oil content and physicochemical characteristics of oils

OIL CONTENT AND PHYSICOCHEMICAL CHARACTERISTICS OF OILS FROM WILD
PLANTS OF KIVU REGION, DEMOCRATIC REPUBLIC OF CONGO
BY
KAZADI MINZANGI
BSc /ANALYTICAL CHEMISTRY/UNIVERSITY OF KINSHASA, D.R. CONGO
A DISSERTATION SUBMITTED TO MAKERERE UNIVERSITY IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF A DEGREE OF
MASTER OF SCIENCE IN ENVIRONMENT AND NATURAL RESOURCES OF
MAKERERE UNIVERSITY
NOVEMBER 2009
1

DECLARATION
I, KAZADI MINZANGI, declare that this work is entirely the product of my own findings and has
never been presented for any academic award.
Sign………………………………………… Date: November …. , 2009
KAZADI MINZANGI
Signed………………………………………. Date: November …. , 2009
DR. ARCHILEO N. KAAYA
Department of Food Science and Technology,
Makerere University
Signed……………………………………. Date: November …. , 2009
PROF. FRANK KANSIIME
Institute of Environment and Natural Resources,
Makerere University
Signed……………………………………. Date: November …. , 2009
DR. JOHN R.S. TABUTI
Institute of Environment and Natural Resources,
Makerere University
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DEDICATION
To my wife Esther MUSHIYA and my dear children Nathan, Nathalie, Mireille, Joël, David, Elisé,
Josué, Esaie and Israël.
3

ACKNOWLEDGEMENT
I thank the Belgian Technical Cooperation of Kinshasa for the fellowship that enabled me to do my
Masters studies at Makerere University. I thank also MacArthur Foundation for additional funding to
cover research expenses. This work would not have been possible without the generous support of
numerous people and institutions. I am glad to extend my profound and cordial gratitude to my
Supervisors Dr. Archileo N. Kaaya, Prof. Frank Kansiime and Dr. John R.S. Tabuti for their skilled
advice and sympathetic attitude during my research work. I also extend my thanks to the following:
Prof. Bashwira Samvura of Department of Chemistry, Insititut Supérieur Pédagogique de Bukavu, in
D.R. Congo for his advices on the work in the home country; Prof. Otto Grahl-Nielsen of
Department of Chemistry, University of Bergen, in Norway for experimental work in gas
chromatography for fatty acids analysis; The Staff of CRSN/Lwiro for all opportunities for the work
in the laboratory of CRSN and all the social and administrative supervision; my laboratory
assistants, Mr. Zabona Muderhwa and Mr. Ndatabaye Lagrissi for their contribution in the field and
the laboratory; Mr. Justus Kwetegyeka, Lecturer to Department of Chemistry of Makerere
University for his valuable comments on my proposal. Finally, thanks to the numerous friends I
made and sympathetic people I met in Uganda who made my stay enjoyable.
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TABLE OF CONTENTS
DECLARATION...……………...………………………………………………………….. i
DEDICATION.....…………………….……………………………………………………... ii
ACKNOWLEDGEMENT ...…...…………………………………………………………… iii
TABLES OF CONTENT.....................................................................….…..……………….. iv
LIST OF TABLES .................................................................................….…..……………….. ix
LIST OF FIGURES ………………………………………………………..…....…..………... x
LIST OF ACRONYMS ………………………………………………....…....…..…………. xi
ABSTRACT...……………………………………………………………....………..………. xii
CHAPTER ONE: INTRODUCTION ……………………………...……………………… 1
1.1. Background...………...………………………………………………………………..…. 1
1.2. Problem Statement ...……………..….……………………………………………..…… 2
1.3. Objectives........................................................................................................................... 3
1.3.1. Overall objective ……………………………………………………………..…………
I.3.2. Specific Objectives ……………………………………..……………………..………. 3
1.4. Hypotheses ………………..……………………………..……………………...……… 3
1.5. Significance of the study.....……………..…………..………………………………….. 4
CHAPTER TWO: LITERATURE REVIEW……………………............................................. 5
2.1. Oil structure ......……..…………………………………………………………………... 5
2.2. Importance of oils.………………………………………………………………………… 5
2.3. Physicochemical Characteristics of plant oil and fatty acid composition ……………….. 7
2.3. 1. Specific gravity ...……………………………………….…………………………….. 8
2.3.2. Melting point ……………………………………………………......……….………… 8
5
3

2.3.3. Saponification value ................................................................……….…………………. 9
22.3.4. Unsaponifiable matter …………….………………………………………………… 9
2.3.5. Oil acidity........................................................................................................................... 9
2.3.6. Fatty acids.....…................................................................................................................. 10
2.3.6.1. Fatty acids nomenclature …….………………………………………………………. 11
2.3.6.2. Fatty acids composition of plant oils ………………………………………………… 13
2.4. A review of wild oil plants that have been studied in this project …………….…….…… 15
2.4.1. Carapa grandiflora Sprague (Meliaceae)...................................................................... 15
2.4.2. Carapa procera DC. (Meliaceae) ...………………………………………………… 15
2.4.3. Cardiospermum halicacabum Linn (Sapindaceae)...…………………………………. 15
2.4.4. Maesopsis eminii Engler (Rhamnaceae).....…………………………………….….…. 16
2.4.5. Millettia dura Dunn (Fabaceae)… …………………………………………………… 17
2.4.6. Myrianthus arboreus P. Beauv. (Cecropiaceae)… …………………………………… 17
2.4.7. Myrianthus holstii Engl. (Cecropiaceae)...….…….………….. ……………………… 17
2.4.8. Pentaclethra macrophylla Benth (Mimosaceae) .......................................................… 18
2.4.9. Podocarpus usambarensis Pilger (Podocarpaceae) ………………………………...…. 19
2.4.10. Tephrosia vogelii Hook. (Fabaceae) ......…………………………………………… 19
2.4.11. Treculia africana Decne (Moraceae)...………….……..…………………………… 19
CHAPTER THREE: MATERIALS AND METHODS …..……………………………….. 22
3.1. Plant collection ………....……………………………………………………………….. 22
3.2. Laboratory analysis.…...………………………………………………………………… 24
3.2.1. Extraction of oil …………………………………………… ………........…………… 24
3.2.1.1. Extraction procedure description …………………………………………………… 25
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3.2.2. Plant seed oil content determination…………………………………………………… 26
3.2.3. Determination of physical and chemical characteristics of oils.…………………
3.2.3.1. Specific gravity ……………………………….....………………………………… 27
3.2.3.2. Melting point ………………………………………….………..………………… 28
3.2.3.3. Saponification value………………………....…………….……………………… 28
3.2.3.4. Percentage of unsaponifiable matter….…………………………………………… 29
3.2.3.5. Acidity index ….. ………..………………………………………………………… 29
3.3. Identification and quantification of fatty acids ……………………………………… 30
3.4. Data analysis …………………....……………………………………………………….. 32
CHAPTER FOUR: RESULTS....………………………………………………………… 33
4.1. Plant seed oil content ....………………………………………………………………… 33
4.2. Physical and chemical characteristics of oils.....………………………………………… 34
4.2.1. Specific gravity ....…………………………………………………………………….. 34
4.2.2. Melting point ....………………………………………………………………….……. 34
4.2.3. Saponification value …………………………………………………………………. 35
4.2.4. Oil unsaponifiable matter content …………….………….………………….……….. 36
4.2.5. Acidity index……… ………………………………………………………………….. 37
4.3. Plant oil fatty acid composition ...….………..…………………………………………. 38
4.3.1. Saturated fatty acids ……......... ……………………………………………………… 40
4.3.2. Unsaturated fatty acids …….....…………...………………………………………… 41
4.3.3. Omega–6 (ω-6) and Omega–3 (ω-3) fatty acids ….....……………………………… 41
4.3.4. Long Chain Fatty acids.....………………………………………………………..…… 42
CHAPTER FIVE: DISCUSSION ….....……..………………………..…………………… 42
7
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5.1. Seed oil content of plants species ...……………………………………………….……. 42
5.2. Physicochemical characteristics of oils ..........................................................................… 43
5.2.1. Specific gravity ……. ....…………………………………………………………….….. 43
5.2.2. Melting point.....……………………………………………………………………..… 44
5.2.3. Saponification value ....…..…………………………………………………………..… 45
5.2.4. Oil unsaponifiable matter content...………………………………….………..……..… 45
5.2.5. Oil acidity …………………………………………………………...……………....... 47
5.3. Plant oil fatty acid composition ...…..……………………………………………..…… 48
5.3.1. α-linolenic acid .........................……………………………………………………… 48
5.3.2. Linoleic acid …………………………………………………………………............. 48
5.3.3. Oleic acid …………………………………………….....…………………………… 49
5.3.4. Saturated fatty acids …….. ....……………………………………………………….... 50
5.3.5. Unsaturated fatty acids …….. ...………….………………………………………….. 50
5.3.6. Omega–6 (ω-6) and Omega–3 (ω-3) fatty acids …….………………………………. 51
5.3.7. Long Chain Fatty acids and Chemotaxonomy Relevant ……………………………… 51
CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS ……………………. 54
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6.1. Conclusions ……………………………………………………………………………. 54
6.2. Recommendations ……………………………………………………………………… 56
REFERENCES ………………. ………………….………………………………………… 58
APPENDICES ………………………………………….……………………..…..……… 69
Appendix 1: Analysis of variance of seed oil content and physicochemical characteristics of
oils from plant species obtained from Kahuzi-Biega National Park and the surrounding areas
in D.R. Congo ………………………………………………………………………………… 69
Appendix 2: Gas Chromatogram of fatty acids methyl esthers of oils from plant species
obtained from Kahuzi-Biega National Park and the surrounding areas in D.R. Congo …… 70
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LIST OF TABLES
Table 1: Oil content and characteristics of oils from some crops plants ……………………… 10
Table 2: Names and descriptions of some fatty acids found in biological materials ………… 12
Table 3: Fatty acid composition of some crops plants ………………………………………… 14
Table 4: Review of oil content and oil characteristics of studied species …………………….. 20
Table 5: Review of oil fatty acid of studied species …………………………………………… 21
Table 6: Wild oil plants identified from Kahuzi-Biega National Park and the surrounding areas 24
Table 7: Chromatographic Equipment and settings …………………………………………… 31
Table 8: SG at 40 and 30 O C of oils from plants obtained from Kahuzi-Biega National Park and
the surrounding areas …………………………………………………………………………… 34
Table 9: Melting point ranges of oils from plants obtained from Kahuzi-Biega National Park
and the surrounding areas ……………………………………………………………………… 35
Table 10: Fatty acid composition of oil from plants of Kahuzi-Biega National Park and
surrounding areas ………………………………………………………………………………. 39
Table 11: Saturated FAs, Monounsaturated FAs, Polyunsaturated FAs, LCFA and Omega FAs
content in oils of plants from Kahuzi-Biega National Park and surrounding areas in D.R.
Congo.………………………………………………………………………………. 41
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LIST OF FIGURES
Figure 1: Location of sampling sites (Irangi, Lwiro, Mugeri and KBNP/Tshibati) in Kahuzi-
Biega National Park and the surrounding areas……………..……………………………… 23
Figure 2: Photography and representation of a Soxhlet extractor…………………………… 26
Figure 3: Schematic representation of a pycnometer ………………………………………… 27
Figure 4: Seed oil content of plant species from Kahuzi-Biega National Park and surrounding
areas…………………………………………………………………………………………. 33
Figure 5: Saponification values of oils from plants of Kahuzi-Biega National Park and
surrounding areas…………………………………………………………………………… 36
Figure 6: Unsaponifiable matter of oils of plants obtained from Kahuzi-Biega National Park
and surrounding areas……………………………………………………… 37
Figure 7: Acidity Index of oils of plants obtained from Kahuzi-Biega National Park and
surrounding areas……………………………………………………………………………… 38
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AI Acidity index
ALA α-linolenic acid
AOCS American Oil Chemists Society
LIST OF ACRONYMS
CRSN Centre de Recherche en Sciences Naturelles (Lwiro)
DHA Docosahexaenoic acid
EFA Essential fatty acid
EPA Eicosapentaenoic acid
FA Fatty acids
FAME Fatty acid methyl ester
KBNP Kahuzi-Biega National Park
LCFA Long-chain fatty acid
MUFA Monounsaturated fatty acid
PUFA Polyunsaturated fatty acid
SFA Saturated fatty acid
SG Specific gravity
SV Saponification value
UFA Unsaturated fatty acid
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ABSTRACT
Many important plant species in Kahuzi-Biega National Park and surrounding areas in Kivu Region,
Democratic Republic of Congo are threatened with extinction. Some of theses plants are harvested
and their oil extracted and used as medicines or as food. Many of theses plants have not been
evaluated to determine their oil content and its characteristics. This study was undertaken to
determine the oil content and its characteristics for selected 11 wild plants species in Kivu Region,
D.R. Congo. These were Carapa grandiflora, Carapa procera, Cardiospermum halicacabum,
Maesopsis eminii, Millettia dura, Myrianthus arboreus, Myrianthus holstii, Pentaclethra
macrophylla, Podocarpus usambarensis, Tephrosia vogelii and Treculia africana. The oils were
extracted using ethyl ether in Soxhlet extractor. To determine the oil physicochemical characteristics
the methods of the American Oil Chemists Society were used. The identification and quantification
of the fatty acids were undertaken using Gas Chromatography.
The seed oil content of these 11 species ranged from 17.2 to 64.4%. The highest oil content was
obtained from P. usambarensis and the lowest from T. vogelii. The oil specific gravity varied from
0.8050 to 0.9854; melting point from -12 to 32 O C; saponification values from 182.5 to 260.9 mg
KOH/g; acidity index from 1.74 to 5.31 mg KOH/g and the unsaponifiable matter from 0.54 to
2.25%. Twenty four fatty acids were found and 18 of these were identified including α-linolenic
acid, linoleic acid, oleic acid, stearic acid and palmitic acid. There were also remarkable occurrences
of long chain fatty acids particularly lignoceric acid (9.8% in P. macrophylla oil) and behenic acid
(7.3% in M. dura oil, 6.3% in P. macrophylla oil and 5.8% in T. vogelii oil).
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The plant seed oil contents reported in this study are high compared to some food crops such as
soybean and olive seed. The oils of the plants studied here have potential for use in food especially
M. eminii, P. usambarensis and T. vogelii oils because of their essential fatty acids content; M.
arboreus and M. holstii oils due to their high linoleic acid content; M. eminii oil because it has fatty
acid with similarities with to that of groundnut oil and C. procera oil which was found to be more
stable as deep frying oil. Some of the oils from the studied wild plants showed good promise in use
in the cosmetic industry and particularly most promising are C. grandiflora, C. procera, M.
arboreus, M. holstii and P. usambarensis seed oil due to their fatty acids profile and high
unsaponifiable matter content.
These plant oils may also have good application as biofuels and the most promising are those of high
density and relative low melting point as T. vogelii, T. africana, M. arboreus, M. holstii, C.
grandiflora, M. dura and C. procera. P. macrophylla, P. usambarensis, M. dura and T. vogelii seed
oils may be sources for long chain fatty acids which have important chemotaxonomic significance.
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1.1. Background
CHAPTER ONE
INTRODUCTION
Many important plant species in Kahuzi-Biega National Park (KBNP) and surrounding areas in the
Democratic Republic of Congo (D.R.C.) are threatened with extinction (Kaleme et al., 2007). The
threats towards these species include habitat conversion and degradation, and unsustainable uses of
the species (Kasereka, 2003). In order to conserve these species, there is need to highlight other
important sustainable exploitations of these species by documenting and validating their uses
(Tabuti, 2003).
One of the important uses of plants is in the production of oil. Oil is a very important resource, much
in demand everywhere in the world and is used in a variety of ways (Pryde and Carlson, 1985). The
sources of oils and fats are diminishing, this means therefore that there is a growing need for the
search of new sources of oil as well as exploiting sources that are currently unexploited in order to
supplement the existing ones (Mohammed et al., 2003). Furthermore, the price of edible oil is
increasing due to the effects of turning edible oil into energy sources (Nyapendi, 2008). Similarly the
biodiesel market is growing (Pioch and Vaitilingom, 2005). The oil for nutritional use and derivate
products like soaps, cosmetics and medicinal products have become more unaffordable for most
people because the sources of these products are small and limited (ABC, 2003).
The world yearly production of oils exceeds 120 millions tons of which about 4/5 are devoted to
food uses, the remainder is used for non food uses, for example in making animal feeds, soap,
15

oleochemicals (Pioch and Vaitilingom, 2005). Plant seeds are important sources of oils of
nutritional, industrial and pharmaceutical importance. The suitability of oil for a particular purpose,
however, is determined by its characteristics and fatty acid (FA) composition (Alvarez and
Rodríguez, 2000). No oil from any single source has been found to be suitable for all purposes
because oils from different sources generally differ in their FA composition (Dagne and Jonsson,
1997).
In previous studies conducted in and around Kahuzi-Biega National Park, DRC more than 40 oil
producing plant species were identified (Kazadi, 1999; 2006). Oil content from a few of these
species was determined (Kazadi, 1999). In this study I build on this past work by determining oil
content, specific gravity, melting point, saponification value, percentage of unsaponifiable, acidity
and FA composition for eleven plant species: Carapa grandiflora, Carapa procera, Cardiospermum
halicacabum, Maesopsis eminii, Millettia dura, Myrianthus arboreus, Myrianthus holstii,
Pentaclethra macrophylla, Podocarpus usambarensis, Tephrosia vogelii and Treculia africana.
1.2. Problem Statement
Many plants have been identified as sources of oil in South Kivu, DRC (Kazadi, 1999; 2006). Some
of the plant species in Kahuzi-Biega National Park and the surrounding forests are harvested and
their oil extracted and used as medicines and food (Kasereka, 2003). However, very few of these
species have their oil characteristics determined (Kazadi, 1999). Adriaens (1944) conducted an
inventory of oilseed plants of the D.R. Congo, but this work and others before him were made using
old analytical methods.
16

Kabele et al. (1975) analyzed FAs from plants species of western D.R. Congo and found that some
oils from these plants were rich in lignoceric and cerotic FAs, while others had highly unsaturated
oils. Foma and Abdala (1985) analyzed seed oil from plants species sampled in north D.R. Congo
and found notably that kernel oils of Panda oleosa, Treculia africana and Desplatzia dewevrei could
be used as edible oil sources because of their relatively high percentage of unsaturated FAs, in
particular the linoleic acid. Kazadi and Chifundera (1993) determined different FAs from Afzelia
pachyloba plant harvested in west of D.R. Congo and found that the oil was rich in acetylenic FAs.
The present work adds to the ongoing work to evaluate plant oils in eastern D.R. Congo.
1.3. Objectives
1.3.1. Overall objective
The overall objective of this study was to determine whether some wild plants from Kivu,
Democratic Republic of Congo are a potential source of quality oils that can be utilized for several
purposes.
1.3.2. Specific Objectives
1. To determine the oil content of selected wild plants from Kivu Region, D.R. Congo,
2. To characterize the oils extracted from these wild plants from Kivu Region, D.R. Congo,
3. To come up with a list of oils with suitable characteristics for utilization by both industry and
home consumption.
1.4. Hypotheses
The seeds of wild oil plants in Kivu region produce oil that differs in quantity, fatty acid composition
17

and chemical and physical characteristics.
1.5. Significance of the study
Exploitation of non-timber forest products, particularly fruits and seeds as a source of oil can help to
reduce oil costs by diversifying the sources for this commodity. This form of exploitation can be
more sustainable than timber extraction, because this is often viewed as a means of sustainable forest
management affecting the structure and function of forests much less than other uses (Forget and
Jansen, 2007). Demonstration of tangible economic values can lay the foundation for rational use
and protection of plant resources, because people tend to conserve plants which they know are
important for their needs. This study sought to establish the importance of some local plants from
Kivu Region by assessing their chemical properties in order, to support rural people’s needs in ways
that are in harmony with environment. Data generated from this study will benefit industries for
production of oils for various purposes. The tree species identified as sources of oil could be
proposed for domestication so that they can be used for oil extraction for food and other uses and at
the same time be conserved which is good for the environment. In addition the content and
composition of fatty acids of plant seed oils can serve as plants taxonomic markers (Bağci and
Şahin, 2004).
18

2.1. Oil and fat structure
CHAPTER TWO
LITERATURE REVIEW
Plant seed oils have a wide variety of structures, because oils do not occur in nature as single pure
entities, but rather as complex mixtures of molecular species in which various fatty acids (FAs) and
glycerin are present in different combinations (Christie, 1989). There are many different kinds of
fats, but each is a variation on the same chemical structure. Triglycerides are the main constituents of
vegetable oils. All fats consist of FAs (chains of carbon and hydrogen atoms, with a carboxylic acid
group at one end) bonded to a backbone structure, often glycerol (a "backbone" of carbon, hydrogen,
and oxygen) (Zamora, 2005). Chemically, this is a tri-ester of glycerol, an ester being the molecule
formed from the reaction of the carboxylic acid and an organic alcohol (Gunstone and Herslöf,
2000). Oils are usually from plants while fats are from animal origin (O'Brien, 1998).
2.2. Importance of oils
Many plant oils are used in food, in medicine, cosmetics and as fuels. They are consumed directly,
or are used as ingredients in the preparation of food (O'Brien, 1998). Fat and oil are the most
concentrated kind of energy that humans can use (Odoemelam, 2005). They provide 9 kilocalories
per gram of oil (Gurr, 1999) while the other two types of energy that humans can use i.e.
carbohydrates and proteins provide 4 kilocalories per gram each (Lawson, 1995). The Food and
Agriculture Organization (FAO) and the World Health Organisation (WHO) have listed the
important functions of dietary oils as a source of energy, cell structure and membrane functions,
source of essential FAs, vehicle for oil-soluble vitamins and for control of blood lipids (Alvarez and
Rodriguez, 2000).
19

Yaniv et al. (1999) made an assay of Citrullus colocynthis utilized for oil production, especially in
Nigeria. Its oil contains a large amount of linoleic acid (C18:2) which is important for human
nutrition (Yaniv et al. 1996). Such oil composition resembles safflower oil and is very beneficial in
human diets (Pioch and Vaitilingom, 2005). For treating some conditions, such as rheumatoid
arthritis or diabetic neuropathy, one may try oils high in gamma linolenic acid, such as primrose oil.
Here the oil is used as a medication to treat symptoms of a disease with both positive and negative
effects (Athar and Nasir, 2005). Consuming oils high in polyunsaturated FAs can lower blood
cholesterol levels and thereby decrease the risk of cardiovascular diseases (Dagne and Jonsson,
1997). Some oils have medicinal properties (Aubourg et al., 1993) while others can make excellent
excipients in pharmaceutical and cosmetical preparations (Alvarez and Rodriguez, 2000).
Although many plant oils have good structural values, it is not yet clear whether they can be safely
consumed because toxicity has been associated with some oils. Even the most common commercial
plant oils, such as canola (rapeseed), soybeans, cottonseed or castor oils in their crude form are not
fit for human consumption without further processing (Cˇmolík and Pokorny´, 2000). These
processes include filtration, neutral and physical refining, fractionation, bleaching and deodorizing.
Refining removes undesirable impurities of oil whereas fractionation separates oils and fats on a
commercial scale into two or more components (Gurr, 1999). Fractionation increases oils range of
use, shelf life and adds value (Ferris et al., 2001). The deodorizing makes possible the production of
neutral flavor food product i.e. tasteless and odorless oil (Gunstone and Herslöf, 2000) while the
colorless oil production is the obvious result of bleaching (Lawson, 1995).
Plant oils are used to make soaps, skin products, candles, perfumes and other cosmetic products
20

(Dawodu, 2009; Ferris et al., 2001). High unsaturated oils are suitable as drying agents, and are used
in making paints and other wood treatment products (Giuffre, 1996). They are also increasingly
being used in the electrical industry as insulators since they are non-toxic to the environment,
biodegradable if spilled and have high flash and fire points (Oommen et al., 1999).
Many plant oils have similar fuel properties to those of diesel fuel and may substitute for this fuel,
most significantly as engine fuel or for home heating oil (Schwab et al., 1987). Crop plant oils
already used as fuels include canola, sunflower, soybean and palm oils (Athar and Nasir, 2005).
They can be used in pure form but they are often blended with regular diesel (Pioch and Vaitilingom,
2005).
2.3. Physicochemical characteristics of plant oil and FA composition
To evaluate the suitability of plant oils for a given purpose, it is necessary to determine their
physicochemical characteristics and FA composition (Bettis et al., 1982). Plant oils vary in their
physicochemical properties. These include specific gravity, melting point, saponification value,
percentage of unsaponifiable, acidity and FA composition.
2.3. 1. Specific gravity
The specific gravity (SG) indicates the FAs average molecular weight of oil (Gunstone and Herslöf,
2000). It is the heaviness of a substance compared to that of water, and it is expressed without units
(Eren, 2000). The SG of plant oils is usually about 0.920 at 25°C (Elert, 2000). The SG is
proportional to the FAs mean chain-length of the oil, as the FA chain-length is proportional to the
FA molecular mass. As the temperature increases, the SG of the oil decreases (Lawson, 1995). The
21

common edible oils have SG from 0.88 to 0.94 (Toolbox, 2005) while oils used for fuel range from
0.82 to 1.08 (CSG, 2008).
2.3.2. Melting point
The melting point may contribute to the palatability and appearance of oil for edible products
(Brinkmann, 2000). The complete melting point (Cmp) of oil is the temperature at which solid fat
becomes liquid oil. Oils melt over a range of temperatures, and do not have a sharp melting point
(mpt). However, pure FA has specific mpts that are related to their FA chain length (Holley and
Phillips, 1995). The Cmp is affected by the average chain length of the FAs, the positioning of the
FAs on the glycerol molecule, the relative proportion of saturated to unsaturated FAs and the
processing techniques such as the degree of selectivity of hydrogenation process (Holley and
Phillips, 1995). Many edible oils have Cmp ranging from – 23 to about 2 O C; butter and other fats
range between 28 to 48 O C (Rossel, 1987).
2.3.3. Saponification value
Saponification value (SV) is defined as the number of milligrams of potassium hydroxide required to
saponify 1gram of oil (AOCS, 1993). It is an indicator of molecular weight or size as a function of
the chain lengths of the constituent FAs (Agatemor, 2006). The saponification value of around 195
indicates that oil contains mainly FAs of high molecular mass. For example, the saponification value
of palm oil ranges from 196 to 205, that of olive oil from 185 to 196, linseed oil from 193 to 195,
cotton seed from 193 to 195 and that of soy oil is around 193 (Pearson, 1981). One the other hand
oils having high saponification value (around 300) have mainly FAs of low molecular mass and are
useful for soapmaking (Alabi, 1993).
22

2.3.4. Unsaponifiable matter
The Unsaponifiable matter (USM) of oil contains minor compounds comprising sterols and fat-
soluble vitamins (Ayo et al., 2007). It is a small portion of oil around one percent which is extracted
by organic solvent after the oil is saponified by an alkali (Hartman et al., 1968). Common edible oils
have USM ranging from 0.2 to 2.5% (Rossel, 1987). Oil with high unsaponifiable matter content
have efficacy as skin products (Alvarez and Rodríguez, 2000)
2.3.5. Oil acidity
The acidity of oil is given by the quantity of FAs derived from the hydrolysis of the triglycerides i.e.
separation between FA and the glycerol in the triglyceride (Gurr, 1999). This alteration occurs under
unsuitable conditions of treatment and preservation of the oil. The oil acidity, can therefore,
indicates the purity of the oil (Pérez-Camino et al., 2000). The oil acidity is expressed either as the
percentage of free FAs or in terms of the number of milligrams of KOH required to neutralize one
gram of sample (mg KOH/g) (Gunstone and Herslöf, 2000). Numerically the acidity of ordinary fats
and oils is approximately twice the percentage of free FAs (Dawodu, 2009).
Most unrefined oils contain high levels of acidity (Pioch and Vaitilingom, 2005), e.g. soybean oil
when unrefined or in crude form has an acidity level ranging between 1.2 and 2.8 mg KOH\g
(Orthoefer and List, 2007) and crude palm oil between 6 and 12 mg KOH/g (Egbe et al., 2000). Oils
required for use in food have an acidity level less than 0.1 mg KOH/g (FAO, 1993) whereas a high
acidity is preferred in biofuels (Pioch and Vaitilingom, 2005). The oil content and physicochemical
characteristics of oils for some common plant crops are shown in Table 1.
23

Table 1: Oil content and characteristics of oils from some crops plants
Oil source Oil
%
Mp ( O C) SV Uns. % Oil acidity Reference
Canola 30 -9 168-181 0.2-2.0 - Rossel, 1987
Cocoa - 33 188-198 0.1-1.2 - Rossel, 1987
Coconut 35.3 24 248-265 0-0.5 - Rossel, 1987
Corn 4.0 -11 187-193 0.5-2.8 - Rossel, 1987
Cotton 36 0 193-195 0.2-1.5 - Rossel, 1987
Grape - -10 188-194 - - Rossel, 1987
Olive ? -1 185-196 0.7-1.5 - Rossel, 1987
Palm - 37 196-205 0.3-1.2 6 -12 Egbe et al., 2000;
Pearson, 1981
Palm kernel 40 25 230-254 0.2-0.8 - Rossel, 1987
Peanut 49 - - - - Rossel, 1987
Safflower 59 -15 186-198 0.3-1.3 - Rossel, 1987
Sesame 49 - 2 187-195 0.9-2 - Rossel, 1987
Soybean 17.7 - 21 188-195 0.5-1.6 1.2– 2.8 Orthoefer and List,
2007; Rossel, 1987
Sunflower 44 -17 188-194 0.3-1.3 - Rossel, 1987
2.3.6. Fatty acids
FAs are composed of carbon, hydrogen and oxygen arranged in a carbon chain skeleton with a
carboxyl group (-COOH) at the alpha position. FAs in biological systems usually contain an even
number of carbon atoms, typically between 8 and 24. The FAs with 16- and 18-carbons are more
frequent (GCRL, 2008). Fatty acids differ from each other by the number of carbon atoms and the
number and placement of their double bonds. There are three classes of FAs: Saturated FA (SFA),
monounsaturated FA (MUFA) and polyunsaturated FA (PUFA) (Christie, 1989). SFAs have carbon
atoms containing all the hydrogen atoms that they can hold. MUFAs have carbon chain containing
one double bond. PUFAs contain two or more double bonds.
24

There are two families of polyunsaturated FAs, the omega-3 and the omega-6 family (NCPA, 2006).
FAs have many physiological roles (Gurr, 1999). Essential FAs (EFA) are those polyunsaturated
FAs that are required in the human diet for growth and proper functioning of the body (Erasmus,
1993). They include omega-3 FA such as α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) (Wijendran and Hayes, 2004). Long-chain FAs (LCFA) are FAs
having 20 or more carbons in their chains as the case of arachidonic (20:4n6) and docosapentaenoic
(22:5n3) acids (Simopoulos, 1998).
2.3.6.1. Fatty acid nomenclature
In chemical nomenclature the carbon of the carboxyl group is carbon number one. Greek numeric
prefixes such as di, tri, tetra, penta, hexa, etc., are used as multipliers and describe the length of
carbon chains containing more than four atoms. Thus, "9,12-octadecadienoic acid" indicates that this
is an 18-carbon chain (octa-deca) with two double bonds (di-en) located at carbons 9 and 12, with
carbon 1 constituting a carboxyl group (oic acid) (Zamora, 2005). FAs are frequently represented by
a notation such as 18:2 that indicates that the FA consists of an 18-carbon chain and 2 double bonds
(Beare-Rogers et al., 2001; Moss, 1976).
In biochemical nomenclature the terminal carbon atom is called the omega (ω) carbon atom. The
term "omega-3 or omega-6" signifies that their double bond occurres at carbon number 3 or 6,
respectively counted from and including the omega carbon. This makes it possible to classify PUFA
in families: Omega-3 and Omega-6. For example the acid eicosapentaenoïc 20:5 ω-3 (omega-3) has
20 carbon atoms and 5 non-saturations (20: 5) and the first non-saturation is on carbon 17 (20 - 3 =
17). Also the arachidonic acid (omega-6) is called “acid 20: 4 ω-6”. The first non-saturation is on
25

C14 carbon (20-6=14). The “ω” can be replaced by a “∆” or “n” (GCRL, 2008). The names and
descriptions of some FAs found in biological materials are presented in Table 2.
Table 2: Names and descriptions of some fatty acids found in biological materials
Common name Scientific name
No. of
double
bonds
Lauric acid Dodecanoic acid 0 12:0
26
Carbons Nr &
Scientific
symbol
Reference
Beare-Rogers et al.,
2001
Myristic acid Tetradecanoic acid 0 14:0 Christie, 1989
Palmitic acid Hexadecanoic acid 0 16:0
Beare-Rogers et al.,
2001
Palmitoleic acid 9-Hexadecenoic acid 1 16:1n-7
Beare-Rogers et al.,
2001
Stearic acid Octadecanoic acid 0 18:0
Beare-Rogers et al.,
2001
Vaccenic Acid 11-Octadecenoic Acid 1 18:1 n-7 Christie, 1989
Oleic acid 9-Octadecenoic acid 1 18:1n-9 Christie, 1989
Linoleic acid
9,12-Octadecadienoic
acid
2 18:2n-6
Christie, 1989
α-linolenic acid
9,12,15-
Octadecatrienoic acid
3 18:3n-3
Christie, 1989
Arachidic acid Eicosanoic acid 0 20:0 Zamora, 2005
Gadoleic Acid 11-eicosenoic acid 1 20:1n-9 Zamora, 2005
Eicosadienoic
Acid
11,14-Ecosadienoic
Acid
2 20:2 n-6
Christie, 1989
Eicosatrienoic
Acid
11,14,17-
Eicosatrienoic Acid
3 20:3 n-3
Christie, 1989
Arachidonic acid
AA
8,11,14,17-
Eicosatetraenoic acid
4 20:4n-3
Zamora, 2005
Arachidonic acid
AA
5,8,11,14-
Eicosatetraenoic acid
4 20:4n-6
Christie, 1989
EPA
5,8,11,14,17-
Eicosapentaenoic acid
5 20:5n-3
Christie, 1989
Behenic acid docosanoic acid 0 22:0 Zamora, 2005
Erucic Acid 13-Docosenoic Acid 1 22:1 n-9 Christie, 1989
DHA
4,7,10,13,16,19-
Docosahexaenoic acid
6 22:6n-3
Christie, 1989
Lignoceric acid tetracosanoic acid 0 C24:0 Zamora, 2005
Nervonic Acid
15-Tetracosaenoic
Acid
1 24:1 n-9
Christie, 1989

2.2.6.2. Fatty acid composition of plant oils
Plant oils’ characteristics are related to their fatty acid (FA) composition (Allena et al., 2004). The
FA composition depends on the sources of the oils. The FA composition of plant oils vary,
depending on factors such as location of plants, growth area, soil conditions and climate (Lawson,
1995). FA compositions of some commonly used edible plant oils have oleic acid ranging from 40 to
about 70%, linoleic acid from 22 to about 50% and linolenic acid from 1 to 10% (Lawson, 1995).
The FAs compositions of some common oils from some crop plants are presented in Table 3.
27

Table 3: Fatty acid composition of some crop plants
Crops name ω6/ω3 C8-C14 Palmitic Stearic Oleic Linoleic ALA
28
Arachidic Reference
Canola Oil 4 - - 7.0 54.0 30.0 7.0 - Erasmus, 1993
Cocoa Butter - - 25.1 36.4 34.1 2.8 0.2 36.4 Dubois et al., 2007
Coconut oil - - 91.0 - 6.0 3.0 - - Erasmus, 1993
Corn Oil - - - 17.0 24.0 59.0 - - Erasmus, 1993
Cottonseed oil - - - 25 21 50 - - Erasmus, 1993
Grape seed oil - - - 12.0 17.0 71.0 - - Erasmus, 1993
Olive oil - - 12.1 2.6 72.50 9.40 0.6 0.4 Dubois et al., 2007
Palm oil - 1.7 43.8 4.4 39.1 10.2 0.3 0.3 Dubois et al., 2007
Palm olein - 1.00 37.00 4.00 46.00 11.00 - - Dubois et al., 2007;
Palm kernel oil - 71.6 8.4 1.6 16.4 3.1 - - Dubois et al. 2007
Peanut oil - 0.1 10.4 3.00 48.00 30.30 0.4 1.2 Dubois et al. 2007
Safflower oil - - - 12 13.0 75.00 - - Erasmus, 1993
Sesame oil - - - 13 42 45.00 - - Erasmus, 1993
Soybean oil 7 - 9.0 6.0 26.0 50.0 7.0 - Erasmus, 1993
Sunflower oil - - 6.4 4.5 22.1 65.6 0.5 0.3 Dubois et al., 2007;
Aleurites
moluccana
- - - - 18.80 46.86 25.43 -
Zulberti, 1988
Giuffre et al., 1996

2.4. A review of wild oil plants that have been studied in this project
2.4.1. Carapa grandiflora Sprague (Meliaceae)
Common name in eastern D.R. Congo: Ewechi. This is a tree that grows up to 30 m high. Habitat: in
montane forest. Distribution: Cameroon, Congo, D.R. Congo and Uganda (Burkill, 1995). In D.R.
Congo it is found in the forests of Kivu and Ituri Regions (Adriaens, 1944). Stembark and fruits are
eaten by chimpanzees and gorillas (Tuttle, 1986). Local people in Bwindi, in Uganda extract oil
from the seeds and use it as a substitute for vaseline (Naluswa, 1993). Samples from Uganda were
found with seed oil content of 30%. The oil has a SG value of 0.9261, its melting point range of 15-
23 O C and has saponification value of 198.1 mgKOH/g (Adriaens, 1944).
2.4.2. Carapa procera DC. (Meliaceae)
Its common name in Eastern D.R. Congo is Ewechi, English names: Tallicoonah oil tree; kunda oil
tree; monkey kola (Ghana). It is a very important species prized not only for the production of high
grade timber but also a number of non-timber forest products (Raquel, 2002). In some countries the
seeds are used to produce oil that is used medicinally for various ailments which include arthritis,
rheumatism, and parasitic infection. It is also used as emetics, febrifuges, leprosy, pulmonary
troubles and venereal diseases (Burkill, 1995; Butler, 2006). Adriaens (1944) reported 62% of oil in
seed kernel; the oil has SG value of 0.9238; the melting point is around 37 O C and saponification
value of about 198.
2.4.3. Cardiospermum halicacabum Linn (Sapindaceae)
Common name in Eastern D.R. Congo: Mubogobogo (Mashi). It is a perennial climbing plant with a
large ornamental seed pod that resembles a balloon. It has been mainly found in tropical India,
29

America and many parts of Africa. It is used in several tropical applications and has been found to
have anti-arthritic effect (Babu and Krishnakumari, 2006). The seeds from Holland were found to
have oil with 11-eicosenoic (gadoleic) acid (42%) as the major FA. Other FAs found in the species
are palmitic 3%, linolenic 8%, linoleic 8%, oleic 22%, stearic 2%, and arachidic 10% (Chisholm and
Hopkins, 1958). Occurrence of large amounts of C20 acids was confirmed and cyanolipid
constituents of four different types found in oil of seed samples from Maryland in USA
(Mikolajczak et al., 1970). Seeds of this species from Pakistan had a very high proportion of
unsaturated FAs particularly C20:1 (Ahmad, 1992).
2.4.4. Maesopsis eminii Engler (Rhamnaceae)
Common name in Eastern D.R. Congo: Omugaruka (Mashi). Trade name: Musizi (Katende et al.,
1995). It is a large African forest tree introduced to many parts of the tropics and grown in
monoculture plantations as a fast growing timber tree (Binggeli, and Hamilton, 1993). The heavily
felled and encroached forests within the fringes of the Lake Victoria zone and the River Nile are
characterized by Maesopsis eminii and Abizia spp as colonizers (Andrua, 2002). Analyses of seeds
from Karnakata in India, showed that the kernel contains 40-45% of oil. The oil contains 35.91%
saturated and 62.26% unsaturated FAs and 1.82% of an unidentified acid. The major components
were stearic 26.48%, oleic 47.49% and linoleic acids 14.79% (Theagarajan et al., 1986).
30

2.4.5. Millettia dura Dunn (Fabaceae)
Common name in English: Millettia; Nshunguri in eastern D.R. Congo. It is a small tree up to 13 m
tall. The specific epithet ‘dura’ reflects the locality from where the first botanical collection was
made, the Dura River in Kibale forest Uganda. Its geographic distribution is DR Congo, Ethiopia,
Kenya, Rwanda, Tanzania and Uganda (ICRAF. 2008). Its fruits are reported as elephant food in
Kibale National Park, Uganda (Rode et al., 2006). No information has been reported on oil from this
plant, however, related species, Millettia pinnata has been cultivated as a source of lamp oil, for
biodiesel and for natural medicine in India (Earth Equity, 2008). Perrett et al. (1995) have reported
the oil of Millettia thonningii samples from Ghana to be effective in preventing subsequent
establishment of infection on skin. Ezeagu et al. (1998) reported that Millettia thonningii samples
from Nigeria had a seed oil content of 30.66%, and the linolenic (23.05%) and linoleic acids
(18.19%). They also found behenic (8.93%), lignoceric (2.49%) and gadoleic acids (1.73%).
2.4.6. Myrianthus arboreus P. Beauv. (Cecropiaceae)
Called Bwamba in eastern D.R. Congo, it is a tree that grows to about 20 m high, much-branched
with yellowish-white wood soft, fibrous and difficult to work. Its distribution is the forest zone from
Guinea and Sierra Leone to West Cameroon, and extending across Africa to Sudan, Tanganyika and
Angola (Aluka, 2008). Myrianthus arboreus has been found to have oil-rich seed, which is about 1
cm long eaten after cooking from Côte d’Ivoire to D.R. Congo. This oil consists almost exclusively
of linoleic acid (93%) (Okafor, 2004).
2.4.7. Myrianthus holstii Engl. (Cecropiaceae)
Common name in Eastern D.R. Congo: Chamba, in English: Giant yellow, in Uganda: Mugunga and
Omufe (Cunningham, 1996 and Katende et al., 1995). It is a medium sized tree that grows up to 10
31

m with a short bole and large branches. Its fruit is around 4 cm of diameter. The seeds are
surrounded with edible acid pulp (Katende et al., 1995). Its fruits are reported as food of
chimpanzees inhabiting the montane forest of Kahuzi, D.R. Congo and in Bwindi Impenetrable
National Park, Uganda (Basabose, 2002; Rothman et al., 2006). According to Cunningham (1996) in
Uganda the use of edible Myrianthus holstii fruits is generally limited to famine periods. Myrianthus
holstii has high prospects as new crop plant and has been proposed by FAO for planting and fruit
production (Naluswa, 1993). It seems no information on the oil of this plant has been reported.
2.4.8. Pentaclethra macrophylla Benth (Mimosaceae)
Common name in East of D.R. Congo: Lubala (Kirega), in English: Bean tree,
in Wolof (Senegal): ataa, atawa. It is a tree which grows to about 21 m in height. The pods are 40-
50 cm long and 5-10 cm wide. It has been cultivated in Nigeria since 1937 and for many years in
other West African countries where its seed is relished as a food (ICRAF, 2008). Naturally P.
macrophylla occurs from Senegal to Angola and also to the Islands of Principe and Sao Tome. Its
geographic distribution is Cameroon, Cote d'Ivoire, DR Congo, Ghana, Niger, Nigeria, and Togo.
The seed is a source of edible oil (ICRAF, 2008 and Neuwinger, 1996). Ikhuoria et al. (2008)
reported 47.90% oil content in seed and stated that processing the seeds for oil would be economical
and the oil has some domestic and industrial potentials. Linoleic acid was found as a major FA in
this plant, and two long-chain FAs not commonly found in plant oils were identified. These were
shown to be hexacosanoic (C26:0) and octacosanoic (C28:0) acids (Foma and Abdala, 1985; Jones
et al., 1987).
32

2.4.9. Podocarpus usambarensis Pilger (Podocarpaceae)
Common name in Eastern D.R. Congo: Omufa (Mashi). Trade and common name: Podo (English),
Musenene (Luganda). This is a large evergreen tree, up to 60 m high, with thin pulp surrounding one
seed (Katende et al., 1995). Oil from this plant seems to be not yet studied, however related species,
Podocarpus nagera contains 24% of podocarpic acid (5c11c14c-20:3) (Berger and Jomard, 1998).
2.4.10. Tephrosia vogelii Hook. (Fabaceae)
Its common names in English include fish bean and Mukulukulu in eastern D.R. Congo. It is a
potential source of rotenone, an important nonresidual insecticide, and also a fish poison
(Blommaert, 1950 and Lambert et al., 1993). Samples from Western D.R. Congo were found with
about 14% of oil in seed (Adriaens, 1944).
2.4.11. Treculia africana Decne (Moraceae)
Treculia africana Decne is a large evergreen tropical food tree species (Onyekwelu and Fayose,
2007). Its common name in Eastern D.R. Congo: Bushingu, in English: African Breadfruit and wild
jackfruit, in Luganda: Muzinda. It is an evergreen tree of 15-30 m high bearing large fruits up to 30
cm of diameter, containing many orange seeds (Katende et al., 1995). Treculia africana
geographical distribution includes Angola, Benin, Cameroon, Central African Republic, Congo,
Cote d'Ivoire, D.R. Congo, Uganda and Zambia (ICRAF. 2008). The seeds are widely consumed and
many rural dwellers in Nigeria and Cameroon are engaged in collection, processing and sale of T.
africana seeds as a means of livelihood (Onyekwelu and Fayose, 2007).
33

Table 4: Review of oil content and oil characteristics of studied species
Plant name Oil % SG Mp
( O SV AI Uns.
Reference
C)
%
C. grandiflora 30 0.9261 15-23 198 x x Adriaens, 1944;
C. procera 62 0.9238 37 198 x x Adriaens, 1944;
C. procera 62 0.9043 x x x x Kabele, 1975
C. procera 66.4 x x x x x Oldham et al. 1993
C. procera 57.3 x 15-46 1.5 Lewkowitsch, 1909
C. procera 35 x x x x x Heckel, 1908
C. halicacabum x x 206 11.7 0.4 Chisholm and Hopkins, 1958
M. eminii 42.5 x x x x x Theagarajan et al., 1986
M. thonningii 30.7 x x x x x Ezeagu et al., 1998
P. macrophylla 47.9 x x 171 3.25 Ikhuoria et al., 2008
P. macrophylla 45 x x 187 x x Adriaens, 1944; Foma and
Abdala, 1985; Kabele, 1975
P. macrophylla 53.6 0.8600 x x x x Akindahunsia, 2004
P. macrophylla x x x 209 2.8 3.6 Akubugwo et al., 2008;
Odoemelam, 2005
T. vogelii 14 x x x x x Adriaens, 1944;
T. africana 12 0.8363 x x x x Foma and Abdala,1985
T. africana 0.9078 x 197 x x Kabele, 1975
T. africana 14.6 0.8450 x 113 1.96 x Dawodu, 2009;
T. africana x x x 213 8.41 x Akubugwo et al., 2008
x = no data available for the species
Dawodu (2009) reported 14.62% of liquid brownish yellow oil with 0.845 specific gravity, 1.96 acid
value and 112.5 saponification value. Foma and Abdala (1985) have found oleic acid followed by
linoleic and palmitic acids as predominant FAs in this plant. A review of plant seed oil content, oil
characteristics and FA compositions of oils from these plant species are presented in Tables 4 and 5.
34

Table 5: Review of oil fatty acid of studied species
Plant name Palmitic Stearic Oleic Linoleic ALA Arachidic Eicosenoic Behenic Lignoceric
Reference
16:0 18:0 18:1 18:2 18:3 20:0 20.1 22.0 24.0
C.guianensis x x x 9 x x x x x Taylor,2005
C.procera 26.4 8 48.9 14.4 x x x x x Kabele, 1975
C.procera 26.6 11.8 9.9 25.2 10.5 9.4 x x x Oldham et al. 1993
C.halicacabum 3 2 22 8 8 10 42 x x Chisholm and Hopkins, 1958
M. eminii x 26.48 47.49 14.79 x x x x x Theagarajan et al., 1986
M. thonningii x x x 18.19 23.05 x 1.73 8.93 2.49 Ezeagu et al., 1998
M. arboreus x x x 93 x x x x x Okafor, 2004
P. macrophylla 3.7 2.3 31.3 40.4 2.5 2.3 8.5 8.8 Foma and Abdala, 1985
P. macrophylla x x 16.1 56.6 x x x x 10.5 Jones et al., 1987
P. nagera x x x x x x 24 x x Berger and Jomard, 1998
T. africana 25.7 14.2 32.7 25.8 x x x x x Foma and Abdala, 1985
x = no data available for the species
35

3.1. Plant collection
CHAPTER THREE
MATERIALS AND METHODS
Seed plant samples from eleven species listed in Table 6 were collected from Kahuzi-Biega National
Park (KBNP) and surrounding areas. Kahuzi-Biega National Park is located in Kivu region, Eastern
D.R. Congo (2°30'S and 28°45’E, Figure 1). The area is a typical tropical forest in two zones: high
mountain range and a wide area of low mountains (Kasereka, 2003). The dominant tree species in
KBNP include Podocarpus usambarensis, Symphonia globulifera, and Carapa grandiflora in the
primary forest; Hagenia abyssinica, Myrianthus holstii, and Vernonia spp. in the secondary forest;
Hypericum revolutum and Rapanea melanophloeos in the swamp and Symphonia globulifera and
Syzigium parvifolium in and around swamp areas (Basabose, 2004).
Mature seed samples were collected from beneath the trees and kept in plastic bags. Only entire
seeds whose kernels were protected by seed coat were collected to avoid contamination. At least 500
g of seeds were collected for each plant species. Voucher specimens of these plant species were
brought to Herbarium of CRSN/Lwiro (Centre de Recherche en Sciences Naturelles de Lwiro) in
South Kivu, D.R. Congo and to Herbarium of Department of Botany of Makerere University to
confirm their identity.
36

Figure 1: Location of sampling sites (Irangi, Lwiro, Mugeri and KBNP/Tshibati) in Kahuzi-
Biega National Park and the surrounding areas in D.R. Congo at coordinates of
2°30'S and 28°45’E
37

Table 6: Species of wild oil plants studied in this project. All plant species were collected from
the sites shown, in Kahuzi-Biega National Park and the surrounding areas
Scientific name Family Local name Locality
Carapa grandiflora Meliaceae Igwerhe KBNP/Tshibati
Carapa procera Meliaceae Ewechi Irangi
Cardiospermum halicacabum Sapindaceae Mubobogo Mugeri
Maesopsis eminii Rhamnaceae Omuguruka Lwiro
Millettia dura Fabaceae Nshunguri KBNP/Tshibati
Myrianthus arboreus Moraceae Bwamba Irangi
Myrianthus holstii Moraceae Chamba KBNP/Tshibati
Pentaclethra macrophylla Mimosaceae Lubala Irangi
Podocarpus usambarensis Podocarpaceae Omufu KBNP/Tshibati
Tephrosia vogelii Fabaceae Mukulukulu Lwiro
Treculia africana Moraceae Bushingu Irangi
3.2. Laboratory analysis
Seed samples were taken to Phytochemistry laboratory of CRSN where they were sun dried before
drying them in the oven at 105 O C following the procedure described by Odoemelam (2005). After
this, shelling was made by hand and the seeds were crushed to produce fine seed flour from which
oil was extracted. To crush the seed a coffee-mill (model Corona 01 Landers & CIA. SA) was used.
The physical and chemical characteristics were determined following the American Oil Chemists
Society official methods (AOCS, 1993). The identification and quantification of FAs were
undertaken using Gas Chromatography (Christie, 1989).
3.2.1. Extraction of oil
Oil from the plant species was extracted by repeated washing (percolation) with petroleum ether
(boiling range between 40-60 O C) using the Soxhlet's procedure (Barthet et al., 2002).
38

3.2.1.1. Extraction procedure description
At the start of the procedure, the seed flour was placed inside a porous cellulose thimble made from
thick filter paper. The thimble containing the seed flour was loaded into the main chamber of the
Soxhlet extractor glassware (4 on Figure 2) and the extraction chamber placed above a flask
containing petroleum ether (1 and 2 on Figure2). The whole set up was placed below a condenser (9
on Figure 2). The principle of the method is that the solvent is heated to boiling; the condenser
returns it to drip steadily into the chamber housing the thimble containing the seed flour. The
chamber containing the seed flour slowly fills with warm petroleum ether. Oil then dissolves in the
warm petroleum ether. When the chamber containing the flour is almost full with warm petroleum
ether, it rises up in the siphon (6 and 7 on Figure 2) until this overflows. Thus the chamber is
automatically emptied by this siphon side arm, with the solvent running back down to the bottom
flask sweeping oil with it.
This cycle was allowed to repeat several times and during each cycle, a portion of oil dissolves in the
solvent. After many cycles the oil is concentrated in the extraction flask. After eight hours, the flask
was removed, the hot oil dissolved in petroleum ether solvent filtered on filter paper (Whatman no.
1) and the solvent evaporated under vacuum using a rotary evaporator. The remaining solvent traces
were removed by heating the flask containing the oil in the water bath. The oil obtained was stored
in hermetically closed bottles and kept in a refrigerator for analysis.
39

Figure 2: Set up of the Soxhlet extractor.
Source, Wikipedia (2009)
1: Stirrer bar, 2: extraction flask, 3: Distillation path, 4: Thimble, 5: plant seed flour, 6: Siphon top,
7: Siphon exit, 8: Expansion adapter, 9: Condenser, 10: Cooling water getting in, 11: Cooling water
out
3.2.2. Plant seed oil content determination
Ten grams of seed flour were mixed with 10 g of fine sand and then extracted in Soxhlet. After eight
hours the solvent was evaporated from the oil-solvent mixture in water bath and the solvent traces
remaining removed in oven at 105 O C in a period of 20 minutes. The mass of the remaining oil was
measured and the percentage of oil in the initial sample calculated using following formula:
⎛ P ⎞
% Oil = ⎜ ⎟x100 , where P = mass of oil and M = mass of plant seed flour used.
⎝ M ⎠
40

3.2.3. Determination of physical and chemical characteristics of oils
3.2.3.1. Specific gravity
The specific gravity (SG) of the extracted oils was determined ffrom the ratio of the mass of a
specified volume of oil in pycnometer to the mass of an equal volume of water, at temperatures of
40 O C in water bath. Pycnometers (Figure 3) are small glass or metal (around 2 ml) containers with a
determined volume (Eren, 2000). They are used to determine the density of liquids and their specific
gravity.
Figure 3: Representation of a pycnometer
Source: Eren (2000)
The empty pycnometer was weighed (mass ‘a’). Then the pycnometer was filled with distilled water
and kept at 40 O C until it reached this temperature and then weighed (mass ‘b’). The same
pycnometer was then filled with oil and kept in thermostatted bath at 40 O C until it reached this
temperature and also weighed (mass ‘c’). The formula used to calculate the SG
⎛ c − a ⎞
was SGt = ⎜ ⎟xDo , where a = mass of the empty pycnometer, b = mass of the pycnometer full of
⎝ b − a ⎠
water at 40 0 C, c = mass of the pycnometer full of plant oil at 40 O C and Do = density of water at 40 O C
(0.9922 g/ml). The values of SG obtained at 40 O C were also converted to temperature of 30 O C
because SG decreases linearly with temperature. Thus according to following regression equation
41

SGt 2 −0.
0006X
+ SGt1
= (Wan Nik et al., 2007), where X is the variation of temperature in degrees.
This linearity is more reliable around the interval of 10 degrees (Kabele, 1975). This will allow
comparing plant oil SG with different products such as fuel.
3.2.3.2. Melting point
The complete melting point (Cmp) of the oils was determined using a fusiometer (Baur, 1995). The
mpt is defined as the temperature at which a column of oil in an open capillary tube moves up the
tube when it is subjected to controlled heating in a water bath (APOC, 2004). To determine the Cmp,
a melting point capillary tube was filled with oil from the open end of tube. The capillary was placed
in the melting point apparatus through one of the side tubes so that the sealed end of the capillary
was touching the front of the mercury reservoir of the thermometer and begins to heat the apparatus
with a micro burner. Thereafter burner was placed under the back end of the oil bath of the
apparatus. The melting range was recorded when oil start dropping until when all oil has empty the
capillary tube. The Cmp is recorded when the oil empty the capillary tube.
3.2.3.3. Saponification value
Two grams of the oil sample were introduced in a flask containing 30ml of ethanolic KOH (0.5 M)
and heated under a reversed condenser for 30 minutes to ensure that the sample was fully dissolved.
The sample was then cooled. One ml of phenolphthalein was added and titrated with 0.5 M HCl
until a pink endpoint was reached. The same procedure was repeated with a blank, i.e. a flask with
30ml of ethanolic KOH (0.5 M) but without oil. The saponification value was computed using the
C1
− C2
formula: SV X 28
M
= , where C1 = number of ml of the Hydrochloric acid (0.5 M) used in
blank, C2 = number of ml of the Hydrochloric acid used in the assay, M = mass in g of oil used.
42

3.2.3.4. Percentage of unsaponifiable matter
To determine the percentage of unsaponifiable matter (USM), the oil sample was saponified with
alcoholic potassium hydroxide (0.5 M). The USM was extracted using diethyl ether in a glassware
separating funnel. After washing off the crude extract with water, the organic solvent was evaporated
and the USM dried in oven at 105 O C and weighed to calculate the percentage of USM using
P
following formula, % Uns . = X100
, where P = mass of the extracted USM in g and M = mass of
M
the oil sample saponified.
3.2.3.5. Acidity index
Five grams of oil were dissolved in a solvent mixture of 75 ml of ethanol 96% and 75 ml of diethyl-
ether. One ml of phenolphthalein was added and the solution titrated with potassium hydroxide
(0.5N) until a pink endpoint was reached. The formula used to calculate acidity index
CXNX 56.
1
was AI = , where C = number of ml of the potassium hydroxide used, N = normality of
M
potassium hydroxide used and M = mass in g of oil used.
43

3.3. Identification and quantification of fatty acids
The identification and quantification of FAs was undertaken using Gas Chromatography by Prof.
Otto Grahl-Nielsen, in the laboratory of the Department of Chemistry, University of Bergen, in
Norway. Oil samples of approximate 50 mg were transferred to thick-walled 15 ml glass tubes,
taking care to avoid water contamination. The tubes were prepared with an accurately determined
amount of the saturated FA, nonadecanoic acid (C19:0; Nu Chek Prep, Elysian, Minn., USA) as
internal standard. This was added to the tubes by pipetting 50.0 µl of a solution of C19:0 chloroform
into the tubes, and then allowing the chloroform to evaporate. This pipetting was carried out using a
handystep electronic, motorized repetitive pipette. 750 µl anhydrous methanol containing hydrogen
chloride were added to the methanol as dry gas, in a concentration of 2 mol/l to allow the hydrolysis
of oil triglycerides. The tubes were securely closed with teflon-lined screw caps. After keeping the
tubes in an oven at 90°C for two hours, the samples were methanolysed by the replacement of
glycerol in the triglyceride by methanol. In this way all FAs were converted to FA methyl esters
(FAMEs). After cooling to room temperature, approximately half the methanol was evaporated by
bubbling nitrogen-gas through the mixture, 0.5 ml distilled water was then added.
The FAMEs were extracted from the methanol/water-phase with 2 x 1.0 ml hexane by vigorous
shaking by hand for one minute, followed by centrifugation at 3000 rpm. The FAMEs extracted
were recovered in a 4 ml vial with teflon-lined screw cap. One µl of the FAMEs extracted was
automatically injected splitless (the split was opened after 4 min), on a capillary column. Details of
chromatographic equipment and settings are given in Table 7. Samples were analysed in random
order with a standard solution, GLC 68D from Nu Chek Prep (Elysian, Minn., USA) containing 20
FAMEs.
44

Table 7: Chromatographic Equipment and settings
Chromatograph Hewlett-Pakard 5890A
Auto-sampler Hewlett-Pakard 7673A
Column 25m × 0.25mm fused silica capillary with polyethylene
glycol as the stationary phase with a thickness of 0.2 µm (CP-
WAX 52CB from Chrompack)
Carrier gas Helium at 1,7ml/min at 40°C
Injector Split/Splitless kept at 260°C
Detector Flame ionization kept at 330°C
Temperature program 90°C for 4 min, 90°C to 165°C with 30°C/min, 165°C to
225°C with 3°C/min, 225 O C for10 min, total run time 43 min,
cooling included
Labdata system Chomeleon from Thermo LabSystems
The quantitatively most important FAs were identified in the samples, using a standard mixture
basing on previous experience of relative retention times of FAMEs and mass spectrometry. The
smallest peaks, that is those with areas of less than 0.1% of the total area of all peaks, were not
considered (Appendix 2). The peaks were integrated by Chromeleon software and the resulting area
values exported to Excel, where they were corrected by response factors. These empirical response
factors, relative to 18:0, were calculated from the 20 FAMEs, present in known proportions in the
standard mixture. An average of 10 runs of the standard mixture was used for these calculations. The
response factors for the FAMEs for which there were no standards, were estimated by comparison
with the standard FAMEs which resembled each of those most closely in terms of chain length and
number of double bonds. The relative amount of each FA in a sample was expressed as percentage
of the sum of all FAs in the sample.
The determination of oil content, specific gravity, melting point, saponification value, percentage of
45

unsaponifiable, acidity and FA composition were performed with 3 replicates except for FAs
analysis of Millettia dura (8 replicates) and Myrianthus arboreus (4 replicates). This was done to
acquire the analytical variance for FA composition of these two species oils.
3.4. Data analysis
Mean values and their standard deviations (mean ± SD) were calculated using GenStat computer
package programme, GenStat release 7.1 of 2003. Analysis of variance (ANOVA) was performed
for oil content, specific gravity, melting point, saponification value, percentage of unsaponifiable,
acidity and FA composition of all species analyzed. The least significant differences of means (LSD)
test at 5% probability level were also carried out.
46

4.1. Plant seed oil content
CHAPTER FOUR
RESULTS
The total oil content of the plant seeds ranged from 17.2 – 64.4% in the studied species (Figure 4).
Podocarpus usambarensis had the highest oil content followed by Maesopsis eminii (56.67%) and
Pentaclethra macrophylla (55.79%) while Tephrosia vogelii had the lowest oil content. The mean
values of oil content of different species were significantly different (p< 0.001).
% Seed oil content
70
60
50
40
30
20
10
0
P.
usambarensis
M. eminii
P.
macrophylla
M. arboreus
C. procera
47
C.
grandiflora
C.
halicacabum
Figure 4: Oil content of seeds of plant species from Kahuzi-Biega National Park and
surrounding areas in D.R. Congo. Error bars are indicated in the figure. LSD (5%)
= 1.133
M. holstii
T. africana
M. dura
T. vogelii

4.2. Physical and chemical characteristic of oils
4.2.1. Specific gravity
The Specific Gravity (SG) in oil of analyzed species ranged from 0.8050 to 0.9854 (Table 8).
Tephrosia vogelii had the highest specific gravity and Treculia africana the lowest. Six of species
studied had specific gravity greater than 0.9000. The mean values of SG of different plant species
analyzed were significantly different (p< 0.001).
Table 8: SG at 40 and 30 O C of oils from plants obtained from Kahuzi-Biega National Park and
the surrounding areas in D.R. Congo
Plant scientific name SG (40 O C) SG (30 O C)
Treculia africana 0.8050±0.0033 0.8110
Podocarpus usambarensis 0.8324±0.0009 0.8384
Pentaclethra macrophylla 0.8615±0.0006 0.8675
Myrianthus arboreus 0.8781±0.0019 0.8841
Myrianthus holstii 0.8793±0.0007 0.8853
Maesopsis eminii 0.9059±0.0008 0.9119
Cardiospermum halicacabum 0.9239±0.0003 0.9299
Carapa grandiflora 0.9311±0.0003 0.9371
Millettia dura 0.9397±0.0009 0.9457
Carapa procera
0.9403±0.0006 0.9463
Tephrosia vogelii 0.9854±0.0013 0.9914
LSD (5%) 0.002242 0.002256
4.2.2. Melting point
The result for the melting point (mp) of the oils analysed are presented Table 9. The mp ranged from
-12 to 32 O C. The oil of most plants analyzed is liquid at room temperature. The oils of Myrianthus
were liquids even below 0 O C. one the other hand, Carapa procera and Cardiospermum halicacabum
oils were solid at room temperature, while Pentaclethra macrophylla and Carapa grandiflora oils
were semi-solid at room temperature.
48

Table 9: Melting point ranges of oils from plants obtained from Kahuzi-Biega National Park
and the surrounding areas in D.R. Congo
Plant name Mp ( O C)
Carapa procera 27 - 32
Cardiospermum halicacabum 23 - 25
Carapa grandiflora 21 - 25
Pentaclethra macrophylla 15 - 20
Maesopsis eminii 11 - 15
Podocarpus usambarensis 8 - 13
Millettia dura 3 - 7
Tephrosia vogelii 2 - 6
Treculia africana 1 - 5
Myrianthus arboreus -5 - - 2
Myrianthus holstii -12 - -1
4.2.3. Saponification value
The saponification values found ranged from 182.5 to 260.9 mg KOH/g (Figure 5). Myrianthus
holstii, Maesopsis eminii and Myrianthus arboreus oils had the highest values around 260 while
Podocarpus usambarensis and Tephrosia vogelii had the lowest (182.5 and 184.3 respectively). The
saponification values of oils obtained from the different plant species were significantly different (p
< 0.001).
49

Saponification values
300
250
200
150
100
50
0
M. arboreus
M. eminii
M. holstii
M. dura
T. africana
50
P.
macrophylla
C.
halicacabum
C. procera
C.
grandiflora
T. vogelii
P.
usambarensis
Figure 5: Saponification values of oils from plants of Kahuzi-Biega National Park and
surrounding areas in D.R. Congo. Error bars (SD) are indicated in the figure. LSD
(5%) = 5.154
4.2.4. Oil unsaponifiable matter content
The oils unsaponifiable matter content ranged from 0.48 to 2.25% (Figure 6). Podocarpus
usambarensis oil had the highest values while Maesopsis eminii had the lowest. Myrianthus
arboreus, Myrianthus holstii and Maesopsis eminii had fairly similar amounts of unsaponifiable
matter of 0.54, 0.51 and 0.48 % respectively. All values obtained from oils of plant species analyzed
were statistically different (p

% Unsaponif. matter
3.0
2.5
2.0
1.5
1.0
0.5
0.0
P.
usambarensis
P.
macrophylla
M. dura
C.
grandiflora
C. procera
51
T. africana
C.
halicacabum
Figure 6: Unsaponifiable matter of oils of plants obtained from Kahuzi-Biega National Park
and surrounding areas in D.R. Congo. Error bars are indicated in the figure. LSD
(5%) = 0.1998
4.2.5. Acidity index
The Acidity indices (AI) found ranged from 1.74 to 6.36 mg KOH/g (Figure 7). Treculia africana oil
had the highest value while Myrianthus holstii had the lowest. The AI of oils obtained from the
different plant species were significantly different (p < 0.001).
T. vogelii
M. arboreus
M. holstii
M. eminii

Acidity Index
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
T. africana
P.
macrophylla
C.
grandiflora
M. arboreus
C.
halicacabum
52
C. procera
M. dura
T. vogelii
M. eminii
P.
usambarensis
Figure 7: Acidity Index of oils of plants obtained from Kahuzi-Biega National Park and
surrounding areas in D.R. Congo. Error bars are indicated in the figure. LSD (5%)
= 1.009, N = 3
4.3. Plant oil fatty acid composition
Twenty four FAs were determined in the studied plant species. Eighteen of these FAs were identified
(Table 10). The composition levels of the FAs from the oils were significantly different (p

Table 10: Fatty acid (FA) composition (wt. % of total of FA) of oil from plants of Kahuzi-Biega National Park and
surrounding areas in D.R. Congo. All FAs showed significant variation among species (p

The highest content (21.2%) of the essential FA α-linolenic acid (18:3n3) was found in Millettia
dura. Linoleic acid was the predominant FA in the plant seed oil out of the four analyzed plant
species; M. holstii seed oil (80.2%), M. arboreus seed oil (77.2%), P. macrophylla seed oil (40.6%)
and T. vogelii seed oil (40.3%). Oleic acid was the predominant FA in the seed oil of the six
analyzed plant species, i.e. C. grandiflora seed oil (41.2%), P. usambarensis seed oil (39.9%), C.
halicacabum seed oil (37.5%), M. eminii seed oil (37.0%), T. africana seed oil (30.8%), and M. dura
seed oil (32.3%). Stearic acid was the major FA in Carapa procera seed oil (52.3%) with oleic acid
making a significant contribution (42.5%).
Unidentified FAs make up 0.1 – 8% of the oils with the highest value (8.0%) in oil of Podocarpus
usambarensis, followed by Millettia dura (4.4%) and Treculia africana (3.0%) oils. The oil from
Podocarpus usambarensis seeds is distinct from the others because of this higher level of
unidentified FAs, particularly the Und5 and 6 (Table 10).
4.3.1. Saturated fatty acids
The saturated fraction ranged from 7.1 to 54.4%. The highest was from Carapa procera and the
lowest from Myrianthus arboreus. The saturated FA constituted more than 50% of the total FAs
analyzed in Carapa procera oil and more than quarter of total FAs of six of the other plant species
studied (Tables 10 and 11). Palmitic (16:0) and stearic (18:0) were the major saturated FAs analyzed
while arachidic (20:0), behenic (22:0) and lignoceric (24:0) acids were minor. Maesopsis eminii and
Tephrosia vogelii had about the same amount of saturated FA (respectively 29.3 and 29.2%).
Between Cardiospermum halicacabum and Pentaclethra macrophylla also there is about the same
amount of saturated FA (25.1 and 24.9%).
54

Table 11: Saturated FAs, Monounsaturated FAs, Polyunsaturated FAs, LCFA and Omega
FAs content in oils of plants from Kahuzi-Biega National Park and surrounding
areas in D.R. Congo
SFA MUFA PUFA LCFA ω-3 ω-6
FA FA
Carapa procera 54.4 42 27.9 2.5 1.4 26.4
Carapa grandiflora 30 43 2.3 0.5 0.7 1.6
Cardiospermum halicacabum 25.1 38.1 35.9 6.6 0.8 35.1
Maesopsis eminii 29.3 39.2 31.3 6 4.4 26.9
Millettia dura 18.1 35.5 42 14.4 21.6 20.4
Myrianthus arboreus 7.1 13 78.5 1.5 1 77.5
Myrianthus holstii 7.7 10.4 81.1 1.2 0.7 80.4
Pentaclethra macrophylla 24.9 33.6 41 21.3 0.2 40.8
Podocarpus usambarensis 8.3 42 41.8 6 9.3 32.5
Tephrosia vogelii 29.2 20.6 48.2 10.2 7.8 40.4
Treculia africana 34.3 31.2 31.4 1.8 1.3 30
4.3.2. Unsaturated fatty acids
The monounsaturated fractions in analyzed oils are from 10.4 to 43%. The highest is from Carapa
procera and the lowest from Myrianthus holstii. The monounsaturated FAs content in the extracted
oils constituted more than 30% of the total FAs in the 8 plant species studied (Table 11). Myrianthus
arboreus and Myrianthus holstii had their FAs basically constituted by polyunsaturated FAs which
had around 80% of their total FAs. Besides these, seven other studied species had more than 30% of
their total FAs constituted by polyunsaturated FAs (Table 11).
4.3.3. Omega–6 (ω-6) and Omega–3 (ω-3) fatty acids
In analyzed plant oils, three essential FAs Omega-3 i.e. α-linolenic acid (ALA), Eicosapentaenoic
acid (EPA) and Docosahexaenoic acid (DHA) were found at about the quarter of percentage of the
total FAs in Millettia dura oil and around 5% in Tephrosia vogelii and Maesopsis eminii oils (Table
55

11). ALA and EPA were found in all plant species analyzed from 0.1 to 21.2% for ALA and 0.1 to
0.4% for EPA. DHA was detected only in Millettia dura (0.1%) and Myrianthus arboreus (0.1%)
oils. The essential FAs Omega-6 found in analyzed oils are linoleic acid and eicosadienoic (20:2n6).
This last was found in significant amount (2.9%) only in Podocarpus usambarensis oil.
4.3.4. Long chain FAs
The Long chain FAs (LCFAs) fractions in analyzed oils ranged from 0.5 to 21.3%. The highest is
from Pentaclethra macrophylla and the lowest from Carapa procera. Oils of Pentaclethra
macrophylla, Millettia dura and Tephrosia vogelii had respectively, 21.3, 14.5 and 10.2% of their
total FAs constituted of acids of chain length higher than 18 atoms of carbon.
5.1. Seed oil content of plant species
CHAPTER FIVE
DISCUSSION
Determination of oil content in plants is important because it predicts the profitability of given plants
as potential source of oil. High oil content in plant seeds implies that processing them for oil would
be economical (Ikhuoria et al., 2008). The oil yield found in the studied plants which ranged from
17.2 to 64.4% compares favourably well with the oil yield reported for some commercial plant oils
such as cotton seed (36%), sesame (44%), olive (17%), groundnut (40%), sunflower (44%),
soybeans (18%), palm kernel 40%, canola 40% and corn 3.4% (Rossel, 1987).
Millettia dura oil content (27.81%) is close to that of related species Millettia thonningii (30.66%).
Carapa grandiflora oil content was 41.6% which is higher than that (30%) reported by Adriaens
56

(1944). For Carapa procera there are divergent results reported in literature. The samples from west
of D.R. Congo (Adriaens, 1944; Kabele, 1975) were found to have around 62% of oil. Oldham et al.
(1993) reported 66.4%, Lewkowitsch (1909) reported 57.3% and Heckel (1908) 35%. As for the two
species of Carapa, results of this study showed that Carapa grandiflora had less oil content than the
Carapa procera. This is comparable to results reported by Adriaens (1944). This can be already used
as criterion of distinction between these two species. Concerning the two species of Myrianthus
results showed Myrianthus arboreus to have oil content (52.38%) which was higher than that of
Myrianthus holstii (35.16%).
Regarding Maesopsis eminii, seeds from India were reported to have oil content lower (Theagarajan
et al., 1986) than the results obtained in this study. As far as the oil content of Pentaclethra
macrophylla is concerned, divergent results are reported in literature (Table 4). The analyzed
samples of Pentaclethra macrophylla in this study contained more than the majority but were a bit
close to those of Akindahunsia (2004) from Nigeria. The oil content found for Treculia africana is
more than what was reported in previous studies (Table 4). The percentage of Tephrosia vogelii from
western D.R. Congo was found to contain about 14% of oil in seed (Adriaens, 1944), which was
slightly less than the findings of the current study (17.2%).
5.2. Physicochemical characteristic of oils
5.2.1. Specific gravity
Generally oils are lighter than water, but some are heavier, especially those which contain larger
amounts of oxygenated constituents of the aromatic series (StasoSphere, 2007). The remarkably high
SG of Tephrosia vogelii oil (0.9854) may be explained from this fact; this plant is rich in rotenone
57

(Lambert et al., 1993) a molecule that has many aromatic groups and oxygen atoms (Fang and
Casida, 1999). Akindahunsia (2004) reported that Pentaclethra macrophylla Nigeria’s sample the oil
specific gravity (0.8600) exactly close to that reported in this study (0.8615±0.0006). In the current
study result on specific gravity of Treculia africana (0.8050) is close to that found by Dawodu
(2009) on samples from Nigeria (0.8363) but more lower than that (0.9078) found by Kabele (1975)
on samples from western D.R. Congo. For Carapa procera a value of specific gravity obtained in
this study was 0.9403, which is similar to that (0.9043) reported by Kabele (1975).
Most popular plant oils have specific gravity ranging from 0.9100 to 0.9400 and specific gravity of
0.92 is considered a pretty good number for any cooking oil (Elert, 2000). Some authors have stated
that the specific gravity suitable for edible oils range from 0.8800 to 0.9400 (Toolbox, 2005) and for
oils used for fuel from 0.8200 to 1.0800 at 15.6 O C (CSG, 2008). These SGs ranges compared to
those of current study in Table 8 indicate that crude oils from Carapa grandiflora, Cardiospermum
halicacabum, Maesopsis eminii and the two species of Myrianthus are in the range of common
cooking oils in regard of their SGs values (0.8714 to 0.9314).
5.2.2. Melting point
The high viscosities of plant oils, compared to those of fuels, limits their direct use as bio-fuel. The
viscosity of given plant oil decrease in proportion to its melting point. Thus the low melting point
suit best for bio-fuel use because it correspond to low oil viscosity (Krisnangkura et al., 2006). Most
common edible oils have Cmp that range from – 23 to 2 O C while butter and other fats range from 28
to 48 O C (Rossel, 1987). Thus as indicated in Table 9, Tephrosia vogelii, Treculia africana, Millettia
dura, Myrianthus arboreus, Myrianthus holstii have oils in melting range of edible oil. Adriaens
58

(1944) had reported melting range of 15-23 O C from oil of Carapa grandiflora samples from
Uganda. Lewkowitsch (1909) have found melting range of 15-46 O C from oil of C. procera from
Sierra Leone, and Adriaens (1944) reported melting range around 37 O C for samples from west D.R.
Congo.
5.2.3. Saponification value
Many edible oils have saponification values between 193 and 200 (Pearson, 1981). Among the plant
species studied, Carapa grandiflora, Carapa procera, Pentaclethra macrophylla, Cardiospermum
halicacabum and Treculia africana oils have saponification values that are within this range. The
oils having high saponification value (around 300) are useful for soapmaking (Alabi, 1993). No such
oil was found in this study.
Chisholm and Hopkins (1958) found the Cardiospermum halicacabum seed oil to have
saponification value of 206. This is not far from the current study value (195 - 202). About
Pentaclethra macrophylla Kabele (1975) reported 187, Akubugwo et al. (2008) 209.4 and Ikhuoria
et al. (2008) from Nigeria also found 171.1. The result in current study (199.7) is more close to that
of Akubugwo et al. (2008) from Nigeria. As for oil from Treculia africana Dawodu (2009) had
found in samples from Nigeria the saponification value of 112.5, and Akubugwo et al. (2008) 212.9.
Kabele (1975) in samples from western parts of D.R. Congo has found 196.5 – 198.
5.2.4. Oil unsaponifiable matter content
The minor substances of the oil contained in unsaponifiable matter have antioxidant and other health
benefits in animals and in human subjects (Gunstone and Herslöf, 2000; Kochhar et al., 2001). Some
compounds of unsaponifiable matter have superior moisturizing effect on the upper layer of the skin
59

and reduce scars (Goreja, 2004). Shea butter; avocado, sesame, soybean and olive oils have high
unsaponifiable fractions and from this they are known in cosmetics as having efficacy on dry and
damaged skins (Alvarez and Rodríguez, 2000; Dhellot et al., 2006; Mellerup et al., 2007).
Phytosterols products of unsaponifiable matter are known to be effective in reducing cholesterol in
blood serum (SCF, 2002). Tocopherols and tocotrienols have both vitamin E and antioxidant activity
helping furthermore for oil stability. Tocopherols produced for sale are from soybean, sunflower,
palm and other plant unsaponifiable matter (Beare-Rogers et al., 2001, Gunstone and Herslöf, 2000).
The most of the world supply of corticosteroids and sex hormones is produced from soybean oil
unsaponifiable matter (Clark, 1996).
In C. procera oil samples from Sierra Leone, Lewkowitsch (1909) reported unsaponifiable matter of
1.51%, i.e. about the same as in current study (1.19%). Chisholm and Hopkins (1958) have found for
the Cardiospermum halicacabum seed oil the unsaponifiable matter of 0.4% what is about the half of
that was found in current study (0.85). In oil of Pentaclethra macrophylla samples from Nigeria
Odoemelam (2005) reported 3.6% of unsaponifiable matter, also about the half of that was found in
current study (1.95). From all other eight species it seems to be no previous information.
Quantitatively the value of unsaponifiable matter content of Maesopsis eminii is close to that of
coconut oil. Myrianthus holstii and Myrianthus arboreus oils have unsaponifiable matter content
comparable to that of groundnut oil and Tephrosia vogelii and Cardiospermum halicacabum oils
have unsaponifiable matter content comparable to those of cottonseed, palm and sunflower seed oils
(Rossel, 1987). Unsaponifiable matter content of oils of Treculia africana, Carapa procera, Carapa
grandiflora and Milletia dura fall in the range of unsaponifiable matter content of soybean oil, while
60

those of Pentaclethra macrophylla and Podocarpus usambarensis are near of the one of olive
(Rossel, 1987).
5.2.5. Oil acidity
Oil that is low in acidity is suitable for consumption (Alabi, 1993). They must have acidity level less
than 0.1 mg KOH/g (FAO, 1993). Four of the plant species (Tephrosia vogelii, Pentaclethra
macrophylla, Carapa grandiflora, and Myrianthus arboreus) had oils with relatively high acidities
close to that of crude palm oil while those of Myrianthus holstii, Podocarpus usambarensis and
Maesopsis eminii oils are relatively low and close to that of crude soybean oil (Rossel, 1987). With
all these levels of acidity, all these oils require refining to minimize their acidity before to envisage
eventual food use (Orthoefer and List, 2007).
Chisholm and Hopkins (1958) found the Cardiospermum halicacabum seed oil to have oil acidity of
11.7. This is higher than that found in the currently studied samples (4.02±0.48). About Pentaclethra
macrophylla Akubugwo et al. (2008) and Ikhuoria et al. (2008) from Nigeria found respectively
2.81±0.01 and 3.25±0.20. The current studied result (5.31±0.54) is much higher than all those. As
for oil from Treculia africana, in samples from Nigeria Dawodu (2009) has reported the oil acidity
of 1.96 and Akubugwo et al. (2008) reported very higher than that (8.41). The result in current study
(6.36±0.43) is close to that of Akubugwo et al. (2008).
61

5.3. Plant oil fatty acid composition
The world production of FAs from the hydrolysis of natural fats and oils totals about 4 million
metric tons per year. FAs are consumed in a wide variety of end-use industries as food, medicine,
rubber, plastics, detergents, cosmetics and others (Gunstone, 1996).
5.3.1. α-linolenic acid
Linolenic acid is an important FA in the diet, a health nutrient and a therapeutic agent (Baúci et al.
2003). The linoleic and α-linolenic acids are essential in the diet and without them we would die
(Gurr, 1999). The α-linolenic acids (ALA) content, Milletia dura seed oil (21.2%) is about three
times richer than soybean oil (7.8%) and canola oil (7.9%) which are common crops providing this
acid (MacLean et al., 2005). Oil from related species, Millettia thonningii, is known to be effective
in preventing establishment of infection on skin (Perrett et al., 1995).
5.3.2. Linoleic acid
Linoleic acid is a major dietary FA (Wijendran and Hayes, 2004). Dietary fats, rich in linoleic acid,
prevent cardiovascular disorders, atherosclerosis and high blood pressure (Dagne and Jonsson,
1997). LA is known to lower cholesterol levels and provides anticancer benefits (Taylor, 2005).
Some deficiency symptoms of linoleic acid are eczema, loss of hair, liver degeneration, behavioral
disturbances, sterility in males, miscarriage in females and kidney degenerations. Prolonged absence
of linoleic acid from diet is fatal (Erasmus, 1993). In cosmetics, oil high in linoleic acid helps to
regenerate and moisturize damaged dry skin (Athar and Nasir, 2005). All these deficiency symptoms
can be reversed by adding linoleic acid to the diet (Erasmus, 1993).
62

About Myrianthus holstii, the current study seems to be the first on its FAs composition. The
concentrations of linoleic acid found in Myrianthus arboreus and Myrianthus holstii (77.2 and
80.2% respectively) are higher to that found in common edible oils, such as cottonseed, grape seed,
canola oil, soybean, sunflower, safflower and corn oil (Dubois et al., 2007), and may indicate that
these oils can be used as alternatives for commercial oils for nutritional use. They may thus be,
alternative sources of this acid which is one of the industrially desirable FAs (Bagcı, 2007).
Furthermore Myrianthus species oil contained small amounts (7.0 - 7.8 %) of total saturated FAs
(Table 11), which may be of advantage in view of the fact that diets low in saturated fats may benefit
patients with cardiovascular diseases (Akoh and Nwosu, 1992). Okafor (2004) has reported that oil
of Myrianthus arboreus seed from Ivory-Cost contained exclusively 93% of linoleic acid.
5.3.3. Oleic acid
Oils rich in monounsaturated FAs (e.g. oleic acid) are generally more stable to oxidative rancidity
and stable as deep frying oils (Mohammed et al., 2003). They have many applications such as plant-
based lubricants and as feedstock for the oleochemical industry (Gunstone, 1996). As a fat, oleic oil
is one of the better known ones for consumption and from a health standpoint, it exhibits further
benefits of low total cholesterol, to slow the development of heart disease and promotes the
production of antioxidants (Pérez-Jiménez et al., 2002). In addition oleic acid is part of a number of
products as soap and cosmetics in which it seems to be a great moisturizer (Ellis-Christensen, 2009).
Thus, Carapa grandiflora and Carapa procera can be new cheap source of oleic acid (40.2 and
42.5% respectively) in comparison to palm oil and palm olein, commodities of high economic
importance which comprise respectively 39.1 and 46.0% oleic acid (Dubois et al., 2007). There
63

appears to be no previous reported work on FA composition of Carapa grandiflora, but for Carapa
procera, Kabele (1975) has reported 48.9% of oleic acid in samples from western D.R. Congo.
Oldham et al. (1993) found from Carapa procera 9.9% of oleic acid. A related species, Carapa
guianensis was found oil rich source of usual FAs including oleic acid (Taylor, 2005).
5.3.4. Saturated FAs
Carapa procera oil is the richest in SFA among all analyzed plant species. Its FAs profile is similar
to those of other saturated oils as shea butter (Vitellaria paradoxa), cocoa butter (Theobroma cacao)
(Lipp and Anklam, 1998) and mango (Mangifer indica) oils (Solis-Fuentes and Duran-de-Bazua,
2004; Udayasekhara, 1994). They have stearic acid as their main component followed by oleic acid.
Cocoa butter is mostly used in the food industry, while shea butter is mainly seen in cosmetics,
although it has also been authorized as a cocoa butter alternative in chocolate products (Lipp and
Anklam, 1998). So all these oils, including that from Carapa procera seed, contain around half of
saturated FAs and due to their high melting point, it is obvious that they are more “butters” than oils
and they belong to vegetable butter oils group (Nawar, 1996). Their nutritional interest lies in the
neutrality of stearic and oleic acids with regard to plasma lipid composition (Kris–Etherton and Yu,
1997).
5.3.5. Unsaturated FAs
All studied plants except Carapa procera have more than 60% of their FA content unsaturated
(Table 11). From this they may have a high potential nutritional value (Arnaud et al., 2004). Taken
on the whole the unsaturated fraction (Table 11) in Myrianthus arboreus and Myrianthus holstii is
very near to 92% (91.6 and 91.5).
64

5.3.6. Omega–6 (ω-6) and Omega–3 (ω-3) fatty acids
In all analyzed plant species, the general trend of increase of ω-3 FAs is inversely proportional to the
increase in ω-6 FAs (Table 11). Epidemiological and clinical studies have established that the ω-6
FAs, i.e. linoleic acid and the ω-3 FAs, i.e. ALA, EPA, and DHA collectively protect against
coronary heart disease (Wijendran and Hayes, 2004). Adequate ω-3 FA consumption has been
associated with a significant reduction in the incidence of Alzheimer’s disease (MacLean et al.,
2005). Nutritional experts suggest that consumption of 6% of linoleic acid, 0.75% of ALA, and
0.25% of EPA and DHA represents adequate and achievable intakes for most healthy adults. These
proportions correspond to a ω-6/ω-3 ratio of 6 (Wijendran and Hayes, 2004). However,
recommendations for a ω-6/ω-3 ratio are variable depending on individual need (Simopoulos, 2003).
Maesopsis eminii and Podocarpus usambarensis seed oils followed by Tephrosia vogelii seed oil are
nature’s most balanced oil as dietary and health oils, because among the analyzed plants they have
the most complete profile of essential FAs (Table 11). They have also equilibrated ω-6/ω-3 ratio
(table 10) more near the recommended ratio than canola and soybean oils (Table 3). Tephrosia
vogelii seed oil having poisonous products (Lambert et al., 1993), its use as food can only be
considered after extensive refining (Orthoefer and List, 2007).
5.3.7. Long Chain fatty acids and Chemotaxonomy Relevant
High amounts of individual FAs may be useful to assess chemotaxonomic relationships among plant
taxa (Dagne and Jonsson, 1997), but unusual FAs are even more useful and important to elucidate
chemotaxonomic relationships between some genera and families, because the occurrence of unusual
FAs in plant seeds is often correlated to plant families (Aitzetmuller, 1993). The usual FAs regularly
met in plant oil include lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic
65

acid. The LCFAs (more than 18 carbons atoms) are commonly found in fish oil rather than in plants
(Gurr, 1999). Unusual FAs patterns found can be considered as consistent and chemotaxonomically
significant, if the same pattern was found in several species of the given genus (Tsevegsüren and
Aitzetmüller, 1997).
In current study Pentaclethra macrophylla seed oil had the highest fraction (21.3%) of LCFAs
(Table 11) including lignoceric (9.8%), behenic (6.3%) and eicosenoic acids (2.4%) (Table 10).
Milletia dura seed oil had the second high fraction (21.3%) of LCFAs including lignoceric acid
2.6%, gadoleic acid 2.4%, erucic acid 0.7% and remarkably high percentage (7.3%) of behenic acid.
This Behenic acid was also reported by Ezeagu et al., (1998) in related species Milletia thonningii
seed oil at content (8.93%) nearly similar to that found from Milletia dura seed oil in current study.
Millettia pinnata has been cultivated in India as a source of lamp oil and a natural medicine for
3,000 years (Earth Equity, 2008). Ezeagu et al. (1998) had found from Millettia thonningii samples
from Nigeria FAs profile very alike to that of analyzed samples of Milletia dura in current study.
The behenic, lignoceric and gadoleic acid seem to be characteristic to this Milletia genus. The
LCFAs fraction of Tephrosia vogelii seed oil (10.2 %) contained notably behenic acid 5.8% and
lignoceric acid 1.5%.
Groundnut oil which has arachidic acid content ranging from 1.1 to 2.3% (Davis et al., 2008) is
known as vegetable source of this FA (Zamora, 2005). In analyzed plant species C. grandiflora, C.
halicacabum, M. eminii, P. macrophylla and T. vogelii had arachidic acid content alike that of
groundnut oil with respectively 1.1, 2.4, 2.3, 2.0, and 2.0%. Also, the FA profile of Maesopsis eminii
oil analyzed had found resembles to that of peanut cultivars oil as reported by Davis et al. (2008).
66

In Cardiospermum halicacabum seed oil more than 6% of total FAs are constituted of FAs of LCFA
(Table 11). Surprisingly, I did not find the 11-eicosenoic (gadoleic) acid as major FA as reported in
previous works in samples originating in Brazil, Holland and Pakistan (Ahmad, 1992; Chisholm and
Hopkins, 1958; Hopkins and Swingle, 1967; Mikolajczak et al., 1970). Nevertheless, Chisholm and
Hopkins (1958) had reported other previous works where this same FA was absent in C.
halicacabum seed oil. These differences may be due seasonal variation or soil (Akoh and Nwosu,
1992) because in one species of plant it can have meaningful difference in quantity and quality of
substances restrained according to the different soil properties (Calvaruso et al. 2006). Further
investigations on this species in current study area are necessary.
In Myrianthus arboreus and Myrianthus holstii oils the high amount of linoleic acid may be useful
as chemotaxonomic criteria for this genus. The Myrianthus species characterized in this study
showed similar FA composition both qualitatively and quantitatively. They all have linoleic as
predominant acid respectively at 77.2 and 80.2% followed by oleic acid 12.3 and 9.8%, palmitic acid
3.9 and 4%, and stearic acid 2.8 and 3.3%. These two Myrianthus species are harvested in
geographically distinct localities, Irangi and Tshibati (Table 6), situated more than 100 Km
apart(Figure 1). Nevertheless this resemblance is only about usual acids i.e. linoleic acid, oleic acid,
palmitic acid, stearic acid. On the contrary, the LCFA are quantitatively different between two
species. This is so for EPA with respectively 0.372 and 0.071 %, arachidic acid 0.001 and 0.122%,
erucic acid 0.171 and 0.001% and DHA 0.089 and 0.001%.
67

6.1. Conclusions
CHAPTER SIX
CONCLUSIONS AND RECOMMENDATIONS
The seeds of all eleven plants analyzed had higher oil contents than those of olive seed, soybean and
palm walnut. With this comparison it can be concluded that, based on their oil content these plants
have the potential to be domesticated as an economic source of oil.
Although the plant oils extracted and characterized had good structural values, it is not yet clear
whether they can be consumed because of possible toxicity. Refining could be one of the measures
to improve utilization of these oils. For example Tephrosia vogelii seed contains recognized
poisonous substances and must be adequately refined before food use. However, according to oils
physicochemical characteristics and fatty acid composition established, the findings of the current
study can be summarized as follows:
• Maesopsis eminii, Podocarpus usambarensis and Tephrosia vogelii seed oils have dietary
and medicinal value, because of their profile of essential fatty acids omega-3 and omega-6.
• Myrianthus arboreus and Myrianthus holstii seeds oils are nutritionally valuable due to their
high linoleic acid and the small amounts of total saturated fatty acids which may be an
advantage for patients with cardiovascular diseases.
• Myrianthus seed oils due to their low content of saturated fatty acids may be used in cold
kitchen and stored in cool, dry and dark place to avoid oxidation.
• Maesopsis eminii oil because of similarities between its fatty acid profile with that of
groundnut oil can be used as a substitute to this expensive oil.
68

• Carapa procera seed oil rich in monounsaturated and saturated fatty acids, is more stable to
oxidative rancidity and stable as deep frying oils. It can also be used as for butter or
margarine. The bitterness of oils from all Carapa species can limit its alimentary use if it can
not be eliminated enough by refining.
• Oils from studied plants show good promise in use in the cosmetic industry and particularly
most promising are Carapa grandiflora, Carapa procera, Myrianthus arboreus, Myrianthus
holstii and Podocarpus usambarensis seed oil due to their fatty acids profile and high
unsaponifiable matter.
• Milletia dura, Maesopsis eminii, Podocarpus usambarensis, and Tephrosia vogelii seed oils
may be source of α-linolenic acid valuable for use in the diet, as healthy nutrient and a
therapeutic agent.
• Myrianthus species oils may be alternative sources of linoleic acid while Carapa species can
be cheap alternative to palm oil as source of oleic acid valuable as feedstock for the
oleochemicals industry and as part of cosmetics products in which it is a great moisturizer.
• Carapa grandiflora, Cardiospermum halicacabum, Maesopsis eminii, Pentaclethra
macrophylla and Tephrosia vogelii have arachidic acid content alike that of groundnut oil
and can be alternative as sources of that acid.
• The long chain fatty acids found in Pentaclethra macrophylla, Podocarpus usambarensis,
Milletia dura and Tephrosia vogelii seed oils may have important chemotaxonomic
significance.
• The analyzed plant oils may have good application as biofuels and the most promising are
those of high density and relative low melting point as Tephrosia vogelii, Treculia africana,
69

Myrianthus arboreus, Myrianthus holstii, Carapa grandiflora, Millettia dura and Carapa
procera.
• Myrianthus arboreus and Myrianthus holstii oils are highly unsaturated to may be used to
make varnishes for timber as unsaturated oil are suitable to be used in paint manufacture.
6.2. Recommendations
Based on the findings of the study, the following recommendation can be made in order to extend
the range of use of the oil from wild plants in Kivu Region of D.R. Congo:
• The oils extracted from selected wild plants in Kivu, D.R. Congo should be refined and
determine the characteristics and FA composition of these refined oils, because refining bring
improvement in the quality of the oil by removing variable impurities,
• There is need to carry out wet fractionation of crude and refined oils using organic and
alcoholic solvents and to determine the characteristics and fatty acid composition of the
initial oils and resulting fractions, because fractionation modifies the technological properties
of oils and increases the oil use range , shelf life and added value,
• There is need to carry out dry fractionation of crude and refined oils and to determine the
characteristics and fatty acid composition of the initial oils and resulting fractions because
dry fractionation processing costs are low, while wet fractionation using solvents is
expensive but cleans better the resulting fractions.
• As for Myrianthus arboreus and Myrianthus holstii, additional studies are necessary by
increasing sampling to improve the understanding of statistic resemblance and differences of
fatty acid profiles and other criteria of these species to elucidate their possible
chemotaxonomic significant.
70

• Further investigations on Cardiospermum halicacabum in current study area could help to
clear the fact that the sample from this study area contained as trace 11-eicosenoic acid
which is reported as major fatty acid in various anterior works in samples from elsewhere.
71

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APPENDICES:
Appendix 1: Analysis of variance of seed oil content and physicochemical characteristics of oils
from plant species obtained from Kahuzi-Biega National Park and the
surrounding areas in D.R. Congo.
Source of variation d.f. s.s. m.s. v.r. F pr.
%oil Between Groups 10 6117.7345 611.7735 1367.53

Appendix 2: Gas Chromatogram of fatty acids methyl esthers of oils from plant species
obtained from Kahuzi-Biega National Park and the surrounding areas in D.R.
Congo
1 1A
250
mV
18:1n9
200
150
100
50
16:0
18:0
18:2n6
19:0
9
14:0
16:1n7
18:1n7
12
18:3n3 20:0
20:1n9 20:2n6 5 20:3n3 20:5n3 22:0 22:1n9
24:024:1n9 min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
Carapa grandiflora
250
200
150
100
1 2A
mV
18:0
18:1n9
50
19:0
16:0
9
18:1n7 18:2n6 2 3 4 20:020:1n9 22:0 24:022:6n3 24:1n9 min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
Carapa procera
84

Appendix 2: Gas Chromatogram of fatty acids methyl esthers (Continued)
200
1
mV
18:1n9
3A
18:2n6
150
100
50
16:0
18:0
19:0
9
16:1n7
18:1n712
18:3n3
20:0
20:1n9
5
22:0
20:5n3
24:0
24:1n9 min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
Cardiospermum halicacabum
220
1 4A
mV
18:1n9
150
100
16:0
18:0
18:2n6
50
19:0
3
20:020:1n9
9
14:0
16:1n7
18:1n7 12 4
20:2n6 20:3n3 20:5n3
min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
22:022:1n9 24:0
Maesopsis eminii
85

Appendix 2: Gas Chromatogram of fatty acids methyl esthers (Continued)
1 5A
180
mV
18:1n9
150
125
100
75
18:2n6
18:3n3
50
22:0
16:0
19:0
18:0
20:1n9
24:0
9
14:0
18:1n712
3 4
20:0
20:2n6 5
22:1n9
20:5n3
22:6n3 24:1n9
min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
Millettia dura
86

Appendix 2: Gas Chromatogram of fatty acids methyl esthers (Continued)
300
250
200
150
100
1 6A
mV
18:2n6
50
18:1n9
9
14:1n5
16:0
16:1n7
18:018:1n7
12
19:0
318:3n3 20:020:1n9 20:2n6 5 20:3n3 6 20:5n3 22:022:1n9 24:0 22:6n3 24:1n9 min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
Myrianthus arboreus
350
300
250
200
150
100
1 7A
mV
18:2n6
16:0
18:1n9
18:0
19:0
9
16:1n7
18:1n7 12 18:3n3 20:020:1n9 20:2n6 5 20:3n3 6 20:5n3 22:0 22:1n9 24:0 min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
Myrianthus holstii
87

Appendix 2: Gas Chromatogram of fatty acids methyl esthers (Continued)
1 8A
220
mV
18:2n6
150
100
18:1n9
50
16:0
19:0
18:0
20:0
9
14:1n5
16:1n7
18:1n712
18:3n3
min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
20:1n9
24:0
22:0
20:2n6 20:5n3 22:1n9
24:1n9
Pentaclethra macrophylla
200
1 9A
mV
18:1n9
150
100
50
18:2n6
18:3n3
16:0
18:0
19:0
20:1n9
9
18:1n712
3 4 20:0
min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
20:2n6
5
6
20:3n3 20:5n3 22:0 22:1n9
24:0
Podocarpus usambarensis
88

Appendix 2: Gas Chromatogram of fatty acids methyl esthers (Continued)
160
125
100
75
50
1 10A
mV
16:0
18:0
18:1n9
18:2n6
18:3n3
19:0
9
14:0
16:1n7
18:1n712
3 4
20:0
20:1n9 20:2n6 5 20:5n3 22:1n9
24:0
min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
Tephrosia vogelii
180
1 11A
mV
18:2n6
150
125
100
75
50
16:0
18:0
18:1n9
19:0
9
14:0
16:1n7
18:1n712
3
4
20:0
20:1n9 20:2n6 5
20:5n3 22:022:1n9 24:0
min
8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0
Treculia africana
89
22:0