the inhibitory properties of and mode of action of plant essential oils ...

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Gal/GalNAc: D-galactose/N-acetyl-D-galactosamine G-C FITCmAb: Fluorescein isothiocyante conjugated monoclonal antibody for Giardia cysts and Cryptosporidium oocysts GC-MS: Gas chromotography – Mass spectroscopy GIT: Gastrointestinal tract GLC: Gas liquid chromatography gPDI: Giardia protein-disulphide isomerase GRAS: Generally Recognised as Safe h: Hour HAART: Highly active antiretroviral therapy HBSS: Hanks balanced salt solution HCl: Hydrochloric acid HCT-8: Human colorectal adenocarcinoma cell line HIV: Human immunodeficiency virus HK: Heat killing HL-60 cells: Human lymphocyte-60 cells HMEM: Hanks minimal essential medium HPLC: High performance liquid chromatography hsp: Heat shock protein [ 3 H] thymidine: Tritiated thymidine HSV: Herpes simplex virus IC50: 50% Inhibitory Concentration (that quantity of drug required to inhibit 50% in the growth or viability of a population) IC90: 90% Inhibitory Concentration (that quantity of drug required to inhibit 90% in the growth or viability of a population) ID50: 50% Infectious Dose (dose required to cause infection in 50% of test subjects) IgG: Immunoglobulin type G I.M.: Intramuscular IMS: Immunomagnetic separation IOWA: Iowa isolate of Cryptosporidium parvum from BGF I.P.: Intraperitoneal I.V.: Intravenous kDa: Kilo Daltons k-DNA: Kinetoplast DNA kg: Kilogram kg -1 : Per kilogram LB: Lysis buffer LD50: 50% Lethal Dose (the dose which kills 50% of the animals tested) Lim.: Limonene µg: Microgram µl: Microlitre µm: Micrometre µM: Micromolar MD: Isolate of Cryptosporidium parvum from MRI Metro: Metronidazole MIC: minimum inhibitory concentration min: Minute ml: Millilitre v
ml -1 : Per millilitre MgCl2: Magnesium chloride mg/kg/day: Milligrams per kilogram per day MLC: minimum lethal concentration mm: Millimetre MPC-6: magnetic particle concentrator-6 MRI: Moredun Research Institute MRSA ref lab: Methicillin Resistant Staphylococcus aureas reference laboratory MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MW: Molecular Weight (marker) NaOH: Sodium hydroxide nm: Nanometres OM: Outer membrane Optimem: Modified Eagle’s Minimum Essential Medium PC: Phosphatidyl choline PCR: Polymerase chain reaction PEO: Plant essential oil PES: Phenazine ethosulphate PFOR: pyruvate:ferredoxin oxidoreductase PGE2: prostaglandin E2 Pin.: α-pinene PRBE: Polyphenol-rich blueberry extract PRFE: Polyphenol-rich fruit extract PV: Peripheral vesicle PVP: Polyvinylpyrrolidone QMU: Queen Margaret University RFLP: Restriction fragment length polymorphism RNA: Ribonucleic acid rRNA: Ribosomal Ribonucleic acid RPMI 1640: Basal media RT: Room temperature SCRI: Scottish Crop Research Institute SDS-PAGE: Sodium dodecylsulphate – Polymerised acrylamide gel electrophoresis Sec: Seconds SEM: Scanning electron microscopy SHARP: Soutra Hospital Archaeoethnopharmacological Research Project SI: Selectivity index Sp.: Sporozoites SPDL: Scottish Parasite Diagnostic Laboratory TAE buffer: Tris-acetate-EDTA buffer TBMM: Trypan blue, motility and morphological assay TEM: Tunnelling electron microscopy TEMED: N,N,N’, N’-Tetramethylethylenediamine TRAP-C1: Thrombospondin-related adhesive protein 1 TTO: Tea tree oil UK: United Kingdom vi
Page 1 and 2: A thesis submitted in partial fulfi
Page 3 and 4: ABSTRACT The main aims and objectiv
Page 5: ABBREVIATION LIST 1DE: 1-dimensiona
Page 9 and 10: FIGURE AND TABLE LIST CHAPTER 1 - I
Page 11 and 12: Figure 4.9 Incubation of T. cruzi e
Page 13 and 14: Life cycle.........................
Page 15 and 16: Effect of oil constituents on viabi
Page 17 and 18: 4:3.3 Epimastigote culture analysis
Page 19 and 20: 7:1 ABSTRACT.......................
Page 21 and 22: CHAPTER 1 Introduction 1
Page 23 and 24: ecome resistant to both chloroquine
Page 25 and 26: Leptomonas colosoma, Crithidia fasc
Page 27 and 28: viability of a population) determin
Page 29 and 30: changes in phospholipid composition
Page 31 and 32: killed by garlic (Zenner et al. 200
Page 33 and 34: Table 1.2 Plant oils that demonstra
Page 35 and 36: arouse, until the stomach was clear
Page 37 and 38: Dermatological reactions Many plant
Page 39 and 40: 2002). With industrialised nations,
Page 41 and 42: elongation and joining of large cyt
Page 43 and 44: the ER is the formation and folding
Page 45 and 46: of the cysts through the stomach ex
Page 47 and 48: Figure 1.5 Life-cycle of G. duodena
Page 49 and 50: Cryptosporidium is an apicomplexan
Page 51 and 52: Table 1.4 - Continued Species of Cr
Page 53 and 54: Upon excystation, sporozoites attac
Page 55 and 56: MacKenzie et al. 1994b). However, a
Page 57 and 58:
Figure 1.7 Life-cycle of C. parvum.
Page 59 and 60:
identified by the relative position
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Amastigote These replicative stages
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Life Cycle The sylvatic cycle start
Page 65 and 66:
Acute phase An intense and localise
Page 67 and 68:
inflammatory reactions induced by t
Page 69 and 70:
1:8 Research Hypothesis Little scie
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able to identify them molecularly a
Page 73 and 74:
CHAPTER 2 Giardia duodenalis The in
Page 75 and 76:
2:2 INTRODUCTION The protozoan para
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example, the understanding of antig
Page 79 and 80:
Jassim & Naji 2003) and more recent
Page 81 and 82:
2:3 MATERIALS AND METHODS 2.3.1 Par
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concentration equivalent to 0.02% v
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whole oil respectively. Elemi oil c
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Plus Protein standard; unstained; 1
Page 89 and 90:
2:4 RESULTS Ethanol and DMSO titrat
Page 91 and 92:
Figure 2.1 In vitro viability of G.
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and elemi was 0.005% although > 80%
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Trypan Stained / Non-viable Trophoz
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(10.90%). Whilst both the main cons
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Incubation of G. duodenalis trophoz
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Trophozoite protein profiling by SD
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used in this protein analysis exper
Page 105 and 106:
MIC of 0.005%. This phenomenon was
Page 107 and 108:
Effect of elemi oil on viability as
Page 109 and 110:
0.05). This effect though, was less
Page 111 and 112:
kDa 50- 37- 50- 37- 50- 37- MW G HK
Page 113 and 114:
2:5 DISCUSSION These experiments we
Page 115 and 116:
Using microscopic evaluation of par
Page 117 and 118:
cell death may have been initiated,
Page 119 and 120:
Using this information, investigati
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flavanones, anthocyanidins and isof
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protein was also abolished in elemi
Page 125 and 126:
CHAPTER 3 Cryptosporidium parvum In
Page 127 and 128:
3:2 INTRODUCTION The enteric parasi
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disease in extraintestinal location
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constituents and speed of activity.
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(empty), partially excysted and int
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and incubated for a further 1, 4 or
Page 137 and 138:
3:4 RESULTS 3:4.1 Acidification exp
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When compared with MD, the IOWA iso
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with incubation at 5ºC (-0.5 ± 1.
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constituent in palmarosa oil which
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Spontaneous Excystation Rate (%) 10
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3:5 DISCUSSION Interventions in the
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When these acid treated oocysts wer
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PEOs augment the effect of temperat
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spontaneous excystation within 4 h.
Page 155 and 156:
Trans-anethole is used as a flavour
Page 157 and 158:
CHAPTER 4 Trypanosoma cruzi The act
Page 159 and 160:
4:2 INTRODUCTION The disease caused
Page 161 and 162:
It is clear that alternative therap
Page 163 and 164:
analyse the activity of 12 differen
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µl of parasites. One 96 well plate
Page 167 and 168:
Incubation of T. cruzi epimastigote
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4:4 RESULTS Comparison of incubatio
Page 171 and 172:
MTS Absorbance (492nm) 1 0.9 0.8 0.
Page 173 and 174:
Page 175 and 176:
A B Absorbance (492nm) Trypan Stain
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Titration of PEOs As all the PEOs a
Page 179 and 180:
0.02% 0.01% 0.005% 0.0025% 0.00125%
Page 181 and 182:
Page 183 and 184:
Trypan Stained / Non-viable Epimast
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published concerning the trypanocid
Page 187 and 188:
using the MTS assay, where a lack o
Page 189 and 190:
With myrtle, 2 constituents working
Page 191 and 192:
mixed with inert carrier oils such
Page 193 and 194:
5:1 ABSTRACT The protozoan parasite
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37ºC over a 24 h period, in the ab
Page 197 and 198:
against Cryptosporidium with any su
Page 199 and 200:
polyphenol content of PRBE was 920
Page 201 and 202:
5:4 RESULTS Polyphenolic content of
Page 203 and 204:
Figure 5.1 A Viable and dead G. duo
Page 205 and 206:
Effect of FB, BB and PRBE on the sp
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5:5 DISCUSSION Here it is shown tha
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spontaneous excystation observed (F
Page 211 and 212:
certain concentration is exceeded (
Page 213 and 214:
6:1 ABSTRACT Current conventional t
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Flavonoids and phenolic acids form
Page 217 and 218:
efore any dilutions in TYI-S-33 cou
Page 219 and 220:
6:4 RESULTS Incubation of G. duoden
Page 221 and 222:
Trypan Stained / Non-viable Trophoz
Page 223 and 224:
6:5 DISCUSSION It was demonstrated
Page 225 and 226:
vitro, is not due to the degradatio
Page 227 and 228:
CHAPTER 7 The examination of latrin
Page 229 and 230:
7:2 INTRODUCTION The examination of
Page 231 and 232:
7:3 MATERIALS AND METHODS 7:3.1 Sou
Page 233 and 234:
Figure 7.2 Excavation of Soutra Ais
Page 235 and 236:
7:3.2 Sample preparations Formol et
Page 237 and 238:
magnet. The tubes were rocked throu
Page 239 and 240:
Using the methods of Smith et al. (
Page 241 and 242:
incubated at 55°C for 3 h in a wat
Page 243 and 244:
RNA Cryptosporidium gene to determi
Page 245 and 246:
concentration recommended in publis
Page 247 and 248:
7:4 RESULTS Microscopic examination
Page 249 and 250:
Figure 7.6 Fluorescence staining of
Page 251 and 252:
These slurries were subjected to IM
Page 253 and 254:
Figure 7.10 PCR analysis for Crypto
Page 255 and 256:
Pike 1967 & 1968; Jones 1982; Aspoc
Page 257 and 258:
the digestion enzymes DraI and AseI
Page 259 and 260:
CONCLUSIONS This series of experime
Page 261 and 262:
distortion, blebbing and increased
Page 263 and 264:
trophozoites and Trypanosoma epimas
Page 265 and 266:
Reduviid bug is a means of entry fo
Page 267 and 268:
anticryptosporidial activity may be
Page 269 and 270:
Historically, many PEOs and extract
Page 271 and 272:
REFERENCES Abdo, K.M., Cunningham,
Page 273 and 274:
Anthony, J-P., Fyfe, L. Smith, H.V.
Page 275 and 276:
Best, M., Sattar, S.A., Springthorp
Page 277 and 278:
in the chronic phase of experimenta
Page 279 and 280:
entidade morbida do homem. Memorias
Page 281 and 282:
Current, W.L., Upton, S.J. and Hayn
Page 283 and 284:
Elliot, B.C., Wisnewski, A.V., John
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Ferreira, L.F., Britto, C., Cardoso
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Gillin, F.D., Reiner, D.S. and Bouc
Page 289 and 290:
Hashmey, R., Smith, N.H., Cron, S.,
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Hunter, P.R. and Syed, Q. 2001. Com
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Kim, H.C. & Healey, J.M. 2001. Effe
Page 295 and 296:
Le Bourhis, B. 1968. Preliminary re
Page 297 and 298:
ornidazole. Transactions of the Roy
Page 299 and 300:
Moo-Puc, R.E., Mena-Rejon, G.J., Qu
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length polymorphism assay. Applied
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Pecevski, J., Savkovic, D., Radivoj
Page 305 and 306:
Reduker, D.W. & Speer, C.A. 1985. F
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Rothhammer, F., Allison, M., Nuñez
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Sgambatti de Andrade, A.L., Zicker,
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Sosa Estani, S., Segura, E.L., Ruiz
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Tyler, K.M. and Engman, D.M. 2001.
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Wang, M., Kikuzaki, H., Lin, C.C.,
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Yasunaka, K., Abe, F., Nagayama, A.
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Family: Burseraceae, Commiphora myr
Page 321 and 322:
Family: Lamiaceae, Pogostemon cabli
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Family: Rutaceae, Amyris balsamifer
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Family: Ericaceae, Vaccinium vitis-
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Family: Rosaceae, Rubus fruticosus
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Family: Rosaceae, Sorbus aucuparia
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Appendix 5. SDS-Polyacrylamide gel
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5. When the glass plates are in pla
Page 355 and 356:
Appendix 6: Incubation of G. duoden
Page 357 and 358:
Appendix 8. Agarose gel electrophor
Page 359:
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