SHORT REVIEW

Potential use of flavonoid-rich Euphorbia hirta L. extracts for the management of COVID-19 novel coronavirus infections

Cruz, P. A. C.¹, Farol, A. N. C.¹, Ynion, G. P. Q.¹ & Cruz, P. S.¹

¹Herbanext Laboratories, Inc. , Bago City, Negros Occidental, Philippines

ABSTRACT

An increased number of infection cases coupled with a limited number of possible treatments in the light of the 2019 novel coronavirus (COVID-19) outbreak has greatly driven the demand for traditional medicine as a form of treatment or prevention of the disease. In this brief communication, we present standardized extracts of the popular Philippine medicinal herb Euphorbia hirta L. or “tawa-tawa” as a potential form of management for the disease. Although still in need of further clinical validation, the anti-viral activity noted from literature on some of the active compounds of this plant warrants enough rationale to look into its application. This is further elaborated in the overview presented by this report.

 

Key Words: COVID-19, coronavirus, Euphorbia hirta, tawa-tawa

 

INTRODUCTION

In light of the many cases of infection arising globally due to the spread of the 2019 novel coronavirus, COVID-19, an increased demand for possible formulations for the treatment or prevention of the disease is expected. Many groups are currently addressing this issue through the development of vaccines and the repurposing of existing drugs.¹ Concurrently, an increase in the demand for traditional medicine has also been observed, with many consumers seeking popularly-used remedies in Traditional Chinese Medicine (TCM) as a form of prevention against the disease.²

 

The World Health Organization (WHO), which formally recognizes traditional medicine as a potential form of treatment, has encouraged a scientific approach to test the efficacy of these products, as well as a standardized design for all studies.^1,2 Hence, a rational selection of candidates for traditional medicine is greatly warranted.

 

Among the numerous plants being screened for their potential activity against COVID-19, Euphorbia hirta L. shows particular promise. E. hirta, also known as tawa-tawa or gatas-gatas, is a common herb of the family Euphorbiaceae that has been used in Philippine folk medicine as a form of treatment against dengue viral infection.³ The plant grows abundantly along fields and roadsides, and has been commercialized much throughout the country by sale in the form of herbal capsules containing powdered tawa-tawa or tawa-tawa extracts.³

 

Much of tawa-tawa’s bioactivity has been attributed to its high content of flavonoids—compounds which have long been studied for their antiviral activity.⁴ This report briefly discusses the importance of several major bioactive flavonoids obtained from tawa-tawa to emphasize the rational selection of this plant as a form of traditional medicine with potential applications in the management of COVID-19.

DISCUSSION

Flavonoids are notably one of E. hirta’s most abundant chemical constituents, amounting to approximately 0.10% of the plant’s aqueous extractable fraction.⁵ The most abundant flavonoid, quercetin (QUE), comprises around 60% of all flavonoids in E. hirta, followed by kaempferol (KMF) at 25%, and rutin (RUT) at 15%.⁵ Other phenolic compounds such as gallic acid (GA), quercitrin (QTR), and myricitrin (MTR) have also been obtained from extracts of the aerial parts of E. hirta.^6,7 The application of these compounds for the management and  treatment of viral diseases is no nuance in itself. In silico molecular docking studies using Autodock with QUE, KMF, and GA against several types of viral proteases and replication machinery (see Table 1) have suggested the potential of these compounds as antivirals. ⁸

 

Table 1. Predicted inhibition constants (IC₅₀) of several flavonoids from E. hirta extracts from in silico molecular docking studies using Autodock.⁸

Compound	Viral Protein	IC50 (Autodock), uM
Quercetin	Influenza neuraminidase (1L7F) Dengue NS2b/NS3 protease (2FOM) Chikungunya nsP2 protease (3TRK)	2.83 6.64 2.68
Kaempferol	Influenza neuraminidase (1L7F) Dengue NS2b/NS3 protease (2FOM) Chikungunya nsP2 protease (3TRK)	4.35 12.30 2.39
Gallic acid	Dengue NS5 Methyltransferase (2P40) HIV-1 protease (1ODY)	23.86 4.10

 

Further testing using in vivo models for viral replication has allowed for the translation of this molecular docking data to evidence at the cellular level. For instance, one study on QUE against dengue virus type-2 (DENV-2) infected cultures of Vero cells using the Foci Forming Unit Reduction Assay (FFURA) and qRT-PCR affords an IC₅₀ of 28.9 ug/mL for the compound.⁴ The antiviral activity of QUE against the hand, foot, and mouth disease-causing Picornavirus enterovirus 71 (EV71) has also been assessed in vivo by phenotype screening in human rhabdomyosarcoma (RD) cells, coupled with molecular docking studies to extrapolate the mechanism of action of the compound. The study yielded an EC₅₀ of 12.1 uM for QUE against EV71 with low cytotoxicity (CC₅₀ > 200 uM), and predicted that QUE works by blocking substrate recognition by binding to the EV71 3C^pro and inhibiting its activity.⁹

 

Apart from the aforementioned examples, multiple putative mechanisms of flavonoid action against viruses have been proposed, depending on the flavonoid and the virus studied. Table 2 lists a few examples of the results of several in vivo studies using flavonoids against particular viruses.

 

Table 2. In vivo studies on the use of QUE, KMF, and GA against some common viruses.

Compound	Virus Assayed	Test Method	Results
Quercetin10	Hepatitis C virus	Infection assay, Replication assay, DGAT localization assay	85% reduced replication 92% reduced infectious particles, 65% reduced infectivity
,Kaempferol11	Japanese encephalitis	MTS reduction assay using BHK21 cells	EC50 = 12.6 uM CC50 = 230 uM inhibits viral replication and protein expression
Gallic acid12	Influenza A (H1N1)	Plaque assay, qRT-PCR, neuraminidase assay, immunofluorescence	EC50 = 2.6 uM CC50 = 22.1 uM inhibits viral mRNA replication and plaque formation

 

The activities of these compounds are multiple across a broad range of viral targets. Most notably, one may appreciate the efficacy of these compounds against neglected tropical diseases such as dengue and Zika virus.¹³ Consequently, the data from in vivo studies on flavonoids, particularly QUE, have translated well into both pre-clinical and clinical studies. A meta-analysis on the clinical usage of flavonoids in managing viral upper respiratory tract infections (URTI) concluded that supplementation with flavonoids potentially decreases the incidence of URTIs in a population of healthy individuals. The classification of URTIs in this meta-analysis includes several types of viral infections including those of parainfluenza viruses, adenoviruses, picornaviruses, and coronaviruses.¹⁴ The outcomes of these clinical trials were greatly dependent on the type and amount of flavonoid supplemented to the subjects, and no clear dosing scheme was concluded from consolidating the data; however, a dosage between 0.2~1.2 g/d was utilized in all studies included in the analysis.¹⁴ In fact, the use of flavonoid supplementation to mitigate the risk of incurring URTIs has also been discussed as a potential strategy for maintaining the health of athletes participating in the incoming 2020 Tokyo Olympics.¹⁵

 

Focusing on coronaviruses, there exists no shortage of data supporting the potential application of flavonoids against COVID-19. One in vivo study on the use of Houttuynia cordata Thunb. extracts rich in quercetin, quercetrin, and cinanserin against the murine coronavirus, murine hepatitis virus (MHV), exhibited phenotypic neutralization of cells 2 days post-infection (2dpi) at  a minimum inhibitory concentration (MIC) of 0.98 ug/mL without apparent cytotoxic effects on CCL9.1 cells.¹⁶ Earlier studies on QUE conducted close to the height of the 2003 Severe Acute Respiratory Syndrome (SARS-CoV) epidemic have established its potential to inhibit the entry of SARS-CoV in Vero E6 host cells using  an HIV-luc/SARS pseudotyped virus infection model measured by frontal affinity chromatography-mass spectrometry (FAC/MS).¹⁷ QUE was able to do so with an EC₅₀ of 83.4 uM and a low cytotoxicity (CC₅₀ = 3.32 mM), indicating its clinical safety, further supported by its existing registration under the US FDA.¹⁷

 

An in vitro study on the enzymatic inhibition of QUE against recombinantly-expressed SARS-CoV 3C-like protease (3CL^pro) suggests another mechanism of action for QUE against SARS-CoV in comparison to the aforementioned study. Here QUE was able to maximally inhibit SARS-CoV 3CL^pro activity at 82%, and exhibited an EC₅₀ of 73 uM.¹⁸ Given the high sequence similarity between the genomes of SARS-CoV and SARS-CoV-2 (causing COVID-19),  it may be inferred that a similar effect may be observed in SARS-CoV-2 3CL^pro when treated with the same compound.¹⁹ Lastly, KMF in another in vivo study has also been shown to exhibit a different mechanism of action compared to QUE against SARS-CoV. In that study, heterologously-expressed SARS-CoV 3a channel protein in Xenopus oocytes was effectively blocked by KMF and its glycosidic derivatives at effective concentrations as low as IC₅₀ = 2.3 uM.²⁰

 

CONCLUSION

Given the direct association between the presence of the flavonoids and phenolics in E. hirta and the potential bioactivities of these phenolics and flavonoids against different viruses—coronaviruses in particular—there exists a significant rationale to study the application of this plant and its standardized extracts to provide a greater plethora of possible treatments in the midst of the current COVID-19 epidemic.

 

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