Journal of Pharmacy & Pharmacognosy Research

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Volume 1, Issue 2 (Nov-Dec), 2013 ISSN 0719-4250

Content:

Pages

IFC (Journal of Pharmacy & Pharmacognosy Research).

i

1.- Review Pandurangan Perumal and Vijaya P. Pandey (2013) Antimicrobial peptides: the role of hydrophobicity in the alpha helical structure.

39-53

2.- Original article Alejandrina Alucema, Nicole Chavarría, Marisela Valdés (2013) Patrones de automedicación en clientes de una farmacia comunitaria de la ciudad de Antofagasta. | [Patterns of self-medication in customers of a community pharmacy in the Antofagasta city].

J Pharm Pharmacogn Res JPPRes

54-63

© 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.

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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.

© 2013 Journal of Pharmacy & Pharmacognosy Research, 1 (2), 39-53 ISSN 0719-4250 http://jppres.com/jppres Review | Revisión

Antimicrobial peptides: The role of hydrophobicity in the alpha helical structure [Los péptidos antimicrobianos: El papel de la hidrofobicidad en la estructura helicoidal alfa] Pandurangan Perumal*and Vijaya P. Pandey Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, Tamilnadu, India. * E-mail: [email protected]

Abstract

Resumen

The antimicrobial peptides (AMPs) are a class of molecule obtained from plants, insects, animals, and humans. These peptides have been classified into five categories: 1. Anionic peptide, 2. Linear alpha helical cationic peptide, 3. Cationic peptide, 4. Anionic and cationic peptides with disulphide bonds, and 5. Anionic and cationic peptide fragments of larger proteins. Factors affecting AMPs are sequence, size, charge, hydrophobicity, amphipathicity, structure and conformation. Synthesis of these peptides is convenient by using solid phase peptide synthesis by using FMOC chemistry protocol. The secondary structures of three synthetic peptides were determined by circular dichroism. Also, it was compared the stability of the α-helical structure and confirmed the percentage of helix of these peptides by using circular dichroism. Some of these AMPs show therapeutic properties like antimicrobial, antiviral, contraceptive, and anticancer. The formulations of some peptides have been entered into the phase I, II, or III of clinical trials. This article to review briefly the sources, classification, factors affecting AMPs activity, synthesis, characterization, mechanism of action and therapeutic concern of AMPs and mainly focussed on percentage of α-helical structure in various medium.

Los péptidos antimicrobianos (AMP) son una clase de molécula obtenida a partir de plantas, insectos, animales y seres humanos. Estos péptidos han sido clasificados en cinco categorías: 1. Péptido aniónico, 2. Péptido alfa lineal catiónico helicoidal, 3. Péptido catiónico, 4. Péptidos aniónicos y catiónicos con enlaces de disulfuro, y 5. Fragmentos de péptidos aniónicos y catiónicos de proteínas más grandes. Los factores que afectan a los AMP son secuencia, tamaño, carga, hidrofobicidad, anfipaticidad, estructura y conformación. La síntesis de estos péptidos es conveniente mediante el uso de síntesis de péptidos en fase sólida, mediante el protocolo de química FMOC. Las estructuras secundarias de tres péptidos sintéticos se determinaron por dicroísmo circular. También ha sido comparada la estabilidad de la estructura α-helicoidal y confirmado el porcentaje de hélice de estos péptidos mediante el uso de dicroísmo circular. Algunos de estos AMP muestran propiedades terapéuticas como antibióticas, antivirales, anticonceptivas y anticáncer. Las formulaciones de algunos péptidos se encuentran en fases I, II o III de ensayos clínicos. Este artículo revisa brevemente las fuentes, clasificación, factores que afectan a la actividad de los AMP, la síntesis, caracterización, mecanismo de acción y la acción terapéutica de los AMP y se centra principalmente en el porcentaje de la estructura de α-helicoidal en diversos medios.

Keywords: Anticancer; dichroism; contraceptive.

Palabras Clave: Anticáncer; anticonceptivo; antiviral; dicroismo circular; péptido antimicrobiano.

antimicrobial

peptide;

antiviral;

circular

List of Abbreviations: AMPs - antimicrobial peptides; BOC - acid labile tertiary butyl oxycarbonyl group; CA-P1,2 or 3 - cecropin-A peptide 1,2 or 3; CD - circular dichroism; DCM – dichloromethane; DIPEA - diiso-propyl ethylamine; DMA - Dimethyl acetamide; DMF - N-N-dimethyl formamide; EL absorption coefficient of left circularly polarized light; ER - absorption coefficient of right circularly polarized light; FMOC - base labile fluorenyl methyl oxycarbonyl group; HBTU - tetramethyl uranium hexafluorophosphate; HF - hydrogen fluoride; HOBT – Hydroxy benzotriazole; LPPS – Liquid phase peptide synthesis; NPS – 2-nitro phenyl sulfenyl group; PAA – Poly acryl amide; PEG – Poly ethylene glicol; PS – Polystyrene; SDS - sodium dodecylsulphate; SPPS – Solid phase peptide synthesis; TFA - trifluoro acetic acid; TFE – trifluoroethanol; TFMSA - trifluoro methane sulphonic acid. ARTICLE INFO Received | Recibido: November 16, 2013. Received in revised form | Recibido en forma corregida: December 8, 2013. Accepted | Aceptado: December 15, 2013. Available Online | Publicado en Línea: December 31, 2013 Declaration of Interests | Declaración de Intereses: The authors declare that they have no conflict of interests. Funding | Financiación: none stated.

_____________________________________ This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. (http://creativecommons.org/licenses/bync-nd/3.0/ ) which permits to copy, distribute and transmit the work, provided the original work is properly cited. You may not use this work for commercial purposes. You 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. Este es un artículo de Acceso Libre bajo los términos de una licencia “Creative Commons Atribucion-No Comercial-No trabajos derivados 3.0 Internacional” (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.es) Usted es libre de copiar, distribuir y comunicar públicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los créditos de la obra de la manera especificada por el autor o el licenciador (pero no de una manera que sugiera que tiene su apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar o generar una obra derivada a partir de esta obra. Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor Nada en esta licencia menoscaba o restringe los derechos morales del autor.

Perumal & Pandey

INTRODUCTION Peptides are a class of compounds of low molecular weight, which yield amino acids on hydrolysis. The living organisms are constantly exposed to the potentially harmful pathogens through contact, ingestion and inhalation (Hultmark, 2003). While during pathogenic invasion the first line of defence involves the innate immunity followed by acquired immunity (Fearon and Locksley, 1996). In contrast of acquired immune mechanism, endogenous peptides (which are in the gastrointestinal, respiratory, and genitourinary tracts), which are induced a fast and effective defence against pathogens. This group of molecules is termed as ‘antimicrobial peptides’ (AMPs). These peptides were found in plants, insects, animals, and humans (Maróti et al., 2011). The AMPs are short peptides, generally between 12 to 50 amino acids present in the sequence of the peptides and these peptides are potent, broad spectrum antibiotics, which exhibited potential as novel therapeutic agents (Zanetti et al., 2002; McPhee and Hancock, 2005; Koczulla and Bals., 2003). These peptides are also called as host defence peptides (Tossi et al., 2005). AMPs demonstrated to be effective against Gram positive and Gram negative bacteria, mycobacteria (including Mycobacterium tuberculosis), fungus, viruses and cancer cells

Antimicrobial peptides: hydrophobicity and alpha helical structure

membrane permeabilization to cytoplasm. Ribosomally-synthesized AMPs, containing only natural amino acids can be grouped into linear, alphahelical peptides (such as cecropins, magainins, and mellitin), peptides characterized by enrichment in one or two amino acids (proline arginine-rich PR39, indolicidin), and peptides containing disulfide bonds (e.g., defensins, protegrins). Large number of peptides with potent antimicrobial activity that were synthesized extra-ribosomally or contain substantial post-translational modifications, for example lipopeptides (polymyxin, dermaseptin) and the lantibiotics, which contain non-native amino acids or non-peptide backbone structures. In addition, a wide variety of synthetic AMPs have been developed utilizing either a combinatorial synthesis approach (Lu et al., 2006; Eckert et al., 2006; Gottler and Ramamoorthy, 2009). The aim of this article to review briefly the sources, classification, factors affecting AMPs activity, synthesis, characterization, mechanism of action and therapeutic concern of AMPs. The second version of the antimicrobial peptide database (ADP2) contains detailed structural information (Wang and Wang, 2004). Fig. 1 shows the secondary structure of antimicrobial peptides such as α-helix and antiparallel β-sheets.

(Toke, 2005; Mader and Hoskin, 2006; Suttmann et al., 2008; Da Silva et al., 2008; Wang et al., 2008; Thennarasu and Nagaraj, 1999) and contains more than 50% of

hydrophobic residues and positively charged residues (Devine and Hancock, 2002). AMPs are believed to have a mechanism of action entirely distinct from those of current clinically-used antibiotics, and there is a great interest in their development for treatment of drug-resistant infections (Ge et al., 1999; Zhang and Falla, 2009; Liu et al., 2007; Glukhov et al., 2005).

SECONDARY STRUCTURE AMPs can be broadly classified based on secondary structure and composition. The secondary structures of these peptides consist of four parts: i) alpha-helical, ii) beta-stranded, iii) betahairpin or loop, and iv) extended. These peptides contain a variety of antimicrobial activities from http://jppres.com/jppres

Figure 1. Secondary structure of the antimicrobial peptides.

SOURCES More than 900 AMPs have been identified in various organisms from plants, insects, animals, and humans (Iwanaga et al., 1998; Selsted et al., 1993; Schnapp et al., 1998). These peptides have been grouped based on their primary structure, amino acid composition and their size. Tables 1-4 show the detailed information of AMPs obtained from plants, insects, animals, and humans. J Pharm Pharmacogn Res (2013) 1 (2): 40

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Antimicrobial peptides: hydrophobicity and alpha helical structure

Antimicrobial peptides from plants

(Bulet et al., 1999)

The AMPs were obtained from various plants (Castro et al., 2005) and its examples with details are given in Table 1. Antimicrobial peptides from insects The AMPs were obtained from various insects

given in Table 2.

and its examples with details are

Antimicrobial peptides from animals The AMPs were obtained from various animals

(Iwanaga et al., 1998; Nakamura et al., 1998; Rosa and Barracco, 2010) and its examples with details are

given in Table 3.

Table 1. Details of the antimicrobial peptides from plants. Peptide name

Source

Hevein

Latex of rubber trees

Purothionins

Amino acid number

Antimicrobial activity

43

Wheat endosperm

F +

45 +



G ,G

-

F - Fungus; G - Gram positive; G - Gran negavive.

Table 2. Details of the antimicrobial peptides from insects. Peptide name

Source

Amino acid number

Antimicrobial activity

Acaloleptin

Acalolepta luxuriosa

71

G ,G

Andropin

Drosophila melanogaster

34

G

Apidaecin IA

Apis mellifera

18

G

-

Cecropin

Hyalophora cecropia

37

G

-

Defensin- α

Aedes aegypti

40

G ,G

Drosomycin

Drosophila melanogaster

44

+

-

+

+

-

F +

-

+

-

+

-

+

-

Holotricin

Holotrichia diomphalia

43

G ,G

Sapecin-α

Sarcophaga peregrina

40

G ,G

Tenicin 1

Tenebrio molitor

43

G ,G

Thanatin

Podisus maculiventris

21

G ,G

F- Fungus; G+- Gram positive; G- - Gran negavive.

Table 3. Details of the antimicrobial peptides from animals. Peptide name

Source

Amino acid number

Antimicrobial activity

Androctonin

Androctonus australis

25

F, G , G

Bactenecin

Bovine neutrophils

12

G ,G

Brevinin

Rana brevipora porsa

24

Cupiennin

Cupiennius salei

35

G ,G

Dermaseptin S1

Phyllomedusa sauvagii

34

Lycotoxin

Lycosa carolinensis

27

G ,G

Tachyplesins

Tachypleus tridentatus (horseshoe crab)

17

G

-

+

-

G,G

-

+

+

-

G,G

-

+

+

-

+

-

F- Fungus; G+- Gram positive; G- - Gran negavive.

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J Pharm Pharmacogn Res (2013) 1 (2): 41

Perumal & Pandey

Antimicrobial peptides: hydrophobicity and alpha helical structure

Antimicrobial peptides from humans

Sequence

The AMPs were obtained from humans (Jenssen et al., 2006; Schroder and Harder, 1999; Zanetti et al., 1997)

Peptides contain the basic amino acid residues like lysine or arginine, the hydrophobic residues like tryptophan, alanine, phenylalanine, leucine, isoleucine, tyrosine and valine. Ratios of hydrophobic residues to charged (cationic or anionic) residues can vary from 1:1 to 2:1.

and its examples with details are given in Table 4. CLASSIFICATION

AMPs are classified into five categories and its examples with details are given in Table 5. FACTORS AFFECTING ANTIMICROBIAL PEPTIDES ACTIVITY The factors affecting the antimicrobial activity (Chen et al., 2007) such as sequence, charge, conformation and structure, size, hydrophobicity, amphipathicity and its details as follows:

Charge Anionic peptides contain more amount of aspartic acid and glutamic acids. The cationic peptides contain more amounts of arginine, histidine and lysine. Anionic peptides were complexed with zinc. Highly cationic peptides have more chance for complexation with zinc.

Table 4. Details of the antimicrobial peptides from humans. Peptide name

Source

Amino acid number

Antimicrobial activity

Cathelicidins

Human neutrophils

30

F, G , G

α Defensins

Human neutrophils

12-80

F, G G

Human Histatin 8

Homo sapiens

12

F, G G

LL37

Neutrophils

37

F, G G

-

+

-,

+

-,

+

-,

+

(Homo sapiens) F- Fungus; G+- Gram positive; G- - Gran negavive.

Table 5. Antimicrobial peptides classification. Classes

Characteristics

Examples

Anionic peptides

Rich in glutamic and aspartic acids

Maximin H5 from amphibians

Linear alpha helical cationic peptides

Lack in cysteine

Cecropins from insects, magainin and dermaceptin from amphibians, LL37 from humans

Cationic peptides

Rich in proline, arginine, phenylalanine, glycine, tryptophan

Indolocidin fromcattle, prophenin from pigs

Anionic and cationic peptides that contain disulphide

Cysteine

Peptides with 1 disulphide bond – brevinins, 2 disulphide bonds – protegrin and 3 disulphide bonds – drosomycin and defencins

Anionic and cationic peptide fragments of larger proteins

Tryptophan, lysine, leucine, histidine, proline, arginine, valine

Haemoglobin from humans, lysozyme, ovalbumin and lactoferricin from lactoferrin

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J Pharm Pharmacogn Res (2013) 1 (2): 42

Perumal & Pandey

Conformation and structure Antimicrobial peptides can assume a variety of secondary structures including alpha-helices, relaxed coils and anti-parallel beta-sheet structures. Amphipathic alpha-helical peptides are more active than peptides with less-defined secondary structures. Peptides with a gamma-core motif (two anti-parallel beta-sheets with an inter-posed short turn) are very active. Size The size of antimicrobial peptides differs from six amino acid residues for anionic peptides to more than 60 amino acid residues. Even di- and tri-peptides with antimicrobial activity were reported. Hydrophobicity Water-soluble (hydrophilic) antimicrobial peptides create the partition into the membrane lipid bi-layer. Amphipathicity Peptides contain hydrophilic amino acid residues in one side and hydrophobic amino acid residues in the opposite side of a helical molecule. Quantification of hydrophobic residues is less easy in the non helical peptides. ANTIMICROBIAL PEPTIDE SYNTHESIS To synthesize AMPs can be used two important methodologies: Solid phase peptide synthesis and liquid phase peptide synthesis. Solid phase peptide synthesis Solid phase peptide synthesis (SPPS) was introduced by Bruce Merrifield in 1963 (Stewart and Young, 1984; Fields, 1994; Fields and Fields, 1994; Date et al., 1998). The most of the peptide synthesized by SPPS

usually contain less than 20 amino acids. Synthesis of such peptides is routine and straightforward without significant complications. SPPS is based on addition of the N terminal amino group (depends upon the sequence of the peptide) with C terminal of the side chain protecting amino acid residues to an insoluble http://jppres.com/jppres

Antimicrobial peptides: hydrophobicity and alpha helical structure

polymeric support. The acid labile tertiary butyl oxycarbonyl (BOC) group or base labile fluorenyl methyl oxycarbonyl (FMOC) group is used for protect the functional group of amino acid (nalpha protection). The second protected amino acid is added after deprotection, using either a coupling reagent. The resulting peptide is attached to the resin through C terminals and cleaved to yield a amide, depending on the coupling agent used in the side chain protecting groups are selected so as to be cleaved both at the same time with detachment of the peptide from the resin. Deprotection of the BOC protecting group is achieved by 20% trifluoro acetic acid (TFA) in dichloromethane (DCM) and the FMOC protecting group by 20% piperidine in N-N-dimethyl formamide (DMF). Cleavage of the BOC amino acid containing peptide is achieved by liquid hydrogen fluoride (HF) and trifluoro methane sulphonic acid (TFMSA). DCM and DMF are the primary solvents used for resin deprotection, coupling and washing of peptide. In the continuous flow method the resin is contained in a column through, which reagents and solvents are pumped continuously again under manual or automatic control. FMOC strategy is 100% compatible with the continuous flow method, which depending on the instrument used for real time spectrophotometric monitoring of the progress of coupling and deprotection. Cleavage of the FMOC amino acid containing peptide and side chain deprotection requires TFA. Advantages • Simple filtration. • Synthesis can be carried out in one container • In SPPS, synthesize a large peptide (more amino acid present in the sequence of the peptide). • All the reactions involved in the synthesis should be carried 100% to completion. • All the laborious purification at intermediate steps in the synthesis is eliminated. Solid support The solid support is more appropriated in describing the insolubility of the polymer, which J Pharm Pharmacogn Res (2013) 1 (2): 43

Perumal & Pandey

allows filtration or centrifugation and separation of reactants from the peptides. Variety of solid support has been developed for SPPS. The characteristics of solid support is as follows: • It should be physically stable. • It must be inert. • It must be swell extensively in the solvents using synthesis. • It should be attaching the first entity either amino acid or organic molecules by the formation of covalent bond. Types of solid support • • • • •

Brush polymers. Composites. Gel type supports. Supported gels. Surface-type supports.

Gel type supports It contains four types of resins: • Polystyrene (PS) resins. • Poly acryl amide (PAA) resins. • Poly ethylene glycol (PEG) grafted resins. • PEG- based resins. Protective groups in peptide synthesis The reagents which are used to protect the functional group (amino group) present in the amino acid are termed as protective groups. Types of protective groups • Acid labile. • Base labile. • Other protecting groups. Acid labile It contain BOC group. The BOC group itself is sufficiently stable that the amino acid derivatives can be stored at room temperature. The deprotection of BOC amino acid can be obtained by 30 min treatment with 0.2% TFA in DCM.

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Antimicrobial peptides: hydrophobicity and alpha helical structure

Base labile It contains FMOC amino acid. Base labile alpha protecting is a main role in the solid phase peptide synthesis. This derivative removed from amino acid and peptide by treating with the solution of secondary amine (mostly piperidine) in DMF. FMOC amino acids provide a desirable orthogonal system for solid phase peptide synthesis and it is used for the synthesis of several peptides. The FMOC group loss may produce undesirable short N terminal sequences of the peptide being synthesized due to attachment of amino acid to the exposed hydroxyl groups thus starting new peptide chains. This problem is usually overcome by the use of an acid-labile resin, such as ether resin. When FMOC amino acids are used in coupling reaction the reaction is slow. Since the acid-labile ether resin should be used for FMOC solid phase peptide synthesis final cleavage of the peptide can be carried out by treatment with 25% TFA in DCM. Other protecting group Among the other types of alpha-protecting groups available, the 2-nitro phenyl sulfenyl (NPS) group has been used to some extent in solid phase peptide synthesis. This group can be removed from amino acid by very dilute anhydrous acid or nucleophiles. Treatment of NPS peptide or NPS amino acid with HCL causes formation of NPS chloride as byproduct this is essentially a reversal of the reaction used for the synthesis of NPS derivatives. Peptide protocol

synthesis

using

BOC-chemistry

The mostly used solvent is DCM. It can be purified by refluxing over phosphorous pentoxide (30 min) and normal distillation. TFA is the deprotection reagent. It can be bought in two purifies reagent grade and biograde, and should be stored in glass. Its boiling point is 71-73°C and a simple distillation is sufficient. BOC chemistry can be easily found in SPPS.

J Pharm Pharmacogn Res (2013) 1 (2): 44

Perumal & Pandey

Antimicrobial peptides: hydrophobicity and alpha helical structure

The purity of the best peptide and made by BOC chemistry was comparable with that of the best made by FMOC chemistry. Another interesting alpha-protecting group removable by nucleophillic attack is the di-thiosuccinyl group proposed. This may be very useful protecting group for solid phase peptide synthesis. Some protecting group for amino acids, acetyl, benzoyl, benzyl, butyl, ter-butyl, oxycarbonyl, 2,6,dichlorobenzyl. Peptide synthesis protocol

using

FMOC-chemistry

In the FMOC chemistry (Abatino and Papini, 2008) the most popular solvent is dimethyl foramide (DMF). DMF can be purified by refluxing over ninhydrin and distillation under reduced pressure it should be stored over for molecular sieves. Dimethyl acetamide (DMA) is used in solid phase peptide chemistry. It is slower decomposition than DMF and stock solution of tetramethyl uranium hexafluorophosphate (HBTU), diisopropyl ethylamine (DIPEA) are more stable in this solvent; DMA has a boiling point of 165-167°C. N-methyl pyrrolidine-one has excellent solvating properties and can improve coupling rates on the resin by reducing folding and aggregation of the glowing peptide chain. It can be used in combination with DMF on its own. It has a boiling point of 202-204°C. It can distill under high vacuum. Hydroxy benzotriazole (HOBT) is added during the coupling steps. Piperidine is used for the deprotection of the FMOC group. It can be distilled over KOH. It has boiling point of 104-106°C. A major problem in SPPS is the peptide chain aggregation due to either hydrophobic interactions or interchain hydrogen bonding. This occurs between 5-15 residues from the C-terminus and can lead to incomplete coupling and deprotection. Liquid phase peptide synthesis Liquid-phase peptide synthesis (LPPS) (Chan and is an old method still used for largescale synthesis. This method is slow, because the product has to be manually removed from the reaction solution after each step and requires another chemical group to protect the C-terminus of the first amino acid.

White, 2000)

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Advantage • The product is purified after each step. • Side reactions are easily detected. • Separate peptides are synthesized and then coupled together to create the larger peptides. Disadvantages • Synthesis cannot be carried out in one container, hence the product wastage is high. • In LPPS, synthesis of a large peptide is not possible. • Laborious purification at intermediate steps is complicated. CHARACTERIZATION The secondary structure of the AMPs can be characterized (Merrifield, 1963; Corsini et al., 2010) by circular dichroism (CD) and its detailed discussion is as follows: Circular dichroism The secondary structures (helical structure) of the antimicrobial peptides were characterized by circular dichroism. Here we discussed the following criteria regarding the basics of the circular dichroism in detailed way. Circularly polarized light is produced by passing a plane polarized light through a bi-refringent plate (it is a z-direction plate) which splits the light into two planepolarized beams oscillating along different axis (x axis and y axis). When one of the beams is retarded by 90°, then the two beams which are 90° out of phase are added together, final result is circularly polarized light of one direction. The two axes are inverted to produce circularly polarized light of the other direction. Finally, adding the right and left circularly polarized that passes through the optically active sample is elliptically polarized light, this is termed as circular dichroism. It is equivalent to ellipticity I. When the plane polarized light passed through the optically active medium, it changes the left and right circularly polarized right refractive index with diverese the rotation speed. J Pharm Pharmacogn Res (2013) 1 (2): 45

Perumal & Pandey

The medium is called circularly bi-refringent. In circular birefringence effect in addition to the speeds of the absorption coefficient of left circularly polarized light (EL) and the absorption coefficient of right circularly polarized light (ER) is also possible that these two components get absorbed to different extents. The absorption coefficient of the left polarized light is not equivalent to the absorption coefficient of the right polarized light and the absorption coefficient will not oscillate alone for extended. The medium is said to exhibit CD and the transmitted light would become elliptically polarized. The percentage of peptide helix can be calculated by the following formula:

Antimicrobial peptides: hydrophobicity and alpha helical structure

intra-molecular hydrogen bonding between peptide back-bone amide bonds. In the presence of TFE, a solvent known to promote and stabilize αhelix, CA-P2 displayed more α-helical content as seen in Fig. 3.

Molar ellipticity = 2000/-33000 • 100 Where, 2000 – Base line of CD spectrum. -33300 – Standard value of 100% helix. CD spectroscopy has been extensively used for the determination of secondary structures of proteins and peptides. We have used three media of different polarity to assess the conformational flexibility of the peptides. The peptide CA-P1 was largely unordered in water, the polar solvent. The peptide failed to adopt any regular structure because of hydrogen bonding between water and peptide back-bone. However, in the presence of sodium dodecyl sulphate (SDS) micelles, a condition that mimics bio-membrane, the peptide folds into α-helical conformation, which was stabilized by intra-molecular hydrogen bonding between peptide back-bone amide bonds. In the presence of trifluroethanol (TFE), a solvent known to promote and stabilize α-helix, CA-P1 displayed more α-helical content as seen in Fig. 2. CD spectroscopy was used for the determination of secondary CA-P2 peptide. Were used three media of different polarity to assess the conformational flexibility of the peptides. The peptide CAP2 was largely unordered in water, the polar solvent. The peptide fails to adopt any regular structure because of hydrogen bonding between water and peptide back-bone. However, in the presence of SDS micelles, the peptide folds into αhelical conformation, which was stabilized by http://jppres.com/jppres

Figure 2. Circular dichroism spectrum of cecropin-A peptide 1 (CA-P1) derivative in the medium of buffer, sodium dodecylsulphate (SDS) and trifluoroethanol (TFE).

CD spectroscopy was used for the determination of secondary CA-P3 peptide. Were used three media of different polarity to assess the conformational flexibility of the peptides. The peptide CAP3 was largely unordered in water, the polar solvent. The peptide failed to adopt any regular structure because of hydrogen bonding between water and peptide back-bone. However, in the presence of SDS micelles, the peptide folded into α-helical conformation, which was stabilized by intra-molecular hydrogen bonding between peptide back-bone amide bonds. In the presence of trifluroethanol, a solvent known to promote and stabilize α-helix, CA-P3 displayed less α-helical content as compared to CA-P2 (see Figs. 4 and 5). Fig. 5 shows an overlay of CD profiles of all three peptides viz., CA-P1, CA-P2 and CA-P3 in TFE-water (70:30). As it is clear from the traces, the peptide CA-P2 in more ordered than CA-P1. The peptide containing the modified tryptophan CA-P3 displays the lowest α-helical content, and is more likely to be non-toxic to human cells. The helix percentage of the CA-P3 was very less as compared with remaining two peptides due to more hydrophobicity. The parameters are shown in the Table 6. J Pharm Pharmacogn Res (2013) 1 (2): 46

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Antimicrobial peptides: hydrophobicity and alpha helical structure

[ɵ]MRE (deg. cm 2.dmol-1)

Cec-P2 1 = Buf 2 = SDS 3 = TFE

1 2 3

Wavelength (nm) Figure 3. Circular dichroism spectrum of octyl cecropin-A peptide 2 (CA-P2) derivative in the medium of buffer, sodium dodecylsulphate (SDS) and trifluoroethanol (TFE).

Table 6. The helix percentage of the peptides. Peptide name CA-P1

CA-P2

CA-P3

Solvent name

Ranges

Wavelength (nm)

Helix (%)

Sodium dodecyl suplhate

-6808

222

14.43

Trifluoroethanol

-9040

222

21.14

Sodium dodecyl suplhate

-11420

222

28.28

Trifluoroethanol

-14022

222

36.10

Sodium dodecyl suplhate

-7730

222

17.20

Trifluoroethanol

-6273

222

12.83

CA-P1 - cecropin-A peptide 1; CA-P2 - cecropin-A peptide 2; CA-P3 - cecropin-A peptide 3.

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Antimicrobial peptides: hydrophobicity and alpha helical structure

[ɵ]MRE (deg. cm 2.dmol-1)

Cec-P3 1 = Buf 2 = TFE 3 = SDS

1 3 2

Wavelength (nm) Figure 4. Circular dichroism spectrum of modified cecropin-A peptide 3 (CA-P3) derivative in the medium of buffer, sodium dodecylsulphate (SDS) and trifluoroethanol (TFE).

[ɵ]MRE (deg. cm2.dmol-1)

Cec-P1 1 = Buf 2 = SDS 3 = TFE

Cec-P2 1 = Buf 2 = SDS 3 = TFE

1

1

2 3

2

Cec-P3 1 = Buf 2 = SDS 3 = TFE

1 3 2

3

Wavelength (nm)

Figure 5. Overlay of circular dichroism profiles of all three peptides viz., cecropin A-peptide 1 (Cec-P1), cecropin A-peptide 2 (Cec-P2) and cecropin A-peptide 3 (Cec-P3).

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Antimicrobial peptides: hydrophobicity and alpha helical structure

MECHANISM OF ACTION The AMPs are divided into two types of mechanisms (Lee et al., 2011; Brogden, 2005; Mangoni and Shai, 2011). • Transmembrane pore–forming mechanisms. • Intracellular killing mechanisms.

Intracellular killing mechanisms It contains seven types of mechanisms and their examples are shown in Table 8. Table 8. Intracellular killing mechanisms of some peptides.

Transmembrane pore–forming mechanisms

Type of mechanism

Peptide

Reference

It contains three types of model and their examples are shown in Table 7.

Flocculation of intracellular contents

Human histatin

Andreu & Rivas, 1998

Alters cytoplasmic membrane septum formation

Indolicidin

Subbalakshmi & Sitaram, 1998

Inhibits cell-wall synthesis

Mersacidin

Brotz et al., 1998

Binds nucleic acids

Tachyplesin

Yonezawa et al., 1992

Inhibits nucleic-acid synthesis

Dermaseptin

Patrzykat et al., 2002

Inhibits protein synthesis

Indolicidin

Subbalakshmi & Sitaram, 1998

Inhibits enzymatic activity

Apidaecin

Otvos, 2000

Table 7. Transmembrane pore–forming mechanisms. Model name

Peptides examples

References

Barrel stave

Alamethicin

Bechinger, 1999

Carpet

Cecropin

Gazit et al., 1995

Toroidal pore

LL-37

Henzler Wildman et al., 2003

The barrel-stave model of antimicrobial peptide induced killing In this model, the attached peptides aggregate and insert into the membrane bilayer so that the hydrophobic peptide regions align with the lipid core region (Bechinger, 1999; Yang et al., 2001) and the hydrophilic peptide regions form the interior region of the pore. The carpet model of antimicrobial peptide induced killing In this model, the peptides disrupt the membrane by orienting parallel to the surface of the lipid bilayer (Hallock et al., 2003) and forming an extensive layer or carpet. Toroidal model of antimicrobial peptide induced killing The attached peptides aggregate and induce the lipid monolayers to bend continuously through the pore so that the water core was lined by both the inserted peptides and the lipid head groups (Nissen-Meyer et al., 2010; Matsuzaki et al., 1993).

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THERAPEUTIC CONCERN The therapeutic concern of the AMPs is clearly explained about the broad spectrum activity of AMPs against bacteria, fungus, virus and active against respiratory infections, sexually transmitted infections. AMPs were used for contraceptive potentials. It is also explained about AMPs in clinical trials with recent updates. The AMPs were particularly much active against resistance multidrug pathogens including bacteria, fungus, virus and protozoa. Antimicrobial peptides in anti-microbials The membrane active peptides were active against gram positive and gram negative organisms (Giacomeitti et al., 1998). Most of the peptides were lytic types. The antimicrobial activity of the peptides and their examples with details are given in Table 9 (Miyakawa et al., 1996).

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Antimicrobial peptides: hydrophobicity and alpha helical structure

Table 9. Antimicrobial activity of AMPs inducing lysis. Peptide

Source

Target

Defensin NP-1

Rabbit granulocyte

Cryptococcus neoformans

Defensin NP-2

Rabbit granulocyte

Aspergillus fumigatus

Human defensin

Human neutrophil

Mycobacterium tuberculosis

Magainin-2

Xenopus laevis

Candida albicans

Tripticin

Human

Aspergillus flavus

Antimicrobial peptides against sexually transmitted infections causing pathogens The AMPs were active against the various sexually transmitted infection causing pathogens and their examples are given in Table 10 (Zhang et al., 2002). Table 10. Antimicrobial peptides against transmitted infections causing pathogens.

sexually

Peptide

Mode of action

Target

Cecropin

Cytotoxic to the pathogen

Chlamydia trachomatis

Human α defensin 1, 2 and 3

CD8 antiviral factor secreted by CD8 T cells

Human immunodeficiency virus

Mellitin

Suppression of viral transcription

Human immunodeficiency virus

Protegrin

Membrane disruption of bacteria

Neisseria gonorrhoeae

Protegrin

Prevents uptake of elementary bodies by target cells

Chlamydia trachomatis

Antimicrobial potency

peptides

for

contraceptive

Magainin-A caused 100% sperm immobilization in rat (50 µg/ml), rabbit (400 µg/ml) and monkey and human (800 µg/ml) (Reddy et al., 2004). Antimicrobial peptides in clinical trials P-113 a derivative of histatin, a human salivary peptide is undergoing phase I/II trials to treat oral candidiasis (Paquette et al., 2002). Indolicidin analogue, MBI-549 is in Phase II trials for http://jppres.com/jppres

treatment of acne infections (Fella and Hancock, 1997). Pexiganan (Gaenera, USA), a 22-aminoacid analogue of magainin 2, was the first antimicrobial peptide to undergo commercial development as an antibiotic cream for the topical treatment of diabetic foot ulcers named LocilexTM. In 1999 FDA approval was denied because Pexiganan showed insufficient evidence of efficacy despite unanimous agreement about the drug’s performance in phase II trials at which stage most drugs (60-70%) fail due to low efficacy (Lamb and Wiseman, 1998). P113 (developed by Periodontix, USA, then acquired by Demegen, USA) is a 12-amino-acid cationic peptide based on histatins, naturally occurring AMPs in the saliva (Gordon et al., 2005) that demonstrated excellent in vitro activity against Candida albicans and common Grampositive and Gram-negative pathogens. Demegen licensed P113 to Pacgen (Canada) for treatment as a mouth rinse for oral candidiasis in HIV patients (approval for Phase I/II clinical study received on March 2006). AM-Pharma (The Netherlands) was focused on the development of lactoferricin-based peptides (11-mer peptide from the N-terminus of human lactoferricin, hLF-11) for the prevention of infections in patients undergoing hematopoietic stem cells transplantation (Phase I completed). Protegrin-1 is undergoing phase II/III trials to treat ventilator associated pneumonia. rBPI-21 derived from a human neutrophil peptide is undergoing phase II/III trials for treatment of severe paediatric meningococcaemia and Crohn’s disease. Role of antimicrobial peptides in host defence against vaccinia virus, trial estimated enrollment: 311, study started on June 2005, study completion on February 2010, clinical trials gov identifier is NCT00407069. CONCLUSIONS Antimicrobial peptides are a class of molecule of innate host defence obtained from plants, insects, animals, and humans. Synthesis of the peptides is more easy and convenient by using the methodology of solid phase peptide synthesis as compared with liquid phase peptide synthesis. J Pharm Pharmacogn Res (2013) 1 (2): 50

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© 2013 Journal of Pharmacy & Pharmacognosy Research, 1 (2), 54-63 ISSN 0719-4250 http://jppres.com/jppres Original article | Artículo original

Patrones de automedicación en clientes de una farmacia comunitaria de la ciudad de Antofagasta [Patterns of self-medication in customers of a community pharmacy in the Antofagasta city] Alejandrina Alucema*, Nicole Chavarría, Marisela Valdés Departamento de Ciencias Farmacéuticas, Facultad de Ciencias, Universidad Católica del Norte, Angamos 0610, Antofagasta, Chile. * E-mail: [email protected]

Abstract

Resumen

Context: Self-medication is the use of unprescribed drugs; this means that people obtain drugs on their own initiative and without the assistance of health professionals. This is an increasingly common practice among the population that can cause numerous problems, becoming a public health problem.

Contexto: La automedicación es el uso de medicamentos sin prescripción médica, es decir, por iniciativa propia de las personas, y sin la asistencia de profesionales de la salud. Esta es una práctica cada día más común entre la población que puede causar numerosos problemas, llegando a convertirse en un problema de salud pública.

Aims: To determine the patterns of self-medication in customers who frequented a community pharmacy in the city of Antofagasta.

Objetivos: Determinar los patrones de automedicación en clientes de una farmacia comunitaria de la ciudad de Antofagasta.

Methods: A survey was conducted to 297 users over 18 years and with adequate mental and communicative power to answer the questions, and attending the pharmacy to buy a drug without a prescription.

Métodos: Se aplicó una encuesta a 297 personas mayores de 18 años, con la facultad mental y comunicativa adecuada para responder las preguntas, y que asistieron a la farmacia para comprar un fármaco sin receta médica.

Results: The study revealed that of the 297 people who practiced selfmedication, 41% do it with a frequency of at least once a month. The woman practiced in 64% and the men in 36%. The range of age of the studied group was between 31-50 years. The main reason to practice the self medication was to recognize symptoms (33%), within the most notable was the headache (11%). The therapeutic group of the most requested medicines was the NSAIDs (20%), and of them the paracetamol was the most used. Conclusions: The results reveal that a high percentage of customers surveyed self-medicate, which shows this practice as a real problem, so it is necessary to educate people. Keywords: Self-medication; pharmacoepidemiology; drug use.

Resultados: El estudio reveló que de las 297 personas que practicaban la automedicación, el 41% lo hacían con una frecuencia de al menos una vez al mes. Las mujeres lo hacían con el 64% y los hombres con el 36%. El rango de edad del grupo estudiado se encontraba entre los 31-50 años de edad. El principal motivo para incurrir en la automedicación fue el reconocer síntomas leves (33%), dentro de los cuales el más señalado fue la cefalea (11%). El grupo terapéutico de medicamentos más solicitados fueron los AINE (20%), y de ellos, el paracetamol fue el más usado. Conclusiones: Los resultados revelan que un alto porcentaje de los clientes encuestados se automedican, lo que evidencia esta práctica como un problema real, por lo que se hace necesario educar a la población. Palabras Clave: Automedicación; farmacoepidemiología; utilización de medicamentos.

ARTICLE INFO Received | Recibido: November 16, 2013. Received in revised form | Recibido en forma corregida: December 13, 2013. Accepted | Aceptado: December 16, 2013. Available Online | Publicado en Línea: December 31, 2013 Declaration of Interests | Declaración de Intereses: Los autores declaran no tener conflicto de interés. Funding | Financiación: no declarada.

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Alucema et al.

INTRODUCCIÓN La automedicación es una práctica habitual y hoy en día forma parte de una problemática de carácter tanto nacional como mundial. La automedicación es una práctica recurrente en todos los países del mundo. En estudios realizados en Canadá, España e India se observó que la prevalencia de automedicación fue de 70%, 63% y 59%, respectivamente (Palacios, 2006). En América Latina también se ha demostrado que esta práctica es muy frecuente. En Brasil, un estudio realizado señaló que la automedicación fue el motivo más frecuente, entre 40% y 43%, para la adquisición de medicamentos en las farmacias (Haak, 1988). En Ecuador, se llevó a cabo un estudio en el que el 72% de las ventas se hizo sin prescripción alguna (Lalama, 1999). En México la utilización de medicamentos de libre acceso fue de 71% (Lezama et al., 1999). En Chile, la automedicación es un problema actual que ha ido en incremento. Por ello, el propósito de este trabajo fue conocer la causa de automedicación, los factores que influyen en ésta, y con qué fármacos se automedican las personas que acuden a una de farmacia comunitaria de la ciudad de Antofagasta. En el presente estudio se determinaron las caracteristicas sociodemograficas y los patrones de automedicación del grupo estudiado. MATERIALES Y MÉTODOS Descripción del estudio Se realizó un estudio descriptivo, transversal en una farmacia comunitaria ubicada en el sector centro-sur de la ciudad de Antofagasta. Para el desarrollo del trabajo se recolectaron los datos, durante los meses de junio a noviembre de 2012. Los clientes-pacientes seleccionados cumplieron con los criterios de selección establecidos. Estos fueron informados sobre la naturaleza del estudio y se les pidió su consentimiento para formar parte de este.

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Patrones de automedicación en Antofagasta

Criterios de selección La selección de los pacientes se basó en criterios de inclusión y exclusión, los cuales se listan a continuación: Criterios de inclusión. • Pacientes mayores de 18 años y menores de 78 años, de ambos sexos. • Pacientes que solicitaron medicamentos por iniciativa propia y sin prescripción médica. Criterios de exclusión • Pacientes con dificultad para comunicarse de manera verbal y escrita. • Pacientes que solicitaron fármacos con su respectiva prescripción médica. • Menores de 18 años, mayores de 78 años. • Pacientes que solicitaron un medicamento natural. • Pacientes que antes de solicitar un medicamento, realizaron una consulta con el Químico Farmacéutico. Tamaño de la muestra Se cálculo teniendo en cuenta la prevalencia de la automedicación en Chile, que es de 50% (MINSAL, 2004), el 90% de nivel de confianza y el 0,05 de margen de error. El tamaño de muestra inicial fue de 270. Considerando un error del 10% por omisión de respuestas, se calculó como muestra final 297 clientes. Instrumento de recolección de datos Se utilizó una encuesta para recolectar los datos. Esta constó de 11 preguntas y se aplicó en forma verbal por una de las investigadoras de este estudio. El instrumento consideró las siguientes variables, de acuerdo a Fuentes Albarrán (2006). Características sociodemográficas •

Género: clasificación en hombre o mujer basada en características anatómicas.

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• Edad: años cumplidos en el momento de la encuesta. • Nivel educacional: nivel de enseñanza educacional alcanzada. Patrones de automedicación. • Tipo de automedicación: se clasificó en “no responsable”, como aquellas circunstancias en las que el paciente obtuvo un medicamento sin receta médica, por iniciativa propia para aliviar algún síntoma, y “responsable”, como aquellas situaciones en las que, pese a que el paciente adquirió un medicamento sin prescripción médica, presentaba alguna patología crónica (diabetes, hipertensión, etc.) o situación médica cuyo tratamiento era permanente, y controlado por un profesional de salud. • Motivo de automedicación: permitió conocer cuál o cuáles fueron los motivos por los que el paciente solicitó un medicamento sin realizar una visita médica previa. • Síntomas principales: permitió saber cuál fue la dolencia o condición que presentó el paciente y que lo indujo a solicitar un medicamento sin prescripción médica. • Medicamento solicitado: permitió identificar el nombre comercial o nombre genérico del medicamento solicitado por el paciente para tratar el síntoma o condición. Posteriormente, estos medicamentos se agruparon según su acción terapéutica. • Recomendación del medicamento: permitió determinar cuáles fueron las fuentes de recomendación, o los factores que condujeron al cliente a solicitar un medicamento. Dentro de las fuentes o factores se tomaron en cuenta: auto-recomendación; recomendación de personas allegadas (familiares, amigos, compañeros de trabajo o vecinos); publicidad ofrecida en la farmacia y una antigua prescripción. Esta última se refiere a la conducta del paciente a recurrir al mismo medicamento prescrito con anterioridad. • Frecuencia de automedicación: permitió establecer si fue o no la primera vez que el paciente solicitó el medicamento sin prescripción médica y con qué frecuencia (cada http://jppres.com/jppres

Patrones de automedicación en Antofagasta

15 días, una vez al mes, cada dos a cuatro meses o cada cinco meses o más). • Lectura del prospecto: permitió conocer si el paciente que solicitó un medicamento sin prescripción médica, poseía el hábito de informarse a través de la lectura del prospecto. • Reconocimiento del Químico Farmacéutico: permitió comprobar el nivel de conocimiento por parte del cliente, sobre la labor de este profesional, y si el paciente demandaba una atención del farmacéutico, del vendedor, o simplemente no necesita algún tipo de consulta. Validación del instrumento Una vez elaborado el instrumento de trabajo la encuesta se aplicó previamente a 30 clientes para determinar si las preguntas eran comprendidas por éstos y si recogía los datos que precisaba la investigación. Posteriormente, se modificaron aquellas preguntas que resultaron de difícil comprensión para los clientes y se analizaron los datos recogidos, comprobándose que se obtenía la información requerida para el estudio. Análisis estadístico El análisis de datos se realizó con el paquete estadístico del programa Microsoft Office Excel 2011, con el cual se calcularon las medias, frecuencias y porcentajes. RESULTADOS Características sociodemográficas Distribución de pacientes según género Durante la etapa de estudio, se realizaron 297 encuestas a clientes de una farmacia comunitaria en la ciudad de Antofagasta. Del total de la población encuestada 64% fueron mujeres y 36% hombres. Distribución de pacientes según rango etario El promedio de edad del total de la población estudiada fue de 40 años. De los cuatro grupos J Pharm Pharmacogn Res (2013) 1 (2): 56

etarios, el que más incurrió en la automedicación fue el de 31-50 años (Fig. 1).

Patrones de automedicación en Antofagasta Motivos de automedicación

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Comodidad

7

Conocimiento

7

Fácil acceso a medicamentos

Falta de tiempo

Motivos económicos

1

No le gusta ir al médico

Reconocimiento de síntomas (leves)

0

15

13 6 10

20

30

40

Porcentaje (%)

50

51

Figura 2. Motivos que condujeron a los encuestados a la automedicación.

Distribución de la muestra según nivel educacional Se constató que la automedicación predominó en personas con estudios universitarios (54%), seguida del grupo con estudios de enseñanza media completa (24%) y estudios universitarios incompletos (17%). Los grupos de encuestados que no completaron su enseñanza media o que tenían enseñanza básica completa registraron el 2% cada uno, mientras que sólo el 1% de los clientes no habían completado la enseñanza básica. Patrones de automedicación Tipo de automedicación En relación con el tipo de automedicación que practicaban los pacientes, el 80% lo realizó de forma “no responsable”, mientras que el 20% lo efectuó de manera “responsable”. Esta última pese a no presentar una prescripción médica, era de un medicamento de uso permanente ya sea para una patología crónica o situación médica (anticonceptivos) controlada. Motivo de la automedicación Las razones que llevaron a los encuestados a esta práctica se muestran en la Fig. 2. Más de la mitad de ellos refirieron como motivo principal que los condujo a la automedicación fue el “reconocimiento de síntomas (leves)”.

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Síntomas principales La Fig. 3 muestra los síntomas, agrupados por tipos de padecimientos o situación médica, que condujeron a los clientes a la automedicación. Los síntomas predominantes fueron los neuralgicos (31%), siendo la cefalea el síntoma más recurrente. Le siguieron los respiratorios (25%), de los cuales los más citados fueron aquellos asociados al resfrío y congestión nasal. El 14% de los encuestados manifestaron haber presentado problemas gastrointestinales, predominando la acidez estomacal. La anticoncepción fue la condición médica más referida (11%), seguida de la cardiovascular (6%), específicamente la hipertensión. El resto de los síntomas no superó el 10%. Padecimientos o situación médica

Figura 1. Distribución de la muestra según grupo etario

Respiratorios Oseos Oftalmológicos Neurologicas Neurálgicos Ginecológicos Gastrointestinales Endocrinológicos Dermatológicos Cardiovasculares Anticoncepción

1 1

2

2

0

25

3

4

6

31

14 11

10 20 Porcentaje (%)

30

40

Figura 3. Padecimientos o situación médica que indujeron a los clientes a la automedicación.

Clasificación de los medicamentos El 75% del total de los medicamentos que solicitaron los clientes en la farmacia comunitaria resultaron ser de marcas registradas, mientras que sólo el 25% fueron genéricos. J Pharm Pharmacogn Res (2013) 1 (2): 57

60

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Patrones de automedicación en Antofagasta

Grupos terapéuticos Los grupos terapéuticos más solicitado fueron los analgésicos antiinflamatorios no esteroidales (AINE), con el 21% del total de medicamentos, seguido de los antigripales (18%) y los anticonceptivos (12%). Con porcentajes entre el 2-10%, se identificaron 11 grupos de fármacos (Fig. 4). En la clasificación “Otros”, se agruparon los grupos de fármacos que fueron solicitados en el 1% o menos, conformado por: antiflatulentos, antisépticos bucofaríngeos, antiepilépticos, antieméticos, antitusivos, antiulcerosos, antivirales, calciterápicos, corticoides, hipolipemiantes, hormonoterapia, relajantes musculares y aquellos utilizados para el colon irritable. 2 2 2 2

Grupos terapéuticos

Otros Oftálmico Mucolítico expectorante Hipoglicemiante Broncodilatador Antimicótico Antijaquecoso Antihistamínico Antihipertensivo Antigripal Antiespasmódico Antidiarreico Anticonceptivo Antiácido AINE

11

3 4

2 0

5 5

5

10

10

12 15

Porcentaje (%)

20

Frecuencia

Porcentaje (%)

1

2

3

5

3

5

1

2

3

5

Celecoxib Clonixinato de lisina Ketorolaco Piroxicam Ácido acetilsalicílico Ácido mefenámico Diclofenaco

1

2

7

12

Ibuprofeno

12

20

Ketoprofeno Metamizol sódico Naproxeno

4

7

1

2

4

7

Paracetamol

19

31

Fármacos 21

25

Figura 4. Grupos terapéuticos solicitados por los clientes.

Dentro del grupo terapéutico de los AINE, los tres fármacos más solicitados fueron: paracetamol (31%), ibuprofeno (20%) y diclofenaco (12%). En la Tabla 1 se pueden apreciar otros medicamentos dentro de este grupo, solicitados en porcentajes inferiores al 10%. En la Tabla 2 se muestran los productos antigripales solicitados por los clientes. En ella se constata que los más requeridos fueron: Tapsin limonada (42%) y Trioval día/noche (23%). Los productos más demandados dentro del grupo de los anticonceptivos, fueron: Yazmin y Anulete (11% cada uno). El 42% de las solicitudes correspondieron a “Otros”, en esta clasificación se incluyeron a 15 fármacos que su frecuencia de solicitud fue de una vez, durante el periodo de investigación (Tabla 3).

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Fármaco

Tabla 2. Fármacos solicitados por los clientes en el grupo antigripales (N=53).

18

4

Tabla 1. Fármacos solicitados por los clientes en el grupo de los AINE (N=62).

Frecuencia

Porcentaje (%)

Nastizol

6

12

Nastul compuesto

5

10

Tapsin dia/noche

7

13

Tapsin limonada

22

42

Trioval día/noche

12

23

Tabla 3. Productos demandados en el grupo de los anticonceptivos (N=35). Fármaco

Frecuencia

Porcentaje (%)

Anuar

2

6

Anulette

4

11

Ciclidon

2

6

Nordette

2

6

Nuvaring

2

6

Vexa cd

2

6

Yaz

2

6

Yazmin

4

11

Otros

15

42

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Recomendación del medicamento La mayoría de los medicamentos solicitados por los pacientes (45%) derivó de una “antigua prescripción”. La influencia de terceros en la recomendación del medicamento estuvo marcada por familiares (20%) y amigo o vecino (13%). La “publicidad de la farmacia” influyó en la compra del medicamento en el 17% de los clientes. Mientras que la “iniciativa propia” fue manifestada por el 5 % del grupo estudiado. Frecuencia de automedicación Al preguntar a los encuestados si era la primera vez que compraban el medicamento sin prescripción médica, el 77% aceptó haberlo adquirido en otras ocasiones de la misma forma, mientras que el 23% respondieron que era la primera vez que lo solicitaban sin una prescripción médica. En cuanto a la frecuencia en que las personas que admitieron haber adquirido el medicamento en otras ocasiones y practicaban la automedicación, el 41% de los clientes se automedicó cada mes, el 29% cada cinco meses o más, el 26% cada dos a cuatro meses y sólo el 4% cada 15 días. Lectura del prospecto informativo El 64% de las personas encuestadas manifestaron estar acostumbradas a leer el prospecto que trae el medicamento; mientras que el 36% declaró no leerlo. Solicitud de información al adquirir el medicamento en la farmacia Se le consultó al paciente si al momento de comprar los medicamentos sin prescripción médica, solicitaba información de éstos y a quién se la requería. El 31% dijo ayudarse con el auxiliar de farmacia, el 45% refirió que al momentos de solicitar el medicamento y presentaba una duda, pedían la presencia del Químico Farmacéutico y el 24% señaló no necesitar ayuda. Cuando se le consultó al encuestado si sabía quién era el profesional encargado de una farmacia, el 81% señaló reconocer al Químico Farma-

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Patrones de automedicación en Antofagasta

céutico como tal, mientras que el 19% señalaron no saber de la existencia de éste. DISCUSIÓN Al analizar las encuestas realizadas en la farmacia comunitaria, se demostró que la mayor parte de las solicitudes de medicamentos sin prescripción médica fueron efectuadas por personas del sexo femenino. Esto refleja la preocupación de las mujeres por su salud y también por la de su entorno familiar, ya que son ellas quienes tienen más tendencia a comprar medicamentos por iniciativa propia y también tienen el hábito de almacenar continuamente medicamentos. Además, las mujeres tienen la facilidad de reconocer y expresar que tienen algún problema de salud, mientras que los hombres no lo afrontan y evitan tomar algún medicamento. Es de señalar que las mujeres tienen posibles síntomas asociados al periodo menstrual, que requieren frecuentemente el consumo de algún determinado medicamento como analgésicos, antiinflamatorios no esteroidales. Los resultados de este estudio coinciden con los obtenidos recientemente, los cuales concluyeron que los motivos por los cuales las mujeres se automedican más que los hombres, se asemejan a los señalados anteriormente (Wazaify et al., 2005; Figueras et al., 2000). La práctica de automedicación más frecuente en el grupo etario 31-50 años podría estar asociada a la disponibilidad de tiempo, por razones de trabajo cuentan con menos tiempo para consultar un profesional de la salud. Similar comportamiento se observó en el estudio realizado en una localidad de Bogotá, en el que los rangos etarios que predominaron fueron de 32-45 y de 46-58 años (López et al., 2009). Con respecto al nivel educacional, en este estudio se evidenció un elevado porcentaje (54%) de automedicación en aquellos clientes que tenían formación universitaria. Este hecho podría deberse a que estas personas saben cómo manejar y acceder a información sobre medicamentos, mediante consultas a través de internet u otras fuentes de información, o a un sentimiento de independencia y autonomía mayor, que podría hacerles obviar la visita médica. Este resultado J Pharm Pharmacogn Res (2013) 1 (2): 59

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coincide con un estudio efectuado en Colombia, en el que se encontró el mayor porcentaje de automedicación en las personas con nivel de escolaridad alto, para bachilleres (45,2%) y universitarios (34,6%) (Peñuela et al., 2002). En relación con el tipo de automedicación que practican los pacientes, se evidenció que un elevado porcentaje (80%) lo efectuaba de manera “no responsable” y en menor proporción (20%) en forma “responsable. Las automedicación “no responsable” podría atribuirse a la falta de educación sanitaria que presenta Chile, específicamente respecto a medicamentos, así como también, a la falta de conciencia por parte de la población respecto de su salud. Mientras que aquellas personas que refirieron automedicarse de manera responsable, reconocen su situación de salud y la importancia de seguir las recomendaciones médicas. Este comportamiento se corresponde con lo planteado por la Organización Mundial de la Salud (OMS), al referir que la automedicación responsable requiere de condiciones que sean auto reconocibles por el individuo o también para condiciones crónicas o recurrentes (luego de un diagnóstico médico inicial) (Wilbur et al., 2010). Los motivos que condujeron a las personas encuestadas a la automedicación son variados. En estudios realizados en distintos países de Latinoamérica, incluido Chile (López et al., 2009; Moreno y Apablaza, 2009; Riedemann et al., 2001; Centeno, 1993), se consideró como principal motivo la

economía, ya que cuando las personas poseen dificultades de acceso al sistema público de salud y además ingresos de recursos insuficientes, se les hace muy difícil acceder a cuidados médicos, recurriendo a la automedicación como solución a sus enfermedades. Cabe destacar que, el motivo “problemas económicos” en este estudio reflejó sólo el 1%, lo que haría suponer que éste no fue uno de los factores determinantes para la automedicación del grupo en estudio. No obstante, se debe tomar en cuenta que la farmacia en donde se realizaron las encuestas atiende principalmente a clientes con nivel socioeconómico elevado, lo cual se respalda con el valor del vale promedio del local, el cual fue alrededor de los CLP 8.000 ($US 15.00), por lo que podrían http://jppres.com/jppres

Patrones de automedicación en Antofagasta

esperarse resultados diferentes en otros sectores de la cuidad. El motivo que más se destacó en este trabajo fue la capacidad de “reconocer síntomas” que los describían como leves, por lo que consideraban que no era necesario acudir a una consulta médica; además eran síntomas que habían manifestado en otras ocasiones y tratados con anterioridad, por lo que los encuestados preferían automedicarse con el mismo medicamento que le fue indicado. Este resultado concuerda con un estudio realizado en Perú, en el que las principales motivaciones para automedicarse estuvieron relacionadas con el "conocimiento suficiente" para manejar las molestias presentadas (Llanos et al., 2001). La influencia de terceros en la recomendación del medicamento alcanzó el segundo lugar (20% familiares y 13% amigo). Al contrario de lo obtenido en nuestro estudio, un trabajo realizado en Ecuador evidenció que las recomendaciones por parte de familiares y amigos fueron el motivo más señalado ante la solicitud de un medicamento (Moreno y Apablaza, 2009). Por otra parte, la recomendación más influyente en este estudio, correspondió a la repetición de una antigua prescripción médica. Los clientes referían presentar los mismos síntomas o patología que la última vez, por lo que recurrían al mismo medicamento que le indicaron en dicha ocasión. Si bien con esta práctica se puede lograr el efecto esperado de recuperar la salud, no es la mejor forma, ya que los síntomas pueden variar en gravedad pudiendo requerir más o menos dosis, o bien un medicamento distinto al utilizado anteriormente. En Argentina, un estudio similar también encontró que la “prescripción antigua” fue uno de los motivos más frecuentes que conllevaron a la automedicación, en el que el 44,7% de las personas encuestadas emplearon medicamentos prescritos anteriormente (Blanco et al., 2010). Se sabe que la propaganda ofrecida al consumidor en las farmacias genera un aumento de las ventas de los productos anunciados. En este estudio se constató que la publicidad no fue la motivación que más influyó en la automedicación de los clientes. J Pharm Pharmacogn Res (2013) 1 (2): 60

Alucema et al.

En este estudio se evidenció un mayor consumo de medicamentos de marcas registradas, lo que puede deberse a diferentes razones. Una de ellas puede ser el potenciamiento de la farmacia a la venta de un medicamento de marca por sobre uno genérico o bien al nivel socioeconómico de la población estudiada, esto puede asociarse al sector donde se sitúa la farmacia, el cual es habitado por personas de recursos económicos elevados. Otra razón puede ser la propaganda de los laboratorios, ya sea por la televisión, radio o de forma escrita, lo que permite mantener en la memoria de la gente los medicamentos patrocinados. Ante un dolor de cabeza, una leve molestia muscular o un picor en la garganta las personas suelen tomar, sin prescripción médica previa, algún medicamento que alivie aquellos síntomas, sin darse cuenta que pueden estar enmascarando el verdadero problema. Para las personas aliviar sus síntomas rápidamente es la primera necesidad, por lo que recurren a la automedicación. El síntoma más tratado en este estudio fue el dolor, esto coincide con la mayoría de los estudios revisados en los que el síntoma más tratado fue el dolor de cabeza, seguido por otros dolores (Moreno y Apablaza, 2009; Celosari et al., 2004). Otro de los síntomas más recurrentes fueron los padecimientos respiratorios. Esto pudiera explicarse por el periodo en que se recolectaron los datos, que fue en época de invierno del 2012, en la que los síntomas de resfrío son de mayor incidencia. Los AINE fueron los medicamentos más consumidos por los encuestados, resultados que coinciden con los obtenidos en estudios realizados en España (Guillem et al., 2010; Asenjo Segura y Sáenz Calvo, 2008; Baos, 2000). El consumo de AINE tiene una coincidencia razonable en relación con la frecuencia de síntomas que referían los pacientes al momento de la encuesta, que fue el dolor. Resulta preocupante el uso de este tipo de medicamentos, ya que se ha comprobado que su uso prolongado desarrolla graves efectos secundarios, los más frecuentes son los problemas gastrointestinales, entre los cuales se pueden señalar la úlcera gástrica y hemorragia gastrointestinal alta, como los más complejos. Estos efectos se deben fundamentalmente a la capahttp://jppres.com/jppres

Patrones de automedicación en Antofagasta

cidad de los AINE de inhibir la enzima COX-1 Así, uno de cada 1.000 consumidores de AINE por año presenta una complicación gastrointestinal grave, en los que la prevalencia es muy superior en sujetos mayores de 60 años que en individuos jóvenes (Quintero, 2000). Los AINE también pueden producir toxicidad renal, producida por una inhibición de la síntesis de las prostaglandinas. Además, se ha visto un aumento de toxicidad hepática, debido al uso sin prescripción médica del paracetamol (Duarte, 2010). Los antigripales fueron el segundo grupo más utilizado (18%), en los que el más adquirido fue Tapsin limonada, probablemente porque es uno de los más promocionados en televisión y porque constantemente es parte de las ofertas de la farmacia. Cabe destacar que, aunque sean de venta directa se debe tener precaución con sus contraindicaciones, principalmente las personas hipertensas, debido a su contenido de pseudoefedrina. Los resultados analizados reflejan que la práctica de automedicarse es habitual en los clientes, ya que más de la mitad de los encuestados admitieron que no era la primera vez que se automedicaban. Entre quienes señalaron consumir algún medicamento sin receta, lo hacían con una frecuencia de por lo menos una vez al mes. Al contrario de lo obtenido en este estudio, un trabajo realizado en Ecuador evidenció que el 58% de la población estudiada se automedica cada 2 a 4 meses, refiriendo que fue el periodo en el que se presentaban síntomas gripales (Moreno y Apablaza, 2009). La automedicación es una conducta cada vez más frecuente en la población, lo cual se puede explicar porque es una práctica accesible, cómoda y económica para el paciente. Sin embargo, las personas no consideran que el utilizar un medicamento sin un diagnóstico previo, puede conllevar un problema de salud mayor al que originó la automedicación. Al analizar los resultados de la lectura del prospecto, el 64% de la población encuestada dijo que acostumbraba a leerlo, mientras que sólo el 36% señaló lo contrario. Dentro de las razones de por qué no leían el informativo se señalaron: letra (Hardman et al., 2001).

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muy pequeña, demasiada información escrita o pereza. Este resultado refleja que la mayor parte de la población trata de informase por iniciativa propia sobre los medicamentos que va a utilizar. De esta misma manera lo señala un estudio realizado en Argentina, en el que el 66% de los encuestados declaró leer los prospectos que contenían los medicamentos (Nounou et al., 2009). Cabe destacar que el prospecto que trae el medicamento es una herramienta importante para el proceso de automedicación, ya que entrega información general sobre el fármaco solicitado, pero no es suficiente. Si bien leer la información es un acto responsable es necesario tener en cuenta que no todas las personas pueden utilizar los mismos medicamentos, ya que muchas veces es necesario tener ciertas precauciones o realizar ajustes especiales en cada paciente, de ahí la importancia de no automedicarse y siempre considerar como primera opción acudir al médico. El nivel educacional es un factor influyente en esta práctica de leer el prospecto, ya que las personas con un nivel de enseñanza superior, como se presenta en este estudio, tienen el hábito de informarse sobre lo que van a consumir. En lo que concierne a la solicitud de información del medicamento, el 45% del grupo estudiado refirió que acudía al Químico Farmacéutico. Se cree muchas veces que el rol del Químico Farmacéutico no es bien conocido por la población, por lo que el asesoramiento que este puede ofrecer no es relevante. Sin embargo, los resultados de este estudio reflejaron que el grupo de encuestado conocía el papel del Químico Farmacéutico, no sólo como un dispensador de medicamentos si no también, como aquel que lo puede asesorar sobre la calidad, seguridad y eficacia de los medicamentos. El resultado de pedir ayuda al Químico Farmacéutico respalda el reconocimiento de éste como profesional de salud, ya que el 81% de las personas conocían su labor y la encontraban útil, además lo identificaban entre los demás funcionarios. Esto concuerda con los resultados obtenidos por el estudio “Responsible Self-Medication in Latin America”, en el que el 88% de los encuestados consideraron útil y necesario el rol del Quíhttp://jppres.com/jppres

Patrones de automedicación en Antofagasta

mico Farmacéutico en la Farmacia Comunitaria (Bolaños, 2005). CONCLUSIONES El estudio puso de manifiesto que la automedicación es una práctica frecuente en el grupo de estudio, propiciado por el fácil acceso y disponibilidad de los medicamentos para tratar síntomas leves auto-reconocidos. Un elevado porcentaje de los pacientes-clientes no es consciente de los peligros que enfrentan al automedicarse de manera no responsable para aliviar las dolencias, lo que se evidenció por el amplio uso de fármacos dentro del grupo de los AINE. Es de notar que se encontró como fuente principal de información sobre medicamento la lectura del prospecto, acción que puede considerarse como un acto de responsabilidad del paciente. Este comportamiento está asociado al elevado porcentaje de encuestados con nivel educacional alto. Además, se constató que la mayor proporción de los clientes encuestados reconoce al Químico Farmacéutico como profesional de la salud, por lo que solicitan su orientación para el uso del medicamento, situación que refuerza el rol clave de la profesión en esta área de desarrollo laboral. Los hallazgos del estudio se basan en una muestra de clientes de una farmacia comunitaria, por tanto, los resultados de este no pueden generalizarse a toda la ciudad de Antofagasta. Más investigaciones de este tipo deben llevarse a cabo en pacientes-clientes de otras farmacias y población en general, para comprender mejor las diversas características sociodemográficas y los patrones de automedicación. Resulta necesario establecer estrategias que permitan controlar y mejorar la automedicación, con miras a incrementar conductas responsables de la población durante esta práctica y educar para un uso más racional, seguro y eficaz de los fármacos. En este aspecto, el papel del Químico Farmacéutico resulta importante para ofrecer al paciente información y asesoramiento acerca de los medicamentos más utilizados en la automedicación. Ya que solamente mediante pacientes informados se podría disminuir una J Pharm Pharmacogn Res (2013) 1 (2): 62

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automedicación desordenada y lograr lo que la OMS pretende, una automedicación responsable. CONFLICTO DE INTERÉS Las autoras declaran no poseer conflicto de interés. AGRADECIMIENTOS Las autoras agradecen a la farmacia comunitaria por permitir realizar el estudio en su local y a los clientes que participaron en este. REFERENCIAS Ausejo Segura M, Sáenz Calvo A (2008) ¿Cómo mejorar la automedicación de nuestros pacientes? Aten Primaria 40(5): 221-223. Baos V (2000) Estrategias para reducir los riesgos de la automedicación. Inf Ter Sist Nac Salud 24(6): 147-152. Blanco M, Olmos B, Quijano M, Arizaga D (2010) Automedicación y autoprescripción en pacientes que concurren a centros de salud de la ciudad de Barranqueras. Rev Posgrado VIa Cátedra de Medicina 201: 3-7. Bolaños H (2005) Responsible self-medication in Latin America. Drug Inf J 39(1): 99-106. Centeno M (1993) Automedicación en el distrito de Cuzco: estudio del nivel y factores asociados. Soc Peru Epidemiol 1(6): 46-50. Cesolari JA, Calvi B, Garrote N, Pérez B, Busmail L (2004) Automedicación, un problema de educación médica. Rev Med Rosario 70: 139-145. Duarte A (2010) Reacciones de hipersensibilidad a los antiinflamatorios esteroideos. Tesis doctoral. Facultad de Medicina. Universidad de Murcia. Figueiras A, Caamano F, Gestal-Otero JJ (2000) Sociodemographic factors related to self medication in Spain. Eur J Epidemiol 16: 19–26. Fuentes Albarrán KA (2006) Análisis y cuantificación de los patrones de automedicación en usuarios de farmacias SalcoBrand de Valdivia. Tesis de grado para título de Químico Farmacéutico. Escuela de Química y Farmacia, Facultad de Ciencias. Universidad Austral de Chile. Guillem P, Francès F, Gimenez F, Sáiz C (2010) Estudio sobre automedicación en población universitaria española. Rev Clin Med Fam 3(2): 99-103. Haak H (1988) Pharmaceuticals in two Brazilian villages: lay practices and perceptions. Soc Sci Mod 27: 1415-1427. Hardman JG, Limbird LE, Gilman AG (2001) Goodman & Gilman's The Pharmacological Basis of Therapeutics (10 edición). New York: McGraw-Hill. pp. 1825.

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Patrones de automedicación en Antofagasta Lalama M (1999) Perfil de consumo de medicamentos en la ciudad de Quito, Ecuador. Educ Med Contin 67: 7-9. Lezama M, Faba G, Martínez JA (1999) Automedicación responsable en la República Mexicana. International Workshop on Responsible Self- Medication in Latin America in the Global Society Information, México. Sep. 23-24. Llanos LF, Contreras CE, Velásquez JE, Mayca JA, Lecca L, Reyes R, Peinado J (2001) Automedicación en cinco provincias de Cajamarca, Perú. Rev Med Hered 12: 127133. López JJ, Dennis R, Moscoso S (2009) Estudio sobre la automedicación en una localidad de Bogotá. Rev Salud Pública 11(3): 432-442. MINSAL. Gobierno de Chile (2004) Política Nacional de Medicamentos en la reforma de salud. Ministerio de salud. 2 de abril 2004. Moreno A, Apablaza P (2009) Determinación de los patrones de automedicación en las poblaciones amazónicas de Yacuambi y El Pangui. Tesis de grado para título de Médico. Centro Universitario Loja. Nounou B, Cattaneo M, Salmon R, Palasezze L, Boccaleri J, Cestona E, Bedecarrás F, Ranieri F, Talevi A, Muñoz SM (2009) Estudio sobre consumo y la automedicación con antibióticos en la cuidad de La Plata, Buenos Aires, Argentina. Lat Am J Pharm 28(4): 544-551. Palacios F (2006) Automedicación psiquiátrica en una muestra del barrio Batahola norte durante agosto a octubre 2006 en Nicaragua. Tesis para título de especialista en Psiquiatría. Facultad de Ciencias Médicas. Universidad Nacional Autónoma de Nicaragua. Peñuela M, Espriella A, Escobar E, Velásquez MV, Sánchez J, Arango A, Gómez O (2002) Factores socioeconómicos y culturales asociados a la autoformulación en expendios de medicamentos en la ciudad de Barranquilla. Salud Uninorte. Barranquilla 16: 30-38. Quintero E (2000) Efectos de los AINEs sobre la mucosa gastrointestinal. Rev Española Reumatol 27: 19-24. Riedemann JP, Illesca M, Droghetti J (2001) Automedicación en individuos de la Región de la Araucanía con problemas músculo-esqueléticos. Rev Med Chile 129(6): 1-7. Wazaify M, Shield E, Hughes CM, McElnai JC (2005) Societal perspectives on over the counter (OTC) medicines. Fam Pract 22: 170-176. Wilbur K, El Salam S, Mohammadi E (2010) Patient perceptions of pharmacist roles in guiding selfmedication of over-the counter therapy in Qatar. Patient Preference Adherence 4: 87-93.

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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.

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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.

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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

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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

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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

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

 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]

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

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

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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.

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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

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

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.

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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

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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

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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.

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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.

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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.

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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

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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

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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

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 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

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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

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 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.

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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

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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

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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

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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

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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.

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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

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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.

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