PHYSICOCHEMICAL PROPERTIES, ANTIOXIDANT AND

ANTIMICROBIAL ACTIVITIES OF ROSEMARY

(RosmarinusofficinalisL.) LEAF OIL

M.Sc THESIS

TSEGAYE GUCHALE

JANUARY, 2021
HARAMAYA UNIVERSITY, HARAMAYA

ii
Physicochemical Properties and Biological Activities of Rosemary
(RosmarinusofficinalisL.) Leaf oil

A Thesis Submitted to Postgraduate Program Directorate

School of Biological Science and Biotechnology
HARAMAYA UNIVERSITY

In Partial Fulfillment of the Requirements of the Degree of

MASTER OF SCIENCE IN BIOLOGY

TsegayeGuchale

January, 2021

HaramayaUniversity, Haramaya
 APPROVAL SHEET
HARAMAYA UNIVERSITY
POSTGRADUATE PROGRAM DIRECTORATE

As thesis Research advisors, we hereby certify that we have read and evaluated this Thesis,
prepared, under our guidance by TsegayeGuchale, entitled Physicochemical Properties and
Biological Activities of Rosemary (RosmarinusofficinalisL.) Leaf oil. We recommend that
it be submitted as fulfilling the thesis requirement.

Zekeria Yusuf (PhD) ________________ _______________

Major Advisor Signature Date

MulugetaDesta (PhD) _______________ ________________

Co- Advisor Signature Date

As member of the Board of Examiners of the M.Sc. Thesis Open Defense examination, we
certify that we have read and evaluated the Thesis prepared by TsegayeGuchale, the
candidate. We recommend that the thesis be accepted as fulfilling the thesis requirements for
the degree of Master of Science in field of Biological Sciences.

________________ ________________ ________________

Chairperson Signature Date

________________ ________________ ________________

Internal examiner Signature Date

________________ ________________ _______________

External examiner Signature Date

Final approval and acceptance of the Thesis is contingent upon the submission of its final
copy to the council of Graduate Studies (CGS) through the candidate's department or school
graduate committee (DGC or SGC).

ii
 DEDICATION

This thesis manuscript is dedicated to Mr. NigussieGuchale my brother for his unbound love,
patience and strength that helped me come to this level.

iii
 STATEMENT OF THE AUTHOR

By my signature below, I declare and affirm that this Thesis is my own work. I have followed
all ethical and technical principles of scholarship in the preparation, data collection, data
analysis and completion of this thesis. All scholarly matter that is included in the thesis has
been given recognition through citation. I affirm that I have cited and referenced all sources
used in this document. Every serious effort has been made to avoid any plagiarism in the
preparation of this thesis.

This Thesis is submitted in partial fulfillment of the requirement for advanced Msc degree in
science in biology atHaramaya University and is deposited at the Haramaya University
Library to be made available to borrowers under rules and regulations of the library. I
solemnly declare that this Thesis is not submitted to any other institution anywhere for the
award of any academic degree, diploma, or certificate.

Brief Quotations from this Thesis are allowable without special permission provided that
accurate acknowledgment of source is made. Requests for permission for extended quotation
from reproduction of this thesis in whole or in part may be granted by the Head of the
Department or the Dean of the School of Postgraduate Program Directorate when in his or her
judgment the proposed use of the material is in the interest of scholarship. In all other
instances, however, permission must be obtained from the author.

Name: TsegayeGuchale
Signature ------------------------
Place: Haramaya University
Date of Submission: January 2021

iv
 BIOGRAPHICAL SKETCH

The author was born in North Shoa zone on January 1992. He attended his primary education
at Zeram general Primary and Secondary education at Molale Secondary and Preparatory
School. Up on the successful completion of his secondary education, he joined DebreBerhan
University under College of natural and Computational sciences and graduated with B.sc.
degree in Biology with CGP of 3.72 in 2014 . Right after graduation, he was assigned by
Ministry of education to teach biology in Molale (North Shoa zone, Menz Mama
Midirworeda) senior secondary school and to attend his PGDT Program at DebreBerhan
University under College of Natural and computational Sciences and graduated with CGP of
3.56 in 2015 to teach Biology at High school level. Then, he was employed as Biology
teacher at North Shoa, Molale Senior Secondary and Preparatory School. Soon after, he
joined Haramaya University for Postgraduate Studies in 2017 to pursue his MSC study in
Teaching Biology.

v
 ACKNOWLEDGMENTS

First, priceless praise is to God for His blessing and guidance throughout my life. Next, I
would like to express my extraordinary gratitude and reverence to my thesis major advisor,
Zekaria Yusuf (PhD). His guidance, brainstorming, scaffolding, and stimulation all made this
thesis possible. His unreserved investment of his expertise and time in this thesis contributed
to my successful completion. As the first reader of my many drafts, he patiently tolerated my
writing and provided me valuable input for improvement.

Equal gratitude goes to my co-advisor MulugetaDesta (PhD). His great effort, enthusiasm,
guidance and understanding from the very beginning of my thesis writing. His expertise and
invaluable comments helped greatly in the completion of this thesis.

I would like also to extend my special gratitude to my beloved father atoAysa Adare for his
supporting me throughout my study. Because, his love, unwavering faith and confidence in
me is what has shaped me to be the person I am today.Last but not least, I greatly
acknowledge all staff teachers of Yoale senior Secondary and Preparatory School for their
readiness to support by their time and knowledge, all laboratory technicians of Haramaya
university main campus, Without their support, my study would not have been possible.

vi
 ABBREVIATIONS/ACRONYMS

AA Ascorbic acid
AChE Acetylcholine –cholinesterase
ACV Acid value
AOX Antioxidants
BChE Butyrylcholinesterase
BHA Butylated hydroxyanisole
BHT Butylated hydroxytolune
DGAT diacylglycerol acyltransferase
DPPH 2, 2- diphenyl-1-picrylhydrazyl
EO Essential Oil
FFA free fatty acids
FST Forced swimming test
HPLC High pressure Liquid Chromoatography
HPSA Hydrogen peroxide scavenging activity
LDL low- density lipoproteins
MBC Minimum bacterial inhibitory concentration
MFC Minimum fungal inhibitory concentration
MIC Minimum inhibitory concentration
NGF Nerve growth factor
PV Peroxide value
Spgr Specific gravity
TST Tail suspension test

vii
 TABLE OF CONTENTS

APPROVAL SHEET ii

DEDICATION iii

STATEMENT OF THE AUTHOR iv

BIOGRAPHICAL SKETCH v

ACKNOWLEDGMENTS vi

ABBREVIATIONS/ACRONYMS vii

LIST OF TABLES xi

LIST OF FIGURES Error! Bookmark not defined.

LIST OF TABLES IN THE APPENDIX xii

LIST OF FIGURES IN THE APPENDIX xiii

ABSTRACT xiv

1. INTRODUCTION 1

2. LITERATURE REVIEW 5

2.1. Major chemical constituents of Rosmarinus officinalis (L)volatile oil 5

2.2. Medicinal uses 5
2.2.1. Brain and nervous system conditions 5
2.3. Pharmacological activities 6
2.3.1. Toxicology 6
2.3.2. Clinical pharmacology 7
2.3.3. Antioxidant activity 7
2.3.4. Antimicrobial activity 9
2.3.5. Anticancer activity 10
2.3.6. Antidiabetic activity 10
2.3.7. Antidepressant activity 10
2.3.8. Neuroprotective activity 11

viii
 2.3.9. Anti-inflammatory activity 12
2.3.10. Anti-obesity activity 12
2.3.11. Adverse Reactions and Contra Indications of Rosemary Oil 12
3. MATERIALS AND METHODS 14

3.1. Description of study area 14

3.2. Plant Material and Extract Preparation 14
3.3. Research Design 14
3.4. Oil Extraction 15
3.5. Data collection 15
3.5.1. Determination of oil yield and specific gravity 15
3.5.2. Determination of Acid Value 15
3.5.3. Estimation of Free Fatty Acid 16
3.5.4. Determination of Peroxide Value 16
3.5.5. DPPH Radical Scavenging Activity 16
3.5.7. Ascorbic Acid Analysis 17
3.6. Antimicrobial activity of the oil extracts 18
3.6.1. Test Pathogens 18
3.6.2. Media Preparation and Standardization of Inoculum 18
3.6.3. Disc diffusion Method 19
3.6.4. Inoculation of Mueller Hinton Agar (MHA) plates 19
3.6.5. Determination of Minimum Inhibitory Concentration (MIC) 20
3.6.6. Determination of minimum bactericidal (MBC) and fungicidal concentrations
(MFC) 20
3.7. Data Analysis 21
4. RESULTS AND DISCUSSION 22

4.1. Physicochemical properties of Rosemary (Rosmarinus officinalis L.) leaf oil 22

4.2. Antioxidant activities of Rosmarinus officinalis (L.)of leaf oil extract 23
4.3. Antimicrobial Activities of Rosemary (Rosmarinus officinalis L.) leaf oil 24
4.3. Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC), minimum fungicidal concentration (MFC) of Rosmarinus
officinalis (L.) leaf oil 26

ix
5. SUMMARY, CONCLUSION AND RECOMMENDATION 29

5.1. Summary 29
5.2. Conclusion 31
5.3. Recommendation 31
6. REFERENCES 33

7. APPENDICES 42

x
 LIST OF TABLES

Table Page

1. Physicochemical properties of solvent extracted Rosemary (Rosmarinus officinalis L.) leaf

oil 22

2. Antimicrobial activity Rosmarinus officinalis (L) leaf oil based on diameter of zone of
inhibition 25

3. Minimum inhibitory concentration (MIC), Minimum Bactericidal Concentration (MBC),

minimum fungicidal concentration (MFC) of Rosmarinus officinalis (L.) leaf oil oil extract 27

xi
 LIST OF TABLES IN THE APPENDIX

Appendix Table Page

1. Raw data for physicochemical properties and antioxidant activities 43

2. Raw data for antibacterial activity based on diameter of zone of inhibition 43

3. Raw data for antifungal activity 44

xii
 LIST OF FIGURES IN THE APPENDIX

Appendix Figure 1. Sample preparation 45

Appendix Figure 2. Sample extracted oil 46

Appendix Figure 3. Antimicrobial activity 47

Appendix Figure 4. Measuring inhibition zone during antimicrobial activity test 48

xiii
Physicochemical Properties and Biological Activities of Rosemary
(RosmarinusofficinalisL.) Leaf oil

ABSTRACT

Medicinal plants were used for centuries as remedies for human diseases because they
contain components of therapeutic value, there is an increasing interest in phytochemicals as
new sources of natural antioxidant and antimicrobial agents. Microbial infections have
become one of the major problems of public health in the world and the development of
resistance to the available antibiotics has lead researchers to investigate the antimicrobial
activity of medicinal plants. Essential oil extracts of plants have many applications in
medicines, cosmetics, food stuff and pharmaceutical industries. The present study was aimed
to examine physicochemical properties, biological activities (as antioxidant and
antimicrobial) of oil extract from rosemary leaves. The oil extraction was done in Soxhelt
apparatus using hexane as a solvent. The result of physicochemical properties and
antioxidant activities of Rosmarinusofficinalis (L.) leaf oil presented oil yield (38.5%),
specific gravity (0.84), acid value (1.26 mg/g), free fatty acid (0.63%), peroxide value
(4.70%). The assessment of antioxidant activities of R.officinalis (L) leaf oil extract was
recorded high ascorbic acid content ( 20.43%), DPPH (6.75%), and hydrogen peroxide free
radical scavenging activity (1.50%). The diameter of inhibition zone for R. officinalis (L.) leaf
oil recorded the mean zone of inhibition at highest concentration (2µl/ml) against bacterial
test pathogens ranged from 10.50±0.71mm to 14.50±0.63mm, while 13.65±0.50 to
21.45±0.64 mm against fungal test pathogens. The strongest antibacterial activity with
maximum zone of inhibition (14.50mm) at highest concentration (2µl/ml) of the oil was
recorded againstE. coli. On the other hand, the strongest antifungal activity with maximum
zone of inhibition (21.45mm) was recorded against C. albicans. The oil from R. officinalis
has also exhibited strongest bactericidal activity with Minimum inhibitory concentration
(MIC, 0.06µl/ml) and the corresponding minimum bactericidal concentration (MBC, 0.12
µl/ml) against E. coli while the weakest bactericidal activity with MIC (0.75 µl/ml, the largest
value) and MBC (1.12 µl/ml) was recorded against S. pyogenes indicating that E. coli was the
most susceptiblet antifungal activity with MIC (0.03µl/ml, the least value) and minimum
fungicidal concentration (MFC, 0.05µl/ml) against C. albicans. The rosemary leaf oil extract
demonstrated remarkable potency against test pathogens. Thus promising as an alternative
medicinal therapy against the broad-spectrum drug resistant pathogens.

Keywords: Antimicrobial activity, Free radical scavenging activity, Hexane, Minimum

inhibitory concentration, Minimum bactericidal concentration.

xiv
 1. INTRODUCTION

Medicinal plants were used for centuries as remedies for human diseases because they contain
components of therapeutic value, there is an increasing interest in phytochemicals as new
sources of natural antioxidant and antimicrobial agents (Narayana et al., 2001).
Phytochemicals are naturally occurring compounds of plant kingdom, such as medicinal
plants, vegetables, fruits, that work with nutrients and fibbers to act against diseases or more
specifically, provides protection against diseases (Shah et al., 2011). Microbial infections
have become one of the major problems of public health in the world and the development of
resistance to the available antibiotics has lead researchers to investigate the antimicrobial
activity of medicinal plants (Demissew and Dange, 2001).

Essential oils have gained enormous interest as sources of natural products. They have been
screened for their potential uses as alternative remedies for the treatment of many infectious
diseases (Prabuseenivasanet al., 2006). RosmarinusofficinalisL. belongs to the Lamiaceae
family. This plant contains volatile oil, which has many potentialities in traditional medicine,
cosmetics and flavoring agent in foods. Rosemary essential oil antangonistic activity as
antibacterial effect for both Gram negative and positive bacteria, it's also antifungal,
antioxidant, antimutagenic and shows cytotoxic activity] (Hussain et al., 2010). Rosemary
essential oil also possesses analgesic, anti-inflammatory, antioxidative, anti-tumor, anti-
ulcerogenic and hepatoprotective properties (Minaiyanet al.,2011) and as anti cyanobacterial
(Najemet al., 2015).

Rosemary is a powerful herb belonging to the family Lamiaceae that originates from the
Mediterranean region. It is derived from the Latin word ros(dew) and marinus(sea) which
means „dew of the sea‟ (All about Rosemary, 2012). It has been named the Herb of the Year
in 2001 by the International Herb Association. Rosemary is regarded as the herb of
faithfulness as Elizabethan sweethearts carried a twig of rosemary as its sign. Today market
demand of the plant is growing, as it is used in several commercially available products.
Rosemary is composed of pine-like leaves, which is the heart of all medicinal and other
benefits that are derived from the use of its oil (Rosemary, 2012). The synonyms of the plant
include Garden Rosemary, Polar Plant, Compass-Weed and Compass Plant. It is known in
 2

several vernacular names like Alecrim, common rosemary, echterRosmarin, encensier, garden
rosemary, rosmariin, rosmarina, Rosmarin, rosmarini, rosmarino, rosemary, tresmarino
(Farnsworth, 2005; Kokateet al., 2010).

Rosmarinusofficinalis (L.) is an evergreen perennial shrub commonly known as rosemary, is

garnering considerable attention from the food and medical industries due to its wide array of
biological activities (Hussain et al., 2010). Rosemary has been used as a source of traditional
medicine for centuries, but its potential as a natural food preservative has only recently been
investigated (Orhanet al., 2008). Studies have shown that the strong antioxidant and
antimicrobial activities of the plant‟s extracts make rosemary an ideal replacement for more
toxic artificial food additives (Tavassoliet al., 2011). As concern over these potentially
harmful additives has grown, so has the demand for natural preservatives such as rosemary.
While rosemary is beginning to be used as a preservative around the globe, more can be done
to maximize the plant‟s potential. Additional research is needed to identify the extracts and
essential oils of rosemary that have the most potent preservative activities. Toxicity levels and
potential side effects of rosemary must also be constantly monitored. Assuming it remains
less toxic, R. officinalisshould continue to replace artificial additives in many preservative
applications, thereby reducing risk to consumers (Gachkaret al., 2007).

Rosmarinusofficinalishas also displayed many intriguing therapeutic properties. Rosemary‟s

antimicrobial, antidepressant, neuroprotective, anti-inflammatory, and antiobesity activities all
have potential in the treatment against various diseases. However, it is the anticancer and
antidiabetic mechanisms of rosemary that warrant the most attention. Oxidative stress has
been shown to contribute to the progression of diabetes mellitus and many forms of cancer
(Abdel et al., 2011). It is not surprising, then, that the strong antioxidant activity of rosemary
extract and essential oil represents a promising new strategy in the treatment of both diseases.
The plant‟s ability to promote insulin secretion in diabetic patients and induce apoptosis
within cancer cells is particularly intriguing. While these initial results have been
encouraging, more clinical studies are needed before rosemary can be regularly used in
humans. If it continues to display such promising antidiabetic and anticancer activities with
few side effects, R. officinaliscould eventually provide an innovative treatment in the fight
against these two serious diseases (Bulbul et al., 2012).
 3

Essential oils of plants have many applications in medicines, cosmetics, food stuff and
pharmaceutical industries (Chematet al., 2006). They are present in plants at low
concentration which would require high performance extraction techniques in order to achieve
high yield. Generally, essential oils are produced by different methods, including solvent
extraction, supercritical fluid extraction, hydro-distillation, steam distillation, use of
superheated steam and combinations of the previous techniques with others such as
ultrasound and microwave-assisted processes (Masango, 2005; Chematet al., 2006).

Rosemary leaves contain 1,2-cineole, α-pinene, apigenin, betulin, betulinic acid, caffeic acid,
camphor, carnosic acid, carnosol, carnosol isomer, methyl carnosate, cirsimaritin, diosmin,
hesperidin, limonene, luteolin 3'-O-beta-D-glucuronide, luteolin 3'-O-(3"-O-acetyl)-beta-D-
glucuronide, oleanolic acid, rosmadial, rosmanol, rosmarinic acid, scutellarein, thymol,
ursolic acid. A diterpene, rosmariquinone, has been isolated from a methanolic extract of
Rosmarinusofficinalis(Cantrell et al., 2005). The leaves contain 0.5 to 2.5 % of a volatile oil,
consisting of 0.8-6 % esters and 8-20 % free alcohols. The essential oil is a colourless or pale
yellow liquid with a camphoraceous taste and contains monoterpenes, phenols,
sesquiterpenes, monoterpenoid ethers, monoterpenoid ketones, monoterpernoid alcohols, and
monoterpenoid esters, camphor, eucalyptol, α-pinene, borneol. It contains 1,8-cineole (20–50
%), α-pinene (15-25 %), camphor (10-25 %), bornyl acetate (1-5 %), borneol (1-6 %),
camphene (5-10 %) and α-terpineol (12-24 %), limonene, β-pinene, β-caryophyllene and
myrcene (Chematet al., 2006).On account of such justification the present study was aimed to
examine physicochemical properties, biological activities (as antioxidant and antimicrobial)
of oil extract from rosemary leaves.

Objectives

General Objectives

 To study physicochemical properties, antioxidant and antimicrobial activities

of oil extract from rosemary leaves.
 4

Specific Objectives

 To determine oil yield of oil extracted from rosemary leaves;

 To assess physicochemical properties of the oil extract with respect to specific
gravity, acid value, free fatty acids, acid and peroxide values;
 To determine antioxidant activities of oil extracted from rosemary leaves;
 To evaluate antibacterial and antifungal activities with respect to diameter of zone
of inhibition, minimum inhibitory concentrations and minimum bactericidal and
fungicidal concentrations of the oil extracts.
 5

2. LITERATURE REVIEW

2.1. Major chemical constituents of Rosmarinusofficinalis (L)volatile oil

The chief constituents of rosemary oil are: camphor (5-31%), 1,8-cineol (15-55%), pinene (9-
26%), borneol (1.5-5.0%), camphene (2.5-12.0%), pinene (2.0--9.0%), limonene (1.5-5.0%),
verbenone (2.2-11.1%), caryophyllene (1.8-5.1%) and myrcene (0.9-4.5%) (Williams, 2009).
To 2.5% of essential oil, the chief constituents of which are camphor (5-21%), 1,8-cineole
(15-55%), pinene (9-26%), borneol (1.5-5.0%), 297 camphene (2.5-12.0%), pinene (2.0-
9.0%) and limonene (1.5- -5.0%). Phenolic compounds are represented by flavonoids with a
methylated aglycone (e.g. genkwanin) and by phenolic acids (>3%), particularly by
rosmarinic, chlororenic and caffeic acids. Also present are tricyclic diterpenes such as
rossmaridiphenol, carnosol, carnosic acid and rosmanol, and diterpenes, including seco-
hinokio (Cantrell et al., 2005).

2.2. Medicinal uses

It is used as carminative, rubifacient, stimulant and as flavouring agent for liniments, hair
lotions, inhaler, soaps and cosmetics (Kokateet al., 2010). Rosemary leaves have many
traditional uses based on their antibacterial and spasmolytic actions. Used orally for the
treatment of dyspeptic complaints (British Herbal, 1996), and in external applications for
supportive management of rheumatic complaints and circulatory disorders (Blumenthal,
1998). AetheroleumRosmarinicrude drug may enhance cognition. It is used as a
cholagogue,diaphoretic, digestant, diuretic, esmmenagogue, laxative and tonic (Farnsworth,
2005) also used in the management of headache, menstrual disorders, nervous menstrual
complaints, tiredness, defective memory, sprains and bruises (Hagers, 2003).

2.2.1. Brain and nervous system conditions

In general debility long-term nervous or physical illness, improves the memory, insomnia,
mental fatigue, nervous anxiety and tension, nervous depression, nervous disorders,
restorative effect on the nervous system, soothes the nerves, stimulates the brain and
nervoussystem, tension headaches, and migraines (Farnsworth, 2005). It improves circulation,
raises blood pressure, and stimulates the weak heart subject to palpitation when consumed in
small doses. In conditions of bad breath, and stomach upset. Promotes proper digestion,
 6

toning and calming effect on the digestion, dropsy, regulates the menstrual cycle, promotes
liver function, promotes the production of bile, stimulates the sexual organs, treatment of
colds, & colic, eases cramps, expels morbid matter from the system, failing eyesight,
headache. Externally, it is used to treat bites, stings. In aromatherapy the essential oil is used
as a decongestant, as an inhalant, for exhaustion, for headaches, to enhance memory and clear
concentration (Del Banoet al., 2003).

The oil is usedin oils/lotions for Arthritis, bruises, eczema, gout, muscular pain, neuralgeia,
revitalizing paralyzed limbs, rheumatism, rheumatoid arthritis, sciatica, scrofulous sores,
wounds and rubbed into hair for stimulating the hair bulbs to renewed activity and to prevent
premature baldness, as perfume in ointments, shampoos and soaps. The flowers are laid in
clothes and cupboards to destroy moths. The leaves are crushed into meats, fish, potato salads,
etc. to prevent food poisoning (Makino et al., 2002).

2.3. Pharmacological activities

The plant is scientifically proved to possess antiinflammatory activity (Lo, 2002), antioxidant
activity (Del Banoet al., 2003), antihepatotoxic activity (Fahimet al., 1999), antinephrotoxic
activity (Makino et al., 2002), antimicrobial activity (Mangena and Muyima, 1999),
antitrypanosomal activity (Abe et al., 2002), antitumour activity (Singletary and Nelshoppen
1991), antiulcer activity (Dias et al., 2000), diuretic effects (Halouiet al., 2007),
antispasmodic effects (Lis-Balchin, 1996), osteoclastic effects (Muhlbaueret al., 2003),
enzyme induction (Debersacet al., 2001), estrogenic effects (Zhu et al., 1998), immune
stimulant activity (Huret al., 2004), carcinogenesis, mutagenesis, impairment of fertility
(Alkofahiet al., 1997).

2.3.1. Toxicology

The embryotoxic effects of d-camphor were investigated in rats and rabbits after intragastric
administration for the treatment of hypotonic circulatory dysregulations (Leuschner, 1997).
 7

2.3.2. Clinical pharmacology

A clinical study to assess the olfactory impact of the essential oils of lavender
(Lavandulaangustifolia) and rosemary (Rosmarinusofficinalis) on cognitive performance and
mood in healthy volunteers was performed (Sanders et al., 2002).

2.3.3. Antioxidant activity

A mixture of α-tocopherol and rosemary extract, as additives each at 0.035% (total 0.07%)
expressed very strong antioxidant activity in sardine oil stored at 300C, 500C and dried sardine
meat at 5oC (Wada and Fang, 1994). Four diterpenoids (carnosic acid, rosmanol, camosol and
epirosmanol) isolated from the leaves of Rosmarinusofficinalis by bioassay-directed fraction,
inhibited superoxide anion production in the xanthine/xanthine oxidase system, showing to be
protective against oxidative stresses (Haraguchiet al., 1995). Inhibition of the growth of six
strains of food associated bacteria and yeasts by carnosol and ursolic acid, was achieved at
concentrations of 150µg/ml for carnosol to the greatest extent. Butylated hydroxyanisole
(BHA) proved a superior inhibitor to ursolic acid which itself was more effective than
butylated hydroxytolune (BHT) (Collins and Charles, 1987).A study on the variability of
rosemary and sage and their volatile oils on the British market has been performed. The
antioxidative properties of the various samples were determined and found to be variable
based on the geographical location and type of processing (Svoboda, 1992).

The concentration of phenolic diterpenes in commercially available extracts of

Rosmarinusofficinalisdetermined by HPLC with electrochemical detection ranged from 2.8 to
22.5 %. Antioxidant activity of extracts under simultaneous storage and thermal stress
depended directly on the concentration of phenolic diterpenes. Differences in rates of
degradation of individual phenolic diterpenes at different temperatures were obtained
(Schwarz et al., 1992).
Tateo et al. (1988) made the comparison of the antioxidant power between two dry rosemary
extracts obtained by a simplified extraction process, a commercial rosemary extract and BHA,
and an evaluation of the mutagenic effect of four different dry rosemary extracts. Four
conclusions were reached by the authors: the antioxidant activity of the extracts was
comparable; the extraction treatment by supercritical CO2, which is as efficient for
 8

deodorizing as the traditional method of steam flow distillation, gives an antioxidant product
with an activity comparable to the product deoleated by steam flow distillation; the
antioxidant activity of rosemary extracts in general is less evident in regards to soy oil, even at
considerably higher concentrations than those active in solid fat; the antimutagenic activity is
higher for the rosemary extract obtained by hydroalcoholic extraction (ethyl alcohol 50 %
v/v).
Various experimental models were used for the characterisation of the antioxidant activity of
fourcommonly consumed herbs belonging to the Lamiaceae family, i.e. oregano (Origanum
vulgaris L).rosemary (RosmarinusofficinalisL.), sage (Salvia officinalisL.) and thyme
(Thymus vulgaris L.),including iron reduction capacity, 2.2-diphenyl-1-picrylhydrazyl DPPH_
, ABTS_+ and _OH radicalscavenging activities and the capacity of the extracts to inhibit
copper-induced oxidation of human low- density lipoproteins (LDL) ex vivo. The extracts
showed varying degrees of reductive and radicalscavenging capacity, and were capable of a
marked prolongation of the lag-time in the LDL oxidation assay. The hierarchy of the
observed antioxidant activity of the extracts was dependent on the type of assay used. The
observed antioxidant characteristics were not fully related to the total phenoliccontents of the
extracts in any of the assays, but were presumably strongly dependent on rosmarinicacid, the
major phenolic component present in this type of Lamiaceae extract (Dorman et al., 2003).

The DPPH radical scavenging method, Folin–Ciocaulteu method and HPLC chromatography
were usedto study the distribution and levels of antioxidants (AOXs). A good correlation
between the AOXactivities and total phenol content in the extracts was found. All rosemary
extracts showed a highradical scavenging activity (Moreno et al., 2006).On other study,
Kuhlmann et al. (2006) were able to demonstrate that the DPPH radical-scavengingactivity of
the extracts of rosemary depended on the amount of all three phytochemicals, carnosic
acidcarnosol, and rosmarinic acid, in particular the last one. The anti-inflammatory character
of theextracts was mainly based on their carnosic acid content.

R. officinalisL. essential oil showed greater activity than three of its main components (1,8-
cineole, α-pinene, β-pinene) by means of DPPH assay and β-carotene bleaching test. The
antioxidant activities ofall the tested samples were mostly related to their concentrations
(Wang et al., 2008).The results of a study performed with methanolic extracts from the leaves
 9

of Rosmarinusofficinalisharvested from different locations of Turkey at four different times of

the year, were analyzed byHPLC, and their radical scavenging capacities and antioxidant
activities were studied by various assays.The results indicated that the plants harvested in
September possess higher levels of active constituents and had superior antioxidant capacities
compared to those collected at other times (YesilcCeliktas et al., 2007).

2.3.4. Antimicrobial activity

Rosmarinusofficinalishas demonstrated potent antibacterial and antifungal activities in

multiple studies. Like its antioxidant activity, the antimicrobial activity of rosemary depends
on the chemical composition of its essential oil, which can vary greatly depending on
location, climate, and time of harvest. The antimicrobial activity of an oil is also determined
by the interactions between its components (Jordánet al., 2013). Rosemary has been shown to
inhibit the growth of bacteria such as Escherichia coli, Listeria monocytogenes, and
Staphylococcus aureus (Marinas et al., 2012). The significance of rosemary‟s antibacterial
effect does not end there, However, according to one study, rosemary has the potential to
inhibit the drug resistance of some bacteria by overcoming and reducing the impermeability
of these bacteria‟s membranes (Oluwatuyiet al., 2004). This represents an innovative strategy
for containing and eliminating resistant strains of bacteria. Rosemary essential oil can also
increase the susceptibility of certain bacteria to standard antibiotics (Marinas et al., 2012).
This impressive antibacterial activity makes R. officinalisa strong defense against common
food pathogens and a promising new preservative that could replace artificial additives
(Tavassoliet al., 2011).

In addition to its antibacterial properties, Rosmarinusofficinalishas several antifungal

mechanisms. The plant‟s essential oil has been shown to inhibit the adhesion of Candida
albicansby denaturing cellular structures and altering membrane permeability (Cavalcantiet
al., 2011). According to one study, rosemary can even prevent the development of highly
resistant fungal biofilms. By coating nanoparticles with rosemary essential oil, a
nanobiosystem was produced that significantly inhibited the adherence and biofilm
development of Candida fungal strains (Chifiriuc et al., 2012). Both these new strategies are
necessary alternatives to traditional medicine in the treatment against drug-resistant fungi.
 10

The ability to inhibit the growth and aflatoxin production of many fungi contributes to
rosemary‟s potential as an effective food preservative (Rasooliet al., 2008).

2.3.5. Anticancer activity

Many studies have reported on the anticancer mechanisms of Rosmarinusofficinalis.

Rosemary has displayed significant antiproliferative activities against several human cancer
cell lines. Major compounds in the plant‟s extract, such as carnosic acid, carnosol, and
rosemarinic acid, have been shown to induce apoptosis within these cancer cells, possibly
through the production of nitric oxide (Dilaset al., 2012). Carnosic acid Rosemary extract also
has intriguing antitumorigenic activity. In one study, the extract was found to strongly inhibit
skin tumorigenesis in mice by preventing carcinogens from binding to epidermal DNA. This
anticarcinogenic effect is caused by the extract‟s antioxidant activity (Al-Sereitiaet al., 1999).

2.3.6. Antidiabetic activity

Diabetes mellitus is a growing worldwide disorder. By 2025, an estimated 300 million people
will be diabetic, and global costs of treating the disease could reach U.S. $1 trillion annually
(Bakirelet al., 2008). The development of diabetes is often fostered by high oxidative stress;
pancreatic β-cells are especially vulnerable to reactive oxygen species, leading to decreased
insulin secretion and higher blood glucose levels. This information has prompted new
diabetes treatments to focus on natural antioxidants, particularly those found in plants. Not
surprisingly, multiple studies have identified Rosmarinusofficinalisas a promising antidiabetic
agent. Rosemary‟s antioxidant properties execute several antidiabetic and hypoglycemic
mechanisms. In one study, rosemary extract lowered blood glucose levels in normoglycemic,
hyperglycemic, and diabetic rabbits. By inhibiting lipid peroxidation and activating
antioxidant enzymes, the extract also promoted insulin secretion (Bakirelet al., 2008).
Rosemary was also found to alleviate delayed wound healing, a serious complication of
diabetes (Abu-Al-Basal, 2010). These antidiabetic activities are attributable to the body‟s
improved antioxidant status after administration of rosemary (Khalil et al., 2012).

2.3.7. Antidepressant activity

The potential use of Rosmarinusofficinalisas an antidepressant was the focus of many

research articles reviewed for this project. The majority of these studies involved two tests
 11

that are used to model antidepressant-like effects in mice–the tail suspension test (TST) and
forced swimming test (FST). The administration of rosemary continuously decreased the
immobility time of mice in both the TST and the FST, indicating an antidepressant-like effect
(Machado et al., 2013). Rosemary‟s antidepressant potential was further bolstered when it
was found to decrease exploratory and anhedonic-like behavior in bulbectomized mice
(Machado et al., 2012). There is much evidence that the antidepressant activity of R.
officinalisdepends on interactions with the monoaminergic system. Rosemary is believed to
enhance dopaminergic, serotonergic, noradrenergic, and cholinergic functions within the
brain, possibly explaining its antidepressant effects. Rosemary has also been found to increase
the concentration of neurotransmitters in the brains of mice. Several compounds in rosemary
extract and essential oil are responsible for its antidepressant activity, including carnosol,
betulinic acid, ursolic acid, and polyphenols (Machado et al., 2013).

2.3.8. Neuroprotective activity

Remarkably, Rosmarinusofficinalishas demonstrated significant neuroprotective effects

against neurodegenerative diseases such as Alzheimer‟s disease and dementia. Rosemary has
displayed inhibitory activities against the two enzymes in the brain responsible for the
breakdown of acetylcholine –cholinesterase (AChE) and butyrylcholinesterase (BChE). These

anti-AChE and anti-BChE activities are likely caused by rosemarinic acid and terpene
compounds in the plant‟s essential oil (Adewusiet al., 2010). By increasing total choline
levels in the brain, rosemary could attenuate not just Alzheimer‟s disease, but also memory
loss, anxiety, and depression (Ozarowskiet al., 2013). Two more studies highlight the
neuroprotective ability of R. officinalis. In the first, polyphenols present in rosemary extract
were found to inhibit stress proteins, which play a role in the neurodegenerative process
Omriet al., 2012). The second study concluded that rosemary promotes the production of
nerve growth factor (NGF), a protein vital to the growth and maintenance of nerve tissue.
Increased NGF levels can help alleviate Alzheimer‟s disease, dementia, and other
neurodegenerative diseases. Both these studies clearly demonstrate rosemary‟s growing
potential as a neuroprotective agent (Omriet al., 2010).
 12

2.3.9. Anti-inflammatory activity

Rosmarinusofficinalisdisplayed potent anti-inflammatory mechanisms in several of the

reviewed studies. Rosemary essential oil and extract were found to significantly inhibit
leukocyte migration in vivo (De Meloet al., 2011). This reduced the number of leukocytes
(white blood cells) at the site of inflammation, resulting in an anti-inflammatory response
(Mengoniet al., 2011). Rosemary extract also inhibited other pro-inflammatory substances,
such as nitric oxide and inflammation-associated genes. While carnosol and carnosic acid
appear to be particularly important, the anti-inflammatory activity of rosemary most likely
depends on a synergistic mechanism between many of its components (Yu et al., 2013).
These studies suggest that the antiinflammatory effect of R. officinalisis rather strong; in fact,
the anti-inflammatory activities of pure carnosol and carnosic acid were found to be nine
times higher than that of indomethacin, a common anti-inflammatory drug (Mengoniet al.,
2011).

2.3.10. Anti-obesity activity

While only three studies reported anti-obesity activities of Rosmarinusofficinalis, their

findings are very noteworthy. All three found rosemary to effectively limit weight gain, but
each study identified a different mechanism to explain this response. In one study, extracted
carnosic acid was found to suppress adipocyte differentiation. This inhibition of adipogenesis
can promote sustainable weight loss (Gaya et al., 2013). In another study, rosemary extract
prevented weight gain by limiting lipid absorption in the intestine. This was made possible
through the inhibition of pancreatic lipase activity (Ibarra et al., 2011). Finally, the third study
found rosemary extract to inhibit lipid synthesis through the suppression of diacylglycerol
acyltransferase (DGAT), the main enzyme responsible for the production of triglycerides. The
results of all three studies indicate that R. officinalishas great potential as an effective
treatment against obesity and other metabolic disorders (Cui et al., 2012).

2.3.11. Adverse Reactions and Contra Indications of Rosemary Oil

Inhalation can occasionally cause irritation and very rarely laryngospasm (Blumenthal, 1998).
External use may worsen bronchospasm. Rarely hypersensitivity reactions of the skin may
occur. Photoaggravated allergic contact dermatitis and cheilitis have been reported.
 13

AetheroleumRosmarini(Armisenet al., 2003). AetheroleumRosmariniis contraindicated in

cases of hypersensitivity or allergy to the plant material (Blumenthal et al., 2000). It should
not be used in patients suffering from bronchial asthma or bronchitis or on damaged skin,
such as in cases of burns, lesions or skin rashes (Cui et al., 2012).
 14

3. MATERIALS AND METHODS

3.1. Description of study area

The study was conducted at Haramaya University main campus, which is located at about 510
km East of Addis Ababa, between Dire Dawa and Harar cities. The University is located at
09.00N and 42.00E and at an altitude of 1950 meters above sea level. Oil extraction was
carried out in Animal Nutrition Laboratory, while oil quality analysis was carried out in
Biotechnology laboratory under the School of Biological Sciences and Biotechnology,
Haramaya University.

3.2. Plant Material and Extract Preparation

The rosemary plant sample was collected from Home garden, Debreberhan City, Ethiopia.
The leaf sample was manually washed with distilled water and residual moisture was
evaporated at room temperature. Thereafter, the leaf samples were ground in a grinder for 2
min, the process was stopped for 15 sec to avoid heating of sample using the standard
methods of the Association of the Analytical Chemists (AOAC, 2000). Hexane was used as a
solvent for oil extraction.

3.3. ResearchDesign

The oil extraction was done in Soxheltapparatus using hexane as a solvent. Then,
physicochemical properties of the oil extract were done based on determination of oil content,
specific gravity, acid value, percent free fatty acid and peroxide value. The antioxidant
activities were investigated based on free radical scavenging activities of DPPH and hydrogen
peroxide. The antimicrobial experiment was arranged as 1x1x7 [1 source extracts (oil
extractfrom leaves ofat three concentration levels), 1 solvent system i.e hexane, 7 test
organisms (4 bacteria: Escherichia coli,Staphylococcus aureus, Salmonella typhi, and
Streptococcus pyogenes; three fungi (Aspergillus versicolor, A. Niger, Candida albicans)]
completely randomized factorial design in two replications. The antimicrobial activities using
disc diffusion method and broth dilution method. In addition, the least concentration of extract
that show antimicrobial activity was selected for further determining the minimum inhibitory
 15

concentration (MIC): minimum bactericidal (MBC) and minimum fungicidal concentrations

(MFC).

3.4. Oil Extraction

The Extraction of the oil was done using solvent extraction method in soxhelt apparatus using
hexane as a solvent. For this purpose two flasks were kept in hot air oven for 2 hours. Then
the flask were kept in desiccators for 30 min to cool at room temperature. 20gm of powdered
leaf samples were dissolved in 120ml hexane solution and kept in Soxhlet apparatus for 8
hours. The crude extract was concentrated in water bath by adding sodium sulfate.

3.5. Data collection

Data were recorded for oil content, specific gravity, acid value, free fatty acid value, peroxide
value, DPPH and hydrogen peroxide scavenging activities, ascorbic acid(vitamin C), and
antimicrobial activities including zone of inhibitions, MIC, MBC and MFC values.

3.5.1. Determination of oil yield and specific gravity

The % oil yield of the sample was determined as:

oil weight (OW )

Oil yield = sample x 100
weight (SW )

Where, oil weight= W2-W1: W1= Weight of the extraction flask (g); W2=Weight of the
extraction flask plus the dried crude fat (g).

Specific gravity: the specific gravity of the oil was determined gravimetrically by employing
the weight ratio of the oil to the equivalent amount of water according to the following
𝐖𝟐
formula: Specific gravity =
𝐖𝟏

Where, W2 and W1 are the weights of oil and equivalent amount of water respectively.

3.5.2. Determination of Acid Value

The acid value was determined as per AOAC (1990) method. Briefly 2g of oil sample was
weighed into a 250ml conical flask and then, 25ml diethyl ether mixed with 25ml alcohol and
1ml of 1% phenolphthalein indicator were added to the oil sample. The conical flask was then
 16

placed on a hot water bath until the oil was completely dissolved in the solvent. The hot
solution was then titrated with 0.1M KOH until a pink colour which persisted for 15 seconds
was noticed. The acid value was calculated as:

Ti tre ml x 5.61
Acid value = Weight of sample used

Acid value expressed as Acid value (mg KOH /g of oil).

3.5.3. Estimation of Free Fatty Acid

The percentage free fatty acid (%FFA) was estimated by multiplying the acid value with the
factor 0.503. The %FFA = 0.503×Acid value.

3.5.4. Determination of Peroxide Value

To a weighed sample (1.0g) in a flask, powdered potassium iodide (1.0g) and solvent mixture
(2: 1, glacial acetic acid:chloroform v/v) were added. The resulting solution was then placed
on a water-bath to dissolve properly and 5% potassium iodide (20cm3) was then added. The
sample solution was then titrated with 0.002N sodium thiosulphate using starch as an
indicator. The peroxide value of the samples was calculated using equation (Nielsen, 2002).

PV = 2 X V

Where: PV = Peroxide value, V = Volume of Sodium thiosulphate used, 2 = (N x 1000) / W,

N = Normality of Sodium thiosulphate used, W = Weight of sample used.

3.5.5. DPPH Radical Scavenging Activity

The radical scavenging activity (RSA) of the oil extract is used to measure antioxidant
activity using the DPPH (2, 2- diphenyl-1-picrylhydrazyl) method(Loizzoet al., 2016).
Briefly, 2 mL of oil extract (100µg/ml) was added to 2mL of DPPH (0.1 mM) solution. The
mixtures was kept aside in a dark area for 30 min and absorbance was measured at λmax 517
nm against an equivalent amount of DPPH and methanol as a standard. The percentage of
DPPH radical scavenging activity (RSA %) was estimated using the equation:
 17

Acontrol − Asample
DPPH radical scanveging activity % = x100
Acontrol

where𝐀 𝐜𝐨𝐧𝐭𝐫𝐨𝐥 is the absorbance of the standard solution and 𝐀 𝐬𝐚𝐦𝐩𝐥𝐞 is the absorbance in the
presence of the sample.

3.5.6. Hydrogen Peroxide Scavenging Activity

The radical scavenging activity of the oil extract was determined using the H 2O2 method
(Bozinet al., 2008). Briefly, 2 mL of oil extract solution (100µg/mL) was added to 4.0 mL of
H2O2 (20 mM) solution in phosphate buffer (pH 7.4). After 10 min, the absorbance was
measured at λmax 230 nm against the phosphate buffer control solution. The percentage
scavenging of H2O2 was calculated using the equation:

(A control −A sample )
% scavenging of H2O2 = x100
A control

Where Acontrol = absorbance of the blank (phosphate buffer with H2O2) and Asample =
absorbance of the oil sample.

3.5.7. Ascorbic Acid Analysis

The ascorbic acid content was determined using the 2, 6- dichlorophenol indophenol (DCPIP)
dye method (AOAC, 2000). Accordingly, 5ml of the standard ascorbic acid solution was
pipetted into a 100 ml conical flask and then 5ml of the 3% HPO3 solution was added. The
ascorbic acid solution was titrated with the dye solution to a pink colour, that persisted for
15sec. The titre value was recorded. Dye factor was expressed as mg of ascorbic acid per ml
of the dye. Since 5ml of the standard ascorbic acid solution contains 0.5 mg ascorbic acid:

0.5mg
Dye factor (mg ascorbic acid per dye) = titrant volume

One ml of the extracted oil was diluted to 5ml with 3% metaphosphoric acid in a 50 ml
volumetric flask. The aliquot was then centrifuged (Model, Z300, 580W, 3052 Nm, German)
for 15 minutes and titrated with the standard dye to a pink end point (persisting for 15
seconds). The ascorbic acid content was calculated from the titration value, dye factor,
dilution and volume of the sample as:
 18

ABRsample x dye factor x volume of initial test solution

% A.A = x 100%
volume of test solution titrated

Where: A.A=Ascorbic Acid; ABR= Average Burette reading

3.6. Antimicrobial activity of the oil extracts

The experiment was arranged as 1 x1x7 factorial designs (i.e. one solvent, one oil extracts,
and 7 test pathogenic microbes (including 4 bacterial and 3 fungal pathogens) in three
replications. A completely randomized design (CRD) was used to determine the antimicrobial
activities using disc diffusion and broth dilution methods. In addition, the least concentration
of oil extract that show antimicrobial activity was selected for further determination of the
minimum inhibitory concentration (MIC): minimum bactericidal concentration (MBC) and
minimum fungicidal concentrations (MFC).

3.6.1. Test Pathogens

Seven test pathogens including four bacteria: Escherichia coli,Staphylococcus aureus,

Salmonella typhi, and Streptococcus pyogenes; three fungi (Aspergillus versicolor, A. Niger
and Candida albicans) were obtained from Ethiopian Food and Health Institution. The fungal
and bacterial pathogens were subcultured and maintained on Potato Dextrose Agar (PDA) and
Nutrient Agar, respectively. Then, the fungal and bacterial cultures were incubated for 72 h at
27 ºC and for 18-24 h at 37 ºC, respectively.

3.6.2. Media Preparation and Standardization of Inoculum

Nutrient Agar (NA), Potato Dextrose Agar (PDA), and Muller Hinton agar (MHA) were used
for sub-culturing of bacterial test organism, fungal test organism, and determination of
antimicrobial activities, respectively. These media were prepared and sterilized using an
autoclave according to the manufacturers‟ instructions. Two to three bacterial colonies on the
plate were picked up with a sterile inoculating loop and transferred into a test tube containing
sterile normal saline and vortexes thoroughly. The spores of the test fungi were harvested by
washing the surface of the fungal colony using 5mL of sterile saline solution. This procedure
repeated until the turbidity of each bacterial and fungal spore suspension matched the
turbidity of 0.5 McFarland Standards as described by the Clinical Laboratory Standards
 19

Institute (CLSI, 2015). The resulting suspension was used as inoculums for the pathogens in
the antimicrobial susceptibility test.

3.6.3. Disc diffusion Method

Discs of 6 mm diameter was prepared from sterile filter paper cut into small, circular pieces of
equal size by a perforator and then impregnated each of them with 0.1 ml of the prepared test
extract. The extract impregnated discs were placed onto MHA plates evenly inoculated with
test pathogens (Hudzicki, 2009).

3.6.4. Inoculation of Mueller Hinton Agar (MHA) plates

After adjusting the turbidity of the suspension of inoculums within 15 minutes, a sterile cotton
swab was dipped into adjusted suspension and rotated several times by pressing firmly on the
inside wall of the tube above the fluid level. This removes excess fluid from the swab. Then,
the dried surface of Mueller Hinton Agar plates were inoculated by streaking using the swab
three times over the entire surface and rotating the MHA plates approximately 60° each time
to ensure an even distribution of the inoculum. Then, the MHA plates were left open for three
to five minutes to allow for any excess surface moisture to be absorbed (CLSI, 2012).

Following this step, the impregnated discs were dispensed onto the surface of the inoculated
agar plates using sterile forceps. Each disc was pressed down to ensure complete contact with
the agar surface. The discs were distributed evenly so they were not closer than 24 mm from
center to center (CLSI, 2015). Discs of commercial gentamycin (1µl/disc) and fulconazole
(1µl/disc) were used as positive controls for bacterial and fungal pathogens, respectively and
the pure solvent (hexane) impregnated discs were used as negative controls.

Then the MHA plates were sealed with parafilm and incubated at 37°C for 24 hrs and 27°C
for 72 hrs for bacterial and fungal pathogens, respectively. After incubation, the diameters of
the zone of inhibition around each disc were measured to the nearest millimeter along two
axes (i.e. 90° to each other) using a transparent ruler and the means of the two readings were
be recorded. For each selected pathogen the experiment was carried out in three replications.
 20

3.6.5. Determination of Minimum Inhibitory Concentration (MIC)

The oil extracts that showed significant antimicrobial activity in the antimicrobial activity
tests were selected for determination of MIC based on the method used by Morshedet al
(2012) with slight modifications. The MIC of the oil extracts of rosemary leaf was determined
by broth dilution method. In the broth dilution method, the oil extract solution for example at
1µl/ml was serially diluted in a two-fold dilution as 1 µl/ml, 0.50 µl/ml, and 0.25 µl/ml, 0.125
µl/ml, 0.0625 µl/ml concentrations. Two mililiter of nutrient broth and potato dextrose broth
for bacteria and fungi respectively were added into all test tubes and 0. 1 ml of the prepared
concentration of each oil extract was mixed with the nutrient broth and potato dextrose.
Thereafter, standardized inoculums of 0.1 ml of the respective test pathogens were dispensed
into the test tubes containing the suspensions of the broth and the oil extract. Then, all test
tubes were properly corked and incubated at 37°C for 24 hrs for bacteria and 27°C for 72 hrs
for fungi. After that, they were observed for absence or presence of visible growth. The
lowest concentration at which no visible growth of organisms were regarded as the MIC. The
experiment was carried out for each test organism in triplicates.

3.6.6. Determination of minimum bactericidal (MBC) and fungicidal concentrations

(MFC)

For the determination of the MBC and MFC, fresh nutrient agar and potato dextrose agar
plates were inoculated with one loop full of culture taken from each of the broth cultures that
showed no growth in the MIC tubes. That is MBC/MFC values were determined by
subculturing from respective MIC values if for example MIC=0.50 µl/ml (v/v) subculturing
was performed as 0.50 µl/ml, 1.00 µl/ml, 1.50 µl/ml, 2.00 µl/ml up to four acceptable
concentration levels. Since antibacterial agents are usually regarded as bactericidal if the
MBC is no more than four times the MIC (CLSI, 2012). MBC/MFC is the amount of the
extract that kills microbial growth. While MBC assay plates were incubated for 48 h, MFC
assay plates were incubated for 3 days. After the incubation periods, the lowest concentration
of the extract that did not allow any bacterial or fungal growth on solid medium was regarded
as MBC and MFC for the extract (CLSI, 2015). This observation was matched with the MIC
test tube that did not show evidence of growth after 48 h of incubation for bacteria or spore
germination after 3 days of incubation for fungi. Fulconazole (1µl/disc) disc was applied as
 21

positive control for incubation of fungi while gentamycin (1µl/disc) was served as positive
control for bacterial pathogens.

3.7. Data Analysis

All data were entered into Microsoft excel. Mean comparison and Analysis of variance
(ANOVA) was carried out using SAS version 20 software package. Statistically significant
differences was indicated by p<0.05.
 22

4. RESULTS AND DISCUSSION

4.1. Physicochemical properties of Rosemary (RosmarinusofficinalisL.) leaf oil

The physicochemical properties and antioxidant activities of Rosmarinusofficinalis (L.)leaf oil

as in Table 1.The physicochemical properties presented oil yield (38.5%), specific gravity
(0.84), acid value (1.26 mg/g), free fatty acid (0.63%), peroxide value (4.70). Similar study
was conducted byKokateet al (2010) who suggested thatrosemary oil is colourless to pale
yellow with characteristic flavour and camphoraceous taste. The specific gravity is 0.894-
0.912, refractive index 1.464-1.476 and has an optical rotation of 5-10°. The oil is insoluble in
water, soluble in 10 volumes of 80% of alcohol. The acid value is not more than 1.0.Acid
Value (ACV) and Free Fatty Acid (FFA) are analytically used to detect the level of
unesterified fatty acid in a lipid sample to define its quality. Peroxide value (PV) measures the
degree of lipid oxidation in fats and oils but not their stability. PV measures a transient
(temporary) product of oxidation. A low value may represent early or advanced oxidation;
which can be distinguished with time. The low peroxide value indicates the absence of
rancidity in the oil samples (Abdulkadir and Jimoh, 2013).

Table 1. Physicochemical properties of solvent extracted Rosemary (RosmarinusofficinalisL.)

leaf oil
Parameters Values (%)

Oil yield 38.5

Specific gravity 0.84

Acid value mg/OH/g 1.26

Free fatty acid (as oleic acid) 0.63

Peroxide value mg/kg 4.70

DPPH 6.75

Hydrogen peroxide free radical scavenging activity 1.50

Ascorbic acid 20.43

 23

4.2. Antioxidant activities of Rosmarinusofficinalis(L.)of leaf oil extract

In the present study the antioxidant activities were assessed based on ascorbic acid content,
DPPH, and hydrogen peroxide free radical scavenging activities (Table 1). The
Rosmarinusofficinalis (L)leaf oil extract was recorded high ascorbic acid content ( 20.43%),
DPPH (6.75%), and hydrogen peroxide free radical scavenging activity (1.50%). As the
concentration of phenolic compounds or degree of hydroxylation of the phenolic compounds
increases, their DPPH radical scavenging activity also increases and can be defined as
antioxidant activity (Mohdalyet al., 2011). The oxidative damage to cellular components is
believed to be associated with the development of degenerative diseases including
cardiovascular disorders, cancer, arthritis, immune system suppression, brain dysfunction,
cataract, and others (Lee et al., 2004). The therapeutic effect of many plants and herbs is
caused by the presence of antioxidant constituents such as flavonoids (Rice-Evans, 2004),
phenolic acids, and phenolic diterpenes (Shahidi&Wanasundara, 1992).

A good correlation between the antioxidant activities and total phenol content in the extracts
was found. All rosemary extracts showed a highradical scavenging activity (Moreno et al.,
2006). On other study, Kuhlmann et al. (2006) were able to demonstrate that the DPPH
radical-scavenging activity of the extracts of rosemary depended on the amount of all three
phytochemicals, carnosicacidcarnosol, and rosmarinic acid, in particular the last one. The
anti-inflammatory character of the extracts was mainly based on their carnosic acid content.

The yield chemical composition and quality oils in general, varies with the origin of the
vegetal material used, cultivation conditions, environmental conditions and the obtaining
method, among other factors. For fatty acids, the acid value, in conjunction with the
saponification value, can be used to give a measure of the amount of neutral fat present. The
free fatty acids (FFA) is normally expressed as oleic acids. The exceptions are coconut and
palm kernel oils, which are calculated as lauric acids. Where the FFA is expressed as oleic
acids, the acid value, for all the intents and purposes, is double the FFA value (Milwidsky and
Gabriel, 1982).
 24

Unlike most of its biological activities, rosemary‟s antioxidant activity is directly attributable
to chemical compounds in the plant‟s essential oils and extracts. While synergistic
mechanisms between many oil components likely contribute to the antioxidant activity,
phenolic diterpenes such as carnosic acid, carnosol, and rosemarinic acid have been identified
as the strongest antioxidants present in rosemary essential oil (Zaoualiet al., 2010).

Rosmarinusofficinalisexerts its antioxidant effect through several metabolic pathways. For

example, rosemary essential oil and extract have been shown to destroy and prevent free
radicals (Abdel et al., 2011). Rosemary is also capable of preventing lipid peroxidation, a
destructive process that is caused by oxidative stress (Bulbul et al., 2012). In addition to
reducing the amount of reactive species in the body, rosemary has been found to increase the
activity of antioxidant enzymes (Afonsoet al., 2013). All these effects augment the body‟s
defense against harmful reactive species and oxidative damage. Oxidative stress contributes to
the development of many diseases. A lack of antioxidant defenses and the resulting oxidative
damage have been shown to cause both diabetes and cancer (Dilaset al., 2012). By limiting
oxidative stress in the body, rosemary helps prevent diseases such as these that depend on the
accumulation of free radicals and other reactive species. Therefore, the strong antioxidant
activity of R. officinalisis vital to its therapeutic potential. Indeed, its antioxidant activity is
fundamental to nearly all of rosemary‟s other therapeutic applications (Al-Sereitia et al.,
1999).

4.3. Antimicrobial Activities of Rosemary (RosmarinusofficinalisL.) leaf oil

The diameter of inhibition zone, measured based on disc diffusion, for

Rosmarinusofficinalis(L.) leafoil as in Table 2. Significant antimicrobial activities were
obtained for R. offcinalis leaf oil extracts against tested bacteria and fungi. The mean zone of
inhibition at highest concentration (2µl/ml) against bacterial test pathogens ranged
from10.50±0.71mm to 14.50±0.63mm, while 13.65±0.50 to 21.45±0.64 mm against fungal
test pathogens. The strongest antibacterial activity with maximum zone of inhibition
(14.50mm) at highest concentration (2µl/ml) of the oil was recorded againstE. coli while the
weakest antibacterial activity (10.50mm) was observed against S. aureus indicating that E.
coli was the most susceptible while S. aureus was the least susceptible. On the other hand, the
 25

strongest antifungal activity with maximum zone of inhibition (21.45mm) was recorded
against C. albicans;However,the weakest antifungal activity with minimum zone of inhibition
(13.65mm) was observed against A. nigersuggestingR. offcinalis leaf oil extract has wide
range of antimicrobial activities.

Table 2. Antimicrobial activity Rosmarinusofficinalis(L) leaf oil based on diameter of zone of

inhibition

Test pathogens Concentration of the oil extract Gentamycin

1µl/ml 1.25µl/ml 2µl/ml (1µl/ml)

E. coli 9.50±0.71aC 11.50±0.57aC 14.50±0.63aB 18.35±0.22aA

S. typhi 8.75±0.35abD 9.75±0.42bC 12.75±0.50bB 18.40±0.28aA

S. pyogenes 7.80±0.28bD 9.65±0.50bC 12.25±0.35bB 18.80±0.42aA

S. aureus 0.00±0.00cD 6.25±0.35cC 10.50±0.71cB 18.40±0.14aA

fulconazole

(1µl/ml)

A. niger 9.50±0.71bC 11.20±0.28bC 13.65±0.50bB 17.50±0.71aA

A. versicolor 9.75±0.35abC 11.25±0.27bC 15.50±0.71bB 18.75±0.35aA

C. albicans 11.50±0.71aD 19.75±0.35aB 21.45±0.64aA 17.75±0.35aC

Means followed by same letter within a column were not significantly different at 0.05
probability level based on LSD (Least Significance difference) test. Small letters: significance
within column; capital letters: significance across row. E. coli: Escherichia coli, S. aureus:
Staphylococcus aureus, S. typhi: Salmonella typhi, and S. pyogenes:Streptococcus pyogenes;
A. niger: Aspergillus niger; C. albicans: Candida albicans.

The significant difference among the zone of inhibition may be associated to variation
physiology, metabolism, nutrition, genetic composition and biochemistry of the isolates under
study (Kigigha and Kalunta, 2017). Furthermore, age of the plants, type of solvents, extract
protocol, environmental condition of the area the plant was cultivated, time of harvesting may
affect the sensitivity of the plants against some microbial isolates. The synergy showed
 26

apparently increase in sensitivity level compared to the independent extracts of ginger and
lemon grass (Izah et al., 2018).Likeits antioxidant activity, the antimicrobial activity of
rosemarydepends on the chemical composition of its essential oil,which can vary greatly
depending on location, climate, andtime of harvest. The antimicrobial activity of an oil is
alsodetermined by the interactions between its components (Jordan et al., 2013).

4.3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration

(MBC), minimum fungicidal concentration (MFC) ofRosmarinusofficinalis(L.) leaf oil

The effectiveness of R. officinalis leaf oil against pathogenic microbes was further assessed
using MIC, MBC and MFC as in Table 3. The oil from R. officinalis has exhibited strongest
bactericidal activity with MIC (0.06µl/ml) and the corresponding MBC (0.12 µl/ml) against
E. coli while the weakest bactericidal activity with MIC (0.75 µl/ml, the largest value) and
MBC (1.12 µl/ml) was recorded against S. pyogenes indicating that E. coliwas the most
susceptible while S. pyogenes was the least susceptible to the R. officinalisleaf oil extract.

By contrast, R. officinalisoil has presented strongest antifungal activity with MIC (0.03µl/ml,
the least value) and MFC (0.05µl/ml) against C. albicanswhereas the weakest antifungal
activity with MIC (0.20µl/ml) and MFC (0.25µl/ml) was recorded against A. nigershowing
that C. albicans was the most susceptible to the oil extract while A. nigerwas the least
susceptible to the R. officinalisleafoil. Similar study was conducted by Doukkali et al. (2018)
who suggested the antibacterial activity increased with increasing volume of rosemary oil.
Indeed, it was observed that the zones of inhibition were greater with a greater volume of the
rosemary leaf oil. This is may be due to the presence of more active compounds in a higher
volume of essential oils. This antibacterial activity can be attributed to the antibacterial
properties of some compounds in the oil extract.

Wang et al. (2015) were investigated the antibacterial activity of rosemary essential oil on K.
Pneumoniae in aiding of MIC technique, the results exhibited a remarkable impact on the
biofilm of this bacteria by inhibition the initial cell attachment. Essential oil of rosemary
showed antagonistic activity against 60 strains of multidrug resistant E.coli isolated from
many specimens: the respiratory tract, abdominal cavity, urinary tract, skin and from hospital
equipment. tested the antibacterial activity of 10 plant extracts included rosemary against 14
 27

pathogenic microorganisms included E. coli, P. aeruginosa, K. Pneumoniae, S. aureus and

MIC values for each were evaluated, the plant extract of rosemary showed no antagonistic
activity against all the tested microorganisms except Bacillus subtilis and Candida
albicanswhich were susceptible to the rosemary extract (Sienkiewicz et al., 2013).

Table 3. Minimum inhibitory concentration (MIC), Minimum Bactericidal Concentration

(MBC), minimum fungicidal concentration (MFC) of Rosmarinusofficinalis(L.) leaf oiloil
extract
Test pathogens MIC (µl/ml) MBC/MFC (µl/ml)

E. coli 0.06 0.12

S. typhi 0.25 0.50

S. pyogenes 0.75 1.12

S. aureus 0.40 0.75

A. niger 0.20 0.25

A. versicolor 0.04 0.50

C. albicans 0.03 0.05

According to Lopez et al (2005) oils from Ocimumbasilicumand

Rosmarinusofficinalisexhibited an antibacterial activity against the Gram-positive bacteria S.
aureus, Enterococcus faecalisand, Listeriamonocytogenesand against Gram-negative bacteria
E. coli, Yersinia enterocolitica, Salmonella choleraesuis and P. aeruginosa as food borne
bacterial strains. The rosemary essential oil strongly inhibits E. coli ATCC 25922 (Lopez et al
2005) and another study results showed that rosemary oil has an antibacterial effect on a
number of microorganisms responsible for respiratory infections, isolated from clinical
specimens, such as Streptococcus pyogenes, S. agalactiae, S. pneumoniae and K.
pneumoniae, S. aureus and Stenotrophomonasmaltophilia(Fabio et al., 2007). The essential
oil from Rosmarinustournefortiipossesses antimicrobial activity against Gram-negative E. coli
 28

and P. aeruginosa and Gram-positive S. aureus (Bendeddouche et al., 2011). A number of

studies show that essential oils and their constituents possess useful properties concerning
human health. Many of them may be applied in anticancer therapy, cardiovascular and
nervous system disorders to reduce the level of cholesterol, to regulate the glucose level or to
stimulate hormone production (Edris et al., 2007).
 29

5. SUMMARY, CONCLUSION AND RECOMMENDATION

5.1. Summary

Medicinal plants were used for centuries as remedies for human diseases because they contain
components of therapeutic value, there is an increasing interest in phytochemicals as new
sources of natural antioxidant and antimicrobial agents. Microbial infections have become one
of the major problems of public health in the world and the development of resistance to the
available antibiotics has lead researchers to investigate the antimicrobial activity of medicinal
plants.

Essential oil extracts of plants have many applications in medicines, cosmetics, food stuff and
pharmaceutical industries. They are present in plants at low concentration which would
require high performance extraction techniques in order to achieve high yield. Generally,
essential oils are produced by different methods, including solvent extraction, supercritical
fluid extraction, hydro-distillation, steam distillation, use of superheated steam and
combinations of the previous techniques with others such as ultrasound and microwave-
assisted processes.On account of such justification the present study was aimed to examine
physicochemical properties, biological activities (as antioxidant and antimicrobial) of oil
extract from rosemary leaves.

The oil extraction was done in Soxhelt apparatus using hexane as a solvent. Then,
physicochemical properties of the oil extract were done based on determination of oil content,
specific gravity, acid value, percent free fatty acid and peroxide value. The antioxidant
activities were investigated based on free radical scavenging activities of DPPH and hydrogen
peroxide. The antimicrobial experiment was arranged as 1x1x7 ( 1 source extracts (oil extract
from leaves ofat three concentration levels), 1 solvent system i.e. hexane, 7 test organisms [4
bacteria: Escherichia coli,Staphylococcus aureus, Salmonella typhi, and Streptococcus
pyogenes; three fungi (Aspergillus versicolor, A. Niger, Candida albicans)] completely
randomized factorial design in two replications. The antimicrobial activities using disc
diffusion method and broth dilution method.
 30

The result of physicochemical properties and antioxidant activities of Rosmarinusofficinalis

(L.)leafoil presentedoil yield (38.5%), specific gravity (0.84), acid value (1.26 mg/g), free
fatty acid (0.63%), peroxide value (4.70).

The assessment of antioxidant activities of Rosmarinusofficinalis (L)leaf oil extract was

recorded high ascorbic acid content ( 20.43%), DPPH (6.75%), and hydrogen peroxide free
radical scavenging activity (1.50%).

The diameter of inhibition zone, measured based on disc diffusion, for

Rosmarinusofficinalis(L.) leafoil recorded the mean zone of inhibition at highest
concentration (2µl/ml) against bacterial test pathogens ranged from 10.50±0.71mm to
14.50±0.63mm, while 13.65±0.50 to 21.45±0.64 mm against fungal test pathogens. The
strongest antibacterial activity with maximum zone of inhibition (14.50mm) at highest
concentration (2µl/ml) of the oil was recorded againstE. coli while the weakest antibacterial
activity (10.50mm) was observed against S. aureus indicating that E. coli was the most
susceptible while S. aureus was the least susceptible. On the other hand, the strongest
antifungal activity with maximum zone of inhibition (21.45mm) was recorded against C.
albicans, but the weakest antifungal activity with minimum zone of inhibition (13.65mm) was
observed against A. nigersuggestingR. offcinalis leaf oil extract has wide range of
antimicrobial activities.

The effectiveness of R. officinalis leaf oil against pathogenic microbes was further assessed
using MIC, MBC and MFC. The oil from R. officinalis has exhibited strongest bactericidal
activity with MIC (0.06µl/ml) and the corresponding MBC (0.12 µl/ml) against E. coli while
the weakest bactericidal activity with MIC (0.75 µl/ml, the largest value) and MBC (1.12
µl/ml) was recorded against S. pyogenes indicating that E. coli was the most susceptible while
S. pyogenes was the least susceptible to the R. officinalisleaf oil extract.

By contrast, R. officinalis oil has presented strongest antifungal activity with MIC (0.03µl/ml,
the least value) and MFC (0.05µl/ml) against C. albicanswhereas the weakest antifungal
activity with MIC (0.20µl/ml) and MFC (0.25µl/ml) was recorded against A. nigershowing
that C. albicans was the most susceptible to the oil extract while A. nigerwas the least
susceptible to the R. officinalisleaf oil.
 31

5.2. Conclusion

The physicochemical properties and antioxidant activities of rosemary leaf oil extract
demonstrated the quality and stability of the oil extract. The oil extract has exhibited
significant antimicrobial activities against the growth of test bacteria and fungi and have both
cidal and static activity. E. coli was the most susceptible while S. aureus was the least
susceptible. On the other hand, the strongest antifungal activity with maximum zone of
inhibitio was recorded against C. albicans, but the weakest antifungal activity with minimum
zone of inhibition was observed against A. nigersuggestingR. offcinalis leaf oil extract has
wide range of antimicrobial activities.The rosemary leaf oil extract demonstrated remarkable
potency against test pathogens. Thuspromising as an alternative medicinal therapy against the
broad-spectrum drug resistant pathogens.

5.3. Recommendation

Based on the scope and results of the present study, the following recommendations were
forwarded:

 The finding of this study has indicated the oil extract of rosemary leaf was potential
for treatment of different species of bacteria and fungi. Therefore; studies are
required to assess the application of the oil extract as food preservative and
pharmacological application against various drug resistant microbes.
 Rosemary leaf oil will be a better source of further extraction and purification of oil
for large scale and therapeutic use.
 Further studies are needed to identify the specific biologically active compounds and
to evaluate the efficiency of the compound against pathogenic microorganisms
associated with various human diseases.
 Effective isolation and detailed characterization of the different physicochemical
properties and in vivo antibacterial activity against selected pathogens using model
animals should be conducted.
 The chemical composition of essential oils in general, varies with the origin of the
vegetal material used, cultivation conditions, environmental conditions, and the
obtaining method, among other factors. Further, studies should have to be done to
 32

assess biological activities of rosemary leaf oil and identify potential areas
recommended for rosemary leaf oil quality production.
 33

6. REFERENCES

Abdel W. K. G. E, El-Shamy K. A, El-Beih N. A. E, Morcy F. A, Mannaa F. A. E 2011.

Protective effect of a natural herb (Rosmarinusofficinalis) against hepatoxicity in
male albino rats. Comunicata Scientiae 2: 9-17.
Abdulkadir, A.G. and Jimoh, W.L.O. 2013. Comparative Analysis of Physico-Chemical
Properties of Extracted and Collected Palm Oil and Tallow. Chem Search J. 4(2): 44-
54.
Abe F., Yamauchi T., Nagao T., Kinjo J., Okabe H., Higo H., Akahane H. 2002. Ursolic acid
as a trypanocidal constituent in rosemary. Biol. Pharm. Bull. 25:1485-1487.
Abu-Al-Basal M. A. 2010. Healing potential of Rosmarinusofficinalis L. on full-thickness
excision cutaneous wounds in alloxan-diabetic BALB/c mice. Journal of
Ethnopharmacology, 131: 443-450.
Adewusi E. A, Moodley N, Steenkamp V. 2010. Medicinal plants with cholinesterase
inhibitory activity: A Review. African Journal of Biotechnology 9: 8257-8276.
Afonso M. S., De O Silva A. M., Carvalho E. B., Rivelli D. P., Barros S. B. et al. 2013.
Phenolic compounds from Rosemary (Rosmarinusofficinalis L) attenuate oxidative
stress and reduce blood cholesterol concentrations I diet-induced hypercholesterolemic
rats. Nutrition & Metabolism, 10: 19.
Alkofahi A., Batshoun R., Owais W., Najib N., 1997. Biological activity of some Jordanian
medicinal plant extracts. Part 2. Fitoterapia 68: 163-168.
Al-Sereitia M. R., Abu-Amerb K. M., Sena P. 1999. Pharmacology of rosemary (Rosmarinus
officinalis Linn.) and its therapeutic potentials. Indian Journal of Experimental
Biology, 37: 124-131.
AOAC, (2000). Official Methods of Analysis of the AOAC International (2000). 7th Edition,
Volume II. Gaithersburg, Maryland, USA.
AOAC. (1990). Association ofOfficial Methods of Analysis, 15th ed. Association of Official
Chemists‟ Washington DC.
Bakirel T, Bakirel U, Keleş OU, Ülgen SG, Yardibi H (2008). In vivoassessment of
antidiabetic and antioxidant activities ofrosemary (Rosmarinusofficinalis) in alloxan-
diabetic rabbits.Journal of Ethnopharmacology 116: 64-73.
 34

Bendeddouche M. S., Benhassaini H., Hazem Z. and Romane A. 2011. Essential oil analysis
and antibacterial activity of Rosmarinustournefortiifrom Algeria. Nat. Prod.
Commun, 6: 1511–1514.
Blumenthal M., 1998. The complete German Commission E Monographs: Therapeutic guide
to herbal medicines. Am. Bot. Coun. Austin.
Boulos L., 1983. Medicinal plants of North Africa. St. Clair River Drive Algonac, Michigan.
Bozin, B.; Mimica-Dukic, N.; Samojlik, I.; Goran, A.; Igic, R. 2008. Phenolics as antioxidants
in garlic (Allium sativum L., Alliaceae). Food Chem. 111: 925–929.
British Herbal Pharmacopoeia, 1996. Exeter, British Herb. Med. Assoc. London.
Bulbul A, Bulbul T, Biricik H, Yesilbag D, Gezen S. S. 2012. Effects of various levels of
rosemary and oregano volatile oil mixture on oxidative stress parameters in quails.
African Journal of Biotechnology 11: 1800-1805.
Cantrell C.L., Richheimer S.L., Nicholas G.M., Schmidt B.K., Bailey D.T., 2005. Seco-
hinokiol, a new abietanediterpenoid from Rosmarinusofficinalis. J. Natural Prod. 68
(1): 98-100.
Cavalcanti Y. W, Leopoldina F. A, Wilton W. N. 2011. Anti-adherent activity of Rosmarinus
officinalis essential oil on Candida albicans: an SEM analysis. Rev OdontoCienc 26:
139-144.
Chemat, F., Lucchesi, M., Smadja, J., Favretto, L., Colnaghi, G., and Visinoni, F. 2006.
"Microwave accelerated steam distillation of essential oil from lavender: A rapid,
clean and environmentally friendly approach", AnalyticaChimicaActa, 555: 157.
Chifiriuc C., Grumezescu V, Grumezescu A. M, Saviuc C, Lazăr V, et al. 2012. Hybrid
magnetite nanoparticles/Rosmarinusofficinalis essential oil nanobiosystem with
antibiofilm activity. Nanoscale Research Letters 7: 209.
Clinical and Laboratory Standards Institute (CLSI) (2015). Document M45. Methods for
Antimicrobial Dilution and Disk Susceptibility of Infrequently Isolated or Fastidious
Bacteria; Approved Guideline. Third Edition. CLSI, 940 West Valley Road, Suite
1400, Wayne, Pennsylvania 19087-1898, USA.
Clinical and Laboratory Standards Institute (CLSI, 2012). Performance standards for
antimicrobial disk susceptibility tests; approved standards – Eleventh Edition. CLSI
 35

document M02-A11. Clinical and Laboratory Standards Institute, 950 West Valley
Road, Suite 2500, Wayne, PA, 19087 USA, 2012.
Cui L, Kim M. O, Seo J. H., Kim I. S., Kim N. Y. et al., 2012. Abietanediterpenoids of
Rosmarinusofficinalis and their diacylglycerol acyltransferase-inhibitory activity.
Food Chemistry, 132: 1775-1780.
De Melo G. A. N, Grespan R, Fonseca J. P, Farinha T. O. 2011. Rosmarinusofficinalis L.
Essential Oil Inhibits In Vivo and In Vitro Leukocyte Migration. Journal of Medicinal
Food 14: 944-949.
Debersac P., Heydelb J.M., Amiotc M.J., Goudonnetb H., Arturb Y., Suscheteta M., Siessa
M.H., 2001. Induction of cytochrome P450 and/or detoxication enzymes by various
extracts of rosemary: description of specifi c patterns. Food Chem. Toxicol. 39: 907-
918.
Del Bano M.J., Lorente J., Castillo J., Benavente-García O., Del Río J. A., Ortuño A., Quirin
K.W., Gerard D., 2003. Phenolic diterpenes, flavones, and rosmarinic acid
distribution during the development of leaves, flowers, stems, and roots of Rosmarinus
officinalis: antioxidant activity. J. Agric. Food Chem. 51: 4247-4253.
Demissew, S., Dange, E. 2001. Basic and Applied Research on Medicinal Ethiopia, In:
Proceedings of national workshop on conversation and sustainable Use of Medicinal
plants in Ethiopia, Addis Ababa, 2001, 29.
Dias P.C., Foglio M.A., Possenti A., Carvalho J.E., 2000. Antiulcerogenic activity of crude
hydroalcoholic extracts of RosmarinusofficinalisL. J. Ethnopharm. 69: 57-62.
Dilas S., Knez Z., Cetojevic-Simin D., Tumbas V., Škerget M. et al. 2012. In vitro antioxidant
and antiproliferative activity of three rosemary (Rosmarinusofficinalis L.) extract
formulations. International Journal of Food Science and Technology 47: 2052-2062.
Doukkali L., Tahiri A., Tazi B., Guenoun F. 2018. Chemical Composition and Antibacterial
Activity of two Essential Oils of rosemary against Erwiniaamylovora, the causal
agent of fire blight. J. Mater. Environ. Sci., 9 (10): 2913-2918.
Edris A. E. 2007. Pharmaceutical and therapeutic potentials of essential oils and their
individual volatile constituents: A review. Phytother. Res., 21: 308–323.
 36

Fabio A., Cermelli C., Fabio G., Nicoletti P. and Quaglio P. 2007. Screening of the
antibacterial effects of a variety of essential oils on microorganisms responsible for
respiratory infections. Phytother. Res., 21, 2007, 374–377.
Fahim F. A., Esmat A.Y., Fadel H. M., Hassan K. F., 1999. Allied studies on the effect of
RosmarinusofficinalisL. on experimental hepatotoxicity and mutagenesis. Int. J.
Food Sci. Nutr. 50: 413-427.
Farnsworth N. R., 2005. NAPRALERT Database. University of Illinois at Chicago, Chicago
IL [An online database available directly through the University of Illinois at Chicago
or through the Scientific and Technical Network [STN] of Chemical Abstracts
Services, 30.06.2005].
Gachkar L, Yadegari D., Rezaei B., Taghizadeh M., Astaneh S. A. et al. 2007. Chemical and
biological characteristics of Cuminumcyminum and Rosmarinusofficinalis essential
oils. Food Chemistry, 102: 898-904.
Gaya M, Repetto V, Toneatto J, Anesini C, Piwien PG, et al. 2013. Antiadipogenic effect of
carnosic acid, a natural compound present in Rosmarinusofficinalis, is exerted
through the C/EBPs and PPARγ pathways at the onset of the differentiation program.
BiochimicaetBiophysicaActa1830: 3796-3806.
HagersHandbuch der Drogen [CD ROM]. 2003. Springer Heidelberg.
Haloui M., Louedec L., Michel J.B., Lyoussi B., 2007. Experimental diuretic effects of
Rosmarinusofficinalisand Centauriumerythraea. J. Ethnopharm. 71: 465-472.
Hudzicki J. 2009. Kirby-Bauer Disk Diffusion Susceptibility Test Protocol. American
Society for Microbiology. pp 1.23.
Hur Y.G., Yun Y., Won J., 2004. Rosmarinic acid induces p56lck-dependent apoptosis in
Jurkat and peripheral T cells via mitochondrial pathway independent from Fas/Fas
ligand interaction. J. Immunol. 172: 79-87.
Hussain A. I, Anwar F, Chatha S. A. S, Jabbar A, Mahboob S, et al. 2010. Rosmarinus
officinalis essential oil: antiproliferative, antioxidant and antibacterial activities.
Brazilian Journal of Microbiology, 41: 1070-1078.
Hussain, A., Anwar, F., Chatha, A., Jabbar, A., Mahboob, S. and Nigam, P. 2010.
Rosmarinusofficinalis essential oil: antiproliferative, antioxidant and antibacterial
activities. Brazilian Journal of Microbiology, 41: 1070-1078.
 37

Ibarra A, Cases J, Roller M, Chiralt B. A., Coussaert A, et al. 2011. Carnosic acid-rich
rosemary (Rosmarinusofficinalis L.) leaf extract limits weight gain and improves
cholesterol levels and glycaemia in mice on a high-fat diet. British Journal of
Nutrition, 106: 1182-1189.
Izah S. C and Aseibai E. R. 2018. “Antibacterial Efficacy of Aqueous Extract ofMyristica
fragrans(Common Nutmeg)”. EC Pharmacology andToxicology 6.4: 291-295.
Jordan M. J., Lax V., Rota M. C., Lorán S., Sotomayor J. A. 2013. Effect of bioclimatic area
on the essential oil composition and antibacterial activity of Rosmarinusofficinalis L.
Food Control, 30: 463-468.
Khalil O. A, Ramadan K. S, Danial E. N, Alnahdi H. S, Ayaz N. O. 2012. Antidiabetic
activity of Rosmarinusofficinalis and its relationship with the antioxidant property.
African Journal of Pharmacy and Pharmacology 6: 1031-1036.
Kigigha LT and Kalunta CG. 2017. “Antimicrobial efficacy of leaf extractsof Piper nigrum
against Escherichia coli,Staphylococcus aureus and Candida albicans”. Journal of
Basic Pharmacologyand Toxicology 1.2 (2017): 32-36.
Kokate C. K., Purohit A.P., Gokhale S.B., 2010. Pharmacognosy. NiraliPrakashan Pune.
Kuhlmann A. and Rohl C. 2006. Phenolic antioxidant compounds produced by in vitro.
Cultures of Rosemary (Rosmarinusofficinals) and their anti-inflammatory effect on
lipopolysaccharide-activated microglia. Pharmaceuticl Biology, 44 (6): 401-410.
Lee J, Koo N, Min DB. (2004). Reactive oxygen species, aging, andantioxidative
nutraceuticals. Comp Rev Food Sci Food Safety, 3, 21–33.
Leuschner J., 1997. Reproductive toxicity studies of D--camphor in rats and rabbits.
Arzneimittelforschung 47:124-128.
Lis-Balchin M., 1996. Comparison of the pharmacological and antimicrobial actions of
commercial plant essential oils. J. Herbs Spices Medic. Plants 4: 69-82.
Lo A. H., Liang Y. C., Lin-Shiau S. Y., Ho C. T., Lin J. K. 2002. Carnosol, an antioxidant in
rosemary, suppresses inducible nitric oxide synthase through down-regulating nuclear
factor-B in mouse macrophages. Carcinogenesis 23: 983-991.
Loizzo, M.R., Bonesi, M., Menichini, F., Tenuta, M.C., Leporini, M., Tundis, R., 2016:
Antioxidant and carbohydrate-hydrolysing enzymes potential of Sechiumedule
 38

(Jacq.) Swartz (Cucurbitaceae) peel, leaves and pulp fresh and processed. Plant
Food Hum. Nutr.71: 381-387.
Lopez P., Sanchez C., Battle R. and Nerin C. 2005. Solid- and vapor-phase antimicrobial
activities of six essential oils: Susceptibility of selected foodborne bacterial and fungal
strains. Agric. Food Chem. 53: 6939–6946.
Machado D. G, Cunha M. P, Neis V. B, Balen G. O, Colla A. et al. 2013. Antidepressant-like
effects of fractions, essential oil, carnosol and betulinic acid isolated from Rosmarinus
officinalis L. Food Chemistry 136: 999-1005.
Machado D. G, Cunha M. P, Neis V. B, Balen G. O, Colla A. R, et al. 2012. Rosmarinus
officinalis L. hydroalcoholic extract, similar to fluoxetine, reverses depressive-like
behavior without altering learning deficit in olfactory bulbectomized mice. Journal of
Ethnopharmacology 143: 158-169.
Makino T., Ono T., Liu N., Nakamura T., Muso E., Honda G., 2002. Suppressive effects of
rosmarinic acid on mesangioproliferative glomerulonephritis in rats. Nephron. 92:
898-904.
Mangena T., Muyima N.Y.O., 1999. Comparative evaluation of the antimicrobial activities of
essential oils of Artemisia afra, Pteroniaincana and Rosmarinusofficinalison selected
bacteria and yeast strains. Lett. Appl. Microbiol. 28: 291-296.
Marinas, I., Grumezescu A. M, Saviuc C, Chifiriuc C., Mihaiescu D. et al. 2012. Rosmarinus
officinalis essential oil as antibiotic potentiator against Staphylococcus aureus. Nano
Bio Sci 2:271-276.
Masango, P. 2005. "Cleaner production of essential oils by steam distillation", J. Cleaner
Production, 13: 833.
Mengoni E. S, Vichera G., Rigano L. A, Rodriguez P. M. L. 2011. Suppression of COX-2,
IL-1β and TNF-α expression and leukocyte infiltration in inflamed skin by bioactive
compounds from Rosmarinusofficinalis L. Fitoterapia82: 414-421.
Milwidsky, B.M. and Gabriel, D.M. (1982). Detergent analysis. (A Handbook for cost-
effectivequality control). Pp 187-234
Minaiyan, M., Ghannadi, A. R., Afsharipour, M., Mahzouni, P. 2011. .Effects of extract and
essential oil of RosmarinusofficinalisL. on TNBS-induced colitis in rats. Res
Pharm Sci,6: 13-21.
 39

Mohdaly, A., Smetanska, I., Ramadana, M.,Sarhan, M. and Mahmoud, A. 2011.Antioxidant

Potential of Sesame (Sesamumindicum) Cake Extract in Stabilization ofSunflower
and Soybean Oils. Ind. CropsProd., 34: 952-959.
Moreno S., Scheyer T., Romano C. S., Vojnow A. 2006. Antioxidant and antimicrobial
activities of rosemary extracts linked to their polyphenol composition. Free radical
research. 40:2, 223-231.
Morshed, H., Islam, M. S., Parvin, S., Ahmed, M. U., Islam, M. S., Mostofa, A G. M. and
Bin Sayeed, M. S. 2012. Antimicrobial and Cytotoxic Activity of the Methanol
Extract of PaederiafoetidaLinn. (Rubiaceae). Journal of Applied Pharmaceutical
Science 02 (01); 77-80.
Muhlbauer R. C., Lozano A., Reinli A., 2003. Common herbs, essential oils, and
monoterpenes potently modulate bone metabolism. Bone 32: 372-380.
Najem, A. M., Abed, I. J. and Al Haidarry, A. M. 2015. Evaluation the anti Cyanobacterial
effect of Rosemary (Rosmarinusofficinalis L.) Essential oil. Iraqi Journal of
Biotechnology, 15: 1: 97-102.
Narayana, K. R., Reddy, M. S., Chaluvadi, M. R., and Krishna, D. R. 2001. Bioflavonoid
classification, pharmacological, Biochemical effects and therapeutic potent. Indian J
pharmol, 33: 2-16.
Nielsen, S.S. (2002): Introduction to the ChemicalAnalysis of Foods. Pp 183-204.
Oluwatuyi M., Kaatz G. W, Gibbons S. 2004. Antibacterial and resistance modifying activity
of Rosmarinusofficinalis. Phytochemistry 65: 3249-3254.
Omri A. E. L, Han J, Abdrabbah M. B, Isoda H. 2012. Down regulation effect of Rosmarinus
officinalis polyphenols on cellular stress proteins in rat pheochromocytoma PC12
cells. Cytotechnology 64: 231-240.
Omri AE, Han J, Yamada P, Kawada K, Abdrabbah MB, et al. (2010) Rosmarinusofficinalis
polyphenols activate cholinergic activities in PC12 cells through phosphorylation of
ERK1/2. Journal of Ethnopharmacology 131: 451-458.
Orhan I., Aslan S., Kartal M., Şener B., Başer K. H. C 2008. Inhibitory effect of Turkish
Rosmarinusofficinalis L. on acetylcholinesterase and butyrylcholinesterase enzymes.
Food Chemistry, 108: 663-668.
 40

Ozarowski M, Mikolajczak P. L, Bogacz A, Gryszczynska A, Kujawska M, et al. (2013)

Rosmarinusofficinalis L. leaf extract improves memory impairment and affects
acetylcholinesterase and butyrylcholinesterase activities in rat brain.Fitoterapia 91:
261-271.
Prabuseenivasan, S., Jayakumar, M. and Ignacimuthu, S. 2006. In vitro antibacterial activity
of some plant essential oils. BMC Complement Altern Med, 6: 1-8.
Rasooli I, Fakoor M. H, Yadegarinia D, Gachkar L, Allameh A, et al. 2008.
Antimycotoxigenic characteristics of Rosmarinusofficinalis and Trachyspermum
copticum L. essential oils. International Journal of Food Microbiology, 122: 135-139.
Rice-Evans C. (2004). Flavonoids and isoflavones: Absorption,metabolism, and bioactivity.
Free RadicBiol Med, 36, 827–828.
Rosemary(Rosmarinusofficinalis). 2012.[online], http:// www. gits4u.
com/agri/agri5rosmeri.htm.
Sanders C., Diego M., Fernandez M., Field T., Hernandez--Reif M., Roca A., 2002. EEG
asymmetry responses to lavender and rosemary aromas in adults and infants. Int. J.
Neurosci. 112: 1305-1320.
Shah, S. M., Khan, F. A., Shah, S. M. H., Chishti, K. A., Pirzada, S. M., Khan, M. A., and
Farid, A. 2011. Evaluation of phytochemicals and antimicrobial activity of white and
blue capitulum and whole plant of Silybummarianum. World ApplSci J, 12: 1139-
1144.
Shahidi F, Wanasundara PK. (1992). Phenolic antioxidants. Crit RevFood SciNutr, 32, 67–
103.
Sienkiewicz M., Łysakowska M., Pastuszka M., Bienias W. and Kowalczyk E. 2013. The
Potential of use basil and Rosemary Essential Oils as Effective Antibacterial Agents.
Molecules, 18: 9334-9351.
Singletary K. W., Nelshoppen J. M., 1991. Inhibition of 7,12-dimethylbenz[c]anthracene
(DMBA)-induced mammary tumorigenesis and of in vivo formation of mammary
DMBA-DNA adducts by rosemary extract. Cancer Lett. 60:169-175.
Tavassoli S. K, Mousavi S. M, Emam D. Z, Razavi S. H. 2011. Chemical composition and
evaluation of antimicrobial properties of Rosmarinusofficinalis L. essential oil.
African Journal of Biotechnology 10: 13895-13899.
 41

Tavassoli S. K, Mousavi S. M, Emam D. Z, Razavi S. H. 2011. Chemical composition and

evaluation of antimicrobial properties of Rosmarinusofficinalis L. essential oil.
African Journal of Biotechnology, 10: 13895-13899.
Wang H., Liang F. and Zhang L. 2015. Composition and Anti-cyanobacterial Activity of
Essential Oils from Six Submerged Macrophytes. Pol. J. Environ. Study, 24(1), 333-
338.
Williams C.E., 2009. Trease and evanspharmacognosy. Saunders Elsevier Edinburg.
Yu, M., Choi J., Chae I., Im I., Im H. et al. (2013) Suppression of LPS induced inflammatory
activities by Rosmarinusofficinalis L. Food Chemistry 136: 1047-1054.
Zaouali, Y, Bouzaine T, Boussaid M. 2010. Essential oils composition in two Rosmarinus
officinalisL. varieties and incidence for antimicrobial and antioxidant activities. Food
and Chemical Toxicology, 48: 3144-3152.
Zegura B, Dobnik D, Niderl MH, Filipič M (2011) Antioxidant andantigenotoxic effects of
rosemary (Rosmarinusofficinalis L.)extracts in Salmonella typhimurium TA98 and
HepG2 cells.Environmental Toxicology and Pharmacology 32: 296-305.
Zhu B. T., Loder D.P., Cai M.X., Ho C. H. T., Huang M. T., Conney A. H., 1998. Dietary
administration of an extract from rosemary leaves enhances the liver microsomal
metabolism of endogenous estrogens and decreases their uterotropic action in CD-1
mice. Carcinogen. 19: 1821-1827.
 42

7. APPENDICES
 43

Appendix Table 1. Raw data for physicochemical properties and antioxidant activities

Oil rep Oil spgr Acv FFA PV DPPH PVsc AA

extract yield
leaf 1 40 0.89 1.4025 0.71 5 6.8 6.8 18.75
leaf 2 37 0.80 1.122 0.56 4.4 6.7 6.9 22.11
Spgr: specific gravity; ACV: acid value; FFA: free fatty acid value; PV: peroxide value;
DPPH: 2, 2- diphenyl-1-picrylhydrazyl; PVSC: peroxide scavenging activity; AA: ascorbic
acid.

Appendix Table 2. Raw data for antibacterial activity based on diameter of zone of inhibition
Test pathogens Rep Concentration of the oil extract Gentamycin
1µl/ml 1.5µl/ml 2µl/ml (1µl/ml)
E. coli 1 9 12 15 18.19
E. coli 2 10 11 14 18.5
S. typhi 1 9 10 13 18.2
S. typhi 2 8.5 9.5 12.5 18.6
S. aureus 1 0 6.5 11 18.3
S. aureus 2 0 6 10 18.5
S. pyogenes 1 8 10 12 18.5
S. pyogenes 2 7.6 9.3 12.5 19.1
 44

Appendix Table 3. Raw data for antifungal activity

Test pathogens Rep Concentration of the oil extract Fulconazole
1µl/ml 1.5µl/ml 2µl/ml (1µl/ml)
A. niger 1
9 11 13.3 17
A. niger 2
10 11.4 14 18
A. versicolor 1
10 11 15 19
A. versicolor 2
9.5 11.5 16 18.5
C. albicans 1
11 20 21.9 17.5
C. albicans 2
12 19.5 21 18
 45

Appendix Figure 1. Sample preparation

 46

Appendix Figure 2. Sample extracted oil

 47

Appendix Figure 3. Antimicrobial activity

 48

Appendix Figure 4. Measuring inhibition zone during antimicrobial activity test