1. 1
Chemical and Pharmacological Studies on Khat
(Catha edulis, Celastraceae)
Thesis submitted by Muna Ismail
2010
A thesis submitted to King‟s College London for the degree of
Doctor of Philosophy in Pharmacy
Department of Pharmacy,
School of Biomedical Sciences,
Franklin Wilkin‟s Building,
King‟s College London.
2. 2
Certificate
This is to verify that the research work embodied in this thesis entitled ‘Chemical
and Pharmacological Studies on Khat (Catha edulis, Celastraceae)’ has been
carried out by me under supervision and guidance of Prof Peter Houghton and
Dr Sarah Salvage.
Muna Ismail
3. 3
Abstract
The aim of this present project is to review the phytochemistry of the cathedulin
alkaloids in Catha edulis and study their dopaminometic effect in vitro in comparison
with cathamine alkaloids (-)-cathinone and (-)-norephedrine using aqueous and
methanolic extracts of Herari and Mira types.
Catha edulis extracts from different geographical areas; i.e. the Herari and Mira, have
been known to differ in the levels of their constituent (-)-cathinone and (-)-
norephedrine. Hence the objective was to explore if the cathedulin alkaloids also have
variations within the different types of the plant and whether there were differential
effects based on the alkaloid compositions between the two types of Catha edulis, the
cathedulin alkaloids extract fractions and the pure cathamines alkaloids [(-)-cathinone
and (-)-norephedrine)] on:
1. The release of 3
H-DA from rat striatal slices.
2. D1 and D2 receptor displacement of 3
H-SCH 23390 and 3
H-
Spiperone.
In the phytochemical profiles of the Catha edulis extracts analysed by LC-ESI-MS, it
was shown that, in terms of the number of the cathedulin pyridine alkaloids there was
no significant differences the Herari and the Mira Catha edulis except in the ratios of
relative abundance between the various cathedulin compounds. There were 62
alkaloids identified in this group by this study, including the fifteen previously known
cathedulin compounds. In the DA release study both the total extracts of the two types
of Catha edulis increased the release of 3
H-DA from rat striatal slices. The pattern of
release by the total extracts suggested that of the two Catha edulis types, the Mira
extracts exhibits a higher DA releasing effect than the Herari extracts. The studies
investigating the displacement of binding to D1 and D2 receptors suggested that
components of both the aqueous and methanolic total extracts were more potent in
their ability to bind to D2 dopamine receptor compared to the D1 receptor. Also (-)-
cathinone was observed to contribute most of the effect as it potently displaced both
3
H-SCH 23390 and 3
H-spiperone binding to D1 and D2 receptors respectively.
4. 4
Acknowledgements
My appreciation and gratitude goes to my supervisors, Professor Peter Houghton and
Dr Sarah Salvage for their supervision during the course of my research and the
writing up of the thesis. I am indebted by their kindness and understanding of the
challenges that I faced. Their guidance throughout my PhD course was invaluable.
They have shown unparalleled patience and dedication to the progression of my work.
The pharmacology sections of this thesis could not have been done without the
excellent mentoring supervision and the expertise of Dr Salvage. I would like to
express my deepest thanks to her for the continuous support and encouragement she
gave me from the initiation of the project to its conclusion. She is a great mentor and I
am fortunate to have been her student. My deepest thanks also go to Dr Geoff Kite of
the Royal Botanic Gardens, Kew, for his continuous help and expertise in the LC/MS
work of Catha edulis extracts. I am grateful for the support of Professor Monique
Simmonds of the Royal Botanic Gardens, Kew who allowed me the opportunity to do
the LC/MS extract analyses at the Jodrell laboratories. My gratitude are also extended
to Dr. Dennis Donovan and his colleagues at the Alcohol and Drug Abuse Institute
(ADAI) in the University of Washington, Seattle, for the IT and library support they
provided for the writing of my thesis. I would also like to acknowledge Dr. Richard
Smith (ex-Director of the charity Mind in Tower Hamlets) for the initial
encouragement to begin the Catha edulis research and the study leave he negotiated on
my behalf; Dr. Eleni Plazidou (Consultant Psychiatrist at East London Foundation
NHS University Trust) for her input on the clinical mental health issues with Catha
edulis abuse; Ms. Val Harding for securing funding from the Home Office for part of
this research project. Most importantly, my love and gratitude goes to my parents
(Ismail A Ismail and Amina A Duale) for their devotion and wise advice without
which I could not have completed this research and my brother Ibrahim Ismail. I am
also grateful for the support of my cousin, Ali Mahdi and my dearest friends; Hawa
Elmi and Yasin Id during the writing up stage.
Dedication: I dedicate this work. to my old friends, researchers and students of the
Chemistry Faculty in the previous National University of Somalia (Mogadishu) who
either lost their lives or were disabled as a result of the Somali civil war. In particular
to the memory of Professor Abuckar Dhalow, who introduced me to Pharmacognosy
and Natural Product Chemistry.
6. 6
Table of Contents
CHAPTER 1.................................................................................................. 19
General Introduction .......................................................................
1.1 Overview of Catha edulis .......................................................... 20
1.2 History of Catha edulis use ........................................................ 27
1.3 Catha edulis in the UK ............................................................... 29
1.4 Botany of Catha edulis ............................................................... 33
1.5 Chemistry of Catha edulis .......................................................... 36
1.5.1 The Alkaloids originally isolated................................................ 36
1.5.2 Phenylalkylamine alkaloids ........................................................ 37
1.5.3 Cathedulin alkaloids.................................................................... 44
1.5.4 Constituents other than alkaloids................................................ 46
1.6 Experimental Pharmacology and Pharmacodynamics of Catha
edulis........................................................................................... 51
1.6.1 Pharmacology of cathinone and related phenylalkylamines ...... 51
1.6.2 Experimental models of addiction .............................................. 56
1.6.3 Pharmacokinectic effects of cathamines in Catha edulis ........... 57
1.7 Dependency issues...................................................................... 58
1.8 Catha edulis use and psychiatric co-morbidity........................... 61
1.9 Rationale for this research .......................................................... 65
1.9.1 The link between chemical composition and activity in different
varieties of Catha edulis ............................................................. 66
1.10 Hypothesis underlying this thesis ............................................... 68
CHAPTER 2.................................................................................................. 69
Phytochemical Investigation of Herari and Mira Catha edulis
2.1 Introduction................................................................................. 70
2.2 Hypothesis................................................................................... 72
2.3 Aims of the study reported in this chapter.................................. 72
2.4 Materials and Methods................................................................ 73
2.4.1. Methods of extraction ................................................................. 74
7. 7
2.4.1.1 Maceration .................................................................................. 74
2.4.1.2 Soxhlet extraction ....................................................................... 75
2.4.1.3 Extractions for alkaloids ............................................................. 76
2.4.2. Developing a method for removal of tannins ............................. 78
2.4.3 Methods of Analysis ................................................................... 79
2.4.3.1 Thin Layer Chromatography (TLC) ........................................... 79
2.4.3.2 High Performance Liquid Chromatography (HPLC) ................. 81
2.4.3.3 Flash Chromatography parallel C18 Columns ............................. 82
2.4.3.4 Liquid Chromatography Electospray Ionization Mass
Spectrometry (LC-ESI-MS)........................................................ 84
2.5 Results......................................................................................... 89
2.5.1 Thin Layer Chromatography preliminary Analysis.................... 89
2.5.2 HPLC quantification of (-)-cathinone and cathedulin K11 in
extracts of samples of Herari and Mira...................................... 91
2.5.3 Isolation of cathedulin fractions by flash chromatography ........ 94
2.5.4 LC-ESI/MS/MS analysis of total Herari and Mira extracts....... 97
2.5.4.1 Analyses of extracts obtained by maceration ........................... 103
2.5.4.2 Analysis of extracts from the soxhlet extraction ...................... 110
2.5.4.3 Analysis of fractions by alkaloid extraction method ................ 113
2.5.4.4 Analysis of extracts treated to remove the tannins ................... 113
2.5.4.5 Analysis of cathedulin contents in fresh extracts after 6 months ...
................................................................................................... 117
2.6 Discussion................................................................................. 123
2.6.1 MS/MS analysis for structural assignments of cathedulin alkaloids
................................................................................................... 123
2.6.1.1 High Molecular Mass Cathedulins ........................................... 123
2.6.1.2 High Molecular Mass Cathedulins with open C-bridge ........... 124
2.6.1.3 Medium Molecular Mass Cathedulins with only an E-bridge.. 134
2.6.1.4 Medium Molecular Mass Cathedulins with only C-bridge ............
(open or closed)......................................................................... 138
2.6.1.5 Cathedulin 44 (M=1118 Da)..................................................... 142
2.6.1.6 Lower molecular weight cathedulins........................................ 142
8. 8
2.6.2 Summary of findings................................................................. 146
2.7 Conclusion ................................................................................ 147
CHAPTER 3................................................................................................ 153
The effect of Catha edulis, cathamines alkaloid and cathedulin
alkaloid fractions on 3
H-dopamine release from rat striatal slices .
3.1 Introduction............................................................................... 154
3.2 Hypothesis................................................................................. 155
3.3 Aims of the study reported in this chapter................................ 156
3.4 Materials and Methods.............................................................. 157
3.4.1 Sample material ........................................................................ 157
3.4.2 Experimental Animals .............................................................. 158
3.4.3 Tissue superfusion .................................................................... 158
3.4.4 Preparation of tissue slices........................................................ 158
3.4.5 Loading of 3
H-DA radio-labeled neurotransmitter................... 159
3.4.6 Study of Release of 3
H-DA....................................................... 160
3.4.7 The effect of drug treatment on 3
H-DA release from rat striatal
slices.......................................................................................... 160
3.4.7.1 Investigation of the effect of fresh aqueous total extract of Catha
edulis on 3
HDA release from rat striatal slices......................... 161
3.4.7.2 Comparison of the effects of freeze dried and fresh total extracts
of Catha edulis on 3
H-DA release from rat striatal slices......... 161
3.4.7.3 Investigating the effects of freeze dried aqueous and methanolic
extracts of Catha edulis on 3
H-DA release from rat striatal slices
................................................................................................... 161
3.4.7.4 The effect of (-) cathinone and (-) norephedrine on DA release162
3.4.7.5 The effect of cathedulin extract fractions on 3
H-DA release from
rat striatal slices......................................................................... 162
3.4.8 Data Analysis............................................................................ 162
3.4.9 Statistical analysis..................................................................... 162
3.5 Results....................................................................................... 164
9. 9
3.5.1 The effect of fresh extracts of Mira and Herari on 3
H-DA release
................................................................................................... 164
3.5.2 Comparison between the effects of fresh and freeze-dried extracts
................................................................................................... 164
3.5.3 Comparison effect between the freeze dried Herari and Mira
aqueous and methanolic extracts .............................................. 169
3.5.4 Comparison of total 3
H-DA release between aqueous and
methanolic extracts of Mira and Herari ................................... 169
3.5.5 Comparison of the total 3
H-DA release between the two types of
Mira and Herari extracts .......................................................... 169
3.5.6 The effects of (-) cathinone and (-) norephedrine on 3
H-DA
release ....................................................................................... 171
3.5.7 Comparison between the effects of the cathedulin methanolic and
aqueous fractions on 3
H-DA release......................................... 174
3.6 Discussion................................................................................. 176
3.6.1 The effects of extracts of Herari and Mira Catha edulis on 3
H- DA
release ....................................................................................... 176
3.6.2 The effect of (-)-cathinone and (-)-norephedrine on 3
H-DA release
................................................................................................... 180
3.6.3 The effects of cathedulin sesquiterpene pyridine alkaloids on 3
H-
DA release................................................................................. 181
3.7 Conclusion ................................................................................ 182
CHAPTER 4................................................................................................ 183
Binding of Catha edulis extracts and its alkaloids: cathinone,
norephedrine and. the cathedulin pyridine alkaloids to D1 and D2
dopamine receptorsin rat striatum
4.1 Introduction............................................................................... 184
4.2 Hypothesis................................................................................. 185
4.3 Aims of the study reported in this chapter................................ 186
4.4 Material and Methods ............................................................... 187
4.4.1 Dopamine receptors .................................................................. 187
10. 10
4.4.2 D1 receptor family.................................................................... 188
4.4.3 D2 receptor family.................................................................... 188
4.4.4 Theory of in vitro ligand binding studies.................................. 190
4.4.4.1 3
H-SCH23390 binding to D1 receptors .................................... 190
4.4.4.2 3
H-Spiperone binding to D2 receptors...................................... 191
4.4.5 Radioligand Binding studies..................................................... 192
4.4.5.1 Dissection and preparation of brain tissue................................ 192
4.4.5.2 Saturation binding studies of 3
H-SCH23390 and 3
H-Spiperone193
4.4.5.3 Analyses of pure alkaloid and extract displacement of 3
H-
SCH23390 binding.................................................................... 193
4.4.5.4 Analyses of pure alkaloid and extract displacement of 3
H-
Spiperone binding ..................................................................... 194
4.4.6 Binding analysis........................................................................ 194
4.4.7 Data Analysis for displacement binding................................... 195
4.5 Results....................................................................................... 198
4.5.1 Displacement of 3
H-SCH23390 by aqueous and methanolic Catha
edulis extracts............................................................................ 198
4.5.2 Displacement of 3
H-Spiperone by freeze dried aqueous and
methanolic Catha edulis extracts.............................................. 200
4.5.3 Displacement of 3
H-SCH23390 by (-)-cathinone and (-)-
norephedrine ............................................................................. 202
4.5.4 Displacement of 3
H-spiperone by (-)-cathinone and (-)-
norephedrine ............................................................................. 202
4.5.5 Displacement of 3
H-SCH23390 by cathedulin alkaloids fractions
................................................................................................... 204
4.5.6 Displacement of 3
H-Spiperone by Cathedulin alkaloids fractions
................................................................................................... 204
4.6 Discussion................................................................................. 207
4.6.1 The ability of extracts of Catha edulis, its cathamines, (-)-
cathinone and (-)-norephedrine, and cathedulin alkaloid fractions
to bind to D1 and D2 dopamine receptors in vitro. .................. 207
11. 11
4.6.2 The differential effect of the two types of Catha edulis, Herari
and Mira, on D1 and D2 receptor binding................................ 208
4.6.3 The contribution of the constituent alkaloids to the binding of two
types of Catha edulis, Herari and Mira, to D1 and D2 receptors
................................................................................................... 208
CHAPTER 5................................................................................................ 213
General Discussion
5.1 Summary of the findings........................................................... 214
5.2 Are the effects seen on DA release and receptor binding
translated in vivo ....................................................................... 217
5.3 The effects of chronic Catha edulis use in man....................... 224
5.4 Critical evaluation of the studies reported in this thesis........... 226
5.5 Contributions made by the findings in this thesis..................... 228
CHAPTER 6................................................................................................ 231
References
Appendix 1 ...........................................................................................................240
Appendix 2 ........................................................................................... 241
12. 12
List of figures
Figure 1-1: Geographical proximity of the three main cultivating countries in
known as the Catha edulis-belt countries............................................ 21
Figure 1-2: Catha edulis shrub (source: bbc.co.uk)............................................... 22
Figure 1-3: A bundle of Herari Catha edulis, the leaves and soft tips are only
chewed................................................................................................. 23
Figure 1-4: ¾ of the length of a typical twig of Mira Catha edulis as normally
chewed................................................................................................. 23
Figure 1-5: Typical size of a bundle of Mira Catha edulis wrapped in banana leaves
............................................................................................................ 25
Figure 1-6: Distribution network of Catha edulis via UK (ACMD, 2005)............ 32
Figure 1-7: Summary of the all the identified alkaloid groups in Catha edulis..... 41
Figure 1-8: Oxidation/reduction of (-) cathinone in leaves and in extracts
(Brenneissen et al., 1985 ; Krizevski et al., 2007) .............................. 43
Figure 1-9: Some of the experimental pharmacology studies on cathinone and
Catha edulis ............................................................................................
52
Figure 1-10: WHO ICD10 six criteria for the classifications of dependency on drugs
............................................................................................................ 60
Figure 2-1: An overview of extraction and analytical used for mapping Catha edulis
alkaloids in total plant extracts. .......................................................... 77
Figure 2-2: Procedure for the alkaloid extraction method using fresh plant material.
............................................................................................................ 78
Figure 2-3: Jones Chromatography FlashMaster Parallel™ columns used for the
collection of cathedulin alkaloid fractions. ........................................ 83
Figure 2-4: Diagrammatical form of Electronspray Ionization (ESI) chamber .........
............................................................................................................ 85
Figure 2-5: LC/MS/MS diagram from injection to detection of ions (Neissen, 1999)
............................................................................................................ 86
Figure 2-6: Catha edulis TLC chromatogram of total freeze-dried and fresh extracts
of Mira and Herari ............................................................................. 90
Figure 2-7: Catha edulis TLC chromatogram of total freeze-dried and fresh extracts
of Mira and Herari with cathedulin K11 and K19 for reference material
Silica gel GF254. DCM-EtOAc-MeOH-NH4OH (10%, 0.1M) in
30:40:20:10 ratios................................................................................ 90
Figure 2-8: HPLC chromatogram of fresh fMM extract analysed by LiChrosorb
column (5µm, length x I.D: 250mmx4.6mm, id.).Isocratic elution of
13. 13
92% DCM 8% MeOH, 0.5% HAC and 0.25% DET. Rt 3.28 minutes is
K11 peak and 11.34 minutes is the (-)-cathinone peak. ...................... 92
Figure 2-9: Concentrations of cathinone in four extracts stored over 14 days shows
approximately 50% reduction in its concentration in all extracts. The
results are presented as mean ± SEM, n=3.......................................... 93
Figure 2-10: Concentrations of cathedulin K11 in four extracts stored over14 days
shows not much reduction in their levels. The results are presented as
mean ± SEM, n=3................................................................................ 93
Figure 2-11: Cathedulin 5A2meth. fraction collected from methanolic Mira extract
............................................................................................................ 95
Figure 2-12: Cathedulin 5A2aq fraction collected from aqueous Mira extract ....... 95
Figure 2-13: Cathedulin 5CA1 leaf-only aqueous fraction collected from Mira extract
............................................................................................................ 96
Figure 2-14: Base peak chromatogram from LC/MS analysis of total methanolic
Mira extract showing 62 cathedulins. . ............................................. 104
Figure 2-15: ESI-MS [M+H]+
peak ion for cathedulin K11 .................................. 105
Figure 2-16: Cathedulin K11 [M+H]+
base ion and its [M+2H]+
........................ 105
Figure 2-17: LC/MS profile of fresh Herari MeOH extract ................................. 106
Figure 2-18: LC/MS Profile of fresh Herari Et2O extract .................................... 106
Figure 2-19: Profile of fresh Herari EtOAc extract.............................................. 107
Figure 2-20: Chromatogram of fdHA extract analysis.......................................... 108
Figure 2-21: Chromatogram of fdHM extract analysis.......................................... 108
Figure 2-22: fdMA Rt 12-27minutes showing cathedulin alkaloid........................ 109
Figure 2-23: Chromatogram of fdMM extract analysis ........................................ 109
Figure 2-24: LC/MS profile of fresh fMA extract ................................................ 111
Figure 2-25: LC/MS profile of fresh fHA extract ................................................. 111
Figure 2-26: Profile of compounds present in Mira following soxhlet extraction with
MeOH to the description (section 2.4.1.2) ........................................ 112
Figure 2-27: Profile of aqueous acid (AA) of Herari extract from the alkaloid
extraction method.............................................................................. 114
Figure 2-28: Profile of the CHCl3 layer (AC) of Herari extract from the alkaloid
extraction method.............................................................................. 115
14. 14
Figure 2-29: Chromatogram of methanolic sample extract that has been eluted
through PVPP stationary column ...................................................... 116
Figure 2-30: Chromatogram of methanolic sample extract that has been eluted
through Sephadex stationary column ................................................ 116
Figure 2-31: Chromatogram of profile of compounds in MeOH extract of Herari that
has been through clean-up with C18 stationary phase........................ 117
Figure 2-32: Chromatogram of fresh aqueous Mira extract................................... 119
Figure 2-33: Chromatogram of 6 months old aqueous Mira extract...................... 119
Figure 2-34: Cathamine and cathedulin calibration curves for quantitativeanalyses
120
Figure 2-35: [M+2H]2+
CID spectrum of high molecular cathedulin K11 40 ....... 125
Figure 2-36: [M+2H]2+
CID spectrum of cathedulin K12 ..................................... 127
Figure 2-37 Theoretical protonation of diacid cathate and evoninate fragments.. 131
Figure 2-38: CID spectrum of [M+2H]2+
cathedulin 12 with one possible structure of
its isomer cathedulin 8....................................................................... 137
Figure 2-39: Possible structure of newly identified cathedulin 31 by MS/MS analysis
of its [M+H]+
ion............................................................................... 140
Figure 2-40 : Possible structure of novel cathedulin 18 structure by MS/MS analysis
of its [M+2H]2+
ion. .......................................................................... 141
Figure 2-41: CID spectrum of [M+H]+.
of cathedulin E2 .............................................
.......................................................................................................... 143
Figure 2-42: Prevoiusly known lower molecular mass cathedulins and possible
structures proposed for the newly identified cathedulins 56 and 61
(isomers) and 57 ............................................................................... 145
Figure 3-1 Sketch of a typical chamber in the superfusion system..................... 159
Figure 3-2: Time course of treatment of striatal slices with extract and alkaloids
derived from Catha edulis or vehicle (Krebs)................................... 160
Figure 3-3 The effect of fMA and fHA on 3
H-DA release from rat striatal slices.
Slices were superfused with (A) fMA (25-100mg/ml) or (B) fHA (25-
100mg/ml). ....................................................................................... 166
Figure 3-4 Comparison between the fMA and fHA effects on 3
H-DA release... 166
Figure 3-5: Comparison of AUC between freeze-dried and fresh aqueous extracts..
.......................................................................................................... 167
15. 15
Figure 3-6 Concentration response curves of the effects by freeze-dried Mira
aqueous (A), methanolic (B), and Herari aqueous (C), methanolic (D)
extracts............................................................................................... 168
Figure 3-7: Comparison of (A) fdMM and fdMA, (B) fdHM and fdHA, (C) fdHA
and fdMA and (D) fdHM and fdMM on 3H-DA release from rat striatal
slices. ................................................................................................. 171
Figure 3-8: Time course response curve for (A) [(-)-cathinone, 0.1-10 ug/ml] and
(B) [(-)-norephedrine, 0.5-20 ug/ml] 172
Figure 3-9: AUC for each concentration were calculated for (C) [(-)-cathinone]
and (D) [(-)-norephedrine] 172
Figure 3-10 Concentration response curves of the three cathedulin alkaloids fraction
fractions (A) = aqueous fraction; (B) = methanolic fraction and (C) =
leaf-only aqueous fraction................................................................. 173
Figure 3-11: Comparison of AUC 3
H-DA release by cathedulin fractions........... 175
Figure 4- 1: Picture of 48-cell Brandel® Harvester used in binding assay analyses.
www.brandel.com.............................................................................. 196
Figure 4-2: Saturation binding for [A] 3
H-SCH23390 (Kd 0.55 and Bmax 127.9
pmole/g) and [B] 3
H-Spiperone (Kd 0.11nM and Bmax 31.3 pmole/g)
binding to striatal membrane fractions that was used for this study. 197
Figure 4- 3: Displacement of 3
H-SCH23390 (0.25nM) binding to rat striatal
membrane (A) fdHA and fdMA and (B) fdHM and fdMM. ......... 199
Figure 4-4: Displacement of 3
H-Spiperone (0.20 nM) binding to rat striatal
membrane [A] fdHA and fdMA and [B] fdHM and fdMM. ......... 201
Figure 4-5: Displacement of [A]3
H-SCH23390 (0.25nM) and [B] 3
H-Spiperone
(0.20nM) binding to rat striatal membrane (-)-cathinone and (-)-
norephedrine .................................................................................... 203
Figure 4-6: Displacement of [A] 3
H-SCH23390 and [B] 3
H-Spiperone binding to rat
striatal membrane fractions by cathedulin alkaloid fractions 5A2meth,
5A2aq and 5CA1 extract. . ................................................................ 205
16. 16
List of Tables
Table 1-1: Some of names of Catha edulis in the three major cultivating
countries…………………………………………………
1
Table 2-1 Voucher record of Catha edulis material in the present
study………………………………………………………… 73
Table 2-2 Reference codes used for the plant material extracts used in
this study…………………………………………………… 75
Table 2-3 Summary of cathedulins found in the three 5A2meth, 5A2
aq. and 5CA1fractions…
96
Table 2-4 Retention times of all the cathedulin alkaloids identified in
Catha edulis by Kite et al., (2003) 98
Table 2-5 Summary of the previously identified cathedulin alkaloids by
Crombie et al., 1980 99
Table 2-6 Quantification in µg/mg (mean ±SEM) of cathamine [(-)-
cathinone and (-)-norephedrine] and cathedulin K11 and K19
in Mira and Herari extracts by LC-ESI-MS.
122
Table 2-7 The m/z values and relative intensities (% base peak) of
diagnostic ions observed in CID [M+2H]2+
of high mass
cathedulins with E-bridge and open or closed C-bridge 132
Table 2-8 The m/z values and relative intensities (% base peak) of
diagnostic ions observed in [M+2H]2+
CID spectra of
cathedulins with an E-bridge and nicotinoyl group. 135
Table 2-9 New medium mass cathedulins detected in Catha edulis by
MS/MS analysis
136
Table 2-10 Summary of acylation patterns proposed for cathedulins
detected by LC/MS analysis of Catha edulis 150
Table 3-1 The effect of freeze drying on the 3
HDA releasing activity of
Mira and Herari extracts of Catha edulis from rat striatal
slices
166
Table 4-1 EC50 values of Catha edulis extracts, cathedulin alkaloid
fractions, (-) cathinone and (-)-norephedrine standards from
D1 and D2 displacement in vitro assay using 3
H-SCH23390
and 3
H-Spiperone
206
Table 4-2 The contribution of the cathamine alkaloids to the
displacement of 3
H-SCH23390 and 3
H-spiperone from rat
striatal slices by freeze dried extract of Catha edulis.
210
Table 5-1: Concentrations of cathinone, cathine and norephedrine in
plasma during random tests of 19 cases
220
17. 17
List of Abbreviations
Ac = acetyl
ACMD = Home Office advisory committee on misuse of drugs
ANOVA = analysis of variance
BBB = blood brain barrier
Bmax = receptor density
CaCl2 = calcium chloride
CID = collision induced dissociation
CHCl3 = chloroform
cm = centimetre
conc. = concentration
CPM = counts per minute
°C = degree Celsius
CNS = Central nervous system
DA = dopamine
DAT = Dopamine transporter
DCM = dichloromethane
DEA = drug enforcement agency in USA
DOPA = 3,4-dihydroxyphenylalanine
DOPAC = -3,4-dihydroxyphenylacetic acid
DPM = disintegration per minutes
EC50 = dose of drug inducing half maximal effect
ESI = electrospray ionization mass spectrometry
EtOAc = ethyl acetate
Et2O = diethyl ether
fmol = femto molar
g = gram
GABA = Gamma amino butyric acid
h = hour
HPLC = High pressure liquid chromatography
HCl = hydrogen chloride
ip = intraperitoneal
Kd = dissociation constant
KCl = Potassium chloride
LC/MS = liquid chromatography mass spectrometry
MgCl2 = magnesium chloride
ml = millilitre
M+
= molecular ion
Me = methyl
MeOH = methanol
18. 18
mg = milligram
min = minute
ml = millilitre
mm = millimetre
mM = millimolar
MS = mass spectrometry
m/z = mass to charge ratio
NA = noradrenalin
NaCl = sodium chloride
Na2CO3 = sodium carbonate
NaHCO3 = sodium hydrogen carbonate
pmol = pica molar
µg = microgram
µl = microlitre
µM = micromolar
nM = nanomolar
Rf = retardation factor
RP C18 = reverse phase octadecylsilane stationary phase
rpm = revolutions per minute
Rt = retention time
SD = standard deviation
SEM = standard error of the mean
SCH23390 = 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-
1H-3-benzazepine
TLC = thin-layer chromatography
VLC = vacuum liquid chromatography
VMAT = vesicular monoamine transporters
v/v = volume by volume
w/v = weight by volume
5HT = serotonin or 5-hydroxytryptamine
6-OHDA = 6-hydroxydopamine
20. 20
1.1 Overview of Catha edulis
Since time immemorial humans have sought the use of drugs for inducing pleasurable
feelings, as well as for altering mood and treating sickness; indeed many drugs that are
abused either produce feelings of well being and euphoria or reduce the aversion to the
perceived environment. Stimulant drugs including cocaine and amphetamine are but
two examples among the variety of drugs within this group, while cannabis and heroin
are popular candidates in the latter classification.
Cocaine, heroin and cannabis are all well known drugs of natural origin that have
crossed continents and transcended different cultures for centuries. Out of the many
old World plants Catha edulis, the general name given to describe Catha edulis species
which is increasingly becoming important. The leaves are chewed for their pleasurable
stimulant effect; has been historically but until recently were fairly unknown outside of
the area of its original habitat.
The Catha edulis plant, Catha edulis (Vahl.) Forssk, ex Endl., is a member of the
Celastraceae, which includes about 350 species of trees and shrubs in 15 genera, and
grows wild, but often cultivated, in the east of a region extending from Yemen in the
Arabian Peninsula to much of eastern and southern Africa. It also grows in Uganda,
Tanzania, Rwanda, and Madagascar. In central Asia the plant largely grows in the wild
and its distribution extends as far as Afghanistan and Turkistan (Krikorian, 1984).
Catha edulis grows at an altitude of 1670-2600 meters adapting to a range of soil and
climatic conditions. Catha edulis tree is hardly ever affected by diseases, takes two
years to be ready for harvest and can live up to 75-100 years (Kennedy et al., 1983).
Figure 1-1 shows the geographical area of the Catha edulis-belt countries from Yemen
across the Red Sea to eastern Africa. In Kenya it grows well on fairly moist slopes of
between 1220-2750 meters on Nyambene Hills in the northeast of Mount Kenya
(Carrier, 2005). In Ethiopia it is farmed in almost every region, although traditionally
cultivation was confined to Harerghe area in eastern Ethiopia (Lamessa, 2001) and on
either semi-humid lowlands or lower highland forests 1400-2000 meters above sea
level.
22. 22
In Yemen it is cultivated all over the country in mountainous and flat lands but not in
coastal area with hot climate (Al-Motarreb et al., 2002).
Although several varieties are cultivated within each of the Catha edulis-belt countries
(Figure 1-1), Catha edulis grown in different cultivation areas with different climate
conditions differs in appearance; in the colour of the leaves and twigs that is either red
or green; in the length of the twig (Geisshusler and Brenneisen, 1987a) and in the size
of the leaves as well as in taste (Al-Motarreb et al., 2002). Furthermore, Catha edulis
(Figure 1-2) is a shrub or small tree that grows to between 1.5 meters and 20 meters
tall, depending on region and rainfall, with evergreen leaves 5–10 cm long and 1–4 cm
broad.
Figure 1-2: Catha edulis shrub (source: www.bbc.co.uk).
23. 23
Figure 1-3: A bundle of Herari Catha edulis, the leaves and soft tips are only
chewed.
Figure 1-4: ¾ of the length of a typical twig of Mira Catha edulis as normally
chewed.
24. 24
Figure 1-3 shows the typical twigs of the Ethiopian Catha edulis, Herari sold as an
article of commerce. From consumer perspective, Catha edulis type from Kenya, the
Mira (Figure 1-4) appears to be generally shorter with slender and softer twigs. The
leaves are also smaller and softer than the Herari type, from Ethiopia, which often has
a bigger twig size and larger, slightly leathery, leaves.
Catha edulis is harvested in the same way regardless of type or growing location.
During harvesting only young shoots (stems and leaves) are picked then bundled
together (200-300 g equivalent to 20-30 stems) and wrapped in green, dry banana
leaves (Figure 1-5). This is done in order to preserve freshness while the plant is en
route to the market destination.
The commonest mode of administration is by chewing the plant through mastication,
with some buccolingual absorption of active material. However, in countries where
Catha edulis is illegal, e.g. USA a dried or a freeze-dried form of the plant known
otherwise as garaabo is used. Garaabo is boiled and the resulting infusion is used as a
tea (DEA, 2007). Flowers are also used amongst Catha edulis users to prepare a tea
with stimulating properties (Krizevski et al., 2007).
Catha edulis chewing involves picking leaves (one by one) from each twig and
chewing them thoroughly, while the juice extracted is swallowed. Altogether, each
person takes some 200 to 400g of the leaves; with young leaves being most favoured,
mainly because they are more potent, but also because they are tenderer to chew. When
chewing, the macerated quid material is pushed back and kept in the cheek as a bolus
that is later spat out. During the session, the group may smoke from water pipes or
smoke cigarettes and there is usually a generous supply of beverages.
The gathering context, place and the time for the actual slow consumption of Catha
edulis are known as a Catha edulis session. Male and female users congregate
separately and the typical session lasts for 5-8 hours. Great effort is often made by
Catha edulis users to create appropriate ambience to aid the quality of the Catha edulis
session: Wearing traditional clothes, burning incense, reciting poetry, listening to folk
music, smoking and
25. 25
Figure 1-5: Typical size of a bundle of Mira Catha edulis wrapped in banana leaves
(http://thisisbristol.co.uk).
drinking hot tea or soft drinks, all belong to the complex ritual of a Catha edulis
chewing setting (Nencini et al., 1986). Another feature of this ritual that is common
involves the shutting of all windows to exclude draughts and increase temperature in
the room where Catha edulis is being chewed, in order to speed up optimum stimulant
effect.
As Catha edulis is used in its natural form without processing, it is almost exclusively
required to be used fresh for an optimum stimulating effect. It is usually not considered
acceptable if harvested more than 4 days before use. For these reasons Catha edulis
consumption in the past was limited to areas close to where it grew, because the leaves
and soft shoots, which are the parts used, lose their potency within short time. When
Catha edulis was not available beyond its geographical cultivation areas, its use was
unknown. However, in recent years the advent of a rapid air transport network and
distribution facilities have made fresh Catha edulis to be available in distant places
such Europe and America (Kalix, 1991).
The regular gathering rooms where Catha edulis chewing sessions take place are
called Muffraj (Arabic), Mafrish1
(Somali) or Bercha in Amharic. These rooms are
1
Mafrish is a room where Khat chewing sessions are held and chewers (most likely) adult
men congregate. It‟s a socializing space as well as a shop for selling Khat to passers-by.
26. 26
often part of either within household complex, or specifically designed buildings for
public Catha edulis sessions, as is normally the case in Yemen. As the price of Catha
edulis in market varies according to its taste (level of tannic acid content) and potency;
the gathering places are also status-orientated according to the social positions of the
users. While chewing involves passive enjoyment for relaxation in parts of Ethiopia,
Somali and Yemen, in other places mostly in Kenya chewing Catha edulis is also
common during the working hours of the day for better productivity by individuals.
Catha edulis is known by a variety of different names in the areas it grows. Within
each of the main cultivation countries i.e. Kenya, Ethiopia and Yemen it has many
local and tribal names attesting the widespread as well as the old knowledge of the
plant by the natives of these countries.
Kenya Mira, Miraa, Miruingi, Giza, Gangetta, laari, Gisa
bum
Ethiopia Jaad, Tchaat, Qat, Tohai, Abyssinian Tea, Kaad,
Herari
South Africa (grows wild) Bushman’s tea
Yemen As much as 40 different type names according to
locality. General name of Qat in Arabic applies.
Other local names include: Ta’izi, Ghorbani,
Harazi, Sawti and Dula’ee.
Table 1-1 Some of the local names of Catha edulis in the three major cultivating
countries.
It is estimated that over 20 million people (Saha and Dollery, 2006) chew Catha edulis
in the world today, while its consumption is not only increasing but also rapidly
spreading (Kalix, 1992) across continents to Europe (Nencini and Ahmed, 1989),
Australia (Jager and Sireling, 1994), USA and Canada. Following the migration in the
last two decades of communities originating from war-torn areas of the Horn of Africa,
Mafrishs in UK are normally small rooms that are packed with 20-30 men all sitting and
reclining against the walls. Tea and cigarettes are also sold in the Mafrish so that chewers
hardly have the need to go outside.
27. 27
Catha edulis has become a widely available commodity in the West. Levels of Catha
edulis use in cultivating countries are said to be comparable. In Somalia a large survey
found 31% of respondents admitting current use, in Ethiopia this was 50%, and in
Yemen 82% of men and 43% of women (ACMD report 2005).
However there are variations in cultural attitudes towards usage, even amongst the two
major Catha edulis consumer societies; the Somalis and the Yemenis, where Catha
edulis use is culturally sanctioned and common practice. Nonetheless use has been
evolving along with the dynamics in societal psychosocial changes. For the Yemenis,
Catha edulis consumption is a deeply rooted tradition. A recent large survey of 2500
people estimated current adult users in Yemen at 61.1% of total population (Ali et al.,
2004). In comparison, for Somalis, habitual Catha edulis use is fairly new and has only
been developed as rapid cultural phenomena, growing out of urban communities in the
1970s, and later, as a result of the civil war of the 1990s, it spiraled into deviant
patterns of misuse.
1.2 History of Catha edulis use
There is much speculation about the early history of the plant. (Al-Hebshi and Skaug,
2005a) cite Kennedy (1983) who mentions a theory by Cotterville-Girandet that
suggested Catha edulis was known to ancient Egyptians. (Kennedy et al., 1983)also
reported another account from an Arabic source which indicated that the plant was
used for medicinal purposes as early as the beginning of the 11th
century in Turkistan
Afghanistan. This report is by the Persian Physician Bin Ahmed Al-Biruni [973-1051
AD] in his book Kitab al-Saidana fi al-Tibb, an 11th century work on pharmacy and
materia-medica, (Krikorian, 1984) in which he mentions correspondence that he had
with the famous Persian physician Ibn Sina or Avicenna, [980-1037 A.D.]. In one of
his later manuscripts Al-Biruni, who was unfamiliar with Catha edulis describes it as:
"a commodity from Turkestan. It is sour to taste and slenderly made in the manner of batan-
alu. But qat is reddish with a slight blackish tinge. It is believed that batan-alu is red, coolant,
relieves biliousness, and is a refrigerant for the stomach and the liver." (Hamarneh, 1972).
28. 28
According to Krikorian (1984), d‟Hericourt was the first European to mention the
existence of Catha edulis in his manuscripts on Arabia Felix (present day Yemen)
which cites „planting of Catha edulis was introduced from Abyssinia into Yemen
about 1424 by Sheikh Abou Zerbin‟. El Mahi, (1962) on the other hand suggests that
„the current names of Coffee and Catha edulis are etymologically derived from the
place name „Kafa‟ in Ethiopia where they flourished‟. There has been also some
debate as to the origin of the Catha edulis plant. According to most researchers Catha
edulis is believed to originate in Ethiopia (Getahun and Krikorian, 1973) and was
introduced to Yemen in 525 AD during the Ethiopian occupation (Al-Motarreb et al.,
2002). Most of these early entries about Catha edulis use were written in Arabic and
mainly focus on areas bordering the Red Sea, hence relate to accounts documented up
until mid 16th
century, before European travel to Southern Arabia began. The earliest
monograph about the effects of Catha edulis was that by Ibn Hajar al-Haytami (1504-
1567) „The Authoritative Warning against the Use of Kafta and Kat’.
A translation of this work by the Arabist Frans Rosenthal at Yale University was later
reviewed by Hess (1976), and gives the description that some of the effects Al-
Haytami attributed to Catha edulis were worse than those of hashish (Cannabis).
Accounts of Catha edulis use or its effect appears to have started to filter through
slowly to the scholarly circles of Europe from 17th
century. Several historical
publications point out that it was partly access to earlier colourful writings depicting
Catha edulis as an exotic and mysterious plant that could have been the catalyst to
many of the early European expeditions to Yemen (Al-Hebshi and Skaug, 2005b). The
earliest entry in the European literature about Catha edulis use in Southern Arabia was
by the French orientalist Barthelemy d‟Herbelot [1625-1695] who described three
different beverages commonly used in the area. The first he called Cahuat al Catiat [a
corruption of what is presumed to be kat coffee] or Caftah; the second Cahuat al
Caschriat [kishr, a beverage from the mesocarp or husks of coffee fruit] and the third,
Cahuat al Bunniat [bunn, coffee from the seed or beans]. Following these earlier
accounts, Achille Richard [1811-1860], a Professor of Botany at the University of
Paris reassigned plants collected from Tigre and Shoa regions of Ethiopia to the genus
Catha and rejected Martin Vahl‟s earlier contention that the plant should be in the
genus Celastrus (Krikorian, 1984).
29. 29
In the late 19th
century interest had also been generating amongst scientist and
pharmacists in Europe to sell Catha edulis as a pharmaceutical preparation for use in
various medical disorders (Krikorian 1984). In the early and mid twentieth century,
and during the British colonial era in Eastern Africa and Yemen, the story of Catha
edulis use amongst the various societies in these regions became common knowledge;
along with various legal attempts to prohibit its widespread use (Carrier, 2005). In the
latter part of the last century Catha edulis use began and grew in countries in Europe
and North America whereas its trade due to the advent of modern transportation made
it possible for it to be sold across contents.
1.3 Catha edulis in the UK
Catha edulis, as a plant material, is not illegal in the UK, but two of its alkaloids,
cathinone and cathine, are controlled substances under the Misuse of Drugs Act 1971.
The consumption of Catha edulis as recreational drug substance has proliferated in the
past decade (Gebissa, 2008). According to ACMD (Advisory Committee on the
Misuse of Drugs) report on Catha edulis (2005), 6 tons of Catha edulis arrives in the
UK per week, mostly by air from Kenya although an earlier report commissioned by
the Home Office (Griffiths et al., 1997) puts this figure slightly higher at 7 tons per
week. The bulk of the imported Catha edulis in the UK is in transit to other countries
for example Canada and Scandinavian countries, USA; all of which are places where
Catha edulis is illegal. Figure 1-6 summarizes the transit routes via UK and modes of
transportation to markets.
Nowhere is the mass phenomenon of acculturation in Catha edulis use more apparent
than in the largely displaced Somali communities living in Britain‟s inner cities
(Hennessey, 1994). The pattern of misuse varies between the different Catha edulis-
using migrant groups, and according to the ACMD report (2005), Catha edulis use
appears less prevalent among the Somali community living in the UK than the
population in Somalia.
However, among immigrants living in Great Britain, where there are substantial
minorities of East African and Yemeni origin, the levels of Catha edulis use have
increased so that it is termed endemic in parts of larger cities e.g. London,
Birmingham, Sheffield, Cardiff (NACRO 2005). Although existing evidence suggests
30. 30
(Klein, 2008) that Catha edulis consumption is wide spread in the UK and prevalent2
amongst immigrant communities from the Horn of Africa and Arabia, there is no real
evidence that it has crossed over to other communities. Concern about habitual Catha
edulis use has progressed to become a major concern for health and social care
providers.
The Home Office has commissioned various surveys and pilot projects within the
voluntary sector to look into the extent of levels of misuse and the provision of
specialist services to provide health promotion and treat Catha edulis-related adverse
effects. In response to calls to ban Catha edulis, the Home Office asked ACMD to
consider evidence on Catha edulis concerning its harmful effects (ACMD report on
Catha edulis 2005). Two other separate pieces of research were also commissioned by
the Home Office and were carried out by the organizations Turning Point (2004) and
NACRO (2005) respectively.
On the basis of the evidence presented, ACMD recommended that Catha edulis should
not be controlled under the Misuse of Drugs Act 1971, on the grounds that its use is
very limited to specific communities within the UK (ACMD report on Catha edulis
2005). Furthermore available scientific evidence presented suggested that Catha edulis
has not, ., nor does it appear likely to, spread to the wider community.
However the ACMD acknowledged that the use of Catha edulis is not without
detrimental effects to health and well being of communities who use it, and made the
following five specific recommendations discouraging its use in the wider sense:
1. That Catha edulis be not controlled under the Misuse of Drugs Act 1971.
2. That there is a need for emphasis on education for primary health care
professionals and health promotion for communities about the health risk
associated with Catha edulis use and on treatment options.
2
current prevalence data range from 34%-67% (Klein 2008) within the Somali community of
those who identify as current users.
31. 31
3. That Drug Action teams to be formed to focus treatment options based on
education and prevention; with interventions that should include family
involvements.
4. That government and local relevant authorities should explore the possibility of a
voluntary agreement amongst retailers of Catha edulis on excluding sale of
Catha edulis to those under 18 years old.
5. That a campaign be started to raise awareness on the health and safety
implications of chewing Catha edulis in poorly ventilated Mafrishs and their
“unhygienic nature”. Community leaders and Mafrishs‟ owners should adhere to
current health and safety regulations on such matters as ventilation, lighting, fire
escapes.
32. 32
Figure 1-6: Distribution network of Catha edulis via UK (ACMD, 2005).
Yemen
Kenya
Largest Catha
edulis
exporter to
the UK
Ethiopia
UK
Average of 7
tonnes
arrive per day
Holland
& other
European
airports
North
America
Illegal and
expensive
Wholesalers
Local shops
Retailers selling
£3/bundle
Mafrish
Selling £3/bundle
of 200g
Mobile
Traders
Street selling at
£2.50/bundle
VViiaa
bbooaatt
SSmmuugggglleedd
bbyy aaiirr
SSmmuugggglleedd
bbyy aaiirr
AAiirr ffrreeiigghhtt
BByy
RRooaadd
33. 33
1.4 Botany of Catha edulis
The Catha edulis plant, Catha edulis (Vahl.) Forssk, ex Endl., is a member of the
Celastraceae, which includes about 350 species of trees and shrubs in 15 genera. The
early classification of Catha edulis was based on that entered by Forsskal, the botanist
who started with Neibuhr a mission into Yemen in 1762. He described the plant under
the name Catha edulis and classified it along with an additional species then
designated as spinosa. Forsskal noted the cultivation of Catha edulis, along with
coffee, and reported that the Arabs in Yemen ate the green leaves and ascribed
medicinal virtues to the plant.
Niebuhr‟s book was based on an earlier work of an ill-fated expedition to Arabian
Felix [present day Yemen] by the botanist Forsskal who classified Catha edulis as a
Catha species. It was not until twenty years later following Forsskal‟s death that
Martin Vahl, (a professor of Botany at the University of Copenhagen) rearranged the
collection that Forsskal made and placed Catha in the genus Celastrus hence naming
Catha edulis as Celastrus edulis (Krikorian, 1984).
Previously the opinion held amongst botanists was that the genus Catha consisted of
three different species; Catha edulis (Vahl) Forssk. ex Endl., Catha transvaalensis
(Codd) and, Catha abbottii. (Van Wyk & Prins). Catha edulis was also believed to be
the type specimen of the genus Catha Forssk. (Robson, 1965) and prior to 1966 Catha
edulis was considered to be the sole representative of the genus Catha.
In 1971 Lydenburgia cassinoides which is confined to a relatively small area in the
north eastern Transvaal and had been originally identified by Robson (1965), was
renamed C. transvaalensis (Codd, 1977). Catha abbottii endemic to South Africa‟s
southern Natal/Pondoland sandstone was described by Van Wyk and Prins (1987) with
assertion that it was a Catha species. C. abbottii was suggested to be related to C.
transvaalensis, although it is separated by its scaly bark and fewer leaf crenations
(Codd 1977). As a result of these studies the prevailing opinions held at the time
recognized Catha as three-species genus.
However, on the basis of molecular phylogenetic analysis with morphological
characters, the present day botanical classification places the genus Catha in
34. 34
Celastraceae and recognises only one species, Catha edulis Forssk. A review study by
(Simmons et al., 2008) on the DNA of Catha edulis resolved its genome as the nearest
to Allocassine, Cassine, Lauridia, and Maurocenia in Celastraceae. Furthermore,
Gymnosporia cassinoides, which is reportedly chewed as a stimulant in the Canary
Islands, was resolved as a derived member of Gymnosporia, more closely related to
Lydenburgia than to Catha. Celastrus edulis, seen in some publications, is a misnomer
from an obsolete classification by Vahl in 1790.
Macroscopical features
In spite of its being regarded as one species, there is a wide variation in the appearance
and taste of samples of C. edulis from different regions, due to the cultivation of the
plant as different cultivars and under varying ecological conditions. The leaves have an
astringent taste depending on the levels tannic of acid present. Catha edulis is said to
have very few morphological and anatomical features that could be used for the
purpose of sample identification. (Nordal, 1980) described the plant in two parts; the
shoots of the plant as young part (stems and leaves) and flowers and fruits as the old.
Depending on the area or region of cultivation, the plant grows between 1.5- 20metres
in height and produces small flowers that are white in colour. The fruit is smooth and
an oblong shaped with three halves containing 1–3 seeds each. The seeds (~3mm long)
have thin brown wings at the base and reddish brown when matured (WHO, 1980).
The stem is grayish, straight and slender, the bark has different colours depending on
the variety and age of the stem and branches (Lamessa, 2001). The colour of the
branches also range from green in older leaves to crimson pink in young branches and
the root system grows to 3-5 meters in depth (Gebissa, 2008).
The leaves of Catha edulis are oval-lanceolate in opposite and alternate arrangements,
and may vary considerably in size, colour and shape, according to geographical origin.
While the colour of the leaves in the Ethiopian type is often green (or crimson green);
the Kenyan type has a mixture of both red (on the tips) and green leaves. Catha edulis
leaves range between 4-11cm long and 1.8-5cm wide, with a clear serrated margin.
The tip of the leaves show a pinnate venation while in the main part of the lamina there
are visible lateral veins leaving the midrib at a fairly wide angle and immediately
curving upwards.
35. 35
Microscopical features
An earlier anatomical study Shadan and Shellard, (1962) produced one of the few
descriptions of the microscopic features of the leaf of Catha edulis 3
. The leaves of
both the Yemen and Ethiopian Catha edulis were shown to have a similar cellular
structure with three different cells; i.e. upper epidermal, ‟Y‟ funnel shaped mesophyll
cells and lower epidermal. The epidermal cells are single layer cells with polygonal
shape and cell walls containing crystals of calcium oxalate are thickened with
cellulose. The mesophyll has no distinctive cell inclusions apart from chloroplasts in
the top layer, but the lower row, which are in groups of 2-3 cells, are „Y‟ shaped and
are suggested to be the collecting cells of the upper row. The mesophyll cells also
contain crystals of calcium oxalate as well as tannins in resinous form, hence their
classification as tanniferous cells. The lower epidermis is a single layer of thick-walled
cells with polygonal shape and there are no cells containing crystals of calcium
oxalate.
Cultivation
Catha edulis is propagated from seedlings or root cultivars and grows into a shrub in 1-
5 years. It then develops into a small evergreen tree of 5-8 meters in height that has
rounded clusters of bending branches with opposite oval leaves. Older trees can grow
up to 25 meters in height. Catha edulis is a fast growing and drought tolerant plant that
grows easily in any type of soils without the need for treatment during cultivation
(Krikorian, 1984). The method of cultivation is either credited with its environmental
friendliness, or said to cause depletion of underground water reservoirs. In Ethiopia
and Kenya Catha edulis cultivation plays an important role in minimizing soil erosion
and the threat of deforestation in intensive farming. However, in Yemen 40% of the
country's water supply goes towards irrigating Catha edulis trees, and with 10-15 %
increase in consumption every year, this is causing a huge drain on groundwater levels
for large urban cities which are estimated to run dry by 2017 (Horton 2010).
Catha edulis is planted interspersed with other food crops in rows on hillsides along
terraces. Catha edulis growing requires well drained red-brown or sandy soil (pH of
6.0-8.2) with a low percentage of clay and medium to high amounts of total nitrogen,
3
Shadan and Shellard studied only Yemen and Ethiopian Khat. The study excluded the
Kenyan type of Catha edulis , which has different surface leaf structure.
36. 36
organic matter, available phosphorus, calcium, potassium and magnesium (Lamessa
2001). Catha edulis can be harvested year round at any time of the day, generally 2-3
times a year. The method of harvest is by breaking off the young branches from the
main branches and trimming them to about 40-cm. Young and soft shoots are detached
with the bare hands, while hardy shoots are cut off by hand tools.
1.5 Chemistry of Catha edulis
1.5.1 The Alkaloids originally isolated
The history for the search of the active compounds in Catha edulis dates back as far as
1887, when Fluckiger (1887) established the presence of an alkaloid he called “katin”.
Serious research began with the advent of ephedrine in the western medicine. Wolfes
(1930), was the first scientist to identify (+)-norpseudoephedrine 1 which is also
known as cathine, from Catha edulis. Wolfes however did not report any
pharmacological activity that could be attributed to the new substance and whether or
not it could account for why people used the plant.
1
OH
CH3
NH2
2
CH3
NH2
O
3
CH3
NH2
OH
37. 37
Brücke (1941) was the first to make the bold suggestion that the fresh plant of Catha
edulis contained a more potent substance than (+)-norpseudoephedrine, and that this
was corroborated by the preference shown by Catha edulis consumers for the fresh
plant rather than the tea (concoction and infusion) made out of the dried plant.
Hoffmann and co-workers (1955) subsequently concluded from their work on
synthetic norpseudoephedrine isomers that the presence of natural (+)
norspseudoephedrine in Catha could explain its usage.
Paris and Moyse 1967 also isolated (+)-norpseudoephedrine and this was followed by
(Alles et al., 1961) whose work quantified the levels of (+) norpseudoephedrine from
various grades of dried Herari samples and other samples of the plant grown in Florida
and California.
1.5.2 Phenylalkylamine alkaloids
Catha edulis alkaloids comprise two groups of alkaloids, the phenylpropylamines
widely known as the cathamines, and the cathedulins. The cathamines consist of the
phenylalkylamines of which there are four compounds in both Ethiopian and Kenyan
types, i.e. (+)-norpseudoephedrine or cathine 1 (-)-cathinone 2, (-)-norephedrine 3, and
three other phenylpentylamines known as (+)-merucathinone 4, (+)-merucathine 5 and
(-)- pseudomerucathine 6 (Brenneisen et al., 1984) which are found only in the Kenyan
type. The cathedulin group is composed of sesquiterpene pyridine alkaloids polyester
based on either polyhydroxylated dihydroagarofuran 7 or euonyminol 8 cores. They
are more numerous than the cathamines and have been classified according to their
molecular weight into high, medium and low (Crombie, 1980). Figure 1-7 shows an
illustrative chart of these two Catha edulis alkaloid groups.
4
O
NH2
CH3
39. 39
Pharmacological studies mostly concentrated on the phenylalkylamine group. Early
work commenced with (+)-norpseudoephedrine indicating there could be more
powerful stimulant present in the fresh Catha edulis material that accounted for the
symptoms produced. Schorno and co-workers group at the University of Berne,
Switzerland also isolated cathinone 2 [(-) aminopropiophenone] (Schorno and
Steinegger, 1979). It was also shown that cathinone is a labile compound which
degrades upon drying or storage of fresh plant due to an enzymatic reduction. It is
transformed into the less active stimulant cathine isomers, (+)-norpseudoephedrine 1
and (-)-norephedrine 3 (Schorno et al., 1981).
Latter investigations established that cathinone is an intermediate product in the
biosynthesis of cathine, and accumulates in young (but not adult) leaves ((Brenneisen
and Geisshusler, 1985). Subsquently cathinone, was also isolated in the UN Narcotic
laboratory from fresh Catha edulis (Szendrei, 1980) and was shown to have strong
potency in stimulating the Central Nervous System. Other pharmacological
investigations on cathinone named the compound “Natural Amphetamine” (Kalix and
Khan, 1984), due to the fact that cathinone had not only an analogous structure, but
also a close pharmacological profile, to the symphatomimetic effects induced by (+)
amphetamine 9.
Efforts to review the differences of alkaloid content in Catha edulis from different
origins led (Geisshusler and Brenneisen, 1987a) to isolate two other cathamines which
were unsaturated phenylpentylamines from fresh Catha edulis originating in the Meru
region of Kenya. These were found only in significant amounts in the Mira type of
Catha edulis and were named merucathinone 4 and merucathine 5. It was found that (-)
cathinone can be oxidized (Figure 1-8) to give 1-phenyl-1, 2-propandione 10
(Brenneisen et al., 1986); a compound recently also identified in the volatile fraction of
Catha edulis extract (Abdulsalam et al., 2004), as well as dimers, such as 3, 6-
dimethyl-2,5-diphenylpyrazine 11, which were artifacts arising during isolation.
41. 41
Figure 1-7: Summary of the all the identified alkaloid groups in Catha edulis.
High mol. mass
(1000-1170 Da)
Medium mol. mass
(765-891 Da)
Low mol. mass
(595-700 Da)
Alkaloids in Catha edulis
Cathedulins
Cathamines
Phenylpropylamines Phenylpentylamines
(-)-cathinone
Merucathinone
Merucathine
Pseudomerucathine
(+)-norpseudoephedrine (-)-norephedrine
42. 42
The (-)-cathinone found in young leaves was found to be as much as 70% of the total
phenylpropylamine fraction of an extract analyzed by TLC. In contrast, fully
developed leaves contained no (-)-cathinone 2, but high levels of (+)-cathine 1 and its
diastereomer (-)-norephedrine 3 (Guantai and Maitai, 1982; Kalix, 1990a). This
explains why young fresh leaves are preferred in chewing Catha edulis, since older or
dried leaves would have little cathinone and so have not such a strong effect. In the
living plant cathinone occurs in very low concentrations in the stem and bark.
Relatively higher levels of (-)-cathinone have also been found recently in the
inflorescence, in comparison to the older leaves and bark (Krizevski et al., 2007).
Pharmacokinetic studies on cathinone indicated that the compound is metabolised via a
keto reduction pathway. Oral administration of naturally occurring (-) cathinone 2,
gave results, from the urinary excretion, which showed that it was converted into (-)-
norephedrine 3, and its (+)-diastereomer into corresponding (+)-norpseudoephedrine 1
(Brenneisen et al., 1986). This is particularly important when analyzing the blood
plasma levels of the phenylalkylamines, since concentrations of these isomers may not
reflect the original levels in the plant material. Other Catha edulis phenylalkylamine
alkaloids i.e. the diastereoisomers (+)-norpseudoephedrine 1 and (-)-norephedrine 3,
were also verified to be common to all samples from a variety of ecological origins
(Geisshusler et al., 1987).
Krizevski et al., (2007) also reported that in the biosynthesis of (-)-cathinone, 1-
phenyl-1,2-propanedione 10, is the precursor whose distribution is the highest in young
Catha edulis leaves, but in the presence of the enzymatic catalyzing activity of
NADPH, (-)-cathinone 2 is then further reduced to (+) norpseudoephedrine 1 and (-)
norephedrine 3 in leaves or in extracts. It is well known that origin, cultivation, harvest
affect cathamine content (Schorno et al., 1981).
Geisshusler et al 1987 compared the differences in the phenylalkylamine content, i.e.
cathinone, norephedrine and norpseudoephedrine, in samples from markets in
Ethiopia, Kenya, North Yemen and Madagascar. In many samples it was found that the
actual price of the Catha edulis type correlated with the level of cathinone, which was
the marker for the quality criterion.
43. 43
Figure 1-8: Oxidation/reduction of (-)-cathinone in leaves and in extracts
(Brenneissen et al., 1985; Krizevski et al., 2007).
ReducedReduced
Oxidised
Reduced
Dimerized
(-)-norephedrine(+)-norpseudoephedrine
CH3
NH2
OH
CH3
NH2
O
(-)-cathinone
O
O
CH3
N
NH3C
CH3
3, 6-dimethyl-2, 5-diphenylpyrazine
1-phenyl-1, 2-propanedione
O
OH
CH3
Benzoyl ethanol
OH
CH3
NH2
44. 44
Morghem et al., (1983) also identified forty different varieties grown within Yemen
alone, and in Kenya the giza variety is distinguishable from and more highly prized
than, the gangeta variety. The most important criterion of discrimination that is used as
a marker for type characteristics is the colour (red or white) and the height (short or
long) of the stem.
1.5.3 Cathedulin alkaloids
Earlier scientific studies on Catha edulis and attempts in the search to elucidate the
active constituents of the plant led to the separation of various and similar water
insoluble and weakly basic compounds under the names of “cathinin”, “cathidine”, and
“celestrin”. The evidence obtained from these studies showed that these substances
were probably the early representatives of the cathedulin type polyester alkaloids.
Baxter and co workers (1979) at the University of Nottingham (in liaison with the UN
group) investigated alkaloids in Catha edulis and this led to the discovery and the
identification of the cathedulin sesquiterpene pyridine alkaloids. In continuation of
these studies, Crombie (1980) successfully elucidated the structures of many of the
known cathedulins in Catha edulis from samples originating in Ethiopia, Kenya and
Yemen. The cathedulins consists of polyesters or lactones of a sesquiterpene polyol
core. Crombie assigned the cathedulins, according to their structures, into three groups
on the basis of their molecular weights and the number of the esterifying acids on the
different hydroxyl positions of the sesquiterpene core (WHO, Bulletin on Narcotics,
1980).
The lower molecular weights cathedulins (595-700 Da) are described as simple esters
of pentahydroxydihydroagarofuran; the medium molecular weight (765-891 Da) have
a euonyminol core with a dilactone bridge, and the higher molecular weight group
(1000-1170 Da) consists of more complex esters of an euonyminol core with one or
two dilactone bridges. Higher and medium molecular weight cathedulins are amongst
the most abundant in this group of Catha edulis pyridine alkaloids. Their structures are
discussed in detail in Chapter 2. The esterifying acid functions of cathedulin alkaloids
include benzoic 12, nicotinic 13 2-hydroxyisobutyric 14, tri-O-methylgallic 15, and
edulinic acids 16. Dilactone bridges are formed from the dibasic acids evoninic 17 and
cathic 18 acids.
46. 46
N
O
OH
HO
17
H3C
CH3
O
N
OO
O
H3C
O
O
OH
CH3 HO
18
1.5.4 Constituents other than alkaloids
In addition to the phenylalkylamines and the cathedulins alkaloids many different
compounds are found in Catha edulis, including, terpenoids, flavonoids, sterols,
glycosides, amino acids, minerals and others (Kalix and Braenden, 1985;Nencini et al.,
1989). El Sissi et al., (1966), identified three flavonols: kaempferol 19, quercetin 20
and myricetin 21 in hydrolyzed Catha edulis extracts.
48. 48
Dihydromyricetin and its 3-O-rhamnoside 22 was isolated from crude flavonol mixture
of Catha edulis (Gellert et al., 1981) and it was also highlighted that of tannins are
present in Catha edulis. Tannins is a term often used to cover a complex mixture of all
phenolics and Catha edulis was found to contain a considerable amount of tannins; 7-
14% by weight, in dried leaves (Al-Motarreb et al., 2002). Catha edulis also contains a
significant amount of ascorbic acid (vitamin C). An early study by Mustard (1952)
measured the vitamin C content in both leaves and branch tips and found higher
amounts in leaves (325mg/100g) than branch tips (136mg/100g).
Catha edulis is known to have a characteristic slightly pungent odor with a particular
aromatic twist in taste which has been attributed to its volatile oil which consists of α-
thujone 23 and, other monoterpenes including fenchone 24, that are found in fairly low
levels (Qedan.S, 1972).
Crombie (1980) isolated from root bark of the plant, a series of red triterpene quinones;
celestrol 25, pristimerin 26, iguesterin 27 and tingenone A and B 28. Similar products
isolated from leaf part of Catha edulis included β-sitosterol 29 and its glycoside 30. It
was also reported that iguesterin 27 found in C. edulis is common to two species in
Celastraceae that are endemic to the Canary and Madeiran archipelagos, namely
Maytenus canariensis; sometimes chewed by Canary Islanders to combat fatigue, and
Maytenus umbellate (Gonzalez et al., 1986).
OHO
OH O
OH
OH
OH
22
O Rhamnoside
51. 51
1.6 Experimental Pharmacology and Pharmacodynamics of Catha
edulis
Some of the experimental pharmacology conducted on Catha edulis and constituent
alkaloids to date are summarized in Figure 1-9.
1.6.1 Pharmacology of cathinone and related phenylalkylamines
The symptoms of Catha edulis use are said to be reminiscent of those induced by
amphetamine. The chewing of Catha edulis produces a range of sympathomimetic
effects which are largely attributed to the mechanisms of actions of its most potent
alkaloid constituent; cathinone 2 on central and peripheral nervous system. The
following paragraphs provide critical review on cathinone and related
phenylalkylamine alkaloids, particularly with respect to the differences between the
actual biological mechanisms of action of these individual alkaloids and the effects of
Catha edulis itself.
Since the discovery of cathinone as a potent alkaloid in Catha edulis (Szendrei 1980),
most of the scientific investigations have focused on its biochemical properties as an
explanation for the underlying pharmacological action of the plant. It was established
that (-) cathinone 2 is structurally analogous to (+) amphetamine 9 and has
sympathomimetic effects similar to the neurochemical responses induced by
amphetamine. As aromatic amines, phenylalkylamines are naturally psychomimetic in
that they interact with the catecholamine neurotransmitters by interference with their
storage, release and metabolism.
Cathinone activity has been linked to the release of monoamine oxides ((Kalix and
Braenden, 1985). It was also observed that (-)-cathinone in vivo in humans, has both
euphorigenic and psychostimulant effects which closely resemble those of (+)-
amphetamine (Brenneisen et al., 1990). Cathinone of natural origin has been shown to
be the (-) enantiomer, which has the same configuration as dexamphetamine and is
three times more potent than its (+) enantiomer of cathinone.
52. 52
Figure 1-9: Some of the experimental pharmacology studies on cathinone and Catha edulis [from (Feyissa and Kelly, 2008)].
Neurochemical effects of cathinone and Catha edulis
Wagner et al. (1982)
Kalix (1981,
1982, 1983)
Cleary and Docherty
(2003)
Cleary and Docherty
(2003)
Mereu et al. (1983)
Banjaw et al. (2006)
Banjaw and Schmidt
(2005)
Not examined
Not examined
Not examined
Not examined
Not examined
DA in PFC, PO 200mg/kg,
10 days
↓5-HT& 5-HIAA in PFC
anterior and posterior striatum
†Release and inhibition of uptake of [³H] DA,
1-100M, CATH: AMPH (1-1.6)
Enchased release of [³H] 5-HT, 12µM †efflux
and inhibition of uptake of [³ H]DA, 3-9µM,
CATH: AMPH (1:2-6X)
Inhibits [³H] nisoxetine binding to NA transporter,
2.5µM, CATH ≈ COC ≈ MDMA
Efflux of [³H] NA and inhibition of uptake of [³H]NA,
1.2-2µM, CATH ≈ COC ≈ MDMA
Inhibition of the firing rate of nigral DA neurons,
0.4 mg/kg (IP), CATH ≈ AMPH ↓ extracellular DOPAC
(NAc, SL), IP, 6 mg/kg ↓5-HT&5-HTP (NA, SL), IP 6mg/kg
†DA (PFC), ↓DOPAC (PFC, NAc, CP) after 1.5 mg/kg (PO) for 10
days
↓5-HT&5-HIAA in PFC and anterior and posterior striatum,
after 1.5 mg/kg (PO) for 4 weeks
In vitro study
Synaptosmal preparations
Rabbit/Rat striatal tissue
Rat cerebral cortex
Rat atrial/ventricular strip
In vitro study in rats
Postmortem analysis in rats
ReferenceCatha edulis extractCathinoneNature of study
IP, intrapertonal; PO, oral; AMPH, (+)amphetamine; COC, cocaine; CATH, cathinone; NAc, nucleus accumbens; SL, septi lateralis; CP, caudate Putamen; NA,
Noradrenaline; DA, dopamine; 5-HT, 5-hydroxytryptamine; DOPAC, Dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindolamineacid; PFC, prefrontal cortex.
53. 53
The very first report describing the effects of cathinone stated that the alkaloid induces
hypermobility in rats (Kalix, 1980b). A similar study, confirmed that cathinone
produces locomotor stimulation in mice and comparable stereotypy in rats qualitatively
similar to amphetamine, although it is approximately half as active (Zelger et al.,
1980). Subsequent investigations (Zelger and Carlini, 1981) also found that, in using
the rotational test after lesions of substantia nigra with 6-hydroxydopamine in rats,
cathinone produced circling behaviour comparable to that induced by amphetamine. In
neurochemical studies using striatal slices, cathinone inhibited the reuptake of
dopamine but enhanced the release of 3
H-DA in a similar manner to amphetamine
(Kalix, 1983).
In one important study Wagner and co-workers showed that cathinone exerted its
characteristic behavioral effects through brain dopamine neurons and that cathinone
mimicked (+)-amphetamine‟s long-term toxic effect on brain DA terminals (Wagner et
al., 1982). However, although the regional distribution of DA terminal damage,
following dosing with (-) cathinone and d-amphetamine appeared to be the
nigrostriatal dopamine DA terminals, their neurotoxic effect was selective, as
evidenced by the lack of long-term effect on regional norepinephrine and serotonin
levels on following repeated administrations. The findings in this study point out that
doses of 100 mg/kg (-)- cathinone are needed to induce dopamine neurotoxicity i.e. an
interval of over 12 hours at a time for each dose administered.
With reference to the actual practice of Catha edulis chewing, it is doubtful that
humans ever ingest the high doses of cathinone extrapolated from this work,
considering both the rate limiting method of mastication itself as well as the shelf half
life of the alkaloid. However the results of the findings at least raise the possibility of
dopamine DA nerve terminal damage in the event of chronic Catha edulis and
cathinone abuse.
Clearly we need to understand the pharmacology of cathinone in order to explain the
psychotropic effects of the drug. Early studies showed that both cathinone and
amphetamine reduced rat striatal levels of DOPAC suggesting an effect of cathinone
on dopamine release (Mereu et al., 1983). In a later study, using microdialysis, Pehek
et al., (1990) reported that cathinone increased extracellular dopamine in the striatum,
giving further evidence to this concept. Mereu et al., (1983) also showed that
54. 54
intravenous injection of cathinone reduced the electrical activity of dopaminergic cells
in the substantia nigra of rats, suggesting a negative feedback of dopamine on neuronal
activity. This was reversed by haloperidol-induced blockade of presynaptic D2
receptors.
In similar experiments on rabbit caudate nucleus (Kalix, 1983b), it was observed that
the catecholamine reuptake inhibitors benztropine, nomifensine and mazindol were
able to block the (-) cathinone-induced release, indicating that the alkaloid has to
penetrate to intraneuronal sites in order to evoke release. It was therefore concluded
that the pharmacological similarity between (-) cathinone and (+) amphetamine
extends to the cellular level and that the behavioral effects of (-) cathinone are due to
stimulation of release of DA from central catecholamine storage sites.
(-)-Cathinone was found to affect DA release by a mechanism similar to that of (+)-
amphetamine (Kalix, 1985; Kalix, 1983a). The only difference between (-)-cathinone
and amphetamine effects were pointed out to be one of quantity (Kalix, 1985); e.g. at
5µM (+) amphetamine was observed to be 3-5 times more potent than (-) cathinone in
terms of the quanta of efflux they released from prelabelled rat striatal slices (Kalix,
1985). Furthermore cathine or (+)-norpseudoephedrine in previous studies was
observed to be a weaker releaser of DA i.e. 8 times less than (-) cathinone and it was
suggested that it is not important to the stimulating properties of Catha edulis(Kalix
1983 b). (+)-Norpseudoephedrine and (-)-norephedrine are the two isomeric forms of
the phenylpropylamine alkaloids in Catha edulis.
The effects of racemic cathinone on dopamine (DA) and 5HT-containing neurons in
several regions [in the nuclei caudatus putamen, accumbens, amygdaloideus centralis,
septi lateralis, and hypothalamus] of rat brain in vivo were examined relative to the rate
of synthesis of DA (Nielsen, 1985). By measuring the concentration of DOPA after the
i.p. administration of NSD 1015 (100mg/kg) an inhibitor of aromatic L-amino acid
decarboxylase and using HPLC with an electrochemical detector, it was observed that
the concentration levels of DA, and its metabolite DOPAC varied in the different brain
regions. The findings showed that (-) cathinone decreased levels of DOPAC in a time
and dose-related manner, with peak effect between 30-60 minutes, as well as the as the
accumulations of DOPA in caudatus putamen, accumbens, amygdaloideus centralis,
septi lateralis but with no effect in hypothalamus. The relevance of these findings has
55. 55
been validated by Pehek using in vivo microdialysis to measure the extracellular levels
of dopamine and metabolites in the anterior caudate putamen and the nucleus
accumbens, after i.p. administration (Pehek and Schechter, 1990). It was shown in this
study that 0.8, 1.6 or 3.2 mg/kg of either (-) cathinone or (+) amphetamine increased
the levels of DA, but decreased the levels of its metabolite, DOPAC. At 1.6mg/kg the
same difference was seen in the nucleus accumbens while at a higher dose of 3.2
mg/kg, amphetamine had a greater effect than cathinone on DA release in both regions.
In studies to compare the behavioral effects of (-) cathinone and amphetamine in mice,
pretreatment with the DA antagonist, haloperidol was used to produce significant
reduction of the alertness, locomotor activity, licking and biting induced by either of
these stimulants (Connor et al., 2002). In other earlier studies pretreatment with the
monoamine antagonist reserpine had shown moderate reduction of the locomotor
effect of cathinone (Valterio and Kalix 1982). In another behavioral experiment
(Knoll, 1979) it was observed that cathinone reduced the food intake of test animals.
Subsequently Zelger et al., (1980) confirmed that, while chronic intraperitoneal
administration of cathinone to rats had an anorectic effect; it was in comparison less
potent than amphetamine.
The role of DA in the motor effects of cathinone was also reported (Calcagnetti and
Schechter, 1992). He observed that, in rats, intra-cerebroventricular or bilateral
microinjection of the drug into the nucleus accumbens produced an increase of
locomotor activity which was dose-related. In another study, the conditioned place
preference (CPP) effect of cathinone in rats was also investigated, and it was found
that cathinone administration increased the time the rats spent in a non-preferred
environment and that the effect was more marked in rats previously trained to
recognize cathinone (Calcagnetti and Schechter, 1993). However pretreatment with a
dopamine release inhibitor attenuated the CPP effect associated with Catha edulis.
In a later study Catha edulis extracts have been showed to increases locomotor activity
in rats (Connor et al., 2002) The motor effects of Catha edulis (containing an
unspecified amount of alkaloids) were compared with that of amphetamine. It was
found that Catha edulis extracts induced the locomotor activity in a dose-related form
but also induced head twitches. In comparison (+) amphetamine was only able to
produce such locomotor activity in high doses in a dose dependent manner when
56. 56
competing behaviors, such as intense grooming, sniffing, and other stereotypes usually
replace the voluntary locomotion.
1.6.2 Experimental models of addiction
There are few studies which have looked into the post-synaptic dopaminergic effects
of Catha edulis and there appears generally an information gap on the basic effects of
the plant extract and its constituents at DA receptor binding level. It must be
emphasized that in the last four decades there has been an ongoing debate about
whether Catha edulis and its constituent cathamines are addictive, causing
dependency, or whether the bulky nature of the plant material downgrades the severity
of dependence. However, in the drug dependency milieu, chronic exposure to
reinforcing drugs can lead to drug addiction, which is also characterized by excessive
drug-seeking behaviour. Indeed cravings and tolerance to the increases of CNS effects
are both common amongst habitual Catha edulis chewers (Kalix, 1985; Kalix, 1990a).
Self-administration of psychoactive drugs in experimental animal models is one area
that has been proved to be highly predictive of the risk of abuse by humans
(Richardson and Roberts, 1996). (-)-Cathinone; which is characterized for its high-
abuse potential, has also been reported to be a reinforcing drug that maintains self-
administration in monkeys (Foltin and Schuster, 1983). Similarly, Catha edulis itself
has been suggested to have a higher potential for dependency than amphetamine
because of its less aversive nature (Goudie, 1987). There is a well-established key role
of dopaminergic transmission in the positive reinforcing effects of psychostimulants,
effects of drug self-administration. In a comparative study of (-)-cathinone and
cocaine, it was observed that (-)-cathinone is a potent reinforcer in rats and that
pretreatment with the D1 selective antagonist SCH-23390 at 10mg/kg caused a
significant increase in (-)-cathinone self administration, whereas the D2 antagonist
Spiperone caused only a slight increase, suggesting a role for the D1-type DA receptor
in mediating the reinforcing effects of the alkaloid (Gosnell et al., 1996).
Furthermore it has been shown that rats develop tolerance to discriminative effects of
(-)-cathinone within 10 days of chronic administration (Schechter, 1986a) but the
pretreatment with the D2 dopaminergic antagonist haloperidol significantly decreased
the effect (Schechter, 1986b). Such a finding as this could also be an indication that D2
57. 57
receptors play a role in mediating the stimulating effects of Catha edulis and its
alkaloids. These studies created momentum for further studies on Catha edulis and its
alkaloids to see if its effects are possibly mediated through the dopamine receptor.
1.6.3 Pharmacokinectic effects of cathamines in Catha edulis
The dopaminergic activity of cathinone has been studied intensively to explain the
psycho-stimulant effects that Catha edulis induces. These investigations focus on
cathinone as a compound, rather than as a constituent of a complex of other
compounds in varying concentrations in the plant (see section 1.5.2 to 1.5.4)
Prior to the 1990s, few studies were conducted on the pharmacokinetics of cathamines
since these were the focus of all pharmacological studies. To correlate blood
concentrations of cathamine alkaloids with the pharmacological effects produced by
Catha edulis intake in double-blind random study was conducted on six healthy
volunteers (Brenneisen et al., 1990). It was found that administration of cathinone 0.5
mg/kg (corresponding to the ingestion of 100 g of Catha edulis) in gelatin capsules
gave a peak plasma concentration of approximately 100 ng/mL after 72 ± 33 minutes.
When a standardized Catha edulis dose (equivalent to cathinone 0.8 mg/kg) was
ingested by the peak plasma concentration of (-)-cathinone was delayed up to 127
minutes, the time most likely needed for the extraction of the alkaloid from Catha
edulis (Widler et al., 1994). The study showed the concentration peak of (-)-cathinone
was 127 ng/mL, with a 4 hours elimination half-life and an area under the curve 0–9
hours of 415 ng/mL. This was slightly different to the earlier finding by (Halket et al.,
1995) which found the maximum plasma levels of cathinone to range from 40 to 140
ng/ml (mean 83 ng/ml) after one hour chewing of 60 g fresh Catha edulis leaves per
subject (n=3).
(-)-cathinone is known to metabolize to (-)-norephedrine 3 and (-)-norpseudoephedrine
1 (Figure 1-8) but also (+)-norpseudoephedrine can be found unchanged in urine in
nearly 24 hours (Maitai and Mugera, 1975; Widler et al., 1994). As these two
metabolites have a longer half life, their levels remain constant for more than 9 hours
from ingestion and their pharmacological effect is perpetuated. In comparison with (-)-
cathinone which gives a mean peak plasma concentration after 1.5 hours (Widler et al.,
58. 58
1994) need not be the only cathamine responsible for the sum effects of Catha edulis.
In view of this, both (-)-norephedrine 3 (Wellman, 1990), and (-)-norpseudoephedrine
1 (Nencini and Ahmed, 1989; Rothman et al., 2003) were suggested to be
pharmacologically active compounds, which contribute to the general effects of Catha
edulis chewing.
The levels of cathamines absorption were measured during a Catha edulis session in 4
non habitual users each chewing 0.6 g/kg (b.w.) plant material (Toennes et al., 2003)
and it was found that (-)-cathinone had a half-time elimination of 1.5 ± 0.81 hours.
However the study demonstrated that (+)-norpseudoephedrine was detectable in blood
samples up to 10 hours from ingestion and have a longer half-time elimination of 5.22
± 3.36 hours. No data could be determined for (-)-norephedrine which is both a
constituent of Catha edulis alkaloid as well as cathinone metabolite. In another
pharmacokinetic study measuring blood concentration of the phenylalkylamines
(cathinone, cathine and norephedrine) in 19 random cases of men suspected of driving
under the influence of Catha edulis it was found that the highest plasma
concentrations of cathinone was 173 µg/L while the norephedrine level was 250µg/L
(Toennes and Kauert, 2004).
Although the finding of the study may not be meaningful in terms of the variety of
Catha edulis chewed and the amount taken, as well as time lapse between intake and
measurement all of which are unknown it gives an indication as to the sort of levels of
cathamines which might be found in plasma.
1.7 Dependency issues
As noted above in 1.6.1 (-)-cathinone 2 is analogous to (+)-amphetamine 9 in structure
and in many of its effects are similar (Kalix et al., 1985). Generally it has observed that
drugs of abuse (e.g. Opiates, Cocaine and Amphetamine-types) circumvent the
negative feedback in the mesocorticolimbic dopamine system which is the key that
mediates the action of rewards for craving and reinforcement of drug-related stimuli
(Di Chiara, 1988).
59. 59
In vivo experimental models have shown that tolerance develops in rats after 10-15
days oral administration of Catha edulis extract (Schechter, 1990) and (-)cathinone has
been found to cause an increase in the level of dopamine in a dose dependent manner,
although of lesser magnitude than amphetamine (Pehek et al., 1990). With repeated
administration, cathinone and Catha edulis, like amphetamine, were found to produce
prolonged behavioural sensitization in rats, and chronic dependency on Catha edulis
extract has been shown to decrease levels of dopamine in the basal ganglia (Banjaw et
al., 2005).
Both cathinone and Catha edulis have also been shown to induce stereotyped
movements in rats including biting, licking, pawing, sniffing, head twitches, and
rearing (Zelger et al., 1980; Al-Meshal et al., 1991, Connor et al., 2002; Banjaw et al.,
2006, 2005). The prominent psychological effects of Catha edulis include euphoria,
elation, logorrhea, and an alleged increase in concentration (Kalix, 1984). Hence,
although there are actual physical limitations as to the nature of how much plant
material can be consumed by chewing, the less aversive nature of Catha edulis
(Gaudie et al., 1985) has led to the suggestion that the plant itself could have the
potential for higher dependency than amphetamine (Kalix, 1990).
Catha edulis taking is a complex behavoiur that is not only dependant on the
reinforcing psychostimulant, but also on the compulsive drive of users to secure their
daily fix (Feyisa et al., 2008) which is sanctioned by the cultural norm of khat-
chewing. Chronic use has been reported to induce continual psychological dependence
in humans (Nencine et al., 1989). However, while cravings and tolerance to the
increases of CNS effects are both common amongst habitual Catha edulis chewers,
there are no severe withdrawal symptoms reported other than lethargy, mild
depression, slight trembling and recurrent bad dreams (Kalix, 1985; 1990).
It is known that drugs which have a fast method of action have a high potential for
being addictive (Samaha, 2000). According to ICD 10 (the International Manual for
the Classification of Mental Disorders) dependence on drugs or alcohol is defined as a
syndrome and dependence on such substances is based on meeting three criteria out of
a list of six, in Figure 1-10. A person may use a substance for many years without
becoming dependent, if use does not result in harm, and they are in control of their
drug use.
60. 60
The effects of chewing Catha edulis i.e. the pleasurable effects that it affords have
been viewed as addiction fulfilling at least four out of the six ICD 10 criteria listed
above. Of note is the strong inducement for many users to procure by any means the
necessary supplies at least once a day and to repeat or to prolong the periods of
chewing, often at the expense of vital needs such as food.
1. A strong desire or compulsion to take the substance
2. Difficulties in controlling substance-taking behaviour
3. Physiological withdrawal state upon cessation of substance use
4. Evidence of tolerance to a substance
5. Neglect of alternative interests due to time spent using the substance
6. Persisting with substance use despite evidence of harmful consequences
Figure 1-10: WHO ICD 10 six criteria for the classifications of dependency on drugs
(http://www.who.int/classifications/icd/en/).
There is also the multi-drug use aspect that accompanies Catha edulis, particularly
cigarette smoking this is widely used to enhance the stimulant effect from chewing,
and alcohol is used increasingly to offset the “high” after chewing.
Catha edulis use behaviour has been seen as a manifestation of psychological
dependence, and an earlier expert group of the World Health Organization has come to
the conclusion that Catha edulis may induce moderate but often persistent
psychological dependence (WHO, 1980). Accordingly, discontinuation of its use after
prolonged use is said to produce minor symptoms of withdrawal such as slight
trembling, loss of energy and ambition, lethargy and mild depression, an increased
desire to sleep and nightmares with predominantly paranoid content (Halbach, 1972;
Luqman and Danowski, 1976; Kennedy et al. 1980; Kennedy, 1987; Cox et al., 2003).
Furthermore, although there is a limitation as to the amount of Catha edulis chewed in
a given time due to the bulkiness of the plant material, it has been suggested that
because of the craving coupled with the quasi-withdrawal symptoms and the tolerance
that develops to the inherent symapthomimetic effects, dependency on Catha edulis
61. 61
use is no different to that seen with amphetamine (Graziani et al., 2008).
In the NACRO report (2005) some subjects reported they would do „whatever was
necessary‟ to get a supply of Catha edulis if they ran out. However by far the majority
reported they would „go without‟ (44%) or „do other things‟ (40%), and only 4% of
respondents said they would drink alcohol or use other drugs. Asked if recent users
thought their Catha edulis use was out of control, 5% of men and 15% of women
responded in the affirmative, 27% and 50% respectively expressing a desire to stop
using Catha edulis.
1.8 Catha edulis use and psychiatric co-morbidity
There are two main factors that make it difficult to evaluate the CNS effects of Catha
edulis especially since the actions of its alkaloids and other constituents are not yet
fully investigated. However since Catha edulis is a natural substance:
1. It is not standardized and has a moderate effect in comparison with drugs like
cocaine
2. It has a dose-limiting absorption rate since after peak blood plasma
concentration in the initial 1.5-3 hours, the concentration can not be increased
(Wilder et al., 1996).
Catha edulis users report various subjective effects as a result of chewing the plant,
including increased levels of alertness, high energy flow, heightened self esteem and
sensations of elation. Habitual use of Catha edulis over a number of years is said to
lead to personality disorders and to an impairment of mental health (Kalix et al., 1985).
Frequent and excessive Catha edulis use (or abuse) may precipitate acute psychiatric
problems in certain individuals and the CNS-stimulating effect may result in agitation
and aggressiveness as well as manic behavior. In exceptional cases, Catha edulis
consumption was reported to cause psychosis (Giannini et al. 1992).
In a case report a patient who had grown Catha edulis for his own use in his own
home, and used it both excessively and frequently, was examined and said to have
