a study of the quality of a local herbal tea and volatiles of parinari ...

A STUDY OF THE QUALITY OF A LOCAL HERBAL TEA AND 

VOLATILES OF PARINARI CURATELLIFOLIA (MOBOLA PLUM), 

STRYCHNOS COCCULOIDES (MONKEY ORANGE), FADOGIA 

ANCYLANTHA, MANIHOT ESCULENTA (CASSAVA) AND 

COLOCASIA ESCULENTA (COCOYAM) GROWING IN MALAWI 

MASTER OF SCIENCE (APPLIED CHEMISTRY) THESIS 

BY 

TINOTENDA SHOKO 

UNIVERSITY OF MALAWI 

CHANCELLOR COLLEGE 

APRIL, 2012

A STUDY OF THE QUALITY OF A LOCAL HERBAL TEA AND 

VOLATILES OF PARINARI CURATELLIFOLIA (MOBOLA PLUM), 

STRYCHNOS COCCULOIDES (MONKEY ORANGE), FADOGIA 

ANCYLANTHA, MANIHOT ESCULENTA (CASSAVA) AND 

COLOCASIA ESCULENTA (COCOYAM) GROWING IN MALAWI 

MASTER OF SCIENCE (APPLIED CHEMISTRY) THESIS 

BY 

TINOTENDA SHOKO 

BSc Food Science and Technology-University of Zimbabwe 

A thesis submitted to the Department of Chemistry, Faculty of Science in fulfillment of 

the requirements for the degree of Master of Science (Applied Chemistry) by Research 

UNIVERSITY OF MALAWI 

CHANCELLOR COLLEGE 

APRIL, 2012

DECLARATION BY THE CANDIDATE 

I the undersigned hereby declare that this thesis is my own original work which has not 

been submitted to any other institution for similar purposes. Where other people’s work 

has been used acknowledgements have been made. 

Full Legal Name 

Signature 

Date 

i

CERTIFICATE OF APPROVAL 

The undersigned certify that this thesis represents the student’s own work and effort and 

has been submitted with our approval. 

Signature: Date: 

Saka J.D.K., PhD (Professor) 

Main Supervisor 

Signature: Date: 

Monjerezi M., M.Eng (Lecturer) 

Member, Supervisory Committee 

Signature: _______________________ Date: ____________________________ 

Mwatseteza J.F., PhD (Senior Lecturer) 

Head of Department 

ii

DEDICATION 

I dedicate this thesis to my parents who taught me the value of education and to my sister 

for her support in so many different endeavors. I am greatly indebted to them for their 

continued love and support. 

iii

ACKNOWLEDGEMENTS 

It is a pleasure to thank my supervisory team, Prof J.D.K. Saka and Mr M. Monjerezi for 

their guidance throughout my research. I also want to thank Prof Z.Apostolides of the 

University of Pretoria, Prof A.Viljoen and Dr G. Kamatou of Tshwane University of 

Technology for technical support. I am also thankful for the technical support and 

guidance provided by Mr Kamanula of Mzuzu University. My acknowledgements would 

not be complete without the mention of colleagues, members of the academic and 

technical staff in the Chemistry department at University of Malawi. 

This work was co-funded by the Carnegie Regional Initiative in Science Education 

(Carnegie-RISE) through the Southern Africa Biochemistry and Informatics for Natural 

Products (SABINA) network and the ACP EU through the POL-SABINA network. This 

support is gratefully acknowledged. 

Above all, I thank the Lord God Almighty for my life, wellbeing and for granting me the 

opportunity and capacity to do this work. 

iv

ABSTRACT 

This study concerned the quality of a local herbal tea (Fadogia ancylantha Schweinf) and 

volatile constituents of indigenous fruits (Strychnos cocculoides and Parinari 

curatellifolia) and vegetables (cocoyam and cassava) in Malawi. Tea quality parameters 

were determined by extracting tea samples with organic solvents and calculating 

thearubigin and theaflavin contents from optical density measured at 360 nm. Total 

colour and brightness were calculated from absorbance measurements at 460 nm. Volatile 

constituents were isolated using distillation and headspace methods, and subsequently 

identified by GC and GC-MS. 

The total theaflavin content in F. ancylantha tea (0.37 %) was significantly (p

Esters were the dominant group of compounds identified in P. curatellifolia fruit pulp. 

Both the SPME and CFMD methods identified ethyl butyrate, ethyl isovalerate, phenyl 

acetonitrile and phenyl alcohol in P. curatellifolia. The use of CFMD in ripe S. 

cocculoides identified 1-oxiranylethanone, ethyl-2-methylbutyrate and 2-butoxy ethanol. 

In the unripe S. cocculoides, only 2-butoxyethanol was identified using CFMD. 

Hydrodistillation and solvent extraction from the hydrosol of fresh cocoyam leaves gave 

oxoisophorone, citronellyl formate, 2-butoxy ethanol, linalool, citronellol, nerol, geraniol 

and phenol. Compounds such as 2-hexanal, benzaldehyde, geranyl acetone, β-ionone, 

β-ionone epoxide and phenyl ethyl alcohol were identified in the dried leaves using 

CFMD and the hydrodistillation then solvent extraction methods. 

Fatty acids and carbonyls were the only classes of compounds identified in fresh cassava 

leaves after steam and hydrodistillation subsequently followed by solvent extraction. 

Both these two methods also extracted geranyl acetone, β-ionone, methyl palmitate, 

ethyl palmitate and methyl linolenate. Using hydrodistillation and solvent extraction of 

the hydrosol, fresh and dried cassava leaves gave carbonyls as the dominant class of 

compounds. The compounds, 2-hexanal, 1-hexanol, trans linalool oxide and benzyl 

alcohol were identified using the CFMD from both fresh and dried leaves. 

The presence of volatile constituents of S. cocculoides fruits, F. ancylantha, cocoyam and 

cassava leaves creates an opportunity for their utilisation in flavour, fragrance and 

cosmetic formulations. 

vi

TABLE OF CONTENTS 

DECLARATION BY THE CANDIDATE ......................................................................... i 

CERTIFICATE OF APPROVAL ....................................................................................... ii 

DEDICATION ................................................................................................................... iii 

ACKNOWLEDGEMENTS ............................................................................................... iv 

ABSTRACT .........................................................................................................................v 

LIST OF FIGURES .......................................................................................................... xii 

LIST OF TABLES ........................................................................................................... xiii 

ABBREVIATIONS ......................................................................................................... xiv 

CHAPTER 1: INTRODUCTION ........................................................................................1 

1.1 Background ..............................................................................................................1 

1.2 Problem statement and justification .........................................................................3 

1.3 General and specific objectives ...............................................................................5 

CHAPTER 2: LITERATURE REVIEW .............................................................................6 

2.1 Uses of plant natural products..................................................................................6 

2.2 Natural products contributing to the quality of tea ..................................................6 

2.3 Fruit and vegetable aroma ......................................................................................10 

2.4 Biogenesis of aroma compounds ...........................................................................11 

vii

2.4.1 Metabolism of fatty acids...........................................................................11 

2.4.2 β-Oxidation Pathway .................................................................................12 

2.4.3 Lipoxygenase (LOX) Pathway ..................................................................14 

2.4.4 Metabolism of carbohydrates (isoprenoid pathway) ..................................15 

2.4.5 Metabolism of amino acids ........................................................................17 

2.4.6 Shikimic acid Pathway ...............................................................................18 

2.5 Determination of volatile flavour constituents ......................................................20 

2.5.1 Extraction methods ....................................................................................20 

2.5.1.2 Distillation methods .............................................................................21 

2.5.1.2.1 Steam distillation ................................................................................22 

2.5.1.2.3 Hydrodistillation .................................................................................23 

2.5.1.3 Headspace Methods .............................................................................24 

2.5.1.3.1 Solid-phase micro extraction (SPME) ................................................24 

2.5.1.3.2 Cold finger molecular distillation .......................................................25 

2.5.2 Separation, identification and quantification .............................................26 

2.6 Fruits, vegetables and tea plants ............................................................................28 

2.6.1 P. curatellifolia ..........................................................................................28 

2.6.2 S. cocculoides .............................................................................................29 

2.6.3 C. esculenta (L.) Schott .............................................................................30 

2.6.4 M. esculenta Cranz.....................................................................................32 

viii

2.6.5 F. ancylantha Schweinf .............................................................................33 

2.6.6 Camelia sinensis ........................................................................................34 

CHAPTER 3: MATERIALS AND METHODS .............................................................36 

3.2 Sample Treatment ..................................................................................................38 

3.3 Chemicals, reagents and apparatus ........................................................................38 

3.4 Preparation of chemicals and reagents ...................................................................39 

3.4.1 Sodium hydrogen carbonate (aq) solution (2.5 %, m/v) ............................39 

3.4.2 Saturated aqueous oxalic acid solution ......................................................39 

3.4.3 Methanol solution (60 %, m/v) ..................................................................39 

3.5 Determination of moisture content of F. ancylantha and Lipton 

control teas.............. ..................................................................................39 

3.6 Determination of theaflavins, thearubigins, colour and brightness of 

F. ancylantha and Lipton control teas ..........................................................40 

3.6.1 Preparation of tea samples .........................................................................40 

3.6.2 Extraction of theaflavins and thearubigins ................................................40 

3.6.3 Calculations of total theaflavins, total thearubigins, total colour and 

brightness. .....................................................................................................42 

3.7 Extraction of volatile constituents of F. ancylantha tea leaves .............................44 

3.7.1 Hydrodistillation and solvent extraction of F. ancylantha tea leaves ........44 

3.7.2 Hydrodistillation and solvent extraction from hydrosol of dried 

F. ancylantha tea .....................................................................................44 

3.7.3 Cold finger molecular distillation of F. ancylantha tea leaves ..................45 

ix

3.8 Extraction of volatile constituents of cassava and cocoyam leaf vegetables .........46 

3.8.1 Steam distillation and solvent extraction of fresh cassava leaves..............46 

3.8.2 Hydrodistillation and solvent extraction from hydrosol of dried cassava 

leaves…………………………………………………………………......46 

3.8.3 Hydrodistillation and solvent extraction from hydrosol of fresh cassava 

leaves……..................................................................................................47 

3.8.4 Hydrodistillation and solvent extraction from hydrosol of fresh 

cocoyam leaves...................... ....................................................................47 


cocoyam leaves................... .......................................................................48 

3.8.6 Hydrodistillation and solvent extraction of fresh cassava leaves ..............49 

3.8.7 Cold finger molecular distillation of fresh cassava leaves.........................49 

3.8.8 Cold finger molecular distillation of dried cassava leaves ........................50 

3.8.9 Cold Finger Molecular Distillation of dried cocoyam leaves ....................50 

3.9 Extraction of volatile constituents in P. curatellifolia and S. cocculoides fruits ...51 

3.9.1 Solid phase micro extraction (SPME) of P. curatellifolia and 

S. cocculoides fruit pulp........... ..................................................................51 

3.9.2 Cold finger molecular distillation of ripe S. cocculoides fruit pulp ...........51 

3.9.3 Cold finger molecular distillation of unripe S. cocculoides fruit pulp .......52 

3.9.4 Cold finger molecular distillation of P. curatellifolia fruit pulp ...............52 

3.10 Identification of volatile constituents.....................................................................53 

x

3.10.1 GC and GC-MS conditions for SPME .......................................................53 

3.10.2 GC and GC-MS conditions for hydrodistillation, steam distillation and 

CFMD samples ..........................................................................................53 

3.11 Data analysis ..........................................................................................................54 

CHAPTER 4: RESULTS AND DISCUSSION .................................................................55 

4.1 Quality of F. ancylantha tea .................................................................................55 

4.2 Identity of volatile constituents of F. ancylantha tea leaves .................................57 

4.3 Volatile constituents of S. cocculoides and P. curatellifolia ................................61 

4.3.1 Identity of compounds extracted from P. curatellifolia fruits using SPME . 

and CFMD methods ...................................................................................61 

4.3.2.1 Identity of volatile compounds extracted from S. cocculoides fruits 

using SPME and CFMD methods ........................................................64 

4.3.2.2 Effect of ripening on volatile constituents of S. cocculoides ...............66 

4.4 Volatile constituents of cocoyam and cassava leaves ............................................66 

4.4.1. Identity of volatile constituents of cocoyam leaves ......................................66 

4.4.2 Identity of volatile flavour constituents in cassava leaves ............................69 

CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS .....................................75 

5.1 Conclusions ............................................................................................................75 

5.2 Recommendations ..................................................................................................77 

REFERENCES ..................................................................................................................78 

xi

LIST OF FIGURES 

Scheme 2.1: Enzymatic degradation of fatty acids by the β-oxidation cycle and 

formation of various types of aroma compounds in fruits and vegetables ..... 13 

Scheme 2.3:Isoprenoid pathway: Metabolic pathways leading to the formation of 

terpenes in citrus fruit.....................................................................................16 

Scheme 2.4:Schematic pathway for the formation of volatiles from amino acids ............ 18 

Scheme 2.5:Pathway for the formation of phenolic acids and phenol esters in banana .... 19 

Scheme 2.6: Processing of F.ancylantha tea in Karonga Malawi ...................................... 34 

Figure 2.1: Ripe P. curatellifolia fruits .............................................................................. 28 

Figure 2. 2: Ripe S. cocculoides fruits ................................................................................ 29 

Figure 2.3: C. esculenta leaves ........................................................................................... 30 

Figure 2.4: M. esculenta leaves .......................................................................................... 32 

Figure 2.5: F. ancylantha herb ........................................................................................... 33 

Figure 3.1: Map of Malawi showing three locations from which samples 

were collected............................................................................................... 37 

Figure 4.1:Structures of compounds identified in F. ancylantha tea leaves after 

extraction using the hydrodistillation methods ............................................... 60 

Figure 4.2: Structures of compounds extracted from the headspace of S. cocculoides 

fruit pulp using SPME .................................................................................... 65 

Figure 4.3: Structures of compounds identified in fresh cassava leaves after 

extraction using both the HS and the SS methods. ......................................... 70 

Figure 4.4: Structure of compounds identified in both fresh and dried cassava 

leaves following hydrodistillation and solvent extraction from the hydrosol. 72 

Figure 4.5: Structures of compounds identified in fresh and dried cassava leaves 

following CFMD extraction ............................................................................ 73 

xii

LIST OF TABLES 

Table 4.1: Quality parameters of Lipton green, Lipton black and F. ancylantha teas ....... 55 

Table 4.2: Volatile constituents of F. ancylantha tea extracted using 

hydrodistillation and solvent extraction from the hydrosol ............................ 58 

Table 4.3: Volatile constituents of F. ancylantha tea extracted using the 

hydrodistillation and solvent extraction method ............................................. 59 

Table 4.4: Presence and identity of P. curatellifolia volatiles extracted using 

the SPME and the CFMD methods. ................................................................ 62 

Table 4.5: Presence and identity of volatile compounds identified in ripe and 

unripe S. cocculoides fruits following SPME and CFMD extraction ............ 64 

Table 4.6: Compounds identified after hydrodistillation and solvent extraction 

from the hydrosol of fresh cocoyam leaves .................................................... 67 

Table 4.7: Volatile constituents obtained from dried cocoyam leaves 

using hydrodistillation and solvent extraction from the hydrosol 

(HSH) and CFMD ........................................................................................... 68 

Table 4.8: Volatile compounds from hydrodistillation and solvent extraction 

(HS) and steam distillation and solvent extraction (SS) of fresh 

cassava leaves ................................................................................................. 69 

Table 4.9: Compounds identified after hydrodistillation and solvent extraction 

from the hydrosol of fresh and dried cassava leaves ...................................... 71 

Table 4.10: Compounds identified after CFMD of fresh and dried cassava leaves ........... 74 

xiii

ABBREVIATIONS AND ACRONYMS 

CFMD - Cold finger molecular distillation 

GC - Gas chromatography 

GC-MS - Gas chromatography-mass spectrometry 

HS - Hydrodistillation and solvent extraction 

HSH - Hydrodistillation and solvent extraction of the hydrosol 

RI - Retention Indices 

RPM - Revolutions per minute 

SDE - Simultaneous distillation extraction 

SPME - Solid phase microextraction 

SS - Steam distillation and solvent extraction 

xiv

1.1 Background 

CHAPTER 1: INTRODUCTION 

Plants are essential for food security, health, social and economic welfare of rural 

communities (Agbidye et al., 2009). Indigenous fruit trees provide a major part of the 

food and nutritional requirements of people living in sub-Saharan Africa (Chirwa and 

Akkinifesi, 2008). Local fruits such as Parinari curatellifolia (maula) and Strychnos 

cocculoides (kabeza) have unique flavours and provide nutritional value (Saka et al., 

2008; Saka et al., 1994). Cocoyam leaves (masamba ya zicheche or masamba ya 

masimbi) and cassava (chigwada) leaves are used as traditional leaf vegetables in 

Malawi. The vegetables have a pleasant flavour and nutritional value; they contain 

essential and non essential amino acids (Mkambankhani, 2009). Cassava leaves are rich 

in crude fibre, protein, water, vitamin A, C, E and several minerals (Ebuchi et al., 2005). 

Flavour is the combined characteristics of a food material taken in the mouth, perceived 

principally by the senses of taste and smell as well as the general pain and tactile 

receptors in the mouth and interpreted by the brain (Burdock and Fenaroli, 2005). The 

aroma, taste, texture and mouth feel of the food account for the major stimuli that 

constitute flavour (Taylor, 1996). Taste is concerned with the sensations of sweet, sour, 

salty, bitter and savoury which are associated with receiptors on the tongue (Reineccius, 

1993). In contrast, texture and consistency produce physical impressions like touching, 

1

pressing, resistance against grinding or a viscous smooth pudding like feeling (Rothe, 

1988). Mouth feel responses include the heat of spices and the cooling of menthol 

(Reineccius, 1993). Aroma, which is made up of thousands of volatile components is 

mainly responsible for the characteristic flavours of foods (Grosch, 2004). Generally, 

flavour chemistry is, most often associated with the volatile component of flavour 

(aroma) (Reineccius, 1993). 

Plants add flavour, taste, colour and aesthetic appeal to what would otherwise be a 

monotonous diet (Mepba, 2007). Aromatic fruits and leaves are used to flavour soups and 

stews, and are sometimes put in beer and ale to improve flavour and increase forming 

(Fern, 2007). Lemongrass leaves are widely used as a lemon flavour ingredient in herbal 

teas and other formulations (Skaria et al., 2007). Monkey orange is traditionally used to 

flavour cereal porridge and the pulp has a sweet taste (Saka et al., 2008; Mwamba, 2005). 

A local plant in Malawi, F. ancylantha Schweinf (masamba ya muthondo) is used as a 

herbal tea and is strongly preferred by the Vundakulwayo community in the Northern 

region of Malawi. It has no caffeine (Darvish, 2004). Tea is probably the most widely 

consumed beverage in the world (Reilly, 2002). It contains volatile oils, vitamins, 

minerals, purines and polyphenols which include catechins, theaflavins, and thearubigins 

(Ferrara et al., 2001; Frei and Higdon, 2003). The quality of tea largely depends on the 

components in the leaves and aroma components are one of the most important factors to 

influence the flavour, taste and quality of tea such as Pu-erh (Liang et al., 2005b). 

2

Considering the benefits associated with herbal teas and increasing interest in 

complementary and alternative medicines, it is important to know about the parameters 

which determine the quality of the local herbal teas such as F. ancylantha. Information on 

volatile constituents in some local fruits and vegetables found in Malawi would open new 

doors for utilisation of these plants. 

1.2 Problem statement and justification 

Studies have shown that some indigenous plant species are rich in amino acids 

(Mkambankhani, 2009). In rural Africa, wild fruits provide diversity of foods, vitamins 

and minerals in the diet (Harris and Mohammed, 2003). Apart from their nutritional 

value, plants are also eaten because of their flavour (Mepba, 2007). 

Indigenous fruits such as mobola plum and monkey orange have unique flavour. P. 

curatellifolia has a sweet and strong pineapple smell (Williamson, 1975; Maundu et al, 

1999). Joulain et al (2004) identified 2-phenylethanol as the major component of the 

volatile fraction of mobola plum using a vacuum heaspace concentration method. 

Monkey orange is traditionally used to flavour cereal porridge and the pulp has a sweet 

taste (Saka et al., 2008). Unlike P. curatellifolia the volatile constituents of S. 

cocculoides have never been studied before. 

The leaves of Manihot esculenta (cassava) and Colocasia esculenta (cocoyam) are used 

as vegetables in Malawi. Volatile constituents of C. esculenta tuber have been 

determined. Major components were palmitic acid and linoleic acid (Wong et al., 1998). 

In another study, cooked taro (C. esculenta) corms were extracted using the Lickens 

3

Nickerson method followed by a low temperature-high vacuum concentration, 62 

components were identified (Macleod, 1990). Dougan et al (1983) obtained some volatile 

components of fresh cassava root using a simultaneous distillation extractraction method. 

Fourty compounds were identified in freshly cooked cassava. The major classes of 

compounds were aromatic hydrocarbons, aliphatic carboxylic acids, aliphatic and 

aromatic carbonyl compounds. Since cassava leaves are better flavoured than the tuber, 

the associated soup had better aroma than other vegetable soups tested (Mepba, 2007). 

Volatile constituents of cocoyam and cassava leaves have not been studied. 

Aroma volatiles in food samples cover a wide range of polarities, solubilities, functional 

groups, vapour pressures, concentrations and volatilities. There is no perfect sample 

preparation technique to use for flavour research (G1Innovations, 2003). In flavour 

extraction, the extraction method used should provide an extract with sensory 

characteristics as close as possible to the complete product (Sarrazin et al., 2000). Since 

volatiles in a food sample have different chemical characteristics and cannot be extracted 

using a single method, different extraction methods should be used to capture the widest 

range of volatiles from a food material. 

Information regarding the flavour of food is critical in developing a variety of pleasurable 

aromas in foods (Nadine, 2009). Knowledge of the type and composition of aromatic 

volatiles in the fruits and vegetables is required to expand the formulation of new 

flavours in the food industry. 

4

A local herbal tea made from F. ancylantha Schweinf is strongly preferred by the 

Chilumba community in Karonga region of Malawi. The use of associated genotype in 

Zimbabwe as a tea is well documented (Mencherini et al., 2010). Prices of various teas 

are quite variable and are dependent on the quality of the tea which is traditionally 

assessed by the tea taster. The tea taster trade have a language to describe various quality 

attributes of tea infusion which is sometimes difficult to understand to the consumer 

(Liang et al., 2005a). There has been need to identify objective and reliable chemical tea 

quality parameters to augment the current subjective organoleptic evaluation method in 

the assessment of value of tea (Okinda Owour, 2005). It is therefore important to 

develop precise chemical or physical methods for the objective estimation of the quality 

of tea (Liang et al., 2005a). The quality of F. ancylantha tea leaves has not been 

previously established. 

1.3 General and specific objectives 

The main objective of this study was to investigate the quality of a local herbal tea and 

the flavour volatiles of some edible indigenous fruit and vegetable species in Malawi. 

The specific objectives are two fold: 

i). To determine the content of theaflavins, thearubigins, colour, brightness and volatile 

constituents of a local herbal tea. 

ii). To establish the identity of volatile constituents of two edible fruits (mobola plum and 

monkey orange fruits) and leaf vegetables (cocoyam and cassava). 

5

CHAPTER 2: LITERATURE REVIEW 

2.1 Uses of plant natural products 

Plants are a source of natural chemicals and materials such as medicines, flavorings, 

fragrances, dyes, oils, wood, fiber and rubber (Grace, 2006). Recognition of the 

biological properties of myriad natural products has catalysed the search for new drugs, 

antibiotics, insecticides and herbicides (Croteau et al., 2000). Natural products have 

served as a major source of drugs for centuries, and about half of the pharmaceuticals in 

use today are derived from natural products (Clark, 1996). Tea is a natural product with 

flavouring properties (Sikorski, 2002) and therefore it is used both as a fresh drink and 

also a traditional herb for human health (Quan et al., 2007). 

2.2 Natural products contributing to the quality of tea 

The quality of tea largely depends on the components in the leaves (Liang et al., 2005a; 

Liang et al., 1996). Chemical composition of tea is complex and not completely 

understood (Hamilton-Miller, 1995). Usually, tea is classified as green, black, oolong, 

yellow, white and dark compressed tea (Liang et al, 2005a). Green tea differs from black 

tea in that an oxidation step (called “fermentation”) occurs in the processing of the latter 

tea but not the former (Hamilton-Miller, 1995). If the fermentation is partially carried out, 

oolong tea is obtained (Liang et al., 2005a). Black tea has many more components than 

6

green tea, partly because of the oxidation processes that occur during fermentation 

(Hamilton-Miller, 1995). Tea quality is greatly influenced by the tea polyphenols, amino 

acid and caffeine contents in tea leaves and the colour differences of tea infusions (Liang 

et al., 2005b). Theaflavins, residual catechin compositions, thearubigins, sensory 

characteristics of total colour, brightness and briskness of tea are used as quality 

indicators of tea (Obanda et al., 2001). The total quality score of pu-erh tea was 

significantly correlated to the concentration of amino acids, volatile constituents and 

colour (Liang et al., 2005b). 

It has been reported that tea has various bioregulatory activities such as anticarcinogenic, 

antimetastatic activities, antioxidative activity, antihypercholesterolemic activity, 

antidental caries activity, antihypertensive activity, antibacterial activity, and interstinal 

flora amelioration activity. Major principles for these activities were shown to be 

catechins, a group of polyphenolic compounds (Maeda-Yamamoto et al., 2004). In green 

tea leaves, catechins are present in relatively high amounts of up to 30 % of dry matter 

(Schulz et al., 1999). Green tea has the highest catechin content, and therefore provides 

more health benefits. Consequently, total polyphenols and total catechins content in green 

tea are two important factors in evaluating the quality of green tea (Quan et al., 2007). 

The major tea catechins are (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin 

(EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC) (Zuo et al., 2002). 

In producing black tea, the tea leaves are withered, crushed, and allowed to undergo an 

enzyme-mediated oxidation commonly referred to as fermentation (Lambert et al., 2005). 

7

During fermentation, enzymatic oxidation of catechins takes place leading to the 

formation of theaflavins and thearubigins which are responsible for the characteristics of 

black tea infusions (Liang et al., 2005a). Thus, depending on the extent of fermentation 

the colour of teas will vary accordingly, leading to the colour difference between 

different samples of tea types, such colour differences could be used to differentiate 

between various teas irrespective of the type of processing (Liang et al., 2005a). 

Confirmation was been obtained that theaflavin content is an extremely important factor 

in determining quality in black tea (Robert and Smith, 1963). Separate studies have 

shown that theaflavins are useful black tea quality parameters, which when used properly, 

can give objective estimate of plain black tea quality irrespective of geographical area of 

production (Okinda Owour, 2005). Theaflavins and thearubigins contribute to the 

astringency/ brightness/ briskness and colour/strength/mouthfeel respectively of black tea 

(Sud and Baru, 2000). Theaflavins, found predominantly in black tea, combine with 

caffeine to form a substance known as cream thereby modulating the bitterness and 

stringency of the individual compounds and giving tea its flavour (Hamilton-Miller, 

1995). Theaflavin (together with its mono- and di-galloyl derivatives) are constituents of 

the polyphenol content of tea not only influencing its colour but also contributing to the 

characteristic taste (Charlton et al., 2000). 

Caffeine (1,3,5-trimethylxanthine), a mild addicting drug though used for medicinal 

purposes is the active ingredient that makes tea valuable to humans (Wanyika et al., 

2010). Caffeine, theophylline and theobromine are important quality factors in green and 

black teas (Schulz et al.,1999; Sharma et al., 2004). Caffeine is present in tea at an 

8

average level of 3 % along with very small amounts of the other common 

methylxanthines (theobromine and theophylline)(Graham, 1992). Caffeine has a bitter 

taste (Charlton et al., 2000). 

The amino acid theanine (5-Nethylglutamine) is unique to tea (Graham, 1992). It is the 

main amino acid component in tea and usually constitutes between 1 and 2 % of the dry 

weight of the tea leaves (Ekborg-Ott et al., 1997). Free amino acids in tea leaves are 

important chemical constituents that considerably influence the quality of tea, especially 

that of green tea (Ruan et al., 1998). Theanine is as prevalent in tea as all the other free 

amino acids combined and both enantiomers of theanine were found to have similar 

sweet taste with little or no aftertaste (Ekborg-Ott et al., 1997). 

Volatile components make up a very small fraction of flush and tea leaf (10 to 20 ppm) 

but play an important part on its flavour (Hamilton-Miller, 1995). These flavour 

components are one of the most important factors to influence the flavour, taste and 

quality of Pu-erh tea (Liang et al., 2005b). Volatile constituents of green tea gyokuro (C. 

sinensis) were investigated; seventy-nine compounds were positively identified. Major 

constituents were identified as 2,6,6-trimethyl-2-hydroxycyclohexanone, linalool, 

geraniol, cis-jasmone, β-ionone and cyclohexanone, 5,6-epoxy-β-ionone, indole, and 

caffeine (Yamaguchi and Shibamoto,1981). In fennel tea (Foeniculum vulgare Mill.), 

fenchone and trans-anethole were demonstrated to play an important role in the tea 

flavour (Zeller and Rychlic, 2006). 

9

2.3 Fruit and vegetable aroma 

Fruit aroma which is a complex mixture of a large number of volatile compounds 

contributes to the overall sensory quality of fruit specific to species and cultivar (Sanz et 

al., 1997). Most volatile constituents in fruits contain aliphatic hydrocarbon chains, or 

their derivatives and esters as the major volatiles in fruits. For example, apples which 

have a prominent odour largely produce esters of relatively low molecular weight. In 

contrast, pear odour which is more subtle contains esters of higher molecular weight 

(Fisher and Scott, 1997). 

Vegetables contain flavour compounds which are mostly too low to obtain essential oils 

(Sikorski, 2002). The flavour compounds in vegetables are diverse and include fatty acid 

derivatives, terpenes, sulphur compounds as well as alkaloids (Berger, 2007). This 

diversity is partially responsible for the unique flavours found in different species of 

vegetables. Most vegetable aromas develop during cellular disruption, which allows 

enzymes that act upon non volatile precursors which are chemically bound to precursor 

handles such as sugars for example terpene glycosides and amino acids (Fisher and Scott, 

1997). 

Essential oils are the volatile secondary plant products which are responsible for the 

aroma and flavour characteristic to the plant (Salisbury and Ross, 1992). These are found 

throughout the plant cellular tissue or in special cells, glands or ducts located in several 

parts of the plant like leaves, barks, roots, flowers, fruits or seeds (Heath, 1978). The 

constituents of essential oils are terpenes, terpenoids (oxygenated terpenes) including 

10

alcohols, aldehydes, esters, ethers, ketones, phenols, oxides and other specific 

compounds containing either sulphur or nitrogen (Heath, 1978). While monoterpenes 

(C10) are the lower boiling point fraction of essential oils, sesquiterpenes (C15) are mainly 

found in the higher boiling point fraction. The diterpenes (C20) and triterpenes (C30) are 

generally eliminated by distillation (Croteau, 1980). While terpenes contribute negligibly 

to the flavour profile, they act as a carrier of less volatile constituents and thus 

influencing odour (Heath, 1978). 

2.4 Biogenesis of aroma compounds 

Many flavours, especially those in fruits and vegetables, are the secondary products of 

various metabolic pathways (Fisher and Scott, 1997). There are three main classes of 

compounds in relation to aroma biogeneration; these are fatty acids, amino acids, and 

carbohydrates (Sanz et al., 1997). Secondary metabolites act as an interface between the 

producing organism and its environment, they may be produced to combat infectious 

diseases, to attract pollinators and to discourage or encourage herbivores (Fisher and 

Scott, 1997). Several pathways are involved in aroma biosynthesis; these have not been 

fully described but appear to be common for different fruits (Sanz et al., 1997). 

2.4.1 Metabolism of fatty acids 

Fatty acids seem to be the major precursors of volatile compounds responsible for aroma 

of most plant products (Sanz et al., 1997). The character impact compound for a 

particular flavour or aroma is a unique chemical substance that provides the principal 

sensory identity (McGorrin, 2002). Many character impact compounds responsible for 

the fresh, green, and fruity notes of fruits and vegetables are derived from fatty acids 

(Berger, 2007). Natural plant volatiles, such as aliphatic esters, alcohols, acids and 

11

carbonyls, can be derived from fatty acid metabolism (Fisher and Scott, 1997). In 

general, fatty acids are catabolised through two main oxidative pathways, β-oxidation and 

the lipogenase (LOX) pathway (Sanz et al., 1997). The LOX pathway produces 

compounds known as Green Leaf Volatiles (GLVs) which are C6 or C9-aldehydes and 

alcohols. GLVs are commonly used as flavours to confer a fresh green odour of vegetable 

to food products (Gigot, 2010). 

2.4.2 β-Oxidation Pathway 

β-oxidation typically occurs in intact tissue during ripening of fruits and vegetables 

(Berger, 2007). Most unripe fruits produce a variety of fatty acids which, during ripening 

are converted into esters, ketones and alcohols. The enzyme complex catalysing β- 

oxidation pathway is located in the mitochondria and is inactivated during disruption of 

plant cells (Fisher and Scott, 1997). 

12

Scheme 2.1: : Enzymatic degradation of fatty acids by the β-oxidation oxidation cycle and 

formation of various types of aroma compounds in fruits and vegetables 

(Berger, 2007) 

In the β-oxidation oxidation cycle shown in Scheme cheme 2.1 above fatty acids specifically acyl-CoA acyl 

derivatives are metabolized tto 

shorter chain acyl-CoAs by losing sing two carbons at every 

round of the cycle (Sanz et al., ., 1997). This sequence is repeated several times until the 

complete breakdown of the compound. Depending on many factors, the breakdown can 

be stopped, resulting in the liberation of medium medium-chain-length length or short-chain-length 

short 

volatile compounds. This can lead to variety of volatile compounds such as saturated and 

unsaturated lactones, esters, alcohols ketones and acids (Berger, 2007). Aroma 

differences in varieties of apples are related to the proportion of alcohols and esters which 

depends upon competing reaction rates in the β-oxidation oxidation pathway of the various fatty 

acids (Fisher and Scott, 1997). 

13

2.4.3 Lipoxygenase (LOX) Pathway 

During cell breakdown that occurs naturally in fruit ripening and especially when 

vegetative cells are mechanically broken, oxidation of unsaturated fatty acids is 

catabolised by lipoxygenase (Fisher and Scott, 1997). The enzyme converts 

polyunsaturated fatty acids mostly linoleic and linolenic acids into hydroperoxides, these 

break down to give aldehydes, alcohols and esters (usually with the help of other 

enzymes-hydroperoxide hydroperoxide ly lyases, ases, alcohol dehydrogenases, isomerases and esterases). Each 

type of plant has its own set of lipoxygenases, pH and medium conditions. These factors 

influence the path of the break down resulting in different volatile products from the 

same polyunsaturated d fatty acid starting material (Fisher and Scott, 1997). 

Scheme 2.2: : Lipoxygenase pathway: Reaction scheme for enzymatic splitting of 

linolenic acid into aldehydes and oxo acids (Tressl and Drawert, 1973) 

14

In Scheme 2.2 linolenic acid is transformed into the 13- or 9-hydroperoxy acid by 

lipoxygenase. These intermediate products might be split into aldehydes and oxo acids by 

an enzyme called aldehyde lyase (Tressl and Drawert, 1973). 

2.4.4 Metabolism of carbohydrates (isoprenoid pathway) 

Terpenes of fruit and vegetable aromas are a class of volatile compounds that constitute 

the characteristic components of many essential oils, and contribute to, and in some cases 

determine the aroma of many fruits (Sanz et al., 1997). There are two main types of 

terpenoids that may contribute significantly to the flavour of vegetables and fruits, these 

are monoterpenes and sesquiterpenes. Tissue disruption does not normally alter the 

profile of monoterpenes and sesquiterpenes in the raw product, although changes in the 

concentration of some monoterpenes may occur due to oxidation and release of 

glycoside-bound oxygenated terpenoids (Berger, 2007). 

15

Scheme 2.2: : Isoprenoid pathway: Metabolic pathways leading to the forma formation forma of 

terpenes in citrus fruits (Reineccius, 1998) 

In Scheme 2.2 the six-carbon carbon compound mevalonic acid is transformed into the five – 

carbon phosphorylated isoprene units in a series of reactions beginning with 

phosphorylation of the primary alcohol group. Two different ATP ATP-dependant dependant enzymes 

are involved, resulting in mevalonic acid diphosphate and decarboxylation 

decarboxylation-dehydration 

decarboxylation 

then follows to give isopentenyl pyrophosphate (IP (IPP). P). IPP is isomerised to the other 

isoprene unit, (Dimethylallyl pyrophosphate) DMAPP by an isomerase enzyme. 

Conversion of IPP into DMAPP generates a reactive electrophile and therefore, a good 

16

alkylating agent. DMAPP possesses a good leaving group the diphosphate (Dewick, 

2009). DMAPP acts as a `primer` molecule and undergoes condensation with IPP to give 

the C10 intermediate geranyl pyrophosphate (GPP) precursor of monoterpenes (Britton, 

1983). Geraniol is a result of addition of water to the geranyl cation. Linalyl PP and neryl 

PP are isomers of GPP, and are likely to be formed from GPP by ionization to the allylic 

cation, allowing a change in attachment of the diphosphate group. These compounds by 

relatively modest changes can give rise to a range of linear monoterpenes found as 

components of volatile oils used in flavouring and perfumery. Neryl PP or linalyl PP are 

immediate precursors of the monocyclic menthane system, generating a carbocation 

(termed menthyl or α-terpinyl). The newly generated menthyl cation could be quenched 

by attack of water, in which case the alcohol α-terpineol would be formed, or it could 

lose a proton to give limonene (Dewick, 2009). Successive addition of two further 

molecules of IPP to GPP gives the C15 farnesyl pyrophosphate (FPP), precursor to 

sesquiterpenes, steroids and triterpenes, and the C20 (Geranyl geranyl pyrophosphate) 

GGPP. The chain-lengthening process may continue to give long-chain polyprenols, or 

the GGPP may be used to form C20 diterpenes, including phytol, the sidechain of 

chlorophyll, or to give the C40 carotenoids (Britton, 1983). 

2.4.5 Metabolism of amino acids 

Metabolism of amino acids acting as direct precursors generates alcohols, carbonyls, 

acids and esters, aliphatic, branched, or aromatic (Sanz et al., 1997). Enzymes remove 

both amine and carboxyl groups from the amino acid to produce aldehydes. The 

aldehydes are either enzymatically oxidized or reduced and then esterified (Fisher and 

Scott, 1997). 

17

Scheme 2.3: : Schematic pathway for the formation of volatiles from amino acids 

(Sanz et al., 1997) 

In Scheme 2.3 above leucine is converted into labeled 33-methylbutanol, 

methylbutanol, 3-methylbutanoic 

3 

acid and 3-methylbutyl methylbutyl esters (Tressl and Drawert, 1973). The initial l step is deamination 

of the amino acid followed by decarboxylation (Reineccius, 1998). Various reductions 

and esterifications then lead to volatiles which are significant to fruit flavour (Reineccius, 

1998). 

2.4.6 Shikimic acid Pathway 

The aromatic amino acids, phenylalanine and tyrosine, are formed by the shikimic acid 

pathway. Many aromatic flavour compounds from spices such as eugenol (cloves), 

18

cinnamaldehyde and coumarin (cinnamon), are generated from these amino acids via 

deamination and oxidation or reduction (Fisher her and Scott, 1997). Scheme 2.4 below 

shows generation of aroma compounds from aromatic amino acids phenylalanine and 

tyrosine formed by the shikimic acid pathway. 

These pathways described are mainly responsible for the formation of the wid wide wid range of 

volatile constituents which are available in plants. 

Scheme 2.4: : Pathway for the formation of phenolic acids and phenol esters in 

banana (Tressl and Drawert, 1973) 

19

2.5 Determination of volatile flavour constituents 

Aroma volatiles in food samples can be heterogeneous, covering a wide range of 

polarities, solubilities, functional groups, vapour pressures, concentrations and volatilities 

(G1Innovations, 2003). In order to study the flavour of a food, it is first necessary to 

isolate the volatiles from the complex of non-volatile material (Takeoka and Full, 1997). 

Ideally, the isolation method should not discriminate between polar and non-polar 

compounds, not cause thermal degradation of aroma compounds, oxidation, reduction, 

pH changes or loss of highly volatile compounds (Da Costa and Eri, 2005). There is no 

one perfect sample preparation technique to use for flavour research (G1Innovations, 

2003). Ideally the extraction method used should provide an extract with sensory 

characteristics as close as possible to the complete product (Sarrazin et al., 2000). Based 

on the principle of aroma compound isolation, sample preparation methods can be 

broadly grouped into solvent extraction methods, steam distillation methods, headspace 

techniques and sorptive techniques (Da Costa and Eri, 2005). 

2.5.1 Extraction methods 

2.5.1.1 Solvent extraction 

Solvent extraction is an effective method of isolating highly water-soluble flavour 

constituents which are typically poorly recovered by distillation and headspace 

techniques (Takeoka and Full, 1997) The method uses the concept of transfer of the 

aroma chemicals from the sample to the organic solvent (Da Costa and Eri, 2005). 

Solvent extraction commonly involves the use of pentane, dichloromethane, diethyl ether 

or some other volatile organic solvent (G1Innovations, 2003). The extract is obtained by 

20

mixing and agitating a liquid or solid sample with organic solvent, allowing separation 

and collection of the solvent phase (Da Costa and Eri, 2005). The major limitation of this 

method is that it is most useful on foods that do not contain any lipids, if the food 

contains lipids the lipids will also be extracted along with the aroma constituents 

(Reineccius and Heath, 2006). 

2.5.1.2 Distillation methods 

Distillation methods take advantage of the volatility of flavour components and non 

volatility of the major food constituents (Hui et al., 2010). When foods are boiled in 

water at 100 0 C for the cooking process, volatilisable compounds are released or created 

and evaporated (steam distilled) into the headspace where they condense or evaporate 

into the atmosphere (Self, 2005). Aroma components, by definition must be volatile to 

make a contribution to odour. Distillation utilises the creation of two or more coexisting 

phases, generally two phases, vapour and liquid phases at the same temperature and 

pressure which differ in composition (Hui et al., 2010). The mixture of condensed water 

and essential oil is collected and separated by decantation. Elevated temperatures and 

prolonged extraction time can cause chemical modifications of the essential oil 

components and often a loss of the most volatile molecules (Sawamura, 2010). 

Distillation involves the transfer of material from one phase to another phase (Hui et al., 

2010). During distillation, fragrant plants exposed to boiling water or steam release their 

essential oils through evaporation. Recovery of the essential oil is facilitated by 

distillation of two immiscible liquids, namely, water and essential oil, based on the 

principle that, at boiling temperature, the combined vapour pressure equals the ambient 

pressure (Sawamura, 2010). According to Dalton`s law of partial pressures, a mixture 

21

oils when the sum of partial pressures equals the external pressure (Self, 2005). Thus 

essential oil ingredients, for which boiling points normally range from 100 0 C to 300 0 C 

are evaporated at a temperature close to that of water (Sawamura, 2010). 

2.5.1.2.1 Steam distillation 

Distillation using water vapour is called steam distillation (Hui et al., 2010). Water is 

heated in a separate boiler and steam generated is fed into the food sample in the 

distillation flask (Self, 2005). Volatile constituents pass with the steam into a condenser 

where both oil and steam will be cooled. Oil and water then separate, the oil which is less 

dense can be collected (Hay and Waterman, 1993). Essential oils are released from the 

plant material when the steam bursts sacs containing the oil molecules (Sawamura, 2010). 

Steam distillation can be used when the material to be distilled has a high boiling point or 

decomposition can occur if direct distillation is employed (Hui et al., 2010). Food aromas 

are steam distilled during the cooking process, thus steam distillation is used as an 

extraction medium for cooked flavour analysis (Self, 2005). 

2.5.1.2.2 Steam distillation-solvent extraction 

The distillate from steam distillation can be a very dilute solution of volatile flavours and 

water. Flavour must be isolated from the aqueous solution. This is generally done by 

extracting the distillate with an organic solvent. The organic solvent containing the 

flavour isolate is dried via the addition of anhydrous MgSO4 or Na2SO4 filtered and 

concentrated for instrumental analysis. A major disadvantage of steam distillation-solvent 

extraction is that of an additional solvent step (solvent extraction of the distillate). A 

method whose isolation efficiency depends on volatility has been combined with a 

22

method whose isolation efficiency depends on extraction efficiency. The second step 

adds additional selectivity and error to the method (Reineccius, 1998). 

2.5.1.2.3 Hydrodistillation 

Plant material is immersed in the water which is kept boiling while contents are 

mechanically stirred (Heath, 1978). Steam from boiling water carries the volatile oils 

with it. Cooling and condensation subsequently separate the oil from the water 

(Sawamura, 2010). Condensation conditions are the similar to those of steam distillation 

but the condensate water may be returned to the still by way of a special trap (Heath, 

1978). Apart from its slowness, the disadvantage of this technique is that both materials 

and scent deteriorate from constant heat exposure (Sawamura, 2010). Not all oils can be 

processed by hydrodistillation because boiling water and steam can have deteriorating 

influence upon delicate flavour substances, moreover, certain raw materials yield no oil 

during hydrodistillation (Hui et al., 2007). Hydrolysis of certain components of the 

essential oils and decomposition caused by heat are always present in the 

hydrodistillation process. As such, hydrodistillation cannot be applied to substances 

which, even at low temperatures react with water or are hydrolysed by water for example 

esters (Guenther, 2008). Although hydrolysis is an unavoidable reaction, typically the 

intensity of hydrolysis is low under the conditions normally used. Decomposition by 

degradation of some substances can cause interference with the obtained oil odour. 

Hydrodistillation to extract essential oils should be used keeping the temperature as low 

as possible (Hui et al., 2010). Solubility in water lowers the vapour pressure of the 

compound, reduces its capability for vaporisation and impedes the separation of the oil 

23

from the distillate. For this reason, the aroma of many flowers cannot be isolated by 

distillation (Guenther, 2008). 

2.5.1.3 Headspace Methods 

Headspace analysis, as the name implies, is a way to monitor the composition of the 

gaseous space above a liquid or solid sample in a sealed container (Burgard and 

Kuznicki, 1990). It is especially useful for matrices that give off a lot of odour, such as 

flowers and fruits (Da Costa and Eri, 2005). For matrices that are not odorous, gentle 

heating can be applied to aid in release of volatiles (Rowe, 2005). Due to the fact that 

headspace techniques detect highly volatile compounds, they can be used to help identify 

compounds that may be hidden by solvent peaks in liquid extracts (Da Costa and Eri, 

2005). The composition of the headspace is a function of the air/sample partition 

coefficients for the volatile compounds in the sample (Burgard and Kuznicki, 1990). 

2.5.1.3.1 Solid-phase micro extraction (SPME) 

SPME uses a fused-silica fibre that is coated on the inside with an appropriate stationery 

phase, analytes in the sample are directly extracted to the fibre coating (Vas and Vekey, 

2004). The choice of sorbent is essential, it must have a strong affinity for the target 

organic compounds, the most reported stationery phase for SPME is 

polydimethylsiloxane (PDMS)(Dean, 2009). Essentially, there are two discrete steps: 

solute absorption from the sample matrix into a thick layer of silicone or related 

adsorptive material and transfer of the analytes into a chromatography inlet system by 

gaseous or liquid means (Hinshaw, 2003). Two sampling methods which are immersion 

or headspace sampling can be used with SPME depending on the placement of the fiber 

relative to the sample (G1Innovations, 2003). In the immersion sampling, the fiber is 

24

immersed directly into the sample solution and the analytes are transferred directly from 

the sample matrix to the extracting phase (Tankeviciute et al., 2004). In SPME headspace 

analysis, a fiber is placed in the headspace above the sample (G1Innovations, 2003). 

SPME is simple and easy to use and does not require elaborate and expensive instrument 

accessories (Hinshaw, 2003). It reduces the time necessary for sample preparation, 

decreases purchase and disposal costs of solvents and can improve detection limits 

(Vas and Vekey, 2004). It decouples sample from matrix effects that would distort 

sample composition or disturb the chromatographic separation (Hinshaw, 2003). The 

SPME technique can be routinely used in combination with gas chromatography and 

capillary electrophoresis and places no restriction on mass spectrometry (Vas and Vekey, 

2004). SPME has also been used with HPLC (Dean, 2009). 

2.5.1.3.2 Cold finger molecular distillation 

Molecular distillation is also called high vacuum distillation or short path distillation. 

This method is used to distil compounds having very high boiling points and which can 

get decomposed at high temperatures (Ahluwalia et al., 2005). In flavour analysis, 

retention of the full range of volatiles requires low temperature trapping, dry ice-acetone 

or liquid nitrogen are useful coolants (Self, 2005). The condensers can also be cooled 

with ice water (Reineccius, 1998). In conventional distillation, molecules leaving the 

surface of the boiling liquid may undergo many collisions with residual air molecules. In 

molecular distillation, all air is removed from the system by vacuum. The condensing 

surface is located close to the boiling liquid in such a way that no molecular collisions 

take place. The method is particularly useful for distillation of aromatic materials having 

a molecular weight between 400 and 1200 and distilling between 50 0 C and 150 0 C 

25

(Heath, 1981). The main feature of the distillation unit is the short direct path between a 

heated liquid surface and the cooled condensing area (Ahluwalia et al., 2005). The 

advantages include minimal decomposition of thermo labile components and elimination 

of any oxidative reactions (Heath, 1981). A major problem with cryogenic trapping is 

that water is the most abundant volatile in most foods and, therefore the trap condensate 

is primarily water. An additional step is generally necessary to extract the flavour 

constituents from the water. The condensates are typically pooled and then extracted with 

diethyl ether, dichloromethane, or some other organic solvent. The solvent extract is 

dried with anhydrous magnesium sulphate or sodium sulphate and then concentrated for 

GC analysis. While headspace analysis using cryogenic trapping meets the objective of 

sampling a large headspace volume, it requires 2-4 hours of analysis time and subsequent 

solvent extraction of the aqueous distillate. The need for solvent extraction adds concerns 

for solvent impurities, efficiency of extraction and flavour losses during solvent 

concentration. High- vacuum stripping (molecular distillation) of oils is often used for the 

isolation of flavours from lipid based foods (Reineccius, 1998). 

2.5.2 Separation, identification and quantification 

The development of chromatography, especially gas chromatography-mass spectrometry 

(GC-MS) made identification much easier (Da Costa and Eri, 2005). Gas 

chromatography (GC) is a highly sensitive and selective separation technique capable of 

resolving hundreds of components in a short time with part-per-million (ppm, one part in 

10 6 ) or better sensitivity (Hinshaw, 2003). A combination of chromatographic and 

spectrophotometric methods (GC-MS) is used for identification of volatile flavour 

constituents (Jordan et al., 2001). The retention index (RI) was first proposed by Kovats 

26

(1958) as a parameter for identifying solutes from chromatograms (Skoog et al., 1998). 

Retention indices calculated from a series of n-alkanes are used to identify the presence 

of volatile constituents (Gazim et al., 2008). The retention index (RI) for any given 

compound can be derived from a chromatogram of a mixture of that solute with at least 

two normal alkanes having retention times that bracket that of the solute (Skoog et al., 

1998). A mass spectrometer (MS) separates rapidly moving ions on the basis of their 

mass to charge ratios, m/z (Skoog et al.,1998). Mass spectrometer data and RI are 

compared to confirm the identity of a compound (Viljoen et al.,2008). Identifications 

made from mass spectral data alone could be inconclusive as those made from retention 

index libraries. In an extensive retention index library several components can have 

identical retention index values, therefore mass spectral data is needed to confirm 

tentative identification by retention indices (Steward and Pitzer, 1988). To illustrate this 

for the compound ethyl butyrate, using retention indices calculated from a series of C6- 

C24 alkanes the retention index is 1051. This is considered to be tentative identification 

and if after mass spectrometry the compound is also identified to be ethyl butyrate then 

confirmation has been obtained from these two identification methods. 

In conclusion, the choice of method of extraction will determine the type of volatile 

constituents to be obtained from the food material studied. Each method has its own 

strengths and limitations, a combination of these methods in extraction will provide a 

better understanding of the type of volatiles in the food material. 

27

2.6 Fruits, vegetables and tea plants 

2.6.1 P. curatellifolia 

Mobola plum tree, P. curatellifolia Planchon ex Benth. (Chrysobalanaceae) is a large 

evergreen tree which grows up to 15 m high with a tall straight trunk, erect branches and 

dense rounded crown (Mbuya et al., 1994). It is distributed along the eastern coast of 

South Africa and extends into the Limpopo province. It is also found in the southern parts 

of Zimbabwe and is reported to grow in parts of Mozambique and Malawi (Joulain et al., 

2004). The oval (50 mm X 25 mm), russet-yellow, pitted fruits (Figure 2.1) have a yellow 

flesh with a pleasant taste (Joulain et al., 2004). 

Figure 2.1: Ripe P. curatellifolia fruits (local name: maula) 

Mobola fruit is a wild fruit, it has a sweet and has a strong pineapple smell (Williamson, 

1975; Maundu et al., 1999). Joulain et al (2004) isolated volatile flavour components of 

P. curatellifolia obtained from Venda in South Africa by a vacuum headspace 

concentration method and analysed them using hyphenated gas chromatographic 

techniques. A total of 88 components were identified, of which 12 contain nitrogen, 

including 2-aminobenzaldehyde and phenylacetaldoxime, which were detected for the 

first time in an edible fruit. Optically active (2-nitrobutyl) benzene, which is a new 

28

natural product was identified. The main component of the volatile fraction was 2-phenyl 

ethanol (12-15 ppm) and the bulk of its esters represent about 25 % of the volatile 

fraction. 

2.6.2 S. cocculoides 

S. cocculoides belongs to the family Loganiaceae (Mwamba, 2005). In Malawi the fruits 

are known as “kabeza.” In Southern Africa the tree grows naturally in Brachystegia 

woodlands, mixed forests, deciduous woodlands and lowlands (Chirwa and Akkinifesi, 

2008). Fruits are 6 to 12 cm in diameter, round, blue-green mottled white when young, 

mottled green-yellow to yellow orange when ripe (Figure 2.2), with a granular skin, hard 

shelled (shell about 2 - 5 mm wide) containing irregularly curved flattened seeds 

(Mwamba, 2005). 

Figure 2.2: Ripe S. cocculoides fruits (local name: kabeza) 

Seeds are numerous, they have a hard coat and are compressed to 2 cm in diameter and 

are embedded in a fleshy pulp that is juicy and yellow when ripe (Chirwa and Akkinifesi, 

2008). Pulp from ripe fruits is either eaten fresh or dried and stored for later use (Oppelt, 

29

2004). The pulp has a sweet taste (Mwamba, 2005). Saka et al (2001) reported that 

S. cocculoides provides good juices of comparable quality to exotic fruit juices and that 

the organoleptic properties of the juices appear not to be dependant on the provenance 

and hence on the source of the collection. S. cocculoides is used to add sour flavour to 

cereal porridge (Saka et al., 2008). S. cocculoides juice was preffered to juices of 

U. kirkiana, S. cocculoides, Adansonia digitata and Mangifera indica after a sensory 

evaluation test (Saka et al., 2007). The inner skin of fruit shells contains valuable oils that 

have potential in cosmetics. Unripe fruits and seeds are used to induce vomiting and to 

treat snakebite victims (Oppelt, 2004). 

2.6.3 C. esculenta (L.) Schott 

Figure 2.3: C. esculenta leaves (local name: masamba ya masimbi) 

C. esculenta (Taro) leaves (Figure 2.3), are used as a traditional leaf vegetable in Malawi 

where they are known as “masamba ya masimbi”. The tubers are starchy, with a rather 

sweet flavour, however, the leaves are considered to be better flavoured than the tuber 

30

(Larkcom, 2007). Taro leaves and leaf stalks are used as a leafy vegetable and potherb for 

soups and sauces, or as relish (Safo, 2004). Soups prepared with fresh cocoyam leaves 

had higher scores for flavour/aroma as compared to other leaf vegetable soups tested 

(Mepba, 2007). C. esculenta leaves were recommended as a cheap source of plant protein 

(Lewu et al., 2009). In Mauritius boiled young leaves are eaten to treat arterial 

hypertention and liver affections, whereas juice is applied externally to treat eczema 

(Safo, 2004). Using Lickens Nickerson Apparatus and combined GC-MS volatile 

components of taro corms were analysed for the first time. A total of 62 components 

(c.95 % of the isolate) were positively identified, and a further component (c.2.3 %) was 

partially characterized. The most abundant component was octane (21.10 %) and pyridine 

was present at the unusually high level of 18.6 % (Macleod, 1990). Volatile constituents 

of taro corms were also isolated by steam distillation with subsequent extraction of the 

distillate with dichloromethane. Sixty compounds among which aliphatic acids (50.3 %), 

heterocyclic compounds (16.6 %), phenols (9.3 %) and alcohols (7.8 %) were 

quantitatively extracted and determined with capillary GC and GC/MS (Wong et al., 

1998). 

31

2.6.4 M. esculenta Cranz 

M. esculenta leaves (Figure 2.4) are eaten as a vegetable in Malawi where they are 

known as “chigwada”. The leaves are also used as a vegetable in some parts of sub- 

Saharan Africa such as Democratic Republic of Congo (DRC), Uganda, Nigeria and 

some Asian countries, the Philipines, Indonesia, Malaysia and Senegal (Mathieu and 

Meissa, 2007). Production of cassava leaves as a vegetable for human consumption in 

Indonesia is estimated at 0.5-0.7 million tones/year (Wargiono et al., 2007). High 

potential of cassava leaf as an unconventional protein resource for both humans and 

animals was indicated (Fasuyi, 2005). Organoleptic evaluation rated M. esculenta leaf 

soup equal to Amaranthus leaf soup in terms of taste, flavour and overall acceptability 

(Awoyinka et al., 1995). The leaves can also be used to treat boils (Matieu and Meissa, 

2007). Dougan et al (1983) obtained some volatile components of fresh cassava root by 

combined steam distillation/solvent extraction. Aroma-grams for the freshly cooked root 

and for two processed products were identified by GC-MS techniques. Fourty compounds 

were identified in freshly cooked cassava. The major classes of compounds were 

aromatic hydrocarbons, aliphatic carboxylic acids, aliphatic and aromatic carbonyl 

compounds. 

Figure 2.4: M. esculenta leaves (local name: chigwada) 

32

2.6.5 F. ancylantha Schweinf 

F. ancylantha Schweinf, synonym name Temnocalyx abovatus (N.E.Br) Robyns. It 

belongs to the family Rubiaceae. F. ancylantha leaves are used as a tea and medicinal 

plant in Karonga region of Malawi where it is known as “masamba ya muthondo’’ which 

means tea from the bush. Its traditional uses in this region are claimed to include curing 

stomach ulcers, curing abdominal pains, reducing pain during birth, increasing blood 

volume, lowering blood pressure and increasing growth rate in children. The herb is 

known as “makoni” or “marange” in Zimbabwe where it grows mainly in the Eastern 

Highlands (Mencherini et al., 2010). Makoni herbal tea can assist in boosting the immune 

system, growth, insulin production and is helpful for healthy sex organs (Darvish, 2004). 

An ethanol-water extract of F. ancylantha and its phenolic constituents showed 

significant free-radical-scavenging and antimicrobial activities (Mencherini et al., 2010). 

Traditional uses of the herb in Zimbabwe include calming abdominal and menstrual 

pains, reducing backaches and chest pains, strengthening bones, building stamina, soothe 

coughs, flu, including whooping cough and as an antidote for snakebite poisoning 

(Darvish, 2004). 

Figure 2.5: F. ancylantha herb (masamba ya muthondo) 

33

Fresh leaves 

Smoke/Sun /Shade drying (2-4days) 

Pounding dried leaves 

Seive 

Leaves stored in closed containers 

Scheme 2.5: Processing of F. ancylantha tea in Karonga Malawi 

The processing of F. ancylantha tea in Vundakulwayo district in Karonga (Scheme 2.5) 

involves harvesting leaves before flowering and drying in the sun and them either in the 

sun, using smoke or in the shade. Drying is conducted until the leaves turn brownish; this 

is achieved within 2 - 4 days depending on the method. The leaves are pounded, sieved 

and stored in closed containers ready for making tea. The presence of a fermentation step 

(during drying) produces a black tea. 

2.6.6 Camelia sinensis 

C. sinensis, a member of the Theaceae family, is an evergreen shrub or tree that can grow 

to a height of 30 feet, but is usually clipped to a height of 2-5 feet in cultivation (Brown, 

1999). Leaves alternate, exstipulate, lanceolate to obovate, up to 30 cm long, 2 - 5 cm 

broad, pubescent, sometimes becoming glabrous, serrate, acute or acuminate; flowers 1- 

3, in axillary or subterminal cymes, deflexed, 2 - 5 cm broad, aromatic, white or pinkish, 

actinomorphic, sepals and petals 5 - 7, pedicels 5 - 15 mm long; stamens numerous ovary 

3-5 carpellate, each carpel 4-6 ovulate; capsules depressed-globose, brownish, lobate, to 

34

2 cm broad, valvate, with 1-3 subglobose seeds in each lobe; approximately 500 seeds/kg 

(Ferrara et al., 2001). The shrub is heavily branched with dark-green leaves cultivated 

and preferentially picked as young shoots (Brown, 1999). The properties of the plant 

were known 4000 years ago and since then traditional Chinese medicine has 

recommended this plant for headaches, body aches and pains, digestion, depression, 

detoxification, as an energiser and in general to prolong life (Ferrara et al., 2001). 

Chinese green tea has been planted and drunk worldwide for more than a dozen centuries 

(Zhu et al., 2004). Green and black teas are derived from C. sinensis, it is the production 

process which differentiates the two types of tea (Brown, 1999). 

In conclusion, F. ancylantha tea, mobola plum fruits, monkey orange, cocoyam and 

cassava leaves contribute a wide variety of flavours to the community and apart from the 

aesthetic appeal associated with them, they have several health benefits which the 

community can benefit from. A study of the volatile constituents of these plants may 

provide an opportunity to unravel the benefits of these plants leading to their full 

exploitation. 

35

3.1 Sample Collection 

CHAPTER 3: MATERIALS AND METHODS 

Samples of a shrub used as tea leaves in Karonga region were collected from 

Vundakulwayovillage (Figure 3.1) and taken to the national herbarium for identification. 

The plant was identified as F. ancylantha Hiern, synonym name Temnocalyx abovatus 

(N.E.Br) Robyns and belongs to the family called Rubiaceae. F. ancylantha tea leaves 

processed by the villagers by shade drying were collected from Vundakulwayo village in 

Karonga district. Lipton green and Lipton black teas which were used as controls were 

bought from a Supermarket in South Africa. 

Ripe S. cocculoides and P. curatellifolia fruit samples were collected from Sanga in 

Nkhata Bay (Figure 3.1). Unripe S. cocculoides samples were also collected from the 

same provenance. Samples were cleaned, packed in polythene bags and refrigerated at 

temperatures below -10 0 C to minimise changes in flavour. 

Cocoyam and cassava leaf samples were collected from Sogoja village in Zomba (Figure 

3.1) and taken to the National Herbarium for identification. A portion of the leaves were 

refrigerated at 5 0 C and another portion was shade dried in the laboratory at 25 0 C for 10 

days. 

36

Fruits and vegetables were placed in cooler boxes which were kept at approximately 0 0 C 

and tea leaves were stored in plastics before being transported to South Africa for further 

analysis. On arrival, the tea samples were stored at room temperature, fruit and vegetable 

samples were stored at temperatures below -10 0 C. 

Figure 3.1: Map of Malawi showing three locations from which samples were 

collected 

37

3.2 Sample Treatment 

Fruit and vegetable samples were cleaned after collection to reduce the microbial load. 

Selection was done to remove damaged, discoloured and contaminated samples. Some of 

the cocoyam and cassava leaves were dried at 24 0 C for 10 days and stored in sacks. S. 

cocculoides fruits were cracked just before analysis to collect the pulp. P. curatellifolia 

fruits were peeled and the pulp was scrapped off using a sharp knife. 

3.3 Chemicals, reagents and apparatus 

Chemicals and reagents used in this study were of analytical grade. n-Alkane standards 

were purchased from Sigma Aldrich (Pretoria, South Africa) whereas dichloromethane, 

methanol, n-hexane, sodium chloride and Sodium hydrogen carbonate were purchased 

from Merck Chemicals (Johannesburg, South Africa). Oxalic acid and sodium sulphate 

were purchased from Saarchem (Johannesburg, South Africa) and antibumping granules 

from BDH Chemicals (Johannesburg, South Africa). 

The following apparatus and equipment were used; Clevenger apparatus purchased from 

Sigma Aldrich (Johannesburg, South Africa), Cold Finger glassware purchased from 

Sigma Adrich (Johannesburg, South Africa), Knf Neuberger laboport vacuum pump 

(1.0bars) (Johannesburg, South Africa), steam distillation unit purchased from Sigma 

Aldrich (Pretoria, South Africa), 30cm vigreux column, Solid Phase Microextraction 

(SPME) Device (5mm, 100µm) PDMS (SUPELCO) purchased from Sigma Aldrich 

(Pretoria, South Africa), gas chromatograph-mass spectrometer (GC-MS) (GC Agilent 

6890N system coupled directly to a 5973 MS detector). The ELISA machine used was a 

Microtiter plate reader (Multiskan Ascent; Thermo Labsystems; USA) 

38

3.4 Preparation of chemicals and reagents 

3.4.1 Sodium hydrogen carbonate (aq) solution (2.5 %, m/v) 

Sodium hydrogen carbonate (NaHCO3) (2.5 g), placed in a 100 ml volumetric flask and 

made up to mark with triple distilled water. 

3.4.2 Saturated aqueous oxalic acid solution 

Oxalic acid (10 g) was weighed and placed in a 100ml volumetric flask. The solution was 

made up to the mark with triple distilled water and stored at room temperature. 

3.4.3 Methanol solution (60 %, m/v) 

Methanol (60 ml) was diluted to 100 mL with triple distilled water and shaken thoroughly 

to ensure homogeneity of the solution. From this solution the following methanol 

solutions were prepare as follows: 

Solution 1: Thirty millilitres of methanol (60 %, m/v) were mixed with triple distilled 

water (16 mL) in a 100 ml Schott bottle. 

Solution 2 was made by adding 30 ml of 60% Methanol to 12 ml of triple distilled water 

and 4 ml of oxalic acid in 100 ml Schott bottle. 

3.5 Determination of moisture content of F. ancylantha and Lipton control teas 

Moisture was determined using the (ISO Tea determination loss in mass at 103 0 C: 1980) 

method. Determination of moisture content was done in duplicate. Crucibles were dried 

in the oven overnight at 103 0 C, cooled in a desiccator and weighed to at least 0.001 g. 

Tea sample (5 g) was placed in each crucible in duplicate. Crucibles and tea leaves were 

placed in the oven for 12 hours at 103 0 C, cooled in a desiccator and accurately weighed. 

The percentage of moisture content of the teas was calculated by subtracting the loss in 

mass of the tea leaves after drying and expressing it as a percentage of the initial mass. 

39

3.6 Determination of theaflavins, thearubigins, colour and brightness of 

F. ancylantha and Lipton control teas 

3.6.1 Preparation of tea samples 

The water bath was set to 90 ºC and tea samples (0.200 g) were weighed in a 10ml glass 

extraction tube. Tripple distilled water (1L) was heated in a 90 ºC water bath for 30 min 

for equilibration. An extraction tube containing tea sample was placed in a water bath and 

triple distilled water (5 ml) was added. A stopper was placed on the extraction tube and 

mixing was done on a vortex. The mixture was kept in a water bath for 10 min and 

vortexed after 5 min and after 10 min. Tubes were removed from the water bath and left 

for 5 min to cool down to room temperature. The stopper was removed and mixture was 

centrifuged for 10 min at 3500 rpm. The supernatant was decanted into a 10 ml 

volumetric flask and set aside. The process was repeated and the supernatant was added 

to the previously collected supernatant and made up to 10 ml with cold triple distilled 

water. The extracted tea supernatant was filtered using a 0.22 µM filter coupled to a 

10 ml syringe. 

3.6.2 Extraction of theaflavins and thearubigins 

Determination of the content of thearubigins, theaflavins, colour and brightness was done 

by the method of Robert and Smith (1963). 

Theaflavins and thearubigins contribute to brightness, colour and strength of tea (Sud and 

Baru, 2000). Thearubigins has been broken down into three fractions, fraction SII 

precipitated by ether from methanol solution, fraction SI precipitated by ether from 

acetone solution, and a filtrate fraction left in solution after successive precipitations of SI 

40

and SII (Robert and Smith, 1963). Theaflavins are extracted from tea liquor using 

isobutyl methyl ketone (IBMK) which does not extract thearubigins of SII type; other 

thearubigins are partially extracted when present as free acids, although their potassium 

and calcium salts are not extracted. The thearubigins extracted by IBMK are soluble in 

sodium hydrogen carbonate solution, whereas the theaflavins are almost insoluble 

(Robert and Smith, 1963). A solution of sodium hydrogen carbonate is then used to 

separate theaflavins and thearubigins and thereby forming the basis of quantification of 

these compounds (Robert and Smith, 1963). 

Tea supernatant (1 ml) was mixed with isobutylmethylketone (IBMK) (1 ml), the mixture 

was put on vortex four times for 30 seconds each time. The aqueous (bottom) layer was 

separated from the top (IBMK layer). Methanol solution 1 (184 µL) was added to 16 µL 

of the aqueous layer, to make solution B. Methanol solution 2 was added to another 16 

µL of the aqueous layer to make solution D. Methanol (60 %, m/v) (168 µL) was added 

to IBMK (32 µL) layer to make solution A. Another portion of the IBMK layer (500 µL) 

was mixed with 2.5 %NaHCO3 (500 µL) for 30 seconds. The mixture was separated and 

the bottom layer (NaHCO3) was discarded. Methanol (60 %, m/v) (168 µL) was mixed 

with the IBMK layer (32 µL) to make solution C. All solutions were prepared in 

Eppendorph tubes and were vortexed. Solutions were transferred to ELISA plates. 

Absorbance of each solution (EB, ED, EA, EC) was measured at 380 nm and at 460 nm. 

41

3.6.3 Calculations of total theaflavins, total thearubigins, total colour and 

brightness 

The theaflavin and thearubigin contents were calculated from the following relationships: 

i). % theaflavins = 6.25 x Ec x f1 

ii). % thearubigins = [(12.5ED + 6.25(EA – EC)] x f2 

Where f1 and f2 are the corresponding spectrophotometric conversion factors. The value 

of the factor f1 used for calculating the percentage theaflavin (as in equation i) as 

anhydrous theaflavin gallate of the sample of tea from the reading obtained at 380nm is 

f1 at 380nm = 0.02 X 856.7 X 375 

2.225 X 892.7 X 9 

= 0.36 

Where the (E(1cm) 0.02 % ) value for theaflavin gallate dehydrate of molecular weight 

892.7 is 2.225 which is the infusion being prepared from 9 g of tea and 375 ml of water. 

The value of f1 used for calculating the percentage of theaflavin (as anhydrous theaflavin 

gallate) from the reading obtained at 460 nm is 

f1 at 460nm = 0.02 X 856.7 X 375 

= 1.07 

0.747 X 892.7 X 9 

42

Where the (E(1cm)0.02 %) value for theaflavin gallate dihydrate being 0.747 at 460 nm. 

The factors for converting absorbance to percentage thearubigins f2 from the value 

obtained at 380 nm is 

f2 at 380nm = 0.02 X 375 

0.733 X 9 

= 1.19 

Where the average E 0.02% value for SI and SII thearubigin fractions is 0.733. 

The EB values have also been used, with the EA values, for obtaining data on the colour 

and brightness of tea infusions. The sum of the absorbancies of solutions A and B can be 

used to measure the colour and brighteness of tea infusions. Thus, the sum of the optical 

densities of the A and B solutions can be used to measure the colour of the infusion and 

the value is the total colour. 

iii). Total colour =6.25 X (EA+2EB) 

This is virtually the same as the absorbance of the original tea infusion. 

Brightness is due to theaflavins, and a measure of ‘brightness’ is obtained by expressing 

the extinction value at 460 nm due to theaflavins as a percentage of the total extinction. 

Thus, iv) % Brightness= (100 X EB)/(EA+2EB) 

Optical density was measured at two wavelengths (380nm and 460nm) because 

theaflavins absorb at 380nm and thearubigens absorb at 460nm. 

43

3.7 Extraction of volatile constituents of F. ancylantha tea leaves 

In each extraction method, extraction was done until an adequate amount of sample 

required for analysis was collected. 

3.7.1 Hydrodistillation and solvent extraction of F. ancylantha tea leaves 

Dried F. ancylantha leaves (50 g) were placed in the still (1 L), double distilled water 

(500 ml) and antibumping granules added to the still. Distillation was carried out in a 

clevenger apparatus for 4 hrs and 22 minutes. Leaves were removed after distillation and 

replaced with fresh leaves (50 g). The water was also replaced with double distilled water 

(500 ml). Essential oil was not removed since the quantity was too small. Distillation was 

done for 3 hours and 5 minutes with the essential oil collecting in the same clevenger 

apparatus as the first oil. The leaves were removed after distillation and replaced with 

fresh leaves (50 g). Water was also replaced with double distilled water (500 ml). 

Distillation was done for 3 hours and 5 minutes while the essential oil was also collecting 

in the same appatus. Distillation was carried out three times on separate leaf samples so 

as to collect more essential oils from the plant. Redistilled n-hexane (0.1 ml) was added 

to the collecting section for essential oils. Anhydrous sodium sulphate (Na2SO4) was 

added to the collected essential oil in the vial. The extract was stored at 5 0 C before GC- 

MS analysis (Saim et al., 2008) 


F. ancylantha tea 

Dried F. ancylantha tea leaves (50 g) were placed in the still (1 L), double distilled water 

(500 ml) with antibumping granules were added to the still of a clevenger apparatus. 

44

Distillation was carried out for 4 hrs and 33 minutes at reduced temperature because of 

forming. The hydrosol (600 ml) was placed in a separatory flask, dichloromethane 

(30 ml) and a saturated solution of NaCl (30 ml) were added. The mixture was swirled to 

allow mixing and extraction of the compounds. The organic layer was separated and 

collected in a separate container. Extraction with dichloromethane (30 mls) was done 

twice as above. The three portions of organic extracts were collected, mixed and dried 

with anhydrous Na2SO4. Filtration with glass wool was done to remove particles of 

Na2SO4. The extract was concentrated on a vigreux column (30 cm) to approximately 2 

ml. Further concentration was done by blowing a stream of nitrogen over the sample to 

about 0.1 ml. The extract was stored at 5 0 C before GC-MS analysis (Saim et al., 2008). 

3.7.3 Cold finger molecular distillation of F. ancylantha tea leaves 

Dried F. ancylantha tea leaves (50 g), distilled water (200 ml) and NaCl (30 g) were 

placed in a flat bottomed flask (1 L) which was then closed with a cold finger. The cold 

finger was connected to a Knf Neuberger laboport vacuum pump (1.0 bars). Vacuum 

pump was connected and the cold finger was filled with liquid nitrogen. The experiment 

was run for 3 hours with the distillate condensing on the cold finger trap. The distillate 

was extracted with 5 ml of dichloromethane. The aqueous layer was separated from the 

organic layer and the organic layer was dried with Na2SO4. Concentration to about 0.1 ml 

was done by blowing a gentle stream of nitrogen over the extract. The concentrate was 

stored at 5 0 C before GC-MS analysis (Gazim et al., 2008). 

45

3.8 Extraction of volatile constituents of cassava and cocoyam leaf vegetables 

3.8.1 Steam distillation and solvent extraction of fresh cassava leaves 

Fresh cassava leaves (60 g) were placed in the still, double distilled water (500 ml) and 

antibumping granules were placed in the boiling flask (1 L). Distillation was carried out 

for 3 hrs and 5 minutes. To increase the amount of essential oil collected, leaves were 

removed from the still and replaced with fresh ones (60 g). The water was replaced with 

fresh double distilled water (500 ml) and new antibumping granules were placed in the 

boiling flask. Distillation was done for 3 hours and 27minutes. Leaves were removed 

after distillation and replaced with fresh leaves (60 g). Water was also replaced with 

double distilled water (500 ml). Distillation was done for 3 hours and 13 minutes with the 

essential oil collecting in the same portion as that of the first and second distillations. 

Redistilled n-hexane (0.1 ml) was added to the collecting section for essential oils. The 

extract was dried with anhydrous Na2SO4 and stored at 5 0 C (Saim et al., 2008). 


cassava leaves 

Dried cassava tea leaves (60 g) were placed in the still (1 L), double distilled water (500 

ml) with antibumping granules were added to the still. Distillation was carried out for 3 

hrs and 12 minutes using a clevenger apparatus. Double distilled water (200 ml) was 

being added at hourly intervals. The hydrosol (610 mls) was placed in a separatory flask, 

dichloromethane (30 ml) and a saturated solution of NaCl (30 ml) were added. The 

mixture was swirled to allow thorough mixing and extraction of the compounds. The 

organic layer was separated and collected in a separate container. Extraction with 

dichloromethane (30 mls) was done twice as above. The three portions of organic extracts 

were collected, mixed and dried with anhydrous Na2SO4. Filtration with glass wool was 

46

done to remove particles of Na2SO4. The extract was concentrated on a vigreux column 

(30 cm) to approximately 2 ml. Further concentration was done by blowing a stream of 

nitrogen over the sample to about 0.1 ml. The extract was stored at 5 0 C (Saim et al., 

2008). 


cassava leaves 

Fresh cassava leaves (100 g) were placed in the still (1 L), double distilled water (500 ml) 

with antibumping granules were added to the still. Distillation was carried out for 

2 hrs and 51 minutes using a clevenger apparatus. Double distilled water (200 ml) was 

being added at hourly intervals. The hydrosol (600 mls) was placed in a separatory flask; 


mixture was swirled to allow mixing and extraction of the compounds. The organic layer 

was separated and collected in a separate container. Extraction with dichloromethane (30 

ml) was done twice as above. The three portions of organic extracts were collected, 

mixed and dried with anhydrous Na2SO4. Filtration with glass wool was done to remove 

particles of Na2SO4. The extract was concentrated on a vigreux column (30 cm) to 

approximately 2 ml. Further concentration was done by blowing a stream of nitrogen 

over the sample to about 0.1 ml. The extract was stored at 5 ⁰C (Saim et al., 2008). 


cocoyam leaves 

Fresh cocoyam leaves (100 g) were placed in the still (1 L), double distilled water (500 


hours and 57 minutes. Double distilled water (200 ml) was being added at hourly 

intervals. The hydrosol (610 ml) was placed in a separatory flask, dichloromethane 

47

(30 ml) and a saturated solution of NaCl (30 ml) were added. The mixture was swirled to 

allow mixing and extraction of the compounds. The organic layer was separated and 

collected in a separate container. Extraction with dichloromethane (30 ml) was done 

twice as above. The three portions of organic extracts were collected, mixed and dried 

with anhydrous Na2SO4. Filtration with glass wool was done to remove particles of 

Na2SO4. The extract was concentrated on a vigreux column (30 cm) to approximately 2 

ml. Further concentration was done by blowing a stream of nitrogen over the sample to 

about 0.1 ml. The extract was stored at 5 ⁰C (Saim et al., 2008). 


cocoyam leaves 

Dried cocoyam leaves (50 g) were placed in the still (1 L), double distilled water (500 


hours and 3 minutes. The hydrosol (200 mls) was placed in a separatory flask, 


mixture was swirled to allow mixing and extraction of the compounds. The organic layer 

was separated and collected in a separate container. Extraction with dichloromethane (30 

ml) was done twice as above. The three portions of organic extracts were collected, 

mixed and dried with anhydrous Na2SO4. Filtration with glass wool was done to remove 

particles of Na2SO4. The extract was concentrated on a vigreux column (30 cm) to 

approximately 2 ml. Further concentration was done by blowing a stream of nitrogen 

over the sample to about 0.1 ml. The extract was stored at 5 0 C (Saim et al., 2008). 

48

3.8.6 Hydrodistillation and solvent extraction of fresh cassava leaves 

Fresh cassava leaves (60 g) was placed in the still (1 L), double distilled water (500 ml) 

with antibumping granules were added to the still. Distillation was carried out for 3 hrs 

and 2 minutes. To collect more essential oil, leaves were removed after distillation and 

replaced with fresh leaves (60 g). Water was also replaced with double distilled water 

(500 ml). Distillation was done for 3 hours and 5 minutes. Leaves were removed after 

distillation and replaced with fresh leaves (100 g). Water was also replaced with double 

distilled water (500 ml). Distillation was done for 3 hours. Leaves were removed after 

distillation and replaced with fresh leaves (100 g). Water was also replaced with double 

distilled water (500 ml). Distillation was done for 3 hours and 2 minutes, the essential oil 

for the first, second and final distillations were collecting in the same portion. Redistilled 

N-hexane (0.1 ml) was added to the collecting section for essential oils. Anhydrous 

Na2SO4 was added to the collected essential oil in the vial. The extract was stored at 5 0 C 

(Saim et al., 2008). 

3.8.7 Cold finger molecular distillation of fresh cassava leaves 

Fresh cassava tea leaves (100 g), distilled water (100 ml) and NaCl (30 g) were placed in 

a flat bottomed flask (1 L) then closed with a cold finger. The cold finger was connected 

to a Knf Neuberger laboport vacuum pump (1.0 bars). Vacuum pump was connected and 

the cold finger was filled with liquid nitrogen. The flask and the contents were heated at 

about 45 0 C throughout the experiment. Heat was applied for the 1 hour 22 minutes and 

stopped to remove the distillate which had completely covered the cold finger. The 

experiment was continued for 43 minutes and stopped to remove the distillate. 

Experiment was done for another 1 hour and stopped to remove the distillate. The three 

distillates were combined and extracted with 5 ml of dichloromethane. The inorganic 

49

layer was separated from the organic layer and the organic layer was dried with Na2SO4. 

Concentration to about 0.1 ml was done by blowing a gentle stream of nitrogen over the 

extract. The concentrate was stored at 5 0 C (Gazim et al., 2008). 

3.8.8 Cold finger molecular distillation of dried cassava leaves 

Dried cassava tea leaves (50 g), distilled water (300 ml) and NaCl (30 g) were placed in a 

flat bottomed flask (1 L) then closed with a cold finger. The cold finger was connected to 

a Knf Neuberger laboport vacuum pump (1.0 bars). Vacuum pump was connected and 





Experiment was done for another 1 hour 14 minutes and stopped to remove the distillate. 

The three distillates were combined and extracted with 5 ml of dichloromethane. The 

inorganic layer was separated from the organic layer and the organic layer was dried with 

Na2SO4. Concentration to about 0.1 ml was done by blowing a gentle stream of nitrogen 

over the extract. The concentrate was stored at 5 0 C (Gazim et al., 2008). 

3.8.9 Cold Finger Molecular Distillation of dried cocoyam leaves 

Dried cocoyam leaves (500 g), distilled water (200 ml) and NaCl (30 g) were placed in a 

flat bottomed flask (1 L) then closed with a cold finger. The cold finger was connected to 

a Knf Neuberger laboport vacuum pump (1.0 bars). Vacuum pump was connected and 




50


Experiment was done for another 1 hour and stopped to remove the distillate. The three 

distillates were combined and extracted with 5 ml of dichloromethane. The inorganic 

layer was separated from the organic layer and the organic layer was dried with Na2SO4. 

Concentration to about 0.1 ml was done by blowing a gentle stream of nitrogen over the 

extract. The concentrate was stored at 5 0 C (Gazim et al., 2008). 

3.9 Extraction of volatile constituents in P. curatellifolia and S. cocculoides fruits 

3.9.1 Solid phase micro extraction (SPME) of P. curatellifolia and 

S. cocculoides fruit pulp 

Headspace analysis was carried out on the fruit pulp of S. cocculoides and 

P. curatellifolia. A SPME (SUPELCO) device consisting of a fused silica fiber, coated 

with 100 µm polydimethylsiloxane (PDMS) polymeric adsorbent was used. The pulp was 

placed in separate vials and tightly closed. The SPME fiber (5 mm) was inserted into the 

headspace for 20 min.The fiber was removed from the vial and directly inserted into the 

injection port of the GC-MS for desorption (Viljoen et al., 2008). 

3.9.2 Cold finger molecular distillation of ripe S. cocculoides fruit pulp 

Ripe S. cocculoides fruit pulp (326 g) and NaCl (30 g) were placed in a flat bottomed 

flask (1 L) which was then closed with a cold finger. The cold finger was connected to a 

Knf Neuberger laboport vacuum pump (1.0 bars). Vacuum pump was connected and the 

cold finger was filled with liquid nitrogen. The experiment was done for 3 hours and 1 

minute with the distillate condensing on the cold finger trap. The distillate was extracted 

with 5 ml of dichloromethane. The inorganic layer was separated from the organic layer 

51

and the organic layer was dried with Na2SO4. Concentration to about 0.1 ml was done by 

blowing a gentle stream of nitrogen over the extract. The concentrate was stored at 5 0 C 

(Gazim et al., 2008). 

3.9.3 Cold finger molecular distillation of unripe S. cocculoides fruit pulp 

Unripe S. cocculoides fruit pulp (500 g) and NaCl (30 g) were placed in a flat bottomed 



cold finger was filled with liquid nitrogen. The experiment was done for 3 hours and 3 

minutes with the distillate condensing on the cold finger trap. The distillate was extracted 

with 5 ml of dichloromethane. The inorganic layer was separated from the organic layer 

and the organic layer was dried with Na2SO4. Concentration to about 0.1 ml was done by 

blowing a gentle stream of nitrogen over the extract. The concentrate was stored at 5 0 C 

(Gazim et al., 2008). 

3.9.4 Cold finger molecular distillation of P. curatellifolia fruit pulp 

Ripe P. curatellifolia fruit pulp (300 g) and NaCl (30 g) were placed in a flat bottomed 



cold finger was filled with liquid nitrogen. The experiment was done for 3 hours with the 

distillate condensing on the cold finger trap. The distillate was extracted with 5 ml of 

dichloromethane. The inorganic layer was separated from the organic layer and the 

organic layer was dried with Na2SO4. Concentration to about 0.1 ml was done by blowing 

a gentle stream of nitrogen over the extract. The concentrate was stored at 5 ⁰C (Gazim 

et al., 2008). 

52

3.10 Identification of volatile constituents 

3.10.1 GC and GC-MS conditions for SPME 

The head-space volatiles were analysed by GC-MS (Agilent 6890N GC system coupled 

directly to a 5973 MS). The splitless injection was carried out manually at 24.79 psi and 

an inlet temperature of 250 0 C. The GC system equipped with a HP-Innowax 

polyethylene glycol column (60 m x 250 µm i.d., 0.25 µm film thickness was used. The 

oven temperature program was 60 0 C for the first 10 min, rising to 220 0 C at a rate of 4 

0 C/min and held for 10 min and then rising to 240 0 C at a rate of 1 0 C / min. Helium was 

used as a carrier gas at a constant flow rate of 1.2 ml/min. Spectra were obtained on 

electron impact at 70 eV, scanning from 35 to 550 m/z. n- Alkanes (C6-C24) were used 

as reference points in calculation of retention indices (RI). Component identifications 

were made by comparing mass spectra and retention indices. Library searches were 

carried out using NIST, Mass Finder, Flavour and the Baser library of essential oil 

constituents by comparing mass spectra and retention indices (Viljoen et al., 2008). 

3.10.2 GC and GC-MS conditions for hydrodistillation, steam distillation 

and CFMD samples 

Extracts from hydrodistillation, steam distillation and cold finger molecular distillation 

were analysed using the following conditions: A GC-MS (Agilent 6890N GC system 

coupled directly to a 5973 MS) was used. A volume of 1µl was injected (splitless) using 

an autosampler at 24.79 psi, an inlet temperature of 250 0 C. The GC system equipped with 

a HP-Innowax polyethylene glycol column (60 m x 250 µm i.d x 0.25 µM film thickness) 

was used. The ion source (electron impact ionization and the GC-MS interface 

temperature) was 260 0 C. The oven temperature program was 60 0 C for the first 10 min, 

rising to a rate of 4 0 C/min and held for 10 min and then rising to 240 0 C at a rate of 

53

1 0 C/min. Helium was used as a carrier gas at a constant flow of 1.2 ml/min. Spectra were 

obtained on electron impact of 70 eV, scanning from 35 to 550 m/z. n-Alkanes (C6-C24) 

were used as reference points in the calculation of retention indices (RI) Library searches 

were carried out using NIST, Mass Finder, Flavour and the Baser library of essential oil 

constituents by comparing mass spectra and retention indices (Kamatou et al., 2010). 

The retention index RI for an individual compound RIC was calculated using the formula: 

RIc = 100Z +100((log t`RC – log t`RZ)/(log t`R(Z+1)- log t`RZ)) 

where t`RC, tRZ and t`R(Z+1) are the corrected retention times for the compound and the 

alkanes eluting before and after the compound, respectively (Braithwaite and 

Smith, 1996). 

3.11 Data analysis 

One way analysis of variance (ANOVA) was performed using Genstat 13 th edition to 

establish the differences in the mean levels of quality parameters studied between 

F. ancylantha tea and the control teas. The significant differences among the means were 

tested at 95% confidence interval. 

54

CHAPTER 4: RESULTS AND DISCUSSION 

4.1 Quality of F. ancylantha tea 

The quality parameters of locally processed F. ancylantha tea and those of commercially 

available Lipton green and black are presented in Table 4.1. The results showed that there 

were significant (p

The difference in total theaflavin content between F. ancylantha and Lipton black tea 

could be attributed to the fermentation process employed in the herbal tea processing. In 

Vundakulwayo village in Karonga district, drying of the tea leaves is continued “until the 

leaves turn brownish”. This allows fermentation to proceed and thearubigins increase at 

the expense of theaflavins (Srilakshmi, 2003). The herbal tea had higher thearubigin 

content than Lipton black while theaflavins were higher in Lipton black than in 

F. ancylantha tea. According to Hara (2004), high theaflavin content usually implies 

good manufacturing practices. The presence of high thearubigins in the herbal tea means 

improvement in the processing of the herbal tea is necessary. Inorder to improve the 

quality of this traditional tea, the fermentation time should be reduced to minimise 

thearubigins and thus achieve adequate theaflavins. When compared with the content of 

theaflavins in black tea (0.3 - 2.0 %) the values for Lipton black (0.48 %) and 

F. ancylantha (0.37 %) are within the acceptable range of theaflavins for black teas 

(Banerjee, 2005). The presence of theaflavins in Lipton green (0.22 %) means some 

fermentation occurred during processing of the Lipton green tea. This is because green 

tea may also contain constituents commonly found in black tea such as theaflavins and 

thearubigins and therefore some oxidation and condensation of catechins occured 

(Brown, 1999). 

Significant (p

Significant (p

Table 4.2: Volatile constituents of F. ancylantha tea extracted using hydrodistillation and 

solvent extraction from the hydrosol 

RI Compounds Total number of 

compounds 

1212 2-Hexanal 

1333 6-Methyl-5-hepten-2-one 

1457 Furfural 

1492 2,4-Heptadienal 

1534 Benzaldehyde 

1570 5-Methyl furfural 

1637 Safranal* 

1937 β-Ionone 

1989 β-Ionone epoxide 

1849 Geranyl acetone 

2190 3.4-Dimethyl-5-pentyl-5H-furan-2-one 

Total carbonyls 11 

1376 3-Hexanol 

1395 2-Butoxy ethanol 

1441 Trans Linalool oxide 

1471 Cis Linalool oxide 

1541 Linalool 

1701 α-Terpineol 

1822 Geraniol 

1885 Benzyl alcohol 

1903 Phenyl ethyl alcohol 

1993 Phenol 

2041 Nerolidol 

2077 Cresol 

2188 Eugenol 

Total alcohols 13 

1653 Methyl salicylate 

1893 Geranyl butyrate 

2359 Dihydroactinidiolide 

Total Esters 3 

Total number of compounds 27 

*, identified using MS 

58

Using the hydrodistillation and solvent extraction method, 16 compounds were obtained 

(Table 4.3). It is evident that the relative number of class of compounds identified were; 

alcohols (41.7 %), carbonyls (25 %), total fatty acids (16.7 %), sesquiterpenes (8.3 %) 

and diterpenes (8.3 %). Thus, alcohols remained the most dominant class of compounds. 

Table 4.3: Volatile constituents of F. ancylantha tea extracted using the 

hydrodistillation and solvent extraction method 

RI Compounds Total number of 

compounds 

1849 Geranyl acetone 

1937 β-Ionone 

2117 6,10,14-Trimethyl pentadecan-2-one 

Carbonyls 3 

1541 Linalool 

1822 Geraniol 

1954 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 

2288 Isophytol 

2615 Phytol 

Alcohols 5 

2028 2-epi (E)-β-caryophyllene 

Sesquiterpenes 1 

1980 Neophytadiene 

Diterpenes 1 

2225 Methyl palmitate 

2559 Methyl linolenate 

Fatty acid esters 2 


59

In contrast, the he CFMD method extracted only the compound 2-Butoxy y ethanol. ethanol 

An analysis of the data in Table 4.2 and 4.3 indicated that ffatty 

acid id esters were extracted 

using the hydrodistillation and solvent extraction method only. . This was due to the use of 

the non polar solvent (hexa (hexane) used for extraction in this method. . In the hydrodistillation 

and solvent extraction action from the hydrosol and the CFMD method, 

(dichloromethane) was used, hence, fatty acids were not extracted. 

The compounds linalool, geraniol, β-ionone and geranyl acetone were obtained from 

F. ancylantha tea leaves using both hydrodistillation methods (Figure 4.1). 4.1) Linalool has 

been shown to have anticancer effects (Usta et al., 2009). While geranyl eranyl acetone is 

known to be a mosquito to repellent (Innocent et al., , 2010). Thus, the use of F. ancylantha 

leaves as a repellent against the mosquito, A. gambie s.s. should be investigated and 

exploited. 

60 

a polar solvent 

Figure 4.1: Structures of compounds identified in F. ancylantha tea leaves after 

extraction using the hydrodistillation methods

4.3 Volatile constituents of S. cocculoides and P. curatellifolia 

Since S. cocculoides and P. curatellifolia are fruits and are mostly eaten without cooking, 

it was appropriate to investigate their volatile constituents using methods which do not 

include heating so as to avoid generation of new compounds during the extraction 

process. Consequently, the use of the headspace methods SPME and CFMD was made. 

4.3.1 Identity of compounds extracted from P. curatellifolia fruits 

using SPME and CFMD methods 

The volatile constituents of compounds extracted from P. curatellifolia using the SPME 

and CFMD are given in Table 4.4. Using the SPME, eleven compounds were identified 

from P. curatellifolia fruit pulp. While seven compounds were identified using CFMD. 

61

Table 4.4: Presence and identity of P. curatellifolia volatiles extracted using the 

SPME and the CFMD methods. 

RI Compound M.W 

62 

(g/mol) 

SPME 

1051 Ethyl butyrate 116.16 Y Y 

1066 Ethyl isovalerate 130.19 Y Y 

1142 Ethyl valerate 130.19 Y Y 

1228 Ethyl hexanoate 144.21 Y X 

1670 Ethyl benzoate 150.18 Y X 

1298 Isoamyl Isovalerate 172.26 Y X 

Total number of Esters 6 3 

1917 Phenyl alcohol 108.14 Y Y 

1395 2-Butoxyethanol 118.18 X Y 

1915 2.6-Diter butyl-4-methyl-phenol 220.35 Y X 

Total number of Alcohols 2 2 

1940 Phenyl acetonitrile 117.15 Y Y 

Total number of Heteroatoms 

1 1 

1281 2-Butanol-3-one 88.11 X Y 

Total number of carbonyls X 1 

1570 α-Bergamotene 204.35 Y X 

1665 β-Farnesene 204.35 Y X 

Total number of sesquiterpenes 2 X 

Total number of compounds 11 7 

Y- compound was extracted using this method 

X- compound was not identified using this method. 

CFMD

The SPME method identified 11 compounds from the P. curatellifolia headspace and 

these comprised esters (54.5 %), alcohols (18.2 %), sesquiterpenes (18.2 %) and 

heteroatoms (9.1 %). Upon using the CFMD, the relative number of class of identified 

compounds were esters (42.9 %), alcohols (28.6 %), heteroatoms (14.2 %) and carbonyls 

(14.2 %). These results revealed that in P. curatellifolia fruits, using both methods the 

compounds extracted most were esters. This is because esters are the prime components 

of the characteristic aroma of fruits (Sanz et al., 1997). 

The esters ethyl hexanoate, isoamyl isovalerate and ethyl benzoate were extracted using 

the SPME but not with the CFMD method. This could be due to the high molecular 

weights of these compounds which are above (144g/mol). The vacuum (1.0 bars) used in 

the CFMD may not have been able to suck these high molecular weight esters. The 

chemical composition of the SPME fiber may also have contributed to the adsorption of 

these compounds and hence enabling their extraction. The sesquiterpenes α-bergamotene 

(204.35 g/mol), β-Farnesene (204.35 g/mol) and alcohol 2.6-diter butyl-4-methyl-phenol 

(220.35 g/mol) were extracted using the SPME method and not by the CFMD method. 

This is not surprising because of their high molecular weights and the chemical 

composition of the PDMS fiber used in SPME. Both methods extracted the compounds 

ethyl butyrate, ethyl isovalerate, phenyl acetonitrile and phenyl alcohol. Joulain et al., 

(2004) also established the presence of ethyl butyrate in P. curatellifolia collected from 

Thohoyandu, Venda South Africa using a vacuum headspace concentration method and 

was the third most abundant compound after 2-phenylethanol and ethyl pentanoate. 

63

4.3.2.1 Identity of volatile compounds extracted from S. cocculoides fruits 

using SPME and CFMD methods 

The volatile constituents of ripe and unripe S. cocculoides fruits extracted using SPME 

and CFMD methods are provided in Table 4.5 below. Six compounds were obtained in 

ripe S. cocculoides using SPME. In contrast, upon using the CFMD method, the same 

ripe S. cocculoides produced three compounds. Unripe S. cocculoides gave only 2-butoxy 

ethanol after CFMD. 

Table 4.5: Presence and identity of volatile compounds identified in ripe and unripe 

S. cocculoides fruits following SPME and CFMD extraction 

RI Compounds 

Ripe fruits Rip 

extracted 

using SPME 

Ripe fruits Unripe fruits 

extracted extracted using 

using CFMD CFMD 

1037 Isobutyl acetate Y X X 

1071 Ethyl 2 methyl butyrate Y Y X 

1141 2-Methyl-butyl-acetate Y X X 

1190 Butyl-2-methyl butyrate Y X X 

1758 Geranyl acetate Y X X 

Total number of esters 5 1 0 

1395 2-Butoxy ethanol X Y Y 

1915 2,6-Di-terbutyl-4-methyl-phenol Y X X 

Total number of alcohols 1 1 1 

1013 1-Oxiranylethanone* X Y X 

Total number of carbonyls X 1 Y 

Total number of compounds 6 3 1 

*tentative identification MS only 

Y-compound was extracted using this method 

X- compound was not extracted using this method 

64

Esters constituted (83.3 % %) while alcohols s comprised (16.7 %) of the total number of 

compounds identified in S. cocculoides upon using SPME. In contrast, using the CFMD 

method, resulted into ester esters (33.3%), alcohols (33.3 %) and carbonyls s (33.3 %) in ripe 

fruits. The unripe fruits provided only one alcohol when CFMD was used. used 

Figure 4.2 Structures of compounds extracted from the headspace of S. cocculoides 

fruit pulp using SPM SPME 

Using the he SPME method, isobutyl acetate, 22-methyl 

butyl acetate, , ethyl 2 methyl 

butyrate, 2.6 di-ter butyl-4-methyl-phenol, 

butyl-2-methyl butyrate, and geranyl acetate 

were identified (Figure 4.2 4.2). The CFMD method provided ethyl 2 methyl butyrate, 

2 butoxy ethanol and 1-oxiranylethanone. 

It is interesting that ethyl 2-methyl 2 butyrate 

was identified in S. cocculoides using both the SPME and the CFMD method. Due to its 

low odour threshold and fruity aroma aroma, ethyl 2-methyl methyl butyrate is considered consider to be one of 

the important contributors to the characteristic sweet, apple apple-like like odour of California 

skullcap (Scullaria Scullaria californica A. Gray) flowers (Takeoka et al., ., 2008), and Rambutan 

fruit (Nephelium Nephelium lappaceum L.) aroma (Ong et al.,1998). The presence e of geranyl acetate 

in S. cocculoides using SPME has been hitherto unreported unreported. 

65

4.3.2.2 Effect of ripening on volatile constituents of S. cocculoides 

Volatile constituents of ripe and unripe S. cocculoides obtained using the CFMD are 

provided in Table 4.5 above. Using the CFMD, the ripe S. Cocculoides showed the 

following volatile constituents; 1-Oxiranylethanone, ethyl-2-methyl-butyrate, and 

2-butoxyethanol. While in unripe S. cocculoides the same method gave only the 

compound 2-butoxy ethanol. This is probably because most of the important odour-active 

volatiles are not produced until later stages of ripening and maturation (Yahia et al., 

1990). 

4.4 Volatile constituents of cocoyam and cassava leaves 

4.4.1. Identity of volatile constituents of cocoyam leaves 

Using hydrodistillation and solvent extraction from the hydrosol, fresh cocoyam leaves 

produced eight (8) compounds (Table 4.6). These compounds constituted carbonyls 

(12.5 %), esters (12.5 %) and alcohols 75 %. The compounds identified were 

oxoisophorone, citronellyl formate, 2-butoxyethanol, linalool, citronellol, nerol, geraniol 

and phenol. Linalool is an important compound which has anticancer effects (Usta et al., 

2009). Thus, cocoyam leaves should also be investigated for such activity. 

66

Table 4.6: Compounds identified after hydrodistillation and solvent extraction from 

the hydrosol of fresh cocoyam leaves 

RI Compounds 

67 

Carbonyls 

1703 Oxoisophorone 

Total number of carbonyls 1 

Esters 

1609 Citronellyl formate 

Total number of esters 1 

Alcohols 

1395 2-Butoxyethanol 

1541 Linalool 

1765 Citronellol 

1800 Nerol 

1822 Geraniol 

1822 Phenol 

Total number of alcohols 6 


Dried cocoyam leaves produced fifteen compounds using the hydrodistillation and 

solvent extraction method (Table 4.7). The same leaves produced ten compounds using 

CFMD. The compounds, 2 hexanal, benzaldehyde, geranyl acetone, β-ionone, β-ionone 

epoxide and phenyl ethyl alcohol were identified in the dried leaves using both methods. 

Identification of these compounds using two different methods confirms their presence in 

dried cocoyam leaves.

Table 4.7: Volatile constituents obtained from dried cocoyam leaves using 

hydrodistillation and solvent extraction from the hydrosol (HSH) and 

CFMD 

RI Compounds HSH CFMD 

1212 2-Hexanal Y Y 

1457 Furfural Y X 

1534 Benzaldehyde Y Y 

1660 Phenyl acetaldehyde Y X 

1703 Oxoisophorone Y X 

1849 Geranyl acetone Y Y 

1937 β-Ionone Y Y 

1989 β-Ionone epoxide Y Y 

2197 3.4-Dimethyl-5-pentyl-5H-furan-2-one Y X 

Total number of carbonyls 9 5 

1340 1-Hexanol X Y 

1376 Cis-3-hexanol X Y 

1390 3-Hexen-1-ol Y X 

1395 2-Butoxy ethanol Y X 

1438 2-Hexen-1-ol Y X 

1441 trans Linalool oxide Y X 

1442 1-Octen-3-ol X Y 

1885 Benzyl alcohol X Y 

1903 Phenyl ethyl alcohol Y Y 

Total number of alcohols 5 5 

2359 Dihydroactinidiolide Y X 

Total number of esters 1 


Y- compound has been extracted by this particular method 

X- compound has not been extracted by this particular method 

The compounds identified using HSH from dried cocoyam leaves were comprised of 

carbonyls (60 %), alcohols (33.3 %) and esters (6.7 %). Using the CFMD, carbonyls 

constituted 50 % of the total number of compounds while alcohols made up the 

remaining 50 %. The presence of the lipid derived compounds such as benzaldehyde, 3- 

hexen-1-ol, 1-hexanol and cis-3 hexanol shows that cocoyam leaf lipids may be 

significant aroma precursors (Macleod, 1990). 

68

4.4.2 Identity of volatile flavour constituents in cassava leaves 

Compounds which were identified in fresh cassava leaves after extraction with steam 

distillation followed by solvent extraction (SS) and hydrodistillation followed by solvent 

extraction (HS) are provided in Table 4.8. 

Table 4.8: Volatile compounds from hydrodistillation and solvent extraction (HS) 

and steam distillation and solvent extraction (SS) of fresh cassava leaves 

RI Compounds HS SS 

1849 Geranyl acetone Y Y 


1616 Cyclocitral Y X 

1637 Safranal Y X 

Total carbonyls 4 2 

2209 Methyl palmitate Y Y 

2242 Ethyl palmitate Y Y 

2559 Methyl linolenate Y Y 

2594 Ethyl linolenate Y X 

Total fatty acid esters 4 3 


Y- the compound has been extracted using this particular method 

X- the compound has not been extracted using this particular method 

The results indicated that only fatty acid esters and carbonyls were identified. Extraction 

of fatty acid esters was due to the non polar solvent (n-hexane) used in the solvent 

extraction step. Steam distillation and solvent extraction of fresh cassava leaves 

identified, fatty acids and carbonyls which constituted 60 % and 40 % respectively of the 

compounds identified. Hydrodistillation and solvent extraction of the same leaves 

69

esulted in the identification of fatty acids (50 %) and carbonyls (50 %) of the 

compounds. Clearly, these methods identified the same classes of compounds, compounds this can be 

attributed to both methods being based on distillation. 

Figure 4.3: Structures uctures of compounds identified in fresh cassava leaves after 

extraction using both the HS and the SS methods. 

Both methods extracted geranyl acetone, β-ionone, methyl palmitate, ethyl palmitate 

and methyl linolenate (Figure 4.3) 4.3). Extraction of fatty acid esters seems to occur when a 

non polar solvent (n-hexane) hexane) is used for solvent extraction. 

70

Table 4.9: Compounds identified after hydrodistillation and solvent extraction from 

the hydrosol of fresh and dried cassava leaves 

RI Compounds HSH of dried leaves HSH of fresh 

leaves 

Carbonyls 


1290 3-Hydroxy-2-butanone X Y 

1390 Nonanal Y X 

1457 Furfural Y X 

1520 Heptadienal Y X 

1534 Benzaldehyde X Y 

1628 Isophorone Y Y 

1640 Phenylacetaldehyde X Y 

1643 Benzeneacetaldehyde X Y 

1703 Oxoisophorone Y Y 

1856 Dihydropseudoinone X Y 


1989 β-Ionone epoxide Y X 


1155 Penten-3-ol X Y 

1320 2-Penten-1-ol* X Y 

1340 1-Hexanol X Y 

1376 3-Hexanol Y X 

1395 Butoxyethanol Y Y 

1441 Trans Linalool oxide X Y 

1471 Cis- Linalool oxide X Y 

1541 Linalool X Y 

1795 Butoxy ethoxy ethanol X Y 

1880 Guiacol X Y 

1885 Benzyl alcohol Y Y 

1903 Phenyl ethyl alcohol Y Y 

1941 Phenol X Y 

2020 Ethy guiacol X Y 

2-Phenyl butanol X Y 

2077 Cresol X Y 

2188 Eugenol X Y 

2190 4-Ethyl phenol X Y 

2212 Vinyl guaiacol Y Y 

Total alcohols 5 18 

2359 Dihydroactinidiolide Y Y 

Total Esters 1 1 

Hexadecanoic acid X Y 

Total Alkanoic acids X 1 


*tentative identification MS only 

Y- the compound has been extracted using this particular method 

X- the compound has not been extracted using this particular method 

71

Hydrodistillation and solvent extraction of the hydrosol (HSH) of fresh cassava leaves 

produced twenty-nine nine compounds which comprised alkanoic acids (3.4 %), alcohols 

(62.1 %), carbonyls (31.1 %) and esters (3.4 %) (Table 4.9). Using the same method, method 

fourteen compounds which were made up of carbonyls (57.1 %), alcohols lcohols (35.7 %) and 

esters (7.2 %) were extracted from the dried leaves leaves. . Thus, carbonyls were the most 

dominant class of compounds extracted from fresh and dried cassava leaves using the 

HSH method. 

Figure 4.4: Structure of compounds identified in both fresh and dried cassava leaves 

following hydrodistillation and solvent extraction from the hydrosol. 

Fresh cassava leaves produced more volatile constituents than the dried leaves. This 

could imply that some flavour compounds are lost during the drying process. The 

compounds 2-hexanal, hexanal, isophorone, oxoisophorone, β-ionone, ionone, butoxyethanol, benzyl 

alcohol, phenyl ethyl alcohol, vinyl guiacol and dihydroactinidiolide were extracted from 

both fresh and dried leaves using the HSH method (Figure 4.4). The presence of these 

compounds in both fresh and dried cassava leaves shows that dried cassava leaves retain 

some of the flavour which is in fresh cassava leaves. 

72

Seven compounds made up oof 

f carbonyls (28.6 %), alcohols (57.1 %) and alkenes 

(14.3 %) were extracted from fresh leaves oof 

cassava using CFMD (Table 4.10). In dried 

cassava leaves the same method extracted ttwenty 

three constituents. . Esters E constituted 

4.3 %, alcohols 60.9 % an and d carbonyls comprised 34.8 % of the compounds. compounds The 

compounds 2 hexanal, 1-hexanol, 

trans linalool oxide and benzyl alcohol were identified 

in both fresh and dried leaves (Figure 4.5). 22-Hexanal 

gives a pleasant greenish gre to green 

teas and plays an importan important t role in the quality of green teas (Kato and Shibamoto, 2001). 

Figure 4.5: : Structures of compounds identified in fresh and dried cassava leaves 

following CFMD extraction 

The presence of 3-hexen hexen-1-ol, 2-hexanal and 1-hexanol hexanol (green leaf volatiles) volatiles in dried 

cassava leaves indicates cates that the leaves confer a fresh green odour even after they have 

been dried. 

73

Table 4.10: Compounds identified after CFMD of fresh and dried cassava leaves 

RI Compounds CFMD of fresh 

leaves 


1337 6-methyl-5-heptene-2-one X Y 

1465 2,4 Heptadienal X Y 

1534 Benzaldehyde Y X 

1628 Isophorone X Y 

1849 Geranyl acetone X Y 

1937 β-Ionone X Y 

1989 β-Ionone epoxide X Y 

2141 Hexadecanal X Y 


1155 Penten-3-ol X Y 

1237 Amyl alcohol X Y 

1340 1-Hexanol Y Y 

1382 3-Hexen-1-ol X Y 

1395 2-Butoxy ethanol X Y 

1420 2-Hexen-1-ol X Y 

1441 trans Linalool oxide Y Y 

1442 1-Octen-3-ol X Y 

1464 6-methyl-5-hepten-2-ol X Y 

1480 2-Ethyl-1-hexanol X Y 

1541 Linalool X Y 

1885 Benzyl alcohol Y Y 

1903 Phenyl ethyl alcohol X Y 

1914 Phenyl ethanol Y X 

1630 β-Cyclocitral* X Y 

74 

CFMD of dried 

leaves 

Total alcohols 4 14 

2359 Dihydroactinidiolide X Y 

Esters 1 

1471 Heptadiene Y X 

Alkenes 1 0 


*tentative identification (MS only) 

Y-compound has been extracted by this particular method 

X- compound has not been extracted by this particular method

CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 

5.1 Conclusions 

Using the quality parameters, the quality of the local herbal tea was lower than that of 

Lipton black tea. Compounds identified in F. ancylantha tea depend on the extraction 

method used. The compounds linalool, geraniol, β-ionone and geranyl acetone were 

extracted using both hydrodistillation methods while the CFMD method achieved only 2- 

butoxy-ethanol from the tea. Alcohols were the major group of compounds extracted 

from the herbal tea. 

In the indigenous fruits, different classes of compounds were also obtained; esters 

constituted the dominant class of compounds. Both SPME and CFMD methods both 

identified ethyl butyrate, ethyl isovalerate, and phenyl acetonytrile and phenyl alcohol in 

P. curatellifolia. The use of CFMD for S. cocculoides resulted in 1-oxiranylethanone, 

ethyl 2 methyl butyrate and 2-butoxy ethanol being identified. Ethyl-2-methylbutyrate 

was also obtained in ripe S. cocculoides upon using SPME. In the unripe S. cocculoides, 

2-butoxy ethanol was identified using the CFMD. 

Using hydrodistillation and solvent extraction from the hydrosol of fresh cocoyam leaves, 

the compounds identified were dominated by alcohols, constituting 75 %. The identified 

compounds were oxoisophorone, citronellyl formate, 2-butoxy ethanol, linalool 

75

citronellol, nerol, geraniol and phenol. Compounds, 2-hexanal, benzaldehyde, geranyl 

acetone, β-ionone, β-ionone epoxide and phenyl ethyl alcohol were identified in the dried 

leaves using hydrodistillation followed by solvent extraction and CFMD methods. 

Alcohols were the dominant class of compounds in fresh cocoyam leaves while carbonyls 

were dominant in dried cocoyam leaves. 

Fatty acids and carbonyls were obtained from fresh cassava leaves after steam distillation 

or hydrodistillation and subsequently solvent extraction. Both these two methods also 

extracted geranyl acetone, β-ionone, methyl palmitate, ethyl palmitate and methyl 

linolenate. Using hydrodistillation and solvent extraction of the hydrosol, fresh and dried 

cassava leaves gave carbonyls as the dominant class of compounds. In contrast, CFMD 

of the fresh and dried cassava leaves provided alcohols as the major group of compounds. 

The compounds, 2-hexanal, 1-hexanol, trans linalool oxide and benzyl alcohol were 

identified using this method from both fresh and dried leaves. 

76

5.2 Recommendations 

In order to exploit the different volatiles in the food and cosmetic industries, it is 

important that further work be carried out to: 

i) determine quantitatively the composition of volatile constituents in the plant 

materials; 

ii) exploit the volatile compounds from S. cocculoides, F. ancylantha, cocoyam 

and cassava leaves in unique formulations for household and industrial 

applications, and 

iii) determine the value of the herbal tea through processing inorder to expand its 

utilisation in Malawi and the region. 

77

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