CHAPTER-IV
Effect of active compounds on structure and biochemistry of T.rubrum
4.1 Introduction
Trichophyton rubrum is one of the most common fungi which causes
dermatophytosis,
mycosis that affect humans and animals around the wor ld.
Researches aiming new products with antifungal activity become necessary to
overcome difficulties on treatment of these infections. Fungi comprise one of the five
major kingdoms of organisms characterized by a unique specialized chitino us cell wall
(Deason, 1997). These eukaryotic non motile organisms afford a diverse range of clinical
manifestations including allergy, toxic reactions and infections (mycoses) in human and
animals (Ajello and Hay, 1998). In recent years, a remarkable increase has been reported
in the incidence of different mycoses due to aggressive cancer chemotherapy, widespread
use of broad-spectrum antibiotics, increasing in the number of immunosuppressive
diseases and highly effective immune suppressants for organ transplantation (Anaissie et
al., 2003). Because of huge similarities between fungal and mammalian cells, there is a
limited selective target for designing new antifungal formulations (Barrett, 2002;
Georgopapadakou and Walsh, 2002). On the other hand, availab le drugs, especially
polyenes and azoles, suffer from a number of limitations, which can cause some
difficulties in their applications. In this regard, host toxicity, drug resistance, drug-drug
interactions, fungistatic mode of actions, and limited routes of applications were found to
be considered in the treatment with antifungal agents (Georgopapadakou et al., 1996).
There is thus an urgent need for new antifungals with new modes of action broad
fungicidal spectrum of action and fewer doses – limited side effects (Graybill, 1996;
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Maertens and Boogaerts; 2000). In recent years the attempts for understanding the mode of
actions of plants with known antimicrobial properties has markedly increased which led in
some extents to finding out new effective compounds including low- molecular weight
compounds, peptide and proteins (Sagar, 2008).
In this area there was no reports documented on the morphological changes of
fungi grown in the presence of compounds. This study was undertaken to the study of
morphological changes of Trichophyton rubrum under restraint with compounds 07 like
AR-1, AS-1, CR-1, ET-1, FR-1, P-1 and VN-1 in order to find out the site of action of the
compounds.
In the present study 07 isolated compounds were treated with T. rubrum under
restraint with compounds in order to find out the site of action of the compound. The
structural and biochemical changes of T. rubrum were recorded.
Main morphological changes on the T.rubrum fungal mycelium and hyphae was
observed in SEM studies observed and reported in previous chapter. The biochemical
changes of common dermatophytic fungi T. rubrum was in need to observe. Because the
incidence of severe fungal infections caused by opportunistic moulds has risen in recent
years, especially in immunocompromised patients. The National Committee for Clinical
Laboratory Standards (NCCLS) has devoted a great deal of effort in developing a
reference method for in vitro antifungal susceptibility testing of moulds that could serve as
guidance for clinical treatment. Recently, a reference method was proposed in which the
inoculum consists of conidial suspensions (NCCLS, 1998). However, considering that the
invasive form of mould infection is hyphal, the use of conidia as the starting inoculum may
be questioned. Some comparative studies on MICs obtained with hyphae and conidia have
been performed and the reported data are controversial (Koenig and Kremer, 1979; Regli
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et al., 1980; Bezjak, 1985; Guarro, 1997; Manavathu et al., 1999). Guarro et al., (1997)
found important discrepancies between MICs obtained with both forms of inoculum. On
the other hand, Manavathu et al., (1999) reported practically identical results with
germinated and ungerminated conidia of Aspergillus fumigatus.
Inspite of the great advances which have been made in the field of bacterial
metabolism, similar studies with the filamentous fungi is limited. Only such well- studied
molds such as Penicillium chrysogenum, Aspergillus niger, and Neurospora crassa, have
received
significant biochemical attention,
whereas the
metabolic changes of
dermatophytes are comparatively unknown. Most studies on the chemical composition of
fungi have shown that the percent total protein (Suskind and Bonner, 1960), increased in
young mycelia and decreased in older mycelia. Howe ver, these studies did not determine
the composition of mycelia from the lag phases of growth. Gottleib and Van Etten (1964)
studied the entire growth cycle of Pseudomonas atrovenetum and found that the
percentage of protein, ribonucleic acid (RNA), and total nitrogen decreased with age and
that total carbohydrate increased with cellular age.
The inhibitory effects of pelargonic and capric acid on Microsporum gypseum were
examined by Chadeganipour and Haims (2001). Solid and liquid Sabouraud glucose media
containing different concentrations of pelargonic and capric acid were separately prepared
and inoculated with the suspension of mycelium and spores of M. gypseum and incubated
at 25° C for 1 month. The culture media were examined periodically for fungal growth and
the minimum inhibitory concentration (MIC) of each fatty acid was determined. The MIC
for capric acid was 0.02 mg/ml and for pelargonic acid 0.04 mg ml for capric acid and
0.05 mg ml for pelargonic acid in the liquid media. There are also repor ts of losses of
many enzymatic activities in addition to decrease in endogenous respiration during
starvation (Swanson and Stock 1966).
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There are no reports on chemical changes in components of dermatophytes in the
presence of isolated compounds 07 like AR-1, AS-1, CR-1, ET-1, FR-1, P-1 and VN-1.
4.2 Review of literature
The antifungal activity of Thymus pulegioides was evaluated for its main
components. To clarify its mechanism of action on yeasts and filamentous fungi, flowcytometric studies of cytoplasmic membrane integrity were performed and effect on the
amount of ergosterol was investigated. The results showed that T. pulegioides essential oil
exhibited a significant activity against clinically relevant fungi, mainly due to lesion
formation in the cytoplasmic membrane and a considerable reduction of the ergosterol
content (Eugenia Pinto et al., 2006).
The microscopical observations showed that the mycelium of P. digitatum treated
with LfcinB developed alterations of growth, sporulation and chitin deposition, and
permeation of hyphal cells (Munoz and Marcos, 2006). The study of morphological
alterations in toxigenic Aspergillus parasiticus exposed to neem leaf and seed aqueous
extract by Mehdi Razaghi- Abyaneh et al., (2005) revealed that at high concentrations i.e
50% v/v of resulted in vacuolation of the mycelial cytoplasm and vesicle deformation
causing attenuation of cell wall at variable intervals.
Herniation of the cytoplasmic
contents that was protruding from the mycelium was associated with deformation of the
mycelium. Some mycelia showed a cleft between the cell wall and cytoplasm. In the
microscopic observation of the antifungal activity from leaf extracts of Cassia alata,
Cassia fistula and Cassia tora revealed that the treated hyphae and macroconidia with leaf
extracts were shrunken and collapsed, which might be due to cell fluid leakage (Souwalak
Phongpaichit et al., 2004).
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Romagnoli et al., (2005) evaluated antifungal efficacy of two main compounds,
piperitone and piperitenone from Tagetes patula and ultrastructural modification in
mycelia were observed via electron microscopy, evidencing large alternations in hyphal
morphology and a multisite mechanism of action. Masoomeh Shams Ghahfarokhi et al.,
(2004) studied morphological alterations, in T. rubrum and T. mentagrophytes, where
morphological changes occurred to a less content for T. rubrum compared with
T.
mentagrophytes.
According to Mares et al., (2005) the extracts from roots of the common vegetable
Cichorium intybus L., were ineffective on geophilic species and on tested phytopathogens,
with the exception of Pythium ultimum, whereas they inhibited the growth of zoophilic and
anthropophilic dermatophytes, in particular Trichophyton tonsurans var. sulfureum, whose
treatment caused morphological anomalies, here observed by scanning electron
microscopy. This behaviour was discussed on the basis of the presence in the chicory
extract of the two main sesquiterpene lactones, 8-deoxylactucin and 11 beta, 13dihydrolactucin.
The effect of Brazilian propolis on the germ tube formation and cell wall of Candida
albicans was studied by Andre Marinho et al., (2006). The ulrastructural findings (TEM)
revealed hyperplasia and changes in the cell surface at 0.43 μg/ml and they suggested that
the antifungal activity of propolis is due to changes in the cell wall leading to an increase
of volume and membrane rupture.
The observations on TEM on colonies of B. cinerea and L. sedotiosum revealed
several autophagic- like vacuoles, morphological alterations on lomasome and lipid
accumulations in the apical zone of hyphae of both fungi. Observations on spore
germination of B. cinerea revealed the presence of strongly stained lipid accumulations
retained by vacuoles at the cell periphery of young hyphae (Savluchinske Feio et al.,
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2002). SEM observations of dermatophytes demonstrated irregular hyphae with
discontinuous cell wall, which after longer exposure time to the plant extract, became
totally broken with severe changes (Berdicevsky et al., 2001). Similarly, the microscopical
observations by Yasuo Yamada and Keizo Azuma (1977) revealed that allicin induced
morphological abnormalities in hyphae of T. mentagrophytes Morita. Superficial and
ultrastructural alterations were induced by Econazole nitrate on Microsporum canis by
Dominique Mazabrey et al., (1985).
Arya MitndSajb Ghosal, (2014) reported a compartive Sorological proteins
of partially
purified
Heamagglutinins
from an Anthrophophilic dermatophytes
(Trichophyton rubrum) and Zoophile dermatophyte (Trichophyton mentagrophytes).
Many works have been carried out on ultrastructral changes in Trichophyton
mentagrophytes. Vannini et al., (1976), reported that in T. mentagrophytes, the most active
compound induced as unusual increase of the plasma membrane with production of intro
and extracytoplasmic complexes, a deterioration of nuclear and mitochondrial membranes
and a formation of autophagic- like vacuoles. The ultrastructural study of T.
mentagrophytes by Park et al., (2009) reveal, a light modification of hyphal morphology,
i.e. a waving of the hyphal surface, thus supports the observation that morphological
changes of T. mentagrophytes caused by amorolfine were associated with its growthinhibitory and killing activity, which depended on the drug concentration and treatment
time.
The optical and electron microscopy of T. rubrum treated in vitro wit 1-amio-6methyl-4-phenylpyrazolo (3, 4-d)-1, 2, 3-triazole showed that the treatment suppressed the
various forms of saprophytic conidia, induced the formation of chlamydospores and
accelerated the formation of arthroconidia. (Donatella Mares et al., 1999).
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The action of allylamine antifungal agents on Epidermophyton floccosum was
studied by Butty (1992), using scanning electron microscopy. Lesions observed after 24 h,
3 and 7 days of contact were mainly on the structure and rigidity of the mycelial and
macroconidial wall. They were characterized by hyphal balloo ning and twisting and by
apical bulbous bulges. Deterioration of macroconidia was characterized by wall
exfoliation. Mares and Fasulo (1990), in their in vitro study of effect of protoanemonin
(PrA) on the ultrastructural studies on Epidermophyton floccosum and Trichophyton
mentagrophytes revealed that wave like hyphae with distorted apical tips were frequently
observed and wall formation was variously affected as revealed by the deposition of
incomplete septa and the accumulation of lomasome- like infoldings.
Effects of drug treatment (Ketoconazole) on T. mentagrophytes were investigated
by scanning electron microscopy by Scott et al., (1985). They found that in the absence of
imidazoles, hyphae were straight and regular; ketoconazole treated hyphae were short,
irregular and branching; miconazole treatment produed extensive debris and spores
forming within the hyphae were visible. Again, Osumi et al., (1984) in their investigations
on morphological changes on hyphae of Trichophyton mentagrophytes treated with a new
azole (Mycospor) revealed several findings: development of curled hyphae; occasional
formation of swollen cells often arranging in chain.
The effect on the changes in the hyphae of Trichophyton mentagrophytes treated
with Naftifine (0.01-0.5 mg/ml) were studied by light and electron microscopy The most
striking changes observed following treatment with this new antimycotic agent were bulbshaped thickenings at the hyphal tips and dose-dependent, spherical, or drop-shaped
depositions of varying size within the cells (Meingassner et al., 1981).
Effects of Pycnoporellus fulgens (Fr.) Donk crude extract on Candida glabra
ultrastructure was investigated by Joelle Moulin Trafort et al., (1999) and TEM revealed
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that the extract acted on cell envelope (cell wall and plasmalemma). Cell divisions were
also affected by thickening of the septum (50 μg/ml) and a deficiency in the daughter cell
wall texture. The extent of the antifungal effect clearly depended on the extract
concentration.
Scanning and transmission electron microscopy showed that a ceiling quantity
(1.56 mcg) of antifungal antibiotic Pyrroinitrin caused heavy damage to dermatophyte
Microsporon audouinii Gruby CBS 313-54 (Nicola and Silvano, 1974).
In the studies by Romagnoli, et al., (2004), the imidazo-pyrazole and pyrazolothiazoles were not particularly effective, while the two pyrazole-thiocyanates proved
highly active Epidermophyton floccosum and Trichophyton rubrum, the most active 5amino-3-methyl-1-phenylpyrazolo-4-thiocyanate was chosen to perform SEM and TEM
morphological studies on both fungi. Both SEM and TEM observations revealed
interesting alterations on the two dermatophytes, particularly involving the endomembrane
system. The minimum inhibitory concentrations (MICs) of three known irreversible
inhibitors
of
polyamine
synthesis,
alpha-difluoromethylornithine
(DFMO)
and
monofluoromethyldehydroornithine methylester (MFMOme), inhibitors of ornithine
decarboxylase (ODC) and alpha-difluoromethylarginine (DFMA), an inhibitor of arginine
decarboxylase (ADC), were determined for 10 species of dermatophytic fungi.
Trichophyton species were generally more sensitive to these inhibitors than Microsporum
species. The ultrastructure of cells cultured in the presence of either DFMO or DFMA was
similar and revealed disruption of calcium metabolism, an increase in mitochondrial
number and alterations to membrane systems. DFMA and DFMO also inhibited
sporulation in Microsporum gypseum (Gruhn and Boyle, 1991).
In the in vitro study on fungitoxicity of the essential oil of Syzygium aromaticum
was studied by Arina Zafar and Iqbal Ahmad (2002). It was found that minimum
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279
fungistatic concentration was found to be 0.05%. Above this concentration, lysis of conidia
and inhibition of mycelial growth were detected in Alternaria alternata. Studies by
Vannini et al., (1981) in an electron microscopic observation undertaken by using the most
active compound, revealed that in Candida albicans mitochondria were the only cell
plasmalemma and main cytoplasmic organelles were damaged in various degrees, where
as in the dermatophytes cell wall, plasmalemma and main cytoplasmic organelles were
damaged in various degrees. The clinical outcome and the effects on morphogenesis and
cell infrastructure induced by a 1% ciclopiroxolamine solution in six patients with proven
pityriasis versicolor were studied and TEM techniques showed extensive internal
disruption, mainly severe necrosis of the cytoplasm, 3 and 7 days after the start of
treatment (Del Palacio and Guarro Artigasp, 1990).
The investigations of Keisuke Fujita et al., (1978) found that both the whole- leaf
powder and the high- molecular-weight component powder of Aloe arborescens Mill sub
sp. natalensis Berger induced various morphological abnormalities in spores and hyphae
by the inhibition of spore germination and development of hyphae. Effects of laser
irradiation on Trichophyton rubrum growth and ultrastructure was reported by Xu ZL et
al., (2012). Tao Liu et al., (2014) were observed gene expression changes in Trichophyton
rubrum after Skin interaction. Fillipe de Oliveira Pereira et al., (2011) was investigated the
antifungal activity of essential oil from Cymbopogon winterianus against the dermatophyte
T. rubrum. Luciana Arantes Soares et al., (2013) provided a brief review on anti
dermatophytic therapy - Prospects for the discovery of new drugs from natural products,
Berenice Aguilar-Guadarrama et al., (2009) was reported active compounds against tinea
pedis dermatophytes from Ageratina pichinchensis var. bustamenta. Karin Giddey et al.,
(2007) have given a detail protein profile by performing SDS page. In vitro antifungal
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activity of Limonene against Trichophyton rubrum was observed by Hee Youn Chee et al.,
(2009).
4.3 Materials and Methods
4.3.1 Effect of active compounds on structural (morphological) changes in
Trichophyton rubrum
The square samples (10 by 10 mm) of T. rubrum grown in the presence of 07
compound like AR-1, AS-1, CR-1, ET-1, FR-1, P-1 and VN-1 at concentration 300 μg/ml
was cut off from plates and mounted in lactophenol cotton blue and examined
microscopically for morphological changes.
Scanning Electron Microscopy
Fungal material obtained from cultures grown either in presence of compounds like
AR-1, AS-1, CR-1, ET-1, FR-1, P-1 and VN-1 or in absence was processed for
morphological studies according to Bozzola and Russel (1999). Mycelial samples were
recovered from 15 days old cultures. The pathologic changes of compounds like AR-1,
AS-1, CR-1, ET-1, FR-1, P-1 and VN-1 isolated from the selected 07 medicinal plants
treated mycelia were analyzed after comparing with control groups grown in the absence
of the compounds. The samples were processed by fixing in 3% (w/v) glutaraldehye in 0.1
M sodium phosphate buffer (pH 7.5) for 3 h at room temperature in Philips SEM (model
515). Fixed materials were infiltrafted with 2% molten agar after thoroughly washing in
phosphate buffer and then post fixed in 1% aqueous osmium tetroxide for 3 h at room
temperature. The samples were dehydrated in a graded water acetone series (10% steps
from 30% to 90% each of 60 min, 100% for 180 min and finally polymerized in spurr’s
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resin at 45o C for 24 h and 65o C for 72 h. the dried particles were then coated with a god
layer in an argon medium and attached on the microscope supported with silver glue.
4.3.2 Effect of active compounds on biochemistry of T.rubrum
Bioche mistry in Trichophyton rubrum treated with compounds like AR-1, AS-1, CR1, ET-1, FR-1, P-1 and VN-1
Polyacrylamide gel electrophoresis
Reagents
1. 30% Acrylamide: 29.2 g acrylamide and 0.8 g n, N + methylen bis acrylamide
solublised in distilled water and made up to 100 ml, filtered and stored in brown
bottles.
2. 1.5 M Tris- Hcl, pH 8.8
3.
1.5 M Tris- Hcl, pH 6.8
4.
10% (w/v) Ammonium per sulfate (APS)
5.
10% (v/v) TEMED.
Separating gel solution
10% gel
15 ml (Total volume)
Distilled water
5.9 ml
30% Acrylamide
5.0 ml
1.5 M Tris-HCl, pH 8.8
3.8 ml
APS
150 μl
TEMED
6 μl
Stacking Gel Solution
10% gel
10 ml (Total volume)
Distilled water
6.8 ml
30% Acrlamide
1.7 ml
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1 M Tris-HCl, pH 8.8
1.25 ml
APS
100 μl
TEMED
10 μl
Sample buffer
1 M Tris-HCl, pH 6.8
0.625 ml
Glycerol
1.0 ml
Bromophenol blue
0.05 g
Electrophoresis buffer
Tris
3.0 g
Glycine
14.3 g
Distilled water
1 litre
The glass plates and spacers were thoroughly cleaned, dried and then assembled
with the help of bulldog clips. The assembly was sealed from inside using molten 1% agar
and allowed to solidify. All the reagents used to for separating gel were taken in a clean
beaker and mixed well. APS and TEMED were added together, just before pouring the gel.
The mixture was added in between two cleaned plates using a 10 ml pipette, up to 3/4th of
the space. Distilled water was added above the separating gel to get uniform surface.
Water was drained off after the gel had solidified. Similarly, stacking gel was prepared and
added over the separating gel. A comb was introduced in to the stacking gel and it was
allowed to set. After setting of the gel the lower spacer and comb were removed and the
assembly was placed in electrophoresis chamber. Protein samples were loaded in the wells
with the help of syringe. Electrophoresis buffer was added to upper and lower tanks.
Electrodes were connected to power pack and gel was run to 50 V until the tracking dye
reached the bottom. Gel plates were separated carefully after removing the spacers and gel
was put in respective staining solution.
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4.4 Experimental results
Effect of compounds on structural changes in Trichophyton rubrum
Effect of seven compounds on hyphae of T. rubrum was observed as follows;
On normal SDA medium, the germ tubes grew rapidly; they were long and
regularly branched, and their tips appeared normal. But on SDA medium containing
300μg/ml, spherical hyphae or bamboo- like-joint hyphae occurred after 7 days of
incubation (Plate-4.1). Hyphae developing from the abnormally swollen microconidia
were spherical or ellipsoid and shorter and thicker than those developing from normal
microconidia.
Microscopic observation on the effect of compounds on hyphae of T. rubrum was
performed by scanning electron microscopy study (Plate-4.1). The concentration 300μg/ml
of compounds seven caused morphological changes in T. rubrum. The most prominent
change seen in T. rubrum treated with 300 μg/ml of compounds was characterized by
several findings: (a) irregular hyphae with discontinuous cell wall, which after longer
exposure time became totally broken with dramatic changes; development of wavy or
shrunken hyphae; (b) excretion of fibrillar materials; (c) partial exfoliation of hyphae
walls. However, in the control the hyphae were arranged in beads form, joined to one
another in the form of chain.
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Table 4.1: Effect of acti ve 07 compounds on structure changes in T.rubrum.
Sl.
Name of the B otanical
Compound
Test
Control
Standar
Treated 7 da ys culture at
no
name
code
dermatophyte
d
300μg/ml
1.
Annona reticulata L.
AR-1
T.rubru m
N
e
A
ac
2.
Annona squamosa L.
AS-1
T.rubru m
N
e
A
ab
3.
Corchorus olitorius L.
CR-1
T.rubru m
N
e
C
bc
4.
Euphorbia tirucalli L.
ET-1
T.rubru m
N
e
B
bc
5.
Ficus racemosa L.
FR-1
T.rubru m
N
e
A
ac
6.
Pongamia pinneta L.
P-1
T.rubru m
N
e
C
ac
7.
Vitex negundo L.
VN-1
T.rubru m
N
e
B
ac
(N) Normal.
(A) Spherical hyphae or bamboo- like-joint hyphae.
(B) Hyphae developing from the abnormally swollen microconidia were spherical or
ellipsoid.
(C) Shorter and thicker than those developing from normal microconidia.
(a) Irregular hyphae with discontinuous cell wall, which after longer exposure time
became totally broken with dramatic changes; development of wavy or shrunken
hyphae.
(b) Excretion of febrile materials.
(c) Partial exfoliation of hyphae walls. However, in the control the hyphae were arranged
in beads form, joined to one another in the form of chain.
(e) Fully irregular shrunken hyphae.
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Plate 4.1: Effect of acti ve 07 compounds on structure changes in T.rubrum S EM observations.
Protein profile
The electrophoretic pattern of soluble proteins dermatophytes grown in the
presence of 07 compounds is presented in figure 4.1. The electrophoretic pattern was
altered when the fungi were grown in the presence of 07 different compounds.
Quantitatively, it was found soluble proteins in the test fungi were significantly lower than
those in control set. Enzyme pattern of the treated fungi differed distinctively from patterns
of control samples.
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Many bands appeared in the treated and control fungi, in electrophoretic gels, but
differences in protein-band patterns were noted; some bands present in control samples
were absent in samples treated with 07 compounds and vice-versa.
Figure-4.1: Protein profiles of test dermatophyte by SDS -PAGE.
Lane C= Trichophyton rubrum (Control); Lane A= T.rubrum treated with AR-1; Lane B=
T.rubrum treated with AS-1;Lane C= T.rubrum treated with CR-1;Lane D= T.rubrum
treated with ET-1;Lane E= T.rubrum treated with FR-1;Lane F= T.rubrum treated with P1;Lane G= T.rubrum treated with VN-1;Lane H= MW= molecular weight marker 1=37
kD, 2=50 kD, 2=75 kD, 2=100 kD.
4.5 Discussion
Over the last decade, the demand for safe and effective antifungal agents has
dramatically been increased in parallel with the expanding number of immune-deficient
patients at risk for fungal infections (Barrett, 2002). Plant derived compounds are of
interest in treating the widespread occurrence of dermal infections caused by
dermatophytes. They comprise safer or even more effective substitutes than synthetically
produced antimicrobial agents. Inspite of the identification of various plant species with
antifungal properties and some of their active components, such as defensins, little
documented data have been presented on their mechanisms of action (Barrrett, 2002).
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In this investigation, the isolated 07 compounds from different extract of
selected plants have strongly affected the hyphae of T. rubrum at 300 μg/ml. The spherical,
shorter and thicker hyphae reveal the decomposition of compounds in the hyphal region of
T. rubrum.
The scanning electron microscopy results strongly suggest that sub inhibitory
concentrations of compounds profoundly affected the normal growth and induced
degenerative changes of the hyphae of T. rubrum probably by affecting some essential
metabolism or structure of the fungal cell. It seems that these dramatic changes in hyphae
are due to a severe damage in the fungal cell coat, or perhaps some alteration in the
membrane permeability, resulting in the loss of cytoplasm or by severe depletion of hyphal
contents. Ultrastructural findings for compounds treated fungus showed that it primarily
targets the hyphal cell membrane. The results of this investigation demonstrated that
damages took place by breaking down of the cell wall and degradation of cell organelles.
Totally, it seemed that compounds acts on the hyphal cell wall. It appears that the
cell wall is the main target of compounds. P lasmolysis and finally cell death appeared to
be the final event of such treatment. The anomalous extrusion of the materials out the wall
observed by SEM in T. rubrum treated with 07 compounds is similar to that previously
observed in other dermatophytes treated with plant derived antimycotics (Mares et al.,
2006). Although all these compounds have different modes of action, this anamoly can be
seen as a generalized reaction to a treatment which affect the normal assembly of the
various parietal components.
Trichophyton rubrum is an anthropophilic fungus with a worldwide distribution
which causes inflammatory or chronic non- inflammatory finely scaling lesion of skin,
nails and scalp. T. rubrum is high contagious and temporary exclusion from school until
appropriate treatment has commenced has long been considered a part of treatment. T.
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288
rubrum is the causative agent of tinea corporis gladiatorum, a fungal infection of wrestlers
and spread among wrestling teams worldwide (Poisson et al., 2005). Effects of laser
irradiation on Trichophyton rubrum growth and ultrastructure was reported by Xu ZL et
al., (2012). Tao Liu et al., (2014) were observed gene expression changes in Trichophyton
rubrum after Skin interaction. Fillipe de Oliveira Pereira et al., (2011) was investigated the
antifungal activity of essential oil from Cymbopogon winterianus against the dermatophyte
T. rubrum. Luciana Arantes Soares et al., (2013) provided a brief review on anti
dermatophytic therapy - Prospects for the discovery of new drugs from natural products
This investigation is a contribution to the area of antifungal chemotherapy which
can be used in the cure of tinea corporis caused by T. rubrum. But, before the use of these
compounds clinical trials should be made in order to prove its antidermatophytic potential.
Protein profile
The proteinase assay performed in this study were primarily aimed at determining
if relative enzyme activities revealed by microorganisms can serve as indicators of
virulence. Varying activity of enzymes in this biochemical assays of all the test fungi in
the presence of compounds may imply that compounds at high concentrations retarded the
production of enzymes, like it is seen in T. rubrum, which has produced least amount of
enzymes at 300 μg/ml.
Dermatophytes invade hair, nail and skin to cause a superficial mycosis. The
hydrolysis of keratin by proteinases (Keratinases) is very likely an important aspect of
fungal pathogenesis. Keratin is an insoluble macromolecule requiring the secretion of
extracellular enzymes for its degradation. However, the production of proteinases differs in
various dermatophytes. The virulence of the pathogenic fungi can be to some extent
identified by its electrophoretic pattern.
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In dermatophytes and fungal infection; there is clear evidence suggesting that
proteinases produced by the dermatophytes play an important role in the pathogenesis of
tissue invasion. Following invasion to the host, cells of the fungal pathogens, are exposed
to unfavorable environmental conditions, this starts the stress response of the fungal cells
(Plesofsky et al., 1993). Adaptation of the microorganism to new environment is necessary
if it is survived in the host. Frequently, this adaptation is linked with the changes of cell
shapes. Reversible transition between mycelial and yeast phases were described in many
significant mycopathogen e.g. Candida albicans, Paracoccidioides brasiliensis and
Histoplasma capsulatum (Szaniszlo et al., 1985). In this study, the compoundsmight have
produced stress to the test fungi and hence there was reduced or sometime nil growth
depending upon the MICs and the antistress protein has been secreted by the fungi in order
to combat the stress. The test dermatophytes in this investigation have failed to adapt to the
stress condition created by comp-2, as adaptation is one of the necessary factor for the
existence of fungi in the host organism and it presumes expression of stress proteins.
Biochemical transformation includes deletion of some protein (including enzymes),
intensification of others, and/or production of new components as evidenced by
quantitative and qualitative changes in bands appearing in electrophoretic gels. These
changes in band pattern indicate generally that biochemical mechanisms operative in hostpathogen interrelationships function efficiently and systematically in favor of the fungus.
The results in this study have supported the above statement that biochemical alterations
were found in samples treated with 07 compounds. The biochemical alterations in the
treated group of fungi in our study may partly play role in appearance of new bands in
gels. In the study by Hearn et al., (1992) on the antigenic activity of detergent extracts of
intact organisms viz., Trichophyton rubrum and Trichophyton interdigitale was analysed
following SDS-PAGE revealed the differences in protein-band patterns; some bands
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present in control samples were absent in azole-treated samples and vice versa. These
differences were reflected in antigenic band patterns also. In this investigation there was
difference in protein bands in T. rubrum (control and treated). Similarly, Fachin et al.,
(2001) in their study of effect of sub-MICs of antimycotics on expression of intracellular
esterase of Trichophyton rubrum, found that the electrophoretic pattern of the intracellular
esterase of the dermatophyte was altered when this fungus was grown in the presence of
sub inhibitory concentrations of the antimycotics tioconazole or griseofulvin. The presence
/ absence of bands in treated/ control group of fungi in this study suggest that the stress
enzymes are produced inside the cell and may be a nonspecific response to cellular stress,
or may participate in cellular detoxification process in the presence of the 07 compounds.
The similar detail protein profile report from other plant extract was given by Karin
Giddey et al.,(2007). Therefore, the protein pattern in this study has shown that few bands
were present in treated samples and absent in control samples and vice-versa. Thus
produced proteins are observed in electrophoretic pattern, but with different kDa values.
This investigation is a contribution to the area of antifungal chemotherapy which
can be used in the cure of tinea corporis caused by T. rubrum. But, before the use of these
compounds clinical trials should be made in order to prove its antidermatophytic potential.
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