Cinnamomum cassia exhibits antileishmanial activity against Leishmania donovani infection in vitro and in vivo.

BACKGROUND: There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia. METHODOLOGY: Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS). PRINCIPAL FINDINGS: CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 µg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 µg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect. CONCLUSIONS: Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial compounds.

Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis.

Visceral leishmaniasis (VL) is a fatal, vector-borne disease caused by the intracellular protozoa of the genus Leishmania. Most of the therapeutics for VL are toxic, expensive, or ineffective. Sesquiterpenes are a new class of drugs with proven antimicrobial and antiviral activities. Artemisinin is a sesquiterpene lactone with potent antileishmanial activity, but with limited access to infected cells, being a highly lipophilic molecule. Association of artemisinin with liposome is a desirable strategy to circumvent the problem of poor accessibility, thereby improving its efficacy, as demonstrated in a murine model of experimental VL. Nanoliposomal artemisinin (NLA) was prepared by thin-film hydration method and optimized using Box-Behnken design with a mean particle diameter of 83±16 nm, polydispersity index of 0.2±0.03, zeta potential of -27.4±5.7 mV, and drug loading of 33.2%±2.1%. Morphological study of these nanoliposomes by microscopy showed a smooth and spherical surface. The mechanism of release of artemisinin from the liposomes followed the Higuchi model in vitro. NLA was free from concomitant signs of toxicity, both ex vivo in murine macrophages and in vivo in healthy BALB/c mice. NLA significantly denigrated the intracellular infection of Leishmania donovani amastigotes and the number of infected macrophages ex vivo with an IC50 of 6.0±1.4 µg/mL and 5.1±0.9 µg/mL, respectively. Following treatment in a murine model of VL, NLA demonstrated superior efficacy compared to artemisinin with a percentage inhibition of 82.4%±3.8% in the liver and 77.6%±5.5% in spleen at the highest dose of 20 mg/kg body weight with modulation of cell-mediated immunity towards protective Th1 type. This study is the first report on the use of a liposomal drug delivery system for artemisinin as a promising alternative intervention against VL.

Immunotherapeutic Potential of Eugenol Emulsion in Experimental Visceral Leishmaniasis.

BACKGROUND: The therapy of visceral leishmaniasis (VL) is limited by resistance, toxicity and decreased bioavailability of the existing drugs coupled with dramatic increase in HIV-co-infection, non-availability of vaccines and down regulation of cell-mediated immunity (CMI). Thus, we envisaged combating the problem with plant-derived antileishmanial drug that could concomitantly mitigate the immune suppression of the infected hosts. Several plant-derived compounds have been found to exert leishmanicidal activity via immunomodulation. In this direction, we investigated the antileishmanial activity of eugenol emulsion (EE), complemented with its immunomodulatory and therapeutic efficacy in murine model of VL. METHODOLOGY/PRINCIPAL FINDINGS: Oil-in-water emulsion of eugenol (EE) was prepared and size measured by dynamic light scattering (DLS). EE exhibited significant leishmanicidal activity with 50% inhibitory concentration of 8.43±0.96 µg ml-1 and 5.05±1.72 µg ml─1, respectively against the promastigotes and intracellular amastigotes of Leishmania donovani. For in vivo effectiveness, EE was administered intraperitoneally (25, 50 and 75 mg/kg b.w./day for 10 days) to 8 week-infected BALB/c mice. The cytotoxicity of EE was assessed in RAW 264.7 macrophages as well as in naive mice. EE induced a significant drop in hepatic and splenic parasite burdens as well as diminution in spleen and liver weights 10 days post-treatment, with augmentation of 24h-delayed type hypersensitivity (DTH) response and high IgG2a:IgG1, mirroring induction of CMI. Enhanced IFN-γ and IL-2 levels, with fall in disease-associated Th2 cytokines (IL-4 and IL-10) detected by flow cytometric bead-based array, substantiated the Th1 immune signature. Lymphoproliferation and nitric oxide release were significantly elevated upon antigen revoke in vitro. The immune-stimulatory activity of EE was further corroborated by expansion of IFN-γ producing CD4+ and CD8+ splenic T lymphocytes and up-regulation of CD80 and CD86 on peritoneal macrophages. EE treated groups exhibited induction of CD8+ central memory T cells as evidenced from CD62L and CD44 expression. No biochemical alterations in hepatic and renal enzymes were observed. CONCLUSIONS: Our results demonstrate antileishmanial activity of EE, potentiated by Th1 immunostimulation without adverse side effects. The Th1 immune polarizing effect may help to alleviate the depressed CMI and hence complement the leishmanicidal activity.

Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo.

Visceral leishmaniasis (VL) is a life-threatening protozoal infection chiefly impinging the rural and poor population in the tropical and sub-tropical countries. The deadly affliction is rapidly expanding after its association with AIDS, swiftly defying its status of a neglected disease. Despite successful formulation of vaccine against canine leishmaniasis, no licensed vaccine is yet available for human VL, chemotherapy is in appalling state, and the development of new candidate drugs has been painfully slow. In face of lack of proper incentives, immunostimulatory plant preparations owing antileishmanial efficacy bear potential to rejuvenate awful antileishmanial chemotherapy. We have earlier reported profound leishmanicidal activity of Piper nigrum hexane (PNH) seeds and P. nigrum ethanolic (PNE) fractions derived from P. nigrum seeds against Leishmania donovani promastigotes and amastigotes. In the present study, we illustrate that the remarkable anti-promastigote activity exhibited by PNH and PNE is mediated via apoptosis as evidenced by phosphatidylserine externalization, DNA fragmentation, arrest in sub G0/G1 phase, loss of mitochondrial membrane potential and generation of reactive oxygen species. Further, P. nigrum bioactive fractions rendered significant protection to L. donovani infected BALB/c mice in comparison to piperine, a known compound present in Piper species. The substantial therapeutic potential of PNH and PNE was accompanied by elicitation of cell-mediated immune response. The bioactive fractions elevated the secretion of Th1 (INF-γ, TNF-α, and IL-2) cytokines and declined IL-4 and IL-10. PNH and PNE enhanced the production of IgG2a, upregulated the expression of co-stimulatory molecules CD80 and CD86, augmented splenic CD4(+) and CD8(+) T cell population, induced strong lymphoproliferative and DTH responses and partially stimulated NO production. PNH and PNE were devoid of any hepatic or renal toxicity. These encouraging findings merit further exploration of P. nigrum bioactive fractions as a source of potent and non-toxic antileishmanials.

Corrigendum: Leishmanicidal activities of Artemisia annua leaf essential oil against Visceral Leishmaniasis.

[This corrects the article on p. 626 in vol. 5, PMID: 25505453.].

Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a fatal vector-borne parasitic syndrome attributable to the protozoa of the Leishmania donovani complex. The available chemotherapeutic options are not ideal due to their potential toxicity, high cost and prolonged treatment schedule. In the present study, we conjectured the use of nano drug delivery systems for plant-derived secondary metabolite; artemisinin as an alternative strategy for the treatment of experimental VL. Artemisinin-loaded poly lactic co-glycolic acid (ALPLGA) nanoparticles prepared were spherical in shape with a particle size of 220.0±15.0 nm, 29.2±2.0% drug loading and 69.0±3.3% encapsulation efficiency. ALPLGA nanoparticles administered at doses of 10 and 20mg/kg body weight showed superior antileishmanial efficacy compared with free artemisinin in BALB/c model of VL. There was a significant reduction in hepatosplenomegaly as well as in parasite load in the liver (85.0±5.4%) and spleen (82.0±2.4%) with ALPLGA nanoparticles treatment at 20mg/kg body weight compared to free artemisinin (70.3±0.6% in liver and 62.7±3.7% in spleen). In addition, ALPLGA nanoparticle treatment restored the defective host immune response in mice with established VL infection. The protection was associated with a Th1-biased immune response as evident from a positive delayed-type hypersensitivity reaction, escalated IgG2a levels, augmented lymphoproliferation and enhancement in proinflammatory cytokines (IFN-γ and IL-2) with significant suppression of Th2 cytokines (IL-10 and IL-4) after in vitro recall, compared to infected control and free artemisinin treatment. In conclusion, our results advocate superior efficacy of ALPLGA nanoparticles over free artemisinin, which was coupled with restoration of suppressed cell-mediated immunity in animal models of VL.

Apoptosis mediated leishmanicidal activity of Azadirachta indica bioactive fractions is accompanied by Th1 immunostimulatory potential and therapeutic cure in vivo.

BACKGROUND: Exploration of immunomodulatory antileishmanials of plant origin is now being strongly recommended to overcome the immune suppression evident during visceral leishmaniasis (VL) and high cost and toxicity associated with conventional chemotherapeutics. In accordance, we assessed the in vitro and in vivo antileishmanial and immunomodulatory potential of ethanolic fractions of Azadirachta indica leaves (ALE) and seeds (ASE). METHODS: A. indica fractions were prepared by sequential extraction of the powdered plant parts in hexane, ethanol and water. Erythrosin B staining was employed to appraise the anti-promastigote potential of ALE and ASE. Cytostatic or cytocidal mode of action was ascertained and alterations in parasite morphology were depicted under oil immersion light microscopy. Study of apoptotic correlates was performed to deduce the mechanism of induced cell death and anti-amastigote potential was assessed in Leishmania parasitized RAW 264.7 macrophages. In vivo antileishmanial effectiveness was evaluated in L. donovani infected BALB/c mice, accompanied by investigation of immunomodulatory potential of ALE and ASE. Adverse toxicity of the bioactive fractions against RAW macrophages was studied by MTT assay. In vivo side effects on the liver and kidney functions were also determined. Plant secondary metabolites present in ALE and ASE were analysed by Gas chromatography-mass spectrometry (GC-MS). RESULTS: ALE and ASE (500 µg ml(-1)) exhibited leishmanicidal activity in a time- and dose-dependent manner (IC50 34 and 77.66 µg ml(-1), respectively) with alterations in promastigote morphology and induction of apoptosis. ALE and ASE exerted appreciable anti-amastigote potency (IC50 17.66 and 24.66 µg ml(-1), respectively) that was coupled with profound in vivo therapeutic efficacy (87.76% and 85.54% protection in liver and 85.55% and 83.62% in spleen, respectively). ALE exhibited minimal toxicity with selectivity index of 26.10 whereas ASE was observed to be non-toxic. The bioactive fractions revealed no hepato- and nephro-toxicity. ALE and ASE potentiated Th1-biased cell-mediated immunity along with upregulation of INF-γ, TNF-α and IL-2 and decline in IL-4 and IL-10 levels. GC-MS analysis revealed several compounds that may have contributed to the observed antileishmanial effect. CONCLUSION: Dual antileishmanial and immunostimulatory efficacy exhibited by the bioactive fractions merits their use alone or as adjunct therapy for VL.

Th1-biased immunomodulation and therapeutic potential of Artemisia annua in murine visceral leishmaniasis.

BACKGROUND: In the absence of vaccines and limitations of currently available chemotherapy, development of safe and efficacious drugs is urgently needed for visceral leishmaniasis (VL) that is fatal, if left untreated. Earlier we reported in vitro apoptotic antileishmanial activity of n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) against Leishmania donovani. In the present study, we investigated the immunostimulatory and therapeutic efficacy of AAL and AAS. METHODOLOGY/PRINCIPAL FINDINGS: Ten-weeks post infection, BALB/c mice were orally administered AAL and AAS for ten consecutive days. Significant reduction in hepatic (86.67% and 89.12%) and splenic (95.45% and 95.84%) parasite burden with decrease in spleen weight was observed. AAL and AAS treated mice induced the strongest DTH response, as well as three-fold decrease in IgG1 and two-fold increase in IgG2a levels, as compared to infected controls. Cytometric bead array further affirmed the elicitation of Th1 immune response as indicated by increased levels of IFN-γ, and low levels of Th2 cytokines (IL-4 and IL-10) in serum as well as in culture supernatant of lymphocytes from treated mice. Lymphoproliferative response, IFN-γ producing CD4+ and CD8+ T lymphocytes and nitrite levels were significantly enhanced upon antigen recall in vitro. The co-expression of CD80 and CD86 on macrophages was significantly augmented. CD8+ T cells exhibited CD62Llow and CD44hi phenotype, signifying induction of immunological memory in AAL and AAS treated groups. Serum enzyme markers were in the normal range indicating inertness against nephro- and hepato-toxicity. CONCLUSIONS/SIGNIFICANCE: Our results establish the two-prong antileishmanial efficacy of AAL and AAS for cure against L. donovani that is dependent on both the direct leishmanicidal action as well as switching-on of Th1-biased protective cell-mediated immunity with generation of memory. AAL and AAS could represent adjunct therapies for the treatment of leishmaniasis, either alone or in combination with other antileishmanial agents.

Leishmanicidal activities of Artemisia annua leaf essential oil against Visceral Leishmaniasis.

Visceral leishmaniasis (VL), the second-most dreaded parasitic disease after malaria, is currently endemic in 88 countries. Dramatic increases in the rates of infection, drug resistance, and non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents from natural sources. In this study, we showed the leishmanicidal effect of essential oil from Artemisia annua leaves (AALEO) against Leishmania donovani in vitro and in vivo. AALEO was extracted by hydrodistillation and characterized by GC-MS, the most abundant compounds were found to be camphor (52.06 %) followed by ß-caryophyllene (10.95 %). AALEO exhibited significant leishmanicidal activity against L. donovani, with 50 % inhibitory concentration of 14.63 ± 1.49 µg ml(-1) and 7.3 ± 1.85 µg ml(-1), respectively, against the promastigotes and intracellular amastigotes. The effect was mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA nicking by TdT-mediated dUTP nick-end labeling assay, dyskinetoplastidy, cell cycle arrest at sub-G0-G1 phase, loss of mitochondrial membrane potential and reactive oxygen species generation in promastigotes and nitric oxide generation in ex vivo model. AALEO presented no cytotoxic effects against mammalian macrophages even at 200 µg ml(-1). Intra-peritoneal administration of AALEO (200 mg/ kg.b.w.) to infected BALB/c mice reduced the parasite burden by almost 90% in the liver and spleen with significant reduction in weight. There was no hepato- or nephro-toxicity as demonstrated by normal levels of serum enzymes. The promising antileishmanial activity shown by camphor-rich AALEO may provide a new lead in the treatment of VL.