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Evaluation of Antiviral Activity of Selected Anise Oil as An Essential Oil Against Bovine Herpes Virus Type -1 In vitro

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Abstract

Abstract: In this study, the antiviral activity of anise oil against bovine herpes virus type-1 (BHV-1) was investigated in the cell culture. Anise oil was found to be non-toxic to MDBK cells up to concentration 100μg/ml and inhibited the growth and development of BHV-1 in a dose-dependent manner in MDBK cells. In order to study the possible mechanism of the antiviral activity of anise oil, MDBK cells were treated with anise oil as a pre, simultaneous and post viral infection treatment assay. At maximum non-cytotoxic concentrations of anise oil, the inhibition of virus yield showed that the treated cells with anise oil for 1 hour after infection can significantly reduce the virus titer from 106 to 103 TCID /ml but the inhibitory effect of anise 50 oil was not significant in other cases. Although the precise mechanism has not yet to be defined, our work indicated that anise oil could inhibit growth and development of BHV-1 in in vitro cultured cells
Global Veterinaria 10 (5): 496-499, 2013
ISSN 1992-6197
© IDOSI Publications, 2013
DOI: 10.5829/idosi.gv.2013.10.5.7338
Corresponding Author: Fatma, M. Abdallah, Department of Virology, Faculty of Veterinary Medicine,
Zagazig University, Egypt.
496
Evaluation of Antiviral Activity of Selected Anise Oil as
An Essential Oil Against Bovine Herpes Virus Type -1 In vitro
Fatma, M. Abdallah, H. Sobhy and G. Enan
1 22
Department of Virology, Faculty of Veterinary Medicine, Zagazig University, Egypt
1
Department of Botany and Microbiology, Faculty of Science, Zagazig University, Egypt
2
Abstract: In this study, the antiviral activity of anise oil against bovine herpes virus type-1 (BHV-1) was
investigated in the cell culture. Anise oil was found to be non-toxic to MDBK cells up to concentration
100µg/ml and inhibited the growth and development of BHV-1 in a dose-dependent manner in MDBK cells.
In order to study the possible mechanism of the antiviral activity of anise oil, MDBK cells were treated with
anise oil as a pre, simultaneous and post viral infection treatment assay. At maximum non-cytotoxic
concentrations of anise oil, the inhibition of virus yield showed that the treated cells with anise oil for 1 hour
after infection can significantly reduce the virus titer from 10 to 10 TCID /ml but the inhibitory effect of anise
63
50
oil was not significant in other cases. Although the precise mechanism has not yet to be defined, our work
indicated that anise oil could inhibit growth and development of BHV-1 in in vitro cultured cells.
Key words: Bovine Herpes Virus Type-1 (BHV-1) Anise oil Antiviral activity MDBK cell TCID50.
INTRODUCTION compounds have encouraged more detailed studies of
Bovine herpes virus type-1 (BHV-1) are DNA viruses components posses pharmacological effects,
belonging to the family herpesviridae and are responsible demonstrating anti inflammatory, antioxidant and anti
for a variety of mild to severe disease which are cancerogenic properties [6]. Medicinal plants produce a
sometimes life threatening [1], BHV-1 has a great variety of chemical constituents with the potential to
importance in veterinary medicine as it classified in a list inhibit viral replication and compounds from natural
B disease by the office International des Epizooties (OIE) sources are of interest as possible sources to control viral
[2]. infection. The anti herpes activity of several essential oils
In Egypt, since 1960s attention was drawn to BHV-1 of different plant sources as well as some constituents
as one of the most significant causes of great economic of essential oils had been demonstrated previously [7].
losses in feedlot and dairy farms; mainly due to deaths The application of anise oil as an essential oil met the
from pneumoentritis, abortion and prolonged feeding standard demands of current pharmacopeias [8]. This
periods programs [3]. A very effective treatment for essential oil is rich in trans-anethole and consist of about
BHV-1 is available since the introduction of acyclovir in 80% of this phenyl propanoid [9]. Phenyl propanoids
the 1970s and it is the most commonly used chemotherapy present in these essential oils contribute to their antiviral
[4]. Antiviral agents might produce toxic side effects. activity [10]. Therefore, the present study was undertaken
In addition, the emergence of virus strains resistant to for the evaluation of antiviral activity of selected anise oil
commonly used anti herpes virus drugs [5]. The as an essential oil against bovine herpes virus type-1.
development of viral resistance toward antiviral agents
enhances the need for new effective compounds against MATERIALS AND METHODS
viral infection. In the recent years, there has been an
increasing interest in the use of natural products and Anise Oil: Anise oil was purchased from (Sigma-
some questions concerning the safety of synthetic Aldrich). Anise oil was dissolved in ethanol and was
plant resources. Various essential oils and their
Global Veterinaria, 10 (5): 496-499, 2013
497
further diluted in medium for cell culture experiments in For determination of antiviral activity of the anise oil at
different concentrations as 20, 40, 60, 80, 100, 120, 140, simultaneous and post BHV-1 infection; the anise oil was
160, 180, 200 µg /ml [11]. added together with the virus and after 1, 4, 8 and 12 hour
BHV-1 and Cell Culture: BHV-1 was kindly obtained performed in antiviral susceptibility studies. Each assay
from Virology Department, Faculty of Veterinary was run in three replicates. Wells containing medium with
Medicine, Zagazig University. Continuous cell line of 1% ethanol but no anise oil was also included on each
Madin Darby Bovine Kidney (MDBK) cells were plate as controls. Virus titer was determined by the
purchased from VACSERA Company, Agoza, Giza, Egypt. endpoint dilution method and expressed as TCID /ml by
MDBK cells were grown in monolayer culture with comparison with virus control [16].
Dulbecco s modified Eagle s medium supplemented with
''
8% fetal calf serum (FCS), 100 IU/ml penicillin, 100 µg/ml RESULTS
streptomycin and amphotericin B (0.025µg/ml) and
maintained at 37°C with 5% CO2 for 24 hours for Cell Viability Test: The assessment of cytotoicity was
confluency. MDBK cells were cultured onto 96- well and performed with anise oil serially diluted in DMEM with
6 -well culture plates for cytotoxicity and antiviral assays, different concentrations as 10, 20, 40, 60, 80, 100, 120, 140,
respectively and propagated in monolayer culture with 160, 180, 200 µg/ml. The maximum non-cytotoxic
Dulbecco s modified Eagles medium (DMEM) concentration of anise oil was about 100µg/ml and this
''
supplemented with 2% fetal calf serum at 37°C in an concentration did not cause any change in MDBK cells
atmosphere of 5% of CO [12]. Stock virus was and was used in further experiments in this work.
2
propagated and titrated and stored at -80°C until used as However, cell lysis and degeneration were observed with
previously described [13]. Virus titer was obtained by limit increasing intensity at higher concentrations of the anise
dilution method and expressed as 50% tissue culture oil (Figure 1).
infectious dose per ml (TCID /ml) [14]. The infection titer
50
of the stock virus solution was 10 TCID / ml. Virus Yields Inhibition Assay: The potent antiviral
6
50
Cell Viability Test: For cytotoxicity assays, MDBK cells in vitro. The inhibition of virus yield by the anise oil was
were seeded onto 96- well plates and incubated for evaluated by TCID /ml assay in MDBK cells. When host
24 hours at 37°C. The medium was removed and fresh cells were pretreated with anise oil prior to infection,
Dulbecco's modified Eagles medium (DMEM) containing none of the tested oils showed significant effects on viral
'
the appropriate dilutions of anise oil were added onto infection as well as simultaneous infection treatment
subconfluent cells in three replicates for each assay (Figure 2). On the other hand at postinfection
concentration of anise oil. Wells containing medium treatment assay, the infectivity of BHV-1was inhibited
without anise oil were also included on each plate as especially at 1 hour post viral infection, where the virus
controls. Cell destruction was evaluated during titer of the supernatant dropped from 10 to 10 TCID /ml.
microscopic examination. Cytoxicity was expressed as the The inhibitory effect of anise oil was not significant
50% cytotoxic concentration (CC ), that is, the at 4, 8 and 12 hour post infection treatment assay where
50
concentration of anise oil required to reduce viral BHV-1 replicated normally and the viral titers recovered
cytopathogenicity by 50% (within micro tray well) [15]. from supernatant of infected cultures were ranged from
Virus Yields Inhibition Assay: The maximum non
cytotoxic concentration of anise oil was always used to DISCUSSION
evaluate the mode of antiviral action. In order to study the
possible mechanism of the antiviral activity of anise oil, Despite the successes in the treatment of some virus
MDBK cells were treated with anise oil as a pre, diseases during the past three decades, the search for
simultaneous and post BHV-1 infection. Cell monolayers new antiviral drugs remains an area of active investigation
were pretreated with anise oil prior to inoculation with [4]. Effective treatment is not available for many viral
BHV-1 by adding the oil to medium followed by infections. Moreover, the selection of resistant and
incubation for 1 hour at 37°C before viral infection. cross-resistant mutants caused partially by the narrow
post viral infections at 37°C, respectively as typically
50
activity of anise oil was determined against BHV-1
50
63
50
10 - 10 TCID / ml as virus control.
5.5 5.9
50
Global Veterinaria, 10 (5): 496-499, 2013
498
Fig. 1: Cytotoxicity effect of Anise oil on MDBK cells (X100). A-Normal MDBK cell cultures showing a confluent
monolayer sheet of cells. B-MDBK cells treated with high concentrations of anise showing cell rounding and
clumping
Fig. 2: Antiviral activity of the anise oil (pre, suitable animal models.
simultaneous and post BHV-1 infection treatment
assay) on virus yield in MDBK cells REFERENCES
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... It is an evergreen tree abundant in upper east Vietnam and southwest China. This tree is 6-8 m in height, dull gritty hued bark, green branchlets, glabrous; with all parts of the tree having a satisfying sweet-smell (Abdallah et al., 2013;Benmalek et al., 2013). The essential components that account for the awesome sweetsmelling aromas are essentially terpenes, monoterpenes and linalool (Muazzam et al., 2020;Bagewadi et al., 2019). ...
... The essential components that account for the awesome sweetsmelling aromas are essentially terpenes, monoterpenes and linalool (Muazzam et al., 2020;Bagewadi et al., 2019). In a previous study, EOs obtained from I. verum were evaluated for antiviral activity versus bovine herpes virus Type 1, in vitro and yielded promising results (Abdallah et al., 2013). ...
... The phytochemicals present in the Illicium verum essential oil can be used successfully to treat the multi-drug resistant and highly virulent MRSA. Recently, due to the irrational employment of antibiotics in the management of infectious diseases as well as patient non-compliance has led to the emergence of multi-drug resistant strains and further over utilization of the antimicrobials can lead to more serious outcomes (Abdallah et al., 2013;Benmalek et al., 2013;Muazzam et al., 2020). The WHO has effectively warned about the harmful impacts due to the misuse of antimicrobial agents, which not only has increased the incidence of antimicrobial resistance, but also led to antibiotic-induced adverse drug reactions. ...
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Objective: The Illicium verum is an evergreen Chinese plant, from which a culinary spice named star anise is obtained and is predominantly used in Asian countries. Besides its flavor, it possess high medicinal value. Illicium verum possesses good antimicrobial properties and in the past utilized to treat versatile ailments of microbial origin. Illicium verum is commonly known as star anise. Methods: This study is an attempt to resurrect the ancient forgotten traditional spice Illicium verum by analyzing its essential oils (EOs) for the phyto-biochemical properties using GC-MS, IR studies and various standard biochemical tests to record the presence of the chemical compounds based on its test reactions and also to measure its anti-microbial vulnerability testing towards the MRSA clinical isolates, by deploying the standard anti-microbial assay in the aseptic microbiological laboratory conditions. Results: The data procured from the standard antimicrobial assay of Illicium verum essential oil were compared with that of the standard antibiotic e-strips towards the same set of MRSA clinical isolates. The antimicrobial assay test results of Illicium verum essential oil against the clinical isolates of the MRSA showed promising results when compared to that of standard antibiotic e-strips. The results showed that all the MRSA clinical isolates were susceptible to the tested drug with inhibition zone diameter ranging from 18 mm to 28 mm (Mean 24.05 mm). The MBC and MIC results confirmed that the tested extract was potent against the highly virulent Methicillin-Resistant Staphylococcus aureus (MRSA) clinical isolates with MBC and MIC of 0.97 mg/mL and 0.70 mg/mL, which was lower compared to MBC and MIC values of the standard antibiotics. Conclusion: The results obtained from this study is a motivation to rediscover the ancient phytochemical compounds as an added asset to the standard available drugs in dealing with the upcoming hazardous diseases and pandemics. Thus, it relishes us to perform more of such studies to enhance the phyto-pharmacological industry. Ó 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
... It is an evergreen tree abundant in upper east Vietnam and southwest China. This tree is 6-8 m in height, dull gritty hued bark, green branchlets, glabrous; with all parts of the tree having a satisfying sweet-smell (Abdallah et al., 2013;Benmalek et al., 2013). The essential components that account for the awesome sweetsmelling aromas are essentially terpenes, monoterpenes and linalool (Muazzam et al., 2020;Bagewadi et al., 2019). ...
... The essential components that account for the awesome sweetsmelling aromas are essentially terpenes, monoterpenes and linalool (Muazzam et al., 2020;Bagewadi et al., 2019). In a previous study, EOs obtained from I. verum were evaluated for antiviral activity versus bovine herpes virus Type 1, in vitro and yielded promising results (Abdallah et al., 2013). ...
... The phytochemicals present in the Illicium verum essential oil can be used successfully to treat the multi-drug resistant and highly virulent MRSA. Recently, due to the irrational employment of antibiotics in the management of infectious diseases as well as patient non-compliance has led to the emergence of multi-drug resistant strains and further over utilization of the antimicrobials can lead to more serious outcomes (Abdallah et al., 2013;Benmalek et al., 2013;Muazzam et al., 2020). The WHO has effectively warned about the harmful impacts due to the misuse of antimicrobial agents, which not only has increased the incidence of antimicrobial resistance, but also led to antibiotic-induced adverse drug reactions. ...
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Objective The Illicium verum is an evergreen Chinese plant, from which a culinary spice named star anise is obtained and is predominantly used in Asian countries. Besides its flavor, it possess high medicinal value. Illicium verum possesses good antimicrobial properties and during olden days utilized to treat versatile ailments of microbial origin. Illicium verum is commonly known as star anise. Methods This study is an attempt to resurrect the ancient forgotten traditional spice Illicium verum by analyzing its essential oils (EOs) for the phyto-biochemical properties using GC-MS, IR studies and various standard biochemical tests to record the presence of the chemical compounds based on its test reactions and also to measure its anti-microbial vulnerability testing towards the MRSA clinical isolates, by deploying the standard anti-microbial assay in the aseptic microbiological laboratory conditions. Results The data procured from the standard antimicrobial assay of Illicium verum essential oil were compared with that of the standard antibiotic e-strips of the available drug towards the same set of MRSA clinical isolates. The antimicrobial assay test results of Illicium verum essential oil against the clinical isolates of the MRSA showed promising results when compared to that of standard antibiotic e-strips. The results showed that all the MRSA clinical isolates were susceptible to the tested drug with inhibition zone diameter ranging from 18mm to 28mm (Mean 24.05 mm). The MBC and MIC results confirmed that the tested extract was potent against the highly virulent Methicillin-Resistant Staphylococcus aureus (MRSA) clinical isolates with MBC and MIC of 0.97 µ/mL and 0.70 µ/mL, which was lower compared to MBC and MIC values of the standard antibiotics. Conclusion The result obtained from this study is a motivation to rediscover the ancient phytochemical compounds as an added asset to the standard available drugs in dealing with the upcoming hazardous diseases and pandemics. Thus it relishes us to perform more of such studies to enhance the phyto-pharmacological industry.
... On the other hand, plant-derived antiviral extract are of increasing interest in the development of new, non-toxic, more effective and specific anti-herpesviruses. Indeed, several trials using plant extracts have shown in vitro anti-BHV-1 activities at early and/or late stages of the viral replication such as: Phyllanthus orbicularis [3], Erythroxylum deciduum, Lacistema hasslerianum (chodat), Xylopia aromatica [4], Heteropteris aphrodisiaca [5], Acacia nilotica (gum arabic tree) [6], Lippia graveolens (Mexican oregano or redbrush lippia) [7], Guettarda angelica (Velvetseed) [8], Prunus myrtifolia (West Indian cherry), Symphyopappus compressus [9], and Pimpinella anisum (Anise) [10]. A large number of plant extracts from Lamiaceae were also examined for their potential antiviral activity against herpesvirus, such as: Melissa officinalis (lemon balm), Mentha piperita (pepper-mint), Prunella vulgaris (prunella), Rosmarinus officinalis (rosemary), Salvia officinalis (sage) and Thymus vulgaris (thyme)11121314. ...
... The thyme EO revealed higher antiviral activity and an SI of 14.49, whereas EE and AE revealed lower SIs (table 2). The anti-herpes activity of several EOs of different plant sources as well as of some constituents of EOs was reported previously, e. g. peppermint oil, thyme oil and anise oil [10, 24, 25]. The presence of thymol and β-caryophyllene in EO may contribute strongly to their antiviral effect which is in agreement with our results and described by others [14, 26]. ...
... On the other hand, plant-derived antiviral extracts are of great interest for the development of new, non-toxic, more effective and specific anti-herpesvirus active molecules. Indeed, several trials using plant extracts have shown in vitro anti-BHV-1 activities at early and/or late stages of the viral replication, such as: Phyllanthus orbicularis [3], Erythroxylum deciduum, Lacistema hasslerianum (chodat), Xylopia aromatica [4], Heteropteris aphrodisiaca [5], Acacia nilotica (gum arabic tree) [6], Lippia graveolens (Mexican oregano or redbrush lippia) [7], Guettarda angelica (Velvetseed) [8], Prunus myrtifolia (West Indian cherry), Symphyopappus compressus [9], and Pimpinella anisum (Anise) [10]. ...
... The anti-herpes virus activities of several EOs of different plant sources as well as of some constituents of EOs were reported previously, e.g. peppermint oil, thyme oil and anise oil [10,25,26]. The presence of thymol and β-caryophyllene in EO may strongly contribute to their antiviral effect which is in agreement with our results and as described by others [14,27]. ...
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... Yang et al. (2010) introduced I. verum as natural antimicrobials for the treatment of antibiotic pathogens. Abdallah et al. (2013) investigated the antiviral activity of anise oil against bovine herpes virus type 1-(BHV-1) in cell culture and they found that anise oil is non-toxic to MDBK cells upto 100 ug/ml, and also inhibited the growth and development of BHV-1. Sung et al. (2012) recommended star anise in the treatment of inflammatory diseases. ...
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Star anise (Illicium verum Hook. f.) is an important herb in traditional Chinese medicine as well as traditional Asian medicine. The fruit is aromatic and has a strong, pungent and mildly sweet taste. Star anise is one of the many species that contain bioactive compounds as well as a number of phenolic and flavonoid compounds, having antioxidant, preservative and antimicrobial properties. All relevant papers in the English language from researchers of different countries were collected. The keywords of Chinese star anise, anise, traditional Chinese medicine and modern pharmaceutical science were searched in Google Scholar, Scopus, Research Gate and PubMed. Its seeds are good source of minerals like calcium, iron, copper, potassium, manganese, zinc, and magnesium. The essential oil of Chinese star anise contains anethole which has shown several functional properties including antimicrobial, antioxidant, hypoglycemic, hypolipidemic and oestrogenic properties. Due to all positive characteristics, such as antidiabetic, hypolipidemic, antioxidant activities, anticancer and antimicrobial properties, both seeds and essential oils of anise is promising for safe use as super food supplements and raw constituents in the both pharmaceutical and food industries. Anise seed oil contains anethol, estragole, eugenol, pseudisoeugenol, methyl chavicol and anisaldehyde, coumarins, scopoleting, umbelliferon, estrols, terpene hydrocarbons, and polyacetylenes as the major compounds. The plant oil has both pharmacological and clinical effects. The pharmacological effects consist of antimicrobial, hepatopreotective, anticonvulsant, anti-inflammatory, antispasmodic, bronchodilator, estrogenic, expectorant and insecticidal effects, and clinical effects such as nausea, constipation, menopausal period, virus, diabetes, obesity and sedative action. Combined with a nutritious diet, other traditional Chinese and Asian medicine, Chinese star anise and ansie seed may improve various aspects of health. More clinical studies are necessary to uncover the numerous substances and their effects in ginseng that contribute to public health.
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Keyvani and star anise essential oil by supercritical CO2 F. Bokharaei-Salim, 2012. Evaluation of in vitro extraction herpes simplex virus type-1. African Journal of 10
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Herpes Simplex Virus Type Screening for antiviral activites of isolated compounds from essential oils. Evidence Based Complementary and Alternative Medicine
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Inhibitory Effect of Essential Oils Against 18. Astani, A., J. Reichling and P. Schnitzler, 2011. Herpes Simplex Virus Type, 2. 15(1-2): 71-78. Screening for antiviral activites of isolated compounds from essential oils. Evidence Based Complementary and Alternative Medicine. Article ID 253643, pp: 8-15.