In vitro Anti-schistosomal Activity of "Plectranthus tenuiflorus" on Miracidium, Cercaria and Schistosomula Stages of Schistosoma mansoni
I.Z. Abdel Aziz,
Ayman A. El-badry
Sabah H. El-Gayed
Human schistosomiasis is the second most prevalent tropical disease after malaria and has considerably public health and socioeconomic burden in developing world. The only drug available for treating schistosomiasis is praziquantel, however it lacks efficacy against schistosomulae and there are already reports of resistance to its use in treatment, making it necessary to explore and upsurge for new compounds to combat schistosomiasis. This study was designed to evaluate anti-Schistosoma activity of the methanolic extract of Plectranthus tenuiflorus (Vatke) Agnew; an herb on different stages of Schistosoma mansoni. Parasite viability and morphological changes were assessed after incubation with different dilutions of the plant extract using inverted as well as scanning electron microscopy. Plectranthus tenuiflorus (Vatke) Agnew extract showed moderate anti-cercaria and anti-schistosomulae activity with calculated IC50 12.29 and 17.39, respectively compared to lesser anti-miracidium activity with IC50 24.37 mg/100 mL. This is the first study to assess anti-schistosoma potency of Plectranthus tenuiflorus (Vatke) Agnew extract and may suggest a promising medicinal constituent of Plectranthus tenuiflorus (Vatke) Agnew extract importantly against cercaria infective stage and the significantly ambulant invasive Schistosomulae inside human body which impose further studies to evaluate different extracts and fractions of this plant.
to cite this article:
I.Z. Abdel Aziz, Ayman A. El-badry and Sabah H. El-Gayed, 2011. In vitro Anti-schistosomal Activity of "Plectranthus tenuiflorus" on Miracidium, Cercaria and Schistosomula Stages of Schistosoma mansoni. Research Journal of Parasitology, 6: 74-82.
Received: April 08, 2011;
Accepted: July 06, 2011;
Published: December 02, 2011
Schistosomiasis is the one of the most important communicable disease caused
by a blood born fluke of the genus schistosoma. It is the second most
prevalent tropical disease in Africa after malaria. It is of relatively low
mortality; however, it is of high burden in terms of chronic pathogenicity and
disability (WHO, 1998).
Praziquantel is the only drug available for the treatment and control of schistosomiasis
with good safety and efficacy profile against all schistosome species parasitizing
humans (Doenhoff et al., 2008; Black
et al., 2009) however, praziquantel lack the efficacy against schistosomulae,
the young developing stages of the parasite. This may be explained the low cure
rates and rapid re-infection rates in areas of heavy transmission of schistosomiasis,
where patients may be concurrently infected with juvenile and adult parasites
(NGoran et al., 2003). In addition tolerance
or resistance to praziquantel may already exist or might be developing with
the increasing use of praziquantel (Cioli and Pica, 2003;
Keiser and Utzinger, 2007).
Medicinal plants are being used for hundreds of years as therapeutics worldwide.
Being natural products are safer than their synthetic equivalent (Elujoba
et al., 2005). The family Lamiaceae contains several genera with
a rich diversity of ethnobotanical uses. It has an important role as a source
of medicinal plants and as an aromatic of commercial importance. Plectranthus
is a large genus found in Tropical Africa, Asia and Australia and containing
about 300 species, 62 of them were reported to be used as medicines, ornamentals,
foods, flavors and fodder. Genus Plecteranthus (P.) which belongs
to family Lamiaceae is represented in Saudi Arabia by six species: P. arabicus,
P. cylindraceys, P. tenuiflorus, P. barbatus, P. lanuginosus
and P. asirensis. Plecteranthus tenuiflorus (P. tenuiflorus);
"Shaar" in Arabic, is one of the medicinal plants in the genus Plecteranthus
(Colenett, 1998). Ryding and Paton
(2001) reported that Plecteranthus aegyptiacus is the correct name
of P. tenuiflorus. P. tenuiflorus essential oil as its crude extract
showed an anti-phytoviral against different plant viruses like Tobacco Necrosis
Virus (TNV) (Othman and Shoman, 2004; Lukhoba
et al., 2006).
Thus, our study was designed in attempt to seek out for new natural anti-schistosomal chemotherapy, one plant of the family Lamiaceae, Plectranthus tenuiflorus (P. tenuiflorus) (Vatke) Agnew was selected to evaluate its methanol leaves extract activity against miracidium, cercaria and schistosomula stages of Schistosoma mansoni in vitro.
MATERIALS AND METHODS
Plant material: Plant was collected in March 2007 and April 2008 from
different region in south and east Saudi Arabia around Jeddah and al Taeif,
done by author while a scientific mission stay in Saudi Arabia.
Preparation of extract: Fresh herb of P. tenuiflorus was cut into small pieces, dried in shade for fifteen days and finally in oven below 60. The dried plant material (1 kg) was ground into fine powder and exhaustively extracted with methanol. The extract was concentrated under vacuum at 40. The dark green viscous residue (65 g) was kept away from light in a refrigerator at 4°C different concentrations of this extract was suspended in 100 mL of methanol and shaken automatically for 30 min then centrifuged. The clear supernatant solution was used to carry out the different experiments.
Preparation of parasite stages: Preparation of Egyptian strain of
Schistosoma mansoni stages miracidium, cercaria and syringe shed schistosomulae
was performed freshly just before testing the plant extract by Schistosome Biological
Supply Center SBSC team, Theodor Bilharz Research Institute, Imbaba. In brief,
collection of eggs from murine intestines was performed according to the method
of Liang and Kitikoon (1980), collected viable eggs
were pipetted into a small Petri dish (1.5x6 cm) and the dish was placed under
ceiling illumination for hatching of miracidia. The entire procedure takes approximately
15 min and miracidia usually appear within 5 min. Biomphalaria glabrata snails
used in this study were bred and infected with Schistosoma mansoni in
SBSC lab. The snails were induced to shed cercariae under electric light for
nearly 10-20 min, infectious cercaria were examined and counted per one mL.
The entire procedure takes less than one hour. This procedure dramatically reduces
the mortality among snails due to prolonged shedding time (Liang
et al., 1987). Some of the recently shed infectious cercariae were
subjected to sheering stress through a syringe with a 0.8 mm needle. The cercariae
were passed through the needle 20 times or more, until they had lost their tail
and were transformed into schistosomulae. This is a rough method compared to
transformation of cercariae to schistosomulae by passing through a mouse skin
(Clegg and Smithers, 1972). Nevertheless, under careful
microscopic examination Schistosomulae were counted and were assured
that only healthy schistosomulae were used in the experiment, only 50 cercariae
were added in each well. In control wells only 500 μL of cell culture medium
was added to the same number of each parasite stage.
In vitro evaluation of anti-Schistosomal activities of medicinal
plants: In vitro experiments were carried out in flat bottomed microliter
plates with 24 wells. The 400 mg/100 mL was attempted as the basic concentration
in the first well, knowing that this may not include all plant constituents
equally. Four lower concentrations of plant extract 300, 250, 50 and 25 mg/100
mL were used; dilution was done using cell culture medium (100 mL, RPMI 1640
supplemented with glutamin, antibiotics and 5% fetal calf serum) and served
as incubation media for all Schistosoma mansoni stages in our experiments.
To facilitate counting and testing each stage separately, each dilution was
prepared then divided into three identical aliquots for the three tested stages.
Each concentration was tested in triplicate and at least two experiments were
performed on separate occasions. The parasite stages in each well were carefully
examined and counted. Since Schistosomulae is a stage that only occurs in
vivo and survives shortly when produced by this sheering method (Molgaard
et al., 2001), as well as the cercariae are known to lose infectivity
rapidly, examination of plant effect was carried out after ½, 2, 4 and
6 h of incubation at 37°C and not prolonged to 24 or 48 h. Parasite viability
was determined by direct microscopy at 40x magnification using inverted microscope
(Nikon). For determination of the extract effect; parasites in each well was
counted in duplicate, with the live parasite identified as freely swimming with
no structural changes; while dead parasite was recognized by complete loss of
motility and was laying at the bottom of the well (Holtfreter
et al., 2011).
Scanning electron microscopy for parasite stages morphological alteration:
Four hours after incubation with plant extract in different concentrations,
parasite stages were processed for electron microscopic examination by the help
of the staff in Electron Microscopy Department, Theodor Bilharz Research Institute,
Imbaba. In brief; specimen were fixed with 2.5% (v/v) glutaraldehyde in phosphate-buffered
saline (PBS, pH 7.4) for 30 min at 22-24°C (room temperature). After rinsing
three times with PBS, specimen post fixed with 1% (v/v) Osmium tetraoxide in
phosphate buffered saline for 30 min at 4°C, afterward was washed thrice
with double distilled water at room temperature, dehydrated in ascending ethanol
concentration and finally examined and image captured in a high resolution SEM
(Philips FEI Inspect) (Robards and Wilson, 1993).
Statistical analysis: The rank sum Mann-Whitney U test was used to compare
two nonparametric unpaired groups. Also the dose-response curve was applied
to measure an agonist/inhibitors potency for a particular active component.
So Graphprism5 software was used to produce nonlinear fitting curve (log inhibitor
vs. normalized response-variable slope curve, with calculation of IC50
value). The IC50 simply defines the concentration of inhibitor required
to provoke a half response between the baseline and maximum responses.
Variable inhibitory effect of plant extract on S. mansoni stages:
This study concentrated on evaluating anti-helminthic effect of methanolic extract
of P. tenuiflorus (Vatke) Agnew on different stages of Schistosoma
mansoni. Detection of motile viable parasites observed by inverted microscopic
examination, methanolic plant extract exhibited statistically significant decrease
in viability of S. mansoni stages (Fig. 1a). When this
plant extracts inhibitory effect was stratified against different time
intervals, it confirmed the previous finding, furthermore indicated that the
strongest inhibitory effect was on schistosomulae rather than on cercariae
or miracidium shown in (Fig. 1b). Furthermore; calculated
P. tenuiflorus (Vatke) Agnew methanol extract concentration that gives
a half efficient response IC50 against miracidium, cercaria and schistosomula
was 24.37, 17.39 and 12.29 mg/100 mL, respectively and demonstrated dose-response
downhill curve (Fig. 2) with attached table, ensuring that
P. tenuiflorus (Vatke) Agnew extract had lesser effect on miracidium
stage, on the other hand it has more inhibitory effect on cercaria and schistosomula
|Fig. 1 (a-b):
||The inhibitory effect of methanol extract of P. tenuiflorus
(Vatke) Agnew on tested S. mansoni stages. (a) show statistically
significant inhibitory effect of methanol extract of P. tenuiflorus (Vatke)
Agnew on all S. mansoni stages when compared to negative control,
line in bars denote the Median with asterisks for standard deviation and
(b) showing effect of the plant extract after different time intervals,
with the lowest viability detected when S. mansoni schistosomula
stages were incubated to the methanolic plant extract. Mann-Whitney U test
used to compare two groups. p value (*) is significant when between 0.01-0.05
||The calculated (IC50) of P. tenuiflorus (Vatke)
Agnew extract on S. mansoni stages viability. Lines indicate the
fitted dose-response curve assuming a non-linear effect of Log concentration
of plant extract, solid line for effect on miracidium, dashed line for effect
on cercaria and dotted line for effect on Schistosomula. Values of calculated
(IC50), half inhibitory efficient concentration on different
Schistosoma stages are shown in small attached table, equation applied
Y = 100/(1+10^((LogIC50-X)*HillSlope)). X = Log conc, Y = response, Hillslope
= Slope factor
Tegument morphological alterations detected by scanning electron microscopy:
Typical signs of injury were S. mansoni stages tegument changes which
often consisted of vesiculations, thickening, wrinkling or perforations. As
the tegument has a vital function, the parasite stages would often be weakened
or die as a result of these changes. Obvious changes were detected in cercaria
and schistosomula stages when compared to control specimen. Schistosoma mansoni
cercaria tegument showed major changes represented in crumpling and wrinkling
which led to obvious overall shortening of the cercaria Fig. 3a
and b compared to Fig. 3e and f,
furthermore clearly perforation were observed at the tail tegument Fig.
3c compared to Fig. 3g, with vesiculations/blebs at the
forked distal part of the tail Fig. 3d compared to Fig.
Furthermore; Schistosoma mansoni schistosomula tegument exhibited the
same overall shortening of the schistosomula Fig. 4b,
when incubated in P. tenuiflorus (Vatke) Agnew extract compared to negative
control Fig. 4a, in addition to observable swelling with higher
concentration of extract Fig. 4c, this swelling obscured partially
wrinkling or crumpling that observed in lower concentration.
|Fig. 3 (A-H):
||Scanning Electron Microscope (SEM) showing Schistosoma mansoni
cercaria.(A) Showing the observed morphological changes due to 4 hour incubation
in plant extract 250 mg/100 mL. Both body or tail tegument showed visibly tegument shrinkage and thickening compared to negative control (E), with remarkable wrinkling at tail just blow the body seen in (B) and compared
with (F), Tail teguments showed multiple holes/perforations (arrows) seen clearly
in(C) compared to (G). Unmistaken vesicles/blebs (arrows) were observed at
the distal forked part of the tail in (D), compared to (H)
|Fig. 4 (A-C):
||Scanning Electron Microscope (SEM) showing effect of P. tenuiflorus (Vatke) Agnew extract on Schistosoma mansoni schistosomula after 4 h incubation.(A) show negative control, unaffected stage is 114 µ in length. (B) Show
the tegument wrinkling or crumpling due to 250 mg/100 mL plant extract that led to overall shortening compared to negative control. In (C) further morphological
alteration when incubated in higher plant extract 400 mg/100 mL, observed
as further shortening with tegument swelling that masked wrinkling or crumpling shown in (B)
Schistosomiasis is a severe snail-borne disease affects more than 200 million
people worldwide (WHO, 1998). Among different species
of the genus Schistosoma known to infect humans Schistosoma mansoni
is the major transmitted one and endemic in 54 countries and territories
in South America, Africa and the Caribbean and the eastern Mediterranean regions
(WHO, 1998). Schistosomiasis remains a major health problem
despite the efforts to control the disease with significant disabling and financial
burdens (Chitsulo et al., 2000). One of the main
requirements for treating schistosomiasis is the development of an efficient
chemotherapeutic against schistosomulae.
Plants represent an important source of therapeutics from which 25% of the
currently used pharmaceuticals has been derived (Farnsworth
and Bingel, 1977). Medicinal plants represent natural therapeutic alternatives
against pathogenic microorganisms, being safer than their synthetic counterparts.
In a search for natural anti-schistosomal products, the anti-schistosomal activity
of ethanol extracts of leaves of one of folk and traditional medicinal plant;
P. tenuiflorus (Vatke) Agnew against Schistosoma mansoni miracidia,
cercaria and schistosomula was investigated in vitro. P. tenuiflorus
is one of the Lamiaceae family, it's essential oil has antimicrobial activity
against microorganisms including human pathogenic bacteria, yeast and fungi
(Al-Garni and Kabli, 2005). It is commonly used as herb
for non-specific treatment of ear ache and inflammation of middle ear (Hedberg,
1979) sore throat and laryngitis (Mossa et al., 1987;
Rahman et al., 2004), respiratory system infections
(Abulfatih, 1987) and digestive and genitourinary disorders
(Pakia and Cooke, 2003). P. tenuiflorus was able
to promote wound healing in rat module through stimulation of fibroblast proliferation
both in rat model (in vivo) and in culture (in vitro) (Khorshid
et al., 2010). Due to its anti-inflammatory and anti-microbial activity
it might be a candidate to test its anti-Schistosomal activity. To our knowledge
P. tenuiflorus extracts and essential oil has been not tested for their
anti-parasitic activity before and this might be the first study to assess anti-Schistosoma
potency of this plant extract.
In our present study we demonstrated significant and reproducible anti-schistosomicidal
activity of P. tenuiflorus methanol leaf extracts against miracidium,
cercaria and schistosomula (Fig. 1-4). Interestingly,
the in vitro effects of P. tenuiflorus methanol leaf extracts against
Schistosoma mansoni miracidium, cercaria and schistosomula were due to
both the effect on muscular function (loss of motor activity) and the extract
induced tegumental changes (Fig. 3, 4).
Both Schistosoma mansoni cercariae and schistosomula were most susceptible
to P. tenuiflorus methanol leaf extracts with an IC50 of 12.29
and 17.39 mg/100 mL, respectively (Fig. 2), while miracidia
being least susceptible with IC50 of 24.37 mg/100 mL (Fig.
2). The observed morphological alterations to cercarial tegument in this
study were comparable to SEM findings after in vitro praziquantel exposure,
as disruption of tegument, blebs formation and vacuolization (Oliveira
et al., 2006; Lofty et al., 2009).
These finding were proposed to occur at the base of the of the syncytial layer,
increase in size, protrude above the surface and result in final bursting of
the blebs (Brindley and Sher, 1987), in a process suggested
to be accompanied by increased exposure of Schistosoma antigens at the
parasite surface rendering it more susceptible to host immune system (Harnett
and Kusel, 1986).
To date the majority of the phytochemical studies of the plant have concentrated
on the isolation of diterpenoids and although these compounds have been shown
to have potent antimicrobial activity few have been tested in bioassays that
are directly related to the traditional uses of the species the compounds were
isolated from. Natural product chemists have mostly reported on novel diterpenoids
in species of Plectranthus. Beside diterpenoids other monoterpenes and
sesquiterpenes including β-caryophyllene, p-cymene and thymol were isolated
from Plectranthus aegyptiacus in considerable constituents and many have
antimicrobial activity (Smith et al., 1996),
where the plant extract lack alkaloids, steroids, anthraquinones and flavonoides
This study may suggest a promising efficacy of P. tenuiflorus (Vatke) Agnew methanol extract against cercaria which is the infective stage, as well as against the significantly ambulant invasive Schistosomulae stage inside human body. Thus, there is a scientific case for further studies to be undertaken on the activity and distribution of different extracts and to identify active compounds of this plant, along with weighing up their biosafety.
Special thanks are extended to Prof. Soheir S. Mahmoud at Schistosome Biological Supply Center SBSC, Theodor Bilharz Research Institute, Imbaba, Cairo for her sincere cooperation.
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