Pomegranate Peel Extract as an Inhibitor of SARS-CoV-2 Spike Binding to Human ACE2 Receptor (in vitro): A Promising Source of Novel Antiviral Drugs

Abstract

Plant extracts are rich in bioactive compounds, such as polyphenols, sesquiterpenes, and triterpenes, which potentially have antiviral activities. As a consequence of the coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, thousands of scientists have been working tirelessly trying to understand the biology of this new virus and the disease pathophysiology, with the main goal of discovering effective preventive treatments and therapeutic agents. Plant-derived secondary metabolites may play key roles in preventing and counteracting the rapid spread of SARS-CoV-2 infections by inhibiting the activity of several viral proteins, in particular those involved in the virus entry into the host cells and its replication. Using in vitro approaches, we investigated the role of a pomegranate peel extract (PPE) in attenuating the interaction between the SARS-CoV-2 Spike glycoprotein and the human angiotensin-converting enzyme 2 receptor, and on the activity of the virus 3CL protease. Although further studies will be determinant to assess the efficacy of this extract in vivo, our results opened new promising opportunities to employ natural extracts for the development of effective and innovative therapies in the fight against SARS-CoV-2.

Keywords: ACE2; COVID-19; SARS-CoV-2; polyphenols; pomegranate (Punica granatum L.) peel extracts; pomegranate peels.

Figure 1

Spike/ACE2 binding in the presence of PPE, used at three concentrations, compared with control and antibody inhibitor AC384. The results are the averages of three independent experiments, expressed as percentages respect to control arbitrarily set as 100%. The error bars represent SDs and the asterisks indicate statistically significant values (^*p-value is between 0.01 and 0.05; ^**0.001 and 0.01) according to T-test. ACE2, angiotensin-converting enzyme 2; PPE, pomegranate peel extract.

Figure 2

MST. The binding curves were obtained incubating PPE with the RBD Spike, Spike FL protein, and ACE2. ACE2, angiotensin-converting enzyme 2; MST, microscale thermophoresis; PPE, pomegranate peel extract; RBD Spike, Spike receptor-binding domain; Spike FL, Spike full-length.

Figure 3

Infection rate of Spike SARS-CoV-2 pseudo-typed lentivirus in HK-2, determined by GFP fluorescence measure. The results are the averages of six independent experiments, expressed as percentages respect to control arbitrarily set as 100%. The error bars represent SDs and the asterisks indicate statistically significant values (^***p-value is between 0.0001 and 0.001) according to the T-test. GFP, green fluorescent protein; HK-2, human kidney-2 cells (HK-2); SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.

Figure 4

Gene expression analysis in HK-2 cells treated with PPE for 72 h. The results are the averages of three independent RT-PCR experiments. The values are expressed as percentages respect to control arbitrarily set as 100%. The error bars represent standard deviations and the asterisks indicate statistically significant values (^**p-value is between 0.001 and 0.01; ^***0.0001 and 0.001) according to T-test. HK-2, human kidney-2 cells (HK-2); PPE, pomegranate peel extract; RT-PCR, real-time reverse transcription polymerase chain reaction.

Figure 5

5α-Reductase activity in HFDPC stimulated with testosterone 600 nM and treated with either PPE or finasteride 100 nM. The results are the averages of three independent experiments, expressed as percentages respect to testosterone stimulated cells, arbitrarily set as 100%. The error bars represent SDs and the asterisks indicate statistically significant values (^**p-value is between 0.001 and 0.01) according to T-test. HFDPC, human follicle dermal papilla cells; PPE, pomegranate peel extract.

Figure 6

3CL protease activity in the presence of PPE, the main extract compounds (PC, EA, and GA) or GC376 used as positive control. The results are the averages of three independent experiments, expressed as percentages respect to control arbitrarily set as 100%. The error bars represent SDs and the asterisks indicate statistically significantly values (^*p-value is between 0.01 and 0.05; ^**0.001 and 0.01; ^***0.0001 and 0.001) according to T-test. PPE, pomegranate peel extract; PC, punicalagin; EA, ellagic acid; GA, gallic acid.