Edison Osorio

In Vitro and In Vivo Cytotoxicities and Antileishmanial Activities of Thymol and Hemisynthetic Derivatives

ABSTRACT The in vitro and in vivo antileishmanial and cytotoxic activities of thymol and structural derivatives in comparison to those of Glucantime were studied. The results showed here suggest that thymol and hemisynthetic derivatives have promising antileishmanial potential and could be considered as new lead structures in the search for novel antileishmanial drugs.

Influence of cultivar and ripening time on bioactive compounds and antioxidant properties in Cape gooseberry (Physalis peruviana L.)

BACKGROUND Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued for its organoleptic properties and bioactive compounds. Considering that the presence of phenolics and ascorbic acid could contribute to its functional capacity, it is important to investigate the quality parameters, bioactive contents and functional properties with respect to genotype and ripening time. In this study the genotype effect was evaluated in 15 cultivars for two different harvest times. Changes during maturation were recorded in two commercial cultivars within seven levels of maturity. RESULTS Multivariate statistical analysis suggested that phenolic content and ORAC value were mainly affected by harvest time and that ascorbic acid content and DPPH level were mainly affected by genotype. In addition, acidity, phenolic content, ORAC value and inhibition of LDL oxidation decreased with maturity, but soluble solids content, ascorbic acid content, β-carotene content and DPPH-scavenging activity were higher in mature fruits. CONCLUSION The phenolic content, ascorbic acid content and antioxidant properties of Cape gooseberry fruit were strongly affected by cultivar, harvest time and maturity state. Consequently, the harvest time must be scheduled carefully to gain the highest proportion of bioactive compounds according to the specific cultivar and the environment where it is grown.

Chapter 2 Alkaloids with Antiprotozoal Activity

Publisher Summary This chapter discusses the alkaloid natural products that are particularly active against Leishmania sp ., Trypanosomacruzi, Trypanosomabrucei, and Plasmodium falciparum . The alkaloids with antiprotozoal activity are grouped according to their structures or origin in several categories such as quinoline and isoquinoline alkaloids, indole alkaloids, steroidal alkaloids, alkaloids from marine organisms, and other alkaloids. A discussion on structure-antiprotozoal activity relationships is included, and the mechanism of action of several of these metabolites is described in the chapter. Recent developments, as well as new experimental strategies in discovery and development of antiprotozoal compounds, are also discussed in the chapter. Some of the in vitro antitrypanosomal or cytotoxic activities have been transformed into molar concentrations to allow a better comparison, independent of their molecular weight. Nevertheless, direct comparison remains complex because of the different assay procedures used in various laboratories. Alkaloids are one of the most important groups of natural products, already providing many drugs for human use. Although they can be seriously toxic for the host, alkaloid-containing plants and their biosynthesized alkaloids have a remarkable potential to provide pharmaceutical and biological agents contributing to the development of future antiparasitic drugs. Natural alkaloids will continue to play an important role in the development of a new generation of antiprotozoal drugs.

Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning.

Alkaloid metabolite profiles by GC/MS and acetylcholinesterase inhibitory activities with binding-mode predictions of five Amaryllidaceae plants

Acetylcholinesterase (AChE) enzymatic inhibition is an important target for the management of Alzheimer disease (AD) and AChE inhibitors are the mainstay drugs for its treatment. In order to discover new sources of potent AChE inhibitors, a combined strategy is presented based on AChE-inhibitory activity and chemical profiles by GC/MS, together with in silico studies. The combined strategy was applied on alkaloid extracts of five Amaryllidaceae species that grow in Colombia. Fifty-seven alkaloids were detected using GC/MS, and 21 of them were identified by comparing their mass-spectral fragmentation patterns with standard reference spectra in commercial and private library databases. The alkaloid extracts of Zephyranthes carinata exhibited a high level of inhibitory activity (IC50 = 5.97 ± 0.24 μg/mL). Molecular modeling, which was performed using the structures of some of the alkaloids present in this extract and the three-dimensional crystal structures of AChE derived from Torpedo californica, disclosed their binding configuration in the active site of this AChE. The results suggested that the alkaloids 3-epimacronine and lycoramine might be of interest for AChE inhibition. Although the galanthamine group is known for its potential utility in treating AD, the tazettine-type alkaloids should be evaluated to find more selective compounds of potential benefit for AD.

Neuroprotective activity and acetylcholinesterase inhibition of five Amaryllidaceae species: A comparative study

AIMS Amaryllidaceae alkaloids exhibit a wide range of physiological effects, of which the acetylcholinesterase (AChE) inhibitory activity is the most relevant. However, scientific evidence related to their neuroprotective effectiveness against glutamate-induced toxicity has been lacking. Thus, the purpose of this study was to conduct a comparative study of the neuroprotective activity and the AChE inhibitory activity of species of Amaryllidaceae. MAIN METHODS The neuroprotective activity against glutamate-induced toxicity was measured in rat cortical neurons and the Ellman method was employed for the quantification of acetylcholinesterase inhibitory activity of alkaloidal extracts of five species of Amaryllidaceae (Crinum jagus, Crinum bulbispermum, Hippeastrum barbatum, Hippeastrum puniceum and Zephyranthes carinata). The alkaloid Amaryllidaceae patterns based on GC/MS analyses were also investigated. KEY FINDINGS The results showed that the alkaloidal extract from C. jagus presented a high neuroprotective activity in both pre- and post-treatments against a glutamate excitotoxic stimulus. Furthermore, the alkaloid extracts from C. jagus and Z. carinata revealed an inhibitory activity of AChE from the electric eel with IC50 values of 18.28±0.29 and 17.96±1.22μg/mL, respectively. In addition, 46 alkaloids were detected by GC/MS, and 20 of them were identified based on their mass spectra and retention index. The results suggest that the neuroprotective effects might be associated with lycorine and crinine-type alkaloids, whereas the acetylcholinesterase enzyme inhibitory activity could be related to galanthamine and lycorine-type alkaloids, although not based on synergistic processes. SIGNIFICANCE In summary, Amaryllidaceae species are sources of alkaloids with potential use for Alzheimers disease.

Inhibition of the toxic effects of Bothrops asper venom by pinostrobin, a flavanone isolated from Renealmia alpinia (Rottb.) MAAS

ETHNOPHARMACOLOGICAL RELEVANCE Renealmia alpinia has been traditionally used to treat snakebites by indigenous Embera-Katíos tribes belonging to the regions of Antioquia and Chocó, Colombia, and it has been shown to inhibit the enzymatic and biological activities of Bothrops venoms and their purified phospholipase A2 (PLA2) toxins. In addition to its common local usage against snakebites, Renealmia alpinia is commonly used to treat pain. To evaluate the inhibitory ability of pinostrobin, the main compound in the dichloromethane extract of Renealmia alpinia, on the toxic effects of Bothrops asper venom through in vitro and in vivo models and to evaluate its activity against pain and edema. MATERIALS AND METHODS Pinostrobin was isolated from the dichloromethane extract of Renealmia alpinia leaves. The protective properties of the extract and of pinostrobin against the indirect hemolytic, coagulant and proteolytic effects of Bothrops asper venom were evaluated in vitro, and the anti-hemorrhagic and anti-inflammatory activity were evaluated in vivo. RESULTS Renealmia alpinia extract significantly inhibited the proteolytic activity and indirect hemolytic activity of Bothrops asper venom at a venom:extract ratio of 1:20. Moreover, the present data demonstrate that pinostrobin may mitigate some venom-induced local tissue damage due to hemorrhagic effects, and the compound is also responsible for the analgesic and anti-inflammatory activity of the extract from Renealmia alpinia. This is the first report to describe pinostrobin in the species Renealmia alpinia and its properties in vitro against Bothrops asper venom. CONCLUSION Our studies of the activity of Renealmia alpinia against the venom of Bothrops asper have confirmed that this species possesses inhibitory effects against Bothrops asper venom in both in vitro and in vivo models and that these effects may be due to pinostrobin, supporting the traditional usage of the plant. Additionally, pinostrobin may be responsible for the anti-hemorrhagic and analgesic activity (peripheral analgesic activity) of Renealmia alpinia.

The biflavonoid morelloflavone inhibits the enzymatic and biological activities of a snake venom phospholipase A2

The biflavonoid morelloflavone has been reported as inhibitor of secretory PLA2s (phospholipases A2 from human synovial and bee venom sources); however, its capacity to interact and inhibit snake venom PLA2 activities has not been described. In this work we tested the inhibitory ability of morelloflavone on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA2 isolated from Crotalus durissus cumanensis venom. The biflavonoid displayed IC50 values of 0.48 mM (95% Confidence intervals: 0.45-0.51) and 0.38 mM (95% Confidence intervals: 0.36-0.40) on the PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. In addition, morelloflavone inhibited in a time-dependent manner and irreversibly the PLA2 enzymatic activity. When mice were injected with PLA2 preincubated (preincubation assay) with 0.13, 0.63 and 1.26 mM of the biflavonoid, the myotoxic activity induced by the PLA2 was inhibited up to 63%. Nevertheless, these values decreased up to 38% when the morelloflavone was injected into muscle after PLA2. Moreover, morelloflavone inhibited, in a concentration-dependent manner, edema-forming activity of the PLA2 in the footpad. Morelloflavone also inhibited the anticoagulant activities of the PLA2 in concentration-dependent mode. In order to have insights on the mode of action of morelloflavone, intrinsic fluorescence studies were performed. Results of these assays suggest that morelloflavone interacts directly with the PLA2. These findings were supported by molecular docking results, which suggested that morelloflavone forms hydrogen bonds with residues Gly33, Asp49, Gly53 and Thr68 of the enzyme. In addition, our results suggested a π-π stacking interaction between rings A of morelloflavone with that of the residue Tyr52, and Van der Waals interactions with Gly32, His48 and Ala56. Our molecular modeling results suggest that morelloflavone may occupy part of substrate binding cleft of the PLA2. Morelloflavone is a candidate for the development of inhibitors to be used in snakebite envenomation.

Passiflora tarminiana fruits reduce UVB-induced photoaging in human skin fibroblasts

Skin aging is a complex process that is strongly affected by UV radiation, which stimulates the production of reactive oxygen species (ROS) in the epidermis and dermis and subsequently causes skin damage. Among the major consequences are increased collagen degradation and reduced collagen synthesis. Previous reports have demonstrated the beneficial effects of polyphenols for healthy skin. Passiflora tarminiana Coppens & V.E. Barney, a species of the Passifloraceae family, is widely distributed in South America and is rich in flavonoids. We show that UVB radiation increases metalloproteinase 1 (MMP-1) and reduces procollagen production in human dermal fibroblast (HDF) cells in a dose- and time-dependent manner. We examined the antioxidant and antiaging effects of the extract and fractions of P. tarminiana fruits. The fractions showed high polyphenol content (620mg EAG/g) and antioxidant activity, as measured by ORAC (4097μmol ET/g) and ABTS (2992μmol ET/g) assays. The aqueous fraction drastically inhibited the collagenase enzyme (IC50 0.43μg/mL). The extract and fractions presented photoprotective effects by reducing UVB-induced MMP-1 production, increasing UVB-inhibited procollagen production, and decreasing ROS production after UVB irradiation in HDF. Finally, the polyphenol contents of the extracts and fractions from P. tarminiana were analyzed by HPLC-DAD-ESI-MSn, and procyanidins and glycosylated flavonoids were identified.

GC/MS method to quantify bioavailable phenolic compounds and antioxidant capacity determination of plasma after acute coffee consumption in human volunteers

Coffee, a source of chlorogenic acids (CGAs), is recognized for preventing chronic diseases associated with oxidative stress. Therefore, sensitive, selective and easy access methods for the determination of the bioavailability and antioxidant function in vivo are required in clinical studies. The aim of this work was to validate a GC/MS method to quantify caffeic acid (CA) and ferulic acid (FA) and to apply different methodologies to determine the antioxidant capacity of plasma after the acute consumption of 420mg of CGAs provided by 400mL of coffee. The intervention was performed in 20 adults (6 men and 14 women) with a mean±SD age of 35.7±9.0 and body mass index of 22.1±1.6kg who were assigned to 2 groups: a control group and a group that consumed coffee. The validated analytical GC/MS method was exact, precise and selective. The selected derivatizing reagent was N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tertbutyldimethylchlorosilane (MTBSTFA+1% TBDMSCl). The method was reproducible, and the limit of detection (LOD) was 3nM for CA and 5nM for FA. CA and FA were detected in plasma 1h after coffee intake and were undetectable in the control group. Compared to the baseline values, the antioxidant capacity of plasma significantly increased when it was measured by ferric reducing antioxidant power (FRAP) (6.67%; P<0.001) and by oxygen radical absorbance capacity (ORAC) (7.16%; P<0.05). The in vitro and ex vivo experiments on plasma with CA and FA showed a significant increase of the antioxidant activity (P<0.05) as well as delay of LDL oxidation (P<0.001). The method validated by GC/MS was proposed as an alternative for evaluating the bioavailability of ACG after acute coffee intake. The need for in vitro methodologies was demonstrated to determine the antioxidant activity.