Classifications

• • 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
  • A61K36/06—Fungi, e.g. yeasts
  • A61K36/07—Basidiomycota, e.g. Cryptococcus
  • A61K36/074—Ganoderma
• • 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
  • A61K36/06—Fungi, e.g. yeasts
  • A61K36/07—Basidiomycota, e.g. Cryptococcus
• • 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
• • 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
  • A61P31/04—Antibacterial agents
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Antimicrobial composition comprising an extract of a Polyporaceae and a process for making the same.
• the present invention is directed to a fungal extract comprising antimicrobial activity. More specifically, the fungus used belongs to the family Polyporaceae, especially genus Trametes, namely Trametes sp. 11E4.
• the extract demonstrated activity against bacteria such as R. sonalacearum, E. coli, S. aureus, B. cereus and S. anatum. It also may be used in compositions, such as pest control compositions, which would be useful in agriculture, i.e. treating bacterial wilt and medicine.
• Pesticidal compounds have long been used to increase yields and extend agricultural production capabilities into new areas. They have also been extremely important tools for ameliorating season-to-season differences in yield and quality caused by weather-driven variations in disease pressure.
• R. solanacearum is an important phytopathogen with worldwide distribution and with a big number of hosts, more than 200 species in 50 families.
• Some of the most important hosts include tomato (Lycopersicum esculentum Mill.), pepper (Capsicum frutescens L), potato (Ipomoea sp.), tobacco (Nicotiana tabac ⁇ m L), banana (Musa sp.), peas (Pisum sativum L), peanut (Arachis hypogaea L), cashew (Anacardium occidentale L), papaya (Carica papaya L) and olive (Olea europaea L)
• Several strategies for pest control include resistant host plants, trangenic plants, soil correction and biological control.
• Biological agents used for controlling tomato bacterial wilt includes arbuscular mycorrhizae, non-virulent mutants of R solanacearum, antagonistic bacteria genetically engineered and some naturally occurring antagonistic rhizobacteria such as Bacillus spp., Pseudomonas spp., Streptomyces spp.
• Chemical pesticides have provided an effective method of control; however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water and the environment. Stringent new restrictions on the use of chemicals and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling pests. In addition, the regular use of chemical toxins to control unwanted organisms can select for resistant strains.
• Moura et al. evaluated the antagonistic potential of 190 actinomycetes isolates, obtained from different soils, rhizosphere, rhizoplane and internal tissues from healthy plants against isolates of R. solanacearum. Tomato seeds were treated with actinomycetes propagules under stirring for 10 minutes, and grown in pathogen infested soil. Eighteen isolates provided a 100% control.
• Document WO 2006/065985 discloses compositions for fighting bacterial wilt in crops by using an extract of Yucca plants.
• the present invention differs from this prior art by using a fungal extract rather than a plant extract to control bacterial wilt.
• Document EP 935918 discloses compositions of 3-(3-indolyl)-butanoic acid capable of selectively suppressing the growth of Ralstonia solanacearum in crops.
• the present invention differs from this prior art by using a fungal extract rather than a synthesized substance to control bacterial wilt.
• Document EP 1 762 613 discloses insertion elements that have the ability of recombining with the genetic material of R. solanacearum, preventing further infection from this bacteria in crops.
• the present invention differs from this prior art by using a fungal extract rather than an insertion element to control bacterial wilt.
• Document WO 2006/06133708 discloses polysaccharides obtained from several fungi species containing biological activity such as antineoplasic.
• One suitable genus is Trametes sp.
• the present invention differs from this prior art by using an extract and filtered cultures against bacterial wilt, and not cancer.
• the present invention comprises a step of polysaccharides precipitation.
• Document US 2007/093387 discloses a composition comprising a laccase with antimicrobial/antiviral properties, wherein said laccase can be obtained from several microorganisms, such as fungus from Trametes genus.
• the present invention differs from this prior art in that it uses extracts rather than isolated enzymes as antimicrobial agents.
• an antimicrobial composition comprising: a) an extract from a fungus belonging to Polyporaceae family; and b) an acceptable vehicle. It is a further objective of the invention a process for production an extract of a fungus belonging to Polyporaceae family comprising the steps of: a) growing in a liquid medium a fungus belonging to Polyporaceae family; and b) removing the mycelia from the liquid medium.
• It is a further objective of the invention a method for protecting organisms from pathogenic microorganisms infection comprising the steps of: a) contacting the plant with an antimicrobial composition comprising: a. an extract from a fungus belonging to Polyporaceae family; and b. an acceptable vehicle.
• the process comprises the additional steps of further contacting the liquid medium with an adequate organic solvent.
• the fungus is chosen from the genus Trametes. More specifically, the fungus is Trametes sp. 114E.
• the extract is a filtered culture from a liquid medium growth.
• the adequate organic solvents can be hexane, dichloromethane, ethyl acetate and butanol.
• Figure 1 shows the procedure for inoculating the bacteria in solid medium.
• Figure 2 shows the scores given according to bacterial growth in Petri dishes with solid medium.
• Fungus belonging to Polyporaceae family Suitable fungi according to the present invention include fungi belonging to the Polyporaceae family. Examples of fungi belonging to said family include, without limitation genera Ganoderma, Coriolus, Trametes and other related fungi. In a preferred embodiment, the fungus is chosen from genus Trametes, namely Trametes sp. 11 E4.
• pathogenic microorganisms include, without limitation, bacteria, fungi, viruses and protozoa.
• the microorganism may also be a phytopathogenic microorganism.
• Suitable bacteria include, without limitation, Gram-positive and Gram- negative bacteria belonging to the following genera: Staphylococcus, Ralstonia, Escherichia, Salmonella, Pseudomonas, Bacillus, Streptococcus and mixture thereof.
• the composition of the present invention has a special selectivity for E. coli, S. aureus, B. cereus, S. anatum and R. solanacearum. Organisms
• Suitable organisms according to the invention include, without limitation, plants and animals.
• Suitable animals include, without limitation mammals, such as humans.
• Suitable plants include, without limitation, seeds, corms, bulbs, flowers, stems, leaves, exposed roots, and fruits of plants including, but not limited to, grapes, pears, apples, peaches, nectarines, grapefruit, cherries, apricots, lemons, limes, oranges, mangos, bananas, pineapple, and tangerines. Any plant which is susceptible to bacterial wilt disease caused by R solanacearum is encompassed within the scope of the methods of the present invention.
• the plants which may be treated further include, but are not limited to, tomatoes, potatoes, bananas, peas, alfalfa, cabbage, clover, kale, lentil, soybean, sweet potato, radish, grape, cotton, sunflower, rape, chicory, chickpea, sorghum, onion, coconut, lily, sugarcane, cucumbers, squash, zucchini, eggplant, chili pepper, bell pepper, tobacco, groundnut, lettuce, cantaloupes, ginger, rice, corn, wheat, oats, barley, rye, millet, and other cereals, turfgrasses, and flower crops, including geraniums, other ornamental plants, nightshades, and almonds.
• the antimicrobial composition according to the present invention comprises: a) an extract from a fungus belonging to Polyporaceae family; and b) an acceptable vehicle.
• the antimicrobial composition can be incorporated in cosmetic, pharmaceutical and/or agricultural compositions, in order to impart antimicrobial properties to the parent compositions.
• the antimicrobial composition is added to an agricultural composition useful for crop protection, in order to protect crops from infections such as bacterial wilt.
• Example 1 Microorganisms for antimicrobial activity
• the pathogenic bacteria used in the assays were Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella anatum and Ralstonia solanacearum.
• the R. solanacearum was originally isolated from several plants, such as tomatoes, cucumber, banana, eggplant, capsicum and moringa.
• the bacteria were cultured in specific culture media: R. solanacearum was purified in medium LPGA (5 g/L yeast extract, 5 g/L peptone, 5 g/L glucose 15 g/L agar, water, pH 7.0) and grown in 10 mL of LPG medium, at 28 0 C for 48 hours.
• LPGA 5 g/L yeast extract, 5 g/L peptone, 5 g/L glucose 15 g/L agar, water, pH 7.0
• anatum all were purified in nutrient agar (3 g/L meat extract, 10 g/L peptone, 5 g/L sodium chloride, 20 g/L agar, water, pH 7.0) and grown in 10 mL of liquid medium LB (10 g/L peptone, 5 g/L NaCI, 10 g/L yeast extract, pH 7.0), incubated at 37°C for 24 hours with no agitation.
• nutrient agar 3 g/L meat extract, 10 g/L peptone, 5 g/L sodium chloride, 20 g/L agar, water, pH 7.0
• liquid medium LB 10 g/L peptone, 5 g/L NaCI, 10 g/L yeast extract, pH 7.0
• BDA potato, 20 g/L dextrose, 15g/L agar
• GPY 20 g/L glucose, 10 g/L peptone, 2 g/L yeast extract, 15 g/L agar, water; pH 6.6
• the fungus was previously cultured in solid GPY (20 g/L glucose, 10 g/L peptone, 2 g/L yeast extract, 15 g/L agar, water; pH 6.6) and incubated at 28°C for 10 days. After the solid GPY culture, three mycelia dishes were inoculated into 16
• Example 4 Evaluation of antimicrobial production during mycelia growth of Trametes sp.
• the antimicrobial production was evaluated during the eight weeks of culture.
• the lineage R. solanacearum V55 was chosen as test microorganism.
• the bacterium was cultured in liquid LPG, under stirring, for 24 hours. Then several and sequential dilutions of the bacterium culture were performed in MgSO 4 2 g/L, varying from 10 "1 to 10 "7 . Samples from the bacterial suspension (1 mL containing 10 5 bacteria) plus a volume of the fungus filtered culture were put into test tubes. As a control, a tube with MgSO 4 2 g/L was used, and all tubes were incubated at 30 0 C for 24 hours. The table below gives details of each test tube.
• Bacteria with a grade higher than 3.06 was considered as tolerant. The plates were incubated for 24 hours and then given the score for the growth. These scores were converted to % of bacterial growth, where a score of 4.0 corresponds of 100% growth.
• the fungal filtered culture grown during 30 days, was diluted in sequential dilutions of a solution of MgSO 4 2 g/L, from 10 "1 to 10 ⁇ 6 .
• the table below gives details of each test tube.
• a 50 mL sample of the filtered culture was added to 150 mL cool (8°C) acetone and put to rest at 4°C for 24 hours for polysaccharides precipitation. After that, two phases were observed, a white phase at the bottom and a colorless at the top. The colorless phase, containing acetone plus the metabolites was transferred to a new container, and lyophilized.
• Example 7 Detection of antibacterial metabolites by diffusion 6 mm diameter paper-filter discs were wet with the filtered culture, ca. 10 ⁇ l_, and placed over LPGA medium (for phytopathogenic bacteria) and LB medium (for pathogenic bacteria), both media containing bacterial samples spread in the surface.
• LPGA medium for phytopathogenic bacteria
• LB medium for pathogenic bacteria
• the dishes were incubated at 4°C for 4 hours, for metabolites diffusion in the medium. Then were incubated at 30 0 C for 48 hours (for phytopathogenic bacteria) and at 37°C for 24 hours (for pathogenic bacteria).
• Antibacterial activity was determined by the measurement of the inhibition zone diameter (in mm) and the average by the number of repetitions.
• Example 8 Detection of antibacterial metabolites by cup-plate technique Eleven lineages of R. solanacearum were tested. Bacteria was cultured in solid LPGA for about 48 to 72 hours and then grown in 50 mL of liquid LPG and incubated at 28°C for 24 hours under stirring. Dilutions were made by removing 1 mL samples and adding 9 mL of sterilized saline solution of MgSO 4 2 g/L 1 mL of each dilution was transferred to sterilized Petri dishes with LPGA at 40 0 C. After solidification of the medium, cup-plate were made with 6 mm diameter test tubes.
• Partition tests were performed in 2 L separation flasks, containing 300 mL of filtered culture and 300 mL hexane. The flask was gently stirred and opened for liberation of vapors. The system was put at rest until phase separation was evident. The hexane phase was separated from the aqueous phase. The aqueous phase was put in a flask containing 300 ml_ of dichloromethane. The flask was gently stirred and opened for liberation of vapors. The system was put at rest until phase separation was evident. The dichloromethane phase was separated from the aqueous phase. The process was repeated with different solvents with increasing polarity.
• Example 10 Biological assays with the extracts from the filtered culture The extracts from the filtered culture were biologically tested in order to verify which fraction contains the active principle against R.solanacearum detected into the filtered culture.
• the lineage R. solanacearum V55 was cultured in LPGA and 28°C for 24 hours. Then it was transferred to a flask containing 50 ml_ of LPG, which was incubated for 24 hours at 28°C under stirring.
• the controls used were discs with water, methanol, dichloromethane and dry paper-filter disc.
• the crude and semi-purified extracts presented antibacterial activity against R. solanacearum.
• the inhibition halo was greater for the hexanic fraction (10.7 mm diameter) when compared with the dichloromethanic fraction (7.4 mm diameter).
• Example 11 Extracts chemical constituents analysis by comparative thin layer chromatography (comparative TLC) The extracts obtained from the partition experiments were analyzed by comparative thin layer chromatography. For each extract the suitable solvent for its solubilization was determined. The process begins with low polarity solvents until a clean extract with no particles in suspension is obtained.
• the samples were applied in silica gel GF 254 Merck chromatopapers (5 x 3 cm) and tested different solvents for choosing an adequate elution system.
• the plates were observed under UV lamp (254 and 366 nm) and then revealed with sulfuric vanillin and heating (110 0 C) until the observation of color.
• the samples were solubilized in adequate solvents and applied at the surface of a KBr pellet.
• the reading was performed in the range of from 4000 to 400 cm "1 of infrared spectra.
• the IR spectra of the filtered culture presented typical regions of a mixture of substances, suggesting, among other functions, alcohols and carbonyls.
• the spectra from the acetate and butanol phases were similar.
• the spectra from the hexane and dichloromethane phases were also similar, showing the presence of fatty acids and absence of proteins.

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• 2007
  • 2007-12-28 US US12/810,380 patent/US20100285054A1/en not_active Abandoned
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