Evaluation of in-vitro antiviral activity of nanocurcumin and nanoeugenol against goat pox

Introduction

Goatpox is often a great threat to goats and sheep and characterized by pyrexia and generalised pock lesions with lymphadenopathy causing high mortality (50-100%) and morbidity upto 100% (Bhauprakash et al., 2006a; Babiuk et al., 2008) [2,7] . It is more severe in young ones compared to adults (Joshiet al., 1996). The disease is not distinguishable from sheeppox serologically but possible by molecular technique (Hosamaniet al., 2004a) [4,6] . Indigenous sheep and goat exhibit some natural immunity, while the European breeds of sheep and goats are more susceptible to infection with these viruses (Heine et al., 1999). Since its first report by Hansen in 1879 from Norway, goatpox remains to be enzootic in Southwest and Central Asia, Northern and Central Africa, and Indian subcontinent (Hosamani et al., 2004b) [4,6] and recently in Vietnam, Mongolia and Greece indicating emerging nature of the disease (Babiuk et al., 2008) [2,7] . In India, first reported case was in 1934 (Ramesh et al., 2009). In India, goatpox is enzootic like other pox viral infections and regular outbreaks have been reported from most of the states (Bhanuprakash et al., 2005) [9] . Major control stretagies against goatpox include restriction of movement, test and slaughter and vaccination (Carn et al. 1995) [10] . Treatment strategies are almost nil due to lack of effective therapeutics for most of the viral diseases, emergence of the antiviral drug resistance, more of side effects and high cost of some antiviral therapies necessitate finding new effective antiviral compounds (Lemonie et al., 2013) [11] . Hence there is an increasing requirement for new effective antiviral substances of indigenous nature with low toxicity and economically feasible. It has been demonstrated that medicinal plants have wide range of antiviral activity against different viruses. Among them International Journal of Advanced Biochemistry Research 2026; 10(8): 34-46 We have focused on curcumin and eugenol for their antiviral activities. Curcumin is extracted from turmeric (Curcuma longa) plant, a perennial herb belonging to the family Zingiberaceae (Priyadarshini et al., 2014) [36 ], which is cultivated extensively in south and south-east tropical Asia. Eugenol, a phytogenic bioactive component with welldefined functional characteristics, is widely found in a variety of herbal plants. Eugenol's ability to scavenge free radicals, prevent the production of reactive oxygen and nitrogen species, increase cyto-antioxidant potential, and disrupt microbial DNA and proteins are some of the main principle mechanistic approaches connected to its therapeutic potential (Khalil et al., 2001) [13] . The body quickly eliminates curcumin and eugenol in their natural form, and it has very little ability to penetrate organs. According to Tsai et al. (2011) [14] , eugenol and curcumin have a noticeably better penetration and distribution rate in their nano form. In order to evaluate the in-vitro antiviral activity of nanocurcumin and nanoeugenol combined against goatpox, the current investigation was carried out.

Materials and Methods

Preparation of nanocurcumin

Curcumin was purchased from HIMEDIA. All the reagents used were of technical grade. N-Hexane (HPLC grade) and ethanol (99.5-99.8%) were obtained from Mercks.

Evaporative precipitation of nanosuspension

According to the procedure outlined by Siekmann et al. (1996) [15] , curcumin was dissolved in ethanol before being mixed with an antisolvent (hexane) to create nanocurcumin. The rotary evaporator was used to quickly evaporate the solvent and antisolvent under vacuum to produce the drug particles in the nanosuspension. The nanoparticles were then vacuum dried to thoroughly evaporate all of the liquids. The Solvent to Ant Solvent (SAS) ratios employed ranged from 1:10, 1:15, and 1:20 (v/v), and the drug doses used were 5, 10, and 15 mg/mL. 200-400 ml of hexane were used for every 20 ml of the medication solution.

Preparation of nanoeugenol

Eugenol was procured from SIGMA ALDRICH and Tween-80 was procured from CDH was used for the preparation of nanoeugenol.

Ultrasonication with little modifications

Nanoeugenol was prepared using the method described by Kamel et al., 2015 [16] , the eugenol oil (99%, MW164.21; Fluka code 46100), and non-ionic surfactant (Tween 80) were added slowly under gentle stirring until a homogeneous mixture formed. Then water was added drop wise at 37 0 C and stirred. This mixture was sonicated using an Ultrasonicator (MLI, Germany) for 10 min at 470 W. The particle size was analyzed by Zeta-sizer (IASST, Guwahati). [17] .

Experimental protocol and procedure Experimental animal

Cell lines

African green monkey kidney cell line i.e; VERO cell line (Passage no. 51) obtained from cell line repository, Department of Microbiology, College of Veterinary Science, Khanapara, Guwahati, Assam was used in the present study.

Maintainence of vero cells

Vero cells passage 51 were obtained from cell line repository, Dept of Microbiology, College of Veterinary Science, A.A.U, Khanapara, Guwahati and propagated using minimum essential media with Earle's salts (EM/EBSS, CELL-lone), supplemented with 8% Fetal Bovine Serum (FBS; Sigma Aldrich). Antibiotic solution (combination of penicillin, Streptomycin, Amphotericin B; Hyclone) was added to avoid contamination of the growth medium @ of 100 units per ml for maintaining the cells. L-glutamine at 3% was added to Growth Media for better maintenance of cell morphology. For maintaining the cells and infected cells, maintenance medium containing MEM with 2% FBS, 3% L-glutamine and 100 units per ml antibiotic was used.

Subculture of vero cell lines

Cell culture flask (25cc or 75cc) with 100% confluent monolayer of Vero cells were selected for subculturing. The growth media was discarded and the monolayer was washed with EMEM. Then 750µl for 25cc and 2.5µl for 75cc of 0.25% trypsin with 2% EDTA solution was added and kept at room temperature for 2-3 minutes. Later trypsin was discarded and the flask was gently tapped to detach the cells. The cells were split in 1:2 ratio from 25cc and 1:3 ratio from 75cc flask. [37] with slight modifications.

Virus

Determination of cell cytotoxicity50 (CC50/CyC50) Dye exclusion method

Concentration of active principle and nanoparticles (i.e, curcumin, nanocurcumin and eugenol, nanoeugenol) inducing 50% of the cell lysis or cell death (CC50 or CyC50) was determined as per Tolo et al. (2006) [38] . Total, viable cells and viable cell percentage were calculated using the following formulas.

Total cells/ml = total cells in a square × dilution factor × 10 4

Viable cells/ml = viable cells in a square × dilution factor × 10 4 % of viable cells = 100 × (viable cell count/ total cell count) Dilution factor = 2

The concentration of the curcumin, nanocurcumin and eugenol, nanoeugenol reducing the cell viability by 50% (CC50 or CyC50) was determined from a curve colligating percent cell viability to concentration of the nanoparticles.

Virus estimation assay

Determination of tissue culture infective dose 50 (TCID50) TCID50 was performed following guidelines of virology laboratory manual. Cell culture fluids were collected from Vero cell adapted positive field isolates. A volume of 0.9 ml of media was dispensed into 5 tubes and labeled. 0.1 ml of the positive cell culture fluid was added to the first tube. The content of the first tube were mixed by repeated pipetting. Then 0.1 ml was withdrawn and transferred to the second tube and same procedure was followed until all the dilutions (10 -1 to 10 -5 ). Vero cell monolayer was grown in 24 well cell culture plate for 24 hours. 100µl of inoculum from different dilutions were added to all the wells from row 1-5 and row 6 was kept as control. The plate was kept in CO2 incubator and maintained. It was monitored for 4-5 days and gradual development of CPE was recorded in the following manner:

• About 50% of cells exhibiting CPE = (+)

• About 75% of cells exhibiting CPE = (++)

• The monolayer was totally destroyed = (+++) TCID50 was calculated by Karber's method (Veterinary virology, 1981) as per the following formula:

Virus inhibition assays Cytopathic Effect (CPE) reduction assay

Antiviral activity was determined by reduction of virus titres using TCID50 determinations according to Goncalves et al. (2005) [37] . TCID50 was calculated by Karber's method (Veterinary virology, 1981) as per the following formula: Log10TCID50 = L-d (s-0.5), (L= Log10 of most concentrated virus dilution tested d = Log dilution factor, s = Sum of proportions). 1. The experiment was carried out in triplicates. 2. The antiviral activity was expressed as percentage of inhibition (PI) using antilogarithm values of TCID50. PI = 1 (T antilogarithm/ C antilogarithm). The active principle and nanoparticles was considered active when the PI is >80%, at its MNTC.

Effective concentration50 (EC50)

Modified end point assay was followed as described by Berghe et al. (1986) for nanocurcumin, nanoeugenol and their combination. TCID50 was calculated by Karber's method (Veterinary virology, 1981) as per the following formula: Log10TCID50 = L-d (s-0.5), (L= Log10 of most concentrated virus dilution tested. d = Log dilution factor, s = Sum of proportions). Effective concentration reducing CPE by 50% (EC50) in comparison with virus controls was estimated from data plots by plotting different percentage of inhibition, according to Serkedjieva et al. (1998) [39] and expressed as the mean of 3 independent experiments. Therapeutic Index (TI) was calculated as the ratio of CC50/EC50.

One step growth curve

To determine the stage at which the extract has effect on the replication of GTPV (Yamasaki and Tayaga, 1980). PI was calculated with the formula PI = 1 (T antilogarithm/ C antilogarithm).

Dose-response study

Vero cell monolayer (48 hr old) were washed with EMEM and infected with 10 fold dilution of GTPV at 50µl/well (log 10) with each dilution in triplicates. Then various concentration of extract in 50µl/well were added to each virus dilution in triplicates and appropriate controls were maintained. Reduction in titre in the treated and untreated virus controls was determined.

Result

The present study was conducted to evaluate the antiviral effect of nanocurcumin and nanoeugenol against goatpox invitro. The nanocurcumin was prepared from active principle curcumin by Evaporative precipitation of nanosuspension method, had the particle size of 64.22nm, while original curcumin had the particle size of 4706nm. The nanoeugenol was prepared from active principle eugenol by Ultrasonication and particle size 97.65nm, while original eugenol was 7.27nm.

Acute toxicity studies of Nanocurcumin and Nanoeugenol

The acute oral toxicity potential of nanocurcumin & nanoeugenol was conducted on female mice (300mg/kg and 2000mg/kg doses) did not show any clinical signs of toxicity and mortality for 24 hours. There were no significant changes in the clinical signs, body weights, gross pathological changes and mortality in the period of study. These results suggested that it is safe.

Maximum non-toxic concentration/dose (MNTC/MNTD)

The maximum non-toxic dose (MNTD) was determined by confirming no visual changes in the Vero cells i.e, granulation of the cell, cell integrity, and morphology and cell death. The Table 1 represents the MNTDs of various treatment groups. The MNTD for Curcumin was found to be 39.06µg/ml, Nano-curcumin was 140.62µg/ml, Eugenol was 78.12µg/ml, Nano-eugenol was 156.25µg/ml and combination of Nanocurcumin and Nanoeugenol was 296.875µg/ml. The MNTDs of the each suspension were used for the antiviral efficacy testing.

Cytotoxic concentration 50 (CC50)

The cell death was determined by rounding, granulations, loss of intra-cellular matrix, clumping and finally detachment of the cells from the surface were the major manifestations. The Cytotoxic Concentration50 (CC50) was determined by measuring the cell viability using trypan blue and the concentration showing 50% of the cell death was considered (Fig. 1 Nano-Curcumin CC 50 The TCID50 for Goatpox virus sample no. 106 was calculated by Spearman-Karber's method. The CPE was confirmed by rounding of the cells, loss of intracellular matrix and clumping, and vacuole formation and graded accordingly by the extent of CPE exhibited (Fig. 6). The log TCID50 for this particular sample was 4.75 and this was taken as virus control for the further antiviral studies.

Virus inhibition assay / cytopathic effect reduction assay

The MNTDs of each suspension were used for the antiviral efficacy testing. Antiviral activity was determined by reduction of virus titers using antilogarithm of treatment and control TCID50 values as per the protocol. The virus titre in virus control was 4.75 and the reduction in the virus titre was seen when it was treated with curcumin, nanocurcumin, eugenol, nanoeugenol and combination of nanocurcumin and nanoeugenol. The reduction in virus titre with curcumin was 3.5 with 74.88 percentage of inhibition which was significant at P<0.05. The reduction in virus titre with nanocurcumin was 2.5 with 97.48 percentage of inhibition which was significant at P<0.05. The reduction in virus titre with eugenol was 3 with 79.43 percentage of inhibition which was significant at P<0.05. The reduction in virus titre with nanoeugenol was 1.75 with 99.55 percentage of inhibition which was significant at P<0.05. The reduction in virus titre with combination of nanocurcumin and nanoeugenol was 1.25 with 99.85 percentage of inhibition which was significant at P<0.05 which is represented in Table 3.

Effective concentration50 (EC50)

The doses used were within and around MNTD of each suspension for the antiviral efficacy testing. The concentration at which the drug inhibited 50% of the viral CPE was considered as EC50 and was determined using Dose-Response Curve. The Table 4 represents the EC50 of various treatment groups. The EC50 of Nanocurcumin was found to be 92.47±1.586µg/ml, Nanoeugenol was found to be 54.93±1.1220µg/ml and combination of Nano-curcumin+Nano-eugenol was found to be 52.48±1.1481µg/ml (Fig. 7).

Therapeutic index (TI)

The Table 5 represents the therapeutic index of various treatment groups. The therapeutic index of Nanocurcumin was found to be 15.81, Nanoeugenol was found to be 7.877, combination of Nanocurcumin+Nanoeugenol was found to be 24.94.

Nanocurcumin

The nanocurcumin was administered at different hours of pre and post-infection to ascertain the stage of viral inhibition. Treatment with nanocurcumin was given preinfection with goatpox virus and 100% inhibition was seen.

Treatment with nanocurcumin at 0 th hour post infection, 99.85% inhibition was seen. The rest 24, 48, 72, 120, 144 hour post infection the inhibition was nil.

Nanoeugenol

The nanoeugenol was administered at different hours of pre and post-infection to ascertain the stage of viral inhibition.

Treatment with nanoeugenol was given pre-infection with goatpox virus and 100% inhibition was seen. Treatment with nanoeugenol was given in 0 th hour post infection and 99.74% inhibition was seen. The 24 h.p.i 98.58% was seen and 48 and 72 h.p.i, 95.53 and 79.431% was seen respectively.No inhibition at 120 and 144 hour post infection.

Combination of Nanocurcumin+Nanoeugenol

Nanocurcumin

Percentage Inhibition (PI) was represented in Fig. 9 as 8.58% at 80µg/ml, followed by 79.431% at 100µg/ml, 99.85% at 120µg/ml and 100% at 140µg/ml.

Log conc PERCENTAGE OF INHIBITION

Combination of Nanocurcumin+Nanoeugenol

Percentage Inhibition (PI) was represented in Fig. 11 as 8.58% at 50µg/ml, followed by 79.431% at 100µg/ml, 95.53% at 150µg/ml, 98.58% at 200µg/ml, 99.74% at 250µg/ml and 100% at 300µg/ml.

Discussion

The present study was conducted to evaluate the anti-viral effect of the curcumin, nanocurcumin, eugenol, nanoeugenol and combination of nanocurcumin+ nanoeugenol in-vitro against Goatpox virus in Vero cell lines. The resultant nanocurcumin was prepared by Evaporative Precipitation of Nanosuspensions (EPN) method from the curcumin had the particle size of 64.22nm, while the original curcumin had the particle size of 4706nm and the nanoeugenol was prepared by ultrasonication of eugenol with surfactant Tween-80 had the particle size of 7.27nm, while the original eugenol had the particle size of 97.65nm. The nanocurcumin with various advantages in drug delivery and medicinal values were compared wi who Siekmann et al. (1996) [15] studied nanoparticles by two methods: Antisolvent Precipitation With A Syringe Pump (APSP) and Evaporative Precipitation of Nanosuspension (EPN) and resultant particle size was 330nm and 150nm respectively. Tsai et al. (2011) [14] prepared Curcuminloaded PLGA nanoparticles (C-NPs) by the high-pressure emulsification-solvent evaporation method. The resultant nanoeugenol with better drug delivery and various medicinal values especially antiviral effect were compared with Kamel et al. (2015) [16] who prepared eugenol oil emulsion with commercial eugenol (>99%) with non-ionic surfactant (Tween 20) and water by ultrasonication for 10min at 700W and the resultant particle size was in range of 50-110nm (Z-avg:80nm) and shape was spherical. Acute oral toxicity studies were conducted in the mice for nanocurcumin and nanoeugenol at 300mg/kgbwt and 2000mg/kgbwt. There were no mortality and no change in behaviour and body weight reported in the 14 day study period. The safe nanocurcumin on oral treatment was compared with the studies of Gopi et al. (2016) [17] where they conducted acute oral toxicity on curcuminoids and concluded it to be safe. Bisht et al. (2007) [18] concluded that the highest dose 2000mg/kgbwt and lowest dose 50mg/kgbwt of nanocurcumin did not show any signs of toxicity and mortality and it's safe for oral administration. Reddy et al., (2005) [19] conducted acute oral toxicity on mice with nanocurcumin and concluded its safety at 2000mg/kgbwt. The safe nanoeugenol on oral treatment was compared with Reddy et al., (2005) [19] found no signs of toxicity, behavioral changes and death when rats were treated with eugenol of doses 200, 600, and 2000 mg/kgbwt. The Maximum Non-Toxic Dose (MNTD) was determined by confirming no visual changes in the Vero cells. The calculated MNTD for Curcumin, Nano-curcumin, Eugenol, and Nano-eugenol was found to be 39.06µg/ml, 140.62µg/ml, 78.12µg/ml, 156.25µg/ml respectively, while the combination of Nanocurcumin and Nanoeugenol was 296.875µg/ml. These doses did not produce any morphological changes and cell cytotoxicity in the Vero cells and hence this dose was considered for the further antiviral studies. Use of curcumin, nanocurcumin, eugenol and nanoeugenol against goatpox is very scanty and very feeble work have been done in this regard. Hence our results were compared with Flores et al. (2016) [20] who tested curcumin on against Herpes Simplex Virus 1 and 2 in Verocells and MNTC was found to be 30µM and below. Hence the doses below 30µM were chosen for anti-viral studies. Gandapu et al., (2011) [21] described that both cucrcumin and nano-curcumin were non-toxic in the lower concentration but at 25µM concentration sol-curcumin was extremely toxic while the nanocurcumin at the same concentration was less toxic significantly. In our present study we have considered the active principles rather than the aqueous or ethanolic extraction of the plants and it is used on a different cell lines. This could be the probable reason for the change in the calculated MNTD values from the range described in the other research. It is proven that curcumin dissolved in ethanol is more toxic to the cells when compared to the nanocurcumin which was prepared by EPN method which was supported by Gandapu et al., (2011) [21] who said curcumin is more toxic on cell lines than nanocurcumin. The same reason can be quoted for the eugenol being more toxic than the nanoeugenol. There are very scanty or no work have been done with the active principle of curcumin and eugenol, and nanocurcumin and nanoeugenol antiviral activity against goatpox. Hence in our present study we found that all the treatments chosen caused very less adverse effects and cell cytotoxicity to the vero cells and can be considered as an antiviral drugs at their particular MNTDs for the further evaluation of the antiviral potency. The Cytotoxic Concentration50 (CC50) was determined by measuring the cell viability using trypan blue and the concentration showing 50% of the cell death was considered. The calculated CC50 values of cucrumin and nanocucrumin were compared with Khosropanah et al., (2016) [22] who reported that the proliferation of MDA-MB231 cell line and the overall 50% cell death occurred at 53.18µg/ml and 23.25µg/ml with 48hr treatment for curcumin and nanocurcumin respectively. The IC50 curcumin after 24hr, 48hr and 72hr for MDA-MB231 cell line was 79.58µg/ml, 53.18µg/ml and 30.78 µg/ml, whereas for nanocurcumin was 37.75 µg/ml, 23.25 µg/ml and 12.99 µg/ml respectively. Zandi et al. (2010) [23] found the 50% cytotoxic concentration (CC50) of the curcumin, galliumcurcumin and Cu-curcumin on Vero cell line showed to be 484.2𝜇𝜇g/mL, 255.8𝜇𝜇g/mL and 326.6 𝜇𝜇g/mL respectively which was tested for antiviral activity against herpes simplex virus type 1 (HSV-1). Lin et al. (2012) [40] demonstrated IC50 levels of nanoencapsulated curcumin ranged from 7.9µM (CT26 cells) to 30µM (A549 cells) and the curcumin resulted in lower IC50 for all the cell lines ranging from 1.0 µM to 1.7 µM. Much wider range of safety was observed with the nanoencapsulated curcumin. In case of curcumin resistant cells, the IC50 of nanoencapsulated curcumin for 2 parental cell lines rose from 8 µM to more than 20 µM. Singh et al. (2010) [24] studied di-Otryptophanylphenylalanine curcumin and di-O-decanoyl curcumin revealed remarkable antiviral activity against VSV and FIPV/FHV with EC50 values of 0.011 𝜇𝜇M and 0.029𝜇𝜇M, respectively. The calculated CC50 values of eugenol and nanoeugenol were compared with Bhanuprakash et al., 2006b [1] tested O. sanctum for its antiviral activity against GTPV in vero celllines and found CC50 value to be 3749±1.2µg/ml (Dye exclusion method) and 3849.7±0.50µg/ml (OD540/630). The MNTDs of the each suspension were used for the antiviral efficacy testing. Antiviral activity was determined by reduction of virus titers using antilogarithm of treatment and control TCID50 values as per the protocol. The virus titre in virus control was 4.75 and the reduction in the virus titre was seen when it was treated with curcumin, nanocurcumin, eugenol, nanoeugenol and combination of nanocurcumin and nanoeugenol and the titre was found to be 3.5, 2.5, 3, 1.75 and 1.25 with 74.88, 97.48, 79.43, 99.55 and 99.85 percentage of inhibition respectively which was significant at P<0.05. This results were supported by antiviral actions of curcumin, nanocurcumin, eugenol, nanoeugenol and the combination of nanocurcumin and nanoeugenol in various different viruses. Barthelemy et al. (1998) [27] studied Curcumin and its derivatives (reduced curcumin, allyl-curcumin, and tocopheryl-curcumin) revealed 70% to 85% inhibition in Tat protein trans-activation of HIV-1 LTR measured by 𝛽𝛽galactosidase activities of HeLa cells which in HIV-1 LTR was fused to the indictor of lacZ gene. Tocopheryl-curcumin demonstrated the most inhibition activity with 70% inhibition at 1nM compared to 35% inhibition of curcumin at this concentration. The doses used for the antiviral efficacy testing were within and around MNTD of the each suspension. The concentration at which the drug inhibited 50% of the viral CPE was considered as EC50 and was determined using Dose-Response Curve. These results were compared with the antiviral effect and EC50 of curcumin and eugenol, since there almost no works on antiviral effect of nanocurcumin and nanoeugenol and their combination. Kutluay et al. (2008) [26] Plaque assay was performed for the anti-viral activity of the curcumin. Curcumin reduced the number of plaques on vero cell monolayer by 3 folds. Mean plaque size after 3 days was 0.64±0.10mm when compared with untreated 0.73±0.13mm. In vitro replication of HSV-2 could be decreased by curcumin with ED50 value of 0.32mg/mL. Therapeutic index is the ratio between cytotoxicity and the inhibitory capacity of the substance (CC50/EC50). These results were compared with Ichsyani et al. (2017) [41] who found the SI of curcumin to be 2.56 when treated for Dengue on Huh7it-1 cells. Padilla et al. (2014) found the SI to be 4.8 when used on the Vero cells for antiviral study of curcumin on Dengue virus type-2. The preparations was administered at different hours of pre and post-infection to ascertain the stage of viral inhibition. Treatment with nanocurcumin was given pre-infection with goatpox virus and 100% inhibition was seen. Treatment with nanocurcumin at 0 th hour post infection, 99.85% inhibition was seen. The rest 24, 48, 72, 120, 144 hour post infection the inhibition was nil. Curcumin has anti-viral effect, but their biochemical mechanisms of anti-viral effect has been incompletely defined (Li et al., 1993; Sui et al., 1993; Bruke et al., 1995; Mazumder et al., 1995 and Bourne et al., 1999) [93] . Maximum PI (100% and 99.83%) of GTPV was observed, when the extract was added simultaneously and after 0 hpi. This indicated that the nanocurcumin might have the direct virucidal activity, when it is administered simultaneously and can also inhibit one or more steps of viral replication after the adsorption/entry.Treatment with nanoeugenol was given pre-infection with goatpox virus and 100% inhibition was seen. Treatment with nanoeugenol was given in 0 th hour post infection and 99.74% inhibition was seen. The 24 h.p.i 98.53% was seen and 48 and 72 h.p.i, 95.53 and 79.431% was seen respectively. No inhibition at 120 and 144 hour post infection. Bhanuprakash et al. (2006a) [1] studied one step growth curve for A. Arabica and E. jambolana on vero cells against goatpox virus and found they are virucidal and inhibited the viral replication after entry upto some extent. This indicated that the nanoeugenol might have the direct virucidal activity, when it is administered simultaneously and can also inhibit one or more steps of viral replication after the adsorption/entry upto 72 hours of post infection. Treatment with Combination of Nanocurcumin+Nanoeugenol was given pre-infection with goatpox virus and 100% inhibition was seen.

Treatment with Combination of Nanocurcumin+Nanoeugenol was given in 0 th hour post infection and 100% inhibition was seen. The 24 h.p.i 100% was seen and 48 and 72 h.p.i, 95.53 and 79.431% was seen. No inhibition at 120 and 144 hour post infection. This indicated that the nanoeugenol might have the direct virucidal activity, when it is administered simultaneously and can also inhibit one or more steps of viral replication after the adsorption/entry upto 72 hours of post infection. This proves that all our preparations have a potent virucidal activity and can be considered as potent antiviral agent against goatpox virus. There was graded increase in response i.e, Percentage of Inhibition (PI) of virus replication as the concentration of the treatments were increased. Percentage Inhibition (PI) of nanocurcumin was 8.58% at 80µg/ml, followed by 79.431% at 100µg/ml, 99.85% at 120µg/ml and 100% at 140µg/ml and naoeugenol was 8.58% at 60µg/ml, followed by 79.431% at 80µg/ml, 98.58% at 100µg/ml, 99.85% at 120µg/ml and 100% at 140µg/ml and 160µg/ml, while the combination of nanocurcumin and nanoeugenol was 8.58% at 50µg/ml, followed by 79.431% at 100µg/ml, 95.53% at 150µg/ml, 98.58% at 200µg/ml, 99.74% at 250µg/ml and 100% at 300µg/ml. Such graded increase in dose response of all the treatments were supported with the reports by Mokbel et al. (2017) who tried different concentration of EON (125, 250, 500µg/ml) on banana bunchy top virus, which inhibited the virus in graded response (94.81%, 95.5% and 96.66% respectively). PI was 36.9 at 31.25µg/ml, 60.1 at 62.5µg/ml, 84.2 at 125µg/ml, 96.9 at 250µg/ml, 93.9 at 500/1000µg/ml and 99.68 at 2000µg/ml for E. jambolina against goatpox was reported by Bhanuprakash et al. (2006a) [1] . In the present study, CPE assays indicated the potential inhibition of the goatpox virus replication by all the preparations. The dose response study as anticipated, signaled that the higher concentration has more inhibitory effects than the lower ones.

Acknowledgement

AcknowledgementWe are thankful to Dept of Pharmacology & Toxicology, College of Veterinary Science, Assam Agricultural University, and Khanapara for providing facility for carrying out research work.

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