Nathalia Luiza Andreazza

HPLC-ESI-MS/MS of Imidazole Alkaloids in Pilocarpus microphyllus

Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MSn was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus.

Berberine as a photosensitizing agent for antitumoral photodynamic therapy: Insights into its association to low density lipoproteins

Recent years have seen a growing interest in Berberine, a phytochemical with multispectrum therapeutic activities, as anti-tumoral agent for photodynamic therapy (PDT). In this context, low density lipoproteins (LDL) play a key role in the delivery of the photosensitizer in tumor cells. We correlate the physicochemical parameters of the berberine association to LDL with the influence of LDL-delivery on its accumulation in a glioma cell line and on its photo-induced activity in view of antitumor PDT. Our results evidence an important binding of 400 berberine molecules per LDL. Changes in berberine and apoprotein fluorescence suggest different fixation types, involving various LDL compartments including the vicinity of the apoprotein. The berberine association to LDL does not affect their recognition by the specific B/E receptors, of which over-expression increases the cellular uptake of LDL-preloaded berberine. Fluorescence microscopy evidences the mitochondrial labeling of the glioma model cells, with no significant modification upon LDL-delivery. Moreover, the cellular delivery of berberine by LDL increases its photocytotoxic effects on such cells. So, this research illustrates the potential of berberine as a photosensitizing agent for PDT, in particular due to their behavior towards LDL as plasma vehicles, and gives insights into its mechanisms of cell uptake.

Bioactivity and chemical composition of the essential oil from the leaves of Guatteria australis A.St.-Hil

Essential oil from the leaves of Guatteria australis was obtained by hydrodistillation, analyzed by Gas Chromatography coupled to Mass Spectromery (GC–MS) and their antiproliferative, antileishmanial, antibacterial, antifungal and antioxidant activities were also evaluated. Twenty-three compounds were identified among which germacrene B (50.66%), germacrene D (22.22%) and (E)-caryophyllene (8.99%) were the main compounds. The highest antiproliferative activity was observed against NCI-ADR/RES (TGI = 31.08 μg/ml) and HT-29 (TGI = 32.81 μg/ml) cell lines. It also showed good antileishmanial activity against Leishmania infantum (IC50 = 30.71 μg/ml). On the other hand, the oil exhibited a small effect against Staphylococcus aureus ATCC 6538, S. aureus ATCC 14458 and Escherichia coli ATCC 10799 (MIC = 250 μg/ml), as well as small antioxidant activity (457 μmol TE/g) assessed through ORACFL assay. These results represent the first report regarding chemical composition and bioactivity of G. australis essential oil.

Antiprotozoal and antioxidant alkaloids from Alternanthera littoralis

Five alkaloids, in addition to hydroxytyrosol and uridine, were isolated from aerial parts of Alternanthera littoralis P. Beauv. Among the isolated compounds, alternamide A was an unusual tricyclic alkaloid with a bridged benzoazepine core. All isolated alkaloids have a catechol moiety, indicating a possible common biosynthetic route. Their structures were established by 1D and 2D NMR spectroscopy in combination with extensive tandem MS experiments by collisional induced dissociation (CID). The antiprotozoal activity of the isolated compounds was assayed against trypomastigote forms of Trypanosoma cruzi and amastigotes of Leishmania amazonensis. Alternamine A was the most active compound, reducing markedly the viability of both parasites. Antioxidant capacities evaluated by ORACFL assay showed that the isolated alkaloids (mainly alternamide B) contributed to the high activity recorded for the ethanolic crude extract; possibly, the catechol moiety present in all structures plays a central role in this result.

Photodynamic Inactivation of Yeast and Bacteria by Extracts of Alternanthera brasiliana

This study was undertaken to evaluate the effect of Alternathera brasiliana (Amaranthaceae) extracts as photosensitizing agents in photodynamic antimicrobial therapies (PACT) against Staphylococcus aureus, Staphylococcus epidermidis and Candida dubliniensis. The crude hexane and ethanol extracts were obtained from A. brasiliana whole plant and showed absortion from 650 to 700 nm. Also, singlet molecular oxygen (1O2) production (type II photosensitization reaction) was examined, and the results show that 1,3-diphenylisobenzofuran photodegradation was greatly enhanced in the presence of the A. brasiliana extracts. One plate in each assay was irradiated while the other was not irradiated, the number of colony-forming units per milliliter (CFU/mL) was obtained, and data analyzed by the Tukey test. The chemical composition of the extracts was determined by chromatographic and spectrometric techniques; steroids, triterpenes, and flavonoids were identified. Laser irradiation alone at 685 nm using diode laser, output power of 35 mW, and energy of 28 J/cm2, or non-irradiated crude extracts in sub-inhibitory concentration did not reduce the number of CFU/mL significantly, whereas irradiated hexane and ethanol extracts, in sub-inhibitory concentrations, inhibited the growth of these microorganisms. The photoactivation of hexane and ethanol extracts of A. brasiliana, in sub-inhibitory concentrations, using red laser radiation at 685 nm had an antimicrobial effect.

Antimicrobial photodynamic effect of extracts and oxoaporphine alkaloid isomoschatoline from Guatteria blepharophylla.

Photodynamic Therapy, a tumor therapy idealized at the beginning of the last century, emerges nowadays as a promising treatment alternative against infectious diseases. In this study we report a bioguided study of Guatteria blepharophylla phytoderivatives for antimicrobial PDT. Crude extracts and fraction from the species bark were obtained and further fractionated for substances isolation. All samples were evaluated in relation to their photophysical (absorbance and fluorescence) and photochemical properties (1,3-DPBF bleaching method). Then, bioassays were conducted using as biological models bacteria and yeast strains and a diode laser as a light source. Phytochemical analyses lead to the isolation of 5 isoquinoline alkaloids from oxoaporphine subclass, denominated GB1 to GB5. Photophysical and photochemical analysis showed that extracts, fraction and GB1 (isomoschatoline) presented absorption profile with bands at 600-700nm and were positive for singlet oxygen production. Photobiological assays indicate that these samples presented photodynamic antimicrobial activity against both gram-positive and gram-negative bacterial and some Candida ssp. yeast strains at sub-inhibitory concentrations. The susceptibility of gram-negative bacteria was significantly enhanced when CaCl2 or MgCl2 were employed. Greater energy doses and double samples dosage also decreased microbial survival. It is suggested that GB1 photodynamic activity happens through both types I and II photochemical mechanisms, but with a predominance of the latter. Phytoderivatives of G. blepharophylla promoted antimicrobial effect, however more detailed study concerning chemical composition of the crude extracts and fractions as also photophysical and photochemical characteristics of GB1 are necessary to ensure their potential as photosensitizers at antimicrobial photodynamic inactivation.

Photodynamic antimicrobial effects of bis-indole alkaloid indigo from Indigofera truxillensis Kunth (Leguminosae)

Multidrug-resistant microbial infections represent an exponentially growing problem affecting communities worldwide. Photodynamic therapy is a promising treatment based on the combination of light, oxygen, and a photosensitizer that leads to reactive oxygen species production, such as superoxide (type I mechanism) and singlet oxygen (type II mechanism) that cause massive oxidative damage and consequently the host cell death. Indigofera genus has gained considerable interest due its mutagenic, cytotoxic, and genotoxic activity. Therefore, this study was undertaken to investigate the effect of crude extracts, alkaloidal fraction, and isolated substance derived from Indigofera truxillensis in photodynamic antimicrobial chemotherapy on the viability of bacteria and yeast and evaluation of mechanisms involved. Our results showed that all samples resulted in microbial photoactivation in subinhibitory concentration, with indigo alkaloid presenting a predominant photodynamic action through type I mechanism. The use of CaCl2 and MgCl2 as cell permeabilizing additives also increased gram-negative bacteria susceptibility to indigo.

Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.

Investigation of plant cell culture as experimental model applied to the study of coumarin and chlorogenic acid biosynthesis pathway in Mikania glomerata and Mikania laevigata

Mikania glomerata and Mikania laevigata (guaco) are used as treatment for respiratory system diseases. Both are indistinctively used, however studies have shown differences between their chemical profiles. Therefore, there is a need to investigate the biosynthetic pathway of these metabolites in both species as way to guarantee the quality of these products. Plant cell culture can be an useful tool for studying changes in secondary metabolism as it provides a completely controlled environment and also allows manipulation of biosynthetic pathways for a more efficient production of metabolites. This study showed that plant cell cultures are viable experimental models and can be used to the study of biosynthetic pathways of coumarin and chlorogenic acid in both species.