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Ajayi et al. European Journal of Biomedical and Pharmaceutical Sciences
49
ANTIMICROBIAL PROPERTIES OF AFRAMOMUM MELEGUETA SEEDS ON
STAPHYLOCOCCUS AUREUS AND STREPTOCOCCUS SPECIES
Ajayi A. O.1*, Fadeyi T. E.1 and Olumekun V. O.2
1Department of Microbiology, Adekunle Ajasin University, P.M.B. 01, Akungba-Akoko, Ondo State, Nigeria.
2Department of Plant Science & Biotechnology, Adekunle Ajasin University, P.M.B. 01, Akungba-Akoko, Ondo State,
Nigeria.
Article Received on 07/06/2016 Article Revised on 28/06/2016 Article Accepted on 19/07/2016
INTRODUCTION
Alligator pepper (Aframomum melegueta) has vast
medicinal value in treating and managing some chronic
diseases characterized by inflammatory conditions
(Kumar et al., 2015). It belongs to the family
Zingiberaceae. The plant is herb native to the tropics
commonly found in swampy habitats of Nigeria, Uganda,
Angola, Benin, The Gambia, Ghana, Guinea, Cote
d’Ivoire (Ivory Coast), Liberia, Sierra Leone, Togo,
Cameroon, Congo, Gabon, Guinea-Bissau in West Africa
and as far as the Democratic Republic of the Congo. It is
also widely cultivated in other parts tropical Africa and
South America. It possesses tufted leafy stem that can be
up to 1.5m high. The leaves are simple, alternate and
lanceolate. The mature leaves can grow as long as 40cm
and 12cm-15cm wide. It produces purple coloured
flowers which develop into pods that can be as long as
8cm and about 3cm wide. The pod contains numerous
reddish-brown seeds (can be as many as 300 seeds in one
pod) (Anjah et al., 2015, Tropilab INC. (2015). The
sharp and peppery taste of the seeds is caused by the
aromatic ketones; 6-paradol, 6-gingerol and 6-shogaol
present in it (Sugita et al., 2013). The fruits are fleshy,
indehiscent and produce spikes.
Alligator pepper is widely used by many cultures in
Nigeria for the treatment of various types of diseases
including the aetiologic agents of enteric diseases like
Escherichia coli, as demonstrated by Kigigha, et al.,
(2015). Socially, it is served along with kolanuts (Kola
spp.) to guests for entertainment, and for religious rites
by diviners for invoking spirits. It is a common
ingredient in pepper soup, a spicy delight in many parts
of West Africa. Concoctions made of alligator pepper are
often used by traditional doctors as medications for
various ailments (personal communication). Pregnant
women are not excluded from eating this widely used
substance. The constituents of an essential oil,
extractable by hydro-distillation from the seeds of
Aframomum melegueta include two sesquiterpene
hydrocarbons, humelene and caryophyllene, their oxides
and a few non-terpenoids. This oils are partly beneficial
SJIF Impact Factor 3.881
Research Article
ejbps, 2016, Volume 3, Issue 8, 49-55.
European Journal of Biomedical
AND Pharmaceutical sciences
http://www.ejbps.com
ISSN 2349-8870
Volume: 3
Issue: 8
49-55
Year: 2016
Corresponding Author: Dr. Ajayi A. O.
Department of Microbiology, Adekunle Ajasin University, P.M.B. 01, Akungba-Akoko, Ondo State, Nigeria.
ABSTRACT
Plants sources have been investigated for their antimicrobials in the search for novel compounds for the treatment
and control of diseases. This study therefore investigated the potential of Aframomum melegueta seeds which have
been long known for the ethno-botanical uses, as a source of antimicrobials. Analysis of the seeds used in this
study showed that the ethanolic extract had the highest antimicrobial effect on Staphylococcus aureus at 0.6g/ml
with the highest zone of inhibition at 18mm. It was also observed that the ethanolic extract had the highest
antimicrobial effect on Streptococcus species with the concentration 0.6g/ml giving the highest zone of inhibition
at 15.0mm. Petroleum ether extract of the seeds had lesser antimicrobial effect while the water extract had the
least antimicrobial effect on Streptococcus species. Phytochemical screening indicated the presence of tannin,
phenol, alkaloid, flavonoids, oxalate, saponin and phytate; some of which are recognised as bioactive components
while the mineral components (per 100g) were sodium (Na), potassium (K), calcium (C), magnesium (Mg), zinc
(Zn), iron (Fe), lead (Pb), copper (Cu), manganese (Mn) and phosphorus (P). This study highlights the
antimicrobial property of Aframomum melegueta seeds and demonstrates the potential for the control of disease
agents such as Staphylococus aureus and Streptococcus spp. It is expected that the information obtained will be
helpful in medicinal formulations.
KEYWORDS: Aframomum melegueta; Seeds; Antimicrobial properties; Staphylococus aureus; Streptococcus
species.
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Ajayi et al. European Journal of Biomedical and Pharmaceutical Sciences
50
to repel some destructive insect pest of stored grains like
Rhyzopertha dominica (Fabricius)
(Coleoptera:Bostrichidae) (Ukeh, 2008) and against
some inflammatory conditions (Doherty et al., 2010; Ilic
et al., 2014).
The spice is used in West Africa for the purposes of
alleviating stomach ache and diarrhoea (Ilic, et al.,
2010), as well as hypertension, diabetes, tuberculosis and
a remedy for snakebites and scorpion stings (Kumar et
al., 2015; Lans, 2001). These seeds are also used for
culinary reasons (due to the pungency of the seeds, they
are common used as food seasoning).
In the development of herbal medicines, the quality of
base material used for formulating the products is a
prerequisite. Since the materials used in herbal drugs are
traded mostly as roots, bark, twigs, flowers, leaves, fruits
and seeds, visible authentication of the material used is
difficult and this has led to a high level of adulteration.
To identify and authenticate the materials, the
availability of detailed morphological, histological and
pharmacognostic information is essential.
Standardisation of natural products is a complex task due
to their heterogeneous composition, which is in the form
of whole plants, plant parts or extracts obtained thereof.
To ensure quality reproduction of herbal products, proper
control of starting material is essential. The first step
towards ensuring quality of starting material is
authentication. Thus, in recent years there has been a
rapid increase in the standardisation of selected
medicinal plants of potential therapeutic significance
(Reddy et al., 1999; Venkatesh et al., 2004). This study
therefore set out to evaluate the phytochemical
properties, pharmaceutical values and the effectiveness
of Aframomum melegueta on two common pathogens,
Staphylococcus aureus and Streptococcus spp.
MATERIALS AND METHODS
Source of plant materials
Seeds of Alligator pepper (Aframomum melegueta) were
obtained from the local market of Akungba-Akoko,
Ondo State, Nigeria. The plant seeds were authenticated
at the Department of Plant Science and Biotechnology,
Adekunle Ajasin University, Akungba-Akoko, Nigeria.
Source of Micro-organisms
The test organisms, Staphylococcus aureus and
Streptococcus spp., were obtained from the stock culture
of previously identified isolates of the Microbiology
Laboratory of the University. The bacteria were
maintained on nutrient agar slant and stored in the
refrigerator at 4oC for further tests.
Extraction Methods
The seeds of Aframomum melegueta were washed with
distilled water, dried ground to powder. Two hundred
grammes (200g) of the powder were separately soaked in
400ml of 95% ethanol, distilled water and petroleum
ether in a 500ml reagent bottle and stoppered. This was
allowed to stand for 14 days to permit full extraction of
the active ingredients. The fluids were then filtered using
Whatman No.1 filter paper. The extracts were dried in a
rotary evaporator to obtain the concentrate and kept in
the refrigerator prior to use. A 2.0µg/ml solution of each
extract (Brian LaBreque, Horizon Technology, Inc.,
Salem, NH, 2016; Naseer et al., 2016). was prepared
with DMSO (dimethyl sulfoxide) and fractionated into
0.6µg/ml, 0.4µg/ml and 0.2µg/ml concentrations needed
for the bioassay.
Sterility Test of the Plant Extracts
The aqueous and the ethanolic extracts were tested for
growth or contamination by inoculating 1ml each on
nutrient agar and incubated at 37oC for 24hours. The
plates were observed for growth. No growth in the
extract after incubation indicated that the extracts were
sterile. The extracts were then assessed for antimicrobial
activity.
Antimicrobial Assay
The antimicrobial properties of the extracts were
determined using the agar diffusion method and the
diffusion disc method (CLSI, 2012; Kaiser, 2012). In the
agar diffusion method, twenty-four hour old broth
cultures of the test organisms were swabbed onto a
sterile Mueller Hinton agar in Petri dishes using sterile
cotton swab. A sterile cork borer of 6mm diameter was
used to punch wells on the agar on each of the Petri
dishes. The holes were filled with 0.5ml of the extracts.
Control experiments were also carried out where the
holes were filled with 0.5ml metronidazole. Each hole
was labelled representing a particular concentration
(Mohanty et al., 2010).
In the disc diffusion method, the Petri dishes containing
Mueller Hinton agar were seeded throughout with the
twenty-four hour old test organisms. Diffusion discs used
were then impregnated with corresponding
concentrations to that of extracts on the agar diffusion
method which is 0.6µg/ml, 0.4µg/ml and 0.2µg/ml and
also with 0.5ml of metronidazole as the control. The
discs were then evenly dispensed and lightly pressed
onto the agar surface. The process was carried out for
each extract and the inoculated Petri dishes were left for
few minutes for extract to diffuse into agar. The plates
were incubated at 370C for 24 hours (not done in an
inverted position) after which the zones of inhibition (if
any) were measured (Mohanty et al., 2010).
Determination of Minimum Inhibitory Concentration
(MIC)
The minimum inhibitory concentration was determined
against bacteria after the antimicrobial test had been
performed. Minimum inhibitory concentration (MIC) is
the lowest concentration of an antimicrobial that will
inhibit the visible growth of a microorganism after
overnight incubation. The MIC was determined using
Mueller Hinton agar based on the modified agar
diffusion method described by Kirby and Bauer (Kaiser,
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Ajayi et al. European Journal of Biomedical and Pharmaceutical Sciences
51
2012). Sterile cork borer of diameter 6mm was used to
bore holes on the plates after seeding the plates with the
bacterial strain concerned, left for one hour at room
temperature and incubated at 37oC. Results were read
after 24hours of incubation using a calibrated ruler to
measure the zone of inhibition.
Qualitative Method of Analyses
Filtrates were prepared by boiling 20g of the fresh plant
in distilled water for 5 mins. The solution was filtered
through a vacuum pump. The filtrates were used for
screening for flavonoids, tannins, saponins, alkaloids,
reducing sugars, anthraquinones and anthocyanosides.
(i)Test for Alkaloids
About 0.2ml of filterate was warmed with 2% of H2SO4
for two minutes, filtered and few drops of Dragendoff's
reagent were added. Orange red precipitate indicated the
presence of alkaloids (Trease and Evans, 1989; Evans,
2002).
(ii) Test for Tannins
One millilitre of the filtrate was mixed with 2m1 of FeCl.
A dark green colour indicated the presence of tannins.
(iii) Test for Saponins
One millilitre of each filtrate was diluted with 2ml of
distilled water and the mixture was vigorously shaken
and left to stand for 10mins during which time, the
development of foam on the surface of the mixture
lasting for more than 10mm, indicates the presence of
saponins.
(iv) Test for Anthraquinones
A Borntranger test (Indigo Pharma, 2009), was
performed whereby one millilitre of the plant filtrate was
shaken with 10ml of benzene; the mixture was filtered
and 5 ml of 10% (v/v) ammonia solutions was added
shaken (Mohammed et al., 2014). A pinkish or violet
solution indicated a positive test (Indigo Pharma, 2009).
(v) Test for Anthocyanosides
One millilitre of the plant filtrate was mixed with 5m1 of
dilute HCl; a pale pink colour indicated a positive test.
(vi) Test for Flavonoids
One millilitre of filtrate was mixed with 2m1 of 10%
lead acetate; a brownish precipitate indicated a positive
test for phenolic flavonoids. For Flavonoids, 1m1 of the
filtrate was mixed with 2m1 of dilute NaOH; a golden
yellow colour indicated the presence of flavonoids
(Edeoga et al., 2005).
(vii)Test for Reducing Sugars
Two solutions are needed for this purpose: Fehling's "A"
uses 7 g CuSO4.5H2O dissolved in distilled water
containing 2 drops of dilute sulfuric acid. Fehling's "B"
uses 35g of potassium tartrate and 12g of NaOH in 100
ml of distilled water. One millilitre of the filtrate was
mixed with Fehling's A and Fehling's B separately; a
brown colour with Fehling's B and a green colour with
Fehling's A indicated the presence of reducing sugars.
(Brilliant biology Student, 2015).
(viii) Test for Cyanogenic glucosides
This was carried out adding 0.5g of the extract to 10ml
sterile water. This is filtered and sodium picrate was
added to the filtrate that is heated to boiling point.
(ix) Test for Cardiac glucosides
The killer-kiliani and legal test and was adopted for the
purpose of cadiac glucosides examination. 0.5g of the
extract was added to 2ml of acetic anhydrate plus H2S04.
Quantitative Method of Analyses
(i) Saponins
a) 1 ml solution of extract was diluted with distilled
water to 20 ml and shaken in a graduated cylinder for 15
minutes. Development of stable foam suggests the
presence of saponins.
b) 1ml extract was treated with 1% lead acetate
solution. Formation of white precipitates indicates the
presence of saponins (Devmurari, 2010). Alternatively,
Twenty grammes each of dried plant samples were
ground and, put into conical flasks after which 100ml of
20% aqueous ethanol was added. Each mixture was
heated in a water bath at about 55OC for 4hours with
continuous stirring, filtered and the residue re-extracted
with a further 200ml of 20% ethanol. The combined
extracts were reduced to 40 ml over a water bath at about
90°C. Each concentrate was transferred into a 250ml
separatory funnel and 20ml of diethyl ether was added
and vigorously shaken. The aqueous layer was recovered
while the ether layer was discarded. The purification
process was repeated three times and 60ml of n-butanol
was added. The combined n-butanol extracts were
washed twice with 10m1 of 5% aqueous sodium chloride
and the remaining solution was heated in a water bath.
After evaporation, the samples were dried in the oven to
a constant weight; the saponin content was calculated as
percentage of the starting material.
(ii) Flavonoids
Ten (10) grammes of each plant sample was extracted
repeatedly with 100 ml of 80% aqueous methanol, at
room temperature and the solution was filtered through
Whatman's filter paper No 42. The filtrates were later
transferred into a crucible and evaporated into dryness
over a water bath; the resultant content was dried to a
constant weight.
(iii) Cardiac glucosides
Legal test and the keller-Kiliani was adopted, 0.5g of the
extract were added to 2ml of acetic anhydrate plus H2S04
(Trease and Evans, 1989; Evans, 2002).
(iv) Tannins
About 500mg of the plant sample was weighed into a
50ml plastic bottle. Fifty milliliters of distilled water was
added and shaken for 1 hour on a mechanical shaker.
This was filtered into a 50ml volumetric flask and made
up to the marked level. Five milliliters (5ml) of the
filtrate was transferred into a test tube and mixed with
2ml of 0.1 M FeCl in 0.1 M HCl and 0.008 M potassium
ferrocyanide. The absorbance was measured at 120nm
within 10minutes. The tannins content was calculated
using a standard curve.
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Ajayi et al. European Journal of Biomedical and Pharmaceutical Sciences
52
(v) Alkaloids
Five grammes (5g) of the plant sample was weighed into
a 250ml beaker into which 200ml of 10% acetic acid in
ethanol was added. The reaction mixture was covered
and allowed to stand for 4 hours. This was filtered and
the extract was concentrated on a water bath to one-
quarter of the original volume. Concentrated ammonium
hydroxide was added drop-wise to the extract until the
precipitation was complete. The whole solution was
allowed to settle and the precipitate was collected,
washed with dilute ammonium hydroxide and then
filtered. The residue being the alkaloid, was dried to a
constant mass and weighed (Trease and Evans, 1989;
Evans, 2002).
(vi) Phlobatannins
About 0.5grams of each Aframomum spp., seed extracts
was dissolved in distilled water and filtered. The filtrate
was boiled in 2% HCl, red precipitate show the present
of phlobatannins. (Adegoke and Adebayo-tayo, 2009)
RESULTS
This study elucidated the activity of Aframomum
melegueta on Staphylococcus aureus and Streptococcus
spp. which are known to be specific aetiologic microbes.
The ethanolic extract of Aframomum melegueta had
highest antimicrobial effect on Staphylococcus aureus
(Table 1) with the 0.6g/ml concentration of the extract
giving the highest zone of inhibition (18.0mm).
Petroleum ether extract, although effective, had lesser
antimicrobial effect while the aqueous extract of had the
least antimicrobial effect on Staphylococcus aureus. In
the same vein, the ethanolic extract of Aframomum
melegueta had the highest antimicrobial effect on
Streptococcus species (Table 2) with 0.6g/ml
concentration giving the highest zone of inhibition
(15.0mm). Petroleum ether extract also had lesser
antimicrobial effect while the aqueous extract had the
least antimicrobial effect on Streptococcus species.
Qualitative analysis showed that Aframomum melegueta
is composed of various phytochemicals such as
Alkaloids, Cyanogenic Glucosides, Steroids,
Anthraquinones, Phenols, Tannins, Saponins and
Flavonoids as presented in Table 3.
Quantitative Analyses of mineral elements (Table 4)
showed the presence of Sodium (Na), Potassium (K),
Calcium (Ca), Magnesium (Mg), Zinc (Zn), Iron (Fe),
Copper (Cu), Manganese (Mn) and Phosphorus (P) but
Lead (Pb) was not detected. Data presented show that
phosphorus content was highest in comparison with other
minerals analysed. Analyses also indicated that the plant
material had all the anti-nutrients presented in Table 5,
with saponin having the highest percentage composition.
The results presented in Table 6 also show that the seeds
were composed largely of carbohydrates.
Table 1: Antibacterial effect of Aligator Pepper (Aframomum melegueta) seed extracts on Staphylococcus aureus
(in mm inhibition zone).
0.2g/ml
0.4g/ml
0.6g/ml
Control
Aframomum melegueta
14.0
13.0
18.0
12.0
Ethanolic extract (mm)
Aqueous extract (mm)
5.0
3.0
6.0
8.0
Petroleum ether extract (mm)
11.0
12.0
12.0
10.0
Table 2: Antibacterial effect of Aligator Pepper (Aframomum melegueta) seed extracts on Streptococcus species (in
mm inhibition zone).
0.2g/ml
0.4g/ml
0.6g/ml
Control
Ethanolic extract (mm)
5.0
8.0
7.0
15.0
Aqueous extract (mm)
3.0
1.0
3.0
10.0
Petroleum ether extract (mm)
5.0
4.0
4.0
12.0
Table 3: Phytochemical Screening of Aframomum melegueta
Samples
Alkaloid
C. Glucoside
Steroid
Anthraquinone
Phenol
Tannins
Saponins
Flavonoids
+ve
+ve
±ve
+ve
+ve
+ve
+ve
+ve
Keys: +ve= Presence of constituents, -ve =Absence of constituents , ±ve = Slightly present.
Table 4: Quantitative Analyses of Aframomum melegueta minerals composition (mg/100g)
Na
K
Ca
Mg
Zn
Fe
Pb
Cu
Mn
P
Composition
(mg/100g)
21.37
30.54
23.55
19.67
17.58
10.21
ND
0.03
25.37
97.65
Key: ND- Not Detected
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Ajayi et al. European Journal of Biomedical and Pharmaceutical Sciences
53
Table 5: Quantitative Analyses of Anti–nutrients present in Plant Extracts (%)
Anti–nutrients
% Composition
Tannins
2.10
Phenols
3.41
Phylates
1.87
Oxalates
2.09
Saponins
4.00
Flavonoids
3.89
Alkaloids
3.00
Table 6: Quantitative analyses of proximate nutrient composition of plant extracts (%)
Ash
Moisture
Content
Crude
Protein
Fat
Fibre
Carbohydrate
% Composition
10.67
8.00
11.93
5.92
9.68
40.56
DISCUSSION
The prevalence of antibiotic resistance has become
problematic in disease management hence the
widespread search for novel compounds. Usually, natural
sources are preferred hence the need to screen plant
resources for beneficial activity against common disease
causing agents. This study has confirmed the potential of
Aframomum melegueta as a source of antimicrobials as
previously reported (Ramadurai et al., 1999). The results
show that the mode of extraction is crucial in the efficacy
of the extract. As presented in Table 1, it was observed
that the ethanolic extract of Aframomum melegueta had
the highest antimicrobial effect on Staphylococcus
aureus. Petroleum ether extract also had antimicrobial
effect but was observed to be less efficacious and the
aqueous extract had the least antimicrobial effect. This
corroborates the findings of Kigigha, et al. (2015) who
reported low antimicrobial effect on some
Staphylococcus aureus strains compared with relatively
high activity on Escherichia coli and Bacillus species in
their study. The aqueous extract also had very low
antimicrobial effect on Streptococcus species (Table 2).
Analysis of the phytochemical constituents of
Aframomum melegueta confirmed the presence of
Alkaloids, Cyanogenic Glucoside, Steroids,
Anthraquinone, Phenols, Tannins, Saponins and
Flavonoids (Table 3) and these could act as bioactive
agents (Devmurari, 2010). Analysis of mineral elements
(Table 4) indicated the presence of Sodium (Na),
Potasium (K), Calcium (Ca), Magnesium (Mg), Zinc
(Zn), Iron (Fe), Copper (Cu), Manganese (Mn) and
Phosphorus (P), with phosphorus being the most
prevalent while Lead (Pb) was not detected.
Quantitative Analyses of Anti–nutrients (Table 5)
indicated the presence of Saponins, Tannins, Phylates,
Oxalates, Flavonoids, Phenols and Alkaloids, with
Saponins having the highest percentage. Similarly, as
shown in Table 6, the seeds were made up of Ash, Crude
Protein, Fat, Fibre and Carbohydrate with carbohydrates
having the highest percentage composition.
This study confirms the potential of Aframomum
melegueta in the development of new chemotherapeutic
agents. The first step towards this goal is the in vitro
antibacterial activity assay (Tona et al., 1998; Samy and
Ignacimuthu, 2000; Palombo, and Semple, 2001). Some
of these observations have helped in identifying the
active ingredients responsible for such activities as a
prelude to the development of drugs for therapeutic use
in humans. However, there are limited reports on the
exploitation of the antifungal or antibacterial properties
of plant extracts in the development of commercial
formulations for crop protection. Adopting this approach
may therefore be valuable in the treatment of plant
diseases too. The major benefit of this study however is
that it will be helpful in developing valuable medications
that can be incorporated into products for sustainable
treatment of skin and intestinal aetiologic diseases whose
causative agents are of Staphylococcus aureus and
Streptococcus spp. related origin.
ACKNOWLEDGEMENTS
We acknowledge the support of the Environmental
Research Unit, Department of Microbiology, Adekunle
Ajasin University, Akungba-Akoko, Nigeria, for the
successful completion of this work.
Statement of Conflict of Interest: There was no
conflict of interest.
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