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African Journal of Traditional, Complementary and Alternative Medicines
African Ethnomedicines Network
ISSN: 0189-6016
Vol. 1, Num. 1, 2004, pp. 55-62
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African Journal. Traditional, Complementary and Alternative Medicines Vol.
1, Num. 1, 2004, pp. 55- 62
Research Paper
ANTITRICHOMONAL ACTIVITY OF 1,3-DIARYL-2-PROPEN-1-ONES ON TRICHOMONAS
GALLINAE
A.O.Oyedapo1, V. O. Makanju1, C. O. Adewunmi1,
E.O.Iwalewa2, and T. K. Adenowo3.
1Drug Research and Production Unit, 2Department of Pharmacology,
Faculty of Pharmacy, 3Department of Anatomy and Cell Biology, College
of Health Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria. E-mail:cadewumi@yahoo.com
Code Number: tc04006
Abstract
1,3-diaryl-2-propen-1-ones were synthesized by the Claisen - Schmidt condensation
method. T. gallinae parasites isolated from domestic pigeon were cultured in
vitro. The in vitro susceptibility of T. gallinae was evaluated
in multi-well plates at 37oC. Four of the synthetic compounds produced
significant antitrichomonal activity against T. gallinae. The minimal
lethal concentrations (MLCs) (produced by) 2'-hydroxy-4-methoxychalcone,
2'-hydroxy-2,4'-dimethoxychalcone, 2'-hydroxy-4-chlorochalcone,
3,4,4'-Trimethoxychalcone and 4-hydroxcychalcone were 100.0, 0.78, 50.0,
50.0 and 3.13 µg/ml respectively. The results indicate that 2'-hydroxy-2,4'-dimethoxychalcone
and 3 other synthetic 1,3-diaryl-2-propen-1-ones possess potent antitrichomonal
activity against T. gallinae. However, the studies on cytotoxicity effect
showed that all the active chalcones demonstrated a very low haemagglutination
titre
values ranging between 0.57 -4.06 suggesting their low toxicity profile.
Keywords: Trichomonas gallinae, 1,3-diaryl-2-propen-1-ones, in
vitro activity
Introduction
Trichomonas gallinae is a flagellated protozoan living in the upper digestive
tract and in various organs of different avian groups, especially columbiformes
(doves and pigeons). The domestic pigeon, Columba livia, (Anth) is the
primary host of this parasite as well as certain dove species such as white-wing
doves. T. gallinae is the causative agent of canker in pigeons and causes
a variety of pathologic manifestations depending on the strain of the parasite
and species of bird it infects (Baker, 1986; Cooper and Petty, 1988; Henderson
et al., 1988; Jessup, 1980). The pathologic changes associated with T. gallinae infection
of the upper digestive tract of birds range from mild inflammation of the mucosa
to large caseous lesions that block the lumen of the esophagus. Pigeons however
are more susceptible to secondary organ invasion (liver, air sacs, lung, and
brain) by virulent strains of the parasite. Necrotic lesions develop in these
organs leading to the death of the host (Stabler, 1954).
The effectiveness of a drug (2-amino-5-nitrotiazole), against pigeon trichomonosis
caused by the protozoan Trichomonas gallinae has been reported (Stabler
and Mellentin, 1951), and this led to the discovery of the activity of a nitroimidazole
derivative (metronidazole) against T. vaginalis (Cosar and Julou, 1959).
The latter resulted in the opportunity for a significant improvement in the
treatment of trichomonad infections in humans (Forsgren and Forssman, 1979).
Metronidazole was also shown to be effective against T. gallinae (Bussieras
et al., 1961). Metronidazole has been the drug of choice for treatment of human
urogenital trichomonosis since its discovery (Krieger et al., 1985). In avian
veterinary medicine, several nitroimidazoles including metronidazole, dimetridazole,
ronidazole and carnidazole, have been developed as effective drugs against T.
gallinae (Franssen and Lumeij, 1992). In man, cases of clinical resistance
to nitroimidazoles are rare despite their extensive use worldwide (Edwards,
1993). However, resistance to drugs in avian trichomonosis management has been
reported using three isolates from wild birds (Munoz et al., 1998).
In our
continued efforts at examining synthesized chalcone and related compounds
for possible biological activities, a number of chalcones [2-9] and
1,3-diaryl-2-propen-1-ones [10-11] were examined for antitrichomonal
activities. (Figure 1)
Chalcones and other biogenetically related compounds are collectively called
flavonoids and many of their derivatives have been tested for biological activities
which includes molluscicidal (Adewunmi et al, 1987) and antimicrobial
(Gabor et al, 1967) activities. They have also been found to exhibit
anti-inflammatory (Viana et al, 2003; Pushkar and Balawant, 2001; Tewtrakul et
al, 2003), anti-oxidant (Miranda et al, 2000; Repetlo and Lesuy,
2002) and analgesic (Viana et al, 2003; Azarifar and Ghasemnejad, 2003)
activities. An interesting chalcone from natural product 2',6'-dihydroxy-4'-methoxychalcone
isolated from Piper aduncum (Piperacea)
produced significant antiprotozoal activity against Leishmania amazonensis (Torres-Santos
et al., 1999). There have been no reports of chalcones or any of their derivatives
for anti-trichomonal activity. In view of the problems associated with drug
resistance in the treatment of avian T. gallinae, some synthesized chalcones
and related compounds were evaluated as potential anti-trichomonal agents.
Material and Methods
Synthesis of 1,3-diaryl-2-propen-1-ones
Using Claisen - Schmidt condensation method (Adewunmi et al., 1987), appropriate
acetophenones were condensed with benzaldehydes to give 3, 4,- methylenedioxy-21-
hydroxychalcone[2], 21-hydroxy-4-methoxychalcone [3],
21-hydroxy-3,4,41-
trimethoxychalcone [4], 21-hydroxy-2,41-dimethoxychalcone
[5], 21-hydroxy-4-chlorochalcone
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[6], 2-methoxy-41-hydroxychalcone
[7], 3, 4, 4-trimethoxychalcone
[8], 4-hydroxychalcone [9], 1-Phenyl-3-(2-thienyl)-2-propen-1-one
[10]
and 1-Phenyl-3-(2-furyl)-2-propen-1-one [11] in 65-81% yield.
Stuctural
elucidations of the compounds were carried out using Gallenkamp apparatus
for melting point determination. The IR spectra were run on a Pye Unicam SP
3-300
IR spectrophotometer as potassium bromide pellets.1Hmr spectra were taken on a
Bruker WM 300 nuclear magnetic resonance using tetramethylsilane (TMS) as internal
standard and deuteriodimethyl sulphoxide as solvent. Mass spectra were determined
using MAT 44S spectrometer at 70ev while the elemental analyses were obtained
with Carlo Erba 1106 elemental analyzer.
Biological assays
Samples of Trichomonas gallinae from the buccal cavity and oesophagus
of the local pigeon (C. livia) were taken with a cotton swab to detect
the presence of the protozoa in the birds' upper digestive tract. The parasites
were cultured xenically in vitro, in Locke-egg (LE) medium (NIH modification
of Boeck and Drbohlav's medium) (Von Brand et al., 1943) without antibiotics,
pH 7.2, supplemented with 10% heat inactivated bovine serum, at 37 °C.
Previous studies have shown that the presence of antibiotics in the culture
medium affected the pathogenicity level and decreased the haemolytic activity
of T. gallinae isolates (Stabler, 1954). Isolates were subcultured every
48 h. The trichomonads in the logarithmic phase of growth and subcultured every
48 h exhibited more than 95% mobility and normal morphology.
The method used in the in vitro assay was essentially as previously described
(Meingassner and Thurner, 1979). Sterile, multi-well plates were used to incubate
the isolates with the corresponding drug dilutions. Different assays, with
each compound were performed in triplicate. The plates were incubated for 48
h at 37oC under aerobic conditions. The wells were examined after
24 and 48 h of incubation with an inverted microscope. The MLCs for each drug
were thus obtained by means of the observation with the microscope. These MLCs
were defined by the lowest drug concentrations in which no motile organisms
were seen or no growth was detected after 24 and 48 h of cultivation, respectively.
Cytotoxicity assay
The cytotoxicity of the 10 chalcones were monitored by haemagglutination activity
using formaldehyde fixed bovine erythrocytes as described by Peumans et al.,
(1982), Sadique et al., (1989) and Wang et al., (1995).
Preparation and Fixation of bovine erythrocyte:
Bovine (Bos taurus) erythrocytes fixed with formalin were prepared according
to the modified procedures of Sadique et al., (1989). Fresh blood sample was
collected from N'Dama, (a representative of B. taurus breed) into
a sterile conical flask containing 3.8% (w/v) trisodium citrate and mixed thoroughly.
Then, 20 ml of blood was centrifuged at 4000 rpm for 10 min. on a Gallenkamp
centrifuge. The packed red blood cells were washed with 10 mM phosphate buffer
saline (PBS) pH 7.2 until a clear supernatant
was
obtained. The washed packed RBC were suspended in (5%v/v) formaldehyde - phosphate
buffer saline (1:12.3 v/v) solution. The mixture was left at room temperature
for 24 h. The final fixed RBC were washed and centrifuged with PBS 3 times,
and preserved with 0.1% methyl parabene to prevent microbial growth and stored
at 4o C.
Heamagglutination assay:
100 µl of PBS was pipetted into 96 well microtitre plates. The first row
was used as control without the compounds. The compounds (100 µl) were
added into the first well of the second row, and a 2-fold serial dilution was
made until the last well in row three. Then 50 µl of fixed bovine erythrocytes
was added to all the wells. They were incubated at room temperature for 1 h.
The presence of buttons in the centre of the well indicates no agglutination
and the heamagglutination titre value of the drugs were estimated as the reciprocal
of the last dilution showing agglutination
Chemicals
All the chemicals were obtained from various sources, all are of Analar grade.
Formaldehyde ( Sigma), Ethanol (BDH), Methanol (BDH), Sodium tri citrate (BDH),
DMSO -Dimethyl sulfoxide (Sigma), DPPH (Sigma), Ascorbic acid (BDH Laboratories),
Methyl paraben (Sigma), Sodium chloride (BDH), Na2HPO4 and NaH2PO4.
Results
Eight of the ten 1,3-diaryl-2-propen-1-ones examined were chalcones
[2-9] while two were 1-Phenyl-3-(2-thienyl)-2-propen-1-one [10]
and 1-Phenyl-3-(2-furyl)-2-propen-1-one [11]. The results of the effects
of the synthetic compounds on T. gallinae are shown in Table
1 The cytotoxicity/haemagglutination
assay results are presented in Table
2. All the compounds exhibited very low
haemagglutination (HA) titre values with a wide range of concentrations at
which agglutination occur on fixed bovine erythrocytes.
According to Table 2, the chalcones and the reference drugs produced various
degrees of protection on the bovine RBC membrane and exhibited very low haemagglutination
(HA) titre values ranging between 0.57 -4.06. The order of protection
of these agents was: 2'-Hydroxy-4-chlorochalcone [6] > 3,4,4'-Trimethoxychalcone
[8] > 2'-Hydroxy-4-methoxychalcone [3] = Acetylsalicylic
acid [ASA] > 2'-Hydroxy,2,4-dimethoxychalcone[5] = Metronidazole
[MTZ] > 4-Hydroxychalcone [9]. (This was shown in the following concentrations
through which agglutination occur in decreasing order respectively: 1.75 +
0.56, 1.50 + 0.00, 1.25 + 0.34, 1.25 + 0.34, 0.375 + 0.00, 0.375 + 0.00 and
0.25 + 0.06).
Discussion
The chalcones examined were those with hydroxyl substitutent
at position 2' [2-6] and those without substituent at position
2' [7-9].
Five of the chalcones (3, 5, 6, 8 and 9) showed antitrichomonal
activity at concentrations equal to and below 100 mg/ml.
It is interesting to observe that 1-phenyl-3-(2-thienyl)-2-propen-1-one [10]
and 1-phenyl-3-(2-furyl)-2-propen-1-one [11] were not active. This suggests
that 1-thienyl, and 1-furyl substitutents do not enhance anti-trichomonal activities
of 1,3-diaryl-2-propene-1-ones. The observed activity of chalcones (3, 5 and 6)
is not surprising because of the 2'-hydroxyl substituent which could
form flavones or isoflavones through intra-molecular cyclisation leading to
enhanced activity. Compound [4] showed little anti-trichomonal activity
compared with compound [5] which is the most active product. The difference
in potency may be due to the intramolecular formation of methylenedioxy substituent
by the 3,4-dimethoxy substituent on [4] as witnessed in the inactivity
of 2'-hydroxy-3,4-methylenedioxychalcone [2].
The five synthesized
chalcones, metronidazole and acetylsalicylic acid were subjected to the cytotoxicity
abilities on bovine fixed RBC to investigate any correlation between their
cytotoxic abilities through haemagglutination titre assay and anti-trichomonal
activities. We therefore use this method to explain the safety and efficacy
of synthesized chalcones against trichomonal infection. It appears that the
low cytotoxicity ability of [3], [6], [8] and acetylsalicylic
acid has contributed to their ineffetiveness against T. gallinae. In
this way, they have very low titre values, an indication of low cell destructive
property thereby leaving these organisms without lethal effects. However,
metronidazole (MTZ), [5] and [9] produced high HA values indicating
higher cytotoxicity than that of [3], [6], [8] and acetylsalicylic
acid. In order words, there appears to be some correlation between the anti-trichomonal
activity of the compounds and the haemagglutination titres.
The most active compound, 5, was almost as potent as metronidazole against T.
gallinae. Although the active compounds were not tested against metronidazole-resistant
strains of T. gallinae, the targets of these compounds may be different from
that of metronidazole. Whether or not this renders cross-resistance unlikely
must be tested in future studies. More work needs to be done on other derivatives
of 1,3-diaryl-2-propene-1-ones to decide whether they can be successfully developed
for use in chemotherapy of trichomonosis. In particular, the nature of the
substitutions at R1, R2, R3 and R4, should be studied
further.
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