Therapeutic Potential of Medicinal Plants against Dengue Infection: A Mechanistic Viewpoint.

Dengue is a tropical disease caused by the Dengue virus (DENV), a positive-sense, single stranded RNA virus of the family Flaviviridae, which is transmitted by Aedes mosquitoes. The occurrence of dengue has grown dramatically around the globe in recent decades, and it is rapidly becoming a global burden. Furthermore, all four DENV serotypes cocirculate and create a problematic hyperendemic situation. Characteristic symptoms range from being asymptomatic, dengue fever to life-threatening complications such as hemorrhagic fever and shock. Apart from the inherent virulence of the virus strain, a dysregulated host immune response makes the condition worse. Currently, there is no highly recommended vaccine or therapeutic agent against dengue. With the advent of virus strains resistant to antiviral agents, there is a constant need for new therapies to be developed. Since time immemorial, human civilization has utilized plants in traditional medicine to treat various diseases, including infectious viral diseases. With the advancement in molecular biology, cell biology techniques, and bioinformatics, recent studies have tried to provide scientific evidence and determine the mechanism of anti-dengue activity of various plant extracts and plant-derived agents. The current Review consolidates the studies on the last 20 years of in vitro and in vivo experiments on the ethnomedicinal plants used against the dengue virus. Several active phytoconstituents like quercetin, castanospermine, α-mangostin, schisandrin-A, hirsutin have been found to be promising to inhibition of all the four DENV serotypes. However, novel therapeutics need to be reassessed in relevant cells using high-throughput techniques. Further, in vivo dose optimization for the immunomodulatory and antiviral activity should be examined on a vast sample size. Such a Review should help take the knowledge forward, validate it, and use medicinal plants in different combinations targeting multiple stages of virus infection for more effective multipronged therapy against dengue infection.

Introduction

Dengue is a widespread viral infection caused by the four dengue virus serotypes, DENV1, DENV2, DENV3, and DENV4. DENV is a member of the genus Flavivirus, family Flaviviridae, and is transmitted to humans via biting of an infected female Aedes aegypti mosquito, the primary vector species.[1] In the mosquito, the virus replicates in the midgut cells and then is released into the hemocoel and disseminates to other tissues via hemolymph, ultimately reaching the salivary glands and releasing virions in the next bite to the human host. The person gets symptoms of dengue fever 5 days after being bitten by an infected mosquito, and these symptoms might last a week or more.[2] Before 1970, only nine countries had experienced severe dengue epidemics. At present, the disease is endemic in more than 100 countries. According to a recent estimate, around 390 million dengue infections could occur every year, of which 96 million manifest clinically. The Dengue virus infection poses a threat to 3.9 billion individuals in 128 nations.[3] Almost 75% of the global population exposed to dengue live in Asia-Pacific.[4] Early symptoms of a dengue-infected individual include fever, headache, rash, nausea, and joint and musculoskeletal pain.[5] The primary manifestations of the disease include capillary leak syndrome or plasma leakage due to endothelial cell dysfunction, hemorrhagic tendencies, leukopenia, and thrombocytopenia, which is mainly seen in dengue hemorrhagic fever (DHF).[6] The most severe Dengue shock syndrome (DSS) stage occurs during or shortly after symptom onset and is accompanied by transient hypotension or a weak pulse (less than 20 mmHg) with cold, clammy skin (in the early stage of shock), later being fatal in a few cases.[7] The WHO guidelines for 2009 state that a rapid decline or platelet count below 150 000/mm3 of blood is one of the indicators of clinical dengue worsening.[8]

Virus Structure, Function, and Life Cycle

DENV is enveloped, positive-sense, single-stranded RNA virus which has four closely related serotypes (DENV-1 to -4) that are antigenically distinct. Each DENV serotype is subdivided into different genotypes and clades based on the divergence of viral gene sequences.[9,10] Around 10.9 kb of the DENV genome encodes one open reading frame (ORF) with three structural (capsid, precursor membrane, and envelope) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural protein envelope glycoprotein (E) is the main target for neutralizing antibodies and involves receptor binding and entry to the host cell by fusion.[11]

The first step of virus infection involves attachment of DENV to the host cell surface and entry by receptor-mediated endocytosis. The DIII domain of E protein is the receptor-binding domain that binds to the host receptor, like DC-SIGN, mannose receptor, heparin sulfate, and many others. In the endosome, acidic pH results in activation and trimerization of the E protein in the virion, resulting in the fusion of the viral and cell membranes.[12] The nucleocapsid containing the RNA genome is released into the cytoplasm, where it gets translated into a single polyprotein that is processed cotranslationally and post-translationally by viral NS3 protein and host proteases. Replication of the genome occurs on intracellular membranes. Assembly of immature virus particles occurs on the surface of the endoplasmic reticulum.[13] Noninfectious, immature viral, and subviral particles are transported through the trans-Golgi network. The immature virion particles are then cleaved by the host protease furin, resulting in mature, infectious particles, which are subsequently released from the host cell by exocytosis.[14] In the immature virus, the prM protein forms protruding trimers with E, which creates a “spiky” appearance, whereas in the mature virion, the membrane protein (M) sits below the E protein with a “smooth” surface.[15]Figure illustrates the stages of the DENV life cycle and points where drugs can inhibit DENV infection.

Figure 1

Depiction of the life cycle of DENV and potential antidengue drug target sites. (1) Attachment: The E protein of DENV binds to a specific receptor of host cells. (2) Endocytosis: the virus is taken up by clathrin-mediated endocytosis. (3) Fusion: DENV E encounters acidic pH in the endosome, which changes the conformation of E protein catalyzing fusion of virus and endosome membrane and releasing DENV RNA into host cytoplasm. (4) Virus RNA translation and replication. (5) Assembly: virus proteins assemble with viral genome. (6) Maturation: E protein in immature virus in TGN is cleaved by host furin protease resulting in mature DENV. (7) DENV release by budding. (8) Host immune response.

Host Immune Response and Virus Pathogenesis

Viral RNA is sensed by two host sensors, one belonging to cytoplasmic retinoic acid-inducible gene I (RIG-I) like receptors (RLR) and another belonging to endosome toll-like receptors (TLR-3 and TLR-7). RIG-I and melanoma differentiation-associated protein 5 (MDA5) recruits the adaptor mitochondrial antiviral-signaling protein (MAVS) pathway to activate within the cytoplasm on dengue infection. This MAVS activation triggers phosphorylated TANK-binding kinase1 (TBK1) and IκB kinase-ε (IKKε) complex to phosphorylate interferon regulatory factors 3 and 7 (IRF3 and IRF7).[16] The phosphorylated IRF3 and IRF7 enter the nucleus to trigger the production of type I interferons (IFNs) such as IFN-β. The interferon (IFN) system is the primary host defensive mechanism by which the innate immune defense system gets activated against viruses. The IFN system consists of type I interferons (IFN-α and IFN-β), type II interferon (IFN-γ), and type III interferons (IFN-λ1–4), also known as IL-28A–C, IL-29. Type I IFNs (IFN-α and IFN-β) are the primary IFNs generated in almost all nucleated cells within hours of viral infections.

Meanwhile, dsRNA formed during viral replication activates endosomal primary TLR-3, which thereby causes phosphorylation of TIR-domain-containing adapter inducing IFNβ, interacting with TNF-receptor-associated factor 3 (TRAF3) and TBK1/IKKε to induce IFNα/β-stimulating genes (ISGs) and chemokines.[17] TLR7 recognition of ssRNA, including DENV genomic fragments, uses the myeloid differentiation primary response gene 88-dependent signal pathway (MyD88) by recruiting TRAF6 to activate inhibitor of nuclear factor-κB kinases (IKKα/IKKβ/IKKγ) and trigger nuclear factor-κB (NF-κB) to ultimately produce IFNα, IFNβ, and proinflammatory cytokines (TNFα and IL6).[15] Secreted type-I/III IFNs bind to their receptors IFNAR1/2, which further activates Janus kinase (Jak)–signal transducer and activator of transcription (STAT)-mediated signaling pathway leading to transcription of ISGs, which inhibit virus infection. The stimulation of IFN genes (STING), which function downstream of MAVS and upstream of TBK1, plays a vital role in activating IRF3 and nuclear factor-kappa B (NF-κB)[18] (Figure ).

Figure 2

Steps of human innate immune response after sensing dengue viral RNA inside the cell. (A) RIG-I mediated cytoplasmic response (B) Toll-like receptor (TLR-3 and TLR-7) mediated endosomal response. Abbreviations: ssRNA = single-stranded RNA; dsRNA = double stranded RNA; RIG-I = retinoic acid-inducible gene I receptor; MAVS = mitochondrial antiviral-signaling protein; sfRNA= subgenomic flavivirus RNA; IFN = Interferon; IRF = interferon regulatory factor; TRIF = TIR-domain-containing adapter-inducing IFNβ; TRAF = TNF-receptor-associated factor; MyD88 = Myeloid differentiation responsive gene 88; IKK = IκB kinase complex (α, β, γ, and ε); TBK = TANK-binding kinase; NF-Κb = Nuclear factor kappa B.

To counteract the host antiviral actions, DENV has evolved strategies targeting various immune defense system steps, from sensing the foreign DNA/RNA to the induction, signaling, and manipulation of the IFN system.[19] Dengue disease manifestation ranges from mild to severe cases. Several studies indicate that excessive inflammation contributes to the pathogenesis of severe dengue disease. DHF/DSS is thought to manifest in the context of amplified and unbalanced production of cytokines termed as cytokine storm. Dysregulated cytokines ultimately target the vascular endothelium and eventually lead to a transient increase in vascular permeability, the occurrence of hemorrhagic manifestations, and hemoconcentration, a hallmark of DSS. Many inflammatory cytokines and chemokines, especially IL-6, IL-8, IL-1β, IP-10, CCL8, CXCL9, CXCL16, and MCP-1, and immunosuppressive cytokines, especially IL-10, are elevated, resulting in increased vascular permeability in severe dengue cases[20] (Figure ). These complex interaction networks of several cytokines regulate the pathogenesis of dengue, and their fine modulation determines the disease outcome. The immunomodulation of the virus-induced hyperinflammation-caused severe cases of dengue are being explored for therapeutic intervention.

Figure 3

Immune response observed in mild and severe dengue disease.

Antiviral candidates are tested in vitro using various biochemical and cell-based assays, indicating their potential as therapeutic. A few of the most used assays are discussed below and with illustration in Figure .

Figure 4

Assays used for assessing antiviral activity.

Anti-Viral Assays

Cell-Based Assays

(21)

Cytopathic Effect

The cytopathic effect (CPE) refers to cellular changes induced by a virus that can be microscopically visible. The infected monolayer cells gradually deteriorate with the changes like swelling, shrinkage of cells, syncytia (cell–cell fusion), and inclusion bodies. The cytopathic effect of any agent is measured as the median tissue culture infectious dose (TCID 50). TCID 50 is defined as diluting a virus that can infect 50% of the given cell culture.

Plaque Assay

This is a classical virology technique used to quantify infectious viral particles and is based on the fact that viruses can induce cell lysis. In a plaque assay, serial dilution of the virus is inoculated to susceptible cells for a few hours. Cells are then covered with a nutrient medium containing agar or methylcellulose, restricting viruses released from infecting neighboring cells. Each infectious particle generates a plaque, a circular zone of infected cells. The plaque eventually becomes large enough to be seen with the naked eye. Dyeing agents for living cells are frequently utilized to improve the contrast between the living cells and the plaques, which will be transparent. Plaque assays are reported as plaque-forming units (PFU) per measure volume. The conventional plaque assay is time-consuming and labor-intensive, with low throughput. Therefore, a faster version of these assays has been developed, such as the focus forming assay.[22]

Focus Forming Assay

The focus forming assay relies on detecting viral proteins by immune staining techniques, offering the unique advantage of detecting viruses but not producing cell damage. The procedure is similar to plaque assay until overlaying stage. Focus assay utilizes fluorescently labeled antibody-based staining and may detect infected but not necessarily dead cells. For more quantitative analysis, flow cytometry is used to measure the ability of a virus to infect cells. Based on the same principle, microneutralization assays based on an ELISA, flow cytometry, and ELISPOT enhance sensitivity by using antibodies against DENV proteins inside host cells.[23−25]

Viral Replication

The inhibitor effect is also assessed by quantifying viral replication. Viral RNA is extracted from cells or supernatants using a viral RNA extraction kit. cDNA is synthesized using the reverse transcriptase (RT) enzyme. The cDNA is amplified using DENV-specific primers, and a fluorescent dye that intercalates between the DNA bases and binds within double-stranded DNA molecules. Real-time PCR is a quantitative PCR method because the magnitude of fluorescence can be detected at the end of each amplification cycle. A real-time-PCR read-out is given as Ct (cycle threshold) value, the number of PCR cycles for the fluorescent signal to cross the threshold value. Ct values are inversely proportional to the amount of nucleic acid present.[26,27]

In order to understand which step of viral lifecycle is blocked by the inhibitor, time of drug addition (TDA) experiments are performed. At different time points during virus infection, an antiviral agent is administered to the virus or host cells.[28] (1) Before viral infection; cells are pretreated with an antiviral agent to see whether the drug can block the viral receptor and inhibit viral attachment to the host cells. (2) Pretreatment of the virus with an antiviral substance, followed by inoculation of the treated virus to the cells, assesses the virucidal activity of the antiviral substance. The virucidal assay is performed to determine whether a test compound inactivates virus outside of cells or if the compound inactivates the virus before it infects the cells. (3) Cells are cotreated with virus and antiviral drug to investigate the antiviral effect on the virus entrance processes, including virucidal (neutralizing) activity and inhibition of viral attachment and penetration to the cells. (4) Treatment of antiviral agent after virus infection examines the antiviral effect during the postentry steps, such as translation and replication, virion assembly, and release from the cells.[29]

Biochemical Assays

NS3 and NS5 are DENV enzymes that have critical role in the DENV life cycle. Candidates with anti-NS5 or NS3 activity can be a potential therapeutic agent. These biochemical enzymatic assays are easy to perform in a test tube without requiring a cell culture facility or live virus and produce precise interpretable results.[30]

NS3 Activity

NS3 together with cofactor, NS2B, constitute a serine protease that cleaves viral polyprotein to individual proteins. The NS2B/NS3 protease complex is essential for viral replication and is a primary target for developing antidengue drugs. NS3 protein can be expressed in E. coli or baculovirus system and purified. NS3 activity is measured using a synthetic fluorogenic/chromogenic substrate containing amino acid sequence derived from the NS2B/NS3 site. The rate and amount of cleavage are evaluated spectroscopically. IC50 values are determined for inhibitors from substrate titration experiments performed in the presence of increasing inhibitor concentration using Dixon plots.[31,32]

NS5 Polymerase Activity

NS5 is a RNA-dependent RNA polymerase (RdRp) which replicates viral RNA. Recombinant NS5 RNA polymerase activity is evaluated by the elongation assay. The most frequent method used is the scintillation proximity assay (SPA). In this assay, a biotin-labeled primer is annealed to a poly rC template, and the primer extension is initiated in the presence of 3H-GTP and NS5 polymerase. The newly synthesized RNA incorporating radioactive GTP is captured through biotin binding to streptavidin-coupled SPA beads, and a liquid scintillation counter detects the captured radioactivity. Other variations give fluorescence-based read-out instead of radioactivity.[33]De novo RNA synthesis of NS5 is measured using a DENV-2 sub genomic RNA template.

Apart from evaluating the direct effect of the inhibitor on virus infection, there is a search for inhibitors that could modulate DENV-induced hyperimmune response, thus alleviating the “cytokine storm” seen in severe dengue cases. In order to test whether an inhibitor has an immunomodulatory effect, cytokine levels of the host in the presence of an inhibitor on dengue infection are measured by cytokine assay. The levels of cytokines in virus-infected supernatant or patient serum are quantified by cytokine sandwich ELISA which detects and quantifies the concentration of soluble cytokine and chemokine proteins. Cytokine gene transcription levels inside cells can be measured by RT-PCR using cytokine-specific primers.[34]

In Vivo Studies

Since there is no appropriate animal model to imitate dengue sickness, particularly the severe forms (DHF/DSS), it is a major technical impediment to understanding the etiology and testing of any prospective candidate for DENV infection. Nonhuman primates show viremia on dengue infection but do not show any clinical signs. Also, the use of primate species has limitations, like the high cost and challenges of managing them. There is no appropriate mouse model for dengue since rodents do not exhibit dengue virus replication efficiently. The ICR suckling mouse was the initially used model obtained by serial passaging of DENV in the brain (intracranial), resulting in DENV-induced encephalitis and paralysis.[35] It is still used; however, the biological relevance is questionable since DENV primarily infects the brain, whereas in reality, DENV brain infection is rare. Dengue infection in immunocompetent mice like Balb/C, A/J, and C57BL/6 is rare. However, inoculation with high viral load (VL) in C57BL/6 detects virus in serum, liver, and the brain, though the viral load is low. Artificial viremia induced in mice by inoculation with K562-infected cells has been reported and used.[36] Humanized mice animal models transplanted with human cells targeted explicitly by the virus during natural infection have developed clinical signs of DF like fever, erythema, and thrombocytopenia.[37] DENV subverts interferon signaling in humans, which is not achieved in mice, and therefore, it is unable to replicate. AG129 mice are deficient in IFN alpha/beta and gamma signaling, so when infected with DENV, clinical isolates show VL in serum, liver post 3 days infection (DOI), and death on day 7 with paralysis because there is a high viral load in the CNS.[38]

Medicinal Plants in Dengue Therapeutic Research

Medicinal plants have been widely used throughout the world, especially in developing countries, to treat various infectious and noninfectious ailments. Plant-based natural medicinal product usage is becoming popular even in developed countries. As per World Health Organization (WHO) estimates, 80% of the world’s population fulfills their healthcare needs from phytomedicinal sources.[39] This is because plants and their derived products are often seen as natural, readily available, and having few side effects. Natural products obtained from plants have long been one of the most important sources of “lead” compounds for the pharmaceutical industry, with up to 40% of modern medicines obtained from natural sources employing either the natural substance or a synthetic version. Medicinal plants include a variety of chemical compounds with a variety of biological capabilities, including the potential to inhibit the replication cycle of various types of viruses. Until now, in the treatment of dengue, no licensed drug is available that targets the virus. The emergence of viral resistance to antiviral medicines necessitates discovering new efficient antiviral drugs. Various plants are reported in various traditional medicine systems to combat the dengue disease.

Traditional plants like Carica papaya and Euphorbia hirta have been used in different countries based on interpretation of the results they have found for generations.[40] However, proof of concept and knowledge of the mode of action is lacking. Today for any agent to be used as any useful therapeutic product, it should be adequately quantified for its toxicity and efficacy and validated using modern lab methods to establish the dosage, the toxicity, and the mode of action. Also, the validation should be done in vivo and ex vivo in animal models mimicking the disease. In the last two decades, various studies using plants and their derived compounds have been conducted for their antiviral activity, including the dengue virus. Most of the research has been done in cell lines, with only a few in vivo validations. In vitro cell culture models are the primary screening point for the search for any inhibitor. The following sections describe extracts or/and isolated compounds from various families of medicinal plants that have been reported with antidengue activity. Table summarizes the studies conducted in cell lines and a biochemical assay system. Table presents studies of medicinal plants conducted on in vivo systems, and Figure illustrates the active compound with antidengue activity discussed in this paper.

Table 1

Medicinal Plants and Their Isolated Compound Exhibiting Anti-Dengue Activity Performed in Cell Culture in Vitro System with the Assay System Used and Probable Mode of Action

s. no.	plant name/part used/family	extract used/active constituents	assay method/mechanism of action	targeted DENV serotype and cell line used	study parameters	references
1	Androgra phis panic ulata, aerial part, Acanthaceae	methanolic extract	assay: CPE	DENV-1, Vero cells	MNTD = 0.050	(43)
2	Androgra phis paniculat a whole plants, Acanthaceae	andrographolides	assay: flow cytometry, RT- PCR, TDA	DENV-2, DENV-4, HepG2 and HeLa cells	EC50 = 21.3 μM	(70)
			MOA: tnhibition at the postinfection stage, virus budding and secretion
3	Acacia catecu, herb powder, Leguminosae	peptide extract	assay: FFU assay, TDA assay	DENV-1, DENV-2, DENV-3, DENV-4, Vero cells and Huh7 cells	IC50 = 0.18 μg/mL	(57)
			MOA: binding of the peptide to the virus inhibition at the early step of infection.
4	Acoruscalams, leaves, Acoraceae	methanolic extract	assay: FFU assay	DENV-2, Huh7it-1 cells	CC50 = 424.93 μg/mL	(48)
5	Anacolosa pervilleana, leaves, Asclepiadaceae	methanolic extract	assay: NS5 polymerase assay	DENV-1, DENV-2	IC50 = 3 μM	(82)
			MOA: inhibited DENV NS5 polymerase activity
6	Arrabidaea pulchra, leaves, Bignoniaceae	ethanolic extract	assay: MTT assay	DENV-2, Vero and LLCMK-2 cells	EC50 = 46.8 ± 1.6 μg/mL	(52)
7	Azadarachta indica, leaves, Meliaceae	azadirachtin	assay: CPE and viral RNA RT- PCR	DENV-2, C6/36 cells	MNTD = 1.897 mg/mL	(51)
		aqueous extract
8	Alternanthera philoxeroides, Amaranthaceae	petroleum ether	assay: MTT assay and CPE	DENV-2, C6/36 cells	TD50 = 47.43	(42)
9	Allium sativum, Amaryllidaceae	diallyl disulfide (DADS)	assay: viral RNA RT-PCR, cytokine ELISA	DENV-2, Huh-7 cells	reduced inflammatory cytokines (TNF- α, IL-8, IL-10) in DENV-2 infection	(75)
			MOA: immunomodulatory activity
10	Basilicum polystach yon, Lamiaceae	-	assay: plaque assay	DENV-1, DENV-2	CC50 = 96 ± 17 μM	(83)
				Vero cells	IC50 = 1.4 ± 2/1 μ M
11	Boesenbergia rotunda, rhizomes	methanolic extract	assay: NS2b/NS3 protease assay	DENV-1	Ki = 21 and 25 μM for 4- hydroxypan duratin A and panduratin A, respectively	(84)
			MOA: inhibit NS3 protease activity
12	Castanospermum austral, seeds	castanospermine	Assay: plaque, flow cytometry, subgenomic replicon assay	DENV-1, DENV-2, DENV-3, DENV-4, BHK-21 and Huh-7 cells	IC50 = 85.7 μM (Huh-7 cells), IC50 = 1 μM (BHK-21 cells)	(73)
			MOA: inhibit secretion of virus particles
13	Cissampelos pareira, aerial part	methanolic extract	assay: plaque assay, NS1 ELISA, cytokine ELISA, MOA: Virucidal effect	DENV-1, DENV-2, DENV-3, DENV-4, Vero, LLCMK, and C6/36 cells	IC50 ≤ 25 μg/mL	(76)
14	Cladosiphonokamuranus	Fucoidan	assay: FFU assay, dye-labeled virus binding assay	DENV-2, DENV-3, DENV-4, BHK-21 cells	IC50 = 4.7, 500, and 365 μg/mL for DENV-2, 3, and 4, respectively	(58)
			MOA: inhibition of virus entry
15	Coptis chinensis	-	assay: MTT assay	DENV-2, Vero cells	EC50 = 26.4 μM	(64)
			MOA: blocking the activity of NS3 protease,
16	Cryptocar ya chartacea, bark	chartaceones A–F	assay: NS5 polymerase assay	DENV-2	IC50 = 1.8 −4.2 μM	(85)
			MOA: inhibit NS5 polymerase activity
17	Cryptone mia crenulata, seaweed	ethanolic extract	assay: plaque assay and TDA	DENV-2, DENV-3, DENV-4, Vero cells	IC50 = 1 μg/mL (DENV-2), IC50 = 13.9–14.2 μg/mL (DENV-3) and IC50 = 29.3–50 μg/mL (DENV-4)	(67)
			MOA: inhibit virus adsorption and host cell internalization
18	Cymbopogon citratus, root	methanolic extract	assay: FFU assay	DENV-2, Huh7it-1 cells	CC50 = 183.74, EC50 = 29.37 μg/mL	(48)
19	Distictellaelonge, leaves, fruit	ethanolic extract	assay: MTT assay, antioxidant activity	DENV-2, Vero and LLCMK2 cells	EC50 = 11.1 ± 1.6 μg/mL (fruit extract)	(52)
			EC50 = 9.8 μg/mL (leaf extract)
20	Flacourtia ramontchi, stem bark	betulinic acid 3β-caffeate	viral RNA replication/RNA dependent RNA polymerase (RdRp) polymerase assays	DENV RNA	IC50 = 0.85 ± 0.1 μM	(82)
				polymerase, Vero cells
21	Ficus septica, fruit hartwood stem, and leaves	methanolic extract	assay: immunofluorescence	DENV-1 and DENV-2, A549, HepG2, HuH7.1 cells	IC50 = 3.05–100 μg/mL	(44)
22	Gastrodia elata, WSS45-sulfated derivative of α-d-glucan	-	assay: MTT, RT PCR, plaque assay, TDA	DENV-2, BHK-cells	EC50= 0.68 ± 0.17 μg/mL	(64)
			MOA: inhibit early stage of virus life cycle
23	Gymnogo ngrus torulosus, red seaweeds	dl-galactan hybrid	assay: plaque assay and TDA	DENV-2, Vero cells	IC50 = 0.19–1.7 μg/mL	(59)
			MOA: virucidal effect, inhibit early stage of virus infection- binding of the virus to receptor
24	Garcinia mangosta na, pericarp of fruit	ethanolic extract, α-mangostin	assay: immunofluorescence, flow cytometry, cytokine transcription	DENV-1, DENV-2, DENV-3, DENV-4, HepG2, Huh-7 cells	IC50 = 20 μM; also significant reduction of cytokine (IL-6 and TNF-α) and chemokine (RANTES, MIP-1β, and IP-10)	(78)
			MOA: immunomodulation
25	Hedyotis auricularia, Hemigrap hisreptans, leaves stem and roots	ethanolic and methanolic extracts	assay: plaque assay, RT-PCR, NS3 protease activity	DENV-2 NS2B-NS3 protease, Vero cells	IC50 ≤ 100 μg/mL	(56)
			MOA: inhibited NS3 protease activity
27	Hippophae rhamnoides, leaves	ethanolic extract	assay: plaque assay, cytokine ELISA,	DENV-2, human macrophages	IC50 = 50 μg/mL; also significant decrease in DENV infection and the release of cytokines	(86)
			MOA: immunomodulation, decreased proinflammatory cytokines, TNF-α and IL-10
28	Houttuyni a cordata, whole plants	aqueous extract	assay: viral RNA RT PCR, plaque assay, TDA assay	DENV-2, HepG2 and LLC- MK2	EC50 = 0.8 μg/mL	(62)
			MOA: decreased viral RNA production, virucidal and early stages of infection
29	Leucaena leucocephala, seeds	aqueous extract	assay: MTT assay and immunofluorescence	DENV-1, C6/36 cells	at 37 mg/L, a 100-fold decrease in virus titer	(45)
30	Laurentia longiflora, leaves, stems, and roots	ethanolic and methanolic extracts	assay: plaque assay, RT-PCR, NS3 protease activity	DENV-2, Vero cells	IC50 ≤ 100 μg/mL	(56)
			MOA: inhibited NS3 protease activity
31	Mimosa scabrella, seeds	sulfated galactomannans	assay: MTT, assay and immunofluorescence	DENV-1, C6/36 cells	at 347 mg/L, a 100-fold decrease in virus titer	(45)
32	Momordic a charantia, roots and entire fruits	methanolic extract	assay: MTT and CPE	DENV-1, Vero and E6 cells	MNTD = 0.20 mg/mL	(43)
33	Myrtopsis corymbosa leaves and barks	ethyl acetate extract	viral RNA replication,	DENV-2	at 1 μg/mL, inhibition of NS5 RdRp activity by 87%	(87)
			assay: NS5 polymerase activity
			MOA: inhibited RNA dependent RNA polymerase (DENV-NS5 RdRp) activity
34	Mammea Americana, Seed	ethanolic extract coumarin[18] A and B	assay: real-time PCR, TDA	DENV-2, Vero cells	EC50 = 9.6 μg/mL (coumarin A) EC50 = 2.6 μg/mL (coumarin B)	(88)
			MOA: effective at both pre and post-treatment
35	Tabernaemontana cymosa, seed	ethanolic extract lupeol acetate and voacangine	assay: real-time PCR, TDA	DENV-2, Vero cells	EC50 = 37.5 μg/mL and 10.1 μg/mL, for lupeol acetate and voacangine, respectively	(88)
			MOA: effective at post-treatment
36	Myristica fatua	methanolic extract	assay: FFU assay	DENV-2, HuH7it-1cells	EC50 = 25.33 μg/mL	(48)
37	Nephelium lappaceum, rind	geraniin	assay: MTT assay, plaque assay, time-of-addition assay, virucidal assay	DENV-2, Vero cells	IC50 = 1.75 μM	(68)
			MOA: inhibits binding of the virus to the receptor at early stages of infection
			ELISA competitive binding assay confirmed geraniin interaction with rE-DIII with high affinity
38	Ocimum sanctum	methanol extracts	assay: CPE, plaque assay	DENV-1, HepG2 cells	MNTD = 23.44 μg/mL there was 75% inhibition	(53)
39	Phyllanthus amarus, whole plant	methanolic extract	assay: plaque assay, NS1 ELISA, cytokine ELISA	DENV-1, DENV-2, DENV-3, DENV-4, Vero, LLCM, and C6/36 cells	IC50 ≤ 25 μg/mL	(76)
			MOA: Virucidal effect
40	Psidium guajava, bark	ethanolic extracts catechin	assay: FFU assay, TDA	DENV-2, Vero cells	CC50 = 1000.0;	(89)
			MOA: catechin inhibited both at early and late stage of infection		EC50 = 7.8
41	Polygonum cuspidatum, rhizomes	methanolic extract	assay: flow cytometry-based viral infection assay, cell–cell spread assay, TDA	DENV-2, Vero, and Huh-7 cells	CC50 = 227.7 ± 1.1 μg/mL, E50 = 8.1 ± 1.0 μg/mL, SI = 28.1	(66)
			MOA: virucidal activity, Block the viral attachment and entry/fusion
42	Persea Americana, fruit	(2 R,4 R)-1,2,4-trihydroxyheptadec-16-yne (THHY) extracted from fruit	assay: DENV RT PCR, Western blot of cytokine protein, ISER- driven luciferase reporter assay	DENV-1, DENV-2	EC50 = 10.98 ± 1.9 μM	(81)
			MOA: immunomodulation	DENV-3
				DENV-4, Vero cells
43	Quercus lusitanica, gall	crude methanol extracts/methyl gallate	assay: MTT, CPE and NS2b/NS3 protease assay	DENV-2, NS3 protease, C6/36 cells	MNTD = 100 μg/mL showed a 96% inhibition at TCID50 = 1000 showed more than 98% inhibition at 0.3 mg/mL	(47)
			MOA: inhibition of NS3 protease activity
44	Senna angustifolia, plant leaves and stems	ethanolic extract	assay: Plaque assay, RT-PCR, NS3 protease activity	DENV-2 NS2B-NS3 protease, Vero cells	IC50 = 30.1 μg/mL	(56)
			MOA: inhibited NS3 protease activity
45	Schisandra chinensis	schisandrin-A	assay: viral RNA RT-PCR, plaque assay, cytokine RT-PCR	DENV-1, DENV-2, DENV-3, DENV-4, Huh-7 cells	EC50 = 28.1 ± 0.42 μM	(77)
			MOA: immunomodulation increased STAT1/2 phosphorylation, thereby increasing IFN-α gene expression
46	Syzygium samarangense, dried leaves	aqueous extract 5-hydroxy- 7-methoxy- 6-methylfla vanone (FN5Y)	assay: plaque, TDA, fusion inhibition assay	DENV-2 DENV- 4, Vero cells, and LLCMK-2 cells	EC50 = 15.99 ± 5.38 μM	(69)
			MOA: inhibition early stage of infection most likely at fusion stage		SI > 6.25 (LLCMK2 cells), EC50 = 12.31 ± 1.64 μM S I = 2.23 (Vero cells)
47	Trigonoste moncherrie ri, bark and wood	trigocherrin A1, A, and B	assay: NS5 polymerase assay	not mentioned	IC50 = 12.7, 3.1, and 16 μM for trigocherrin A1, A and B, respectively	(90)
			MOA: inhibit NS5 activity
48	Tridax procumbers, stem	ethanolic extract	assay: plaque assay, RT-PCR, NS3 protease activity	DENV-2	IC50 = 25.6 ± 3.8 μg/mL for NS3 protease activity	(56)
			MOA: inhibited NS3 protease activity
49	Tripterygiu m wilfordii, root	root extracts, celastrol	assay: Viral RNA RT PCR, host gene RT PCR, phosphoprotein Western blotting, plaque assay, cytokine ELISA	DENV-1, DENV-2, DENV-3 DENV-4, Huh-7 cells	IC50 = 0.08–0.19 μM	(91)
			MOA: immunomodulation, celastrol induces antiviral IFN-a gene expression and protein secretion
50	Uncaria tomentosa, stem barks	alkaloid fraction, hydroethanolic extract	assay: flow cytometry, cytokine ELISA	DENV-2, human peripheral blood monocytes	at 1 μg/mL, significant reduction in TNF and IFN production and a reduction in DENV infection	(79)
			MOA: immunomodulation, reduced TNF-α, IL-10, IFN- α production levels
51	Uncaria rhynchophylla	hirsutine	assay: FFU, TDA	DENV-1, DENV-2, DENV- 3, DENV-4, BHK-21, and A549 cells	EC50 = 10 μM	(72)
			MOA: inhibits the viral particle assembly, budding, or release step
52	Vernoniacinera, leaves	methanolic extract	assay: plaque assay, RT-PCR, NS3 protease activity	DENV-2	IC50 = 23.7 ± 4.1 μg/mL	(56)
			MOA: inhibited NS3 protease activity	NS3 protease
53	Zostera marina	zosteric acid, CF-238	assay: FFU, TDA	DENV-1, DENV-2, DENV-3 DENV-4, Vero cells	IC50 = 2.3 Mm (ZF)	(65)
			MOA: inhibition at an entry step in the viral life cycle		IC50 = 14–47 μM (CF 238),

Table 2

Medicinal Plants and Their Isolated Compound Exhibiting Anti-Dengue Activity and Study Parameters Performed in an In Vivo System

s. no.	plant species	extract/compound used	DENV serotype	study parameters	animal strain	references
1	Azadirachta indica	leaves’ extract	DENV-2	at MNTD of 120–30 mg/mL, the leaf extract inhibited DENV replication as observed by the absence of viral RNA by RT-PCR and DENV related clinical symptoms of mice	suckling mice	(51)
2	Carica papaya	freeze-dried Carica papaya leaf	DENV-2	DENV2 infected mice treated with 500 and 1000 mg/kg freeze-dried Carica Papaya leaf juice resulted in a decrease in inflammatory cytokines in the liver (CCL6, CCL8, CCL12, CCL17, IL1R1, IL1RN/IL1Ra, NAMPT and PF4/CXCL4)	AG129 mice	(92)
3	Castanos permum australe	castanospermine	DENV-1, DENV-2, DENV-3, DENV-4	A/J mice were infected with mouse-adapted DEN-2 via the intracranial route daily for 10 days. Mice were treated with a range of doses of 0.2, 1, 5 mg of castanospermine, gives survival rates of 25%, 90% and 85% in mice	A/J mice	(73)
4	Cissampel os pareira	methanolic extract of aerial part	DENV-1, DENV-2, DENV-3, DENV-4	mice infected with brain-adapted DENV were administered intraperitoneally with methanol free Cipa extract twice a day for 5 days; compared with the placebo-treated group, the level of protection (median survival time) by the 250 mg/kg dose was statistically significant (p = 0.021)	AG129 mice	(76)
5	Lonicera japonica	honeysuckle aqueous extract	DENV-2	treatment of aqueous honeysuckle extract before or after intracranial injection with DENV2 showed decreased NS1 RNA and protein expression levels accompanied by alleviated disease symptoms, decreased virus load, and prolonged survival time	ICR suckling mice	(93)
6	Schisandra chinensis	schisandrin A	DENV-2	schisandrin A decreases the mortality of DENV-infected ICR suckling mice, and the survival rate of DENV-infected mice treated with schisandrin A reached 80%	ICR suckling mice	(77)
7	Tripterygi um wilfordii	celastrol	DENV-2	celastrol at a concentration of 0.1 mg/kg protected 80% of the mice against infection-induced lethality and related illness; celastrol induced an antiviral interferon response with significant increase in IFN-α-2, IFN-α-5, gene expression levels	ICR suckling mice	(91)
8	Persea americana	1,2,4-trihydroxyheptadec-16-yne (THHY)	DENV-2	THHY (5 mg/kg) significantly decreased clinical scores (about 40%) and increased the survival rate (60%) of DENV infected mice as compared to control mice	ICR suckling mouse	(81)

Figure 5

Molecular structure of some phytoconstituents or active compounds with already known antidengue activity.

There are various reports with plant extracts and isolated compounds exhibiting inhibition of dengue infection. The following plants and their isolated compounds have been shown to have antidengue activity as assessed by CPE, plaque assay, or FFU assay, but the precise stage or mode of action was not investigated.

(family Acanthaceae) is a native herb of Southern and Southeastern Asia and is traditionally used for the common cold, throat infection, osteoarthritis, and ulcerative colitis.[41] A study by Tang et al. reported that a methanolic extract of the aerial part of A. paniculate at a maximum nontoxic dose (MNTD)- 0.050 mg/mL was able to show a 75% inhibitory effect against DENV-1 in Vero cells by the CPE assay.[42]

, a native species found in South America, are rich in coumarin. The coumarin extract [TD (50) = 535.91] and petroleum ether [ED (50) = 47.43] showed an inhibitory effect against DENV (serotype not mentioned) in the MTT assay.[42]

(bitter melon) belongs to the family Cucurbitaceae and is predominantly found in Asia, Africa, and the Caribbean. Leaves and green fruits have been used to fight cancer, diabetes, and many infectious diseases. A methanolic extract of Momordica charantia showed an inhibitory effect against DENV-1 at MNTD of 0.20 mg/mL in Vero E6 cells.[43]

, family Moraceae, is traditionally used as a folk medicine for headache, fever, rheumatoid, cold, cough, bacterial, and fungal diseases. Experiments conducted by Huang et al. demonstrated that methanol extracts of fruit, heartwood, leaves, and stem from Ficus septica inhibited DENV-1 and DENV-2 infection, with an IC50 of 3.05 ± 10.75uM.[44]

Ono and group reported that two galactomannans extracted from seeds of (BRS) and (LLS) exhibited antidengue activity against DENV-1 in C6/36 cells. BRS at a concentration of 347 mg/1 and LLS at 37 mg/L could reduce viral infection about 100-fold.[45]

(family Quercaceae) is a small tree or shrub found in the Mediterranean area, whose galls have been shown to have medicinal properties such as astringent and antidiabetic antipyretic and anti-Parkinsonian activities.[46] In C6/36 cells, crude methanol extracts of Q. lusitanica inhibited DENV-2 infection at a TCID50 of 1–1000. At a TCID50 of 1000, methyl gallate isolated from fractionated crude extracts of Q. lusitanica showed a 96% inhibition at the MNTD of 100 μg/mL.[47]

(family Acoraceae) has been used in traditional medicine for centuries to treat digestive disorders and pain. , commonly known as lemongrass, is native to Maritime Southeast Asia. (family Myristicaceae) seeds are used in traditional medicine to treat headaches and other sicknesses. A study by Rosmalena et al. revealed that the methanolic extracts of A. calamus, C. citratus, and M. fatua at a dose of 20 μg/mL completely inhibited DENV-2 infection. C. citratus and M. fatua had EC50 values of 29 and 25 μg/mL, respectively.[48]

(Cham.) Sandwith, found in savannas, is reported as anti-inflammatory, astringent, antisyphilitic, and antidiarrheal.[49] The ethanolic extract of leaves showed 80% inhibition at 100 μg/mL toward DENV-2 in Vero and LLCMK-2 cells in the MTT assay.[49] Bioguided fractionation of this extract revealed that arylpropanoid glycoside derivatives, verbascoside, caffeoyl callerianin, and a terpenoid, ursolic acid, are responsible for this antiviral activity.

or Neem (family Meliaceae) is mainly found in the Indian subcontinent. Traditionally it is used as an anti-inflammatory, antiarthritic, antipyretic, antifungal, and antibacterial agent.[50] An aqueous neem leaves’ extract at its MNTD of 1.897 mg/mL inhibited DENV-2 in C6/36 cells by 100–10,000 TCID50.[51] A bioguided isolation of fruit ethanolic extract of (Bignoniaceae) revealed that a mixture of pectolinarin and acacetin-7-O-rutinoside showed anti-DENV-2 activity with an EC50 11.1 ± 1.6 μg/mL.[52]

, popularly known as “holy basil”, has long been used to treat and prevent ailments like cough, fever, and ulcers. The methanolic extract exhibited inhibitory activity against DENV- 1 with a MNTD of 23.44 μg/mL and showed around an 8-fold reduction in plaque assay.[53] Stachyonic acid derived from displayed antidengue activity with an IC50 = 1.4 μM in Vero cells.[54]

belongs to the family Rubiaceae and is distributed in India. The bark of P. tomentosa is used to treat visceral blockages in children, either as a decoction or in pulverized form. Decoctions of leaves are used to relieve the aches of hemorrhoids. P. tomentosa acetone leaf extract showed inhibitory activity against DENV-1 in C6/36 cells at CC50 = 125 ug/mL using MTT assay.[55]

A study by Rothan et al. showed that a methanolic extract of leaves, an ethanol extract of stems, and to less extent an ethanolic extract of leaves at 50 μg/mL were able to reduce the CPE of the DENV-2-infected cells effectively. and extracts showed a considerable reduction in plaque formation of about 80.6% ± 6.1 and 64.0% ± 9.4, respectively.[56]

The secondary metabolites of medicinal plants comprise various compounds with a wide range of biological activities. The active compounds have been identified in a few studies and showed a wide range of activity against DENV. The isolated products belonged to various chemical classes such as sulfated polysaccharides, flavonoids, quercetin, alkaloids, and terpenes. The chemical structures of the compounds from plants mentioned here are shown in Figure . Recent studies with plant extracts and isolated compounds have explored the mechanism of action for its infection inhibitory activity. Employing a time-of-drug-addition assay, it is possible to understand the possible point of inhibition in a virus life cycle.

Plants and Their Phytoconstituents Inhibiting Virus Attachment or Entry to Cell

The virus’s attachment to the host cell membrane and its entry into the host cell is the earliest stage of any viral infection. Inhibitors acting at this stage can be most efficient in inhibiting virus infection.

(Family: Mimosaceae) is primarily found in India and Southeast Asia. It is used as a therapeutic for anti-inflammatory and antidiarrheal purposes. A study by Panya et al. reported that a crude peptide extract at 1.25 μg/mL could reduce virus production by 100-fold. Two isolated bioactive peptides at 50 μM could inhibit DENV foci formation by more than 90% and were found to be inhibitory against all four DENV serotypes. A time-of-drug-addition assay further revealed that inhibition occurred at the early stages of virus infection. The most effective peptide (designated Pep-RTYM) inhibited DENV infection with a half-maximal inhibition concentration of 7.9 μM.[57]

belongs to the family Chordariaceae. Fucoidan derived from Cladosiphon okamuranus was found to inhibit DENV-2 infection in BHK-21 cells by FFU assay.[58] A dye-labeled virus binding assay revealed that fucoidan inhibited the binding of the virus to the cell. Pujol and group revealed that the dl-galactan hybrids extracted from the red seaweed, , exhibits anti-DENV-2 activity in Vero cells with an IC50 of 0.19–1.7 μg/mL using plaque assay.[59] A time-of-drug-addition assay revealed that the compounds have virucidal activity and inhibit the binding of the viral protein to the receptor.

is a vegetable consumed by people in East and Southeast Asia. Its therapeutic use includes chronic sinusitis and allergy.[60,61] Aqueous extracts from Houttuynia cordata at concentrations as low as 10–40 μg/mL exhibited a significant protective effect by directly blocking the virus (virucidal) and decreasing virus replication.[62]

A sulfated derivative of an α-d-glucan, WSS45, from the significantly reduced DENV-2 infection in BHK cells with an EC50 value of 0.68 ± 0.17 μg/mL. A time-of-drug-addition assay revealed that drug inhibition occurred at the early stages of DENV infection.[63,64]

Zosteric acid (ZA) and an active compound CF-238, isolated from (marine eelgrass), exhibit antidengue activity. ZA displayed an IC50 of 2.3 mM against DENV-2. CF 238 showed IC50 values of 24, 46, 14, and 47 μM against DENV-1, DENV-2, DENV-3, and DENV- 4, respectively. A time-of-drug-addition assay with CF 238 showed inhibition at an early stage of the viral life cycle.[65]

(family Polygonaceae) is used to treat cough, hepatitis, jaundice, amenorrhea, leucorrhea, arthralgia, hyperlipidemia, scald, bruises, and snake bites. The methanolic extract from rhizomes (PCME) at 30 μg/mL inhibited DENV-2 infection without causing significant cytotoxicity. PCME displayed the virucidal activity of free virus particles and blocked the viral attachment and early entry/fusion events.[66]

Two sulfated polysaccharides, the kappa/iota/nu carrageenan G3d and the dl-galactan hybrid C2S-3 from the red seaweeds, and , exhibited antidengue activity in a plaque assay. The IC50 values were 1.0 μg/mL, 14 μg/mL, and 29.3–50 μg/mL with DENV-2, DENV-3 and DENV-4, respectively. A time-of-drug-addition assay demonstrated that G3d and C2S-3 inhibited only at the early stage of infection.[67]

of the family Sapindaceae is found in tropical countries, such as Malaysia, Indonesia, the Philippines, Thailand, and Southeast Asian regions. Geraniin is the primary compound and is an ellagitannin. Geraniin inhibited DENV-2 infectivity with an IC50 of 1.75 μM in plaque reduction assay. The mode of action was suggested to be during the attachment of the virus to the host cell at the early stages of infection. Geraniin binds to recombinant DIII of DENV envelop protein using competitive ELISA, and it was found that at the concentration of 26.3 μM, it was able to completely inhibit the attachment.[68] Novel flavanone derivative, 5-hydroxy-7-methoxy-6-methylflavanone (FN5Y), from the leaves of rose, inhibited DENV-2 infectivity with an EC50 and selectivity index (SI) of 15.99 ± 5.38 (SI > 6.25) μM and 12.31 ± 1.64 (SI = 2.23) μM in LLC/MK2 and Vero cell lines, respectively, and inhibited DENV-4 at 11.70 ± 6.04 (SI > 8.55) μM. A time-of-drug-addition study revealed that the maximal efficacy was achieved at an early stage of infection, which corresponded with pH-dependent fusion.[69]

Plants Inhibiting Virus Secretion or Release

Virus release from the host cell to infect more neighboring cells is critical to spreading infection. Inhibitors that could inhibit the assembly or secretion of the virus outside the host cell should contain the infection. Andrographolide derived from displayed a significant reduction in cellular infection and virus output with EC50 values of 21.3 and 22.7 mM for HepG2 and HeLa cells, respectively. Andrographolide’s activity was limited to postinfection stages like the viral particle assembly, budding, or release steps but not at the viral translation and replication steps in the DENV life cycle.[70,71]

Hirsutine from inhibited infection by all DENV serotypes at 10 μM. A time-of-drug-addition assay suggested that inhibition does not suppress viral RNA synthesis or translation but occurs at assembly, budding, or release of virions.[72] Castanospermine is a natural alkaloid obtained from the tree of the (black bean or Moreton Bay chestnut). Treatment of cells with castanospermine inhibited the yield of infectious DENV-2 in Huh-7 cell line with an IC50 of 85.7 μM and an IC50 of 1.0 μM in BHK-21 cells. There was reduced infectivity as measured by plaque assay but no change in replication as measured by RT-PCR thus confirming that inhibitory action was due to the reduction of secretion of viral particles from the host cell.[73]

Plants Inhibiting Virus Infection Due to the Immunomodulatory Effect

An uncontrolled and generalized inflammatory response called “cytokine storm” is observed in severe dengue patients (Figure ). Apart from directly decreasing dengue infection, controlling inflammatory responses through immunomodulation can be one way to prevent severe disease.

(Family Elaeagnaceae) is present in Europe and Asia. The alcoholic extracts of Hippophae rhamnoides leaves at 50 μg/mL significantly inhibited the DENV-2 infectivity in blood-derived human macrophages as assessed by plaque assay.[71,74] Furthermore, cytokine ELISA showed that there was also reduction in the secretion of inflammatory cytokines, TNF-alpha and IL-10.

or garlic (Family Amaryllidaceae) is used in high blood pressure, hyperlipidemia, and artherosclerosis.[74] Organosulfur compounds isolated from garlic, diallyl disulfide (DADS), diallyl sulfide (DAS), and alliin were able to significantly reduce inflammatory cytokines (TNF-α, IL-8, IL-10) on DENV-2 infection in Huh-7 and U937 cells. Lipid peroxidation and iNOS, an indicator of oxidative damage, was also reduced on compound treatment.[75]

A methanolic extract of (Cipa extract) and inhibited infection of all four DENV serotypes. Phyllanthus amarus exhibited IC50 of 3–20 μg/mL, and Cissampelos pareira displayed an IC50 of 1.2–11 μg/mL against all DENV serotypes. Pretreatment of DENV with an increasing concentration of Cipa extract revealed that inhibition is due to its direct virucidal effect. Further, Cipa extract efficiently suppressed the secretion of TNF-α and IL-1β levels, thus acting as an immunomodulating agent.[76]

(Turcz.) Baill is a widely used herbal medicine. It is mainly used as a sedative, analgesic, and antipyretic agent. It is also used to treat hyperlipidemia, heart conditions, and neurodegenerative diseases. Experiments by Yu et al. revealed that Schisandrin A could inhibit the replication of all four serotypes of DENV. Schisandrin A was most effective against DENV-2 with an EC50 of 28.1 ± 0.42 μM.[77] Schisandrin A significantly enhanced IFN-α gene expression in DENV infected cells. Schisandrin A also increased STAT1/2 phosphorylation. Downregulation of STAT1/2 using specific shRNAs restored DENV replication. Schisandrin A could increase the promoter activity of pISRE-Luc reporter plasmid carrying IFN-stimulated response element (ISRE)-driven firefly luciferase in the presence of DENV infection, thus confirming that Schisandrin A induces the antiviral interferon-stimulated gene expression for inhibition of DENV replication.

Bioactive compound α-mangostin (α-MG) from the pericarp of the mangosteen fruit (Garcinia mangostana Linn) inhibited DENV production and cytokine expression of HepG2 cells. Treatment of DENV-infected cells with α-MG (20 μM) significantly reduced the infection rates of all four DENV serotypes by 47–55%. Furthermore, α-MG could markedly reduce cytokine (IL-6 and TNF-α) and chemokine (RANTES, MIP-1β, and IP-10) transcription.[78]

(a woody vine) found in Amazon and Central American rainforests has been traditionally used to treat several diseases like gastritis, gastric ulcers, cancer, arthritis, asthma, and inflammatory disorders. Crude hydroethanolic extract and alkaloid fraction inhibited DENV-2 infection in human monocytes. A multiplex biometric immunoassay was employed for cytokine measurement. The alkaloidal fraction at 100 μg/mL showed immunomodulatory function inhibiting proinflammatory cytokines, TNF-α and IFN-α.[79]

Celastrol (quinone methide triterpene) from root extracts of (family Celastraceae) inhibited replication of all 4 DENV serotypes in the range of 0.08–0.19 μM. Celastrol is well-known for its immunomodulatory properties that boost the expression of IFN-2, IFN-5, and IFN-17 genes. Celastrol elevated STAT1/2 phosphorylation as measured by Western blot assay. A Jak1-specific inhibitor effectively attenuated the anti-DENV effect of celastrol. Silencing of STAT1 by shRNA resulted in decreased DENV replication. Celastrol induced antiviral IFN response through the JAK/STAT pathway.[77] (family Lauraceae), also known as avocado, is mainly found in tropical and subtropical regions. The fruit, stem, and leaf of avocado are widely used in ethnomedicine.[80] A study conducted by Wu and their group revealed that t(2 R,4 R)-1,2,4-trihydroxyheptadec-16-yne (THHY), extracted from avocado () fruit, inhibited replication of all DENV serotypes. THHY significantly suppressed DENV-2 RNA and protein synthesis, with an EC50 = 10.98 ± 1.9 μM. THHY treatment also increased STAT1 and STAT2 phosphorylation levels in the presence of DENV-2 replication.[81] Using a NF-κB promoter-based reporter assay it was revealed that THHY dose-dependently induced NF-κB promoter activity upon DENV infection. Further, THHY induced IFN-α RNA level and secreted IFN-α in DENV-2-infected cells.

Plants Inhibiting Virus at Both Entry and Exit of Host Cell

Some of the inhibitors act at multiple points of the virus lifecycle. An ethanolic extract from the bark of exhibited antidengue activity with CC50 1.0 mg/mL and EC50 7.8 μg/mL.[89] Isolated bioactive compounds from the bark are gallic acid, quercetin, and catechin (50 μg/mL), which inhibited the production of infectious DENV-2 viral particles. The compound catechin was an efficient inhibitor of both pre- and post-treatment stages.[89] Coumarin A and B isolated from seeds could inhibit DENV-2 infection at EC50 values of 9.6 and 2.6 μg/mL, respectively. The two compounds, lupeol acetate and voacangine, derived from the seeds, also significantly inhibited infection by DENV-2 in Vero cells with EC50 values of 37.5 and 10.1 μg/mL, respectively. Time-of-drug-addition assays revealed that coumarin A and B were effective as inhibitors in both pre and post-treatment experimental strategies, whereas lupeol acetate only significantly inhibited the DENV infection during the post-treatment strategy.[88]

As discussed previously, NS3 and NS5 are viral enzyme proteins critical for dengue virus replication and infectivity. Various compounds from medicinal plants have been studied for their inhibitory NS5 and NS3 activity.

Plants with NS3 Protease Inhibitory Activity

Compounds derived from were tested for inhibitory activities toward DENV-2 NS3 protease activity, using fluorogenic peptide substrate Boc-Gly-Arg-Arg-MCA. 4-Hydroxypanduratin A and panduratin A are cyclohexenyl chalcone derivatives that inhibited DENV-2 NS3 protease activity at Ki values of 21 and 25 μM amounts, respectively.[84] Pinocembrin and cardamonin were also able to inhibit NS3 protease activity though to a lesser degree, with Ki values of 345 ± 70 and 377 ± 77 μM, respectively. When used in combination, better inhibitory activity was seen, suggesting a synergistic effect. A mechanistic evaluation revealed that pinostrobin and cardomonin inhibited the NS3 protease via a noncompetitive mechanism while panduratin A and 4-hydroxypanduratin A inhibition was via competitive inhibition.

Rothan et al. screened around 19 extracts of different medicinal plants for potential NS3 inhibitory activity. Different extracts of , , and exhibited inhibitory activities against DENV-2 NS2B-NS3 protease with all displaying IC50 values <100 μg/mL. An ethanolic extract of leaves, a methanolic extract of leaves, and ethanolic extracts of stems were found to have the highest inhibitory activity compared with other extracts with IC50 values of 30.1 ± 3.4, 23.7 ± 4.1 and 25.6 ± 3.8 μg/mL, respectively.[56]

Plants with NS5 Polymerase Inhibitory Activity

(Family Rutaceae) are plants found in New Caledonia. Ethyl acetate crude extracts of barks and leaves of M. corymbose were found to have anti NS5 activity as assayed by monitoring the incorporation of radiolabeled guanosine into a homopolymeric cytosine RNA template.[87] Leaf and bark extracts at 10 μg/mL inhibited 78 and 92% viral RNA polymerase activity, respectively.

belongs to the family Olacaceae and is found throughout the tropics. Traditionally, it is used to treat schistosomiasis against syphyllis and general weakness. Active compounds were identified from leaf extracts showing anti NS5 polymerase activity. Acetylenic acids, the octadeca-9,11,13-triynoic acid, (13E)-octadec-13-en-9,11-diynoic acid, (13E)-octadec-13-en-11-ynoic acid exhibited significant inhibition of NS5 activity with IC50 values around 3 μM.[82]

Highly oxygenated daphnane diterpenoid orthoesters (DDO) with chlorinated moiety, Trigocherrin A and trigocherriolides A and B, isolated from the bark and the wood of , showed significant NS5 inhibitory activity with IC50 values of 12.7, 3.1, and 16.0 μM, respectively in NS5 polymerase assay.[90] An in vitro screening of bark of (new Caledonian plant) was done to identify compounds showing significant dengue virus NS5 inhibiting activity. Mono- and dialkylated flavanones, chartaceous A–F, along with pinocembrin exhibited significant NS5 inhibitory activity. Chartaceones C–F exhibited the most significant NS5 inhibiting activity, with IC50 ranging from 1.8 to 4.2 μM.[85]

Plants with Antidengue Activity: In Vivo System

After being tested in vitro, only few plant products have been taken forward for validation in in vivo models. (neem leaves) extract at its MNTD 120–30 mg/mL with 100 LD50 doses of DENV-2 inoculated intracerebrally in suckling mice, resulting in inhibition of the virus replication as exhibited by the absence of dengue-related clinical symptoms (characteristic Dengue specific weight loss, slow gait, inability to suck mother’s milk, and flaccid paralysis followed by death) and absence of virus-specific 511 bp amplicon in RT-PCR.[51]

A study conducted by Norahmad et al. revealed that freeze-dried leaf (FCPLJ) extract in AG129 mice has an immunomodulatory effect. The AG129 mice infected intraperitoneally with DENV-2 (2 × 106 PFU) were fed orally with FCPLJ (500 mg/kg of mouse body weight) for 3 consecutive days after 24 h of dengue virus inoculation.[92] The sign of illness was scored on the basis of lethargy, ruffled fur, moribund or paralyzed with limited activity, and inability to reach food-water. Plasma cytokines quantitation by immune assay showed that the treatment increased the plasma CCL2/MCP-1 level during the peak of viremia. RT-PCR gene expression analysis of liver showed downregulation of mice inflammatory cytokine genes- CCL6/MRP-1, CCL8/MCP-2, CCL12/MCP-5, CCL17/TARC, IL1R1, IL1RN/IL1Ra, NAMPT/PBEF1, and PF4/CXCL4 in FCPLJ treated infected mice compared with untreated.[92] A/J mice infected intracranially with 105 PFU of a mouse-adapted DENV-2 strain developed hind limb paralysis and succumbed to fatal central nervous system infection within 11 days of inoculation. Castanospermine isolated from seeds of was shown to have a protective effect in the mouse model. Castanospermine treatment prevented mortality in a mouse model of dengue virus infection, with doses of 10, 50, and 250 mg/kg (of mouse body weight) per day being highly effective at promoting survival (P < 0.0001).[73] AG129 mice were challenged with 106 PFU of DENV-2, which would be lethal 25 days postchallenge. Illness preceding death involved lethargy, ruffled hair, and hindlimb paralysis. Two hours after infection, a Cipa treatment of 10 mL/kg/dose was commenced and continued twice daily for 5 days. The median survival time (MST) of challenged mice orally treated with Cipa extract (methanol-free) twice a day for 5 days postchallenge was increased in a dose-dependent manner. The level of protection provided by the 250 mg/kg dose was statistically significant (p = 0.021) when compared with the placebo-treated group.[76]

Honeysuckle () aqueous extract before or after intracranial injection with DENV-2 in ICR suckling mice decreased clinical score significantly and prolonged survival time. In the study performed by Lee and group, ICR suckling mice were pretreated with aqueous extract of honeysuckle on day 4 after birth (twice a day) followed by intracranial (i.c.) injection of DENV-2 (2.5 × 105 PFU) on day 7, and all mice continuously drank aqueous honeysuckle extract until day 13. A decrease in viral load (42%) in the brain tissue and prolonged survival time was observed in the infected mice compared with the DENV-2-infected mice without extract treatment. Also, pretreatment of honeysuckle suppressed DENV-2 NS1 RNA and protein expression by 20% and 68%, respectively, in the brain tissue. In the post-treatment experiment, suckling mice were intracutaneously injected with DENV-2 (2.5 × 105 PFU) on day 6, followed by honeysuckle treatment starting on day 7, twice a day for 4 days. Honeysuckle treatment after DENV-2 infection significantly decreased the clinical scores of the infected mice on days 4 and 6 compared to the DENV-2-infected mice without honeysuckle from day 8 to day 10. Honeysuckle treatment resulted in a 27% reduction in NS1 RNA expression, a 52% reduction in NS1 protein expression, and a 30% viral titer in the brain tissue of the infected mice.[93]

Schisandrin, a bioactive compound derived from the fruit of , was found to reduce DENV-infected mice’s illness symptoms and mortality effectively. Schisandrin A treatment showed a lower clinical score than the control mice. The survival rate of DENV-infected mice treated with schisandrin A reached 80% at 6 dpi compared to control. Schisandrin A increased IFN-α-2 and IFN-α-5 RNA levels, thus suggesting that schisandrin A inhibits DENV replication by stimulating IFN-mediated antiviral responses in vivo.[77]

Celastrol isolated from roots of showed inhibition of DENV infection in vivo. Six-day-old ICR suckling mice (intracerebrally infected with 2.5 × 105 pfu of DENV-2) were treated with or without celastrol. Severe illness leading to death within 4–6 days dpi was noted in the DENV group. Celastrol at a concentration of 0.1 mg/kg protected 80% of the mice against life-threatening DENV-2 infection compared with the noncelastrol-treated mice. As observed in the in vitro studies, celastrol also showed IFN-modulating activity in vivo.[91]

(2 R,4 R)-1,2,4-trihydroxyheptadec-16-yne (THHY) extracted from Avocado fruit when infected in a DENV-infected ICR suckling mouse model resulted in an increased survival rate. Six-day-old ICR suckling mice infected with DENV-2 by intracerebral injection were treated with 5 mg/kg of THHY or saline. THHY significantly decreased clinical scores (about 40%) and increased the survival rate (60%) of DENV infected mice as compared with control mice.[81]

Molecules with anti-dengue activity belonged to all different classes of compounds, including phenolic derivatives (4-hydroxy panduratin, geraniin, αmangostin, methyl gallate), alkaloids (castanospermine, hirsutine, palmatine), flavonoids (chartaceones, pectolinarinandacactein-7-O-rotinosides, catechin, 5-hydroxy-7-methoxy-6-methyl flavanone), terpenoids (betulinic acid 3β-caffeate, lupeol acetate, celastrol, andrographolide), and polysaccharides (fucoidan, carrageenan, galactomanns).

Conclusion and Future Direction

There has been increased interest in plant products for their use in various ailments. Literature survey indicates that many recent studies report scientific evidence on their role and mechanism of inhibiting dengue virus infection in vitro and in vivo. The majority of studies have reported antidengue activity by decreasing dengue infection. Insight into the mode of action of antiviral agents is now almost a prerequisite for clinical development. Further, knowledge of the mechanism of action can aid in using a combination of plants that could be tested for inhibiting virus infection at various points. Taking these extracts or compounds forward in combination is essential for checking a synergistic effect. Most Southeast Asian countries are hyperendemic, with all four DENV serotypes circulating. Only few studies report the effect of plants using all four serotypes, and it would be of value if future studies were validated with all four DENV serotypes. In vitro methods are very cumbersome in virology, and there is a need to develop high-throughput assay systems where multiple products can be checked simultaneously, and there will be uniformity in the read-out. Also, antiviral assays should be done in more cell lines of physiological relevance like human monocytes. Apart from checking the effect on virus infection and host immune response, there are models available that check in vitro the vascular permeability before going to the in vivo model. Since dengue complication is because of hemorrhagic manifestation, such assays would indicate if any natural product can protect against vascular damage. Many in silico studies have been done with plant products on their potential binding and inhibition of dengue viral proteins. It is highly recommended that these compounds be validated in vitro and later in vivo.