In Vitro Antiviral Activity of Cinnamomum cassia and Its Nanoparticles Against H7N3 Influenza A Virus

Abstract

Nanoparticles have wide-scale applications in various areas, including medicine, chemistry, electronics, and energy generation. Several physical, biological, and chemical methods have been used for synthesis of silver nanoparticles. Green synthesis of silver nanoparticles using plants provide advantages over other methods as it is easy, efficient, and eco-friendly. Nanoparticles have been extensively studied as potential antimicrobials to target pathogenic and multidrug-resistant microorganisms. Their applications recently extended to development of antivirals to inhibit viral infections. In this study, we synthesized silver nanoparticles using Cinnamomum cassia (Cinnamon) and evaluated their activity against highly pathogenic avian influenza virus subtype H7N3. The synthesized nanoparticles were characterized using UVVis absorption spectroscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy. Cinnamon bark extract and its nanoparticles were tested against H7N3 influenza A virus in Vero cells and the viability of cells was determined by tetrazolium dye (MTT) assay. The silver nanoparticles derived from Cinnamon extract enhanced the antiviral activity and were found to be effective in both treatments, when incubated with the virus prior to infection and introduced to cells after infection. In order to establish the safety profile, Cinnamon and its corresponding nanoparticles were tested for their cytotoxic effects in Vero cells. The tested concentrations of extract and nanoparticles (up to 500 μg/ml) were found non-toxic to Vero cells. The biosynthesized nanoparticles may, hence, be a promising approach to provide treatment against influenza virus infections.

Introduction

New applications of nanomaterials and nanoparticles are emerging rapidly [13]. Nanoparticles can be prepared by a variety of methods such as chemical reduction [11], bioreduction [50], electrochemical reduction [22,24], photochemical reduction [45], and heat evaporation [55,58]. Biological methods for the synthesis of nanoparticles using enzymes [18], microorganism [9,10], and plants or plant extracts [1,47] can be advantageous over chemical and physical methods as they are eco-friendly and cost effective, and can be scaled up easily for large-scale production. Nanoparticles are being explored extensively in the field of medicine. As the size of nanoparticles is similar to that of biological molecules, this makes them a promising candidate for application in both in vivo and in vitro research [31]. Metallic nanoparticles have been studied as potential antimicrobials to target highly pathogenic and multidrug-resistant microorganisms. Their applications are being extended further to the development of antivirals to inhibit numerous viruses [16,33].

Influenza viruses are enveloped, negative-sense RNA viruses belonging to family Orthomyxoviridae. These viruses are prevalent in nature and can infect all species of birds, many mammalian species such as horses, pigs, and seals, and humans [60]. Confirmed cases of human infections caused by various subtypes of avian influenza viruses such as H5N1, H7N7, and H9N2 have been reported [32,53]. These viruses pose a high risk to human and animal health. Avian influenza viruses usually contain hemagglutinin (HA) having Gln226 and Gly228 residues, which form a narrow receptor binding pocket that prefers 2,3-sialic acid binding. Human species generally contain Leu226 and Ser228, forming a broad pocket that favors 2,6-sialic acid binding [15,41]. Avian influenza viruses need a switch in preferential binding of the HA protein from 2,3-sialic acid to 2,6-sialic acid, to induce a pandemic.

Antiviral drugs offer the primary line of defense for an influenza virus pandemic, where vaccines might be not accessible in time [40]. Currently approved anti-influenza drugs are inhibitors of viral M2 ion channel (amantadine and rimantadine) and viral neuraminidase (oseltamivir or zanamivir) [61]. These drugs are often limited owing to their toxicity or the appearance of mutant forms of the virus resistant to drugs [28]. Given the limited capability of currently available anti-influenza drugs, there is a need to develop new drugs that would exploit alternate modes of action and offer broad-spectrum cross-strain therapeutic cover.

Extensive work has been carried out to develop newer drugs from both natural and synthetic sources. However, the drugs from natural origin are considered to be safer because of their minimal adverse drug reactions. Hundreds of thousands of plant species have been studied for their medicinal properties [26,42]. Many plants have been shown to possess anti-influenza activity. These include Allium fistulosum [34], Pinus thunbergia [20], Ephedrae herba [49], Sambucus nigra [14], Alpinia katsumadai [29], and Psidium guajava [2].

Plant-based silver nanoparticles (AgNPs) are a likely source of new antiviral agents because of their multi-targeting mechanism of action. Plants are readily available, have low cost, are easy to handle and nontoxic, and have a variety of metabolites that can assist reduction of silver ions [59]. Several groups reported the synthesis of Au, Ag, and Pd nanoparticles using plant extracts such as Geranium leaf [44], Aloe vera [46], lemongrass [8], tamarind leaf [13], and others [4,19]. Metal nanoparticles have been studied for their antiviral potential and have proven to be antiviral agents against hepatitis B virus [27], respiratory syncytial virus [35], human immunodeficiency virus type 1 (HIV-1) [62], herpes simplex virus type 1 [25,30], Tacaribe virus [63], monkeypox virus [54], and influenza virus [39,43].

Emerging and re-emerging influenza infections demand additional antiviral strategies against influenza. Cinnamon has a long history both as a medicine and spice [3]. Little is known about the anti-influenza activity of Cinnamon and synthesis of AgNPs from Cinnamon have not been reported earlier. In the present study, green synthesis of AgNPs using aqueous extract of Cinnamon bark is reported. Furthermore, the activity of Cinnamon bark and its corresponding nanoparticles was evaluated against highly pathogenic avian influenza virus subtype H7N3 in Vero cells.

 

Materials and Methods

Viral Propagation in Eggs

The field isolates of avian influenza virus H7N3 were obtained from the Disease Diagnostic Section, Poultry Research Institute, Rawalpindi, Pakistan. The virus was propagated in 9-day-old embryonated chicken eggs. After 24 h of inoculation, the allantoic fluid was harvested and a hemagglutination test was performed to quantify the virus. The virus was then used for the infectivity assay.

Vero cells were obtained from the Microbiology Laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and incubated at 37℃ under 5% CO2. Cells were monitored daily for confluency and passaged into a new tissue culture flask when they reached >90% confluency. They were used for antiviral assay on the 14th passage.

Preparation of Plant Extract and Nanoparticles

Commercially available, highest grade Cinnamon bark was obtained from Fateh Food International, Pakistan. Cinnamon bark was grinded and passed through a sieve of 50/80 mesh to obtain fine powder. Then, 25 g of powder was weighed and added into 100 ml of deionized water. The Cinnamon aqueous extract was filtered with Whatman No. 1 filter paper (pore size 25 μm) and 10 ml of this extract was added into 90 ml of 1 mM silver nitrate (AgNO3) solution and kept at room temperature for 5 h for reduction of silver (Ag+) ions. AgNPs synthesis in the reaction mixture containing AgNO3 and Cinnamon extract was observed by color change.

Physicochemical Characterization of Nanoparticles

A small aliquot of the prepared silver nanoparticles was diluted with deionized water and subjected to UV-Vis spectral analysis on a spectrophotometer (Model UVD-2950; USA) in the wavelength range of 250-550 nm. The absorbance of Cinnamon aqueous extract and corresponding NPs was measured and plotted against wavelength. The AgNP suspension was centrifuged at 16,350 ×g for 15 min. The AgNP pellet was washed with deionized water three times to remove impurities and dried.

The shape and size of the nanoparticles were determined by scanning electron microscopy (SEM; JSM-6490; USA). A drop of NP suspension was placed on a specimen chamber and coated with an ultra-thin layer of gold by a sputter coater and mounted on a specimen stub in the scanning electron microscope. Images were captured at 50,000× magnification. Then, Fourier transform infrared spectroscopy (FTIR) measurements were performed to obtain information about the possible chemical groups responsible for the reduction of ions and stability of AgNPs. Cinnamon aqueous extract and Cinnamon-based AgNPs were heated with potassium bromide (KBr) in 100:1 ratio at 110℃ for 10-15 min. KBr pellets were made by a hydraulic press and placed in a sample chamber of a FTIR spectrometer (Perkin Elmer Spectrum, USA). Measurements were carried out by scanning in the spectral range of 500-4,000 cm-1 with a resolution of 1 cm-1.

Determination of 50% Tissue Culture Infectious Dose

Vero cells were seeded into a 96-well plate (100 μl/well) at a concentration of 5 × 104 cells/ml and incubated for 24 h at 37ºC under 5% CO2. H7N3 influenza virus was diluted serially in 10-fold from 10-1 to 10-9 and each dilution was titrated into a 96-well plate with six wells per dilution. A few wells were left as negative control having no virus. Plates were incubated at 37℃ under 5% CO2 and the cytopathic effect in each well was observed for 7 days. The 50% tissue culture infectious dose (TCID50) was calculated by the Reed and Muench method [5].

Cytotoxicity Assay

In order to perform the cytotoxicity analysis, 100 μl of cells was seeded in each well of 96-well plates at a density of 5 × 104 cells/ml. After 24 h of incubation, these cells were treated with 100 μl of various concentrations of Cinnamon bark extract and its silver nanoparticles (500, 250, 125, 62.5, 31.25, and 15.62 μg/ml). A 100 μl volume of 2% FBS-DMEM was taken as the negative control. Cells were incubated at 37℃ under a 5% CO2 environment for an additional 48 h. Then, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) was prepared at a concentration of 5 mg/ml and 100 μl was added to each well. Plates were incubated at 37℃ under 5% CO2 for 4 h. Next, the reagent was removed and 100 μl of DMSO was added to each well in order to dissolve purple-color formazan crystals. Then, the optical density was measured at 540 nm using a microplate reader (ELx 800; BioTek). The cell viability was determined from the optical density values using the formula

The 50% cytotoxic concentration (CC50) values of the Cinnamon bark extract and corresponding silver nanoparticles were calculated by regression analysis. CC50 is defined as the concentration required to reduce cell viability by 50%.

Infectivity Assay

Vero cells were treated with various concentrations of Cinnamon bark extract (0, 31.25, 62.5, 125, 250, and 500 μg/ml) and corresponding nanoparticles (0, 1, 10, 50, 100, and 200 μg/ml) during and after influenza virus infection. For pre-penetration exposure, 104 TCID50 virus suspension was incubated at 37℃ with Cinnamon bark extract and its nanoparticles, and 100 μl of the mixture was then added to the Vero cells cultured in the 96-well microtiter plate. Cells were incubated for 2 h at 37℃ under 5% CO2. Following incubation, the supernatant was removed and cells were washed with PBS. Thereafter, 100 μl of medium was added to each well. For post-penetration exposure, 104 TCID50 virus suspension was added to the Vero cell culture and incubated for 2 h at 37℃ under 5% CO2. Following incubation, cells were washed and 100 μl of Cinnamon bark extract and its nanoparticles was added to each well. Along with treated wells, virus-infected control and mock-infected control were also maintained. All plates were incubated for 48 h at 37℃. Then the MTT assay was carried out. The viability of infected and uninfected cells was determined from optical density values, and the percentage of protection was calculated using the following formula:

Statistical Analysis

All experiments of cytotoxicity and virus infectivity were conducted in triplicate and three independent experiments were carried out. Data are presented as the mean ± Standard deviation (SD). The data were evaluated statistically using one-way analysis of variance (ANOVA) to compare the difference in the means of treated and untreated samples. A p-value of <0.05 was considered to be significant. GraphPad Prism program (ver. 5.03) was used for determination of the IC50 and CC50 values.

 

Results

Synthesis of Cinnamon-Based AgNPs

Silver nanoparticles were synthesized using an aqueous extract of Cinnamon bark. When the extract was mixed with AgNO3 solution and incubated at room temperature, change in color was observed within 5-6 h. The color of AgNPs synthesized using Cinnamon bark changed to dark brown. The change in color indicates the reduction of silver ions into silver particles.

Characterization of Cinnamon-Based AgNPs

The synthesized silver nanoparticles were characterized by UV-Vis spectrophotometric analysis. In our study, absorption spectra of silver NPs formed from Cinnamon showed a single broad peak at 410 nm (Fig. 1). To get more information about the size and shape of the particles, the colloidal suspensions of AgNPs were diluted and analyzed using a scanning electron microscope. SEM analysis showed that the NPs were spherical in shape. The average size of Cinnamon-reduced NPs was approx. 42nm, with size ranged from 25 to 55 nm (Fig. 2).

Fig. 1.UV-Vis spectral analysis of silver nanoparticles synthesized from bark extract of Cinnamon.

Fig. 2.SEM image of silver nanoparticles synthesized by bark extract of Cinnamon, at 50,000× magnification.

FTIR analysis was done to get information about the transformation of functional groups due to the reduction process. FTIR absorption spectra in the region of 500-4,000 cm-1 of Cinnamon bark extract before and after reduction of silver ions are shown in Fig. 3. Absorbance peaks of Cinnamon bark extract were observed at 688, 1634, 2,077, and 3,445 cm-1. The Cinnamon-based silver NPs showed absorption peaks at 674, 1,637, and 3,433 cm-1. Comparison between FTIR spectra of Cinnamon and Cinnamon-based AgNPs revealed minor changes in magnitude and positions of the absorption peaks. The wavenumbers varied typically about ±1–15 cm-1.

Fig. 3.FTIR spectrum analysis of Cinnamon bark extract before and after synthesis of silver nanoparticles.

Cytotoxic Effect of Cinnamon and Cinnamon-Based AgNPs

Safety is one of the major prerequisites for any potential antiviral agent. Thus, the toxicity of the various concentrations of Cinnamon bark extract and Cinnamon bark-derived silver nanoparticles was tested in Vero cells in order to determine their safety profile. Vero cells were treated with various concentrations of Cinnamon bark extract and its nanoparticles (500, 250, 125, 62.5, 31.25, and 15.6 μg/ml). After 24 h incubation, cells were observed under a microscope. No cytopathic effects were found, showing that none of the tested concentrations of the Cinnamon extract and its nanoparticles were toxic to cells. Furthermore, by means of MTT colorimetric assay, the viability of cells was also determined. In viable cells, the mitochondrial dehydrogenase reduces MTT to formazan. This conversion can be measured spectrophotometrically. It was confirmed by MTT assay that there was no significant difference in the viability of treated cells (Cinnamon bark extract and its AgNPs) and untreated control (Fig. 4). The CC50 of Cinnamon bark extract was 14.7 mg/ml, and the cytotoxicity of Cinnamon-derived silver nanoparticles was increased with CC50 of 4.9 mg/ml against Vero cells. CC50 values were greater than the highes concentration tested. In this case, CC50 was the theoretical value obtained by graphical extrapolation of the results.

Fig. 4.Cytotoxicity effect of Cinnamon bark extract and its nanoparticles in Vero cells.

Antiviral Activity of Cinnamon and Cinnamon-Based AgNPs

In order to determine the protective effects of Cinnamon bark extract and its AgNPs, Vero cells were treated with five different non-cytotoxic concentrations of Cinnamon (31.25, 62.5, 125, 250, and 500 μg/ml) and its nanoparticles (1, 10, 50, 100, and 200 μg/ml). Influenza virus was incubated with Cinnamon and the corresponding NPs before infection to cells (pre-penetration exposure) and cells were treated with cinnamon extracts and its nanoparticles after infection with the virus (post-penetration exposure).

Antiviral activity of cinnamon bark extract showed that it was effective against influenza virus. All the tested concentrations of the extracts (31.25, 62.5, 125, 250, and 500 μg/ml) showed significant antiviral activity in comparison with the control, as shown in Figs. 5A and 5B. Comparison of the Cinnamon bark effectiveness against virus in pre-treatment and post-treatment exposures showed Cinnamon bark extract was more effective against viral infection when the virus was pre-treated with the extract before their introduction to cells (IC50 242 μg/ml) as compared with that of treatment of cells with extract after virus infection (IC50 316 μg/ml). Furthermore, at the high concentrations of 250 and 500 μg/ml, the infection inhibition in pre-penetration and post-penetration exposures showed no noticeable difference of antiviral activity of Cinnamon bark extract, whereas at lower concentrations (31.250, 31.25, 62.5, and 125 μg/ml) extracts showed more efficient inhibition of virus infection when it was preincubated with virus prior to infection. The Cinnamon-derived NPs increased the antiviral activity of the Cinnamon, as evident in Figs. 6A and 6B. The concentration of NPs at which infectivity was inhibited by 50% (IC50) was 101 μg/ml and 125 μg/ml for treatment of cells during and after viral infection, respectively. The treatment of cells with Cinnamon NPs after viral infection did not show any significant inhibition (p values > 0.05) at lower concentrations (1 and 10 μg/ml). When virus was incubated with same concentrations of NPs prior to inoculating the cells, significant inhibition of the virus was observed. Higher concentrations of Cinnamon NPs (50, 100, and 200 μg/ml) showed statistically significant antiviral activity in both pre-penetration and post-penetration exposures. However, this effectiveness was increased when the virus was pretreated with the NPs prior to infection.

Fig. 5.Effect of Cinnamon bark extract on inhibition of H7N3 infection in Vero cells.

Fig. 6.Effect of Cinnamon bark-based silver nanoparticles on inhibition of H7N3 infection in Vero cells.

 

Discussion

Avian influenza virus is a respiratory pathogen distributed throughout the world. It possesses the ability to switch to a new host and to escape antiviral measures [37]. Therefore, there is an urgent need to develop new antiviral agents for the treatment and control of influenza [56]. Medicinal plants have a variety of natural compounds having antiviral activity. Plant-based AgNPs can further improve the therapeutic applicability of plants and are a likely source of new antiviral agents [16]. These are safer and, because of their multivalent functions, less probable to encounter resistant viruses. Here, we report green synthesis of silver nanoparticles using Cinnamon bark and their application in inhibition of H7N3 infection.

Many researchers have explained the efficient method of green synthesis of silver NPs using various plant extracts. Our results are in agreement with Geethalakshmi and Sarada [7], who reported the formation of AgNPs within 5 h of incubation. Another study stated the reduction of silver ions to NPs within 8 min using Ocimum sanctum leaf extract [17]. The differences in the rate of bioreduction may be due to variability in the plants used for synthesis of nanoparticles. The reduction was ascribed to the polysaccharides, phenolics, terpenoids, and flavone compounds present in the plant extract [19]. The procedure of bioreduction is not fully known and needs to be explored.

The optical absorption spectrum of metal NPs is highly influenced by the size and shape of nanoparticles [64]. Metal NPs ranging 2-100 nm show strong and broad peaks [4]. In our study, the absorption spectrum of silver NPs formed from Cinnamon showed a single broad peak at 410 nm. The number of peaks in the absorption spectra is a blueprint for the shape of the NPs. It has been shown that spherical nanoparticles generate a single peak. Broadening of the peak indicates that the particles are polydispersed [65]. Further SEM analysis confirmed that NPs were spherical in shape, and the average size of Cinnamon-reduced NPs was 42 nm with size ranged from 25 to 55 nm. Several reports have shown that the silver nanoparticles are generally spherical in shape and variable in size. Silver nanoparticles produced by Morinda pubescens and Leptadenia reticulate were 15-20 nm and 50-70 nm, respectively [6,57]. The size and shape of the silver nanoparticles can have an impact on their application [12].

FTIR analysis was carried out to identify the possible potential biomolecules in the Cinnamon bark extract responsible for the reduction of silver ions to silver nanoparticles. The FTIR spectra of Cinnamon-based AgNPs revealed few changes in the position and magnitude of the bands. The band at 688 cm-1 was shifted to a lower wave number at 674 cm-1, suggesting alkyl halides involvement in AgNP formation. After reduction of AgNO3, the band at 1,634 cm-1 characteristic of C=C- or aromatic groups vibrations showed increased transmittance and decreased absorbance, indicating these groups might be involved in stabilization and formation of the silver NPs. The band at 2,077 cm-1 related to C=O stretching vibrations disappeared, which showed that the carbonyl functional group of aldehydes, ketones, and carboxylic acids might be involved in reduction of silver ions. The increased transmittance of the band at 3,445 cm-1 corresponding to the NH group of amides or OH group of alcohol or phenol showed that these might be responsible for the formation of AgNPs. Previously, it has been reported that formation of AgNPs increased the transmittance of bands corresponding to NH and OH stretching [21,38].

The tested concentrations of Cinnamon bark and its NPs did not show cytotoxic effect in Vero cells. There was no difference in cell number and morphological characteristics between the treated and untreated cells. The MTT assay was performed to determine the cell viability, as the assay has a high sensitivity and rapid response as compared with lactate dehydrogenase and trypan blue exclusion assays [52]. The anti-influenza effects of Cinnamon and its NPs were evaluated in Vero cells. Although both Cinnamon and its corresponding silver nanoparticles inhibited H7N3 influenza virus infection in Vero cells, results showed the Cinnamon-based AgNPs are more effective against the virus.

Cinnamon has been used as medicine around the world because of its health benefits. The major constituents of Cinnamon are cinnamaldehyde, trans-cinnamaldehyde, cinnamic acid, essential oils, and eugenol. Study reported by Hayashi et al. [51] showed that trans-cinnamaldehyde of cinnamon could inhibit influenza A/PR/8 virus propagation in vitro and in vivo. In our study, incubation of virus with Cinnamon bark extract before influenza infection to cells reduced infectivity by up to 45% at a concentration of 500 μg/ml. It is therefore speculated that interaction of Cinnamon components with the virus possibly blocked HA function and resulted in inhibition of viral entry into the cell. In addition, treatment of cells with Cinnamon after viral entry also showed reduction of infection by up to 45% at a concentration of 500 μg/ml. It might be due to interactions of Cinnamon components with certain factors or pathways essential for viral replication.

We synthesized silver nanoparticles using Cinnamon bark for reduction of AgNO3. According to our results, biosynthesis of silver nanoparticles using Cinnamon enhanced the antiviral activity in both pre-penetration and post-penetration exposures. AgNPs showed more significant inhibition of viral infection when incubated with virus prior to infection, where the infection level was reduced by up to 24% at a concentration of 200 μg/ml. It has been reported that silver or gold nanoparticles exhibit antiviral activity against numerous viruses such as herpes simplex virus [25], hepatitis B [27], and H1N1 influenza A virus [48,36]. Although the antiviral mechanism of action has not been determined, the antiviral activity of silver NPs against several types of viruses is likely due to binding of NPs to viral envelope glycoproteins, thereby hampering viral penetration into the host cell [27,62]. It has been shown that silver nanoparticles inhibit HIV adsorption to the host cells [23]. In our study, treatment of cells with Cinnamonbased AgNPs after viral entry reduced infection by up to 29% at a concentration of 200 μg/ml. It revealed that besides the interaction with viral glycoproteins directly, NPs may get access into the cell and show their antiviral activity through interactions with the viral genome (RNA or DNA), cellular factors, or pathways of host cells that are essential for viral replication [48].

In the present study, silver nanoparticles were successfully developed by a green synthetic approach using bark extract of Cinnamon. Cinnamon-reduced silver nanoparticles exhibited an enhancement of antiviral activity against H7N3 influenza virus as compared with Cinnamon bark aqueous extract, in both pre-penetration and post-penetration exposures. Cinnamon and its corresponding nanoparticles were found nontoxic against Vero cells. The safe and multi-target benefits of plant-based silver nanoparticles give hope where there are no treatments for highly mutating viruses. The mechanism of action of nanoparticles needs to be further investigated to develop better antiviral therapeutics. In vivo studies to show the effectiveness of Cinnamon-based nanoparticles against influenza are under way.