Antiviral and Cytoprotective Effect of Zinc (Yasad Bhasma) Based Nanoformulations Against Bovine Coronavirus

Abstract

Ayurvedic medicine utilizes metal-based preparations, known as bhasmas, to treat various health conditions. Yasad bhasma (YB), a zinc-based ayurvedic preparation, shows promise as a potential candidate for developing zinc-based nanomedicines with anti-inflammatory and antioxidant properties. In this study, we synthesized a formulation combining YB and hydroxychloroquine (HC) as a zinc ionophore (YBHC) and investigated its biocompatibility and antiviral effects against buffalo calf coronavirus (BCoV) in Vero cells. Our results demonstrated that the formulation exhibited good conformity and enhanced cell proliferation compared to untreated cells. Additionally, no cytopathic effects were observed in BCoV-infected Vero cells treated with YBHC and YB, while infected control cells exhibited cytopathic effects. YB showed cytoprotection by promoting epithelial tissue turnover. We further explored whether YB/YBHC exerted a lysosomotropic effect to produce antiviral effects on coronavirus-adapted Vero cells, but no cell internalization was observed. In addition to the synergistic antiviral effect of YB and HC, YB may play a vital role in rejuvenating affected tissues.

Introduction

The significance of metals in maintaining mammal health is well-established in the scientific literature [1]. Metals play a crucial role in metabolic processes, and their deficiency can lead to various diseases [2]. Ayurvedic medicine employs metal-based preparations, such as bhasmas (submicronic particles or nanoparticles), to address diverse health conditions. Rasashastra, a branch of Ayurveda, focuses on the preparation and therapeutic applications of nanomedicines, particularly metallic and mineral nanoparticles. The process involves processing these metals and minerals with various organic materials through repeated heating. The resulting metal or mineral ashes are claimed to be organically structured nanoparticles and are referred to as Bhasma in Ayurveda [3]. These preparations are considered safe for human use and have been reported in various biological activities [4].

Yasad bhasma (YB), an ayurvedic preparation made from zinc, is considered a potential candidate for drug development and the creation of zinc-based nanomedicines. Zinc, the main component of YB also known as zinc calx or zinc oxide, exhibits anti-inflammatory and antioxidant properties, which are believed to assist in reducing inflammation and lung damage in conditions like asthma, bronchitis, and chronic obstructive pulmonary disease (COPD). Additionally, zinc, an essential mineral for the immune system, has shown antiviral effects against certain viruses, including the common cold virus and the herpes simplex virus [5–7]. Its antiviral properties stem from its impact on the viral replication cycle and its ability to prevent viral binding to host cells [8]. While there is limited scientific research available on the antiviral effects of YB, preliminary studies suggest its potential in this regard. However, it is crucial to note that further research is needed to establish the safety and efficacy of YB for respiratory infections.

Hydroxychloroquine also possesses antiviral activity and acts as a zinc ionophore. Recently, we have demonstrated the antiviral effect of our zinc-based hydroxychloroquine formulation and its lysomotropic effect [9]. However, in the present study, we use YB in combination with hydroxychloroquine to synthesize the formulation. The formulations were characterized for size, morphology, surface charge, and interaction of various chemicals used during synthesis. Cytotoxicity and efficacy against bovine coronavirus isolated from a buffalo calf (BCoV) were also evaluated. Furthermore, we also explored whether YB possesses a lysomotropic effect to produce an antiviral effect on bovine coronavirus-adapted Vero cells either alone or with the use of zinc ionophore hydroxychloroquine.

Materials and Methods

Materials

Yasad bhasam was purchased from Baidyanath, India. Zinc sulfate was procured from Qualigens Fine Chemicals Pvt. Ltd., Mumbai, India. Hydroxychloroquine was purchased from Ipca laboratories (India). The FluoZin-3 AM probe and LysoTracker probe were purchased from Life Technologies Co. (Carlsbad, CA). Other chemicals and reagents mentioned in this study were also of analytical grade and purchased from Sigma-Aldrich Chemicals Private Ltd. (Bangalore). All glassware used in this study was from M/S Borosil (India).

Synthesis of Yasad Bhasam and Hydroxychloroquine Blended Composites

Yasad bhasam (10 ml; 7.5 mg/ml) and 300 µg HC were mixed thoroughly and filtered through a 0.22 µM membrane and stored at 4 °C. We also synthesized dispersible ZnO NPs via a pulse on and off using the microwave-assisted rapid method as described previously using zinc sulfate as a precursor [10].

Characterization: Morphology, Particle Size and Surface Charge Determination (ζ) and, Fourier Transform Infrared Spectroscopy

Dynamic light scattering (DLS) was used to measure the average particle size and size distribution (polydispersity index) of formulated YB, YBHC NPs using the Zetasizer nano ZS (Malvern Instruments, Malvern, UK). The zeta potential of the samples was determined using the laser light scattering technique to predict the stability of colloid dispersions. The measurements were conducted in an aqueous dip cell in an automatic mode by diluting the samples in distilled water and adjusting the cell position. The morphology of the ZnO NPs and YBHC NPs was studied using scanning electron microscopy (SEM) with an EDAX detector. The samples were mounted on metal stubs and recorded at an accelerating voltage of 13 kV.

The constituents used during the synthesis of YBHC were determined using Fourier transform infrared spectroscopy (FTIR) using an IR spectrophotometer (Perkin Elmer Spectrum BX II, Massachusetts, USA).

Cytotoxicity of Nanoformulations

The cytotoxicity of the NPs was evaluated on Vero cell lines using the resazurin dye method [10]. Vero cells were obtained from the National Centre of Veterinary Type Collection Centre, NRCE, Hisar. Around 70% of confluent cells were treated with different concentrations of the ZnO NPs, HC, and YBHC NPs for 24 h at 37 °C under 5% CO₂. After the addition of resazurin dye, the reduction of the blue to pink color indicated viable cells, and the absorbance was measured at 590 nm using an ELISA plate reader. The cytotoxicity was calculated as the percentage reduction in absorbance compared to the untreated control cells.

Biocompatibility in Embryonated Chicken Eggs

The biocompatibility of YBHC was assessed in embryonated chicken eggs for 10–12 days. YBHC or phosphate-buffered saline (PBS; negative control) was inoculated into the eggs, and embryo viability was monitored for up to 5 days.

Viruses

Bovine cornavirus strain, (BCoV) isolated from a diarrheic buffalo calf from Haryana, India, kindly provided by Dr. Minakshi Prasad, LUVAS, Hisar, India, and propagated in Vero cells. Briefly, the cells were infected with BCoV and incubated at 37 °C. After 1 h, the inoculum was removed and replaced with FBS-supplemented DMEM growth media. After 4–6 days, the conditioned medium obtained was collected after centrifugation and filtration to remove cells and debris. Vero cells were then infected with BCoV using this conditioned medium, for one hour at 37 °C. Following washing the cells thrice with PBS, lysates were prepared by rapid freeze–thaw method. Virus stocks were stored at -80 °C.

RT-PCR

RNA was extracted from viral infected, subsequently treated and untreated cells using Trizol (Invitrogen), as per the manufacturer’s instruction. Extracted RNA from each sample using an RNA extraction kit (QiagenGmBH, Hilden, Germany) \was further transcribed with MMLV reverse transcriptase enzyme (Biorad). BCoV M gene was amplified from cDNA by polymerase chain reaction (PCR) with thermal conditions of 5 min at 94 °C and 35 cycles of denaturation at 94 °C for 30 s, annealing 55 °C for 30 s, extension at 72 °C for 30 s and finally 72 °C for 7 min. The amplicons observed on 1.5% agarose gel were purified, and got sequenced.

Effects of Formulations on African Green Monkey Kidney Cells (Vero Cells)

Vero cells were exposed to BCoV in triplicates. After 1 h, the inoculum was removed and replaced with growth media and subsequently treated with different concentrations of ZnONPs, YB, HC, and YBHC NPs.

After 24, 48 h, and 72 h the effect of treatment was observed on the morphology, proliferation, and CPE.

Intracellular Zinc Detection

Lysosomal and intracellular free zinc ions were detected using LysoTracker and FluoZin-3 probes under fluorescent microscopy, following the manufacturer's instructions. Vero cells were seeded in a 12-well plate and treated with various concentrations of ZnO nanoparticles, HC, YB, and YBHC for 30 min. After the treatment period, the culture medium was replaced with a fresh medium containing LysoTracker and/or FluoZin-3, and the cells were further incubated for 30 min. Subsequently, the medium was removed, and the cells were washed before being observed under a Nikon Eclipse TE2000-U microscope. The acquired images were utilized to quantify the presence of lysosomes and zinc ions within the cells using image analysis software.

Statistical Analysis

The results of the experiments were expressed as the mean ± standard deviation (SD) of at least three independent trials. The statistical analysis was conducted using the student's t-test, and the differences between the experimental groups were determined through Graphpad software online. A p-value of less than 0.05 was considered statistically significant [11].

Results and Discussion

Characterization

Size, Morphology and Charge

The particle size of the YB was shown as microparticles whereas the YBHC showed a majority of the particles from 1 to 100 nm ranges (Fig. 1A). YB was mostly observed as microparticles with PDI 0.0158 suggesting better dispersion. ZnO NPs showed negative polarity, whereas the polarity of YB, and YBHC was positive.

Fig. 1.

Characterization of Yasad Bhasam hydroxychloroquine formulation. A Particle size of YB, YBHC as determined by particle size analyzer. B Scanning electron microscope of images YB, YBHC C energy dispersive X-ray spectra (EDS) of ZnO NPs and YB with elemental quantification D Fourier transform infrared spectroscopy (FT-IR) of YBHC

Scanning electron microscopy (SEM) of YB shows individual crystalline structures in aggregated form, whereas porous structure was observed for YBHC in SEM images (Fig. 1B). The energy-dispersive X-ray spectra (EDS) of ZnO NPs and YBHC NPs with elemental quantification are shown in Fig. 1C.

The concentration of Zn was observed as 73.37% and 43.00% in YBHC and ZnO NPs respectively whereas ‘O’ was observed higher in ZnO NPs and then YB. We also observed ‘S’ in ZnO NPs which is due to zinc sulfate used in the synthesis of NPs.

Fourier Transform Infrared Spectroscopy

Fourier Transform Infrared Spectroscopy (FT-IR) is a powerful analytical technique that is commonly used to study the chemical and physical properties of materials, including nanoparticles. FT-IR works by measuring the absorption of infrared radiation by a sample, which can provide information about the chemical bonds and functional groups present in the material.

FT-IR was used to study the interaction of various chemicals used during the synthesis of ZnO NPs and YBHC NPs. The FT-IR spectra of YBHC (a combination of YB and HC) were obtained and compared. The FTIR spectrum of YB is generally pure and very few peaks were observed (Fig. 1D). This suggests that YB is a relatively simple material with few functional groups or chemical bonds that can absorb infrared radiation. It is also mentioned that the very small amount of HC blended in YB did not show marked shifts of peak. This suggests that the interaction between YB and HC was relatively weak and did not result in significant changes to the chemical structure or functional groups of either material.

Cytotoxicity Studies on African Green Monkey Kidney Cells (Vero Cells)

The cytotoxic activities of ZnO NPs, HC, YB, and YBHC NPs were assessed by colorimetric assay using resazurin dye. Our results revealed that ZnO NPs were more toxic than HC followed by YB and YBHC NPs at concentrations more than 1000 µg/ml (Fig. 2). Metallic nanoparticles tend to disperse and release ions when exposed to a biological milieu. The difference in toxic effects between ZnO NPs and other formulations at higher concentrations was found to be statistically significant (10 µg/ml and 5 µg/ml, p < 0.05). However, the toxicity at lower concentrations was statistically non-significant.

Fig. 2.

Cytotoxicity of ZnO NPs, YB, and YBHC at different concentrations on the metabolic activity on vero cells after 24 h incubation. At the end of the incubation period, mitochondrial function was determined by the resazurin assay. The graphs show the cytotoxic percentage for a. ZnO NPs, YB, YBHC treated cells. Data are presented as mean ± standard deviation

Biocompatibility in SPF Chick Embryos

The early stages of chickens’ rapid embryonic growth provide a sensitive model for investigating toxicity. SPF embryonated chicken eggs, in triplicates, were inoculated with YBHC NPs (containing 1.87 mg/ml of YB) per egg in 100 μl quantity through an allantoic route. The eggs were visualized daily up to the 5th-day post-inoculation for mortality of the embryos. None of the embryos showed any sign of toxicity suggesting the biocompatibility of synthesized YBHC NPs formulations (Fig. 3).

Fig. 3.

Biocompatibility in SPF chick embryos of 10–12 days. YBHC or phosphate-buffered saline (PBS; negative control) were inoculated into the eggs, and embryo viability was monitored for up to 5 days

Virus on African Green Monkey Kidney Cells (Vero Cells)

Vero cells were infected with BCoV and incubated at 37 °C. After one hour, the inoculum was replaced with growth media containing fetal bovine serum (FBS) and antibiotics. The conditioned medium was then harvested, centrifuged, and sterile-filtered to remove cells and debris. The cell lysates were prepared using a rapid freeze–thaw method and stored at -80 °C. The experiment was conducted in triplicates.

Effects of Formulations on African Green Monkey Kidney Cells (Vero Cells)

Three PBS washes were performed on Vero cells after they had been infected with BCoV for an hour at 37 °C. Untreated cells and virus-infected cells were preserved as controls. The virus-infected Vero cells were treated with ZnO NPs, HC, YB, and YBHC NPs in two-fold dilutions (2000–62.5 g/ml). Vero cells' response to nanoformulations was studied (Fig. 4). The formulation YBHC showed the highest level of cell growth, followed by YB and ZnO NPs, HC. After 24 and 72 h post-treatment, the cell morphology of the formulation (YBHC, YB) treated cells showed better proliferation than that seen with untreated cells. The HC-treated cells showed some morphological and detrimental effects on the cells. Virus control cells showed cytopathic effects. We did not observe CPE on the cells treated with YB, YBHC, ZnO NPs as well as uninfected ones.

Fig. 4.

Influence of ZnO NPs, HC, YB and YBHC on BCOV adapted vero cells after 24 and 72 h. A Photomicrograhs (400X) showing percent cell confluency of cells treated with ZnO NP, HC, YB, YBHC, Uninfected cells, virus infected and untreated control. B Graphical presentation of the cell proliferation. Mean + SD

Bovine Coronavirus-Infected Vero Cells Exhibited M Protein

We intended to determine whether the Vero cells have adapted to the BCoV after the infection. We extracted the RNA from BCoV-infected cells treated with different formulations and uninfected control, transcribed by reverse transcription and amplified M protein as per the details given earlier in the methodology section. The most prevalent viral M protein controls coronavirus assembly, binds to the nucleocapsid, and gives the viral envelope a particular form [12]. The M–M interaction supports the viral scaffold as a whole. Only virus-infected samples were used as the positive control. The treated samples produced a 519 bp product unique to the specific M protein of coronavirus (Fig. 5). The amplification in the negative control was not visible. The representative sample was sequenced and submitted to Gen Bank NCBI USA (accession number MW592691). This further verified the adaptation of the BCoV. In the treated cells in supernatants, we were unable to uncover any evidence of virus replication, either because there was none or because the quantity of the virus was too meager to detect the virus replication if any. This proved the effectiveness of our formulation.

Fig. 5.

Virus-infected samples yielded 519 bp product specific to M-protein

Zinc Uptake in Vero Cells

Chloroquine is well-known as a lysosomal targeting agent [13–15]. We examined the intracellular distribution of zinc after treating Vero cells with ZnO NPs, HC, YB, and YBHC NPs. Several researchers reviewed applications of Zinc oxide nanoparticles, cellular uptake of zinc ions, and possible toxicity in biological systems [16–18]. As shown in Fig. 6, our ZnO NPs-treated cells showed an accumulation of intracellular zinc ions. However, we did not observe any fluorescence or zinc internalization in HC, YB, or YBHC-treated cells. Among the treatments, YBHC-treated cells exhibited the highest cell proliferation, followed by YB. However, these formulations do not exhibit internalization or a lysomotropic effect, even after using HC as a zinc ionophore. Other factors facilitating cell proliferation and antiviral activity need to be explored in further studies. YB has been shown to exhibit cytoprotective activity by promoting the turnover of epithelial tissues in various organs, inhibiting lipid peroxidation, and preventing DNA fragmentation in target tissues [19]. In addition, it is effective in stabilizing the body's exaggerated immune and inflammatory responses and promoting healthy body tissues that are more receptive to treatment. It is also believed that YB acts through immune stimulation. Studies have demonstrated that Yashada bhasma's immunomodulatory and free radical scavenging activities can prevent systemic damage by reducing oxidative stress [20]. HC also contributes to antiviral activity. In addition to the synergistic antiviral effect of YB and HC, YB can play a vital role in rejuvenating the tissues affected by the virus. The findings from this study provide insights into the biocompatibility of YB and YBHC and, highlight the potential of these nanoparticles as a novel approach to drug discovery. Nevertheless, we have provided a delivery vehicle or an alternative to zinc oxide and chloroquine to combat their adverse or toxic effects.

Fig. 6.

African monkey kidney cells (Vero cells) in a 96-well plate were treated with A ZnO NPs, HC, YB, YBHC (containing 0.94 mg/ml of ZnO NPs/YB in respective formulations). The FluoZin-3 at indicated concentration was added and the fluorescent signals were measured by excitation and emission at 490/20 nm and 555/28 nm for Fluozin respectively and observed under fluorescent microscope. B Shows the same field observed under normal microscope (200px)

In summary, we synthesized a formulation using YB and HC and investigated its biocompatibility and antiviral effects. The findings demonstrated that there were no cytopathic effects in YBHC-treated BCoV infected-Vero cells but virus-control cells demonstrated CPE. Even the formulation had superior cell proliferation and conformity than untreated cells. The study investigated whether YB/YBHC has a lysosomotropic effect in bCoV-adapted Vero cells to induce antiviral effects. YB/YBHC-treated cells did not show any cell internalization. In comparison to the other treatments, the YBHC-treated cells showed the highest levels of cell growth. By encouraging the replacement of epithelial tissues in numerous organs and preventing lipid peroxidation, YB showed cytoprotective effects. YBHC played a significant role in regenerating the damaged tissues. The findings point to the possibility of the YBHC formulation as an alternate therapy against BCoV.

Data Availability

The datasets of M gene generated during the current study is available in the Gen Bank NCBI USA with accession number MW592691.