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Antigiardial Activity of some Cucurbita Species and Lagenaria Siceraria

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This study was carried out to evaluate antigiardial activity of Cucurbita maxima D, Cucurbita pepo L and Lagenaria siceraria. Variety supreme court seeds petroleum ether and methanolic extracts in vitro tests were perform using three concentrations (1000 ppm, 500 ppm and 250 ppm). The highest activity against Giaradia lambelia, with respect to time, was obtained from C. maxima seeds petroleum ether extract which exhibited 100% mortality within 48 giving IC50 of 548.80 ppm (with a concentration of 1000 and 500 ppm) followed by L. siceraria petroleum ether extract which exhibited 100% mortality within 72 hours with IC50 of 95.65 ppm whereas Metronidazol, a pure compound, (positive control) showed 100% mortality within 96 hours. On the other hand the lowest antigiardial activity was recorded by C. pepo petroleum ether extract (83.67% mortality with 500 ppm concentration within 96 hours ) giving IC50 of 60671.32ppm whereas C. maxima and L. siceraria methanol extract exhibited 100 % mortality within 96 hours with IC50 35.6ppm and 120 hours with IC50 8.9ppm respectively (with 1000ppm concentration). The best result was obtained by C. maxima petroleum ether extract at 250 ppm (up to 100% mortality within 72 hours) with IC50 1 ppm. This result will approve that this species (C. maxima) is a promising species in treating Giardia lambelia and agree with traditional claims.
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JOURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(4), 43-47 ISSN:23254513(PRINT) ISSN 2325 - 453X (ONLINE)
RESEARCH ARTICLE 43
Abstract This study was carried out to evaluate antigiardial
activity of Cucurbita maxima D, Cucurbita pepo L and Lagenaria
siceraria. Variety supreme court seeds petroleum ether and
methanolic extracts in vitro tests were perform using three
concentrations (1000 ppm, 500 ppm and 250 ppm). The highest
activity against Giaradia lambelia, with respect to time, was
obtained from C. maxima seeds petroleum ether extract which
exhibited 100% mortality within 48 giving IC50 of 548.80 ppm
(with a concentration of 1000 and 500 ppm) followed by L.
siceraria petroleum ether extract which exhibited 100% mortality
within 72 hours with IC50 of 95.65 ppm whereas Metronidazol, a
pure compound, (positive control) showed 100% mortality within
96 hours. On the other hand the lowest antigiardial activity was
recorded by C. pepo petroleum ether extract (83.67% mortality
with 500 ppm concentration within 96 hours ) giving IC50 of
60671.32ppm whereas C. maxima and L. siceraria methanol
extract exhibited 100 % mortality within 96 hours with IC50
35.6ppm and 120 hours with IC50 8.9ppm respectively (with
1000ppm concentration). The best result was obtained by C.
maxima petroleum ether extract at 250 ppm (up to 100%
mortality within 72 hours) with IC50 1 ppm. This result will
approve that this species (C. maxima) is a promising species in
treating Giardia lambelia and agree with traditional claims.
Index Terms Cucurbita maxima D, Cucurbita pepo L and
Lagenaria siceraria, Gairdia lameblia
I. INTRODUCTION
edicinal plants are still invaluable source of safe, less
toxic, lower price, available and reliable natural resources
of drugs all over the world. People in Sudan and in other
developing countries have relied on traditional herbal
preparations to treat themselves. Therefore, it is useful to
investigate the potential of local plants against these disabling
diseases [1and 2].
The treatment of giardiasis consists of the use of one or more
drugs, with metronidazole being the first choice. Other
nitroimidazolic derivatives (secnidazole, tinidazole, and
ornidazole), benzimidazoles (albendazole, mebendazole),
* Corresponding author: wasyko2002@yahoo.com
furazolin, quinacrine and paromomycin have also been
employed in therapeutic regimens. However, these drugs have
adverse effects including gastrointestinal disturbances, nausea,
headache, leucopoenia, myopia, neuralgia, and allergic
dermatitis and an unpleasant taste in the mouth. Furthermore,
they can lead to neurotoxic effects, ataxia, convulsions and
vertigo, bringing about the interruption of treatment. In addition,
mutagenic and carcinogenic effects have been described in
laboratory animals [3-8].
Thus the need of alternative drugs to reduce their burden of
purchasing the synthetic drugs especially after the problem of
getting resistant to many clinical patients against metronidazole
[9 and10] and thus new antigiardial drugs are probably required.
Cucurbits are well recognized source of secondary metabolites
such as alkaloids, flavonoids, phenols, saponins, tannins and
cucurbitacins (tetracyclic triterpenoids) which impart a bitter
flavor to many Cucurbits [11and 12]. Terpenoids which are rich
in oxygen, are of potent antigiardial activity [13].
From ancient time the seeds of the genus Cucurbita and
Lagenaria were used in treating intestinal parasites.
Experimental research was carried out at the Parasitology and
Chemistry laboratories of the Jorge Basadre Grohmann
National University, in Tacna, for testing Cucurbita maxima as
antiparasitic agent against canine tape worms in vitro and in
vivo using albino mice, It was found that the MIC of 23 gr. of
pumpkin seeds in 100 ml. of distilled water can produce an
antihelminthic effect [14].
With the purpose of searching for new antigiardial agents, in the
present work Cucurbita pepo, Cucurbita maxima and lagenaria
siceraria which are used traditionally for treatment of clinical
signs associated with giardiases were selected to evaluate the
activity of their petroleum ether and methanolic crude extracts
against Giardia lamblia trophozoites in vitro.
Antigiardial Activity of some Cucurbita
Species and Lagenaria Siceraria
Ihsan Mohamed Elhadi 1, Waleed S. Koko2*, Mahmoud M. Dahab2, Yahia Mohamed El Imam3,
Mona Abdu Elmonem Abdu El Mageed1
1Depart of pharmacognosy, Faculty of Pharmacy, Omdurman Islamic University, Sudan.
2Medicinal and Aromatic Plant Research Institute, National Center for Research, Khartoum, Sudan.
3Faculty of Pharmacy, The National Ribat University.
(Received April 09, 2013; Accepted July 04, 2013)
M
JOURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(4), 43-47 ISSN:23254513(PRINT) ISSN 2325 - 453X (ONLINE)
44
II. MATERIALS AND METHODS
Plant materials
The seeds of Cucurbita pepo L, Cucurbita maxima D, and
Lagenaria siceraria variety supreme court were collected
between April 2008 and August 2008. The seeds of Cucurbita
pepo, Cucurbita maxima were collected from Khartoum state
whereas Lagenaria siceraria variety supreme court was
gathered from Saudi Arabia. The plants were identified and
authenticated by the taxonomists Dr, Abdu Elgabar Nasir
Gumaa, Department of Biology, Faculty of Education,
Khartoum University. The seeds were air-dried and coarsely
ground to poweder.
Preparation of Crude extracts
30 grams of the coarsely ground material of the seeds were
successively extracted by Soxhlet apparatus using petroleum
ether, and methanol.
The extracts were then filtered and evaporated under reduced
pressure using rotatory evaporator.
Parasite isolate
G.lambelia used in all experiments were taken from patient. All
positive samples were examined by wet mount preparation.
Trophozoites of G. lambelia were performed at 37 1C in
RPMI 1640 medium containing 5% bovine serum. The
trophozoites were maintained for the assays and were employed
in the log phase of growth. Parasites were counted under the
microscope by haemocytometer chamber.
In vitro susceptibility assays
In vitro susceptibility assays used the sub- culture method of
Cedilla et al., [15]. This is highly stringent and sensitive method
for assessing the anti-protozoal effects (gold standard)
particularly in Entamoba histolytica, Gairdia intestinalis and T.
vaginalis [16].
5 mg from each extract was dissolved in 50 µl of dimethyl
sulfoxzide (DMSO) at eppendorf tube containing 950 µl D.W in
order to reach concentration of 5 mg/ml (5000ppm). The
concentrates were stored at -20°C for further analysis.
Sterile 96-well microtite plate was used for different plant
extracts, positive control and negative control.
Three out of 8 columns of microtitre plate wells ( 8 columns ×
12 rows ) were chosen for each extract, 40 μl of an extract
solution (5 mg/ml) were added to the first column wells C-1: On
the other hand , 20 μl of complete RPMI medium were added to
the other wells of` the second column and third column ( C-2
and C-3) . Serial dilutions of the extract were obtained by taking
20 µl of extract to the second column wells and taking 20 μl out
of the complete solution in C-2 wells to C-3 wells and
discarding 20 μl from the total solution of C-3 to the remaining
20 μl serial solutions in the successive columns. 80 μl of culture
medium was complemented with parasite and added to all wells.
The final volume in the wells was 100 μl.
Each test included metronidazole pure compound
[(1-(2-hydroxyethl)-2-methyl-5 nitroimidazole], a drug was
used as positive control in concentration 312.5 gml, whereas
untreated cells were used as a negative controls (culture
medium plus trophozoites). Samples were taken for counting at
0, 24, 48, 72, 96, and 120 hours.
For counting, the samples were mixed with Trypan blue in equal
volumes The final number of parasites was determined with
haemocytometer in triplicate.
The mortality % of parasite for each extract activity was carried
out according to the following formula:
Mortality of parasite (%) = Control negative- tested sample with extract×100
Control negative
Statistical analysis
All data were presented as means ± S.D. Statistical analysis for
all the assays results were done using Microsoft excel program.
Student t test was used to determine significant difference
between control and plant extracts at level of P < 0.05.
III. RESULTS AND DISCUSSION
Out of six extract investigated, 5 extracts (83.33) exhibited
100% mortality of parasite within 120 hours or less (Figure 1, 2,
3, 4, 5 and 6).
Out of 5 active extracts, one showed 100% mortality within 48
hours, one within 72 hours whereas one within 96 hours and two
within 120 hours.
Three extracts (60%) attained 100% mortality by
concentrations: (1000, 500 and 250ppm) and two extracts (40%)
attained 100% mortality only at 1000ppm.
Figure 1. In vitro activity of Cucurbita maxima seeds
petroleum ether extract against Giardia lambelia; mortality
percentage in relation to time
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45
Figure 2. In vitro activity of Cucurbita maxima seeds
methanolic extract against Giaradia lamblia
Figure 3. In vitro activity of Cucurbita pepo seeds petroleum
ether extract against Giaradia lambelia: ; mortality percentage
in relation to time
Figure 4. In vitro activity of Cucurbita pepo seeds methanolic
extract against Giaradia lambelia
Figure 5. In vitro activity of Lagenaria siceraria seeds pet
ether extract against Giaradia lambelia: ; mortality percentage
in relation to time
Figure 6. In vitro activity of Lagenaria siceraria seeds
methanol extract against Giaradia lambelia: ; mortality
percentage in relation to time
Giardia lamblia is an important cause of acute and chronic
gastrointestinal disease throughout the world and has been
identified as the etiologic agent in numerous waterborne
outbreaks of diarrheal disease. Although G. lamblia is among
the most prevalent enteric protozoal infections in humans, it is
relatively recently that improvements in t he in vitro cultivation
of this organism have allowed reliable, reproducible tests to
assess the in vitro activity of therapeutic agents against G.
lambelia [17].
Calzada et al.[18] reported that methanolic extracts of nineteen
plant species of Mexican origin, distributed among thirteen
families, and described potent giardicidal activity in six species
(Acalypha phleoides, Cnidoscolus tehuacanensis, Geranium
nievum, Hellianthella quinquenervis, Heliopsis longipes and
Teloxys graveolens), with IC50 values less than or equal to 20.64
μg/mL.
The results represented in Figures 1, 2, 3, 4. 5 and 6, revealed
that, the highest activity against Giaradia lambelia, with respect
JOURNAL OF FOREST PRODUCTS & INDUSTRIES, 2013, 2(4), 43-47 ISSN:23254513(PRINT) ISSN 2325 - 453X (ONLINE)
46
to time, was obtained from C. ma. seeds petroleum ether extract
which exhibited 100% mortality within 48 hrs giving IC50 of
548.80ppm (with a concentration of 1000 and 500ppm)
followed by L. s. petroleum ether extract which exhibited 100 %
mortality within 72 hours with IC50 of 95.65ppm whereas
Metronidazol, a pure compound, (positive control) showed
100% mortality within 96 hours. On the other hand the lowest
antigiardial activity was recorded by C. p. petroleum ether
extract (83.67% mortality with 500 ppm concentration within
96 hours ) giving IC50 of 60671.32ppm whereas C. ma. and L. s.
methanol extract exhibited 100 % mortality within 96 hours
with IC50 35.6ppm and 120 hours with IC50 8.9ppm respectively
(with 1000ppm concentration).
It had been clearly noticed that all studied extracts reached
100 % mortality except C. p. petroleum ether extract. The best
result was obtained by C. ma. petroleum ether extract at 250
ppm (exhibited 100% mortality within 72 hours) with IC50
1ppm. This result will approve that this species (C. ma) is a
promising species in treating Giaradia lambelia better than
synthetic antigiardial drugs.
The antigiardial activity of C. ma. and other studied species
could be due to the presence of triterpene (Cucurbitacins) as has
been demonstrated by Loiy et al., [19] who investigated the
antigiard [20] ial activities of Citrullus lanatus var. citroides
(wild watermelon) fruits petroleum ether, ethyl acetate, butanol
crude extracts as well as Cucurbitacin E and Cucurbitacin L
2-O-β-glucoside pure isolated compounds from C. lanatus var.
citroides. Cucurbitacin E and Cucurbitacin L 2-O-β-glucoside
were revealed to have strong potent antigiardial activity against
Giardia lamblia in vitro with IC50= 2 and 5 µg/ml after 5 days
respectively. It could be due to the presence of essential oil of
the seeds as had been demonstrated by Marisa et al., who
evaluated the anti-Giardia activity of phenolic-rich essential oils
obtained from Thymbra capitata, Origanum virens, Thymus
zygis subsp. Sylvestris chemotype thymol, and Lippia
graveolens aromatic plants. The tested essential oils inhibited
the growth of Giardia lamblia at IC50 (71 257) μg/ml since the
first hour of incubation and were able to kill almost 50% of the
parasites population in a time-dependent manner.
The phytochemical screening of C. ma. and the other studied
species revealed the presence of triterpene in the seeds
petroleum ether extract as well as methanolic extract, and GC-
Ms analysis of seeds oil declared the presence of Myristic,
Stearic, Palmitic, Linolenic, ω6, Arachidic acid and other fatty
acids [21]. All these constituents could be the causative factors
of antigiardial activity for the above investigated species.
IV. CONCLUSION
It has been concluded that the seeds of the studied species can
solved the problem of diarrhea that caused by Giardia
intestinalis instead of Metronidazol which has been
demonstrated to have side effects and they can be used
traditionally or can be formulated
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... We thought that cinnamon might have affected trophozoites attachment leading to their slipping and disintegration while with ginger many trophozoites were still attached in-situ and passed out after intestinal wash. The reduction of both fecal cyst and intestinal trophozoites was nearly similar to those in previously published reports (10,21,22,28,29,30,31). ...
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... We thought that cinnamon might have affected trophozoites attachment leading to their slipping and disintegration while with ginger many trophozoites were still attached in-situ and passed out after intestinal wash. The reduction of both fecal cyst and intestinal trophozoites was nearly similar to those in previously published reports (10,21,22,28,29,30,31). ...
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