ABSTRACT
Raphia africana (Otedoh) is a member of the palm family Arecaceae. The plant was collected from banks of the creeks Ukoko and Uertsor in Buruku Local Government Area of Benue State. The fruit mesocarp was extracted and subjected to phytochemical and antimicrobial screenings. The ethyl acetate extracts, obtained via different extraction protocols (soxhlet extraction, microwave assisted extraction and maceration) were subjected to Artemia salina lethality assay. Extracts (ethyl acetate and methanol) were subjected to Gas Chromatography Mass Spectrometry (GCMS) and High-Performance Liquid Chromatography (HPLC) as part of pre-isolation analysis. Phytochemical screening showed the presence of steroids, triterpenes, alkaloids, anthroquinones, cardiac glycosides and flavonoids. Antimicrobial screening of the extracts showed the ethyl acetate extract to have the broadest and highest activity against the test microbes, having lower minimum inhibitory concentration (0.625 mg/mL) and a wider zone of inhibition diameter (27 mm) on Klebsiella pneumoniae. The ethyl acetate extract also showed good minimum bactericidal/fungicidal concentration values on Staphylococcus aureus (2.5 mg/mL), Klebsiella pneumoniae (1.25 mg/mL) and Candida albicans (2.5 mg/mL). The hexane extract was the least active against the test microbes with chloroform and methanol extracts showing intermediate activity. Extracts obtained by soxhlet extraction and microwave assisted extraction showed toxicity at a concentration of 269.2 mg/mL, while the extract obtained through maceration showed the lowest toxicity (467.7 mg/mL). The GCMS profiles showed the extracts to consist of lipids and diterpene alcohols while the selectively targeted HPLC showed traces of sterols (campesterol, cholestanol, cholesterol, ß-sitosterol, stigmasterol and tocopherol) and flavonoids (isovitexin,
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kaempferol and orientin). Crude extracts were extensively purified using vacuum liquid chromatography (VLC) and gel filtration (GF). Fractions from VLC and GF sessions were characterised using Nuclear Magnetic Resonance Spectroscopy (NMR) and found to be mainly lipids. Extensive chromatography allowed the isolation of saturated fatty acids (margaric and myristic acids), a monounsaturated fatty acid methyl ester (methyl oleate), triglycerides, a sterol methyl ether, tentatively identified diosgenin-type steroidal saponins, tentatively identified monogalactosyl diacylglycerides, monoacyl glycerides and diacylglycerides. This is the first time these substances have been identified in the mesocarp of this plant. The isolated compounds were tested against microbes and they showed higher but similar activity compared to the crude extracts. Ja 83 (a monogalactosyldiacylglyceride) showed the largest zone of inhibition against Staphylococcus aureus (30 mm), against Klebsiella pneumoniae (31 mm) and 32 mm against Shigella dysenteriae while showing the least zone against Microsporum sp. Isolated compounds were inactive towards Aspergillus fumigatus and Aspergillus niger. Relatively mild behaviour of the crude extracts against Artemia salina, and the lipidic nature of the phytoconstituents reaffirm its use as a food source and it is recommended that this, now increasingly neglected tropical fruit tree, be actively cultivated for human use provided the toxicity studies allow them for such purpose.
TABLE OF CONTENTS
CERTIFICATION PAGE ………………………………………………………………………………….. iv
ACKNOWLEDGEMENT …………………………………………………………………………………… v
DEDICATION…………………………………………………………………………………………………. viii
LIST OF TABLES ……………………………………………………………………………………………. xv
LIST OF FIGURES ………………………………………………………………………………………… xvii
LIST OF APPENDICES …………………………………………………………………………………… xx
ABBREVIATIONS ………………………………………………………………………………………. xxviii
ABSTRACT …………………………………………………………………………………………………… xxix
CHAPTER ONE ………………………………………………………………………………………………… 1
1.0 INTRODUCTION……………………………………………………………………………………. 1
1.1.1 Justification of the Study ……………………………………………………………………………… 2
1.1.2 Aim and Objectives ……………………………………………………………………………………… 2
1.1.3 Objectives of the Research: ………………………………………………………………………….. 2
CHAPTER TWO ……………………………………………………………………………………………….. 4
2.0 LITERATURE REVIEW ………………………………………………………………………… 4
2.1 Man and Medicinal Plants ……………………………………………………………………………… 4
2.1 Plant Secondary Metabolites…………………………………………………………………………… 8
2.1.1 Alkaloids ……………………………………………………………………………………………………… 8
2.1.2 Terpenes and Terpenoids …………………………………………………………………………….. 8
2.1.3 Flavonoids …………………………………………………………………………………………………. 14
2.1.4 Saponins ……………………………………………………………………………………………………. 15
2.1.5 Steroids ……………………………………………………………………………………………………… 16
2.2 ARECACEAE…………………………………………………………………………………………….. 18
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2.2.1 The Genus Raphia ……………………………………………………………………………………… 18
2.2.2 Traditional Usage of Raphia ………………………………………………………………………. 21
2.2.3 Phytochemistry of Raphia ………………………………………………………………………….. 22
2.2.4 Raphia africana (Description and Geographical Spread) ………………………………. 23
2.2.5 Vernacular names of Raphia africana …………………………………………………………. 27
2.3 Vacuum Liquid Chromatography ………………………………………………………………….. 27
2.3.1 Gel filtration or Gel permeation Chromatography ………………………………………. 29
2.4 Hyphenated Techniques in Compound Isolation ……………………………………………… 30
2.4.1 High Performance Liquid Chromatography (HPLC) ………………………………….. 30
2.4.2 Gas Chromatography – Mass Spectrometry (GC-MS) ………………………………… 31
2.5 Spectroscopic Techniques …………………………………………………………………………….. 31
2.5.1 Nuclear Magnetic Resonance Spectroscopy (NMR) …………………………………….. 31
2.5.2 Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR) of Lipids ………. 33
2.5.3 Infrared Spectroscopy of Lipids …………………………………………………………………. 35
2.6 Microwave Assisted Extraction (MAE)………………………………………………………….. 36
2.7 Lipids and Lipid Biosynthesis ………………………………………………………………………. 37
2.7.1 Fatty Acids ………………………………………………………………………………………………… 38
2.8 Bacteria ……………………………………………………………………………………………………… 38
2.8.1 Shigela dysenteriae ……………………………………………………………………………………… 39
2.8.2 Salmonella typhae ………………………………………………………………………………………. 40
2.9 Fungi …………………………………………………………………………………………………………. 40
2.10 Brine Shrimp Lethality Assay ……………………………………………………………………….. 41
3.0 MATERIALS AND METHODS ………………………………………………………………….. 42
3.1 Materials ……………………………………………………………………………………………………. 42
3.2 Equipment ………………………………………………………………………………………………….. 42
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3.3 Sample Collection ……………………………………………………………………………………….. 43
3.4 Extraction Protocol ……………………………………………………………………………………… 44
3.4.1 Extraction 1: Cold Maceration …………………………………………………………………… 44
3.4.2 Extraction 2: Microwave Assisted Extraction (MAE) ………………………………….. 44
3.4.3 Extraction 3: Soxhlet Extraction. ………………………………………………………………… 45
3.5 Sample Analysis………………………………………………………………………………………….. 45
3.5.1 Pre-isolation Analyses ………………………………………………………………………………… 45
3.5.2 Phytochemical Screening ……………………………………………………………………………. 48
3.6 Biological Studies ……………………………………………………………………………………….. 51
3.6.1 Brine Shrimp Lethality Test ……………………………………………………………………….. 51
3.6.2 Antimicrobial Screening …………………………………………………………………………….. 52
3.7 Chromatographic Purification of compounds ………………………………………………….. 55
3.7.1 Vacuum Liquid Chromatography (VLC) ……………………………………………………. 55
3.7.2 Thin Layer Chromatography (TLC) …………………………………………………………… 57
3.8 Spectroscopic Analyses of Isolated Compounds ……………………………………………… 58
3.8.1 Nuclear Magnetic Resonance Spectroscopy …………………………………………………. 58
3.9 Isolation profile: ………………………………………………………………………………………….. 59
4.0 RESULTS ……………………………………………………………………………………………… 60
4.1 Extraction …………………………………………………………………………………………………… 60
4.2 Phytochemical Screening of the Mesocarp of Extracts of R. africana ………………… 61
4.2.1 Fehling’s test for free reducing sugars ………………………………………………………… 61
4.2.2 Test for anthraquinone glycosides ………………………………………………………………. 61
4.2.3 Test for saponin glycosides …………………………………………………………………………. 62
4.2.4 Tests for steroids and terpenoids ………………………………………………………………… 62
4.2.5 Test for tannins ………………………………………………………………………………………….. 62
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4.2.6 Test for alkaloids ……………………………………………………………………………………….. 62
4.2.7 Test for cardiac glycosides ………………………………………………………………………….. 63
4.2.8 Test for flavonoids ……………………………………………………………………………………… 63
4.3 Antimicrobial Assay of the Mesocarp of Extracts of R. africana……………………….. 64
4.4 Minimum Inhibitory Concentration for the Mesocarp extracts of R. africana ……… 64
4.5 Minimum bactericidal concentration and minimum fungicidal concentration for the Mesocarp extracts of R. africana …………………………………………………………….. 66
4.6 Brine Shrimp Lethality Assay ……………………………………………………………………….. 66
4.7 Thin Layer Chromatography of Crude Extracts ………………………………………………. 70
4.8 HPLC analysis of methanol and ethyl acetate extracts of Raphia africana …………. 70
4.9 GC-MS Analysis of Ethyl acetate extract of Raphia africana …………………………… 72
4.10 Sample (VLC fractions) coding for Antimicrobial and NMR Studies ………………… 73
4.11 Antimicrobial assay for isolated compounds …………………………………………………… 84
4.11.1 Antimicrobial sensitivity and diameter of zone of inhibition for isolated compounds ……………………………………………………………………………………………… 84
4.11.2 Minimum inhibitory concentration of isolated compounds ………………………….. 85
4.11.3 Minimum Bactericidal/Fungicidal concentration ………………………………………… 86
4.11.4 1H NMR Spectrum of Ja 64 ………………………………………………………………………… 94
4.11.5 1H NMR Spectrum of Ja 65 ………………………………………………………………………… 94
4.11.6 NMR Spectra of Ja 66 ………………………………………………………………………………… 94
4.11.7 1H NMR Spectrum of Ja 67 ………………………………………………………………………… 95
4.11.8 1H NMR Spectrum of Ja 68 ………………………………………………………………………… 95
4.11.9 NMR Spectra for Ja69 ……………………………………………………………………………….. 95
4.11.10 1H NMR Spectrum of Ja75 ………………………………………………………………………. 96
4.11.11 1H NMR Spectrum of Ja76 ………………………………………………………………………. 96
4.11.12 NMR Spectra for Ja77 …………………………………………………………………………….. 96
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4.11.13 NMR Spectra for Ja78 …………………………………………………………………………….. 97
4.11.14 1H NMR Spectrum of Ja79 ………………………………………………………………………. 97
4.11.15 1H NMR Spectrum of Ja80 ………………………………………………………………………. 98
4.11.16 1H NMR Spectrum of Ja81 ……………………………………………………………………… 98
4.11.17 1H NMR Spectrum of Ja82 ………………………………………………………………………. 98
4.11.18 1H NMR Spectrum of Ja83 ………………………………………………………………………. 99
4.11.19 1H NMR Spectrum of Ja95 ………………………………………………………………………. 99
4.11.20 1H NMR Spectrum of Ja96 …………………………………………………………………….. 100
4.11.21 1H NMR Spectrum of Ja 97 ……………………………………………………………………. 100
4.11.22 1H NMR Spectrum of Ja 98 ……………………………………………………………………. 100
4.11.23 1H NMR Spectrum of JSEA2 …………………………………………………………………. 100
4.11.24 1H NMR Spectrum of JSEA30 ………………………………………………………………. 101
4.11.25 1H NMR spectrum for JSEA46 ………………………………………………………………. 101
5.0 DISCUSSION ……………………………………………………………………………………………. 102
5.1 Phytochemistry …………………………………………………………………………………………. 102
5.2 Antimicrobial activity of crude extracts ……………………………………………………….. 103
5.3 Artemia salina lethality assay ……………………………………………………………………… 103
5.4 GC-MS profile of R. africana and profiles of some raphia species …………………… 104
5.5 HPLC Profile of R. africana ……………………………………………………………………….. 104
5.6 NMR Studies …………………………………………………………………………………………….. 105
5.7 Characterisation of compounds isolated from R. africana using NMR methods … 105
5.7.1 Characterization of Ja 64, Ja 65, Ja 68, and Ja 76 as Heptadecanoic acid …… 108
5.7.2 Characterization of Ja69 as hexadecanoic acid ………………………………………….. 115
5.7.3 Characterization of JSEA2 ………………………………………………………………………. 115
5.7.4 Characterization of JSEA46 ……………………………………………………………………… 123
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5.7.5 Characterization of Ja96 …………………………………………………………………………… 124
5.7.6 Identification of Ja 66 as a Triglyceride …………………………………………………….. 125
5.7.7 Characterization of Ja 67 ………………………………………………………………………….. 126
5.7.8 Characterization of Ja75 …………………………………………………………………………… 127
5.7.9 Characterization of Ja77 …………………………………………………………………………… 132
5.7.10 Characterization of Ja78 …………………………………………………………………………… 134
5.7.11 Characterization of Ja79 …………………………………………………………………………… 135
5.7.12 Characterization of Ja80 …………………………………………………………………………… 135
5.7.13 Characterization of Ja81 …………………………………………………………………………… 135
5.7.14 Characterization of Ja82 …………………………………………………………………………… 138
5.7.15 Characterization of Ja83 as a digalactosyldiacylglyceride ………………………….. 139
5.7.16 Characterization of Ja95 …………………………………………………………………………… 140
5.7.17 Characterization of Ja97 …………………………………………………………………………… 142
5.7.18 Characterization of Ja98 …………………………………………………………………………… 143
5.7.19 Characterization of JSEA30 ……………………………………………………………………… 143
5.10 Antimicrobial Activity of Isolated Compounds ……………………………………………… 144
6.0 CONCLUSION AND RECOMMENDATION ……………………………………………. 149
6.1 Conclusion ………………………………………………………………………………………………… 149
6.1 Recommendations ………………………………………………………………………………… 151
REFERENCES ……………………………………………………………………………………………….. 153
APPENDICES ………………………………………………………………………………………………… 181
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CHAPTER ONE
1.0 INTRODUCTION
Thousands of plants have already been investigated for metabolites/phytochemicals by scientists (Dias et al., 2012). The number of novel compounds isolated from plants has steadily fallen, from its peak in the heyday of isolation (1960s and 1970s). Many new applications are being discovered every day for previously isolated compounds (Dias et al., 2012). Many neglected tropical diseases have no viable cure yet (Lawal et al., 2010). For instance, the best drugs for treatment of trypanosomiasis kill about 5% of patients (Pépin et al., 1995; Kayser et al., 2003). All anti-malarials are hepatotoxic (Alkadi, 2007; German et al., 2007) while human immunodeficiency virus induced Acquired Immune Deficiency Syndrome (HIV/AIDS) is still ravaging millions world-wide. Worse yet, a new crop of epidemics from Zika, Chikungunya, Yellow fever, Ebola haemorrhagic fever, to Lassa fever are ravaging the third world; disturbingly, these epidermics have no viable cure (Musso and Gubler, 2016; Phillips and Neyland, 2016).
Species of Raphia like R. farinifera, R. hookeri, R. regalis and R. sudanica are used for treating mouth ulcers, stomach problems and for treating heart disease (heart pain). A search on internet databases, like Elsevier, National Center for Biotechnology Information (NCBI) yielded no result on R. Africana; offline resources like Kashim Ibrahim Library (KIL) Ahmadu Bello University Library showed that no scientific study investigating Raphia africana’s ethnomedicinal applications had been reported prompting further interest in the plant.
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1.1.1 Justification of the Study
There is lack of scientific information on Raphia africana. This study seeks to investigate the traditional claims on Raphia africana (Otodeh) Arecaceae with a view to contributing to the knowledge base in the fight against tropical diseases like mouth ulcers.
1.1.2 Aim
The aim of this research is to authenticate or otherwise, the claims by traditional medicine practioners on the fruit mesocarp of Raphia africana by isolating and characterising some of the compounds from it.
1.1.3 Objectives of the Research:
The aim will be achieved through the following objectives: i. Extraction of the powdered plant material using different solvents based on the eluotropic series i.e. non-polar pet-ether (60-80 OC), chloroform, ethyl acetate and methanol (polar), ii. phytochemical screening of the crude solvent extracts, iii. antibacterial and antifungal screening of the various extracts,
iv. lethality assay of the crude extracts using Brine shrimp (Artemia salina),
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v. separations and purifications of the crude extracts using several steps of chromatographic techniques, vi. spectroscopic analyses (NMR, IR, GCMS) of the isolated compounds.
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