In vitro Anticoccidial, Antioxidant Activities and Cytotoxicity of Psidium guajava Extracts

Full text

OPEN ACCESS Research Journal of Parasitology
ISSN 1816-4943
DOI: 10.3923/jp.2018.1.13
Research Article
In vitro Anticoccidial, Antioxidant Activities and Cytotoxicity of
Psidium guajava Extracts
1Yamssi Cedric, 1Vincent Khan Payne, 1Noumedem Anangmo Christelle Nadia, 2Norbert Kodjio,
1Etung Kollins, 1Leonelle Megwi, 2Jules-Roger Kuiate and 1Mpoame Mbida
1Research Unit of Biology and Applied Ecology, Faculty of Science, University of Dschang, Dschang, Cameroon
2Research Unit of Microbiology and Antimicrobial Substances, Faculty of Science, University of Dschang, Dschang, Cameroon
Abstract
Background and Objective: Coccidiosis remains one of the most important infectious cause of digestive disorders in rabbits.
The aim of this study was to evaluate
in vitro
anticoccidial and antioxidant activities of
Psidium guajava
(
P. guajava
) extracts.
Materials and Methods: Sporulation inhibition bioassay was used to evaluate the activity of
Psidium guajava
extracts on sporulation
of
Eimeria flavescens
,
Eimeria stiedae
,
Eimeria intestinalis
and
Eimeria magna
oocysts and sporozoites. The set up was examined after
24 and 48 h for the oocysticidal activities and after 12 and 24 h for anti-sporozoidal activities. The antioxidant activity was determined
by measuring FRAP (ferric reducing-antioxidant power), 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and nitric oxide
(NO) radical scavenging. The cytotoxicity of the most active extract was determined against animal cell lines fibroblast L929, HEPG2 and
HeLa cells using MTT assay. The impact of the toxicity was established by analyzing the selectivity index (SI) values. Data obtained were
analyzed using one-way analysis of variance (ANOVA) and were determined by Waller-Duncan test using SPSS. Results: The highest
efficacy of tested plant extracts was recorded after 24 h, which varied according to different concentrations of the tested extracts. The
highest efficacy was 88.67±2.52% at the concentration of 30 mg mLG1 of the methanolic extract against
E.
intestinalis.
Most extracts
including the aqueous extract exhibited good anti-sporozoidal activities against
E. flavescens
,
E. stiedae
,
E. intestinalis
and
E. magna
sporozoites at 1000 µg mLG1. The highest viability inhibitory percentage was 97.00±1.73% at a concentration of 1000 µg mLG1 of
P. guajava
methanolic extract against
E. intestinalis
sporozoites. These results also showed that methanolic and ethyl acetate extract,
possessed strong antioxidant activities (IC50<20 µg mLG1). The methanolic extract of
P. guajava
exhibited CC50 of >30 µg mLG1 against
selected cell lines, suggesting that the compounds were not toxic. Phytochemical screening of the most active extract showed presence
of alkaloids, flavonoids, saponins and phenols. Conclusion: These results provide confirmation to the usage of
Psidium guajava
agains t
coccidiosis by Agricultural farmers in Cameroon.
Key words:
Psidium guajava
, anticoccidial activity, antioxidant,
Eimeria
species, Cameroon
Citation: Yamssi Cedric, Vincent Khan Payne, Noumedem Anangmo Christelle Nadia, Norbert Kodjio, Etung Kollins, Leonelle Megwi, Jules-Roger Kuiate
and Mpoame Mbida, 2018.
In vitro
anticoccidial, antioxidant activities and cytotoxicity of
Psidium guajava
extracts. Res. J. Parasitol., 13: 1-13.
Corresponding Author: V.K Payne, Department of Animal Biology, Faculty of Science, University of Dschang, P.O. Box 067, Dschang, Cameroon
Tel: (237) 677365519
Copyright: © 2018 Yamssi Cedric
et al
. This is an open access article distributed under the terms of the creative commons attribution License, which permits
unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Competing Interest: The authors have declared that no competing interest exists.
Data Availability: All relevant data are within the paper and its supporting information files.

Res. J. Parasitol., 13 (1): 1-13, 2018
INTRODUCTION
In recent years, there has been increasing commercial
production of rabbits as a source of protein. The consumers
prefer rabbits for their low cholesterol and fat contents and
high levels of essential amino-acid1. In addition to this
commercial value, these animals are used as very important
models for medical research and as pets2. Therefore, rabbit
production become one of the important animal resources in
the world1. However, coccidiosis remains one of the most
important infectious causes of digestive disorders in rabbits3.
According to a recent estimate4, coccidiosis may cost the US
rabbit industry about $127 million annually and likewise
similar losses may occur worldwide.
Coccidiosis is caused by intracellular protozoon parasites
of the genus
Eimeria
and causes significant mortality in
domestic rabbits. Coccidiosis is one of the most frequent and
prevalent parasitic diseases, accompanied by weight loss, mild
intermittent to severe diarrhea with faeces containing mucus
or blood and results in dehydration, decreased rabbit
breeding5. The disease is seen most often in rearing
establishments where sanitation is poor. So far, 15 species of
Eimeria
in rabbits have been identified6. Today 14 species of
Eimeria
are known to infect the intestine while one was
located in the biliary duct of the liver. Two types of coccidiosis,
intestinal and hepatic were described in rabbits. The intestinal
coccidial species which cause weight reduction, diarrhoea and
mortality due to villi atrophy leads to malabsorption of
nutrients, electrolyte imbalance, anaemia, hypoproteinemia
and dehydration7. The rabbit intestinal coccidia parasitize
distinct parts of the intestine and at different depths of the
mucosa 8. Thus coccidiosis was probably the most expensive
and wide spread infectious disease in commercial rabbit
systems.
Most of the current anti-coccidial drugs show low efficacy
and cause deleterious side effects. The extensive use of
chemical anti-coccidial drugs in controlling this disease has
led to the development of drug-resistant parasites9. Parasite
resistance and the side effects of some of the anti-coccidial
drugs have serious consequences on disease control. In the
surrounding environment, commonly used disinfectants
include some phenolic products such as ammonia, methyl
bromide and carbon disulfide. Toxic effects of these products
represent a danger to the staff and health of animals and
therefore their use has been restricted10. Because of
widespread drug resistance constraints11, residual effects of
drugs in meat of animals and toxic effects of disinfectants,
scientists all over the world are shifting towards alternative
approaches for the control of parasitic problems12.
In various physiological and pathological conditions, the
systemic amount of free radicals and reactive oxygen species
are higher than normal. Free radical oxidative species are
known to be produced during the hosts cellular immune
response to invasion by
Eimeria
species13, which plays an
important role in defending against parasitic infections.
Another free radical oxidative species, nitric oxide
promotes vasodilation and hemorrhage in coccidian infections
which could be toxic to both parasites as well as to host cells
harboring the coccidian parasite14.
Georgieva
et al
.15 observed that
E. acervulina
oocysts
motivate lipid peroxidation, increase oxidative damage and
imbalance in the antioxidant status in infected animals by
disturbing the oxidative balance. Therefore, to alleviate or
reduce the oxidative stress, natural (e.g., Vitamin E, Se) and
synthetic (e.g., butylated hydroxytoluene) antioxidants as feed
supplements are commonly used in the poultry industry.
The use of antioxidants as anticoccidial remedies,
therefore, holds promise as an alternative in the control of
coccidiosis. Today, the use of antioxidant- rich plant extracts
has gained special importance because of restriction in the
use of synthetic compounds against coccidial infections due
to emergence of resistance and their drug residues16.
Naidoo
et al
.17 also described antioxidant rich plant extracts as
potential candidates in controlling coccidiosis in poultry.
Therefore, the use of natural antioxidants may alleviate
difficulties related to synthetic drugs, as they are not only
natural products but may comprise new molecules to which
resistance has not yet developed.
Psidium guajava
is a medicinal plant used in tropical and
subtropical countries to treat many health disorders. It has
been reported that
Psidium guajava
leaf extract has a wide
spectrum of biological activities such as anticough,
antibacterial, haemostasis18,19, antidiarrhoeal narcotic20 and
antioxidant properties21. This study was therefore, aimed at
evaluating the anticoccidial and antioxidant activities of crude
extracts of
P. guajava
in order to justify its usage by
Agricultural farmers as an anticoccidial drug.
MATERIALS AND METHODS
Plant material: The leaves of
Psidium guajava
were collected
in Menoua Division, Western Region of Cameroon and
identified by Mr. NGANSOP Eric, a botanist at the Cameroon
National Herbarium (Yaounde) using a voucher specimen
registered under the Reference No 2884/SRF.
2

Res. J. Parasitol., 13 (1): 1-13, 2018
Preparation of extract: Methanol, hexane and ethyl acetate
extracts were obtained using the procedure described by
Pone22. Briefly, 100 g of stored powder were macerated in
1.5 L of each of the organic solvents. This helped to remove
the principal natural compounds of the plants23. The mixture
was stirred daily and 72 h later, these solutions were then
filtered using Whatman paper No. 3. The filtrate was
concentrated by evaporating the solvent at 75EC using a
rotatory evaporator (Buchi R-200) to obtain the extracts.
For the aqueous extract (Infusion), a similar procedure
was carried out except for the fact that distilled water was
heated at 100EC and 100 g of the stored powder were poured
into 1.5 L of hot distilled water. The mixture was stirred and
the solution filtered using a tea sieve and filter paper. The
methanolic, hexane, ethyl acetate and aqueous extracts
obtained were kept in a refrigerator at 4EC for further
processing.
Anticoccidial activities of the extracts
Preparation of culture media
Potassium dichromate (K2Cr2O7): About 2.5% potassium
dichromate were prepared by dissolving 2.5 g of potassium
dichromate in 100 mL of distilled water. This culture medium
was stored and used to prepare plant extract concentrations.
Preparation of Hanks buffered salt solution (HBSS):
CBuffer HBSS: KCl 0.4 g
CKH2PO40.06 g
CNaCl 8.0 g
CNaHCO30.35 g
CNa2HPO40.048 g
CD-glucose 1.0 g
Water was added up to 1 L and the buffer frozen for
storage.
Preparation of the excystation solution: About 125 mL of
HBSS were added to 0.32 g of trypsin, 0.25 g bile salt and
0.3 g of taurocholate and the pH was adjusted to 7.6 using
NaOH.
Preparation of sporulated oocysts: Field isolates of
Eimeria
flavescens
oocysts were collected from the large intestine
while oocysts of
E. stiedae
were collected from the gall
bladders and necrotic hepatic lesions of naturally infected
rabbits (The floatation technique was used to determine that
they were naturally infected). These oocysts were washed and
concentrated by the flotation method24. The sporulated
o oc ys t s w er e st o re d i n 2. 5 % p ot a s si u m d i ch r om a te a t 4 EC until
they were used for experimental infections.
Eimeria intestinalis
and
Eimeria magna
were kindly provided by Alisson
Niepceron (INRA, BASE, Tours, France). The
Eimeria flavescens
,
Eimeria intestinalis
,
Eimeria magna
and
E. stiedae
field
isolates were maintained by periodic passage through young
rabbits in the Laboratory of Biology and Applied Ecology.
Preparation of stock solutions: For the aqueous extracts,
1200 mg of each extract were weighed using an electric scale
balance and then 20 mL of distilled water introduced into the
mortar. After homogenization, the mixture was transferred
into a beaker. For the organic extract, a stock solution was
equally prepared and the same amount of dry extract was first
mixed with 0.3 mL of dimethyl sulfoxide (DMSO) to facilitate
dissociation of the organic extract with water. Stock solutions
with a concentration of 40 mg mLG1 were thus obtained. By
successive dilutions, obtained solut ions of con cent ration
40, 20, 10 and 5 mg mLG1 for the oocysticidal evaluation. For
the anti sporozoidal evaluation, a working stock solution of
2000 µg mLG1 of the plant extract solution was prepared by
weighing 20 mg of crude extract and dissolving it in 10 mL of
distilled water. This was well mixed and serial dilution was
carried out to obtain solutions of concentration 1500, 1000,
500, 250 µg mLG1.
In vitro
oocysticidal effect of extracts: Pe tr i di sh e s w er e us ed
to evaluate
in vitro
disinfectant activities. Each well contained
a total volume of 2 mL of each concentration of the extracts
(2.5, 5, 10, 20 and 30 mg mLG1) inoculated with equal number
of unsporulated oocysts and incubated at 28EC. For
comparison, phenol was used as the reference disinfectant.
The set up was examined after 24 and 48 h. The number of
sporulated and non-sporulated oocysts was counted and the
percentage of sporulation was estimated by counting the
number of sporulated oocysts in a total of 100 oocysts. The
sporulation inhibitory percentage was calculated as follows:
Sp % of control Sp % of extract
Sporulation (sp) inhibition 100
percentage (%) Sp % of control 


In vitro
anti-sporozoidal effect of extracts: Stored oocysts in
K2Cr2O7 were washed several times with HBSS (pH 7.2) until
the K2Cr2O7 was completely removed. The oocysts were then
incubated in a water bath at 41EC and shaken during
incubation for 60 min. The suspension was centrifuged at
3,000-5,000 rpm 10 min and resuspended in HBSS. Liberated
sporozoites were washed with HBSS. The sporozoites were
counted using the malassez counting chamber.
3

Res. J. Parasitol., 13 (1): 1-13, 2018
Petri dishes were used to evaluate the
in vitro
sporocidal
activities. Each well contained a total volume of 2 mL of each
concentration of the extracts (125, 250, 500, 750 and
1000 µg mLG1) and inoculated with equal number of
sporozoites. For comparison, amprocox was used as the
reference drug. The set up was examined after 12 and 24 h.
The number of viable and non-viable sporozoites was counted
and the percentage of viability was estimated by counting the
number of viable sporozoites in a total of 100 sporozoites.
The viability inhibitory percentage was calculated as
follows:
Vi % of control Vi % of extract
Viability (Vi) inhibition (%) 100
Vi % of control


Antioxidant activities
1,1-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
assay: The radical scavenging activities of crude extracts
were evaluated spectrophotometrically using the
1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical25. When
DPPH reacts with an antioxidant compound which can donate
hydrogen, it was reduced. The changes in colour were
measured at 517 nm under UV/Visible light
spectrophotometer (Jenway, Model 1605). Pure methanol was
used to calibrate the counter. The extract (2000 µg mLG1) was
two fold serially diluted with methanol. One hundred
microliters of the diluted extract were mixed with 900 µL of
0.3 mM 1,1-Diphenyl-1-picrylhydrazyl (DPPH) methanol
solution, to give a final extract concentration range of
12.5-200 µg mLG1 (12.5, 25, 50, 100 and 200 µg mLG1). After
30 min of incubation in the dark at room temperature, the
optical densities were measured at 517 nm. Ascorbic acid
(Vitamin C) was used as control. Each assay was done in
triplicate and the results, recorded as the mean±standard
deviation (SD) of the three findings, were presented in tabular
form. The radical scavenging activity (RSA, in %) was
calculated as follows:
Absorbance of DPPH Absorbance of sample
RSA 100
Absorbance of DPPH


The radical scavenging percentages were plotted against
the logarithmic values of concentration of test samples and a
linear regression curve was established in order to calculate
the RSA50 or IC50 which was the concentration of the sample
necessary to decrease by 50% the total free DPPH radical26.
Ferric reducing/antioxidant power (FRAP) assay: The ferric
reducing power was determined by the Fe
3+-Fe2+
transformation in the presence of the extracts. The Fe2+ was
monitored by measuring the formation of Perls Prussian blue
at 700 nm. Different volumes (400, 200, 100, 50, 25 µL) of
methanolic extracts prepared at 2090 µg mLG1 were mixed
with 500 µL of phosphate buffer (pH 6.6) and 500 µL of 1%
potassium ferricyanide and incubated at 50EC for 20 min. Then
500 µL of 10% trichloroacetic acid was added to the mixture
and centrifuged at 3000 rpm for 10 min. The supernatant
(500 µL) was diluted with 500 µL of water and mixed with
100 µL of freshly prepared 0.1% ferric chloride. The
absorbance was measured at 700 nm. All the tests were
performed in triplicate and the results were the average of
three observations. Vitamin C was used as a positive control.
Increased absorbance of the reaction mixture indicated a
higher reduction capacity of the sample27.
Nitric oxide (NO) radical scavenging assay: The method
reported by Chanda and Dave28 was used with slight
modification. To 0.75 mL of 10 mM sodium nitroprusside in
phosphate buffer was added 0.5 mL of extract or reference
compounds (Vitamin C and butylated hydroxytoluene (BHT))
in different concentrations (62.5 -1000 µg mLG1). The resulting
solutions were then incubated at 25EC for 60 min. A similar
procedure was repeated with methanol as blank which served
as negative control. To 1.25 mL of the incubated sample
1.25 mL of Griess reagent (1% sulfanilamide in 5% phosphoric
acid and 0.1% N-1-naphthylethylenediamine dihydrochloride
in water) were added. A final concentration range of
12.5-200 µg mLG1 (12.5, 25, 50, 100 and 200 µg mLG1) was
obtained. After 5 min of incubation in the dark at room
temperature, absorbance of the chromophore formed was
measured at 540 nm. Percent inhibition of the nitrite oxide
generated was measured by comparing the absorbance
values of control and test samples. The percentage of
inhibition was calculated according to the following equation:
1
0
A
Inhibition (%) = 1- 100
A





Where:
Al= Absorbance of the extract or standard
A0= Absorbance of the negative control
Total phenol contents (TPC): The amount of total phenols
was determined by Folin-Ciocalteu Reagent method. The
reaction mixture consisted of 20 µL of extract (2000 µg mLG1),
1380 µL of distilled water, 200 µL of 2N FCR (Folin Ciocalteu
Reagent) and 400 µL of a 20% sodium carbonate solution. The
mixture was incubated at 40EC for 20 min. After cooling, the
4

Res. J. Parasitol., 13 (1): 1-13, 2018
absorbance was measured at 760 nm. In the control tube, the
extract volume was replaced by distilled water. A standard
curve was plotted using gallic acid (0-0.2 µg mLG1). The tests
were performed in triplicate and the results expressed as
milligrams of gallic acid equivalents (mg GAE) per gram of
extract.
Total flavonoid content (TFC): The amount of total flavonoids
was determined by the aluminum chloride method.
Methanolic solution of extracts (100 µL, 2000 µg mLG1) was
mixed with 1.49 mL of distilled water and 30 µL of a 5% NaNO2
solution. After 5 min, 30 µL of 10% AlCl3.H2O solution were
added. After 6 min, 200 µL of 0.1 M sodium hydroxide
and 240 µL of distilled water were added. The solution
was well mixed and the increase in absorbance was
measured at 510 nm using a UV-Visible spectrophotometer.
Total flavonoid content was calculated using the
standard catechin calibration curve. The results were
expressed as milligrams of catechin equivalents (mg CE) per
gram of extract.
Evaluation of plant extracts cytotoxicity: The cytotoxicity of
the most active extract was evaluated on animal cell lines
fibroblast L929, HEPG2 and HeLa cells using MTT assay as
described by Mosmann29. Briefly, cells (104 cells/200 mL/well)
were seeded into 96-well flat-bottom tissue culture plates in
complete medium (10% foetal bovine serum, 0.21% sodium
bicarbonate (Sigma, USA) and 50 mg mLG1 gentamicin). After
24 h, plant extracts at different concentrations were added
and plates incubated for 48 h in a humidified atmosphere at
37EC and 5% CO2. About 10% DMSO (v/v) was used as a
positive inhibitor. Thereafter, 20 µL of a stock solution of MTT
(5 mg mLG1 in 1X phosphate buffered saline) were added to
each well, gently mixed and each plate incubated for another
4 h. After spinning the plates at 1500 rpm for 5 min,
supernatants were removed and 100 mL of 10% DMSO were
added in each well to stop the reaction of extracts. Formation
of formazon obtained after transformation of tetrazolium was
read on a microtiter plate reader at 570 nm. The 50% cytotoxic
concentration (CC50) of plant extract was determined by
analysis of dose-response curves, according to the cytotoxicity
gradient of plant extracts established by Malebo
et al
.30. Also,
the selectivity index (SI) was calculated using the following
formula:
50
50
CC
SI CC

Phytochemical screening: The most active extract was tested
for the presence of phenolic compounds, alkaloids, flavonoids,
polyphenols, tannins, saponin, triterpenes and steroids using
standard procedures described by Builders
et al
.31.
Statistical analysis: The data obtained were analyzed using
one-way analysis of variance (ANOVA) and presented as
mean±standard deviation (SD) of 3 replications. The levels of
significance, considered at p<0.05, were determined by
Waller-Duncan test using the statistical package for social
sciences (SPSS) software version 12.0.
RESULTS
Anticoccidial activities
In vitr o
oocysticidal activities of
P. gua java
extracts: The
in vitro
oocysticidal activity of different extracts from the
plants against
Eimeria
intestinalis
,
Eimeria magna,
Eimeria
flavescens
and
Eimeria stiedae
strains is summarized in
Table 1. It can be seen from Table 1 that about 90% of oocysts
of
Eimeria
sp., managed to sporulate in the control
incubations containing oocysts and DMSO or K2Cr2O7. The
highest efficacy of tested plant extracts were recorded after
24 h post exposure which varied according to different
concentrations of the tested extracts. Concerning
P. guajava
extracts, the highest efficacy was 88.67±2.52% at the
concentration of 30 mg mLG1 of methanolic extracts against
Eimeria intestinalis
. On the contrary the lowest efficacy was
7.00±4.36% at the concentration of 2.5 mg mLG1 of the hot
water extract on
Eimeria flavescens
after 48 h of incubation.
Passing through the other used concentrations of
P. guajava
extracts (2.5, 5, 10 and 20 mg mLG1), they showed reduced
efficacy depending on species of
Eimeria
tested.
In vitro
anti-sporozoidal activities of
P. guajava
extracts:
Different concentrations of
P. guajava extracts
showed
concentration dependent inhibition for viability of coccidial
sporozoites of different
Eimeria
species as compared to
control groups Control-I (DMSO) and Control-II (HBSS) as
shown in Table 2. According to current study results, most
extracts including aqueous extracts exhibited good anti
sporozoidal activities against
E. flavescens
,
E. stiedae
,
E. intestinalis
and
E. magna
strains at 1000 µg mLG1. The
highest viability inhibitory percentage was 97.00±1.73% at a
concentration of 1000 µg mLG1 of
P. guajava
methanolic
extract against
E. intestinalis
strain (Table 2). The lowest
efficacy was 8.67±2.08% at a concentration of 125 µg mLG1 of
the infusion extract against
E. magna
.
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Res. J. Parasitol., 13 (1): 1-13, 2018
Table 1: Sporulation inhibition percentage of
P. guajava
extracts on different
Eimeria
strains
Incubation time and
Eimeria
strains
--------------------------------------------------------------------------------------------------------------------------------------------------------------------
24 h 48 h
Concentration ------------------------------------------------------------------------------- -------------------------------------------------------------------------------
(mg mLG1) Extract
E. intestinalis E. magna E. flavescens E. stiedae E. intestinalis E. magna E. flavescens E. stiedae
2.5 IF 17.00±11.53ab 9.00±2.65a7.67±3.06a19.67±3.10a9.00±5,57a8.00±3.61a7.00±4.36a16.00±1.73a
HE 13.33±1.16a18.00±3.00b10.33±2.08b11.00±2.65ab 12.33±1.16a16.67±1.53b9.67±1.53a7.00±3.61a
EA 21.67±2.52ab 20.00±2.00b27.00±6.56c21.33±1.53ab 20.33±3.06b18.67±2.52b25.67±6.03b18.33±3.06a
ME 31.33±4.16b21.67±1.53b27.00±6.56d23.67±1.53b23.67±2.89b20.00±1.00b25.67±6.03b19.00±1.00a
5 IF 15.00±1.00a11.67±2.52a13.33±1.53a25.33±3.22a12.33±1.53a10.00±3.00a12.00±1.00a21.00±2.65a
HE 36.67±3.22b23.33±3.21b31.00±6.56b13.67±2.52b34.67±3.22b22.00±2.65b29.33±6.66b9.00±3.00b
EA 38.33±4.04b28.33±2.08b48.33±6.35b30.33±2.08c37.33±3.51b26.33±3.06b47.67±6.66b25.33±3.06b
ME 53.33±6.11c38.33±5.51c48.33±6.35c39.67±4.51d47.67±8.51c36.67±5.51c47.67±6.66c36.33±6.51c
10 IF 38.00±4.00a26.00±4.00a31.00±2.00a38.00±4.36a35.67±4.51a24.33±4.04a29.33±2.08a34.67±4.04a
HE 47.00±4.58b36.33±4.16b40.67±1.53b27.33±3.06b45.67±4.51b35.00±4.58ab 39.33±2.08b24.00±5.00ab
EA 46.00±2.00b39.67±2.52b50.67±1.53b41.67±2.52c44.00±2.65b38.00±2.00b50.33±0.58b37.00±2.00ab
ME 51.33±2.52b47.33±1.53c50.67±1.53c48.33±3.06c48.67±2.08b45.00±1.73c50.33±0.58c45.33±0.58b
20 IF 56.33±6.66a42.00±4.00a53.67±8.02a49.00±2.00a55.67±6.81a41.00±5.00a52.00±7.55a45.00±4.00a
HE 59.33±9.07a47.33±2.56a57.00±4.58a44.00±4.00a57.00±6.58a45.67±3.51ab 55.67±4.73a40.00±5.00a
EA 67.00±3.00ab 54.33±2.52b71.00±1.00ab 56.00±2.65a65.00±2.65ab 52.67±3.51b69.67±1.16ab 52.00±3.61a
ME 73.33±2.52b67.00±2.65c71.00±1.00b68.67±3.22a71.67±2.08b66.00±2.65c69.67±1.16b65.33±2.08b
30 IF 69.67±6.51a56.67±4.16a65.67±6.66a64.33±3.51a68.67±6.51a55.67±3.21a64.33±6.51a62.00±4.36a
HE 51.33±38.42a63.33±3.79ab 68.00±4.59a58.33±4.04a71.67±3.79a62.00±4.00ab 67.33±4.04a55.00±3.46a
EA 77.00±1.00a68.33±3.79b80.67±2.52ab 70.00±4.36a75.67±4.16a67.00±4.36b79.67±1.53ab 64.67±3.79a
ME 90.00±1.73a76.00±3.00c80.67±2.52b78.00±3.00b88.67±2.52b76.00±1.00c79.67±1.53b75.00±1.00b
Negative control DMSO+K2Cr2O78.00±3.61 8.00±2.00 8.00±1.00 8.33±0.58 5.33±2.08 6.33±1.53 6.67±0.58 6.33±0.58
K2Cr2O710.33±2.10 9.33±1.53 10.33±1.53 10.33±0.58 8.67±1.53 8.00±1.73 8.33±1.52 9.00±1.00
Positive control 5% 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 86.67±10.69 86.67±10.69 84.00±1.00 82.00±1.00
ME: Methanolic extract, HE: Hexane extract, EAE: Ethyl acetate extract, IF: Infusion extract, DMSO: Dimethyl sulfoxide and K2Cr2O7: Potassium dichromate. The results
are the mean±SD of triplicate tests evaluated after 24 and 48 h of incubation at room temperature. For the same column same concentrations, values carrying the
same superscript letter are not significantly different at p> 0.05 (Student-Newman-Keuls test)
Table 2: Viability inhibitory percentage of
P. guajava
extracts on different
Eimeria
strains
Incubation time and
Eimeria
strains
---------------------------------------------------------------------------------------------------------------------------------------------------------------------
12 h 24 h
Concentration ------------------------------------------------------------------------------- ----------------------------------------------------------------------------------
(mg mLG1) Extract
E. intestinalis E. magna E. flavescens E. stiedae E. intestinalis E. magna E. flavescens E. stiedae
125 IF 7.67±3.06a15.00±2.65a3.00±4.36a13.00±1.73a24.00±11.53a8.67±2.08a18.67±3.06a24.67±3.06a
HE 9.33±1.15a24.00±3.00b5.67±1.53a4.00±3.61b20.33±1.15ab 9.33±1.15a21.33±2.08a16.00±2.65b
EA 17.33±3.06b26.00±2.00b14.00±4.36b15.33±3.06b28.67±2.52ab 17.33±3.06b29.67±4.04b26.33±1.53b
ME 20.67±2.89b27.67±1.53b21.67±6.03b16.00±1.00b38.33±4.16b20.67±2.89b38.00±6.56c28.67±1.53b
250 IF 9.33±1.53a17.67±2.52a8.00±1.00a18.00±2.65a22.00±1.00a9.33±1.53a24.33±1.53a30.33±3.21a
HE 31.67±3.21b29.33±3.21b25.33±6.66b6.00±3.00b43.67±3.21b31.67±3.21b42.00±6.56b18.67±2.52b
EA 34.33±3.51b34.33±2.08b32.00±3.61b22.33±3.06b45.33±4.04b34.33±3.51b47.67±4.51b35.33±2.08b
ME 44.67±8.50c44.33±5.51c43.67±6.66c33.33±6.51c60.33±6.11c44.67±8.50c59.33±6.35c44.67±4.51c
500 IF 32.67±4.51a32.00±4.00a25.33±2.08a31.67±4.04a45.00±4.00a32.67±4.51a42.00±2.00a43.00±4.36a
HE 42.67±4.51b42.33±4.16b35.33±2.08b21.00±5.00b54.00±4.58b42.67±4.51b51.67±1.53b32.33±3.06b
EA 41.00±2.61b45.67±2.52b38.33±4.16b34.00±2.00b53.00±2.00b41.00±2.65b55.00±3.61b46.67±2.52b
ME 45.67±2.08b53.33±1.53c46.33±0.58c42.33±0.58c58.33±2.52b45.67±2.08b61.67±1.53c53.33±3.06c
750 IF 52.67±6.81a48.00±4.00a48.00±7.55a42.00±4.00a63.33±6.66a52.67±6.81a64.67±8.02a54.00±2.00a
HE 54.00±6.56a53.33±2.52a51.67±4.73a37.00±5.00ab 66.33±9.07a54.00±6.56a68.00±4.58a49.00±4.00a
EA 62.00±2.65ab 60.33±2.52b57.33±1.53ab 49.00±3.61b74.00±3.00ab 62.00±2.65ab 74.33±1.53ab 61.00±2.65b
ME 68.67±2.08b73.00±2.65c65.67±1.15b62.33±2.08c80.33±2.52b68.67±2.08b82.00±1.00b73.67±3.21c
1000 IF 65.67±6.51a62.67±4.16a60.33±6.51a59.00±4.36a76.67±6.51a65.67±6.51a76.67±6.66a69.33±3.51a
HE 68.67±3.79a69.33±3.79ab 63.33±4.04a52.00±3.46b58.33±38.42a68.67±3.79a79.00±4.58a63.33±4.04ab
EA 72.67±4.16a74.33±3.79b69.33±3.22ab 61.67±3.79b84.00±1.00a72.67±4.16a86.00±3.00ab 75.00±4.36b
ME 85.67±2.52b82.00±3.00c75.67±1.53b72.00±1.00c97.00±1.73a85.67±2.52b91.67±2.52b83.00±3.00c
Negative control DMSO 00.00±00 00.00±00 00.00±00 00.00±00 00.00±00 00.00±00 00.00±00 00.00±00
HBSS 00.00±00 00.00±00 00.00±00 00.00±00 00.00±00 00.00±00 00.00±00 00.00±00
Positive control 50 µg mLG179.00±1.00 83.67±10.69 81.00±1.00 78.00±1.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00
ME: Methanol extract, HE: Hexane extract, EAE: Ethyl acetate extract, IF: Infusion extract DMSO: Dimethyl sulfoxide, HBSS: Buffer Hanks buffered salt solution and
K2Cr2O7: Potassium dichromate. The results are the mean±SD of triplicate tests evaluated after 12 and 24 h of incubation at room temperature. For the same column
same concentrations, values carrying the same superscript letter are not significantly different at p>0.05 (Student-Newman-Keuls test)
6

Res. J. Parasitol., 13 (1): 1-13, 2018
Table 3: DPPH radical-scavenging activities of
P. guajava
Concentration of extract (µg mLG1) and scavenging activity (%)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Extracts 12.5 25 50 100 200 IC50
IF 42.074±1.42bcd 46.074±0.33ab 50.370±0.78b55.555±2.65b70.518±1.96b102.831±22.78ab
HE 42.592±3.17bcd 47.037±1.28ab 56.666±1.55c63.407±4.20c86.296±3.90d37.969±13.59a
EA 44.66±1.99cd 70.518±2.11cd 88.518±2.21e90.296±0.49e91.925±0.61e2.879±0.20a
ME 47.185±0.66d78.740±4.25cd 86.296±4.10e92.074±1.33e94.592±0.32e2.168±0.27a
Vitamin C 76.178±6.69e86.186±0.62e87.262±0.75e90.157±1.03e93.465±0.37e1.295±0.14a
The results are the Mean±SD of triplicate tests. For the s ame column, values carryi ng the same su perscript let ter are not sig nificantly dif ferent at p>0.05
(Student-Newman-Keuls test). ME: Methanolic extract, HE: Hexane extract, EAE: Ethyl acetate extract, IF: Infusion extract
Table 4: Ferric reducing power activities of
P. guajava
extracts
Concentrations (µg mLG1) and absorbance (700 nm)
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Extracts 12.5 25 50 100 200
IF 0.632±0.08d0.642±0.05d0.802±0.07d0.999±0.06ab 1.285±0.06b
HE 0.783±0.03e0.782±0.03e0.940±0.03d1.317±0.03b1.691±0.02c
EA 0.625±0.06d1.331±0.04f1.354±0.04f1.810±0.02c2.317±0.07e
ME 1.691±0.07g1.940±0.03h2.31±0.03h2.517±0.05d2.908±0.07g
Vitamin C 0.028±0.00a0.044±0.00a0.056±0.02a2.510±0.65d6.339±0.09h
The results are the mean±SD of triplicate tests. For the same column, values carrying the same superscripts letter are not significantly different at p $ 0.05 (Student-
Newman-Keuls test). ME: Methanolic extract, HE: Hexane extract, EAE: Ethyl acetate extract, IF: Infusion extract
In vitro
antioxidant activities of
P. guajava
extracts
Effects of
P. guajava
extracts on the DPPH radical: The
DPPH radical scavenging activity of different extracts of
P. guajava
were evaluated and the results are shown in
Table 3. All the extracts of
P. guajava
exhibited stronger
antioxidant activities, compared to that of the standard
antioxidant molecule (Vitamin C) used. The hot water extract
showed the lowest activity at any concentrations with an
inhibition percentage of 70.52% at 200 µg mLG1, while the
methanolic extract showed the highest activity (94.59%) at the
concentration 200 µg mLG1. However, there was no significant
(p>0.05) difference between the activity of Vitamin C and that
of the methanolic and ethylacetate extracts of
P. guajava
at
the concentration 200 µg mLG1.
The concentrations which inhibited 50% of DPPH (IC50)
are presented in Table 3. These results show that the hot
water extract had a high IC50 (low activity). The ethyl acetate
and the methanol extract of
P. guajava
had the lowest
IC50 (i.e., had the highest activity). The methanol extract of
P. guajava
had the lowest IC50 (i.e., the highest activity).
Ferric reducing/antioxidant power (FRAP) of
P. guajava
extracts: The reducing power was determined by the
Fe3+-Fe2+ transform ation in th e presence of the ex tracts of
P. guajava
and the results obtained are shown in Table 4. The
hot water extract showed the lowest reducing power while
the standard (Vitamin C) exhibited the highest reducing power
at the concentrations of 100 and 200 µg mLG1. At 100 µg mLG1,
there was no significant difference between the reducing
power of Vitamin C (2.510±0.65) and the methanolic extract
of
P. guajava
(2.517±0.01). However, the hot water extract
showed the lowest optical densities (i.e., lowest reducing
power) at every concentration. The remaining extracts
exhibited varied activities from one extract to another at each
concentration.
Effects of
P. guajava
extracts on Nitric oxide: The results of
the scavenging capacity against nitric oxide were recorded in
terms of percentage inhibition as presented in Table 5. The
extracts of
P. guajava
showed considerable antioxidant
potential. The methanolic and ethylacetate extracts revealed
the highest percentage inhibition indicating the best nitric
oxide scavenging activity. However, hexane extracts of
P. guajava
showed the lowest scavenging activity at every
concentration.
Total phenolic content of
P. guajava
extracts: The total
phenolic content of
P. guajava
extracts were determined in
this study using Folin-Ciocalteu Reagent method and the
results are presented in Table 6. The concentration of phenolic
compounds in the methanolic extract (18.536 mgGAE mgG1)
was higher than in all other extracts. The methanolic and
ethyl acetate had relatively the same concentration
(p>0.05) and the lowest concentration of phenolic
compounds was observed in the infusion extract
(8.380 mgGAE mgG1).
7

Res. J. Parasitol., 13 (1): 1-13, 2018
Table 5: Nitric oxide (NO) radical scavenging of
P. guajava
extracts
Concentrations (µg mLG1) and percentage inhibition (%)
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Extracts 12.5 25 50 100 200
IF 86.295±0.147a89.23±0.327ab 89.591±0.269ab 89.634±0.374bc 89.787±0.274ab
HE 81.029±0.211a81.978±2.037a84.003±0.546ab 84.349±0.473b86.738±3.725ab
EA 83.271±4.231b88.594±0.725ab 89.425±0.798ab 89.627±0.385bc 90.734±0.672c
ME 85.849±1.725b86.257±0.725c89.647±0.258ab 88.464±11.151bc 92.349±0.729c
Vitamin C 92.427±3.627c94.595±2.032c94.595±1.339b96.556±0.895c96.556±0.298c
BHT 94.946±0.800c96.429±0.110d97.274±0.526c97.624±0.027d99.410±0.055d
The results are the Mean±SD of triplicate te sts. For the same colum n, values car rying the same superscript l etter are not significantly different at p>0.05
(Student-Newman-Keuls test). ME: Methanolic extract, HE: Hexane extract, EAE: Ethyl acetate extract, IF: Infusion extract
Table 6: Total phenolic and flavonoid contents of
P. guajava
extracts
Extracts Phenols (mgGAE mgG1) Flavonoids (mg CE mgG1)
Infusion 8.380±0.80bc 0.494±0.00ab
Hexane 10.461±1.20cd 1.720±0.13d
Ethyl acetate 15.328±2.13ef 1.881±0.03d
Methanol 18.536±2.17f1.991±0.18d
The results are the Mean±SD of triplicate tests. Along each column, values with
the same superscripts are not significantly different, Waller Duncan (p>0.05)
Table 7: Selectivity index, CC50 on L929, HEPG2 and HeLa cells of
P. guajava
methanolic extracts
CC50 Sporozoidal Selectivity
Plants Cell line (µg mLG1)IC
50 (µg mLG1) index (µg mLG1)
P. guajava
L929 cells 148.83 94.99 20.64
HEPG2 cells 96.24 1.01
HeLa cells 129.29 1.36
Table 8: Phytochemical screening of
P. guajava
methanolic extracts
Chemical groups/plant extract
P. guajava
Alkaloids +
Flavonoids +
Polyphenols -
Tannins +
Saponins +
Steroids +
Terpenoids -
+: Present, -: Absent
Total flavonoid content of
P. guajava
extracts: The total
flavonoid contents of the various extracts are presented in
Table 6. The result obtained showed that the methanol extract
had the highest flavonoid content (1.991 mg CE mgG1) while
the infusion extract showed the lowest value of flavonoid
content.
Cytotoxicity test: In order to evaluate the cytotoxicity effect,
L929, HEPG2 and HeLa cells were exposed to
P. guajava
methanolic extract, for 48 h and cell grown inhibition was
accessed using MTT assay. In this study, the methanolic extract
exhibited CC50 of >30 µg mLG1 against (Table 7) the selected
cell lines, suggesting that the compounds are not toxic.
Selectivity index: The selectivity index of the methanolic
extract was then evaluated using the MTT assay on L929,
HEPG2 and HeLa cells in order to check that their toxicity was
specific to the parasite (Table 7). The impact of toxicity was
established by analysing the selectivity index (SI) values. In this
study, selectivity index values for the tested extract ranged
between 1.01-20.64 µg mLG1. The methanolic extract of
P. guajava
showed the highest selectivity index value of
20.64 µg mLG1, on L929 cells which was noteworthy as the
extracts from this plant showed good anticoccidial activity.
Phytochemical analysis: Phytochemical screening of the
mo s t a c ti v e e x tr a ct s wa s co n si st e nt w it h d e te c ti on o f a lk a lo i ds ,
flavonoids, saponins, steroids and tannins, whereas, the
absence of polyphenols and terpenoids were noticed
(Table 8).
DISCUSSION
According to this study results, most extracts including
aqueous extracts exhibited good oocysticidal activity against
Eimeria
intestinalis
,
Eimeria magna
,
Eimeria flavescens
and
Eimeria stiedae
strains. The
P. guajava
extract showed
maximum sporulation inhibition activity at 30 mg mLG1 and
was observed to be more effective against
Eimeria intestinalis
.
Similar to present findings, Molan
et al
.32 also observed
in vitro
sporulation inhibition with aqueous extracts of pine bark
(
Pinus radiata
) in three species of avian coccidia. Since extracts
have been shown to inhibit endogenous enzyme activities33,
then it is possible that
P. guajava
extract reduced the
proportion of sporulation by inhibiting or inactivating the
enzymes responsible for the sporulation process as in
helminth eggs34. Jones
et al
.35 suggested that extracts may
penetrate the cell wall of oocysts and cause a loss of
intracellular components. In the present study, the
P. guajava
extracts might have penetrated the wall of the oocysts and
damaged the cytoplasm (sporont) as evidenced by the
appearance of abnormal sporocysts in oocysts exposed to
higher concentrations. The differences between the four
extracts in inhibiting sporulation of coccidia oocysts may be
due to differences in chemical composition.
8

Res. J. Parasitol., 13 (1): 1-13, 2018
The percentage of cells viability under control
circumstances (DMSO and HBSS) in this study was comparable
with other studies using
Eimeria
species36, therefore, the
method used may be considered an acceptable model.
According to the knowledge, this is the first study to evaluate
the effects of
P. guajava
as inhibitors of
Eimeria
intestinalis
,
Eimeria magna
,
Eimeria flavescens
and
Eimeria stiedae
sporozoites
in vitro
. This study findings confirm the results of
another study on the inhibitory effect of curcumin on the
activity of
E. tenella
sporozoites
36. The mechanism of
inhibition is unknown, but may be linked to osmotic effects
attributed to extracts37. Schubert
et al
.38 had demonstrated
that extracellular calcium and Ca2+ signaling are essential for
the invasion of
E. tenella
sporozoites into host cells. Extracts
have been shown to activate and desensitize receptors in
calcium channels39. It is possible that
P. guajava extracts
contribute to the observed inhibition of sporozoite viability by
disrupting calcium-mediated signaling in the sporozoites.
Since multiple characteristic reactions and mechanisms
are involved in the so-called oxidative stress, using a single
test is not sufficient to evaluate the antioxidant potential of
plant natural compounds or extracts40. Therefore, many
antioxidant assays such as DPPH radical scavenging activity,
ferric reducing/antioxidant power and nitric oxide scavenging
activity methods were chosen in order to evaluate the
antioxidant properties of
P. guajava
extracts.
The DPPH assay has been used widely to determine the
radical scavenging activity of antioxidant substances41,42. The
DPPH free radical scavenging activity was significantly
(p<0.05) higher in the methanol extract followed by ethyl
acetate, while the infusion and the hexane extracts had the
least DPPH free radical scavenging activity. This method is
based on the reduction of DPPH in methanol solution in the
presence of a hydrogen-donating antioxidant due to
formation of the non-radical form DPPH-H43. The extracts
significantly inhibited the activity of DPPH radicals in a
dose-dependent manner and the maximum scavenging
activities were observed at the concentration of 200 mg mLG1.
The effect of antioxidants on DPPH radical has been thought
to be due to their hydrogen donating ability. Hence, DPPH is
usually used as a substrate to evaluate antioxidative or free
radical scavenging activity of antioxidant agents. In this
experiment, the high DPPH radical scavenging activities of
some extracts were comparable to the standard antioxidant,
V it am i n C , s u gg e st i ng t ha t th e e x tr a ct s h a ve s om e c o mp ou n ds
with high proton donating ability and could therefore, serve
as free radical inhibitors. However, the organic extract of
P. guajava
demonstrated a more remarkable anti-radical
activity with IC50<20 µg mLG1. In fact, according to
Souri
et al
.44, th e antiox idant acti vitie s of plan t extrac ts
are significant when IC50<20 µg mLG1, moderate when
20 µg mLG1<IC50<75 µg mLG1 and weak when
IC50>75 µg mLG1. There was no significant difference (p>0.05)
between IC50 values of the organic extracts and ascorbic acid.
The higher radical scavenging activity observed in
P. guajava
leaves is perhaps attributed to the higher condensed tannins
content in these leaves. In the present study, the condensed
tannins content and the radical scavenging activity of
P. guajava
leaves are likely to show a good relationship.
Previous studies had also reported the relationship between
the high level of polyphenolic compounds and radical
scavenging activity45,46. On the other hand, the higher DPPH
free radical scavenging activity of
P. guajava
extracts may be
due to the potential and effective condensed tannins source
because of reactions between condensed tannins molecules
and radicals resulting in the scavenging of radicals by
hydrogen donation47.
Antioxidants can be reductants and inactivation of
oxidants by reductants can be described as oxido-reduction
reactions48. The presence of reductants such as antioxidant
substances in the samples causes reduction of the ferric to the
ferrous form which can be monitored by measuring the
formation of Perlis prussian blue at 700 nm. The FRAP assay,
therefore, provides a reliable method to study the antioxidant
activity of various extracts. In this study, the infusion extracts
had moderate reducing power, the highest activity was
obtained with the methanol extract and the lowest activity
was obtained with the infusion. These data suggest that the
extract of
P. guajava
may contain several compounds with
intermediate polarity. The methanol extract of
P. guajava
showed significantly (p<0.05) higher reducing ability
compared to other extracts. Reducing power is associated
with antioxidant activity and may serve as a significant
reflection of the antioxidant activity. The methanol extract of
P. guajava
exhibited a higher reducing power. The reducing
power of
P. guajava
is mainly correlated to the presence of
reductones like ascorbic acid and guava is reported to be rich
in ascorbic acid49. In the present study observed a
concentration-dependent decrease in the absorbance of the
reaction mixture for all the extracts and ascorbic acid. The
reducing capacity of extracts is much related to the presence
of biologically active compounds (condensed tannins) with
potent donating abilities may therefore, serve as an indicator
of its potential antioxidant activity50. The observed reducing
ability of
P. guajava
extracts in the present study could be
attributed to the presence of condensed tannins as reported
by Omoruyi
et al
.51. Previous studies of Omoruyi
et al
.51 and
Park and Jhon52 correlated the reducing power ability of plant
9

Res. J. Parasitol., 13 (1): 1-13, 2018
extracts to the presence of phenolic content. The antioxidant
potential and effectiveness of condensed tannins is generally
proportional to the number of hydroxyl (‒OH) groups present
on the aromatic ring (s) as well as arrangement of the hydroxyl
groups and extraction processes.
It is well documented that during chicken coccidiosis, the
generation of pro-inflammatory mediators, together with the
oxidative and nitric oxide (NO) species, contribute principally
to inflammatory injury, diarrhea, mortality and weight loss53.
Therefore, substances that generate oxidative stress or have
antioxidant properties such as n-3 fatty acids, g-tocopherol,
curcumin, essential oil blends and green tea extracts
demonstrated certain coccidiostat effects54. It seems that after
parasite invasion, free radicals, together with high levels of
NO production, are the major factors that compromise the
cellular antioxidant defense system. Compounds that are
meeting the demands of antioxidant defense system or
directly interfere with free radicals, such as tannins, may
restore the balance of oxidants/antioxidants, leading to
improvement in intestinal integrity and performance during
subclinical coccidiosis 17. Antioxidants act by scavenging the
NO radicals28. Nitric oxide radical scavenging activity is
correlated to the presence of phenolic compounds55. There
was a significant decrease in the NO radical due to the
scavenging ability of extracts and ascorbic acid. The increased
nitric oxide radical scavenging activity was observed in every
extract of the tested plants. The ethyl acetate extracts showed
better scavenging capacity compared to methanolic extract.
The nitric oxide scavenging potential may be due to
antioxidant principle in the extract which competes with
oxygen to react with nitric oxide and thus inhibit the
generation of nitrites.
Phenolic compounds exhibit antioxidant activity by
inactivating free radicals or preventing decomposition of
hydroperoxide into free radicals56. Flavonoids protective
effects in biological systems are linked to their ability to
transfer electrons to free radicals, chelate metals, activate
antioxidant enzymes and reduce radicals of alpha-tocopherol
or to inhibit oxidases56. The results obtained in this study
showed that antiradical scavenging activity was related to
the phenolic content. Then, the methanolic crude extract of
P. guajava
was found to have high phenolic contents with
18.536 mgGAE mgG1 and which may be one of the reasons
explaining its high antioxidant activity with an IC50 of
2.168±0.27 (DPPH radical-scavenging activity) and
absorbance of 2.908±0.07 at 200 µg mLG1 (ferric reducing
power activity). There was a positive linear correlation
between antioxidant activity index and total phenolic content
fo r al l th e ex trac ts. Thes e re su lts sug ges t tha t th e phenolic
compounds contribute significantly to the antioxidant
capacity of the investigated plant species. In addition, these
results are consistent with the findings of many researchers
w ho re p or te d su c h p os i ti v e c or r el a ti on b et we e n t o ta l p h en o li c
content and antioxidant activity57. However, Bajpai
et al
.58
disproved the correlation between phenolic compounds and
antioxidant activity.
Cytotoxicity screening is the
in vitro
toxicological
assessment of specific adverse effects of drugs. Assessment of
the cytotoxicity
P. guajava
revealed that the CC50 of the
methanol extract on L929, HEPG2 and HeLa cell lines were
above 30 µg mLG1 indicating the overall safety of
P. guajava.
According to Malebo
et al
.30, plants were classified by
their cytotoxicity potential as:
CHigh cytotoxicity (CC50 <1.0 µg mLG1 )
CModerate (CC50 1.0-10.0 µg mLG1)
CMild (CC50 10.0-30.0 µg mLG1 )
CNontoxic (CC50>30 µg mLG1)
This study realize that the tested extract was found to be
non-cytotoxic or with very low toxicity on L929, HEPG2 and
HeLa mammalian cell lines. It has been reported that
P. guajava
leaf extracts demonstrated no cytotoxicity in
clinical trials with humans59. In a separate study, Ling
et al
.60
reported that some ethanolic e xtracts inclu ding that of
P. guajava
lack cytotoxicity in assays involving 3T3 and 4T1
cells.
CONCLUSION
Due to widespread development of resistance to
anticoccidial drugs, there is shift to reduce the use of these
chemical compounds. Efforts have been made to develop new
strategies for control of rabbit coccidiosis. These efforts
include a search for new agents with anticoccidial activity such
as naturally occurring compounds that are considered most
effective and safe. The control of oxidative damage caused by
reactive oxygen species and free radicals produced within the
cell is a major field of study nowadays. Latest research on
natural antioxidants including herbal antioxidants have
proved their health benefits against oxidative stress which is
involved in the pathology of several diseases in living
organisms including coccidiosis in rabbits. They can be
considered as best substitutes to chemical anticoccidials.
However further experimental studies are required to explore
the efficacy of
P. guajava
anticoccidials, antioxidants and their
modes of action.
10

Res. J. Parasitol., 13 (1): 1-13, 2018
SIGNIFICANCE STATEMENTS
This study discovered
in vitro
anticoccidial and
antioxidant activities of
Psidium guajava
extracts. These
results also showed that methanolic and ethyl acetate extract,
possessed strong antioxidant and anticoccidial activities.
The methanolic extract of
P. guajava
exhibited CC50 of
>30 µg mLG1 against selected cell lines, suggesting that the
compounds are not toxic. This study will help the researcher
to uncover critical areas of coccidiosis and oxidative stress that
many researchers were not able to explore. Thus a new theory
on the usage of
Psidium guajava
against coccidiosis by agro
pastoral farmers in Cameroon may be arrived at.
ACKNOWLEDGMENTS
The authors wish to thank Alisson Niepceron
(INRA, BASE, Tours, France) who kindly provided
Eimeria
intestinalis
and
Eimeria magna
strains used in the study and
Dr. Michal Pakandl for his expertise in rabbit coccidiosis.
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