DAODU, JOHN OLABANJI

PHA/P/14/15/0006
(MPHIl./Ph.D.)

SUPERVISOR:
Prof. L.S. Kasim
 PHYTOCHEMICAL
SCREENING,
ANTIOXIDANT AND
ANTIMICROBIAL
ACTIVITIES OF
AcalyphafimbriataSchumchTho
nn.
 ABSTRACT
 The tendency for microorganisms to grow despite exposure to
antimicrobial agents that are specifically designed to inhibit
their growth (antimicrobial resistance, AMR), is a challenge
that demands urgent and tactical approach. A remedial measure
through the use of medicinal plants has steadily increased
worldwide in recent years.
 The present study was carried out to analyse the phytochemical
constituents of Acalypha fimbriata as well as its antioxidant and
antimicrobial activity.
 The collected plant sample from Agricultural farm, Abadina,
Ibadan, Oyo state, was dried, grounded to powder and
subjected to sequential extraction using a Soxhlet apparatus
with n-Hexane, Ethylacetate and Methanol solvents in the order
of increasing polarity. The extracts were concentrated using a
rotary evaporator (Rotavapor-R, India) and tested for
antioxidant and antimicrobial activity.
 The phytochemical screening indicated the presence of
alkaloids, glycosides, saponins, flavonoids, tannins and
terpenes. The phenolic content, total flavonoids content,
saponin and alkaloid contents were determined. The
ethylacetate demonstrated a strong antimicrobial activity
while the antioxidant result also revealed that the
ehtylacetate fraction scavenge DPPH(IC50 96.35mg/ml)
and methanol fraction (IC5045.10mg/ml) radicals in a
concentration-dependent manner. The presence of the
secondary metabolites was indicative of varied
pharmacological activities inherent in the plant, while
the antimicrobial and antioxidant activity observed
justify some of ethnobotanical claims of the plant.
Further work on the plant sample is still on to produce
new antimicrobial drug of natural source to boast health
care delivery.
INTRODUCTION
 Antioxidants are man-made or natural substances that
protect cells from the damage caused by unstable
molecules known as free radicals by interacting with them
to achieve stability. Although, oxidation reactions are
crucial for life, they can also be damaging as they can
produce free radicals which start chain reactions that
damage cells. Plants and animals maintain complex
systems of multiple types of antioxidants such as
glutathione, vitamins C,A, and E, as well as enzymes such
as catalase, superoxide, dismutase and various peroxides.
Traditional herbal medicine and dietary foods were the
source of antioxidant for ancient people that protected
them from the damage caused by free radicals.
Antioxidants are widely used in dietary supplements and
have been investigated for the prevention of diseases such
as cancer, coronary heart disease and even altitude
sickness.
 Although initial studies suggested that antioxidant
supplements might promote health, later large clinical
trials of antioxidant supplements including beta
carotene, vitamins A and vitamin E, singly or in
combinations suggest that supplementation has no effect
on mortality or possibly increases it. They are also used
in the food industry in the form of preservatives in foods
and cosmetics and prevent the degradation to rubber and
gasoline.
 Acalypha fimbriata Schumach Thonn., of the family
Euphorbiaceae, commonly called Acalypha is a
dicotyledonous plant, very similar to Acalyphacilita but
consistently differing in the teeth of the female bracts,
which are up to 1.5mm long, falcate – lanceolate, curved
at the apex, often lying alongside each other and almost
contiguous. It is widespread in TropicalAfrica and
Northern South Africa (Hyde, et-al, 2018).
 AcalyphafimbriataSchumachThonn., of the family
Euphorbiaceae, commonly called Acalypha is a
dicotyledonous plant, very similar to Acalyphacilita but
consistently differing in the teeth of the female bracts, which
are up to 1.5mm long, falcate – lanceolate, curved at the
apex, often lying alongside each other and almost
contiguous. It is widespread in TropicalAfrica and Northern
South Africa (Hyde, et-al, 2018).
 The genius is represented by sixteen species in Nigeria and
they are abundant in the forest and savannah ecosystems
where they show preferences for distributed terrestrial
habitats such as farmland, gutter – walls, roadsides, floor
crevices and garden (Sofowora, 1993). The plant is used
traditionally for the treatment of microbial and fungi
infections, such as syphilis and other ailments such as worm
infections, asthma, ulcer, rheumatism etc. (Odugbemi,
2016).
MATERIALS AND METHODS
General Materials
 Soxhlet Apparatus, Rotary evaporator, Distilled water,
cork borer, incubator, weigh balance, Meuller Hinton
Agar, Petridishes, n-Hexane, Ethylacetate, methanol,
Gentamycin, Aluminium chloride (AlCl3), Gallic acid,
Folin – Ciocalteu’sPhenol reagent, sodium carbonate
(NaCO3) 1-1- Diphenyl – 2 – picrylhydrazyl (DPPH),
quercetin, Trichloroacetic acid (TCA), potassium
ferricyanide, butylated hydroxytoluene (BHT),
vitamin C, tannic acid, Iron III chloride (FeCl3),
staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa, Bacillus subtilis.
All chemicals and reagents were of
analytical grade and were obtained
differently from BDH chemicals Ltd. UK
and Merck, South Africa.
The plant was collected at Agricultural Farm,
Abadina Ibadan, Oyo State, Nigeria in
March, 2017 and identified with a herbarium
specimen at the herbarium Department of the
forestry Research Institute of Nigeria (FRIN)
Ibadan. It was then dried and powdered.
EXTRACTION
The powdered leaf sample was
subjected to sequential extraction
using a soxhlet apparatus with
solvents of different polanity, n-
Hexane, Ethylacetate and methand.
The extracts were concentrated using
a rotary evaporator (Rotavapor – R,
India) and preserved in the
refrigerator for antimicrobial
screening.
 ANTIMICROBIAL SCREENING OF THE EXTRACTS
 Agar Cup Diffusion Method as described by Kasim, et al.
(2012).
 A volume of 0.1ml of overnight broth cultures of the
organisms was transferred into a 9.9ml of sterile distilled
water and 0.1ml of the solution containing organism was
seeded into 20ml of melted and cooled Mueller Hinton
Agar. The inoculated agar was allowed to set for about
15minutes, a 8mm diameter sterile cork-borer was used to
bored 6 wells at equidistant to each other which was added
0.1ml solution of each extracts reconstituted with 100%
methanol to final concentration of 100mg/ml, 25mg/ml,
12.5mg/ml,6.25mg/ml and methanol as negative control.
The plates were left for one hour to allow
pre-diffusion and the plates were then
incubated at 370C for 24 hours after which
diameters of zones of growth inhibition
were measured. Gentamycin (10mg/ml)
was used as the positive control.
Determination of Minimum Inhibitory
Concentration(MIC)
The MIC for the bioactive methanol and N-
hexane extract was determined by agar dilution
method. Different concentration of the extracts
were prepared to final concentration in the range
of 100mg/ml, 50mg/ml, 25mh/ml, 12.5mg/ml
and 6.25mg/ml. two milliliters(2ml) of the
extract from each dilution was mixed with 18ml
of molten sensitivity test agar medium and
poured into sterile Petri plate allowing the agar
to set.
The surface of the agar was allowed
to dry before streaking with overnight
broth culture of the test organisms.
The plate were incubated accordingly
after which the lowest concentration
preventing visible growth was taken
as minimum Inhibitory
Concentration(MIC) of the extracts.
All procedures were performed in
triplicate.
PHYTOCHEMICAL ANALYSIS
Thephytochemical test was
carried out on the powdered leaf
of A. fimbriata using standard
procedure (Trease and Evans,
2002) showing the presence of
alkaloids, glycosides, saponins,
flavonoids, tannins and terpenes
with total contents determined as
follows:
DETERMINATION OF TOTAL PHENOLICS

Total phenolic contents were evaluated with

Folin-Ciocalteu’s phenol reagent (Adedapo et al.,
2009; Nabaviet al., 2008). 1ml of the extract
solution in methanol was mixed with 1ml folin-
Ciocalteureagemt previously diluted with water
(1: 9 v/v). After 5 minutes, 0.8 ml of 7% Na 2CO3
solution was added with mixing. The tubes were
vortexed for 5 seconds and allowed to stand for
30 min at 400C for color development.
Absorbance was then measured at 765 nm using
the Hewlett Packard UV- vis spectrophotometer.
Samples of extract were evaluated at a
final concentration of 0.1 mg/ml. total
phenolic content was expressed as mg/g
tannic acid equivalent using the following
equation based on the calibration curve: y
= 0.1216x, R2 = 0.9365, where y was the
absorbance x was the concentration. Each
test was done in triplicate.
DETERMINATION OF TOTAL FLAVONOIDS

Colorimetric aluminum chloride method

was used for flavonoid determination
(Ebrahimzadeh et al., 2001: Nabaviet al.,
2008). 1. 5 ml solution of each plant
extract in methanol was separately mixed
with 1. 5 ml of 2% aluminum chloride.
After one hour at room temperature, the
absorbance was measured at 420 nm. A
yellow color indicated the presence of
flavonoids.
Extract samples were evaluated at a
final concentration of 0. 1 mg/ml.
total flavonoid content were
calculated as quercetin equivalents
(mg/g) using the following equation
based on the calibration curve: y =
0.0255x, R2 = 0.9812, where y was
the absorbance and x was the
concentration. Each test was done in
triplicate.
SAPONIN DETERMINATION
The saponin content in the plant extracts was
estimated as described by kim et al. (2003). 5g
of the powdered sample was placed in 100 ml
of 20% ethanol. The suspension was heated in
water bath at 550C for 4 hours with continuous
stirring. The mixture was filtered and residue
was re-extracted as above. The combined
extracts were reduced to 40ml over a water
bath at 900 C. the concentrate was transferred
into a 250ml separator funnel and 20ml diethyl
ether was added and shaken vigorously.
The ether layer was discarded, while the
purification process was repeated. 60 ml
of n-butanol was added and the extracts
were washed twice with 10 ml of 5%
aqueous sodium chloride. The remaining
solution was heated in a water bath for
evaporation. After which, the sample was
dried in the oven to a constant weight.
The saponin content was calculated
according to the equation: amount of
saponin (mg/g) = weight of
residue/weight of sample.
Alkaloids determination
25 mg of the powdered sample was
weighed into 10 ml of 20% acetic acid in
ethanol and allowed to stand for 4 hours.
This was filtered and the extract was
concentrated using a water bath at 55 0C to
one-quarter of the original volume.
Concentrated ammonium hydroxide was
added drop wise into the extract until
precipitation was complete.
The whole solution was allowed to settle
and the precipitate collected was washed
with dilute ammonium hydroxide solution
and then filtered. The residue which is the
crude alkaloid was weighed and
calculated according to equation: amount
of alkaloid (mg/g) = weight of
precipitate/weighed sample (Obadoni and
Ochuko, 2001).
Antioxidant assays (Assay of DPPH scavenging
activity)

The DPPH radical-scavenging activity of

the test extracts was examined using the
method of Ebrahimzadeh et al. (2001).
Different concentrations (0.7 – 500
mg/ml) of each extract were added, at an
equal volume, to methanolic solution of
DPPH (100mM).
The mixture was allowed to react at room
temperature in the dark for 30 minutes,
the Vitamin C was used as standard
control. Three replicates were made for
each test sample. After 30 minutes, the
absorbance (A) was measured at 518 nm
and converted into percentage antioxidant
activity using the following equation.
%Inhibition= {[ABScontrol -
ABSTest]/ABScontrol} x 100
ABScontrol is the absorbance of the negative
control; ABStest is the absorbance of test
samples.
IC50 values denote the concentration of sample
which is required to scavenge 50% of DPPH
free radicals. The IC50 values were calculated
using graphPab Prism.
RESULTS
Table 1.0: percentage yield of extracts

EXTRACT n-Hexane Ethylacetate Methanol

% YIELD
3.5% 1.65% 7.1%
Results of antimicrobial activity
and the minimum inhibitory
concentration of the extracts are
shown in tables 2.0 and 3.0.
respectively.
Results of the phytochemical
analysis and antioxidant assays are
tabulated in tables 4.0, 5.0, 6.0, 7.0,
and 8.0 respectively.
 TABLE 2.0: ANTIMICROBIAL ACTIVITY
(DIAMETER OF ZONES OF GROWTH
INHIBITIONS) OF COMPOUNDS OF A.
FIMBRIATA
Ethylacetate fraction
Microorganisms Methanol fraction N-hexane fraction
(mg/ml)
(mg/ml) (mg/ml)

+co
12.2 -ve
100 50 25 12.5 6.25 ntr 100 50 25 6.25 +control 100 50 25 12.5 6.25 +control
5 control
ol

Staphylococcus
aureus
14 12 10 - - 16 14 12 10 10 - 16 12 10 - - - 16 -

Escherihia Coli

10 - - - - 20 16 14 12 12 10 20 - - - - - 20 -

Pseudomonas
aeruginosa
16 14 14 12 10 - 16 12 12 12 10 - 12 10 - - - - -

Bacillus subtilis

14 12 10 10 - 22 12 10 - - - 22 16 14 12 10 10 22 -
Key: Gentamicin 4mg/ml= +ve control
N-hexane 100mg/ml =ve control only for
N-hexane extract only because of its
naturally inherent anti-microbial activity.
 Table 3.0 Minimum Inhibitory
Concentration of Methamol, ethylacetate
and N-hexane
Ethylacetate fraction Methanol fraction N- hexane fraction
Microorganisms
(mg/ml) (mg/ml) (mg/ml)

100 50 25 12.5 6.25 100 50 25 12.5 6.25 100 50 25 12.5 6.25

Staphylococcus aureus

- - - (-) + - (-) + + + - (-) + + +

Escherihia Coli

- (-) + + - - - (-) + - (-) + + +

+

Pseudomonas
aeruginosa
- (-) + + + - - - (-) + - (-) + + +

Bacillus subtilis

- (-) - + + - - - (-) + - (-) + + +

 Key: values in bracket is the MIC of each organism for each

extract.
Table 4.0 Total Flavonoid content of A.
fimbriata
ABSORBANCE

Second extract A. FirmbriataMeOH A. Firmbriata H2O

ABS 0.438 1.658 0.126

ABS 0.577 1.62 0.124

AVERAGE ABS 0.5075 1.639 0.125

Mg/g quercetin 19.90196 64.27451 4.901961

 Table 5.0 Total phenol content of A.
fimriata
ABSORBANCE

Second extract A. FirmbriataMeOH A. Firmbriata H2O

ABS 0.221 0.221 0.15

ABS 0.286 0.286 0.162

AVERAGE ABS 0.2535 0.2535 0.156

Mg/g tannic acids 2.084704 2.084704 1.282895

 Table 6.0 Total Alkaloids content of A. fibmbriata

Initial conc. ABSORBANCE

Second extract A A
FirmbriataMeOH Firmbriata H2O

0.0022 0.0252 0.0298

0.0023 0.0272 0.0286

AVERAGE ABS 0.00225 0.0262 0.0286

% alkaloids 18% 0.6298% 0.6875%

Table 7.0 Total Saponnin content of A. fimbriata

Initial conc. ABSORBANCE

Second extract A A
FirmbriataMeOH Firmbriata H2O

4.17 0.0116 0.0069 0.0619

0.0109 0.0017 0.0639

AVERAGE 0.01125 0.0043 0.0629

% saponin 0.27% 0.1075% 1.51%

 Table 8.0 DPPH Scavenging activity of A.fimbriata
Second extract A firmbriataMeOH A fimbriata H2O

Conc %DPPH Conc %DPPH Conc %DPPH

0.023438 74.399294 0.023438 78.80795 0.125 46.69604

0.011719 55.84989 0.011719 68.87417 0.0625 36.12335

0.005859 15.89404 0.005859 78.1457 0.03125 28.19383

0.00293 16.55629 0.00293 62.91391 0.015625 24.00881

0.001465 10.37528 0.001465 60.92715 0.007813 23.56828

0.000732 10.59603 0.000732 59.8234 0.003906 22.90749

IC50 0.009635 0.004510 0.03485

DISCUSSION AND CONCLUSION
The dual factor of antimicrobial resistance
and limited scope of orthodox medicine
globally, it has become imperative that
global health care be sustained by
indigenous herbal medicines (Lifongo, et.al.
2014). Consequently, the recent renewed
interest in exploiting herbal medicine as
sources of potentially important new
pharmaceutical substances is a move in the
right direction (Adebayo and Krettli, 2011).
 The presence of the secondary metabolites; alkaloids,
glycosides, saponins, flaronoids, tannins, phenols and
terpenes showed by the powdered leaf of a firmbirata
was indicative of varred pharmacological activities of
the extracts appeared to be broad spectrum since the
gram positive and gram negative bacteria were sensitive
to the extracts of different, but varied concentrations.
Pseudomoras aerations and Bacillus subfilis obtained
from wound infections was found to be susceptible to
ethyl acetate and methanol extract, but if was resistant to
concentration of 50-100mg/ml. Escherichia coil was
highly susceptible to methanol extracts but it was
resistant completely to n-hexane and ethyl acetate
extracts while Staphylococcus aureus obtained from
superlative infection elicited susceptibility to methanol
and ethyl acetate. Extracts but pronounced resistant to n-
hexane extract as shown in the Table 2.0.
 The ethyl acetate extract had highest antimicrobial
activity on Pseudomonas aeruginosa, followed by
methanol extract while the n-hexane extracts was
recorded to be the lowest in the MIC’s ratio values of
6.25mg/ml: 12.5mg/ml: 50mg/ml respectively. Ethyl
acetate extracts also had the highest values against
Escherichia coil and Bacillus suvtilis but a lowest
activity on n-hexane extract of the compound, 50mg/ml
MIC were recorded as elicited in table .
 Some of these bacteria have been implicated in diseases
such as diarrhea, wound and gastrointestinal infections.
The strong antimicrobial activities demonstrated
especially ethyl acetate extract may therefore justifies
some of the ehno pharmacological claims about this
compound in the treatment of the aforelisted infections.
The ethyl acetate fraction was observed to
scavenged DOPH (IC50 96.35 mg/ml) while the
methanol fraction scavenged DPPH (IC50 45.10
mg/ml) radicals in a concentration dependent
manner, which was indicative that A firmbriata
could be a natural source of antioxidant for t5he
treatment of infections diseases particularly, that
has to do with oxidative consequences. Research
findings on the plant sample is still on-going for
isolation and characterization of these discovered
bioactive activities for the possible synthesis of a
novel pharmoceeutical drug moreso with the
broad spectrum of activity showed by the ethyl
acetate fraction against the fest organisms.
REFERENCES
 Adebayo J.O, Kretti, A.U, (2011) Potential antimalarials
from Nigerian Plants: A review. Journal of
Ethnopharmacology. 133: 289-302
 Adedapo, A.A, Jimoh, F.O, Koduru, S., Masika, J.P,
Afolayan, A.J. (2009). Assessment of the medicinal
potentials ofs the manthanol extracts of the leaves and
stems of BuddlejaSaligra. Brrac Comp/Altern. 9: 9-21
 Anuji, Y., Rewa, K., Ashwani Y., and Shashi .P (2016).
 Resources of Environmental and life sciences.9(11).
 Cowan, na.na. (1999). Plant products as antimicrobial
agents.Clicnical microbiological Review 12: 564 - 82
 Ebrahimzadeh, MoA.,Nabiri S.F., Nabiri S.M., (2001).
Antioxidant activities of methanol extract of
sambucusebulus L. flower. Pakistan Journal of Biological
Sciences. 12: 447 – 450.
 Fehintola O.A (2014). Antioxidant and
Antimicrobial activites of Acalyphafombriata.A
Department of pharmaceutical and medicinal
chemistry, faculty of pharmacy, Olabisi Onabanjo
University.P. 2.
 Gurbin – Fakim, A (2006). Medicinal plants:
traditions of yesterday and Drugs of tomorrow.
Moleculer Aspects of medicine.27: 1- 93.
 Hyde, M.A, Wursten, B.T., Bailings, P and Coates,
P.M. (2018) flora of Botswana flora.Com/species
data/species.Php? Species id = 135080.
 Kasim, L.S., Olatunde, A, Effedua, H.I., Adejumo,
O.E., Ekor M. and Fajemirokun, T.O.
(2012).Antimicrobial activity of six selected plants
against some strains of pathogenic
 Obadoni B.O., Ochuko, P.O. (2001). Phytochemical
studies and comparative efficacy of the crude
extracts of some homeostatic plants in Edo and
Delta State of Nigeria.Glob. Journal of Pure and
Applied Sciences 8: 203 – 208.
 Sies H. (1997). Oxidative stress: Oxidants and
antioxidants. Experimental physiology 82 (2) : 291
–5.
 Sofowora A. (1993). Medicinal plants and
Traditional Medicine in Africa.1st Edition.John
Wiley and sons, Manchester, New York. ISBN-10:
0471103675. P. 256
 Trease, G. E and Evans, W. C.
(2002).Pharmacognogy.15th Edition. Alden Press.
Oxford, UK. 241 – 260.
Van Vuuren, S.F. (2008).
Antimicrobial Activity of South
African Medicinal Plants.Journal of
Ethnopharmacology. 119: 462 – 72
Willcox, M.L. Bodeker G. (2004).
Traditional herbal medicines for
malaria.BMJ.329:1156 –9.