Pharmacological Potential of Three Berberine-Containing Plant Extracts Obtained from Berberis vulgaris L., Mahonia aquifolium (Pursh) Nutt., and Phellodendron amurense Rupr.

Three berberine-containing plant extracts were investigated for their pharmacological properties. The stems and leaves of Berberis vulgaris, Mahonia aquifolium, and Phellodendron amurense were characterized through scanning electron microscopy. The plant extracts obtained from fresh stem barks were further analyzed through high-performance liquid chromatography, revealing berberine concentrations, among berbamine and palmatine. The plant extracts were further tested for their anticancer potential against 2D and 3D human skin melanoma (A375) and lung adenocarcinoma (A549) cell lines. The concentrations at which 50% of the cells are affected was determined by the viability assay and it was shown that B. vulgaris, the plant extract with the highest berberine concentration, is the most efficient inhibitor (0.4% extract concentration for the 2D model and 3.8% for the 3D model). The membrane integrity and nitrate/nitrite concentration assays were consistent with the viability results and showed effective anticancer potential. For further investigations, the B. vulgaris extract was used to obtain silver nanoparticles, which were characterized through transmission electron microscopy, energy dispersive spectroscopy, and X-ray diffraction. The formed nanoparticles have a uniform size distribution and are suited for future investigations in the field of biomedical applications, together with the B. vulgaris plant extract.

Remodeling tumor microenvironment by liposomal codelivery of DMXAA and simvastatin inhibits malignant melanoma progression.

Anti-angiogenic therapies for melanoma have not yet been translated into meaningful clinical benefit for patients, due to the development of drug-induced resistance in cancer cells, mainly caused by hypoxia-inducible factor 1α (HIF-1α) overexpression and enhanced oxidative stress mediated by tumor-associated macrophages (TAMs). Our previous study demonstrated synergistic antitumor actions of simvastatin (SIM) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on an in vitro melanoma model via suppression of the aggressive phenotype of melanoma cells and inhibition of TAMs-mediated angiogenesis. Therefore, we took the advantage of long circulating liposomes (LCL) superior tumor targeting capacity to efficiently deliver SIM and DMXAA to B16.F10 melanoma in vivo, with the final aim of improving the outcome of the anti-angiogenic therapy. Thus, we assessed the effects of this novel combined tumor-targeted treatment on s.c. B16.F10 murine melanoma growth and on the production of critical markers involved in tumor development and progression. Our results showed that the combined liposomal therapy almost totally inhibited (> 90%) the growth of melanoma tumors, due to the enhancement of anti-angiogenic effects of LCL-DMXAA by LCL-SIM and simultaneous induction of a pro-apoptotic state of tumor cells in the tumor microenvironment (TME). These effects were accompanied by the partial re-education of TAMs towards an M1 phenotype and augmented by combined therapy-induced suppression of major invasion and metastasis promoters (HIF-1α, pAP-1 c-Jun, and MMPs). Thus, this novel therapy holds the potential to remodel the TME, by suppressing its most important malignant biological capabilities.

Phytochemical Analysis of Anti-Inflammatory and Antioxidant Effects of Mahonia aquifolium Flower and Fruit Extracts.

Oxidative stress and inflammation are interlinked processes. The aim of the study was to perform a phytochemical analysis and to evaluate the antioxidant and anti-inflammatory activities of ethanolic Mahonia aquifolium flower (MF), green fruit (MGF), and ripe fruit (MRF) extracts. Plant extract chemical composition was evaluated by HLPC. A DPPH test was used for the in vitro antioxidant activity. The in vivo antioxidant effects and the anti-inflammatory potential were tested on a rat turpentine oil-induced inflammation, by measuring serum nitric oxide (NOx) and TNF-alpha, total oxidative status (TOS), total antioxidant reactivity (TAR), oxidative stress index (OSI), 3-nitrothyrosine (3NT), malondialdehyde (MDA), and total thiols (SH). Extracts were administrated orally in three dilutions (100%, 50%, and 25%) for seven days prior to inflammation. The effects were compared to diclofenac. The HPLC polyphenol and alkaloid analysis revealed chlorogenic acid as the most abundant compound. All extracts had a good in vitro antioxidant activity, decreased NOx, TOS, and 3NT, and increased SH. TNF-alpha was reduced, and TAR increased only by MF and MGF. MDA was not influenced. Our findings suggest that M. aquifolium has anti-inflammatory and antioxidant effects that support the use in primary prevention of the inflammatory processes.

Alternative fluorimetric-based method to detect and compare total S-nitrosothiols in plants.

Nitric oxide (NO) is an important signaling molecule occurring in virtually all organisms, whose mechanism of action relies mainly on its interaction with proteins or peptides by nitrosylation, forming compounds such as S-nitrosothiols (SNO). The Saville reaction and the ozone-based chemiluminescence method are the main techniques used for nitrosylated protein quantification. While the Saville assay is not very sensitive, the ozone-based chemiluminescence is expensive and time-consuming. Here we propose a method in which the protein-bound NO groups are exposed to UV light, cleaving the bond and allowing the quantification of the derived NO molecules by diamino-rhodamine (DAR) dyes. The DAR-based method was shown to be specific in plant tissues by pre-treatment of the samples with reducing agents and parallel EPR analysis. Spike-and-recovery assays revealed 72% recovery after a GSNO spike. Moreover, the method was significantly more sensitive than the Saville reaction, and this increase in sensitivity was crucial for detecting the reduced levels of nitrosylated proteins in plant species other than Arabidopsis. The method presented here is a suitable alternative to compare plant samples, allowing simple and fast detection of nitrosylated proteins.

An assay for pro-oxidant reactivity based on phenoxyl radicals generated by laccase.

A transient species may be detected with UV-vis and EPR spectroscopy during turnover of a laccase with quercetin; this species is assigned as a quercetin-derived radical, based on EPR spectra as well the observed UV-vis similarities (a 540nm centred band) with previously reported data. The rates of formation and decay of this species correlate well (r=0.9946) with the pro-oxidant reactivity manifested by flavonoids in the presence of laccase. An assay for the pro-oxidant reactivity of natural products is hence proposed based on the results reported here; its application is demonstrated for a series of pure compounds as well as for several propolis extracts. This assay has the advantages of using a biologically relevant process (haemoglobin oxidation), and not requiring the addition of oxidising agents such as peroxide or superoxide. Correlations, or the lack thereof, between the pro-oxidant parameters and the redox potentials, antioxidant capacities and lipophilicities, were analysed. The laccase employed in our study does display reactivity-related similarities to a range of other proteins, including human plasma ceruloplasmin.

Ecosystem discrimination and fingerprinting of Romanian propolis by hierarchical fuzzy clustering and image analysis of TLC patterns.

The fingerprinting capacity of thin layer chromatography (TLC) and image analysis in the case of propolis samples collected in different area in Romania has been investigated. Fuzzy divisive hierarchical clustering approach was used as a powerful tool of samples discrimination and fingerprinting according to the geographical origin and local flora. The fuzzy partition and patterns obtained by membership degrees plot were in a very good agreement with floral origin and geographic location of Romanian propolis samples, and clearly illustrate the fuzziness concerning their similarities and difference. The results obtained strongly support that TLC via image analysis can be successfully employed in the fingerprinting methodologies if they are combined with appropriate fuzzy clustering method. The method developed in this paper might be also extended in the authenticity and origin control of fruits, herbs or derived products.

Multivariate analysis of reflectance spectra from propolis: geographical variation in Romanian samples.

The present study described reflectance spectroscopy as a suitable analytical tool to discriminate the floral origin of 39 Romanian propolis samples. Relevant differences between the UV-vis reflectance spectra of the investigated propolis samples within the 220-850nm spectral range were found. The results obtained applying cluster analysis, principal component analysis and linear discriminant analysis to the digitized data of zero order, zero order normalized and first order derivative spectra support the reliability of this technique. In addition, the application of the linear discriminant analysis to the score matrices corresponding to the first principal components appeared to be an illuminating solution. Generally, the samples have been assigned to two large groups in a good agreement with their vegetal sampling location, samples originating from predominant forest area and samples originating from meadows. Within the first group, two subgroups were identified according to the dominant type of the forest, deciduous or resinous, while within the last group three subgroups were found according to the extend and variety of the meadow.

Redox reactivity in propolis: direct detection of free radicals in basic medium and interaction with hemoglobin.

Propolis is one of many natural products with known antioxidant properties. The present work aims to investigate the intimate molecular-level mechanisms of this antioxidant reactivity. Electron paramagnetic resonance (EPR)-detectable free radical signals are described here for the first time in propolis extracts. The shape of these signals and the conditions in which they were obtained, point to polyphenolic flavonoids as the sites of the radicals. An inverse correlation between antioxidant capacity and free radical signal intensity is shown. The free radical reactivity of propolis is also illustrated by the effect it exerts on the biologically-relevant peroxide reactivity of hemoglobin. A new test of antioxidant ability in natural extracts such as propolis is proposed, based on modulation of the ascorbate peroxidase activity of hemoglobin (HAPX). Results of this test correlate well with those obtained via traditional methods such as those based on DPPH (2,2-diphenyl-1-picrylhydrazyl), or on ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid).

Quantitative determination of some food dyes using digital processing of images obtained by thin-layer chromatography.

A high-performance thin-layer chromatographic method combined with image processing of scanned chromatograms was developed for the determination of some food dyes (tartrazine, azorubine and Sunset Yellow) in different products. Porous silica gel with 3-aminopropyl functional groups attached to the matrix was used as stationary phase and a mixture of isopropanol, diethyl ether and ammonia (2:2:1, v/v/v) formed the mobile phase. Quantitative evaluation was performed using special-purpose software. The linearity of the analytical procedure was sustained by the numerical parameters such as correlation coefficient (0.9952-0.9980) and standard error of determination (0.03-0.20). The limits of detection were found to be within the range of 5.21-9.34 ng/spot, and the limits of quantification between 10.21 and 18.09 ng/spot. Recovery studies performed on two levels of concentration gave values between 96.39 and 102.76%. These results show that the regression approach provides rigorous and realistic detection and quantification limits and as a consequence can be routinely applied to other analytical systems. This method does not require expensive analytical instruments compared with classical densitometry and provides a reliable quantitative evaluation with minimum of time.