ABSTRACT
Three
compounds 3β-lup-20
(29)-en-3-ol (lupeol, LS 1),
(3S,8S,9S,10R,13R,14S,17R)-17-[(E,2R,5S)-5-ethyl-6-methylhept-3-en-2-yl]-10,13-dimethyl-2,
3, 4, 7, 8, 9, 11, 12, 14, 15, 16, 17-dodecahydro-1H-cyclopenta [a]
phenanthren-3-ol (Stigmasterol, LS 2) and 3, 5,
7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl) chromen-4-one (isorhamnetin, LS 3)
were isolated from the hexane and dichloromethane soluble fractions of the
stembark of Lonchocarpus sericeus Poir
Kunth EX DC (Papilionaceae), a medicinal plant commonly used traditionally in
the treatment of inflammation and pains, using column, thin layer and
preparative thin layer chromatographic techniques. The chemical structure of
these isolated compounds were elucidated by spectroscopic methods including
GC-MS, ESI-MS, IR, 1H, 13C, 2D NMR experiments and by
direct comparison with literature values. Preliminary phytochemical screening
revealed that the stembark of L. sericeus
is rich in alkaloids, cardiac glycosides, flavonoids, saponins, tannins and
terpenes. Quantitative determinations of some bioactive constituents of the
plant showed a higher flavonoid content (0.52 ± 0.02 mg/ 100 g) compared to
alkaloids (0.36 ± 0.02 mg/100 g) and saponins (0.49 ± 0.03 mg/). Acute toxicity
test (LD50) revealed very low level of toxicity (3,100 mg/kg, i. p
mice). This implies the decoction of this plant part at this dose regime is not
expected to produce any adverse effect. The anti-inflammatory and analgesic
studies revealed that the methanol extract, hexane, dichloromethane, aqueous
fractions as well as the isolated compounds ( LS 1 and LS 2 ) effectively
reduced oedema caused by egg albumin and xylene and exhibited high analgesic
properties in inhibiting pain induced by acetic acid and heat. These reductions
were dose-dependent and statistically significant (P < 0.05 - 0.001) when
compared to distilled water and similar to prototype drugs employed. This study
has therefore identified 3β-lup-20(29)-en-3-ol (LS1) and stigmasterol (LS2) as
the active anti-inflammatory and analgesic principles in the stembark of L. sericeus.
The result further validates the use of L.
sericeus in the treatment of inflammation and management of pain in
folkloric medicine. This is the first report of the isolation of stigmasterol
and 3, 5, 7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl) chromen-4-one
(isorhamnetin) from the stembark of this leguminous tree Lonchocarpus sericeus.
TABLE OF CONTENTS
Title
Page i
Declaration ii
Certification
iii
Dedication iv
Acknowledgements v
Abstract
vii
Table
of Contents viii
List
of Tables xi
List
of Figures xiii
List
of Schemes xiv
List
of Plates xv
List
of Appendices xvi
Abbreviations
Used xviii
CHAPTER 1: INTRODUCTION
1.1 Background to the Study 1
1.2 The Plant Lonchocarpus sericeus 6
1.3 Statement of the Problem 9
1.4 Justification of Study 10
1.5 Aim of Study 10
1.6 Specific Objectives of Study 11
CHAPTER
2: LITERATURE REVIEW
2.1
Lonchocarpus
sericeus 12
2.2 Review of Work Done on the Plant Genus 22
2.3 Medicinal Plants as Anti-inflammatory and
Analgesic Agents 39
2.4 Inflammation 55
2.5 Pain 59
2.6 Chromatographic Technique 64
2.7 Methods of identification and
characterization 67
CHAPTER
3: MATERIALS AND METHODS
3.1 General Methods 71
3.2 Reagents and Chemicals 72
3.3 Plant Materials 72
3.4 Preparation of Extracts 73
3.5 Phytochemical Analyses 73
3.6 Partitioning and Isolation of the Crude
Extract 77
3.7 Pharmacological Investigations 83
3.7.1 Animal Stock 83
3.7.2
Preparation of Drugs for Study 83
3.7.3 Acute Toxicity Test (LD50) 84
3.7.4 Evaluation of Anti-inflammatory Activity 85
3.7.5 Evaluation of Analgesic Activity 86
3.8 Data Analyses 87
CHAPTER
4: RESULTS AND DISCUSSION
4.1 Results 88
4.2 Discussion 116
4.3 Spectroscopic Analyses 121
CHAPTER
5: SUMMARY, CONCLUSION AND RECOMMENDATIONS
5.1 Summary 127
5.2 Conclusion 129
5.3 Recommendations 130
References 131
Appendices
LIST OF TABLES
Page
2.1: Some Medicinal plants with anti-inflammatory and
analgesic activities 54
4.1: Result of phytochemical screening of
methanol stembark extract of L. sericeus 89
4.2: Quantitative determination of some
bioactive compounds of stembark extract
of Lonchocarpus sericeus 90
4.3a: The median lethal dose (LD50) of
methanol stembark extract of L. sericeus 91
4.3b: The median lethal dose (LD50) of
methanol stembark extract of L. sericeus
92
4.4:
Effect of methanol extract and
fractions of L. sericeus stem on
Egg-albumin- induced right hind paw oedema in mice 94
4.5:
Effect of isolated compounds (LS1 and
LS2) of L. sericeus stem on
Egg-albumin- induced right hind paw oedema
in mice 95
4.6:
Effect of methanol stembark extract
and fractions of L. sericeus on
xylene- induced ear oedema in
mice. 96
4.7:
Effect of isolated compounds from L.
sericeus on xylene-induced ear oedema in mice.
97
4.8: Effect of stembark extract and fractions of
L. sericeus on acetic acid –induced writhing in mice. 99
4.9: Effect of isolated compounds (LS1 and LS2)
of L. sericeus on acetic acid – induced writhing in mice.
100
4.
10: Effect of methanol extract and
fractions of L. sericeus stem on
thermally-induced pain in mice. 102
4.
11: Effect of isolated compounds (LS1 and LS 2) of L. sericeus stem on thermally-induced pain in mice. 103
4.12:
Characteristics of isolated compounds (LS1, LS 2 and LS3). 110
4.13:
1H NMR and 13C NMR (CDCl3) Data for LS1, δ in
ppm and J in Hz 111
4.14:
1H NMR and 13C NMR (CDCl3) Data for LS2, δ in
ppm and J in Hz 113
4.15:
1H NMR and 13C NMR (CDCl3) Data for LS3, δ in
ppm and J in Hz 115
LIST OF FIGURES
Page
1.1: Some
useful drugs of plant origin 4
1.2: Some
useful drugs of plant origin 5
2.1: Some
examples of NSAIDs 62
2.2: Some
examples of opioid analgesics 63
4.1: Structure
of 3β-lup-20(29)-en-3-ol) (LS 1)
109
4.2: Structure of stigmasterol (LS2) 112
4.3:
Structure of 3, 5,
7-trihydroxy-2-(4-hydroxy-3- methoxy
phenyl) chromen-4-one (LS 3) 114
4. 4: Formation of the fragment
ions with m/z 207 and m/z 189 in LS1 121
4.5: Formation of the fragment
ions with m/z 255 and m/z 271 in LS 2 123
LIST OF SCHEMES
page
3.1: Flowchart for the partitioning of stembark extract of Lonchocarpus sericeus 79
3.2: Procedure for isolation
of LS 1 80
3.3: Procedure for isolation of LS 2 81
3.4: Procedure for isolation of LS 3 82
LIST OF PLATE
Page
1: A segment of Lonchocarpus
sericeus 8
2a: TLC Profile of LS1 and LS2 developed in
Hexane/Ethyl acetate (3:1) and sprayed
with 1 % Anisaldehye in 10 % H2SO4 and activated at 110 0C.
105
2b: TLC Profile of LS1 and LS2 developed in
Hexane/Ethyl acetate (3:1) and sprayed
with 80 % H2SO4 and activated at 110 0C 105
3a: TLC Profile of LS1 and LS2 developed in
Hexane/DCM (1:5) and sprayed
with 1 % Anisaldehye in 10 % H2SO4
and activated at 110 0C. 106
3b: TLC Profile of LS1 and LS2 developed in
Hexane/DCM (1:5) and sprayed
with 80 % H2SO4
and activated at 110 0C. 106
4a: TLC Profile of LS3 developed in
Hexane/DCM (1:5) and sprayed with 1 % Anisaldehye in 10 % H2SO4
and activated at 110 0C. 107
4b: TLC Profile of LS3 developed in Hexane/DCM
(1:5) and sprayed with 80 % H2SO4
and activated at 110 0C 107
5a: TLC Profile of LS3 developed in
Hexane/Ethylacetate (5:1) and sprayed with 1
% Anisaldehye in 10 % H2SO4 and activated at 110 0C. 108
5b: TLC Profile of LS3 developed in
Hexane/Ethylacetate (5:1) and sprayed with 80
% H2SO4 and activated at 110 0C 108
LIST OF APPENDICES (Pages 148-194)
1: IR spectrum of LS1
2: Mass spectrum of LS1
3: m/z values and intensities of LS1
4: m/z values and peak intensities of LS1
5: 1H NMR spectrum of LS1
6: 13C NMR spectrum of LS1
7: 13C NMR spectrum (expansion) for LS1
8: HSQC spectrum of LS1
9: HSQC spectrum of LS1
10: HSQC spectrum of LS1
11: IR spectrum of LS 2
12: Mass spectrum of LS 2
13: m/z values and peak
intensities of LS 2
14: m/z values and peak
intensities of LS 2
15: 1H NMR spectrum of
LS 2
16: 13C NMR spectrum
of LS 2
17: HMQC spectrum (a) of LS 2
18: HMQC spectrum (b) of LS2
19: HMQC spectrum (c) of LS 2
20: IR spectrum of LS 3
21: ESI-MS/MS (negative mode) of
LS 3
22: 1HNMR spectrum of
LS 3
23: 13C NMR spectrum
of LS 3
24: COSY spectrum of LS 3
25: HMQC spectrum of LS 3
26: Ethical approval letter for
use of laboratory animals
27: Statistical computations for
anti-inflammatory activity using
egg-albumin model
28: Statistical
computations for analgesic activity with acetic acid writhing model
29: Statistical computations for
analgesic activity using hot plate model
30: Statistical computations for
anti-inflammatory activity using
xylene ear oedema model
ABBREVIATIONS USED
2D Two dimensional
CC Column Chromatography
CDCl3 Deuterated Chloroform
COSY COrrelation
SpectroscopY
DCM Dichloromethane
ESI-MS Electrospray Ionization
Mass Spectrometer
FT-IR Fourier Transformed
Infra-Red
GC-MS Gas Chromatography-Mass
Spectrometer
HMQC Heteronuclear Multiple
Quantum Coherence
HSQC Heteronuclear Single
Quantum Coherence
Hz Hertz
m/z Mass to Charge
ratio
MeOH Methanol
MS Mass Spectroscopy
NMR Nuclear Magnetic
Resonance
ppm Parts Per Million
TLC Thin Layer
Chromatography
MHz Megahertz
δ Chemical shift (ppm)
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND TO THE STUDY
It is a truism that
during the past decades, traditional systems of medicine have attained global
prominence. Twenty years ago, the World Health Organization indicated that many
people in developed countries had begun to look in the direction of alternative
or complimentary therapies, including medicinal herbs (WHO, 1999). In the
last eight years specifically, the global market of products derived from
plants was estimated at 83 billion United States Dollars and is expected to
continuously grow in the future years (WHO, 2011). For instance, it is
estimated that approximately 25 % of modern drugs and as many as 60 % of
antitumor drugs are natural products-based (Brower, 2008; Newman and Cragg,
2012).
A medicinal plant has
been defined by World Health Organization consultative group as any plant in
which one or more of its organs contains substances that can be used for
therapeutic purposes or which are precursors for chemo-pharmaceutical semi
synthesis. (Das, 2003). Interestingly, between 65 % and 80 % of the populations
of developing countries presently use medicinal plants as remedies (WHO, 2011).
The development of new products from natural sources is also invigorated
because it is estimated that of the 300,000 plant species that exist in the
world, only 15 % have been assessed to determine their pharmacological
capacities (De Luca et al., 2012). Studies to establish the
effectiveness and importance of medicinal plants are being carried out globally
in many countries that cover a wide array of developmental phases (Aslanargun et
al., 2012; Butnariu and Coradini, 2012; Coghlan et al., 2012;
Gurib-Fakim, 2006; Mankga et al., 2013).
It has also been
established that population rise, inadequate supply of drugs, prohibitive cost
of treatment, side effects of several drugs and development of resistance to
currently used drugs for infectious diseases, have led to renewed emphasis on
the use of plant materials as a source of medicines for a wide range of human
ailments. Global estimates indicate that a large percentage of the population
cannot afford the products of the Western pharmaceutical industry and have to
rely upon the use of traditional medicines which are mainly derived from plant
materials (Joy et al., 2001).
Green plants synthesize
and preserve a variety of biochemical products, many of which are extractable
and can be utilized as chemical feed stocks and as raw materials for scientific
investigations. Also, plants can offer biologically active molecules and lead
structures for the development of modified derivatives with enhanced activity and
reduced toxicity. Many secondary metabolites of plant origin are commercially
important and find extensive use in a number of pharmaceutical compounds and
formulations. However, a sustained supply of the source material often becomes
difficult owing to factors like environmental changes, cultural practices,
diverse geographical distribution, labour cost and selection of superior plant
stock and over exploitation by pharmaceutical industry. The small proportion of
flowering plants investigated has yielded several therapeutic agents of known
structures.
Some of these useful
plant drugs (and their uses) include: vinblastine (anticancer) (1);
reserpine (tranquilizer) (2); artemesinin (antimalarial) (3);
taxol (antitumor) (4); quassinoid (antiprotozoal) (5); allicin
(antifungal) (6); magnolol (peptic ulcer) (7) ; forskolin
(hypotensive) (8); berberine (for leishmaniosis) (9); pilocarpine
(antiglaucoma) (10); emetin (amoebicide) (11); morphine
(analgesic) (12); codeine (analgesic) (13) and quinine
(antimalarial) (14). Thus, it becomes quite imperative that the
isolation and identification of active principles and the elucidation of
mechanism of action of the drugs from medicinal plants is of paramount
importance (Iwu, 1999; Joy et al., 2001).
Numerous publications
exist on the traditional uses of medicinal plants in the management of
diseases. Some of these medicinal plants may function as analgesic (Okokon et
al., 2012; Tatiya et al., 2017), anti-malarial (Okokon et al.,
2016; Kweyamba et al., 2019), antiplasmodial (Bagavan et al., 2011;
Sangian et al., 2013; Toure et al., 2018) and anti-inflammatory
agents (Conforti et al., 2008; Okokon et al., 2011; Chua, 2014;
Maione et al., 2016; Oguntibeju, 2018; Zhang et al., 2019).
Others may serve as
antimicrobial agents (Igwe and Echeme, 2013; Sharifi-Rad et al., 2017),
antihypertensive (Rawat et al., 2016; Balogun and Ashafa, 2019),
antidiabetic/antioxidative agents (Antia et al., 2015; Dar et al.,
2017) and anti-tuberculosis agents (Oladosu et al., 2011; Sivakumar and
Jayaraman, 2011; Gupta et al., 2017) to mention but only a
few. Ethnobotanical information in the Niger Delta region of Nigeria has
revealed that Lonchocarpus sericeus Poir (Papilionaceae) is a medicinal
plant used in the treatment of inflammation and pain (Etukudoh, 2003), hence
the choice of this plant for studies.
Fig. 1.1: Some useful drugs of plant origin
Fig. 1.2: Some useful drugs of plant origin
1.2 THE
PLANT Lonchocarpus sericeus
Lonchocarpus sericeus Poir (Papilionaceae) is
a leguminous plant which is known as Senegal lilac or Cube root. It is also
known as Farir Sansami, shunin biri, or furen ‘yar sarki (Hausa), Njasi (Igbo),
Apapo (Yoruba) and Ayara awa (Ibibio). It is a dry deciduous tree that can grow
from 10 to 16 meters high and flowers with dense hanging racemes of purple
flowers which makes it perfect for display purposes. The flowers have a marked
smell similar to vanilla. It is frequently planted in villages as a shade tree
and in gardens. The wood is clear yellow, sometimes marbled, with heart-wood
and olive-green (Kojs et al., 2004 and Adewuyi et al.,
2012).
The leaves are used for
general healing while the bark is used for treatment of body pains, arthritis,
rheumatism, cutaneous and subcutaneous parasitic infection, malnutrition,
debility, paralysis, epilepsy, convulsions and spasm. It is also used as
fish-poisons and laxatives. The roots are used for treatment of leprosy. The
fruit and seeds are used as insect repellants and arachnicides (Burkill, 1985).
Classification
Kingdom:
Plantae
Sub-Kingdom:
Tracheobionta
Superdivision:
Spermatophyta
Division:
Magnoliophyta
Class:
Magnoliopsida
Sub-class:
Rosideae
Order:
Fabales
Family:
Fabaceae
or Papilionaceae
Genus:
Lonchocarpus
Species:
Sericeus
1.2 THE
PLANT Lonchocarpus sericeus
Lonchocarpus sericeus Poir (Papilionaceae) is
a leguminous plant which is known as Senegal lilac or Cube root. It is also
known as Farir Sansami, shunin biri, or furen ‘yar sarki (Hausa), Njasi (Igbo),
Apapo (Yoruba) and Ayara awa (Ibibio). It is a dry deciduous tree that can grow
from 10 to 16 meters high and flowers with dense hanging racemes of purple
flowers which makes it perfect for display purposes. The flowers have a marked
smell similar to vanilla. It is frequently planted in villages as a shade tree
and in gardens. The wood is clear yellow, sometimes marbled, with heart-wood
and olive-green (Kojs et al., 2004 and Adewuyi et al.,
2012).
The leaves are used for
general healing while the bark is used for treatment of body pains, arthritis,
rheumatism, cutaneous and subcutaneous parasitic infection, malnutrition,
debility, paralysis, epilepsy, convulsions and spasm. It is also used as
fish-poisons and laxatives. The roots are used for treatment of leprosy. The
fruit and seeds are used as insect repellants and arachnicides (Burkill, 1985).
Classification
Kingdom:
Plantae
Sub-Kingdom:
Tracheobionta
Superdivision:
Spermatophyta
Division:
Magnoliophyta
Class:
Magnoliopsida
Sub-class:
Rosideae
Order:
Fabales
Family:
Fabaceae
or Papilionaceae
Genus:
Lonchocarpus
Species:
Sericeus
Plate 1: A segment of Lonchocarpus
sericeus stem showing the bark.
Extensive literature
survey has revealed previous studies on the leaf, root and seed of this plant
species but information on works relating to the stembark is quite scanty
(Fellows et al., 1977; Fellows et al., 1979; Evans et
al., 1983; Elbein et al., 1984; Evans et al., 1985; Mahmoud
and Waterman, 1986; De-Andrade Cunha, 2003; Fontenele et al., 2005;
Napimoga et al., 2007; Agbonon and Gbeassor, 2009; Oyedeji et al.,
2015). A few reports that relate to the stembark are the isolation of a
pentacyclic triterpenoid lupeol from the stembark of Lonchocarpus sericeus (Abdullahi
et al., 2013) and the phytochemical and anticonvulsant activity of
methanol extract of the stembark of Lonchocarpus sericeus (Musa et al.,
2006).
1.3 STATEMENT
OF THE PROBLEM
Organic chemists have
realized that plants contain a bewildering diversity of secondary metabolites
that are important in the discovery of new drugs. The numerous challenges in
drug discovery and development have led to renewed search for new drugs from natural
sources. Some of these challenges include various side-effects posed by the use
of synthetic drugs and pharmaceuticals , resistance of some diseases or
ailments to existing drugs, inability to find cure for some diseases (pain,
oxidative stress, diabetes, HIV-AIDS, cancer and sickle cell, asthma and
chronic obstructive pulmonary disease), limited life span of some drugs
(antibiotics), occurrence of new diseases and concerns about environmental
impact and the potential health risks related to the use of synthetic drugs and
pharmaceuticals (Cowan, 1999; Abad et al., 2007). Hence there is a great
demand for novel drugs and pharmaceuticals belonging to a wide range of
structural classes selectively acting on new targets with fewer side-effects
(Abad et al., 2007).
Since natural products
either as pure compounds or as standardized plants extracts, provide new drug
leads because of the matchless availability of chemical diversity, then one
approach in the testing and characterization of plants traditionally used for
their bioactivities as potential sources for drug discovery and development.
These reasons have initiated this research interest which is aimed at
evaluating the chemical constituents from Lonchocarpus sericeus Poir and
its potential health benefit in the management of body pain and inflammation.
1.4 JUSTIFICATION
OF STUDY
Lonchocarpus sericeus is widely distributed in
Nigeria. They are used traditionally by the Ibibio-speaking people of the Niger
Delta region of Nigeria in various decoctions for the treatment of various
ailments such as body pains, arthritis, rheumatism, cutaneous and subcutaneous
parasitic infection, malnutrition, debility and paralysis. It is also used as
fish-poisons and laxatives (Etukudoh, 2003). The roots are used for treatment
of leprosy (Etukudoh, 2003). However, from extensive literature survey,
scientific evaluation of this plant has been restricted to the leaf, seed and
root. Thus, the isolation of chemical constituents, subsequent structural
characterization and biological activity profiles of isolated compounds from
the stembark of the medicinal plant are imperative for validation of use in the
management of body pains and diseases related to inflammation.
1.5 AIM
OF STUDY
This research work seeks
to isolate, purify and characterize the structures of chemical compounds from
the stembark of Lonchocarpus sericeus and determine their
anti-inflammatory and analgesic potentials.
1.6
SPECIFIC OBJECTIVES OF STUDY
The specific objectives
of the study are:
a.
Obtain the crude methanol extract of stembark of Lonchocarpus
sericeus.
b.
Subject the methanol stembark extract to both preliminary
phytochemical screening and
quantification of some bioactive compounds from the plant part using standard procedures.
c.
Fractionate the methanol crude extract using a combination of
separation methods followed by
isolation and purification to obtain pure compounds.
d.
Assess the acute toxicity profile (LD50) of the Lonchocarpus
sericeus stembark.
e.
Isolate and purify the chemical compounds from the stembark of L.
sericeus using column
chromatography as well as preparative thin layer chromatographic (TLC) techniques using appropriate solvent
mixtures.
f.
Evaluate the biological activity of the pure compounds
(Anti-inflammation and analgesic
activity shall be investigated using two standard models in each case).
g.
Elucidate the structure (s) of the pure compound (s) isolated
using a combination of spectroscopic
techniques including FT-IR, GC –MS, ESI-MS, NMR (1H and 13C including 2D NMR) and by
direct comparison with literature data.
h.
Validate the use of this plant in folkloric medicine based on (b)
– (f) above.
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