Carica papaya Leaf Juice for Dengue: A Scoping Review

The potential therapeutic effect of Carica papaya leaf juice has attracted wide interest from the public and scientists in relieving dengue related manifestations. Currently, there is a lack of evaluated evidence on its juice form. Therefore, this scoping review aims to critically appraise the available scientific evidence related to the efficacy of C. papaya leaf juice in dengue. A systematic search was performed using predetermined keywords on two electronic databases (PubMed and Google Scholar). Searched results were identified, screened and appraised to establish the association between C. papaya and alleviating dengue associated conditions. A total of 28 articles (ethnobotanical information: three, in vitro studies: three, ex vivo studies: one, in vivo study: 13, clinical studies: 10) were included for descriptive analysis, which covered study characteristics, juice preparation/formulations, study outcomes, and toxicity findings. Other than larvicidal activity, this review also reveals two medicinal potentials of C. papaya leaf juice on dengue infection, namely anti-thrombocytopenic and immunomodulatory effects. C. papaya leaf juice has the potential to be a new drug candidate against dengue disease safely and effectively.


Introduction
The use of medicinal plants in disease prevention and treatment has been around for many generations worldwide and some of them have been scientifically proven. Various medicinal plants, especially those with antiviral activity have drawn interest from researchers to formulate new medicinal drugs for infectious diseases around the world [1]. Like other plants, Carica papaya L. has a high content of phytochemicals that not only have beneficial food nutritional values but also medicinal potential. Its leaves contain alkaloids [2][3][4][5], flavonoids [3,[6][7][8], phenolic acids [6][7][8], saponin [9], amino acids [6], organic acids [6], vitamins [8,10], minerals [10], carbohydrates [6] and carotenoids [8]. Traditionally, this plant is used to lower blood pressure and blood sugar levels by ingesting boiled young leaves [11]. Infusion or decoction of the leaves taken orally not only regulate blood pressure but can also treat overweight condition. This plant is very famous for its edible fruit to relieve constipation. Its seeds can be pounded and then ingested to treat intestinal worms. Toothache, corns and warts can be treated by topically applying its sap [12]. Recently, the C. papaya, particularly its leaf part, has attracted wide attention for its potential use in dengue treatment.
Dengue, an Aedes mosquito-borne viral infection, has become a public health risk that demands the world's focus, especially in tropical countries [13]. This infection is one of the crucial health concerns in Malaysia because of its worrying statistical value. There were more than 900,000 dengue cases with over a thousand deaths reported in Malaysia from the year 2009 to present and more than 125,000 dengue cases were reported in 2019 alone [14]. The dengue virus (DENV) belongs to the virus family Flaviviridae and consists of five serotypes (DENV-1 to 5). Compared to other serotypes, the fifth serotype has only been circulated between non-human primates and mosquitoes (also known as the sylvatic cycle) with only one new human infected case reported in 2013 [15,16]. The ideal vector for DENV-5 is mosquito Aedes niveus, whilst the female Aedes aegypti mosquito is the common vector in DENV transmission [16,17]. Compared to the male type, the female mosquito requires external blood as a nutrient supply, particularly iron mineral, for its eggs production and development [18]. Due to the very low transmission rate of the DENV-5 and a lack of scientific information, at present the World Health Organization upholds that the dengue disease is a public health concern mainly caused by the other four serotypes [19].
There are primary and secondary dengue infections. Individuals who have recovered from primary infection of a serotype have lifelong immunity against the same serotype but the risk of developing severe dengue would be higher following infection by any other of the three serotypes (secondary infection) [20,21]. Severe dengue, also known as dengue hemorrhagic fever, is potentially fatal. Despite of the life-threatening complication, there are still no specific antiviral medications for dengue infection [22]. Nevertheless, scientists are persistently striving to find the cure by diverging their attentions to develop a therapeutic drug that can disrupt or cease targeted DENV proteins' functions [23]. A licensed dengue vaccine commercially available in some countries is not effective against all four common serotypes of DENV, and it can cause individuals unexposed to DENV to be at greater risk of developing severe disease [24,25]. These situations highlight the research need in identifying potent compounds with promising anti-dengue activities via in depth understanding of dengue pathogenesis. There are currently no systematic scoping reviews focused on C. papaya leaf juice. Therefore, this scoping review was conducted to gather and highlight the available scientific evidence for the use of C. papaya leaf, particularly using juice form in treating dengue infection.

Materials and Methods
This scoping review was designed based on framework adapted from Arksey and O'Malley's study (2005) [26]. A systematic search was conducted by two independent investigators using combination of keywords on PubMed and Google Scholar electronic databases. The keywords used at PubMed were 'carica papaya' AND ('leaf' OR 'leaves') AND 'juice' AND 'dengue' used in all fields. While the search setting used in Google Scholar was: (1) with exact phrase of 'carica papaya'; (2) with all the words of 'leaf, 'leaves', 'juice', and 'dengue'; and (3) include citations and anywhere in the article. The search period was in the default setting for both electronic databases until 1 March 2022. The search result was manually screened and selection of the included articles was limited to: (1) English language journal article; (2) full text accessibility; (3) conducted study on the juice derived from C. papaya leaf; (4) related to anti-dengue activity; and (5) article about ethnobotanical information, in vitro, in vivo and human studies. The definition of juice for this review referring to any liquid form originated from the C. papaya by crushing, pounding, pressing, cutting, squeezing and/or blending its leaves. A bibliographic manager (EndNote version 20, Clarivate Analytics, Philadelphia, PA, USA) was used to manage the search results. Data extraction for included studies was performed independently by two authors using a customized data extraction table (Table 1). Any disagreement was reviewed by a third author.

Study Inclusion
From a total of 1030 records identified from keyword searches on the selected online databases, a final 28 articles were included in this scoping review, as presented in the preferred reporting items for systematic review and meta-analysis (PRISMA) chart ( Figure 1).

Study Inclusion
From a total of 1030 records identified from keyword searches on the selected online databases, a final 28 articles were included in this scoping review, as presented in the preferred reporting items for systematic review and meta-analysis (PRISMA) chart ( Figure 1).

Study Characteristics
The 28 articles included in the review reported ethnobotanical information (n = 3), in vitro findings (n = 3), ex vivo findings (n = 1), in vivo findings (n = 13) and clinical findings (n = 10). These findings are summarized in Table 2. The three surveys compiling ethnobotanical information were in the regions of Bangladesh and the Philippines [27][28][29]. The three included in vitro studies used blood cells from human subjects and laboratory rats
Seven out of the 10 clinical studies allowed conventional treatment (such as fluid replacement, antipyretics, antibiotics, antimalarial drugs, immunosuppressant and antiemetics) for the dengue patients on top of the intervention treatment. Briefly, two studies treated the patients using conventional treatment before starting the intervention treatment [45,46]. Four studies allowed conventional treatment together with intervention treatment [47][48][49]53], whilst only one study treated the patients using conventional treatment after completion of intervention treatment period [52]. Only one study used a combination of the intervention, Ayurveda therapy and conventional treatment [47].

Ethnobotanical Findings
Traditionally, limited published material has reported the use of C. papaya leaf juice in dengue treatment. Out of the 28 included studies, only three showed evidence of traditional use of interest. All three articles are collective survey studies, which involved 92 local residents (the majority were traditional health practitioners) in the selected regions of Bangladesh [27] and the Philippines [28,29]. However, certain information cannot be found from all three articles, such as the amount of leaf juice being used, juice preparation and consumption period (Tables 2 and 3).

In Vitro Findings
Three included studies investigated the effect of C. papaya leaf juice at different maturity using cells isolated from healthy and dengue patients and healthy rodents, respectively ( Table 2). According to Ranasinghe et al., (2012), no significant difference was found on hemolysis inhibition level (p > 0.05) between three different maturities of leaves on both types of heat-induced hemolysis erythrocytes (healthy: 31.8-38.5%, dengue: 25.7-32.5%), respectively, compared to the aspirin control group (healthy: 45.9%, dengue: 43.6%). Testing on hypotonicity-induced hemolysis erythrocytes showed the inhibition level was also not significantly different (p > 0.05) in the intervention-treated group (partly mature leaves) on both types of erythrocytes (healthy: 31.57%, dengue: 57.03%) compared to the indomethacin control group (healthy: 47.67%, dengue: 65.35%) [30].
Chinnappan et al., (2016) investigated the efficacy of C. papaya leaf juice on adenosine diphosphate-induced platelet aggregation on plasma rich platelets and plasma poor platelets obtained from healthy volunteers and dengue patients. They found that platelet aggregation was significantly lower (p < 0.05) in both intervention-treated healthy and dengue plasma rich platelets than the untreated group. The same observation occurred (p < 0.05) for intervention-treated healthy plasma rich platelets (which was pre-infected with intervention pre-treated dengue plasma poor platelets) compared to the untreated group [31].
Beneficial uses of C. papaya leaf juice on key clinical manifestations in dengue infection were shown by in vitro findings through the inhibition of hemolysis erythrocytes was comparable with aspirin and indomethacin drugs in stabilizing the plasma membrane of dengue patients. Therefore, the risk of patients having plasma leakage due to increased vascular permeability and capillary fragility can be reduced. The DENV non-structural protein 1 was not only able to induce platelet activation but also aggregation through toll-like receptor 4 [55]. Interestingly, the leaf juice was found to be able to inhibit the excessive aggregation of platelets, which suggests thrombocytopenia and hemorrhage conditions triggered by dengue infection can be minimized. Effect on phagocytic activity and cytokine release also provide an insight on the potential of C. papaya leaf juice in modulating functional and non-functional immune responses which are triggered when a pathogen invades the host's body.

Ex Vivo and In Vivo Findings
There are 13 included studies using mouse and rat models, whilst there is only one study using mosquitoes to investigate the potential use of C. papaya leaf juice as a method to combat dengue ( Table 2). Vector control via the use of larvicidal is a straightforward way of controlling mosquito vector borne diseases. The common vector for dengue is Aedes aegypti mosquitoes and it has four life stages (egg, larvae, pupa and adult). It only takes up to 10 days for the eggs to evolve into adults [56]. Therefore, it is critical to stop the emergence of the adult mosquitoes as early as possible. However, from the article search targeting only juice form, the C. papaya leaf juice was found to have an insecticidal effect on the larvae in one study. Rubio (2016) found that mosquito larvae such as Aedes sp. died 5 to 30 minutes after the addition of the leaf juice into the artificial trap containing clean water [33].
Compared to the procedures used in Jayasinghe et al.'s study, one study isolated the cells only after treating the healthy Wistar rats with leaf juice for three days. Platelet counts (increased by 68%), differential white blood cells (monocytes: 44.67%; lymphocytes: 10%) and bone marrow cells (35%) in the intervention-treated group were better than in the distilled water-control group (p < 0.05). Similarly, phagocytic activity of peritoneal macrophages was also measured, and it significantly increased (p < 0.05) in the interventiontreated group (109%) compared to distilled water-control group [34].
Out of the 12 studies using a rodent model, there were four studies using a thrombocytopenia rat model (Table 2). Akhter and team (2015) found that after three days of treatment, platelet counts significantly increased (p < 0.05) in the intervention-treated group (7.83 × 10 5 /µL) compared to the hydrocortisone control group (4.05 × 10 5 /µL) [35]. Pure leaf juice administration (0.18 mL/100 g body weight, for five days) significantly increased (p < 0.05) the red blood count on the fifth day of treatment compared to the untreated control group. Bleeding time was significantly shorter (p < 0.05) in the intervention-treated groups on the second and fifth day of treatment compared to the untreated control group [36].
It has been reported that there was a dose-dependent efficacy of the leaf juice when compared to the untreated control group after 14 days of treatment. Bleeding and clotting times were significantly shorter (p < 0.05) in the intervention-treated groups since the eighth day of treatment. Levels of cellular malondialdehyde (p < 0.001) and serum thrombopoietin cytokine (p < 0.05) were downregulated whilst levels of cellular antioxidant enzyme (SOD) and GSH (p < 0.001) were upregulated in the intervention-treated groups on the 14th day of treatment. In contrast, platelet count significantly increased (p < 0.001) in the interventiontreated groups starting from the eighth day of treatment when compared to the water control group. Without a noticeable dose dependent effect, promising findings such as shorter prothrombin times and higher expression level of MPL-CD110 receptor (p < 0.05) were observed in the intervention-treated group on the 14th day of treatment compared to the untreated control group. Mature megakaryocytes with high cellularity and erythroblast cells in bone marrow were observed in the intervention-treated group compared to the untreated group [37].
Using a similar animal model and same time length of intervention administration, Anjum et al., (2017) found that not only did the platelet counts increase (p < 0.01) starting from day seven of treatment in the intervention-treated groups, but the levels of monocytes, basophils, eosinophils, lymphocytes and neutrophils also increased (p < 0.01) in the intervention-treated groups (after 14 days of treatment), compared to the saline control group. Clotting and bleeding time were significantly shorter (p < 0.01) in the interventiontreated groups compared to the saline control group [38].
Apart from using rats (Table 2), Anjum et al., (2017) also administered the same juice preparation to thrombocytopenic mouse model (the condition was re-induced on the fifth to seventh day after three days of intervention treatment) to investigate immunomodulatory activity against the untreated control group. Inflammation level was significantly lower (p < 0.01) in the intervention-treated group after 48-h of treatment. Total leukocyte counts were significantly higher (p < 0.05) in the intervention-treated group. Phagocytic index, mean antibody titer and level of TNF-α (a pro-inflammatory cytokine) were significantly lower (p < 0.05) in the intervention-treated group [38]. Myelosuppression mice were administered with the leaf juice at two different doses (5 and 10 mL/kg body weight), respectively, for 21 days. There was no significant difference (p ≥ 0.005) on platelet count increase from day 7 to 21 between male (719-950 × 10 9 /L) and female (700-979 × 10 9 /L) plant variety of both doses of the intervention-treated groups. Platelet count significantly increased (p < 0.001) in the intervention-treated groups (in a dose dependent manner) starting from day seven (700-793 × 10 9 /L) compared to the distilled water-control group (78 × 10 9 /L) [39].
Mohd Ridzuan and teams (2018-2021) used mouse model of DENV-infection (also known as AG129 mouse) in their four studies to investigate the therapeutic effect of leaf juice with a three-day treatment period. Three studies used New Guinea strain-DENV-2 and another one used the Malaysian clinical DENV-2 strain. In their first study, no significant difference (p > 0.05) on plasma antigen level was found between the interventiontreated group (1000 mg/kg body weight) on the last day of intervention treatment as well as on second and fourth day after the treatment period, compared to the distilled water group. Similar findings were reported for the plasma viral RNA of interventiontreated group on the second and fourth day after the treatment period [40]. Subsequently, they reported that the same preparation affected four gene expressions (CCL2, ITGB3, FN1, ICAM1) of endothelial cell biology in the liver of the intervention-treated group compared to the untreated group [41]. Their teams also found that the expressions of eight genes (CCL6/MRP-1, CCL8/MCP-2, CCL12/MCP-5, CCL17/TARC, IL1R1, IL1RN/IL1Ra, NAMPT/PBEF1 and PF4/CXCL4) were downregulated (p < 0.05) a day after the treatment period compared to the untreated group [42].
Based on their latest published study, the same leaf juice preparation on mice infected with a different strain of DENV-2 provided some other findings. Total white blood cell count and neutrophils in the intervention-treated group (1000 mg/kg body weight) significantly increased (p < 0.05) by 1.44-fold compared to the untreated group. Five plasma cytokines (GM-CSF, GRO-α, IL-6, MCP-1, MIP-1β) significantly decreased (p < 0.05) in the intervention-treated group (both doses) compared to the untreated group. Whereas levels of IL-1β (as plasma cytokine) and IL-6 (as intracellular cytokine expression in the liver) were only significantly lower (p < 0.05) for 500 mg/kg for the intervention-treated group compared to the untreated group. Dengue viral RNA level in the liver was significantly lower (p < 0.05) in intervention-treated group (1000 mg/kg body weight) compared to the untreated group [43]. Out of seven included in vivo studies using a mouse model, only one study investigated the efficacy of C. papaya leaf juice (seven-day administration) on healthy mice. Levels of platelet and red blood cells were significantly higher (p < 0.05) starting from a day after the treatment period in the intervention-treated group compared to the water control group [44].
Developing a safe and effective antiviral drug is challenging because viruses use host's cells in their replication. The pathogenesis and severity of dengue are linked to immune response interruption caused by the DENV. The DENV not only suppresses bone marrow level, but also attacks by binding to the platelets, which are tiny disc-shaped cells that are produced by megakaryocytes (mature white blood cells) in bone marrow before entering the blood bloodstream and spleen. The generated antibodies for these DENV will then identify and flag the infected-platelets as foreign matter to be exposed to the host's body lines of defense. Infected vascular endothelial cells will have platelets aggregated in them and the generated antibodies eventually kill these cells and platelets too. Such an immune response certainly reduces the platelet counts in the dengue patients. Whilst the bone marrow suppression would lead to anemia and hemorrhagic events [57,58]. Therefore, a drug that protects bone marrow, stimulates platelet production and shortens bleeding and clotting time would be ideal.
Evaluated in vivo evidence in this review mostly induced a thrombocytopenia condition in the animal model and several experiments using a dengue specific animal model to mimic the dengue's key clinical manifestations as much as possible to provide better confidence in the research findings. A gene expression profile of inflammatory cytokines and receptors found to be associated with the presence of dengue infection. Interestingly, the degree of liver damage has been proposed to be correlated to DENV infection because the liver is surrounded with sinusoidal endothelium and during the DENV invasion, endothelial permeability increases. Consequently, irregular levels of liver function enzymes, histopathological lesion and traces of dengue antigen can be detected in liver tissue. Both dengue infected and healthy animal models used in the included studies imply the pharmacological role of the C. papaya leaf juice on functional (activation of phagocytosis and cell proliferation) and non-functional (regulation of immune cell level and release of inflammatory cytokines) immune responses.

Clinical Findings
Patients were confirmed with dengue infection after checking their blood parameters for the presence of dengue immunoglobulin G antibody, dengue immunoglobulin M antibody, and/or non-structural protein 1 antigen [45,[47][48][49][51][52][53][54], whilst some patients were diagnosed without any antigen or antibody test but solely based on low platelet counts (not more than 150,000/µL) and clinical symptoms such as high fever and body ache [46,50].

Case Reports
The different juice preparations used were documented in three case reports (Table 2). These dengue patients, aged 23-51 years old, were also receiving standard treatment associated with dengue symptoms such as fluid replacement, antipyretics, antibiotics, antimalarial drugs, immunosuppressant and antiemetics [45][46][47]. With no symptom improving after receiving five-day standard treatment in the ward, a male patient started to consume the intervention (150 mL) once daily with alternative sip of commercially made fruit juice for the next five days of hospitalization. The platelet count showed some progress (increased by 1.10 × 10 5 /µL), as did the level of white blood cells (by 4.8 × 10 4 /µL) and level of hemoglobin (by 0.5 g/µL) [45]. With the same five-day duration of intervention treatment but consumption frequency increased to twice daily, Ahmad et al., (2011) found that the intervention (25 mL) upregulated the levels of platelets, white blood cells and neutrophil in the male patient starting on the second day of treatment and returned to a normal range on the fifth day of treatment compared to the starting the intervention after five days of standard treatment [46]. Along with receiving standard treatment together with the intervention (25 mL twice daily) for five days, this male patient also received Ayurveda treatment for a further three days together with the intervention. The platelet counts (increased by 1.73 × 10 5 /µL) and white blood cell counts (by 7.3 × 10 4 /µL) showed a good sign of recovering. The patient was discharged after completing the eight-day intervention treatment period [47].

Cross-Sectional Study
A cross-sectional study ( Table 2) involving 214 dengue patients aged ≥18 years old who were admitted to Hospital Universiti Sains Malaysia between January 2014 and December 2015 was conducted to collect information on the use of traditional and complementary medicines (T&CM), which they believe can treat dengue. There were 131 respondents that consumed C. papaya leaf juice at least once daily for three days while receiving standard treatment throughout their hospitalization. Another two T&CMs reported to be commonly used were crab soup (174 respondents) and isotonic drinks (126 respondents) [48].

Quasi Experiment
Comparing before and after treatment effect (Table 2), Hettige (2008) investigated the efficacy of C. papaya leaf juice both adult dengue patients (eight subjects aged 12-55 years old, three females and five males) and children (four subjects aged 5-8 years old, three females and one male), who also received standard oral treatment. After a one-day intervention treatment period, their platelet counts increased by 3000-58,000/µL and white blood cell counts increased by 350-3400/µL. Five of the patients no longer experienced a hemorrhagic skin rash after consuming the intervention. All 12 patients recovered with no hospital admission [49]. Prakash (2012) found that platelet count of all five dengue patients (19-52 years old) increased by 8000-11000/µL after consuming the leaf juice (two tablespoons, three times for one day) [50].
Solanki et al., (2020) studied a larger sample (100 dengue patients: 42 females and 58 males in the intervention group vs. 50 patients: 20 females and 30 males in the control group) who received the leaf juice treatment (adult: 10 mL, children: 5 mL) thrice daily for three days together with one kiwi fruit. Platelet count in the intervention-treated group significantly increased (p < 0.05) by 6.31 × 10 5 /µL, whilst it only increased by 2.64 × 10 3 /µL in the control group, compared to before receiving treatment. Compared to the levels before treatment, the increase in white blood cells (p < 0.05) was higher in the intervention-treated group (1.03 × 10 3 /µL) than the control group (1.73 × 10 2 /µL) [51]. In a clinical study investigating the effect of a six-day leaf juice treatment (5 mL thrice daily), it was reported that not only did platelets and total white blood cell counts increase (p < 0.05) on day 6, but also all nine dengue patients (six females and three males) recovered from lethargy, fatigue and fever [52].

Randomized Controlled Trial
Two open-labelled randomized controlled trials were included in this review ( Table 2). Subenthiran et al., (2013) assessed the platelet count in 18-60 years old dengue patients (111 patients: 20 females and 91 males in the intervention group vs. 117 patients: 14 females and 103 males in control group) after consuming 30 mL of C. papaya leaf juice for three days as well as receiving standard treatment. Compared to 8-h hospitalization, the platelet count after 40-h hospitalization was higher (p = 0.019) (mean difference = −7.890) whilst the platelet count in the control group had a significantly higher value (−7.703) only after 48-h hospitalization. Arachidonate 12-lipoxygenase (ALOX12) and platelet-activating factor receptor (PTAFR) genes highly expressed in intervention-treated groups, respectively, (∆Ct mean = 16.02, fold change = 15.00; ∆Ct mean = 14.87, fold change = 13.42) compared to control group [53]. Another similar trial reported significant findings on other outcome parameters in 16-60 years old in-ward dengue patients (43 patients: 7 females and 36 males in the intervention group vs. 76 patients: 15 females and 61 males in the control group) that consumed 20 mL of the leaf juice twice daily until discharged. The total duration of illness, fever and hospitalization was significantly shorter (p < 0.05) than the control group. Episodes of pleural effusion were also lesser (p < 0.05) in the intervention-treated group (two subjects) compared to the control group (12 subjects) [54].
Dengue hemorrhagic fever is a potentially life-threatening complication found among dengue patients. Clinically, two common severe dengue manifestations are a rapid drop of platelet count and severe hemorrhage (caused by plasma leakage). The mechanism underlying these phenomena correlate to the devastation of infected-platelets and bone marrow suppression during DENV invasion, as mentioned earlier. Certain genes can regulate different biological processes, such as ALOX12 and PTAFR. The ALOX12 gene is highly expressed in platelet, megakargocytes and epidermis, and able to produce 12(S)-hydroxyeicosatetraenoic acid (HETE), which is an essential inflammatory signaling molecule. Therefore, the ALOX12 gene is involved in regulating platelet activation, cell apoptosis, endothelial cell migration and cell proliferation [59]. Platelet activating factor (PAF) is a phospholipid activator and mediator of white blood cells, platelet aggregation, inflammation and anaphylaxis. A G-protein coupled receptor 1 family binds to the PAF (and then forms PTAFR). Therefore, during virus invasion into body, high expression of the PTAFR genes could indicate an active inflammation response to release more platelets [60]. Gathered clinical evidence in this review clearly shows the likelihood of C. papaya in leaf juice form improving the thrombocytopenia condition and modulating the immune response during dengue infection. Apart from these, the leaf juice also seems to be able to relieve dengue symptoms such as fever, skin rash, lethargy, fatigue, pleural effusion, sick period and hospitalization period. Two clinical studies appraised in this review did not recruit dengue patients diagnosed with dengue hemorrhagic fever and/or with an irregular level of liver enzyme and/or low level of creatine kinase. These exclusion criteria were possibly to standardize the severity level of dengue infection among the recruited patients in order to accurately assess the effectiveness of intervention. There are reports that correlated hepatic dysfunction to dengue infection and even proposed the possibility of using the level of liver enzymes as a reference point in predicting severity of dengue infection [61][62][63]. Therefore, not recruiting dengue patients with underlying liver-related problems minimizes the confounding factors that could influence the study outcome.

Safety Findings
Apart from reporting dengue related clinical manifestations, several included studies also shared safety-related observations. Two three-day oral acute toxicity studies using 0.72 mL C. papaya leaf juice per 100 g body weight of healthy rats neither showed hepatoxicity or nephrotoxicity [32,34], but a cytotoxicity effect was observed in healthy bone marrow cells and splenocytes treated with 500 and 1000 µg/mL of leaf juice [32]. Nandini et al., (2021) found that healthy rats treated with 5-2000 mg/kg body weight of leaf juice had a significantly lower level of alanine aminotransferase (p < 0.05) compared to the untreated control group; however, fewer lesions were found on the liver and kidney in the intervention-treated group [37]. Cyclophosphamide-induced thrombocytopenia rats treated with the leaf juice (50 and 150 mg/kg body weight) had fewer histological changes on their livers and spleens compared to the saline-treated group [38]. In addition, Mohd Ridzuan and his team (2018) found that body weights of both mock infected AG129 mice groups: one group treated with 1000 mg/kg body weight of leaf juice, whilst another group treated with distilled water for three days, remained unchanged until the seventh day after the treatment period [40]. In his later study using AG129 mice treated with 500 and 1000 mg/kg body weight of leaf juice for three days, there was no significant body weight changes (p > 0.05) between the intervention-treated and untreated mock infected groups. Spleen size of the mice were also not significantly different (p > 0.05) between the intervention-treated dengue infected group and untreated mock infected group [43].
With the current available information on dengue pathogenesis, several organs (such as the liver (as mentioned earlier) and spleen (one site produces platelets)) and cells (such as platelets, bone marrow cells, splenocytes) are found to be the target of infection. Based on the above reported safety findings, treatment of C. papaya leaf juice, up to certain doses and treatment duration, seems to have a protective effect on the organs of dengue infected subjects.

Discussion
In summary, this is the first review on juice form of C. papaya leaf consisting of 28 included studies that focused on efficacy of this herbal preparation on dengue related parameters. The doses of juice used and treatment duration were varied. Interestingly, the findings from the included studies seemed to be associated with the use of C. papaya leaf juice. Therefore, regardless of variation in dose and treatment duration, this suggests the potential use of the leaf juice in treating dengue manifestation. This review found that C. papaya leaf juice does not demonstrate a linear dose-response relationship in the measured study parameters [32,[36][37][38]43]. Nevertheless, s hormesis dose-response relationship was observed where beneficial effects were observed at low doses instead of high dose. Such a dose-response model has been reported to occur in some therapeutic agents such as antiviral drugs [64]. The DENV replication in a host's body would only be successful by inhibiting the signals of interferon in the body. In conjunction with this factor, a reliable mouse research model in dengue research was established. The AG129 mouse used in dengue research lacks α-, βand γ-interferon receptors making the introduced DENV unable to communicate with the host's interferon and therefore the DENV can successfully replicate in the host's body [65,66]. The use of such a laboratory model could mimic clinical manifestation of human dengue infection. Another team of scientist also discovered the association between endothelial permeability of liver tissue and DENV and suggested that the liver can be the virus replication site [67]. Despite the efforts investigating larvicide(s) in reducing the number of mosquitoes as a disease vector, there was very few studies pertaining to the larvicidal effect of the leaf juice.
The choice of efficacy studies for medicinal plants is commonly determined by the ethnobotanical information. Ethnobotany is a field of study related to traditional knowledge on use of plants such as for medicinal use. This type of valuable information is mostly based on years of belief and observation; and richer in countries with big ethnic diversity as these regions would have more communities, such as indigenous people, who used or tried alternative healthcare treatment, such as using plants to heal health conditions. Several drugs prescribed in conventional medicine originated from naturally occurring substances and are plant-based such as digoxin extracted from Digitalis lanata for hearth problem and morphine (from Papaver somniferum) for pain control. Despite the credit of ethnobotany in drug discovery and development, consumers should always keep in mind that not all traditional knowledge of medicinal plants has been therapeutically investigated to establish a safe dose and therefore they should be more cautious in using it [68].
Lately, an extensive review about the safe use of different formulations of C. papaya leaves highlighted some safety concerns. Apart from the mild gastrointestinal side effects, interaction with co-administered drugs, such as certain hypoglycemic agents, anti-malarial, cardiovascular drugs, and antibiotics, has either increased or decreased the efficacy of the drugs. It revealed that consumption of C. papaya leaves (up to 21 days) affected the reproductivity in both male and female animals. The possibility of consuming products containing C. papaya leaves which cause dysregulation of liver enzymes and lesions on the liver, was suggested from three in vivo studies with long treatment duration (up to 35 days). However, no liver function-related side effect was reported in the appraised clinical data, except there were two case reports that documented a plausible association between consumption of C. papaya leaf extract and irregular level of liver enzymes but the impact from confounding factors, such as any concomitant drug and/or underlying health condition of the patients, was not described in the reports and therefore such association is still questionable [69].
Despite these unfavorable findings reported in the safety review, there are other aspects that shall be addressed too. Firstly, there is no clinical evidence that reports the adverse effect of C. papaya leaves on the human reproductive system, either long or short term. Secondly, as compared to a long consumption period, neither hepatoxicity nor other toxic-related effects were found on the animals treated with a single dose of the leaves aqueous extract within a 14-day study duration [70]. In addition, the safety findings from this review imply protective role of C. papaya leaf juice on the organs of dengue infected subjects. Thirdly, the type of solvents used in plant extraction is a key factor that may influence the efficacy and/or level of toxicity observed, especially when toxic chemical solvents are used [71,72].
A series of rodent toxicity studies using freeze-dried C. papaya leaf juice, from 14 days of single dosing up to 90 days of daily dosing over a range of doses, showed minimal toxicity findings and the no-observed-adverse-level was 2000 mg/kg body weight [70,73,74]. Hence, a human equivalent dose for the 2000 mg/kg body weight by considering a safety factor of 10 is 32.26 mg/kg body weight, which is equivalent to 2.26 g of the freeze-dried C. papaya leaf juice taken by a 70 kg human. The treatment regime prescribed in the included RCTs was 5-30 mL of the juice for adult consumption (vs. children: 2.5-5 mL) for one to six days [49][50][51][52]54].
Flavonoids have already been studied for their antiviral effect on human viruses, such as herpes simplex, polio, parainfluenza and respiratory syncytial viruses [75]. Flavonoids derived from plants have been shown to inhibit dengue viral replication where the in vitro inhibitory effect was observed using plaque assay [76]. Flavanones isolated from Boesenbergia rotunda (L.) Mansf. Kulturpfl. showed competitive inhibition towards NS3 protease [77]. Using inhibition kinetics study, docking and protease assay, certain flavonoids were found to inhibit one of DENV enzymes non-structural proteins (NS2B/NS3 protease) at an allosteric site [78]. Other than the protease complex, a few studies also showed commercial flavonoids and flavonoids isolated from different plants inhibited another DENV enzyme (NS5-RNA-dependent RNA polymerase) activity [79][80][81][82]. Other quercetin and kaempferol analogues showed potential for inhibition on DENV enzymatic activities. Both structures, respectively, formed multi-hydrogen bonds with amino acid residues, which enhanced the binding strength of the compounds at the target site [83][84][85][86][87].
The ability of flavonoids to interact with the cell membrane surface that protects the lipid bilayer against harmful agents, such as free radicals, has been discovered [88,89]. Therefore, the reported erythrocyte membrane stabilizing effect could correlate to the high content of flavonoids in the C. papaya leaf juice. Similarly, scientists also found an association between flavonoid and immunomodulation activity, such as T helper cell differentiation into inflammatory and regulatory cells via mTOR pathway [90]. Interestingly, in this review, the C. papaya leaf juice was also found to be modulating the functional and non-functional immune responses.
As one of the biggest phenolic groups [91] and also the most abundant content found in C. papaya leaf juice, flavonoids have also been reported to modulate platelet aggregation (a clinical manifestation that could happen to dengue patients) through few pathways such as inhibition of arachidonic acid, suppression of cytoplasmic calcium ion, blockage of degranulation and integrin signaling mediated by αIIbβ3 integrin, inhibition of platelet granule secretion, and inhibition of thromboxane formation [92]. One study shows the protecting role of bone marrow by C. papaya leaf extract in regulating protein carbonyl and glutathione contents within bone marrow, less severity of histology lesion found on the lead-induced oxidative damage bone marrow and promotes production of blood cells and platelet in the bone marrow [93]. Similar prominent findings were also reported by the included studies in protecting or enhancing the production of bone marrow cells and splenocytes.
In Malaysia, C. papaya trees are planted for food consumption and commercial purposes. Two papaya varieties are popular in Malaysia, i.e., 'eksotika' and 'sekaki'. Both varieties can be commonly found as hermaphrodite and there are slight differences in their physical appearance [94]. Currently, only one metabolite profiling analysis was conducted on the fruit part of both varieties and a distinct metabolite profile was found between the 'eksotika' and 'sekaki' [95]. Similar extensive profiling on the leaf part to investigate content difference for both varieties is still lacking.
The phytochemicals detected in the C. papaya leaf juice, such as the flavonoids, should be given attention to as they could be potential DENV inhibitors. These points of discussion provided insights on the potential of the C. papaya leaf found in this review; for example, recovery of platelet count to minimize risk of bleeding among thrombocytopenic dengue patients. Compared to the three plausible mechanisms of action of C. papaya leaf on dengue infection (anti-thrombocytopenic effect, immunomodulatory effect and antiviral effect) suggested by Bok et al., (2020) [96], the findings from this review focusing on leaf juice, emphasize the effect of larvicidal activity, anti-thrombocytopenia and immunomodulation.
Based on the gathered scientific evidence in this review, not only the larvicidal effect, but the C. papaya leaf juice also has the potential in relieving dengue manifestations (antithrombocytopenic effect and immunomodulatory effect), which are preferable for only a short consumption period, such as the short treatment regime in treating dengue patients.

Review Limitation
The findings from this scoping review are restricted by several factors. Firstly, only English articles with accessibility of full text were included. Secondly, the therapeutic use of C. papaya leaf was only limited to juice form. Thirdly, there were insufficient clinical trials that met inclusion criteria. Fourthly, several included studies provided experimental values in graphic form and thus the authors were unable to compare the findings in one study to another study that measured the same parameter. Consequently, the authors were unable to perform a meta-analysis to draw solid conclusion on anti-dengue activity of C. papaya leaf juice. However, given the limitations faced in conducting this scoping review, it is unlikely that any missed data would possibly amend the conclusion drawn based on this review due to a clear focus on juice form obtained from only the leaf part and the electronic database search was performed to include citations and anywhere the keywords appeared in the article.