ABSTRACT
Humanity is faced with numerous health challenges ranging from severe to chronic disease conditions, such health challenges common to mankind among others include pains, arthritis, inflammations and rheumatism. Newbouldia laevis P. Beauv (farm Bignoniaceae) is used in traditional herbal medicine for the treatment of the health challenges mentioned above. We are motivated by the use of N. laevis leaves in traditional medicine to extract, isolate and characterize the bioactive components which may be responsible for its analgesic properties. Pulverized leaves sample of N. laevis was extracted by cold maceration using methanol. The extract was concentrated in vacuo to yield a reddish brown solid 120.191 g. The crude methanol extract was partitioned into n-hexane 0.1 g, dichloromethane 2.5 g, ethlylacetate 4.6 g, and methanol 10.0 g, fractions via coarse chromatography. The phytochemical screening reveals the presence of secondary metabolites as 21.73±0.36 % alkaloids, 40.78±0.27 % flavonoids, 15.99±0.044 % saponins, 6.088±0.06 % Tannins, 3.086±0.03 % Trepenoids and 12.13±0.01 % Cardiac Glycosides, with alkaloids, flavonoids and saponins having the highest percentage. The methanol fraction gave the highest yield; and it was subjected to further purification using repeated column chromatography to yield pure components namely NLM 24 (Rf 0.48), EAc:n-hex:MeOH (4:5:1) and NLM 19 (Rf 0.47), EAc:n-hex:MeOH respectively. These pure fractions were subjected to 1H NMR, 13C, COSY, HSQC and HMBC spectroscopy. Pheophytin A and β-amyrin were proposed as the structures of the isolated compounds. Acute toxicity test and analgesic test were done using the crude methanol extract and fractions. The acute toxicity test shows no death of the white whisker rats administered with low dose (1000 mg/kg) and high dose (6000 mg/kg), meaning that the leaves are edible. The analgesic activities of the crude methanol extract and fractions on white whisker rats showed that at low dose of 4000 mg/kg of the extract and the fractions have percentage inhibition of pains as; methanol crude (63.48±4.62), methanol fraction (79.14±7.39), dichloromethane fraction (60.79±6.69), ethylacetate fraction (23.26±9.75), and n-hexane fraction (64.82±9.75). At high dose of 8000 mg/kg, the percentage inhibition of pain are 5200±2.00, 71.87±7.04, 80.31±6.20, 45.61±12.60, 43.87±8.13 percent respectively; a statistical analysis at (P>0.05), confidence level the methanol fraction had the highest analgesic activities while the ethylacetate fraction had the least analgesic activity. The non-toxic effect of the extract on the rats showed why the leaves of N. laevis are cooked and consumed for pain relief in folk medicine. Even though the pure fractions were not used for analgesic activity, literature work reveals that pheophytin A & β-amyrin are potent analgesics. This may be the first time pheophytin A and β-amyrin are isolated from N. laevis leaves P. Beauv.
TABLE OF CONTENTS
Page
Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgments v
Table of Contents vi
List of Tables ix
List of Figures x
List of Plates xi
List of Appendices xii
Abstract xiii
CHAPTER 1: INTRODUCTION 1
1.1 Background of the Study 1
1.2 Statement of the Problem 4
1.3 Aim and Objectives of the Study 5
1.4 Justification of the Study 5
1.5 Significance of the Study 5
1.6 Scope and Limitations of the Study 6
CHAPTER 2: LITERATURE REVIEW 7
2.1 General Botanical Characteristics of Newbouldia laevis 8
2.2 Chemical Constituents of Newbouldia laevis P. Beauv 11
2.2.1 Phytochemicals 11
2.2.2
Alkaloids 12
2.2.3 Flavonoids 14
2.2.4 Saponins 16
2.2.5 Steroids 18
2.2.6 Tannins 21
2.2.7 Cardiac glycosides 23
2.2.8 Terpenoids 25
2.3 Analgesics 27
2.3.1 Synthetic natural analgesic 27
2.3.2 Natural analgesics 30
2.3.3 Analgesic effects of Newbouldia laevis P. Beauv 32
2.4 Acetic Acid Induced Writhing 32
2.5 Activities Attributed to Newbouldia laevis leaves 33
2.6 Chromatography 35
2.6.1
Thin layer chromatography (TLC) 36
2.6.2 Column chromatography 36
2.7 Current Trend in Structure Elucidation of
Organic Compounds 37
2.8 Spectroscopy 37
CHAPTER 3: MATERIALS/EQUIPMENT 43
3.1 Materials for Extraction 43
3.2.0 Methods 45
3.2.1 Summary of experimental procedure 45
3.2.2 Collection and preparation of plant material 47
3.2.3 Extraction and fractionation 47
3.2.4 Column chromatography 48
3.2.5 Thin layer chromatography (TLC) 49
3.2.6 Phytochemical screening 50
3.4.1 Toxicity test 53
3.4.2 Analgesic test 54
3.4.3 Spectroscopic analysis 55
CHAPTER 4: RESULTS AND
DISCUSSIONS 56
4.1 Result of Quantitative Phytochemical
Screening 57
4.2 Result of Column Chromatography 60
4.3 Thin layer Chromatography (TLC) Result 65
4.4 Spectral Analysis Result 67
4.5
Spectroscopic Analysis of Isolated
Compounds 69
4.5.1 Characterization of NLM 24 as pheophytin A 69
4.5.2 Characterization of NLM19 as ᵦ-Amyrin 75
CHAPTER 5: CONCLUSIONS
AND RECOMMENDATIONS 83
5.1
Conclusions 83
5.2
Recommendations 84
5.3 Contribution to Knowledge 84
REFERENCES 86
APPENDICES 97
LIST OF TABLES
Page
2.1: Active compounds Isolated from Natural
Sources 30
2.2: Activities Attributed to Newbouldia Laevis leaves 33
3.1: Chemicals and Reagents 44
3.2: Equipment, Samples Obtained and the
Laboratory where the Experiments was Done 55
4.1: The Extracts NLM, NLE and NLDCM with their
yields and appearance 56
4.2: Result of
Qualitative and Quantitative Phytochemical Screening of Methanol Crude
Extract of N.laevis
leaves 57
4.3:
Result of Column Chromatography result of Newbouldia laevis Leaves Extract 60
4.4:
1H(400MHZ) and 13CNMR (100MHZ) data of NLM 24 and
Literature data in CDCL3 67
4.5:
1H(500MHZ) and 13C(125MHZ) NMR Data of NLM19 and
Literature Data in CDCL3 74
4.6:
Acute Toxicity Study of Methanol leave Extract of Newbouldia leavis LD50 result of the Extract 79
4.7: Analgesic
Effects of the Crude Methanol Extract and its Fractions of the Leaf Extracts
of Newbouldia
leavis on Whisker Albino Rat. 81
LIST OF FIGURES
Page
1. Withasomine 13
2. 4-Hydroxynewbouldine 13
3. 3- Hydroxynewbouldine -
Lapachone 15
4. 5- Hydroxynewbouldine - Lapachone 15
5. Lapachol 15
5. Oleanolic Acid 17
6. Glycyrrhizin 17
7. Ginsenosides 17
8. Beta-sitosterol 20
9. Stigmasterol 20
10. 2,3,4,6-tetra-o-galloyl-D- glucopyronose 22
11. Ellagitannins 22
12. Digoxin 24
13. Ouabain 24
14. Oleandrin 24
15. Ursolic acid 26
16. Canthic acid 26
17. Summary of Experimental procedure 45
18. Flow Chart of Purification using Flash
Chromatography 46
19. Structure of pheophytin A 73
21. Structure of (ᵦ-amyrin) 78
22. The percentage analgesic activities of
different doses of the extracts 81
LIST OF PLATES
Page
1. Newbouldia laevis
leaves 10
2. TLC
chromatogram for NLM 24 and 19 66
LIST OF APPENDICES
Page
1H-NMR spectrum
for NLM 24 (Pheophytin A)
97
13C spectrum for
NLM 24 (pheophytin A) 98
COSY NMR Spectrum
for NLM 24 (Pheophytin A) 99
HSQC NMR Spectrum
for NLM 24 (Pheophytin A) 100
HMBC
NMR Spectrum for NLM 24 (Pheophytin A) 101
1H
NMR Spectrum for NLM 19 (ᵦ-amyrin) 102
13C NMR Spectrum for NLM 19 (ᵦ-amyrin) 103
COSY
NMR Spectrum for NLM 19 (ᵦ-amyrin) 104
HSQC
NMR Spectrum for NLM 19 (ᵦ-amyrin) 105
HMBC
NMR Spectrum for NLM 19 (ᵦ-amyrin) 106
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND TO THE STUDY
Nature has provided vegetation to mankind,
such vegetation comprises of abundant medicinal plants of different diversities
such as herbs, shrubs, sub-shrubs, grasses and plants located in different
habitats like tropical Rain Forest, Savanna Grass Land, Sahel Savanna and
desert regions of the world. The various medicinal plants parts like the root,
bark, stem, flower, leaves, fruits and seeds could be used in both folklore and
allopathic medicine to fight against numerous health challenges so as to
enhance healthy living of the populace.
Presently,
humanity is faced with different challenges due to proliferation of diseases
caused by organisms found everywhere in the environment.
Ainooson,
(2009) opined that the use of plants in the management and treatment of
diseases is an age long practice. According to him, it is no doubt that in the
recent times with good number of research, it has been found that many plants
possesses medicinal value and different parts of these plants have been useful
for the synthesis of medicines used in hospitals, clinics, and other health
care centers. Tietz (2000) stated that traditional medicine is leading the
front line research for finding solution to many health challenges which are so
common in the society. In Nigeria, many diseases were treated and are still
being treated with the use of folk medicine and most of them have so far shown
positive results. These diseases among others include pains, muscle inflammation,
ulcers, convulsions, diarrhea, bacterial and fungal infections, asthma,
malaria, diabetes, and typhoid fever. Some of the numerous medicinal plants
already in use in Nigeria are: Garcina
kola, which is used for the treatment of asthma, Carica papaya used for hypertension, Ocinum basilicum for typhoid fever, Senna occidentalis for skin diseases and Newbouldia laevis for pains, (Burtis, et al, 2003). WHO (2005), recognized the place of herbal or folks
medicine in primary healthcare with greater interest in different developing
countries of the world. (WHO) further encouraged member nations to develop
national policies for identification, sustainable exploitation, scientific
development and appropriate use of herbal medicines so as to salvage their situations.
The
efficacy of these medicinal plants are based primarily on their diverse
phytochemical constituents. In the light of this, plants contain an array of
phytochemicals that are certified to be pharmacologically active and have been
mostly utilized in the treatment of many diseases wrecking both man and animals
Schneidar, (2004).
Oliver
Bever, (1986), stated that a very greater area of Nigeria ecological zones is
made up of numerous plant species which have found their usefulness to the
healthcare of the populace. According to Bruneton (1994), the medicinal uses of
most of these plants are numerous and cannot be exhausted in respect of oral
traditions and folklores from the time immemorial that have continued to
increase the medicinal potencies of these plants and their crude extracts. He
further stated that there are different bioactive principles embedded in the
medicinal plants such as flavonoids, alkaloids, tannins, saponins, glycosides,
etc. These bioactive principles obtainable from a wide range of
pharmaceutically derived medications consist of components which are obtained from
phytochemicals in plants. The bioactive principles in medicinal plants possess
the healing property attributed to such plant (Alves et al., 2007).
The
medicinal plant secondary metabolites are believed to be intermediates in
metabolic processes found in nature and are usually small molecules. Primary
metabolites are involved directly in normal growth, development and
reproduction for example, (ethanol, acetic acid, citric acid, and lactic acid)
and cell components (lipids, vitamins and polysaccharides). Secondary
metabolites on their own are not directly involved in those processes and
usually has a duty not important for the organisms (e.g. antibiotics, proteins
and carotenoids).
sa
One
of such medicinal plants abundant in nature is Newbouldia laevis P.Beauv (Bignoniaceae). The plant is known to
possess great potency in the cure and treatment of elephantiasis, syphilis, rheumatic
swellings, and as a vermifuge to roundworms. The plant has also been identified
to be useful in the treatment of sore feet, ear ache, chest pain, children’s’
convulsion and epilepsy (Akunyili, 2000). The leaf, stem, and fruits have been
reportedly used for febrifuge, stomach ache and wound dressing Iwu, (2000). A
lot of ethno medicinal activities have been reported to be associated to Newbouldia laevis leaves extract such as
uterine stimulant, treatment of arthritis and rheumatism, gastro-intestinal
treatment and all kinds of body pain (Bafor and Sanni, 2009).
Investigations
from Sofowara, (1993) revealed a number of phytochemicals are embedded in Newbouldia laevis. The leaves of N.laevis P.Beauv (Bignoniaceae) is used
in folks medicine for the treatment of various ailments like body pain,
rheumatism, arthritis, inflammations and others, the crude extract has been
found potent on some diseases, this is why the decoction of the leaves are
consumed locally as a remedy to many health challenges, there are important
drugs isolated from N. laevis leaves
such as digoxin,and digitoxin which are useful in the treatment of congestive
heart failure (Thompsons, 2008). Taxol is another drug extracted from the
plant. Taxol is used as a cancer chemotherapeutic drug, (Stierle,et al.1993). Newbouldine is another drug
for pain relief and antimalarial (https://doi.org/10-1016/j.tet.2010.05.085).
Also lysergic is a drug produced from ursolic acid obtained from newbouldia laevis (Halpern et al.,2014).
According
to Liu (2004), phytochemicals are chemical substances that are found naturally
in plants, some of the phytochemicals are responsible for colour and
organoleptic properties such as the deep purple of blueberries and the smell of
garlic, while a good number of others are the principles responsible for much
of the protection conferred on fruits, plants, vegetables, cereals, beans, and
plant based beverages like tea and wine. Phytochemical screening of the
methanolic- extract showed the presence of flavonoids, tannins, glycosides,
alkaloids, terpenes and steroids (Sofowara, 1993; Trease and Evans, 2002).
Research
done recently by (Seed et al 2008),
revealed that the leaf crude methanol extract using white whisker albino rats
showed no toxic effect at LD50>6000 mg/kg. Similar research was conducted by the
researcher on the same laboratory animals and the plant was found to be a very
active analgesic at all concentrations and doses. Despite a whole lot of
research in human medicine, challenges are still encountered, one of those
leading challenges is body pain which arises from other ailments and as
symptoms of a particular sickness; the effect of such medicinal disease
condition is prevalent in Nigeria; this is due to relative unavailability of
medicines with a promising efficacy and proliferation of adulterated analgesic
drugs (Sagganuwan and Gulumbe, 2006). This research mainly focuses attention on
extraction of the bioactive principles of the plant, elucidation of their
structures and evaluation of their analgesic efficacy on some laboratory
animals.
1.2 STATEMENT OF THE PROBLEM
The
inefficiency of analgesic drugs like Ibumol, Paracetamol, Ibuprofen and
Diclofenac on pain relief has made it necessary for the discovery of new
organic compounds that will be more efficacious for analgesic purposes, the
extraction of bioactive- principles from Newbouldia
laevis and essential elucidation of their structures may lead to the
discovery of new organic molecules that may be more effective for pain relief
without much side effect.
1.3 AIM AND OBJECTIVES OF THE STUDY
The
research is aimed at the isolation and characterization of secondary
metabolites from the leaves of N. laevis.
This aim is hoped to be achieved through the following specific objectives;
i.
to isolate bioactive
compounds in the leaf of Newbouldia
laevis.
ii.
to elucidate the
structures of the bioactive compounds.
iii.
to access the analgesic
activities attributed to the leaf of Newbouldia
laevis.
iv.
to access the toxicity
profile of Newbouldia laevis.
v.
to isolate the
phytochemicals embedded in the leaf of Newbouldia
laevis.
1.4 JUSTIFICATION OF THE STUDY
Newbouldia laevis
leaf is utilized in orthodox medicine in different Africa countries for the
treatment of various diseases such as pains, cancer, eye and ear diseases,
bacteria infection, cough, heartburn, snake bite, constipation, vermifuge,
ophthalmia, septic wounds, diarrhea, oedema, rheumatism and arthritis, malaria,
conjunctivitis, headache among others. Though the leaf of this plant is
utilized in the treatment of pain locally, there is no scientific proof of its
pain-killer properties. Results from this research would add to the database of
information on the leaf of Newbouldia
laevis.
1.5 SIGNIFICANCE OF THE STUDY
This
research on the leaves of N. laevis P. Beauv (Bignoniaceae), is important from both literature work and
experimental analysis. For the literature aspect, the research is built on
previous work done so far on the leaves of the plant which gives the insight on
the ethnomedicinal uses of the plant. On the part of experimentation, the
findings may be of relevant to researchers, pharmaceutical industries for
production of more potent analgesic drugs and also to traditional herbalists
for effective cure of rheumatic pain, arthritic pain, inflammations, menstrual
pains e.t.c. the findings will equally help researchers by providing the basic
empirical information to do further work on the plant.
1.6 SCOPE AND LIMITATIONS OF THE STUDY
The
study covers a wider area of scientific research such as plant collection and
identification, extraction, phytochemical screening, partitioning followed by
chromatography, spectra analysis Bioassay (acute toxicity test and analgesic
activity). The study is limited to chemical characterization and analgesic
activity of extracts from N. laevis
leaves.
Click “DOWNLOAD NOW” below to get the complete Projects
FOR QUICK HELP CHAT WITH US NOW!
+(234) 0814 780 1594
Login To Comment