Therapeutic effects of Lucilia sericata larval excretion/secretion products on Leishmania major under in vitro and in vivo conditions.

BACKGROUND: Leishmaniasis is a neglected infectious disease caused by protozoa of the genus Leishmania. The disease generally manifests as characteristic skin lesions which require lengthy treatment with antimonial drugs that are often associated with adverse side effects. Therefore, a number of studies have focused on natural compounds as promising drugs for its treatment. This study aimed to evaluate the effects of larval excretion/secretion products (ES) of Lucilia sericata in crude and fractionated forms on Leishmania major, by using in vitro and in vivo models. METHODS: The in vitro experiments involved evaluation of ES on both promastigotes and macrophage-engulfed amastigotes, whereas the in vivo experiments included comparative treatments of skin lesions in L. major-infected mice with Eucerin-formulated ES and Glucantime. RESULTS: The half maximal inhibitory concentrations of the crude ES, > 10-kDa ES fraction, < 10-kDa ES fraction, and Glucantime were 38.7 µg/ml, 47.6 µg/ml, 63.3 µg/ml, and 29.1 µg/ml, respectively. Significant differences were observed between percentage viabilities of promastigotes treated with the crude ES and its fractions compared with the negative control (P < 0.0001). The crude ES was more effective on amastigotes than the two ES fractions at 300 µg/ml. The macroscopic measurements revealed that the reduction of lesion size in mice treated with the crude ES followed quicker cascades of healing than that of mice treated with Glucantime and the ES fractions. CONCLUSIONS: The present study showed that the larval ES of L. sericata in both crude and fractionated forms are effective for both intracellular and extracellular forms of L. major. Also, the ES exert both topical and systemic effects on mice experimentally infected with L. major.

In vitro and in vivo effects of natural honey on Leishmania major.

Leishmaniosis is an insect-borne disease whose clinical manifestations range from skin ulcer to visceral disease. Antimony compounds are currently known to be the main treatment for leishmaniosis, but there are limitations to their use. This study was performed to determine the in vitro and in vivo efficiency of honey on a standard strain of Leishmania major parasite in comparison with glucantime and amphotericin as the first line treatment. Leishmania major was exposed to different concentrations of honey extract at 400, 200, 100, 50, 25, 12.5, 6.25 µg/ml. The effectiveness of honey concentrations was determined by counting the parasite by Neubauer's chamber. Then, using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method, for promastigotes and macrophages then IC50 was calculated. A flow cytometry test was performed and necrosis and apoptosis diagrams were drawn. Next, the effect of the honey on the amastigotes inside macrophage cells was investigated. Finally, for the in vivo experimentation, the parasite was injected in the base of BALB/c mice tails and the resulting wounds were treated with honey. The results of all tests showed that the honey extract at 400 µg/ml concentration had the best effects on all stages. The honey has lethal effects on Leishmania parasite in vitro as well as therapeutic effects on wounds caused by the parasite. Further experiments are recommended to evaluate the performance of the extract on the parasite in volunteer human models.

A study on the in vitro and in vivo effects of aqueous and alcoholic extracts of Salvia mirzayanii on Leishmania major.

Salvia mirzayanii contains anti-hyperglycemic, antimicrobial, antioxidant, anti-inflammatory, and neuroprotective effects. The purpose of this study was to evaluate the anti-leishmanial efficacy of aqueous and alcoholic extracts of S. mirzayanii (both in vitro and in vivo) against Leishmania major. Aqueous and alcoholic extracts of S. mirzayanii were prepared and tested on L. major promastigotes and amastigotes. MTT test was used to evaluate the cytotoxicity of the plant against L. major. Flow cytometry was performed to assay apoptosis induced by 50 and 100 µg/ml of extracts on the promastigotes and macrophages. For the in vivo assay, the therapeutic effects of aqueous and alcoholic extracts of S. mirzayanii were tested in BALB/c mice. After 72 h, the IC50 value of aqueous and alcoholic extracts of S. mirzayanii against L. major promastigotes was 6.04 and 4.47, respectively. The inhibitory concentration (IC50) of aqueous and alcoholic extracts of S. mirzayanii to amastigotes were determined to be 47.78 µg/ml and 33.58 µg/ml, respectively. Flow cytometry revealed that the apoptosis of promastigotes using 100 µg/ml of aqueous and alcoholic extracts of S. mirzayanii was 5.81% and 5.39%, respectively, while apoptosis induced at 200 µg/ml were 5.09% and 70.71%, respectively. Lesion size was significantly decreased in in vivo experiments, and the survival rate of the treated mice improved in contrast to the control group. Given the efficacy of aqueous and alcoholic extracts of S. mirzayanii on promastigotes both in vitro and in vivo condition, the plant could be considered as a candidate source for the treatment of leishmaniosis.

Investigating in Vivo and in Vitro Effects of Ethanolic and Aqueous Extracts of Myrtle (Myrtus communis) on Leishmania major.

BACKGROUND: The extract of myrtle plant contains polyphenolic compounds that show antibacterial, antiviral, and anti-parasitic properties. We aimed to investigate the therapeutic effect of aqueous and ethanolic myrtle extract against leishmaniasis caused by L. major in vivo and in vitro conditions. METHODS: This study was carried out in Tarbiat Modares University, Tehran, Iran in 2018. Aqueous and ethanolic extract of myrtle plant at 6.25 to 400 mg/ml concentrations were tested on Leishmania major promastigotes, non-infected macrophages, and macrophages infected with amastigotes in vitro using counting, MTT and flow cytometry techniques. Then, BALB/c mice were treated with ethanolic, aqueous and a mixture of both extracts of myrtle plant. The treatment was carried out for four weeks. Then, the effectiveness of the herbal medicine was assessed by measuring wounds diameters, mice weights and their mortality rate on weekly basis. RESULTS: The IC50 values of aqueous and ethanolic extracts for promastigotes were 7.86 and 11.66 µg/mL respectively. The IC50 values of the aqueous and ethanolic extracts for amastigotes were 12.5 and 47.2 µg/mL respectively. Flow cytometry indicates 62.88% and 60.16% apoptosis induced by ethanolic and aqueous extract of myrtle plant respectively. The lowest parasitic load was seen in the group treated with ethanolic extract. CONCLUSION: The lesion sizes for treated groups with extracts were similar to those treated with glucantime. Oral administration instead of injection is another advantage of myrtle plant over glucantime, which makes the herb easy and more practical.

In-vitro and in-vivo comparative effects of the spring and autumn-harvested Artemisia aucheri Bioss extracts on Leishmania major.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia aucheri Bioss contains flavonoid, coumarin and santonin with antioxidant, antimicrobial and antileishmanial effects. The current study was aimed to comparatively evaluate the effects of spring and autumn extracts of A. aucheri Bioss on Leishmania major both in-vitro and in-vivo conditions. METHODS: HPLC analysis was used to evaluate the percentages of compounds in spring and autumn extracts of A. aucheri. For in-vitro assay, the effect of different concentrations of spring and autumn extracts of A. aucheri was tested on L. major promastigotes and amastigotes. MTT and flow cytometry methods were used to evaluate the cytotoxicity and probable apoptosis of A. aucheri extracts on L. major promastigotes. On the other hand, for in-vivo assay, the extracts were used as ointments to treat lesions developed on BALB/c mice after 28 days post inoculation of L. major. The diameter of lesions and the survival rates of infected BALB/c mice were measured weekly for a period of two months. RESULTS: The HPLC analysis showed the substance Quercitrin was present in the spring A. aucheri extract but not in the autumn extract. The mean numbers of amastigotes in each treated macrophage with the spring and autumn A. aucheri extracts were 1.2 and 1.8 respectively, which showed statistically significant differences (P < 0.05). Flow cytometry revealed that the spring and autumn A. aucheri extracts caused about 32% and 3.78% apoptosis respectively. The inhibitory concentration (IC50) of spring and autumn A. aucheri extracts to amastigotes were determined to be 90 µg/mL and 183 µg/mL respectiovely. In-vivo, the diameter of lesions treated with the spring A. aucheri extract was significantly less (P < 0.05) compared to those treated with the autumn extract (2.6 and 7.8 mm respectively). Also, mice treated with spring A. aucheri extract had higher survival rates compared to control group. CONCLUSION: Given the above results, it can be concluded that spring A. aucheri extract has a greater fatality effect on L. major promastigotes in-vitro compared to the autum extract. In addition, the spring extract has stronger therapeutic effect on lesions caused by L. major in BALB/c mice than the autum extract.

Mechanism of protein-z-mediated inhibition of coagulation factor xa by z-protein-dependent inhibitor: a molecular dynamic approach.

Protein Z is a plasma protein functioning as a carrier for ZPI. Protein Z also accelerates inhibitory effect of ZPI on factor Xa by 1000-fold. Inhibition of coagulation cascade via FXa by ZPI and other serpins is very important safety factor for normal homeostasis protecting human life against unwanted thrombosis. In the present work using native structure of PZ, ZPI, FXa and in a dynamic simulation, using NAMD software, the ternary complex was studied in an up to 10 nanoseconds protocol. Rely on trajectory analyses, we postulated that PZ binds ZPI by using its SP-like domain and through noncovalent forces. PZ then transfers ZPI through-out the blood, and by using its GLA domain and a bivalent cation of calcium, PZ binds to phospholipid bilayers (e.g., platelet) where the FXa is preallocated. In case of PZ-ZPI binding to plasma membrane, a series of complementary interactions take place between FXa, and PZ-ZPI complex including interactions between RCL loop of ZPI and catalytic site of FXa and some take place between long arm of PZ (composed of GLA, EGF1, and EGF2 domains) and GLA domain of FXa. In our claim these complementary interactions lead PZ to bind correctly to prelocated FXa.