Anthelmintic activity of Punica granatum ethanol extract against

paramphistomes in infected sheep

Keywords:
Punica granatum, paramphistomosis, EPG, FECR %
079-086 | JRB | 2014 | Vol 2 | No 1
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www.janimalsciences.com
Journal of Research in Animal Sciences
An International Scientific
Research Journal
Authors:
Lalhmingchhuanmawii K
1
,
Veerakumari L
1*
and
Raman M
2
.

Institution:
1. PG and Research
Department of Zoology,
Pachaiyappas College,
Chennai - 600 030, Tamil
Nadu, India.

2. Programme Director
(Vaccine), Translational
Research Platform for
Veterinary Biologicals,
TANUVAS, Chennai
600 007, Tamil Nadu, India.



Corresponding author:
Veerakumari L.














Email:




Web Address:
http://janimalsciences.com/
documents/AS0025.pdf.
Dates:
Received: 06 Feb 2014 Accepted: 25 Feb 2014 Published: 14 Mar 2014
Article Citation:
Lalhmingchhuanmawii K, Veerakumari L and Raman M.
Anthelmintic activity of Punica granatum ethanol extract against paramphistomes in
infected sheep.
Journal of Research in Animal Sciences (2014) 2(1): 079-086
An International Scientific Research Journal
Original Research
Journal of Research in Animal Sciences
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ABSTRACT:

Parasitic diseases remain a major threat to livestock production around the
world, particularly in India. Paramphistomosis caused by paramphistomes are one of
the most common and economically important diseases of livestock. The high
incidence of resistance to chemotherapeutics, toxicity and side effects has urged the
necessity of finding alternative plant-based anthelmintics against helminth parasites.
Therefore, the present investigation was aimed to assess the anthelmintic effect of
the rind of Punica granatum Ethanol Extract (PgEE) against paramphistomes in
infected sheep. Infected sheep were treated orally with 30 and 50 mg/ml
concentrations of PgEE. Eggs Per Gram (EPG) count on faeces, haematological and
biochemical parameters of sheep were investigated. In PgEE-treated sheep, the egg
count reduced significantly in the faeces and the reduction was proportional to
dosage and duration after treatment. The maximum reduction (97.95 %) was
observed on day 21 post-treatment with 50 mg/ml concentration of PgEE. In infected
sheep, the haemoglobin and protein content were below standard physiological
values. Improvement of haematobiochemical profile was observed in sheep after
treatment with PgEE.


INTRODUCTION
Gastrointestinal helminth infection is one of the
common and economically important diseases of grazing
livestock (Perry et al., 2002). The clinical symptoms of
GI helminth infections are decreased appetite, weight
loss, reduction of meat and milk production, foul-
smelling diarrhoea and dehydration (Githigia et al.,
2005). Constant competition for the nutrients and tissue
damage caused by the immature flukes during feeding
and migration leads to oedema, anorexia and anaemia
which ultimately results in the morbidity and mortality of
host animals particularly in young, aged and
immunosuppressed animals (Eysker and Pleoger, 2003).
Among helminth parasites, paramphistomes are the most
common and pathogenic found in the rumen and
reticulum of sheep, goats, cattle and buffaloes (Manna et
al., 1994). In domestic ruminants, high mortality rate is
due to immature amphistomes (Hassan et al., 2005).
Synthetic anthelmintics are used to control
helminth infections in livestock. However, all sheep and
goat farming countries reported anthelmintic resistance
(Jackson and Coop, 2000; Sangster and Dobson, 2002;
Kaplan, 2004). The resistance to commercially available
anthelmintic drugs and the entry of drug residues in the
food chain have instigated the search for alternative
drugs such as medicinal plants. Medicinal plants have its
origin in ethnoveterinary medicine and have been used
for the prevention and treatment of gastrointestinal
parasitism in many parts of the world (Athanasiadou
et al., 2007).
Punica granatum commonly known as
pomegranate has been the focus of classical reviews for
more than 100 years (Li et al., 2002) and various parts of
the bark, leaves, immature fruits, and fruit rind have
some therapeutic properties. In Ayurvedic medicine, the
bark and root have shown to possess anthelmintic and
vermifuge properties (Wang et al., 2010). Active
constituents isolated from the stem bark are the alkaloids
viz. pelletierine, isopelletierine, pseudopelletierine and
methyl isopelletierine (Neuhofer et al., 1993).
An experiment on a living subject is more
preferable because it provides more insight in the aspects
of the studies, therefore, in vivo studies is frequently
employed over in vitro studies. The in vivo assay such as
the faecal egg count reduction test is suitable for the
evaluation of all types of anthelmintics (Verma et al.,
2006). Furthermore, in vivo observations on the
haematological and biochemical profiles of the animals
can offer more insights into the safety and protective
efficacy of the plant extracts. Haematological parameters
such as haemoglobin (Hb), packed cell volume (PCV),
total erythrocyte count (TEC), total leucocyte count
(TLC) and differential count (DC) are important
diagnostic tools for assessing healthiness of host animals.
Biochemical profiles such as serum glucose, total serum
protein (TSP), albumin (A), globulin (G), aspartate
aminotransferase (AST) and alanine amino transferase
(ALT) are likely to be affected because of infection and
therefore may exhibit quantitative changes prior to and
after the treatment (Priya et al., 2013).
In view of the above, the present study was
undertaken to assess the efficacy of Punica granatum
Ethanol Extract against paramphistomes in naturally
infected sheep based on the changes in the number of
paramphistome eggs in the faeces, haematological and
biochemical profiles.

MATERIALS AND METHODS
The present study was undertaken on a sheep
farm in Thiruthavalli, 30 km south of Chennai, Tamil
Nadu, India. A total of 200 sheep (Madras Red) were
randomly selected for the study. The study animals were
put out to graze in a fenced area within the farm
premises.
Preparation of Punica granatum ethanol extract
About 2 kg of the rind of Punica granatum were
cleaned, shade dried and coarsely powdered. Successive
solvent extraction was done by cold percolation method
Lalhmingchhuanmawii et al., 2014
080 Journal of Research in Animal Sciences (2014) 2(1): 079-086
by soaking in hexane, chloroform, ethyl acetate and
ethanol successively in an aspirator bottle for 48 h. After
48 h, they were filtered by Whatman Filter paper No.1.
The solvent was removed by distillation using Evator
Rotary Evaporator and the extracts were concentrated
and dried in Lyodel Freeze Dryer.
The stock solution (100 mg/ml) of PgEE was
then serially diluted with water to obtain 30 and 50 mg/
ml concentrations.
Experimental animals
The experimental trial was conducted in
naturally infected sheep. Out of 200 sheep, twenty four
sheep of either sex aged six months and above naturally
infected with paramphistomes were selected based on
egg counts. Animals having faecal egg count more than
500 eggs per gram were selected and divided into four
groups of six animals each.
Experimental design
Twenty four naturally paramphistome infected
sheep were divided into four groups - group I, group II,
group III and group IV. Group I animals were dewormed
prior to the experimental period with albendazole (at the
manufacturer-recommended dose) served as uninfected
(healthy) control. Group II served as infected-untreated
control. Group III and IV were treated orally with PgEE
at two different concentrations, 30 and 50 mg/ml
respectively on day 0.
Faecal examination
Faecal samples for examination were collected
per rectum at day 0 (before treatment) and days 7, 14 and
21 post treatment. The McMasters method was
employed for the quantitative estimation of eggs per
gram (Soulsby, 1982). The percentage reduction in faecal
egg count (FECR %) in groups III and IV were
calculated.
Haematological and biochemical analysis
Blood samples were drawn from the jugular
veins with the help of a sterilized dry syringe and
transferred to two disposable blood collecting tubes, BD
Vacutainer Sodium Heparin 68 USP units and BD
Vacutainer Serum on day 0 before treatment and on days
7 and 21 post treatment from all the four groups. Blood
from BD Vacutainer Sodium Heparin 68 USP unit was
used for the evaluation of haematological studies such as
Hb, PCV, TEC, TLC, neutrophils (N), lymphocytes (L)
and eosinophils (E) (Jain, 1986). Blood from BD
Vacutainer Serum was allowed to coagulate at 37 C for
30 min. The serum was separated by centrifugation at
3000 rpm for 10 min and was used for the biochemical
assay. Serum samples were analysed for TSP, albumin,
globulin, A/G ratio, glucose, AST and ALT.
Statistical analysis
All the data obtained in the present study were
statistically analysed using the statistical software SPSS
version 16.0. One-way Anova using Bonferroni test was
applied to find out the significant difference between
healthy, infected-untreated and PgEE-treated sheep.

RESULTS AND DISCUSSION
The mean EPG and FECR % in healthy, infected
-untreated and PgEE-treated sheep were given in Table
1. Throughout the study period, the faeces of healthy
sheep (Group I) contain no eggs of paramphistomes. In
contrast, faeces of infected-untreated (Group II) showed
absolutely high counts during the whole period of
observation. In PgEE-treated sheep (Group III and IV),
the egg counts reduced significantly on day 7 post-
treatment and continued to decline further till day 21 post
-treatment. The maximum reduction was recorded as
97.95 % happening on day 21 after treatment with
50 mg/ml of PgEE. The EPG counts and FECR % on
days 0, 7, 14 and 21 are significantly different (P < 0.01).
The statistical analysis (Bonferroni test) of
Table 1 showed that the values of all the four groups
(I-IV) differ significantly on days 0, 7, 14 and 21
(P <0.01). The values of group II on the different days of
observations (0, 7, 14 and 21) do not differ significantly
(P > 0.05). There is significant difference (P <0.05)
Lalhmingchhuanmawii et al., 2014
Journal of Research in Animal Sciences (2014) 2(1): 079-086 081


between values of group III and IV on all days of
observations. There is no significant difference between
the egg counts of all the three groups (II-IV) on day 0.
The results of the haematological indices were
given in Table 2. The Hb, PCV, TEC and TLC in healthy
sheep (Group I) were in a range of 12-13 g %, 33-34 %,
910
5
/cmm and 9-1110
3
/cmm respectively throughout
the period of investigation whereas the Hb, PCV and
TEC level of the infected sheep was lesser than the
healthy sheep. High level of TLC was observed in
infected sheep. Treatment with 30 and 50 mg/ml
concentrations of PgEE gradually improved the Hb, PCV
and TEC to normal range and the improvement level was
dose and time dependent. The TLC level was also
brought down to a count comparable to normal range
after treatment with 30 and 50 mg/ml concentrations of
PgEE on 21 days PT. Significant reduction of neutrophil
and eosinophil was observed after treatment with PgEE.
Infected sheep treated with 30 and 50 mg/ml
concentrations of PgEE increased the lymphocyte level
in groups III and IV gradually.
The statistical analysis (Bonferroni test) of
Table 2 revealed that the values of all the four groups
(I-IV) differ significantly on days 0, 7 and 21 (P < 0.01).
There is significant difference (P < 0.05) between values
of group III and group IV on all days of observations.
The values of group II on the different days of
observations (0, 7 and 21) do not differ significantly
(P >0.05). There is no significant difference (ns) from
the respective group I values (P > 0.05).
The observations on the biochemical profiles of
paramphistomes infected sheep before and after
treatment with 30 and 50 mg/ml concentrations of PgEE
are presented in Table 3. The TSP level of healthy sheep
(Group I) was approximately 7 g/dl throughout the
period of investigation whereas the TSP level of the
infected-untreated sheep (Group II) was significantly
less, about 4 g/dl. The TSP level of the infected sheep
significantly increases following treatment with PgEE.
The albumin, globulin and A/G ratio of infected sheep
was found to be lower than the healthy sheep, but
showed a similar trend of increase following treatment
with PgEE. Treatment of infected sheep with 30 and 50
mg/ml concentrations of PgEE had no significant effect
on the glucose level. High levels of AST and ALT were
observed in the infected sheep but the levels returned to
normal after treatment with PgEE.
Lalhmingchhuanmawii et al., 2014
082 Journal of Research in Animal Sciences (2014) 2(1): 079-086
Groups
Pre-treatment
(0 day)
Days post-treatment
EPG count FECR %
7 14 21 0 7 14 21
I 0 0.00 0 0.00 0 0.00 0 0.00 - - - -
II 564 34.29 552 32.79 564 30.75 576 25.17 - - - -
III 576 48.00 144 15.17 120 09.79 72 05.36 - 75.00 79.16 87.50
IV 588 27.79 48 09.79 24 06.19 12 04.89 - 91.83 95.91 97.95
Table 1 EPG count and FECR % in healthy (Group I), infected-untreated (Group II)
and PgEE-treated (Groups III and IV) sheep
Each value represent mean SD of n = 6
Group I - Healthy sheep
Group II - Infected-untreated sheep
Group III - Infected sheep treated with 30 mg/ml PgEE
Group IV - Infected sheep treated with 50 mg/ml PgEE
The statistical analysis (Bonferroni test) of Table
3 exposed that the values of all the four groups (I-IV)
differ significantly on days 0, 7 and 21 (P < 0.01) except
for glucose content which does not show any significant
difference (P > 0.05). There is significant difference
(P < 0.05) between values of group III and group IV on
all days of observations. The values of group II on the
different days of observations (0, 7 and 21) do not differ
significantly (P > 0.05). There is no significant
difference (ns) from the respective group I values (P >
0.05).
In the present investigation, the EPG count of
paramphistome eggs were significantly reduced
following oral administration of PgEE. Priya et al.,
Lalhmingchhuanmawii et al., 2014
Parameters Days
Groups
I II III IV
Hb (g%)
0 13.02 0.81 07.48 0.33 07.78 0.54 07.12 0.39
7 12.59 0.47 07.15 1.03 08.41 1.19
ns
10.22 0.43
21 12.68 0.50 07.41 0.75 09.72 1.21
ns
12.69 0.93
ns

PCV (%)
0 33.55 0.99 23.64 0.53 25.22 1.07
ns
23.22 0.41
7 34.09 0.44 24.00 0.85 29.24 0.61 34.85 0.42
ns

21 34.59 0.53 23.33 0.25 32.44 0.48 35.19 0.32
TEC
(10
5
/cmm)
0 09.05 0.49 05.68 0.35 06.26 0.34 05.23 0.35
7 09.29 0.28 06.18 0.14 07.70 0.37 09.30 0.39
ns

21 09.06 0.39 05.92 0.51 09.18 0.67 09.30 0.36
TLC
(10
3
/cmm)
0 09.25 0.34 14.36 0.38 14.37 0.36 15.05 0.68
ns

7 10.48 0.14 15.24 0.38 09.68 0.48 11.33 0.44
21 11.82 0.47 14.58 0.34 11.28 0.56
ns
11.64 0.12
N (%)
0 28.95 0.45 42.11 0.52 43.29 0.41 42.61 0.51
7 30.48 0.76 42.74 0.57 34.38 0.44 31.91 0.67
21 33.25 1.06 43.39 0.57 31.95 0.70
ns
29.43 0.59
ns

L (%)
0 57.20 0.44 42.36 0.85 41.81 0.73
ns
43.81 0.27
7 58.11 0.95 40.91 0.54 57.23 0.57
ns
59.10 0.50
ns

21 59.21 0.42 43.11 0.30 58.21 0.37 60.25 0.28

E (%)
0 01.90 0.28 05.40 0.14 05.30 0.37 05.40 0.29
7 01.40 0.17 05.60 0.14 03.40 0.26 02.20 0.57
ns

21 02.30 0.35 05.90 0.25 02.80 0.20 01.70 0.17
Table 2 Haematological parameters of healthy (Group I), infected-untreated (Group II)
and PgEE-treated (Groups III and IV) sheep
Each value represent mean SD of n = 6
Group I - Healthy sheep
Group II - Infected-untreated sheep
Group III - Infected sheep treated with 30 mg/ml PgEE
Group IV - Infected sheep treated with 50 mg/ml PgEE

Standard physiological values Hb (8-16 g%); PCV (24-40 %); TEC (8-15 10
5
/cmm);
TLC (4-12 10
5
/cmm); N (25-35 %); L (60-70 %); E (1-3 %)
Journal of Research in Animal Sciences (2014) 2(1): 079-086 083


(2013) reported a similar reduction in faecal egg count of
sheep infected with paramphistomes after treatment with
aqueous extract of pods of Acacia concinna. Faecal egg
count reduction of gastrointestinal nematodosis in Garole
sheep treated with ivermectin, levamisole and
albendazole was also reported by Pandit et al., (2009).
The present findings are further supported by the studies
of Verma et al., (2006) and Diaz et al., (2006), who
Lalhmingchhuanmawii et al., 2014
Parameters Days
Groups
I II III IV
Total serum
protein (g/dl)
0 07.12 00.55 04.87 00.39 05.14 00.21 04.74 00.40
7 07.25 00.53 04.49 00.24 06.39 00.34 06.84 00.32
21 07.54 00.25 04.84 00.32 07.27 00.31 07.65 00.27
Albumin (g/dl)
0 03.03 00.62 01.03 00.30 01.23 00.28 01.17 00.38
7 03.16 00.32 01.28 00.31 03.16 00.43
ns
03.25 00.40
ns

21 03.67 00.11 01.46 00.16 03.06 00.32
ns
03.56 00.19
Globulin (g/dl)
0 04.24 00.38 02.35 00.06 02.39 00.19 02.70 00.40
ns

7 05.06 00.23 03.08 00.20 03.61 00.17 04.12 00.25
21 04.68 00.55 02.72 00.33 04.32 00.51
ns
04.73 00.39
A/G ratio
0 00.71 00.15 00.43 00.13 00.51 00.13
ns
00.43 00.15
ns

7 00.62 00.05 00.40 00.08 00.87 00.14
ns
00.78 00.09
21 00.78 00.09 00.54 00.08 00.71 00.10 00.75 00.04
Glucose (g/dl)
0 40.50 01.47 41.70 00.64 42.50 00.38 43.70 00.44
ns

7 43.40 00.40 42.60 00.49 40.60 00.43 44.90 00.43
21 45.10 00.43 45.70 00.44 44.20 00.44
ns
45.20 0.58
ns

AST (U/L)
0 139.01 00.56 166.92 00.50 168.12 00.29 165.74 00.35
7 136.32 00.44 169.14 00.24 153.71 00.61
ns
149.68 00.44
21 140.39 00.42 170.08 00.71 142.12 00.27 138.89 00.74
ns


ALT (U/L)
0 28.40 00.42 35.20 00.31 36.24 00.61
ns
39.24 00.36
7 29.16 00.35 40.30 00.63 33.14 00.32 31.16 00.67
ns

21 32.24 00.29 38.42 00.83 30.09 00.54
ns
29.11 00.65
ns

Table 3 Biochemical profile of healthy (Group I), infected-untreated (Group II)
and PgEE-treated (Groups III and IV) sheep
Each value represent mean SD of n = 6
Group I - Healthy sheep
Group II - Infected-untreated sheep
Group III - Infected sheep treated with 30 mg/ml PgEE
Group IV - Infected sheep treated with 50 mg/ml PgEE

Standard physiological values Total serum protein (6-7.9 g/dl); Albumin (2.4-3.9 g/dl);
Globulin (3.5-5.7 g/dl); A/G ratio (1-1.2); Glucose (40-80 g/dl); AST (98-278 U/L); ALT (24-83 U/L)
084 Journal of Research in Animal Sciences (2014) 2(1): 079-086
reported a significant reduction in egg count in buffaloes
and cattle naturally infected with paramphistomes after
treatment with chemical anthelmintics.
A decrease in Hb, PCV, TEC, lymphocytes, TSP,
albumin, globulin, A/G ratio and increase in TLC,
neutrophils, eosinophils, AST and ALT levels were
observed in the infected sheep prior treatment. The fall in
Hb, PCV and TEC probably implies that the animals
were suffering from anaemia, due to the haemorrhage
caused by the immature parasites. Verma et al., (2006)
and Diaz et al., (2006) also reported a decrease in Hb,
PCV and TEC in cattle naturally infected with
paramphistomes. A lower value of TSP, albumin and
globulin and high values of AST and ALT in cattle and
buffaloes infected with paramphistomes were also
reported by Bharti and Prasad (2001).
The fall in the protein level observed in the
present study might be due to haemodilution, a
compensatory mechanism for intestinal haemorrhage
caused by migrating flukes and later on due to loss of
large quantities of serum protein in the gut through
exudation. Hypoproteinemia in helminthosis is primarily
the result of hypoalbuminaemia. Hypoproteinemia,
coupled with loss of appetite, seems to be the most
important pathophysiological consequence of
paramphistomosis (Priya et al., 2013). The changes in
serum biochemical constituents during Fasciola/
Paramphistomum infection reflect disturbances in liver
function caused due to tissue damage and fluid loss
caused by the parasites in situ. Recovery from the
damages of this vital organ following treatment of the
infected animals may result in the resumption of liver
functions and restoring the body fluid balance (Bharti
and Prasad, 2001). The levels of AST and ALT reduced
after treatment with 30 and 50 mg/ml concentrations of
PgEE and the decline in AST and ALT level was
proportional to dosage and duration. A similar result of
the decline in AST and ALT was reported by Bharti and
Prasad (2001) in cattle following treatment with
oxyclozanide.
Due to PgEE treatment, the levels of Hb, PCV,
TEC, TLC, neutrophils, lymphocytes, eosinophils, TSP,
albumin, globulin, A/G ratio, AST and ALT approached
normal values in paramphistome infected sheep. This
study clearly revealed the anthelmintic efficacy of PgEE
against paramphistomes. Similar findings on the
improvement in haematological and biochemical profiles
of treated animals using synthetic anthelmintic drugs
have been reported by several researchers (Bharti and
Prasad, 2001; Diaz et al., 2006; Verma et al., 2006).
Priya et al., (2013) also reported a similar outcome on
the haematological and biochemical profiles of the sheep
infected with paramphistomes following treatment with
aqueous extract of pods of Acacia concinna.

CONCLUSION
Observations, in the present study, on EPG count
and haemato-biochemical parameters among healthy,
infected-untreated and PgEE-treated sheep revealed that
PgEE is highly effective against paramphistomes causing
an improvement in the health status of the infected host.
Therefore, PgEE could be successfully used as an
anthelmintic to treat paramphistomosis in livestock.

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