442 Asian Pac J Trop Dis 2014; 4(6): 442-446

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Disease

journal homepage: www.elsevier.com/locate/apjtd

Document heading doi:10.1016/S2222-1808(14)60603-4 襃 2014 by the Asian Pacific Journal of Tropical Disease. All rights reserved.

Efficacy of fresh leaf extracts of Spondias mombin against some clinical

bacterial isolates from typhoid patients
Olukemi Aromolaran*, Omotola Kikelomo Badejo
Department of Biological Sciences, Wesley University of Science and Technology, PMB 507 Ondo, Ondo State, Nigeria

PEER REVIEW ABSTRACT

Peer reviewer Objective: To evaluate the phytochemical properties and antibacterial activity of methanol,
D r. M ojisola O . E dema, F ederal acetone, ethanol and aqueous extracts of fresh leaves of Spondias mombin (S. mombin) on some
U niversity of T echnology, A kure, clinical bacterial isolates.
Nigeria. Methods: Clean and fresh leaves of S. mombin were collected in Ondo, Southwestern Nigeria.
Tel: +2347033779957 The leaves were blended, extracted with methanol, acetone, ethanol and water. The extracts
E-mail: moedemao@gmail.com were evaporated to dryness using rotary evaporator and tested for the presence of saponins,
tannins, cardiac glycoside, terpenoids, flavonoids, reducing sugars, volatile oils, alkaloids and
Comments glycoside. The extract were tested against Gram-negative bacteria Klebsiella pneumonia, Serratia
T he concept of the work is good, marcescens, Salmonella typhi and Enterobacter aerogens; Gram-positive bacteria Staphylococcus
though similar studies have been aureus by observing the zones of inhibition using agar well diffusion assay.
carried out on the antimicrobial of the Results: The study showed that the leaves contained saponins, tannins, flavonoids, alkaloids and
dry leaf extract against some bacteria, glycoside. All the solvent extracts showed activity against all the test bacteria. The methanol
this work focused on the antibacterial extract also showed the highest activity against Enterobacter aerogens, zone of diameter (15.00
efficacy of the fresh extract against 依1.89) mm, while the ethanol extract showed the highest activity against Staphylococcus aureus
bacteria of clinical origin. with zone of diameter (12.50依1.50) mm. The acetone extract showed the highest activity against
Details on Page 445 Salmonella typhi, zone of diameter (17.50依0.29) mm followed by methanol extract showing zone
of diameter (15.67依1.01) mm. The acetone extract showed the highest activity against Klebsiella
pneumonia (15.17依0.67) mm, while the aqueous extract shows the highest activity against Serratia
marcescens (14.67依2.68) mm. The minimum inhibitory concentration of the leaf extracts ranged
between 10-90 mg/mL.
Conclusions: This study showed that the aqueous and organic solvents extract of fresh leaves of S.
mombin has anti-microbial activity against all the tested organisms.

KEYWORDS
Phytochemicals, Antibacterial, Spondias mombin, Extracts

1. Introduction medicine is not cheap and a large number of people depend

on medicinal plants for their health care needs. Medicinal
Infectious diseases account for almost 50 000 deaths every plants are cheap, readily available and are known to
day and are the world’s major threat to human health[1]. contain certain substances that are toxic to bacteria[3]. They
P athogenic bacteria causing different kinds of life- have played significant roles in maintaining human health
threatening infections have increased and it is becoming and improving the quality of human life for many years.
an important cause of death in immune-compromised Medicinal and aromatic plants contain biologically active
patients, especially in developing countries[2]. Conventional chemical substances and free radical scavenging molecules
*Corresponding author: Olukemi Aromolaran, Department of Biological Sciences, Article history:
Wesley University of Science and Technology, Ondo Nigeria. Received 16 May 2014
Tel: +2348062225745 Received in revised form 23 May, 2nd revised form 29 May, 3rd revised form 5 Jun 2014
E-mail: kemiaromolaran@gmail.com Accepted 10 Jun 2014
Available online 28 Dec 2014
 Olukemi Aromolaran and Omotola Kikelomo Badejo/Asian Pac J Trop Dis 2014; 4(6): 442-446
443

such as saponins, tannins, essential oils, flavonoids, 200 g of the fine blended fresh leaves into 300 m L of
alkaloids, anthocyanins, carotenoids, dietary glutathione distilled water. The ethanol extract was also prepared by
and other chemical compounds that are rich in antioxidant suspending 200 g of the blended leaves into 200 mL of 98%
activities[4-6], and they have demonstrated their contribution ethanol. The methanol extract was prepared by suspending
to the treatment of diseases such as HIV/AIDS[7], malaria, 300 g into 500 mL of 98% methanol and the acetone extract
cataract[8], diabetes[9], skin disease[10], sickle-cell anaemia, was prepared by suspending 80 g into 90 mL of 98% acetone
mental disorders[11], and microbial infections[3,12,13]. for 24 h. Thereafter, the suspensions of the fresh leaves were
Spondias mombin Linn (S. mombin) is a fructiferous tree filtered into sterile beakers separately using sterile muslin
that thrives in the rainforest and coastal areas of Africa. It cloth.
belongs to the family Anacardiaceae. The plant is readily The extracts were subsequently concentrated to dryness
used in folk medicine and it is known by various names. in vacuum at 40 °C for the organic solvents and 80 °C for the
The fruit is called iyeye or yeye in the Yoruba language, aqueous solvent using a rotary evaporator. They were then
ngulungu in Igbo and isadaa in Hausa[14,15]. Other names placed in a sterile container, labelled and stored in the
may include hog plum, true yellow mombin, golden apple desiccator for phytochemical screening and antimicrobial
and java plum. analysis.
All the parts of the tree are important in traditional
medicine. The fruits are popular for eating and the extracted 2.3. Collection and identification of test organisms
juice is used to prepare ice cream, cool beverages, jam
and wine like V iro de T aperiba [16]. T he fruit juice is The test organisms used for this study were bacterial
drunk as a diuretic and febrifuge. The decoction of the isolates from typhoid patients at the Medical Microbiology
astringent bark serves as an emetic, a remedy for diarrhoea, L aboratory, F ederal M edical C entre, O wo, O ndo S tate,
dysentery, haemorrhoids and a treatment for gonorrhoea and N igeria. T hey are four G ram-negative and one G ram-
leucorrhoea[17]. All the parts of the plant are reported to be positive bacteria. The Gram-negative isolates are Klebsiella
medicinally useful and their traditional uses in reproduction pneumoniae ( K. pneumoniae ) , Serratia marcescens ( S.
have been reported. It has also been reported that the leaves marcescens), Salmonella typhi (S. typhi) and Enterobacter
serves as an antidiarrheal, antimicrobial, anti-viral and also aerogens (E. aerogens). Staphylococcus aureus (S. aureus)
contains vitamin C[17,18]. Infusion of its leaves has been used served as the Gram-positive isolate.
for a long time without any report of collateral effects due to
its activity. Its uses show that it is a medicinal plant with a 2.4. Standardization of test organisms
lot of potential, valuable, untapped resource of active drugs
for treating diseases. This present study aimed to investigate Pure colonies of the identified isolates were inoculated
the phytochemical properties and antimicrobial potential into sterile nutrient agar slants, incubated for 24 h at 37 °C
of fresh leaf extracts of S. mombin against some clinical and kept at 4 °C till further use.
bacteria isolates from typhoid patients. All inoculums were standardized using the Mcfarland
nephelometer method. Test-tubes of varying concentrations
of barium chloride and sulphuric acid were used. The
2. Materials and methods standard that was used for this work corresponds to 15伊108/
bacterial suspension per millimetre.
2.1. Plant material collection and identification
2.5. Antibacterial susceptibility screening
Fresh and healthy leaves of S. mombin were collected
around the vicinity of Wesley University of Science and The agar well diffusion technique was used to determine
Technology, Ondo. The identification and authentication was the antibacterial activity of the plant extracts[21]. About 0.1
carried out at the Department of Botany, Obafemi Awolowo mL of the different standardized organisms were introduced
University, Ile-Ife with herbarium number 16856. separately and thoroughly mixed with 20 mm of Mueller
Hinton agar each in a sterile Petri dish and allowed to set. A
2.2. Plants extract preparation sterile 5 mm cork borer was then used to punch holes in the
seeded agar. The wells were filled with each of the extracts
The fresh leaves of S. mombin were extracted with four and antibiotic (streptomycin) serves as the positive control
solvents: ethanol, methanol, acetone and aqueous solvent which were labelled accordingly while two wells contained
using modified methods[19-21]. the extractant (i.e. the solvent used for the extraction) to
Healthy and fresh plant leaves of S. mombin collected serve as negative control. These were then left on the bench
were grounded into fine texture using a sterilized electric for 2 h for adequate diffusion of the extracts and incubated
blender. The fresh water extract was prepared by suspending at 37 °C for 24 h. After incubation, the diameters of the zones
444 Olukemi Aromolaran and Omotola Kikelomo Badejo/Asian Pac J Trop Dis 2014; 4(6): 442-446

of inhibition around each well were measured to the nearest glycosides, alkaloids and flavonoids, while terpenoids,
mm and the mean of the reading were calculated. A ll reducing sugars, volatile oils and cardiac glycosides were
experiments were done in triplicates. not detected (Table 1).

Table 1
2.6. Antibiotic sensitive test
Phytochemical screening of S. mombin leaves.
Ethanol Methanol Acetone Aqueous
Antibiotic susceptibility test was carried out on the test Phytochemicals
extract extract extract extract
bacteria using multi-disc diffusion method as described[22]. Saponins + + + +
The test organism was introduced on the surface sterile Tannins + + + +
Cardiac glycosides - - - -
nutrient agar in Petri dish. The multi-disc antibiotic was
Terpenoids - - - -
then placed on the culture plates aseptically, incubated at Reducing sugars - - - -
37 °C for 24 h. After incubation, the diameter of the zone of Volatile oils - - - -
inhibition around each well was measured. Flavonoids + + + +
Alkaloids + + + +
Glycosides
2.7. Determination of minimum inhibitory concentration + + + +
+: present, -: absent.
(MIC)

The minimum bacterial growth inhibition of the various All the examined extracts showed varying degrees of
extracts was determined using the broth dilution technique. antibacterial activities against the pathogens. T able 2
Different increasing concentration of the extracts was used. shows the results of the antibacterial susceptibility test of
About 2 mL of Mueller Hinton broth was prepared into test the extracts against the test isolates. From the results, the
tubes for each extract, and 0.5 mL of 10-90 mg/mL of each methanol extract also showed the highest activity against
extract was added to the different test tubes containing the E. aerogens, zone of diameter (15.00依1.89) mm, while the
Mueller Hinton broth. This was prepared for each organism. ethanol extract showed the highest activity against S. aureus
A colony of 24 h cultured-organism was inoculated into with zone of diameter (12.50依1.50) mm. The acetone extract
test tubes containing 1 mL of sterile distilled water to form showed the highest activity against S. typhi, zone of diameter
turbidity of 0.5 McFarland standards and was thereafter (17.50依0.29) mm followed by methanol extract showing zone
dispensed into the test tubes containing the suspension of of diameter (15.67依1.01) mm. The acetone extract showed the
the Mueller Hinton broth and the different extracts. This highest activity against K. pneumonia (15.17依0.67) mm, while
was done for all the organisms at the tested concentrations. the aqueous extract shows the highest activity against S.
All test tubes were properly corked and incubation at 37 °C marcescens (14.67依2.68) mm.
for 24 h, after which they were observed for the absence or
Table 2
present of visible growth. The lowest concentration without
Antibacterial activity of extracts of S. mombin leaves against some clinical
visible growth (turbidity) of organisms was regarded as the isolates.
MIC. Zone of diameter (mm)
Organisms Aqueous Ethanol Acetone Methanol
Streptomycin
2.8. Phytochemical screening extract extract extract extract
K. pneumoniae 12.83依3.42 11.50依1.15 15.17依0.67 12.83依2.46 12.33依1.09
S. aureus 10.67依2.62 12.50依1.50 11.17依3.44 11.83依3.18 08.67依3.98
The extracts were analysed to test for the presence of the S. marcescens 14.67依2.68 11.50依3.21 11.33依2.89 10.17依0.88 11.00依4.31

alkaloids, saponins, tannins, terpenoids, flavonoids, cardiac S. typhi 12.17依0.33 12.00依1.00 17.50依0.29 15.67依1.01 12.67依1.42
E. aerogenes 13.17依1.17 13.50依0.87 13.33依0.88 15.00依1.89 12.33依3.22
glycosides, volatile oils and reducing sugars[23].
Data are expressed as mean依SEM.

2.9. Statistical analysis

Table 3 shows the susceptibility of the test organisms to
The data obtained from the study were analysed by the different antibiotics. All the test bacteria were inhibited by
use of Two-way analysis of variance with replicates (ANOVA) at least one antibiotic except for S. marcescens and S. aureus
to determine the significance antimicrobial activity of S. which were resistant to all the antibiotics. S. typhi and E.
mombin leaves. The values were expressed in mean依SEM. aerogens were susceptible to ofloxacin, ciprofloxacin and
pefloxacin.
The methanol and ethanol extracts showed the lowest
3. Results MIC of 10 mg/mL against all the tested isolates (Table 4).
There was no inhibition at all the tested concentration for
T he phytochemical screening of the fresh leaves of the aqueous extract except with MIC of 50 mg/mL on K.
S. mombin showed the presence of saponins, tannins, pneumoniae.
 Olukemi Aromolaran and Omotola Kikelomo Badejo/Asian Pac J Trop Dis 2014; 4(6): 442-446
445
Table 3
Antibiotic susceptibility of the test organisms.
Organisms Antibiotic susceptibility Resistant
K. pneumoniae NIT, GEN, COT,OFL, AMX, CPX, TET, PFX CRO, AUG
S. aureus No susceptibility CRO, NIT, GEN, COT, OFL, AMX, CPX, TET, PFX, AUG
S. marcescens No susceptibility CRO, NIT, GEN, COT, OFL, AMX, CPX, TET, PFX, AUG
S. typhi OFL, CPX, PFX CRO, NIT, GEN, COT, AMX, TET, AUG
E. aerogens OFL, CPX, PFX CRO, NIT, GEN, COT, AMX, TET, AUG
CRO: Ceftriazone (30 µg), NIT: Nitrofurantoin (200 µg), GEN: Gentamicin (10 µg), COT: Co- trimoxazole (25 µg), OFL: Ofloxacin (5 µg), AUG: Augumentin (30 µg), AMX:
Amoxicilin (25 µg), PFX: Pefloxacin (5 µg), TET: Tetracycline (30 µg), CPX: Ciprofloxacin (10 µg).

Table 4 Conflict of interest statement

MIC of various extracts of fresh leaves of S. mombin (mg/mL).
Aqueous Methanol Acetone Ethanol
We declare that we have no conflict of interest.
Organisms extract extract extract extract
10 50 90 10 50 90 10 50 90 10 50 90
K. pneumonia + - - - - - + - - - - -
S. aureus + + + - - - - - - - - - Acknowledgements
S. marcescens + + + - - - + - - - - -
S. typhi - - - - - - - - -
+ + +
The authors are thankful to the management of Wesley
E. aerogens + + + - - - - - - - - -
University of Science and Technology, Ondo Nigeria for
+: growth, –: no growth.
providing the laboratory facilities needed for the research
work, Mr. Ojo FK for the authentication of plant (Botany
Department, Obafemi Awolowo University, Ile-ife Nigeria),
4. Discussion Mr. Akinyemi IA for the bacterial isolates, technical support
provided by Mrs. Adamolekun Oluwatosin and Mr. Badejo
The therapeutic value of medicinal plants lies in the VO for financial support of the project.
various chemical constituents it contains. The bioactivity of
plant extracts is attributed to phytochemical constituents.
Plants rich in tannins have antibacterial potential due to Comments
their character that allow them to react with proteins to form
stable water soluble compounds thereby killing the bacteria Background
by directly damaging its cell membrane. In a similar study, The study evaluated the phytochemical properties and
it was reported that S. mombin contains tannins, saponins antibacterial activities of methanol, acetone, ethanol and
and anthraquinone glycosides that showed significant aqueous extracts of fresh leaves of S. mombin on some
antimicrobial activity[24,25]. Most of the effects observed with clinical bacterial isolates viz. K. pneumonia, S. marcescens,
extract of S. mombin may be attributed to the constituent S. typhi, E. aerogens, and S. aureus by observing the zones of
compounds of phenols, tannins, anthraquinones and inhibition using agar well diffusion assay. This study showed
flavonoids presence in the plant. that the aqueous and organic solvents extract of fresh leaves
There may be several factors that will predispose bacteria of S. mombin has anti-microbial activity against all the
to antibacterial agents such as previous encounters with the tested organisms.
agent or the nature of the medium used which may affect the
ability of the agent to diffuse[22]. The fact that the extracts Research frontiers
were active against both Gram-negative and Gram-positive S. mombin contains a number of phytochemicals such as
bacteria indicated a broad spectrum of activity. tannins, saponins and anthraquinone glycosides that showed
The presences of bioactive substance in plant extracts are significant antimicrobial activity. Studies are in the area of
responsible for the antibacterial activities and the difference isolating and characterizing new antimicrobial agents from
in the efficacy of the organic extracts may have different such plants in order to develop novel therapeutic agents.
polarities for different solvents[18,21,26].
This study demonstrated that folk medicine can be as Related reports
effective as modern medicine to combat infectious diseases. T his study investigated the use of different organic
The use of this plant in folk medicine suggests that they solvents (aqueous, ethanol, methanol and acetone) of fresh
represent an economic alternative to treat diseases. The leaf extracts of S. mombin against clinical isolates instead
antimicrobial activity of the plants may be due to the of only the ethanol and aqueous dry leaf extract in previous
presence of various active constituents in the leaves. Further report of Ajao et al. (1985) against some bacterial isolates.
studies are needed to isolate and characterize the properties The report of this study confirms the antimicrobial ability of
to develop new antimicrobial agents. the leaf.
446 Olukemi Aromolaran and Omotola Kikelomo Badejo/Asian Pac J Trop Dis 2014; 4(6): 442-446

Innovations & breakthroughs 67-71.

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clinical bacterial isolates. [11] Okigbo RN, Mmeka EC. An Appraisal of phytomedicine in Africa.
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