EP1945237A2 - Use of extracts of myrtle and other mediterranean plant species, as antimycotic agent towards yeast and yeast-like microorganisms - Google Patents

Use of extracts of myrtle and other mediterranean plant species, as antimycotic agent towards yeast and yeast-like microorganisms

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Publication number
EP1945237A2
EP1945237A2 EP06810032A EP06810032A EP1945237A2 EP 1945237 A2 EP1945237 A2 EP 1945237A2 EP 06810032 A EP06810032 A EP 06810032A EP 06810032 A EP06810032 A EP 06810032A EP 1945237 A2 EP1945237 A2 EP 1945237A2
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Prior art keywords
yeast
extracts
myrtle
deriving
species
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German (de)
French (fr)
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Patrizia Pinelli
Benedetta Turchetti
Giovanni Nieddu
Annalisa Romani
Pietro Buzzini
Innocenza Chessa
Flavia Franconi
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/22—Anacardiaceae (Sumac family), e.g. smoketree, sumac or poison oak
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics

Definitions

  • the ecosystem Mediterranean maquis is recognized as "excellent of plant biological diversity. A great variability is also expressed at intraspecies level, due to the plant species adaptability to the heterogeneous environmental conditions, which characterize the territories around the Mediterranean Basin. Among the distinctive plant species, the following are regarded as a valuable opportunity for* the sustainable development of many areas in the Mediterranean Basin:
  • flavonoids seems to phlogosis or inflammation control (Katiyar et al., 1992), the platelet function (Duffy et al., 2001), the vascular tone (Duarte et al., 2001 ; Burs et al., 2000), the angiogenesis (Cao e Cao, 1999), the arteriosclerosis (Stenvold et al., 1992), and the carcinogenesis (Y an 9. 1997).
  • the different flavonoids can express different biological activities: sometirhes, the single flavonoid molecule is characterized by a less significant activity than the mixture of different compounds, thus suggesting the existence of a (possible) synergistic activity.
  • Enzymes inhibited by the polyphenols occurring in green tea composition are associated with the pathogenesis of some cardiovascular diseases (Cahilly et al., 2000; Griendling et al., 2000).
  • catechins and the epigallocatechins, as well as a*number of other bioflavonoids act by directly neutralizing the ROS (whose role on phenomena of apoptosis and expression of stress protein in cell of cardiovascular apparatus is well documented).
  • the invention genetically relates to utilization of natural extracts of myrtle, cistus, arbutus, lentisk and other Mediterranean plant species containing galloyl derivatives and hydrolysable tannins deriving from artisan or industrial manufactures as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
  • FIG. 1 shows in Figs. 1 ;and 2 diagrams relating to antimycotic spectrum of myrtle extracts: killing curve towards Candida glabrata DBVPG 3828.
  • the invention relates to the preparation of myrtle extracts (standardized in the content of polyphenols) from different plant tissues and from by-products deriving from artisan or industrial manufactures carried out over the same species and their utilization as antimycotic agents active towards yeast and yeast-like microorganisms of biomedical interest.
  • the present invention has considered the following phases: I) Selection of myrtle genotypes, their propagation and cultivation. The adoption of crop management systems characterized by a low-environmental impact is recommended. The growing of selected varieties will assure the ripening control and the reproducibility of the mean quality characteristics from one year to the other and from different growing locations. The aims of a direct domestication of wild plants species and the crop management are essentially directed to standardize the final product quality and to lessen the energy input, as well as to reduce environmental and operational costs.
  • Type strain - T type strains belonging to species considered as known or emerging pathogens for humans and animals.
  • type strain - T type strains belonging to species considered as known or emerging pathogens for humans and animals.
  • myrtle extracts (above cited) as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered known or emerging pathogens for humans and animals.
  • the preliminary determination of antimycotic activity obtained through the combined use of myrtle extracts (above cited) with Amphotericin B has been carried out by using the Agar Diffusion Well Bioassay (ADWB) (Hammond e Lambert, 1978) .
  • ADWB Agar Diffusion Well Bioassay
  • V derivatives and/or hydrolysable tannins in association with cistus, arbutus, lentisk, and phillyrea extracts in order to obtain mixed preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
  • extracts obtained through aqueous or low-temperature hydroalcoholic extraction procedures) from all above Mediterranean plants (in all possible combinations) as mixed preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
  • the invention has also foreseen the use of a set of yeasts and yeast-like microorganisms including type strains (type strain - T) belonging to species considered known or emerging pathogens for humans and animals.
  • type strains type strain - T
  • the type strain represents its reference strain internationally recognized and conserved in all most important world collections of microbial germplasm. Accordingly, since the biological variability observed within strains belonging to a same species, results obtained by using worldwide available strains (e.g. type strains) gave the advantage of their high comparability within and among laboratories.
  • the invention also foresees the combined use of extracts of myrtle, cistus, arbutus, lentisk, and phillyrea, as well as commercial extracts of plants of the genera Vitis and Pinus, among them or in association with Amphotericin B or Ketoconazole, in order to induce a synergistic effect among different active principles. This should be interesting in reducing the risk of overexposition of patients to high dosages of antibiotics. Analogously to above we also foresee the combined use of polyene antibiotics other than Amphotericin B (characterized by synthetic or biological origin).
  • type strain - T type strains belonging to species considered known or emerging pathogens for humans and animals. Accordingly, the use of type strains as target microorganisms may be considered an additional innovative point.
  • the type strain of a given species represents its internationally recognized reference strain. Type strains are conserved in all worldwide collections of microbial germplasm. Accordingly, since the biological variability observed within strains belonging to a same species, results obtained by using worldwide available strains (e.g. type strains) gave the advantage of their high comparability within and among laboratories.
  • MIC Minimal Inhibitory Concentration
  • Myrtle extract exhibited the ability to inhibit growth of of strains belonging to the species Candida albicans, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Clavispora lusitaniae, lssatchenkia orientalis, Kluyveromyces marxianus, Pichia guillermondi, Saccharomyces cerevisiae, Cryptococcus laurentii, Filobasidiella neoformans, Prototheca wickeramii, and Prototheca zopfii: values ranging from 2 to 10 mg extract/mL substrate have been observed (Table 3, Fig.
  • MIC Minimal Inhibitory Concentration
  • MIC Minimal Inhibitory Concentration
  • One interesting aspect of the invention is that related to the possibility to obtain plant tissues from selected genotypes of myrtle, propagated and cultivated through cultural models characterized by a low-environmental impact and with the possible control of harvesting time.
  • the present supply of leaves and berries obtained through harvest of spontaneous plants causes ecological damages determining the reduction of plant biodiversity of "Mediterranean maquis"), and represents a limit in view of the potential exploitation of myrtle leaves in a industrial scale.
  • the seasonal variation of plant productivity don't warrant a quality of plant raw material (leaves) suitable for industrial exploitation.
  • the possibility to use plant biomass for alternative utilization e.g.
  • Prototheca zopfii DBVPG 8830 mastitis milk Italy Table 2: Antimycotic spectrum of myrtle extracts determined by the ADWB. Ketoconazole and Amphotericin B have been used as control antibiotic.
  • Myrtle extracts Ketoconazole Amphotericin B (30 mg/ml) (100 ⁇ g/ml) (100 ⁇ g/ml)
  • Table 3 MICs of myrtle extracts. Amphotericin B has been used as control antibiotic.
  • Cryptococcus laurentii DBVPG 6265 4.4 2.0 Filobasidiella neoformans DBVPG 6010 3.2 1.5

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  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
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Abstract

The present invention is directed to the preparation of natural extracts of myrtle, cistus, arbutus, lentisk and other Mediterranean plant species extracts (standardized in the content of polyphenolic molecules), isolated both from different plant tissues and by-products deriving from artisan or industrial manufactures carried out on the same species. The utilization of the above extracts as antimycotic agents is related to its activity towards yeast and yeast-like microorganisms of biomedical interest.

Description

USE OF. EXTRACTS OF MIRTLE AND OTHER MEDITERRANEAN PLANT SPECIES STANDARDIZED IN THE CONTENT OF POLYPHENOL MOLECULES, AS A NOVEL ACTIVE ANTIMYCOTIC AGENT TOWARDS YEAST AND YEAST-LIKE MICROORGANISMS OF BIOMEDICAL INTEREST
DESCRIPTION
The ecosystem Mediterranean maquis is recognized as" wealthy of plant biological diversity. A great variability is also expressed at intraspecies level, due to the plant species adaptability to the heterogeneous environmental conditions, which characterize the territories around the Mediterranean Basin. Among the distinctive plant species, the following are regarded as a valuable opportunity for* the sustainable development of many areas in the Mediterranean Basin:
Myrtus communis L. (myrtle) - Cistus monspelientis L. and Cistus spp. (cistus)
Arbutus unedo L. (arbutus or strawberry-tree) Pistacia lentiscus L. (lentisk)
Phillyrea latifolia L. and Phillyrea angustifolia L. (phillyrea) In order to protect the Mediterranean maquis vegetation, at ecosystem, species and gene level, researches are in progress to describe, characterize and collect the Mediterranean plant genetic resources.
Taking into account their potential exploitation, the mentioned species have been so far proposed for the following uses: i) for naturalistic engineering purposes (e.g. the recovery of degraded or highly desertified areas) (Tattini et a!., 2001 ; Romani et al., 2002); ii) for ornamental purposes; iii) in traditional food preparations (e.g. lentisk oil, spices, marmalades, alcoholic beverages, etc.), and iv) for phytotherapy (hypotensive, antioxidant) and cosmetic use. Concerning myrtle, the traditional liqueur produced in the Mediterranean island of Sardinia (Italy) has lately turned into a large-scale marketed product with a wide national and international distribution.
Nowadays, the raw plant materials for processing are hand-picked from spontaneous plants. Thus, a selective ecological pressure over wild plants in the context of Mediterranean maquis will derive. Besides the risk for the species survival, the indiscriminate harvest will reduce their genetic base. Threats come also from the grazing of wild and domestic herbivorous, as well as fires and woodcutting. To increase the constant availability of the plant raw materials of high quality the direct domestication and cultivation of wild plants has been suggested, providing also a mean of environment protection. Accordingly, studies aimed at selecting suitable varieties for all different uses are in progress (Mulas et ah, 1999; Mulas et al., 2002, Chessa and Mulas, 2005). The characterization of different wild population and selected genotypes, applying the molecular markers technique (RAPD and microsatellites) is directed to: i) acquire knowledge on the species genetic variability and relatedness among and within population from different growing areas, ii) to identify genotypes showing useful morphological traits for utilization, and iii) as a tool in the selection of the genetic material within wild populations to be propagated for cultivation (Nieddu et al., 2005) Despite the use of above plants as food or pharmaceutical preparations is known since antiquity (Craig, 1999, Eisenberg et al., 1993), only recent studies aimed at characterizing their phytochemical composition and biological activity have allowed to discover the role of some potentially beneficial molecules. As a result, a few studies have been focused on polyphenols, particularly on bioflavonoids (Hollmaan, 1996). The qualitative and quantitative occurrence of such compounds is related to some factors: i) plant taxonomy; ii) phenological phase, and iii) environmental factors or stress conditions (Dixon e Paiva, 1995).
Particularly, a great amount of galioyl derivatives (esters of quinic acid, galloylglucosides and elagitannins) and myricetin derivatives has been observed in leaves of myrtle and lentisk (Romani et al., 2004). On the basis of in vitro experiments, these molecules have been able to express an antioxidant action (Bouchet et al, 1998). Secoiridoids (e.g. oleuropein), idroxycinnamic derivatives (e.g. verbascoside), and flavones (e.g. luteolin- and apinenin-glucosides) have been identified in the composition of tissues of leaves of phillyrea. On the other hand, although its phytochemical characterization is still incomplete, tanrfins and flavonols (e.g. quercetin) have been identified in the composition of leaves of arbutus. The antioxidant and antiradical activity expressed on metabolic processes by some components of plant tissues has been recently compared with analogous biological activity exerted by such molecules on animal biological systems: these finding has thus correlated with health potentialities possessed by a few compounds (Craig, 1999). Hence, a low frequency of cardiovascular diseases in a given population has been correlated with its flavonoid-rich diet (Hertog et al., 1993; KeIi et al., 1996; Hertog et al., 1995). In in vitro experimental models, flavonoids seems to phlogosis or inflammation control (Katiyar et al., 1992), the platelet function (Duffy et al., 2001), the vascular tone (Duarte et al., 2001 ; Burs et al., 2000), the angiogenesis (Cao e Cao, 1999), the arteriosclerosis (Stenvold et al., 1992), and the carcinogenesis (Yan9. 1997). The different flavonoids can express different biological activities: sometirhes, the single flavonoid molecule is characterized by a less significant activity than the mixture of different compounds, thus suggesting the existence of a (possible) synergistic activity. Galloyl derivatives, particularly catechins and epigallocatechins occurring in green tea composition, apparently act by reducing the activity of inducible nitroxyde sintetase (iNOS). Additional activity of these classes of molecules are: i) the neutralization of the peroxynitrite; ii) the neutralization of the activity of ROS-producing NADPH oxydase of the blood vessels; and iii) the reduction of the activity of metalloproteinase (which is considered a key enzyme for remodeling). Myricetin is considered a potent inhibitor of Ca- ATPase. Enzymes inhibited by the polyphenols occurring in green tea composition are associated with the pathogenesis of some cardiovascular diseases (Cahilly et al., 2000; Griendling et al., 2000). In addition, catechins and the epigallocatechins, as well as a*number of other bioflavonoids, act by directly neutralizing the ROS (whose role on phenomena of apoptosis and expression of stress protein in cell of cardiovascular apparatus is well documented). References
- Ahearn D.G. et al. J. Clin. Microbiol. 19, 412, 1984
- Bouchet N. et al.; Pharmaceut Biol 36,63,1998
- Burs J. et al. ; J Agric Food Chem, 48, 220, 2000
- Cahilly C. et al.; Circ. Res. 86, 391 , 2000 - Cao Y e Cao Y Nature, 398, 381 , 1999
- Chen YC J.; Cell Biochem, 2001
- Chessa I., Mulas M. ltalus Hortus. 12, 37, 2005
- Craig WS Am J Clin Nutr, 70s, 491 , 1999 - Demeule M. et al BBA.; 1478, 51 , 2000
- Dick J.D. et al. Ann. Intern. Med. 102, 67, 1985
- Dixon R. et Paiva NL. ; Plant Cell 7, 1085, 1995
- Duffy SJ. et al. Arterioscl Thromb Vase Biol, 21 , 1084, 2001
- Duarte J. et al. Br J Pharmacol 117, 24, 2001 - Eisenberg DM et al, N Eng J Med, 328, 246, 1993
- Griendling KK. et al.; Cir Res 86, 494, 2000
- Hammond S.M., Lambert P.A. Stud. BioL90, 8, 1978.
- Hertog MLG. et al., Lancet 342, 1007, 1993
- Hertog MLG et al Arch Intern Med 158, 381 , 1995 - Hollmaan PCH.; Eur J Clin Nutr 51 , s66, 1996
- Katiyar SK. et al. Cancer Res, 14, 361 , 1992
- KeIi SO. et al., Arch Intern Med 156, 637, 1996
- Malinowski E. et al. Bull. Vet. Inst. Pulawy 46, 295, 2002
- Morre JD. et al. Biochem Pharmacol 60, 937, 2000 - Mulas et al., Acta Hort. 502, 85-88, 1999
- Mulas et al., Jour. Herbs, Spices & Med. Plants, 9(2/3): .127-131 , 2002
- National Committee for Clinical Laboratory Standards. Approved standard M27-A...1997 .
- Nieddu M., et al.,. ltalus Hortus ((n press). 2005 - Paraquay JB.; J Agric Food Chem 48, 5768,2000
- Pinelli P. et al. XX international Conference on Polyphenols, Freising- Weihestephan, Germany 11 -15 /Sept, 2000
- Rex J.H. et al. Ant. Ag. Chem. 39, 1 , 1995
- Romani A. et al.; Chromatographic, 49, 17, 1999 - Romani A. et al.; Phytochem. An., 2002, 13 (2),79-86
- Romani A. et al.; Free Rad. Res., 38(1), 97-103, 2004
- Romani et al.; Bioorg. Med. Chem. Lett., 15, 4000-4003, 2005.
- Stenvold I. et al.; Prev Med 21 , 546, 1992 - Tattini M. et al.; New Phytologist. 2001 , 148, 69
- Thiyagarajah P. et al.; Biochem Pharmacol 41 , 669, 1991
- Wang HK.; Exp Opin Invest Drugs, 9, 2103, 2000
- Yang CS.; Nature, 389, 134, 1997 The invention genetically relates to utilization of natural extracts of myrtle, cistus, arbutus, lentisk and other Mediterranean plant species containing galloyl derivatives and hydrolysable tannins deriving from artisan or industrial manufactures as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
Other characteristics are defined in the attached claims following this description.
The drawing shows in Figs. 1 ;and 2 diagrams relating to antimycotic spectrum of myrtle extracts: killing curve towards Candida glabrata DBVPG 3828.
In a particular embodiment the invention relates to the preparation of myrtle extracts (standardized in the content of polyphenols) from different plant tissues and from by-products deriving from artisan or industrial manufactures carried out over the same species and their utilization as antimycotic agents active towards yeast and yeast-like microorganisms of biomedical interest.
At present, neither studies, nor application (on artisan or industrial scale) of solid or liquid extracts of myrtle exhibiting an antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals exist. The present invention has considered the following phases: I) Selection of myrtle genotypes, their propagation and cultivation. The adoption of crop management systems characterized by a low-environmental impact is recommended. The growing of selected varieties will assure the ripening control and the reproducibility of the mean quality characteristics from one year to the other and from different growing locations. The aims of a direct domestication of wild plants species and the crop management are essentially directed to standardize the final product quality and to lessen the energy input, as well as to reduce environmental and operational costs. We propose i) the control of phenological phases by means of appropriate cultivation techniques, in order to assure the regular availability of the raw material; H) the selection of specific myrtle varieties showing high uniformity and quality of the product, fitting with the definite objective; iii) the application of biomolecular procedures for the description of selected genotypes suitable to agamic propagation and cultivation; and iv) the selection of specific myrtle varieties and the application of molecular markers for the identification and biotyping of the intraspecific genetic diversity within and among myrtle populations from different geographical habitats.
II) High-temperature aqueous extraction (use of high -temperature steams) of polyphenols from myrtle leaves or from wastes (byproducts of artisan or industrial manufactures) and standardization of their polyphenol content. The production of lyophilised extracts for commercial use is thus hypothesized. We propose the use of lyophilised products (in a partially purified stage) for the production of lavage, cream and gel for antimycotic use in cosmetic, pharmaceutical and veterinary use.
III) Total hydro alcoholic or low-temperature aqueous extractions of polyphenol components from myrtle leaves or from wastes (byproducts of manufactures) and standardization of their polyphenol content. We propose the use of lyophilised products
(in a partially purified stage) for the production of lavage, cream and gel for antimycotic use in cosmetic, pharmaceutical and veterinary use.
IV) Individuation of a set of yeasts and yeast -like microorganisms including type strains (type strain - T) belonging to species considered as known or emerging pathogens for humans and animals. We used of twenty-four strains of yeasts (belonging to thirteen species of nine genera) and three strains of yeast -like microorganisms (Prototήeca spp.) (Table 1) as target microorganisms for the tests of antimycotic activity carried out by using the above extracts. Some of the above strains have proven to be resistant to polyene (Amphotericin B) and azole antibiotics (Ketoconazole) currently used in clinical set. All above strains are conserved in the Industrial Yeast Collection DBVPG of the Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali e Zootecniche, Sezione di Microbiologia Agroalimentare ed Ambientale, http//www.agr.unipg.it/dbvpg, University of Perugia, Italy. The Collection DBVPG is nowadays the sole service collection of microbial germplasm existing in
Italy (since 1996 DBVPG has been included within the IDA - International Depository Authority - network). Determination of antimycotic spectra (under form of inhibition halos of microbial grpwth) exhibited by myrtle extracts (characterized by different polyphenol contents) towards yeasts and yeast-like microorganisms pathogen for humans and animals. This step has been carried out by using the Agar Diffusion Well Bioassay (ADWB) in Petri plates (Hammond and Lambert, 1978). Further assessment of the antimycotic activity exhibited by myrtle extracts has been done through the determination of the Minimal Inhibitory Concentration (MIC) (NCCLS, 1997). On the basis of the results obtained, we propose the use of myrtle extracts (above cited) as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered known or emerging pathogens for humans and animals. Utilization of above myrtle extracts (characterized by different polyphenol contents) in combination with polyene antibiotics (characterized by synthetic or biological origin) in order to induce synergistic actions between active principles. The preliminary determination of antimycotic activity obtained through the combined use of myrtle extracts (above cited) with Amphotericin B has been carried out by using the Agar Diffusion Well Bioassay (ADWB) (Hammond e Lambert, 1978) . Further assessment of the antimycotic activity towards yeast and yeast- like microorganisms has been obtained through the determination of the Minimal Inhibitory Concentration (MIC) of Amphotericin B used in combination with myrtle extracts . (NCCLS, 1997). The aim of such combined use is that to obtain a mixed preparation characterized simultaneously by a higher antimycotic activity and by a reduced concentration of polyene antibiotics (Amphotericin .B or Ketoconazole). This appears to be interesting for reduce the risk of overexpositioή of patients to high amounts of antibiotics. On the basis of the results obtained, we propose the use of myrtle extracts (above cited) as a mixed preparations with Amphotericin B in order to obtain a product characterized by a high antimycotic activity towards yeast and yeast-like microorganisms, but containing reduced concentration of Amphotericin B .
VII) Utilization of natural extracts of cistus (obtained via aqueous or low-temperature hydroalcohoiic extraction) containing galloyl derivatives and hydrolysable tannins as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
VIII) Utilization of natural arbutus extracts of (obtained via aqueous or low-temperature hydroalcohoiic extraction) containing galloyl derivatives and hydrolysable tannins as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
IX) Utilization of natural extracts of lentisk (obtained via aqueous or low-temperature hydroalcohoiic extraction) containing galloyl derivatives and hydrolysable tannins as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals. 9 ' . . - .- .
X) Utilization of natural extracts of myrtle (obtained via aqueous or low-temperature hydroalcoholic extraction) containing galloyl
V derivatives and/or hydrolysable tannins in association with cistus, arbutus, lentisk, and phillyrea extracts in order to obtain mixed preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals. In addition, we consider the combined use of extracts obtained (through aqueous or low-temperature hydroalcoholic extraction procedures) from all above Mediterranean plants (in all possible combinations) as mixed preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals. Xl) Combined use of natural extracts of myrtle, cistus, arbutus, lentisk, and phillyrea (obtained via aqueous or low-temperature hydroalcoholic extraction), containing galloyl derivatives and hydrolysable tannins, with polyene or azole antibiotics (characterized by synthetic or biological origin) as mixed preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals. In addition, we consider the combined use of all above extracts obtained (through aqueous or low-temperature hydroalcoholic 1 extraction procedures) from above Mediterranean plants (in all possible combinations) with polyene or azole' antibiotics (characterized by synthetic or biological origin) as mixed preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganism's belonging to species considered as known or emerging pathogen for humans and animals.
XII) The invention also relates to other plant species selected varieties as sources of raw material for the production of extracts characterized by a high concentration of hydrolysable tannins for antimycotic use. In addition to myrtle, cistus, arbutus, lentisk, and phillyrea will benefit from the application of farming methods with low and energetic inputs. In the case of intensive crop growing the use of traditional cultivation practices is suggested, or a cultivation system based on the agroforestry techniques is hopeful to maintain the dynamic natural balance of the plant growing environment. Furthermore, we consider the possible supply of leaves and berry from wild plants. The regulation of harvesting time and methods is recommended to increase the supply of biomass, at trie same time avoiding the plant over- exploitation and the habitats disturbance.
XIII) We also consider the possibility to use wastes and by-products deriving from artisan or industrial manufactures as a source of extracts containing galloyl derivatives and hydrolysed tannins exhibiting antimycotic agents. In addition, we claim the utilization of above wastes and by-products to obtain innovative products in the field of food, cosmetic and pharmaceutical industries. The invention foresee the possibility for above industries to achieve added value through the commercialisation of products characterized by a high degree of innovation, low environmental impact, also in view of obtain environmental certification (respect to UNI EN. ISO standard, ecolabel etc) both for the process.and for the product. . .
XIV) The invention also foresees the use of extract deriving from leaves of Vitis vinifera L. (grape seed, skin, and marc), Pinus. marittima L and other species of the genus Pinus containing condensed and hydrolysed tannins as raw preparations exhibiting antimycotic activity for cosmetic, pharmaceutical and veterinary use.
XV) The invention has also foreseen the use of a set of yeasts and yeast-like microorganisms including type strains (type strain - T) belonging to species considered known or emerging pathogens for humans and animals. Conventionally, for a given species, the type strain represents its reference strain internationally recognized and conserved in all most important world collections of microbial germplasm. Accordingly, since the biological variability observed within strains belonging to a same species, results obtained by using worldwide available strains (e.g. type strains) gave the advantage of their high comparability within and among laboratories.
XVI) Results supporting the invention reported that extracts of myrtle, cistus, arbutus, lentisk, and phillyrea, as well as commercial extracts of plants of the genera Vitis and Pinus, exhibited the ability to inhibit the growth of yeasts and yeast-like microorganisms belonging to species considered known or emerging pathogens for humans and animals (Candida albicans, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Clavispora lusitaniae, lssatchenkia orientalis, Kluyveromyces marxianus, Pichia guillermondi, Saccharomyces cerevisiae, Cryptococcus laurentii, Filobasidiθlla neoformans, Prototheca wickeramii e Prototheca zopfii). This ability has been experimentally assessed through the determination of the Minimal Inhibitory Concentration (MIC) (Table 3, Fig. 1).
XVII) The invention also foresees the combined use of extracts of myrtle, cistus, arbutus, lentisk, and phillyrea, as well as commercial extracts of plants of the genera Vitis and Pinus, among them or in association with Amphotericin B or Ketoconazole, in order to induce a synergistic effect among different active principles. This should be interesting in reducing the risk of overexposition of patients to high dosages of antibiotics. Analogously to above we also foresee the combined use of polyene antibiotics other than Amphotericin B (characterized by synthetic or biological origin).
XVIII) The invention foresees, as above reported, the utilization of myrtle extracts (as well as of the other above reported
Mediterranean plants) containing galloyl derivatives and hydrolysable tannins as antimycotic agents. At present, neither studies, nor application (on artisan or industrial scale) of solid or liquid extracts of myrtle extracts (containing galloyl derivatives and hydrolysable tannins) exhibiting an antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals exist. XIX) One the advantages of the invention is represented by the use of wastes and by-products deriving from artisan or industrial manufactures as a source of extracts containing galloyl derivatives and hydrolysed tannins exhibiting antimycotic agents. The present economical interest of myrtle is exclusively focused on the production of (r^d or white) spirits of myrtle. Although their traditional food utilization has been initially restricted to its original geographical niche (Sardinia, Italy), they are nowadays characterized by a worldwide spreading. In this context, the production of innovative products (exhibiting antimycotic activity) as an alternative utilization of wastes and by-products deriving from artisan or industrial manufactures focused at the production of spirits, may constitute an added value. In addition, the reduction of the environmental impact could be favoured by the use of above wastes, otherwise difficult to dispose of. XX) We also consider the utilization of a set of yeasts and yeast -like microorganisms including type strains (type strain - T) belonging to species considered known or emerging pathogens for humans and animals. Accordingly, the use of type strains as target microorganisms may be considered an additional innovative point. Conventionally, the type strain of a given species represents its internationally recognized reference strain. Type strains are conserved in all worldwide collections of microbial germplasm. Accordingly, since the biological variability observed within strains belonging to a same species, results obtained by using worldwide available strains (e.g. type strains) gave the advantage of their high comparability within and among laboratories.
XXI) The determination of the Minimal Inhibitory Concentration (MIC) of myrtle extracts towards yeasts and yeast-like microorganisms belonging to species considered known or emerging pathogens for humans and animals has been carried out. Myrtle extract exhibited the ability to inhibit growth of of strains belonging to the species Candida albicans, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Clavispora lusitaniae, lssatchenkia orientalis, Kluyveromyces marxianus, Pichia guillermondi, Saccharomyces cerevisiae, Cryptococcus laurentii, Filobasidiella neoformans, Prototheca wickeramii, and Prototheca zopfii: values ranging from 2 to 10 mg extract/mL substrate have been observed (Table 3, Fig. 1). Such results appear to be extremely interesting in view of the potential exploitation of above extract as pharmacological preparation: this finding represents one of the innovative point of this invention. Indeed, at present, neither studies, nor application (on artisan or industrial scale) of solid or liquid extracts of myrtle extracts (containing galloyl derivatives and hydrolysable tannins) exhibiting an antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals exist. Particularly, one additional innovative aspect of this invention is also related to the observed activity of myrtle extracts towards some yeasts resistant to concentrations higher than 100 μg of Amphotericin B /mL of substrate. This finding appear to be of particular interest and worthwhile, also in view of its possible use as pharmacological preparation exhibiting antimycotic activity towards yeast and yeast-like strains exhibiting genotypes resistant to antibiotics currently used in clinical set. At this point it is important to underline that the phenomenon of antibiotic resistance is becoming greater and greater also in veterinary practice: outbreaks of antibiotic resistance has been recently observed in strains of Candida spp. and Prototheca spp. (Rex et al., 1995; Malinowski et al., 2002). Accordingly, above findings could be considered extremely interesting for their potential exploitation in cosmetic, pharmacological, and veterinary industry. Also in this case, nowadays, neither studies, nor application (on artisan or industrial scale) of solid or liquid extracts of myrtle extracts exhibiting an antimycotic activity towards yeasts and yeast-like microorganisms responsible of phenomena of mycosis in humans and animals.
XXII) One additional interesting result obtained by the invention has been the determination of the Minimal Inhibitory Concentration (MIC) of Amphotericin B used in combination with above myrtle extracts (characterized by different polyphenol contents) (Fig. 2) towards yeasts and yeast-like microorganismsm belonging to species considered as known or emerging pathogen for humans and animals. Since the ever increasing phenomenon of antibiotic resistance in yeast and yeast-like microorganisms, researches aimed at discovering and developing novel agents able to increase the antimycotic activity of already known antibiotic molecules become of strategic relevance for cosmetic, pharmaceutical and veterinary industry. Accordingly, the Minimal Inhibitory Concentration (MIC) of Amphotericin B towards strains of Candida glabrata (sometimes responsible of phenomena of mycoses in humans and animals) have been compared with that obtained towards the same strains by using this antibiotic in combination with myrtle extracts. The combined use of myrtle extracts + Amphotericin B gave values of MIC significantly lower than those observed by using AmB alone (Fig. 2). Such results demonstrate that myrtle extracts may stimulate a synergistic activity with Amphotericin B, in the terms of a considerable increase of its antimycotic activity. Accordingly, the combined use of myrtle extracts + Amphotericin B may be potentially exploitable through the use of preparations containing a mixture of both active principles (extract + antibiotic) characterized by a reduced concentration of Amphotericin B, but by an improved antimycotic activity of polyene antibiotics currently used in clinical practice. The polyphenols (galloyl derivatives occurring in the composition of myrtle leaves) may represent the class of molecules responsible of synergistic activity between the . myrtle extract and Amphotericin B, in agreement with published studies reporting the synergistic interaction between synthetic di-galloyl glucosides and Amphotericin B (Romani et al., 2005). Therefore, the extracts obtained by tissues of myrtle, as well as by tissues of above cited Mediterranean plants, could be utilized for phytotherapy exploitations in order to considerably increase
(through the induction of synergistic effects) the antimycotic activity of Amphotericin B towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals. A potential exploitation of antimycotic activity expressed by myrtle extracts
(containing high concentrations of polyphenols of the ciass of galloyl derivatives) could be thus represented by their use under form of preparations containing a mixture of both active principles (extract + Amphotericin B) where myrtle extracts may act as coadjuvant component of natural origin for the utilization of lower amounts of Amphotericin B. The use of reduced concentration of commercial antibiotic (e.g. Amphotericin B) may so potentially reduce the risk of overexposition of patients to high dosages of antibiotics. As above reported, also this evidence, which may be of particular interest in view of its potential exploitation by cosmetic, pharmaceutical and veterinary industry, represents undoubtedly an additional aspect of innovation. Indeed, at present, neither studies, nor application (on artisan or industrial scale) of above synergistic activities between myrtle extracts (characterized by different concentrations of polyphenols) and antibiotic molecules of commercial interest
(e.g. Amphotericin B) exist. XXIII) One interesting aspect of the invention is that related to the possibility to obtain plant tissues from selected genotypes of myrtle, propagated and cultivated through cultural models characterized by a low-environmental impact and with the possible control of harvesting time. The present supply of leaves and berries obtained through harvest of spontaneous plants causes ecological damages determining the reduction of plant biodiversity of "Mediterranean maquis"), and represents a limit in view of the potential exploitation of myrtle leaves in a industrial scale. In addition, the seasonal variation of plant productivity don't warrant a quality of plant raw material (leaves) suitable for industrial exploitation. The possibility to use plant biomass for alternative utilization (e.g. creation of innovative products) determines the inevitability to cultivate above plants under "domesticated" form, through the utilization of selected genotypes aηd the definition of suitable agronomic practices for their innovative use. The aims of above models of cultivation are moreover related to the possibility to obtain above preparations from plant raw materials deriving from selected genotypes propagated and cultivated through cultural models characterized by lower energy and environmental costs and by reduced operating costs. The control of both phenological phases and cultivation techniques which allow a high uniformity degree of the product, as well as by a quality fitting with the definite objective (production of extracts characterized by a high concentration of hydrolysable tannins for antimycotic use) constitute a further innovative point of the invention. Additional innovative elements, fine tuned in the course of the selection of myrtle varieties, are represented by the application of molecular methodologies' for the identification, as well as the evaluation of intraspecific genetic variability for typing myrtle populations originating from different natural environments and for the selection of genotypes suitable to be propagated and cultivated. Table 1 : Strains utilized as target microorganisms: all strains are conserved in the Industrial Yeast Collection DBVPG (www.agr.unipg.it/dbvpg). Each strain is labelled by a different (and unique) number. T = Type strain; NT = Neotype
Species Strain status isolation source Isolation place human cutaneous
Candida albicans DBVPG 6133 T mycosis Uruguay
Candida albicans DBVPG 6157 NT of Candida stellatoidea unknown unknown
Candida glabrata DBVPG 7212 . unknown unknown
Candida glabrata DBVPG 3828 T* human faeces unknown
Pichia guilliermondii DBVPG 6140 T of Candida guilliermondii sputum unknown
Candida parapsilosis DBVPG 6150 T human mycosis Puerto Rico
Candida tropicalis DBVPG 3982 T bronchitic patient unknown
Candida zeylanoides DBVPG 6163 NT human mycosis unknown
Clavispora lusitaniae DBVPG 6142 T of Candida lusitaniae caecum of pig Portugal
Clavispora lusitaniae DBVPG 6148 sputum Norway lssatchenkia orientalis DBVPG 6782 fruit juice unknown
Kluyveromyces marxianus DBVPG 6141 T of Candida kefir kefyr grains The Netherlands
Saccharomyces cβrevisiae DBVPG 6173 T baker yeast The Netherlands
Saccharomyces cerevisiae DBVPG 6497 unknown unknown
Saccharomyces cerevisiae DBVPG 6500 unknown unknown
Yarrowia iipolytica DBVPG 6053 T maizeprocessing plant USA
Cryptococcus laurentii DBVPG 3883 tuberculotic patients . unknown
Cryptococcus laurentii DBVPG 4272 human oral cavity Italy
Cryptococcus laurentii DBVPG 6265 T palm wine Congo
Filobasidiella neoformans DBVPG 3428 unknown unknown
Fiiobasidieiia T of Cryptococcus neoformans DBVPG 6010 neoformans fermented fruit juice unknown
Filobasidiella - neoformans DBVPG 6225 human spinal, fluid USA
Filobasidiella human cerebro-spinal neoformans DBVPG 6981 fluid USA
Filobasidiella neoformans DBVPG 6982 droppings of cuckoo . Thailand
Prototheca wickerhamii DBVPG 8879 unknown unknown
Protoiheca zopfii DBVPG 8880 unknown unknown
Prototheca zopfii DBVPG 8830 mastitis milk Italy Table 2: Antimycotic spectrum of myrtle extracts determined by the ADWB. Ketoconazole and Amphotericin B have been used as control antibiotic.
Diameter of inhibition halos
Species Strains (mm)
Myrtle extracts Ketoconazole Amphotericin B (30 mg/ml) (100 μg/ml) (100 μg/ml)
Candida albicans DBVPG 6133 15.8 60.8 22.5
Candida albicans DBVPG 6157 11.4 73.6 16.4
Candida glabrata ■ DBVPG 7212 22.9 17.0
Candida glabrata DBVPG 3828 23 18.7
Pichia guilliermondii DBVPG 6140 19.5 64.5
Candida parapsilosis DBVPG 6150 20.6 65.6
Candida tropicalis DBVPG 3982
Candida zeylanoides DBVPG 6163 21.6 58.8 18.0
Clavispora lusitaniaβ DBVPG 6142 67.7
Clavispora lusitaniae DBVPG 6148 20 64.6 lssatchenkia orientalis DBVPG 6782 18.5 31.1
Kiuyveromyces marxianus DBVPG 6141 17.9 72.4
Saccharomyces cerevisiae DBVPG 6173 12.3
Saccharomyces cerevisiae DBVPG 6497 19.7
Saccharomyces cerevisiae DBVPG 6500 22
Yarrowia lipolitica DBVPG 6053 46.0
Cryptococcus laurentii DBVPG 3883 45.2 14.2
Cryptococcus laurentii DBVPG 4272 31.1 27.7
Cryptococcus laurentii DBVPG 6265 47.9 15.7
Filobasidiella neoformans DBVPG 3428 56.6 20.5
Filobasidiella neoformans DBVPG 6010 19.6 50.4 15.9
Filobasidiella neoformans DBVPG 6225 16.4 50.8 13.1
Filobasidiella neoformans DBVPG 6981 22.8 43.5 16.2
Filobasidiella neoformans DBVPG 6982 19.8 53.4 19.0
Prototheca wickerhamii DBVPG 8879 19.8 16.6
Prototheca zopfii DBVPG 8880
Prototheca zopfii DBVPG 8830 22.7
Table 3: MICs of myrtle extracts. Amphotericin B has been used as control antibiotic.
Species Strains MIC (mg/ml)
* Myrtle extracts Amphotericin B (mg/ml) (μg/ml)
Candida albicans DBVPG 6133 10 1.0
Candida glabrata DBVPG 3828 6.8 1.6
Candida tropicalis DBVPG 3982 9.2 > 100 lssatchenkia orientalis DBVPG 6782 4.2 > 100
Yarrowia lipolytica DBVPG 6053 2.0 > 100
Cryptococcus laurentii DBVPG 6265 4.4 2.0 Filobasidiella neoformans DBVPG 6010 3.2 1.5

Claims

1. Utilization of natural extracts of myrtle, cistus, arbutus, lentisk and other Mediterranean plant species containing galloyl derivatives and hydrolysable tannins deriving from artisan or industrial manufactures as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
2. Utilization according to claim 1 , of natural extracts of myrtle containing galloyl derivatives and hydrplysable tannins deriving from artisan or industrial manufactures as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
3. Utilization according to claim 1 , of natural extracts of cistus containing galloyl derivatives and hydrolysable tannins deriving from artisan or industrial manufactures as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
4. Utilization according to claim 1 , of natural extracts of arbutus containing galloyl derivatives and hydrolysable tannins deriving from artisan or industrial manufactures as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
5. Utilization according to claim 1 , of natural extracts of lentisk containing galloyl derivatives and hydrolysable tannins deriving from artisan or industrial manufactures as preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
6. Combined use of natural extracts of myrtle according to claim 1 or 2, containing galloyl derivatives and hydrolysable tannins with cistus, arbutus, lentisk, and phillyrea, deriving from artisan or industrial manufactures as mixed preparations exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
7. Combined use according to claim 6, of natural extracts of myrtle, cistus, arbutus, -lentisk, and phillyrea, galloyl derivatives and hydrolysable- tannins with polyene antibiotics deriving from artisan or industrial manufactures as mixed preparations; exhibiting antimycotic activity towards yeasts and yeast-like microorganisms belonging to species considered as known or emerging pathogen for humans and animals.
8. Use of natural extracts, according to at least one of above claims, originating from "Mediterranean maquis" and obtained through aqueous extraction procedures in combination with polyene or azole antibiotics.
9. Use of natural extracts, according to at least one of above claims, originating from "Mediterranean maquis" and obtained through hydroalcoholic extraction procedures in combination with polyene or azole antibiotics.
10. Use of natural extracts, according to the above claims 8 and 9, in agro-food applications.
11. Use of natural extracts, according to the above claims 8 and 9, in cosmetic applications.
12. Use of wastes and by-products deriving from artisan or industrial manufactures as a source of galloyl derivatives and hydrolysed tannins exhibiting antimycotic agents.
13. Use of wastes and by-products deriving from artisan or industrial manufactures for obtain innovative products for food, cosmetic and pharmaceutical industries.
14. Use of extracts deriving from leaves of Vitis vinifera L. (grape seed, skin and marc), Pinus marittima L and other species of the genus Pinus containing condensed and hydrolysed tannins as raw preparations exhibiting antimycotic activity for cosmetic, pharmaceutical and veterinary use.
15. Combined use of extracts deriving from leaves of Vitis vinifera L (grape seed, skin and marc), Pinus rηarittima L. and other species of the genus Pinus (containing condensed and hydrolysed tannins) among them and with polyene (e.g. Amphotericin B) antibiotics as mixed preparations for inducing a synergistic effect among them in order to allow for the utilization of lower dosages of antibiotics.
16. Utilization of polyphenols (galloyl-derivatives occurring in the composition of leaves of myrtle and of other Mediterranean plant species) in phytotherapy as molecules responsible of the induction of a synergistic activity with Amphotericin B (increase via synergy of the antimycotic activity of Amphotericin B), also at low dosages, towards yeast and yeast-like microorganisms of biomedical interest.
17. Utilization of the products as above described.
EP06810032A 2005-10-20 2006-10-17 Use of extracts of myrtle and other mediterranean plant species, as antimycotic agent towards yeast and yeast-like microorganisms Withdrawn EP1945237A2 (en)

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