Abstract
Viscum album (Mistletoe) is a semi-parasitic plant which grows on the stems of other tree species. This present study was carried out to investigate the phytochemical composition, acute toxicity, analgesic, antiinflammatory and tonic properties of Viscum album, which was soxhlet-extracted using ethanol as solvent.Qualitative phytochemical analysis of ethanol extract of Viscum album showed the presence of saponins, alkaloids, flavonoids, phenolics, tannins, terpenes, glycosides and steroids. Quantitative screening yielded high result for flavonoids (6.93mg/100g), followed by alkaloids, saponins, tannins, steroids phenols, terpenes and glycosides yielding (6.39, 5.81, 4.38, 3.12, 2.76, 1.95 and 0.94)mg/100g respectively. Acetic acid-induced writhing model was employed for analgesic activity assay and aspirin was used as standard control. Oral administration of (400 and 800mg/kg b.w) significantly reduced the nociceptive responses induced by acetic acid by (80.34±0.67 and 58.95±2.25) % respectively. The antiinflammatory activity was screened using a phlogistic agent, egg albumin-induced rat paw edema, using aspirin (100mg/kg) as reference control. It was observed that administration of (200 and 400mg/kg) of extract significantly inhibited inflammatory response by (58.22±8.24 and 58.73±4.40) % respectively at the end of 120min of the assay. The tonic profile of the extract was screened on laboratory water bath, using an isolated rabbit jejunum. First, the effects of different concentrations of ethanol extract of Viscum album(300, 600 and 1200) µg/ml were assayed and it was found to reduce the basal contractions (15.00±0.41, 14.00±0.41 and 16.00±0.58)mm to (2.75±0.48, 2.00±0.41 and 1.75±0.25)mm respectively, which represent (81.74, 85.68 and 88.93)% respectively. Then the effects of same concentration of extract 300µg/ml was examined on different concentrations of acetylcholine (3.33, 6.66 and 13.32)µg/ml and was shown to inhibit the excitatory activity of acetylcholine from (17.00±0.41, 21.75±0.48 and 21.75±0.48)mm to (2.25±0.25, 6.50±0.29 and 6.50±0.29)mm, which represent (86.75, 79.29 and 70.05)% respectively. Furthermore, results of the effects of same concentration of atropine on acetylcholine induced contraction had no significant effect, but began inhibiting acetylcholine activity upon increased concentrations of atropine. The reduction in the contractile response to acetylcholine, following supposed blockade of cholinergic receptors by plant extract was far more considerable than atropine in rabbit jejunum. These results indicate that the ethanol extract of Viscum album is a potent analgesic, antiinflammatory and tonic compound. These findings further suggest that the analgesic, antiinflammatoryeffects and the relaxing effects of theethanol extract of Viscum album on jejunal contractions may in fact be due to the presence of the phytoconstituents present. However, further research on the isolation and characterization of the responsible phytochemicals is pertinent.
TAB
LE OF CONTENTS
Title Page i
Declaration
ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables xi
Abstract xii
CHAPTER 1: INTRODUCTION
1.1 Background of the Study 1
1.2 Aim of the Study 3
1.3 Objectives of the Study 3
1.4 Statement of the Problem 4
1.5 Justification 5
CHAPTER 2: LITERATURE
REVIEW
2.1 Pain 6
2.1.1 Acute pain 7
2.1.2 Chronic Pain 8
2.1.3 Special
Types Of pain 10
2.1.3.1 Neuropathic pain 10
2.1.3.2 Phantom pain 11
2.1.3.3 Central pain 12
2.1.3.4 Allodynia 13
2.1.3.5 Nociceptive
pain 13
2.1.3.6 Breakthrough
pain 14
2.1.3.7 Psychogenic
pain 14
2.1.4 Pathways
of pain 15
2.1.5 Regulators
of pain 16
2.1.6 Pain management and treatment 17
2.1.6.1 Assessment of pain 18
2.1.6.11 Physiological
assessment 18
2.1.6.12 Pain
scales 18
2.1.7 Treatment of pain 19
2.1.7.1 Non-pharmacological approach 20
2.1.7.11Acupuncture: 20
2.1.7.12Heat and cold 20
2.1.7.12.1 Transcutaneous
electrical nerves stimulation (TENS) 20
2.1.7.2 Pharmacological
approach 21
2.1.7.21
Analgesics and anesthesia 21
2.1.7.211 Paracetamol (acetaminophen) 21
2.1.7.212 NSAIDs 22
2.1.7.213 Opioids 23
2.1.7.214Gabanoids,
gabapentin, and gregabalin 24
2.2
Overview of inflammation 25
2.2.1 Types of
inflammation 27
2.2.1.1 Acute
inflammation 27
2.2.1.2 Chronic
inflammation 28
2.2.2 Etiology of inflammation 29
2.2.3 Pathophysiology 30
2.2.4 Inflammatory mediators 30
2.2.5 Treatment
of inflammation 32
2.2.6
Inflammatory
diseases 34
2.3 Smooth Muscles: introduction 35
2.3.1 Core function of smooth muscles 36
2.3.2 Organs involved in smooth muscles
functionality 36
2.3.3 Smooth muscle 37
2.3.4 Mechanism of smooth muscle functionality 38
2.3.5 Pathophysiology of smooth muscle 39
2.3.6 Important modulators of smooth muscle tone 41
2.3.6.1 Structural modulators 41
2.3.6.2 Organic chemical modulators 41
2.3.6.21 Neurotransmitters
as organic modulators 42
2.3.6.211
Acetylcholine 43
2.3.6.22Classification of neurotransmitters 44
2.3.6.23 Drugs that influence
neurotransmitters 45
CHAPTER 3: MATERIALS
AND METHODS
3.1 Materials 47
3.1.1 Plant material 47
3.1.2 Animals 47
3.1.3 List of instruments/equipment 47
3.1.4 Preparation of reagents for phytochemical assay 49
3.2 Methods 51
3.2.1 Extractionof plant material 51
3.2.2 Qualitative phytochemical analysis of
ethanol extract of Viscum album 51
3.2.2.1 Test for saponins 52
3.2.2.2 Test for flavonoids 52
3.2.2.3 Test for alkaloids 53
3.2.2.4 Test for terpenes 53
3.2.2.5 Test for tannins ` 53
3.2.2.6 Test for phenols 54
3.2.2.7 Test for steroids 54
3.2.2.8 Test for glycosides 54
3.2.3 Quantitative phytochemical analysis of ethanol
extract of Viscum album 54
3.2.3.1 Determination of saponins 54
3.2.3.2 Determination of alkaloids 55
3.2.3.3 Determination of tannins 56
3.2.3.4 Determination of flavonoids 56
3.2.3.5 Determination of phenolic content 57
3.2.3.6 Determination
of cardiac glycosides 57
3.2.3.7
Determination of steroids 58
3.2.3.8 Determination of terpenes 58
3.2.4 Acute toxicity and lethal dose test (LD50) 59
3.2.5 Evaluation of analgesic activity 59
3.2.5.1 Experimental animals 60
3.2.5.2 Experimental procedure 60
3.2.6 Evaluation of antiinflammatory activity 61
3.2.6.1 Induction of a hind paw edema 61
3.2.6.2 Experimental animals 61
3.2.6.3 Procedure 62
3.2.7 Evaluation of tonic effect 62
3.2.7.1 Reagents for tyrode solution 63
3.2.7.2 Calculation of drug volumes and concentrations
for tonic activity 63
3.2.7.3 Experimental protocol 64
3.2.8 Statistical analysis 65
CHAPTER 4 : RESULTS AND DISCUSSION
4.1
Results 66
4.1.1 Phytochemical assay 66
4.1.1.1 Qualitative phytochemical report 66
4.1.1.2 Quantitative phytochemical Report 67
4.1.2 Acute toxicity profile 69
4.1.3 Analgesic effect 70
4.1.4 Antiinflammatory effect 71
4.1.5 Tonic effect 73
4.2 Discussion 76
CHAPTER 5: CONCLUSION
AND RECOMMENDATION
References 84
Appendix 92
LIST OF TABLES
4.1:
Phytochemical components of ethanol
extract of Viscum album
(Mistletoe) 65
4.2: Quantitative phytochemicals content of extract
of Viscum album
(Mistletoe) 66
4.3: Acute toxicity results of ethanol extract
of Viscum album 68
4.4: Analgesic effect of Viscum album on acetic acid induced writhing in experimental albino
rats. 69
4.5: Results for antiinflammation 70
4.6: Change in paw diameter and % change in paw
diameter of antiinflammation 70
4.7a: Effect of the extract Viscum album on isolated rabbit jejunum 72
4.7b: Effects of Ach (Acetylcholine) on isolated
rabbit jejunum 72
4.7c: Effects of atropine on acetylcholine induced
contraction 73
4.7d: Effects of extract on acetylcholine
administration on isolated rabbit jejunum 74
CHAPTER 1
INTRODUCTION
1.1
BACKGROUND
OF THE STUDY
The benefits of medicinal plants cannot be
overemphasized. A plethora of health challenges have been remedied by recent
advancements in their research and applications. Plant derived substances have
constantly remained significant in drug development, especially in the discovery
of novel analgesic and antiinflammatory drugs. They not only help as
aforementioned, but also greatly contribute to understanding the complex
pathway of pain transmission, receptor types and endogenous ligands which play
critical role in its transmission. The search for new naturally occurring
analgesic, antiinflammatory and compounds is taking aggressive dimensions due
to their efficacy, reduced contraindications, creating significant leads in the
development of highly efficacious synthetic molecules (Qurqn, 2009).
Viscum album
belongs to the family Loranthaceae.
It is a small greenish plant, known as mistletoe in English. Mistletoe (Viscum album) is a semi-parasitic plant which
thrives on varieties of host trees which includes pine, oak, birch and apple.
The plant is preponderantly found in Northern areas of Pakistan on the hills in
Chitral and Dir Upper. The plant has a record length growth of up to 100 cm
with leathery leaves alternate on the stems. Fruit of Viscum album is a waxy white gelatinous berry. The term semi-parasitic
implies that the mistletoe plant undergoes photosynthetic process
independently, while feeding off the host’s water and minerals endowment. There
are several varieties of mistletoe, which are defined by the host that they
parasitize. European mistletoe is dioecious, that is each plant is unique with
either male or female flowers. They are pollinated by insects and bear small
white berries on evergreen foliage, other varieties bear red berries. Alkaloids,
glycosides, phenylpropanoids, tannins, lignins sugars and viscotoxinsare some
of the several chemical components found in Viscum
album. Several subcomponents of flavonoids like quercetin, kaempferol and
rarely naringenin have also been found to be present. Methyl derivatives of
quercetin and kaempferol have also been reported from this plant (Ame et al., 2010)
MISTLETOE preparations have been widely used in Europe
and other parts of the world including Africa for centuries to treat epilepsy,
infertility, hypertension, cardiovascular disorders and arthritis. Medicinal
use of Viscum albumh as also been
recorded in ancient Greek practice. The treatment of diseases associated with
spleen disorders and menstruation using preparations of Viscum album have been documented by Hippocrates (460–377 BC). In
folklore medicine, mistletoe has been used as a panacea for a wide range of
diseases such as sciatica, neuralgia, rheumatic deafness and asthma, diabetes
mellitus, chronic cramps, stroke, epilepsy, stomach upsets, as antihypertensive
and discomforts associated with menopause. Medicinally, Viscum album is recorded to be highly useful in diuretics, as antispasmodic
and relieving cardiac problems (Ohiri et al., 2003)
Chemical constituents isolated from Viscum album (mistletoe) have been tentatively
recorded to possess the potential for the treatment of several cancer types,
including bladder, breast, pancreas and many more, based on the observation
that the plant, like cancer, is parasitic and lethal to its host. Albeit, there
is lack of quality clinical trials and no significant evidence of an effect
(Ernst, 2006; Hong et al., 2014; Von
Schoen-Angerer et al., 2015).
However, not so much findings are there to
show that extract and compounds isolated from Viscum album reduce the activity of smooth muscle. Further studies
however are needed. In folk medicine, it has been utilized for its
cardiovascular properties; clinical efficacy has not been established.
Injectable mistletoe extract is widely used in Europe but have not yet been
ratified for use in the United States. Several experimental studies have shown
to imply that administration of extracts from mistletoe could prove beneficial
in alleviating critical medical conditions or cases of degenerative joint
diseases/osteoarthritis, diarrhea, arteriosclerosis, epilepsy, gastrointestinal
disorders, heart conditions, high blood pressure, infertility, indigestion,
tachycardia and bachycardia.
1.2 AIM OF THE STUDY
The aim of this study was to assay the phytochemical
composition, analgesic, antiinflammatory and tonic properties of extract of Viscum album (Mistletoe).
1.3 OBJECTIVES OF THE STUDY
a) To
evaluate the qualitative and quantitative phytochemical compositions such as tannins,
flavonoids, saponins, phenolic compounds, alkaloids and terpenes of the extract
of Viscum album.
b) To
determine the acute toxicity (LD50) of the extract of Viscum album in experimental Wistar
rats.
c) To
evaluate the analgesic effect of the extract of Viscum album on experimental albino rats.
d) To
evaluate the anti-inflammatory effect of the extract of Viscum album on selected experimental wistar rats.
e) To
determine in vivo, the tonic potential of the extract of Viscum album on isolated rabbit jejunum.
f) To
determine the mechanism of the tonicity potential of the extract of Viscum album on isolated rabbit jejunum.
1.4 STATEMENT OF THE PROBLEM
Mistletoe (Viscum
album) is widely utilized in traditional practice for remedy in cases of infertility, hypertension, and arthritis, mostly
in the southern part of Nigeria. The plant grows in diverse parts of the
African rainforests and has long been employed in villages and urban
metropolis, to alleviate certain physiological diseases and dysfunction. Though
numerous therapies for pain, inflammatory and tonic disorders.
The World Health Organization (WHO) also encourages
research studies pertaining to the treatment and/or prevention of the above
mentioned maladies using traditional medical practices as there is the worldwide
belief, that herbal remedies are safer and less harmful to the human and animal
systems, than the widely used synthetic drugs (Meite et al., 2009)
A tenable solution is to find and develop new
traditional remedies for the cure, treatment and possible prevention of the
maladies both in children and in adults.
1.5 JUSTIFICATION FOR THE STUDY
The use of herbal remedies, according to WHO, to treat
diseases and disorders would provide best sources from which new varieties of
medications could be developed (Akuodor et
al., 2011). The traditional application of this specific plant for the cure
of a plethora of pain, inflammatory and tonicity-related dysfunctions and
maladies underpins this research study, designed to assay the composition,
analgesic, anti-inflammatory and tonic tendencies of the ethanol extract of the
leaves of Viscum album (mistletoe) on
selected experimental albino rats.
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