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Inhibitory Activity of Illicium verum Extracts against Avian Viruses

Inhibitory Activity of Illicium verum Extracts against Avian Viruses Hindawi Advances in Virology Volume 2020, Article ID 4594635, 8 pages https://doi.org/10.1155/2020/4594635 Research Article Inhibitory Activity of Illicium verum Extracts against Avian Viruses 1,2 2 2 Mohammed S. Alhajj , Mahmood A. Qasem, and Saud I. Al-Mufarrej Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, amar University, Dhamar, Yemen Department of Animal Production, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia Correspondence should be addressed to Mohammed S. Alhajj; alhaj12004@gmail.com Received 20 August 2019; Accepted 2 January 2020; Published 25 January 2020 Academic Editor: Jay C. Brown Copyright © 2020 Mohammed S. Alhajj et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (is study aimed at screening the inhibitory activity of Illicium verum extracts against avian reovirus, infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), and infectious laryngotracheitis virus (ILTV). (e cytotoxic and antiviral actions of 3 extracts, absolute methanol (100MOH), 50% methanol (50MOH), and aqueous extracts (WA.), were evaluated by MTT assay. (e Illicium verum extracts were added to the cultured chick embryo fibroblast (CEF) with tested viruses in three attacks, preinoculation, postinoculation, and simultaneous inoculation. (e three extracts showed antiviral inhibitory activity against all tested viruses during simultaneous inoculation and preinoculation except 100MOH and 50MOH that showed no effect against IBDV, thereby suggesting that the extracts have a preventive effect on CEF against viruses. During postinoculation, the extracts exhibited inhibitory effects against NDV and avian reovirus, while no effect against IBDV recorded and only the 100MOH showed an inhibitory effect against ILTV. (e initial results of this study suggest that Illicium verum may be a candidate for a natural alternative source for antiviral agents. (H1N1) [3], Newcastle disease virus [4], rotavirus [5], and 1. Introduction herpes simplex virus type 1 [6]. Natural resources such as herbs In spite of tremendous progress in the means of care, using and their extract were investigated for the biological action that medicine and vaccination to control infectious diseases, gets them an encouraging prospect for research to control such there is still a significant threat to the poultry industry. disease. Star anise (Illicium verum) represents one of the me- Viruses are one of the biggest causative agents of poultry dicinal herbs, especially after the popularity it achieved as an diseases and are considered the principal risk to the poultry antiflu drug. Shikimic acid extracted from the fruits of star anise industry resulting in enormous economic losses to the being used for the production of Tamiflu [7]. Illicium verum poultry industry worldwide. Vaccination remains an im- plant is an evergreen plant, usually recognized as Chinese star portant strategy to combat infection of poultry diseases. anise, which has star-shaped fruits and originates in China and Vietnam, traditionally practiced as a spice and herb. Also, it has However, the infectious disease occurs despite vaccination against these diseases while antiviral agents are not used in medicinal properties that have significant health benefits [8]; poultry diseases due to toxicity and high costs of antiviral star anise extracts showed antiviral activity against human compounds [1, 2]. (erefore, it is necessary to find other immunodeficiency virus (HIV) [9], herpes simplex virus type 1 ways to control viral diseases. (HSV-1) [10], herpes simplex virus type 2 (HSV-2) [11], and Currently, attention is increased in searching for new ef- bovine herpes virus type 1 (BHV-1) [12]. However, the data fective and safe compounds to control viral diseases in human available about its activity against avian viruses are limited. (is beings and animals. A number of investigations were carried study was planned to evaluate the antiviral activity of the three out to test different plant extracts against influenza virus extracts from Illicium verum against four avian viruses. 2 Advances in Virology tetrazolium bromide) method [15]. Confluent monolayers of 2. Material and Methods CEF cells in 96-well tissue culture plates were incubated with 2.1. e Plant. Illicium verum dry fruits were purchased different concentrations of Illicium verum extracts from a local herb store in Riyadh, Saudi Arabia. (e dry (250–0.48 mg/ml) diluted with M199 medium and incu- fruits of star anise were ground into a fine powder. bated at 37 C with 5% CO ; cells without the extracts were applied as the control. After 72 hours of incubation, 20 μl of MTT solution (5 mg/ml PBS) (Sigma) was added to each 2.2. Preparation of Star Anise Extracts. (e extraction of well, the plates were further incubated at 37 C for 4 hours, Chinese star anise was carried out using three solvents: and then, the medium was removed and 100 μl of dimethyl absolute methanol, 50% methanol, and sterile distilled water. sulfoxide (DMSO) was added to each well. (e plates were For each extract, 100 g of anise powder was added separately shaken in a microplate shaker for 5 minutes, and the ab- to 500 ml of the corresponding solvent and mixed well in sorbance was read at a wavelength of 570 nm using a tightly sealed flasks and then placed in a water bath at 37 C microplate reader. (e experiments were performed in for 24 hours with intermittent shaking. (e supernatant was duplicate and repeated three times. collected and filtered (Whatman filter paper no. 1). (e residue was kept in the flasks and the extraction process was repeated 3–5 times until a clear solution is obtained. After 2.7. Infection and Viral Growth Assay. (e inhibitory anti- that, the extract solutions were collected and filtered again viral activity of the extracts was evaluated by the ability of the (Whatman no. 1). (e extract was allowed to dry in the oven extracts to inhibit CPE of viruses in CEF cells. According to at 45 C. (e dry extract was weighed and either diluted to a the outcome of the cytotoxicity assay, the extracts were diluted final concentration of 500 mg/ml or kept in fastened capped into different concentrations starting from the maximum bottles in the refrigerator for subsequent usage. (e diluted noncytotoxic concentration with M199 (3.9–0.24 mg/ml). (e extracts were centrifuged and filtered through a 0.22 μm antiviral activity of Illicium verum extracts against avian Millipore membrane filter (Whatman, Kent, UK). reovirus, NDV, IBDV, and ILTV was evaluated in three approaches of addition following the method of Fan et al. [4] and Xu et al. [16]. In the first method, simultaneous inocu- 2.3. Cells and Viruses. Chick embryo fibroblast cells (CEF) lation, 100 TCID of virus suspensions was incubated to- were used in this study, and the cells were prepared from 9- to 50 gether with Illicium verum extracts outside of cell culture for 1 11-day-old embryo chicks. Virus vaccine strains, avian reovirus hour at 4 C, and then the treated virus was added to the cell (S1133), NDV (Clone 30), IBDV (D78), and ILTV (LT-IVAX) culture and incubated for 1 hour at 37 C. After that, virus (Intervet Inc., Omaha NE68103 USA), were used in this study suspensions were removed and the cells were washed with to assess the antiviral action of Chinese star anise extracts. PBS and new M199 was added to the plates, and all plates were incubated at 37 C with 5% CO ; positive control wells contain 2.4. Preparation of Chick Embryo Fibroblast Culture. CEF cells and virus without the extracts, negative control wells cells were organized from 9- to 10-day-old chick embryo included only cells in the medium, and blank wells contain following the standard protocol described previously [13]. only the medium. (e cells were examined daily using an inverted optical microscope. In the preinoculation method, the Illicium verum extracts were incubated with the CEF cells 2.5. Propagation of Viruses. CEF monolayers grew in M199 for 2 hours at 37 C and 5% CO , and then the extracts were medium supplemented with 10% newborn fetal serum and removed and the treated cells were washed with PBS, and then incubated at 37 C with 5% CO . When the confluent CEF 100 TCID of virus suspensions was incubated with the monolayers were obtained, they were subcultured using treated cells for 1 h. After that, virus suspensions were re- trypsin/EDTA solution. Once the new CEF grew to 85% moved and maintenance medium was added to the plates, and confluence in T75 flasks (Corning), the growth medium was the plates were incubated at 37 C in a humidified atmosphere removed and 1 ml of each virus suspension stock was in- and 5% CO . (e plates were then examined daily for CPE. In oculated into the tissue culture flasks; the inoculated flasks the postinoculation method, 100 TCID of the virus was ° 50 were incubated for 1 hour at 37 C to allow viral adsorption. incubated with monolayers of the CEF in 96-well plates and (e virus suspensions were removed and M199 supple- incubated for 1 hour at 37 C to allow attachment. After that, mented with 5% newborn calf serum was added and then the the medium was removed and the cells were washed with PBS, inoculated cells were incubated at 37 C and 5% CO for and then different concentrations of the extracts seven days and tested daily for cytopathic effect (CPE) using (3.9–0.24 mg/ml) were added to each well of the plates. (e an inverted optical microscope (Nikon, Japan). (e viruses MTT assay was used to measure the livability of CEF, and the were passaged five times to ensure adoption to CEF. Virus test is done when active control cells showed 80% CPE. (e titration was done according to the method of Reed and experiments were repeated three times. (e virus inhibitory Muench [14]. rate was calculated based on the following formula: virus inhibitory rate � ((extract + virus − virus control)/(cell con- 2.6. Cytotoxicity Assay. Cytotoxicity of Illicium verum ex- trol − virus control))∗ 100. (e absorbance values and virus tracts was evaluated in vitro using a cell viability assay inhibitory rate were considered as indicators of antiviral known as MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl activity. Advances in Virology 3 Table 1: Absorbance means of the cytotoxicity of Illicium verum 2.8. Statistical Analysis. Data were analyzed by ANOVA extracts. using the GLM model of SAS [17]. (e Duncan multiple range test was used to compare differences among treatment Star anise extracts Concentration (mg/ml) means (P≤ 0.05) when significant. Percentage data were 100MOH 50MOH WA subjected to arcsine transformation before analysis. All c d c 62.5 0.152 0.198 0.379 experiments were repeated three times. d e e 31.25 0.126 0.141 0.293 d e f 15.62 0.108 0.124 0.247 d c g 3. Results and Discussion 7.8 0.116 0.258 0.127 a a d 3.9 0.390 0.361 0.324 a b a In the cytotoxicity assay using MTT, the absorbance value of 1.9 0.382 0.324 0.458 a b b cells is used as an indicator of the number of living cells and 0.97 0.389 0.327 0.410 b b c 0.48 0.353 0.307 0.373 related to cell growth [18], so that high absorbance value a a bc Cell control 0.402 0.380 0.395 means more living cells. When the absorbance values of the SEM 0.007 0.009 0.009 treated cells were not significantly lower than cell control, it P value <0.0001 <0.0001 <0.0001 indicated that the extract had no cytotoxicity on cells, and Means within a column with different subscripts are significantly different the corresponding concentration could consider as the (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol maximal safety level. (e results of the cytotoxicity test of extract; WA � water extract. Illicium verum extracts are listed in Table 1. (e results showed that the 100MOH and 50MOH extracts at 3.9 mg/ml and WA extract at 1.9 mg/ml were not significantly lower and WA presented the highest (P≤ 0.05) inhibitory rates of 56% and 67%, respectively. (e 50MOH extract showed the than those of the cell control group. (erefore, these con- centrations were considered as the maximal safety levels. (e lowest inhibitory rate (32%), while in postinoculation, no in- hibitory effect was detected in the three extracts, and only the 100MOH and 50MOH extracts at high concentrations (62.5 to 7.8 mg/ml) were toxic to CEF as indicated by the lower WA extract showed an inhibitory effect against IBDV in preinoculation mode (51%). Based on these results, the WA value of absorbance compared to cell control. (e aqueous extract has a significant antiviral effect in two adding methods extract at a concentration of 62.5 mg/ml showed no sig- nificant difference in comparison with cell control. (e high (preinoculation and simultaneous), while the other extracts have a significant antiviral effect only in simultaneous process, absorbance value may be due to the darker color of the aqueous extract at this high concentration (62.5 mg/ml), but and hence, the antiviral activity of WA extract against IBDV was better than that of other extracts. at the following levels (31.25–3.9 mg/ml), the absorbance values were significantly lower than the control group. On (e antiviral activity of Illicium verum extracts against ILTV is illustrated in Table 3. During preinoculation, the 100MOH the other side, the aqueous extract at the concentrations 1.9 and 0.97 mg/ml exhibited significantly larger values extract at 0.48 and 0.24 mg/ml and WA extract at 0.97 to 0.24 mg/ml concentrations showed significantly (P≤ 0.05) (P≤ 0.05) than the control group that may indicate that this extract could promote the cell growth within these dose higher values compared to the virus control group, while no significant difference was found between the 50MOH extract ranges [4]. For facilitating the comparison between the and virus control group (P≤ 0.05), which indicated that the extracts at the same levels, the maximal safety concentration of the extracts was considered at 3.9 mg/ml. 100MOH and aqueous extracts could prevent ILTV infection at a particular concentration. In postinoculation, the 50MOH and (e antiviral activity of Illicium verum extracts against IBDV is presented in Table 2. In preinoculation, the WA extracts showed no significant difference with the corre- sponding virus control group at all levels, while the 100MOH 100MOH and 50MOH extracts showed no significant dif- ference from the corresponding virus control group at all exhibited significantly (P≤ 0.05) higher value compared to the virus control group, which revealed that the 100MOH extract concentrations. (e aqueous extract at 0.48 and 0.24 mg/ml exhibited significantly (P≤ 0.05) more considerable value could treat ILTV infection. In simultaneous inoculation, the 100MOH extract at 3.9 and 1.9 mg/ml, 50MOH at 3.9 to than the virus control group, which indicated that the 0.48 mg/ml, and WA extract at all concentrations were sig- aqueous extract could prevent IBDV infection at a particular nificantly (P≤ 0.05) higher than those of the corresponding concentration. During postinoculation, the three extracts virus control groups, which indicated that the three extracts showed no significant difference from the corresponding virus control group at all levels, which indicated that the could have virucidal activity against ILTV at a particular dose. (e virus reduction rates of the extracts against ILTV are extracts have no treatment effect against IBDV after in- fection. In simultaneous inoculation, the values of 100MOH shown in Figure 2. During preinoculation, WA extract exhibited the highest (P≤ 0.05) virus inhibitory rate (60%), followed by extract at 0.48 and 0.24 mg/ml, 50MOH at 0.48 mg/ml, and WA extracts at 1.9 to 0.48 mg/ml were significantly the 100MOH with 32% inhibitory rate, while the 50MOH showed the lowest inhibitory rate (8%) against ILTV. During (P≤ 0.05) larger than those of the corresponding virus control group, which indicated that the three extracts could postinoculation modes, only 100MOH exhibited inhibitory activity against ILTV with the highest reduction rate (85%). In have virucidal effect against IBDV at specific dose. simultaneous inoculation, WA extract presented the most ele- (e virus inhibitory rates of the extracts against IBDV are illustrated in Figure 1. (e extracts showed inhibitory activity vated (P≤ 0.05) inhibitory rate (69%) while no significant differences were found between 100MOH (52%) and 50MOH against IBDV during simultaneous inoculation, and 100MOH 4 Advances in Virology Table 2: Absorbance means of the antiviral activity of Illicium verum extracts against infectious bursal disease virus in the three adding methods. Preinoculation Postinoculation Simultaneous inoculation Concentrations (mg/ml) 100MOH 50MOH WA 100MOH 50MOH WA 100MOH 50MOH WA b b c b b b d d d 3.9 0.181 0.185 0.185 0.183 0.164 0.172 0.205 0.198 0.277 b b c b b b d d c 1.9 0.183 0.195 0.193 0.192 0.169 0.191 0.187 0.206 0.338 b b c b b b cd d bc 0.97 0.184 0.183 0.220 0.197 0.178 0.181 0.226 0.216 0.343 b b b b b b b b b 0.48 0.195 0.190 0.303 0.168 0.167 0.168 0.356 0.325 0.391 b b b b b b b bc d 0.24 0.216 0.229 0.321 0.181 0.191 0.189 0.374 0.292 0.288 b b c b b b c c d Virus control 0.226 0.226 0.226 0.211 0.211 0.211 0.265 0.265 0.265 a a a a a a a a a Cell control 0.412 0.412 0.412 0.409 0.409 0.409 0.458 0.458 0.458 P value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 SEM 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 Means within a column with different subscripts are significantly different (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. –20 Preinoculation Postinoculation Simultaneous inoculation 100MOH 50MOH WA Figure 1: Virus inhibitory rate of Illicium verum extracts against infectious bursal disease virus using three adding methods. 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. Table 3: Absorbance means of the antiviral activity of Illicium verum extracts against infectious laryngotracheitis virus in the three adding methods. Preinoculation Postinoculation Simultaneous inoculation Concentrations (mg/ml) 100MOH 50MOH WA 100MOH 50MOH WA 100MOH 50MOH WA c b c b b b b b 3.9 0.187 0.181 0.208 0.256c 0.217 0.195 0.349 0.308 0.325 c b c b b c b b 1.9 0.186 0.185 0.193 0.353b 0.199 0.217 0.271 0.317 0.357 c b b b b d b b 0.97 0.194 0.187 0.270 0.335b 0.203 0.206 0.206 0.303 0.358 b b b b b d b b 0.48 0.249 0.196 0.301 0.374ab 0.217 0.196 0.196 0.318 0.369 b b b b b d c b 0.24 0.260 0.204 0.316 0.265c 0.201 0.198 0.173 0.246 0.325 c b c b b d c c Virus control 0.186 0.186 0.186 0.204d 0.204 0.204 0.198 0.198 0.198 a a a a a a a a Cell control 0.412 0.412 0.412 0.409a 0.409 0.409 0.458 0.458 0.458 P value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 SEM 0.02 0.01 0.02 0.02 0.01 0.01 0.02 0.02 0.02 Means within a column with different subscripts are significantly different (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. extracts (47%). (ese results showed that the 100MOH extract (e antiviral activity of Illicium verum extracts against had a significant (P≤ 0.05) antiviral activity against ILTV in the NDV is summarized in Table 4. All extracts showed a three adding methods, WA extract has antiviral activity in two preventive effect against NDV infection at specific con- adding models (preinoculation and simultaneous) while centrations at preinoculation. (e 100MOH extract at 50MOH extract had such activity only in simultaneous process. 0.24 mg/ml, 50MOH at 0.48 mg/ml, and WA extract at all (erefore, the antiviral activity of 100MOH and WA extracts concentrations showed significantly higher values compared against ILTV was better than that of 50MOH extract. Fur- to the virus control group (P≤ 0.05). Also, there was no thermore, 100MOH had to treat effect after ILTV infection, significant difference between the WA extract at 0.48 and while the other extracts did not show such effect. 0.24 mg/ml compared to the cell control group, which Virus inhibitory rate (%) Advances in Virology 5 ab 40 b b b Preinoculation Postinoculation Simultaneous inoculation 100MOH 50MOH WA Figure 2: Virus inhibitory rate of Illicium verum extracts against infectious laryngotracheitis virus using three adding methods. 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. Table 4: Absorbance means of the antiviral activity of Illicium verum extracts against Newcastle disease virus in the three adding methods. Preinoculation Postinoculation Simultaneous inoculation Concentrations (mg/ml) 100MOH 50MOH WA 100MOH 50MOH WA 100MOH 50MOH WA bc c b d b d d d c 3.9 0.311 0.297 0.361 0.174 0.209 0.193 0.188 0.190 0.233 bc bc b c b bc c cd c 1.9 0.289 0.319 0.342 0.216 0.197 0.278 0.269 0.227 0.230 bc b c b b b bc b 0.97 0.320 0.301c 0.361 0.249 0.206 0.314 0.349 0.281 0.330 bc b ab b a ab bc b b 0.48 0.322 0.384 0.387 0.318 0.348 0.323 0.298 0.312 0.359 b bc ab b a c d b b 0.24 0.356 0.320 0.402 0.317 0.352 0.249 0.196 0.316 0.363 c c c c b cd cd c c Virus control 0.255 0.255 0.255 0.237 0.237 0.237 0.242 0.242 0.242 a a a a a a a a a Cell control 0.454 0.454 0.454 0.367 0.367 0.367 0.445 0.445 0.445 P value 0.0004 0.0004 0.0007 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0003 SEM 0.02 0.02 0.02 0.01 0.01 0.01 0.02 0.02 0.02 Means within a column with different subscripts are significantly different (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. indicated a high protective effect of the WA extract at these concentrations against the inactivity of NDV. Similar results were found in postinoculation modes, and the three extracts b a exhibited a treatment effect against NDV. After infection, 60 b the 100MOH, 50MOH (0.48 and 0.24 mg/ml), and WA extracts (0.97 and 0.48 mg/ml) showed significantly (P≤ 0.05) higher difference compared with the virus control group. In simultaneous inoculation, the values of 100MOH extract at 0.97 mg/ml, 50MOH at 0.48 and 0.24 mg/ml, and WA extracts at 0.97 to 0.24 mg/ml concentrations were Preinoculation Postinoculation Simultaneous higher (P≤ 0.05) than those of the corresponding virus inoculation control groups which indicated that the three extracts could 100MOH have virucidal activity against NDV at certain doses. 50MOH (e virus inhibitory rates of the three extracts against NDV WA are presented in Figure 3. In preinoculation, WA and 50MOH Figure 3: Virus inhibitory rate of Illicium verum extracts against extracts demonstrated the highest (P≤ 0.05) virus inhibitory Newcastle disease virus using three adding methods. rate (75% and 66%, respectively), and the 100MOH showed the 100MOH � absolute methanol extract; 50MOH � 50% methanol lowest inhibitory rate (49%) against NDV. In the postinocu- extract; WA � water extract. lation method, 50MOH exhibited the highest (P≤ 0.05) re- duction rate (88%), while 100MOH and WA extracts showed a similar reduction effect, 60%, and 63%, respectively. After si- showed that the extracts had significant antiviral activity multaneous inoculation, the WA (60%) and 100MOH (53%) against NDV in three inoculation methods. extracts exhibited the highest reduction rate, while 50MOH (e results of the antiviral activity of Illicium verum extract showed the lowest inhibitory rate (38%). (ese results extracts against avian reovirus are illustrated in Table 5. (e Virus inhibitory rate (%) Virus inhibitory rate (%) 6 Advances in Virology Table 5: Absorbance means of the antiviral activity of Illicium verum extracts against the avian reovirus in the three adding methods. Preinoculation Postinoculation Simultaneous inoculation Concentrations (mg/ml) 100MOH 50MOH WA 100MOH 50MOH WA 100MOH 50MOH WA c bc b d d c c d d 3.9 0.294 0.326 0.356 0.182 0.205 0.228 0.243 0.199 0.184 a b b d d c bc c c 1.9 0.434 0.388 0.354 0.188 0.200 0.225 0.301 0.288 0.336 a b b b d b b b bc 0.97 0.412 0.349 0.358 0.285 0.195 0.277 0.322 0.356 0.356 ab b b c c b bc b ab 0.48 0.387 0.378 0.367 0.233 0.241 0.303 0.262 0.382 0.411 bc bc bc c b b bc b a 0.24 0.330 0.332 0.331 0.242 0.291 0.304 0.242 0.371 0.440 c c c c c c bc c c Virus control 0.282 0.282 0.282 0.235 0.235 0.235 0.301 0.301 0.301 a a a a a a a a a Cell control 0.454 0.454 0.454 0.367 0.367 0.367 0.445 0.445 0.445 P value 0.0007 0.001 0.006 <0.0001 <0.0001 <0.0001 0.0009 <0.0001 <0.0001 SEM 0.02 0.01 0.02 0.01 0.009 0.01 0.02 0.02 0.02 Means within a column with different subscripts are significantly different (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. three extracts revealed preventive action against reovirus infection at specific concentrations during preinoculation modes. (e 100MOH extract and 50MOH at 1.9–0.48 mg/ml and WA extract from 3.9 to 0.48 mg/ml showed significantly (P≤ 0.05) higher values compared to the virus control group 60 (P≤ 0.05). (e 100MOH extract at 1.9 to 0.48 mg/ml showed no significant difference compared to the control cell group, indicating a high protective effect on CEF against avian reovirus infection. During postinoculation modes, the three extracts exhibited a treatment effect against avian reovirus infection at a particular dose, and the values of 100MOH at Preinoculation Postinoculation Simultaneous 0.97 mg/ml, 50MOH at 0.24 mg/ml, and WA extracts at inoculation 0.97–0.24 mg/ml were significantly (P≤ 0.05) higher than 100MOH those of the virus control group. In simultaneous inocula- 50MOH tion, the values of 50MOH at 0.48 mg/ml and WA extracts at WA 0.24 mg/ml concentrations were significantly higher Figure 4: Virus inhibitory rate of Illicium verum extracts against (P≤ 0.05) than those of the virus control groups, which avian reovirus using three adding methods. 100MOH � absolute indicated that these extracts could have a virucidal effect on methanol extract; 50MOH � 50% methanol extract; WA � water avian reovirus at an appropriate dose. extract. (e virus inhibitory rates of the three extracts against avian reovirus are presented in Figure 4. In preinoculation, during before virus adsorption or attachment to CEF cells. 100MOH extract revealed the highest (P≤ 0.05) virus in- During preinoculation, similar findings were mentioned hibitory rate (P≤ 0.05) against avian reovirus (91%), fol- except 100MOH and 50MOH extract against IBD, which lowed by 50MOH with a 63% reduction rate, while the WA may indicate that the extracts are interfering with virus extract showed the lowest inhibitory rate (49%). During the attachment and penetration of host cells, suggesting that the postinoculation method, the WA extract exhibited the extracts have a preventive effect on CEF against viruses. highest reduction rate (51%), while 100MOH and 50MOH However, in postinoculation, the results showed the dif- extracts showed a similar reduction effect with inhibitory ferent impact of the extracts in each virus; the extracts rates of 38% and 42%, respectively. After simultaneous exhibited antiviral effect against NDV and reovirus while no inoculation, the WA extract presented the highest (P≤ 0.05) effect against IBDV and only the 100MOH extract showed reduction effect against avian reovirus (95%), 50MOH ex- activity against ILTV; these results indicate that anise ex- tracts showed 55% reduction rate, and 100MOH extract tracts have virucidal properties and may prevent the virus showed the lowest inhibitory rate (14%). (ese results in- replication after infection. (e differences among viruses in dicated that both WA and 50MOH extracts had significant response to Illicium verum extracts in this study may be antiviral activity against avian reovirus in the three adding attributed to the differences in the levels of active compo- methods while the 100MOH extract had high antiviral ac- nents in the extracts and variations among the tested viruses. tivity against avian reovirus during the preinoculation mode. For example, IBDV is a nonenveloped and highly resistant In general, the three adding methods of the extracts and virus [19]. It was proposed that the antiviral action of plant tested viruses refer to the stages of the virus infection cycle. extracts may be ascribable to an interaction with the viral Results showed that the extracts exhibited antiviral activity envelopes [20, 21]. However, the mechanism of action is still against all tested viruses during simultaneous inoculation not clear, whether the inhibitory effect of anise extracts is with respect to reduction rate among each extract on each due to interference with cellular membrane proteins or virus virus, indicating that the extracts have an inhibitory effect Virus inhibitory rate (%) Advances in Virology 7 receptors involved in host cell adsorption and penetration, References consequently preventing virus infection of the cells [11, 22]. [1] R. Elizondo-Gonzalez, D., Ricque-Marie, L. E. 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Oliveira et al., “Screening and 26-methyl ester extracted from Illicium verum roots of Brazilian medicinal plants for antiviral activity against displayed moderate anti-HIV activity [9]. Illicium verum rotavirus,” Journal of Ethnopharmacology, vol. 141, no. 3, essential oil inhibited viral infectivity by 99%, while phe- pp. 975–981, 2012. nylpropanoids reduced the HSV infectivity by 60–80% and [6] V. Lipipun, M. Kurokawa, R. Suttisri et al., “Efficacy of (ai medicinal plant extracts against herpes simplex virus type 1 sesquiterpenes inhibited the infectivity by 40–98% [10]. In infection in vitro and in vivo,” Antiviral Research, vol. 60, another study, anise oil showed dose-dependent antiviral no. 3, pp. 175–180, 2003. activity against HSV-2 [25]. (ere was no inhibitory effect [7] P. M. Dewick, “(e shikimate pathway: aromatic amino acids when the essential oils were added to the cells before in- and phenylpropanoids,” in Medicinal Natural Products: A fection or after the adsorption period. (e authors con- Biosynthetic Approach, P. M. Dewick, Ed., pp. 137–186, John cluded that essential oils may interact with the virus Wiley & Sons, Chichester, UK, 3rd edition, 2009. envelope and prevent the adsorption of the virus. [8] G.-W. Wang, W.-T. Hu, B.-K. Huang, and L.-P. Qin, “Illicium In conclusion, the three extracts showed antiviral ac- verum: a review on its botany, traditional use, chemistry and tivity against all tested viruses during simultaneous inoc- pharmacology,” Journal of Ethnopharmacology, vol. 136, no. 1, ulation and preinoculation except 100MOH and 50MOH pp. 10–20, 2011. [9] W.-Y. Song, Y.-B. Ma, X. Bai et al., “Two new compounds and that showed no effect against IBD, thereby indicating that anti-HIV active constituents from Illicium verum,” Planta the extracts have a prophylactic effect on CEF against vi- Medica, vol. 73, no. 4, pp. 372–375, 2007. ruses. Nevertheless, in postinoculation, the extracts [10] A. Astani, J. Reichling, and P. Schnitzler, “Screening for exhibited inhibitory effects against NDV and avian reo- antiviral activities of isolated compounds from essential oils,” virus, while no outcome found against IBDV and only the Evidence-Based Complementary and Alternative Medicine, 100MOH showed an inhibitory effect against ILTV. (e vol. 2011, Article ID 253643, 8 pages, 2011. initial results of this study suggest that Illicium verum may [11] C. Koch, J. Reichling, J. Schneele, and P. Schnitzler, “Inhib- be a candidate for a natural alternative source for antiviral itory effect of essential oils against herpes simplex virus type agents. However, more in vivo trials are required to con- 2,” Phytomedicine, vol. 15, no. 1-2, pp. 71–78, 2008. firm these findings. [12] M. S. Abdallah, H. Sobhy, and G. Enan, “Evaluation of an- tiviral activity of selected anise oil as an essential oil against bovine herpes virus type -1 in vitro,” Global Veterinaria, Data Availability vol. 10, no. 5, pp. 496–499, 2013. [13] S. K. Amir Hossain, S. Nasir Uddin, M. S. Rahman, A. Wadud, (e data used to support the findings of this study are and M. H. Khan, “Propagation of infectious bursal disease included within the article. virus (IBDV) in chicken embryo fibroblast cells,” Journal of Biological Sciences, vol. 6, no. 1, pp. 146–149, 2006. [14] L. J. Reed and H. Muench, “A simple method of estimating Conflicts of Interest fifty percent endpoints12,” American Journal of Epidemiology, vol. 27, no. 3, pp. 493–497, 1938. (e authors declare that they have no conflicts of interest. [15] T. Mosmann, “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays,” Journal of Immunological Methods, vol. 65, no. 1-2, Acknowledgments pp. 55–63, 1983. [16] J. Xu, X. Song, Z. Q. Yin et al., “Antiviral activity and mode of (e authors would like to extend their sincere appreciation action of extracts from neem seed kernel against duck plague to the Deanship of Scientific Research at King Saud Uni- virus in vitro,” Poultry Science, vol. 91, no. 11, pp. 2802–2807, versity and King Abdul-Aziz City for Science and Tech- nology (KACST) for funding this research. 8 Advances in Virology [17] SAS Institute, SAS User’s Guide, SAS Institute, Cary, NC, USA, 1999. [18] S. Patel, N. Gheewala, A. Suthar, and A. Shah, “In-vitro cy- totoxicity activity of Solanum nigrum extract against hela cell line and vero cell line,” International Journal of Pharmacy and Pharmaceutical Sciences, vol. 1, pp. 38–46, 2009. [19] F. Ingrao, F. Rauw, B. 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Hayashi, “Antiviral and immunostimulating effects of lignin-carbohydrate-pro- tein complexes from Pimpinella anisum,” Bioscience, Bio- technology, and Biochemistry, vol. 75, no. 3, pp. 459–465, 2011. [24] P. Schnitzler, A. Schuhmacher, A. Astani, and J. Reichling, “Melissa officinalis oil affects infectivity of enveloped her- pesviruses,” Phytomedicine, vol. 15, no. 9, pp. 734–740, 2008. [25] C. Koch, J. Reichling, R. Kehm et al., “Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase- positive and thymidine-kinase-negative herpesviruses,” Journal of Pharmacy and Pharmacology, vol. 60, no. 11, pp. 1545–1550, 2008. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Advances in Virology Hindawi Publishing Corporation

Inhibitory Activity of Illicium verum Extracts against Avian Viruses

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Hindawi Advances in Virology Volume 2020, Article ID 4594635, 8 pages https://doi.org/10.1155/2020/4594635 Research Article Inhibitory Activity of Illicium verum Extracts against Avian Viruses 1,2 2 2 Mohammed S. Alhajj , Mahmood A. Qasem, and Saud I. Al-Mufarrej Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, amar University, Dhamar, Yemen Department of Animal Production, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia Correspondence should be addressed to Mohammed S. Alhajj; alhaj12004@gmail.com Received 20 August 2019; Accepted 2 January 2020; Published 25 January 2020 Academic Editor: Jay C. Brown Copyright © 2020 Mohammed S. Alhajj et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (is study aimed at screening the inhibitory activity of Illicium verum extracts against avian reovirus, infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), and infectious laryngotracheitis virus (ILTV). (e cytotoxic and antiviral actions of 3 extracts, absolute methanol (100MOH), 50% methanol (50MOH), and aqueous extracts (WA.), were evaluated by MTT assay. (e Illicium verum extracts were added to the cultured chick embryo fibroblast (CEF) with tested viruses in three attacks, preinoculation, postinoculation, and simultaneous inoculation. (e three extracts showed antiviral inhibitory activity against all tested viruses during simultaneous inoculation and preinoculation except 100MOH and 50MOH that showed no effect against IBDV, thereby suggesting that the extracts have a preventive effect on CEF against viruses. During postinoculation, the extracts exhibited inhibitory effects against NDV and avian reovirus, while no effect against IBDV recorded and only the 100MOH showed an inhibitory effect against ILTV. (e initial results of this study suggest that Illicium verum may be a candidate for a natural alternative source for antiviral agents. (H1N1) [3], Newcastle disease virus [4], rotavirus [5], and 1. Introduction herpes simplex virus type 1 [6]. Natural resources such as herbs In spite of tremendous progress in the means of care, using and their extract were investigated for the biological action that medicine and vaccination to control infectious diseases, gets them an encouraging prospect for research to control such there is still a significant threat to the poultry industry. disease. Star anise (Illicium verum) represents one of the me- Viruses are one of the biggest causative agents of poultry dicinal herbs, especially after the popularity it achieved as an diseases and are considered the principal risk to the poultry antiflu drug. Shikimic acid extracted from the fruits of star anise industry resulting in enormous economic losses to the being used for the production of Tamiflu [7]. Illicium verum poultry industry worldwide. Vaccination remains an im- plant is an evergreen plant, usually recognized as Chinese star portant strategy to combat infection of poultry diseases. anise, which has star-shaped fruits and originates in China and Vietnam, traditionally practiced as a spice and herb. Also, it has However, the infectious disease occurs despite vaccination against these diseases while antiviral agents are not used in medicinal properties that have significant health benefits [8]; poultry diseases due to toxicity and high costs of antiviral star anise extracts showed antiviral activity against human compounds [1, 2]. (erefore, it is necessary to find other immunodeficiency virus (HIV) [9], herpes simplex virus type 1 ways to control viral diseases. (HSV-1) [10], herpes simplex virus type 2 (HSV-2) [11], and Currently, attention is increased in searching for new ef- bovine herpes virus type 1 (BHV-1) [12]. However, the data fective and safe compounds to control viral diseases in human available about its activity against avian viruses are limited. (is beings and animals. A number of investigations were carried study was planned to evaluate the antiviral activity of the three out to test different plant extracts against influenza virus extracts from Illicium verum against four avian viruses. 2 Advances in Virology tetrazolium bromide) method [15]. Confluent monolayers of 2. Material and Methods CEF cells in 96-well tissue culture plates were incubated with 2.1. e Plant. Illicium verum dry fruits were purchased different concentrations of Illicium verum extracts from a local herb store in Riyadh, Saudi Arabia. (e dry (250–0.48 mg/ml) diluted with M199 medium and incu- fruits of star anise were ground into a fine powder. bated at 37 C with 5% CO ; cells without the extracts were applied as the control. After 72 hours of incubation, 20 μl of MTT solution (5 mg/ml PBS) (Sigma) was added to each 2.2. Preparation of Star Anise Extracts. (e extraction of well, the plates were further incubated at 37 C for 4 hours, Chinese star anise was carried out using three solvents: and then, the medium was removed and 100 μl of dimethyl absolute methanol, 50% methanol, and sterile distilled water. sulfoxide (DMSO) was added to each well. (e plates were For each extract, 100 g of anise powder was added separately shaken in a microplate shaker for 5 minutes, and the ab- to 500 ml of the corresponding solvent and mixed well in sorbance was read at a wavelength of 570 nm using a tightly sealed flasks and then placed in a water bath at 37 C microplate reader. (e experiments were performed in for 24 hours with intermittent shaking. (e supernatant was duplicate and repeated three times. collected and filtered (Whatman filter paper no. 1). (e residue was kept in the flasks and the extraction process was repeated 3–5 times until a clear solution is obtained. After 2.7. Infection and Viral Growth Assay. (e inhibitory anti- that, the extract solutions were collected and filtered again viral activity of the extracts was evaluated by the ability of the (Whatman no. 1). (e extract was allowed to dry in the oven extracts to inhibit CPE of viruses in CEF cells. According to at 45 C. (e dry extract was weighed and either diluted to a the outcome of the cytotoxicity assay, the extracts were diluted final concentration of 500 mg/ml or kept in fastened capped into different concentrations starting from the maximum bottles in the refrigerator for subsequent usage. (e diluted noncytotoxic concentration with M199 (3.9–0.24 mg/ml). (e extracts were centrifuged and filtered through a 0.22 μm antiviral activity of Illicium verum extracts against avian Millipore membrane filter (Whatman, Kent, UK). reovirus, NDV, IBDV, and ILTV was evaluated in three approaches of addition following the method of Fan et al. [4] and Xu et al. [16]. In the first method, simultaneous inocu- 2.3. Cells and Viruses. Chick embryo fibroblast cells (CEF) lation, 100 TCID of virus suspensions was incubated to- were used in this study, and the cells were prepared from 9- to 50 gether with Illicium verum extracts outside of cell culture for 1 11-day-old embryo chicks. Virus vaccine strains, avian reovirus hour at 4 C, and then the treated virus was added to the cell (S1133), NDV (Clone 30), IBDV (D78), and ILTV (LT-IVAX) culture and incubated for 1 hour at 37 C. After that, virus (Intervet Inc., Omaha NE68103 USA), were used in this study suspensions were removed and the cells were washed with to assess the antiviral action of Chinese star anise extracts. PBS and new M199 was added to the plates, and all plates were incubated at 37 C with 5% CO ; positive control wells contain 2.4. Preparation of Chick Embryo Fibroblast Culture. CEF cells and virus without the extracts, negative control wells cells were organized from 9- to 10-day-old chick embryo included only cells in the medium, and blank wells contain following the standard protocol described previously [13]. only the medium. (e cells were examined daily using an inverted optical microscope. In the preinoculation method, the Illicium verum extracts were incubated with the CEF cells 2.5. Propagation of Viruses. CEF monolayers grew in M199 for 2 hours at 37 C and 5% CO , and then the extracts were medium supplemented with 10% newborn fetal serum and removed and the treated cells were washed with PBS, and then incubated at 37 C with 5% CO . When the confluent CEF 100 TCID of virus suspensions was incubated with the monolayers were obtained, they were subcultured using treated cells for 1 h. After that, virus suspensions were re- trypsin/EDTA solution. Once the new CEF grew to 85% moved and maintenance medium was added to the plates, and confluence in T75 flasks (Corning), the growth medium was the plates were incubated at 37 C in a humidified atmosphere removed and 1 ml of each virus suspension stock was in- and 5% CO . (e plates were then examined daily for CPE. In oculated into the tissue culture flasks; the inoculated flasks the postinoculation method, 100 TCID of the virus was ° 50 were incubated for 1 hour at 37 C to allow viral adsorption. incubated with monolayers of the CEF in 96-well plates and (e virus suspensions were removed and M199 supple- incubated for 1 hour at 37 C to allow attachment. After that, mented with 5% newborn calf serum was added and then the the medium was removed and the cells were washed with PBS, inoculated cells were incubated at 37 C and 5% CO for and then different concentrations of the extracts seven days and tested daily for cytopathic effect (CPE) using (3.9–0.24 mg/ml) were added to each well of the plates. (e an inverted optical microscope (Nikon, Japan). (e viruses MTT assay was used to measure the livability of CEF, and the were passaged five times to ensure adoption to CEF. Virus test is done when active control cells showed 80% CPE. (e titration was done according to the method of Reed and experiments were repeated three times. (e virus inhibitory Muench [14]. rate was calculated based on the following formula: virus inhibitory rate � ((extract + virus − virus control)/(cell con- 2.6. Cytotoxicity Assay. Cytotoxicity of Illicium verum ex- trol − virus control))∗ 100. (e absorbance values and virus tracts was evaluated in vitro using a cell viability assay inhibitory rate were considered as indicators of antiviral known as MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl activity. Advances in Virology 3 Table 1: Absorbance means of the cytotoxicity of Illicium verum 2.8. Statistical Analysis. Data were analyzed by ANOVA extracts. using the GLM model of SAS [17]. (e Duncan multiple range test was used to compare differences among treatment Star anise extracts Concentration (mg/ml) means (P≤ 0.05) when significant. Percentage data were 100MOH 50MOH WA subjected to arcsine transformation before analysis. All c d c 62.5 0.152 0.198 0.379 experiments were repeated three times. d e e 31.25 0.126 0.141 0.293 d e f 15.62 0.108 0.124 0.247 d c g 3. Results and Discussion 7.8 0.116 0.258 0.127 a a d 3.9 0.390 0.361 0.324 a b a In the cytotoxicity assay using MTT, the absorbance value of 1.9 0.382 0.324 0.458 a b b cells is used as an indicator of the number of living cells and 0.97 0.389 0.327 0.410 b b c 0.48 0.353 0.307 0.373 related to cell growth [18], so that high absorbance value a a bc Cell control 0.402 0.380 0.395 means more living cells. When the absorbance values of the SEM 0.007 0.009 0.009 treated cells were not significantly lower than cell control, it P value <0.0001 <0.0001 <0.0001 indicated that the extract had no cytotoxicity on cells, and Means within a column with different subscripts are significantly different the corresponding concentration could consider as the (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol maximal safety level. (e results of the cytotoxicity test of extract; WA � water extract. Illicium verum extracts are listed in Table 1. (e results showed that the 100MOH and 50MOH extracts at 3.9 mg/ml and WA extract at 1.9 mg/ml were not significantly lower and WA presented the highest (P≤ 0.05) inhibitory rates of 56% and 67%, respectively. (e 50MOH extract showed the than those of the cell control group. (erefore, these con- centrations were considered as the maximal safety levels. (e lowest inhibitory rate (32%), while in postinoculation, no in- hibitory effect was detected in the three extracts, and only the 100MOH and 50MOH extracts at high concentrations (62.5 to 7.8 mg/ml) were toxic to CEF as indicated by the lower WA extract showed an inhibitory effect against IBDV in preinoculation mode (51%). Based on these results, the WA value of absorbance compared to cell control. (e aqueous extract has a significant antiviral effect in two adding methods extract at a concentration of 62.5 mg/ml showed no sig- nificant difference in comparison with cell control. (e high (preinoculation and simultaneous), while the other extracts have a significant antiviral effect only in simultaneous process, absorbance value may be due to the darker color of the aqueous extract at this high concentration (62.5 mg/ml), but and hence, the antiviral activity of WA extract against IBDV was better than that of other extracts. at the following levels (31.25–3.9 mg/ml), the absorbance values were significantly lower than the control group. On (e antiviral activity of Illicium verum extracts against ILTV is illustrated in Table 3. During preinoculation, the 100MOH the other side, the aqueous extract at the concentrations 1.9 and 0.97 mg/ml exhibited significantly larger values extract at 0.48 and 0.24 mg/ml and WA extract at 0.97 to 0.24 mg/ml concentrations showed significantly (P≤ 0.05) (P≤ 0.05) than the control group that may indicate that this extract could promote the cell growth within these dose higher values compared to the virus control group, while no significant difference was found between the 50MOH extract ranges [4]. For facilitating the comparison between the and virus control group (P≤ 0.05), which indicated that the extracts at the same levels, the maximal safety concentration of the extracts was considered at 3.9 mg/ml. 100MOH and aqueous extracts could prevent ILTV infection at a particular concentration. In postinoculation, the 50MOH and (e antiviral activity of Illicium verum extracts against IBDV is presented in Table 2. In preinoculation, the WA extracts showed no significant difference with the corre- sponding virus control group at all levels, while the 100MOH 100MOH and 50MOH extracts showed no significant dif- ference from the corresponding virus control group at all exhibited significantly (P≤ 0.05) higher value compared to the virus control group, which revealed that the 100MOH extract concentrations. (e aqueous extract at 0.48 and 0.24 mg/ml exhibited significantly (P≤ 0.05) more considerable value could treat ILTV infection. In simultaneous inoculation, the 100MOH extract at 3.9 and 1.9 mg/ml, 50MOH at 3.9 to than the virus control group, which indicated that the 0.48 mg/ml, and WA extract at all concentrations were sig- aqueous extract could prevent IBDV infection at a particular nificantly (P≤ 0.05) higher than those of the corresponding concentration. During postinoculation, the three extracts virus control groups, which indicated that the three extracts showed no significant difference from the corresponding virus control group at all levels, which indicated that the could have virucidal activity against ILTV at a particular dose. (e virus reduction rates of the extracts against ILTV are extracts have no treatment effect against IBDV after in- fection. In simultaneous inoculation, the values of 100MOH shown in Figure 2. During preinoculation, WA extract exhibited the highest (P≤ 0.05) virus inhibitory rate (60%), followed by extract at 0.48 and 0.24 mg/ml, 50MOH at 0.48 mg/ml, and WA extracts at 1.9 to 0.48 mg/ml were significantly the 100MOH with 32% inhibitory rate, while the 50MOH showed the lowest inhibitory rate (8%) against ILTV. During (P≤ 0.05) larger than those of the corresponding virus control group, which indicated that the three extracts could postinoculation modes, only 100MOH exhibited inhibitory activity against ILTV with the highest reduction rate (85%). In have virucidal effect against IBDV at specific dose. simultaneous inoculation, WA extract presented the most ele- (e virus inhibitory rates of the extracts against IBDV are illustrated in Figure 1. (e extracts showed inhibitory activity vated (P≤ 0.05) inhibitory rate (69%) while no significant differences were found between 100MOH (52%) and 50MOH against IBDV during simultaneous inoculation, and 100MOH 4 Advances in Virology Table 2: Absorbance means of the antiviral activity of Illicium verum extracts against infectious bursal disease virus in the three adding methods. Preinoculation Postinoculation Simultaneous inoculation Concentrations (mg/ml) 100MOH 50MOH WA 100MOH 50MOH WA 100MOH 50MOH WA b b c b b b d d d 3.9 0.181 0.185 0.185 0.183 0.164 0.172 0.205 0.198 0.277 b b c b b b d d c 1.9 0.183 0.195 0.193 0.192 0.169 0.191 0.187 0.206 0.338 b b c b b b cd d bc 0.97 0.184 0.183 0.220 0.197 0.178 0.181 0.226 0.216 0.343 b b b b b b b b b 0.48 0.195 0.190 0.303 0.168 0.167 0.168 0.356 0.325 0.391 b b b b b b b bc d 0.24 0.216 0.229 0.321 0.181 0.191 0.189 0.374 0.292 0.288 b b c b b b c c d Virus control 0.226 0.226 0.226 0.211 0.211 0.211 0.265 0.265 0.265 a a a a a a a a a Cell control 0.412 0.412 0.412 0.409 0.409 0.409 0.458 0.458 0.458 P value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 SEM 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 Means within a column with different subscripts are significantly different (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. –20 Preinoculation Postinoculation Simultaneous inoculation 100MOH 50MOH WA Figure 1: Virus inhibitory rate of Illicium verum extracts against infectious bursal disease virus using three adding methods. 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. Table 3: Absorbance means of the antiviral activity of Illicium verum extracts against infectious laryngotracheitis virus in the three adding methods. Preinoculation Postinoculation Simultaneous inoculation Concentrations (mg/ml) 100MOH 50MOH WA 100MOH 50MOH WA 100MOH 50MOH WA c b c b b b b b 3.9 0.187 0.181 0.208 0.256c 0.217 0.195 0.349 0.308 0.325 c b c b b c b b 1.9 0.186 0.185 0.193 0.353b 0.199 0.217 0.271 0.317 0.357 c b b b b d b b 0.97 0.194 0.187 0.270 0.335b 0.203 0.206 0.206 0.303 0.358 b b b b b d b b 0.48 0.249 0.196 0.301 0.374ab 0.217 0.196 0.196 0.318 0.369 b b b b b d c b 0.24 0.260 0.204 0.316 0.265c 0.201 0.198 0.173 0.246 0.325 c b c b b d c c Virus control 0.186 0.186 0.186 0.204d 0.204 0.204 0.198 0.198 0.198 a a a a a a a a Cell control 0.412 0.412 0.412 0.409a 0.409 0.409 0.458 0.458 0.458 P value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 SEM 0.02 0.01 0.02 0.02 0.01 0.01 0.02 0.02 0.02 Means within a column with different subscripts are significantly different (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. extracts (47%). (ese results showed that the 100MOH extract (e antiviral activity of Illicium verum extracts against had a significant (P≤ 0.05) antiviral activity against ILTV in the NDV is summarized in Table 4. All extracts showed a three adding methods, WA extract has antiviral activity in two preventive effect against NDV infection at specific con- adding models (preinoculation and simultaneous) while centrations at preinoculation. (e 100MOH extract at 50MOH extract had such activity only in simultaneous process. 0.24 mg/ml, 50MOH at 0.48 mg/ml, and WA extract at all (erefore, the antiviral activity of 100MOH and WA extracts concentrations showed significantly higher values compared against ILTV was better than that of 50MOH extract. Fur- to the virus control group (P≤ 0.05). Also, there was no thermore, 100MOH had to treat effect after ILTV infection, significant difference between the WA extract at 0.48 and while the other extracts did not show such effect. 0.24 mg/ml compared to the cell control group, which Virus inhibitory rate (%) Advances in Virology 5 ab 40 b b b Preinoculation Postinoculation Simultaneous inoculation 100MOH 50MOH WA Figure 2: Virus inhibitory rate of Illicium verum extracts against infectious laryngotracheitis virus using three adding methods. 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. Table 4: Absorbance means of the antiviral activity of Illicium verum extracts against Newcastle disease virus in the three adding methods. Preinoculation Postinoculation Simultaneous inoculation Concentrations (mg/ml) 100MOH 50MOH WA 100MOH 50MOH WA 100MOH 50MOH WA bc c b d b d d d c 3.9 0.311 0.297 0.361 0.174 0.209 0.193 0.188 0.190 0.233 bc bc b c b bc c cd c 1.9 0.289 0.319 0.342 0.216 0.197 0.278 0.269 0.227 0.230 bc b c b b b bc b 0.97 0.320 0.301c 0.361 0.249 0.206 0.314 0.349 0.281 0.330 bc b ab b a ab bc b b 0.48 0.322 0.384 0.387 0.318 0.348 0.323 0.298 0.312 0.359 b bc ab b a c d b b 0.24 0.356 0.320 0.402 0.317 0.352 0.249 0.196 0.316 0.363 c c c c b cd cd c c Virus control 0.255 0.255 0.255 0.237 0.237 0.237 0.242 0.242 0.242 a a a a a a a a a Cell control 0.454 0.454 0.454 0.367 0.367 0.367 0.445 0.445 0.445 P value 0.0004 0.0004 0.0007 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0003 SEM 0.02 0.02 0.02 0.01 0.01 0.01 0.02 0.02 0.02 Means within a column with different subscripts are significantly different (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. indicated a high protective effect of the WA extract at these concentrations against the inactivity of NDV. Similar results were found in postinoculation modes, and the three extracts b a exhibited a treatment effect against NDV. After infection, 60 b the 100MOH, 50MOH (0.48 and 0.24 mg/ml), and WA extracts (0.97 and 0.48 mg/ml) showed significantly (P≤ 0.05) higher difference compared with the virus control group. In simultaneous inoculation, the values of 100MOH extract at 0.97 mg/ml, 50MOH at 0.48 and 0.24 mg/ml, and WA extracts at 0.97 to 0.24 mg/ml concentrations were Preinoculation Postinoculation Simultaneous higher (P≤ 0.05) than those of the corresponding virus inoculation control groups which indicated that the three extracts could 100MOH have virucidal activity against NDV at certain doses. 50MOH (e virus inhibitory rates of the three extracts against NDV WA are presented in Figure 3. In preinoculation, WA and 50MOH Figure 3: Virus inhibitory rate of Illicium verum extracts against extracts demonstrated the highest (P≤ 0.05) virus inhibitory Newcastle disease virus using three adding methods. rate (75% and 66%, respectively), and the 100MOH showed the 100MOH � absolute methanol extract; 50MOH � 50% methanol lowest inhibitory rate (49%) against NDV. In the postinocu- extract; WA � water extract. lation method, 50MOH exhibited the highest (P≤ 0.05) re- duction rate (88%), while 100MOH and WA extracts showed a similar reduction effect, 60%, and 63%, respectively. After si- showed that the extracts had significant antiviral activity multaneous inoculation, the WA (60%) and 100MOH (53%) against NDV in three inoculation methods. extracts exhibited the highest reduction rate, while 50MOH (e results of the antiviral activity of Illicium verum extract showed the lowest inhibitory rate (38%). (ese results extracts against avian reovirus are illustrated in Table 5. (e Virus inhibitory rate (%) Virus inhibitory rate (%) 6 Advances in Virology Table 5: Absorbance means of the antiviral activity of Illicium verum extracts against the avian reovirus in the three adding methods. Preinoculation Postinoculation Simultaneous inoculation Concentrations (mg/ml) 100MOH 50MOH WA 100MOH 50MOH WA 100MOH 50MOH WA c bc b d d c c d d 3.9 0.294 0.326 0.356 0.182 0.205 0.228 0.243 0.199 0.184 a b b d d c bc c c 1.9 0.434 0.388 0.354 0.188 0.200 0.225 0.301 0.288 0.336 a b b b d b b b bc 0.97 0.412 0.349 0.358 0.285 0.195 0.277 0.322 0.356 0.356 ab b b c c b bc b ab 0.48 0.387 0.378 0.367 0.233 0.241 0.303 0.262 0.382 0.411 bc bc bc c b b bc b a 0.24 0.330 0.332 0.331 0.242 0.291 0.304 0.242 0.371 0.440 c c c c c c bc c c Virus control 0.282 0.282 0.282 0.235 0.235 0.235 0.301 0.301 0.301 a a a a a a a a a Cell control 0.454 0.454 0.454 0.367 0.367 0.367 0.445 0.445 0.445 P value 0.0007 0.001 0.006 <0.0001 <0.0001 <0.0001 0.0009 <0.0001 <0.0001 SEM 0.02 0.01 0.02 0.01 0.009 0.01 0.02 0.02 0.02 Means within a column with different subscripts are significantly different (P≤ 0.05). 100MOH � absolute methanol extract; 50MOH � 50% methanol extract; WA � water extract. three extracts revealed preventive action against reovirus infection at specific concentrations during preinoculation modes. (e 100MOH extract and 50MOH at 1.9–0.48 mg/ml and WA extract from 3.9 to 0.48 mg/ml showed significantly (P≤ 0.05) higher values compared to the virus control group 60 (P≤ 0.05). (e 100MOH extract at 1.9 to 0.48 mg/ml showed no significant difference compared to the control cell group, indicating a high protective effect on CEF against avian reovirus infection. During postinoculation modes, the three extracts exhibited a treatment effect against avian reovirus infection at a particular dose, and the values of 100MOH at Preinoculation Postinoculation Simultaneous 0.97 mg/ml, 50MOH at 0.24 mg/ml, and WA extracts at inoculation 0.97–0.24 mg/ml were significantly (P≤ 0.05) higher than 100MOH those of the virus control group. In simultaneous inocula- 50MOH tion, the values of 50MOH at 0.48 mg/ml and WA extracts at WA 0.24 mg/ml concentrations were significantly higher Figure 4: Virus inhibitory rate of Illicium verum extracts against (P≤ 0.05) than those of the virus control groups, which avian reovirus using three adding methods. 100MOH � absolute indicated that these extracts could have a virucidal effect on methanol extract; 50MOH � 50% methanol extract; WA � water avian reovirus at an appropriate dose. extract. (e virus inhibitory rates of the three extracts against avian reovirus are presented in Figure 4. In preinoculation, during before virus adsorption or attachment to CEF cells. 100MOH extract revealed the highest (P≤ 0.05) virus in- During preinoculation, similar findings were mentioned hibitory rate (P≤ 0.05) against avian reovirus (91%), fol- except 100MOH and 50MOH extract against IBD, which lowed by 50MOH with a 63% reduction rate, while the WA may indicate that the extracts are interfering with virus extract showed the lowest inhibitory rate (49%). During the attachment and penetration of host cells, suggesting that the postinoculation method, the WA extract exhibited the extracts have a preventive effect on CEF against viruses. highest reduction rate (51%), while 100MOH and 50MOH However, in postinoculation, the results showed the dif- extracts showed a similar reduction effect with inhibitory ferent impact of the extracts in each virus; the extracts rates of 38% and 42%, respectively. After simultaneous exhibited antiviral effect against NDV and reovirus while no inoculation, the WA extract presented the highest (P≤ 0.05) effect against IBDV and only the 100MOH extract showed reduction effect against avian reovirus (95%), 50MOH ex- activity against ILTV; these results indicate that anise ex- tracts showed 55% reduction rate, and 100MOH extract tracts have virucidal properties and may prevent the virus showed the lowest inhibitory rate (14%). (ese results in- replication after infection. (e differences among viruses in dicated that both WA and 50MOH extracts had significant response to Illicium verum extracts in this study may be antiviral activity against avian reovirus in the three adding attributed to the differences in the levels of active compo- methods while the 100MOH extract had high antiviral ac- nents in the extracts and variations among the tested viruses. tivity against avian reovirus during the preinoculation mode. For example, IBDV is a nonenveloped and highly resistant In general, the three adding methods of the extracts and virus [19]. It was proposed that the antiviral action of plant tested viruses refer to the stages of the virus infection cycle. extracts may be ascribable to an interaction with the viral Results showed that the extracts exhibited antiviral activity envelopes [20, 21]. However, the mechanism of action is still against all tested viruses during simultaneous inoculation not clear, whether the inhibitory effect of anise extracts is with respect to reduction rate among each extract on each due to interference with cellular membrane proteins or virus virus, indicating that the extracts have an inhibitory effect Virus inhibitory rate (%) Advances in Virology 7 receptors involved in host cell adsorption and penetration, References consequently preventing virus infection of the cells [11, 22]. [1] R. Elizondo-Gonzalez, D., Ricque-Marie, L. E. 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Advances in VirologyHindawi Publishing Corporation

Published: Jan 25, 2020

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