Browsing by Author "Espinoza Castro, Karla Esther"

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Comparación de la aplicabilidad de las técnicas DPPH (1,1-difenil-2-picril-hidrazilo) y Peróxidos para extractos resinosos y no resinosos en metanol y diclorometano
(Universidad de Cuenca, 2015-06-02) Espinoza Castro, Karla Esther; López Domínguez, María Belén; Wilches Arizábala, Isabel María
In the present research, the antioxidant capacity present in methanol and di-chloro methane extracts obtained from leaves of Jungia rugosa Less and Desmodium molliculum was evaluated. Desmodium molliculum, a plant traditionally named “hierba del infante” was collected into the Azuay province and was botanically characterized at Herbarium Azuay (HA) into the Universidad del Azuay. Dried extracts were obtained by means of percolation, using methanol and dichloromethane as solvents for extraction. A fraction of those extracts was used for their phytochemical characterization using a general screening method based on Thin Layer Chromatography (TLC). Results show the presence of phenolic compounds (flavonols and flavone) and terpenoids as the more important compounds. To evaluate antioxidant activity, two different techniques were applied: discoloration of radical 2,2-diphenyl-1-picrilhidrazilo (DPPH) and the hydrogen peroxide scavenging assay, ascorbic acid was used as a standard, IC50 value for standard was determined as 2.68 ug/ml by the first method and 58.63 ug/ml by the second method. In the DPPH assay, IC50 value for methanolic extracts of Jungia rugose Less was between 40 – 129 µg/ml and for dichloromethane extracts was between 74 – 340 µg/ml. IC50 of Desmodium molliculum was 48 µg/ml in methanolic extract and 80 ug/ml in dichloromethane extract. When hydrogen peroxide assay was developed, the IC50 value of Desmodium molliculum methanolic extract was 61 µg/ml, and between 177 – 349 µg/ml for Jungia rugosa Less. Antioxidant activity of dichloromethane extracts was not evaluated by this technique due to incompatibility with the reagents used. The findings suggest that “Jungia” and “Desmodium” have antioxidant activity, which could be related to the presence of phenolic compounds.
Identificación y clonación de secuencias codificantes de peroxidasas de granos inmaduros de fréjol (Phaseolus vulgaris)
(Universidad de Cuenca, 2020-12-09) Espinoza Castro, Karla Esther; Vallecillo Maza, Antonio Javier
Peroxidases are hemoproteins whose catalytic activity is used in diagnostic tests, development of biosensors and bioremediation, the most used are Horseradish (Armoracia rusticana) peroxidase or HRP and Soybean (Glycine max) peroxidase or SBP. Currently these enzymes have a high cost, so it is necessary to find alternatives for obtaining them and have immediate availability in the country for application in the development of diagnostic tools. In the search for alternatives to commercial peroxidase, a literature review was carried out to identify previously described sequences of SBP, the localized sequences were used to identify orthologous coding sequences in Phaseolus vulgaris (Common bean), with the identified sequences were designed oligonucleotides for the generation and amplification of the respective cDNAs. After obtaining total RNA from immature grain cuticle, synthesis of the cDNA and generation of PCR products was carried out. PCR products sequencing results showed that it has been possible to obtain sequences encoding the orthologue peroxidase of Ep (NP_001238315.1, NM_001251386 .1) and Prx2 (NP_001237601.1, NM_001250672.2) from common bean. The sequences obtained in this work have a high identity with the reported sequences in Ph. vulgaris, Ep (PHAVU_006G129900g) with 99.08 % and 99.69 % for Prx2 (PHAVU_003G078600g). Cloned coding peroxidases sequences in pET15b vector were induced Escherichia coli Origami (DE3) and total protein extract obtained showed enzymatic activity on 3,3′- diaminobenzidine, 4-chloro-1-naphthol and syringaldazine peroxidases substrates. Finally, the results obtained allow us to conclude that it was possible to obtain two protein coding sequences with peroxidase activity from Phaseolus vulgaris.