**1. Introduction**

Virus infections are now more clearly than ever a severe hazard to human health on a worldwide scale. SARS-CoV-2 triggered one of the deadliest pandemics in human history, with over 500 million confirmed cases of infection by worldometer (https://www. worldometers.info/coronavirus/) (accessed on 29 June 2022). The SARS-CoV-2 virus causes COVID-19 disease, which has a wide range of symptoms ranging from mild and asymptomatic cases to respiratory infections with fatal consequences. In addition to the deaths of over 6 million people worldwide, this pandemic imposed a new strain on all countries, causing local healthcare systems to collapse. Recent research studies have

**Citation:** Suruˇci´c, R.; Radovi´c Selgrad, J.; Kundakovi´c-Vasovi´c, T.; Lazovi´c, B.; Travar, M.; Suruˇci´c, L.; Škrbi´c, R. In Silico and In Vitro Studies of *Alchemilla viridiflora* Rothm—Polyphenols' Potential for Inhibition of SARS-CoV-2 Internalization. *Molecules* **2022**, *27*, 5174. https://doi.org/10.3390/ molecules27165174

Academic Editor: Vincenzo De Feo

Received: 29 June 2022 Accepted: 10 August 2022 Published: 14 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

provided a detailed explanation of the SARS-CoV-2 virus's entrance into the host cell [1]. This is a complex process since it requires multiple enzymatic structures from the host cell to be activated in stages. Coronavirus' S glycoprotein is a structural component required for interaction with the host receptor. Previous research has shown that entry glycoproteins are typically split into two subunits before being internalized by the host cell. SARS-CoV-2 S glycoprotein is made up of two subunits: S1 is in charge of making contact with ACE2 and S2 attaches virus glycoprotein to the host cell's membrane [2]. Then, this initiates a multi-step process that involves, furin convertase and transmembrane protease serine 2 [3]. However, it became obvious that an alternative method of internalization exists once it was shown that viruses may infiltrate host cells without using the ACE2 receptor. This method for virus internalization has recently been discovered to include neuropilin-1 receptors [4].

The anti-SARS-CoV-2 activity was studied in a number of medicinal plants with a documented history of antiviral use in traditional medicine [5]. One of their shared properties is the abundant presence of compounds with polyphenol chemical moiety. Plant polyphenols are a diverse group of molecules, and their substantial presence in plant tissue is associated with many medicinal plants' health-beneficial properties (antioxidant, antidiabetic, antibacterial, etc.) [6,7]. It has been shown that phenolic compounds can block viral attachment to the human angiotensin-converting enzyme 2 (ACE2) receptor by interacting with the spike (S)-glycoprotein's receptor-binding region [8]. One of the most promising modes of action for natural compounds has been identified as the interaction between S-glycoprotein and the ACE2 receptor [9]. In fact, numerous naturally occurring substances with known antiviral properties, such as hesperidin, punicalin, and punicalagin demonstrated potent anti-SARS-CoV-2 activity in a variety of in vitro and in silico studies. This was attributed to contact interference between the virus and the ACE2 receptor on a host cell [10–13].

The traditional medical usage of many *Alchemilla* species for treating viral infections has been supported by recent studies that demonstrated virucidal activity against influenza and orthopoxviruses [14,15]. Recent investigations have revealed that *Alchemilla viridiflora* Rothm. (Figure 1) polar extract possessed a strong ACE inhibitory effect, with particular components, such as miquelianin, being emphasized for their individual contributions to this activity [16].

**Figure 1.** *Alchemilla viridiflora* Rothm: (**a**) plant at natural habitat; (**b**) magnified flowering parts.

This could be yet another link between *A. viridiflora* constituents and SARS-CoV-2 given that the incidence of COVID-19 disease requiring hospital admission is significantly reduced while taking ACE inhibitors [17]. Even though immunization is the most effective strategy to avoid SARS-CoV-2 infection, there are some circumstances where people are unable to get vaccinated due to medical reasons. Therefore, there is a need to develop alternative strategies to prevent and treat SARS-CoV-2 infection in these individuals. To avoid infection or at least reduce viral load, one strategy is to use natural compounds in appropriate pharmaceutical dosage forms to block early contact between the virus and ACE2 and NRP1 receptors.

To the best of the authors' knowledge, *Alchemilla* isolates have not yet been investigated for their capacity to prevent SARS-CoV-2 infection despite being a rich source of bioactive polyphenols with demonstrated antiviral activity. The overall aim of this study is to clarify *A. viridiflora* methanol extract's real potential for SARS-CoV-2 internalization through two main mechanisms by applying in silico and in vitro studies.
