FIELD OF THE INVENTION
This invention relates to a novel anticandidal sulphur
' containing molecule from Emericella Sp. in particular to the isolation
and identification of a new biomolecule and its chemical & biological
properties having biomedical significance.
BACKGROUND OF THE INVENTION
Medicinal plants and soil borne micro-organisms are known to
produce a plethora of antibiotics. Due to chemical redundancy in
structure there is a need to discover new natural niches for isolation
of novel and designer molecules. Endophytes are microorganisms
that spend part or all of their life cycle colonizing either inter- or
intra-cellular in the healthy tissues of plants without causing any
immediate overt effects (Shen et aE., 201 1). In addition to the plant
bioactive molecules endophytes are also known to accumulate a
wealth of other biologically active and structurally diverse natural
products that are unexplored (Tan and Zou, 2001; Strobe1 and Daisy
2003; Strobe1 et al., 2004; Gunatilaka, 2006; Zhang et al., 2006;
Verma et al., 2009). These compounds are of importance for drug
discovery or as lead compounds for agriculture, food and
pharmaceutical industries (Strobe1 2006 a, b; Mitchell et al., 2008).
Neem (Azadirachta indica A. Juss) is an indigenous medicinal
plant in India and Africa. It is an evergreen tree of the tropics and
. sub-tropics and has adaptability to a wide range of climatic,
topographic and edaphic factors including stress conditions when
compared to other species (Chari et al., 1996). The polar and
nonpolar extracts of this plant have been observed to show
antibacterial activity (Mahfuzul hoque et al., 2007et al., 2007;
Thakurta et al., 2007, Maragathavalli et al., 20 12) and anticandidal
activity (Menon et aL, 2005, Grover et al., 201 1). In accordance with
the present disclosure Emericella sp. (HE5848 10) reported as an
Page 2 of 22
endophyte from Azadirachta indica of semiarid regions of Rajasthan,
India (data unpublished) has been reported to produce a sulphur
containing mticandidal molecule identified as N-[(2S,3aR,6S,7aS-)6 -
acetamido-octahydro- 1,3-benzothiazol-2-yl12-adamanta1n--y l)
acetamide. Sulphur containing amides are known from plant species
belonging to Glycosmis species exhibiting antibacterial and
antitrypanosomal properies (Greger et al., 1994, Rahmani et al.,
2004, Astelbauer et al., 2010). To the best of our knowledge this is
the first report of sulphur containing acetamide of microbial origin
having anticandidal properties.
Candida species are the leading cause of nosocomial blood
stream infections. Although synthetic natural drugs are available in
the market, but there is always a call for new natural cost effective
drugs. The past years have seen a continuous effort toward the
synthesis of new antifungal agents. Most of them belong to the Nsubstituted
irnidazole and triazole. Another interesting series of
antifungals are the allylamines. Biochemically both the azole
derivatives and the ally1 amines belong to the class of ergosterol
biosynthesis inhibitors revealed that ergoserol depletion is the
consequence of the interaction of the azole derivatives with the
cytochrome P-450 involved in the 14-alpha demethylation of
lanosterol which affect the membranes and membrane bound
enzymes of yeast and fungi. Because of the specific anticandidal
activity of the compound, it can be used as a lead molecule to design
a drug against candidiasis.
Efforts are undertaken to provide a molecule for inhibiting or
preventing the human pathogenic disease caused by C. albicans.
Further, provides evidence that the novel metabolite is of fungal
origin and is effective to inhibit the growth of pathogenic C. albicans
ATCC 1023 1. The present invention provides substantially purified
Page 3 of 22
metabolite identified by GCMS, to be used as lead molecule in
developing a drug for candidiasis.
OBJECTIVES OF THE INVENTION:
The main object of the invention is to provide an isolated
culture of a strain of Emericella sp. from Azadirachta indica.
Another object of this invention is to provide an isolated strain
Emericella sp. wherein the ITS sequence is deposited in EMBL
database.
Yet another object of this invention is to devise a culture
wherein the strain is capable of producing anticandidal metabolite
identified as N-[(2S,3aR,6S,7aS)-6-acetamido-octahydr1o,-3 -
benzothiazol-2-yl12- (adamantan- 1 -yl) acetarnide.
The foregoing has outlined some of the ~e&ent objectives of
the invention. These objectives should not be construed to be merely
illustrative of some of the more prominent features and applications
of the intended invention. Many other beneficial results can be
obtained by applying the disclosed invention in a different manner or
modifjmg the invention within the scope of disclosure.
. Accordingly, ~therob jectives and a full understanding of the
invention and the detailed description of the preferred embodiment
in addition to the scope of invention are to be defmed by the claims
, undertaken.
These and other objectives and advantages of the invention
will be apparent from the ensuing description.
Page 4 of 22
BRIEF DESCRIPTION OF THE DRAWINGS:
. Further objectives and advantages of this invention will be
more apparent from the ensuing description when read in
conjunction with the accompanying drawings wheyein:
Figure 1. : Anticandidal Activity .of Hexane, Ethyl
acetate, Ethanolic, Methanolic and
Aqueous Extracts of Fungal Biomass.
Figure 2a. : TLC profile of Crude on BEA (90: 10).
Figure 2b. : Bioautogram of crude on BEA (90: 10).
Figure 3a. : TLC profile of residual spot from BEA.
Figure 3b. : Bioautogram of residual spot from BEA
on TE (60:40) BEA on TE (60:40).
Figure 4. : IR spectra of the compound eluted from
TLC plate.
Figure 5a. : Gas Chromatogram of the active band
eluted from TLC.
Figure 5b. : Fragmentation pattern of the compound
obtained at RT 28.38.
Figure 5c. : 2D structure of the compound isolated
from Emericella sp.
While the invention is described in conjunction with the
illustrated embodiment, it is understood that it is not intended to
limit the invention to such embodiment. On the contrary, it is
intended to cover all alternatives, modifications and equivalents as
may be included within the spirit and scope of the invention
disclosure as defined by the claims.
Page 5 of 22
STATEMENT OF INVENTION
According to this invention there is provided a Sulphur
containing anticandidal molecule with a general formula A isolated
from a strain of Emeiicella Sp.
General Formula A
DETAILED DESCRIPTION OF THE INVENTION:
At the outset of the description, which follows, it is to be
understood that the ensuing description only illustrate a particular
form of the invention. However, such a particular form is only an
exemplanary embodiment and the teachings of the invention are not
intended to be taken restrictively.
. For the purpose of promoting an understanding of the
principles of the invention, reference is now to be made to the
embodiments illustrated and the specific language would be used to
. describe the same. It is nevertheless to be understood that no
limitations of the scope of the invention is hereby intended, such
alterations and further modifications in the illustrated bag and such
further applications of the principles of the invention as illustrated
Page 6 of 22
therein being contemplated as would normally occur to one skilled in
the art to which the invention relates.
The present invention is further described in light of the
following steps which are set forth for illustration purpose only and
not to be constructed for limiting the scope of the disclosure.
STEP 1: ISOLATION AND CHARACTERIZATION OF Emericella sp.:
Experimental organism: Emericella sp (Accession number
HE5848 10) was isolated from twigs of Neem Azadirachta indica
STEP 2: CULTIVATION OF Emericella sp. AND EXTRACTION OF THE
ACTIVE METABOLITES
The fungus was grown in Liquid Wickerham media (3 gm
Yeast extract, 3 gm Malt extract, 5 gm Peptone, 10 gm Glucose 1-1,
pH 6.8) (Amal et al., 2008) for 28 days at 22.S°C under static
condition with intermittent shaking. After the incubation period, the
mycelial mass was separated by filtration through Whatman no 1
filter paper and the biomass was subjected to extraction. The fungal
biomass obtained after cultivation of Emericella sp. was filtered and
air dried under laminar air flow hood and soaked in hexane with
continuous stirring for 2 hrs (Lu et al., 201 1). The mass was again
filtered, dried and soaked in ethyl acetate and re-filtered. After
filtration it was again soaked in ethanol and the ethanolic phase was
collected, filtered and concentrated under reduced pressure using
vacuum evaporator at 40°C (Ramos et a1.,20 10; Cao and Cardy 20 11;
Santiago 20 12). Mass was also subjected to methanolic and aqueous
extraction by the same procedure (Weber et al., 2007).
STEP 3: ANTIMICROBIAL ASSAY:
Page 7 of 22
Only the ethanolic extract of biomass exhibited antimicrobial
activity specifically against Candida tested by cup plate method
(Figure 1 ., Table 1). Commercially available antibiotic discs (Himedia)
Ketoconazole 32-0.002 pg/disc, Itraconazole 32-.002yg/disc,
Fluconazole 256-0.0 16 pgldisc, Arnphotericin B 32-0.002 yg/disc,
100 units, Nystatin 100 units, Clotrimazole lOpg were used as
positive controls for Candida albicans.
Table 1 Sensitivitv profile of test pathogens against the ethanolic
extract of biomass
STEP 4: ISOLATION AND PUNFICATION OF THE ACTIVE
METABOLITEf S) BY TLC [THIN LAYER
CHROMATOGRAPHY):
Pathogens tested
Staphylococcus aureus
Salmonella typhimurium
Escherichia mli
Pseudomonas aeruginosa
Aspergillus niger
C. albicans
The TLC analysis of the ethanolic extract of biomass was
Sensitivity against ethanolic extract
of fungal biomassf
R
R
R
R
R
S
carried out on 20x20 cm silica gel plates (Merck). 100 pl of the
extract dissolved in DMSO was spotted with a band length of 185 cm
with the help of CAMAG Linomat automated spotter. The spot was
dried at room temperature for 1 hour and developed in two different
solvent systems. The Rr values were calculated using CAMAG
Reprostar 3 (Mabrouk et al., 2008).
Step 4.1: lMobile phase: Benzene:Ethvl acetate 90: 101:
Page 8 of 22
Eight bands were observed at Rf 0.93, 0.90, 0.78, 0.69, 0.4 1,
0.33, 0.25, 0.10 at 366 nm when Benzene and ethyl acetate (90: 10)
was used as mobile phase (Figure 2a). All the eight bands were
scraped and eluted in ethanol. The elute was evaporated and the
residue was dissolved in lOOyl DMSO. Each band and the scraping
from the point of application were checked for anticandidal potential
by .cup plate method (Strobe1 et al., 1996). In addition to the
anticandidal activity of individual bands, bioautograms were also
developed for the TLC plate against Candida albicans to confirm the
location of the active band by the method described by Nithya and
Muthumary (2010). However, the active component was retained at
the point of application as determined by eluants from the plate and
bioautogram (Figure 2b). As the anticandidal activity was retained at
the point of application of the sample, the same was scraped and
eluted in ethanol and was run on second mobile phase.
Step 4.2: LToluene: Ethanol, 60:40):
The second mobile phase of Toluene and ethanol (60:40) was
used for further separation of compounds from the residual band
after elution. The mobGe phase led to the separation of 3 bands at Rf
0.72, 0.79 and 0.89 at 366nm. However, the anticandidal activity
was detected for band with Rf 0.72 (Figure 3a and 3b). The
bioautogram was performed. The active band confirmed by
bioautogram was scraped, eluted in ethanol and evaporated to
dryness. This dried powder was subjected to GCMS and IR. This
band was eluted, evaporated to dryness and subjected to IR and
GCMS for identification of the molecule.
STEP 5: GAS CHROMATOGRAPHY:
GCMS: The sample was analysed by GCMS (Schimadzu GC
2010 and GCMS QP-2010 plus) coupled with mass
spectrophotometer. The GC column dimensions'used were 30m x
Page 9 of 22
0.25mm x 0.5mm AB35 MS fused silica capillary column. For GC the
injector temperature was set at 250°C, column temperature kept
isothermal at 50°C then programmed to rise up to 150°C with a rate
of 30°C/min and be held at this temperature for 3 min, then again
with the rate of 3°C /min, the temperature rose to 260°C and held
up for 1 min. The ion source temperature was 200°C and the
interface temperature was 250°C. Helium gas was engaged as carrier
gas at the rate of lrnl/min. The spectra were obtained in the EI mode
with 70eV ionization energy. The run time for sample was 45 min
with scanning speed of 1 1 1 1 and m/z scanning range was from 50
m/z to 500 m/z. Single peak was obtained at RT of 28.384 min when
the compound was subjected to GCMS. On the basis of GCMS the
molecular weight of the bioactive molecule is 383 and molecular
formula is C21H25N302S (Fig 5a and 5b). The GCMS Library results
depicted the compound as N-[(2S,3aR,6S,7aS)-6-acetamidooctahydro-
l,3-benzothiazol-2-yl12-(adamantan1--y l) acetamide with
90% similarity.
STEP 6: RESULTS OBTAINED
IR spectra for analysis of functional groups: The functional
. groups present in the metabolite were identified by Fourier
Transform Infra Red (FTIR) spe,ctrophotometer (Schimadzu 8400 S) . ,
The dried powder from TLC elute (lmg) was mixed with 300 mg of
dried, finely powdered IR grade KBr so as to make a smooth powder.
A disc of 13 mm diameter was prepared by spreading the mixture
uniformly in a die set. The die set was compressed under vacuum at
a pressure of about 800MPa. The resultant disc was mounted in the
holder of the spectrophotometer to check percentage transmittance
of the disc. A disc with 75% transmittance was prepared. The holder
was adjusted into the scanning slot. The detection range was set in
the range of 400 - 400Ccm-1.
Page 10 of 22
IR SPECTRAL INTERPRETATION:
(Figure 4; Figure 5a;Figure 5b; Figure 5c.)
The IR spectra of the metabolilte: (KBr) Vm, cm -1 34 17-32 18
(NH2), 2957-2853 (CH3 attached to keto group), 1659 (Streching of
NH2-C=O), 1562 (-NH bending of secondary amide), 1383 (-CH3
bending), 1125 (-CH of Aromatic rings). Presence of sulphur in the
molecule is depicted by a weak absorption between 670-715 cm-1.
CONCLUSIONS:
Endophytes from new niches can be explored for development
of novel and designer molecules of indus'trial applications.
Anticandidal compounds known till date belong to azole, allylamine
and polyene class of antifungal agents. Here a new molecule with
sulphur is found inhibitory specifically towards Candida sp. , hence
this molecule can be used as a lead molecule to design new
anticandidal drugs.
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All documents cited in the description are incorporated herein
by reference. The present invention is not to be limited in scope by
the' specific embodiments and examples which are intended as
illustration of a number of aspects of the scope of this invention.
Those skilled in the art will know or to be able to ascertain using no
more than routine experimentation many equivalents to the specific
embodiments of the invention described herein.
It is to be noted that the present invention is susceptible to
modifications, adaptations and changes by those skilled in the art.
Such variant embodiments employing the concepts and features of
this invention are intended to be within the scope of the present
invention, as set forth under the claims hereunder.
Page 15 of 22

WE CLAIM:
'1. A Sulphur containing anticandidal molecule with a general
formula A isolated from a strain of Emericella Sp.
General Formula A
2. A sulphur containing anticandidal molecule as claimed in
claim 1 wherein a strain of Emericella Sp is isolated from
Azadirachta Indica.
3. A sulphur containing anticandidal molecule as claimed in
claim 1 & claim 2 wherein ITS sequence of the isolated strain
of Emericella Sp is deposited EMBL database with the
accession number HE5848 10.
'4. A sulphur containing anticandidal molecule as claimed in
preceding claims which is capable of producing anticandidal
metabolite identified as N- [2S7 3aR7 6S, 7aS)-6-acetamidooctahydro-
l,3-benzothiazol-2-y 112-(adamantan- 1 -y 1)
acetamide.
Page 16 of 22
5. A sulphur containing anticandidal molecule as substantially
described herein with reference to Examples and
accompanying figures.