Exploring plant-based dengue therapeutics: from laboratory to clinic

Abstract

Dengue virus (DENV) is a mosquito-borne virus that causes dengue fever, a significant public health concern in many tropical and subtropical regions. Dengue is endemic in more than 100 countries, primarily in tropical and subtropical regions of the world. Each year, up to 400 million people get infected with dengue. Approximately 100 million people get sick from infection, and 40,000 die from severe dengue. Unfortunately, dengue vaccine development is also marred with various complicating factors, as the forefront candidate vaccine performed unsatisfactorily. Moreover, the only licensed vaccine (Dengvaxia) for children 9 through 16 years of age is available in just a few countries. The treatment difficulties are compounded by the absence of an effective antiviral agent. Exploring plant-based therapeutics for dengue from the laboratory to clinical application involves a multi-stage process, encompassing various scientific disciplines. Individual investigators have screened a wide range of plant extracts or compounds for potential antiviral activity against DENV. In vitro studies help identify candidates that exhibit inhibitory effects on viral replication. Some of the most promising medicinal plants showing in vitro activity against DENV include Andrographis paniculate, Acorus calamus, and Cladogynos orientalis. Further laboratory studies, both in vitro and in animal models (in vivo), elucidate the mechanisms of action by which the identified compounds exert antiviral effects. Medicinal plants such as Carica papaya, Cissampelos pareira, and Ipomea batata exhibited potent platelet-enhancing activities while Azadirachta indica and Curcuma longa showed promising effects in both in vitro and in vivo studies. Based on positive preclinical results, researchers design clinical trials. This involves careful planning of trial phases, patient recruitment criteria, ethical considerations, and endpoints. The most important medicinal plants showing efficacy and safety in clinical trials include Carica papaya and Cissampelos pareira. This review suggests that several promising medicinal plants exist that have the potential to be turned into clinical drugs to treat dengue infection. However, in addition to developing synthetic and plant-based therapies against dengue infection, vector management strategies should be made robust, emphasizing the need to focus on reducing disease incidence.

Introduction

Viruses put an enormous burden on humanity and are the single most important source of infectious disease morbidity and mortality throughout the world [7, 31, 61]. Dengue virus (DENV) is a member of the Flaviviridae family and the flavivirus genus with four genetically related but different serotypes, DENV 1, 2, 3, and 4. Any one of the four DENV serotypes (DENV-1 to DENV-4) can cause an infection. The symptoms of dengue shock syndrome (DSS), which can result in major hemorrhage and shock and carry a 20% mortality risk if left untreated, are similar to those of dengue fever, including fever, nausea, vomiting, rash, and body pains [44]. DENV is spread by mosquitoes and affects millions of people each year. It is endemic in more than 100 countries, primarily in tropical and subtropical regions of the world. Almost 7.6 million dengue cases were reported to the World Health Organization (WHO), including 3.4 million confirmed cases with 16,000 severe cases, and over 3000 deaths. A notable rise has been observed in the Americas, where the number of cases crossed 7 million by the end of April 2024, as compared to the annual high of 4.6 million cases in 2023. Additionally, vector control and surveillance cost approximately 500 million dollars [107, 123].

Aedes aegypti, the primary vector, is a daytime mosquito that may infect multiple victims at once, and nests in artificial water-collection containers. Aedes albopictus, a less efficient vector, is expanding its geographic range into temperate and tropical areas. The main reasons for the development of Aedes mosquitoes and the increased incidence of dengue are population growth, high population density, rural-to-urban migration, deteriorated urban environments, lack of dependable piped water, and inadequate mosquito control programs [104]. The mobility of humans has risen as a result of modern transportation, which has contributed to the rapid spread of dengue viruses around the world [45]. The likelihood of dengue outbreaks in temperate zones is raised by both global warming and the spread of Aedes mosquitoes [128].

Vaccine development for diseases like dengue faces significant challenges, including the issue of Antibody-Dependent Enhancement (ADE), where antibodies from a previous infection may worsen a subsequent infection with a different virus type [117]. As far as antiviral agents are concerned, direct-acting antivirals (DAAs) are a promising alternative, but they are prone to resistance development. Host-directed antivirals (HDAs) offer another potential strategy by interfering with cellular pathways to disrupt the virus's replication environment. However, a narrower therapeutic window and interference with normal physiological function make it a less desirable candidate [117]. The alarming rise in dengue cases and glaring treatment gaps for its management are compelling researchers to look for plant-based therapeutic agents. In this review, many promising plant-derived treatments have been mentioned, like Andrographis paniculate, Carica papaya, and Curcuma longa [131]. These studies should be scaled up and replicated to combat the burgeoning threat of dengue, as climate change is only going to exacerbate this menace [12].

Epidemiology of dengue infection

Aedes mosquitoes are responsible for the transmission of dengue, a disease that affects over 3 billion people worldwide and is particularly prevalent in tropical and subtropical areas [132]. As of 30 April 2024, over 7.6 million dengue cases were reported to the WHO, including 3.4 million confirmed cases. Moreover, 16,000 severe cases and over 3000 deaths have also been reported. A notable rise in dengue cases has been observed at the global level in the last half a decade, particularly in the Americas, where the number of cases had already exceeded 7 million by the end of April 2024, going beyond the annual high of 4.6 million cases in 2023. Currently, 90 countries have known active dengue transmission in 2024, not all of which have been captured in formal reporting [123] affecting 5 billion people, and some places have mortality rates are extremely high. More than 100 countries are affected by dengue infections, including the United States, and Europe [92, 125]. In Pakistan, the incidence of dengue has risen dramatically over the past twenty years [10, 52, 137] becoming a serious public health issue due to high mortality, morbidity, and loss of productivity. The country has frequently reported severe dengue outbreaks, including in 1994, 1995, 1997, 2006, 2007, 2011, and 2019 [75]. The climate conditions of Pakistan provide a favorable breeding ground for DENV replication and transmission. The loss of human capital linked with the aftermath of the dengue epidemic is growing. 16,580 confirmed dengue cases, with 257 deaths, were registered in 2010. Whereas in 2019, 47,000 dengue cases and 75 deaths were recently reported [75]. Until November 2021, the country recorded a total of 24,146 dengue cases and 183 associated deaths [122]. Notably, the record-breaking rainfall in August of 2022 and subsequent floods catalyzed the increase in dengue cases in October 2022 [54, 113].

DENV genome, its proteins & their role in dengue infection

It is proposed that the cell and tissue tropism of DENV could profoundly influence the prognosis of DENV infections. In vitro, and autopsy studies indicate the critical role played by three organ systems in dengue pathogenesis: the liver, the immune system, and endothelial cell (EC) linings of blood vessels [74]. DENV has a single-strand positive-sense RNA genome of 11 kb (Fig. 1). The genome encodes a polyprotein that is translated into seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) and three structural proteins (capsid protein—CP, envelope protein—EP, membrane protein- MP). Non-structural proteins play vital roles in virus entry, replication, assembly, and pathogenesis. Moreover, non-structural proteins regulate DENV replication, influence immunological responses, induce vascular leakage, and assist in viral RNA synthesis and polyprotein cleavage. [60]. According to [19] Dengue severity arises from a complex interplay involving the virus, host genes (HLA, and IL-10), and immune responses (ADE, memory T cells).

Fig. 1

Dengue Virus Structure and Genomic Processing. An overview of the Dengue Virus (DENV), showing its structural anatomy with the RNA genome, capsid, envelope, and E & M proteins. It outlines the positive-sense RNA genome's division into regions coding for structural and non-structural proteins and details the translation and processing of these proteins from a polyprotein precursor into individual components crucial for the virus's life cycle

DENV is presumably injected following the mosquito bite into the epidermis and dermis of an organism. This results in the infection of immature Langerhans cells, also known as epidermal dendritic cells (DC) and keratinocytes [71, 129]. Moreover, DENV was found to not only activate the DCs but also disrupt their physiological dermal migration process [71]. Afterward, the infected cells leave the site of infection to the lymph nodes, initiating the recruitment of monocytes and macrophages, which fall prey to the infection. This leads to the spread of infection to viral dissemination through the lymphatic system. This primary surge in viremia infects several cells of the mononuclear lineage, including blood-derived monocytes [35], myeloid DC [24], and liver and splenic macrophages [23, 47]. It is noteworthy that during secondary infections with heterologous DENV, high concentrations of DENV-specific immunoglobulin G (IgG) form a complex newly produced virus, which adheres to and is subsequently taken up by the mononuclear cells [74]. Following infection, mononuclear cells predominantly die by apoptosis [36]. Abortively infected or bystander-DC releases several mediators that are involved in inflammatory [73] and hemostatic [48] responses of the host. In order to undermine the integrity of the endothelial cell monolayer in blood vessels, the DENV NS1 protein interacts with Toll-Like Receptors 4 (TLR4) activating macrophages and peripheral blood mononuclear cells (PBMCs). Apoptotic pathways are triggered and platelets are destroyed when NS1 proteins from cells infected with DENV interact with TLR4 on the platelets' plasma membrane. This happens because P-selectin is expressed more often [30]. Furthermore, stromal cells of bone marrow also demonstrated a susceptibility to DENV infection [82]. Some of the important players involved in dengue infection are outlined in Fig. 2.

Fig. 2

Pathophysiology of Dengue-Induced Inflammation and Shock. The pathophysiological mechanisms triggered by Dengue virus infection that lead to inflammation and potentially to cytokine storm and shock. Upon infection, the virus activates platelets and monocytes, leading to the release of inflammatory cytokines (IL-6, IL-10, IL-12, IL-18) and TNF-α. Macrophages produce nitric oxide, while mast cells release VEGF and secretory Phospholipase A2 (sPLA2). These mediators contribute to endothelial activation and NF-κB production, further promoting inflammation. The combined effects result in increased vascular permeability (plasma leakage), contributing to the development of shock

Important anti-dengue drug targets

Broadly, anti-viral targets for dengue treatment are classified as viral entry inhibitors, fusion inhibitors, capsid (C) protein storage inhibitors, viral assembly inhibitors, and viral maturation inhibitors [2]. Several studies have identified both structural and nonstructural proteins as important drug targets including the structural proteins (C-protein, E-protein, prM), as well as nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) [86].

Targeting structural proteins, the building blocks of viruses enervates and eventually kills the virus. Therefore, they can be promising drug targets for treating DENV infections [9]. Notably, E-protein has been the major target for vaccine development owing to its vital role in virus attachment and penetration into host cells [13, 70]. Another important drug target is prM. It is believed to be a chaperon for E-protein to ensure its conformational changes during different steps of the secretory pathway and to produce infectious viral particles, particularly blocking membrane fusion [136].

C-protein, located in the cytosol, is the first protein translated by the viral genome followed by prM. However, due to the least conservation of C-protein across DENV serotypes, this target is less favored [83, 103].

Non-structural NS1 is an enigmatic multifunctional, dengue glycoprotein. Due to the high conservation of NS1 among all DENV serotypes it is a favorable target for drug and vaccine development [80, 98]. Similarly, NS3 the extensively studied, multifactorial protein is among the most conserved DENV proteins. Notably, 77% of amino acids of NS3 are similar in all four serotypes, making it a potential target for drug and vaccine development [18]. NS4B, which is involved in host membrane modification, and essential for replication, emerges as one of the most promising drug targets as novel inhibitors targeting NS4B and its interaction network are entering clinical studies [68, 130]. Similarly, NS5 remains a crucial protein for DENV replication and pathogenesis. Targeting NS5 offers a strategic avenue for designing effective antiviral drugs [83].

Current treatments and associated challenges

The therapeutic vacuum in treating dengue infection is being filled by symptomatic treatment and fluid resuscitation methods due to the absence of a potent anti-dengue medicine. This makes dengue management more challenging. At the moment, vector control is the sole method for preventing disease, despite a poor cost–benefit ratio. Supportive care, including antipyretics, analgesics, and bed rest is the basic treatment for dengue patients. Urgent resuscitation with intravenous fluids is necessary for DSS patients, with Ringer's lactate being effective for mildly serious dengue and starch or dextran being preferred for more critical cases [96]. Severe dengue, characterized by plasma leakage, severe bleeding, or organ impairment, entails significant morbidity and mortality if not treated timely. Multi-organ impact of dengue can lead to problems like fluid overload and poor metabolism of analgesics, especially for patients with comorbidities [119].

The most commonly used antipyretic and analgesic is acetaminophen, which comes with its own set of challenges like liver and kidney toxicity [39]. Inhibitors of cyclooxygenase 2 may be helpful as anti-inflammatory and antioxidants, but they may also inhibit platelets [8, 15]. Moreover, ribavirin has been effective in treating a number of RNA viruses, however, it has not been successful in treating DENV, and it has a cytostatic impact on DENV-infected cells. Similarly, a promising alternative therapy called RNA interference (RNAi) destroys unnecessary genetic material to shield the host from viral infections. Notably, the RNAi machinery can boost HCV replication, and flavivirus RNA is resistant to RNAi [65]. Furthermore, prophylactic platelet transfusion is prohibited and is reserved for managing severe cases. In addition to the high costs of platelet transfusion, blood-safety concerns create more complications. Numerous studies have documented transfusion-transmitted dengue, with the first cases being reported in China in 2002 and Singapore in 2008 [78].

Dengue vaccine development is also marred by various complicating factors. Unfortunately, the forefront candidate vaccine performed unsatisfactorily, and its efficacy depends on serostatus and age factors. The observations from clinical studies depicted ambiguity regarding the efficacy of the dengue vaccine [50]. Moreover, the only licensed vaccine (Dengvaxia) for children 9 through 16 years of age is available in just a few countries. Despite its licensure for clinical use, the vaccine has performed below par efficacy in children and dengue-naive individuals and notably resulted in an increased risk of developing severe dengue in young, vaccinated recipients [94, 115]. In addition to absence of a suitable animal model, another major hurdle in vaccine development is ADE. In the case of ADE, antibodies generated after the first infection can actually facilitate the entry of the virus into cells when a person is infected with a second, different type of DENV, making the infection more severe [58]. ADE enhances DENV NS1 production, an endothelial toxin, increasing vascular permeability that leads to severe, even fatal, dengue [40, 43].

Direct-acting antivirals (DAA), which interact with viral proteins to exert antiviral function, offer a promising approach with lower toxicity and a broad therapeutic window. However, a downside of DAA is a higher risk for viral resistance [117]. The most extensively studied structural protein and non-structural antiviral drug targets are the E protein, and NS5 and NS3, respectively. Despite numerous DAA identified, a few are validated through in vivo studies. Furthermore, only one DAA, balapiravir, has been tested in clinical trials but no remarkable difference in cytokine profile, plasma viral load, and fever clearance time between the placebo and balapiravir groups was reported [25].

Another potential approach is interference with cellular pathway to prevent viral commandeering of cellular machinery to create a favourable environment for its replication. The compounds capable of such intervention are called as host-directed antivirals (HDA). HDA not only presents a promising avenue for broad-spectrum treatment but also has a reduced risk of resistance development [59]. In comparison to DAA, HDA typically have a narrow therapeutic and toxicity window, as they may also interfere with cell homeostasis[4]. The most studied cellular target is α-glucosidase, which facilitates proper protein folding and maturation. One of the Few studied HDA in clinical trials is the α-glucosidase inhibitor UV-4B. This drug exhibited a good safety profile up to a concentration of 1000 mg [85]. However, a follow-up study regarding the pharmacokinetics of UV-4B in healthy individuals was terminated.

Furthermore, a live-attenuated tetravalent DENV vaccine has been developed by Takeda (TAK-003). The vaccine is comprised of a DENV-2 backbone and 3 chimeric viruses with pre-membrane and envelope protein genes from three DENV strains, DENV 1, 3, and 4 [88]. Notably, the vaccine is getting licensed for use around the world, like in Brazil, Argentina, Indonesia, Colombia, Thailand, the European Union, and the United Kingdom. Clinical trials have demonstrated, that upon giving 2 subcutaneous injections 3 months apart, TAK-003 is well-tolerated and immunogenic not only in healthy adults from dengue-nonendemic areas but also in adults and children in dengue-endemic regions [20, 21]. However, in Takeda's February 2024 4–5-year QDENGA efficacy report, out of 3174 patients vaccinated and seronegative aged 4–16 years, only 11 were hospitalized with DENV 3 infections, while from 1832 seronegative patients on placebos, only 3 were hospitalized, giving a negative efficacy of 11·6%, similar to that of Dengvaxia [43, 110]. Moreover, overall vaccine efficacy (VE) was 80.2% against virologically confirmed dengue (VCD) caused by any serotype from 30 days after the second dose to the end of year 1 [22]. The overall VE against hospitalized dengue patients from 30 days after the second dose to the end of 18 months was 90.4% [20]. At 4.5 years postvaccination mark, the cumulative VE from the first dose against VCD was 61.2%, while 53.5% in baseline seronegative participants against all 4 dengue serotypes, and 64.2% in baseline seropositive subjects for any dengue serotype [116]. The evaluation study suggested that irrespective of the baseline serostatus, the vaccine is efficacious against DENV 1 and 2. On the other hand, VE against DENV 3 and 4 was only evident in baseline seropositive subjects. As for baseline seronegative recruits, data suggested no satisfactory efficacy against DENV 3, due to the low number of DENV 4 cases a proper assessment of VE could not be made [116]. The Strategic Advisory Group of Experts on Immunization recently recommended the use of QDENGA in dengue virus-endemic countries in settings of high transmission, for children aged 6–16 years irrespective of serostatus [124]. The approval was given in light of studies reporting an absence of a statistically significant number of severe DENV 3 infections in QDENGA vaccinated seronagative patients [124].

Medicinal plants as remedies for dengue infection

The use of medicinal plants in treating dengue infections is an area of growing interest, especially given the limited availability of specific antiviral treatments for dengue. Medicinal plants have been used for centuries to treat various human illnesses [5, 49, 53, 102]. Various plants have been studied for their potential to alleviate symptoms or affect the virus directly. Some plants have been found to possess antiviral properties that may be effective against the dengue virus. These plants can inhibit the replication of the virus or interfere with its ability to enter host cells. Certain plants are known for their ability to alleviate symptoms associated with dengue, such as fever, pain, and inflammation. They may possess analgesic, antipyretic, or anti-inflammatory properties [42, 99]. Some medicinal plants can strengthen the immune system, helping the body to fight off the virus more effectively. In many cultures, specific plants have been used traditionally to treat symptoms similar to those of dengue fever. These traditional uses often guide scientific research into the potential of these plants as dengue remedies.

Rural populations around the world employ a variety of herbs from the Lamiaceae family to cure DENV infections. As shown in Table 1, other widely used species include Carica papaya (Caricaceae) and Euphorbia hirta (Euphorbiaceae). Several plant components are employed, including the leaves, roots, stems, and flowers, in decoctions, infusions, and leaf juice for treating dengue infection. In this section, we describe and discuss medicinal plants with in vitro and in vivo activities against DENV. We also discuss clinical trials in which medicinal plants were employed for treating dengue infection.

Table 1 In vitro studies for pharmacologically active medicinal plants DENV infection treatment

Medicinal plants with in vitro activity

In vitro screening of drugs for dengue treatment plays a crucial role in the early stages of drug development. In vitro screening allows researchers to test a wide range of compounds for their antiviral activity against the dengue virus. This initial screening helps identify potential drug candidates that exhibit inhibitory effects on viral replication. In this section, we describe and discuss some of the most important medicinal plants showing promising anti-dengue potential as tested in vitro (Table 1 and Fig. 3).

Fig. 3

Several medicinal plants have shown activities against various DENV targets. Extracts of plants or phytocompounds isolated from them were effective asNS1 inhibitor (Quersus lucitanica), NS5 inhibitor (Acorus calamus), vRNA inhibitor (Houttuynia cordata), and viral entry inhibitor (Orthosiphon stamineus)

Alternanthera philoxeroides

Alternanthera philoxeroides, commonly known as alligator weed, belongs to the Amaranthaceae family. Various polarity-based extracts of the whole plant were assessed using C6/36 cell lines. Cytotoxicity was only exhibited by the concentrations exceeding 320 μg/ml. All four extracts demonstrated inhibitory effects on the dengue virus, however, petroleum ether extracts exhibited the strongest inhibitory effects on the dengue virus (ED₅₀ = 47.43) among the four extracts [57].

Andrographis paniculata

Andrographis paniculata, commonly known as the King of Bitters, belongs to the family Acanthaceae. Ethanolic and aqueous extracts of A. paniculata at a concentration above 500 μg were toxic to Vero cells; also, they exhibited antiviral effects, with 55–97% cell viability recorded in dengue-infected cells. The study determined the maximal non-toxic dose (MNTD) and highlighted the potential of A. paniculata as an anti-dengue agent, showing 75% viral inhibition [97].

Acorus calamus

Methanolic extract of Acorus calamus (Acoraceae), prepared from A. calamus leaves, was subjected to in vitro laboratory experiments combined with in-silico analysis. The results indicated that the extract was non-toxic to Huh7it-1 cells while demonstrating a significant inhibition of DENV-2. Molecular docking analysis revealed that artesunic acid in A. calamus is potential inhibitor of the NS5 protein of the dengue virus [101].

Cladogynos orientalis

Cladogynos orientalis (Family Rubiaceae) exhibited significant inhibitory activity against DENV-2 in Vero cells. Ethanol extracts C. orientalis showed 34.85% inhibitory activity, at a concentration of 12.5 µg/ml. Additionally, C.orientalis, exhibited the ability to inactivate viral particles by 52.9% at a concentration of 100 µg/ml [62].

Euphorbia hirta

Euphorbia hirta (Euphorbiaceae) is traditionally used in the Philippines to combat dengue virus. The ethyl acetate layer of the plant extract exhibited the most potent antiviral activity against dengue virus serotypes 1 and 2. It also significantly reduced the plaque-forming capacity of the dengue virus, suggesting potential antiviral properties [114].

Flagellaria indica

The ethanol extract of Flagellaria indica (Flagellariaceae) at a concentration of 12.5 µg/ml showed 45.52% inhibitory effects on DENV-2 replication. However, the identity of the active constituents and underlying mechanism was not explored by the investigators [62].

Flacourtia ramontchi

Flacourtia ramontchi, colloquially known as ramontchi, governor's plum, and Indian plum, belongs to the Salicaceae family. The stem bark extract of F. ramontchi exhibited significant anti-dengue activity. Bioassay-guided isolation led to the identification of six new phenolic glycosides, including flacourtosides A–F (1–6), scolochinenoside D, and betulinic acid 3β-caffeate. It was observed that betulinic acid 3β-caffeate, flacourtosides A and E (1 and 5), and scolochinenoside D exhibited significant inhibition in the DENV RNA polymerase assay. Notably, betulinic acid 3β-caffeate demonstrated an IC₅₀ of 0.85 ± 0.1 μM, highlighting its potential as a candidate for further exploration as an anti-dengue therapeutic agent [26].

Faramea bahinensis

A methanol extract from the leaves of Faramea bahiensis, a plant belonging to the Rubiaceae family, demonstrated in vitro non-cytotoxicity and significant anti-DENV-2 activity in human hepatocarcinoma (HepG2) cell lines. Further fractionation led to the isolation of a new flavanone glycoside, 5-hydroxy-4′-methoxy-flavanone-7-O-ß-d-apiofuranosyl-(1 → 6)-ß-d-glucopyranoside, in addition to known flavanone glycosides. The treatment of DENV-2-infected HepG2 cells with the new flavanone glycoside resulted in the control of viral replication and subsequent reduction in the number of infected cells (12%), infectious particles in the culture supernatant (97%), and the number of DENV-2 RNA copies in HepG2 cells (67%) [84].

Houttuynia cordata

An aqueous extract of aerial stems and leaves of Houttuynia cordata exhibited inhibitory effects on intracellular DENV-2 RNA production and dengue protein expression in HepG2 cells. The extract exhibited a protective effect on virion release from the infected monkey kidney cell line (LLC-MK2), with hyperoside identified as the predominant bioactive compound. Importantly, the extract was found to be non-genotoxic, further supporting its potential safety for consumers [66]. An ethanolic extract of Houttuynia cordata was obtained by soaking the herb in 80% ethanol, followed by filtration, concentration, and freeze-drying. The ethyl acetate (EA) fraction of H. cordata, particularly quercetin exhibited significant anti-DENV-2 activity, while the combination of quercetin and quercitrin showed a synergistic effect without inducing cytotoxicity. The acute oral toxicity assessment of the EA fraction in mice revealed no signs of toxicity or adverse effects [33].

Faramea hyacinthina and Faramea truncate

The in-vitro assays employed the defatted fractions of methanol extracts from the leaves of Faramea hyacinthina and Faramea truncate (family Rubiaceae) exhibited non-cytotoxicity and demonstrated anti-DENV2 activity in HepG2 cells. Several antiviral compounds were isolated including the antiviral flavanone (2S)-isosakuranetin-7-O-β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside (1), diastereoisomeric epimers (2S) + (2R) of 5,3′,5′-trihydroxyflavanone-7-O-β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside (2a/2b), narigenin-7-O-β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside (3), rutin (4), quercetin-4′-β-d-O-glucopyranosyl-3-O-rutinoside (5), kaempferol-3-O-rutinoside (6), erythroxyloside A (7), and asperuloside (8). Compounds 4 – 8 were reported for the first time in Faramea species [17].

Justicia adhatoda

Aqueous extracts of Justicia adhatoda (family, Acanthaceae), were encapsulated in phytoniosomes (PN) and their antiviral activity against DENV-2 was evaluated. The PN formulations, particularly AGN4, showed significant inhibition of plaque formation in in-vitro conditions, suggesting the potential of J. adhatoda leaf extract in combating dengue virus. However, the mechanism of inhibition was not investigated [126].

Piper retrofractum

Several Thai medicinal plants, including Piper retrofractum belonging to the Piperaceae family, were investigated in in-vitro anti-dengue activity. In the study, dichloromethane and ethanol extracts from P. retrofractum demonstrated inhibitory activity against DENV-2 in Vero cells. At a concentration of 12.5 µg/ml, the ethanol extract exhibited a significant inhibitory activity of 53.53%. Additionally, at a concentration of 100 µg/ml, the ethanol extract of P. retrofractum showed an 84.93% inhibitory activity against DENV-2 viral particles. The cytotoxicity evaluation revealed a CC₅₀ of 625 µg/ml for the ethanol extract of P. retrofractum, indicating its safety at this concentration. The mechanisms underlying its antiviral effects were not elucidated in the study [62].

Psidium guajava

An in vitro study model involving VERO cells was employed to assess the cytotoxicity and anti-DENV effects of ethanolic extracts from Psidium guajava, a plant belonging to the Myrtaceae family [118]. The most promising fraction, Pg-YP-I-22C, exhibited high. Further analysis revealed several phytocompounds, including catechin, gallic acid, hesperidin, naringin, and quercetin. In-vitro evaluations demonstrated significant antiviral effects, with catechin exhibiting 100% inhibition with a pre-treatment strategy and 91.8% with a post-treatment strategy. Molecular docking studies indicated favorable interactions, particularly with the viral envelope protein. The study sheds light on guava's potential as a source of antiviral compounds, with catechin emerging as a promising candidate for further investigation.

Phyllanthus amarus

An aqueous cocktail extract of Phyllanthus amarus (Phyllanthaceae family), excluding roots, was prepared, and processed into extracts using water, diethyldithiocarbamic acid, and formic acid. The study used in vitro experiments utilizing Vero cells as the model. Notable phytcompounds identified included gallic acid, geraniin, syringin, and corilagen. The MNTD on Vero cells was determined to be 250 μg/ml. Moreover, in vitro antiviral experiments against DENV2 revealed that Phyllanthus amarus exhibited significant activity, particularly in simultaneous treatment, resulting in an 83–95% reduction of virus inhibition. Proteomic analysis revealed altered expression of various host and viral proteins involved in crucial processes such as viral entry, replication, and cellular metabolism, suggesting Phyllanthus amarus as an important source of inhibitors against dengue virus [67].

Quersus lucitanica

Quersus lusitanica, commonly known as the Portuguese oak, belongs to the Fagaceae family. A crude extract derived from Q. lusitanica seeds investigated against DENV-2 using C6/36 cells, a cloned cell line derived from Aedes albopictus larvae, revealed dose-dependent antiviral activity. At its MNTD of 0.25 mg/ml, the Q. lusitanica extract completely inhibited various virus concentrations, demonstrating its effectiveness against cytopathic effects. Proteomic analysis showed the extract's impact on the NS1 protein expression, a glycoprotein crucial for flavivirus viability. The results exhibited down-regulation of NS1 protein expression in infected C6/36 cells upon treatment with Q. lusitanica extract. The promising inhibitory effects of Q. lusitanica extract on DENV-2 replication, showcase its potential as an antiviral agent in both conventional cell culture and proteomic analyses [79].

Rhizophora apiculata

Rhizophora apiculata is a mangrove plant belonging to the family Rhizophoraceae. An ethanol extract of R. apiculata plant exhibited a significant inhibitory activity against DENV-2 strain 16,681, in Vero cells with 56.14% inhibition at a concentration of 12.5 µg/ml. Moreover, R. apiculata extract demonstrated the ability to inactivate viral particles. The results suggest an anti-dengue potential of R. apiculata [62].

Spondias mombin & Spondias tuberosa

The plant extracts obtained from the leaves of two Spondias species of Anacardiaceae family, namely Spondias mombin and Spondias tuberosa, indigenous to Brazil, showed promising activity against DENV-2 evaluated in vitro using C6/36 cells, a cloned cell line derived from larvae of Aedes albopictus [109]. The study focused on the identification of phenolic compounds, including quercetin, rutin, and ellagic acid, through HPLC analysis. The extracts and compounds demonstrated non-toxicity to the cells at concentrations up to 1000 µg/mL. The antiviral activity revealed a significant inhibition of virus replication by rutin and quercetin at a concentration of 500 µg/mL. Notably, rutin exhibited a lower IC50 value (362.68 µg/mL) compared to quercetin (500 µg/mL), suggesting its greater potency. The study underscores the potential of Spondias species, particularly rutin and quercetin, as candidates for the development of anti-DENV agents.

Uncaria tomentosa

Uncaria tomentosa (family: Rubiaceae), commonly known as cat's claw, is a large woody vine native to the Amazon and Central American rainforests. Given the plant’s rich history of medical use, a hydro-alcoholic extract of U. tomentosa, as well as its pentacyclic oxindole alkaloid-enriched and non-alkaloid fractions were investigated utilizing human monocytes infected with DENV-2. Results indicated that both the extract and the alkaloidal fraction exhibited inhibitory activity against DENV-2, reducing the rates of DENV-2 antigen-positive cells. Additionally, the alkaloidal fraction indicated strong immunomodulation by decreasing levels of pro-inflammatory cytokines TNF-α and IFN-α, while modulating anti-inflammatory cytokine IL-10. These findings suggest that the pentacyclic oxindole alkaloids from U. tomentosa hold promise as potential candidates for clinical application in Dengue Fever treatment [100].

Annona muricata

An aqueous leaf extract of Annona muricata, commonly known as soursop, belonging to the family Annonaceae, was evaluated on Vero cells, revealing a CC₅₀ of approximately 2.5 mg/mL and a 50% Effective Concentration (EC₅₀) of about 0.20 mg/mL. Notably, the SI of the extract against DENV-2 was more than 10, indicating its potential as an antiviral agent. A. muricata demonstrated greater efficacy in inhibiting viral replication in post-treatment compared to pre-treatment. The phytochemical analysis identified over 200 chemical compounds, including alkaloids, phenols, and acetogenins. The study suggests that A. muricata aqueous leaves extract holds promise as a nature-based antiviral drug, particularly effective in the early stages of viral replication. However, the active compounds responsible for the observed effects were not identified in this study [120, 121].

Catharanthus roseus

Catharanthus roseus of the family Apocynaceae, commonly known as Madagascar periwinkle, is a medicinal plant with a rich history of traditional use for treating various diseases. A methanol extract from the leaves of C. roseus showed a CC₅₀ of 0.13 mg/mL in vitro cytotoxicity tests using Vero cells. The antiviral activity against DENV-2 was also assessed through foci-forming unit reduction assays (FFURA), including post-treatment, pre-treatment, and virucidal tests. The results indicated that C. roseus extract exhibited a concentration-dependent antiviral effect, with more than 60% reduction in foci observed in the post-treatment assay at the highest concentration (0.078 mg/mL). The extract showed weak activity in pre-treatment and mild virucidal activity. The EC₅₀ against DENV-2 was 0.025 mg/mL, resulting in a SI of 5.2 [1].

Orthosiphon stamineus

An aqueous leaf extract of Orthosiphon stamineus (Lamiaceae), commonly known as cat's whiskers or Java tea, revealed a CC₅₀ value of 5 mg/ml, indicating non-cytotoxicity to the cells when tested against DENV-2 using Vero cells [120, 121]. The antiviral activity against DENV-2 was demonstrated through morphological changes and confirmed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, showing a selective index of 13. The study suggests that the extract from O. stamineus contains phytochemical compounds with non-cytotoxic and potential antiviral activities. This plant species holds promise as a candidate for further activity-monitored fractionation to identify active principles.

Hippophae rhamnoides

A leaf extract of Hippophae rhamnoides, of the Elaeagnaceae family, demonstrated a significant anti-dengue effect at a concentration of 50 μg/ml, comparable to Ribavirin, a commercially available anti-viral drug. The treatment with H. rhamnoides leaf extract led to a significant decrease in TNF-α and an increase in IFN-γ production in dengue-infected cells. Additionally, plaque assay results indicated a reduction in plaque numbers following the treatment, confirming its significant anti-dengue activity [55].

Faramea bahinensis, Faramea hyacinthina & Faramea truncate

An investigation centered on the methanolic extracts of three Faramea species – Faramea bahiensis, F. hyacinthina, and F. truncata, hailing from the Rubiaceae family revealed important anti-DENV2 activities. Utilizing online high-performance liquid chromatography-diode array detector-electrospray ionization tandem mass spectrometry (HPLC–DAD-ESI–MS/MS), the study identified 31 phenolic compounds, including flavonoid O- and C-glycosides, phenolic acids, and one coumarin. Notably, the stem extracts of F. hyacinthina and F. bahiensis exhibited significant anti-DENV2 activity comparable to their leaves. However, F. truncata demonstrated increased cytotoxicity [127]

Garcinia mangostana

Α-mangostin, derived from the fruit of G. mangostana (Guttiferae) [28], showed promising dengue treatment potential. Notably, at the concentrations of 20 and 25 μM, α-mangostin reduced DENV production by more than tenfold and 1,000-fold, respectively. Moreover, α-mangostin effectively inhibited the infection of immature monocyte derived dendritic cells (moDCs) by all four serotypes of DENV. α-mangostin, at 25 μM inhibited DENV at the early stage of replication. In addition, the phytocompound significantly reduced cytokine/chemokine (TNF-α, CCL4, CCL5, CXCL10, IL6, IL1β, IL10, and IFN-α) transcription in DENV-infected immature moDCs. This makes α-mangostin a suitable candidate for dengue treatment, as it demonstrates a distinct ability to thwart DENV-induced ‘cytokine storm’ [134].

Hystrix brachyuran

Traditionally, porcupine dates are used in dengue fever treatment. To study the anti-dengue compounds and their activity, two porcupine dates, black date and powdery date, from H. brachyuran (Hystricidae) were investigated. Methanol crude extracts were prepared from these dates to isolate tannin-rich fractions. The results revealed that all the extracts exhibited antiviral activity against DENV-2 in Vero cells. The IC₅₀ of black date and powdery date tannin-rich fractions were 25 µg/mL and 11 µg/mL, respectively. Similarly, te fractions were highly virucidal (IC₅₀ 11 µg/mL) than methanol crude extracts with an IC₅₀ value of 52–66 µg/mL. Notably, all the extracts inhibited the attachment of DENV-2 by at least 80% [95].

Medicinal plants with in vivo activity

In vivo screening of drugs for dengue treatment is a critical step in the drug development process, after the in vitro studies. In vivo studies involve testing potential drug candidates in living organisms, typically animal models, and provide valuable insights into the drug's efficacy, safety, and pharmacokinetics. The following plants showed potent in vivo activities in enhancing platelet, and thrombocyte count as well as attenuating the cytokine storm via downregulatio of pro-inflammatory cytokines.

Alternanthera sessillis

A study investigated the platelet augmentation activity of the leaf extract from Alternanthera sessilis (Amaranthaceae) to assess their potential as a therapy for thrombocytopenia. Platelet reduction was induced in Sprague–Dawley rats using anagrelide, and subsequent administration of plant extracts showed significant platelet augmentation activity. Notably, the percentage increase of mean platelet counts with A. sessilis was found to be 93.17% (p = 0.0001) [14].

Carica papaya

Carica papaya, commonly known as papaya, which belongs to the Caricaceae family (Table 2). An investigation used a novel approach involving a suspension of powdered C. papaya leaves in palm oil. Conducted in mice, the in vivo study administered 15 mg of powdered leaves/ kg body weight to different groups. The results demonstrated a significant increase in thrombocyte counts at multiple time points for the C. papaya leaf formulation group [105]. In a separate study, the authors aimed to evaluate the platelet augmentation activity of C. papaya extract in thrombocytopenic disorders. The in vivo study involved inducing platelet reduction in Sprague–Dawley rats and administering the plant extracts for nine days. C. papaya leaf extract demonstrated a notable increase in mean platelet counts (125.87%) [14].

Table 2 In vivo & in vivo/in vitro studies for pharmacologically active medicinal plants DENV infection treatment

Ipomea batatas

In another study, [14] investigated the platelet augmentation activity of Ipomoea batatas (Convolvulaceae), commonly known as sweet potato, in the context of thrombocytopenia. The study was conducted using Sprague–Dawley rats with induced platelet reduction through the administration of anagrelide. The aqueous extracts of I. batatas, particularly the violet and green varieties, demonstrated significant platelet augmentation activity, with a percentage increase of mean platelet counts by 106.07% and 107.88%, respectively. Further exploration is recommended to identify the specific phytochemical constituents responsible for this activity and to establish the safety profile of the plant extract.

Crocus sativus

C. sativus (Iridaceae) is renowned for immunomodulatory and anti-inflammatory properties. A study looking into C. sativus anti-dengue effects found that crocetin (C. sativus-derived natural compound) reduced DENV-induced apoptosis in DENV-infected mice at the dose of 50 mg/kg. Moreover, it significantly down regulated the pro-inflammatory cytokine expression, in addition to modulating antioxidant status in DENV-infected mice. Furthermore, crocetin demonstrated the ability to reduce the nuclear translocation of NF-kB. Another notable finding was that crocetin improved host responses that reduces liver injury in DENV-infected mice [111].

Medicinal plants in clinical trials

Clinical trials for drugs intended for dengue treatment are essential for several reasons, and their importance lies in advancing our understanding of the drug's safety, efficacy, optimal dosage, and overall impact on patients. Following are some of the important clinical trials that we have summarized.

Carica papaya

In Sri Lanka, twelve patients, who met the eligibility criteria of the clinical trial but declined hospital admission, were administered C. papaya leaf extract (CPLE) in the form of two 5 ml doses at an 8-h interval for adults and two 2.5 ml doses for children under 10 years. All patients showed an increase in platelet counts within 24 h of leaf extract administration, with no hospital admissions required. Some patients with elevated SGPT levels experienced a reduction, and those with low platelet counts and itching hemorrhagic skin rash showed improvement [46].

In a separate case report, C. papaya, was tested in a 45-year-old man bitten by a carrier mosquito. In the investigation, 25 mL of aqueous extract of C. papaya leaves was administered twice daily for five consecutive days. Before the administration, blood samples were analyzed, revealing decreased platelet, white blood cells, and neutrophil count. After the leaves extract administration, a notable increase in platelet (from 55 × 10^3/μL to 168 × 10^3/μL), WBC (from 3.7 × 10^3/μL to 7.7 × 10^3/μL), and neutrophils (from 46.0% to 78.3%) count was observed [6].

In a Malaysian clinical study, C. papaya leaves juice (CPLJ) was investigated for its platelet count-elevating property in patients with dengue fever, and DHF. It was an open-labeled randomized controlled trial involving 228 patients. During the clinical trial, approximately half of the patients received CPLJ for three consecutive days, and the other half served as controls receiving standard management. The results exhibited a significant rise in mean platelet count in the intervention group compared to the control group after 40 and 48 h of admission. Additionally, gene expression studies on ALOX 12 and PTAFR genes revealed high expression in patients on CPLJ, suggesting its potential role in platelet production and aggregation [112].

In another clinical study conducted in Indonesia, investigators used CPLE capsules to assess its platelet improvement potential. In the trial, 80 patients, who met the inclusion criteria, were enrolled for the trial and they were split into two groups (40 patients per group). One cohort served as an intervention cohort, while the other was labeled as a control group. A total of 24 capsules were given to these subjects, divided into twice-daily dosing schedules. The results showed a significantly improved platelet count (p < 0.05), maintained hematocrit stability, and shortened hospital stay of dengue patients in the intervention group [135].

A multicentered clinical trial conducted in Pakistan, Malaysia, Sri Lanka, and other Asian countries investigated CPLE capsules for dengue management. This randomized clinical trial enrolled and split the participants into two groups: a study group receiving CPLE capsules (500 mg) along with routine supportive treatment and a control group receiving only routine supportive treatment. Following the treatment, on the 3rd, 4th, and 5th days, the platelet count in the study group was significantly higher than the control group, in addition to preventing complications of thrombocytopenia in dengue fever [38].

In India, a clinical trial was conducted aimed at assessing the efficacy and safety of CPLE in managing severe thrombocytopenia (≤ 30,000/μL) in adult dengue patients [106]. The extract was utilized in a double-blind, placebo-controlled, randomized, and prospective clinical trial, involving 51 laboratory-confirmed adult dengue patients randomly assigned to either the treatment CPLE group or the placebo group. CPLE-treated patients exhibited a significantly increased platelet count by day 3 compared to the placebo group. Notably, the treatment group also required fewer platelet transfusions and showed a faster recovery to a platelet count of ≥ 50,000/μL. The study suggested that CPLE was safe and well-tolerated, with no significant decrease in mean hospitalization days. Additionally, plasma cytokine profiling indicated potential immunomodulatory effects and a faster clearance kinetics of viral NS1 antigenemia was observed in the CPLE group.

Euphorbia hirta

A clinical trial conducted in Lahore; Pakistan investigated the potential therapeutic effects of the herbal water derived from Euphorbia hirta (Euphorbiaceae) in dengue fever. The trial involved 125 confirmed dengue fever patients. The patients were categorized into two groups based on age. After 24 h of E. hirta herbal water administration, results demonstrated that over 70% of patients exhibited improvement in platelet count, fever, and flu-like symptoms [77] (Table 3).

Table 3 Clinical trials/studies for pharmacologically active medicinal plants DENV infection treatment

Medicinal plants in mix-models (in-vivo and in-vitro)

The following plants have shown anti-dengue effects both in vitro and in vivo studies.

Azadirachta indica

Azadirachta indica, commonly known as Neem, of the family Meliaceae, has been investigated both in vitro and in vivo for anti-dengue activities. Crude aqueous extract of A. indica leaves and pure neem compound (Azadirachtin) were used. The aqueous extract at an MNTD concentration of 1.897 mg/ml completely inhibited the virus in vitro. The in vivo protection studies revealed it inhibited viral replication, as confirmed by the absence of dengue-related clinical symptoms in suckling mice at 120–30 mg/ml [90].

Curcuma longa

In vitro assays of Curcuma longa (Zingiberacea) extract, using Huh7it-1 cells, revealed an IC₅₀ value of 17.91 μg/mL. The selectivity index (SI) calculated from these values was 4.8, indicating a potentially safe and effective antiviral compound [51]. In the in vivo study conducted on ddY mice, oral administration of C. longa extract at a dose of 0.147 mg/mL significantly reduced the viral load after 24 h of DENV-2 infection. Histopathological examination of liver and kidney tissues showed no specific abnormalities and biochemical observations revealed no significant increase in SGPT, SGOT, urea, and creatinine levels. These results suggest that C. longa could serve as a promising antiviral agent against DENV with low cytotoxicity and effective inhibition, highlighting its potential for further exploration and development [51] (Table 2).

Discussion

Plants and Phytocompounds represent a major source of novel therapeutic agents in drug discovery research. Numerous drugs, currently in use, have been fashioned from plant sources. Recently, pharmaceutical companies have started to pour more resources into phytocompound discovery and development owing to technological advances [81]. However, the transition from allopathic to herbal remedies is plagued by various challenges. For instance, naturally sourced drugs have bioavailability issues due to reasons such as poor aqueous solubility and high molecular weight. Moreover, herbal drugs must be validated for their safety and toxicity profile.

With an aim to promote and ensure efficacy, safety and quality of plant-based drugs, the WHO launched a strategy on conventional medicine (2014–23) [56]. In order to optimize the use of and address the drug-delivery challenges associated with phytocompounds, computational screening techniques, such as in silico docking of the therapeutic compound and structure based drug design should be employed [72, 76]. Most of the phytoconstituents revelaed by these approaches have only been evaluated in vitro. To advance the drug development process of phytoconstituents, initially antiviral activity should be tested in a panel of human cell lines that are crucial during natural DENV infection. For example, some of the significant cellular targets during natural dengue virus infection which coule be used for therapeutic evaluation iclude primary human peripheral blood mononuclear cells, hepatic cell lines like Huh7 cells or U2OS cells, and human monocyte-derived macrophages [32].

Moreover, by integrating newly developed organoid cultures, such as 3 dimensional liver organoids, into drug development process could prove to be instrumental to assess the antiviral activity in complex in vitro model [64, 91]. Furthermore, ideally a phytochemical or drug should be active against all serotypes and preferably confirmed for multiple DENV strains within a serotype [69]. Nevertheless, anti-dengue agents or DENV inhibitors which are protective towards two or three serotypes should be promoted to further testing stage. Impotantly, minimal cytotoxicity should be exhibited by these compounds [16]. Novel techniques like cellular thermal shift assays and 3D organoids of the liver may help to gain a more complex and specific insight into the cytotoxicity profile of novel drugs [27, 37].

Lastly, the stability of a phytocompound-based drug is critical for optimal pharmacokinetic profile as limited stability could lead to premature degradation of a therapeutic agent in the gastrointestinal tract or in the liver, significantly impacting the bioavailability [11, 117]. For this purpose, hepatic microsome assay can prove to be more helpful. In this assay, a drug is subjected to subcellular liver fractions with drug-metabolizing enzymes to investigate its metabolic degradation [34, 41, 63]. Ostensibly, it is challenging to identify compounds with the proper characteristics but with thorough research in the virus–host interactions, occurring while viral infection and/or chemical manipulation of identified compounds may accelerate the development of compounds that fit the required criteria [117].

As previously mentioned, lack of proper in vivo testing model also significantly hampers the drug development process. For in vivo testing, The AG129 model is considered the best model to study DENV infection, due to the lack of more representative small animal DENV disease models. AG129 mice are deficient in the interferon I and II receptors, producing high DENV viremia levels and high levels of pro-inflammatory cytokines, leading to thrombocytopenia, vascular leakage, and death [29]. There are two distinct AG129 mouse models, the lethal and the non-lethal mouse model. This is based on the virus strain used and the dose applied [29]. However to study the antiviral efficacy the non-lethal AG129 model is preferred as it allowed to study the time needed to resolve viremia, which was more representative for the clinical situation [117].

It is noteworthy that from 200 viruses identified in humans, only nine can be treated with approved antiviral compounds [29], including human immunodeficiency virus, hepatitis B virus, hepatitis C virus, etc. Numeorous approved regimens are based on combinational therapies, a cocktail of drugs that target different steps of the virus replication cycle [133]. By adding the phytocompounds, especially those that boost platelet count like A. sessillis, C.papaya, more effective anti-dengue regimens can be designed [89]. It is challenging to develop a safe and effective antiviral compound with pan-protective antiviral properties, low toxicity, low chance of viral resistance, and proper stability to ensure absorption and distribution. Many of the plants and phytocompounds, like C.papaya [38, 106] and E. hirta [77] are proving to be effective for dengue treatment in clinical settings by alleviating platelet count. Furthermore, by integrating phytocompounds into the dengue management plans one can hope to not only enhance the therapeutic efficacy but also drive down the overall cost of the treatment [3, 87, 93, 108].

Conclusions

Integrating research on medicinal plants for dengue treatment into a broader agenda is crucial. Efforts are ongoing to establish evidence-based care guidelines for using medicinal plants in dengue infection. It's also important to highlight that the efficacy and safety of medicinal plants in treating dengue virus infection are still areas of ongoing research, and results may vary. The safety and efficacy of these herbal preparations for DSS need more research. A proposed collaborative platform in endemic regions could facilitate studies on fluid therapy and antiviral drug testing of these medicinal plants. Improved surveillance systems in dengue-endemic countries are essential for effective prevention and control. In addition to more research in finding potent cures for dengue infection, vector management strategies should be robust, emphasizing the need to focus on reducing disease incidence.