Journal of Pharmacy & Pharmacognosy Research

Volume 5, Issue 2 (Mar-Apr), 2017 ISSN 0719-4250

Content:

Pages

IFC (Journal of Pharmacy & Pharmacognosy Research).

i

1.- Original article Kambham Venkateswarlu (2017) Evaluation of glibenclamide microspheres for sustained release.

78-87

2.- Original article Jorge S. Amador, Juan P Carrasco, Álvaro A. Morales, Claudia P. Cortes (2017) Evaluación terapéutica de infusiones prolongadas de antibióticos β-lactámicos en el tratamiento y manejo del paciente crítico. | [Therapeutic evaluation of prolonged infusions of β-lactam antibiotics in the treatment and management of critically ill patients].

88-95

3.- Original article Nilia de la Paz, Dania Pérez, Mirna Fernández, Caridad M. García, Vivian Martínez, Antonio Nogueira, Oscar García (2017) In vitro release of dibucaine hydrochloride from chitosan semisolid vehicles: emulsion and hydrophilic gels.

96-105

4. Original article Pilar A. Soledispa Cañarte, Migdalia Miranda Martínez, Viviana García Mir (2017) Validación de una técnica por cromatografía líquida de alta resolución para la determinación de isoflavonas totales. | [Validation of a technique by high-performance liquid chromatography for the determination of total isoflavones].

106-113

5. Original article Marcos Cobaleda-Velasco, Ruth E. Alanis-Bañuelos, Norma Almaraz-Abarca, Marlon RojasLópez, Laura S. González-Valdez, José A. Ávila-Reyes, Sara Rodrigo (2017) Phenolic profiles and antioxidant properties of Physalis angulata L. as quality indicators.

114-128

6. Original article Edebi N. Vaikosen, Gideon O. Alade (2017) Determination of heavy metals in medicinal plants from the wild and cultivated garden in Wilberforce Island, Niger Delta region, Nigeria.

J Pharm Pharmacogn Res JPPRes

129-143

© 2016 Journal of Pharmacy & Pharmacognosy Research

ISSN 0719-4250

http://jppres.com/jppres

JOURNAL OF PHARMACY & PHARMACOGNOSY RESEARCH For specialists working in the pharmaceutical and herbal fields

The Journal of Pharmacy & Pharmacognosy Research (JPPRes) is an international, specialized and peer-reviewed open access journal, which publishes studies in the pharmaceutical and herbal fields concerned with the physical, botanical, chemical, biological, toxicological properties and clinical applications of molecular entities, active pharmaceutical ingredients, devices and delivery systems for drugs, vaccines and biologicals, including their design, manufacture, evaluation and marketing. This journal publishes research papers, reviews, commentaries and letters to the editor as well as special issues and review of pre-and post-graduate thesis from pharmacists or professionals involved in Pharmaceutical Sciences or Pharmacognosy. JPPRes has an acceptance rate of 50%. The average time between submission and final decision is 45 days and the average time between acceptance and final publication is 15 days. Manuscripts submitted to JPPRes are only accepted on the understanding that they are subject to editorial review and that they have not been, and will not be, published in whole or in part in any other journal.

Journal of Pharmacy & Pharmacognosy Research (JPPRes) es una revista internacional, especializada y con revisión por pares, la cual publica estudios científicos en los campos farmacéuticos y farmacognósticos, relacionados con la física, botánica, química, propiedades biológicas, toxicológicas y aplicaciones clínicas de entidades moleculares, ingredientes farmacéuticos activos, dispositivos y sistemas de administración de medicamentos, vacunas y productos biológicos, incluyendo su diseño, fabricación, evaluación y comercialización. Esta revista publica artículos de investigación, revisiones, comentarios y cartas al editor, así como ediciones especiales y las reseñas de libros y tesis de pre y postgrado de los farmacéuticos o los profesionales que intervienen en Ciencias Farmacéuticas y Farmacognosia. JPPRes tiene una tasa de aceptación de 50%. El tiempo promedio entre la presentación del manuscrito y la decisión final es de 45 días y el tiempo medio entre la aceptación final y la publicación es de 15 días. Los manuscritos presentados a JPPRes se aceptan solamente en el entendimiento de que son objeto de revisión editorial y que no han sido, ni serán, publicados en su totalidad o en parte en cualquier otra revista.

Editorial Office (for submission queries and papers under review, according with the JPPRes Instructions): [email protected] Executive Editor (for accepted and published papers only): [email protected]

EDITOR-IN-CHIEF

Gabino Garrido, Departamento de Ciencias Farmacéuticas, Universidad Católica del Norte, Antofagasta, Chile. E-mail: [email protected]

EXECUTIVE EDITOR

Marisela Valdés González, Departamento de Ciencias Farmacéuticas, Universidad Católica del Norte, Antofagasta, Chile. E-mail: [email protected]

EDITORIAL AND DESIGN MANAGER

Xavier Garrido Valdés. Universidad Santo Tomás, Antofagasta, Chile.

EDITORIAL BOARD MEMBERS

Carla Delporte (Santiago, Chile) Damaris Silveira (Brasília, Brazil) Douglas S. de A. Chaves (Rodovia, Brazil) Edgar Pastene (Concepción, Chile) Etile Spegazzini (Buenos Aires, Argentina) Farid Chemat (Avignon, France) Fidel O. Castro (Chillán, Chile) Guilherme N. L. do Nascimento (Tocantins, Brazil) Jacqueline Sepúlveda (Concepción, Chile) Jelena Nadinic (Buenos Aires, Argentina)

REVIEWERS

José H. Isaza-Martínez (Cali, Colombia) Juan C. Sepúlveda-Arias (Pereira, Colombia) Madan M. Gupta (Trinidad & Tobago, West Indies) Mahomoodally M. Fawzi (Réduit, Mauritius) María Inés Isla (Tucumán, Argentina) Marisol Fernández Alfonso (Madrid, España) Mayank Gangwar (Varanasi, India) Silvia Debenedetti (Buenos Aires, Argentina) Vikash Kumar Ruhil (Haryana, India) Yasser Shahzad (Huddersfield, United Kingdom)

The reviewers will be recruited among researchers and clinicians with high international reputation in Pharmacy and Pharmacognosy and supported by members of the editorial board. Abstracted/indexed in: Web of Science™ Core Collection Emerging Sources Citation Index (ESCI), Scopus, LATINDEX, Directory of Open Access Journal, Qualis CAPES, REDIB, Google Scholar, PERIODICA, BIBLAT, HINARI, Chemical Abstract, SHERPA/RoMEO, ResearchGate.

© 2017 Journal of Pharmacy & Pharmacognosy Research, 5 (2), 78-87, 2017 ISSN 0719-4250 http://jppres.com/jppres Original Article | Artículo Original

Evaluation of glibenclamide microspheres for sustained release [Evaluación de microesferas de glibenclamida para liberación sostenida] Kambham Venkateswarlu* Department of Pharmaceutics, JNTUA Oil Technological and Pharmaceutical Research Institute, Ananthapuramu, Andhra Pradesh-515001, India. *E-mail: [email protected]

Abstract

Resumen

Context: Sustained release drug delivery systems are more preferred than the conventional drug delivery systems due to its enhanced bioavailability and patient compliance. Earlier studies reported on glibenclamide (GBCM) were not clear and hence, the step has been taken to explore the sustained release drug delivery system of GBCM.

Contexto: Los sistemas de suministro de fármacos de liberación sostenida son más preferidos que los sistemas convencionales de administración debido a su mejor biodisponibilidad y al cumplimiento del paciente. Los estudios anteriores sobre glibenclamida (GBCM) no fueron claros, por lo que se decidió explorar el sistema de liberación sostenida de fármacos de GBCM.

Aims: To evaluate the sustained release microspheres obtained of GBCM. Methods: Microspheres were prepared by ionic gelation method using the polymers like Eudragit RS 100 and xanthan gum. Polymers can sustain the drug release from microspheres. The prepared microspheres were subjected to micromeritic studies like Carr’s index, Hausner’s ratio and angle of repose. Results: Micromeritic studies confirmed that the microspheres possessing acceptable flow properties. It was observed from the in vitro release studies, formulations F8 and F9 showed sustained drug release for desired time of 12 h and when compared to F9, formulation F8 showed maximum drug release for 12 h. Conclusions: Results confirmed the formulation F8 consist of the polymers such as Eudragit RS 100 about 150 mg and xanthan gum about 100 mg showed desired sustained release of 12 h with 96.07% and kinetic studies confirmed that the release from microspheres followed nonFickian diffusion mechanism. Due to its sustained release property, it could enhance the bioavailability of drug thereby improves the patient compliance and expect better treatment than conventional dosage forms.

Keywords: Eudragit RS 100; glibenclamide; ionic gelation method; sustained release; xanthan gum.

Objetivos: Evaluar las microesferas de acción sostenida obtenidas de GBCM. Métodos: Las microesferas se prepararon mediante un método de gelificación iónica con polímeros como Eudragit RS 100 y goma xantano. Los polímeros pudieron sostener la liberación del fármaco a partir de microesferas. Las microesferas preparadas se sometieron a estudios micromeríticos como el índice de Carr, la relación de Hausner y el ángulo de reposo. Resultados: Los estudios microméríticos confirmaron que las microesferas poseen propiedades de flujo aceptables. Se observó, a partir de los estudios de liberación in vitro, que las formulaciones F8 y F9 mostraron una liberación sostenida del fármaco durante un tiempo deseado de 12 horas y cuando se comparó con F9, la formulación F8 mostró liberación máxima del fármaco durante 12 h. Conclusiones: Los resultados confirmaron que la formulación F8 constituida de polímeros tales como Eudragit RS 100 (150 mg) y goma xantano (100 mg) mostró una liberación sostenida deseada de 12 h con 96,07% y estudios cinéticos confirmaron que la liberación de microesferas seguía mecanismo de difusión no Fickiano. Debido a su propiedad de liberación sostenida, esta podría mejorar la biodisponibilidad del fármaco, por lo tanto, mejorar el cumplimiento del paciente y se esperaría un mejor tratamiento que las formas de dosificación convencionales. Palabras Clave: Eudragit RS 100; glibenclamida; goma xantano; liberación sostenida; método de gelificación iónica.

ARTICLE INFO Received | Recibido: September 2, 2016. Received in revised form | Recibido en forma corregida: October 18, 2016. Accepted | Aceptado: October 26, 2016. Available Online | Publicado en Línea: November 11, 2016. Declaration of interests | Declaración de Intereses: The authors declare no conflict of interest. Funding | Financiación: The author confirms that the project has no funding or grants. Academic Editor | Editor Académico: Gabino Garrido.

_____________________________________

Venkateswarlu

In vitro studies of glibenclamide microspheres

INTRODUCTION

Compatibility studies

Oral drug delivery system is the most preferred route of drug administration due to its ease of administration, stability of formulation and improved patient compliance (Thirumalesh et al., 2016a). In the case of formulation development categories, sustained or controlled systems are more preferred than conventional systems due to its better treatment, efficacy and patient compliance (Thirumalesh et al., 2016b). There are a lot of systems developed for sustained or controlled drug release and amongst those, the prominent systems are matrix systems (Venkateswarlu and Shanthi, 2012), floating systems (Venkateswarlu and Chandrasekhar, 2016a) and microspheres (Vijayabhaskar et al., 2016). Diabetes mellitus is a group of syndromes and a chronic metabolic disorder characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and proteins because of a lack of or ineffective use of the hormone insulin (Venkateswarlu and Shanthi, 2012). GBCM has actions and uses similar to that of sulfonylureas and it is more potent than tolbutamide on a weight basis, but the maximal hypoglycemic effect is similar to that of other sulfonylureas. GBCM stimulates the secretion of insulin but also increases peripheral sensitivity to insulin by a post-receptor mechanism. Inhibition of hepatic glucose production is an important factor in glycemic control (Rajkumar et al., 2010). Literature reported that emulsion solvent evaporation technique for the preparation of GBCM microspheres or microparticles (Kumar et al., 2013; Rashmi et al., 2014). Hence, the present study was aimed to develop the sustained release microspheres of GBCM by ionic gelation method using Eudragit RS 100, xanthan gum and sodium alginate.

Compatibility of the drug with excipients used in the formulation was known by Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC).

MATERIAL AND METHODS Materials GBCM was a gift sample from Hetero Drugs Pvt. Ltd., India. Xanthan gum, Eudragit RS 100, sodium alginate and Tween 80 were obtained from Yarrow Chem. Products, India. Remaining chemicals used were of analytical grade. http://jppres.com/jppres

Fourier Transform Infrared Spectroscopy studies In order to check the integrity (compatibility) of drug with excipients was done by FTIR (Shimadzu FT-IR 8400 spectrophotometer, Japan) studies using KBr disc method. The samples were thoroughly blended with dry powdered potassium bromide crystals, compressed to form a disc, placed in a sample holder and then the spectrum was recorded from 4000 to 400 cm-1. The IR spectrum of the pure drug was compared with the IR spectrum of the physical mixtures (Venkateswarlu and Chandrasekhar, 2016b). Differential Scanning Calorimetry studies The pure drug and optimized formulation were subjected to differential scanning calorimeter equipped with an intra cooler (NETZSCH, Japan). Indium/zinc standards were used to calibrate the DSC temperature and enthalpy scale. The samples were sealed in aluminum pans and heated at a constant rate of 10°C/min over a temperature range of 50-400°C. An inert atmosphere was maintained by purging nitrogen gas at a flow rate of 50 mL/min (Ashok and Desai, 2016). Preparation of alginate microspheres GBCM microspheres were prepared by ionic gelation method and ingredients were specified in Table 1. Sodium alginate and xanthan gum were dissolved in purified water (80 mL) to form a homogeneous polymer solution. GBCM was dissolved in methanol to form 5 mg/mL solution and to this solution; calculated amount of Eudragit RS 100 was added. Twenty mL of GBCM solution was added into the above polymer solution and mixed thoroughly with a magnetic stirrer to form a viscous dispersion. The resulting dispersion was then added drop by drop with the help of syringe and needle to the calcium chloride (10% w/v) solution and the addition was done with continuous stirring. The J Pharm Pharmacogn Res (2017) 5(2): 79

Venkateswarlu


added droplets were retained in the calcium chloride solution for 1 h to complete the curing reaction and to produce rigid spherical microspheres. The microspheres were collected by decantation and the product thus separated was repeatedly washed with water to remove excess calcium impurity and air dried (Bindu et al., 2009).

Drug entrapment efficiency Microspheres equivalent to 10 mg of GBCM was weighed, crushed and then suspended in 100 mL of pH 7.4 phosphate buffer. After 24 h, the solution was filtered, 1 mL of the filtrate was pipette out, diluted to 10 mL and analyzed for the drug content using a UV-visible spectrophotometer (Elico, India) at 229 nm (Omar et al., 1987).

Characterization of the prepared microspheres

Swelling studies

Derived properties of GBCM alginate microspheres

The swellability of microspheres in physiological media was determined by swelling them in the phosphate buffer. Accurately weighed 100 mg of microspheres were immersed in little excess (25 mL) of phosphate buffer for 12 h, washed and the degree of swelling was calculated (Patil et al., 2012).

The flowability of the microspheres was determined by subjecting the microspheres to tapped density, bulk density (Venkateswarlu et al., 2016c), Hausner’s ratio (Venkateswarlu et al., 2016d), Carr’s index (Venkateswarlu et al., 2016e) and angle of repose (Thirumalesh et al., 2016c) studies and performed according to the standard methods reported in earlier studies.

Particle size analysis Particle size of the prepared microspheres was determined by optical microscopy. The optical microscope was fitted with an ocular micrometer and a stage micrometer. The eye piece micrometer was calibrated. The particle diameter of 200 microspheres was measured randomly by optical microscope (Tejraj et al., 1999).

Percentage yield The practical percentage yield was calculated from the weight of dried microspheres recovered from each batch in relation to the sum of the initial weight of starting materials (Vijayabhaskar et al., 2016).

Table 1. Formulation of glibenclamide (GBCM) microspheres. Formulation

GBCM

Sodium alginate (g)

Eudragit RS 100 (mg)

Xanthan gum

(mg)

(mg)

Total weight (g)

F1

100

2

50

50

2.20

F2

100

2

50

100

2.25

F3

100

2

50

150

2.30

F4

100

2

100

50

2.25

F5

100

2

100

100

2.30

F6

100

2

100

150

2.35

F7

100

2

150

50

2.30

F8

100

2

150

100

2.35

F9

100

2

150

150

2.40


J Pharm Pharmacogn Res (2017) 5(2): 80

Venkateswarlu


Shape and surface morphology

RESULTS AND DISCUSSION

The shape and surface characteristics of prepared microspheres were evaluated by means of scanning electron microscopy (SEM) (JEOL-JSM840A, Japan). The samples for SEM were prepared by gently sprinkling the microspheres powder on a double adhesive tape, which was stuck to an aluminum stub. The stubs were then coated with gold using a sputter coater (JEOL Fine coat JFC 1100E, ion sputtering device) under high vacuum and high voltage to achieve a film thickness of 30 nm. The samples were then imaged using a 20 KV electron beam (Tejraj et al., 1999).

In the present study controlled delivery system of microspheres of GBCM was prepared by Ionic gelation method using different polymers like xanthan gum, Eudragit RS 100 and sodium alginate. The pre and post formulation parameters for all the formulations were evaluated. Compatibility studies were performed by FTIR spectrophotometer. The IR spectrum of the pure drug with its IR spectrum of the physical mixture was studied. The characteristic absorption peaks of GBCM were obtained at wave numbers 3315, 3367, 2989, 2931, 2848, 1715, 1618, 1523, 1450, 1341, 1306, 1244, 1159, 1035, 820, 685 cm-1 and corresponding to the functional groups of N-H stretching of CONH, N-H stretching of CONH, C-H stretching of CH3, C-H stretching of CH2, C-H stretching of OCH3, C=O stretching, C=C ring stretching, C=C ring stretching, C-H bending, C-H bending, SO2, SO2, C-O, C-O-C, 1,4-disubstituted benzene, C-Cl, respectively. The obtained characteristic peaks of pure drug correlates with its peaks of physical mixture (Table 2). Hence, it was confirmed that the drug was compatible with the excipients used in the formulation. The DSC thermo gram study for drug and its formulations was also utilized for establishing physical characteristics. The DSC thermo gram of pure drug showed its sharp melting point at the temperature of 178.9°C, which indicates its sharp melting point. The DSC thermo gram of xanthan gum showed exothermic peak at 285.8°C, Eudragit RS 100 at 412°C and sodium alginate at 242.5°C. The DSC thermo gram of the best formulation (F8) showed drug peak at 176.1°C. Even though slightly differs in the nature and appearance, this endothermic peak is almost all near to 178.9°C. The comparative study of these two thermo grams, i.e. drug and its best formulation F8 showed the endothermic peak corresponding to the melting point of the drug. Hence, it indicated that there was no interaction between the drug with polymer and other excipients in best formulation (Fig. 1).

In vitro drug release studies The in vitro release profile of the microspheres was evaluated by USP type II dissolution test apparatus (paddle type assembly) (Lab India 8 basket dissolution apparatus, India) using phosphate buffer (pH 7.4) and maintained at 37 ± 0.5°C with the speed of agitation at 100 rpm. Accurately weighed the amount of microspheres equivalent to 10 mg of drug were placed in a vessel containing dissolution medium and the experiment was performed. At the prefixed time of intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 h), 5 mL of solution was withdrawn and the same volume was replaced with pH 7.4 phosphate buffer. After suitable dilution, samples were assayed spectrophotometrically for the drug content at 229 nm using a UV-Visible spectrophotometer (Elico, India). Kinetic studies For determination of in vitro drug release mechanism, the obtained drug release data was fitted to zero order, first order, Higuchi’s and KorsmeyerPeppas models (Venkateswarlu, 2013). Statistical analysis One-way and two-way ANOVA was applied as the test of tool using GraphPad Prism 6 and significance was set at p CIM). Para obtener concentraciones óptimas en los antibióticos β-lactámicos se utilizan las infusiones prolongadas o continuas, de esta manera la exposición del fármaco sobre el patógeno se logra en un mayor tiempo a través de concentraciones óptimas.

Aims: To evaluate the therapeutic response of β-lactam antibiotics in critically ill patients with prolonged infusions by applying the model PK/ PD. Methods: Prospective observational study (concurrent cohort), taking as a control non-concurrent historic cohort, conducted for a period of seven months in the intensive care unit of the Hospital Clínico San Borja Arriarán (HCSBA), Santiago, Chile. Results: It was found a significant difference in number of days of hospitalization in ICU for the group bolus versus infusion group (12.5 ± 5.4 vs. 18 ± 9.7 days, IC: 1.5-9.5; p = 0.009). Conclusions: This study suggests that there would be a therapeutic advantage in the use of prolonged infusion in ICU stay duration.

Objetivos: Evaluar la respuesta terapéutica de antibióticos β-lactámicos en el paciente crítico, con infusiones prolongadas, mediante la aplicación del modelo PK/PD. Métodos: Se realizó un estudio observacional prospectivo (cohorte concurrente), por un periodo de siete meses tomando como control una cohorte no concurrente histórica, en la unidad de pacientes críticos del Hospital clínico San Borja Arriarán (HCSBA), Santiago, Chile. Resultados: Se obtuvo una diferencia significativa en días cama de hospitalización en la unidad de cuidados intensivos (UCI), a favor del grupo infusión versus el grupo bolo (12,5 ± 5,4 vs. 18 ± 9,7 días, IC: 1,5-9,5; p = 0,009). Conclusiones: Este estudio sugiere que existe una ventaja terapéutica en lo que respecta a la infusión prolongada, principalmente en días cama UCI.

Keywords: critically ill patients; β-lactam antibiotics; prolonged infusion.

Palabras Clave: antibióticos β-lactámicos; infusión prolongada; paciente crítico.

ARTICLE INFO Received | Recibido: September 2, 2016. Received in revised form | Recibido en forma corregida: October 15, 2016. Accepted | Aceptado: October 21, 2016. Available Online | Publicado en Línea: December 8, 2016. Declaration of interests | Declaración de Intereses: The authors declare no conflict of interest. Funding | Financiación: The authors confirm that the project has no funding or grants. Academic Editor | Editor Académico: Gabino Garrido.

_____________________________________

Amador et al.

INTRODUCCIÓN Los antimicrobianos son los fármacos más comúnmente prescritos en el manejo de los pacientes en estado crítico, en el caso de sepsis o choque séptico el uso apropiado y temprano de estos medicamentos ha demostrado disminuir la morbimortalidad (Ibrahim et al., 2000; Vargese et al., 2011). En el paciente crítico, se han descrito alteraciones en todas las fases de la farmacocinética modificando la respuesta a fármacos (Pea et al., 2005; Smith et al., 2012). Entre estas alteraciones se encuentran la absorción intestinal disminuida debido a alteraciones hemodinámicas producidas por el shock y el uso de drogas vasoactivas; el volumen de distribución aumentado por la compensación de la hipovolemia y fuga de proteínas desde la sangre a tejidos; el metabolismo variado debido a alteraciones en la actividad enzimática, concentración de proteínas séricas y flujo hepático, traduciéndose en un menor aclaramiento de medicamentos que requieran este órgano y; la eliminación variable que ocurre en pacientes con quemaduras extensas y etapa temprana de la sepsis, en los que se genera un aumento del aclaramiento renal con disminución de los niveles séricos de fármacos. Mientras que en trauma, falla multiorgánica, choque cardiogénico o hipovolémico y etapas posteriores de la sepsis, pueden generar fallas o injurias renales que lleva a que el aclaramiento se vea disminuido. Estas variables farmacocinéticas deben ser consideradas en todo momento e individualizadas para cada paciente, ya que, de lo contrario, se tiende a estandarizar un mismo régimen de dosificación que por lo general es sub-terapéutico, contribuyendo de esta manera a la presión antibiótica y resistencia bacteriana (Roberts et al., 2008; Mounton et al., 2012). Dentro del marco de la resistencia, la ausencia de nuevos antimicrobianos y las variaciones en la concentración de fármacos en el paciente crítico, surge el modelo FC/FD o Farmacocinética/Farmacodinamia (más conocido por sus siglas en inglés PK/PD) (Park et al., 1998). Dicho modelo separa a los antimicrobianos en tres clases, según las concentraciones libres de antimicrobiano que se deben lograr para obtener una máxima eficacia, según tres índices farmacocinéticos: Concentración Máxima (Cmáx), Área Bajo la Curva (ABC) y Tiempo de exhttp://jppres.com/jppres

Evaluación de infusiones prolongadas en paciente crítico

posición del fármaco (T); y uno farmacodinámico: Concentración inhibitoria mínima bacteriana (CIM) (Robert et al., 2006). En los antibióticos concentración dependiente (Cmáx/CIM) es necesario alcanzar una alta concentración de fármaco en dosis única. En general, los fármacos pertenecientes a este grupo presentan un marcado efecto post-antibiótico que permite ejercer su efecto farmacológico a pesar de que las concentraciones sanguíneas estén bajo la CIM bacteriana. Dentro de esta clase destacan los aminoglucósidos, en los que el uso de dosis única diaria de esta familia, ejemplifica este modelo (Robert et al., 2006). Los antibióticos área bajo la curva dependiente (ABC/CIM) se comportan tanto como concentración y tiempo dependiente, en los que se necesita un valor específico de ABC, según la sensibilidad bacteriana para lograr el efecto máximo. Los glicopéptidos, como la vancomicina, se encuentran dentro de esta clasificación (Robert et al., 2006). En los antibióticos tiempo dependiente (Tiempo/CIM) se requiere un tiempo de exposición determinado a través de un régimen de dosificación, para que la concentración del fármaco esté por sobre la concentración inhibitoria mínima bacteriana. El tiempo necesario es característico para cada antimicrobiano. El grupo de los antibióticos βlactámicos son la familia más representativa de esta clase (Robert et al., 2006). Dentro de los antibacterianos, los antibióticos βlactámicos son los más frecuentemente prescritos, teniendo indicación en infecciones de la piel y tejidos blandos, infecciones de las vías respiratorias, endocarditis bacteriana, infecciones del sistema nervioso central, intra-abdominales, del tracto urinario, osteoarticulares, sepsis de origen no definido, neutropenia febril, entre otras (Suarez y Gudiol, 2009). Según el modelo, para lograr su máximo efecto bactericida, es necesario que la concentración libre de esta familia de antibióticos se mantenga constante por un amplio intervalo de tiempo sobre la CIM. Este objetivo es posible lograrlo con la utilización de infusiones continuas y/o prolongadas (Goue et al., 2009; Vargese et al., 2011; Abdul-Aziz et al., 2012). A pesar de existir mucha literatura que respalda el uso de infusiones continuas y/o prolongadas, no se encontraron estudios publicados con las mismas J Pharm Pharmacogn Res (2017) 5(2): 89

Amador et al.

características a nivel local, por lo que nace el interés de conocer la realidad local en un hospital clínico docente asistencial de Chile. Debido a la limitada data clínica, al amplio uso de los agentes antimicrobianos, su enorme variabilidad en la farmacocinética en el paciente crítico y a la necesidad de usar correctamente los antibióticos, se estableció como pregunta de investigación: Si en pacientes en UCI con indicación de terapia antibiótica con β-lactámicos, la administración por infusión prolongada, en comparación a la administración en bolo, presenta mejor respuesta farmacológica con respecto a mortalidad y días camas de hospitalización (Roberts et al., 2009; Gonzalves-Pereira y Póvoa, 2011; Sinnollareddy et al., 2012). MATERIALES Y MÉTODOS Se realizó un estudio observacional prospectivo (cohorte concurrente: que se mencionará como “grupo cohorte”), desde diciembre de 2012 hasta julio de 2013, en la Unidad de Paciente Crítico del HCSBA, Santiago, Chile. Se recolectaron datos de pacientes con indicación médica de tratamiento antibiótico con fármacos β-lactámicos. Dichos antibióticos fueron infundidos según el protocolo de administración por infusión prolongada, el que indicó la forma en que debía reconstituirse el medicamento a utilizar, la dilución y en cuánto tiempo debían ser administrados. Los antimicrobianos β-lactámicos que fueron infundidos en tres horas fueron cefazolina, ceftazidima, cefepime, imipenem/cilastatina, meropenem y ertapenem, y en cuatro horas para piperacilina/tazobactam. El grupo cohorte se comparó con controles históricos (cohorte no concurrente: que se mencionará como “grupo control”). Para este último, se recopiló información sobre pacientes previo a la implementación del protocolo de infusión prolongada, entre los años 2010 - 2011, revisando las terapias de enfermería almacenadas en la UCI y/o unidad de archivos del HCSBA. Para los criterios de inclusión/exclusión de pacientes, utilización y recolección de datos de documentos clínicos, se contó con la aprobación del Comité Ético-Científico del Servicio de Salud Metropolitano Central, bajo Resolución Exenta Nº 1303, certificado y aprobado en sesión plenaria. Se manhttp://jppres.com/jppres


tuvo la confidencialidad de la identidad de los pacientes y los datos en todo momento. En los criterios de inclusión se tuvieron en cuenta los pacientes ingresados por más de 48 h en la UCI, mayores de 16 años con uso e indicación médica de antibióticos β-lactámicos a lo menos por 48 h en la unidad, con microorganismo aislado y sensible al antibiótico prescrito. Los criterios de exclusión comprendieron a pacientes con insuficiencia renal crónica o aguda en hemodiálisis (aclaramiento de creatinina según Cockcroft Gault < 30 mL/min), pacientes con bacteria aislada resistente al antibiótico utilizado y pacientes que hubieran usado antibióticos en infusión prolongada previo al estudio. El ajuste de dosificación, según la función renal, fue realizado de acuerdo a las guías estándares de monitorización utilizadas por los tratantes. Con los datos obtenidos se analizaron las siguientes variables: sexo, edad, diagnóstico de ingreso, APACHE II, días cama UCI, mortalidad general, mortalidad a 30 días, antibióticos utilizados y microorganismos aislados. Análisis estadístico Los datos fueron tabulados y analizados mediante el programa SPSS v17.0. Para determinar la distribución de los valores se utilizó la prueba de normalidad (Kolmogorov-Smirnov y Shapiro-Wilk). Dada la distribución de los datos se utilizó un test paramétrico para comparar medias (t de Student). Las variables cualitativas se analizaron con el programa Open-epi utilizando la prueba de Fisher de 2 colas. Se consideró como significativo un valor de p0,99

Sepsis y choque séptico

26,7%

20,0%

0,76

Postoperatorio

16,7%

16,7%

-

Síndrome convulsivo

10,0%

6,7%

>0,99

Infección urinaria

6,7%

10,0%

>0,99

Cardiovascular

0,0%

3,3%

-

Otros

16,7%

16,7%

-

Motivo de ingreso UCI

Valor p: 95 % confianza, test exacto de Fisher 2 colas

tratamiento de antibióticos β-lactámicos por infusión prolongada, de los cuales solo 30 cumplieron los criterios de inclusión. En el grupo control se identificó la presencia de 70 pacientes con terapia de antibióticos β-lactámicos en el periodo de dos años, previamente definidos. Luego de revisar las fichas clínicas médicas y de enfermería se obtuvieron los datos de 30 pacientes que cumplieron los criterios de inclusión. La Tabla 1 muestra las características generales de los pacientes incluidos en el estudio, tales como la distribución por sexo en cada grupo, mediana de edad, clasificación de gravedad APACHE II, días cama de hospitalización en UCI y criterios de ingreso a la unidad. El ingreso por complicaciones respiratorias incluyó los criterios de necesidad de ventilación mecánica invasiva, neumonía adquirida en la comunidad y asociada a ventilación mecánica. El ingreso por infecciones urinarias involucró a infecciones complicadas del tracto urinario alto. Los pacientes post-operados incluyeron complicaciones abdominales, principalmente. El mayor porcentaje de ingresos en el estudio correspondió a pacientes con sepsis, ingresos de causa respiratoria y post quirúrgicos, siendo mayor a 63,4% de los motivos de ingreso para ambos grupos estudiados. http://jppres.com/jppres

La mayoría de las infecciones tratadas fueron neumonías (55% vs. 44%, p=0,44. Data no mostrada) e infecciones urinarias (24% vs. 25%, p=0,99. Data no mostrada). El microorganismo aislado más común fue Pseudomonas aeruginosa, el que correspondió al 35% de todas las bacterias encontradas en ambos grupos. Dentro de los antibióticos, no se observaron diferencias entre los grupos en lo que respecta a terapia empírica, concomitante, ni en los -lactámicos utilizados. Piperacilina/tazobactam fue el más utilizado (45% vs. 40%, p=0,88). El análisis estadístico de días cama y mortalidades se muestra en la Tabla 2, dividido en la estadística de toda la población estudiada y el subgrupo pareado. Se observa en la población una diferencia significativa en lo que respecta a los días cama, teniendo un promedio de estadía en UCI de 5,5 días menos en el grupo con infusión prolongada (12,5 vs. 18; IC: 1,5 - 9,5; p=0,009). Se observa también una diferencia significativa en el grupo etario, siendo mayor en los pacientes del grupo bolo (48,8 vs 59,7; IC: 1,3 20,5; p = 0,027). En relación a la mortalidad en UCI, esta fue mayor en el grupo control (27%) comparada con el 10% del grupo infusión (IC: 0,1-1,3; p=0,18). El análisis multivariado arrojó que los factores que influyeron J Pharm Pharmacogn Res (2017) 5(2): 91

Amador et al.


en la mortalidad fueron el criterio de ingreso, sepsis y choque séptico, el número de bacterias aisladas y la edad. En el grupo pareado por edad se observó que las edades no difirieron estadísticamente con 54,8 vs. 54,1 (IC: -13,25 - 11,95; p=0,76), pero se observó una diferencia estadísticamente significativa en los valo-

res de días cama UCI con 11,9 vs. 18,4 días (IC: 1,1 11,9; p=0,027). El costo de hospitalización diario en la UCI del HCSBA (sin considerar insumos, medicamentos y valor hora del personal de salud) fue de, aproximadamente, CLP 300.000. Con la diferencia de 5,5 días cama se generó un ahorro promedio de CLP 1.650.000.

Tabla 2. Datos estadísticos de la muestra estudiada. Parámetro

Control

Cohorte

IC95%

p

RR

DR

NNT

Días cama UCI

18 ± 9,7

12,5 ± 5,4

1,5 - 9,5

0,009

_

_

_

Edad (años)

59,7 ± 17,7

48,8 ± 19,6

1,3 - 20,5

0,027

_

_

_

Mortalidad en UCI

27 %

10%

0,1 - 1,3

0,18

0,37

0,17

6

Mortalidad a 30 días

14 %

4%

0,03 - 2,7

0,33

0,27

0,09

10

Datos estadísticos de la muestra pareada por edad Días cama UCI

18,4 ± 10,3

11,9 ± 6,1

1,1 - 11,9

0,027

_

_

_

Edad (años)

54,1 ± 18,9

54,8 ± 20,6

-13,25 – 1,95

0,76

_

_

_

Mortalidad en UCI

20%

10%

0,1 – 2,4

0,66

0,5

0,1

10

Mortalidad a 30 días

6,3%

0%

_

0,48

-

0,06

16

Los datos son expresados como medias ± desviación estándar. IC 95%: intervalo de confianza del 95%, RR: riesgo relativo, DR: diferencia de riesgo, NNT: número necesario a tratar. p: calculado por la prueba exacta de Fisher.

DISCUSIÓN En este estudio se excluyó la ceftriaxona y ampicilina-sulbactam, también de uso común en la unidad. La justificación recae en que la ceftriaxona difiere en sus características farmacocinéticas con los demás miembros de su familia (Perry et al., 2011) y no presenta reportes con respecto a la administración por infusión prolongada, pero sí por infusión continua (Roberts et al., 2007a;b). Por otra parte, la ampicilina-sulbactam es utilizada en la unidad para tratar principalmente infecciones por Acinetobacter baumanni multiresistente, donde la sensibilidad está dirigida hacia el inhibidor de betalactamasas y no para el antibiótico (Betrosian et al., 2008), por lo que no existen investigaciones sobre qué forma de administración presenta mayores ventajas para el sulbactam. Los antibióticos estudiados fueron adquiridos a los mismos proveedores a lo largo del tiempo por lo http://jppres.com/jppres

que se asume la misma preparación y composición química de los fármacos y excipientes. Si bien algunos fármacos tienen la estabilidad suficiente para ser administrados por infusión continua, la dificultad de plantear este método en pacientes críticos recae en la necesidad de una vía exclusiva para este propósito. La infusión prolongada evita esta problemática puesto que da tiempo suficiente para que otros medicamentos utilicen la misma vía venosa. Es por ello que se privilegió esta forma de administración en el protocolo de infusión prolongada. Según los resultados de esta investigación, no existen diferencias estadísticamente significativas en los grupos muestreados según sexo, motivos de ingreso ni utilización de antibióticos. Observándose una ventaja en el uso de las infusiones prolongadas de los antibióticos β-lactámicos, principalmente en días cama de hospitalización en UCI, sin obtener ventajas significativas de mortalidad en la unidad ni en mortalidad a 30 días. J Pharm Pharmacogn Res (2017) 5(2): 92

Amador et al.


Los días cama promedios en la UCI del hospital por neumonías, sepsis y complicaciones postquirúrgicas, son 21 días. Como estos ingresos representan la mayoría de los pacientes recopilados (63,4%), se evidencia un alto promedio de días cama en los grupos. Lodise et al. (2007) demostraron una disminución, tanto en días cama como en mortalidad, para la infusión extendida de piperacilina/tazobactam en pacientes con una clasificación de gravedad APACHE II >17, infectados por Pseudomonas aeruginosa. Si bien se mantiene en el presente trabajo la disminución en días cama, la mortalidad en UCI no logró ser significativa. A pesar que la piperacilina/tazobactam fue el antibiótico β-lactámico más utilizado como tratamiento empírico nosocomial, debido a que se encuentra aprobado por el comité de infecciones asociadas a la atención en salud del HCSBA, es importante destacar que también fueron utilizados ceftazidima, cefepime, imipenem y meropenem en infusiones prolongadas. Existen varios trabajos con cefalosporinas y carbapenémicos, los que han demostrado mejor respuesta terapéutica a través de modelos farmacocinéticos por la administración de infusiones prolongadas y/o continuas, incluso para distintos gérmenes (Lomaestro y Drusano, 2005; Deryke et

El período de estacionalidad pudo también afectar los resultados, ya que en la cohorte los pacientes estuvieron en la temporada verano-otoño, mientras que el control fue más representativo por abarcar dos años. El número necesario de pacientes en un estudio ideal para lograr una potencia estadística de 80% y confirmar los resultados obtenidos es de 92 pacientes por grupo, lo que no es una cifra imposible de lograr en un estudio de seguimiento más prolongado o en su defecto incorporando más centros reclutadores. Finalmente, es importante señalar que las últimas y más importantes publicaciones lideradas por los principales investigadores a nivel internacional, siguen respaldando el individualizar los tratamientos antibióticos, usar infusiones prolongadas y/o extendidas, así como considerar las variables farmacocinéticas y farmacodinámicas sobre todo en el manejo de los pacientes críticamente enfermos. Estas estrategias respaldan la optimización de la antibiótico-terapia, ayudan a disminuir la resistencia bacteriana por presión antibiótica y contribuiría a prolongar la vida útil de los antimicrobianos, considerando el cada vez más restringido arsenal terapéutico debido a la generación de microorganismos multi-resistentes (Arnold et al., 2013; Bauer et al., 2013;

al., 2007; Sakka et al., 2007; Nicolau, 2008; Hubert et al., 2009; Kiratisin et al., 2013). Sin embargo, ninguno de ellos

Dulhunty et al., 2013; 2015; Falagas et al., 2013; MacVane et al., 2014; Roberts et al., 2014; Teo et al., 2014; Abdul-Aziz et al., 2016).

tuvo un impacto similar, desde el punto de vista de morbi-mortalidad y económico de manera simultánea, como la piperacilina/tazobactam (Lodise et al., 2007). Dentro de los sesgos de este estudio, la edad fue la variable confundente más importante de acuerdo a los resultados entre ambos grupos, por lo que se tomó una muestra de la población considerando solo 20 pacientes por cada grupo pareándolos por edad. Para ello, se seleccionaron pacientes con similares características o en su defecto los que tuvieran una clasificación de gravedad APACHE II mayor. Debido a esto se pudo disminuir la diferencia etaria a costa de una menor potencia estadística de 39% a 14%. Cabe mencionar que si bien el pareamiento es un método útil para eliminar sesgos en los estudios, la selección de los pacientes genera una idealización lo que se traduce en un grupo menos representativo de la población real. http://jppres.com/jppres

CONCLUSIONES Este estudio piloto sugiere que existiría una ventaja terapéutica en lo que respecta a la infusión prolongada de antibióticos β-lactámicos en pacientes críticos, principalmente en ocupación de días cama. Sin embargo, se hace necesario realizar un estudio prospectivo aleatorizado con un número más amplio de pacientes, idealmente multi-céntrico. De este modo, se podrían confirmar estos resultados con respecto a la optimización de la antibióticoterapia, relacionado directamente a la morbimortalidad en el paciente crítico. CONFLICTO DE INTERÉS Los autores declaran que no existe conflicto de interés.


Amador et al.

AGRADECIMIENTOS Los autores agradecen al Dr. Juan Fulla, Urólogo y Magíster en Biología de la Reproducción, por su colaboración en el análisis de los datos estadísticos.

REFERENCIAS Abdul-Aziz MH, Dulhunty JM, Bellomo R, Lipman J, Roberts JA (2012) Continuous beta-lactam infusion in critically ill patients: the clinical evidence. Ann Intensive Care 2(1): 37. Abdul-Aziz MH, Sulaiman H, Mat-Nor MB, Rai V, Wong KK, Hasan MS, Abd Rahman AN, Jamal JA, Wallis SC, Lipman J, Staatz CE, Roberts JA (2016) Beta-Lactam Infusion in Severe Sepsis (BLISS): a prospective, two-centre, openlabelled randomised controlled trial of continuous versus intermittent beta-lactam infusion in critically ill patients with severe sepsis. Intensive Care Med 42(10): 1535-1545. Arnold HM, Hollands JM, Skrupky LP, Smith JR, Juang PH, Hampton NB, McCormick S, Reichley RM, Hoban A, Hoffmann J, Micek ST, Kollef MH (2013) Prolonged infusion antibiotics for suspected gram-negative infections in the ICU: a before-after study. Ann Pharmacother 47: 170180. Bauer KA, West JE, O'Brien JM, Goff DA (2013) Extendedinfusion cefepime reduces mortality in patients with Pseudomonas aeruginosa infections. Antimicrob Agents Chemother 57: 2907-2912. Betrosian AP, Frantzeskaki F, Xanthaki A, Douzinas EE (2008) Efficacy and safety of high-dose ampicillin/sulbactam vs colistin as monotherapy for the treatment of multidrug resistant Acinetobacter baumannii ventilator-associated pneumonia. J Infect 56(6): 432-436. Deryke CA, Kuti JL, Nicolau DP (2007) Re-evaluation of current susceptibility breakpoints for gram-negative rods based on pharmacodynamic assessment. Diagn Microbiol Infect Dis 58: 337-344. Dulhunty JM, Roberts JA, Davis JS, Webb SA, Bellomo R, Gomersall C, Shirwadkar C, Eastwood GM, Myburgh J, Paterson DL, Lipman J (2013) Continuous infusion of betalactam antibiotics in severe sepsis: a multicenter double blind, randomized controlled trial. Clin Infect Dis 56: 236244. Dulhunty JM, Roberts JA, Davis JS, Webb SA, Bellomo R, Gomersall C, Shirwadkar C, Eastwood GM, Myburgh J, Paterson DL, Starr T, Paul SK, Lipman J; BLING II Investigators for the ANZICS Clinical Trials Group (2015) A multicenter randomized trial of continuous versus intermittent β-lactam infusion in severe sepsis. Am J Respir Crit Care Med 192: 1298-1305. Falagas ME, Tansarli GS, Ikawa K, Vardakas KZ (2013) Clinical outcomes with extended or continuous versus short-term intravenous infusion of carbapenems and piperacillin/tazobactam: a systematic review and meta-analysis. Clin Infect Dis 56: 272-282. Gonzalves-Pereira J, Póvoa P (2011) Antibiotics in critically ill patients: a systemic review of the pharmacokinetics of βlactam. Critical Care 15: R206. http://jppres.com/jppres


Goue DG, Jun L, Fahima N (2009) Antimicrobial breakpoint estimation accounting for variability in pharmacokinetics. Theor Biol Med Model 6: 10. Hubert D, Le Roux E, Lavrut T, Wallaert B, Scheid P, Manach D, Grenet D, Sermet-Gaudelus I, Ramel S, Cracowski C, Sardet A, Wizla N, Deneuville E, Garraffo R (2009) Continuous versus intermittent infusions of ceftazidime for treating exacerbation of cystic fibrosis. Antimicrob Agents Chemother 53: 3650-3656. Ibrahim EH, Sherman G, Ward S, Fraser VJ, Kollef MH (2000) The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the UCI setting. Chest 118 (1): 146-55. Kiratisin P, Keelb RA, Nicolau D (2013) Pharmacodynamic profiling of doripenem, imipenem and meropenem against prevalent Gram-negative organisms in the Asia-Pacific región. Int J Antimicrob Agents (41): 47– 51. Lodise TP Jr, Lomaestro B, Drusano GL (2007) Piperacillintazobactam for Pseudomonas aeruginosa infection: Clinical implications of an extended-infusion dosing strategy. Clin Infect Dis 44(3): 357-63. Lomaestro BM, Drusano GL (2005) Pharmacodynamic evaluation of extending the administration time of meropenem using a Monte Carlo simulation. Antimicriob Agents Chemother 49: 461-463. MacVane SH, Kuti JL, Nicolau DP (2014) Prolonging β-lactam infusion: a review of the rationale and evidence, and guidance for implementation. Int J Antimicrob Agents 43: 105113. Mounton JW, Brown DFJ, Apfalter P, Cantón R, Giske CG, Ivanova M, MacGowan AP, Rodloff A, Soussy CJ, Steinbakk M and Kahlmeter G (2012) The role of pharmacokinetics/pharmacodynamics in setting clinical MIC breakpoints: the EUCAST approach. Clin Microbiol Infect 18: E37-45. Nicolau D (2008) Pharmacodynamic optimization of β-lactams in the patient care setting. Crit Care 12(suppl4): S2. Park K, Verotta D, Gupta SK, Sheiner LB (1998) Use of pharmacokinetic/pharmacodynamic model to design an optimal dose input profile. J Pharmacokinet Biopharm 26(4): 471-492. Pea F, Viale P, Furlanut M (2005) Antimicrobial therapy in critically ill patients: A review of pathophysiological conditions responsible for altered disposition and pharmacokinetic variability. Clin Pharmacokinet 44(10): 1009-1034. Perry TR, Schentag JJ (2001) Clinical use of ceftriaxone, a pharmacokinetic-pharmacodynamic perspective on the impact of minimum inhibitory concentration and serum protein binding. Clin Pharmacokinet 40(9): 685-694. Roberts JA, Boots R, Rickard CM, Thomas P, Quinn J, Roberts DM, Richards B, Lipman J (2007b) Is continuous infusion ceftriaxone better than once-a-day dosing in intensive care? A randomized controlled pilot study. J Antimicrob Chemother 59(2): 285-291. Roberts JA, Kruger P, Paterson DL, Lipman J (2008) Antibiotic resistance — What’s dosing got to do with it? Crit Care Med 36: 2433–2440. Roberts JA, Lipman J (2006) Antibacterial dosing in intensive care, pharmacokinetics, degree of disease and J Pharm Pharmacogn Res (2017) 5(2): 94

Amador et al.


pharmacodynamics of sepsis. Clin Pharmacokinet 45(8): 755-773. Roberts JA, Paratz J, Paratz E, Krueger WA, Lipman J (2007a) Continuous infusion of beta-lactam antibiotics in severe infections: a review of its role. Int J Antimicrob Agents 30(1): 11-18. Roberts JA, Paul SK, Akova M, Bassetti M, De Waele JJ, Dimopoulos G, Kaukonen KM, Koulenti D, Martin C, Montravers P, Rello J, Rhodes A, Starr T, Wallis SC, Lipman J; DALI Study (2014) DALI: defining antibiotic levels in intensive care unit patients: are current beta-lactam antibiotic doses sufficient for critically ill patients? Clin Infect Dis 58: 1072-1083. Roberts JA, Webb S, Paterson D, Ho KM, Lipman J (2009) A systemic review on clinical benefits of continuous administraction of β-lactam antibiotics. Crit Care Med 37(6): 2071-2078. Sakka SG, Glauner AK, Bulitta JB, Kinzig-Schippers M, Pfister W, Drusano G, Sorgel F (2007) Population Pharmacokinetics and pharmacodynamics of continuous versus short-

term infusion of imipenem-cilastatin in critically III patients in a randomized, controlled trial. Antimicrob Agents Chemother 51: 3304–3310. Sinnollareddy MG, Roberts MS, Lipman J, Roberts JA (2012) βlactam pharmacokinetics and pharmacodynamics in critically ill patients and strategies for dose optimization: A structured review. Clin Exp Pharmacol Physiol 39(6): 489496. Smith BS, Yogaratnam D, Levasseur-Franklin KE, Forni A, Fong J (2012) Introduction to drug pharmacokinetics in the critically ill patient. Chest 141(5): 1327-1336. Suárez C, Gudiol F (2009) Antibióticos betalactámicos. Enferm Infecc Microbiol Clin 27(2): 116-129. Teo J, Liew Y, Lee W, Kwa AL (2014) Prolonged infusion versus intermittent boluses of β-lactam antibiotics for treatment of acute infections: a meta-analysis. Int J Antimicrob Agents 43: 403. Varghese J, Roberts J, Lipman J (2011) Antimicrobial PK and PD issues in the critically ill with severe sepsis and septic shock. Crit Care Clin 27(1): 19-34.

_________________________________________________________________________________________________________________

Author contributions: Contribution

Amador JS

Concepts or Ideas

X

Design

X

Definition of intellectual content

X

X

Morales AA

Cortes CP

X

X

X

X

Literature search Clinical trial

X

Experimental studies

X

X X

X X

X

Data acquisition Data analysis

Carrasco JP

X

Statistical analysis

X

X

X

X

Manuscript preparation

X

X

Manuscript editing

X

X

Manuscript review

X

X

Citation Format: Amador JS, Carrasco JP, Morales AA, Cortes CP (2017) Evaluación terapéutica de infusiones prolongadas de antibióticos β-lactámicos en el tratamiento y manejo del paciente crítico. [Therapeutic evaluation of prolonged infusions of β-lactam antibiotics in the treatment and management of critically ill patients]. J Pharm Pharmacogn Res 5(2): 88-95.



© 2017 Journal of Pharmacy & Pharmacognosy Research, 5 (2), 96-105, 2017 ISSN 0719-4250 http://jppres.com/jppres Original Article | Artículo Original

In vitro release of dibucaine hydrochloride from chitosan semisolid vehicles: emulsion and hydrophilic gels [Liberación in vitro del clorhidrato de dibucaína desde vehículos semisólidos con quitosana: emulsionados y geles hidrofílicos] Nilia de la Paz1, Dania Pérez2, Mirna Fernández2, Caridad M. García1, Vivian Martínez1, Antonio Nogueira1, Oscar García3 1Centro

de Investigación y Desarrollo de Medicamentos (CIDEM). Ave. 26 # 1605 e/ Puentes Grandes y Boyeros. La Habana, Cuba. de Farmacia y Alimentos (IFAL). Universidad de la Habana. Calle 23 No. 21425 e/ 214 y 222. La Habana. Cuba. 3Empresa Laboratorio Farmacéutico Roberto Escudero Díaz. Calle 20 de Mayo No. 540 esq. Marta Abreu. La Habana, Cuba. 2Instituto

*E-mail: [email protected]

Abstract

Resumen

Context: Chitosan has received attention as a functional, sustainably renewable, nontoxic and biodegradable biopolymer for pharmaceutical applications.

Contexto: La quitosana ha recibido gran atención al ser un biopolímero funcional, biodegradable, renovable y no tóxico con múltiples aplicaciones farmacéuticas.

Aims: To evaluate the release of dibucaine hydrochloride from semisolid vehicles of oil/aqueous type emulsion and aqueous gels, stabilized by using chitosan (CH) or chitosan acetate (CHAc).

Objetivos: Evaluar la liberación del clorhidrato de dibucaína desde vehículos semisólidos emulsionados aceite/agua y geles acuosos, estabilizados con quitosana (CH) o acetato de quitosana (CHAc).

Methods: Emulsions were developed by varying the emulsifying agent: polysorbate 80, CH or CHAc and by combining CH with polysorbate 80 or CHAc with polysorbate 80. The hydroxypropylmethyl cellulose F4M was added as a stabilizing agent in gel formulations. The release rates of model drug from semisolid vehicles were measured by using a dialysis sac. Drug release was also quantified by using a validated UV-VIS spectrophotometric method.

Métodos: Las emulsiones fueron elaboradas variando el agente emulsificante: polisorbato 80, CH o CHAc, o las combinaciones de CH o CHAc con polisorbato 80, respectivamente. La hidroxipropilmetil celulosa F4M se adicionó como viscosante en el gel. La liberación del fármaco modelo, se realizó empleando bolsas de membranas de diálisis. En la cuantificación del fármaco se utilizó un método espectrofotométrico validado.

Results: The pH values showed minimal changes for emulsion and gel formulations. The drug is a cationic salt, and it is not able to bind polymer cations by electrostatic repulsion. The rheological property of the vehicle type emulsion was adjusted to plastic and pseudo-plastic fluid to the gels. The drug release was independent of the viscosity of vehicles. Dibucaine release from both types of formulation was found to follow a square-root-of-time kinetic model, but a higher rate of release was obtained from gel formulations.

Resultados: Los valores de pH mostraron variaciones mínimas en los sistemas emulsionados y geles acuosos. Al ser el fármaco una sal catiónica existe repulsión electrostática con el biopolímero. Los vehículos emulsionados mostraron comportamiento de flujo plástico mientras que los geles pseudoplástico. La liberación de la dibucaína fue independiente de la viscosidad de los vehículos semisólidos. El perfil de liberación, desde ambos sistemas, se ajustó al modelo cinético de la raíz cuadrada del tiempo, siendo la velocidad mayor desde los geles acuosos.

Conclusions: It was shown that chitosan was adsorbed to the surface of polysorbate 80-coated droplets, and that the electrostatic attraction between the non-ionic surfactant and the drug retarded its release from a semisolid system. The multilayer emulsions showed more influence of the release of drug than CH or CHAc single layer emulsion.

Conclusiones: Se demostró que la quitosana fue adsorbida en la superficie de las gotículas cubiertas con polisorbato 80, y la interacción electrostática entre el surfactante no iónico y el fármaco retardó su liberación desde los sistemas semisólidos. Las combinaciones de emulgentes mostraron mayor influencia sobre la liberación del fármaco que los estabilizados con CH o CHAc.

Keywords: dibucaine hydrochloride; chitosan; chitosan acetate; release; semisolid vehicles.

Palabras Clave: acetato de quitosana; clorhidrato de dibucaina; liberación; quitosana; vehículos semisólidos.

ARTICLE INFO Received | Recibido: September 8, 2016. Received in revised form | Recibido en forma corregida: November 17, 2016. Accepted | Aceptado: December 10, 2016. Available Online | Publicado en Línea: December 13, 2016. Declaration of interests | Declaración de Intereses: The authors declare no conflict of interest. Funding | Financiación: The authors confirm that the project has no funding or grants. Academic Editor | Editor Académico: Gabino Garrido.

_____________________________________

De la Paz et al.

INTRODUCTION Chitosan has received considerable attention as a functional, sustainably renewable, nontoxic and biodegradable biopolymer for diverse applications, especially in pharmaceutics, food and cosmetics (Kumar et al., 2004). Natural polymers are the product of living organisms and are often readily available, sustainably renewable, and possess better biocompatibility and biodegradability, low or no toxicity, and a higher modification capability compared to various synthetic materials (Korkiatithaweechai et al., 2011). At relatively low pH (< 6.5), it is positively charged and tends to be soluble in dilute aqueous solutions, but at higher pH it tends to lose its charge and may precipitate from solution. Because of its polymeric cationic characteristics can interact with negatively charged molecules or polymers (Boonsongrit et al., 2006; Rani et al., 2010; Geetha et al., 2011; Starýchová et al., 2014).

Several papers describe the stabilization of o/w emulsion where chitosan is adsorbed on oil surfaces through interaction with an added anionic surfactant or protein (Ogawa et al., 2003a;b; Payet and Terentjev, 2008). Owing to its cationic properties is able to interact with anionic agents and form a waterinsoluble barrier that participates in the release of a drug (Avila et al., 2010; Szymańska and Winnicka, 2012; Starýchová et al., 2014). Chitosan has been shown to be useful in the preparation of stable emulsions without any other surfactant (Schulz et al., 1998; Del Blanco et al., 1999; Rodríguez et al., 2002), together with anionic or nonionic surfactants (Jumaa and Müller, 1999; Jumaa et al., 2002). Under certain conditions, chitosan adsorption promotes droplet flocculation by acting as a “bridge” that links two or more droplets together, and interacts with the adsorbed surfactant to form interfacial complexes that improve emulsion stability (Mun et al., 2005). Is an amphiphilic polyelectrolyte, which combines two stabilization mechanisms of emulsions: electrosteric and viscosifying effect. Its emulsification properties are proportional to chitosan concentration (Rodríguez et al., 2002). Our previous studies involved the optimization of the alkaline N–deacetylation process to obtain chitosan from lobster chitin (Panulirus argus), http://jppres.com/jppres

Dibucaine release from chitosan semisolid vehicles

which physical-chemical and microbiological characteristics were in good agreement with the limits of chitosan-like pharmaceutical excipients (de la Paz et al., 2012; 2013). We demonstrated that chitosan can be successfully salified with acetic and lactic acids by means of spray drying (Fernández Cervera et al., 2011) showing an adequate stability (de la Paz et al., 2015). Dibucaine hydrochloride is a strong cationic salt with anesthetic activity. It is available in rectal, creams and ointments formulations (Martindale, 2009; USP 36, 2013). Dibucaine hydrochloride was chosen as the model drug for the release study, considering that it is a cationic drug and it is employed in semisolid dosage forms of topical use. The objective of this study was to evaluate the release of dibucaine hydrochloride from semisolid vehicles of oil/aqueous type emulsion and hydrophilic gels, by using chitosan or chitosan acetate as an emulsifying and viscosity-increasing agent. There is no history of similar studies with chitosan derived from lobster chitin. MATERIAL AND METHODS Chemicals Chitosan (CH), deacetylation degree (DD) of 79.90%, was prepared on an industrial scale in a Cuban facility. The DD was determined using a previously-validated potentiometric method (de la Paz et al., 2012). Spray-dried chitosan acetate (CHAc) with a deacetylation molar degree of 57.10% was produced at an industrial plant spray dryer (de la Paz et al., 2015). Dibucaine hydrochloride (DH) (Dolder AG., Switzerland), acetic and citric acids and disodium hydrogen phosphate were purchased from Merck, Germany. Polysorbate 80 (Dizhong, China), propylene glycol (Basf, Germany), cetyl alcohol (Ecogrem, Spain), mineral oil (Merkur, Germany) and hydroxypropylmethyl cellulose F4M (HPMC F4M) (Blanver, Brasil). Semisolid delivery vehicles preparation The release of DH was evaluated from semisolid vehicles of oil/aqueous type emulsion at a propor-


De la Paz et al.

tion of 20/80 and hydrophilic gels. The semisolid vehicles (SV) developed can be seen in Table 1. SV1 to SV5 were developed by varying the emulsifying agent: non-ionic (polysorbate 80), cationic (CH or CHAc), the combination of CH with polysorbate 80 or CHAc with polysorbate 80. DH, emulsifying agent and 15% (w/w) of propylene glycol composed the aqueous phase. The discontinuous phases consisted of 15% (w/w) of cetyl alcohol and 5% (w/w) of mineral oil. Five formulations present the same lipid content (20%). The semisolid preparation technique was the simultaneous mixture phases. The aqueous/emulsifying and oil phases were weighted separately and heated to the same temperature, about 75°C. The oil phase was gradually added to the aqueous/emulsifying phase and stirred with high shear dispersing system model Ultra Turrax (IKA Werke, Staufen, Germany), equipped with an 18 mm diameter dispersing tool at 13 500 min-1 for 5 min. DH and polysorbate 80 were dissolved separately in distilled water at room temperature. Hydrophilic gels (SV6 to SV8) were developed varying the viscosity-increasing agent: non ionic (HPMC F4M) and cationic (CH or CHAc). DH, viscosity-increasing agent and 15% (w/w) of propylene glycol composed the gel. Three formulations present equal aqueous content (80%). In SV6, the HPMC F4M was added as a stabilizing agent to form a gel formulation. It was dispersed in propylene glycol and added to the distilled water. SV7 and SV8 were formulated, adding CH or CHAc to the mixture of propylene glycol and distilled water. The stirring was kept constant until a homogeneous mixture was obtained. DH was dissolved separately in distilled water, CH was dissolved in 1% of acetic acid solution and CHAc was dissolved in distilled water, at room temperature. Freshly prepared formulations were transferred into glass test tubes and kept at 30°C ± 2°C to the evaluation. The pH measurements were performed at 25°C using a pH meter (Mettler Toledo, Switzerland). An average of three measures was considered. A viscometer with controlled stress MCR 302 rheoplus (Anton Paar, Germany) equipped with plate-plate geometry (PP25) was used. The plate diameter was 24.980 mm, the sample volume was http://jppres.com/jppres


0.49 mL, and measuring position was 1 mm. Measurements were performed at 30°C by increasing the share rate from 0 to 100 s-1 for 1 min. The data obtained were processed using RheoPlus Software version 3.6x. In vitro release of dibucaine hydrochloride As part of the experiment, 1.0 g of SV was placed in a dialysis sac with pore size of 12000-14000 Da (Spectra/dialysis membrane 4, diameter: 20.4 mm, nominal flat width: 32 mm), and the sac was immersed in a constantly stirred receiver vessel containing a desired aqueous buffer (pH = 5.5) at 37°C. At designated periods, the sample (5 mL) was removed from the receiver vessel and replenished with fresh buffer. The total experiment time to collect the material aliquots was 8 h. The samples were analyzed using a spectrophotometer (Genesys 10S, USA) and the release profile was observed. Results are the average of three determinations. The DH quantification was carried out by spectrophotometric method. UV-VIS spectroscopy method and validation The test solution was prepared by using exact weights; 10 mg of the DH were dissolved in buffer and diluted to 100.0 mL with the same solvent, stirring vigorously for 10 minutes. The buffer solution was composed of 0.1 mol/L citric acid and 0.2 mol/L disodium hydrogen phosphate (pH= 5.5), and 10 mL of this solution was diluted to 25.0 mL with buffer. Absorbance was measured at 254 nm. The same buffer was used for sample preparation. The spectrophotometric method was previously validated including selectivity, linearity, accuracy, precision and limits of detection and quantification (USP 36, 2013). The influence of SV excipients was analyzed to determine the selectivity of the method. The absorption spectra was recorded and compared with the spectra obtained from the 100% reference substance of DH. Five DH concentrations in triplicate were analyzed within a range of 50 to 150% of the stated theoretical quantity. Results were statistically processed and the following parameters were determined: r (coefficient of linear correlation), r2 (coefficient of determination), a (intercept) and b J Pharm Pharmacogn Res (2017) 5(2): 98

De la Paz et al.


(slope). The limits of detection and quantification were analyzed by performing a calibration curve below the curve linearity of the system, using concentrations of 2, 4, 8, 12 and 16 µg/mL. A recovery curve (Y) was plotted of the recovered percentage vs. the percentage added (X) of points equivalent to 80, 100 and 120%, analyzed in triplicate at each level of concentration. The percentage of recovery (R) and the coefficient of total variation (CV) were calculated. Repeatability: Samples were evaluated six times, with a concentration equivalent to 100%. Determinations were performed by the same analyst, under the same working conditions. Intermediate precision: At the same laboratory, two researchers carried out analyses over two days. Triplicate analyses were performed in each case for samples that were equivalent to 100%. Total CV was calculated. Statistical analysis Results were presented in tables or figures and expressed as mean ± SD. The level of significance was tested using One-way ANOVA followed by Duncan Multiple Range Test. Results were regarded as significant when p 5. The nondetection of Ni at top soil may be due to leaching from run-off and its migration from top soil to bottom soil over time (for where they were found). However, the presence of Ni in Vernonia amygdalina leaf tissues (3.5 ± 0.00 µg/g) has been reported in samples collected from the wild in Ghana, with non-detection in Ocimum gratissimum and Jatropha gossypiifolia foliage (Annan et al., 2010). The non-detection of Ni in Vernonia amygdalina and other foliage in spite of its presence in the bottom soil in the CHS environment may be due to geochemical properties such as pH, total organic carbon (TOC), clay content, among others. Geochemical properties of the soil and the selective inhibition of the accumulation of Ni by Talinum triangulare and Zea mays have been reported (Abu-Darwish, 2009). Clayey soils are reported to have high adsorption

capacity and this would restrict desorption of bound metals in soil for up-take by plant through the root route. In addition, the uptake of heavy metals from soil by plants through the roots and subsequent translocation to the aerial parts of a plant is usually through the xylem or/and phloem in solubilized form. For example Ni would be easily absorbed as Ni2SO4.7H2O from the soil and translocated to the leaves. Nickel is a constituent of the enzyme urease and small quantities are essential for some plant species (Takishima et al., 1988); it is an essential micronutrient, which is required by urease for hydrolyzing urea. High concentrations may be toxic to plants. Extremely high concentrations of nickel have left some farmland unsuitable for growing crops. Its toxic effects have been frequently reported, such as inhibition of mitotic activity of Cajanus cajan, reduction in germination of cabbage and adverse effects on fruit yield and quality of wheat.

Table 2. Physico-chemical characteristics of soils. Parameters

CHS medicinal garden

*

*

CHS Quarters

0 – 15 cm

15 – 30 cm

0 – 15 cm

15 – 30 cm

PH

6.43 ± 0.02

6.01 ± 0.01

5.41 ± 0.01

5.47 ± 0.02

Conductivity (µs/cm)

184 ± 4.00

142 ± 4.00

116 ± 3.00

117 ± 2.00

Salinity (‰)

0.05 ± 0.00

0.04 ± 0.00

0.03 ± 0.00

0.03 ± 0.00

Sulphate (mg/kg)

172 ± 4.00

305 ± 5.00

496 ± 6.00

381 ± 5.00

Total organic carbon (TOC) (%)

2.43 ± 0.12

1.09 ± 0.11

1.27 ± 0.09

0.80 ± 0.01

Total nitrogen (TN) (%)

0.19 ± 0.02

0.10 ± 0.01

0.11 ± 0.01

0.08 ± 0.00

C:N Ratio

13:1

11:1

11:1

10:1

Total organic matter (TOM) (%)

4.33 ± 0.21

1.94 ± 0.20

2.26 ± 0.16

1.42 ± 0.02

CEC (meq/100g)

30.59 ± 0.98

20.82 ± 1.31

23.74 ± 1.05

19.81 ± 1.19

Sand (%)

58.0

59.5

63.1

61.3

Silt (%)

17.4

19.2

14.8

18.1

Clay (%)

24.6

21.3

22.1

20.6

Particle size distribution

*

Composited soil samples. C:N – Carbon:Nitrogen ratio; CEC: Cation exchanging capacity; CHS: College of Health Sciences.

The soil samples are basically acidic.



Vaikosen and Alade

Heavy metals in medicinal plants from Wilberforce Island, Nigeria

Table 3. Percent recovery and relative standard deviation for analyte metals in matrixes. Plant

Cr

Cd

Pb

Ni

%R

%RSD

%R

%RSD

%R

%RSD

%R

%RSD

Vernonia amygdalina

96.8 ± 2.2

2.3

93.6 ± 4.7

5.0

97.0 ± 2.0

2.0

93.0 ± 3.9

4.2

Talinum triangulare

99.8 ± 3.2

3.2

93.3 ± 1.9

2.1

97.0 ± 5.8

6.0

96.2 ± 3.2

3.4

Jatropha tanjorensis

94.7 ± 3.2

3.4

95.3 ± 3.9

4.1

93.2 ± 2.0

2.2

96.7 ± 4.7

4.9

Ocimum gratissimum

96.4 ± 3.4

3.6

94.9 ± 2.0

2.1

99.0 ± 4.0

4.0

94.6 ± 2.1

2.2

Celosia argentea

96.7 ± 1.6

1.7

98.4 ± 5.4

5.5

96.1 ± 1.2

1.3

100.2 ± 2.4

2.4

Corchorus olitorius

94.9 ± 4.1

4.3

97.3 ± 0.7

0.8

97.1 ± 2.0

2.1

101.5 ± 5.2

5.1

Colocasia esculenta

97.8 ± 2.4

2.4

95.8 ± 2.1

2.2

95.7 ± 0.8

0.8

96.5 ± 1.6

1.7

Ipomoea batatas

95.6 ± 3.8

4.0

103.4 ± 5.8

1.6

98.1 ± 1.1

1.1

98.0 ± 3.8

3.9

Zea mays

95.6 ± 1.6

1.7

98.1 ± 4.0

4.1

96.8 ± 3.2

3.3

98.6 ± 3.8

3.8

Top soil (0 - 15 cm)

98.7 ± 2.1

2.1

95.8 ± 4.1

4.3

95.8 ± 3.9

4.1

95.6 ± 2.4

2.5

Bottom soil (15 - 30 cm)

94.4 ± 3.4

3.6

95.4 ± 2.4

2.5

95.7 ± 2.2

2.3

97.3 ± 1.2

1.3

Top soil (0 - 15 cm)

96.6 ± 0.9

0.9

98.3 ± 2.2

2.2

99.0 ± 1.3

1.3

96.7 ± 2.8

2.9

Bottom soil (15 - 30 cm)

98.5 ± 4.4

4.5

95.8 ± 2.2

2.3

96.2 ± 6.1

6.3

96.1 ± 4.3

4.5

Soil Medicinal garden

CHS quarters

%R = Percent recovery; %RSD = Relative standard deviation.

A

B

Figure 2. Percentage detection frequency of heavy metals in (A) herbal plants and (B) soils. The number of times each of the heavy metals were detected in herbal plants and in soils at the CHS quarters and FoP (CHS) garden were computed into percentage detection frequency (i.e. ratio of number of times metal was detected to total no. of samples analyzed per location multiplied by 100). Cd had the highest detection frequency in both soil and leaf samples.



Vaikosen and Alade


Table 4. Levels of heavy metals in the leaves of plant species. Plant species

Concentration (µg/g) Cr

Cd

Pb

Ni

Vernonia amygdalina

ND

0.54 ± 0.01

ND

ND

Talinum triangulare

ND

0.23 ± 0.01

ND

ND


ND

2.44 ± 0.08

ND

ND

Ocimum gratissimum

ND

0.41 ± 0.01

ND

ND

Celosia argenta

ND

0.66 ± 0.02

ND

ND

Corchorus olitorius

ND

1.23 ± 0.03

4.31 ± 0.15

ND

Colocasia esculenta

ND

0.87 ± 0.02

ND

ND

Ipomoea batata

ND

1.72 ± 0.03

ND

ND

Zea mays

ND

0.92 ± 0.02

ND

ND

ND (Not detected) < 0.001 µg/g.

Table 5. Distribution of heavy metals in soils. Medicinal plant

Concentration in soil profiles (µg/g)

Species

Top soil (0 – 15 cm)

Bottom soil (15 – 30 cm)

Cr

Cd

Pb

Ni

Cr

Cd

Pb

Ni

Vernonia amygdalina

ND

0.95 ± 0.08

ND

ND

ND

0.26 ± 0.01

ND

0.38 ± 0.01

Talinum triangulare

ND

0.20 ± 0.02

ND

ND

3.90 ± 0.11

0.19 ± 0.02

ND

2.04 ± 0.05


ND

0.00 ± 0.00

ND

ND

ND

0.19 ± 0.01

ND

ND

Ocimum gratissimum

ND

0.00 ± 0.00

ND

ND

ND

ND

ND

ND

Corchorus olitorius

ND

0.21 ± 0.01

ND

ND

ND

0.47 ± 0.04

ND

ND

Celosia argentea

ND

0.89 ± 0.04

1.63 ± 0.09

ND

ND

0.22 ± 0.01

ND

ND

Colocasia esculenta

ND

0.50 ± 0.02

ND

ND

ND

0.57 ± 0.02

ND

ND

Ipomoea batata

ND

0.39 ± 0.01

ND

ND

ND

0.68 ± 0.02

ND

ND

Zea mays

ND

0.37 ± 0.01

ND

ND

5.32 ± 0.09

0.80 ± 0.04

ND

3.03 ± 0.06

Cadmium (Cd) Cadmium was found in all medicinal plant foliar tissues, with residual levels ranging from 0.23 ± 0.01 µg/g (Talinum triangulare) to 2.44 ± 0.08 µg/g (Jatropha tanjorensis) (Table 2). The order of residual concentration was Jatropha tanjorensis > Ipomea batatas > Celosia argenta > Zea mays > Colocasia esculenta > Corchorus olitorius > Vernonia amygdalina > Ocimum gratissimum > Talinum triangulare. Sobukola et al. (2010) reported much lower concentrations for bitter leaf (Vernonia http://jppres.com/jppres

amygdalina) and water leaf (Talinum triangulare) purchased from selected markets in Lagos, Nigeria. Also, residual values reported in foliar part for Ocimum gratissimum and Vernonia amygdalina sampled from Road Side and Physique Garden in Kwame Nkrumah University of Science and Technology and natural habitat respectively in Ghana (Annan et al., 2010; 2013), were significantly higher than level found in the same plant species cultivated in FoP medicinal plant garden. All plant species except Talinium triangulare exceeded the 0.3 µg/g maxiJ Pharm Pharmacogn Res (2017) 5(2): 138

Vaikosen and Alade

mum allowable limit stipulated by WHO for raw medicinal plant material (WHO/FAO, 2011). Also, in soil, cadmium was found at all corresponding locations where plant species were collected, except for Ocimum gratissimum, and Jatropha tanjorensis at both depths and 0 - 15 cm depth, respectively. The level of residual Cd concentration ranged from ND (Ocimum gratissimum and Jatropha tanjorensis) to 0.95 ± 0.08 µg/g (Vernonia amygdalina) at depth 0 – 15 cm, while depth 15 - 30 cm ranged from ND (Ocimum gratissimum) to 0.80 ± 0.04 µg/g (Zea mays). These concentration ranges were comparable to values reported in soils on which four medicinal plant species were collected in Karak District, Khyber Pakhtunkhwa, Pakistan (Shah et al., 2013), but significantly lower than residual concentrations in Haridwar, India (Maharia et al., 2010) and high traffic urban areas in Aba city, Nigeria, where Ocimum gratissimum and Vernonia amygdalina foliage were harvested (Princewill-Ogbonna and Ogbonna, 2011). The presence of Cd on Ocimum gratissimum leaf tissues and its non-detection in surrounding soils (top and bottom profiles) implied that the Cd found on Ocimum gratissimum foliage may have been through aerial deposition and not by uptake from the soil through the root via translocation. However, the residual concentrations of Cd in all soils were below the stipulated permissible limit of 3 µg/g by FAO/WHO (2011). Cadmium has been reported to have adverse effect on sperm count and motility (Kumar et al., 2005; Ige et al., 2012) and also implicated in renal dysfunction and anaemia. Xu et al. (2001) reported the significant reduction in sperm count and motility seven days after administration in rats. In plants, it has caused profound reduction in the production yield at concentrations ≥ 5 mg/kg (Hussain et al., 2005). Cd as a potential toxic metal has been found widely in water, soil, milk, dietary products, medicinal plants and herbal products. The major sources leading to accumulation of cadmium in soil and plants are phosphate fertilizers, non-ferrous smelters, lead mines, sewage sludge application and combination of fossil fields. Cadmium is used as a catalyst in the manufacturing of products such as margarine and peanut butter; wrapping papers and plastics in packaging http://jppres.com/jppres


refined foods; as plating on equipment or inks, dyes and lubricants used on and around food processing equipment (Wilson, 2012). Chromium (Cr) Residual Cr was not detected in any of the medicinal plant leaf tissues and soils at 0 -15 cm depth. However, at depth 15 – 30 cm, residual concentrations were 1.95 ± 0.05 µg/g and 2.66 ± 0.06 µg/g at Talinum triangulare and Zea mays sampling locations respectively, while other sampling location were below detection limit. The non-detection of Cr in the leaf tissues of Talinum triangulare and Zea mays may be due to the non-availability of the Cr metal in solubilized form for easy uptake from the soil. In addition, the non-availability of Cr may be due to its adsorption to the surrounding soil particulate. The interaction between heavy metals and soil particulates are influenced by variables such as organic carbon (OC), clay, temperature and pH have been reported (Basta et al., 2005). The presence of high percentage clay and OC tends to enhance adsorption of metals such as Cr to soil particulates; thereby making it unavailable for uptake by plant species. The concentrations of Cr in all soils were significantly below the level of maximum permissible level of 50 µg/g for agricultural soil (MAFF, 1992). Sources of contamination may be due to solid waste disposal of items such as plastic materials, paint containers, polythene bags and paper waste at the residential area of the CHS. Chromium is used on a large scale in industries such as metallurgical, electroplating, paints and pigments, tanning, wood preservation, pulp and paper (Zayed and Tery, 2003). The maximum residue level (MRL) in raw medicinal plant stipulated by WHO is 2 µg/g (WHO, 2005). Chromium has been reported to elicit reduction in motility and sperm morphology (Kamel, 2011) caused 75% reduction in sperm count at 10 mg/kg in rats (Li et al., 2001). Chromium is regarded as one of the most toxic pollutants in the world and is released by tanneries, steel industries and sewage sludge applications. The order of bulk soil contamination for total metal [i.e sum (∑) of Cr, Cd, Ni, Pb] at sampling locations were as follows: Zea mays > Talinum triangulare > Vernonia amygdalina > Celosia argenta > Colocasia esculenta > Ipomea batatas > Corchorus J Pharm Pharmacogn Res (2017) 5(2): 139

Vaikosen and Alade

olitorius > Jatropha tanjorensis > Ocimum gratissimum (Fig. 3); while the order of residual concentration of heavy metals in bulk soils was Cr > Cd > Ni > Pb. Bioaccumulation factor Calculated bioaccumulation factor (BCF) for Cr, Cd, Pb and Ni in nine medicinal plants is presented in Fig. 4 (values were computed as ratio of metal concentrations in leaf tissue to bulk soil – average of both depths). BCF values ranged from 0 – 25.93 for all foliar tissues of medicinal plants and heavy metals. BCF value was 0 (zero) for Cr, Pb and Ni in all medicinal leaf tissues, except for Celosia argenta, which recorded a value of 0.19 for Pb, while values for Cd were as follows: Jatropha tanjorensis (25.93), Ipomea batatas (3.23), Celosia argenta (2.21), Corchorus olitorius (1.94), Colocasia esculenta (1.62), Zea mays (1.57), Talinum triangulare (1.17), Vernonia amygdalina (0.89) and Ocimum gratissimum (0) in decreasing order. Effect of pH, clay and organic matter contents on metal availability The behavior of metals in soils are affected by principal factors such as pH, texture and quantity of clays with the oxides present, organic matter and the type of humic substances (Basta et al., 2005; Wahba and Zagloul, 2007). Cr, Cd and Ni were detected in


soils at Vernonia amygdalina, Talinium triangulare and Zea mays sampling locations. Order of heavy metal residual concentrations was Ni > Cd (Vernonia amygdalina); Cr > Ni > Cd (Talinium triangulare and Zea mays) (Table 4). Only Cd was found in all three plant species. This implies that Cd was most available for uptake. Higher Cd residual levels relative to other metals in Theobroma cocao nibs have been reported for contaminated soil (Nartey et al., 2012). At low pH, Cd exhibits higher mobility in soils relative to other heavy metals (Kabata-Pendias and Pendias, 1992; Alloway, 1995); thus greater availability in soil solution for plant uptake. The pH of soils in this study ranged from 5.41 - 6.43. This was acidic enough for its preferential up-take to Ni and Cr. The presence of Cd and non-detection of Ni and Cr in corresponding plant species with respect to sampling locations may also be due to metal-soil properties relationship (Tables 2 - 5). Clay is usually nutrient rich and are too tightly bound to be easily released and absorbed by plant roots (Kerrigan and Nagel, 1998) for subsequent translocation to the shoots and leaves. Soil properties such as clay and organic matter contents have been reported to favor greater affinity for Ni than Cd (Chorom et al., 2013), hence stronger adsorption of Ni to soil and nonavailability for plant uptake. These phenomena may have contributed largely to the uptake of Cd by all plant species investigated in this study.

Figure 3. Heavy metal distribution in bulk soil. Average concentration of each heavy metals in top soil (0 -15 cm depth) and sub-soil (15 -30 cm) were computed to give concentration of heavy metals in bulk soil for sampling point where each of the nine herbal plants were collected. Zea mays had the highest bulk soil contamination. A:Vernonia amygdalina; B:Talinum triangulare; C: Jatropha tanjorensis; D: Ocimum gratissimum; E: Corchorus olitorius; F: Celosia argenta; G: Colocasia esculenta; H: Ipomea batata; I: Zea mays



Vaikosen and Alade

Phyto-toxic assessment of plant species The potential risk of residual heavy metals in leafy medicinal plant species was evaluated by comparing residual levels in medicinal plant foliage, with stipulated limits for heavy metals in raw medicinal plant materials or/and leafy vegetables by the WHO and the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The WHO maximum residue limits (MRL) for cadmium in raw medicinal plant is 0.3 µg/g, while for leafy vegetables, stipulated JECFA maximum permissible limit is 0.2 µg/g (WHO, 2005; FAO/WHO, 2011). The residual Cd concentrations in all nine medicinal plant species investigated in this study ranged from 0.23 to 2.44 µg/g. This implies that 100% of the plant species exceeded the JECFA maximum limit, while > 88% exceeded the WHO limit for medicinal plant species. Residual Pb was found only in Corchorus olitorius, with a mean concentration of 4.31 µg/g. This value exceeded the JECFA permissible maximum limit of 0.3 µg/g for Pb when Corchorus olitorius is consumed as vegetable (FAO/WHO, 2011). However, the WHO maximum limit as medicinal plant is 10 µg/g (WHO, 2005). The provisional tolerable monthly intake (PTMI) of Cd for adult is 25 μg/kg body weight (FAO/WHO, 2011). The predicted quantity of plant species that would be equivalent to the PTMI of Cd for adult with body weight of 60 kg are as follows; Vernonia amygdalina (2.79 kg), Talinium triangulare (6.67 kg), Jatropha tanjorensis (0.62 kg), Ocimum gratissimum (3.64 kg), Ipomea batatas (0.87 kg), Celosia argenta (2.26 kg), Corchorus olitorius (1.22 kg), Colocasia esculenta (1.73 kg), Zea mays (1.63 kg). This depicts that the consumption of these plant species above the predicted PTMI in our diets or herbal medications is likely to be harmful. CONCLUSIONS The present study has demonstrated that the variation in heavy metal concentrations in medicinal plants is dependent on the collection sites, plant species and physico-chemical properties of soil such as pH, clay and organic matter contents. Cd exhibited the greatest bioavailability in the investigated soils and also recorded the highest rate of phytotoxicity assessment. Medicinal plants growing withhttp://jppres.com/jppres


in the CHS residential areas in the wild were more contaminated due to the loitering of solid waste in this environment. Cd and Pb found in plant foliage were due to uptake from soil and aerial deposition respectively, while > 88% exceeded the WHO limit for medicinal plant species. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENT The authors express their gratitude to the Faculty of Pharmacy, Niger Delta University, for access granted to the Faculty’s Medicinal Garden.

REFERENCES Abu-Darwish MS (2009) Essential oils yield and heavy metals content of some aromatic medicinal plants grown in AshShoubak Region, South of Jordan. Adv Environ Biol 3(3): 296-301. Alloway BJ (1995) Soil processes and the behavior of heavy metals. In: Alloway BJ (eds). Heavy Metals in soils. New York: Blackie, pp. 11-37. Annan K, Dickson RA, Isaac K, Amponsah IK, Isaac K, Nooni IK (2013) The heavy metal contents of some selected medicinal plants sampled from different geographical locations. Pharmacognosy Res 5(2): 103–108. Annan K, Kojo IA, Cindy A, Samuel A, Tunkumgnen BM (2010) Profile of heavy metals in some medicinal plants from Ghana commonly used as components of herbal formulations. Pharmacognosy Res 2(1): 41–44. Baldantoni D, Alfani A, Di Tommasi P, Bartoli G, De Santo AV (2004) Assessment of macro and microelement accumulation capability of two aquatic plants. Environ Pollut 130: 149-156. Basta NT, Ryan JA, Chaney RL (2005) Trace element chemistry in residual-treated. soil: Key concepts and metal bioavailability. J Environ Qual 34: 49–63. Burkill HM (1994a) A revision of Dalziel’s the useful plants of nd West Africa. 2 Edition. Vol. 1. pp. 53-54, 501–503, 535-537. Kew: Royal Botanical Gardens. Burkill HM (1994b) A revision of Dalziel’s the useful plants of nd West Africa. 2 Edition. Vol. 2. pp. 378–384. Royal Botanical Gardens. Burkill HM (1994c) A revision of Dalziel’s the useful plants of nd West Africa. 2 Edition. Vol. 3. Pp. 25-27. Kew: Royal Botanical Gardens. Burkill HM (1994d) A revision of Dalziel’s the useful plants of nd West Africa. 2 Edition. Vol. 4. pp. 472. Kew: Royal Botanical Gardens. Burkill HM (1994e) A revision of Dalziel’s the useful plants of nd West Africa. 2 Edition. Vol. 5. pp. 194-196. Kew: Royal Botanical Gardens. Chorom M, Karkaragh RM, Kaviani B, Kalkhajeh YK (2013) Monometal and competitive adsorption of Cd, Ni, and Zn J Pharm Pharmacogn Res (2017) 5(2): 141

Vaikosen and Alade

in soil treated with different contents of cow manure. Appl Environ Soil Sci 2013: 1-9. EDQM - European Directorate for the Quality of Medicine and Healthcare (2007) Guide for the elaboration of monographs on herbal drugs and herbal drug preparations (of the European Pharmacopoeia). Council of Europe, 67075 Strasbourg Cedex, France. Fabricant DS, Farnsworth NR (2001) The value of plants used in traditional medicine for drug discovery. Environ Health Perspect 109(1): 69–75. FAO/WHO - JECFA (Joint FAO/WHO Expert Committee on Food Standard Programme Codex Committee on Contaminants in Foods) (2011) Fifth Session, at The Hague, The Netherlands, 21-25 March 2011. Gopal NM, Tejaswini J, Mantry S, Kumar SA (2014) International standards of medicinal plants. Int J Inn Pharm Sci Res 2(10): 2498-2532. Guala SD, Vega FA, Covelo EF (2010) Heavy metal concentrations in plants and different harvestable parts: A soil-plant equilibrium model. Environ Pollut 158: 2659-2663. Hu Y, Liu X, Bai J, Shih K, Zeng EY, Cheng H (2013) Assessing heavy metal pollution in the surface of soils of a region that had undergone three decades of intense industrialization and urbanization. Environ Sci Pollut Res 20: 6150– 6159. Hussain I, Khan L, Metmood T, Khan I, Ullah W, Khan H (2005) Effect of heavy metals on the growth and development of Silybum marianum in various polluted areas of Peshawar Pakistan. J Chem Soc Pak 27(4): 367-373. Ige SF, Olaleye SB, Akhigbe RE, Akanbi TA, Oyekunle OA, Udoh UAS (2012) Testicular toxicity and sperm quality following cadmium exposure in rats: Ameliorative potentials of Allium cepa. J Hum Reprod Sci 5(1): 37-43. Kabaka-Pendias A, Pendias H (1992) Trace elements in soils nd and plants. 2 edition, Boca Raton, Florida: CRC Press, pp. 365. Kamel MM, El Razek AHA, Ahmed KA, Kamel GM (2011) Exposure of adult male rats to cadmium: Assessment of sexual behaviour, fertility, aggression as well as anxiety like behaviour with special reference to biochemical and pathological alterations. Life Sci J 8(2): 106-119. Kerrigan J, Nagel M (1998) Soils. http://www.hcs.ohiostate. du/mg/manual/soil.htm. [Accessed on 31 December, 2015]. Kumar S, Sathwara NG, Gautam AK, Agarwal K, Shah B, Kulkarini PK, Saiyed H (2005) Semen quality of industrial workers occupationally exposed to chromium. J Occup Health 47: 424–430. Lewington A (1993) Medicinal Plant and Plant Extracts: A Review of Their Importation into Europe. Cambridge, UK: Traffic International. Li H, Chen Q, Li S, Yao W, Li L, Shi X, Wang L, Castranova V, Vallyathan V, Ernst E, Chen C (2001) Effect of Cr (VI) exposure on sperm quality:human and animal studies. Ann Occup Hyg 45(7): 505-511. MAFF (Ministry of Agriculture, Fisheries and Food) and Welch Office Agriculture Department (1992) Code of Good Agriculture Practice for the Protection of Soil. Draft Consultation Document, MAFF, London



Maharia RS, Dutta RK, Acharya R, Reddy AVR (2010) Heavy metal bioaccumulation in selected medicinal plants collected from Khetri copper mines and comparison with those collected from fertile soil in Haridwar, India. J Environ Sci Health B 45: 174–181. Mendhan J, Denney RC, Barnes JD, Thomas NJK (2006) Vogel’s textbook of quantitative chemical analysis, 6th Edition, Pearson Education, Ltd, and Dorling Kindersley Publishing Inc., pp 704–710. Moore F, Shakeri A, Modabberi S (2009) Heavy metal contamination and distribution in the Shiraz industrial complex zone soil, South Shiraz, Iran. World Appl Sci J 6: 413-425. Nartey VK, Haizel M, Doamekpor LK, Dankyi E (2012) Studies on the contribution of fertilizers to heavy metal levels in soils and cocoa from some cocoa farms in the Western Region of Ghana. J Nat Sci Res 2(8): 111 -120. Omobuwajo OR, Alade GO, Akanmu MA, Obuotor EM, Osasan SA (2011) Microscopic toxicity studies on the leaves of Jatropha tanjorensis. Afr J Pharm Pharmacol 5(1): 12-17. Osundiya MO, Ayejuyo OO, Olowu AR, Bamgboye OA, Ogunlola AO (2014) Bioaccumulation of heavy metals in frequently consumed leafy vegetable grown along NigeriaBenin Seme Border, West Africa. Adv Appl Sci Res 5(1): 1-7. Princewill-Ogbonna LI, Ogbonna CP (2011) Heavy metal content in soil and medicinal plants in high traffic urban area. Pakistan J Nutri 10(7): 618-624. Schippmann U, Leaman DJ, Cunningham AB (2002) Impact of Cultivation and Gathering of Medicinal Plants on Biodiversity: Global Trends and Issues. FAO publication on; Biodiversity and the Ecosystem Approach in Agriculture, Forestry and Fisheries. Satellite event on the occasion of the Ninth Regular Session of the Commission on Genetic Resources for Food and Agriculture. Rome, 12-13 October 2002. Inter-Departmental Working Group on Biological Diversity for Food and Agriculture. Rome. Serafim A, Company R, Lopes B, Rosa J, Cavaco A, Castela E, Olea N, Bebianno MJ (2012) Assessment of essential and non-essential metals and different metal exposure biomarkers in the human placenta in a population from the South of Portugal. J Toxicol Environ Health 75(13-15): 867877. Sethy SK, Ghosh S (2013) Effects of heavy metals on germination of seed. J Nat Sc Biol Med 4(2): 272–275. Shah A, Niaz A, Ullah N, Rehman A, Akhlaq M, Zakir M, Khan MS (2013) Comparative study of heavy metals in soil and selected medicinal plants. J Chem 2013: 1–5. Smirjiakova S, Ondrasovicova O, Kaskova A Lakicova K (2005) The effect of cadmium and lead pollution on human and animal health. Folia Vet 49(3): 531-532. Sobukola OP, Adeniran OM, Odedairo AA, Kajihausa OE (2010) Heavy metal levels of some fruits and leafy vegetables from selected markets in Lagos, Nigeria. Afr J Food Sci 4(2): 389–393. Stoltz E, Greger M (2002) Accumulation properties of As, Cd, Cu, Pb, and Zn by four wetland plant species growing on submerged mine tailings. Environ Exp Bot 47: 271-280. Takishima K, Suga T, Mamiya G (1988) The structure of jack bean urease. The complete amino acid sequence, limited


Vaikosen and Alade


proteolysis and reactive cysteine residues. Eur J Biochem 175: 151–165. Tangahu BV, Abdullah SRS, Basri H, Idris M, Anuar N, Mukhlisin M (2011) A review of heavy metals (As, Pb and Hg) uptake by plants through phytoremediation. Int J Chem Eng 2011: Article ID 939161. The Lancet (1994) Pharmaceuticals from plants: great potential, few funds. The Lancet 343: 1513-1515. USEPA - US Environmental Protection Agency (1996) Soil screening guidance: Technical Rep. 540/R-95/128. Washington DC: US Gov. Print office. Vaikosen EN, Alade GO (2011) Evaluation of pharmacognostical parameters and heavy metals in some locally manufactured herbal drugs. J Chem Pharm Res 3(2): 88-97. van Seters AP (1997) Forest based medicines in traditional and cosmopolitan health care Part I: General Articles covering Global Issues. Non-wood forest products series -11, Medicinal plants for forest conservation and health care. Edited by Gerard Bodeker. FAO - Global initiative for traditional systems (gifts) of health. Food and Agriculture Organization of the United Nations. Rome, 1997.

Wahba MM, Zaghloul AM (2007) Adsorption characteristics of some heavy metals by some soil minerals. J Appl Sci Res 3(6): 421-426. WHO - World Health Organization (2005) Quality control methods for medicinal plants materials reused draft update. QAS/05, 131/Rev. 1, 22-27. Wilson L (2012) Cadmium, the pseudo-masculine mineral and a death mineral - The Center for Development. www.drlwilson.com/Article/cadmium.htm. [Accessed, 12, September, 2014]. Xu LC, Wang SY, Yang XF, Wang XR (2001) Effects of cadmium on rat sperm motility evaluated with computer assisted sperm analysis. Biomed Environ Sci 14(4): 312-317. Yang H, Shen Z, ZhoS, Wang W (2008) Heavy metals in wetland plants and soils of Lake Taihu, Chaina. Environ Toxicol Chem 27 (1): 38-42. Zayed AM, Terry N (2003) Chromium in the environment: factors affecting biological remediation. Plant Soil 249: 139. Zhuang PB, Zou NY, Li ZA (2009) Heavy metal contamination in soils and food crops around Dabaoshan mine in Guangdong, China: Implication for human health. Environ Geochem Health 31: 707-715.

_________________________________________________________________________________________________________________

Author contribution: Contribution

Vaikosen EO

Alade GO

Concepts or Ideas

X

X

Design

X

X

Definition of intellectual content

X

X

Literature search

X

X

Experimental studies

X

X

Data acquisition

X

X

Data analysis

X

X

Statistical analysis

X

X

Manuscript preparation

X

X

Manuscript editing

X

X

Manuscript review

X

X

Citation Format: Vaikosen EO, Alade GO (2017) Determination of heavy metals in medicinal plants from the wild and cultivated garden in Wilberforce Island, Niger Delta region, Nigeria. J Pharm Pharmacogn Res 5(2): 129-143.



© 2016 Journal of Pharmacy & Pharmacognosy Research ISSN 0719-4250 http://jppres.com/jppres

AUTHOR’S GUIDELINES Index AUTHOR’S GUIDELINES .................................................................................................................................................................................................................. 2 GENERAL........................................................................................................................................................................................................................................ 2 OPEN ACCESS ............................................................................................................................................................................................................................... 2 LANGUAGE .................................................................................................................................................................................................................................... 3 AUDIENCE...................................................................................................................................................................................................................................... 3 AREAS.............................................................................................................................................................................................................................................. 3 EDITORIAL POLICY .................................................................................................................................................................................................................. 4 ETHICS ............................................................................................................................................................................................................................................ 4 SUBMISSION OF MANUSCRIPTS ......................................................................................................................................................................................... 5 PREPARATION OF THE MANUSCRIPT ............................................................................................................................................................................ 6 1- Covering Letter .............................................................................................................................................................................................................. 7 2- Title page........................................................................................................................................................................................................................... 7 3-Abstract and key words .............................................................................................................................................................................................. 9 4- Introduction ..................................................................................................................................................................................................................10 5- Materials and methods ............................................................................................................................................................................................10 6- Results ..............................................................................................................................................................................................................................13 7- Discussion .......................................................................................................................................................................................................................13 Conclusions..........................................................................................................................................................................................................................13 8- Acknowledgements ...................................................................................................................................................................................................14 9- Conflict of interest ......................................................................................................................................................................................................14 10- References ...................................................................................................................................................................................................................14 11- Tables .............................................................................................................................................................................................................................16 12- Figures ...........................................................................................................................................................................................................................17 What type of manuscript you want to send to JPPRes? ......................................................................................................................................18 a- Original article .............................................................................................................................................................................................................18 b- Critical Review .............................................................................................................................................................................................................18 c- Short communication................................................................................................................................................................................................18 d- Letter to the Editor.....................................................................................................................................................................................................19 e- Case Report ....................................................................................................................................................................................................................19 f- Book or Thesis Review ..............................................................................................................................................................................................19 CONFLICTS OF INTEREST ....................................................................................................................................................................................................20 PLAGIARISM...............................................................................................................................................................................................................................21 Article withdrawal .................................................................................................................................................................................................................21 Article Retraction ...................................................................................................................................................................................................................22 Article Removal: Legal limitations ................................................................................................................................................................................22 Article Replacement ..............................................................................................................................................................................................................22 ACKNOWLEDGEMENTS ........................................................................................................................................................................................................23 Check List for Submitting a Manuscript......................................................................................................................................................................23 Important e-mail IDs and URLs .......................................................................................................................................................................................23 Templates ...................................................................................................................................................................................................................................24 REFEREES ....................................................................................................................................................................................................................................24 REVISED MANUSCRIPT.........................................................................................................................................................................................................26 PROOFS.........................................................................................................................................................................................................................................26 Errata/Corrigenda .................................................................................................................................................................................................................27 Offprints.......................................................................................................................................................................................................................................27 Copyright.....................................................................................................................................................................................................................................27

© 2016 Journal of Pharmacy & Pharmacognosy Research ISSN 0719-4250 http://jppres.com/jppres Author’s Guidelines

AUTHOR’S GUIDELINES GENERAL The Journal of Pharmacy & Pharmacognosy Research (JPPRes) is an international, specialized and peer-reviewed open access journal, which publishes studies in the pharmaceutical and herbal fields concerned with the physical, botanical, chemical, biological, toxicological properties and clinical applications of molecular entities, active pharmaceutical ingredients, devices and delivery systems for drugs, vaccines and biologicals, including their design, manufacture, evaluation and marketing. This journal publishes research papers, reviews, commentaries and letters to the editor as well as special issues and review of pre-and post-graduate thesis from pharmacists or professionals involved in pharmaceutical sciences or pharmacognosy. This journal is peer-reviewed and emphasizes scholarly publication and communication among pharmacists, researchers, students and other health care professionals. The focus is multi-dimensional: international pharmacy and pharmacognosy issues, pharmacy and pharmacognosy practice and education, clinical practice, drug information, commentaries and editorials. Manuscripts submitted to JPPRes are only accepted on the understanding that they are subject to editorial review and that they have not been, and will not be, published in whole or in part in any other journal. All manuscripts are subjected to an assessment made by experts (peer review), outside the Editorial Committee of the JPPRes, which conducts an assessment of the items of single-blind. The URL of the journal website is jppres.com/jppres. The e-mails are [email protected] or [email protected]

OPEN ACCESS All articles published by Journal of Pharmacy & Pharmacognosy Research are made freely and permanently accessible online immediately upon publication, without subscription charges or registration barriers.

P a g e 2 | 27


Authors of articles published in Journal of Pharmacy & Pharmacognosy Research are the copyright holders of their articles and have granted to any third party, in advance and in perpetuity, the right to use, reproduce or disseminate the article, according to the Document of Copyright and Ethics agreement of this journal.

LANGUAGE JPPRes accepts, in Spanish or English, review articles, articles for educational forum, reviews of book and thesis of pre- or post-grade, original research articles (full length and short communications), letter to editor, and case reports. Articles concerning all aspects of Pharmacy & Pharmacognosy will be considered. Articles of general interest (e.g. methods, therapeutics, medical education, interesting websites, new drug information and commentary on a recent topic) are also welcome. Manuscripts in which language is difficult to understand may be returned to the author for revision before scientific review.

AUDIENCE Biochemists, Biotechnologists, Botanists, Chemical Engineers, Clinical Pharmacologists, Medical Scientists, Medicinal Chemists, Natural Product Chemists, Pharmaceutical Scientists, Pharmacologists, Pathologists, Plant Scientists, and Toxicologists, among others.

AREAS                

Alternative and Complementary Medicine Analytical Toxicology Biochemical Pharmacology Biologicals Chinese Medicine Resources Clinical Pharmacology Cosmetic Sciences Drug Delivery System Drug Information Drug Metabolism Education in Pharmacy, Pharmacology and Pharmacognosy Ethnobotany Ethnopharmacology Food-Drug, Herbal-Drug, DrugDrug Interactions Formulations Functional Foods and Nutraceuticals

                 

Health and Medical Informatics Herbal Medicine Immunopharmacology Medical Anthropology Medication Management Medicinal Chemistry Molecular Medicine Molecular Modeling Molecular Pharmacology Neuropsychopharmacology Nutrition Pharmaceutical Care Pharmaceutical Marketing Pharmaceutical Microbiology Pharmaceutical Raw Material Science Pharmaceutical Research Pharmaceuticals Pharmaceutics and Pharmaceutical Microbiology

                  

Pharmaceutical Raw Materials Pharmacodynamics Pharmacoepidemiology Pharmacogenetics Pharmacogenomics Pharmacokinetics Pharmacology Pharmacotherapy Pharmacy Informatic Pharmacy Practice Pharmacognosy Physical Pharmaceutics Phytochemistry Phytomedicine Phytotherapy Social and Administrative Pharmacy Toxicology Traditional Medicine Vaccines P a g e 3 | 27




Veterinary Pharmacology



Zoopharmacognosy

EDITORIAL POLICY JPPRes considers only original contributions submitted exclusively to the journal. Prior and duplicate publications are not allowed. Publication of abstract under conference proceedings will not be considered as prior publication. It is the duty of the authors to inform the JPPRes about all submissions and previous reports that might be regarded as prior or duplicate publication. Manuscripts for publication will be considered on their individual merits. All manuscripts will be subjected to peer review. Normally manuscripts will be sent to at least two reviewers and their comments along with the editorial board’s decision will be forwarded to the contributor for further action.

ETHICS The JPPRes insists on ethical practices in both human and animal experiments. Evidence for approval by a local Ethics Committee must be supplied by the authors on demand. Animal experimental procedures should be as humane as possible and the details of anaesthetics and analgesics used should be clearly stated. The ethical standards of experiments must be in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and EU Directive 2010/63/EU for animal experiments. The journal will not consider any paper which is ethically unacceptable. A statement on ethics committee permission and ethical practices must be included in all research articles under the ‘Materials and Methods’ section. Uniform Requirements for manuscripts submitted to Biomedical Journals must be observed. Authors must be careful when they reproduce text, tables or illustrations from other sources. Plagiarism will be viewed seriously. Please see instructions below on this subject. All accepted papers are subject to editorial changes. JPPRes adopts the COPE guidelines (http://publicationethics.org/) on publication ethics. Authors of JPPRes must confirm the following:  Submitted manuscripts must be the original work of the author(s). P a g e 4 | 27


 Only unpublished manuscripts should be submitted.  It is unethical to submit a manuscript to more than one journal concurrently.  Any conflict of interest must be clearly stated.  Acknowledge the sources of data used in the development of the manuscript.  All errors discovered in the manuscript after submission must be swiftly

communicated to the Editor. Reviewers of JPPRes must confirm the following:  That all manuscripts are reviewed in fairness based on the intellectual content of the

paper regardless of gender, race, ethnicity, religion, citizenry nor political values of author(s).  That any observed conflict of interest during the review process must be communicated to the Editor.  That all information pertaining to the manuscript is kept confidential.  That any information that may be the reason for the rejection of publication of a manuscript must be communicated to the Editor. Editors of JPPRes must confirm the following:  That all manuscripts are evaluated in fairness based on the intellectual content of the

paper regardless of gender, race, ethnicity, religion, citizenry nor political values of authors.  That information pertaining manuscripts are kept confidential.  That any observed conflict of interest pertaining manuscripts must be disclosed.  The Editorial Board takes responsibility for making publication decisions for submitted manuscripts based on the reviewer’s evaluation of the manuscript, policies of the journal editorial board and legal restrain acting against plagiarism, libel and copyright infringement.

SUBMISSION OF MANUSCRIPTS Editorial Office (for submission queries and papers under review, according with the Instructions below): [email protected]

P a g e 5 | 27


Executive Editor (for accepted and published papers only): [email protected] or [email protected]

PREPARATION OF THE MANUSCRIPT Authors should keep their manuscripts as short as possible. Manuscripts should be typed double spaced in a single column in Letter size only. It should be paginated on the upper right hand corner of each page, beginning with the title page. The language of manuscript must be simple and explicit. If needed, the authors should consult those experienced in scientific writing and communication. Recent issues of the Journal of Pharmacy & Pharmacognosy Research should be reviewed for the general format adopted in respect to various elements of a paper. Identity of the author(s) must NOT appear anywhere in the manuscript (except on the first page file). These may either be a full length research article or a short communication. These papers should be arranged into the following sections: 1. Covering letter 2. Title page 3. Abstract and key words 4. Abbreviation list (if necessary, in alphabetical order) 5. Introduction 6. Materials and Methods 7. Results 8. Discussion 9. Acknowledgment 10. Conflict of interest 11. References 12. Tables 13. Figures

P a g e 6 | 27


Manuscripts in general should be organized in the following order:

1- Covering Letter In addition to the general details (name, address, contact details including mobile number of the corresponding author), it should mention in brief what is already known about this subject and what new is added by the submitted work.

2- Title page It should be paginated as page 1 of the paper. It should include the title, authors’ names and affiliations, running title, address for correspondence including e-mail address and also the total number of pages, figures and tables.

Title Must be informative, specific, short, clear, concise, and unambiguous reflect the paper’s contents. It should not exceed 150 characters. It must be write in English and Spanish.

Name(s) of author(s) The names of authors and their affiliations should be given. Family name, First name, and initial(s) of the middle name(s) of each author should be written. It should be made clear which address relates to which author. The corresponding author should be identified with an asterisk (*). When there are two or more authors and they belong to more than one affiliation, the connection between each author and his or her affiliation should be indicated by italicized superscripts a, b, c… placed after each author’s name and before each affiliation. Authorship credit should be based only on: 1. Substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2. drafting the article or revising it critically for important intellectual content; and 3. final approval of the version to be published. Conditions 1, 2, and 3 must all be met. Acquisition of funding, the collection of data, or general supervision of the research group, by themselves, do not justify authorship.

P a g e 7 | 27


Running title It is a short title printed in the journal at the right top corner of right hand page of the article (except the lead page). It should be not more than 50 characters in length.

Address for correspondence The corresponding author’s address should be given on the title page. The e-mail ID of the corresponding author or the contact e-mail ID must also be provided.

Affiliations include the name of department (if any), institution, city and state or country where the work was done, indicating which authors are associated with which affiliation.

E-mail address of the corresponding author As all correspondence, including proofs, should be sent only to him.

Abbreviations Abbreviations and their explanations should be collected in a list, arranged alphabetically. Abbreviations should generally be used sparingly. Non-standard abbreviations must be defined in the text following their first use. Provide a list of all nonstandard abbreviations after the keywords. Abbreviations and their explanations should be collected in a list, arranged alphabetically. However, the following need not be defined: ADP (adenosine 5’diphosphate), AIDS (acquired immunodeficiency syndrome), AMP (adenosine 5’monophosphate or adenylic acid), ATP (adenosine 5’-triphosphate), cAMP (adenosine 3_,5_cyclic monophosphate), cDNA (complementary DNA), CoA (coenzyme A), DNA (deoxyribonucleic acid), ED50 (50% effective dose), ESR (electron spin resonance), FAB-MS (fast atom bombardment mass spectrometry), FAD (flavin adenine dinucleotide), GC-MS (gas chromatography-mass spectrometry), GLC (gas-liquid chromatography), GMP (guanosine 5_-monophosphate), HPLC (high-performance liquid chromatography, highpressure liquid chromatography), IC50 (inhibitory concentration, 50%), IR (infrared), LC (liquid chromatography), LC/MS (liquid chromatography/mass spectrometry), LD50 (50% lethal dose), mRNA (messenger RNA), MS (mass spectrum), NMR (nuclear magnetic P a g e 8 | 27


resonance), P450 (as in cytochrome P450), RNA (ribonucleic acid), TLC (thin-layer chromatography), tRNA (transfer RNA), UV (ultraviolet).

Symbols and Units International standardized abbreviations should be used, for example: length (m, cm, mm, µm, nm, Å), mass (kg, g, mg, µg, ng, pg, mol, mmol, µmol), volume (L, mL, µL), time (s, min, h, d), temperature (°C, K), radiation (Bq, dpm, Gy, Sv), and concentration (M, mM, mol/L, mmol/L, mg/mL, µg/mL, %, % (v/v), % (w/v), ppm, ppb), acceleration due to gravity (g) need not be defined. Other abbreviations should be defined the first time that they are used in the text (i.e., the specific term should be followed by its abbreviation in parentheses), and they should be used consistently thereafter. Preferably, SI units should be used.

3-Abstract and key words Abstract The Abstract should be informative and completely self-explanatory, briefly present the topic, state the scope of the experiments, indicate significant data, and point out major findings and conclusions. The Abstract should not exceed 250 words in length (150 words for Case Report). Complete sentences, active verbs, and the third person should be used, and the abstract should be written in the past tense. For Original Articles, it must be in a structured form (Context, Objectives, Methods, Results and Conclusions) and explain briefly what was intended, done, observed and concluded. The conclusions and recommendations not found in the text of the manuscript should not be given in the abstract. Standard nomenclature should be used and abbreviations should be avoided. No literature should be cited. It must be write in English and Spanish.

Key Words At least three and not more than six in alphabetical order will be listed in English and Spanish, which will help readers or indexing agencies in cross-indexing the study. The words found in title need not be given as key words. Use terms from the latest Medical Subject Headings (MeSH) list of Index Medicus. A more general term may be used if a suitable MeSH term is not available.

P a g e 9 | 27


4- Introduction Briefly review important prior publications and state the reasons for the investigation being reported. It should start on a new page. Essentially this section must introduce the subject and briefly say how the idea for research originated. Give a concise background of the study. Do not review literature extensively but provide the most recent work that has a direct bearing on the subject. Justification for research aims and objectives must be clearly mentioned without any ambiguity. The purpose of the study should be stated at the end.

5- Materials and methods Description of methods, equipment and techniques (including statistical treatments used in the research). This section should deal with the materials used and the methodology (how the work was carried out). The procedure adopted should be described in sufficient details to allow the experiment to be interpreted and repeated by the readers, if desired. The number of subjects, the number of groups, the study design, sources of drugs with dosage regimen or instruments used, statistical methods and ethical aspects must be mentioned under the section. The data collection procedure must be described. If a procedure is a commonly used, giving a previously published reference would suffice. If a method is not well known (though previously published) it is better to describe it briefly. Give explicit descriptions of modifications or new methods so that the readers can judge their accuracy, reproducibility and reliability. The nomenclature, the source of material and equipment used, with details of the manufacturer in parentheses, should be clearly mentioned. Drugs and chemicals should be precisely identified using their non-proprietary names or generic names. If necessary, the proprietary or commercial name may be inserted once in parentheses. The first letter of the drug name should be small for generic name (e.g., dipyridamole, propranolol) but capitalized for proprietary names (e.g., Persantin, Inderal). New or uncommon drug should be identified by the chemical name and structural formula. The doses of drugs: should be given as unit weight per kilogram body weight e.g., mg/kg and the concentrations should be given in terms of molarity e.g., nM or mM. The routes of administration may be abbreviated, e.g., intra-arterial (i.a.), intracerebroventricular (i.c.v.), intra-gastric gavage (i.g.), intramuscular (i.m.), intraperitoneal (i.p.), intravenous (i.v.), per os (p.o.), subcutaneous (s.c.), transdermal (t.d.), etcetera. P a g e 10 | 27


Documentation of plants and other organisms or starting materials: Use the correct scientific nomenclature. For plants, the Index Kewensis (electronic Plant Information Centre ePIC, Royal Botanic Gardens, Kew, UK: http://www.kew.org/epic), and/or the International Code of Botanical Nomenclature (www.bgbm.fu-berlin.de/iapt/nomenclature/code/tokyoe/default.htm) should be followed. Give the scientific name (in italics), the author of this name and the family, i.e. Mangifera indica Linneo (Anacardiaceae). Indicate who identified the material. The manuscript must include references to voucher specimens of the plants (deposited in a major regional herbarium) or the material examined including their registration number(s). It should be mentioned which plant parts have been used. Description of the preparation of extracts and isolation of compounds: Extraction and isolation should be described in detail. The kind and amount of starting material, solvents and extraction methods must be indicated. The description of chromatographic systems should contain the quantitative information that allows the reader to repeat the work. Column dimensions, elution volumes, fraction sizes, etc. should be reported. Analytical studies: Key data on method validation must be provided and should typically include information on specificity, linearity, limit of detection, limit of quantification, accuracy, precision, intermediate precision, and some robustness studies. Information on the purity of reference compounds and on the methods used for the determination of purity must be given. Recoveries of extraction and sample pre-purification steps have to be indicated. Adequate statistical treatment of data is required. For more information regarding validation issues, prospective authors should also refer to ICH guidelines. Pharmacological investigations: JPPRes will consider manuscripts in which conclusions are based on adequate statistics that incorporate the appropriate tests of significance, account for the type of data distribution and are based on the number of experimental observations required for the application of the respective statistical method. In each case positive controls (reference compounds) should be used and the dose/activity dependence should be shown. Manuscripts describing animal experiments should be conducted in accordance with the experimental animal guidelines of the institution as well as the appropriate government guidelines. Only manuscripts of experiments conducted in accordance with the appropriate guidelines will be eligible for publication. When working with experimental animals, reference must be made to principles of laboratory animal care or similar regulations and to approval by the local ethical committee. The approval number and the corresponding date

P a g e 11 | 27


must be provided. It must clearly indicate that appropriate measures were taken to minimize pain or discomfort, and details of animal care should be provided. Biological screening: Biological activities should be reported by listing IC50 values, or a dose-response relationship should be shown by using at least two test concentrations. Positive controls (reference compounds) should be included. Clinical studies: Clinical studies must be designed, implemented and analyzed in a manner to meet current standards of randomised controlled trials. For guidelines see the following reviews: Begg et al. (1996) JAMA 276: 637-639 and Moher et al. (2001) BMC Medical Research Methodology 1:2. Reference must be made to approval of the study by the local ethical committee. The approval number and the corresponding date must be provided. All methods and variables used in a trial should be described; the data must be based on adequate statistics. For manuscripts dealing with scientific investigations involving human subjects and/or human tissues, the experiments should be performed in accordance with the ethical principles for medical research outlined in the Declaration of Helsinki 1964 as modified by subsequent revisions (http://www.wma.net/en/30publications/10policies/b3/). If approval was obtained from an Ethics Committee the authors should indicate this, as well as any approval/reference number. Written informed consent must be obtained from study participants and the existence of this consent must be stated in the article. Patients have a right to privacy: Any information that might result in identification of individuals must be omitted, especially if it is not directly clinically relevant. Patient age, sex, admission dates and co-morbidities should be removed as far as possible. If it is possible that a patient could be identified, the authors must obtain written informed consent from the individual(s) concerned and state that this has been obtained in the article. Publication consent forms should be retained by the authors and not supplied to the Journal. If the patient is deceased the next of kin should be contacted. If consent cannot be obtained the authors must explain the circumstances briefly in the article, as well as in detail in the covering letter. In rare circumstances where relevant clinical details mean that the patient can be identified, the patient/next of kin must be shown the manuscript before submission and made aware as part of the informed consent process that the article may appear on the internet.

P a g e 12 | 27


Statistical Analysis The variation of data should be expressed in terms of the standard error of mean (S.E.M) or the standard deviation (S.D.), along with the number of observations (n). The details of statistical tests used and the level of significance should be stated. If more than one test is used it is important to indicate which groups and parameters have been subjected to which test.

6- Results The results should be stated concisely without comments. Efforts should be made to avoid jargon, to spell out all non-standard abbreviations the first time they are mentioned and to present the contents of the study as clearly and concisely as possible. Results should be presented in logical sequence in the text with appropriate reference to tables and/or figures. The data given in tables or figures should not be repeated in the text. The same data should not be presented in both tabular and graphic forms. Simple data may be given in the text itself instead of figures or tables. Avoid discussions and conclusions in the results section.

7- Discussion (may be combined with the Results section). This section should deal with the interpretation, rather than recapitulation of results. It is important to discuss the new and significant observations in the light of previous work. Discuss also the weaknesses or pitfalls in the study. New hypotheses or recommendations can be put forth. Avoid unqualified statements and conclusions not completely supported by the data. Repetition of information given under Introduction and Results should be avoided.

Conclusions It must not reiterate any discussion or introductory comments, they must be genuine conclusions drawn from the results of the study. Conclusions must be drawn considering the strengths and weaknesses of the study. Make sure conclusions drawn should tally with the objectives stated under Introduction.

P a g e 13 | 27


8- Acknowledgements Acknowledge only those who have contributed to the scientific content or provided technical support. Sources of financial support may be mentioned.

9- Conflict of interest The authors should declare if exist or not conflict of interest with the data contained in the paper.

10- References It should begin on a new page. The number of references should normally be restricted to a maximum of 30 for a full paper. Majority of them should preferably be of articles published in the last 5 years. Papers which have been submitted and accepted but not yet published (“in press”) may be included in the list of references with the name of the journal and indicated Digital object identifier (DOI) number. Information from manuscript “submitted” but “not yet accepted” should not be included. Avoid using abstracts as references. The “unpublished observations” and “personal communications” may not be used as references but may be inserted (in parentheses) in the text. Authors are fully responsible for the accurate citing of the references. In the text, a reference identified by means of an author‘s name should be followed by the date of the reference in parentheses. When there are more than two authors, only the first author‘s name should be mentioned, followed by “et al.”. In the event that an author cited has had two or more works published during the same year, the reference, both in the text and in the reference list, should be identified by a lower case letter like ’a‘ and ’b‘ or ‘c’ after the date to distinguish the works.

Examples: Gonzalez (2010) or Glasgow et al. (2012) or (Garcia, 2009) or (Crystal and Roll, 2003) or (Hernandez, 2007; Moon, 2009 a, b, c; Tell, 2008, 2011) or (McGregor et al., 2013) The references must be verified by the author(s) against the original documents.

P a g e 14 | 27


References to books, journal articles, articles in collections and conference or workshop proceedings, and technical reports should be listed at the end of the article in numbered order. References to books should include the author’s name; year of publication; title; publisher; place of publication, in the order given in the example below. 1. AOAC (2007) Official Methods of Analysis, 18th edn. Washington, DC: Association of Official Analytical Chemists. References to articles in an edited collection should include the author’s name; year of publication; article title; editor’s name; title of collection; first and last page numbers; publisher; place of publication, in the order given in the example below. 2. Abrams R, Gonzalez E (2013) Pharmacological activities of cuprum nanoparticles. In: Gosen A, Perez J (eds), The Biological Activity of Nanoparticles. New York: Springer, pp 2130. References to articles in conference proceedings should include the author’s name; year of publication; article title; editor’s name (if any); title of proceedings; first and last page numbers; place and date of conference; publisher and/or organization from which the proceedings can be obtained; place of publication, in the order given in the example below. 3. Green R, Red N (2013) Topical effects of garlic cream. In: Brown C, Blue G (eds), Proceedings of an International Conference on Pharmacology of Garlic, Santiago, Chile, June 28-30, pp 23-27. References to articles in periodicals should include the author’s name; year of publication; article title; abbreviated title of periodical; volume number (issue number where appropriate); first and last page numbers, in the order given in the example below. 4. Rodriguez A, Schulz M (2013) Anti-inflammatory activity of an aqueous extract from Olea europaea L. seeds. J Pharm Pharmacogn Res 43: 329-336. References to technical reports or doctoral dissertations should include the author’s name; year of publication; title of report or dissertation; institution; location of institution, in the order given in the example below. 5. Martinez V (2013) Peels and seeds from Solanum spp. as a fiber source for human food. PhD Thesis, Department of Dietectics, Bellohuracan University, San Miguel, Chile. P a g e 15 | 27


References to Proceedings of Congress if there is not official Abstracts book: Pillon P, Guerra A, War W (2013) Ethopharmacological study and immunological activity of Solanum quitoense. Communication to the Ethno-Pharmaceutical Conference 2013 (EthnoPharmaceutical Society of Chile, Valparaiso, Chile, 14-16 April). With official Abstracts book: Pillon P, Guerra A, War W (2013) Ethopharmacological study and immunological activity of Solanum quitoense. Communication to the EthnoPharmaceutical Conference 2013 (Ethno-Pharmaceutical Society of Chile, Valparaiso, Chile, 14-16 April) p.37 or Pillon P, Guerra A, War W (2013) Ethopharmacological study and immunological activity of Solanum quitoense. J Pharm Pharmacogn 5(Suppl. 1): 82. References to Patents: Guest B, Verde A, Negrin M (2012) Pharmaceutical compositions of naproxen with antiviral effects. US Patent No. 2,922,101B2. CSIR, March 12. References to Websites: Diaz AR (2000) Parmaceuticals containing molybdenum. http://www.mhra.gov.uk/home/idcplg?IdcService=GET_FILE&dDocName=con1045821&Revi sionSelectionMethod=Latest [Consulted October 16, 2012]. or CNN. Cuba’s health care manages despite seizure. http://www.cnn.com/TRANSCRIPTS/0108/18/yh.00.html [Consulted October 15, 2012]. IMPORTANT: Web pages that have no scientifically recognized entity that takes responsibility for the above information will be censored.

11- Tables Tables should be kept to a minimum and be designed to be as simple as possible. Each table should be on a separate page, numbered consecutively in Arabic numerals (1, 2, etc.) and supplied with a heading and a legend if any. Such explanatory footnotes, identified by superscript letters, should be placed immediately below the table. Tables should be selfexplanatory without reference to the text. The details of the methods used in the

P a g e 16 | 27


experiments should preferably be described in the legend instead of in the text. The same data should not be presented in both table and graph form or repeated in the text. Check list for Tables          

Serially numbered in Arabic numerals? Short self explanatory heading given? Columns have headings? Units of data given? ‘n’ mentioned? Mean ± SD or Mean ± SEM given? Statistical significance of groups indicated by asterisks or other markers? P values given? Rows and columns properly aligned? Appropriate position in the text indicated?

12- Figures All photographs, graphs and diagrams should be referred to as a ‘Figure’ and they should be numbered consecutively in Arabic letters (1, 2, etcetera). Multi-part figures ought to be labeled with lower case letters (a, b, etcetera). Please insert keys and scale bars directly in the figures. Relatively small text and great variation in text sizes within figures should be avoided as figures are often reduced in size. Figures may be sized to fit approximately within the column(s) of the journal. Provide a detailed legend (without abbreviations) to each figure, refer to the figure in the text and note its approximate location in the margin. Please place the legends in the manuscript after the References. Each figure must be numbered and a short descriptive caption must be provided. A computer drawn figure with good contrast is acceptable. Sometimes, raw data for graphs may be required in Excel sheet when the article is accepted for publication. Graphic files for diagrams and figures may be converted to *.tiff, *.jpg, *.gif format. These files should not exceed 2 MB in size. Figure legends should have not more than 40 words. Information given in legends should not be repeated in the text. Check list for Figures  Serially numbered? Self explanatory caption given?  X and Y axes graduated?  X and Y axes titled (legend)? P a g e 17 | 27


 Units mentioned (if necessary)?  Different symbols/markers for different groups given?  SD or SEM represented (graphically)?  Statistical significance indicated?  Approximate position in the text marked?

What type of manuscript you want to send to JPPRes? a- Original article Original articles are the result of research studies describe a highly significant advancement in the particular field of Pharmacy or Pharmacognosy research. All papers are judged according to originality, novelty, quality of scientific content and contribution to existing knowledge. An Original Article may describe instrumental developments, innovative applications or strategies for problem solving with a multidisciplinary approach. Articles dealing with known analytical methods should offer a highly significant original application of the method, or results for novel analytes. References to the established technique must be given in the manuscript. Articles on fundamentals of measurement sciences may be theoretical in approach. There is no strict page limit, but we advise a maximum length of up to 6000 words including 20-30 references, plus 4-6 figures and 1-3 tables. Most importantly, paper length and content must be appropriate. Extensive tables, procedures, computer programs or animated graphics should be presented in form of Electronic Supplementary Material.

b- Critical Review JPPRes only accepts Critical Review. For a Critical Review the expectation is to present and critically evaluate the current state of the field, with illustrative examples (not only from the author’s own work), to point the reader to trends and likely future developments and to give a selection of important references to the current literature. Simple literature surveys are not accepted. For a critical review we advise a length of approx. 9000 words, plus figures, tables, and references.

c- Short communication While other things remain the same as described above, these papers should be considerably small in contents. P a g e 18 | 27


A short communication is for a concise, but independent report representing a significant contribution to Pharmacy or Pharmacognosy fields. Short communication is not intended to publish preliminary results. Only if these results are very original, of high interest and likely to have a significant impact will be considered for publication. Although JPPRes welcomes the submission of this type of article, fragmentation of a substantial body of work into a number of short publications is strongly discouraged. Unnecessary fragmentation is a valid reason for rejection of a Short Communication. JPPRes reserves the right to edit a suggested Original Article manuscript as Short Communication, according to the quantity and quality of the study results. It should be no more than 2500 words, and could include two figures or tables. It should have at least eight references.

d- Letter to the Editor This may either be a small research communication or a commentary on a contemporary issue or remarks/queries on a recently published article in JPPRes. It should be restricted to about 500 words excluding the references.

e- Case Report Interesting clinical cases (with pharmacologic or toxicological significance) may be considered for publication. Those with photographs stand a better chance. The case reports should have an unstructured abstract, introduction, case history and a brief discussion. It should be restrict to about 1000 words excluding the references and abstract.

f- Book or Thesis Review The book or thesis review should not exceed 1000 words and it can be written in Spanish or English. Aspects to consider:  Leading the review with full details of the work including the author’s name, title of

book or work of creation. For books, indicate ISBN, place of publication, printing, date and number of pages. Example: Thompson, Judith E. A Practical Guide to Contemporary Pharmacy Practice. Philadelphia: Lippincott Williams & Wilkins; Third edition, 2009. 760 pp. ISBN 978-0-7817-8396-5. P a g e 19 | 27


 Adopt throughout the review ‘s text an evaluative perspective of the author against

the work reviewed, identifying the contributions of this to the discipline(s) in that is inserted the central theme of the work.  Introduce the topic and the central problem in the beginning of the introduction.  Specify the readers or potential readers to whom it is directed the work.  Present the structure (chapters and sections) of the work with a complete synthesis of the content.  Mention the existence of glossaries, appendices or annotated bibliographies.  To link the work reviewed with other work on the same subject: What place does this work in the context of others in the same field? What this study adds to those are already published? What are the positive aspects and what are the negatives?  Place the work in the context of time and place in which it appears. Please focus on evaluating the work and its relevance; you do not take up too much in pointing out minor typographical errors. In the comments, keep a professional tone, avoid personal attacks.

CONFLICTS OF INTEREST Conflicts of interest have the potential to affect authors, referees and Editors (including Executive Editor and the Editor-in-Chief). JPPRes has the following systems in place to deal with conflicts of interest:

Authors: Authors are required to include a Document for Copyright & Ethics in every submission to the Journal.

Referees: When invited to act, and again when they agree to act, referees are reminded to consider whether they have any potential conflicts of interest. Referees are asked to discuss any perceived potential conflict with the Editor of the article who will reach a decision as to whether it is appropriate that the referee acts on the article or whether they should withdraw.

Editors: When an article is assigned to a Executive Editor or an Editor they are reminded to consider whether there are any potential conflicts of interest, and if so, to discuss them with the handling Executive Editor or the Editor-in-Chief, who will come to a decision as to whether it is appropriate for them to act on the article, or whether it should be reassigned.

P a g e 20 | 27


PLAGIARISM Authors should note that:  Copying verbatim text, tables or illustrations from any source (journal article, book,

monographs, thesis, Internet/any electronic media or any other published or unpublished material) and passing it as one’s own is considered plagiarism whether or not a reference to the copied portion is given.  Listing the source of copied material under ‘References’ does not absolve the authors of plagiarism.  If a few lines of text are to be reproduced from any source, ‘the author’ and ‘the source’ must be clearly indicated in the text. The reproduced lines must be in italics and given within quotes. If it is a paragraph it must be slightly indented also. To reproduce large portions of text, permission from the copyright owner(s) must be obtained and submitted to the JPPRes.  To reproduce tables or illustrations, permission from the copyright owner(s) must be obtained and a copy of the permission letter must be submitted to the journal. The source must be clearly acknowledged below the table or illustration as required by the copyright owner(s).  JPPRes defines plagiarism as a case in which a paper reproduces another work with at least 20% similarity and without citation.  If evidence of plagiarism is found before or after acceptance or after publication of the paper, the authors will be offered a chance to defense their paper. If the arguments are not found to be satisfactory, the manuscript will be retracted and the authors found to have been guilty of plagiarism will no longer have papers accepted for publication in JPPRes.  JPPRes has plagiarism detection systems.

Article withdrawal Only used for Articles in Press which represent early versions of articles and sometimes contain errors, or may have been accidentally submitted twice. Occasionally, but less frequently, the articles may represent infringements of professional ethical codes, such as multiple submission, bogus claims of authorship, plagiarism, fraudulent use of data or the like. Articles in Press (articles that have been accepted for publication but which have not been formally published and will not yet have the complete volume/issue/page information) that include errors, or are discovered to be accidental duplicates of other published P a g e 21 | 27


article(s), or are determined to violate the JPPRes ethics guidelines in the view of the editors (such as multiple submission, bogus claims of authorship, plagiarism, fraudulent use of data or the like), may be “Withdrawn” from JPPRes. Withdrawn means that the article content (HTML and PDF) is removed and replaced with a HTML page and PDF simply stating that the article has been withdrawn according to the JPPRes Policy on Article in Press Withdrawal with a link to the current policy document.

Article Retraction Infringements of professional ethical codes, such as multiple submission, bogus claims of authorship, publish without the consent of the co-authors, plagiarism, fraudulent use of data or the like. Occasionally a retraction will be used to correct errors in submission or publication. The retraction of an article by its authors or the editor under the advice of members of the scholarly community has long been an occasional feature of the learned world. Standards for dealing with retractions have been developed by a number of library and scholarly bodies, and this best practice is adopted for article retraction by JPPRes: •

• • • •

A retraction note titled “Retraction: [article title]” signed by the authors and/or the editor is published in the paginated part of a subsequent issue of the journal and listed in the contents list. In the electronic version, a link is made to the original article. The online article is preceded by a screen containing the retraction note. It is to this screen that the link resolves; the reader can then proceed to the article itself. The original article is retained unchanged save for a watermark on the .pdf indicating on each page that it is “retracted.” The HTML version of the document is removed.

Article Removal: Legal limitations In an extremely limited number of cases, it may be necessary to remove an article from the online database. This will only occur where the article is clearly defamatory, or infringes others’ legal rights, or where the article is, or we have good reason to expect it will be, the subject of a court order, or where the article, if acted upon, might pose a serious health risk. In these circumstances, while the metadata (Title and Authors) will be retained, the text will be replaced with a screen indicating the article has been removed for legal reasons.

Article Replacement In cases where the article, if acted upon, might pose a serious health risk, the authors of the original article may wish to retract the flawed original and replace it with a corrected P a g e 22 | 27


version. In these circumstances the procedures for retraction will be followed with the difference that the database retraction notice will publish a link to the corrected republished article and a history of the document.

ACKNOWLEDGEMENTS All messages and reviews sent electronically will be acknowledged automatically upon receipt. Note: Do not send hard copies/CDs, until you receive e-mail request from Editorial office. A timely submission, however, is not a guarantee that your work will be accepted for forthcoming publication. All submissions are peer reviewed by the editorial board and a select group of reviewers. Please make sure that all guidelines are followed carefully. All the accepted articles will be queued for publication and will appear in the futures issues based on the priorities set by the editorial board. The manuscript should be submitted through e-mail. Hard copies are not accepted.

Check List for Submitting a Manuscript Covering letter (first page)  Copyright & Ethics Forms (Scanned) (Download)  Manuscript  Illustrations or Tables (if any) 

Important e-mail IDs and URLs     

Queries: [email protected] or [email protected] Other correspondence: [email protected] or [email protected] Editor in Chief: [email protected] Submissions: [email protected] or [email protected] Journal Website: jppres.com/jppres

P a g e 23 | 27


Templates These ready to use templates are made to help the contributors write as per the requirements of the Journal. Save the templates on your computer and use them with a word processor program. Click open the file and save as the manuscript file.     

Template for Original Articles (.DOT file) (Download) Template for Case Reports. (.DOT file) (Download) Template for Review Articles. (.DOT file) (Download) Template for Letter to the Editor. (.DOT file) (Download) Template for Document of Copyright & Ethics (.DOT file) (Download)

REFEREES All manuscripts are subjected to an assessment made by experts (peer review), outside the Editorial Committee of the JPPRes, which conducts an assessment of the items of singleblind. After preliminary examination of the submission by Editorial Office staff to check that all the necessary elements are present, the paper is passed to the Editor-in-Chief. The Editor-in-Chief then assigns the paper to an appropriate Executive Editor. The Executive Editor is then responsible for selecting an Editor to handle the article. Articles can be rejected immediately by the Editor-in-Chief, a Executive Editor or an Editor without further peer review. The assigned Editor is responsible for selecting referees and obtaining referee reports. These reports will be communicated anonymously to authors. The usual number of referees is two, however, the Editors reserve the right to make a decision on a paper on the basis of one referee report, or seek the opinion of more than two referees if they judge this to be necessary or desirable. Leading articles and Correspondence are not routinely sent for external refereeing, but the Editor-in-Chief, Executive Editor and Editors reserve the right to seek the opinion of one or more external referees if they judge this to be necessary or desirable. Executive Editor, Editors and referees are asked to consider whether they have any conflicts of interest when they are assigned a paper, and if necessary to decline to handle the paper. See the section ‘Conflicts of interest’ for more information on this subject. If an Editor decides upon rejection of a paper, it is passed back to the handling Executive Editor for approval of this decision. All rejection correspondence therefore originates from P a g e 24 | 27


the Executive Editor. Authors should regard rejection as final and only resubmit if they have been invited to do so. Papers may be rejected because of they may be of only peripheral interest and perhaps more suitable for submission to a different journal; they may be, in the opinion of the reviewers, scientifically flawed; they may be unclear or overly long; or they may not make a significant contribution to the literature. Requests that a revised version of a paper be submitted for consideration are sent direct to the corresponding author from the Editor responsible. Any revised version should be submitted within 6 weeks of the revision request or the Journal reserves the right to consider the manuscript as a new submission that may be subject to further refereeing. The Editor-in-Chief, Executive Editor and Editors reserve the right to request more rounds of revision and resubmission/refereeing, or reject a paper outright, if they judge that any revised version does not adequately address the concerns raised by the referees and the Editor. Once the Editor is satisfied that a revised version has adequately dealt with any points raised they may accept the paper. Authors can appeal against a decision by contacting the handling Executive Editor, but unless there has been a gross misunderstanding of the submitted article by the Editor and referees, rejection appeals are not likely to be successful. Authors should appreciate that if they resubmit an article that has been rejected without substantially modifying it in line with the suggestions of the Editor and referees, it is almost certain to be rejected again. After acceptance the paper is sent for copy-editing and typesetting prior to production of proofs for author correction. The Journal maintains the right to edit any paper to the extent necessary to achieve clarity and precision of expression and to conform to English usage and the Journal’s conventions. Please note that if authors ignore requests to conform to Journal style at the revision stage, these changes may be enforced during copy-editing and proof production. Please submit the revised manuscript no later than six weeks from the date of notification of manuscript revision by the editor. A manuscript that is not revised within six weeks may be rejected.

P a g e 25 | 27


REVISED MANUSCRIPT The authors should revise the manuscript immediately after receipt of the comments from the JPPRes. A note mentioning the changes incorporated in the revised text as per referee’s comments (point by point) should be sent. The revised manuscript has to be submitted online within the stipulated time. Calling for revision does not guarantee acceptance. A revised manuscript which underwent major changes is likely to be sent to referees for rereview. If the authors have substantial reasons that their manuscript was rejected unjustifiably, they may request for reconsideration. Note: e-mails with vicious language, offensive writings to Editors will lead to rejection of the manuscript.

PROOFS JPPRes reserves the right to edit a manuscript for grammar, house style, scientific and statistical clarity, and overall length, while maintaining the scientific accuracy of the report. Authors may be asked to incorporate editorial amendments of spelling, grammar, house style and to check minor inconsistencies in the text or reference list, together with scientific and/or statistical corrections, before returning a revised manuscript for final approval by the Editor. Failure to make either scientific/statistical or editorial amendments could result in delayed acceptance and publication. Proofs are sent to the corresponding author by e-mail as a PDF. Acrobat Reader will be required in order to read this file. This software can be downloaded (free of charge) from the following website: www.adobe.com/products/acrobat/readstep2.html. Proofs should be read carefully, paying particular attention to any tables, figures and references, and corrections should be sent to the JPPRes Editorial Office as soon as possible. Corresponding author will enable the file to be opened, read on screen and edited electronically. You may prefer to print the PDF and add your corrections offline. Further instructions will be sent with the proof. To avoid publication delays proofs should be checked immediately and returned by email. Authors should pay particular attention that they check any dosage directions, owing to the seriousness of any error entering the printed record. Extensive changes at the proof stage are not permitted. Authors are advised that they are responsible for proofreading of the text, references, tables and figures for absolute accuracy. New material cannot be accepted at this stage and substantial rewriting of paragraphs is not permitted. P a g e 26 | 27


Errata/Corrigenda Authors should advise the publisher ([email protected] or [email protected]) of corrections, apologies and retractions to the publisher who will publish them as the next available opportunity. The Editor-in-Chief may be consulted as appropriate.

Offprints Free access to the final PDF offprint of your article will be available via JPPRes website:jppres.com as an Open Access Article.

Copyright JPPRes is an open access journal distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. (http://creativecommons.org/licenses/by-nc-nd/3.0/) which permits to copy, distribute and transmit the work, provided the original work is properly cited. Authors may not use the JPPRes information for commercial purposes. Authors may not alter, transform, or build upon this work. Any of these conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. Authors are required to include a Document for Copyright & Ethics in every submission to JPPRes.

Last Update: November 16, 2015.

P a g e 27 | 27