3.6.3. Flavonoids against SARS-CoV-2 PLPro

Potentially active flavonoids, whose activities against SARS-CoV-2 PLPro were in the order of baicalin > hesperidin > naringen > flemiflavanone D > Euchresta flavanone A on the basis of their binding energy (−10.82, −10.61, −10.17, −10.07, −9.95 kcal/mol, respectively) and dissociation constant (0.01, 0.02, 0.04, 0.04, 0.05 µm, respectively) using molecular docking and simulation studies [147]. The active site residues of PLPro, which are found common with almost all the flavonoids were Lys157, Leu162, Gly163, Asp164, and Glu167, and therefore, all these residues are critical for the ligand interaction.

### 3.6.4. Flavonoids against SARS-CoV-2 RdRp

An interesting protein–ligand blind docking approach proposed that the SARS-CoV-2 RNA replication can be inhibited by targeting its RdRp protein. Theaflavin inhibits the viral RNA replication by interfering with the RdRp catalytic pocket with the binding energy of −8.8 kcal/mol [157]. Shawan et al. identified luteolin as a potential inhibitor of ACE2 with the binding energy of −10.1 kcal/mol, which is very close to the binding energy of FDA-approved remdesivir (−10.0 kcal/mol) [158]. Fayyaz et al. identified three potentially active flavonoids against SARS-CoV-2 RdRp were in the order of hesperidin > baicalin > naringen based on their binding energy (−9.53, −9.01, −8.54 kcal/mol, respectively) and dissociation constant (0.1, 0.25, 0.55 µm, respectively) using molecular docking and simulation studies [147].

#### 3.6.5. Flavonoids against SARS-CoV-2 Helicase

Along with Mpro, PLPro, RdRp, and spike protein, Fayyaz et al. identified two potential flavonoids: hesperidin and baicalin based on binding energies (−8.93 and −8.9 kcal/mol, respectively) and dissociation constant (0.283 and 0.29 µM, respectively). Hesperidin and baicalin are the only flavonoids that interact and inhibit all the main targets of SARS-CoV-2, such as MPro, PLPro, RdRp, and helicase with excellent binding energies; therefore, both of these flavonoids are considered as multi receptor/protein targets for COVID-19.

#### 3.6.6. Flavonoids against SARS-CoV-2 ACE2

Luteolin is a potential inhibitor of ACE2 with the binding energy of −10.1 kcal/mol, which is very close to the binding energy of FDA-approved remdesivir (−10.0 kcal/mol) [158]. Using virtual screening via Autodock vina, studies identified various flavonoids such as Myritilin, myricitrin, δ-Viniferin, TaiwanhomoflavoneA, Afzelin Biorobin, and Nympholide A that can inhibit the ACE2 [159]. Similarly, Hesperetin, Baicalin, Scutellarin against ACE2 using virtual screening and molecular docking studies [160]. Tangeretin, Nobiletin, Naringenin, Brazilein, Brazilin, Galangin also inhibit ACE2 receptors [161].

#### *3.7. Clinical Trials and Future Prospects*

Many in vitro studies exploring the anti-SARS-CoV-2 action of flavonoids were published over the last two years since the advent of the COVID-19 pandemic. These studies were guided by in silico studies and the further in vitro or in vivo research of anti-SARS-CoV and anti-MERS-CoV activities of various flavonoids over the last decade owing to the two epidemics caused by these respective.

Until 17 August 2021, 13 clinical trials were reported and explored the effect and efficacy of various flavonoids and a few other polyphenols and their extracts on COVID-19 patients (Table 5 and Table S2). Of these, the most promising and popular flavonoid is the flavonol quercetin, furthering our findings. In particular, the study (NCT04401202) exploring the effect of *Nigella sativa* seed oil, which is rich in quercetin and kaempferol [162], found in its phase 2 trial that 62.1% of the intervention group which received *Nigella sativa* oil 500mg soft gel capsules orally twice daily recovered within 14 days, compared to only 36% in the control group. Following their progress can prove highly beneficial to clinicians around the globe in identifying potential COVID-19 therapeutic agents at the earliest.


**Table 5.** Some flavonoids and their natural source extracts are currently in clinical trials on COVID-19 patients. Extracted on 17 August 2021 from https://clinicaltrials.gov, accessed on 3 June 2021.


NLR: Neutrophil to Lymphocyte Ratio; hs-CRP: high-sensitivity C-reactive protein; ARDS: acute respiratory distress syndrome; PDI: Protein disulfide-isomerase; \* participants ≥ 18 yrs.

#### **4. Conclusions**

The results of our review of in vitro and in silico studies are encouraging. Considering that no internationally accepted effective therapeutic intervention exists for COVID-19, there is an urgent need for more extended studies and further in vivo and clinical trials to confirm these results and promote the synthesis of more efficient drugs using the SARs outlined in previous sections.

In this review, we report that the flavonols quercetin, myricetin, and their derivatives, the flavones baicalin and baicalein, the flavan-3-ol EGCG, and finally, tannic acid, have the most promising scope for further evaluation using both in vivo and consequently clinical studies. Unfortunately, the tendency of flavonoids to aggregate and their limited bioavailability limit their therapeutic interventions. The use of flavonoids in combination with synthetic and commercially produced drugs showed promising results, but more research is needed to prove their synergistic effects. Looking at the growing concern of antiviral resistance, naturally occurring flavonoids are a promising alternative.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms222011069/s1.

**Author Contributions:** A.C. designed the study, critically supervised the project, revised the text, and wrote some parts of the review; D.B. edited and reviewed the manuscript; V.D.D. and S.K. wrote a part of the manuscript; R.K. and P.P. equally carried out most of the study, wrote most of the manuscript and generated the tables and the graphs. All authors have read and agreed to the published version of the manuscript.

**Funding:** D.B. was supported by a National Priorities Research Program grant (NPRP 11S-1214- 170101) from the Qatar National Research Fund (QNRF, a member of Qatar Foundation) Otherwise, this research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study.

**Acknowledgments:** The publication of this article was funded by the Weill Cornell Medicine—Qatar Distributed eLibrary.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

