INVESTIGATION OF THE IN VITRO ANTIMICROBIAL ACTIVITY OF ROSMARINUS
OFFICINALIS (ROSEMARY) ESSENTIAL OIL AGAINST MICROORGANISMS IN
BURN WOUND INFECTIONS
BY : POOJA NEIL LUMB
U29/37241/2011
B. PHARM IV
SCHOOL OF PHARMACY
SUPERVISOR: DR. B. AMUGUNE
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
UNIVERSITY OF NAIROBI
A DISSERTATION IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD
OF THE DEGREE OF BACHELOR OF PHARMACY OF UNIVERSITY OF NAIROBI.
SEPTEMBER 2015
DECLARATION
Investigator
I, Pooja Neil Lumb, hereby declare that this work is my original work and has not been submitted
elsewhere for examination, award of a degree or publication. Where other people’s work or my own
work has been used, this has properly been acknowledged and referenced in accordance with the
University of Nairobi’s requirements.
Signature: Date:
Supervisor
This proposal has been submitted for evaluation and examination purposes with my approval as the
supervisor.
Dr. B. Amugune, PhD
Senior Lecturer – Department of Pharmaceutical Chemistry
University of Nairobi
Signature: Date:
ii
DECLARATION OF ORIGINALITY
Name of Student: POOJA NEIL LUMB
Registration Number: U29/37241/2011
College: College Of Health Sciences
School: School Of Pharmacy
Course Name: Bachelor Of Pharmacy
1. I understand what Plagiarism is and I am aware of the University’s policy in this regard.
2. I declare that this project is my original work and has not been submitted elsewhere for
examination, award of a degree or publication. Where other people’s work, or my own work
has been used, this has properly been acknowledged and referenced in accordance with the
University of Nairobi’s requirements.
3. I have not sought or used the services of any professional agencies to produce this work.
4. I have not allowed, and shall not allow anyone to copy my work with the intention of
passing it off as his/her own work
5. I understand that any false claim in respect of this work shall result in disciplinary action, in
accordance with University Plagiarism Policy.
Signature: Date:
iii
DEDICATION
Oh God! Thou art the Giver of Life,
Remover of pain and sorrow,
The Bestower of happiness,
Oh! Creator of the Universe,
May we receive thy supreme sin-destroying light,
May Thou guide our intellect in the right direction.
- A translation of the Gayatri Mantra,
A Hindu Prayer
I would like to dedicate this project to my parents. To my mother, who has always guided,
supported and strengthened me, and to my Late father, whose presence I felt throughout the course
of the project as he had planted the Rosemary bush that provided the focus of my project. Thank
you both. I wish to make you proud parents. May God always continue to watch over us.
iv
ACKNOWLEDGEMENTS
“The will to win, the desire to succeed, the urge to reach your full potential... these are the keys that
will unlock the door to personal excellence,” - Confucius.
This project was successful because of the support and aid of those around me.
My heartfelt gratitude goes to my supervisor Dr. Beatrice Amugune for her invaluable guidance and
input on the matters experimental aspects and the writing of the dissertation of this project. Her
approachability and patience are both highly appreciated.
Special thanks extended to Dr. Alex Okaru, lecturer of the department of Pharmaceutical Chemistry
for support in carrying out the experimental procedures involved in Gas Chromatography. His
willingness to share his time and insight is greatly valued.
More appreciation goes to Mr. H.N. Mugo and Mr. J. Nyamatari of the department of
Pharmaceutical Chemistry, as well as to Mr. J. Mwalukumbi and Mr. R. Ingwela, of department of
Pharmacology and Pharmacognosy, University of Nairobi, for their assistance in laboratory work.
To my paternal uncle Mr. R. K. Lumb, I will treasure him for not only wholeheartedly supporting
my education but also being a father figure.
I am indebted to my friends F. S. Gulamhussein and T. P. Patel for their vital contribution and
encouragement.
To my classmates, B. Pharm class of 2015, I wish to express my gratitude for their support in all my
ventures as a student as well as being their class representative. I wish all of them success ahead.
v
TABLE OF CONTENTS
TITLE....................................................................................................................................................i
DECLARATION..................................................................................................................................ii
DECLARATION OF ORIGINALITY................................................................................................iii
DEDICATION.....................................................................................................................................iv
ACKNOWLEDGEMENTS.................................................................................................................v
TABLE OF CONTENTS....................................................................................................................vi
LIST OF FIGURES.............................................................................................................................ix
LIST OF TABLES................................................................................................................................x
LIST OF ABBREVIATIONS..............................................................................................................xi
ABSTRACT.......................................................................................................................................xii
CHAPTER ONE – INTRODUCTION................................................................................................1
1.1 Burns..........................................................................................................................................1
1.2 Burn wound infections...............................................................................................................1
1.3 Micro-organisms involved.........................................................................................................2
1.4 Plants as a source of drug products............................................................................................3
CHAPTER TWO – LITERATURE REVIEW.....................................................................................4
2.1 Rosmarinus officinalis...............................................................................................................4
2.2 Uses of Rosemary......................................................................................................................5
2.2.1 Traditional uses and folklore..............................................................................................5
2.2.2 Culinary uses......................................................................................................................6
2.2.3 Cosmetic, fragrance and industrial uses.............................................................................6
2.3 Precautions, side effects and interactions..................................................................................7
2.4 Previous studies done on Rosemary..........................................................................................7
2.5 Previous studies on burns..........................................................................................................9
2.6 Current burn wound treatment options....................................................................................10
2.7 Traditional medicines used for burn wounds and research for alternatives.............................10
2.8 Justification..............................................................................................................................11
2.9 Research question....................................................................................................................11
2.10 Hypothesis..............................................................................................................................11
2.11 Objectives..............................................................................................................................12
2.11.1 General objectives..........................................................................................................12
2.11.2 Specific objectives..........................................................................................................12
vi
CHAPTER THREE – EXPERIMENTAL..........................................................................................13
3.1 Plant collection and identification...........................................................................................13
3.2 Extraction of oil.......................................................................................................................13
3.3 Procedure for screening...........................................................................................................15
3.3.1 Materials and apparatus...................................................................................................15
3.3.2 Preparation of standards...................................................................................................16
3.3.3 Subculturing of microorganisms......................................................................................16
3.4 Microtitre dilution screening using 96-well plate....................................................................16
3.4.1 Preparation of test solutions.............................................................................................16
3.4.2 Screening..........................................................................................................................17
3.5 Antimicrobial screening by disk diffusion...............................................................................18
3.5.1 Preparation of test solutions.............................................................................................18
3.5.2 Screening..........................................................................................................................19
3.6 Phytochemical tests..................................................................................................................20
3.6.1 Drying and milling...........................................................................................................20
3.6.2 Reagents and materials.....................................................................................................20
3.6.3 Test for alkaloids..............................................................................................................20
3.6.4 Tests for glycosides..........................................................................................................20
3.6.5 Test for tannins.................................................................................................................21
3.6.6 Test for saponins..............................................................................................................21
3.7 Gas chromatographic analysis.................................................................................................22
3.7.1 Gas chromatography using a flame ionization detector...................................................22
3.7.2 Gas chromatography using a mass spectrometer detector...............................................22
CHAPTER FOUR – RESULTS.........................................................................................................23
4.1 Nature and volume of oil collected..........................................................................................23
4.2 Percentage yield.......................................................................................................................23
4.3 Results for microtitre dilution screening.................................................................................23
4.4 Antimicrobial screening in Petri dish.......................................................................................25
4.5 Results of phytochemical tests.................................................................................................27
4.6 Gas Chromatography...............................................................................................................27
4.6.1 Gas chromatography using a flame ionization detector...................................................27
4.6.2 Gas chromatography using a mass spectrometer detector...............................................29
CHAPTER FIVE – DISCUSSION....................................................................................................31
CHAPTER SIX – CONCLUSION.....................................................................................................33
vii
6.1 Conclusion..........................................................................................................................33
6.2 Recommendations...............................................................................................................33
REFERENCES...................................................................................................................................34
viii
LIST OF FIGURES
Page
Figure 1 – Photographs showing Rosmarinus officinalis plant 5
Figure 2a – Photograph showing set up used in oil extraction 14
Figure 2b – Photograph showing Clevenger-like apparatus and layer of oil formed
during extraction
15
Figure 3 – General lay-out of samples on the Petri dishes 19
Figure 4a – Photograph of 96-well plates before incubation with MTT dye – Plate 1
(right), Plate 2 (left)
24
Figure 4b – Photograph of 96-well plates after incubation with MTT dye – Plate 1
(right), Plate 2 (left)
24
Figure 5 – Photographs of Petri-dishes showing zones of inhibition created by
Rosemary oil
25
Figure 6a – Gas chromatogram of Rosemary oil 28
Figure 6b – Gas chromatogram of American variety of Rosemary oil 28
Figure 6c – Mass spectrum gas chromatogram of Rosemary oil 30
Figure 6d – GC-MS total ion current of Rosemary oil 30
ix
LIST OF TABLES
Page
Table 1 – Taxonomic classification of Rosmarinus officinalis 4
Table 2 – Pharmacological actions and therapeutic potential of Rosemary 8
Table 3 – Weight of plant material used in oil extraction 13
Table 4 – Dilutions of Rosemary oil used in microtitre dilution screening 16
Table 5 – Arrangement of Plate 1 – Antibacterial activity of Rosemary oil 17
Table 6 – Arrangement of Plate 2 – Antifungal activity of Rosemary oil 18
Table 7 – Key for Tables 5 & 6 18
Table 8 – Dilutions of Rosemary oil used in Petri dish screening 19
Table 9 – Percentage and average yield of Rosemary oil 23
Table 10 – Diameters of zones of inhibition 26
Table 11 – Relative potency of Rosemary oil dilutions compared to positive control 26
Table 12 – Results of phtyochemical tests 27
Table 13 – List of constituents of Rosemary oil (Based on order of elution) 29
x
LIST OF ABBREVIATIONS
μl – microlitres
μm – micrometres
cm – centimetres
DMSO – Dimethylsulphoxide
in – inches
KeV – kilo electrovolts
m/z – Mass per charge ratio
m – metres
ml – millilitres
mm – millimetres
MTT – Tetrazolium GR
SDA – Sabouraud Dextrose Agar
SDB – Sabouraud Dextrose Broth
TSA – Tryptone Soya Agar
TSB – Tryptic Soy Broth
USA – United States of America
WHO – World Health Organization
xi
ABSTRACT
Burn wound infections are a major health concern both locally and internationally. Its current form
of management has several drawbacks, urging research for alternatives, including from plant
sources. Rosemary was chosen for this project due to its relative abundance, prominent use in
traditional medicine and promising results in previous studies.
All the Rosemary plant material used in the project was obtained from a single bush from a private
residence in South B area of Nairobi, identified by Mr. J. Mwalukumbi at the Department of
Pharmacognosy, School of Pharmacy, University of Nairobi.
Rosemary essential oil was extracted using a Clevenger-like apparatus. The average yield from two
extractions was obtained to be 0.43%.
The in vitro antimicrobial activity of the oil obtained was used tested against Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus and Saccharomyces
cerevisiae, microorganisms that represent those involved in burn wound infections, in a microtitre
dilution screening carried out in a 96-well plate. The results showed that Rosemary essential oil of
the Kenyan variety indeed possesses antimicrobial activity against the microbes, however to
appreciate the activity better, a second screening was carried out on Petri dishes using a disk
diffusion method. The second screening showed that Rosemary essential oil has appreciable activity
however is not as potent as broad spectrum antibiotics.
Gas chromatographic analysis carried out on the oil indicated the presence of 27 different
constituent compounds, from which pinene, camphene, eucalyptol, limonene and caryophyllene
were most abundant. The antimicrobial activity of the oil may be attributed to the presence of the
compound alpha-pinene.
xii
CHAPTER ONE – INTRODUCTION
1.1 Burns
A burn is an injury to the skin or other organic tissue primarily caused by heat or due to radiation,
electricity, friction or contact with chemicals. The most common form of burns is thermal (heat)
burns, that occur when some or all of the cells in the skin or other tissues are destroyed by either hot
liquids (scalds), hot solids (contact burns) or flames (flame burns) [1].
According to statistics compiled by WHO in 2014, an estimated 265,000 deaths every year are
caused by burns, the vast majority of which occur in low and middle-income countries, and that
burn injuries are increasing annually. In Kenya, a retrospective study carried out at Gertrudes's
Garden Children's Hospital over a period of five years (2003 – 2007) found that 19.2 % of all
patients admitted had burn injuries. A similar study at Kenyatta National Hospital, showed that of
all patients admitted due to injuries, 34.8% were admitted with burn injuries [1, 2].
Burns that are non-fatal are a leading cause of morbidity due to burn wounds becoming infected and
thus leading to delayed recovery and prolonged hospitalization in the short term. Disfigurement and
disability occur in the long term, which often results in stigma and rejection towards burn survivors
Burns are among the leading causes of disability-adjusted life-years (DALYs) lost in low-income
and middle-income countries and are also the fifth most common cause of non-fatal childhood
injuries worldwide [1, 3].
Burn injuries also pose an economic threat, for example, in 2000, the direct costs for care of
children with burns in the USA exceeded US$ 211 million, while in 2007, in Norway, costs for
hospital burn management exceeded €10.5 million [1].
Statistics show that people living in low-income and middle-income countries are at higher risk for
burns than people living in high-income countries, however, within all countries, the risk of burn
injuries correlates with socioeconomic status [1].
1.2 Burn wound infections
Infection in a burn patient is a leading cause of morbidity and mortality and is a challenging
concern for the burn teams in clinical settings. Burn wound infections occur due to the loss of the
protective barrier of the skin and accompanying thrombosis of the subcutaneous blood vessels. The 1
resulting avascular wound bed allows for a medium that can support the growth of microorganisms
as well as prevent the penetration of systemically administered antimicrobial drugs [4].
Studies have shown that immediately after burns, the wound is sterile, but within a very short time
bacteria contaminating the wound surface begin to multiply and proliferate in the area of the burn
wound leading to extensive bacterial colonization. Hence the burn wound will always be colonized
with organisms until wound closure is achieved. It is also noted that as the size of the wound
increases, so does the risk of infection [5].
Thermal injury to the skin also causes a massive release of humoral factors, including cytokines,
prostaglandins, vasoactive prostanoids, and leukotrienes. Accumulation of these factors at the site of
injury results in “spillover” into the systemic circulation, giving rise to immunosuppression, which
promotes the occurrence of burn wound infection [5].
Burn wound infections can be local or invasive. Local wound infections are characterized by
redness or cellulitis, purulent, drainage, graft loss, fever >38.5°C and leukocytosis. Invasive wound
infections are characterized by conversion of partial-thickness to full-thickness injury, rapid eschar
separation, necrosis of small blood vessels, oedema, redness, and tenderness at the wound edges.
Systemically, the patient may be hypothermic or hyperthermic, hypotensive, have a decreased urine
output and illeus. Laboratory results will reveal leukocytosis or leukopenia, thrombocytopenia,
positive blood cultures, hyperglycemia and invasion of organisms into viable tissue on
histopathologic examination of the wound. Generally, in the event of a large uncovered burn surface
getting infected, the patients face a higher morbidity than mortality, due to long periods of
dressings, leading to deformities and contractures [4, 5].
1.3 Micro-organisms involved
The colonizing micro-organisms on burn wounds can be sourced from the patient’s own
endogenous (normal) flora, from exogenous sources in the environment, and from healthcare
personnel. Exogenous organisms from environment of the hospital tend to be more resistant to
antimicrobial agents than endogenous organisms. The common microorganisms associated with
infection in burn patients include gram-positive bacteria, gram-negative bacteria and yeast/fungal
organisms [4].
The distribution of micro-organisms changes over time in the individual patient. The typical burn
2
wound is initially colonized predominantly with gram-positive microorganisms, which are fairly
quickly replaced by antibiotic-susceptible gram-negative microorganisms, usually within a week of
the burn injury. If wound closure is delayed these microorganisms may be replaced by yeasts, fungi,
and antibiotic-resistant bacteria. Systemic antimicrobials are indicated to treat infections, such as
pneumonia, bacteremia and urinary tract infection secondary to invasive wound infections.
Systemic antimicrobials will not eliminate colonizations burn wounds, due to poor vascularization,
but rather promote emergence of resistant organisms [4, 6].
Therefore, there is a need to broaden the field of drugs available to patients dealing with burn
wounds.
1.4 Plants as a source of drug products
Plants have been a source of medication for people worldwide for many millennia. Although a large
portion of current conventional medicine has its roots in plant sources, it can be described as being
just the tip of the ice-berg, in terms of the potential they have as a source of bio-active compounds.
Research on plants as sources of medicinal compounds is therefore constantly taking place. From
common kitchen vegetables, such as garlic, to rare, exotic plants such as Japanese knotweed, plants
are a rich source of bioactive compounds, and can be used as building blocks for potential drugs [7].
There has, in addition, been an increase in consumer demand for plant based medicinal products.
This is due to several factors that include the increasing awareness of the harmful adverse effects
posed by synthetic drugs, the relative safety of plant-based medication and the relatively lower costs
of plant-based medications [8].
There is also a growing concern of resistance being acquired by invasive microorganisms towards
drugs currently used. An infamous example is the methicillin-resistant Staphylococcus aureus
(MRSA), a form of bacteria that is resistant to numerous antibiotics used including methicillin,
amoxicillin, penicillin and oxacillin, thus causing challenges in treatment of the infection. Hence
new drug developments are encouraged to stay ahead of microorganisms in the race against drug
resistance [9].
In this study, we have looked into the plant product of the essential oil of Rosemary, Rosmarinus
officinalis, as a potential antimicrobial agent against microorganisms that are commonly involved in
burn wounds infections.
3
CHAPTER TWO – LITERATURE REVIEW
2.1 Rosmarinus officinalis
Rosmarinus officinalis, commonly known as Rosemary, is an evergreen, perennial shrub, of the
Lamiaceae family, characterized by a unique aromatic odour. A native of the Mediterranean region,
Rosemary is now cultivated worldwide for its aromatic, medicinal and ornamental properties [10,
11].
Rosemary can grow to a height of 1m to 2m in favourable settings, with its erect stems dividing into
numerous long, slender branches that have an ash-coloured, scaly bark. Its rigid, opposite leaves are
about 3.5 cm long and 4 mm wide, appear dark green on top and pale grey-green on the underside
with a distinctive mid vein, and the leaves curl inward along the margins. Its numerous trichomes
make the lower leaf surface grey and woolly, while the typical labiate glandular hairs contain the
volatile oils. Its flowers are small and its colouring ranges from white, pale blue, deep blue to purple
[10, 11, 12, 13].
Other common names for the herb include polar plant, compass-weed, compass plant, dew of the
sea, garden rosemary, incensier, rusmari, Mary’s mantle, mi-tieh-hsiang, herb of crowns and old
man [14].
Figure 1 shows photographs of a Rosemary bush. Table 1 outlines the taxonomic classification of
Rosmarinus officinalis [14].
Table 1 – Taxonomic classification of Rosmarinus officinalis
Kingdom Plantae
Division Magnoliophyta
Class Magnoliopsida
Order Lamiales
Family Lamiaceae
Genus Rosmarinus
Species R. officinalis
Binomial name Rosmarinus officinalis
4
Figure 1 – Photographs showing Rosmarinus officinalis plant
Fresh material yields about 1-2 % of volatile oil containing 0.8-6 % esters and 8-20 % alcohols. At
least 20 compounds have been identified with its principal constituents as 1,8-cineole, borneol,
camphor, bornyl acetate and monoterpene hydrocarbons. It also contains rosmarinic acid and
several flavanoids [11, 12].
Spain is the largest exporter of rosemary. In the late 1940s, commercial development of rosemary
oils was attempted in Kenya, however political, social and economic factors in the early decades
after independence hampered the project along with other similar projects such as for those oils of
indigenous hardwoods (cedarwood, sandalwood) [15].
2.2 Uses of Rosemary
2.2.1 Traditional uses and folklore
Rosemary has long been used in the treatments of various ailments and thus was a favoured herb in
early apothecary gardens. Ancient Greek scholars would wear garlands of rosemary in their hair,
when engaged in study as an aid to increase their memory and concentration. It was believed that its
smell improved alertness and its aroma could make one feel more confident.
In the 14th century, Queen Isabella of Hungary used an alcohol extract of the flowering herb to treat
gout, and the formed concoction was named ‘Queen of Hungary’s Water’. This concoction was used
for centuries to treat dandruff, gout, skin problems and to prevent baldness. In France, rosemary
5
was hung in hospitals and sickrooms as healing incense and as a disinfectant. Also in France, the
‘Vinegar of Four Thieves,” a potion used by grave robbers for protection against plague, contained
Rosemary. Gypsy travellers sought rosemary for its use as a rinse for highlighting dark hair, or as a
rejuvenating facial wash [13, 16, 17, 18].
In Ayurvedic medicine, Rosemary essential oil was used as one of the standard inhalations for
treating respiratory disorders, sinusitis and gall bladder problems, depression, fear and fatigue.
Rosemary was often recommended especially for cases of low blood pressure. It is also effective in
stimulating menstrual flow and as an abortifacient [14, 19].
Orally, rosemary has also been taken for symptomatic relief of dyspepsia and mild spasmodic
disorders of the gastrointestinal tract [20].
In traditional Jordanian medicine, Rosemary has been used in the management and treatment of
skin wounds [21].
2.2.2 Culinary uses
Rosemary is most well-noted for its culinary use as a common household spice and a condiment. Its
leaves, both fresh and dried, are used in traditional Mediterranean cuisine. Their bitter, astringent
taste and highly aromatic nature complements a wide variety of foods, most popularly lamb.
Commercially, its fragrance is added to products such as frozen desserts, candy, alcoholic and non-
alcoholic beverages, puddings and various other similar goods [11, 12, 14].
The antibacterial and antioxidant activity of rosemary is used to extend the keeping quality of fats
and meat, while an antioxidant prepared from both sage and rosemary improves the stability of soy
oil and potato chips [11, 22].
2.2.3 Cosmetic, fragrance and industrial uses
Due to its aromatic nature, Rosemary is a major ingredient used in the preparation of Eau-de-
Cologne. Rosemary and its essential oil are also used as an ingredient in soaps, lotions, facial and
body creams, deodorants, hair tonics, and shampoos. One of the best known uses of rosemary oil is
that it serves as an extremely effective mouthwash. It is also used in many household cleaners,
candles and air fresheners and it can be included in potpourris or scented sachets or also burnt as
incense [11, 14].
6
It is a major constituent of some organic pesticides and is used as a ground-cover and garden plant.
It can be planted as hedge and is a good source of nectar for bees [23].
2.3 Precautions, side effects and interactions
Rosemary should be avoided in medicinal preparations during pregnancy or breast-feeding,
although it is safe to use in cooking in small quantities. Rosemary extract has shown to slightly
decrease the likelihood of conception but does not necessarily interfere with normal development of
the foetus after implantation. People with high blood pressure, epilepsy or diverticulosis, chronic
ulcers, or colitis, should not take the herb internally for medicinal purposes [11].
Relatively few interactions between Rosemary and conventional pharmaceutical products have been
reported. A notable interaction is that of Rosemary and doxorubicin, a cytotoxic used in cancer
management, where it appears to increase the effects of doxorubicin. Although further studies are
necessary, as of 2002, patients taking doxorubicin are advised to consult their physicians before
taking Rosemary [12].
An overdose of essential oil of Rosemary may lead to deep coma, vomiting, spasms, uterine
bleeding, gastroenteritis, kidney irritation, and even death, although no such cases have ever been
reported. Rosemary essential oil may be irritating to skin and eyes and some people may experience
hypersensitivity reactions. They could present with nausea and vomiting [20].
2.4 Previous studies done on Rosemary
Rosemary has been featured in numerous previous studies and has shown great potential as a source
of bioactive compounds. In a study by Al-Sereitia et al 1999, the pharmacological effects of the
aqueous extracts and essential oil were noted and therapeutic potential based on the observations
were suggested. These are outlined in Table 2 [24].
Further studies have shown that rosemary and its extracts possess hepatoprotective, antithrombotic,
diuretic, antidiabetic, antiinflammatory, antioxidant and anticancer effects [25, 26 27, 28, 29, 30].
The antimicrobial effects of the essential oil and extracts Rosemary, have also been studied. In a
study by Tanja Rožman et al, 2009, two selected rosemary extracts were tested for antimicrobial
activity against different species of Listeria using two commonly used methods: disk diffusion
7
method and broth dilution method. It was established that the resistance of Listeria species against
rosemary extracts depends on: selected extract, selected concentration, various species and strain of
Listeria [31].
Table 2 – Pharmacological actions and therapeutic potential of Rosemary
Pharmacological actions Therapeutic potential
1. Relaxation of bronchial smooth muscle Bronchial asthma
2. Relaxation of intestinal smooth muscle Antispasmodic
3. Reduction of leukotrienes and increase PGE2
production
Bronchial asthma, Peptic ulcer, Inflammatory
diseases
4. Inhibition of lipid peroxidation Hepatotoxicity Atherosclerosis and Ischaemic
heart disease, Inflammatory diseases,
Asthenozoospermia
5. Inhibition of the complement Inflammatory diseases
6. Prevention of the carcinogen-DNA adduct
formation
Cancer (protection)
In 2011, a study by Yang Jiang et al, on the time–kill dynamic processes of α-Pinene and Rosemary
essential oil were tested against three Gram-positive bacteria (Staphylococcus epidermidis,
Staphylococcus aureus and Bacillus subtilis), three Gram-negative bacteria (Proteus vulgaris,
Pseudomonas aeruginosa and Escherichia coli) and two fungi (Candida albicans and Aspergillus
niger). The essential oil showed pronounced antibacterial and antifungal activity compared to α-
Pinene against all of the tested microbes [32].
Fernanda Villas Boas Petrolini et al, in 2013, evaluated the antibacterial activity of the crude
hydroalcoholic extracts of rosemary against bacteria that cause urinary tract infections, i.e.
Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Enterobacter aerogenes, Pseudomonas
aeruginosa, Staphylococcus saprophyticus, Staphylococcus epidermidis and Enterococcus faecalis.
It was found that the extract led to promising results in the case of Gram-positive bacteria, resulting
in a considerable interest in the development of reliable alternatives for the treatment of urinary
infections [33].
8
Studies have also been carried out to investigate the healing properties of Rosemary of skin wounds.
In 2010, Abu-Al-Basal et al, demonstrated that Rosemary extracts were active in healing diabetic
wounds [21].
In 2012, Rahşan Yilmaz et al, compared the rate of wound healing in rabbits when treated with
Rosemary extract, povidone-iodine and isotonic saline solution and concluded that the wounds tend
to heal faster when treated with Rosemary extracts [34].
A similar study in 2014 by Nejati et al, showed that cutaneous wounds on rats had improved
healing when Rosemary essential oil was topically applied on the wounds [35].
A study on the effects of Rosemary and Chamomile extracts on burn wounds on rabbits also showed
accelerated healing. It is thought that the antioxidant properties of Rosemary essential oil aids in
accelerated healing [36].
Gas chromatographic analysis has been carried out on the American species of Rosemary. It has
been found to contain 21 constituents, the major ones being α-pinene, camphene, β-pinene,
eucalyptol, limonene, camphor and caryophyllene [37].
2.5 Previous studies on burns
Data from 1234 burn wound infections collected by National Nosocomial Infections Study System,
Centers for Disease Control and Prevention, USA, between the years 1980–1998, showed that
23.0% contained isolates of Staphylococcus aureus, Pseudomonas aeruginosa isolates made up
19.3%, Enterococci 11.0%, Enterobacter 9.6%, Escherichia coli 7.2% and Candida albicans made
up 3.5% of isolates [6].
In 2009, a study of patients with burn wounds at a Plastic and Reconstructive Surgery Clinic in
Bosnia-Herzegovina showed that 84.5% of patients had infected burn wounds. The most frequent
causes of infection in the control group of patients were Staphylococcus epidermidis (27.4%),
Staphylococcus aureus (21.6%), Pseudomonas aeruginosa (19.6%). It was also noted that the
presence of infection and antibiotic resistance of the isolated bacteria were the cause of a prolonged
hospitalisation as well as increased treatment costs of the patients with burn injuries [38].
9
A study in India, in 2010, concluded that burn wound infection is primarily caused by bacteria
(70%) followed by fungi (20–25%), anaerobic and virus (5–10%). However, a prior study from a
largest burn centre in Asia done on 220 burn patients gave a 42% positivity rate for isolation of
Candida species and a 10% as fungal wound infection [39].
In a Ugandan hospital, a similar study was carried out in 2011, where out of 103 burn patients
recruited in the study, 51 patients (49.5%) had fungal colonized burn wounds and histological
evidence of fungal infection was seen in 7 patients (6.8%). Aspergillus species was isolated from
35.3% and Candida Albicans in 31.4%. Other species included Candida Tropicalis (25.5%), other
non-Albican Candida (15.7%) and Penicillium (5.9%) [3].
2.6 Current burn wound treatment options
Current clinical guidelines for the care of burn wound surfaces include application of silver
sulphadiazene on burn surfaces as a topical antiseptic cream. The non-pharmacological
management techniques include, cleaning the wound with water or normal saline, frequently
changing wound dressings and early surgical debridement for dead tissue [40].
Silver-based compounds have been a major part of topical burn care since the early 1960s. They are
beneficial in that they aid to compress the inflammatory events in wounds and facilitate the early
phases of wound healing [5].
However, their use has been problematic in that they are known to cause various adverse drug
reactions. These include delayed wound healing, direct silver induced renal toxicity, transient
leucopenia which occurs within several days of the initiation of therapy and argyria that has been
reported after prolonged use. Hypersensitivity reactions may prove a contraindication for their use
in some patients [41].
2.7 Traditional medicines used for burn wounds and research for alternatives
Traditional burn wound dressings used in parts of India include boiled potato peel and banana leaf,
placed directly on the wound surfaces. An Ayurvedic dressing for burn wounds involves use of
alkanet pounded with oil & mixed with dried earthworms. Honey and Aloe vera extracts are also
popular home remedies for treating burn wounds. Current research for alternatives to silver
10
sulphadiazine have led researchers to believe that orange oil, tree tea oil and cinnamon oil have
potential to be used as antimicrobial agents to use in burn wounds [5, 42, 43, 44, 45, 46, 47, 48].
2.8 Justification
Burn wound infections are a major health problem faced by a large number of people, both locally
and worldwide. Its current forms of management need to be improved in several aspects, including
ultimately reducing the patients' hospitalization time and in the long term to reduce the occurrence
of disability and disfigurement, and to reduce the cost of treatment. The current predominant use of
silver sulphadiazine, presents its own set of challenges as discussed in the literature review, hence
the search for a better alternative is warranted.
Rosemary's usefulness as a traditional medicine encourages more research to be undertaken on the
plant. Previous studies have showed antifungal and antibacterial activity, however no studies have
been carried out in the Kenyan species. By carrying out the study, it can provide the backbone for
further investigations on the plant, including in vivo studies. This will help broaden the field of
drugs available to patients, much to their benefit.
Rosemary being a hardy plant that easily grows around the country, making it a viable raw material
for prospective drugs, as it is easy to access and its essential oil is easy to extract. It will also
provide as a cheaper alternative to currently used drugs, which is especially relevant considering
burn risk correlates with socioeconomic status.
2.9 Research question
Does the Kenyan Rosemary essential oil have antibacterial and antifungal activity against common
microorganisms involved in burn wound infections?
2.10 Hypothesis
Rosemary essential oil has antibacterial and antifungal activity against common microorganisms
involved in burn wound infections.
11
2.11 Objectives
2.11.1 General objectives
The major objective of this work was to evaluate the antimicrobial activity of the Kenyan
Rosmarinus officinalis variety against bacteria and fungi commonly found in burn wound
infections.
2.11.2 Specific objectives
1. To extract Rosemary essential oil from fresh leaves of the plant using a Clevenger-like
apparatus
2. To screen for the in vitro antimicrobial activity of the oil against bacteria and fungi
commonly found in burn wounds infections.
3. To carry out gas chromatography on Rosemary oil to ascertain its constituents.
12
CHAPTER THREE – EXPERIMENTAL
3.1 Plant collection and identification
The leaves of Rosmarinus officinalis were collected from a private residence in South B, Nairobi
and identified by Mr. J. Mwalukumbi from the Department of Pharmacology and Pharmacognosy,
School of Pharmacy, University of Nairobi.
3.2 Extraction of oil
Rosemary essential oil was extracted in two separate batches, both using fresh Rosmarinus
officinalis leaves picked from the same bush from a private residence in South B, Nairobi. The first
batch was prepared on the 15th of April 2015, using about 412g of fresh leaves and 700 ml of
distilled water. The leaves had been picked the previous evening and had been refrigerated
overnight before the extraction. The oil collected was used in the microtitre dilution screening.
The second batch of oil was extracted on the 11th of August 2015. The leaves were picked in the
morning of the extraction. About 522g of fresh leaves and 800ml of distilled water was used. The
oil of this batch was used to carry out screening by disk diffusion in Petri dishes and for gas
chromatography.
Both extractions used the same procedure where the fresh Rosemary leaves were filled into a round
bottomed flask and distilled water was added. The flask was connected to a Clevenger-like
apparatus which was in turn connected to a condenser with a cold water inlet and warm water
outlet. The set-up was transferred onto a heating mantle (Electrothermal, Staffordshire, England)
and heated gently for one hour. Oil was collected from the oil collection tap into an amber coloured
bottle with a tight lid. It was stored in a refrigerator.
Figures 2a and 2b show the set up of apparatus involved in oil extraction.
The actual weights of fresh plant material used to extract the oil are shown in Table 3.
Table 3 – Weight of plant material used in oil extraction
Weight of Plant
material + Bag (g)
Weight of Empty
Bag (g)
Weight of Plant
material (g)
1st Extraction – April 2015 422.91 10.87 412.04
2nd Extraction – August 2015 545.32 23.11 522.21
13
Figure 2a – Photograph showing set up used in oil extraction
14
Figure 2b – Photograph showing Clevenger-like apparatus and layer of oil formed during
extraction
3.3 Procedure for screening
3.3.1 Materials and apparatus
For the microtitre dilution screening, working cultures of the microorganisms Saccharomyces
cerevisiae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and
Staphylococcus aureus were used. Tryptic Soy Broth (TSB) (Scharlau, Barcelona, Spain) was used
as culture media for the bacteria, while Sabouraud Dextrose Broth (SDB) (Sigma-Aldrich, Buchs,
Switzerland) was used as culture media for Saccharomyces cerevisiae. Nystatin standard of
concentration 0.3 mg/ml was used as a positive control against the fungi, while gentamicin standard
of 0.5 mg/ml concentration was the positive control against the bacteria. Tetrazolium GR (MTT
dye) (Loba Chemie, Mumbai, India) was used in later stages of the screening for visualisation. The
screening was carried out in two 96-well plates (Becton Dickinson Labware, Massachusetts, USA).
For the screening using disk diffusion in Petri dishes, working cultures of the microorganisms
Saccharomyces cerevisiae, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus
to create subcultures. Tryptone Soya Agar (TSA) (Fluka, Lausanne, Switzerland) and Sabouraud
Dextrose Agar (SDA) (Oxoid, Hampshire, England) were used as culture media for the bacteria and
the fungi respectively. Nystatin standard of concentration 0.3 mg/ml and gentamicin standard of
concentration 0.3 mg/ml were used as positive controls. It also required the use of a handheld
vernier calliper for measuring the diameters of the zones of inhibition.15
Both procedures required the use of a top loading electronic balance (Mettler, Toledo, Switzerland)
for weighing the dry culture media, alongside a portable autoclave (Winconsin Aluminium Foundry
Co., Manitowoc, USA) for sterilizing the culture media and glassware, a 20μl micropipette
(Eppendorf, Hamburg, Germany) and a 50μl micropipette (Thermo Labsystems, Hanover,
Germany) together with micropipette filter tips for introducing samples. Pure DMSO (100%) was
used as the negative control in both screenings.
3.3.2 Preparation of standards
To prepare the positive control standards, 0.0024g of gentamicin sulphate standard powder was
dissolved in 0.5 ml of distilled water to obtain an equivalent concentration of 5mg/ml gentamicin.
The nystatin standard was prepared by dissolving 0.0031g of nystatin standard powder in 1ml of
dimethylsulphoxide (DMSO) to get a concentration of approximately 3mg/ml.
The positive controls for the disk diffusion screening involved dissolving 0.0061g of gentamicin
sulphate standard in 2 ml of distilled water to obtain an equivalent concentration of 3mg/ml of
gentamicin whereas the nystatin standard was prepared by dissolving 0.0027g of nystatin standard
powder in 1ml of DMSO to get a concentration of approximately 2.7mg/ml.
3.3.3 Subculturing of microorganisms
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and Staphylococcus
aureus were subcultured on TSA and were incubated overnight at 37°C, while Saccharomyces
cerevisiae was subcultured on SDA and incubated overnight at 25°C.
3.4 Microtitre dilution screening using 96-well plate
3.4.1 Preparation of test solutions
Five dilutions of the Rosemary oil were used in the screening. They were made using 20μl and 50μl
volume micropipettes and were stored in air-tight amber glass bottles. The various dilutions were
prepared using DMSO as shown in Table 4.
Table 4 – Dilutions of Rosemary oil used in microtitre dilution screening
100% 80% 60% 40% 20%
Volume Of Oil (μl) 300 240 180 120 60
Volume Of DMSO (μl) 0 60 120 180 240
Total Volume (μl) 300 300 300 300 300
16
3.4.2 Screening
A sterile broth was prepared by dissolving 3.212g of TSB and 1.223g of SDB each in 100ml of
distilled water, and autoclaved for 2 hours. Once cooled to 55°C, the 10ml of broth was transferred
each into 5 sterile bottles and 100μl of the respective inoculum was added. The inoculum was
prepared by adding 5ml of sterile distilled water to each of the subculture containing test tubes.
200μl of the inoculated broth was then transferred to wells in the 96-well plate. 50μl of each of the
dilutions of the Rosemary oil/standards were added to the selected wells. The plates were incubated
overnight, the bacteria-containing plate, Plate 1, was incubated at 37°C, while the fungi-containing
plate, Plate 2, was incubated at 25°C. The following day, 50μl of MTT dye, of concentration
2.5mg/ml in DMSO, was pipetted into the wells, incubated for 2 hours and later observed. All the
steps involving microorganisms were carried out aseptically under laminar air-flow cabinet. The
lay-out of the wells of the plates was as arranged in Tables 5 to 7.
Table 5 – Arrangement of Plate 1 – Antibacterial activity of Rosemary oil
100% Oil 80% Oil 60% Oil 40% Oil 20% Oil - Positive
Control
(Gentamicin)
Negative
Control
(DMSO)
Staphylococcus
aureus
Staphylococcus
epidermidis
Pseudomonas
aeruginosa
Escherichia coli
17
Table 6 – Arrangement of Plate 2 – Antifungal activity of Rosemary oil
100%
Oil
80%
Oil
60%
Oil
40%
Oil
20%
Oil -
Positive
Control
(Nystatin)
Negative
Control
(DMSO)
Saccharomyces
cerevisiae
Sterile broth
Table 7 – Key for Tables 5 & 6
Well
Colour
Well Contents
Empty well
Well containing only inoculated broth
Well containing inoculated broth + Oil
Well containing inoculated broth + Positive Control (Gentamicin/Nystatin)
Well containing inoculated broth + Negative control (DMSO)
Well containing only sterile broth
Well containing sterile broth + Oil
Well containing sterile broth + Positive Control (Nystatin)
Well containing sterile broth + Negative control (DMSO)
3.5 Antimicrobial screening by disk diffusion
3.5.1 Preparation of test solutions
Three dilutions of the Rosemary oil were prepared using 50μl volume micropipettes and stored in
18
air-tight amber glass bottles. The oil was diluted using DMSO as described in Table 8.
Table 8 – Dilutions of Rosemary oil used in Petri dish screening
100% 50% 25%
Volume Of Oil (μl) 300 150 75
Volume Of DMSO (μl) 0 150 225
Total Volume (μl) 300 300 300
3.5.2 Screening
The culture media were first prepared by weighing out 4.012g of TSA and suspending it in 100ml of
distilled water, and weighing out 3.305g of SDA and suspending it in 50ml of distilled water. The
media was then autoclaved for 2 hours and allowed to cool to 55°C. Inoculum was prepared by
adding 5ml of sterile and distilled water to the subcultured test tubes. The culture media was
divided into portions of 20ml and inoculated with 0.2ml of inoculum. The inoculated agar was
poured into a Petri dish and allowed to set. Five wells were cut out from the set agar and using a
50μl micropipette, were filled with the oil sample/standard. The dishes were incubated overnight.
The following morning the diameter of the zones of inhibition were measured. The general lay-out
of the samples on the Petri dishes is shown in Figure 3.
Figure 3 – General lay-out of samples on the Petri dishes
19
3.6 Phytochemical tests
The phytochemical tests were carried out as prescribed by B.Pharm Third Year Pharmacognosy
practical manual, Department Of Pharmacology and Pharmacognosy, School Of Pharmacy,
University of Nairobi.
3.6.1 Drying and milling
Approximately 100g of Rosemarinus officinalis leaves were dried in an oven (Memmert,
Schwabach, Germany) two days at 30°C. A Dade DFT-50 (Bean product, Shanghai, China) grinding
machine was used to pulverize the dried leaves into coarse powder.
3.6.2 Reagents and materials
For testing the presence of alkaloids, 10% sulphuric acid solutions were used, along with Mayer's
reagent. Mayer’s reagent was obtained by mixing about 1.36 g of mercuric chloride and about 5g of
potassium iodide in 100 ml of water.
The presence of glycosides was determined using 70% alcohol, 10% sulphuric acid and lead sub
acetate solution to initially extract the glycosides. The reagent, 2% 3, 5-dinitrobenzoic acid in 90%
alcohol was used to determine the presence of unsaturated lactone ring of the aglycone moiety of
glycosides. Glacial acetic acid containing trace quantities of ferric chloride was used to determine
the presence of 2-deoxy sugar moiety in glycosides. Cyanogenic glycoside presence was
determined using sodium nitrate paper.
Lead sub acetate, ferric chloride, potassium dichromate and atropine solutions were used during the
testing procedure for tannins. Distilled water was used to determine the presence of saponins.
3.6.3 Test for alkaloids
To one gram of powdered Rosmarinus officinalis, 10 ml of 10% sulphuric acid was added, and
warmed for 5 minutes over a water bath. This was then filtered and a portion tested with the
addition of 2 drops of Mayer's reagent. Precipitation would indicate the presence of alkaloids.
3.6.4 Tests for glycosides
About one gram of the powdered Rosmarinus officinalis was extracted with 10 ml of 70 % alcohol ,
heated for 2 minutes in a water bath and allowed to cool, then filtered. To the filtrate, 10 ml of water
20
and 5 drops of strong solution lead sub acetate was added, filtered and later, 10 % sulphuric acid
was added. This was then filtered and extracted using chloroform. The chloroform extract was then
divided into two parts to be used in the Kedde and Keller-Killian tests.
i. Kedde test – Test for unsaturated lactone ring of the aglycone
The chloroform extract was evaporated to dryness, one drop of 90 % alcohol and 2 drops of
2 % 3, 5-dinitrobenzoic acid in 90 % alcohol was added followed by 20 % sodium
hydroxide. A purple colour was expected if unsaturated lactone ring was present.
ii. Keller-killian test – Test for 2-deoxy sugar
The chloroform extract was evaporated to dryness, about 0.4 ml of glacial acetic acid
containing trace quantities of ferric chloride was added followed by 0.5 ml of concentrated
sulphuric acid. A green-blue colour was expected in the upper acetic acid layer if deoxy
sugars were present.
iii. Test For cyanogenic glycosides
About 1.5g of Rosmarinus officinalis powder was place with a few drops of water in a
stoppered test tube containing a strip of sodium nitrate paper. The test tube was warmed
gently and change of colour of the sodium nitrate paper from yellow to red-brown would
indicate the presence of cyanogenic glycosides.
3.6.5 Test for tannins
In about 20 ml of water, 2g of the powdered Rosemary sample was boiled, cooled and then filtered.
To separate samples of about 2 ml of the filtrate, a few drops of ferric chloride solution was added,
a green precipitate was expected if tannins were present. About 1 ml solution of lead sub acetate
solution was added. A white precipitate was expected if tannins were present. About 1 ml of
potassium dichromate solution was added, an orange precipitate was expected to give positive
results for tannins. About 1 ml of atropine solution was added, a white solution was expected to
give positive results for tannins.
3.6.6 Test for saponins
A little of the powdered drug was shaken vigorously with water. Persistent frothing would indicate
the presence of saponins.
21
3.7 Gas chromatographic analysis
3.7.1 Gas chromatography using a flame ionization detector
Gas chromatography was carried out on the sample of Rosemary essential oil extracted in August
2015 using a GCMS-QP2010 ultra chromatograph (Shimadzu corporation, Tokyo, Japan). The
conditions of chromatography included using ZB-WAX Plus® capillary column (Phenomenex,
USA) 60m length, 0.25mm internal diameter and 0.25μm film thickness. The oven profile started at
45ºC for 2 minutes, ramped to 130ºC at 8ºC/min, then ramped to 200º at 30ºC/min and held for 2
minutes. The carrier gas used was nitrogen with a total flow of 77ml/min. The injection was
splitless, and the amount injected was 1μl and the oil was diluted in dichloromethane. The injector
temperature and detector temperature was 200ºC. A flame ionization detector (FID) was used. The
procedure was run for 14.96 minutes. This procedure was similar to that used in analysing the
American variety of Rosemary oil.
3.7.2 Gas chromatography using a mass spectrometer detector
The same batch of oil was also subjected to a different conditions and detector. The oven profile
started and was held at 60ºC for 1 minute, then ramped to 190ºC at 10ºC/min, held for 10 minutes,
then ramped again by 10ºC/min to 220º at for 15 minutes. The total analysis time was 37 minutes.
The carrier gas used was helium with a total flow of 93ml/min. The injection was splitless, and the
amount injected was 1μl and the oil was diluted in dichloromethane. The detector used was a mass
spectrometer. The injector temperature, ion source temperature and interface temperature was
240ºC. The ion source used electron impact method at 70KeV. The scan range was between m/z 35
to m/z 500. This method is known as the Cacheca method.
The data from both runs was analysed using the GCMS Solution software (Shimadzu corporation,
Tokyo, Japan).
22
CHAPTER FOUR – RESULTS
4.1 Nature and volume of oil collected
The oil from the first extraction had a sharp odour and was pale yellow in colour. The volume of oil
collected was measured using a graduated 5ml plastic syringe. It was found to be 1.1 ml.
The oil from the second extraction had a sharp odour and was clear in appearance. Its volume was
3.1 ml.
4.2 Percentage yield
The percentage yield was calculated using the formula as:
Yield = Volume Of Oil x 100%
Weight Of Leaves Used
The values of yield were used to calculate the average yield using the following formula:
Average Yield = Yield of first extraction + Yield of second extraction
2
The calculated yields are shown in the Table 9.
Table 9 – Percentage and average yield of Rosemary oil
Yield
First Yield (April 2015) 0.26%
Second Yield (August 2015) 0.59%
Average Yield 0.43%
4.3 Results for microtitre dilution screening
The positive control wells had clear yellow colour indicative of no viable microbes’ presence, while
deep purple was observed in the negative control wells indicating the presence of viable microbes.
Figure 4a shows the plates before incubation with MTT dye, while Figure 4b while captured the
varied shades of yellow-purple wells. Plate 1 contained bacteria, while Plate 2 contained the fungi,
the arrangement of their wells is described in Tables 5 – 7.
23
Figure 4a – Photograph of 96-well plates before incubation with MTT dye – Plate 1 (right),
Plate 2 (left)
Figure 4b – Photograph of 96-well plates after incubation with MTT dye – Plate 1 (right),
Plate 2 (left)
24
4.4 Antimicrobial screening in Petri dish
All the dilutions of Rosemary oil and the positive control produced clear zones of inhibition in the
inoculated agar within the Petri dishes, whereas the negative control produced no clear zones as
shown in Figure 5. The diameters of these zones were measured using a vernier calliper and
diameters of the zones of inhibition are shown in Table 10.
Figure 5 – Photographs of Petri-dishes showing zones of inhibition created by Rosemary oil
25
Table 10 – Diameters of zones of inhibition
Inhibition Zone Diameter (mm)
100% Oil 50% Oil 25% Oil Positive
control
Negative
control
Saccharomyces
cerevisiae
18.36 13.60 10.88 25.84 0.00
Staphylococcus
aureus
11.53 9.71 8.50 21.25 0.00
Pseudomonas
aeruginosa
12.04 10.62 9.92 24.79 0.00
Escherichia coli 11.53 12.14 10.93 19.42 0.00
By comparing the diameters of the zones produced by the various dilutions of oil to those produced
by the positive controls at the tested concentrations, the relative potency of the oil dilutions was
calculated as shown in Table 11.
Table 11 – Relative potency of Rosemary oil dilutions compared to positive control
Potency Relative To Positive Control (%)
100% Oil 50% Oil 25% Oil Positive
control
Negative
control
Saccharomyces
cerevisiae
71.06 52.63 42.12 100 0.00
Staphylococcus
aureus
54.28 45.71 40.00 100 0.00
Pseudomonas
aeruginosa
48.57 42.86 40.00 100 0.00
Escherichia
coli
59.38 62.49 56.25 100 0.00
26
4.5 Results of phytochemical tests
The results of the phytochemical tests are shown in Table 12.
Table 12 – Results of phtyochemical tests
Test Observation Inference
1) Test for alkaloids using Mayer’s reagent
No precipitate formedNo alkaloid were present
2) Test for saponins No persistent frothing seen Saponins were not present
3) Test for tannins using:
Tannins were present
Ferric chloride Green precipitate was observed
Lead sub acetate White precipitate was formed
Potassium dichromate No precipitate observed
Atropine No precipitate observed
4) Test for glycosides:
Trace glycosides including cyanogenic glycosides were present.
Kedde Test Light purple colour seen
Keller-killian Test No colouring seen
Cyanogenic glycoside Brown colouring of sodium nitrate paper seen
4.6 Gas Chromatography
4.6.1 Gas chromatography using a flame ionization detector
The chromatogram produced using a flame ionization detector indicated the presence of 23
compounds in the Rosemary essential oil. Five of these compounds produced major peaks as shown
in Figure 6a. It can be compared to Figure 6b that shows a gas chromatogram of the American
variety of Rosemary oil using a mass spectrum detector that yielded 21 constituents.
27
5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min
0.0
1.0
2.0
3.0
4.0
5.0
uV(x1,000,000) Chromatogram
Figure 6a – Gas chromatogram of Rosemary oil
Figure 6b – Gas chromatogram of American variety of Rosemary oil
The chromatographic conditions both analysis procedures were similar. These included using a
chromatographic column of 60m length, 0.25mm internal diameter and 0.25μm film thickness. The
oven profile started at 45ºC for 2 minutes to 130ºC at 8ºC/min to 200º at 30ºC/min for 2 minutes.
The carrier gas used was nitrogen with a total flow of 77ml/min. The injection was splitless, and the
amount injected was 1μl and the oil was diluted in dichloromethane. The injector temperature and
detector temperature was 200ºC.
28
4.6.2 Gas chromatography using a mass spectrometer detector
The chromatogram produced using the mass spectrometer detector indicated the presence of 27
compounds. Using a virtual library the constituents were named and were compared to the
constituents of the American variety in Table 13. Figure 6c shows the mass spectrum chromatogram
produced. Figure 6d shows the gas chromatogram – mass spectrum total ion current of the oil.
Table 13 – List of constituents of Rosemary oil (Based on order of elution)
Constituent
Number
Constituent of Kenyan variety of
Rosemary oil
Constituent of American variety of
Rosemary oil
1 Tricyclene Tricyclene
2 alpha-Thujene alpha-Thujene
3 alpha-Pinene alpha-Pinene
4 Camphene Camphene
5 beta-Pinene beta-Pinene
6 4-terpenenyl acetate beta-Myrcene
7 cis-pinen-3-ol Eucalyptol
8 beta-Myrcene Limonene
9 alpha-Phellandrene Terpinene
10 alpha-Terpineol Terpinolene
11 alpha-Terpinene Linalool
12 D-Limonene Camphor
13 Eucalyptol Isoborneol
14 gamma-Terpinene Borneol
15 Cymene 4-Terpineol
16 (+)4-Carene Terpineol
17 beta-Terpineol Bornyl acetate
18 D-Linalool Eugenol
19 Bornanone Copaene
20 Yomogi alcohol Caryophyllene
21 3-Pinanone alpha-Caryophyllene
22 Isobornyl acetate
23 4-Terpineol
24 Caryophyllene
25 Myrcenol
26 Borneol
27 L-Verbinone
29
Figure 6c – Mass spectrum gas chromatogram of Rosemary oil
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
(x10,000,000)TIC
Figure 6d – GC-MS total ion current of Rosemary oil
By comparing the contents of both oil varieties made in Table 13, it was deduced that the
compounds common to both the Kenyan and American varieties include tricyclene, alpha-thujene,
alpha-pinene, camphene, beta-pinene, beta-myrcene, eucalyptol, limonene, terpinene, linalool,
borneol, 4-terpineol, terpineol, bornyl acetate, caryophyllene and alpha-caryophyllene.
The compounds present only in the Kenyan variety include 4-terpenenyl acetate, cis-pinen-3-ol,
alpha-phellandrene, cymene, carene, bornanone, yomogi alcoho, l3-pinanone, isobornyl acetate,
myrcenol and l-verbinone, while those found solely in the American variety include copaene,
eugenol, isoborneol, camphor and terpinolene.
30
CHAPTER FIVE – DISCUSSION
The obtained average yield of 0.43% is very small when compared to American and European
varieties whose fresh material yields about 1-2 % essential oil. This means it might not be very
economical to extract Rosemary oil from the Kenyan variety of the species.
However, the yield from the second extraction, carried out in August, was more than double than
the yield obtained in the first extraction in April. This can be attributed to seasonal variations or the
different timings at which the plant material was harvested.
From phytochemical tests it was found that Rosemary contained tannins and glycosides. It
contained no saponins or alkaloids.
The choice of microorganisms was based on studies carried out on burns discussed in the literature
review. It was noted that Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis
and Staphylococcus aureus were among the leading causal bacteria of infections in burn wounds.
Hence the four were chosen for the project, and also they represent both Gram negative and Gram
positive bacteria. The leading fungal cause of infections is the Candida species. The unavailability
of its working culture during the study period led to the use of Saccharomyces cerevisiae to
represent an example of the fungi that cause infections in burn wounds.
The microtitre dilution screening was carried out with an objective of clearly observing either wells
containing viable microbes (dyed purple by MTT dye) or wells containing no viable microbes
(yellow). However, after incubation, it became apparent that the Rosemary oil dilutions did not
produce an all-or-nothing type of response but instead produced a degree of activity relative to the
positive standards' activity. This could not be elucidated by the naked eye, hence needed to be
analysed by a UV spectrophotometer. The spectrophotometer however was not functional during the
study period. The disk diffusion technique in Petri dishes was then employed to screen for activity
that could easily be visualised by the naked eye as zones of inhibition.
The microtitre dilution screening was initially chosen because of its advantages over the disk
diffusion method, in that it could simultaneously test the activities of several dilutions of oil and
standards against a number of different microorganisms within the same plate, with minimum
chances of contamination.
31
The inhibition due to the oil samples was seen as clear zones in the inoculated agar comparable to
those produced by the positive standards. By comparing the diameters of the zones produced by the
dilutions of oil to those produced by the positive controls at the tested concentrations, the relative
potency of the oil dilutions was calculated. The relative potencies showed that Rosemary oil has a
fraction of the potency of broad spectrum antibiotics. The lower potency could be undesirable as it
may promote resistance in micro-organisms. However it is promising as natural products do tend to
have a lesser degree of activity compared to commercial antibiotics hence warranting further study.
Gas chromatographic analysis of Rosemary oil of the Kenyan variety showed that the oil has 27
different compounds present in comparison to 21 found in the American variety. Notably, camphor,
a major constituent of the American variety was not present in the Kenyan variety.
The major constituents of the Kenyan variety of Rosemary oil include, pinene, camphene,
eucalyptol, limonene and caryophyllene.
Previous studies discussed in the literature review show that the antibacterial and antifungal activity
of Rosemary oil has been attributed to alpha-pinene. The compound is also present in the Kenyan
variety hence maybe responsible for its antimicrobial activity.
32
CHAPTER SIX – CONCLUSION
6.1 Conclusion
Rosemary oil was extracted from fresh plant material using a Clevenger-like apparatus. It was
shown to have antimicrobial activity against bacteria and fungi that commonly cause burn wounds
infections, showing highest activity against the fungi Saccharomyces cerevisiae.
Gas chromatographic analysis showed that the Kenyan variety of Rosemary oil is made of 27
constituent compounds, most notably, pinene, camphene, eucalyptol, limonene and caryophyllene.
The antimicrobial activity of the oil may be due to the presence of pinene.
6.2 Recommendations
The study proved that the Kenyan variety of Rosemary essential oil possesses antimicrobial activity
against microbes that cause burn wound infections. An in vivo assay is recommended to ascertain
effectiveness of the oil on living tissue.
It is also recommended that various formulations be made using the oil, such as incorporating it in a
cream or aerosol for application on the wound.
Based on the traditional use of Rosemary in French hospitals as a healing and disinfecting incense,
Rosemary oil could be used as a disinfectant spray in rooms of burn patients, to control the
microorganisms present in the environment around the patients.
33
REFERENCES
1. http://www.who.int/mediacentre/factsheets/fs365/en/ WHO | Burns - accessed on 31st March
2015
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