Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species.

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF-MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.

Influence of 5-amino-3-methyl-4-isoxazolecarbohydrazide on selective gene expression in Caco-2 cultured cells.

The 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide (HIX) is a synthetic isoxazole derivative with a potential for development as an anti-inflammatory drug candidate. The goal of this study was to explore in vitro autoimmune and inflammatory gene modulation by HIX in human Caco-2 cultured cells. The effect of low dose of HIX was tested on the expression level of RNA in 24 h Caco-2 cultures using the QIAGEN Th17 for Autoimmunity & Inflammation RT2 Profiler PCR Array. We choose the PCR technology as the most reliable and sensitive gene expression profiling method for analyzing specific gene regulatory networks. In all experiments, Leflunomide (5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide), an immuno-suppressive disease-modifying antirheumatic drug was used, as a reference to clinical utility of the isoxazole derivatives. Changes in RNA levels were analyzed and differentially expressed genes with at least 2-fold change were identified. For the majority of genes tested, the effects of HIX and Leflunomide were similar, including up-regulation of CX3CL1 and IL-17F, and down-regulation of IL-10 and TLR4. However twelve genes were were differently regulated by the two compounds: interleukins (IL) IL-1B, IL-6 and a chemokine CCL22 were upregulated by HIX and significantly supressed by Leflunomide. In contrary, IL-2 and IL-27 were upregulated by Leflunomide and suppressed by HIX. The network search by Ingenuity Pathway Analysis showed, that majority of differentially expressed genes were involved in cellular inflammatory responses. These results suggest that 5-amino-3-methyl-4-isoxazolecarbohydrazide has a potential for future clinical developments with structure modification as a disease modifying agent in different than Leflunomide applications.

IL-6 mediates 11ßHSD type 2 to effect progression of the mycobacterial cord factor trehalose 6,6'-dimycolate-induced granulomatous response.

Granulomatous structures are highly dynamic during active mycobacterial infection, with accompanying responsive inflammation contributing to modulation of pathology throughout the course of disease. The heightened inflammatory response coinciding with initiation and maintenance of newly developing granulomatous structures must be limited to avoid excessive damage to bystander tissue. Modulating the cellular bioavailability of glucocorticoids by local regulation of 11ßHSD enzymes within responding tissue and parenchyma would allow controlled inflammatory response during infection. Mycobacterial glycolipid trehalose 6,6'-dimycolate was used to induce strong pulmonary granulomatous inflammation immunopathology. Pulmonary corticosterone was significantly increased at days 3 and 5 after administration. An inverse relationship of 11ßHSD1 and 11ßHSD2 message correlated with pathology development. Immunohistochemical analysis also demonstrated that 11ßHSD2 is expressed in proximity to granulomatous lesions. A role for pro-inflammatory IL-6 cytokine in regulation of converting enzymes to control the granulomatous response was confirmed using gene-disrupted IL-6-/- mice. A model is proposed linking IL-6 to endocrine-derived factors which allows modification of active corticosterone into inert 11-dehydrocorticosterone at the site of granuloma formation to limit excessive parenchymal damage.

Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression.

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications.