Cyclotide nanotubes as a novel potential Drug-Delivery System: Characterization and biocompatibility.

Cyclotides are a class of cyclic peptides that can be self-assembled. This study aimed to discover the properties of cyclotide nanotubes. We performed differential scanning calorimetric (DSC) to characterize their properties. Then, we incorporated the coumarin as a probe and identified the morphology of nanostructures. The stability of cyclotide nanotubes after 3 months of keeping at -20 °C was determined by field emission scanning electron microscopy (FESEM). The cytocompatibility of cyclotide nanotubes was evaluated on peripheral blood mononuclear cells. In vivo, studies were also conducted on female C57BL/6 mice by intraperitoneally administration of nanotubes at 5, 50, and 100 mg/kg doses. Blood sampling was done before and 24 h after nanotube administration and complete blood count tests were conducted. DSC thermogram showed that the cyclotide nanotubes were stable after heating until 200 °C. Fluorescence microscopy images proved that the self-assembled structures of cyclotide can encapsulate the coumarin. FESEM proved that these nanotubes were stable even after 3 months. The results of the cytotoxicity assay and in-vivo study confirmed that these novel prepared nanotubes were biocompatible. These results suggested that the cyclotide nanotubes could be considered as a new carrier in biological fields while they are biocompatible.

In Vitro Study of the Leishmanicidal Activity of Perovskia Abrotanoides Terpenoid-Rich Fractions Against Leishmania Major (MRHO/IR/75/ER).

Background: Cutaneous leishmaniasis (CL) is an ulcerative skin disease caused by some species of the genus Leishmania. Evidence shows that Perovskia abrotanoides is an important herbal medicine against Leishmania. This study was conducted to investigate the killing effect of terpenoid-rich fractions on promastigotes of L. major (MRHO/IR/75/ER). Material and Method: The eluates of reverse phased medium pressure liquid chromatography (RP-MPLC) of the extract were subjected to thin-layer chromatography (TLC) and categorized into six final fractions. Primary proton nuclear magnetic resonance (H-NMR) spectroscopy confirmed fractions' nature. Fractions 4, 5, and 6 (F4, F5, F6) were identified as terpenoid-rich content. Two concentrations of 50 and 100 µg/ml were prepared to test leishmanicidal activity. Followed by treating promastigotes of L. major by the fractions in incubation times of 12, 24, and 48 hours, their viability was determined using a cell proliferation MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Result: F4, F5, and F6 showed significant killing activity on promastigotes of L. major in a concentration-dependent manner. The viability of promastigotes was significantly reduced at a concentration of 100 µg/ml compared to 50 µg/ml (P-value <0.05). Also, over time a significant decreasing trend in the viability of promastigotes confirmed the time-dependent manner of the fractions (P-value <0.01). Furthermore, F5 had the highest leishmanicidal activity at the first incubation time compared with other fractions. Conclusion: Terpenoid-rich fractions of the P. abrotanoides have a leishmanicidal activity that depends on time and concentration. Among them, F5 has the highest potency that may contain potent terpenoid constituents.

The Effectiveness of Perovskia abrotanoides Extract Topical Formulation on the Cutaneous Leishmaniasis: A Randomized Controlled Clinical Trial.

Objective: Despite many attempts to treat leishmaniasis, new approaches are necessary to reduce the burden of disease. Perovskia abrotanoides (Brazambel) has shown significant effects against Leishmania parasites in some studies. This study aimed to investigate the effects of P. abrotanoides extract topical formulation on cutaneous leishmaniasis. Methods: In this randomized controlled clinical trial, patients with cutaneous leishmaniasis were assigned to experimental (n = 18) and control (n = 18) groups. Both groups received intralesional meglumine antimoniate (Glucantime®). The experimental group also received 5% Brazambel extract ointment once a day. The interventions continued until the complete healing of the lesions (reepithelialization) for a maximum of 8 weeks. The clinical response, defined as complete response (reepithelialization >75%), partial response (reepithelialization 50%-75%), or treatment failure (reepithelialization <50%), was compared between the groups. Findings: The percentage of reepithelialization in the experimental group (4th week: 64.44 ± 25.13; 8th week: 83.85 ± 11.54) was higher than the control group (4th week: 53.97 ± 25.88; 8th week: 76.27 ± 21.67); however, the differences were not statistically significant (P = 0.252 and 0.494, respectively). Moreover, there was no significant difference between the experimental and control groups regarding the rate of complete healing (88.9% vs. 72.2%, respectively). Conclusion: The use of P. abrotanoides extract 5% topical formulation does not affect the healing of cutaneous leishmaniasis.

Effects of Asparagus officinalis on immune system mediated EAE model of multiple sclerosis.

Background: About 5 to 10 percent of the population in developed countries are affected by autoimmune diseases. One of the most important autoimmune disease with high prevalence rate is Multiple sclerosis in which there is currently no definitive cure for it, and most medications such as interferons are used only to limit the disease. The present study aims to investigate the effect of using Asparagus Officinalis fractions in an immune system mediated model of multiple sclerosis. Material and Methods: Fractionation was performed by maceration using n-hexane, chloroform, chloroform-methanol (9: 1), n-Butanol and methanol solvents from aerial parts of Asparagus Officinalis. Thin layer chromatography, NMR and phenolic component measurement were done and two fractions were selected for checking in MS induced in vivo model. Results: It was observed that chloroform-methanolic and N-Butanol fractions had higher content of saponin in comparison of other extracts. Also, it was showed that the methanolic and n-Butanol extracts contains the highestportion of glycosylic steroid saponins in comparison to other fractions. Regarding experimental autoimmune encephalomyelitis (EAE) score, Butanolic and methanolic fractions with doses higher that 100mg/kg showed a potent supportive effects as long as locomotor activity protection even in lower dose in comparison to phosphate buffered saline (PBS) group. Conclusion: Considering the proved different effects of saponin compounds on the immune system we observed that those fractions altered the circulatory peripheral blood cells and also remit the clinical signs after EAE induction along with enhanced myelin sheath content in the median region of corpus callusom. It could be inferred that this fractions are promising candidates for further investigation as dose-dependent immune system regulating compounds in multiple sclerosis patients.

Fabrication and optimization of electrospun polymeric nanofibers loaded with 5-fluorouracil and rosemary extract.

BACKGROUND: Topical 5-fluorouracil [5FU] is one of the mostly prescribed medications for different types of skin cancer; however, it is associated with drug resistance and adverse effects. Rosemary extract has promising dose-dependent antitumor effects, as well as a synergistic effect in combination with 5-fluorouracil besides sensitizing the 5-FU-resistant cells. OBJECTIVE: Polymeric nanofibers loaded with 5FU and rosemary extract were optimized to combine both ingredients in one controlled release drug delivery system, aiming to enhance the efficacy while retaining the adverse effects. METHOD: Polymeric nanofibers loaded with 5-FU and rosemary were fabricated via electrospinning technique. Design expert software was utilized to study the effect of independent variables including polymer concentration, voltage, and feeding rate on the characteristics of the resulting nanofibers. Afterwards, the FTIR spectrum and release kinetic of the drug and extract from the optimized nanofibers and their cytotoxic effect against A375 cell line were investigated. RESULTS: The formulation composed of 6.65% PVA electrospun at 1 mL.h-1 and 17.5kV was chosen as the optimum fabrication condition. The mean diameter of the optimized nanofibers was 755 nm. The drug and rosemary extract contents were 75.38 and 93.42%, respectively. The fabrication yield was 100%, bioadhesion force was 1.28 N, and bead abundance was 10 per field. The cytotoxicity of the optimized formulation was significantly higher than the control groups. CONCLUSION: According to the appropriate loading percent, release efficiency and release kinetics, bioadhesion force, and cytotoxicity, these nanofibers could be further investigated as a topical treatment option to increase the efficacy of 5-FU.

Immunomodulatory effects of cyclotides isolated from Viola odorata in an experimental autoimmune encephalomyelitis animal model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that causes chronic inflammation. Cyclotides are small plant proteins with a wide range of biological activity, making them a target for researchers to investigate. This study was conducted to investigate the possible effects of cyclotide-rich fractions from Viola odorata as an immunomodulatory agent in an experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: At room temperature, the plant materials were subjected to maceration in methanol: dichloromethane (1:1; v/v) for 3 days. The extraction was repeated 3 times, and the final concentrated extract was partitioned 3 times by 1/2 volume of double-distilled water. The aqueous phases were separated and freeze-dried. Finally, the crude extract was fractionated by C18 silicagel using vacuum liquid chromatography, with mobile phases of 30%, 50% and 80% of ethanol: water, respectively. The 50%, and 80% fractions were analyzed by HPLC and MALDI-TOF analysis and administrated intraperitoneally to forty-five female C57BL/6 EAE-induced mice, at 5, 25, and 50 mg/kg doses. After 28 days, the animals were evaluated using EAE clinical scoring which was done every 3 days, cytokine levels, and myelination level. RESULTS: The results confirmed the presence of cyclotides in V. odorata based on their retention time and the composition of mobile phase in HPLC and the molecular weight of the peaks in MALDI-TOF analysis. It was observed that cyclotides, especially in the 80% fraction group at the dose of 50 mg/kg significantly reduced the clinical scores, inflammation, and demyelination in EAE mice compared with the normal saline group (P<0.05), and the results of this group were comparable with fingolimod (P>0.05). CONCLUSION: It could be concluded that V. odorata is a rich source of cyclotides which they could be extracted by an easily available process and also, they could be used as immunomodulatory agents in MS, with similar effects to fingolimod.

Isolation and identification of 6-methoxy parillin and coniferin from the bulbs of Allium affine L.

Background and purpose: Phytochemically, Allium species are a rich source of important secondary metabolites especially steroidal saponin and sapogenins, flavonoids, and sulfur compounds. As a member of this genus, Allium affine, which is locally known as "tareh kouhi", is an endemic plant of middle Asian countries. Experimental approach: Bulbs of A. affine were collected and air-dried in the shade. The chloroform-methanol (9:1) extract of the sample was subjected to purification by MPLC and HPLC. Structure elucidation of isolated compounds was done using comprehensive spectroscopic methods including 1D-NMR, 2D-NMR, and MS. Findings/Results: A steroidal saponin structurally related to parillin and a phenylpropanoid glycoside (coniferin) were isolated and identified from the plant chloroform-methanol extract. Conclusion and implication: To the best of our knowledge isolation of these potentially medicinal compounds from A. affine was reported for the first time in this study.

Isolation of dioscin-related steroidal saponin from the bulbs of Allium paradoxum L. with leishmanicidal activity.

Alliums are rich sources of steroidal saponins, flavonoids, and sulphoric compounds of which steroidal saponins have recently received more attention due to their important pharmacological activities. Allium paradoxum L. is a common edible vegetable in north regions of Iran, especially in Mazandaran province, where it is named "Alezi" and considerably used as a raw vegetable, to make dishes, and as a medicinal plant. Phytochemical investigation of chloroform-methanol extract of the plant resulted in the isolation and identification of a dioscin related steroidal saponin, using comprehensive spectroscopic methods including 1D and 2D NMR, its chemical structure was determined as (25R)-spirost-5-en-3b-ol,3-O-α-rhamnopyranosyl-(1→4)-α-rhamnopyranosyl-(1→4)-[a-rhamnopyranosyl-(1→2)]-glucopyranoside. Investigation of in vitro antileishmanial activity of the isolated compound, in 10 and 50 µg/mL concentrations, exhibited significant leishmanicidal effects (P < 0.001) against the promastigotes of Leishmania major. The results established a valuable basis for further studies about A. paradoxum and anti-parasitic activity of steroidal saponins.