Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice.

Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches.

Antileishmanial lead structures from nature: analysis of structure-activity relationships of a compound library derived from caffeic Acid bornyl ester.

Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization.

Pomegranate pericarp extract enhances the antibacterial activity of ciprofloxacin against extended-spectrum ß-lactamase (ESBL) and metallo-ß-lactamase (MBL) producing Gram-negative bacilli.

A methanolic extract of Punica granatum (pomegranate) fruit pericarp (PGME) was tested in combination with ciprofloxacin against extended-spectrum ß-lactamase (ESBL) producing Escherichia coli, Klebsiella pneumoniae, and metallo-ß-lactamase (MBL) producing Pseudomonas aeruginosa, which were screened for their resistance profile against fluoroquinolone antibiotics. The minimum inhibitory concentrations (MIC) of ciprofloxacin and PGME, alone, were determined, and synergy of ciprofloxacin-PGME combinations evaluated by checkerboard assay and fractional inhibitory concentration (FIC). Nineteen out of forty-nine strains exhibited synergy with ciprofloxacin (FIC of 0.125-0.5 for ciprofloxacin) further verified by agar-well assay. This could be due to the bacterial efflux pump inhibitor (EPI) activity of the polyphenolic constituents of PGME. However, the isolates exhibiting a high level of ciprofloxacin resistance did not respond to ciprofloxacin-PGME combinations, which could be due to target site modification not influenced further by EPI activity of PGME. Again, some strains were sensitive or weakly resistant to ciprofloxacin, which exhibited 'indifference' to the combination, probably due to a lack of over-expressed efflux mechanism. Thus, a synergy of a ciprofloxacin-PGME combination was demonstrated for the first time against ESBL- and MBL-producing Gram-negative bacilli, and the efficacy of an existing drug improved with the help of an inexpensive alternative therapy.

Antileishmanial activity of cryptolepine analogues and apoptotic effects of 2,7-dibromocryptolepine against Leishmania donovani promastigotes.

Cryptolepine (5-methyl-10H-indolo [3, 2-b] quinoline), an indoloquinoline alkaloid (1) isolated from a medicinal plant traditionally used in Western Africa for treatment of malaria, has been shown to possess broad spectrum biological activity in addition to its antiplasmodial effect. Here, the antileishmanial properties of 11 synthetic derivatives of cryptolepine against Leishmania donovani parasites have been evaluated for the first time. 2,7-Dibromocryptolepine (8; IC50 0.5 ± 0.1 µM) was found to be the most active analogue against the promastigote form of a classical L. donovani strain (AG83) in comparison to the natural alkaloid, cryptolepine (1; IC50 1.6 ± 0.1 µM). Further, 8 was found to substantially inhibit the intracellular amastigote forms of two clinical isolates, one of them being an SbV-resistant strain of L. donovani. Moreover, the toxicity of 8 against normal mouse peritoneal macrophage cells was markedly lower than that of 1 (IC50 values: 9.0 ± 1.2 and 1.1 ± 0.3 µM, respectively), indicating 8 to be a prospective "lead" towards novel antileishmanial therapy. This was supported by studies on the mechanism of cytotoxicity induced by 8 in L. donovani promastigotes (AG83), which revealed the cytoplasmic and nuclear features of metazoan apoptosis. Light microscopic observation demonstrated a gradual decline in the motility, cell volume, and survival of the treated parasites with increasing incubation time. Flow cytometric analysis of phosphatidylserine externalization and distribution of cells in different phases of cell cycle confirmed the presence of a substantial percentage of cells in early apoptotic stage. Disruption of mitochondrial membrane integrity in terms of depolarization of membrane potential, and finally degradation of chromosomal DNA into oligonucleosomal fragments - the hallmark event of apoptosis - characterized the mode of cell death in L. donovani promastigotes.

Valeriana wallichii root extracts and fractions with activity against Leishmania spp.

Leishmanial diseases, posing a public health problem worldwide, are caused by Leishmania parasites with a dimorphic life cycle alternating between the promastigote and amastigote forms. Promastigotes transmitted by the vector are transformed into amastigotes residing in the host tissue macrophages. Presently, new antiparasitic agents are needed against Leishmania donovani and Leishmania major, the respective organisms causing visceral and cutaneous leishmaniasis, since the available treatments are unsatisfactory due to toxicity, high cost, and emerging drug resistance. Over the years, traditional medicinal flora throughout the world enriched the modern pharmacopeia. Hence, roots of 'Indian Valerian' (Valeriana wallichii DC) were studied for its antileishmanial activity for the first time. The methanol and chloroform extracts showed activity against L. donovani promastigotes and both promastigotes and amastigotes of L. major. The most active fraction, F3, obtained from the chloroform extract, showed IC(50) at ∼ 3-7 µg/ml against both the promastigotes and 0.3 µg/ml against L. major amastigotes. On investigation of the mechanism of cytotoxicity in L. donovani promastigotes, the 'hall-mark' events of morphological degeneration, DNA fragmentation, externalization of phosphatidyl serine, and mitochondrial membrane depolarization indicated that F3 could induce apoptotic death in leishmanial cells. Therefore, the present study revealed a novel and unconventional property of V. wallichii root as a prospective source of effective antileishmanial agents.