PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY OF

ACALYPHA WILKESIANA AND MAERUA ANGOLENSIS

Amina Sada Yusuf, Ibrahim Sada, Yusuf Hassan*

Department of Chemistry, Umaru Musa Yar’adua University, Katsina, Nigeria

*
Corresponding author: [email protected]

ABSTRACT

The crude ethanol extract from Acalypha wilkesiana and Maerua angolensis were fractionated
with n-hexane, chloroform, distilled water, ethylacetate and methanol to afford five soluble
fractions, respectively. The fractions were tested for antibacterial activity using agar diffusion
method. All the fractions showed good activities against the bacteria, particularly the methanol
soluble fraction. Phytochemical analysis showed the presence of alkaloids, saponins, tannins, and
flavanoids.

Keywords: Acalypha wilkesiana, Maerua angolensis, phytochemicals, antibacterial activity

INTRODUCTION

Antibiotics continued to provide the basis for the treatment of bacterial infections. Unfortunately
however, there are several reports of antibiotic resistance of human pathogens to the available
antibiotics [1]. This is largely due to the high genetic variability of bacteria which enables them
to quickly circumvent the action of the antibiotics by developing resistance. Hence, the need for
new and efficient antibiotics is of paramount importance [2]. Plants have proved to be the source
of many pharmaceutical products that are currently used as therapy for various diseases [3-5].
The inspiration which led to the discovery of those plant-derived drugs has always been based on
their traditional use as remedy by different cultures around the globe [6]. Acalypha wilkesiana is

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an evergreen shrub which grows in tropical and sub-tropical regions, its ointment is widely used
in Nigeria to treat fungal skin diseases [7]. Maerua angolensis is a tall tree that grows in tropical
Africa and arid regions, its parts are widely used traditionally to treat skin rashes, sores, womb
cleansing, and sexually transmitted diseases [8]. The efficacy of these plants in the cure of such
infections has attracted our attention to further establish more scientific basis for their
application.

MATERIALS AND METHODS

Plant Material
Acalypha wilkesiana and Maerua angolensis were collected from Katsina metropolis and its
outskirt, respectively. The plants were identified and authenticated at the Department of Biology,
Umaru Musa Yar’adua University, Katsina, Nigeria.

Extraction
The ground samples of Acalypha wilkesiana and Maerua angolensis (200g each) were extracted
by percolation with 800ml of re-distilled ethanol for a period of two weeks. Each extract was
concentrated and evaporated to dryness on a rotary evaporator at 40°C to afford ethanol extract
[9].

Fractionation of Crude Extracts

The crude extract was prepared as mentioned above and label as F 1. The residue, F1 was
macerated four times with 20ml of n-Hexane and the soluble fraction evaporated to afford n-
Hexane fraction, F2. The insoluble residue was further macerated three times with 20ml each of
distilled water and chloroform. The chloroform soluble fraction; F 3, the water soluble fraction;
F4, were separately evaporated to dryness. The chloroform and water insoluble residue was
macerated four times with 20ml of ethylacetate. The ethylacetate soluble fraction, F 5, was also
evaporated to dryness. While the insoluble residue was further macerated four times with 20ml
of methanol and the soluble fraction, F6, evaporated to dryness.

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Phytochemical Screening
Test for Alkaloids
Each extract (0.5g) was stirred with 5ml of 1 per cent aqueous hydrochloric acid on a steam bath;
1ml of the filtrate was treated with a few drops of Mayer’s reagent and a second 1ml portion was
treated similarly with Dragendorff’s reagent. Turbidity or precipitation with either of these
reagents was taken as evidence for the presence of alkaloids in the extract being evaluated [10].

Test for Saponins

Each extract (0.5g) was shaken with water in a test tube. Frothing which persists on warming
was taken as evidence for the presence of saponins [11].

Test for Tannins

Each extract (0.5g) was stirred with 10ml of distilled water. This was filtered and ferric chloride
reagent was added to the filtrate, a blue-black precipitate was taken as evidence for the presence
of tannin [12].

Test for Flavonoids

A portion of each extract was heated with 10ml of ethylacetate over a steam bath for 3min. The
mixture was filtered and 4ml of the filtrate was shaken with 1ml of dilute ammonia solution. A
yellow coloration was taken as evidence for the presence of flavonoids [13].

Test for Anthraquinones

0.5g of crude extract was shaken with 10ml of benzene and was filtered. 0.5ml of 10% ammonia
solution was added to the filtrate and the mixture was shaken well and the presence of the violet
colour in the layer phase indicates the presence of the anthraquinones [10].

Sources of Microorganisms
Two bacterial strains were used in this study; one gram negative and one gram positive, namely
staphylococcus aureus and Escherischia coli respectively. All the tested strains were obtained
from Federal Medical Centre, Katsina and brought to the Department of Microbiology, Umaru
Musa Yar’adua University, Katsina. These bacterial cultures were maintained in nutrient agar
slant for further investigation.

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Antibacterial Susceptibility Test
Three concentrations for each fraction of the plants extract were prepared as 500mg/ml,
250mg/ml and 125mg/ml. Thus, the stock solution of 500mg/ml of the plant extract was firstly
prepared by dissolving 1g of each fraction in 2ml Dimethylsulphoxide (DMSO). Subsequently,
250mg/ml and 125mg/ml were prepared by taking 0.6ml and 0.2ml of the stock solution and then
dissolved in 0.4ml and 0.8ml of DMSO, respectively. These concentrations were used for the
antibacterial susceptibility test against the selected organisms.

Sensitivity of different bacterial strains to various extracts was measured in terms of zone of
inhibition using agar diffusion assay described by Bauer et al. [14]. The plates containing
nutrient agar were spread with 0.2 ml of the inoculum. Wells were cut out from agar plates using
a sterilized stainless steel borer and filled with 0.1 ml of the extract. The plates inoculated with
different bacteria were incubated at 37°C up to 48 h and diameter of any resultant zone of
inhibition was measured.

RESULTS AND DISCUSSION

Phytochemical screening was employed as a guide in describing the large number of secondary
metabolites found in the various extracts of plant. The results (Table 1) revealed the presence of
alkaloids, saponins, tannins and flavanoids in all the fractions. While alkaloids were found to be
present in all the fractions of both plants, anthraquinones were found only in one fraction of
Maerua angolensis. Saponins, tannins and flavanoids were found present in some fractions and
absent in some for both plants.

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Table 1. Result of Phytochemical Screening
Plant (part) Fraction Constituents
Alk Sap Tan Ant Fla
F1 + - + - +
Acalypha F2 + - - - -
wilkesiana F3 + - - - -
(whole plant) F4 + + + - -
F5 + + + - +
F6 + - + - +

F1 + + + - +
Maerua F2 + + - - +
angolensis F3 + + - + -
(leaves) F4 + + + - +
F5 + + + - +
F6 + - - - +

Alk = Alkaloids, Sap = Saponins, Tan = Tannins, Ant = Anthraquinones, Fla = Flavonoids
F1= ethanol F2= n-Hexane, F3= chloroform, F4= water, F5= ethylacetate, F6= methanol
+ = Present, - = Absent

Antibacterial susceptibility test (Table 2 and 3) showed the zones of inhibition measured in
millimetre (mm) on the bacteria susceptible to the plant extract. The methanol soluble fraction
(F6) and chloroform soluble fraction (F3) of Acalypha wilkesiana were found to be more active
against Staphylococcus aureus at all the three concentrations (500mg/ml, 250mg/ml, 125mg/ml).
While the chloroform soluble fraction (F3) and methanol soluble fraction (F6) of Maerua
angolensis showed best activity against Staphylococcus aureus at two concentrations (500mg/ml
and 250mg/ml).

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Table 2. Anti Staphylococcus aureus activity

Diameter Zones of inhibition (mm)

Disc potency (mg/disc)
Plant Part Fraction 500 250 125
F1 18 12 NA
Acalypha F2 14 12 NA
Wilkesiana F3 16 15 11
(whole plant) F4 16 11 NA
F5 12 12 NA
F6 16 16 10

F1 12 NA NA
F2 14 14 12
Maerua
F3 16 11 NA
angolensis
F4 NA NA NA
(leaves)
F5 10 NA NA
F6 14 10 NA

F1 = ethanol F2 = n-Hexane F3 = chloroform F4 = water

F5 = ethylacetate F6 = methanol NA= not active

Table 3. Anti Escharischia coli activity

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 Diameter Zones of inhibition (mm)
Disc potency (mg/disc)
Plant Part Fraction 500 250 125
F1 16 12 NA
Acalypha F2 12 09 NA
Wilkesiana F3 13 NA NA
(whole plant) F4 12 10 NA
F5 22 10 NA
F6 14 10 NA

F1 13 12 NA
Maerua F2 12 10 NA
angolensis F3 14 13 09
(leaves) F4 NA NA NA
F5 14 10 NA
F6 16 12 NA

F1 = ethanol F2 = n-Hexane F3 = chloroform F4 = water

F5 = ethylacetate F6 = methanol NA= not active

CONCLUSIONS
The promising result displayed by these extracts both in the antibacterial bioassay and
phytochemical screening justified the use of the plants in traditional medicine. This indicates that
the fractions contained antibacterial agent(s) that could be effective in treatment of skin
infections caused by bacteria whose chemotherapeutic index may exceed the drugs in used.
Based on the interesting result displayed by these plants extracts, particularly the methanol
soluble fraction which was found to be the most active fraction and hence isolation of the active
agent(s) is recommended.

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ACKNOWLEDGEMENTS

The authors are grateful to Fatima Abdulkadir for assisting in the collection of Acalypha
wilkesiana, Mustapha Haruna for the identification of the plants and Mannir Kabir for the
bioassay.

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