DOCTOR OF PHILOSPHY IN CHEMISTRY

native medicinal plants
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSPHY
PAKISTAN
2
2015
DECLARATION
I hereby declare that the contents of the thesis, “Chemical composition, biological
and pharmaceutical potential of essential oils from native medicinal plants” is
product of my own research and no part has been copied from any published
source (except the references, standard mathematical or genetic models/ equations/
formulate/ protocols etc). I further declare that this work has not been submitted
for the award of any other diploma/ degree. The University may take actions if the
information provided is found inaccurate at any stage.
--------------------------------------
Faisalabad
“We, the supervisory committee, certify that the contents and form of thesis submitted
by Arfaa Sajid Regd. No. 2007-ag-71 have been found satisfactory and
recommend that it be processed for evaluation by the External Examiner(s) for the
award of degree”.
Affection
5
ACKNOWLEDGEMENTS
In the name of almighty Allah, the merciful, the beneficent, most merciful, the lords of
the lords, who guide us in difficulties and blessed me with courage and power to complete
this thesis.
I would like to take this opportunity to convey my cordial gratitude, and appreciation to
my kind, respected and worthy supervisor Dr. Raja Adil Sarfraz, Assistant professor,
Department of Chemistry, University of Agriculture, Faisalabad for his valuable and skilled
guidance which always helped me to carry in with my research work. I would like to pay
my deepest gratitude and appreciation to one of the member of my supervisory committee
Dr. Muhammad Asif Hanif Assistant professor, Department of Chemistry, University of
Agriculture, Faisalabad. I am also extremely thankful to Dr. Muhammad Shahid
Assistant professor, Department of Biochemistry, University of Agriculture, Faisalabad for
his generous corporation and providing valuable suggestions during accomplishment of my
PhD studies.
I ardently extend my special thanks to Professor Dr Bharat B. Aggarwal for his warm
hospitality, dedicated cooperation and encouragement during the part of my PhD in
Experimental therapeutic laboratory, MD Anderson cancer center, university of Texas,
Houston, USA. I am also very thankful to Qaisar Manzoor, Dr. Amit k Tyagi, Dr.
Sahdeo Parsad and Dr. Shinjini Singh to be very much cooperative and supportive during
my stay in USA.I am very grateful to my friends, to be very much cooperative, meanwhile
thanks to other laboratory fellows and all Hi-Tech staff for their cooperation.
I am most earnestly obliged to my Mother and Father for the strenuous efforts done by
them in enabling me to join the higher ideas of life and always wished to see me glittering
high on the skies of success like glories. I am very thankful to my husband, Qaisar
Manzoor and my sisters Anam Sajid and Najum Fatima for their ingenious cooperation
and guidance during my whole study period.
Finally thanks to all those who taught me ever one word in life.
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Chapter 2 REVIEW OF LITERATURE 9
2.1 Pinus roxburghii 10
2.1.2 Chemical composition 11
2.1.3 Biological testing 11
2.1.4 Anticancer activity 13
2.2 Lantana camara 13
2.3 Cymbopogon flexuosus 17
2.4 Citrus pseudolimon 20
2.5 Thuja orientalis 23
2.6.1 Ethno botanical uses 27
7
2.8 Callistemon viminalis 32
2.9 Anethum graveolens 35
3.1 Materials 39
3.1.2 Instruments 39
3.1.4 Strains of microorganisms utilized to access the antimicrobial activity
of essential oils
41
3.1.5 Cancer cell lines used to detrmine the anticancer potential of essential
oils
42
3.3 Physical analyses 42
3.5.1 Evaluation of antioxidant activity of essential oils 43
3.5.1.1 DPPH• radical scavenging activity 43
3.5.1.2 β-Carotene/linoleic acid bleaching assay 44
3.5.1.3 Determination of total phenolic content 44
3.5.2 Evaluation of anticancer potential of essential oils 41
3.5.2.1 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
3.5.2.3 Clonogenic assay 46
3.5.2.4 Apoptosis assay 46
3.5.2.6 Western blot analysis 47
3.5.2.7 Electrophoretic Mobility Shift Assay (EMSA) for Nuclear Factor-kB 48
3.5.3 Assessment of antimicrobial activity of essential oils 49
3.5.3.1 Disc diffusion assay 49
3.5.3.2 Agar well-diffusion assay 49
3.5.3.3 Determination of minimum inhibitory concentration (MIC) 49
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Chapter 4 RESULTS AND DISCUSSION 51
4.1 Yield (g 100g-1) and physical properties of essential oils 51
4.2 Total phenolic contents in essential oils 52
4.3 Evaluation of antioxidant activity of essential oils 54
4.3a DPPH• radical scavenging activity 54
4.3b β-Carotene/linoleic acid bleaching ability 56
4.4 Evaluation of anticancer potential of essential oils 57
4.4.1 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
4.4.3 Essential oils induce apoptosis in colon cancer cells 72
4.4.4 Effect of essential oils on colony formation 82
4.4.5 Propidium Iodide (PI) Staining for Apoptotic Cells 87
4.4.6 Essential oils down modulate the gene expression associated with
Survival, Proliferation and Metastasis
4.4.7 Essential oils suppress NF-κB activation in tumour cells 94
4.5 Assessment of antimicrobial activity of essential oils 98
4.6 Chemical composition of essential oils by GC/MS 107
4.6.1 Pinus roxburghii 107
4.6.2 Lantana camara 110
4.6.3 Cymbopogon flexuosus 113
4.6.4 Citrus pseudolimon 116
4.6.5 Thuja orientalis 118
4.6.7 Schinus terebinthifolius Raddi 123
4.6.8 Callistemon viminalis 126
4.6.9 Anethum graveolens 128
Chapter 5 SUMMARY 131
Chapter 6 REFRENCES 133
Table Title Page 2.1 Biological classification of Pinus roxburghii 12
2.2 Biological classification of Lantana camara 14
2.3 Biological classification of Cymbopogon flexuosus 18
2.4 Biological classification of Citrus pseudolimon 21
2.5 Biological classification of Thuja orientalis 24
2.6 Biological classification of Alpinia allughas 26
2.7 Biological classification of Schinus terebinthifolius 29
2.8 Biological classification of Callistemon viminalis 33
2.9 Biological classification of Anethum graveolens 35
3.1 Instruments used with their model and company 40
3.2 Plants studied in present research 41
4.1.1 Yield and physical properties of essential oils of selected medicinally
active plants 51
4.3.1 Antioxidant activity of of essential oils of selected medicinally
active plants 54
4.6.7 Chemical composition of Schinus terebinthifolius essential oil 123 4.6.8 Chemical composition of Callistemon viminalis essential oil 126 4.6.9 Chemical composition of Anethum graveolens essential oil 128
10
LIST OF FIGURES
Figure Title Page
no. 1.1 Possible route for EOs-mediated cancer cell death 6 2.1 Pinus roxburghii 10 2.2 Lantana camara 13
2.3 Cymbopogon flexuosus 17
2.4 Citrus pseudolimon 20
2.5 Thuja orientalis 23
2.6 Alpinia allughas 26
2.7 Schinus terebinthifolius 29
2.8 Callistemon viminalis 32
2.9 Anethum graveolens 35
4.4.1.1 Cytotoxicity (percent cell viability) of Pinus roxburghii essential oil against
different human cancer cell lines by MTT 57
4.4.1.2 Cytotoxicity (percent cell viability) of Lantana camara essential oil against
4.4.1.3 Cytotoxicity (percent cell viability) of Cymbopogon flexuosus essential oil
against different human cancer cell lines by MTT 59
4.4.1.4 Cytotoxicity (percent cell viability) of Citrus pseudolimon essential oil against
4.4.1.5 Cytotoxicity (percent cell viability) of Thuja orientalis essential oil against
4.4.1.6 Cytotoxicity (percent cell viability) of Alpinia allughas essential oil against
different human cancer cell lines by MTT
62
63
against different human cancer cell lines by MTT
64
different human cancer cell lines by MTT
65
4.4.2.1 Cytotoxicity (percent cell death) of Pinus roxburghii essential oil against
KBM-5 cells lines by Trypan Blue Exclusion assay
67
4.4.2.2 Cytotoxicity (percent cell death) of Lantana camara essential oil against
KBM-5 cells lines by Trypan Blue Exclusion assay 68
4.4.2.3 Cytotoxicity (percent cell death) of Cymbopogon flexuosus essential oil against
4.4.2.4 Cytotoxicity (percent cell death) of Citrus pseudolimon essential oil against
4.4.2.5 Cytotoxicity (percent cell death) of Thuja orientalis essential oil against
4.4.2.6 Cytotoxicity (percent cell death) of Alpinia allughas essential oil against
70
11
70
71
4.4.2.9 Cytotoxicity (percent cell death) of Anethum graveolens essential oil against
71
oil in Live/Dead assay 73
4.4.3.1b Percentage Apoptosis by Pinus roxburghii essential oil against
HCT-116 cancer cells in Live/Dead assay
73
oil in Live/Dead assay 74
4.4.3.2b Percentage Apoptosis by Lantana camara essential oil against
74
essential oil in Live/Dead assay 75
4.4.3.3b Percentage Apoptosis by Cymbopogon flexuosus essential oil against
75
4.4.3.4b Percentage Apoptosis by Citrus pseudolimon essential oil against
76
4.4.3.5b Percentage Apoptosis by Thuja orientalis essential oil against
77
4.4.3.6b Percentage Apoptosis by Alpinia allughas essential oil against
78
4.4.3.7b Percentage Apoptosis by Schinus terebinthifolius essential oil against
79
4.4.3.8b Percentage Apoptosis by Callistemon viminalis essential oil against
80
4.4.3.9b Percentage Apoptosis by Anethum graveolens essential oil against
81
4.4.4.1 Effect of Pinus roxburghii essential oil on colony formation in
HCT-116 cancer cells 82
4.4.4.2 Effect of Lantana camara essential oil on colony formation in
12
4.4.4.3 Effect of Cymbopogon flexuosus essential oil on colony formation in
4.4.4.4 Effect of Citrus pseudolimon essential oil on colony formation in
4.4.4.5 Effect of Thuja orientalis essential oil on colony formation in
HCT-116 cancer cells
84
4.4.4.6 Effect of Alpinia allughas essential oil on colony formation in
HCT-116 cancer cells
85
4.4.4.7 Effect of Schinus terebinthifolius essential oil on colony formation in
4.4.4.8 Effect of Callistemon viminalis essential oil on colony formation in
4.4.4.9 Effect of Anethum graveolens essential oil on colony formation in
4.4.5.1 Effect of Pinus roxburghii essential oil on cell cycle study in KBM-5
cancer cells 87
4.4.5.2 Effect of Lantana camara essential oil on cell cycle study in KBM-5
cancer cells
88
4.4.5.3 Effect of Cymbopogon flexuosus essential oil on cell cycle study in KBM-5
cancer cells
88
4.4.5.4 Effect of Citrus pseudolimon essential oil on cell cycle study in KBM-5
cancer cells 89
4.4.6.1 Effect of Pinus roxburghii essential oil on expression of different
proteins in KBM-5 cancer cells 90
4.4.6.2 Effect of Lantana camara essential oil on expression of different
4.4.6.3 Effect of Cymbopogon flexuosus essential oil on expression of different
4.4.6.4 Effect of Citrus pseudolimon essential oil on expression of different
proteins in KBM-5 cancer cells
92
essential oil
essential oil 95
essential oil 96
essential oil 97
4.6.1 Typical GC-MS chromatogram of Pinus roxburghii essential oil
showing the separation of chemical components
109
112
essential oil showing the separation of chemical components 115
13
showing the separation of chemical components 117
4.6.5 Typical GC-MS chromatogram of Thuja orientalis essential oil
4.6.6 Typical GC-MS chromatogram of Alpinia allughas essential oil
4.6.7 Typical GC-MS chromatogram of Schinus terebinthifolius essential oil
125
127
129
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Abstract
This project was designed to evaluate the chemical composition, antioxidant,
antimicrobial, anti-inflammatory and anticancer activities of the essential oils of nine plants. The
essential oils were isolated through steam distillation and characterized by GC and GC-MS
techniques. Different antioxidant assays were used to examine the antioxidant potential of
essential oils. The highest activity was observed against Lantana camara essential oil with IC50
5.45 μg mL-1 in DPPH assay and 78.95% inhibition in case of β-carotein linoleic acid assay.
Antimicrobial potential was determined by disc diffusion assay and Minimum inhibitory
concentration (MIC). All essential oils possessed good antimicrobial potential while among all
tested oils Cymbopogon flexuosus essential oil showed the highest activity (27.9 and 32.5 mm)
against Staphylococcus aureus and Helmenthosporium solani in case of bacterial and fungal
strains respectively. The anticancer potential was checked by different assays, Lantana camara,
Pinus roxburghii, Cymbopogon flexuosus and Citrus pseudolimon essential oils showed excellent
cytotoxic potential against HCT-116 (colon cancer), KBM-5 (myelogenous leukemia), U-266
(multiple myeloma cells), MiaPaCa-2 (pancreatic cancer cells), A-549 (lung carcinoma cells)
and SCC-4 (squamous cell carcinoma) human cancer cell lines. In addition, they induced the
inhibition on nuclear factor κB (NF-κB) in a dose-dependent manner. Flow cytometric analysis
indicated that treatment with essential oils resulted in Go/ G1 cell cycle arrest in a dose-
dependent manner. GC-MS analysis revealed that α-Pinene (27.11%) in Pinus roxburghii,,
Caryophyllene oxide (19.63) in Lantana camara , trans-citral (21.65%) in Cymbopogon
flexuosus while limonene (58.01%) in Citrus pseudolimon were the major components.
Presented results suggest that selected essential oils are good sources of phytochemicals with
antioxidant and antimicrobial activities and can play an important role in cancer prevention.
1
INTRODUCTION
Oxidation and reduction reactions characterize the transfer of electrons from an electron
donor to an electron acceptor species. Oxidation is crucial to various living organisms for the
production of energy to fuel biological processes. Reactive oxygen species (ROS) are produced
in the body during aerobic respiration and other redox processes (Dar et al. 2012). These ROS
can damage several important biomolecules e.g. lipids, enzymes, proteins, RNA and DNA by
inhibiting their normal function and changing their structures, leading to cellular smash up and
plays a main role in aging process. To frustrate oxidative stress, human body has some defensive
mechanisms including antioxidant enzymes e.g. catalase (CAT) and superoxide dismutase
(SOD), or chemical compounds e.g. ascorbic acid, α-tocopherol, carotenoids, glutathione and
polyphenols (Niki et al. 1994). But, the excessive production of free radicals and reactive oxygen
species in the body make the organism incapable to scavenge all the ROS and give rise to
oxidative stress. Oxidative stress has been reported to play a significant role in the development
of several diseases, including cardiovascular diseases, diabetes, aging, nephritis, rheumatism,
neurodegenerative diseases and cancer (Santhoshkumar et al. 2014).
Cancer is an “old-age disease” that has an “age-old” history. Cancer is one disease that
fits the paradigm that ‘‘more we know, less we understand its intricacies’’ (Aggarwal et al.
2009). Cancer is referred as a generic term for more than 200 different diseases that can affect
any part of the body, and is characterized by the uncontrolled development and proliferation of
normal healthy tissues and multiplication of cells (Yarbro et al. 2005; Singh et al. 2015).
According to 2012 World Health Organization reports, cancer becomes the most important
reason of premature deaths worldwide, accounting for 7.6 million deaths just in 2008 that is
approximately 13% of total deaths. The overall deaths from different types of cancer worldwide
are anticipated continuously going up, reaching probably 13.1 million in 2030 (WHO 2013). In
2008 the worldwide cancer cases reached up to 12.7 million and in 2030 it is expected to
increase up to 21 million (Ferlay et al. 2010).
2
Cancer is becoming a solemn health risk in numerous Asian pacific countries and now it
has become the foremost reason of death in Asian countries (Park et al. 2008). Pakistan is the
sixth most populated country in the world with a population of 190,291,129 in 2012. According
the GLOBOCAN 2012 data new cases of cancer in Pakistan is 148,000 and total deaths 101,000.
The ratio of incidences 112/ 100,000 and deaths is 80/100,000 in Pakistan (Singh et al. 2015).
It is a potentially hazardous disease that might be spread in three dissimilar ways those
are inaccurate diet, from the environmental factors (Chemicals, X-rays, ultraviolet light, viruses
and tobacco products) and inherited predilection. 90-95% cancers are connected to lifestyle and
environmental factors while only 5-10% caused by inheritance of the mutated genes and due to
somatic mutations. Approximately 30% of total cancers have been accredited to tobacco smoke,
35% due to unbalanced diet, 14-20% to obesity, 18% to different infections and 7% from
radiation and environmental pollutants (Alison, 2001).
Treatments for cancer, including surgery, radiotherapy, immunotherapy, hormone therapy
and chemotherapy, are the treatments which has not the capacity to treat the affected part
completely, expensive and produce adverse side effects such as alopecia, vomiting, diarrhea,
constipation, myelosuppression, cardiac, neurological, pulmonary and renal toxicity (Cho et al.
2007). On the other hand, treatments like resection surgery procedures are responsible for
functional deficiencies or aesthetic discomfort. All these side effects decrease the quality of life
and play a discouraging role in patients to continue these medical treatments, which promote the
progression of cancer and complications with them (Castro et al. 2011). Therefore, there is an
urgent need for the development of alternative anticancer drugs, which may be more selective,
potent, and less toxic as compared to the currently drugs that are in practice (Wang et al. 2012).
The exploration and the development for safe and effective treatment modalities and
supplementary therapies for early and advanced stages of cancer is now an important research
goal (Zhu et al. 2005).
Natural products are the secondary metabolites, which is produced by the organisms in a
response to exterior stimuli e.g. infection, nutritional changes and competition. Natural products
that are produced by plants, fungi, bacteria, insects, protozoans and animals have been isolated as
biologically active pharmacophores (Strohl, 2000). It is important more than 62% of the
anticancer drugs approved from 1983 to 1994 are either natural products or natural product
analogues (Anthwal et al. 2014). Nature blessed us with many potent anticancer agents, some of
3
them derived from microorganisms e.g. doxorubicin and dactinomycin, others from plants (Ruffa
et al. 2002). There are above one thousand plants which have been documented to have
significant anticancer properties. topotecan, irinotecan, Taxol, vincristine, colchicine,
vinblastine, ellipticine, podophyllotoxin, lepachol, camptothecin, etoposide, irinotecan and
paclitaxel are examples of plant derived compounds which are found to have broad applications
in cancer therapeutics (Yadav et al. 2012). According to the WHO, 80% of Earth’s population is
dependent on the traditional way of medicine to fulfill their primary health care desires,
relatively caused by high prices of Western pharmaceuticals (Kim et al. 2012). Medicinal plants
and their products are major source of the health care throughout the earth for thousands of
years, due to the availability of medicinal plants all over the world. So, 75-80% of the world
population especially in developing countries is using herbal drugs (Gahlaut and Chhillar, 2013).
Natural products from plants are recognized to be effective, chemically balanced and least
harmful with none and to a great extent reduced side effects as compared to synthetic medicines
(Awal et al. 2004).
Aromatic plants are being used since ancient times due to their medicinal and
preservative properties, and they give flavor and aroma in to food. These pharmaceutical
properties of aromatic plants are partially accredited to the essential oils. The word ‘essential oil’
first time was used by Paracelsus von Hohenheim in the 16th century, who named the active
component of a drug, ‘Quinta essential’ (Edris 2009; Roger, 1997). There are almost 17,500
aromatic plant species and approximately 3000 essential oils are identified out of which 300 are
commercially vital for pharmaceutical, perfume and cosmetics industries (Tripathi et al. 2009).
The ability of plants to produce essential oils is relatively high in both Gymnosperms and
Angiosperms, even though commercially the most important essential oil plant sources are
belonged to the Angiosperms. Most of the aromatic plants and essential oil supplies in terms of
world trade related to the Lamiaceae, Compositae and Umbelliferae families (Teixeira da Silva,
2004; Celiktas et al. 2007; Anwar et al. 2009).
Essential oils (EOs) are volatile, aromatic, concentrated, and hydrophobic oily liquids
which are obtained from a variety of plant parts such as flowers, seeds, buds, bark, leaves, twigs,
woods, roots and fruits (Tabassun and Vidyasagar, 2013). Essential oils are secondary
metabolites, which plants produced for their own needs other than for nutrition. The aromatic
character of EOs is responsible for various functions in the plants including repelling or
4
attracting insects, shielding themselves from cold or heat and utilizing chemical ingredients in
the oil as protection materials (Ebadollahi, 2013; Nazzaro et al. 2013).
Essential oils are extracted by using a number of techniques e.g. hydrodistillation, steam
distillation, microwave assisted distillation, organic solvent extraction, high pressure solvent
extraction, microwave hydro diffusion and gravity, supercritical CO2 extraction (SCE), solvent
free microwave extraction and ultrasonic extraction. Though, the characteristics of the essential
oils isolated by these methods have been different depending on the technique used (Okoh et al.
2010).
Essential oils are a complex mixture of organic compounds, primarily monoterpenes,
sesquiterpenes and their oxygenated derivatives (ketones, alcohols, aldehydes, esters, ethers,
phenols,and oxides) that provide characteristic odor and flavor to leaves, barks, flowers, seeds,
fruits and rhizomes. Additional volatile compounds in EOs are phenyl propenes and specific
sulphur or nitrogen containing substances (Coisin et al. 2012). Biological activity of EOs may be
as a result of one compound or due to the whole mixture (Djilani and Dicko, 2012).
The action of essential oils begins by entering the human body via three possible different ways:
1. Direct absorption through skin: EOs constituents are fat soluble. Therefore, they have
the capability to permeate the skin membranes earlier than being trapped by the
microcirculation and drained in to the systemic circulation that reaches every target
organs (Baser and Buchbauer, 2010).
2. Inhalation: Essential oils are volatile in nature, so they can be inhaled simply by lungs
and the respiratory tract that can allocate them in to the blood stream (Moss et al. 2003).
3. Diffusion through the skin tissue: Oral intake of EOs needs attention because several
oils found to be toxic on oral intake. Ingested essential oil components and their
metabolites might then be immersed and drained to the other parts of the body through
blood stream. Once EOs molecules are in the body, they interconnect with the
physiological functions through three distinct ways:
I. Biochemical: EOs molecules interact with the blood stream and chemically
interact with enzymes and hormones such as farnesene.
II. Physiological: By acting on specific physiological function, e.g. the fennel
essential oil contains an estrogen like compounds that may be efficient in female
problems e.g. menstruation and lactation.
5
III. Psychological: Through inhalation the olfactory region of the brain (limbic
system) undergoes an action activated by the EOs molecules after that chemical
and neurotransmitter messengers bring changes in the emotional and mental
behavior of the person. Lemon and Lavender oils are examples for their relaxant
and sedative properties (Shibamoto et al. 2010).
Essential oils have been used for their biological activities including antiseptic, analgesic,
sedative, anti-inflammatory, antimicrobial, spasmolytic, and locally anesthetic properties.
Furthermore, they are used in aromatherapy for health improvement due to their sedative or
stimulant properties (Russo et al. 2015).The miscellaneous therapeutic potential of EOs has
drawn the interest of the researchers to trial them for their anticancer potential, taking benefit of
the reality that they have different mechanism of action than the typical cytotoxic
chemotherapeutic agents (Rajesh et al. 2003). Essential oils have been reported to improve the
quality of life of cancer patients by lowering the level of their agony (Gautam et al. 2014).
The monoterpenes mode of action is based upon two major approaches, chemotherapy
and chemoprevention. Chemotherapy works through the promotion phase, that includes
inhibition of the cancer cell proliferation, acceleration of the rate of cancer cell death and
induction of the cancer cell differentiation. Although, chemoprevention occurs in the initiation
phase of the carcinogenesis to stop the contact of chemical carcinogens with the DNA, through
induction of phase I and phase II enzymes by detoxifing the carcinogens (Edris, 2007). Different
mechanisms implicated in cancer treatment are modulation of DNA repair signaling, activation
of detoxification enzymes, anti-angiogenesis and anti-metastasis. Many pathways are involved in
the anti-proliferative activity demonstrated by the EOs in the cancer cells. Various targets of EOs
for cancer prevention are represented in Figure 1.1. This makes EOs suitable anticancer agents
with no large apparent effects being displayed on the normal cells (Gautam et al. 2014).
6
Fig. 1.1. Possible route for EOs-mediated cancer cell death (Gautam et al. 2014)
Figure 1.1 explained the multi-targeted role of EOs towards cancer prevention. The EOs-
mediated anticancer strategies identified so far including cell cycle arrest, apoptosis, and DNA
repair mechanisms. EOs reduces cancer cell proliferation, metastasis, and MDR which make
them potential candidates towards adjuvant anticancer therapeutic agents.
Pakistan is blessed with a marvelous climate and agricultural land quality. In Pakistan the
agro and physical climatic conditions are very favorable for the growth and cultivation of various
kinds of the essential oil bearing crops (Hussain et al. 2008). Northern regions of Pakistan are
well- known for the production of lots of useful medicinal plants. The home climatic and soil
conditions modify the chemo types such as chemical composition of EOs in plants which makes
the essential oil more desirable. In fact, various aromatic plants and herbs are cultivated in the
different areas of the country as well as grown in the wild plane and hilly regions of Pakistan
(Hussain et al. 2006) In Pakistan 60% of the people, particularly in villages are getting health
care through traditional practitioners (Hakims), who recommend herbal preparations. There are
many herbs and aromatic plants that have the ability to be employed as natural tonic in native
medicine systems (Erdemgil et al. 2007). The Pakistani market for EOs is minuscule and
represents less than 1% of the world market, for that reason, the producers of EOs in Pakistan
would most likely competing in the world market (Gilani et al. 2001; Ahmed et al. 2004).
7
Aims and Objectives:
The current study was undertaken with the main objective to isolate and characterize the
essential oils of selected plants growing in Pakistan for their detailed chemical constituents,
anticancer attributes, antioxidant potential and antimicrobial activities.
The present project was designed with the following principle objectives:
1. Extraction of essential oils from medicinal and aromatic plants (nine) indigenous to
Pakistan
2. To explore the anticancer activity of essential oils by in vitro assays (Cell Proliferation
assay, apoptosis assay, clonogenic assay and Propidium iodide (PI) staining assay)
3. To dermine the protein expression in cancer cells (Western blot analysis and EMSA)
4. To investigate the antioxidant and antimicrobial (antibacterial and antifungal) activities of
essential oils
5. Studying the profiles of bioactive constituents of essential oils using modern
chromatographic / spectroscopic techniques
REVIEW OF LITERATURE
Aromatic plants have been used from thousands of years due to their medicinal
applications (Bakkali et al. 2008). For many years, volatile oils have been used to alleviate
various human maladies such as pneumonia, bronchitis, pharyngitis, diarrhea, periodontal
disease, wounds, and many other diseases. Essential oils from medicinal plants have been
renowned to possess biological activities, mainly antifungal, antibacterial, antioxidant and
antitumor properties (Sharma et al. 2009). Bioactivity of EOs depends on their chemical
composition which is determined through the plant genotype and is intensely influenced by a
number of factors for instance geographical origins, environmental and agronomic situations
(Celikel and Kavas, 2008).
Due to vast medicinal applications of plants, in the present study we have been selected nine
essential oils bearing plants. This selection of plants have been done on the basis interviews of
traditional practitioners (Hakims) and ethnopharmacologic information obtained from literature
survey.
7) Schinus terebinthifolius Raddi
Figure 2.1 Pinus roxburghii
Pinus roxburghii Sarg named after William Roxburgh usually known as the Chir Pine,
belongs to family Pinaceae. It has 110 to 120 species that are dispersed throughout temperate
regions of the Northern Hemisphere (Parasharami et al. 2006). It normally presents at lower
altitudes as compared other pines in the Himalaya, from 500-2000 m, sporadically up to 2300 m.
Its range extends from northern Pakistan (North-West Frontier Province, Azad Kashmir), across
northern India (Jammu and Kashmir, Himachal Pradesh,Punjab, Sikkim Uttrakhand) and Nepal
to Bhutan (Sanjay et al. 2006). The biological classification of Pinus roxburghii is shown in the
table 2.1.
Kingdom Plantae
Division Coniferophyta
Class Pinopsida
Order Pinales
Family Pinaceae
Genus Pinus
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P. roxburghii has a long history of ethno botanical applications in various cultures;
numerous ethnic groups regarded it as a treatment for all ailments. Resin, wood, gum, seeds, oil,
bark and needles from the plant are used in the medical preparations all over the region where
this plant is found (Kaushik et al. 2013). In Asian sub-continent, Pinus roxburghii is also
traditionally used in the treatment of gastrointestinal diseases, disorders of liver and spleen, ear,
throat, skin, bronchitis, diaphoresis, giddiness, ulcer, inflammation, itching and for snake bite
(Sinha, et al. 2013; Chaudhary et al. 2014).
2.1.2. Chemical composition
The literature survey from Pakistan revealed that the chemical composition of stem
essential oil of Pinus roxburghii analyzed by GC-MS contained α-pinene, 3-carene and
caryophyllene as the major components (Hassan and Amjid, 2009) and in needles essential oils
of Pinus roxburghaii α-pinene, caryophyllene, 3-carene, α-terpineol, caryophyllene oxide were
the dominant constituents (Iqbal et al. 2011). Satyal et al. (2013) also demonstrated the chemical
composition of needle, bark, and cone essential oils of P. roxburghii. The results revealed that
major component in needle, bark and cone was (E)-caryophyllene with different percentage
composition. A report from Egypt showed that the major constituents in wood oil of Pinus
roxburghaii were caryophyllene, thunbergol, 3-carene and cembrene, while in bark and needle
they were α-pinene and 3-carene (Salem et al. 2014). Qadir and Shah, (2014) from India reported
that the principle constituents of the oil were α-pinene, β-pinene, limonene, camphene,
betapinene, β-caryophyllene and α-terpinol.
2.1.3. Biological testing
Iqbal et al. (2011) from Pakistan studied the antimicrobial potential of Pinus roxburghaii
needles essential oil. The antibacterial activity of this plant oil indicated that Staphylococcus
aureus and Bacillus subtilis were the most sensitive strains whereas no effect was noted against
Enterobacter aerogenes, Escherichia coli and Salmonella typhi. Moreover, antifungal property
showed a significant and dose dependent inhibition against Aspergillus terrus, Aspergillus flavus
and Trichoderma viride and no activity was found against Aspergillus niger Aspergillus candidus
and Aspergillus vessicolor. Chaudhary et al. (2012) from India compared the antibacterial and
antifungal activities of volatile oil, chloroform extract and methanol extract of the woods of the
Cedrus deodara and Pinus roxburghii plants. It was indicated that the essential oil and the
chloroform extracts possessed the good antibacterial potential whereas the methanolic extracts
11
exhibited the least antibacterial potential. The higher values for Minimum Inhibitory
Concentration of oils and extracts against fungus revealed that the plants had less antifungal
potential. Kaushik et al. (2012) studied the analgesic and anti-inflammatory effect of alcoholic
extract of Pinus roxburghii. Analgesic property was measured by tail immersion and acetic acid-
induced writhing assays in the Swiss albino mice. Anti-inflammatory potential was measured by
cotton pellet granuloma and carrageenan-induced paw oedema in Wistar albino rats. In this
study, the P. roxburghii alcoholic bark extract showed good anti-inflammatory and analgesic
potential in the tested Wistar albino rats.
Satyal et al. (2013) from Nepal studied the antimicrobial potential of needle and cone
essential oils of P. roxburghii. Results revealed that both oils exhibited no antibacterial potential
against all tested strains (B. cereus, S. aureus P. aeruginosa and E. coli) while possessed
significant antifungal activity against A. niger. Qadir and Shah (2014) from India tested the
antioxidant and antibacterial activity of Pinus roxburghii essential oil by 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay and agar well diffusion method respectively. The results showed
that Pinus roxburghii essential oil had negligible antioxidant activity whereas possessed good
antibacterial potential against all tested strains. The highest activity (32 and 30mm) was observed
against P. vulgaris and E. coli respectively. Salem et al. (2014) from Egypt compared the
antibacterial and antioxidant properties of P. roxburghii wood, bark and needle essential oil
isolated through steam distillation. Almost all of the essential oils were active against human
pathogens bacteria while in case of plant pathogenic bacteria only bark and needle essential oils
were active. The needle oil showed less (50 ± 2.24%) total antioxidant activities (TAA%) as
compared to the bark (85 ± 1.24%) and wood (82 ± 2.12%) essential oil.
2.1.4. Anticancer activity
Jo et al. (2012) from Korea determined the anticancer activity of Pinus densiflora leaf
essential oil in human oral squamous carcinoma cells (YD-8). Particularly, treatment with P.
densiflora leaf oil led to the production of ROS, caspase-9 activation, phosphorylation of the
JNK-1/2 and ERK-1/2, cleavage of PARP, and Bcl-2 down-regulation, thus has anti-survival,
anti-proliferative and pro-apoptotic actions on cancer cells. A report from Nepal showed that
needle and bark essential oil of Pinus roxburgii possessed good anticancer potential (70.9 ±
12
1.4%) at 100µg/mL against Human MCF-7 breast adenocarcinoma cells by MTT assay (Satyal et
al. 2013). In another report from Korea Cho et al. (2014) investigated the anticancer potential of
Pinus koraiensis against HCT-116 cells by using BrdU and crystal violet assays and concluded
that P. koraiensis oil significantly reduced migration and proliferation of the human colorectal
cancer cells. Qadir and Shah (2014) from India reported the anticancer activity of Pinus
roxburgii essential oil against A549, C6, T47D, MCF, and TH-1 human cancer cell lines by
MTT assay and found that the oil was active against all the five cancer cell lines tested. Kaushik
et al. (2015) from India compared the anticancer potential of petroleum ether, chloroform, ethyl
acetate and ethanolic extract of Pinus roxburgii Sarg. by SRB assay method on IMR-32 Human
Neuroblastoma cancer cell line. Results indicated that petroleum ether and chloroform fractions
showed a promising anti-cancer activity.
2.2. Lantana camara
Figure 2.2 Lantana camara
The genus Lantana camara belonged to family verbenaceae, as described by the
Linnaeus in 1753 consist seven species; six belongs to South America and one from Ethiopia.
Lantana camara L., usually known as wild or red sage, is the most widespread species of this
genus. It is growing splendidly at a height up to 2000 m in temperate, tropical and sub-tropical
regions (Ghisalberti, 2000; Kuhad et al. 2010). The biological classification of Lantana camara
is shown in the table 2.2.
Table 2.2 Biological classification of Lantana camara:
Kingdom Plantae
Species L. camara
2.2.1. Ethnobotanical uses
Lantana possesses various medicinal properties and it has been used in folk medicine
with antimicrobial, antipyretic, antimutagenic, carminative, antispasmodic, antirheumatic and
anticancer properties were reported (Mello et al. 2005; Pasha et al. 2007; Verma and
Balasubramanian, 2014). In Asian countries (India, Pakistan and China), its leaves were used in
the treatment of stomach-ache, cuts, ulcers, rheumatisms, leprosy, scabies, fever and for the
treatment of hypertension, asthma, and high blood pressure (Ghisalberti, 2000).
hydroxy olean-12-en-28-oic acid (Misra and Laatsch, 2000), camaric acid and oleanolic acid
(Qamar et al. 2005) camarolic acid and lantrigloylic acid (Begum et al. 2008), flavonoids
Linaroside and lantanoside (Begum et al. 2008) and lantaninilic acid and lantoic acid (Begum et
al. 2014) have been reported to isolate from L. camara plant.
Marongiu et al. (2007) from Italy reported that curcumene, α-humulene, α-zingiberene
and caryophyllene were the major components in L. camara leaf essential oil extracted by
supercritical carbon dioxide extraction. Sousa et al. (2010) discussed that bicyclogermacrene,
germacrene, valencene and isocaryophyllene were the major constituents in leaf oil of L. camara.
Saikia and Sahoo, (2011) from India showed that main components in L. camara leaf oil were α-
humulene, β-caryophyllene, sabinene and bicyclogermacrene. In another report from Brazil,
Passos et al. (2012) compared the leaf essential oil of Lantana camara and Lantana radula. The
major constituents of L. camara essential oil were E-caryophyllene and germacrene-D, whereas
in L. radula essential oil the major ones were E-nerolidol, E-caryophyllene and phytol. A report
14
from Iran showed that the main components of L. camara leaf essential oil were α-humelene and
cis-caryophyllene (Sohani et al. 2012).
Deena and Thoppil, (2000) from India tested the L. camara essential oil against various
microorganisms and showed a broad spectrum of antifungal and antibacterial activities. Begum
et al. (2008) reported that two flavonoids Linaroside and lantanoside isolated from Lantana
camara exhibited antimycobacterial activity. Kirimuhuzya et al. (2009) reported the anti-
mycobacterial properties of Lantana camara methanol and chloroform extracts to treat
respiratory tract infections. Methanolic extracts showed more potential (18.0-22.5 mm) against
M. tuberculosis than that of chloroform extracts. Nayak et al. (2009) evaluated the wound
healing property of leaf extracts L. camara. A report from India showed the adulticidal activity
of Lantana camara leaves essential oil against different mosquitoes species (Dua et al. 2010). In
another study from India Saikia and Sahoo, (2011) reported the antibacterial potential of Lantana
camara essential oil. The essential oil showed promising antibacterial potential against Bacillus
cereus, Staphylococcus aureus, Bacillus subtilis and Escherichia coli. Saraf et al. (2011) studied
the antimicrobial activity of crude methanolic and acetone extracts of Lantana camara. Both L.
camara extracts inhibited the growth of Staphylococcus aureus. Kamaraj et al. (2012) reported
the antimalarial activities of ethyl acetate and methanolic extracts of Lantana camara.
Kazmi et al. (2012) isolated a new stearoyl glucoside of ursolic acid, urs-12-en-3β-ol-28-
oic acid 3β-D-glucopyranosyl-4′-octadecanoate and some other constituents from Lantana
camara leaves and reported that these compounds appreciably lowered the glucose level in the
blood of streptozotocin-induced diabetic rats. A report from Egypt showed a comparison of
antioxidant and antibacterial properties of leaves essential oils from Lantana camara, Cupressus
sempervirens and Syzygium cumini. The L. camara oil exhibited less activity as compared to S.
cumini essential oil (Elansary et al. 2012).
A group from Malaysia isolated Lantadene A, a Pentacyclic Triterpenoid from the leaves
of L. camara plant. Lantadene A possessed good antioxidant activity assessed by different in
vitro tests including 2,2-diphenyl-1-picryl-hydrazyl, hydroxyl radical, ferric reducing antioxidant
power, superoxide anion scavenging activities nitric oxide radical, and ferrous ion chelating
assay (Lynn et al. 2012). In another report from Malaysia Pour et al. (2012) reported the
antioxidant potential of methanolic extracts of various parts of Lantana camara. The extracts
15
were screened for antioxidant potential by oxidase inhibition activity, free radical scavenging
assay (DPPH•), xanthine and Griess-Ilosvay test. The leaves extract had more potential than that
of other parts.
Remya et al. (2013) compared the bioactivities of the chloroform, diethyl ether, methanol
and hot water extracts of Lantana camara roots. The antioxidant potential was measured by
reducing power assay, diphenyl-2-picryl-hydrazyl (DPPH) radical assay, and Nitric oxide radical
scavenging method. The highest antioxidant activity was observed by methanolic extract as
compared to chloroform, diethyl ether and hot water extracts. The antibacterial activity
determined by agar well diffusion assay and methanolic extract was found to be effective against
all tested bacteria. El Baroty et al. (2014) evaluated the antioxidant and antimicrobial activities
of leaves and flowers essential oils of Egyptian Lantana camara. Both oils possessed moderate
antioxidant and antimicrobial activities. Kumar et al. (2014) from India evaluated the total
phenolics and antioxidant potential of different varieties of Lantana camara leaves and
concluded that Lantana camara leaves methanolic extracts possessed excellent antioxidant
potential.
2.2.4 Anticancer activity
Badakhshan et al. (2009) from Malaysia studied the cytotoxic activity of Lantana
camara's root and leaf methanol extracts against Jurkat leukemia cell line by MTT assay. Result
implicated that both root and leaf extracts had remarkable anticancer potential. A report from
Brazil also showed the antiproliferative activity of methanolic extracts of 14 plants by MTT
method against the laryngeal cancer (HEp-2) and lung cancer (NCI-H292) cell lines. Results
indicated that Lantana camara was the second most active plant among all the tested plants
(Melo et al. 2010). Bisi-Johnson et al. (2011) studied the cytotoxicity of methanolic extracts of
African medicinal plants against hepatocarcinoma cell line (Huh-7) by using a modified MTT (3-
(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide) assay and revealed that Lantana
camara had moderate activity.
Cymbopogon flexuosus (lemongrass) belongs to the genus Cymbopogon of the
Poaceae/Gramineae family. It is an aromatic perennial herb growing to 1 m in height, mostly
distributed around the world, especially in tropical and subtropical regions like India, Sri Lanka,
Burma, and Thailand (Sharma et al. 2009). The biological classification of Cymbopogon
flexuosus is shown in the table 2.3.
Table 2.3 Biological classification of Cymbopogon flexuosus:
Kingdom Plantae
Division Magnoliophyta
Class Liliopsida
Subclass Commelinidae
Order Poales
Family Poaceae
Genus Cymbopogon
Species C. flexuous
Cymbopogon species are generally used in folk medicine to treat skin disorders,
conjunctiva, headaches and hepatitis, rheumatism, cold, flu, fevers, intestinal parasites, snake-
bite and of digestive and menstrual problems (Abena et al. 2007).
17
Saeio et al. (2011) from Thailand explored the chemical composition of Cymbopogon
citrates and found that neral and geranial were the prominent components. Adukwu et al., (2012)
from UK studied the chemical composition of Cymbopogon flexuosus and found neral and
geranial as the major constituents. A report from Belgium showed the variations in the chemical
compositions among four Cymbopogon species in. The major constituents in Cymbopogon
citrates were geranial, neral, β-pinene and cis-geraniol, in Cymbopogon giganteus were cis-p-
mentha-1(7),8-dien-2-ol, trans-p-mentha-1(7),8-dien-2-ol, cis-p-mentha-2,8-dienol, trans-p-
citronellol, elemol and limonene and in Cymbopogon schoenantus were piperitone, (þ)-2-carene,
limonene, elemoland β-eudesmol (Kpoviessi et al. 2014). Ntonga et al. (2014) characterized the
essential oils of Cymbopogon citrates by using GC-FID and GC-MS and concluded that
essential oil was rich in geranial, 1,8-cineole and linalool.
Doran et al. (2009) reported that geranium and lemongrass EOs individually and blended
inhibited the growth of antibiotic resistant and antibiotic sensitive bacteria (MRSA, VRE
(vancomycin- resistant Enterococci), Clostridium difficile and Acinetobacter baumanii) and
reduced surface and airborne levels of bacteria. Jeong et al. (2009) from Korea reported the
antimicrobial potential of Cymbopogon citrates against Pectobacterium carotovorum,
Colletotrichum gloeosporioides, and Aspergillus niger and found it very effective against all
tested strains. A report from Brazil showed the antinociceptive potential and Redox properties of
Citronellal, an essential oil found in lemongrass (Junior et al. 2011). Costa et al. (2011) reported
the in vivo anxiolytic-like effect of Cymbopogon citratus essential oil. Saeio et al. (2011)
compared the antioxidant and antityrosinase properties of essential oils of twenty edible Thai
plants. Cymbopogon citrates oil possessed the highest antityrosinase activity while Ocimum
sanctum had the highest antioxidant potential. Adukwu et al. (2012) compared the antimicrobial
and anti-biofilm action of Citrus paradisi and Cymbopogon flexuosus essential oils against five
strains of the Staphylococcus aureus. C. flexuosus exhibited strong anti-biofilm activity while C.
paradisi did not show any effect. Dzeufiet et al. (2014) evaluated the antihypertensive potential
of the aqueous extract obtained from the mixture of stems and fresh leaves of Cymbopogon
citratus, fresh leaves of Persea americana, fruits of Citrus medica. Ntonga et al. (2014)
compared the larvicidal and anti-plasmodial potential of Cymbopogon citrates, Ocimum canum
18
and Ocimum basilicum essential oils. C. citrates oil was found to be most active against the
Plasmodium falciparum and Anopheles funestus larvae.
Kumar et al. (2008) from India reported that Cymbopogon flexuosus (CFO) and its main
chemical component isointermedeol (ISO) a sesquiterpene, induced apoptosis in HL-60 human
leukaemia cells. Apoptosis was checked by different end-points, such as DNA laddering,
apoptotic bodies formation, annexinV binding and also an increase in hypo diploid sub-G0 DNA
content during this early 6 h phase of study. At the same time, both amplified the expression of
mitochondrial cytochrome c protein with its concomitant release into cytosol that causes
activation of caspase-9, predicting thus the association of both extrinsic and intrinsic pathways of
apoptosis. Apart from this, Bax translocation, and decrease in the expression of nuclear NF-κB
suggesting multi-target actions of ISO and CFO whereas both showed same signaling apoptosis
routes. A study from India also investigated the in vivo and in vitro anticancer potential of
Cymbopogon flexuosus. The results revealed that tested oil had a significant anticancer potential
and reduced the viability of tumor cells through activating the apoptosis as cleared by electron
microscopy (Sharma et al. 2009). Halabi and Sheikh (2014) from Malaysia compared the
antiproliferative effect of Cymbopogon citratus methanol, ethanol and aqueous extracts and
tested against five different cancer cells: breast carcinoma (MDA-MB-231 and MCF-7), ovarian
carcinoma (COAV and SKOV-3), human colon carcinoma (HCT-116) and normal liver cell line
(WRL 68) using MTT assay. The results revealed that ethanolic extracts showed good
antiproliferative efficacy. Kpoviessi et al. (2014) determined the cytotoxicity of Cymbopogon
citrates, Cymbopogon giganteus, Cymbopogon nardus and Cymbopogon schoenantus against
Chinese Hamster Ovary (CHO) cells and WI38 (human non cancer fibroblast) cell lines by MTT
test. Cymbopogon citrates showed toxicity against both cell lines. Thangam et al. (2014)
reported that a Cymbopogon citratus polysaccharide fraction activates the intrinsic apoptotic
signaling pathway in Siha and LNCap cancer cells.
2.4. Citrus pseudolimon:
Figure 2.4 Citrus pseudolimon
Citrus pseudolimon locally known as Galgal, belongs to family Rutaceae. It consists of about
160 genera and citrus is the most important genus of this family (Janoti et al. 2014). Galgal is a
medium sized 5-6.5 m tall tree, with an uneven and loose crown and a trunk of 28 cm in
diameter. Citrus are mostly grown in areas with temperate summers and mild winters, mainly in
Mediterranean countries like Brazil, Japan, Argentina, USA and Australia (Kamal et al. 2013).
Citrus fruit yield in Pakistan is 9.5 tons ha-1 and 1.28 million tons per season (Balal et al. 2011).
Pakistan with respect to annual production of citrus fruits stands among the top ten citrus
producing countries of the world (Ashraf et al. 2013). The biological classification of Citrus
pseudolimon is shown in the table 2.4.
Table 2.4 Biological classification of Citrus pseudolimon:
Kingdom Plantae
Class Magnoliopsida
Subclass Rosidae
Order Sapindales
Family Rutaceae
Genus Citrus
20
Citrus crop is an ancient crop; which dates back to 2100 BC (Moore, 2001) and have
many pharmacological applications including antioxidant, anti-carcinogenic, anti-inflammatory,
antimicrobial and anxiolytic properties (Choi et al. 2000; Ghasemi et al. 2009; Vasudeva and
Sharma, 2012).
The genus Citrus have many species and varieties, and their essential oil profile have
been reported (Singh et al. 2010). Vasudeva and Sharma, (2012) studied the chemical profile of
Citrus limettioides Tanaka essential oil and found β-myrcene, limonene, (±)-linalool, α-pinene,
(E)-citral as prominent constituents. Kamal et al. (2013) investigated the chemical profile of
three Pakistani Citrus essential oils using GC and GC-MS. β-myrcene, limonene and linalool,
were found as the major components in Citrus reticulate, Citrus sinensis and Citrus paradisii
respectively. Patil et al. (2009) from India demonstrated that Citrus aurantifolia essential oil
contained the following main constituents: D-Limonene, D-Dihydrocarvone, α-Pinene. Janoti et
al. (2014) had conducted a comparative study for the chemical profile of the Citrus limettioides
and Citrus pseudolimon leaves essential oils and reported that limonin, linalool, terpineol were
higher in C. limettioides oil while Citronellol content was found to be higher in C. pseudolimont.
Ghasemi et al. (2009) studied the antioxidant potential, phenol and flavonoid contents of
methanolic extracts of thirteen Citrus species peels and tissues. Omran and Esmailzadeh, (2009)
reported the anti-Candida potential of, pennyroyal, thyme and lemon essential oils. Shahzad et
al. (2009) from Pakistan demonstrated the antifungal, antibacterial and antioxidant activities of
Citrus reticulate fruit essential oil. Singh et al. (2010) studied the antioxidant antifungal and
antiaflatoxigenic activity of Citrus sinensis and Citrus maxima Burm. essential oils and one of its
major component limonene. Both oils may be regarded as safe plant based antioxidants as well
as antimicrobials for increasing the shelf life of food commodities by testing their aflatoxin
production, fungal infestation, as well as lipid peroxidation. Mathur et al. (2011) examined the in
vitro antimicrobial and antioxidant potential of peel and pulp of some citrus fruits and reported
remarkable antimicrobial and antioxidant activities. Janoti et al. (2014) from India studied the
antioxidant potential of Citrus Pseudolimon and Citrus limettioides leaves essential oils by
DPPH assay and found that Citrus limettioides have good antioxidant potential (IC50=15.35 ul)
as compared to Citrus Pseudolimon (IC50=18.43 ul).
21
Fayed, (2009) from Egypt compared the anticancer effect of Citrus reticulate and
Pelargonium graveolens essential oils against two cancer cell lines (HL-60 and NB4) by trypan
blue assay. The results revealed that Pelargonium graveolens essential oil had more cytotoxic
potential with the LC50 values of 62.50 μg/ml in NB4 cell line and 86.5 μg/ml in HL-60 cell
line, than Citrus reticulate (LC50 values of 85.05 μg/ml in NB4 cell line and 105.73 μg/ml in
HL-60 cell line). Patil et al. (2009) determined the proliferation inhibition of Citrus aurantifolia
volatile oil. C. aurantifolia oil induced apoptosis against SW-480 (human colon cancer cells).
Murthy et al. (2012) from USA studied the chemical components of essential oil from Citrus
sinensis and found the possible mechanisms of inhibition of colon cancer cell proliferation and
reported that D-limonene enriched oil from Citrus sinensis showed dose-dependent inhibition of
cancer cell proliferation and induced apoptosis in the cancer cells. In Italy various studies were
done on Citrus bergamia for their anticancer potential and to check their possible routes for
inhibition of cancer cells proliferation against SH-SY5Y neuroblastoma cells (Berliocchi et al.
2011; Celia et al. 2013).
2.5. Thuja orientalis:
Figure 2.5 Thuja orientalis
Thuja orientalis commonly known as Morpankh, is a small genus of the Cupressaceae family
consisting five extant species. T. orientalis is an evergreen, monoecious trees or shrubs growing
to 10-60 feet tall. The shoot are flat, leaves are scale like. The leaves are arranged in flattened fan
shaped growing with resin glands (Farjon, 2005). It grows naturally in China, Korea, Japan and
Iran and widely cultivated as a common ornamental plant that is often planted in hedges, even in
22
rural areas (Shah and Qadir, 2014). The biological classification of Thuja orientalis is shown in
the table 2.5.
Kingdom Plantae
Subdivision Spermatophyta
Division Coniferophyta
Class Pinopsida
Order Pinales
Family Cupressaceae
Genus Thuja
Species T. Orientalis
Thuja orientalis has been used as a folk medicine for treatment of gout, hemorrhages,
dermatitis, and chronic tracheitis, enuresis, cystitis, psoriasis, uterine cancer, amenorrhea,
antipyretic, antitussive, astringent, diuretic, and rheumatism (Kim et al. 2011; Srivastava et al.
2012).
The main constituents of Thuja orientalis essential oils from Iran and India were reported
to be α-pinene, limonene, sabinene, delta-3-carene (Nickavar et al. 2003; Guleria et al. 2008).
From Thuja orientalis grown in Austria (Chizzola et al. 2004), five major chemotypes have been
reported, α-thujone, fenchone, β-thujone, beyerene and camphor. Tsiri et al. (2009) examined the
chemical profile of essential oils using GC and GC-MS of four Thuja species (T. occidentalis
‘aurea’, T. occidentalis ‘globosa’, T. plicata ‘gracialis and T. plicata) cultivated in Poland and
observed that major components in all samples were the monoterpenes ketones fenchone,
sabinene and α- and β-thujone, as well as the diterpenes rimuene and beyerene. Three new
diterpenoids, 18-formyloxy-8 β-hydroxysandaracopimar-15-ene, 15(R)-N-butoxypinusolidic acid
and 15,16-dihydro-15,16-dimethoxylambertianic acid, were isolated from Thuja orientalis leaves
extract (Kim et al. 2012). Shah and Qadir, (2014) from India reported that α-pinene, sabinene
23
and 3-carene were the major constituents of Thuja orientalis fruits essential oil isolated by
hydrodistillation.
Singh and Singh, (2009) from India compared the Molluscicidal activity of ethanolic
extracts of Thuja orientalis and Saraca asoca against the fresh water snail Lymnaea acuminate.
T. orientalis leaf ethanolic extracts were more effective than S. asoca. Tsiri et al. (2009) reported
the antimicrobial potential of four thuja species cultivated in Poland. The volatile oils of the two
T. plicata species showed good antimicrobial effect. Hudson et al. (2011) studied the
antimicrobial properties of Thuja plicata volatile oil and concluded that Thuja plicata oil were
active against all tested strains. Shah and Qadir, (2014) reported the antioxidant and antibacterial
activities (B.subtilis, K.pneumoniae, E.coli, P.aeroginosa and P.vulgaris) of Thuja orientalis
fruits essential oil. The oil showed good antibacterial potential against all tested strains while
maximum activity was observed against P.vulgaris and E.coli with highest inhibition zones of 24
and 22 mm respectively.. Na et al. (2013) compared the antiviral potential of the plant extracts of
Aster spathulifolius, Pinus thunbergii and Thuja orientalis against the Influenza Virus
A/PR/8/34. T. orientalis extract possessed highest anti-influenza A/PR/8/34 potential as
compared to other plant extracts. Jasuja et al. (2013) compared the antibacterial and antioxidant
potential of Thuja orientalis leaves extracts (E1) ethyl acetate: chloroform: ethanol (40: 30: 30)
and (E2) methanol: distilled water (70:30).The results showed that E2 extract showed good
antioxidant potential while both extracts showed good antibacterial potential against
Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Agrobacterium tumefaiens. Zhang
et al. (2013) reported the hair growth-enhancing potential of Thuja orientalis hot water extract.
These extract enhanced the hair growth to initiate the anagen phase in the resting hair follicles
and it might be an effective hair growth- enhancing agent.
Ju et al. (2010) from Korea evaluated the protective effect of Thuja orientalis leaves on
6-Hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells using MTT assay.
Thuja orientalis leaves ethanolic extract had potential to protect SH-SY5Y cells from 6-OHDA
by downregulating the oxidative stress and mitochondrial-mediated apoptosis, and by regulating
pERK. Kim et al. (2011) indicated that methylene chloride fraction from leaves of the Thuja
orientalis inhibited the in vitro inflammatory biomarkers through inhibiting the NF-κB and p38
24
MAPK signaling and protects the mice from fatal endotoxemia. Mukhrejee et al. (2012) from
India investigated that Thuja Occidentalis ethanolic leaf extracts inhibited proliferation of A549
cancer cells and caused apoptosis in a dose dependant way.
2.6. Alpinia allughas (retzius) Roscos:
Figure 2.6 Alpinia allughas
Alpinia is one of the largest genera in faimly Zingiberaceae, comprised of 1,200 species
belonging to 49 genera and it is widely distributed in the Southeast Asian region (Shen et al.
2012). Alpinia allughas (retzius) Roscos commonly known as Lachii is an aromatic herb with an
elongate leafy stem and horizontal root stocks, with oblong or lanceolate leaves. The flowers are
present in terminal, racemes or panicles, braceolate, large, sometimes enveloping the buds
(Prakash et al. 2007). The biological classification of Alpinia allughas is shown in the table 2.6.
Table 2.6 Biological classification of Alpinia allughas (retzius) Roscos:
Kingdom Plantae
Division Pinophyta
Class Liliopsida
Subclass Zingiberidae
Order Zingiberales
Family Zingiberaceae
Genus Alpinia
Alpinia species are commonly used in folk medicine because of its anti-hypertensive,
aphrodisiac properties (Santos et al. 2011) and the decoction is extensively used in treating of
cough, bronchitis, diabetics, respiratory ailments, arthritis and asthma (Arambewela et al. 2010).
Prakash et al. (2007) from India studied the chemical profile Alpinia allughas rhizome
and leaves essential oil by GC and GC-MS. The major components of the rhizome oil were α and
β-pinene while in the leaf oil 1, 8-cineole, α-humulene and β-pinene were the major ones.
Padalia et al. (2010) examined the essential-oil constituents of flowers, rhizomes and leaves of
Alpinia calcarata Rosc., Alpinia galanga, Alpinia allughas and Alpinia speciosa K. Schum.
using capillary GC and GC/MS. Ibrahim et al. (2014) characterized the essential oils of Alpinia
mutica and Alpinia latilabris from Peninsular, Malaysia. Both the volatile oils were found to be
rich in camphor camphene, -pinene, and transfarnesol.
Arambewela et al. (2010) evaluated the antifungal and antioxidant properties of Alpinia
calcarata Roscoe rhizomes essential oil. Essential oil of Alpinia calcarata possessed moderate
antioxidant and significant antifungal activity. Prakatthagomol et al. (2011) studied the
bactericidal activity of Alpinia galanga essential oil on food-borne bacteria and concluded that
oil had strong effect on gram negative and positive bacteria. Cunha et al. (2013) reported that
methanolic fraction of the essential oil of Alpinia zerumbet possessed vasorelaxant and
antihypertensive activities. Sattatr et al. (2013) compared the antioxidant activities of ethanol,
acetone, methanol and n-hexane extracts of Z. officinale Roscoe and A. allughas Roscoe
rhizomes. Total phenolics and flavonoid contents were evaluated by the Folin-Ciocalteu and the
aluminum chloride colorimetric methods respectively and radical scavenging effect was
expressed by DPPH assay. Both spices possessed good antioxidant potential and were good
sources of phytochemicals. Wang et al. (2013) reported the in vitro antioxidant and cytotoxic
potential of Alpinia oxyphylla fruits ethanolic extract. The total phenolic contents and antioxidant
activity of the extracts were determined by Folin-Ciocalteu reagent, Trolox equivalent
antioxidant capacity, 1, 1-diphenyl-2-picrylhydrazyl (DPPH•) and reducing power assay
respectively. Cytotoxicity of the extracts was tested on six human cancer cell lines (lung
adenocarcinoma, breast cancer, cervix carcinoma, liver carcinoma, gastric cancer and colon
26
cancer cell lines) using the SRB assay. The ethanolic extract of A. oxyphylla fruit, especially the
ethanolic fraction, was found to possess potent antioxidant and anticancer potential.
Alpinia officinarum and Alpinia blepharocalyx were tested for their anticancer potential by
different assays involving mechanisms of inhibition of cancer cell proliferation (An et al. 2008;
Tbata et al. 2009). Du et al. (2012) isolated Alpinetin from Alpinia katsumadai Hayata, a novel
plant flavonoid, found to exhibit strong anticancer activity against BxPC-3 pancreatic cancer
cells. Raj et al. (2012) from India reported the anticancer effect of Alpinia purpurata against
human ovarian cancer cell lines (PA1) using MTT method. The extracts showed significant
anticancer potential in a dose dependant manner. Chourasiya et al. (2013) isolated
phenylpropanoids from the methanolic extracts of Alpinia galanga rhizomes and determined
their anticancer potential against the human cancer cell lines A549 (lung cancer), Colo-205
(colon cancer), A431 (skin cancer), HT-29 (colon cancer), PC-3 (prostate cancer) and NCI H460
(lung cancer). Ghil, (2013) from Korea examined the anti-proliferative potential of Alpinia
officinarum extract against MCF-7 (human breast cancer cell line). A. officinarum extract
exhibited an anticancer effect in breast cancer cells through inducing apoptosis and cell cycle
arrest in S-phase.
Schinus terebinthifolius Raddi also known as Brazilian pepper, Aroeira, Florida holly, Rose
pepper, or Christmas berry belongs to the Anacardiaceae family of plants and is originated from
South America, mainly from Brazil, Paraguay, and Argentina (Gundidza et al. 2009; Cole et al.
2014). S. terebinthifolius is an evergreen, medium sized tree that may grow up to a height of 30
ft (Pritchard et al. 2000). The biological classification of Schinus terebinthifolius is shown in the
table 2.7.
Kingdom Plantae
Class Magnoliopsida
Subclass Rosidae
Order Sapindales
Family Anacardiaceae
Genus Schinus
In traditional medicine it has been used for the treatment of inflammations, diuretic,
astringent, antibacterial, digestive, antiviral, stimulant, tonic, antiseptic and wound healing
(Gazzaneo et al., 2005; Molina-Salinas et al., 2006; Carvalho et al. 2013). In the Brazilian
Amazon, a bark tea is used as a laxative and a bark-and-leaf tea is used as an antidepressant and
stimulant. In Argentina, a decoction is made with the dried leaves and is used for menstrual
disorders and is also used for urinary and respiratory tract infections (Schmourlo et al. 2005).
Gundidza et al. (2009) from South Africa reported the chemical profile of leaves essential
oil of Schinus terebinthifolius isolated by hydrodistillation method. It was demonstrated that
major constituents of the essential oil were sabinene, α-pinene, terpinene-4-ol, α-phellandrene, β-
pinene, trans-β-ocimene and myrcene. Richter et al. (2010) isolated three sesquiterpene
hydrocarbons from fruits essential oil of Schinus terebinthifolius. Literature revealed that various
28
reports from Brazil were presented for the chemical composition of Schinus terebinthifolius leaf
and fruit essential oil. In fruit essential oil δ-3-carene, followed by limonene, α-phellandrene and
α-pinene while in leaf oil p-cymen-7- ol, 9-epi-(E)-cariophyllene, carvone and verbenone were
the major components (Silva et al. 2010; Cole et al. 2014).
Lima et al. (2006) investigated the antibacterial properties of twenty five medicinal
Brazilian plants against Escherichia coli, two resistant strains of the Staphylococcus aureus and a
susceptible strain of the Staphylococcus aureus. S. terebinthifolius stem barks ethanol extract
presented good antibacterial potential against Staphylococcus aureus. Machado et al. (2008)
evaluated the anti-allergic properties of Schinus terebinthifolius leaves ethyl acetate fraction in
IgE induced mice paw edema and pleurisy. El-Massry et al. (2009) evaluated the
antioxidant/antimicrobial activities of essential oil, ethanol and dichloromethane extract from the
Schinus terebinthifolius leaves grown in Egypt. The antioxidant potential of ethanol extract was
higher than that of essential oil and dichloromethane extract in β-carotene/bleaching test and 2,2-
diphenylpicrylhydrazyl assay. The dichloromethane extract showed the highest antimicrobial
potential against six strains, followed by ethanolic extract and then essential oil. Bendaoud et al.
(2010) compared the antioxidant properties of Schinus terebinthifolius Raddi. and Schinus molle
L berries essential oils. The in vitro antioxidant and antiradical scavenging activities of the tested
oils were measured using DPPH and ABTS test. S. terebinthifolius oil exhibited greatest
antioxidant potential in ABTS assay as compared to S. molle. Johann et al. (2010) determined the
antifungal potential of schinol and a new biphenyl component extracted from the Schinus
terebinthifolius hexane and dichlomethane fractions from leaves and stems against the
pathogenic fungus Paracoccidioides brasiliensis. Costa et al. (2012) studied the antimicrobial
and cytotoxicity of several plants used as medicinal on an indigenous reserve in Riodas Cobras,
Parana, Brazil. The extracts were evaluated against strains of Bacillus subtilis, Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, Candida tropicalis,
Candida parapsilosis, Leishmania amazonensis, Poliovirus and HSV-1. Cytotoxicity test were
also conducted by using VERO cells. The stronger anti-Leishmania and anti-candida activity was
expressed with Schinus terebinthifolius and Zanthoxylum rhoifolium. All plants showed good
cytotoxic effect except Zanthoxylum rhoifolium. Various reports were present for the wound
29
healing property of Schinus terebinthifolius raddi oil in rats and in beef cattle (Santos et al. 2012;
Lipinski et al. 2012; Estevao et al. 2013).
Melo et al. (2014) conducted an experiment with an alcoholic extract of the inner bark of
the Schinus terebinthifolius raddi to determined its effect on autogenously fecal peritonitis in the
Wistar rats. The effect of Schinus terebinthifolius raddi alcoholic extract was considered
extremely positive and hopeful as natural native antiseptic against much severe peritonitis in the
Wistar rats. Miyasato et al. (2014) demonstrated the antimutagenic and antigenotoxic potential of
methanolic extracts of Schinus terebinthifolius Raddi (MEST) in Allium cepa and the Swiss
mice. The antimutagenic and antigenotoxic effects in peripheral blood were measured by using
the comet and micronucleus tests, respectively. The percentage of the damage reduction was
considered to compare the results of A. cepa and mice. The results indicated that MEST can act
as a chemopreventive agent that enhanced the cellular genome integrity through desmutagenic
and bioantimutagenic properties in the vegetal and the animal models.
Queires et al. (2006) also purified polyphenols from Schinus terebinthifolius, Raddi and
investigated their anticancer effect on the androgeninsensitive DU145 human prostatic
carcinoma cell line. The polyphenol fraction was considered to induce G0/G1 cell growth arrest
and caused cell apoptosis. Matsuo et al. (2011) from Brazil investigated the α-Pinene extracted
from the Schinus terebinthifolius Raddi induced apoptosis in a melanoma skin cancer cells.
Santana et al. (2012) investigated the cytotoxic potential of Schinus terebinthifolius leaves
essential oil against cervical carcinoma (HeLa), human melanoma (A2058), murine melanoma
cell line (B16F10-Nex2), breast adenocarcinoma (MCF7) and leukemia (human leukemia (HL-
60) cell lines. The essential oil showed anticancer potential in various cell lines, especially in
leukemia and the human cervical carcinoma.
2.8. Callistemon viminalis:
Figure 2.8 Callistemon viminalis
The Callistemon is a genus of 34 species belongs to family Myrtaceae. It is present all over the
world, mostly distributed in the humid tropical regions that include tropical Asia Australia and
South America. Callistemon viminalis (Bottle brush) is a small tree or shrub with pendulous
foliage, even though some forms are more pendulous than others (Spencer and Lumley, 1991;
Wheeler, 2005). The biological classification of Callistemon viminalis is shown in the table 2.8.
Table 2.8 Biological classification of Callistemon viminalis:
Kingdom Plantae
Class Magnoliopsida
Subclass Rosidae
Order Myrtales
Family Myrtaceae
Genus Callistemon
Species C.viminalis
The genus Callistemon is known in folk medicine for its expectorant, anti-bronchitis, and
insecticidal effects and its volatile oils have been used as anti-microbial and anti-fungal agents.
C. viminalis, was used in the traditional medicines for healing hemorrhoids, gastroenteritis,
diarrhea and skin infections (Goyal et al. 2012).
31
Salem et al. (2013) from Egypt investigated the chemical constituents of Callistemon
viminalis leaves essential oil using GC/MS and reported fourteen components present in it with
1,8-cineole and α-pinene as major constituents. In another report from Egypt Gohar et al. (2013)
isolated a new constituent 3,4-dihydro-2-(hydroxymethyl)-4-methyl-2H-pyrrol-2-ol from bark
and fruits of the C. viminalis. Oliveira et al. (2014) from Brazil extracted the essential oil by
hydrodistillation and determine their chemical constituents by GC-MS. The prominent
components of the Callistemon viminalis flowers essential oil were limonene, α-pinene and 1,8-
cineole. Oyedeji et al. (2009) performed a comparative study for the chemical composition of the
Callistemon viminalis and Callistemon citrinus from South Africa and found that major
components of both species were 1,8-cineole and α-pinene.
Nazreen et al. (2012) from India isolated two flavones a) 5,7-dihydroxy-6,8-dimethyl- 4′
-methoxy flavone b) 8-(2-hydroxypropan-2-yl)-5-hydroxy-7-methoxy-6-methyl-4′-methoxy
flavone from aerial parts of Callistemon lanceolatus and both flavones lowered the glucose level
in the blood diabetic rats. Salem et al. (2013) evaluated the antibacterial, antioxidant properties
and total flavonoid and phenolic contents in Callistemon viminalis leaves essential oil and
extracts. The results revealed that extracts possessed more antioxidant potential than essential oil
whereas essential oil, methanol and ethyl acetate leaves extracts exhibited significant
antibacterial properties. Zandi-Sohani et al. (2013) demonstrated the insecticidal and repellent
properties of the Callistemon citrinus essential oil against the Callosobruchus maculates. Zubair
et al. (2013) from Pakistan reported that methanolic extract from the leaves of Callistemon
viminalis enhanced the oxidative stability of sunflower oil. Jamzad et al. (2014) compared the
antioxidant activities of hydromethanolic extract from flower leaves and stem of Callistemon
citrinus skeels. Results indicated the best antioxidant activity for flower extracts. Chitemerere
and Mukanganyama, (2014) studied the antimicrobial properties of ethanolic leaf extracts of two
plants Callistemon citrinus and Vernonia adoensis from Zimbabwe against Staphylococcus
aureus and Pseudomonas aeruginosa. Both plants extract possessed good inhibitory potential
against tested bacterial strains. Gohar et al. (2013) discovered the molluscicidal activity of
32
Callistemon viminalis methanol extract of fruits, bark and leave against Biomphalaria
alexandrina Snails.
Park et al. (2010) isolated C-methylflavonoids from Callistemon lanceolatus and
examined its anticancer potential against PC12 cells. Results showed that this compound caused
apoptotic cell death. Ali et al. (2011) from Pakistan studies the cytotoxicity of Callistemon
citrinus methanol extract by Brine shrimp assay. The results revealed that methanol extract was
safe at 250 mg/ml or below concentration and results of this brine shrimp cytotoxicity assay
showed that Callistemon citrinus might be a good source of cytotoxic agents.
2.9. Anethum graveolens:
Figure 2.9 Anethum graveolens
Anethum graveolens also known as Dill belongs to family Apiaceae (Umbelliferae), which is
native to Mediterranean countries, southeastern Europe and West Asia. Dill is a short-lived
perennial aromatic herb growing to a height of 1.5 m with tiny yellow flowers. The plant
cultivated in Pakistan, Afghanistan, India, Middle East, Egypt Russia and Iran (Babri et al. 2012;
Dahiya and Purkkayastha, 2012). The biological classification of Anethum graveolens is shown
in the table 2.9.
Kingdom Plantae
The use of this plant for medicinal and consumption reasons have been recorded to dating
back to the Greek and the Egyptian civilizations (Ramadan et al. 2013). A. graveolens was
reported as ‘‘brain tonic’’ in the 17th century in Europe (Orhan et al. 2013). Traditionally dill has
been used for gastrointestinal diseases e.g. stomach-ache colic, indigestion, flatulence and to
tract intestinal gas, pain in stomach and intestines, rheumatism, bladder inflammation, dyspepsia,
liver diseases, insomnia and cramps (Grosso et al. 2008; Kaur and Arora, 2009).
Radulescu et al. (2010) from Romania showed the chemical profile of different plant
parts of Anethum graveolens. The main components in leaves were α-phellandrene, limonene
and anethofuran; in flowers were α-phellandrene, limonene and anethofuran. Cis-carvone and
limonene are the major constituents of seeds volatile oil. A report from Pakistan showed that
essential oil of seeds of Anethum graveolens were contained carvone, apiol, limonene and trans-
dihydrocarvone as the main components (Babri et al. 2012). Ramadan et al. (2013) from Egypt
reported the chemical composition of dried fruit of Anethum graveolens essential oil by GC-MS.
The main volatile components were 7-α-hydroxy manool, l-carvone, limonene, epi-α-bisabolol,
α-terpinene, α-phellandrene, p-cymene, sabinene and α-pinene were the minor constituents. A
report from Tajkistan showed that major components of the aerial part of dill oil were carvone,
trans-dihydrocarvone, dill ether, α-phellandrene and limonene (Sharopov et al. 2013). Kazemi
and Abdossi, (2015) from Iran investigated the volatile constituents of the leaves of Anethum
graveolens. The major constituents of the volatile oil were found to be α-Phellandrene, limonene,
dill ether, α-pinene, n-tetracosane, sabinene, neophytadiene, n-eicosane, n-docosane, n-
nonadecane, n-tricosane, n-heneicosane, α-tujene and β-myrcene.
34
Kaur and Arora, (2009) studied the antibacterial effect of organic and aqueous seed
extracts of Anethum graveolens, Foeniculum vulgare and Trachyspermum ammi. The
antibacterial activity was measured by agar well diffusion assay, minimum inhibitory
concentration and the viable cell count tests. Acetone and hot water seed extracts of three plants
indicated remarkable good antibacterial potential against all tested bacteria except Pseudomonas
aeruginosa and Klebsiella pneumoniae. Jinesh et al. (2010) compared the antioxidant properties
of ethanolic leaves extracts of edible and non-edible leaves of Anethum graveolens. The green
leaves possessed more antioxidant potential than dry leaves. Zeng et al. (2011) evaluated the in
vivo and in vitro anti-Candida properties of Anethum graveolens. The results revealed that the
tested essential oil was effective against vulvovaginal candidiasis in immunosuppressed mice.
Tian et al. (2012) investigated the mechanism of antifungal Action of dill essential oil against
Aspergillus flavus. The antifungal potential of dill essential oil reported its ability to destroy the
permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced
ROS accumulation in the A. flavus. Ramadan et al. (2013) determined the hepatoprotective and
nephroprotective activity against free radicals generated by paracetamol of the aromatic herb
Anethum graveolens. The present study revealed that A. graveolens has antihepatotoxic
properties that could minimize the deleterious effects generated by hepatotoxin paracetamol, and
therefore it can be used as a potent antihepatotoxic agent. Kazemi, (2015) determined the
phenolic contents, antioxidant ability and anti-inflammatory potential of Anethum graveolens L.
essential oil. A. graveolens oil expressed a stronger activity in each antioxidant system especially
in reducing power and β-carotene bleaching test. The TLC-bioautography screening and
fractionation resulted in the separation of the major antioxidant constituents, that were identified
as sabinene and limonene.
Plengsuriyakarn et al. (2012) compared the anticancer potential of eighteen Thai
medicinal plants for their anticancer potential in animal models against cholangiocarcinoma. All
plants showed good anticancer potential and good chemotherapeutic agents for the treatment of
cholangiocarcinoma. In another report from Thailand Peerakam et al. (2014) studied the
chemical profile and anticancer activity of Anethum graveolens L. seed essential oil on KB-Oral
cavity and MCF7-Breast cancer cells using Resazurin micro plate assay (REMA). Results
demonstrated that Anethum graveolens essential oil possessed good anticancer potential.
35
The review of past studies disscused in this chapter, showed that essential oils obtained
from many plants have gained much attention among food scientists and researchers due to their
numerous biological properties. Although plenty of studies have been carried out and lot of
results have been obtained regarding chemical characterization and biological activities of
aromatic plants, however, to the best of our knowledge there are no detailed findings on
chemical characterization and biological activities of essential oils of selected plants, native to
Pakistan.
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CHAPTER 3
MATERIALS AND METHODS The rese

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DOCTOR OF PHILOSPHY IN CHEMISTRY