Research Article http://www.alliedacademies.

org/parasitic-diseases-diagnosis-therapy/
ISSN: 2591-7846
In vitro anticoccidial, antioxidant activities and cytotoxicity of Psidium guajava
extracts.
Yamssi Cedric1*, Vincent Khan Payne1, Noumedem Anangmo Christelle Nadia1, Norbert Kodjio2, Etung
Kollins1, Leonelle Megwi1, Jules-Roger Kuiate2, Mpoame Mbida1
1
Research Unit of Biology and Applied Ecology, Faculty of Science University of Dschang, Cameroon
2
Research Unit of Microbiology and Antimicrobial Substances, Faculty of Science, University of Dschang, Cameroon

Abstract
Background: Coccidiosis remains one of the most important infectious causes of digestive
disorders in rabbits. The aim of this study was to evaluate in vitro anticoccidial and antioxidant
activities of Psidium guajava extracts.
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 h and 48 h for the oocysticidal
activities and after 12 h 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 Hella cells using MTT assay. The impact of the toxicity was established by analysing
the Selectivity Index (SI) values.
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/ml 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/ml. The highest viability inhibitory
percentage was 97.00 ± 1.73% at a concentration of 1000 µg/ml 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/ml). The methanolic extract of P.
guajava exhibited CC50 of>30 µg/ml against selected cell lines, suggesting that the compounds
are 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 against
coccidioses by Agricultural farmers in Cameroon.

Keywords: Psidium guajava, Anticoccidial activity, Antioxidant, Eimeria species, Cameroon.

Accepted October 13, 2017

Introduction prevalent parasitic diseases, accompanied by weight loss,

mild intermittent to severe diarrhea with faeces containing
In recent years, there has been increasing commercial production
mucus or blood and results in dehydration, decreased rabbit
of rabbits as a source of protein. The consumers prefer rabbits
breeding [5]. The disease is seen most often in rearing
for their low cholesterol and fat contents and high levels of
establishments where sanitation is poor. So far, 15 species
essential amino-acid [1]. In addition to this commercial value,
of Eimeria in rabbits have been identified [6]. Today 14
these animals are used as very important models for medical
species of Eimeria are known to infect the intestine while
research and as pets [2]. Therefore, rabbit production become
one is located in the biliary duct of the liver. Two types
one of the important animal resources in the world [1]. However,
of coccidiosis, intestinal and hepatic are described in
coccidiosis remains one of the most important infectious causes
rabbits. The intestinal coccidial species which cause weight
of digestive disorders in rabbits [3]. According to a recent
reduction, diarrhoea and mortality due to villi atrophy
estimate [4], coccidiosis may cost the US rabbit industry about
leads to malabsorption of nutrients, electrolyte imbalance,
$127 million annually and likewise similar losses may occur
anaemia, hypoproteinemia and dehydration [7]. The rabbit
worldwide.
intestinal coccidia parasitize distinct parts of the intestine
Coccidiosis is caused by intracellular protozoon parasites and at different depths of the mucosa [8]. Thus coccidiosis
of the genus Eimeria and causes significant mortality in is probably the most expensive and wide spread infectious
domestic rabbits. Coccidiosis is one of the most frequent and disease in commercial rabbit systems.

14 J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2

Citation: Cedric Y, Payne VK, Nadia NAC, et al. In vitro anticoccidial, antioxidant activities and cytotoxicity of Psidium guajava extracts. J Parasit Dis
Diagn Ther. 2017;2(2):14-24

Most of the current anti-coccidial drugs show low efficacy and Division, Western Region of Cameroon and identified by
cause deleterious side effects. The extensive use of chemical Mr. NGANSOP Eric, a botanist at the Cameroon National
anti-coccidial drugs in controlling this disease has led to the Herbarium (Yaoundé) using a voucher specimen registered
development of drug-resistant parasites [9]. Parasite resistance under the Reference No 2884/SRF.
and the side effects of some of the anti-coccidial drugs have
Preparation of extract
serious consequences on disease control. In the surrounding
environment, commonly used disinfectants include some Methanol, hexane and Ethyl Acetate extracts were obtained
phenolic products such as ammonia, methyl bromide and using the procedure described by Wabo Poné et al. [22]. Briefly,
carbon disulfide. Toxic effects of these products represent a 100 g of stored powder were macerated in 1.5 L of each of the
danger to the staff and health of animals and therefore their use organic solvents. This helped to remove the principal natural
has been restricted [10]. Because of widespread drug resistance compounds of the plants [23]. The mixture was stirred daily
constraints [11], residual effects of drugs in meat of animals and 72 h later, these solutions were then filtered using Whatman
and toxic effects of disinfectants, scientists all over the world Paper N 3. The filtrate was concentrated by evaporating the
are shifting towards alternative approaches for the control of solvent at 75°C using a rotatory evaporator (Buchi R-200) to
parasitic problems [12]. obtain the extracts.
In various physiological and pathological conditions, the For the aqueous extract (Infusion), a similar procedure was
systemic amount of free radicals and reactive oxygen species carried out except for the fact that distilled water was heated at
are higher than normal. Free radical oxidative species are known 100°C and 100 g of the stored powder were poured into 1.5 L
to be produced during the host’s cellular immune response to of hot distilled water. The mixture was stirred and the solution
invasion by Eimeria species [13], which plays an important role filtered using a tea sieve and filter paper. The methanolic,
in defending against parasitic infections. hexane, Ethyl Acetate and aqueous extracts obtained were kept
in a refrigerator at 4ºC for further processing.
Another free radical oxidative species, nitric oxide promotes
vasodilation and hemorrhage in coccidian infections which Anticoccidial activities of the extracts
could be toxic to both parasites as well as to host cells harboring
Preparation of culture media
the coccidian parasite [14].
Dichromate (K2Cr2O7) Potassium: 2.5% Potassium dichromate
Georgieva et al. [15] observed that E. acervulina oocytes
were prepared by dissolving 2.5 g of potassium dichromate in
motivate lipid peroxidation, increase oxidative damage and
100 ml of distilled water. This culture medium was stored and
imbalance in the antioxidant status in infected animals by
used to prepare our plant extract concentrations.
disturbing the oxidative balance. Therefore to alleviate or
reduce the oxidative stress, natural (e.g. Vitamin E, Se) and Preparation of hanks buffered salt solution (HBSS):
synthetic (e.g. butylated hydroxytoluene) antioxidants as feed
Buffer HBSS: KCl …………………….0.4 g
supplements are commonly used in the poultry industry.
KH2PO4 ……………… .0.06 g
The use of antioxidants as anticoccidial remedies, therefore,
holds promise as an alternative in the control of coccidiosis. NaCl ……………………8.0 g
Today, the use of antioxidant- rich plant extracts has gained NaHCO3 ……….……….0.35 g
special importance because of restriction in the use of synthetic
compounds against coccidial infections due to emergence of Na2HPO4 ……………….0.048 g
resistance and their drug residues [16]. Naidoo et al. [17] also D-glucose ………………1.0 g
described antioxidant rich plant extracts as potential candidates
in controlling coccidiosis in poultry. Therefore, the use of natural Water was added up to 1L and the buffer frozen for storage
antioxidants may alleviate difficulties related to synthetic drugs, Preparation of the excystation solution: 125 ml of HBSS
as they are not only natural products but may comprise new were added to 0.32 g of trypsin, 0.25 g Bile Salt and 0.3 g of
molecules to which resistance has not yet developed. taurocholate and the pH was adjusted to 7.6 using NaOH.
Psidium guajava is a medicinal plant used in tropical and Preparation of sporulated oocysts: Field Isolates of Eimeria
subtropical countries to treat many health disorders. It has been flavescens oocysts were collected from the large intestine while
reported that Psidium guajava leaf extract has a wide spectrum occysts of E. stiedae were collected from the gall bladders and
of biological activities such as anticough, antibacterial, necrotic hepatic lesions of naturally infected rabbits. These
haemostasis [18,19], antidiarrhoeal narcotic [20], and antioxidant oocysts were washed and concentrated by the flotation method
properties [21]. This work was therefore aimed at evaluating the [24]. The sporulated oocysts were stored in 2.5% potassium
anticoccidial and antioxidant activities of crude extracts of P. dichromate at 4°C until they were used for experimental
guajava in order to justify its usage by Agricultural farmers as infections. Eimeria intestinalis and Eimeria magna were
an anticoccidial drug. kindly provided by Alisson Niepceron (INRA, BASE, Tours,
Materials and Methods France). The Eimeria flavescens, Eimeria intestinalis, Eimeria
magna and E. stiedae field isolates were maintained by periodic
Plant material passage through young Rabbits in the Laboratory of Biology
The leaves of Psidium guajava were collected in Menoua and Applied Ecology.

J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2 15

 Cedric/Payne/Nadia/et al.

Preparation of stock solutions: For the aqueous extracts, at 517 nm under UV/Visible light spectrophotometer (Jenway,
1200 mg of each extract were weighed using an electric scale Model 1605). Pure methanol was used to calibrate the counter.
balance and then 20 ml of distilled water introduced into the The extract (2000 μg/mL) was twofold serially diluted with
mortar. After homogenization, the mixture was transferred methanol. One hundred microliters of the diluted extract were
into a beaker. For the organic extract, a stock solution was mixed with 900 μL of 0.3 mM 2,2-diphenyl-1-picrylhydrazyl
equally prepared and the same amount of dry extract was first (DPPH) methanol solution, to give a final extract concentration
mixed with 0.3 ml of Dimethyl sulfoxide (DMSO) to facilitate range of 12.5 - 200 μg/mL (12.5, 25, 50, 100 and 200 μg/mL).
dissociation of the organic extract with water. Stock solutions After 30 min of incubation in the dark at room temperature,
with a concentration of 40 mg/ml were thus obtained. By the optical densities were measured at 517 nm. Ascorbic acid
successive dilutions, we obtained solutions of concentration 40, (Vitamin C) was used as control. Each assay was done in triplicate
20, 10 and 5 mg/ml for the oocysticidal evaluation. For the anti and the results, recorded as the mean ± standard deviation (SD)
sporozoidal evaluation, a working stock solution of 2000 µg/ml of the three findings, were presented in tabular form. The radical
of the plant extract solution was prepared by weighing 20 mg of scavenging activity (RSA, in %) was calculated as follows:
crude extract and dissolving it in 10 ml of distilled water. This
Absorbance of DPPH - Absorbance of sample
was well mixed and serial = dilution was carried out to obtain RSA × 100
Absorbance of DPPH
solutions of concentration 1500, 1000, 500, 250 µg/ml.
The radical scavenging percentages were plotted against the
In vitro oocysticidal effect of extracts: Petri dishes were used
logarithmic values of concentration of test samples and a linear
to evaluate in vitro disinfectant activities. Each well contained
regression curve was established in order to calculate the RSA50
a total volume of 2 ml of each concentration of the extracts
or IC50 which is the concentration of the sample necessary to
(2.5, 5, 10, 20 and 30 mg/ml) inoculated with equal number of
decrease by 50% the total free DPPH radical [26].
unsporulated oocysts and incubated at 28°C. For comparison,
phenol was used as the reference disinfectant. The set up was Ferric reducing/antioxidant power (FRAP) assay: The
examined after 24 h and 48 h. The number of sporulated and ferric reducing power was determined by the Fe3+ - Fe2+
non-sporulated oocysts were counted and the percentage of transformation in the presence of the extracts. The Fe2+ was
sporulation was estimated by counting the number of sporulated monitored by measuring the formation of Perl’s Prussian
oocysts in a total of 100 oocysts. The sporulation inhibitory blue at 700 nm. Different volumes (400, 200, 100, 50, 25 μL)
percentage was calculated as follows. of methanolic extracts prepared at 2090 μg/mL were mixed
Sp % of control − Sp % of extract with 500 μL of phosphate buffer (pH 6.6) and 500 μL of 1%
Sporulation
= ( sp) inhibition percentage (%) × 100
Sp % of control potassium ferricyanide and incubated at 50°C for 20 min. Then
In vitro anti-sporozoidal effect of extracts: Stored oocysts in 500 μL of 10% trichloroacetic acid was added to the mixture
K2Cr2O7 were washed several times with HBSS (pH 7.2) until and centrifuged at 3000 rpm for 10 min. The supernatant (500
the K2Cr2O7 was completely removed. The oocysts were then μL) was diluted with 500 μL of water and mixed with 100 μL
incubated in a water bath at 41oC and shaken during incubation of freshly prepared 0.1% ferric chloride. The absorbance was
for 60 min. The suspension was centrifuged at 3,000 – 5,000 measured at 700 nm. All the tests were performed in triplicate
x g 10 min and resuspended in HBSS. Liberated sporozoites and the results were the average of three observations. Vitamin
were washed with HBSS. The sporozoites were counted using C was used as a positive control. Increased absorbance of the
the malassez counting chamber. reaction mixture indicated a higher reduction capacity of the
sample [27].
Petri dishes were used to evaluate the in vitro sporocidal
activities. Each well contained a total volume of 2 ml of each Nitric oxide radical scavenging (NO) assay: The method
concentration of the extracts (125, 250, 500, 750 and 1000 reported by Chanda and Dave [28] was used with slight
μg/ml) and inoculated with equal number of sporozoites. For modification. To 0.75 mL of 10 mM sodium nitroprusside in
comparison, amprocox was used as the reference drug. The set phosphate buffer was added 0.5 mL of extract or reference
up was examined after 12 h and 24 h. The number of viable compounds (Vitamin C and Butylated hydroxytoluene (BHT))
and non-viable sporozoites were counted and the percentage in different concentrations (62.5 - 1000 μg/mL). The resulting
of viability was estimated by counting the number of viable solutions were then incubated at 25°C for 60 min. A similar
sporozoites in a total of 100 sporozoites. procedure was repeated with methanol as blank which served
The viability inhibitory percentage was calculated as follows. as negative control. To 1.25 mL of the incubated sample 1.25
Vi % of control − Vi % of extract
mL of Griess reagent (1% sulfanilamide in 5% phosphoric
Viability
= (Vi ) inhibition percentage (%) × 100 acid and 0.1% N-1-napthylethylenediamine dihydrochloride in
Vi % of control
water) were added. A final concentration range of 12.5 - 200 μg/
Antioxidant activities mL (12.5, 25, 50, 100 and 200 μg/mL) was obtained. After 5
The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical min of incubation in the dark at room temperature, absorbance
scavenging assay: The radical scavenging activities of crude of the chromophore formed was measured at 540 nm. Percent
extracts were evaluated spectrophotometrically using the inhibition of the nitrite oxide generated was measured by
1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical [25]. When comparing the absorbance values of control and test samples.
DPPH reacts with an antioxidant compound which can donate The percentage of inhibition was calculated according to the
hydrogen, it is reduced. The changes in color were measured following equation:

16 J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2

Citation: Cedric Y, Payne VK, Nadia NAC, et al. In vitro anticoccidial, antioxidant activities and cytotoxicity of Psidium guajava extracts. J Parasit Dis
Diagn Ther. 2017;2(2):14-24

saponin, triterpenes and steroids using standard procedures

inhibition
%= (1- ( A1 A0 ) ) × 100
described by Builders et al. [31].
Where, Al=absorbance of the extract or standard and
Statistical analysis
A0=absorbance of the negative control.
The data obtained were analyzed using one-way analysis of
Total phenol contents (TPC): The amount of total phenols was
variance (ANOVA) and presented as mean ± standard deviation
determined by Folin-Ciocateu Reagent method. The reaction
(SD) of three replications. The levels of significance, considered
mixture consisted of 20 μL of extract (2000 μg/mL), 1380 μL
at P<0.05, were determined by Waller-Duncan test using the
of distilled water, 200 μl of 2N FCR (Folin Ciocalteu Reagent)
Statistical Package for Social Sciences (SPSS) software version
and 400 μL of a 20% sodium carbonate solution. The mixture
12.0.
was incubated at 40°C for 20 min. After cooling, the absorbance
was measured at 760 nm. In the control tube, the extract volume Results and Discussion
was replaced by distilled water. A standard curve was plotted
Results
using Gallic acid (0-0.2 μg/mL). The tests were performed in
triplicate and the results expressed as milligrams of Gallic Acid Anticoccidial activities
Equivalents (mgGAE) per gram of extract. In vitro oocysticidal activities of P. guajava extracts: The
Total flavonoid content (TFC): The amount of total flavonoids in vitro oocysticidal activity of different extracts from the
was determined by the Aluminum chloride method. Methanolic plants against Eimeria intestinalis, Eimeria magna, Eimeria
solution of extracts (100 μL, 2000 μg/ml) was mixed with 1.49 flavescens and Eimeria stedai strains is summarized in (Table
mL of distilled water and 30 μL of a 5% NaNO2 solution. After 1). It can be seen from Table 1 that about 90% of oocysts of
5 min, 30 μL of 10% AlCl3H2O solution were added. After 6 Eimeria sp managed to sporulate in the control incubations
min, 200 μl of 0.1 M sodium hydroxide and 240 μl of distilled containing oocysts and DMSO or K2Cr2O7. The highest efficacy
water were added. The solution was well mixed and the increase of tested plant extracts was recorded after 24 h. post exposure
in absorbance was measured at 510 nm using a UV-Visible which varied according to different concentrations of the
spectrophotometer. Total flavonoid content was calculated tested extracts. Concerning P. guajava extracts, the highest
using the standard catechin calibration curve. The results were efficacy was 88.67 ± 2.52% at the concentration of 30 mg/ml of
expressed as milligrams of Catechin Equivalents (mgCE) per methanolic extracts against Eimeria intestinalis. On the contrary
gram of extract. the lowest efficacy was 7.00 ± 4.36% at the concentration of 2.5
mg/ml of the hot water extract on Eimeria flavescens after 48
Evaluation of plant extracts cytotoxicity h of incubation. Passing through the other used concentrations
of P. guajava extracts (2.5, 5, 10 and 20 mg/ ml), they showed
The cytotoxicity of the most active extract was evaluated on
reduced efficacy depending on species of Eimeria tested.
animal cell lines fibroblast L929, HEPG2 and Hella cells
using MTT assay as described by Mosmann [29]. Briefly, In vitro anti-sporozoidal activities of P. guajava extracts:
cells (104 cells/200 ml/well) were seeded into 96-well flat- Different concentrations of P. guajava extracts showed
bottom tissue culture plates in complete medium (10% foetal concentration dependent inhibition for viability of coccidial
bovine serum, 0.21% Sodium Bicarbonate (Sigma, USA) and sporozoites of different Eimeria species as compared to control
50 mg/ml gentamicin). After 24 h, plant extracts at different groups Control-I (DMSO) and Control-II (HBSS) as shown
concentrations were added and plates incubated for 48 h in a in (Table 2). According to our results, most extracts including
humidified atmosphere at 37ºC and 5% CO2. 10% DMSO (v/v) aqueous extracts exhibited good antisporozoidal activities
was used as a positive inhibitor. Thereafter, 20 µl of a stock against E. flavescens, E. stiedae, E. intestinalis and E. magna
solution of MTT (5 mg/mL in 1X phosphate buffered saline) strains at 1000 µg/ml. The highest viability inhibitory percentage
were added to each well, gently mixed and each plate incubated was 97.00 ± 1.73% at a concentration of 1000 µg/ml of P.
for another 4 h. After spinning the plates at 1500 rpm for 5 min, guajava methanolic extract against E. intestinalis strain (Table
supernatants were removed and 100 ml of 10% DMSO were 2). The lowest efficacy was 8.67 ± 2.08% at a concentration of
added in each well to stop the reaction of extracts. Formation 125 µg/ml of the infusion extract against E. magna.
of formazon obtained after transformation of tetrazolium was In vitro Antioxidant activities of P. guajava extracts
read on a microtiter plate reader at 570 nm. The 50% cytotoxic
concentration (CC50) of plant extract was determined by analysis Effects of P. guajava extracts on the DPPH radical: The DPPH
of dose–response curves, according to the cytotoxicity gradient radical scavenging activity of different extracts of P. guajava
of plant extracts established by Malebo et al. [30]. Also, the was evaluated and the results are shown in (Table 3). All the
Selectivity Index (SI) was calculated using the following extracts of P. guajava exhibited stronger antioxidant activities,
formula: compared to that of the standard antioxidant molecule (Vitamin
C) used. The hot water extract showed the lowest activity at any
CC 50 concentrations with an inhibition percentage of 70.52% at 200
SI =
CC 50 µg/ml, while the methanolic extract showed the highest activity
Phytochemical screening (94.59%) at the concentration 200 µg/ml. However, there
was no significant (p>0.05) difference between the activity of
The most active extract was tested for the presence of phenolic Vitamin C and that of the methanolic and ethylacetate extracts
compounds, alkaloids, flavonoids, Polyphenols, tannins, of P. guajava at the concentration 200 µg/ml.

J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2 17

 Cedric/Payne/Nadia/et al.

Table 1. Sporulation inhibition percentage of P. guajava extracts on different Eimeria strains.

Incubation time and Eimeria strains
Conc mg/ml Extract 24 h 48 h
E. intestinalis E. magna E.flavescens E. stedai E. intestinalis E. magna E. flavescens E. stedai
IF 17.00 ± 11.53ab 9.00 ± 2.65a 7.67 ± 3.06a 19.67 ± 3.10a 9.00 ± 5,57a 8.00 ± 3.61a 7.00 ± 4.36a 16.00 ± 1.73a
HE 13.33 ± 1.16a 18.00 ± 3.00b 10.33 ± 2.08b 11.00 ± 2.65ab 12.33 ± 1.16a 16.67 ± 1.53b 9.67 ± 1.53a 7.00 ± 3.61a
2.5
EA 21.67 ± 2.52 ab
20.00 ± 2.00 b
27.00 ± 6.56 c
21.33 ± 1.53 ab
20.33 ± 3.06 b
18.67 ± 2.52 b
25.67 ± 6.03 b
18.33 ± 3.06a
ME 31.33 ± 4.16 b
21.67 ± 1.53 b
27.00 ± 6.56 d
23.67 ± 1.53 b
23.67 ± 2.89 b
20.00 ± 1.00 b
25.67 ± 6.03 b
19.00 ± 1.00a
IF 15.00 ± 1.00 a
11.67 ± 2.52 a
13.33 ± 1.53 a
25.33 ± 3.22 a
12.33 ± 1.53 a
10.00 ± 3.00 a
12.00 ± 1.00 a
21.00 ± 2.65a
HE 36.67 ± 3.22b 23.33 ± 3.21b 31.00 ± 6.56b 13.67 ± 2.52b 34.67 ± 3.22b 22.00 ± 2.65b 29.33 ± 6.66b 9.00 ± 3.00b
5
EA 38.33 ± 4.04b 28.33 ± 2.08b 48.33 ± 6.35b 30.33 ± 2.08c 37.33 ± 3.51b 26.33 ± 3.06b 47.67 ± 6.66b 25.33 ± 3.06b
ME 53.33 ± 6.11c 38.33 ± 5.51c 48.33 ± 6.35c 39.67 ± 4.51d 47.67 ± 8.51c 36.67 ± 5.51c 47.67 ± 6.66c 36.33 ± 6.51c
IF 38.00 ± 4.00 a
26.00 ± 4.00 a
31.00 ± 2.00 a
38.00 ± 4.36 a
35.67 ± 4.51 a
24.33 ± 4.04 a
29.33 ± 2.08 a
34.67 ± 4.04a
HE 47.00 ± 4.58 b
36.33 ± 4.16 b
40.67 ± 1.53 b
27.33 ± 3.06 b
45.67 ± 4.51 b
35.00 ± 4.58 ab
39.33 ± 2.08 b
24.00 ± 5.00ab
10
EA 46.00 ± 2.00 b
39.67 ± 2.52 b
50.67 ± 1.53 b
41.67 ± 2.52 c
44.00 ± 2.65 b
38.00 ± 2.00 b
50.33 ± 0.58 b
37.00 ± 2.00ab
ME 51.33 ± 2.52b 47.33 ± 1.53c 50.67 ± 1.53c 48.33 ± 3.06c 48.67 ± 2.08b 45.00 ± 1.73c 50.33 ± 0.58c 45.33 ± 0.58b
IF 56.33 ± 6.66a 42.00 ± 4.00a 53.67 ± 8.02a 49.00 ± 2.00a 55.67 ± 6.81a 41.00 ± 5.00a 52.00 ± 7.55a 45.00 ± 4.00a
HE 59.33 ± 9.07a 47.33 ± 2.56a 57.00 ± 4.58a 44.00 ± 4.00a 57.00 ± 6.58a 45.67 ± 3.51ab 55.67 ± 4.73a 40.00 ± 5.00a
20
EA 67.00 ± 3.00 ab
54.33 ± 2.52 b
71.00 ± 1.00 ab
56.00 ± 2.65 a
65.00 ± 2.65 ab
52.67 ± 3.51 b
69.67 ± 1.16 ab
52.00 ± 3.61a
ME 73.33 ± 2.52 b
67.00 ± 2.65 c
71.00 ± 1.00 b
68.67 ± 3.22 a
71.67 ± 2.08 b
66.00 ± 2.65 c
69.67 ± 1.16 b
65.33 ± 2.08b
IF 69.67 ± 6.51 a
56.67 ± 4.16 a
65.67 ± 6.66 a
64.33 ± 3.51 a
68.67 ± 6.51 a
55.67 ± 3.21 a
64.33 ± 6.51 a
62.00 ± 4.36a
30 HE 51.33 ± 38.42a 63.33 ± 3.79ab 68.00 ± 4.59a 58.33 ± 4.04a 71.67 ± 3.79a 62.00 ± 4.00ab 67.33 ± 4.04a 55.00 ± 3.46a
EA 77.00 ± 1.00a 68.33 ± 3.79b 80.67 ± 2.52ab 70.00 ± 4.36a 75.67 ± 4.16a 67.00 ± 4.36b 79.67 ± 1.53ab 64.67 ± 3.79a
ME 90.00 ± 1.73a 76.00 ± 3.00c 80.67 ± 2.52b 78.00 ± 3.00b 88.67 ± 2.52b 76.00 ± 1.00c 79.67 ± 1.53b 75.00 ± 1.00b
DMSO
Negative 8.00 ± 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
+K2Cr2O7
Control
K2Cr2O7 10.33 ± 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
5% 100.00 ± 0.00 100.00 ± 0.00 100 ± 0.00 100.00 ± 0.00 86.67 ± 10.69 86.67 ± 10.69 84.00 ± 1.00 82.00 ± 1.00
Control

ME: Methanolic extract, HE: Hexane extract, EAE: Ethyl acetate extract, IF: Infusion extract, DMSO: Diméthylsulfoxide 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).

The concentrations which inhibited 50% of DPPH (IC50) are Total phenolic content of P. guajava extracts: The total
presented in (Table 3). These results show that the hot water phenolic content of P. guajava extracts were determined in
extract had a high IC50 (low activity). The ethyl acetate and the this study using Folin-Ciocateu Reagent method and the results
methanol extract of P. guajava had the lowest IC50 (i.e. had the are presented in (Table 6). The concentration of phenolic
highest activity). The methanol extract of P. guajava had the compounds in the methanolic extract (18,536 mgGAE/mg)
lowest IC50 (i.e. the highest activity). was higher than in all other extracts. The methanolic and Ethyl
Acetate had relatively the same concentration (p>0.05) and the
Ferric reducing/antioxidant power (FRAP) of P. guajava
lowest concentration of phenolic compounds was observed in
extracts: The reducing power was determined by the Fe3+- Fe2+
the infusion extract (8.380 mgGAE/mg).
transformation in the presence of the extracts of P. guajava, and
the results obtained are shown in (Table 4). The hot water extract Total flavonoid content of P. guajava extracts: The total
showed the lowest reducing power while the standard (Vitamin flavonoid contents of the various extracts are presented in
C) exhibited the highest reducing power at the concentrations (Table 6). The result obtained showed that the methanol extract
of 100 and 200 µg/ml. At 100 µg/ml, there was no significant had the highest flavonoid content (1,991 mgCE/mg) while the
difference between the reducing power of Vitamin C (2,510 ± infusion extract showed the lowest value of flavonoid content.
0,65) and the methanolic extract of P. guajava (2,517 ± 0,01).
Cytotoxicity test: In order to evaluate the cytotoxicity effect,
However, the hot water extract showed the lowest optical
L929, HEPG2 and Hella cells were exposed to P. guajava
densities (i.e. lowest reducing power) at every concentration.
methanolic extract, for 48 h and cell grown inhibition was
The remaining extracts exhibited varied activities from one
accessed using MTT assay. In our current study, the methanolic
extract to another at each concentration.
extract exhibited CC50 of >30 µg/ml against (Table 7) the
Effects of P. guajava extracts on Nitric oxide: The results selected cell lines, suggesting that the compounds are not toxic.
of the scavenging capacity against nitric oxide were recorded
Selectivity index: The selectivity index of the methanolic
in terms of percentage inhibition as presented in (Table 5).
extract was then evaluated using the MTT assay on L929,
The extracts of P. guajava showed considerable antioxidant
HEPG2 and Hella cells in order to check that their toxicity was
potential. The methanolic and ethylacetate extracts revealed
specific to the parasite (Table 7). The impact of toxicity was
the highest percentage inhibition indicating the best nitric oxide
established by analysing the selectivity index (SI) values. In
scavenging activity. However, hexane extracts of P. guajava
our study, selectivity index values for the tested extract ranged
showed the lowest scavenging activity at every concentration.

18 J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2

Citation: Cedric Y, Payne VK, Nadia NAC, et al. In vitro anticoccidial, antioxidant activities and cytotoxicity of Psidium guajava extracts. J Parasit Dis
Diagn Ther. 2017;2(2):14-24

Table 2. Viability inhibitory percentage of P. guajava extracts on different Eimeria strains.

Incubation time and Eimeria strains
Conc µg/ml Extract 12 h 24 h
E. intestinalis E. magna E. flavescens E. stedai E. intestinalis E. magna E. flavescens E. stedai
IF 7.67 ± 3.06a 15.00 ± 2.65a 3.00 ± 4.36a 13.00 ± 1.73a 24.00 ± 11.53a 8.67 ± 2.08a 18.67 ± 3.06a 24.67 ± 3.06a
HE 9.33 ± 1.15a 24.00 ± 3.00b 5.67 ± 1.53a 4.00 ± 3.61b 20.33 ± 1.15ab 9.33 ± 1.15a 21.33 ± 2.08a 16.00 ± 2.65b
125
EA 17.33 ± 3.06 b
26.00 ± 2.00 b
14.00 ± 4.36 b
15.33 ± 3.06 b
28.67 ± 2.52 ab
17.33 ± 3.06 b
29.67 ± 4.04 b
26.33 ± 1.53b
ME 20.67 ± 2.89 b
27.67 ± 1.53 b
21.67 ± 6.03 b
16.00 ± 1.00 b
38.33 ± 4.16 b
20.67 ± 2.89 b
38.00 ± 6.56 c
28.67 ± 1.53b
IF 9.33 ± 1.53 a
17.67 ± 2.52 a
8.00 ± 1.00 a
18.00 ± 2.65 a
22.00 ± 1.00 a
9.33 ± 1.53 a
24.33 ± 1.53 a
30.33 ± 3.21a
HE 31.67 ± 3.21b 29.33 ± 3.21b 25.33 ± 6.66b 6.00 ± 3.00b 43.67 ± 3.21b 31.67 ± 3.21b 42.00 ± 6.56b 18.67 ± 2.52b
250
EA 34.33 ± 3.51b 34.33 ± 2.08b 32.00 ± 3.61b 22.33 ± 3.06b 45.33 ± 4.04b 34.33 ± 3.51b 47.67 ± 4.51b 35.33 ± 2.08b
ME 44.67 ± 8.50c 44.33 ± 5.51c 43.67 ± 6.66c 33.33 ± 6.51c 60.33 ± 6.11c 44.67 ± 8.50c 59.33 ± 6.35c 44.67 ± 4.51c
IF 32.67 ± 4.51 a
32.00 ± 4.00 a
25.33 ± 2.08 a
31.67 ± 4.04 a
45.00 ± 4.00 a
32.67 ± 4.51 a
42.00 ± 2.00 a
43.00 ± 4.36a
HE 42.67 ± 4.51 b
42.33 ± 4.16 b
35.33 ± 2.08 b
21.00 ± 5.00 b
54.00 ± 4.58 b
42.67 ± 4.51 b
51.67 ± 1.53 b
32.33 ± 3.06b
500
EA 41.00 ± 2.61 b
45.67 ± 2.52 b
38.33 ± 4.16 b
34.00 ± 2.00 b
53.00 ± 2.00 b
41.00 ± 2.65 b
55.00 ± 3.61 b
46.67 ± 2.52b
ME 45.67 ± 2.08b 53.33 ± 1.53c 46.33 ± 0.58c 42.33 ± 0.58c 58.33 ± 2.52b 45.67 ± 2.08b 61.67 ± 1.53c 53.33 ± 3.06c
IF 52.67 ± 6.81a 48.00 ± 4.00a 48.00 ± 7.55a 42.00 ± 4.00a 63.33 ± 6.66a 52.67 ± 6.81a 64.67 ± 8.02a 54.00 ± 2.00a
HE 54.00 ± 6.56a 53.33 ± 2.52a 51.67 ± 4.73a 37.00 ± 5.00ab 66.33 ± 9.07a 54.00 ± 6.56a 68.00 ± 4.58a 49.00 ± 4.00a
750
EA 62.00 ± 2.65ab 60.33 ± 2.52b 57.33 ± 1.53ab 49.00 ± 3.61b 74.00 ± 3.00ab 62.00 ± 2.65ab 74.33 ± 1.53ab 61.00 ± 2.65b
ME 68.67 ± 2.08 b
73.00 ± 2.65 c
65.67 ± 1.15 b
62.33 ± 2.08 c
80.33 ± 2.52 b
68.67 ± 2.08 b
82.00 ± 1.00 b
73.67 ± 3.21c
IF 65.67 ± 6.51 a
62.67 ± 4.16 a
60.33 ± 6.51 a
59.00 ± 4.36 a
76.67 ± 6.51 a
65.67 ± 6.51 a
76.67 ± 6.66 a
69.33 ± 3.51a
63.33 ±
1000 HE 68.67 ± 3.79a 69.33 ± 3.79ab 63.33 ± 4.04a 52.00 ± 3.46b 58.33 ± 38.42a 68.67 ± 3.79a 79.00 ± 4.58a
4.04ab
EA 72.67 ± 4.16a 74.33 ± 3.79b 69.33 ± 3.22ab 61.67 ± 3.79b 84.00 ± 1.00a 72.67 ± 4.16a 86.00 ± 3.00ab 75.00 ± 4.36b
ME 85.67 ± 2.52b 82.00 ± 3.00c 75.67 ± 1.53b 72.00 ± 1.00c 97.00 ± 1.73a 85.67 ± 2.52b 91.67 ± 2.52b 83.00 ± 3.00c
Negative DMSO 00 ± 00 00 ± 00 00 ± 00 00 ± 00 00 ± 00 00 ± 00 00 ± 00 00 ± 00
Control HBSS 00 ± 00 00 ± 00 00 ± 00 00 ± 00 00 ± 00 00 ± 00 00 ± 00 00 ± 00
Positive 100.00 ±
50µg /ml 79.00 ± 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
Control 0.00

ME: Methanol extract, HE: Hexane extract, EAE: Ethyl acetate extract, IF: Infusion extract DMSO: Diméthylsulfoxide, 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).
Table 3. DPPH radical-scavenging activities of P. guajava.
Concentration of extract (µg/mL) and scavenging activity (%)
Extracts IC50 (µg/ml)
12.5 25 50 100 200
IF 42.074 ± 1.42 bcd
46.074 ± 0.33 ab
50.370 ± 0.78 b
55.555 ± 2.65 b
70,518 ± 1,96 b
102.831 ± 22.78ab
HE 42.592 ± 3.17 bcd
47.037 ± 1.28 ab
56.666 ± 1.55 c
63.407 ± 4.20 c
86,296 ± 3,90 d
37.969 ± 13.59a
EA 44.66 ± 1.99 cd
70.518 ± 2.11 cd
88.518 ± 2.21 e
90.296 ± 0.49 e
91,925 ± 0,61 e
2.879 ± 0.20a
ME 47.185 ± 0.66d 78.740 ± 4.25cd 86.296 ± 4.10e 92.074 ± 1.33e 94,592 ± 0,32e 2.168 ± 0.27a
Vitamin C 76.178 ± 6.69e 86.186 ± 0.62e 87.262 ± 0.75e 90.157 ± 1.03e 93.465 ± 0.37e 1,295 ± 0,14a

For the same column, values carrying the same superscript 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.
Table 4. Ferric reducing power activities of P. guajava extracts.
Concentrations (µg/ml) et absorbance (à 700 nm)
Extracts
12.5 25 50 100 200
IF 0.632 ± 0.08d 0.642 ± 0.05d 0.802 ± 0.07d 0.999 ± 0.06ab 1.285 ± 0.06b
HE 0.783 ± 0.03e 0.782 ± 0.03e 0.940 ± 0.03d 1.317 ± 0.03b 1.691 ± 0.02c
EA 0.625 ± 0.06d 1.331 ± 0.04f 1.354 ± 0.04f 1.810 ± 0.02c 2.317 ± 0.07e
ME 1.691 ± 0.07g 1.940 ± 0.03h 2.31 ± 0.03h 2.517 ± 0.05d 2.908 ± 0.07g

Vitamin C 0.028 ± 0.00a 0.044 ± 0.00a 0.056 ± 0.02a 2.510 ± 0.65d 6.339 ± 0.09h

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.

between 1.01 to 20.64 µg/ml. The methanolic extract of P. flavonoids, Saponines, Steroids and Tannins, whereas, the
guajava showed the highest selectivity index value of 20.64 µg/ absence of polyphenols and terpenoids were noticed (Table 8).
ml, on L929 cells which was noteworthy as the extracts from
this plant showed good anticoccidial activity. Discussion
Phytochemical analysis: Phytochemical screening of the most In Cameroon as in all developing countries, plants are regularly
active extracts were consistent with detection of alkaloids, solicited by farmers to treat recurrent coccidioses. In this study,

J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2 19

 Cedric/Payne/Nadia/et al.

Table 5. Nitric oxide (NO) radical scavenging of P. guajava extracts.

Extracts Concentrations (µg/ml) et pourcentage d’inhibition (%)
  12.5 25 50 100 200
IF 86.295 ± 0.147a 89.23 ± 0.327ab 89.591 ± 0.269ab 89.634 ± 0.374bc 89.787 ± 0.274ab
HE 81.029 ± 0.211a 81.978 ± 2.037a 84.003 ± 0.546ab 84.349 ± 0.473b 86.738 ± 3.725ab
EA 83.271 ± 4.231b 88.594 ± 0.725ab 89.425 ± 0.798ab 89.627 ± 0.385bc 90.734 ± 0.672c
ME 85.849 ± 1.725 b
86.257 ± 0.725 c
89.647 ± 0.258 ab
88.464 ± 11.151 bc
92.349 ± 0.729c
Vitamine C 92.427 ± 3.627 c
94.595 ± 2.032 c
94.595 ± 1.339 b
96.556 ± 0.895 c
96.556 ± 0.298c
BHT 94.946 ± 0.800 c
96.429 ± 0.110 d
97.274 ± 0.526 c
97.624 ± 0.027 d
99.410 ± 0.055d

For the same column, values carrying the same superscript letter are not significantly different at p ≥ 0.05 (Student-Newman-Keuls test). ME:
Methanolic extract, HE: Hexane extract, EAE: Ethyl acetate extract.
Table 6. Total phenolic and flavonoid contents of P. guajava extracts. the wall of the oocysts and damaged the cytoplasm (sporont) as
Extracts Phenols (mgGAE/mg) Flavonoids (mgCE/mg) evidenced by the appearance of abnormal sporocysts in oocysts
Infusion 8.380 ± 0.80bc 0.494 ± 0.00ab exposed to higher concentrations. The differences between the
Hexane 10.461 ± 1.20cd 1.720 ± 0.13d four extracts in inhibiting sporulation of coccidia oocysts may
Ethyl Acetate 15.328 ± 2.13ef 1.881 ± 0.03d be due to differences in chemical composition. The observation
Methanol 18.536 ± 2.17 f
1.991 ± 0.18d
that K2Cr2O7 could not inhibit sporulation could be explained by
Along each column, values with the same superscripts are not the fact that since it is a bactericidal drug as well, it might have
significantly different, Waller Duncan (P>0.05).
killed the bacteria present thereby enhancing the sporulation
Table 7. Selectivity index, CC50 on L929, HEPG2 and Hella cells of P. of oocysts. Potassium dichromate killed bacteria in a sample
guajava methanolic extracts. containing coccidian oocysts thereby enhancing sporulation of
Plants Cell line
CC50 Sporozoidal Selectivity index coccidia oocysts. Therefore it could be that bacteria if present,
(µg/ml) IC50 (µg/ml) (µg/ml)
could have interfered with the sporulation of oocysts, possibly
L929 cells 148.83 20.64
by competing for nutrients and/or feeding on the oocysts.
P. guajava HEPG2 cells 96.24 94.99 1.01
Hella cells 129.29 1.36 The percentage of cells viability under control circumstances
(DMSO and HBSS) in this study was comparable with other
Table 8. Phytochemical screening of P. guajava methanolic extracts. studies using Eimeria species [35,36], therefore the method used
Chemical groups/Plant extract P. guajava may be considered an acceptable model. To our knowledge, this
Alkaloids + is the first study to evaluate the effects of P. guajava as inhibitors
Flavonoids + of Eimeria intestinalis, Eimeria magna, Eimeria flavescens and
Polyphenols -
Eimeria stedai sporozoites in vitro. Our findings confirm the
Tannins +
results of another study on the inhibitory effect of curcumin
Saponines +
on the activity of E. tenella sporozoites [36]. The mechanism
Steroids +
of inhibition is unknown, but may be linked to osmotic effects
Terpenoids -
attributed to extracts [37]. Schubert et al. [38] had demonstrated
we evaluated the anticoccidial and antioxidant activities of that extracellular calcium and Ca2+ signaling are essential for the
crude extracts of one African traditional medicinal plant. The invasion of E. tenella sporozoites into host cells. Extracts have
observations that P. guajava extract concentrations had an effect been shown to activate and desensitize receptors in calcium
on the sporulation of coccidia oocysts indicates that P. guajava channels [39]. It is possible that P. guajava extracts contribute
extracts are able to kill or inhibit growth and development of to the observed inhibition of sporozoite viability by disrupting
oocysts. The finding that P. guajava had the highest sporulation calcium-mediated signaling in the sporozoites.
inhibition at 30 mg/ml suggests that it is more effective in The antioxidative profile of various extracts of P. guajava
treating coccidiosis. According to our results, most extracts is a prelude to finding agent(s) that could be used to reduce
including aqueous extracts exhibited good oocysticidal activity oxidative stress associated with coccidioses. Since multiple
against Eimeria intestinalis, Eimeria magna, Eimeria flavescens characteristic reactions and mechanisms are involved in the so-
and Eimeria stedai strains. The P. guajava extract showed called oxidative stress, using a single test is not sufficient to
maximum sporulation inhibition activity at 30 mg/ml and was evaluate the antioxidant potential of plant natural compounds or
observed to be more effective against Eimeria intestinalis. extracts [40]. Therefore, many antioxidant assays such as DPPH
Similar to present findings, Molan et al. [32] also observed in- radical scavenging activity, ferric reducing/antioxidant power
vitro sporulation inhibition with aqueous extracts of pine bark and nitric oxide scavenging activity methods were chosen
(Pinus radiata) in three species of avian coccidia. Since extracts in order to evaluate the antioxidant properties of P. guajava
have been shown to inhibit endogenous enzyme activities [33], extracts.
then it is possible that P. guajava extract reduced the proportion
of sporulation by inhibiting or inactivating the enzymes The DPPH assay has been used widely to determine the
responsible for the sporulation process as in helminth eggs [34]. radical scavenging activity of antioxidant substances [41,42].
Jones et al. [34] suggested that extracts may penetrate the cell The DPPH free radical scavenging activity was significantly
wall of oocysts and cause a loss of intracellular components. In (P<0.05) higher in the methanol extract followed by Ethyl
the present study, the P. guajava extracts might have penetrated Acetate; while the infusion and the hexane extracts had the least

20 J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2

Citation: Cedric Y, Payne VK, Nadia NAC, et al. In vitro anticoccidial, antioxidant activities and cytotoxicity of Psidium guajava extracts. J Parasit Dis
Diagn Ther. 2017;2(2):14-24

DPPH free radical scavenging activity. This method is based on may therefore, serve as an indicator of its potential antioxidant
the reduction of DPPH in methanol solution in the presence of activity [50]. The observed reducing ability of P. guajava
a hydrogen-donating antioxidant due to formation of the non- extracts in the present study could be attributed to the presence of
radical form DPPH-H [43]. The extracts significantly inhibited condensed tannins as reported by Omoruyi et al. [51]. Previous
the activity of DPPH radicals in a dose-dependent manner studies of Omoruyi et al. [51] and Park and Jhon [52] correlated
and the maximum scavenging activities were observed at the the reducing power ability of plant extracts to the presence of
concentration of 200 mg per ml. The effect of antioxidants on phenolic content. The antioxidant potential and effectiveness of
DPPH radical has been thought to be due to their hydrogen condensed tannins is generally proportional to the number of
donating ability. Hence, DPPH is usually used as a substrate hydroxyl (-OH) groups present on the aromatic ring (s) as well
to evaluate ant oxidative or free radical scavenging activity of as arrangement of the hydroxyl groups and extraction processes.
antioxidant agents. In our experiment, the high DPPH radical
It is well documented that during chicken coccidiosis, the
scavenging activities of some extracts were comparable to the generation of pro inflammatory mediators, together with the
standard antioxidant, Vitamin C, suggesting that the extracts oxidative and Nitrous Oxide (NO) species, contribute principally
have some compounds with high proton donating ability and to inflammatory injury, diarrhea, mortality and weight loss [53].
could therefore serve as free radical inhibitors. However, the Therefore, substances that generate oxidative stress or have
organic extract of P. guajava demonstrated a more remarkable antioxidant properties such as n-3 fatty acids, g-tocopherol,
anti-radical activity with IC50<20 µg/ml. In fact, according curcumin, essential oil blends and green tea extracts
to Souri et al. [44], the antioxidant activities of plant extracts demonstrated certain coccidiostat effects [54]. It seems that after
are significant when IC50 <20 µg/ml, moderate when 20 µg/ parasite invasion, free radicals, together with high levels of NO
ml≤IC50 ≤ 75 µg/ml and weak when IC50>75 µg/ml. There production, are the major factors that compromise the cellular
was no significant difference (p>0.05) between IC50 values antioxidant defense system. Compounds that are meeting the
of the organic extracts and ascorbic acid. The higher radical demands of antioxidant defense system or directly interfere
scavenging activity observed in P. guajava leaves is perhaps with free radicals, such as tannins, may restore the balance
attributed to the higher condensed tannins content in these of oxidants/antioxidants, leading to improvement in intestinal
leaves. In the present study, the condensed tannins content and integrity and performance during subclinical coccidiosis [17].
the radical scavenging activity of P. guajava leaves are likely to Antioxidants act by scavenging the NO radicals [28]. Nitric
show a good relationship. Previous studies had also reported the oxide radical scavenging activity is correlated to the presence
relationship between the high level of polyphenolic compounds of phenolic compounds [55]. There was a significant decrease
and radical scavenging activity [45,46]. On the other hand, the in the NO radical due to the scavenging ability of extracts and
higher DPPH free radical scavenging activity of P. guajava ascorbic acid. The increased nitric oxide radical scavenging
extracts may be due to the potential and effective condensed activity was observed in every extract of the tested plants.
tannins source because of reactions between condensed tannins The ethyl acetate extracts showed better scavenging capacity
molecules and radicals resulting in the scavenging of radicals by compared to methanolic extract. The nitric oxide scavenging
hydrogen donation [47]. potential may be due to antioxidant principle in the extract
Antioxidants can be reductants, and inactivation of oxidants by which competes with oxygen to react with nitric oxide and thus
reductants can be described as oxido-reduction reactions [48]. inhibit the generation of nitrites.
The presence of reductants such as antioxidant substances in the Phenolic compounds exhibit antioxidant activity by inactivating
samples causes reduction of the ferric to the ferrous form which free radicals or preventing decomposition of hydroperoxide into
can be monitored by measuring the formation of Perlis prussian free radicals [56]. Flavonoids’ protective effects in biological
blue at 700 nm. The FRAP assay, therefore, provides a reliable systems are linked to their ability to transfer electrons to free
method to study the antioxidant activity of various extracts. In radicals, chelate metals, activate antioxidant enzymes and
this study, the infusion extracts had moderate reducing power; reduce radicals of alpha-tocopherol or to inhibit oxidases
the highest activity was obtained with the methanol extract [56]. The results obtained in this study showed that antiradical
and the lowest activity was obtained with the infusion. These scavenging activity was related to the phenolic content. Then,
data suggest that the extract of P. guajava may contain several the methanolic crude extract of P. guajava was found to have
compounds with intermediate polarity. The methanol extract of high phenolic contents with 18,536 mgGAE/mg and which may
P. guajava showed significantly (P<0.05) higher reducing ability be one of the reasons explaining its high antioxidant activity
compared to other extracts. Reducing power is associated with with an IC50 of 2,168 ± 0,27 (DPPH radical-scavenging
antioxidant activity and may serve as a significant reflection activity) and absorbance of 2,908 ± 0,07 at 200 µg/ml (Ferric
of the antioxidant activity. The methanol extract of P. guajava reducing power activity). There was a positive linear correlation
exhibited a higher reducing power. The reducing power of P. between antioxidant activity index and total phenolic content
guajava is mainly correlated to the presence of reductones for all the extracts. These results suggest that the phenolic
like ascorbic acid and guava is reported to be rich in ascorbic compounds contribute significantly to the antioxidant capacity
acid [49]. In the present study we observed a concentration- of the investigated plant species. In addition, these results are
dependent decrease in the absorbance of the reaction mixture consistent with the findings of many researchers who reported
for all the extracts and ascorbic acid. The reducing capacity of such positive correlation between total phenolic content and
extracts is much related to the presence of biologically active antioxidant activity [57]. However, Bajpai et al. [58] disproved
compounds (condensed tannins) with potent donating abilities the correlation between phenolic compounds and antioxidant

J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2 21

 Cedric/Payne/Nadia/et al.

activity. The results of antioxidant assays further suggest against oxidative stress which is involved in the pathology of
that these extracts contain powerful free radical scavenging several diseases in living organisms including coccidiosis in
phytochemicals that could be used to fight against free radical rabbits. They can be considered as best substitutes to chemical
upsurge, as well as oxidative stress; and consequently might anticoccidials. However further experimental studies are
ameliorate oxidative stress-associated metabolic disorders. required to explore the efficacy of P. guajava anticoccidials,
antioxidants and their modes of action.
Cytotoxicity screening is the in vitro toxicological assessment
of specific adverse effects of drugs. Assessment of the Conflict of Interest Statement
cytotoxicity P. guajava revealed that the CC50 of the methanol
We declare that we have no conflict of interest.
extract on L929, HEPG2 and Hella cell lines were above 30 µg/
ml indicating the overall safety of P. guajava. Acknowledgments
According to [30], plants were classified by their cytotoxicity The authors wish to thank Alisson Niepceron (INRA, BASE,
potential as: Tours, France) who kindly provided Eimeria intestinalis and
(a) high cytotoxicity (CC50<1.0 μg/ml), Eimeria magna strains used in the study and Dr. Michal Pakandl
for his expertise in rabbit coccidioses.
(b) moderate (CC50 1.0–10.0 μg/ml)
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*Correspondence to:
Yamssi Cedric
Department of Biology and Applied Biology
Faculty of Science
University of Dschang
PO Box 067, Dschang, Cameroon
Tel: (237) 677365519
E-mail: [email protected]

24 J Parasit Dis Diagn Ther 2017 Volume 2 Issue 2