A biotechnology perspective of fungal proteases.

Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

A biotechnology perspective of fungal proteases

Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

Determination of rutin in Erythroxylum suberosum extract by liquid chromatography: applicability in standardization of herbs and stability studies / Determinación de rutina en el extracto Erythroxylum suberosum por cromatografía líquida: aplicación de la normalización de las hierbas y los estudios de estabilidad

In this study, a reverse phase-high performance liquid chromatography (RP-HPLC) technique for determination of rutin in Erythroxylum suberosum extract was developed and validated. A regression analysis was performed, with the observation of good linearity (r = 0.9997). The values obtained for precision and accuracy determination are in agreement with ICH guidelines. The detection and the quantitation limits of rutin were 0.19 ug/mL and 0.60 ug/mL, respectively. The results demonstrated that the developed method is a reliable HPLC technique for determination of rutin in E. suberosum extract. In addition, the applicability of this method in stability studies and standardization of herbs was investigated.

En este estudio, la técnica de cromatografía líquida de alta resolución en fase reversa para la determinación de la rutina en el extracto Erythroxylum suberosum fue desarrollada y validada. Se realizó un análisis de regresión, con la observación de una buena linealidad (r = 0,9997). Los valores obtenidos para la precisión y la determinación de la precisión están de acuerdo con las directrices ICH. La detección y cuantificación de los límites de la rutina fueron 0,19 ug / mL y 0,60 ug / mL, respectivamente. Los resultados demostraron que el método desarrollado es una técnica fiable de HPLC para la determinación de la rutina en el extracto de E. suberosum. Además, se investigó la aplicabilidad de este método en los estudios de estabilidad y la estandarización de hierbas.

A regulação sanitária brasileira e a Hemobrás / Brazilian sanitary regulation and Hemobrás

A Hemobrás é uma empresa criada para possibilitar o acesso, pela população,aos medicamentos produzidos a partir do plasma humano e oriundos de processos biotecnológicos. Todo o ciclo produtivo de tais medicamentos é regulado por legislações sanitárias, as quais serão apresentadas e discutidas neste trabalho. Observamos que muitas destas normas são antigas e precisam de reformulação para atender melhor o grande avanço dos últimos anos nesta área, pois não condizem mais com o novo cenário brasileiro e mundial. Além disso, a Hemobrás tem exercido um papel relevante para o amadurecimento e o progresso das normas que regulamentam o setor de medicamentos no Brasil.

The Hemobrás is a company created to facilitate access by the population to medicines made from human plasma and derived from biotechnological processes. Throughout the production cycle of these drugs is regulated by sanitary laws, which were presented and discussed in this paper. We note that many of these standards are old and need reshaping to better meet the great advance in recent years in this area, because more do not fit with the new Brazilian and global scenario. Furthermore, Hemobrás has played an important role for the maturation and advancement of standards governing the drug industry in Brazil.

Plants from Brazilian Cerrado with potent tyrosinase inhibitory activity.

The increased amount of melanin leads to skin disorders such as age spots, freckles, melasma and malignant melanoma. Tyrosinase is known to be the key enzyme in melanin production. Plants and their extracts are inexpensive and rich resources of active compounds that can be utilized to inhibit tyrosinase as well as can be used for the treatment of dermatological disorders associated with melanin hyperpigmentation. Using in vitro tyrosinase inhibitory activity assay, extracts from 13 plant species from Brazilian Cerrado were evaluated. The results showed that Pouteria torta and Eugenia dysenterica extracts presented potent in vitro tyrosinase inhibition compared to positive control kojic acid. Ethanol extract of Eugenia dysenterica leaves showed significant (p<0.05) tyrosinase inhibitory activity exhibiting the IC50 value of 11.88 µg/mL, compared to kojic acid (IC50 value of 13.14 µg/mL). Pouteria torta aqueous extract leaves also showed significant inhibitory activity with IC50 value of 30.01 µg/mL. These results indicate that Pouteria torta and Eugenia dysenterica extracts and their isolated constituents are promising agents for skin-whitening or antimelanogenesis formulations.

Effect of ultraviolet filters on skin superoxide dismutase activity in hairless mice after a single dose of ultraviolet radiation.

Organic sunscreens may decrease their protective capability and also behave as photo-oxidants upon ultraviolet radiation (UVR) exposure. The present study investigated the effect of a cream gel formulation containing the UV filters benzophenone-3, octyl methoxycinnamate, and octyl salicylate on skin superoxide dismutase (SOD) after a single dose of UVR (2.87 J/cm(2)). The retention of these UV filters was first evaluated in vivo using hairless mice to guarantee the presence of the filters in the skin layers at the moment of irradiation. The in vivo effect of the UV filters on skin SOD was then assayed spectrophotometrically via the reduction of cytochrome c. The cream gel formulation promoted the penetration of the three UV filters into the epidermis and the dermis at one hour post-application. A significant decrease in SOD activity was observed in irradiated animals treated with sunscreen formulation. However, no effect on SOD activity in skin was observed by the isolated presence of the sunscreens, the formulation components, or the exposure to UVR. The sunscreens may have formed degradation products under UVR that may have either inhibited the enzyme or generated reactive species in the skin.

Efficacy of marigold extract-loaded formulations against UV-induced oxidative stress.

The present study investigated the potential use of topical formulations containing marigold extract (ME) (Calendula officinalis extract) against ultraviolet (UV)B irradiation-induced skin damage. The physical and functional stabilities, as well as the skin penetration capacity, of the different topical formulations developed were evaluated. In addition, the in vivo capacity to prevent/treat the UVB irradiation-induced skin damage, in hairless mice, of the formulation with better skin penetration capacity was investigated. All of the formulations were physically and functionally stable. The gel formulation [Formulation 3 (F3)] was the most effective for the topical delivery of ME, which was detected as 0.21 µg/cm(2) of narcissin and as 0.07 µg/cm(2) of the rutin in the viable epidermis. This formulation was able to maintain glutathione reduced levels close to those of nonirradiated animals, but did not affect the gelatinase-9 and myeloperoxidase activities increased by exposure to UVB irradiation. In addition, F3 reduced the histological skin changes induced by UVB irradiation that appear as modifications of collagen fibrils. Therefore, the photoprotective effect in hairless mice achieved with the topical application of ME in gel formulation is most likely associated with a possible improvement in the collagen synthesis in the subepidermal connective tissue.

Evaluation of the Potential of Brazilian Propolis against UV-Induced Oxidative Stress.

This study investigated the potential use of topically and orally administered propolis extracts to prevent UV irradiation-induced oxidative stress in skin. The results illustrated that green propolis extract (GPE) contained greater amounts of polyphenols, coumaric acid, drupanin, baccharin and artepillin C than did brown propolis extract (BPE). GPE showed higher antioxidant activity than BPE when the IC(50) (concentration that caused 50% inhibition) values were compared. Interesting, the oral treatment of hairless mice demonstrated a recovery of 30.0% for GPE and 22.8% for BPE with respect to UV irradiation-induced GSH depletion. The topical pretreatment of animals with both propolis extract solutions recovered around 14.0% of the depleted GSH. However, the employed treatments did not inhibit the increase of cutaneous proteinase secretion/activity caused by irradiation. These findings indicate that despite differences in composition and antioxidant properties, GPE and BPE both successfully prevent UV-induced GSH depletion in vivo and are both promising antioxidant systems against oxidative stress in skin. Based on these findings, complementary studies should be performed to enhance our understanding of the protective effects of propolis extracts in skin.

Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: evaluation of reduced glutathione levels and matrix metalloproteinase secretion.

BACKGROUND AND PURPOSE: Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. METHODS: Firstly, the physico-chemical composition of marigold extract (ME) (hydroalcoholic extract) was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. RESULTS AND CONCLUSIONS: The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL. However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin.