ABSTRACT
This study investigated the phytochemical constituents and antibacterial potentials of leaves of Cannabis sativa and Azadirachta indica in its aqueous and ethanolic extract against some clinical organisms. The in-vitro antibacterial potentials of the plants extracts were screened using the paper disc diffusion and broth dilution techniques at varying concentrations. The ethanolic and aqueous extracts showed synergism activity against at least one of the test organisms with the ethanol extract of A. indica and C. sativa showing maximum synergism activity against Staphylococcus aureus with zones of inhibition in the range of 8.5±0.70 to 15.0±1.41mm and 9.0±1.41mm to 12.5±0.70mm respectively. The crude ethanolic extract of the plants, Cannabis sativa and Azadirachta indica showed remarkable synergistic activity against the growth of Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa at 100mg/ml concentrations. With diameter zone inhibition of 14.5mm, 13.0mm and 11.5mm respectively. The aqueous extract of the leaf of these plants, showed least activity when compared with the ethanol extracts. The aqueous extracts were only active at 100mg/ml concentrations. Azadirachta indica extract showed an MIC and MBC values in the range of 12.5 to 50mg/ml. Similarly, Cannabis sativa showed MIC and MBC within 25mg/ml and 50mg/ml concentrations. The Phytochemical screening of samples revealed the presence of secondary metabolites such as Alkaloids, Flavonoids, quinines, Cardiac glycosides and Tannins. This study establishes a good support to the use of plant in herbal remedies and as base for development of new drugs
TABLE OF CONTENTS
Certification i
Title page ii
Dedication iii
Acknowledgements iv
Table of
Contents v
List of Tables vii
Abstract viii
CHAPTER ONE
1.0 Introduction 1
1.1
Aims and Objectives 4
CHAPTER TWO
2.0 Literature review 6
2.1 Cannabis
sativum 6
2.2 Cannabis
toxicity in humans 7
2.2.1 Cannabis
and the central nervous system (CNS) 8
2.2.1.1 Cognition and coordination 8
2.2.2.2 Psychosis and schizophrenia 9
2.2.2.3 Pulmonary disease and lung cancer 10
2.2.2.4
Vascular conditions: 10
2.2.2.5
Cannabis and carcinogenic
potential: 10
2.2.2.6
Fertility 11
2.2.2.7 Cannabis hyperemesis syndrome 11
2.3 Cannabis
during development: the adolescent and prenatal periods 12
2.3.1 Fetus 12
2.3.2 Adolescence 12
2.4 Cannabis
and society 14
2.4.1 Cannabis and driving 14
2.4.2 Cannabis, employment, the workplace 14
2.4.3 Cannabis and advanced education 15
2.5 Dependence, abuse and cannabis use disorder
(cud) 15
2.5.1 Psychological or physiological dependence 15
2.5.2 Globally 16
2.6 The
use of cannabis for medical purposes 18
2.6.1
Neurological diseases or symptoms 18
2.6.2 Multiple sclerosis 19
2.6.3 Neuropathic pain 19
2.6.4 Randomized
controlled trials with cannabis: 20
2.6.5 Alzheimer’s disease 21
2.7 Azadirachta
indica: neem tree 22
2.8 Medicinal uses 23
2.9 Phytochemicals 25
2.10 Antimicrobial
activity of Azadirachta indica
(neem tree) 27
2.10.1 Antibacterial and anti-fungal activities 28
CHAPTER THREE
3.0 Materials and Methods 30
3.1
Collection of plant materials 30
3.2 Extraction of plant
material 30
3.3 Preparation
of extract 31
3.3.1 Screening
of the Extracts for Antibacterial Activity 31
3.3.2 Determination of MIC and MBC 32
3.4 Qualitative phytochemical screening 33
3.4.1 Tests for Alkaloids (Mayer’s Test) 33
3.4.2 Test
for Flavonoids (Alkaline reagent test) 33
3.4.3 Test
for Tannins 33
3.5.3 Tests for Glycosides 33
3.6 Biochemical
identification of the test organisms 34
3.6.1 Gram
staining 34
3.6.2 Catalase
Test 34
3.6.3 Coagulase
Test ` 34
3.6.4 Citrate Utilisation Test 35
3.6.5 Hydrogen Sulphide (H2S)
Production Test 35
3.6.6 Indole Test 35
CHAPTER FOUR
4.0 Results 36
CHAPTER FIVE
5.0
Discussion, conclusion and recommendation 43
5.1
Discussion 43
5.2
Conclusion 46
5.3
Recommendation 47
References
LIST OF TABLES
Table Title Page
1 Diameter zone of Inhibition (mm) produced by ethanol and
aqueous extracts of A. indica 37
2 Diameter zone of Inhibition (mm) produced by ethanol and
aqueous extracts of C. sativa 38
3 MIC and MBC values (mg/ml) of ethanol extract of Cannabis sativa and A. indica plants against
the test isolates 39
4 MIC and MBC values (mg/ml) of aqueous extract of Cannabis sativa and A. indica
plants against the test
isolates 40
5 Shows the
Phytochemical analysis of extracts of Cannabis
sativum and Azadirachta indica 41
CHAPTER ONE
1.1 INTRODUCTION
Nature has provided a complete store house of remedies to cure all
ailments of mankind. The natural or herbal remedies are still the backbone of
medicines. The use of medicinal plants as a source for relief from illness can
be traced back over five millennia to written documents of the early civilization,
but it is doubtless an art as old as mankind. Phytotherapy is a medicinal
practice based on the use of herbal plants and their extracts. These herbs or
plants and their active ingredients are used in traditional herbal remedies.
The easy availability, low cost and negligible side effects, natural products
are popular in the nowadays in the world (Albinu et al., 2010). All the herbs produced bewildering variety of
phytochemicals like primary metabolites (carbohydrates, fats, proteins) and
secondary metabolites (Alkaloids, flavonoids, steroids, saponins, polyphenols,
etc.) for their normal metabolic activities (Nair and Chanda, 2010). These
secondary metabolites showed various biological activities and act in plant
defense mechanisms. The chemical profile of a single plant may vary over time
as it reacts to changing conditions. The secondary metabolites have therapeutic
actions, which produced drugs (Lai,
2004).
Biologically active compounds present in the medicinal plants have
always been of great interest to scientists working in this field. In recent
years this interest to evaluate plants possessing antibacterial activity for
various diseases is growing (Kaushik et
al., 2003). Plant materials remain an important resource to combat serious
diseases in the world. The traditional medicinal methods, especially the use of
medicinal plants, still play a vital role to cover the basic health needs in
the developing countries (Satish et al.,
2008). The medicinal value of these plants lies in some chemical active
substances that produce a definite physiological action on the human body
(Ceylan and Fung, 2004).
Plants produce a diverse range of bioactive molecules, making them
rich source of different types of medicines. The most important of these
bioactive constituents of plants are alkaloids, tannins, flavonoids and
phenolic compounds. Phytomedicines derived from plants have shown great promise
in the treatment of various diseases including bacterial and viral infections
(Duke, 1992).
Azadirachta indica Neem
is used in traditional medicine as a source of many therapeutic agents in the
Indian culture and grows well in the tropical countries. Its twigs provide a
chewing stick and are widely used in the Indian sub-continent. Earlier studies
on neem have showed that it contains active substances with multiple medicinal
properties (Maragatharavlli et al., 2012). Neem leaves has antibacterial
properties and could be used for controlling airborne bacterial contamination
in the residential premise (El-Mahmood et al., 2010). Administration of
alcoholic extract of neem flower disrupts the estrous cycle in Sprague Dawley
rats and causes a partial block in ovulation and has the potential of an ideal
antifertility agent (Gbotolorun et al., 2008). The popularity of the
plant products is increasing because of their biodegradability, least
persistence and least toxic to non-target organisms, economic and easy
availability. Today about 200 plants with insecticidal activities are known.
Among the natural products, one of the most promising natural compounds is
Azadirachtin, an active compound extracted from the Azadirachta indica,
whose antiviral, antifungal, antibacterial and insecticidal properties have
been known for several years. Chemical investigation on the products of the
neem tree was extensively undertaken in the middle of the isolation of nimbin,
the first bitter compound isolated from neem oil, more than 135 compounds have
been isolated from different parts of neem (Ganguli, 2002). Neem elaborates a
vast array of biologically active compounds that are chemically diverse and
structurally complex. Medicinal properties of the plant Azadirachta indica were
studied by several workers. Azadirachta indica is a wonder plant with
valuable economic and health significance attached to all its parts. In fact,
it is a well know versatile medicinal plants with wide spectrum of biological
activities (Siddique et al., 2004).
For example, its leaf, back, roots, fruit coat, seed and flowers (Atawodi and
Atawodi, 2009) have been demonstrated to exhibit immunomodulatory (Haque et al., 2006), anti-inflammatory
(Akihisa et al., 2011),
anti-hyperglycaemic and antidiabetic (Bhat
et al., 2011), antiulcer (Chattopadhyay
et al., 2004), antimalarial (Isah et
al., 2003), antifungal (Natarajan et
al., 2003), antibacterial (Thakurta et
al., 2007), antiviral (Parida et al.,
2002) anticarcinogenic (Kumar et al.,
2006) and spermicidal (Khillare and Shrivastav, 2003) properties and
antifertility agent ( Gbotolorun et al., 2008). (Bandyopadhyay et al., 2002). Boiled neem leaf
water makes an excellent antiseptic to clean wounds, soothes, swellings and
eases skin problems (Bonjar and Holland, 2004).
The 'Cannabis sativa' plant is perhaps one of the most
easily identifiable plants of the plant kingdom. The Cannabis sativa plant,
more commonly known to us as marijuana falls under the taxon of wild or feral
plants. Cannabis sativa is an angiosperm belonging to the Cannabaceae
family and Cannabis genus (Borhade, 2013). Cannabis is grown and
processed for many uses. Many plant parts are used as medicine for humans and
livestock, whole seeds and seed oil are eaten by humans, seeds and leaves are
fed to animals, seeds oil and stalks are burned for fuel. Whole plants, leaves
and wood have environmental uses, bark, fiber and seeds are also of ritual
importance (Clarke, 2002). Renewed interest into cannabis in the last few
decades balanced between excitement from all kinds of newly discovered
pharmacologically desired effects and fear from abuse and risky behavior in
society (Pomahacova et al., 2009).
Cannabinoids interact with various neurotransmitters and neuromodulators, such
as gamma-aminobutyricacid (GABA), histamine, serotonin, dopamine, glutamate,
norepinephrine, prostaglandins and opioid peptides (Grotenhermen, 2002). The
use of Cannabis sativa (Cannabis) extracts as medicine was described in
China and India before the birth of Christ (Fankhauser, 2002). The therapeutic
use of Cannabis was introduced in Western medicine in the first half of
the 19th century and reached its climax in the last two decades of the same
century. At the turn of the century, several pharmaceutical companies were
marketing Cannabis extracts and tinctures which were prescribed by
doctors for many different complaints including pain, whooping cough and
asthma, and as a sedative/hypnotic agent (Fankhauser, 2002).
The leaves of 'Cannabis sativa' plant possess good
antimicrobial activity against Mycobacterium tuberculosis,
Gram-negative bacteria of the Escherichia
coli, Pseudomounas aeruginosa, Proteus vulgaris, Enterococccus
faecalis, acid-fast bacteria, yeast like fungi, filamentous fungi and
dermatophyt (Wasim et al., 1995). Cannanbinoids have strong
antileishmanial activity and effective to killing Candida albicans (Whittakar
et al., 2004). The contact of both herpes simplex virus type 1 and
herpes simplex virus type 2 to various absorptions of delta-9-
tetrahydrocannabinol present a plaque assay utilizing confluent monkey cells
that have possible mechanisms for antiviral activity and that this activity is
modified by the presence of serum proteins (Lancz et al., 1991).
1.2
AIMS AND OBJECTIVES
1.
To investigate
the antibacterial activities of aqueous and ethanolic extracts of Cannabis sativa and Azadirachta indica in synergism on some
pathogenic organisms;
2.
To determine
the Minimum Inhibitory Concentration (MIC) of the aqueous and ethanolic
extracts of Cannabis sativa and Azadirachta indica in synergism on some
pathogenic organisms;
3.
To determine
the Minimum Bactericidal Concentration (MBC) of the aqueous and ethanolic
extracts of Cannabis sativa and Azadirachta indica in synergism on some
pathogenic organisms;
4.
To screen the
leaves of Cannabis sativa and Azadirachta indica for different phytochemical
constituents.
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