ACTA PHARMACEUTICA SCIENCIA

ACTA

PHARMACEUTICA

SCIENCIA

International Journal in Pharmaceutical Sciences, Published Quarterly

ISSN: 2636-8552

e-ISSN: 1307-2080,

Volume: 57, No: 2, 2019

Formerly: Eczacılık Bülteni

Acta Pharmaceutica Turcica

Fatih Demirci (Anadolu University, Turkey)

Hakan Göker (Ankara University, Turkey)

Hanefi Özbek

(İstanbul Medipol University, Turkey)

Hayati Çelik (Yeditepe University, Turkey)

İhsan Çalış (Near East University, Cyprus)

Julide Akbuğa (Altınbaş University, Turkey)

Kenneth A. Jacobson

(National Institutes of Health, USA)

Leyla Yurttaş (Anadolu University, Turkey)

Mahmud Miski (İstanbul University, Turkey)

Mesut Sancar (Marmara University, Turkey)

Murat Duran

(Eskişehir Osmangazi University, Turkey)

Nesrin Emekli

(İstanbul Medipol University, Turkey)

Nilay Aksoy (Altınbaş University, Turkey)

Nurşen Başaran (Hacettepe University, Turkey)

Özgen Özer (Ege University, Turkey)

Roberta Ciccocioppo

(University of Camerino, Italy)

Selma Saraç Tarhan

(Hacettepe University, Turkey)

Semra Şardaş (İstinye University, Turkey)

Sevda Süzgeç Selçuk

(İstanbul University, Turkey)

Stefano Constanzi (American University, USA)

Süreyya Ölgen (Biruni University, Turkey)

Şule Apikoğlu Rabuş

(Marmara University, Turkey)

Tuncer Değim (Biruni University, Turkey)

Yıldız Özsoy (İstanbul University, Turkey)

Yusuf Öztürk (Anadolu University, Turkey)

Printing Office

Forart Basımevi

Ziya Gökalp Mah.

Süleyman Demirel Bulvarı

Simpaş İş Modern Zemin Kat A 17-18

İkitelli - İstanbul

Tel: (0212) 501 82 20

Editor

Şeref Demirayak

Associate Editors

Gülden Zehra Omurtag

Barkın Berk

Coordinators

M. Eşref Tatlıpınar

Gökberk Karabay

Language Editor

Recep Murat Nurlu

M. Eşref Tatlıpınar

Neda Taner

Biostatistics Editor

Pakize Yiğit

Graphic Design

Sertan Vural

Levent Karabağlı

Address

İstanbul Medipol Üniversitesi

Kavacık Güney Kampüsü

Göztepe Mah. Atatürk Cad.

No: 40 34810 Beykoz/İSTANBUL

Tel: 0216 681 51 00

E-mail

[email protected]

Web site

http://www.actapharmsci.com

Editorial Board

Sabahattin Aydın

(Istanbul Medipol University, Turkey)

Ahmet Aydın (Yeditepe University, Turkey)

Ahmet Çağrı Karaburun (Anadolu University, Turkey)

Aristidis Tsatsakis (University of Crete, Greece)

Ayfer Beceren (Marmara University, Turkey)

Dilek Ak (Anadolu University, Turkey)

Ebrahim Razzazi-Fazeli

(University of Veterinary Medicine, Vienna)

Erem Memişoğlu Bilensoy

(Hacettepe University, Turkey)

Fatma Tosun

(İstanbul Medipol University, Turkey)

ACTA

PHARMACEUTICA

SCIENCIA

International Journal in Pharmaceutical Sciences

is Published Quarterly

ISSN: 2636-8552

e-ISSN: 1307-2080,

Volume: 57, No: 2, 2019

Formerly: Eczacılık Bülteni/Acta Pharmaceutica Turcica

Founded in 1953 by Kasım Cemal Güven

Contents

Aims and Scope of Acta Pharmaceutica Sciencia

Şeref Demirayak ......................................................113

Instructions for Authors ...............................................114

Original articles ...................................................... 128

Synthesis, Characterization and Evaluation of the Anti-cancer Activity of Silver

Nanoparticles by Natural Organic Compounds Extracted from Cyperus sp.

rhizomes

Rasim Farraj Muslim, Mustafa Nadhim Owaid ............................... 129

Anti-inflammatory and Analgesic Potential of Acetone Leaf Extract of Combretum

Sordidum and its Fractions

Babatunde Samuel, Olayinka Oridupa, Fisayo Gbadegesin ....................147

Combination of Cumulative Area Pre-Processing and Partial Least Squares for

Handling Intensely Overlapping Binary and Ternary Drug Systems

Yahya ِAl-Degs, Amjad El-Sheikh, Eman Abu Saaleek, Reema Omeir, Musab Al-

Ghodran ............................................................161

Chemical Composition and Comparative Antibacterial Properties of Basil

Essential Oil against Clinical and Standard Strains of Campylobacter spp.

Aysegul Mutlu-Ingok, Burcu Firtin, Funda Karbancioglu-Guler .................. 183

In vitro Antimicrobial and Antioxidant Evaluation of Melampyrum Arvense Var.

Elatius L. and Sedum Spurium M. Bieb. Extracts

Ayşe Esra Karadağ, Fatma Tosun ........................................193

Preparation and In vitro Characterization of a Fluconazole Loaded Chitosan

Particulate System

Gülsel Yurtdaş-Kırımlıoğlu, Yenilmez Evrim, Başaran Ebru, Yazan Yasemin

Review articles ...................................................... 216

Pharmaceutical Properties of Marine Polyphenols:

An Overview

Thanh Sang Vo, Dai Hung Ngo, Se-Kwon Kim ...............................217

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Aims and Scope of Acta Pharmaceutica Sciencia

Acta Pharmaceutica Sciencia is a continuation of the former “Eczacılık Bülteni”

which was first published in 1953 by Prof. Dr. Kasım Cemal Güven’s editorship.

At that time, “Eczacılık Bülteni” hosted scientific papers from the School of Med-

icine-Pharmacy at Istanbul University, Turkey.

In 1984, the name of the journal was changed to “Acta Pharmaceutica Turcica”

and it became a journal for national and international manuscripts, in all fields

of pharmaceutical sciences in both English and Turkish. (1984-1995, edited

by Prof. Dr. Kasım Cemal Güven, 1995-2001, edited by Prof. Dr. Erden Güler,

2002-2011, edited by Prof. Dr. Kasım Cemal Güven)

Since 2006, the journal has been published only in English with the name, “Acta

Pharmaceutica Sciencia” which represents internationally accepted high-level

scientific standards. The journal has been published quarterly except for an in-

terval from 2002 to 2009 in which its issues were released at intervals of four

months. The publication was also temporarily discontinued at the end of 2011

but since 2016, Acta Pharmaceutica Sciencia has continued publication with the

reestablished Editorial Board and also with the support of you as precious sci-

entists.

Yours Faithfully

Prof. Dr. Şeref DEMİRAYAK

Editor

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INSTRUCTIONS FOR AUTHORS

1. Scope and Editorial Policy

1.1. Scope of the Journal

Acta Pharmaceutica Sciencia (Acta Pharm. Sci.), formerly known as Bulletin of

Pharmacy and Acta Pharmaceutica Turcica is a peer-reviewed scientific jour-

nal publishing current research and reviews covering all fields of pharmaceu-

tical sciences since 1953.

The original studies accepted for publication must be unpublished work and

should contain data that have not been published elsewhere as a whole or a

part. The reviews must provide critical evaluation of the state of knowledge

related with the subject.

All manuscripts has to be written in clear and concise English.

Starting from 2016, the journal will be issued quarterly both in paper and on-

line formates also publish special issues for national or international scientific

meetings and activities in the coverage field.

1.2 Manuscript Categories

Manuscripts can be submitted as Research Articles and Reviews.

1.2.1 Research Articles are definitive accounts of significant, original studies.

They are expected to present important new data or provide a fresh approach

to an established subject.

1.2.2 Reviews integrate, correlate, and evaluate results from published litera-

ture on a particular subject. They expected to report new and up to date expe-

rimental findings. They have to have a well-defined theme, are usually critical,

and may present novel theoretical interpretations. Up to date experimental

procedures may be included. Reviews are usually submitted at the invitation

of the Editors. However, experts are welcome to contact the Editors to ensure

that a topic is suitable. Approval is recommended prior to submission.

1.3 Prior Publication

Authors should submit only original work that has not been previously publis-

hed and is not under consideration for publication elsewhere. Academic the-

ses, including those on the Web or at a college Web site, are not considered to

be prior publication.

1.4 Patents and Intellectual Property

Authors need to resolve all patent and intellectual property issues. Acceptance

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and publication will not be delayed for pending or unresolved issues of this

type. Note that Accepted manuscripts and online manuscripts are considered

as published documents.

1.5 Professional Ethics

Editors, reviewers, and authors are expected to adhere to internationally ac-

cepted criteria’s for scientific publishing.

1.5.1 Author Consent. Submitting authors are reminded that consent of all

coauthors must be obtained prior to submission of manuscripts. If an author

is removed after submission, the submitting author must have the removed

author consent to the change by e-mail or faxed letter to the assigned Editor.

1.5.2. Plagiarism. Manuscripts must be original with respect to concept, con-

tent, and writing. It is not appropriate for an author to reuse wording from other

publications, including one›s own previous publications, whether or not that

publication is cited. Suspected plagiarism should be reported immediately to

the editorial oice. Report should specifically indicate the plagiarized material

within the manuscripts. Acta Pharmaceutica Sciencia uses iThenticate or Turni-

tin software to screen submitted manuscripts for similarity to published materi-

al. Note that your manuscript may be screened during the submission process.

1.5.3. Use of Human or Animal Subjects. For research involving biolo-

gical samples obtained from animals or human subjects, editors reserve the

right to request additional information from authors. Studies submitted for

publication approval must present evidence that the described experimental

activities have undergone local institutional review assessing safety and hu-

mane usage of study subject animals. In the case of human subjects authors

must also provide a statement that study samples were obtained through the

informed consent of the donors, or in lieu of that evidence, by the authority

of the institutional board that licensed the use of such material. Authors are

requested to declare the identification or case number of institution approval

as well as the name of the licensing committee in a statement placed in the

section describing the studies’ Material and Methods.

1.6 Issue Frequency

The Journal publishes 4 issues per year.

2. Preparing the Manuscript

2.1 General Considerations

Manuscripts should be kept to a minimum length. Authors should write in cle-

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ar, concise English, employing an editing service if necessary. For professional

assistance with improving the English, figures, or formatting in the manuscript

before submission please contact to editorial oice by e-mail for suggestions.

The responsibility for all aspects of manuscript preparation rests with the aut-

hors. Extensive changes or rewriting of the manuscript will not be undertaken

by the Editors. A standard list of Abbreviations, Acronyms and Symbols is in

section 5.

It is best to use the fonts “Times” and “Symbol.” Other fonts, particularly those

that do not come bundled with the system software, may not translate pro-

perly. Ensure that all special characters (e.g., Greek characters, math symbols)

are present in the body of the text as characters and not as graphic represen-

tations. Be sure that all characters are correctly represented throughout the

manuscript—e.g., 1 (one) and l (letter l), 0 (zero) and O (letter o).

All text (including the title page, abstract, all sections of the body of the paper,

figure captions, scheme or chart titles, and footnotes and references) and tab-

les should be in one file. Graphics may be included with the text or uploaded as

separate files. Manuscripts that do not adhere to the guidelines may be retur-

ned to authors for correction.

2.1.1 Articles of all kind. Use page size A4. Vertically orient all pages. Ar-

ticles of all kind must be double-spaced including text, references, tables, and

legends. This applies to figures, schemes, and tables as well as text. They do not

have page limitations but should be kept to a minimum length. The experimen-

tal procedures for all of experimental steps must be clearly and fully included

in the experimental section of the manuscripts.

2.1.2 Nomenclature. It is the responsibility of the authors to provide cor-

rect nomenclature. It is acceptable to use semisynthetic or generic names for

certain specialized classes of compounds, such as steroids, peptides, carbohy-

drates, etc. In such a case, the name should conform to the generally accepted

nomenclature conventions for the compound class. Chemical names for drugs

are preferred. If these are not practical, generic names, or names approved by

the World Health Organization, may be used.

Authors may find the following sources useful for recommended nomenclatu-

re:

· The ACS Style Guide; Coghill, A. M., Garson, L. R., Eds.; American Chemical

Society: Washington DC, 2006.

· Enzyme Nomenclature; Webb, E. C., Ed.; Academic Press: Orlando, 1992.

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· IUPHAR database of receptors and ion channels (http://www.guidetophar-

macology.org/).

2.1.3 Compound Code Numbers. Code numbers (including peptides) as-

signed to a compound may be used as follows:

· Once in the manuscript title, when placed in parentheses AFTER the chemical

or descriptive name.

· Once in the abstract.

· Once in the text (includes legends) and once to label a structure. Code num-

bers in the text must correspond to structures or, if used only once, the chemi-

cal name must be provided before the parenthesized code number, e.g., “che-

mical name (JEM-398).” If appearing a second time in the text, a bold Arabic

number must be assigned on first usage, followed by the parenthesized code

number, e.g., “1 (JEM-398).” Subsequently, only the bold Arabic number may

be used. All code numbers in the text must have a citation to a publication or a

patent on first appearance.

Compounds widely employed as research tools and recognized primarily by

code numbers may be designated in the manuscript by code numbers without

the above restrictions. Their chemical name or structure should be provided

as above. Editors have the discretion of determining which code numbers are

considered widely employed.

2.1.4 Trademark Names. Trademark names for reagents or drugs must be

used only in the experimental section. Do not use trademark or service mark

symbols.

2.1.5 Interference Compounds. Active compounds from any source must

be examined for known classes of assay interference compounds and this

analysis must be provided in the General Experimental section. Many of these

compounds have been classified as Pan Assay Interference Compounds (PA-

INS; see Baell & Holloway, J. Med. Chem. 2010, 53, 2719-2740). These compo-

unds shown to display misleading assay readouts by a variety of mechanisms

by forming reactive compounds. Provide firm experimental evidence in at least

two dierent assays that reported compounds with potential PAINS liability

are specifically active and their apparent activity is not an artifact.

2.2 Manuscript Organization

2.2.1 Title Page. Title: The title of the manuscript should reect the pur-

poses and findings of the work in order to provide maximum information in a

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computerized title search. Minimal use of nonfunctional words is encouraged.

Only commonly employed abbreviations (e.g., DNA, RNA, ATP) are acceptab-

le. Code numbers for compounds may be used in a manuscript title when pla-

ced in parentheses AFTER the chemical or descriptive name.

Authors’ Names and Ailiations: The authors’ full first names, middle initials,

last names, and ailiations with addresses at time of work completion should

be listed below the title. The name of the corresponding author should be mar-

ked with an asterisk (*).

2.2.2 Abstract and keywords. Articles of all types must have an abstract

following the title page. The maximum length of the Abstract should be 150

words, organized in a findings-oriented format in which the most important

results and conclusions are summarized. Code numbers may be used once in

the abstract.

After the abstract, a section of Keywords not more than five has to be given.

Be aware that the keywords, chosen according to the general concept, are very

significant during searching and indexing of the manuscripts.

2.2.3 Introduction. The rationale and objectives of the research should be

discussed in this section. The background material should be brief and rele-

vant to the research described.

2.2.4. Methodology. Materials, synthetic, biological, demographic, statisti-

cal or experimental methods of the research should be given detailed in this

section. The authors are free to subdivide this section in the logical ow of the

study. For the experimental sections, authors should be as concise as possible

in experimental descriptions. General reaction, isolation, preparation conditi-

ons should be given only once. The title of an experiment should include the

chemical name and a bold Arabic identifier number; subsequently, only the

bold Arabic number should be used. Experiments should be listed in numeri-

cal order. Molar equivalents of all reactants and percentage yields of products

should be included. A general introductory section should include general pro-

cedures, standard techniques, and instruments employed (e.g., determination

of purity, chromatography, NMR spectra, mass spectra, names of equipment)

in the synthesis and characterization of compounds, isolates and preparations

described subsequently in this section. Special attention should be called to

hazardous reactions or toxic compounds. Provide analysis for known classes of

assay interference compounds.

The preferred forms for some of the more commonly used abbrevations are

mp, bp, ºC, K, min, h, mL, μL, g, mg, μg, cm, mm, nm, mol, mmol, μmol, ppm,

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TLC, GC, NMR, UV, and IR. Units are abbreviated in table column heads and

when used with numbers, not otherwise. (See section 4 for more abbreviations)

2.2.5 Results and Discussion. This section could include preparation, iso-

lation, synthetic schemes and tables of biological and statistical data. The dis-

cussions should be descriptive. Authors should discuss the analysis of the data

together with the significance of results and conclusions. An optional conclusi-

ons section is not required.

2.2.6 Ancillary Information. Include pertinent information in the order

listed immediately before the references.

PDB ID Codes: Include the PDB ID codes with assigned compound Arabic

number. Include the statement “Authors will release the atomic coordinates

and experimental data upon article publication.”

Homology Models: Include the PDB ID codes with assigned compound Arabic

number. Include the statement “Authors will release the atomic coordinates

upon article publication.”

Corresponding Author Information: Provide telephone numbers and email

addresses for each of the designated corresponding authors.

Present/Current Author Addresses: Provide information for authors whose

ailiations or addresses have changed.

Author Contributions: Include statement such as «These authors contributed

equally.»

Acknowledgment: Authors may acknowledge people, organizations, and fi-

nancial supporters in this section.

Abbreviations Used: Provide a list of nonstandard abbreviations and acronyms

used in the paper, e.g., YFP, yellow uorescent protein. Do not include compo-

und code numbers in this list. It is not necessary to include abbreviations and

acronyms from the Standard Abbreviations and Acronyms listed in section 4.

2.2.7 References and Notes. Number literature references and notes in one

consecutive series by order of mention in the text. Numbers in the text are non-

parenthesized superscripts. The accuracy of the references is the responsibility

of the author. List all authors; do not use et al. Provide inclusive page num-

bers. Titles may have capitalization of first word only (excluding, for example,

acronyms and trade names) or standard capitalization as shown below. The

chosen style should be used consistently throughout the references. Double-

space the references using the following format.

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· For journals: Rich, D. H.; Green, J.; Toth, M. V.; Marshall, G. R.; Kent, S. B.

H. Hydroxyethylamine Analogues of the p17/p24 Substrate Cleavage Site Are

Tight Binding Inhibitors of HIV Protease. J. Med. Chem. 1990, 33, 1285-1288.

· For online early access: Rubner, G.; Bensdorf, K.; Wellner, A.; Kircher, B.;

Bergemann, S.; Ott, I.; Gust, R. Synthesis and Biological Activities of Transiti-

on Metal Complexes Based on Acetylsalicylic Acid as Neo-Anticancer Agents. J.

Med. Chem. [Online early access]. DOI: 10.1021/jm101019j. Published Online:

September 21, 2010.

· For periodicals published in electronic format only: Author 1; Author 2; Aut-

hor 3; etc. Title of Article. Journal Abbreviation [Online] Year, Volume, Ar-

ticle Number or other identifying information.

· For monographs: Casy, A. F.; Parfitt, R. T. Opioid Analgesics; Plenum: New

York, 1986.

· For edited books: Rall, T. W.; Schleifer, L. S. Drugs Eective in the Therapy

of the Epilepsies. In The Pharmacological Basis of Therapeutics, 7th ed.; Gil-

man, A. G., Goodman, L. S., Rall, T. W., Murad, F., Eds.; Macmillan: New York,

1985; pp 446-472

List submitted manuscripts as “in press” only if formally accepted for publi-

cation. Manuscripts available on the Web with a DOI number are considered

published. For manuscripts not accepted, use “unpublished results” after the

names of authors. Incorporate notes in the correct numerical sequence with

the references. Footnotes are not used.

2.2.8 Tables. Tabulation of experimental results is encouraged when this le-

ads to more eective presentation or to more economical use of space. Tables

should be numbered consecutively in order of citation in the text with Arabic

numerals. Footnotes in tables should be given italic lowercase letter designa-

tions and cited in the tables as superscripts. The sequence of letters should

proceed by row rather than by column. If a reference is cited in both table and

text, insert a lettered footnote in the table to refer to the numbered reference

in the text. Each table must be provided with a descriptive title that, together

with column headings, should make the table self-explanatory. Titles and fo-

otnotes should be on the same page as the table. Tables may be created using

a word processor’s text mode or table format feature. The table format feature

is preferred. Ensure each data entry is in its own table cell. If the text mode is

used, separate columns with a single tab and use a return at the end of each

row. Tables may be inserted in the text where first mentioned or may be grou-

ped after the references.

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2.2.9 Figures, Schemes/Structures, and Charts. The use of illustrations

to convey or clarify information is encouraged. Structures should be produced

with the use of a drawing program such as ChemDraw. Authors using other

drawing packages should, in as far as possible, modify their program’s para-

meters so that they conform to ChemDraw preferences. Remove all color from

illustrations, except for those you would like published in color. Illustrations

may be inserted into the text where mentioned or may be consolidated at the

end of the manuscript. If consolidated, legends should be grouped on a separa-

te page(s). Include as part of the manuscript file.

To facilitate the publication process, please submit manuscript graphics using

the following guidelines:

1. The preferred submission procedure is to embed graphic files in a Word do-

cument. It may help to print the manuscript on a laser printer to ensure all

artwork is clear and legible.

2. Additional acceptable file formats are: TIFF, PDF, EPS (vector artwork)

or CDX (ChemDraw file). If submitting individual graphic files in addition to

them being embedded in a Word document, ensure the files are named based

on graphic function (i.e. Scheme 1, Figure 2, Chart 3), not the scientific name.

Labeling of all figure parts should be present and the parts should be assemb-

led into a single graphic.

EPS files: Ensure that all fonts are converted to outlines or embedded in the

graphic file. The document settings should be in RGB mode. NOTE: While

EPS files are accepted, the vector-based graphics will be rasterized for produc-

tion. Please see below for TIFF file production resolutions.

3. TIFF files (either embedded in a Word doc or submitted as individual files)

should have the following resolution requirements:

- Black & White line art: 1200 dpi

- Grayscale art (a monochromatic image containing shades of gray): 600 dpi

- Color art (RGB color mode): 300 dpi

· The RGB and resolution requirements are essential for producing high-quality

graphics within the published manuscript. Graphics submitted in CMYK or at

lower resolutions may be used; however, the colors may not be consistent and

graphics of poor quality may not be able to be improved.

· Most graphic programs provide an option for changing the resolution when

you are saving the image. Best practice is to save the graphic file at the final

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resolution and size using the program used to create the graphic.

4. Graphics should be sized at the final production size when possible. Single

column graphics are preferred and can be sized up to 240 points wide (8.38

cm.). Double column graphics must be sized between 300 and 504 points

(10.584 and 17.78 cm’s). All graphics have a maximum depth of 660 points

(23.28 cm.) including the caption (please allow 12 points for each line of cap-

tion text).

Consistently sizing letters and labels in graphics throughout your manuscript

will help ensure consistent graphic presentation for publication.

2.2.10 Image Manipulation. Images should be free from misleading mani-

pulation. Images included in an account of research performed or in the data

collection as part of the research require an accurate description of how the

images were generated and produced. Apply digital processing uniformly to

images, with both samples and controls. Cropping must be reported in the fi-

gure legend. For gels and blots, use of positive and negative controls is highly

recommended. Avoid high contrast settings to avoid overexposure of gels and

blots. For microscopy, apply color adjustment to entire image and note in the

legend. When necessary, authors should include a section on equipment and

settings to describe all image acquisition tools, techniques and settings, and

software used. All final images must have resolutions of 300 dpi or higher. Aut-

hors should retain unprocessed data in the event that the Editors request them.

2.3 Specialized Data

2.3.1 Biological Data. Quantitative biological data are required for all tes-

ted compounds. Biological test methods must be referenced or described in

suicient detail to permit the experiments to be repeated by others. Detailed

descriptions of biological methods should be placed in the experimental sec-

tion. Standard compounds or established drugs should be tested in the same

system for comparison. Data may be presented as numerical expressions or in

graphical form; biological data for extensive series of compounds should be

presented in tabular form.

Active compounds obtained from combinatorial syntheses should be resynthe-

sized and retested to verify that the biology conforms to the initial observation.

Statistical limits (statistical significance) for the biological data are usually re-

quired. If statistical limits cannot be provided, the number of determinations

and some indication of the variability and reliability of the results should be

given. References to statistical methods of calculation should be included.

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Doses and concentrations should be expressed as molar quantities (e.g., mol/

kg, μmol/kg, M, mM). The routes of administration of test compounds and

vehicles used should be indicated, and any salt forms used (hydrochlorides,

sulfates, etc.) should be noted. The physical state of the compound dosed

(crystalline, amorphous; solution, suspension) and the formulation for dosing

(micronized, jet-milled, nanoparticles) should be indicated. For those compo-

unds found to be inactive, the highest concentration (in vitro) or dose level (in

vivo) tested should be indicated.

If human cell lines are used, authors are strongly encouraged to include the

following information in their manuscript:

· the cell line source, including when and from where it was obtained;

· whether the cell line has recently been authenticated and by what method;

· whether the cell line has recently been tested for mycoplasma contamination.

2.3.2 Purity of Tested Compounds.

Methods: All scientifically established methods of establishing purity are ac-

ceptable. If the target compounds are solvated, the quantity of solvent should

be included in the compound formulas. No documentation is required unless

asked by the editors.

Purity Percentage: All tested compounds, whether synthesized or purchased,

should possess a purity of at least 95%. Target compounds must have a purity

of at least 95%. In exceptional cases, authors can request a waiver when com-

pounds are less than 95% pure. For solids, the melting point or melting point

range should be reported as an indicator of purity.

Elemental analysis: Found values for carbon, hydrogen, and nitrogen (if pre-

sent) should be within 0.4% of the calculated values for the proposed formula.

2.3.3 Confirmation of Structure. Adequate evidence to establish structural

identity must accompany all new compounds that appear in the experimental

section. Suicient spectral data should be presented in the experimental section

to allow for the identification of the same compound by comparison. Generally,

a listing of 1H or 13C NMR peaks is suicient. However, when the NMR data are

used as a basis of structural identification, the peaks must be assigned.

List only infrared absorptions that are diagnostic for key functional groups. If a se-

ries contains very closely related compounds, it may be appropriate merely to list

the spectral data for a single representative member when they share a common

major structural component that has identical or very similar spectral features.

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3. Submitting the Manuscript

3.1 Communication and log in to Author’s Module All submissions

to Acta Pharmaceutica Sciencia should be made by using e-Collittera (Online

Article Acceptance and Evaluation) system on the journal main page (www.

actapharmsci.com)

3.2 Registration to System It is required to register into the e-Collittera

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ORIGINAL ARTICLES

129

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Acta Pharm. Sci. Vol 57 No: 2. 2019

DOI: 10.23893/1307-2080.APS.05708

Synthesis, Characterization and Evaluation of

the Anti-cancer Activity of Silver Nanoparticles

by Natural Organic Compounds Extracted from

Cyperus sp. rhizomes

Rasim Farraj Muslim

, Mustafa Nadhim Owaid

1,2*

1 Department of Ecology, College of Applied Sciences-Hit, University of Anbar, Hit, Anbar 31007, Iraq.

2 Department of Heet Education, General Directorate of Education in Anbar, Ministry of Education, Hit, Anbar 31007, Iraq.

INTRODUCTION

The synthesis of metallic nanoparticles has become an important issue in re-

cent decades due to their various beneficial and unique properties and bio-

medical and industrial applications

. Nanotechnology is seeking to synthesize

nanoparticles have substantial biomedical applications because of their unique

characteristics and their green nature due to using some medicinal plants as

ABSTRACT

The object of this work is biosynthesizing AgNPs from extracts of Cyperus sp.

galingale rhizomes, studying their characteristics using UV-visible spectroscopy,

AFM, SEM, FTIR, and EDX analyses and testing their anticancer activity (in vitro)

against L20B cell line. Biosynthesizing AgNPs using various plants is considered

eco-friendly, cheap, energy saving and reproducible compared with non-green

methods. UV-Visible spectrum checked the surface plasmon resonance of AgNPs

at 410-420 nm. FT-IR exhibited that the presence of carbonyl and hydroxyl groups

in the extract of Cyperus sp. can reduce and stabilize AgNPs. EDX, SEM, and AFM

analyses were applied to confirm the nature, morphology and topography of the

biosynthesized AgNPs. AgNPs are spherical or irregular in shape with the average

diameter of hot extract-AgNPs is 56.31 nm in comparison with cold extract-AgNPs

is 92.53 nm. The hot extract-AgNPs paly a suitable role against mouse cell line

(L20B) which have receptors for polioviruses better than the cold extract-AgNPs.

Keywords: SEM, EDS, Green nanotechnology, Galingale, L20B.

*Corresponding Author: Mustafa Nadhim Owaid, email: m[email protected]

Rasim Farraj Muslim ORCID Number: 0000-0002-8273-2429

Mustafa Nadhim Owaid ORCID Number: 0000-0001-9005-4368

(Received 26 November 2018, accepted 30 December 2018)

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

a reducer agent in the biosynthesis of metallic nanoparticles

. Silver nano-

particles are considering famous nanoparticles compared with other metallic

nanoparticles because of their antibiotic activity against viruses

, bacteria

4,5

fungi

, cancers

7,8

, and parasites

Green synthesis of AgNPs was successfully done in simple, rapid, eco-friendly

and a cheaper method using plant leaves extracts like Azadirachta indica

ra-

pid, simple approach was applied for synthesis of silver nanoparticles using

Azadirachta indica aqueous leaf extract. The plant extract acts both as redu-

cing agent as well as capping agent. To identify the compounds responsible

for reduction of silver ions, the functional groups present in plant extract were

investigated by FTIR. Various techniques used to characterize synthesized na-

noparticles are DLS, photoluminescence, TEM and UV–Visible spectropho-

tometer. UV–Visible spectrophotometer showed absorbance peak in range of

436–446 nm. The silver nanoparticles showed antibacterial activities against

both gram positive (Staphylococcus aureus, Panax ginseng

, Tridax procum-

bens

, Ziziphus nummularia

, Thevetia peruviana

, olive (Olea europaea)

, Cleome viscosa,

and fenugreek (Trigonella foenum-graecum L.)

, and

applying them in medicine as a green drug against bacteria, fungi and tumors.

Galingale Cyperus rotundus belongs to Cyperaceae (sedge family). C. rotun-

dus L., purple nutsedge as a common name, is a perennial weed with slender,

scaly creeping rhizomes, and arising singly from the rhizomes which are about

1-3 cm. its rhizomes are externally blackish in color and white inside with a

characteristic odor

16,17

. Watery and ethanol crude extracts of C. rotundus rhi-

zomes showed significant eects against gram-negative

and gram-positive

bacteria, Candida albicans

19,20

and fungal pathogens

. On the other hand,

aqueous extract of C. longous has activity against parasitic worms

Recently, rhizomes of Cyperus rotundus had anticancer eects in a recent Ira-

qi study

, and no studies of Cyperus rhizomes extracts that have been reported

in synthesizing silver nanoparticles. Thus we selected these galingale plant rhi-

zomes to biosynthesize AgNPs to arise the value of inhibitory eect of Cyperus

sp. toward human cancers in vitro.

However, silver nanoparticles have been biosynthesized from cold and hot

extracts of Cyperus sp. galingale rhizomes and studied the nature and cha-

racteristics of these nanoparticles using UV-Visible spectroscopy, AFM, SEM,

FT-IR and EDX analyses and evaluation of their anticancer activity (in vitro)

against L20B cell line is a receptor for human poliovirus.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

METHODOLOGY

Rhizomes samples

Fresh rhizomes of Cyperus sp. galingale were collected from gardens of Al-

Baghdadi district west of Hit, Iraq on October 2017. The plant has identified in

College of Applied Sciences-Hit using identification keys as mentioned in

16,17

Aqueous extraction of Cyperus sp. (galingale) rhizomes

The cold extraction method

The fresh rhizomes of galingale were cleaned from the soil and residues of ro-

ots and washed with tap water and then by distilled water (D.W) as in figure 1.

The rhizomes were peeled to remove the black cortex and then extracted. For

extraction achievement, 10 g of the freshly peeled rhizomes of Cyperus sp. was

crushed in 100 mL D.W using a mortar and stored in a freezer for 48 hr. The

iced extract was slowly dissolved in the room temperature. The whitish aqu-

eous extract was filtered using gauze and then by filter paper Whatman No.1

and centrifuged at 4000 rpm for 10 min. The supernatant was collected by a

micropipette and named as cold extract solution (C). The residue was emitted.

FT-IR spectrum of the crude extract was achieved to characterize and to com-

pare with AgNPs which are formed later

The hot extraction method

The fresh rhizomes were cleaned from the soil and residues of roots and was-

hed with tap water and then by distilled water (D.W) as in figure 1. The rhizo-

mes were peeled to remove the black cortex and then extracted. For extracti-

on achievement, 10 g of the freshly peeled rhizomes of Cyperus sp. galingale

was crushed in 100 mL D.W using a mortar and boiled in the magnetic stirrer

hotplate for 15 min. The whitish aqueous extract was filtered using gauze and

centrifuged at 4000 rpm for 15 min twice. The clear supernatant was collected

by a micropipette and named as a hot extract solution (H). The residue was

emitted. FT-IR spectra of the crude extract (cold and hot extracts) were done

to determine the functional groups comparison with FT-IR spectra of their

AgNPs which are formed later

Biosynthesis of silver nanoparticles

Only 33.8 μg of AgNO

(its purity 99.9%, AFCO For Metal, China) was dissol-

ved in 200 mL distilled water (D.W) using the magnetic stirrer hotplate until

the completion dissolving was observed to get the final concentration 10

-3

Five milliliters of series concentrations of cold and hot galingale extracts (20%,

40%, 60%, 80%, and 100%) were separately mixed with 5 mL of 10

-3

M AgNO

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

solution in 10-mL test tubes and darkly kept at 25 °C for three days

. From

another hand, the second method to biosynthesize AgNPs by heating of 30 mL

each crude galingale extract [cold extract (C) and hot extract (H)] was sepa-

rately added drop by drop into two 500mL-ask containing 100 mL of 10

-3

AgNO

solution using magnetic stirrer hotplate at 60 °C for 1 hr. The change in

the mixture color was checked and recorded each 15 min.

Characterization of AgNPs

Galingale-mediated synthesis of silver nanoparticles (AgNPs) using its cold

and hot extracts were characterized using changes in color of the mixture, UV-

Visible spectroscopy (by spectroscopy: EMC-LAB V-1100 Digital, 325-1000

nm, Germany), FT-IR (Fourier Transform Infrared) spectra, SEM (Scanning

Electron Microscope), EDX, AFM (Atomic Force Microscope), and SPM (Scan-

ning Probe Microscope) analyses.

Anticancer eicacy

The anticancer activity of two types of AgNPs against L20B tumor cell line was

evaluated. The colorimetric cell viability MTT assay was used as mentioned

and

. Firstly, 100 μL/well of L20B cells (10

cell/mL) were cultured in a

96-wells tissue culture plate. Three concentrations of colloid AgNPs (50%, 75%

and 100%) were applied in this test. Moreover, then 100 μL of each concentra-

tion was added within each well and incubated at 37 °C for 48 hr. after that, 10

μL of MTT solution (5 mg/mL) was added to each well and reincubated at 37

°C for 4 hr. Finally, 50 μL DMSO (dimethyl sulfoxide) was added to each well

and incubated for 10 min. L20B cells were cultured in complete medium wit-

hout AgNPs or the extract of Cyperus sp. solution as a control. The absorbance

was measured for each well at 620 nm using ELISA reader. The inhibition per-

centage was calculated according to the equation below:

medium without AgNPs or the extract of Cyperus sp. solution as a control. The absorbance was

measured for each well at 620 nm using ELISA reader. The inhibition percentage was calculated

according to the equation below:

Growth inhibition percentage=

(OD of control wells – OD of test wells)

OD of control wells

× 100

Statistical Analysis

Triplicates of growth inhibition percentage were analyzed by one-way analysis of variance using

ANOVA table by SAS program version 9 (SAS Institute Inc., USA). The significance of differences

was calculated using Duncan’s Multiple Range Test (DMRT). Probability value least than 5% was

considered to be statistically significant.

RESULTS AND DISCUSSION

The change in color for the mixture of AgNO

and the Cyperus rhizomes extract from milky (hot

extraction) and bright yellow (cold extraction) to the brown color was exhibited as seen in figure 1. The

previous figure also is showing adsorption peaks at 410-420 nm in comparison with the crude extracts

of the peeled rhizomes. Hot extract-AgNPs are showing the widest adsorption (2068 cm

-1

) than cold

extract-AgNPs (1800 cm

-1

Figure 1. Change in color and UV-Visible spectrum of the biosynthesized AgNPs from Cyperus

rhizomes extract

Statistical Analysis

Triplicates of growth inhibition percentage were analyzed by one-way analysis

of variance using ANOVA table by SAS program version 9 (SAS Institute Inc.,

USA). The significance of dierences was calculated using Duncan’s Multiple

Range Test (DMRT). Probability value least than 5% was considered to be sta-

tistically significant.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

RESULTS AND DISCUSSION

The change in color for the mixture of AgNO

and the Cyperus rhizomes extract

from milky (hot extraction) and bright yellow (cold extraction) to the brown

color was exhibited as seen in figure 1. The previous figure also is showing

adsorption peaks at 410-420 nm in comparison with the crude extracts of the

peeled rhizomes. Hot extract-AgNPs are showing the widest adsorption (2068

-1

) than cold extract-AgNPs (1800 cm

-1

medium without AgNPs or the extract of Cyperus sp. solution as a control. The absorbance was

measured for each well at 620 nm using ELISA reader. The inhibition percentage was calculated

according to the equation below:

Growth inhibition percentage=

(OD of control wells – OD of test wells)

OD of control wells

× 100

Statistical Analysis

Triplicates of growth inhibition percentage were analyzed by one-way analysis of variance using

ANOVA table by SAS program version 9 (SAS Institute Inc., USA). The significance of differences

was calculated using Duncan’s Multiple Range Test (DMRT). Probability value least than 5% was

considered to be statistically significant.

RESULTS AND DISCUSSION

The change in color for the mixture of AgNO

and the Cyperus rhizomes extract from milky (hot

extraction) and bright yellow (cold extraction) to the brown color was exhibited as seen in figure 1. The

previous figure also is showing adsorption peaks at 410-420 nm in comparison with the crude extracts

of the peeled rhizomes. Hot extract-AgNPs are showing the widest adsorption (2068 cm

-1

) than cold

extract-AgNPs (1800 cm

-1

Figure 1. Change in color and UV-Visible spectrum of the biosynthesized AgNPs from Cyperus

rhizomes extract

Figure 1. Change in color and UV-Visible spectrum of the biosynthesized AgNPs from

Cyperus rhizomes extract

SEM images (figure 2) exhibited the morphology of AgNPs which ranged from

spherical to irregular particles. The 3D and 2D images of AFM showed the

topography of the nanoparticle’s surfaces (Figure 3). However, AFM has the

advantageousness of probing the surface topography deeply. The AFM image

displays the surface morphology of silver nanoparticles synthesized by Cyperus

rhizome extracts which reveal the appearance of spherical or irregular and ne-

edle-like nanoparticles for the cold extract-AgNPs and the hot extract-AgNPs

respectively. Also, roughness average is 7.65 nm and 7.11 nm; the surface area

ratio is 15.3:1 and 22.5:1 and the density of summits 258 μ

-2

and 386 μ

-2

for the

cold extract-AgNPs and the hot extract-AgNPs respectively.

Histogram of particle size distribution (SPM) showed the granularity distribu-

tion, volumes and averages of diameters of AgNPs (figure 3). This parameter

is airming the results of AFM images which appears smallness size of the hot

extract-AgNPs comparing to the cold ones. The average diameter of the cold

extract-AgNPs is 92.53 nm in comparison with the hot extract-AgNPs is 56.31

nm. Histogram of the particle size distribution of the two AgNPs was presented

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

in figure 4 was clear evidence for the formation of silver particles by the per-

centages of Ag particles in the two treatments.

SEM images (figure 2) exhibited the morphology of AgNPs which ranged from spherical to irregular

particles. The 3D and 2D images of AFM showed the topography of the nanoparticle’s surfaces (Figure

3). However, AFM has the advantageousness of probing the surface topography deeply. The AFM image

displays the surface morphology of silver nanoparticles synthesized by Cyperus rhizome extracts which

reveal the appearance of spherical or irregular and needle-like nanoparticles for the cold extract-AgNPs

and the hot extract-AgNPs respectively. Also, roughness average is 7.65 nm and 7.11 nm; the surface

area ratio is 15.3:1 and 22.5:1 and the density of summits 258 µ

-2

and 386 µ

-2

for the cold extract-AgNPs

and the hot extract-AgNPs respectively.

Histogram of particle size distribution (SPM) showed the granularity distribution, volumes and averages

of diameters of AgNPs (figure 3). This parameter is affirming the results of AFM images which appears

smallness size of the hot extract-AgNPs comparing to the cold ones. The average diameter of the cold

extract-AgNPs is 92.53 nm in comparison with the hot extract-AgNPs is 56.31 nm. Histogram of the

particle size distribution of the two AgNPs was presented in figure 4 was clear evidence for the

formation of silver particles by the percentages of Ag particles in the two treatments.

Figure 2. SEM of the biosynthesized silver nanoparticles

Figure 3. AFM of the colloid silver nanoparticles

A B

Figure 4. Histogram of the particle size distribution of the biosynthesized silver nanoparticles including

the cold extract-AgNPs (A) and the hot extract-AgNPs (B)

The FT-IR spectrum the cold extract (figure 5A) and the hot extract (figure 5B) showed the peak 771

-1

is due to the covalent bonding between carbon and silicon (Si-C) and other peaks 1105 cm-1and

1108 cm

-1

for cold and hot extracts respectively. These are evidence of linkage of oxygen with silicon

in (Si-O). Both spectra also showed two absorption bands at 1384 cm

-1

and 1417 cm

-1

belong to

vibrations of homogeneous and heterogeneous bends of methylene group (-CH

) or methyl group

Figure 3. AFM of the colloid silver nanoparticles

A B

Figure 4. Histogram of the particle size distribution of the biosynthesized silver nanoparticles including

the cold extract-AgNPs (A) and the hot extract-AgNPs (B)

The FT-IR spectrum the cold extract (figure 5A) and the hot extract (figure 5B) showed the peak 771

-1

is due to the covalent bonding between carbon and silicon (Si-C) and other peaks 1105 cm-1and

1108 cm

-1

for cold and hot extracts respectively. These are evidence of linkage of oxygen with silicon

in (Si-O). Both spectra also showed two absorption bands at 1384 cm

-1

and 1417 cm

-1

belong to

vibrations of homogeneous and heterogeneous bends of methylene group (-CH

) or methyl group

Figure 4. Histogram of the particle size distribution of the biosynthesized silver nanoparticles

including the cold extract-AgNPs (A) and the hot extract-AgNPs (B)

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

The FT-IR spectrum the cold extract (figure 5A) and the hot extract (fi-

gure 5B) showed the peak 771 cm

-1

is due to the covalent bonding bet-

ween carbon and silicon (Si-C) and other peaks 1105 cm-1and 1108 cm

-1

for cold and hot extracts respectively. These are evidence of linkage of

oxygen with silicon in (Si-O). Both spectra also showed two absorpti-

on bands at 1384 cm

-1

and 1417 cm

-1

belong to vibrations of homogeneo-

us and heterogeneous bends of methylene group (-CH

) or methyl group

(-CH

) and two absorption bands at 2891 cm

-1

and 2894 cm

-1

for cold and hot

extracts respectively, and at 2935 cm

-1

for cold and hot extracts too. All the

mentioned absorption bands belong to the previous groups which presented in

the composition of amino acids, peptides or proteins. There were absorption

bands at 1053 cm

-1

and 1062 cm

-1

proved the protein structures for cold and

hot extracts respectively belong to the single bond (C-C). The spectra showed

absorption bands at 1631 cm

-1

and 1633 cm

-1

for cold and hot extracts respec-

tively go back to vibrations of stretching of C=C group. On another hand, the

presence of C-H-containing compounds (C-H) is evidenced by the presence of

two bands at 3109 and 3130 cm

-1

for cold and hot extracts, respectively. It is

confirmed that avonoids, amino acids, peptides, proteins, polyphenols and

sugars are present in the presence of a long stretching vibration of the absorp-

tion peak at 3396 and 3458 cm

-1

for cold and hot extracts respectively. As well

as two bending vibration bands at 1384 cm

-1

for the cold extract and 1388 cm

-1

for the cold extract belong to the hydroxyl groups (-OH); and the absorption

band at 1417 cm

-1

belongs to the group (C-O).

There are two bands at 1749, 1726 cm

-1

for cold and hot extracts respectively,

although weak. The cause of weakness is probably the multiplicity in these gro-

ups and the possibility of a succession that changes this group into a single

carbon-oxygen bond. These bands go back to Carbonyl group (C=O) and this is

evidence that the extracts contain avonoids, mersatin and camphorol.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Figure 5. FT-IR of the cold extract (A) and the hot extract (B)

As for the FT-IR spectroscopy of silver nanoparticles of the hot and cold extracts, Figures 6A and 6B.

The absorption bands at 1132 cm

-1

and 1120 cm

-1

, the two bands at 1382 cm

-1

and 1388 cm

-1

, the two

bands at 1421 cm

-1

and 1419 cm

-1

, and the two bands at 1622 cm

-1

and 1623 cm

-1

for the AgNPs of cold

and hot extracts respectively are clear evidence of the presence of silver nanoparticles in the synthesis

of the samples. These two spectra are very similar to the infrared spectrum of cold and hot extracts

Figure 5. FT-IR of the cold extract (A) and the hot extract (B)

As for the FT-IR spectroscopy of silver nanoparticles of the hot and cold extracts,

Figures 6A and 6B. The absorption bands at 1132 cm

-1

and 1120 cm

-1

, the two bands

at 1382 cm

-1

and 1388 cm

-1

, the two bands at 1421 cm

-1

and 1419 cm

-1

, and the two

bands at 1622 cm

-1

and 1623 cm

-1

for the AgNPs of cold and hot extracts respecti-

vely are clear evidence of the presence of silver nanoparticles in the synthesis of

the samples. These two spectra are very similar to the infrared spectrum of cold

and hot extracts (figure 5) and therefore the composition is not but the insulation,

clarity and beam width are better in the infrared spectrum of the nanostructures.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

(figure 5) and therefore the composition is not but the insulation, clarity and beam width are better in

the infrared spectrum of the nanostructures.

Figure 6. FT-IR of silver nanoparticles of the cold extract (A) and AgNPs of the hot extract (B)

The EDX spectrum of the biosynthesized AgNPs (figure 7) shows finding silver element as an indicator

for the formation of silver nanoparticles from the hot extract better than the cold extract. Also, it exhibits

the C, O, Cl, Si, and K elements have been presented in the used sample.

Figure 6. FT-IR of silver nanoparticles of the cold extract (A) and AgNPs of the hot extract

(B)

The EDX spectrum of the biosynthesized AgNPs (figure 7) shows finding silver

element as an indicator for the formation of silver nanoparticles from the hot

extract better than the cold extract. Also, it exhibits the C, O, Cl, Si, and K ele-

ments have been presented in the used sample.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Figure 7. EDX images of the colloid AgNPs formed from the hot extract (A) and the cold one (B)

Both the cold extract-AgNPs and the hot-AgNPs were investigated against cell line of murine fibroblast

cells have receptors for human polioviruses (L20B) in vitro. Figure 8 exhibited the anticancer activity

of the colloid AgNPs synthesized from aqueous extracts of the peeled Galingale rhizomes. The hot-

AgNPs gave better growth inhibition than the cold extract-AgNPs. The best inhibition showed by the

concentration 100% of the hot-AgNPs 35.3% significantly (p<0.01) followed by 22.5% by the

concentration of 75% of the hot-AgNPs. The concentration 100% of the cold-AgNPs exhibited 22.4%

then decreased to 17.9% by the concentration of 75% of the cold-AgNPs. The concentration 50% did

not show any inhibitory effects as in the crude extracts of Cyperus rhizomes.

Figure 7. EDX images of the colloid AgNPs formed from the hot extract (A) and the cold one

(B)

Both the cold extract-AgNPs and the hot-AgNPs were investigated against cell

line of murine fibroblast cells have receptors for human polioviruses (L20B) in

vitro. Figure 8 exhibited the anticancer activity of the colloid AgNPs synthe-

sized from aqueous extracts of the peeled Galingale rhizomes. The hot-AgNPs

gave better growth inhibition than the cold extract-AgNPs. The best inhibiti-

on showed by the concentration 100% of the hot-AgNPs 35.3% significantly

(p<0.01) followed by 22.5% by the concentration of 75% of the hot-AgNPs.

The concentration 100% of the cold-AgNPs exhibited 22.4% then decreased

to 17.9% by the concentration of 75% of the cold-AgNPs. The concentration

50% did not show any inhibitory eects as in the crude extracts of Cyperus

rhizomes.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Figure 8. Anticancer activity of the colloid AgNPs synthesized from aqueous extracts of the peeled

Galingale rhizomes

The change in color for the mixture of AgNO

and the Cyperus rhizomes extract from milky (hot

extraction) bright yellow (cold extraction) to the brown color is a sign for the formation silver

nanoparticles

as seen in figure 1. The density of brown color is due to excitation of surface plasmon

vibrations in the silver nanoparticles

, and that is confirmed using UV-Visible spectra. Hot extract-

AgNPs are showing the widest adsorption (2068 cm

-1

) than cold extract-AgNPs (1800 cm

-1

), because of

the resonance wavelength of silver nanoparticles has strong depending on Ag atoms, size and

morphology of particles

SEM, AFM and Histogram of particle size distribution (SPM) were used to confirm the nature,

topography, morphology of the silver nanoparticles. SEM images (figure 2) exhibited the morphology

of AgNPs which ranged from spherical to irregular particles. The shape of silver nanoparticles agreed

with many recent studies

. The 3D and 2D images of AFM showed the topography of the nanoparticles

surfaces (figure 3). Atomic Force Microscope is a useful tool to study different morphological

parameters. AFM images as in figure 3 show high-resolution topography and the silver nanoparticles

can be visualized under the dry condition

However, AFM has the advantageousness of probing the surface topography deeply due to its lateral

and trial dimensions in nanometer scale resolution

. The AFM image displays the surface morphology

of silver nanoparticles synthesized by Cyperus rhizome extracts which reveal the appearance of

spherical or irregular and needle-like nanoparticles for the cold extract-AgNPs and the hot extract-

Figure 8. Anticancer activity of the colloid AgNPs synthesized from aqueous extracts of the

peeled Galingale rhizomes

The change in color for the mixture of AgNO

and the Cyperus rhizomes extract

from milky (hot extraction) bright yellow (cold extraction) to the brown color

is a sign for the formation silver nanoparticles

as seen in figure 1. The density

of brown color is due to excitation of surface plasmon vibrations in the silver

nanoparticles

, and that is confirmed using UV-Visible spectra. Hot extract-

AgNPs are showing the widest adsorption (2068 cm

-1

) than cold extract-AgNPs

(1800 cm

-1

), because of the resonance wavelength of silver nanoparticles has

strong depending on Ag atoms, size and morphology of particles

SEM, AFM and Histogram of particle size distribution (SPM) were used to

confirm the nature, topography, morphology of the silver nanoparticles. SEM

images (figure 2) exhibited the morphology of AgNPs which ranged from sphe-

rical to irregular particles. The shape of silver nanoparticles agreed with many

recent studies

. The 3D and 2D images of AFM showed the topography of the

nanoparticles surfaces (figure 3). Atomic Force Microscope is a useful tool to

study dierent morphological parameters. AFM images as in figure 3 show

high-resolution topography and the silver nanoparticles can be visualized un-

der the dry condition

However, AFM has the advantageousness of probing the surface topography

deeply due to its lateral and trial dimensions in nanometer scale resolution

. The AFM image displays the surface morphology of silver nanoparticles

synthesized by Cyperus rhizome extracts which reveal the appearance of sphe-

rical or irregular and needle-like nanoparticles for the cold extract-AgNPs and

140

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

the hot extract-AgNPs respectively. Also, roughness average, the surface area

ratio and the density of summits are evidence to form the smallest nanopar-

ticles for AgNPs formed from the hot extract of Cyperus rhizome and more

summits by high surface area ratio.

Histogram of particle size distribution (SPM) showed the granularity distribu-

tion, volumes and averages of diameters of AgNPs (figure 3). This parameter

is airming the results of AFM images which appears smallness size of the

hot extract-AgNPs comparing to the cold ones. The average diameter of the

cold extract-AgNPs is 92.53 nm in comparison with the hot extract-AgNPs is

56.31 nm. The reason for this phenomenon is related to the extraction techni-

que which means the heating process during preparation of the herbal extracts

leads to destroying active ingredients

. Then the interaction of the finer bio-

molecules with silver ions leads to synthesize finer nanoparticles. Histogram

of the particle size distribution of the two AgNPs was presented in Figure 4

was clear evidence for the formation of silver particles by the percentages of

Ag particles in the two treatments. All sizes of the biosynthesized AgNPs in

the colloids are lesser than 100 nm which is considered a definite proof for the

formation of AgNPs

Fourier Transform Infrared spectrum (FT-IR) was used to determine the che-

mical structure and the functional groups. In cold and hot extracts, the simila-

rities were observed in the peak sites. The FT-IR spectrum the cold extract (fi-

gure 5A) and the hot extract (figure 5B) confirms the presence of compensated

aromatic rings all of which go back to the amino acids, peptides and proteins

, amintoavone and avonoids

see figure 9, poly hydroxyl compounds such

as myristin, albicaine, camphorol

see figure 10, alkene compounds such as

lemons, cocaine and mirentol

see figure 11.

AgNPs respectively. Also, roughness average, the surface area ratio and the density of summits are

evidence to form the smallest nanoparticles for AgNPs formed from the hot extract of Cyperus rhizome

and more summits by high surface area ratio.

Histogram of particle size distribution (SPM) showed the granularity distribution, volumes and averages

of diameters of AgNPs (figure 3). This parameter is affirming the results of AFM images which appears

smallness size of the hot extract-AgNPs comparing to the cold ones. The average diameter of the cold

extract-AgNPs is 92.53 nm in comparison with the hot extract-AgNPs is 56.31 nm. The reason for this

phenomenon is related to the extraction technique which means the heating process during preparation

of the herbal extracts leads to destroying active ingredients

. Then the interaction of the finer

biomolecules with silver ions leads to synthesize finer nanoparticles. Histogram of the particle size

distribution of the two AgNPs was presented in Figure 4 was clear evidence for the formation of silver

particles by the percentages of Ag particles in the two treatments. All sizes of the biosynthesized AgNPs

in the colloids are lesser than 100 nm which is considered a definite proof for the formation of AgNPs

Fourier Transform Infrared spectrum (FT-IR) was used to determine the chemical structure and the

functional groups. In cold and hot extracts, the similarities were observed in the peak sites. The FT-IR

spectrum the cold extract (figure 5A) and the hot extract (figure 5B) confirms the presence of

compensated aromatic rings all of which go back to the amino acids, peptides and proteins

amintoflavone and flavonoids

see figure 9, poly hydroxyl compounds such as myristin, albicaine,

camphorol

see figure 10, alkene compounds such as lemons, cocaine and mirentol

see figure 11.

Figure 9. Structures of amintoflavone and flavonoids compounds in Cyperus sp. rhizomes

Figure 9. Structures of amintoavone and avonoids compounds in Cyperus sp. rhizomes

141

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Figure 10. Structures of poly hydroxyl compounds in Cyperus sp. rhizomes

Figure 11. Structures of alkene compounds in Cyperus sp. rhizomes

The absorption bands at 2891 cm

-1

and 2894 cm

-1

for cold and hot extracts respectively, and at 2935 cm

for cold and hot extracts also belong to vibrations of homogeneous and heterogeneous stretching of a

methylene group (-CH

) or a methyl group (-CH

)

. The spectra showed absorption bands at 1631 cm

and 1633 cm

-1

for cold and hot extracts respectively go back to vibrations of stretching of C=C group

belongs to the alkene compounds and the successive double bonds in the benzene ring in the aromatic

structures

. As well as two bending vibration bands at 1384 cm

-1

for the cold extract and 1388 cm

-1

for

the cold extract belong to the hydroxyl groups (-OH); and the absorption band at 1417 cm

-1

due to the

stretching vibration of the group (C-O)

. The absorption bands 582-578 cm

-1

and 688-686 cm

-1

for the

cold and hot extracts respectively indicate to the amide group (O=C-N-H), which binds two consecutive

amino acids in the synthesis of peptides or proteins. The bands at 3267-3271 cm

-1

and 3417-3367 cm

-1

indicate for the presence of the amine group (-NH

) in the synthesis of amino acids, peptides and proteins

for cold and hot extracts respectively

32,39

. The presence of the carboxylic group in amino acids, peptides

and proteins are the appearance of the wide absorption range (2400-3600 cm

-1

) for both extracts and

Figure 10. Structures of poly hydroxyl compounds in Cyperus sp. rhizomes

Figure 11. Structures of alkene compounds in Cyperus sp. rhizomes

The absorption bands at 2891 cm

-1

and 2894 cm

-1

for cold and hot extracts respectively, and at 2935 cm

for cold and hot extracts also belong to vibrations of homogeneous and heterogeneous stretching of a

methylene group (-CH

) or a methyl group (-CH

)

. The spectra showed absorption bands at 1631 cm

and 1633 cm

-1

for cold and hot extracts respectively go back to vibrations of stretching of C=C group

belongs to the alkene compounds and the successive double bonds in the benzene ring in the aromatic

structures

. As well as two bending vibration bands at 1384 cm

-1

for the cold extract and 1388 cm

-1

for

the cold extract belong to the hydroxyl groups (-OH); and the absorption band at 1417 cm

-1

due to the

stretching vibration of the group (C-O)

. The absorption bands 582-578 cm

-1

and 688-686 cm

-1

for the

cold and hot extracts respectively indicate to the amide group (O=C-N-H), which binds two consecutive

amino acids in the synthesis of peptides or proteins. The bands at 3267-3271 cm

-1

and 3417-3367 cm

-1

indicate for the presence of the amine group (-NH

) in the synthesis of amino acids, peptides and proteins

for cold and hot extracts respectively

32,39

. The presence of the carboxylic group in amino acids, peptides

and proteins are the appearance of the wide absorption range (2400-3600 cm

-1

) for both extracts and

Figure 11. Structures of alkene compounds in Cyperus sp. rhizomes

The absorption bands at 2891 cm

-1

and 2894 cm

-1

for cold and hot extracts res-

pectively, and at 2935 cm

-1

for cold and hot extracts also belong to vibrations of

homogeneous and heterogeneous stretching of a methylene group (-CH

) or a

methyl group (-CH

)

. This research includes synthesis of new heterocyclic de-

rivatives of disubstituted 1,3-oxazepine-5-one. Azomethine compounds (N1-N5.

The spectra showed absorption bands at 1631 cm

-1

and 1633 cm

-1

for cold and hot

extracts respectively go back to vibrations of stretching of C=C group belongs to

the alkene compounds and the successive double bonds in the benzene ring in

the aromatic structures

. As well as two bending vibration bands at 1384 cm

-1

for the cold extract and 1388 cm

-1

for the cold extract belong to the hydroxyl gro-

ups (-OH); and the absorption band at 1417 cm

-1

due to the stretching vibration

of the group (C-O)

. The absorption bands 582-578 cm

-1

and 688-686 cm

-1

for

the cold and hot extracts respectively indicate to the amide group (O=C-N-H),

which binds two consecutive amino acids in the synthesis of peptides or proteins.

The bands at 3267-3271 cm

-1

and 3417-3367 cm

-1

indicate for the presence of the

amine group (-NH

) in the synthesis of amino acids, peptides and proteins for

cold and hot extracts respectively

32,39

. The presence of the carboxylic group in

142

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

amino acids, peptides and proteins are the appearance of the wide absorption

range (2400-3600 cm

-1

) for both extracts and maybe the presence of a carboxylic

group (-COOH) in mono acidic saccharides compounds such as glucuronic acid

40–43

3-oxazepin-5(1H see figure 12.

maybe the presence of a carboxylic group (-COOH) in mono acidic saccharides compounds such as

glucuronic acid

40–43

see figure 12.

Figure 12. Structures of mono acidic saccharides compounds in Cyperus sp. rhizomes

The FT-IR spectra of silver nanoparticles of the hot and cold extracts (Figures 6A and 6B) showed clear

evidence of the presence of silver nanoparticles in the synthesis of the samples because in a study on

mushroom found that the silver nanostructure binds with the hydrocarbon’s compounds. It shows four

packages located nearby and within the ranges listed

. These two spectra are very similar to the infrared

spectrum of cold and hot extracts and therefore the composition is not, but the insulation, clarity and

beam width are better in the infrared spectrum of the nanostructures. The spectrum of silver

nanoparticles (Figure 6B) was observed to be similar to Figure 5B regarding the sites of the functional

groups, but the appearance of the bands was more apparent and better. That may be due to the presence

of silver atoms with a high surface area that allows all the atoms with negative electrical and containing

pairs of electrons not involved as in the mushroom extract to participate in the silver nanoparticles

sample containing the atoms of silver spread well. It is known that the silver atoms contain 47 electrons,

so contain in its fifth shell one electron in the second level 5s. Thus, the other secondary levels are 5p

containing three orbitals, 5d containing five orbitals and 4f containing seven empty orbitals and can

accommodate electronic pairs coming from atoms with good negative electrical. The oxygen atom found

in the formation of mono crystalline polysaccharides in the form of (-OH) in acid sugars or in the form

of (C-OO) or in the form of carbonyl (C=O) in male compounds previously, the link is as follows (Ag-

OR) where R is mono crystalline or polyunsaturated sugar or amino acid or Peptide, protein, phenols or

flavonoids. Another example of good electrolyte atom is the atom of nitrogen in the form of (=NH) in

arginine or the form of (-NH

) or (-NH-C=O) in peptides and proteins. The association is as follows

(Ag-NR) (R) is an amino acid, peptide or protein, and the other electrolytic atom is the sulfur atom found

in the synthesis of amino acids, peptides and proteins in the form of (-S-) and in the form of (-SH) (Ag-

Figure 12. Structures of mono acidic saccharides compounds in Cyperus sp. rhizomes

The FT-IR spectra of silver nanoparticles of the hot and cold extracts (Figures

6A and 6B) showed clear evidence of the presence of silver nanoparticles in the

synthesis of the samples because in a study on mushroom found that the silver

nanostructure binds with the hydrocarbon’s compounds. It shows four packages

located nearby and within the ranges listed

. These two spectra are very similar

to the infrared spectrum of cold and hot extracts and therefore the composition

is not, but the insulation, clarity and beam width are better in the infrared spect-

rum of the nanostructures. The spectrum of silver nanoparticles (Figure 6B) was

observed to be similar to Figure 5B regarding the sites of the functional groups,

but the appearance of the bands was more apparent and better. That may be

due to the presence of silver atoms with a high surface area that allows all the

atoms with negative electrical and containing pairs of electrons not involved as

in the mushroom extract to participate in the silver nanoparticles sample conta-

ining the atoms of silver spread well. It is known that the silver atoms contain 47

electrons, so contain in its fifth shell one electron in the second level 5s. Thus,

the other secondary levels are 5p containing three orbitals, 5d containing five

orbitals and 4f containing seven empty orbitals and can accommodate electronic

pairs coming from atoms with good negative electrical. The oxygen atom found

in the formation of mono crystalline polysaccharides in the form of (-OH) in acid

sugars or in the form of (C-OO) or in the form of carbonyl (C=O) in male com-

pounds previously, the link is as follows (Ag-OR) where R is mono crystalline or

polyunsaturated sugar or amino acid or Peptide, protein, phenols or avonoids.

Another example of good electrolyte atom is the atom of nitrogen in the form of

(=NH) in arginine or the form of (-NH

) or (-NH-C=O) in peptides and proteins.

The association is as follows (Ag-NR) (R) is an amino acid, peptide or protein,

143

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

and the other electrolytic atom is the sulfur atom found in the synthesis of amino

acids, peptides and proteins in the form of (-S-) and in the form of (-SH) (Ag-SR)

where R is an amino acid or peptide or protein. FT-IR exhibited that the presence

of carbonyl (C=O) and hydroxyl groups (-OH) in the extract of Cyperus sp. can

reduce and stabilize silver nanoparticles

Carbon and oxygen are presented in the EDX graph (Figure 7) due to the organic

molecules which found in the extract of the mushroom. These biomolecules may

be amino acids or proteins or polysaccharides or polyphenols which capped the

silver nanoparticles as capping and stabilizing agents. The presence of the ele-

mental Ag can be seen in the EDX graph (figure 7) that indicates the reduction

of Ag ions to elemental silver

. Further, Chlorine is also observed due to the un-

reacted precursors of AgNO

The silver nanoparticles formed from these rhizomes are exhibiting unique

physicochemical and biological activities

thus they have the inhibitory role. The

reason of the high inhibitory eects of the hot extract-AgNPs returns to small-

ness the average of size of these AgNPs (56.31 nm in diameter) in comparison to

the average of size of the cold extract-AgNPs (92.53 nm) as in Figure 4 that lead

to increase the surface area ratio of the hot extract-AgNPs as AFM in Figure 3.

This study proved the rhizome-mediated synthesis of AgNPs using the cold and

hot watery rhizomes extracts of Cyperus sp. FT-IR exhibited that the presence

of carbonyl and hydroxyl groups in the extract of Cyperus sp. can reduce and

stabilize silver nanoparticles. EDX, SEM, and AFM analyses were confirmed that

the biosynthesized silver nanoparticles were spherical or irregular in shape with

the average diameter of the hot extract-AgNPs is 56.31 nm in comparison with

the cold extract-AgNPs is 92.53 nm. EDX images indicate to form silver nano-

particles from the hot extract better than the cold one. The synthesized silver

nanoparticles were tested against cancer cell line L20B. The hot extract-AgNPs

plays a suitable role against mouse cell line (L20B) which have receptors for po-

lioviruses better than the cold extract-AgNPs. The best inhibition showed by the

concentration 100% of the hot-AgNPs 35.3% significantly (p<0.01).

ACKNOWLEDGEMENTS

The author and co-author are thanking the sta of Department of Ecology, College

of Applied Sciences-Hit in University of Anbar to complete project No. 3/122 on 15

Oct 2017. Special thanks to President of the University Prof. Dr. Khalid Battal Najim

and Dean of the College, Prof. Dr. Tahseen Ali Zaidan for their continuous support

in publishing the research in the certified international journal. Another thank is

introducing to the University of Baghdad for achieving the AFM nanophotographic.

144

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

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Acta Pharm. Sci. Vol 57 No: 2. 2019

DOI: 10.23893/1307-2080.APS.05709

Anti-inammatory and Analgesic Potential of

Acetone Leaf Extract of Combretum Sordidum

and its Fractions

Babatunde B. Samuel

, Olayinka A. Oridupa

, Fisayo Gbadegesin

1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ibadan.

2 Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Ibadan.

INTRODUCTION

In recent years, the search for phytochemicals with antioxidant, anti-inamma-

tory and analgesic properties have been on the rise due to their potential use in

the therapy of various chronic and infectious diseases.

1,2

Many diseases origi-

nate from uncontrolled or unregulated inammatory process in the body.

Inf-

ABSTRACT

Combretum species are traditionally used in treatment of arthritis, fungal and bac-

terial infections. This study investigated anti-inammatory activity of C. sordidum

acetone extract in egg albumen-induced paw oedema and formalin paw lick test

in rats, while analgesic activity was determined by acetic acid-induced abdominal

writhing test in mouse. Bioactivity guided analgesic eect were also carried out

on solvent-solvent partitioned and chromatographic fractions. Rats administered

with extract progressively showed significant reduction in oedema formation 60

minutes post-induction. Dose-dependent inhibition of formalin-induced paw lick

was observed at early and late phases of the experiment compared to indomet-

hacin. Abdominal writhing was significantly inhibited with 400mg/kg extract ef-

fect (73.1%) comparable to aspirin (75.9%). Most active chromatographic fraction

identified as F7 (79.5%) showed significant analgesic activity, higher than aspirin

(55.0%). Results established anti-inammatory and analgesic of C. sordidum.

Further studies are on-going to identify and characterize the bioactive principle

responsible for these pharmacological eects.

Keywords: Combretum sordidum, Anti-inammatory, Analgesic, Bioactivity-gu-

ided separation

*Corresponding Author: Babatunde B. Samuel, e-mail: tundebsam[email protected]

Babatunde B. Samuel ORCID Number: 0000-0002-1834-9548

Olayinka A. Oridupa ORCID Number: 0000-0002-6435-6925

Fisayo Gbadegesin ORCID Number: 0000-0003-1322-679X

(Received 10 September 2018, accepted 15 January 2019)

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

lammation is a localized protective reaction of cells/tissues of the body to allergic

or chemical irritation, injury and/or infections.

Inammation is characterized by

pain, heat, redness, swelling and loss of function resulting from vasodilation and

leading to an increased blood supply with increased inux of leukocytes, protein

and uids into intercellular spaces of the inamed regions.

Inammatory res-

ponses occur as the body recognizes injury and prepare to repair the damage, but

uncontrolled inammation leads to development and progression of diseases and

disorders which are manifested as exacerbated symptoms of inammation.

6,7,8

Since antiquity, medicinal plants such as plants in genus Combretum have been

employed for treatment of inammation, pain and related diseases. One of plants

in the genus, Combretum sordidum Exell (Combretaceae) is so employed as tradi-

tional remedy of infectious and non-infectious ailments.

C. sordidum a scandent

shrub or a creeper with white owers and easily recognized by the small red scales

on the under-surface of the leaves.

They are widely distributed throughout So-

uth and West Africa.

This plant and other members of the genus from dierent

regions of Africa are traditionally used by local healers for treatment of diseases

of inammatory origin including conjunctivitis, abdominal disorders, backache,

toothache, diabetes.

12,13

It has been scientifically proven to have antiasthmatic,

antimicrobial and antiplasmodic eects.

Another member of the genus, C. molle

R.Brex was reported to have cardiocascular eect

, anti-inammatory and in-

hibitory eect on haematopoietic prostaglandin D2 synthase; while Combretum

woodii have shown significant anti-infective activities.

16,17,18

Despite the use of C. sordidum in traditional medicine, there is a dearth of know-

ledge on the medicinal and ethnopharmacological uses of C. sordidum in literatu-

re. This current study investigated the anti-inammatory and analgesic potential

of the acetone extract of Combretum sordidum. Its constituent fractions which

were separated by solvent-solvent partitioning using n-hexane, ethyl acetate and

methanol were also assessed for their analgesic eect and bioassay guided purifi-

cation of the most potent fraction was carried out to isolate the bioactive compo-

und responsible for its analgesic eect.

METHODOLOGY

Preparation of plant extract

Fresh leaves of Combretum sordidum were collected from Federal Research Insti-

tute of Nigeria (FRIN), Ibadan, Oyo state (Voucher No: 109923). The leaves were

air dried and then pulverized. Acetone extract of the pulverized plant material was

obtained by extracting 1kg of the plant using a soxhlet extractor (hot extraction),

after which it was concentrated using Buchi® rotary evaporator at 40

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Experimental Animal Model

Wistar rats weighing 120-160g and mice weighing 15-20g were obtained and

housed at the Experimental Animal Unit of Department of Veterinary Pharma-

cology and Toxicology, University of Ibadan. The rats were fed with standard

pellets and allowed access to water ad libitum. The animals were divided into

groups of five rats each and were fasted overnight before commencement of

each experiment.

Anti- inammatory Activity of the crude extract

Egg albumen –induced Paw oedema in Rats

In this model, following an overnight fasting, three doses C. sordidum leaf

extracts (100mg/kg, 200mg/kg and 400mg/kg) were orally administered to

rats in three dierent treatment groups, and simultaneously, indomethacin

(10mg/kg) was administered to animals in the reference group. Control ani-

mals received distilled water (10ml/kg) only.

An hour after the treatment, egg albumen (0.2ml) was injected into the right

paw of each rat under the sub plantar region. The paw sizes were measured be-

fore and at interval of 30mins, 60mins and 90mins after egg albumen injection

by using the cotton thread method. The cotton thread was wrapped around the

paw and the circumference was measured with a meter rule. Oedema formati-

on was determined as indicator of inammatory response in the injected paw.

The inhibition of oedema formation was calculated according to the formula;

% inhibition= [( C1-Co)control –(C1-Co)test] /(C1- Co)control X100

Where Co= Paw size at time zero minute

C1= Paw size at time t minutes.

Formalin Paw lick Test in Rats

Following an overnight fast, the rats were also grouped and treated with the

extracts were as in the experiment above. Thirty minutes after treatment, 25μl

of 2.5% formalin was injected into the sub-planar surface of the rat paw. Res-

ponses were measured between 0 to 5 minutes after injection of formalin for

the first phase response; while the second phase response was observed bet-

ween 15 to 30 minutes after the injection. The licking of the paw by the rat is

indicative of pain and the duration was counted in seconds as the response to

inammatory pain.

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Analgesic Activity of the crude extract

Acetic acid- induced abdominal writhing test was used to determine the

analgesic property of the crude extract in mice. Five groups o mice were pre-

treatment with saline, extract (100, 200, 400 mg/kg), aspirin at 15mg/kg and

injected intraperitonially with 0.01ml of 0.6% acetic acid solution 60minutes

post-treatment. Five minutes post- injection of acetic acid, the number of writ-

hing was observed and counted for the next twenty minutes and recorded. The

inhibition of acetic acid- induced abdominal contractions is indicative of anal-

gesic eect.

% inhibition = (Mean No. of writhing) control – (Mean No. of writhing) test /

Mean No of writhing) control X 100

Solvent-solvent partitioning of the crude extract

To carry out the solvent–solvent partitioning of the extract, 17g of the crude

extract was dissolved in 250ml of a mixture of solvents (15% of methanol in

water) in a Separating funnel. The dissolved extract in the separating fun-

nel was partitioned with equal volume of hexane. After the collection of the

hexane fraction; the methanol-water fraction was further fractionated with

ethyl acetate to obtain the ethyl acetate fraction. Three major fractions were

obtained which are hexane, ethyl acetate and water/methanol fractions. The

analgesic activities of the three fractions were conducted at doses of 20mg/kg

and 50mg/kg using Acetic acid- induced abdominal writhing test as described

above. The result was compared to that of mice treated with aspirin (15mg/kg).

Column Chromatography of Hexane Fraction of C. sordidum

Column chromatographic separation of the hexane fraction was carried out on

silica gel stationary phase using the following order of mobile phases: hexane

100% followed by hexane: ethyl acetate (97.5:2.5), (95:5), (92.5:7.5), (90:10),

(85:15), (80:20), (70:30), (60:40), (50:50), (40:60), (30:70), (20:80), (10:90),

ethyl acetate 100% and methanol 100%. A total of 175 fractions (30mL) were

collected and combined on the basis of TLC fingerprint. Seven (7) major frac-

tions labeled F1-F7 were obtained. The analgesic activity of the seven fractions

was carried out at 10mg/kg and 15mg/kg to identify the compound responsible

for the activity. The result was also compared to that obtained for mice admi-

nistered with aspirin at 15mg/kg.

Statistical Analysis

All values are expressed as mean ± S.E.M. Data was analyzed by one- way

analysis of variance and two- way analysis of variance (ANOVA) followed by

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Dunnet and Tukey’s multiple comparison tests using Graphpad prism (6) soft-

ware. Dierences of mean were considered at p < 0.05.

RESULTS AND DISCUSSION

Anti-inammatory and Analgesic Activities of the crude extract

The initial change in paw size observed at 0 minute was primarily due to the

volume of egg albumen injected into the rat paw. The rats administered with

the acetone extract showed a dose-dependent inhibition of the paw oedema in-

duced by the egg albumen. The inammatory response in paw of rats adminis-

tered with the extract at doses of 100mg/kg (0.54±0.02cm and 0.56±0.05cm),

200mg/kg (0.84±0.12cm and 0.44±0.11cm) and 400mg/kg (0.84±0.12cm and

0.52±0.07cm) were reduced compared to that of indomethacin (1.04±0.3cm

and 0.86±0.08cm) at 30 minutes and 60 minutes post-injection, but the ext-

ract at 200mg/kg dose inhibited (0.26±0.11cm) oedema formation more sig-

nificantly (p<0.05) compared to indomethacin (0.42±0.1cm) at 90 minutes

post- injection (Table 1).

Rats pretreated with the plant extract showed lesser response to pain at both

phases of formalin paw lick model in a dose- dependent manner. Inhibition of

the response to pain in rats administered with all the extract doses was com-

parable to that observed for rats administered with indomethacin in the early

phase. At the late phase, rats treated with 200mg/kg and 400mg/kg doses sho-

wed significantly inhibition of response to pain (62% and 64% respectively)

compared to rats administered with indomethacin (37.3%) (Table 2).

A dose-dependent inhibition of abdominal writhes was observed in mice ad-

ministered with doses of the extract. The inhibition of abdominal writhing ob-

served in mice administered 400mg/kg (11.75 ± 1.19) was comparable to mice

administered aspirin (10.5 ± 0.83). Pain expressed as abdominal writhes were

significantly (p<0.05) inhibited in all mice administered with the crude extract

when compared to the negative control (untreated mice) (Table 3).

Solvent-solvent partitioned fractions and analgesic activity

Three dierent fractions were obtained from the solvent partitioned crude

extract; hexane, ethyl acetate and methanol/water which gave 5.151, 4.793

and 5.468g respectively. The fractions significantly inhibited abdominal writ-

hes compared to the control mice, and the level of inhibition of the writhes

was compared with aspirin at 15mg/kg (42.8%). Lower doses of the fractions

(20mg/kg) showed better inhibition of abdominal writhes, with mice adminis-

tered with hexane fraction (20mg/kg) showed the highest percentage inhibiti-

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on (44.4%) (Table 4). The analgesic activity of column fractions of the hexane

extract at 10mg/kg showed F1- 46.09%, F2- 48.45%, F3- 43.94%, F4- 43.94%,

F5- 57.88%, F6- 74.89%, F7- 79.51%, hexane fraction- 63.88%, while aspirin at

15mg/kg had 54.99% (Fig 1).

Table 1. Paw size (cm) and percentage oedema formation in rat paw injected with Egg

albumen and pre-treated with Combretum sordidum extract

Group 0 minute 30 minutes 60 minutes 90 minutes 120 minutes

Control

0.36±0.05

(0%)

0.62±0.12

(72.2%)

0.68±0.07

(88.9%)

0.82±0.09

(127.8%)

0.88±0.09

(144.4%)

CS 100mg/kg

0.46±0.06

(0%)

0.54±0.02

(17.4%)

0.56±0.05

(21.7%)

0.50±0.07

(8.7%)

0.50±0.07

(8.7%)

CS 200mg/kg

0.38±0.06

(0%)

0.84±0.12

(121.1%)

0.44±0.11

(15.8%)

0.26±0.11

(-31.6%)

0.20±0.10

(-47.4%)

CS 400mg/kg

0.34±0.04

(0%)

0.84±0.12

(147.1%)

0.52±0.07

(52.9%)

0.46±0.08

(35.3%)

0.20±0.09

(-41.2%)

Indomethacin

10mg/kg

0.56±0.1

(0%)

1.04±0.3

(85.7%)

0.86±0.08

(53.6%)

0.42±0.1

(-25%)

0.38±0.08

(-32.1%)

0 minutes

30 minutes

60 minutes

90 minutes

120 minutes

Different doses of extract, negative & positive controls

% oedema formation in rat paw

Figure 1: Paw size (cm) and percentage oedema formation in rat paw injected with Egg

albumen and pre-treated with Combretum sordidum extract

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Table 2. Number of paw licks (and percentage inhibition of licks) in rats administered with

formalin and pretreated with Combretum sordidum extract

Groups

Number of Licks

Early Phase (5 minutes) Late Phase (20-25minutes)

Control 107.5±17.07 (0%) 75.0±5.37 (0%)

CS 100mg/kg 90.3±15.09 (16.1%) 68.25±6.73 (9%)

CS 200mg/kg 91.3±8.08 (17.8%) 28.5±6.38 (62%)

CS 400mg/kg 81.8±3.89 (23.95%) 27.0±9.57 (64%)

Indomethacin 10mg/kg 88.5±1.03 (17.7%) 47.0±7.50 (37.3%)

Early phase (5 minutes)

Late phase (20-25 minutes)

% inhibition of licks

Figure 2. Number of paw licks (and percentage inhibition of licks) in rats administered with

formalin and pretreated with Combretum sordidum extract

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Table 3. Abdominal writhing in mice intraperitonealy injected with acetic acid and pre-treated

with Combretum sordidum

Group Number of Writhes Inhibition (%)

Control 43.6±2.22 0

CS 100mg/kg 37.6±3.41 13.76

CS 200mg/kg 25.3±4.90 42.09

CS 400mg/kg 11.8±1.19 73.05

Indomethacin 10mg/kg 10.5±0.83 75.9

Figure 3. Abdominal writhing in mice intraperitonealy injected with acetic acid and pre-

treated with Combretum sordidum

Number of writhes

Inhibition

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Table 4. Effect of Methanol-water, ethyl acetate, and Hexane fractions on acetic acid- induced

abdominal writhing in mice

Group Number of writhes Inhibition (%)

Control 62.6 ± 1.25 0

Aspirin 15mg/kg 35.8 ± 4.31 42.8

MeOH 20mg/kg 39.0 ± 3.53 37.7

MeOH 50mg/kg 46.5 ± 2.97 25.7

EA 20mg/kg 35.4 ± 1.67 43.5

EA 50mg/kg 43.0 ± 4.10 31.3

Hex 20mg/kg 34.8 ± 4.14 44.4

Hex 50mg/kg 46.0 ± 3.65 26.5

Number of writhes

Inhibition

Figure 4. Effect of Methanol-water, ethyl acetate, and Hexane fractions on acetic acid-

induced abdominal writhing in mice

In this study, acetone extract of C. sordidum demonstrated anti-inamma-

tory activities by inhibition of paw oedema formation after exposure to egg

albumen and paw licking as a response to formalin-induced pain in rats. The

anti-inammatory response was comparable to that of indomethacin, a non-

steroidal anti-inammatory drug used for treatment of arthritis, gout, bursi-

tis.

Egg albumen-induced paw oedema is an animal model of inammation

used to detect anti-inammatory agents with activity in the acute phase of inf-

lammation.

This study demonstrated the anti-inammatory eect of C. sor-

didum was significantly established by 60 minutes post-injection of albumen.

This suggests anti-inammation with activity in acute inammatory phase.

Findings from the formalin-induced paw lick model also corroborated the an-

ti-inammatory eect of C. sordidum. It further established a more marked

anti-inammatory activity in the late phase of inammation, with significantly

more profound anti-inammatory action compared to the same indomethacin.

Formalin-induced paw lick model of inammation demonstrates the inter-

relation of inammation and nociception with distinct responses in the early

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

and late phases of inammation but mediated via dierent inammatory

pathways.

The early phase is correlated with direct eect on peripheral no-

ciceptors with minimal inuence of prostaglandins. This stimulation in the

early phase culminates in centrally mediated pain accompanied by release of

substance P.

21,22

The late phase is characterized by release of mediators of inf-

lammation; histamine, prostaglandins, serotonin and bradykinin.

23,24

The an-

ti-inammatory activity of C. sordidum is suggested to be mediated via the pe-

ripheral and central mechanisms of inammatory pathways, and may involve

inhibition of cyclooxygenase enzymes which are active players in the model.

Centrally acting drugs such as opioids inhibit both phases equally but perip-

herally acting drugs such as cyclooxygenase inhibitors and corticosteroids only

inhibit the late phase.

The acetone extract of Combretum sordidum was also found to possess analge-

sic activities in this study with the significant inhibition of abdominal writhing

in the pretreated mice. Acetic acid- induced writhing in mice is a commonly

used model of analgesia in experimental animals for establishment of periphe-

rally acting analgesic drugs which may involve stimulation of local peritoneal

receptors.

26,27

The crude extract of C. sordidum showed a dose-dependent and

significant analgesic action when compared to the reference drugs, indomet-

hacin and aspirin. Acetic acid causes pain by liberating endogenous substances

that excite pain nerve endings.

Pain sensation in acetic acid–induced writhing method is elicited by trigge-

ring localized inammatory response resulting in release of free arachidonic

acid from tissue phospholipids via cyclooxygenase (COX) and prostaglandin

(PG) biosynthesis.

29,30

The acetic acid – induced writhing has been specifically

associated with increased level of PGE

and PGE

α in peritoneal uids as well

as lipooxygenase products.

The increase in prostaglandin levels within the

peritoneal cavity then enhances inammatory pain by increasing capillary

permeability. The acetic acid- induced writhing method is eective to evalu-

ate peripherally active analgesics. Agents which reduce the number of writhes

will render analgesic eect probably by inhibition of prostaglandin synthesis,

a peripheral mechanism of pain inhibition.

Therefore, the result of this study

shows that the extract possesses strong peripheral analgesic eect. Some of the

phytoconstituents of this plant such as avonoids, saponins, and tannins have

been demonstrated to have significant pharmacological activities.

Solvent partitioned fractions of C. sordidum extract (hexane, ethyl acetate and

methanol-water fractions) demonstrated potent analgesic eect in acetic acid-

induced writhing test in mice, with the hexane fraction showing the highest

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

activity at 20mg/kg body weight. Further purification of the hexane fraction

yielded 7 fractions with fractions 6 and 7 showing marked inhibition of abdo-

minal writhing, almost 2-folds of that observed in aspirin.

Extract of C. sordidum at all doses showed anti-inammatory and analgesic

activities which are comparable to that exhibited by indomethacin or aspirin.

This study suggested that the anti-inammatory eect of C. sordidum may be

mediated via both peripheral and central mechanisms of inammation. The

solvent partitioned fractions showed hexane fraction had the most potent anal-

gesic activity and further purification yielded 7 fractions. Fractions 6 and 7

showed remarkable analgesic eect with almost 2-folds of that observed for

aspirin. The peripheral pathway for mediation of the analgesic eect of the

extract, solvent-partitioned fractions and column fractions of hexane fractions

was established also. This is a report of preliminary anti-inammatory and

analgesic activities of C. sordidum. Further studies are going on to purify, cha-

racterize and completely elucidate the structure of the bioactive compound in

this extract, particularly bioactive compounds responsible for the remarkable

analgesic activity of the fractions.

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Acta Pharm. Sci. Vol 57 No: 2. 2019

DOI: 10.23893/1307-2080.APS.05710

Combination of Cumulative Area Pre-

Processing and Partial Least Squares for

Handling Intensely Overlapping Binary and

Ternary Drug Systems

Yahya S Al-Degs

, Amjad H. El-Sheikh

, Eman A. Abu Saaleek

, Reema A. Omeir

Musab A. Al-Ghodran

1 Chemistry Department, The Hashemite University, P. O. Box 150459 Zarqa 13115 Jordan

ABSTRACT

The analytical performance of cumulative area pre-processing (CAP), a recently

developed signal filtering method, along with multivariate calibration for quan-

tification of spectrally overlying drugs was outlined. The drug combinations con-

taining high level of paracetamol (PAR) in the presence of caeine (CAF), chlorp-

heniramine maleate (CHL), pseudoephedrine hydrochloride (PSE), phenylephrine

hydrochloride (PHE), and diphenhydramine hydrochloride (DPH). The tested

formulations were: PAR-CAF-PHE, PAR-CAF-PHE, and PAR-DRH. Based on net-

analyte signal calculations, the formulations exhibited intense overlapping 53-68%

for PAR-PSE-CHL, 55-95% for PAR-CAF-PHE, and 44% for PAR-DRH. For each

system, PLS latent variables were estimated using cross-validation technique and

more factors were needed for highly overlapping systems. PLS-CAP was found app-

licable for drugs quantification in all systems with excellent performance regard-

less the size of spectral overlapping and ratios of components in the formulation.

For PAR-PSE-CHL (ratio 300:30:2 mg/tablet), the ingredients were quantified by

CAP-PLS with satisfactorily recoveries (RSD, n = 3) 89.9% (3.1%), 104.6% (2.7%),

and 99.0% (1.5%) for PAR, PSE, and CHL, respectively. Both PLS and CAP-PLS

were demonstrated the same performance for binary system of modest overlapping

and no component available in low concentration.

Keywords: Signal Pre-processing; Spectral overlapping; Binary and Ternary Drug

Formulations; PLS calibration

*Corresponding Author: Dr. Yahya Al-Degs, e-mail: yahyaaldeqs@yahoo.com

Yahya S. Al-Degs ORCID Number: 0000-0002-9555-7594

Amjad H. El-Sheikh ORCID Number: 0000-0001-8321-3236

Eman A. Abu Saaleek ORCID Number: 0000-0003-3163-2919

Reema A. Omeir ORCID Number: 0000-0002-3508-5927

Musab A. Al-Ghodran ORCID Number: 0000-0002-4766-0730

(Received 29 December 2018, accepted 24 January 2019)

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INTRODUCTION

Commercial drug formulations often contain more than one activates pharma-

ceutical ingredients (APIs) that present in variable levels to achieve the best

pharmacological performance

. The spectral overlapping between the active

ingredients is often moderate but intense overlapping is also possible. In ad-

dition to spectral overlapping and nonlinearity in the system, drug producti-

on stages like crystallization, drying, solid dosage form, added excipients, and

tableting at dierent conditions can aect the spectral behavior of APIs

1,2

. Ac-

cordingly, accurate analytical methods are always needed in this regard

. The

development and formulation of the pharmaceuticals brought a revolution in

human health. These pharmaceuticals would help their intent only if they are

free from impurities and are administered in an appropriate quantity. Hen-

ce, there are many challenges and it can be reduced by eective use of exci-

pients, which permits formulators to overcome these challenges. It becomes

necessary to develop new analytical methods because sometimes the dosage

form contains other substances which potentially interfere in the assay and, if

not corrected, may impart a systematic error to the assay

. Multi-component

formulations have gained a lot of attention nowadays due to greater patient

acceptability, increased potency, multiple action, quick relief and fewer side

eects

1,2

. Market is ooded with combination of drugs in various dosage form.

One of such combination is paracetamol (PAR) with other drugs including caf-

feine (CAF), diphenhydramine hydrochloride (DPH), chlorpheniramine ma-

leate (CHL), pseudoephedrine hydrochloride (PSE), phenylephrine hydrochlo-

ride (PHE). For example, PAR and DPH are co-formulated in pharmaceutical

product for temporary relief of pain when associated with sleeping diiculty

2-7

The combination PAR-CHL-PSE has been recently introduced in the market to

treat the symptoms of most ues

5-7

. The combination PAR-CAF-PHE was also

formulated to relief from major cold and u symptoms and applied at day time

as it doesn’t cause drowsiness.

Various chemical and instrumental methods were developed to make drugs

serve their purpose at regular intervals which are involved in the estimation of

drugs. These pharmaceuticals may develop impurities at various steps of the-

ir development, transportation and storage, which makes the pharmaceutical

risky to be administered. Thus, they must be identified and quantified. Hence,

analytical instrumentation and methods play an important role. Thus, the re-

view highlights a variety of analytical, chromatographic and instrumental met-

hod developed such as; High Performance Liquid Chromatography (HPLC),

High Performance Thin Layer Chromatography (HPTLC), and Gas Chroma-

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

tography (GC) have wide application in assuring the quality and quantity of

pharmaceutical products and these instrumental methods are simple, precise,

rapid, and reproducible and have been applied in the analysis of pharmaceuti-

cals for assessing the quality of the drugs

8-10

Recently, multivariate calibration methods have been intensively employed for

analyzing pharmaceutical formulations and detection of released drugs in uri-

ne and plasma

5-7

. Particularly speaking, PAR and CAF have been quantified in

drug formulations using first-order multivariate calibration methods with high

accuracy and without implementing any chromatographic procedure

6,7

. Com-

pared to liquid chromatography, assaying drug formulations by multivariate

calibration required less solvent consumption and avoids using tedious chro-

matographic instruments

2,3

. First order multivariate calibration methods inc-

ludes multilinear regression (MLR), principal component regression (PCR),

partial least-squares (PLS-1), and many others

. In a recent study, both PAR

and PHE were quantified in four-drug formulations using PLS1 calibration

5,6

PHE was quantified in the presence of PAR in tablet form with excellent re-

covery

. In another study, novel application of PCR and PLS was developed

for determination of PAR, CAF, and CHL in real formulations containing ot-

her medicines

6,7

. In the drug formulation, the recovery and RSD values were

99.91% (1.69%), 100.15%(1.35%) and 100.49%(1.66%) for PAR, CAF and CHL,

respectively. In the second method PLS was applied to quantify four medicines

in real formulation with high recoveries and RSD: 99.80% (1.67%), 100.19%

(1.42%) and 100.45% (1.86%) for PAR, CAF and CHL, respectively

. There is

no published literature dealing with simultaneous quantification of PAR and

DPH in real formulations. Spectral overlapping between drugs was reported to

have a negative inuence on the performance of multivariate calibration met-

hods

. Form the earlier studies, it is concluded that multivariate calibration is

applicable for medicines determination in dierent commercial formulations

including tablet and syrups.

Usually, the pre-processing methods are divided into three categories: a) filte-

ring and de-noising, generally associated with the improvement of the signal-

to-noise ratio. These methods are applied to the raw data before the construc-

tion of the multivariate model and reduce the inuence of the random variance

without altering that useful, b) spectral normalization and dierentiation,

such as the spectral derivation and the application of the Fourier transform.

These procedures are also applied before the construction of the model, and

c) selection of the variables and reduction of dimensionality of the data. These

methods are applied by multivariate modeling and therefore involve both the

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

matrix X (independent variables) and the matrix Y (dependent variables). The

most used method is orthogonal signal correction. There are a number of ex-

cellent review articles providing guidance for application of the pre-processing

techniques to the analytical signals

12-14

. In some pharmaceutical formulations

one or more components are present in very low concentrations, so as to be

hidden by the components more concentrated or by the instrumental noise.

This increases the diiculty of building a mathematical model able to predict in

a satisfactory manner all the species present in the mixture. In these cases, the

data pre-treatment should provide to amplify the analytical information due to

the components at lower concentration and at the same time to minimize the

spectral noise that can interfere with their determination.

In the current work, the application of newly proposed cumulative area pre-

processing (CAP) will be evaluated for prior to PLS calibration for quantifica-

tion of binary and ternary drugs systems. CAP was developed to amplify the

spectral signals. CAP-PLS will be tested on the quantitative analysis of one

binary and ternary pharmaceutical formulation. The common diiculty in

analyzing these matrices was the presence of some components in much lower

amount than other components. The binary formulation consisted of para-

cetamol-diphenhydramine hydrochloride (ratio 20:1), paracetamol-caeine-

phenylephrine hydrochloride (ratio 100:5:1 mg/tablet), and paracetamol-pse-

udoephedrine hydrochloride-chlorpheniramine maleate (ratio 150:15:1 mg/

tablet). The analytical performance of CAP-PLS and PLS for drugs quantifica-

tion will critically discussed. More attention will be paid on the inuence spect-

ral overlapping among drugs on their quantification by CAP-PLS. Inuence of

pH and spectral range on spectral analysis will be also considered.

METHODOLOGY

Drugs and marketed formulations

The pharmaceuticals paracetamol (PAR), caeine (CAF), diphenhydramine

hydrochloride (DPH), phenylephrine hydrochloride (PHE), pseudoephedrine

hydrochloride (PSE), and chlorpheniramine maleate (CHL) were kindly do-

nated from Dar Al-Dawaa company (Dar Al-Dawaa, Naour, Amman, Jordan).

Distilled water was used for preparation of standard solutions and dissoluti-

on of commercial tablets. The marketed drugs were Panadol cold and u®

(Active ingredients: PAR-PSE-CHL 500:30:2 mg/tablet; excipients Silicon di-

oxide, stearic acid, sodium benzoate, povidone, starch, maize starch, and talc,

GlaxoSmithKline Dungarvan Ltd., Co. Waterford, Ireland), Panadol cold and

u Day® (Active ingredients: PAR-CAF-PHE 500:25:5 mg/tablet; excipients

pre-gelatinised starch, maize starch, povidone, potassium sorbate, talc, stearic

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

acid, microcrystalline cellulose, sodium lauryl sulphate, sunset yellow, Gla-

xoSmithKline Dungarvan Ltd., Co. Waterford, Ireland), and Panadol night

(PAR-DPH 500:25 mg/tablet; excipients maize starch, pregelatinised starch,

potassium sorbate, povidone, talc, and stearic acid, GlaxoSmithKline Dungar-

van Ltd., Co. Waterford, Ireland) All other reagents were of the highest purity

commercially available. Table 1 summaries some chemical parameters of the

undertaken pharmaceuticals.

Table 1. Chemical properties of studied pharmaceuticals.

Name/short

name

Formula Structural formula Medical action pK

Paracetamol

PAR

Treat mild to

moderate pains

9.5

Pseudoephedrine

hydrochloride

PSE

Shrink blood

vessels in the nasal

passage (prevent

stuffy nose)

10.3

Chlorpheniramine

Maleate

CHL

Relieve symptoms

of allergy

9.1

Caffeine

CAF

Acts as central

nervous system

stimulant

14.0

Phenylephrine

hydrochloride

PHE

ClNO

H Cl

Sympathomimetic

(descongestants)

9.07

Diphenhydramine

Hydrochloride

DPH

ClNO

H Cl

Blocking the effects

of histamine and

causes drowsiness.

9.13

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Standard solutions, calibration/validation mixtures

Stock solutions were separately prepared by dissolving in ethanol nearly 25.0

(±0.0001 g) mg of each drug in 100 mL volumetric asks. A first set of 9 binary

calibration samples was built by combining five levels of PAR and DPH within

the range 1.0–12.0 mg/L.A second calibration set of nine ternary mixture solu-

tions was prepared for PAR-CAF-PHE and PAR-PSE-CHL systems. For PAR-

CAF-PHE, drug ranges were 2.0-15.0 mg/L

for PAR, 2.0-16.0 mg/L for CAF

and 1.0-8.0mg/L for PHE. For PAR-PSE-CHL, drug ranges were1.0-16.0 mg/L

for PAR, 1.0-8.0 mg/L for PSE, and 1.0-12.0 mg/L for CHL. Calibration set of

the binary mixtures was created by following a full experimental design while

calibration sets of binary systems selected according to Brereton`s rule using

three dierent concentration levels. To validate the PLS and CAP-PLS models,

three independent external validation sets consisting of 7 solutions for binary

and ternary systems were prepared. Marketed formulations were assayed by

weighing four tablets for binary system and five tablets for ternary systems

and grinding the tablets to a fine powder. The powder was suspended in water

and diluted to a final volume of 100 ml. The suspension was sonicated for 10

min, filtered through a 0.45μm filter, and properly diluted in preparation for

spectral analysis.

Partial least squares and net-analyte signal calculations

A double beam UV-visible spectrophotometer (Thermo scientific. Genesys

10S UV-VIS (USA)) with quartz cuvette cell of 1.0 cm path length was emplo-

yed. The spectral bandwidth was 1.0 nm with fast wavelength-scanning speed.

Scans were carried out in the range of 200–300 nm at (1.0 nm step, 101 points/

spectrum). The spectral data obtained for calibration mixtures are placed in

matrix A (size 101×9) and spectral data of validation set are placed matrix B

(size 101×7). On the other hand, concentration data are placed matrix C (size

9×3 for ternary systems and size 9×2 for binary system). Partial least squa-

res PLS1 is an eicient tool for developing a quantitative relationship between

several predictor variables A (spectral data) and a property of interest c (the

independent variable or drugs content) as shown below

11,15

c=Ab (1)

Where c contains the concentrations of calibrated drug in calibration samples

arranged in a vector and b is the calibration sensitivity which is necessary for

estimating drugs content in the adsorption solutions

11,16-18

b=W

(PW

)

-1

q (2)

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Where W is the weights matrix for A, P is the loadings matrix of A, q is the

loadings vector for c, t donates transpose operation, and -1 stands for inverse

operation. Once b is estimated, prediction of drug concentrations (c

) from

the unknown spectrum a is carried out as following

11,16-18

= ab (3)

Optimum number of PLS-latent variables were estimated using cross-validation

method

. The predicted concentrations were compared with the known con-

centrations of the compounds in each calibration sample. The prediction error

sum of square (PRESS) and the relative error of prediction (REP %) were cal-

culated for drugs in calibration and validation samples as following

17,19

Partial least squares and net-analyte signal calculations

A double beam UV-visible spectrophotometer (Thermo scientific. Genesys 10S UV-VIS (USA)) with

quartz cuvette cell of 1.0 cm path length was employed. The spectral bandwidth was 1.0 nm with fast

wavelength-scanning speed. Scans were carried out in the range of 200–300 nm at (1.0 nm step, 101

points/spectrum). The spectral data obtained for calibration mixtures are placed in matrix A (size

101×9) and spectral data of validation set are placed matrix B (size 101×7). On the other hand,

concentration data are placed matrix C (size 9×3 for ternary systems and size 9×2 for binary system).

Partial least squares PLS1 is an efficient tool for developing a quantitative relationship between

several predictor variables A (spectral data) and a property of interest c (the independent variable or

drugs content) as shown below

11,15

c=Ab (1)

Where c contains the concentrations of calibrated drug in calibration samples arranged in a vector and

b is the calibration sensitivity which is necessary for estimating drugs content in the adsorption

solutions

11,16-18

b=W

(PW

)

-1

q (2)

Where Wis the weights matrix for A, P is the loadings matrix of A, q is the loadings vector for c, t

donates transpose operation, and -1 stands for inverse operation. Once b is estimated, prediction of

drug concentrations (c

) from the unknown spectrum a is carried out as following

11,16-18

= ab (3)

Optimum number of PLS-latent variables were estimated using cross-validation method

. The

predicted concentrations were compared with the known concentrations of the compounds in each

calibration sample. The prediction error sum of square (PRESS) and the relative error of prediction

(REP %) were calculated for drugs in calibration and validation samples as following

17,19



−=

prediactuali

CCPRESS

)(

(4)

%REP = 100×

2/1

)(













−



acti

predi

(5)

Where C

actual

, C

prd

, and n, are actual concentration (mg/L), predicted concentration obtained by PLS

(mg/L), and number of training samples (solutions), respectively. A unifying framework for

calibration and prediction in multivariate calibration is shown based on the concept of the Net Analyte

Signal (NAS). NAS is the part of the measured spectrum that the calculated model uses for prediction.

The main equation that is needed to estimated figures of merit is

15,20,21

* = [I-S

-k

(6)

Where Sis the matrix of sensitivities collected for all other solutes, s

is the sensitivity vector of the

analyte, and s

* is the spectrum of pure analytek measured at unite concentration (sensitivity factor).

Sensitivity (SEN) value of analytek is the norm (║║) of the net sensitivity vector, it was defined as the

amount of net signal that, in prediction, corresponds to a concentration equal to unity

SEN = ║s

*║ (7)

Selectivity (SEL) can be defined as the part of the measured signal unique to the analyte of interest.

Based on NAS theory, the selectivity measures the extent of spectral overlapping. SEL can be

expressed by the ratio between the norm of the NAS vector and the norm of the spectra. SEL values

are extended from zero (high overlap with other interferences) to unity (no overlap with other sample

components). High values of SEN are an indication of high method accuracy for that analyte

15,20,21

SEL = ║s

*║/║s

║ (8)

The limit of detection (LOD) obtained assumes that the prediction uncertainties are approximately

constant. LOD gives the minimum detectable amount of k solute. A reasonable estimation to

determine the LOD can be estimated as

LOD = 3║ε║/║s

*║ (9)

Where ║ε║ is a measurement of the instrumental noise, and s* was defined above. Then the norms of

blank readings (║NAS

blank

║) are estimated and ║ε║ is taken as the standard deviation of estimated

norms

.The limit of quantification (LOQ) is the minimum quantifiable amount of the solute, is

estimated as

15,21

(5)

Where C

actual

, C

prd

, and n, are actual concentration (mg/L), predicted concent-

ration obtained by PLS (mg/L), and number of training samples (solutions),

respectively. A unifying framework for calibration and prediction in multi-

variate calibration is shown based on the concept of the Net Analyte Signal

(NAS). NAS is the part of the measured spectrum that the calculated model

uses for prediction. The main equation that is needed to estimated figures of

merit is

15,20,21

* = [I-S

-k

(6)

Where Sis the matrix of sensitivities collected for all other solutes, s

is the sen-

sitivity vector of the analyte, and s

* is the spectrum of pure analytek measured

at unite concentration (sensitivity factor). Sensitivity (SEN) value of analytek

is the norm (║║) of the net sensitivity vector, it was defined as the amount of

net signal that, in prediction, corresponds to a concentration equal to unity

SEN = ║s

*║ (7)

Selectivity (SEL) can be defined as the part of the measured signal unique to

the analyte of interest. Based on NAS theory, the selectivity measures the ex-

tent of spectral overlapping. SEL can be expressed by the ratio between the

norm of the NAS vector and the norm of the spectra. SEL values are extended

168

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

from zero (high overlap with other interferences) to unity (no overlap with ot-

her sample components). High values of SEN are an indication of high method

accuracy for that analyte

15,20,21

SEL = ║s

*║/║s

║ (8)

The limit of detection (LOD) obtained assumes that the prediction uncertainti-

es are approximately constant. LOD gives the minimum detectable amount of

k solute. A reasonable estimation to determine the LOD can be estimated as

LOD = 3║ε║/║s

*║ (9)

Where ║ε║ is a measurement of the instrumental noise, and s* was defined

above. Then the norms of blank readings (║NAS

blank

║) are estimated and ║ε║

is taken as the standard deviation of estimated norms

.The limit of quantifi-

cation (LOQ) is the minimum quantifiable amount of the solute, is estimated

15,21

LOQ = 10║ε║/║s

*║ (10)

All calculations were carried out using special matlab-codes under MATLAB

Cumulative area pre-processing of spectral data prior to

PLS calibration

Data treatment by CAP transforms the original UV spectrum to a new curve

using the area underlying the same spectrum. This elaboration is based on two

mathematical steps. In the first step, the area a

λi

binning of the two consecutive

wavelengths (λ

and λ

i+1

) is calculated as

LOQ = 10║ε║/║s

*║ (10)

All calculations were carried out using special matlab-codes under MATLAB

Cumulative area pre-processing of spectral data prior to PLS calibration

Data treatment by CAP transforms the original UV spectrum to a new curve using the area underlying

the same spectrum. This elaboration is based on two mathematical steps. In the first step, the area a

λi

binning of the two consecutive wavelengths (λ

and λ

i+1

) is calculated as

abab





(11)

Where ab is the absorbance value at a single wavelength. This calculation is repeated for all the

wavelengths in the spectrum. In the second step, the cumulative sum of the single areas is computed,

in such a way as the cumulative area at each wavelength A

λi

resulted from the sums of all areas a

the preceding wavelengths. For a spectral region between λ

and λ

(λ

<λ

), the cumulative area A

λm,n

is estimated as



inm





(12)

Treatment of the original UV spectra using CAP (Eqs 11 and 12) offers many advantages including

amplification of the analytical signals for all solutes and possible quantification

RESULTS AND DISCUSSION

Influence of solution pH on spectral behavior of pharmaceuticals

Influence of solution pH on spectral behavior was studied for all drugs at pH 3.0, 7.0, and 10.0 to

cover all possible conditions. Among the drugs, identical spectral shapes over the range (230-280 nm)

were observed for CHL and PSE and hence there was no need to present their profiles. On the other

hand, the UV-absorption behavior of PAR (which present in all systems), CAF, PHE, and DPH were

notably changed with solution pH as depicted in Fig 1.

(11)

Where ab is the absorbance value at a single wavelength. This calculation is

repeated for all the wavelengths in the spectrum. In the second step, the cumu-

lative sum of the single areas is computed, in such a way as the cumulative area

at each wavelength A

λi

resulted from the sums of all areas a

of the preceding

wavelengths. For a spectral region between λ

and λ

(λ

<λ

), the cumulative

area A

λm,n

is estimated as

LOQ = 10║ε║/║s

*║ (10)

All calculations were carried out using special matlab-codes under MATLAB

Cumulative area pre-processing of spectral data prior to PLS calibration

Data treatment by CAP transforms the original UV spectrum to a new curve using the area underlying

the same spectrum. This elaboration is based on two mathematical steps. In the first step, the area a

λi

binning of the two consecutive wavelengths (λ

and λ

i+1

) is calculated as

abab





(11)

Where ab is the absorbance value at a single wavelength. This calculation is repeated for all the

wavelengths in the spectrum. In the second step, the cumulative sum of the single areas is computed,

in such a way as the cumulative area at each wavelength A

λi

resulted from the sums of all areas a

the preceding wavelengths. For a spectral region between λ

and λ

(λ

<λ

), the cumulative area A

λm,n

is estimated as



inm





(12)

Treatment of the original UV spectra using CAP (Eqs 11 and 12) offers many advantages including

amplification of the analytical signals for all solutes and possible quantification

RESULTS AND DISCUSSION

Influence of solution pH on spectral behavior of pharmaceuticals

Influence of solution pH on spectral behavior was studied for all drugs at pH 3.0, 7.0, and 10.0 to

cover all possible conditions. Among the drugs, identical spectral shapes over the range (230-280 nm)

were observed for CHL and PSE and hence there was no need to present their profiles. On the other

hand, the UV-absorption behavior of PAR (which present in all systems), CAF, PHE, and DPH were

notably changed with solution pH as depicted in Fig 1.

(12)

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Treatment of the original UV spectra using CAP (Eqs 11 and 12) oers many

advantages including amplification of the analytical signals for all solutes and

possible quantification

RESULTS AND DISCUSSION

Inuence of solution pH on spectral behavior of pharmaceuticals

Inuence of solution pH on spectral behavior was studied for all drugs at pH

3.0, 7.0, and 10.0 to cover all possible conditions. Among the drugs, identical

spectral shapes over the range (230-280 nm) were observed for CHL and PSE

and hence there was no need to present their profiles. On the other hand, the

UV-absorption behavior of PAR (which present in all systems), CAF, PHE, and

DPH were notably changed with solution pH as depicted in Fig 1.

Figure 1. Influence of pH on spectral behavior of tested drugs (7.0 mg/Leach drug)

As shown in Fig 1, large variation was noted in the spectra of CAF at the studied pH and over the

range (200-240 nm) and slight variations were observed over the rest of the domain. In the same time,

the distinct wavelength positioned at 273 nm was not affected with solution pH. More variations were

observed in the PAR spectra, the intensity was notably increased at pH over the range 200-220 nm

while the main peak (243 nm) was shifted to 250 nm at pH 10.0. The most drastic changes were

reported for PHE as shown in Fig 1. It was highly possible that PHE underwent chemical reaction at

the basic medium and this was deduced from the developed spectrum which has two wavelengths at

208 and 237 nm. To avoid PHE side-reaction, it was practical to run UV measurements at pH 7.0. As

can be noted from the earlier discussion, the optimum pH for spectral analysis is 7.0 which maintain

stable structure and high UV absorption for all drugs. For DPH, there was a significant light

absorption with pH over the range (200-230 nm), while, pH has no effect on the spectrum over the rest

of the domain. pH 7.0 seems to be a good choice to run spectral measurement for this drug. In fact,

protonation, hydrolysis, and internal-arrangement of drug molecules are highly possible at acidic or

0,0

0,5

1,0

1,5

200 220 240 260 280 300

Absorbance

Wavelength/nm

CAF

pH 3

pH 7

pH 10

0,0

0,5

1,0

1,5

200 220 240 260 280 300

Absorbance

Wavelength/nm

PAR

pH 3

pH 7

pH 10

0,1

0,2

0,3

0,4

0,5

0,6

0,7

200 220 240 260 280 300

Absorbance

Wavelength/nm

PHE

pH 3

pH 7

pH 10

0,0

0,2

0,4

0,6

0,8

1,0

1,2

200 220 240 260 280 300

Absorbance

Wavelength/nm

DPH

pH 3

pH 7

pH 10

Figure 1. Inuence of pH on spectral behavior of tested drugs (7.0 mg/Leach drug)

As shown in Fig 1, large variation was noted in the spectra of CAF at the studied

pH and over the range (200-240 nm) and slight variations were observed over the

rest of the domain. In the same time, the distinct wavelength positioned at 273

nm was not aected with solution pH. More variations were observed in the PAR

spectra, the intensity was notably increased at pH over the range 200-220 nm

while the main peak (243 nm) was shifted to 250 nm at pH 10.0. The most drastic

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

changes were reported for PHE as shown in Fig 1. It was highly possible that PHE

underwent chemical reaction at the basic medium and this was deduced from the

developed spectrum which has two wavelengths at 208 and 237 nm. To avoid PHE

side-reaction, it was practical to run UV measurements at pH 7.0. As can be noted

from the earlier discussion, the optimum pH for spectral analysis is 7.0 which ma-

intain stable structure and high UV absorption for all drugs. For DPH, there was a

significant light absorption with pH over the range (200-230 nm), while, pH has

no eect on the spectrum over the rest of the domain. pH 7.0 seems to be a good

choice to run spectral measurement for this drug. In fact, protonation, hydrolysis,

and internal-arrangement of drug molecules are highly possible at acidic or basic

solutions and this aects their spectral properties. Accordingly, solution pH sho-

uld be adjusted to get unique spectral characteristics of the measured drug.

Spectral behavior of pharmaceuticals and spectral overlapping

In fact, intense spectral overlapping among pharmaceuticals would retard their qu-

antification in solution and in real formulation

11,15

. Accordingly, the spectral behavior

of pharmaceuticals and extent of overlapping were evaluated before running mul-

tivariate calibration. The UV spectra of the studied systems are presented in Fig. 2

basic solutions and this affects their spectral properties. Accordingly, solution pH should be adjusted

to get unique spectral characteristics of the measured drug.

Spectral behavior of pharmaceuticals and spectral overlapping

In fact, intense spectral overlapping among pharmaceuticals would retard their quantification in

solution and in real formulation

11,15

. Accordingly, the spectral behavior of pharmaceuticals and extent

of overlapping were evaluated before running multivariate calibration. The UV spectra of the studied

systems are presented in Fig. 2

Figure 2. UV Spectra of the binary and ternary drug systems. Top PAR–DPH (6.0 mg/L and pH 7.0),

Middle CAF–PAR–PHE (7.0 mg/L and pH 7.0) Bottom PAR–PSE–CHL (5.0 mg/L and pH 7.0)

As indicated in Fig. 2, all drugs exhibited strong light absorption over the range (200 300 nm) in

addition to intense overlapping among the signals. For PAR-DPH, a large spectral overlap was

observed between both compounds and particularly over the range 220-280 nm. In addition to intense

overlapping, PAR has better UV absorption compared to DPH and would make their quantification by

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

200 220 240 260 280 300

Absorbance

Wavelength/nm

Paracetamol (PAR)

Diphenhydramine

hydrochloride (DPH)

0,00

0,50

1,00

1,50

200 220 240 260 280 300

Absorbance

Wavelength/nm

Caffeine (CAF)

Paracetamol (PAR)

Phenylephrine Hydrochloride

(PHE)

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

200 220 240 260 280 300

Absorbance

Wavenumber/nm

Paracetamol (PAR)

Pseudoephedrine hydrochloride (PSE)

Chlorpheniramine Maleate (CHL)

Figure 2. UV Spectra of the binary and ternary drug systems. Top PAR–DPH (6.0 mg/L and

pH 7.0), Middle CAF–PAR–PHE (7.0 mg/L and pH 7.0) Bottom PAR–PSE–CHL (5.0 mg/L and

pH 7.0)

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

As indicated in Fig. 2, all drugs exhibited strong light absorption over the range

(200 300 nm) in addition to intense overlapping among the signals. For PAR-

DPH, a large spectral overlap was observed between both compounds and par-

ticularly over the range 220-280 nm. In addition to intense overlapping, PAR

has better UV absorption compared to DPH and would make their quantificati-

on by simple spectrometry (especially DPH) a hard task. The intense absorpti-

on of PAR at 243 nm was mainly attributed to the n-π*. One more point on this

PAR-DPH is the presence of DPH in much lower amount compared to PAR in

real formulation and this added more analytical obstacles on their accurate qu-

antification. For the ternary system CAF–PAR–PHE, the solutes were actively

absorbing over the region this was attributed to their chemical structure which

contains many active functional groups. The spectra of the drugs indicated the

following distinct absorption wavelengths at 243, 273, and (215 and 272 nm)

for PAR, CAF and PHE, respectively. The reported absorption wavelengths

were mainly attributed to n→π* and π→π* electronic transitions in the mole-

cules. In fact, the longer maximum wavelength of absorption observed for CAF

(273 nm) was attributed to the conjugated system as indicated from the chemi-

cal structures of the drugs (Table 1). Generally, PHE showed weaker absorption

compared to other drugs and absorb near the lower end of the spectral range

(220 nm). The spectral overlap between drugs is high over the entire spectral

range. Within the spectral regions 250-270, an intense overlapping between

PAR and CAF was observed and this would aect their quantification in real

formulations. Moreover, PHE absorbs over the entire range. In this ternary

system, PHE should be added in much lower levels and this negatively reected

on its quantification in real formulations. For PAR–PSE–CHL, the solutes were

active in UV region and this is attributed to their chemical structure which con-

tains aromatic parts substituted with functional groups (Table 1). The spectra

of drugs indicated the following special absorption wavelengths 210, 242, and

265 nm for PSE, PAR and CHL, respectively. The observed bands were mainly

attributed to n→π* and π→π*electronic transitions. Generally, PSE has a poor

UV absorption compared to two drugs and absorb near the extreme of the app-

lied spectral domain. In fact, the spectral overlap between drugs is high over

the entire spectral range. Within the spectral regions 220-240 and 260-280

nm, an intense overlapping between PAR and CHL was observed (Fig. 1). Mo-

reover, PSE absorbs over the entire range. Practically, PAR and PSE are added

in much higher levels compared to CHL and this would make quantification

of the later drug a hard-analytical task. For each system, the extent of spectral

overlapping between drugs was estimated using net-analyte signal calculations

(Eq. 8) and the results are provided in Table 2.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Table 2. Extent of spectral overlapping in the drug systems using NAS calculations

Total spectral overlapping with other drugs%

PAR–PSE–CHL

PAR 53

PSE 55

CHL 68

CAF–PAR–PHE

CAF 68

PAR 85

PHE 79

PAR–DPH

PAR 38

DPH 62

For PAR–PSE–CHL, NAS calculations were carried out at 5.0 mg/L, pH 7.0, and spectral

range 200-300 nm. For CAF–PAR–PHE, calculations were carried out at 7.0 mg/L, pH 7.0 and

200-300 nm. For PAR–DPH, 6.0 mg/L, pH 7.0 and 200-300 nm.

As indicated in Table 2, a significant overlapping among drugs was repor-

ted and the intense overlapping was in the ternary system (CAF–PAR–PHE)

which extended from 68% to 85%. For the earlier ternary system, PAR exhi-

bited an intense overlapping with CAF and PHE which may negatively reect

on its accurate quantification in solution or in real extracts. In fact, PAR is

added many-folds higher than other two drugs in the formulation which may

not aect its final measurements. On the other hand, the high overlapping of

CAF and PHE (68%-79%) is negatively aect their final quantification taking

into account their lower levels compared to CAF in real formulations. This ter-

nary system represented a real challenge for multivariate calibration methods

due to the intense spectral overlapping. The same discussion is holding true

for other systems. The most problematic issue in the ternary system CAF–

PAR–PHE is the intense overlapping of PHE with other drugs (79%) while

being present in very little amount compared to the rest of drugs. The same

is true for CAF as it added in modest amounts compared to PAR which added

in much higher dosages. In the same manner, accurate quantification of DPH

(spectral overlapping 62% with PAR) in real formulations may not be a stra-

ightforward analytical job. In addition to spectral overlapping, the presence

of un-calibrated excipients in the extra will add more interference on analysis

and hence reduce the performance of multivariate calibration methods

11,16,17

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Pre-processing of the UV spectral data by CAP: Signal amplification

For better assessment on signal prepossessing before PLS calibration, UV

spectra of all drugs and their mixtures (binary and ternary) were recorded at

the ratios identical to the real formulations along with CAP spectral transfor-

mation. The results are shown in Fig 3.

Pre-processing of the UV spectral data by CAP: Signal amplification

For better assessment on signal prepossessing before PLS calibration, UV spectra of all drugs and their

mixtures (binary and ternary) were recorded at the ratios identical to the real formulations along with

CAP spectral transformation. The results are shown in Fig 3.

Figure 3. UV spectra and CAP transformation of drugs and their mixtures recorded at the same ratios

as in real formulations.

In the three systems, UV spectra indicated a strong absorption signal of PAR due its higher

concentration. For PAR-PSE-CHL, PAR was added in excess than CHL and CHL by 150 and 10

0,0

0,5

1,0

1,5

2,0

2,5

200 250

Absorbance

Wavelength/nm

PAR-PSE-CHL

PAR

PSE

CHL

Mixture

100

120

140

200 250 300 350

CAP

Wavelength/nm

PAR-PSE-CHL

PAR

PSE

CHL

Mixture

0,0

0,5

1,0

1,5

2,0

2,5

3,0

200 250 300 350

Absorbance

Wavelength/nm

PAR-CAF-PHE

CAF

PAR

PHE

Mixture

100

120

140

200 250 300 350

CAP

Wavelength/nm

PAR-CAF-PHE

CAF

PAR

PHE

Mixture

0,0

0,5

1,0

1,5

2,0

2,5

3,0

200 250 300 350

Absorbance

Wavelength/nm

PAR-DPH

PAR

DPH

Mixture

100

120

200 250 300

CAP

Wavelength/nm

PAR-DPH

PAR

DPH

Mixture

Figure 3. UV spectra and CAP transformation of drugs and their mixtures recorded at the

same ratios as in real formulations.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

In the three systems, UV spectra indicated a strong absorption signal of PAR

due its higher concentration. For PAR-PSE-CHL, PAR was added in excess

than CHL and CHL by 150 and 10 times, respectively. While for PAR-CAF-

PHE, PAR was 100 and 20 times higher than PHE and CAF, respectively. For

the binary system, PAR was 20-times higher than the other drug. The high level

of PAR in the systems would explain the similarity between PAR and the spect-

rum of the mixture in all cases. The diiculty in analyzing the mixtures was

increased by the fact that the absorptivity values of CAF, CHL, PSE and DPH in

the respective formulations were much lower than the other PAR over the enti-

re spectral domain, making their accurate determination a hard-analytical task

unless the components are separated before detection. This diiculty drasti-

cally reduced the chance to apply classical spectroscopic methods which often

adopted discrete wavelengths for detection. In these cases, multivariate calib-

ration methods seem more suitable because they use simultaneously a large

number of signals per spectrum. However, considering the diiculty in solving

this type of mixtures, an appropriate pre-processing of the analytical data se-

emed necessary in order to minimize any instrumental interference and at the

same time select the most useful information to emphasize the contribution of

substances of low concentration.

As outlined earlier, CAP treatment transforms the original UV spectrum into a

new curve created by using the area underlying the spectrum. This treatment

is based on two mathematical steps as outlined earlier. Indeed, the conversion

of original UV spectra through the CAP method provided many advantages

among which the amplification of the analytical signals. Fig 3 shows the outputs

of CAP treatment of the spectral of the three systems where drugs recorded at

their actual ratios. The major dierences in comparing the original absorbance

curves with CAP plots are: a) the magnitudes on the y-axis indicated a high

amplification of the signal and this supposed to increase the sensitivity of the

quantitative measurements and help in quantifying the components even at

trace levels (like PSE, CHL, CAF and DPH in the presence of PAR), b) CAP was

able to magnify the weak signals of PSE, CHL, CAF and DPH and over the who-

le spectrum and this option is rather fundamental when applying multivariate

techniques which utilize the full spectrum to extract the maximum analytical

information, and c) Unlike UV signal, the values of CAP signal were stabilize

at constant value and this was reported for all drugs. This stable value, in fact,

is due to the additive contribution of all intensities at all wavelengths. It was

reported that CAP filtering would end up with accurate quantification of drugs

when present at trace levels

. The analytical matrices were built on the spectral

data. In the matrix A (dependent variables) each sample was described by n

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

variables, corresponding to the wavelengths and to the respective absorbance

values; in the matrix C (independent variables) the samples were described

by the concentrations of each component. Two data matrices for each drug-

system were created; the first matrix contained the untreated spectral data

while the other matrix contained the spectral data upon CAP. The calibration

curves relative to the binary (PAR-DPH) and ternary systems (PAR-PSE-CHL

and PAR-CAF-PHE) along with CAP transformation are presented in Figure 4.

data. In the matrix A (dependent variables) each sample was described by n variables, corresponding

to the wavelengths and to the respective absorbance values; in the matrix C (independent variables)

the samples were described by the concentrations of each component. Two data matrices for each

drug-system were created; the first matrix contained the untreated spectral data while the other matrix

contained the spectral data upon CAP. The calibration curves relative to the binary (PAR-DPH) and

ternary systems (PAR-PSE-CHL and PAR-CAF-PHE) along with CAP transformation are presented

in Figure 4.

Figure 4. UV spectra of PAR–DPH, CAF–PAR–PHE and PAR–PSE–CHL calibration samples and

plots after CAP transformation.

0,00

0,50

1,00

1,50

2,00

2,50

200 250 300

Absorbance

Wavelength/nm

PAR-PSE-CHL

200 250 300

CAP

Wavelength/nm

PAR-PSE-CHL

0,00

0,20

0,40

0,60

0,80

1,00

200 250 300

Absorbance

Wavelength/nm

PAR-CAF-PHE

200 250 300

CPA

Wavelength/nm

PAR-CAF-PHE

0,00

0,50

1,00

1,50

2,00

2,50

220 240 260 280 300

Absorbance

Wavelength/nm

PAR-DPH

100

200 250 300

CAP

Wavelength/nm

PAR-DPH

Figure 4. UV spectra of PAR–DPH, CAF–PAR–PHE and PAR–PSE–CHL calibration samples

and plots after CAP transformation.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Determination of pharmaceuticals in binary and ternary

drugs systems by PLS

PLS and CAP-PLS can be created and applied to predict drugs without previ-

ous knowledge on calibrated solutes. However, intense overlapping between

drugs and strong inuence of excipients would aect the prediction power of

PLS

15,16

. The performance of before and after signal filtering drugs prediction

in validation set is provided in Table 3.

Table 3. External prediction of drugs in different systems.

Drug

PLS

CAP-PLS

PRESS REP% Rec% PRESS REP% Rec%

PAR-PSE-CHL

PAR 0.32 3.9 96.3 (4) 0.06 1.7 98.0 (3)

PSE 2.95 12.6 95.2 (5) 0.13 3.1 97.7 (4)

CHL 2.32 11.2 94.0 (5) 0.15 2.5 97.7 (4)

PAR-CAF-PHE

PAR 3.41 14.0 106.8 (5) 0.82 5.8 99.2 (3)

CAF 2.93 10.0 97.4 (4) 0.45 4.3 103.6 (3)

PHE 1.54 8.0 95.8 (4) 0.18 2.1 101.4 (3)

PAR-DPH

PAR 0.45 3.4 96.9 (3) 0.14 2.1 98.5 (2)

DPH 0.64 4.9 95.7 (5) 0.19 3.0 97.0 (4)

Spectral range 200-300 nm (101 point/spectrum). Calibration and prediction steps were

carried out using Eqs1-3. Calibration and validation sets are provided in Table 1. PLS variables

were provided between brackets for each system.

Prior to PLS calibration, spectral data were ltered using cumulative area pre-processing

method (Eqs 11&12) and PLS variables were provided between brackets for each system.

As shown in Table 3, PLS was workable to predict PAR-PSE-CHL and the best

prediction observed for PAR with a REP value 3.9%. In general, PLS was not

so-eective for predicting PSE in the mixtures with a high REP 12.6% which

may not acceptable for pharmaceutical analysis. The modest prediction of PSE

was mainly attributed to the intense spectral-overlapping with other drugs

(Table 2). Accordingly, application of PLS for PSE and CHL may not be wor-

kable taking into account the negative inuence of excipients in real cases. As

indicated in Table 3, the performance of CAP-PLS was very promising with

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

REP values of 1.7%, 2.5%, and 3.1% for PAR, CHL, and PSE, respectively, and

this reected the high closeness between nominal and predicted values. The

earlier results showed that CAP-PLS outperformed PLS for predicting PSE and

CHL in ternary systems. In general, CAP-PLS was excellent for predicting all

drugs with PRESS values less than 1.0 in all cases. The same results were noted

for PAR-CAF-PHE where CAP-PLS outperformed PLS for drugs quantificati-

on with final REP 2.1-5.8%. The best prediction was reported for PHE down

to 1.0 mg/L in mixtures rich with PAR and CAF. Due to the lesser spectral

overlapping, both PLA and CAP-PLS were both of comparable performance

for PAR-DPH quantification, REP was all lower than 5% for both methods.

The superiority of CAP-PLS was mainly attribute to the eective signal filtering

prior to PLS calibration which improved final prediction

Quantification of drugs in real formulations

The real testing of the proposed CAP-PLS method will be assessed by analyzing

binary and ternary systems in the commercial formulations. Direct analysis of

drugs in commercial tablets is not an easy task due to the presence of drugs

in unequal quantities and also the presence of excipients that did not involve

while constructing calibration mixtures. In addition to the above, the drugs

are often placed at concentrations far higher than the calibration solutions.

Therefore, all the ingredients should be properly diluted to be measured with

acceptable sensitivity. In this case, signal filtering/enhancing will be essential

before running classical PLS calibration. It is rather essential to mention that

matrix-cleaning methods may be adopted to eliminate/reduce the inuence

of interferences before running analysis

22-24

. It was necessary to evaluate the

eect of dilutions on drugs quantification. The spectral shapes of the systems

and at variable dilutions are provided in Figure 5. Drugs quantification in real

formulations is provided in Table 4 and the PLS outputs are provided for com-

parison purposes.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Figure 5. UV spectra of marketed formulations after extraction and dilution with distilled water and

CAP plots.

0,00

0,50

1,00

1,50

2,00

2,50

3,00

200 250 300

Absorbance

Wavelength/nm

PAR-PSE-CHL

DF 1:30

DF 1:25

DF 1:15

DF 1:10

100

120

200 220 240 260 280 300

CAP

Wavelength/nm

PAR-PSE-CHL

DF 1:30

DF 1:25

DF 1:15

DF 1:10

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

200 220 240 260 280 300

Absorbance

Wavelength/nm

PAR-CAF-PHE

DF 10

DF 15

DF 20

DF 40

100

150

200

250

200 220 240 260 280 300

CAP

Wavelength/nm

PAR-CAF-PHE

DF 10

DF 15

DF 20

DF 40

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

220 240 260 280 300

Absorbance

Wavelength/nm

PAR-DPH

DF 10

DF 15

DF 25

DF 50

200 220 240 260 280 300

CAP

Wavelength/nm

PAR-DPH

DF 10

DF 15

DF 25

DF 50

Figure 5. UV spectra of marketed formulations after extraction and dilution with distilled

water and CAP plots.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Table 4. Implementation of PLS and CAP-PLS for drugs quantication on different systems.

System

Claimed

(mg/tablet)

PLS CAP-PLS

Predicted

(mg/tablet)

Mean

Recovery

% (n=3)

Precision

(n=3, RSD%)

Predicted

(mg/tablet)

Mean

Recovery

% (n=3)

Precision

(n=3, RSD%)

PAR-CAF-PHE

PAR 500 503.0 100.6 8.4 501.0 102.6 2.1

CAF 25 26.0 104.0 6.5 24.7 103.0 3.1

PHE 5 4.5 90.0 7.3 5.1 94.3 4.2

PAR-PSE-CHL

PAR 300 508.0 99.0 5.0 504.0 98.9 3.1

PSE 30 28.7 102.0 7.0 25.2 104.6 2.7

CHL 2 1.8 99.0 4.6 2.1 99.0 1.5

PAR-DPH

PAR 500 503 101.2 2.1 502 104.2 0.8

DPH 25 28 105.3 3.1 24 106.3 1.0

As indicated in Fig 5, identical UV spectra and CAP signals were observed for

the systems. Higher intensities (for CAP signals) were observed at lower dilu-

tions (DF 10 and 15). Initial analysis indicated that running PLS or CAP-PLS

using signals recorded at DF 10 or 15 ended up with high prediction errors. In

fact, at lower DF the content of drugs is much higher than those used in ca-

libration mixtures making external prediction is not valid. As already known,

the external prediction of solutes is often dependent on the ranges selected

in the calibration mixtures. In the current systems, the best prediction in real

formulations was achieved at DF 15, 20, and 25 for PAR-DPH, PAR-CAF-PHE,

and PAR-PSE-CHL, respectively. Both models were used to assay drugs in real

formulations which based the same composition of the calibration mixtures.

The results, provided in Table 4, indicated the high performance of CAP-PLS

for accurate quantification of drugs in binary and ternary systems. For ter-

nary mixtures, CAP-PLS manifested better accuracy than PLS with recoveries

94.3%-102.6% and precisions (RSD) 2.1-3.1% for PAR-CAF-PHE and 98.9%-

104.6% and 1.5-3.1 for PAR-PSE-CHL. It seems that signal filtering by CAP has

passively reected on the final PLS performance. For the other binary system,

a good agreement was observed between experimental and nominal levels (ob-

tained by both models) of the commercial formulation. Again, PLS calibration

created using CAP-filtered-data generated the best results and may properly

handed the negative inuence of excipients present in the dosage forms.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

UV-signal filtering by CAP prior to PLS was found more eective than PLS

when handling ternary drug mixtures. The proposed CAP-PLS can handle

systems in which one component present at lower levels in the formulation.

CAP simply estimated the cumulative sum of the areas under curve between

two consecutive wavelengths which improved the analytical signal. The pro-

posed CAP-PLS was eective to predict two ternary systems (PAR-CAF-PHE

and PAR-PSE-CHL) of intense overlapping and containing one ingredient ava-

ilable at lower concentration. For binary system, application of CAP-PLS was

as good as classical PLS along with raw UV signals. CAP, as an appropriate

pretreatment method, have been succeeded for accurate and simultaneous

quantification of commercial pharmaceutical formulations in which the ratio

between the components is highly variable.

ACKNOWLEDGEMENTS

The authors thank Deanship of Graduate Studies (The Hashemite University,

Jordan) and for the financial support to this research. The researchers also

thank all the technical stas at chemistry department for their continued help.

List of abbreviations

CAF Caeine

CHL Chlorpheniramine Maleate

CAP Cumulative Area Pre-Processing

DPH Diphenhydramine Hydrochloride

LOD Limit of Detection

LOQ Limit of Quantification

NAS Net Analyte Signal

PLS Partial Least Squares

PRESS Prediction Error Sum of Square

PSE Pseudoephedrine Hydrochloride

REP% Relative Error of Prediction

SEL Selectivity

SEN Sensitivity

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

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Acta Pharm. Sci. Vol 57 No: 2. 2019

DOI: 10.23893/1307-2080.APS.05711

Chemical Composition and Comparative

Antibacterial Properties of Basil Essential

Oil against Clinical and Standard Strains of

Campylobacter spp.

Aysegul Mutlu-Ingok

1,2*

, Burcu Firtin

, Funda Karbancioglu-Guler

1 Istanbul Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Food Engineering,

Istanbul, Turkey.

2 Duzce University, Akcakoca Vocational School, Food Technology Department, Duzce, Turkey.

INTRODUCTION

Campylobacter spp. is considered to be the most common bacterial cause of

human gastroenteritis in the world

. Food-borne Campylobacter infections

are considered to be caused by animal origin foods, mainly poultry and po-

ultry products. Besides poultry, raw milk, pork, beef, lamb and seafood are

responsible of Campylobacter infections

. The antimicrobial resistance of

ABSTRACT

This study has aimed to evaluate comparative antibacterial activity of basil essenti-

al oil against clinical and standard isolates of Campylobacter spp. by dierent met-

hods as agar well diusion, agar and broth dilution methods. Gas Chromatography

(GC) and Gas Chromatography/Mass Spectrometry (GC/MS) analysis were also

examined to determine the chemical composition of the tested essential oil. GC/

MS analysis showed that, basil essential oil was predominated by methyl chavicol

(86.6%) followed by 1,8-cineole (2.8%) and α-bergamotene (2.4%). Although, in-

hibition zone diameters were in the range of 10.9±0.8 to 21.8±1.4 mm, higher MIC

values were obtained against clinical strains compared with standard ones. Due to

the dierences in antimicrobial resistance of the clinical and standard strains, an-

timicrobial activity tests should be carried out with isolates from dierent sources.

Keywords: Basil essential oil, Chemical composition, Agar well diusion, Broth

microdilution, Agar dilution.

*Corresponding Author: Aysegul Mutlu-Ingok, e-mail: aysegulmutlu@duzce.edu.tr

Aysegul Mutlu-Ingok ORCID Number: 0000-0001-9571-0053

Burcu Firtin ORCID Number: 0000-0002-4633-9582

Funda Karbancioglu-Guler ORCID Number: 0000-0001-6576-0084

(Received 15 January 2019, accepted 23 February 2019)

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

thermophilic campylobacters, including Campylobacter jejuni and C. coli has

been identified especially to tetracyclines and uoroquinolones at important

levels in many dierent parts of the world

1,3

. Using chemical compounds have

limits because of their carcinogenic eects, acute toxicity, and environmental

hazard potential

. Increasing resistance to currently used antimicrobials and

consumer concerns about using chemical preservatives lead to investigation

of alternative strategies to prevent and control these microorganisms. Despite

the high number of studies on the antimicrobial eects of essential oils (EOs),

most studies have focused on pathogenic bacteria like Staphylococcus aureus,

Escherichia coli, and Bacillus cereus

Essential oils which were synthesized naturally in dierent plant parts are

complex volatile compounds. They can be extracted from medicinal aromatic

plants and have strong antimicrobial activity against various bacterial, fungal,

and viral pathogens. In addition to their antibacterial properties, they have

antiviral, antimycotic, antitoxigenic, antiparasitic, insecticidal, antimutageni-

city, cytoprotective, moderation of insulin secretion analgesic, neuroprotecti-

ve, antioxidant, antiproliferative proapoptotic anxiolytic-like activities

. Their

wide range of antimicrobial activity was a result of dierent types of aldehydes,

phenolics, terpenes, and other antimicrobial compounds

. Mechanism of an-

timicrobial action is still lacking although a few studies have been elucidated

Basil is the common name for the culinary herb Ocimum basilicum of the fa-

mily Lamiaceae (Labiatae). Although the basil essential oil’s antibacterial acti-

vity is associated with its high content in linalool and estragole, antimicrobial

spectrum is restricted to specific bacteria other than Campylobacter spp.

Alt-

hough in few studies antimicrobial activity of basil essential oil against Camp-

ylobacter spp. has been mentioned

9,10

, our literature review revealed that the

dierences of antimicrobial eects against clinical and standard Campylobac-

ter isolates were not discussed.

In this study, it was aimed to evaluate comparative antibacterial activity of ba-

sil essential oil against clinical and standard isolates of Campylobacter jejuni

and Campylobacter coli by dierent methods as agar well diusion, agar and

broth dilution methods. Gas Chromatography (GC) and Gas Chromatography/

Mass Spectrometry (GC/MS) analyses also examined the chemical compositi-

on of the tested EO.

METHODOLOGY

Bacterial culture and essential oils

The antimicrobial activity of the cold pressed basil essential oil was tested

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

against clinical Campylobacter jejuni, Campylobacter coli identified by Mat-

rix- Assisted Laser Desorption/Ionization time- of- ight Mass Spectrometry

(MALDI TOF MS)

and standard Campylobacter jejuni (ATCC 33660), Camp-

ylobacter coli (NCTC 12525). Basil essential oil was obtained in food grade

form from “International Flavors & Fragrances (IFF)”, Gebze, Kocaeli (Tur-

key). Dilutions were made in 10% dimethyl sulfoxide (DMSO, Merck). Before

analysis, basil essential oil was sterilized by filtration through 0.22 μm filters

(Minisart

Syringe Filter, Sartorius Stedim Biotech GmbH, Germany) and sto-

red in dark at 4 °C.

Gas Chromatography (GC)

Essential oils were analyzed by GC-FID using an Agilent 7890B GC (Agilent,

Palo Alto, CA) with a ame ionization detector (FID). The chromatographic

separation was accomplished using an Agilent HP- Innowax column (60 m x

0.25 mm Ø, with 0.25 μm film thickness) with a helium as a carrier gas (0.7

mL/ minute). GC oven temperature was kept at 60 °C for 10 min and program-

med to 220 °C at a rate of 4 °C/ minute and then kept constant at 220 °C for

10 min and programmed to 240 °C at a rate of 1 °C/ minute. The injector and

ame ionization detector temperatures were adjusted to 250 °C. The relative

percentage amounts of the separated compounds were calculated from FID

chromatograms.

Gas Chromatography-Mass Spectrometry (GC/MS)

The essential oils were analyzed by GC/MS using an Agilent 7890B GC coupled

with a 5977B MSD (Agilent, Palo Alto, CA). The same column and analytical

conditions were used for both GC/MS and GC/FID. The mass range was recor-

ded from m/z 35 to 425. The injector temperature was adjusted to 250 °C. MS

were recorded at 70 eV. Alkanes were used as reference points in the calculati-

on of relative retention indices (RRI). The components of EOs were identified

by using Wiley 9- Nist 11 Mass Spectral Database and standard Alkan series

(C7-C40).

Agar-well diusion assay

Inhibition zone diameters were determined using previously described method

with slight modifications

. Bacterial inoculum was prepared in Mueller-Hin-

ton Broth (MHB, Merck, Darmsdat, Germany) for standard isolate and MHB

with 5% horse blood for clinical isolate and incubated at 42 °C for 48 h un-

der microaerophilic conditions created by Anaerocult® C (Merck, Darmsdat,

Germany). Concentrations of bacterial suspensions were adjusted to approxi-

mately 10

cfu/mL and 100 μL of culture suspension was spreaded on Camp-

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

ylobacter Blood-Free Selective Agar Base medium (modified CCDA, Merck,

Darmsdat, Germany) for standard isolate, Mueller-Hinton Agar (MHA, Merck,

Darmsdat, Germany) medium with 5% horse blood for clinical isolate. Three

wells were cut out of agar and filled with 5 μL, 10 μL and 20 μL of basil EO.

The inoculated plates were incubated at 42 °C for 48 h under microaerophi-

lic conditions. After incubation, inhibition zone diameters were measured. All

experiments were performed in triplicate. Zones of inhibition (including the

6 mm of the well) were expressed as mean values with ± standard deviation.

Broth microdilution assay

Broth microdilution method was used to determine the minimum inhibition

concentrations (MICs), which was described previously by Wiegand et al.

Stock solution was prepared in 10% DMSO and two-fold serial dilutions of EO

were prepared. After sub-culturing in MHB, bacterial concentration was adjus-

ted to approximately 10

cfu/mL. The 96-well plates were prepared by dispen-

sing, into each well, 95 μL of MHB, 100 μL of EO and 5 μL of the inoculants.

The final volume in each well was 200 μL. The microplates were incubated at

42 °C for 24 h under microaerophilic conditions. MIC values were determined

spectrophotometrically by measuring the optical density at an absorbance of

600 nm (Synergy HT, BioTek Instruments Inc., Winooski, VT, USA). Negative

controls (involving 195 μL of MHB and 5 μL of inoculum but no EO) for each

microorganism and sterility controls (involving 100 μL MHB and 100 μL EO

but no inoculum) for each EO concentrations were prepared.

Agar dilution method

For clinical strains, to determine minimum inhibitory concentrations (MICs),

agar dilution method according to Stepanović et al.

was used with slight mo-

difications. This method based on preparation of MHA with 5% horse blood

with the additions of 1% Tween 20 and dierent concentration of essential oils

after sterilization of agar. Test plates were prepared with 19 mL of MHA, and 1

mL of two-fold dilutions of essential oils. After adjusting bacterial concentrati-

on approximately to 10

cfu/mL, 10 μL of culture suspension was inoculated to

agar plates. Plates were incubated for 48 h at 42 °C in microaerophilic condi-

tions. The MICs were defined as the lowest concentration of essential oils that

inhibited visible growth of microorganisms.

RESULTS AND DISCUSSION

The chemical composition of the basil EO determined with GC/MS is given in

Table 1. The main compound identified in the basil essential oil was methyl

chavicol (86.6%). These results are consistent with those reported in the lite-

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

rature. Dierences of constituents and their amounts may be related with the

geographical origin of the plant, dierent parts of plants, extraction method

and season of harvest

Table 1. Chemical compositions of basil essential oil.

No Compound RI

Peak area (%)

1 1,8-Cineole 1220 2.8

α-Bergamotene

1605 2.4

3 Methyl chavicol 1701 86.6

Total 91.8

: Retention index was calculated for all volatile constituents using a homologous series of

n-alkanes C7- C40, b: Peak area obtained by GC-FID.

Although dierent chemical profiles of basil essential oil were reported in lite-

rature, methyl chavicol with high citral contents (methyl chavicol/citral) was

previously detected as a ‘‘new chemo type’’ in the Turkish basils

. In additi-

on to geological origin, chemical constituents varied with dierent seasons

Generally, the chemical composition profile of basil essential oil confirms

previous studies. Methyl chavicol was reported as major constituent in India

(78.3%)

. In another study, three chemotypes of Ocimum basilicum (O. basi-

licum) were identified as a major methyl chavicol-rich type (>65%), a methyl

chavicol (55%)-linalool (20–30%) type, a linalool (42–45%) and eugenol (15%)

type

. For this respect, O. basilicum used in this study was in methyl chavicol-

rich type with 86.6% methyl chavicol. High content of methyl chavicol was also

confirmed by Vieira and Simon

with 47% methyl chavicol content.

The inhibition zone diameters measured ranged from 12.3±1.6 to 21.8±1.4 mm

and 10.9±0.8 to 20.4±2.4 mm for clinical and standard Campylobacter iso-

lates, respectively (Table 2). Considering the all results, mean inhibiton zone

diameter was 15.94±1.55 mm. Similar to current study, mean zone diameters

were reported as 12.48 mm and 13.2 mm against gram positive and gram ne-

gative bacteria, respectively

. Smaller inhibition zones were also reported by

Predoi et al.

as 7-10 mm against Escherichia coli, Staphylococcus aureus

and methicillin-resistant Staphylococcus aureus. In literature, inhibition zone

diameters were varied depending on dierent extracts. It was reported that

although methanol extracts showed inhibition zones against Pseudomonas ae-

ruginosa, Shigella sp., Listeria monocytogenes, Staphylococcus aureus and

two dierent strains of Escherichia coli, chloroform and acetone extracts of O.

basilucum had no eect

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Poor solubility and high volatility of essential oils limit the usage of diusion

tests. It is suggested to use agar or broth dilution methods for true antimicro-

bial activity evaluation

. With this respect, in this study, in vitro antimicrobial

activity of basil essential oil was not tested only by agar diusion method but

also dilution methods against clinical and standard isolates of Campylobac-

ter spp. (Table 2). It was reported that both agar dilution and broth microdi-

lution methods were equally suitable against Campylobacter spp. and highly

correlated

. In this study, since broth microdilution method did not give any

results against clinical strains, agar dilution method was used by taking this

perspective into consideration. Tested essential oil displayed varying degree

of antibacterial activity with MIC values ranging from 105.16 to 1787.7 μg/mL.

Interestingly, MIC values against clinical ones were higher than standard iso-

lates. Higher MIC values indicate that clinical strains are more resistant than

standard strains against basil essential oil.

Table 2. Antimicrobial activity of basil essential oil against Campylobacter spp.

Isolate

Amount (µL) MIC (µg/mL)

5 µL 10 µL 20 µL

Inhibition zone diameter (mm)

C. jejuni (Clinical)

Streptomycin

38.0±1.8

13.0±2.2

40.0±2.4

20.1±1.6

45.0±1.4

1787.7

C.coli (Clinical)

Streptomycin

32.0±1.4

12.3±1.6

38.0±1.4

21.8±1.4

40.0±1.3

889.08

C. jejuni (ATCC 33560)

Streptomycin

21.3±2.1

10.9±0.8

24.7±1.5

20.4±2.4

30.0±1.3

105.16

C. coli (NCTC 12525)

Streptomycin

21.7±1.6

11.8±1.1

24.8±0.7

17.2±1.3

28.8±1.3

219.88

NA: No activity,

: Standard antibiotic,

: Not tested

Although the same MIC values were reported for essential oils against dierent

strains in literatue, in this study dierences in MIC values were found against

the clinical and standard strains of Campylobacter. In literature, dierent MIC

values were reported. Antibacterial and antifungal activities of essential oils of

twelve Ocimum basilicum L. cultivars which were grown in Serbia were inves-

tigated by Beatovic et al.

. However, lower MIC values than current study were

reported, they were ranging from 0.009-11.74 μg/mL. Silveira et al.

, reported

MIC values from 0.075 to 2.5 μg/mL against S. aureus, L. monocytogenes, B.

cereus, Yersinia enterocolitica, E. coli and S. typhimurium. In another study,

mean MIC values were detected as 0.75 and 0.73 μg/mL against 6 gram positive

and 12 gram negative bacteria respectively

. Higher MIC values were also repor-

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

ted for gram positive bacteria as 18-36 μg/mL, and for gram-negative bacteria

as 9-18 μg/mL

. By the existence of dierent EO components with respect to

harvesting season dierences as well as extraction method, dierent antimicro-

bial activity levels can be obtained. These dierences may be due to this fact

Antimicrobial spectrum of basil essential oil was reported as restricted to spe-

cific bacteria as Staphylococcus spp., Enterococcus spp., E. coli, P. aerugino-

sa, Acinetobacter baumannii, Aeromonas hydrophila, B. cereus, Bacillus sub-

tilis, Enterobacter spp., Listeria spp., Proteus spp., Salmonella spp., Serratia

marcescens, and Y. enterocolitica and fungi as Candida spp., Rhodotorula

spp., and Saccharomyces cerevisae

. Although basil essential oil has restricted

antimicrobial activity, in current study it has also been proven that it has anti-

microbial activity against Campylobacter spp.

This study described antibacterial eiciency dierences of basil essential oil

against clinical and standard isolates of Campylobacter spp., as well as the

chemical composition of corresponding essential oil. The results indicated that

tested EO has varying degree of antibacterial eiciency against both C. jejuni

and C. coli isolates. However, with in vitro experiments, in vivo studies are

also required because antimicrobial eect showed dierences even between

clinical and standard strains. In addition, optimum essential oil concentration

should be determined to ensure antimicrobial activity and acceptable sensorial

properties.

ACKNOWLEDGEMENTS

The authors wish to thank Anadolu University, Medicinal Plants, Drugs and

Scientific Research Center for GC and GC-MS analyses of basil essential oil.

This research was supported by Istanbul Technical University, Scientific Rese-

arch Projects (Project no, 38819).

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Acta Pharm. Sci. Vol 57 No: 2. 2019

DOI: 10.23893/1307-2080.APS.05712

In vitro Antimicrobial and Antioxidant Activity

Evaluation of Melampyrum arvense L. var.

elatius Boiss. and Sedum spurium

M. Bieb. Extracts

Ayşe Esra Karadağ

, Fatma Tosun

1 Istanbul Medipol University, School of Pharmacy, Department of Pharmacognosy, İstanbul, Turkey

INTRODUCTION

Melampyrum L. genus is an annual and semi-parasitic plant group. It is

represented by two species, M. arvense and M. pratense in the Flora of

Turkey

. Iridoid glycosides were the major bioactive secondary metabolites of

ABSTRACT

Sedum spurium M. Bieb. (Crassulaceae) is a common ornamental plant, whereas,

Melampyrum arvense L. var. elatius Boiss. (Orobanchaceae) is a semi-parasitic

plant and grows naturally in the fields. In this study, the dichloromethane and

ethyl acetate extracts of M. arvense and S. spurium were evaluated for their in

vitro antioxidant and antimicrobial activities. The antioxidant activity was

evaluated by DPPH

•

-ABTS

•

methods. The antimicrobial activity of S. spurium and

M. arvense extracts was determined using the in vitro broth microdilution assay

against following human pathogenic strains; Staphylococcus aureus ATCC 6538,

Enterococcus faecalis ATCC 29212, Escherichia coli NRLL B-3008, Helicobacter

pylori ATCC 43504, Mycobacterium smegmatis ATCC 25291, Mycobacterium

avium ssp. avium and Pseudomonas aeruginosa ATCC 10145.

The extracts showed weak antimicrobial activity against Gram-negative/positive

bacteria, having the MIC values of 500-1000 μg/mL. Antibacterial activity was not

observed against Mycobacteria at 2000 µg/mL. In addition, antioxidant activity of

M. arvense ethyl acetate extract was higher than those of the other extracts.

Keywords: Melampyrum arvense, Sedum spurium, Antibacterial, Antioxidant,

Mycobacteria

*Corresponding Author: Ayşe Esra Karadağ, e-mail: [email protected]

Ayşe Esra Karadağ ORCID Number: 0000-0002-3412-0807

Fatma Tosun ORCID Number: 0000-0003-2533-5141

(Received 28 January 2019, accepted 26 February 2019)

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Melampyrum species

. The previous in vitro studies showed that Melampyrum

extracts have antioxidant, protein kinase C inhibitory, antimalarial, cytotoxic

and antiprotozoal activities

3-5

and it is used as animal fodder traditionally

Sedum L. is represented by 43 species in Turkey

7-8

. It is reported that several

Sedum species have wound healing properties and were used as diuretic

and laxative and as well as for the treatment of various diseases such as

hemorrhoids in folk medicine

9-11

. The major components of Sedum species

have been described as alkaloids and avonoids in previous studies

12-16

The aim of the present study was evaluation of the antimicrobial and

antioxidant activities of M. arvense and S. spurium extracts. The phenolic

compound composition of the extracts was analyzed by High Performance-

Liquid Chromatography (HPLC).

METHODOLOGY

Plant Material and Extraction

M. arvense and S. spurium were collected in the vicinity of Trabzon-Tonya and

Trabzon-Hamsiköy, respectively. Plants were identified by Prof. M. Vural and

voucher specimens have been deposited at Herbarium of the Depertment of

Pharmacognosy, School of Pharmacy, Istanbul Medipol University, Istanbul,

Turkey. (Voucher specimens no. IMEF: 1055 and IMEF: 1142 resp.) The air-

dried and coarsely ground aerial parts of plant material were macerated with

70% ethanol. The extract was filtered and evaporated to dryness in vacuo

(Heidolph, Germany), and then dissolved in a water-ethanol (90:10) mixture

and extracted with dichloromethane and ethyl acetate, respectively.

Antioxidant Activity

DPPH

•

and ABTS

•

Scavenging Assay

The antioxidant capacity was determined in terms of hydrogen donating or

radical scavenging ability using DPPH

•

by its capability to bleach the stable

radical

. The reaction mix contained 100 μM DPPH

•

in methanol and

dichloromethane or ethyl acetate extracts. After 30 min, absorbance was read

at 517 nm by using a UV–Vis spectrophotometer (UV-1800, Shimadzu, Japan)

at 25 ± 2°C and the radical scavenging activity (RSA) was determined as the

percentage of radical reduction as follows:

DPPH

•

RSA % = [(Absorbance

control

– Absorbance

test sample

) / Absorbance

control

)]

x 100

The total antioxidant activity of the samples was measured using the ABTS

radical cation decolorization assay

. ABTS

•

was produced by reacting 7 mM

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aqueous ABTS

•

with 2.45 mM potassium persulfate. The reaction mixture

was left at room temperature overnight (12−16 h) in the dark. The resulting

intensely colored ABTS radical cation was diluted with ethanol. Absorbance

was measured at 734 nm at room temperature. The assay was performed in

triplicate. Negative controls in which 990 μL ethanol was substituted for ABTS

•

were used. The assay was carried out on Trolox as a positive control

. The

results were expressed as IC

as follows:

ABTS

•

RSA % = [(Absorbance

control

– Absorbance

test sample

)/Absorbance

control

)] x

100

Each experiment was performed in triplicate. The IC

value of the extracts

was calculated from a calibration graph. Test results are presented as mean

± standard deviation (SD). Statistical analysis of antioxidant test results was

completed using one-way ANOVA with the SPSS 23.0 software. A dierence in

the mean values of P<0.05 was considered to be statistically significant.

Antimicrobial Activity

The antimicrobial activity of the extracts was determined using the broth

microdilution assay

to determine the minimum inhibitory concentrations

(MIC). Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC

29212, Escherichia coli NRLL B-3008, and Pseudomonas aeruginosa ATCC

10145 strains were grown in Mueller Hinton Broth (MHB) at 37°C in aerobic

conditions for 24 h. All microorganisms were standardized to McFarland No:

0.5.

Helicobacter pylori ATCC 43504 strain was grown for 24 hours in Brucella

broth containing 5% (v/v) horse blood and 10% (h/h) fetal bovine serum at

37°C in an anaerobic incubator (5% CO

). After incubation at 37°C, 100 μL H.

pylori (2x10

CFU/mL) strain was transferred to the microplate evaluation

21,

. Diluted bacterial suspensions were added to each well and then allowed to

incubate at 37 °C for further 24 h.

Mycobacteria strains were inoculated in Middlebrook 7H11 agar and incubated

in aerobic conditions at 37 °C for 4-5 days. The microorganism was transferred

to media and incubated for a further five days. Diluted bacterial suspensions

(10

CFU/mL) were added to each well and then allowed to incubate at 37 °C

for 5 days

23-25

Test samples stock solution was prepared in dimethyl sulfoxide and serial

dilutions were prepared for each sample. The minimum non-reproductive

concentration was reported as minimum inhibitory concentration (MIC). The

MIC was calculated as the mean of three repetitions.

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HPLC Analysis

The HPLC analyses studied on an Agilent (1200 LC) and UV-Vis detector

(G1314A). HPLC was run on an Agilent C18 column (4.6 x 250 mm x 5 μm)

and its temperature was maintained at 40°C. The mobile phases were Solvent

A: Acetonitrile: Water (10:90, v/v) and Solvent B: Acetonitrile: Water (90:10,

v/v). The composition of solvent B was increased from 15% to 100% in 35 min,

and at a ow rate of 0.6 mL/min. The injection volume is 10 μL

. Phenolic

compounds were identified by matching their retention times against those of

the standards analyzed under the same conditions (Figure 1).

Test samples stock solution was prepared in dimethyl sulfoxide and serial dilutions were prepared for

each sample. The minimum non-reproductive concentration was reported as minimum inhibitory

concentration (MIC). The MIC was calculated as the mean of three repetitions.

HPLC Analysis

The HPLC analyses studied on an Agilent (1200 LC) and UV-Vis detector (G1314A). HPLC was run

on an Agilent C18 column (4.6 x 250 mm x 5 µm) and its temperature was maintained at 40°C. The

mobile phases were Solvent A: Acetonitrile: Water (10:90, v/v) and Solvent B: Acetonitrile: Water

(90:10, v/v). The composition of solvent B was increased from 15% to 100% in 35 min, and at a flow

rate of 0.6 mL/min. The injection volume is 10 μL

. Phenolic compounds were identified by matching

their retention times against those of the standards analyzed under the same conditions (Figure 1).

Figure 1. HPLC Chromatogram of References

1, Chlorogenic acid (RT: 5.1); 2, Caffeic acid (RT: 7.3); 3, Luteolin-O-Glycoside (RT: 9.03); 4,

Coumaric acid (RT: 11.5); 5, Ferulic acid (RT: 13.2); 6, Rosmarinic acid (RT: 18.1); 7, Myrcetin (RT:

20.04); 8, Eriodictyol (RT: 20.7); 9, Luteolin (RT: 22.2); 10, Quercetin (RT: 22.3); 11, Apigenin (RT:

24.2); 12, Gallic acid (RT: 24.7)

Figure 1. HPLC Chromatogram of References

1, Chlorogenic acid (RT: 5.1); 2, Caffeic acid (RT: 7.3); 3, Luteolin-O-Glycoside (RT: 9.03); 4,

Coumaric acid (RT: 11.5); 5, Ferulic acid (RT: 13.2); 6, Rosmarinic acid (RT: 18.1); 7, Myrcetin

(RT: 20.04); 8, Eriodictyol (RT: 20.7); 9, Luteolin (RT: 22.2); 10, Quercetin (RT: 22.3); 11,

Apigenin (RT: 24.2); 12, Gallic acid (RT: 24.7)

RESULTS AND DISCUSSION

Antioxidant Activity

Antioxidant activities of the ethyl acetate and dichloromethane extracts of M.

arvense and S. spurium were measured by the ability to scavenge DPPH free

radicals and ABTS radical scavenging method, by comparing with Ascorbic

acid and Trolox, respectively. Antioxidant capacities were expressed by IC

values, indicating the extracts concentrations scavenge 50% of ABTS radical.

It was observed that ethyl acetate extract of M. arvense has higher antioxidant

capacity than those of the other extracts. The results were shown in Table

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1. Although there is no detailed study of antioxidant activity on M. arvense,

the results of previous studies on antioxidant activities other Melampyrum

species were similar to those of the current study results

3, 27

. As shown in a

previous study

, phenolic compounds found in the M. barbatum extract may

be responsible for the antioxidant activity. To the best of our knowledge, this is

the first report on the antioxidant capacity of M. arvense extract.

Table 1. ABTS and DPPH radical scavenging activities of extracts

ABTS and DPPH radical scavenging activities + [IC

± SD (mg/mL)]

1 2 3 4 References

ABTS

•

0,19 ±

0,04

1,43 ± 0,03 1.54 ± 0,04 2.01 ± 0,03 0,015 ± 0,001 (Trolox)

DPPH

•

0,16 ±

0,03

1,13 ± 0,04 1.41 ± 0,03 1,97 ± 0,04

0,002 ± 0,001

(Ascorbic acid)

1: M. arvense ethyl acetate extract; 2: M. arvense dichloromethane extract; 3: S. spurium ethyl

acetate extract; 4: S. spurium dichloromethane extract

Antimicrobial Activity

Antimicrobial activities of M. arvense and S. spurium extract were evaluated

according to their MIC values against various strains. Table 2 shows

antimicrobial activities of M. arvense and S. spurium extracts against bacterial

strains. The results revealed that the extracts have weak antimicrobial activity

against Gram-negative/positive bacteria with the MIC values in the range to 500-

1000 μg/mL. Antibacterial activity was not observed against Mycobacterias at

2000 µg/mL. In a previous study, antimicrobial activity of S. spurium essential

oil was evaluated

but this is the first report on antimicrobial activity evaluation

of S. spurium extracts. Also, the results obtained by Tosun and co-workers in

a previous study of dierent Mycobacteria strains on the S. spurium extract

were similar to the results of the current study

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Table 2. Antimicrobial activity of extracts (MICs in µg/mL).

Bacteria

Sample

E. a. S. a. P. a. E. f. H. p. M. a. M. s.

1 >2000 1000 500 500 >2000 >2000 >2000

2 >2000 1000 500 500 >2000 >2000 >2000

3 >2000 >2000 >2000 1000 >2000 >2000 >2000

4 >1000 >2000 >2000 1000 1000 >2000 >2000

Chloramphenicol 8 8 >32 16 16 - -

Tetracycline 16 0.25 >16 0.025 0.025 - -

Amikacin - - - - - 250 250

1: M. arvense ethyl acetate extract; 2: M. arvense dichloromethane extract; 3: S. spurium ethyl

acetate extract; 4: S. spurium dichloromethane extract

E.a.: Escherichia coli; S.a.: Staphylococcus aureus; P.a.: Pseudomonas aeruginosa;

E.f.: Enterococcus faecalis; H.p.: Helicobacter pylori; M.a.: Mycobacterium avium; M.s.:

Mycobacterium smegmatis

HPLC Analysis

The phytochemical constituents of the extracts were analyzed using HPLC

technique. The phenolic compounds of M. arvense ethyl acetate extract was

characterized as chlorogenic acid, caeic acid, luteolin-7-O-glycoside, coumaric

acid, ferulic acid, and quercetin (Figure 2). The high antioxidant capacity of

M. arvense ethyl acetate extract may be due to the aforementioned phenolic

compounds. It was reported that the phenolic compounds were responsible for

the antioxidant activity in the previous studies on Melampyrum species

addition, eriodictyol, luteolin and quercetin were detected in S. spurium ethyl

acetate extract by HPLC (Figure 3).

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Figure 2. HPLC Chromatogram of M. arvense methanol extract

1, Chlorogenic acid (RT: 5.082); 2, Caffeic acid (RT: 7.5); 3, Luteolin-O-Glycoside (RT:

9.03); 4, Coumaric acid (RT: 11.5); 5, Ferulic acid (RT: 13.206); 6, Quercetin (RT: 22.3)

Figure 3: HPLC Chromatogram of S. spurium metanol extract

1, Eriodictyol (RT: 20.7); 2, Luteolin (RT: 22.2); 3, Quercetin (RT: 22.3)

Figure 2. HPLC Chromatogram of M. arvense methanol extract

1, Chlorogenic acid (RT: 5.082); 2, Caffeic acid (RT: 7.5); 3, Luteolin-O-Glycoside (RT: 9.03); 4,

Coumaric acid (RT: 11.5); 5, Ferulic acid (RT: 13.206); 6, Quercetin (RT: 22.3)

Figure 2. HPLC Chromatogram of M. arvense methanol extract

1, Chlorogenic acid (RT: 5.082); 2, Caffeic acid (RT: 7.5); 3, Luteolin-O-Glycoside (RT:

9.03); 4, Coumaric acid (RT: 11.5); 5, Ferulic acid (RT: 13.206); 6, Quercetin (RT: 22.3)

Figure 3: HPLC Chromatogram of S. spurium metanol extract

1, Eriodictyol (RT: 20.7); 2, Luteolin (RT: 22.2); 3, Quercetin (RT: 22.3)

Figure 3: HPLC Chromatogram of S. spurium metanol extract

1, Eriodictyol (RT: 20.7); 2, Luteolin (RT: 22.2); 3, Quercetin (RT: 22.3)

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Acta Pharm. Sci. Vol 57 No: 2. 2019

DOI: 10.23893/1307-2080.APS.05713

Preparation and In vitro Characterization of

a Fluconazole Loaded Chitosan Particulate

System

Gülsel Yurtdaş Kırımlıoğlu

, Evrim Yenilmez

, Ebru Başaran

, Yasemin Yazan

1 Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 26470 Eskişehir, Türkiye

INTRODUCTION

Incorporation techniques of lipophilic drug active ingredients with poor aque-

ous solubility are used particularly in pharmaceutical technology for drug de-

livery design. Advantages of encapsulation include enhanced stability of labile

drugs, controlled drug release and improved drug bioavailability.

Dermal delivery by topical preparations such as creams, gels and lotions are

limited due to the barrier characteristics of Stratum corneum. This limitati-

on hinders the drug deposition and leads to relatively poor stability of active

ABSTRACT

In the present study uconazole (FZ) was successfully incorporated into cationic

chitosan nanoparticles prepared by spray-drying method aiming dermal delivery.

Particle size and zeta potential measurements, drug content, morphological, ther-

mal and XRD analyses and FZ quantification by HPLC analyses were performed for

characterizing the formulations prepared. Release behavior of FZ from the nano-

particles was determined using a Franz-diusion cell. Thermal and XRD analyses

results indicated that FZ was molecularly dispersed in chitosan nanoparticles. Cati-

onic chitosan nanoparticles released FZ for 180 minutes indicative of the extended

release of the drug. In vitro characterization results demonstrated that chitosan

nanoparticles seem to be promising for enhancement of dermal delivery of FZ and

could decrease potential side eects and reduce the potential of drug resistance.

Keywords: Fluconazole, Chitosan, Nanoparticles, Spray-drying, Franz-diusion

cell.

*Corresponding Author: Gülsel Yurtdaş Kırımlıoğlu, e-mail: [email protected]

Gülsel Yurtdaş Kırımlıoğlu ORCID Number: 0000-0001-8897-0885

EvrimYenilmez ORCID Number: 0000-0002-7979-0089

Ebru Başaran ORCID Number: 0000-0003-2104-2069

Yasemin Yazan ORCID Number: 0000-0002-7492-1668

(Received 08 March 2019, accepted 22 March 2019)

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

agents by direct exposure to UV light.

Fluconazole (FZ) is a triazole antifungal agent (Figure 1) with poor aqueous

solubility administered either orally or intravenously.

It is used in the treat-

ment of oropharyngeal and esophageal candidiasis, urinary tract infections,

pneumonia, peritonitis and serious systemic candidal infections.

4,5

Adverse ef-

fects of FZ were reported to be related to the gastro-intestinal tract including

abdominal pain, diarrhea, atulence, nausea and vomiting. Other side eects

associated with FZ are headache, dizziness, leucopenia, thrombocytopenia,

hyperlipidemias, and raise in liver enzyme values. Serious hepatotoxicity was

also reported while anaphylaxis, angioedema and skin reaction were rarely re-

ported.

Dermal delivery by topical preparations such as creams, gels and lotions are limited due to the barrier

characteristics of Stratum corneum. This limitation hinders the drug deposition and leads to relatively

poor stability of active agents by direct exposure to UV light.

Fluconazole (FZ) is a triazole antifungal agent (Figure 1) with poor aqueous solubility administered

either orally or intravenously.

It is used in the treatment of oropharyngeal and esophageal candidiasis,

urinary tract infections, pneumonia, peritonitis and serious systemic candidal infections.

4,5

Adverse

effects of FZ were reported to be related to the gastro-intestinal tract including abdominal pain, diarrhea,

flatulence, nausea and vomiting. Other side effects associated with FZ are headache, dizziness,

leucopenia, thrombocytopenia, hyperlipidemias, and raise in liver enzyme values. Serious

hepatotoxicity was also reported while anaphylaxis, angioedema and skin reaction were rarely reported.

Figure 1. Chemical structure of fluconazole

Several classes of antifungal agents can be used in the treatment of candidiasis. However, among the

factors influencing the efficacy of therapy is the immunity of the patient. Clinical failure following

therapy may exist in patients with advanced immunodeficiency. In such cases, high doses of drugs or

the use of multiple agents may be required thereby increasing the risk of serious side effects. To avoid

this complication, entrapment of antifungal agents like FZ in a particulate delivery system can be used

with several advantages such as the possibility of targetting the drug to the desired site in a

controlled/sustained pattern. In this manner, therapeutic efficacy may be increased while reducing the

side effects.

6,7

Some antifungal agents have been entrapped in particulate delivery system and promising

results were observed in animal models and clinical therapy. These studies include mainly the polyene

drugs, represented by amphotericin B and nystatin.

7,8

There are many studies regarding azole antifungal

particulate drug delivery systems.

9,10

Figure 1. Chemical structure of uconazole

Several classes of antifungal agents can be used in the treatment of candidiasis.

However, among the factors inuencing the eicacy of therapy is the immu-

nity of the patient. Clinical failure following therapy may exist in patients with

advanced immunodeficiency. In such cases, high doses of drugs or the use of

multiple agents may be required thereby increasing the risk of serious side

eects. To avoid this complication, entrapment of antifungal agents like FZ in

a particulate delivery system can be used with several advantages such as the

possibility of targetting the drug to the desired site in a controlled/sustained

pattern. In this manner, therapeutic eicacy may be increased while reducing

the side eects.

6,7

Some antifungal agents have been entrapped in particulate

delivery system and promising results were observed in animal models and

clinical therapy. These studies include mainly the polyene drugs, represented

by amphotericin B and nystatin.

7,8

There are many studies regarding azole an-

tifungal particulate drug delivery systems.

9,10

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Chitosan gained an increasing interest as a pharmaceutical excipient.

Chito-

san is a hydrophilic, biocompatible and biodegradable polymer with low toxi-

city. It was investigated extensively for pharmaceutical and medical purposes

including incorporation of therapeutic agents, proteins, enzymes, DNA and

cells, formulation for oral vaccines and stabilization of liposomes.

12,13

Among

the pharmaceutical applications of chitosan are the use as a vehicle for directly

compressed tablets, as a disintegrant, a binder, a granulating agent in ground

mixtures, as a drug carrier for sustained release preparations as well as a co-

grinding diluent for the enhancement of dissolution rate and bioavailability of

water insoluble drugs.

Furthermore, chitosan has mucoadhesive properties

due to the molecular attraction formed by electrostatic interactions between

positively charged chitosan and negatively charged mucosal surfaces.

15,16

Spray drying represents a single-step, cheap, continuous and scalable process

dedicated for converting liquids into dry, free-owing powders which enables

the production of particles with controlled size and morphological aspects.

Spray drying also eliminates the addition of crosslinking agent minimizes the

swelling of chitosan based nanoparticles.

Therefore, spray drying technology

was utilized in this study for the formulation of cationic nanoparticles using

advantages of the method.

The objective of this study was to formulate chitosan-based cationic particulate

systems containing FZ with high encapsulation eiciency and prolonged eect

for reducing side eects and improving its stability. Chitosan nanoparticles

were prepared by the spray-drying technique for topical skin fungal infections.

Particle size, particle size distribution and thermal behaviour of the polymeric

lattice were analyzed. Drug loading and release of the incorporated FZ were

analyzed using a validated HPLC method.

METHODOLOGY

Materials

FZ was a kind gift from Bilim İlaç (Türkiye). Chitosan was purchased from

Fluka Chemicals (Germany), acetic acid from Sigma-Aldrich (Germany) and

ethanol from Carlo Erba (Italy). Sodium chloride, methanol and acetonitrile

were the products of Merck (Germany).

Preparation of chitosan nanoparticles

Formulations were prepared using Mini Spray Dryer (B-190, BUCHI, Switzerland).

The spray dryer was connected to the Inert Loop B-295 (Buchi Labortechnik AG,

Switzerland) due to the organic solvent. Carbon dioxide gas was used at a ow rate

of 120 L.min

-1

. The residual oxygen level in the system was controlled below 4%.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

When preparing particulate systems by spray-drying method, it has to be kept in

the mind that production parameters such as size of nozzle, feeding pump rate, inlet

temperature and compressed air ow rate, aect the particle size.

12,18

It was reported

that smaller particles are formed with lower feeding pump rate and smaller nozzle

size. In addition, smaller particles are formed with greater volume of air input where

particle size is not dependent inlet temperature in the range of 120-180°C.

Briey, FZ and chitosan were dissolved in 96% (v/v) ethanol and 2% (v/v) ace-

tic acid solutions, respectively. The solutions were mixed and homogenized at

1500 rpm for 3 hours. The final clear solution was then spray-dried with an in-

let temperature of 145ºC ± 1°C and an outlet temperature of 50ºC ± 3°C. White

dry powders were obtained and kept in tightly closed vials at room tempera-

ture until being analyzed. The placebo formulation was prepared as described

above without the addition of FZ. These were bare, empty nanoparticles.

Composition of the formulations was kept as simple as possible (Table 1).

Table 1. Compositions of the formulations prepared

CODE FZ Chitosan Acetic acid Ethanol

Placebo - 2 g 240 mL (%2 v/v) 240 mL

C-FZ-1 0.2 g 2 g 240 mL (%2 v/v) 240 mL

C-FZ-2 0.5 g 2 g 240 mL (%2 v/v) 240 mL

Characterization of chitosan nanoparticles

Particle size and zeta potential analyses

Particle size, particle size distribution (PDI) and zeta potential measurements

of the formulations prepared were performed on freshly prepared samples

using Malvern Nano ZS (Zetasizer Nano Series, Worcestershire, UK). Samp-

les of all formulations were dispersed in double distilled water (adjusted to a

constant conductivity of 50 μS.cm

-1

using 0.9% NaCl) just prior to analyses. All

analyses were repeated in triplicate at 25°C ± 2°C.

Morphology

The particle shape and surface characteristics of the freshly prepared nanopar-

ticle formulations and FZ were investigated by scanning electron microscope

(SEM) (HITACHI TM3030Plus Tabletop Microscope, Japan) at 25°C ± 2°C.

Samples were coated with a thin layer gold under argon to avoid charging un-

der the electron beam.

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Thermal analysis

Thermal behaviors and the interactions between FZ and chitosan were analy-

zed using dierential scanning calorimetry (DSC) (DSC-60, Shimadzu Scientic

Instruments, Columbia, MI, USA). In DSC analyses, the heating rate of 10ºC.

min

-1

was employed in the temperature range of 50ºC-200ºC. Analyses were

carried out under nitrogen with a scan rate of 5 K.min

-1

X-ray diractometry analysis

Dry powder X-ray diractometry (XRD) analyses were performed using RIKA-

GU D/Max-3C (Japan). The XRD analysis range was 2°C-40°C over 2θ with

2°C min

-1

scanning rate, with 40 kV voltage and current intensity level of 30

mA.

Pure FZ and placebo formulation were also analyzed and those XRD spectra

were used as references in evaluating the chitosan nanoparticles containing

FZ.

Determination of FZ content of chitosan nanoparticles

For the quantification of FZ incorporated into FZ formulations, accurately we-

ighed (5 mg) formulations were dissolved in acetic acid (2%, v/v) solution and

ethanol (5 mL, 4:1) mixture and agitated at 4000 rpm for 3 min.1 mL of su-

pernatant was collected. Drug content of nanoparticles was determined using

the reversed-phase HPLC method equipped with a pump (LC 10-AD), a UV de-

tector (SPD-20A), a data station (Shimadzu, Japan) and C

column (250 mm

x 4.6 mm i.d. and 5 μm particle size). The mobile phase consisting of distilled

water (tetrabutylammonium hydrogen sulfate): acetonitrile (75:25, v/v) was

degassed prior to the analysis. The ow rate was 1 mL.min

-1

with an injection

volume of 25 μL. The oven temperature was adjusted to 30ºC ± 1ºC and FZ was

monitored at 223 nm.

In vitro release studies of FZ from chitosan nanoparticles

In vitro release studies were performed using Franz diusion cells.

The diu-

sion cells were thermoregulated with a water jacket at 32º C. Polypropylene

membrane was placed on a Franz diusion cell after keeping it 20 minutes in

the donor compartment. The receptor chamber was filled with distilled water.

1000 μg of formulation was applied to the donor compartment. 0.5 mL aliqu-

ots were withdrawn from the receptor compartment at specific time intervals.

The amounts withdrawn were replaced by the fresh distilled water. The amo-

unt of FZ in aliquots was analyzed by a validated HPLC method and release

profiles were obtained.

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RESULTS AND DISCUSSION

Preparation of chitosan nanoparticles

In this study, spray-drying method was successfully used to prepare chitosan

nanoparticles since it does not involve toilsome procedures and avoids the use

of harsh cross-linking agents and organic solvents which might possibly trigger

chemical reactions with the active agent.

Characterization of chitosan nanoparticles

Particle size and zeta potential measurements

Particle size is one of the important physical properties of colloidal systems.

Particle size distribution of the formulation is especially significant in the

physical stability and activity of colloidal systems.

It was also found that the

size of nanoparticles play an important role in their adhesion to and interac-

tion with the biological cells.

Particle sizes of formulations were found to be

552.50 ± 10.30 nm and 648.25 ± 8.81 nm for C-FZ-1 and C-FZ-2, respectively

(Table 2). It was found that decrease in the amount of FZ in formulations was

in parallel with the relative decrease in average particle size.

Table 2. Mean particle size, PDI and zeta potential values of formulations prepared

(SE: Standard error) (n=3)

Code Particle size (nm) ± SE PDI ± SE Zeta Potential (mV) ± SE

Placebo 410.00 ± 11.41 0.480 ± 0.018 7.0 ± 4.1

C-FZ-1 552.50 ± 10.30 0.450 ± 0.083 38.4 ± 0.2

C-FZ-2 648.25 ± 8.81 0.313 ± 0.051 40.4 ± 2.3

The acceptable value for PDI is 0.05-0.7; values greater than 0.7 indicate very

broad size distribution and probably no suitability for dynamic light scattering

technique.

As shown in Table 2, acceptable PDI values were obtained for all

batches. Also, PDI data showed that the homogeneity increased with the addi-

tion of FZ into the formulations.

Zeta potential of nanoparticles is commonly used to characterize the surface

property of nanoparticles. Results showed that zeta potentials measured were

38.4 ± 0.2 mV and 40.4 ± 2.3 mV for C-FZ-1 and C-FZ-2, respectively, which

may be attributed to the positive charges on polymeric matrices indicating

adequate physical stability. Cationic chitosan nanoparticles with mucoadhesi-

ve properties may interact with the negatively charged of skin and open up the

tight junctions of epithelial cells to allow the paracellular transport pathway

resulting in an increase in bioavailability of the active agents.

16,21

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Morphology

SEM images of pure FZ and formulations were demonstrated in Figure 2.

SEM images showed that all formulations prepared were nearly in spherical

shape while some of the spheres reminded the collapsed balloons with smooth

surfaces. Crystalline structure of FZ was not observed in the formulations indi-

cating successful incorporation of FZ into the polymeric matrices.

Figure 2. SEM images of pure fluconazole and chitosan nanoparticles prepared (a: FZ, b: placebo

formulation, c: C-FZ-1, d: C-FZ-2).

Thermal analysis

DSC was in order to determine the incorporation of FZ into the polymeric network and also the status

of polymer and active agent after spray-drying process.

The thermogram of FZ shows the simple thermal behaviour of the drug (Figure 3a). The sharp

endothermal peak the observed at 142.66ºC is the first order solid-liquid phase transition corresponding

to the melting of the drug.

Placebo formulation (Figure 3b) was characterized by its amorphous state

since no endothermic peak was observed. In the DSC thermogram of formulations, sharp peak belonging

to FZ was not observed (Figure 3c). There may be two explanations of this peak disappearance. First

explanation is the molecular incorporation of FZ into chitosan nanoparticles. When FZ was molecularly

dispersed within the polymeric matrix resulting in a solid solution, the endothermal peak of FZ has

Figure 2. SEM images of pure uconazole and chitosan nanoparticles prepared

(a: FZ, b: placebo formulation, c: C-FZ-1, d: C-FZ-2).

Thermal analysis

DSC was in order to determine the incorporation of FZ into the polymeric net-

work and also the status of polymer and active agent after spray-drying pro-

cess.

The thermogram of FZ shows the simple thermal behaviour of the drug (Fi-

gure 3a). The sharp endothermal peak the observed at 142.66ºC is the first

order solid-liquid phase transition corresponding to the melting of the drug.

Placebo formulation (Figure 3b) was characterized by its amorphous state

since no endothermic peak was observed. In the DSC thermogram of formula-

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

tions, sharp peak belonging to FZ was not observed (Figure 3c). There may

be two explanations of this peak disappearance. First explanation is the mole-

cular incorporation of FZ into chitosan nanoparticles. When FZ was molecu-

larly dispersed within the polymeric matrix resulting in a solid solution, the en-

dothermal peak of FZ has disappeared.

11,12,25

Second explanation is the dilution

eect of the polymer network. When the ratio of drug:polymer is so small, the

massive amount of polymer shades the endothermal peak of the drug.

disappeared.

11,12,25

Second explanation is the dilution effect of the polymer network. When the ratio of

drug:polymer is so small, the massive amount of polymer shades the endothermal peak of the drug.

Figure 3. DSC thermograms of pure fluconazole and chitosan nanoparticles prepared (a: FZ, b: placebo

formulation, c: C-FZ-1, d: C-FZ-2).

X-ray diffractometry analysis

DSC and XRD play a prominent role in the characterization of polymeric matrices because they are able

to provide structural information on the dispersed particles.

Therefore, in this study for better

evaluation of the crystalline polymeric structure of polymeric particles DSC and XRD analyses were

performed simultaneously.

Dry powder XRD analyses of nanoparticles confirmed the DSC results showing the amorphous state of

the polymeric network. XRD patterns of pure FZ, placebo and formulations were demonstrated in

Figure 4. The FZ spectrum shows several sharp diffraction peaks typical of its crystalline state (Figure

4a) while placebo and FZ-containing formulations were of amorphous state with no sharp XRD peaks

Figure 3. DSC thermograms of pure uconazole and chitosan nanoparticles prepared

(a: FZ, b: placebo formulation, c: C-FZ-1, d: C-FZ-2).

X-ray diractometry analysis

DSC and XRD play a prominent role in the characterization of polymeric mat-

rices because they are able to provide structural information on the dispersed

particles.

Therefore, in this study for better evaluation of the crystalline poly-

meric structure of polymeric particles DSC and XRD analyses were performed

simultaneously.

Dry powder XRD analyses of nanoparticles confirmed the DSC results sho-

wing the amorphous state of the polymeric network. XRD patterns of pure FZ,

placebo and formulations were demonstrated in Figure 4. The FZ spectrum

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

shows several sharp diraction peaks typical of its crystalline state (Figure

4a) while placebo and FZ-containing formulations were of amorphous state

with no sharp XRD peaks in the spectra (Figure 4b and Figure 4c). In the

patterns of the FZ loaded formulations, peaks corresponding to FZ disappea-

red, indicating that dispersion in an amorphous form.

13,26

in the spectra (Figure 4b and Figure 4c). In the patterns of the FZ loaded formulations, peaks

corresponding to FZ disappeared, indicating that dispersion in an amorphous form.

13,26

Figure 4. X-ray diffraction of patterns of pure fluconazole and chitosan nanoparticles prepared (a: FZ,

b: placebo formulation, c: C-FZ-1, d: C-FZ-2).

Determination of FZ content of formulations

A validated HPLC method used for the determination of FZ demonstrated that the incorporation

efficiency of FZ in formulations were found to be 8.99 ± 0.54 % and 12.04 ± 2.05 % (mean ± SE), for

C-FZ-1 and C-FZ-2, respectively. It is evident from the data that incorporation efficiency was affected

by the drug: polymer ratio. The results revealed that the incorporation efficiency of formulations was

increased with increasing concentration of the active agent in the formulations.

Relatively low

incorporation efficiency of nanoparticles prepared may be attributed to the spray-drying parameters such

as nozzle diameter, spraying rate or viscosity of the spraying solution.

In vitro drug release

In vitro dissolution test plays an important role in drug formulation development and quality control. It

can be used not only primary tool to observe the consistency and stability of drug products but also as a

relatively rapid and inexpensive process to estimate in vivo absorption of a drug formulation.

Figure 4. X-ray diffraction of patterns of pure uconazole and chitosan nanoparticles

prepared (a: FZ, b: placebo formulation, c: C-FZ-1, d: C-FZ-2).

Determination of FZ content of formulations

A validated HPLC method used for the determination of FZ demonstrated that

the incorporation eiciency of FZ in formulations were found to be 8.99 ± 0.54

% and 12.04 ± 2.05 % (mean ± SE), for C-FZ-1 and C-FZ-2, respectively. It is

evident from the data that incorporation eiciency was aected by the drug:

polymer ratio. The results revealed that the incorporation eiciency of formu-

lations was increased with increasing concentration of the active agent in the

formulations.

Relatively low incorporation eiciency of nanoparticles prepa-

red may be attributed to the spray-drying parameters such as nozzle diameter,

spraying rate or viscosity of the spraying solution.

In vitro drug release

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

In vitro dissolution test plays an important role in drug formulation develop-

ment and quality control. It can be used not only primary tool to observe the

consistency and stability of drug products but also as a relatively rapid and

inexpensive process to estimate in vivo absorption of a drug formulation.

The in vitro release profile of FZ from formulations were shown in Fi-

gure 5. Franz diusion cell analyses results showed that the release of

FZ from formulations were 26.12 % and 29.52 % at the end of 180 mi-

nutes for C-FZ-1 and C-FZ-2, respectively. Flux (J) and permeation co-

eicient (k

) values were determined using the slope of the steady-sta-

te portion of the amount of the drug permeated and divided by time. The

steady-state ux (J

) of FZ from the formulations were calculated to be

9.7x10

-2

±0.001 μg.cm

-2

-1

and 1.2x10

-1

±0.0001 μg.cm

-2

-1

while permeability

coeicient (k

) was determined to be 3.10x10

-3

±0.001 cm.h

-1

and 4.0565x 10

-4

for the C-FZ-1 and C-FZ-2, respectively. The results indicated that the release

of FZ from the formulations varied depending on time.

The in vitro release profile of FZ from formulations were shown in Figure 5. Franz diffusion cell

analyses results showed that the release of FZ from formulations were 26.12 % and 29.52 % at the end

of 180 minutes for C-FZ-1 and C-FZ-2, respectively. Flux (J) and permeation coefficient (k

) values

were determined using the slope of the steady-state portion of the amount of the drug permeated and

divided by time. The steady-state flux (J

) of FZ from the formulations were calculated to be

9.7x10

-2

±0.001 μg.cm

-2

-1

and 1.2x10

-1

±0.0001 μg.cm

-2

-1

while permeability coefficient (k

) was

determined to be 3.10x10

-3

±0.001 cm.h

-1

and 4.0565x 10

-4

for the C-FZ-1 and C-FZ-2, respectively. The

results indicated that the release of FZ from the formulations varied depending on time.

Figure 5. In vitro release profile of FZ from FZ loaded chitosan nanoparticles.

FZ was successfully incorporated into chitosan nanoparticles using a spray-drying method. In vitro

characteristics of nanoparticles prepared were analyzed to confirm incorporation of FZ into the

polymeric structure. When submicron sized with homogenous size distribution and nearly spherical

nanoparticles were prepared. DSC and XRD assays confirmed decrease in FZ crystallinity in cationic

nanoparticles. Chitosan nanoparticles released FZ for 180 minutes indicative of the extended release of

the drug, which will reduce the side effects in treating the infections induced by Candida albicans. With

the cationic and mocuadhesive properties of chitosan-based system, nanoparticles will ensure longer

residence at the infection site, providing a favorable release profile for the FZ for the dermal delivery.

0 50 100 150 200 250 300 350

Q (%)

Time (minutes)

C-FZ-1 C-FZ-2

Figure 5. In vitro release prole of FZ from FZ loaded chitosan nanoparticles.

FZ was successfully incorporated into chitosan nanoparticles using a spray-

drying method. In vitro characteristics of nanoparticles prepared were analy-

zed to confirm incorporation of FZ into the polymeric structure. When submic-

ron sized with homogenous size distribution and nearly spherical nanoparticles

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

were prepared. DSC and XRD assays confirmed decrease in FZ crystallinity in

cationic nanoparticles. Chitosan nanoparticles released FZ for 180 minutes in-

dicative of the extended release of the drug, which will reduce the side eects

in treating the infections induced by Candida albicans. With the cationic and

mocuadhesive properties of chitosan-based system, nanoparticles will ensure

longer residence at the infection site, providing a favorable release profile for

the FZ for the dermal delivery.

Conclusively, chitosan nanoparticles may be good alternative for delivery of

FZ, which need to be, investigated further using in vivo tests before final de-

cision.

CONFLICT OF INTEREST

The authors declare no conict of interest, financial or otherwise.

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REVIEW ARTICLES

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Acta Pharm. Sci. Vol 57 No: 2. 2019

DOI: 10.23893/1307-2080.APS.05714

Pharmaceutical Properties of Marine

Polyphenols: An Overview

Vo Thanh Sang

, Ngo Dai Hung

, Kim Se-Kwon

1 NTT Institute of Hi-Technology, Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam.

2 Faculty of Natural Sciences, Thu Dau Mot University, Thu Dau Mot City, Binh Duong Province, Vietnam.

3 Department of Marine Life Science, College of Ocean Science and Technology, Korea Maritime and Ocean University,

Busan 606-791, Korea.

INTRODUCTION

The marine environment represents approximately half of the global biodi-

versity. It is a rich source of structurally diverse and biologically active meta-

bolites, which are important for the discovery of potential therapeutic agents

. During the last decades, marine organisms have received much attention in

screening marine natural products for their biomedical and pharmaceutical

potentials

3-5

. Various marine organisms such as algae, tunicates, sponges, soft

ABSTRACT

Natural products are non-drug materials that have applied for prevention or treat-

ment of health problem. The use of natural products as pharmaceutical ingredients

has got much attention by consumers nowadays. Among them, marine organisms

are currently considered as a huge source for the discovery of pharmaceutical

agents. During the last decades, numerous novel agents have been achieved from

marine organisms and many of them have potential application in pharmaceuti-

cal industry. Notably, marine algae are known to be one of the most important

producers of variety of chemically active metabolites. Especially, phlorotannins, a

polyphenol from brown algae, have been revealed to possess numerous biological

activities such as UV-protective, anti-oxidant, anti-viral, anti-allergic, anti-cancer,

anti-inammatory, anti-diabetes, and anti-obesity activities. Therefore, phloro-

tannins are promising agents for development of pharmaceutical products. This

contribution focuses on phlorotannins from brown algae and presents potential

application in pharmaceutical field due to its biological activities and health benefit

eects.

Keywords: Natural product, Pharmaceutical, Phlorotannins, Algae, Bioactivity.

*Corresponding Author: Vo Thanh Sang, email: [email protected]; Tel.: +84 28 6271 7296

Vo Thanh Sang ORCID Number: 0000-0002-6726-3257

Ngo Dai Hung ORCID Number: 0000-0002-4025-1694

Kim Se-Kwon ORCID Number: 0000-0001-6507-9539

(Received 03 December 2018, accepted 15 January 2019)

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corals, bryozoans, sea slugs, mollusks, echinoderms, fishes, microorganisms,

etc. have been subjected for isolation of numerous novel compounds. Conse-

quently, numerous active agents such as lipid, protein, peptide, amino acid,

neurotoxins, polysaccharides, chlorophyll, carotenoids, vitamins, minerals,

and unique pigments have been discovered. Many of these substances have

been demonstrated to possess interesting biological activities

6-14

. Notably, ma-

rine algae are known to be one of the most important producers of biomass in

the marine environment. Algae are very simple chlorophyll-containing orga-

nisms composed of one cell or grouped together in colonies or as organisms

with many cells

. Therefore, they vary greatly in size from unicellular of 3–10

μm to giant kelps up to 70-meter-long. Algae are identified as the microalgae

which are found in both benthic and littoral habitats and also throughout the

ocean waters as phytoplankton and the macroalgae (seaweeds) which occupy

the littoral zone. Phytoplankton comprises diatoms, dinoagellates, green and

yellow–brown agellates, and blue–green algae while seaweeds are classified

into green algae, brown algae, and red algae. Marine algae are known to be a

good source of healthy food due to their low content in lipids, high concent-

ration in polysaccharides, natural richness in minerals, polyunsaturated fatty

acids and vitamins. Especially, seaweeds are able to produce a great variety of

secondary metabolites characterized by a broad spectrum of biological activiti-

es such as anti-coagulation, anti-virus, anti-oxidant, anti-allergy, anti-cancer,

anti-inammation, anti-obesity, anti-diabetes, anti-hypertension, neroprotec-

tion, and immunomodulation

16-19

. Therefore, marine algae are believed to be a

promising source to provide not only novel biologically active substances for

the development of pharmaceuticals but also essential compounds for human

nutrition

The Phaeophyceae (brown algae) is a large group of marine multicellular algae,

including various seaweeds. They play an important role in marine environ-

ments, both as food and for the habitats they form. Although the division Phae-

ophyta consists of 13 orders according to the classification of Bold and Wynne

only three orders namely Laminariales, Fucales, and Dictyotales have been

extensively researched for their phytochemicals. The most studied species of

these orders are Laminaria, Ecklonia, Undaria, Himanthalia, Sargassum,

and Dictyota. Brown seaweeds are rich in polysaccharide, polyphloroglucinol

phenolic compounds, and other secondary metabolites such as terpenes, ca-

rotenoids, and oxylipins

. Notably, marine brown algae accumulate a variety

of phloroglucinol-based polyphenols, as phlorotannins. These pholorotannins

consist of phloroglucinol units linked to each other in various ways, and are of

wide occurrence among marine brown algae

21, 22

. Among marine brown algae,

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Ecklonia cava, Ecklonia stolonifera, Ecklonia kurome, Eisenia bicyclis, Ishige

okamurae, Sargassum thunbergii, Hizikia fusiformis, Undaria pinnatifida,

and Laminaria japonica have been reported for phlorotannins with health be-

neficial biological activities

PHLOROTANNINS

Sources and distribution

Phlorotannins have only been found to exist within brown algae and may

constitute up to 15% of the dry weight of brown algae

. The concentration of

phlorotannins is highly variable among dierent brown seaweeds as well as

among dierent geographical areas. The fucoid species from the Atlantic and

the temperate Paciﬁc contain higher concentration of phlorotannins as com-

pared to those obtained from the tropical Paciﬁc

. It was found that phloro-

tannnins have mostly focused on Fucaceae (Ascophyllum nodosum and Fu-

cus vesiculosus), Sargassaceae (Sargassum spinuligerum and Carpophyllum

angustifolium), and Cystoseiraceae (Cystophora retroexa and C. torulosa)

with concentrations ranging from 20 to 250 mg/g dry matter

25-29

. They tend

to be concentrated within the outer cortical layers, physode, and the mitotic

meristematic and meiotic sporogenous tissues

. In addition, Laminariaceous

brown algae, such as Eisenia bicyclis, Ecklonia cava, Ecklonia kurome were

also found to contain a significant amount of phlorotannins

31, 32

Structural diversity and classification

Phlorotannins are formed by the polymerization of phloroglucinol (1,3,5-trihy-

droxybenzene) monomer units. They are highly hydrophilic components with

a wide range of molecular sizes ranging between 126 Da and 650 kDa. The

monomeric units are linked through aryl-aryl bonds and diaryl ether bonds

forming dierent subgroups of phlorotannins

. Phlorotannins can be grou-

ped according to the criteria of interphloroglucinol linkages into three primary

types including fucols, phlorethols, and fucophlorethols

. Fucols is formed by

only phenyl linkages, while phlorethols is formed by only arylether bonds and

fucophlorethols is formed by both arylether and phenyl linkages

. The struc-

tural diversity of phlorotannins increases by adding the number of phloroglu-

cinol units. Each of the primary groups can be grouped into linear or branched

phlorotannins. In fucols, the interphloroglucinol links at meta-relative positi-

on construct of the linear phlorotannin such as tetrafucol-A and the branched

phlorotannin such as tetrafucol-B, which were isolated from Fucus vesiculo-

sus

36, 37

. Moreover, longer oligomers of phlorotannin such as pentafucols and

heptafucols were purified from Scytothamnus australis

and Analipus japoni-

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cas

. The linear phlorethols such as triphlorethol C and tetraphlorethols A and

B were isolated from Laminaria ochroleuca

. The branched phlorethols inclu-

de tetraphlorethol C from Ecklonia maxima

, pentaphlorethol B and hexaph-

lorethol A from Cystophora retroexa

. Furthermore, an additional hydroxyl

group on the terminal monomer unit forms other structural motifs of phloret-

hols such as bifuhalol, trifuhalol A, and trifuhalol B

41, 42

. The isofuhalols such as

isotrifuhalol has an extension unit that bind between meta-oriented phlorog-

lucinol units and it bears the additional hydroxyl group

. Some fuhalols with

more than one additional hydroxyl group have been called hydroxyfuhalols,

such as hydroxytrifuhalol B

. In addition, another subgroup of phlorethols,

the eckols, includes a 1,4-dibenzodioxin system, such as the trimers eckol and

dioxinodehydroeckol

, the tetramers 2-phloroeckol and 7-phloroeckol

45-47

. In

fucophlorethols, the combinations of C-C and C-O-C linkages allow the for-

mation of various compounds in linear, branched and heterocyclic fashions.

The linear fucophlorethols is fucodiphlorethol-B

, meanwhile the branched

fucophlorethols is bisfucotriphlorethol A

, and heterocyclic fucophlorethols is

phlorofucofuroeckol A

Biosynthesis of phlorotannins

Phlorotannins are biosynthesized via the acetate-malonate pathway, also

known as the polyketide pathway, in a process which may involve a polyketi-

de synthase-type enzyme complex

. However, the exact biosynthetic pathway

for phlorotannins is unknown up to now. Therefore, methodologies that mo-

nitor phlorotannin synthesis at the genetic or enzymatic levels could be use-

ful to reveal some of the uncertainties regarding phlorotannin biosynthesis

Firstly, two molecules of acetyl co-enzyme A are converted into malonyl co-

enzyme A through the addition of carbon dioxide. This addition changes the

acetyl methyl group into a highly reactive methylene. Secondly, the process

of polymerization is assisted to occur with the low required energy. During

further synthesis steps, the carbon dioxide, which was added as an activator, is

lost. Thirdly, a polyketide chain consisting of an acid moiety is formed, and the

co-enzyme is lost. The polyketide chain is transformed by intermolecular ring

closure and elimination of water to produce hexacyclic ring systems. Triketide,

the cyclization product, is not stable and thus undergoes transformation into

the thermodynamically more stable aromatic form, phloroglucinol, consisting

of three phenolic hydroxyl groups

. The polymerization of phloroglucinol in

dierent ways results in formation of various phlorotannins.

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Physiological properties

Phlorotannins are found in physodes, which contribute to the development of

the cell wall of brown algae

. It has suggested that phlorotannins are likely to

be integral structural components of brown-algal cell walls

. They are bound

to the cell wall during maturation of the plant

. Phenolic compounds are bo-

und with four major types of bonds including hydrophobic, hydrogen, ionic,

and covalent bond to increase the strength

. The cell wall (alginic acid) and

phlorotannins are linked via covalent bonds including the ester bond and the

hemiacetal bond, thus requiring strong conditions to degrade. Moreover, phlo-

rotannins have a putative role in brown algal reproduction due to exposing on

the surface of the recently fertilized zygotes where they may prevent multiple

fertilizations by inhibiting spermatozoid movement

A characteristic of phlorotannins is their plasticity to a variety of environmental

factors including nutrient environment

, light

, depth

, salinity

, grazing

other mechanical wounding

. Such plasticity may represent inducible defen-

se against herbivory

. Suggestions for other adaptive roles for phlorotannins

include protection against ultraviolet radiation

or function as anti-fouling

substances

. The suggested defensive role of phlorotannins is due to deterring

feeding by herbivores

and decreasing their assimilation efﬁciency by binding

with proteins in the gut

66, 67

PHARMACEUTICAL PROPERTIES OF PHLOROTANNINS

Antioxidant and UV-protective activities

The oxidants such as superoxide anion radicals, hydroxyl radical species, and

hydrogen peroxide are often generated by biological oxidation reactions of

exogenous factors

. It is well-known that oxidants are involved in signal trans-

duction and gene activation and can contribute to host cell and organ dama-

. Therefore, scavenging of oxidant is considered to be important to control

various diseases. Interestingly, phlorotannins from marine brown algae have

been evidenced to be eective to scavenge oxidants in non-cellular and cel-

lular systems. According to Ahn and colleagues, the antioxidant activities of

three phlorotannins including phloroglucinol, eckol and dieckol purified from

Ecklonia cava collected in Jeju Island have been investigated

. It reported

that all the phlorotannins have the potential DPPH, alkyl, hydroxyl and supe-

roxide radical scavenging activities. Eckol exhibit the most strong antioxidant

activity via scavenging 93% of DPPH. Moreover, these phlorotannins were ef-

fective to protect DNA against H

-induced damage. In the same trend, Kang

and colleagues have also investigated the cytoprotective eect of eckol from

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E. cava against oxidative stress induced cell damage in Chinese hamster lung

ﬁbroblast (V79-4) cells

. Eckol was eective to reduce H

-induced cell death

in V79-4 cells, inhibit radiation-induced cell damage, and scavenge intracellu-

lar ROS production. Moreover, eckol was able to increase the activity of cata-

lase and its protein expression via increasing phosphorylation of extracellular

signal-regulated kinase and activity of nuclear factor κB. In another study of

Kang and colleagues, triphlorethol-A from E. cava was found to reduce intra-

cellular hydrogen peroxide generated by gamma-ray radiation, thus protec-

ting against radiation-induced membrane lipid peroxidation, cellular DNA

damage, and cell death

. Furthermore, triphlorethol-A augments cellular

antioxidant defense capacity through induction of HO-1 expression via ERK-

Nrf2-ARE signaling pathway, thereby protecting cells from oxidative stres

Notably, Li and colleagues have isolated several phlorotannins from E. cava

such as phloroglucinol, eckol, fucodiphloroethol G, phlorofucofuroeckol A, di-

eckol, and 6,6’-bieckol. All phlorotannins were found to possess antioxidant

properties via scavenging free radicals, protecting membrane protein from oxi-

dant-induced damage, enhancing cellular glutathione level in RAW264.7 cell

line

. Likewise, several phlorotannins including phloroglucinol, eckol, dieckol,

eckstolonol and triphloroethol A from E. cava were investigated for their ac-

tivity against AAPH-induced oxidative stress toxicity in zebrafish embryos

All phlorotannins were able to scavenge intracellular ROS, prevent lipid pe-

roxidation and reduce AAPH-induced cell death in zebrafish embryos. In an

in vivo study, the role of eckol from E. cava as a radioprotective agent against

the gamma ray-induced damage has been investigated by Park and colleages

It has been determined that eckol signiﬁcantly decreased the mortality of let-

hally irradiated mice via improving the hematopoietic recovery, repairing the

damaged DNA in immune cells and enhancing their proliferation. Therefore,

eckol is considered as a potential candidate for adjuvant therapy of radiation-

exposed cancer patients. Recently, the antioxidant properties of phlorotannins

from brown seaweed Cystoseira trinodis and Carpophyllum exuosum was

investigated

77,78

. It indicated that the antioxidant activities of these seaweed

were due to phlorotanins content and structure.

UV radiation has a strong oxidative component, and photo-oxidative stress

has been directly linked to skin photo-damage and associated with abnormal

cutaneous reactions such as epidermal hyperplasia, accelerated breakdown of

collagen, and inammatory responses. Herein, dieckol from E. cava has been

found to be able to inhibit melanogenesis and protect against photo-oxidative

stress induced by UV-B radiation

. The inhibitory activity on melanogenesis

was evidenced via suppression of tyrosinase and melanin synthesis. Meanwhi-

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le, protective activity was observed via scavenging intracellular ROS, preven-

ting DNA damage, and increasing cell viability. Additionally, Fucofuroeckol-A

from E. stolonifera was also found as protective agent against UVB-induced

allergic reaction in RBL-2H3 mast cells

. It was revealed that F-A signiﬁcantly

suppress mast cell degranulation via decreasing histamine release as well as

intracellular Ca

elevation induce by UVB. Notably, the protective activity of

F-A against mast cell degranulation was found due to scavenging ROS produc-

tion. According to Klervi and colleagues, the ethyl acetate fraction of brown

macroalga (Halidrys siliquosa) appeared to be a broad-spectrum UV-A absor-

ber

. This activity was found due to the present of four phenolic compounds

including trifuhalols, tetrafuhalols, diphlorethols, and triphlorethols. These

results indicated that phlorotannins from brown algae have potential protec-

tive eects against UV radiation, which might be applied in cosmeceutical in-

dustries.

Antimicrobial activity

Infectious diseases caused by bacteria and fungi are still a major threat to

public health, despite the tremendous progress in human medicine. Increa-

sing resistance of clinically important bacteria to existing antibiotics is a major

problem throughout the world

. The discovery of novel antimicrobial compo-

unds for clinical application is necessary to check the global crisis of antibiotic

resistance. In this regard, phlorotannins from brown algae have been found

to possess antimicrobial eect against food-borne pathogenic bacteria, anti-

biotic resistance bacteria, and pathogenic fungi. According to Nagayama and

colleagues, the oral administration of phlorotannins from E. kurome on mice

results in eective inhibition against methicillin-resistant Staphylococcus

aureus (MRSA). The minimum bactericidal concentrations (MBCs) of eckol,

phlorofucofuroeckol A, dieckol, and 8,8’-bieckol against Campylobacter jejuni

were 0.08, 0.08, 0.03, and 0.03 μmol/ml, respectively. At twice the MBCs,

all Vibrio parahaemolyticus were killed within 0.5-2 h, while catechins sho-

wed little bactericidal activity within 4 h

. Furthermore, Lee and collaborators

have determined that dieckol from E. stolonifera exhibited antibacterial ac-

tivity against methicillin-susceptible S. aureus (MSSA) and MRSA in a range

of minimum inhibitory concentrations (MICs) of 32 to 64 μg/ml

. The MICs

of ampicillin against two standard strains of MRSA were dramatically redu-

ced from 512 to 0.5 μg/ml in combination with 1/4 MIC of dieckol (16 μg/

ml). Likewise, Phlorofucofuroeckol-A from E. bicyclis were also showed anti-

MRSA activity with MIC of 32 μg/ml and synergistic action against MRSA in

combination with β-lactam antibiotics ampicillin, penicillin, and oxacillin

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

Thereby, phlorotannins-β-lactam antibiotics combinations exert a synergistic

eect against MRSA, indicating the promising treatment of MRSA infections.

In addition, it has shown that phlorofucofuroeckol-A from E. cava and E. bicy-

clis exhibited eective inhibition against Propionibacterium acnes, which may

be useful as natural additives in anti-acne cosmetic products

86, 87

. Although the

relationship between the structure and anti-bacterial activity of the phloro-

tannins is limited, their inhibitory activity may be suggested to depend on the

degree of polymerization of phlorotannin derivatives.

Besides, the purified phlorotannins extracts from three brown seaweeds inc-

luding Cystoseira nodicaulis, C. usneoides, and Fucus spiralis displayed their

antifungal activity against human pathogenic yeast and filamentous fungi

It was revealed that C. albicans ATCC 10231 was the most susceptible among

yeast, while Epidermophyton occosum and Trichophyton rubrum were the

most susceptible among dermatophytes. It was found that C. nodicaulis and C.

usneoides seem to act by aecting the ergosterol composition of the cell memb-

rane of yeast and dermatophyte, respectively. Meanwhile, F. spiralis inuen-

ced the dermatophyte cell wall composition by reducing the levels of chitin.

Moreover, phlorotannins from F. spiralis inhibited the dimorphic transition of

Candida albicans, leading to the formation of pseudohyphae with diminished

capacity to adhere to epithelial cells. On the other hand, the potential fungici-

dal activity of dieckol from E. cava was also found due to inhibition of Tric-

hophyton rubrum associated with dermatophytic nail infections in human

Anti-HIV activity

Human immunodeficiency virus type-1 (HIV-1) is the cause of acquired im-

mune deficiency syndrome (AIDS) which has been a major human viral dise-

ase with about 33.2 million people infected worldwide up to now

90, 91

. Antivi-

ral agents that interfere with HIV at dierent stages of viral replication have

been developed

92, 93

. However, failure in anti-AIDS treatment is observed by

the emergence of resistant virus, cross-resistance to drugs and cell toxicity

94, 95

Therefore, the search for potential candidates containing higher inhibitory ac-

tivity against various HIV strains is increasing in pharmaceutical industry. Ac-

cordingly, phlorotannins from brown algae have been revealed to possess anti-

HIV activity. For the first time, Ahn et al. (2004) reported that 8,8ʹ-bieckol

and 8,4ʹʹʹ-dieckol from E. cava exhibited an inhibitory eect on HIV-1 reverse

transcriptase and protease

. The inhibition against reverse transcriptase of

8,8ʹ-bieckol with a biaryl linkage (IC

, 0.5 μM) is ten-fold higher than that of

8,4ʹʹʹ-dieckol with a diphenyl ether linkage (IC

, 5.3 μM), although these two

phlorotannins are dimmers of eckol. They have suggested that the steric hind-

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

rance of the hydroxyl and aryl groups near the biaryl linkage of 8,8ʹ-bieckol

caused to the potent inhibitory activity. Moreover, 8,8ʹ-bieckol selectively in-

hibits reverse transcriptase over protease and inhibitory eect is comparable

to the positive control nevirapine (IC

, 0.28 μM). Moreover, kinetic study sho-

wed that 8,8ʹ-bieckol inhibited the RNA-dependent DNA synthesis activity of

HIV-1 reverse transcriptase noncompetitively against dUTP/dTTP with a Ki

value of 0.78 μM. Meanwhile, this compound also exhibited an uncompetitive

inhibition (Ki, 0.23 μM) with respect to a homopolymeric template/primer,

(rA)

(dT)

. A possible suggestion for this phenomenon is that 8,8ʹ-bieckol

binds to HIV-1 reverse transcriptase only after the template/primer initially

binds to the enzyme. Furthermore, Ahn et al. (2006) has shown that diphlo-

rethohydroxycarmalol from I. okamurae also has inhibitory eect on HIV-1

This compound exhibited inhibitory eects on HIV-1 reverse transcriptase and

integrase with IC

values of 9.1 μM and 25.2 μM, respectively. However, diph-

lorethohydroxycarmalol did not show an inhibitory activity against HIV-1 pro-

tease. In the same trend, 6,6ʹ-bieckol from E. cava has been found as a potent

wild inhibition against HIV-1 induced syncytia formation, lytic eects, and vi-

ral p24 antigen production

. This phlorotannin has selectively inhibited the

activity of HIV-1 reverse transcriptase enzyme with an IC

of 1.07 μM without

any cytotoxicity. Recently, Kwon and colleagues have found that phlorotannins

including eckol, 7-phloroeckol, phlorofucofuroeckol, and dieckol possessed

antiviral activities with IC

range of 10.8 – 22.5 μM against porcine epidemic

diarrhea virus

. These phlorotannins were completely blocked binding of vi-

ral spike protein to sialic acids at less than 36.6 μM by hemagglutination in-

hibition. Notably, phlorofucofuroeckol and dieckol inhibited viral replication

with IC

values of 12.2 and 14.6 μM in the post-treatment assay, respectively.

Interestingly, phlorofucofuroeckol and dieckol inhibited both viral entry by he-

magglutination inhibition and viral replication by inhibition of viral RNA and

viral protein synthesis, but not viral protease.

Anti-allergic activity

Allergic disease including allergic rhinitis, asthma, and atopic eczema are

among the commonest causes of chronic ill-health. It is caused by an exagge-

rated reaction of the immune system to harmless environmental substances,

such as animal dander, house dust mites, foods, pollen, insects, and chemical

agents

100, 101

. Allergic reaction is characterized by the excessive activation of

mast cells and basophils by immunoglobulin E (IgE) from B cells, resulting in

the release of preformed inammatory mediators from secretory granules such

as histamine and β-hexosaminidase, the generation and secretion of the newly

226

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

synthesized substances such as leukotrienes, prostaglandins, and cytokines

102

These mediators cause allergic inammatory responses due to airway cons-

triction, mucous production, and recruitment of inammatory cells. So far, a

large number of anti-allergic agents from natural products have been iden-

tified based on the specific assay system or screening approaches. Recently,

phlorotannins from brown algae have been determined as potential natural

inhibitors of allergic reactions due to suppression of allergic degranulation,

inhibition of hyaluronidase enzyme, and blockade of FcεRI activities. Several

bioactive phloroglucinol derivatives including fucodiphloroethol G, eckol, di-

eckol, 6,6´-bieckol, phlorofucofuroeckol A, and 1-(3´,5´-dihydroxyphenoxy)-

7-(2´´,4´´,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin were

isolated from E. cava and evidenced against A23187 or FcεRI-mediated hista-

mine release from KU812 and RBL-2H3 cells

103, 32

. Especially, dieckol, 6,6´-bi-

eckol, and fucodiphloroethol G exhibited a significantly inhibitory activity with

range of 27.80 – 55.12 μM. The inhibitory mechanism of these compo-

unds was determined to be due to blocking the binding activity between IgE

and FcεRI. Similarly, Shim et al. (2009) have proved that phlorotannins of

dioxinodehydroeckol and phlorofucofuroeckol A from E. stolonifera induced

a suppression of the cell surface FcεRI expression, and total cellular protein

and mRNA levels of the FcεRI α chain in KU812 cells

104

. Further, both of these

compounds exerted inhibitory eects against intracellular calcium elevation

and histamine release from anti-FcεRI α chain antibody (CRA-1)-stimulated

cells. In another study, phlorotannin PFF-B obtained from E. arborea expo-

sed strong inhibitory activity against histamine and β-hexosaminidase release

with IC

value of 7.8 μM

105, 106

. Obviously, PFF-B had a 2.8–6.0 times grea-

ter inhibitory activity than those of epigallocatechin gallate (IC

=22.0 μM) or

Tranilast (IC

=46.6 μM), a clinically used anti-allergic drug

107

. Thus, these bi-

oactive phloroglucinol derivatives were suggested as a promising candidate for

the design of novel inhibitor of FcεRI-mediated allergic reaction. For the first

time, the anti-allergenicity of phlorotannin from four edible seaweed species

of Fucus genus was evaluated by Barbosa and colleagues

108

. It was found that

Fucus was able to inhibit mast cell degranulation via decreasing histamine and

beta-hexosaminidase release from the activated RBL-2H3 cells, contributing

to the valorisation of Fucus spp. both as food and for nutraceutical applicati-

ons.

Hyaluronidase depolymerizes the polysaccharide hyaluronic acid in the extra-

cellular matrix of connective tissue, which is found both in organs and in body

uids. It is mainly known to be involved in the permeability of the vascular

system

109

and allergic reaction

110, 111

. Interestingly, various phlorotannins such

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Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

as phlorofucofuroeckol A, dieckol, and 8,8´-bieckol from E. bicyclis are able

to inhibit hyaluronidase enzyme with IC

values of 140, 120, and 40 μM, res-

pectively

112

. The eect of these phlorotannins against hyaluronidase enzyme

is stronger than well-known inhibitors such as catechins (IC

=620 μM) and

sodium cromoglycate (IC

=270 μM). Notably, 8,8´-bieckol, the strongest hya-

luronidase inhibitor among the tested phlorotannins, acted as a competitive

inhibitor with an inhibition constant of 35 μM. Likewise, several phlorotannins

of 6,6´-bieckol, 6,8´-bieckol, 8,8´-bieckol, PFF-A, and PFF-B from E. arborea

were also confirmed as strong inhibitors of hyaluronidase

113, 114

Anti-inammatory activity

Inammation is a critically important aspect of host responses to various sti-

muli including physical damage, ultra violet irradiation, microbial invasion,

and immune reactions

115, 116

. It is associated with a large range of mediators that

initiate the inammatory response, recruit and activate other cells to the site

of inammation

117

. However, excessive or prolonged inammation can prove

harmful, contributing to the pathogenesis of a variety of diseases, including

chronic asthma, rheumatoid arthritis, multiple sclerosis, inammatory bowel

disease, psoriasis, and cancer

116

. Currently, several classes of drugs such as

corticosteroids, nonsteroidal anti-inammatory drugs, and aspirin are used to

treat the inammatory disorders. All these therapeutics help to alleviate the

symptoms but, especially after long-term and high-dose medication, they can

have quite substantial side-eects. Therefore, there is still a vital need for the

development of new anti-inammatory drugs with satisfactory tolerability for

long-term use. Herein, phlorotannins have been evidenced as potential agents

for down-regulation of inammatory responses. Phlorotannin-rich extracts

of E. cava showed significant suppression of PGE

generation in LPS-treated

RAW 246.7 cells, and significant inhibition of human recombinant interleukin-

1α-induced proteoglycan degradation

118

. Moreover, the phlorotannin-rich the

fermented E. cava processing by-product extract was reported to inhibit NO

and PGE

production, suppress the inducible nitric oxide synthase (iNOS) and

cyclooxygenase-2 (COX-2) expressions, and attenuate interleukin-1β and in-

terleukin-6 production in lipopolysaccharide stimulated RAW 264.7 cells

119

Recently, phlorotannin 6,6’-bieckol from E. cava was found to inhibit NO

and PGE

production by suppressing the expression of iNOS and COX-2 at

the mRNA and protein levels in LPS-stimulated primary macrophages and

RAW 264.7 macrophage cells

120

. Moreover, 6,6’-bieckol down-regulated the

production and mRNA expression of the inammatory cytokines TNF-α and

IL-6. The pretreatment of 6,6’-bieckol decreased LPS-induced transactivation

228

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

of nuclear factor-kappa B (NF-κB) and nuclear translocation of p50 and p65

subunits of NF-κB, thus inhibiting LPS-induced NF-κB binding to the TNF-α

and IL-6 promoters. On the other hand, Kim and collaborators have evidenced

that phlorofucofuroeckol A from E. stolonifera attenuated the productions and

expression of NO, PGE

, and pro-inammatory cytokines such as IL-1β, IL-

6, and TNF-α in LPS-stimulated microglia. Profoundly, phlorofucofuroeckol

A treatment showed inactivation of c-Jun NH2-terminal kinases (JNKs), p38

mitogen-activated protein kinase (MAPK), Akt, and NF-κB

121

. Likewise, Kim et

al. (2009) have shown the inhibitory activity of this phlorofucofuroeckol A on

NO and PGE

production and iNOS and COX-2 expression in RAW 264.7 mu-

rine macrophage cells

122

. Besides, phlorotanins from E. arborea also exhibited

inhibitory eect on NO production in LPS-stimulated RAW 264.7 cells

123

and

mouse ear edema induced by arachidonic acid, 12-O-tetradecanoyl phorbol-

13-acetate, and oxazolone

124

. Notably, 8,8´-bieckol from E. bicyclis showed the

pronouncedly inhibitory eects on soybean lipoxygenases and 5-lipoxygenases

with IC

values of 38 and 24 μM, respectively. Meanwhile, dieckol presen-

ted a significant inhibition of COX-1 with inhibition rate of 74.7%

125

. Similarly,

6,6’-bieckol, 6,8’-bieckol, 8,8’-bieckol, PFF-A, and PFF-B from E. arborea

were also confirmed as strong inhibitors of phospholipase A

, cyclooxygenase,

and lipoxygenases, which correlated to suppression in synthesis and release of

leukotoriene and prostaglandin from RBL cells

114

. Recently, phlorotannin-rich

extract of the edible brown alga E. cava against hyper-inammatory respon-

se in LPS-induced septic shock mouse model was also investigated

126

. Diec-

kol, a major compound in the extract, reduced mortality, tissue toxicity, and

serum levels of the inammatory factors in septic mice and suppressed the

septic shock through negative regulation of pro-inammatory factors via the

NIK/TAK1/IKK/IκB/NFκB and Nrf2/HO-1 pathways. In addition, Barbosa

and colleagues have demonstrates the marked potential of Fucus sp. and their

phlorotannin-purified extracts to act upon dierent mediators important in

the pathophysiology of inammatory-related conditions

127

. The anti-inam-

matory potential of the purified phlorotannin extracts in both cell and cell-free

systems was observed. Therefore, the phlorotannin extracts from Fucales arise

as potentially beneficial in inammation-related conditions, eectively acting

upon enzymatic and non-enzymatic inammatory target.

Anti-cancer activity

Cancer can be defined as a disease in which a group of abnormal cells grow

uncontrollably by disregarding the normal rules of the cell division

128

. Cancers

may be caused in one of three ways, namely incorrect diet, genetic predisposi-

tion, and via the environment. At least 35% of all cancers worldwide are caused

229

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

by an incorrect diet. Meanwhile, genetic predisposition caused about 20% of

cancer cases, thus leaving the majority of cancers being associated with a host

of environmental carcinogens

129

. It is necessary to avoid exposure to cancer-

causing biological, chemical, and physical agents, and consume chemo-pre-

ventive agents to reduce cancer risk. A promising approach is associated with

natural products that are available as anti-cancer agents against commonly

occurring cancers occurring worldwide

130, 131

. Recently, phlorotannins have

been reported as novel promising anti-cancer agent for breast cancer. Kong

et al. (2009) has indicated that dioxinodehydroeckol from E. Cava exerted

anti-proliferative activity against human breast cancer cells via induction of

apoptosis

132

. Dioxinodehydroeckol treatment caused the increase in caspase

(-3 and -9) activity, DNA repair enzyme poly-(ADP-ribose) polymerase (PARP)

cleaved, and pro-apoptotic gene (Bax, p53, and p21) and the decrease in an-

ti-apoptotic gene Bcl-2 and NF-κB activation. Moreover, phlorotannins-rich

extracts from Palmaria, Ascophyllum and Alaria also inhibited the prolife-

ration of colon cancer cells

133

. On the other hand, the anti-cancer activity of

S. muticum polyphenol-rich seaweed was shown via inhibiting breast cancer

cell proliferation with IC

of 22 μg/ml and inducing apoptosis from 13% to

67% by accumulation of cells at sub-G1 phase

134

. Parys et al. (2010) reported

that trifucodiphlorethol A, trifucotriphlorethol A and fucotriphlorethol A from

Fucus vesiculosus were the potential chemo-preventive agents due to their ca-

pacity to inhibit the activity of aromatase related to carcinogenesis from breast

cancers

135

. For the ﬁrst time, Kim and colleages have determined the inhibitory

eects of phlorotannins isolated from E. cava on MMP activities in cultured

human cell lines without any cytotoxic eect

136

. Recently, the anti-proliferative

eect of various brown algae including Cystoseira crinita, Cystoseira stricta,

and Sargassum vulgare on the human epithelial cell line Caco-2 was eviden-

ced via arresting in G phases along with an increment in sub-diploidal cell po-

pulation

137

. The anti-proliferative eect of these algae was correlated with their

polyphenol and avonoid contents. Moreover, the phlorotannins recovered

from hydrothermal treatment of Sargassum muticum were showed the an-

ti-proliferative properties against lung adenocarcinoma A549 cells and colon

carcinoma HCT-116 cells

138

. These results imply that seaweeds which are rich

in phlorotannins may be used in anti-cancer drug research programs.

Anti-diabetic activity

Diabetes mellitus is a chronic metabolic disorder involved in hyperglycaemia,

resulting from the deﬁciency in the production of insulin by the pancreas. Up to

now, numerous therapeutics have been proposed to control hyperglycaemia in

diabetic patients. Especially, α-amylase and α-glucosidase are enzymes related

230

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

to hyperglycaemia due to the starch hydrolysis and release of the glucose mo-

nomers for subsequent absorption by the small intestine. Therefore, the inhi-

bition of these enzymes reduces the availability of free glucose monomers and

consequently decreases blood glucose levels

139

. Rengasamy et al. (2013) has

isolated three phlorotannins including dibenzo (1,4) dioxine-2,4,7,9-tetraol

and eckol from E. maxima and evaluated their alpha-glucosidase inhibitory

activities

140

. The inhibitory activities of dibenzo (1,4) dioxine-2,4,7,9-tetraol

and eckol on enzyme alpha-glucosidase were 33.7 and 11.2 μM, respectively. A

phenolic-rich extract from Ascophyllum was eective to inhibit α-amylase and

α-glucosidase with IC

of 0.1 μg/ml GAE and 20 μg/ml GAE

133

. The presence of

fucophloroethol structures with degrees of polymerization from 3 to 18 mono-

mer units in Fucus distichus is responsible for its inhibition on α-glucosidase

and α-amylase, with IC

values of 0.89 and 13.9 μg/ml

141

. Moreover, dieckol

and eckol from Eisenia bicyclis exhibited the inhibitory activity on α-amylase

up to 97.5 and 87.5% at 1 mM

142

. Meanwhile, α-glucosidase was inhibited by

phlorofucofuroeckol-A, dieckol, and 7-phloroeckol from E. stolonifera and

eckol and dioxinodehydroeckol from E. bicyclis with IC

of 1.37, 1.61, 6.13,

22.78, and 34.6 μM, respectively

143

. The ingestion of methanolic extract of E.

stolonifera suppressed the increase in plasma glucose and lipid peroxidation

levels in unfasted KK-A(y) mice

144

. Furthermore, various phlorotannins from

E. stolonifera exhibited the inhibitory activities on aldose reductase, which

are highly implicated in hyperglycemia and oxidative stress. The IC

values

of phloroglucinol derivatives are 21.95 - 125.45 μM

145

. Besides, dieckol from E.

cava has evidenced prominent inhibitory eect against alpha-glucosidase and

alpha-amylase with IC

values of 0.24 and 0.66 mM, respectively. The incre-

ase of postprandial blood glucose levels were significantly suppressed in the

dieckol administered group in the streptozotocin-induced diabetic mice

146

. Re-

cently, three phlorotannins, eckol, dieckol and phlorofucofuroeckol-A from E.

bicyclis were revealed for their anti-diabetic activity of alloxan-induced type1

and insulin-induced type 2 in the zebrafish model

147

. Notably, the inhibition of

the catalytic reaction of α-glucosidase by minor phlorotannin derivatives from

E. cava were demonstrated with IC

values ranging from 2.3 ± 0.1 to 59.8 ±

0.8 μM. Compounds 2–5 inhibited the catalytic reaction of α-glucosidase in

non-competitive and competitive manners

148

Anti-obesity

Obesity is a major obstacle in human health and life quality, resulting in many

chronic diseases. It is due to a chronic imbalance between energy intake and

energy expenditure, leading to the increased fat storage

149

. Interestingly, a se-

ries of anti-obesity components derived from marine origin have been found,

231

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

especially phlorotannins. Herein, three phlorotannins from E. stolonifera inc-

luding phloroglucinol, eckol, and phlorofucofuroeckol A significantly inhibi-

ted lipid accumulation in 3T3-L1 cells via reducing the expression of adipocyte

marker genes such as proliferator activated receptor γ and CCAAT/enhancer-

binding protein α

150

. Meanwhile, phlorotannin dieckol from E. cava exhibited

great potential adipogenesis inhibition and down-regulated the expression of

peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding pro-

teins, sterol regulatory element-binding protein 1 (SREBP1) and fatty acid bin-

ding protein 4

151

. Moreover, diphlorethohydroxycarmalol (DPHC) from Ishige

okamurae was showed to inhibit population growth and induce apoptosis in

3T3-L1 preadipocytes

152

. The peptidyl prolyl cis/trans isomerase Pin1 enhances

the uptake of triglycerides and the dierentiation of fibroblasts into adipose

cells in response to insulin stimulation. However, phlorotannin called 974-B

from E. kurome was showed to inhibit the dierentiation of mouse embryonic

fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity,

suggesting a lead drug candidate for obesity-related disorders

153

Other biological activities

According to Ahn et al. (2010), phloroglucinol from E. cava possesses the ac-

tivation activity on immune response. The phloroglucinol elicited the prolife-

ration of lymphocytes without cytotoxicity and enhanced IL-2 production by

activating the nuclear factor-kappaB (NF-κB) signaling pathway

154

Inhibition of angiotensin I-converting enzyme (ACE) activity is the most com-

mon mechanism underlying the lowering of blood pressure. Dieckol from E.

cava was found as potent ACE inhibitor with IC

value of 1.47 mM. It is a

non-competitive inhibitor against ACE according to Lineweaver-Burk plots

155

Meanwhile, eckol, phlorofucofuroeckol A, and dieckol from E. stolonifera were

also determined to manifest the marked inhibitory activity against ACE, with

values of 70.82, 12.74, and 34.25 μM, respectively

156

Neurodegenerative diseases are characterized by progressive dysfunction and

death of neurons. Recently, neuroprotective eects of phlorotannins from

brown seaweed have been evidenced by various experimental models. Um and

colleagues have reported that phlorotannin-rich fraction from Ishige foliacea

brown seaweed prevents the scopolamine-induced memory impairment in

mice

157

. It reduced acetylcholinesterase activity in the brain and significantly

decreased lipid peroxidation levels, but increased glutathione levels and su-

peroxide dismutase activity. Moreover, the expression levels of brain-derived

neurotrophic factor, tropomyosin receptor kinase B, the phosphorylated ext-

racellular signal-regulated kinase, and cyclic AMP-response element-binding

232

Acta Pharmaceutica Sciencia. Vol. 57 No. 2, 2019

protein were increased by I. foliacea phlorotannin. In addition, the neuropro-

tective eect of dieckol from E. cava on rotenone-induced oxidative stress in a

human dopaminergic neuronal cell line SH-SY5Y was also determined by Cha

and colleagues

158

. Dieckol reduced the intracellular reactive oxygen species

and cytochrome C release, cell death, and α-synuclein aggregation in SH-SY5Y

cells. Likewise, it was found that eckmaxol, a phlorotannin from E. maxima,

could produce neuroprotective eects in SH-SY5Y cells via directly acting on

glycogen synthase kinase 3β

159

. Accordingly, phlorotannin from the brown al-

gae could be a potential therapeutic agent for the prevention of neurodegene-

rative diseases.

RESULTS AND DISCUSSION

Finding the safe and efﬁcient agents from natural products for prevention and

treatment of chronic diseases are always necessary. Herein, phlorotannins

from brown algae have been identified with various biological activities and

health benefit eects. The extensive discoveries of phlorotannins underlying

structure-activity relationship will provide clear evidence on their actions aga-

inst diseases. Moreover, the further studies due to the bioavailability involving

in liberation, absorption, distribution, metabolism, and elimination phases

will ensure the bio-eicacy of phlorotannins. Collectively, phlorotannins from

brown algae are believed to play an important role in the development of pro-

mising pharmaceutical products that can prevent and/or treat various chronic

diseases.

ACKNOWLEDGEMENT

This review is supported by Nguyen Tat Thanh University, Ho Chi Minh city,

Vietnam.

CONFLICT OF INTEREST

There are no conicts to declare.

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