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The design, preparation and evaluation of Artemisia Afra and placebos in tea bag dosage form suitable for use in clinical trials.

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University of the Western Cape

Artemisia Afra, a popular South African traditional herbal medicine is commonly administered as a tea infusion of the leaves. However, clinical trials proving it safety and efficacy are lacking mainly due to the absence of good quality dosage forms and credible placebos for the plant. The objectives of this study were to prepare a standardized preparation of the plant leaves and freeze-dried aqueous extract powder of the leaves, in a tea bag dosage form and to design and prepare credible placebos for these plant materials.

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THE DESIGN, PREPARATION AND EVALUATION OF ARTEMISIA

AFRA AND PLACEBOS IN TEA BAG DOSAGE FORM SUITABLE

FOR USE IN CLINICAL TRIALS

A. DUBE

A thesis submitted in partial fulfilment of the requirements for the degree of

Magister Pharmaceuticiae in the School of Pharmacy, University of the

Western Cape, Bellville, South Africa.

Supervisor: Prof. James A. Syce

June 2006

THE DESIGN, PREPARATION AND EVALUATION OF ARTEMISIA

AFRA AND PLACEBOS IN TEA BAG DOSAGE FORM SUITABLE

FOR USE IN CLINICAL TRIALS

A. DUBE

KEY WORDS

Artemisia afra

Standardized plant material

Plant freeze-dried aqueous extract

Tea bag dosage form

Infusion from tea bag

Luteolin

HPLC

Herbal stability

Herbal placebo

Clinical trials

Summary

The design, preparation and evaluation of Artemisia afra and placebos in tea bag dosage

form suitable for use in clinical trials.

A. Dube

M. Pharm. Thesis: School of Pharmacy, University of the Western Cape, Bellville, South

Africa.

Artemisia afra, a popular South African traditional herbal medicine is commonly

administered as a tea infusion of the leaves. However, clinical trials proving its safety and

efficacy are lacking mainly due to the absence of good quality dosage forms and credible

placebos for the plant.

The objectives of this study were to prepare a standardized preparation of the plant leaves

and freeze-dried aqueous extract powder of the leaves, in a tea bag dosage form and to

design and prepare credible placebos for these plant materials. It was hypothesised that

the intra-batch variation in the plant flavonoid constituents would be lower in the

standardized A. afra leaves and freeze-dried aqueous extract powders compared to the

traditionally used non-standardized leaves, that the tea bag would be a pharmaceutically

suitable dosage form for the traditionally used plant leaves, that the infusion profiles of

luteolin (the flavonoid marker), from the tea bag would be similar to that of the loose

leaves traditional form and that credible placebos for the plant materials devoid of

pharmacological activity could be prepared.

To realise the objectives, different batches of plant materials were blended and an

aqueous decoction and freeze-drying method used to prepare the standardized leaves and

freeze-dried aqueous extract powder of the plant leaves, respectively. These plant

materials were packed in 36cm2 tea bags which were pharmaceutically evaluated using

the European Pharmacopoeia criteria, subjected to stability testing and their infusion

profiles determined using the British Pharmacopoeia dissolution apparatus I (basket) and

a modification of the BP apparatus II (paddle) incorporating a holding cell, for the loose

leaves and the tea bag preparations, respectively, and the infusion profiles compared

using the f1 and f2 mathematical method. The A. afra leaves were exhaustively solvent

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extracted and inert in-organic salts blended to prepare the placebos for the leaves and

freeze-dried aqueous extract powder, respectively. Finally, the placebos were evaluated

for lack of pharmacological activity using the isolated guinea pig tracheal muscle

preparation.

The A. afra standardized dried leaves and freeze-dried aqueous extract powder, contained

a total luteolin content of 2.065 ± 0.2347 and 13.870 ± 1.2460µg/mg, respectively, with

an intra-batch variation (% R.S.D) of 11.36% and 6.70%, respectively, reduced from an

initial %R.S.D of 21.24%, 30.00% and 16.77%, for the three separately collected plant

batches used. The tea bag of the A. afra standardized dried leaves was stable under room

temperature and humidity conditions for 6 months, while the freeze-dried aqueous extract

tea bag was not and both tea bag preparations were unstable at conditions of 40˚C/ 75%

RH. The f1 and f2 values for the infusion of luteolin from the leaves in tea bag compared

to the loose leaves were 73.52% and 13.85%, respectively, indicating that the profiles

were not similar. Finally, the placebo materials prepared closely resembled the respective

plant materials and the placebo of the A. afra leaves possessed only slight muscle relaxant

activity while the placebo for the extract powder was pharmacologically inert.

In summary, the results showed that the tea bag was a suitable dosage form for the A. afra

standardized dried leaves, but not the freeze-dried aqueous extract powder and that the

tea bag preparation did not have similar infusion profiles to the loose leaves, but could

still be used in clinical trials provided that adjustments in the dose preparation and

administration methods are made. Finally, credible placebos for the plant materials

suitable for use in clinical trials were prepared.

DECLARATION

I declare that the thesis The design preparation and evaluation of Artemisia afra and

placebos in tea bag dosage form suitable for use in clinical trials is my own work, that it

has not been submitted before for any degree or examination in any other University and

that all the sources I have used or quoted have been indicated and acknowledged by

complete references.

A. Dube June 2006

Signed UWC, Bellville.

ACKNOWLEDGEMENTS

I would like to gratefully acknowledge the following;

Prof. James A. Syce my supervisor, for his guidance, support and the role he has played

in my personal development.

The National Research Foundation of South Africa (NRF) for providing the funding for

this project.

Coffee, Tea and Chocolate Company (Pvt) Ltd for donating the tea bag paper used in this

study.

A.R. Agencies for donation of the colourant chemical used in this study.

Mr B. Mouers and Mr F. Weitz for assistance with the identification and preparation of

voucher specimens of the plant material.

Mr V. Jeaven for his friendship and assistance with laboratory equipment and materials.

Mr. L. Cyster for assistance with the freeze-drying techniques.

Mr Y. Alexander for assistance with laboratory equipment.

Ms. Lorraine, Tanya Mapfumo, my girlfriend and wife to be, for her unending love and

support and assistance especially with the proof reading of this thesis.

Ms L. Manthata for assistance with the organ bath experiments.

My colleagues and friends; Hai Qiu Ma, Max Ojangole, Sizwe Mjiqiza, Caroline Kinyua,

James Mukinda, Joseph Kabatende, Kenechukwu Obikeze, Emmanuel Molosiwa, Doris

Tshambuluka, Ian Backman, Denis Chopera, Chido Ruwodo, Natasha Katunga, for your

invaluable friendship and support that made the completion of this thesis possible.

Finally, the entire school of pharmacy staff for making my study pleasant.

Thank you all.

vii

DEDICATION

I dedicate this master’s thesis to my parents Mr and Mrs Dube for their love,

support and encouragement that has got me where I am today.

viii

Contents

Title page i

Summary ii

Declaration iv

Acknowledgements v

Dedication vii

List of tables xiii

List of figures xiv

List of graphs xvi

List of appendices xvi

Chapter 1: Introduction 1

Chapter 2: Literature review 3

2.1 Introduction 3

2.2 Artemisia afra – An important indigenous medicinal plant 3

2.2.1 Vernacular names 3

2.2.2 Botanical classification, and morphology of Artemisia afra 4

2.2.3 Geographical distribution of Artemisia afra 4

2.2.4 Traditional uses and dosage forms of Artemisia afra 5

2.2.5 Shortcomings of the traditional dosage formulations 6

2.3 The need for safety and efficacy data 8

2.3.1 Clinical trials 9

2.3.1.1 Placebos 10

2.4 Teas 11

2.4.1 Instant teas 12

2.4.2 Tea bags 13

2.4.2.1 Infusion from tea bags 13

2.4.2.2 Advantages of tea bags as a dosage form 15

2.5 Phytochemical constituents of Artemisia afra 16

2.6 The flavonoids 16

2.7 Quality considerations for herbal materials 19

2.7.1 Standardization of herbal materials 19

2.7.2 Chemical fingerprints of herbal materials 20

2.8 Quality assessment of herbal materials 20

2.8.1 Quality assessment of herbal starting materials 21

2.9 Quality assessment of herbal preparations 21

2.9.1 Dissolution / infusion testing 22

2.9.2 Dissolution / Infusion profile comparison methods 23

Chapter 3: Work Plan 26

3.1 Study objectives 26

3.2 Hypothesis 26

3.3 Study approach 27

3.3.1 Why Artemisia afra? 27

3.3.2 Why a tea bag dosage form? 27

3.3.3 Why luteolin? 28

Chapter 4: Preparation and evaluation of Artemisia afra plant material and design

of placebos 29

4.1 Introduction 29

4.2 Equipment and materials 29

4.3 Methods 30

4.3.1 Collection and preparation of the plant material 30

4.3.2 Standardization of the Artemisia afra dried leaves 31

4.3.3 Preparation of the freeze-dried aqueous extract powder 31

4.3.4 Irradiation of the plant materials 32

4.3.5 Determination of the plant material organoleptic characteristics 32

4.3.5.1 Determination of plant material particle size and shape 33

4.3.5.1.1 The sieve method 33

4.3.5.1.2 Determination of the plant material particle shape 34

4.3.5.2 Determination of the plant material colour 34

4.3.5.3 Determination of the plant material odour 34

4.3.5.4 Determination of the plant material bitterness value 35

4.3.6 Determination of the plant material ash values 36

4.3.7 Determination of the plant material moisture content 37

4.3.8 Determination of microbial contamination of the materials 37

4.3.9 Development and validation of the HPLC assay 38

4.3.10 Quantitation of luteolin in the plant materials 39

4.3.11 Design of the plant material placebos 41

4.3.11.1 Design of placebo for the standardized dried leaves 41

4.3.11.2 Design of placebo for the freeze-dried aqueous extract powder 43

4.3.11.3 Matching of odour between the placebos and the plant materials 44

4.3.11.4 Matching of taste between the placebos and the plant materials 46

4.4 Results and discussion 47

4.4.1 Preparation of the plant materials 47

4.4.2 Organoleptic properties of the A. afra plant materials 48

4.4.2.1 Particle size and shape of the A. afra plant materials 49

4.4.2.2 Bitterness value of the A. afra plant materials 49

4.4.3 Ash values of the A. afra plant materials 49

4.4.4 Moisture content of the A. afra plant materials 50

4.4.5 Microbial contamination of the plant materials 51

4.4.6 Development and validation of the HPLC assay 52

4.4.7 Quantitation of luteolin in the A. afra plant materials 54

4.4.8 Design of the placebos for the plant materials 57

4.4.8.1 Design of placebo for the A. afra standardized dried leaves 57

4.4.8.2 Matching of odour of the placebo and plant materials 60

4.4.8.3 Matching of taste of the placebo and plant materials 60

4.4.8.4 Design of placebo for the freeze-dried aqueous extract powder 60

4.5 Conclusions 61

Chapter 5: The preparation and evaluation of tea bags and placebos of

Artemisia afra 63

5.1 Introduction 63

5.2 Equipment and materials and animals 63

5.3 Methods 64

5.3.1 Preparation of the tea bags 64

5.3.1.1 Determination of the amount of plant material for the tea bags 65

5.3.1.2 Determination of the appropriate tea bag size 65

5.3.1.3 Selection of the appropriate tea bag paper 66

5.3.1.4 Preparation of the Artemisia afra and placebo tea bag dosage forms 66

5.3.2 Pharmaceutical evaluation of the tea bag dosage forms 67

5.3.2.1 Determination of uniformity of mass of the tea bag dosage forms 67

5.3.2.2 Determination of the infusion profile of the A. afra dosage forms 67

5.3.2.2.1 Determination of the infusion profile of the A. afra loose leaves 68

5.3.2.2.2 Determination of the infusion profile of the A. afra tea bag dosage

forms 69

5.3.2.2.4 Sample preparation, analysis and comparison of the infusion profiles 71

5.3.2.3 Determination of the stability of the dosage forms upon storage 72

5.3.3.4 Comparison of the A. afra tea bag dosage forms and their placebos 73

5.3.4 Pharmacological evaluation of the placebo for the plant leaves 73

5.3.5.1 Preparation of solutions used in the tracheal muscle experiments 74

5.3.5.2 Procedure for evaluation of the effect of the A. afra and placebo

materials on the isolated guinea pig tracheal muscle 72

5.7 Results and discussion 76

5.7.1 Preparation of tea bags containing the A. afra plant material 76

5.7.2 Uniformity of mass of the dosage forms 77

5.7.3 Infusion profile of the A. afra loose leaves 78

5.7.4 Infusion profile of the tea bags containing the A. afra leaves 82

5.7.5 Infusion profile of the tea bags containing the freeze-dried aqueous

extract of A. afra 84

xii

5.7.6 Comparison of the infusion profiles of the A. afra loose leaves and tea

bags containing the leaves and freeze-dried aqueous extract powder 85

5.7.7 Results of the stability studies conducted on the dosage forms 87

5.7.7.1 Organoleptic and chromatographic evaluation: Conditions at site A 87

5.7.7.2 Organoleptic and chromatographic evaluation: Conditions at site B 90

5.7.7.3 Organoleptic and chromatographic evaluation: Conditions at site C & D 91

5.7.7.4 Infusion profile characteristics of the dried leaves tea bag stored under

the conditions at site A and B 92

5.7.7.5 Infusion profile characteristics of the freeze-dried aqueous extract

powder in tea bag stored under conditions at site C and D 94

5.7.8 Comparison of tea bags of the A. afra plant material and their placebos 95

5.7.9 Results of test for absence of activity of the placebo for the A. afra

dried leaves 97

5.8 Conclusions 99

Chapter 6: Conclusion 101

References 103

Appendices 116

Vitae 136

xiii

List of the Tables

Table 4.1 Various excipients and their proportions used in different formulations

in the design of the placebo for the A. afra freeze-dried aqueous

extract powder. 44

Table 4.2 Organoleptic characteristics of the A. afra standardized dried leaves and

freeze-dried aqueous extract powder. 48

Table 4.3 Residual moisture content of the A. afra standardized dried leaves 50

Table 4.4 Residual moisture content of the A. afra freeze-dried aqueous extract

powder 51

Table 4.5 Accuracy and recovery of the luteolin standard 54

Table 4.6 Intra-day and inter-day precision of the assay 54

Table 4.7 The series of solvent extractions employed to produce the placebo material

of the A. afra leaves 58

Table 4.8 Excipients and quantities used in the formulation of the placebo for the

A. afra freeze-dried aqueous extract powder 61

Table 5.1 Uniformity of mass of the prepared tea bags of the A. afra standardized

dried leaves 78

Table 5.2 Uniformity of mass of the prepared tea bags of the freeze-dried aqueous

extract powder of A. afra 78

Table 5.3 Mean percentage release values (n = 6) of luteolin from the A. afra

loose leaves and leaves in tea bag 86

Table 5.4 Comparison of the infusion profiles of luteolin from the A. afra leaves

in tea bag (test) against that from the loose leaves (reference) 86

Table 5.5 Loss on drying of the A. afra dried leaves after 180 days stability testing at

Site A 89

Table 5.6 Loss on drying of the A. afra dried leaves after 90 days stability testing at

Site B 90

Table 5.7 Percentage released by the tea bags of the A. afra dried leaves

at 180 minutes from tea bags stored at storage sites A and B 93

Table 5.8 Luteolin content (µg/ml) infused after 5 minutes infusion of the tea

xiv

bags of the freeze-dried aqueous extract powder stored under

conditions at site C and D 94

Table 5.9 Physicochemical comparison of tea bags of the A. afra leaves and

placebo for the leaves 96

Table 5.10 Physicochemical comparison of tea bags of the A.afra freeze-dried

aqueous extract and placebo for the extract powder 96

Table 5.11 Percentage relaxation produced by cumulative concentrations of the

A. afra standardized dried leaves and placebo of the leaves on the

methacholine-induced contraction of the isolated guinea pig tracheal

muscle 97

List of Figures

Figure 2.1 Artemisia afra plant in its natural habitat at the Montagu museum 4

Figure 2.2 Geographical distribution of Artemisia afra in South Africa 5

Figure 2.3 Schematic diagram showing the biosynthesis of the flavonoids 17

Figure 2.4 The flavonoid nuclear structure and the structure of luteolin (3′,4′,5,7-

tetrahydroxyflavone) 17

Figure 4.1 Sampling positions used in the quantitation of luteolin in the A. afra plant

materials 40

Figure 4.2 Chemical structure of linalool 46

Figure 4.3 The standardized dried leaves of A. afra plant and the freeze-dried

aqueous extract powder of the A. afra standardized dried leaves 47

Figure 4.4 Retention times and peak heights of morin and luteolin

standards injected for generation of the standard curve 53

Figure 4.6 Chromatogram showing the retention of morin and luteolin within the

plant matrix 54

Figure 4.7 Concentrations of the different forms of luteolin at various sampled

positions in the A. afra plant materials 57

Figure 4.8 Placebo leaves produced by repeated solvent extractions of the A. afra

dried leaves 56

Figure 4.9 A. afra standardized dried leaves chromatographic fingerprint after first

solvent extraction 59

Figure 4.10 A. afra leaves chromatographic fingerprint after final solvent extraction 60

Figure 5.1 The holding cell devised to contain the A. afra tea bags during the infusion

process and a schematic diagram of the modified dissolution apparatus II

used for the determination of the infusion profile of the tea bags

containing the A. afra plant materials 70

Figure 5.2 The organ bath system used for evaluation of the activity of the plant

materials 75

Figure 5.3 A close up picture of a single organ bath 75

Figure 5.4 Prepared tea bag containing 4g of the standardized dried leaves of

A. afra and that containing 0.55g of the A. afra freeze-dried aqueous

extract powder 77

Figure 5.10 A tea bag of the A. afra dried leaves at day 0 and at day 180, stored

under conditions of normal room temperature and humidity 89

Figure 5.11 A tea bag containing the A. afra freeze-dried aqueous extract

powder at day 0 and at day 28, stored under conditions of normal

room temperature and humidity 89

Figure 5.12 A tea bag of the A. afra leaves at day 0 and after 90 days storage

at conditions of 40ºC/75% relative humidity 90

Figure 5.13 The tea bag of the A. afra freeze-dried aqueous extract powder at

day 0, after day 90 stored under conditions at site C and after 90 days

storage at the conditions at site D 91

Figure 5.14 The A. afra standardized dried leaves in tea bag and the A. afra dried

leaves placebo in tea bag 96

Figure 5.15 The freeze-dried aqueous extract powder of A. afra in tea bag, the

placebo of the freeze-dried aqueous extract powder of A. afra in

tea bag and the colours of the teas produced by A. afra placebo

and A. afra freeze-dried extract powder 97

Figure 5.16 Percentage relaxation versus log concentration of the A.afra

xvi

standardized dried leaves and placebo effect on the isolated guinea pig

tracheal muscle 98

List of Graphs

Figure 4.5 The standard curve and linear regression line values used in the

quantitation of luteolin 53

Figure 5.5 The infusion profiles of the A. afra loose leaves and the percentage release

profile of the A. afra loose leaves determined using UV spectroscopic

analysis at 220 and 349nm 79

Figure 5.6 The infusion profiles of luteolin release and the percentage release profiles

of luteolin from the A. afra loose leaves 79

Figure 5.7 The infusion profiles of the A. afra and the percentage release profiles of

the tea bags containing the A. afra leaves determined using UV

spectroscopic analysis at 220 and 349nm 82

Figure 5.8 The infusion profiles of luteolin release from the A. afra tea bag and the

percentage release profiles from the tea bags containing the A. afra

leaves 82

Figure 5.9 The infusion profiles and the percentage release profiles of the tea bags

containing the A. afra freeze-dried aqueous extract, determined using UV

spectroscopy analysis at 220 and 349nm 84

List of Appendices

Appendix A1 Table showing results of the quantitative determination of percentage of

leaves, flowers and stems which constitute Artemisia afra plant and the

average loss on drying of the A. afra leaves after dyring in an oven for 3

days at 30ºC 116

Appendix A2 Figure of certificate of irradiation for the Artemisia afra dried leaves and

freeze-dried aqueous extract powder 117

Appendix A3 Table of the extractions conducted and resultant yields obtained in

xvii

the preparation of the freeze-dried aqueous extract of Artemisia afra 118

Appendix A4 Tables of the results of the particle size classification of the A. afra plant

materials 118

Appendix A5 Method and results obtained for the determination of the bitterness

value of A. afra dried leaves and freeze-dried extract powder 119

Appendix A6 Results of the ash value determinations on the A. afra plant materials 121

Appendix A7 The microbiological testing methods used and the results obtained

in the determination of the microbial contamination on the

A. afra standardized dried leaves and freeze-dried aqueous

extract powders before and after irradiation of the plant materials 123

Appendix B The data and results of the determinations of the luteolin

content and variation in the plant material batches 126

Appendix C The specifications for the DynaporeTM 117/7/0 tea bag

paper used for manufacture of the A. afra plant material tea bags 128

Appendix D1 The results of the organoleptic evaluation conducted on the tea bags

of the A. afra leaves subjected to different storage conditions 129

Appendix D2 Chromatographic fingerprint and UV spectral overlays of the

samples from the A. afra plant materials in tea bag after storage at the

various conditions 131

Appendix E The pen recordings (data) from the pharmacological evaluation

of the placebo material using the isolated guinea pig trachea model 135

Chapter 1

Introduction

Artemisia afra is a popular traditional herbal medicine widely used in South Africa for

the treatment of a variety of ailments (Roberts, 1990; Van Wyk & Gerike, 2000).

Infusions of the plant leaves are commonly prepared as teas for treatments ranging from

asthma, malaria to diabetes, and the steam from such infusions may also be inhaled for

headaches and colds (Hutchings et al., 1996; Thring & Weitz, 2006).

Despite the popularity and widespread use of this herb, clinical trials demonstrating its

safety and efficacy in humans are lacking. For traditional herbal medicines in general,

several reasons have been given to account for this state of affairs and include the

variability in the preparation methods and poor quality of the traditional dosage forms,

the variation in the phytochemical composition of the plant materials used and the lack of

credible placebos for use in the clinical trials (Wolsko et al., 2005). For example, in the

Artemisia afra traditional preparations, it is commonly directed that a quarter cup or a

double handful of the wet or dried leaves be infused as a tea (Roberts, 1990) and from

this it may be anticipated that such non-specific directions to measurement may lead to

variations in dose each time a treatment is prepared. In addition, the use of wet leaves in

the dosage preparations may result in poor product quality, as the presence of moisture

may encourage microbial growth. The A. afra plant materials used may also be subject to

factors that may affect their phytochemical composition such as variations in the

geographic source of the materials, time of harvest, drying processes and storage

conditions and as a result, variations in the observed therapeutic effects may be expected

(Graven et al., 1990; Zidorn et al., 2005). Collectively, the above issues, together with the

fact that there is currently no placebo material for A. afra plant, makes it difficult to

conduct good quality clinical trials on the herb.

To address some of the above issues and therefore enable clinical trials on the herb, it was

proposed to standardize the plant leaves and prepare them in a tea bag dosage form. It

was anticipated that such a preparation would be of suitable pharmacopoeial quality and

would have release profiles of the actives that were comparable to those observed in the

traditional infusions (and therefore could be used in place of the traditional preparation in

clinical trials). In addition, the process of standardization would reduce the inherent

variability in the phytochemical constituents of the plant materials, guarantee

reproducible clinical effects and therefore enable good quality safety and efficacy data for

the herb to be generated. Furthermore, in order to obtain an even more uniform and

constant material composition, a freeze-dried aqueous extract powder of the plant leaves

was to be prepared in the tea bag dosage form, for administration as an instant tea, with

the added advantage of rapid preparation for the patients (Wichtl, 1994). Finally, it was

intended to design placebo materials which looked, smelt and tasted similar to the A. afra

plant materials and were devoid of pharmacological activity.

Suitable analytical methods such as HPLC were used to characterise the quality of the

plant materials and of the dosage preparations. Methods were designed for the

preparation of the placebo materials and their pharmacological activity was evaluated

using the isolated guinea pig tracheal muscle preparation.

Chapter 2

Literature Review

2.1 Introduction

In the present chapter, an overview of Artemisia afra plant is given and this includes its

description, uses and traditional methods of preparation. The shortcomings of the

traditional preparations shall also be highlighted and the proposed methods of addressing

them discussed. The need for safety and efficacy data, including the general aspects of

clinical trials and quality aspects, for herbal products shall also be discussed. Finally, a

review of the methods used for dissolution profile comparisons shall be presented.

2.2 Artemisia afra – An important indigenous medicinal plant

2.2.1 Vernacular names

Artemisia afra Jacq. Ex Willd, is one of the oldest known and most widely used of

medicinal plants in South Africa (Roberts, 1990; Watt & Breyer-Brandwijik, 1962).

Several of the indigenous ethnic groups of South Africa have a long tradition of use of

this plant for various ailments, and as such different names relate to it some of which are

given below (Watt & Breyer-Brandwijik, 1962):

Xhosa : Umhlonyane

Zulu : Mhlonyane

Sotho : Lanyana

Tswana : Lengana

English : African Wormwood

Afrikaans : Wildeals

2.2.2 Botanical classification and morphology of Artemisia afra

Artemisia afra belongs to the;

Division : Magnoliphyta

Class : Magnoliopsida

Sub class : Asteridae

Order : Asterales

Family : Asteraceae

Genus : Artemisia

Species : A. afra

Artemisia afra is an erect growing, shrubby, woody, perennial plant growing up to 2 m

tall with a leafy and hairy stem (Van Wyk et al., 1997). Its leaves grow up to 8 cm long

and 4 cm wide. The leaf shape is narrowly ovate, bi-pinnatipartite, feathery and finely

divided. The ultimate segments which are linear in shape with an acute tip and smooth or

toothed margin, grow up to 10 mm long and 2 mm wide. It has a pectinated midrib with

similar lobes, a smooth or glandular-punctated upper surface and a canescent (grayish)

lower surface. The petiole is up to 2 cm long, dilated at the base with a pair of simple or

divided leaf-like stipules. The inflorescence is subglobose with nodding, leafy, terminal,

racemose panicles up to 40 cm long (Hilliard, 1977).

The plant is easily identifiable by its characteristic aromatic odour. It flowers between

January and June, producing yellow coloured flowers. The fruits are about 1mm long,

somewhat 3-angled and slightly curved with a silvery-white coating. In winter, the

branches die back but rapidly regenerate from the base (Hilliard, 1977; Van Wyk et al.,

1997).

2.2.3 Geographical distribution of Artemisia afra

Artemisia afra is found growing abundantly in Namibia, Zimbabwe, and in the mountain

regions of Kenya, Tanzania and Uganda. It is also found as far up as Ethiopia, in the east

Fig. 2.1: Artemisia afra plant.

and south of tropical Africa and down to the eastern part of South Africa including

Swaziland and Lesotho (Van Wyk et al., 1997; Hilliard, 1977).

In South Africa, the plant can be found growing in regions from Stellenbosch, the

Western Cape, Aliwal North and the Graaff Reinet Mountains in the North. It is also

widespread in Natal from the coast to the Drakensberg (Hilliard, 1977). Its geographical

distribution in South Africa is shown in the Figure 2.2 below.

2.2.4 Traditional uses and dosage forms of Artemisia afra

Artemisia afra is a herb used for the treatment of several ailments (Van Wyk et al., 1997).

It is a well-known treatment for coughs, colds, croup, whooping cough, colic, heartburn,

flatulence and gout (Roberts, 1990; Thring & Weitz, 2006). It is also commonly used for

the treatment of asthma, acute bronchitis, hay fever, bladder and kidney disorders,

convulsions, diabetes, fever, headache, inflammation, rheumatism, stomach disorders and

worms (Felhaber, 1997; Thring & Weitz, 2006). The herb is also used for the treatment of

malaria (Watt & Breyer-Brandwijik, 1962).

In traditional Zulu medicinal use, leaf infusions are taken as teas or administered as

enemas and the steam from the infusions is commonly inhaled for the treatment of

Figure 2.2: Geo

raphical distribution of

Artemisia afra in South Africa

(SATMERG, 1999).

headaches and colds (Hutchings et al., 1996). Enemas made from ground plants

suspended in water or milk are administered for constipation or intestinal worms in

children. Decoctions are also taken as blood purifiers for acne, boils, measles and

smallpox (Bryant, 1966). In a survey of medicinal plant use in the Bredasdorp/ Elim

region of the Southern Overberg in the Western Cape Province of South Africa,

Artemisia afra was found to be the plant with the greatest use value amongst the

respondents. In other words, Artemisia afra was used the most for the treatment of

various ailments more than any other traditional plant medicine among the people

surveyed (Thring & Weitz, 2006).

The usual dosage preparation of the plant is in the form of a tea or a decoction (Roberts,

1990; Felhaber, 1997; Van Wyk et al., 1997). Commonly, a quarter cup of fresh leaves to

one cup of boiling water is allowed to stand and steep for 10 minutes, then strained and

sweetened with honey before drinking (Roberts, 1990). This preparation may be

administered orally for the relief of most of the ailments previously mentioned. An

infusion may also be prepared, by pouring 2 litres of boiling water over 1 cup of fresh

leaves and stems and allowed to draw for an hour before being strained and this may be

used as a bath for measles, wounds, bites and stings (Felhaber, 1997). A strong brew of

the herb may be prepared using a half-cup of leaves to 11/2 or 2 cups of boiling water,

allowed to draw for 10 minutes and then strained. This can be used as a mouthwash for

gumboils, mouth ulcers or for the relief of earache (Bryant, 1966). Apart from these

orally administered dosage forms, poultices, vapours for inhalation and enemas are

prepared for the treatment of a variety of ailments (Roberts, 1990; Felhaber, 1997).

2.2.5 Shortcomings of the traditional dosage formulations

As stated above, the common method of dosage preparation is that of a decoction which

involves the use of wet leaves immersed in boiling water to make a tea. The traditional

method of preparation has several disadvantages attributed to it. Firstly, the use of wet

leaves is undesirable. This is because the presence of moisture in the leaves may promote

microbial growth and accelerate degradation of the product (McCutcheon, 2002). The

microbes may however be killed by use of boiling water during the decoction process, but

in some circumstances the microbial load may still be outside the acceptable limits as set

by the European Pharmacopoeia for herbal medicinal products (EP, 2002c).

Under the traditional method, the directions for dosage preparation are often obscure.

Often the directions are stated as ‘a double handful’, ‘cup of boiling water’ or ‘quarter

cup of leaves’ (Roberts, 1990). It is difficult to accurately quantify a double handful or

measure a quarter cup consistently. Furthermore the size of ‘cups’ varies and this may

lead to variations in the actual dose the patient receives.

It is well known that plants grown in different geographic locations may contain different

compositions of the active principles (Zidorn et al., 2005; Gilani & Atta-ur-Rahman,

2005). The chemical constituents may vary depending on time of harvest, plant origins,

drying processes, storage times and other factors (Liang et al., 2004; Yang et al., 2005).

Phenolic compounds such as flavonoids in plants have been shown to increase

proportionally with increases in altitude and this has been attributed mainly to their

function as UV-B protective agents (Zidorn et al., 2005). Graven et al (1990) have

reported significantly higher oil yields from Artemisia afra when the crop was harvested

during anthesis and early seed set as opposed to earlier or later harvesting in the

reproductive period. The aforementioned observations may be expected to affect the plant

materials used in the traditional setting, resulting in variations in the phytochemical

composition of the plant and consequently the amount of actives the patient receives in a

given dose.

In the traditional method of preparation, there is no mention of the expected degree of

comminution of the leaves used. In the extraction of constituents from plant matrices, i.e.

in the decoction or infusion process, the degree of comminution (leaf size) is of crucial

importance as the amount of constituents released into the tea per given time period

increases as the degree of comminution increases and the highest yields are often

obtained with smaller leaf sizes (Wichtl, 1994). In addition to the leaf size, the method of

preparation or drying/ storage of the plant material may affect the kinetics of release of

the active principles due to changes in the internal leaf structure (Jaganyi & Price, 1999).

2.3 The need for safety and efficacy data

According to the World Health Organization (WHO), about 65-80% of the world

population rely on herbal medicines for their primary health care needs (WHO, 1993). A

marked growth in worldwide use of phytotherapies has occurred over the last 20 years

and in Germany, it is estimated that about 80% of physicians prescribe herbs and sales of

herbal medicines worldwide are in the region of hundreds of billions of dollars a year

(Gilani & Atta-ur-Rahman, 2005). However, the boom in use and popularity has not been

followed by an increase in supporting scientific data and insufficient safety and efficacy

data exists for most plants to support their widespread use (Bombardelli, 2001; Calixto,

2000; Chang, 2000). This state of affairs has been attributed mainly to the lack of patent

protection and the diversity and relatively small-scale of the industry involved, which has

seen many of them unable to meet the financial demands of efficacy and safety studies

(Mills, 1998). In addition, quality issues such as lack of plant material standardization,

good quality dosage forms and thorough characterization of the traditional plant materials

has also hampered clinical trial research or where conducted, has led to poor quality

studies (Wolsko et al., 2005). Finally, the use of different dosages and variations in the

duration of treatment (as administered in the traditional setting) makes comparison and

analysis of the clinical data difficult (Kroes & Walker, 2004).

However, it is important that claims for safety and efficacy of herbal medicines be

scientifically validated. Once the herb has been scientifically validated, it can gain the

acceptance of conventional medicine (Calixto, 2000). Ispaghula, Garlic, Ginseng,

Gingko, St. John’s Wort and Saw Palmetto are a few examples of herbs that have gained

popularity and approval by physicians as a result of scientific validation through clinical

studies (Gilani & Atta-ur-Rahman, 2005). In addition, clinical studies help reveal the

toxic effects, risks and inherent side effects of the plants for safer and more effective use

of the medicines (Calixto, 2000).

Once scientifically validated, the herb may be approved by drug regulators and be

marketed for public use. Although preliminary assessments of efficacy can be obtained

through in vitro testing and experiments on animals, authorities licensing new medicines

for public use require evidence of their effect on human beings and clinical trials, with

minimum bias, are able to satisfy these requirements (Mills, 1998).

2.3.1 Clinical trials

A clinical trial is any systematic study of a medicinal product in human subjects whether

in patients or in non patient volunteers in order to discover or verify the effects of and/ or

identify any adverse reaction to the investigational product and/ or study their absorption,

distribution, metabolism and excretion in order to ascertain the efficacy and safety of the

product (EEC, 1997).

Clinical trials on herbal medicines may have one of two types of objectives. One is to

evaluate the safety and efficacy that is claimed for a traditional herbal medicine and the

other is to develop new herbal medicines or examine a new indication for an existing

herbal medicine or change of dose formulation, or route of administration. In some cases,

trials may be designed to test the clinical activity of a purified or semi purified compound

derived from herbal medicines (EEC, 1997; WHO, 1998).

Clinical trials are generally divided into 4 phases. Phase I trials are carried out on a small

number of healthy volunteers or patients suffering from the disease for which the

medicine is intended. The main purpose of this type of trial is to observe tolerance to the

medicine and therefore get an indication of the dose that may be used safely in

subsequent studies (EEC, 1997; WHO, 1998). Phase II studies are conducted on a limited

number of patients to determine clinical efficacy and further confirm safety. Such studies

are usually randomized, double-blind, controlled studies, using for control groups either

an existing alternative treatment or a placebo. The dosage schedules used in such studies

are then used for more extensive clinical studies (EEC, 1997; WHO, 1998). Phase III

studies are performed on larger patient groups usually situated at several study centres

using a randomized double blind design to validate the preliminary evidence of efficacy

obtained in earlier studies. Normally such studies are conducted in conditions which

mimic the anticipated or normal conditions of use as closely as possible (WHO, 1998).

Finally, phase IV studies are performed after the dosage form is available for public use

and the main purpose of such studies is to detect toxic events that may occur so rarely

that they are not detected in earlier studies (WHO, 1998).

Clinical trials are required to conform to the standards of Good Clinical Practice (GCP),

which essentially, is an international ethical and scientific quality standard for designing,

conducting, recording and reporting trials. Compliance with this standard provides public

assurance that the rights, safety and well-being of trial subjects are protected, consistent

with the principles in the Declaration of Helsinki, and that the clinical trial data is

credible (EEC, 1997).

2.3.1.1 Placebos

A placebo, also commonly referred to as a dummy pill, is in theory a pharmacologically

inert substance. The placebo may be defined as any therapy or component of therapy that

is deliberately used for its non-specific, psychological, or psychophysiological effect, or

that is used for its presumed specific effect, but is without specific activity for the

condition being treated (Emilien et al., 1998).

Historically, placebos began to be utilized in therapeutic research around the early

nineteenth century in response to accusations that the efficacy observed had to do with

just the appearance of a treatment, as opposed to any real effect from the therapy itself

(Kaptchuck et al., 2001). As a result, placebos are now included in clinical research in

order to function as a mechanism for controlling and ensuring that any observed

improvement in the condition under investigation is due to the effects of the treatment

alone. The requirements for a credible placebo are that factors such as the appearance, the

volume or quantity, the number of intakes, preparation and route of administration be

similar to the treatment under investigation (Emilien et al., 1998).

Herbal materials are known to present significant challenges in designing credible

placebos. This is mainly due to the nature of plant materials with respect to their

appearance, smell and taste, which is difficult to mimic using synthetic materials or

chemicals (Kaptchuck et al., 2001). In literature, few herbal clinical trials employing the

use of a placebo actually discuss the ingredients used in the formulation of the placebo

and from the studies found, none were identified as using a placebo of the plant parts

themselves, i.e. the leaves, bark, roots, etc. All studies found used extracts of the herbs

which were formulated as tablets or capsules. One example is that of a placebo of a

Chinese herbal preparation, that contained 78.2% calcium hydrogen phosphate, 19.6%

soy fibre, 0.3% cosmetic brown, 0.5% cosmetic yellow, 0.01% edicol blue 0.09%

identical liquorice dry flavour, and 0.03% bitter flavour (Bensoussan et al., 1998).

2.4 Teas

Tea was first processed as a beverage in China over 3000 years ago and today millions of

cups of tea are consumed everyday around the world making tea one of the most popular

beverages in the world (Peterson et al., 2004).

Teas vary by type (i.e. black, oolong, green and pu’er), variety (i.e. blended and

unblended), and processing (i.e. conventional, cut, tear, and curl). Unblended teas are

named by their country of origin (e.g. Assam, China, Darjeeling), while blended teas are

named generically (Earl Gray, Irish Breakfast), rather than by the teas they contain

(Peterson et al., 2004).

In brewing a tea, several factors are involved in influencing the characteristics of the final

brew. Apart from the quality of the tea leaf itself, the quality of the water used is

important in determining the brew (Jaganyi & Wheeler, 2003). Bottled or filtered water is

normally preferred and this has to do mostly with the influence on the resultant flavour of

the tea (Anonymous, 2006a). The temperature of the water used for the brew is also

important and it is recommended that black teas be brewed with boiling water, oolongs

with water just below boiling point and green teas with water around 80˚C. Again, the

temperature affects the flavour as well as the constituents extracted (Anonymous, 2006a).

The other important factor in brewing teas is the steeping time. Steeping times depend on

the type of leaf and its grade. Black teas are normally steeped from 3 - 7 minutes while

oolongs are steeped from 1 ½ - 4 minutes and greens from 2 - 3 minutes. Steeping time

affects the amount of constituent extracted, which ultimately affects the flavour of the tea.

Longer steeping times normally result in bitter teas (Anonymous, 2006a).

Flavonoids are known to be extracted from teas during the tea making process. The

resultant flavonoid content in a cup of tea may depend on one of the two factors, i.e. the

characteristics of the tea leaf itself and the brewing characteristics, as described above

(Peterson et al., 2005). Currently, there is much interest in the type and amount of

flavonoids extracted from teas and herbal infusions. This is largely due to the reported

health benefits of teas such as antioxidant, artherosclerotic and anti- carcinogenic benefits

and flavonoids are believed to be mainly responsible for these actions (Wang &

Helliwell, 2001; Atoui et al., 2005).

2.4.1 Instant teas

An instant tea is produced as a result of an exhaustive extraction of the tea leaves/herb

using water or in some instances water/ ethanol mixtures. The resultant powders are then

mixed with hot water for consumption as a tea (Wichtl, 1994). Generally, two types of

instant teas are available resulting from differences in the manufacturing process. Spray-

dried extracts are dry and hollow pellets produced as the extract is sprayed through a

nozzle and forms fine droplets in a current of warm air. The result is a low-density highly

soluble powder. However, these extracts are often hygroscopic with caking as a major

problem (Wichtl, 1994). Tea granules are granular or cylindrical aggregates produced

when the extract is sprayed onto a carrier (usually saccharose) and dried with heating.

These granular extracts are often very soluble in water and slightly hygroscopic.

However, they contain a small percentage of drug extract, as carrier and filler substances

occupy a large proportion of the granule (Wichtl, 1994).

Generally, spray- dried instant teas are considered the ideal for a tea for medicinal

purposes. They have the advantage of rapid preparation, which can be of convenience for

the patients. They also have the added advantage of a uniform and constant composition,

which is important in order to produce reproducible pharmacological effects (Wichtl,

1994; Bombardelli, 2001).

2.4.2 Tea bags

Millions of cups of tea are consumed everyday around the world and most of these are

brewed using tea bags (Jaganyi & Ndlovu, 2001). Different shapes and sizes exist on the

market, all with the aim of capturing the attention of the consumer. Apart from the

traditional square and rectangular shapes, round and pyramidal shaped tea bags are also

common (Jaganyi & Ndlovu, 2001). Tea bag papers used are commonly manufactured

from nonwoven fibres usually based on cellulose from the seeds of cotton or stem fibres

of hemp, jute or abaca trees (Anonymous, 2006b). These may be oxygen bleached or be

processed unbleached to form the tea bag paper. The fibres may also be processed in

combination with synthetic polymers to form papers that are heat sealable (Schoeller &

Hoesch, 2005). High strength at the sealing joints, good cut-ability, high wet strength,

good particle/dust retention and flavour infusion are some of the requirements for a good

quality tea bag paper (Schoeller & Hoesch, 2005).

2.4.2.1 Infusion from tea bags

It has been reported in literature that when infused in water, the tea leaf swells by a factor

of about 4.25 (Spiro & Price, 1985) and therefore, depending on the size of the tea bag,

some hindrance occurs to the swelling of the tea leaf and this affects the rate of infusion

of the constituents (Jaganyi & Mdletshe, 2000). In experiments conducted on the effect of

tea bag size and shape on the rate of caffeine extraction from Ceylon orange pekoe tea

(Jaganyi & Ndlovu, 2001), it was observed firstly, that the infusion of caffeine from loose

tea was much faster than from tea in the tea bag. Secondly, significant increases in the

rate of extraction were observed with increases in tea bag size. From a 16 to 36 cm2 tea

bag area, an increase of 25% was observed and smaller increases in the rate of extraction

were observed from tea bags larger than 36 cm2 up to an area of 64 cm2 (Jaganyi &

Ndlovu, 2001).

The above observations have been attributed to increases in the space available for the

swelling and movement of the tea leaf inside the tea bag. The theory behind this

explanation assumes that when the tea leaf comes into contact with water, it swells first

and then infusion follows. In the case of smaller sized tea bags, the tea leaves after

swelling a compacted together. This makes penetration of water to the leaf and to the

centre of the tea bag, more difficult. Moreover, the extracted constituents from the middle

of the tea leaf to the bulk of the solution have to follow a more tortuous passage within

the leaf particles; hence, a slower infusion process results (Spiro & Lam, 1995; Jaganyi &

Ndlovu, 2001).

The hindrance to infusion can also be explained from the point of view of Nernst

diffusion layers (Jaganyi & Ndlovu, 2001). A Nernst diffusion layer by definition is a

fictitious layer above the true concentration profile or diffusion layer (IUPAC, 1997).

This diffusion layer is present both inside and outside the tea bag membrane. The Nernst

diffusion layer on the inside decreases with increase in the tea bag size because of an

increase in free movement of the tea leaves and solution inside the bag. This explains the

observed increase in rate of infusion with tea bag size. It also explains why the infusion is

faster from loose leaves than from a tea bag as the Nernst diffusion layer in the former is

zero (Spiro & Jaganyi, 2000). It also follows that, any motion which decreases the

thickness of the inner and outer Nernst layers adjacent to the tea bag paper membrane,

will increase the rate of tea brewing and this explains empirical findings among tea

drinkers that stirring the brew around the tea bag, moving the tea bag up and down and

jiggling the tea bag all speed up the brewing of the tea (Spiro & Jaganyi, 2000).

Other factors which affect the rate of tea brewing in a tea bag include the transfer of the

constituents through the membrane, diffusion towards and away from the membrane plus

through the swollen leaf, method of manufacture/preparation and storage of the tea leaves

and composition and temperature of the extracting medium (Spiro & Jaganyi, 2000;

Jaganyi & Mdletshe, 2000).

2.4.2.2 Advantages of tea bags as a dosage form

Tea bags offer several advantages compared to the traditional tea preparation. Firstly, the

fact that the correct amount (dose) is already accurately measured out offers both safety

(from over- or under- dosing) and convenience in preparation for the patient. This

advantage is more apparent in the case of mixed herbal teas, where the correct

proportions of the herbs (with similar particle size) are already measured into the tea bag

and therefore the patient does not have to scoop and measure each constituent herb as

would be necessary with loose teas (Wichtl, 1994). Secondly, the considerable degree of

comminution, which is required to pack into tea bags, also allows for a better extraction

of the constituents compared to the uncut herb. The amount of constituents in the tea has

been shown to increase as the degree of comminution increases and higher yields are

often obtained using powdered drugs (Wichtl, 1994).

Tea bag paper material has been shown to retard the rate of infusion by about 29% when

compared to loose tea (Jaganyi & Mdletshe, 2000) and this may offer an advantage if one

wishes to control the amount of actives the patient receives within a given time period.

However, in practice, because of the considerable degree of comminution these smaller

leaves infuse faster inside the tea bags such that the overall infusion rate is increased over

and above the larger-sized loose tea leaves (Jaganyi & Mdletshe, 2000).

Finally, tea bags also offer practical advantages. For example, the tea is easier to handle

and simpler and less messy to dispose of as there is no residue left in the cup (Jaganyi &

Mdletshe, 2000).

2.5 Phytochemical constituents of Artemisia afra

Literature sources contain some information on the phytochemical constituents of

Artemisia afra. The essential oil of A. afra is known to contain the compounds α-pinene,

γ-terpinene, camphene, ρ -cymene, 1.8-cineole, α-thujone, β- thujone, camphor, borneol,

Artemisia ketone and sesquiterpene- 1-3 (Graven et al., 1990; Van Wyk et al., 1997;

Piprek, 1982). In addition to these compounds found in the essential oil, other

compounds have been detected within the leaves and these include the tannins, saponins,

terpenoids of the eudesmadien and germacratien types, triterpenes α- and β- amyrin, the

friedelin alkanes ceryl cerotinate and n- nonacosane as well as the coumarins and

acetylenes (SATMERG, 1999; Van Wyk et al., 1997).

However, little is documented in the literature concerning the flavonoid constituents of A.

afra. Investigations in our laboratory by Waithaka (2004), Komperlla (2005) and

Mukinda (2006) on leaf infusions and aqueous extracts of the plant leaves have revealed

the presence of the flavonoids luteolin, kaempferol, apigenin and quercetin.

2.6 The flavonoids

Flavonoids are a class of natural compounds present in all vascular plants. They occur in

virtually all plant parts including the leaves, roots, wood, bark, pollen, flowers, berries

and seeds (Markham, 1982). They are the pigments responsible for the hues and various

colours observed in flowers and are also required for the normal growth, development

and defense mechanisms in plants (Di Carlo et al., 1999; Harborne & Williams, 2000).

Flavonoids are biosynthesized via a combination of the shikimic acid and

acylpolymalonate pathways. A cinnamic acid derivative synthesized from shikimic acid

acts as the starting compound in a polyketide synthesis in which additional acetate

residues are incorporated into the structure (Figure 2.3). This is followed by ring closure

and through subsequent hydroxylations and reductions, plants are then able to form

different classes of flavonoids (Markham, 1982; Di Carlo et al., 1999).

ure 2.3: Schematic dia

ram showin

the bios

nthesis of the flavonoids

(

Di Carlo et al.

1999

)

Structurally, flavonoids consist of a fifteen-carbon atom basic nucleus (C6-C3- C6

configuration) with several phenolic groups and, in some instances, attached sugars to

form glycosides. They possess two benzene rings namely A and B, which are connected

by an oxygen-containing heterocyclic ring C (Figure 2.4). Flavonoids are grouped

according to the presence of different substituents on the rings and by the degree of ring

saturation. Flavonoids containing a pyran ring i.e. a hydroxyl group in position C3 of the

C ring are classified as 3-hydroxyflavonoids (i.e. the flavonols, anthocyanidins and

catechins) and those lacking a hydroxyl group in position C3 as 3-desoxyflavonoids i.e.

the flavonones (e.g. hesperetin, naringenin) and flavones (e.g. luteolin, apigenin)

(Markham, 1982; Heim et al., 2002).

Figure 2.4: The flavonoid nuclear structure (on the left) and the structure of luteolin ( 3′,4′,5,7-

tetrahydroxyflavone) (on the right).

Flavonoids may occur as aglycones (consisting of a benzene ring condensed with a six

member ring which possesses a phenyl ring at the 2 position), glycosides (that carry one

or more sugar residues on the ring) or as their methylated derivatives. Linking to various

sugar residues increases the variation in structure and polymerization of the flavonoid.

For example, quercetin alone is known to have over 179 glycosides and of the 2 x 103

different flavonoids already identified, up to 2 x 106 are thought to exist (Molnar-Perl &

Fuzfai, 2005).

Flavonols and flavones are the most widely occurring flavonoids; of these luteolin (figure

2.4), quercetin, kaempferol, myricetin, chrysin and apigenin are widely distributed. The

flavanones, flavanols, dihydroflavones and dihydrochalcones, are considered minor

flavonoids because of their limited natural distribution (Di Carlo et al., 1999).

The starting step in the analysis of flavonoids, normally involves extraction or isolation

of the flavonoids. Extraction with methanol or solid phase extractions are the commonly

used methods. Optimum extracting conditions, however, vary and depend on the matrix

to be isolated from e.g. plasma, urine or herbal material. Chromatographic techniques

such as HPLC are the methods of choice in the separation, identification and quantitation

of flavonoids. (Molnar-Perl & Fuzfai, 2005; de Rijke et al., 2006). In the analysis

procedures, it is accepted practice to first acid hydrolyse the glycosides and then identify

or quantify the released aglycones. This is mainly due to the large number of flavonoid

conjugates present and the few numbers of commercially available reference compounds

for them (Crozier et al., 1997). The hydrolysis procedure may also be used as a step to

reduce the number of compounds to be determined, resulting in better resolution and

improved characterization of the flavonoid constituents (Molnar-Perl & Fuzfai, 2005).

A wealth of information exists in the scientific literature concerning the pharmacological

properties of flavonoids. Antioxidant, anti-mutagenic, anticancer, anti-hypertensive, anti-

inflammatory, anti-diabetic, anti-allergy, anti-asthma, anti-ischaemic, anti-ulcer,

antibacterial, antiviral and immune stimulating properties (Havsteen, 2002). Some of the

reported pharmacological actions of the flavonoids are similar to those reported for A.

afra, which may give a lead as to the possible active compounds within the plant.

2.7 Quality considerations for herbal materials

Quality may be defined as the sum of variable characteristics that may significantly

impact upon a product. For herbal medicines, such variable characteristics include the

origins of the herb, botanical identity, purity, potency, stability and content of specified

marker compounds (McCutcheon, 2002). Apart from these, issues of Good Agricultural

Practices and Good Manufacturing Practices are also important and have a direct bearing

on the final quality of the product (EMEA, 2001).

2.7.1 Standardization of herbal materials

Standardization refers to measures taken to ensure that there is a consistent quantity of a

defined marker compound within a herbal material, as herbal materials are known to be

highly variable in their make up (Gilani & Atta-ur-Rahman, 2005). Intrinsic factors (e.g.

genetics) and extrinsic factors (e.g. growing, harvesting, storage and drying processes)

may lead to variations in the chemical profiles of the herb (McCutcheon, 2002; Yang et

al., 2005; Zidorn et al., 2006). In order to achieve reproducible biological data in terms of

safety and efficacy, it is recommended that the herbal material be standardized to the

active ingredients when they are known, or to specific markers when the actives are not

yet known (Bombardelli, 2001).

Standardization is commonly achieved through blending different batches of the plant

material (McCutcheon, 2002; Bombardelli, 2001). The assumption is that the content of

the other constituents will also vary in proportion to the marker compound, and that if

each batch contains the same amount of marker compound, other constituents will also be

relatively consistent (Bombardelli, 2001). Alternatively, normalization may be employed.

This is the process of adjusting the extraction ratio and/or adding fillers to achieve the

targeted marker content. However, this is usually acceptable within narrow limits and

large adjustments are only permissible in cases where it has been established that the

marker is responsible for the pharmacological activity (McCutcheon, 2002). Certain

authorities consider this method similar to adulteration, particularly if this is not declared

on the product label and consequently this method of standardization is not encouraged

(McCutcheon, 2002).

2.7.2 Chemical fingerprints of herbal materials

Chemical fingerprints are commonly used to confirm the identity, authenticity and lot-lot

consistency of a plant (Fan et al., 2006). Currently chromatographic and/or

electrophoretic techniques are used to generate chromatographic patterns of plant

materials (Springfield et al., 2005; Fan et al., 2006). Chemical fingerprints allow most of

the phytochemical constituents of the herb to be determined and in this way, the full

herbal plant can be considered as the active as opposed to the use of a single constituent

(which may or may not be responsible for clinical efficacy). In this way, a form of

chemical standardization can thus be applied as a quantitative ratio among the various

constituents can be established, i.e. fixing a quantitative relationship between classes of

compounds, the active principles or some characteristic compounds present in the plant

(Bombardelli, 2001). It is generally accepted that materials with similar chromatographic

fingerprints are likely to possess similar properties and as a result, the fingerprints allow

the concept of phytoequivalence to be applied. This is the comparison of the chemical

fingerprints for a material or product under question with that of a clinically proven

reference product and is meant to guarantee patient safety as well as protection from

adulterated products (Liang et al., 2004).

2.8 Quality assessment of herbal materials

Monographs exist for some of the commonly used and popular herbal medicines and the

European Pharmacopoeia contains over 130 monographs to which the respective herbs

must comply. However where monographs are not available, quality assessment is

usually based on the results of test procedures performed on the plant material itself

(Springfield et al., 2005). Such tests include analysis of the starting material, tests on

microbial quality or contaminants such as pesticides and fumigation agents. Quantitative

determination of marker compounds with known activity is also assessed as a quality

criterion (EMEA, 2001).

2.8.1 Quality assessment of herbal starting materials

An assessment of the quality of the starting material and excipients is required. Firstly,

information on the site of collection, time of harvesting, stage of growth, drying and

storage conditions should be documented (WHO, 2004) and in the case of herbal drugs

with constituents with known activity, assays of their content using validated methods are

required. This content must be stated as a range in order to ensure reproducibility

(EMEA, 2001). Where the constituents are not known, suitable marker compounds may

be selected and used (WHO, 2004; EMEA, 2001).

Generally, herbal materials must be tested for microbial contamination, pesticides and

fumigation agents, toxic metals and other likely contaminants and adulterants.

Acceptance criteria and limits exist but are diverse and there appears to be lack of

consensus on these (WHO, 1998). For instance, the limits for some pesticides published

in the Pharmeuropa 1993 are more restrictive than the WHO limits. In addition, the

limits specified for microbial contamination in the European Pharmacopoeia 2002 are

more restrictive than the WHO 1998 limits.

2.9 Quality assessment of herbal preparations

Guidelines require that the particulars of the characteristics, identification tests and purity

tests for the product be established (EMEA, 2001). These may include details of tests on

the performance of the dosage form such as dissolution or infusion. Chemical fingerprints

can be used to trace the stability of the herbal preparation. Since the whole herbal drug or

preparation may be considered to be the active, a determination of the stability of a single

marker compound may not suffice; an analysis of the whole herbal material may be more

appropriate (EMEA, 2001). Heigl et al in a study to examine possible changes in the

flavonoid pattern of common herbal drugs during long term and stress testing storage

conditions, used HPLC fingerprint comparisons to demonstrate differences in stability of

individual flavonoid components (Heigl et al., 2003). Such comparisons may allow

determinations on substances present in the herbal preparations with respect to their

stability and proportions, for quality purposes (Bombardelli, 2001).

2.9.1 Dissolution / infusion testing

In vitro drug release from dosage forms may be characterized as a quality control

procedure as well as to establish in- vitro in- vivo correlations. Compendial methods such

as the BP or USP apparatus I (basket), II (paddle) and III (reciprocating cylinder) may be

used to evaluate the in vitro release characteristics of the dosage form.

Several factors are known to influence drug dissolution and include the effective surface

area of the drug, dissolution coefficient of the drug, thickness of the diffusion layer, the

saturation solubility of the drug, volume of the dissolution media and the amount of drug

in the solution (Missaghi & Fassihi, 2005). Generally, the compendial methods of

dissolution testing have been designed to test for dissolution from conventional

pharmaceutical dosage forms and although a test for dissolution from medicated patches

exists in the British Pharmacopoeia 2000, utilizing an extraction cell to hold the patch in

place, by and large the methods are intended for tablets and capsule dosage forms.

In characterizing the infusion of tea bags, which are both floating and swelling matrices

(a non conventional dosage form), the selection of an appropriate dissolution method

requires special considerations in order to attain sensitive and reproducible dissolution

data. Missaghi and Fassihi (2005) evaluated the effect of various hydrodynamic

conditions on drug release from a floating and eroding matrix. The USP apparatus I, II,

III and a modified apparatus II (paddle over mesh) were evaluated for similarity over

various agitation speeds and the results from the determinations of these investigations

showed that the paddle apparatus at 50 revolutions per minute (rpm) has similar

hydrodynamics to the paddle over a mesh at 50 rpm and to the basket apparatus at 100

rpm. These results may aid in the selection of an appropriate dissolution method and

respective agitation rate, for similar non-conventional dosage forms such as tea bags.

2.9.2 Dissolution / Infusion profile comparison methods

Dissolution/infusion profile comparison methods have been developed in order to answer

questions regarding the similarity in performance of pharmaceutical dosage forms. These

comparisons also aid the development of in- vitro in- vivo correlations, which can help

reduce costs, speed up product development and reduce the need to perform costly

bioavailability/ bioequivalence studies. They also have applications in establishing final

dissolution specifications for pharmaceutical dosage forms (O’Hara et al., 1998; Freitag,

2001).

A dissolution profile may be defined as the measured fraction (or percentage) of the

labelled amount of drug that is released from a dosage unit at a number of predetermined

time points when tested in a dissolution apparatus (O’Hara et al., 1998). The methods to

compare dissolution profile data can be categorized as either, exploratory data analysis

which includes graphical and numerical summaries of the data; mathematical methods

that typically use a single number to describe the difference between the profiles and

statistical and modelling methods which take both the variability and underlying

correlation structure in the data into account in the comparison.

Exploratory data analysis methods as stated above, involve graphical and numerical

analysis. The data may be illustrated graphically by plotting the mean dissolution profile

data for each formulation with error bars extending to two standard errors at each

dissolution time point. The dissolution profiles may be considered to differ significantly

from each other if the error bars at each time point do not overlap (O’Hara et al., 1998).

As a complement to the graphical summary, the data may be summarized numerically by

presenting the mean and standard deviation at each time point. In addition, the difference

between the mean profiles and a 95% confidence interval for the difference in the mean

profiles may be presented. If the 95% confidence interval does not contain zero, then the

difference at that time point are considered significantly different at the 5% significance

level (O’Hara et al., 1998). The exploratory method is not currently endorsed by the

United States Food and Drug Administration (FDA) and another deficiency is that it is

difficult to definitively conclude that profiles are different if the error bars overlap only at

some time points and not at others, or if the 95% confidence interval for the difference in

the profiles contains zero only at some time points. In addition, if several formulations

are being compared, graphical and numerical illustrations may become too cluttered, with

all the error bars and columns in the tables, making the data evaluation difficult (O’Hara

et al., 1998).

Two mathematical comparison methods are described in the literature. The first described

by Moore and Flanner and the second by Roscigno. Only the Moore and Flanner method

shall be discussed here, as it is the most popular and accepted of the two. Moore and

Flanner described two equations; a ‘difference factor’ f1 (equation 2.1) and a ‘similarity

factor’ f2 (equation 2.2).

Where n is the number of dissolution time point, Rt and Tt are the reference and test

dissolution values at time tt , respectively and wt is an optional weighting factor (Freitag,

2001).

The f1 equation is the sum of the absolute values of the vertical distances between the test

and reference values. The f1 equation is zero when the profiles are similar and increases

proportionally as the difference between the profiles increases and values between 0 and

15 mean similarity between profiles (FDA, 1997). The f2 equation is a logarithmic

transformation of the average of the squared vertical distances between the test and

Eqtn: 2.1

Eqtn: 2.2

reference dissolution values at each time point. The f 2

value approaches 100 when the

profiles are identical and values between 50 and 100 ensure similarity between the

profiles (FDA, 1997).

This mathematical method is the most popular of the comparison methods as it is easy to

compute and is accepted and recommended by the FDA. The main disadvantage is that it

does not take into consideration the variability or correlation structure in the data.

However, the FDA implies that the f2 equation should only be used when the within-

batch variation, in terms of coefficient of variation, is less than 15% (FDA, 1997). The f1

and f2 are also sensitive to the number of time points used and it has been criticized that

the basis of the criteria for deciding the difference or similarity between the dissolution

profiles is unclear. In other words, the difference between the dissolution profiles at

which the difference is considered to be of practical importance or likely to affect in vivo

performance is not clear (O’Hara et al., 1998; Saranadasa & Krishnamoorthy, 2005).

Statistical methods include the one-and two-way analysis of variance (ANOVA)

methods, mixed effects model (multivariate methods), modelling-based methods and

Chow and Ki’s methods (O’Hara, 1998). Statistical methods take into consideration the

variability and correlation structure of the data. However, they possess several

disadvantages which limit their use. For example, ANOVA comparisons may only be

statistically significant at some dissolution time points and not at others, thus making it

difficult to conclude whether there is any difference. As a result, ANOVA methods are

only recommended in the case of immediate release data at a single dissolution time point

(O’Hara, 1998; Freitag, 2001). The other statistical methods also have similar pitfalls

which limit their use, i.e. ambiguous in interpretation, tedious to perform, difficulty in

implementation using standard statistical software, sometimes inefficient and a lack of

regulatory endorsement. However, the multivariate method is recommended by the FDA

when the within-batch variability has a coefficient of variation greater than 15%, but this

method also suffers from some of the pitfalls mentioned above (FDA, 1997; O’Hara et

al., 1998).

Chapter 3

Work Plan

3.1 Study objectives

The objectives of the study were:

• To prepare standardized dried leaves and freeze-dried aqueous extract powder of

A. afra plant leaves and to characterise the physiochemical properties of the

materials,

• To develop and validate an analytical method for the detection and quantitation

of luteolin in the A. afra plant materials,

• To quantitate and determine the inter- and intra batch variation in the luteolin

content of the plant materials,

• To prepare the standardized dried leaves and freeze-dried aqueous extract powder

in tea bag dosage form and determine and compare the infusion profiles (using

luteolin as a marker compound), of the tea bag dosage form and the loose leaves

(traditional dosage form),

• To design and evaluate credible placebos for the dried leaves and the freeze-dried

aqueous extract powder of A. afra.

3.2 Hypotheses

It was hypothesised that:

• The intra batch variation in the luteolin levels decreases in the order dried leaves

> standardized dried leaves > freeze-dried aqueous extract,

• The tea bag dosage form is a suitable dosage form for the traditional plant leaves

and will meet pharmacopoeial quality requirements,

• The infusion profile of luteolin from the tea bag dosage form will be similar to

that of the loose leaves (traditional form) and therefore be suitable for use in

clinical trials and,

• It is possible to design credible placebos for the Artemisia afra plant materials

devoid of pharmacological activity.

3.3 Study approach

This study intended to enable clinical trials on the plant to be conducted by, firstly,

standardizing the A. afra plant materials and preparing a freeze-dried aqueous extract

powder of the plant leaves and secondly, by preparing the plant materials in a tea bag

dosage form and determining the similarity of the infusion profiles of the tea bags and the

loose leaves (in order to ensure interchangeability) and finally, by designing credible

placebos for the plant materials devoid of pharmacological activity.

3.3.1 Why Artemisia afra?

Artemisia afra (Wilde als) has several uses attributed to it in literature. Several authors

describe it as being one of the most widely used and popular plant medicines in South

Africa. It is therefore important that such a popular medicinal plant be validated in terms

of its safety and efficacy using clinical trials. In addition, its many uses, widespread

distribution throughout Africa and several different methods of preparation and

administration made the plant an ideal candidate to model as a plant standardized and

developed into a suitable dosage form for evaluation in clinical trials following which,

regulatory approval for marketing can be granted.

3.3.2 Why a tea bag dosage form?

Traditionally A. afra is administered in the form of a tea. This tea is commonly prepared

by infusing the loose leaves in hot water (Roberts, 1990). Therefore, in order to mimic

the traditional method of preparation, which would be necessary for the evaluation of the

safety and efficacy of the plant as used traditionally, a dosage form which closely

resembles the loose leaves, yet overcomes the disadvantages associated with the loose

leaves was needed. Previous studies have attempted to manufacture tablet dosage forms

of the plant leaves (Komperlla, 2005). These have by and large proved unsuitable due to

the hygroscopic nature of the extracts used to manufacture the tablets. In addition, these

extracts may not contain all the constituents found within the leaves. Therefore, a tea bag

containing the A. afra leaves would allow the complete plant leaves to be administered to

the patient and closely resemble the way the plant is used traditionally.

3.3.3 Why luteolin?

It is generally recommended that marker compounds be used to identify and characterize

the quality and stability of plant materials especially where the active ingredient is

unknown (EMEA, 2005). In this study, the flavonoid luteolin was chosen as a marker

compound for Artemisia afra leaves. This flavonoid has been found consistently in

various batches of A. afra plant leaves, and was found to be stable in assay procedures

applied (Waithaka, 2004). In addition, several literature sources describe the

pharmacological effects of luteolin, e.g. anti-oxidant, anti-inflammatory, anti-allergic,

anti-cancer and blood glucose modifying effects (Shimoi et al, 2004; Mino et al, 2004)

and these effects, in relation to the traditional uses of A. afra, may suggest that this

compound may play a role in the observed therapeutic effects of the herb. Also, much

was known on the extraction, detection and quantitation of flavonoids in plant matrices,

making the flavonoids and luteolin in particular, suitable marker compounds to

characterise plant materials.

Chapter 4

Preparation and evaluation of Artemisia afra plant material and design

of placebos

4.1 Introduction

In this chapter, the methods used in the preparation of the standardized dried leaves and

freeze-dried aqueous extract powder (herewith referred to as SDL and FDAE,

respectively), of Artemisia afra leaves are presented. The development and validation of

the HPLC assay used for evaluation of the plant materials is also described in detail.

Finally, methods used in the design of the placebos as well as the results obtained are

presented and discussed.

4.2 Equipment and materials

The following equipment were used;

-85˚C freezer (Lozone CFC Freezer, Model U855360, New Brunswick Scientific,

USA), balance (Scaltec SPB42, Model SPB71, Scaltec Instruments, Heiligenstadt,

Germany), centrifuge (Labofuge 200, Germany), furnace (Naber Model L47T.

Industrieofenbau 2804, Lilienthal/Bremen, West Germany), freeze-drier (Virtis

Freeze Mobile 72SL, The Virtis Company Gardner, New York, USA), light

microscope (Nikon Monocular Model Sc, Japan), HPLC filter unit (Millipore

Cameo 25 AS, DDA 02025So MSI: Micro separation Inc., USA), solvent filter

paper (47mm filter membrane 0.45µm PVDF, Millipore, USA), filtration system

(SUPELCO) connected to vacuum pump (Medi-Pump Model 1132-2, Thomas

Industries, Inc., USA), filter paper (Whatman No. 41 & Whatman No.1,

Whatman, England), laboratory blender (Waring Commercial Laboratory

Blender 8010, Model 32BL79, New Hartford, Connecticut, USA), oven (Model

Memmet 854 Schwabach, West Germany), pH meter (Basic 20 Crison

Instruments, S.A, Italy), spectrophotometer (DU 640 spectrophotometer,

Beckman, USA), test sieve shaker (Endecott sieve shaker, E.F.L. 1MK11,

Endecotts (test sieve) LTD, London, England), vortex machine (Vortex-2, G-

560E, Scientific Industries, Inc. Bohemia, N.Y. 11716 USA), water bath (Labcon,

CDH 110, Maraisburg, South Africa).

The HPLC system used consisted of an auto sampler (Beckman Gold Module 507), a

programmable binary gradient pump (Beckman Gold Module 126 series), a diode array

detector (DAD) (Beckman Gold Module 168 series) with a 32-KaratTM-software package

and a Synergy® Hydro- reverse phase column (Phenomenex, USA) having 4μm particle

size and a column length of 250 x 4.60 mm.

The following materials were used;

Artemisia afra plant, quinine hydrochloride dihydrate Ph Eur (Fluka Chemie

GmbH, Germany), acetonitrile (Burdick & Jackson, M.I. USA), ethyl acetate CP

(Saarchem, South Africa), luteolin, morin hydrate, hesperetin (Sigma Aldrich,

Germany), sodium hydroxide (UnivAR, Saarchem, South Africa), methanol AR,

hydrochloric acid, potassium hydrogen phosphate (Riedel-de Haen AG,

Germany), lactose (UniLab, Saarchem, South Africa), D.C brown (AR Agenicies

cc, South Africa), sodium starch glycollate (UniLab, Saarchem, South Africa),

potato starch (Saarchem, South Africa), calcium chloride, microcrystalline

cellulose, di-sodium hydrogen phosphate heptahydrate (Merck, South Africa),

sodium saccharin (Supelco. Bellefonte, USA), linalool (Sigma-Aldrich Chemie

GmbH, Germany), silica gel (BDH, South Africa), dimethyl sulphoxide (Sigma-

Aldrich, Germany).

4.3 Methods

4.3.1 Collection and preparation of the plant material

Whole Artemisia afra plants were collected from the Montagu district of the Western

Cape Province of South Africa in February 2005. A sample of the collected plants was

filed as a voucher specimen (Voucher No: 6735) in the herbarium of the Botany

Department of the University of the Western Cape. This collected plant material was

considered as batch 001. Additional plant material batches 002 and 003 were collected

from the same district in the months of March and April 2005, respectively. The freshly

collected plant material was separated from earthy and other foreign material and the

leaves (for use in the study) manually separated from the stems and flowers of the plant.

The collected leaves were then weighed before being dried in an oven at a temperature of

30oC for 3 days (Komperlla, 2005). After drying, the leaves were weighed again to obtain

the final dry mass. Finally, the dried leaves were cut up into smaller fragment sizes using

a laboratory blender and the resultant leaf fragments stored sealed in plastic bags in a cool

dark place away from direct light.

4.3.2 Standardization of the Artemisia afra dried leaves

There are various ways of achieving standardization of plant materials. The most

common and accepted method is blending different batches of plant material together

(McCutcheon, 2002), and in this study, the 3 different batches of A. afra plant leaves

were blended together. Equal portions of each plant batch were separately weighed and

placed together in a large plastic bag before being mixed by shaking vigorously for 5

minutes. The resultant leaves blend was then stored in sealed plastic bags in a cool dry

place, protected from direct light.

4.3.3 Preparation of the freeze-dried aqueous extract powder.

The extraction procedure used to prepare the extract, as far as possible, mimicked the

methods described in the literature for the preparation of the traditional dosage.

Commonly, a quarter cup quantity of A. afra leaves to a cup of boiling water is allowed to

stand and seep for 10 minutes (Roberts, 1990). Therefore, the aqueous extract was

prepared by adding boiling distilled water to the appropriate mass of standardized dried

leaves (SDL) in 1:35 ratio of leaves to solvent (Komperlla, 2005) and the extraction

mixture left to seep and draw for 10 min. After this period, the mixture was filtered using

Whatman no. 1 filter paper and the filtrate frozen at -85oC in a freezer. The frozen extract

was then dried at -44oC under vacuum over 3 days using a freeze drier. The resultant

extract powders were then combined and weighed and the percentage yield calculated.

The FDAE powder was then placed in amber glass containers and these stored in

desiccators until used (Komperlla, 2005).

4.3.4 Irradiation of the plant materials

There is a requirement for microbiological decontamination of materials of natural origin

due to the high levels of microbes inherent in these materials. However, methods of

decontamination are few and are often restricted. For example the use of ethylene oxide is

forbidden, and the efficacy of some other methods has not been well established (WHO,

1998). Ionizing irradiation is a well established decontamination method which has been

proved effective and safe, having been used for decontamination of spices and seasonings

for several years (Razem & Katusin-Razem, 2002).

In this study, the SDL as well as the FDAE powder of A. afra were decontaminated using

ionizing radiation from a Cobalt (Co) source at a level of 18 kGy. Pre- and post-

irradiation microbial tests were conducted to check and confirm the effectiveness of the

procedure in the decontamination of the plant materials.

4.3.5 Determination of the plant material organoleptic characteristics

Organoleptic characteristics refer to the appearance, colour, odour, and taste of a

substance. An examination to determine these characteristics is normally the first step

towards establishing the identity and degree of purity of materials (WHO, 1998; EMEA,

2005). Characterization of these properties is also a primary step in setting of

specifications, as well as setting a standard to which a placebo should resemble if it is to

be credible. In addition, changes in the stability of a material may be recognized by

changes in the organoleptic properties. Sweeteners, flavorants and aroma chemicals may

be added to mask objectionable odours and tastes present in a material (Zheng & Keeney,

2006).

For this study, the appearance, colour, odour and taste of the SDL and FDAE powder of

A. afra were characterized using the human sensory evaluation (i.e. by eye, nose and

tongue).

4.3.5.1 Determination of the plant material particle size and shape.

Particle size and shape have an effect on the physico-chemical properties of a dosage

form. They play a great role in producing homogeneity of dosage forms and, in the case

of tea bags, influence the rate of infusion of the soluble constituents. Smaller fragment

sizes are known to infuse faster than larger sized ones (Wichtl, 1994; Jaganyi &

Mdletshe, 2000) and this difference in the rate of infusion may affect the dose the patient

receives for a given tea brewing time. The size and shape of the particles also has a direct

impact on mixing and significant deviations in the particle size can result in the

segregation of the mixture, with smaller fragments settling at the bottom and larger

fragments at the top of the mixture (Fayed & Otten, 1984). Sieve and microscopic

methods are the commonly used methods to determine particle size and shape and were

the methods used in this study.

4.3.5.1.1 The sieve method

The degree of coarseness or fineness of a powder is classified according to the nominal

aperture size expressed in micrometers of the mesh of the sieve through which the

powder will pass (BP, 2000a). Using this criterion, powders are divided into ‘coarse’,

‘moderately fine’, ‘fine’, and ‘very fine’. The degree of fineness of the powder is

expressed as a weight-to-weight percentage of the powder passing through different size

sieves. If a single sieve number is given, not less than 95% of the powder passes through

the sieve of that number, unless otherwise indicated.

For the evaluation of the A. afra plant materials, sieves having numbers of 1400, 355,

180, 125 and 90 µm were assembled in a descending order, i.e. 1400 µm size sieve on top

and 90 µm at the bottom. The assembled set of sieves was placed on a test sieve shaker

before 10 g of the SDL or FDAE powder was placed onto the top sieve, and the assembly

shook for 30 minutes. Thereafter the powder collected on each of the sieves was weighed

and the percentage (w/w) of each fraction determined.

4.3.5.1.2 Determination of the plant material particle shape

The microscopic method allows the determination of the shape of the particles and a

Nikon light microscope was used to determine the particle shape of the plant materials

used. A few milligrams of the SDL or FDAE powder were sprinkled onto a slide and

viewed under the microscope. The particle shapes were observed and described as either

irregular, round, cylindrical or rectangular.

4.3.5.2 Determination of the plant material colour

Colour can be used as a means of identifying a particular substance. Several

Pharmacopoeias include the colour of the substance as part of the substances monograph.

In this study, the colour of the A. afra SDL and FDAE powder as well as the colour of the

hues produced when these materials are infused in hot water to produce teas was

determined. This information was crucial for the design of the placebos which should be

similar in appearance to the plant materials.

In determining the colour of the materials, 1 g of the material being examined was placed

against a white background and its colour described. In determining the colour of the

hues produced, 4 g of the SDL and 1g of the FDAE powder were infused in 200 ml of

distilled water at a temperature of 80˚C, stirred for 2 minutes, and allowed to stand for 8

minutes. The resultant hue was then visually examined and described accordingly.

4.3.5.3 Determination of the plant material odour

Odour can be used as a quick method for identification of a substance. The presence or

absence of odour in A. afra plant material was determined using the method prescribed in

the British Pharmacopoeia 2000. One gram of the SDL or the FDAE powder of A. afra

was placed on a watch glass about 5 cm in diameter. The material was then allowed to

stand for 15 minutes, smelt and the presence or absence of odour from the material noted

(BP, 2000b).

4.3.5.4 Determination of the plant material bitterness value

The bitterness value is defined as the reciprocal of the dilution of a compound, liquid or

extract that still has a bitter taste (EP, 2002a). The bitter properties of plant materials are

determined by comparing the threshold bitter concentration of a preparation of the

material with that of a dilute solution of quinine hydrochloride. The threshold bitter

concentration is defined as the lowest concentration at which the material continues to

provoke a bitter sensation 30 seconds after tasting it. The bitterness value is expressed in

units equivalent to the bitterness of a solution containing 1g of quinine hydrochloride, the

bitterness value of which is set at 200 000 (WHO, 1998). In the literature A. afra is

described as having an extremely bitter taste (Van Wyk et al., 2000; Roberts, 1990). In

order to aid effective design of the herbal placebos it was necessary to characterize the

bitterness value of the plant materials.

For the bitterness value determinations, the SDL and FDAE of A. afra were dispersed in

boiling distilled water at a concentration of 0.01 g/ml, stirred and allowed to stand for 10

minutes before being filtered using Whatman No. 1 filter paper. The solutions were

allowed to cool before tasting. Serial dilutions of quinine hydrochloride and the plant

materials, i.e. 0.00001 g/ml and 0.001, 0.0001, 0.00002, 0.00001 g/ml respectively, were

then prepared as described in the European Pharmacopoeia 2002 (EP, 2002a).

A panel comprising of 6 tasters was set up. In order to correct for individual differences

in tasting bitterness, a correction factor for each taster was established using quinine

hydrochloride. The panel members then tasted serially diluted concentrations of the SDL

and the FDAE solutions, starting with the most dilute concentration. The bitterness value

was determined as prescribed in the European Pharmacopoeia 2002 using the following

equation:

Bitterness Value = Y x k Eqtn: 4.1

X x 0.1

Where; Y = Dilution factor of threshold bitter concentration

k = Correction factor for each panel member

X = Number of ml of threshold bitter concentration which when diluted to

10 ml still has a bitter taste.

4.3.6 Determination of the plant material ash values

Ash values can be considered as quality standards to indicate identity, purity or possible

adulteration of a herbal material. The ash remaining following ignition of medicinal plant

materials is determined by different methods which measure total ash, acid-insoluble ash

and water-soluble ash. The total ash method is designed to measure the total amount of

material remaining after ignition. This includes both “physiological ash”, which is

derived from the plant tissue itself, and “non-physiological” ash, which is the residue of

the extraneous matter (e.g. sand and soil) adhering to the plant surface (WHO, 1998).

Acid-insoluble ash is the residue obtained after boiling the total ash with dilute

hydrochloric acid, and igniting the remaining insoluble matter. This measures the amount

of silica present, especially as sand and siliceous earth. Water-soluble ash is the

difference in weight between the total ash and the residue remaining after treatment of the

total ash with water (EMEA, 2005).

Total ash and acid-insoluble ash determinations were performed on the standardized dried

leaf material and FDAE powder of A. afra according to the prescribed methods in the

British Pharmacopoeia 2000. Water-soluble ash determination is a non-pharmacopoeial

test and was performed by adding 25 ml of water to the crucible containing the total ash

and boiling for 5 minutes. Thereafter, the insoluble matter was collected in a sintered-

glass crucible, washed with hot water, ignited in a crucible for 15 minutes at a

temperature of 450°C and the remaining ash retrieved. The difference in weight (in mg)

of this residue from the weight of the total ash was the water-soluble ash (WHO, 1998).

4.3.7 Determination of the plant material moisture content

The moisture content of the plant materials was determined using the method prescribed

in the European Pharmacopoeia 2002 which involves the use of a drying oven. For this,

0.5g quantities of the SDL and the FDAE powder of A. afra were placed in a flat-

bottomed dish about 50mm in diameter and dried in an oven for 3 hours at a temperature

range of 100-105ºC (EP, 2002b). The moisture content was calculated as a mass

percentage using the following formula:

% Moisture = Initial weight (Wet mass) – Final weight (Dry mass) x 100 Eqtn: 4.2

Initial Weight

4.3.8 Determination of microbial contamination of the materials

Medicinal plant materials normally carry a great number of bacteria and moulds, often

originating from the soil. Bacterial and fungal contamination is of serious concern

especially in terms of health risks (EMEA, 2005). Unacceptable levels of microbial

contamination are often due to poor agricultural practices or improper drying and storage

of the harvested plant material and typically result in degradation of the plant materials.

In some cases microbial contamination may render the plant material toxic either by

transforming benign chemicals in the plant into harmful substances, or through the

microbes’ production of toxic compounds (McCutcheon, 2002).

The European Pharmacopoeia provides guidance on which microbes are to be tested for

as well as their minimum acceptable limits (EP, 2002c). Following this guidance, the

total viable aerobic count, total yeasts and moulds, absence of Escherichia coli,

Salmonella and enterobacteria were consequently tested for in the SDL and FDAE

powder of A. afra. In addition to this, tests not specified in the pharmacopoeia were also

conducted. These were tests for absence of Staphylococcus aureus and Pseudomonas

aeruginosa. Microbiological testing on the materials was conducted before and after

irradiation of the materials, using standard microbial test methods as described in

Appendix A7.

4.3.9 Development and validation of the HPLC assay

For the quantitation of luteolin in the plant materials, a reversed phase HPLC method

using an internal standard was developed. The compounds hesperetin, p-coumaric acid,

mefenamic acid, morin hydrate, cinnamic acid and salicylic acid were evaluated as

possible internal standards. The requirements were that the compound should not be

present in the A. afra plant materials, should absorb strongly at the luteolin absorption

wavelength maximum (349 nm), be extractable in ethyl acetate, have similar

physiochemical characteristics to luteolin, and have a chromatographic retention time that

did not interfere with other peaks from the plant.

Different chromatographic conditions were evaluated for the separation of the luteolin

peak from the internal standard peak and other possible interfering peaks. Three different

types of reverse phase chromatographic columns (i.e. silica C-18 (250 x 4.6 mm, 5 µm),

Luna ® C-18 (150 x 4.60 mm, 5 µm), Luna® C-18 (250 x 4.6 mm, 5 µm)), different

mobile phase compositions and flow rates were evaluated and optimized. The mobile

phases consisted of either methanol or acetonitrile, with a mixture of acetic acid or formic

acid in water or a phosphate buffer at different concentrations to control the pH and to

reduce peak tailing. To validate the assay, the linearity, recovery, limit of detection

(LOD), limit of quantitation (LOQ) and intra- and inter-day precision, for the assay were

determined.

In performing the validation, firstly, stock solutions of luteolin and morin hydrate

(1mg/ml) were prepared in dimethyl sulphoxide (DMSO), wrapped with aluminium foil

to protect from light and stored at -21˚C. Standard solutions were freshly prepared each

day by appropriate dilution of the stock solutions with the mobile phase (acetonitrile

30%, phosphate buffer 70% at pH 2). Concentrations of 2.5, 5, 10, 20, 50 and 100 µg/ml

of luteolin and 100 µg/ml of morin were prepared.

For the preparation of the calibration curve, the stock solutions were diluted with mobile

phase to produce the concentrations described above. To each concentration of luteolin,

50 µg of morin hydrate was added. 50 µl of each standard was injected onto the column

and the peak height ratios between luteolin and morin plotted against the corresponding

concentrations of the injected luteolin. The complete procedure was repeated over 3

consecutive days.

The limit of detection (LOD) was obtained by successively decreasing the concentration

of luteolin as long as a signal to noise ratio of 3: 1 appeared. The limit of quantitation

(LOQ) was defined as the lowest concentration where an accuracy better than 20 % was

achieved (Ruckert et al., 2004).

Finally, for the determination of intra-day precision and accuracy, 6 replicates of the

standards were analysed on the same day. The precision and the accuracy for the inter-

day analysis were determined on 3 different days.

4.3.10 Quantitation of luteolin in the plant materials

The flavonoid luteolin was used as a marker compound for the A. afra plant materials. As

part of the characterization of the plant materials, different batches of the plant leaves, the

SDL and the FDAE were evaluated for the average content of total, free and glycosidic or

conjugated luteolin using the validated HPLC assay. In addition, the intra- and inter-batch

variation in the content of the different forms of luteolin in the various plant materials,

were also determined.

A defined procedure was used for sampling from the various plant material batches. This

entailed randomly taking 20g of the plant material under evaluation from the batch bulk

and spreading it on a tile 10 x 10cm. From here, 6 sampling positions were defined (as

shown in Figure 4.1) and 250 mg samples were removed from each position.

The sampled plant material was then prepared following the common traditional method

for the preparation of the tea. The 250 mg sample was placed in 10 ml of hot (80˚C)

distilled water (25 mg/ml), vortexed for 2 minutes and then allowed to stand for 8

minutes before being filtered using Whatman no. 1 filter paper. The filtrate was separated

into two 1 ml portions for analysis of the total luteolin (hydrolysed) and free luteolin

(unhydrolysed).

For the total luteolin assay, the 1 ml sample was transferred to a tube containing 4.8 ml of

2N HCl and an appropriate amount of internal standard. The mixture was vortexed for 30

seconds, before being left to ‘hydrolyse’ in a water bath at 80˚C for 40 minutes.

Following this, the sample was allowed to cool, 5 ml of ethyl acetate added, vortexed for

2 minutes (to ensure the luteolin was extracted into the ethyl acetate layer) and then

centrifuged at 3000 rpm for 10 minutes to separate the ethyl acetate from the aqueous

1 2

3 4

5 6

10cm

Figure 4.1: Sampling positions used in the quantitation of

luteolin in the A. afra plant materials.

layer. Finally, the top layer of ethyl acetate was pipetted off into a clean tube and

evaporated to dryness under a cool stream of nitrogen.

For unhydrolysed (free) luteolin assay, the 1 ml sample was transferred to a tube

containing 5ml of ethyl acetate and internal standard. This was vortexed for 2 minutes

before being centrifuged at 3000 rpm for 10 minutes. The top layer of ethyl acetate was

then removed and the sample evaporated to dryness as described above.

To quantify the amount of luteolin in the extracted samples, the dry residues were

reconstituted in 1ml of the mobile phase. 50 µl of the solution was then injected onto the

column and isocratically eluted at a flow rate of 1 ml/min at room temperature with a run

time of 30 minutes using a mobile phase of acetonitrile 30% and phosphate buffer 70% at

pH 2). Detection was conducted at a wavelength of 349 nm and the luteolin in the

samples quantitated from a standard curve based on the peak height ratio of luteolin and

internal standard.

4.3.11 Design of the plant material placebos

Designing credible herbal placebos is known to present special challenges especially with

regard to achieving and maintaining concealment. This is mainly due to the fact that

herbal medicines are more elaborate in their composition compared to the simple

chemical structures of drugs commonly encountered in pharmaceutical placebo design

(Kaptchuck, 2001). In this study, the objective was to design placebos which looked,

smelt and tasted similar to the A. afra SDL and FDAE.

4.3.11.1 Design of placebo for the standardized dried leaves

The SDL of A. afra were used to produce the placebo. Since the phytochemical

constituents, in particular the flavonoids, were thought to be responsible for the observed

therapeutic effects of the plant leaves, it was inferred that A. afra leaves without these

phytochemical constituents or with very low levels of them, would be devoid of

pharmacological activity. Therefore, to produce the placebo of the dried leaves, the

phytochemical constituents were to be extracted out of the A. afra leaves as much as

possible.

A method utilising exhaustive solvent extraction with hydrolysis was developed to extract

as much as possible of the flavonoids (as well as any other compounds soluble in the

extracting solvents) within the leaves. In selecting appropriate solvents, flavonoid

solubilities were taken into consideration. For example, for the extraction of water-

soluble flavonoids, a more polar solvent was selected. The safety or hazardous potential

of the solvents were also taken into consideration. Various solvents were evaluated for

their effectiveness in extracting the constituents using the minimal number of extractions.

Based on the above considerations, a total of 15 extractions using a combination of either

water, methanol/ water (50/50, v/v), methanol or ethyl acetate, (depending on the

particular extraction step) including a hydrolysis and neutralisation step (to free up the

sugar bound flavonoids for optimal extraction), were conducted. In all extractions a mass

to solvent ratio of 1:20 was used. In conducting the extractions, the leaf and solvent

mixture was stirred for 2 minutes then allowed to stand for 10 minutes before being

filtered using Whatman no. 1 filter paper. The leaf to solvent ratio was kept constant in

all extractions. From the supernatant, a 1 ml sample was retained for analysis using UV

spectrophotometry and to the remaining residue of leaves, additional solvent was added.

UV spectrophotometry was conducted at the luteolin absorbance maxima (349 nm) as

well as the absorbance maxima for the plant material extract. The process was repeated

using the next solvent in the procedure, while monitoring the UV absorbance of the

extract, until the absorbance was acceptably low. Samples of the initial and final extracts

were also analysed using the validated HPLC assay in order to obtain a chromatographic

fingerprint of the material as well as obtain a measure of the extraction process through a

comparison of the pre- and post extraction chromatograms.

4.3.11.2 Design of the placebo for the freeze-dried aqueous extract powder

There are few clinical trial studies in the literature, where placebos of herbal extracts have

been employed (Pan et al., 2000). In many of these trials, extracts in capsules or tablets

were used with the result that the patient could not see the extract itself. In making these

placebos, elaborate formulas of inorganic salts were employed to mimic the extract as

well as impart good flow properties to the powder to aid the manufacture of the tablets or

capsules. One example is that of a placebo of a Chinese herbal preparation containing

78.2% calcium hydrogen phosphate, 19.6% soy fibre, 0.3% cosmetic brown, 0.5%

cosmetic yellow, 0.01% edicol blue 0.09% identical liquorice dry flavour and 0.03%

bitter flavour (Bensoussan et al., 1998).

In the design of the placebo of the A. afra FDAE, various selected inorganic salts were

blended together. These salts were chosen based on their organoleptic properties relative

to the organoleptic properties of the FDAE. In addition, in order to match the hue

produced when the A. afra FDAE is dissolved in water, a colourant was included in the

placebo formulation. The appropriate proportions of inorganic salts to be added were

determined on a trial and error basis, where a formulation was evaluated against the

aqueous extract and modified as needed. The various formulations and proportions of

each salt tried are shown in Table 4.1 below.

Table 4.1: Various excipients and their proportions used in different

formulations in the design of the placebo for the A. afra freeze-dried

aqueous extract powder.

Amount of various excipients in the various formulations (g)

Excipient 1 2 3 4 5 6

Potassium dihydrogen

phosphate

0.275 --- --- 0.200 0.400 0.475

Microcrystalline

cellulose

0.225 0.300 0.300 --- --- ---

Sodium starch glycollate

--- 0.100 --- 0.100 --- ---

Potato starch

--- --- 0.130 0.130 0.050 0.03

Lactose

--- --- 0.050 --- 0.035 0.025

Calcium chloride

---- --- --- 0.050 --- ---

DC Brown 0.050 0.015 0.020 0.020 0.02 0.02

Di-sodium hydrogen

phosphate heptahydrate

--- 0.135 --- 0.050 --- ---

Total 0.55 0.55 0.55 0.55 0.55 0.55

4.3.11.3 Matching of odour between the placebos and the plant materials

A. afra is a highly aromatic plant. The dried leaves and infusions from the leaves

(aqueous extract) also possess a characteristic odour. In order to make the odours similar

between the placebos and the A. afra plant materials, an aroma chemical was employed to

provide an aroma to the placebos as well as to spike the A. afra plant materials so that the

plant material odours are matched.

An appropriate aroma chemical was selected from a range of commercially available

synthetic aroma chemicals. In selecting the appropriate chemical, the fragrance

characteristics, flavour properties, toxicity, fixative properties and substantivity (which

refers to the ability of the perfume to last on a specific surface) of the chemical was taken

into consideration. A chemical with a more natural or green fragrance with a high

tenacity and substantivity was preferred in order to resemble the herbal smell of A. afra.

The aroma chemical selected to mask the odour of the A. afra plant materials was linalool

(Figure 4.2). This chemical possesses a fruity, herbal woody, rosewood odour (BASF,

2006). Linalool is a substance widely used in functional and alcoholic perfumery. It is

also frequently used in fruit imitations (peach, apricot, pineapple, grape, berry flavours

etc), and for chocolate and spice complexes (BASF, 2006). It is accepted by the Council

of Europe for use in foods as an artificial flavouring and is considered Generally

Recognised as Safe (GRAS) by US Food and Drug Administration (OECD, 2004). Based

on this, the aroma chemical was deemed suitable for inclusion in the formulations.

In order to determine the appropriate concentration of the aroma chemical sufficient to

mask the smell of an A. afra tea solution, a modification of the European Pharmacopoeia

method for the determination of bitterness of materials was used. Serial dilutions of the

aroma chemical were prepared, i.e. 0.0042, 0.0048, 0.0052, 0.0056 and 0.0058 g/ml and

mixed with equal proportions of a 20 mg/ml hot water extract of A. afra. Using a panel of

four evaluators, the natural sense of smell was used to discriminate and evaluate the

odours. The concentration of the aroma chemical at which the odour of the A. afra in the

mixed solution could not be determined was taken to be the threshold masking

concentration. This concentration capable of masking the characteristic odour of A. afra

was therefore used in matching the smell properties of the materials.

In order to incorporate the aroma chemical into the materials, a method was devised

whereby a silica gel adsorbent would adsorb the aroma chemical and be able to release

the aroma when in solution. The aroma chemical was dissolved in 1ml of ethyl acetate

and the solution added to 0.125 g of silica gel powder. The ethyl acetate was then

evaporated off under a stream of nitrogen to leave a dry silica gel powder containing the

aroma chemical. Appropriate amounts of the aroma-impregnated silica gel were then

added to the A. afra SDL and FDAE, as well as the placebo materials.

Figure 4.2: Chemical structure of linalool.

4.3.11.4 Matching of taste between the placebos and the plant materials

A. afra is an extremely bitter tasting plant (Roberts, 1990). Therefore the inclusion of a

sweetening agent was considered in order to improve the patient acceptance of the plant

medicine as well as to match the taste properties of the plant with that of the placebo

(Zheng & Keeney, 2006). In the selection of an appropriate sweetener, the safety profile

and classification of the sugar compound was taken into consideration. Sodium saccharin,

a synthetic sweetener was preferred as opposed to a nutritive caloric natural sweetener

such as glucose. This was to avoid possible confounding of results if the preparation was

to be used in a trial such as a diabetes trial.

In order to determine the concentration of sodium saccharin required to mask the

bitterness of the plant materials, a method was devised based on a modification of the

European Pharmacopoeia bitterness value determination method. Serial dilutions of the

chemical were prepared and mixed with equal proportions of a 20 mg/ml hot water

extract of A. afra. Using a panel of 4 tasters, the natural sense of taste was used to

establish the solution in which the bitter taste could no longer be tasted and the solution

that was determined to be sweet. The amount of sodium saccharin in this solution was

then adsorbed onto the silica gel adsorbent as described in section 4.3.11.3 above and

incorporated into the A. afra and placebo materials.

4.4 Results and Discussion

4.4.1 Preparation of the plant materials

For this study, the leaves were removed from each of the 3 collected A. afra plant

batches. On average 43.5% of the total A. afra plant weight consisted of the leaves.

Drying of these collected leaves in the oven for 3 days at 30˚C in resulted in an average

57.36 ± 7.51% loss in weight. This result therefore indicates that approximately 50% of

the mass of harvested A. afra leaves is in the form of moisture. This information may be

useful to researchers in future studies on the plant, planning the amount of plant material

required in order to attain a certain dry mass of leaves. The complete data for the

collection and preparation are presented in Appendix A1.

To prepare the FDAE powder, a mass ratio of leaves to solvent of 1:35 was used. Several

extractions of the SDL were conducted and a summary of the yields obtained for each

extraction are presented in Appendix A3. The average yield for the FDAE powder

obtained was 21.96 ± 1.97%. This value was similar to that obtained previously by other

investigators, viz 27.88% and 19.9% by Komperlla (2005) and Mukinda (2006),

respectively. Figure 4.3 shows an image of the SDL and the resultant FDAE powder.

Figure 4.3: The standardized dried leaves of A. afra (left) and the freeze-dried aqueous extract

powder of the A. afra standardized dried leaves (right).

4.4.2 Organoleptic properties of the A. afra plant materials

The results of the organoleptic determinations on the A. afra SDL and FDAE powder are

shown in Table 4. 2 below. The leaves were observed to be grey-green in colour, while

the FDAE powder resulting from them was light brown in colour (see Figure 4.3). This

difference in colour may point to a possible breakdown or change in the compounds

present in the leaves occurring during the infusion process. When the FDAE was allowed

to stand for 15 minutes, the extract became darker in colour and the powder clumped

together. This observation was similar to that made by Komperlla (2005) and was

attributed to the hygroscopic nature of the extract. The taste, odour and colour of the teas

(hue) of the leaves and FDAE were however similar. The A. afra leaves and FDAE

powder were also both bitter in taste. The bitterness of the leaves is described in the

literature and in some traditional preparations sugar or honey is added to the preparations

to mask the bitterness and improve the patient acceptance of the medicine (Van Wyk &

Gerike, 2000; Roberts, 1990). The above information gathered during the organoleptic

evaluation was used to set specifications for the plant materials and with these

specifications, set standards for the placebo materials to meet.

Table 4.2: Organoleptic characteristics of the A. afra standardized dried leaves and

freeze-dried aqueous extract powder.

Organoleptic

characteristic

A. afra

Standardized dried leaves

A. afra

freeze-dried aqueous extract

powder

Appearance

Colour of material

Colour of hue

produced

Odour

Taste

Particulate leaves, finely divided,

hydrous, with a rough texture.

Grey-green.

Light brown to golden brown hue.

Characteristic odour, highly aromatic.

Odour still present after 15mins on

watch glass.

Extremely bitter.

Brittle, free flowing, small particulate

powder, which clumps together to

form a solid mass on prolonged

exposure to air.

Light brown powder changing to dark

brown on prolonged exposure to air.

Light brown to golden brown hue.

Characteristic odour, aromatic. Odour

still present after 15mins on watch

glass.

Extremely bitter.

4.4.2.1 Particle size and shape of the A. afra plant materials

Under the microscope, the particles of the SDL and FDAE powder were both observed to

be irregular in shape. The results of the particle size determinations on the materials are

presented in Appendix A4. Based on the BP 2000 classification system, both the SDL

and FDAE were classified as being coarse powders. The particles of the SDL passing

through the 1400 µm but retained by the 355 µm sieves i.e. those in the particle size

range 1400-355 µm were retained for use in this study, while the FDAE powder was used

un-sieved.

4.4.2.2 Bitterness value of the A. afra plant materials

The results of the determination and calculation of the bitterness values are presented in

Appendix A5. The bitterness value for both the A. afra SDL and FDAE was calculated to

be 600 000. From this, it meant that the A. afra leaves and FDAE were 3 times more

bitter than quinine hydrochloride which is known to be an extremely bitter tasting

compound. To our knowledge this is the first estimate of the bitterness of A. afra leaves

and FDAE powder, and this result can be added to the list of already known

specifications for A. afra plant materials. The fact that the bitterness values for the dried

leaves and the FDAE were similar, indicated that the compounds responsible for the

bitterness were extracted from the leaves during the infusion process and were probably

present in similar amounts in the two preparations. This therefore implied that in the

formulation of the placebo materials, the placebo of the FDAE powder and dried leaves

needed to be of similar taste. In addition, as a result of the bitterness, a sweetener

chemical was required in order to mask the bitter taste of the materials and therefore

improve the patient acceptance of the medicines.

4.4.3 Ash values of the A. afra plant materials

The complete data sets and results for the ash value determinations on the A. afra

materials are presented in Appendix A6. The average percentage of total ash, acid

insoluble ash and water soluble ash for the SDL were 9.9832 ± 0.0617%, 0.9308 ±

0.0012% and 4.4052 ± 0.0012%, respectively. These results are comparable to the

8.558% and 1.341% previously reported by Komperlla (2005) for the total ash and acid-

insoluble ash, respectively, of A. afra dried leaves.

The average percentage of total ash, acid insoluble ash and water-soluble ash for the

FDAE was found to be 21.820 ± 0.0886%, 0.0094 ± 0.0793% and 16.5952 ± 0.6760%,

respectively. These results were also comparable to those obtained by Komperlla (2005),

viz. 17.436% and 0.027% for total ash and acid insoluble ash, respectively. To the best of

our knowledge, values for water-soluble ash of A. afra have not previously been reported

and the values above are the first such for A. afra plant leaves and FDAE. These values

may be added to the current list of specifications for the plant materials.

Collectively, the ash values, in addition to the organoleptic characteristics results helped

to confirm the identity of the A. afra plant materials used. It was noted that the A. afra

materials used by the previous investigators mentioned above was collected from the

same source as that used in this study. It may therefore be inferred that A. afra plants

growing in similar conditions (similar climate and soil conditions) possess similar

organoleptic and physiochemical properties.

4.4.4 Moisture content of the A. afra plant materials

The results of the moisture content determinations on the A. afra SDL and FDAE powder

are shown in Tables 4.3 and 4.4, respectively.

Table 4.3: Residual moisture content of the A. afra standardized dried leaves

Sample

Initial mass of leaves (g) Final mass of leaves (g) Loss on drying

(g)

Percentage

moisture content

0.5253

0.5191

0.5206

0.5213

0.5149

0.4700

0.4650

0.4722

0.4613

0.4558

0.0553

0.0541

0.0484

0.0600

0.0591

10.53

10.42

9.38

11.51

11.48

Ave 0.5202 0.4649 0.0554 10.66

S. D 0.0038 0.0066 0.0046 0.88

Table 4.4: Residual moisture content of the A. afra freeze-dried aqueous extract

powder.

Sample

Initial mass of aqueous

extract (g)

Final mass of aqueous

extract (g)

Loss on drying

(g)

Percentage of

moisture

0.5039

0.5071

0.5145

0.5081

0.5020

0.4609

0.4645

0.4695

0.4642

0.4577

0.0430

0.0426

0.0450

0.0439

0.0443

8.53

8.40

8.75

8.64

8.82

Ave 0.5071 0.4634 0.0438 8.62

S. D 0.0048 0.0044 0.0010 0.17

The average moisture content for the SDL and FDAE powder of A. afra were 10.66 ±

0.88% and 8.62 ± 0.17%, respectively. Moisture content determinations are necessary,

particularly for materials known to be hygroscopic (EMEA, 2005). In the present study

the moisture content determinations were conducted immediately following the drying of

the plant materials and therefore were an indication the residual moisture present in the

materials at that stage. The values obtained in this part of the study were therefore used as

the moisture content specifications for the materials and were used in the later part of the

study to determine moisture absorption by the materials upon storage.

4.4.5 Microbial contamination of the plant materials

Microbial contamination tests were conducted before and after irradiation of the plant

materials. The tests conducted before irradiation included tests for total microbial

activity, and for absence/presence of Escherichia coli, Pseudomonas and Staphylococcus

aureus. All testing was conducted externally by a company contracted to do the testing.

The results of the microbial contamination tests are presented in Appendix A7. Before

irradiation, considerable contamination was detected within the dried leaves, i.e. a total

microbial activity of more than 106 and the presence of more than 104 and 300 colony

forming units (CFU) per gram, of Pseudomonas and E. coli bacteria respectively. Based

on the European Pharmacopoeia 2002 guidelines, which state that not more than 200

CFU/gram E. coli and not more than 107 bacteria should be present (EP, 2000c), the dried

leaves did not comply with the specification on microbial contamination of herbals. The

presence of E. coli in the plant leaves may have reflected the quality of the growing and

harvesting practices at the source of the material, as E. coli bacteria are not normally

found as part of the flora and fauna of the soil (WHO, 1998). The tests on the FDAE

powder before irradiation however indicated the presence of a reduced total microbial

activity of 984 000 and the absence of all the other organisms previously detected in the

dried leaves. This implies that hot water extractions, and presumably also the traditional

decoction/infusion process, reduce the microbial load present in the plant materials.

After irradiation of the plant materials, no contamination was detected for the tests

conducted (i.e. absence of total microbial activity, yeasts & moulds, E. coli, Salmonella

and Enterobacteriaceae) demonstrating the efficacy and usefulness of the irradiation

process. From these results, it was thus assured that the plant materials were suitable for

use in the manufacture of the medicinal dosage forms.

4.4.6 Development and validation of the HPLC assay

A reversed-phase HPLC method, using morin as internal standard, was developed for the

quantitation of luteolin in A. afra plant materials. The optimal conditions of separation

and detection were achieved on a Synergy® Hydro - reverse phase column, packed with a

4 μm particle size resin and a column length of 250 x 4.60 mm using a mobile phase of

acetonitrile (30%) and phosphate buffer 100mM (70%, pH 2) at a flow rate of 1 ml/min

and UV detection at 349 nm.

The standard curve of peak height ratio versus concentration of luteolin was linear in the

range of 2.5 - 100 µg/ml (i.e. 125 – 5000 ng luteolin on column) (Figures 4.4 and 4.5),

and was described by the equation Y = 0.03407X + 0.008233 (where Y = peak height

ratio, X = luteolin concentration in µg/ml) and had a regression correlation coefficient, r

value of 0.9996. The concentration of internal standard used was 50 µg/ml and the HPLC

injection volume was 50 µl.

Figure 4.4: Retention times and peak heights of morin and luteolin standards injected for

generation of the standard curve. The average retention times for morin and

luteolin were 11.90 ± 0.06 minutes and 15.86 ± 0.05 minutes, respectively.

025 50 75 100

Conc (μg/ml)

Peak Height Ratio

Figure 4.5: The standard curve and linear regression line values, used in the quantitation of

luteolin.

The results of the accuracy and recovery of the assay are shown in table 4.5 and the inter-

and intra- day precision results are presented in Table 4.6. The mean recovery of the

method was 94.70 ± 8.62%. The concentration of 2.5 µg/ml had accuracy greater than

20.0% and was therefore considered to be the limit of quantitation (LOQ) for the assay

(Ruckert et al., 2004). The LOD was found to be 50 ng on column, from an injected

volume of 20 µl corresponding to a concentration of 2.5 µg/ml. The average intra- and

inter-day precision were found to be 2.497 ± 0.6470% and 3.123 ± 1.068%, respectively.

Collectively, these values indicated the good validity and reproducibility of the assay.

Best-fit values

Slope 0.03407 ± 0.0004926

Y-intercept 0.008233 ± 0.02296

X-intercept -0.2416

1/slope 29.35

95% Confidence Intervals

Slope 0.03271 to 0.03544

Y-intercept when X=0; 0.05550 to 0.07196

X-intercept when Y=0; -2.171 to 1.587

Goodness of Fit

R 0.9996

Sy.x 0.04172

Minutes

0246810 12 14 16 18 20

mAU

100

150

200

250

mAU

100

150

200

250

Det 168-349nm

st 2.5mg/ml Det 168-349nm

st 5mg/ml D et 168-349nm

st 10mg/ml Det 168-349nm

st 20mg/ml D et 168-349nm

st 30mg/ml Det 168-349nm

st 50mg/ml Det 168-349nm

st 100mg/ml

morin

luteolin

Table 4.5: Accuracy and recovery of the luteolin standard.

Given (µg/ml) Found, mean ± S.D (µg/ml) Accuracy (%

deviation)

% Recovery

2.5

5.0

10.0

20.0

50.0

100.0

1.810 ± 0.196

4.772 ± 0.177

9.467 ± 0.169

20.470 ±1.040

48.110 ± 0.308

99.010 ± 0.403

-22.639

-4.071

-3.386

-1.100

-3.779

-0.540

77.361

98.703

96.614

99.853

96.221

99.460

Table 4.6: Intra-day and inter-day precision of the assay.

Intra-day Inter-day Given (µg/ml)

Precision

(R.S.D., %)

Accurac

(% deviation)

Precision

(R.S.D., %)

Accurac

(% deviation)

10.0

50.0

100.0

1.79

3.06

2.64

-3.386

-3.779

-0.540

4.06

3.35

1.96

-2.563

+1.542

-2.384

4.4.7 Quantitation of luteolin in the A. afra plant materials

The data sets and results of the quantitation of luteolin at each sampled position for each

plant batch are presented in Appendix B. Figure 4.6 shows a chromatogram of luteolin

and the internal standard morin within the plant matrix.

Stability t esting of A.afra t ea bags

Minutes

02 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

100

120

140

mAU

100

120

140

3.300 303588

3.717 92753

4.417 135373

5.217 361549

6.383 219069

7.750 6336

9.950 13613

12.800 29384

14.233 2651

16.200 3349

26.283 3164

Det 168-349nm

T3U6 -R ep2

Retention Time

Height

Figure 4.6: Chromatogram showing the retention of morin and luteolin within the plant matrix.

The A. afra leaves batches 001, 002 and 003 contained 0.9879 ± 0.2494, 0.8141 ± 0.1825

and 1.0980 ± 0.0991 µg/mg of free luteolin, respectively. The levels of

conjugated/glycosidic luteolin were 0.5597 ± 0.2706, 0.9829 ± 0.4317 and 1.5780 ±

morin

luteolin

0.5743 µg/mg for batches 001, 002 and 003 respectively, and the levels of total luteolin

content were 1.548 ± 0.3287, 1.7967 ± 0.5391 and 1.7350 ± 0.2909 µg/mg, respectively.

From a one-way, non parametric analysis of variance (ANOVA) of the luteolin content

for the different plant batches, the results showed that there was no significant inter-batch

difference (p values; 0.2010, 0.0949 and 0.5471 for free, conjugated and total luteolin,

respectively (alpha < 0.05)) between the luteolin content of the different plant batches i.e.

the luteolin content present in each plant batch was similar. This similarity may be

attributed to the similar method of preparation and storage of the plant materials and the

fact that the plant materials were harvested from the same geographic location. The time

of harvest was however, separated by a period of one month for each batch and from the

results obtained, it can be concluded that this separation period had no effect on the levels

of luteolin in the plant batches.

The SDL contained 0.8628 ± 0.0991, 1.203 ± 0.1862 and 2.065 ± 0.2347 µg/mg of free,

conjugated and total luteolin, respectively. A one way ANOVA of this data and that

obtained for batches 001, 002 and 003, showed that there was no significant difference in

the content of free and total luteolin for the plant batches, i.e. p values were 0.1766 and

0.1399 for free and total luteolin, respectively (alpha < 0.05). This result is as expected,

as the standardized batch was produced by blending together the 3 plant batches.

The FDAE powder contained 7.0880 ± 0.4751, 6.781 ± 1.152 and 13.870 ± 1.2460 µg/mg

of free, conjugated and total luteolin respectively. These values obtained showed that the

freeze-dried extract contained approximately 7 times more luteolin on a weight for

weight basis than the A. afra SDL. This may be attributed to the extraction and freeze-

drying process, whereby the constituents of the leaves are concentrated into a given

volume of extracting solvent and then evaporated under vacuum. Therefore, dosage forms

containing the FDAE powder should contain 7 times less material on a weight for weight

basis compared to those containing the SDL. The amount of total luteolin found in the

FDAE was comparable to the 6.965 ± 0.024 µg/mg determined in a previous

investigation by Waithaka (2004).

There was however, considerable intra-batch variation in the luteolin levels of the plant

materials as indicated by the percentage relative standard deviation for each batch. For

instance, the % R.S.D for the total luteolin in the batches 001, 002 and 003 were 21.24,

30.00 and 16.77%, respectively. This intra-batch variation could be attributed to

inconsistencies in the plant material make up, i.e. some batches contained a certain

proportion of twigs and branches (as foreign matter), and also could have been due

possibly to inconsistencies in the sample preparation, i.e. there could have been variation

in the weighing or infusion of the leaves, across the batches. However, the % R.S.D for

the total luteolin in the SDL was considerably less (% R.S.D = 11.36), and indicated a

reduced variation within that batch attributable to the fact that this batch represented the

‘mean’ of the 3 batches having being produced by blending of batches. Similar trends

were also observed for the conjugated and free luteolin content for each of the plant

batches. Moreover, the intra-batch variation was much less (i.e. total luteolin % R.S.D =

6.70) for the FDAE powder compared to that for the SDL, and this may be attributed to

the decoction process used to produce the extract, as the solution from which the latter

resulted was homogeneous. It could therefore be concluded that standardization of the

plant leaves reduced the inherent variation in the levels of luteolin and this variation was

further reduced through preparation of a FDAE of the leaves. The low % R.S.D present

for the FDAE and SDL, therefore showed that the FDAE powder was a more consistent

material and that these materials were suitable for use in the manufacture of the medicinal

dosage forms. To further highlight the inter and intra-batch variation in the luteolin

content, the results obtained for the different plant material batches are also presented in

bar graph form in Figure 4.7 below.

1 2 3 4 5 6

0.0

0.5

1.0

1.5

2.0

2.5

Free luteolin

Total luteolin

Conjugated luteolin

Sampling position

Conc (

g/mg)

123456

0.0

0.5

1.0

1.5

2.0

2.5

Free luteolin

Total luteolin

Conjugated luteolin

Sampling position

Conc (

g/mg)

1 2 3 4 5 6

0.0

0.5

1.0

1.5

2.0

2.5

Free luteolin

Total luteolin

Conjugated luteolin

Sampling position

Conc (

g/mg)

123456

0.0

0.5

1.0

1.5

2.0

2.5

Free luteolin

Total luteolin

Conjugated luteoli n

Sampling position

Conc (

g/mg)

123456

0.0

2.5

5.0

7.5

10.0

12.5

15.0

Free luteoli n

Total luteolin

Conjugated luteolin

Sampling position

Conc (

g/mg)

4.4.8 Design of the placebos for the plant materials

4.4.8.1 Design of placebo for the A. afra standardized dried leaves

Using the 15 extractions, it was possible to reduce the absorbance of an aqueous solution

extract of the leaves (at UV wavelength 220 nm) from an initial value of 4.8851 to a final

value of 0.0854, i.e. a reduction of approximately 98% in absorbance was attained. The

solvent extractions conducted and the resultant absorbances for a single batch is shown in

Table 4.7 below. The process developed had an average recovery of 41%, i.e. from an

initial mass of leaves of 10 g, 4.1 g of placebo leaves were obtained. This low recovery

may be attributed to the several extraction steps involved, which may have contributed to

the high loss in material.

Figure 4.7: Concentrations of the different forms of luteolin at various sampled positions in the A. afra

plant materials. Graphs (1), (2), (3), (4) and (5), show the concentrations of luteolin in plant

batches 001, 002, 003, the standardized leaves batch and the freeze-dried aqueous extract

powder, respectively.

3 4

Table 4.7: The series of solvent extractions employed to produce the placebo

material of the A. afra leaves. The UV results presented are from a

single manufactured 10 g batch.

UV Absorbance Extraction

step

Procedure/ 10g leaves

349nm 220nm

1 200ml cold water, stir for 2 minutes, allow to stand 10 minutes,

then filter. To residue conduct step 2.

3.7798 4.1132

2 200ml hot water (80˚C), stir 2 minutes, allow to stand 10

minutes, then filter. To residue conduct step 3.

3.9441 4.2586

3 200ml hot water (80˚C), stir for 2 minutes, allow to stand 10

minutes, then filter. To residue conduct step 4.

3.8926 4.1577

4 200ml methanol/water (50/50 v/v), stir 2 minutes, allow to stand

10 minutes, then filter. To filter residue conduct step 5.

4.8851 4.9241

5 200ml methanol/water (50/50v/v) + 40ml 2N HCl (place in water

bath for 120 minutes at 80˚C) then neutralise with 5N NaOH.

Filter. To residue conduct step 6.

5.0400 5.2291

6 200ml methanol. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 7.

4.1889 4.2348

7 200ml methanol. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 8.

3.6138 3.8694

8 200ml methanol. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 9.

3.3656 3.7540

9 200ml methanol. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 10.

1.3457 1.5540

10 200ml methanol. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 11.

0.4295 0.5399

11 200ml ethyl acetate. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 12.

0.1923 0.2811

12 200ml ethyl acetate. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 13.

0.1326 0.2281

13 200ml ethyl acetate. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 14.

0.1192 0.1251

14 200ml ethyl acetate. Stir for 2 minutes, allow to stand for 10

minutes. Filter. To residue conduct step 15.

0.0976 0.0907

15 200ml ethyl acetate. Stir for 2 minutes, allow to stand for 10

minutes. Filter.

0.0707 0.0854

Figure 4.8 shows an image of the placebo of the dried leaves obtained after the repeated

solvent extractions of A. afra leaves. The resultant placebo material was slightly darker

than the original A. afra leaves (with a burnt appearance) and had a rougher texture than

the A. afra leaves. This material also lacked the characteristic odour and bitter taste of the

A. afra leaves.

The HPLC chromatographic fingerprints of the plant leaves before and after the solvent

extractions are shown in Figures 4.9 and 4.10, respectively. As indicated by the reduced

number of peaks, it is evident that most of the phytochemical constituents had been

removed and therefore, the placebo produced may be expected to posses little

pharmacological activity compared to the A. afra leaves. However, further evaluation in

the form of pharmacological tests was warranted in order to prove the inertness of the

material.

Figure 4.9: A. afra standardized dried leaves chromatographic fingerprint after first solvent

extraction.

Minutes

02 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

200

400

600

800

1000

mAU

200

400

600

800

1000

Det 168-220 nm

Figure 4.8: Placebo leaves (on the left) produced b

repeated solvent extractions of

A. afra dried leaves (on the right).

Figure 4.10: A. afra leaves chromatographic fingerprint after0 final solvent extraction.

4.4.8.2 Matching of odour of the placebo and plant materials

The amount of linalool capable of masking the A. afra smell was found to be 100 mg.

This amount was adsorbed onto 0.125 g of silica gel as described in section 4.3.11.3 and

included in the A. afra plant and placebo materials.

4.4.8.3 Matching of taste of the placebo and plant materials

0.15 g of sodium saccharin was found to mask the bitterness of the A. afra SDL and

FDAE solutions. This amount was included in the preparations of the plant and placebo

materials.

4.4.8.4 Design of placebo for the freeze-dried aqueous extract powder

The formula for the placebo for the FDAE is presented in Table 4.8 below. The placebo

contained similar amounts of the aroma chemical and sweetener as the dried leaves

preparations. The concentrations of linalool and sodium saccharin of 0.125 g and 0.15 g

in the formulation constituted percentages of 0.063 and 0.075%, respectively. These

levels are in line with the recommended safety levels of these chemicals. Linalool is

generally approved for use as in fragrances up to a level of 1.5% and up to 500 mg/body

weight/day of sodium saccharin may be ingested by humans without harm (OECD, 2004;

SCF, 1997). 0.02 g of the synthetic colourant D.C. Brown was found to match the colour

of hue produced by the A. afra plant materials. The chemicals included in the formulation

(except for D.C. Brown) have been used in studies in the literature, (in similar amounts as

Minutes

02 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

100

200

300

400

500

600 Det 168-220 nm

A.afra placebo 20mg/ml

well as with other chemicals), to make placebo formulations which have been evaluated

and considered pharmacologically inert (Bensoussan et al., 1998; Kaptchuck, 2000).

Based on this, no further pharmacological evaluation for the FDAE placebo was

warranted, and as such the extract was considered suitable for use in clinical trials.

Table 4.8: Excipients and quantities used in the formulation of the placebo for

the A. afra freeze-dried aqueous extract powder.

Excipient Quantity (g) Use levels per 200 ml tea

solution (%)

Potassium hydrogen phosphate

Lactose

Potato starch

D.C. Brown

0.475

0.025

0.03

0.02

0.238

0.013

0.015

0.010

Total 0.55 0.275

Sodium saccharin

Silica gel (100mg linalool)

0.15

0.125

0.075

0.063

4.5 Conclusions

The chapter addressed certain objectives of the study, viz. the preparation of the SDL and

FDAE of A. afra, the characterization of the physicochemical properties of the plant

materials, development and validation of an analytical method for use in the quantitation

of luteolin in the materials, and the design of placebos for the plant materials.

From the results obtained, the following conclusions could be drawn;

• Approximately 50% of the mass of harvested A. afra leaves is due to moisture.

• Ionizing irradiation is an effective tool for the microbiological decontamination of

the A. afra plant materials.

• Some new physicochemical specifications for the A. afra plant leaf materials were

established, viz. water soluble ash values and bitterness values, which were

16.5952 ± 0.6760% and 16.5952 ± 0.6760%, for the dried leaves and freeze dried

aqueous extract powder, respectively and the bitterness value of 600 000 for both

materials.

• A suitable and reproducible assay method for the quantitation of luteolin in the A.

afra plant materials using morin hydrate as internal standard was developed and

validated.

• There is significant intra-batch variation but insignificant inter-batch variation in

the luteolin levels of A. afra plant leaves collected from the same geographic

location. A time of harvest, separated by a period of one month for each batch has

no effect on the levels of luteolin in the plant batches.

• The FDAE powder contains approximately 7 times more total luteolin on a weight

for weight basis than the SDL material.

• Standardization of the A. afra leaves reduces the inherent intra-batch variation in

luteolin levels. This intra-batch variation is further reduced by preparation of a

FDAE powder of the plant leaves.

• Placebo materials which resemble the SDL and FDAE powder can be produced

using repeated solvent extractions and mixing of inorganic salts, respectively.

Linalool, sodium saccharin and D.C brown can mask the odour, taste and match

the colour of tea produced, respectively, of the A. afra plant materials.

In summary, the above results obtained showed that plant materials with reduced intra-

batch variation in luteolin content and with acceptable pharmacopoieal microbial load

had been produced, and were therefore suitable for the manufacture of the tea bag dosage

form.

Chapter 5

The preparation and evaluation of tea-bags and placebos of Artemisia

afra

5.1 Introduction

This chapter presents the materials and methods used in the preparation and evaluation of

tea bags containing the standardized dried leaves (SDL), freeze-dried aqueous extract

(FDAE) powder and placebos of the plant materials. The results obtained are also

presented and discussed.

5.2 Equipment, materials and animals

In this part of the study, the following equipment were used;

balance (Scaltec SPB42 Model SPB71, Scaltec Instruments, Heiligenstadt,

Germany), centrifuge (Labofuge 200, Germany), climatic chamber (Labcon,

FSIE-H 20, Labdesign Engineering, South Africa), dissolution test apparatus

(Hanson Research, Northridge, CA, USA), HPLC filter unit (Millipore Cameo

25 AS, DDA 02025So MSI: Micro separation Inc., USA), solvent filter paper

(47mm filter membrane 0.45µm PVDF,Millipore, USA), organ bath, with

transducer (custom made) and recorder (Rikadenki R-03, Tokyo, Japan),

spectrophotometer (DU 640 spectrophotometer, Beckman, USA), filtration

System (SUPELCO) connected to vacuum pump (Medi-Pump Model 1132-2,

Thomas Industries, Inc, USA), filter paper (Whatman No. 41 & Whatman No.1,

Whatman, England), pH meter (Basic 20 Crison Instruments, S.A, Italy.), vortex

(Vortex-2, G-560E, Scientific Industries, Inc. Bohemia, N.Y. 11716 USA), and a

water bath (Labcon, CDH 110 Maraisburg, South Africa).

The HPLC system used consisted of an auto sampler (Beckman Gold Module 507), a

programmable binary gradient pump (Beckman Gold Module 126 series), a diode array

detector (DAD) (Beckman Gold Module 168 series) with a 32-KaratTM-software package

and a Synergy® Hydro-reverse phase column (Phenomenex, USA) having 4 μm particle

size and a column length of 250 x 4.60 mm.

The following materials were used;

Artemisia afra plant material, tea bag filter paper (Dynapore 117/7/0, Schoeller &

Hoesch, Germany), morin hydrate, luteolin, isoprenaline, sodium pentobarbitone

(Sigma-Aldrich, Germany), methacholine (Fluka Chemie GmbH, Germany), high

density polyethylene (HDPE) storage container, silica gel (BDH, South Africa),

sodium chloride (UnivAR, Saarchem, South Africa), potassium hydrogen

phosphate (Riedel-de Haen, Germany), glucose, sodium hydrogen carbonate

(UniLab, Saarchem, South Africa), calcium chloride, potassium chloride (Merck,

South Africa), magnesium sulphate (UnivAR, Saarchem, South Africa),

acetonitrile HPLC grade (Burdick & Jackson, M.I. USA), ethyl acetate CP

(Saarchem, South Africa), hydrochloric acid (Kimix, South Africa).

The following animals were used;

Male and female Dunkin Hartley guinea pigs of between 400 g and 700 g weight,

obtained from the animal facility at the University of Cape Town, a few days

before use. The animals were kept in an environmentally controlled animal room

with temperature control and a 12 hr light darkness cycle. Ethics clearance was

obtained for the use of the guinea pigs from the UWC Senate Ethics committee.

5.3 Methods

5.3.1 Preparation of the tea bags

The primary objective of the study was to prepare the SDL and FDAE powder of A. afra

in a tea bag dosage form containing amounts of the materials equivalent to those used in

the traditional infusions.

5.3.1.1 Determination of the amount of plant material for the tea bags

The most common traditional method of preparation directs that a quarter cup of leaves

be infused in hot water, allowed to seep for 10 minutes and the resultant tea drunk

(Roberts, 1990). Based on this, a 200 ml tared cup was loosely filled to the brim with

dried A. afra leaves, the weight recorded and divided by 4 to obtain the amount

equivalent to a quarter cup (i.e. dose of leaves required).

To determine the amount of FDAE powder to be incorporated in the tea bags, the total

luteolin content present in the SDL was used. It was known (section 4.4.7), that the

FDAE powder, on a weight-to-weight basis, contained approximately 7 times more total

luteolin than the standardized A. afra leaves and therefore, the amount of FDAE

incorporated into the tea bag was a seventh of that contained in the dried leaves tea bag.

5.3.1.2 Determination of the appropriate tea bag size

It has been reported in the literature, that various sizes of tea bags result in different rates

of extraction of the soluble species (Jaganyi & Ndlovu, 2001). In fact, the rate of

extraction increases with increases in tea bag size and in one study, viz. an investigation

of the effect of tea bag size on the extraction of caffeine from Ceylon Orange Pekoe tea,

the results showed that beyond a mass to surface area ratio of 1:9, only small increments

in the rate of extraction were observed (Jaganyi & Ndlovu, 2001). This ‘threshold ratio’

of 1: 9 was therefore adopted for the manufacture of the A. afra plant material and

placebo tea bags, i.e. the tea bags had a content mass to surface area ratio of 1:9. Tea bags

containing the FDAE powder also had a content mass to surface area ratio of 1:9,

although the material in these bags was only a seventh of the mass of the dried leaves

used. This was done in order to increase the mass to surface area ratio beyond the

threshold and therefore ensure an almost instant infusion and in addition, it was

anticipated that depending upon the clinical trail design, the FDAE powder may be

evaluated against the dried leaves and therefore the two preparations needed to be similar

in size to facilitate clinical trial blinding.

5.3.1.3 Selection of the appropriate tea bag paper

In selecting the tea bag paper with the appropriate specifications, particle retention (pore

size), wet strength and paper smell of the material were taken into consideration.

Consequently, tea bag filter paper with high particle retention (and therefore suitable for

containing the FDAE powder), high wet strength, absence of paper odour, made from

oxygen-bleached raw materials and with heat sealable properties was selected (see

Appendix C for the specifications). The oxygen-bleached tea bag paper was preferred in

the hope that the bleaching process would have oxidised any pharmacologically active

phytochemical constituents within the paper material and therefore the tea bag paper

would be pharmacologically inert.

5.3.1.4 Preparation of the Artemisia afra and placebo tea bag dosage forms

Generally tea bags may be filled using an automated method, where the tea material

flows through a hopper and is wrapped around by the tea bag paper and the latter sealed

to enclose its contents. Typically, heated rods on either side of the tea bag paper will

press together to seal the tea bag before it is cut into the desired shape (Anonymous,

2006a).

In this study, the tea bags of the A. afra plant materials were prepared and sealed

manually. From the tea bag paper roll, strips 7 cm wide were cut, folded over, and cut to

make squares 7 cm x 7 cm. Two of the three open sides of the square were then sealed

closed, 0.5 cm into each side, using a hot iron. The appropriate amount of the sieved SDL

of A. afra (particle size range of 0.35 - 1.40 mm) was then accurately weighed and

transferred into the bag via the remaining open side and thereafter the latter side sealed,

0.5 cm into the side. This therefore meant that the available surface area for the plant

material was 36 cm2 (6 cm x 6 cm). The above procedure was repeated for the

preparation of the tea bags containing the FDAE powder and the placebo materials.

5.3.2 Pharmaceutical evaluation of the tea bag dosage forms

Few quality control tests exist in the pharmacopoeia for the specific evaluation of tea bag

dosage forms. Control tests are typically conducted on the herbal or tea material itself and

include identity and microbiological evaluation tests. However, the test for uniformity of

mass is specified in the European Pharmacopoeia 2002 to be conducted on herbal teas in

sachets (EP, 2002d). The tea bag dosage forms of A. afra plant material were

consequently evaluated on the latter in addition to other quality tests as described below.

5.3.2.1 Determination of uniformity of mass of the tea bag dosage forms

Twenty tea bags were randomly selected from each of the manufactured batches

containing the A. afra SDL and FDAE powder. Each tea bag was weighed and then

completely emptied of its contents and any remaining particles removed using a brush.

To obtain the mass of the tea bag contents, the mass of the empty tea bag was subtracted

from the initial mass of the tea bag (EP, 2002d) and from masses of the 20 tea bags, the

average mass and deviation in the individual tea bags content was calculated.

5.3.2.2 Determination of the infusion profile of the A. afra dosage forms

Few studies are available in the literature on the release profiles of flavonoids in tea

infusions. The majority of studies have focused on the influence of temperature,

manufacturing method or composition of the extracting media on the infusion and

equilibria of the tea infusions (Jaganyi & Mdletshe, 2000; Jaganyi & Wheeler, 2003;

Spiro & Jaganyi, 2000).

To establish the infusion profiles of the A. afra dosage forms, the British Pharmacopoeia

dissolution apparatus I (basket apparatus) at 100 revolutions per minute (rpm) and a

modified apparatus II (paddle apparatus) at 50 rpm were used for the loose leaves and tea

bag preparations, respectively. In the experiments, water, as the dissolution media, at a

temperature of 80˚C was used (Jaganyi & Wheeler, 2003; Spiro & Lam, 1995), as it is the

temperature closest to that normally used in the preparation of teas (Anonymous, 2006a).

At various times, samples from the infusions were collected and analysed for dissolved

plant constituents.

The flavonoid luteolin was used as a marker compound to characterize the infusion

profiles of the A. afra dosage forms and the validated HPLC assay described in section

4.4.6 was used to quantitate the amount of luteolin released over time. Infusion samples

were acid hydrolysed yielding total luteolin (i.e. free aglycones plus the aglycones

initially in glycosidic linkages) and unhydrolysed yielding free luteolin and the sugar

moieties of luteolin, at each sampling time point and in addition, the amount of glycosidic

luteolin present at each sample time point was calculated (i.e. total luteolin – free

luteolin). Luteolin is known to be present in plants as luteolin-7- O-glucuronide, luteolin-

5-O-glucoside, luteolin-3’- glucuronide, luteolin 3′-O- β - D -glucuronide and/or luteolin-

3’,4’-diglucuronide forms (Markham, 1982; Heitz et al., 2000) and these were the likely

glycosidic forms of luteolin to be quantitated at each time point.

The samples collected from the infusion of the dosage forms were also analysed using a

UV spectrophotometric assay, which provided a profile of the infusion of all the soluble

plant constituents over time.

5.3.2.2.1 Determination of the infusion profile of the A. afra loose leaves

The British Pharmacopoeia dissolution apparatus I (basket method) was used for the

infusion profile characterization of the loose leaves. The dissolution apparatus was fitted

with a heater on the water bath capable of attaining and maintaining a temperature of

80˚C within a range of ±5˚C. One gram of the sieved SDL (particle size range 0.35-1.40

mm) was accurately weighed and loosely filled into the apparatus basket. The dissolution

apparatus vessel containing 900 ml of de-aerated distilled water was weighed with its

contents, allowed to equilibrate with the water bath temperature and once equilibrated,

the basket containing the leaves lowered into the vessel and rotated at 100 rpm. Sampling

was conducted through a fixed glass rod inserted through the cover disc at a depth of 5

cm into the vessel, at a position 1 cm from the wall of the vessel.

Samples of 3 ml each were withdrawn from the vessel using a plastic syringe. 1 ml was

transferred into a tube containing 5 ml of distilled water for UV spectrophotometric

analysis, 1 ml into a tube for hydrolysed luteolin analysis using HPLC and the other 1 ml

into a tube for unhydrolysed luteolin analysis using HPLC. Each sample withdrawn was

immediately replaced with an equal volume of de-aerated distilled water. For UV and

HPLC analysis, a total of 8 samples for each were withdrawn at the sampling time points:

0.0 (blank), 5.0 10.0, 30.0, 60.0, 120.0, 180.0 and 240.0 minutes. At the end of the run,

the apparatus vessel was again weighed with its contents and the difference in weight

taken as ∆V the volume lost through evaporation and sampling (assuming a specific

gravity of 1 for the lost media). This was used to calculate the rate of loss for correction

of the concentrations (Spiro & Lam, 1995).

5.3.2.2.2 Determination of the infusion profile of the A. afra tea bag dosage

forms

The British Pharmacopoeia dissolution apparatus II (paddle method) was used for the

infusion profile characterization of the A. afra tea bag dosage forms, i.e. tea bags

containing the A. afra SDL and FDAE powder. However, because tea bags have a

propensity to float in aqueous medium thus affecting the hydrodynamic conditions within

the dissolution vessel, a holding cell was devised to hold the tea bag and maintain it in a

fixed position within the vessel. The holding cell devised for this study incorporated a

wire mesh cage and two stainless steel weight rods and was a modification of the double

ring mesh assembly designed by Durig and Fassihi (2000) in investigating the dissolution

of floating and sticking delivery systems and that used by Jaganyi and Mdletshe (2000),

where stainless steel bolts were attached to the tea bag to prevent floating.

The holding cell devised and shown in Figure 5.1, was made from netting wire (wire

specifications 900 x 13 x 0.71 mm), which was cut and folded to make a holding cell 7

cm x 7 cm, with a height of 1 cm. To ensure that the holding cell would not move during

the infusion process, weights were added in the form of two stainless steel rods, on either

side at the bottom of the holding cell. The rods had dimensions of 6.5 x 1 cm and the

complete holding cell had a weight of 115 g. The shape of the dissolution vessel and the

dimensions of the holding cell ensured that the holding cell would only go a certain

distance within the vessel (i.e. 2.5 cm from the bottom of the vessel) and no further. The

tea bag was placed inside the holding cell, such that it lay flat and the wire mesh did not

hinder any swelling of the tea bag (see Figure 5.1).

In determining the infusion from the tea bags, once the media had been weighed and

allowed to equilibrate to the water bath temperature (as described in section 5.3.2.2.1),

the holding cell containing the tea bag was placed inside the vessel taking care to ensure

that water did not splash out of the vessel during this process. The dissolution apparatus

paddles were immediately lowered into the vessel up to a distance of 3.0 cm above the

holding cell and rotated at a speed of 50 rpm. For UV and HPLC analysis, a total of 12

samples for each analysis method were withdrawn at the sampling time points: 0.0

(blank), 5.0 10.0, 30.0, 60.0, 120.0, 180.0, 240.0, 300.0, 420.0, 480.0 and 540.0 minutes.

At the end of the run, the dissolution vessel was weighed with its contents and the

difference in weight taken as ∆V, the volume lost through evaporation and sampling for

use in calculation of the concentrations.

Sampling tube

Rotating paddle

Holding cell

Tea bag

Figure 5.1: The holding cell devised to contain the A. afra tea bags during the infusion process (on the

left) and on the right, a schematic diagram of the modified dissolution apparatus II, used for

the determination of the infusion profile of the tea bags containing the plant materials.

For the determination of the infusion profile of the tea bags containing the FDAE, the

same procedure as described above was used. However, in this case, the infusion samples

for UV spectrophotometric analysis were withdrawn at the time points: 0 (blank), 1.0,

2.0, 3.0, 4.0, 5.0, 10.0 and 15.0 minutes. From the infusion profile generated using the

spectrophotometric analysis, the time point at which 85% of the extract powder had

infused was determined. This point was then used as a sampling time point for a single

point dissolution assay for luteolin using HPLC (FDA, 1997) and total, free and

conjugated luteolin quantified at this time point.

5.3.2.2.4 Sample preparation, analysis and comparison of the infusion profiles

Samples collected from the infusion experiments were hydrolysed and extracted using the

procedure described in section 4.3.10 and assayed for total (hydrolysed) and free

(unhydrolysed) luteolin using the validated HPLC assay. The quantity of total, free and

the calculated conjugated luteolin concentrations at each time point were plotted against

time to generate the infusion profile for the dosage forms.

For the UV spectrophotometric analysis, the 1 ml sample withdrawn was placed in a tube

containing 5 ml of distilled water and immediately cooled, by placing in a cooler box, to

prevent evaporation of the sample as well as any unknown degradation of the sample

which could occur. The UV absorbance of the samples were measured at 349 nm

(luteolin λ max) and at 220 nm, the λ max for the infusion samples, and the infusion

profiles generated by plotting the absorbance measurements against time.

The infusion profiles of the A. afra loose leaves and the tea bag dosage forms generated

from the UV spectroscopic and the HPLC quantitation of the different forms of luteolin

were compared by calculating the similarity factor (f2) and the difference factor (f1)

between the profiles.

5.3.2.3 Determination of the stability of the dosage forms upon storage

The tea bags containing the A. afra SDL and FDAE powder were subjected to real time

and accelerated stability testing. Real time stability testing was conducted at normal room

temperature and normal room humidity for a period of 6 months i.e. at Site A and

accelerated stability testing conducted at 40˚C, 75% relative humidity for a period of 3

months i.e. at Site B. In addition, the effect of temperature alone on the stability of the

FDAE tea bag preparation was evaluated. For this, tea bags containing the FDAE powder

were placed in a secondary packaging comprising a high density polypropylene (HPDE)

container containing a 5 g sachet of silica gel desiccant. These containers were placed

under 2 different conditions of temperature, viz. room temperature and 40˚C, for a period

of 3 months (i.e. at Site C and D respectively).

The stability of the tea bags upon storage were evaluated based on their organoleptic

properties, i.e. appearance, odour, leakage of tea bag contents, moisture content of tea bag

contents, infusion profile and chromatographic fingerprint of the tea bag contents. The

latter was assessed using HPLC with diode array detection and the chromatographic

fingerprints of the plant materials obtained, at 14 and 30-day intervals for the FDAE and

dried leaves respectively, compared to determine the stability of the plant material as a

whole. To obtain the chromatographic fingerprints, 25 mg samples were withdrawn from

the preparations under storage, placed in 1ml of distilled water at 80˚C, vortexed for 2

minutes and left to stand for 8 minutes before being filtered using Whatman no. 1 filter

paper. Fifty microliters of the filtrate was injected directly onto the HPLC column, under

the conditions described in section 4.3.10. The chromatographic fingerprints obtained

were overlaid onto each other in order to show any changes that may have occurred. The

UV spectra of the HPLC assayed samples were also overlaid, and compared, at a

wavelength and retention time corresponding to the peak with maximum absorbance.

This comparison would show the purity and identity of the material throughout the period

of the stability test, i.e. any degradation or change could be observed through the UV

spectra (Springfield et al., 2005). Finally, the procedure for determining the organoleptic

properties, moisture content and infusion profile of the preparations was as described in

section 4.3.5, 4.3.7 and 5.3.2.2, respectively.

5.3.3.4 Comparison of the A. afra tea bag dosage forms and their placebos

The organoleptic properties of the placebos of the A. afra leaves and FDAE powder were

compared to that of the A. afra leaves and FDAE in tea bags, respectively. This was in

terms of the overall appearance of the tea bags, colour and taste of the teas produced and

odour of the product. For this evaluation, the natural senses of sight, taste and smell were

used.

5.3.4 Pharmacological evaluation of the placebo for the plant leaves

In designing a placebo, it is essential that the placebo be evaluated for lack of

pharmacological activity before it can be considered inert and suitable for use in a clinical

trial. It therefore follows that the placebo should be evaluated against the ‘active’ for

pharmacological activity, which the ‘active’ possesses. Bioassays utilizing either whole

animals, isolated organs and tissues, blood and its components, tissue culture cells or

subcellular cells can be used for evaluating the efficacy of the test material (WHO, 1993).

In selecting an appropriate test system, consideration should be given to the sensitivity,

reproducibility and general acceptance of the test system. Dose selection may be based on

doses administered traditionally or clinically and results are often presented as dose-

response relationships (WHO, 1993).

In this study, the muscle relaxant activity of the A. afra placebo and plant materials was

evaluated using an isolated guinea pig tracheal muscle mounted in an organ bath system.

This part of the study was ethically approved by the Ethics Committee of the UWC

Senate Research Committee, as part of another more comprehensive project viz. ‘The

investigation of the mechanism for the respiratory smooth muscle relaxant effect of a

flavonoid-containing aqueous extract of Artemisia afra’ (M. Pharm research project of L.

Manthata, School of Pharmacy, UWC), i.e. the animal tissue was isolated for use in the

afore-mentioned project and after an experimental run, was used for the present

evaluation. The procedures used for the preparation of the test solutions and animal tissue

to establish the activity of the A. afra plant material and placebos is described below.

5.3.5.1 Preparation of solutions used in the tracheal muscle experiments

For the evaluation of the muscle relaxant activity, similar concentrations of the placebo

and A. afra leaves, i.e. 5, 10, 20, 50 and 100 mg/ml in distilled water, were prepared. For

this, the appropriate mass of material was placed in a volume of hot (80˚C) distilled

water, vortexed for 2 minutes, allowed to stand for 8 minutes and then filtered using

Whatman no. 1 filter paper before being used. Stock solutions of 60 mM potassium

chloride, 10-1M methacholine and 1M isoprenaline for use in the experiments, were

prepared in distilled water. Krebs Henseleit solution for sustaining the tissues was made

by dissolving 35.05 g NaCl, 1.78 g KCl, 816.55 mg KH2PO4, 1.48 g MgSO4.7H2O, 10.50

g NaHCO3, 1.84 g CaCl2.2H2O in distilled water, and adding 9.90 g of glucose just

before use.

5.3.5.2 Procedure for evaluation of the effect of the A. afra and placebo

materials on the isolated guinea pig tracheal muscle

For this study, the tracheal muscle was isolated for mounting in the organ bath as follows;

firstly, the guinea pig was weighed and anaesthetised with an intra-peritoneal injection of

sodium pentobarbitone (190 mg/kg body weight). The trachea was then removed from the

animal, placed in a petri-dish containing cold Krebs Henseleit solution, the excess tissue

and fat trimmed off using a razor blade and the muscle opened by cutting longitudinally

through the cartilage rings, diametrically opposite the trachealis muscle. The flat tissue

was then pinned onto a corkboard (still immersed in the cold Krebs Henseleit solution)

and cut into zig-zag strips by making transverse slits at equal intervals in the tissue

(Uhlig, 1998). Finally, the strip was divided into 4 equal pieces, cotton threads inserted at

each end and the strips mounted in the isolated organ bath system.

The isolated organ bath system used in this study, and shown in Figures 5.2 and 5.3,

consisted of (i) a circulating water bath, (ii) 4 custom made water-jacketed, 15 ml glass

organ baths each connected, with tubing and tap, to (iii) a reservoir containing Krebs-

Henseleit solution, (iv) a force transducer and amplifier system and (v) a 4-pen strip chart

recorder. The organ baths were filled with pre-warmed medium from the reservoir via

glass tubing located in the water-jacketed glass systems, the flow into the bath being

controlled via an inlet tap and flow out via a drain tap at the bottom of the bath. The

contents of the bath and the glass perfusion lines were kept at 37ºC by pumping water

through the glass jackets of the organ bath and glass systems using the circulating water

bath. The contents in the organ bath were also continuously aerated with 5%CO2 / 95%O2

via an inlet at the bottom of the bath. The tissue was mounted in the bath by hooking one

cotton thread to a hook at the bottom of the bath and the other end to the force transducer.

The transducer converted the mechanical movements of the contracting/relaxing muscle

into an electrical signal that was amplified and the result eventually recorded on a chart

recorder.

Figure 5.3: A close up picture of a sin

an bath. A shows cotton

thread used to tie the tissue to

the transducer, B is the Krebs’

inlet tap, C shows the suspended

tissue, D shows the bath, E

shows the gas inlet, F, shows one

of the pipes to the water-bath

that transport warm water to

maintain the temperature, G

shows the Krebs’ outlet tap and

H shows the transducer.

Figure 5.2 The organ bath system used

for evaluation of the activity

of the plant materials. A is the

bath reservoir containing

Krebs Henseleit solution, B is

one of the water-jacketed

glass reservoirs C is the water

bath temperature controller,

D is the 4-pen strip chart

recorder and E is the signal

amplifiers.

Once mounted in the organ bath the tissue was placed under a tension of 2 g and allowed

to equilibrate for 1.5 to 2 hours, while being continuously irrigated and aerated with

Krebs Henseleit solution at a temperature of 37 ºC (that was replaced at 15 min intervals)

and a mixture of 95% O2 and 5% CO2. Thereafter, the tissue was depolarised with 2 doses

of 0.1 ml potassium chloride (60 mM) injected into the bath and after each depolarising

dose, and sufficient time allowed for the response, the tissue was washed and allowed

time to recover before the next dose was given. Following this, the tissue was contracted

using 0.1 ml methacholine (10-1M), the contraction left to stabilise for 20 minutes and the

response recorded on the strip chart recorder. The methacholine was then washed out and

another methacholine dose administered, i.e. the process repeated in order to check the

reproducibility of the tissue response. After the contractions had stabilized, cumulative

0.1ml doses of the placebo leaves solutions (i.e. 5, 10, 20, 50, and 100 mg/ml) were

injected into the organ bath starting with the lowest concentration and allowing enough

time for its effect to stabilise before the subsequent dose was injected. After the injection

and stabilisation of the effect of the last dose (i.e. 0.1 ml of 100 mg/ml), 0.1ml of 1x10-

1M isoprenaline was injected to obtain the maximal relaxation response. Throughout, the

responses of the tissue to the injected solutions were continuously recorded on the chart

recorder. The traced responses (i.e. the change from base-line or maximum contraction),

were then measured and the relaxant effect of the injected solutions expressed as a

percentage of the maximum relaxation induced by the isoprenaline. A log dose response

curve of the percent relaxation vs. log dose was then plotted.

The same procedure was repeated for solutions of the A. afra plant leaves.

5.7 Results and discussion

5.7.1 Preparation of tea bags containing the A. afra plant material

The mass of a quarter cup of the A. afra dried leaves, as determined in section 5.3.1.1 was

found to be 4 g and this was thus taken as the amount of material in one traditional dose

and therefore the amount to be incorporated in one tea bag. Based on the ratio of the total

luteolin content in the SDL to that in the FDAE powder, found in section 4.4.7, a seventh

of the A. afra leaves quantity i.e. 0.55 g, was taken as the amount of FDAE powder to be

incorporated in the tea bags. These dose quantities used were in agreement with those

obtained by previous investigators, viz. Mukinda (2006), in determining the amount of

dried leaves that were equivalent to the traditional dosage preparation, found a quantity of

3.5 g, while Komperlla (2005) found that 0.60 g of the FDAE powder was equivalent to

the traditional dried leaves preparation

Four grams of the SDL and 0.55 g of FDAE powder were thus, separately prepared in

Dynapore 117/7/0 tea bag paper to produce tea bags having 36 cm2 surface area. Images

of the prepared tea bags containing the dried leaves and FDAE powder are shown in

Figure 5.4. Although equivalent in luteolin content and tea bag surface area, the two

products were clearly distinguishable from each other, with the A. afra leaves completely

filling and extending the bag while the extract powder only occupied a small portion of

the tea bag.

5.7.2 Uniformity of mass of the dosage forms

The uniformity of mass data for the manufactured tea bags containing the A. afra SDL

and FDAE powder, is shown in Tables 5.1 and 5.2, respectively.

Figure 5.4: Prepared tea bag containing 4g of the standardized dried leaves of A. afra (on the

left) and that containing 0.55g of the A. afra freeze-dried aqueous extract powder

(on the right).

Table 5.1: Uniformity of mass of the prepared tea bags of the A. afra

standardized dried leaves.

Mass of tea bag

contents (g)

Mass of tea bag

contents (g)

Mass of tea bag

contents (g)

Mass of tea bag

contents (g)

4.0020

4.0039

4.0038

4.0004

4.0002

4.0004

3.9995

3.9992

4.0002

3.9992

3.9995

4.0075

4.0032

4.0085

3.9998

4.0008

4.0021

4.0206

4.0023

3.9997

Average tea bag content mass ± S.D = 4.003 ± 0.0013

% R.S.D = 0.12

Table 5.2: Uniformity of mass of the prepared tea bags of the freeze-dried

aqueous extract powder of A. afra.

Mass of tea bag

contents (g)

Mass of tea bag

contents (g)

Mass of tea bag

contents (g)

Mass of tea bag

contents (g)

0.5564

0.5679

0.5609

0.5574

0.5645

0.5487

0.5421

0.5539

0.5618

0.5585

0.5533

0.5604

0.5645

0.5681

0.5544

0.5531

0.5663

0.5501

0.5522

0.5543

Average tea bag content mass ± S.D = 0.557 ± 0.0069

% R.S.D = 1.24

The A. afra tea bags containing the SDL had an average content of 4.003 ± 0.0013 g and

those containing the FDAE powder, an average of 0.557 ± 0.0069 g of material. The

percent deviation in the content of the manufactured tea bags was well within the

European Pharmacopoeia 2002 requirements (i.e. R.S.D of 0.12% and 1.24 %) for

uniformity of mass of herbal teas in sachets. The latter requires a percent deviation of not

more than 7.5% for an average mass more than 2.0 g and a percent deviation not more

than 15% for a mass less than 1.5 g. Taken together with the results obtained in the

identity and microbial tests conducted in Chapter 4, it was thus concluded that the

manufactured tea bags complied with the European Pharmacopoeia 2002 requirements.

5.7.3 Infusion profile of the A. afra loose leaves

The infusion profiles of the A. afra loose leaves are presented below and Figure 5.5

shows the UV infusion profiles and the percentage of infused plant material released from

the A. afra loose leaves. Figure 5.6 shows the infusion profiles of the luteolin release

(free, conjugated and total luteolin) and the percentage luteolin released from the A. afra

loose leaves. The results shown were corrected for 4 g of plant material, since only 1 g of

plant material, the maximum that could be loosely filled in the apparatus basket, was

used. The results were also corrected to allow for evaporation and sampling losses.

050 100 150 200 250

0.0

2.5

5.0

7.5

10.0

12.5

349nm

220nm

Time ( mins )

Abs

050 100 150 200 250

100

349nm

220nm

Time ( mins )

% released

050 100 150 200 250

100

Total luteolin

Free luteolin

Conjugated luteolin

Time (mins)

050 100 150 200 250

100

Conjugated luteoli n

Total luteolin

Free luteoli n

Time (mins)

From the UV and HPLC generated infusion profiles, it was apparent that the profiles

generated from the two methods were qualitatively similar, suggesting that luteolin was a

suitable marker to use to characterise the infusion profiles of the A. afra leaves and

Figure 5.5: The infusion profiles of the A. afra loose leaves (on the left) and the percentage release profile

(on the right), determined using UV spectroscopic analysis at 220 (-●-) and 349 (-○-) nm.

Conc. luteolin

(µg

/ml

)

Percentage released

Figure 5.6: The infusion profile of luteolin (total -●-, conjugated -○- and free -▲-) release (on the left)

and the percentage release profile of luteolin (on the right) from the A. afra loose leaves.

therefore the following discussion of the infusion results for this dosage form is based on

the HPLC luteolin data only.

The infusion profiles obtained demonstrated a characteristic pattern of release, i.e. an

initial burst release phase followed by a controlled release phase which, from the

literature, is known to be typically associated with swelling (of the leaves). The initial

burst phase normally accounts for 50% of the release and the swelling phase from 50%

up to the point where 100% release is reached (Missaghi & Fassihi, 2005).

From the results obtained, insight into the amount and form of luteolin present in the

traditional teas over the infusion period can be gained. Commonly, the traditional method

of preparation allows the tea to stand and seep for 10 minutes before being consumed

(Roberts, 1990). At this time point, based on the present results, an average of 35.01 ±

10.09 µg/ml of free luteolin (aglycone) and 24.20 ± 11.14 µg/ml of conjugated luteolin

(glycosidic) were present in the tea and this corresponded to an average total luteolin

concentration of 52.24 ± 16.04 µg/ml and an average percentage release of 31.35%,

47.39% and 34.61% of the free, conjugated and total luteolin, respectively. The final

concentrations of free, conjugated and total luteolin present in the tea at steady state (i.e.

after 100% release) were 63.30± 10.93 µg/ml, 47.35 ± 16.72 µg/ml, and 113.90 ± 11.18

µg/ml, respectively.

From the percentage released plots, it was evident that the different forms of luteolin

were released into solution at different rates. The glycosidic or conjugated luteolin was

released into solution faster than the aglycone form viz. 47.39% vs. 31.35% released in

the first 10 minutes, respectively. This observation was expected as it is known that

glycosylation renders flavonoids more water soluble (Markham, 1982). The conjugated

luteolin was released into solution rapidly and reached an asymptote in concentration

within 40 minutes, i.e. approximately 100% of the glycosidic luteolin content of the

leaves was released within 40 minutes. On the other hand, the free luteolin aglycone was

released at a slower rate and only reached an asymptote in concentration at approximately

180 minutes. A possible explanation for this could be that the conversion of the

conjugated luteolin to the aglycone (free) form, occurred in the tea solution, such that the

concentration of conjugated luteolin did not increase further (an equilibrium existed

between the released and converted conjugated luteolin), as the conjugated luteolin in

solution was converted to free luteolin resulting in a gradual increase in free luteolin and

no increase in conjugated luteolin.

The total luteolin content present in the A. afra SDL (section 4.4.5) was found to be 2.065

± 0.235 µg/mg of plant material and this was expected to translate (under the infusion

experiment volume conditions) to a total luteolin concentration of 9.18 µg/ml after a 10-

minute period of infusion. However, instead an average concentration of 52.24 µg/ml (i.e.

approximately 5 fold higher) was actually obtained after 10 minutes infusion. This could

be attributed to differences in the experimental conditions, as in the quantitation

determinations (section 4.3.10), the leaves were stirred for only 2 minutes before being

allowed to stand and seep for 8 minutes, while in the infusion experiments (section

5.3.2.2.1), the leaves were continuously rotated at 100 rpm for 10 minutes and beyond.

Under the latter conditions it could be expected that a greater amount of luteolin would

infuse from the leaves and, therefore, the value for the total luteolin released after 250

mins (i.e. at steady state conditions) found in the infusion study may be the more correct

quantity of total luteolin present in the standardised A. afra leaves.

In summary, the results obtained suggested, firstly that luteolin was a suitable marker for

the characterization of the infusion of the A. afra loose leaves. Secondly, that the different

forms of luteolin were released into solution at different rates, with conjugated luteolin

being released at a faster rate than the aglycone form. Thirdly, that the method used to

determine the amount of luteolin present in the plant materials may not give a correct

estimation of the true luteolin content present. Finally, the amount of luteolin released

over time in the traditional infusions has for the first time been characterised and the

results obtained could be used to calculate the actual amounts of luteolin subjects would

ingest when using A. afra teas brewed for different periods of time. Such information

would be critical, when conducting a clinical trial to assess the efficacy of this traditional

preparation.

5.7.4 Infusion profile of the tea bags containing the A. afra leaves

The UV infusion profile of the A. afra SDL in tea bags is shown in Figure 5.7 below,

while Figure 5.8 shows the infusion profiles and percentage of luteolin released. All the

results shown were corrected to allow for evaporation and sampling losses.

0100 200 300 400 500 600

349nm

220nm

Time (mins)

Abs

0100 200 300 400 500 600

100

349nm

220nm

Time (mins)

0100 200 300 400 500 600

Conjugated luteoli n

Free l uteoli n

Total luteolin

Time (mins)

0100 200 300 400 500 600

100

Conjugated luteolin

Free luteolin

Total luteolin

Time (mins)

Conc. luteolin (µg/ml)

Percentage released

Figure 5.7: The infusion profiles (on the left) and the percentage release profile (on the right) of the

tea bags containing the A. afra leaves determined using UV spectroscopic analysis at

220 (-●-) and 349 (-○-) nm.

Figure 5.8: The infusion profile of luteolin (total -●-, conjugated -○- and free -▲-) release (on the left) and the

percentage release profile of luteolin (on the right) from the tea bags containing the A. afra leaves.

As was the case for the loose leaves, the infusion profiles determined using the HPLC

and UV methods were similar and the discussion below will again be based on the HPLC

luteolin results only.

The infusion profiles for the A. afra leaves in the tea bag also demonstrated a

characteristic pattern of release, i.e. an initial burst followed by a controlled release

phase. However, in this case, the initial burst phase encompassed release up to

approximately 35% of the total amount released at steady state and the swelling phase

from 35% onwards. As a result of the short burst release phase and longer controlled

release phase compared to that of the loose leaves, the rate of release in this case was

expected to be slower. Using the same infusion time point as used for the loose leaves (of

10 minutes), an average of 1.66 ± 0.64 µg/ml of free luteolin and 2.71 ± 0.66 µg/ml,

conjugated and 4.81 ± 0.55 µg/ml of total luteolin were present in the tea and at this time

point, an average of only 8.88%, 10.90% and 10.08% of free, conjugated and total

luteolin, respectively, were released. A comparison of these values with those obtained

for the loose leaves at the same point, showed that the initial rate of infusion from the tea

bags was approximately 3.63 times slower than that of the loose leaves. As the only

difference between the two preparations was the tea bag membrane, the difference in the

rate must have been due to this, i.e. the tea bag paper retarded the infusion of luteolin

from the leaves by a factor of 3.63.

As was the case for the loose leaves, the glycosidic luteolin was released into solution

faster than the aglycone form. Steady state conditions for luteolin release were reached

after 540 minutes of brewing and this showed that a longer infusion time was required for

the release of luteolin (and presumably the other phytochemical constituents of A. afra

leaves) from the tea bags compared to that from the loose leaves. In fact, the infusion

time to steady state was 3 times longer than that required for the loose leaves (i.e. 540 vs.

180 mins), clearly indicating the rate retarding effect of the tea bag paper.

Collectively, the results of the A. afra tea bag infusion study suggested that although the

pattern of luteolin release was similar to that for the standardized leaves alone, the rate of

luteolin release was retarded (3 times slower) by the presence of the tea bag paper and it

therefore might be worthwhile, in future studies, to investigate the effect of different

types of tea bag papers on the rate of infusion of the plant leaves.

5.7.5 Infusion profile of the tea bags containing the freeze-dried aqueous extract of

A. afra

Figure 5.9 shows the UV infusion profile of the FDAE powder of A. afra in tea bag

dosage form and from the profile it can be observed that the powder was rapidly

dissolving with an asymptote in concentration being reached within 10 minutes of

infusion. Also, the rapid infusion was also quite evident, with 85% of the constituents

being released within 5 minutes and 100 % released after 15 minutes of brewing.

0.0 2.5 5.0 7.5 10.0 12.5 15.0

349nm

220nm

Time (min)

Abs

0.0 2.5 5.0 7.5 10.0 12.5 15.0

100

349nm

220nm

Time (min)

At the infusion time point of 85% release, i.e. at 5 minutes, quantitation of the luteolin

content using HPLC, found an average of 4.8510 ± 0.9048 µg/ml, 2.352 ± 0.4387 µg/ml

and 2.499 ± 0.4661 µg/ml of total, free and conjugated luteolin, respectively, present in

the tea solution. Based on the results of the determination of total luteolin content in the

FDAE powder (section 4.4.7), a total luteolin content in solution after 100% release of

9.18 µg/ml was expected. Therefore, the result of the luteolin content present after 5

minutes showed that the tea bag containing the FDAE powder was capable of releasing

52.84% of the total luteolin content within a period of 5 minutes, thus demonstrating that

Figure 5.9: The infusion profile (on the left) and percentage release profile (on the right) of the tea bags

containing the A. afra freeze-dried aqueous extract determined using UV spectroscopy

analysis at 220 (-●-) and at 349 (-○-) nm.

Percentage released

this tea bag was capable of releasing high amounts of luteolin within a much shorter

period of time compared to the loose SDL and SDL in the tea bags. This finding was

expected, as the FDAE was a finer particulate powder and highly soluble in water (as it

was prepared using water). In addition, the fact that the powder was prepared from a 10-

minute infusion of the dried leaves meant that the preparation contained the desired

luteolin content (for a 10-minute brewing time) and this may be a distinct advantage of

this preparation as in a clinical trial this tea bag could be allowed to infuse until 100%

release is obtained, without one having to stop the infusion process as would be necessary

with the tea bags or the loose leaves. Also, the rapid release of constituents within a short

period of time compared to that found for the loose leaves and tea bags may make this

preparation more suitable for use in clinical trials evaluating the loose leaves. Finally, the

luteolin concentrations obtained at the 5-minute infusion time point served the purpose of

setting a value against which the stability of the tea bags containing the extract powder

could be evaluated.

In summary, the results showed that the contents of this dosage form were highly soluble

and rapidly dissolving, with 85% of the constituents released within 15 minutes and as a

result this dosage form could suitable for use in clinical trials evaluating the loose leaves.

5.7.6 Comparison of the infusion profiles of the A. afra loose leaves and tea bags

containing the leaves and freeze-dried aqueous extract powder.

Table 5.3 gives a summary of the mean percentage release values obtained for the

infusion of luteolin from the loose leaves and leaves in tea bag and in Table 5.4, a

comparison of the infusion profiles based on the mean percentage release values is

presented, i.e. the calculation of the f1 (difference factor) and f2 (similarity factor). In the

calculation of the factors, the loose leaves were considered the reference product and the

leaves in tea bag the test.

Table 5.3: Mean percentage release values (n = 6) of luteolin from the A. afra

loose leaves and leaves in tea bag

Infusion of the A. afra loose leaves (% ± S.D) Infusion of A. afra leaves in tea bag (% ± S.D) Time

(mins)

Free luteolin Conjugated

luteolin

Total luteolin Free luteolin Conjugated

luteolin

Total luteolin

120

180

240

480

540

18.300±9.051

31.346±10.574

60.481± 7.395

78.145 ± 6.629

91.097 ± 8.078

96.375 ± 8.910

99.269 ± 9.372

30.952±8.663

47.388±8.331

73.904±5.960

85.545±5.246

92.650±6.059

95.264±6.627

96.636±6.916

19.452±8.434

34.610±8.871

60.061±6.754

75.523±5.560

86.343±6.176

90.633±6.991

92.958±7.882

2.100±0.768

8.881± 2.122

15.150±4.643

27.239±6.882

46.249±7.949

59.549±7.290

68.887±6.685

91.367±9.895

94.635±11.166

1.517± 1.070

10.908±3.008

11.230±6.844

20.698±10.347

36.588±13.510

48.505±12.915

57.285±11.931

80.113±17.092

83.639±19.719

1.774±0.861

10.08±2.401

12.500±5.357

24.785±8.382

40.844±9.819

53.325±9.207

62.281±8.434

84.667±12.417

88.014±14.033

Table 5.4: Comparison of the infusion profiles of luteolin from the A. afra leaves

in tea bag (test) against that from the loose leaves (reference).

Form of luteolin f1 (%) f2 (%)

Free

Conjugated

Total

65.51

78.20

73.52

18.41

9.48

13.85

Dissolution or infusion f1 values between 0 and 15 and f2 values between 50 and 100 are

regarded by the FDA as indicating similarity between profiles (FDA, 1997). From the

values obtained in this study, i.e. f1 more than 15 and f2 less than 50, it was determined

that the infusion profiles of the two preparations were not similar. This lack of similarity

therefore meant that the tea bag dosage form did not have comparable release profiles to

the loose leaves and therefore could not be used as a replacement for the loose leaves in

clinical trials. However, although the infusion profiles were not similar, the tea bag

dosage form could still be used in clinical trials if adjustments in the dose preparation and

administration methods were done. For example, the final desired luteolin concentration

in the tea could be achieved by allowing the tea bag to seep and draw 3 times longer than

the loose leaves would be allowed, or alternatively, adjustments in the amount of tea

consumed could be done, e.g. the patient could consume 10 ml of the tea bag tea solution

equivalent to consuming 1 ml of the loose leaves tea in order to achieve the desired doses.

The above suggestions therefore meant that in this case, the infusion profiles needed not

be similar in order for the tea bags to be used in place of the loose leaves in clinical trials.

By observation of the infusion profiles of the loose leaves and the FDAE powder in tea

bag, it was determined that the profiles were also not similar based on the fact that the

infusion of the extract tea bag was complete within 15 minutes, while that of the leaves

required 240 minutes and moreover, due to the very different time points, it was not

possible to calculate f1 and f2 values. However, the tea bag containing the FDAE powder,

released a total luteolin content corresponding to 52.84% of the luteolin present in the tea

bag within 15 minutes compared to the loose leaves and leaves in tea bag which only

reached this level after approximately 30 minutes and 180 minutes of infusion,

respectively. As a result of this rapid release, this dosage form could be a suitable

replacement for the traditional dosage form for use in clinical trials even though the

infusion profile is not similar to that of the loose leaves.

In summary, the results obtained showed that the infusion profiles for the tea bags

containing the leaves and FDAE powder, were not similar to that of the loose leaves.

However, they could still be used in clinical trials if adjustments in the preparation and

administration methods were done.

5.7.7 Results of the stability studies conducted on the dosage forms

5.7.7.1 Organoleptic and chromatographic evaluation: Conditions at site A

The results obtained in the organoleptic evaluation of the tea bags subjected to the storage

conditions at site A (room temperature and normal humidity) are presented in Appendix

D1. The A. afra dried leaves in tea bag showed no changes in the appearance, odour, or

leakage of the tea bag contents over the 6-month storage period. Figure 5.10 shows

images of the tea bags at the beginning and at the end of the stability study at this site.

At the end of the stability study, the moisture content of the SDL in tea bag was 11.45 ±

1.02% from an initial average percentage of 10.13 ± 1.51% (see Table 5.5 below) and this

showed that there were no significant increases in moisture in the leaves (p value 0.4145

at alpha < 0.05) and it could therefore be concluded that the SDL in tea bag do not absorb

significant amounts of moisture under these test conditions

The HPLC chromatographic fingerprint analysis collected at 30-day periods, over the

stability test period (see Appendix D2 for the overlaid chromatograms and UV spectra)

also showed no changes occurring within the phytochemical constituents of the plant

leaves as represented by the similar peaks. This lack of change and therefore similar

identity of the plant materials was further validated by means of the UV spectra which

were conducted at the peak with the maximum absorbance, which was at 220 nm and at

an average retention time of 3.52 minutes. From this, it was observed that no changes in

the UV spectral plots occurred indicating the similarity of the peaks throughout the

stability period.

Tea bags of the FDAE powder stored under the conditions at site A, however, showed

significant changes in the organoleptic properties after 7 days of storage, i.e. the extract

became a dark brown, sticky paste, which leached out of the tea bag (see Figure 5.11),

however, no changes in the odour of the material were observed. Changes were also

observed in the chromatographic fingerprints, which showed that almost all the peaks

initially present were no longer present after the 7-day period (see Appendix D2).

In summary, the results showed that the tea bags containing the SDL were stable under

normal room temperature and humidity conditions, but that the tea bags of the FDAE

were not stable under these conditions.

Table 5.5: Loss on drying of the A. afra dried leaves after 180 days stability

testing at site A.

Sample No Initial mass of

leaves (g)

Final mass of

leaves (g)

Loss on drying

(g)

Percentage

moisture content

0.5052

0.5021

0.5013

0.4466

0.4402

0.4494

0.0586

0.0619

0.0519

11.60

12.38

10.35

Ave 0.5029 0.4454 0.0575

11.45

S. D 0.0021 0.0047 0.0051 1.023

5.7.7.2 Organoleptic and chromatographic evaluation: Conditions at site B

The tea bags of the dried leaves subjected to accelerated storage testing conditions of

40ºC /75% relative humidity for a period of 3 months, were found to show no noticeable

changes in the physiochemical properties up to a period of 30 days. However, after 30

Figure 5.10: A tea bag of A. afra dried leaves at day 0 (on the left) and on the right, a tea bag of A. afra

leaves at day 180, stored under conditions of normal room temperature and humidity.

Figure 5.11: A tea bag containing the A. afra FDAE powder at day 0 (on the left) and on the right, a tea

bag containing the FDAE powder at day 28, stored under conditions of normal room

temperature and humidity.

days, a green colouration on tea bag surface was noticed, and this colouration increased

in intensity up to the end of the stability study. Figure 5.12 shows images of the tea bags

at day zero and at the end of the stability study at this condition.

At the end of the storage period, the moisture content of the dried leaves from the stored

tea bags had changed significantly from an initial loss on drying of 10.13 ± 1.51%, to

16.60 ± 1.01% after 90 days (p value 0.010 at alpha < 0.05) (see Table 5.6 below). The

preparation thus absorbed significant amounts of moisture under the storage conditions

and this may have been responsible for the green colouration observed on the tea bag

paper as the high humidity wetted the leaves within the tea bag. Despite this change, the

HPLC chromatographic fingerprint analysis and UV scan at 220 nm, showed no

differences in the peaks, indicating that the tea bag leaf constituents did not change in

composition over the test period.

Table 5.6: Loss on drying of the A. afra dried leaves after 90 days stability testing

at site B.

Sample No Initial mass of

leaves (g)

Final mass of

leaves (g)

Loss on drying

(g)

Percentage

moisture content

0.5019

0.4995

0.5028

0.4140

0.4157

0.4248

0.0879

0.0838

0.0780

17.51

16.78

15.51

Ave 0.5014 0.4182 0.0832

16.60

S. D 0.0017 0.0058 0.0050 1.012

Figure 5.12: Shows, on the left, a tea bag containing the A. afra leaves at da

0 and on the ri

ht, a tea

bag containing the A. afra leaves after 90 days storage at conditions of 40ºC /75% relative

humidity. Note the green colouration on the tea bag surface after 90 days.

Nevertheless, the above results showed that the tea bags containing the SDL were not

stable under the high temperature and humidity storage condition as shown by the

greenish colouration on the tea bag surface.

5.7.7.3 Organoleptic and chromatographic evaluation: Conditions at site C and D

Tea bags containing the freeze-dried extract powder were also evaluated under conditions

of room temperature or 40ºC and zero humidity, i.e. at site C and D, respectively. The

results of the organoleptic evaluation of the tea bags stored under these conditions are

presented in Appendix D1. No changes in organoleptic properties were observed from

day 0 to day 28 at both storage conditions, however, after 28 days the powders were no

longer free flowing and became hard solid masses within the tea bags, with those under

the higher temperature condition harder than those at the lower temperature condition (as

the former tended to crumble easily). Figure 5.13 shows images of the tea bags at the

beginning and end of the storage under the two storage conditions.

The chromatographic fingerprint analysis of samples of the extract powder from the tea

bags under the lower temperature condition, showed no changes over the entire test

period (see Appendix D2). However, the fingerprint analysis of the samples from the tea

bags at the higher temperature condition, showed a change after 60 days, i.e. peaks,

which had previously been observed at 8 minutes and 23 minutes, were no longer present

(see Appendix D2).

Figure 5.13: A tea bag containing the A. afra FDAE at da

0 (on the left), in the middle, a tea ba

containin

the FDAE at da

90 stored under conditions at site C and on the ri

ht, a tea ba

containing the FDAE powder after 90 days storage at the conditions at site D.

From the results, it was clear that the temperature affected the stability of the FDAE

powder in tea bag and from the data obtained the most suitable storage conditions for the

tea bags containing the FDAE powder would appear to be at zero humidity and normal

room temperature.

5.7.7.4 Infusion profile characteristics of the dried leaves tea bags stored

under the conditions at site A and B.

Determinations of the percentage of plant material infused at a single time point for the

preparations subjected to the real time and accelerated stability testing was conducted and

the results obtained are presented below. Table 5.7 shows the percentage of the

phytochemical constituents released after 180 minutes of infusion (determined from the

UV spectroscopic analysis) of the tea bags containing the A. afra SDL stored at the

storage sites A and B.

Table: 5.7 Percentage released by the tea bags of the A. afra dried leaves at 180

minutes from tea bags stored at storage sites A and B.

Percentage released by the A. afra dried leaves tea bag at 180 minutes ± S. D

Storage condition site

Day 0 Day 90

Day 180

53.7929 ± 5.5370%

53.8849 ± 5.3210%

23.3427 ± 3.6160%

57.6955± 6.520%

From the comparison of the infusion of the tea bags under the conditions of normal room

temperature and humidity, no significant differences in the percentage of phytochemical

constituents infused at the 180 minute time point, for the duration of the stability study,

was found (p value 0.9733 at alpha < 0.05). This suggested that the preparation remained

stable and within specification under this storage condition.

However, there was a significant difference in the mean percentage material infused from

the tea bags containing the SDL after 90 days of storage under the accelerated storage

conditions (p value 0.0001 at alpha < 0.05). Initially, a mean value of 53.80% was

released within 180 minutes and after 90 days the mean value at this time point was

found to be 23.34%, which represented a reduction of approximately 50%. This reduction

in the rate and extent of the infusion suggested that this condition of high temperature and

humidity, adversely affected the stability of the preparation over the time period

evaluated. Taken together with the changes in organoleptic properties observed, i.e. that

the leaves became wet (increased moisture content) and stuck together, it could be

possible that the storage conditions created a more tortuous passage for the leaf

constituents to infuse through (through sticking together of the leaves), as well as made it

difficult for the media to wet the leaves and this could explain the observed reduction in

the rate of infusion.

Collectively, the results obtained from the stability evaluations (i.e. the infusion profile

analysis, chromatographic fingerprint analysis, loss on drying and organoleptic tests),

suggested that only normal room temperature and humidity were suitable storage

conditions for the tea bags containing the dried leaves of A. afra.

5.7.7.5 Infusion profile characteristics of the freeze-dried aqueous extract

powder in tea bag stored under conditions at site C and D.

Determinations of the amount of luteolin infused after 5 minutes from the tea bags stored

under conditions of zero humidity and normal room temperature or 40ºC for 3 months,

i.e. at site C and D, respectively are presented in Table 5.8.

Table: 5.8 Luteolin content (µg/ml) infused after 5 minutes infusion of the tea

bags containing the freeze-dried aqueous extract powder stored under

conditions at site C and D.

Luteolin content (µg/ml) infused

after 5 minutes from the tea bags

stored under conditions at site C

Luteolin content (µg/ml) infused after 5

minutes from the tea bags stored under

conditions at site D

Form of luteolin

Day 0

Day 90

Day 0

Day 90

Free

Conjugated

Total

2.3520 ± 0.4387

2.4990 ± 0.4661

4.8510 ± 0.9048

2.8690 ± 0.2640

0.0225 ± 0.0061

2.8915 ± 0.9048

2.3520±0.4387

2.4990±0.4661

4.8510±0.9048

1.0250 ± 0.0567

0.0827 ± 0.1113

1.1040 ± 0.0603

For the tea bags containing the FDAE powder subjected to storage under conditions of

zero humidity and room temperature (site C), resulted in a significantly less (p value

0.0065 at alpha < 0.05) amount of luteolin released after 5 minutes of infusion. Initially,

4.8510 µg/ml of total luteolin was released and after 90 days storage, 2.8915 µg/ml was

released representing an almost 50 % reduction. This difference may be attributed to the

organoleptic observation that the FDAE became hard and crusty mass and this may have

reduced the rate of dissolution by making it more difficult for the extract to dissolve in

the solution. Furthermore, there was a significant decrease in the conjugated luteolin

content observed (p value 0.0001 at alpha < 0.05) from the preparations stored at this site

and from this it may be concluded that the FDAE in tea bags are not stable when stored

under these conditions as a result of the decrease in the conjugated luteolin and the

reduction in the rate of infusion.

The infusion of the tea bag preparations at under zero humidity and 40˚C (site D) also

showed a significant decrease in the amount of luteolin infused after 5 minutes (p value

0.0006 at alpha < 0.05) and a reduction in the rate of infusion of approximately 4 times

(4.8510 vs. 1.1040 µg/ml released after 5 minutes). Organoleptic evaluations at this site

(section 5.7.7.3) revealed that the extract powder solidified and this could have been

responsible for the observed reduction in the luteolin infused. Changes in conjugated

luteolin content were also observed and the changes were similar to those that occurred

under stability site C and therefore it could be concluded that the FDAE powder in tea

bag undergoes a reduction in the conjugated luteolin content when stored.

Collectively, the results obtained from the stability evaluations (i.e. the infusion profile

analysis, chromatographic fingerprint analysis, loss on drying and organoleptic tests), on

the FDAE powder in tea bag, suggested that the preparations were not stable under

conditions of normal room temperature, 40ºC and zero humidity as a reduction in the rate

of infusion and in the conjugated luteolin and as a result, these tea bags cannot be used in

clinical trials as a replacement for the traditional loose leaves preparation.

5.7.8 Comparison of tea bags of the A. afra plant material and their placebos

The organoleptic properties of the placebos for the A. afra SDL and FDAE powder in tea

bag were compared against the plant material tea bags and Tables 5.9 and 5.10 give a

summary of the characteristics of the tea bags and placebos prepared.

The addition of the aroma chemical and sweetener to the placebo and A. afra plant

materials resulted in preparations with similar aroma and taste. Also the colour of the teas

produced by both the placebos and plant preparations were found to be similar (see

Figure 5.15c). From an observation of the images (Figure 5.14 and 5.15a and b) it can be

observed that there were no apparent differences between the tea bag containing the plant

leaves and that containing the placebo for the leaves. On close inspection of the tea bags

containing the placebo and the FDAE powder, it could be observed that the placebo

preparation was slightly more reddish in colour compared to the tea bag containing the

FDAE powder. However, the teas resulting from the powder preparations were of similar

intensity, (crucial in the clinical trials where teas will be prepared) indicating that the

FDAE placebo could still function as a credible placebo.

In summary, the placebo preparations were judged to match the A. afra plant materials

(spiked with inert aroma and sweetener chemicals) and as such, the placebo preparations

could be used in clinical trials evaluating the plant materials.

Table 5.9: Physicochemical comparison of tea bags of the A.afra leaves and

placebo for the leaves

Parameter A.afra leaves in tea bag Placebo for A.afra leaves in tea bag

Description

Taste/flavour

Odour

Hue produced by tea

7x7cm square grey tea bag containing

material with rough texture.

Sweet, citrus

Citrus, herbal woody, rosewood.

Reddish brown clear hue

7x7cm square grey tea bag containing

material with rough texture.

Sweet, citrus

Citrus, herbal woody, rosewood.

Reddish brown clear hue

Table 5.10: Physicochemical comparison of tea bags containing the A.afra freeze-

dried aqueous extract and extract placebo.

Parameter A. afra FDAE in tea bag Placebo of the A. afra FDAE in tea bag

Description

Taste/flavour

Odour

Hue of tea produced

7x7cm square grey tea bag containing

a brittle, free flowing, brown,

particulate material.

Sweet, citrus

Citrus, herbal woody, rosewood.

Reddish brown clear hue

7x7cm square grey tea bag containing a

brittle, free flowing, reddish, particulate

material.

Sweet, citrus

Citrus, herbal woody, rosewood.

Reddish brown clear hue

Figure 5.14: Shows the A. afra standardized dried leaves in tea bag on the left and the A. afra dried

leaves placebo in tea bag on the right.

5.7.9 Results of test for absence of activity of the placebo for the A. afra dried

leaves.

The results of the pharmacological evaluation of the A. afra placebo and plant leaves are

shown in tabular and graphic form in Table 5.11 and Figure 5.16, respectively.

Table 5.11: Percentage relaxation produced by cumulative concentrations of the

A. afra standardized dried leaves and placebo of the leaves on the

methacholine-induced contractions of the isolated guinea pig tracheal

muscle.

Conc.

(mg/ml)

Cumulative

conc. in bath

(mg/ml)

Average %

Relaxation ± SD

EC50 Max. relaxation ±

PLANT 5

100

0.0333

0.7000

0.8333

1.1666

1.8333

3.3710 ± 0.8720

3.7780 ± 0.3449

49.7760 ± 2.5100

236.18 49.776 ± 2.5100

Conc.

(mg/ml)

Cumulative

conc. in bath

(mg/ml)

Average %

Relaxation ± SD

EC50 Max. relaxation ±

PLACEBO 5

100

0.0333

0.7000

0.8333

1.1666

1.8333

0.660 ± 1.143

1.9610 ± 1.698

3.9550 ± 0.9622

- 3.955 ± 0.9622

Figure 5.15: (A) Shows the FDAE of A. afra in tea bag and (B) shows the placebo of the FDAE of A. afra

in tea bag. (C) Shows the colours of the teas produced by A. afra placebo (on the left) and A.

ra freeze-dried extract

owder

(

on the ri

)

0.0 0.5 1.0 1.5 2.0 2.5

A.afra leaves

Placebo leaves

log conc

% relaxation

Figure 5.16: Percentage relaxation versus log concentration of the A.afra standardized dried

leaves (-●-) and placebo (-○-) effect on the isolated guinea pig tracheal muscle.

Cumulative concentration of the A. afra plant material (up to 185 mg/ml or 1.833 mg/ml

in the organ bath) injected onto the guinea pig trachea produced a maximal relaxation of

49.776 ± 2.51%, relative to the isoprenaline maximal relaxation, while the same

cumulative doses of the placebo material produced a maximum relaxation of only 3.955 ±

0.962%. Generally, the traditional teas would be prepared as 4 g of leaves in 200 ml of

hot water, which is equivalent to a 20 mg/ml solution and this concentration

corresponded to 0.133 mg/ml in the organ bath. At this concentration the A. afra leaves

were found to produce an average percentage relaxation of 0.804%, while the placebo

had an average percentage relaxation of only 0.068%. Furthermore, depending on the

volume of the tea the patient ingests, increased concentrations of the tea solution may be

absorbed. For example, if a patient consumed 15 ml or a tablespoon of the tea, this would

correspond to a concentration of approximately 1.833 mg/ml in the organ bath (assuming

100% absorption), which would result in a percentage relaxation of 46.206% and 3.921%

from the plant material and placebo, respectively. The above results therefore showed

that the plant material was active at the concentrations used traditionally, while the

placebo was only slightly active at the same concentrations.

With the experimental design, it was difficult to conclude whether the slight relaxation

seen with the placebo was due to the natural relaxation of the tissue or due to the actual

effect of the placebo. Also, it was not possible to determine whether this slight effect

would have any observable therapeutic effect in humans and therefore further studies of

the effect of this preparation in humans are required. The relaxation seen from the A. afra

dried leaves was however apparent and validated the traditional use of A. afra leaves in

the treatment of asthma and other bronchial conditions requiring bronchodilation.

5.8 Conclusions

Collectively, as for the results obtained in the preparation and evaluation of tea bags and

placebos of Artemisia afra, the following conclusions could be drawn;

• The manufactured tea bags containing 4 g and 0.55 g of A. afra SDL and FDAE

powder, respectively, were equivalent to the traditional dose and met the

European Pharmacopoeia 2002 quality requirements.

• Luteolin is a suitable marker compound to use to characterise the infusion of the

A. afra dosage forms.

• The method used to quantitate the luteolin content of the A. afra leaves does not

give a correct estimation of the true luteolin content present in the material.

• The rate of infusion of luteolin from the manufactured tea bags is three times

slower than that from the loose leaves.

• The tea bags containing the SDL and those containing the FDAE powder do not

have comparable release profiles of luteolin to the loose leaves. However, they

could still be used in clinical trials if adjustments in the tea preparation and

administration methods are done.

• Tea bags containing the SDL are stable under conditions of normal room

temperature and normal room humidity. However, tea bags containing the FDAE

powder are not stable and as such this dosage form is not suitable for the FDAE

powder.

• It is possible to design credible placebos for the A. afra SDL and FDAE powders.

However, the placebo for the SDL possesses slight muscle relaxant activity.

In summary, the results suggested, firstly, that the tea bag was a suitable dosage form for

the A. afra standardized dried leaves, but not for the freeze-dried aqueous extract powder

and secondly, that the tea bags containing the standardized dried leaves do not have

100

comparable release profiles to the loose leaves traditional preparation and finally, that it

is possible to prepare credible placebos for the plant material, which possess little

pharmacological activity compared to the plant material.

101

Chapter 6

Conclusion

The primary objectives of the study were; to prepare standardized A. afra plant leaves and

freeze-dried aqueous extract powder, to prepare these materials in a tea bag dosage form

and compare the infusion profiles of the tea bag and the traditional loose leaves dosage

forms and to design credible placebos for the A. afra leaves and freeze-dried aqueous

extract powder in tea bags, suitable for use in clinical trials.

It was hypothesised that; the variation in the luteolin levels in standardized plant leaves

and freeze-dried aqueous extract powder would be lower than that in the traditionally

used non-standardized leaves, that a tea bag would be a suitable dosage form replacement

for the traditional plant leaves and would meet pharmacopoieal quality requirements, that

the infusion profiles of luteolin from the tea bag dosage form and traditional loose leaves

will be similar and, finally, that credible placebos for the A. afra plant materials, devoid

of pharmacological activity could be designed and prepared.

From the results obtained, the following conclusions could be drawn;

• Standardization of the A. afra leaves reduces the intra-batch variation in luteolin

content present and this variation can be further reduced by preparation of a

freeze-dried aqueous extract powder of the plant material.

• The tea bag is a suitable dosage form for standardized dried A. afra leaves, but not

for the freeze-dried aqueous extract powder of the plant.

• The infusion profile of luteolin from tea bags containing standardized dried A.

afra leaves is not similar to that of the loose leaves, however, the tea bags can still

be used in clinical trials provided appropriate adjustments in the dose preparation

and administration methods are made.

• Credible placebos for A. afra plant material tea bags which appear, smell and taste

similar to the plant materials and possess little pharmacological activity and thus

suitable for use in clinical trials were made.

102

In summary, A. afra plant materials with reduced intra-batch variation in luteolin content

were prepared and manufactured in a tea bag dosage form, of which only the standardized

dried leaves were stable upon storage. The release profiles of luteolin from the tea bag

were not similar to that from the loose leaves, however, the tea bags could still be used in

clinical trials if adjustments in the dose preparation and administration of the tea bag tea

are made. Finally, credible placebos of the plant dosage form were prepared and can be

used in clinical trials.

From the results, several areas for further research were noted. Firstly, there is a need to

further evaluate the effect of the geographical source and period of harvest of the plant

materials and different storage conditions, on the intra- and inter-batch luteolin content.

Secondly, the effect of various leaf sizes, tea bag sizes and types of tea bag paper on the

rate of infusion and infusion profiles of the plant tea bag dosage form requires further

investigation. Finally, characterization of the phytochemical constituents still present in

the placebo materials and further evidence of their pharmacological inertness is required.

Notwithstanding the aforementioned further studies, the tea bags of the A afra

standardized dried leaves and placebo materials can still be used in clinical trials with

human subjects.

103

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116

APPENDICES

APPENDIX A1

Artemisia afra collected from the Montagu district of the Western Cape, was analysed to

determine the proportion of the plant that constituted the leaves, flowers and

stems/branches.

Table showing results of the quantitative determination of percentage of leaves,

flowers and stems which constitute Artemisia afra plant.

Table showing the average loss on drying of the A. afra leaves after drying in an

oven for 3 days at 30˚C.

Batch 001 (%) Batch 002 (%) Batch 003 (%) % Average ± S.D

Leaves

Flowers

Stems/branches

43.47

30.14

23.90

48.54

29.58

25.67

41.57

32.16

24.29

44.53 ± 3.60

30.63 ± 1.36

24.62 ± 0.93

Batch 001 Batch 002 Batch 003

Initial mass (g)

Final mass (g)

Loss on drying (g)

% Loss on drying

352.51

202.20

150.31

42.64

2787.11

1305.35

1481.76

53.16

6128.20

2613.00

3515.20

57.36

% Average ± S.D 51.05 ± 7.58

117

APPENDIX A2

Figure showing the certificate of irradiation for the Artemisia afra dried leaves and

freeze-dried aqueous extract powder.

118

APPENDIX A3

Table of the extractions conducted and resultant yields obtained in the

preparation of the freeze-dried aqueous extracts of Artemisia afra.

Sample

Volume of aqueous

extract freeze dried

(ml)

Mass of freeze

dried extract

obtained (g)

Yield of freeze-

dried aqueous

extract (%)

300

350

380

350

200

450

380

300

2.20

2.76

2.70

2.30

1.60

3.20

2.70

2.60

2.30

2.25

21.28

22.87

20.61

19.10

23.05

20.62

24.87

25.13

21.52

20.58

Average ± SD 21.96 ± 1.97

APPENDIX A4

Particle size classification of the A. afra plant materials

Table of the results of the particle size classification of the A. afra dried leaves.

Sieve Number Av. Weight

passing through

(g)

Av. % w/w

passing through

Particle size

classification (BP

2000)

1400

355

180

125

9.80

2.00

0.37

0.23

0.17

96.41

19.71

3.64

2.32

1.66

Coarse powder

119

Table of the results of the particle size classification of the A. afra freeze-dried

aqueous extract.

Sieve Number Av. Weight

passing through

(g)

Av. % w/w

passing through

Particle size

classification (BP

2000)

1400

355

180

125

9.63

5.13

2.83

1.93

0.93

95.73

50.99

28.14

19.20

9.28

Coarse powder

APPENDIX A5

Method and results obtained for the determination of bitterness value of Artemisia

afra dried leaves and freeze-dried extract powder (EP 2002a).

1. Determination on A. afra standardized dried leaves stock solution 0.01 g/ml

• Correction factor using Quinine Hydrochloride 0.01 mg/ml (Bitterness value 200

000)

Q1(0.042mg) Q2 (0.044mg) Q3 (0.046mg) k (n/5.00)

Taster 1 B B 0.72

Taster 2 B B 0.72

Taster 3 B B 0.72

Taster 4 B B 0.72

Taster 5 B B 0.72

• The threshold bitter concentration for tasting quinine is at 0.042mg. The number

of ml of stock solution in this concentration was 3.6. Therefore the correction

factor k, (which is calculated as this number of ml divided by 5 was 0.72 for all

tasters)

C4 (DF 100 000) C3A (DF 50 000) C3 (DF 10 000)

Taster 1 B

Taster 2 B

Taster 3 B

Taster 4 B

Taster 5 B

• The solution with the threshold bitter concentration for the tasters is solution is

C4, with dilution factor 100 000 (Y).

• Upon tasting 1.2 ml (X) of C4 + 8.8 ml of water, the solution was found to be

bitter by all tasters.

120

• Bitterness value for tasters;

(Y x k) / (X x 0.1)

= 100 000 x 0.72 = 600 000.

1.2 x 0.1

Where Y is the dilution factor for the threshold bitter concentration.

X is the volume of bitter concentration judged to be bitter after addition of water.

k is the correction factor for each taster.

2. Determination of A.afra freeze-dried extract stock solution 0.01g/ml

C4 (DF 100 000) C3A (DF 50 000) C3 (DF 10 000)

Taster 1 B

Taster 2 B

Taster 3 B

Taster 4 B

Taster 5 B

The threshold bitter concentration is C4.

• After tasting 1.2 ml of C4 + 8.8 ml of water, the solution was found to be bitter by

all tasters.

• Bitterness value for all tasters = (Y x k) / (X x 0.1)

= 600 000

121

APPENDIX A6

Results of the ash value determinations on the A. afra plant materials

Table showing the data and results of the total ash and acid-insoluble ash value

determinations for the Artemisia afra dried leaves.

Sample

Mass of

the dried

leaves

powder (g)

Mass of

carbon free

ash (g)

Percentage

of Total ash

Mass of Acid-

insoluble ash (g)

Percentage of

Acid-insoluble

ash

2.0006

2.0017

2.0003

2.0009

2.0021

2.0022

2.0001

2.0036

2.0006

2.0001

0.1986

0.1991

0.1994

0.2014

0.1986

0.2020

0.1983

0.2009

0.1998

0.2000

9.9270

9.9465

9.9551

10.065

9.9196

10.088

9.9145

10.0270

9.9870

9.9995

0.0145

0.0192

0.0194

0.0213

0.0156

0.0221

0.0178

0.0191

0.0183

0.0190

0.7248

0.9592

0.9685

1.0645

0.7792

1.1038

0.8899

0.9533

0.9147

0.9499

Ave 2.001 0.1998

9.9831 0.0186 0.9308

SD 0.0115 0.0127

0.0617 0.0230 0.0012

Table showing the data and results of the total ash and water-soluble ash value

determinations for the Artemisia afra dried leaves.

Sample

Mass of

the dried

leaves

powder (g)

Mass of

carbon free

ash (g)

Percentage

of Total ash

Mass of Water-

soluble ash (g)

Percentage of

Water-soluble

ash

2.0005

2.0050

2.0035

2.0094

2.0015

2.0027

2.0025

2.0001

2.0028

2.0045

0.2013

0.2023

0.1977

0.2015

0.2018

0.1988

0.1969

0.1987

0.1986

0.1996

10.039

10.089

9.8677

10.027

10.082

9.9266

9.8327

9.9345

9.9161

9.9576

0.0874

0.0876

0.0941

0.0901

0.0887

0.0850

0.0866

0.0888

0.0876

0.0867

4.3689

4.3691

4.6968

4.4839

4.4317

4.2443

4.3246

4.4338

4.3739

4.3253

Ave 2.003 0.1997

9.9675 0.0886 4.4052

SD 0.0267 0.0188

0.0886 0.0248 0.0012

122

Table showing the data and results of the total ash and acid-insoluble ash value

determinations on the Artemisia afra freeze-dried extract powder.

Sample

Mass of

aqueous

extract (g)

Mass of

carbon free

ash (g)

Percentage

of Total ash

Mass of Acid-

insoluble ash (g)

Percentage of

Acid-insoluble

ash

2.0044

2.0013

2.0016

2.0008

2.0006

2.0011

2.0012

2.0014

2.0001

2.0009

0.4350

0.4357

0.4406

0.4469

0.4366

0.4461

0.4514

0.4231

0.4198

0.42571

21.7023

21.7708

22.0124

22.3361

21.8235

22.2927

22.5565

21.4020

20.9889

21.2759

0.0002

0.0014

0.0037

0.0034

0.0007

0.00998

0.06995

0.1849

0.1699

0.0349

Ave 2.001 0.4361

21.820 0.0019 0.0094

SD 0.0116 0.0106

0.0886 0.0016 0.0793

Table showing the data and results of the total ash and water-soluble ash value

determinations on the Artemisia afra freeze dried extract powder.

Sample

Mass of

aqueous

extract (g)

Mass of

carbon free

ash (g)

Percentage

of Total ash

Mass of Water-

soluble ash (g)

Percentage of

Water-soluble

ash

2.0044

2.0013

2.0016

2.0008

2.0006

2.0011

2.0012

2.0014

2.0001

2.0009

0.4350

0.4357

0.4406

0.4469

0.4366

0.4461

0.4514

0.4231

0.4198

0.42571

21.7023

21.7708

22.0124

22.3361

21.8235

22.2927

22.5565

21.4020

20.98895

21.2759

0.108

0.0987

0.0970

0.0947

0.1074

16.8957

17.6244

16.2936

16.2542

15.9083

Ave 2.001 0.4361

21.820 0.1012 16.5952

SD 0.01159 0.01062

0.08862 0.0061 0.6760

123

APPENDIX A7

The microbiological testing methods used and the results obtained in the

determination of the microbial contamination on the A. afra standardized dried

leaves and freeze-dried aqueous extract powders before and after irradiation of the

plant materials.

124

Figure showing the results of microbial contamination tests on the A. afra

standardized dried leaves and freeze-dried aqueous extract powders before

irradiation of the plant materials.

125

Figure showing the results of microbial contamination tests on the A. afra

standardized dried leaves and freeze-dried aqueous extract powders after

irradiation of the plant materials.

126

APPENDIX B

The data and results of the determinations of the luteolin content and variation in

the plant material batches.

Table showing the data and results of the determinations of the free luteolin content

and variation in the plant material batches.

Concentration of free luteolin in plant batches (µg/mg)

Sampling

Position

Plant Batch

001

Plant Batch

002

Plant Batch

003

Plant Batch

Standardized

leaves

Freeze

dried

aqueous

extract

1.0339

1.4071

1.0976

0.7313

0.8765

0.7808

0.9882

1.0052

0.8175

0.8784

0.5680

0.6269

0.7040

0.9145

1.0557

0.9407

1.3524

1.6180

0.8533

0.7854

0.9970

0.7267

0.9428

0.8719

6.5789

7.2001

7.7892

7.1435

7.2922

6.5220

Average 0.9879 0.8141 1.0980 0.8628 7.0880

S.D 0.2494 0.1825 0.3318 0.0991 0.4751

% R.S.D 25.25 22.42 30.23 11.48 6.70

127

Table showing the data and results of the determinations of the conjugated/

glycosidic luteolin content and variation in the plant material batches.

Table showing the data and results of the determinations of the total luteolin content

and variation in the plant material batches.

Concentration of conjugated luteolin in plant batches (µg/mg)

Sampling

Position

Plant Batch

001

Plant Batch

002

Plant Batch

003

Plant Batch

Standardized

leaves

Freeze

dried

aqueous

extract

0.1393

0.6781

0.3666

0.5316

0.8052

0.8372

0.8649

1.4333

1.3835

0.8975

1.0698

0.2483

0.7714

0.8168

0.2745

0.9909

0.4736

0.4977

0.8585

1.1186

1.3119

1.2706

1.3291

1.3265

8.3880

5.2185

7.2968

6.4871

7.4305

5.8680

Average 0.5597 0.9829 0.6375 1.203 6.781

S.D 0.2706 0.4317 0.2658 0.1862 1.152

% R.S.D 48.35 43.92 41.69 15.49 16.98

Concentration of total luteolin in plant batches (µg/mg)

Sampling

Position

Plant Batch

001

Plant Batch

002

Plant Batch

003

Plant Batch

Standardized

leaves

Freeze

dried

aqueous

extract

1.1732

2.0852

1.4642

1.2629

1.6817

1.6180

1.8531

2.4385

2.2010

1.7759

1.6378

0.8752

1.4754

1.7313

1.3302

1.9316

1.8260

2.1157

1.7118

1.9040

2.3089

1.9973

2.2719

2.1984

14.9669

12.4186

15.0860

13.6306

14.7227

12.3900

Average 1.548 1.7967 1.735 2.065 13.87

S.D 0.3287 0.5391 0.2909 0.2347 1.246

% R.S.D 21.24 30.00 16.77 11.36 8.98

128

APPENDIX C

The specifications for the Dynapore 117/7/0 tea bag paper used for manufacture of

the A. afra plant material tea bags.

Parameter Specification

Grammage

Thickness

Apparent density

Tensile force MD

Tensile force CD

Wet tensile force CD

Sand fall out

Air permeability

Heat-seal CD

16.75g/m2

64.40pm

0.260g/cm3

12.90N/15mm

3.80N/15mm

1.28N/15mm

8.67%

1.561 Akustron (1/ms2)

1.34N/15mm

129

APPENDIX D1

The results of the organoleptic evaluation conducted on the tea bags of the A. afra

leaves subjected to different storage conditions.

The results of the organoleptic evaluation conducted on the tea bags of the A. afra

leaves subjected to stability testing at sites A and B. Changes occurring are

highlighted in italics.

Organoleptic characteristic Condition

& Sample

Days Appearance Odour Leakage

Site A

120

180

Grey square tea bag with

material of rough texture.

Grey square tea bag with

material of rough texture

Grey square tea bag with

material of rough texture

Grey square tea bag with

material of rough texture

Grey square tea bag with

material of rough texture

Grey square tea bag with

material of rough texture

Odour characteristic

of A. afra

Odour characteristic

of A. afra

Odour characteristic

of A. afra

Odour characteristic

of A. afra

Odour characteristic

of A. afra

Odour characteristic

of A. afra

No leakage

Site B

Grey square tea bag with

material of rough texture.

Grey square tea bag with

material of soft texture.

Green/grey square tea bag

with material of soft texture.

Green/grey square tea

bag with material of soft

texture.

Odour characteristic

of A. afra

Odour characteristic

of A. afra

Odour characteristic

of A. afra

Odour characteristic

of A. afra

No leakage

Green

colouration on

tea bag paper

Green colouration

on tea bag paper

130

Table showing the results of the organoleptic evaluation conducted on the tea bags

of the freeze-dried aqueous extract subjected to stability testing at sites at sites A, C

and D. Changes occurring are highlighted in italics.

Parameter Site &

Sample

Days Appearance Odour Leakage

Site A

Grey square tea bag with dark,

free flowing powder material.

Dark brown stained tea bag with

sticky material inside.

Odour characteristic of

A. afra

Odour characteristic of

A. afra

No leakage

Leakage of

contents out of the

tea bag

Site C

Grey square tea bag with dark,

free flowing powder material.

Grey square tea bag with dark,

free flowing powder material.

Grey square tea bag with dark,

free flowing powder material.

Grey square tea bag with dark,

hard crusted powder.

Grey square tea bag with dark,

hard crusted powder.

Grey square tea bag with dark,

hard crusted powder.

Grey square tea bag with dark,

hard crusted powder.

Odour characteristic of

A. afra

Odour characteristic of

A. afra

Faint odour of A. afra

No leakage

Site D

Grey square tea bag with dark,

free flowing powder material.

Grey square tea bag with dark,

free flowing powder material.

Grey square tea bag with dark,

hard crusted powder.

Grey square tea bag with dark,

hard crusted powder.

Grey square tea bag with dark,

hard crusted powder.

Grey square tea bag with dark,

hard crusted powder.

Grey square tea bag with dark,

hard crusted powder.

Odour characteristic of

A. afra

Faint odour of A. afra

No leakage

131

APPENDIX D2

Chromatographic fingerprint and UV spectral overlays of the samples from the A.

afra plant materials in tea bag after storage at the various conditions.

Figure showing the chromatographic overlays of the A. afra dried leaves (in tea bag)

over a 180 day stability testing period at site A.

Figure showing the UV spectra overlay of A. afra dried leaves at day 0 and day 180

at site A. (λmax 220nm, retention time 3.52 minutes).

Figure showing the chromatographic overlay of A. afra freeze-dried aqueous extract

in tea bag subjected to stability testing at site A.

Minutes

02 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

100

200

300

400

500

100

200

300

400

500

Det 168-220 nm

RT Leaves day 0 Det 168-220 nm

rt leaves day 30 Det 168-220 nm

rt leaves day 60 Det 168-220 nm

rt leaves day 90 Det 168-220 nm

rt leaves day 180

200 250 300 350 400 450 500 550 600

mAU

200

400

600

3.52 Min

RT A. afra leaves day 0 Leaves Rt Day 180.spc - 3. 52 Min

132

Figure showing the UV spectra overlay of A. afra freeze-dried aqueous extract in tea

bag at the beginning and end of stability testing at site A. (λmax 220nm, retention

time 3.52 minutes).

Figure showing the chromatographic overlays of the A. afra dried leaves (in tea

bag) subjected to stability testing at site B.

Figure showing the UV spectra overlay of the A. afra dried leaves in tea bag

subjected to stability testing at site B. (λmax 220nm, retention time 3.52 minutes).

Minutes

02 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

100

200

300

400

500

600

700

100

200

300

400

500

600

700

Det 168-349nm

Aq Ex Day 0.dat Det 168-220 nm

aq ex rt d 7

200 250 300 350 400 450 500 550 600

mAU

200

400

600

3.52 Min

RDAY0EXTRACT RT Day 28 extract.spc - 3.52 Min

Minutes

02 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

-50

100

150

200

250

300

350

400

mAU

-50

100

150

200

250

300

350

400

Det 168-220 nm

Leaves Accelarated Day 0 Det 168-220 nm

leaves accelarated d ay 30 Det 168-220 n m

leaves accelarated d ay 60 Det 168-220 n m

leaves accelarated d ay 90

133

Figure showing the chromatographic overlays of the A. afra dried leaves (in tea

bag) subjected to stability testing at site C.

Figure showing the UV spectra overlay of the A. afra dried leaves in tea bag

subjected to stability testing at site C. (λmax 220nm, retention time 3.52 minutes).

Figure showing the chromatographic overlays of the A. afra freeze-dried aqueous

extract (in tea bag) subjected to stability testing at site D.

Minutes

02 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

250

500

750

1000

1250

1500

1750

2000

mAU

250

500

750

1000

1250

1500

1750

2000

Det 168-220 nm

Aq Ex Day 0.dat Det 168-220 nm

aq ex real ti me day 14 D et 168-220 nm

aq ex real ti me day 28 D et 168-220 nm

aq ex real ti me day 42 D et 168-220 nm

aq extract day 60

134

Figure showing the UV spectra overlay of the A. afra dried leaves in tea bag

subjected to stability testing at site D. (λmax 220nm, retention time 3.52 minutes).

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mAU

200

400

600

800

1000

1200

1400

mAU

200

400

600

800

1000

1200

1400

Det 168-220 nm

Aq Ex Acc Day 0 Det 168-220 nm

aq ex acc day 14 Det 168-220 nm

aq ex acc day 28 Det 168-220 nm

aq ex acc day 42 Det 168-220 nm

aq ex acc day 60 Det 168-220 nm

aq extract acc day 74 Det 168-220 nm

aq ex acc day 90

135

APPENDIX E

The pen recordings (data) from the pharmacological evaluation of the placebo

material using the isolated guinea pig trachea model. Cumulative concentrations of

the placebo and the A. afra plant materials were injected onto the perfused trachea.

The 100% tissue relaxation was obtained using 0.1ml of 1x10-2M isoprenaline. The

‘p’ and ‘l’, markings on the charts represent the injected concentrations of the

placebo and A. afra leaves solutions respectively.

Figure showing pen chart recordings from recorder A.

Figure showing pen chart recordings from recorder B.

136

Vitae

The author was born in Harare, Zimbabwe on the 1st of March in 1980. His primary and

high school education was done in Harare and in July 2003 he obtained a Bachelor of

Pharmacy (Hons) degree from the University of Zimbabwe. After completing his

pharmacy internship and working briefly in the area of medicines regulation in

Zimbabwe, the author enrolled for a M. Pharm. degree at the University of the Western

Cape, South Africa, in February 2005.

Artemisia afra

Chapter

Full-text available

Jan 2023

Artemisia afra Jacq. ex Willd. (Asteraceae), referred to as African wormwood, is an aromatic, perennial shrub that can grow up to 2 m in height. The finely divided leaves have a silver-green colour due to the presence of fine hairs. The plant is found in the mountainous regions of Africa, as far north as Ethiopia. In southern Africa, it occurs in Namibia, Zimbabwe, Swaziland (Eswatini), Lesotho and South Africa, where it is widespread and abundant. The plant is mainly used in the form of decoctions, infusions, poultices, or as an inhalant, to treat a range of ailments including coughs, colds, headaches, chills, colic, asthma, malaria, diabetes, influenza, convulsions, fever and rheumatism. In vitro and in vivo studies of the plant have indicated that it has promising antimicrobial, anti-oxidant, anti-inflammatory, antidiabetic and antimycobacterial activities. The essential oil composition has been reported to be highly variable, and several (artemisia ketone, 1,8-cineole, α-thujone, β-thujone, camphor and myrcenone) chemotypes have been described. Chemical profiles of A. afra essential oils, obtained by hydro-distillation were obtained using semi-automated high-performance thin-layer chromatography (HPTLC) and gas chromatography coupled to mass spectroscopy (GC–MS). The HPTLC plate, viewed under white light after derivatisation with anisaldehyde, confirms variation in the composition of the oils. The use of UPLC–MS for non-volatile qualitative analysis enabled the identification of scopolin, rutin, scopoletin, chrysoeriol, 3,5-di-O-caffeoylquinic acid and acacetin in the acetone:methanol extracts. The use of HPTLC indicated considerable variation in the phenolic compound composition of samples from different populations.

An integrated approach to enable learners to reach their full potential within the Wellness Sector DOCTOR OF NATURAL MEDICINE And COUNSELLING At STAFFORD UNIVERSITY

Thesis

Full-text available

May 2024

DECLARATION By submitting this dissertation electronically, I hereby declare that this work in its entirety is my own original work and personal experience for which I have carried the total cost. As the sole author of this dissertation, the publication and/or reproduction thereof or part thereof, will infringe on my intellectual property unless written consent of such action has been given. 3 ACKNOWLEDGEMENTS The researcher would like to sincerely thank my Mentor and Promotor Professor M.D. Herholdt for his many years of guidance, encouragement and counselling which has ultimately transformed experience, knowledge, and passion into this manuscript. The researcher would also like thank Pierre my husband, companion and for continuous support and council-much of this work should also be attributed to him. 4 CONTENTS

Formulation of a herbal tea bag with potential in vitro thrombolytic activity using Adhatoda vasica Linn (Pawatta), Vitex negundo Linn (Nika), and Caesalpinia bonduc Linn (Kumburu)

Article

Full-text available

Dec 2022

Introduction: Adhatoda vasica Linn, Vitex negundo Linn, and Caesalpinia bonduc Linn are medicinal plants belongs to Acanthaceae, Verbenaceae, and Caesalpiniaceae families respectively. This study aimed to develop a herbal tea bag from a combination of the three medicinal plants and assess its thrombolytic effect in vitro using the clot lysis assay. Methods: Phytochemical profiles of the selected medicinal plants were determined. Aqueous extract (AE) of leaves of each plant was added (100 μL) at concentrations ranging from (125-500 mg/mL) into microcentrifuge tubes containing pre-weighed blood clots. After 90 minutes of incubation at 37 ºC, the supernatants containing disintegrated blood clots were discarded. The tubes were weighed again, and the percentage of clot lysis was determined. Streptokinase was utilized as the positive control, while distilled water as the negative control. The thrombolytic effect of several plant combinations was investigated and the most effective combination was formulated into a tea bag. Results: Alkaloids, tannins, saponins, flavonoids, diterpenoids, cardiac glycosides, phenolic compounds, proteins, amino acids, and carbohydrates were detected. AE of C. bonduc, V. negundo and A. vasica leaves showed maximum thrombolytic activity of 33.32% (p= 0.001), 28.16% (p=0.007) at the concentration of 500mg/ mL, and 22.02 %, at the concentration of 125mg/ mL respectively (p= 0.031). Streptokinase, the most effective combination (1:4:4) and the tea bag demonstrated 88.50% (p=0.000), 31.01% (p=0.003) and 13.35% (p=0.04) of clot-dissolving activity respectively. Conclusion: The presence of modest thrombolytic activity in the AE of A.vasica, V.negundo, and C.bonduc leaves, both individually and in combination, was demonstrated in this study. Stability testing and further development of the herbal tea bag are recommended in future studies.

Formulation of a herbal tea bag with potential in vitro thrombolytic activity using Adhatoda vasica Linn (Pawatta), Vitex negundo Linn (Nika), and Caesalpinia bonduc Linn (Kumburu)

Article

Dec 2022

Introduction: Adhatoda vasica Linn, Vitex negundo Linn, and Caesalpinia bonduc Linn are medicinal plants belongs to Acanthaceae, Verbenaceae, and Caesalpiniaceae families respectively. This study aimed to develop a herbal tea bag from a combination of the three medicinal plants and assess its thrombolytic effect in vitro using the clot lysis assay. Methods: Phytochemical profiles of the selected medicinal plants were determined. Aqueous extract (AE) of leaves of each plant was added (100 µL) at concentrations ranging from (125-500 mg/mL) into microcentrifuge tubes containing pre-weighed blood clots. After 90 minutes of incubation at 37 ºC, the supernatants containing disintegrated blood clots were discarded. The tubes were weighed again, and the percentage of clot lysis was determined. Streptokinase was utilized as the positive control, while distilled water as the negative control. The thrombolytic effect of several plant combinations was investigated and the most effective combination was formulated into a tea bag. Results: Alkaloids, tannins, saponins, flavonoids, diterpenoids, cardiac glycosides, phenolic compounds, proteins, amino acids, and carbohydrates were detected. AE of C. bonduc, V. negundo and A. vasica leaves showed maximum thrombolytic activity of 33.32% (p= 0.001), 28.16% (p=0.007) at the concentration of 500mg/ mL, and 22.02 %, at the concentration of 125mg/ mL respectively (p= 0.031). Streptokinase, the most effective combination (1:4:4) and the tea bag demonstrated 88.50% (p=0.000), 31.01% (p=0.003) and 13.35% (p=0.04) of clot-dissolving activity respectively. Conclusion: The presence of modest thrombolytic activity in the AE of A.vasica, V.negundo, and C.bonduc leaves, both individually and in combination, was demonstrated in this study. Stability testing and further development of the herbal tea bag are recommended in future studies. Keywords: Clot lysis, Herbal tea bag, Thrombolytic activity, Adhatoda vasica Linn, Vitex negundo Linn, Caesalpinia bonduc Linn

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Importance and prior considerations for development and utilization of tea bags: A critical review

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J FOOD PROCESS ENG

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Article

Full-text available

Apr 2009
S AFR J BOT

The genus Artemisia consists of about 500 species, occurring throughout the world. Some very important drug leads have been discovered from this genus, notably artemisinin, the well known anti-malarial drug isolated from the Chinese herb Artemisia annua. The genus is also known for its aromatic nature and hence research has been focussed on the chemical compositions of the volatile secondary metabolites obtained from various Artemisia species. In the southern African region, A. afra is one of the most popular and commonly used herbal medicines. It is used to treat various ailments ranging from coughs and colds to malaria and diabetes. Although it is one of the most popular local herbal medicines, only limited scientific research, mainly focussing on the volatile secondary metabolites content, has been conducted on this species. The aim of this review was therefore to collect all available scientific literature published on A. afra and combine it into this paper. In this review, a general overview will be given on the morphology, taxonomy and geographical distribution of A. afra. The major focus will however be on the secondary metabolites, mainly the volatile secondary metabolites, which have been identified from this species. In addition all of the reported biological activities of the extracts derived from this species have been included as well as the literature on the pharmacology and toxicology. We aim at bringing together most of the available scientific research conducted on this species, which is currently scattered across various publications, into this review paper.

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There are over 500 species of the genus Artemisia in the Asteraceae family distributed over the globe, with varying potentials to treat different ailments. Following the isolation of artemisinin (a potent anti-malarial compound with a sesquiterpene backbone) from Artemisia annua, the phytochemical composition of this species has been of interest over recent decades. Additionally, the number of phytochemical investigations of other species, including those of Artemisia afra in a search for new molecules with pharmacological potentials, has increased in recent years. This has led to the isolation of several compounds from both species, including a majority of monoterpenes, sesquiterpenes, and polyphenols with varying pharmacological activities. This review aims to discuss the most important compounds present in both plant species with anti-malarial properties, anti-inflammatory potentials, and immunomodulating properties, with an emphasis on their pharmacokinetics and pharmacodynamics properties. Additionally, the toxicity of both plants and their anti-malaria properties, including those of other species in the genus Artemisia, is discussed. As such, data were collected via a thorough literature search in web databases, such as ResearchGate, ScienceDirect, Google scholar, PubMed, Phytochemical and Ethnobotanical databases, up to 2022. A distinction was made between compounds involved in a direct anti-plasmodial activity and those expressing anti-inflammatory and immunomodulating activities or anti-fever properties. For pharmacokinetics activities, a distinction was made between compounds influencing bioavailability (CYP effect or P-Glycoprotein effect) and those affecting the stability of pharmacodynamic active components.

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Article

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Julie Laplante

This study highlights current global health biopolitics in conjunction with politics of indigeneity. The illustration is achieved by looking at the politics of knowledge at play in the pre-clinical trial of a traditional medicine in Cape Town, South Africa. The instance reunites healers and scientists in a common project to test the efficacy of a local wild shrub through the process of a randomised controlled trial, the current golden scientific standard to determine truth about the efficacy of medicines. How traditional forms of knowledge translate, or not, into this process is the first point I develop with relation to the trial. Inevitable reassessments of efficacy awakened in this process open a debate between representational learning of standardized scientific knowledge, and learning through embodied forms of knowledge in negotiations of how best to heal with medicines. I argue that the dynamics of these politics of knowledge within the trial lead on the one hand towards an optimal management of life at the molecular level, and on the other hand towards broader relational politics of life anchored in sounds and embodied forms of knowledge.

Compositae in Natal

Article

Jan 1978

Selection, preparation and pharmacological evaluation of plant materials

Article

Jan 1996

Some Potentially Important Indigenous Aromatic Plants for the Eastern Seaboard Areas of Southern Africa

Chapter

Jan 1982

The Ciskei Essential Oils Project (CENTOIL project) was established in 1974 in an effort to discover, investigate and develop labour intensive crops which could contribute meaningfully to the development of the agricultural resources and the uplift of the tribal communities of rural Ciskei.

Biopharmaceutical characterisation of herbal medicinal products

Article

Jan 2001

Plant extracts can be classified according to the knowledge on their composition and efficacy into 3 categories (A, BI and B2). Herbal medicinal products containing extracts of categories A or BI should comply with the Note for Guidance on the Investigation of Bioavailability and Bioequivalence. In order to justify waiver from bioequaivalence the following characteristics should be considered. a) Characteristics related to the active substance Risk of therapeutic failure or adverse reactions Risk of bioinequivalence Solubility (highest dosage strength in 250 ml of each of three pharmacopoeial buffers (preferably at pH 1.0, 4.6, 6.8) Pharmacokinetic properties b) Characteristics related to the medicinal product Rapid dissolution (85 % are dissolved within 15 minutes) Excipients manufacture HMPs containing extracts of category B2 need not comply with the above Note for Guidance. In case of high solubility of the extracts or equivalent solubility behaviour bioavailability, bioequivalence or clinical studies may be waived. The Note for Guidance may be applied in the case of extreme differences in solubility of the extracts.

The isolated perfused lung

Chapter

Jan 1998

S. Uhlig

The lung has developed dedicated structures and cell types to accomplish its particular functions. An elaborate exchange of both information and matter takes place between the lung and other organs of the body, which in total is a system of utmost complexity. In order to be able to study this perplexing system at least some of the complexity has to be eliminated. In this sense the isolated perfused lung represents a system much less complicated than the whole animal while preserving most of the integrity of the organ. As an experimental approach the isolated perfused lung (IPL) stands in between experiments in vivo with whole animals and in vitro with cultured cells. As such it combines assets and shortcomings of both. The present chapter will describe a set-up for the isolated per-fused rat lung that allows to study four important aspects of lung physiology: respiratory mechanics, vessel mechanics, gas exchange and edema formation. Here, we will focus on the perfused rat lung, however, most of the equipment can also be used for isolated lungs from rabbits [1] or guinea pigs [2]. In a number of aspects the present text supplements our previous papers on the method of the rat IPL [3, 4]. A good compilation of earlier papers on perfused lungs is given in the introduction of the paper by Kröll et al. [2]. Previous reviews on the method of the isolated lung can be found in [5–9]. In addition, more specialized systems such as single lung perfusion [10] or perfusion through the bronchial artery [11] have also been described.

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A lot of factors such as temperature, light exposure, water availability, nutritients, period and site of collection, age and part of plant collected, method of collection, processing (drying, chemical/radioactive treatment,...), storage and distribution of starting herbal material, etc., can greatly affect the quality and consequently the therapeutic value of herbal medicines. Therefore, it is essential to produce starting material under Good Agriculture and Collection Practice (GACP) conditions. Because of plant's genetic and chemical content variations, the site and date should be recorded for each collection. The production of starting materials by cultivation would normally lead to more consistent herbal products due to greater genetic uniformity. Furthermore, cultivation permits monitoring of active constituents and defining of the best period for harvesting. Whether field collected from the wild or produced by cultivation, botanical authentication of plant species and identification by their binomial Latin names are necessary to insure that the correct herbal material is obtained. Also, care must be taken to free the targeted starting material of undesirable plant parts, soil, insects, animal excreta and other contaminants. During post-collection treatments microbial, agrochemical residues and toxic metals contamination should be reduced to a minimum. The harvested starting material must be processed to produce the finished herbal product under Good Manufacture Practices (GMPs) which are similar to those employed for the manufacture of conventional drugs. Frequent variability in the composition of herbally based medicines can be avoided by proper standardization of herbal drugs/preparations and their quality control using pharmacopoeial and/or validated testing methods. In the cases where the active principles are unknown, marker substance(s) should be established for analytical purposes. Independently on traditional use of HMP, all data concerning the quality »from the seeds to the final product« should be provided in the application dossier for marketing authorisation. Post-marketing surveillance by regulatory agencies is further pivotal step in the permanent quality assurance of HMP.

Zulu medicine and medicine-men

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