Therapeutic Potential of Folk Plants Used Against Pathogenic and Opportunistic Free Living Amoeba- Juniper Publishers
Juniper Publishers- Journal of cell Science
Free living amoebae (FLA) are opportunistic protozoa
which are widely spread in our environment. Among these, three genera
Acanthamoeba, Balamuthia and Naegleria are responsible for life
threatening human brain infections involving central nervous system
(CNS) with poor diagnosis. This is also contributed to poor
understanding of the pathogenicity of FLA and limited availability of
therapeutic options against them. The purpose of this review is to
document the medicinal plants used against free living amoeba and
critically review the literature on the anti-FLA properties of folk
plants against emerging FLA infections. Information regarding FLA was
collected from scientific databases like PubMed and scientific
literatures based on published papers from Elsevier, Springer, Wiley
publishers. An in-depth analysis of previous studies was undertaken and
future prospective is considered. Acanthamoeba has been found the mostly
studied FLA so far with regards to folk plant extract trails and
literature does not describe a single report against Balamuthia and
Naegleria. Among all folk plants AHium sativum, Peganum harmala, Origanum syriacum, Arachis hypogaea L., Salvia caespitose, and Melissa officinalis
have shown optimal anti-Acanthamoeba effects in vitro. Folk plants and
their products could be a potential candidate for the therapeutic drug
discovery against FLA. To the best of our knowledge, this document is
the first extensive review regarding the utilization of folk plants
against FLA in literature.
Keywords:
Free living amoeba; Acanthamoeba; Balamuthia; Naegleria; Granulomatous
amoebic encephalitis; Balamuthia amoebic encephalitis; Primary amoebic
meningoencephalitis; Folk plants
Abbreviations:
BAE: Balamuthia Amoebic Encephalitis; CNS: Central Nervous System; DNA:
Deoxyribonucleic Acid; ER: Endoplasmic Reticulum; FLA: Free Living
Amoeba; HCEC: Human Corneal Epithelial Cells; HIV: Human
Immunodeficiency Virus; GAE: Granulomatous Amoebic Encephalitis; MIC:
Minimal Inhibitory Concentration; PAM: Primary Amoebic
Meningoencephalitis; RNA: Ribosomal Ribonucleic Acid
Introduction
Free living amoebae (FLA) are opportunistic protozoa
and ubiquitous which are widely spread in our environment ultimately
present in various types of soil, water and air. Among these, three
genera Acanthamoeba, Balamuthia and Naegleria can potentially cause life
threatening human brain infections involving central nervous system
(CNS) in humans and animals [1-3].
Because most of the infections caused by these amoebae are ultimately
fatal, diagnosis is often made at autopsy, even in developed countries
where sophisticated diagnostic facilities are readily available.
However, in Africa and Asia, where HIV/ AIDS is epidemic, it is quite
possible that majority of cases have gone undetected. This may be due to
a number of reasons like (i) lack of expertise about these amoebae (ii)
cultural and financial barriers that prevent autopsies. The actual
incidence of protozoan diseases is therefore not really known.
In 1930, Acanthamoeba were discovered as eukaryotic
cell culture contaminants of yeast and were sited in the genus
Acanthamoeba [4-6].
Acanthamoeba is facultative pathogen and well-known as an agent of
granulomatous amoebic encephalitis (GAE), a fatal disease of the central
nervous system which always lead to death [7,8].
In addition it may cause keratitis, a painful sight-threatening disease
mostly related to contact lens wearer. Acanthamoeba sp is widely spread
in our surroundings like air, water and soil thus play a pivotal
predatory role in ecosystem. Acanthamoeba consists of two stages in its
life cycle cyst and trophozoites (Figure 1a).
Under favourable conditions it survives as a trophozoite thus feeds to
live and reproduce. It is transformed into a dormant cyst under
unfavourable conditions [7].
Acanthamoeba keratitis is differentiated by photophobia, ophthalmalgia,
blue-red vision and blood extravasations. Improper use of contact
lenses or corneal trauma results in keratitis. Fever headaches,
neurological disorders, such as disorientation, hallucinations and
vision disorders and coma are clinical symptoms of human GAE [2].
For the first time in 1986, Balamuthia mandrillaris
was isolated from remains of a mandrill baboon (Papio sphinx) brain
tissue that died due to a neurological disease at the San Diego Zoo Wild
Animal Park in California, USA. Later on in 1991, Balamuthia mandrillaris was linked with deadly human infections (Balamuthia amoebic encephalitis) which involved the CNS [9,10].
It has two stages of life cycle known as cyst and trophozoite. Cysts,
become visible as a double walled, the outer wall being wavy and the
inner wall round, when examines under a light microscope. However,
ultra-structural studies have shown that cyst wall possess three layers:
1. ectocyst (an outer thin and irregular layer), 2. endocyst (an inner
thick layer) and 3. mesocyst (a middle amorphous fibrillar layer).
Balamuthia mandrillaris has been isolated from soil so far [11].
Under unfavorable ecological conditions, for example extremes in
temperature or pH, lack of nutrients, excess of waste products or
overcrowding of cells, trophozoites convert into dormant or cysts
through a process named as encystment. Encystment guarantees the
survival of the amoeba in unfavorable ecological conditions (Figure 1b & 1d).
Balamuthia amoebic encephalitis (BAE) is a chronic
disease which may last from 3 months to 2 years, and always end up to
the patient's death. Unlike, Acanthamoeba encephalitis, BAE has been
reported both in immunocompromised people and those with normal
immunity, suggesting the virulent potential of this amoeba [11-13].
BAE has been identified in patients suffering from human
immunodeficiency virus (HIV)-infected patients, cancer, diabetes, or
alcohol and drugs abusers. So far, about 120 cases worldwide have been
identified, but the exact number of BAE cases may never be recognized
which may be due to a lack of awareness about infection and amoeba, poor
diagnosis methods and poor public health systems, particularly in under
developed countries. Mixtures of drugs are being used for the treatment
against this amoeba with very partial success and this is a growing
concern in the current medical profession [14].
The patients may show such symptoms from several weeks to months. Some
patients sometimes display hemi-paresis and weakness on one part of the
face or body, along with restrictions in patient's movement. The
patient’s conditions started to deteriorate further with lack of
response to stimuli along with pulmonary oedema or pneumonia, focal
seizures, photophobia, and finally resulting in death [15,16].
The genus Naegleria comprised of a group of FLA found in various habitats all over the world. Naegleria
spp. has been isolated from various environmental recourses like
domestic water supplies, ponds, freshwater lakes, swimming pools,
thermal pools, soil, and dust [17-19]. Naegleria fowleri
may cause primary amebic meningoencephalitis (PAM), a fast deadly
disease of the CNS which mostly identified among children and young
adults with a history of water sports. Naegleria sp have three
morphological stages in the life cycle of,
First human infection caused by Naegleria fowleri was described in South Australia by Fowler and Carter [20]. In addition to that PAM cases have also been connected to domestic water supplies [21].
PAM hits in immune-competent individuals and draw out a more rapid
disease course. Naegleria infection is commenced by the introduction of
water holding amoeba into the nasal cavity of the host. Amoebae attaches
to the nasal mucosa, travel along with the olfactory nerves, pass
through the cribriform plate, and enter into the brain. Once Naegleria
is in that compartment, the amoebae cause massive tissue damage and
inflammation. PAM can be distinguished by severe frontal headache,
fever, nausea and vomiting, stiff neck, and rarely seizures.
Furthermore, the acute hemorrhagic necrotizing meningoencephalitis
followed by the invasion of the CNS usually results in death 7-10 days
post-infection [1,2,22,23].
1). a trophozoite, 2). a flagellate, and 3). a cyst (1) (Figure 1C) .
There have been a number of reports showing folk
plants and their extracts have been used in vitro trails against
Acanthamoeba. But this is quite surprising that there is not a single
report of any folk plant used against Balamuthia, Naegleria and Sappinia
which may be due to many reasons. According to our understanding the
other amoeba are very difficult to culture (fastidious organisms) as
compared to Acanthamoeba. Secondly Balamuthia has only specie reported
so far. Furthermore Naegleria has a complex life cycle consisting of
three life stages which make this organism difficult to handle and work
with. In contrast 24 species and 17 genotypes of Acanthamoeba [24]
has been reported and very easy to culture in laboratory environment
that will be one of the reason of the researcher choice for folk
medicine trail.
Interest of scientific community to study
Acanthamoeba has developed enormously. Acanthamoeba has gained attention
from the large scientific community investigating their biochemistry,
environmental biology, physiology, cellular microbiology, molecular
biology and cellular interactions. This is because of their adoptable
characteristics in natural environment. It acts as vectors and
reservoirs for pathogenic bacteria and cause serious and life threaten
human diseases such as blinding keratitis and deadly encephalitis
respectively. Therefore the current review will be mainly focused on
Acanthamoeba as in literature only folk plants were used against this
amoeba. This review will summarize the amoebicidal activity of folk
plants which have been studied so far against Acanthamoeba in vitro.

Research Trends in Amoebagenesis
During the past few decades, a gradual increase in
scientific interest among the scientific community has been observed
regarding FLA (Figure 2).
Among three protozoa, Acanthamoeba has obtained great attention among
scientists. Moreover, all the folk plants were used against Acanthamoeba
in the literature (Figure 3a & 3b).
The major reason behind its fame is its vast frequency in our
environment. Based on its 18S rRNA sequence, 24 species which belong to
three morphological groups and 17 genotypes have been identified to
date. Furthermore, so far about 400 victims of Acanthamoeba infections
have been identified all over the world. Recently for the first time
both pathogenic and non-pathogenic genotypes have been isolated from
various water supplies in Pakistan [24]. All the features of FLA are described in detail in Table 1.
For advancement in the outcome of the diagnosis and management of
protozoan infections requires new therapeutic approaches. In this regard
a better understanding of the amoebic pathogenic mechanisms is
required. In the present review we will discuss the current status of
knowledge of the new therapeutic approaches (folk plants) used to
evaluate their effects against protozoa. We analyzed and documented the
effects of folk plants in selected clinically important protozoa
Acanthamoeba.



Therapeutics
Unlike other two protozoa, various treatments have
been reported to cure Acanthamoeba infections like successful
chemotherapy has been done with extremely toxic drugs commonly used for
disinfection, e.g. chlorhexidine derivatives but the percentage of
success is very low as mostly patients die during chemotherapy [25,26].
Some of the successful treatments of these two infections in
immunocompetent individuals have been documented with combined therapy,
only if started at initial stage of the disease as ineffectiveness
increases in later stages [27].
Successful treatments have been documented with use of a mixture of
cationic antiseptics (polyhexamethylene biguanide, chlorhexidine) which
obstruct the membrane functions, aromatic diamidines (propamidine
isethionate, hexamidine, pentamidine) which reduce DNA synthesis,
aminoglycosides (neomycin, paromomycin) which ultimately slow down the
protein synthesis, and imidazoles (clotrimazole, itraconazole,
ketoconazole, fluconazole, miconazole) which weaken cell walls and
polyenes, such as amphotericin B [28].
There are certain limitations in the treatment of Acanthamoeba
infections. The main problem with this parasite is its eradication from
the site of infection as under hostile conditions it encysts itself and
resists various chemicals. Thus medical treatment is mostly less
effective against cysts as compared to trophozoites. The rigidness of
cysts is because of its double-layered wall which formulates it
extremely resistant to anti-amoebic drugs and it can cause relapse of
the disease [29].
Furthermore, the risk of drug resistance, expensiveness of medical
treatment and its frequent adverse side effects are major restrictions.
Peganum harmala, Ricinus communis and Melia azedarach
Recently Peganum harmala, Melia azedarach, and Ricinus communis has been used in vitro to check the anti-amoebicidal activity [30]. This study described that Peganum harmala exhibited maximum amoebicidal effects followed by Ricinus communis and Melia azedarach
at 1.5mg/ml. It was also shown that even after 24h incubation
Acanthamoeba could not damage human corneal epithelial cells (HCEC)
cells layer in the existence of all extracts dilutions tested. On the
other hand when amoeba was incubated with HCEC in the absence of
extracts, amoeba completely destroyed the HCEC cells layer.
Acanthamoeba normally exhibits more than 90% adhesion
to HCEC which was drastically reduced with all the extracts tested.
Interestingly among all Peganum harmala have demonstrated the
highest inhibition (>50%) of amoebic adhesion to HCEC cells layer at
maximum dose 1.5mg/ml. Furthermore studies have shown that Acanthamoeba
produces maximum HCEC cell death (> 80 %) within 24h which was
drastically inhibited in the presence of plant extracts at maximum dose
tested (1.5mg/ ml). This was further supported by haematoxylin staining
which demonstrated that all plants extracts safeguard the HCEC cells
layer from amoeba, while the Acanthamoeba alone without extracts with
HCEC destroy the whole cell layer.
Another interesting finding of the study was that
Acanthamoeba could not multiply in number when incubated with extracts
even after a week time suggesting crude extracts reduces Acanthamoeba
number raise. On the other hand Acanthamoeba displayed maximum growth in
growth medium without HCEC cells.
Allium sativum
This study [31] for the first time described the activity of non-polar methanolic extracts of Allium Sativum
on the proliferation of Acanthamoeba castelanii cysts and trophozoites
at various doses (ranging from 0.78 to 62.50mg/mL) for different time
intervals. This study further elaborates that due to presence of extract
after first hour all the trophozoites were killed with 62.50mg/mL dose
while the trophozoites were viable at lower concentration 0.78 to
31.25mg/mL for some time and disappeared after 72h. On the other hand
extracts could not show significant effect on Acanthamoeba cysts after 3
hours which demonstrated the strong resistance of the cyst to chemicals
as compare to the trophozoite. After 6 hours, the only dose showed cyst
inhibition was 62.40mg/mL while low doses 0.78-1.95mg/ mL had no effect
even after 72h. Cytotoxic effects to corneal cells were also evaluated
in the presence of difference concentration of extact. It was reported
the extract showed cytotoxic inhibition with dose dependent manner with
maximum activity on maximum concentration. Thus, Acanthamoeba
trophozoites are more sensitive to A. sativum extracts than cysts. A. scrodoprosum
subsp. rotundum exhibited outstanding amoebicidal effect on
Acanthamoeba and might be used as a new natural agent against
Acanthamoeba.
Origanum syriacum and Origanum laevigatum
Two species of genus Origanum Syriacum and Origanum Laevigatum have been tested for amoebicidal activity on both cysts and trophozoites [32].
Different doses of methanolic extracts (1 to 32mg/ml) have been used to
evaluate the activity with exponential increase in the time period.
Effectiveness of Origanum laevigatum has been reported to be less than that of Origanum Syriacum. Within 3h, extract of O. Syriacum
with 32mg/mL dose has been proved to be effective against trophozoites
only. After 72h trophozoites have been inhibited with 32 and 16mg/ml
only. On the other hand the cyst showed significant resistance at
different time intervals except the high dose (32mg/mL) which ultimately
killed cysts while no inhibition was observed with other doses. Origanum Syriacum has very less effect on cysts of Acanthamoeba which shows the resistance of cysts against this herb.
Curcuma longa L., Arachis hypogaea L. and Pancratium maritimum L
Curcuma longa L: Antiparasitic activity of
Curcuma longa has also been studied against various parasites like
Schistosoma, Leishmania, Plasmodium, Trypanosoma and more usually
against other cosmopolitan parasites like Babesia, Coccidia, nematodes,
Giardia and Sarcoptes [33]. One of the recent study conducted by El-Sayed et al. [28] showed an inhibitory effect of C. longa L
on Acanthamoba castellanii cyst increase with MIC of 1g and 100mg/ml
after 48 and 72h, respectively. Concentration of 10mg/ml extract failed
to cause entire blockage of Acanthamoeba castellanii cyst growth. After
72h, 90.2% of maximal inhibition has been observed while 1mg/ml and
0.1mg/ml doses demonstrated growth decrease by 60-83.1% in all times
intervals tested. These findings have been recognized to the curcumin
which is the main component responsible for its biological activities.
In previous studies [34] it has been described as anti-malarial agent by showing its cytotoxic effect on mitochondrial and nuclear DNA of (P. falciparum).
Arachis hypogaea L: In a study conducted by [28] a notable inhibitory effect was observed on Acanthamoeba castellanii cysts with the ethanol extract of A. hypogaea L.
The dodes 0.1 and 0.01mg/ml exhibited growth decline by 64.4%-82.6% in
all incubation periods while more inhibition was observed with MIC of 1,
10 and 100mg/ml after 24, 48, and 72h, respectively. These findings
have been recognized to quercetin and flavonoids which characterize a
key component responsible for its natural actions. One more biologically
active compound flavonoid has been identified as anti-leishmania
activity [35].
Quercetin in this plant has been reported as an inhibitor of DNA
production and detained cell cycle progression in Leishmania donovani
promastigotes, which ultimately lead to apoptosis [36].
Pancratium maritimum L: This plant contain
metabolite like Pancratistatin which has a wide range of therapeutic
benefits such as anti-viral, anti-neoplastic and antiparasitic effect
against Encephalitozoon intestinalis [37,38].
The main active compounds alkaloids, phenolic acids and flavonoids in
this plant are well known for their anti-malarial, anti-fungal and
cytotoxic properties [39-42].
Efficacy of P. maritimum against A. castellanii cyst growth was observed by El-Sayed et al. [28].
This study reveled that MIC of 200mg/ml after 72h affects the growth
while the 20 and 2mg/ml doses exhibited optimal growth decrease by 94.3%
and 85.5%, respectively with in 72h. However, the lower doses of 0.2
and 0.02 mg/ml demonstrated growth decrease by 34-74.8 in all time
intervals. The findings in the study have been attributed to
pancratistatin and 7-deoxynarciclasine which are cyclin kinase
inhibitors as cyclin-dependent kinase and their controllors are well
known for their contribution in the reproduction and expansion of the
eukaryotes. Thus, these enzymes characterize striking possible targets
for anti-parasitic chemotherapy [37].
Moreover, cytotoxic phenolic composites like flavonoids and phenolic
acids have also been attributed to this anti-parasitic activity.
Croton
Anti-amoebic activity against Acanthamoeba polyphaga
(ATCC 30461) of three species that is Croton Pallidulua, Croton
Ericoides, and Croton Isabelli activity and cytotoxic effect in mammalian cells has been studied by [43].
Croton Isabelli: The oil of C. isabelli
has shown low amoebicidal activity. The dose of 10mg/ml could only
killed 4 % of the Acanthamoeba polyphaga trophozoites. Very low action
was recorded for the oil of C. isabelli, in which no monoterpenes were
found. It has also been shown that oils of C. isabelli were lethal to 98
% when tested against Vero cell line [43].
Croton pallidulus: The oils of C. pallidulus
in the final doses of 2.5, 5, and 10mg/ml have been identified as
trophozoiticidal. These oils showed only 29 % inhibition of trophozoites
with 0. 5mg/mL concentration. These oils could not show hydrocarbon
monoterpenes but 16.2 % of oxygenated monoterpenes that perhaps
contributed for the recorded activity. It has also been observed that
the oils of C. pallidulus were toxic up to 98 % against Vero cell line [43].
Croton ericoides: Trophozoites have been killed by C. ericoides
essential oils with doses 2.5, 5, and 10mg/ml. These extracted oils are
extra active and ableed to kill 87% of trophozoites at the dose of
0.5mg/ml. The greater activity of C. ericoides perhaps due to improved percentage of monoterpenes, composites with known organic characteristics. C. ericoides
exhibited the optimal cytotoxicity being to disrupt 100 % of the Vero
cell line, and after the experiment no active/live cells were observed [43].
Pterocaulon
Pterocaulum polystachyum: In a study conducted by [44], the extract of Pterocaulum polystachyum
taken in hexane demonstrated amoebicidal property against a virulent
strain of Acanthamoeba (50492). When the amoebicidal activity of extract
of P polystachyum and extracts of dichloromethane, hexane and
methanol fractions at the dose of 5mg/ml were analysed, all the doses
exhibited additional activity than the crude methanolic extract. It was
also observed that, among all fractions, the increase in the activity
was directly proportional to the lipophilic character of the sample. The
activity of fraction obtained from hexane was dose dependent and after
48 and 72h almost 66-69% trophozoites were killed with this fraction.
Along with the inhibition of trophozoites, this fraction also prevented
the encystment of the trophozoites. Ergosterol is the major component of
the outer walls of majority of the fungi and thus the procedure of
action of the antifungal agents is totally based on the relation or on
the inhibition of it [45].
Some drugs like amphotericin B (Polyenic derivative) and fluconazole
(azoles) are used as anti-protozoals for their stimulation on the cell
membrane. Hence, the excisting of ergosterol and other sterols in the
Acanthamoeba's membrane might explain the sensitivity of these amoebas
to these drugs. So, it is assumed that, coumarins presence in the
fraction obtained by hexane treatment could interrelate with the
ergosterol of the Acanthamoeba's membrane. Furthermore, it has also been
reported that, compounds of this reduce few enzymes involved in the
synthesis of sterol [46].
In a study conducted by [47] P polystachyum
demonstrated that amoebicidal action against trophozoites of a virulent
strain of Acanthamoeba polyphaga (ATCC 30461). With 5, 2.5 and
1.25mg/ml, the extracted oil was capable of killing 81.1%, 71.6% and 60%
of the trophozoites, respectively. Within 24h, the inhibition of
trophozoites has been observed with the doses of 20 and 10 mg/ml.
Laboratory tests have also showed the inhibition of all trophozoites
with the doses of 20 and 10mg/ ml, while with 5, 2.5 and 1.25mg/ml, the
oil killed 80.7%, 46.3% and 46% trophozoites, respectively. This
activity is directly proportional to the increase of the dose. This oil
also prevents encystment of the trophozoites. When this oil was
subjected to cytotoxic test it showed high cytotoxicity against Vero
cells. However, its contribution for the inhibition of trophozoites and
prevention of encystment can't be neglected so this oil can be utilized
in contact lens cleaning solutions after the elimination of its
hazardous effects on mammalian cells.
Saliva staminae and Salvia caespitose
Salvia staminae: Goze and co worker in 2009 [48]
tested different doses of methanolic extract of S. staminea was tested
against both Acanthamoeba castellanii trophozoites and cysts. Different
doses from 1-32mg/ml were prepared to test their effect along with an
increase in the time period. After 6 hours, no active/live trophozoites
were seen with 32mg/mL concentration. All the trophozoites were
inhibited by all doses after 72 hours. When cysts were observed after 6
hours, there was a decrease in number of cysts but complete inhibition
was observed after 12 hours. After 72 hours, doses with concentration
32, 16 and 8mg/ml killed all the cysts. There is an increase in number
of trophozoites and cysts with the decrease in concentrations of the
extracts.
Salvia caespitose: Complete inhibition
of trophozoites was observed after 12h with 32mg/ml concentration. After
24-48 hours, two doses i.e. 16 and 32mg/ml inhibited the trophozoites
completely. When results were observed after 72h, three doses i. e. 8,
16 and 32 mg/ml showed an inhibition. Resistance of cysts can be
estimated by analyzing the observed data in which complete inhibition
was observed with 32mg/ml concentration after 48h and after 72h, two
doses 16 and 32mg/ml showed the same results.
S. staminea extracts obtained by methanolic
treatment have shown no cell death at the dose of 16 mg/ml. But
non-cytotoxic effects were observed at 32mg/ml for S. caespitosa
extracts obtained by methanolic treatment. No decolonization zone was
observed around the samples. Even though the Acanthamoeba were in direct
contact with the Salvia species extracts obtained by methanolic
treatment in the growth medium, there were no signs of damage to their
morphological properties and seemed similar to controls trails.
Pouzolzia indica and virkon®
Pouzolzia indica: In an investigation conducted by [49] light, scanning and electron microscopic studies were performed to check the activity of methanolic extract Pouzolzia indica
on Acanthamoeba cysts. Six dilutions were used to check the amoebicidal
activity. After treating with minimum concentration of 1:4, light
micrographs of cysts showed the double walls of cyst with crumple
ectocyst and even endocyst. The space between the endocyst and cell
membrane increased due to cutering of inside cell. Organelles were
difficult to identify and few cells got excluded as many unfilled cells
could be recognized. When cysts were observed under a transmission
electron microscope, it showed the deterioration of the cyst walls,
become visible as the exposed electron dense myelin-like structure which
surrounds the cytoplasm. Within the cytoplasm appeared as clumps, with
the anonymous plasma membrane. Various micrographs showed an unfilled
and rip open cyst wall due to expulsion of the cytoplasm to the
environment. A series of deterioration of cyst's morphology was observed
under scanning electron microscope. Firstly, the oval cysts became flat
and ectocyst walls collapsed after shrinkage. In some cyst, dropsical
appearance was observed due to damaged opercula. After the entrance of
fluid inside the cyst wall the dropsical cyst wall torn into pieces [49]. It has been reported that there is a great amount of phenolic and sugar substances in extracted Pouzolzia indica
Benn. Phenolic compounds injure the plasma membrane which resulted into
the leakage of intracellular components of the cell. Phenolic compounds
perform as oxidizing agents which, cause cell membrane injury by
responding with cellular carbohydrates, lipids, proteins and nucleic
acids [50-53].
Virkon: Roongruangchai and co-workers in 2010 [54] compared the activity of methanolic extracts of Pouzolzia indica
and Virkon against Acanthamoeba cysts. Cysts treated with both showed
same morphological changes like cytoplasmic clumps, unfilled double
walled cysts and injured cyst walls under a light microscope.
Transmission electron microscope showed the exposed and broke cyst walls
along with injured cytoplasmic organelles. Shrinkage and destruction of
the cytoplasmic machinery inside the cyst wall occurred. Scanning
electron micrographs (SEM) exposed crushed crumpled strips with inflamed
and integrated cyst walls. Virkon activity is due to the presence of an
active ingredient i.e. potassium peroxymonosulphate. Virkon is a tough
oxidizing agent and having a highest surfactant which responds with some
proteins of the cell cytoplasm or cell wall [55,56].
This study concludes the destruction of cyst in a
three step process as, firstly, the cyst walls contract the opercula are
damaged and the cyst wall divides, along with cytoplasmic clusters and
eject out of the cyst wall. In a second step, the cysts become dropsical
due to passing of the solution into the cell, causing decreased
wrinkling of the cyst wall. In the last step, the cyst walls tear and
break into small pieces.
Cananga odorata, Kaempferia galangal and Ipomoea sp.
Chu and co-workers in 1998 [57]
selected plants from Southeast Asia to check amoebicidal activity.
Amoebae cultured in plant extract were investigated for encystment or
lysis. Two hundred plant extracts were examined during this study. The
extracts acquired from three plants Cananga odorata, Kaempferia galangal and Ipomoea sp., were discovered to have amebicidal activity against 3 species of Acanthamoeba in vitro, whereas a 4th extract obtained from Gastrochilus panduratum
showed amebicidal activity against Acanthamoeba polyphaga but not
against Acanthamoeba castellanii or Acanthamoeba culbertsoni. Some of
the plant extracts also exhibited tumoricidal activity but did not
injure the primary macrophage cultures. Natural composites obtained from
plant extracts could provide as a new foundation of anti-amoebic drugs
for the treatment of amoebic keratitis, carcinomas and GAE.
Peucedanum species
Methanolic extracts of Peucedanum caucasicum, Peucedanum chryseum, Peucedanum longibracteolatum and Peucedanum palimbioides were used to investigate the amoebicidal activity in vitro by [58]. Dose ranged from 1 to 32mg/ml was checked for 72 hours. P longibracteolatum
have shown the strongest amoebicidal effect on both trophozoites and
cysts. While at maximum dose of extract (32mg/ml) no alive Acanthamoeba
trophozoites or cysts were found from 24 to 72h. The same results were
obtained from 16mg/ml dose of extract on trophozoites. The amount of
live cysts was numbered as 10.6±2.1 with the same concentration within
24 hour. While in the existence of extract dose (8mg/ml), no live
trophozoites were observed after 48 hour. Furthermore, at the same dose
cysts (51%) were destroyed by the extract after 72h. It was observed
cysts showed more resistant to the extracts as compared to trophozoites.
Inula oculus-christi and Pastinaca armenea
In another study conducted in Turkey, by [59] Inula oculus- christi and Pastinaca armenea
were used in vitro to estimate the amoebicidal potential of their
aqueous extracts. The numbers of the live Acanthamoeba cysts and
trophozoites were reduced with doses ranged from 1 to 32mg/ml. The study
further elaborates that Inula oculus-christi exhibited strongest
amoebicidal effect on both cysts and trophozoites. When 32mg/ml extract
dose was incubated within culture media, no live trophozoites were
observed within 24h. Besides, when 16mg/ml dose of Inula oculus-christi
extract were used, no live trophozoites were found after 72 h. While a
restricted effects of Inula oculus-christi extract was observed on
cysts. On the other hand at 32mg/ml extract dose, only 25.3% of the
total cysts were destroyed successfully. While, in the presence of
32mg/ml extract of Pastinaca armenea, 23% cysts and 40.3% trophozoites were destroyed after 72h time interval.
Satureja cuneifolia and Melissa officinalis
Methanolic extracts of Melissa officinalis and Satureja cuneifolia were used to investigate in vitro amoebicidal activity [60].
When methanolic extracts (ranging from 1 to 32mg/ml) were used the
numbers of the viable Acanthamoeba cysts and trophozoites were reduced.
Amoebicidal action was time and dose dependent. Among two extracts
trails Satureja cuneifolia exhibited the optimal amoebicidal
effect on both cysts and trophozoites. After 24h, in the presence of 32
mg/ml extract no live trophozoites were found. While at the same dose,
the extract showed effecacy against the cysts at a rate of 46.3% after
72h. On the other hand Melissa officinalis exhibited restricted
amoebicidal effect on Acanthamoeba. The extract dose (32mg/ml) has shown
44.3% trophozoites and 30% cysts were destroyed.
Teucrium chamaedrys and Teucrium polium
Methanolic extracts of Teucrium chamaedrys and Teucrium polium were used [61]
to investigate amoebicidal activity. Both extracts (from 1 to 32mg/ml)
demonstrated amoebicidal potential on both cysts and trophozoites of
Acanthamoeba by time and dose dependent manner. Teucrium chamaedrys
demonstrated the optimal amoebicidal effect on the trophozoites. At
16mg/ml extract concentration no live trophozoites were found after 48h.
Similarly at 32mg/ml extract concentration no live trophozoites were
found in the case of Teucrium polium. As usual cysts were found more resistant than trophozoites at 48h.
Pueraria lobata, Rubus chamaemorus, Solidago graminifolia and Solidago virgaurea
This study was conducted by Derda and co-workers in 2009 [62] to investigate the amoebicidal or amoebistatic activity of potential of extracts obtained from Pueraria lobata, Rubus chamaemorus, Solidago graminifolia and Solidago virgaurea. It was found that extracts from Pueraria lobata, Rubus chamaemorus, and Solidago virgaurea
exhibited chemotherapeutic characteristics in doses of about 0.010.05mg
extract/ml in vitro. Furthermore at doses of 0.35|ig/ml expressed in
ellagic acid for Rubus chamaemorus and 0.053|ig/ ml expressed in puerarin for Pueraria lobata.
Therefore in vivo trails, it was also observed that, following therapy
using the above mentioned extracts, trail animals infected with
Acanthamoeba Table 2:
Summary of potential folk plants used against free living amc sp. were
survived for some extra time i.e., 2.5-3 times longer. It was also
assessed that extracts obtained from the selected plants could be used
both internally and externally especially in a combined therapy for
acanthamoebiasis. It is noteworthy that the plant extracts tested during
this study was found to be non toxic to animals.
Conclusion
There is a need to develop therapies with low cost,
greater efficacy and minor side effects. In this context, in the past
few years the trend has been shifted towards the use of medicinal plants
against Acanthamoeba (Table 2).
Acanthamoeba is widely spread in air, water and soil etc this is
because of their adoptable characteristics in natural environment.
Furthermore Acanthamoeba has been reported to be cultured comparatively
easier in laboratory conditions as compared to Balamuthia and Naegleria
which could be one of the reasons of the researcher choice for folk
plants extract for in vitro trails. Although this is not a new concept
as plant extracts were believed to be significant for diverse ailments
by the early civilizations [63].
Many natural products have been identified as antiparasitic agents with
astonishing efficacy and selectivity such as plant- derived terpenes,
alkaloids and phenolics [64].
In developing countries, the utilization of medicinal plants by people
is very popular because these products are safe, easily accessible and
commonly available at low cost. In conventional medicine, the use of
plants in the form of their various parts crude extracts, plasters or
infusions is an extensively practiced to cure common infections in
various parts of the country. In spite of this, there is still a
shortage of scientific studies authenticate the possible antibiotic
potential of a great number of these therapies. The plants reported in
literature used against Acanthamoeba are given below in detail [28-62].

For the past few years, FLA has gained much attention
by scientific community because of its vast prevalence in our
environment and thus causing deadly infections in humans. The main
hindrance in the treatment of infections is the formation of cyst which
results in the failure of treatment with chemotherapeutic agents. The
life cycle of FLA consists of an infective trophozoites and cyst forms.
Under harsh conditions, trophozoites differentiate into a cyst form.
Cysts are double walled, consisting of an outer ectocyst and an inner
endocyst. Both walls meet at points known as arms or rays. Furthermore,
amoeba cysts can survive up to several years while maintaining their
pathogenicity [65].
These characteristics suggest that the key functions of cysts are to
withstand the adverse conditions and spreading of amoebae throughout the
environment. Amoebae cysts are metabolically inactive and resistant to
many chemotherapeutic drugs resulting in the recurrence of the disease.
For instance, Ficker and co-workers in 1990 [66]
observed the development of propamidine resistance during the course of
therapy for Acanthamoeba keratitis, which led to recurrence of the
infection. This is of particular concern with the availability of very
limited drugs for Acanthamoeba infections. The process of encystment in
Acanthamoeba is triggered under harsh environmental conditions. This is a
reversible change and is dependent on the environmental conditions.
Moreover, these chemotherapeutic agents also produce
side effects with lower efficacy. In this perspective, the trend has
been shifted towards the use of medicinal plants which have higher
efficacy and less side effects. The utilization of medicinal plants by
the community in developing countries is widespread because the products
achieved from these plants are widely reachable at low cost, safe and
easily available. In conventional medicine, the plants utilization in
the form of infusions, crude extracts or plasters is a common practice
to nurse common infections in various parts of the world. In spite of
this, still not much scientific reports validating the promising
antibiotic potential of a vast number of these therapies. In vitro
antimicrobial screening methods may provide the required initial
interpretation to choose, among the crude plant products, those with
possible valuable characteristics for further pharmacological and
chemical studies.
However, a complete understanding of FLA pathogenesis
is crucial to develop therapeutic interventions to design preventative
measures. Folk plants and their products could be a potential candidate
for the therapeutic drug discovery against FLA. Among all folk plants
Allium sativum, Peganum harmala, Origanum syriacum, Arachis hypogaea L.,
Pancratium maritimum L., Pterocaulum polystachyum, Salvia caespitose,
and Melissa officinalis have shown optimal anti-Acanthamoeba effects in
vitro. Present review suggests the possibility of further research with
purified fractions of plant extracts to identify the active ingredients
and to elucidate the mechanism of action of the effective compounds both
in vitro and in vivo which may provide a new series of potential
chemotherapeutic agents against FLA. To the best of our knowledge, this
report is the first extensive review regarding the utilization of folk
plants against FLA in literature.
Acknowledgment
The authors are indebted to Dr. Suk-Yul Jung, South
Korea; Ambreen Gul Muazzam, Canada; Eleni Pavlopoulou, England; Hafiz
Muhammad Shohaib, Abida Akbar, Pakistan for assistance. Furthermore, we
are also very thankful to Ed Jarroll, Boston, for providing us TEM
images of Balamuthia mandrillaris.
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