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

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Statistical analysis........................................................................................180 5:4 RESULTS..........................................................................................................181 Polyphenolic content of FP, BB and PRBE..............................................................181 Survival of G. duodenalis trophozoites in FB, BB and PRBE..................................181 Effect of FB, BB and PRBE on the spontaneous excystation of C. parvum oocysts.185 5:5 DISCUSSION....................................................................................................187 CHAPTER 6 - The effect of polyphenol-rich fruit extracts on Giardia duodenalis viability.................................................................................................192 6:1 ABSTRACT......................................................................................................193 6:2 INTRODUCTION............................................................................................194 6:3 MATERIALS AND METHODS.....................................................................196 6:3.1 Parasite culture...............................................................................................196 Source of G. duodenalis trophozoites...........................................................196 Preparation and analysis of polyphenol contents of PRFEs........................196 Preparation of ellagic acid..........................................................................196 6:3.2 Experimental treatments of parasites............................................................197 Incubation of G. duodenalis trophozoites with PRFE..................................197 Incubation of G. duodenalis trophozoites with metronidazole.....................197 6:3.3 Parasite culture/experimental analysis..........................................................198 G. duodenalis cell count assay......................................................................198 Statistical analysis........................................................................................198 6:4 RESULTS..........................................................................................................199 Incubation of G. duodenalis trophozoites with PRFE..............................................199 Screening of PRFEs......................................................................................199 Titration of the most active PRFEs...............................................................200 Incubation of trophozoites with ellagic acid............................................................200 6:5 DISCUSSION....................................................................................................203 CHAPTER 7 - The examination of latrine and cess pit sediments from 2 different localities in Britain and eras of history for protozoan parasites, with a special emphasis on Cryptosporidium spp.............................................................207 xvii
7:1 ABSTRACT......................................................................................................208 7:2 INTRODUCTION............................................................................................209 7:3 MATERIALS AND METHODS.....................................................................211 7:3.1 Source of archaeological samples..................................................................211 Soutra cess pit samples.................................................................................211 York latrine samples.....................................................................................212 7:3.2 Sample preparation........................................................................................215 Formol ether concentration of samples........................................................215 Trisodium phosphate rehydration and resuspension of samples..................216 Immunomagnetic separation (IMS) of samples............................................216 Fluorescence staining of samples.................................................................218 DNA extraction from oocyst positive slides or from IMS beads-oocyst complexes......................................................................................................219 Sample preparation for gel electrophoresis.................................................221 7:3.3 Sample analysis...............................................................................................221 Microscopic examination of sample concentrates – light microscopy.........221 Microscopic examination of sample concentrates – Fluorescence stained samples..........................................................................................................222 Polymerase chain reaction (PCR) of samples..............................................222 Oligonucleotide primers...............................................................................224 Enzymatic digestion of positive secondary PCR products and RFLP analysis.........................................................................................................225 Gel electrophoresis.......................................................................................226 7:4 RESULTS..........................................................................................................227 Microscopic examination of sample concentrates – light microscopy.....................227 Microscopic examination of sample concentrates – Fluorescence stained samples228 PCR analysis of samples...........................................................................................230 Enzymatic digestion of positive secondary PCR products and RFLP analysis........231 7:5 DISCUSSION....................................................................................................234 CONCLUSIONS.....................................................................................................239 xviii
Page 1 and 2: A thesis submitted in partial fulfi
Page 3 and 4: ABSTRACT The main aims and objectiv
Page 5 and 6: ABBREVIATION LIST 1DE: 1-dimensiona
Page 7 and 8: ml -1 : Per millilitre MgCl2: Magne
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: 4:3.3 Epimastigote culture analysis
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
Page 61 and 62: Amastigote These replicative stages
Page 63 and 64: 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
Page 71 and 72:
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
Page 77 and 78:
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
Page 83 and 84:
concentration equivalent to 0.02% v
Page 85 and 86:
whole oil respectively. Elemi oil c
Page 87 and 88:
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.
Page 93 and 94:
and elemi was 0.005% although > 80%
Page 95 and 96:
Trypan Stained / Non-viable Trophoz
Page 97 and 98:
(10.90%). Whilst both the main cons
Page 99 and 100:
Incubation of G. duodenalis trophoz
Page 101 and 102:
Trophozoite protein profiling by SD
Page 103 and 104:
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
Page 121 and 122:
flavanones, anthocyanidins and isof
Page 123 and 124:
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
Page 129 and 130:
disease in extraintestinal location
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constituents and speed of activity.
Page 133 and 134:
(empty), partially excysted and int
Page 135 and 136:
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.
Page 143 and 144:
constituent in palmarosa oil which
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Spontaneous Excystation Rate (%) 10
Page 147 and 148:
3:5 DISCUSSION Interventions in the
Page 149 and 150:
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
Page 165 and 166:
µl of parasites. One 96 well plate
Page 167 and 168:
Incubation of T. cruzi epimastigote
Page 169 and 170:
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
Page 177 and 178:
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
Page 185 and 186:
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
Page 195 and 196:
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
Page 207 and 208:
5:5 DISCUSSION Here it is shown tha
Page 209 and 210:
spontaneous excystation observed (F
Page 211 and 212:
certain concentration is exceeded (
Page 213 and 214:
6:1 ABSTRACT Current conventional t
Page 215 and 216:
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
Page 285 and 286:
Ferreira, L.F., Britto, C., Cardoso
Page 287 and 288:
Gillin, F.D., Reiner, D.S. and Bouc
Page 289 and 290:
Hashmey, R., Smith, N.H., Cron, S.,
Page 291 and 292:
Hunter, P.R. and Syed, Q. 2001. Com
Page 293 and 294:
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
Page 301 and 302:
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
Page 307 and 308:
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.
Page 315 and 316:
Wang, M., Kikuzaki, H., Lin, C.C.,
Page 317 and 318:
Yasunaka, K., Abe, F., Nagayama, A.
Page 319 and 320:
Family: Burseraceae, Commiphora myr
Page 321 and 322:
Family: Lamiaceae, Pogostemon cabli
Page 323 and 324:
Family: Rutaceae, Amyris balsamifer
Page 325 and 326:
Family: Ericaceae, Vaccinium vitis-
Page 327 and 328:
Family: Rosaceae, Rubus fruticosus
Page 329 and 330:
Family: Rosaceae, Sorbus aucuparia
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Appendix 5. SDS-Polyacrylamide gel
Page 353 and 354:
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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