The current vaccines approved for use have been shown to reduce disease severity and lower fatality rates. However, novel therapeutics are still required for treatment and management of disease. This is important as possible breakthrough infections can occur in previously infected and vaccinated individuals. In addition, there is a possibility that variants may emerge that evade the immune response. Traditional medicinal plants have been used previously to treat various respiratory infections, as they are believed to have promising antiviral capacities11 15 16. Different countries have tested potential indigenous plants for immune-modulating capabilities against SARS-CoV-27 8 9 and compounds in plant extracts with the potential to have antiviral activity against SARS-CoV-2 have been identified using in silico molecular docking17. Traditional medicinal plants that have previously been shown to be efficient against viral infection are one approach for identifying cost-effective treatments during outbreaks/pandemics when no effective treatments are available.
This study evaluated the activity of PHELA and its components against SARS-CoV-2 using mammalian cell lines overexpressing the ACE-2 receptor. PHELA is under development as an immune booster in South Africa, with recent studies showing its effectiveness in the treatment of gram-positive Staphylococcus aureus18 and possible use for the treatment of Alzheimer's disease19. An in vitro investigation of PHELA showed over 90% inhibition of SARS-CoV-2 and SARS-CoV infections at 0.005 mg/ml to 0.03 mg/ml20. Additionally, PHELA demonstrated close to 100% inhibition of MERS-CoV infection at concentrations ranging from 0.1 mg/ml to 0.6 mg/ml. This study aimed to assess the effect on viral load pre-and post-treatment with plant extracts and cytokine release associated with cytokine storms after treatment with the plant extracts. Based on Ct values for viral loads, treatment of cells with plant extracts did not appear to inhibit viral replication. There was no statistical significance in Ct values between cells treated with plant extracts and the virus-infected control cells, suggesting that the plant extracts may not have the ability to inhibit SARS-CoV-2 replication. However, the concentration of the plant extracts used may have also been too low to impact virus replication. Although not significant, post-treatment with PHELA resulted in higher Ct values compared to virus-infected control cells and cells treated with other plant extracts. Post-treatment of cells with PHELA resulted in Ct values of 31.49, 34.22, and 32.42 for the E, RdRp, and N genes, respectively (Supplementary Tables). Ct values of cells treated with the components of PHELA pre- and post-treatment resulted in Ct values between 12 and 19, and virus-infected control cells had Ct values between 16 and 19. These results may suggest that post-treatment with PHELA caused a delay in viral RNA replication, suggesting that post-treatment of cells with PHELA may have potentially interfered with viral replication.
There were clear differences in levels of cytokines due to treatment with plant extracts. Multiple studies have reported on the dysregulated production of inflammatory cytokines in patients with severe COVID-19 disease21 22 23. Release of these cytokines has been shown to be an essential indicator of the severity of disease for SARS-CoV-2, associated mainly with organ failure rather than viral load. Previous studies have also associated the release of IL-6, IL-1β, and TNF-α with the severity of the disease24 25. In this study, key inflammatory cytokines, including IL-1β, IL-2Rα, IL-6, TNF-α, and IFN-γ, that have been associated with cytokine storm were included in our analysis. Some cytokines, including TNF-α, IL-1β, and IL-2Rα, showed significantly higher release in virus-infected cells than those treated with plant extracts. The pretreatment of cells with plant extracts significantly reduced TNF-α at 48 hours post-infection in cells pretreated with CG, RM, and PHELA, but not SO before infection. Moreover, at 72 hours post-infection, CG and RM demonstrated a significant reduction in TNF-α release, while SO and PHELA did not show the same effect. Pretreatment significantly reduced IL-1β in cells treated with CG, RM, SO, and PHELA at 48 hours post-infection. However, a significant reduction was seen only with GC, RM, and SO at 72 hours post-infection, with PHELA being significantly higher than the control of virus-only infected cells. Post-treatment of cells with immune modulators resulted in a significant reduction of IL-1β, IL-2Rα, and TNF-α at 48 and 72 hours post-infection of PHELA and all its components, suggesting a potential role of these plant extracts in modulating the immune response by reducing TNF-α release during SARS-CoV-2 infection. IFN-γ was not detected in cells treated with PHELA, all its components, and the uninfected cells. In contrast, high levels of IFN-γ were detected in h1299-hACE2-E3 virus-infected untreated cells at 48 and 72 hours post-infection. Results also show a better response post-treatment than pretreatment, suggesting a role in managing cytokine storms associated with severe symptoms of COVID-19 disease. The results show the complexity of innate immune response to viral infections and the potential of PHELA and components of PHELA in modulating innate immune responses. Understanding the underlying mechanisms through which these compounds interact with the immune system could provide valuable insights into possible treatments for SARS-CoV-2 and related viruses to manage the dysregulated immune responses in severe cases. Previous studies have associated IL-6 with severity of disease26, with high viral loads being associated with increased levels of IL-6. However, in this study, IL-6 was not detectable in cells treated with plant extracts or the controls, even though infected cells demonstrated high viral loads27. This could possibly be due to the ELISA methods not being sensitive enough to detect low levels of these cytokines. Another reason for the IL-6 being undetectable could be the early sampling post-infection, the timing of sample collection in relation to the progression of the infection may influence the detectability of the proinflammatory cytokine. Majority of individuals in which high levels of IL-6 is detected are usually in the severe stage of the disease with disease severity usually occurring five days post-infection, in our case, sampling was only performed for up to 72 hours in infected cells.
In summary, PHELA and three out of its four component plants demonstrated a potential to reduce the upregulation of proinflammatory cytokines associated with severe SARS-CoV-2 disease. However, treating cells with plant extracts did not result in reduced viral loads post-infection with SARS-CoV-2 Omicron virus, suggesting that they may have a limited role in reducing viral load. Whether the observed effects could be relevant for use in humans needs to be further investigated. These results warrant further studies on the effect of traditional medicinal plants on modulating cytokine release during viral infections. The limitation of the study is that the small design and sample number warrant repeating the experiments with more samples and additional parameters and using gene expression studies or microarrays to further characterize the effect on the release of cytokines. Additionally, the results obtained in in vitro studies are not readily transferrable to a clinical setting to test in humans. However, they do provide the potential these extracts may have against SARS-CoV-2. Cytokine storms are not unique to SARS-CoV-2 and can occur in response to various viral infections and other inflammatory conditions. If PHELA or its components prove effective in modulating cytokine responses, they could have broader applications in treating diseases characterized by excessive immune activation. These findings provide valuable insights into understanding the immunological aspects of viruses and pave the way for further exploration of plant-based therapies for managing COVID-19 and related inflammatory responses.