*2.3. In Vitro SARS-CoV-2 Internalization Inhibition Assays*

The binding inhibitory effects of an extract of *A. viridiflora* on S glycoprotein-ACE2 and S glycoprotein-NRP were explored as a mechanism of their anti-SARS-CoV-2 potential in this work. Umifenovir and quercetin served as positive controls for the S glycoprotein-ACE2 inhibition assay, and brevifolin carboxylic acid served as a positive control for the S glycoprotein-NRP inhibition assay, both at the same concentrations as the samples. The extract was tested at concentrations ranging from 0.0625 to 1.00 mg/mL. The results indicated that the tested *A. viridiflora* extract was able to inhibit S-glycoprotein interactions with both receptor targets in a dose-dependent manner. The inhibition values for S-glycoprotein binding to NRP1 and ACE2 were 56.3% and 87.1%, respectively, at the highest tested concentration of *A. viridiflora* methanol extract (1.00 mg/mL). Positive controls umifenovir and quercetin inhibited S-glycoprotein and ACE2 contact at the highest tested concentrations (1 mg/mL), with inhibition values of 5.22% and 2.10%, respectively, whereas brevifolin carboxylic acid at the same concentration inhibited contact between S-glycoprotein and NRP1 by 63.07%. These in vitro results are consistent with the docking study simulation, which indicated that umifenovir had a higher inhibtion potential than quercetin, another positive control and that *A. viridiflora* methanol extract could have an even more potent antiviral effect considering 12 constituents with a higher affinity for the same target than umifenovir. Brevifolin carboxylic acid suppressed S-glycoprotein and NRP1 interactions more effectively than *A. viridiflora* extract, whose constituent it is. Possible cause could be its lower concentration in extract relative to less potent polyphenols. Using the OriginPro v. 9.8.0.200 program (OriginLab Corp.), the doses that resulted in a 50% inhibition of binding interactions between S-glycoprotein and receptors for internalization were determined to be 0.18 and 0.22 mg/mL for NRP1 and ACE2, respectively (Figure 6).

**Figure 6.** Concentration-inhibition curves of *A. viridiflora* methanol extract on S-glycoprotein-ACE2 (black line) and S-glycoprotein-NRP1(red line) contacts.

Results obtained in vitro are consistent with recently published research that investigated the potential of pomegranate ellagitannin-rich extracts to inhibit the interaction between S-glycoprotein and ACE2 [11,13]. Ellagitannin polyphenols from pomegranate peel extract synergistically inhibited contact between virus S-glycoprotein and ACE2 receptor. Tellimagrandin I and brevifolin carboxylic acid, two of the ellagitannins found in *A. viridiflora*, are frequently present in different pomegranate extracts. The significant potential of *A. viridiflora* extract for inhibiting SARS-CoV-2 internalization via ACE2 receptors may be explained by these complementary ellagitannins and other compounds from the same class [16,30]. Additionally, it was discovered that urolithin A, a common ellagitannin metabolite in humans, is a potent inhibitor of SARS-CoV-2 binding to the ACE2 receptor [13]. Liu et al. (2020) in their in vitro study demonstrated that quercetin, *A. viridiflora* major flavonoid representative has potency for the recombinant human ACE2 receptor inhibition, at physiologically relevant dosages [31]. This is a further strong indication that two classes of polyphenols have synergistic inhibitory effects on the internalization of SARS-CoV-2 through the ACE2 host receptor. However, the study's findings donot support a firm conclusion in this way and instead can serve as the foundation for some additional investigation. In order to determine whether NRP1 was a host factor for SARS-CoV-2, Daly et al. (2020) employed the small ligand EG00229. This ligand was a confirmed NRP1 antagonist and it was shown to be bound to NRP1 with a Kd of 5.1 and 11.0 μM at pH 7.5 and 5.5, respectively [32]. It was also previously determined that EG00229 in 3 μM concentration selectively inhibits 50% of vascular growth endothelial factor A binding to a purified NRP1 b1 domain [33]. However, despite doing a thorough literature search, the authors were unable to find any additional studies that examined a natural small ligand as an NRP1 antagonist.
