Mature leaf concentrate of Sri Lankan wild type Carica papaya Linn. modulates nonfunctional and functional immune responses of rats

Pharmacological manipulation of the immune system is emerging as a novel approach in managing human diseases. There is a recent spurt in the interest of identification of immunomodulatory leads of herbal origin which are safe, efficient and economical in chronic usage.

The immune system is an ensemble of complex defense mechanisms functioning at different levels, from an individual cell to the whole organism [1]. Hence, imbalance of the immune homeostasis is implicated in the onset and progression of chronic ailments [2]. An overactive immune system is associated with autoimmunity, chronic inflammatory diseases, systemic vasodilatation and carcinogenesis [3, 4].Conversely, immunosupression increases the susceptibility to infection and is implicated in tumor development [5, 6].

The clinical concept of immunomodulation focuses on stimulating both innate and adaptive arms of immunity or suppressing excessive immune function using exogenous therapeutic agents [2]. Synthetic cytotoxic drugs (cyclophosphamide, methotrexate and 5-fluorouracil), recombinant cytokine therapy, and antibody therapy are established in allopathic medicine as immunotherapy against cancers and autoimmune diseases [7]. However, the occurrence of adverse events and high costs of these have generated barriers to successful therapeutic applications [8]. In this regard, plant based immunomodulators are advantageous considering their pleotropic activity, less side effects and cost effectiveness.

Since ancient times, medicinal plants have been used virtually in all cultures as sources of medicines to improve the immune system. Recently, advancements in immunological techniques enabled the characterization of medicinal plants in unprecedented detail. Numerous plant species such as Aloe vera, Withania somnifera, Allium sativum, Ocimum sanctum, Azadirachita indica and many others have been scientifically justified for their immunomodulatory activity together with possible mechanistics [9].

Carica papaya, (Common name- paw paw) belongs to family Caricaceae and is one of the most popular and economically important plants in the world as a food source and as a herbal medicine [10]. It is native to the tropics of the Americas but now is cultivated worldwide as a fruit crop [10].

Different parts of C. papaya has been traditionally claimed as therapeutics for a broad range of diseases [10]. Particularly the leaf extract/concentrate has been scientifically investigated for pharmacological properties such as anti-microbial, anti-parasitic (antimalarial) activity, anti-cancer, anti-inflammatory and membrane stabilization properties [10].

In Sri Lankan traditional medicine the leaf juice of C. papaya is claimed as a powerful remedy for modulating the immune system (Personal communication- Dr. Pathum Jayaweera, Sri Lankan traditional medical practitioner). Thus far several in vitro and in vivo experiments have been carried out to investigate the immuno pharmacological properties of the leaf concentrate of C. papaya.

Our group for the first time, established the platelet and total white blood cell (WBC) increasing activity of the mature leaf concentrate of C. papaya of the red lady cultivar grown in Sri Lanka using hydorxyurea induced thrombocytopenic Wistar rat model as well as in normal counterpart rats [11]. Concurrently, we observed pronounced anti-inflammatory activity of this preparation indicating immunomodulatory potential [11]. Similarly, oral gavage of papaya leaf extract exhibited a significant platelet and WBC increasing activity in immunosuppressive rats using cyclophosphamide [12] and ansgrelide (thrombocytopenic drug) [13].

Dharmarathna et al. [14] observed a marked platelet count elevation in none thrombocytopenic mice following the oral treatment of C. papaya extract for seven days [14]. Another study conducted using non thrombocytopenic rats reported a marked increment in platelet counts, mean cell haemoglobin (MCH) and mean corpuscular volume (MCV) following the oral treatment with this leaf formulation for 7 days [15].

In addition to animal experimentation, the platelet increasing activity of papaya leaf extract has been validated in thrombocytopenia associated dengue patients. An open labeled randomized controlled trial carried out with 228 patients (Treatment = 111, Control = 117) with dengue fever (DF) and dengue haemorrhagic fever (DHF) revealed a significant increase of platelet counts after 40 and 48 h of oral administration of C. papaya leaf juice for 3 consecutive days [16]. A pilot study reported an increase of platelet and WBC counts of dengue patients after 24 h of administration of papaya leaf juice and patients recovered without hospital admission [17]. A similar effect was also observed in an open labeled randomized controlled trial with 30 dengue patients treated with a tablet (Caripill) prepared from C. papaya leaf extract [18]; A significant increase of platelet counts was observed following oral treatment with this tablet three times daily for five days and exerted fewer side effects and good tolerability.

Although, a wealth of information is available on platelet increasing activity of different extracts of C. papaya leaves, a comprehensive account on immunomodulatory potential is limited. Otsuki et al. [19] reported immunomodulatory potential of the aqueous extract of C. papaya leaf using in vitro culture of human peripheral blood mononuclear cells (PBMCs).

When PBMCs were treated in vitro with C. papaya aqueous extract, Interleukin 2 and 4 (IL-2 and IL-4) secretion were attenuated whereas that of Interleukin 12p40, 12p70 (IL- 12p40, IL-12p70), Interferon Gamma (IFN-γ) and Tumor Necrosis Factor alpha (TNF-α) was enhanced without growth inhibition [19]. Moreover, microarray analyses showed the expression of 23 immunomodulatory genes of PBMC [20].

Tomar et al. [21] reported oral administration of alcoholic extract of papaya leaf (50–200 mg/kg) had stimulated both innate and humoral immune responses of Bagg Albino laboratory bred (BALB/c) mice.

Although, the immunomodulatory potential of different C. papaya extracts (aqueous and alcohol) was adequately demonstrated in the above in vitro and in vivo studies, a comprehensive account of the effect of the mature leaf concentrate of C. papaya leaf both on nonfunctional and functional immune responses may strongly warrant the development of a potential drug lead.

Thus, the present study specifically aimed at investigating the effect of MLCC on nonfunctional (immune cell counts and cytokine levels) and functional (cell proliferation and phagocytosis) immunological parameters of rats. Further, liver and kidney function parameters were examined to envisage the safety of oral administration of the MLCC. Moreover, the chemical profile of the MLCC was investigated using liquid chromatography-mass spectrometry (LC-MS) and Gas chromatography-mass spectrometry (GC-MS).

A preliminary study on platelet increasing activity revealed that the Sri Lankan wild type C. papaya variety possessed more potent platelet increasing activity than the hybrid cultivars such as red lady (unpublished data). Thus, leaves of the Sri Lankan wild type cultivar of C. papaya were used in this study.

The oral administration of MLCC for 3 consecutive days had significantly ameliorated rat immune cell counts i.e. platelets, WBC and BMCs, where platelet increasing activity was pronounced. Increase of both platelets and WBCs were consistent with previous studies established with non-thrombocytopenic [14, 15] as well as thrombocytopenic murine models [12, 13].

The immunostimulatory potential of the MLCC is well illustrated in the numerical increment of total WBCs as well as of sub populations of monocytes and lymphocytes. Significant increase in the percentage of circulating mononuclear cells (lymphocytes and monocytes) indicated the effectiveness of the MLCC on both innate and adaptive arms of the immune system [1]. Previously, aqueous leaf extract of C. papaya treated peripheral blood mononuclear cells (PBMCs) elicited an up regulation of 23 genes mainly including monocyte chemo-attractant protein-1, 2 and 3 (MCP-1, MCP-2 and MCP-3) [19]. These proteins regulate the migration and infiltration of monocytes/macrophage [27]. Thus, activation of monocytes in numbers and function by the papaya leaf constituents raise the possibility of enhancing immunological surveillance.

The mechanism of the papaya leaf constituents induced platelet and leukocyte increment continues to be explored. Augmentation of bone marrow hematopoiesis is the most debated mechanism in literature [28]. Tham et al., [29] established that the leaf extract of C. papaya can reverse lead acetate induced oxidative damage by regeneration and stimulation of hematopoiesis, particularly of myeloblasts and megakaryocytes as evident in histopatholoigcal examination [29]. Moreover, the unaltered levels of SC counts which are mature lymphocytes also highlight the preference of the MLCC on hematopoietic stem cells. Therefore, it may be posited that the MLCC specifically stimulates hematopoietic stem cells to propagate and differentiate into precursor cells of megakaryocytes and those of leukocyte origin which will be mobilized in peripheral blood. In addition to the stimulation of BMCs, a rapid elevation of platelets in thrombocytopenic rats following oral gavage of MLCC was surmised to the release of stored platelets from the spleen [11]. An observation of platelet increment by the low dose of MLCC irrespective of significant upsurge of BMCs agrees with the probable release of stored platelets in the spleen.

Previously, upregulated synthesis of IL-6, a major thrombopoietic cytokine, was observed in human peripheral blood lymphocytes and stem cells from exfoliated deciduous teeth following the treatment with unripe papaya extract rich in papain [30]. This finding prompted us to investigate the effects of the oral gavage of MLCC on rat cytokine levels which may regulate the immune responses.

The present study established, in contrast to Aziz et al. [30], that plasma levels of IL-6 were significantly reduced in rats treated with the MLCC. Although, papaya based extracts are known to induce IL-6 levels in in vitro stem cell cultures, oral administration of the MLCC had reduced the systemic IL-6 levels. This paradox may be due to different experimental systems and pleiotropic activity of IL-6 [31].

Similar to that of IL-6, TNFα levels were significantly reduced by the oral treatment of MLCC. Conversely, the reduction of IFNγ levels was not significant. Since, IL-10 levels were lower than the detection levels of the ELISA kits used, the influence of MLCC on IL-10 cannot be predicted. However, inhibition of TNFα indicated anti-inflammatory property of the MLCC which was established previously [11, 32].

The MLCC, administered orally to rats enhanced phagocytosis of peritoneal macrophages and suggested a modulation of functional immunity of rats. Consistent with our findings, Tomar et al. [21] in 2012 demonstrated that oral treatment of an alcohol extract of C. papaya leaf significantly increased carbon clearance by the rat reticular-endothelial system that provided evidence for enhanced phagocytosis [21].

The in vitro phagocytic activity based on NBT dye reduction assay, revealed an enhancement of phagocytosis by the MLCC treatment. However, precise dose dependency was not observed. Increased phagocytosis in both in vitro and in vivo experiments signifies the immunostimulatory potential of the MLCC.

Increased phagocytic activity of the PMs was correlated with Th1 type cytokine response. IFNγ enhances phagocytosis and increases protection towards pathogens [33]. The MLCC mediated IL-10 may stimulate the anti-inflammatory responses [34] and may lessen the consequences of inflammation.

The functional immunological assays based on ex vivo proliferation provide more concrete evidence for immunomodulatory potential of the MLCC. The proliferation ability of the MLCC was compared with LPS, a bacterial endotoxin that revealed paradoxical activity i.e. low concentrations of the MLCC stimulated the proliferation while higher concentrations exhibited cytotoxic activity. Such a biphasic dose responsive effect is termed “hormesis” [35]; A comparable response pattern was observed in in vitro membrane stabilization potential of papaya leaf extract [36]. Thus, we assume bioactivities of the papaya leaf constituents may follow this type of dose response.

The significant proliferation of BMCs and SCs by the low concentration of the MLCC may be attributed to the presence of single or several types of mitogens or growth factors. Plant mitogens such as lectins are glycoproteins that nonspecifically bind to cell surfaces and stimulate cells to undergo mitosis [37]. However, these are selective in triggering T or B cell populations. Previously, several mitogens such as Phytohaemagglutinin(PHA), concanavalin A (Con A) and Pokeween Mitogen (PWM) have been identified from plants and these mostly impacted on the proliferation of T lymphocytes [37].Conversely, polysaccharides, polyneucleotides or lipoproteins from bacterial cell wall favor B cell proliferation. There is a high possibility that fresh plant extracts such as the MLCC may contain bacteria and their products (endotoxins) which may provide false positive results. To eliminate such contaminations the MLCC was filtered using 0.22 μm filter and furthermore, contamination of bacterial endotoxins was ruled out due to negative results obtained from the LAL test, confirming that the mitogenic activity indeed resulted due to phytoconstituents of the MLCC.

Previously, human PBMC cultures treated with C. papaya aqueous leaf extract revealed an enhanced production of cytokines, such as IL-12p40, IL-12p70, IFN-γ and TNF-α without growth inhibition [19]. A similar pattern of IFNγ release was observed in both rat cell types (BM and SC) we tested, but in contrast to Ostuski et al. [19] we observed significant cell inhibition at high concentrations (500 and 1000 μg/ml) of the MLCC. This disparity may be due to different types of preparation of the papaya leaf, different papaya cultivars used, and cell types used in these two studies.

IFNγ has been specifically reported as a hematopoietic inhibitor as it attenuates human bone marrow colony formation and inhibits CD34+ bone marrow cells [38]. Thus, IFNγ may have played a major role in the cell inhibition observed in high concentrations of the MLCC (500 and 1000 μg/ml). Similarly, elevated level of IFNγ was observed in SCs upon treatment with the MLCC and higher IFNγ levels were correlated with the inhibition of SCs counts. Thus, MLCC induced higher IFNγ may contribute to the inhibition of both BMCs and SCs under in vitro conditions.

Both in vivo and in vitro murine models verified the remarkable immunomodulatory potential of the MLCC. In vivo immunomodulation indicated that the active constituents of the MLCC are indeed bioavailable and effective against the ethno -pharmacologically accepted oral route [39].

The ethno - pharmacological significance of the MLCC was further emphasized by its safety. Though, the MLCC exerted in vitro cytotoxicity at higher concentrations against cultured cells it was well tolerated by rats showing no overt signs of toxicity, stress, aversive behavior or behavioral changes. Further, hepato and renal toxicity were also ruled out. Also, the MLCC failed to alter the body weights and the weights of vital organs of the test rats.

The chemical profile data obtained from LC-MS and GC-MS chromatograms revealed the presence of several bioactive compounds. The LC-MS profile exhibited several peaks and it is presumed these peaks resemble both mitogenic and anti-inflammatory compounds. Among them phenolics and flavonoids may be prominent as previous studies reported phenolic and flavonoids were abundant in the papaya leaf [40]. Further, studies are required to identify the compounds obtained from the LC-MS analysis.

Several potent anti-inflammatory compounds such as Azelaic acid [41], 1-Hexadecanoic acid [42], antioxidant compounds; 2,4-Di-tert-butylphenol [43], were elucidated in the GC-MS analysis. Hence, reported immunomodulatory properties of the MLCC could be attributed to these compounds. In addition, the GC-MS profile revealed the presence of several cytotoxic compounds such as 9-Octadecenoic acid methyl ester [44], 2,4-Di-tert-butylphenol [43] and Benzhydrazide [45]. Thus, these compounds may have contributed to the MLCC induced cytotoxicity observed in higher MLCC concetrations in in vitro cultures of PM, BMC and SCs and warrant the investigation of anticancer activity of the MLCC.

The remarkable ability of differential modulation of the immune system was previously reported for a few plants such as Echinacea angustifolia and Pelargonium sidoides Echinacea [39]. However some preparations are active only in in vitro systems. Nevertheless, the MLCC is an orally active, safe, and readily available preparation which has the potential to develop a therapeutic lead, with immense benefit to individuals suffering from immune disorders such as infections, autoimmune diseases and cancers.

The present study in toto established that the MLCC effectively modulates the nonfunctional and functional immune responses such as stimulation of immune cell proliferation, increase of phagocytosis activity and modulates cytokine responses. Several active secondary metabolites with immunomodulatory properties were identified.

Collectively, when administered orally, the MLCC is safe (non-toxic) for a period of 3 days and is orally active, effectively modulates the immune response and inhibits pro-inflammatory cytokines which overly justify claims of traditional medicine. Hence, the MLCC may be a potential candidate for further research leading to the development of a herbal therapeutic agent for modulating the immune system in numerous diseases.