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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