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Antimicrobial, Phytochemical and Proximate Analysis of Four Nigerian

Medicinal Plants on some Clinical Microorganisms

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Current Research in Microbiology and Biotechnology
Vol. 2, No. 5 (2014): 457-461
Research Article
Open Access
ISSN: 2320-2246

Antimicrobial, Phytochemical and Proximate

Analysis of Four Nigerian Medicinal Plants on some
Clinical Microorganisms
Osuntokun Oluwadare Temitope1 and Ajayi Ayodele O2*
1 Department of Microbiology, Adekunle Ajasin University, Akungba Akoko, Nigeria
2 Department of Microbiology, Federal University Oye Ekiti, Oye Ekiti, Nigeria

* Corresponding author: Ajayi Ayodele; email: [email protected]

Received: 07 August 2014 Accepted: 18 August 2014 Online: 01 September 2014

ABSTRACT
The increasing incidence of resistance to antibiotics among bacteria currently poses serious clinical and public
health threat and has propelled efforts for newer alternatives to alleviate the problem. Extensive search and
testing for different plants with potentials for antimicrobial properties is presently considered one of the most
feasible options as substitutes to the current regimen of antimicrobials. We tested the antibacterial characteristic
and the phytochemical composition of four candidates: Momordica balsamina, Pavetta crassipes, Phyllanthus
amarus and Aloe vera. Extractions were carried out using distilled water, ethanol, ethyl acetate and hexane.
Generally, all extracts showed considerable antibacterial properties against Salmonella typhi, Klebsiella
pneumoniae, Staphylococcus aureus and Escherichia coli. Antibacterial activities for each extract of the plants
increased with increasing concentration. The aqueous and ethanolic extracts showed the highest antibacterial
activity while hexane extracts showed the least activity against the test organisms. The plants contained essential
phytochemicals and minerals were detected except lead. This study further confirmed the antimicrobial activity
of these plants and efforts should be intensified in the search for novel plants with medicinal and antimicrobial
potentials.

Keywords: antimicrobial, phytochemicals, microorganisms

INTRODUCTION
For decades, microbial infections in humans have aureus [8] and extended spectrum beta-lactamase
posed significant public health problems in developing producing Enterobacteriacaea [9,10].
and developed countries with high mortality and
morbidity [1]. Consequently, antibiotics are massively Due to the imperativeness of reducing the growing
deployed to treat bacterial infections, control them incidence of antibiotic resistant infections and their
with the ultimate intent of reducing the morbidities public health impact, alternatives to the current
and mortalities; and by implication, reduce the burden regimens of antibiotics are currently being sought
of disease mostly associated with infectious diseases in globally [11]. An example of such approach is the
Africa and globally [2]. More specifically, antibiotic utilization of bioresources, especially plants that
consumption has been reported to increase globally by possess suspected and slightly proven medicinal
a factor of approximately 36% between 2000-2010 [3]. potentials with limited uses [12, 13]. Although green
Antibiotics and antimicrobial agents have historically plants have historically occupied a common place in
been subjected to abuses and overuse. As a result, traditional medicine for various preventive and
many bacterial etiological agents that cause infectious therapeutic uses, deliberate research efforts are still
diseases have since developed resistance to the being focused on underutilized plants in order to
antibiotics [4, 5]. The most prominent antibiotic unravel their antibacterial properties with a view
resistant infections include vancomycin resistant deploying them for use in humans to reduce bacterial
enterococci [6, 7] methicillin resistant Staphylococcus infections [11, 14].

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The following are some of the plants with confirmed room temperature for about two weeks to ensure that
medicinal properties: Momordica balsamina, Pavetta the samples lose most of their moisture content. The
crassipes, Phyllanthus amarus and Aloe vera. The following extractions were carried out: aqueous,
Phyllanthus amarus Schum and Thonn is a small herb ethanolic, hexane and ethyl acetate. For each
reputed for its various medicinal properties [15]. extraction, 250g of each dried plant material was
Momordica balsamina is abundantly grown in Nigeria weighed separately into conical flasks containing
and it is suspected to possess antitumour, hypotensive 750ml each of distilled water, ethanol, hexane and ethyl
and purgative properties [16]. Furthermore, Pavetta acetate. The mixtures were initially shaken rigorously
crassipes exists as a shrub commonly found in the and left for 9 days. All mixtures were filtered using
savannah belt of Nigeria and it is utilized for some sterile Whatman filter papers and the filtrates were
respiratory and abdominal disorders [17]. Aloe vera is collected directly into sterile crucibles. All filtrates
reputed for its various biological properties and its obtained were introduced into sterile reaction tubes
extensive use in traditional medicine [18]. and heated continuously in water bath at the following
temperatures: 78OC for ethanol extraction, 55OC for
In this present study, we investigated the antibacterial Ethyl acetate, 105OC for distilled water and room
properties of the four medicinal plants: Momordica temperature for hexane extracts. The residues obtained
balsamina, Pavetta crassipes, Phyllanthus amarus and were kept at room temperature.
Aloe vera herbs against four clinical strains of
Salmonella typhi, Staphylococcus aureus, Klebsiella Antimicrobial assay of plants’ extracts
pneumonie and Escherichia coli. All antibacterial assays for the plant extracts were
carried out by well diffusion technique. All the test
MATERIALS AND METHODS organisms were sub-cultured onto sterile Mueller
Collection of plant sample Hinton Agar plates and incubated at 37OC for 18-24h.
Around June 2014, fresh leaves and bark of four Five distinct colonies for each organism were
selected plants: Momordica balsamina, Pavetta inoculated onto sterile Mueller Hinton broth and
crassipes, Phyllanthus amarus and Aloe vera , were incubated for 3-4h. All innocula were standardized
collected from Owo reserved forest along Ute road accordingly to match the 0.5 McFarland standard and
,Owo, Ondo State, southwest Nigeria. The plants were this standard was used for all susceptibility tests. All
authenticated at the herbarium unit, Department of the extracts were reconstituted accordingly into the
Environmental Biology, Faculty of Science, Adekunle following concentrations: 20mg/ml, 40mg/ml and
Ajasin University, Akungba Akoko, Ondo State, Nigeria. 60mg/ml; using the Dimethyl Sulphoxide (DMSO).
The barks and leaves were washed thoroughly with Twenty five milliliters of Mueller Hinton agar was
distilled water, stored in air tight containers and kept poured into sterile petri dishes and allowed to set.
at room temperature prior to use [19] Standardized test bacterial culture for each organism
was inoculated into the sterile Kliger Iron agar plate
Test organisms using sterile cotton swab. Three wells with 6mm
The test bacteria used uin this study were Salmonella diameter were punched on the medium. Each
typhi, Klebsiella pneumoniae, Staphylococcus aureus and concentration of the extracts was dispensed into each
Escherichia coli. They were obtained from the Federal well. A central well containing only DMSO was used as
Medical Center, Owo, Ondo State, Nigeria. They were control for all tests. The plates were incubated at 37OC
isolated on sterile nutrient agar slants and taken to the for 18-24 hours after which the zones of inhibition
microbiology laboratory of the Adekunle Ajasin were measured.
University, Akungba Akoko, Nigeria. All slants of test
organisms were kept at 4OC prior to bioassay of the Phytochemical screening methods and
extracts. Extensive biochemical tests were carried out determination of mineral content
to further confirm all the test bacterial strains [20]. Qualitative analyses were carried out for the presence
of the following phytochemical components: alkaloid,
Sources and collection of plant materials glycosides, tannins, saponins, anthraquinones,
The roots of the following selected plants were anthocyanosides, flavonoids, reducing sugars and
obtained from their respective locations Momordica cyanogenic compounds [21, 22 and 23]. Quantitative
balsamina and Pavetta crassipes K. Schum, Phyllanthus determination of the mineral content was also carried
amarus Schumach and the branch of Aloe verafrom the out accordingly [24].
reserved forest of Ipaho/OkeAko, in Ikole local
Government of Ekiti State, Nigeria. The plant roots RESULTS AND DISCUSSION
were authenticated by a certified botanist at the All extracts showed increasing antibacterial activity
herbarium unit of Department of Environmental with gradual increase in the concentration of the
Biology, Adekunle Ajasin University, Akungba Akoko, extracts. The aqueous extracts of M. balsamina showed
Ondo State, Nigeria. the highest activity against the test organisms except
Escherichia coli (Table 1). Among the ethanolic extracts,
Preparation of plant extracts P. amarus showed the highest activity against the test
All the plant materials obtained were first washed bacteria and it was observed that S. typhi showed the
thoroughly with sterile distilled water and air dried at highest susceptibility to the ethanolic extracts of P.

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 OO. Temitope and O. Ajayi Ayodele / Curr Res Microbiol Biotechnol. 2014, 2(5): 457-461

amarus (Table 2). It was also observed that the ethyl plants also showed some activity against the test
acetate extracts showed the least antibacterial activity organisms but E. coli appeared to be the most sensitive
compared with the aqueous and ethanolic extracts. The to the hexane extracts of the plants (Table 4). Another
ethyl acetate extracts of M. balsamina showed the major observation was that all the plants’ extracts
highest activity against the S. aureus and E. coli while contained essential phytochemical components (Table
that of P. amarus showed the highest activity against S. 5) and essential minerals were detected except lead
typhi and E. coli (Table 3). The hexane extracts of the (Table 6).

Table 1. Antibacterial effect of aqueous extracts at different concentrations

Selected Clinical Salmonella typhi Staph aureus Klebsiella pneumoniae Escherichia coli
organism
Extract 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/ 20mg 40mg/ 60mg/
ml ml ml ml ml ml ml ml ml /ml ml ml
Momordica 10.0 12.0 13.0 10.0 12.0 14.0 10.0 12.0 13.0 4.0 7.0 10.0
balsamina
Pavetta 6.0 7.0 10.0 4.0 6.0 10.0 5.0 7.0 10.0 3.0 6.0 7.0
crassipes K.
Schum
Aloe vera 7.0 8.0 11.0 6.0 7.0 11.0 6.0 8.0 10.0 7.0 8.0 10.0
Phyllanthus 9.0 10.0 11.0 7.0 8.0 10.0 5.0 8.0 9.0 3.0 6.0 7.0
amarus
Schumach

Table 2. Antibacterial effect of ethanol extracts at different concentrations.

Selected Clinical Salmonella typhi Staph aureus Klebsiella pneumoniae Escherichia coli
organism
Extract 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/
ml ml ml ml ml ml ml ml ml ml ml ml
Momordica 7.0 8.0 10.0 5.0 6.0 10.0 5.0 8.0 9.0 4.0 8.0 10.0
balsamina
Pavetta 5.0 6.0 10.0 7.0 8.0 10.0 2.0 6.0 7.0 4.0 7.0 9.0
crassipes K.
Schum
Aloe vera 5.0 7.0 8.0 7.0 8.0 10.0 3.0 5.0 7.0 3.0 6.0 9.0
Phyllanthus 5.0 10.0 12.0 7.0 8.0 10.0 3.0 7.0 10.0 7.0 9.0 11.0
amarus
Schumach

Table 3. Antibacterial effect of ethyl acetate extracts at different concentrations.

Selected Clinical Salmonella typhi Staph aureus Klebsiella pneumoniae Escherichia coli
organism
Extract 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/
ml ml ml ml ml ml ml ml ml ml ml ml
Momordica 2.0 3.0 5.0 7.0 9.0 10.0* 3.0 4.0 5.0 7.0* 8.0 10.0
balsamina
Pavetta 2.0 4.0 7.0 1.0 5.0 7.0 3.0 5.0 7.0 3.0 6.0 8.0
crassipes K.
Schum
Aloe vera 4.0 8.0 9.0* 1.0 4.0 8.0 4.0* 8.0 9.0 1.0 4.0 8.0
Phyllanthus 2.0 5.0 7.0 3.0 5.0 10.0 3.0 6.0 10.0 2.0 7.0 8.0
amarus
Schumach

Table 4. Antibacterial effect of hexane extracts at different concentrations.

Selected Clinical Salmonella typhi Staph aureus Klebsiella pneumoniae Escherichia coli
organism
Extract 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/ 20mg/ 40mg/ 60mg/
ml ml ml ml ml ml ml ml ml ml ml ml
Momordica 1.0 3.0 5.0 0.0 2.0 6.0 2.0 6.0 8.0 3.0 5.0 8.0
balsamina
Pavetta 5.0 6.0 8.0 4.0 8.0 9.0 5.0 6.0 9.0 4.0 8.0 10.0
crassipes K.
Schum
Aloe vera 4.0 6.0 8.0 6.0 9.0 10.0 5.0 6.0 7.0 4.0 8.0 10.0
Phyllanthus 2.0 5.0 6.0 1.0 6.0 8.0 8.0 12.0 5.0 5.0 6.0 10.0
amarus
Schumach

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Table 5. Qualitative phytochemical screening of the medicinal plant samples

Plant Cardiac Tannins Phenol Alkaloid Saponin Philobatain Steroid flavoniods
Samples glucoside
Momordica +ve +ve +ve +ve +ve +ve +ve +ve
balsamina
Pavetta crassipes K. ±ve +ve +ve +ve +ve +ve -ve +ve
Schum
Aloe vera +ve +ve +ve +ve +ve +ve -ve +ve
Phyllanthus amarus +ve +ve +ve +ve +ve +ve +ve -ve
Schumach
Keys: +ve = Present, -ve = Absent, ±ve = Slightly present.

Table 6. Minerals composition in plant extracts (mg/100g)

S/N Na K Ca Mg Zn Fe Pb Cu Mn P
Momordica balsamina 23.73 30.12 32.47 25.05 27.04 6.77 ND 0.01 5.39 26.52
Pavetta crassipes K. Schum 14.47 33.65 17.50 32.47 40.00 3.50 ND 0.01 4.45 79.00
Aloe vera 11.68 19.54 12.75 22.64 20.50 7.48 ND 0.01 17.33 103.21
Phyllanthus amarus Schumach 18.02 25.13 32.45 21.06 24.03 5.75 ND 0.01 6.19 25.10
Key: ND= Not detected

Table 7. Percentage of Anti-nutrient in plant extracts

Parameters Momordica balsamina Pavetta crassipes K. Schum Aloe vera Phyllanthus amarus
Schumach
a b a b a b a b
Ash (%) 12.55 12.61 9.30 9.20 8.73 8.70 10.00 10.12
MC (%) 9.33 9.30 3.71 3.70 7.39 7.32 9.11 9.00
CP (%) 13.37 12.83 14.30 14.70 16.24 16.11 14.45 12.01
Fat (%) 6.42 6.45 7.99 6.83 5.31 5.41 6.55 6.55
Fiber (%) 11.43 11.48 4.22 4.44 8.20 8.52 10.36 10.12
CHO (%) 46.64 47.18 60.30 60.91 53.70 53.33 42.47 41.17
NB: This was carried out in duplicates for each plant a and b

The recent upsurge in global incidence of resistance to some inhibitory effect against a variety of organisms
antibiotics among clinically significant bacteria has re- [28].
invigorated the interest for the deliberate search and
screening of plants for possible novel antimicrobial and There is well documented evidence of the biological
medicinal properties. The present study reiterates the activities of P. crassipes which include activities against
antibacterial properties of Phyllanthus amarus, protozoans and tumour cells [29]. Mustapha and Bala
Momordica balsamina, Pavetta crassipes and Aloe vera. (2010) [30] reported the activities of chloroform and
These plants are widespread in the tropical habitats ethanol extracts of P. crassipes against some bacterial
across the globe and the ease of access to these plants respiratory pathogens. Comparatively, the antibacterial
could be an additional motivation for their anticipated activity they observed for this plant was observed to be
use as cheap alternatives to the current regimens of lower than what we observed in our study and this may
antibiotics currently used in clinical medicine. be attributed to different climatic conditions where the
plants were found. The antibacterial activity of the P.
The results obtained on the antibacterial activity of crassipes has been strongly linked with the presence of
these plants are consistent with previous studies. For bioactive compounds especially the flavonoids, which is
instance, Usie et al., (2013) [25] reported the one of the principal phytochemical components of the
antimicrobial activity of P. amarus against some gram plant and which was also detected in this study [31].
positive and gram negative bacteria. Oluwafemi and The compound conferred activity against P. aeruginosa,
Debiri (2008) [26] reported that the ethanolic extracts K. pneumonia, S. pyogenes and E. coli – a finding which
of P. amarus showed strongest antibacterial activity also aligns with our present observations. Aliyu et al.,
against E. coli and S. typhi, compared to the hot and cold (2008) [32] also confirmed a high antibacterial activity
water extracts of the plants. It has been confirmed that of M. balsamina against methicillin resistant strains of
the antimicrobial activity of P. amarus is due mainly to Staphylococcus aureus in Northern Nigeria.
the presence of a chemical called the phyllantin [27].
The Aloe vera has a huge reputation for its global
Momordica balsamina demonstrated potent activity acceptability and recognition of the numerous
against the test bacterial strains. Similar studies have medicinal uses. Our present study further confirms the
also confirmed that the plant showed activity against potency of A. vera against common bacterial pathogens.
gram-positive and gram-negative bacteria [16]. The disk diffusion method for antibacterial activity
Different parts of the M. balsamina plant have been confirmed that ethanolic extracts of dried Aloe vera gels
confirmed to contain resins, alkaloids, flavonoids and possess demonstrable antibacterial activity against E.
other phytochemicals that confer specific antimicrobial coli, Enterococcus faecalis and S. aureus. In agreement
and medicinal properties on the plants. In addition, this with the results we obtained in our present study, the
plant has been found to contain mormodin that has ethanolic extract of the A. vera also showed the highest
activity against test organisms and the various

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 OO. Temitope and O. Ajayi Ayodele / Curr Res Microbiol Biotechnol. 2014, 2(5): 457-461

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