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Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
28
American Journal of
Biomedical Sciences
ISSN: 1937-9080
nwpii.com/ajbms
Antimicrobial Activities of Chrysophyllum Albidum Leaves, Fruits and
Seeds
GEORGE, Olajide A.1; ADENIPEKUN, Eyitayo O.2; FASOGBON, Samuel Ayobami3;
OPARANOZIE Jude A.4
1 Medical Laboratory Science Council of Nigeria, South-South Zonal office, Benin city, Nigeria
2 Department of Medical Laboratory Science, College of Medicine, University of Lagos, Nigeria
3,4 Public Health In-vitro Diagnostic Control Laboratory, Medical Laboratory Science Council of Nigeria, Yaba-
Lagos, Nigeria.
*Corresponding Author
George, Akintunde Olajide
Medical Laboratory Science Council of Nigeria
73, Murtala Mohammed Way, Benin City,
Edo State,
Nigeria.
E-mail: georgeolajide2016@gmail.com
Cell phone: +2348062234415
Received:31 January 2018 ; | Revised:01 March 2018; |Accepted: 20 March 2018
Abstract
Background: Chrysophyllum albidum is a medicinal plant that belongs to the Sapotaceae family. This
study was carried out to determine the antimicrobial activities of the leaves, seeds and fruits of
Chrysophyllum albidum.
Materials and Methods: The fruits, leaves and seeds of Chrysophyllum albidum were extracted with
distilled water, Seaman's Schnapps, Methanol and Petroleum ether using cold extraction. These extracts were
tested on various organisms. Antimicrobial activities of the various extracts were determined using the agar
well diffusion technique to detect the Minimum Inhibitory Concentration (MIC).
Results and Discussion: Methicillin Resistant Staphylococcus aureus was inhibited by all the extracts at
MIC between 32µg/ml to 512µg/ml except Seaman's Schnapps leaves and petroleum ether seed extract. The
fruit extracts had a high antimicrobial activity against all the tested organisms. The extracts from Seaman's
Schnapps had little activity while the aqueous extracts had MIC ranging from 64- 512µg/ml on most of the
organisms tested. The statistical analysis of the extracts indicated P-value of 0.122 for Aqueous extracts,
which was statistically insignificant compared to 0.003 for Methanolic extracts and 0.001 for Seaman's
Schnapps extracts in the inhibition pattern of the extracts. Aqueous extracts showed more inhibitory activity
than Methanolic and Seaman's Schnaps extracts.
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
29
Conclusion: Chrysophyllum albidum possess antimicrobial properties that can also be developed as
antimicrobial for Multidrug resistant (MDR) organisms. Chyrsophyllum albidum is therefore recommended
to be developed into new antimicrobials in readily available forms for use in Medical Microbiology
Laboratory.
Keywords: Chrysophyllum albidum;Antimicrobial; MIC;Extracts.
1. Introduction
Chrysophyllum albidumis a medicinal plant
that belong to the Sapotaceae family which has up
to 800 species and make up almost half of the order
Ericales [1]. Chrysophyllum is a genus of about 70-
80 species of tropical trees growing rapidly to 10-
20m or more in height. The generic name is derived
from the Greek word Chrysos meaning ‘gold' and
Phyllos meaning ‘leaf' [2].The genus is native to
tropical regions throughout the world, with the
greatest number of species in northern South
America. Chrysophyllum albidum is a dominant
canopy tree of lowland mixed rain forest,
sometimes riverine. It is widely distributed from
West Africa to the Sudan with an eastern limit in
Kakamega forest, Kenya [2]. Chrysophyllum
albidum, is distributed throughout the Southern part
of Nigeria where it is called ‘Agbalumo' (Yoruba) ;
‘Udara' (Igbo), while in the Northern Nigeria, it is
called ‘Khada'(Hausa) [3,4]. Tannins, flavonoids,
terpenoids, protein, carbohydrates, and resins are
the phytochemicals that have been reported in
Chrysophyllum albidum [5]. Chrysophyllum albidum
has antioxidant properties by scavenging free
radicals, decreasing lipid peroxidation and
increasing the endogenous blood antioxidant
enzymes level [6]. The leaves of Chrysophyllum
albidum was shown to reveal the presence of
Alkaloids, Cardiac glycoside, Anthraquinones,
Flavonoids, Terpenoids, and Steriods, which are
useful substances that have medicinal and
physiological activities. Vitamin D which is also
believe it contain have also been linked with breast
cancer [7-8].
In recent times, there have been an increased
antibiotic resistant strains of clinically important
pathogens, which has led to the emergence of new
bacterial strains that are multi-resistant [9,10,
11].Therefore, this has led to the search for more
effective antimicrobial agents among materials of
plant origin, with the aim of discovering potentially
active ingredients that can serve as a source for the
synthesis of new antimicrobial drugs [12].
Medicinal plants use date back to several
centuries in Africa and other regions of the world. It
is about the first form of traditional medicine known
and reported, and the practice is still popular [13, 4].
The traditional preparations comprise medicinal
plants, minerals and organic matter. Herbal drugs
constitute only those traditional medicines which
primarily use medicinal plant preparation for
therapy [14], however the search for agent to cure
infectious disease began long before people were
aware of the existence of microbes. These early
attempts used natural substances, usually native
plants or their extracts and many of these herbal
remedies proved successful [15]. A number of studies
have been done on the advantages or potency of
herbs as cure for example, capsule Artimin from
plant, and is a vegetarian capsule and dietary
supplement that helps maintain the physiological
well being of joints. It contains extracts of Salix
alba (willow), Harpagophytum procumbens
(Harpagus),Boswellia serrata (Boswelia), Malpighia
punicifolia (Acerola) and Uncaria tormentosa
(Uncaria) [16, 17]. Therefore, more plants have to be
checked for antimicrobial properties, so the need for
this study.
Several workers have carried out the in vitro
effects of extracts from plants with claimed
medicinal use. Several experimental approaches are
used. Usually water is used for extraction just as is
used traditionally [18]. Other solvents used for
extraction apart from water include chloroform,
petroleum ether, ethanol, methanol, acetone etc.
These extracts were tested against a wide range of
microorganisms and their efficacy results well
documented. It is not at all times however that such
result confirms the claims of the tested herbs [19].
There is paucity of data on the antimicrobial
screening of Chrysophyllum albidum leaves, fruits
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
30
and seed against multidrug resistant organisms,
including Extended Spectrum Beta Lactamase
producers and Methicilin Resistant Staphylococcus
aureus. This study investigated the antimicrobial
properties of Chrysophyllum albidum leaves, fruits
and seed against multidrug resistant organisms.
2. Materials and Methods
2.1 Area of Study
This study was carried out at Department of
Medical Microbiology, College of Medicine
University of Lagos, Lagos State, Nigeria.
2.2 Ethical Consideration
Approval for this research work was obtained
from the Research Ethics Committee of College of
Medicine University of Lagos, Lagos State, Nigeria
2.3 Collection and Preparation of Plant Extract
The plants was identified and authenticated at
the herbarium unit and Pharmacognosy Laboratory,
College of Medicine, University of Lagos. The
leaves and fruits of Chrysophyllum albidum
(Agbalumo-Yoruba, Udara-Igbo and Khada-Hausa)
were purchased at the Mushin Market in Lagos,
Nigeria. The seed were obtained from the fruit pulp.
(Plate 1 and 2)
Plate 1 Chrysophyllum albidum Leaves
Plate 2 Chrysophyllum albidum Fruits
2.4 Processing of Plant Materials
The fresh leaves and fruits were properly
washed in tap water and the seed were removed
manually and washed in tap water. The plant leaves,
fruits and seeds were allowed to dry for two weeks.
The plants materials were pulverized into powder
using an electric blender.
2.5 Extraction of Plant Materials
Distilled water, Seaman's Schnapps, Methanol
and petroleum ether were used for the extraction of
plant extracts. 100 grams of the dried and grounded
leaves were suspended in 500ml of distilled water,
Seaman's Schnapps and Methanol for extraction.
100 grams of the dried and grounded fruits were
suspended in 500ml of Distilled water, Seaman's
Schnapps and Methanol for extraction while 80
grams of the dried and grounded seed was soaked in
distilled water and Petroleum ether. This was left to
soak at room temperature for 72hours with agitation
at intervals. The extracts were filtered with
Whatman filter paper No. 42 (125mm). The filtrate
were concentrated using the rotary evaporator at
450C.The concentrated extracts from each solvent
were taken for lyophilization at the Department of
Biochemistry, College of Medicine, University of
Lagos. Each solid extract/ paste obtained after
lyophilization was reconstituted in their respective
solvents to obtain a stock solution of 512µg/ml [25,
26]. The stock solutions were stored in sterile
capped bottles and labeled as follows:
1. Aqueous Leave Extract (ALE)
2. Aqueous Fruit Extract (AFE)
3. Aqueous Seed Extract (ASE)
4. Seaman's Schnapps Leave Extract (SSLE)
5. Seaman's Schnapps Fruit Extract (SSFE)
6. Methanolic Leave Extract (MLE)
7. Methanolic Fruit Extract (MFE)
8. Petroleum ether Seed Extract (PSE)
The stock solutions were stored at 4 0C -80C.
2.6 Test Organisms
The organisms used were Shigella flexneri,
Klebsiella pneumoniae (ESBL), Pseudomonas
aeruginosa (MDR), Escherichia coli,
Staphylococcus aureus(MRSA), Enterococcus
faecalis and Candida albicans. The test organisms
were obtained from the research laboratory of
Medical Microbiology and Parasitology of College
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
31
of Medicine, University of Lagos and Lagos
University Teaching Hospital Laboratory.
2.7 Control Organisms
Control strains of Staphylococcus aureus
ATTC 25923 and Escherichia coli ATTC 25922
were used and tested along with the test organisms
as control.
2.8 Identification of Microorganisms
Microorganisms collected were authenticated
for identification by Analytical Profile index 20E
which is a standardized identification system for
Enterobacteriaceae and other non-fastidious, Gram-
negative rods.
The Analytical profile index 20E (API 20E,
Biomerieux) strip consists of 20 micro tubes
containing dehydrated substrates. These strips are
inoculated with a 0.5 McFarland standard bacterial
suspension that reconstitutes the media. They were
then incubated at 37oC overnight .The reactions are
recorded and the identification is obtained by
referring to the Analytical profile index
identification software/flow chart.
2.9 Reading the Strip
Identification of the organisms is made by
looking up the seven /nine digit profile in the
Analytical Profile Index (API20E).The index is
divided into two sections, the seven digit profiles
read after 18-24hours of incubation and the nine
digit profiles read after 36-48hours of incubation.
The seven digit profile was used in this experiment.
The flow chart was followed in the identification of
the organisms.
2.10 Phenotypic Detection of extended spectrum
beta-lactamase
The presence of Extended Spectrum Beta-
Lactamase (ESBL) was detected by the double disc
synergy test (DDST).A 0.5 McFarland Standard
suspension of the test organism was inoculated on
Mueller-Hinton agar. A disc containing 30µg
Amoxicillin plus clavulanic acid disc was placed
centrally on the plate (Plate 5).Disc containing
Cefotaxime (30µg),Ceftazidime (30µg) and
Ceftriaxone (30µg) were placed round the
Amoxicillin plus Clavulanic acid disc at a distance
of 15mm [10].The plates were incubated overnight at
37oC for 24 hours. Isolates that exhibited a distinct
shape/size with an increased zone of inhibition
towards Amoxicillin plus Clavulanate disc were
considered as Extended spectrum beta-lactamase
producers [10].
2.11 Detection of Methicillin Resistant
Staphylococcus aureus (MRSA)
The presence of MRSA was detected using the
modified Kirby-Bauer method (Bauer, et
al.,1966).Isolated colonies from an 18hours culture
was used to prepared a direct inoculum equivalent
to a 0.5 McFarland Standard. Suspension of the test
organism was inoculated on Mueller Hinton Agar
using a sterile swab. An Oxacillin disc containing
(1µg) and Cefoxitin (30µg) were placed on the agar.
The Mueller Hinton Agar on which Oxacillin discs
was applied were supplemented with 2% Sodium
Chloride (NaCl) to enhance the detection of MRSA
and incubated aerobically for 24hours at 37oC
while the one on which Cefoxitin was placed was
not supplemented with sodium chloride(NaCl) but
incubated aerobically at 37oC for 24hours(Plate
6)The strains were reported as sensitive, or resistant,
to Oxacillin/ Cefoxitin with inhibition zone
diameter equal or more than 13mm and less than or
equal to 10mm respectively [27].
2.12 Standardization of Inoculum
Test organisms were sub-cultured onto fresh
plates of Mueller Hinton agar and Sabouraud
Dextrose Agar for18-24 hours at 37oC for bacteria
and fungi respectively. Colonies from these were
suspended in sterile normal saline to a turbidity
matching 0.5 McFarland standard containing 1 x
108cfu/ml for bacteria and fungi respectively [25].
2.13 Antimicrobial Assay
Suspension of the microorganisms were made
in sterile normal saline and adjusted to 0.5
McFarland standards. The resulting suspension
contains 1 x 108cfu/ml. Each labeled medium Plates
were uniformly seeded with a test organism by
means of sterile swab rolled in the suspension and
streaked on the plate surface. Well of 5mm in
diameter and about 2cm apart were punched in the
culture media with sterile cork-borer. Then10µl of
the concentrations of the Chrysophyllum albidum
extracts ranging from 4µg/ml to 512µg/ml were
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
32
placed in each well [28, 26]. Ciprofloxacin, Distilled
water, Seaman's Schnapps, methanol and petroleum
ether solvents were placed in a well on each plate
seeded with test organisms as controls. Each plate
was kept in the refrigerator at 40C for 1 hour to
allow the extracts to diffuse into the culture medium
while the immediate growth of the organism was
stopped from taking place [25].These plates were
then incubated at 370C for 24 hours. The zones of
inhibition around the wells were measured in
millimeter.
2.14 Reading of Results
A ruler was placed across the well and zone of
inhibition was measured in millimeter. This was
recorded as positive bioactivity. Control strains of
organism were set up and measured as well.
2.15 Antifungal Assay
Suspension of the Candida albicans were made
in sterile normal saline and adjusted to 0.5
McFarland standards. The resulting suspension
contains 1 x108cfu/ml. Each labeled medium Plates
were uniformly seeded with the Candida albicans
by means of sterile swab rolled in the suspension
and streaked on the plate surface. Well of 5mm in
diameter and about 2cm apart were punched in the
culture media with sterile cork-borer. The
concentrations of the extracts ranging from 4µg/ml
to 512µg/ml were dropped in each well to fullness
[28, 26]. Each plate was kept in the refrigerator at 40C
for 1 hour to allow the extracts to diffuse into the
culture medium while the immediate growth of the
organism was stopped from taking place. [25].These
plates are then incubated at 370C for 48 hours. The
zones of inhibition around the wells were measured
in millimeter.
2.16 Determination of minimum inhibitory
concentration (MIC)
To measure the MIC values, various
concentrations of the stock, 512µg/ml, 256µg/ml,
128µg/ml, 64µg/ml, 32µg/ml, 16µg/ml, 8µg/ml, and
4µg/ml were assayed against the test bacteria. The
minimum inhibitory concentration was defined as
the lowest concentration able to inhibit any visible
bacterial growth on the culture plates [29, 28, 26].
Table 1 Zone of inhibition (mm) of organisms to aqueous leave extract (ale) of chrysophyllum albidum
TEST ORGANISMS
CONCENTRATION (µg/ml)
512
256
128
64
32
16
8
4
CIP
H20
MIC
S.aureus ATCC 25923
14
11
10
-
-
-
-
-
15
-
128
S. aureus
18
17
-
-
-
-
-
-
15
-
256
S.aureus(MRSA)
15
12
-
-
-
-
-
-
-
-
256
E.coli ATCC 25922
11
8
-
-
-
-
-
-
30
-
256
E.coli (ESBL)
12
10
-
-
-
-
-
-
29
-
256
Shigella flexneri
11
8
-
-
-
-
-
-
-
-
256
E.feacalis
12
10
-
-
-
-
-
-
-
-
256
Klebsiella pneumonia
15
10
-
-
-
-
-
-
-
-
256
Pseudomonas
aeruginosa(MDR)
16
8
-
-
-
-
-
-
-
-
256
Candida albicans
10
-
-
-
-
-
-
-
-
-
512
Key
H2O = Water
MRSA = Methicillin resistant Staphylococcus aureus
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
33
- = No activity
CIP = Ciprofloxacin control
MIC = Minimum Inhibitory Concentration
ALE = Aqueous leave extract.
MDR = Multi drug resistance
Table 2 Zone of inhibition (mm) of organisms to aqueous fruit extract (afe) of chrysophyllum albidum
TEST ORGANISMS
CONCENTRATION (µg/ml)
512
256
128
64
32
16
8
4
CIP
H20
MIC
S.aureus ATCC 25923
16
14
-
-
-
-
-
-
16
-
256
S. aureus
31
24
20
11
-
-
-
-
24
-
64
S.aureus(MRSA)
30
23
16
14
-
-
-
-
15
-
64
E.coli ATCC 25922
-
-
-
-
-
-
-
-
28
-
-
E.coli (ESBL)
-
-
-
-
-
-
-
-
20
-
-
Shigella flexneri
-
-
-
-
-
-
-
-
-
-
-
E.feacalis
-
-
-
-
-
-
-
-
-
-
-
Klebsiella pneumonia
16
10
-
-
-
-
-
-
-
-
256
Pseudomonas
aeruginosa(MDR)
15
13
11
10
-
-
-
-
-
-
64
Candida albicans
14
13
8
-
-
-
-
-
-
-
128
Key
H2O = Water
MRSA = Methicillin resistant Staphylococcus aureus
- = No activity
CIP = Ciprofloxacin control
MIC = Minimum Inhibitory Concentration
AFE = Aqueous fruit extract
MDR = Multi drug resistance
3. Results
The results in Table I showed that all the tested
organisms were inhibited by the aqueous leave
extracts of Chrysophyllum albidum with Minimum
Inhibitory Concentration (MIC) ranging between of
128- 512µg/ml. S. aureus ATCC 25923 had the
lowest MIC.
Ciprofloxacin was used as control antibiotics
for all tests carried out as well as the solvents used
for extraction.
The aqueous fruit extract inhibited six
organisms with MIC range between 64-256µg/ml
(Table2 and Plate 3).
Escherichia coli ATCC 25922, Shigella
flexneri, ESBL Escherichia coli and Enterococcus
faecalis were resistant to the aqueous fruit extract of
Chrysophyllum albidum. Methicillin Resistant
Staphylococcus aureus, Staphylococcus aureus and
Pseudomonas aeruginosa had the lowest MIC of
64µg/ml respectively (Table 2)
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
34
The aqueous seed extract of Chrysophyllum
albidum showed inhibition on all the organisms
tested except Pseudomonas aeruginosa and
Enterococcus faecalis which were resistant. The
MIC range is between128-512µg/ml. Methicillin
Resistant Staphylococcus aureus had the lowest
MIC of 128µg/ml (Table 3).
Plate 3 Showing the zone of inhibition of Methicillin Resistant Staphylococcus
aureus to the aqueous leave, fruit and seed extracts of Chrysophyllum albidum
Table 3 Zone of inhibition (mm) of organisms to aqueous seed extract (ase) of chrysophyllum albidum
TEST ORGANISMS
CONCENTRATION (µg/ml)
512
256
128
64
32
16
8
4
CIP
H20
MIC
S.aureus ATCC 25923
18
13
-
-
-
-
-
-
18
-
256
S. aureus
16
15
-
-
-
-
-
-
24
-
256
S.aureus(MRSA)
22
12
11
-
-
-
-
-
14
-
128
E.coli ATCC 25922
14
-
-
-
-
-
-
-
40
-
512
E.coli (ESBL)
6
-
-
-
-
-
-
-
15
-
512
Shigella flexneri
15
10
-
-
-
-
-
-
35
-
256
E.feacalis
-
-
-
-
-
-
-
-
-
-
-
Klebsiella pneumonia
13
-
-
-
-
-
-
-
-
-
512
Pseudomonas
aeruginosa(MDR)
-
-
-
-
-
-
-
-
-
-
-
Candida albicans
16
12
-
-
-
-
-
-
-
-
256
Key
H2O = Water
MRSA = Methicillin resistant Staphylococcus aureus
- = No activity
CIP = Ciprofloxacin control
MIC = Minimum Inhibitory Concentration
ASE = Aqueous seed extract
MDR = Multi drug resistance
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
35
The results in Table 4 showed that
Pseudomonas aeruginosa had (MIC) of 64µg/ml
with the Seaman's Schnapps leaf extract (Plate 4).
Shigella flexneri was sensitive with MIC of
256µg/ml while other organisms were resistant to
the Seaman's Schnapps leaf extract (Table 4).
The Seaman's Schnapps fruit extract exhibited
antimicrobial activity against all the organisms. The
MIC range is between 32-512µg/ml. S.aureus
(MRSA), E.coli (ESBL), Pseudomonas aeruginosa
(MDR) and Candida albicans had MIC of 64µg/ml
respectively. The control strains S.aureus ATCC
25923, S.aureus, and E.faecalis had MIC of
512µg/ml. The lowest MIC was exhibited by
Klebsiella pneumonia with MIC of 32µg/ml (Table
5).
The methanolic leaf extract of Chrysophyllum
albidum exhibited antimicrobial activities against
S.aureus (MRSA), Pseudomonas aeruginosa and
Shigella flexneri whichhad MIC of 512µg/ml
respectively (Table 6).
Table 4 Zones of inhibition (mm) of organisms to seaman's schnapps leaf extract (ssle) of chrysophyllum
albidum
Key
SS = Seaman's Schnapps
MRSA = Methicillin resistant Staphylococcus aureus
- = NO activity
CIP = Ciprofloxacin control
MIC = Minimum Inhibitory Concentration
MDR = Multi drug resistance
TEST ORGANISMS
CONCENTRATION (µg/ml)
512
256
128
64
32
16
8
4
CIP
SS
MIC
S.aureus ATCC 25923
-
-
-
-
-
-
-
-
20
-
--
S. aureus
-
-
-
-
-
-
-
-
17
-
-
S.aureus (MRSA)
-
-
-
-
-
-
-
-
-
-
-
E.coli ATCC 25922
-
-
-
-
-
-
-
-
31
-
-
E.coli (ESBL)
-
-
-
-
-
-
-
-
-
-
-
Shigella flexneri
12
10
-
-
-
-
-
-
35
-
256
E.feacalis
-
-
-
-
-
-
-
-
8
-
-
Klebsiella pneumonia
-
-
-
-
-
-
-
-
-
-
-
Pseudomonas
aeruginosa(MDR)
15
13
16
15
-
-
-
-
20
-
64
Candida albicans
-
-
-
-
-
-
-
-
-
-
-
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
36
Plate 4 Showing the zone of inhibition of Pseudomonas aeruginosa to
the Seaman's schnapps leave extracts of Chrysophyllum albidum
Table 5 Zones of inhibition (mm) 0f organisms to seaman's schnapps fruit extract (ssfe) of chrysophyllum
albidum
TEST ORGANISMS
CONCENTRATION (µg/ml)
512
256
128
64
32
16
8
4
CIP
SS
MIC
S.aureus ATCC 25923
15
-
-
-
-
-
-
-
24
-
512
S. aureus
16
-
-
-
-
-
-
-
-
-
512
S.aureus(MRSA)
15
14
12
10
-
-
-
-
-
-
64
E.coli ATCC 25922
14
12
11
-
-
-
-
-
23
7
128
E.coli (ESBL)
14
13
12
10
-
-
-
-
36
-
64
Shigella flexneri
16
9
7
-
-
-
-
-
34
-
128
E.feacalis
15
-
-
-
-
-
-
-
23
-
512
Klebsiella pneumonia
15
10
9
7
6
-
-
-
-
32
Pseudomonas
aeruginosa(MDR)
17
16
14
11
-
-
-
-
32
-
64
Candida albicans
13
12
10
9
-
-
-
-
-
-
64
Key
SS = seaman's schnapps
MRSA = methicillin resistant Staphylococcus aureus
- = NO activity
CIP = Ciprofloxacin control
MIC = Minimum Inhibitory Concentration
MDR = Multi drug resistance
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
37
The methanolic fruit extract exhibited
antimicrobial activity against all the tested
organisms with MIC range between 16-256µg/ml of
C.albidum.
Staphylococcus aureus had the lowest
minimum inhibitory concentration of 16mg/ml.
S.aureus (MRSA) and Shigella flexneri both had
MIC of 32µg/ml. E.coli, E.faecalis,
K.pneumoniaewere susceptible at MIC of 64µg/ml
respectively. E.coli ATCC 25922 and Pseudomonas
aeruginosa both showed MIC of 128µg/ml
respectively. Staphylococcus aureus ATCC 25923
and Candida albicans showed MIC of 256µg/ml
(Table7).
Enterococcus faecalis with MIC of 8µg/ml
showed high sensitivity to the seed oil extract
followed by Shigella flexneri with MIC of 64µg/ml.
Other organisms were resistant to the seed oil
extract. (Table 8)
Table 6 Zones of inhibition (mm) 0f organisms to methanolic leaf extract (mle) of chrysophyllum albidum
TEST ORGANISMS
CONCENTRATION (µg/ml)
512
256
128
64
32
16
8
4
CIP
METH
MIC
S.aureus ATCC 25923
-
-
-
-
-
-
-
-
-
-
-
S. aureus
-
-
-
-
-
-
-
-
20
-
-
S.aureus(MRSA)
6
-
-
-
-
-
-
-
-
-
512
E.coli ATCC 25922
-
-
-
-
-
-
-
-
14
-
-
E.coli (ESBL)
-
-
-
-
-
-
-
-
35
-
-
Shigella flexneri
8
-
-
-
-
-
-
-
17
-
512
E.feacalis
-
-
-
-
-
-
-
-
18
-
-
Klebsiella pneumonia
-
-
-
-
-
-
-
-
-
-
-
Pseudomonas
aeruginosa(MDR)
10
-
-
-
-
-
-
-
25
6
512
Candida albicans
-
-
-
-
-
-
-
-
-
-
-
Key
METH. = Methanol
MRSA = Methicillin resistant staphylococcus aureus
- = No activity
CIP = Ciprofloxacin control
MIC = Minimum Inhibitory Concentration
MDR = Multi drug resistance
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
38
Table 7 Zones of inhibition (mm) 0f organisms to methanolic fruit extract (mfe) of chrysophyllum albidum
TEST ORGANISMS
CONCENTRATION (µg/ml)
512
256
128
64
32
16
8
4
CIP
METH.
MIC
S.aureus ATCC 25923
16
13
-
-
-
-
-
-
27
-
256
S. aureus
22
20
16
10
8
7
-
-
-
10
16
S.aureus(MRSA)
21
16
15
10
9
-
-
-
-
-
32
E.coli ATCC 25922
17
14
10
-
-
-
-
-
37
-
128
E.coli (ESBL)
19
17
10
7
-
-
-
-
32
-
64
Shigella flexneri
20
15
13
12
11
-
-
-
33
-
32
E.feacalis
16
11
10
8
-
-
-
-
20
-
64
Klebsiella pneumonia
15
14
11
10
-
-
-
-
-
-
64
Pseudomonas aeruginosa(MDR)
14
13
8
-
-
-
-
-
25
7
128
Candida albicans
12
10
-
-
-
-
-
-
-
-
256
Key
METH.= Methanol
MRSA = Methicillin resistant Staphylococcus aureus
- = No activity
CIP = Ciprofloxacin control
MIC = Minimum Inhibitory Concentration
MDR = Multi drug resistance
Table 8 Zones of inhibition (mm) 0f organisms to petroleum ether seed extract (pse) of chrysophyllum albidum
TEST ORGANISMS
CONCENTRATION (µg/ml)
512
256
128
64
32
16
8
4
CIP
PET
MIC
S.aureus ATCC 25923
-
-
-
-
-
-
-
-
27
-
-
S. aureus
-
-
-
-
-
-
-
-
-
-
-
S.aureus(MRSA)
-
-
-
-
-
-
-
-
-
-
-
E.coli ATCC 25922
-
-
-
-
-
-
-
-
38
-
-
E.coli (ESBL)
-
-
-
-
-
-
-
-
-
16
-
Shigella flexneri
17
15
12
10
-
-
-
-
33
-
64
E.feacalis
25
24
23
20
18
17
15
-
20
-
8
Klebsiella pneumonia
-
-
-
-
-
-
-
-
-
-
-
Pseudomonas aeruginosa(MDR)
-
-
-
-
-
-
-
-
37
-
-
Candida albicans
-
-
-
-
-
-
-
-
-
-
-
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
39
Key
PET. = Petroleum ether.
MRSA = Methicillin resistant Staphylococcus aureus
- = No activity
CIP = Ciprofloxacin control
MIC = Minimum Inhibitory Concentration
MDR = Multi drug resistance
4. Discussion
The organisms used included Shigella flexneri,
Klebsiella pneumoniae (ESBL), Pseudomonas
aeruginosa (MDR- multi drug resistant),
Escherichia coli, Staphylococcus aureus,
Enterococcus faecalis, Methicilin Resistant
Staphylococcus aureus, Escherichia coli(ATCC
25922), Staphylococcus aureus(ATCC 25923) and
Candida albicans and these gave a wider range of
comparison of antimicrobial properties among
several bacteria unlike other medicinal plants
studies where only four to six organisms were used
[20, 22].
A research reported that water and methanolic
extracts of Chrysophyllum albidum leaves used
against Staphylococcus aureus, Escherichia coli,
Salmonella typhi, and Shigella species suggested
that water and methanolic extracts from the leaves
may contain active agent(s), which make them
susceptible to the tested organisms [20]. Another
study reported also that ethanolic extract of
Chrysophyllum albidum leaves and stem used
against Pseudomonas aeruginosa, Staphylococcus
aureus, Escherichia coli, Salmonella typhi,
Klebsiella species, Bacillus subtilis, and Candida
albicans were susceptible to plant ethanolic extract
[21] [7].
Another research also reported that the
aqueous extracts of the leave, stem, root and seed of
Chrysophyllum albidum was used against
Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa Bacillus subtilis,
Clostridium tetani and Candida albicans. It was
reported that Chrysophyllum albidum stem and root
extracts inhibited Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa Bacillus
subtilis, Clostridium tetani and Candida albicans [22].
The seed cotyledon only showed inhibition on
Candida albicans, while the leaves did not inhibit
any of these pathogens. Antimicrobial resistance is
resistance of a microorganism to an antimicrobial
drug to which it was originally sensitive. Resistant
organisms (bacteria, fungi, viruses and some
parasites) are able to stop the antimicrobial
medicines from working, such as antibiotics,
antifungals, antivirals and anti-malarials, so that
standard treatments become ineffective and
infections persists thereby increasing risk of spread
to others [23]. It is the ability of a microorganism to
withstand the effects of an antibiotic [24].
Of note is the activity of Chrysophyllum
albidum aqueous leave extract which inhibited all
multi drug resistant organisms which was similar to
a report by [25], who reported that the lime oil (neat)
showed a high antimicrobial activity against all
organisms tested which include Pseudomonas
aeruginosa, Staphylococcus aureus, Escherichia coli
and Candida albicans.
The aqueous seed extract of Chrysophyllum
albidum inhibited all the organisms tested except
Enterococcus faecalis and Pseudomonas aeruginosa
which were resistant. Methicillin Resistant
Staphylococcus aureus had the lowest MIC of
128µg/ml.
The aqueous fruit extract of Chrysophyllum
albidum did not inhibit Escherichia coli ATCC
25922, ESBL Escherichia coli, Shigella flexneri and
Enterococcus faecalis.
However, Staphylococcus aureus,
Pseudomonas aeruginosa and Methicillin Resistant
Staphylococcus aureus had MIC of 64 µg/ml
respectively. This is similar to the study by [30], in
which it was reported that the aqueous extract of
lemon grass showed inhibition to Escherichia coli
ATCC 25922, Shigella flexneri, Escherichia
coli ,and Salmonella paratyphi.
Notable in this study also was activity of
Seamans Schnapps leaves extract on Shigella
flexneri and Pseudomonas aeruginosa (MDR)
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
40
which had MIC of 256µg/ml and 64µg/ml
respectively showing the possibility of the extract as
an agent that can be used in treating infections
caused by Pseudomonas aeruginosa. This is similar
to the report by [21] where Pseudomonas aeruginosa,
S.aureus, E.coli, and Klebsiella species were
susceptible to the ethanolic leaf extract of
C.albidum. Unlike the work by [22], who reported
that the ethanolic leaf extract showed no sensitivity
to Pseudomonas aeruginosa. This may be due to the
time of harvest of plants and age of plants [25].
Methicilin Resistant Staphylococcus aureus
(MRSA), Shigella flexneri and Pseudomonas
aeruginosa were the only organisms susceptible to
the methanolic leaf extract. This supports the report
of [20] that S.aureus, E.coli, S.typhi and Shigella
species were susceptible to aqueous extract and
methanolic leaf extract of Chrysophyllum albidum
unlike the work by [26] on Albrus precatorius
methanolic extract that showed no inhibition to
Pseudomonas aeruginosa. This may be as a result of
the difference in concentration and solvent for the
extracts or time of collection or age of plant
according to [31]. The minimum inhibitory
concentration (MIC) of Chrysophyllum albidum
Seaman's Schnapps fruit extracts varied with respect
to the test organisms. At 32µg/ml the growth of
Klebsiella Pneumoniae was inhibited. At 64µg/ml
concentration, S.aureus (MRSA), E.coli (ESBL),
Pseudomonas aeruginosa and Candida albicans
were inhibited. At 512µg/ml, S.aureus (ATCC
25923), S.aureus, and Enterococcus feacalis were
inhibited. Staphylococcus aureus was inhibited by
the methanolic fruit extract which is similar to the
work done by [26] on methanolic extract of Abrus
precatorius (L) fabaceae in which it was reported
that Staphylococcus aureus was the most sensitive
organism. [22] reported that only Candida albican
showed inhibition to the aqueous seed extract of C.
albidum, however, this study showed inhibition of
S.aureus, S.aureus (MRSA) and S.aureus ATCC
25923, E.coli, ESBL E.coli, Shigella flexneri,
Klebsiella pneumonia and Candida albicans to the
seed extract of Chrysophyllum albidum. This can be
due to the age of leaves, time of harvesting and
handling after harvesting [32]. Petroleum ether seed
extract of Chrysophyllum albidum did not inhibit
S.aureus, S.aureus (MRSA) and S.aureus ATCC
25923, although,it inhibited Shigella flexneri and
Enterococcus faecalis showing the oil of C. albidum
has antimicrobial properties on some organisms.
However, this is in contrast to [26], where they
reported that the seed oil extract of Abrus
precatorius (L) Fabaceae was the most potent and
had greater zones of inhibition against most of the
Staphylococcus species used. The aqueous leaves
extract in comparison to the aqueous fruit extract
showed that the aqueous fruit extract had more
antimicrobial potency than the leaves
extract .Though the aqueous leaves extract inhibited
all the tested organisms with MIC range between
128-512µg/ml, aqueous fruit extract with MIC
range between 64-256µg/ml, was more potent in
antimicrobial activities. This probably was due to
time of harvesting and the use since plants from
vegetative parts are less effective in therapy [33].
Aqueous leaves inhibited all the tested
organisms when compared with Seaman's Schnapps
leaves extract which inhibited only Pseudomonas
aeruginosa and Shigella flexneri which may be due
to the solvents inability to extract the necessary
ingredients or due to drug interaction [33]. Aqueous
leaves extract of Chrysophyllum albidum inhibited
all the tested organisms and has more active
antimicrobial activities than Methanolic leaves
extract which inhibited MRSA, Shigella flexneri
and Pseudomonas aeruginosa different from the
study by [26] who reported that methanolic extract of
Abrus precatorius(L) Fabaceae was more potent
than Aqueous leave extract.
It is interesting to note that the Aqueous
leaves extract and the Methanolic fruit extract both
inhibited all the tested organisms, however,
Methanolic fruit extract exhibited more
antimicrobial potency against the tested organisms
similar to the study by [26].The methanolic leave and
Petroleum ether seed extract of Chrysophyllum
albidum has no antibacterial effect on
Staphylococcus aureus and Escherichia coli which
was in contrast to the methanolic seed extract of
Daucus carota which showed antibacterial effect on
the organisms as reported by [34].The aqueous seed
extract of Chrysophyllum albidum inhibited
S.aureus, MRSA, E.coli, Shigella flexneri,
Klebsiella pneumonia and Candida albicans which
were similar to the studies by [35] who reported that
the ethanolic and methanolic seed extracts of
Mangifera indica L (Mango) inhibited these
Am. J. Biomed. Sci. 2018,10(1), 28-44; doi:10.5099/aj180100028 © 2018 by NWPII. All rights reserved
41
organisms except Klebsiella pneumonia which was
resistant.
The aqueous leave extract of Chrysophyllum
albidum had antibacterial effect on S.aureus, E.coli,
Shigella flexneri and Pseudomonas aeruginosa
unlike studies by [36] in which it was reported that
the crude leaf extract of Mangifera indica Linn had
no activity on these bacteria.
The methanolic crude extract of the fruit and
leaves of Momordica charantia (Bitter melon)
inhibited S.aureus, E.coli, Pseudomonas aeruginosa,
Klebsiella pneumoniae and Candida albicans as
reported by [37], which is similar to the result
obtained with methanolic fruit extract of
Chrysophyllum albidum in this study.[38] reported
that the aqueous extracts of Carrot, Amla and
Pomegranate inhibited S.aureus, E.coli,
Pseudomonas aeruginosa and Shigella flexneri
which is similar to the result obtained with the
aqueous leave extract of Chrysophyllum albidum in
this study.
The result was different with the aqueous
extract of Honey which inhibited Escherichi coli
and Shigella flexneri. Aloe vera extract inhibited
Escherichia coli, Pseudomonas aeruginosa and
Shigella flexneri [38].
The methanolic fruit extract of Chrysophyllum
albidum inhibited all the organisms used in this
study which was similar to the study by [39], who
reported that the methanolic extract of
Ocimumsanctum, Azadirachita indica and
Phyllanthus amarus inhibited Staphylococcus
aureus and Pseudomonas aeruginosa.
The lowest Minimum Inhibitory Concentration
of 8µg/ml was exhibited by the petroleum seed
extract against Enterococcus faecalis, which was
closely followed by methanolic fruit extract against
Staphylococcus aureus while the Seaman's
Schinapps fruit extract which had the lowest MIC of
32µg/ml against Klebsiella pneumoniae which was
similar to the study by [40], who reported that all the
organisms used including Klebsiella pneumonia
were susceptible to the ethanolic extract of Unripe
banana(Musa sapientum L).
The aqueous leaves extract shows more
antimicrobial activities than the Seaman's Schinapps
and methanolic leaves extracts. However,
methanolic fruit extract was the best for the
extraction of the active ingredients in
Chrysophyllum albidum because it inhibited all the
tested organisms.
Methicillin Resistant Staphylococcus aureus is
known to be of clinical importance due to its
resistance pattern. The aqueous and methanolic
leave extracts of Chrysophyllum albidum inhibited
MRSA which was significant, except Seaman's
Schnapps leaves and Petroleum ether seed extracts
which showed resistant to the organism. A study by
[26] showed that methanolic extract of Abrus
precatorius (L) Fabaceae was more potent than
aqueous leave extract.
Multi-drug resistance in Gram negative
organisms is becoming a global threat and most
available drugs are not active [9]. Chrysophyllum
albidum had activity on these organisms and unlike
studies by [36], which had no activity on bacteria
isolates.
5. Conclusion
This study revealed that Chyrsophyllum
albidum leaves, fruits and seed extracts are active
against Multidrug Resistant (MDR) organisms.
Aqueous extract of Chrysophyllum albidum possess
antimicrobial properties. Chyrsophyllum albidum is
therefore recommended to be developed into new
antimicrobials in readily available forms for use in
Medical Microbiology Laboratory.
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