Phytochemical study of Seriphidium khorassanicum (syn. Artemisia khorassanica) aerial parts: sesquiterpene lactones with anti-protozoal activity.

Two new eudesmane-type sesquiterpene lactones, 1ß,3α,8α-trihydroxy-11ß,13-dihydroeudesma-4(15)-en-12,6α-olide (1) and 1ß,4α,8α-trihydroxy-11ß,13-dihydroeudesma-12,6α-olide (2), and an unprecedented elemane-type sesquiterpene lactone, 1ß,2ß,8α-trihydroxy-11ß,13-dihydroelema-12,6α-olide (3) along with a known eudesmanolide artapshin (4) were isolated from Seriphidium khorassanicum. Structures were elucidated by NMR, HR-ESI-MS, and ECD spectral data analysis. The anti-protozoal activity was evaluated against Leishmania major promastigotes and amastigote-infected macrophages. They showed dose- and time-dependent activity against L. major amastigotes with IC50 values in the range of 4.9 to 25.3 µM being favourably far below their toxicity against normal murine macrophages with CC50 values ranging from 432.5 to 620.7 µM after 48 h of treatment. Compound 3 exhibited the strongest activity and the highest selectivity index (SI) with IC50 of 4.9 ± 0.6 µM and SI of 88.2 comparable with the standard drug, meglumine antimoniate (Glucantime), with IC50 and SI values of 15.5 ± 2.1 µM and 40.0, respectively.

Synthesis, biological assessment, and computational investigations of nifedipine and monastrol analogues as anti-leishmanial major and anti-microbial agents.

Leishmaniasis includes a range of parasitic diseases caused by numerous types of the protozoan kinetoplastid parasite. Fungal and bacterial pathogens have led to infectious illnesses causing some main public health problem in current years. A series of dihydropyridine and tetrahydropyrimidine derivatives having fluoro, bromo, and nitro substituents at para-phenyl ring on C4 of dihydropyridine and tetrahydropyrimidine rings were synthesized. Then, anti-leishmanial and antimicrobial potencies of compounds were assessed. All compounds were synthesized via Hantzsch and Biginelli reactions. All derivatives were evaluated for their anti-leishmanial and antimicrobial activities. Moreover, docking and molecular dynamics simulation calculations of the compounds in PRT1 binding site were performed to report the results of anti-leishmanial and antimicrobial activities. Compounds 4a and 4b showed the highest anti-amastigote and anti-promastigote activities. Compound 4a revealed the highest antimicrobial activity against E. coli, P. aeruginosa, and C. albicans strains. In addition, compound 4c showed the highest activity against S. aureus. The fluoro, bromo, and nitro substituents in para-position of phenyl group at C4 of dihydropyridine and tetrahydropyrimidine moieties as well as the bulk and length of the chain linking to the ester moieties are essential for anti-leishmanial and anti-microbial activities of these derivatives. Low cytotoxicity was shown by most of derivatives against macrophages. The molecular docking studies were in agreement with in vitro assay. Moreover, hydrogen binds, RMSF, RMSD, and Rg, strongly showed the steady binding of 4a and 4b compounds in PRT1 active site.

Preparation, optimization, and in vitro-in vivo evaluation of sorafenib-loaded polycaprolactone and cellulose acetate nanofibers for the treatment of cutaneous leishmaniasis.

The most common form of leishmaniasis is cutaneous leishmaniasis (CL). The major difficulties in the treatment of leishmaniasis include emergence of resistance, toxicity, long-term treatment, and the high cost of the current drugs. Although the therapeutic effect of sorafenib (SF) has been demonstrated in both in vitro and in vivo models of Leishmania infection, the therapeutic applications are limited due to severe drug-related toxicity; this is, in turn, due to non-specific distribution in the body. Thus, topical delivery has the advantage of the site directed delivery of SF. This research study evaluated SF-loaded hybrid nanofibers (NFs) which were composed of polycaprolactone (PCL) and cellulose acetate (CA) for the CL topical treatment. Accordingly, SF-loaded hybrid NFs were prepared using the electrospinning method. Formulation variables including total polymer concentration, drug/polymer ratio, and CA concentration were optimized using a full factorial design. The prepared SF-loaded NFs were then characterized for morphology, diameter, encapsulation efficiency (EE)%, drug loading (DL) %, and percentage of release efficiency during a 24-h period (RE24h%); the mechanical characteristics were also considered. The physical state of the drug in the optimized NF was evaluated by the X-ray diffraction analysis. Finally, its in vivo efficacy was determined in L. major-infected mice. The optimized formulation had a smooth, cylindrical, non-beaded shape fiber with a diameter of 281.44 nm, EE of 97.96%, DL of 7.48%, RE of 51.05%, ultimate tensile strength of 1.08 MPa, and Young's moduli of 74.96 MPa. The XRD analysis also demonstrated the amorphous state of SF in NF. Further, the in vivo results displayed the higher anti-leishmanial activity of the SF-loaded hybrid NF by efficiently healing lesion and successfully reducing the parasite burden. This, thus, indicated the potential of the clinical capability of the SF-loaded hybrid NF for the effective treatment of CL.

Sb(V) Kaempferol and Quercetin Derivative Complexes: Synthesis, Characterization and Antileishmanial Activities.

Background: Recent studies on Leishmaniasis treatment have confirmed the antiparasitic effects of flavonols and organic antimony pentavalent [(Sb(V)] complexes. Objectives: This study aimed to identify new Sb(V) complexes by combining the benefits of antimonials and flavonols as well as by optimizing their properties. Methods: Kaempferol and quercetin peracetate were prepared using acetic anhydride in pyridine. By performing regioselective synthesis, 7-O-paramethylbenzyl as an electron-donating group and 7-O-paranitrobenzyl as an electron-withdrawing group were added to quercetin, and, then, the synthesis of Sb(V) kaempferol and quercetin derivative complexes were performed using SbCl5 solution in glacial acetic acid. The structures were confirmed by UV, ESI mass, IR, 1H-, and 13C-NMR spectral data, and the Stoichiometry of the ligand-metal complex by the mole ratio method. Computational molecular modeling was conducted using the Gaussian program. Results: The structures were confirmed based on the results from UV, nuclear magnetic resonance (NMR), and electrospray ionization (ESI) mass analyses (3-12). Among the produced compounds, 11 and 12 as newly described, and other compounds as pre-defined compounds were identified. According to the results from biological test, kaempferol triacetate with more lipophilicity showed the highest anti-promastigote activity with an IC50 value of 14.93 ± 2.21 µM. As for anti-amastigote activity, despite the differences, all antimony complexes showed anti-amastigote effects in vitro with IC50 values of 0.52 to 14.50 µM. Conclusions: All flavonol Sb(V) complexes showed higher activity compared to meglumine antimonate in anti-amastigote effect. Inside the host macrophages, by breaking down the complex into antimony and quercetin or kaempferol analogs, the observed antiparasitic effects may have been related to both Sb(V)/Sb(III) conversion and flavonoid antileishmanial activities.

Synthesis and Biological Evaluation of Tetrahydropyrimidine and Dihydropyridine Derivatives Against Leishmania Major.

PURPOSE: A number of tetrahydropyrimidines and their bioisosteric dihydropyridines bearing chloro substituent at various positions of phenyl ring in C4 of main scaffolds were designed, synthesized and evaluated for antileishmanial activity. METHODS: The antileishmanial activity of the synthesized compounds was evaluated against promastigote and amastigote forms. Moreover, molecular docking studies of the compounds in pteridine reductase 1 (PTR1) pocket were carried out to describe the results of biological experiments. RESULTS: The compounds exhibited moderate to good antileishmanial activity against promastigote and amastigote forms. Among the screened compounds, 1d and 2c were found as the most potent compounds against promastigote form with EC50 values of 15.5 and 10.5 µM, respectively. Compounds 2a and 2c were the most potent compounds against amastigote form with EC50 values of 5.4 and 2.2 µM, respectively. CONCLUSION: According to structure-activity relationship (SAR) studies, the chloro substituent in different positions of phenyl ring at C4 of 1,2,3,4-tetrahydropyrimidine (THPM) and 1,4-dihydropyridine (DHP) rings and also the length of the chain belonging to the ester groups could be important for antileishmanial activity of these compounds. Most of these compounds exhibited low cytotoxicity against macrophages. Compounds 1 h, 2a, 2b and 2c revealed higher activity than glucantime while all compounds showed lower activity toward amphotericine B. Docking studies showed that the synthesized compounds were fit well in the PTR1 pocket. Compounds 1 h and 2c indicated the highest score docking among screened compounds in PTR1 enzyme.

Preparation and characterization of itraconazole loaded nanomicelles based on dextran-behenic acid for cutaneous leishmaniasis treatment.

Cutaneous leishmaniasis is known as the most prevalent clinical form of leishmaniasis. It needs the development of new therapies due to the serious side-effects promoted by taking the current drugs. In the present study, dextran-behenic acid (DEX-BA) based nanomicelles were developed to direct the delivery of itraconazole (ITZ) to the macrophages and enhance its toxic effects against Leishmania parasites. DEX-BA was synthesized through the esterification of dextran with behenic acid. The critical micelle concentration of the newly developed conjugate was evaluated using pyrene as the fluorescent probe. The nanomicelles were generated by the dialysis method; then they were optimized by applying a Box-Behnken design. The effects of the dialysis temperature, polymer content, and sonication time on the characteristics of micelles were subsequently studied. Furthermore, in vitro efficacy against Leishmania major promastigotes and parasite-infected macrophages was evaluated. The optimized formulation showed the particle size of 195.16 ± 3.06 nm, the polydispersity index of 0.39 ± 0.01, the zeta potential of -16.29 ± 0.89 mV, the encapsulation efficiency % of 56.11 ± 4.9, and the release efficiency % of 51.29 ± 1.97. According to scanning electron microscopy, the nanomicelles were found to be nearly spherical in shape. ITZ-loaded nanomicelles showed the strongest anti-leishmanial activities when compared with the free ITZ and drug-free nanomicelles. It could be, therefore, concluded that ITZ-loaded nanomicelles might be useful as an alternative therapy for the treatment of cutaneous leishmania.

Synthesis and antileishmanial activity of antimony (V) complexes of hydroxypyranone and hydroxypyridinone ligands.

A novel series of antimony (V) complexes with the hydroxypyranone and hydroxypyridinone ligands were synthesized and characterized by 1HNMR, FT-IR and electron spin ionization mass spectroscopic (ESI-MS) techniques. The synthesis process involved protection of hydroxyl group followed by the reaction of the intermediate with primary amines and finally deprotection. All compounds were evaluated for in vitro activities against the amastigote and promastigote forms of Leishmania major. Most of the synthesized compounds exhibited good antileishmanial activity against both forms of L. major. IC50 values of the most active compounds; 9d, 9d and 9e, after 24, 48 and 72 h against amastigote model were 15, 12.5 and 5.5 µg/mL, respectively. 9e, 11 and 9e inhibited the promastigote form of parasite after 24, 48 and 72 h with IC50 values of 10, 2 and 1 µg/mL, respectively.

Chitosan-titanium dioxide-glucantime nanoassemblies effects on promastigote and amastigote of Leishmania major.

The purpose of the present study was to design nanoassemblies of chitosan-titanium dioxide (TiO2) nanoparticles (NPs) loaded with glucantime for using their synergistic effects and enhancing the toxic effects of glucantime on Leishmania parasites. The nanoassemblies were prepared by electrostatic interactions and optimized by a response surface central composite design. The effects of glucantime, chitosan and TiO2 NPs amounts were studied on the particle size, zeta potential, loading efficiency, and release efficiency of drug from nanoassemblies. The conjugation of TiO2/chitosan-glucantime was verified by UV spectroscopy and changes in surface charge of NPs. The anti-promastigots effect of glucantime loaded in TiO2/chitosan nanoassemblies was studied by tripan blue dye test and their anti-amastigotes effect by counting the average number of parasites per infected J774 macrophages in 100 cells. The optimized formulation obtained by using 12.5mg glucantime, 25mg chitosan and 6mg TiO2 NPs. Although TiO2 NPs alone were effective more than negative control in reduction of promastigots and amastigotes but they didn't show significant difference compared with free glucantime (p>0.05). However, at the concentration of 50µg/mL and after 72h exposure nanoassemblies decreased the proliferation of L. major promastigotes and amastigotes 13 and 4-fold, respectively compared with glucantime alone.

Identification of etiologic agents of cutaneous Leishmaniasis in palpebral position followed by effective therapy.

CONTEXT: Cutaneous Leishmaniasis (CL) is a self-healing lesion but prevention of complications and involvement of vital organs such as palpebra requires proper treatment. AIMS: The main objectives were to detect agents of CL in palpebral region and estimate the proportion of palpebral lesion also possible complications among CL patients in a zoonotic CL endemic area. SETTINGS AND DESIGN: The study was performed from April 2012 to March 2013 in a total of 1613 CL suspected patients by interview and physically exam. SUBJECTS AND METHODS: The samples were used for direct smear using Giemsa stain method, or cultured in Novy-McNeal-Nicol medium. For further checked, nested polymerase chain reaction (PCR) was used for negative palpebral samples. Of the 1613 examined samples, 848 (81.4%) by direct smear, 188 (18%) by culture and 6 (0.6%) by nested PCR were positive, respectively. A total of 233 of the patients showed lesions on the face of whom 15 (male = 5, female = 10, 1.43% of all and 6.43% of facial lesions) presented with palpebral CL. The results of nested PCR indicated that all the palpebral cases were due to Leishmania major. CONCLUSIONS: About 93% of the patients with palpebral lesion were under 5 years old and were infected in the hyper endemic regions, but no ocular complication was seen in any of them. Based on the results, it seems that early referring to the medical center for proper diagnosis and treatment is the main reason for prevention of further complications.

In vivo/In vitro immune responses to L. major isolates from patients with no clinical response to Glucantime.

BACKGROUND: Leishmaniasis is a major health problem in some endemic areas of tropical and subtropical areas of the world. Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are essential cytokines associated with initiation of Th1 response. The main objective of this study was to evaluate of the type of immune response to L. major isolates from patients with no clinical response to antimonite (Glucantime). MATERIALS AND METHODS: This experimental study was carried out during 2013-2014. In the current study Leishmania major were isolated from 10 CL patients with a history of at least one course of treatment with Meglumine antimonate (Sb5). The isolates were used to evaluate in vitro and in vivo response to Sb5. J774 murine macrophage cell line was used for in vitro tests and Balb/c mice was used for in vivo studies. IL-12 gene expression was evaluated using Real-time PCR and IFN-γ serum level was quantified using ELISA technique. SPSS (version: 20), analysis of Covariance (ANCOVA) was used for statistical analysis. RESULTS: PCR results confirmed that all 10 isolates were L. major. The mean of IL-12 gene expression in vitro, in vivo and IFN-γ serum levels (pg/ml) after 2 and 3 weeks treatment in vivo, increased significantly following the treatment with Glucantime in the two groups of Balb/c mice infected either with patients' isolates or standard L. major. No significant difference was seen between the patients' isolates and standard species. CONCLUSIONS: Although the L. major were isolated from patients with active lesion and no clinical response to Glucantime after at least one courses of Glucantime treatment but in vivo and in vitro immune response of L. major isolates showed no difference between the patients' isolates and standard L. major.

Anti-Leishmania Activity of Osthole.

BACKGROUND: Treatment of cutaneous leishmaniasis (CL) is occasionally highly resistant to pentavalent antimonials, the gold standard in pharmacotherapy of CL. Since there is no effective vaccine, the discovery of natural antileishmanial products as complementary therapeutic agents could be used to improve the current regimens. OBJECTIVE: In this study in vitro and in vivo antileishmanial activities of osthole, a natural coumarin known to possess antibacterial and parasiticidal activities are evaluated. MATERIALS AND METHODS: Leishmania major infected J774.A1 macrophages were treated with increasing concentrations of osthole. CL lesions of BALB/c mice were treated topically with 0.2% osthole. RESULTS: Osthole exhibited dose-dependent leishmanicidal activity against intracellular amastigotes with IC50 value of 14.95 µg/ml. Treatment of CL lesions in BALB/c mice with osthole significantly declined lesion progression compared to untreated mice (P < 0.05), however did not result in recovery. CONCLUSION: Osthole demonstrated remarkable leishmanicidal activity in vitro. Higher concentrations of osthole may demonstrate the therapeutic property in vivo. SUMMARY: In vitro and in vivo antileishmanial activities of osthole, a pernylated coumarin extracted from Prangos asperula Boiss., are studied against Leishmania major.

Antigenic profile of heat-killed versus thimerosal-treated Leishmania major using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

BACKGROUND: Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM) and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM). In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). MATERIALS AND METHODS: L. major (MRHO/IR/75/ER) from pre-infected Balb/c mice was isolated with modified Novy-MacNeal-Nicolle (NNN) medium and then subcultured in liquid RPMI 1640 medium supplemented with fetal calf serum (FCS) 20% for mass production. Two preparations of KLM and ALM were produced by Razi Vaccine and Serum Research Institute, Iran, under WHO/TDR supervision. Electrophoresis was performed by SDS-PAGE method and the gel was stained by Coomassie brilliant blue dye. The resultant unit bands were compared using standard molecular proteins. RESULTS: Electrophoresis of the two preparations produced many bands from 10 kDa to 100 kDa. KLM bands were much like those of freshly harvested intact L. major. CONCLUSION: It is concluded that although there are similar bands in the three forms of Leishmania antigens, there are some variations which might be considered for identification and purification of protective immunogens in a total crude antigen, and detection of their stability is essential for the production and marketing of a putative vaccine.

Identification of Leishmania isolates from Varzaneh city, Isfahan province, Iran using nested polymerase chain reaction method.

BACKGROUND: Leishmaniasis, a parasitic disease, is caused by the Leishmania genus, a protozoan parasite transmitted by sand fly arthropods. Cutaneous leishmaniasis (CL) in old world is usually caused by L. major, L. tropica, and L. aethiopica complexes. One of the most important hyper endemic areas of CL in Iran is Isfahan province. Varzaneh is a city in the eastern part of Isfahan province. Due to different biological patterns of parasite strains which are distributed in the region, this study was design to identify Leishmania species from human victims using Kinetoplastid DNA as templates in a molecular PCR method. MATERIALS AND METHODS: Among 186 suspected cases, 50 cases were confirmed positive by direct microscopy after Giemsa staining. Species characterization of the isolates was done using Nested- PCR as a very effective and sensitive tool to reproduce mini circle strands. RESULTS: After Nested-PCR from all 50 cases, 560 bp bands were produced which according to products of reference strains indicate that the infection etiologic agent has been L. major. 22 (44%) of patients were females and 28 (56%) of them were males. Their age ranges were between 7 months and 60 years. CONCLUSION: According to the results of the study and the particular pattern of infection prevalent in the region, genetic studies and identification of Leishmania parasites are very important in the disease control and improvement of regional strategy of therapy protocols.

Effect of integrated pest management on controlling zoonotic cutaneous leishmaniasis in Emamzadeh Agha Ali Abbas (AS) District, Isfahan province, 2006-2009.

BACKGROUND: Cutaneous leishmaniasis (CL) is still considered as a health problem in the world. Several methods of control in different regions, together with obtaining integrated information on its natural foci, are needed to decrease its prevalence. This study was designed to evaluate the effects of simultaneous interventions on CL control. MATERIALS AND METHODS: A standard questionnaire was used to identify patients among pilgrims to Emamzadeh Agha Ali Abbas (Isfahan Province, Iran). Subsequently, three methods of controlling the disease, including, spraying residential buildings with Baygon, baiting with zinc phosphide poisons, changing the vegetative cover of the region, improving the environment, and mounting a mesh on all doors and windows of buildings in residential areas were used. The control measures were then evaluated by comparing the number of pilgrims affected by CL after and before the interventions. RESULTS: While 23 pilgrims (1.4%) were affected with CL before the intervention (pretest), five (0.3%) persons were found to have CL after taking control measures. The Chi-square test did not indicate any significant difference in the relative frequency of CL (P = 0.731). CONCLUSION: The only scientific method for preventing and controlling zoonotic CL (ZCL) is a combination of the control methods (improving the environment and fighting off the disease districts and vectors) together with changing the vegetative cover of the region. Any measure for controlling this disease must be taken and programmed in accordance with the relevant experts' views, in coordination with the participation of other organizations and the society.

The knowledge, attitude, and prevention practices of students regarding cutaneous leishmaniasis in the hyperendemic region of the Shahid Babaie Airbase.

INTRODUCTION: The Shahid Babaie Airbase is one of the most endemic areas of cutaneous leishmaniasis (CL) in Isfahan. Community training on CL prevention could have a critical role in controlling CL in endemic areas. Because of the high incidence of disease among youth, this survey was designed to assess the knowledge, attitude, and prevention practices (KAP) of students regarding CL in this endemic area. MATERIALS AND METHODS: This study consisted of a questionnaire that was filled out while interviewing students attending middle and high school on the Shahid Babaie Airbase of Isfahan. The questionnaire contained questions about KAP of students regarding CL. The total scores in each field were categorized as weak, intermediate, or strong. RESULTS: Four hundred fifty students participated in this study, which included 245 high-school students and 205 middle-school students. The total knowledge score of the students was 17.47 (range, 0-30), which indicates an intermediate level of CL knowledge in this population. The students' attitude toward CL was intermediate, with a score of 37 and a range of 13-52. Additionally, practice of prevention was weak (score of 1.8; range, 0-6). There was a significant correlation between gender and both the attitude and knowledge of the students; both scores were higher in female students. Specific knowledge about CL symptoms, carriers, and reservoirs was higher than knowledge about preventative methods. The study revealed that 47.2% of students believed in fortune as a factor involved in acquisition of CL infection. Although 97.9% of students were aware that sandflies carry CL, only 28.6% were able to identify a sandfly. CONCLUSIONS: The results of this study further emphasize the importance and necessity of educating this at-risk population by planning direct, in-person training, which is an essential step in improving attitudes and preventative practices toward CL and in controlling CL in endemic areas.