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1.
Molecules ; 26(20)2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34684722

RESUMO

Avobenzone, one of the most commonly used UV filters in topical sunscreens, is susceptible to photodegradation with a consequential reduction of its UV absorbing properties. This loss of function may lead to skin irritation, photodermatosis, and photoallergic reactions caused by photodegradation byproducts. In this work, we aim to address this issue with a substance named methoxy-monobenzoylmethane (MeO-MBM), which is neither a UVB nor a UVA filter, but which converts to avobenzone, a known and approved UVA filter, under mainly UVB light irradiation. The antioxidant and intracellular radical formation properties of MeO-MBM were compared to the ones of avobenzone. The UV irradiation of MeO-MBM led to an increase in UV absorption primarily in the UVA range after conversion, both in vitro and in vivo. HPTLC and UHPLC studies illustrate the conversion of MeO-MBM to avobenzone in vitro after irradiation at 250 kJ/m2, reaching a conversion rate of 48.8%. A stable molecular antioxidant activity was observed, since 100-µM MeO-MBM was measured to be 11.2% in the DPPH assay, with a decrease to 9.7% after irradiation. In comparison, the molecular antioxidant activity of 100-µM avobenzone was determined to be 0.8%. In keratinocytes, MeO-MBM reduces the intracellular ROS by 90% and avobenzone by 75% with tBHP as the inducer and by 53% and 57%, respectively, when induced by pyocyanin, indicating the redox scavenging capacity of both these molecules. These results indicate that MeO-MBM functions initially as an antioxidant material and as a photoantioxidant during its conversion process to avobenzone. This research provides insight into the development of active ingredients for topical applications with dynamic functionalities. Using this approach, we demonstrate the possibility to extend the UV protection offered to skin cells while combating cellular stress in parallel.


Assuntos
Benzoatos/farmacologia , Metano/análogos & derivados , Protetores Solares/farmacologia , Antioxidantes , Estabilidade de Medicamentos , Humanos , Queratinócitos/efeitos dos fármacos , Metano/farmacologia , Fotólise , Propiofenonas/química , Propiofenonas/farmacologia , Substâncias Protetoras , Pele/efeitos dos fármacos , Protetores Solares/química , Raios Ultravioleta
2.
Mediators Inflamm ; 2021: 6652791, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34557056

RESUMO

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

3.
Biotechnol Prog ; 37(4): e3147, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33742790

RESUMO

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.

4.
J Ethnopharmacol ; 270: 113741, 2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33359867

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Waltheria Indica L. is traditionally used in Africa, South America and Hawaii to treat pain, anemia, diarrhea, epilepsy and inflammatory related diseases. AIM OF THE STUDY: This study aimed to identify extraction parameters to maximize tiliroside yield and to quantitative secondary metabolite composition of Waltheria Indica under various extraction conditions. The extracts were tested for COX-2 inhibition and their activity correlated with the type and quantity of the secondary metabolites. Insight was gained about how extraction parameters influence the extract composition and thus the COX-2 enzymatic inhibitory activity. MATERIALS AND METHODS: Powdered leaves of Waltheria Indica were extracted using water, methanol, ethyl acetate and ethanol at different temperatures. Tiliroside was identified by HPLC-HRMS n and quantified using a tiliroside standard. The compound groups of the secondary metabolites were quantified by spectrometric methods. Inhibitory potential of different Waltheria extracts against the COX-2 enzyme was determined using a fluorometric COX-2 inhibition assay. RESULTS: The molecule, tiliroside, exhibited a COX-2 inhibition of 10.4% starting at a concentration of 15 µM and increased in a dose dependent manner up to 51.2% at 150 µM. The ethanolic extract at 30 °C and the ethyl acetate extract at 90 °C inhibited COX-2 with 37.7% and 38.9%, while the methanolic and aqueous extract showed a lower inhibition of 21.9% and 9.2% respectively. The results concerning phenol, alkaloid and tiliroside concentration in the extracts showed no dependence on COX-2 inhibition. The extracts demonstrated a direct correlation of COX-2 inhibitory activity with their triterpenoid-/steroidal-saponin concentration. COX-2 inhibition increased linearly with the concentration of the saponins. CONCLUSION: The data suggest that Waltheria Indica extracts inhibit the key inflammatory enzyme, COX-2, as a function of triterpenoid- and steroidal-saponin concentration and support the known efficacy of extracted Waltheria Indica leaves as a traditional treatment against inflammation related diseases.


Assuntos
Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Malvaceae/química , Malvaceae/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Alcaloides/análise , Alcaloides/química , Flavonoides/análise , Flavonoides/química , Flavonoides/farmacologia , Imunidade/efeitos dos fármacos , Fenóis/análise , Fenóis/química , Folhas de Planta/química , Folhas de Planta/metabolismo , Saponinas/análise , Saponinas/química , Saponinas/farmacologia , Metabolismo Secundário , Esteroides/análise , Esteroides/química , Triterpenos/análise , Triterpenos/química
5.
Biotechnol Prog ; 37(3): e3117, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33372404

RESUMO

Events of viral contaminations occurring during the production of biopharmaceuticals have been publicly reported by the biopharmaceutical industry. Upstream raw materials were often identified as the potential source of contamination. Viral contamination risk can be mitigated by inactivating or eliminating potential viruses of cell culture media and feed solutions. Different methods can be used alone or in combination on raw materials, cell culture media, or feed solutions such as viral inactivation technologies consisting mainly of high temperature short time, ultraviolet irradiation, and gamma radiation technologies or such as viral removal technology for instance nanofiltration. The aim of this review is to present the principle, the advantages, and the challenges of high temperature short time (HTST) technology. Here, we reviewed effectiveness of HTST treatment and its impact on media (filterability of media, degradation of components), on process performance (cell growth, cell metabolism, productivity), and product quality based on knowledge shared in the literature.

6.
J Biotechnol ; 186: 110-8, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-25014403

RESUMO

Fed-batch culture bioprocesses are currently used predominantly for the production of recombinant proteins, especially monoclonal antibodies. In these cultures, concentrated feeds are added during cultivation to prevent nutrient depletion, thus extending the cellular growth phase and increasing product concentrations. One limitation in these bioprocesses arises from the low solubility or stability of some compounds at high concentrations, in particular amino acids. This study describes the synthesis and evaluation of a phosphotyrosine disodium salt as a tyrosine source in fed-batch processes. This molecule is highly soluble in concentrated feeds at neutral pH. Mechanistic studies demonstrated that the molecule is cleaved in the cell culture supernatant after processing by released phosphatases, leading to phosphate and free L-tyrosine which can be taken up by the cells. No intact phosphotyrosine was detected intracellularly or incorporated into the sequence of the monoclonal antibody. The use of this new molecule allows the simplification of fed-batch processes in large scale manufacturing via the implementation of neutral pH, highly concentrated feeds.


Assuntos
Anticorpos Monoclonais/metabolismo , Reatores Biológicos , Meios de Cultura/química , Fosfotirosina/química , Proteínas Recombinantes/metabolismo , Sódio/química , Animais , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus , Meios de Cultura/metabolismo , Fosfotirosina/metabolismo , Proteínas Recombinantes/química , Sódio/metabolismo
7.
Methods Enzymol ; 533: 25-30, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24182915

RESUMO

In proteomics research, one essential step among enrichment techniques is subcellular fractionation. This is of special importance for analyzing intracellular organelles and multiprotein complexes. Subcellular fractionation is a flexible and adjustable approach to reducing sample complexity and is most efficiently combined with high-resolution 2-D gel/mass spectrometry analysis as well as with gel-independent techniques.


Assuntos
Fracionamento Celular/métodos , Proteômica/métodos , Animais , Fracionamento Celular/instrumentação , Mamíferos , Proteômica/instrumentação , Frações Subcelulares
8.
Methods Enzymol ; 533: 31-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24182916

RESUMO

In proteomics research, one essential step among enrichment techniques is subcellular fractionation. This is of special importance for analyzing intracellular organelles and multiprotein complexes. Subcellular fractionation is a flexible and adjustable approach to reducing sample complexity and is most efficiently combined with high-resolution 2-D gel/mass spectrometry analysis as well as with gel-independent techniques.


Assuntos
Fracionamento Celular/métodos , Proteômica/métodos , Leveduras/citologia , Tampões (Química) , Fracionamento Celular/instrumentação , Proteômica/instrumentação , Protoplastos/citologia , Frações Subcelulares
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