Evaluation of antiviral activity of Ocimum sanctum and Acacia arabica leaves extracts against H9N2 virus using embryonated chicken egg model

Evaluation of antiviral activity of Ocimum sanctum and Acacia arabica leaves extracts against... Background: In the view of endemic avian influenza H9N2 infection in poultry, its zoonotic potential and emergence of antiviral resistance, two herbal plants, Ocimum sanctum and Acacia arabica, which are easily available throughout various geographical locations in India were taken up to study their antiviral activity against H9N2 virus. We evaluated antiviral efficacy of three different extracts each from leaves of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) against H9N2 virus using in ovo model. Methods: The antiviral efficacy of different leaves extracts was systematically studied in three experimental protocols viz. virucidal (dose-dependent), therapeutic (time-dependent) and prophylactic (dose-dependent) activity employing in ovo model. The maximum non-toxic concentration of each herbal extracts of O. sanctum and A. arabica in the specific pathogen free embryonated chicken eggs was estimated and their antiviral efficacy was determined in terms of reduction in viral titres, measured by Haemagglutination (HA) and real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays. Results: All the extracts of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) showed significant virucidal activity, however, crude extract and terpenoid ocimum ocimum showed highly significant to significant (p <0.001–0.01) decrease in virus genome copy numbers with lowest dose tested. Similarly, therapeutic effect was observed in all three extracts of O. sanctum in comparison to the virus control, nevertheless, crude extract and terpenoid maintained this effect for longer period of time (up to 72 h ocimum ocimum post-incubation). None of the leaves extracts of A. arabica had therapeutic effect at 24 and 48 h post-incubation, however, only the crude extract and polyphenol showed delayed therapeutic effect (72 h post-inoculation). acacia acacia Prophylactic potential was observed in polyphenol with highly significant antiviral activity compared to virus acacia control (p <0.001). Conclusions: The crude extract and terpenoid isolated from the leaves of O. sanctum and polyphenol from A. arabica has shown promising antiviral properties against H9N2 virus. Future investigations are necessary to formulate combinations of these compounds for the broader antiviral activity against H9N2 viruses and evaluate them in chickens. Keywords: O. sanctum, A. arabica,Avian influenza H9N2, in ovo testing, HA, Real time RT-qPCR * Correspondence: richa.bhatia0609@gmail.com National Institute of High Security Animal Diseases, Anand Nagar, Bhopal, Madhya Pradesh, India Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 2 of 10 Background properties against Peste des petits ruminants (PPR) virus Influenza A viruses (IAVs), members of the family [18], along with inhibition of Goatpox virus replication Orthomyxoviridae are characterized by a single stranded, [19]. However, antiviral H9N2 influenza activity of differ- segmented negative-sense RNA genome. Among the ent extracts derived from the leaves of these two plants IAVs, avian influenza (AI) H9N2 has become endemic in has not been studied. terrestrial poultry in several countries of the Eurasian For evaluation of the antiviral properties of medicinal continent including India in recent years [1–4]. The plants against IAVs, three methods viz. tissue culture spread of AI H9N2 has resulted in significant economic [20, 21], laboratory/experimental animals [22] and in ovo losses in poultry mainly because of reduced egg produc- model [23] have been used frequently. Each method has tion and high mortality associated with co-infection with its pros and cons. We preferred in ovo model, which is other respiratory pathogens [5]. Although AI H9N2 does at the borderline of in vitro and in vivo studies and thus not fall under the definition of highly pathogenic avian in- does not conflict with either ethical or legal aspects of fluenza (HPAI) viruses, there has been ever increasing animal protection [23, 24]. Therefore, keeping in view speculation about pandemic potential of H9N2 viruses [6]. the endemicity and pandemic potential of H9N2, this So far, a total of 28 laboratory-confirmed cases of human study was taken up to assess the H9N2 inhibitory poten- infection with avian influenza A (H9N2) viruses, however tial of various extracts derived from leaves of O. sanctum none fatal, have been detected globally (http://www.who.int/ and A. arabica using in ovo model. influenza/human_animal_interface/Influenza_Summary_ IRA_HA_interface_25_02_2016.pdf). Among them, the Methods recent one includes from China [7]. In addition, sero- Virus and embryonated chicken eggs logical evidences of AI H9N2 virus exposure to human Avian Influenza virus, A/chicken/CL/15–12/103075 (H9N2) have been reported on several occasions from Iran, China was collected from one of the major water body of and India [8–11]. The higher human infection capability Maharashtra state of India during an ongoing surveillance of these viruses was provided by the fact that H9N2 binds programme. Specific pathogen free (SPF) embryonated to α-2,6 sialic acid receptors that are abundant in the hu- chicken eggs (ECEs) were obtained from SPF Unit of man upper respiratory tract while H5N1 chiefly bind to ICAR- National Institute of High Security Animal human receptors in the lower respiratory tract [12]. The Diseases, Bhopal, India. This virus was passaged two recently emerged influenza A (H7N9) and (H10N8) in- times in the allantoic fluid of 10-days-old SPF ECEs to fecting humans had acquired gene segments from H9N2 make seed virus stock. The virus seed stock was titrated virus [13, 14]. The potential of genetic reassortment of by the haemagglutination test as per OIE protocol [25]. IAVs, due to segmented genome, from different animal The allantoic cavities of 10-day-old SPF ECEs were inocu- species is thought to be a mechanism for the emergence lated with 0.2 ml of seed virus suspension (1:100 diluted of influenza viruses with pandemic potential [14]. Endem- in 0.01 M phosphate-buffered saline, pH 7.2) and incu- icity of H9N2 circulation in poultry especially in India bated for 72 h at 37 °C followed by chilling at 4 °C over- could further aggravate the current situation. Among the night. The allantoic fluid was harvested, clarified by control measures, existing vaccines are unable to keep up centrifugation and then stored at − 80 °C. The ten-fold with the mutation rates of viruses. New vaccine develop- serially diluted virus was inoculated in the allantoic cavity ment takes a long time and at the same time, viruses are of SPF ECEs and the virus titer was estimated as the egg also developing resistance to the currently used drugs infective dose 50% per ml (EID /ml). [15]. Hence, there is no immediate response drug to the newly emerging virus infections/outbreaks. To address Plant extracts and antiviral drugs this problem, there is an exigent need for the development Two plants, O. sanctum (commonly known as Tulsi) and of a new paradigm preventive and therapeutic agent to A. arabica (commonly known as Babul), native to the control the immediate spread of viral outbreaks. In this Indian subcontinent were used in this study. The leaves scenario, traditional herbal medicines have been postu- of these plants were collected from Madhya Pradesh, lated to prove effective due to fewer side effects, relatively India and identified by Dr. S.S. Ghoke through standard low cost and easy availability [16]. morphological and anatomical techniques. Plants were Two plants, Ocimum sanctum and Acacia arabica are authenticated and voucher numbers for O. sanctum widely distributed and easily available throughout vari- (Specimen#: vi p-1050313) and A. arabica (Specimen#: ous geographical locations in India. The efficacy of O. vi p-174629) were obtained. Crude extracts of these two sanctum as inhibitory compound has been documented plant leaves were prepared in hydro-methanol (1:1 dilu- against several viruses like Newcastle Disease virus, tion with water) by standard hot continuous extraction Vaccinia virus and Infectious Bursal Disease virus [17]. method using Soxhlet apparatus [26]. Two class com- Similarly, A. arabica has been explored for its virucidal pounds each in identified plants, O. sanctum (terpenoid Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 3 of 10 and polyphenol) and A. arabica (flavonoid and polyphenol) of different plant leaves extracts (three extracts each from were isolated and purified by ultraviolet–visible prep HPLC two different plants leaves) was incubated with 500 EID / technique [27]. Antiviral drugs, Amantadine hydrochloride 0.1 ml of H9N2 virus separately at 37 °C for 2 h. The drug (Sigma, USA) and Oseltamivir (FLUVIR, Hetero Drugs control group included amantadine hydrochloride Ltd., India), the currently approved drugs for influenza [28] (MNTC-16 μg/0.1 ml) incubated with 500 EID /0.1 ml of were used as drug control and hydro-methanol as vehicle H9N2 virus and the virus control group included 500 control group. EID /0.1 ml of H9N2 virus incubated with 0.1 ml PBS for 2 h at 37 °C. The vehicle control group comprised of Toxicity assay of plant extracts and antiviral drugs 0.1 ml of hydro-methanol incubated with 500 EID / To determine the maximum non-toxic concentration 0.1 ml of H9N2 virus. Afterwards, 0.1 ml of each of the (MNTC) of all the extracts of O. sanctum (crude virus-extract mixture was inoculated into the allantoic extract ,terpenoid , polyphenol )and cavity of 10-day-old SPF ECEs (5 each) separately and in- ocimum ocimum ocimum A. arabica (crude extract , flavonoid and cubated at 37 °C for 72 h. The allantoic fluid was acacia acacia polyphenol ) the guidelines prescribed by the harvested from each of the treatment groups 72 h acacia , Organization for Economic Co-operation and Develop- post-incubation. The presence of virus in the allantoic ment (OECD) were followed [29] Average egg biomass of fluid was assessed by haemagglutination (HA) test as per 10 SPF ECEs which included the embryo along with its al- methods described [30] and quantified by real time lantoic sac and yolk was measured and the maximum dose RT-qPCR [31](Fig. 1). for each of the herbal extract groups that could be used without inducing toxicity to the embryo was calculated as Therapeutic, time-dependent activity 135 mg/kg of egg biomass [29]. Afterwards, the MNTC Eight treatments groups (except the vehicle control was again assessed in the in ovo model. 0.1 ml of two-fold group) as formulated for testing of virucidal antiviral dilution of each of the plant extracts (200, 175, 150, 135, activity were made for testing the therapeutic efficacy in 100, 75, 50, 25, 10 and 5 mg/0.1 ml, diluted in extraction a time dependent manner. In each of the treatment medium hydro-methanol) were inoculated into the allan- groups, 500 EID /0.1 ml of H9N2 virus was inoculated toic cavity of 10-days-old SPF ECEs in triplicates. The SPF into the allantoic cavity of 10-day-old SPF ECEs (6 eggs ECEs inoculated with 0.1 ml of hydro-methanol alone for each group) and incubated at 37 °C for 2 h. Then, served as vehicle control. The MNTCs of amantadine and the MNTC dose (135 mg/0.1 ml) of each plant extract oseltamivir antiviral drugs were determined by inoculating was injected into the sharp pole of albumen of these 5.33, 8 and 16 μg/0.1 ml and 1.75, 2.66 and 5.25 μg/ ECEs separately and incubated at 37 °C. This route has 0.1 ml, respectively into the allantoic cavity of 10-days-old been suggested as an improved model for testing anti- SPF ECEs separately in triplicates. All the inoculated SPF viral drugs especially if the bioavailability of the drug is ECEs were incubated at 37 °C till hatching and were can- poor [32]. In the drug control group, oseltamivir dled twice daily for checking the viability. (MNTC–2.66 μg/0.1 ml) was injected through albumen route 2 h post-virus inoculation. The allantoic fluid was Assessment of efficacy of herbal extracts against H9N2 harvested from each of the treatment group at 24, 48 virus using in ovo model and 72 h post-inoculation (Fig. 1). The presence of virus The antiviral activity of each plant crude extract and its in the allantoic fluid was tested by HA test and quanti- isolated class compounds were assessed in three differ- fied by real time RT-qPCR. ent formats viz; virucidal (dose-dependent), therapeutic (time-dependent) and prophylactic (dose-dependent). Prophylactic, dose-dependent activity For testing of prophylactic efficacy, same eight treatment Dose-dependent virucidal activity groups as mentioned in above testing methodologies A total of nine treatment groups (each treatment group were used to examine the effect of extracts in a dose with five 10-days-old SPF ECEs) were made for the dependent manner. In each of the treatment groups, the determination of virucidal activity of herbal extracts two-fold dilution of MNTC (135 mg; 67 mg; 33 mg/ against A/chicken/CL/15–12/103075 (H9N2). Of the nine, 0.1 ml) of different plant extracts (three extracts each six treatment groups corresponded to the respective six from two plants leaves) was inoculated into the allantoic herbal extracts (crude extract , terpenoids , cavity of 10-day-old SPF ECEs (5 eggs for each group) ocimum ocimum polyphenol ; crude extract , flavonoid , and incubated at 37 °C for 2 h. Then, 500 EID /0.1 ml ocimum acacia acacia 50 polyphenol ) used in this study. The remaining three of H9N2 virus was inoculated into the allantoic cavity of acacia treatment groups were the drug control, the vehicle con- each of these eggs and incubated at 37 °C for 72 h trol and the virus control group. 0.1 ml of two-fold dilu- (Fig. 1). In the drug control group, amantadine hydro- tion of pre-calculated MNTC (135 mg, 67 mg, and 33 mg) chloride (MNTC-16 μg/0.1 ml) was given through Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 4 of 10 Fig. 1 Experimental protocols for the assessment of the antiviral activity of Ocimum sanctum and Acacia arabica leave extracts against H9N2 virus using in ovo model allantoic cavity 2 h post-virus inoculation. The presence Results of virus in the allantoic fluid was detected by HA test Toxicity assay of plant extracts and antiviral drugs and quantified by real time RT-qPCR. In a first set of experiments, MNTC was calculated for each herbal extracts and antiviral drugs in the SPF ECEs. The criterion of non-toxicity of herbal extracts was iden- Real-time quantitative reverse transcription PCR tified as the absence of death of embryos up to hatching Viral RNA from the respective allantoic fluids was ex- of all the eggs (three per drug concentration) at various tracted using QIAamp Viral RNA mini kit (Qiagen, concentrations (200, 175, 150, 135, 100, 75, 50, 25, 10 Germany) as per the recommendations of the manufac- and 5 mg/0.1 ml). Irrespective of the different herbal ex- turer. One step RT-qPCR was carried out in duplicates tracts, all the ECEs that had received 200 mg/0.1 ml of as per standard method [31]. The PCR assay was con- extract died within 48 h of post-inoculation. The ducted on Light Cycle® 480 Real-Time PCR, Roche, mortality of 50–15% was observed in ECEs given 175 USA. The fluorescence reading was noted at the end of and 150 mg/0.1 ml extract, while those that received each extension step. No template control and no probe 135 mg/0.1 ml or less showed no evidence of mortality control were included in each run. till hatching. The concentrations of antiviral drugs tested were non-toxic to the embryos at the dose of 16 μg/ Statistical analysis 0.1 ml and 2.66 μg/0.1 ml for amantadine hydrochloride The infectivity titer of the H9N2 virus (A/chicken/CL/ and oseltamivir, respectively and thus have been used as 15–12/103075) was expressed as EID /ml and was cal- MNTC throughout the study. culated as per the standard method [33]. The data is presented as mean ± standard deviation. For the analysis Antiviral activity on the basis of haemagglutination test of the significance of differences among the different The aim of this experiment was to identify the most ef- treatment groups in terms of the virus genome copy fective extract in inhibiting the H9N2 virus replication number, two ways analysis of variance (ANOVA) with in the SPF ECEs. The virus challenge dose was identified Tukey post hoc test was used. P values equal to or less on the basis that there was no mortality in the eggs up than 0.05 were considered statistically significant. to 72 h, in any of the virus challenge groups made, for Analyses were performed using GraphPad Prism version evaluating the antiviral activities for different doses as 6.0 (GraphPad Software, SanDiego, CA, USA). well as time intervals. Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 5 of 10 Dose-dependent virucidal activity inhibiting the H9N2 virus replication. The crude All the herbal extracts treatment groups including drug extract when administered at the dose of 135 mg/ ocimum control did not show any HA titer irrespective of de- 0.1 ml significantly inhibited the virus replication com- creasing dose (135, 67, 33 mg/0.1 ml) treatments, indi- pared to virus control group (p < 0.001). At the same cating that even the low dose of all extracts were dose (135 mg/0.1 ml), the other four extracts; effective in inhibiting the replication of H9N2 virus. Al- terpenoid , polyphenol , flavonoid and ocimum ocimum acacia lantoic fluid collected from these treatment groups were polyphenol also inhibited the virus replication acacia given two consecutive passages in SPF eggs to ascertain significantly (p < 0.05–0.01) (Fig. 3). On reducing the the absence of H9N2 replication which did not show dose (67 mg/0.1 ml), only crude extract and ocimum any HA titer and reconfirmed the virucidal activity. terpenoid were effective (p < 0.05). Further redu- ocimum However on further two-fold reduction in dose to cing the dose (33 mg/0.1 ml), crude extract ocimum 17.5 mg/0.1 ml for each extract, we found that crude remained effective (p < 0.05) (Fig. 3). Non-significant ef- extract , terpenoid , crude extract and ficacy was recorded within both groups of herbal ex- ocimum ocimum acacia flavonoid treatment groups showed a mean HA tracts of O. sanctum and A. arabica in the present study. acacia 4.2 5 4.2 5.3 titer of 2 ,2 ,2 and 2 , respectively which on fur- ther two fold reduction of dose to 8.75 mg/0.1 ml Therapeutic, time-dependent activity All the three ex- remained almost same as above indicating loss of viru- tracts of O. sanctum (crude extract ,terpenoid ocimum ocimum cidal activity at lower doses. However, polyphenol , and polyphenol ) were significantly effective in ocimum ocimum polyphenol and the drug control amantadine treat- limiting the H9N2 virus replication in the SPF ECEs at acacia ment groups did not show any HA titer even at the low- different time intervals (24, 48 and 72 h post-inoculation) est dose (8.75 mg/0.1 ml) tested indicating a high (p <0.001–0.0001). Within the O. sanctum treatment virucidal activity of these class compounds (Fig. 2). group, crude extract was found to have significantly ocimum high efficacy in comparison to terpenoid (p <0.05) ocimum and polyphenol (p < 0.01) at 48 h post-inoculation ocimum Time-dependent therapeutic activity as indicated by decrease in viral genome copy numbers Among the extracts of O. sanctum, only crude extract ocimum (Fig. 4). However, terpenoid showed significantly ocimum treatment group showed absence of HA titer in the allan- high inhibitory activity compared to crude extract ocimum toic fluid harvested at all the three time intervals (24, 48 (p < 0.0001) and polyphenol (p <0.001) at 72 h ocimum and 72 h) post-inoculation. The drug control group oselta- post-inoculation. mivir also showed absence of HA titer indicating no viral In the case of A. Arabica, none of the extracts were ef- replication. The other two extract treatment groups, fective initially at 24 and 48 h post-inoculation (p >0.05), terpenoid and polyphenol however had mean ocimum, ocimum 4.8 3.8 however, at 72 h post-inoculation, crude extract acacia HA titers 2 and 2 , respectively at 72 h post-inoculation (p < 0.001) and polyphenol (p < 0.01) depicted thera- acacia (Fig. 2). peutic activity as indicated by significant decrease in the virus genome copy numbers as compared to virus control Dose-dependent prophylactic activity group (Fig. 4). However, differences in the time dependent All the three extracts of O. sanctum at doses 135 and therapeutic effect among the extracts of A. arabica were 67 mg/0.1 ml showed prophylactic potential as revealed non-significant (p > 0.05). On comparison of antiviral by the absence of HA titers. However, on further reduc- treatment between the two groups it was found that all tion of dose to 33 mg/0.1 ml, all the three extracts (crude three extracts of O. sanctum treatment group had signifi- extract ,terpenoid and polyphenol ) ocimum ocimum ocimum cantly higher (p <0.05–0.0001) antiviral efficacy than the 5.9 5.6 3.6 showed a mean HA titer of 2 ,2 and 2 respectively. crude extract or polyphenol extracts. acasia acacia In the case of A. arabica, MNTC dose (135 mg/0.1 ml) of all the three extracts showed absence of HA titer. Lower Prophylactic, dose-dependent activity The prophylac- doses (67 and 33 mg/0.1 ml) of any of the extracts of A. tic potential of all the herbal extracts were determined arabica were not effective in preventing viral replication. against H9N2 virus in the SPF ECEs. In the O. sanctum The drug control group showed complete absence of HA group, only crude extract and polyphenol ocimum ocimum titer (Fig. 2). treated ECEs showed significant (p < 0.05) decrease in viral genome copy numbers at dose of 135 mg/0.1 ml Antiviral activity on the basis of viral quantification by (Fig. 5). The decrease in viral genome copy numbers was real time RT-qPCR non-significant at lower doses of 67 and 33 mg/0.1 ml in Virucidal, dose-dependent activity all the three extracts of O. sanctum groups. In A. arab- The aim of this experiment was to quantify the viral ica group, only polyphenol was more successful in acacia amount and identify the most effective extract in reducing the viral genome copy numbers at doses of 135 Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 6 of 10 Fig. 2 Measurement of antiviral activity of O. sanctum and A. arabica leave extracts against H9N2 virus using HA assay. a Dose-dependent virucidal activity; b Time-dependent therapeutic activity; c Dose-dependent prophylactic activity. The virus control group included 500 EID /0.1 ml of H9N2 virus incubated with 0.1 ml PBS for 2 h at 37 °C. The HA titer were expressed as log HA titer (p < 0.0001) and 67 mg/0.1 ml (p < 0.001) while it be- being considered as an emerging pandemic threat in view came ineffective at low dose of 33 mg/0.1 ml (p > 0.05) of the clinical and confirmed cases of H9N2 in China, (Fig. 5). The other two extracts of A. arabica, crude Hong Kong, Bangladesh and Egypt [14]. This reaffirms the extract and flavonoid were not effective as need for search of new compounds with antiviral activity. acacia acacia prophylactic extract at any of the doses (p > 0.05). With increase in drug resistance to synthetic antivirals, nat- ural products remain an important alternative for the con- Discussion trol infectious diseases. In the present study, the in ovo Spread of H9N2 subtype has demonstrated an increased model has been used for studying antiviral activities of dif- theoretical threat to humans because of the potential ferent leaves extracts of Ocimum sanctum and Acacia arab- emergence of novel subtypes of avian influenza [13]. It is ica against low pathogenic avian influenza (LPAI) H9N2. Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 7 of 10 Fig. 3 Assessment of virucidal activity of the O. sanctum and A. arabica leaves extracts in a dose-dependent manner by Real Time RT-qPCR assay. Each error bar represents standard deviation (SD) from the 5 ECEs. The virus control group included 500 EID /0.1 ml of H9N2 virus. Statistical significance was assessed by two ways analysis of variance (ANOVA) with Tukey post-hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) In our previous work we were able to demonstrate flavonoid and polyphenol) against LPAI H9N2 using 100% virucidal activity of cold aqueous extract of bark of in ovo model. A. arabica at a concentration of 0.625 mg/ml against In dose dependent virucidal activity, all the extracts of HPAI H5N1 using in vitro model. Mild therapeutic ac- both the plants in doses (135, 67 and 33 mg/0.1 ml) dis- tivity was also demonstrated up to 48 h in cold aqueous played absence of HA titer indicating that all the ex- extract of Ocimum tenuiflorum when tested against tracts had inhibitory potential. On further reduction of HPAI H5N1 [20]. dose rate in double dilution, polyphenol and poly- acasia In the present work, we had selected two plants, phenol only maintained virucidal activity up to a Ocimum Ocimum sanctum and Acacia arabica for studying their low dose rate of 8.75 mg/0.1 ml which was confirmed by efficacy against avian influenza H9N2 virus because absence of HA in the harvested allantoic fluid of ECEs these plants are widely distributed and easily available in the next two passages also. This virucidal effect could throughout various geographical locations in India and be due to masking/blocking of HA protein of the virus have strong record in the literature for their antiviral ac- by the herbal extracts during the incubation period, tivity against different viruses [16, 17]. Three different which might have inhibited the H9N2 virus replication. approaches viz. virucidal (dose dependent), therapeutic Catechin compounds in polyphenols are known to have (time dependent) and prophylactic (dose dependent) antiviral activity mediated by preventing adsorption of were employed to systematically explore the potential of viruses to cells [34, 35]. Highly significant to significant extracts of leaves of O. sanctum (crude extract, terpen- decrease in the viral genome copy numbers was recorded oid and polyphenol) and A. arabica (crude extract, in all the extracts at the MNTC dose (135 mg/0.1 ml) Fig. 4 Assessment of therapeutic activity of the O. sanctum and A. arabica leaves extracts in a time-dependent manner by Real Time RT-qPCR assay. Each error bar represents standard deviation (SD) from the 6 ECEs. The virus control group included 500 EID /0.1 ml of H9N2 virus. Statistical significance was assessed by two ways analysis of variance (ANOVA) with Tukey post-hoc test (*p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 8 of 10 Fig. 5 Assessment of prophylactic activity of the O. sanctum and A. arabica leaves extracts in a dose-dependent manner by Real Time RT-qPCR assay. Each error bar represents standard deviation (SD) from the 5 ECEs. The virus control group included 500 EID /0.1 ml of H9N2 virus. Statistical significance was assessed by two ways analysis of variance (ANOVA) with Tukey post-hoc test (*p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) except crude extract (P <0.05–0.001). Although the to the decrease in concentration of the active ingredient acacia virucidal effect was seen more prominently in the HA test which might have allowed the virus to replicate because where the virus HA titer was absent in comparison to the of single dose regimen followed in the experiment. In 7.3 virus control (mean HA titer 2 )and vehicle control addition, other possible reason might be the high sensi- 5.8 (mean HA titer 2 ), the quantification of the virus by real tivity of real time PCR assay. Surprisingly, in A. arabica time RT-qPCR also corroborated with the HA results es- treated groups, crude extract and polyphenol acacia acacia pecially at higher extract doses. The results of both the as- showed significant decrease in viral genome copy num- says clearly indicated that crude extract in particular bers at a later time interval of 72 h post-inoculation ocimum is a potent inhibitor of H9N2 virus replication. (P < 0.01). Since the route of administration was via Our study is further supported by the fact that individ- albumen, slow movement/distribution of A. arabica ex- ual plant extracts were less effective compared to crude tracts from albumen to allantoic cavity might be the rea- extracts which may due to synergistic effect of individual son for delayed effect. Variable bioavailability of drugs, component and some of the uncharacterised compo- oseltamivir and ribavirin via albumin route has been nents [36]. Earlier studies for evaluation of virucidal ac- established previously also [39]. tivity against H9N2 virus by crude extract and In the case of dose dependent prophylactic activity as- echinecea crude extract against H9N2 virus has also been sessment, only the high doses of crude extract , sambucus ocimum ascribed to blocking of the influenza virus entry via at polyphenol and polyphenol were effective as ocimum acacia least two virion targets, HA and NA on the surface of observed by the absence of HA titer and significant de- virus [37]. Mentofin® (combination of eucalyptus oil and crease in viral genome copy numbers. Lower doses of peppermint) in another study has been reported to have these extracts failed to provide sufficient prophylactic ac- complete virucidal activity against H9N2 virus in the tivity. Activity of different polyphenolic compounds in presence of organic matter (skimmed milk) [38]. plants worked their best when added to the cells just On assessing the therapeutic potential, we were able to around or before the time of virus adsorption using in demonstrate that all the extracts of O. sanctum (crude vitro model. They do possess high binding affinities with extract , terpenoid and polyphenol ) sig- viral HA and NA which might be a reason for their ocimum ocimum ocimum nificantly reduced the viral genome copy numbers prophylactic activity [34, 40–42]. Thus, the therapeutic or (P < 0.0001) at all the tested time intervals of 24, 48 and prophylactic efficacy testing using in ovo model could be 72 h post-inoculation. However, crude extract and improved by considering the multiple dose regimens and ocimum terpenoid had higher therapeutic potential at 48 monitoring the active ingredients diffusion of plant ex- ocimum and 72 h post-inoculation, respectively. Although terpe- tracts through suitable methods. Moreover, since these ex- noid and polyphenol treated groups showed tracts shows no adverse effect, a clinical pilot study would ocimum ocimum 5.1 4.8 a mean HA titer of 2 ,2 , respectively at 48 h give further information on the potency of these extracts post-inoculation and a slightly decreased mean HA titer in protecting against H9N2 virus infection in chickens. 4.8 3.8 of 2 ,2 , respectively at 72 h post-inoculation, the genome copy numbers in these treated groups were sig- Conclusions nificantly reduced in comparison to virus control The treatment with the crude extract derived from the (P < 0.0001). The possible reason for this could be due leaves of Ocimum sanctum leads to the significant H9N2 Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 9 of 10 virus reduction in assessing the all the three; virucidal, Author details National Institute of High Security Animal Diseases, Anand Nagar, Bhopal, therapeutic and prophylactic activities using in ovo Madhya Pradesh, India. Indian Veterinary Research Institute, Izatnagar, model. At this stage, we conclude that crude extract 3 ocimum Bareilly, Uttar Pradesh, India. Present Address: Department of Veterinary could be a promising extract for developing safe and effi- Epidemiology and Preventive Medicine, COVAS, Udgir, Latur, Maharashtra, India. cacious antiviral compound against H9N2 virus. The pro- tective efficacy of crude extract might be attributed ocimum Received: 9 November 2017 Accepted: 23 May 2018 to multiple mechanisms of action, e.g. specific inhibition of a stage in viral intracellular multiplication and non-specific interference with virus-cell interactions like References 1. Lee DH, Song CS. 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Evaluation of antiviral activity of Ocimum sanctum and Acacia arabica leaves extracts against H9N2 virus using embryonated chicken egg model

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Medicine & Public Health; Complementary & Alternative Medicine; Internal Medicine; Chiropractic Medicine
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Abstract

Background: In the view of endemic avian influenza H9N2 infection in poultry, its zoonotic potential and emergence of antiviral resistance, two herbal plants, Ocimum sanctum and Acacia arabica, which are easily available throughout various geographical locations in India were taken up to study their antiviral activity against H9N2 virus. We evaluated antiviral efficacy of three different extracts each from leaves of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) against H9N2 virus using in ovo model. Methods: The antiviral efficacy of different leaves extracts was systematically studied in three experimental protocols viz. virucidal (dose-dependent), therapeutic (time-dependent) and prophylactic (dose-dependent) activity employing in ovo model. The maximum non-toxic concentration of each herbal extracts of O. sanctum and A. arabica in the specific pathogen free embryonated chicken eggs was estimated and their antiviral efficacy was determined in terms of reduction in viral titres, measured by Haemagglutination (HA) and real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays. Results: All the extracts of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) showed significant virucidal activity, however, crude extract and terpenoid ocimum ocimum showed highly significant to significant (p <0.001–0.01) decrease in virus genome copy numbers with lowest dose tested. Similarly, therapeutic effect was observed in all three extracts of O. sanctum in comparison to the virus control, nevertheless, crude extract and terpenoid maintained this effect for longer period of time (up to 72 h ocimum ocimum post-incubation). None of the leaves extracts of A. arabica had therapeutic effect at 24 and 48 h post-incubation, however, only the crude extract and polyphenol showed delayed therapeutic effect (72 h post-inoculation). acacia acacia Prophylactic potential was observed in polyphenol with highly significant antiviral activity compared to virus acacia control (p <0.001). Conclusions: The crude extract and terpenoid isolated from the leaves of O. sanctum and polyphenol from A. arabica has shown promising antiviral properties against H9N2 virus. Future investigations are necessary to formulate combinations of these compounds for the broader antiviral activity against H9N2 viruses and evaluate them in chickens. Keywords: O. sanctum, A. arabica,Avian influenza H9N2, in ovo testing, HA, Real time RT-qPCR * Correspondence: richa.bhatia0609@gmail.com National Institute of High Security Animal Diseases, Anand Nagar, Bhopal, Madhya Pradesh, India Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 2 of 10 Background properties against Peste des petits ruminants (PPR) virus Influenza A viruses (IAVs), members of the family [18], along with inhibition of Goatpox virus replication Orthomyxoviridae are characterized by a single stranded, [19]. However, antiviral H9N2 influenza activity of differ- segmented negative-sense RNA genome. Among the ent extracts derived from the leaves of these two plants IAVs, avian influenza (AI) H9N2 has become endemic in has not been studied. terrestrial poultry in several countries of the Eurasian For evaluation of the antiviral properties of medicinal continent including India in recent years [1–4]. The plants against IAVs, three methods viz. tissue culture spread of AI H9N2 has resulted in significant economic [20, 21], laboratory/experimental animals [22] and in ovo losses in poultry mainly because of reduced egg produc- model [23] have been used frequently. Each method has tion and high mortality associated with co-infection with its pros and cons. We preferred in ovo model, which is other respiratory pathogens [5]. Although AI H9N2 does at the borderline of in vitro and in vivo studies and thus not fall under the definition of highly pathogenic avian in- does not conflict with either ethical or legal aspects of fluenza (HPAI) viruses, there has been ever increasing animal protection [23, 24]. Therefore, keeping in view speculation about pandemic potential of H9N2 viruses [6]. the endemicity and pandemic potential of H9N2, this So far, a total of 28 laboratory-confirmed cases of human study was taken up to assess the H9N2 inhibitory poten- infection with avian influenza A (H9N2) viruses, however tial of various extracts derived from leaves of O. sanctum none fatal, have been detected globally (http://www.who.int/ and A. arabica using in ovo model. influenza/human_animal_interface/Influenza_Summary_ IRA_HA_interface_25_02_2016.pdf). Among them, the Methods recent one includes from China [7]. In addition, sero- Virus and embryonated chicken eggs logical evidences of AI H9N2 virus exposure to human Avian Influenza virus, A/chicken/CL/15–12/103075 (H9N2) have been reported on several occasions from Iran, China was collected from one of the major water body of and India [8–11]. The higher human infection capability Maharashtra state of India during an ongoing surveillance of these viruses was provided by the fact that H9N2 binds programme. Specific pathogen free (SPF) embryonated to α-2,6 sialic acid receptors that are abundant in the hu- chicken eggs (ECEs) were obtained from SPF Unit of man upper respiratory tract while H5N1 chiefly bind to ICAR- National Institute of High Security Animal human receptors in the lower respiratory tract [12]. The Diseases, Bhopal, India. This virus was passaged two recently emerged influenza A (H7N9) and (H10N8) in- times in the allantoic fluid of 10-days-old SPF ECEs to fecting humans had acquired gene segments from H9N2 make seed virus stock. The virus seed stock was titrated virus [13, 14]. The potential of genetic reassortment of by the haemagglutination test as per OIE protocol [25]. IAVs, due to segmented genome, from different animal The allantoic cavities of 10-day-old SPF ECEs were inocu- species is thought to be a mechanism for the emergence lated with 0.2 ml of seed virus suspension (1:100 diluted of influenza viruses with pandemic potential [14]. Endem- in 0.01 M phosphate-buffered saline, pH 7.2) and incu- icity of H9N2 circulation in poultry especially in India bated for 72 h at 37 °C followed by chilling at 4 °C over- could further aggravate the current situation. Among the night. The allantoic fluid was harvested, clarified by control measures, existing vaccines are unable to keep up centrifugation and then stored at − 80 °C. The ten-fold with the mutation rates of viruses. New vaccine develop- serially diluted virus was inoculated in the allantoic cavity ment takes a long time and at the same time, viruses are of SPF ECEs and the virus titer was estimated as the egg also developing resistance to the currently used drugs infective dose 50% per ml (EID /ml). [15]. Hence, there is no immediate response drug to the newly emerging virus infections/outbreaks. To address Plant extracts and antiviral drugs this problem, there is an exigent need for the development Two plants, O. sanctum (commonly known as Tulsi) and of a new paradigm preventive and therapeutic agent to A. arabica (commonly known as Babul), native to the control the immediate spread of viral outbreaks. In this Indian subcontinent were used in this study. The leaves scenario, traditional herbal medicines have been postu- of these plants were collected from Madhya Pradesh, lated to prove effective due to fewer side effects, relatively India and identified by Dr. S.S. Ghoke through standard low cost and easy availability [16]. morphological and anatomical techniques. Plants were Two plants, Ocimum sanctum and Acacia arabica are authenticated and voucher numbers for O. sanctum widely distributed and easily available throughout vari- (Specimen#: vi p-1050313) and A. arabica (Specimen#: ous geographical locations in India. The efficacy of O. vi p-174629) were obtained. Crude extracts of these two sanctum as inhibitory compound has been documented plant leaves were prepared in hydro-methanol (1:1 dilu- against several viruses like Newcastle Disease virus, tion with water) by standard hot continuous extraction Vaccinia virus and Infectious Bursal Disease virus [17]. method using Soxhlet apparatus [26]. Two class com- Similarly, A. arabica has been explored for its virucidal pounds each in identified plants, O. sanctum (terpenoid Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 3 of 10 and polyphenol) and A. arabica (flavonoid and polyphenol) of different plant leaves extracts (three extracts each from were isolated and purified by ultraviolet–visible prep HPLC two different plants leaves) was incubated with 500 EID / technique [27]. Antiviral drugs, Amantadine hydrochloride 0.1 ml of H9N2 virus separately at 37 °C for 2 h. The drug (Sigma, USA) and Oseltamivir (FLUVIR, Hetero Drugs control group included amantadine hydrochloride Ltd., India), the currently approved drugs for influenza [28] (MNTC-16 μg/0.1 ml) incubated with 500 EID /0.1 ml of were used as drug control and hydro-methanol as vehicle H9N2 virus and the virus control group included 500 control group. EID /0.1 ml of H9N2 virus incubated with 0.1 ml PBS for 2 h at 37 °C. The vehicle control group comprised of Toxicity assay of plant extracts and antiviral drugs 0.1 ml of hydro-methanol incubated with 500 EID / To determine the maximum non-toxic concentration 0.1 ml of H9N2 virus. Afterwards, 0.1 ml of each of the (MNTC) of all the extracts of O. sanctum (crude virus-extract mixture was inoculated into the allantoic extract ,terpenoid , polyphenol )and cavity of 10-day-old SPF ECEs (5 each) separately and in- ocimum ocimum ocimum A. arabica (crude extract , flavonoid and cubated at 37 °C for 72 h. The allantoic fluid was acacia acacia polyphenol ) the guidelines prescribed by the harvested from each of the treatment groups 72 h acacia , Organization for Economic Co-operation and Develop- post-incubation. The presence of virus in the allantoic ment (OECD) were followed [29] Average egg biomass of fluid was assessed by haemagglutination (HA) test as per 10 SPF ECEs which included the embryo along with its al- methods described [30] and quantified by real time lantoic sac and yolk was measured and the maximum dose RT-qPCR [31](Fig. 1). for each of the herbal extract groups that could be used without inducing toxicity to the embryo was calculated as Therapeutic, time-dependent activity 135 mg/kg of egg biomass [29]. Afterwards, the MNTC Eight treatments groups (except the vehicle control was again assessed in the in ovo model. 0.1 ml of two-fold group) as formulated for testing of virucidal antiviral dilution of each of the plant extracts (200, 175, 150, 135, activity were made for testing the therapeutic efficacy in 100, 75, 50, 25, 10 and 5 mg/0.1 ml, diluted in extraction a time dependent manner. In each of the treatment medium hydro-methanol) were inoculated into the allan- groups, 500 EID /0.1 ml of H9N2 virus was inoculated toic cavity of 10-days-old SPF ECEs in triplicates. The SPF into the allantoic cavity of 10-day-old SPF ECEs (6 eggs ECEs inoculated with 0.1 ml of hydro-methanol alone for each group) and incubated at 37 °C for 2 h. Then, served as vehicle control. The MNTCs of amantadine and the MNTC dose (135 mg/0.1 ml) of each plant extract oseltamivir antiviral drugs were determined by inoculating was injected into the sharp pole of albumen of these 5.33, 8 and 16 μg/0.1 ml and 1.75, 2.66 and 5.25 μg/ ECEs separately and incubated at 37 °C. This route has 0.1 ml, respectively into the allantoic cavity of 10-days-old been suggested as an improved model for testing anti- SPF ECEs separately in triplicates. All the inoculated SPF viral drugs especially if the bioavailability of the drug is ECEs were incubated at 37 °C till hatching and were can- poor [32]. In the drug control group, oseltamivir dled twice daily for checking the viability. (MNTC–2.66 μg/0.1 ml) was injected through albumen route 2 h post-virus inoculation. The allantoic fluid was Assessment of efficacy of herbal extracts against H9N2 harvested from each of the treatment group at 24, 48 virus using in ovo model and 72 h post-inoculation (Fig. 1). The presence of virus The antiviral activity of each plant crude extract and its in the allantoic fluid was tested by HA test and quanti- isolated class compounds were assessed in three differ- fied by real time RT-qPCR. ent formats viz; virucidal (dose-dependent), therapeutic (time-dependent) and prophylactic (dose-dependent). Prophylactic, dose-dependent activity For testing of prophylactic efficacy, same eight treatment Dose-dependent virucidal activity groups as mentioned in above testing methodologies A total of nine treatment groups (each treatment group were used to examine the effect of extracts in a dose with five 10-days-old SPF ECEs) were made for the dependent manner. In each of the treatment groups, the determination of virucidal activity of herbal extracts two-fold dilution of MNTC (135 mg; 67 mg; 33 mg/ against A/chicken/CL/15–12/103075 (H9N2). Of the nine, 0.1 ml) of different plant extracts (three extracts each six treatment groups corresponded to the respective six from two plants leaves) was inoculated into the allantoic herbal extracts (crude extract , terpenoids , cavity of 10-day-old SPF ECEs (5 eggs for each group) ocimum ocimum polyphenol ; crude extract , flavonoid , and incubated at 37 °C for 2 h. Then, 500 EID /0.1 ml ocimum acacia acacia 50 polyphenol ) used in this study. The remaining three of H9N2 virus was inoculated into the allantoic cavity of acacia treatment groups were the drug control, the vehicle con- each of these eggs and incubated at 37 °C for 72 h trol and the virus control group. 0.1 ml of two-fold dilu- (Fig. 1). In the drug control group, amantadine hydro- tion of pre-calculated MNTC (135 mg, 67 mg, and 33 mg) chloride (MNTC-16 μg/0.1 ml) was given through Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 4 of 10 Fig. 1 Experimental protocols for the assessment of the antiviral activity of Ocimum sanctum and Acacia arabica leave extracts against H9N2 virus using in ovo model allantoic cavity 2 h post-virus inoculation. The presence Results of virus in the allantoic fluid was detected by HA test Toxicity assay of plant extracts and antiviral drugs and quantified by real time RT-qPCR. In a first set of experiments, MNTC was calculated for each herbal extracts and antiviral drugs in the SPF ECEs. The criterion of non-toxicity of herbal extracts was iden- Real-time quantitative reverse transcription PCR tified as the absence of death of embryos up to hatching Viral RNA from the respective allantoic fluids was ex- of all the eggs (three per drug concentration) at various tracted using QIAamp Viral RNA mini kit (Qiagen, concentrations (200, 175, 150, 135, 100, 75, 50, 25, 10 Germany) as per the recommendations of the manufac- and 5 mg/0.1 ml). Irrespective of the different herbal ex- turer. One step RT-qPCR was carried out in duplicates tracts, all the ECEs that had received 200 mg/0.1 ml of as per standard method [31]. The PCR assay was con- extract died within 48 h of post-inoculation. The ducted on Light Cycle® 480 Real-Time PCR, Roche, mortality of 50–15% was observed in ECEs given 175 USA. The fluorescence reading was noted at the end of and 150 mg/0.1 ml extract, while those that received each extension step. No template control and no probe 135 mg/0.1 ml or less showed no evidence of mortality control were included in each run. till hatching. The concentrations of antiviral drugs tested were non-toxic to the embryos at the dose of 16 μg/ Statistical analysis 0.1 ml and 2.66 μg/0.1 ml for amantadine hydrochloride The infectivity titer of the H9N2 virus (A/chicken/CL/ and oseltamivir, respectively and thus have been used as 15–12/103075) was expressed as EID /ml and was cal- MNTC throughout the study. culated as per the standard method [33]. The data is presented as mean ± standard deviation. For the analysis Antiviral activity on the basis of haemagglutination test of the significance of differences among the different The aim of this experiment was to identify the most ef- treatment groups in terms of the virus genome copy fective extract in inhibiting the H9N2 virus replication number, two ways analysis of variance (ANOVA) with in the SPF ECEs. The virus challenge dose was identified Tukey post hoc test was used. P values equal to or less on the basis that there was no mortality in the eggs up than 0.05 were considered statistically significant. to 72 h, in any of the virus challenge groups made, for Analyses were performed using GraphPad Prism version evaluating the antiviral activities for different doses as 6.0 (GraphPad Software, SanDiego, CA, USA). well as time intervals. Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 5 of 10 Dose-dependent virucidal activity inhibiting the H9N2 virus replication. The crude All the herbal extracts treatment groups including drug extract when administered at the dose of 135 mg/ ocimum control did not show any HA titer irrespective of de- 0.1 ml significantly inhibited the virus replication com- creasing dose (135, 67, 33 mg/0.1 ml) treatments, indi- pared to virus control group (p < 0.001). At the same cating that even the low dose of all extracts were dose (135 mg/0.1 ml), the other four extracts; effective in inhibiting the replication of H9N2 virus. Al- terpenoid , polyphenol , flavonoid and ocimum ocimum acacia lantoic fluid collected from these treatment groups were polyphenol also inhibited the virus replication acacia given two consecutive passages in SPF eggs to ascertain significantly (p < 0.05–0.01) (Fig. 3). On reducing the the absence of H9N2 replication which did not show dose (67 mg/0.1 ml), only crude extract and ocimum any HA titer and reconfirmed the virucidal activity. terpenoid were effective (p < 0.05). Further redu- ocimum However on further two-fold reduction in dose to cing the dose (33 mg/0.1 ml), crude extract ocimum 17.5 mg/0.1 ml for each extract, we found that crude remained effective (p < 0.05) (Fig. 3). Non-significant ef- extract , terpenoid , crude extract and ficacy was recorded within both groups of herbal ex- ocimum ocimum acacia flavonoid treatment groups showed a mean HA tracts of O. sanctum and A. arabica in the present study. acacia 4.2 5 4.2 5.3 titer of 2 ,2 ,2 and 2 , respectively which on fur- ther two fold reduction of dose to 8.75 mg/0.1 ml Therapeutic, time-dependent activity All the three ex- remained almost same as above indicating loss of viru- tracts of O. sanctum (crude extract ,terpenoid ocimum ocimum cidal activity at lower doses. However, polyphenol , and polyphenol ) were significantly effective in ocimum ocimum polyphenol and the drug control amantadine treat- limiting the H9N2 virus replication in the SPF ECEs at acacia ment groups did not show any HA titer even at the low- different time intervals (24, 48 and 72 h post-inoculation) est dose (8.75 mg/0.1 ml) tested indicating a high (p <0.001–0.0001). Within the O. sanctum treatment virucidal activity of these class compounds (Fig. 2). group, crude extract was found to have significantly ocimum high efficacy in comparison to terpenoid (p <0.05) ocimum and polyphenol (p < 0.01) at 48 h post-inoculation ocimum Time-dependent therapeutic activity as indicated by decrease in viral genome copy numbers Among the extracts of O. sanctum, only crude extract ocimum (Fig. 4). However, terpenoid showed significantly ocimum treatment group showed absence of HA titer in the allan- high inhibitory activity compared to crude extract ocimum toic fluid harvested at all the three time intervals (24, 48 (p < 0.0001) and polyphenol (p <0.001) at 72 h ocimum and 72 h) post-inoculation. The drug control group oselta- post-inoculation. mivir also showed absence of HA titer indicating no viral In the case of A. Arabica, none of the extracts were ef- replication. The other two extract treatment groups, fective initially at 24 and 48 h post-inoculation (p >0.05), terpenoid and polyphenol however had mean ocimum, ocimum 4.8 3.8 however, at 72 h post-inoculation, crude extract acacia HA titers 2 and 2 , respectively at 72 h post-inoculation (p < 0.001) and polyphenol (p < 0.01) depicted thera- acacia (Fig. 2). peutic activity as indicated by significant decrease in the virus genome copy numbers as compared to virus control Dose-dependent prophylactic activity group (Fig. 4). However, differences in the time dependent All the three extracts of O. sanctum at doses 135 and therapeutic effect among the extracts of A. arabica were 67 mg/0.1 ml showed prophylactic potential as revealed non-significant (p > 0.05). On comparison of antiviral by the absence of HA titers. However, on further reduc- treatment between the two groups it was found that all tion of dose to 33 mg/0.1 ml, all the three extracts (crude three extracts of O. sanctum treatment group had signifi- extract ,terpenoid and polyphenol ) ocimum ocimum ocimum cantly higher (p <0.05–0.0001) antiviral efficacy than the 5.9 5.6 3.6 showed a mean HA titer of 2 ,2 and 2 respectively. crude extract or polyphenol extracts. acasia acacia In the case of A. arabica, MNTC dose (135 mg/0.1 ml) of all the three extracts showed absence of HA titer. Lower Prophylactic, dose-dependent activity The prophylac- doses (67 and 33 mg/0.1 ml) of any of the extracts of A. tic potential of all the herbal extracts were determined arabica were not effective in preventing viral replication. against H9N2 virus in the SPF ECEs. In the O. sanctum The drug control group showed complete absence of HA group, only crude extract and polyphenol ocimum ocimum titer (Fig. 2). treated ECEs showed significant (p < 0.05) decrease in viral genome copy numbers at dose of 135 mg/0.1 ml Antiviral activity on the basis of viral quantification by (Fig. 5). The decrease in viral genome copy numbers was real time RT-qPCR non-significant at lower doses of 67 and 33 mg/0.1 ml in Virucidal, dose-dependent activity all the three extracts of O. sanctum groups. In A. arab- The aim of this experiment was to quantify the viral ica group, only polyphenol was more successful in acacia amount and identify the most effective extract in reducing the viral genome copy numbers at doses of 135 Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 6 of 10 Fig. 2 Measurement of antiviral activity of O. sanctum and A. arabica leave extracts against H9N2 virus using HA assay. a Dose-dependent virucidal activity; b Time-dependent therapeutic activity; c Dose-dependent prophylactic activity. The virus control group included 500 EID /0.1 ml of H9N2 virus incubated with 0.1 ml PBS for 2 h at 37 °C. The HA titer were expressed as log HA titer (p < 0.0001) and 67 mg/0.1 ml (p < 0.001) while it be- being considered as an emerging pandemic threat in view came ineffective at low dose of 33 mg/0.1 ml (p > 0.05) of the clinical and confirmed cases of H9N2 in China, (Fig. 5). The other two extracts of A. arabica, crude Hong Kong, Bangladesh and Egypt [14]. This reaffirms the extract and flavonoid were not effective as need for search of new compounds with antiviral activity. acacia acacia prophylactic extract at any of the doses (p > 0.05). With increase in drug resistance to synthetic antivirals, nat- ural products remain an important alternative for the con- Discussion trol infectious diseases. In the present study, the in ovo Spread of H9N2 subtype has demonstrated an increased model has been used for studying antiviral activities of dif- theoretical threat to humans because of the potential ferent leaves extracts of Ocimum sanctum and Acacia arab- emergence of novel subtypes of avian influenza [13]. It is ica against low pathogenic avian influenza (LPAI) H9N2. Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 7 of 10 Fig. 3 Assessment of virucidal activity of the O. sanctum and A. arabica leaves extracts in a dose-dependent manner by Real Time RT-qPCR assay. Each error bar represents standard deviation (SD) from the 5 ECEs. The virus control group included 500 EID /0.1 ml of H9N2 virus. Statistical significance was assessed by two ways analysis of variance (ANOVA) with Tukey post-hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) In our previous work we were able to demonstrate flavonoid and polyphenol) against LPAI H9N2 using 100% virucidal activity of cold aqueous extract of bark of in ovo model. A. arabica at a concentration of 0.625 mg/ml against In dose dependent virucidal activity, all the extracts of HPAI H5N1 using in vitro model. Mild therapeutic ac- both the plants in doses (135, 67 and 33 mg/0.1 ml) dis- tivity was also demonstrated up to 48 h in cold aqueous played absence of HA titer indicating that all the ex- extract of Ocimum tenuiflorum when tested against tracts had inhibitory potential. On further reduction of HPAI H5N1 [20]. dose rate in double dilution, polyphenol and poly- acasia In the present work, we had selected two plants, phenol only maintained virucidal activity up to a Ocimum Ocimum sanctum and Acacia arabica for studying their low dose rate of 8.75 mg/0.1 ml which was confirmed by efficacy against avian influenza H9N2 virus because absence of HA in the harvested allantoic fluid of ECEs these plants are widely distributed and easily available in the next two passages also. This virucidal effect could throughout various geographical locations in India and be due to masking/blocking of HA protein of the virus have strong record in the literature for their antiviral ac- by the herbal extracts during the incubation period, tivity against different viruses [16, 17]. Three different which might have inhibited the H9N2 virus replication. approaches viz. virucidal (dose dependent), therapeutic Catechin compounds in polyphenols are known to have (time dependent) and prophylactic (dose dependent) antiviral activity mediated by preventing adsorption of were employed to systematically explore the potential of viruses to cells [34, 35]. Highly significant to significant extracts of leaves of O. sanctum (crude extract, terpen- decrease in the viral genome copy numbers was recorded oid and polyphenol) and A. arabica (crude extract, in all the extracts at the MNTC dose (135 mg/0.1 ml) Fig. 4 Assessment of therapeutic activity of the O. sanctum and A. arabica leaves extracts in a time-dependent manner by Real Time RT-qPCR assay. Each error bar represents standard deviation (SD) from the 6 ECEs. The virus control group included 500 EID /0.1 ml of H9N2 virus. Statistical significance was assessed by two ways analysis of variance (ANOVA) with Tukey post-hoc test (*p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 8 of 10 Fig. 5 Assessment of prophylactic activity of the O. sanctum and A. arabica leaves extracts in a dose-dependent manner by Real Time RT-qPCR assay. Each error bar represents standard deviation (SD) from the 5 ECEs. The virus control group included 500 EID /0.1 ml of H9N2 virus. Statistical significance was assessed by two ways analysis of variance (ANOVA) with Tukey post-hoc test (*p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) except crude extract (P <0.05–0.001). Although the to the decrease in concentration of the active ingredient acacia virucidal effect was seen more prominently in the HA test which might have allowed the virus to replicate because where the virus HA titer was absent in comparison to the of single dose regimen followed in the experiment. In 7.3 virus control (mean HA titer 2 )and vehicle control addition, other possible reason might be the high sensi- 5.8 (mean HA titer 2 ), the quantification of the virus by real tivity of real time PCR assay. Surprisingly, in A. arabica time RT-qPCR also corroborated with the HA results es- treated groups, crude extract and polyphenol acacia acacia pecially at higher extract doses. The results of both the as- showed significant decrease in viral genome copy num- says clearly indicated that crude extract in particular bers at a later time interval of 72 h post-inoculation ocimum is a potent inhibitor of H9N2 virus replication. (P < 0.01). Since the route of administration was via Our study is further supported by the fact that individ- albumen, slow movement/distribution of A. arabica ex- ual plant extracts were less effective compared to crude tracts from albumen to allantoic cavity might be the rea- extracts which may due to synergistic effect of individual son for delayed effect. Variable bioavailability of drugs, component and some of the uncharacterised compo- oseltamivir and ribavirin via albumin route has been nents [36]. Earlier studies for evaluation of virucidal ac- established previously also [39]. tivity against H9N2 virus by crude extract and In the case of dose dependent prophylactic activity as- echinecea crude extract against H9N2 virus has also been sessment, only the high doses of crude extract , sambucus ocimum ascribed to blocking of the influenza virus entry via at polyphenol and polyphenol were effective as ocimum acacia least two virion targets, HA and NA on the surface of observed by the absence of HA titer and significant de- virus [37]. Mentofin® (combination of eucalyptus oil and crease in viral genome copy numbers. Lower doses of peppermint) in another study has been reported to have these extracts failed to provide sufficient prophylactic ac- complete virucidal activity against H9N2 virus in the tivity. Activity of different polyphenolic compounds in presence of organic matter (skimmed milk) [38]. plants worked their best when added to the cells just On assessing the therapeutic potential, we were able to around or before the time of virus adsorption using in demonstrate that all the extracts of O. sanctum (crude vitro model. They do possess high binding affinities with extract , terpenoid and polyphenol ) sig- viral HA and NA which might be a reason for their ocimum ocimum ocimum nificantly reduced the viral genome copy numbers prophylactic activity [34, 40–42]. Thus, the therapeutic or (P < 0.0001) at all the tested time intervals of 24, 48 and prophylactic efficacy testing using in ovo model could be 72 h post-inoculation. However, crude extract and improved by considering the multiple dose regimens and ocimum terpenoid had higher therapeutic potential at 48 monitoring the active ingredients diffusion of plant ex- ocimum and 72 h post-inoculation, respectively. Although terpe- tracts through suitable methods. Moreover, since these ex- noid and polyphenol treated groups showed tracts shows no adverse effect, a clinical pilot study would ocimum ocimum 5.1 4.8 a mean HA titer of 2 ,2 , respectively at 48 h give further information on the potency of these extracts post-inoculation and a slightly decreased mean HA titer in protecting against H9N2 virus infection in chickens. 4.8 3.8 of 2 ,2 , respectively at 72 h post-inoculation, the genome copy numbers in these treated groups were sig- Conclusions nificantly reduced in comparison to virus control The treatment with the crude extract derived from the (P < 0.0001). The possible reason for this could be due leaves of Ocimum sanctum leads to the significant H9N2 Ghoke et al. BMC Complementary and Alternative Medicine (2018) 18:174 Page 9 of 10 virus reduction in assessing the all the three; virucidal, Author details National Institute of High Security Animal Diseases, Anand Nagar, Bhopal, therapeutic and prophylactic activities using in ovo Madhya Pradesh, India. Indian Veterinary Research Institute, Izatnagar, model. At this stage, we conclude that crude extract 3 ocimum Bareilly, Uttar Pradesh, India. Present Address: Department of Veterinary could be a promising extract for developing safe and effi- Epidemiology and Preventive Medicine, COVAS, Udgir, Latur, Maharashtra, India. cacious antiviral compound against H9N2 virus. The pro- tective efficacy of crude extract might be attributed ocimum Received: 9 November 2017 Accepted: 23 May 2018 to multiple mechanisms of action, e.g. specific inhibition of a stage in viral intracellular multiplication and non-specific interference with virus-cell interactions like References 1. Lee DH, Song CS. 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BMC Complementary and Alternative MedicineSpringer Journals

Published: Jun 5, 2018

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