Demystifying therapeutic potential of medicinal plants against chikungunya virus.

Viral infections are posing a great threat to humanity for the last few years. Among these, Chikungunya which is a mosquito-borne viral infection has produced enormous epidemics around the world after been rebounded. Although this infection shows a low mortality rate, patients suffer from fever, arthralgia, and maculopapular rashes, which reduce the quality of life for several weeks to years. The currently available treatments only provide symptomatic relief based on analgesics, antipyretics, and anti-inflammatory drugs which are nonspecific without satisfactory results. Medicinal plants are a widely accepted source of new molecules for the treatment of infectious diseases including viral infections. The scientific reports, primarily focusing on the anti-chikungunya activity of plant extracts, natural origin pure compounds, and their synthetic analog published from 2011 to 2021, were selected from PubMed, Google Scholar, and Scopus by using related keywords like anti-chikungunya plants, natural antivirals for Chikungunya. The present review decodes scientific reports on medicinal plants against chikungunya virus (CHIKV) infection and demystifies the potential phytoconstituents which reveals that the screening of flavonoids containing plants and phytochemicals showing efficacy against other arbovirus infections, may prove as a potential lead for drug development against CHIKV. The present article also outlines pathogenesis, clinical aspects, molecular virology, and diagnostic approaches of CHIKV infection.

Introduction

Infectious diseases have also evolved during human species evolution. The new infections emergence and recurrence of existing ones are posing a threat to society. Among these, viral infections account for several casualties each year and constitute serious health issues worldwide because of the nonspecificity of antiviral therapies.[12]

Chikungunya virus (CHIKV) that belongs to the Togaviridae family is a re-emerging mosquito-borne infection, transmitted by the bite of infected female Aedes mosquitoes.[1] It was firstly identified in the Makonde plateau (Tanzania) in 1952 and named after the word “kungunyala,” meaning “that which bends up,” denoting the bent pose of CHIKV infected person due to severe joint pain. From the time of the first 1952 report, it caused infrequent outbreaks mostly in Africa and Southeast Asia until its global re-emergence which started in 2004 from Kenya and spreads through neighbor islands to several Indian Ocean islands and finally to India.[34] During these epidemics, it has infected more than 6 million people.[45] Further, it has approximately infected one million people in the American continent by 2014.[16] All these outbreaks related to CHIKV take place in Africa, Southeast Asia, India, and recently in the American continent have established chikungunya a major global health problem that continues to expand at an alarming rate.[7] The disease is rarely fatal, but persistent severe polyarthritis affects the quality of life and reduces the workability of patients.[8] Unfortunately, treatment is only confined to relieving symptoms by analgesics, antipyretics, corticosteroids, and anti-inflammatory drugs, but does not cure a viral infection. Furthermore, there is no approved CHIKV vaccine or antiviral therapy for treatment and prophylaxis yet.[6] Thus it is prudent to find novel anti-chikungunya drugs that may be used either as therapies or as preventative measures to combat the deadly epidemics.

Almost all civilizations have used and continue to use medicine from natural sources. Among all-natural sources, medicinal plants have been the most widely used source of safe and effective medicine.[9] The chemical constituents of medicinal plants have served as a rich source of new therapeutics during the process of drug development and discovery. Almost 50% of currently available therapeutics is obtained from natural sources, using either natural products or natural products synthetic analogs. Medicinal plants have been used to control infectious diseases since time immemorial. During the last few years, several studies have reported some natural products[1011] and approved drugs[12] having in vitro anti-chikungunya activity. Studies have also revealed the antiviral potential of medicinal plants against the CHIKV.[113]

The research articles, published from 2011 to 2021, are selected from Google Scholar, MEDLINE, and PubMed by using relevant keywords including the anti-CHIKV potential of plants, the antiviral activity of plants against Chikungunya, plant and natural products used in CHIKV infection, virology of CHIKV. The research articles primarily focusing on the anti-CHIKV activity of plant extracts, pure compounds of natural origin, and their synthetic analog are included in the study.

Pathogenesis

Among its symptoms, severe joint pain may continue for several weeks to years in 10% of the infected population.[14] Based on the duration of symptoms, the disease can be divided into the acute, postacute, and chronic phases. The acute phase of the disease includes the first 3 weeks of infection which consists of incubation periods of 4–7 days, after which symptoms such as fever, arthralgia, and maculopapular rash last for 5–10 days.[1516] The postacute phase begins from the 4th week of infection and lasts up to the end of the 3rd month. In this phase severity of the acute phase, complications increased. When symptoms persist after the 3rd month, the disease enters its chronic phase which results in persistent severe polyarthritis almost in 60% of infected patients.[1] Dengue and Zika viruses show similar symptoms of CHIKV like fever, arthralgia, maculopapular rashes, and usually co-circulate in the epidemic regions of CHIKV, thereby they can easily co-infect the patients.[171819] Therefore, specific diagnostic tests for early-phase infection diagnosis are important. Based upon specimen collection time, a diagnostic method is selected from the various available virological, genetic, and serological methods.[17]

Virology

CHIKV is spherical, positive-sense single-stranded RNA encapsidated within icosahedral capsid which is enveloped with lipid-bilayer membrane. The icosahedral capsid of a virus is approximately 65 nm diameter and the genome is around 12 kb long which has two open reading frames one on each end of RNA. The 5' end of RNA encodes four nonstructural proteins (nsP1 to nsP4) and 3' end encodes five structural proteins (1 capsid protein, 2 glycoproteins E1 and E2, 2 cleavage products E3 and 6K).[2021] The different genotypes of the virus have distinct antigenic characteristics and named according to their geographical sources.[22]

Plant Extracts Targeting chikungunya virus Infection

Ipomoea aquatica

This is a semi-aquatic tropical plant, commonly called “Water Morning Glory” grown extensively as a leaf vegetable throughout the world; chemically consists of alkaloids, carbohydrates, flavonoids (nicotiflorin and ramnazin-3-O-rutinoside), and phenolic compounds.[23] Chan et al.[13] evaluated the anti-CHIKV activity of the aerial parts extracted with six solvents of different polarities [Table 1]. Among different extracts, only ethyl acetate extract strongly inhibited the cytopathic effect of CHIKV in Vero cell lines exhibiting 82% cell viability (at 320 μg/ml) by the neutral red uptake assay. Chloroquine was used as a positive control. Further quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of the above extract showed a reduction in viral load by 65% (at 320 μg/ml) as compared to virus control.

Table 1

Cytotoxicity and anti-chikungunya activity of plant extracts in Vero cells by cytopathic effect inhibition assay[13]

Plant name	Nature of extract	CC50	IC50	Selective index (CC50/IC50)	Mechanism of action
Ipomoea aquatica	Ethyl acetate	>640 µg/ml	227	>2.8	Not reported
Persicaria odorata	Ethanol	455 µg/ml	132	3.4	Not reported
Rhapis excelsa	Chloroform	161	29	5.4	Direct virucidal effect
Tradescantia spathacea	Chloroform	238	60	4.0	Direct virucidal and inhibit viral replication
	Ethanol	ND	202	ND
	Methanol	ND	179	ND
Vernonia amygdalina	Ethyl acetate	165	32	5.1	Direct virucidal effect
	Ethanol	272	67	4.0
	Methanol	485	200	2.4

ND= Not determined as no significant cytotoxicity found at the highestconcentration (640 μg/ml) tested.

Persicaria odorata

Persicaria odorata commonly called “Vietnamese coriander” in Malaysia, consists of steroids, terpenoids, flavonoids, tannins and phenolic compounds, carbohydrates and exhibits anti-infective activities.[24] Chan et al.[13] examined the anti-CHIKV activity of six different solvent extracts of the aerial parts [Table 1]. Ethanolic extract strongly inhibited the cytopathic effect of CHIKV with cell viability of more than 70% while metabolic extract showed moderate inhibition exhibiting cell viability <50%. Chloroquine was used as the positive control and the cell viability was measured by the neutral red uptake assay. In quantitative RT-PCR, ethanol extract reduced the Chikungunya viral load by 90% (at 160 μg/ml) in comparison to virus control. Hence, ethanol-soluble extractives could be a good source of anti-CHIKV drugs.

Rhapis excelsa

Methanolic extract of leaves of Rhapis excelsa, commonly known as “lady palm” contains 4 flavonoids: Apigenin-8-C-glucoside (vitexin), Apigenin-6, 8-Di-C-βglucopyranoside (vicenin-2), Luteolin-6-C-glucoside (iso-orientin), and Luteolin-8-C-glucoside (orientin).[25] In a study performed by Chan et al.,[13] leaves extracted with solvents of a wide polarity range and screened for their anti-CHIKV activity. Out of six extracts, chloroform extract (30 μg/ml) exhibited the strongest inhibition of the cytopathic effect of the CHIKV without significant cytotoxicity. The strong anti-chikungunya activity of the extract is further confirmed by quantitative RT-PCR analysis in which extract reduced the viral load by 98% in comparison to virus control. These results suggest that this extract might have direct virucidal effects on CHIKV.

Tradescantia spathacea

This is a tropical plant, commonly known as “oyster plant” used as laxatives, a blood thinner, antibiotic and anti-malaria; consist of alkaloids, flavonoids, glycosides, saponin, and tannins.[26] Chan et al.[13] investigated in vitro antiviral activity of Tradescantia spathacea leaves extracts as a powerful anti-chikungunya plant. The ethanol and methanol extracts, at concentration 512 μg/ml, exhibited strong inhibitory activity against the CHIKV with cell viabilities 93% and 91%, respectively, while chloroform extract showed 89% cell viability at concentration 80 μg/ml. The results of quantitative RT-PCR revealed that the percentage reduction in viral load of ethanol, methanol, and chloroform extracts were 53%, 46%, and 84%, respectively. The above results suggest that active extracts of this plant might inhibit the virus to infect other cells and also exhibit the direct virucidal effect.

Vernonia amygdalina

The leaves of Vernonia amygdalina, commonly called “Bitter leaf” contain alkaloids, glycosides, saponins, quercetin, epicatechin, steroids, coumarins, polyphenolic compounds, mucilage, pectin, lipids, and terpenoids.[27] Chan et al.[13] investigated the antiviral activity of V. amygdalina leaves extract against CHIKV in vitro [Table 1]. Out of six extracts only ethyl acetate, ethanol, and methanol extract exhibited strong cytopathic effect inhibitory activity with cell viability of more than 70%. The acetate extract had the lowest cytotoxicity among all six extracts. In quantitative RT-PCR analysis, the percentage reduction in viral load of ethyl acetate, ethanol, and methanol extracts were 95, 83, and 82 at a concentration of 80, 160, and 320 μg/ml, respectively. Treatment with ethyl acetate extract resulted in the highest reduction in virus load using the lowest concentration.

Andrographis paniculata

It is a traditional medicinal plant, commonly known as King of bitters or Kalmegh; used to treat infections; mainly contain diterpenoid: Andrographolide. Sharma et al.[28] evaluated the anti-CHIKV potential of silver nanoparticles of aqueous leaf extract by calculating the cell viability of virus-infected Vero cells after treatment with extract nanoparticles. In this study, the viability of untreated infected Vero cells which was 25%, increased to about 81% when treated with a maximum nontoxic dose (MNTD) 31.25 μg/ml of silver nanoparticles synthesized from aqueous leaf extract of the plant. The results indicate that it has significant anti-CHIKV potential [Table 2].

Table 2

Antichikungunya activity of plant extracts in Vero cells by cytopathic effect inhibition assay

Plant name	Nature of extract	Concentration (µg/ml)	Viral inhibition (%)	Mechanism of action	Reference
Andrographis paniculata (leaves)	Aqueous	31.25	>75	Inhibit viral genome replication	[28]
Phyllanthus niruri (leaves)	Aqueous	125	>25	Inhibit virus entry	[28]
Tinospora cordifolia (stem)	Aqueous	250	>1	Inhibit virus entry	[28]
Oroxylum indicum (leaves)	Aqueous, methanol	150	10	Inhibit viral replication	[29]
Cynodon dactylon (whole plant)	Ethanol	5025	9888	Inhibit viral replication	[30]
Psidium guajava (leaves)	Aqueous	15	60	Inhibit viral replication	[1]

Phyllanthus niruri

Phyllanthus niruri, commonly called “stone breaker,” is used as antiulcer, hypolipidemic, antiviral and antioxidant in Ayurveda formulations. Sharma et al.[28] prepared the silver nanoparticles of aqueous leaf extract of P. niruri which showed MNTD 125 μg/ml in Vero cells determined by using MTT assay. The FTIR spectra indicate that these nanoparticles consist of flavonoids, polyols, terpenoids, and polyphenols. In the present investigation, the cell viability of untreated infected Vero cells which was 25%, increased to about 30% only when treated with MNTD. The results indicate that it does not have significant anti-CHIKV potential [Table 2].

Tinospora cordifolia

Tinospora cordifolia commonly known as “Guduchi” is renowned in Ayurveda literature for its uses in the therapy of different diseases and is a source of bioactive components like alkaloids, terpenoids, glycosides, and steroids. However, FTIR spectra also indicate the presence of flavonoids, polyols, and polyphenols.[2832] In a study performed by Sharma et al.,[28] silver nanoparticles prepared by treatment of 1 mM aqueous solution of AgNO3 with aqueous extract of this plant stem showed antiviral activity against CHIKV in Vero cell lines. The cell viability of untreated infected Vero cells which was 25%, increased to about 75% when treated with MNTD (250 μg/ml) of these nanoparticles and exhibited about 49% of inhibition activity against the virus. The results indicate that it has significant anti-CHIKV potential [Table 2]. However, in another study, when aqueous extract of stem evaluated for anti-CHIKV activity by Plaque reduction assay, Helicase assay and Protease assay, it did not show antiviral activity in any assay.[33]

Oroxylum indicum

It is a threatened medicinal plant, commonly called the Indian trumpet tree, extensively cultivated in Southeast Asia, India, and China; chemically contains several flavonoids.[34] Mohamat et al.[29] evaluated the anti-CHIK potential of leaves extracted with methanol and water [Table 2]. In cytotoxicity assay, the MNTD (cell viability similar to negative control) of both methanol and aqueous extract was 625 μg/ml. The value of CC50 (cell viability 50%) for methanol and aqueous extracts in Vero cell lines was 1875 and 7700 μg/ml, respectively. Although both extracts, aqueous and methanol, had a poor anti-CHIKV effect comparatively aqueous extract demonstrated higher antiviral potential and reduced viral titer 10% when assessed with a concentration of 150 μg/ml. Although both extracts did not show a direct virucidal effect significantly the aqueous extract affected virus entry as suggested by the Plaque reduction assay.

Cynodon dactylon

It is commonly called Bermuda Grass, family Poaceae. It consists of glycosides, phytosterols, polysaccharides, flavonoids, terpenes, and alkaloids as bioactive constituents. In a study performed by Murali et al.,[30] fractions of ethanolic extract of fresh plant material were separated using chromatographic techniques and then evaluated for anti-CHIV activity in Vero cells [Table 2]. The flavonoid-rich fraction exhibited potent anti-chikungunya activity (98% inhibition) at the concentration of 50 μg/ml and it was found safe to use even up to 250 μg/ml concentration as determined by cytotoxicity studies. The active compounds of this fraction were further identified by Reverse Phase-high-performance liquid chromatography and gas chromatography-mass spectrometry. The flavonoids luteolin and apigenin have shown promising results as anti-CHIKV agents.

Picrorhiza kurroa

This is commonly called Katuki; consists of kutkin (kutkosides and iridoid glycosides) and apocynin (catechol) as major bioactive constituents. The aqueous extract (10 μg/ml) of rhizomes of this plant inhibited CHIKV more than 80% without causing cytotoxicity in plaque reduction assay suggesting extract a viral attachment inhibitor. The plant extract also inhibited helicase enzyme activity in helicase assay suggesting it as a viral replication inhibitor. Further, it was subjected to a virus replication inhibition assay in which it showed 38.3% and 67% viral replication inhibition at concentrations 100 μg/ml and 200 μg/ml respectively [Table 3].[33]

Table 3

Antichikungunya activity of plant aqueous extracts in Vero cells[33]

Plant name	Assay employed	Concentration (µg/ml)	Viral inhibition (%)	Mechanism of action
Picrorhiza kurroa (rhizome)	Replication inhibition assay	200	67	Inhibit viral replication
Ocimum tenuiflorum (whole plant)	Replication inhibition assay	200	100	Inhibit viral replication
Terminalia chebula (fruit)	Replication inhibition assay	200	100	Inhibit viral replication
Zingiber officinale (rhizome)	Plaque reduction assay	10	>80	Inhibit viral replication
Commiphora wightii (resin)	Replication inhibition assay	200	31.6	Inhibit viral attachment
Cedrus deodara (bark)	Replication inhibition assay	200	36.6	Inhibit viral attachment

Ocimum tenuiflorum

This is a holy plant, commonly known as “Shyama Tulsi” consist of mainly phenyl derivative compounds, methyl eugenol, and other terpenoids.[35] Raghavendhar et al.[33] demonstrated the complete antiviral ability of aqueous extract of the whole plant and recommended it for further investigations like in vivo studies. In this study, extract exhibited complete anti-CHIKV ability by inhibiting virus in plaque reduction (more than 80% inhibition), helicase, and protease assay. Results demonstrated the ability of the extract to inhibit CHIKV attachment, its entry and replication. In a replication inhibition assay, the Ocimum tenuiflorum extract exhibited a positive effect against CHIKV by 28.3%, 53.3%, 85%, and 100% at experimental concentrations of 25, 50, 100, and 200 μg/ml, respectively [Table 3].

Terminalia chebula

It is commonly known as Haritaki; consists of tannins, flavonoids (flavonol glycosides), terpenoids, saponin, sterols.[36] Raghavendhar et al.[33] demonstrated the complete antiviral ability of aqueous extract of the fruit. This extract exhibited complete antiviral potential against CHIKV by inhibiting virus in plaque reduction (more than 80% inhibition), helicase, and protease assay. Results demonstrated the ability of the extract to inhibit CHIKV attachment, its entry, and replication. The extract demonstrated dose-dependent inhibition in replication inhibition assay and showed 23%, 51.6%, 83.3%, and 100% inhibition of CHIKV at concentration 25, 50, 100, and 200 μg/ml [Table 3].

Zingiber officinale

Rhizomes consist of bioactive constituents like terpenoids, steroids, gingerols, gingerones, shogaols, tetra, and hexa-hydrocurcumin; commonly called Saunth.[37] Raghavendhar et al.[33] evaluated the anti-CHIKV potential of aqueous extract of rhizomes of this plant by plaque reduction, helicase, and protease assay. The extract (10 μg/ml) exhibited anti-CHIKV activity by inhibited RNA helicase enzyme only in helicase assay suggesting it as a viral replication inhibitor but it did not inhibit virus in plaque reduction assay and protease assay [Table 3]. In another study, the aqueous extracts of fresh dried rhizomes exhibited anti-chikungunya potential by reducing the cytopathic effect of CHIKV in Vero cell lines, and increased the survival of cells by 51.05% and 35.10%, when pretreated with MNTD 62.5 μg/ml and half of MNTD, respectively. Likewise, the survival of Vero cells increased by 52.90% and 49.02% when co-treated with MNTD and half of MNTD, respectively.[38]

Commiphora wightii

The plant gum resin is commonly used for arthritis, high blood cholesterol, atherosclerosis, and skin disease; mainly consists of terpenoids, steroids, guggulstetrols, flavonoids, Guggulsterones E and Z.[39] Raghavendhar et al.[33] evaluated the anti-CHIKV potential of the resin obtained from this plant by plaque reduction, helicase and protease assay. The resin exhibited viral plaque inhibition at concentration 10 μg/ml in plaque reduction assay suggesting resin as a viral attachment inhibitor. It also inhibited virus replication 31.6% at concentration 200 μg/ml in virus replication inhibition assay [Table 3].

Cedrus deodara

It is commonly called Devdaru; bark contains bioactive principles such as cedeodarin, α-pinene, β-pinene, myrcene, cedrin. The aqueous extract of bark of this plant exhibited viral plaque inhibition at concentration 10 μg/ml in plaque reduction assay suggesting it as a viral attachment inhibitor. However, it also suggested as a CHIKV replication inhibitor in virus replication inhibition assay in which it inhibited virus replication 36.6% at concentration 200 μg/ml [Table 3].[33]

Alpinia officinarum

The aqueous, ethanolic and aqueous-ethanolic extracts of rhizomes of this plant showed antiviral activity against Asian CHIKV in Vero cell lines at concentrations of 72.5 μg/ml, 125 μg/ml, and 72.5 μg/ml respectively. The selectivity indices of the extracts were 3.44, 2, and 6. These extracts did not inhibit the growth of the ECSA lineage of CHIKV in the same experimental conditions.[40]

Melia azedarach

In Vero cell lines, an ethanolic extract of this plant's leaves showed anti-CHIKV efficacy against Asian and ECSA strains at concentrations of 31.25 g/ml and 125 g/ml, respectively. The selectivity indices of the extract for Asian and African strains were 8 and 2, respectively.[40]

Azadirachta indica

The aqueous and aqueous ethanolic extracts of leaves of this plant inhibited the growth of Asian CHIKV in Vero cell lines at concentrations of 62.5 μg/ml and 32.25 μg/ml. The selectivity indices of the extracts were 1 and 2. These extracts did not inhibit the growth of the ECSA lineage of CHIKV in the same experimental conditions.[34] In another study, the aqueous extract of bark of the plant did not show anti-CHIKV activity in the Plaque reduction assay, Helicase assay, and Protease assay.[33]

Psidium guajava

The aqueous extracts of leaves and their green synthesized silver nanoparticles reduced the cytopathic effect of CHIKV in Vero cell lines and increased the survival of cells by 60% and 40% in comparison to the virus control, respectively. This indicates the antiviral potential of this plant against CHIKV. In cytotoxicity assay, both extract and nanoparticles showed MNTD 15 μg/ml in Vero cells determined by using MTT assay. Docking studies found that the phytochemicals; longifollen and quercetin interact with a replication essential cysteine protease (nsP2) of CHIKV which may be the mechanism of antiviral activity.[31]

Phytochemicals Targeting chikungunya virus Infection

Epigallocatechin gallate

Among catechins present in green tea extract, Epigallocatechin gallate (EGCG) is the most important, concerning antiviral properties against many viruses including HIV, influenza, and hepatitis C. EGCG was reported to reduce the infection rate of CHIKV 40% at a concentration of 10 μg/ml which clearly shows significant inhibition. The compound inhibited the entry of CHIKV pseudo-particles into the target cell.[41]

Curcumin

It is a bioactive constituent of turmeric rhizomes which are used as a spice in food and herbal supplement. In a study performed by Mounce et al.[42] Curcumin, a component of turmeric rhizomes, exhibited anti-chikungunya activity by inhibiting viral replication and cell binding of the virus in a dose-dependent manner. In further investigation, Curcumin directly reduced the infectivity of the virus in a time and dose-dependent manner.

Harringtonine and its analog homoharringtonine

Cephalotaxus harringtonia mainly consists of alkaloids and terpenoids. Harringtonine, an alkaloid from this plant and its analog homoharringtonine exhibited in vitro anti-CHIKV activity in BHK21 cells. The results of time-of-addition studies suggested that Harringtonine inhibited viral replication after entry into the cell but did not have any effect on the binding and entry of the virus into a cell.[43]

Silymarin

It is an extract from the seeds of Silybum marianum commonly called Milk Thistle; consists of several flavonolignans and a few flavonoids. Lani et al.[10] investigated the silymarin, quercetin, and kaempferol for anti-CHIKV activity using a CHIKV replicon cell line. Silymarin at 100 μg/ml significantly inhibited the CHIKV mediated cytotoxicity in the CPE inhibition assay and resulted in about 70% inhibition of CHIKV infection. In the postadsorption assay, silymarin at this concentration inhibited CHIKV replication about 99%, in dose-dependent mode. Both quercetin and kaempferol at 200 μg/ml exhibited only 25% CPE inhibition. These findings showed that silymarin had potent antiviral action against CHIKV in vitro conditions, reducing viral replication in the virus's postentry stage in a dose-dependent manner.

Andrographolides

Andrographolide, a major bioactive phytochemical, is extracted from the leaves of Andrographis paniculata which is traditionally used to treat infections. Based on this, Wintachai et al.[44] evaluated the antiviral potential of andrographolide against ECSA genotype of CHIKV in human liver cell lines HepG2. Andrographolide showed potent anti-chikungunya activity in plaque assay, with 50% inhibitory concentration 77 μM without cytotoxicity. In this study, andrographolide reduced viral infection in a dose-dependent manner, with 80% inhibition at 100 μM as determined by flow cytometry and confirmed by confocal microscopy. Time of addition and RNA transfection studies suggested that it inhibited viral genome replication at the postentry step.

Nobiletin

The active ingredients of citrus fruits are well known for their wide range of pharmacological activities. The peels of Citrus reticulate consist of flavonoid Nobiletin as a bioactive ingredient in large quantity. Lin et al.[45] evaluated the anti-CHIKV potential of four natural flavonoids: Nobiletin, phlorizin, resveratrol, and oxyreveratrol in Vero E6 cells by neutral red uptake assay. Among these flavonoids, Nobiletin and resveratrol exhibited significant antiviral activity against the CHIKV, but oxyreveratrol and phlorizin did not. The data of MTT assay and lactate dehydrogenase assay revealed that nobiletin (at 250, 200, 150, and 100 μM) reduced viral cytotoxicity, viral yield, and maintained cell proliferation. Besides, it reduced the CPE of the virus within the host cell after 48 h of infection.

Flavaglines

Flavaglines are class of natural compounds derived from plants of genus Aglaia, family Meliaceae; have potent anti-cancer and neuroprotective potential. Wintachai et al.[46] investigated the anti-chikungunya activity of three synthetic flavaglines in the human embryonic kidney cell line Hek293T/17. Three compounds show the inhibition of CHIKV entry and production when cells were treated for only 15 min before infection but not when treated after infection. One compound exhibited moderate antiviral activity by inhibiting CHIKV replication.

Phytochemicals of Tectona grandis Lin

In a study, phytochemicals from the leaves of Tectona grandis were isolated and evaluated for the anti-CHIKV potential.[47] Three compounds were isolated: 2-(butoxycarbonyl) benzoic acid, 3,71115-tetramethyl-1-hexadecanol, and benzene-1-carboxylic acid-2-hexadeconate. These compounds were evaluated for antiviral potential against Asian and African strains of CHIKV by MTT assay using ribavirin as a positive control. These compounds showed selectivity indices 136, 156, 116, and 11.88, 2.11, 4.66 to Asian and African strains, respectively.

Baicalein, fisetin, and quercetagetin

Lani et al.[48] evaluated the antiviral activity of these flavonoids against the ECSA genotype of the CHIKV in Vero cells using various antiviral assays. Baicalein, fisetin, and quercetagetin exhibited strong antiviral activity, with IC50 values of 1.891 mg/ml, 8.444 mg/ml, and 13.85 mg/ml, respectively, and without significant cytotoxicity. The results of different antiviral assays revealed that baicalein and quercetagetin mostly reduced virus attachment to Vero cells in a dose-dependent manner, indicating extracellular anti-CHIKV action [Table 4]. The Western blotting data suggested that fisetin inhibited the translation of nonstructural proteins which reduced the production of replicas units. The western blotting analysis also provided pieces of evidence of intracellular anti-CHIKV activity of these flavonoids.

Table 4

Antichikungunya activity of baicalein, fisetin, and quercetagetin in Vero cells[48]

Compound	MNTD (µg/ml)	CC50 (µg/ml)	IC50 (µg/ml)	Selectivity index
Baicalein	183.2	356.3	1.891	188.4
Fisetin	>100	194.4	8.444	23.02
Quercetagetin	173.5	226.7	13.85	16.3

MNTD=Maximum nontoxic dose

Polyherbal Formulation

Nilavembu Kudineer

Nilavembu Kudineer is a polyherbal formula with A. paniculata as its main herb. The other component plants are Cyperus rotandus, Mollugo cerviana, Piper nigrum, Santalum album, Trichosanthes cucumerina, Vetiveria zizanioides and Zingeber officinale. Ethanolic extract of Nilavembu Kudineer showed a protective effect against CHIKV infection in Vero cells.[49]

Discussion

The newly emerging arboviral diseases like Dengue, CHIK, Zika, have raised a serious health concern over the years.[4050] Cross countries travel and climate change increases the chances to further import these infections in new areas thus need necessary attention and research to avoid serious human threats.[21] The scientific community all over the world is trying to develop a solution through different mechanisms. In these efforts, many medicinal plant extracts and their phytochemicals have demonstrated potential efficacy through in-vitro models depicting inhibition of CHIKV via different antiviral modes of action.[13282930333151]

Despite numerous efforts made to examine the antiviral activity, still, efficacy and toxicity assessment demand further investigations and evidence. As exhibited in Tables 1–3 and Figure 1, efficacy and toxicity fluctuate significantly relying upon the solvent extracts and assay methods. Some solvent extracts like alcoholic and aqueous extracts (mostly containing flavonoids) found more efficacious in comparison to other solvent extracts almost in all the studies [Figure 1]. Only two chloroform and two ethyl acetate extracts showed significant anti-CHIKV activity. The efficacy and toxicity are estimated based on % viral inhibition and selectivity index (SI) which in turn are significant for choosing extracts or phytochemicals to analyze in-vivo. So far, only a SI value ≥10 is typically considered for animal studies and a characterized cut-off value yet to be decided.[6] Future research on a more thorough validation of evaluation parameters for determining efficacy and toxicity of extracts or phytochemicals in in-vitro studies (e.g. multiple assay methods) might help in the success of them in-vivo assays and accelerate the drug development for CHIKV infection.

Figure 1

Comparison of the anti-chikungunya activity of extracts of different solvent

Although extracts are a rich source of chemical diversity, as all the flavonoid-rich extracts[2830] and even some isolated flavonoids like silymarin,[10] nobiletin,[45] baicalein, fisetin, and quercetagetin[48] exhibited potent anti-CHIKV activity, it is believed that the presence of flavonoids in extracts would have contributed significantly to anti-CHIKV activity. This belief is further supported by the fact that flavonoids show antiviral against other arboviruses (eg., CHIKV, Dengue, and Zika virus).[10454852] Therefore, screening of flavonoids containing plants would be more fruitful for the discovery of new leads for anti-CHIKV drug development. Further, it will also be prudent to screen many other antiviral phytochemicals such as silymarin, nobiletin, Curcumin, and resveratrol which have already been approved for various viral sicknesses.[104245]

Aside from showing antiviral action against CHIKV, silymarin, resveratrol, baicalein, fisetin, Curcumin, and ECGC additionally show antiviral action against Dengue or Zika virus or both in vitro.[425253545556] Hence, further research on these phytochemicals would be ideal as CHIKV usually co-circulates and simultaneously co-infects with these arboviruses (e.g. Dengue or Zika virus) which are also transmitted by the same vector (Aedes species).[181957]

Conclusion

Scientific studies have shown the therapeutic effectiveness of medicinal plants and few phytochemicals with sufficient future potential against the CHIKV. Further studies for their therapeutic efficacy in animal models, safety profiles, and mechanisms of action at the molecular level may lead to finding out a suitable remedy.

Financial support and sponsorship

As mentioned above.

Conflicts of interest

There are no conflicts of interest.