ABSTRACT
The roots of Dennettia tripetala and Milicia excelsa were analyzed for the presence of phytochemicals. Five different solvents, which include methanol, ethanol, ethyl acetate, butanol and water, were used for the extraction of the phytochemicals.The root of Dennettia tripetala contained alkaloids, terpenoids, flavonoids, saponins, phenols, steroids and glycosides in varying degrees of abundance in the different solvents with tannins not detected in all the solvents. Milicia excelsa contained all the phytochemicals in Dennettia tripetala, in addition to tannins, in different degrees of abundance in the various solvents. The root of Dennettia tripetala contained 1.83 % alkaloids, 3.64 % flavonoids, 1.41 % saponins, 0.67 %phenols, 0.36 % steroids and 0.08 % glycosides whereas that of Milicia excelsa contained 2.19 %alkaloids, 6.40 % flavonoids, 0.87 % saponins, 0.34 % phenols, 0.36 % tannins, 0.15 % steroids and 0.09 % glycosides. Results of the Principal Component Analysis (PCA) of the phytochemicals revealed that, in Dennettia tripetala, there was strong positive correlation between alkaloids and glycosides (0.995)and also phenols and saponins (1.000) while the strong negative correlations were between alkaloids and flavonoids (-0.980), flavonoids and glycosides (-0.956), phenols and steroids (-1.000) and also saponins and steroids (-1.000). In Milicia excelsa, the strong positive correlations were between alkaloids and flavonoids (0.908), phenols and saponins, glycosides (0.866) and also steroids and tannins (1.000) whereas the strong negative correlations were between phenols and steroids (-0.866), saponins and tannins, steroids (-1.000) as well as tannins and phenols (-0.866). An assay of the antioxidant potentials of various extracts of both plants, using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, revealed that the ethanolic extracts of Dennettia tripetala and Milicia excelsa as well as the methanolic extract of Dennettia tripetala showed high percent inhibition ranging from 83.34 to 89.75 at 0.01 mg/mL of the extracts; results which showed to be better than standard ascorbic acid (67.89) at the same concentration. Other extracts of both plants, at higher concentrations, gave percent inhibitions ranging from 35.80 for the butanolic extract of Milicia excelsa at 0.025 mg/mL to 95.96 for the methanolic extract of the same plant at 0.5 mg/mL. A comparison of the half maximal inhibitory concentration (IC50) values of the extracts showed that ethyl acetate extracts of both plants had the best IC50 values at 0.014 and 0.150 for Dennettia tripetala and Milicia excelsa respectively which were better than ascorbic acid standard whose IC50 value was 1.060. All the other extracts of both plants also had IC50 values better than ascorbic acid except for ethanol in both plants and methanol in Milicia excelsa. The extracts were tested for antimicrobial activity against a gram positive cocci, staphylococcus aureus, and a gram negative rod, klebsiella sp. The results revealed that the ethanolic extract of Dennettia tripetala and the butanolic extracts of both plants showed activity against the test organisms at two concentrations, 400 mg/mL and 200 mg/mL, with the inhibition zone diameters (IZD) ranging from 8.2 mm to 12.0 mm and 6.5 mm to 14.0 mm for the ethanolic and butanolic extracts respectively. The minimum inhibitory concentration (MIC) of the extracts ranged from 41.5 mg/mL to 48.3 mg/mL for the ethanolic extracts and 164.8 mg/mL to 111.7 mg/mL for the butanolic extracts. The presence of these secondary metabolites in varying and substantial amounts in the roots of the plants as well as the antioxidant and antimicrobial potentials of the roots of the plants lends scientific credence to the ethnomedicinal use of these plants parts for the treatment of various diseases and ailments.
TABLE OF CONTENTS
Title page i
Certification iii
Dedication iv
Acknowledgement v
Abstract vi
Table of contents vii
List of tables ix
CHAPTER ONE: INTRODUCTION
1.1 General Background 1
1.2 Phytochemicals 2
1.3 Antibiotics 2
1.3.1 Penicillins 2
1.3.2 Cephalosporins 3
1.3.3 Tetracyclines 4
1.4 Antimicrobial Assay 5
1.5 Antioxidants 5
1.5.1 Antioxidant Assays 6
1.6 Botanical Profiles of the Plants 6
1.7 Ethnomedicinal Uses of the Plants 8
1.8 Objectives of the Study 8
1.9 Scope of the Study 9
1.10 Significance of the Study 9
CHAPTER TWO: LITERATURE REVIEW
2.0 General Background 10
2.1 Classes of Phytochemicals 11
2.1.1 Alkaloids 11
2.1.2 Terpenoids 12
2.1.3 Tannins 13
2.1.4 Saponins 13
2.1.5 Flavonoids 14
2.1.6 Steroids 15
2.1.7 Essential Oils 16
2.1.8 Phenolics 16
2.2 Review of Previous Works on Plants and Phytochemicals 17
CHAPTER THREE: EXPERIMENTAL
3.1 Sample Collection 23
3.2 Chemicals and Reagents 23
3.3 Apparatus and Equipment used 24
3.4 Extraction of the Phytochemicals 24
3.5 Identification of the Phytochemicals 25
3.6 Quantification of the Phytochemicals 26
3.7 Antioxidant Assay 31
3.8 Antimicrobial Assay of Extracts 32
3.8.1 Preparation of Turbidity Standard 33
3.8.2 Preparation of Inoculum 33
3.8.3 Inoculation Procedure 34
3.9 Data Analysis, Results and Interpretation 35
CHAPTER FOUR: RESULTS, DISCUSSIONS AND CONCLUSION
4.1 Results of Phytochemical Analysis 36
4.1.1 Percentage Yield of the Various Phytochemicals 36
4.1.2 Qualitative Phytochemical Analysis of Various Extracts of Dennettia
Tripetala and Milicia Excelsa 37
4.1.3 Quantitative Analysis of the Phytochemicals 39
4.1.4 Statistical Correlation of the Phytochemicals 41
4.1.5 Results of Antioxidant Analysis 44
4.1.5.1 Antioxidant Assay of the Various Extracts of Dennettia Tripetala 44
4.1.5.2 Antioxidant Assay of the Various Extracts of Milicia Excelsa 45
4.1.6 IC50 Values for the Various Extracts 58
4.1.7 Results of Antimicrobial Analysis 61
4.1.7.1 Antimicrobial Activities of the Extracts of the Root of Dennettia Tripetala 61
4.1.7.2 Antimicrobial Activities of the Extracts of the Root of Milicia Excelsa 63
4.2 Discussions 69
4.3 Conclusion 76
References 77
Appendix 92
LIST OF TABLES
4.1 Percentage Yield of the Various Phytochemicals 36
4.2 Phytochemical Analysis of the Various Extracts of Dennettia Tripetala 37
4.3 Phytochemical Analysis of the Various Extracts of Milicia Excelsa 38
4.4 Quantitative estimation (g/100 g) of alkaloids, flavonoids, phenols,
saponins, steroids, tannins and glycosides in the roots of Denettia
tripetala and Milicia excelsa. 39
4.5 Correlation Matrix of the Different Phytochemicals in the Roots of
Dennettia Tripetala 41
4.6 Correlation Matrix of the Different Phytochemicals in the Roots of
Milicia Excelsa 42
4.7 DPPH Radical-Scavenging Activity (% inhibition) of Various Extracts
of Dennettia Tripetala 44
4.8 DPPH Radical-Scavenging Activity (% inhibition) of Various Extracts
of Milicia Excelsa 45
4.9 DPPH Radical-Scavenging Activity (% inhibition) of Ascorbic Acid Standard 46
4.10 IC50 Values for Various Extracts of the Root of Dennettia Tripetala 58
4.11 IC50 Values for Various Extracts of the Root of Milicia Excelsa 59
4.12 Inhibition Zone Diameter (IZD) of ethanolic extract of the root of
Dennettia tripetala against tested organisms 61
4.13 Inhibition Zone Diameter (IZD) of butanolic extract of the root of
Dennettia tripetala against tested organisms 62
4.14 Inhibition Zone Diameter (IZD) of butanolic extract of the root of
Milicia Excelsa against tested organisms 63
CHAPTER ONE
INTRODUCTION
1.1 GENERAL BACKGROUND
Plants and plant parts are a known source of herbal medicine and natural health-enhancing products for many centuries. Various plant parts such as leaves, fruits, seeds, bark, flowers, rhizomes and roots have at one time or the other been utilized for medicinal purposes. It is estimated that about 75% of useful bioactive plant-derived pharmaceuticals used globally are discovered by systematic investigation of leads from traditional medicines1.The search for antimicrobial agents have over the years led researchers to in-depth study and analysis of various plants and their parts2, 3.
Over the years, infections caused by strains of bacteria that are resistant to orthodox drugs, also called multi-drug resistant (MDR) bacteria, have either found cure or control by the use of bioactive compounds isolated from plants. These bioactive compounds are known as phytochemicals. They can help prevent the spread of or totally eliminate infections. These phytochemicals can either be used alone as antimicrobial agents or in combination with commercially available antibiotics asstudies have shown that a higher activity against microorganisms can be achieved by combining certain phytochemicals with commercially available antibiotics4.For example, Pseudomonas aeruginosa, a microorganism which has exhibited resistance to 19 different antibiotics was observed for synergistic effects when phytochemical extracts from clove, jambolan, pomegranate and thyme were used together with known antibiotics. Results showed that bacterial growth was inhibited at phytochemical concentrations of 50µg/mL even to as low as 10µg/mL and, interestingly also, for antibiotics that previously did not show any activity by themselves against the microorganism5.
1.2PHYTOCHEMICALS
Phytochemicals are non-nutrient plant chemicals that contain protective, disease-preventing and curative compounds capable of bringing about physiological changes4, 6-8. Woody plants and herbs synthesize and accumulate in their cells large variety of these phytochemicals9-11.Phytochemicals have been isolated and characterizedfrom fruits such as grapes and apples, vegetables such as broccoli and onion, spices such asturmeric, beverages such as green tea and red wine, as well as many other sources12. These phytochemicals in traditional medicinal plants are capable of curing ailments like fever, asthma, constipation, oesophageal cancer and hypertension etc13.Different plant parts and components have been employed in the treatment of infectious pathologiesin the respiratory system, urinary tract, gastrointestinal and biliary systems, as well as onthe skin14, 15.
1.3ANTIBIOTICS
Antibiotics specifically refer to antimicrobial agents that kill or inhibit bacteria. Over 3000 antibiotics have been identified but only a few dozen are used in medicine. Some of them already in use include:
1.3.1 PENICILLINS: These antibiotics were among the first to be effective against many previously serious diseases and infections caused by staphylococci and streptococci. All penicillins are β-lactam antibiotics. They are usually active against gram-positive bacteria. The key structural feature of penicillins is the four-membered β-lactam ring. This structural moiety is essential for penicillins’ antibacterial activity16. Adverse effects associated with the use of penicillins include diarrhea, hypersensitivity, nausea etc.
Figure 1.1: Basic structure of penicillins where R is the variable side chain that differentiates one penicillin from another
1.3.2 CEPHALOSPORINS: These are a class of semi-synthetic antimicrobial drugs related to the structure and activity of penicillin hence they also possess the β-lactam ring. When the β-lactam ring is fused to a five-membered thiazolidine ring or penam, the drug is classified as a penicillin (as in the above structure) but when it is fused to a six-membered dihydrothiazine ring or cepham, it is classified as cephalosporin. Hypersensitivity reactions are among the most serious adverse effects of cephalosporins.
Figure 1.2: Basic structure of cephalosporins where R1 and R2 are aliphatic or aromatic side chains
1.3.3 TETRACYCLINES: These refer to a number of antibiotics of either natural or semi-synthetic origin derived from a system of four linearly annelated six-membered rings with a characteristic arrangement of double bonds16. Adverse effects of tetracyclines include bowel upsets,severe headache and vision problems.
Figure 1.3: Basic structure of tetracyclines
1.4 ANTIMICROBIAL ASSAY
Antimicrobial activities of plants have been reported17-19. The potency or activity of an antimicrobial is an index of the dose of it that inhibits the growth of a suitable susceptible microorganism. A number of techniques are used to find out the activity of antimicrobial agents on microorganisms. These include disc diffusion method and broth dilution assay. The effectiveness of the antimicrobial can be assessed by their ability to kill bacteria, characterized by the minimal lethal concentration (MLC) or by their ability to suppress or hinder bacterial growth which is the characterized by the minimum inhibitory concentration (MIC). The broth dilution assay can be used to determine MIC and MLC while MIC is usually obtained using the Agar diffusion technique20-22.
The Agar diffusion technique involves the application of antimicrobial solutions of different concentrations to cups, wells or paper discs, placed in the surface of or punched onto Agar plates seeded with the test bacterial strain. Antibiotic diffusion from here sources onto the Agarose medium leading to inhibition of bacterial growth in the vicinity of the source and to the formations of clear “zones” without bacterial lawn. The diameter of these zones increases with antibiotic concentration. The values of MIC is determined as the zero intercept of a linear regression of the squared size of the inhibition zones, x, plotted against the natural logarithm of the antibiotic concentration, c.23
1.5 ANTIOXIDANTS
These are molecules which can inhibit the oxidative action of unstable oxidant molecules. These unstable oxidant molecules are called free radicals. Once formed they are highly reactive and can initiate a chain reaction even capable of damaging vital molecules in the body. Antioxidants interactwith these free radicals and terminate the chain reaction before they cause oxidative damage to the body. Micronutrient antioxidants include Vitamin E, beta-carotene and Vitamin C24, 25. Selenium, a trace metal required for proper functioning of one of the body’s antioxidant enzyme systems, can also be part of this category. Phytochemicals have over time proved to possess antioxidant properties26-29.
1.5.1 ANTIOXIDANT ASSAYS
The antioxidant power of substances can be evaluated by their ability to donate hydrogen or scavenge free-radicals. Standards often used as antioxidants include Butylated hydroxylanisole (BHA), α-tocopherol and Ascorbic acid. 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule30. The antioxidant activity of a substance can therefore be determined by studying whether it interacts with a stable free radical such as DPPH.
1.6 BOTANICAL PROFILES OF THE PLANTS.
Dennettia tripetala commonly known as pepper fruit tree is a plant found mostly in tropical Africa and especially in southern, eastern and western Nigeria. The Igbos call it “:mmimi”, Ibibio and Efik people call it “nkaika”, Urhobo people call it “imako” while the Yorubas call it “igberi”. Its fruit appears red when ripe and green when unripe. The leaves, fruits and roots of the plant possess strong pepperish and pungent spicy taste with a characteristic aroma and fragrance33. The fruits and leaves are used as seasonings in preparing foods such as meat, soup and in some local dishes. Wood from D.tripetala plant also serves as a good fuel. The fruit itself is sold for money especially by the rural dwellers. Below is the botanical profile of the plant.
DENNETTIA TRIPETALA31:
Kingdom————-Plantae
Phylum—————Magnoliophyta
Class——————Magnoliopsida
Order——————Magnoliales
Family—————-Annonaceae
Genus—————–Dennettia
Species—————-D.tripetala
Milicia excelsa on the other hand is a large deciduous forest tree of lowland forest and wet savannah. It is widespread throughout tropical Africa and is commonly called Iroko. The Igbos of eastern Nigeria call it “oji”, Hausas call it “Madachi” while the Urhobos call it “uloho”. Wood from M.excelsa is of high quality and has great commercial value. It is very resistant to termite attack. Here is the botanical profile of the plant.
MILICIA EXCELSA32:
Kingdom—————Plantae
Phylum—————–Tracheophyta
Class——————–Magnoliopsida
Order——————-Rosales
Family——————Moraceae
Genus——————-Milicia
Species—————–M.excelsa
1.7 ETHNOMEDICINAL USES OF THE PLANTS
Dennettia tripetala has been reported to be used in the treatment of fever, cough and as an anti-emetic. In folk medicine, the leaves are commonly used by local herbalists in combination with other medicinal plants to treat various ailments including fever, infantile convulsion, typhoid, cough, worm infestation, vomitting and stomach upset34, 35. Its fruit has also been reported to be used in masking mouth odour as well as being used as stimulant34.
Milicia excelsa on the other hand has also been reported to be used in herbal medicine. The powdered bark is used for coughs, heart problems and lassitude. The latex, leaves and ashes are used as anti-tumour agent and also to clear stomach and throat obstructions36.
1.8 OBJECTIVES OF THE STUDY
This study aims to achieve the followings:
- Assessment of the phytochemicals in the root extracts of Dennettia tripetala and Miliciaexcelsa.
- Quantification of the phytochemicals present in the roots of the two plant samples.
- Evaluation of the antioxidant properties of the root extracts of both plants.
- Evaluation of the antimicrobial potentials of the root extracts in various solvents such as methanol, ethanol, ethyl acetate, butanol and water.
1.9 SCOPE OF THE STUDY
The work was limited to phytochemical screening of the roots of Dennettia tripetala and Milicia excelsa, quantification of the phytochemicals, evaluation of the antimicrobial potentials of the root extracts and also their antioxidant properties. Human testing was not part of the work.
1.10 SIGNIFICANCE OF THE STUDY
This study will add to existing knowledge in the field of phytochemistry and pharmaceutical chemistry by increasing antimicrobial and antioxidant options available to man from plants. The work will also serve to give us new information on the nature and concentration of phytochemicals present in the roots of Dennettia tripetala and Milicia excelsa which, if favourable, could be very vital in the search for and synthesis of new drugs that would better combat pathogenic microorganisms thereby improving health.
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