Topical Formulation Comprising Fatty Acid Extract from Cod-liver Oil

103 Pages·2016·4.57 MB·English

Topical Formulation Comprising Fatty Acid Extract from
Cod-liver Oil: Development, Evaluation and Stability Studies

Biljana Ilievska

MS thesis in Pharmaceutical Sciences
Supervisor: Þorsteinn Loftsson
Faculty of Pharmaceutical Sciences
School of Health Sciences, University of Iceland
May 2016
Þróun útvortis lyfs sem inniheldur fitusýrur unnar úr þorskalýsi

Biljana Ilievska

Faculty of Pharmaceutical Sciences

siy of Iceland
tararitgerð í lyfjafræ

Umsjónar
daofoojsvikp4áskóla Íslands
Meistararitgerð í lyfjavísindum
Umsjónarkennari: Þorsteinn Loftsson
Lyfjafræðideild
Heilbrigðisvísindasvið Háskóla Íslands
Maí 2016
This thesis is submitted in partial fulfillment of the requirements of a MS degree in
Pharmaceutical Sciences and may not be reproduced in any form without the written
permission of the author

©Biljana Ilievska 2016

Printing: Háskólaprent
Reykjavik, Iceland 2016
Author Biljana Ilievska

Supervisor Þorsteinn Loftsson
Professor of Physical Pharmacy
Faculty of Pharmaceutical Sciences
University of Iceland

Masters committee Þorsteinn Loftsson
Professor of Physical Pharmacy
Faculty of Pharmaceutical Sciences
University of Iceland

Þórdís Kristmundsdóttir
Professor of Pharmaceutics
Faculty of Pharmaceutical Sciences
University of Iceland

Guðrún Marta Ásgrímsdóttir
Pharmacist at Lipid Pharmaceuticals

iv
List of Contents

Abstract
List of Tables
List of Figures
List of Abbreviations
1 Introduction………………………………………………………………………….1
1.1 Fatty Acids………………………………………………………………........…1
1.1.1 Chemistry of Fatty Acids…………………………………………...……..1
1.1.2 Essential Fatty Acids……………………………………………………...3
1.1.2.1 Metabolism of EFAs in the skin…………………………………..4
1.1.2.2 PUFAs and skin inflammation………………………………….…7
1.1.3 Antibacterial and antiviral effects of PUFAs ……………………………..9
1.1.3.1 Mechanism of antimicrobial action of fatty acids………………..12
1.2 Fish oil………………………………………………………….……………...14
1.2.1 Fish oil and oxidation………………………………………………….…15
1.2.2 Fish oil and health benefits………………………………………...…….18
1.3 Topical and transdermal delivery……………………………………...……….20
1.3.1 Skin structure and function……………………………………...…….…20
1.3.1.1 Lipid composition in stratum corneum…………………………..23
1.3.1.2 Lipid organization in stratum corneum…………………………..24
1.3.2 Dermal drug delivery………………………………...…………….26
1.3.3 Topical formulations…………………………………...…………….….28
1.3.3.1 Semisolid dosage forms………………………………….……....29
1.3.3.2 Waxes……………………………………………………........….30
2. Aims………………………………………………………………………….….32
3. Materials, Equipment and Methods………………………...………………..….33
3.1 Materials………………………………………………………………...…….33
3.1.1 Materials used in ointment compositions………………………………..33
v
3.1.2 Reagents………………………………………………………………...35
3.1.3 Materials that used for in-vitro antibacterial activity testing…...……….…35
3.2 Equipment…………….………………………………………………………….36
3.3 Methods…………………………………………………………………..……....36
3.3.1 Preparations of ointment………………………………………..….............36
3.3.2 Evaluation of in vitro antibacterial efficacy of FFAs……………………...37
3.3.3 Determination of peroxide value (PV)……….……………………........….38
3.3.4 Measurements of pH……………………………………………...…….….39
3.3.5 Physical evaluation of ointment…………………………………...…….…39
3.3.5.1 Spreadability measurement……………………………...……….…39
3.3.5.2 Tube Extrudability………………………………………...…….….40
3.3.6 Sensory examination….………………………………..…..…………….…40
3.3.7 Microscopic evaluation…………………...………………………..…….…41
3.3.8 Stability study …...………………………………………………...…….…41
4. Results and Discussion……………………………………………………………42
4.1 In vitro evaluation of antibacterial efficiency of FFAs extract…...…………..42
4.2 Ointment design.........................................................................................…...44
4.3 Choice of ointment base………………………………………………………46
4.4 Changes in the peroxide value of ointment……………….…………........…..47
4.5. pH determination…………………………..…………………………………51
4.6 Influence of ointment base on spreadability…………………………………..53
4.7 Influence of ointment base on tube extrudability………………………….….55
4.8 Sensory evaluation…………………………...…………………….………….56
4.9 Microscopic evolution……………...……………………………..….……….58
4.10 Stability study…………………………………...…………………………...60
5. Conclusion…………………………………………………………………....…...62
6. Acknowledgement…………………………………………………………...…....63
7. References ………………………………………………………………………..64
Appendix A: PV measurements (mmolO2/kg)
Appendix B: Full data on pH, spreadability and extrudability measurements
vi
Appendix C: Sensory evaluation on ointments
Appendix D: Certificate of analysis of free fatty acids
Appendix E: Paper I published in Journal of Drug Delivery Science and Technology
Manuscript submitted to Marine Drugs

vii
Ágrip
Tilgangur rannsóknarinnar var að þróa lyfjaform úr fiskiolíu sem væri rík af omega-3 fríum
fitusýrum, s.s. EPA og DHA til staðbundinnar notkunar á húð.
Sýnt hefur verið fram á með ótal rannsóknum og það almennt viðurkennt, að dagleg inntaka
þorskalýsis og annarra fiskiolía hefur jákvæð áhrif á heilsufar fólks og því ljóst að líffræðileg
virkni þeirra er hefur ekki verið rannsökuð til fulls.Samkvæmt in vitro rannsóknum eru fríar
fitusýrur (20% (v / v)) mjög öflugar gegn gram-jákvæðum bakteríum á borð við Staphylococcus
aureus, Enterococcus faecalis, Streptococcus pyogenes og Streptococcus pneumoniae og með
því að vinna fjölómettaðar fitusýrur (PUFA) úr þorskalýsi eða öðrum fiskiolíum er möguleiki að
þróa gagnlegt, öruggt og náttúrulegt sýklalyf.
Algengt er að nota ýmis afbrigði af náttúrulegu og tilbúnu vaxi í snyrtivörur, lyf og matvæli til
að bæta áferð og útlit vörunnar. Frá lyfjafræðilegu sjónarmiði, er notkun á vaxi viðurkennt til að
bæta útlit, uppbyggingu og seigjustig húðlyfja auk þess sem vax getur lengt geymslutíma.
Í þessari rannsókn voru þróuð smyrsl þar sem virka efnið er fríar fitusýrur ríkar af omega-3
fitusýrum. Fríar fitusýrur hafa bakteríu-, veiru- og sveppadrepandi áhrif auk þess sem þekkt eru
bólgueyðandi áhrif omega-3 fitusýra. Hjálparefni sem notuð voru eru ýmsar tegundir af
náttúrulegu vaxi.
Áhrif mismunandi tegunda vaxs s.s. carnauba, ozokerite, laurel, býflugnavax, hrísgrjónaklíð,
candelilla og sellulósa vax, í styrkleika frá 1- 5% (w/w), voru metin m.t.t. áferðar, þéttleika og
stöðugleika.
Niðurstöður hvað varðar áferð og skynjun (sensory profile) voru mjög mismunandi háð vaxi og
styrkleika. Ástæðan var rakin til eðli vaxins og keðjusamsetningar. Sellulósavax gaf bestan
árangur en laurelvax, býflugnavax og hrísgrjónaklíðisvax gáfu einnig mjög góða áferð, þ.e.
svipaða skynjun (sensory profile) og viðeigandi seigjustig.

viii
Abstract
The current study was designed to develop a pharmaceutical formulation that contained fish oil
extract rich in free omega 3 FAs such as EPA and DHA for topical use. Although the health
benefits of cod-liver oil and other fish oils taken as a dietary supplement have been vastly
acknowledged and exploited, it is clear that their use could be further extended to cover other
biological properties. Based on in vitro evaluation, 20% (v/v) fish oil extract was found to be
highly potent against gram-positive bacteria such as Staphylococcus aureus, Enterococcus
faecalis, Streptoccoccus pyogenes and Streptoccoccus pneumonia. Therefore, free PUFAs from
cod-liver oil or other fish oils could be useful as safe and natural antibacterial agents.
Today, varieties of natural and synthetic waxes are used in numerous cosmetic, pharmaceutical
and food applications for texture enhancement and benefits in sensory profile. Seen from a
pharmacological aspect, waxes are recognized as a potential for enhancing aesthetics and
maximizing therapeutic benefits on topical formulation by viscosity building and prolonging the
retention time.
In this study, ointment compositions containing FFAs as active antibacterial agents were
prepared by using various natural waxes and characterized. The effects of different waxes, such
as carnauba, ozokerite, laurel, beeswax, rice bran, candelilla and microcrystalline wax, in the
concentration range 1 to 5% (w/w) on the ointment texture, consistency and stability were
evaluated. The results showed significant variations in texture and sensory profile. This was
attributed to the wax nature and chain composition. Microcristalline wax gave the best results but
laurel wax, beeswax and rice bran wax also gave excellent texturing, similar sensory profile and
well-balanced rheological properties.

ix
List of Tables
Table 1. The systematic and trivial names of PUFAs ………………………...………….………3
Table 2. Representative bioactivities of various polyunsaturated fatty acids…………………....10
Table 3. Fatty acids composition of cod liver oil…………………………......………………….33
Table 4. Waxes tested in the ointment preparation………………………………………………34
Table 5. MIC concentration……………………………………………………………………...43
Table 6. Summarized compositions of ointments……………………………………………….45

x