The Journal of Phytopharmacology 2014; 3(3): 193-199

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Research Article Systemic evaluation of antibacterial activity of

ISSN 2230-480X
JPHYTO 2014; 3(3): 193-199
Anacardium occidentale
May- June
© 2014, All rights reserved Belonwu D.C., Ibegbulem C.O. Chikezie P.C.*

Abstract

Background: Evaluation of antibacterial activity of different parts of two varieties of

Belonwu D.C.
Department of Biochemistry, Anacardium occidentale L. tree {red fruited variety (RFV) and yellow fruited variety (YFV)},
University of Port Harcourt, Port namely, the fruit, leaf, stem bark and root extracts on human pathogens (Staphylococcus aureus,
Harcourt, Nigeria Eschericjia coli, Pseudomonas aeroginosa, Salmonella typhi, Proteus mirabilis and Klebsella
Ibegbulem C.O. spp) was carried out in vitro. Materials and Methods: Phytochemical screening and
Department of Biochemistry, preparation of the extracts was by standard methods. Antibacterial activity was measured by the
Federal University of Technology,
agar diffusion methods, which ascertained the diameter of inhibition halos around wells after 24
Owerri, Nigeria
h incubation at 37°C. Results: Alkaloids, saponins, flavonoids, tannins, HCN, phenols and
Chikezie P.C. anthocyanin were present in leaf and stem bark extracts of the RFV and YFV. HCN was absent
Department of Biochemistry, Imo
State University, Owerri, Imo State,
in the fruit and root of both varieties. Aqueous fruit extract of the RFV did not exhibit
Nigeria antibacterial effect on P. aeroginosa, S. typhi, P. mirabilis and Klebsella spp. Likewise,
ethanolic fruit extract of the RFV did not inhibit bacterial activity of S. typhi and P. mirabilis,
whereas S. aureus, E. coli, P. aeroginosa and Klebsella spp were inhibited by ethanolic red fruit
extract in the following corresponding order: E. coli = 65.11% > P. aeroginosa = 64.30% > S.
aureus = 53.01% > Klebsella spp = 46.76%. The zone of inhibition (ZOI) of aqueous leaf
extract of the RFV was between 10.50 ± 0.05 mm and 14.50 ± 0.01 mm halos. Generally,
antibacterial activities of aqueous and ethanolic stem bark, leaf and root extracts of the RFV and
YFV were identical. Conclusion: The results offered precise reference information on
comparative antibacterial activities of various parts of A. occidentale L. tree for possible
optimum exploitation and usage.

Keywords: Anacardium occidentale, Antibacterial activity, Human pathogens,

Phytochemicals, Zone of inhibition.

Introduction
Infectious diseases caused by pathogens such as bacteria, fungi and viruses are major
challenges to public health, in that microbial activity promotes morbidity and mortality
of the human host. Staphylococcus aureus is the causative organism of skin infections,
pneumonia, food poisoning, toxic shock syndrome and bacteriemia. Eschericjia coli are
of several types and haboured in the digestive tracts of human and animals. Although
Correspondence: most E. coli are harmless microbes, some can cause enterohemorrhage, urinary tract
Dr. Paul Chidoka Chikezie infections and anemia. Pseudomonas aeroginosa infection is complicated and recently,
Department of Biochemistry, Imo
State University, PMB 2000, reports showed the wide spread of resistant strains to antibiotics 1,2. Salmonella typhi
Owerri, Imo State, Nigeria and Proteus mirabilis belong to the family of Enterobacteriaceae; S. typhi is the
Tel: +2348038935327 causative organism of typhoid fever. P. mirabilis causes 90% of Proteus infections and
E-mail: p_chikezie@yahoo.com

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can be considered a community-acquired infection, typhi, Proteus mirabilis and Klebsella spp) was carried in
especially in persons with underlying diseases or vitro.
compromised immune systems. Patients with recurrent
infection have increased frequency of diseases caused by Materials and Methods
Klebsella spp and other microbes. For instance, Klebsiella
pneumonia accounts for a significant proportion of Collection and preparation of plant extract
hospital-acquired urinary tract infections, pneumonia,
septicemias and soft tissue infections.3 Different plant parts, namely, fruit, leaf, stem bark and root
were harvested, between the months of February and
The cashew (Anacardium occidentale L.) tree grows up to March, 2012, from two varieties of A. occidentale L. trees
10 m or above in height and has large stripe, oval shaped {red fruited variety (RFV) and yellow fruited variety
leaves, fragrant red or yellow bulbous fruit when ripe, (YFV)} growing in the wild along Uturu/Okigwe Express
called the cashew apple. Bioactive principles derivable Road, Okigwe, Imo State, Nigeria. The specimens were
from cashew nut kernels have been previously reported by authenticated by Dr. F.N. Mbagwu at the Herbarium of the
Trox J et al.4 Evidence of worldwide ethnomedicinal Department of Plant Science and Biotechnology, Imo State
practices showed that extracts of different parts of A. University, Owerri, Nigeria. Voucher specimens were
occidentale L tree have therapeutic potentials for the deposited at the Herbarium for reference purposes. The
treatment of malaria, bronchitis, dyspepsia, eczema, separate plant parts were washed under a continuous flow
psoriasis, syphilis, urinary insufficiency and nasal of tap water to remove dust/debris and air-dried at room
congestion.5 In addition, Chikezie6, mentioned other temperature for 5 h. The specimens were cut into bits and
multipurpose medicinal values of A. occidentale L tree. further dried at 60°C in an oven (WTC BINDER, 7200
Tuttlingen, Germany) for 3 days 18 till sufficiently devoid
The use of herbs and medicinal plants for the alleviation of of moisture and subsequent ground in Thomas-Willey
pathologic conditions is a universal phenomenon. milling machine. Preparation of separate ethanolic and
Medicinal plants contain substances known to modern and aqueous extracts of the various plant parts was, according
ancient civilization for their therapeutic potentials. Plants, to the methods of Ojiako et al.19 Percentage yield (%Y)
because of their biodiversity, are unlimited veritable was calculated based on the ratio of weight of extract to
sources of a variety of bioactive principles with medicinal that of the specimen (%; w/w). Stock solutions of the
and therapeutic values. Notwithstanding the advent and various botanical extracts were prepared by reconstituting
advances in synthetic organic chemistry at the dawn of the the separate extracts in 20 mL of 10% dimethylsulfoxide
19th century, modern pharmaceutics continue to source (DMSO, Merck). The solutions were allowed to stand in a
therapeutic principles from plant materials. More so, 80% thermostatically controlled water bath at 37°C for 30 min
of Africans as well as 60% of the world population rely on with thorough shaking. The final concentration of the
plant-derived medicines.7,8 extracts = 400 mg% in DMSO w/v; were prepared and
used for sensitivity test.
There are increasing incidences of infectious agents
becoming resistant to orthodox antibacterial drugs1,2,9,10 and Phytochemical screening
therefore, it has become exigent to develop alternative
antibacterial drugs in order to mitigate these and other Phytochemical screening was carried out in the presence of
related health care challenges. The increasing quest for alkaloids, saponins, flavonoids, tannins, HCN, phenols and
antibacterial agents has shifted to plant materials and anthocyanin using standard methods.20.
studies have shown that phytomedicine is reliable,
sustainable and a rewarding prospect in this regard.11-17 In Pathogenic microorganisms
the present study, evaluation of antibacterial activity of
different parts of two varieties of Anacardium occidentale Clinical isolates of known human pathogens (S. aureus
L. tree {red fruited variety (RFV) and yellow fruited AX456, E. coli CG673, P. aeroginosa HJ731, S. typhi
variety (YFV)}, namely, the fruit, leaf, stem bark and root MV675, P. mirabilis BR095 and Klebsella spp FA412)
extracts on human pathogens (Staphylococcus aureus, were obtained from National Root Crop Research Institute,
Eschericjia coli, Pseudomonas aeroginosa, Salmonella Umudike, Abia State, Nigeria. The pathogens were
examined for purity by streaking in nutrient agar plates.

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Stock cultures were prepared in Bijioux bottles at Aqueous fruit extracts of the RFV did not exhibit
controlled temperature of 37°C. The microorganisms were antibacterial effects on P. aeroginosa, S. typhi, P. mirabilis
finally maintained in nutrient agar at refrigerated and Klebsella spp, which were contrary to S. aureus and E.
temperature of 4°C until the tests were carried out. coli, representing 47.08% and 47.98% inhibition
respectively (Table 3). Likewise, ethanolic fruit extract of
Antibacterial test the RFV did not inhibit bacterial activity of S. typhi and P.
mirabilis, whereas S. aureus, E. coli, P. aeroginosa and
The agar diffusion test according to the methods of Klebsella spp were inhibited by ethanolic red fruit extract
Oliveira et al.,17 was used to ascertain the antibacterial in the following corresponding order: E. coli = 65.11% >
activity of the various plant extracts. A 10 µL of each P. aeroginosa = 64.30% > S. aureus = 53.01% > Klebsella
cultured microorganism in 1×10-3 UFC/mL was plated in spp = 46.76%. Table 3 showed that both aqueous and
Muller-Hinton agar (MHA, Sigma), which was perforated ethanolic leaf, stem bark and root extract of the RFV
yielding 1 central well surrounded by 10 wells. In the exhibited antibacterial effects on the six experimental
central well, which served for the positive control, 30 µL human pathogens (SEHP). The zone of inhibition (ZOI) of
of ciprofloxacin in DMSO = 16.6 µg/mL was added, aqueous leaf extract of the RFV was between 10.50 ± 0.05
whereas 30 µL of each plant extract was introduced in the mm and 14.50 ± 0.01 mm halos. ZOI of ethanolic leaf
surrounding wells. The 10th well contained 10 μL of extract of the RFV was within the range of 12.00 ± 0.02—
DMSO, which served as negative control. Antibacterial 14.53 ± 0.01 mm halos. A cursory look at Table 3 showed
activity was ascertained by a measure of the diameter of that ethanolic stem bark extract of the RFV showed greater
inhibition halos around the wells after 24 h incubation at capacity to inhibit bacterial growth of the SEHP than the
37°C, which indicated absence of bacterial growth. corresponding aqueous extract. Specifically, the lowest
ZOI was observed when aqueous stem bark extracts of the
Results RFV was incubated with S. aureus. Ethanolic root extract
of the RFV exhibited comparative greater capacity to
A cursory look at Table 1 showed that ethanolic extracts of inhibit the SEHP than the corresponding aqueous extract.
the various specimens gave greater %Y than the
corresponding aqueous extracts. The %Ys of aqueous and Table 4 showed the absence of halos in culture plates of P.
ethanolic extracts of both varieties were within the range aeroginosa, S. typhi, P. mirabilis and Klebsella spp
of: RFVAQ = 2.65—5.89%; RFVETH = 3.04—6.93%; incubated with aqueous fruit extracts of the YFV.
YFVAQ = 2.55—5.66%; YFVETH = 3.12—6.64%. However, antibacterial activity of an aqueous fruit extract
Specifically, in relation to the two extracting solvents, of the YFV incubated with S. aureus and E. coli
aqueous fruit extract of the YFV and ethanolic fruit extract represented 44.20% and 47.98% inhibition compared to
of the RFV gave the lowest %Ys. The highest %Ys of the the positive control (Table 5) respectively. Tables 3 and 4
two extracts were obtained from aqueous and ethanolic showed that aqueous and ethanolic leaf extracts of the
stem bark extracts of the RFV. RFV and YFV as well as ethanolic stem bark extracts of
the RFV and YFV exhibited relatively greater potency in
Phytochemical screening of the samples showed the inhibiting bacterial activity of P. aeroginosa compared to
presence of alkaloids, saponins, flavonoids, tannins, HCN, the standard antibacterial drug-ciprofloxacin (positive
phenols and anthocyanin in the leaf and stem bark extracts control). Likewise, antibacterial activity of aqueous and
of the RFV and YFV. HCN was absent in fruit and root of ethanolic leaf extracts of the RFV and YFV on S. typhi
both varieties. Furthermore, the phytochemical profile were higher than that of the standard antibacterial drug.
revealed that the RFV and YFV were identified (Table 2). For instance, antibacterial activity of ethanolic leaf extract
of the YFV was higher than the standard antibacterial drug
by 12.45%.

Table 1: Percentage yields of aqueous and ethanolic extracts of A. occidentale L.

%Y; w/w ratio
Fruit Leaf Stem Bark Root
RFV YFV RFV YFV RFV YFV RFV YFV
AQ 2.65 2.55 3.98 3.55 5.89 5.45 5.45 5.66
ETH 3.04 3.12 4.22 4.01 6.93 6.45 6.41 6.64
AQ: aqueous extract; ETH: ethanolic extract.

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Table 2: Phytochemicals of red and yellow fruited varieties of A. occidentale L.

Sample Phytochemicals
RFV Alkaloids Saponins Flavonoids Tannins HCN Phenols Anthocyanin
Fruit + + + + - + +
Leaf + + + + + + +
Stem Bark + + + + + + +
Root + + + + - + +
YFV
Fruit + + + + - + +
Leaf + + + + + + +
Stem Bark + + + + + + +
Root + + + + - + +
+: present; -: absent.

Table 3: Halo measurement of antibacterial activity of extracts of red fruited variety of A. occidentale L.
Inhibition Zone Diameter (mm) (X) ± SEM
Fruit Leaf Stem Bark Root
Pathogens DMSO AQ ETH AQ ETH AQ ETH AQ ETH
SA 0.00±0.00 7.99±0.03 8.89±0.04 10.50±0.05 12.09±0.05 9.50±0.01 12.00±0.00 9.00±0.07 11.00±0.00
EC 0.00±0.00 7.00±0.05 9.50±0.02 12.50±0.01 14.07±0.06 11.50±0.05 14.49±0.00 10.50±0.05 12.50±0.05
PA 0.00±0.00 0.00±0.00 8.50±0.02 14.00±0.12 14.53±0.01 12.00±0.00 13.50±0.00 10.50±0.03 11.00±0.05
ST 0.00±0.00 0.00±0.00 0.00±0.00 14.50±0.01 14.50±0.01 12.00±0.00 12.00±0.01 9.50±0.05 11.50±0.01
PM 0.00±0.00 0.00±0.00 0.00±0.00 11.50±0.01 12.00±0.02 11.00±0.00 10.00±0.11 10.00±0.06 12.33±0.32
KS 0.00±0.00 0.00±0.00 8.00±0.08 12.50±0.32 13.50±0.02 14.00±0.00 14.06±0.02 10.00±0.05 12.50±0.01
Number of determinations (n = 3); SA: S. aureus, EC: E. coli, PA: P. aeroginosa, ST: S. typhi, PM: P. mirabilis, KS: Klebsella spp;
AQ: aqueous extract; ETH: ethanolic extract; DMSO (Dimethylsulphoxide) = negative control.

Table 4: Halo measurements of antibacterial activity of extracts of yellow fruited variety of A. occidentale L.
Inhibition Zone Diameter (mm) (X) ± SEM
Fruit Leaf Stem Bark Root
Pathogens DMSO AQ ETH AQ ETH AQ ETH AQ ETH
SA 0.00±0.00 7.50±0.02 7.19±0.15 10.50±0.00 12.50±0.02 9.00±0.06 12.00±0.00 8.50±0.05 11.03±0.03
EC 0.00±0.00 7.00±0.05 9.03±0.03 12.00±0.00 13.50±0.02 11.00±0.28 14.00±0.05 10.00±0.07 11.87±0.15
PA 0.00±0.00 0.00±0.00 8.50±0.01 14.50±0.01 14.00±0.05 11.50±0.03 14.00±0.00 9.00±0.01 9.50±0.01
ST 0.00±0.00 0.00±0.00 12.03±0.03 14.00±0.05 14.00±0.02 11.00±0.12 11.50±0.01 9.00±0.05 11.00±0.02
PM 0.00±0.00 0.00±0.00 7.00±0.11 11.00±0.04 10.50±1.05 12.00±0.04 12.00±0.00 9.00±0.03 11.03±0.30
KS 0.00±0.00 0.00±0.00 8.00±0.02 13.00±0.06 12.50±0.02 14.50±0.03 14.00±0.01 9.50±0.02 13.00±0.05
Number of determinations (n = 3); SA: S. aureus, EC: E. coli, PA: P. aeroginosa, ST: S. typhi, PM: P. mirabilis, KS: Klebsella spp,
AQ; aqueous extract; ETH: ethanolic extract; DMSO (Dimethylsulphoxide) = negative control.

Table 5: Halo measurements of positive controls.

Pathogens Inhibition Zone Diameter (mm) (X) ± SEM

SA 16.97 ± 0.04
EC 14.59 ± 0.03
PA 13.22 ± 0.05
ST 12.45 ± 0.11
PM 15.09 ± 0.15
KS 17.11 ± 0.21
Number of determinations (n = 3); SA: S. aureus, EC: E. coli, PA: P. aeroginosa, ST: S. typhi, PM: P. mirabilis, KS: Klebsella spp.

On the contrary, ethanolic fruit extract of the YFV effects on the SEHP. An overview of Tables 3 and 4
inhibited the SEHP; ZOI ranged between 7.00 ± 0.11 mm showed that the capacity of aqueous and ethanolic leaf,
halo and 12.03 ± 0.30 mm halos. In the same characteristic stem bark and root extracts of the RFV and YFV to inhibit
manner as the RFV, both aqueous and ethanolic leaf, stem bacterial growth of the SEHP were identical, except for
bark and root extracts of the YFV exhibited antibacterial

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ethanolic fruit extract of the RFV, which showed no additive, in that the combinatorial actions of
inhibitory effect on S. typhi, and P. mirabilis. phytochemicals are responsible for their therapeutic
benefits or toxicologic outcomes in animals.32, 33 It is also
Discussion important to note that negative interactions (interferences)
do occur in botanic extracts, in which the interplay and
The present study showed the various capacities of the interactions of composite phytochemicals cause
different parts of A. occidentale L. tree, especially the leaf, attenuation or outright inhibition of maximum biologic
stem bark and root to inhibit microbial activity, which activity of supposedly pharmacologic active plants.33, 34
conformed to those reported by previous researchers Thus, mixture of phytochemicals in botanic extracts may
elsewhere.13, 14, 17, 21 Notable plant secondary metabolites be more or less biologically active than individual
that have been adduced to be responsible for antibacterial bioactive components.
activity of botanic extracts are the phenols, flavonoids,
saponins and tannins.22-25 More so, several other reports The capability of ethanolic and water, fruits extracts of the
have also shown that the production of a large variety of RFV and YFV to inhibit the SHEP was in consequence of
antibacterial molecules generally referred to as the nature of the extracting solvents. Studies have shown
phytoalexins (MW < 500) in plants, especially in wild that solubility of antibacterial principles in polar or non-
types, are responsible for their capability to exterminate polar solvent is critical in order to exert biologic activity in
invasive noxious microorganisms, as well as mitigate vitro.35, 36 Accordingly, the present findings showed that
abiotic stressors and traumas.26, 27 Furthermore, natural ethanolic solvent extracts exhibited greater antibacterial
products such as terpenoids, glycosteroids, flavonoids and activity than the corresponding aqueous extracts, which
polyphenols are antibacterials and have been reported to be conformed to previous assessments by several authors.13, 37-
40
contained within the structural space of phytoalexins.28, 29 Thus, the results presented here suggest that by virtue of
As mentioned by Jørgensen et al.30, inhibition of the %Y index in connection to the two extracting solvents,
phytoalexins biosynthesis by cyanide, prompted by the there were greater tendencies that higher concentrations of
presence of cyanogenic glycosides in Hevea braziliensis antibacterial elements were extracted into ethanol than the
(rubber tree), increased its sensitivity to attack by corresponding aqueous environment as exemplified in
Microcyclis ulei, which confirmed the evidence in support Table 1. In corroboration with the present findings, Bashir
of the protective function of phytoalexins. et al.41, had previously noted that organic plant extracts
exhibited greater antibacterial activity than the
The present study showed there were variations in the corresponding aqueous suspension. However, comparative
capacities of extracts of the various plant parts to inhibit inspections of outcomes of the present investigations
bacterial growth of the SEHP in vitro. Furthermore, the revealed that the antibacterial potential of the RFV and
absence of antibacterial activity, particularly the fruit YFV of A. occidentale L. were of equivalent status. On the
extracts of the RFV on S. typhi and P. mirabilis amongst contrary, the research reports by Ababutain24, mentioned
the other SEHP, was an indication that the antibacterial that disparity in antibacterial activity of the same plant
phytochemicals are not evenly distributed and their species may manifest when variation in climatic factors
absolute cellular concentrations may vary within or even and soil composition exist in locations where the plants are
absent in some plant organs that constitute the plant cultivated in conjunction with the stage of vegetative cycle
system. That leaf, stem bark and root extracts of A. of the plants. Also, Cock42 had reported that genetic
occidentale L. exhibited relatively greater potency than the variation within a plant species could cause dissimilarity in
fruit extracts to inhibit the activity of the SEHP, which was their phytochemical composition, and consequently, affect
an affirmation of the above assertions. Similarly, studies their corresponding pharmacological properties.
by Karima et al.31, corroborate the present observations of
disparities in levels of activity of antibacterial Conclusion
phytochemicals in plant systems. They noted that root
extracts of Carthamus caeruleus L. were more active than The present preliminary in vitro findings offered precise
the leaf extracts against certain strains of gram-negative reference information on comparable levels of antibacterial
and gram-positive bacteria as well as pathogenic fungus. activities of various parts of A. occidentale L. tree for
However, previous studies have provided evidence that possible optimum exploitation and usage. However, it’s
bioactivity of phytochemicals in botanic extracts is essential to extend the frontiers of the present scope of

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The Journal of Phytopharmacology May- June

study to basic and clinical research endeavours in order to 10. Hancock RE, Nijnik A, Philpott DJ. Modulating immunity as
achieve the quest for sourcing antibacterial therapeutics a therapy for bacterial infections. Nat Rev Microbiol 2012;
from A. occidentale L. tree. 10:243–254.

11. Iwu MW, Duncan AR, Okunji CO. New antimicrobials of

Acknowledgement
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Uses. ASHS Press, Alexandria, VA. 1999; pp457 – 462.
The authors are grateful for the technical assistance offered
by Mr. O.A.K. Emenyonu, Chief Academic Technologist, 12. Daciana I, Bara II. Plant products as antimicrobial agents.
Department of Biochemistry, Imo State University, Analele Stiinţifice ale Universitaţii Alexandru Ioan Cuza
Owerri. Secţiunea Genetica si Biologie Moleculara. VIII, TOM. 2007.

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