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UNIVERSITY OF GHANA 
COLLEGE OF HEALTH SCIENCES 
SCHOOL OF PHARMACY 
DEPARTMENT OF PHARMACOLOGY & TOXICOLOGY 
 
 
SCREENING OF SELECTED GHANAIAN MEDICINAL PLANTS 
FOR ANTI-PROSTATE CANCER ACTIVITY 
BY 
JOSEPH KOFI ABANKWA (10382738) 
 
THIS THESIS / DISSERTATION IS SUBMITTED TO THE UNIVERSITY OF 
GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR 
THE AWARD OF MPHIL. PHARMACOLOGY DEGREE. 
 
JULY, 2018 
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DECLARATION 
I, Joseph Abankwa Kofi of Pharmacology and Toxicology Department, University of Ghana, 
Legon, hereby declare that this thesis is the result of my own research work carried out at the 
Noguchi Memorial Institute of Medical Research under the watch of a research assistant Mrs. 
Eunice Dotse and supervised by Prof.  Regina Appiah-Opong and Prof. Alexander Nyarko. 
 
 
…………………………….                       …………………………………. 
Joseph Kofi Abankwa                                                                  Date 
(Student) 
 
 
………………………………                                                  …………………………………. 
Prof. Alexander Nyarko                                                            Date 
 
 
………………………………                                              ……………………………………. 
Prof. Regina Appiah-Opong                                                  Date 
 
 
 
 
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DEDICATION 
I dedicate this work to God whose unfailing love, mercies, unimaginable divine favor and 
blessings saw me through this M.Phil. studies. I also dedicate this project to my father Jacob 
Kofi Abankwa whose encouragement, financial support and advice has brought me to this 
stage of life. I also dedicate this thesis to my mother Leticia Annor and my siblings Grace 
Ama Abankwa and Moses Kwesi Abankwa for all their words of comfort.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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ACKNOWLEDGMENT 
My sincerest appreciation goes to Prof.  Alexander Nyarko and Prof. Regina Appiah-Opong 
for all their invaluable contribution to this work. My special thanks go to Mrs. Eunice Dotse, 
Ms. Abigail Aning and Mr. Isaac Tuffuor for all the meticulous support I received in the 
determination of the total phenols, antioxidant activities and cytotoxic activities of my plant 
extracts. I wish to express my profound gratitude to all the lecturers and staff members of the 
School of Pharmacy, University of Ghana. My utmost thanks go to Mabel Owusu-Bediako 
and Georgina Ataa-Nyarko for their valuable support in this study.  
  
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ABBREVIATION 
A 
ABTS : 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) 
AQ : Aqueous 
ASR : Age-standardized incidence rates 
B 
BHT : Butylated hydroxyl toulene  
BRCA1 : Breast cancer 1 gene 
BRCA2 : Breast cancer 2 gene 
C 
CE : Capparis erythrocarpus 
COX2 : Cyclooxygenase 2 enzyme 
D  
DCM : Dichloromethane  
DMSO : Dimethyl sulfoxide 
DNA : Deoxyribonucleic acid 
DPPH : 2, 2-Diphenyl, 1-picrylhydrazil 
DRE : Digital rectal examination 
 
E 
EA : Ethylacetate 
ERα : Estrogen receptor α 
ETOH : Ethanol 
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F 
FA : Ficus asperifolia 
FAK : Focal adhesion kinase 
FBS : Foetal bovine serum 
FRAP : Ferric reducing ability of plasma 
G 
GAE : Gallic acid equivalent 
GSH : Glutathione 
GST : Glutathione S-Transferase 
 
H 
HPC1 : Hereditary prostate cancer 1 gene 
HPC2 : Hereditary prostate cancer 2 gene 
I 
IC50 : Inhibitory concentration at 50% 
IFN γ : Inteferon gamma 
IL-10 : Interleukin-10 
IL1-α : Interleukin 1 alpha 
IL-6  : Interleukin-6 
IL-8  : Interleukin-8 
 
J 
JC : Jatropha curcas 
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L 
LPS : Lipopolysaccharide 
LS : Latuca sativa  
M 
MIC-1 : Macrophage inhibitory cytokine 
MRI : Magnetic Resonance Imaging 
MTT : 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide 
 
MO : Moringa oleifera 
MP  : Mentha piperita 
N 
NADH : Nicotinamide adenine dinucleotide 
NADPH : Nicotinamide adenine dinucleotide phosphate 
NFKB : Nuclear factor kappa B 
NMIMR : Noguchi Memorial Institute for Medical Research 
NO : Nitric oxide  
O 
OD : Optical density 
P 
PA : Phyllanthus amarus 
PBS : Phosphate buffered saline 
PE : Petroleum ether  
PMSA : Prostate membrane specific antigen 
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PSA : Prostate specific antigen 
PSG : Penicillin Streptomycin 
PTEN : Phosphatase and tensin homolog 
R 
RNA : Ribonucleic acid 
RNS : Reactive nitrogen species 
ROS : Reactive oxygen species 
RPMI : Rosewell Park Memorial Institute 
RUNX  : Runt related transcription factor 2 
S 
SHBG : Sex hormone binding globulin 
SOD : Superoxide dismutase 
T 
TAEC : Trolox equivalent antioxidant capacity 
TNF-α : Tumour necrotic factor alpha 
TNF-β : Tumour necrotic factor beta 
TPC : Total phenolic content 
U 
UV : Ultra violet 
Z 
Zip1 : Zinc Transporter protein 
 
 
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Screening of selected Ghanaian medicinal plants for anti-prostate cancer activity. 
ABSTRACT 
Introduction: Prostate cancer is a major disease that affects adult males.  The high cost of 
current drugs in use, treatment failure/drug resistance as well as toxic side effects warrants the 
development of more effective, less toxic and affordable anti-prostate cancer drugs. The aim 
of the study was to investigate the anti-prostate cancer properties of selected Ghanaian 
medicinal plants used for the management of prostate cancer.  
 
Method: The leaves, fruit and seeds of selected plants claimed to have anti-prostate cancer 
properties were collected, dried and crushed. Each sample was sequentially extracted with 
solvents of increasing polarity (petroleum ether, dichloromethane, ethyl acetate, ethanol and 
aqueous) using cold maceration. The phenolic substances in the plants were determined by the 
Folin-Ciocalteau method. Antioxidant and anti-proliferative activities were determined using 
DPPH and MTT assays, respectively. 
 
Results: Nine medicinal plants namely, Latuca sativa, Tetrapleura tetraptera, Jatropha 
curcas, Capparis erythrocarpus, Ficus asperifolia, Mentha piperita, Carica papaya, Moringa 
oleifera and Phyllanthus amarus were identified. Among them, the ethanolic extract of P. 
amarus recorded the highest (92.71g GAE/100g) level of phenolic substances. The aqueous 
and ethanolic extracts of P. amarus showed the strongest antioxidant activities (EC50values of 
19.32 ± 1.13µg/ml and 60.03 ±1.07µg/ml, respectively). Ethyl acetate extract of Latuca sativa 
and Mintha piperita also showed significant (p<0.05) antioxidant activities (EC50=121.4± 1.07 
µg/ml, 91.13% and EC50=129.7± 1.07 µg/ml, 88.37%, respectively) but did not meet the 100 
µg/ml limit cut off for consideration as strong antioxidants. Most of the plant extracts 
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significantly inhibited proliferation of LNCaP and PC3 prostate cancer cell lines compared to 
normal prostate (PNT2) cells. The ethyl acetate extract of whole Tetrapleura tetraptera fruit 
showed the highest cytotoxic activity and was selective to PC3 (IC50 =5.39 ± 1.24 µg/ml, 
SI=7.72)and LNCaP (IC50=8.38±1.27µg/ml, SI=4.97) 
Conclusion: All the plants contained phenolic substances and had antioxidant activities. All 
the samples demonstrated anti-proliferative activities with ethyl acetate extract ofwhole 
Tetrapleura tetraptera exhibiting the strongest anti-proliferative activity against LNCaP and 
PC3 prostate cancer cell lines.  
Recommendation: Future studies should focus on investigating the phytochemical profile of 
Tetrapleura tetraptera, isolating the bioactive compounds responsible for the anti-prostate 
cancer activity and understanding the mechanisms involved in their anti-proliferative activity 
against LNCaP and PC3 cancer cells.   
 
Keywords: Medicinal plants, Secondary metabolites, DPPH, Antioxidants, Total phenolic 
content. 
 
 
 
 
 
 
  
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Contents 
DECLARATION ............................................................................................................................ ii 
DEDICATION ............................................................................................................................... iii 
ACKNOWLEDGMENT ................................................................................................................ iv 
ABBREVIATION ........................................................................................................................... v 
ABSTRACT ................................................................................................................................... ix 
Chapter 1 ......................................................................................................................................... 1 
1.0 Introduction ............................................................................................................................... 1 
1.1 Problem statement and Justification ...................................................................................... 6 
1.2 Hypothesis ............................................................................................................................. 7 
1.3 Aim ........................................................................................................................................ 7 
1.4 Objectives of the study .......................................................................................................... 7 
CHAPTER 2 ................................................................................................................................... 8 
2.0 Literature review ....................................................................................................................... 8 
2.1 Cancer .................................................................................................................................... 8 
2.1.1 Types and classification of Cancer ..................................................................................... 8 
2.1.2 Cancer risk factors .............................................................................................................. 9 
2.1.3 Prostate Cancer ................................................................................................................. 10 
2.1.4 Description of Prostate Cancer ......................................................................................... 12 
2.1.5 Pathophysiology of Prostate Cancer ................................................................................. 12 
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2.1.6 Clinical Manifestation ...................................................................................................... 13 
2.1.7 Prevalence of prostate cancer ........................................................................................... 13 
2.1.8 Diagnosis of Prostate cancer ............................................................................................ 14 
2.1.9 Treatment options and Limitation .................................................................................... 16 
2.2.0 Plants in the management of Diseases ............................................................................. 19 
2.2.0 .1 Carica papaya .............................................................................................................. 20 
2.2.0 .2 Phyllanthus amarus ...................................................................................................... 21 
2.2.0 .3 Moringa oleifera ........................................................................................................... 22 
2.2.0 .4 Jatropha curcas ............................................................................................................ 23 
2.2.0 .5 Mentha piperita ............................................................................................................ 24 
2.2.0 .6 F. asperifolia ................................................................................................................ 24 
2.2.0 .7 Tetrapleura tetraptera .................................................................................................. 25 
2.2.0 .8 Latuca sativa ................................................................................................................ 25 
2.2.0 .9 Capparis erythrocarpus ............................................................................................... 26 
2.2.1 Phytochemical Compounds .............................................................................................. 26 
2.2.2 Phytochemical compounds with anti-cancer properties ................................................... 27 
2.2.3 Synergic Medicine ............................................................................................................ 28 
2.2.4 Plant derived anti-cancer agents ....................................................................................... 29 
2.2.5 Approaches to Development of Anti-cancer Drugs ......................................................... 29 
2.2.5.1 Application of Tumor Cell Lines .................................................................................. 31 
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2.2.5.2.1 Cell lines used ............................................................................................................ 31 
2.2.5.2.1 Immortalized cell lines ............................................................................................... 32 
2.2.6 Assessment of antioxidant activity ................................................................................... 33 
2.2.6.2 FRAP assay ................................................................................................................... 34 
2.2.6.3 Trolox Equivalent Antioxidant Assay ........................................................................... 35 
CHAPTER 3 ................................................................................................................................. 36 
3.0 Materials and methods ............................................................................................................ 36 
3.1 Source of materials .............................................................................................................. 36 
3.2. Ethnobotanical /Literature Survey and Collection of Plants .............................................. 36 
3.2 Extraction Method ............................................................................................................... 37 
3.4 DPPH radical scavenging assay .......................................................................................... 39 
3.5 Cell Culture ......................................................................................................................... 41 
3.6 Preparation of test samples .................................................................................................. 41 
3.6.1Cell viability assay ......................................................................................................... 41 
3.8 Statistical analysis ............................................................................................................... 44 
3.6 Ethics Statement .................................................................................................................. 44 
Chapter 4 ....................................................................................................................................... 45 
4.0 Results ..................................................................................................................................... 45 
4.1 Total Phenolic Content (TPC) ............................................................................................. 47 
4.2 Antioxidant Activities of the Plant extracts ........................................................................ 56 
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4.3 In Vitro Cell viability Assay ................................................................................................ 64 
5.0 Discussion ............................................................................................................................... 78 
5.1.1 Latuca sativa .................................................................................................................... 80 
5.1.2 Tetrapleura tertraptera .................................................................................................... 81 
5.1.3 Capparis erythrocarpus ................................................................................................... 83 
5.1.4 Ficus asperifolia ............................................................................................................... 84 
5.1.5 Mentha piperita ................................................................................................................ 85 
5.1.6 Carica papaya .................................................................................................................. 86 
5.1.7 Moringa oleifera ............................................................................................................... 88 
5.1.8 Phyllanthus amarus .......................................................................................................... 89 
6.0 Conclusion and Recommendation........................................................................................... 92 
6.1 Conclusion ........................................................................................................................... 92 
6.2 Recommendation ................................................................................................................. 92 
APPENDIX A ............................................................................................................................. 123 
APPENDIX B ............................................................................................................................. 124 
APPENDIX C ............................................................................................................................. 126 
 
 
 
  
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List of Figures 
Figure 1A: Incidence and mortalities of prostate cancer across all ages…………………......14 
Figure 1B: A scheme demonstrating approaches to cancer drug development……………....30 
Figure 1C: DPPH reaction……………………………………………………………………34 
Figure 2A: A schematic diagram showing the procedures in DPPH antioxidant assay……...39 
Figure 2B: A schematic diagram showing the procedures in determining the TPC………….41 
Figure 2C: A schematic diagram showing the procedures used in MTT assay……………....43 
Figure 3A: Standard calibration curve of Gallic acid…………………………………………48 
Figure 3B: Total phenolic contents of P. amarus (A) and T. tetraptera seed (B) extracts…...50 
Figure 3C: Total phenolic contents of T. tetraptera pulp (C) andL. Sativa(D)extracts……..51 
Figure 3D: Total phenolic contents ofM. Piperita(E)and C. erythrocarpus (F)extracts…...52 
Figure 3E: Total phenolic contents ofJ. curcas(G)and F. asperifolia(H)leaf extracts…….....53 
Figure 3F: Total phenolic contents ofM. Oleifera(I)andwhole T. tetraptera fruit 
(J)extracts………………………………………………………………………………………..
.54 
Figure 3G:  Total phenolic contents of C. papaya extracts……………………………….......55 
Figure 4A:  Antioxidant activities of aerial parts of P. amarus (A) and whole T. tetraptera 
fruit (B) sequentially extracted using various solvents……………………………………….61 
Figures 4B: Antioxidant activities of C. papaya leaves extract (C), T. tetraptera seed extract 
(D), M. piperita leaves extract (E)and L. sativa leaves(F)sequentially extracted using various 
solvents……………………………………………………………………………………….62 
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Figures 4C: Antioxidant activities of F. asperifolia leaves (G), C. erythrocarpus (H), T. 
tetraptera pulp(I), J. curcas leaves(J) and M. oleifera leaves (K) sequentially extracted using 
various solvents (PE, DCM, EA, ETOH and AQ)…………………………………………...63 
Figure 5A: Representative plot of the cytotoxic profile of P. amarus extracts on PC3(Figure 
A. and Figure B.) and LNCaP (Figure C. and Figure D.) prostate cancer cell 
lines……………………………………………………………………………………….…..64 
Figure 6A: Anti-proliferative activities of Curcumin (A), P. amarus extracts (B) and C. 
erythrocarpus (C) extracts on PC3 prostate cancer cell line………………………………….69 
Figure 6B: Anti-proliferative activities of J.  curcasextracts (D), L. sativa extracts (E), M. 
piperita  extracts (F) and M. oleifera extracts (G) extracts on PC3 prostate cancer cell line...70 
Figure 6C: Anti-proliferative activities of F. asperifoliaextracts(H), C. papaya 
extracts(I),whole T. tetrapteraextracts(J) , T. tetraptera  pulp extracts (K)  and T. tetraptera 
seed extracts on PC3 prostate cancer cell line.……………………………………………....71 
Figure 7A: Anti-proliferative activities of Curcumin (A), P. amarus extracts (B) and C. 
erythrocarpus (C) extracts on LNCaP prostate cancer cell line………...……………………72 
Figure 7B: Anti-proliferative activities of F. asperifoliaextracts (D), L. sativa extracts (E), M. 
piperita extracts(F) and J. curcas (G) extracts on LNCaP prostate cancer cell line…………73 
Figure 7C: Anti-proliferative activities of M. oleifera (H), C. papaya (I) , whole T. tetraptera 
(J), T. tetraptera pulp (K) and T. tetraptera seed extracts on LNCaP prostate cancer cell 
line…………………………………………………………………………………………….74 
Figure 8A: Anti-proliferative activities of Curcumin (A), C. erythrocarpus extracts (B) and C. 
papaya (C) extracts on PNT2 normal prostate cell line…………………………………........75 
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Figure 8B: Anti-proliferative activities of F. asperifolia extract (D), J. curcas extract (E), L. 
sativa extracts (F), M. oleifera extracts(G), T. tetraptera pulp extract (H) and whole T. 
tetraptera fruit extract (I) on PNT2 normal prostate cell line……………...…………………76 
Figure 8C: Anti-proliferative activities of M. piperita leaf extract (J), P. amarus leaf extract 
(K) and T.tetraptera seed extract (L) and on PNT2 normal prostate cell line………………77 
  
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LIST OF TABLES 
Table 1: Ethnobotanical information of medicinal plants........................................................................ 5 
Table 2: Identityof medicinal plants and their voucher specimen number ............................................ 45 
Table 3: Percentage yield of medicinal plants ....................................................................................... 46 
Table 4: EC50 antioxidant values of plant extracts ................................................................................. 60 
Table 5: Cytotoxic activities and selective indices of plant extracts for cancer and normal cell lines. . 67 
 
 
 
 
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Chapter 1 
1.0 Introduction 
Cancer refers to the uncontrollable proliferation of a cell with the ability to metastasize to 
other areas of the body (Soladoye et al., 2010). Earlier reports have suggested that cancerous 
growth is parasitic and thrives well at the cost of the human body. Elledge (2009) has 
indicated that cancer cells have hall marks which include resistance to cell death, unlimited 
proliferative activity and are self-sufficing in cell growth signaling. Globally, various forms of 
cancers of the breast, liver, cervical, prostate and many more have been acknowledged and 
reported to have a great negative impact onour economy today. Jemal et al.(2011) has 
reported that cancer is the leading cause of deaths in many economically developed 
countries,killing about 7.6 million people in 2008. In a period of 9 years (1991-2000), the 
Korle bu Teaching Hospital, one of the biggest hospitals in Ghana, reported about 3659 deaths 
related to cancer (Wiredu and Armah, 2006). Wiredu and Armah (2006) reported the major 
types of cancer with high mortality rateamong females to be breast cancer (17.25%) and 
heamatopoietic malignancies (14.69%)and liver (21.15%) and prostate cancer (17.35%) being 
the highest in men. 
 
Prostate cancer has been described to be the growth of malignant tumor in the prostate gland 
of the reproductive system in males (Alotaibi et al., 2017). It is reported to be the succeeding 
principal cause of cancer associated death after hepatocellular carcinoma in Ghanaian men 
(Wiredu and Armah, 2006). A few of the attributed risk factors to cancer as listed by the 
National Cancer Institute include old age, genetics, race and genealogy of prostate cancer 
(Pienta and Esper, 1993). Also, Bostwick et al.(2004) have reported lifestyle practices such as 
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smoking, excessive alcohol intake, lack of exercise and eating of meat to be some risk factors 
to prostate cancer. Pham-Huy et al. (2008) have revealed that free radicals and related reactive 
species generated from some of such lifestyle practices are implicated in prostate cancer hence 
attracting a great deal of alertness for a therapeutic strategy. It is noteworthy that, the reactive 
oxygen species (ROS) and reactive nitrogen species (RNS) are induced in the body through 
various exogenous sources and endogenous processessuch as oxygen metabolism, 
mitochondria oxidative phosphorylation, protein glycation and lipid peroxidation(Valko et al., 
2007; Pacher et al., 2007). These reactive radicals react with protein, genes, DNA and cause 
specific tumor suppressors and proto-oncogenes to become deregulated at organ sites (Taylor 
et al., 2010; Genestra, 2007; Baca et al., 2013; Barbieri et al., 2012). In Ghana, prostate 
cancer is documented to have the second highest mortality rate of 17.35% among 286 males 
(Wiredu and Armah, 2006). Laryae et al. (2014) also indicated that prostate cancer is the 
second most regular cancer case recording 13.2% among 76 males at the Komfo Anokye 
Teaching Hospital (KATH). Different reports have shown that, prostate cancer can be more 
aggressive and may advance to a castration-resistant disease which is mostly common in 
blacks (Gueye, 2003; Yarney, 2013 and Clegg-Lamptey, 2009). Besides, inheriting a faulty 
tumor suppressor gene (BRCA 2, BRCA 1, HPC 1, HPC 2 and p53) can make the disease 
severe (Benafif and Eeles, 2016; Yeboah et al., 2016). 
 
Available treatment options for prostate cancer include surgery, chemotherapy, radiation 
therapy and hormone therapy.The choice of treatments largely depends on the stage and the 
gleanson score of the prostate cancer. Oral chemotherapy which is usually administered by a 
medical oncologist often consists of giving the anti-cancer agent a specific number of cycles 
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over a set period of time. In general, standard chemotherapy begins with docetaxel (Docefrez, 
Taxotere) combined with a glucocorticoid prednisone. However, some of the cancers tend to 
resistant to the anti-cancer agents as some prostate cancer cells continue to grow (Petrylak et 
al., 2004; Tannock et al., 2004). Other substitutes such as mitoxantrone and carbazitaxel 
(Jevtana) have been reported to be toxic and do not improve lifespan (Tannock et al., 2004). 
In general, the side effects may depend on the individual, the type of chemotherapy, the dose 
used and the length of treatment. Prostate cancer treatment is expensive and some such as 
radiation and chemotherapies have toxic effects on non-targeted tissues (Fadeyi et al., 2013). 
Desai et al.(2008) have reported inherent problems such as myelotoxicity, renal toxicity, 
cardiotoxicity and many more associated with oral chemotherapy. Due to these unwanted side 
effects, the search of other natural sources of drugs for treatment is of interest to many 
researchers. 
 
Nature has blessed mankind with medicinal plants which are believed to have reduced side 
effects (Greenwell, 2015). Okwu (2004) has reported that these medicinal plants provide a 
rich supply of antioxidants which are able to scavenge these free radicals. Many therapeutic 
benefits of the medicinal plants are due to the high contents of superoxide dismutase, vitamin 
A, vitamin C, catalase, glutathione reductase, glutathione peroxidases and Vitamin E 
(Hercberg et al., 2004; Halliwell, 2005). Also, Giustarini et al.(2009) have indicated that 
antioxidants could be used in the treatment and management of oxidative stress associated 
diseases like prostate cancer. In addition, Raman (2006) has indicated that medicinal plants 
also contained anti-cancer therapeutic phytochemical compounds which include alkaloids, 
saponins, tannins, flavonoids, anthraquinones, cardiac glycosides and terpenoids. 
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Africa hosts about 57,704 plant species (Bramwell, 2002). Although there are volumes of oral 
histories of the therapeutic benefits of these plants, the scientific evidence of the claims are 
lacking compared to Chinese and Indian herbal medicines (Cordell et al., 1991). Hence, it is 
useful to review the claims that have been made and assess the quality of evidence available 
for better documentation of claims for research and development. 
 
In Ghana, herbalist use medicinal plants and other herbal products for the management and 
treatment of prostate cancer (Mshana et al., 2000). For example, Ghanaian medicinal plants 
such as Croton membranaceous and Zanthoxylum xanthoxyloides have been shown to possess 
high cytotoxic activities against cancer cell lines and their use as anti-cancer agents are 
supported (Ayim et al., 2007). In this study, an ethnobotanical survey was conducted to 
identify other medicinal plants claimed to be useful for management of prostate cancer in 
Ghana. The plants identified are Latuca sativa, Tetrapleura tetraptera, Jatropha curcas, 
Capparis erythrocarpus, Ficus asperifolia, Mentha piperita, Carica papaya, Moringa oleifera 
and Phyllanthus amarus. Information gathered on the plants are summarized in Table 1. 
 
 
 
 
 
 
  
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Table 1: Ethnobotanical information of medicinal plants 
 
 
  
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1.1 Problem statement and Justification 
In many developed countries, cancer cases are rising and cancers are principal causes of death 
due to lifestyle changes, late reporting of the disease and treatment problems (Jemal et al., 
2011; Segbefia et al., 2013). Globocan reports in 2012 indicated that globally, diagnosis stood 
at about 14.9 million cases with about 8.2 million deaths (Globocan, 2012). Also, the cost of 
orthodox medicine is often prohibitive to many individuals. The World Cancer Report valued 
cancer-related expenses at $1.16 trillion per annum as of 2010 (World Health Organization, 
2014). 
 
In Ghana, prostate cancer is prevalent among adult men and has led to the death of many 
(Wiredu and Armah, 2006; Yeboah et al., 2014). Currently, cancer drugs in use have been 
reported to be expensive, toxic and have not fulfilled their therapeutic expectations (Zhang et 
al., 2009; Hirose et al., 2011). Additionally, existing plant derived anti-cancer drugs such as 
vinblastine, taxol, paclitaxel have been shown to have side effects, ineffective due to drug 
resistance and are very expensive. Eid et al. (2015) have reported that 30-80% of cancers are 
resistant to anti-cancer drugs. This major setback necessitates the search for other sources for 
the treatment and management of the disease. Hence, the need to search the Ghanaian plant 
diversity for more effective, less toxic, affordable and readily available anti-prostate cancer 
drugs. Information obtained from the ethnomedical survey in Table 1 indicate that some of the 
selected plants are reported to have anti-cancer activity against cancer cell lines such as breast 
cancer, leukemia, lung cancer but not prostate cancer. Although, P.amarus is reported to have 
anti-proliferative activity on metastatic prostate cancer cells (DU 145), its effects on androgen 
dependent prostate cancer cell line has not yet been assessed. The same is true for Carica 
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papaya. Furthermore, though some of the selected plants are reported to have antioxidant 
properties, their cytotoxic potential against prostate cancer cell lines have not been assessed. 
Therefore, there is need to evaluate the effectiveness of the nine plants as antioxidants and 
anti-prostate cancer agents. 
 
1.2 Hypothesis 
The selected Ghanaian medicinal plants may have strong antioxidant and anti-prostate cancer 
properties. 
1.3 Aim 
The aim of the study was to investigate medicinal plants used in Ghana for the management of 
prostate cancer and determine their anti-cancer activity. 
1.4 Objectives of the study 
1. To identify and select plants used in Ghana for the management of prostate cancer. 
2. To determine the total phenolic content of crude extracts of the identified plants using 
the Folin-Ciocalteau reagent. 
3. To evaluate the total antioxidant activities of the extracts using the free radical 
scavenging assay. 
4. To screen the selected plant extracts for cytotoxic activities against human prostate 
cancer cell lines PC3 and LNCaP using the tetrazolium-based colorimetric (MTT) 
assay. 
  
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CHAPTER 2 
2.0 Literature review 
2.1 Cancer 
Cancer is the rapid growth of malignant cells and can move to various parts of the body 
(Gbadamosi and Erinoso, 2016). Bolarin (2009) has indicated that malignant tumors grow by 
invasion, infiltration and have the tendency to reappear after surgical treatment. Additionally, 
Hanahan (2011) reported that cancer cells can evade apoptosis by altering the apoptotic 
signaling pathway. Globocan reports indicated that about 14.1 million new cases and 8.2 
million deaths occurred around the world (Globocan, 2012). It is estimated that the number of 
cancer related deaths may rise to 12 million by 2030 (Gbadamosi and Erinoso, 2016). The 
Globocan (2012), reports indicated that there were about 850,000 new cancers and almost 
600,000 deaths recorded in Africa. 
 
Studies in Ghana, have indicated that the age-standardized incidence rates (ASR) of cancers 
for males was 10.9/100,000 and 22.4/100,000 for females (Laryae et al., 2014). The reports 
indicate that cancer is a global burden. 
2.1.1 Types and classification of Cancer 
Cancer can be categorized based on the symptoms presented, cellular make up and the 
locations of the cells from which they originated. Classification may be based on categories 
reported by Sorensen et al. (1998), which include lymphomas, carcinomas, sarcomas and 
myelomas.  
 
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Lymphoma is a group of blood cancer that develop from lymphocytes and presents signs 
which include enlarged lymph nodes, fever, unintended weight loss and drenching sweats. 
Carcinoma is another type of cancer that occurs in cells that line the tissues of an organ such 
as the kidney, liver and prostate. 
 
Sarcomas are known to arise from transformed cells of the mesenchymal connective tissue 
origin such as bone and cartilage whiles melanomas were reported to develop from the 
pigment-containing cells known as melanocytes particularly in the skin and can spread to 
other organs in the body. 
 
Myeloma is a type of cancer of the plasma cells that prevent the normal production of 
antibodies, therefore compromising the body’s immune system and making it susceptible to 
infection. Glioma is a form of cancer that occurs in glial cells in the spine and the brain. 
 
Histological classification of cancers is established on determining the kind of cells present 
and their origin (Goldstein, 2010). Reports by Jemal et al. (2005) have indicated that, the most 
prevalent sites of cancer in men are prostate, lung, colon and rectal while cancer of the breast, 
cervix and ovary has been reported in women. 
2.1.2 Cancer risk factors 
The proliferation of cancer cells is undesirable, parasitic and occurs rapidly in the human body 
(Olapade-Olaopa et al., 2014). Numerous studies show that malignant neoplasm can arise 
from a broad group of diseases such as hepatitis C and B and human papilloma virus (Perz, 
2006; Trichopoulos, 1996). Viruses, bacteria and parasites are the common infectious agents 
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that cause DNA damage and invoke mutations causing irregularities in the gene function 
(Trichopoulos, 1996). Reports by Gbadamosi and Erinoso (2016) indicated that, risk factors to 
cancer in Africa may be attributed to numerous factors such as occupation, weather, lifestyle, 
rural area habitation, trauma, virus, food, pressure, drink and drugs. Other established factors 
include sex, age, obesity and race (Feigelson et al., 2004). Additionally, Danaei et al. (2005) 
have identified some general environmental factors that may give rise to cancer related death. 
These include radiation (both ionizing and non-ionizing), environmental pollutants, excessive 
tobacco smoking, fatty diets, infections and stress. These carcinogenic agents lead to the 
production of free radicals that induce oxidative stress. Additionally, the induced oxidative 
stress is very harmful and can damage the DNA, lipids and protein, leading to an increased 
risk of cancer development (Iweala, 2015). 
2.1.3 Prostate Cancer 
Prostate cancer is a form of cancer that occurs in the cells of the prostate gland and has the 
ability to metastasize to other areas of the body. The prostate is an exocrine gland that rest 
under the bladder of the male reproductive system. Early growth of prostate cancer has been 
documented to be androgen driven and normally occurs in the seminal prostate gland cells 
(Huggins and Hodges, 1941). The growth and development of the male reproductive system is 
primarily influenced by androgens which includes testosterone. Research shows that, early 
stages of prostate cancer growth require 5α-dihydrotestosterone derived from testosterone 
produced by the 5α-reductase enzyme system. On the other hand, other studies have shown 
that aromatase enzyme is able to convert testosterone to estradiol which binds to the estrogen 
receptor α (ERα) and act as transcription factors for prostate cancer cell proliferation (Hu et 
al., 2012). Amazingly, hormonal therapy or androgen ablation has been used for the treatment 
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and management of androgen dependent prostate cancer (Paul and Bruel, 2000). Yet, health 
complications have been associated with the use of androgen deprivation therapy. These 
complications include genetic damage from carcinogenic estrogen metabolites and increase 
incidence of osteoporosis (Kim and Freeland, 2010; Wibowo et al., 2010). Also, prostate 
cancer cells are able to adapt to the androgen deprived environment by generating hyper 
active receptors with broadened activation spectra (Hsieh, 2008). 
 
A report by Laufer et al. (2000) has indicated that some prostate cancer cells can be androgen 
independent after prolonged clinical remission from the disease. Hence, hormonal 
intervention becomes futile and void as a result of mutations in the androgen receptor and 
amplification or loss of receptors (Wang and Uchida, 1997; Henshall et al., 2001; Kinoshita et 
al., 2000). 
 
There are several types of unusual proliferation of cells in the prostate tissue. Generally, most 
prostate cancers are classified as adenocarcinoma (Bostwick, 1989). The few forms of cancers 
in the surrounding tissues of the prostate gland may include transitional cell (urothelial) 
cancer, ductal adenocarcinoma, squamous cell cancer, sarcomas and carcinoid. Also, several 
studies have shown that chronic inflammation is implicated in prostate cancer cell (Gurel et 
al., 2014; De Nunzio et al., 2011). Furthermore, some innate and adaptive immune cells such 
as inflammatory cytokines (IL-8 and IL-1α) and cyclooxygenase 2 enzyme (COX2) producing 
prostaglandins, have been reported to play a role in prostate cancer progression (Salazar et al., 
2013; Shiao et al., 2016).  
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2.1.4 Description of Prostate Cancer 
Studies have shown that, prostate cancer is heterogeneous in terms of its biological and 
molecular characteristics, ploidy, grade, oncogene expression and tumor suppression gene 
expression (Ahmed, 2012). In addition, many prostate cancer cells have been reported to 
break off and invade neighboring tissues (especially local lymph nodes and skeletal bone) and 
they grow faster than normal healthy cells (Gbadamosi and Erinoso, 2016). 
2.1.5 Pathophysiology of Prostate Cancer 
In adenocarcinoma, the normal semen-secreting prostate gland cells mutate into cancer cells, 
multiply and spread to the surrounding tissue of the prostate. In this case, the prostate that 
surrounds part of the urethra grows rapidly and eventually blocks or affect urination and 
ejaculation. Also, in prostate cancer some genes that suppress tumour growth are silenced. For 
example, the tumour suppressor gene product for the gene SLC39A1 (ZIP1) which transport 
and accumulates zinc in the prostate for production of citrate, a major component in semenare 
silenced (Costello and Franklin, 2006). Furthermore, there is upregulation of RUNX2 (a 
transcription factor that prevents apoptosis) and macrophage inhibitory cytokine-1 (MIC-1) 
which stimulates the focal adhesion kinase (FAK) signaling pathway which leads to survival 
and growth of prostate cancer (Senapati et al., 2010). Also, the downregulation of a tumour 
suppressor PTEN gene, E-cadherin and CD 44 have been observed. Prostate specific 
membrane antigen has been reported to increase available folates by hydrolyzing glutamated 
folates which stimulates the development of prostate cancer. 
 
Oxidative stress caused by the presence of free radicals (ROS and RNS) have also been 
implicated in some adenocarcinoma (Valko et al., 2007; Parker et al., 2007). The free radicals 
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have been reported to increase as one ages due to the decreased levels of vitamin D, 
glutathione and tumor suppressor protein (p53)(Minelli et al., 2009). Also, inflammation has 
been shown to increase free radicals whiles modulation of androgen have been reported to 
produce hydrogen peroxide (Minelli et al., 2009). Increased oxidative stress has been related 
to prostate cancer and that supplementation using antioxidants have the potential to protect the 
prostate gland against prostate cancer (Khandrika et al., 2009; Chen et al., 2001; Wang et al., 
2015). 
2.1.6 Clinical Manifestation 
The severity of prostate cancer disease increases with age and it is more prevalent in older 
individuals between 40 years and 80 years (Stangelberger et al., 2008; Yeboah et al., 2016). 
The various reported symptoms of prostate cancer include swelling of the prostate gland, 
blood in urine, blood in semen, nausea, pain, and difficulty urinating (Talcott, 2003). Also, 
pains in the bones, loss of bladder control and weak urinary stream have been implicated in 
prostate cancer. However, these symptoms may also arise due to other issues.  
2.1.7 Prevalence of prostate cancer 
It has been reported that 75% of all new prostate cancer cases are diagnosed in developed 
countries with global prostate cancer occurrence accounting for about 899,000 new cases, and 
about 258,000 deaths each year (Alotaibi et al., 2017). Also, cancer is projected to rise to 24 
million by 2035, with consequent increase in expected deaths (Alotaibi et al., 2017). Figure 1 
shows the mortality and incidence of prostate cancer across all ages in different areas of the 
world. The chart shows high incidence with low mortalities due to prostate in Australia / New 
Zealand and more developed countries. In contrast, though in Western Africa the incidence of 
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prostate cancer was lower, mortality rates associated with prostate cancer is high (Globocan, 
2012; Hassanipour-Azgomi,2016). 
 
Figure 1A: The figure above showsthe incidence and mortalities of 100,000 individuals 
suffering from prostate cancer across the ages in different areas of the world (Globocan, 
2012).  
 
In Ghana, 17.35% (n=286) of male cancer death is related to the prostate (Wiredu and Armah, 
2006). The prevalence of the disease has been reported to be high among Ghanaian men 
(Yeboah et al., 2016). Reports by Laryae et al. (2014) also indicated that prostate cancer is the 
second most common cancer cases recording13.2% (out of 76 males) at the Komfo Anokye 
Teaching Hospital (KATH).  
2.1.8 Diagnosis of Prostate cancer 
Diagnosis of prostate cancer helps in determining and managing the disease through various 
tests and therapies. Some of the approved test include digital rectal examination (DRE), 
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biomarker test, biopsy, prostate specific antigen test (PSA), prostate cancer antigen 3 (PCA3) 
test, trans-rectal ultrasound test, magnetic resonance imaging and many more. Physical 
examination of the prostate gland using the digital rectal examination (DRE) helps to identify 
abnormal parts of the prostate gland. The presence of prostate specific antigen (PSA) in either 
the urine or the blood is expressed by abnormal prostate gland cells and usually used for the 
screening of prostate cancer (Polascik et al., 1999). High levels of PSA suggest the existence 
of an inflammation in the prostate gland. Recent studies have, however, shown that some 
benign tumours have higher PSA levels (>4 ng/ml) hence PSA levels could have utility 
limitation in determining precisely the state of the tumour as being cancerous or benign (Loeb 
and Schaeffer, 2009). The precise state of the prostate gland as to whether it is cancerous or 
benign can be confirmed by biopsy. Biopsies involve the removal of a small amount of tissue 
for examination under the microscope. Gleanson score and pathological staging are additional 
tests done to identify the aggressiveness and size of the cancer cells in order 
toinformurologists of appropriate treatment plan. 
 
Specific biomarkers such as prostate specific membrane antigen (PSMA) which is produced 
in two different forms can help in differentiating a benign prostate gland tissue from cancer of 
the prostate gland (Su et al., 1995; Chang, 2004). Prostate specific membrane antigen (PSMA) 
is a transmembrane protein which forms immunoconjugates with anti-PSMA monoclonal 
antibodies 7E11 and still remains a therapeutic target (Chang, 2004). Immuno-histochemistry 
expression of PSMA in prostate specimens has been described, hence making it a useful 
diagnostic tool (Bostwicket al., 1998). 
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2.1.9 Treatment options and Limitation 
The rationale behind the treatment is to exterminate cancer. Treatment options include 
radiation therapy, immunotherapy, surgery, hormone therapy, targeted therapy, chemotherapy 
and traditional herbal medicine (Gbadamosi and Erinoso, 2016). Combination chemotherapy 
has been the basis for treating cancer (Waterhouse et al., 2006). In cancer chemotherapy, anti-
cancer agents are used in the treatment of prostate cancer. These chemotherapeutic agents 
include anti-metabolites, alkylating agents and natural products (Dardi et al., 2012). 
Chemotherapy is the use of drug (natural or synthetic) to annihilate or inhibit the growth of 
malignant tumours. Chemotherapy may be administered as a primary therapy or adjuvant 
chemotherapy. Palumbo et al. (2013) has classified cytotoxic agents as anti-metabolites (such 
as methotrexate and cytarabine), alkylating agents (such as cyclophosphamide and 
oxaliplatin), topoisomerase inhibitors (such as etoposide and irinotecan), anti-tumour 
antibiotics (such as doxorubicin and bleomycin) and microtubule stabilizers (such as 
paclitaxel and docetaxel). Drugs used for cancer treatment could be administered orally or 
intravenously. During drug distribution, the bioavailability of the drug act on rapidly growing 
cells, especially metastasized malignant cells. The toxic effects of these drugs are due to the 
inability to target not only cancer cells but affect all cells including normal cells.  
Antimetabolites are chemical compounds that have a similar structure to the actual metabolite, 
such as altered nucleotides, and act to impair DNA replication machinery, thus preventing cell 
proliferation. Leumann (2005) has reported the pharmacodynamic action of anti-metabolites 
as directly integrated into DNA or RNA and halting cell growth by impairing DNA 
replication machinery. 
 
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Alkylating agents are highly reactive compounds that interfere with DNA replication by 
adding alkyl groups to nucleic acids such as guanine base of DNA molecule.  
 
Topoisomerase inhibitors as anti-cancer agents have been shown to block the action of 
topoisomerase I and II which is responsible for unwinding DNA for replication (Vladimir et 
al., 2017). Also, other agents (anti-tumour antibiotics) such as anthracyclines have been 
reported to intercalate with the DNA and indirectly affecting topoisomerase II whiles 
mitomycin-C act more likely as an alkylating agent. The use of these drugs as anti-cancer 
agents has been successful due to different mechanisms of action on highly dividing cells 
during mitosis and the dependence on biomolecules by cancerous cells as compared to normal 
cells. However, these agents have been reported to target normal cells such as bone marrow 
cells, hair generating cells and cells lining the alimentary, which form the basis of their side 
effects such as alopecia, nausea, bone marrow depression resulting in anaemia and vomiting 
(Palumbo et al. 2013). 
The growth of some cancers such as breast and prostate cancers in the body primarily depends 
on hormones. Thus, blocking the hormone concerned is important in treating such cancers. 
For example, the administration of a hormone-like drugs that block receptors (estrogen 
receptor α) or agents that stop the production of hormones in the prostate slows the primary 
growth of prostate cancer(Feldman and Feldman, 2001). In prostate cancer, drugs like 
bicalutamide, prednisone, have been used to block receptors on prostate gland cell from 
receiving testostorene. Although hormonal therapy provides a different therapeutic strategy it 
is not devoid of inherent side effects such as the lack of sexual drive or libido and erectile 
dysfunction. Other side effects include weight loss, osteoporosis and bone fracture.   
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Immunotherapy is a form of therapy which involves the use of biological agents that mimics 
growth signals and restrict tumour growth. These drugs include monoclonal antibodies, T-cell 
therapy and cancer vaccines are able to stimulate the body to produce immune cells to fight 
cancer cells via either by active immunity or passive immunity. Some examples of 
monoclonal antibodies are rituximab, adalimumab, bevacizumab, infliximab among others are 
administered to fight cancer. Sudhakar (2010) has reported the use of Sipuleucel-T as an 
immunotherapy drug which destroys prostate cancer. However, side effects such as 
cardiotoxicity, nausea, congestive heart failure head ache and optic neuritis has been 
implicated with this kind of treatment (Kaltsonoudis et al., 2014; Gasparyan et al., 2012) 
 
Radiation therapy involves the use of high doses of ionizing radiation to effectively and safely 
treat cancer. It can be used to target the malignant tumour and functions by destroying the 
DNA of the cancerous cell. Fuccio et al. (2012) have reported side effects such as diarrhea 
and sore mouth.  
These aforementioned cancer treatments have documented side effects, reduced efficacy, face 
challenges of drug resistance, high cost and lacks patient compliance. Also, experts who 
perform surgical operations and radiation therapies are inadequate in Ghana, making treatment 
of prostate cancer difficult. These limitations have necessitated the search for affordable, 
safer, less toxic and efficacious anti-cancer agents (Fadeyi et al., 2013). 
 
Plant biodiversity has since time immemorial served as sources of medicines and many 
modern drugs have had plants as their source of origin.  For example, in the case of anti-
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cancer agents, vinca alkaloids, vinblastine, and vincristine have been isolated from the plant 
Catharanthus roseus (Gueritte and Fahy, 2005). Medicinal plants like Croton membranaceous 
and Zanthoxylum xanthoxyloides have also shown high cytotoxic activities against cancer 
cells and have potential use as anti-cancer agents (Ayim et al., 2007). However, some of the 
plants derived anti-cancer agents have also been shown to have side effects such as 
cardiotoxicity, kidney toxicities, and liver toxicities (Chen, 2013; Iqbal, 2017).  
2.2.0 Plants in the management of Diseases 
Medicinal plants are of huge significance to human health and are used to alleviate, treat 
disease or modify physiological processes (Rates, 2001). According to the reports by Kong et 
al. (2003) there are more than thirty-five thousand (35,000) plant species used as medicinal 
plants around the world. Medicinal plants have been used since time immemorial and are 
known to have curative properties as a result of their constituent chemical compounds 
(Tapsell et al., 2006). A plethora of traditional medicine references attest to the curative 
power, and scientific validation of these popular uses is in progressto support at least some of 
the claims. These pharmacologically active compounds include metabolites such as alkaloids, 
glycosides, polyphenols and terpenes (Raman et al., 2006). Previous reports have 
demonstrated tremendous role of secondary metabolites serving as antioxidants, anti-
inflammatory agents and anti-cancer agents (Dias et al., 2012; Kim et al., 2011). 
 
Most medicinal plants-derived substances such as vitamins, carotenoids phenolic compounds, 
flavonoids and functional metabolites have antioxidant that inhibit oxygen free radicals 
formation and degrade peroxides (Arabshahi et al., 2007). Medicinal plants are still used in 
the manufacture of many pharmaceutical drugs. Reports by Konczak et al. (2009) indicate that 
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some herbs and fruits have cytoprotective properties and plant-derived polyphenols in diets 
can lessen the threat of prostate cancer (Apostolou, 2013). The ethno-botanical survey for this 
study resulted in the identification of nine plants used for the management of prostate cancer. 
These are: Latuca sativa, Tetrapleura tetraptera, Jatropha curcas, Capparis erythrocarpus, 
Ficus asperifolia, Mentha piperita, Carica papaya, Moringa oleifera and Phyllanthus 
amarus. 
 
2.2.0 .1Carica papaya 
Carica papaya is a fruit mostly grown in the tropics. It is a species often used as medication 
for the treatment of diseases (Mello, 2008). It has a good effect as a remedy in dyspepsia and 
related ailments. The seeds are normally used as a dewormer when chewed. Also, the root and 
the juice are used for the management and treatment of cough, bronchitis, and other 
respiratory diseases. Although, the leaves are normally discarded, they have been used in folk 
medication for the treatment of conditions such as fever, gastric digestion problems, burns and 
amoebic dysentery (Canini et al., 2007; Zunjar, 2011). Papain from crushed leaves is used 
traditionally to tenderize tough meat according to a report by Kadiri (2016). The leaf is used 
to expel parasitic worms. The dried leaves are smoked to alleviate asthma. Sealed packs of 
dried powdered leaves are sold in "health food" store. In Ghana, leaf decoction is administered 
as a purgative for horses. The dried leaf mixture is taken as a purgative and may cause 
abortion. A report by Owoyele et al. (2008) has indicated the anti-inflammatory activity of the 
leaves. Recently, black seeds of Carica papaya have been reported to have anti-prostate 
cancer activity (Alotaibi et al., 2017). 
 
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The cytotoxic and total free radical scavenging activities of pawpaw leaves has been reported 
to be associated with the phytochemical compounds and their secondary metabolites such as 
proanthocyanidins and flavonoids (Pierson et al., 2012). Also, many antioxidant compounds 
such as flavonoid, tannin, anthraquinone, Vitamin A, and Vitamin C, have been found in 
pawpaw leaves (Imaga, 2010). Imaga et al. (2010) has reported the presence of tannins, 
saponins, alkaloids, vitamin B12, glycosides, folic acid and anthraquinones in different extracts 
of C. papaya leaves. 
Also, Subramanian et al. (2014) has also reported the presence of carbohydrates, flavonoids, 
terpenoids, saponins and tannins in different extracts of C. papaya leaves. The phytochemical 
studies as reported by Subramanian indicated the absence of alkaloids, protein and amino 
acids in each solvent extract (methanol, ethanol, ethyl acetate and chloroform). However, 
tannins have been present in methanol and ethanol solvent extract but absent in ethyl acetate 
and chloroform extracts. Also, saponins were present only in methanol extract. In the leaves 
of C. papaya, some phytochemicals present has been reported to have anti-tumor activity and 
this include lycopene, tocopherol, flavonoid, and benzylisothiocyanate (Ching and Mohamed, 
2001; Miean and Mohamed, 2001; Basu and Haldar, 2008; van Breemen and Pajkovic, 2008). 
2.2.0 .2Phyllanthus amarus 
Phyllanthus amarus is a small herb belonging to the family euphorbiaceae. This plant is 
mostly found in countries in the tropics. It is used traditionally in Nigeria and India to treat 
malaria associated symptoms and to ease genitourinary system, stomach pain, and liver, 
kidney, and spleen disorders (Iranloye et al., 2010). Nguyen et al. (2017) has reported that 
methanolic extract of P. amarus possessed more phenolic content than the aqueous extract. 
Previous studies have demonstrated thecytotoxic activity of P. amarus extracts on several 
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human cancer cell lines such as prostate cancer cell line DU145, colon cancer cell line Caco-
2, lung cancer cell lines A549, breast cancer cell line MCF-7, and liver cancer HepG2 
(Lawson-Evi, 2008; Lee et al.,2011; Poompachee and Chudapongse, 2011; Nguyen et al., 
2017). 
An earlier study showed that the aqueous extract of P. amarus inhibits DNA topoisomerase II 
of cell cultures. In addition, inhibition of cell cycle regulators responsible for cancerous 
growth has been reported in extracts of P. amarus (Rajeshkumar, 2002). Also, Kiemer (2003) 
has indicated that extracts of P. amarus inhibited induction of interleukin (IL)-1β, interferon-γ 
(IFN-γ), and interleukin (IL)-10 and reduced tumor necrosis factor (TNF-α) production. 
Kassuya (2005) has also shown that the hexane and ethanol / water extracts of P. amarus can 
lower the expression of COX-2 and endotoxin-induced nitric oxide synthase and prevent the 
onset of nuclear factor NF-κB. 
2.2.0 .3 Moringa oleifera 
The leaves of Moringa oleifera are sometimes eaten raw or used for food preparations. In 
traditional medicine, aqueous extract of the leaves is used to treat several maladies including 
swellings, genito-urinary ailments, hypertension, malaria, diseases of the skin, arthritis and 
diabetes (Konmy et al., 2016; Aji et al., 2002). The leaves possess antioxidants, deficiency of 
which has adverse health implications (Konmy et al., 2016). Moringa oleifera leaves are rich 
in polyphenols, vitamins, saponins, flavonoids, carotenoids, alkaloids, tannins, isothiocyanates 
and glucosinolates (Sultana et al., 2009; Sreelatha and Padma, 2009).  
 
Coppin et al. (2013) has indicated that Moringa oleifera leaves extract is able to inhibit nitric 
oxide (NO) produced by macrophage cells which was treated with bacterial 
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lipopolysaccharide (LPS). Moreover, anti-inflammatory and immunomodulatory effects of 
Moringa oleifera leaves have been reported in many in vivo and in vitro studies (Das et al., 
2013). In addition, ethyl acetate extracts of Moringa oleifera leaves were reported to inhibit 
human macrophage cytokine production (TNF-α, IL-6 and IL-8) induced by extract of 
cigarette smoke (Kooltheat et al., 2014).   
2.2.0.4Jatropha curcas 
Jatropha curcas is a woody plant belonging to the euphorbiaceae family. This medicinal plant 
is mostly used traditionally for the management of malaria, fever, diabetes, night blindness, 
headaches and arthritis (Iwu, 1993; Sandberg et al., 2005). The aqueous extract of the leaves 
is used locally for treating diabetes in Nigeria (Gbolade, 2009). In India, J. crucas leaves are 
used for treating liver troubles and jaundice (Sharma et al., 2010). Additionally, a report by 
Nath and Choudhury (2010) showed that the decoction of J. curcas potent for the treatment of 
wounds, chest inflammation and reducing pain. Uche and Aprioku (2008) have reported that 
methanolic extract of J. curcas leaves possessed anti-inflammatory activity on Wister albino 
rats. A previous study has reported that methanolic extracts of the leave had cytotoxic activity 
against HT 29 cell line (Oskoueian et al., 2011) 
Odogwu et al. (2011) has reported that Jatropha sp contains secondary metabolites including 
alkaloids, sesquiterpenoids, triterpenes, flavonoids, steroid, saponins and phenolic. J. curcas 
leaves contain flavonoids, jatropholone, and tannins (El Diwani et al., 2009; Manpong et al., 
2009). Previous studies have shown that aqueous and ethanolic extracts of the plant also 
possessed antioxidant activity (Igbinosa et al., 2011; El Diwani et al., 2009; Oskoueian et al., 
2011). Additionally, the phosphomolybdate assay revealed high reducing power of methanolic 
 
extract of J. curcas (38.8mg/g Gallic acid equivalent).
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2.2.0.5 Mentha piperita 
Mentha piperita is commonly known as peppermint. The boiled leaf extract is used 
traditionally for treating bronchitis. As a folk medicine, the peppermint extract has been 
employed for centuries for the treatment of anorexia, vomiting and bronchitis (Iscan, 2002; 
Moreno, 2002). Previous studies have reported antifungal, antioxidant activities and anti-
inflammatory properties (Nickavar, 2008; Sousa, 2010).Sun et al. (2014) reported that the 
essential oil of M. piperita has anti-inflammatory properties against SGC-7901, SPC-A-1 and 
K562 cancer cell lines. 
2.2.0 .6 Ficus asperifolia 
The leaf extract of Ficus asperifolia is used as a purgative and for the treatment of helminths. 
Ojo et al. (2014) have reported the presence of saponins, glycosides, tannins, alkaloids and 
phenolic compounds in aqueous crude extract of Ficus asperifolia. Ojo and Akintayo (2014) 
has indicated that the DPPH scavenging ability of aqueous extract of F. asperifolia at the 
concentration of 5mg/ml was determined to be 78.65 ± 1.15%. Furthermore, a previous study 
has indicated that the methanolic extract of F. asperifolia possessed a good amount of total 
phenolic content (GAE: 31.96 mg/ml). Bayor et al. (2007) has reported that the methanolic 
leaves extracts possessed fair cytotoxic effects against breast cancer MCF-7 and malignant 
melanoma cell lines DLD-1. Wu et al. (2002) has indicated that the isolates from the leaves of 
F. septica have strong cytotoxic activity against NUGC and HONE-1 human cancer cell lines. 
Another report by Simon et al. (2001) has also indicated that the extract of F. citrifolia had 
therapeutic potential against multidrug resistance cancer cells.  
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2.2.0 .7 Tetrapleura tetraptera 
Tetrapleura tetraptera is a popular plant locally known as “Prekese” in the Eastern region of 
Ghana. It belongs to the mimosaceae family. It produces a four-winged brown fruit which 
hangs on sprout stalks. Traditionally, the fruit is used as an ingredient in most locally made 
soups for nursing mothers (Nwawu and Akah, 1986). The fruit is used in traditional medicine 
for the management of diseases such as arthritis, epilepsy, diabetes mellitus, and hypertension. 
Earlier investigations of T. tertraptera crude extracts have demonstrated diverse 
pharmacological properties including analgesic and anticonvulsant actions (Nwaiwu and 
Akah, 1986; Ojewole, 2005). The aqueous extract of the fruit of T. tertraptera has also been 
reported to have anti-inflammatory and hypoglycemic activities (Ojewole and Adewunmi, 
2004). A report by Adesina (1982) indicates that caffeic acid from the fruit has anti-tumour 
and anti-inflammatory properties and also inhibits HIV replications. Further, it is claimed to 
be therapeutically useful in the treatment of leprosy and rheumatoid pains (Abii and Elegalam, 
2007). The fruit contain phytochemical such as tannins, saponins, alkaloids, anthraquinones, 
glycosides, flavonoids and other phenolic compounds (Gberikon et al., 2015) as well as 
minerals such as calcium, zinc, potassium and iron (Okwu, 2003). Ethanolic crude extract of 
T. tertraptera has previously been reported to be cytotoxic to Ehrlich ascites carcinoma cells 
(Ozaslan, 2016).  
2.2.0 .8 Latuca sativa 
Lettuce is a vegetable that plays a major role for a healthy life (Rice- Evans et al., 1997). This 
vegetable provides a good source of vitamins and bioactive compounds such as glycosides, 
flavonoids and many other polyphenols. Lettuce is often eaten raw or cooked. The chemical 
composition of lettuce is mostly affected by ecological, agronomical and genetic factors 
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(Llorach et al., 2008). Previous studiesindicated that extract of L. sativa had higher total 
phenolic substances and antioxidant activity (Edziri et al., 2011; Lu and Foo2002). Other 
investigators have reported that methanolic extract of the L. sativa leaves significantly 
decreases cycloxygenase-2 expressionand the liberation of nitric oxide (Pepe et al., 2015) 
2.2.0 .9 Capparis erythrocarpus 
Capparis erythrocarpus is a local plant belonging to the family capparaceae. This plant has 
multiple traditional uses. It is used by Ghanaian traditional medical practitioners in the 
management of inflammatory conditions. Herbalists grind the leaves and use the juice for 
treatment of convulsive fever in children. The dried root powder has been shown to be 
beneficial when added to beverage or soup for the treatment of rheumatoid arthritis or any 
swellings of the joint (Danquah, 2011). Also, the liquid from the core of the root is used 
traditionally for the treatment of inflammation of the conjunctiva, hazy vision and 
inflammation of the ear (Danquah, 2010). The plant is used in the treatment of urinogenital 
pain and mastitis (Abbiw, 1990). Report indicates that extracts of the plant has antinociceptive 
effect as well as anti-arthritic properties (Woode et al., 2009; Danquah et al., 2011).  
2.2.1 Phytochemical Compounds 
Phytochemicals are naturally occurring compounds present in herbs. Some of the 
micronutrients have been documented to have pharmacodynamic properties such as acting as 
antioxidants, anti-cancer, anti-inflammatory and pain relievers (Pierson et al., 2012). The 
phytochemical compounds are the primary and secondary metabolites such as polyphenols 
and flavonoids serve various purposes such as normal development, reproduction and 
protection of the plants against harsh conditions (Shankar, 2015). Humans use these organic 
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phytochemicals as medicines, recreational drugs and flavoring.The phenolic compounds 
possess strong therapeutic benefits (Cao et al., 1996).  
There is a direct correlation between total phenolic content and antioxidant activity in plants 
(Yang et al., 2009; Lu and Foo, 2002). In the field of cancer research, secondary metabolites 
such as brassinosteroids, polyphenols and alkaloids have been documented to modulate cell 
cycle progression, proliferation and cell apoptosis (HemaIswarya and Doble, 2006).  
2.2.2 Phytochemical compounds with anti-cancer properties 
In the view of the toxicity of some chemotherapic agents to normal cells, scientific research is 
ongoing to obtain safer alternative treatments from medicinal plants. Some plant secondary 
metabolites have apoptosis inducing properties on cancer cells (Siriwantanmetanon, 2010). 
The phytochemicals used for anti-cancer treatment include polyphenols, brassinosteroids and 
taxol. Some polyphenols which include tannins, flavonoids, curcumin, gallacatechins and 
resveratrol have various mechanism of action. For example some of the plant polyphenols 
interfere with proteins which promote growth of the cancer cells, resveratrol regulates Cu (II) 
which binds to DNA and causes its degradation (Azmi et al., 2006) and curcumin has been 
shown to cause suppression of tumor necrosis factor (Gupta et al., 2014).Furthermore, 
saponins with foaming characteristicshave been reported to act as anti-cancer agents (Wang et 
al., 2012; Guclu-Ustundag and Mazza, 2007). Additionally, tannins which are phenolic 
compounds containing hydroxyls groups have anti-inflammatory properties, inhibits free-
radicals and have antiviral and antimicrobial activities (Akiyama et al., 2001) which may be 
responsible for the therapeutic benefits of the medicinal plants.  
 
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Alkaloids are natural product that contain heterocyclic nitrogen atoms and are basic in nature.  
Research has indicated that alkaloids possess anti-cancer pharmacological activities. 
Alkaloids with anti-cancer properties include dimeric indoles, vincristine, and vinblastine 
(Jordan and Wilson, 2004; Solowey et al., 2014).  
Flavonoids are phenolic substances found in vascular plants with over 8000 individually 
known compounds. Aside their functional activities in the plants, flavonoids are important in 
food. Previous reports have recognized the pharmacological benefits of flavonoids which 
include antiviral, antiallergenic, vasodilatory actions and anti-inflammatory actions (Amin and 
Buratovich, 2007; Anand, 2016).  
Currently, a report by Otsuki et al. (2010) has indicated that anti-cancer agents derived from 
medicinal plants have turn out to be recognized therapies. These have been grouped into four 
main classes of compounds which include vinca alkaloids (vindesine, vincristine, vinorelbine 
and vinblastine), podophyllotoxin (teniposide and etoposide), taxanes (paclitaxel and 
docetaxel), camptothecin (topotecan and irinotecan). 
2.2.3 Synergic Medicine 
Medicinal plants contain a mixture of compounds. Recent studies have acknowledged the 
synergistic and additive effects of phytochemical compounds via potentiation (Lila, 2005). 
Recent studies have indicated that potentiation can exist between two phytochemicals in a 
single plant extract or different plants (Schmidt, 2008; Wagner and Ulrich-Merzenich, 2009). 
Also, Becker et al. (2004) have indicated that synergism can occur between polyphenols 
exhibiting antioxidant activity through sacrificial oxidation, regeneration reactions and 
exertion of different pharmacodynamic action via different pathways. 
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2.2.4 Plant derived anti-cancer agents 
Many parts of medicinal plants have been reported by Konczak et al. (2009), to have 
cytoprotective properties as they are a rich source of phytochemical compounds. Many of 
these active ingredients extracted are in clinical use all over the world for management of 
various cancers. Well known plant-derived anti-cancer compounds include vinblastine and 
vincristine from Cataranthus roseus (Gueritte and Fahy, 2005), irinotecan and topotecan from 
Camptotheca acuminate (Wall, 1966), paclitaxel and docetaxel have been isolated from 
pacific yew tree and Taxus brevifolia, respectively (Cragg, 1998). Anti-cancer agents which 
interfere with cell cycle include epipodophyllotoxin which was isolated from podophyllum 
peltatum (Unnati et al., 2013). Likewise, epigallacotechin 3 gallate has been isolated from 
green tea (Raza and John, 2005) and used for the management of prostate cancer. A previous 
research has demonstrated the anti-cancer activity of resins from the root of Podophyllum 
hexandrum and Podophyllum peltatum. Also, a report by Lopez-Lazaro (2009) has reported a 
broad pharmacological action of luteolin. 
 
2.2.5 Approaches to Development of Anti-cancer Drugs 
Models 
In vitro and in vivo studies have been used to establish the antioxidants and anti-cancer 
activities of various substances (Fig. 1B). The in vivo model usually involves the induction of 
cancer in mice or rats and treatment with a test agent (Archana, 2013). 
Advantages of in vivo models 
-Cancer of interest could be induced in a living system and the metastatic process 
could be monitored and captured. 
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Disadvantages 
-Use of in vivo models however take longer period of time to induceand have high 
failure rate of cancer induction (Evans, 2016). Also, visualization of the individual 
steps of the metastasis process is challenging and extracting quantitative mechanistic 
data is usually very difficult. 
In vitro models however, involve use of established cancer cell lines for testing the 
cytotoxicity of new compounds (synthetic or natural products) (Carocho and Ferreira, 2013).  
Advantages of in vitro models 
In vitro studies allow for high-throughput testing of several compounds or agents in 
ashort period of time. It involves low cost assays and is more reliable. Also, it allows 
control of most experimental variables and permits quantitative analysis. 
 
Disadvantages of in vitro models 
-In vitrostudies captures only limited aspects of the tumour microenvironment, 
hasreduced physiological relevance and may not directly reflect the in vivo situation 
(Katt, 2016). 
 
Figure 1B: A scheme demonstrating approaches to cancer drug development 
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2.2.5.1 Application of Tumor Cell Lines 
Many human prostate cancer cell lines have been reported. These include androgen-
independent and androgen-dependent prostate cancer cell lines (Claas and van Steenbrugge, 
1983; Horoszewicz, 1980). Androgen independent prostate cancer cell lines such as PC3, 
TSU-Prl and DU 145 have been established from metastatic deposits (Claas and van 
Steenbrugge, 1983). Metastatic sublines of PC-3 have been derived from mice (Wu et al., 
2013). Androgen sensitive prostate cancer cell lines include LNCaP cell line established from 
a metastatic deposit in a lymph node (Horoszewicz, 1980). Likewise, new cell lines have been 
established by culturing LNCaP cells grown in castrated mice (Wu, 1994). Also, MDA PCa 
2a and MDA PCa 2b prostate cancer cell lines were derived from a single bone metastasis in 
an African-American male who had androgen independent prostate cancer (Navone, 1997). 
2.2.5.2.1 Cell lines used 
The LNCaP cell line is commonly used in the field of oncology due to the inherent ability to 
closely mimic human disease progression. They are adherent elongated epithelial cells that 
can grow in either aggregates or as single cells (Horoszewicz, 1983). The LNCaP cell line has 
a mutated androgen receptor, lacks 5α-reductase enzyme and has a silent mutation on the 
tumor suppressor gene p53 (Carroll, 1993). The androgen and estrogen receptors have high 
affinity for 5α-dihydrotestosterone and estradiol respectively.The cells also express prostate 
specific antigen and are able to grow readily in vitro. 
 
The PC3 cell line is also used in anti-prostate cancer drug development studies. 
Morphologically, the cell line appears round under the light microscope. It has a deletion 
mutation of p53 gene and lacks androgen receptor and 5α-reductase enzyme (Carroll, 1993). 
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This human prostate cancer cell line does not respond to androgens, glucocorticoids or 
fibroblast growth factors and has a highly metastatic ability than DU 145 and LNCaP cells 
which have low metastatic potentials (Pulukuri et al., 2005). 
 
The PNT2 cell line on the other hand is a normal prostate epithelium which is employed in 
cancer drug development studies to assess the toxicities of the drug to normal cells. This cell 
line is also adherent and non-tumourigenic in nude mice. They possess a well differentiated 
morphology with the expression of cytokeratin 8, 18 and 19 with the latter being a feature of 
differentiated luminal cells of the glandular prostate. The cell cytotoxic value of the cell line is 
used in the calculation of the selectivity indices of the drug / extract. 
2.2.5.2.1 Immortalized cell lines 
Immortalized cell lines are either normal cells or tumor cells that have been manipulated 
artificially to grow and divide indefinitely in vitro under optimal culture condition. LNCaP 
and PC3cell lines are the most frequently used prostate cancer cell lines for toxicity studies 
(Horoszewicz et al., 1980; Horoszewicz et al., 1983; Kaighn, 1979). These cell lines can 
continuously divide and eventually fill the flask. A small fraction of these multiplying cells 
can be transferred into new flasks to continue proliferation by passaging. Immortalized cell 
lines are homogenous and have genetically identical population. These characteristics of the 
cell line aid in providing reliable and reproducible results. However, these cell lines are not 
normal and can divide indefinitely expressing unique gene patterns not found in any cell type 
in vivo. These prostate cancer cell lines grow in Roswell Park Memorial Institute media 
supplemented with 1% penicillin/ streptomycin and 10% foetal bovine serum (FBS) 
(Alonzeauet al., 2013). 
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2.2.6 Assessment of antioxidant activity 
Crude extracts of medicinal plants have been reported to contain huge variety of compounds 
which possess varying antioxidant activities. Several methods have been adopted in 
measuring the antioxidant capacity of each compound separately (Prasad et al., 2010).The 
accepted methods are based on the ability of the antioxidants in scavenging specific radicals 
by hydrogen atom transfer (DPPH (2, 2-Diphenyl-1-picrylhydrazyl)) free radical-scavenging 
assay), by inhibiting lipid peroxidation or chelating metal ions. The DPPH free radical-
scavenging assay (Zlotek et al., 2015), ferric reducing / antioxidant power assay (FRAP 
assay) (Rajukar and Hande, 2011), Trolox antioxidant equivalent capacity (TAEC) (Prior et 
al., 2005) among others have been widely employed and used in antioxidant determination. 
2.2.6.1 DPPH assay 
The assessment of the antioxidant activity by DPPH assay has been widely employed due to 
its exclusive ability to assessing the total antioxidant activity. The DPPH is a stable radical, 
which forms a dark purple colour in methanolic solution. However, the dark purple colour 
disappears after the DPPH is reduced by antioxidants through hydrogen transfer to DPPH-H 
(non-radical form) (Fig1C). The reduction in absorbance at a wavelength 517 nm is as a result 
of the reaction between the phytochemicals and DPPH. Research has indicated that the 
extraction conditions and procedures can appreciably affect the efficacy of extracts, their 
stability and yield of polyphenols (Zlotek et al., 2015). The total antioxidant activity has been 
associated with the plant phenolic compounds as well as other secondary metabolites. Also, a 
report by Srikanth et al. (2010) has indicated that the total antioxidant activities of bioactive 
components are concentration-dependent. Furthermore, Blois (1958) has reported an inverse 
relationship between the antioxidant activity and the 50% effective concentration (EC50). 
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Hence, the lower the value of EC50, the higher the antioxidant activity. Plants extracts with 
EC50 valueslower than 50 µg/ml are considered very strong antioxidants (Blois, 1958). Those 
with EC50 valuesof 50-100 µg/ml are considered strong antioxidant, whilstplant extracts that 
produce EC50 valuesof 101-150 µg/ml havemedium antioxidant activity and those EC50 greater 
than 150 µg/ml are considered weak antioxidants (Blois, 1958).  
 
Figure 1C: DPPH reaction 
(Lewis, 2012) 
2.2.6.2 FRAP assay 
FRAP assay (Ferric Reducing Antioxidant Power Assay) is commonly used to study the 
antioxidant capacity of crude extract by measuring the reducing potential in a redox-linked 
reaction. The antioxidant capacity of plant extracts is determined by the ability of the 
antioxidants in the extracts to reduce ferric iron to ferrous in the FRAP reagent, which 
consists of 2, 4, 6-tris (1-pyridyl)-5-triazine (TPTZ) prepared in sodium acetate buffer, at low 
pH. The reduction of ferric iron in FRAP reagent (colourless complex) will result in the 
formation of a blue product (ferrous – TPTZ complex) whose absorbance can be read at 593 
nm (Rajukar and Hande, 2011). 
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2.2.6.3 Trolox Equivalent Antioxidant Assay 
Trolox Equivalent Antioxidant Assay is a chemical assay that measures the ability of 
antioxidants to scavenge the stable radical cation ABTS+ (2,2-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid) to form a blue-green chromophore with maximum 
absorption at 734 nm that decreases in its intensity in the presence of antioxidants. The 
antioxidants neutralize the radical by donating an electron or by hydrogen atom donation. The 
extent of discoloration to the blue-green color depends on the duration of action, the intrinsic 
antioxidant activity and concentration in the sample (Prior et al., 2005).
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CHAPTER 3 
3.0 Materials and methods 
3.1Source of materials 
All the reagents such as dimethyl sulfoxide (DMSO), methanol (MEOH), ethanol (ETOH), 
ethyl acetate (EA), dichloromethane (DCM), petroleum ether (PE), isopropanol were 
obtained from Daejung (South Korea) whereas Folin Ciocalteau reagent was obtained from 
Sigma Aldrich (USA). Also, butylated hydroxyl toluene (BHT), 2, 2-diphenyl-1-
picrylhydrazyl (DPPH), gallic acid and Folin-Ciocalteu reagent were obtained from Sigma 
Aldrich (USA). Likewise, Rosewell Park Memorial Institute (RPMI) and Foetal Bovine 
Serum (FBS) were obtained from (USA). Also, sterile costar 96 well plate used for MTT 
assay was obtained from Sigma Aldrich (USA). Serological pipettes (5ml, 10ml and 25 
ml), falcon tubes (15 ml and 50 ml), Finn pipette (Thermo scientific, OH 61352, Finland), 
eppendorf tubes and pipette filler (Thermo scientific, EN61010 Pudong, Shangai, China) 
were used in both DPPH assay and MTT assay. Centrifuge (Tomy LC-200, Japan), 
Biosafety Cabinet airstream (ESCO Biotech EQR/GL-64, Singapore) and incubator 
(Panasonic MCO-18 AC, China).All other reagents and supplies were obtained from 
standard suppliers. 
3.2. Ethnobotanical /Literature Survey and Collection of Plants 
Based on the literature review and ethnopharmacological details, nine (9) plants were 
selected and collected, extracts prepared to be evaluated for their total phenolic substances, 
antioxidants as well as cytotoxic activities. In the ethnobotanical survey, a list of eight 
plants used in the management of prostate cancer was selected after IRB approval to obtain 
information from herbalist. Also, one plant showing promising anti-cancer properties was 
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included after a thorough literature search (Google scholar, Pub Med and Elsevier). The list 
of plants was presented to taxonomists at the Center of Plant and Medicine Research 
Akuapem-Mampong and University of Ghana Herbarium, respectively. The collection was 
done early in the morning in the botanical gardens at Akuapem- Mampong and also at 
University of Ghana, respectively, between March and April 2017. 
The green fresh leaves, seeds and fruit were cleaned and air dried under room temperature 
(26°C). The samples were ground into fine powder using an electric blender and stored in 
appropriately labeled zip lock bag at 26°C. The seeds and the pulp of the T. tetraptera fruit 
were separated, grinded and kept in zip lock bags at room temperature. 
 
3.2 Extraction Method 
Sequential extraction method was used to obtain crude extracts from the powdered parts of 
the plant using solvent with increasing polarity (Prakash and Gupta, 2005). The powdered 
plant parts were sequentially extracted using petroleum ether (PE), dichloromethane 
(DCM), ethyl acetate (EA), ethanol (ETOH) and distilled water (AQ) in increasing polarity 
of solvents. The powdered plant parts were macerated at room temperature in portions of 
50g, with 200 ml of each solvent in air tight conical flasks. The maceration process was 
conducted by placing the conical flask with the mixture on an electric shaker at a speed of 
350 rpm for 3 days. The extracted material was collected by filtration using a double 
layered muslin cloth and Whatmann No 1 filter paper. The residues were then remacerated 
twice using 150 ml of fresh solvent. Hence, the extraction using petroleum ether was 
repeated three times using a total volume of 500 ml of solvent. The residue was dried 
completely to get rid of the first solvent and the dried residue was extracted three times 
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following the above procedure using the remaining solvent (using dichloromethane (DCM), 
ethyl acetate (EA) and ethanol (ETOH), respectively). The filtrates of the same solvents 
were pooled together and evaporated using rotary evaporator (BUCHI). The thick extracts 
obtained were dried in an oven at 50°C to constant weight. The aqueous extracts were 
prepared by boiling the dried residue in 500 ml of distilled water at 80°C. The procedure 
was repeated once again, filtrates were pooled and lyophilized in a freeze-dryer (Lyotrap 
Lf/ LYO/0411, United Kingdom). The percentage yield was calculated as follows: 
final weight of dried extract
percentage yield = × 100 
Initial weight of plant powder
 The dried crude extracts were dispensed into eppendorf tubes and falcon tubes before 
storing at 4°C. 
3.3 Determination of the total phenolic content (TPC) 
Determination of total phenolics  
A concentration of 5 mg/ml was prepared from each of the plant extracts. The total 
phenolic content of the extracts of each plant was assessed using the Folin-Ciocalteau 
method as described by Anim et al. (2016). Each sample was aliquoted (10µl) into a 24 
well plate and was incubated with Folin-Ciocalteau reagent (50µl). Following addition 
of150µl sodium carbonate20% (w/v), it was incubated again for 2 h. The absorbance of the 
blue chromophore formed from the reduction of phosphotungstic phosphomolybdenum was 
assessed at a wavelength of 750 nm using the spectrophotometer (Tecan Infinite M200 Pro 
plate reader, Austria) (see Fig 2A). The total phenolic content was extrapolated from the 
calibration curve prepared from a standard, gallic acid.  The total phenolic content was 
expressed as Gallic Acid Equivalents (GAE) in grams per 100 g dry weight of plant extract. 
All determinations were carried out in triplicates. 
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Figure 2A: A schematic presentation of the procedures in determining the total phenolic 
content of extracts. 
 
3.4 DPPH radical scavenging assay 
The DPPH assay has been used in assessing the free radical scavenging activity of extracts 
because it is simple and fast (Appiah-Opong et al., 2016). It also has the ability to measure 
the total antioxidant oxidant activity without being specific to the kind of antioxidant 
present. The dried extracts and freeze-dried samples were weighed in 1 mg portions using a 
weighing balance (Mettler Toledo.XP 105 Delta Range, China) into different eppendorf 
tubes. After the solubility test, crude extracts of organic solvents (PE, DCM, EA and 
ETOH) dissolved completely in absolute ethanol whiles freeze dried extracts were 
dissolved in distilled water. A volume of 1 ml of absolute ethanol or distilled water was 
then added to each dried sample, vortexed (Taiyo Mixer S-5F) and sonicated (Ultrasonic 
Washer ASONE, China) for 5 min. A stock solution of BHT (1mg/ml in absolute 
methanol) and a two-fold serial dilution of the stock solutions (BHT and extracts) were 
prepared to obtain seven different concentrations. The 0.5 mM DPPH was prepared by 
dissolving a weight of 3.55g in 18ml of methanol to form a purple color. Aliquots of 100μl 
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of each extract or standard (BHT) of concentrations (1-1000μg/ml) were pipetted into a 
properly labeled 96-well plate. Afterwards, a 100μl of 0.5 mM DPPH solution was added 
to each well to initiate the reaction. The 96-well plate was gently shaken to mix the content, 
incubated for 20 min and the absorbance was assessed at wavelength of 517 nm using a 
spectrophotometer (Tecan Infinite M200 Pro plate reader, Grodigi, Austria) (see Fig 2B). 
Ethanol, methanol and water were used as negative control and blank, respectively. The 
BHT was used as standard or positive control. Analysis was carried out in triplicate for 
standard and each extract. Antioxidant activity of each extract was determined by 
calculating the percentage of antioxidant activity using reduction of DPPH absorbance 
(Bedawey, 2010). Background interferences from absolute methanol, absolute ethanol and 
water were deducted from the activities of the corresponding extracts prior to the 
calculation. The results were expressed as percentage free radical scavenging activity 
which was calculated as shown below: 
𝐵𝑙𝑎𝑛𝑘 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 − 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡
% 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = × 100 
𝐵𝑙𝑎𝑛𝑘 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
The 50% inhibition concentration (IC50) of DPPH scavenging activity of each extract was 
determined from the log concentration response curve using a plot of % Antioxidant 
activity or scavenging activity versus concentration of sample tested ×100 using Graphpad 
prism software version 5(GraphPad Software Inc. San Diego,CA 92037). 
 
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Figure 2B: A schematic picture showing the procedures in DPPH antioxidant assay 
 
3.5 Cell Culture 
The PC3, LNCAP cells were cultured in 25mL culture flask whiles PNT2 cells were 
cultured in 75 mL culture flask containing RPMI media. The media was supplemented with 
10% FBS and 1% streptomycin/ penicillin to make it complete. The cells were incubated in 
a humified incubator (Panasonic CO2 Incubator MCO-18 AC, China) at 37 °C in 5% CO2. 
The spent media was changed every three days with complete media and cells were passed 
at 80 - 90% confluence. 
3.6 Preparation of test samples 
3.6.1Cell viability assay 
Cell viability was assessed using the MTT assay (tetrazolium bromide solution in 
phosphate-buffered saline (2.5 mg/ml) as described (Appiah-Opong et al., 2016). The assay 
is based on the reduction of the tetrazolium bromide by the mitochondria dehydrogenase or 
reductases of the viable cells. The cells were seeded at 10,000 cells per well of the 96 -well 
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plate and incubated for 24 h to get cells attached to the well plate. Before treatment, a stock 
solution of each sample was prepared by dissolving in dimethylsulfoxide (100% DMSO) 
except the AQ extracts which were solubilised in distilled water and filtered using a 0.45 
millipore.  A working plant extract solution of 10% DMSO was prepared from the stock 
plant extract solution (100% DMSO) using RPMI media. Ten microlitre aliquots of 
different concentrations (concentration range 0-1000 µg/ml) of each medicinal plant extract 
and compound (0-200 µg/ml) were added to form 1% DMSO in appropriately labeled 96 
4
well plate already containing 1×10  cells / well. Also, 100 µl aliquots of complete medium 
were added to the blank and untreated wells as a negative control. Each well for the 
negative control contained 10,000 cells /well and 1% DMSO. The final 1% DMSO was 
produced in each well by the addition of 10µl of the mixture of 10 % DMSO and 90% 
RPMI to the wells. A concentration of 10 µM curcumin was used as a positive control. The 
cells were then incubated with different concentrations of the test extracts and compound in 
triplicates at 37 °C in 5% CO2.  Following incubation of the plates for 72 h, 20 µl of 2.5 
mg/ml solution of MTT was added to each well and re-incubated for 4 h. The purple 
formazan crystals formed in each well were dissolved in 150 µl of acidified isopropanol. 
Absorbance was read at a wavelength of 570 nm using the spectrophotometer (Tecan 
Infinite M200 Pro plate reader, Austria).The concentrations to exhibit 50% anti-
proliferative activity (IC50 value) were obtained through regression analysis of the results 
of five different concentrations of each extract and compound. The percentage cell viability 
of was evaluated using the formulae, 
 
[ODT0 − ODT1]
% Cell viability = × 100 
[ODU0 − ODU1]
 
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Where ODT0 is the absorbance of wells treated with test extracts or standard compound 
(Curcumin) for all the cell lines; ODT1 is the absorbance of wells with test extracts or 
Curcumin (Positive control); ODU0 is the absorbance of wells with negative control 
(untreated cells) for all cell lines; ODU1 is the absorbance of wells containing blank 
(culture media only). The inhibition concentration at 50% cell survival (IC50) values was 
obtained from the plot of a dose response curve of the percentage cell survival versus 
concentration of the extracts/fractions. The IC50 values obtained were used to determine the 
selectivity of the extracts/fractions for cancer cells. The IC50 values of the cancerous cells 
(PC3 and LNCaP) and 50% cytotoxic concentration (CC50) of normal cells (PNT2) 
obtained were used to determine the selectivity indices. The selective indices of the 
fractions were determined using the formula. 
SI = CC50 of normal cell/ IC50 of cancer cell line. SI values ≥ 2 were considered as 
significant or promising drug candidate (Badisa et al., 2009). 
 
Figure 2C: A schematic diagram showing the procedures used in MTT assay. 
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3.8 Statistical analysis 
Data obtained were expressed as mean ± SD using statistical software Graph pad prism 
version 5 (GraphPad Software Inc. San Diego, CA 92037). Also one-way ANOVA was 
used to determine the level of significance between the extracts, followed by Tukey’s 
multiple comparison test. The results were considered statistically significant at P < 0.05. 
3.6 Ethics Statement 
Ethical clearance for this study was obtained from the Institutional Review Board Noguchi 
Memorial Institute for Medical Research (NMIMR). All procedures were in strict 
accordance and approved by the board members of Noguchi Memorial Institute for 
Medical Research (NMIMR) Intitutitional Review Board (IRB).  
  
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Chapter 4 
4.0 Results 
The ethno-botanical survey resulted in the identification of nine plants, namely: Latuca 
sativa, Tetrapleura tetraptera, Jatropha curcas, Capparis erythrocarpus, Ficus asperifolia, 
Mentha piperita, Carica papaya, Moringa oleifera and Phyllanthus amarus. 
Fresh sample leaves of Latuca sativa, Jatropha curcas, Carica papaya, Moringa oleifera, 
Phyllanthus amaruswere collected and authenticated by a plant taxonomist and kept at the 
University of Ghana Plant and Herbal medicine herbarium. Also, leaves of Capparis 
erythrocarpus, Ficus asperifolia, Mentha piperita and fruit of Tetrapleura tetraptera were 
obtained and authenticated at Centre of Plant and Medicinal Research, Akuapem 
Mampong. 
Table 2: Identityof medicinal plants and their voucher specimen number 
Voucher specimen  
S/N Plant Family Part used number 
TT Tetrapleura tetraptera Mimosaceae  Fruit, seed CPMR 4867,4868,4869 
FA Ficus asperifolia Moraceae  Leaves CPMR 4846,4865,4866 
CP Carica papaya Caricaceae Leaves UGAJK-2017-001 
PA Phylanthus amarus Euphorbiaceae aerial part UGAJK-2017-005 
CE Capparis erythrocarpus Capparaceae Leaves CPMR 4873,4874,4875 
MP Mentha pepirita Lamiaceae Leaves CPMR 4870,4871,4872 
LS Latuca  sativa Asteraceae. Leaves UGAJK-2017-001 
JC Jatropha curcas Euphorbiaceae Leaves UGAJK-2017-002 
MO Moringa oleifera Moringaceae Leaves UGAJK-2017-004 
 
After plant extraction using increasing polarity of solvents, the dried crude extracts were 
obtained and weighed. The results of the yield were tabulated as shown in Table 3.  The 
percentage yield of the various solvent extracts of the different medicinal plants was 
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calculated, and presented in the table below. Among the medicinal plants, the highest yield 
was observed in the ETOH extract of the T. tetraptera pulp (30.38%). Also, the leat yield 
was obtained from the PE extact of the T. tetraptera pulp (0.15%). The percentage yield is 
represented in Table 3 below. 
 
Table 3: Percentage yield of medicinal plants 
Percentage (%) Yield of 50 grams of plant material 
Petroleum Dichloro- Ethyl 
Test plant Ethanol Aqueous 
ether methane acetate 
(ETOH) (AQ) 
(PE) (DCM) (EA) 
 T. tetraptera (pulp)   0.15 0 .30 1 .05  30.38  10.14 
T.  tetraptera (seed) 5.40 1.34 0.25 1.52 7.52 
L. sativa 2.20 2.36 1.36 8.86 9.87 
C.  erythrocarpus 2.28 3.08 3.15 9.53 9.96 
M. piperita 1.73 1.88 1.36 7.78 9.64 
F.  asperifolia 5.04 0.58 1.07 4.40 11.86 
M. oleifera 3.89 0.68 0.70 3.12 13.94 
C. papaya 3.93 1.52 0.62  2.36 13.84 
J. curcas 0.94 0.16 0.17 1.37 6.26 
P. amarus 4.07 1.14 1.15 4.67 11.09 
Whole T. tetraptera  0.38 0.43 0.33 20.32 17.29 
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4.1Total Phenolic Content (TPC) 
The calibration curve obtained from Gallic acid is shown in (Figure 3A). Table 4 shows the 
TPC levels of the various extracts derived from Figure 3A. The total phenolic contents of 
the aqueous and ethanol extract were significantly higher (p<0.05) in P. amarus, whole T. 
tetraptera fruit, F. asperifolia, pulp of T. tetraptera and L. sativa crude extracts when 
compared to their respective petroleum ether (PE) and dichloromethane (DCM) crude 
extracts. The AQ extract of T. tetraptera seeds showed a significantly higher (p<0.05) TPC 
than the rest of the solvent extracts. 
The ETOH extract of C. papaya and M. oleifera showed a significantly higher total 
phenolic content when compared to the rest of their respective solvent extracts. However, 
there were no significant difference (p>0.05) in TPC of the aqueous, ethyl acetate and 
dichloromethane crude extracts of C. papaya and M. oleifera. 
 
Furthermore, there was no significant difference (p>0.05) between ETOH and PE crude 
extracts of C. erythrocarpus. The TPC of ETOH and PE crude extracts of C. erythrocarpus 
were significantly higher (p<0.05) when compared with other solvent extracts. There was 
no significant difference between total phenolic content of aqueous and dichloromethane 
crude extracts.  
The EA extract of M. piperita had a significantly higher total phenolic content when 
compared to the rest of the solvent extracts. The aqueous extract of M. piperita showed a 
significantly higher total phenolic content when compared to the rest of the solvents except 
ethyl acetate. Also, there were no significant difference between the PE extract and DCM 
crude extract of M. piperita. 
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The ETOH extract of J. curcas possessed a significantly higher total phenolic content when 
compared with the rest of the extracts. There was no significant difference between 
aqueous and dichloromethane extracts of J. curcas. Also, the EA extract of J. 
curcasshowed a significantly higher (p<0.05) total phenolic content than the petroleum 
ether extracts. The results indicate that most plants contain more polar phenolic compounds 
than least polar compounds.  
 
 
0.5
y = 0.384x + 0.058
0.4
R² = 0.996
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1
[Gallic acid] mg/ml  
Figure 3A: Calibration curve of Gallic acid 
Where x is Gallic acid concentration and y is the absorbance 
 
  
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Mean absorbance
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Table 4: Total phenolic content of extracts of selected medicinal plants 
Total phenolic content of Extracts (TPC g GAE/100g) 
 Dichloro- Ethyl 
Test Petroleum  methane acetate Ethanol Aqueous 
plant ether(PE) (DCM) (EA) (ETOH) (AQ) 
    a 
TTS 1.30±0.55 1.08±0.56 1.65±0.42 2.27±0.34 7.58±0.47
   a  
LS 2.95±0.28 2.40±0.34 9.87±0.82 13.71±0.65 11.51±0.47
  a   
MP 5.38±0.35 5.20±0.59 12.97±0.47 3.97±0.34 9.01±0.46
a   a  
CE 7.50±0.27 3.47±0.35 1.80±0.40 8.10±0.23 3.86± 0.32
      
   a  
FA 4.38±0.73 8.22±0.16 5.51±0.87 30.77±1.68 8.00±0.39
      
   a  
MO 2.43±0.19 7.58±0.27 5.32±0.53 30.39±2.18 6.51±0.17
      
    a 
TTP   3.06±0.13 2.97±0.24 3.98±0.50 6.46±0.19 22.33±0.27
      
   a  
JC 4.09±1.80 7.97±0.15 9.58±0.85 35.72±3.08 5.72±0.27
      
   a  
 PA 4.89±0.25 7.81±0.25 4.73±0.44 92.71±3.62 23.69±0.97
      
   a  
WTT 2.75 ±0.53 6.49±0.13 4.79±0.20 18.13±0.99 9.58±0.070
      
   a  
CP 5.46±1.45 10.91±0.89 9.08±0.94 41.50±5.78 7.73±0.93
 
Values are given as means ± SD; total phenolic content (TPC g GAE/100g) is given in 
grams Gallic acid equivalent/ 100 g of plant extract. The TPC was assessed on 5mg/ml of 
dried crude plant extract. One-way ANOVA followed with Tukey’s was used to assess the 
level of significance between solvents of the same plant. “a” means the extract was 
significantly higher when compared to the rest of the extracts of the same plant (P<0.05). 
  
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A. 
 
 
 
 
B. 
 
 
Figure 3B: Total phenolic content of PE, DCM, EA, ETOH and AQ extracts of P.amarus 
leaf (A) and T. tertraptera seed (B) expressed as mean ± standard deviation. 
The total phenolic content (TPC mg GAE/100g) was given in milligrams Gallic acid 
equivalent/ 100 g of plant extract. The TPC was assessed on 5mg/ml of dried crude plant 
extract. One-way Anova followed with Tukey’s was used to assess the level of significance 
between solvents of the same plant. “a” means the extract was significantly higher when 
compared to the rest of the extracts of the same plant (P<0.05). 
 
 
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C. 
 
 
 
 
 
D. 
 
Figure 3C: Total phenolic content of PE, DCM, EA, ETOH and AQ extracts of T. 
tertraptera pulp extracts (C) and L. sativa leafextracts (D) expressed as mean ± standard 
deviation. 
 
The total phenolic content (TPC mg GAE/100g) was given in milligrams Gallic acid 
equivalent/ 100 g of plant extract. The TPC was assessed on 5mg/ml of dried crude plant 
extract. One way Anova followed with Tukey’s was used to assess the level of significance 
between solvents of the same plant. “a” means the extract was significantly higher when 
compared to the rest of the extracts of the same plant (P<0.05). 
 
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E. 
 
 
 
 
F. 
 
Figure 3D: Total phenolic content of PE, DCM, EA, ETOH and AQ extracts ofM. piperita 
leaf (E) and C. erythrocarpus leaf (F) expressed as mean ± standard deviation. 
The total phenolic content (TPC mg GAE/100g) was given in milligrams Gallic acid 
equivalent/ 100 g of plant extract. The TPC was assessed on 5mg/ml of dried crude plant 
extract. One-way Anova followed with Tukey’s was used to assess the level of significance 
between solvents of the same plant. “a” means the extract was significantly higher when 
compared to the rest of the extracts of the same plant (P<0.05). 
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G. 
 
 
 
 
 
 
H. 
 
Figure 3E: Total phenolic content of PE, DCM, EA, ETOH and AQ extracts of J. curcas 
leaf (G) and F. asperifolia leaf(H) expressed as mean ± standard deviation. 
The total phenolic content (TPC mg GAE/100g) was given in milligrams Gallic acid 
equivalent/ 100 g of plant extract. The TPC was assessed on 5mg/ml of dried crude plant 
extract. One-way Anova followed with Tukey’s was used to assess the level of significance 
between solvents of the same plant. “a” means the extract was significantly higher when 
compared to the rest of the extracts of the same plant (P<0.05). 
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I. 
 
 
 
J. 
 
Figure 3F: Total phenolic content of PE, DCM, EA, ETOH and AQ extracts ofM. oleifera 
leaf (I) and wholeT. Tertraptera fruit (J) expressed as mean ± standard deviation. 
 
The total phenolic content (TPC mg GAE/100g) was given in milligrams Gallic acid 
equivalent/ 100 g of plant extract. The TPC was assessed on 5mg/ml of dried crude plant 
extract. One-way Anova followed with Tukey’s was used to assess the level of significance 
between solvents of the same plant. “a” means the extract was significantly higher when 
compared to the rest of the extracts of the same plant (P<0.05). 
 
 
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K. 
 
 
Figure 3G: Total phenolic content of PE, DCM, EA, ETOH and AQ extracts of C. papaya 
leaf (K) expressed as mean ± standard deviation. 
 
The total phenolic content (TPC mg GAE/100g) was given in milligrams Gallic acid 
equivalent/ 100 g of plant extract. The TPC was assessed on 5mg/ml of dried crude plant 
extract. One-way Anova followed with Tukey’s was used to assess the level of significance 
between solvents of the same plant. “a” means the extract was significantly higher when 
compared to the rest of the extracts of the same plant (P<0.05). 
 
  
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4.2 Antioxidant Activities of the Plant extracts 
The median effective DPPH scavenging values for extracts obtained from the various 
solvents and plants are recorded in Table 5. The table shows the EC50values obtained for 
antioxidant activities of the crude extracts of selected medicinal plant. All the samples 
exhibited a concentration dependent pattern of free radical scavenging. The AQ extract of 
P. amarus leaves recorded strong antioxidant activity with the lowest EC50 values of 
(19.32±1.13 µg/ml) when compared to the ETOH and EA extracts, with EC50 values 
of60.03 ± 1.07 µg/ml and 202.1 ± 1.10 µg/ml respectively.The ETOH, EA and AQ extracts 
of P. amarus however, recorded highest % antioxidant activities of 86%, 84% and 77%, 
respectively,at the highest concentration tested, whiles the PE extract showed the lowest 
activity of 15 % as shown in figure A. (figure 4A.). 
 
The AQ extract of the whole T. tetraptera fruit had a strong free radical scavenging activity 
with EC50 value of 83.46±1.07 µg/ml when compared with the rest of the other extracts. 
The AQ, EA and ETOH extract showed highest % antioxidant activities of 83.91%, 
66.31% and 40.16% while the PE extract showed the least maximum efficacy values of 
15%, as shown in Figure 4B. The free radical scavenging activity of the AQ extract of the 
whole T. tetraptera fruit was not significantly different (p>0.05) when compared to the 
BHT (positive control). There was no significant difference between the crude extracts 
(p>0.05). 
 
The AQ extract of the pulp of T. tetraptera recorded the lowest EC50 value of 87.50±1.10 
µg/ml when compared to the rest of the organic solvents. The AQ, ETOH and EA extract 
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showed maximum efficacy values of 72.55%, 69.25% and 40.90 % while petroleum ether 
extract showed the least activity of 8 % as shown in figure I (Figure 4C.).  
 In addition, the AQ extract of T. tetraptera seed had the lowest EC50 values of (244.3± 
1.10 µg/ml) when compared with the other crude seed extracts. The AQ, ETOH and EA 
extracts showed a maximum efficacy values of (53.66%, 40.91% and 9.51%) while the 
DCM extract showed the least maximum efficacy values of 12.35 % as shown in figure D 
(Figure 4B.).  
 
The EA extract of L. sativa and M. piperita appeared to show stronger antioxidant activities 
with the lowest EC 50 values of121.4±1.07 µg/ml and 129.7±1.07 µg/ml respectively, when 
compared with their respective solvent extracts. The EA, ETOH and AQ extracts of L. 
sativa showed a maximum efficacy values of 91.80%, 59.56% and 53.46% respectively, 
while PE extract showed the least maximum activity of 16.27 % as shown in figure F 
(Figure 4B.). There was no significant difference (p>0.05) between the crude extracts. 
Additionally, the EA, AQ and DCM extracts of M. piperita showed maximum efficacy 
values of 88.36%, 52.99% and 67.01% respectively, while PE extract showed the least 
maximum activity of 38.72 % as shown in figure E (Figure 4B.). Interestingly, the 
antioxidant activities of EA, AQ and DCM extracts of M. piperita had EC50 values of 
129.7±1.07 µg/ml, 307.5±1.10 µg/ml and 279.5±1.07 µg/ml, respectively. The antioxidant 
activity of BHT was significantly stronger (p<0.05) when compared with DCM, ethanol 
and aqueous extracts of M. piperita. 
 
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Additionally, the EA, AQ and ETOH extracts of J. curcas showed maximum efficacies 
values of 74.59%, 68.21% and 53.28% respectively, while PE extract showed the least 
maximum activity of 44.47 % as shown in figure J (Figure 4C.). This study reports that the 
EC50 of EA, AQ and ETOH extracts of J. curcas have EC50 values of303.5±1.10 µg/ml, 
435.1±1.07 µg/ml and 177.8±1.15 µg/ml, respectively. However, the difference between 
the antioxidant activities of standard BHT and ethyl acetate, aqueous and ethanolic extracts 
of J. curcas was significant (p<0.05). 
 
Furthermore, the AQ, DCM and ETOH extracts of M. oleifera showed a maximum activity 
of 71.68%, 45.46% and 45.02% respectively, while PE extract showed the least maximum 
activity of 30.03 % as shown in figure K (Figure 4C.). Aqueous extract (AQ) of   M. 
oleifera showed antioxidant activity with EC50 value of250.2±1.08 µg/ml. However, the 
antioxidant activity of BHT was significantly stronger than aqueous extracts and rest of the 
solvent extracts of M. oleifera. 
Besides, the DCM, ETOH and AQ extracts of C. erythrocarpus showed maximum efficacy 
values of 77.04%, 39.72% and 36.03% respectively, while the PE extract showed the least 
maximum activitiesof 17.77 % as demonstrated in figure H (Figure 4C.). Dichloromethane 
extracts of C. erythrocarpus exhibited antioxidant activity with EC50 value of (179.6±1.10 
µg/ml). BHT (positive control) was significantly stronger than AQ extracts and rest of the 
solvent extracts. 
 
Moreover, the AQ, ETOH and EA extracts of F. asperifolia showed maximum efficacy 
values of 68.21%, 59.09% and 55.97% respectively, while DCM extract showed the least 
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maximum activity of 41.04% as demonstrated in figure G (Figure 4C.). The AQ, ETOH 
and EA extract of F. asperifolia exhibited EC50 values of177.8±1.15 µg/ml, 303.5±1.10 
µg/ml and 400.6±1.05 µg/ml, respectively. 
 
Also, the AQ, ETOH and DCM extracts of C. papaya showed maximum antioxidant 
activities of 80.23%, 52.02% and 49.55% respectively, while EA extract showed the least 
maximum antioxidant activity of 31.09 % as shown in figure C (Figure 4B.). The AQ and 
ETOH extracts of C. papaya exhibited EC50 values of (125.9 ±1.08 µg/ml and 439.7 ±1.07 
µg/ml, respectively). BHT showed significantly stronger antioxidant activity (p<0.05) than 
rest of the solvent extracts except AQ extract of C. papaya. 
 
The EA extract of M. piperita and L. sativa showed stronger antioxidant activities when 
compared to the rest of the remaining solvent extracts, respectively. However, AQ extracts 
ofthese plants did not show significantly higher antioxidant activity when compared to their 
respective solvent extracts (p>0.05). Also, the aqueous extracts of J. curcas, M. oleifera, C. 
papaya, F. asperifolia, C. asperifolia, M. piperita and L. sativa appeared to have weaker 
antioxidant activities when compared to the standard (BHT). The table below shows values 
of antioxidant activity (EC 50) of the crude extracts of the plants samples. The antioxidant 
activities of crude extracts and standard BHT were assessed at 1mg/ml of crude extracts.  
 
 
 
 
 
 
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Table 4: EC50 antioxidant values of plant extracts 
Potential antioxidant activity of Extracts  (EC50 µg/ml) 
Test Petroleum Dichloro- 
plant ether methane Ethyl acetate Ethanol Aqueous 
12.81± 
BHT 1.07 13.31± 1.07 12.31± 1.07 13.31± 1.07 12.81± 1.07 
# a,# 
TTS >500  >500 >500 424.4± 1.20 244.3±1.10
a # a,# 
LS >500 >500 121.4± 1.07 243.5± 1.12 324.6±1.17
# 
CE >500 179.6± 1.10 >500 >500 >500 
a,# a a,# a,# 
MP >500 279.5± 1.07 129.7± 1.07 430.8± 1.17 307.5±1.10
      
a,# a,# a,# 
FA >500 >500 400.6± 1.05 303.5 ± 1.10 177.8 ±1.15
      
a,# 
MO >500 >500 >500 >500 250.2 ±1.08
      
 a,# a,# a 
TTP >500 >500 333.8 ±1.10 162.3 ±1.15 87.50 ± 1.10
      
# # # 
JG >500 >500 303.5 ±1.10 435.1 ±1.07 177.8±1.15
      
  a 
PA >500 >500 202.1 ± 1.10 60.03 ± 1.07 19.32±1.13
      
# a 
TTW >500 >500 367.6± 1.07 >500 83.46±1.07
      
# a 
CP >500 >500 >500 439.7 ± 1.07 125.9±1.08
Values are given as means ± SD; Potential antioxidant activity of extracts (EC50 µg/ml) is 
the concentration at 50% DPPH inhibition. The antioxidant activity was assessed on 
1mg/ml of dried crude plant extract / compound. One-way Anova followed with Tukey’s 
was used to assess the level of significance between solvents of the same plant. The 
superscript “a” means the extract was significantly high when compared to the rest of the 
remaining extracts in the same row. Extract with # superscript indicated significant 
difference p< 0.05compared to BHT. 
 
 
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Figures 4A: Antioxidant activities of aerial parts of P. amarus (A) and whole T. 
tetraptera fruit (B) sequentially extracted using various solvents.The figure shows a 
concentration dependent curve. Increasing the concentration of the extract increases its 
antioxidant potential.  One milligram per milliliter ofeach extract was tested as described in 
the method section. 
  
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Figures 4B: Antioxidant activities of C. papaya leaves (C), T. tetraptera seed (D), M. 
piperita leaves (E) and L. sativa leaves(F)sequentially extracted using various solvents.The 
figure shows a concentration dependent curve. Increasing the concentration of the extract 
increases its antioxidant potential. One milligram per milliliter of each extract was tested as 
described in the method section. 
  
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Figures 4C: Antioxidant activities of F. asperifolia leaf extracts (G), C. erythrocarpus leaf 
extracts (H), T. tetraptera pulpextracts(I), J. curcas leaf extracts(J) and M. oleifera leaf 
extracts (K) sequentially extracted using various solvent.The figure shows a concentration 
dependent curve. Increasing the concentration of the extract increases its antioxidant 
potential.  One milligram per milliliter of each extract was tested as described in the 
method section. 
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4.3 In Vitro Cell viability Assay 
Fig 5A shows a representative plot of the cytotoxic profile of extracts of P.amarus on PC3 
and LNCaP prostate cancer cell lines. The figure shows that all the fractions inhibited the 
growth of the cancer cells in a concentration dependent fashion. For the extractsof P.amarus, 
the aqueous extract exhibited the strongest growth inhibition of PC3 and LNCaP cells. 
Compared to all the plant extracts, positive control, curcumin demonstrated the strongest 
inhibition of cell growth as expected.  The median inhibitory concentrations, IC50 values,were 
derived from the log-concentration curve of the plots for the various crude extracts of plant 
and are recorded in Table 6. 
 
Figure 5A: Representative plot of the cytotoxic profile of P. amarus extracts on PC3 (Figure 
A. and Figure B.) and LNCaP (Figure C. and Figure D.) prostate cancer cell lines. 
  
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The Table 6 below shows data on the cytotoxic effect of the plant extracts and curcumin on 
PC3, LNCaP and PNT2 cell lines. The EA extract of whole T. tetraptera fruit demonstrated a 
stronger inhibitory effect compared to all solvent extracts of the seed alone or the pulp alone 
and was more selective to PC3 and LNCaP prostate cancer cells with IC50 values of 5.39 
±1.20 µg/ml and 8.38 ±1.11 µg/ml, respectively. The AQ extract of P. amarus leaves showed 
stronger inhibitory effect compared to their remaining solvent extracts and was more selective 
to PC3 and LNCaP prostate cancer cells with IC50 values of 48.2±1.20 µg/ml and 55.54 ±1.11 
µg/ml, respectively when compared with the other solvent extracts. The AQ extract of P. 
amarus thus showed good selective indices (SI) of 5.92 and 5.1 for PC3 and LNCaP, 
respectively. Also, the EA extract of C. erythrocarpus showed strong inhibitory effecton the 
PC3 and LNCaP prostate cancer cells with IC50 values of 29.51±1.24 µg/ml and 15.75±1.23 
µg/ml, respectively. The EA extract of C. erythrocarpus gave good selective indices (SI) of 
16.42 and 28.63 for PC3 and LNCaP, respectively. The AQ extract of J. curcas also 
demonstrated a strong inhibitory activity on the PC3 prostate cancer cells with an IC50 value 
of 48.56±1.29 µg/ml and a selective index value of 20.59. Similarly, the EA extract of J. 
curcas demonstrated a strong inhibitory activity against LNCaP with IC50 value of 35.48±1.11 
µg/ml and a selective index of 3.97. The DCM extract of M. piperita showed a strong 
inhibitory activity on PC3 with IC50 value of 33.17±1.28 µg/ml. However, the selective index 
was less than two (SI<2) suggesting that it possessed strong inhibitory activity against normal 
cells PNT2. The EA extract of M. piperita showed a strong inhibitory effect on LNCaP 
prostate cancer cells with IC50 value of 10.38±1.23 µg/ml and possessed a selective index 
value of 6.22. Furthermore, the AQ extract of M. oleifera possessed good selective indices 
and inhibited growth of PC3 and LNCaP prostate cancer cells with IC50 values of 
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66.72±1.14µg/ml (SI=14.99) and 148.9±1.15µg/ml (SI=6.72),respectively. The DCM extract 
of F. asperifolia showed good selective indices and strong inhibitory effects yielding IC50 
values of 10.15±1.24 µg/ml (SI=14.79) and 25.57±1.33 µg/ml (SI=5.87) on PC3 and LNCaP 
prostate cancer cells, respectively. The AQ extract of C. papaya also possessed good selective 
indices and inhibited growth of PC3 and LNCaP prostate cancer cells. The IC50 values were 
45.68±1.16 µg/ml (SI =25.50) and 72.62±1.31 µg/ml (SI=13.77) for PC3 and LNCaP prostate 
cancer cells, respectively. The AQ extract of the pulp of T. tetraptera showed an anti-
proliferative activity and was more selective forPC3 and LNCaP prostate cancer cells with 
IC50 values of 47.74 ± 1.17µg/ml (SI=16.38) and 27.78 µg/ml (SI=28.15) when compared to 
the other remaining pulp extracts. Furthermore, the EA extract of T. tetraptera seed had 
stronger inhibitory activities on PC3 and LNCaP prostate cancer cells with IC50 values of 
55.28±1.23 µg/ml and 22.29±1.75 µg/ml, respectively when compared to the other remaining 
seed extracts. The IC50 values of the prostate cancer cells and the normal cells are presented in 
Table 6. 
  
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Table 5: Cytotoxic activities and selective indices of plant extracts toward selected cancer 
and normal cell line. 
 
CODES/Compou PC3 Selective LNCaP Selective   PNT2  
nd (IC50 µg/ml) index  (IC50 µg/ml ) index  (CC50µg/ml) 
Curcumin 7.35±1.27 1.37 2.84±1.24 1.89 5.38± 1.12 
 
PE 4 
a a 
65.1±1.58  0.48 2 28.7±1.43  0.99 2 26.3±1.36 
 
a a 
PA  DCM 98.82±1.22 0.54 43.97±1.14 1.22 53.54±1.22 
a a 
 EA 158.6±1.41 0.59 70.17±1.25 1.33 93.09±1.30 
a  
 ETOH 473.1±1.08 >1000 NA 
a  a  AQ 48.2±1.20 5.92 55.54±1.11  5.1 285.5±1.12 
 
PE  
a b,a 
884.4±1.50   634.6±1.52   NA 
a b,a 
CE DCM 118.2±1.30
 1.77 815.2±2.53  0.26 2.09±1.45 
a,* a,* 
 EA 29.51±1.24 16.42 15.75±1.23 28.63 451±1.20 
  
 ETOH >1000 >1000 NA 
a b,a 
 AQ 76.4±1.24  13.09 69.85±1.24  14.32 >1000 
 
  
PE  >1000   >1000   NA 
 
JC  DCM NA 
 NA  NA 
a a 
 EA 60.91±1.40 2 .31 35.48±1.11  3.97 140.9±1.35 
a a 
 ETOH 613.8±1.24 1.63 589.4±1.59 1.70 >1000 
a  
 AQ 48.56±1.29 20.59 >1000 1 >1000 
 
 a a PE 807.8±1.43 0 .68  198.3±1.28  2.75  546.2±1.26 
 
a a 
LS  DCM 204.2±1.20 0.88 161.4±1.16 6.20 180.1±1.26 
a a 
 EA 147.6±1.40 5.16 202.0±1.12 3.77 761.48 
a  
 ETOH 280.7±1.15 3.56 >1000 1 >1000 
a a 
 AQ 59.38±1.19 16.84 321.9±1.16 3.11 >1000 
 
a 
PE  286.4±1.37  1.74 3 61.4±1.39  0.65 2 34.4±1.31 
 
a a 
MP  DCM 33.17±1.28 0.75 116.6±1.17 0.21 24.86±1.27 
a a,* 
 EA 180.2±1.44 0.36 10.38±1.23 6.22 64.61±1.23 
a a 
 ETOH 138.1±1.13 2.09 199.7±1.15 1.45 289.2±1.23 
a a 
 AQ 422.8±1.22 2.37 159.1±1.19 6.29 >1000 
 
  
PE  >1000   >1000   NA 
a a 
MO  DCM 163.8±1.15 2 .37 194.3±1.24  2.0 388.5±1.38 
a 
 EA 161.3±1.20 1.38 129.7±1.33 1.72 222.9±1.20 
      
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CODES/Compou PC3 (IC50 Selective LNCaP  Selective  PNT2  
nd µg/ml) index (IC50 µg/ml ) index (CC50 µg/ml) 
a  
MO   ETOH 846.6±1.29 >1000 NA 
a a 
 AQ 66.72±1.15 1 4.99 148.9±1.14 6 .72 >1000 
  
  
PE  >1000   >1000  N A 
 
a, * a, * 
FA  DCM 10.15±1.24
 14.79 25.57±1.33 5 .87 150.2±1.24 
a a 
 EA 53.96±1.31 0.80 73.41±1.50 0.59 43..34±1.25 
a  
 ETOH 129.7±1.30 >1000
b,a  
 AQ 944.4±1.04  1.06 >1000 1  > 1000 
 
  
PE  >1000 1  > 1000  1 > 1000 
 
a a 
CP DCM 70.87±1.31 14.11 70.87±1.31 14.11 >1000 
a a 
 EA 336.0±1.30 0.57 931.5±1.26 0.21 192.2±1.20 
a  
 ETOH 978.2±1.21 0.89 >1000 0.87 868.8±1.28 
a a 
 AQ 45.68±1.16 25.50 72.62±1.13 13.77 >1000 
  
a  a
PE  336.5±1.33  27.94±1.27    NA 
 
a, * 
DCM 27.16±1.16 4 
a 
TTP .73 36.70±1.13
 3.50 128.4±1.33 
a, * a 
 EA 12.79±1.28 0.66 110±1.38 0.08 8.44±1.28 
a a 
 ETOH 164.3±1.36 0.54 188.2±1.55 0.47 89.22±1.27 
a a, * 
 AQ 47.74±1.17 16.38 27.78±1.13 28.15 532.1±2.81 
 
       
PE >1000 >1000 NA 
 
a  
TTS  DCM 307.3±1.29
 >1000  NA 
a a, * 
 EA 55.28 ±1.23 1 .55 22.29±1.75 3 .84 85.70±1.24 
a a 
 ETOH 136.6±1.12 1.73 418.4±1.51 0.57 236.6±1.26 
 AQ NA NA NA 
   
 a   a PE 539.9±1.29 444.3±1.22   NA 
a, * a, * 
DCM 11.49±1.20  7.95 26.84±1.15  3.40 91..39±1.30 
a, * a,* 
EA 5.39±1.24 7.72 8.38±1.27 4.97 41.61±1.34 
TTW  a, * a 
ETOH 21.04±1.26 1.44 9.51±1.23 3.19 30.36±1.35 
 
a a 
 AQ 241.2±1.19 4.15 649.2±1.24 1.54 >1000 
Values are given as means ± SD;IC50(µg/ml) is the concentration at 50% cytotoxic effect on 
PC3 and LNCaP cell lines. The cytotoxic activity was assessed on 1mg/ml of dried crude 
plant extract. CC50 is the concentration of the 50% cytotoxic effect on normal cell line PNT2; 
SI (selectiveindex) is the ratio CC50/IC50.One way Anova followed with Tukey’s was used to 
assess the level of significance between solvents of the same plant and on the same cell 
line.IC50values with superscript“a” means they havep values <0.05 when compared to the 
untreated wells. * denotes IC50less than 30 µg/ml which could be considered potential anti-
cancer candidates. 
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MTT assay results for PC3 
 
 
Figure 6A: Anti-proliferative activities of Curcumin (A), P. amarus leaf extracts (B) and C. 
erythrocarpus (C) leaf extracts on PC3 prostate cancer cell line.The cytotoxic activity was 
assessed on 1mg/ml of dried crude plant extract. 
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Figure 6B: Anti-proliferative activities of J. curcas leafextracts (D), L. sativa leaf extracts (E), 
M. piperita leaf extracts (F) and M. oleifera leaf extracts (G) on PC3 prostate cancer cell line. 
The cytotoxic activity was assessed on 1mg/ml of dried crude plant extract. 
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Figure 6C: Anti-proliferative activities of F. asperifolia (H),  C. papaya (I) , whole T. 
tetraptera (J), T. tetraptera  pulp (K)  and T. tetraptera seed extracts on PC3 prostate cancer 
cell line. The cytotoxic activity was assessed on 1mg/ml of dried crude plant extract. 
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Mtt Assay results for LNCaP Cells 
 
 
 
Figure 7A: Anti-proliferative activities of Curcumin (A), P. amarus (B), C. erythrocarpus (C) 
extracts on LNCaP prostate cancer cell line. The cytotoxic activity was assessed on 1mg/ml of 
dried crude plant extract. 
 
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Figure 7B: Anti-proliferative activities of extracts of F. asperifolia (D), L. sativa (E), M. 
piperita (F) and J. curcas (G) on LNCaP prostate cancer cell line. The cytotoxic activity was 
assessed at 1milligram/millitre of dried crude extract. 
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Figure 7C: Anti-proliferative activities of M. oleifera (H), C. papaya (I), whole T. tetraptera 
(J), T. tetraptera pulp (K) and T. tetraptera seed extracts (L) on LNCaP prostate cancer cell 
line. The cytotoxic activity was assessed on 1mg/ml of dried crude plant extract. 
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Mtt Assay results For PNT2 normal prostate cells 
 
 
 
Figure 8A: Anti-proliferative activities of Curcumin (A), C. erythrocarpus extracts (B) and C. 
papaya (C) extracts on PNT2 normal prostate cell line. The cytotoxic activity was assessed on 
1mg/ml of dried crude plant extract. 
 
 
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Figure 8B: Anti-proliferative activities of F. asperifolia extract (D), J. curcas extract (E), L. 
sativa extracts (F), M. oleifera extracts(G), T. tetraptera pulp extract (H) and whole T. 
tetraptera fruit extract (I) on PNT2 normal prostate cell line. The cytotoxic activity was 
assessed on 1 mg/ml dried crude extract 
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Figure 8C: Anti-proliferative activities of M. piperita leaf extract (J), P. amarus leaf extract 
(K), and T. tetraptera seed extract (L) on PNT2 normal prostate cell line. The cytotoxic 
activity was assessed on 1 mg/ml dried crude extract. 
  
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Chapter 5 
5.0 Discussion 
Cancer is a serious health problem in the whole world. Not only does it present itself as a 
disease but also comes with burden such as high economic cost as well as the loss of life. 
Some of the available current treatments are faced with resistance and toxicity and these 
necessitate the search for new, safer, moreaffordable and effective medicines. Medicinal 
plants have been used since time immemorial for the treatment of cancer. Some herbalists in 
Ghana prepare herbal decoction or ethanolic extract from these medicinal plants and use them 
for the management of prostate cancer without any scientific credence on safety and toxicity. 
Recent studies have shown that some medicinal plants have constituent phytochemicals that 
have the ability to scavenge free radicals and are cytotoxic to cancer cells (Raman, 2006; Dias 
et al., 2012). In general, the sequential extraction employed was used to extract active 
principles of plants based on their solubility which in turn influence the anti-cancer activities 
of the extract.  
 
The aim of the study was to identify medicinal plants used in Ghana for the management of 
prostate cancer and determine their anti-cancer activities. These medicinal plants used include 
Latuca sativa, Tetrapleura tetraptera (whole fruit, pulp and seed), Jatropha curcas, Capparis 
erythrocarpus, Ficus asperifolia, Mentha piperita, Carica papaya, Moringa oleifera and 
Phyllanthus amarus. High yields of dried crude extracts were obtained for polar solvents than 
non-polar solvents. The highest yield was obtained in ETOH extract of Tetrapleura tetraptera 
fruit and the lowest yield was obtained in PE extract of Tetrapleura tetraptera fruit. 
According to classification of antioxidants by Blois (1958), the aqueous extract of P. amarus 
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showed a very strong antioxidant activity (EC50=19.32±1.13 µg/ml). Similarly the ethanolic 
extract of P. amarus, aqueous extracts of T. tetraptera pulp only and the whole fruit of T. 
tetraptera showed a strong antioxidant activity. The remaining extracts showed weak 
antioxidant activities.  
 
Furthermore, all the extracts possessed phenolic compounds with the highest recorded in the 
ETOH extract of P. amarus. Phenolic compounds such as flavonoids, gallic acid and 
epigallocatechin have been reported to possess good antioxidant activities and anti-cancer 
activities making them good target compounds as therapies. Hence extraction using ethanol 
would be more ideal since most phenols are soluble in the ETOH solvent. Even though some 
phenols become more soluble when heated in AQ extracts, it appeared that the phenolic 
content was not as high as found in ethanolic extract of P. amarus. According to the US 
National Cancer Institute plant screening program, in vitro cytotoxic activity of a crude 
extract should have an IC50 value less than 30 µg/ml before it can be considered as a potential 
anti-cancer agent for further development (Boik, 2001). Plant extracts with IC50 values 
between 30µg/ml and 200 µg/ml have moderate potential to be developed into cancer 
therapeutic agents whilst those above 200 µg/ml are unlikely to be selected for further 
development (Itharat, 2004). Also, a good selectivity index and anti-cancer property is 
conferred on a plant crude extractwhen it possesses high cytotoxic activity in cancer cells 
whiles having low cytotoxicities to normal cells (Al-Rashidi et al., 2011). Badisa et al. (2009) 
considered extracts with a selectivity index value greater than two (SI>2) to be a good drug 
candidate for drug development. Several compounds from medicinal plants have been 
reported to show strong cytotoxic activities (<30 µg/ml). However, these known drugs such as 
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docetaxel and paclitaxel suffer resistance against cancercells and associated toxicities with 
these drugs warrants the investigation of other medicinal plants for source of anti-cancer 
agents.  
5.1.1 Latuca sativa 
In this study, higher yields of dried crude extracts were observed in the more polar solvents 
than the non-polar solvent therefore suggesting that most of the constituents of L. sativa were 
polar. Phenolic compounds in many herbs form a major group of secondary metabolites with 
bioactive pharmacological capacity attributed to anti-prostate cancer and antioxidant 
activities. Lu and Foo (2002) have indicated that antioxidant activity of vegetable extracts is 
associated with phenolic compounds.  
 
A study by Edziri et al. (2011) has indicated that methanolic (MeOH) extract of L. sativa had 
a higher TPC than the AQ extract. The present study also shows that the ETOH extract 
appeared to possess the highest total phenolic content, followed by AQ extract and then EA 
extract (Figure 3C). However, the EA extract showed a potent antioxidant activity followed 
by ETOH and then the AQ extracts. This shows that phenolic substances which may possess 
good antioxidants activity are soluble in polar organic solvents and could be used in the 
management of prostate cancer. There was no correlation between the total phenolic 
compounds in the extract and their respective antioxidant activities. This could be due to the 
fact that not all phenolicshave antioxidant activities.Also, antioxidants within a group can 
vary remarkably such that the same levels of phenolic substances do not necessarily 
correspond to the same antioxidant response.   
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Apart from EA extracts which demonstrated moderate antioxidant activities, all the other 
crude extracts showed weaker antioxidant activities. The present results are consistent with 
previous studies that have shown that L. sativais very rich in antioxidants (Sharma et al., 
2002; Patil et al., 2003). This suggests that EA extract could be a promising agent in the 
management of oxidative stress related diseases like cancer. Indeed, the EA extract ofL. sativa 
possessed the most potent antioxidant activity.  
 
Pepe et al. (2015) have reported that the methanolic extract of the L. sativa leaves 
significantly decreased cycloxygenase-2 expression, reactive oxygen species and the 
liberation of nitric oxide. However, there has not been any documentation of anti-prostate 
cancer activity of extracts of L. sativa. In this study the aqueous extract of L. sativa was found 
to be more selective for PC3 prostate cancer cells with IC50 value of (59.38±1.19 µg/ml, 
SI=16.84). Although, the DCM extract was more cytotoxic to LNCaP prostate cancer cells, it 
appeared to possess low selective index (SI<2). Hence, suggesting that EA extract of L. sativa 
could be the next preferable choice for LNCaP prostate cancer cell treatment compared to the 
DCM extract, but it is unlikely to be selected for further anti-cancer drug development 
(Itharat, 2004). 
5.1.2 Tetrapleura tertraptera 
In this study, the AQ extract of the whole fruit had the most potent antioxidant activity when 
compared to their respective solvent extracts. This suggests that the aqueous extracts 
contained bioactive compound with strong antioxidant activities that could be used to manage 
oxidative stress related disease including prostate cancer. 
 
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The total phenolic compounds in the aqueous extract of the T. tetraptera pulp and seeds were 
very high suggesting the phenolic substances extracted are highly soluble in polar 
solvents.There was positive correlation between the total phenolic contents and antioxidant 
activity. However, the whole fruit showed the converse. The ETOH extract of the whole T. 
tetraptera fruit pulp had the highest content of phenolic substances. Although the AQ extract 
of the pulp and seed had high phenolic content which exhibited a positive correlation to the 
antioxidant activity, the AQ extract of the whole fruit had a lower phenolic content but 
stronger antioxidant activity than the pulp of the fruit only or seed. This suggests that the AQ 
extract of the whole fruit possesses a very polar compound with stronger antioxidant activity. 
The present results are consistent with a previous study that showed that aqueous extract of 
whole T. tetraptera (with seed) possessed the strong antioxidant activity (Josiah et al., 2017). 
 
Ethanolic crude extract of T. tetraptera has previously been reported to be cytotoxic to Ehrlich 
ascites carcinoma cells (Ozaslan, 2016). In this study, it was also shown that all the extracts of 
the whole fruitsand pulp only exhibited anti-proliferative activity of the cancer cells studied in 
a concentration dependent manner. Although, the EA and ETOH extract of the seed exhibited 
anti-proliferative activity of the cancer cells in a concentration dependent manner, the PE 
extract of the seed did not have any activity on the cancer cells. This suggests that the 
phytochemicals present in PE extract of seed do not possess anti-prostate cancer activity. 
The aqueous extract of T. tetraptera pulp was more selective for PC3 and LNCaP with IC50 
values <50 µg/ml, SI values of about 16 and 28, respectively. The DCM, EA and ETOH 
extracts of the whole T. tetraptera met the cut off limit for good anti-cancer extracts (<30 
µg/ml) and could be considered as potential anti-cancer agent for further development (Boik, 
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2001; Itharat, 2004). The EA extract of the whole T. tetraptera plant was more cytotoxic and 
selective to PC3 and LNCaP prostate cancer cells with IC50 values of 5.39±1.24 µg/ml 
(SI=7.72)and 8.38±1.27 µg/ml (SI=4.97),respectively. The whole T. tetraptera fruit (with 
seed) extract showed a more potent anti-prostate cancer activity when compared to either the 
fruit alone or the seeds alone. This result suggests that the seed and the pulp produced a 
synergistic effect. The plant is reported to have phytochemical such as tannins, saponins, 
alkaloids, anthraquinones, glycosides, flavonoids and other phenolic compounds in AQ and 
ETOH extracts of the fruit (Gberikon et al., 2015) as well as minerals such as calcium, zinc, 
potassium and iron (Okwu, 2003). 
5.1.3 Capparis erythrocarpus 
The present study assessed the pharmacological benefits of the leaves of C. erythrocarpus. All 
the extracts of C. erythrocarpus leaves possessed antioxidant properties and phenolic 
substances. The ethanolic crude extract possessed the highest total phenolic content indicating 
that most of the phenols are soluble in ethanol. There was no correlation between the total 
phenolic content and antioxidant activity. The antioxidant activities of the extracts were 
concentration dependent. The DCM extract showed a stronger antioxidant activity when 
compared to the rest of the extracts. 
The result showed that, the ethyl acetate extracts of C. erythrocarpus exhibited a stronger 
cytotoxic activity and was more selective for PC3 and LNCaP prostate cancer cells 
(IC50=29.51±1.24 µg/ml, SI=16.42 and IC50=15.75±1.23 µg/ml, SI=28.63 respectively) than 
normal cells. Strong pharmacological activities of root extracts of the plants have been 
attributed to active phytochemicals such as flavonoids and alkaloids (Danquah et al., 2011). 
Unlike previous studies focused on the roots, this study has shown that the ethyl acetate 
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extracts of C. erythrocarpus leaves have strong anti-proliferative activity against prostate 
cancer cells. 
5.1.4 Ficus asperifolia 
All the extracts showed antioxidant activities but were not that strong when compared to the 
AQ extract. The aqueous extract possessed a stronger antioxidant activity when compared 
tothe remaining extracts of the plant. This suggests that AQ extract of F. asperifolia possessed 
compoundswith strong antioxidant activity and could be a promising agent for the treatment 
and management of oxidative stress related diseases including prostate cancer. Similar 
findings were observed in a study done by Ojo and Akintayo, (2014). They indicated that the 
DPPH scavenging ability of AQ extract of F. asperifolia at the concentration of 5mg/ml was 
78.65 ± 1.15%. Ojo et al. (2014) reported that the therapeutic benefit could be owed to the 
presence of saponins, glycosides, tannins, alkaloids and phenolic compounds in aqueous crude 
extract of Ficus asperifolia. All the extracts possessed phenols at 5 mg/ml. In the study, the 
total phenolic content was highest in ethanolic extracts with values (30.78 ±1.68 g/100g GAE) 
indicating that most of the phenols were soluble in the ethanolic extract. Moreover, the TPC 
values of the AQ extract appeared to be higher than the reported values by Ojo and Akintayo 
(2014). This could be due to the different extraction methods employed and different 
environmental factors affecting the yield of the plant constituents. Furthermore, a previous 
study has indicated that the methanolic extract of Ficus asperifolia possesses a good amount 
of total phenolic content (GAE: 31.96 mg/ml).  
The present results are consistent with results obtained by Ojo and Akintayo (2014) which 
indicated good amounts of total phenolic content in alcoholic solvent extracts. Previous 
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studies focused on other cell lines such as breast cancer MCF-7 and malignant melanoma cell 
lines DLD-1.  This study has confirmed anti-prostate cancer properties against PC3 and 
LNCaP prostate cancer cells. The anti-cancer properties were observed for all the extracts. 
The DCM extract of Ficus asperifolia showed a stronger anti-prostate cancer activity and was 
more selective to PC3 and LNCaP prostate cancer cell lines than the remaining extracts of F. 
asperifolia. The EA extract showed a moderate anti-cancer activity on PC3 and LNCaP 
prostate cell lines but possessed weak selectivity index (SI<2). The dichloromethane extract 
exhibited a stronger cytotoxic activity and was more selective to PC3 and LNCaP prostate 
cancer cells with IC50 values of 10.15±1.24 µg/ml, SI=14.79 and 25.57±1.33 µg/ml,SI=5.87, 
respectively when compared to the rest of the extracts. 
5.1.5 Mentha piperita 
All the extracts showed antioxidant properties with the EA extract exhibiting a stronger 
activity at a concentration of 1 mg/ml. The ETOH extract in this study showed a higher 
phenolic content of 3.97± 0.34g/GAE100g with stronger antioxidant activity (EC50= 
0.43±0.01 mg/ml) than the previous study conducted by Farnad et al. (2014). Farnad et al. 
(2014) reported that the ethanolic extract showed a total phenolic content of 1.99 ± 
0.32g/GAE 100g with a DPPH free radical scavenging activity (80.74 ± 0.83%, IC50= 11.32 ± 
0.71 mg/ml) at a concentration of 20 mg/ml.  
This difference could be due to the different extraction techniques employed in the study and 
the different environmental factors affecting the yield of phenols. Also, all the extracts 
contained phenolic compounds. The ethyl acetate extract gave the highest total phenolic 
content indicating that most of the phenols were soluble in the ethyl acetate solvent. All the 
extracts showed antioxidant activities indicating that they could be used in scavenging 
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reactive free radicals responsible for oxidative stress related ailments like cancer. The ethyl 
acetate extract showed a stronger antioxidant activity when compared to the remaining 
extracts.  
 
Sun et al. (2014) reported that the essential oil of M. piperita has anti-inflammatory properties 
against SGC-7901 gastric cancer cells, SPC-A-1 lung cancer cells and K562 cancer cell lines.  
Unlike previous studies focused on gastric cancer, lung cancer and erythroleukemia, this study 
has also shown that all the extracts of M. piperita possessed anti-prostate cancer activity. 
Among the extracts, the DCM extract of M. piperita showed the strongest cytotoxic activity 
against PC3 with IC50 value of 33.17±1.28 µg/ml while EA extract exhibited the strongest 
cytotoxic activity against LNCaP prostate cancer cells with IC50 value of 10.38±1.23 µg/ml. 
5.1.6 Carica papaya 
In this study, all the extracts of C. papaya possessed phenols. The ethanolic extracts possessed 
more phenols than the rest of the extracts. This shows that most of the phenols are soluble in 
ethanolic solvents.This result confirms similar findings in a study done by Fidrianny et al. 
(2016). Fidrianny et al. (2016) have established that the ethanolic extract possessed a 
significantly higher phenolic content (5.70 g GAE/100 g) whiles the hexane leaf extract 
possessed a lower phenolic content (1.88 g GAE/100 g). However, it did not confirm studies 
done by Vuong (2013) and Mandal (2015) who found the converse. This could be due to the 
different extraction methods employed in the study. Mandal et al. (2015) indicated that total 
phenolic content of aqueous, methanol and petroleum ether of C. papaya leaves extract were 
57.6 ± 3.7 μg GAE/g dry weights, 47.8 ± 2.4 μg GAE/g dry weight and 8.4 ± 0.4 μg GAE/g 
dry weight, respectively. Additionally, Vuong (2013) has indicated that the aqueous extract of 
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C. papaya contained significantly higher polyphenols than ethanolic extract with values 23.06 
± 1.06 mg GAE/ g dry weight and 9.43 ± 0.14 mg GAE/ g dry weight, respectively. 
 
A report by Fidrianny et al. (2016) has indicated that the ethanolic extract of pawpaw leaves 
possess a strong antioxidant activity with EC50 value of 0.84 µg/ml. However, Mandal et al. 
(2015) showed that extracts of pawpaw leaves (aqueous, methanol and petroleum ether) had 
weak antioxidant activities (EC50=247 ± 3.5 µg/ml, 262.2 ± 4.2 µg/ml and 171.5 ± 3.3 µg/ml 
respectively) using DPPH assay.   
 
In this study, all the extracts showed antioxidant activities indicating that they could be used 
in the treatment of oxidative stress related diseases. The aqueous extract of C. papaya showed 
the most potent antioxidant activity. 
 
An earlier study by Otsuki et al. (2010) has reported a strong anti-cancer activity of the 
aqueous extract of C. papaya leaves. Likewise, the aqueous extract was reported to have 
exerted immunomodulatory activity and anti-tumor activity in Jurkat, Molt-4, CCRF-CEM 
and HPB-ALL cell lines (Otsuki et al., 2010).  
 
Unlike previous reports focused on cancers related to blood, this study has shown anti-
prostate cancer activity. All extracts of C. papaya except petroleum ether and ethanolic 
extracts showed anti-proliferative activities against the prostate cancer cells. The aqueous 
extract exhibited a stronger anti-proliferative activity and was more selective to PC3 with IC50 
value of 45.68±1.16 µg/ml, SI=25.50 while the dichloromethane extract demonstrated 
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stronger anti-proliferative activity against LNCaP prostate cancer cell lines with IC50 value of 
70.87±1.31 µg/ml, SI=14.11. Alorkpa (2016) has reported the presence of alkaloids and 
tannins in non-polar solvent extracts of C. papaya whereas phytochemical compounds such as 
flavonoids, glycosides and saponins were reported to be present in polar solvent extract of C. 
papaya. In this current study the anti-prostate activity may be due to the phytochemical 
compounds present.  
5.1.7 Moringa oleifera 
From the results in this study, all the extracts possessed some amount of phenolic substances 
which suggest that it could be used in the management of diseases since several phenols have 
been shown to have pharmacological activities. The ethanolic extracts possessed the highest 
amount of phenols suggesting that most of the phenols were soluble in ethanol. Previous 
reports have indicated that different solvent extracts of moringa leaves contain phenolic 
compounds, hence a great source of polyphenols (Sultana et al., 2009; Sreelatha and Padma, 
2009).Also, all the extracts showed antioxidant activities, suggesting that they could be used 
in the management of oxidative stress related diseases including prostate cancer. Among the 
extracts, the aqueous extract showed the strongest antioxidant activity and did not correlate 
with the phenolic content. This suggests that AQ extract may possess phytochemical 
compounds with stronger antioxidant activities.The percentage scavenging activity of the 
extract against free radicals was concentration dependent. 
A report by Coppin et al. (2013) have indicated that Moringa oleifera leaf extract is able to 
inhibit nitric oxide (NO) produced by macrophage cells which was treated with bacterial 
lipopolysaccharide (LPS). Moreover, anti-inflammatory and immunomodulatory effects of 
Moringa oleifera leaves have been reported in many in vivo and in vitro studies (Das et al., 
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2013). In addition, ethyl acetate extracts of Moringa oleifera leaves was reported to inhibit 
human macrophage cytokine production (TNF-α, IL-6 and IL-8) induced by extract of 
cigarette smoke (Kooltheat et al., 2014). Unlike previous studies focused on anti-
inflammatory and immunomodulatory effects, this study has shown anti-prostate cancer 
activity of Moringa oleifera. In this study, all the extracts except petroleum ether and 
ethanolic extracts exhibited anti-proliferative activity against the prostate cancer cell lines in a 
concentration dependent manner. Among the extracts, the aqueous extract showed the 
strongest anti-proliferative activity and were more selective to PC3 and LNCaP prostate 
cancer cells with IC50 values of 66.72±1.15 µg/ml, SI=14.99 and 148.9±1.14 µg/ml, 
SI=6.72,respectively. 
5.1.8 Phyllanthus amarus 
In this study, all the extracts possessed varying amounts of polyphenols. The ETOH extract 
possessed the highest amount of phenols indicating that they are more soluble in the ethanolic 
solvent. Djeridane et al. (2006) indicated that the solubility of phenolic substances in an 
extract largely depends on the choice of the solvent used, due to the degree of polymerization 
of the phenols and the kind of interactions that exist between them. Also, all the P. amarus 
extracts showed antioxidant activities in a concentration dependent manner. The aqueous 
extract exhibited the most potent antioxidant activitywhich did not correlate with the phenolic 
content.This suggests that antioxidant activity may not be largely contributed by the phenols 
present in the extract. Numerous phytochemical compoundssuch as alkaloids, flavonoids, 
hydrolysable tannins, lignins, polyphenolic compounds and tetracyclic triterpenoids have been 
reported to be present in P. amarusextracts (Verma, 2014). 
 
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Furthermore, this study showed that, apart from ethanolic extract, all the extracts possessed 
anti-proliferative activity against both PC3 and LNCaP prostate cancer cell lines in a 
concentration dependent manner. From Figure 5A, the percentage cell viability decreased as 
the concentration of extract increased.The present results are consistent with the previous 
report, where P.amarus extract significantly inhibited the growth of DU 145 cell line 
(Nguyen, 2017).The aqueous extract showed the highest anti-prostate cancer activity against 
PC3 prostate cancer cells with IC50 value of (48.2±1.20 µg/ml, SI=5.92) and was more 
selective to LNCaP cells with IC50value of (55.54±1.11 µg/ml, SI=5.1). Previous studies have 
reported the presence of phyto-compounds such as hydrolazable tannins, geraniin, gallic acid 
and ellagic acid in aqueous extracts of P.amarus (Verma, 2014; Okuda et al., 1979).  
5.1.9 Jatropha curcas 
The results from this study revealed that all the extracts possessed some amount of phenolic 
substances. The ethanolic extract possessed more phenols than the remaining solvents 
extracts. This suggests that the phenolic compounds present were soluble in ethanolic solvent. 
From the results, Figure 4C. showed that the scavenging ability of all the extracts were 
concentration dependent. The percentage scavenging activity increased as the concentration of 
the extract increased. Although, the ETOH extract possessed the highest content of phenolic 
substances it did not correlate with the antioxidant activity, as the aqueous extract showed a 
more potent antioxidant activity than the remaining extracts. This suggests that the polar 
phytochemicals of AQ extract possessed stronger antioxidant activities. Conversely, previous 
studies have reported that the methanolic extract of J. curcas possessed strong antioxidant 
activity using DPPH assay(Oskoueian et al., 2011; Fu et al., 2014).  
 
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Uche and Aprioku (2008) have reported that methanolic extract of J. curcas leaves possess 
anti-inflammatory activity on Wister albino rats.  A previous study has reported that 
methanolic extracts of the leave had cytotoxic activity against HT 29 colon cancer cell line 
(Oskoueian et al., 2011). Unlike previous studies focused on human colon cancer, this study 
has shown anti-prostate cancer activity.The aqueous extract showed potent anti-cancer 
activities and a good selective index against PC3 prostate cancer cells,whilst the ethyl acetate 
extract showed a stronger anti-cancer activity and a good selective index against LNCaP 
prostate cells when compared to the remaining solvent extracts of J. curcas. Rejila et al. 
(2012) have reported the presence of phytochemical compounds such as kaempferol, 
coumarin, catechin and quercetin in polar solvents extract of J. curcas leaves, which possesed 
anti-cancer activities.   
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CHAPTER 6 
6.0 Conclusion and Recommendation 
6.1 Conclusion 
The study has shown that all the extracts contained phenolic substances and had antioxidant 
activities which may be useful in the management or treatment of oxidative stress related 
diseases such as prostate cancer. Among all the plant extracts, the ethyl acetate extract of the 
whole fruit of T. tetraptera exhibited the strongest anti-prostate cancer activity against LNCaP 
and PC3 prostate cancer cells. Also, other solvent extracts that possessed strong anti-prostate 
cancer activity as well as good antioxidant activitiesinclude AQ extracts of P. amarus, C. 
erythrocarpus, and C. papaya. Furthermore, ethyl acetate extract of C. erythrocarpus showed 
a strong anti-proliferative activity. These findings suggest that, these extracts could be 
potential anticancer agents. Also, these findings lend pharmacological credence to the 
suggested folkloric uses of the fruit and leaves of the plants in the management of prostate 
cancer. 
6.2 Recommendation 
Future studies should include characterization of the phytochemical profile of the plant 
extracts. Also, the toxicological safety of the bioactive fractions in an in vivo animal model 
should be elucidated. The bioactive compounds responsible for the anti-prostate cancer 
activity should be isolated and characterized. Further work involving the determination of the 
targets of intracellular signaling pathways must be done.  
 
 
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APPENDIX A 
Total Phenolic Content 
Preparation of Gallic Acid Standard  
Stock concentration = 1 mg/ml  
Firstly, 1 mg of gallic acid was weighed and first dissolved in 100μL of absolute ethanol and 
then topped up to 1000 μl with 900μl of dH2O.  
Also, a two- fold serial dilutions was done to obtain working concentrations of; 1 mg/ml, 
0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml, 0.3125mg/ml and 0.015625mg/ml.  
Mathematical presentation 
2-fold dilution = 1:1  
Stock concentration = 1mg in 1ml (1000μL) of solution  
For first dilution, volume needed = ½ × 1000μL = 500μL 
Therefore, 500μL of stock solution + 500μL of solvent = 0.5mg/ml of GA.  
Preparation of 0.09 M Sodium bicarbonate (Na2CO3)  
The volume of required Na2CO3 for a plant sample = 150Μl × 96 = 14400μL. 
Ten grams of Na2CO3was dissolved in 40ml of distilled water and boiled. 
The solution was cooled and pure crystals of Na2CO3 were added to induce recrystallization.  
The solution was then filtered and 10 ml of distilled water was added to obtain 5ml of Na2CO3 
solution. 
Sample Preparation  
Stock concentration = 5 mg/ml  
Serial dilutions (2-fold) to obtain working concentrations of; 5mg/ml, 2.5mg/ml, and 
1.25mg/ml. 2-fold dilution = 1:1   
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APPENDIX B 
Free Radical Scavenging Activity (DPPH) 
Sample Preparation  
Stock concentration of crude extracts and standard BHT = 1mg/ml each 
A two-fold serial dilutions was done to obtain working concentrations of; 1mg/ml, 0.5mg/ml, 
0.25mg/ml, and 0.125mg/ml, 0.0625mg/ml, 0.03125mg/ml and 0.0015625mg/ml. 
2-fold dilution = 1:1  
Stock concentration = 1mg in 1ml (1000μL) of solution  
For first dilution, volume needed = ½×1000μL = 500μL  
Therefore, 500μL of stock solution + 500μL of solvent = 0.5mg/ml of sample. 
The procedure above was used to prepare the successive working concentrations  
Preparation of 0.05 DPPH solution 
Preparation of 0.5mM DPPH for five crude extracts of a plant sample 
Volume of DPPH required for free radical scavenging assay for a plant sample = 100μL×164 
wells = 16400 μL 
However, 18000μL (18 ml) of DPPH was prepared for each plant sample to cater for loss.  
1 Molar mass of DPPH = 394.32g/mol 
3
Also, Concentration (C) = number of moles (n)/volume (dm )  
3
This implies, n = C ×V (dm ) 
3
Concentration (C) = number of moles (n)/volume (dm )  
3
Hence, n = C ×V (dm ) 
-3 -6
n = (0.5 × 10 ) ×0.018= 9.0 × 10  moles  
Theoretically, n = mass (m) /Molar mass (M)  
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Therefore, mass (m) = n × M  
-6 -3
m= (9×10 ) ×394.32 g/mol = 3.5489 ×10 g  
m = 3.55 mg DPPH  
Preparation of 0.5mM DPPH solution was done by weighing 3.55 mg of DPPH and dissolving 
in absolute methanol and making up to 18 ml in a 50 ml falcon tube. 
  
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APPENDIX C 
Preparation of Complete RPMI Media 
Materials 
RPMI, 10% FBS( foetal bovine serum), 1% streptomycin-penicillin, serological pipettes (5ml, 
10ml and 25 ml) and falcon tubes (15 ml and 50 ml) 
Procedure 
The complete media was prepared in a sterilized laminar flow hood. A volume of 45 ml RPMI 
culture media (already containing 1% streptomycin-penicillin) was aliquoted into a 50 ml 
falcon tube using a serological pipette and a pipette filler machine. It was then followed by the 
addition of a volume of 5ml FBS and then mixed together gently to form a complete RPMI 
media 
Cell Count 
Material 
Trypan blue, hemocytometer, coverslip, 70% ethanol, microscope, pipette tips (200 µl and 
1000 µl) and cells (PC3, LNCaP or PNT2) 
Method 
5
A cell suspension of 1 x10  cells/mL was prepared after trypsinization of attached cells. A 
five times dilution of the cells was prepared in a 1:4 ratio of the cell suspension to trypan blue 
in an eppedorf tube. It was gently mixed and allowed to stand for 2 minutes. 
Prior to counting, the hemocytometer and coverslip was washed with 70% ethanol and 
allowed to dry. The cell suspension in trypan blue was then transferred unto the 
hemocytometer already covered with a cover slip. The hemocytometer was then mounted on 
the light microscope and the unstained cells (viable cells) were counted at a × 40 
magnification. The unstained cells in each of the 4 quadrant of the hemocytometer were 
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counted as viable. An average of the four different counts was calculated and multiplied by 
4 
the dilution factor of 5 and 10 to obtain the total number of cells/ml as shown below. 
(𝐴+𝐵+𝐶+𝐷)×104×5
Number of viable cells =  
4
where A, B, C, D are the number of viable cells in each of the four quadrantsrespectively.The 
5
cells were seeded at 1 ×10 / 100 µl into each well of the 96 well plate and waited a day after 
to get the cells attached to the well before the curcumin or crude extracts were added 
 
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