RESUMEN
Botanical extracts are standardized to > or = 1 marker compounds (MCs). This standardization provides a certain level of quality control, but not complete quality assurance. Thus, industries are looking for other satisfactory systems to improve standardization. This study focuses on the standardization of herbal medicines by combining 2 parameters: the concentration of the MC and antioxidant capacity. Antioxidant capacity was determined with the oxygen radical absorbance capacity (ORAC) method and the concentrations of the MCs, by high-performance liquid chromatography. Total phenols were also determined by the Folin-Ciocolteau method. The ORAC values, expressed as micromol Trolox equivalents/100 g (ORAC %), of 12 commercial herbal extracts were related to the ORAC values of the respective pure MCs at the concentrations at which the MCs occur in products (ORAC-MC %). The ORAC % values of 11 extracts were higher than those of the respective MCs and the ratios ORAC-MC %/ORAC % ranged from 0.007 to 0.7, whereas in the case of Olea europaea leaves, the same ratio was 1.36. The ORAC parameters and their ratios, as well as the linear relationship between ORAC-MC % and ORAC %, are described and discussed as tools for improving the standardization of herbal products and detecting modifications due to herb processing and storage.
Asunto(s)
Antioxidantes/análisis , Cromatografía Líquida de Alta Presión/métodos , Extractos Vegetales/normas , Liofilización , Fenoles/análisis , Extractos Vegetales/análisisRESUMEN
The resistance to oxidative stress is a multifactorial reaction involving the clustering of transcriptionally regulated genes. Because glucose-6-phosphate dehydrogenase (G6PD), the principal enzyme responsible for reducing power, is highly expressed in the olfactory bulb (OB), it is of interest to verify whether other enzymes utilizing NADPH are also highly expressed. The level and localization of G6PD- and NADPH-consuming enzymes, such as NADPH-cytochrome P450 oxidoreductase (P450R), glutathione reductase (GR), and NADPH-diaphorase (NADPH-d), were analyzed in the rat olfactory bulb (OB) by quantitative histochemistry and immunohistochemistry. The highest concentration of G6PD, P450R, and GR was observed in the olfactory nerve layer (ONL), suggesting a correlation in the expression of these enzymes at the gene level. Correlation in staining intensity between G6PD and NADPH-d activities occurred only in part of the ONL, some glomeruli, and scattered periglomerular cells. This peculiar distribution of NADPH-d could reflect a spatial patterning of the nose to bulb projections. Taken together, these results indicate that G6PD expression in the ONL could be related to the importance of generating a substantial supply of NADPH to sustain the detoxifying systems represented by GR and P450R reactions and, only in discrete zones, by NADPH-d activity.
Asunto(s)
Glucosafosfato Deshidrogenasa/metabolismo , NADP/metabolismo , Neuronas/enzimología , Bulbo Olfatorio/enzimología , Estrés Oxidativo/fisiología , Animales , Metabolismo Energético/fisiología , Glutatión Reductasa/metabolismo , Inmunohistoquímica , Masculino , NADPH Deshidrogenasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Bulbo Olfatorio/citología , Ratas , Ratas Sprague-DawleyRESUMEN
In cerebellum of the adult rat, glucose-6-phosphate dehydrogenase (G6PD) activity is particularly localized in Purkinje cells, showing lower activity in the molecular and granule cell layers. G6PD is the first and rate-limiting step of the hexose monophosphate shunt (HMS), which has the physiological role of providing NADPH for reductive biosynthesis and detoxifying reactions. In this study, we searched for a possible correlation between G6PD and other NADPH-consuming enzymes, such as NADPH-cytochrome P450 reductase (P450R), glutathione reductase (GR) and NADPH-diaphorase (NADPH-d). This study was performed by means of immunohistochemistry and enzyme histochemistry followed by quantitative densitometric and confocal laser scanning microscopic analyses. Our results demonstrated that G6PD, P450R and GR have a similar distribution pattern characterized by the highest concentration of these enzymes in the somata of Purkinje cells, and by lower concentrations in the molecular and the granule cell layers. Moreover, in Purkinje cells, G6PD colocalized with both P450R and GR. NADPH-d activity showed a different distribution pattern when compared to the other enzymes, revealing the highest activity in the molecular layer and the lowest in Purkinje cells. Our results suggest a coordinated regulative mechanism of G6PD, P450R and GR based on the request of NADPH or on specific transcription factors.
Asunto(s)
Corteza Cerebelosa/citología , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión Reductasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADP/metabolismo , Células de Purkinje/enzimología , Análisis de Varianza , Animales , Corteza Cerebelosa/enzimología , Densitometría/métodos , Proteína Ácida Fibrilar de la Glía/metabolismo , Histocitoquímica/métodos , Inmunohistoquímica/métodos , Masculino , Microscopía Confocal/métodos , Células de Purkinje/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
This review describes the variation of glucose-6-phosphate dehydrogenase (G6PD) activity in the main neurons of the molecular and granular layers as well as in the deep nuclei of the cerebellum as observed so far by optical and electron microscopy studies. Light microscopy and semiquantitative microphotometry of histochemical staining showed that the highest G6PD activity was expressed by Purkinje cells and neurons of the deep cerebellar nuclei; the elements of the molecular layer showed a diffuse G6PD staining, while the granular layer displayed only scattered G6PD activity. Electron microscopy analysis showed that the basket and stellate cells, as well as the Golgi cells, have a remarkable G6PD activity, while in the granule cells the enzyme was barely detectable. The results show that cerebellar G6PD activity changes with different neuron types as a function of its role in sustaining NADPH dependent pathways in these cells.
Asunto(s)
Cerebelo/metabolismo , Glucosafosfato Deshidrogenasa/biosíntesis , NADP/metabolismo , Neuronas/metabolismo , Animales , Cerebelo/citología , Cerebelo/enzimología , Humanos , Microscopía/métodos , Neuronas/enzimologíaRESUMEN
Numerous studies have demonstrated a decrease in glucose-6-phosphate dehydrogenase (G6PD) activity during aging in many cell types, including red blood cells, fibroblasts and lens cells. Moreover, the intracellular activity of G6PD has been shown to be regulated by binding to cell organelles. To investigate whether binding of G6PD to cell organelles is related with the decrease in its activity during aging, distribution patterns of G6PD activity and protein were assessed in small (SI) and large (LI) intestine of 3-month-old and 28-month-old rats. Enzyme activity, as measured spectrophotometrically, did not show any significant change with aging in SI or LI. Enzyme histochemistry, performed by subtracting activity staining of 6-phosphogluconate dehydrogenase (6PGD) from that of G6PD, showed a lower net G6PD activity in SI and LI epithelium of old rats in comparison with young rats. G6PD activity did not change significantly with aging in the muscularis externa of SI and LI. Immunoelectron microscopic analysis of G6PD protein allowed us to measure the density of G6PD molecules in cellular compartments, and the fraction of enzyme bound to cell organelles. In SI and LI epithelia, density of G6PD molecules was higher in old rats than in young rats; however, the fraction of enzyme bound to cell organelles also increased with aging. These data suggest that G6PD activity in epithelium of SI and LI decreases with aging due to the accumulation of significant amounts of enzyme bound to cell organelles, a condition which makes it less active than the soluble enzyme.