Amentoflavone is one of the flavonoids that are known for their antiviral effects and many of them are predicted to have inhibitory effects against severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome Coronavirus (MERS-CoV) enzymes 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). Amentoflavone is a biflavonoid found in the herbal extracts of St. John's wort (
- CYP450 inhibitor
Our more than 18-month long fight against the pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has improved our understanding of some characteristics of the atypical respiratory disease coronavirus disease-2019 (COVID-19), but effective treatments to combat it are still far from sight. While vaccines have now passed phase 2 or 3 trials, traditional medicines are still being considered as possible treatments for COVID-19. The Chinese government has recommended traditional Chinese herbal medicine (TCM) as a therapeutic option for the treatment of COVID-19, and reports it to have >90% efficacy in fighting the Wuhan epidemic . The Regional Expert Committee on Traditional Medicine for COVID-19 formed by the World Health Organization (WHO), the Africa Centre for Disease Control and Prevention, and the African Union Commission for Social Affairs have recently endorsed a protocol for phase III clinical trials of herbal medicine for COVID-19 . As such, many natural products and herbal medicines known for their antiviral properties are being tested for effectiveness against the SARS-CoV-2. However, a drawback of these traditional medicines is that they are mixtures of different plant products with many active ingredients, and with no knowledge of their mechanism of action against new diseases like COVID-19. However, molecular docking studies in silico of active ingredients of various natural–herbal products have produced a list of molecules with probable anti-SARS-CoV-2 properties. These molecules can either block viral entry into cells via the angiotensin-converting enzyme 2 (ACE2) receptor and trans-membrane serine protease 2 (TMPRSS2), or inhibit the activity of 3-chymotrypsin-like protease (3CLpro) or RNA-dependent RNA polymerase (RdRp) by binding to their active sites. Of the various molecules tested in silico, the flavonoids top the hit list and show potential to bind to the catalytic site of SARSCoV-2 main protease (Mpro) and thereby inhibit its replication in the host cell [3, 4]. Some of these flavonoids also show the potential to block the interaction of SARS-CoV S-protein with ACE2. Amentoflavone, a naturally occurring biflavonoid, tops the list of potential inhibitors of the SARS-CoV-2 protease [3,4,5,6,7]. A search on PubMed and PubFacts shows that amentoflavone has broad spectrum antiviral activity in addition to its anti-inflammatory and antioxidant effects, and is a naturally occurring human thrombin inhibitor [8, 9]. Here we critically analyze whether amentoflavone has the potential to be used as a therapeutic agent for COVID-19.
Amentoflavone (C30H18O10; 4′,5,7-trihydroxyflavone)-(3′→8)-(4′,5,7-trihydroxyflavone; PubChem CID 5281600) is a biflavonoid of apigenin (3′,8″-bis-apigenin, didemethyl-ginkgetin). It has been isolated from nearly 120 plants used in traditional medicine by the Chinese, Indians, Africans, and Americans . It is an active ingredient found in extracts from
Molecular docking studies predict amentoflavone to have potential to inhibit the SARS-CoV-2 by binding to its Mpro 3CLpro and thereby blocking the replication of the virus [3,4,5,6,7] (
Such studies collectively emphasize the broad spectrum antiviral activity of amentoflavone, which can inhibit viral pathogenicity by targeting the viral proteins essential for its replication. As a functional FAS is essential for viral replication in general , the inhibitory effects of amentoflavone on FAS activity further makes it a promising antiviral therapeutic candidate. Studies with the fluorogenic peptide in vitro and docking studies in silico have shown a probable anti-SARSCoV effect of amentoflavone; however, to our knowledge, studies showing the inhibitory effects of amentoflavone on SARS-CoV 3CLpro in any cellular system remain lacking.
COVID-19 is regarded primarily as an atypical pulmonary disease characterized by severe pneumonia with acute respiratory distress syndrome (ARDS). Emerging data shows that this disease also leads to hyperinflammatory state resulting in other complications such as liver, kidney, and heart injury, and septic shock . The cause of such complications is suggested to be due to “cytokine release syndrome” or a “cytokine storm”  and due to elevated serum cytokines (particularly interleukin (IL) 1β, IL-6, and tumor necrosis factor α (TNF-α)) levels, impaired interferon responses, and peripheral lymphopenia leading to lung injury . Although the molecular mechanisms of coronavirus-induced pathogenesis are not entirely understood, emerging evidence suggests that, similar to the influenza A virus, SARS-CoV-2 infection possibly causes a rapid influx of inflammatory cells, which is followed by increased reactive oxygen species (ROS) production and elevated cytokine expression and release that leads to acute lung injury (ALI). Thus, it seems that inflammatory and immune response signaling along with ROS plays an important role in the pathogenic mechanism. SARS-CoV-2 also infects endothelial cells to cause endothelialitis that results in the activation of a coagulation cascade leading to vasculopathy, multisystem organ failure, and the hypercoagulable state observed in patients with severe cases of COVID-19 . Thus, while considering traditional medicine, natural antiviral agents with added antioxidant and anti-inflammatory actions could prove effective in the treatment of COVID-19.
Studies of the pharmacological functions of amentoflavone suggest it to be anti-inflammatory, antioxidative, and vasoprotective . The scavenging effects with 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), superoxide, and hydroxyl radicals have shown it to have high antioxidant capacity (19.2%–75.5%) . It significantly suppressed lipopolysaccharide induced nitric oxide (NO), ROS, malondialdehyde (MDA) in a rat astrocytoma cell line; NO, prostaglandin E-2 (PGE-2), the nuclear translocation of c-Fos in RAW 264.7 cells; and TNF-α in a human monocytic leukemia cell line, with no other apparent effects on the cells. It inhibited the oxidative burst of neutrophils and damage to human erythrocyte membranes induced in human peripheral blood mononuclear cells by phytohemagglutinin (PHA) by decreasing the levels of IL-6, TNF-α, IL-1β, and PGE2  (
However, certain characteristics of amentoflavone should be noted. Like other flavonoids, the absolute oral bioavailability of amentoflavone is extremely low (0.06% ± 0.04%) and it undergoes rapid glucuronidation and sulfation in vivo . The glucuronidation rate of amentoflavone is significantly higher than sulfation and it is the main metabolite in circulation. The area under curve (AUC0−t) of amentoflavone glucuronides (410.9 ± 62.2 ng/mL h) was significantly higher than the parent compound (194.5 ± 16.9 ng/mL h). Moreover, the antioxidant property of amentoflavone may remain unaltered following glucuronidation . Studies suggest amentoflavone to be a potent inhibitor of cytochrome P450 (CYP) 3A4 and 2C9 [31, 32], cathepsin B , and also a broad spectrum inhibitor of uridine 5′-diphospho (UDP)-glucuronosyltransferases (UGTs)  (
As the uses of both conventional and traditional medications for COVID-19 are being explored, the possibility of herb–drug interactions should also be noted . CYP monooxygenases and UGTs play a role in the metabolism and detoxification of xenobiotics and their excretion via urine or bile [36, 37]. This indicates a possible herb–drug interaction with amentoflavone. Clinical trials have been conducted with
Being an inhibitor of both CYPs and UGT, the use of amentoflavone should be carefully reviewed to avoid the risk of toxicity due to increased bioavailability of the coad-ministered drugs. Administration of amentoflavone should also be calibrated against antithrombotic drugs like warfarin, rivaroxaban, or betrixaban to avoid bleeding. The possibility of administering amentoflavone as replacement of the conventional CYP 3A4 and 2C9, and UGT inhibitors, can also be considered. The currently approved antiviral drug remdesivir is an adenosine analog, which binds to the viral RdRp , and it can be administered with strong inhibitors of CYPs and UGTs . Studies in vitro with HIV protease inhibitors like lopinavir, ritonavir, and darunavir suggest cleavage of 3CLpro and papain-like protease (PLpro) of SARS-CoV-2; however, they have been suggested to have poor selectivity in vivo as they have to be administered at extremely high doses [21, 42]. Thus, administration of anti-viral drugs such as remdesivir, lopinavir, and ritonavir with amentoflavone can result in increased efficacy. However, studies need to be conducted to ascertain the herb–drug interaction of amentoflavone and/or its main metabolites—the glucuronides.
Although our understanding of the antiviral properties and mechanism of action of amentoflavone and its glucuronides is far from complete, the strong binding affinity of amentoflavone to SARS-CoV 3CLpro and spike glycoprotein predicts it be an effective natural therapeutic drug candidate against SARS-CoV-2. Its strong antioxidant property along with anti-inflammatory, thrombin inhibition, and protection against lung injury could aid in fight against the SARS-CoV-2 pathogenic complications like hyperinflammatory and hypercoagulable states that lead to ALI and multiorgan failure.
However, to provide a better understanding of its pharmacological effect against COVID-19 and using amentoflavone as an antiviral adjunct therapy against SARS-CoV-2, preclinical trials involving animal models need to be undertaken to confirm the antiviral activity of this biflavonoid, which, to our knowledge, has been shown only with assays in vitro. Although amentoflavone has been identified and isolated from many known medicinal plants and their herbal extracts that are used as dietary supplements, it should be noted that the content of amentoflavone in these extracts is very low. The highest content has been isolated from
With the advent of COVID-19 vaccines and rise of new and more virulent SARS-CoV-2 mutants, there is an urgent need to undertake pharmaceutical research of natural compounds that show strong binding affinity to the coronavirus in silico. As natural ingredients of