ABSTRACT
The plant Stachytarpheta angustifolia is a medicinal plant which comes from the genus Stachytarpheta of the family Verbenaceae. This study is aimed at isolating the plant extract, characterizing it and determining the phytochemical and analgesic properties of the plant. Extraction was done using chloroform and column chromatography was used for the elution of the crude extract using silica gel as the stationary phase. Solvents used as the mobile phase were petroleum ether, chloroform and methanol. These solvents had differing polarity; from non-polar to polar. A total of 26 fractions were collected after elution. Purity of the fractions were monitored using thin layer chromatography (TLC) on silica gel coated plates. Solvents used were petroleum ether, choroform and methanol. Quantitative and qualitative screening test were both carried out on the crude extract to determine the phytochemical properties of the plant. Analgesic analysis was carried out using acetic-acid writhing reflex method. After TLC, fraction 26 yeilded a single spot with the solvents, petroleum ether, chloroform and methanol in the ration 1:3:6 (Rf =0.55). The structure was elucidated using FTIR, 1HNMR, 13CNMR, DEPT-135 and C-H COSY and the structure had a molecular formula of C28H48O3. The compound was characterized as a triterpenoid. Phytochemical screening using the standard qualitative techniques revealed the presence of flavonoids, phenolics, tannins, steroids and terpenoids. The result showed very strong presence of Alkaloids, Flavonoids and terpenoids. The crude extract also showed good analgesic property. As a follow-up on the study, it is anticipated that the compound can further be explored to reveal its potential applications.
TABLE
OF CONTENTS
Title
Page i
Declaration ii
Certification iii
Dedication vi
Acknowledgements v
Table of Contents vi
List of Tables xi
List
of Figures xii
List
of Plates xiii
Acronyms xiv
Abstract xvi
CHAPTER 1: INTRODUCTION 1
1.1 Background to the Study 1
1.2 Statement of the Research Problem 4
1.3 Aim
and Objectives of the Study 5
1.4 Justification of the Study 6
1.5 Scope and Limitation 6
CHAPTER 2: REVIEW OF
RELATED LITERATURE 7
2.1 Analgesics 7
2.1.1 Sources
of Analgesic drugs 8
2.2 Starchytarpheta Species 9
2.2.1 Stachytarpheta
angustifolia 9
2.3 Phytochemical Studies of Stachytarpheta 15
2.3.1 Alkaloids 17
2.3.2 Saponins 18
2.3.3 Phenols 19
2.3.4 Flavonoids 19
2.3.5 Tannin 21
2.4 Biological
Activity of the Plant 22 2.4.1 Hypoglycemic
activity 22
2.4.2 Antioxidants 24
2.4.3 Anti-microbial 26
2.4.3.1 Antibacterials 28
2.5 Analgesic
Effect of Stachytarpheta 29
2.6. Acetic acid induced writhing 30
2.7 Chromatography 31
2.7.1 Principles of chromatography 32
2.7.2 Chromatography
terms 32
2.7.3 Types of chromatography 33
2.7.3.1 Column chromatography 34
2.7.3.2 Thin layer chromatography (TLC) 35
2.7.3.3 Paper chromatography 37
2.8 Retention Factor 37
2.9 Nuclear
Magnetic Resonance (NMR) Spectroscopy 38
2.9.1 Principle
of NMR 41
2.10 Infra-Red Spectroscopy 42
2.11 Mass Spectroscopy (MS) 43
2.12 Matrix Assisted Laser Desorption Ionization 44
CHAPTER 3: MATERIALS AND METHODS 45
3.1 Materials 45
3.1.1 Experimental animals 45
3.2 Methods 47
3.2.1 Summary of experimental procedures 47
3.2.2 Sample collection 48
3.2.3 Sample processing 48
3.2.4 Extraction 48
3.3 Phytochemical Screening 48
3.3.1 Screening test 48
3.3.1.1 Alkaloids 49
3.3.1.2 Saponins 49
3.3.1.3 Tannins 49
3.3.1.4 Flavonoids 49
3.3.2 Quantitative test 50
3.3.2.1 Tannin 50
3.3.2.2 Saponin 50
3.3.2.3 Alkaloids 51
3.3.2.4 Phenols 51
3.3.2.5
Flavonoids 52
3.4 Pharmacological Screening 53
3.4.1 Acute toxicity test 53
3.4.2 Acetic acid-induced writhing in rat 54
3.3.4 Statistical analysis 54
3.5 Isolation 55
3.5.1 Spectra analysis 56
CHAPTER 4: RESULTS AND DISCUSSION 57
4.1 Phytochemical Screening 57
4.2 Thin Layer Chromatography 60
4.3 Spectral Analysis of Isolated Extract of S. angustifolia 62
4.3.1. FT-IR
analysis of the isolated chloroform extract of S. angustifolia 62
4.3.2 Proton-NMR
of ST 26 65
4.3.3 Carbon-Proton Cosy (HETCOR) 67
4.3.4 Carbon-13 (13 C) NMR 69
4.3.5 Carbon-13(13 C) DEPT NMR 70
4.4 Analgesic
Activity 72
4.4.1 Acetic acid induced writhing reflex 72
CHAPTER 5: CONCLUSION AND
RECOMMENDATION 74
5.1 Conclusion
74
5.2 Recommendations 75
REFERENCES 76
LIST OF TABLES
2.1 Acetic acid-induced writhing in rats / mice 31
3.1 Body weight, Food and water intake of Wister rats 45
3.2 Fractions of S.
angustifolia extract by column chromatography 56
4.1 Phytochemical qualitative screening of
S. angustifolia 59
4.2 Phytochemical quantitative screening
of S. angustifolia 59
4.3 IR absorption of S. angustifolia 64
4.1 S. angustifolia
on Acetic induced Writhing Reflex in Albino Rats 72
LIST OF FIGURES
2.1:
Iridoid glycoside of S. angustifolia
(Compound 1) 12
2.2:
Iridoid Glycoside S. angustifolia
(Compound 2) 13
2.3:
Iridoid glycoside of S. angustifolia (Compound
3) 13
2.4:
Lanostanoid triterpenoid glucoside of
S. jamaicensis (Compound 4)
14
2.5:
Steroidal Glycoside of S. jamaicensis
(Compound 5) 14
2.6:
Nnesodine B (Compound 6) 14
2.8:
Saponin Structure 18
2.9:
Flavone structure 20 2.10: Isoflavan structure 20
2.11:
Neoflavonoid structure 20
2.12:
Structures of some antioxidants 26
3.1: Flow chart of methodology 47
4.1:
FT-IR Spectrum for the S. angustifolia extract 63
4.2: Proton NMR (1H-NMR)
for the S. angustifolia extract 65
4.3: Carbon-Proton COSY NMR for Stachytarpheta
angustifolia extract 67
4.4: 13C NMR of Stachytarpheta angustifolia extract
69
4.5: 13C-DEPT (135) NMR of Stachytarpheta
angustifolia
extract 70
4.6: Proposed structure from isolated
compound of Stachytarpheta angustifolia. 71
LIST OF PLATES
2.1 Stachytarpheta angustifolia 10
4.1 2D TLC showing
a single spot for fraction ST26 62
ACRONYMS
%: Percentage.
3D: Three Dimension
4D: Four Dimension
ASA: Acetyl
Salicylic Acid.
ADME: Absorption, distribution, metabolism and elimination
COX: Cyclooxygenase
COSY: correlation
spectroscopy
DF: Dilution factor
Fig: Figure
FT-IR: Fourier Transform Infrared Spectroscopy
GC – MS: Gas Column
Spectrophotometry.
H2SO4: Sulphuric
acid.
HCl: Hydrochloric
acid.
HETCOR: Heteronuclear
Correlation
HMBC:Heteronuclear
Multi-Bond Connectivity
HMQC:Heteronuclear
Multi-Quantum Coherence
HOESY:HeteronuclearOverhauser
Effect Spectroscopy
HPLC: High Pressure
Liquid Chromatography
HSQC:Heteronuclear Single Quantum Coherence
IR: Infrared
KG: Kilogram.
L: Liter.
LD50: Median lethal
dose.
MIC : Minimum
inhibitory concentration
ML: Mililiter.
MHz: Mega Hertz
MM: Milimeter.
MRS: Magnetic
Resonance Spectroscopy
NMR: Nuclear Magnetic Resonance
N/S: Normal
saline.
NOE: Nuclear Overhauser Effect
NOESY: Nuclear Overhauser Effect Spectroscopy
NSAIDs: Non-steroidal
anti-inflammatory drugs.
Rf: Retention
Factor.
TLC: Thin layer
chromatography.
UV: Ultraviolet
Light.
W/W: Weight per
weight.
WHO: World Health
Organization.
CHAPTER
ONE
INTRODUCTION
1.1 Background
to the Study
1.1.1 Plants with
medicinal Properties
Medicinal plants with high therapeutic index are
usually used for the treatment of many ailments (Chelaiah and Muniappan, 2006).
This has led to an increased interest in their use rather than chemical drugs
(Sasidharan et al., 2011). Research
has shown significant progress in the use of medicinal plants for the treatment
of various ailments such as diabetes, fever, hypertension, and veneral
diseases.
The total population of plant species in the world is estimated to be between
250, 000 and 500,000 and a large proportion has not been explored for medicinal
purposes (Mehesh and Satish, 2008) and (Shinwari et al., 2013). Some of these claims by
the traditional healers have been substantiated with scientific evidence. The active
ingredients are usually extracted from the plant either traditionally or in the
laboratary. The oldest written verification of medicinal plants usage for
preparation of drugs was found on a Sumerian clay slab from Nagpur
approximately 5000 years old (Sumboonnanonda and Lertsithichai, 2004). This
Sumerian clay slab showed 12 recipes for drug preparation referring to over 250
various plants, some of the plants include poppy, henbane, and mandrake. Many
of these recipes were also found in the ancient Chinese book on roots and
grasses “Pen T’Sao,” written by Emperor Shen Nung circa 2500 BC. This book
details 365 drugs from dried parts of medicinal plants, many of which are used
even currently (Tesfahun et al.,
2014).
While people in the past used medicinal plants
primarily in simple pharmaceutical forms (infusion, decoctions and
marcerations), the demand for compound drugs constantly increased with time.
Plants have secondary metabolites which have been utilized by humans for making
medicines (Daniel et al., 2012). According
to World Health Organization (WHO), 80% of people in the world currently rely
on herbs for their primary healthcare, to generate income and for livelihood
improvement (Ozbilgin and Saltan, 2012). This is because herbs are considered
to have a variety of secondary metabolites and are accessible, safer and
affordable compared to the synthesized products which are regarded as having
adverse side effects on humans and the environment (Gunjan et al., 2013). In addition, most of the secondary metabolites from
plants provide protection against infections and are source of plant survival
(Lauren and Peter, 2011).
Pain however is a common ailment
suffered by many people, therefore the need to find drugs that can relief pain.
Many scholars have reported the use of plants in relieving pain (Fabricant and
Farnsworth, 2001; Kennedy, 2004; Scheid, 2007; Khare, 2007). Researchers have
identified a number of plants whose extracts have shown analgesic activity
(Ezeja et al., 2011; Panda et al., 2009; Okwu and Iroabuchi 2009;
Kondangala et al., 2011; Rahul et al., 2011) and this has especially
been prompted by the fact that many synthesized analgesic drugs that relieve
pain such as morphine are known to be associated with adverse effects like
ulceration, gastrointestinal bleeding, addictive potential, respiratory
distress, drowsiness and nausea (Modi et
al., 2012). Ibuprofen, another pain killer can increase the risk of heart
failure (Forman et al., 2005). Analgesic drugs act in various ways
on the peripheral and central nervous systems. There are many analgesic and
synthetic drugs like Paracetamol, non steroidal anti-inflamatory drugs
(NSAIDs), Ibuprofen, diclofenac whose use is limited due to their significant
adverse side effect on the stomach, kidney and other body tissues. This has led
to the need to find natural sources to relieve pain. Many
phytochemicals from herbal plants have been found to provide analgesic
solutions which are safe and broadly effective with fewer side effects
(Sasidharan et al., 2011; Ali et al., 2014). Plants which are rich sources of
analgesics include: Aloevera barbedensis, Andrographis
paniculata, Elettaria cardamomum, Punica granatum, Eugenia caryophyllus,
Curcuma alismatifolia, Phoenix sylvestris, Stachys schtscheglee, Cissus
quadrangularis, menthol, Bunts longifolia, Buxus sempervirens, Burns
sempervirens, Fumaira vaillantii, Rumex crispus, Urtica dioica and Morinda
Citrifolia, Stachytarpheta jamaicensis (Okwu et
al., 2010), Pimenta officinali (Priya
et al., 2012), Andrographis paniculata, Argemone
mexicana, Pimenta racemosa, Monarda didyma, Citrus bergamia, Boswellia
thurifera which have been used as herbal solutions to
relieve pain (Dana, 2010; Remedy, 2015). Providers
of medications from medicinal plants do not always consider fully the harmful
side effects of their prescriptions (Huntsman Cancer Institute, 2013). Although
opioid drugs are normally much safer than synthetic drugs which is the reason
some medical professionals prefer to prescribe more of opioid drugs. However, opoid
drugs still have some side effects which may incluse; cognitive impairment,
respiratory depression, gastrointestinal e.g. constipation, nausea, vomiting
and gastrointestinal bleeding. Rosiglitazone which is used to treat diabetes is
known to increase the risk of heart failure (Martinez, 2006).
Stachytarpheta angustifolia
extract has also been usedfor a number of ailments as an anti-diabetic,
anti-asthmatic, arbotifacient, sedative, antihypertensive, and antifever
(Yakubu et al, 2005). Iridoid
glycosides have also been extracted from Stachytarpheta
angustifolia. They are a large group of naturally occurring Monoterpenoid
with a glucose moiety attached at C-1 in the pyran ring (Song et al.,
2006). They occupy an important position in the field of natural product
chemistry and biology, as they provide a structural link between terpenoids and
indole alkaloids and as well display a broad spectrum of biological activities
(Sticher, 1977). Iridoids are useful phytochemicals in a number of folk
medicinal plants and many of them poses significant biological and
pharmacological activities; some of them are chemo taxonomically useful as markers
of genus in various plant families (Sticher, 1977).
The main hindrance for herbal
therapies nevertheless is the blending of native knowledge with modern medical
practices with little or no scientific data available regarding the safety and
efficacy of the herbal drugs (Ali et al.,
2014).
1.2 Statement of
the Research problem
Pain is an accompaniment of many medical conditions
and pain control is among the important therapeutic priorities (Ezeja et al., 2011). It is mostly a warning
signal that causes discomfort which leads to many adverse effects (Raquibul et al., 2010). Since most of the analgesic
drugs available have serious side effects, it is necessary to explore
biodiversity and heighten the search for new analgesic drugs with minimal side
effects. This study is set to investigate the analgesic property of the
chroloform extract of Stachytarpheta
angustifolia.
1.3
Aim and objectives of the Study
The
concern of this study is to extract, characterize and isolate the active
components in Stachytarpheta angustifolia
plant using chloroform and determine the analgesic properties of the plant. The
aim of this study is achieved by:
1.
identification and collection of samples
of Stachytarpheta angustifolia and
drying under room temperature.
2.
merceration of the pulverized sample using chloroform to obtain the crude
extract for further analysis
3.
conducting phytochemical screening on the chloroform crude extract to determine
the secondary metabolites in the plant extract.
4.
conducting an acetic acid induced screening on the crude chloroform extract to
determine the analgesic properties of the plant.
5.
conducting statiscal analysis to analyze results gotten from phytochemical
screening and acetic acid-induced writhing in rats.
6.
separation of the crude extract into fractions by using column chromatography
7.
isolation of the pure fraction using Thin Layer Chromatography
8.
carrying out spectroscopic analysis to propose a structure for the isolated
pure fraction
1.4 Justification
The
use of compound drugs for relieving pain has led to the development of more
challenging health problems which is of great concern. This may include ulcer,
heart palpitation, hypertension etc. This brings researchers to seek
alternative ways of relieving pain with lesser or milder side effects. This
research is therefore targeted at finding the analgesic effect of the plant
extract of Stachytarpheta angustifolia
in comparison with compound drug aspirin.
1.5 Scope and Limitation
The
study focuses on the isolation, characterization, phytochemical studies as well
as analgesic activity of Stachytarpheta
angustifolia. The analgesic activity of Stachytarpheta
angustifolia was compared with the synthetic drug Aspirin. The study was
limited by the non-availability of Nuclear Magnetic Resonance instruments in
Nigeria which necessitated the quest to obtain spectral analysis from
sophisticated laboratories abroad.
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