GROWTH PERFOMANCE AND PHYTOCHEMICAL PROFILES OF Prunus africana SAMPLED FROM MUGUGA, KOBUJOI AND KARURI, KENYA

Nyamai Dorothy Wavinya (BSc.) I56/24138/2013

A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Biochemistry) in the School of Pure and Applied Sciences of Kenyatta University

March 2016 i

DECLARATION

ii

DEDICATION

To my family, who accorded me support and encouragement during my studies more so to Edith Mwende and Joseph Mutua. iii

ACKNOWLEDGEMENTS

My gratitude goes to my research supervisors Dr. Marion Burugu, Dr. Margaret

Ng’ang’a and Dr. Alice Muchugi for their guidance, encouragement and scholarly suggestions throughout the research. My heartfelt thanks goes to the World

Agroforestry Centre for funding the project and to International Centre for Insect

Physiology and Ecology through Prof. Baldwyn Torto for allowing me to use their facilities for this research. Special thanks to all the staff of Biochemistry and

Biotechnology Department, Kenyatta University for their support and cooperation during this course. My special thanks goes to my friends Robert Kariba, Samuel

Manthi, Vincent Njung’e, Xavier Cheseto, George Mutungi, Ann Mbora and

Agnes Were for their support towards the completion of this thesis. To my colleagues, Godfrey Mutero, Phillip Einstein, Robert Ouko, Wycllife Arika, Rose

Chemutai, Amos Musyoki, Festus Kioko, Carol Kerubo and Hibert Rachuonyo thank you much for your moral support and encouragement during my studies and the development of this thesis. Lastly, to my family for their financial and moral support which enabled me to earn the Masters degree and above all to The

Almighty God for giving me the strength to do this project. iv

TABLE OF CONTENTS DECLARATION ...... i DEDICATION ...... ii ACKNOWLEDGEMENTS ...... iii TABLE OF CONTENTS ...... iv LIST OF FIGURES ...... viii LIST OF TABLES ...... ix LIST OF APPENDICES ...... x ACRONYMS AND ABBREVIATIONS ...... xii ABSTRACT ...... xiii CHAPTER ONE ...... 1 INTRODUCTION ...... 1 1.1 BACKGROUND INFORMATION ...... 1 1.2 PROBLEM STATEMENT ...... 5 1.3 JUSTIFICATION ...... 6 1.4 RESEARCH QUESTIONS ...... 7 1.5 OBJECTIVES ...... 7 1.5.1 General objective ...... 7 1.5.2 Specific objective ...... 7 LITERATURE REVIEW ...... 9 2.1 Biology of Prunus africana ...... 9 2.1.1 Lifecycle and longevity of Prunus africana ...... 9 2.1.2 Flowering and fruition of Prunus africana ...... 10 2.1.3 Reproductive biology ...... 11 2.2 Ecology of P. africana ...... 12 2.2.1 Origin of P. africana in Africa ...... 12 2.2.2 Natural distribution of P. africana in Africa ...... 13 2.2.3 Environmental factors in Prunus africana habitats...... 14 2.2.3.1 Topography ...... 14

2.2.3.2 Climatic factors ...... 14

2.2.3.3 Soils...... 15 v

2.3 Conservation of P. africana ...... 15 2.3.2 Policy and Regulations on P. africana conservation ...... 16 2.3.2.1 National and local regulations on P. africana bark harvesting ...... 16

2.3.2.2 International policy on conservation of P. africana ...... 17

2.3.3 Cultivation of P. africana ...... 19 2.3.3.1 Propagation of P. africana ...... 20

2.4 Prunus africana provenance trial...... 20 2.5 Provenance based phytochemical variation ...... 23 2.6 Economic importance of P. africana ...... 24 2.6.1 Ecosystem functions ...... 24 2.6.2 Uses of P. africana as timber ...... 25 2.6.3 Medicinal uses of P. africana ...... 25 2.6.3.1 Use of Prunus africana in traditional medicine...... 25

2.6.3.2 Use of Prunus africana in modern medicine ...... 26

2.6.4 Benign Prostatic Hyperplasia ...... 27 2.6.4.1 Pharmacotherapy...... 28

2.6.4.1.1 Conventional therapy ...... 28

2.6.4.1.2 Prunus africana as a herbal remedy for BPH ...... 31

2.7 Gas Chromatography-Mass Spectrometry (GC-MS) ...... 36 2.7.1 National Institute of Standards and Technology (NIST) Library analysis ...... 37 CHAPTER THREE ...... 39 MATERIALS AND METHODS ...... 39 3.1 Study site ...... 39 3.2 Sample collection ...... 39 3.3 Preparation of tree rings for age determination ...... 40 3.4 Visual cross dating ...... 41 3.5 Determination of tree density...... 42 vi

3.6 Reagents and reference compounds ...... 42 3.7 Sample Preparation ...... 43 3.7.1 Extraction of essential oils ...... 43 3.7.2 Preparation of extracts for GC-MS analysis ...... 44 3.8 Instrumentation and chromatographic conditions ...... 44 3.8.1 Gas chromatography – Mass Spepectrometry analysis ...... 45 3.8.2 Identification of components ...... 46 3.9 Liquid Chromatography-Mass Spectrometry analysis...... 47 3.9.1 Preparation of samples for LC-MS ...... 47 3.9.2 LC-MS Analysis ...... 47 3.10 Statistical analysis ...... 48 CHAPTER FOUR ...... 49 RESULTS ...... 49 4.1 Morphological characterization of Prunus africana trees from Muguga stand ...... 49 4.2 Visual and statistical cross dating ...... 50 4.3 Relationship between age and growth rate in Muguga population ...... 51 4.4 Relationship between ring number and DBH of trees from Muguga population ...... 51 4.5 Relationship between wood density and DBH of trees from Muguga population ...... 52 4.6 Relationship between wood density (g/cm3) and growth rate (mm) ...... 53 4.7 Crude yield extracts of individual populations ...... 54 4.8 Phytochemical yields in the three populations...... 55 4.8.1 Total essential oils yields of samples from Muguga, Karuri and Kobujoi populations ...... 55 4.8.2 Hexane extract yields of Muguga, Karuri and Kobujoi populations ... 56 4.8.3 DCM extract yields of Muguga, Karuri and Kobujoi populations ...... 58 4.8.4 Methanol extract yields of Muguga, Karuri and Kobujoi populations 59 4.8.5 Aqueous extract yields of Muguga, Karuri and Kobujoi populations . 61 vii

CHAPTER FIVE ...... 64 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ...... 64 5.1 DISCUSSION ...... 64 5.2 CONCLUSION ...... 72 5.3 RECOMMENDATIONS AND SUGGESTIONS FOR FURTHER RESEARCH ...... 73 5.3.2 Suggestions for Further Research ...... 74 REFERENCES ...... 75 APPENDICES ...... 95

viii

LIST OF FIGURES

Figure 4.1: A fitted line plot of Ring number versus Dbh (cm) ...... 52 Figure 4.2: A fitted line plot of wood density (g/cm3) versus Dbh (cm) ...... 53 Figure 4.3: A fitted line plot of Density (g/cm3) versus Growth rate (mm) ...... 54

ix

LIST OF TABLES

Table 4.1: Growth characteristics of trees at Muguga Prunus africana stand ...... 50 Table 4. 2: Relationship between age (years) and growth rate (mm) ...... 51 Table 4.3: Crude yields (g) for organic extracts ...... 55 Table 4.4: Concentration of essential oils in Muguga, Karuri and Kobujoi (mg/Kg) ...... 56 Table 4.5: Concentrations of compounds in hexane extracts of Muguga, Karuri and Kobujoi (mg/Kg) ...... 57 Table 4.6: Concentration of compounds in DCM extracts of Muguga, Karuri and Kobujoi (mg/Kg) ...... 59 Table 4.7: Concentration of compounds in methanol extract from the three populations (mg/Kg) ...... 61 Table 4.8: Concentrations of compounds in aqueous extracts from the three populations (mg/Kg) ...... 63

x

LIST OF APPENDICES

Appendix 1: Correlation and regression of age and growth rate ...... 95 Appendix 2: Abundances of essential oils in Kobujoi population (%) ...... 96 Appendix 3: Abundances of Karuri population essential oils (%) ...... 96 Appendix 4: Abundances of Muguga population essential oils (%)...... 96 Appendix 5: Total ion chromatogram of essential oils ...... 97 Appendix 6: Abundances of compounds in hexane extract of Muguga (%) ...... 98 Appendix 7: Abundances of compounds in hexane extract of Karuri (%) ...... 98 Appendix 8: Abundances of compounds in hexane extracts of Kobujoi (%) ...... 99 Appendix 9: Abundances of compounds in DCM extracts of Muguga (%) ...... 100 Appendix 10: Abundances of compounds in DCM extracts of Karuri (%) ...... 100 Appendix 11: Abundances of compounds in DCM extracts of Kobujoi (%) ..... 101 Appendix 12: Total ion chromatochram of DCM extracts ...... 102 Appendix 13: Abundances of compounds in methanol extract of Muguga (%) . 103 Appendix 14: Abundances of compounds in methanol extracts of Karuri (%) .. 104 Appendix 15: Abundances of methanol extract compounds of Kobujoi (%) ..... 105 Appendix 16: Abundances of aqueous extract compounds in Muguga (%) ...... 106 Appendix 17: Abundances of aqueous extract compounds of Karuri (%) ...... 106 Appendix 18: Abundances of aqueous extract compounds in Kobujoi (%) ...... 107 Appendix 19: Mass spectrum for β-Sitosterol ...... 108 Appendix 20: Mass spectrum for campesterol...... 109 Appendix 21: Mass spectrum for Myristic acid ...... 110 Appendix 22: Mass spectrum for stigmastan-3,5-diene ...... 111 Appendix 23: Mass spectrum of Quercetin-3,3'-dimethylether-4'-glucoside ..... 112 Appendix 24: Mass spectrum of procyanidin B5 ...... 113 Appendix 25: Mass spectrum of prunetrin ...... 114 Appendix 26: Mass spectrum of chlorogenic acid ...... 115 Appendix 27: Mass spectrum and structure of linoleic acid ...... 116 Appendix 28: Mass spectrum and structure of methyl linoleate...... 117 Appendix 29: Mass spectrum of methyl laurate ...... 117 Appendix 30: Mass spectrum of and structure methyl myristate ...... 118 Appendix 31: Mass spectrum and structure of squalene ...... 119 Appendix 32: Mass spectrum and structure of palmitic acid ...... 119 Appendix 33: Mass spectrum and structure of β-sitostenone ...... 120 Appendix 34: Mass spectrum of α-tocopherol ...... 120 xi

Appendix 35: Mass spectrum and structure of (3β, 5α)-stigmast-7-en-3-ol ...... 121 Appendix 36: Mass spectrum of cyanidin-3-o-rutinoside ...... 122 Appendix 37: Mass spectrum of 3-o-feruloyl-quinic acid ...... 123 Appendix 38: Mass spectrum of isochamaejasmin+ ...... 124 Appendix 39: Mass spectrum of cyanidin-o-galactoside ...... 125 Appendix 40: Mass spectrum of ursolic acid ...... 126 Appendix 41: Mass spectrum of robinetinidol-(4-alpha-8)-catechin-(6,4alpha)- robinetinol ...... 127 Appendix 42: Mass spectrum of cinnamtannin A2...... 128 Appendix 43: Muguga Prunus africana stand layout ...... 129

xii

ACRONYMS AND ABBREVIATIONS

ANOVA Analysis Of Variance BPH Benign Prostatic Hyperplasia °C Degrees centigrade CITES Convention of International Trade in Endangered Species DBH Diameter at breast height DNA Deoxyribonucleic acid ESI-MS Electrospray Ionization Mass spectrometry GC-MS Gas Chromatography-Mass Spectrophotometry HP Hewlett Packard HPLC High Pressure Liquid Chromatography IS Internal standard LC-MS Liquid Chromatography-Mass Spectrophotometry ICRAF International Centre for Research in Agroforestry IUCN International Union for Conservation of Nature Mm millimeter MS Mass Spectrum M/z Mass to charge ratio NIST National Institute of Standards and Technology OAB Overactive Bladder RAPD Random Amplified Polymorphism DNA Rpm Revolutions per minute RT Retention time SMT Southern Migratory Tract xiii

ABSTRACT

Prunus africana (Hook.f.) is an evergreen tree that grows in African mountains. The species’ bark and bark extracts are used for the treatment of benign prostate hyperplasia. The pharmacological efficacy of the extracts is said to be due to synergistic effect of several compounds such as phytosterols, pentacyclic triterpenoids and ferulic acid esters. High demand for the bark and bark extracts has led to over-exploitation of natural population of the species. As a result, P. africana is listed as an endangered species in Appendix II of CITES. Conservation of the species can be done through domestication. However, management and growth factors need to be established first to ensure success of on-farm production. Therefore, the World Agroforestry Centre established a P. africana stand at Muguga, Kenya to monitor the species growth and performance. The main objective of the current study was to evaluate and compare growth characteristics and phytochemical profile of trees in the domesticated stand at Muguga, with reference samples from Kobujoi, a wild stand and Karuri a remnant on-farm stand. Extraction of compounds was done using aqueous, hexane, dichloromethane and methanol solvents. Phytochemical analysis was done using Liquid Chromatography and Gas Chromatography-mass spectrometry. Gas Chromatography-Mass Spectrometry data was analyzed using GC Chemstation software version 11. Height of trees in the domesticated stand at Muguga ranged from 3 meters to 14 meters and diameter at breast height from 0.9cm to 104.5cm. Out of the 273 trees in the plantation, 92 (33%) were fruiting at the time of data collection. Evaluation of the crude yields of organic extracts of the three populations showed no significance difference (p>0.05). From the three stands, bark sample essential oils were essentially composed of myristic acid, linoleic acid, lauric acid, methyl myristate, methyl laurate and methyl linoleate. These compounds lower cholesterol levels in prostates of BPH patients. Campesterol, β-sitosterol, lup-20(29)-en- 3-one, palmitic acid, β-sitostenone, (3.β., 5.α)- stigmast-7-en-3-ol, stigmastan-3,5-diene and α-tocopherol were detected in dichloromethane and hexane extracts of the three populations. (3.β., 5.α)- stigmast-7-en-3-ol, β-sitosterol and β-sitostenone increase urine flow and inhibit prostaglandin production in the prostate. Cyanidin-o-galactoside, cyanidin-3-o-rutinoside, procyanidin B5 and robinetinidol-(4-α-8) catechin-(6,4- α)robinetinol are believed to inhibit cell proliferation and have free radical scavenging activity on cancerous cells. Ursolic acid is believed to have anti-inflammatory, antioxidant and anti-proliferative effects on BPH. Karuri population essential oils had significantly (p<0.05) higher amounts of myristic and lauric acids. Muguga population showed significant variation (p<0.05) on the concentration of myristic acid, linoleic acid, methyl myristate and α-tocopherol compared to Karuri and Kobujoi populations. The results demonstrate that domestication does not interfere significantly (p>0.05) with the phytochemical composition of P. africana and thus on-farm planting can be carried out. The morphological and phytochemical data has important implications in drawing strategies for sustainable harvesting, management and conservation of this species through cultivation. 1

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND INFORMATION Prunus africana is an evergreen tree with a height of more than 40 meters and a stem diameter of up to 1 meter (Gachie et al., 2012). It has shining foliage, blackish-brown bark and greenish or white flowers. P. africana is a geographically widespread species restricted to underlying islands like Sao Tome’ and Madagascar and Afromontane forest islands in Africa (Somalia, South Africa,

Kenya, Tanzania, Sudan, Uganda, Malawi, Ethiopia, Zaire, Cameroon,

Zimbabwe, Angola and Malawi) which are above 1500 meters altitude (Hall et al., 2000). The species grows in the humid midlands and in humid and semi- humid highlands.

In Kenya, it grows in the slopes of Mt. Elgon, Mt. Kenya, Cherangani Hills,

Tugen Hills, Aberdares Range, Mau range and Kakamega, Nandi and Timboroa forests (Gachie et al., 2012). P. africana tree is used for timber and also in traditional and modern medicine in Africa. Bark extracts of P. africana are used to treat benign prostate cancer hyperplasia (Stewart, 2003). Prostafx, Tadenan and

Pygenil are the herbal preparations of P. africana available in the market. Extracts from stem and root barks contain phytochemicals with anticancer, anti- inflammatory and antiviral effects (Vinceti et al., 2013). Bark extracts improve urologic symptoms in prostate cancer patients as they have apoptotic and anti- proliferative effect on the prostate (Kadu et al., 2012). 2

The pharmacological efficacy of the wild tree bark extracts is thought to be due to synergistic effect of various compounds. The main compounds are; pentacyclic triterpenoids (ursolic and oleanolic acids) which inhibit glucosyl-transferase activity and have anti-edematous activity (Donovan et al., 1998), phytosterols mainly β-sitosterol and β-sitostenone have anti-inflammatory effect as they suppress the production of prostaglandins and thus prevent swelling of the prostate (Carbin et al., 1990). The bark also has ferulic acid esters (n-tetracosanol and n-docosanol) and their derivatives which have antitumor and hypocholesterolemic activity on the prostate (Kampa et al., 2004). The three compounds work synergistically to counteract the biochemical and structural changes associated with benign prostatic hyperplasia (BPH). Benign Prostate

Hyperplasia is a non-cancerous urologic condition that leads to enlargement of the prostate gland. The bark extracts from P. africana also inhibit bladder hyperactivity. The use of the bark in traditional medicine includes the treatment of chest pain, urinary and bladder infections, stomach aches, kidney disease and malaria. The bark is either chewed or crushed into powder and drunk as tea

(Stewart, 2003).

Chemical composition varies between different plant species due to environmental differences in their habitats, reflecting defined geographical patterns. Knowledge of variation of the chemical constituents of P. africana is important to optimize the use and ensure sustainable conservation of the tree. 3

Studies carried out in Cameroon, Madagascar and Zaire showed that the chemical composition of the extracts of P. africana depends on the habitat of the species

(Gachie et al, 2012). Studies based on nuclear and chloroplasts DNA markers in

P. africana show five distinct regions throughout Africa that reflect divergent population across the continent (Kadu et al., 2011). In “High Africa” extending from 34°S to 12°N, the species is confined in volcanic highlands and mountains.

In equatorial Africa, the species is found at elevation ranges from 1000 meters to

3500 meters with annual rainfall of over 3000 mm in low altitudes and between

500 to 700 mm in high altitudes (Kadu et al., 2011). The species occurrence below the montane zone is mainly at drainage lines and rocky areas (Hall et al.,

2000). In southern Africa, the species occurs in elevation ranges between 600 and

1000 meters above sea level (Geldenhuys, 1993). Geological barriers and environmental changes have effects on the ecosystem, occurrence of species, their genetic diversity and evolutionary processes.

The high demand of the bark extracts of P. africana has caused serious damage to the wild population (Cunningham et al., 1997). More than 3000 tons of bark oand bark extracts are exported to Europe per year for the production of herbal preparations for BPH treatment (Cunningham et al., 1997). This high demand causes devastating effect to the wild population of P. africana which is the main source of the bark. Attempts at cultivation of P. africana are underway in Kenya and other countries. The bark of P. africana can regenerate if bark removal does 4

not interfere with the vascular cambium and thus harvesting is sustainable

(Cunningham & Mbenkum, 1993).

Despite the resilience of P. africana to debarking, in dry areas, bark re-growth is limited and large scale debarking stresses the tree even when complete re-growth occurs. Limited distribution of the species only in the afromontane islands and increased clearing for agriculture increases the threat to P. africana. The species was included as endangered species in Appendix II of the Convention of

International Trade in Endangered Species (CITES) at the ninth conference due to the increasing international demand (Betti, 2008). The species has also been assigned a vulnerable conservation status on the IUCN Red List. Import of the bark from Cameroon into the European Union was banned from November 2007 to December 2010 when CITES declared that the ban be lifted but with reduced quota of 150,000 kg for 2010 and 2011. In some African countries, policies have been established aiming at ensuring sustainable management of forests that contain P. africana species.

However, control problems and enforcement issues persist and there is need to identify and implement sustainable management options including conservation and domestication measures. In order to optimize sustainable conservation of the vulnerable African cherry, knowledge of the regional variation is essential. World

Agroforestry Centre established a P. africana stand at Muguga for determination of the growth performance and phytochemical yield. The seeds for this stand were 5

obtained from a wild stand at Kobujoi, Nandi County. The aim of this study was to evaluate the growth performance including the phytochemical profiles and yields of bark samples of P. africana from Muguga, Kobujoi and Karuri. The information from this study showed no significant differences in these characteristics and thus will help in sustainable management of the domesticated species. The data will also act as a guide on the right time of bark harvesting and the conservation strategies of on-farm P. africana.

1.2 PROBLEM STATEMENT

Prunus africana bark extracts are used in the treatment of benign prostate hyperplasia (BPH), a common medical condition in older men. The bark extracts that are currently used for the herbal medicine are mainly from the natural forests.

Incidence of BPH increases by 10% per decade and thus demand for the bark continues to increase (Iran et al., 2003). In addition, forest clearing continues for urban development, human settlement and farming. Limited distribution of the species only to the Afromontane regions means the demand for bark is focused on a limited area of the forests. The high demand of P. africana for medicine and other uses leads to uncontrolled exploitation and illegal harvesting of natural populations (Stewart, 2003). The demand is projected to increase due to growing confidence in herbal remedies in consumer markets (Cunningham et al., 1997).

Although P. africana bark demand for commercial processing of drugs for cure of benign prostate hyperplasia has been on the rise, bark harvesting from the wild is 6

unsustainable. Most people believe that bark extracts from wild populations are more potent than those from cultivated plants (Canuto et al., 2012). This is as a result of lack of information on phytochemical yields and profiles of domesticated

P. africana. Before this study, there was no study that evaluated the growth performance of on-farm P. africana.

1.3 JUSTIFICATION

Prunus africana is threatened with extinction but can be conserved by domestication initiatives (Cunningham et al., 2005). Domestication and reliance of planted trees will make the pharmaceutical future of P. africana bark and bark extracts more secure (Cunningham et al., 1997). However, for domestication to be scaled up for commercial purposes, there is need to evaluate the performance of domesticated P. africana by analyzing the phytochemical yield and profiles of a domesticated P. africana stand, in order to guide farmers on sustainable management of the plant and the right age for bark harvesting (Cunningham et al.,

2002). This ensures the tree remains healthy after bark harvesting. The information will also help find out if phytochemical yield is related to the growth rate of the stand and environmental factors.

7

1.4 RESEARCH QUESTIONS i) What is the relationship between height, stem shape and diameter at breast height

of trees in the domesticated P. africana stand? ii) What is the relationship between age and growth rate, growth rings and Diameter

at Breast Height and density and DBH of randomly selected trees from Muguga

P.africana stand? iii) How does the phytochemical profile and yields of randomly selected trees from

the domesticated P. africana stand at Muguga compare to those of a wild and on-

farm remnant populations?

1.5 OBJECTIVES

1.5.1 General objective

To evaluate the growth performance and phytochemical profile of a cultivated

Prunus africana stand at Muguga, and wild populations at Kobujoi and Karuri,

Kenya.

1.5.2 Specific objective

i. To determine the growth characteristics (height, stem shape, size and

diameter at breast height) of a domesticated P. africana stand at Muguga,

Kenya

ii. To determine the age, density and growth rate of randomly selected P.

africana trees from the domesticated stand at Muguga, Kenya 8

iii. To evaluate the phytochemical profiles and yields of P. africana stem bark

extracts sampled from Muguga, Kobujoi and Karuri, Kenya.

9

CHAPTER TWO LITERATURE REVIEW 2.1 Biology of Prunus africana

Prunus africana is a medicinal tree indigenous to the montane regions of West,

East, Central and South Africa. The species is a long-lived and evergreen tree, growing to height of up to 40 meters and can attain diameters of more than 1 meter (Gachie et al., 2012). It is the only species of the Prunus genus that is indigenous in Africa. It has heavy, shining foliage composed of alternate, simple leaves. It bears small flowers which are hairy and either white or greenish in color. Fruits are pinkish-brown and bi-lobed with a thin dark red pulp when ripe.

The fleshy seeds are dispersed by mammals and birds (Farwig et al., 2006).

P.africana peaks flowering between November and February in its area of natural distribution. The flowers have both male and female parts. Young trees have smooth reddish bark while older trees have dark resinous bark. P.africana is found at altitudes greater than 1000 meters and is thus confined to a series of montane populations throughout Sub-Saharan Africa. The tree is found in association with species like Celtis africana, Albizia gummifera, podocarpus falcatus and Cassipoure amalosana.

2.1.1 Lifecycle and longevity of Prunus africana

There is no much information about the lifecycle and longevity of P. africana.

White (1983) describes it as a pioneer species while Geldenhuys (1981) described 10

it as an early secondary species. It is fairly fast growing with a mean annual height increment of 0.6 to 0.8 meters (Breitenbach, 1965; Cheboiwo et al., 2015).

Populations in the equatorial regions have been reported to have a mean height growth of 1 to 1.9 meters for planted seedlings (Mbonyimana, 1988). White

(1983) reported flowering of individuals with heights of 4 meters and collection of flowers from individuals less than 8 meters tall indicating that the species may start flowering at an age less than 10 years (Eggeling, 1951). Geldenhuys (1981) reported reduction in diameter size of individuals after attaining a diameter of 30 centimeters.

2.1.2 Flowering and fruition of Prunus africana

In the Equatorial zone, there is no specific flowering season and some individuals of the species flower almost every month of the year (Munjuga et al., 2000). In regions north of 50 N, flowering takes place as from November to January

Munjuga et al (in press) when the temperatures are low and there is no much rainfall. In regions south of 50 N, flowering takes place from April to October when the conditions are cool and dry. Fruiting of the species occurs 2-3 months after flowering and is associated with rainfall. In the Northern zones, fruiting takes place in the relatively wet months. In the southern regions, fruiting occurs when the rains are starting or in the second half of the dry season (Sun et al.,

1996). 11

2.1.3 Reproductive biology

Seeds for planting purpose are collected from dark red to purple fruits as they germinate better than seeds from green fruits. The fruit flesh is removed prior to germination and seed are sown while fresh. Ex-situ seed storage is limited as the seed are intermediate in nature (Jaenicke et al., 2000). Storage can be done if seeds are depupled immediately after harvesting and the seeds stored at 5°C. At these conditions, stored seeds show 35 % germination after 12 months of storage

(Jaenicke et al., 2000). Propagation of the species is also possible by using wildlings. Seedlings germinate well in soil with high humus content and thus nurseries should have decomposed sawdust. Germination occurs after 50- 90 days of sowing. Studies show that the species is predominantly outcrossing (Munjuga et al., 2000).

The species does not have a specific season of flowering and fruiting and the stigma receptivity of individual flowers is short (Munjuga et al., 2000). The species has androgynous flowers with either red or pink petals. The flowers are insect pollinated with bees been reported to forage pollen and nectar from them

(Fichtl & Adi 1994). Hymenoptera, Diptera and nectar-foraging birds have also been reported to visit flowers thus play a role in pollination (Munjuga et al., 2000). Wind pollination is also possible as pollen is reported to light in weight. Pollination takes place continually as flowering also takes place continually. Flowers are 12

perfect with poor dispersal of pollen that falls to the ground (Munjuga et al.,

2000). There is no reference of the chromosome count in P. africana but the base number of other members of Prunea is (Kalkman,

1988).

2.2 Ecology of P. africana

2.2.1 Origin of P. africana in Africa

There are several speculations about the processes leading to the current distributions of P. africana and several migration paths have been inferred based on extant stands (Kalkman, 1988). Aubreville (1976) suggested a Laurasian origin of the prunus considering only the subgenus Laurocerasus which includes P. africana. He suggested a movement through the Middle East in to North-east

Africa. Kalkman (1988) proposed a Gondwanian origin of the species of the tribe

Pruneae (which includes prunus) with northwards movements along a path starting from regions of Africa, South America and Australia. Genetic study using random amplified polymorphic DNA (RAPD) markers concluded that both migration via the SMT and long distance seed dispersal could be responsible for the occurrence of the species in outlying islands (Muchugi et al., 2006). Muchugi et a, (2006) supported Aubreville’s path of migration because of genetic differences observed among eastern and western Kenya populations and proposed the Rift valley as a probable barrier to gene flow.

13

2.2.2 Natural distribution of P. africana in Africa

P. africana is reported in 22 countries mostly on the eastern side of Africa. It is also found in central Africa (Katanga, Congo), in West Africa, Comoros and

Madagascar. It is native to the montane tropical forests of Sub-Saharan Africa and

Madagascar. The range of P. africana has been significantly affected by past climate change and the diversity is expected to decrease significantly in coming years (Griffin, 2002). Unsustainable use of the species, which mainly affects large, reproductively mature trees is likely to reduce gene flow and seed dispersal increasing isolation and reducing viability of existing populations (Navarro-

Cerrillo et al., 2008).

The genetic variation of P. africana is believed to be diverged as the species has a disjunt distribution in highland forests of Africa (Kalkman, 1988). Dawson and

Powell (1999) did analysis on the species from different geographical zones using molecular markers revealing genetic variations among populations. The data on genetic variation among regions indicates the importance of regional approaches for conservation. In Madagascar and Cameroon, genetic analysis shows significant variation among population and among individuals thus the need to develop genetic management strategies that take account of variation at country level. P. africana stands in Kenya were reported to have more variation than stands form Cameroon after using Random Amplified Polymorphic DNA markers 14

(Muchugi et al., 2006). Principal coordinate analysis showed a genetic disjunction between Central and Western Kenyan P. africana stands (Muchugi et al., 2006).

2.2.3 Environmental factors in Prunus africana habitats

2.2.3.1 Topography

Africa is composed of three basic physiographic domains; Low Africa, the Atlas

Mountains and High Africa. Low Africa includes parts of Africa north of Equator, the Sahara and West Africa whose average elevation is less than 900M. High

Africa encompasses southern, eastern and south central Africa and has a minimum elevation of 900M. P. africana is mainly confined to ‘High Africa’ extending from latitude 110 55’ N near the Gulf of Eden to latitude 330 40’ S in

South Africa. In this domain, the species is primarily restricted to volcanic regions or mountains particularly the Eastern and Western Rift valley systems, the Great

Escarpment of south-eastern Africa and the Ethiopian highlands.

2.2.3.2 Climatic factors

Rainfall, temperature and cloud cover play a significant role in the distribution of

P.africana in Africa. The species is associated with areas of Africa where the monthly temperatures are low especially during the warmest periods of the year.

The species is associated with mean monthly temperatures of 11-19 0C and 17-23

0C in the coolest and warmest months respectively (Hall et al., 2000). It is geographically associated with mean annual rainfall from 500 mm (high latitudes) to over 3000 mm (low latitudes) (Kadu et al., 2011). P. africana is thus restricted 15

to parts of Africa that experience temperate climatic conditions with enough moisture supply for the growing season. Distribution of the species is limited to the montane regions with sufficient moisture supply for evapotranspiration but rare in areas characterized by insufficient precipitation and high temperatures during the warmest months (Mbatudde et al., 2012). Moist conditions in these regions trigger occurrence of stem borers and infestation of powdery mildew resulting to exudation of resin through the stem bore holes (Orwa et al., 2009).

Plants growing in lowland areas are more prone to stem borer infestation thus posing a challenge to domesticate the species in these areas (Franzel et al., 2009).

2.2.3.3 Soils

Soils associated with the Afromontane regions on which the species grow have been described from Malawi (Chapman & White, 1970), Tanzania (Lundgren,

1978), Sudan (Jenkin et al., 1977) and Ethiopia (Lundgren, 1971). The species is associated with humic cambisols and humic nitosols which are both fertile soils.

The soils at the surface are typically loam or sandy loam and are light in texture.

In the deeper horizons, the soils are either sandy clays or clay loams with a fine texture (Lundgren, 1978).

2.3 Conservation of P. africana

Over the last 40 years, P. africana bark harvest has shifted from subsistence and local use to large scale commercial use for international trade. Most of the surviving wild stands of P. africana are in national parks and state-owned forest 16

reserves. Due to concerns regarding the sustainability of the bark trade, the species was proposed by Kenya for CITES in 1994 (Cunningham, 2005) and was included in CITES Appendix II in 1995. Clearance of forests for agriculture is a major threat affecting areas where P. africana occurs. Climate change and expansion of agriculture may also interact with other threats in the Afromontane regions, where the species would be pushed at higher elevations. Fire and illegal encroachment into protected areas are also posing threats to the species. In

Zimbabwe, the introduction of exotic Eucalyptus, Acacia and Pinus species has changed the landscape of Afromontane region and increased fire susceptibility in the forest cover (Jimu, 2011). The conservation strategy carried out is dependent on several important determinants like the reproductive biology, genetic variation of the species and policies and regulations in specific countries.

2.3.2 Policy and Regulations on P. africana conservation

2.3.2.1 National and local regulations on P. africana bark harvesting

In Madagascar and Cameroon, where the species exploitation is highest, there is a set of regulations that address the harvesting of P.africana bark (Ndibi & Kay,

1997). In these two countries, the conservation activities could be beneficial if regulations are adhered to but these regulations are not well understood. Licenses for bark harvesting state that bark should only be from a quarter of standing trees but most trees end up dying even after this regulations are followed (Cunningham

& Mbenkum, 1993, Sunderland & Tako, 1999). In addition, lack of adequate 17

inventory data on the sizes of P. africana populations in these countries makes it difficult to determine sustainable harvesting levels (Cunningham & Mbenkum,

1993; Ewusi et al., 1997).

Regulations and quotas for sustainable harvesting of bark are developed but their enforcement is difficult. Harvesting regulations are not adhered to in areas where

P. africana bark is currently exploited due to institutional weaknesses and lack of resources and awareness (Cunningham et al., 1997; Ndibi & Kay, 1997;

Mbenkum & Fisiy, 1992). Formal regulations may not be effective in promoting sustainable management of wild P. africana populations. This resulted in the placing of the species on Appendix II of CITES by European countries to stop commercial trade of the bark.

2.3.2.2 International policy on conservation of P. africana

The Convention of International Trade for endangered species is the main body that influences the conservation strategies of P. africana. When a species is listed on Appendix II of CITES it means that the trade of cultivated and wild material must be licensed at import and export (Cunningham et al., 1997). However, there are challenges in the implementation of these regulations as it is difficult to identify P. africana products in international market. This has led to unreported import and export of the species materials (Cunningham et al., 1997). In addition, 18

most countries have not nominated CITES authorities hence it is not easy to implement the regulations.

Management changes are essential to reduce and eventually eliminate over- exploitation of P. africana (Ingram et al., 2009). Genetic data from different population across the species is available, offering a unique opportunity to base conservation actions on information that is not available for most species. P. africana has a highly disjunct distribution across Afromontane forest ecosystem and thus genetic data constitute a crucial foundation for the identification of priority sites for conservation (Vincenti et al., 2013). Forest management includes protection, utilization, evaluation and ecosystem manipulation and transformation.

Activities concerned with protection require minimum silvi-cultural or ecological knowledge and are easily undertaken. This mainly involves prevention of grazing, fire and encroachment and boundary maintenance. In Cameroon management of the species involves providing more light under seed trees to encourage the survival of seedlings (Ingram et al., 2009). Young trees in disturbed habitats and fallow are disentangled from climbers and undergrowth.

Agroforestry practices that integrate trees with livestock production and crop cultivation have received increased attention as an alternative to support ecosystem functions and livelihood (Garrity, 2004). These practices help support ecological and social resilience and can increase the efficiency of production systems. Tree conservation can be done in three different settings; ex situ, in situ 19

and circa situm. In situ conservation is the maintenance of tree diversity in woodland populations and wild forest. Circa situm involves preservation of remnant and/or planted trees and wildings in farmland where woodland or natural forest containing the same trees was once found (Pinard et al., 2014). Ex situ conservation is the preservation of trees in seed storage, in field gene banks and in field trials or maintained in other locations outside the usual environments or geographic settings of the tree (Koo et al., 2004).

2.3.3 Cultivation of P. africana

The high current levels of demand of P. africana products (bark and timber) cannot be met long-term by the wild populations and thus there is need for domestication of the species. Approximately, 3500 tons of P. africana bark is being traded internationally every year (Cunningham et al., 1997). This demand is projected to increase due to growing confidence in herbal remedies in consumer markets and ageing populations. Bark harvesting from wild populations at existing rates is not sustainable (Cunningham & Mbenkum, 1993). Most of the current bark harvesting is also done using techniques that kill the tree.

Domestication of the species will ensure sustainable and beneficial exploitation and thus reduce the threat of depletion of the wild populations (ICRAF, 1994;

Sunderland & Nkefor, 1997). 20

2.3.3.1 Propagation of P. africana

Domestication of P.africana is limited by the unavailability of viable seeds in sufficient amounts. Seeds must be derived from fruit collected at the right stage of maturation and the pulp is removed prior to storage or sowing (Were & Munjuga,

1998). The seed moisture content during storage should be about 15% (Hall et al.,

2000). Vegetative propagation of P. africana through cuttings has also been found to be successful in Cameroon, Kenya and Madagascar (Tchoundjeu et al., 2002;

Dharani, 2007). Rooting success is higher with sawdust medium than with sand or a mixture of the two. Rooting success is increased by the application of indole butyric acid (Tchoundjeu et al., 2002).

Planting of P. africana was first done in Kenya at Ngong in 1913 as a timber stand (Simons et al., 1998). In Cameroon, Bioko and Equatorial Guinea there have been attempts to replant the species in highly exploited forests (Sunderland

& Tako, 1999). This activity involved relocating wildlings to areas that needed enrichment after a period in the nursery rather than use of plants raised from seeds under nursery conditions. Use of wildlings is an attractive option, given the problems of seed storage although it allows little control of quality and vigor. The wilding stock is also susceptible to water stress.

2.4 Prunus africana provenance trial

The humid tropics of Africa have several indigenous medicinal and fruit tree species (Leakey, 1998; Okafor & Lamb, 1994). Many rural families depend on 21

these resources for food, fruit, medicinal and construction needs (Abbiw, 1990).

The products from medicinal plants are traded internationally and thus contribute significantly to the economy of the countries in this region. Sustainable economic benefits from conservation of useful plants like P. africana create opportunities to improve livelihoods in Africa (Cunningham et al., 2002; Hamilton, 2003).

Production and trade of wild plants can sustain economic and ecological benefits in developing countries (Srivastava et al., 2005). Most of these resources are obtained exclusively from the wild. These resources are being depleted due to rapid population growth that has led to increasing demand for productive land for farming which is met by clearing of forests (Abbiw, 1990).

Agroforestry is advocated to ensure sustenance of the population’s livelihood in these regions (Leakey, 1998). Agroforestry ensures sustainable use of land that will improve the welfare of the community and farm productivity. Traditional agroforestry systems like the cacao (Theobroma cacao) cultivation systems have been practiced in the humid regions of Africa (Okafor & Fernandes, 1987). In most of the simple agroforests, farmers grow medicinal plants and different types of indigenous fruit. However none of the species planted have been selected and bred for quantity and quality. The intercropping systems used currently have not integrated modern science in order to increase investment returns and to optimize resource use efficiently for environmental quality. There is considerable potential for development of intercropping systems as most simple agroforests in West and

Central Africa are under-utilized compared to the complex agroforestry systems 22

in South-east Asia (Duguma et al., 1990). In 1994, World Agroforestry Centre began an initiative to optimize the role of high value indigenous species through intercropping systems in West and Central Africa (ICRAF, 1994).

Almost all the tree germplasm available or used in agroforestry is wild and unimproved. A species is selected for domestication if farmers identify it as important and which can best contribute to research objectives. World

Agroforestry Centre’s domestication programme includes species like Prunus africana, Dacryodes edulis, Irvingia gabonensis, Chrysophyllum albidum and

Garcinia kola (ICRAF, 1987). The first step after selecting the species for domestication is establishing a gene-bank of the chosen species. This serves as a source for the continuous supply of diverse genetic material of the species for further selection.

The natural populations of P. africana have decreased due to high demand and unsustainable methods of bark harvesting in the wild (Cunningham & Mbenkum,

1993). World Agroforestry Centre in collaboration with Limbe Botanic Garden,

United Nations Educational, Scientific and Cultural organization and Cameroon development Corporation have started a programme to investigate feasibility of domesticating P. africana. The aim of the programme is to select populations which produce highest quantities and best quality of bark or bark extracts for medicinal use (ICRAF, 1994). The programme was initiated with survey of the genetic variation of the species in the wild, seed collection and establishment of 23

live genebanks. The seeds for the genebank were collected from Kilum Mountain,

Mendakwe and Mount Cameroon. A genebank was established at Tole near

Limbe from seeds collected from 80 trees and leaves were collected for molecular genetic analysis. Analysis of the gene banks at Limbe showed that the survival rate of the three provenances varied from 60% to 100%. Early plant growth among the various accessions showed significant variations. Mean height of five- month- old plants varied from less than 40 cm to over 100 cm. The provenance of the seeds did not relate directly to the variation of accessions in survival rate and early height development. These variations indicate that there is potential for genetic improvement of P. africana through selection (ICRAF, 1994).

2.5 Provenance based phytochemical variation

Prunus africana bark extracts from different provenances show variation in phytochemical yields (Gachie et al., 2012). Phytochemical analysis of P. africana bark extracts from Madagascar indicates high 3-0-Acetyl oleonic acid compared to bark extracts from Kenya, Congo and Cameroon (Martinelli et al., 1986). Bark extracts from Kenya have the highest concentration of active ingredients compared to bark extracts from Cameroon and Congo (Nkuinkeu & Ndam, 1999).

There is a positive correlation between the bark extract yield and tree age but yield reduces at old age (Simons & Leakey, 2004). In Kenya, bark from plantation forests (21-40cm dbh) show higher yields than extracts from natural forests while bark harvested from farmlands show the least yields (Gachie et al., 24

2012). The concentration of n-Docosanol in sample extracts from Cameroon is reported to be higher than the concentration of the compound in extracts from

Kenya and Tanzanian. The difference in concentration of this compound is known to be affected by genotypic and environmental factors (Irmak et al., 2008;

Dunford and Edwards, 2010). Ferulic acid concentration in extracts from Kinale,

Kenya is reported to be highest compared to the concentration in extracts from

Tanzania and Cameroon (Kadu et al., 2012). Cameroon also had highest concentration of β-sitostenone compared to concentration from Kenya and

Zimbambwe (Kadu et al., 2012).

2.6 Economic importance of P. africana

2.6.1 Ecosystem functions

The African mountain forests in which P. africana is restricted have been named as important conservation targets (Davis et al., 1994). Bwindi impenetrable forest in Uganda is among the most diverse forests in East Africa and hosts half of the world’s endangered mountain gorilla (Cunningham, 1996). Mount Cameroon has

42 plant species that are only found growing in this mountain (Thomas & Cheek,

1992).The forests are under threat for agricultural clearance as there is high population density on the same regions (Wild & Mutebi, 1996; Cunningham et al., 1997). P. africana fruit is eaten by several mammals and birds (Cunningham

& Mbenkum, 1993) but none of these species solely depends on P. africana.

Fichtl & Adi (1994) reports the foraging of pollen and nectar from the flowers of 25

this species by bees. Munjuga et al (in press) also reports on wasps, birds and some butterflies feeding on the pollen and nectar of the species.

2.6.2 Uses of P. africana as timber

P. africana wood was used traditionally for making hoes, axe handles and poles for house construction (Chapman, 1999). In Uganda, P. africana trunks were used to make brewing troughs (Cunningham, 1996). P. africana timber has high resilience and wearing qualities and it is resistance to abrasion making it suitable for construction and in making durable floors. It is also used in making furniture and window or door frames (Chudnoff, 1980; Goldsmith & Carter, 1981). In East

Africa, it is used for railway sleepers on less heavily used sections of track and for bridge decking (McCoy-Hill, 1957; Bryce, 1967).

2.6.3 Medicinal uses of P. africana

2.6.3.1 Use of Prunus africana in traditional medicine.

The bark and leaves of P. africana are the main parts used in traditional medicine in Africa. The use of P. africana in traditional medicine has been documented since mid-nineteenth century (Watt & Breyer-Brandwijk, 1962). Kokwaro (1976) reported use of the bark for relieving of stomachache whereby the bark was pounded in water to produce a red liquid. In Ethiopia, the leaves of the species 26

were used to dress wounds (Kokwaro, 1976). Leaf infusions were used for relieving fever and in increasing appetite (Kokwaro, 1976).

Bark infusions were used in the treatment of chest pains and as a purgative for cattle (Sunderland & Obama, 1999). In Africa, 500 tonnes of bark were harvested for use in traditional medicine annually (Cunningham et al., 1997). The use of the species in pharmaceutical industry commercially began in 1970s. In 1980, only

200 tonnes of P. africana bark were harvested (Cunningham & Mbenkum, 1993).

The demand for the bark in pharmaceutical industry increased from then and by

1997 the demand had risen to 3500 tonnes (Dawson, 1997; Cunningham et al.,

1997). This involved processing and marketing of bark and bark extracts for the treatment of benign prostate hyperplasia (Hall et al., 2000).

2.6.3.2 Use of Prunus africana in modern medicine

Benign prostatic hyperplasia (BPH) is a condition common in most men over 50 and manifests itself as increased frequency in urination, pain in passing urine, inability to empty the bladder and post urinary dribbling (Garnick, 1994).

Allopathic medical therapy for BPH includes drugs and surgical and non-surgical treatments. Drugs used include the anti-androgens terazosin hydrochloride and finasteride which are synthetic inhibitors of the 5-α-reductase enzyme (Bartsch et al., 2000). Non-surgical therapy includes thermotherapy, balloon dilation and stents. Treatment by surgery involves removal of excess tissue. All these methods 27

have a number of side effects and thus phytotherapy is the primary treatment in

European countries.

2.6.4 Benign Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is a progressive non-cancerous enlargement of the prostate gland accompanied by lower urinary tract symptoms (Parsons &

Kashefi, 2008). The enlargement of the prostate compresses the urethra, thus restricting flow of urine from the bladder. Approximately 50% of men develop

BPH-related symptoms at 50 years of age but the condition is not common before age 40. About 70% of men above 50years of age have symptoms arising from

BPH and 20-30% of men at the age of 80 years require surgical intervention to manage BPH (Parsons & Kashefi, 2008). Several mechanisms seem to be involved in the development of and progression of BPH and thus the etiology still remains uncertain in some aspects.

BPH symptoms are known as lower urinary tract symptoms and can be subdivided into storage symptoms and voiding symptoms. Storage symptoms include nocturia, frequency and urgency in passing urine. Voiding symptoms intermittency, hesitancy, dribbling, straining and decreased urine stream. The severity of BPH can be measured by using the International Prostate Symptom

Score (IPSS) questionnaire that has questions about the urinary symptoms and

Quality of Life (QOF) questions about how much the patient is bothered by the symptoms. Most symptoms that lead to constriction of urinary flow are directly 28

attributed to prostatic hyperplasia but some men have concurrent overactive bladder or bladder detrusor over-activity. In addition to the treatment of BPH, these men will therefore require therapy for OAB.

BPH is caused by increased growth of the prostate gland and increased smooth muscle tone of the prostate. Dihydrotestosterone (DHT), a metabolite of testosterone is the main mediator of prostate growth. Dihydrotestosterone is formed by breakdown of testosterone by 5-alpha reductase enzyme in the prostate cell (Lobaccaro et al., 1998).This enzyme is the target for drug therapy aimed at reducing the size of the prostate. Drug therapy for BPH includes 5-alpha reductase inhibitors like Avodart and Propecia. The active ingredient of propecia is finasteride a N-(1,1-dimethylethyl)-3-oxo-,(5-α,17β)- 4-azaandrost-1-ene-17- carboxamide while Avodart contains dutasteride.

2.6.4.1 Pharmacotherapy 2.6.4.1.1 Conventional therapy The two main classes of therapeutic agents used to treat BPH are the 5-alpha reductase inhibitors (5-ARIs) and the alpha blockers. 5-alpha reductase inhibitors act by inhibiting conversion of testosterone to dihydrotestosterone, the main sex hormone in prostate cells and mediator of BPH progression. They slow prostate growth and initiate decrease in prostate size (Roehrborn et al., 2002). The onset of action of alpha blockers is faster than that of 5-ARIs which takes 4 to 6 months. 29

The main 5-alpha reductase inhibitors are dutasteride (Avodart) and finasteride

(Proscar) (Bartsch et al., 2000; Clark et al., 2004).

Dutasteride inhibits type 1 and type 2 5-alpha reductase iso-enzymes while finasteride inhibits the type 2 iso-enzyme. Finasteride lowers DHT production in the prostate by 70% and dutasteride lowers the DHT by over 90% (McConnell et al., 1998). The side effects of 5-alpha reductase inhibitors are ejaculatory dysfunction, erectile dysfunction and gymnecomastia though rare. 5-alpha reductase inhibitors reduce the risk of BPH related surgery and future urinary retention and improve BPH-related symptoms by shrinking the prostate

(Roehrborn et al., 2002). Alpha blockers have no effect on prostate cancer risk and do not affect prostate specific antigen. However, 5-alpha reductase inhibitors lower PSA by 50% after 6 months on therapy (Gormley et al., 1992).

Alpha blockers on the other hand work to relax the smooth muscle at the prostate and bladder neck and mediate cellular hypertrophy by blocking alpha-1a receptors. By relaxing the prostate at the prostate neck, the urinary channel is opened, allowing a less constricted urinary flow. The alpha blockers have a quick onset of action of between 3-5 days. Alpha blockers are classified into two: second generation drugs- doxazosin (cardura) and terazosin (hytrin) and third generation drugs which include alfuzosin, tamsulosin and silodosin (Macdonald &

Wilt, 2005). Doxazosin and terazosin require dose titration because of their anti- hypertensive properties (Macdonald et al., 2004). Silodosin, alfuzosin and 30

tamsulosin have fewer cardiovascular side effects and thus do not require dose titrations. All alpha blockers are equally effective and their side effects include dizziness, headache, weakness, asthenia, retrograde ejaculation and nasal congestion (Bent et al., 2006). Alpha blockers quickly improve urine flow but do not reduce the size of the prostate and thus they do not reduce the need for BPH- related surgery or the risk of future urinary retention (Kaplan et al., 2006).

Patients with severe allergic reaction to sulfur also react to tamsulosin thus they should avoid the drug. Silodosin is a selective drug that blocks alpha-1a receptors and to a much lesser extent alpha-1b and alpha-1d receptors. This selectivity may result in fewer Cardio-vascular side effects which are mainly regulated by alpha-

1b receptors (Kazuki et al., 2006).

Combination therapy with alpha blockers and 5-ARIs is more potent than using each one of the two alone. Combination therapy with finasteride and doxazosin reduces prostate growth, need for BPH-related surgery and provides fast symptom relieve (Marberger, 2006). The benefit is high in patients with large prostates where the alpha blockers relax the smooth muscles of the prostate and 5-ARIs shrinks the prostate. Alpha blockers are sufficient to alleviate urinary symptoms in patients with small prostates.

Minimally invasive surgical therapies are also used as an option if medical therapy does not alleviate urinary symptoms. Transurethral resection of the prostate (TURP) is the most common surgical procedure. The procedure involves 31

removal of the prostatic urethra creating a channel for the patient to void through.

Risks from the surgery include urinary tract infection, permanent sexual side effects and rarely, urinary incontinence.

2.6.4.1.2 Prunus africana as a herbal remedy for BPH P. africana bark extracts are used to make capsules for benign prostatic hyperplasia a condition common in aging men (Cunningham et al., 1997).

Traditionally the bark is powdered and drunk as a tea for inflammation, genito- urinary complaints, kidney disease, malaria, allergies, fever and stomachache. A patent for use of the bark extracts for the treatment of benign prostate hyperplasia was issued in 1966 (Cunningham et al., 1997). Use of the bark extracts is shown to be effective to alleviate BPH symptoms like failure to urinate, frequent urination, nocturnal urination, voiding volume, residual urine, prostate volume and peak flow. Clinical trials using the extracts showed significant reduction of prostate size and symptoms, and clearance of bladder neck urethra obstruction

(Barlet et al., 1990; Andro & Riffaud, 1995). The bark contains three groups of active constituents: pentacyclic triterpenoids (including friedelin, oleanolic and ursolic acids), phytosterols (including beta-sitosterol), and ferulic esters of long- chain fatty alcohols (including ferulic esters of docosanol and tetracosanol) (Kadu et al., 2012).

Initially, the therapeutic effects of P. africana were attributed to β-sitosterol, and its glycoside and to n-docosanol (Barth, 1981; Longo & Tira, 1981). This finding 32

is unlikely to be true given the low amounts of n-docosanol in P. africana extracts and high levels of triterpenes and sterols (Catalano et al., 1984). The therapeutic effects of P. africana bark extracts are now believed to be as a result of a pharmacological combination whereby several compounds act synergistically thus counteracting the functional and biochemical changes that characterize BPH

(Kadu et al., 2012).

The pharmacologically active compounds in the bark extract include pentacyclic triterpenes, phytosterols and ferulic acid esters of long chain unsaturated fatty acids. Two new compounds have been identified: 4-O-ß-D-glucopyranosyl-7,8- dimethoxyisolariciresinol (Scarpato et al., 1998) and 24-O-trans-ferulyl-2", 3"- dihydroxy-urs-12-en-28-oic acid (Fourneau et al., 1996). The phytosterols, mainly

β-sitosterol, have anti-inflammatory effect and inhibits the synthesis of prostaglandins and 5-alpha-reductase activity (Bauer, 1986; Holms & Meyhoff,

1997; Wasson & Watts, 1998). β-sitosterol helps reduce the elevated levels of prostaglandins in BPH patients (Bauer, 1986). These phytosterols also eliminate vasal congestion and excess blood hence reduces the size of prostate adenomas.

The pentacyclic triterpenoids block enzymatic activity thus inhibits inflammation in the prostate (Marcolli et al., 1986, Wasson & Watts, 1998). They also have anti-edema effects and help increase the integrity of capillaries and small veins.

Ferulic acid esters in the bark extracts act by inhibiting the absorption and metabolism of cholesterol (Catalano et al., 1984; Bormbardelli & Morazzoni, 33

1997). Benign Prostate Hyperplasia and other cases of enlarged prostates are characterized by containing abnormally high levels of cholesterol.

Initial in vivo studies to be done with P. africana extracts showed that the extract prevented hyperplasia in rats that had been injected with human prostate tissue and induced prostate secretions in normal tissue. Men without BPH showed similar results though they had insufficient prostate secretion (Clavert et al.,

1986). Administration of P. africana extract orally in rats stimulates secretory processes in cells of bulbourethral gland and in the prostate (Latalski et al., 1979).

The extracts also stimulates seminal vesicle secretion in castrated rats acting as testosterone antagonist in these organs (Thiebolt et al., 1971). In castrated rats that have been adrenalectomised, the extract increases contents of gonadotropins in the pituitary and testosterone activity. P. africana extract is thus believed to be involved with the pituitary gland and adrenal cortex (Thiebolt et al., 1971). The extracts exhibit reduced vascular permeability due to histamine and anti-oedema and anti-inflammatory activity in rats (Marcoli et al., 1986). The extracts also reduces bladder hyperactivity in guinea pigs and have modulating activity on age- related contraction of bladders of rats (Thiebolt et al., 1971).

P. africana extract is a well-tolerated effective drug for the treatment of symptoms associated with BPH (Andro & Riffaud, 1995; Bombardelli &

Morazzoni, 1997). Use of the extracts in treatment of BPH has been demonstrated in open and double-blind placebo controlled clinical trials with most trials 34

showing excellent results. The trials were carried out for treatment periods from 6 weeks to three months and with doses ranging from 75 to 200mg daily. Once and twice daily dosages of P. africana extracts when compared were found to be equally safe and effective (Chatelain et al., 1999). In most of the trials, urine frequency decreased and urine flow increased. In trials with higher doses, prostate size and irritative symptoms decreased (Bartlet et al., 1990). Patients taking

200mg of the extract daily for two months showed a decrease in sexual disorders associated with chronic prostatis or BPH (Carani et al., 1991). Treatment of genital infection or both non-bacterial and bacterial chronic prostatis with the extract is believed to be effective even without administering antibiotics

(Menchini-Fabris et al., 1988). The extract also increases protein secretion and the activity of prostatic acid phosphatase in patients whose activity is low (Lucchetta et al., 1984).

Prunus africana bark extract efficacy is determined by measuring the effects of the herb on numerous parameters, including dysuria, nycturia, frequent urination, abdominal heaviness, residual urine, voiding volume, prostate volume, and peak flow. Consumption of pygeum resulted in significant amelioration of symptoms, reduction in prostate size, and clearance of bladder neck urethral obstruction.

Transient side effects involving gastrointestinal irritation (inducing nausea and abdominal pain) have been reported in clinical trials (Balch et al., 2008).

35

2.6.4.1.3 Serenoa repens (Saw palmetto) as a herbal remedy for BPH

Serenoa repens is an evergreen shrub with horizontal rhizomes and grows to heights up to 20 to 25 feet (Olson & Bames, 1974; Tyler, 1993). The products used for medicinal purpose are derived from the ripe berries of the species. The species is native to the sandy soils of Louisiana, Texas, Georgia and the islands in

Cuba and Bahamas. The berries turn color from green to bluish-black when ripe.

Herbal preparations from saw palmetto are used to improve symptoms of benign prostatic hyperplasia (Barnes et al., 2007). Herbal supplements from the species are also used as alternative or complementary medicine modalities for men with prostate cancer (Bishop et al., 2011). Saw palmetto herbal preparations have advantages over conventional therapy in that they do not change prostate specific antigen (PSA) levels and have no side effects (Lowe & Fagelman, 1999). Drugs like proscar lower PSA levels and may mask prostate cancer as tests for screening prostate cancer involve measuring PSA levels. Double-blind studies have proved the herb to be effective in improving urinary symptoms (Lowe & Fagelman,

1999).

The mechanism of action of the preparations from this species is inhibition of 5- alpha reductase, the enzyme that converts testosterone to dihydrotestosterone and blocking of DHT from binding to prostate (Bayne et al, 2000). Administration of saw palmetto berry extracts reduces the action of dihydrotestosterone androgen

(Talpur et al., 2003; Van Coppenelle et al., 2000). The main chemical compounds in the berries of S. repens are fatty acids, monoacylglycerides, polyphenols and 36

phytosterols. The biologically active components of saw palmetto are believed to be phytosterols and fatty acids. The extracts mainly consist of fatty acids with high quantities of saturated and medium chain myristate (14.0) and laurate (12.0) fatty acids (Schantz et al., 2008).

The fatty acids in saw palmetto extracts are believed to be responsible for the inhibition of 5α-reductase enzyme (Abe et al., 2009). Some studies show that phytosterols in saw palmetto also inhibits 5α-reductase and BPH symptoms

(Scaglione et al., 2008). However, the phytosterols (campesterol, β-sitosterol and stigmasterol) are not unique to the saw palmetto extracts. The beneficial effects of saw palmetto herbal preparations may be due to synergistic effect from both fatty acids and phytosterols.

2.7 Gas Chromatography-Mass Spectrometry (GC-MS)

Gas chromatography-mass spectrometry is a technique that involves combination of two analytical techniques for separation, identification and quantification of volatile and non-volatile compounds. It comprises gas chromatography coupled to a mass spectrometer (Thomas & Chasteen, 1998). The GC vaporizes and separates the components of a sample while MS aids in the structural identification of the individual components (Fulton et al, 1996; Sreevidya &

Mehrotra, 2003). In this work, GC-MS was used as a technique for identification and quantification of the phytochemicals in various extracts of P. africana bark.

This was possible as the mass spectra of the various components eluting from the 37

GC were recorded and the mass spectrum was characteristic of the identity of the compound.

2.7.1 National Institute of Standards and Technology (NIST) Library analysis

The structures and molecular masses of compounds in P. africana stem bark extracts can be determined through searching through a library of low-resolution mass spectra for a match between the compound spectrum and that of the compound in the library. The library stores up to 50,000 electron impact mass spectra (Rose & Johnstone, 1982). Matching of the spectra involves calculation by the data system of a match factor, similarity index or purity between the unknown spectrum and that of the library (reference) spectra. Most scales have a

0 for complete mismatch to 1000 or 1100 for a perfect match. There is a filter or a pre-search which eliminates dissimilar spectra to avoid time consuming calculations for the purity against all entries in a large library. At each m/z in turn, the ration (R) of the mass peak heights in the normalized unknown compound and reference spectra is calculated. The individual R values are summed and the average value is normalized to produce the final purity for a particular reference spectrum.

The entries with highest values are displayed as the results of the library search.

The entries are ranked in order of highest purity, and the fit values for each match calculated (Rose & Johnstone, 1982). Whatever the purity values or fit, the result of library search should be assessed by visual examination of the matching spectra 38

when presented for the highest ranked library entry. All peaks in the reference spectrum should also be present in the spectrum of the sample (Schymanski et al.,

2008). 39

CHAPTER THREE

MATERIALS AND METHODS

3.1 Study site

The Prunus africana stand was established by the World Agroforestry Centre at

Muguga, Kenya. Muguga Regional Research Centre is situated at Kiambu

County, 10 14’ S, 360 38’ E. Muguga is located approximately 2150 meters above sea level and has an average annual rainfall of 1200mm. Muguga population is a domesticated P. africana stand with pruning and clearing of any undergrowth done from time to time. Karuri P. africana population is an on-farm remnant stand intercropped with food crops. Karuri and Muguga sites are both in Kiambu county and share environmental factors. Trees from Karuri were growing in a farm intercropped with food crops. Kobujoi P.africana population is a natural forest population in North Nandi with the species growing together with various other species.

3.2 Sample collection Samples of P .africana stem bark for phytochemical analysis were collected from the P. africana stand at Muguga. Debarking was done using a sharp-edged panga at a height of 1.3 meters from the ground. Reference stem bark samples were collected from Kobujoi, Nandi a natural forest and Karuri, an on-farm remnant stand. Five trees from each population were randomly selected for debarking.

Bark removal was done in a East, West, North and South orientation to avoid 40

biasness. Trees with Diameters at Breast Height less than 20 cm were not sampled. Each sample was labeled and the wet weight was measured using an electronic weighing balance and recorded.

Morphological characteristics evaluated included stem shape, DBH, tree heights and fruition condition. Stem shape was given as a score of one to five while tree heights were recorded in meters. The assessment of the domesticated P. africana growth rate involved the measurement of the diameter at breast height and tree height. Tree age was determined using the ring method where ring widths were measured using the TSAP-WinTm software. The diameter at breast height (DBH) was determined using a DBH meter and recorded in centimeters. Diameter at

Breast Hyperplasia is the diameter of a tree at a height between 1.3 and 2 meters.

In this study, the DBH was taken at a height of 1.3 meters above the ground. The height of individual trees was determined using a Suunto optical height meter.

Morphological data was grouped based on tree heights. For Muguga stand, the assumption was that the trees were planted at the same time and thus were of same age.

3.3 Preparation of tree rings for age determination

Tree ring samples were collected by cutting cross sections from trees that were felled during thinning of Muguga population. Cross sections were obtained at a height of 1.3 meters using a chain saw and all ring samples were labeled. A height of 1.3 meters was used to maximize the estimation of tree age as the number of 41

rings decrease with increase in height. The samples were dried and an orbital power sander was used to prepare each sample surface and to buff out the scratches. Hand sanding of the surfaces was then done using progressively finer sand paper (80, 120, 180, 240 and 360 grit) to increase visibility of the growth rings. The rings were counted from the outermost to the innermost and each ring boundary was marked. Ring width was visualized using a binocular microscope and measured using TSAP-WinTm which was linked to a computer. Samples were continuously adjusted to ensure ring width was measured perpendicularly to avoid under or over estimation of ring width. The ring width measurements were taken along two randomly selected radii.

3.4 Visual cross dating Cross dating is the process of establishing the exact year in which a ring was formed (Stokes & Smiley, 1968; Schweingruber, 2007). Correct cross dating indicates the effect of external factors like environmental factors on the growth pattern of trees (Worbes, 1995). Visual cross dating of samples was done by matching pattens of narrow and wide rings using the skeleton plot method (Stokes

& Smiley, 1968). One vertical line represents one ring on the skeleton plot and each ring was assigned a value on a scale of 1-10 based on the width previously marked. Each ring was compared to the neighbouring ring and the narrowest rings which mainly indicate the dry season were assigned high values close to 10. The wider rings were marked with a B and rings with average width were not scored

(Stokes & Smiley, 1968). The patterns of the wide and narrow widths should 42

align for one to get the composite skeleton plot that will allow dating the rings to the exact year they were formed (Stokes & Smiley, 1968). After ring width measurement and visual cross dating, ring width data was then stored in the computer in a Raw image format ready for analysis.

3.5 Determination of tree density

Samples for the density determination were obtained from cross sections cut at the

DBH of randomly selected trees at Muguga. The fresh weight of each sample was measured using an electronic balance. The wet volume was determined by use of the displacement method with water as the liquid. The samples were then dried in an oven at 100 °C for 24 hours and the dry weight and volume determined immediately. On dipping the wood sample in a measuring cylinder with water, the water displaced was considered equal to the weight of the sample. The density of the samples was then determined using the dry weight and dry volume using

Archimedes principle.

3.6 Reagents and reference compounds All solvents and compounds used were purchased from Sigma Aldrich Chemical

Company limited, California USA unless otherwise stated. The reagents and chemicals used were of purities ranging from 95-100% and were used as supplied by Sigma Aldrich Chemical Co. Ltd unless otherwise stated. Griseofulvin and 1- heptene standards were purchased from Sigma Aldrich Chemical Co. Ltd. Lupeol 43

standard used was extracted from Fagara tessmanii by Ivan Addae-Mensah

(University of Ghana).

3.7 Sample Preparation

The bark samples were dried under a shade for one month after harvesting to bring down the moisture content. The dried samples were then cut into small pieces and milled to powders using a fine mill. 400 grams of powdered bark of each sample from the Muguga, Karuri and Kobujoi was soaked in hexane, dichloromethane and methanol sequentially for 24 hours. 700 mililitres of each solvent was used for extraction. The extracts were then filtered using Whatman filter no. 1 using a vacuum suck pump. The filtrate was then concentrated under a vacuum at 40o C and at reduced pressure using a rotary evaporator. After the organic solvent extraction each extract was soaked in distilled water and heated in a water bath at 60oC for 5 hours. The aqueous extracts were then filtered using

Whatman filter no. 1 and concentrated using SP Scientific AdVantage 2.0 benchtop lyophilizer.

3.7.1 Extraction of essential oils

Three hundred grams of each powdered bark sample were weighed into a distillation flask. To each bark sample, 1.5 litres of distilled water was added heated to boil. On boiling, the temperature was brought down to 70o C and heated for two hours from the time the temperature was adjusted. Five mililitres of hexane were added to the clevenger to dissolve the essential oils as they condense. 44

The condenser was set at a temperature of -15-15o C. The sample was dissolved in hexane and transferred to sample vials. After distillation, the samples were concentrated using short path distillation apparatus and extracted using one milllitre dichloromethane.

3.7.2 Preparation of extracts for GC-MS analysis One milligram of each sample was weighed into a 1.5 milliliter eppendorf tube each tube labeled. One milliliter of dichloromethane was used to dissolve each dichloromethane and hexane extracts and each mixture was vortexed for 30 seconds. The samples were then sonicated for five minutes using Branson 2510E-

DTE sonicator and centrifuged for five minutes at 1300rpm at room temperature.

The samples were then transferred to 2 milliliter sample vials.

3.8 Instrumentation and chromatographic conditions

Gas chromatograph study includes the important optimization process such as,

i) Introduction of sample extract onto the GC Column

ii) Separation of components on an analytical column and

iii) Detection of target analysis using Mass Spectrometric (MS) detector

GC-FID analysis was carried out on a GC-FID (Model: 7890B Agilent) comprising an auto-sampler and gas-chromatograph interfaced to a Flame

Ionization Detector equipped with a HP-5 phenyl methyl siloxane capillary column of 30 m length, 320µm diameter and 0.25 µm film thickness. For GC-

FID detection, an electron ionization system with ionization energy of 70Ev was 45

used. The carrier gas was Hydrogen (99.99%) used at a constant flow rate of

30ml/min, injector and mass transfer line temperature were set at 270 and 280°C respectively, and an injection volume of 1µl was employed (splitless), the oven temperature was programmed from 35°C (isothermal for 5 min), with an increase of 10°C/min to 280°C for 5.4 min, 50°C/min to 285°C for 35 min.

3.8.1 Gas chromatography – Mass Spepectrometry analysis

Lupeol was used as internal standard for the quantification of hexane and dichloromethane extracts in the GC-MS. Essential oils were also identified and quantified by GC-MS using 1-heptene as internal standard. Before analyzing the extract using Gas Chromatography and Mass Spectrometry, the temperature of the oven, the flow rate of the gas used and the electron gun were programmed initially.

GC-MS analysis was carried out on a GC-MS (7683 Agilent Technologies, Inc.,

Beijing, China) comprising a gas-chromatograph interfaced to a mass spectrometer (GC-MS) instrument equipped with a HP-5 MS (5% phenyl methyl siloxane) low bleed capillary column of 30 m length, 0.25 mm diameter and 0.25

µm film thickness. For GC-MS detection, an electron ionization system with ionization energy of 70Ev was used. The carrier gas was helium (99.99%) used at a constant flow rate of 1.25 ml/min, injector and mass transfer line temperature were set at 250°C and 200°C respectively, and an injection volume of 1 µl

(splitless mode) was employed. The oven temperature was programmed from 46

35°C for 5 minutes, with an increase of 10°C/min to 280°C for 10.5 minutes, then

50°C/min to 285°C for 29.9 minutes with a run time of 70 minutes.

The MS operating parameters were as follows: ionization energy, 70eV; ion source temperature, 230°C, solvent cut time, 3.3 min, relative detector gain mode, scan speed 1666 µ/sec; scan range 40-550 m/z, the interface temperature was

250°C. The total running time of GC-MS was 70 min. The relative percentage of the extract was expressed as percentage with peak area normalization. The data was then compared with the compounds already stored in a compactNational

Institute of Standards and Technology library of chemical substances.

3.8.2 Identification of components

Identification was based on the molecular structure, molecular mass and calculated fragments. Interpretation on mass spectrum GC-MS was conducted using the database of National Institute of Standard and Technology (NIST) which has more than 62,000 patterns. The name, molecular weight and structure of the components of the test materials were ascertained. Library–MS searches using NIST Mass spectral library NIST 05, Chemecol.l, NIST 11, and Adams 2.l databases. NIST mass spectral search program Version 2.0 was used for characterization purposes in the GC-MS data system. The area was used for quantification based on the amount of internal standard added.

47

3.9 Liquid Chromatography-Mass Spectrometry analysis

3.9.1 Preparation of samples for LC-MS

Methanol and aqueous sample extracts were weighed into a 1ml Eppendorf and the weight was recorded in milligrams. For methanol extracts, one millilitre of the methanol was added to each sample to re-dissolve it. Aqueous extracts were re- dissolved using 95% methanol and 5% distilled water. The samples were then vortexed for 30 seconds. Sonication was done using a Branson 2510E-DTE sonicator for five minutes. The samples were then centrifuged for 5 minutes at room temperature at a speed of 1300 rpm and then transferred to 1.5 ml sample vials. Methanol and water extract were analyzed using liquid chromatography linked to mass spectrometry.

3.9.2 LC-MS Analysis HPLC separations were conducted on an HP 1100 capillary system with auto- sampler and a micro-pump (Agilent Technologies, Incorporation, Beijing, China).

Griseofulvin was used as an internal standard to quantify compounds in LC-MS.

A Waters symmetry column, 100µm 2.1mm, 3.5 µm was used to separate the compounds. The injection volume was set at 2µL and the compartment of the auto-ampler was set at 4 oC throughout the analysis. The mobile phase consisted of two components, with component A being water and component B methanol.

The solvent gradient was started at 10% B and held for 30 minutes then programmed to 50% in 3 minutes and held for 5 minutes, then to 100% and held for 10 minutes at a flow rate of 200 L/min. The effluent of the first five minutes 48

from the LC before analysis was diverted to waste to minimize ESI source contamination. Positive ion mode ESI-MS was used for the analysis. Pentacyclic triterpenoids present in the extracts were identified using METLIN metabolite data base and literature precedent and quantified using griseofulvin internal standard.

3.10 Statistical analysis

The ring width data was analysed using COFECHA software to help identify the problem segments and verify crossdating before tree growth curves were combined into a chronology (Grissino-Mayer, 2001). The data collected for the wild, on-farm remnant and the domesticated populations was analyzed using

Winks version 7 software. Means for the quantified chemical compounds of the five individual trees of each population were calculated to establish an overview of relationships. The means were separated using Tukey’s studentized Honestly

Significant Difference at 5% level of significance. The coefficients of variation within population were calculated to have a normalized comparison of variation.

Inter-correlations of different constituents and correlation with tree size and environmental conditions were also calculated. ANOVA was used to test for differences between the populations. A Pearson’s correlation analysis was carried out to determine the relationship between tree ages and their growth rates, wood density and DBH and ring number and DBH.

49

CHAPTER FOUR

RESULTS

4.1 Morphological characterization of Prunus africana trees at Muguga stand

The morphological data of P. africana trees in Muguga stand, was grouped based on tree heights and analyzed as shown in table 4.1. It was assumed that the trees were planted at the same time and thus were of the same age. Two trees in the population had heights below five and half meters tall. There were 146 trees with a height of between 5.6 to 10.5 meters. The third group had 124 trees which had a height range of 11.6 to 15 meters tall. The mean height of group one were significantly different from the other groups in Muguga stand (p>0.05). There was no significant difference in the stem shapes among the three groups. The mean

DBH of the three groups was positively correlated to the mean height of the groups. DBH can be used as a measure of growth rate and so is height.

The mean DBH of the three groups ranged from 9.45 to 54.85 centimeters. Group one had significantly lower mean diameter at breast height compared to the other two groups which showed no significance difference in diameter size (p<0.05).

Fruition condition was expressed as percentages of the total number of trees in a particular group. The two trees in group one were not in the fruition stage. Out of the 146 trees in group 2, 40 (27.4%) of them were in the fruiting stage while 106

(72.6%) trees in this group had no fruits. A layout for the Muguga P. africana stand is as shown in appendix 43. This layout shows the position of trees in the 50

stand. Trees next to the Grevillea robusta border experienced more shading

periods as they were on the West side of the plantation compared to trees next to

the grass border.

Table 4.1: Growth characteristics of trees at Muguga Prunus africana stand

Groups No. Height(M) Stem DBH(cm) Fruiting (Height) of Shape trees Rating Yes No Group 1 (0-5) 2 4.20±1.70a 1.50±0.71a 9.45±12.09b 0 2 (100) Group 2 (6-10) 146 9.22±1.16b 1.53±0.60a 40.17±12.19a 40 106 (27.40) (72.60) Group 3 (11-15) 124 11.67±0.80b 1.63±0.64a 54.85±16.26a 52 72 (41.94) (58.06)

Values are expressed as Mean ± SD (n=3). Values followed by the same super script in same columns are not significantly different (p>0.05). Values in parenthesis after groups show the tree heights in each group. Values in parenthesis in fruiting column are expressed as percentages.

4.2 Visual and statistical cross dating

In this study visual cross dating was first done using the the skeleton plot method

(Stokes & Smiley, 1968). The process was carried out using two radii on the cross

sections to enable detection of wedging rings. Fifteen out of twenty three trees

were successfully crossdated and their age ranged from fifteen to twenty six

years.

51

4.3 Relationship between age and growth rate in Muguga population

The age and growth rate in millimeters of seven randomly selected trees from

Muguga is tabulated in Table 4.2. A Pearson’s correlation analysis for age and

average growth rate of trees randomly selected from Muguga population showed

a non-significant relationship (t =-0.916, DF= 16, r2=0.0498, p>0.05). In addition,

age was negatively correlated to the growth rate (r =-0.223).

Table 4. 2: Relationship between age (years) and growth rate (mm) Age(yrs) Growth Age(yrs) Growth t value DF r2 rate(mm) rate(mm 1 3.53±1.69 10 4.78±3.08 -0.916 16 0.0498 2 3.42±1.96 11 2.26±1.37 3 2.38±1.98 12 2.36±1.13 4 1.82±1.14 13 3.18±1.36 5 2.67±2.05 14 1.80±0.73 6 1.38±0.71 15 3.38±1.98 7 1.06±0.27 16 2.00±0.75 8 2.07±1.84 17 1.22±0.70 9 1.69±0.99 18 1.56±0.68

Values are expressed as Mean ± SD (n=7).

4.4 Relationship between ring number and DBH of trees from Muguga population

A regression and Pearson’s correlation analysis of trunk cross section ring number

and DBH of randomly selected tress from Muguga population showed a non-

significant relationship (DF= 14, r2=0.0858, p>0.05, (Figure 4.1). The number of 52

growth rings in the trunk cross section of the selected trees were positively correlated to the DBH of the tree (r =0.293).

Figure 4.1: A fitted line plot of Ring number versus DBH (cm)

4.5 Relationship between wood density and DBH of trees from Muguga population A Pearson correlation and regression analysis of the wood sample density and

DBH of trees randomly selected from Muguga population showed a non- significant relationship (DF= 16, r2=0.0196, p>0.05, (Figure 4.3). The density of wood sections samples from Muguga population was negatively correlated to the tree DBH (r = -0.140). 53

Figure 4.2: A fitted line plot of wood density (g/cm3) versus DBH (cm)

4.6 Relationship between wood density (g/cm3) and growth rate (mm)

A Pearson correlation and regression analysis of the wood sample density and growth rate of trees randomly selected from Muguga population showed a non- significant relationship (DF= 14, r2=0.0279, p>0.05). The density of wood sections samples from Muguga population was positively correlated to the tree growth rate (r = 0.167). 54

Fitted Line Plot Density(g/cm3) = 0.6011 + 0.02392 Growth rate(mm) 0.80 S 0.0669057 R-Sq 2.8% R-Sq(adj) 0.0% 0.75

) 3 0.70

m

c

/

g

(

y

t

i 0.65

s

n

e

D 0.60

0.55

1.5 2.0 2.5 3.0 3.5 Growth rate(mm)

Figure 4.3: A fitted line plot of Density (g/cm3) versus Growth rate (mm)

4.7 Crude yield extracts of individual populations

Dried bark samples from the three populations were extracted using hexane,

DCM, methanol and water. The yields of the organic solvents are as tabulated in table 4.3. Crude bark extracts of methanol, DCM and hexane had no significance difference in the three populations (p>0.05). Karuri population had the highest yield of methanol crude extract while Kobujoi population had the lowest. Kobujoi population had the highest yield in DCM and hexane crude extracts while Karuri population had the lowest yield of hexane extract. Methanol extract had the highest yield (grams) for the three populations. 55

Table 4.3: Crude yields (g) for organic extracts Population Methanol DCM Hexane Muguga 3.58±2.38a 0.59±0.55a 0.56±0.08a

Karuri 4.94±0.50a 0.88±0.41a 0.46±0.11a a a a Kobujoi 2.79±2.97 0.90±0.47 0.82±0.74

Values are expressed as Mean ± SD (n=3). Values followed by the same super script in same column are not significantly different (p>0.05).

4.8 Phytochemical yields in the three populations

4.8.1 Total essential oils yields in Muguga, Karuri and Kobujoi populations

Phytochemical analysis of essential oils of the three populations was done using

GC-MS. The analysis revealed the presence of linoleic acid, lauric acid, methyl laurate, methyl linoleate, methyl myristate and myristic acid among other compounds in the three populations. Analysis of polyunsaturated fatty acids and their methyl esters is tabulated in table 4.4. Muguga population had the highest concentration of linoleic acid, methyl linoleate and methyl myristate in essential oils. Karuri population had the highest concentrations of myristic acid and lauric acid while Kobujoi had the highest methyl laurate concentration in essential oils.

The concentration of linoleic acid of essential oil samples from Muguga, a domesticated stand was significantly different from Karuri, and Kobujoi (p<0.05).

Muguga has the highest level of linoleic acid while Kobujoi had the lowest concentration. Lauric acid concentration for Karuri samples was significantly different from that of Kobujoi and Muguga samples (p<0.05). Karuri had the highest Lauric acid concentration (1152.14) while Kobujoi had the lowest 56

concentration (4.12). The concentrations of methyl laurate in the three populations were not significantly different (p>0.05). Methyl linoleate concentrations in the three populations were significantly different (p<0.05). Muguga had a significantly higher concentration of methyl myristate as compared to Karuri and

Kobujoi (p<0.05). Myristic acid was significantly different in the three populations with Karuri having the highest concentration.

Table 4.4: Concentration of essential oils in Muguga, Karuri and Kobujoi (mg/Kg)

Compound Muguga Karuri Kobujoi b a a Linoleic acid 196.35±3.48 29.13±3.47 28.93±1.98 Lauric acid 382.66±2.61a 1152.14±315.29b 4.12±1.07a Methyl laurate 2.54±0.41a 3.32±0.50a 3.36±0.71a Methyl linoleate 27.82±1.28a 14.83±1.59b 7.51±1.29c Methyl myristate 26.71±0.64b 4.59±0.16a 5.80±5.20a Myristic acid 287.09±1.36a 554.99±22.60b 92.84±1.81c

Values are expressed as Mean ± SD (n=3). Values followed by the same super script along rows are not significantly different (p>0.05).

4.8.2 Hexane extract yields of Muguga, Karuri and Kobujoi populations

Phytochemical analysis of hexane extracts of the three populations was done using GC-MS and revealed campesterol, β-sitosterol, lup-20(29)-en-3-one, palmitic acid, β-sitostenone, (3.β.,5.α)- stigmast-7-en-3-ol, stigmastan-3,5-diene and α-tocopherol compounds. Analysis of these compounds is tabulated in table

4.5. Muguga population had the highest amounts of campesterol, lup-20(29)-en-3- one, palmitic acid, squalene, β-sitostenone, 3β,5.α-stigmast-7-en-3-ol, stigmastan- 57

3,5-diene, myristic acid and α-tocopherol compounds in hexane extracts. Karuri populations had the highest concentrations of lauric acid and β-sitosterol. The concentrations of campesterol, lauric acid, β-sitosterol, squalene, lup-20(29)-en-3- one, β-sitostenone, stigmastan-3,5-diene, 3β,5.α-stigmast-7-en-3-ol, palmitic acid and α-tocopherol in hexane extracts of the three populations were not significantly different (p>0.05). Muguga samples had significantly different concentration of α- tocopherol (p<0.05). The standard deviation for lauric acid and myristic acid were high because one of my replicates showed no presence of these compounds.

Table 4.5: Concentrations of compounds in hexane extracts of Muguga, Karuri and Kobujoi (mg/Kg) Compound Muguga Karuri Kobujoi Campesterol 9.16±1.93a 4.51±3.75a 6.70±3.29a Lauric acid 2.62±1.18a 4.84±7.56a 0.72±0.32a β-Sitosterol 131.04±31.34a 160.05±3.91a 153.36±13.01a Lup-20(29)-en-3-one 13.32±2.81a 10.04±5.29a 7.97±3.61a Palmitic acid 82.24±30.049a 34.28±19.83a 55.13±58.46a Squalene 34.25±14.99a 26.72±3.18a 28.34±19.13a β-sitostenone 36.92±12.05a 19.79±0.49a 27.80±13.87a 3β,5α-Stigmast-7-en-3- 15.63±4.71a 10.46±4.64a 11.23±6.01a ol Stigmastan-3,5-diene 35.99±11.50a 20.42±2.49a 27.21±16.94a Myristic acid 7.21±0.50a 5.65±5.06a 2.02±0.09a α-Tocopherol 13.44±2.71b 1.84±1.15a 4.88±1.38a

Values are expressed as Mean ± SD (n=3). Values followed by the same super script along rows are not significantly different (p<0.05).

58

4.8.3 DCM extract yields of Muguga, Karuri and Kobujoi populations

Phytochemical analysis of DCM extracts of the three populations was done using

GC-MS and revealed campesterol, β-sitosterol, lup-20(29)-en-3-one, palmitic acid, β-sitostenone, (3.β.,5.α)- stigmast-7-en-3-ol, stigmastan-3,5-diene and α- tocopherol compounds. Hexane extracts analysis showed similar phytochemical profile to that of DCM extracts. This is because these two solvent are non-polar thus they extract similar compounds though with different concentrations due to the slight difference in polarity. These compounds were present in the three populations. These compounds are important in the treatment of BPH and their analysis is as shown in table 4.6.

Muguga population showed the highest concentration of campesterol, lup-20(29)- en-3-one, palmitic acid, squalene, β-Sitosterol, β-sitostenone stigmastan-3,5-diene and myristic acid. Karuri population had the highest amount of lauric acid while

Kobujoi population had the highest amount of 3-β,5α-stigmast-7-en-3-ol and α- tocopherol. The concentrations of campesterol, lauric acid, β-sitosterol, lup-

20(29)-en-3-one, β-sitostenone, stigmastan-3,5-diene, squalene, 3-β,5α-stigmast-

7-en-3-ol, palmitic acid and α-tocopherol in DCM extracts of the three populations were not significantly different (p>0.05). DCM extract of Muguga samples had a significantly different concentration of myristic acid compared to

Karuri and Kobujoi samples (p<0.05).

59

Table 4.6: Concentration of compounds in DCM extracts of Muguga, Karuri and Kobujoi (mg/Kg)

Compound Muguga Karuri Kobujoi Campesterol 12.55±3.75a 7.32±0.56a 8.10±2.51a Lauric acid 1.19±0.768a 1.85±0.93a 1.71±1.24a β-Sitosterol 130.20±72.95a 103.59±28.29a 117.16±20.85a Lup-20(29)-en-3-one 14.04±1.89a 9.99±0.43a 8.30±3.502a a a a Palmitic acid 116.63±42.44 65.55±23.54 90.63±67.70 Squalene 34.56±14.55a 22.23±6.35a 28.34±9.90a β-sitostenone 43.21±15.52a 26.51±4.48a 30.62±6.73a 3β,5α-Stigmast-7-en-3-ol 18.39±7.69a 11.59±2.83a 19.57±13.93a Stigmastan-3,5-diene 36.83±15.75a 26.13±3.66a 29.57±11.68a Myristic acid 6.47±0.99b 2.89±1.27a 3.19±1.40a α-Tocopherol 6.80±1.04a 7.67±2.12a 11.08±2.44a

Values are expressed as Mean ± SD (n=3). Values followed by the same super script along rows are not significantly different (p>0.05).

4.8.4 Methanol extract yields of Muguga, Karuri and Kobujoi populations

The phytochemical analysis of methanolic extracts of the three populations was done using LC-MS and revealed procyanidin B5, feruloyl-quinic acid, robinetinidol-(4-α-8)-catechin-(6-α)-robinetinol, prunetrin, quercetin3,3'- dimethylether-4'-glucoside, cyanidin-o-galactoside, chlorogenic acid, ursolic acid, isochamaejasmin+, cinnamtannin A2, isoliquiritin and two unknown compounds.

Cyanidin-3-o-rutinoside was only present in Karuri population but isoliquiritin and isochamaejasmin+ was not present in Karuri population methanol extracts.

Analysis of these compounds is tabulated in table 4.7. Kobujoi population had the highest concentration of feruloyl-quinic acid, chlorogenic acid, procyanidin B5, quercetin3,3'-dimethylether-4'-glucoside, cinnamtannin A2, and isochamaejasmin 60

in methanol extracts. Muguga population had the highest concentration of ursolic acid, isoliquiritin and unknown compound 1 in methanol extracts. Karuri population had the highest amounts of prunetrin, cyanidin-o-galactoside and robinetinidol-(4-α-8)-catechin-(6-α)-robinetinol in methanol extracts. The concentrations of feruloyl-quinic acid, chlorogenic acid, cyanidin-o-galactoside, ursolic acid, procyanidin B5 and unknown compound 2 in methanolic extracts of samples from the three populations were not significantly different (p<0.05).

Karuri population had significantly different concentration of prunetrin from

Muguga and Kobujoi samples whose concentrations were not significantly different (p<0.05). Analysis of Muguga and Kobujoi methanolic extracts did not reveal any presence of cyanidin-3-o-rutinoside. Kobujoi samples had significantly higher concentrations of cinnamtannin A2 (p<0.05). Concentrations of isochamaejasmin+ in the three populations were significantly different (p<0.05) with the compound absent in Karuri samples (p<0.05).

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Table 4.7: Concentration of compounds in methanol extract from the three populations (mg/Kg)

Compound Muguga Karuri Kobujoi Feruloyl-quinic acid 1.89±0.69a 2.21±0.67a 2.56±1.45a Chlorogenic acid 2.05±0.82a 2.07±1.33a 2.36±0.73a Isoliquiritin 7.48±0.65a 0.000b 7.48±0.18a Prunetrin 1.27±0.62a 2.90±0.630b 1.20±0.52a Cyanidin-O-galactoside 9.87±2.79a 10.69±0.25a 7.37±0.85a Ursolic acid 2.39±2.04a 0.78±0.26a 1.57±0.34a Unknown compound 1 16.16±4.93a 12.56±2.90ab 6.98±1.63b Procyanidin B5 1.29±0.63a 0.82±0.33a 3.10±1.60a Cyanidin-3-O-rutinoside 0.000a 11.74±1.74b 0.000a Quercetin3,3'-dimethylether-4'- 1.14±0.35a 0.62±0.18a 20.27±0.71b glucoside Robinetinidol-(4-α-8)catechin- 0.84±0.27b 4.81±0.35a 4.22±2.43ab (6,4-α)robinetinol Unknown compound 2 4.31±0.66a 3.63±0.69a 6.16±2.98a Cinnamtannin A2 0.67±0.14a 0.74±0.03a 2.29±0.49b Isochamaejasmin+ 1.14±0.39a 0.000b 17.92±0.46c

Values are expressed as Mean ± SD (n=3). Values followed by the same super script along rows are not significantly different (p<0.05).

4.8.5 Aqueous extract yields of Muguga, Karuri and Kobujoi populations

Phytochemical analysis of aqueous extracts of the three populations was done using LC-MS and revealed procyanidin B5, robinetinidol-(4-α-8)-catechin-(6-α)- robinetinol, feruloyl-quinic acid, quercetin3,3'-dimethylether-4'-glucoside, cyanidin-o-galactoside, chlorogenic acid, ursolic acid, cyanidin-3-o-rutinoside, cinnamtannin A2, isoliquiritin, prunetrin and two unknown compounds. The analysis is tabulated in table 4.8. Feruloyl-quinic acid and prunetrin was only present in aqueous extracts of Muguga population. Kobujoi aqueous extracts did not show presence of quercetin3,3'-dimethylether-4'-glucoside compound. 62

Muguga population aqueous extracts showed the highest concentration of quercetin3,3'-dimethylether-4'-glucoside, unknown compound 1 and unknown compound 2. Kobujoi aqueous extracts had the highest amounts of chlorogenic acid, cyanidin-o-galactoside, ursolic acid, procyanidin B5 and cinnamtannin A2.

Karuri population aqueous extracts had the highest concentrations of isoliquiritin, robinetinidol-(4-α-8)-catechin-(6-α)-robinetinol and cyanidin-3-o-rutinoside compounds. The concentrations of cyanidin-3-o-rutinoside, procyanidin B5, unknown compound 1 and cinnamtannin A2 and in aqueous extracts of samples from the three populations were not significantly different (p<0.05). Muguga population is the only population that showed presence of prunetrin and feruloyl- quinic acid in aqueous extracts.

Analysis of Karuri aqueous extracts did not reveal the presence of unknown compound 2. Kobujoi samples had significantly higher concentrations of chlorogenic acid than Muguga and Karuri (p<0.05). Concentrations of quercetin-

3, 3'-dimethylether-4'-glucoside in Muguga and Karuri samples were significantly different (p<0.05) while Kobujoi aqueous extract did not have the compound.

Karuri and Kobujoi aqueous extracts did not show significant difference in the concentration of cyanidin-o-galactoside but the concentrations of the compound were significantly different from that of Muguga samples (p<0.05).

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Table 4.8: Concentrations of compounds in aqueous extracts from the three populations (mg/Kg)

Compound Muguga Karuri Kobujoi Feruloyl-quinic acid 5.64±3.24b 0.000a 0.000a Chlorogenic acid 1.93±1.714a 4.06±2.09a 10.06±2.27b Isoliquiritin 18.469±4.77a 30.97±5.11b 17.61±2.56a Prunetrin 1.74±0.91b 0.000a 0.000a Cyanidin-O-galactoside 1.56±1.64b 3.50±0.75ab 5.48±0.56a Ursolic acid 13.73±7.89b 19.31±4.17ab 27.72±0.84a Unknown compound 1 13.53±6.26a 11.75±2.39a 6.21±0.16a Procyanidin B5 5.58±4.18a 6.99±1.44a 11.27±0.32a Cyanidin-3-O-rutinoside 16.53±10.13a 33.03±5.85a 18.09±2.74a Quercetin3,3'-dimethylether- 7.64±2.74b 3.01±1.00a 0.000a 4'-glucoside Robinetinidol-(4-α-8)- 1.34±0.43a 5.605±0.47b 3.72±0.50c catechin-(6,4-α)robinetinol Unknown compound 2 21.45±2.80a 0.000b 11.13±0.43c Cinnamtannin A2 3.06±1.63a 3.54±1.33a 5.50±1.08a Values are expressed as Mean ± SD (n=3). Values followed by the same super script along rows are not significantly different (p<0.05).

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CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 DISCUSSION

The differences in height and DBH among trees in Muguga P. africana stand was probably due to differences in shading periods within the population. It was also observed that trees that were in the eastern side of the plantation were shorter than trees on the opposite side. Competition for sunlight for the trees away from the sunlight may explain this difference in heights. Light is an environmental factor that influences plant growth as it is a crucial requirement for photosynthesis

(Canham et al., 1990; Valladares, 2003). Light gradients can differ both within plant canopies and within the crowns of individual plants thus, all plants are exposed to some degree of shade in their lifetime. High or low sunlight can limit growth characteristics of a plant but sunlight must be present for photosynthesis to take place (Grubb, 1998).

In the Equatorial region, P. africana species has no specific flowering and some individuals of the species flower almost every month of the year (Munjuga et al,

2000). This wide range of flowering period leads to different fruiting conditions observed in the current study. Fruiting of the species occurs 2-3 months after flowering and is associated with rainfall (Munjuga et al., 2000). Growth rate of plant species over short time periods do not strongly correlate with single 65

environmental factors indicating that the growth rates are determined by complex environment-plant interactions (Berman & DeJong, 1997). Age was negatively correlated to the average growth rate of randomly selected P. africana individuals in Muguga stand. This observation was also recorded by Yoder et al., 1994 in

Pinus contorta and Pinus ponderosa. This negative relationship between age and growth rate can be attributed to reduced rate of photosynthesis as age of the species increases (Yoder, et al., 1994).

Change in seasons lead to variation of cambial activity and thus differences in growth ring widths and other phonological patterns of trees. There are no clear seasons in the tropics (Hoadley, 1990), and this may lead to indistinct ring boundaries, however dendrocronology techniques have been used too identify ring bounderies and to determine tree ages. Variation or ring width is due to changes in seasons. Narrow ring indicate the dry seasons while wide rings are an indication of rainy seasons (Fichtler et al., 2004; Trouet et al., 2009). P. africana does not interrupt the dormancy of cambium activity especially in the short rains but it reactivates cambium activity when the long rains beigin (Krepkowski et al.,

2011). Increase in ring width may occur even at periods with no precipitation as reported in Podocarpus falcatus mainly due to secondary cell enlargement and thickening even whe no cambium activity takes place (Deslauriers et al., 2009).

Wood density is used to describe wood quality especially dimensional stability, workability, and mechanical timber properties. Wood density was negatively 66

correlated to the DBH of trees. This negative relationship was also reported in

Picea mariana species (Zhang et al., 1996). There was a positive relationship between growth ring number and DBH of tree at Muguga population. This can be attributed to the increase in cambium activity with increase in age. In this study, wood density was positively correlated to growth rate. Previous studies show that there is no relationship between wood density and growth rate of diffuse-porous hardwoods (Zobel & van Buijtenen, 1989; Zhang, 1995). The same observation was reported in Betula pendula and Prunus serotina (Nepveu & Velling, 1983.,

Koch, 1967). Some work on Populus species reported a weak negative correlation between growth rate and wood density (Hernandez et al., 1998., Pliura et al.,

2007) while some studies showed absence of correlation of the two parameters

(Debell et al., 2002; Zhang et al., 2003).

Prunus africana bark extract has been used to suppress lower urinary tract symptoms by decreasing inflammation, reducing bladder reactivity and prostate size in patients with benign prostate hyperplasia (Andro & Riffaud, 1995; Ishani et al; 2000). The extracts are believed to counter BPH through inhibition of 5-α- reductase, anti-inflammatory activity, inhibition of prolactin levels and inhibition of prostatic fibroblast proliferation in response to growth factors (Capasso et al.,

2003). The compounds analyzed in this study all play part in the treatment of

BPH or alleviating the effects of BPH symptoms (Donovan et al., 1998; Carbin et al., 1990; Kampa et al., 2004). These compounds include polyunsaturated fatty acids, phytosterols, ketones, phenolic compounds and pentacyclic triterpenoids. 67

Polyunsaturated fatty acids and their methyl esters were mainly present in the essential oil and were extracted by hydro-distillation. However, myristic acid and lauric acid were also present in hexane and DCM extracts though at lower concentrations compared to the essential oil. This difference was observed because DCM and hexane extracts were concentrated using evaporation of the solvent and some of the oils might have been lost during the process as they are volatile. Phytosterols were observed in hexane and DCM extracts because these compounds are non-polar to midpolar and thus were easily extracted by these solvents. Pentacyclic triterpenoids and phenolic compounds are polar compounds and thus were present in methanol and aqueous extracts. Phytochemical content information is important when reconciling management practices between the species production and biodiversity conservation. Phytochemical variation in agrofeorestry and cultivated is relevant for conservation as trait diversity, genetic variation and species richness influences delivery of essential ecosystem services

(Cardinale et al., 2012). Pentacyclic triterpenoids present in P. africana bark extracts mainly ursolic acid inhibits glucosyl-transferase activity and have anti- edematous activity (Kokwaro, 1993; Donovan et al., 1998; Mothana et al., 2006).

Previous studies also showed that ursolic acid inhibits growth of melanoma cells and prostate cancer cells (Nataraju et al., 2007).

In this study, there was no signifance variation in the concentration Phytosterols mainly β-sitosterol and β-sitostenone have anti-inflammatory effect as they suppress the production of prostaglandins and thus prevent swelling of the 68

prostate (Raicht et al., 1976; Carbin et al., 1990). The bark extracts also had ferulic acid esters and their derivatives which have antitumor and hypocholesterolemic activity on the prostate (Kampa et al., 2004). Phenolic compounds in P. africana bark have also been reported to have chemo-preventive effect on estrogen dependent breast cancer (Noratto et al., 2009).

The β-sitostenone concentrations in DCM and hexane extracts of the three populations were lower compared to the concentration of β-sitosterol. Catalano et al., (1984) also reported this difference in the concentration of β–sitostenone compared to β-sitosterol in P. africana. In the DCM extracts, β-Sitosterol concentration was highest in Muguga (130.20 mg/kg). The compound is believed to have anti-cancer activity and cholesterol lowering activity (Award & Fink,

2000). β-sitosterol was found in higher concentrations in P. africana as compared to many other species. Avocado is also a rich source of β-sitosterol (Duester,

2001) and the bark values are similar to those of P. africana. Moringa oleifera

(Anwar et al., 2007) and P. spinosa (Wolbiš et al., 2001) have high levels of β- sitosterol and have been used in traditional medicine for their diuretic properties to increase urine flow. In this study, the concentration of β-sitosterol was not dependent on environmental factors. However, in soybeans plants grown in cold areas produced seeds with a lower content of β-sitosterol compared to plants grown in warm areas (Yamaya et al., 2007). Campesterol concentration was also highest in Muguga population. Campesterol, stigmast-7-en-3-β-ol and β-sitosterol were also reported in hypoxis species (Moghadasian, 2000; Pegel, 1979). These 69

phytosterols have anticancer activity (Choi et al., 2003), cholesterol lowering activity and also anti-inflammatory effects (Quilez et al., 2003).

Myristic acid and α-tocopherol concentrations in Muguga population extracts were slightly higher compared to Karuri and Kobujoi populations. This difference in concentration can be attributed to difference in soil types and environmental conditions in the three populations. Soils vary in chemical, physical and biological properties and these can lead to variation in the growth and metabolic mechanisms of plants. Epigenetic factors can be influenced by environmental conditions and thus affect DNA indirectly by switching on or off genes involved in metabolic processes hence determine secondary compounds produced by a plant.

Palmitic acid was in higher concentration than myristic and lauric acid in the three populations. This variation has also been reported previously in P.africana where the two saturated fatty acids had lower concentration compared to other fatty acids (Ganzera et al., 1999; Abe et al., 2009). The three fatty acids have also been reported in saw palmetto and pumpkin seeds (Ganzera et al., 1999). Lauric acid and myristic acid have also been reported in Artocarpus heterophyllus

(Chowdhury et al., 1997) and in P. amygdalus (Munshi & Sukhija, 1984) but at low concentrations. Fatty acids and sterols are believed to reduce prostate size by blocking the conversion of testosterone to dihydrotestosterone. The mechanism of action of these compounds is by inhibition of 5-α-reductase enzyme thus 70

preventing formation of dihydrotestosterone, the modulator of prostate growth

(Edeoga et al., 2005; Bent et al., 2006).

Friedelin was not detected in the three populations though the compound had been reported in P. lusitanica (Sainsbury, 1970) and in P. africana bark extracts

(Catalano et al., 1984). Friedelin is a triterpenoid that has anti-inflammatory activity (Antonisamy et al., 2011). Ursolic acid is also a natural pentacyclic triterpenoid in plants and has been a component in traditional medicine (Amico et al., 2009). Ursolic acid has been reported to have antioxidant, anti-proliferative and anti-inflammatory activities (Nataraju et al., 2007, Amico et al., 2009). It also serves as starting material for the biosynthesis of more potent bioactive compounds like antitumor agents (Ma et al., 2005). Ursolic acid has also been detected in Eriobotrya japonica at concentrations of up to 2000mg/kg (Zhou et al., 2011).

Cyanidin-o-galactoside and cyanidin-3-o-rutinoside are polyphenols and have also been reported in plums (Kim et al., 2003; Usenik et al., 2008). Phenolic compounds have anti-oxidative activity and are thus used as anticancer agents and also have benefits for cardiovascular disease and diabetes (Utsunomiya et al.,

2005; Belkaid et al., 2006; Noratto et al., 2009). Hydroxycinammic acid derivatives like chlorogenic acid and quercetin derivatives have also been identified among the phytochemicals in plums (Raynal et al., 1989; Kim et al.,

2003). Chlorogenic acid has previously been reported in P. domestica, coffee and 71

blue berries (Donovan et al., 1998; Prior & Cao, 2000). Cyanidin-o-galactoside, cyanidin-3-o-rutinoside, procyanidin B5 and robinetinidol-(4-α-8) catechin-(6,4-

α) robinetinol are members of the flavonoid group and their derivatives and are believed to inhibit cell proliferation and have free radical scavenging activity

(Rukunga & Waterman, 1996; Cai et al., 2004; Jacob et al., 2012). Flavonoids have the ability to inhibit topoisomerases and protein kinases in addition to their ability to modulate apoptosis and cell differentiation and their antioxidant activity

(Kuo, 1997; Pinhero & Paliyath, 2001). These properties make flavonoids important compounds in the field of cancer research. Cyanidin-o-galactoside and cyanidin-3-o-rutinoside have also been reported in Japanese plums as part of the anthocyanins found in the fruits of this species (Wu & Prior, 2005). Flavonoids, particularly anthocyanins give most fruits their color (Usenik et al., 2009).

The three populations showed similar profiles and there was no significant variation in the concentration of the compounds in both organic and aqeous extracts. Phytochemical analysis of tea crops from mixed crop fields and agroforests shows more catechin compounds than those sampled from forests

(Ahmed et al., 2013). These differences are attributed to the type of tea management systems. A study on Amburana cereansis showed a similar phytochemical profile of the cultivated and wild species. These study findings support the idea of the utilization of cultivated medicinal plants for the manufacture of herbal preparations (Canuto et al., 2012). This ensures constant 72

and uniform supply of high quality raw materials and conservation of the species in the original biome (Canuto et al., 2012).

The temperatures at Muguga, Kobujoi and Karuri at the time of bark harvest were

9-18 °C, 8.8-23.7 °C and 10-21 °C respectively. Muguga was the coldest site while Kobujoi was the warmest among the three. Temperature regimes can vary in different microsites within a forest (Longman & Jenik, 1987). These temperature differences may cause chilling injury in plants leading to imbalances in metabolism, accumulation of toxic compounds and increased membrane permeability (Gachie et al., 2012). Intraspecies genetic variation may also lead to difference in types and quantities of secondary metabolites in plants of the same population due to genetic variation (Kadu et al., 2012).

5.2 CONCLUSION Shading periods may have an effect on the growth rate of P. africana hence variation in tree height and DBH of trees growing in same habitat. P. africana species had varied flowering periods even for individuals growing in the same habitat leading to difference in fruiting seasons. The growth rate of P. africana is negatively correlated to age maybe due to increase in requirement of energy for respiration and reduction in photosynthesis rate. Habitat of individuals of P. africana species do not significantly affect the crude yields of bark extract as observed in this study. 73

In this study, methanol and aqueous extracts had similar phytochemical profile and so was hexane and DCM but concentrations of individual phytochemicals varied in each solvent. Thus all these solvents are necessary for one to get high yields of the phytochemicals present in the P. africana bark. P. africana trees from wild, domesticated stand and on-farm remnant habitats do not vary significantly in the concentration of most of the compounds related to BPH treatment but some phytochemicals concentration varied with habitat. Thus habitat or whether the species is wild or domesticated should not be a major concern while harvesting bark for medicinal purposes.

5.3 RECOMMENDATIONS AND SUGGESTIONS FOR FURTHER RESEARCH 5.3.1 Recommendations

Domestication initiatives should be advocated to save the threatened P. africana species as it does not significantly affect the phytochemical profiles and yields of the bark. Polar, mid polar and non-polar solvents should be used for extraction of compounds of medicinal value from P. africana. The age of P. africana trees in

Muguga domesticated stand ranged from fifteen to twenty six years. The profiles and yields of compounds from trees in this stand were not significantly different and thus this study advocates for bark harvesting from trees as from fifteen years old.

74

5.3.2 Suggestions for Further Research i) Bioassays should be carried out to establish the efficacy of bark extracts from

domesticated P. africana. ii) Research to be carried out to establish effects of soils, temperature and intra-

species variation on phytochemical profiles and yields. iii) X-ray crystallography to be done to get the identity of the two unknown

compounds in methanol and aqueous extracts of the P. africana bark extracts

which could be identified by mass spectrometry. iv) Phytochemical analysis of P. africana stem bark extracts of younger trees

should be carried out and their efficacy established.

75

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APPENDICES Appendix 1: Correlation and regression of age and growth rate

Linear Regression and Correlation Table ------

Dependent variable is AVARAGE_GROWTH_RATE, independent variables, 18 cases. ------Variable Coefficient St. Error t-value p(2 tail) ------Intercept 2.7501308 .4797372 5.7325781 <.001 AGE -.0405986 .0443202 -.9160291 0.373 ------R-Square = 0.0498

Analysis of Variance to Test Regression Relation

Source Sum of Sqs df Mean Sq F p-value ------Regression .7985737 1 .7985737 .8391094 0.373 Error 15.227072 16 .951692 ------Total 16.025646 17

------MEAN X = 9.5 S.D. X = 5.339 CORR XSS = 484.5 MEAN Y = 2.364 S.D. Y = .971 CORR YSS = 16.026 REGRESSION MS= .799 RESIDUAL MS= .952 ------

Pearson's r (Correlation Coefficient)= -0.2232

The linear regression equation is: AVARAGE_GROWTH_RATE = 2.750131 + -4.059856E-02 * AGE

Test of hypothesis to determine significance of relationship: H(null): Slope = 0 or H(null): rho = 0 (two-tailed test) t = .92 with 16 degrees of freedom p = 0.373

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Appendix 2: Abundances of essential oils in Kobujoi population (%)

Compound Kar1 Kar2 Kar3 Kar4 Kar5 Linoleic acid 1.68 1.57 1.63 1.91 1.86 Lauric acid 62.84 70.50 62.50 61.85 61.06 Methyl laurate 0.20 0.19 0.19 0.22 0.18 Methyl linoleate 0.94 0.75 1.00 0.76 0.97

Methyl myristate 0.31 0.21 0.30 0.28 0.27

Myristic acid 34.03 26.79 34.39 34.98 35.66 Values are expressed as percentages.

Appendix 3: Abundances of Karuri population essential oils (%)

Compound Kob1 Kob3 Kob4 Kob5 Kob6 Linoleic acid 19.58 22.56 20.07 19.03 20.28 Lauric acid 3.22 3.44 1.62 2.86 3.29 Methyl laurate 1.83 2.11 2.35 2.47 2.97 Methyl linoleate 5.78 5.20 3.85 5.62 5.83 Methyl myristate 0.90 1.01 4.08 9.43 4.53 Myristic acid 68.69 65.68 68.02 60.59 63.11

Values are expressed as percentages.

Appendix 4: Abundances of Muguga population essential oils (%)

Compound T223 T9115 T9123 T18249 T20276 Linoleic acid 21.01 21.50 21.52 21.32 20.99 Lauric acid 41.65 41.50 41.13 41.50 41.47 Methyl laurate 0.27 0.22 0.25 0.32 0.32 Methyl linoleate 2.85 2.93 3.18 2.98 3.13 Methyl myristate 2.82 2.89 2.95 2.83 2.98 Myristic acid 31.41 30.95 30.97 31.05 31.11

Values are expressed as percentages.

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Appendix 5: Total ion chromatogram of essential oils

Linoleic acid (1), lauric acid (2), methyl laurate (3), methyl linoleate (4), methyl myristate (5), myristic acid (6) and internal standard (IS)

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Appendix 6: Abundances of compounds in hexane extract of Muguga (%) Compound T223 T9115 T9123 T18249 T20276 Campesterol 3.45 2.08 2.46 2.28 1.84 Lauric acid 1.15 0.71 0.18 0.72 0.77 β-sitosterol 30.97 25.46 33.06 38.82 42.41 Lup-20(29)-en-3-one 4.64 3.27 3.78 3.56 2.34 Palmitic acid 21.26 35.73 19.31 20.77 11.99 Squalene 6.85 7.28 8.89 6.57 14.58 β-sitostenone 13.42 8.67 11.18 11.00 4.57 (3.β.,5.α)-Stigmast-7-en- 3.88 3.62 4.02 3.06 5.72 3-ol Stigmastan-3,5-diene 8.81 8.19 12.27 6.56 10.67 Myristic acid 1.95 2.17 1.74 1.85 1.77 α-Tocopherol 3.62 2.82 3.10 4.81 3.33

Values are expressed as percentages.

Appendix 7: Abundances of compounds in hexane extract of Karuri (%)

Compound Kar1 Kar2 Kar3 Kar4 Kar5 Campesterol 0.71 0.84 3.50 1.45 0.85 Lauric acid 0.07 0.09 0.11 1.77 5.48 β-sitosterol 61.34 59.38 52.74 49.15 47.92 Lup-20(29)-en-3-one 3.29 2.89 6.22 2.10 2.33 Palmitic acid 4.78 7.31 9.37 17.09 16.68 Squalene 10.55 9.96 9.70 8.37 6.69 β-sitostenone 7.67 7.52 6.34 6.02 5.92 (3.β.,5.α)-Stigmast-7- 2.90 3.65 2.79 5.65 2.36 en-3-ol Stigmastan-3,5-diene 7.91 7.83 6.95 4.93 6.99 Myristic acid 0.44 0.22 1.83 2.44 3.96 α-Tocopherol 0.35 0.30 0.45 1.04 0.82

Values are expressed as percentages.

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Appendix 8: Abundances of compounds in hexane extracts of Kobujoi (%) Compound Kob1 Kob3 Kob4 Kob5 Kob6 Campesterol 2.45 1.20 1.83 3.20 1.53 Lauric acid 0.30 0.23 0.11 0.31 0.21 β-sitosterol 33.42 64.93 34.51 65.70 61.54 Lup-20(29)-en-3-one 2.87 2.37 2.39 2.39 2.02 Palmitic acid 23.89 3.83 27.17 2.36 10.50 Squalene 9.42 5.46 11.49 6.76 6.79 β-sitostenone 11.00 7.50 7.92 8.08 7.02 (3.β.,5.α)-Stigmast-7- 4.49 3.97 3.29 3.13 1.93 en-3-ol Stigmastan-3,5-diene 10.17 7.10 9.84 6.17 5.70 Myristic acid 0.48 0.90 0.42 0.82 0.82 α-Tocopherol 1.50 2.50 1.02 1.09 1.94

Values are expressed as percentages

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Appendix 9: Abundances of compounds in DCM extracts of Muguga (%)

Compound T223 T9115 T9123 T18249 T20276 Campesterol 3.55 3.36 2.63 3.61 2.25

Lauric acid 0.40 0.11 0.26 0.22 0.36 β-sitosterol 22.24 29.23 33.51 26.02 38.13 Lup-20(29)-en-3- 4.73 3.69 4.11 3.42 2.09 one Palmitic acid 31.70 23.43 27.57 35.13 22.52 Squalene 11.41 11.72 4.50 6.32 8.25 β-sitostenone 9.31 11.33 11.27 9.57 10.24 (3.β.,5.α)- 3.43 4.53 4.53 4.69 4.41 Stigmast-7-en-3- ol Stigmastan-3,5- 9.44 8.34 7.96 8.19 9.42 diene Myristic acid 2.12 2.13 1.56 1.29 1.17

α-Tocopherol 1.67 2.13 2.10 1.55 1.17 Values are expressed as percentages.

Appendix 10: Abundances of compounds in DCM extracts of Karuri (%)

Compound Kar1 Kar2 Kar3 Kar4 Kar5 Campesterol 3.41 3.35 2.12 2.09 2.40 Lauric acid 0.55 1.19 0.68 0.64 0.22 β-sitosterol 32.54 36.21 37.66 37.53 36.02 Lup-20(29)-en-3-one 4.66 4.00 2.78 2.82 3.98 Palmitic acid 15.09 22.49 24.92 24.65 24.92 Squalene 9.07 7.45 8.28 8.19 5.92 β-sitostenone 10.56 8.70 8.55 8.46 10.86 (3.β.,5.α)-Stigmast-7- 5.93 2.73 3.70 3.66 4.78 en-3-ol Stigmastan-3,5-diene 13.73 10.11 7.85 8.17 7.69 Myristic acid 1.16 1.24 1.44 0.60 0.68 α-Tocopherol 3.29 2.53 2.03 3.20 2.52 Values are expressed percentages.

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Appendix 11: Abundances of compounds in DCM extracts of Kobujoi (%)

Compound Kob1 Kob3 Kob4 Kob5 Kob6 Campesterol 2.14 1.99 2.29 2.37 2.99 Lauric acid 0.43 0.72 0.39 0.24 0.46 β-sitosterol 35.26 25.75 46.65 31.98 37.06 Lup-20(29)-en-3-one 1.21 1.86 2.25 4.38 2.84 Palmitic acid 18.90 39.58 17.21 21.96 21.71 Squalene 11.19 5.74 6.00 9.44 8.89 β-sitostenone 9.87 6.04 8.90 10.98 9.98 (3.β,5.α)-Stigmast-7- 4.12 8.42 5.37 4.70 3.87 en-3-ol Stigmastan-3,5-diene 12.74 6.89 6.92 9.18 7.03 Myristic acid 0.87 0.74 0.38 1.32 1.22 α-Tocopherol 3.27 2.27 3.64 3.44 3.96

Values are expressed as percentages.

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Appendix 12: Total ion chromatochram of DCM extracts

Campesterol (1), lauric acid(2), β-sitosterol (3), lup-20(29)-en-3-one (4), palmitic acid (5), β-sitostenone (7), (3.β.,5.α)-stigmast-7-en-3-ol (8), stigmastan-3,5-diene

(9), myristic acid (10), α-Tocopherol (11), internal standard (IS)

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Appendix 13: Abundances of compounds in methanol extract of Muguga (%)

Compound T223 T9115 T9123 T18249 T20276 Feruloyl-quinic acid 3.87 4.73 2.51 4.87 2.69 Chlorogenic acid 2.98 4.05 4.61 5.46 2.75 Isoliquiritin 20.42 14.45 13.72 11.52 15.57 Prunetrin 4.77 1.49 2.93 2.67 1.04 Cyanidin-o-galactoside 15.94 15.36 19.15 21.54 24.59 Ursolic acid 2.98 1.67 1.59 6.25 10.59 Unknown compound 1 26.08 39.79 39.54 31.87 21.54 Procyanidin B5 2.03 3.38 2.09 1.13 4.38 Quercetin3,3'- 3.89 1.92 1.30 2.00 2.53 dimethylether-4'- glucoside Robinetinidol-(4-α-8)- 2.90 1.04 1.32 1.63 1.69 catechin-(6-α)-robinetinol Unknown compound 2 10.47 7.56 6.83 8.38 9.83 Cinnamtannin A2 1.35 1.20 1.63 1.34 1.11 Isochamaejasmin+ 2.32 3.36 2.79 1.34 1.69 Values are expressed as percentages. 104

Appendix 14: Abundances of compounds in methanol extracts of Karuri (%) Compound Kar1 Kar2 Kar3 Kar4 Kar5 Feruloyl-quinic acid 2.54 4.35 4.05 4.43 5.12 Chlorogenic acid 2.37 3.14 2.76 3.22 7.12 Prunetrin 3.57 5.10 6.38 6.59 5.54 Cyanidin-o-galactoside 19.87 20.14 21.18 21.80 17.41 Ursolic acid 1.13 1.85 2.01 1.83 0.68 Unknown compound 1 24.78 20.18 22.23 20.98 28.10 Procyanidin B5 0.74 1.89 1.88 2.36 0.90 Cyanidin-3-o-rutinoside 26.23 24.75 20.45 21.24 17.44 Quercetin3,3'-dimethylether- 1.13 1.60 0.77 1.21 1.06 4'-glucoside Robinetinidol-(4-α-8)- 9.68 9.17 10.15 8.52 7.63 catechin-(6-α)-robinetinol Unknown compound 2 6.49 6.55 6.59 6.35 7.78 Cinnamtannin A2 1.49 1.28 1.54 1.47 1.22

Values are expressed as percentages. 105

Appendix 15: Abundances of methanol extract compounds of Kobujoi (%) Compound Kob1 Kob3 Kob4 Kob5 Kob6 Feruloyl-quinic acid 4.54 1.14 2.80 4.43 2.04 Chlorogenic acid 4.05 2.76 2.33 2.60 2.28 Isoliquiritin 8.78 9.18 8.53 7.74 11.15 Prunetrin 0.96 1.84 1.90 1.62 0.70 Cyanidin-o-galactoside 6.80 9.24 8.81 8.26 11.76 Ursolic acid 1.45 2.52 1.54 1.64 2.40 Unknown compound 1 9.31 9.39 8.58 7.97 6.12 Procyanidin B5 5.35 2.70 3.72 4.87 1.27 Quercetin3,3'-dimethylether- 22.15 24.40 23.69 22.30 30.40 4'-glucoside Robinetinidol-(4-α-8)- 4.04 4.36 4.67 8.77 2.50 catechin-(6-α)-robinetinol Unknown compound 2 8.87 8.36 8.74 8.26 1.28 Cinnamtannin A2 2.81 2.97 2.97 2.74 2.11 Isochamaejasmin+ 20.89 21.13 21.74 18.81 26.00

Values are expressed as percentages. 106

Appendix 16: Abundances of aqueous extract compounds in Muguga (%)

Compound T223 T9115 T9123 T18249 T20276 Feruloyl-quinic acid 12.00 3.67 2.25 5.08 4.32 Chlorogenic acid 1.29 1.77 0.23 1.30 4.70 Isoliquiritin 21.82 18.40 14.61 14.17 13.99 Prunetrin 2.95 2.09 0.66 0.83 1.59 Cyanidin-o-galactoside 1.08 0.70 0.48 0.82 4.41 Ursolic acid 1.12 16.40 13.33 13.97 14.47 Unknown compound 1 14.02 6.15 15.46 12.37 11.79 Procyanidin B5 3.85 6.71 7.52 2.71 2.05 Cyanidin-3-o-rutinoside 5.16 11.48 21.13 17.53 15.32 Quercetin3,3'- 10.10 7.49 6.51 6.25 3.89 dimethylether-4'-glucoside Robinetinidol-(4-α-8)- 1.78 0.49 0.96 1.55 1.53 catechin-(6-α)-robinetinol Unknown compound 2 19.97 21.54 14.21 22.57 19.95 Cinnamtannin A2 4.87 3.12 2.66 0.85 1.98

Values are expressed as percentages

Appendix 17: Abundances of aqueous extract compounds of Karuri (%)

Compound Kar1 Kar2 Kar3 Kar4 Kar5 Chlorogenic acid 2.87 6.61 2.56 2.01 2.82 Isoliquiritin 22.11 22.45 27.34 28.27 26.43 Cyanidin-o-galactoside 3.60 3.29 2.23 2.12 3.20 Ursolic acid 18.19 17.73 17.86 17.09 9.15 Unknown compound 1 11.22 10.70 6.94 8.29 11.13 Procyanidin B5 5.13 4.38 6.34 6.40 6.28 Cyanidin-3-o-rutinoside 25.11 22.90 27.34 28.27 31.35 Quercetin3,3'-dimethylether- 2.97 2.36 1.91 1.62 3.49 4'-glucoside Robinetinidol-(4-α-8)- 4.76 5.15 5.00 4.49 3.74 catechin-(6-α)-robinetinol Cinnamtannin A2 4.04 4.43 2.47 1.45 2.41 Values are expressed percentages

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Appendix 18: Abundances of aqueous extract compounds in Kobujoi (%)

Compound Kob1 Kob3 Kob4 Kob5 Kob6 Chlorogenic acid 5.37 9.29 9.40 9.78 9.17 Isoliquiritin 14.56 14.09 14.43 14.71 17.41 Cyanidin-o-galactoside 5.52 4.20 4.87 4.68 4.25 Ursolic acid 23.31 25.48 24.54 24.15 21.57 Unknown compound 1 5.54 5.51 5.48 5.46 4.68 Procyanidin B5 10.07 9.77 10.20 9.60 8.75 Cyanidin-3-o-rutinoside 15.38 14.65 14.43 14.71 18.06 Robinetinidol-(4-α-8)- 3.90 2.70 3.32 3.08 2.94 Catechin-(6-α)-robinetinol Unknown compound 2 10.09 9.84 9.09 9.62 9.13 cinnamtannin A2 6.27 4.45 4.23 4.22 4.04 Values are expressed as percentages

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Appendix 19: Mass spectrum for β-Sitosterol

β-sitosterol has a molecular weight of 414. The MS (Figure 4.3) displayed a molecular ion peak at 414 [90%, M+]. The base ion peak occurred at m/z 414

[90%, M].

109

Appendix 20: Mass spectrum for campesterol

Campesterol has a molecular weight of 400. The MS (Figure 4.4) displayed a molecular ion peak at 400 [25%, M+]. The base ion peak occurred at m/z 400

[25%, M].

110

Appendix 21: Mass spectrum for Myristic acid

Myristic acid has a molecular weight of 228. The MS (Figure 4.5) displayed a molecular ion peak at 228 [48%, M+]. The base ion peak occurred at m/z

228[48%, M].

111

Appendix 22: Mass spectrum for stigmastan-3,5-diene

Stigmastan-3,5-diene has a molecular weight of 396. The MS (Figure 4.6) displayed a molecular ion peak at 396 [60%, M+]. The base ion peak occurred at m/z 396[60%, M].

112

Appendix 23: Mass spectrum of Quercetin-3,3'-dimethylether-4'-glucoside

Quercetin-3,3'-dimethylether-4'-glucoside has a molecular weight of 510. The MS displayed a molecular ion peak at 511.146 [100%, M+]. The base ion peak occurred at m/z 511.146[100%]. Analysis was done with the positive mode thus

M is 510.

113

Appendix 24: Mass spectrum of procyanidin B5

Procyanidin B5 has a molecular weight of 578. The MS (Figure 4.8) displayed a molecular ion peak at 579.15 [100%, M+]. The base ion peak occurred at m/z

579.15 [100%]. Analysis was done in the positive mode thus M is 578. 114

Appendix 25: Mass spectrum of prunetrin

Prunetrin has a molecular weight of 468. The MS (Figure 4.9) displayed a molecular ion peak at 469.13 [100%, M+]. The base ion peak occurred at m/z

469.13 [100%]. Analysis was done in the positive mode thus M is 468. 115

Appendix 26: Mass spectrum of chlorogenic acid

116

Appendix 27: Mass spectrum and structure of linoleic acid

117

Appendix 28: Mass spectrum and structure of methyl linoleate

Appendix 29: Mass spectrum of methyl laurate

118

Appendix 30: Mass spectrum of and structure methyl myristate

119

Appendix 31: Mass spectrum and structure of squalene

Appendix 32: Mass spectrum and structure of palmitic acid

120

Appendix 33: Mass spectrum and structure of β-sitostenone

Appendix 34: Mass spectrum of α-tocopherol

121

Appendix 35: Mass spectrum and structure of (3β, 5α)-stigmast-7-en-3-ol

122

Appendix 36: Mass spectrum of cyanidin-3-o-rutinoside

123

Appendix 37: Mass spectrum of 3-o-feruloyl-quinic acid

124

Appendix 38: Mass spectrum of isochamaejasmin+

125

Appendix 39: Mass spectrum of cyanidin-o-galactoside

126

Appendix 40: Mass spectrum of ursolic acid

127

Appendix 41: Mass spectrum of robinetinidol-(4-alpha-8)-catechin- (6,4alpha)-robinetinol

128

Appendix 42: Mass spectrum of cinnamtannin A2

129

Appendix 43: Muguga Prunus africana stand layout

KEY Yellow = border row Red cells = trees to be removed or already dead Green cells = control plots White cells = trees to remain Blue cells = for debarking trials O=Tree still present/alive X=Tree dead or absent Total trees alive=273

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131