Bioassay-based Corchorus capsularis L. leaf-derived ß-sitosterol exerts antileishmanial effects against Leishmania donovani by targeting trypanothione reductase.

Leishmaniasis, a major neglected tropical disease, affects millions of individuals worldwide. Among the various clinical forms, visceral leishmaniasis (VL) is the deadliest. Current antileishmanial drugs exhibit toxicity- and resistance-related issues. Therefore, advanced chemotherapeutic alternatives are in demand, and currently, plant sources are considered preferable choices. Our previous report has shown that the chloroform extract of Corchorus capsularis L. leaves exhibits a significant effect against Leishmania donovani promastigotes. In the current study, bioassay-guided fractionation results for Corchorus capsularis L. leaf-derived ß-sitosterol (ß-sitosterolCCL) were observed by spectroscopic analysis (FTIR, 1H NMR, 13C NMR and GC-MS). The inhibitory efficacy of this ß-sitosterolCCL against L. donovani promastigotes was measured (IC50 = 17.7 ± 0.43 µg/ml). ß-SitosterolCCL significantly disrupts the redox balance via intracellular ROS production, which triggers various apoptotic events, such as structural alteration, increased storage of lipid bodies, mitochondrial membrane depolarization, externalization of phosphatidylserine and non-protein thiol depletion, in promastigotes. Additionally, the antileishmanial activity of ß-sitosterolCCL was validated by enzyme inhibition and an in silico study in which ß-sitosterolCCL was found to inhibit Leishmania donovani trypanothione reductase (LdTryR). Overall, ß-sitosterolCCL appears to be a novel inhibitor of LdTryR and might represent a successful approach for treatment of VL in the future.

Curative efficacy of purified serine protease inhibitor PTF3 from potato tuber in experimental visceral leishmaniasis.

To overcome the drug toxicity and frequent resistance of parasites against the conventional drugs for the healing of human visceral leishmaniasis, innovative plant derived antileishmanial components are very imperative. Fuelled by the complications of clinically available antileishmanial drugs, a novel potato serine protease inhibitor was identified with its efficacy on experimental visceral leishmaniasis (VL). The serine protease inhibitors from potato tuber extract (PTEx) bearing molecular mass of 39 kDa (PTF1), 23 kDa (PTF2) and 17 kDa (PTF3) were purified and identified. Among them, PTF3 was selected as the most active inhibitor (IC50 143.5 ± 2.4 µg/ml) regarding its antileishmanial property. Again, intracellular amastigote load was reduced upto 83.1 ± 1.7% in pre-treated parasite and 88.5 ± 0.5% in in vivo model with effective dose of PTF3. Protective immune response by PTF3 was noted with increased production of antimicrobial substances and up-regulation of pro-inflammatory cytokines. Therapeutic potency of PTF3 is also followed by 80% survival in infected hamster. The peptide mass fingerprint (MALDI-TOF) results showed similarity of PTF3 with serine protease inhibitors database. Altogether, these results strongly propose the effectiveness of PTF3 as potent immunomodulatory therapeutics for controlling VL.

White jute (Corchorus capsularis L.) leaf extract has potent leishmanicidal activity against Leishmania donovani.

In pursuit of effective, safe and affordable antileishmanial drugs, the current study was designed to explore Corchorus capsularis L. leaf extract (CCEx) as an effective leishmanicidal substitute against Leishmania donovani. The leaf extract displays potent antileishmanial activity against L. donovani promastigotes with an IC50 value of 79.00 ±â€¯0.3 µg/ml. CCEx also significantly induces intracellular reactive oxygen species (ROS) with a concomitant decrease in the level of non-protein thiols in virulent parasites. Additionally, CCEx treatment induces substantial morphological alterations in parasites. Moreover, reagent-based phytochemical analysis of the extract revealed the presence of various phytochemical constituents. Further study is underway to identify the bioactive component(s) or fraction(s) of CCEx through bioassay-guided fractionation.

Serine protease inhibitors rich Coccinia grandis (L.) Voigt leaf extract induces protective immune responses in murine visceral leishmaniasis.

Leishmaniasis is a parasite-mediated tropical disease affecting millions of individuals worldwide. The available antileishmanial chemotherapeutic modalities exhibit adverse toxicity, exorbitant price and advent of drug-resistant parasites. Hence, plant-derived products are an alternative preference for the emergence of novel and effective antileishmanial agents that rejuvenate the host immunity with limited toxicity. The present work is complementary to our previous report that revealed the in vitro antileishmanial and immunomodulatory activity of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) rich in serine protease inhibitors. Thus, preliminary objectives of the study were to elucidate the leishmanicidal activity and host effector mechanism in Leishmania donovani infected BALB/c mice treated with Cg-Ex. Oral administration of Cg-Ex significantly reduced the spleen and liver parasite burden at dose-dependently. The parasite elimination was associated with generation of ROS and NO that are interrelated with up-regulation of disease-suppressing Th1 cytokines and down-regulation of disease-promoting Th2 cytokines at both protein and mRNA level. Moreover, Cg-Ex augmented the delayed-type hypersensitivity (DTH) response and serum IgG2a level which are correlated with the diminution of parasite burden with no hepatic and renal toxicity. Additionally, histological analysis of spleen depicted the improvement of structural disorganization of white and red pulp after Cg-Ex treatment. Therefore, our intriguing findings have presented the first indication of in vivo antileishmanial efficacy through activation of pro-inflammatory immune responses of the host by a natural plant leaf extract (Cg-Ex) containing serine protease inhibitors which could have a role as a potential immunomodulator against visceral leishmaniasis.

Coccinia grandis (L.) Voigt Leaf Extract Exhibits Antileishmanial Effect Through Pro-inflammatory Response: An In Vitro Study.

The conventional drugs used for the treatment of human visceral leishmaniasis have concerns about the toxicity and most importantly parasite resistance. To overcome these troubles, more efforts are made for the development of innovative therapeutic agents having effective antileishmanial activity and simultaneously stimulate adaptive immune system of host cells. Hence, search for new leishmanicidal from the natural origin like plants has shown its effectiveness for the treatment of this tropical disease. The aim of this study is to investigate and characterize the antileishmanial efficacy of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) with its immunomodulatory property against Leishmania donovani in an in vitro experimental model. Cg-Ex significantly reduces the intracellular L. donovani parasite load with IC50 value 193 ± 0.78 µg/ml, but it has lower cytotoxicity on the murine RAW 264.7 macrophage cell line. Interestingly, Cg-Ex induces the generation of potent antimicrobials like reactive oxygen species and nitric oxide dose dependently in infected murine macrophages. Moreover, the increased production of Th1 cytokines (IL-12, TNF-α) with a concurrent decrease of Th2 cytokines (IL-10, TGF-ß) was also observed in Cg-Ex-treated infected host macrophages. Our results thus confirm that serine protease inhibitor(s)-rich Cg-Ex exhibits antileishmanial activity in vitro, and this was mediated through the modulation of pro-inflammatory cytokines. On the whole, the present findings first demonstrate the antileishmanial property of Cg-Ex targeting the Leishmania serine protease resulting protection of host cells with Th1 cytokine expression. Thus, these data indicate that C. grandis leaf extract (Cg-Ex) might be considered as a new lead for designing alternative and novel natural therapeutic against visceral leishmaniasis.

Inhibition of pro-/active MMP-2 by green tea catechins and prediction of their interaction by molecular docking studies.

Matrix metalloproteinases (MMPs) play a crucial role in developing different types of lung diseases, e.g., pulmonary arterial hypertension (PAH). Green tea polyphenolic catechins such as EGCG and ECG have been shown to ameliorate various types of diseases including PAH. Our present study revealed that among the four green tea catechins (EGCG, ECG, EC, and EGC), EGCG and ECG inhibit pro-/active MMP-2 activities in pulmonary artery smooth muscle cell (PASMC) culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-2 with the green tea catechins by computational methods. In silico analysis revealed a strong interaction of pro-/active MMP-2 with EGCG/ECG, and galloyl group has been observed to be responsible for this interaction. The in silico analysis corroborated our experimental observation that EGCG and ECG are active in preventing both the proMMP-2 and MMP-2 activities. Importantly, these two catechins appeared to be better inhibitors for proMMP-2 in comparison to MMP-2 as revealed by gelatin zymogram and also by molecular docking studies. In many type of cells, activation of proMMP-2 occurs via an increase in the level of MT1-MMP (MMP-14). We, therefore, determined the interactions of MT1-MMP with the green tea catechins by molecular docking analysis. The study revealed a strong interaction of MT1-MMP with EGCG/ECG, and galloyl group has been observed to be responsible for the interaction.

Inhibition of MMP-9 by green tea catechins and prediction of their interaction by molecular docking analysis.

Green tea polyphenolic catechins have been shown to prevent various types of diseases such as pulmonary hypertension (PAH), cancer and cardiac and neurological disorders. Matrix metalloproteinases (MMPs) play an important role in the development of PAH. The present study demonstrated that among the four green tea catechins (EGCG, ECG, EC and EGC), EGCG and ECG inhibit pro-/active MMP-9 activities in pulmonary artery smooth muscle cell culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-9 with the green tea catechins by computational methods. In silico molecular docking analysis revealed a strong interaction between pro-/active MMP-9 and EGCG/ECG, and galloyl group appears to be responsible for this enhanced interaction. The molecular docking studies corroborate our experimental observation that EGCG and ECG are mainly active in preventing both the proMMP-9 and MMP-9 activities.

Protective inflammatory response against visceral leishmaniasis with potato tuber extract: A new approach of successful therapy.

The increasing number of drug resistance issue of Leishmania donovani strain to common drugs compels to develop new therapeutics against leishmaniasis with minimal toxicity. In this regard, bioactive phytocomponents may lead to the discovery of new medicines with appropriate efficiency. The important roles of Leishmania proteases in the virulence of Leishmania parasite make them very hopeful targets for the improvement of current remedial of leishmaniasis. As part of a hunt for new drugs, we have evaluated in vivo anti-leishmanial activity of serine protease inhibitor rich fraction (PTEx), isolated by sodium bisulfite extraction from potato tuber. The amastigote load of 25mg/kg body weight/day treated BALB/c mice showed 86.9% decrease in liver and 88.7% in case of spleen. This anti-leishmanial effect was also supported by PTEx induced immunomodulatory activity like acute formation of ROS and prolonged NO generation. The Th1/Th2 cytokine balance in splenocytes of PTEx treated animals was estimated and evaluated by ELISA assay as well as by mRNA expression using RT-PCR. Furthermore, significant survival rate (80%) was observed in PTEx treated hamsters. Thus, from the present observations we could accentuate the potential of PTEx to be employed as a new therapeutics from natural source against L. donovani. This might also provide a novel perception of natural serine protease inhibitor from potato tuber as an alternate approach for the treatment of visceral leishmaniasis.

Antiproteolytic and leishmanicidal activity of Coccinia grandis (L.) Voigt leaf extract against Leishmania donovani promastigotes.

In visceral leishmaniasis (VL), development of alternative safe therapeutic strategy is gaining paramount wherein natural components of plant origin have prominence. We explored Coccinia grandis (L.) Voigt, a medicinal plant known in traditional folk medicine, for its antileishmanial efficacy. SDS-PAGE analysis of the C. grandis leaf extract (Cg-Ex) showed few protein bands about 14-66 kDa among which three (64.8, 55.8 and 15.3 kDa) were identified as serine protease inhibitors by reverse zymography. Since the virulence of Leishmania is also attributed by serine proteases, objective of the present study was to evaluate in vitro antileishmanial activity of Cg-Ex, targeting Leishmania donovani serine protease(s). Inhibition study of Cg-Ex in gelatin-zymogram and spectrophotometric assay revealed its strong inhibitory activity against bovine trypsin rather than chymotrypsin, and also showed significant inhibition of L. donovani serine protease(s). Further, studies with Cg-Ex were extended to estimate its antileishmanial efficacy with half maximal inhibitory concentration (IC50) at 308.0 ± 2.42 µg/ml along with significant morphological alterations. The results have demonstrated the potential of the serine protease inhibitor rich fraction of the C. grandis leaf extract against visceral leishmaniasis.

Bioactivity guided fractionation of Moringa oleifera Lam. flower targeting Leishmania donovani.

Leishmaniases is a group of diseases caused by the protozoan parasite belonging to the genus Leishmania. At least 20 species of Leishmania are known to infect humans transmitted by female sandflies, Phlebotomus spp. Leishmania donovani causes visceral leishmaniasis, considered most lethal among the common three forms of leishmaniasis. Lack of appropriate vaccines, emergence of drug resistance and side effects of currently used drugs stress the need for better alternative drugs, particularly from natural sources. Here, we conducted in vitro and in vivo experiments to study the efficacy of different parts of Moringa oleifera Lam. against Leishmania donovani promastigotes. The flower extract of M. oliefera (MoF) was found to be the most potent antileishmanial agent when compared to other parts of the plant like leaf, root, bark and stem. It imparted significant reduction in parasite number in infected macrophages. The bioactivity guided fractionation of MoF showed ethyl acetate fraction (MoE) as the most active and gave significant parasite reduction in the infected macrophages. Further, growth kinetics studies revealed loss of L. donovani promastigotes viability in the presence of MoE in both time and dose dependent manner. In vivo experiment in Balb/c mouse model of leishmaniasis supported the in vitro findings with a remarkable reduction of the parasite burden in both liver and spleen.

In vitro anti-leishmanial efficacy of potato tuber extract (PTEx): leishmanial serine protease(s) as putative target.

Leishmaniasis, a neglected tropical disease (NTD) causes major health problems in the tropical and subtropical world. Most of the antileishmanial modern therapies with different formulations of pentavalent antimonials, Miltefosine, Amphotericin B etc. are not satisfactory in recent times due to high toxicity to the host and present rising strain resistance issues. So there is an urgent need to develop new, safe and cost-effective drugs against leishmaniasis. In this regard, bioactive phytocomponents may lead to the discovery of new medicines with appropriate efficiency. The prominent roles played by Leishmania proteases in the virulence of this parasite make them very promising targets for the development of current therapeutics of leishmaniasis. As part of a search for novel drugs, we have evaluated in vitro anti-leishmanial activity of serine protease inhibitor rich fraction (PTEx) obtained from potato tuber. The extract (PTEx) was prepared by sodium bisulfite fractionation and inhibitors were identified by reverse zymography. Inhibition study of PTEx in gelatin-zymogram and spectrophotometric assay using BApNA and BTpNA as substrate reveal its strong inhibitory activity against trypsin as well as serine proteases present in cell lysate of Leishmania donovani infective strain. The in vitro MTT based colorimetric assay as well as ex vivo L. donovani infected macrophages showed reduced parasite viability and intracellular parasite load with IC50 = 312.5 ± 0.1 µg/ml and IC50 82.3 ± 0.2 µg/ml of PTEx respectively in a concentration dependent manner. This anti-leishmanial effect was also preceded by PTEx induced acute formation of ROS and prolonged NO generation. The PTEx has no significant cytotoxic effect on host macrophages. So taken together, these findings indicate that PTEx has promising leishmanicidal effect and thus this study provides a new perspective of natural serine protease inhibitor from potato tuber on the development of new drug against leishmaniasis.

Role of an aprotinin-sensitive protease in protein kinase Calpha-mediated activation of cytosolic phospholipase A2 by calcium ionophore (A23187) in pulmonary endothelium.

Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 microM)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 microM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 microM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the cell membrane, it did not inhibit the cPLA2 activity and AA release caused by both low and high doses of A23187.

Protective role of magnesium in cardiovascular diseases: a review.

A considerable number of experimental, epidemiological and clinical studies are now available which point to an important role of Mg2+ in the etiology of cardiovascular pathology. In human subjects, hypomagnesemia is often associated with an imbalance of electrolytes such as Na+, K+ and Ca2+. Abnormal dietary deficiency of Mg2+ as well as abnormalities in Mg2+ metabolism play important roles in different types of heart diseases such as ischemic heart disease, congestive heart failure, sudden cardiac death, atheroscelerosis, a number of cardiac arrhythmias and ventricular complications in diabetes mellitus. Mg2+ deficiency results in progressive vasoconstriction of the coronary vessels leading to a marked reduction in oxygen and nutrient delivery to the cardiac myocytes. Numerous experimental and clinical data have suggested that Mg2+ deficiency can induce elevation of intracellular Ca2+ concentrations, formation of oxygen radicals, proinflammatory agents and growth factors and changes in membrane perrmeability and transport processes in cardiac cells. The opposing effects of Mg2+ and Ca2+ on myocardial contractility may be due to the competition between Mg2+ and Ca2+ for the same binding sites on key myocardial contractile proteins such as troponin C, myosin and actin. Stimulants, for example, catecholamines can evoke marked Mg2+ efflux which appears to be associated with a concomitant increase in the force of contraction of the heart. It has been suggested that Mg2+ efflux may be linked to the Ca2+ signalling pathway. Depletion of Mg2+ by alcohol in cardiac cells causes an increase in intracellular Ca2+, leading to coronary artery vasospasm, arrhythmias, ischemic damage and cardiac failure. Hypomagnesemia is commonly associated with hypokalemia and occurs in patients with hypertension or myocardial infarction as well as in chronic alcoholism. The inability of the senescent myocardium to respond to ischemic stress could be due to several reasons. Mg2+ supplemented K+ cardioplegia modulates Ca2+ accumulation and is directly involved in the mechanisms leading to enhanced post ischemic functional recovery in the aged myocardium following ischemia. While many of these mechanisms remain controversial and in some cases speculative, the beneficial effects related to consequences of Mg2+ supplementation are apparent. Further research are needed for the incorporation of these findings toward the development of novel myocardial protective role of Mg2+ to reduce morbidity and mortality of patients suffering from a variety of cardiac diseases.