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UNIVERSITY OF GHANA 
COLLEGE OF BASIC AND APPLIED SCIENCES 
SCHOOL OF PHYSICAL AND MATHEMATICAL SCIENCES 
 
 
 
    
 
 
 
 
 
ESSENTIAL OILS AND COMPOUNDS ISOLATED FROM THE LEAVES AND 
RHIZOMES OF AFRAMOMUM ATEWAE 
 
BY 
COFFIE ERIC 
(10421123) 
 
 
THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON  
IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF   
MPHIL CHEMISTRY DEGREE 
 
 
 
DEPARTMENT OF CHEMISTRY 
 
 
JULY 2019
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DECLARATION 
 
I, Eric Coffie, hereby declare that this thesis was undertaken by me under the supervision and that 
it has not been submitted to any other university for another degree. 
 
 
………………… 
ERIC COFFIE 
(10421123) 
(Candidate) 
 
 
 
…………………… 
PROF. DORCAS OSEI-SAFO 
(Principal Supervisor) 
 
 
…………………… 
PROF. HENOK KINFE 
(Co-supervisor) 
 
 
 
 
 
 
 
 
 
 
 
 
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ABSTRACT 
Aframomum species have been reported to be at an alarming rate of decline due to climate change 
and anthropogenic activities. This work investigated for the first time the chemical composition of 
Aframomum atewae, a less common species of the genus.  
Essential oils of the leaves and rhizomes were obtained either through hydrodistillation or solvent 
extraction followed by chromatographic separation. GC-MS analysis of the constituents revealed 
that the fresh leaf essential oil was rich in monoterpenes (22.4%) while sesquiterpenes dominated 
the fresh rhizome essential oil (24.4%). Steroids and long chain hydrocarbons were the major 
constituents of the dichloromethane-extracted rhizome essential oil.  The results for the petroleum 
ether-extracted rhizome essential oil are pending (due to sample mix up when submitted for GC-
MS analysis). The major constituents identified in the three essential oils were 1-methyl-1-
(methylamino)isobenzofuran-3-one (17.27%), 2,5-ditertbutylhydroxybenzene (7.80%) and 14-
pregnane (56.95 %) for the leaf, rhizome and DCM-extracted essential oils, respectively. To the 
best of my knowledge, this is the first time these compounds have been identified in the genus. 
The antifungal potential of the 4 essential oils was evaluated against Candida albicans and 
Saccharomyces cerevisiae in an Alamar Blue-based broth dilution assay. The fresh rhizome and 
the DCM-extracted rhizome essential oils exhibited fungicidal activity against C. albicans while 
fungistatic activity was observed for the fresh leaf and the PE-extracted rhizome essential oils. 
With the exception of the PE-extracted oil which was fungistatic against S. cerevisiae, the 
remaining essential oils did not exhibit any activity against S. cerevisiae. 
Crude PE, DCM and MeOH extracts of the air-dried pulverized rhizome were prepared by cold 
percolation. The PE and DCM crude extracts tested positive for terpenoids and steroids while the 
MeOH contained alkaloids, flavonoids, cardiac glycosides, saponins, and tannins. Through 
column chromatographic separations of the three extracts, 10 compounds were isolated, out of 
which 4 compounds were identified to be 1(E)-8-methylundec-8-en-1-yl-3-(cyclohexa-2,5-dien-
1-yl)propanoate, 2-(6-oxotetrahydro-2H-pyran-2-yl)ethyl dodec-8-enoate, myristic acid and 
stigmasterol. Characterization of the compounds was achieved through IR, 1D- and 2D NMR, LC-
MS and HR-MS techniques. 
 
 
 
 
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DEDICATION 
To the glory of Almighty God, my parents, siblings and Ms. Princess Lucy Ocran. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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ACKNOWLEDGMENT 
All Glory, Honour and Adoration are due to God Almighty, for His guidance and protection 
throughout this work. 
 I am grateful to my supervisors, Prof.  Dorcas Osei-Safo and Prof. Henok Kinfe for their guidance 
and assistance throughout this project. 
I am very grateful to the South Africa-Ghana flagship program for funding this project.  
My deepest appreciation goes to Dr. Patrick K. Arthur and his team especially, Michael Debrah, 
and Benaiah Abbey of the West African Centre for Cell Biology of Infectious Pathogens 
(WACCBIP), Department of Biochemistry, Cell and Molecular biology, University of Ghana- 
Legon for their time and assistance in the antifungal studies.  
My utmost appreciation goes to the Chemistry Department, (UG) for providing NMR and IR 
instruments and to Mr. Samuel Owusu-Atuah for his assistance in obtaining the IR data. My special 
appreciation also goes to Mr. Bob Essien who was always available to provide me with solvents 
needed for the work. 
I appreciate the efforts of Mr. Henry Onyame and Mr. Samuel Kwain for their guidance in 
elucidating the structures of the compounds. I also appreciate the students and staff of the 
Department of Chemistry, (University of Johannesburg-South Africa, UJ) especially, Mr. Bruce 
Marakalala for welcoming and hosting me during my visit to UJ.  
My family supported me both physically and spiritually. My parents, Mr. Simon Coffie and 
Madam Faustina Armah, to whom I owe all my gratitude in life, I pray for prosperity and good 
health. You are the best parents one can have. I am grateful to my brothers and sisters for their 
prayers, encouragement, love and assistance throughout my life. 
Special appreciation also goes to the following: Elsie Otumfuo Korkor, Magdalene Dzigbordi 
Togoh, Maureen Agbagba, Elizabeth Ahiagba, Esther Barnes and the entire 2021 class of 
chemistry students – UG for their support. 
I am very grateful to Ms. Emmanuella Bema Twumasi and my classmates for their assistance and 
company. 
To all others whose names I have not mentioned, I am grateful to you all and God Almighty bless 
you. 
 
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TABLE OF CONTENT 
DECLARATION i 
DEDICATION iii 
ACKNOWLEDGMENT iv 
LIST OF FIGURES viii 
LIST OF SCHEMES x 
LIST OF TABLES xi 
ABBREVIATIONS xii 
1.0 INTRODUCTION 1 
1.1 Medicinal plants .............................................................................................................................................. 1 
1.2 Aframomum Species ........................................................................................................................................ 2 
1.3 Fungi ................................................................................................................................................................. 2 
1.4 Problem statement ........................................................................................................................................... 2 
1.5 Justification ...................................................................................................................................................... 3 
1.6 Hypothesis ........................................................................................................................................................ 3 
1.7 Aim ................................................................................................................................................................... 3 
1.8 Objectives ......................................................................................................................................................... 3 
CHAPTER TWO 5 
2.0 LITERATURE REVIEW 5 
2.1 Family Zingiberaceae ...................................................................................................................................... 5 
2.2 Genus Aframomum .......................................................................................................................................... 5 
2.3 Chemosystematics of Aframomum species .................................................................................................... 6 
2.4 Pharmacology of Aframomum Extracts ......................................................................................................... 6 
2.4.1 Anticancer activity .................................................................................................................... 6 
2.4.2 Antimicrobial activity ............................................................................................................... 7 
2.4.3 Antiestrogenic activity .............................................................................................................. 7 
2.4.4 Penile erection function ........................................................................................................... 7 
2.5 Ethnobotanical use and Pharmacological action of selected species of Aframomum ................................ 7 
2.5.1 A. melegueta K. Schum ........................................................................................................... 7 
2.5.2 A. danielli (Hook. f.) K. Schum .............................................................................................. 8 
2.5.3 A. citratum (C. Pereira) K. Schum ......................................................................................... 8 
2.5.4 A. sulcatum (Oliv. & Hanb.) K. Schum ................................................................................. 8 
2.5.5 A. kayserianum K. Schum ....................................................................................................... 8 
2.6 Chemical components of Aframomum and their biological activities. ........................................................ 9 
2.6.1 Terpenoids ................................................................................................................................ 9 
2.6.2 Flavonoids .............................................................................................................................. 11 
2.6.3 Arylalkanoids ......................................................................................................................... 12 
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2.7 Fungal infections ........................................................................................................................................... 14 
2.7.1 Control .................................................................................................................................... 15 
2.7.2 Treatment of fungal infections .............................................................................................. 15 
2.7.3 Natural products as antifungal agents .................................................................................. 15 
CHAPTER THREE 17 
3.0 METHODOLOGY 17 
3.1 Plant collection .............................................................................................................................................. 17 
3.2 General experimental methods .................................................................................................................... 18 
3.3 Chemicals and Reagents ............................................................................................................................... 20 
3.3.1 Anisaldehyde spray reagent ................................................................................................... 20 
3.3.2 Acid spray ............................................................................................................................... 20 
3.3.3 Wagner’s Reagent .................................................................................................................. 20 
3.3.4 Mayer’s Reagent..................................................................................................................... 20 
3.3.5 Dragendoff’s Reagent ............................................................................................................ 21 
3.3.6 Iron (III) chloride solution .................................................................................................... 21 
3.3.7 Liebermann-Burchard Reagent ............................................................................................. 21 
3.4 Essential oil distillation of rhizomes and leaves .......................................................................................... 21 
3.5 Antifungal activity ......................................................................................................................................... 22 
3.6 Solvent extraction of rhizomes ..................................................................................................................... 22 
3.7 Phytochemical screening procedures ........................................................................................................... 23 
3.7.1 Keller-Kiliani Test for Cardiac Glycosides ............................................................................ 23 
3.7.2 Test for Anthraquinones and Anthracene Derivatives ......................................................... 24 
3.7.3 Test for Tannins ..................................................................................................................... 24 
3.7.4 Test for Saponins ................................................................................................................... 24 
3.7.5 Test for Terpenoids ................................................................................................................ 24 
3.7.6 Test for alkaloids .................................................................................................................... 24 
3.7.7 Test for Flavonoids ................................................................................................................ 25 
3.7.8 Test for steroids ...................................................................................................................... 25 
3.7.9 Test for polyphenols ............................................................................................................... 25 
3.8 Investigation of the extracts .......................................................................................................................... 25 
3.8.1 Petroleum ether (40-60 °C) extract ........................................................................................ 25 
3.8.2 Dichloromethane Extract ....................................................................................................... 26 
3.8.3. Methanol extract ................................................................................................................... 27 
CHAPTER FOUR 29 
4.0 RESULTS AND DISCUSSION 29 
4.1 Summary of results 29 
4.2 Chemical composition of the Essential Oils 29 
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4.2.1 Chemical composition of AA-L ................................................................................................................... 29 
4.2.2 Chemical composition of the rhizome essential oil .................................................................................... 34 
4.2.3 Chemical composition of DCM-extracted essential oil .............................................................................. 39 
4.3 Antifungal studies of essential oils 44 
4.4 Investigation of extracts from the rhizome of A. atewae 47 
4.4.1 Phytochemical Screening of the PE, DCM and MeOH Crude Extracts ................................................... 47 
4.4.2 Investigation of the Petroleum Ether (PE) Extract .................................................................................... 48 
4.4.2.1 Characterization of AA/R/PE-2 .......................................................................................... 48 
4.4.2.3 Identification of AA/R/PE-5 ............................................................................................... 56 
4.4.2.4 Identification of AA/R/PE-6 ............................................................................................... 58 
4.4.3 Investigation of the Dichloromethane (DCM) Extract .............................................................................. 60 
4.4.3.1 Characterization of AA/R/DCM- 3 ..................................................................................... 60 
CHAPTER FIVE 68 
5.0 Conclusion 68 
5.1 Recommendations 69 
References 70 
APPENDIX 77 
Appendix I ........................................................................................................................................................... 77 
Appendix II .......................................................................................................................................................... 78 
Appendix IIIA...................................................................................................................................................... 79 
Appendix IIIB ...................................................................................................................................................... 80 
Appendix IV ......................................................................................................................................................... 81 
Appendix V .......................................................................................................................................................... 82 
Appendix VI ......................................................................................................................................................... 83 
Appendix VII ....................................................................................................................................................... 84 
Appendix VIII...................................................................................................................................................... 85 
Appendix IX ......................................................................................................................................................... 86 
Appendix X .......................................................................................................................................................... 87 
Appendix XI ......................................................................................................................................................... 88 
Appendix XII ....................................................................................................................................................... 89 
Appendix XIII...................................................................................................................................................... 90 
 
 
 
 
 
 
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LIST OF FIGURES 
Figure 2.1: Some essential oil constituents of Aframomum species 9 
Figure 2.2: Sesquiterpenes from some species of Aframomum 10 
Figure 2.3: Diterpenes from some species of Aframomum 11 
Figure 2.4: Flavonoids from some species of Aframomum 12 
Figure 2.5: Arylalkanoids from some species of Aframomum 12 
Figure 2.6: Examples of clinically used antifungal agents 15 
Figure 2.7: Natural products with antifungal activity 16 
 Figure 3.1a: Flower of Aframomum atewae                                                                             17 
Figure 3.1b: Leaf and rhizome of Aframomum atewae 18 
Figure 3.1c: Leaf and rhizome of Aframomum atewae 18 
  Figure 4.1: Gas chromatogram of leaf essential oil of A. atewae                                              30 
Figure 4.2: MS of 1-methyl-1-(methylamino)isobenzofuran-3-one, (17.66%), MF = 
C10H11NO2, Rt 24.854 33 
Figure 4. 3: MS of Bornyl chloride, (4.93%), MF = C10H17Cl, Rt 16.166 33 
Figure 4.4: MS of 2-chloro-4,4,7-trimethyl-1,2,3,4,5,6,7,8-octahydronapthalene, 
(5.56%),  MF = C13H21Cl,  Rt 36.671 34 
Figure 4.5: Mass fragmentation of 5,6,6-trimethyl-5-(3-oxo-1-butenyl)- 1-
Oxaspiro[2.5]octan-4-one, (9.019%), MF = C14H20O3, Rt 32.627 34 
Figure 4.6: Other major constituents of the leaf essential oil 34 
Figure 4.7: Gas chromatogram of rhizome essential oil of A. atewae 35 
Figure 4. 8: MS of 2,5-ditertbutylhydroxybenzene, (7.80%) MF = C14H22O, Rt 32.386 38 
Figure 4. 9: MS of dibutylphthalate, (4.58%), MF = C16H22O4, Rt 37.716 38 
Figure 4.10: MS of carvacrol, (3.89%), MF = C10H14O, Rt 30.2158 38 
Figure 4.11: MSof palmitic acid, (4.34%), MF = C16H32O2, Rt 41.067 39 
Figure 4.12: MS of diisobutylphthalate, (3.87%), MF = C16H22O4, Rt 35.8189 39 
Figure 4.13: Other major constituents of the leaf essential oil 39 
Figure 4. 15: MS of 14 -pregnane, (58.44%), MF = C21H36, Rt 34.287 42 
Figure 4.16 MS of (-)-cyprene, (2.51%), MF = C14H24, Rt 19.109 42 
Figure 4.17 MS of diisobutylphthalate, (3.87%), MF = C16H22O4, Rt 35.8189 43 
Figure 4.18: Other major constituents of AA/R/DCM 1 43 
Figure 4.19: Graph of results showing activity of essential oils against C. albicans 45 
Figure 4.20: Graph of results showing the activity of essential oils against S. cerevisiae 46 
Figure 4.21: Full 13C NMR Spectrum 48 
Figure 4.22: Full DEPT 135 ° Spectrum 49 
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Figure 4.23: Full 1H NMR Spectrum 50 
Figure 4.24: Expanded HSQC spectrum 51 
Figure 4.25: Expanded COSY NMR Spectrum 52 
Figure 4.26: Expanded HMBC NMR Spectrum 54 
Figure 4.27: LC-MS of AA/R/PE-2 55 
Figure 4.28: Proposed fragmentation pattern for AA/R/PE-2 56 
Figure 4.29: Proposed structure of AA/R/PE 1 (E)-8-methylundec-8-en-1-yl 3-
(cyclohexa-2,5-dien-1-yl)propanoate 56 
Figure 4.30: Structure of Myristic acid 58 
Figure 4.31: HR-MS spectrum of AA/R/DCM-3 60 
Figure 4.32: 13C NMR spectrum of AA/R/DCM-3 61 
Figure 4.33: DEPT 135 spectrum of AA/R/DCM-3 62 
Figure 4.34: 1H NMR of AA/R/DCM-3 63 
Figure 4.35: HSQC spectrum of AA/R/DCM-3 64 
Figure 4.36: COSY spectrum of AA/R/DCM 3 65 
Figure 4. 37: HMBC spectrum of AA/R/DCM 3 66 
Figure 4.38: Structure of AA/R/DCM 3 67 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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LIST OF SCHEMES 
Scheme 3.1:  Preparations of extracts and fractions of A. atewae 23 
Scheme 3.2: Separation of constituents of AA/PE 26 
Scheme 3.3: Separation of constituents of the AA/DCM 27 
Scheme 3.4: Protocol for alkaloid isolation 28 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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LIST OF TABLES 
Table 2.1: Summary of Aframomum species, isolated compounds and their biological 
activities. 13 
Table 4.1: Retention time, name of constituent, percentage composition, molecular formula and 
weight of A. atewae leaf essential oil as shown by GCMS analysis                                                   30 
Table 4.2: Retention time, name of constituent, percentage composition, molecular 
formula and weight of Aframomum rhizome essential oil as shown by GC-MS analysis 35 
Table 4.3: Retention time, percentage composition, molecular formula and molecular 
weight of constituents of A. atewae rhizome DCM-extracted essential oil as shown by 
GCMS analysis 40 
Table 4.4: Classes of terpenes and common constituents of the 3 essential oils 43 
Table 4. 5: Results of phytochemical screening of crude extracts 47 
Table 4.6: Mass and percentage yields of various extracts 47 
Table 4.7: 1H, 13C, COSY and HMBC NMR data for AA/R/PE-2 54 
Table 4.8: 1H, 13C, COSY and HMBC NMR data for AA/R/PE-2 57 
Table 4.9 Comparative 13C-NMR chemical shifts of stigmasterol with literature 59 
Table 4.10  1H, 13C, COSY and HMBC NMR data for AA/R/PE-2 66 
 
 
 
 
 
 
 
 
 
 
 
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ABBREVIATIONS 
DCM    Dichloromethane 
MeOH Methanol 
EtOH Ethanol 
PE   Petroleum ether 
EtOAc, EA                                   E   t h  y  l  a  c e  t a  t e            
DMSO Dimethyl sulfoxide 
CDCl3 Deuterated chloroform 
ID One dimensional 
2D Two dimensional 
1H NMR                                       P  r  o  t o  n    M   N   R                  
13C NMR                                      C   a  r b  o  n    1  3   N   M   R           
COSY Correlation Spectroscopy 
HSQC   Hetero-nuclear Single Quantum Coherence 
HMBC Hetero-nuclear Multiple Bond Correlation 
IR Infrared 
GC-MS Gas chromatography mass spectrometry 
HR-MS                                         H   i g  h   r e  s o  l  u  t i o  n   m   a ss spectrometry 
TLC    Thin layer chromatography 
LC-MS Liquid chromatography Mass spectrometry 
Br Broad 
NB Nutrient broth 
 
 
 
 
 
 
 
 
 
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CHAPTER ONE 
1.0 INTRODUCTION 
1.1 Medicinal plants 
Africa is gifted with rich plant biodiversity including 45,000 species in sub-Saharan Africa1. A 
significant proportion of this natural resource comprises medicinal plants whose therapeutic 
properties have led to a long time of use as traditional remedies for both human and animal health. 
Several communities on the continent, as well as in Asia and Latin America, with over 80 % 
population of the world, still depend heavily on plant-based remedies for their principal healthcare 
needs. This high patronage is mainly due to accessibility to plant parts and the knowledge of their 
traditional use, affordability, ease of preparation and perceived safety. Lately, there has been a 
growing tendency of integrating plant-based medicines into mainstream health systems in many 
countries2. Ghana adopted this policy in 20102. Further, the utilization of medicinal plants for the 
production of phytopharmaceuticals, food supplements, nutraceuticals, and cosmeceuticals are on the 
ascendancy. Through scientific and technological advancement, medicinal plants continue to serve 
as sources of either useful drugs or chemical scaffolds for the semi-synthetic development of new 
drugs in many drug discovery programs.  
The increased interest in medicinal plant material and related products has resulted in a huge global 
trade at both domestic and international levels, providing a handsome source of income for producers, 
collectors and practitioners. The trade commodities include extracts, essential oils, 
phytopharmaceuticals, gums, spices and tannins. In nearly two decades, the total global trade value 
has more than doubled from $ 2.4 billion to $ 6.2 billion between 1996 and 2013 with a yearly 
progress percentage of 10.7 % registered in recent years3. 
Continued bioprospecting for new drugs and commercialization has resulted in increased use and 
high demand for medicinal plants. More often than not, the collection of wild species is conducted 
indiscriminately and in non-sustainable ways, threatening the future of this vital resource. The 
situation is worsened by effects of climate change (drought, extreme heat and erratic rainfall patterns) 
and habitat loss due to surface mining activities, construction and farming. Destruction or degradation 
of wildlands leaves in its wake loss of unique and precious species containing important cures for 
diseases we face now and new ones that may emerge in the future. This is critical because the 
chemical profile and therapeutic properties of many plants remain to be established to define efficacy 
and safety4. Overexploitation for commercialization purposes may also render useful traditional 
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medicinal plant resources inaccessible and unaffordable to local populations and to the rest of the 
world.   
1.2 Aframomum Species 
The Aframomums constitute a group of diverse plants distributed in West and Central Africa 
described by Schumann in 1904 to house the African species of Amomum. Aframomum is the major 
genus of the family Zingiberaceae, consisting of about 80 species including A. melegueta, A. 
giganteum, A. sulcatum, A. danielli and A. longiscapum. They have long been used ethnobotanically 
as spices and ornamental plants and are largely collected for a several medicinal applications such as 
laxative, fever management, inflammatory conditions as well as postpartum haemorrhage5,6. In 
Ghana, A. melegueta (peppery spice) was cultivated for many years as a cash crop and was exported 
to Europe and North America till its importance declined due to export restrictions during World War 
I and competition from cocoa 7. The species are reported to be rich in terpenoids, flavonoids and 
arylalkanoids possessing antiplasmodial, antimicrobial, antioxidant and anticancer activities5.  
1.3 Fungi 
A fungus is a collection of eukaryotic organisms that consist of mushrooms, moulds and yeasts. 
Though several fungi are known to be important and very useful, relatively few fungi species are 
phytopathogenic and produce toxins that cause diseases (infections and allergies) in man and animals. 
Harmful fungi present a common threat to the health of humans and agricultural production. These 
collective few fungi can cause loss of food for consumption, fatal diseases and huge economic losses 
to agriculture, in humans and animals8-10. Examples of infections that are caused by fungi in human 
are candidiasis, mucormycosis and blastomycosis whiles in plants there are diseases such as smut 
disease, leaf spot and chlorosis. 
1.4 Problem statement 
Aframomum species have been cited to be at an alarming rate of decline as a result of climate change 
effects, habitat destruction from various anthropogenic activities, especially illegal mining, and poor 
conservation practices. While many of the species have not yet received the requisite taxonomical 
attention, their pharmacological potential also remains largely under-researched regardless of their 
wide collection. Literature corroborates this observation with A. melegueta and A. giganteum 
characterizing much of the published research on the genus5. The rapid loss of this rich biodiversity 
has necessitated a call for a swift response not only to explore this huge resource for novel drug 
candidates to demonstrate the full utilization of their myriad of biological activities but also to adopt 
effective and sustainable measures to conserve the genus.  
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1.5 Justification 
Aframomum atewae Lock & Hall is a less ordinary species of the genus. It is a herb that grows up to 
about 1.90 m high and has a white flower with indehiscent fruits14. It was first identified at the Atewa 
Forest Range in the Eastern Region of Ghana and was thought to be endemic to that locality. 
However, in follow-up surveys, it was found in several wetter forests in the country and in one 
location in Cote d’Ivoire7. It is known as ‘sensam’ in Ghana, where the rhizome is boiled and drunk 
for constipation5.  
Several plants can resist infections by fungi present in their environments hence such plants can serve 
as a source of antifungal compounds. Recently, Aframomum plants are being exploited as sources of 
biodegradable fungicides in the control of several pathogenic fungi5. 
A thorough literature survey on Scifinder, PubChem, Chemspider, and other search engines revealed 
no documented research information on the species. Against the backdrop of risk of extinction and 
the need for systematic and targeted research on especially the less common species to unravel their 
medicinal potential, the current project focuses on the chemical and biological study of the leaves and 
rhizomes of A. atewae. The investigation was conducted as part of a South Africa-Ghana flagship 
project aimed at identifying gaps in the chemistry and ethnobotany in indigenous selected Ghanaian 
and South African taxa of the Zingiberaceae. 
1.6 Hypothesis 
Aframomum atewae may be a source of novel chemical constituents with potent biological activities.  
1.7 Aim 
To carry out a chemical examination of the leaves and rhizomes of A. atewae, evaluate the antifungal 
potential of the essential oils.  
1.8 Objectives 
1. To identify and collect samples of A. atewae from the Atewa Forest Reserve in the Eastern 
Region of Ghana. 
2. To hydro distil essential oils from the fresh leaves and rhizomes. 
3. To prepare nonpolar and polar extracts of the air-dried pulverized rhizomes. 
4.  To separate the constituents of the different extracts by chromatographic techniques. 
5. To carry out GCMS analysis on the essential oils.  
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6. To elucidate the isolated compounds by means of spectroscopic (IR, NMR) and spectrometric 
methods (MS).  
 
7. To screen the essential oils for antifungal activity against Saccharomyces cerevisiae and 
Candida albicans. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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CHAPTER TWO 
2.0 LITERATURE REVIEW 
2.1 Family Zingiberaceae 
Zingiberaceae (Ginger family) is a family of flowering plants with nearly 50 genera and 1600 known 
species that are found mainly in the tropical parts of the world3. Members of the family are mainly 
used as a spice, ornamental or medicinal plants. Examples of genera that are used as ornamentals are 
Alpinia, Globba, Hedychium and Renealmia while the Amomum, Aframomum, Myoga and Zingiber 
are used as spices. Essential oils obtained from Alpinia and Hedychium are used in the perfume 
industry11. The species that are endemic to Africa are Aframomum and Zingiber. 
Members of the family are perennial, terrestrial and aromatic herbs with rhizomes which have roots 
on them. Their stems are short and are replaced by leaf sheaths which form pseudostems. Their leaves 
are distichous and simple with leaf blade lanceolate to narrowly strap shaped. Flowers are bisexual 
with tubular calyx and corolla. Stamens are in two whorls with inferior ovaries and placentation is 
parietal, basal or axial. The style is found at the apex of the ovary and the fruits are capsule, fleshy 
and sometimes berrylike with few to many seeds12.  
Some members of the family are used in the production of dyes, as well as spices, perfume and 
medicine. 
 
2.2 Genus Aframomum 
Aframomum represents the African Amomum. Aframomum species are dispersed across tropical 
Africa and some islands in the Indian Ocean (Madagascar, Mauritius and Seychelles). While some 
species are found in different countries, for example, A. danielli, other species such as, A. 
longiligulatum is only found in Cameroon whereas the waterlogged forest of DR Congo River Basin 
is overshadowed by A. pseudostipulare.   The genus consists of about 55 species and is one of the 
biggest genera of the family12. They are found in old fields and along roads with most species 
normally found in light opening and forest borders. They are perennial and aromatic when part is 
crushed. Their flowers are brightly coloured and have peduncles which are covered with sterile 
overlapping bracts5. Most species are used as toothache, laxatives and stomachache reliever, 
antidiarrhoea, antihelmintic, fever management, anti-inflammatory, postpartum hemorrhage 
management and tonic for sexual stimulation13. Also, there have been reports of anticancer, antiulcer, 
antimicrobial, antiplasmodial and hepatoprotective activities of certain species14.  
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Several compounds have been isolated from many species since 1970 with their chemistry and 
biological activities reported5, 15. The types of compounds that have been isolated from several species 
include diterpenoids, sesquiterpenoids, arylalkanoids and flavonoids. About 12 species including A. 
melegueta, A. sulcatum, A. danielli, A. giganteum have extensively been studied chemically with 
labdane diterpenoids and flavonoids isolated from almost all these species16, 17. The less common 
species includes A. atewae, A. chrysanthum, A. longiligulatum, A. longiscapum, A. sebericum, and A. 
strabica.  In Ghana, species such as A. atewae (Atewa forest range), A. chrysanthum (Atewa forest 
range), A. stanfieldii (Atewa forest range), A. melegueta, A. longiscapum, A. cordifolium (Bia forest 
reserve), A sulcatum, A strobilaceum (Ankasa forest reserve), and A. geoscapum are distributed in 
various part of the country.  
 
2.3 Chemosystematics of Aframomum species 
Chemosystematics is a way to group organisms, based on demonstrable similarities and differences 
through their biochemical compositions15. Basically, it is known that plants belonging to the equal 
family typically produce similar classes of compounds owing to the existence of comparable types of 
enzymes and thus comparable biosynthetic pathways16. The Aframomum species, as well as other 
members of the family Zingiberaceae are best known to produce flavonoids and labdane diterpenoids. 
Other classes of compounds comprise sesquiterpenoids and arylalkanoids5. From the twelve most 
chemically studied species of Aframomum, no less than eleven of them contain diterpenoids19. 
Flavonoids and labdane diterpenes may epitomize as the chemotaxonomic marker of the genus 
Aframomum. Nevertheless, there have been a few reports of sesquiterpenoids in some species of 
Aframomum from which includes A. arundinaceum is part of the 20,21.  
 
2.4 Pharmacology of Aframomum Extracts 
2.4.1 Anticancer activity 
The methanolic extracts of A. arundinaceum seeds showed milled cytotoxicity against glioblastoma 
U87MGDEGFR and leukaemia CEM/ ADR5000 cells contrasted their corresponding sensitive 
equals U87MG and CEM/CEM cell lines22. A. melegueta chloroform and methanolic seed extracts 
demonstrate cytotoxicity when tested on PANC-1 pancreatic cancer cells in vitro with IC50 equal 50 
47.8 µg/mL and 13.8 µg/mL, respectively23.  
 
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2.4.2 Antimicrobial activity  
The genus Aframomum is reputed to possess antibacterial activities, due to presence of terpenoids 
such as aframodial [7]27. The hexane seed extract of A. sceptrum was reported to demonstrate the 
uppermost percentage inhibition of 60.26% against Hypocrea lixii (IMI 501885) whereas the 
ethanolic extracts have exhibited a percentage inhibition of 52.73% against Fusarium oxysporum f. 
sp. elaeidis. The methanol and acetone seed extracts showed the least percentage inhibitions of 
42.31% against H. lixii and 42.45% against F. oxysporum f. sp. elaeidis, respectively 28. A. melegueta 
has been reported to inhibit Bacillus cereus with an MIC of 31.25 mg/mL and was recommended to 
help in ensuring food safety29.  
 
2.4.3 Antiestrogenic activity 
El-Halawany and Hattori (2012) reported that 100 µg/mL of A. melegueta (methanolic seeds extracts) 
inhibits 56.7 ± 3.4% of estrogenic activity in a yeast assay. Though A. melegueta outperformed other 
herbs in the study, its activity was low compared to the control, tamoxifen (78% inhibition at 10 
µM)27.  
 
2.4.4 Penile erection function  
Kamtchouing et al. 2002, reported that when 115 mg/kg of A. melegueta (aqueous seeds extract) is 
administered for eight days, it causes growth in the penile erection, genital sniffing and occurrence 
of genital grooming, and a rise in mounting frequency by 54% in male rats. There was an increase of 
60% in ejaculation latency, whiles a decrease of 32% in intromission latency was observed, and while 
all these were greater than the control, they were less compared to the activity of P. guineense28. The 
same was detected to increase the secretions of epididymis and seminal vesicle, which are accessory 
sex organs29. 
 
2.5 Ethnobotanical use and Pharmacological action of selected species of Aframomum 
2.5.1 A. melegueta K. Schum 
The leaves, fruits and seeds of A. melegueta are normally used as spices in several food delicacies in 
Africa. Extracts of these plant parts are employed extensively in remedies against diabetes mellitus. 
In Nigeria, the fruit concoction (mostly done in alcoholic solution) is traditionally used in the curing 
of diabetes mellitus30. Pap containing African giant snail and A. melegueta seed is also employed in 
the treatment of diabetes mellitus. Additionally, a mixture made from A. melegueta leaf, pawpaw root 
and Allium cepa dried leaf is applied to cure diabetes mellitus and other metabolic sickness in 
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Nigeria31. A report by Sugita et al (2013) stated that alcoholic seed extract of A. melegueta rouses 
brown adipose tissue and surges body energy outflow in human subjects which is linked with the 
pathogenesis of type two diabetics (T2D)32. In other studies, A. melegueta extracts (fruit, leaf and 
stem) of different solvents established anti-diabetic and anti-oxidative potentials in vitro33-35.   
 
2.5.2 A. danielli (Hook. f.) K. Schum 
A. danielli is used in flavoring traditional Nigerian dishes, relieving ‘postpartum’ pain 36 and as an 
anti-inflammatory agent by rubbing of the alcohol extracts on allergic and eczematous swellings37. It 
also impedes the growth of several bacteria and fungi and relieves thirst during fever38. A. danielli 
has been observed to inhibit the growth of Aspergillus flavus, A. ochraceus, A. parasiticus, 
Salmonella enteriditis and Staphylococcus aureus39. A. danielli have also been reported to prevent 
the growth of Listeria monocytogenes in a dose-dependent manner40.  
 
2.5.3 A. citratum (C. Pereira) K. Schum 
A. citratum is used locally for the curing of bacterial infections, malaria, cancers and as an 
aphrodisiac41. Its leafy stem is used as a steam-bath against fever and intercostal pain while the seed 
is masticated and used as a tonic42. In 2011, it was reported that the methanol extract of the back of 
A. citratum is significantly active against several strains of E. aerogenes, E. coli, K. pneumoniae, E. 
cloacae, P. aeruginosa and P. stuartii with an MIC range of 256 - 1024 μg/mL43.  
 
2.5.4 A. sulcatum (Oliv. & Hanb.) K. Schum 
A decoction of the seeds of A. sulcatum is used against umbilical hernia and as a purgative42. It is 
also used traditionally to treat fevers and widely used as anthelmintic in Cameroon41.  
 
2.5.5 A. kayserianum K. Schum 
A. kayserianum is used in local medicine as a vermifuge, an anti-mumps and for menstrual cramps45. 
The methanol fraction was reported to have activity against E. coli AG100, ATCC 10536, W3110 
and AG100 Atet; P. stuartii ATCC 29914, K. pneumoniae K24 and E. aerogenes EA289 (MIC of 64 
μg/mL)44. Nonpolar acetone extract from the plant was stated to be the potent against various 
pathogens, such as Schizosaccharomyces pombe, Saccharomyces cerevisiae, Sclerotinia libertiana, 
Candida utilis, Hansenula anomala, Penicillium crustosum, Rhizopus chinensis, Mucor mucedo, B. 
Subtilis, Aspergillus niger, S. aureus, P. aeruginosa and E. coli46.  
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2.6 Chemical components of Aframomum and their biological activities. 
The Aframomum species are known to produce terpenoids, arylalkanoids and flavonoids. 
2.6.1 Terpenoids 
Terpenoids are naturally occurring compounds that occur as functionalized terpenes. Their 
functionalization is usually as a result of the presence of oxygen.  Most are multicyclic structures 
which obey the isoprene rule. About 60% of known secondary metabolites are terpenoids47. 
Terpenoids consist of hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), 
diterpenoids (C20), sesterterpenoids (C25), triterpenoids (C30) and tetraterpenoids (C40).   
 
The leaves and rhizomes of many Aframomum species are rich in diterpenoids and relatively few 
sesquiterpenoids. The essential oils however are reported to contain monoterpenes and 
sesquiterpenes. The main constituents of the essential oil from the flower of A. danielli are α-terpineol 
[1] (21.2%), 1,8-cineole [2] (18.6%), caryophyllene oxide [3] (14.4%), β-caryophyllene [4] (11.2%), 
l-linalool [5] (6.1%), and α-humulene [6] (5.6%)36. The monoterpenes of A. danielli have been 
reported to show antimicrobial potency on some microorganisms. For example, α-terpineol is said to 
reduce the population of Candida tropicalis39. Additionally, (+)-limonene, also found in the essential 
oil of the plant has a MIC of 78 μg/mL against A. parasiticus. The essential oils from the leaf and 
flower of A. danielli have antibrowning effect when used for cereal preservation47.  
From A. citratum, the seed essential oil contains compounds such as -thujene [7], camphene [8], 
phellandrene [9], terpinen-4-ol [10], myrtenol [11], 6-methyl-5-hepten-2-one [12] and trans-sabinyl 
acetate6 [13]41. (Figure 2.1).   
 
Figure 2.1: Some essential oil constituents of Aframomum species 
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A few sesquiterpenes have been isolated from some species of Aframomum, these are ()-S-nerolidol 
[14] isolated from A. sceptrum and A. escapum, (-)-−bisabolol [15] and 6,7-epoxy-3(15)-
caryophyllene [16] found in A. arundinaceum, 49-51 (Figure 2.2).  
 
 
Figure 2.2: Sesquiterpenes from some species of Aframomum 
 
Diterpenes containing acid and aldehyde functionalities have also been reported in several species of 
Aframomum including A. danielli, A. melegueta and A. sulcatum. They include aframodial [17], 8β-
17-epoxy-12E-labdene-15,16-dial [18], methyl-14,15-epoxy-8(17),12(E)-labdadiene-16-oate [19] 
and 8β(17)-epoxy-15,15-dimethoxylabd-12(E)-en-16-al [20]. Aframodial [17] was first isolated from 
A. danielli and it is known to be cytotoxic32, the same compound from the seed of A. sulcatum was 
tested to be antihypercholesterolemic5. The aldehyde, 8-17-epoxy-12E-labdene-15,16-dial [18] was 
reported to exhibit strong antifungal activity against Candida albicans48,49. Other diterpenoids include 
aulacocarpinolide [21], aulacocarpin A [22] and aulacocarpin B [23] from A. aulacocarpos. Also, 
galanolactone [24], sulcanal [25], galanal A [26], galanal B [27] and 11,15-epoxy-15-hydroxy-
8(17),12-labdandien-16-al [28] have been isolated from A. sulcatum. Galanolactone, galanal A and 
B have been demonstrated to possess antifungal, cytotoxic and antiplasmodial activities52.  Also, 
galanal B shows activity towards breast adenocarcinoma MDA-MB-231/BCRP cells but are less 
active towards resistant cancer cells19. There have also been report of moderate cytotoxicity of 
galanals A and B with IC50 of 18 µM and 32 µM, respectively to human T lymphoma Jurkat cells has 
also been reported55 (Figure 2.3). 
 
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Figure 2.3: Diterpenes from some species of Aframomum  
 
Diterpenoids and sesquiterpenoids are known to show activity against fungi, bacteria, protozoa and 
virus. 1(10)E,5(E)-germacradien-4-ol, 5E,10(14)-germacradien-1 ,4-diol and sesquiterpenoids 
oplodiol with respective IC50 values of and 1.54, 1.63 4.17 µM are some active metabolites that have 
been isolated from Aframomum species53,54.  
2.6.2 Flavonoids  
Flavonoids are metabolites in plants and are accountable for the most important plant pigments for 
flower coloration. Antioxidant activity of plant extracts tends to increase when there are high 
concentrations of flavonoids and phenolics present and some vital structural feature, for instance, the 
arrangement and the number of hydroxyl groups, the presence of electron-donating/accepting 
substituents on the ring and the extent of structural conjugation of the ring58. Due to their 
effectiveness, inhibition of several oxidative trauma linked ailments such as cancer, plant phenols 
have gained increasing attention5. Flavonoids are hydroxylated phenolic compounds that have 
exhibited good potential in the stoppage of cardiovascular disease and potent against several microbes 
such as polio type 1 and Coxsackie B4 viruses56,57. Compounds such as paradol [29], shogaol [30] 
and gingerol [31] identified in A. melegueta EtOH seed extract have been reported to exhibit toxicity 
after a 4-week treatment in rats against diabetes59. In preliminary studies, seed and leaf aqueous 
extracts of A. melegueta have exhibited blood glucose dropping activity in alloxan diabetic rats60,61. 
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Kaempferol [32] and quercetin [33] isolated from A. giganteum by Vidari et al. 1971, were reported 
to show antibacterial activities62. They also demonstrate potent anti-inflammatory and antiviral 
activities. Further to that, kaempferol and quercetin prevents the release of histamine in rat mast cell, 
and are also good radical scavengers63,64. Methylated derivatives of quercetin were reported to show 
activity against Coxsackie B4 viruses and polio type 1, both in vivo and in vitro 44 (Figure 2.4). 
 
 
Figure 2.4: Flavonoids from some species of Aframomum  
 
2.6.3 Arylalkanoids 
Diarylheptanoids are groups of secondary metabolites which bear a 1,7-diphenylheptane skeleton as 
a unique character5. Haining and She (2012) grouped them into linear and cyclic diarylheptanoids65.   
Diarylheptanoids shows wide bioactivity such as estrogenic, anti-inflammatory, antioxidant, 
leishmanicidal, antihepatoxic, neuroprotective, melanogenesis, antitumour, antibacterial and recently 
a report on their inhibitory potency against NO production in activated murine macrophages66.  
 Tane et al, 2005 isolated (4Z,6E)-5-hydroxy-1,7-bis(4-hydroxyphenyl)hepta-4,6-dien-3-one [34], 
letestuianin A [35], B [36] and C [37] from the seed of  A. letestuianum (Figure 2.5).  
 
 
Figure 2.5: Arylalkanoids from some species of Aframomum 
A summary of compounds that have been isolated from various species of Aframomum with their 
biological activity is presented in Table 2.1 below. 
 
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Table 2.1: Summary of Aframomum species, isolated compounds and their biological activities. 
SPECIES COMPOUNDS BIOLOGICAL ACTIVITY 
Arylalkanoids 
A. letestuianum (4Z,6E)-5-hydroxy-1,7-bis-(4- Anti-inflammatory 
hydroxyphenyl)hepta-4,6-dien-3-one [32]  Antihepatoxic 
Letestuianin A [33]  Antifungal,  
Letestuianin B [34] Antibacterial 
Letestuianin C [35] Antitumour 
A. melegueta Gingerdione  Termite antifeedant 
6-Paradol [31] Antibacterial 
6-Shogaol [30] Antifungal 
trihydroxyphenyl)heptanes  
3-(s)-Acetyl-1–(49-hydroxy-3’,5’-
dimethoxyphenyl) -7-(3’’,4’’,5’’- 
 (4Z,6E)-5-Hydroxy-1,7-bis-(4-
hydroxyphenyl)hepta-4,6-dien-3-one 
6 -Gingerol [29]  
Flavonoids/phenols 
A. handburyii,  3,5,7,4-Tetrahydroflavone  
A. sceptrum 
A. pruinosum 3-Acetoxy-5,4-dihydroxy-4-  
methoxyflavone 
3,5-Dihydroxy-7,4-dimethoxyflavone 
A. letestuianum 3-Acetoxy-5,4-dihydroxy-7-  
A. pruinosum methoxyflavone 
A. handburyii 
A. sceptrum 3-Acetoxy-5,7,4-trihydroxyflavone  
A. pruinosum 
A. handburyii 
A. letestuianum 
A. giganteum kaempferol-3,7,4-trimethylether  Antibacterial, Antiviral, 
Quercetin-3,7,30,4-tetramethylether  Anti-inflammatory 
Quercetin-3,7,4-trimethylether  
Terpenoids 
A. arundinaceum 6,7-Epoxy-3(15)-caryophyllene [30] Antiplasmodial 
(-)--Bisabolol  
A. sceptrum (+)-S-Nerolidol   
A. escapum 
A. zambesiacum 5E,10(14)-germacradien-1,4-diol  
A. longifolius Hemiacetal 15-hydroxy-15- methoxylabda-  
8(17),12(E)-dien-16-al 
A. arundinaceum Methyl 14,15-epoxy-8(17), 12(E)-  
A. danielli labdadiene-16-oate [9] 
Acetal 8(17)-epoxy-15,15-dimethoxylabd-
12(E)-en-16-al 
A. latifolium  Galanals A [26] Antiplasmodial 
 Galanals B [27]  
Narigenin 
1E,5E-germacradien-4-ol 
Norbislabdane sulcanal 
12(E),8(17)-Epoxy-11-hydroxy-12-labden-
15,16-dial-11,15-hemiacetal 
A. arundinaceum Galanolactone Antifungal, cytotoxic 
A. sulcatum 
 
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A. sceptrum 8(17)-Epoxy-3 ,7-dihydroxylabde- Antiplasmodial 
12(E)-en-16,15-olide Tripanosomial 
Methyl 8  (17)-epoxy-3 ,7 ,15-
trihydroxylabd-12(E)-en-16-oate 
3 ,7 ,8 ,12,17-pentahydroxylabdan-
16,15-olide 
Coronarin B 
A. sceptrum Labda-8(17),12-dien-15,16-dial Antifungal, Cytotoxic 
A. longifolius Antiplasmodial 
A. danielli  
A. aulacocarpos Aulacocarpinolide  Antibacterial 
Aulacocarpin A  Antiplasmodial 
Aulacocarpin B  
A. polyanthum Aframodial [14] Cytotoxic 
A. keysenanum  
A. masuianum Antihypercholesterolemic 
A. arundinaeum  
A. sulcatum 
A. longifolius 
A . latifolium 
 
2.7 Fungal infections 
Infectious diseases are caused by microorganism. Infectious diseases are one of the principal causes 
of illness and death in the emerging countries 67. 
Fungal infections are very common in plants and humans. Some fungal infections do not threaten life 
but distress the value of life while invasive fungal infections (IFIs) are life-threatening. Serious fungal 
infections (SFIs), including significant chronic infections, complicated mucocutaneous infections and 
IFIs influence the quality of life67. It has been reported that 4% of Ghanaians are plagued from SFIs 
annually, with over 35,000 affected by life-threatening IFIs. 
The common symptoms of fungal infections include red and possibly cracking, peeling skin or 
itching. Examples of infectious fungal diseases in human include Tinea pedis, ringworm, yeast 
infection (candida), etc.  Candida albicans is the dominant causative organism for fungal infections 
such as oropharyngeal candidiasis67. 
 
In plants, fungal infections can lead to loss of vegetation which can result in huge economic losses 
and loss of food for consumptions. There are several plant infectious diseases caused by fungi in 
Africa and the world.  
 
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2.7.1 Control 
Even though there is a consensus about the best strategy to control fungal disease, prevention and 
control strategies are based on improving one’s lifestyle and being educated on the extent and effect 
of some fungi found in our surroundings. 
 
2.7.2 Treatment of fungal infections 
Treatment of fungal infections depends on their harshness. Normal preventions include tablets, 
creams, or suppositories, which are obtained via prescription. In Ghana, plant-based creams such as 
mercy cream, joy ointment, etc. are used for fungal infections such as ringworm and vaginal 
candidiasis. Also, antifungal treatment drugs such as fluconazole [38], itraconazole [39], topical 
miconazole [40], topical nystatin [41], griseofulvin [42], and terbinafine [43] (Figure 2.6) are 
prescribed upon diagnosis67.  
 
 
Figure 2.6: Examples of clinically used antifungal agents 
 
2.7.3 Natural products as antifungal agents 
Though there are several synthetic fungicides that are used on several plants, these molecules persist 
in the environment. Hence, they pose several risks to plants, individuals and the environment. 
Persistent use of synthetic fungicides can cause phytotoxicity among plants, death of aquatic animals, 
as well as irritation of skin/eyes and respiratory problems of individuals who handle them68. These 
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and other reasons are why there is the need to research into natural products as a source of fungicides 
due to their biodegradability.  
 
A variety of compounds with antifungal activity against several strains of fungi have been isolated 
from plants and are vital to humans in the prevention of diseases. These compounds can be used 
directly or as a forerunner for developing better molecules68.   
 
3-hydroxy-4-geranyl-5-methoxybiphenyl [44], obtained from the fruits Garcinia mangostana was 
reported to have strong antifungal activity69. Steroidal saponins isolated from the roots of Smilax 
medica, showed antifungal activity when tested against the human pathogenic yeasts C. tropicalis, C. 
albicans, and C. glabrata in the range of 25 – 50 mg/mL70. Phytolaccosides B and E [45] from 
Phytolacca tetramera exhibited antifungal activities on a panel of human pathogenic opportunistic 
fungi71. 2’,4’-dihydroxychalcone [46] and 2’,4’-Dihydroxy-3’-methoxychalcone [47] isolated from 
the DCM extract of Zuccagnia punetata ecchibited minimal antifungal activities against the yeasts 
C. albicans, C. neoformans and S. cerevisiae having MIC values of 62.5–250 mg/mL72.  
Antifungal activities of several compounds that have been isolated from Aframomum have been 
examined. Examples include galanolactone, labda-8(17),12-dien-15,16-dial, aframodial, 6-gingerol, 
as well as reports on antifungal activities of essential oils obtained from A. melegueta and A danielli5. 
Due to many terpenes found in Aframomum species, their antifungal activity has been attributed to 
these compounds5. 
 
 
 
Figure 2.7: Natural products with antifungal activity 
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CHAPTER THREE 
3.0 METHODOLOGY 
3.1 Plant collection 
Samples of Aframomum atewae (Figures 3.1a and 3.1b) were collected from the Atewa range Forest 
Reserve, Sekyemase in the Eastern region of Ghana (Figure 3.1c) on Saturday, October 20, 2018. It 
was identified by Mr. Jonathan Dabo of Ghana Forestry Commission. Its white flower, with a purple 
tinge, and glabrous leaves distinguished it from A. chrysanthum which is more abundant at lower 
altitudes of the range. A. chrysanthum bears yellow flowers and the leaves are slightly rough on both 
sides.  A voucher specimen of A. atewae (DJ2018-29) has been kept at the Forestry Research Institute 
of Ghana, Centre for Scientific and Industrial Research (CSIR-FORIG) Herbarium, Kumasi.  
 
 
Figure 3.1a: Flower of Aframomum atewae 
 
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Figure 3.2b: Leaf and rhizome of Aframomum atewae 
 
 
Figure 3.1c: Map showing site of sampling of A. atewae 
 
3.2 General experimental methods 
Essential oils (EO) were hydrodistilled for 12 hours from fresh rhizomes and leaves using the 
Clevenger apparatus. Other constituents were obtained by successive exhaustive percolation of the 
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air-dried pulverized rhizome using petroleum ether (PE), dichloromethane (DCM) and methanol 
(MeOH). Each extraction was done at least four times at 24 hours interval. The extracts after each 
cycle were filtered and concentrated to dryness under reduced pressure on G3 Heidolph Rotary 
Vacuum Evaporator. The recovered solvent was passed through magnesium sulfate (MgSO4) to get 
rid any traces of water present in the recovered solvent before it was reused for the next extraction. 
Each extract obtained was column chromatographed (CC) using silica gel 60 (sigma – aldrich; 130-
270 mesh) as the stationary phase. Elution was done with PE and the polarity of the mobile phase 
increased gradually using ethyl acetate (EtOAc). Thin layer chromatography (TLC) using aluminum 
foil pre-coated with silica gel (0.2 mm thick kieselgel 60F254 Merck type) was employed to check the 
progress of the column chromatography. 
Visualization of TLC spots was done by ultraviolet (UV) light (Spectroline model ENF-240C/ FE 
UV lamp; 254-365 nm), acid stain and anisaldehyde spray reagent. The melting point of pure isolates 
was determined with Stuart Scientific SMP 10 melting point apparatus. Isolated compounds were 
analyzed by their appearance and retardation factor (RF) on TLC in different solvent systems. Nuclear 
Magnetic Resonance (NMR) spectroscopy 1D and 2D data were acquired on a Brüker Ascend 500 
MHz spectrometer at the Department of Chemistry, University of Ghana. The internal reference used 
was Tetramethylsilane (TMS) and CDCl3 was used as a solvent to obtain spectra of samples. Infrared 
(IR) spectra were obtained on a Perkin-Elmer FTIR spectrometer by means of Attenuated Total 
Reflectance which allows the sample to be analyzed directly in the solid-state without further 
preparation. The masses of the compounds were determined using either LC-MS or HR-MS. 
LC separations were carried out on Kinetex Core C18 column (2.6 μM, 3 x 50 mm, 100Å) maintained 
at 40 °C with 10 mM NH4OAc in 90% CH3OH in H2O. HR-MS data was acquired after separation 
by LC (AQUITY UPLC H-class Waters Corporation) coupled to a SYNAPT G2-Si High Definition 
mass detector. Column type was AQUITY UPLC BEH C18 (1.7 m, 2.1 x 50 mm) employing a flow 
rate was 0.5 mL/min. The MS was operated under electrospray ionization (ESI) and Atmospheric 
pressure chemical ionization (APCI). 
The constituents of the essential oils were characterized using GC-MS. The separation was achieved 
on a gas chromatograph (6890N, Agilent technologies network) fixed to an Agilent technologies inert 
XL EI/CI Mass Selective Detector (MSD) (5975B, Agilent Technologies Inc., Palo Alto, CA). 
Separation of essential oil volatiles was done on a polar STABILWAX (60 mm, 0.25 mm ID, 0.25 
µm film thickness) capillary column. The carrier gas used was helium at a flow rate of 2 mL/min. 
The injector temperature was maintained at 240 °C. One L of the samples was injected in splitless 
mode. The oven temperature was programmed as follows: 45 °C for 3 min and ramped up to 250 °C 
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at a rate of 6 °C/min held for 5 minutes. The MSD (Mass Spectrometric Detector) was operated in a 
full scan mode and the source and quad temperatures were maintained at 230 °C and 150 °C, 
respectively. The transfer line temperature was maintained at 250 °C. The mass spectrometer was 
operated under electron impact mode at ionization energy of 70 eV, scanning from 25 to 450 m/z. 
The constituents were characterized by their mass spectra. The mass spectra were compared with 
Wiley mass spectral library.  
The antifungal activity of the EO was evaluated against the common Saccharomyces cerevisiae and 
Candida albicans, utilizing an Alamar Blue-based broth dilution assay.  
 
3.3 Chemicals and Reagents 
3.3.1 Anisaldehyde spray reagent 
About 5 mL of conc. H2SO4 was added to 135 mL of absolute ethanol. Glacial acetic acid (1.5 mL) 
and 3.7 mL p-anisaldehyde were added to the solution and shaken dynamically and cooled. It was 
then preserved in an amber bottle and placed in a fridge. The sprayed TLC plates are heated at 110 
oC for about 5 minutes. 
 
3.3.2 Acid spray 
About 10 mL of H2SO4 was dissolved in 90 mL of absolute ethanol. The sprayed TLC plates are 
heated at 110 oC for about 5 minutes. 
 
3.3.3 Wagner’s Reagent 
A mass of 2.00 g of potassium iodide (KI) and 1.28 g of iodine (I2) were dissolved in a minimum 
amount of water in a 100 mL volumetric flask and the solution was shaken and topped up to 100 mL. 
The formation of a brown precipitate (ppt) when a few drops of the reagent is added to an acidified 
test solution is a sign of the presence of alkaloids. 
 
3.3.4 Mayer’s Reagent 
Mercuric iodide (HgI2) of mass 1.36 g in 60 mL of deionized water was added to a 10 mL solution 
of 5.01 g of potassium iodide (KI) in a 100 mL flask and topped up to 100 mL. The formation of a 
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yellow ppt when a few drops of this reagent is added to a test mixture is diagnostic of the presence of 
alkaloids. 
 
3.3.5 Dragendoff’s Reagent 
Hydrated bismuth nitrate (BiNO3.H2O) of mass 8.04 g was liquified in 20 mL of concentrated nitric 
acid and the resulting mixture was added slowly and gently to a mixture of 27.23 g of KI in 50 mL 
of deionized water while stirring. The mixture was left to stand and then filtered. The filtrate was 
transferred to a 100 mL volumetric flask and filled to the 100 mL mark. Two or three drops of this 
reagent when added to an acidified test solution containing alkaloids results in the formation of a 
reddish-brown ppt. 
 
3.3.6 Iron (III) chloride solution 
A volume of 8.5 mL of FeCl3 was measured into a 100 mL flask and filled to the mark with deionized 
water. The formation of green colour indicates the presence of tannins and phenolic compounds when 
two to three drops of the solution are added to about 3 mL of a test solution. 
 
3.3.7 Liebermann-Burchard Reagent 
Acetic anhydride of volume 50.0 mL and 5.0 mL of conc. H2SO4 were carefully mixed in the fume 
chamber while cooling with ice. The mixture was added gently and carefully to 50 mL of absolute 
EtOH while cooling in ice. The presence of terpenoids in an extract is indicated by the observance of 
pink or red spots when this reagent is sprayed onto a TLC plate after development followed by heating 
in an oven at a temperature of 110 °C for 15 minutes and then examined under a UV lamp. 
 
3.4 Essential oil distillation of rhizomes and leaves  
About 780 g and 360 g of the fresh rhizomes and leaves, respectively were used for the essential oil 
distillation. Both plant parts were cut into pieces and placed in a round bottomed flask. A substantial 
amount of water was added to the material in the round bottomed flask and was placed on a heating 
mantel. The Clevenger apparatus was fixed on the round bottomed flask, clamped for support and 
connected to a water recirculating chiller. The distillation was done for 12 hours and the essential oils 
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that were produced were pipetted using a Pasteur pipette. The essential oils were dried using 
anhydrous MgSO4.  
Approximately 1 mL of DCM was added to the samples, sonicated overnight and analyzed by GC-
MS. 
 
3.5 Antifungal activity 
The fungicidal and static effects of the EO were determined with the metabolic fluorescent sensor 
Alamar Blue (resazurin) that measures residual metabolic activity to determine cell viability. Alamar 
Blue, a blue non-fluorescent dye, is reduced to the pink-colored, highly fluorescent resorufin with 
metabolic activity of the fungal cells. In the presence of antifungal agents, the fluorescence signal 
yields are significantly reduced, suggesting a reduction in metabolic activity.   
 
Stock solutions of the essential oils were prepared by carefully weighing about 1 μg of each oil and 
dissolving it in 100 µL of hexane. Approximately 25 µL of each stock solution was loaded into a 96-
well plate and allowed to dry. Ninety µL of nutrient broth (NB) was then added to the plate followed 
by addition of 10 µL of S. cerevisiae and C. albicans cells to separate wells. The plates were incubated 
at 30 °C for 24 hours, after which each well was stained with Alamar Blue and observed for 
fluorescence intensity mode (excitation = 525 nm, emission = 598 nm) on a VarioskanTM LUX 
microplate reader.  
 
The negative control consisted of 100 µL of NB only, with neither cells nor essential oil in the 96 
well-plates, whereas the positive control contained 90 µL of NB + 10 µL of the cells without the 
essential oil.  Test solutions were determined to be fungistatic when the fluorescent intensity was 
50% or less than that of the positive control. On the hand, when the observed fluorescence was less 
than that of the negative control, the test solution was deemed to be fungicidal to the cells. An 
observed fluorescent intensity that was greater than that of the positive control implied no antifungal 
activity.   
 
3.6 Solvent extraction of rhizomes 
The air-dried pulverized rhizome of the plant (1.86 kg) was extracted successively with PE, DCM 
and MeOH by cold percolation to get the corresponding crude extracts. The TLC profile of the MeOH 
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crude extract indicated many unresolved spots. Therefore, to facilitate separation about 40 g of the 
extract was fractionated by flash chromatography with PE, EtOAc and EtOH (Scheme 3.1). 
 
 
Scheme 3.1:  Preparations of extracts and fractions of A. atewae 
 
3.7 Phytochemical screening procedures 
Approximately 0.2 g of the PE, DCM and MeOH extracts were weighed into a beaker and dissolved 
in 80% EtOH for phytochemical screening. 
 
3.7.1 Keller-Kiliani Test for Cardiac Glycosides 
About 2 mL of EtOH extract was measured into a test tube and 2 mL of glacial acetic acid was added. 
A drop of FeCl3 and then 1 mL of conc. H2SO4 were added cautiously down the wall of each test 
tube. Development of a brown ring indicates a positive of cardiac glycosides. 
 
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3.7.2 Test for Anthraquinones and Anthracene Derivatives 
About 5.0 mL EtOH extract was measured into a boiling tube. The tube was heated in a water bath 
for 10 min. The test solution was then transferred into a separatory funnel and shaken vigorously with 
4 mL of benzene and allowed to equilibrate. The benzene layer was treated with 1.5 mL of conc. NH3 
and allowed to stand. A positive test for anthraquinones and anthracene derivatives was confirmed 
by a red ppt in the NH3 layer. 
 
3.7.3 Test for Tannins 
About 2 mL of EtOH extract was measured into a test tube. Freshly prepared FeCl3 solution was 
added to the solution. The formation of a dark greenish colouration signposted the presence of 
tannins. 
 
3.7.4 Test for Saponins 
Deionized water was added to 0.01 g of the extract in a test tube and shaken vigorously. Formation 
of a persistent foam which disappeared after the solution was left to stand for a while confirmed the 
presence of saponins. 
 
3.7.5 Test for Terpenoids 
TLCs of the crude extracts were developed in 7:3 and 8:2 PE: EtOAc solvent systems. The plates 
were left to dry and then sprayed with Liebermann-Burchard reagent. They were then heated in an 
oven at 110 °C for 15 minutes and examined under a UV lamp. The presence of a pink spot indicated 
that terpenoids were present in all extracts. 
 
3.7.6 Test for alkaloids 
About 4 mL EtOH extract was measured into a boiling tube and 10 mL of 2 M HCl solution was 
added. The solution was warmed on a water bath and filtered. The filtrate was divided into three test 
tubes labelled A, B and C. To portion A, B and C Mayer’s reagent Dragendoff’s and Wagner’s reagent 
were added, respectively. The presence of a brown ppt confirmed the presence of alkaloid. 
 
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3.7.7 Test for Flavonoids 
About 2 mL of EtOH extract was measured into 3 test tubes; A, B and C. Test tube A was used as 
control. To test tube B, 3 pieces of boiling chips were added followed by 0.5 mL of conc. HCl and 
observed for any colour changes after warming. 0.5 mL of conc. HCl was added to test tube C and 
warmed for 5 minutes on a water bath. The change of colour in test tubes B and C indicated the 
presence of flavonoids. 
 
3.7.8 Test for steroids 
About 2 mL of acetic acid was added to 1 mL of the EtOH extract in a test tube. The solution was 
boiled and cooled, then drops of conc. H2SO4 were added on the wall of the test tube. A brown ring 
at a junction between the two layers indicated the presence of steroid. 
 
3.7.9 Test for polyphenols 
About 2 mL of the EtOH extract was measured into a test tube and 3 drops of 10% aqueous FeCl3 
and 3 drops of K4[Fe(CN)6] were added. Formation of a blueish colour indicated the presence of 
polyphenols. 
 
3.8 Investigation of the extracts  
3.8.1 Petroleum ether (40-60 °C) extract  
The petroleum ether extract was labelled as AA/PE. A total of 14.671 g was loaded onto a glass 
column pre-loaded with silica gel (180 g). The column was first eluted with 100% PE, then with PE 
and EtOAc mixtures until 100% EtOAc was used. The column was lastly washed with chloroform. 
Eluates were collected in 15 mL volumes and those with alike TLC profiles were added to give 6 
subfractions coded AA/PE-1 to AA/PE-6. AA/PE-1 was obtained as an essential oil and was analysed 
by GC-MS to identify its constituents. It was further tested for its antifungal activity against S. 
cerevisiae and C. albicans. The remaining fractions precipitated solids from which AA/PE-2, AA/PE-
5 and AA/PE-6 were characterized using NMR, IR spectroscopy and LC-MS. Due to the presence of 
impurities and paucity of solids from AA/PE-3 and AA/PE-4, no further analysis was performed on 
them. Scheme 3.2 present a summary of the work done on AA/PE. 
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Scheme 3.2: Separation of constituents of AA/PE  
 
3.8.2 Dichloromethane Extract  
The dichloromethane extract was labelled as AA/DCM. A total of 15.108 g was column 
chromatographed, eluting with PE and PE/EtOAc 10:1 and gradually increasing polarity to 100% 
EtOAc. The column was finally washed with EtOH. Combination of fractions with similar TLC 
profiles led to 5 subfractions coded AA/DCM-1 to AA/PE-5. AA/DCM-1 was obtained as essential 
oil which was subjected to GCMS analysis to determine its constituents. It was further tested for its 
antifungal activity against S. cerevisiae and C. albicans. The remaining fractions precipitated solids, 
with which AA/DCM-2 was characterized using NMR, IR spectroscopy and HR-MS.  From a 
comparative TLC and NMR of AA/DCM-4 with AA/PE -6 it was observed that the two precipitates 
were the same but were kept separate. Due to the presence purity and paucity of solids AA/DCM 3 
and AA/DCM 5, no further analysis was performed on them.  Scheme 3.3 presents the work done on 
the AA/DCM extract. 
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Scheme 3.3: Separation of constituents of the AA/DCM  
 
3.8.3. Methanol extract  
The methanol fraction was taken through flash chromatography due to difficulties in obtaining good 
separation when subjected to TLC with various solvents of different polarities. PE, PE:EtOAc 1:1, 
EtOAc, and EtOH were used for the fractionation. Based on a comparative TLC with isolates from 
previous extracts, it was observed that some previously isolated compounds were present in fractions 
PE and PE:EtOAc 1:1. Hence, the EtOAc fraction (2.053 g) was selected for column chromatographic 
separation whiles the EtOH was kept for future analysis. TLC showed a total of 5 spots under UV 
and when stained with anisaldehyde spray. About 2.005g of the extract was loaded on a pre-packed 
silica column. Elution of the column was done starting from 10% EtOAc in PE and polarity was 
increased to 100% EtOAc. This led to 10 fractions from which solids were obtained from fraction 5, 
7 and 9. However, the solids were not soluble in most deuterated solvents hence were not 
characterised. 
From phytochemical screening, it was observed that out of the 3 extracts tested only the MeOH 
extract confirmed the presence of alkaloid. Hence an attempt was made to isolate the alkaloids 
present.  
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Approximately 6.423 g of the extract was dissolved in 150 mL of 0.3 M tartaric acid solution. This 
was done to convert the alkaloids present into alkaloid salt which will dissolve in the aqueous layer. 
The aqueous solution of the extract was poured into a separatory funnel and extracted 3 times with 
EtOAc to free the extract of other class of compounds that could be present. The organic layer was 
obtained and concentrated to dryness while the aqueous layer was neutralized with 0.2 M Na2CO3 to 
convert the alkaloid salts into free alkaloids. It was also extracted 3 times with EtOAc to obtain a 
crude alkaloid extract (AK crude). 
About 1.876 g of AK crude was separated through column chromatography by eluting with PE and 
EtOAc to obtain 5 subfractions. Out of the 5 fractions, 3 precipitated solids AK-1, AK-3 and AK-4. 
Compound AK-1 was obtained as a white grain-like solid which dissolved partially in only EtOH. 
When subjected to TLC using EtOH: CHCl3 1:10 as the mobile phase, a blue spot was observed under 
UV. The remaining solids also dissolved partially in EtOH, but TLC analysis suggested that they 
were impure (due to the presence of multiple spots when observed under UV). Due to the small nature 
of these solids, no further analysis could be performed to characterize them. Scheme 3.4 below 
presents the work done in an attempt to isolate and characterize an alkaloid from the MeOH extract. 
 
Scheme 3.4: Alkaloid isolation  
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CHAPTER FOUR 
4.0 RESULTS AND DISCUSSION 
4.1 Summary of results 
The essential oils (EO) obtained by hydrodistillation of the fresh leaves and rhizomes of A. atewae 
were pale yellow in colour and had a pleasant ginger-like disposition.  Oil yields were 0.5% and 0.7%, 
respectively and consisted of monoterpenes, sesquiterpenes, a couple of diterpenes and some non-
terpenoid compounds as revealed by GC-MS analysis. The leaf and rhizome essential oils were 
significantly different in their composition. While 2,5-Di-tert-butylhydroxybenzene (7.80%) formed 
the major constituent of the rhizome EO, the leaf EO consisted mainly of 1-methyl-1-
(methylamino)isobenzofuran-3-one (17.3%).  The DCM extracted oil of the rhizome was also 
analyzed and 14-pregnane (58.44%) was the major constituent. The chemical composition of the 
PE-extracted oil is yet to be confirmed. All the four essential oils exhibited activity against C. albicans 
while only the PE-extracted oil was active against S. cerevisiae. 
On phytochemical analysis, all the three extracts tested positive for terpenoids. Steroids were also 
present in both the PE and DCM extracts. In addition, flavonoids were identified in the DCM and 
MeOH extracts while only the MeOH extract gave positive results for alkaloids. 
Chromatographic separation of the PE extract led to the isolation of 6 solids out of which 3 were 
characterized and identified as 1 (E)-8-methylundec-8-en-1-yl-3-(cyclohexa-2,5-dien-1-
yl)propanoate, stigmasterol and myristic acid. Out of 5 solids obtained from the DCM extract, only 
one was characterized as 2-(6-oxotetrahydro-2H-pyran-2-yl)ethyl dodec-8-enoate. Also, the EtOAc 
fraction of the MeOH extract gave 10 compounds but due to the poor solubility in various NMR 
solvents including DMSO, none of these compounds has yet been fully characterized. 
 
4.2 Chemical composition of the Essential Oils 
4.2.1 Chemical composition of AA-L 
A total of 119 constituents, representing 88.42% of the total peak area, were identified by GC-MS 
analysis of the leaf EO by comparing the mass spectra of the compounds to the Wiley mass spectral 
library (Figure 4.1 and Table 4.1). 
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Figure 4.1: Gas chromatogram of leaf essential oil of A. atewae  
Table 4.1: Retention time, name of constituent, percentage composition, molecular formula and weight of A. atewae leaf 
essential oil as shown by GCMS analysis  
Peak Retention Compound MF  Mwt. Composition 
No. time (min)  (g/mol) (%) 
1 10.754 α-Terpinene  C10H16 136.238 0.0367 
2 11.2363 l-Limonene C10H16 136.238 0.797 
3 11.5484 1,8-Cineole (Eucalyptol) C10H18O 154.249 0.0263 
4 11.6335 (2-Methylprop-1-enyl)-cyclohexa-1,5-diene C10H14 134.218 0.0633 
5 12.3569 γ-Terpinene  C10H16 136.238 0.0711 
6 12.7683 2-methyl-1,3-Dioxolane-2-pentanol C9H18O3 174.240 0.0255 
7 12.9527 p-Cymene  C10H14 134.218 0.5945 
8 13.2648 terpinolene C10H16 136.238 0.0745 
9 14.1159 2-Heptanol C7H16O 116.204 0.0713 
10 14.2152 1-Butylpyrrole C8H13N 123.196 0.8467 
11 14.3287 (Z)-7-Pentadecen-5-yne,  C15H26 206.370 0.0285 
12 14.6549 2-Pentyl-2-cyclopenten-1-one C10H16O 152.237 0.0179 
13 15.0663 Irid-2-ene  C10H18 138.250 0.0397 
14 15.4918 1,4-Dicyclohexylbutane C10H30 222.410 0.0249 
15 15.6195 2,4-Hexadiene C6H10 82.146 0.0265 
16 15.889 p-Mentha-1,5,8-triene C10H14 134.218 0.0337 
17 16.116 Bornyl chloride C10H17Cl 172.695 4.9307 
18 16.3429 2,3-dimethyl-1,2-pentadiene C7H12 92.170 0.0571 
19 16.6267 α-Campholene aldehyde C10H16O 152.233 0.6374 
20 16.7969 o-Allyltoluene C10H12 132.202 0.1582 
21 16.9529 Acetic acid C2H4O2 60.052 0.1247 
22 17.0948 β-Phellandrene C10H16 136.238 0.1894 
23 17.5203 4,5-Bis(hydroxymethyl)-3,6-dimethylcyclohexene C10H18O2 170.250 0.0502 
24 17.6338 Cis-Ocimene  C10H16 136.238 0.1232 
25 17.7473 Hydrocinnamaldehyde  C9H10O 134.175 0.025 
11-Endo- C12H16 160.250 
26 17.8891 methyltetracyclo[5.4.0.0(2,6).0(4,10)]undec-8-ene 0.0403 
27 18.3572 α-Pinene C10H16 136.238 0.1424 
28 18.57 Nonan-2-ol C9H20O 144.258 0.048 
29 18.6551 α-Terpinolene C10H16 136.238 0.1669 
30 18.726 3-Carbomethoxy-4-methylenecyclohex-1-ene C9H12O2 152.000 0.1461 
31 18.9955 Cyperene  C15H24 204.357 0.3681 
32 19.3502 3-Pinanone  C10H16O 152.233 0.1902 
33 19.4353 3-Eicosyne C20H38 278.524 0.0763 
34 19.8183 Pinocarvone C10H14O 150.104 1.0947 
35 20.0169 Caryophyllan-2,6-β-oxide C15H26O 222.370 0.3771 
36 20.0736 Nopinone C9H14O 138.210 0.587 
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37 20.1729 β-Elemene C15H24O 220.351 0.4325 
38 20.414 α-Farnesene C15H24 220.351 0.0722 
39 20.5133 E, E, E-2,4,6-Octatriene C8H12 108.184 0.0512 
40 20.6836 p-Methylstyrene C9H10 118.179 0.1112 
41 21.024 Myrtenal C10H14O 150.221 1.1560 
42 21.1233 Methyl (Z)-5,11,14,17-eicosatetraenoate C21H34O2 318.500 0.0238 
43 21.251 o-Mentha-1(7),5,8-triene C10H14 134.218 1.9593 
44 21.3644 p-Mentha-1,3,8-triene C10H14 134.218 0.0444 
45 21.5063 Cis-Sabinol C10H16O 152.230 0.6221 
46 21.6907 trans-3-methyl-2-(1-methyl-3-butynyl)oxirane C8H12O 124.000 0.0538 
47 21.7191 Safranal C10H14O 150.210 0.0654 
48 21.9176 Isodurene C10H14 134.222 1.5121 
49 22.2439 δ-3-Carene C10H16 136.238 0.0793 
50 22.3858 endo-Borneol C10H18O 154.249 0.0606 
51 22.6411 Eucarvone C10H14O 150.221 0.2137 
52 22.8397 β-Selinene C15H24 204.357 0.42 
53 22.939 Furfural C5H4O2 96.085 0.1142 
54 23.0241 Linderol C10H18O 154.249 0.1783 
55 23.2369 1-Methyl-1-phenyl-guanidine C8H11N3 149.193 0.6842 
1,2,3,4,5,7,8,9-Octahydro-6H-benzocyclohepten-6- C11H16O 164.291 
56 23.478 one 0.3070 
57 23.7475 Ethyl anthranilate C9H11NO2 165.192 0.0295 
58 23.8894 3-Phenylbutanal C10H12O 148.205 0.0447 
59 24.0028 6,6-Dimethylcycloocta-2,4-dien-1-one C10H14O 150.221 0.0736 
60 24.1022 Myrtenol C10H16O 152.237 0.7569 
61 24.3717 2-Valerylfuran C9H12O2 152.190 0.0977 
62 24.8398 1-Methyl-1-(methylamino)isobenzofuran-3-one C10 H11NO2 177.20 17.2655 
63 24.9816 1-methyladamantane C11H18 150.26 0.0517 
64 25.1944 α-Ionone C13H20O 192.30 0.0395 
Cis-1,2-diethenyl-4-(1-methylethylidene)- C13H20 176.3 
65 25.5348 cyclohexane 0.1261 
66 25.6483 (Z)-2,2-Dimethyl 5-decen-3-yne  C12H20 164.29 0.0339 
67 25.776 2-methoxy-3-ethylpyrazine C7H10N2O 138.17 0.064 
68 26.0029 o-Phenetidine C8 H11NO 137.180 0.0663 
69 26.1022 3-ethyl-4,5-dihydro-1H-pyrazole  C5H10N2 98.146 0.1949 
70 26.2015 1(Z)-5(E)-7-Dodecatriene C12H20 164.29 2.6484 
71 26.8115 o-Menth-8-ene C10H18 138.250 0.2318 
72 27.1236 4-(2,2,4-Trimethylcyclopent-3-en-1-yl)but-2-enol C12H20O 180.34 0.8324 
73 27.2229 Caryophyllene oxide C15H24O 220.356 2.1424 
74 27.5349 α-Selinene C15H24 204.357 0.091 
75 27.6626 methylpatchenol C12H20O 180.260 0.0952 
76 27.7335 Capnellane-8-one C15H24O 220.351 0.0689 
77 27.7903 Perillyl alcohol C10H16O 152.237 0.2234 
78 28.386 Viridiflorol C15H26O 222.370 0.2962 
79 28.4428 2-methyl-2-bornene C11H18 150.260 0.1147 
80 28.6272 1-Vinylbenzyl alcohol C9H10O 134.178 1.0063 
81 28.8399 8,8-dimethyl-9-methylene-1,5-Cycloundecadiene C14H22 190.324 0.057 
82 29.0243 Isolongifolene C15H24 204.357 0.8011 
83 29.0811 Germacrene B C15H24 204.357 0.2375 
84 29.2797 α-Patchoulene C15H24 204.357 1.0430 
3,4,4a,5,8,8a-Hexahydro-4a-methyl-2(1H)- C11H16O 164.240 
85 29.6059 naphthalenone 0.9775 
86 29.7052 trans-Caryophyllene C15H24 204.357 0.7922 
87 30.0315 Aromadendrene C15H24 204.357 0.8943 
88 30.5138 (-)-Camphor C10H16O 152.237 0.4346 
89 30.9819 Viridiflorene C15H24 204.357 0.2127 
90 31.3649 α-Guaiene C15H24 204.351 0.0838 
91 31.8755 β-Selinene C15H24 204.357 0.9387 
92 32.0599 Valerenal C15H22O 218.330 0.1644 
93 32.2302 Bicyclo(5.3.1)undec-1-en-9-one C11H16O 164.237 3.8698 
94 32.4288 Caryophyllenol I C15H24O 220.351 0.3742 
95 32.528 3,5-Diisopropenyl-1,2-dimethyl-cyclohexane C14H24 192.340 2.4262 
5,6,6-trimethyl-5-(3-oxo-1-butenyl)- 1- C14H20O3 236.314 
96 32.6273 Oxaspiro[2.5]octan-4-one 9.019 
97 32.7692 Caryophylla-3,8(13)-dien-5α-ol C15H24O 220.351 0.913 
98 33.0387 2,3-Dimethyl-1,5-divinylcyclohexane C12H20 164.290 1.0356 
99 33.2799 Cuminaldehyde C10H12O 148.205 0.3206 
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100 33.4217 1,2-Dimethyl-1,4-cyclohexadiene C8H12 108.184 1.2823 
101 33.6345 (+)-trans-Isolimonene C10H16 136.238 4.9119 
102 34.3437 Torreferol C20H34O2 306.490 0.1529 
103 34.4288 Junipene  C15H24 204.351 0.2619 
104 34.5565 Valerenol C15H24O 220.351 0.1292 
105 34.9962 3-Thujen-2-one C10H14O 150.22 0.1949 
106 35.053 Alloaromadendrene C15H24 204.351 1.1402 
107 35.1381 α-Humulene C15H24 204.351 0.2372 
108 35.6913 1-(2,3-Dimethylphenyl)ethanol C10H14O 150.22 0.1777 
109 35.975 Neoalloocimene C10H16 136.238 0.1028 
110 36.5424 Calarene C15H24 204.351 0.1371 
111 36.741 Eremophilene C15H24 204.351 0.1159 
112 37.6347 β-Caryophyllene C15H24 204.351 0.4406 
113 37.8332 Hinesol C15H26O 222.366 0.2793 
3-chloro-1,1,6-trimethyl-1,2,3,4,5,6,7,8- C13H21Cl 212.76 
114 38.5709 octahydronaphthalene, (3S-cis)- (9CI) 5.6537 
115 39.4361 Cis-Caryophyllene C15H26 206.367 0 .1741 
116 39.8049 (1R,3S)-Cembra-4,7,11,15-tetraen-3-ol C20H32O2 288.468 0.5288 
117 39.961 4α-Isopropenyl-2-carene C13H20 176.312 0.1617 
118 40.6419 Vulgarol B C15H24O 220.351 0.1236 
119 41.0674 Vulgarol A (Labd-13-ene-8,15-diol) C20H36O2 308.500 0.5407 
120 42.0604 5,6,7-Trimethoxy-1-tetralone C13H16O4 252.260 0.0322 
 
Monoterpenes occurring in minor quantities formed the major part of the EO (22.42%) while 
sesquiterpenes consisted of 13.98 %. The identified functional groups were hydrocarbons (57), 
alcohols (23), ketones (15), aldehydes (8) ester (1) and miscellaneous (15).  Eighteen (15.13%) out 
of the 119 constituents had a composition of more than 1%, the major ones being 1-methyl-1-
(methylamino)isobenzofuran-3-one (17.27%); 5,6,6-trimethyl-5-(3-oxo-1-butenyl)-1-
Oxaspiro[2.5]octan-4-one (9.02 %), 2-chloro-4,4,7-trimethyl-1,2,3,4,5,6,7,8-octahydronapthalene 
(5.36 %); bornyl chloride (4.93%); (+)-trans-isolimonene (4.91%); bicycloundec-1-en-9-one 
(4.25%) and 1,2-dimethyl-3,5-bis(1-methylethenyl)-cyclohexane (3.51%). This chemical profile is 
a marked contrast from the reported chemical composition of many of the extensively researched 
Aframomum plant parts, particularly, the leaf. The isobenzofuran-3-one derivative, identified as the 
single major constituent, is being reported as an Aframomum essential oil constituent for the first 
time. A literature survey on Scifinder, NIST and PubChem identified the isobenzofuranone moiety 
as a structural unit of noscapine-type alkaloids which are known for their antitumor and antitussive 
properties73. However, hexahydro-3a-2(3H)-benzofuranone (C8H12O2, 2.1%), has been identified in 
the crude extracts of A. melegueta74. Bicycloundecec-1-en-9-one has not as yet featured in any 
essential oil of Aframomum although the bicycloundecane moiety is a structural feature of several 
organic compounds including the Homo-adamatanyl type compounds isolated from Hypericum 
cohaerens75. The other major constituents, 2-chloro-4,4,7-trimethyl-1,2,3,4,5,6,7,8-
octahydronapthalene; bornyl chloride and 1,2-dimethyl-3,5-bis(1-methylethenyl)-cyclohexane are 
also new EO constituents of Aframomum. Some reported common and abundant Aframomum leaf EO 
constituents include β-pinene (A. elloitti, 44.3%; A. longiscarpum, 42.6%; A. sceptrum, 15.1%; A. 
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geocarpum, 11.3%; A. danielli, 47.6%; A. citratum, A. hanburyi, A. letestuanum and A. pruinosum - 
30-60%76) and β-caryophyllene (A. danielii, A. letestuianum and A. pruinosum  - 18.4-82.4%)76-78.   
β-pinene was not identified in essential oil of the leaves of A. atewae but 1.23% of β-caryophyllene 
was present. The minor constituents including -humulene, -ionone, -pinene, -selinene, -
terpinene, 1,8-cineole, 3-eicosyne, myrtenal, terpinolene, valerenal and vulgarol A are common 
minor EO constituents of many Aframomum species79. Mass fragmentations and some major 
compounds are shown in Figures 4.2-4.6 below 
 
Figure 4.2: MS of 1-methyl-1-(methylamino)isobenzofuran-3-one, (17.66%), MF = C10H11NO2, Rt 24.854 
 
Figure 4. 3: MS of Bornyl chloride, (4.93%), MF = C10H17Cl, Rt 16.166 
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Figure 4.4: MS of 2-chloro-4,4,7-trimethyl-1,2,3,4,5,6,7,8-octahydronapthalene, (5.56%),  MF = C13H21Cl,  Rt 36.671 
 
Figure 4.5: Mass fragmentation of 5,6,6-trimethyl-5-(3-oxo-1-butenyl)- 1-Oxaspiro[2.5]octan-4-one, (9.019%), MF = 
C14H20O3, Rt 32.627 
 
Figure 4.6: Other major constituents of the leaf essential oil 
4.2.2 Chemical composition of the rhizome essential oil 
The rhizome EO on GC-MS analysis led to the identification of 100 constituents making up 88.43% 
of the total peak area. (Figure 4.7 and Table 4.2). This oil contained more sesquiterpenes (24.44%) 
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than monoterpenes (8.08%) as compared to the leaf EO. The functional groups that were identified 
were significantly different in quantity with respect to the leaf essential oil - hydrocarbons (34), 
alcohols (10), ketones (16), aldehydes (7) ester (2) and miscellaneous (31).   
 
 
Figure 4.7: Gas chromatogram of rhizome essential oil of A. atewae 
 
Table 4.2: Retention time, name of constituent, percentage composition, molecular formula and weight of Aframomum 
rhizome essential oil as shown by GC-MS analysis 
Peak Retention Compound MF  Mwt. Composition 
No. time (min)  (g/mol) (%) 
1 12.9385 p-Cymene  C10H14 134.21 0.3111 
2 16.7968 O-Allyltoluene  C10H12 132.202 0.2579 
3 18.9529 8,9-Dehydro-neoisolongifolene C15H22 202.335 0.5782 
4 20.0026 1,2,4-trimethyl(1-methylethenyl)- Benzene C12H16 160.25 0.1878 
5 20.1586 2,4,6-Octatrienal  C8H10O 122.16 0.1063 
6 21.18 -Gurjunene  C15H24 204.357 0.4142 
7 21.2651 -himachalene C15H24 204.357 0.1833 
8 21.9176 -Selinene  C15H24 204.357 0.2422 
9 22.0452 -cadinene  C15H24 204.357 0.148 
10 22.2438 1,2,3,4-tetrahydro-2,3-methano-2,8-dimethoxynaphthalene C13H16O2 204.26 0.7025 
11 22.4708 -Chamigrene  C15H24 204.357 0.2231 
12 22.8254 Neoalloocimene C10H16 136.23 0.4703 
13 22.8963 -Selinene  C15H24 204.357 0.7009 
4,5-dimethyl-11-methylenetricyclo[7.2.1.0 
14 22.9956 (4.9)]dodecane C15H24 204.357 1.9799 
15 23.2935 (5R)-2-cyano-5,9-dimethyldeca-2,8-dienenitrile  C13H18N2 202.30 0.3519 
(E)-(3'-oxo-3',4'-dihydro-2'H-1',4'-benzoxazin-
16 23.4495 2'ylidene)acetic acid  C10H7NO4 204.17 0.2331 
(2-methylcyclopent-1-enyl)(4,4-dimethyl-3-oxocyclopent-
17 23.5205 1-enyl)methane C14H20O 204.31 0.189 
18 23.6623 Isolongifolene  C15H24 204.357 2.0337 
19 23.946 -Patchoulene  C15H24 204.357 0.2967 
20 24.1872 p-Cresol  C7H8O 108.14 0.1512 
21 24.5702 (3E)-2,6-Dimethyl-5-isopropyliden-1,3,6,9-decatetraene C15H22 202.34 0.0908 
22 24.8539 Calamenene  C15H22 202.34 0.7842 
23 24.9674 Pentacyclo[7.5.0.0(2,8).0(5,14).0(7,11)]tetradecane C14H20 118.31 0.153 
24 25.0525 1-chloro-2,4-dimethoxy-3-methylphenol C9H11ClO3 202.63 0.2219 
25 25.2794 Cumic acid  C10H12O2 164.204 0.2994 
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(E)-2-Methyl-4(2',4',4'-trimethylbicyclo[4.1.0]hept-2'-en-
26 25.3787 3'-yl)-1,3-butadiene C15H22 202.34 0.2985 
27 25.9178 Dimethyl sulfone C2H6O2S 93.13 0.1225 
28 26.0738 -Calacorene C15H20 200.32 0.1437 
29 26.4142 1,2,3,6-Tetramethylbicyclo[2.2.2]oct-2-ene C12H20 164.29 0.5323 
30 27.0951 -Terpinene  C10H16 134.238 0.2453 
31 27.1944 4,5-Dehydro-isolongifolene C15H22 200.352 0.2415 
32 27.2795 Nerolidol oxide C15H26O2 238.37 0.3236 
33 27.4355 Methyl allomaltol C6H6O3 126.110 0.1971 
34 27.7476 Bisabolol oxide  C15H26O2 238.371 0.3263 
35 28.0739 2-endo-(Dichloroamino)norbornane C7H11Cl2N 180.07 0.3723 
36 28.7406 Ledene  C15H24 204.357 1.2532 
37 28.8399 isoobtusadiene  C15H21BrO 297.231 0.1255 
38 29.0243 -Guaiene  C15H24 204.357 1.8647 
39 29.2512 4,4-dimethyltricyclo[6.3.2.0(2,5)]trideca-8-ene-1-ol C15H24O 220.351 0.1022 
40 29.3222 2-methyl-2-bornene C11H18 150.261 0.2611 
41 29.464 1-(2,4-dimethylphenyl)-2-phenylethane C16H14 206.28 0.3806 
2,3-Dihydro-4,5-dimethoxy-6-methyl-3-methyleneinden-
42 29.6626 1-one C13H14O3 218.25 0.2747 
(E)-2-methyl-5-[(1,6 ,7 )-3-(hydroxymethyl)-7-
43 29.8186 methylbicyclo[4.1.0]hept-2-en-7-yl]-2-penten-1-ol  C15H24O2 236.35 0.1141 
4-Methyl-1,2,3,4,5,6-hexahydro-1,5-methano-4,1-
44 30.0314 benzazaphosphocine C12H16NP 205.24 0.5923 
45 30.1023 2,5-Dimethoxyacetophenone C10H12O12 180.203 0.3048 
46 30.2158 Carvacrol C10H14O 150.217 3.8917 
1-[2-Hydroxy-4-methyl-3-(1-methylethyl)phenyl]-1-
47 30.3577 propanone  C13H18O2 206.26 1.5648 
48 30.5846 trans-Caryophyllene  C15H24 204.357 0.3864 
49 30.6839 3,4-Dihydro-iso-methyl--ionone  C10H24O 208.34 0.5354 
(R)-4-Methyl-2-(1-ethylethenyl)-1-cyclopentene-1-
50 30.7832 carboxaldehyde  C11H16O 164.24 1.5449 
51 30.8541 N-(Methyl-D2)-aniline C7H7D2N  0.5345 
52 31.1662 2-Chloro-4-nitromesitylene  C9H10ClNO2 199.63 0.4406 
2,3-dihydro-4-oxy-2-prop-2-ylidene-4H-pyrido[2,3-e]1,3-
53 31.4215 thiazine  C10H10N2OS 206.26 0.6415 
54 31.5066 7-methyl- 3,4,5,6,7,8-Hexahydronaphthalen-1(2H)-one C10H16O 164.24 2.2442 
55 31.5776 Cadinene  C15H24 204.357 2.6417 
56 31.7194 Mellitene C12H18 162.276 0.6448 
(E,1RS,2RS,6RS)-2-(3'-methyl-1,3'-butadien-1-yl)-2,4,4-
57 31.9322 trimethylbicyclo[4.1.0]heptan-3-one  C15H22O 218.33 0.27 
58 31.9748 2-(1-methyl-1-propenyl)-(Z)-Naphthalene  C14H14 182.26 0.2849 
59 32.0457 Nootkatone  C15H22O 218.340 0.534 
60 32.1591 Alloaromadendrene C15H24 204.357 1.3663 
61 32.3861 2,5-Di-tert-butylhydroxybenzene C14H22O 206.32 7.8033 
62 32.5421 Ethyl 2-bromopropanoate C5H9BrO2 181.03 3.4835 
63 32.6556 2-(2'-Methyl-2'-nitropropyl)indan-1-one  C13H15NO3 233.26 0.3776 
2,2,4,4-tetramethyl-1-(1-methylethenyl)bicyclo[3.2.1]oct-
64 32.7407 6-en-3-one  C15H22O 218.33 0.449 
cis-3a,4,5,6,7,7a-hexahydro-2-(2'-propenyl)-1H-inden-1-
65 32.84 one  C12H16O 176.25 0.5465 
66 33.0102 8.9-Epoxiacorenon-B  C15H24O2 236.35 0.4942 
67 33.1379 5-Oxo-isolongifolene  C15H22O 218.33 0.4792 
Dimethanonaphthalen-9-ol, decahydro-2-methyl-, 
68 33.294 (1,2,4 ,4a,5 8 .,8a,9R) C13H20O 192.30 0.5578 
8-Allyl-2-amino-6-methylimidazo[1,5-a]-1,3,5-triazin-
69 33.3649 4(3H)-one C9H11N5O 205.22 0.5758 
70 33.5209 8,9-Dehydro-neoisolongifolene C15H22 200.352 1.0386 
71 33.606 Ambrial C16H26O 234.377 1.9507 
2-[(Z)-(3''-Methylbutadien-2''-yl)methylidene]-1,3,3-
72 33.7621 trimethylcyclohexanol C15H24O 220.35 1.1714 
73 33.8897 Valerenal C15H22O 218.33 0.5864 
74 33.9748 Retinal  C20H28O 284.436 0.3308 
cis/trans-7-Bicyclo[4.1.0]hept-7-ylidene-
75 34.1167 bicyclo[4.1.0]heptane C14H20 188.31 1.6085 
76 34.2302 2-Ethyl-3-phenyl-2-butene-1-al  C12H14O 174.24 0.9137 
77 34.3862 -Oplopenone C15H24O 220.35 0.4572 
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78 34.5422 1,3,6-Trimethyl-8-ethyl-2,7-naphthyridine  C13H16N2 200.28 0.9592 
79 34.6983 1-(2'-ethenyl-1'-cyclohexenyl)-2-propen-1-ol  C11H16O 164.24 0.5498 
80 34.8969 -Caryophyllene C15H24 204.357 0.3587 
81 35.0245 1-O-Acetylfructose C8H14O7 222.19 0.9085 
1,1,2,2-tetramethyl-3-[(methyl)methylene]-8-
82 35.0813 oxobicyclo[4.3.0]non-4(5)-ene  C15H22O 218.33 0.522 
Cyclohexanol, 3-(1,3-butadienyl)-4,4-dimethyl-2-
83 35.2089 methylene-, [1,3 (E)] C13H20O 192.30 0.4184 
84 35.3508 8,9,10,12-Tetrahydro-7H-indolo(1,2)(2)-benzazepine  C17H17N 235.32 0.54 
85 35.4501 trichloromethyl- Benzene C7H5Cl3 195.48 0.4867 
2-[(Z)-(3''-Methylbutadien-2''-yl)methylidene]-1,3,3-
86 35.5494 trimethylcyclohexanol C15H24O 220.35 0.667 
87 35.8189 Di-iso-Butyl phthalate  C16H22O4 275.35 3.8654 
88 36.1451 3-Chloro-1-butyne C4H5Cl 88.54 0.6158 
(Z,1'RS,2'SR,4'RS,7'SR)-1-(2',5',5'-trimethyl-3'-
89 36.2586 oxabicyclo[5.1.0.0(2,4)]oct-4'-yl)-3-methyl-1,3-butadiene C15H22O 218.33 0.5926 
90 36.3437 1,4,7,10,13,16-Hexaoxacyclooctadecane  C12H24O6 264.32 1.4553 
1-(1-cyclohexen-1-yl)-1-Amino-3-hydroxyamino-
91 36.7409 isoquinoline C9H9N3O 175.19 0.7621 
92 36.826 1-Acetylcyclohexene C8H12O 124.180 3.1646 
93 37.2799 trans-1,2-Cyclobutanedicarbonyl chloride C6H6Cl2O2 181.01 1.0099 
94 37.7906 Benzene, 1-(chloromethyl)-2,4-dinitro-  C7H5ClN2O4 216.58 0.4938 
95 37.9183 Dibutyl-phtalate  C16H22O4 275.35 4.5759 
96 39.0956 (S)-(-)-2-[(2-Methoxyethoxy)methoxy]propan-1-al C7H11O4 162.18 0.7272 
6-ethyl-1,2,3,4,5,6,7,8-octahydro-6-nitro-5-[(2-
97 39.8333 nitrophenyl)thio]-,(1R,4S,5R,6R)4-Epoxynaphthalene C18H20N2O5S 376.43 0.3185 
(18S,19S)-18,19-Dihydroxy-1,4,7,10,13,16-
98 40.0318 hexaoxocycloencosane C14H28O8 324.37 0.345 
99 40.4574 tricyclo[5.3.2.0(1,7)]dodecan-2-on-11-ene C12H16O 176.26 0.4252 
100 41.0673 Palmitic acid  C16H32O2 256.43 4.3375 
 
Twenty-two constituents had compositions of >1%. Like the leaf EO, the major constituents of the 
rhizome oil 2,5-ditertbutylhydroxybenzene (7.80%), dibutylphthalate (4.58 %), palmitic acid 
(4.34%), carvacrol (3.89 %), diisobutylphthalate (3.87 %), ethyl 2-bromopropanoate (3.48%) and 1-
acetylcyclohexene (3.16%) - were also uncharacteristic of Aframomums. To the best of my 
knowledge phthalates and palmitic acid have not been previously reported as constituents of 
Aframomum oil. These phthalates however, have been reported in Pterocarpus marsupium EtOH 
extract with antimicrobial and antifouling activities80. Other compounds which occurred above 1% 
are cadinene (2.64%) ambrial (1.95%), -guaiene (1.86%), alloaromadendrene (1.37 %), ledene 
(1.25%), and 8,9-dehydro-neoisolongifolene (1.04%). The work of  Zollo et al., (2002) also examined 
the essential oils of the rhizomes and reported mainly oxygenated monoterpenes and oxygenated 
sesquiterpenoids. The major monoterpenoid identified was linalool (A. citratum and A. hanburyi) and 
the major sesquiterpenoid identified was (E)-nerolidol (A. pruinosum and A. letestuanum)79.  
 Mass spectra and corresponding structures of some of major compounds are presented in the Figures 
4.8 - 4.13 
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Figure 4. 8: MS of 2,5-ditertbutylhydroxybenzene, (7.80%) MF = C14H22O, Rt 32.386 
 
Figure 4. 9: MS of dibutylphthalate, (4.58%), MF = C16H22O4, Rt 37.716 
 
 
Figure 4.10: MS of carvacrol, (3.89%), MF = C10H14O, Rt 30.2158 
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Figure 4.11: MS of palmitic acid, (4.34%), MF = C16H32O2, Rt 41.067 
 
Figure 4.12: MS of diisobutylphthalate, (3.87%), MF = C16H22O4, Rt 35.8189 
 
Figure 4.13: Other major constituents of the leaf essential oil 
 
4.2.3 Chemical composition of DCM-extracted essential oil 
Fifty compounds, comprising 84.89% of the DCM-extracted EO, were identified in the GC-MS 
analysis. The major constituents were mainly steroids (59.72%) and long chain hydrocarbons 
(18.06%), a reflection of the different extraction protocol employed. (Figure 4.14 and Table 4.3). 
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Figure 4.5: Gas chromatogram of DCM-extracted essential oil of A. atewae 
 
 
Table 4.3: Retention time, percentage composition, molecular formula and molecular weight of constituents of A. atewae 
rhizome DCM-extracted essential oil as shown by GCMS analysis 
Peak Retention Compound MF  Mwt. Composition 
No. time (min)  (g/mol) (%) 
1 10.9526 4-Methylthiazole C4H5NS 99.16 0.0292 
2 11.3072  Dodecane C12H26 170.33 0.2777 
3 12.3002 1-Octanol, 2-butyl-  C12H26O 186.336 0.0609 
4 12.9385 p-Cymene  C10H14 134.21 0.0457 
5 13.6619 Tridecane  C13H28 184.37 0.0339 
6 16.0024 Tetradecane  C14H30 198.39 0.8036 
7 16.6691 Egitol C2Cl6 236.7 0.0903 
8 16.7968 Benzene, (2-methyl-1-propenyl)-  C10H12 132.206 0.0123 
9 16.9528 1-Octadecene  C18H36 252.486 0.0487 
10 17.6337 −Terpinene  C10H16 134.238 0.0168 
11 18.2295 Pentadecane C15H32 212.421 0.0524 
12 18.499 2-Hydroxy-3-tert-butylacetophenone  C12H16O2 192.254 0.0185 
13 19.109 Cyperene  C15H24 204.357 2.5054 
14 19.6196 Pentadecane, 3-methyl-  C16H34 226.44 0.0461 
15 20.3998 Hexadecane  C16H34 226.44 0.7518 
16 20.797 cyclosativene  C15H24 204.357 0.5392 
17 21.2509 1-Heptadecene  C17H34 238.455 0.2685 
Bicyclo[4.1.0]heptane, 4,4-dimethyl-3-(3-
18 21.563 methyl-3-butenylidene)-2-methylene C15H22 202.34 0.063 
19 21.6339  Pristane  C19H40 268.51 0.0329 
1-(8-Fluoro-2-hydroxynaphthalen-1-
20 21.946 yl)ethanone  C12H9FO 204.20 0.0465 
21 22.0452 2,5-dimethoxy-3-methylnaphthalene C13H14O2 202.25 0.0424 
22 22.3573 -Gurjunene C15H24 204.357 0.0158 
23 22.5134 Ledene  C15H24 204.357 0.1324 
24 22.7545 Germacrene-D C15H24 204.357 0.0184 
25 22.868 Alloaromadendrene  C15H24 204.357 0.3563 
4,5-dimethyl-11-methylenetricyclo[7.2.1.0 
26 22.9389 (4.9)]dodecane C15H24 204.357 0.0669 
27 23.024 -Maaliene  C15H24 204.357 0.0569 
28 23.1233 Azulene C10H8 128.17 0.0648 
29 23.5489 -Amorphene  C15H24 204.357 0.0438 
30 23.6765 Selina-3,7(11)-diene  C15H24 204.357 0.1188 
31 23.9602 -Gurjunene C15H24 204.357 0.0385 
32 24.2297 Octadecane  C18H38 254.494 0.8373 
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33 24.5844 -Curcumene C15H24 204.357 0.0268 
34 24.7262 isospathulenol C15H24O 220.357 0.033 
35 24.8822 (1S)-cis-Calamenene C15H22 202.33 0.2985 
36 25.9745 Nonadecane C19H40 268.5 0.689 
2,3-dicyano-7,7-dimethyl-5,6-
37 26.088 benzonorbornadiene  C15H12N2 220.27 0.0382 
38 26.3575 -Calacorene C15H20 220.39 0.0708 
39 26.7972 Octahydro--camphorene C20H40 280.53 0.0272 
40 27.549 Lemonene  C10H16 136.24 0.1373 
41 27.776 Eicosane  C20H42 282.56 3.7822 
42 28.1023 14-Pregnane C21H36 288.52 58.4384 
43 29.4782 Heneicosane  C21H44 296.58 0.6989 
44 31.9322 Docosane  C22H46 310.60 2.71 
45 32.2726 Docosane, 2,21-dimethyl- C24H50 338.65 1.1801 
46 33.5351 Tricosane C23H48 324.48 2.8882 
47 36.8544 Tetracosane C24H50 338.65 2.5814 
48 39.2375 Aniline, O-3-butenyl- C10H13N 147.22 1.7711 
49 40.0886 6-fluoro-4,6-cholestadien-3-ol  C27H43FO 402.63 1.2596 
50 42.0461 Cholesta-3,5-diene  C27H44 368.64 0.0298 
 
 
The monoterpene and sesquiterpene components of the oil were 3.38% and 2.44%, respectively. This 
observation is not surprising, considering that the plant material was dried and pulverized prior to 
solvent extraction, which would have resulted in the loss of most of the volatile components. Further, 
the open column chromatographic separation process would also contribute to evaporation of volatile 
organic compounds and account for the fewer number of constituents in comparison to the 
hydrodistilled essential oils. The breakdown for the functional groups identified is as follows: 
hydrocarbons (40), alcohols (3), ketones (2) and miscellaneous (5).   
 
14-pregnane (58.44%), formed the bulk of the oil, occurring as a broad peak on the GC 
chromatogram. It did not feature in either of the hydrodistilled essential oils and it is also being cited 
for the first time in Aframomum species. Pondugula et al (2013) reported that both natural pregnane 
(pregnenolone, progesterone, 17α-hydroxypregnenolone, 5β-pregnane-3,20-dione and 17α-
hydroxyprogesterone) and synthetic ( 6,16α-dimethyl pregnenolone, dexamethasone t-butylacetate 
and pregnenolone16-carbonitrile) activate mouse homologue81. Naturally occurring pregnane 
derivatives such as progesterone, 3,20-pregnanediones and 3,20-pregnenediones have been reported 
to protect against pentylenetetrazole (PTZ)-induced seizures in animals as well as the synthetic A-
ring reduced alphaxolone and pregnanes 2β-morpholino-5α,3α-pregnanolone. Metabolites of 
progesterone, 3α,5α- pregnanolone and 3α,5β-pregnanolone, exhibit anticonvulsant activity82. 
Eicosane (3.28), tricosane (2.89%), docosane (2.71%), tetracosane (2.58%), (-)-cyperene (2.51%) 
and 6-fluoro-4,6-cholestadien-3-ol (1.26%). The long chain hydrocarbons are commonly-occurring 
essential oil constituents. Marrufo et al., (2013) reported that Moringa oleifera leaf essential oil is 
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rich in these compounds and showed a strong radical scavenging activity83. The mass spectrum of 
some selected major constituents are shown in Figure 4.15 – 4.18 below.  
 
 
Figure 4. 14: MS of 14-pregnane, (58.44%), MF = C21H36, Rt 34.287 
 
Figure 4.15: MS of (-)-cyprene, (2.51%), MF = C14H24, Rt 19.109 
 
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Figure 4.16: MS of diisobutylphthalate, (3.87%), MF = C16H22O4, Rt 35.8189 
 
Figure 4.17: Other major constituents of AA/R/DCM 1  
Regardless of the major differences in the chemical profile of the three essential oils, they all 
contained some common minor constituents (Table 4.4) The hydrodistilled essential oils were more 
similar in the respect.  
 
Table 4.4: Classes of terpenes and common constituents of the 3 essential oils 
 Leaf (%) Rhizome (%) DCM-extracted rhizome (%) 
Classes of terpenes 
 
Monoterpene hydrocarbons 10.9169 1.2846 3.3845 
Oxygenated monoterpenes 11.4998 6.7907 - 
Sesquiterpene hydrocarbons 6.9138 17.2087 2.4097 
Oxygenated sesquiterpenes 7.0682 7.2345 0.033 
Diterpene 0.5407 - - 
Common constituents 
 
p-cymene 0.5945 0.3111 0.0292 
α-Terpinene 0.0367 0.2453 0.0168 
α-Selinene 0.091 0.7009 - 
o-Allytoluene 0.1582 0.2579 - 
Isolongifolene 0.8011 2.0337 - 
Valerenal 0.1644 0.5864 - 
Ledene 1.2532 - 0.1324 
Alloaromandendrene 1.1402 1.3663 - 
β-caryophyllene 0.4406 0.3587 - 
α-gurjenene 0.4142 - 0.0385 
Cyperene 0.3681 - 2.5054 
 
 
Most essential oil constituents are biologically active molecules and form part of the plants’ defence 
systems and hence are used for crop protection. Due to their biodegradable nature, these botanical 
pesticides are gradually replacing their synthetic counterparts. They also find wide application as 
flavoring agents, fragrance, or as medicines used to treat various human and animal ailments. Since 
the components of essential oils are what provide them with their intrinsic properties, a variation in 
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the distribution of the components modifies the particular properties of essential oils. This variation 
is influenced by extrinsic factors such as soil, climate, elevation and geographical origin. 
 
4.3 Antifungal studies of essential oils 
As indicated in section 3.5, page 23, the four essential oils were evaluated for their activity against 
C. albicans and S. cerevisiae. Figure 4.19 shows the activity of the 4 essential oils against C. albicans. 
The fluorescence observed for the negative control, the positive control, PE-extracted oil, DCM-
extracted oil, rhizome EO and leaf EO were 300, 1450, 600, 250, 250 and 550 nm, respectively. The 
PE-extracted oil and the rhizome EO exhibited fungistatic activity toward C. albicans because the 
fluorescence emitted was greater than that of the negative control but lower than that of positive 
control. Conversely, fluorescence emitted from the DCM-extracted oil and the rhizome EO was lower 
than observed for both the negative and positive controls hence, they were fungicidal to the cells. β-
Caryophyllene, caryophyllene oxide and terpinolene have been reported to restrict biofilm 
development of fungi including C. albicans 84. β-Caryophyllene (0.39%) was identified as a 
constituent of the fresh leaf EO whiles caryophyllene oxide (2.14%), and terpinolene (0.07%) were 
also present in the rhizome EO, hence these constituents can be linked to the observed activities of 
the essential oils against C. albicans. Shin et al (2003) also reported that p-cymene showed activity 
against C. albicans. p-Cymene was identified as a constituent of the 3 oils with compositions - 0.59%, 
0.31% and 0.029% for the fresh leaf EO, fresh rhizome EO and DCM-extracted oil, respectively. 
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Figure 4.18: Graph of results showing activity of essential oils against C. albicans 
 
The activity of the oils against S. cerevisiae is captured in Figure 4.20. Observed fluorescence values 
were 100, 1800, 200, 2100, 2250 and 2600 nm for the negative control, positive control, PE-extracted 
oil, DCM-extracted oil, rhizome EO and leaf EO, respectively. Out of the 4 oils, only the PE-extracted 
oil emitted fluorescence less than that of the positive control but greater than the negative control, 
suggestive of the fungistatic nature of the oil to the cells. The 3 remaining oils, however, did not show 
any activity against S. cerevisiae. In contrast to Adegoke et al. (1996) reporting that -terpinene 
caused growth reduction of S. cerevisiae36, the 3 EOs tested showed no activity potency due to the 
low percentage of -terpinene, (0.017% - 0.037%).  
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Figure 4.19: Graph of results showing the activity of essential oils against S. cerevisiae 
 
Unfortunately, the constituents of the PE-extracted oil could not be determined due to sample mix-
up during GC-MS analysis. Hence the fungistatic activity observed could not be attributed to any 
compound.  
The observed activities for the various oils suggest that the essential oils of Aframomum atewae have 
the potential as effective fungicides.  In Ghana, especially in the cocoa production sector, farmers use 
chemicals that contain nonbiodegradable compounds such as tetraconazole [48] and neonicotinoid 
such as imidacloprid [49] to control plant pathogens. These compounds are able to accumulate in the 
soil and the fruits of cocoa, and could affect the quality of the cocoa beans and also lead to soil 
contamination hence destroying of ecosystem and pollution of water bodies. Due to similarities in 
mechanisms of infection between plant and animal pathogenic fungi88, essential oil from Aframomum 
atewae could be explored as a biodegradable source of plant pathogens control. 
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4.4 Investigation of extracts from the rhizome of A. atewae 
4.4.1 Phytochemical Screening of the PE, DCM and MeOH Crude Extracts  
From the phytochemical screening, it was observed that the MeOH extract contained all the tested 
phytochemicals except anthraquinones, anthracenes and steroids. The PE extract contained mainly 
terpenoids and steroids with the DCM extract containing flavonoids in addition to steroids and 
terpenoids.   
  
Table 4. 5: Results of phytochemical screening of crude extracts 
Class of compounds Petroleum ether Dichloromethane Methanol 
Alkaloids - - + 
Anthraquinones and anthracenes - - - 
Flavonoids - + + 
Cardiac glycosides - - + 
Saponins - - + 
Tannins - - + 
Terpenoids + + + 
Steroid + + - 
Legend: (+) = present and (-) = absent 
 
The mass and percentage yield of crude extracts are summarized in Table 4.6 below.  
Table 4.6: Mass and percentage yields of various extracts   
Extract Mass Yield (%) 
AA/R/PE 0.0164 kg 0.88% 
AA/R/DCM 0.0255 kg 1.37% 
AA/R/MeOH 0.0441 kg 2.37% 
AA/R/MeOH/PE 3.3846 g 8.46% 
AA/R/MeOH/PE:EtOAc 1:1 3.401 g 8.50% 
AA/R/MeOH/EtOAc 5.053 g 12.63% 
AA/R/MeOH/EtOH 8.493 g 21.23% 
 
 
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4.4.2 Investigation of the Petroleum Ether (PE) Extract 
Through chromatographic separation, an essential oil and 3 compounds labelled AA/R/PE-1, 
AA/R/PE-2, AA/R/PE-5 and AA/R/PE-6 were obtained from the PE extract. AA/R/PE-1 was 
characterized by identifying the constituents of the EO using GC-MS (Table 4.3). The antifungal 
property of the oil was also tested against C. albicans and S. cerevisiae (Figures 4.18, 4.19). 
 
4.4.2.1 Characterization of AA/R/PE-2 
AA/R/PE-2 was isolated as a white solid with a melting point of 112-114 °C. When subjected to IR 
spectroscopic analysis (Appendix I), there were absorptions at 2915.15 cm-1 and 2849.10 cm-1 
assigned to C-H (sp3 and sp2, respectively), a C=O at 1730.01 cm-1 and a C=C  at 1630 cm-1str str str . A 
peak at 1471 cm-1 was assigned to a C-Ostr.  
From the 13C NMR spectrum (Figure 4.21), 17 signals between the range δC 14 - 175 were observed. 
Five signals were due to sp2 hybridized carbons and the remaining were attributed to sp3 hybridized 
carbons. The signal at δC 174.1 was assigned to the carbonyl carbon whereas the remaining 4 sp
2 
carbons were assigned to alkene carbons. An sp3 hybridized carbon attached to a heteroatom was 
observed at δC 64.5.  
 
Figure 4.20: Full 13C NMR Spectrum of AA/R/PE-2 
 
The DEPT 135° spectrum (Figure 4.22) revealed 2 quaternary carbons identified at δC 174.1 (C-1) 
and δC 135.0 (C-2). Methine carbons occurred at δC 129.7 (C-3), δC 125.1 (C-4,), δC 124.4 (C-5) and 
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δC 31.0 (C-10). At δC 16.1 (C-16) and δC 14.2 (C-17), the signals were assigned to methyl carbons 
while the remaining 9 signals were identified as methylene carbons.  
 
Figure 4.21: Full DEPT 135 ° Spectrum of AA/R/PE-2 
 
All the signals in the 1H NMR spectrum (Figure 4.23), were observed within the range δH 0.88 - 5.50. 
The signals at δH 0.88 (t, 3H, J = 2.4 Hz) and δH 1.80 (s, 3H) were assigned to protons on the 2 methyl 
carbons identified in the 13C NMR spectrum. Methylene protons were observed at δH 4.05 (t, 2H, J = 
3.7 Hz), δH 2.28 (t, 2H,  J = 2.3 Hz), δH 2.20 (t, 2H,  J = 2.6 Hz), δH 1.98 (t, 2H,  J =3.2 Hz), δH 1.62 
(m, 4H) and δH 1.60 (t, 2H,  J = 2.7 Hz). At δH 1.26 (s), the signal integrated for 8 protons suggesting 
the presence of a continuous methylene chain in the compound. Three olefinic signals occurred at δH 
5.35 (d, 2H, J = 4.1 Hz), δH 5.30 (t, 1H, J = 3.5 Hz), δH 5.12 (dd, 2H, J = 4.1, 2.3 Hz) while a methine 
was identified at δH 2.17 (m, 1H). 
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Figure 4.22: Full 1H NMR Spectrum of AA/R/PE-2 
 
From the HSQC spectrum (Figures 4.24), the following correlations were made: C-3 (δC 129.7) / δH 
5.35, C-4 (δC 125.1) / δH 5.30, C-5 (δC 124.4) / δH 5.12 and C-10 (δC 31.0) / δH 2.17. In the 
1H NMR 
spectrum, the signals at δH 5.35 and δH 5.12 integrated for 2 protons each. Hence, their HSQC 
correlation to C-3 (δC 129.7) and C-5 (δC 124.4), respectively, which in the DEPT 135 ° experiment 
were identified as methine carbons, suggests that each of these two signals represents two sets of 
equivalent carbons. Further correlations were observed for the methyl groups at C-16 (δC 16.1)/ δH 
1.80 and C-17 (δC 14.2)/ δH 0.88. The oxygenated methylene carbon C-6 (δC 64.5) correlated with the 
protons at δH 4.05. There was also a correlation between both C-11 (δC 29.8, br) and C-14 (δC 28.8) 
with δH 1.26 (br s, 8H). From this observation, it can be deduced that the broad signal at δC 29.8 (C-
11) represents 3 methylene carbons. 
 
 
 
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Figure 4.23: Expanded HSQC spectrum of AA/R/PE-2 
 
Four COSY spin systems were observed involving the methine protons H-3 (δH 5.35, d, 2H, J = 4.1 
Hz), H-5 (δH 5.12, dd, 2H, J = 4.1, 2.3 Hz) and the methylene protons H-8 (δH 2.28, t, 2H, J = 2.3 
Hz); H-9 (δH 1.60, 2H, t, J = 2.7) and H-13 (δH 2.20, 2H, t, J = 2.6); H-4 (δH 5.30, t, 1H, J = 3.5 Hz), 
H-16 (δH 1.26, m) and H-18 (δH 0.88, t, 3H, J = 2.4). The last spin system consisted of a cluster of 
contours from which correlations were established at H-6 (δH 4.05, 2H, t, J = 3.7) / H-14 (δH 1.62, m) 
and H-7 δH (1.98, 2H, t, J = 3.2) /H-12 (δH 1.26, m) (Figure 4.25). 
 
 
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Figure 4.24: Expanded COSY NMR Spectrum of AA/R/PE-2 
 
The identification of two sets of equivalent olefinic methine carbons as part of one spin system 
suggested the presence of a 1,4-cyclohexadienyl moiety in the compound.   
 
 
The HMBC correlation C-10 (δC 31.0)/ H-13 (δH 2.20) indicated the position of attachment of a side 
chain to the ring. Another correlation from C-1 (δC 174.1) to H-6 (δH 4.05) buttressed the presence of 
an ester carbonyl in the compound. C-6 showed correlations with H-14 (identified as 2J from H-6/H-
14 COSY splitting) and H-15 (3J). The H-17 tertiary methyl protons (δH 1.80) correlated with the 
quaternary carbon C-2 (δC 135.0), the methylene carbon C-7 (δC 39.9) and the olefinic methine carbon 
C-4 (δC 125.1).  The link between C-4 and C-16 was established by the COSY spin system H-4/H-
15/H-17 and 2J coupling H-17 to C-15, which supported a 2-methyl-2-pentenyl moiety in the 
structure.  
 
 
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The H-7/H-12 spin system also supported another connection between C-7 and C-12 which showed 
long range coupling to the continuous methylene chain alluded to the proton and 13C NMR spectra.  
(Figure 4.26) 
 
 
 
 
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Figure 4.25: Expanded HMBC NMR Spectrum of AA/R/PE-2 
 
Table 4.7 contains a summary of the NMR data for AA/R/PE-2  
Table 4.7: 1H, 13C, COSY and HMBC NMR data for AA/R/PE-2 
Signals δ  13C Cmult δH /(J/Hz) COSY HMBC 
1 174.1 C   6 
2 135.0 C   16 
3 129.7 CH 5.35 (1H, d, J = 4.1) 5  
 129.7 CH 5.35 (1H, d, J = 4.1) 5  
4 125.1 CH 5.30 (1H, t, J = 3.5, 12.1) 15 16 
5 124.4 CH 5.12 (1H, dd, J = 4.1, 2.3) 3, 8  
 124.4 CH 5.12 (1H, dd, J = 4.1, 2.3) 3, 8  
6 64.5 CH2 4.05 (2H, t, J = 3.7) 13 13, 14 
7 39.9 CH2 1.98 (2H, t, J = 3.2) 3 16 
8 34.6 CH2 2.28 (2H, t, J = 2.3) 5  
9 32.0 CH2 1.60 (2H, t, J = 2.7) 10, 12  
10 31.0 CH 2.17 (1H, m) 9 12 
 29.8 CH2 1.26 (2H, m)  11 
11 29.8 CH2 1.26 (2H, m)  7, 11 
 29.8 CH2 1.26 (2H, m) 7 7, 11 
12 29.5 CH2 2.20 (2H, t, J = 2.6) 3  
13 28.8 CH2 1.62 (2H, m) 6  
14 25.2 CH2 1.62 (2H, m)  13 
15 22.8 CH2 1.26 (2H, m) 4, 17 17 
16 16.1 CH3 1.80 (3H, s)  7 
17 14.2 CH3 0.88 (3H, t, J = 2.4) 15 15 
 
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The LC-MS of AA/R/PE-2 (Figure 4.27) gave a signal at m/z 318.2 at a retention time of 1.291 
minutes deduced to be the molecular ion peak with molecular formula C H O •+ 21 34 2 (5 degrees of 
unsaturation).  
 
 
Figure 4.26: LC-MS of AA/R/PE-2 
The proposed structure of AA/R/PE-2 was supported by the molecular formula and further confirmed 
with the daughter ions C •+18H26O2  (m/z 274.2, base peak), C
•+ 
19H30O2 (m/z 290.2), C
•+
18H28O2  (m/z 
264.2) and C H •+ 8 18 (m/z 107.2) (Figure 4.28).   
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Figure 4.27: Proposed fragmentation pattern for AA/R/PE-2 
From the above information, the structure below was proposed and was named as 1 (E)-8-
methylundec-8-en-1-yl 3-(cyclohexa-2,5-dien-1-yl)propanoate (Figure 4.29). 
 
Figure 4.28: Proposed structure of AA/R/PE 1 (E)-8-methylundec-8-en-1-yl 3-(cyclohexa-2,5-dien-1-yl)propanoate 
 
A thorough literature survey on Scifinder, PubChem, Chemspider, and other search engines revealed 
no compound with the name  (E)-8-methylundec-8-en-1-yl 3-(cyclohexa-2,5-dien-1-yl)propanoate 
have been isolated from any species of Aframomum.  
4.4.2.3 Identification of AA/R/PE-5 
AA/R/PE 5 was identified as myristic acid and was obtained as white amorphous solid.  The melting 
point was measured to be 55.3-55.5 °C, compared to the literature value of 54.4 °C. When subjected 
to IR spectroscopic (Appendix II) analysis, there was an absorption band of 2915.82 cm-1 and 
2848.62 cm-1 was assigned to C-H (stretching) and 1699.00 cm-1 assigned to C=O absorption. The 
stretching peaks at 719.91 and 939.23 cm-1 were due to O-H swinging or rocking mode, which are 
characteristics of the aliphatic chain of myristic acid. This characteristic behaviour of the O-H 
functionality of myristic acid has been reported by Rama Mohan et al, 201580.   
The 13C-NMR (Appendix IIIA and Appendix IIIB) showed the presence of 16 carbons with peaks 
occurring between δC 14-178. A peak at δC 178.4 was assigned to the carbonyl carbon of the 
compound whiles the remaining peaks between δC 14 - 34 were due to sp
3 hybridized carbons. From 
the DEPT 135° (Appendix IV) spectrum, a total of 14 methylene, 1 quaternary and 1 methyl carbons 
were observed with the methyl carbon at δC 14.1. The 
1H NMR spectrum (Appendix V), 4 distinct 
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peaks were observed between δH 0.89-2.34 which integrated for a total of 27 protons. Peaks at δH 
0.89 (t, 3H, J = 6.8 Hz) were assigned to methyl hydrogens of the methyl carbon. The methylene 
protons were observed at δH 1.28 (4H), δH 1.64 (t, 2H, J = 7.4 Hz) and δH 2.34 (t, 2H, J = 7.5 Hz), 
and δH 1.26 (s, 16H). The acidic hydrogen was not observed in the proton NMR and this may be as a 
result of an exchange between the compound and the solvent. From the HSQC spectrum (Appendix 
VI), protons δH 1.64 and δH 2.37 were assigned to the methylene carbons at δC 24.7 and δH 33.8, 
respectively while proton δH 1.28 was assigned to carbons δC 22.7 and δC 31.9. The HMBC spectrum 
(Appendix VII), showed the correlation between the carbonyl carbon at δC 178.4 and proton δH 2.34 
which is attached to the methylene carbon at δC 33.8. The methyl proton at δH 0.89 was also coupled 
to the methylene carbon at δC 31.8. From the COSY spectrum (Appendix VIII) there was coupling 
between proton δH 2.34 and δH 1.64 as well as δH 1.28 and δH 0.89.  A summary of the NMR data is 
shown in Table 4.8. 
 
 
 
 
Table 4.8: 1H, 13C, COSY and HMBC NMR data for AA/R/PE-2 
Signals Atom 1 δC δH /(J/Hz) COSY HMBC 
1 C 178.3   H2 
2 CH2 33.8 2.34, t (J = 7.53H) H12  
3 CH2 31.9 1.28  H14 
4 CH2 29.7 1.26   
5 CH2 29.7 1.26   
6 CH2 29.7 1.26   
7 CH2 29.6 1.26   
8 CH2 29.4 1.26   
9 CH2 29.4 1.26   
10 CH2 29.3 1.26   
11 CH2 29.1 1.26  H2, H12 
12 CH2 24.7 1.64 t (7.38Hz) H2 H2 
13 CH2 22.7 1.28 H14 H16 
14 CH3 14.1 0.89, t (6.84 Hz) H13  
 
 
 
 
 
 
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Figure 4.29: Structure of Myristic acid 
Myristic acid is found in coconut essential oil, palm kernel essential oil,  breast milk and animal fat85. 
It has several uses in the beauty industry. It is also used as surfactant, emulsifier and cleansing agent. 
One of its primary use is as a lubricant, due to its high rate of absorption by the skin. Myristic acid 
has been widely used for construction of phase modification materials for heat energy storage 
applications85.  
 
4.4.2.4 Identification of AA/R/PE-6  
AA/R/PE-6 was identified as stigmasterol. The melting point was measured to be 156-158 °C, 
compared to the literature value of 160-162 °C. When subjected to IR spectroscopic (Appendix IX) 
analysis, there was an absorption band of 3430 cm-1 which is characteristic of O-H (stretching), 2868 
cm-1 was assigned to C-H (stretching) and 1667 cm-1 assigned to C=C absorption. The 13C-NMR 
spectrum (Appendix X), showed the presence of 29 carbons with peaks occurring between δC 11-
145. Peaks at δC 121.84 and δC 140.93 for (C5) and (C6), respectively, which are sp
2 hybridized 
carbons, a peak at δC 71.89 which is due to C3 of stigmasterol was also observed. The angular 
methyls, C18 and C19 occurred at δC 11.8 and δC 19.3, respectively. From the DEPT 135 (Appendix 
XI) analysis, the spectrum showed that there were 3 quaternary, 11 methines, 9 methylene and 6 
methyl carbons. The 1H NMR (Appendix XII) showed characteristic steroidal proton signals 
between δH 0.7-2.4. Peaks at δH 0.70 (s, 3H) and δH 1.30 (s, 3H), were characteristic of the hydrogens 
of the angular methyls, C19 and C18, respectively. A signal at δH 3.54 (m) is due to the hydroxy 
proton at C3. The multiplicity of the hydroxy proton is due to coupling with H2 and H4. The signals 
at δH 5.05 (d, 1H), δH 5.18 (dd, 1H) and δH 5.37 (t, 1H) are the protons on C6, C22 and C23, 
respectively (Table 4.9). 
 
 
 
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Table 4.9: Comparative 13C-NMR chemical shifts of stigmasterol with literature 
Position Carbon type PE-6 δC86 
1 CH2 37.3 37.3 
2 CH2 29.2 31.6 
3 CH 71.8 71.8 
4 CH2 45.8 42.3 
5 C 140.8 140.8 
6 CH 121.7 121.7 
7 CH2 31.9 31.9 
8 CH 31.7 31.9 
9 CH 50.2 51.2 
10 C 36.2 36.5 
11 CH2 21.1 21.1 
12 CH2 39.8 39.7 
13 C 42.3 42.3 
14 CH 56.8 59.9 
15 CH2 26.1 24.4 
16 CH2 28.3 28.4 
17 CH 56.1 56.3 
18 CH3 11.9 11.0 
19 CH3 19.4 21.2 
20 CH 40.5 40.5 
21 CH3 19.8 21.2 
22 CH 138.3 138.3 
23 CH 129.3 129.3 
24 CH 51.2 51.2 
25 CH 40.5 31.9 
26 CH3 19.0 21.2 
27 CH3 18.8 19.0 
28 CH2 26.1 25.4 
29 CH3 12.0 12.1 
 
 
Stigmasterol is a plant sterol that has been isolated from several plants and to the best of my 
knowledge, this is the first time it has been isolated from a species of Aframomum. 
 
 Stigmasterol is known to inhibit tumor elevation in two stage carcinogenesis in mice86. Also, like 
cholesterol, it has been identified to regulate the activity of Na+/K+-ATPase in plants52. However, a 
mixture of stigmasterol and sitosterol is known to inhibit the hatching of N. americanus egg with an 
IC  of 9.4 μg/μl7750 . It is also known to have anti-inflammatory activity
87. 
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4.4.3 Investigation of the Dichloromethane (DCM) Extract 
Through chromatographic separation, an essential oil and 2 compounds labelled AA/R/DCM-1 and 
AA/R/DCM-3, respectively, were obtained from the DCM crude extract. AA/R/DCM-1 was 
characterized by identifying the constituent using GCMS (Table: 4.3). The antifungal property was 
also tested using C. albicans and S. cerevisiae (Figures 4.18, 4.19) 
 
4.4.3.1 Characterization of AA/R/DCM- 3 
AA/R/DCM 3 was obtained as a white solid with melting point 77-79 °C. When subjected to IR 
spectroscopy (Appendix XIII), peaks at 2915.60 cm-1, 2848.81 cm-1, 1734.88 cm-1 and 1706.24 cm-
1 were attributed to C-Hstr (sp
3), C-H 2str (sp ), C=Ostr and C=Cstr, respectively. A molecular formula of 
C19H32O4 was deduced from the HR-ESI-MS (Figure 4.31) with an observed m/z 325.2279 [M+H]
 + 
and calculated m/z 325.2376. The double bond equivalence was calculated to be 4.   
 
 
Figure 4.30: HR-MS spectrum of AA/R/DCM-3 
 
The 13C NMR spectrum (Figure 4.32) exhibited 18 signals, comprising 4 sp2 hybridized and 14 sp3 
carbons which occurred between δC 14 – 179. The signals at δC 179.1 and δC 173.3 were assigned to 
carbonyl carbons whereas the remaining 2 sp2 were assigned to alkene carbons. Two sp3 hybridized 
carbons attached to a heteroatom were observed at δC 68.9 and δC 62.1. 
 
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  Figure 4.31: 13C NMR spectrum of AA/R/DCM-3 
 
The DEPT 135 ° spectrum (Figure 4.33) revealed 2 quaternary carbons observed at δC 179.1 (C-1) 
and δC 173.3 (C-2).  Methine carbons occurred at δC130.0 (C-3), δC129 (C-4) and δC 68.6 (C-5).  At 
δC 14.1(-18), the signal was assigned to a methyl carbon 12 methylene and 1 methyl carbons. A 
methylene carbon attached to a heteroatom was observed at δC 62.1 (C-6). The remaining 14 signals 
were also identified as methylene carbons.  
 
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Figure 4.32: DEPT 135 spectrum of AA/R/DCM-3 
 
From the 1H NMR spectrum (Figure 4.34), 9 signals were observed between δH 0.88 - 5.50.  A methyl 
proton signal was observed at δH 0.88 (t, J = 6.9 Hz, 3H) whiles the methine signal were observed at 
δH 5.34 (m, 2H) and δH 5.26 (td, J = 5.1, 4.3, 1.6 Hz, 1H). The methylene protons were observed at 
δH 1.25 (s, 14H), δH 1.63 (m, 2H), δH 2.01 (m, 2H), δH 2.32 (m, 6H), δH 4.29 (dd, J =11.9, 4.3 Hz, 
1H) and δH 4.15 (dd, J = 11.9, 4.3 Hz, 1H).  
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Figure 4.33: 1H NMR of AA/R/DCM-3 
 
From the HSQC spectrum (Figure 4.35), the oxygenated methine carbon C-5 (δC 68.9) was assigned 
to proton δH 5.26 whereas the remaining 2 methine carbons C-3 (δC 130.1) and C-4 (δC 129.7) 
assigned to δH 5.34 (m, 2H).  The methylene carbon attached to an oxygen at C-7 (δC 62.1) was 
assigned to the protons at δH 4.29 and δH 4.15. The signal at δC 19.1 correlated with the proton at δH 
2.01, other methylene carbons at δC 34.0 (C7), δC 33.9 (C8) and δC 31.9 (C9) were assigned to proton 
δH 2.32. the proton signal at δH 1.25 was assigned to carbons C-10 (δC 29.7), C-11 (δC 29.4), C-12 
(δC 29.3), C-13 (δC 29.2), C-14 (δC 29.1) and C-15 (δC 22.7). The proton at δH 0.88 were attached to 
the methyl carbon at δC 14.1 (C18). 
 
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Figure 4.34: HSQC spectrum of AA/R/DCM-3 
From the COSY spectrum (Figure 4.36), 2 spin systems were observed. One spin system was 
between H-9 ( ) H-17 ( ), H-14 ( ) H-5 ( ), H-8 ( ) and H-6 (  and 
 ). The other spin system was identified as the aliphatic system, between protons H-7 
( ) -1 ( ) C-13 ( )  C-11 ( ) C-10 ( )  C-12 ( ) C-4 ( )  C-
3 ( )  C-10 ( ) C-16 ( ) and C-18 ( ) 
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Figure 4.35: COSY spectrum of AA/R/DCM 3 
 
From the HMBC spectrum (Figure 4.37), correlations between C-2/H-9 and C-2/H-5 were observed, 
suggesting a lactone. 
 
Further to this, a correlation between C-5/H-6 supported the first spin system observed in the COSY 
spectrum. 
 
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An HMBC correlation between C-1/H15 was observed coupled with the aliphatic spin system 
observed in the COSY spectrum was concluded to be an aliphatic ester moiety.  
 
The two structural moieties were connected based on the correlation between C1/H-6. 
  
 
Figure 4. 36: HMBC spectrum of AA/R/DCM 3 
The summary of the NMR data is presented in Table 4.10 
Table 4.10  1H, 13C, COSY and HMBC NMR data for AA/R/PE-2 
 δC DEPT δH HMBC COSY 
1.  179.1 C  H6, H15, H8  
2.  173.3 C  H5, H9  
3.  130.0 CH 5.35  H10 
4.  129.7 CH 5.35  H10 
5.  68.9 CH 5.26 H6 H9 
6.  62.1 CH2 4.15,4.29 H5  
7.  34.0 CH2 2.32  H15 
8.  33.9 CH2 2.32   
9.  31.9 CH2 2.32 H17 5,6 
29.7 CH  1.25   2
10.  
29.7 CH2 1.25  H16 
11.  29.4 CH  1.25   2
12.  29.3 CH  1.25   2
13.  29.2 CH2 1.25   
14.  29.1 CH2 1.25   
15.  24.9 CH2 1.62  H7 
16.  22.7 CH2 1.25 H17 H18, H10 
17.  19.1 CH2 2.01   
18.  14.1 CH3 0.88  H17 
 
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From the spectrometric and spectroscopic information (Table 4.10) was deduced to be 2-(6-
oxotetrahydro-2H-pyran-2-yl)ethyldodec-8-enoate (Figure 4.37). 
 
 
Figure 4.37: Structure of AA/R/DCM 3 
 
Due to the truncated nature of the HR-MS data (Figure 4.31), fragmentation patterns of the proposed 
structure could not be proposed. 
From a literature survey, the proposed compound has not been isolated from any species of 
Aframomum and other plant species. Hence for the first time, 2-(6-oxotetrahydro-2H-pyran-2-
yl)ethyldodec-8-enoate has been isolated from a plant. 
 
 
 
 
 
 
 
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CHAPTER FIVE 
5.0 Conclusion 
 In this current research, the constituents and antifungal activity of A. atewae essential oils were 
studied for the first time as well as characterization of compounds isolated from extracts of the 
rhizome. 
The essential oils were obtained by hydrodistillation of fresh plant parts (leaves and rhizomes) and 
chromatographic separation of crude extracts. GC-MS analysis indicated that the essential oils of the 
plant consist of various classes of terpenes and non-terpenes with the leaf EO characterizing 120 
compounds followed by the rhizome EO, 100 compounds and the DCM extract oil, 50 compounds. 
The fresh leaf essential was rich in monoterpenes (22.4%) while sesquiterpenes dominated the fresh 
rhizome essential oil (24.4%). The DCM-extracted oil was composed mainly of steroids (59.72%) 
and long chain hydrocarbons (18.06%) 
Several Aframomum species have been explored for essential oils but for the first time the following 
major constituents have been identified in the essential oils of A. atewae - 1-methyl-1-
(methylamino)isobenzofuran-3-one (17.27%), 2,5-ditertbutylhydroxybenzene (7.80%), and 14-
pregnane (56.95%) for fresh leaf essential oil, fresh rhizome essential oil and DCM-extracted 
essential oil, respectively. This variation in major components identified in A. atewae to previously 
studied Aframomum species may be as a result of extrinsic factors such as soil, climate, elevation and 
geographical origin. 
 
Fungicidal activity was observed for the rhizome EO and the DCM-extracted oil whereas the leaf EO 
and the PE-extracted oil exhibited a fungistatic activity against C. albicans. Apart from the PE-
extracted oil which was fungistatic against S. cerevisiae, the remaining oils were inactive. 
A total of 10 compounds were isolated upon column chromatographic separation of the various 
extracts obtained from air dried pulverized rhizome of A. atewae. Due to poor solubility, only 4 
compounds were characterized as 1(E)-8-methylundec-8-en-1-yl-3-(cyclohexa-2,5-dien-1-
yl)propanoate, myristic acid, stigmasterol and  2-(6-oxotetrahydro-2H-pyran-2-yl)ethyl dodec-8-
enoate using IR, NMR, LC-MS and HR-MS. 
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5.1 Recommendations 
The chemical composition of the PE-extracted oil should be acquired in order to attribute the observed 
activity against S. cerevisiae to a particular compound. Also, the isolated compounds should be tested 
for their antifungal activities.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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hosts. Eukaryotic cell, America Society for Microbiology 5(12), 1941–1949.  
 
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APPENDIX 
Appendix I 
 
 
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Appendix II 
 
 
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Appendix IIIA 
 
 
 
 
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Appendix IIIB 
 
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Appendix IV 
 
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Appendix V 
 
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Appendix VI 
 
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Appendix VII 
 
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Appendix VIII 
 
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Appendix IX 
 
 
 
 
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Appendix X 
 
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Appendix XI 
 
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Appendix XII 
 
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Appendix XIII 
 
 
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