ABSTRACT
Dissotis rotundifolia is used in traditional herbal medicine for the treatment of diseases and infections such as cough, stomach ache, tooth ache, diarrhoea, painful swellings, jaundice and rheumatism. Hence, this research aims to study the chemical constituents of the leaves of the plant. The air dried and pulverized leaves of Dissotis rotundifolia were extracted with distilled n-hexane and ethylacetate, using Soxhlet apparatus. Isolation and purification were carried out using column and thin layer chromatographic techniques. Fraction B14 gave a single spot on thin layer chromatography with a retention factor (Rf) value of 0.57. Characterization of fraction B14 was done using 1H-NMR, 13C-DEPT, COSY, HSQC and HMBC and pheophytin A was proposed as the structure of the compound. The phytochemical screening revealed the presence of alkaloids (110.23±1.23 mg/100 g), tannins (84.73±1.02 mg/100 g), flavonoids (62.40±0.82 mg/100 g), phenols (95.38±1.74 mg/100 g), saponins (3.39 mg/100 g) and glycosides (55.10 mg/100 g), with alkaloids having the highest concentration and saponins the lowest. The proximate analysis revealed that the moisture, dry matter, crude fibre, crude protein, carbohydrate, ash, lipid content and energy value were 10.62±0.15 %, 89.38±0.15 %, 14.76±0.49 %, 18.42±0.31 %, 44.54±0.28 %, 9.82±0.17 %, 1.84±0.11 % and 268.40±1.17 K Cal. respectively. Vitamins present include ascorbic acid (1.45±0.15 mg/100 g), thiamine (3.10±0.08 mg/100 g), riboflavin (0.18±0.01 mg/100 g), niacin (0.22±0.01 mg/100 g) and β-carotene (which is a precursor of vitamin A) (7.28±0.17 mg/100 g), with β-carotene having the highest concentration and riboflavin the lowest. The mineral elements detected include sodium (63.74±0.05 mg/100 g), potassium (264.60±1.10 mg/100 g), magnesium (63.84±1.71 mg/100 g), calcium (82.60±0.04 mg/100 g), phosphorus (29.42±0.82 mg/100 g), iron (5.18±0.18 mg/100 g), zinc (2.76±0.02 mg/100 g), copper (0.38±0.03 ppm) and lead (0.02±0.01 ppm). The antioxidant activity revealed that pheophytin A and the crude extract showed antioxidant activities of 19.51±0.02 % and 40.23±0.02 % at the highest concentration of 4.00 mg/ml respectively. The antimicrobial activity result of the crude extract at minimum and maximum concentrations of 25.00 and 150 mg/ml revealed growth inhibitions of 4.51±0.09 - 24.04±0.01 mm, 4.00±0.02 - 23.02±0.03 mm and 2.72±0.02 - 15.06±0.15 mm for Escherichia coli, Staphylococcus aureus and Streptococcus pneumoniae respectively. However, Salmonella typhi did not show sensitivity to the extract at 25.00 mg/ml but at 150.00 mg/ml, it gave 20.00±0.52 mm. The Antimycobacterium tuberculosis assay result showed that the crude extract inhibited the growth of the test organism in a dose dependent manner, with the highest growth inhibition of 10.75±0.18 mm at 150 mg/ml and the lowest at 3.62±0.25 mm at 25 mg/ml concentration. This investigation suggests the use of pheophytin A and other bioactive compounds present in Dissotis rotundifolia in pharmaceutical and herbal preparations. The ability of the extract from the leaves of D. rotundifolia to exhibit potent antitubercular activity suggests the use of the plant in the prevention and treatment of tuberculosis. Also the bioactive chemical compounds in the leaves might be responsible for its use in the treatment of various diseases and infections in traditional medicine.
TABLE OF CONTENTS
Page
Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of contents vi
List of tables xii
List of figures xiii
List of plates xiv
Abstract xv
CHAPTER 1: INTRODUCTION
1.1 Background of the Study 1
1.2 Statement
of the Problem 3
1.3 Aim and Objectives of the Study 4
1.4 Justification of the Study 4
CHAPTER 2: LITERATURE
REVIEW
2.1 Origin and Geographical Distribution of Dissotis rotundifolia 5
2.2 Description of Dissotis rotundifolia 5
2.3 Ethnomedicinal uses of Dissotis rotundifolia 8
2.4 Chemical Constituents of Dissotis rotundifolia 8
2.5 Medicinal Properties of Dissotis rotundifolia 14
2.5.1 Antidiarrhoeal activity 14
2.5.2 Antitrypanosomal activity 15
2.5.3 Antiplasmodial activity 16
2.5.4 Antioxidant activity 16
2.5.5 Anti-ulcer activity 17
2.5.6 Antibacterial activity 18
2.5.7 Anti-fertility activity 19
2.6 Tuberculosis 19
2.6.1 Transmission of tuberculosis 21
2.6.2 Symptoms of tuberculosis 21
2.6.3 Treatment of tuberculosis 21
2.7 Phytochemicals 22
2.7.1 Flavonoids 23
2.7.2 Tannins 25
2.7.3 Alkaloids 26
2.7.4 Saponins 27
2.7.5 Glycosides 30
CHAPTER 3: MATERIALS AND METHODS
3.1 Plant Collection 31
3.2 Plant Preparation 31
3.3 Extraction of Plant Material 31
3.4 Column Chromatography 32
3.5 Thin layer Chromatography 33
3.6 Quantitative Phytochemical Determination 33
3.6.1 Determination of alkaloids 33
3.6.2 Determination of tannins 34
3.6.3 Determination of saponins 35
3.6.4 Determination of flavonoids 36
3.6.5 Determination of phenols 36
3.6.6 Determination of glycosides 37
3.7 Determination of Proximate Composition 37
3.7.1 Determination of moisture content 37
3.7.2 Determination of total ash 38
3.7.3 Determination of protein content 38
3.7.4 Determination of lipid content 39
3.7.5 Determination of fibre content 40
3.7.6 Determination of carbohydrate content 40
3.7.7 Determination of gross food energy content 40
3.8 Determination of Vitamins 41
3.8.1 Determination of ascorbic acid 41
3.8.2 Determination of thiamine 42
3.8.3 Determination of riboflavin 42
3.8.4 Determination of niacin 43
3.8.5 Determination of carotenoid 43
3.9 Determination of Mineral Element 45
3.9.1 Digestion of minerals 45
3.9.2 Determination of sodium and potassium by flame
photometry 46
3.9.3 Determination of calcium and magnesium by complexiometric
titration 46
3.9.4 Determination of phosphorus 47
3.9.5 Determination of Zn, Fe, Cu and Pb by atomic
absorption spectrophotometer 48
3.10 Antioxidant Activity Determination 49
3.11 Antimicrobial Analysis 49
3.11.1 Antibiotics 50
3.11.2 Standardization of bacterial cell suspension 50
3.11.3 Antimicrobial susceptibity assay using gar well
diffusion method 50
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Phytochemical Constituents of Dissotis rotundifolia 52
4.2 Proximate Composition of Dissotis rotundifolia 56
4.3 Vitamin Composition of Dissotis rotundifolia 59
4.4 Mineral Element Composition of Dissotis rotundifolia 61
4.5 Results of Column Chromatography 64
4.6 Thin – Layer Chromatography Results 67
4.7 Spectral Analysis Results 68
4.7.1 1H – NMR spectra result 68
4.7.2 2 – Dimensional NMR spectral interpretation 72
4.8 Antioxidant Activity Results 84
4.9 Antimicrobial Screening Results 87
4.10 Antimycobacterium Tuberculosis Screening Result 90
CHAPTER 5: CONCLUSION AND RECOMMENDATION
5.1 Conclusion 92
5.2 Recommendations 92
REFERENCES 93
LIST OF TABLES
4.1 Phytochemical Composition of D. rotundifolia 52
4.2 Proximate Composition of D. rotundifolia 56
4.3 Vitamin Compistion of D. rotundifolia 59
4.4 Mineral Elements Composition of D. rotundifolia 61
4.5 Column Chromatography Result of Dissotis rotundifolia Leaves Extract 64
4.5 Zone of Inhibition of Chloroform Extract
of D. rotundifolia Leaves 63
4.6 Zone of Inhibition of D. rotundifolia Leaves Extract 68
4.7.1 1H-NMR Chemical Shift for Fraction
B14 68
4.7.2 13C (DEPT) Chemical Shift for Fraction
B14 72
4.8 Antioxidant Activity of Pheophytin A and Crude
Extract from D. rotundifolia 84
4.9 Zone of Inhibition of Chloroform Extract
of D. rotundifolia 87
4.10 Zone of Inhibition of D. rotundifolia Leaves Extract 90
LIST OF FIGURES
Page
4.1 1H-NMR for Fraction B14 79
4.2 13C (DEPT) NMR for Fraction
B14 80
4.3 1H – 1H – COSY Spectrum
for Fraction B14 81
4.4 1H – 13C HSQC Spectrum
for Fraction B14 82
4.5 1H – 13C HMBC Spectrum
for Fraction B14 83
LIST OF PLATES
Page
Plate
1: Dissotis rotundifolia Leaves 7
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND
OF THE STUDY
The
search for new drugs has increased greatly due to increase in infectious diseases
as well as drug resistance by pathogenic organisms. Man has continued to seek inspiration from plants
due to their diverse medicinal properties (Friday et al., 2018).
Phytomedicine
(the use of plants as medicine) is as old as man and from time immemorial, man
has always depended on plants for survival ranging from food, shelter and
medicine. As far as life is concerned, man must continue to rely on plants
because they have the answers to man’s health problems.
Traditional
medicine also called ethnomedicine, folk medicine, complementary and
alternative medicine is an old and cultural method of healing used by humans to
manage and fight various diseases threatening their existence and survival
(Abdullahi, 2011). The world has been threatened by various diseases whose cure
could be obtained from natural products (medicinal plants).
It
has been reported that about 80 % of the world’s population rely on plants and
about 70 – 95 % of people from developing countries depend on plant medicines as
their primary source of healthcare (Karunamoorthi et al., 2013). The reasons include their lesser side effect, better
compatibility and adaptability with the human body (Oladeji, 2016). Other
reasons why much attention is given to herbal medicine are due to the high cost
of conventional medicines and the fact that majority of the people living in
rural communities does not have access to conventional medicines.
Plants
produce a wide range of organic compounds classified traditionally into primary
and secondary metabolites. The key roles of primary metabolites are in
photosynthesis, respiration as well as plant growth and development. They include
phytosterols, acyl lipids, nucleotides, amino acids and organic acids.
Secondary metabolites are structurally diverse compounds distributed among
plant species such as flavonoids and other polyphenolics, terpenoids and
alkaloids. Some secondary metabolites have a key role of protecting plants from
predators and infections from microorganisms (Crozier et al., 2006). Secondary metabolites obtained from plants are used
as dyes, fibres, oils, flavouring agents, drugs and perfumes and can be used in
the production of new drugs from natural sources, as well as antibiotics and
insecticides (Croteau et al., 2000).
Furthermore,
medicinal plants have played an important role in the discovery and synthesis
of drugs. Natural products and their derivatives make up 50 % of all drugs used
in clinics with about 25 % originating from higher plants (Maridas and De
Britto, 2008). The therapeutic properties of medicinal plants are due to the
presence of wide range of biologically active chemical compounds, hence necessitating
the need for the isolation, purification, characterization and pharmacological
evaluation of individual chemical compounds present in these plants as it would
aid in discovering new drugs.
In
Nigeria, medicinal plants are assuming greater importance in health care
systems. Various plant species are used in different parts of Nigeria as food
and medicine for the management and treatment of various diseases, infections
and health problems.
Dissotis rotundifolia
is one of such medicinal plants that are rich in chemical compounds with
therapeutic uses and have been employed in ethnomedicine for the treatment of
various ailments such as jaundice, diarrhoea, dysentery, rheumatism, stomach ache
and cough (Kokwosa, 1976; Gill, 1992; Mann et
al., 2003). The pharmacotherapeutic activities such as antidiarrhoeal,
antitrypanosomal, antiulcer, antiplasmodial, antibacterial and antioxidant activities
have been reported (Mann et al.,
2003; Abere et al., 2010; Aditorney,
2015; Nondo et al., 2015). However,
there is paucity of information on its antitubercular properties.
1.2 STATEMENT OF THE PROBLEM
The
increase in antibiotics resistance and with the emergence of other chronic
diseases and infections as well as side effects associated with some of the
synthesized antibiotics have become a global problem and have called for great
concern. Hence, there is need for
alternative sources of antibiotics.
Dissotis rotundifolia
has been employed in ethnomedicine for the treatment of various diseases and
infections such as diarrhoea, conjunctivitis, jaundice, trypanosomiasis, ulcer,
stomach ache and cough. The phytoconstituents of this plant which are
responsible for its therapeutic characteristics have not been fully documented.
Only a few isolations have been carried out on the leaves of the plant. The
pharmacological properties of D.
rotundofolia such as the antidiarrhoeal, antiulcer, antitrypanosomal,
antiplasmodial and antioxidant activities have been reported. However, there is
paucity of information on the antitubercular activity of the plant.
1.3 AIM
AND OBJECTIVES OF THE STUDY
This
research is aimed at the isolation and NMR characterization of a secondary
metabolite from the leaves of D.
rotundifolia. This aim is hoped to be achieved through the following
specific objectives
(i)
Determination of the phytochemical
constituents in the leaves of D. rotundifolia.
(ii)
Determination of
the proximate composition of D.
rotundifolia.
(iii)
Determination of the vitamin composition of D. rotundufolia.
(iv)
Determination of mineral composition of D. rotundifolia.
(v)
Determination of the antioxidant activity of the crude
extract and the isolated compounds
(vi)
Evaluation of the antitubercular activity of the crude
extract.
(vii)
Evaluation of the
antimicrobial activity of the crude extract.
1.4 JUSTIFICATION
OF THE STUDY
Dissotis rotundifolia
is used in traditional medicines for the treatment of various diseases and
infections such as cough, diarrhoea, rheumatism, painful swellings, tooth ache,
jaundice, dysentery, stomach ache, eye infections, gonorrhoea, ulcer, conjunctivitis,
fever, arthritis and infertility, in different parts of Africa. Though this
plant is used in the treatment of cough traditionally, there is no scientific
proof of its antitubercular properties. Results from this research would add to
the data base of information on the Leaves of D. rotundifolia.
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