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

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THE INHIBITORY PROPERTIES OF AND MODE OF ACTION OF PLANT ESSENTIAL OILS AND FRUIT EXTRACTS ON PROTOZOAN PARASITES JEAN-PAUL ANTHONY A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy QUEEN MARGARET UNIVERSITY 2008
ABSTRACT The main aims and objectives of this study was to determine if plant essential oils (PEOs) and polyphenol-rich fruit extracts (PRFEs) could reduce the viability of Giardia duodenalis trophozoites, Trypanosoma cruzi epimastigotes and Cryptospordium parvum oocysts in vitro. All PEOs tested reduced epimastigote and trophozoite viability at a concentration of 0.02% v/v, with titrations of the PEOs showing a concentration dependant decrease in viability. The minimum inhibitory concentrations (MICs) of PEOs demonstrated that myrtle and elemi oil were the most active PEOs (trophozoites = 0.005% v/v; epimastigotes = 0.00125% v/v) with the terpenes, α-pinene and limonene, constituents of these oils, being responsible for their action. Incubation of palmarosa oil and its terpene, geraniol, with C. parvum oocysts caused the almost complete excystation of oocysts (in the presence of increased temperature and time), with geranium oil and its terpene, citronellol, being nearly as effective. PRFEs reduce trophozoite viability, with 4 members of the Rosaceae Family causing complete reduction at 167 μg ml -1 , possibly through their ellagitannin content. Cloudberry extract was found to have an MIC comparable to the drug metronidazole (67 μg ml -1 ). The historical use of blueberries for the treatment of diarrhoeal diseases was demonstrated by the ability of blueberry PRFE, pressed juice and drink to kill trophozoites. Protein expression was both inhibited and upregulated in several proteins in whole cell lysates of PEO treated trophozoites, indicating a supplemental intracellular mode of action. Both PEOs and PRFEs cause morphological changes to epimastigotes and trophozoites through flagellar truncation and internalisation, swelling and rounding of the cell body, cytoplasmic condensation and the formation of large membrane protrusions. These indicate an action on the membrane itself with possible changes in osmoregulation. Both PEOs and PRFEs can be considered to be candidates for novel drug discovery for the treatments of cryptosporidiosis, giardiasis and American trypanosomiasis. Key words: Giardia duodenalis; Trypanosoma cruzi; Cryptosporidium parvum; plant essential oils; monoterpenes; terpenic compounds; fruits; polyphenol-rich fruit extracts. ii
Page 1: A thesis submitted in partial fulfi
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 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.
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identified by the relative position
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Amastigote These replicative stages
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Life Cycle The sylvatic cycle start
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Acute phase An intense and localise
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inflammatory reactions induced by t
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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
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2:4 RESULTS Ethanol and DMSO titrat
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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
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MIC of 0.005%. This phenomenon was
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Effect of elemi oil on viability as
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0.05). This effect though, was less
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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
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CHAPTER 3 Cryptosporidium parvum In
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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
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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.
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Trans-anethole is used as a flavour
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CHAPTER 4 Trypanosoma cruzi The act
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4:2 INTRODUCTION The disease caused
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It is clear that alternative therap
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analyse the activity of 12 differen
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µl of parasites. One 96 well plate
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Incubation of T. cruzi epimastigote
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4:4 RESULTS Comparison of incubatio
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MTS Absorbance (492nm) 1 0.9 0.8 0.
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Page 175 and 176:
A B Absorbance (492nm) Trypan Stain
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Titration of PEOs As all the PEOs a
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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
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using the MTS assay, where a lack o
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With myrtle, 2 constituents working
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mixed with inert carrier oils such
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5:1 ABSTRACT The protozoan parasite
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37ºC over a 24 h period, in the ab
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against Cryptosporidium with any su
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polyphenol content of PRBE was 920
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5:4 RESULTS Polyphenolic content of
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Figure 5.1 A Viable and dead G. duo
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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
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certain concentration is exceeded (
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6:1 ABSTRACT Current conventional t
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Flavonoids and phenolic acids form
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efore any dilutions in TYI-S-33 cou
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6:4 RESULTS Incubation of G. duoden
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Trypan Stained / Non-viable Trophoz
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6:5 DISCUSSION It was demonstrated
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vitro, is not due to the degradatio
Page 227 and 228:
CHAPTER 7 The examination of latrin
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7:2 INTRODUCTION The examination of
Page 231 and 232:
7:3 MATERIALS AND METHODS 7:3.1 Sou
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Figure 7.2 Excavation of Soutra Ais
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7:3.2 Sample preparations Formol et
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magnet. The tubes were rocked throu
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Using the methods of Smith et al. (
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incubated at 55°C for 3 h in a wat
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RNA Cryptosporidium gene to determi
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concentration recommended in publis
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7:4 RESULTS Microscopic examination
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Figure 7.6 Fluorescence staining of
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These slurries were subjected to IM
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Figure 7.10 PCR analysis for Crypto
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Pike 1967 & 1968; Jones 1982; Aspoc
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the digestion enzymes DraI and AseI
Page 259 and 260:
CONCLUSIONS This series of experime
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distortion, blebbing and increased
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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.
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Best, M., Sattar, S.A., Springthorp
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in the chronic phase of experimenta
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Current, W.L., Upton, S.J. and Hayn
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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
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Hunter, P.R. and Syed, Q. 2001. Com
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Kim, H.C. & Healey, J.M. 2001. Effe
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Le Bourhis, B. 1968. Preliminary re
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Moo-Puc, R.E., Mena-Rejon, G.J., Qu
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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
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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
Page 353 and 354:
5. When the glass plates are in pla
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Appendix 6: Incubation of G. duoden
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Appendix 8. Agarose gel electrophor
Page 359:
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