|Title of Invention||
A NOVAL ANTIFUNGAL/ANTICANCER COMPOSITION
|Abstract||The present invention relates to a novel antifungal/anticancer composition. More particularly, the present invention relates to methyl-ß-orcinol carboxylate of formula 1 isolated from a lichen (Everniastrum cirrhatum), for treating pathogenic fungal infections of humans that are resistant to polyene and azole antibiotics such as amphotericin B, nystatin, clotrimazole etc.|
|Full Text||Field of the invention
The present invention relates to a novel antifungal/anticancer composition. More particularly,
the present invention relates to methyl-ß-orcinol carboxylate of formula 1 isolated from a
lichen (Everniastrum cirrhatum), for treating pathogenic fungal infections of humans that are
resistant to polyene and azote antibiotics such as amphotericin B, nystatin, clotrimazole etc.
Lichens are symbiotic associations between fungi, green algae and/or cyanobacteria.
They have a varied chemistry and produce many polyketide-derived compounds, including
some, such as depsides and depsidones that are rarely reported elsewhere. Depsides are a
class of compounds, which appear to be unique to the lichens. These compounds are dimeric
esters of variously substituted orsellinic acids and are the major source of the so-called lichen
acids. Although lichens have been appreciated in traditional medicines, their value has largely
been ignored by the modern pharmaceutical industry because difficulties in establishing
axenic cultures and conditions for rapid growth preclude their routine use in most
conventional screening processes.
The association between fungi and algae is specific and selective. The name of the
fungal component is given to the whole lichen and there are >13500 described species,
including almost one-fifth of all known fungi (Hawcksworth and Hill, 1984; The Lichen
Forming Fungi, Mecorquodale Ltd). Although the individual mycobionts and photobionts
(the fungi and the photosynthetic algae or cyanobacteria, respectively) are small and
nondescript if cultured in a laboratory dish, the symbiotic components together in nature
present a full range of varied and beautiful forms, and some such as Ramalina menziesii (the
'fishnet' lichen) can drape entire trees, creating a prominent display (Arvis,W.O.,20QO;
Lichens, Smithsonian Situation Press). They perform a variety of ecological roles such as
colonizing marginal habitat in Antarctica, stabilizing soil in the semi-arid desert of Australia
and contributing to nitrogen turnover in the northern pacific forests of North America.
They produce characteristic secondary metabolites that are unique with respect to
those of higher plants. Lichens produce a wide range of chemical compounds, among which
approximately 350 secondary metabolites have been identified. These mycobiont derived
products usually accumulate as extra cellular crystals on the cell walls of the symbionts, and
account for up to 10% (in exceptional cases, up to 40%) of thallus dry mass (Galun, M. and
Shomer-llan, A. (1988) in CRC Handbook of Lichenology, Vol. Ill; Galun, M., ed.), pp,3-
8.CRC Press); many are unique to lichens. Most lichen secondary compounds are formed by
the polyketide pathway, while others derive from the shikimic acid and mevalonic acid
pathways these are key routes for secondary metabolism in all organisms. Several lichen
extracts have been used for various remedies in folk medicine, and screening test with lichens
have indicated the frequent occurrence of metabolites with antibiotic, antimycobacterial,
antiviral, analgesic, and antipyretic properties.
Furthermore, a distinct class of lichen metabolites is the depsides. These types of
compounds are formed by condensation of two or more hydroxybenzoic acids whereby the
carboxyl group of one molecule is esterified with a phenolichydroxyl group of a second
molecule. Owing to the phenolic nature of their chemical structures, these molecules are
interesting candidates for evaluating their effects on leukotriene biosynthesis, as a major class
of inhibitors often contains a hydroxylated aromatic ring (Fitzsimmons et al 1989). Moreover,
two small-molecule lichen-derived metabolites, protolichesterinic acid and lobaric acid, have
been reported to inhibit 5-LO from porcine leukocytes (Ogmundsdottir et al 1998). The latter
has also been shown to inhibit peptide leukotriene formation (Gissurarson et al 1997). Lichen
depsides have also been described to inhibit prostaglandin biosynthesis (Sankawa et al 1982).
Lichens and lichen products have been used in traditional medicines for centuries and
still hold considerable interest as alternative treatments in various parts of the world. Indeed,
today a variety of lichen-based tonics, lotions and lozenges can be purchased in Iceland,
where they're medicinal. However, lichens have been essentially ignored by the modern
pharmaceutical industry, despite the fact that lichens produce a large number of low
molecular weight molecules with diverse structures and that studies have provided evidence
of biological activity in extracts from whole lichens (Table-1). There are two contributing
reasons for this; (1) lichens are slow growing in nature and (2) they are difficult to propagate
and resynthesize in culture (Ahmadjian , 1993; The Lichen Symbiosis, Blaisdell Publishing
Company). Industrial scale harvests are neither ecologically sensible nor sustainable and for
many species are not feasible. Even if the lichen cultures are established in-vitro they do not
produce the typical lichen substances and the techniques to encourage this are still unknown.
Table-1: Previously described bioactive constituents from different Lichens.
It is thought that most secondary metabolites of lichens are made by the mycobiont
(Huneck, and Yoshimura, (1996) Identification of Lichen Substances, Springer-Verlag). This
is not surprising because fungal compounds are well known in medicine (e.g. penicillin and
cyclosporin). It is possible, however, that the photobionts also contribute to the repertoire of
lichen metabolites. Cyanobacteria produce many bio-active secondary metabolites
(Namikoshi, M. and Rinehart, K.L. (1996) Bioactive compounds produced by cyanobacteria.
J. Ind. Microbiol. 17, 373-143) and there is an example of a patented anti fungal compound
produced by a strain of Nostoc isolated from a lichen (US Pat.No. 4, 946, 835, Merck & Co).
There are compelling reasons for expanding the search for natural-product drugs
because previously reliable standard antibiotics are becoming less and less effective against
new strains of multi drug-resistant pathogens. It has even been suggested that the end of the
antibiotic era is fast approaching. In the past, search for pharmaceutically active molecules
concentrated on the products of microbes that can be cultivated in the laboratory. More
recently synthetic chemical methodologies have attracted a great deal of attention and
combinatorial chemistry has been promoted as a source of molecules for automated highthroughput
screening methods. Although these approaches have provided some lead
molecules there is still a great need to discover novel chemical entities for therapeutic use.
Systemic and superficial fungal infections affect millions of people throughout the
world. Most of these diseases are caused by Candida albicans, Cryptococcus neoformans,
Aspergillus sp., Trichophyton sp., Microsporum gypseum, Epidermophyton floccossum that
are infectious in nature. In India, large number of people are involved in agriculture with
majority of them living in villages where due to the prevailing unhygienic conditions the
incidence of mycotic infections are severe. Fungal infections are also assuming increasing
importance on account of decrease in immune systems mainly because of organ transplant
operations, cancer chemotherapy and acquired immune deficiency syndrome (AIDS).
Moreover the skin infections spread rapidly due to poor hygienic conditions and over
population as well as increasing level of environmental pollution. To counter these infections
only a handful of antifungal agents such as greseofulvine, amphotericin and nystatin are
available in the market, although the available antibacterials are replete. Most of these
antifungals are synthetic derivatives with known side effects to human and animals.
Compounding this problem is the development of resistance towards commonly used drugs
thus rendering the chemotherapy less useful. Therefore new antifungal substances from
natural sources have to be generated to counter the resistance phenomenon. During 1990-96
the world market for antifungals was over US $ 1500 millions representing 1.5% of the total
global anti-infective market. Currently anti-fungals (both topical and systemic) represent
more than 6% of the total anti-infective agents. The world market for antifungals is
expanding at the rate of 20% per annum and is estimated to reach over US $ 600
million/annum. However, many of the synthetic drugs produce side effects in immune
stressed individuals. On the other hand natural products and their formulations made out of
herbal sources will have more acceptances than the synthetic antifungals.
Objects of the invention
The main object of the present invention is to provide a novel antifungal/anticancer
The main object of the present invention to identify Lichen extract, which can
specifically kill the polyene drug resistant fungal infections of humans.
It is also the object of the invention to isolate, characterize and establish the nature of
the bioactive molecule from the active lichen extract by bioactivity-guided fractionation.
Still another object of the invention is to test the ergosterol binding ability of the
bioactive molecule using in-vitro assays.
Summary of the invention
Accordingly the present invention provides a novel antifungal/anticancer composition
comprising a pharmaceutically effective amount of methyl-p-orcinol carboxylate of formula I
and a pharmaceutically acceptable carrier
In one embodiment of the invention, the composition is anti-fungal and the methyl-ßorcinol
carboxylate of formula I is present in a concentration in the range of 10-400 μg/ml.
In another embodiment of the invention, the composition is anticancer and the
methyl-p-orcinol carboxylate of formula I is present in concentration in range of 1-10 μg/ml.
In another embodiment of the invention, the fungus is from the group of yeasts
comprising of Candida sp, exemplified by Candida albicans.
In another embodiment of the invention, the cancer is liver, colon, ovarian or mouth
(oral) cancer of humans.
The invention also relates to a method of treatment of fungal infections in a subject
comprising administering to the subject an anti-fungal composition comprising a
pharmaceutically effective amount of methyl-p-orcinol carboxylate of formula I and a
pharmaceutically acceptable carrier.
In one embodiment of the invention, the methyl-p-orcinol carboxylate of formula I is
isolated from lichen Everniastrum cirrhatum.
In another embodiment of the invention, the fungus comprises a multiple or single
drug resistant strain.
In another embodiment of the invention, the methyl-p-orcinol carboxylate of formula
I is present in a concentration in the range of 10-400 jig/ml.
In a further embodiment of the invention, the fungus is from the group of yeasts
comprising of Candida sp, exemplified by Candida albicans.
In a further embodiment of the invention, the fungus is a polyene drug resistant strain,
the polyene drug being exemplified by nystatin and anphotericin
In yet another embodiment of the invention, the fungus comprises an azole resistant
strain, the azole drug being exemplified by clotrimazole, flucanoazole, itracanoazole and
In yet another embodiment of the invention, the fungus is simultaneously resistant to
both polyene and azole classes of antibiotics.
The subject is preferably human.
The present invention also provides a method for the treatment of cancer in a subject
such as a human being, the cancer being either of liver, colon, ovarian and mouth (oral)
cancer comprising administering to the subject a pharmaceutically effective amount of
methyl-p-orcinol carboxylate of formula I and a pharmaceutically acceptable carrier.
In one embodiment of the invention, the concentration of methyl-p-orcinol
carboxylate of formula I is in the range of 1-10 μg/ml.
The present invention also relates to the use of methyl-p-orcinol carboxylate of
for the treatment of fungal infection or cancer in a subject.
Detailed description of the invention
The present invention relates to a novel biomolecule methyl-p-orcinolcarboxylate of
formula I isolated from a lichen (Everniastrum cirrhatum),
for treating pathogenic fungal infections of humans that are resistant to polyene antibiotics
such as amphotericin B, nystatin etc. However, the biomolecule does not possess ergosterolbinding
Infections due to Candida sp account for about 80% of all major systemic fungal
infections. Candida is now the fourth most prevalent organism found in bloodstream
infections and is the most common cause of fungal infections in immuno-compromised
people. Vaginal candidiasis commonly affects women, including those with normal
immunity, especially after antibiotic use.
Lichens were collected from Narayan Ashram, Pithoragarh; Uttaranchal, India in the
month of April 2002. Subsequently the lichens were identified taxonomically as
Everniastrum cirrhatum. The collected lichen was air dried in shade and ground to fine
powder. The powdered lichen material was used further for chemical analysis. Ethanol
extract was prepared and tested against Candida albicans MTCC 1637 (equivalent to ATCC
18804) the fungi that cause different forms of candidiasis in humans and drug resistant
mutants of the fungi. Amphotericin and nystatin are standard polyene antifungal drugs used
in chemotherapy. Candida albicans isolates resistant to these polyene antibiotics are already
reported. High-level resistance to amphotericin B, seen in all the major Candida species, is
most common in neutropenic patients who have received prolonged courses of amphotericin
B. Such drug resistant infections are clinically difficult to treat and are physician's nightmare.
Hence, we developed such polyene resistant strains of Candida albicans in-vitro and
evaluated the anti-candidial effect of lichen extracts / compounds against them. The extract
and subsequent solvent (hexane and ethyl acetate) fractions were found to be active against
amphotericin and nystatin resistant Candida. Bioactivity guided fractionation of the active
fractions resulted in the isolate of active compounds by column chromatography. The active
compound could be crystallized from 96% hexane: 4% ethyl acetate fraction. The purified
compound was analyzed by spectroscopic techniques using 'H & 13C NMR, LC-MS etc to
decipher the chemical structure. Ccompound was identified as methyl-(3-orcinolcarboxylate,
of formula I. The compound is a colorless crystal with melting temperature of 137°C.
Caccamese et al (1985) have already found that the methyl-(3-orcinolcarboxylate inhibit the
growth of yeast strains such as Saccharomyces cerevisiae. However, in this study we shown a
unique property of methyl-p-orcinolcarboxylate wherein the compound specifically inhibits
the growth of polyene and azole drug resistant strains of Candida albicans and
Saccharomyces cerevisiae. The principal sterol in the fungal cytoplasmic membrane, is the
target site of action of amphotericin B and the azoles. Amphotericin B, a polyene, binds
irreversibly to ergosterol, resulting in disruption of membrane integrity and ultimately cell
death. Therefore, the ability of lichen compounds to bind to ergosterol was also investigated
using in-vitro ergosterol binding assay (Antonio & Molinski 1993; J.Natl.Prod.56:54-6\).
The results indicated that the compounds do not possess any specificity to ergosterol in the
wild type and drug resistant strains of Candida sp.
The present invention therefore provides an antifungal/anticancer composition
comprising a pharmaceutically effective amount of methyl-p-orcinol carboxylate of formula I
and a pharmaceutically acceptable carrier. A concentration of methyl-p-orcinol carboxylate
of formula I in the range of 10-400 μg/ml provides antifungal activity against the group of
yeasts comprising of Candida sp, exemplified by Candida albicam. A concentration of
methyl-p-orcinol carboxylate of formula I in the range of 1-10 μg/ml provides anticancer
activity against liver, colon, ovarian or mouth (oral) cancer of humans.
The methyl-p-orcinol carboxylate of formula I is isolated from lichen Everniastrum
The fungus can be either a multiple drug resistant or single drug resistant strain. For
example, the fungus can be from the group of yeasts comprising of Candida sp, exemplified
by Candida albicans. The drugs in question can be a polyene drug exemplified by nystatin
and anphotericin or a azole drug exemplified by clotrimazole, flucanoazole, itracanoazole and
The following examples are illustrative and should not be construed as limiting the
scope of the invention in any manner.
1.Isolation of polyene drug resistant mutant strains of Candida albicans MTCC 1637
(equivalent to ATCC 18804)
C. albicans was grown to log phase in Sabouraud's dextrose broth (5 ml) for 48 hrs at
37°C in a shaker at 250rpm. The cells were pelleted by centrifugation at SOOOrpm at 4°C and
the pellet was dissolved in 5 ml phosphate buffered saline PBS (6.8pH). The culture was
divided in to five groups of 1 ml each in eppendrof tubes.
Ethyl methane sulfonate (EMS) was added to each of the culture tube @ 0.1% (v/v)
and allowed to grow for 40 min. Then the mutagen was completely washed off thrice by
repeatedly pelleting the cells and re-dissolving in PBS. The mutagenized stocks was then
diluted in Sabouraud's dextrose broth two folds and allowed to grow for 6 hrs at 37°C in a
shaker at 250rpm. Titre of the cells before treatment with EMS and immediately after
treatment with EMS was calculated to obtain the killing percentage in each of the five tubes.
The mutagenized and fixed cultures were then plated in Sabouraud's dextrose agar containing
different concentration of amphotericin, nystatin and clotrimazole.
The colonies found growing after 5thday from each of the five mutagenized stocks
were then purified thrice separately by streaking in the same medium containing the
2. Drug resistance of mutant strains against polyenes and azoles
The drug resistance property of the mutants was studied by standardized disc
diffusion assay (Bauer at al 1966, American Journal of Clinical Pathology 45: 493-496) with
slight modifications. The discs were prepared (5 mm diameter made of Whatman #3 filter
paper) by impregnating 8 μl of test compound and placing them on pre-inoculated agar
A disc containing only the solvent was used as the control. A zone of growth
inhibition surrounding the disc is indicative of the resistant nature of the strains to antibiotics.
As is evident from this example the results indicate that all the mutant strains were highly
resistant to amphotericin and nystatin as the zone of growth inhibition was far less in mutants
than that of the wild type parent strain. However only Amph C7R, Amph C6R, Clo 31R and
Clo 28R were only resistant to clotrimazole indicating of less zone of growth inhibition.
3.Collection and extraction of lichen materials:
Two kg of the lichen (Everniastrum cirrhatum) material were collected from Narayan
Ashram, Pithoragargh, Uttaranchal, during the month of April 2002. They were separated and
air-dried at room temperature (35°C-40°C) in shade. After air drying they were ground and
sieved to fine powder in a mixer grinder. 1.5 kg of the powdered materials were dipped in
absolute ethanol in a percolator for 72 hrs at room temperature (35°C-40°C).
Ethanol extract was filtered using Whatman filter paper No.l and concentrated at the
60°C under reduced pressure. The ethanolic extract was then lyophilized to obtain 15.5 g of
crude extract. Stock of 100 mg/ml was made in DMSO and tested for bio-activity.
4. Bioactivity guided fractionation of the lichen materials
Solvent fractionation of the active crude extracts was undertaken to isolate the active
principle. Ethanolic extract was dissolved in 500ml of hexane. Then it was filtered using
Whatman No. 1 filter paper. The insoluble portion was dissolved in 500ml of ethyl acetate.
All the solvent fractions were concentrated at 40°C under reduced pressure to obtain 3g of
hexane and 1.5g of ethyl acetate extract and tested. The results indicate that both ethyl acetate
and hexane fraction obtained from the crude extract possessed the bioactivity against drug
resistant strains of C.albicans. The hexane fraction was considerably more active than the
ethyl acetate extract.
5. Purification and characterization of the active molecule
The hexane and ethyl acetate fractions thus obtained are mixed together and further
fractionated in a glass column having an internal diameter of 3.0 cm and length of 72.0 cm.
Hexane was used as the initial mobile phase and silica gel (particle size 60-120 mesh) as the
stationary phase. Different fractions of approximately 100 ml were collected and dried under
vacuum. Concentrated fractions were then run on TLC plates and fractions of similar TLC
pattern were pooled together. After about 3 liter of hexane fraction collected the polarity of
the mobile phase was slightly increased from fraction No. 36 by adding ethyl acetate to
hexane (4% of ethyl acetate in final volume). Similarly fractions No.64 to 78 were combined
together based on identical 7-spot bands as appeared in TLC. Above fractions were dissolved
in 50 ml of acetone and kept at room temperature (25-30°C) for crystallization of compounds.
Crystals thus obtained were again properly washed with acetone and TLC of crystals was
carried out by using a mobile phase of benzene 98% plus acetone 2%. TLC plates showed a
single spot on exposing to iodine fume. These TLC plates exhibited a single dark red colored
spot when dipped in bacopa reagent (vaniline 3.5 g, H2SO4 17.8 g, absolute alcohol 332.5 ml)
and heated at 120 ° C for 5 minutes. About 40 mg of the crystal could be collected from the
above run. The melting temperature of crystal thus obtained was found to be 137 ° C.
The active spot obtained by TLC was further purified by repetitive column
chromatography, which can be performed by a person skilled in the art and then analyzed by
H & 13C NMR, LC-MS to determine the structure of the active pure compound. On the basis
of spectroscopic data the compound isolated was identified as Methyl-p-orcinolcarboxylate.
6. Specific anticandidial activity of methyl-p-orcinolcarboxylate acid against polyene
and azole resistant strains
The pure compound isolated was then tested against polyene and azole resistant
strains of Candida albicans. The data described below indicates that the compound methyl-ßorsellinic
acid was able to inhibit the growth of drug resistant strains in a dose dependant
manner whereas it was inactive against the wild type strain. In another experiment welldefined
amphotericin and nystatin resistant strains of Saccharomyces cerevisiae were used in
the assay. These strains designated as erg 1 and erg 6 carry mutations in the ergosterol
biosynthetic pathway and therefore are unable to synthesize ergosterol which are the binding
site of polyene drugs. Therefore absence of ergostreol results in polyene resistance. The
results suggests that methyl-p-orcinolcarboxylate was able to specifically inhibit the growth
of polyene drug resistant Saccharomyces cerevisiae.
7. Anticancer property of methyl p-orsellinic acid against human cancer cell lines
Cytotoxicity testing in vitro was done by the method of Woerdenbag et al.,1993;
J.Nat.Prod. 56 (6): 849-856). 2x103 cells/well were incubated in the 5% CO2 incubator for
24h to enable them to adhere properly to the 96 well polysterene microplate (Grenier,
Germany). Test compounds dissolved in 100% DMSO (Merck,Germany) in atleast five doses
were added and left for 6h after which the compound plus media was replaced with fresh
media and the cells were incubated for another 48h in the CO? incubator at 37°C. The
concentration of DMSO used in our experiments never exceeded 1.25%, which was found to
be non-toxic to cells. Then, 10 μl MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; Sigma M 2128] was added, and plates were incubated at 37°C for 4 h. 100 μl
dimethyl sulfoxide (DMSO, Merck, Germany) were added to all wells and mixed thoroughly
to dissolve the dark blue crystals. After a few minutes at room temperature to ensure that all
crystals were dissolved, the plates were read on a SpectraMax 190 Microplate Elisa reader
(Molecular Devices Inc., USA), at 570 nm. Plates were normally read within 1 h of adding
the DMSO. The experiment was done in triplicate and the inhibitory concentration (1C)
values were calculated as follows: % inhibition =[1-OD (570 nm) of sample well / OD (570
nm) of control well] x 100. IC9o is the concentration μg/mL required for 90% inhibition of
cell growth as compared to that of untreated control. The results described indicate that the
ethanolic crude extract of the lichen and the isolated pure compound methyl-Pore
inolcarboxylate was active against liver (WRL-68); colon (Caco-2); ovarian (MCF-7 &
PA-1) and oral (KB 403) human cancer cell lines.
1. A novel antifungal/anticancer composition comprising a pharmaceutically effective amount of methyl-ß-orcinol carboxylate of formula I and a pharmaceutically acceptable
2. A composition as claimed in claim 1, wherein the composition is anti-fungal and the
methyl-p-orcinol carboxylate of formula I is present in a concentration in the range of 10-
3. A composition as claimed in claim 1 wherein the composition is anticancer and the
methyl-p-orcinol carboxylate of formula I is present in a concentration in the range of 1-
4. A composition as claimed in claim 2 wherein the fungus is from the group of yeasts
comprising of Candida sp.
5. A composition as claimed in claim 3, wherein the cancer is liver, colon, ovarian or mouth
(oral) cancer of humans.
6. An antifungal/anticancer composition substantially as herein described withreference to
the foregoing examples.
|Indian Patent Application Number||3824/DELNP/2005|
|PG Journal Number||8/2010|
|Date of Filing||26-Aug-2005|
|Name of Patentee||COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH|
|Applicant Address||ANUSANDHAN BHAWAN,RAFI MARG,NEW DELHI-110 001,INDIA.|
|PCT International Classification Number||A61K|
|PCT International Application Number||PCT/IN03/00097|
|PCT International Filing date||2003-03-31|