Abstract: In this study, the antiviral effect of holm oak wood extract was investigated against (H9N2) virus, which belongs to the group of influenza (A) viruses that cause serious diseases to human and animal, this in vitro study was carried out in comparison to Amantadine HCl (AMN) in addition to extracts of green tea leaves and pomegranate fruit rinds and arils, as these substances have shown previously a considerable antiviral properties against influenza (A) viruses. Initially, cytotoxicity examination was conducted for the crude aqueous methanol extracts (CAMEs) of Quercus ilex L. (wood), Camellia sinensis (leaves) and Punica granatum (rind), besides crude ethanolic extract (CEE) of P. granatum (arils) on MDCK cell line. This was followed by evaluation for their in vitro antiviral activities against AMN resistant (H9N2) virus infection on the same cell line using MTT based colorimetric assays. Holm oak wood extract Showed visible antiviral effect against (H9N2) influenza virus on MDCK cells, where the virus inhibition percentage exceeded 97% at the viral dose of (50 TCID50) and was equivalent for large extent to the antiviral effect shown by green tea extract against the same virus, despite the comparatively more toxic effect of green tea extract on MDCK cells. On the other hand, the pomegranate extracts unexpectedly exhibited much less Influenza virus inhibitory effects than the former extract. Based on the results revealed in this study, the virus inhibitory effects shown by the holm oak wood extract, suggest this plant as promising one that need more research to find out its active components that may be useful in the development of new effective antiviral agents.
INTRODUCTION
Regarding human and animal health, Influenza type A Viruses (IAV) are the most dangerous pathogens among the five genera of influenza viruses that belong to the family Orthomyxoviridae (Palese and Shaw, 2007), they are responsible for seasonal flu epidemics as well as serious pandemics that are associated with high morbidity and mortality rates (Memoli et al., 2008; Zimmer and Burke, 2009).
The binding of IAV viral hemagglutinin (HA) to sialic acid receptor on the host cell surface initiates the infection course, followed by receptor mediated endocytosis of the virus particles, with subsequent viral uncoating inside cells, After fusion, the virus shuts off cell replication and cell protein synthesis, consequently, infected cells die by apoptosis or cytolysis (Wu et al., 2010).
The M2 inhibitor, Amantadine (AMN) blocks the ion channel activity of the M2 protein, thus interfering with viral uncoating inside cells; hence, it is one of the very few drugs approved by the FDA to treat Influenza A virus infection (Palese 2004; De Clercq, 2006).
Unfortunately, the long history of AMN use was frequently associated with emergence of drug-resistant influenza viral variants (Linhares et al., 1989). By the end of the last decade, the Centers for Disease Control and Prevention (CDC) reported that 100% of the seasonal H3N2 virus isolate tested were resistant to (AMN) and 99.6% of the seasonal H1N1 viruses tested were resistant to oseltamivir, both antiviral drugs are currently in use for both prophylactic and therapeutic treatments of Influenza viruses (CDC, 2009).
Therefore, there has been a continuous need for new antiviral agents to overcome the increasing problem of viral resistance to existing antiviral drugs.
Plants are appropriate candidates for antiviral research programs because they produce a large number of phytochemical substances to adapt themselves to environmental stressors including attack by pathogens and they also have a long history of effective and harmless use as traditional medications against different illnesses including infectious diseases (Dixon, 2001; Guo et al., 2006).
Green tea (C. sinensis) extracts and derived compounds displayed an evident in vitro antiviral activity against different Influenza viruses (Song et al., 2005, 2007), rotavirus, enterovirus (Mukoyama et al., 1991) in addition to Human Papillomavirus (HPV) (Gross et al., 2007; U.S. FDA, 2008).
It was reported that pomegranate (P. granatum) components possess an in vitro antiviral activity against HIV-1 (Neurath et al., 2004) and more recently reported to inhibit Influenza virus replication (Haidari et al., 2009; Sundararajan et al., 2010).
On the other hand, the reported antibacterial activities exposed by the extracts of Q. ilex leaves (Gulluce et al., 2004) and Q. ilex bark (Berahou et al., 2007), is thought to be attributable to the rich polyphenolic content of Quercus species extracts including proanthocyanidins, acylated flavonoid glycosides and tannins (Zhentian et al., 1999; Meng et al., 2001; Ito et al., 2002), However, no information has been reported including antiviral activity of Q. ilex extracts against Influenza viruses.
In this study, we aimed to evaluate the antiviral effect of Q. ilex (wood) extract against H9N2 Influenza virus reference strain (Influenza A/turkey/Wisconsin/1/1966) infection in (MDCK) cells, compared to Amantadine HCl as a Standard anti-Influenza A antiviral drug besides the extracts of C. sinensis and P. granatum which both are two edible plants of previously reported anti-Influenza virus capabilities.
MATERIALS AND METHODS
Cell line: A continuous cell line of MDCK (Madin-Darby Canine Kidney cells), was obtained from cell culture department of the holding company for biological products and vaccines (VACSERA), Agouza, Cairo, Egypt. Cells were grown in DMEM supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), 100 IU mL-1 penicillin G, 100 μg mL-1 streptomycin and 0.25 μg mL-1 amphotericin B. The cells were incubated at 36°C in 5% CO2 humidified atmosphere and were subcultured twice a week.
Virus: A low pathogenic Avian influenza A virus H9N2 reference strain (Influenza A/turkey/Wisconsin/1/1966) was used with stock solution infective titer of 104.8 TCID50 mL-1 (50% tissue culture infective doses), it was kindly provided by Prof. Ahmed EL-Sanousi, Prof. of virology, Faculty of veterinary medicine, Cairo University, Cairo, Egypt. Virus was stored in small aliquots in -70°C freezer until use.
Reagents: DMEM, Dulbecco’s Modified Eagle’s Medium, (Lonza®, Belgium), was used as: Growth medium (+10% Fetal Bovine Serum, FBS), or as maintenance medium (+2% FBS); FBS, (Gibco®, invitrogen™, USA); Antibiotic mix 100 U mL-1 penicillin G, 100 μg mL-1 streptomycin and 0.25 μg mL-1 amphotericin B. (SERVA® Electrophoresis GmbH, Germany).
MTT, [3-(4,5-dimethylthiazol-2ol)-2,5 diphenyl tetrazolium bromide)], (SERVA® Electrophoresis GmbH, Germany). Crystalline Trypsin = 2500 USP U mg-1, solid, (from bovine pancreas), (Sigma Aldrich® Co., St. Louis, USA).
DMSO, distilled Dimethyl SulfOxide (SERVA® Electrophoresis GmbH, Germany).
Standard antiviral: Amantadine Hydrochloride obtained from (Sigma Aldrich® Co., St. Louis, USA) used as a standard antiviral drug against Influenza A virus at dose of 25 μg mL-1 (AI-Jabri et al., 1996).
Plant materials: Holm oak (Quercus ilex), a whole branch of the evergreen tree of Q. ilex grown in El-Orman National Herbarium, El-Orman Gardens, Dokki, Cairo, Egypt.
Green tea, (Camellia sinensis), commercial EL-MAABAD® Chinese pure green tea, purchased from the famous HARRAZ herbal shop, Bab El-Khalck, Cairo, Egypt. Pomegranate, (Punica granatum), the whole fruits, available at conventional fruit shops, Cairo, Egypt.
Extract preparation: The crude aqueous extracts of Q. ilex, C. sinensis and pomegranate rinds were prepared as follows:
Fifty grams of the grinded dried part of the plant (Table 1) was firstly dissolved in 250 mL of de-ionized bi-distilled water and then boiled for 2 h, the outcomes were filtered on filter paper and evaporated to dryness under vacuum at 40°C using rotatory evaporator, the dried aqueous solutions were re-extracted by methanol to get rid of dust residues and undesirable compounds, then extracts were preserved at 4°C until use.
In case of Pomegranate arils, extraction process was done as follows:
Fifty grams of the detached arils were soaked in 250 mL of absolute ethanol to get rid of free sugars for one day, filtered using filter paper, dried under vacuum, preserved at 4°C until use.
Different plant extracts were redissolved later (just before use) in sterile PBS (or in dist. DMSO [max. concentration = 0.2%] in case of Q. ilex wood extract) and subsequently diluted in cell culture medium (Table 1) and sterilized by filtration using (0.22 mm filter) just before testing.
| Table 1: | Four plant extracts used in antiviral assay |
Virus titration: Colorimetric H9N2 virus Titration was done on MDCK cells according to Levi et al. (1995) with minor modifications. Briefly, Cells were seeded in growth medium in 96-well flat bottom Microtiter cell culture plates (cell star®, greiner bio-one®, Germany), at a density of 15000 cells/well and then incubated at 36°C in a humidified atmosphere containing 5% CO2 for 24-48 h, then ten-fold serial dilutions of virus stock were prepared, when cells showed 70-90% confluence, growth medium was removed and cells were infected with 50 μL of each dilution of the virus, after 1-2 h adsorption period, 200 μL of maintenance media (devoid of FBS and supplemented with 2.5 μg mL-1 of Crystalline Trypsin) were added to each well and plates were put in incubator again.
Uninfected cells incubated only with DMEM were used as negative control cells. The cytopathic effects were observed daily for 5-6 days, then MTT assay was performed as described by Mosmann (1983), Changes occurred to the monolayers of cells were detected by measuring the formed (MTT) formazan crystals Optical Density (OD) within each well using microplate ELISA reader (Biotech® international, USA) at a wavelength of 540 nm.
Positive wells were those with an OD value lower than the cutoff value which was determined as the mean OD of the uninfected control wells minus 2xSD (i.e., standard deviation), whereas, negative wells were those with an OD value = The cutoff value according to Levi et al. (1995). The 50% tissue culture infective dose (TCID50) per mL was calculated as described previously by Reed and Muench (1938).
Moreover, a confirmatory Cell culture Heamagglutination virus titration was done for H9N2 virus on MDCK cells as follows:
Cells were seeded in growth media in 96-well U-shaped bottom cell culture plates (cell star®, greiner bio-one®, Germany) at a density of 15000 cells/well and then incubated at 36°C in a humidified atmosphere containing 5% CO2 for 24-48 h, when cells showed 70-90% confluence, growth media were removed, then tenfold serial dilutions of virus stock were prepared and cells were infected with 50 μL of each dilution of the virus, after 1-2 h adsorption period, 200 μL of maintenance media (devoid of FBS and supplemented with 2.5 μg mL-1 of Crystalline Trypsin) were added to each well and plates were put in incubator again.
Uninfected cells incubated only with DMEM were used as a negative control. Throughout incubation, the cytopathic effect was recorded daily for 5-6 days.
Then, 50 μL of 1% chicken RBCs solution was added to each well of the plate to detect the incidence of heamagglutinating influenza viruses within each well and then the plates were left for 15-20 min in room temperature, the end point was the virus dilution which induced (+ve) complete haemagglutination (lattice shape) or (-ve) no haemagglutination (button shape) of RBCs, the 50% tissue culture infective dose (TCID50) per mL was then calculated as described previously by Reed and Muench (1938).
Cytotoxicity assay: The assay was performed using 96-well microtiter plates seeded with 15000 MDCK cells/well and after 24-48 h of incubation at 36°C in a humidified 5% CO2 atmosphere, cells were treated with decreasing concentrations of plant extracts (2 fold serially diluted).
Monolayers of cells incubated with DMSO were considered as solvent controls whereas, monolayers of cells incubated only with DMEM were used as cell controls.
Cytotoxic changes occurred to the monolayers of cells during the next 5-6 days were daily observed microscopically and subsequently detected colorimetrically using MTT assay by measuringthe formed MTT formazan crystals within each well using microplate reader at a wavelength of 540 nm as described above to determine the maximum non-toxic concentration (MNTC) of each extract to MDCK cells.
Antiviral assay: MDCK Cells were seeded in growth media in 96-well flat bottom cell culture plates at a density of 15000 cells/well and then incubated at 36°C in a humidified atmosphere containing 5% CO2 for 24-48 h.
A tenfold serial dilution of virus stock was carried out, when cells showed 70-90% confluence, growth media were removed and cells were infected with 50 μL of each dilution of the virus, after 1-2 h adsorption period, 200 μL of maintenance media containing the MNTC of each of the used extracts were added to each well (or maintenance media without extract were added as negative cell controls) and plates were put in incubator again for 5-6 days.
During incubation, the virus cytopathic effect (CPE) was daily observed microscopically and at the end of incubation period MTT assay was done according to Mosmann (1983). Where viral CPE was detected colorimetrically by measuring the formed XTT formazan crystals (i.e., OD) within each well using microplate reader at a wavelength of 540 nm.
Controls consisted of untreated infected cells (virus control), treated non-infected cells (extract control), untreated non-infected cells (cell control) and cells incubated only with DMSO were considered as (solvent control).
Viral inhibition rate (IP %) was calculated from the equation:
where, ODtv, ODcv and ODcd indicate the optical density of the extract treated infected cells, the optical density of the untreated virus infected control and the optical density of the cell control, respectively.
RESULTS
The results of Cytotoxicity assay of plant extracts on MDCK cells; showed that the maximum non toxic concentrations were 100 μg mL-1, 12.5 μg mL-1, 62.5 μg mL-1 and 250 μg mL-1 for Q. ilex, C. sinensis, pomegranate rinds and pomegranate arils, respectively (Table 2).
Remarkably, the solvent, DMSO at the used concentration (i.e., 0.2%) showed no toxicity on MDCK cells.
The titer of the used H9N2 virus was measured by two methods:
| • | The MTT microculture virus titration (MCVT) assay method (Fig. 1, 2), the result obtained was that the virus used in this experiment has a titer of 10 4.732 TCID50 mL-1 |
| • | The cell culture heamagglutination (CCHA) virus titration method (Fig. 1) the result obtained was that the virus used has a titer of 104.8 TCID50 mL-1 |
| Table 2: | Result of cytotoxicity assay of different plant extract on MDCK cells |
| (MNTC): Maximum non toxic concentration | |
| Fig. 1: | Microplate reader mean absorbance results of virus titration assay of tenfold serially diluted H9N2 virus on MDCK cells (using MTT assay) (OD), absorbance |
| Fig. 2: | Cell culture HA results of virus titration assay of tenfold serially diluted H9N2 virus on MDCk cells (using 1% RBCs solution), button shape (-ve) heamagglutination, lattice shape (+ve) heamagglutination |
| Table 3: | Result of antiviral assay of different plant extracts and Amantadine against H9N2 Influenza virus reference strain (influenza A/turkey/Wisconsin/1/1966) infection in MDCk cells |
| -: IP<25%, +: IP = 25-50%, ++: IP = 50%, +++: IP = 90-96%, ++++: IP≥97%, TCID50: 50% tissue culture infective doses | |
However, an unexpected higher mean absorbance in case of 10-1 dilution compared to the 10-2 dilution and the next higher dilutions was seen (Fig. 1).
Regarding the antiviral assay, both Q. ilex (wood) extract and C. sinensis (leaves) extract showed potent antiviral effects (IP = 97 %) against H9N2 virus at virus dose of 50 TCID50, (Table 3).
However, pomegranate arils extract demonstrated a potent antiviral effect too (i.e., IP = 97%) against H9N2 Influenza A virus, only shown at virus dose of 5 TCID50 (Table 3).
In case of pomegranate rinds extract no touchable antiviral effect was observed (Table 3).
AMN used with the virus infected cells exhibited a very poor antiviral effect (i.e., IP<25%).
DISCUSSION
Although, Investigation of the antiviral potential of various promising plants was difficult in the past, in the last three decades, strategies for the in vitro evaluation of plant derived compounds with biological activity have progressed, along with the development of automated antiviral bioassay that depends on colorimetric quantification of the proliferating cell cultures (Mosmann, 1983; Weislow et al., 1989; Mukhtar et al., 2008).
Plant extracts used in herbal medicine were recently estimated by the WHO to cover the health needs of more than two thirds of the world’s population, including the treatment of viral diseases (Robinson and Zhang, 2011) and they have been suggested to be useful to overcome the growing problem of viral resistance to the available synthetic antiviral drugs (Chung et al., 1995; Vlietinck et al., 1995).
Plant family Fagaceae includes the genus Quercus which contain so many species, some of which are reported to have anthelmintic, antibacterial, antioxidant and antiviral activities (Konig et al., 1994; Hussein et al., 2000; Andrensek et al., 2004; Gulluce et al., 2004; Muliawan et al., 2006; Berahou et al., 2007).
However, no information including antiviral activity of Q. ilex (wood) extracts has been reported against influenza viruses.
The aim of this work was to evaluate in a colorimetric way the in vitro antiviral potential of Q. ilex (wood) extract against H9N2 Influenza virus infection in (MDCK) cells.
The H9N2 virus titer revealed by (CCHA) virus titration method confirms the accuracy of the result of colorimetric MTT (MCVT) method as the titers revealed by both methods were nearly identical; this also prove the previously mentioned findings of Parida et al. (1999) who stated that MTT assay is superior to conventional MCVT method.
However, an unexpected higher mean absorbance in case of 10-1 dilution compared to the 10-2 dilution and the next higher dilutions was seen (Fig. 1), which could be attributable to the presence of large amount of defective interfering particles (Von Magnus particles) that interfere with the infective H9N2 virions leaving the MDCK monolayer more intact than in the next higher virus dilution as mentioned previously by Nakajima et al. (1979).
The pomegranate arils extract was found to have the least toxicity on MDCK cells (Table 2). agreeing with the recent report of Sundararajan et al. (2010) who reported the relative safety of pomegranate extracts on MDCK cells, while on the other hand the highest relative toxicity was seen in case of C. sinensis extract which may be attributable to its different chemical structure, especially tannin content.
Indeed, the extract of pomegranate rinds resulted in more toxicity on MDCK cells in comparison to pomegranate arils extract, this may be due to its higher tannins content, in agreement with Tzulker et al. (2007) who found that pomegranate rinds are much abundant in hydrolysable tannins (mainlypunicalagin) content compared to pomegranate arils.
Remarkably, the solvent, DMSO at the used concentration (i.e., 0.2%) showed no toxicity on MDCK cells.
According to Hussein et al. (2000) and Simoni et al. (2007) viral inhibition percentage, (IP)≥97% is regarded as potent antiviral activity; (IP between 90-96%) is regarded as reasonable antiviral activity and (IP less than 90%) is regarded as poor antiviral activity.
The Q. ilex (wood) extract showed a potent antiviral effect (IP≥97%) against H9N2 virus at virus dose of 50 TCID50, (Table 3), which perhaps attributable to this extract content of polyphenolic compounds (mostly ellagitannin), lignans and other organic compounds, which are abundant in oak wood as previously reported (Conde et al., 1997; Fernandes et al., 2009; Michel et al., 2011).
Similarly, C. sinensis extract showed an output equivalent to that of Q. ilex as it showed a potent antiviral effect (i.e., IP≥97%) against H9N2 virus at challenging dose of 50 TCID50, (Table 3), this antiviral effect apparently agrees with previous studies that described the antiviral properties of green tea against influenza A viruses on MDCK cells (Shimamura and Hara, 1991; Nakayama et al., 1993; Song et al., 2005, 2007).
Notably, despite of showing a potent antiviral effect against H9N2 Influenza A virus equivalent to that of C. sinensis extract, the (wood) extract of Q. ilex has an edge over the former as it exhibited much less cytotoxicity on MDCK cells (Table 2).
However, pomegranate arils extract demonstrated a potent antiviral effect too (i.e., IP≥97%) against H9N2 Influenza A virus, agreeing with the recent reports of Haidari et al. (2009) and Sundararajan et al. (2010) who described the antiviral effects of pomegranate extract on MDCK cells against influenza A viruses, but in actual fact, this antiviral effect is unlike that of Q. ilex and C. sinensis extracts against the same virus as it was only shown at lower H9N2 virus dose = 5 TCID50 (Table 3), this could be caused by different antiviral mode of action of each of these plant extracts which may be caused by their different extract constituents.
Unfortunately, in case of pomegranate rinds extract no antiviral effect was observed (Table 3) disagreeing with the report of Jassim and Naji (2003) who reported the antiviral effect of pomegranate rind extract.
On the other hand, the AMN used with the virus infected cells exhibited a very poor antiviral effect (i.e., IP<25%) demonstrating that the H9N2 virus used in this work was reasonably resistant to AMN, this apparently agrees with the report of Weinstock and Zuccotti (2009) who mentioned that influenza viruses have a confirmed capacity to develop resistance to the AMN and other available anti-influenza medications in addition to the recent report of CDC (2009) which declared that 100% of the seasonal H3N2 virus isolates tested were resistant to the Adamantanes including AMN.
CONCLUSION
This preliminary work is the first report on Q. ilex L (wood) extract antiviral activity against H9N2 Influenza A virus which was found to be as effective as green tea (leaves) extract, while being less toxic to MDCK cells, further research is highly recommended to explore the active components of this plant which may be valuable in the development of new antiviral agents effective against influenza viruses.
ACKNOWLEDGMENTS
We are grateful to Prof. Hamdallah Zidan, Dr. Laila Bassiony and Dr. Eman El Maamon Nasr, (VACSERA), Agouza, Cairo (Egypt), for facilities and support they offered to us during this study.