Category Archives: Enzymes

Ovariectomy-induced hypernociception in rats

J Pharm Pharmacogn Res 3(6): 148-161, 2015.

Original Article | Artículo Original

Ovariectomy-induced chronic abdominal hypernociception in rats: Relation with brain oxidative stress

[Hipernocicepción abdominal crónica inducida por ovariectomía en ratas y su relación con el estrés oxidativo cerebral]

Bárbara B. Garrido-Suárez1, Gabino Garrido-Garrido2, Marian Castro Labrada1, Addis Bellma Menéndez1, Roberto Menéndez Soto del Valle3, René Delgado-Hernández1

1Laboratorio de Farmacología y Toxicología, Centro de Investigación y Desarrollo de Medicamentos. Ave. 26 No. 1605 e/ Boyeros y Puentes Grandes, Nuevo Vedado, Plaza. Apdo. Postal 10600. La Habana, Cuba.
2Departamento de Ciencias Farmacéuticas, Facultad de Ciencias, Edificio Ñ3, Universidad Católica del Norte, Angamos 0610, Antofagasta, Chile.
3Departamento de Farmacología, Centro de Bioproductos Marinos (CEBIMAR). Calle Loma entre 35 y 37, Alturas del Vedado, Plaza de la Revolución, Apdo. Postal. 10400. La Habana, Cuba.
Abstract

Context: Ovarian hormone deficiency observed in menopausal women increases the production of reactive oxygen species, which could be implicated in central sensitization subjacent in chronic functional pain syndromes.

Aims: To examine the hyperalgesic state induced by ovariectomy in adult rats and its relation to some oxidative stress outcomes.

Methods: The female Wistar rats were divided into normal, sham ovariectomized (OVX) and OVX groups, which were tested for mechanical and thermal hypernociception during 6 weeks and a single acetic acid-induced test 6 weeks after surgery. Redox biomarkers determinations of superoxide dismutase (SOD) enzyme activity, glutathione (GSH) and nitrates/nitrites as an indicator of nitric oxide (NO) concentrations were determined in the brain and cerebellum of 6 animals of each group.

Results: Exclusivity OVX rats developed a robust state of mechanical hypernociception and allodynia in the abdomen, hindlimbs and proximal tail. Besides, thermal pain thresholds (hot plate) decreased. That was established 3-4 weeks after OVX and lasted for the 6 weeks of the experiment. Increases in visceral sensitivity were also observed in OVX rats. SOD enzyme activity decreased in OVX rats, which showed major deficit for this enzymatic defense under visceral inflammatory injury. However GSH concentrations were increased in brain of OVX animals that allow the balance during acute inflammation. NO concentrations were raised only in OVX rats exposure to chemical inflammatory injury.

Conclusions: OVX in rats provide a useful model, which mimics the functional pain in females that could be related with brain oxidative stress.

Keywords: Functional pain syndromes; hyperalgesia; hypernociception; ovariectomized rats; ovariectomy; pain.

Resumen

Contexto: La deficiencia de las hormonas ováricas en la mujer menopáusica incrementa la producción de especies reactivas de oxígeno que han sido implicadas en la sensibilización central subyacente en los síndromes de dolor funcional crónico.

Objetivos: Examinar el estado hiperalgésico inducido por ovariectomía en ratas adultas y su relación con algunas variables de estrés oxidativo.

Métodos: Ratas Wistar femeninas fueron divididas en grupos: normal, falsas ovariectomizadas (OVX) y OVX, para la evaluación de hipernocicepción mecánica y termal durante 6 semanas, así como la prueba de contorsiones abdominales inducidas por ácido acético a las 6 semanas tras la cirugía. La actividad de la enzima superóxido dismutasa (SOD), concentraciones de glutatión reducido (GSH) y de nitratos/nitritos como indicador de la producción de óxido nítrico (NO) fueron determinadas en cerebros y cerebelos de 6 animales de cada grupo.

Resultados: Las ratas OVX desarrollaron hiperalgesia mecánica y alodinia en abdomen, patas traseras y cola proximal, así como un descenso de sus umbrales al dolor térmico (plato caliente). Estos cambios fueron establecidos 3-4 semanas post-OVX y mantenidos durante las 6 semanas del experimento. La sensibilidad visceral también fue incrementada. La actividad de SOD disminuyó en las ratas OVX, que mostraron mayor deficiencia para la defensa enzimática ante la injuria inflamatoria visceral. El GSH fue incrementado en el cerebro de los animales OVX, lo cual podría facilitar el balance durante la inflamación aguda. El NO solo incrementó en las ratas OVX expuestas al daño químico inflamatorio.

Conclusiones: OVX en ratas provee un modelo beneficioso que mimetiza el dolor funcional en mujeres y que podría estar relacionado con el estrés oxidativo cerebral.

Palabras Clave: Hiperalgesia; hipernocicepción; dolor; ovariectomía; ratas ovariectomizadas; síndromes de dolor funcional.

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Citation Format: Bárbara B. Garrido-Suárez, Gabino Garrido-Garrido, Marian Castro Labrada, Addis Bellma Menéndez, Roberto Menéndez Soto del Valle, René Delgado-Hernández (2015) Ovariectomy-induced chronic abdominal hypernociception in rats: Relation with brain oxidative stress. J Pharm Pharmacogn Res 3(6): 148-161.

© 2015 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

Control of dental biofilm microorganisms by C. illinoinensis, Q. rubra and S. glyciphylla

J Pharm Pharmacogn Res 3(5): 118-129, 2015.

Original Article | Artículo Original

Effect of Carya illinoinensis, Quercus rubra and Smilax glyciphylla extracts, pectin, and papain on the dental biofilm microorganisms

[Efecto de extractos de Carya illinoinensis, Quercus rubra y Smilax glyciphylla, pectina y papaina sobre los microorganismos de la placa dental]

Elda P. Segura1*, Luis Méndez2, Eréndira Márquez1, Alejandra I. Vargas1, Karla M. Gregorio1, José L. Martínez1, Anna Ilyina1

1Nanobioscience Group, Chemistry School; 2Dentistry School. Coahuila Autonomous University. Blvd. V. Carranza e Ing. J.C. Valdés, CP 25280, Col. República, Saltillo, Coahuila, México.
Abstract

Context: Dental caries is an infectious disease resulting in destruction of tooth structure by acid-forming bacteria found in dental plaque and intraoral biofilms, which are made up of mixed-species microbial communities, and their uncontrolled outgrowth can lead to oral disease.

Aims: To analyze new biological materials (papain, pectin, three plant extracts and their combinations), for prevention, control, and treatment of oral bacteria and biofilm in vitro and in vivo.

Methods: Papain, citric pectin, extracts of Carya illinoinensis, Quercus rubra, and Smilax glyciphylla were applied. In vitro test was performed by means of the spectrophotometric assay and CFU evaluation after treatments application. In vivo tests were performed to evaluate the number of microorganisms presented in dental biofilm: before and 1.5 h after brushing with different treatments; after 10 days of brushing with various treatments in 10 groups of patients, signing an informed consent approved by the Institutional Ethics Committee of the Autonomous University of Coahuila.

Results: In vitro, the plant extracts inhibited the growth of Streptococcus sp. as well as a mixture of microorganisms that form dental biofilms. Papain activity was inhibited by plant extracts. In vivo, brushing of teeth with selected plant extracts reduced the number of bacteria in the dental plaques.

Conclusions: The extracts of Quercus rubra, Carya illinoinensis and Smilax glyciphylla and papain (with or without pectin) had an inhibitory effect on the dental biofilm formation. In vitro test demonstrated the bacteriostatic effect of plant extracts or their mixture.

Keywords: Aqueous extract; Carya illinoinensis; oral microorganisms control; papain; pectin; Quercus rubra; Smilax glyciphylla.

Resumen

Contexto: La caries dental es una enfermedad infecciosa; destruye la estructura dentaria por bacterias formadoras de ácido existentes en la placa dental y la biopelícula intraoral, compuestos por comunidades de distintas especies microbianas, la reproducción descontrolada de dichos microorganismos puede causar diversas enfermedades en la cavidad oral.

Objetivos: Analizar nuevos materiales biológicos (papaína, pectina, tres extractos de plantas y sus combinaciones), para la prevención, control y tratamiento de las bacterias orales y biopelícula bacteriana in vitro e in vivo.

Métodos: Se aplicaron papaína, pectina cítrica, extractos de Carya illinoinensis, Quercus rubra, y Smilax glyciphylla. La prueba in vitro se realizó por espectrofotometría y CFU después de la aplicación de tratamientos. In vivo se evalúo número de microorganismos en la biopelicula; antes, 1.5 h después del cepillado y después de 10 días de cepillado con los diferentes tratamientos en 10 grupos de pacientes, firmando consentimiento aprobado por el Comité Institucional de ética de la Universidad Autónoma de Coahuila.

Resultados: In vitro, los extractos vegetales inhibieron el crecimiento de Streptococcus sp. así como la mezcla de microorganismos que forman la biopelícula bacteriana. La actividad de la papaína fue inhibida por extractos de plantas. In vivo, el cepillado de los dientes con extractos de plantas seleccionadas redujo el número de bacterias en la placa dental.

Conclusiones: Los extractos de Quercus rubra, Carya illinoinensis y Smilax glyciphylla y papaína (con y sin pectina) tuvieron efecto inhibitorio en la formación de la biopelícula dental. Las pruebas in vitro demostraron efecto bacteriostático de los extractos de plantas o sus mezclas.

Palabras Clave: Carya illinoinensis; control de microorganismos orales; extracto acuoso; papaína; pectina; Quercus rubra: Smilax glyciphylla.

 

Download the PDF file .

 

Citation Format: Elda P. Segura, Luis Méndez, Eréndira Márquez, Alejandra I. Vargas, Karla M. Gregorio, José L. Martínez, Anna Ilyina (2015) Effect of Carya illinoinensis, Quercus rubra and Smilax glyciphylla extracts, pectin, and papain on the dental biofilm microorganisms. J Pharm Pharmacogn Res 3(5): 118-129.
This article has been cyted by:
Vargas Segura AI, Ilyina A, Segura Ceniceros EP, Silva Belmares Y, Méndez González L (2015) Etiology and microbiology of periodontal diseases: A review. African Journal of Microbiology Research 9(48): 2300-2306. DOI: 10.5897/AJMR2015.7609

© 2015 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

C 041: THE USE OF ENZYMES FOR POLYMER CONJUGATION

J Pharm Pharmacogn Res 2(Suppl. 1): S115, 2014

Special supplement with the abstract book of LATINFARMA 2013

Conference

C 041: THE USE OF ENZYMES FOR POLYMER CONJUGATION

Pasut G.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, F. Marzolo 5, 35131, Padova, Italy.
Abstract

Biocatalysis represents a promising area of research in organic chemistry. The specificity and selectivity of enzymes can dramatically improve the yields of synthesis, especially for those involving very complex structures. In the field of polymer conjugation, enzymes have also been considered as tools for site-specific conjugation at the level of amino acids not usually addressed with chemical methods. Different enzymatic protocols have been developed and proposed for polymer conjugation to protein. The most advanced is GlycoPEGylation that, applied to coagulation factor IX, yielded a conjugate presently in clinical trials . In this case the method is based on two enzymes to mimic O-glycosylationor N-glycosylation and consequently it has the advantage to couple PEG at the site of naturally occurring glycosylation. Although innovative, this approach involves two enzymes and therefore its feasibility at industrial scale might be limited. Another approach exploits the enzyme Sortase A to couple a polymer at the C-terminus of proteins. Also in this case there are some limitations such as the requirement of a specific C-terminal consensus amino acid sequence as substrate for the enzyme.

A promising alternative is the transglutaminase (TGase) enzymes that catalyze the transfer of a primary amine (amino donor) to the gammacarboxamide group of a glutamine (acyl donor). Interestingly, these enzymes recognize other substrate than ε-amino group of a lysine as amino donor, such as for example a PEG-NH2. My lab focused in microbial TGase (mTGase) from Streptomyces mobaraense, already approved as food additive. Beside the fact that this approach can target an amino acid that otherwise cannot be modified by chemical method another advantage of mTGase is its selectively. Among the several glutamines present in a protein only those inserted in very flexible loops can be a substrate, therefore very homogeneous conjugates can be obtained. Furthermore, mTGase preserves a good level of activity in a wide range of pH and ionic strength values and also in the presence of organic co-solvents (up to 50% v/v). We exploited this feature to develop tailor-made reaction conditions for specific proteins with the aim to stabilize the protein or to reduce the number of glutamines substrate of mTGase, thus obtaining mono-PEGylated forms.

The potential of mTGase-mediated PEGylation has been compared in our lab to another known approach of site selective conjugation: Nterminal PEGylation. The methods were applied to hGH by using a 20kDa PEG, and the results were comparable in terms of yield, and pharmacokinetic/pharmacodynamic. Relevant was the efficacy of the conjugates able to produce a weight growth with a single weekly injection in hypophysectomized rats that was comparable to that obtained with 6 weekly injections of free hGH.

The present and future development of TGase as tool for PEGylation is the development of a immobilized mTGase on resin beads, which will offer the relevant advantage of a fast removal of the enzymes and the possibility of a fast screening of several reactions conditions. Furthermore, we discovered that immobilized mTGase present an increased selectivity towards the Glu inserted in the most flexible loops.

This new era of enzymatic polymer conjugation is at the beginning and it might possess greater potentials of development and feasibility for even more advanced protein conjugates.