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Antimicrobial activity and

pytochemical screening of leaves

extracts fromJatropha curcas

Bartolome, Marino D.

Martin, Shyrl Ann Kay E.

Montalban, Emma Rose T.

Ocampo, Eunice D.

January 21, 2013

Introduction:


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Philippines are blessed to be a home of such a huge variety of naturally

occurring medicinal plants which is known well in Asia and all throughout the

world. Our natures biodiversity projects many species of medicinal plants that

are being utilized by people as an alternative source of medicine.

One of the most promising medicinal plants in our country isJatropha curcas. Itis a small tree or shrub with smooth gray bark, which exudes whitish colored,

watery, latex when cut. Normally, it grows between three and five meters in

height, but can attain a height of up to eight or ten meters under favorable

conditions.

It has large green to pale-green leaves, alternate to sub-opposite, three-to five-

lobed with a spiral phyllotaxis. In winter, all the leaves fall and the shrub is

leafless.

Traditional medicine using plant extracts continues to provide health coverage

for over 80% of the worlds population, especially in the developing world

(WHO, 2002).

Jatropha species belong to the family Euphorbiaceae and are used in

traditional folklore medicine to cure various ailments in Africa, Asia and Latin

America (Burkill, 1994).

Jatropha curcas Linn is commonly called physic nut, purging nut or pig nut.

Previous studies have reported that the plant exhibits bioactive activities for

fever, mouth infections, jaundice, guinea worm sores and joint heumatism

(Irvine,1961; Oliver-Bever, 1986).

Fagbenro-Beyioku (1998) investigated and reported the anti-parasitic activity

of the sap and crushed leaves of J. curcas.

Previous works have shown that many Jatropha species possess antimicrobial

activity (Aiyela-agbe et al.,2000; Aiyelaagbe, 2001).

Several studies have confirmed the antimicrobial efficacy of differentJatropha

species. However, there is insufficient information regarding the antimicrobial

activities of J. curcas. Whatever limited information available on the medicinal

properties ofJ. curcas is mostly on the leaf extracts of the plant.

Thats why it caught our awareness and moved us to study the antimicrobial

property of crude extracts of the leaves ofJ. curcas to be studied as part of the

exploration for new and novel bio-active compounds.


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Objectives :

O To investigate the effectiveness ofJetropha curcas against some selectedmicroorganisms which are known to cause diseases in human beings

O To determine the phytochemicals present inJetropha curcas

O To find out which of the methods of extraction contained the highest

concentration of the active principle ofJetropha curcas

Methodology :

Materials and Method:

Plant materials and preparation of extract:

Fresh leaves ofJatropha curcas will be collect from Binangonan, Rizal in the

month of February 2013.The fresh leaves will be air-dried to constant weight, to

undergo pulverizing and will be in storage at an air-tight container for further

use.

The pulverized plant material will be cold extracted in ethanol and methanol

separately. And the other plant material will also undergo the pulverizing

process in water for 4 days with occasional shaking. Ethanol and methanol will

be used as an analytical grade.

The separated extracted will be then filtered. Filter paper and the ethanol and

methanol filtrate will be separately concentrated to dryness to remove the

ethanol and methanol. The aqueous extract will be lyophilized to obtain a dry

powder extract.

Test microorganisms

The Staphylococcus aureus, Escherichia coli, Candida ablicans, andAspergillus

nigerwill be used as the test microorganisms in this study to be obtain from

the culture collections of Department Of Science and Technology (DOST).

The bacterial isolates will be first sub- cultured in a nutrient agar and incubated

while the fungal isolates will be sub-cultured on potato dextrose agar (PDA).


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Phytochemical analysis of the plant extract

The extracts will be subjected to phytochemical tests for plant secondary

metabolites with little modification.

Antibacterial activity

The antibacterial activity of the crude extracts will be determined in

accordance with the agar-well diffusion method. The bacterial isolates will be

first grown in a nutrient broth before to be used and standardized. Two

hundred microliter of the standardized cell suspensions will be spread on a

nutrient agar. Wells will be then bored into the agar using a sterile cork borer.

The crude extract will be introduce into the wells, and be allowed to stand at

room temperature for about 2 hours and then incubated at 37C.Controls will

be set up in parallel using the solvents that will be used to reconstitute the

extract. The plates will then be observed for zones of inhibition after 24 h. Theeffects will then be compared to the fungal isolates.

Antifungal activity

The fungal isolates will be allowed to grow on a Potato Dextrose Agar (PDA) at

a required temperature until they begin to sporulate. The fungal spores will be

then harvested after sporulation by pouring a mixture of sterile glycerol and

distilled water to the surface of the plate and later scraped the spores with a

sterile glass rod.

The harvested fungal spores and bacterial isolates will be standardized to an

OD600nm of 0.1 sterile 6 mm cork borer and the wells filled with the solution of

the extract taking care not to allow spillage of the solution to the surface of the

agar medium. The plates will then be allow to stand on the laboratory bench for

1 h to allow for proper diffusion of the extract into the media. Plates will be

incubate at 25C for 96 h and later observed for zones of inhibition.

Minimum inhibitory concentration (MIC)

The estimation of MIC of the crude extracts will be carried out. Two-fold

dilutions of the crude extract will then be prepared at 2 ml aliquots of differentconcentrations of the solution will then to be added to 18 ml of pre-sterilized

molten nutrient agar and SDA for bacteria and fungi respectively at 40C to

give final concentration regimes of 0.050 and 10 mg/ml.

The medium will then be poured into sterile Petri dishes and be allowed to set.

The surface of the medium will be allowed to dry under laminar flow before

streaking with the old bacterial and fungal cultures.


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The plates will then undergo to be incubated at 37C for 24 h and at 25C for

up to 72 h for bacteria and fungi respectively, after which we will be examine

for the presence or absence of growth.The MIC will be taken as the lowest

concentration that will prevent the growth of the Staphylococcus aureus and

Escherichia coli thatwill be used as the test microorganisms.

Minimum bactericidal concentration (MBC)

The MBC of the plant extracts will then be determined by a modification of the

method. Samples will be taken from plates with no visible growth in the

MIC assay and sub- cultured on freshly prepared nutrient agar plates and PDA

plates, and will later be incubated at 37C for 48 h and 25C for 72 h for

bacteria and fungi respectively.

The MBC will be taken as the concentration of the extract that did not show any

growth on a new set of agar plates. The MBC will not be determined for water

extract since the antibacterial activity was low.


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