantity of Hager’s reagent (saturated aqueous

solution of picric acid) to filtrate. Formation of yellow coloured precipitate
shows the presence of alkaloids.

4. Wagner’s test: Add few drops of Wagner’s reagent (Iodine in potassium

iodide) in 3 ml of filtrate. Formation of reddish brown coloured precipitate the
presence of alkaloids.
Chemical test for Saponins:

1. Foam test: The drug extracts were vigorously shaken with water. Persistent
foam formation indicates presence of saponins.

Chemical test for Proteins:

1. Biuret test: To about 3 ml of the extract,40% sodium hydroxide solution

and few drops of 1% copper sulphate solution is added. It produces blue
colour.

Chemical test for Amino acids:

1. Ninhydrin test: To the heated 3 ml test solution, add 3 drops of 5%

ninhydrin solution and boil for 10 minutes. Purple or bluish colour appears.
Chemical test for Steroids:
1. Salkowski reaction: Add 2 ml of the extract, add 2 ml of chloroform and 2
ml of concentrated sulphuric acid. It was shaken well. Chloroform layer
appeared red and acid layer showed greenish yellow fluorescence.

Chemical test for cardiac glycosides:

1. Killer-killani test: To the test solution, few drops of Ferric Chloride
solution and concentrated sulphuric acid was added. Formation of two
layers occur, lower layer is reddish brown colour and upper layer of bluish
green colour simultaneously.
Chemical test for Flavonoids:
1. Shinoda test: 5 ml of 95% ethanol was added in the extract and then few
drops of concentrated HCl and 0.5g magnesium turnings were added. Pink
colour shows the presence of flavonoids.

4.4EVALUATION OF ANTI- BACTERIAL ACTIVITY:

Agar disc-diffusion testing developed in 1940, is the official method used in many
clinical microbiology laboratories for routine antimicrobial susceptibility testing.
Nowadays, many accepted and approved standards are published by the Clinical and
Laboratory Standards Institute (CLSI) for bacteria and yeasts testing.
In this well-known procedure, agar plates are inoculated with a standardized inoculum
of the test microorganism. Then, filter paper discs (about 6 mm in diameter),
containing the test compound at a desired concentration, are placed on the agar
surface. The Petri dishes are incubated under suitable conditions. Generally,
antimicrobial agent diffuses into the agar and inhibits germination and growth of the
test microorganism and then the diameters of inhibition growth zones are measured.
The organisms which are used for study are two-gram positive and two-gram negative
micro-organisms and ZOI (Zone of Inhibition). (27,28)

4.4.1Preparation of Nutrient broth:

The nutrient broth may be prepared using the following formula:

Beef Extract 3grams
Peptone 5grams
Sodium chloride 5grams
Distilled water 1000milliliter

Weigh & transfer the solid ingredients into a clean conical flask. Dissolve in the
required volume of water with the aid of heating. Adjust pH to 7.2-7.4 with 5M
NaOH. Heat for ten minutes at about 60°C. Filter & plug with non-absorbent cotton &
cover with kraft paper. Sterilize in autoclave at 121°C for 15 minutes.

4.4.2Preparation of Nutrient agar:

The nutrient agar may be prepared using the following formula:

Beef Extract 3grams

Peptone 5grams
Sodium chloride 5grams
Distilled water 1000milliliter
Agar 15grams

Prepare nutrient broth as in 4.4.1. Add agar powder, heat at about 98°C to dissolve
completely. Adjust pH to 7.2-7.4 using 5M NaOH. Plug with non-absorbent cotton &
cover with kraft paper. Sterilize in autoclave at 121°C for 15 minutes. (29)
4.4.3 Disc-diffusion method:
To inoculate a nutrient agar plate, dip a sterile swab into the inoculum tube and rotate
it to remove excess fluid. Inoculate the plate by streaking the swab three times over
the entire surface, rotating about 60 degrees each time. Rim the plate with the swab to
remove excess liquid. Discard the swab and let the plate sit at room temperature for 3
to 5 minutes, but no more than 15 minutes, for the surface to dry.
Placement of the antibiotic discs:
Dispense antimicrobial-impregnated disks onto the agar surface using forceps.
Sterilize the disks with alcohol pad or isopropyl alcohol before touching them to
ensure complete contact with the agar surface. Do not move the disk once it has
contacted the agar surface, even if it's not in the correct location, as some of the drug
begins to diffuse upon contact. This is crucial before replacing the petri dish lid to
prevent static electricity from causing disks to relocate or adhere to the lid. To add
disks to an agar plate using forceps, place the plate on the template and sterilize the
forceps by cleaning them with alcohol. Remove one disk from the cartridge and
gently press it onto the agar surface. Replace the lid to minimize exposure to room air.
Invert the plates, place them in a 35°C air incubator for 16 to 18 hours, and repeat the
process for each disk. This procedure ensures complete contact with the agar surface.
The study used ethanol as a negative control and streptomycin as a positive control to
evaluate the anti-microbial activity of a plant extract. The sample was then incubated
and the Zone of Inhibition (ZOI) was measured after the incubation. (30)

Fig 4.1 Inoculum of bacteria

RESULTS
 5. RESULT AND DISCUSSION:

Table 5.1 Preliminary Phytochemical analysis of herbal extract:

TEST OBSERVATION INFERENCE

1. Molisch test Violet colour ring is Presence of

developed between the layers. Carbohydrates.

2. Benedict’s test Green-red or yellow Presence of

precipitate is observed. Carbohydrates.

3. Dragendorff’s test orange- brown coloured presence of Alkaloids.

precipitate is seen.

4. Mayer’s test cream coloured precipitate presence of Alkaloids.

5. Foam test Persistent foam formation Presence of saponins.

6. Shinoda’s test Cheery Red colour is Presence of flavonoids.

observed.

7. Biuret test Blue colour is observed. Presence of Proteins.

8. Ninhydrin test Purple or bluish colour Presence of Amino acids.

appears.
9. Salkowski reaction Chloroform layer appeared Presence of Steroids.
red and acid layer showed
greenish yellow fluorescence.
10. Killer-killani test Formation of two layers Presence of Cardio
occur, lower layer is reddish glycosides.
brown colour and upper layer
of bluish green colour
simultaneously.
 Table 5.2 Constituents present in extract:
Tests Results
Carbohydrates Present
Alkaloids Present
Glycosides Present
Flavonoids Present
Proteins Present
Amino acids Present
Saponins Present
Steroids Present

Table 5.3 Zone of Inhibition:

Organism Plant extract Positive control Negative control

(100µl) (Streptomycin (Ethanol-100µl)
0.75mg) 1mg/ml
E. coli 25mm 34mm -

Pseudomonas 24mm 30mm -

aeruginosa

Staphylococcus 16mm 40mm -

aureus

Bacillus 25mm 38mm -

sphaericus
Anti-bacterial activity of Pithecellobium dulce plant sample:

The petri dish showing the anti-bacterial activity against the Pseudomonas aeruginosa,
the ZOI of positive bacteria, negative bacteria, control & plant extract.

The petri dish showing the anti- bacterial activity against the E. coil, the ZOI of
positive bacteria, negative bacteria, control & plant extract.

The petri dish showing the anti- bacterial activity against the Staphylococcus
aureus,the ZOI of positive bacteria, negative bacteria, control & plant extract.

The petri dish showing the anti- bacterial activity

against the Bacillus sphaericus, the ZOI of positive
bacteria, negative bacteria, control & plant extract.

DISCUSSION:
Pithecellobium dulce leaves were collected from
Randgareddy and used for extraction. Hot method
of Extraction is used. Soxhlet method is used to
extract leaves of Pithecellobium dulce, powdered, and placed in a muslin bag. The
solvent is poured into a flask, evaporated, and condensed, then injected into the
chamber to extract the drug. The solvent and extracted plant material return to the
flask when the solvent level in the extraction chamber reaches the top of the siphon.
The process continues until the drug is completely extracted, suitable for partially
soluble and insoluble plant materials, but not thermolabile ones. The percentage yield
is about 60% of raw material taken.
 The preliminary phytochemical analysis for crude drug which was shown the
presence of primary metabolites such as Carbohydrates, Fats, Proteins and the
secondary metabolites such as Alkaloids, Cardiac glycosides, Amino acids, Steriods,
Flavonoids and Saponins.
Nutrient medium was used to evaluate anti-bacterial activity. Anti-bacterial was done
by disc diffusion method. The microbes which were selected for determining the anti-
bacterial activity are two-gram negative microorganism (Escherichia coli,
Pseudomonas aeruginosa) and two-gram positive microorganism (Staphylococcus
aureus, Bacillus subtillis). Ethanol was taken as a negative control and streptomycin
of concentration 1mg/ml was taken as positive control of anti-microbial of plant
extract i.e., sample was evaluated for anti-bacterial activity.
The plant extract of 100µl was tested against four bacteria i.e., Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtillis.
The extract shows about 75% of anti-bacterial activity against Escherichia coli, 83%
of anti-bacterial activity against Pseudomonas aeruginosa ,40% of anti-bacterial
activity against Staphylococcus aureus and 65% of anti-bacterial activity against
Bacillus subtillis when compare to standard,Streptomycin activity.
CONCLUSION:

The Preliminary phytochemical analysis was done for leaf extract of

Pithecellobium dulce and it shows the presence of Alkaloids, Cardiac
glycoside, Amino acid, Saponins, Fats, Proteins, Carbohydrates, Steroids and
Flavonoids.
The anti-bacterial activity against the two-gram negative bacteria (E. coli-
25mm standard of 34mm; Pseudomonas aeruginosa – 24mm standard of
30mm) and gram-positive bacterial (Staphylococcus aureus- 16mm standard
of 40mm; Bacillus subtillis-25mm standard of 38mm).
Anti bacterial activity of Pithecellobium dulce showed that the extract showed
more potent again activity against Pseudomonas aeruginosa when compared
to other microbes in the study i.e.,
The extract showed anti bacterial activity against both gram positive and gram
negative bacteria.
The order of potency of anti-bacterial activity of extract was as follows:
Pseudomonas aeruginosa, followed by E. col, then Bacillus subtillis and
Staphylococcus aureus.
The present study revealed the potency of Pithecellobium dulce which is also
used as neutraceutical can be the natural drug of choice for its anti-bacterial
activity.
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