Accepted Manuscript Title: Anthelmintic activity of ocimum sanctum leaves extract against ovine gastrointestinal nematodes in india Author: Dharmendra Kanojiya, Daya Shanker, Vikrant Sudan, Amit Kumar Jaiswal, Rahul Parashar PII: DOI: Reference:
S0034-5288(15)00042-9 http://dx.doi.org/doi: 10.1016/j.rvsc.2015.01.017 YRVSC 2812
To appear in:
Research in Veterinary Science
Received date: Accepted date:
14-10-2014 28-1-2015
Please cite this article as: Dharmendra Kanojiya, Daya Shanker, Vikrant Sudan, Amit Kumar Jaiswal, Rahul Parashar, Anthelmintic activity of ocimum sanctum leaves extract against ovine gastrointestinal nematodes in india, Research in Veterinary Science (2015), http://dx.doi.org/doi: 10.1016/j.rvsc.2015.01.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Anthelmintic activity of Ocimum sanctum leaves extract against ovine gastrointestinal nematodes in
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India
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Dharmendra Kanojiya, Daya Shanker, Vikrant Sudan*, Amit Kumar Jaiswal, Rahul Parashar
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Department of Parasitology,
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College of Veterinary Sciences & Animal Husbandry,
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U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go
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Anusandhan Sansthan (DUVASU), Mathura- 281001, INDIA.
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*Corresponding authors email: [email protected]; +91 9045414200
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Highlights
In vitro and in vivo efficacy of extracts of Ocimum sanctum on sheep helminthosis Phytochemical studies to know the active ingredients Assessment of blood, enzymes and antioxidant parameters for safety trail Detection of significant anthelminthic efficacy
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ABSTRACT Leaves of Ocimum sanctum have been traditionally used for various ethno-veterinary
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practices as well as medicinal purpose.
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aqueous and hydro-alcoholic extracts of the bulb of Ocimum sanctum was investigated.
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Alkaloids, carbohydrates, steroids and tannins were identified in phytochemical analyses. The
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various blood parameters coupled marker enzymes and anti oxidant status, were also evaluated
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during in vivo trial. Aqueous extract showed better EC50 and EC99 values in comparison with
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methanolic extract in egg hatch assay and larval development test, respectively. However, in
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larval paralysis test, both aqueous and methanolic extracts showed almost similar efficacy. A
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77.64 % reduction in faecal egg output was observed on day 14. No deleterious ill effect was
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found in any of the haematological and biochemical parameters suggesting that the plant could
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be safer for use in sheep.
In vitro ovicidal and larvicidal potential of crude
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Keywords: Anthelminthic activity Gastrointestinal nematodes
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India
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Ocimum sanctum
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Sheep
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1. Introduction
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With the emergence of anthelminthic resistance ( the parasitic control programme
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suffered gradual setback in several countries forcing the researchers to the search for alternative
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control methods (Dhar et al., 1982). Anthelminthic resistance has been reported for almost all the
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commercially available anthelminthics in the Indian market (Jaiswal et al., 2013). Ethno
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veterinary alternatives are considered to be of immense potential in overcoming the
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anthelminthic resistance due to synthetic chemical compounds (Lans et al., 2007). Worldwide
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research has shown that certain plants can effectively be used to reduce the degree of parasitism
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and hence, they can be seen as promising alternatives to the conventional chemical anthelmintics
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(Githiori et al., 2006).
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Ocimum sanctum (commonly known as Tulsi), is native throughout the Eastern World
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tropics and widespread as a cultivated throughout India. The leaves are the most commonly
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medicinally used plant part worldwide for many centuries. In herbal medicine, it is used to
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prevent cancer (Pandey, 2009), used as an immunomodulator (Mukherjee et al., 2005),
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hepatoprotectant and haematinic activities (Chattopadhyay et al., 1992). Besides these, it also 3
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possess some anthelminthic activity against roundworms particularly ascarids (Singh and
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Nagaich, 2000). The present study was designed to assess the anthelminthic potential of various
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extracts of leaves of O. sanctum on naturally occurring gastrointestinal nematodes of sheep using
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standard in vitro and in vivo tests.
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2. Materials and methods
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2.1. Collection and processing of plant material
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Fresh leaves of O. sanctum were collected from from the plants grown within the
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premises of Veterinary University, Mathura, India. The plants were later identified by the
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Department of Botany, Babu Shivnath Agrawal College, Mathura and the specimens were
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recorded in the form of voucher number (DUVASU-2014-OS) for the sake of further references.
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The leaves were cleaned from adulterants and air dried under shade at a well-ventilated place.
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The plant material was pulverized to powder form with a mixer grinder and stored in air tight
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containers.
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2.2. Preparation of crude extracts
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Extracts from the leaves of O. sanctum were prepared in water and methanol (S.D Fine
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Chem. Ltd.). For preparing aqueous extract, 100g of powdered leaves were taken in a beaker
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and suspended in 600ml of water and was continuously stirred using magnetic stirrer for 4 hours.
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Again, for preparing methanolic extract 100 g of powder was soaked in 500 ml of methanol
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solvent to exhaustion (120 h). The extraction was carried out in a percolator by a combination of
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maceration and percolation at room temperature. The filtrates of both the extracts were collected
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through Whatmann filter paper No. 4. The removal of the solvent was performed at a temperature
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below 40°C,
under reduced pressure and a rotation speed of 20 rpm in vacuum rotary evaporator. 4
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The extract was scrapped off, transferred to an air-tight container and stored in a freezer at −20
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°C till subsequent use.
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2.3. Determination of extraction yield (% yield)
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The yield (%, w/w) from all the dried extracts was calculated as:
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Yield (%) = (W1 x 100)/W2
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where, W1 was the weight of the extract obtained after evaporation of solvent; and W 2
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was the weight of the plant powder. Later both the extracts (100mg each) were dissolved in 1ml
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of distilled water to get 100mg/ml concentration and subsequent lower dilutions were prepared
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(100 to 0.781 mg/ml) by using two fold dilution. The extracts were kept in air tight containers at
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40C to avoid loss of any volatile principles or/and activities till further use.
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2.4. Phytochemical analysis of crude extracts
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Both the extracts were tested for the presence of active principle like carbohydrate,
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steroids, tannins, flavonoids, alkaloids, glycoside, fat and proteins using standard procedures
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(Debela, 2002).
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2.5. In vitro tests
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2.5.1. Egg hatch test
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The eggs of GI nematodes were recovered using the method described by Taylor et al.
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(2002) and recommended by the World Association for the Advancement of Veterinary
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Parasitology, WAAVP (Coles et al., 1992). Briefly, about 10 g of faeces were collected directly
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from the rectum of naturally infected sheep and was mixed and homogenized with tap water. The
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mixture was sieved through strainer and centrifuged for 2 minutes at 2000 rpm, the supernatant 5
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was discharged and after the tube was filled with saturated solution, cover slips were put over the
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meniscus for 5 minutes and were later washed to collect the eggs. The egg suspension, with a
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concentration of 100 eggs/0.2 ml, was distributed in 24–multiwell plate (50 μl per well) along
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with 50 μl of extract of each tested concentration. Three replicates
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concentration. The plates were incubated, under humidified conditions, at 27°C for 48 h and later
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a drop of Lugol’s iodine solution was added to each well to stop further hatching. Hatched larvae
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and unhatched eggs were then counted under the microscope.
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2.5.2. Larval development test
were made for each
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The Micro agar larval development test was performed as per the standard protocol of
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Coles et al. (2006). Briefly, 10 µl of individual concentration of each extract was placed in wells
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in triplicates and 150 µl of 2% bacto agar at 45 0C was added in triplicate form into each well
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and allowed to cool at room temperature. Thereafter, 10 µl of egg suspension, diluted in
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Amphoteracin B (1:1) was poured upon the cooled agar. Then, 10 µl of yeast extract, prepared
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in Earle’s salt solution (Hubert and Kerbouf, 1984), was added to it. The plates were sealed with
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parafilm and incubated at 25 0C for 7 days and the number of live L3 in each well was counted
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thereafter.
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2.5.3. Larval Paralysis test
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Larvae were cultured according to MAFF (1986) using Bearmann’s apparatus. 100μl of
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suspension (approx 100 larvae) was added to 100μl of each concentration, in triplicate form, in
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the 96-well plate and were kept at room temperature for 24 h. The live (motile) and dead larvae
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were counted.
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Albendazole at the dose of 0.125 mg/ml was used as a positive control while distilled
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water served as negative control in all the three tests (Kanojiya et al., 2014).
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2.6. In vivo test
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2.6.1. Faecal Egg Count Reduction Test (FECRT)
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The same sheep flock that was used for the collection of eggs for the in vitro trial was
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again used for in vivo trial. The flock consisted of local Chokla breed of sheep that were
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naturally infected with gastrointestinal parasites. Single pooled larval culture, one from each
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group, was done on day 0 in order to find out the species of GI parasites involved. The flock was
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divided into three groups of fifteen animals each. Group 1 served as a negative control and
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received no treatment while Group 2 served as a positive control and was given a single dose of
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Albendazole at the dose of 7.5mg/kg bodyweight. Group 3 was drenched a single oral dose of
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aqueous extract at the dose of 5g/animal by making final volume of 5 ml with water (Kanojiya et
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al., 2014). Fecal samples of animals were individually collected on day 0, 7, 14 and 21 post-
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treatment. These samples were processed for egg per gram (epg) calculation using the McMaster
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method (Soulsby, 1982). FECRT efficacy was calculated according to Dash et al., (1988) using
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the following formula:
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where T is average fecal egg count of treated group; C is average fecal egg count of control
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group; 1 is pre-treatment average fecal egg count ; 2 is post-treatment average fecal egg count .
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Each group of sheep was housed separately but were allowed to graze altogether
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freely during the day time. Again on day 21 post treatment, single pooled larval culture, one
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from each group, was done to identify the species of GI parasites surviving the herbal treatment.
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2.7. Blood sample for haematology
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Blood samples were obtained on day 0 and day 14 from each animal via. Jugular
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vein puncture into 10 ml vacutainer. Different blood parameter were analyzed using
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Haematology analyser (Diatron) and Semi auto chemistry analyzer (Electronics India) using
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serum [Serum Glutamic Pyruvate Transaminase
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Transaminase (SGOT)], whole blood [ Red Blood Cells (RBCs), White Blood Cells (WBCs),
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Haemoglobin (Hb), Haemocrit (HCT), Mean Corpuscular Volume (MCV), Red Blood Cell
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Distribution Width (RDWc), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular
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Haemoglobin Concentration (MCHC), Platelet Count (PLT), Mean Pack Volume (MPV),
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Procalcitonin (PCT), Lymphocyte %, Monocyte % and Granulocyte %) and plasma (Ferric
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Reducing Antioxidant Power FRAP). Total antioxidant activity was measured by FRAP assay
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(Benzie and Strain 1999). Briefly, plasma (100 l) was mixed with 3 ml of working FRAP
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reagent and absorbance was measured at 0 minute after vortexing. Thereafter, samples were
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placed at 37°C in a water bath and absorbance was again measured after 4 min. Ascorbic acid
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standards (100 M-1000 M) were processed in the same way.
FRAP value of sample (mol/L) =
A-B
(SGPT), Serum Glutami cOxaloacetic
X 100
X-Y
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where A is reading of the sample at 0 minute; B is reading of the sample at 4 minute; X
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reading of the standard at 0 minute; Y is reading of the standard at 4 minute; and 100 is FRAP
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value of 100 M standard solution.
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2.8. Statistical analysis
is
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The extract concentration required to inhibit 50% and 90% egg hatching (EC50 and EC90)
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was calculated by using log probit analysisusing SAS software (SAS, 1998). The results of the
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tests and the various haematological parameters were expressed as mean ± standard error of
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mean (S.E.). Means were evaluated by t-test to compare the anthelmintic efficacy at different
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concentrations along with the haematological parameters also. p values ≤ 0.05 were considered
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statistically significant.
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3. Results
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3.1. Extraction yield (% yield) of the various extracts
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Slight variation in yields of the two extracts was observed. The methanolic extract was
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having lower yield (11.25%) in comparison with aqueous (11.69%). extract.
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3.2. Phytochemical analysis of different crude extract
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The aqueous extract was positive for alkaloids, carbohydrates, steroids and tannins while
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the methanolic extract was also positive for alkaloids and tannins only (Table 1).
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3.3. In vitro tests
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3.3.1. Egg Hatch Test
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Both the extracts inflicted significant inhibition of egg hatching in a dose dependent
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manner. The aqueous extract (EC50=5.827 and EC99 =37.144mg/ml) of O. sanctum was found to
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be more effective in having ovicidal properties in comparison to the
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(EC50=6.218 and EC99 =21.32mg/ml).
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3.3.2. Larval Development Test
methanolic extract
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Again a dose dependent relationship was observed in both the extracts. The aqueous
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extract (EC50=7.746 and EC99 =28.199 mg/ml) of O. sanctum was again found to be more
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effective in having larvicidal properties in comparison to the methanolic extract (EC50=12.179
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and EC99 =64.721 mg/ml) in larval development test.
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3.3.3. Larval Paralysis Test
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Similar finding to the findings described above for the other tests, the Larval paralyisis test also
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followed a dose dependent trend.
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=368.628mg/ml) and the methanolic (EC50=12.982and EC99 =358.27mg/ml) counterpart showed
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almost similar efficacy in the Larval paralysis test
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Howewer, the aqueous extract (EC50=16.486 and EC99
Albendazole (0.125mg/ml) that was used as a positive control showed 100% efficacy in
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all the three tests as presented in Tables 1, 2 and 3.
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3.4. In vivo test
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3.4.1. Faecal Egg Count Reduction Test (FECRT)
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Results from coprocultures reveled that on day 0 the flock was mainly infected
with
Haemonchus spp.(85%) followed by Trichostrongylus spp. (7%), Oesophagostomum spp. (5%) 10
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and Strongyloides spp. (3%). Results from the in vivo trial demonstrated that on day 7 pos
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treatment the FECR was 74.12% and 26.47% for albendazole and O. sanctum, respectively.
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However, at day 14 the FECR were 94.4% and 77.64% for albendazole and O. sanctum,
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respectively. On day 21 post treatment, albendozole still had 90.2% effectiveness in reduction of
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faecal egg count while O. sanctum caused 39.41% reduction in faecal egg count. (Table 4). The
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results from coprocultures at day 21, demonstrated a proportion of Haemonchus spp. (92%)
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by Trichostrongylus spp. (4%), Oesophagostomum spp. (3%) and Strongyloides spp. (1%) in O.
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sanctum treated animals. Coproculture of control group showed no variation from the results of
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Day 0 where as coproculture of albendazole treated group demontrated Haemonchus spp.(95%)
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followed by Trichostrongylus spp. (3%), Oesophagostomum spp. (1%) and Strongyloides spp.
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(1%).
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3.5. Blood Haematology
followed
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The details of the blood parameters examined viz., SGPT, SGOT, RBCs, WBCs, Hb,
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HCT, MCV, RDWc, MCH, MCHC, PLT, MPV, PCT, Lymphocyte %, Monocyte%
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Granulocyte % and FRAP are provided in Table 5. Significant changes were observed in SGPT,
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RBCs, Hb, RDWc, PLT, PDWC, Lymphocyte and Granulocyte values, while the rest of
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parameters showed non significant alterations within the normal range in the stipulated time of
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herbal trial (Details given in Table 5).
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4. Discussion
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The aim of the present study was to evaluate the in vitro and in effect of O. sanctum in
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comparison to a reference drug-albendazole against the gastrointestinal nematodes of sheep. 11
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Results revealed both the aqueous and methanolic extracts of O. sanctum effectively targeted
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both the egg and larval developmental stages of the parasite in a dose dependent manner.
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Significant effects in terms of non hatching of eggs, non development of larvae and paralysis
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and/or death of the larvae at different time durations, were observed in in vitro trials. The reason
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for evaluating only
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performance in the in vitro tests in comparison with the methanolic extract.
the aqueous extract of O. sanctum for the in vivo trial was based on its better
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The compound Eugenol, present in O. sanctum essential oil, is attributed to be the
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putative anthelmintic principle of the plant (Asha et al., 2001). In vitro testing of essential oils of
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Ocimum sanctum and purified eugenol showed potent anthelmintic activity in the
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Caenorhabditis elegans model (Asha et al., 2001). The essential oil of O. gratissimum, as well as
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eugenol, was found effective in inhibiting eclodibility of H. contortus eggs (Pessoa et al., 2002).
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Bihari et al. (2010) screened the activity of methanolic extract of O. sanctum against healthy
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adult Indian earthworms, Pheretima posthuma and its efficacy at the same level as albendazole
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when compared using in vitro tests.
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O. sanctum is reported to block the glucose uptake by the nematode parasites, thereby
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altering with their mobility and survivability (Singh and Nagaich, 2000). Alkaloids,
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carbohydrates, steroids and tannins were the main secondary compounds found in the present
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study. There are many reports of reduction in faecal egg count in sheep treated with plants rich in
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tannins (Githiori et al., 2006). Tannins interact with proteins in the nematode cuticle thereby
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considerably changing its chemical and physical properties (Athanasiadou et al., 2001).
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Flavonoids are also known to possess action against H. contortus (Camurça-Vasconcelos et al.,
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2007).
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Toxicity of O. sanctum extract was one of the main concerns, however, no deleterious ill effects
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were found
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plant is safe for use in sheep. Although, significant changes were observed in SGPT, RBCs, Hb,
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RDWc, PLT, PDWC, Lymphocyte and Granulocyte values, the rest all the parameters showed
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non significant alterations and were within the normal range in the stipulated time of herbal trial.
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Increase in SGPT values might be attributed to the consumption of large amount of herbal
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extract.
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haematinic and hepatoprotectant role of O. sanctum in the treated animals (Chattopadhyay et al.,
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1992). Again O. sanctum caused marked decrease in the stress of animals due to parasitism as
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shown by FRAP values. This could be very much contributed to antioxidant properties of the
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plant (Saija et al., 1995). These antioxidant effects are supposed to be due to the presence of
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flavonoids (Saija et al., 1995). O. sanctum appears to modulate both humoral and cell-mediated
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immune responses (Mukherjee et al., 2005).
in any of the haematological and biochemical parameters observed suggesting that the
RBCs, Hb and RDWc values showed significant increase which could be due to
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As a matter of fact, in the majority of the cases, the anthelmintic activity of these
270
ethanopharmacologically important plants has been found to be lower and/ or below that
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reported for synthetic anthelmintics. Again, plants having moderate anthelmintic activity should
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also still be considered, while they are definitely not useful as a sole alternative but may still be
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valuable as part of an integrated approach specifically designed to achieve sustainable parasite
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control in small ruminant production systems. Further studies on the standardization of dose and
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identification of exact mechanism of action of these extracts against nematodes are thereby
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warranted. Although it will take time and expertise to standardize the plant anthelmintics and
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address the recent emergent trends in anthelmintics, the use of herbal remedies as anthelmintics
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based on local plants will at least offer a cheap, reliable and a readily available alternative to 13
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highly expensive and unavailable conventional anthelmintics to the poor farmers worldwide. The
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findings from the present study will surely contribute in the field of plant anthelmintics to
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develop sustainable, effective and safe alternatives to conventional anthelmintics.
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Acknowledgement
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The authors are highly thankful to the Vice Chancellor, DUVASU for the facilities
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provided.
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Conflict of Interest
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The authors declare that they are having no conflict of interest.
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and Applied Biology 9, 47–52.
352 353 354 355
Soulsby , E.J.L., 1982. Helminths, arthropods and protoza of domesticated animals 7th edn. The English Language Book Society and Bailliere Tindall, London, pp234-235. Taylor, M.A., Hunt, K.R., Goodyear. K.L., 2002. Anthelmintic resistance detection methods. Veterinary Parasitology 103, 183–194.
356 357 358 359 360 361 362 363 364 365
17
Page 17 of 24
366 367
Table 1: Chemical constituents in aqueous and methanolic extract of O. sanctum
Steroids
Glycosides
Tannins
Protein
Methanolic
Carbohydrates
Aqueous
Fat
Ocimum sanctum
Nature of extract
Flavonoids
Plant
Alkaloids
Plant constituents found as per the methods of Debela 2002
+
-
-
+
+
-
+
_
+
_
_
_
_
_
+
_
368 369 370 371 372 373 374 375 376 377 18
Page 18 of 24
378 379
Table 2: Percentage efficacy of aqueous and methanolic extracts of O. sanctum using various in vitro tests.
380 381 Ocimum sanctum Egg Hatch Assay
Concentration (mg/ml)
Larval Development Test
Larval Paralysis Test
100
Aqueous extract 100
Methanolic extract 100
Aqueous extract 100
Methanolic extract 100
Aqueous extract 100
Methanolic extract 100
50
100
100
100
100
90.±4.1
92.8±4.7
25
100
100
100
71.6±8.9
53.3±3.3
57.9±5.6
12.5
100
97.5±2.5
72.1±3.7
55.4±7.6
44.9±5.7
52.4±4.1
6.25
52.8±6.2
41.7±9.1
16.5±4.6
11.2±4.1
10.3±4.3
39.9±3.2
3.12
20.1±3.9
11.6±2.9
7.5±3.2
3.1±2.1
8.3±4.3
15.8±4.3
1.562
14.2±3.3
4.6±1.1
0
0
7.1±3.5
9.5±3.3
0.781
0
0
0
0
0
0
Negative control
10.8±3.2
10.8±3.2
10.550±2.3
10.5±2.3
10.6±2.4
10.6±2.4
Albendazole (0.125mg/ml)
100
100
100
100
100
100
382 383 384 19
Page 19 of 24
385 386 387 388 389 390
Table 3: EC50 and EC99 of aqueous and methanolic extracts of Ocimum sanctum using various in vitro tests.
391 392 393 Ocimum sanctum
Test
EC50±SE
EC99±SE
EHT
5.8±0.4
37.1±9.3
LPT
16.4±0.6
368.6±137.5
LDT
7.7±0.3
28.1±3.2
EHT
6.2±0.3
21.3±2.2
LPT
12.9±0.5
358.2±138.8
LDT
12.1±0.8
64.7±10.8
Aqueous extract
Methanolic extract
394
*EC: Effective Concentation; SE: Standard Error
395 20
Page 20 of 24
396 397 398 399 400
Table 4: : Faecal egg count reduction test (FECRT) showing effect of aqueous extract of O. sanctum on epg
401 402 403 Group 1
Group 2
Group 3
Untreated Control
Albendazole
Ocimum sanctum
0days
905+20.3
895+25.8
898+22.3
7days
850+19.7
185+27.9
625+18.6
14days
835+21.1
40+6.6
190+16.3
21days
860+26.6
70+8.1
515+23.6
Mean
862.5a
252.5b
545.0b
Days post treatment
404 405 406 21
Page 21 of 24
407
Table 5: Effect of O. sanctum on different blood parameters and FRAP values along with positive and negative control
SGPT(ALT) IU/L
SGOT(AST) IU/L
RBC ×102/L
Hb g/dl
HCT %
MCV fl
RDWc %
MCH pg
MCHC g/dl
PLT ×103/µl
MPV fl
PCT µg/L
PDWC g/L
WBC ×109/L
LYM %
MON %
GRANULO CYTE %
FRAP µmol/L
0 day
191.4 ±11.2
9.7±0.3
7.4±0.3
25.7±1. 2
23.4±2. 4
22.1±0.3
7.6±0 .1
29.7±0. 4
243.9±19. 1
6.4±0.1
0.2±0. 07
32.2±0.2
11.9±0. 5
46.3±1. 7
4±0.3
49.6±1.8
613.3+3 1.2
14 day
20.3 ±2.2
193.5±1 1
10±0.4
7.4±0.2
25.3±1. 1
26±0.5
22.1±0.3
7.7±0 .2
30.4±0. 5
246.1±18. 6
6.6±0.0 7
0.2±0. 06
32.6±0.2
10.6±0. 4
44.5±0. 8
4.2±0.3
51.2±1.0 1
569.3+2 8.3
Mean
20.5b
192.5
9.8b
7.4b
25.2
24.7
22.1b
7.7
30.1
245.0ab
6.5
0.2
32.4a
11.2
45.4a
4.1
50.4b
591.3
0 day
21.5±2.2
191.2±1 0.7
10.1±0.3
7.4±0.2
24.7±1. 1
25.6±0. 3
22.3±0.3
7.6±0 .1
29.3±0. 4
247.6±18. 9
6.5±o.1
0.2±0. 06
32.1±0.2
10.2±0. 3
46.4±1. 1
4.±0.3
49.5±1.1
593.3+2 6.16
14 day
18.±2.1
199.6±1 7.2
11.6±1.1
8.8±0.9
26.4±1. 2
25.8±0. 3
22.4±0.2
7.5±0 .1
29.4±0. 3
320.4±21. 6
6.4±0.1
0.2±0. 03
33.±0.5
10.5±0. 3
46.5±1. 2
4.1±0.3
49.2±1.2
582.6+3 3.3
Mean
19.8 b
195.4
10.8 ab
8.1 ab
25.5
25.7
22.4ab
7.5
29.3
284.0a
6.4
0.2
32.6 a
10.4
46.5 a
4.09
49.4 b
587.9
22.1±2
191.6±1 1
9.8±0.3
7.3±0. 2
25.4±1 .1
25.1±0 .7
22.4±0.2
7.6± 0.1
29.6±0 .3
246.8±19 .1
6.4±0. 1
0.19± 0.01
32.1±0. 2
10.8±0 .2
45.2±1 .6
4.5±0. 2
50.2±1. 7
593.3+ 34.1
29±1.6
165.1±3 .6
12.4±0. 7
9.5±0. 6
31.2±2 .3
25±0.7
23±0.4
7.2± 0.1
30.9±0 .5
197.7±12 .2
6.1±0. 2
0.11± 0.01
31.3±0. 7
10.5±0 .3
45.5±1 .7
3.9±0. 2
50.5±1. 8
525.3+ 33.2
0 day
20.6 ±2,.2
14 day
Ocimum sanctum
Albendazole
Negative Control
408
22
Page 22 of 24
Mean
25.5 a b
178.3
11.1ab
8.4ab
28.3
25.1
22.74 ab
7.44
30.33
222.25b
6.26
0.15
31.75ab
10.66
45.38a
4.21
50.40 b
559.33
23
Page 23 of 24
24
Page 24 of 24
S0034-5288(15)00042-9 http://dx.doi.org/doi: 10.1016/j.rvsc.2015.01.017 YRVSC 2812
To appear in:
Research in Veterinary Science
Received date: Accepted date:
14-10-2014 28-1-2015
Please cite this article as: Dharmendra Kanojiya, Daya Shanker, Vikrant Sudan, Amit Kumar Jaiswal, Rahul Parashar, Anthelmintic activity of ocimum sanctum leaves extract against ovine gastrointestinal nematodes in india, Research in Veterinary Science (2015), http://dx.doi.org/doi: 10.1016/j.rvsc.2015.01.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Anthelmintic activity of Ocimum sanctum leaves extract against ovine gastrointestinal nematodes in
2
India
3
Dharmendra Kanojiya, Daya Shanker, Vikrant Sudan*, Amit Kumar Jaiswal, Rahul Parashar
4
Department of Parasitology,
5
College of Veterinary Sciences & Animal Husbandry,
6
U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go
7
Anusandhan Sansthan (DUVASU), Mathura- 281001, INDIA.
8
*Corresponding authors email: [email protected]; +91 9045414200
9 10 11 12 13 14 15 16 17 18 19 20 1
Page 1 of 24
21 22 23 24 25 26 27
Highlights
In vitro and in vivo efficacy of extracts of Ocimum sanctum on sheep helminthosis Phytochemical studies to know the active ingredients Assessment of blood, enzymes and antioxidant parameters for safety trail Detection of significant anthelminthic efficacy
28 29 30 31
ABSTRACT Leaves of Ocimum sanctum have been traditionally used for various ethno-veterinary
32
practices as well as medicinal purpose.
33
aqueous and hydro-alcoholic extracts of the bulb of Ocimum sanctum was investigated.
34
Alkaloids, carbohydrates, steroids and tannins were identified in phytochemical analyses. The
35
various blood parameters coupled marker enzymes and anti oxidant status, were also evaluated
36
during in vivo trial. Aqueous extract showed better EC50 and EC99 values in comparison with
37
methanolic extract in egg hatch assay and larval development test, respectively. However, in
38
larval paralysis test, both aqueous and methanolic extracts showed almost similar efficacy. A
39
77.64 % reduction in faecal egg output was observed on day 14. No deleterious ill effect was
40
found in any of the haematological and biochemical parameters suggesting that the plant could
41
be safer for use in sheep.
In vitro ovicidal and larvicidal potential of crude
42 43 44
Keywords: Anthelminthic activity Gastrointestinal nematodes
45
India
46
Ocimum sanctum
47
Sheep
48 49
2
Page 2 of 24
50 51 52 53 54 55 56 57 58
1. Introduction
59
With the emergence of anthelminthic resistance ( the parasitic control programme
60
suffered gradual setback in several countries forcing the researchers to the search for alternative
61
control methods (Dhar et al., 1982). Anthelminthic resistance has been reported for almost all the
62
commercially available anthelminthics in the Indian market (Jaiswal et al., 2013). Ethno
63
veterinary alternatives are considered to be of immense potential in overcoming the
64
anthelminthic resistance due to synthetic chemical compounds (Lans et al., 2007). Worldwide
65
research has shown that certain plants can effectively be used to reduce the degree of parasitism
66
and hence, they can be seen as promising alternatives to the conventional chemical anthelmintics
67
(Githiori et al., 2006).
68
Ocimum sanctum (commonly known as Tulsi), is native throughout the Eastern World
69
tropics and widespread as a cultivated throughout India. The leaves are the most commonly
70
medicinally used plant part worldwide for many centuries. In herbal medicine, it is used to
71
prevent cancer (Pandey, 2009), used as an immunomodulator (Mukherjee et al., 2005),
72
hepatoprotectant and haematinic activities (Chattopadhyay et al., 1992). Besides these, it also 3
Page 3 of 24
73
possess some anthelminthic activity against roundworms particularly ascarids (Singh and
74
Nagaich, 2000). The present study was designed to assess the anthelminthic potential of various
75
extracts of leaves of O. sanctum on naturally occurring gastrointestinal nematodes of sheep using
76
standard in vitro and in vivo tests.
77
2. Materials and methods
78
2.1. Collection and processing of plant material
79
Fresh leaves of O. sanctum were collected from from the plants grown within the
80
premises of Veterinary University, Mathura, India. The plants were later identified by the
81
Department of Botany, Babu Shivnath Agrawal College, Mathura and the specimens were
82
recorded in the form of voucher number (DUVASU-2014-OS) for the sake of further references.
83
The leaves were cleaned from adulterants and air dried under shade at a well-ventilated place.
84
The plant material was pulverized to powder form with a mixer grinder and stored in air tight
85
containers.
86
2.2. Preparation of crude extracts
87
Extracts from the leaves of O. sanctum were prepared in water and methanol (S.D Fine
88
Chem. Ltd.). For preparing aqueous extract, 100g of powdered leaves were taken in a beaker
89
and suspended in 600ml of water and was continuously stirred using magnetic stirrer for 4 hours.
90
Again, for preparing methanolic extract 100 g of powder was soaked in 500 ml of methanol
91
solvent to exhaustion (120 h). The extraction was carried out in a percolator by a combination of
92
maceration and percolation at room temperature. The filtrates of both the extracts were collected
93
through Whatmann filter paper No. 4. The removal of the solvent was performed at a temperature
94
below 40°C,
under reduced pressure and a rotation speed of 20 rpm in vacuum rotary evaporator. 4
Page 4 of 24
95
The extract was scrapped off, transferred to an air-tight container and stored in a freezer at −20
96
°C till subsequent use.
97
2.3. Determination of extraction yield (% yield)
98
The yield (%, w/w) from all the dried extracts was calculated as:
99
Yield (%) = (W1 x 100)/W2
100
where, W1 was the weight of the extract obtained after evaporation of solvent; and W 2
101
was the weight of the plant powder. Later both the extracts (100mg each) were dissolved in 1ml
102
of distilled water to get 100mg/ml concentration and subsequent lower dilutions were prepared
103
(100 to 0.781 mg/ml) by using two fold dilution. The extracts were kept in air tight containers at
104
40C to avoid loss of any volatile principles or/and activities till further use.
105
2.4. Phytochemical analysis of crude extracts
106
Both the extracts were tested for the presence of active principle like carbohydrate,
107
steroids, tannins, flavonoids, alkaloids, glycoside, fat and proteins using standard procedures
108
(Debela, 2002).
109
2.5. In vitro tests
110
2.5.1. Egg hatch test
111
The eggs of GI nematodes were recovered using the method described by Taylor et al.
112
(2002) and recommended by the World Association for the Advancement of Veterinary
113
Parasitology, WAAVP (Coles et al., 1992). Briefly, about 10 g of faeces were collected directly
114
from the rectum of naturally infected sheep and was mixed and homogenized with tap water. The
115
mixture was sieved through strainer and centrifuged for 2 minutes at 2000 rpm, the supernatant 5
Page 5 of 24
116
was discharged and after the tube was filled with saturated solution, cover slips were put over the
117
meniscus for 5 minutes and were later washed to collect the eggs. The egg suspension, with a
118
concentration of 100 eggs/0.2 ml, was distributed in 24–multiwell plate (50 μl per well) along
119
with 50 μl of extract of each tested concentration. Three replicates
120
concentration. The plates were incubated, under humidified conditions, at 27°C for 48 h and later
121
a drop of Lugol’s iodine solution was added to each well to stop further hatching. Hatched larvae
122
and unhatched eggs were then counted under the microscope.
123
2.5.2. Larval development test
were made for each
124
The Micro agar larval development test was performed as per the standard protocol of
125
Coles et al. (2006). Briefly, 10 µl of individual concentration of each extract was placed in wells
126
in triplicates and 150 µl of 2% bacto agar at 45 0C was added in triplicate form into each well
127
and allowed to cool at room temperature. Thereafter, 10 µl of egg suspension, diluted in
128
Amphoteracin B (1:1) was poured upon the cooled agar. Then, 10 µl of yeast extract, prepared
129
in Earle’s salt solution (Hubert and Kerbouf, 1984), was added to it. The plates were sealed with
130
parafilm and incubated at 25 0C for 7 days and the number of live L3 in each well was counted
131
thereafter.
132
2.5.3. Larval Paralysis test
133
Larvae were cultured according to MAFF (1986) using Bearmann’s apparatus. 100μl of
134
suspension (approx 100 larvae) was added to 100μl of each concentration, in triplicate form, in
135
the 96-well plate and were kept at room temperature for 24 h. The live (motile) and dead larvae
136
were counted.
6
Page 6 of 24
137
Albendazole at the dose of 0.125 mg/ml was used as a positive control while distilled
138
water served as negative control in all the three tests (Kanojiya et al., 2014).
139
2.6. In vivo test
140
2.6.1. Faecal Egg Count Reduction Test (FECRT)
141
The same sheep flock that was used for the collection of eggs for the in vitro trial was
142
again used for in vivo trial. The flock consisted of local Chokla breed of sheep that were
143
naturally infected with gastrointestinal parasites. Single pooled larval culture, one from each
144
group, was done on day 0 in order to find out the species of GI parasites involved. The flock was
145
divided into three groups of fifteen animals each. Group 1 served as a negative control and
146
received no treatment while Group 2 served as a positive control and was given a single dose of
147
Albendazole at the dose of 7.5mg/kg bodyweight. Group 3 was drenched a single oral dose of
148
aqueous extract at the dose of 5g/animal by making final volume of 5 ml with water (Kanojiya et
149
al., 2014). Fecal samples of animals were individually collected on day 0, 7, 14 and 21 post-
150
treatment. These samples were processed for egg per gram (epg) calculation using the McMaster
151
method (Soulsby, 1982). FECRT efficacy was calculated according to Dash et al., (1988) using
152
the following formula:
153 154
where T is average fecal egg count of treated group; C is average fecal egg count of control
155
group; 1 is pre-treatment average fecal egg count ; 2 is post-treatment average fecal egg count .
7
Page 7 of 24
156
Each group of sheep was housed separately but were allowed to graze altogether
157
freely during the day time. Again on day 21 post treatment, single pooled larval culture, one
158
from each group, was done to identify the species of GI parasites surviving the herbal treatment.
159
2.7. Blood sample for haematology
160
Blood samples were obtained on day 0 and day 14 from each animal via. Jugular
161
vein puncture into 10 ml vacutainer. Different blood parameter were analyzed using
162
Haematology analyser (Diatron) and Semi auto chemistry analyzer (Electronics India) using
163
serum [Serum Glutamic Pyruvate Transaminase
164
Transaminase (SGOT)], whole blood [ Red Blood Cells (RBCs), White Blood Cells (WBCs),
165
Haemoglobin (Hb), Haemocrit (HCT), Mean Corpuscular Volume (MCV), Red Blood Cell
166
Distribution Width (RDWc), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular
167
Haemoglobin Concentration (MCHC), Platelet Count (PLT), Mean Pack Volume (MPV),
168
Procalcitonin (PCT), Lymphocyte %, Monocyte % and Granulocyte %) and plasma (Ferric
169
Reducing Antioxidant Power FRAP). Total antioxidant activity was measured by FRAP assay
170
(Benzie and Strain 1999). Briefly, plasma (100 l) was mixed with 3 ml of working FRAP
171
reagent and absorbance was measured at 0 minute after vortexing. Thereafter, samples were
172
placed at 37°C in a water bath and absorbance was again measured after 4 min. Ascorbic acid
173
standards (100 M-1000 M) were processed in the same way.
FRAP value of sample (mol/L) =
A-B
(SGPT), Serum Glutami cOxaloacetic
X 100
X-Y
8
Page 8 of 24
174
where A is reading of the sample at 0 minute; B is reading of the sample at 4 minute; X
175
reading of the standard at 0 minute; Y is reading of the standard at 4 minute; and 100 is FRAP
176
value of 100 M standard solution.
177
2.8. Statistical analysis
is
178
The extract concentration required to inhibit 50% and 90% egg hatching (EC50 and EC90)
179
was calculated by using log probit analysisusing SAS software (SAS, 1998). The results of the
180
tests and the various haematological parameters were expressed as mean ± standard error of
181
mean (S.E.). Means were evaluated by t-test to compare the anthelmintic efficacy at different
182
concentrations along with the haematological parameters also. p values ≤ 0.05 were considered
183
statistically significant.
184
3. Results
185
3.1. Extraction yield (% yield) of the various extracts
186
Slight variation in yields of the two extracts was observed. The methanolic extract was
187
having lower yield (11.25%) in comparison with aqueous (11.69%). extract.
188
3.2. Phytochemical analysis of different crude extract
189
The aqueous extract was positive for alkaloids, carbohydrates, steroids and tannins while
190
the methanolic extract was also positive for alkaloids and tannins only (Table 1).
191
3.3. In vitro tests
192
3.3.1. Egg Hatch Test
9
Page 9 of 24
193
Both the extracts inflicted significant inhibition of egg hatching in a dose dependent
194
manner. The aqueous extract (EC50=5.827 and EC99 =37.144mg/ml) of O. sanctum was found to
195
be more effective in having ovicidal properties in comparison to the
196
(EC50=6.218 and EC99 =21.32mg/ml).
197
3.3.2. Larval Development Test
methanolic extract
198
Again a dose dependent relationship was observed in both the extracts. The aqueous
199
extract (EC50=7.746 and EC99 =28.199 mg/ml) of O. sanctum was again found to be more
200
effective in having larvicidal properties in comparison to the methanolic extract (EC50=12.179
201
and EC99 =64.721 mg/ml) in larval development test.
202
3.3.3. Larval Paralysis Test
203
Similar finding to the findings described above for the other tests, the Larval paralyisis test also
204
followed a dose dependent trend.
205
=368.628mg/ml) and the methanolic (EC50=12.982and EC99 =358.27mg/ml) counterpart showed
206
almost similar efficacy in the Larval paralysis test
207
Howewer, the aqueous extract (EC50=16.486 and EC99
Albendazole (0.125mg/ml) that was used as a positive control showed 100% efficacy in
208
all the three tests as presented in Tables 1, 2 and 3.
209
3.4. In vivo test
210
3.4.1. Faecal Egg Count Reduction Test (FECRT)
211 212
Results from coprocultures reveled that on day 0 the flock was mainly infected
with
Haemonchus spp.(85%) followed by Trichostrongylus spp. (7%), Oesophagostomum spp. (5%) 10
Page 10 of 24
213
and Strongyloides spp. (3%). Results from the in vivo trial demonstrated that on day 7 pos
214
treatment the FECR was 74.12% and 26.47% for albendazole and O. sanctum, respectively.
215
However, at day 14 the FECR were 94.4% and 77.64% for albendazole and O. sanctum,
216
respectively. On day 21 post treatment, albendozole still had 90.2% effectiveness in reduction of
217
faecal egg count while O. sanctum caused 39.41% reduction in faecal egg count. (Table 4). The
218
results from coprocultures at day 21, demonstrated a proportion of Haemonchus spp. (92%)
219
by Trichostrongylus spp. (4%), Oesophagostomum spp. (3%) and Strongyloides spp. (1%) in O.
220
sanctum treated animals. Coproculture of control group showed no variation from the results of
221
Day 0 where as coproculture of albendazole treated group demontrated Haemonchus spp.(95%)
222
followed by Trichostrongylus spp. (3%), Oesophagostomum spp. (1%) and Strongyloides spp.
223
(1%).
224
3.5. Blood Haematology
followed
225
The details of the blood parameters examined viz., SGPT, SGOT, RBCs, WBCs, Hb,
226
HCT, MCV, RDWc, MCH, MCHC, PLT, MPV, PCT, Lymphocyte %, Monocyte%
227
Granulocyte % and FRAP are provided in Table 5. Significant changes were observed in SGPT,
228
RBCs, Hb, RDWc, PLT, PDWC, Lymphocyte and Granulocyte values, while the rest of
229
parameters showed non significant alterations within the normal range in the stipulated time of
230
herbal trial (Details given in Table 5).
231
4. Discussion
232
The aim of the present study was to evaluate the in vitro and in effect of O. sanctum in
233
comparison to a reference drug-albendazole against the gastrointestinal nematodes of sheep. 11
Page 11 of 24
234
Results revealed both the aqueous and methanolic extracts of O. sanctum effectively targeted
235
both the egg and larval developmental stages of the parasite in a dose dependent manner.
236
Significant effects in terms of non hatching of eggs, non development of larvae and paralysis
237
and/or death of the larvae at different time durations, were observed in in vitro trials. The reason
238
for evaluating only
239
performance in the in vitro tests in comparison with the methanolic extract.
the aqueous extract of O. sanctum for the in vivo trial was based on its better
240
The compound Eugenol, present in O. sanctum essential oil, is attributed to be the
241
putative anthelmintic principle of the plant (Asha et al., 2001). In vitro testing of essential oils of
242
Ocimum sanctum and purified eugenol showed potent anthelmintic activity in the
243
Caenorhabditis elegans model (Asha et al., 2001). The essential oil of O. gratissimum, as well as
244
eugenol, was found effective in inhibiting eclodibility of H. contortus eggs (Pessoa et al., 2002).
245
Bihari et al. (2010) screened the activity of methanolic extract of O. sanctum against healthy
246
adult Indian earthworms, Pheretima posthuma and its efficacy at the same level as albendazole
247
when compared using in vitro tests.
248
O. sanctum is reported to block the glucose uptake by the nematode parasites, thereby
249
altering with their mobility and survivability (Singh and Nagaich, 2000). Alkaloids,
250
carbohydrates, steroids and tannins were the main secondary compounds found in the present
251
study. There are many reports of reduction in faecal egg count in sheep treated with plants rich in
252
tannins (Githiori et al., 2006). Tannins interact with proteins in the nematode cuticle thereby
253
considerably changing its chemical and physical properties (Athanasiadou et al., 2001).
254
Flavonoids are also known to possess action against H. contortus (Camurça-Vasconcelos et al.,
255
2007).
12
Page 12 of 24
Toxicity of O. sanctum extract was one of the main concerns, however, no deleterious ill effects
256 257
were found
258
plant is safe for use in sheep. Although, significant changes were observed in SGPT, RBCs, Hb,
259
RDWc, PLT, PDWC, Lymphocyte and Granulocyte values, the rest all the parameters showed
260
non significant alterations and were within the normal range in the stipulated time of herbal trial.
261
Increase in SGPT values might be attributed to the consumption of large amount of herbal
262
extract.
263
haematinic and hepatoprotectant role of O. sanctum in the treated animals (Chattopadhyay et al.,
264
1992). Again O. sanctum caused marked decrease in the stress of animals due to parasitism as
265
shown by FRAP values. This could be very much contributed to antioxidant properties of the
266
plant (Saija et al., 1995). These antioxidant effects are supposed to be due to the presence of
267
flavonoids (Saija et al., 1995). O. sanctum appears to modulate both humoral and cell-mediated
268
immune responses (Mukherjee et al., 2005).
in any of the haematological and biochemical parameters observed suggesting that the
RBCs, Hb and RDWc values showed significant increase which could be due to
269
As a matter of fact, in the majority of the cases, the anthelmintic activity of these
270
ethanopharmacologically important plants has been found to be lower and/ or below that
271
reported for synthetic anthelmintics. Again, plants having moderate anthelmintic activity should
272
also still be considered, while they are definitely not useful as a sole alternative but may still be
273
valuable as part of an integrated approach specifically designed to achieve sustainable parasite
274
control in small ruminant production systems. Further studies on the standardization of dose and
275
identification of exact mechanism of action of these extracts against nematodes are thereby
276
warranted. Although it will take time and expertise to standardize the plant anthelmintics and
277
address the recent emergent trends in anthelmintics, the use of herbal remedies as anthelmintics
278
based on local plants will at least offer a cheap, reliable and a readily available alternative to 13
Page 13 of 24
279
highly expensive and unavailable conventional anthelmintics to the poor farmers worldwide. The
280
findings from the present study will surely contribute in the field of plant anthelmintics to
281
develop sustainable, effective and safe alternatives to conventional anthelmintics.
282
Acknowledgement
283
The authors are highly thankful to the Vice Chancellor, DUVASU for the facilities
284
provided.
285
Conflict of Interest
286
The authors declare that they are having no conflict of interest.
287
References
288
Asha, M.K., Prashanth,D., Murali,B., Padmaja,R., Amit,A., 2001. Anthelmintic activity of
289
essential oil of Ocimum sanctum and eugenol. Fitoterapia 72(6), 669–670.
290
Athanasiadou, S., Kyriazakis, I., Jackson, F., Coop, R.L., 2001. Direct anthelmintic effects of
291
condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in
292
vivo studies. Veterinary Parasitology 99, 205–219.
293
Benzie, F., Strain, J.J. 1999. Ferric reducing/ antioxidant power assay: direct measure of total
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antioxidant activity of biological fluids and modified version for simultaneous
295
measurement of total antioxidant power and ascorbic acid concentration. Methods in
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Enzymology 299, 15-23.
297
Bihari, C.G., Shankar, N.B., Kumar, J.P., Keshari, P.S., Ellaiah, P., 2010. Phytochemical
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investigation and screening for anthelmintic activity of leafy extracts of various Ocimum
299
(Tulsi) species. Journal of Pharmacy Research 3(9), 2140-2141.
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Camurça-Vasconcelos, A.L.F., Bevilaqua, C.M.L., Morais , S.M., Maciel, M.V., Costa, C.T.C.,
301
Macedo, I.T.F., Oliveira, L.M.B., Braga, R.R., Silva, R.A., Vieira, L.S., Navarro, 14
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A.M.C., 2007. Anthelminthic activity of Croton zehntneri and Lippia sidoides essential
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oils. Veterinary Parasitology 148, 288–445.
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Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J.,
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1992. World Association for the Advancement of Veterinary Parasitology (WAAVP)
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methods for the detection of anthelmintic resistance in nematodes of veterinary
307
importance. Veterinary Parasitology 44(1-2), 35-44.
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Coles, G.C., Jakson. F., Pomroy, W.E., Prichard, R.K., Silvestre, A., Taylor. M.A., Vercruysse.
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J., 2006. The detection of anthelmintic resistance in nematode of Veterinary importance.
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Veterinary Parasitology 136(3-4), 167-185.
311
Chattopadhyay, R.R., Sarkar, S.K., Ganguly, S., Medda, C., Basu, T.K., 1992. Hepatoprotective
312
activity of O. sanctum leaf extract against paracetamol induced hepatic damage in rats.
313
Indian Journal of Pharmacology 24,163.
314
Dash, K., Hall, K., Barger, I.A., 1988. The role of arithmetic and geometric worm egg counts in
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faecal egg count reduction test and in monitoring strategic drenching programs in sheep.
316
Australian Veterinary Journal 65, 66–68.
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Debela, A., 2002. Manual for Phytochemical Screening of Medicinal Plants. Ethiopian Health and Nutrition Research Institute, Addis Ababa, Ethiopia. pp 35-47. Dhar, D.N., Sharma, R.L., Bansal, G.C., 1982. Gastrointestinal nematodes in sheep in Kashmir. Veterinary Parasitology 11, 271–277.
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Githiori, J.B., Athanasiadou, S., Thamsborg, S.M., 2006. Use of plants in novel approaches in
322
control of gastrointestinal helminthes in live stock with emphasis on small ruminants.
323
Veterinary Parasitology 139, 308–320.
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Hubert, J., Kerboeuf, D., 1984. A new method for culture of larvae used in diagnosis of ruminant
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gastrointestinal strongylosis: comparison with faecal cultures. Canadian Journal of
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Comparative Medicine 48, 63–71.
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Jaiswal, A.K., Sudan, V., Shanker, D., Kumar, P., 2013. Emergence of ivermectine resistance in a semi-organized farm of Mathura District- India. Veterinarski Archiv 83(3), 275-280.
329
Kanojiya, D., Shanker, D., Sudan, V., Parashar, R ., 2014. In vitro and in vivo efficacy of
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extracts of leaves of Eucalyptus globulus on ovine gastrointestinal nematodes.
331
Parasitology Research DOI 10.1007/s00436-014-4169-1
332 333
Lans, C., Turner, N., Khan, T., Brauer, G., Boepple, W., 2007. Ethnoveterinary medicines used
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for ruminants in British Columbia, Canada. Journal of Ethnobiology and Ethnomedicine
335
doi:10.1186/1746-4269-3-11.
336 337 338 339 340 341
M.A.F.F.,1986. Ministry of Agriculture, Fisheries and Food, Manual of veterinary parasitological laboratory techniques. HMSO, London, pp1-152. Mukherjee, R., Das, P.K., Ram, G.C., 2005. Immunotherapeutic potential of Ocimum sanctum Linn. bovine subclinical mastitis. Review in Veterinary Science 79(1),37-43. Pandey G., 2009. An overview on certain anticancer natural products. Journal of Pharmacological Research 2(12),1799-1803.
342
Pessoa , L.M., Morais , S.M., Bevilaqua, C.M.L., Luciano, J.H.S., 2002. Anthelmintic activity of
343
essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus contortus .
344
Veterinary Parasitology 109(1–2), 59–63.
345
S.A.S., 1998. Statistical Analysis System: User’s Guide. Statistical Institute, North Carolina.
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Saija, A., Scalese, M., Lanza, M., Marzillo, D., Bonina, F., Castelli, F., 1995. Flavonoids as
347
antioxidant agents: Importance of their interaction with biomembrane. Free Radical in
348
Biological Medicine 19: 481.
349
Singh, K., Nagaich, S., 2000. Studies on the anthelmintic activity of Allium sativum (Garlic) oil
350
on common poultry worms Ascaridia galli and Hetarakis gallinae. Journal Parasitlogy
351
and Applied Biology 9, 47–52.
352 353 354 355
Soulsby , E.J.L., 1982. Helminths, arthropods and protoza of domesticated animals 7th edn. The English Language Book Society and Bailliere Tindall, London, pp234-235. Taylor, M.A., Hunt, K.R., Goodyear. K.L., 2002. Anthelmintic resistance detection methods. Veterinary Parasitology 103, 183–194.
356 357 358 359 360 361 362 363 364 365
17
Page 17 of 24
366 367
Table 1: Chemical constituents in aqueous and methanolic extract of O. sanctum
Steroids
Glycosides
Tannins
Protein
Methanolic
Carbohydrates
Aqueous
Fat
Ocimum sanctum
Nature of extract
Flavonoids
Plant
Alkaloids
Plant constituents found as per the methods of Debela 2002
+
-
-
+
+
-
+
_
+
_
_
_
_
_
+
_
368 369 370 371 372 373 374 375 376 377 18
Page 18 of 24
378 379
Table 2: Percentage efficacy of aqueous and methanolic extracts of O. sanctum using various in vitro tests.
380 381 Ocimum sanctum Egg Hatch Assay
Concentration (mg/ml)
Larval Development Test
Larval Paralysis Test
100
Aqueous extract 100
Methanolic extract 100
Aqueous extract 100
Methanolic extract 100
Aqueous extract 100
Methanolic extract 100
50
100
100
100
100
90.±4.1
92.8±4.7
25
100
100
100
71.6±8.9
53.3±3.3
57.9±5.6
12.5
100
97.5±2.5
72.1±3.7
55.4±7.6
44.9±5.7
52.4±4.1
6.25
52.8±6.2
41.7±9.1
16.5±4.6
11.2±4.1
10.3±4.3
39.9±3.2
3.12
20.1±3.9
11.6±2.9
7.5±3.2
3.1±2.1
8.3±4.3
15.8±4.3
1.562
14.2±3.3
4.6±1.1
0
0
7.1±3.5
9.5±3.3
0.781
0
0
0
0
0
0
Negative control
10.8±3.2
10.8±3.2
10.550±2.3
10.5±2.3
10.6±2.4
10.6±2.4
Albendazole (0.125mg/ml)
100
100
100
100
100
100
382 383 384 19
Page 19 of 24
385 386 387 388 389 390
Table 3: EC50 and EC99 of aqueous and methanolic extracts of Ocimum sanctum using various in vitro tests.
391 392 393 Ocimum sanctum
Test
EC50±SE
EC99±SE
EHT
5.8±0.4
37.1±9.3
LPT
16.4±0.6
368.6±137.5
LDT
7.7±0.3
28.1±3.2
EHT
6.2±0.3
21.3±2.2
LPT
12.9±0.5
358.2±138.8
LDT
12.1±0.8
64.7±10.8
Aqueous extract
Methanolic extract
394
*EC: Effective Concentation; SE: Standard Error
395 20
Page 20 of 24
396 397 398 399 400
Table 4: : Faecal egg count reduction test (FECRT) showing effect of aqueous extract of O. sanctum on epg
401 402 403 Group 1
Group 2
Group 3
Untreated Control
Albendazole
Ocimum sanctum
0days
905+20.3
895+25.8
898+22.3
7days
850+19.7
185+27.9
625+18.6
14days
835+21.1
40+6.6
190+16.3
21days
860+26.6
70+8.1
515+23.6
Mean
862.5a
252.5b
545.0b
Days post treatment
404 405 406 21
Page 21 of 24
407
Table 5: Effect of O. sanctum on different blood parameters and FRAP values along with positive and negative control
SGPT(ALT) IU/L
SGOT(AST) IU/L
RBC ×102/L
Hb g/dl
HCT %
MCV fl
RDWc %
MCH pg
MCHC g/dl
PLT ×103/µl
MPV fl
PCT µg/L
PDWC g/L
WBC ×109/L
LYM %
MON %
GRANULO CYTE %
FRAP µmol/L
0 day
191.4 ±11.2
9.7±0.3
7.4±0.3
25.7±1. 2
23.4±2. 4
22.1±0.3
7.6±0 .1
29.7±0. 4
243.9±19. 1
6.4±0.1
0.2±0. 07
32.2±0.2
11.9±0. 5
46.3±1. 7
4±0.3
49.6±1.8
613.3+3 1.2
14 day
20.3 ±2.2
193.5±1 1
10±0.4
7.4±0.2
25.3±1. 1
26±0.5
22.1±0.3
7.7±0 .2
30.4±0. 5
246.1±18. 6
6.6±0.0 7
0.2±0. 06
32.6±0.2
10.6±0. 4
44.5±0. 8
4.2±0.3
51.2±1.0 1
569.3+2 8.3
Mean
20.5b
192.5
9.8b
7.4b
25.2
24.7
22.1b
7.7
30.1
245.0ab
6.5
0.2
32.4a
11.2
45.4a
4.1
50.4b
591.3
0 day
21.5±2.2
191.2±1 0.7
10.1±0.3
7.4±0.2
24.7±1. 1
25.6±0. 3
22.3±0.3
7.6±0 .1
29.3±0. 4
247.6±18. 9
6.5±o.1
0.2±0. 06
32.1±0.2
10.2±0. 3
46.4±1. 1
4.±0.3
49.5±1.1
593.3+2 6.16
14 day
18.±2.1
199.6±1 7.2
11.6±1.1
8.8±0.9
26.4±1. 2
25.8±0. 3
22.4±0.2
7.5±0 .1
29.4±0. 3
320.4±21. 6
6.4±0.1
0.2±0. 03
33.±0.5
10.5±0. 3
46.5±1. 2
4.1±0.3
49.2±1.2
582.6+3 3.3
Mean
19.8 b
195.4
10.8 ab
8.1 ab
25.5
25.7
22.4ab
7.5
29.3
284.0a
6.4
0.2
32.6 a
10.4
46.5 a
4.09
49.4 b
587.9
22.1±2
191.6±1 1
9.8±0.3
7.3±0. 2
25.4±1 .1
25.1±0 .7
22.4±0.2
7.6± 0.1
29.6±0 .3
246.8±19 .1
6.4±0. 1
0.19± 0.01
32.1±0. 2
10.8±0 .2
45.2±1 .6
4.5±0. 2
50.2±1. 7
593.3+ 34.1
29±1.6
165.1±3 .6
12.4±0. 7
9.5±0. 6
31.2±2 .3
25±0.7
23±0.4
7.2± 0.1
30.9±0 .5
197.7±12 .2
6.1±0. 2
0.11± 0.01
31.3±0. 7
10.5±0 .3
45.5±1 .7
3.9±0. 2
50.5±1. 8
525.3+ 33.2
0 day
20.6 ±2,.2
14 day
Ocimum sanctum
Albendazole
Negative Control
408
22
Page 22 of 24
Mean
25.5 a b
178.3
11.1ab
8.4ab
28.3
25.1
22.74 ab
7.44
30.33
222.25b
6.26
0.15
31.75ab
10.66
45.38a
4.21
50.40 b
559.33
23
Page 23 of 24
24
Page 24 of 24
