CC BY
0JWPR
Journal of World's Poultry Research
2020, Scienceline Publication
J. World Poult. Res. 10(3): 429-435, September 25, 2020
Research Paper, PII: S2322455X2000049-10 License: CC BY 4.0
DOI: https://dx.doi.org/10.36380/jwpr.2020.49
Effect of Polar and non Polar Extract of Ferula assafoetida Dermanyssus gallinae in vivo and in vitro Conditions
Samere Ghavami1, Keramat Asasi1 and Mostafa Razavi2
'Avian Diseases Research Center, School of Veterinary Medicine, Shiraz University, Shiraz, Iran 2Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
»Corresponding author's Email: asasi@shirazu.ac.ir; ORCID: 0000-0001-6180-4182
Received: 29 Jun. 2020 Accepted: 16 Aug. 2020
ABSTRACT
Dermanyssus gallinae is one of the most common arthropods in layers that affects the quality and quantity of egg production. Although there are different synthetic compounds against this mite, but despite these compounds, drug resistance and the presence of these compounds and synthetic compounds in meat and eggs makes that the use of alternative methods, as well as increased use of herbal extracts and essential oils. In the present study, the N-Hexane and Ethanol extracts of Ferula assafoetida were used. GC-MS analysis revealed the constituents of the two extracts. The lethal properties of the extracts were determined by contact toxicity. In this field study, ethanolic extract of Ferula assafoetida was sprayed on laying hens that infected with red mite. The most available compounds of the Ethanol extract and N-hexan extract of Ferula assafoetida were Diethylpyridine and Aurapten respectively in this study. The LC50 of Ethanol extract of Ferula assafoetida was 16 ^g/cm3 and in vitro study determined that ethanolic extract of Ferula assafoetida has been able to reduce the red mite population. This study indicated Ethanol extract of Ferula assafoetida could use as a substitute compound against red mite.
Key words: Polar and nonpolar extract, Ferula assafoetida, Red mite
Dermanyssus gallinae (red mite) is one of the most important arthropods, especially in laying hens all over the world (Sparagano et al., 2013; Kim et al., 2016; Tabari et al., 2017; Kim et al., 2018) that affects the quality and quantity of egg production, irritation, anemia and, even in the case of severe contamination, causes the death of the bird (George et al., 2009; Spargano et al., 2013). D. gallinae plays an important role in the transmission of rickettsial, viral and bacterial pathogens and occasionally causes skin irritation in humans (George et al., 2009; Na et al., 2011; Spargano et al., 2013).
There are various chemical combinations including organophosphates, pyrethroids and carbamates to counteract this mite. The continuous applications of these compounds have increased the resistance to these compounds in this mite (Marangi et al., 2009; George et al., 2009; Tabari et al., 2015). In addition, the chemical residues of these compounds in meat, eggs and the environment are among the limitations of the use of these compounds (Dalton et al., 2001). Because of this, it
increases the importance of using alternative methods such as extracts and essential oils of plants of Ferula assafoetida to control red mite. The acaricidal properties of many plant extracts and essential oil have been reported against D.gallinae (Kim et al., 2004, 2007; George et al., 2009; Tabari et al., 2015; Nechita et al., 2015; Masoumi et al., 2016; Kim et al., 2016, 2018)
The genus of Ferula belong to the family of Apiaceae that distributed the Mediterranean area and central Asia including Iran and Afghanistan (Bagheri et al., 2010). F. assafoetida is traditionally use for the treatment disease including parasitic disease (Iranshahi et al., 2011). The compounds of this plant are 40-46% resin, 25% gum, 10-17 % volatile oil and 1.5 10% ash. The resin consist of ferulic acid esters, free ferulicacid ,umblliferone and coumarine (Iranshahi et al., 2011). The sesquiterpenes and sesquiterpene coumarins are the most compound of the genus Ferula (Pimenov et al., 1982). The essential oils of F. assafoetida are strong ovicides and larvicides of mosquitoes (Muturi et al., 2018). anthelmintic (Kakar et al., 2013; Upadhyay et al., 2017) antiprotozoal activity (El Deeb et al., 2012; Bafghi et al., 2014; Barati et al., 2014)
To cite this paper: Ghavami S, Asasi K and Razavi M (2020). Effect of Polar and non Polar Extract of Ferula assafoetida Dermanyssus gallinae in vivo and in vitro Conditions. J. World Poult. Res., 10 (3): 429-435. DOI: https://dx.doi.org/10.36380/jwpr.2020.49
are a characteristic of this plant The acaricidal activity of F. assafoetida has not been investigated against D. gallinae. The purpose of this study was to investigate the acaricidal activity of polar (Ethanol) and nonpolar (N-hexan) extracts of F. assafoetida on red mite under in vivo and in vitro conditions.
MATERIALS AND METHODS
Mites source
Between October 2018 and may 2019 D. gallinae samples were collected from a laying poultry farm in Amol, Iran. The mites were placed in dark containers under 25 °C and humidity of 55% transferred to the laboratory in Science and Technology Park of sari, Iran
Essential oil extraction and GC-MS analysis
The aerial parts of F. assafoetida were collected in Mashhad city, Iran and dried on 25°C. The aerial parts of F. assafoetida were ground mechanically using a commercialelectric mill. To provide Extract mill plant was macerated with ethanol and N-hexan in soxhlet apparatus and subsequently, the extract was filtered and solvent was evaporated by using a rotary evaporator and the acquired extract was dried in desiccator (Sonar et al., 2016). The polar extract was obtained by Ethanol solvent. However, the non-polar extract was obtained by N-hexan solvent. To analyze and identify the constituents of the extract Gas chromatography coupled to mass spectrometry (model Shimadzu-QP5050A, Japan) was used. In this study, gas chromatography Agilent-6890 model equipped with DB-5 column with a length of 40 m, an inner diameter of 0.18 mm to 0.25 mm thick layer of stationary phase are used. The column heat program was adjusted from 60 to 210 °C with a gradient of 5 °C/min. The injection chamber temperature was 280 °C and the used detector temperature was 270 °C. Helium gas was used as carrier gas and its speed was 0.9 mm/min and fission ratio of 1 to 43. The injection rate was 0.1 ^l of sample and the source ionization temperature was 230 °C. The electron ionizatin mode and the ionization energy were 70 ev. A series of normal alkanes were also injected under the same conditions to calculate the retention index inhibition index. The sample retention index was calculated using a computer program.
Finally, the essential oil components were identified by comparing the mass spectra obtained with the standard mass spectra in the Wiley 2000 electronic library in Labsolution GC/MS software and computing the standard inhibition index and comparing them with the standard
numbers in the references (Shibamoto, 1987; Adams, 2001).
Contact toxicity
Contact toxicity assay was done according to the method described by Tabari et al. (2015). In this study, treatment groups, control group of solvents, negative control and standard group were considered. 50 mites were added to all studied groups. The number of dead mites were recorded during 24, 48 and 72 hours after spraying the extract, and then the concentration of Lethal Concentration 50 (LC50) were calculated. Two replicates were carried out for all tested groups of mite.
Experimental groups
In the studied groups, the different concentrations were prepared (0.5, 1, 2, 4, 8, 16, 32, 64, 128 ^g/cm3). For dilution of polar extract from Ethanol and for nonpolar extract of N- hexane solvent used. Polar extract diluted in 50 ^l Ethanol and non polar extract diluted in 50 ^l N-hexane, the Wattemn's paper was then embedded with dilutions after three minutes, the paper dried and the paper was loaded on to the plate, about 50 mites were added to each plate. The number of red mite that were lost during 24, 48, and 72 hours after treatment were counted. Negative control group without any treatment was placed with 50 red mites on filter papers at the bottom of the plate. In Ethanol solvent control group, the filter paper was smeared with 50 ^l of ethanol solvent and after two minutes, the filter paper was dried and placed on the bottom of the plate and then 50 red mites added to the plate. In N-hexan solvent control group, the filter paper was smeared with 50 ^l of N-hexan solvent and after 2 minutes, the filter paper was dried and placed on the bottom of the plate and then 50 red mites added to the plate. In standard group, the filter paper impregnated with 50 ^l of diluted cypermethrin solution and then dried at the bottom of the plate, about 50 red mites were added to the plate.
In vitro experiment
Preparation nest
The research was conducted in one of the laying farms of Amol city in may 2019. Each study group contained 20 laying hens (at 40 weeks of age) that reared in cage system (5 laying hens in each cage). Ventilation, lighting and temperature controlled on the basis of breed recommendations (LSL catalog, 2018).
Treatments
In the treatment groups, the ethanolic extracts of F. assafoetida was sprayed on 20 LSL laying hens at 40 weeks of age based on the LC50 concentration obtained in vivo studies. The negative control group (without conflict with the red mite and untreated), positive control group (involved with the red mite untreated), standard (involved with red mite and treatment with Cypermethrin Mahan Chemical Company) was considered. Two replicates were considered for all study groups. In order to create red mite contamination, contaminated fields were collected from laying farm of Amol city, Iran and then in each group about 2000 experimental red mite were generated. According to the LC50 concentration indicated in the in vivo studies, Ethanolic extract of F. assafoetida and cypermethrin toxin were sprayed on the bird's body and repeated for one week more. 24 hours after each spray, the number of dead mites on the floor of each cage was measured and counted using adhesive paper traps.
Statistical analysis
The mortality rates of mites were analyzed using a one-way ANOVA in SPSS software (version 16). Values of P<0.05 were considered significant.
RESULTS
GC-MS analysis
The major constituents of the extract are shown in tables 1 and 2. The most available compounds in Ethanol extract were Diethylpyridine (23.54%), Aurapten (15.58%), Coumarin (5.11%) respectively and N-hexan extract of Ferula assafoetida were Aurapten (16.39%), Lutidine (7.36%) and Ergosten (4.87) respectively.
Table 1. The constituents of Ethanolic extract of Ferula
assafoetida
Compound Retention index Peak area
Coumarin 41.65 5.11
Quinolinium 48.37 1.68
Benzene 48.76 1.96
Ethylene 48.98 2.1
Methoxyindole 49.82 7.19
Phenol 50.11 2.37
Naphthalenone 50.33 3.1
Benzopyran 50.62 2.93
Aurapten 51.16 15.58
Diethylpyridine 51.63 23.54
Costol 52.48 2.63
Lavandulol 53.92 3.72
Table 2. The constituents of N-hexan extract of Ferula assafoetida_
Compound Retention index Peak area
Geranyl 33.467 3.62
a.-Selinene 33.99 3.78
Farnesal 34.81 1.07
Myristoleate 36.9 1.2
Oleic acid 37.03 1.85
Decanone 39.4 1.56
Farnesol 41.89 1.17
Phenyl ethanone 47.45 1.89
Oelsauere 48.15 2.06
Dehydrogingerdione 48.33 1.84
Formamide 48.83 1.27
Isothiocyanate 49 3.69
Trienoic acid 49.54 1.97
Dimethoxyindole 49.76 3.04
Benzenedicarboxylic acid 50.12 2.7
Xanthene 50.42 2.9
Aurapten 51.35 16.39
Lutidine 51.7 7.36
Ergosten 52.89 4.87
Quinoline 53.21 3.91
Squalene 55.91 1.32
Contact toxicity
Generally in all studied timings (After 24 hours, 48 hours and 72 hours), the results indicated that the responses to treatments were the extraction method and dose dependent in contact toxicity assay, Ethanol extracts of Ferula assafoetida was effective than N-hexan extract on red mite (P <0.05). The LC50 was 16 ^g/cm3 for Ethanol extracts (Table 3).
In vitro study
The ethanolic extract of F. assafoetida was effective on red mite under field conditions and the differences between groups were significant (P<0.05). After first spray, the number of dead red mite was not significant between positive and negative control (P>0.05) but mean mortality rates of mites in treatment group with F.assafoetida extract and standard groups significantly higher than the control groups (P <0.05). After the second spray, the number of dead red mites in treatment group with F. assafotida and standard group were significantly higher than the control groups (P <0.05, Table 4).
Table 3. Comparison of lethal effect polar and nonpolar extracts of Ferula assafoetida on red mite at different times
Treatment After 24 hours After 48 hours After 72 hours Total
N-hexan F. assafoetida 128 ^g/cm3 1a 1.33±0.33a 1.33±0.33a 1.22±0.14a
N-hexan F. assafoetida 64 ^g/cm3 N-hexan F. assafoetida 32 ^g/cm3 N-hexan F. assafoetida 16 ^g/cm3 N-hexan F. assafoetida 8 ^g/cm3 N-hexan F. assafoetida 4 ^g/cm3 N-hexan F. assafoetida 2 ^g/cm3 N-hexan F. assafoetida 1 ^g/cm3 N-hexan F.assafoetidaa 0.5 ^g/cm3 Ethanol F. assafoetida 128^g/cm3 Ethanol F. assafoetida 64^g/cm3 Ethanol F. assafoetida 32^g/cm3 Ethanol F. assafoetida 16^g/cm3 Ethanol F. assafoetida 8^g/cm3 Ethanol F. assafoetida 4^g/cm3 Ethanol F. assafoetida 2^g/cm3 Ethanol F. assafoetida 1^g/cm3 Ethanol F. assafoetida 0.5^g/cm3 0a 0.33±0.33a 0.33±0.33a 0.22±0.14a
0.33±0.33a 0.33±0.33a 0.33±0.33a 0.33±0.16a
0a 0.33±0.33a 0.33±0.33a 0.22±0.14a
1±0.57a 1±0.57a 1±0.57a 1±0.28a
1a 0a 0.66±0.33a 1a 0a 0.66±0.33a 1a 0a 0.66±0.33a 1a 0a 0.66±0.16a
0a 41±0.57f 0a 41.33±0.66f 0a 41.33±0.66f 0a 41.22±0.32g
35e 25±0.57d 41±0.57f 33.33±1.6e 41±0.57f 33.33±1.6e 39±1g 30.55±1.55f
16.66±1.66c 24.66±0.88d 24.66±0.88d 22±1.46d
8.33±1.66b 17.66±1.45c 17.66±1.45c 14.55±1.73c
3.33±1.66a 10b 10.33±0.33b 7.88±1.24b
0.33±0.33a 0.33±0.33a 0.66±0.33a 0.44±0.24a
0a 0a 1a 0.33±0.33a 1a 0.33±0.33a 0.66±0.16a 0.22±0.14a
Standard 45.33±0.33g 46.33±0.33a 46.33±0.33a 46.11±0.26h
N-hexan control 1±0.57a 2a 2a 1.66±0.23a
Ethanol control 0.33±0.33a 2a 2a 1.44±0.29a
Pasitive control 0.33±0.33a 1.66±0.33a 2a 1.33±0.28a
Non-anonymous latin letters in each column indicate significant difference (P < 0.05).
Table 4. Comparison of lethality of ethanolic extract of Ferula assafoetida on red mite under field conditions
Treatment Number of dead red mite after first spray Number of dead red mite after second spray
Ethanolic Extract of F. assafoetida 30±2.3b 40±1.52c
Standard 60±3.46c 50±1.15d
Negative control 0a 0a
Positive control 5±1.15a 7±0.57b
Non-anonymous Latin letters in each column indicates significant difference (P<0.05)
DISCUSSION
F. assafoetida is a well-known traditional plant with antiparasite properties. Anti-parasitic activity of this plant against leishmania (Bafghi et al. 2014, Gholami et al. 2013), eestode (Farhadi et al. 2016) culex (Muturi et al. 2018) Giardia (Nazer et al., 2019) and mosquito (Evergetis et al., 2012) has been demonstrated. Despite there are extensive reports concerning excellent insecticidal activity af F. asafoetida, there was no study to evaluate its acaricidal activity on D. gallinae. The present study is one the first in vitro and in vivo acaricidal studies of F. assafoetida against D. gallinae. In present study it was obtained that ethanolic extract was effective on D. gallinae in vitro and in vivo but N-Hexan extract had no significant effect on D. gallinae. This could be due to such active compounds in the ethanolic extract of F. assafoetidae.
The GC-MS analyses indicated that Ethanolic extract of F. assafoetidae was Diethylpyridine (23.54%) but N-Hexan extract of F. assafoetidae was Aurapten (16.39%). The anti parasitic effect of Diethylpyridine has been demonstrated against leshmania (Abdala et al., 2002). Generally, the major components of plant materials play the main role to determine the biological properties, but this point should not be ignored that the potential of the major compositions may be regulated by other minor components and the biological activites of plant materials are on account of synergistic/antagonistic interactions of all constitutes (Szcepanik et al., 2012).
El-Razek et al. (2001) and Pimenov et al. (1982) and Iranshahi et al. (2012) have reported that the most constituent compounds of F. assafoetida are sesquiterpenes and sesquiterpene coumarins. Numerous factors like geographic origin, seasonality, method of oil extraction, year of harvest and even storage conditions could affect the composition of essential oils, so the results from different toxicity studies might not always be adequate (Chalchat et al., 2007).
According to the present study ethanolic extract of F. assafoetidae was effective on red mite and LC50 was obtained 16 ^g/cm3. However N-Hexan extract of F. assafoetidae was not effective on red mite. In a similar study the aquatic and ethanolic extracts of Conocarpus erectus, regarding relative toxic and repellency properties, were used as botanical, safe acaricide and repellent agent for control of D. gallinae in avicultures (Rajabpour et al., 2018). The similar results were reported about Ethanol extract of Syzygium cumini and indicated the most efficient acaricidal activity against Tetranychus urticae
followed by hexane extract, ether and ethyl acetate extracts (Afify et al., 2011). The similar studies reported Ethanolic extracts of Veratrum album and Tanacetum parthenium could be useful to control Tetranychus urticae populations on vegetable plants grown through Integrated Pest Management and organic systems of agriculture (Yildirim et al., 2012).
Present results shows the ethanol extract of F. assafoetidae was effective on red mite in vitro condition. Some studies reported that the neem seed extract acaricide has positive against red mite in vitro coundition (Abdel-ghafar et al. 2008; Locher et al., 2010). Similar findings were reported on garlic (Allium sativum) extract for controlling red mite infestation in a layer farm in Babol, North of Iran (Faghihzade et al., 2014).
CONCLUSION
Present findings indicated that the ethanolic extract of Ferula assafoetidae was effective on red mite and reduced the red mite population in vivo and in vitro condition but N-Hexan extract of Ferula assafoetidae was not effective on red mite, due to active components like Diethylpyridine and LC50 of ethanolic extract that was indicated 16 ^g/cm3.
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