Dengue is a rapidly growing public health problem faced by 50 per cent of the global population1. Currently, there is a single vaccine which is approved for use in certain dengue-endemic countries and two vaccines are under phase III trials2. There are no antivirals for treatment. Progression-to-severe disease in infected individuals is hastened by the host inflammatory response contributed by cytokine storm involving interleukins (IL) – IL-1β, IL-6, IL-10, interferon-γ (IFN- γ) and tumour necrosis factor (TNF)-α3. Severe dengue, characterized by vascular leakage due to disruption of endothelial cell layer in the vascular system3, is preceded by thrombocytopenia (rapid fall in platelet count) indicating disruption of thrombopoiesis. These processes in the pathogenesis indicate the requirement of both immunomodulators and antivirals for treatment. Medicinal plants and herbal medicines, which are known to possess both immunomodulatory and antiviral activities, are now being considered as potential alternatives for the management of dengue worldwide. Among all the potential medicinal plants considered for dengue treatment, Carica papaya has been studied extensively. This perspective is an attempt to summarize the studies on immunomodulatory and antiviral effects of papaya leaf extract on dengue infection along with the essential safety concerns regarding its consumption for medicinal purposes and suggest a way forward regarding the utility of the extract.
C. papaya is used in Ayurveda to treat several digestive disorders. Whole C. papaya plant is used for its antimicrobial, antimalarial, anti-helminthic, hepatoprotective, male and female antifertility and immunomodulatory effects4. Several compounds such as papain, flavonoids, L-tocopherol, ascorbic acid and others present in papaya leaves possess therapeutic properties. The antioxidants present in papaya leaves act by reducing lipid peroxidation, and also exhibit anti-tumour and immunomodulatory effects. Further the leaves contain vitamin B, vitamin A and vitamin C and are rich in minerals such as sodium and potassium. Metabolites such as quinic acid, malic acid, caffeoyl malate, manghaslin (quercetin based), p-coumaroyl malate, clitorin, rutin, feruloyl malate, nicotiflorin and carpaine have been identified in C. papaya leaf extract (CPLE) using advanced phytochemical analytic techniques5. Metabolites such as carpaine and quercetin are known to possess anti-plasmodial and anti-dengue activity6,7. Carpaine is the principal alkaloid component contained in the CPLE6. It comprises 63 per cent of the total alkaloid content8 and has been reported to be a major contributor towards anti-thrombocytopenic properties9. Carpaine also possess antitumour activity and anti-helminthic activity10. In a study by Zunjar et al9, among the two types of phytochemical groups isolated from CPLE, i.e. phenols and alkaloids, only alkaloid fraction exhibited good biological activity. Carpaine exhibited potent activity in busulfan-induced thrombocytopenic mice by sustaining platelet counts up to 555.50±85.17×109/l with no acute toxicity. Papaya leaves also contain pseudocarpaine, and dehydrocarpaine I and II, which impart bitter taste to the leaves11.
Use of CPLE to increase the platelet count in dengue-affected patients is a topic of debate. Whether the natural increase in the platelet count observed in dengue patients after the defervescence phase12 is being mistaken as the effect of papaya leaves, has been addressed by several studies. The anecdotal evidence is supported by studies indicating rapid increase in platelet count in a test group of dengue patients following administration of CPLE in the form of pills/tonic13,14. Thrombocytopenic mice treated with CPLE showed increased levels of IL-6 and thrombopoietin, cytokines which attracts leucocytes at the site of infection and regulates platelet production, respectively15,16. Dengue virus-infected AG129 mice treated with CPLE showed decreased production of IL-1 β, IL-6, monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory proteins (MIP)-1 β in the plasma17. A study carried out in murine model revealed that the oral uptake of CPLE increased platelet and red blood cell counts compared to the control group18. Multiple clinical trials have established platelet-enhancing effects of CPLE in dengue-infected adults and paediatric age group patients14,19-21. A double-blind, placebo-controlled, randomized, prospective study carried out on 51 adult dengue cases with severe thrombocytopenia (≤30,000/μl) revealed that oral uptake of a tablet containing CPLE (1100 mg) three times a day for five days significantly increased platelet counts compared to the placebo group. The time to recovery of platelet count (≥50,000/μl) was two days in the treatment group compared to three days in the placebo group. The same study also revealed that the cytokines responsible for inflammatory response (IL-6, TNF-α and IFN-γ) were suppressed in the treatment group compared to the placebo group19. Another study, which investigated the use of oral administration of CPLE juice (prepared from 50 g of leaves) one time for three days, reported a significant increase in platelet count with the effect more prominent between eight and 48 h of treatment in the intervention group compared to the placebo. Gene expression studies revealed significant increase in the expression of the ALOX12 gene in the treatment group20. In patients with familial thrombocytopenia, decreased expression of the ALOX12 gene has been reported22. ALOX12 gene codes for platelet type 12 lipoxygenase, which influences many platelet functions23. CPLE has also been reported to exert significant inhibition of haemolysis along with erythrocyte membrane stabilization24. Thus, the immunomodulatory effect of CPLE, particularly with regard to the platelet count enhancing effect, has been established9,19,21. The suppressive effect of CPLE on inflammatory cytokine suggests that CPLE could play a role in ameliorating cytokine storm mediated pathogenesis in dengue patients. Demonstration of immunomodulatory effects of CPLE has even resulted in a few patent applications to enable exploration of its therapeutic potential25,26.
An in vitro study with whole CPLE has been shown to exert substantial antiviral effect at a concentration of 2000 µg/ml27. Methanol and freeze-dried CPLE showed no inhibition of dengue virus (DENV)28,29. However, a bioflavonoid quercetin present in papaya leaves has been shown to reduce DENV viral RNA by 75 per cent compared to control with a selectivity index of 8.730. In silico studies have shown the interaction of quercetin with dengue NS2B-NS3 protease31. Treatment of dengue virus infected cells with CPLE reduced the expression of viral E and NS1 protein at 100 µg/ml concentration and a complete inhibition at 200 µg/ml15. Another study reported increase in plasma cytokines involved in macrophage and monocyte recruitment to the site of infection32. These findings suggest that the antiviral role of CPLE is still ambiguous. Selection of proper extraction solvent and the techniques used for extraction play a pivotal role in the proper extraction of bioactive compounds from plants. Use of different solvents (polar/nonpolar) lead to the extraction of a different set of bioactive compounds. Commonly used solvents are water, methanol, ethanol, ethyl acetate, acetone, chloroform, etc. A study conducted to establish the phytochemical profile of CPLE showed that the maximum yield was obtained in aqueous solvent and least was obtained in n-hexane (water>methanol>ethanol>ethyl acetate>dichloromethane>n butanol>n hexane)33. Phytochemical screening revealed that the aqueous extract contains flavonoids, tannins and saponins and the methanol extract contains flavonoids, alkaloids, tannins and saponins as major components. The chloroform extract contains saponins and tannins, while n-hexane extracts contain steroids, flavonoids, saponins and tannins34. Hence, change in extraction solvent may affect antiviral activity. Likewise, techniques for extraction such as supercritical fluid extraction, lyophilized leaf extract, papaya extract-silver nanoparticle and pulverized papaya leaf powder may provide varied results. We evaluated the in vitro antiviral activity (on Vero CCL-81 cells) of certain papaya-based tonics/drugs commercially available in the market to treat thrombocytopenia in DENV infection. The results revealed that there was no significant reduction in virus titre in drug-treated cells compared to virus control35. It is possible that the methods used to prepare these commercial products may not lead to the extraction of possible antiviral agents present in the CPLE but may be sufficient to retain the agents involved in immunomodulation. Moreover, the concentration of the active principle that may act as an antiviral agent in the CPLE might be too low to exert any inhibitory effect. Although there are no studies to suggest that different varieties of C. papaya vary in their effectiveness in increasing the platelet counts, studies have suggested that plants grown in different geographical locations will have varied concentration of the active constituents. Importantly, leaves from young plants have been reported to have higher concentration of carpaine36.
Although Ayurveda/traditional medicines work best as a whole of the extract of an identified plant/part of the plant, to be considered as a phytopharmaceutical drug, the traditional drug preparation or purified extract or fraction should contain at least four defined bioactive compounds which can be assessed qualitatively and quantitatively37. In order to establish the potential of CPLE as a phytopharmaceutical drug, we need to identify and isolate the active principle, evaluate its mechanism of action, side effects, and finally develop a balanced formulation which can be assessed qualitatively and quantitatively. The absorption, distribution, metabolism and excretion profile along with toxicity (ADMET) prediction is important for studying a drug’s pharmacokinetics in human body38. In silico studies have reported that quercetin and luteolin present in CPLE act as lead compounds against NS2B/NS3 protease with good ADMET efficacy31,39. Different parts of the papaya plant have different properties. The ability to increase the platelet count has been attributed only to the leaves and not the fruits or seeds40. While modern drug development involves isolation of active principle from the crude plant, the development of complex formulations involves combining simple extracts with other herbs and further subjecting them to complex processing methods. From biomedical point of view, there is a need to conduct more studies, observations and investigations to gain a thorough understanding of the potential uses of papaya leaves in dengue fever. Noticeably, CPLE does not show cell toxicity even in high doses41, and has been shown to be safe for short-term use in adults. It is suggested that persons with allergic tendencies, liver impairment and pregnant ladies should consume it with caution and gastrointestinal disturbances and potential herb-drug interactions are reported as common side effects42.
There is a need to explore many more herbal formulations which are used locally, followed by proper documentation, and scientific studies of their efficacy, understanding the mechanism of action along with safety profile so that they can be exploited for their potential anti-dengue activity. Thorough examination of candidate herbal plants with promising antiviral activities on similar lines can be helpful. However, adhering to pre-clinical and clinical study guidelines and to other regulatory issues in such efforts will be critical.
Financial support & sponsorship: None.
Conflicts of Interest: None.
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