phytochemical and antimicrobial analysis of the leaf extract of jatropha curcas

CHAPTER 1

1.0 Introduction

1.1 Brief description:

Jatropha curcas is a poisonous, semi-

evergreen shrub reaching a height of 6m (20ft).

It is cultivated in both tropical and

subtropical regions around the world and also

becoming naturalized in some areas. It is a

perennial crop which is resistant to a high

degree of aridity allowing it to grow in

deserts. The plant also bears fruits with seeds

that contain about 27-40% oil with an average of

about 34.4%.

1.2 Scientific classification

:

Fig. 1

This plant belongs to the euphobiaceae family

whose biological classification is shown in the

table below;

Kingdom Plantae

Unranked Angiosperm

Unranked Eudicots

Unranked Rosids

Order Malphigiales

Family Euphobiaceae

Genus Jatropha

Species J. Curcas

Table 1.

The plant has the binomial name as earlier

mentioned Jatropha curcas.

1 . 3 Uses and application of plant products :

Due to its growth in areas with high degree of

aridity, jatropha curcas is used in control of

desertification. The leaf extract of the plant

is used as a remedy for jaundice when applied by

rectal injection. The latex of the stem is mixed

with salt and used as toothpaste. The roots are

use for chest diseases or may be cooked with

gruel and given to the patient suffering from

kidney disease (Okajugu et.al 2006), and

contains also insecticides. The plant extract is

also used as an antidote for snake bites and

also used for the cure of diseases like cancer,

piles, paralysis and dropsy. The water extract

of the plant inhibits Human Immunodeficiency

Virus (HIV) cythopathic effect with low

cytotoxicity. The seed also produces oil which

has a strong purgative action and it is also

widely used for skin disease and to soothe pain

such as the one caused by rheumatism. The oil

also burns with a clear smoke-free flame and it

has been tested successfully as fuel for simple

diesel engine known as Biodiesel.

1 . 4 Metabolites :

Plants consist of metabolites which are

intermediates and products of metabolism. These

metabolites have various functions including;

fuel, structure, signaling, stimulatory and

inhibitory effect on enzymes, catalytic activity

of their own, defense and interaction with other

organisms. The metabolites present in the leaf

extract are refer to as secondary metabolites

because they are not directly involved in the

normal growth, development, or reproduction of

an organism. The absence of secondary

metabolites usually does not result in immediate

death but rather,in long-term impairment of the

organisms fecundity or aesthetic or perhaps no

significant change at all. They often play a

vital role in plants defense against herbivores

and other interspecies defenses. Humans use

secondary metabolites as medicines, flavorings

and recreational drugs. These secondary

metabolites are also refer to as Phytochemicals.

1 . 4 .0 Phtochemicals :

These are secondary metabolites usually

found in plant extracts. They include;

alkaloids, saponins, tannins, triterpenoids,

cardiac glycosides, steroids and flavonoids.

1.4.1 Alkaloids:

These are naturally occurring compounds

containing nitrogen as an hetero-atom usually

found in plant extracts. An example is morphine

Fig. 2

As shown in the example above, some of the

alkaloids may also consist of other hetero-atom

such as oxygen, sulphur and more rarely

chlorine, bromine and phosphorus. They are good

stimulant too.

1.4.2 Saponin:

Saponins are amphipathic glycosides grouped

in terms of phenomenology by the soap-like

foaming they produce when shaken in aqueous

solution and in terms of structure by the

composition of a hydrophilic glycoside moietes

combined with lipophilic tri terpene. Example is

solanine

Fig. 3

1.4.3 Tannins:

These are astringent, bitter plants,

phenolic compounds that binds to and also

precipitate proteins and other organic compounds

including amino acids and alkaloids. The

compounds are widely distributed in many species

of plants. Example is tannic acid

Fig. 4

1.4.4 Triterpenoid:

They are derivatives of terpene molecules.

They have useful anti-cancer properties. They

consist of six isoprene units. An example is

betulinic acid

Fig. 5

1.4.5 Cardiac glycoside:

These are compounds that have structures

which contain a sugar molecule bound to small

organic molecules that are non-carbonhydrate.

Example is salicin

Fig. 6

1.4.6 Steroids:

These are organic compounds that contains a

characteristic arrangement of four cycloalkane

rings that are join to each other(3 cycloalkane

rings and 1 cyclopentane ring) known as the

gonane molecular nucleus. An example is

cholestane

Fig. 7

1.4.7 Flavonoid:

These are the most important plant pigments

for flower coloration producing yellow or

red/blue pigmentation in petals. Example is the

yellow type known as flavones

Fig. 8

Others include phenolic compounds, terpenes,

other classes of glycosides such as cytogenic

glycosides e.t.c.

1.5.0 Antimicrobial activity:

These analysis measures how susceptible a

micro organism when it comes in contact with

antibiotics. The antibiotic for this analysis is

the leaf extract of plants. In other words, this

analysis measures how active the extract can be

as an antibiotic to inhibit the normal behaviour

and growth of micro organisms. It is usually

measured in terms of Minimum Inhibition

Concentration (MIC). The organisms usually

employed in this test include; staphilococcus

aureus, escheria coli, bacillus cereus e.t.c.

1.6.0 Aim of the project:

This project aims at analyzing the presence

of the analytes i.e, phytochemicals present in

the leaf extract of the plant and also, to

measure the extent at which the extract can

inhibit or render micro organisms susceptible to

it which alter its growth and normal activity

also known as anti microbial, antibiotic test or

susceptibility test.

Chapter 2

2.1 Literature review:

Several works have been carried out on the

leaf extract of jatropha curcas.

Some of which include the phytochemical

analytical works of Oluwale I. Oyewale and Peter

F. Akingbala reported that, the methanolic

extract of Jatropha curcas consist of

flavonoids, tannins, triterpenoids, saponins,

phenolic compounds and cardiac glycosides. Also,

according to the works of Oseni, Lateef Adebayor

and Alphonse, Prince Kofi; the ethanolic leaf

extract consist of saponins, tannins, alkaloids

and triterpenoids present. But, the petroleum

ether extract consist of alkaloids only.

Furthermore, the works of Uche F. I. shows that

alkaloids, flavonoids, saponins,tannins and

steroids were found to be present in the

methanolic leaf extract.

Similarly, for the anti-microbial activity

test usually measured in terms of Minimum

Inhibition Concentration (MIC), Ogbonosa et.al

2009 reported that the antibiotic test of the

methanolic leaf extract on staphilococcus

aureus, bacillius cereus, escheria coli and

bacillius substitis were given as 5.00mg/ml,

10.00mg/ml, 5.00mg/ml and 6.25mg/ml

respectively. There zones of inhibitions ranges

between 10-12 2mm.

Furthermore, Oseni and Alphonse 2011

presented the following result of antimicrobial

analysis of the ethanol extract of jatropha

curcas. The analysis was carried out on bacillus

cereus and staphilococcus aureus who’s MIC were

given as 7.00mg/ml and 6.00mg/ml respectively.

Chapter 3

3.0 Methodology:

3.1 Sampling of leaves and preparation of the

extract:

The collection of the representative part

of the leaves of this plant was carried out at

Dogon dutse, Yan trailer and Fudawa village. The

leaves collected were air dried at room

temperature and were later pounded to increase

its surface area for more interaction with the

solvents in order to get reasonable amount of

the extract. A concentrated methanol solution

was used for the extraction. The pounded leaves

were soaked at room temperature for 3 days. The

resulting mixture was filtered. The filtrate was

distillated under normal atmospheric condition

to evaporate the solvent and the extract

removed.

3.2.0 Test for phytochemicals:

50g of the extract was dissolved in 20ml of

methanol and was use for the phythochemical

test.

Methods described by Odebiyi and Sofowara

were used to test for the presence of saponins,

tannins and alkaloids. Liebermann Burchad

reaction as described by herbourne was used to

test for steroids while cardiac glycosides and

phenolic compounds were tested using the

Solowski test.

Test for saponins: 2ml of the extract solution was

tranfered into a test tube. Foaming which

persist on warming was taken as an evidence for

saponins.

Test for tannins: 5ml of the extract was stirred

with 10ml of distilled water and then filtered.

Few drops of 5% FeCl3 reagent were added to the

filterate. A blue-black or green colouration or

precipitation was taken as an indication for the

presence of tannins.

Test for alkaloids: 2ml of the extract was stirred

with 5ml of 1% HCl on a steam bath. The solution

obtain was filtered and 1ml of the filterate was

treated with a few drops of Meyer's reagent. The

turbidity of the filterates shows the presence

of alkaloids.

Test for steroids: 2ml of acetic anhydride was added

to 0.5g of the extract with 2ml of concentrated

sulphuric acid. The colour change from violet to

blue or green indicates a positive result for

steroids.

Test for cardiac glycosides: 0.5g of the extract was

dissolved in 2ml of chloroform. 1ml of

concentrated sulphuric acid was carefully added

to form a lower layer. A redish-brown colour at

the interface indicated the presence of cardiac

glycosides.

Test for flavonoids: 0.2g of the extract was dissolved

in a mixture of 1ml and 2ml NaOH and HCl

respectively. A yellow solution that turns

colourless on dilution indicates the presence of

flavonoids.

3.2 Procedure for antimicrobial test:

The method used here was the Kirby-Bauer

disk diffusion method which proceeds as follows.

A sterile swap was placed into the broth

culture of a specific micro-organism and was

gently pressed to the walls of the test-tube to

remove excess liquid. The swap was then used to

streak the Mueller-Hinton agar plate in one

direction and then the plate was rotated at

about 90 and was streaked again. The process was

repeated for about 3times to obtain uniform

growth on the plate. The plate was allowed to

dry for approximately 5minutes. The disc which

where kept in a cold condition inside the

solutions containing the extracts at various

concentrations. By the use of a sterilized

forcep, each antibiotic disc was gently

transfered and pressed by the forcep to ensure

its attachment to the agar plate. The plate was

incubated overnight in an incubator and was

maintained at a temperature of about 35+2C. The

cleared zones observed around each of the

antibiotic disc if there was any, was measured

by a ruler and recorded.

Chapter 4.

4.0 Result and discussion:

The pytochemical screening of the extract revealed the

presence of saponins, tannins, triterpenoids, alkaloids,

flavonoids and cardiac glycosides as shown in the table below;

Phytochemicals Results

Saponins ++

Tannins +

Triterpenoids +

Alkaloids +

Cardiac glycosides +

Flavonoids +

Phenolic compounds -

Steroids -

Table 2. Results for phytochemical analysis.

Key:

+ = present

++ = present in aboundance

- = absent.

Micro organisms concentration of

extracts (mg/ml)

zones of

inhibition

Table 3. Result for susceptibility test.

Considering table 2, unlike the works of

Adebayor et. al (2009) whose ethanolic extract

consist of saponins, alkaloids, triterpenoids

and tannins only and also, the petroleum ether

extract consist of alkaloids only. This shows

that, the type of solvent used for the

extraction also affects the result. This happens

in the extraction in such a way that, the

phytochemical to solvent extraction is too small

to allow the dissolution of the phytochemicals

in the solvents. But the works of Uche F.I. that

also used the same solvent as mine (i.e

methanol) reported that, the phytochemicals

present are alkaloids, flavonoids,

triterpenoids, saponins, tannins and sterioids.

This result is almost in full agreement with the

result in table 2. The absence of cardiac

glycosides in my result might be due to the

following reasons; climatic condition,

environmental factors, individual variation of

plant species, maturity of the plant or

experinmental error. But from al indications,

methanol is the common solvent that have more

affinity to the phytochemicals in the extraction

compare to other common solvents whose report I

have ever come across in my literature review.

For the antimicrobial screening, the

susceptibility test result was presented in

table 3. It gives the mean zones of inhibition

of the susceptibility test of the methanolic

extract at various concentrations. The Minimum

Inhibition Concentration was traced using the

breakpoints-MIC relationship as shown below.

Zones of

inhibition

(mm)

</= 12 13-14 >/= 15

Results Resistant Intermedia

te

Susceptibl

e

So from the results in table 3., the MIC of

the methanolic leaf extract of J. Curcas on

Staphilococcus aureus is 10mg/ml while that of

Escheria coli is 12mg/ml. This result

corresponds to previous works that has been

carried out in the sense that, Escheria coli is

more resistant to antibiotics compare to

Staphilococcus aureus.

Also, the introduction in chapter 1 reveals

that, medicinal plants are backbones of

traditional medicine and antibacterial

activities. This property of the plants depends

on the components of its extracts which are

classified as antimicrobial compounds Rojas

et.al(2006). In plants,these compounds function

as to attract beneficials and repel harmful

organisms serving as photoprotectants and also

responds to environmental change. In humans,

they have both complementary and overlapping

action which include anti-oxidant effect,

stimulation of immune systems, reduction of

inflamations and including antibacterial and

antiviral effects Johana (2003).

The preliminary phytochemical analysis shows

that saponins, tannins triterpenoids, alkaloids,

cardiac glycosides and flavonoids are present.

This compounds have been reported to inhibit

bacteria growth and also capable of protecting

plants against bacteria infections (Clark 1981);

(Mather & Gonzalez 1982). Alkaloid are known to

be toxic compounds with limited distribution in

the plant kingdom. They are widely reported to

be responsible for antimicrobial activities

(Doughari 2006) which is consistent with that of

De et.al (1999). The mechanism of the

antimicrobial activities of the phytochemicals

may be due to incresing the permeability of the

cell wall and cell membrane, inhibition of

protein and DNA synthesis or by inhibiting the

transport of nutrient across the cell membrane

(Stewart & Baswick, 1979). Saponin also have

detergent properties and also serve as a lytic

agent and exibits anti-inflamatory properties

(Lewis & Elvin Lewis, 2009). While, glycosides

also function with ends of their defence

mechanisms and wet as protective agents against

invading micro organisms.