USES OF HONEY EXTRACT OF ULMO unifloral as bactericide and fungicide.

DESCRIPTIVE MEMORY

The present invention relates to uses of an extract of unifloral Ulmo honey, rich in phenolic compounds, able to inhibit the in vitro growth of human pathogenic bacteria such as Staphylococcus aureus, aeruginosa Pseudomonas, Escherichia coli, among others, in addition to bactericidal activity bacteria. This extract also has fungicidal and fiingistática environments on fungi genera Mucor, Rhizopus, Aspergillus, Candida and Penicillium activity.

The uses of the present invention are surface disinfection by a cleaning household surfaces as topical or systemic application as bactericide and fungicide in living organisms, such as animals and humans beings with a therapeutic purpose.

Furthermore, the method of extraction of phenolic compounds of unifloral Ulmo honey, which will be used as an active ingredient of a sanitizing composition and / or disinfectant is described.

BACKGROUND OF THE INVENTION

Disinfection of environments is a basic need of hygiene in all activities related to the production and handling of food, in workplaces, at home, in hospitals. Places where the risk of transmission of diseases caused by bacteria, fungi and viruses, can not only cause serious problems to human health, but it can also cause economic losses. Numerous examples, such as the detection of Escherichia coli bacteria OI 57, causing hemolytic uremic syndrome in humans, discovered in poultry and meat of a major multinational fast food chain. Product of this detection, closed stores in several countries. Other examples are the hospital (nosocomial) intra- diseases, caused by cross contamination, which can cause death of people. Such is the case that occurred in a hospital in Talca in 2007, with the death of a baby by the bacterium SERRARIA marescens.

The elimination of bacteria in environments is complicated by the emergence and spread of antibiotic resistant strains and disinfecting agents, a growing phenomenon around the world and of great complexity. It is for this reason that the World Health Organization (WHO), through resolution 1998, declared as global public health problem of the phenomenon of antimicrobial resistance. The consequences of microbial resistance are severe, because infections caused by resistant microorganisms can be difficult to manage and may not respond to antimicrobial management, being necessary in some cases even use toxic products for control.

By the assertions described, the search for safe and natural disinfectants that eliminate germs such as viruses, bacteria, fungi and protozoa is a beta of widespread and growing scientific research development. Previous concerning the unifioral antimicrobial activity of the extract honey bee (Apis mellifera) ulmo (Eucryphia cordifolia), only Chilean honey in the world of botanical and geographical origin, allowed the formulation of an aerosol research whose active ingredient corresponds to extracts compounds from such phenolic groups, able to inhibit the in vitro growth of human pathogenic bacteria Staphylococcus aureus, Streptococcus β hemolytic, Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, Salmonella typhi and Candida albicans fungus.

Within analyzed honey extracts, extracts from unifioral ulmo honey possess a spectrum of control as to the ability to inhibit a wider range of pathogens. This extract honey unifioral ulmo and its use as a disinfectant agro-industrial purposes whose uses are the invention is applied for protection in Chile Cl Patent Application 01069-2006 (Montenegro, 2008a); in the US for US Patent Application 797,504 Al (Montenegro, 2008b) and in Europe by the patent application EP 1852017 (Montenegro 2007).

in which defined the presence of synthetic phenolic compounds derived from refining metals and wood industries in drinking water as a pollution hazard, on the other hand, scientific research (. Ren et al, 2003 Castle and Puig, 1997) are known as these products may cause toxicity in humans. Most of these compounds correspond to phenolic liposoluble compounds which do not correspond to the compounds present in the product described in the present patent application, being cytotoxic activity of these compounds in mammalian cell lines HL-60, MCF-7, CCRF -Cem, and CEM / VLB, killing of these cell lines (Selassie et al., 2005).

The US patent 7,582,318 B2 issued dated September 1, 2009 the Chilean equivalent of 1069-2006 application describes a unifloral honey extract ulmo to control bacterial infections in plants and its extraction process.

Unlike the above-mentioned document, the present invention describes a process of extracting a fraction rich in phenolic compounds unifloral ulmo honey, a semi-industrial scale, which avoids the use of solvents such as methanol to collect the phenolic compounds, or as diethyl ether to remove the phenolic compounds from the extract obtained, both toxic compounds, so the method described in the present invention is not only useful to produce larger volumes of extract unifloral ulmo honey, but it reduces the use of toxic solvents to the environment and humans. On the other hand this application presents studies evaluating the disinfecting ability unifloral honey extract ulmo on bacteria and fungi commonly found on hard surfaces, such as tables, shelves, etc., as well as in humans who demonstrate their effectiveness against bacteria and fungi, which differs from the US patent 7,582,318 B2, since this is only useful for bacterial infections in plants.

DESCRIPTION OF THE FIGURES

Figure 1: mining equipment and components 1) Tank feeder 2) celestial PVC tubes corresponding to the chromatographic column with Amberlite XAD-2 resin and polyurethane. 3) solvent Ponds 4) flavonoid collection pond 5) vacuum pump and cooling system 6) electricity board.

Figure 2: Flow diagram of exhaust operation, the steps involved in the process are described. Arrows indicate flow inputs and outputs.

Figure 3: Calibration curve of gallic acid, for quantification of total phenols soluble in the extract

Figure 4: Plates used to determine, S. aureus, E. coli, Candida albicans, S. typhi, Enterobacter aerogenes and Streptococcus β type (MICs) Minimal inhibitory concentration ulmo honey extract in distilled water on P. aeruginosa.

Figure 5: Plaques used to determine the MIC honey extract in distilled water ulmo K. oxytoca, K. pneumoniae, B. cereus, B. subtilis, S. epidermidis, S. marescens and Saccharomyces spp.

Figure 6: Pseudomonas aeruginosa seeded in Petri dishes in Trypticase Soya Agar aerosol propellant sprayed with honey and control, incubated at 37 ° C for 24 hours

Figure 7: Salmonella typhi seeded in Petri dishes in Trypticase Soya Agar aerosol propellant sprayed with honey and control, incubated at 37 ° C for 24 hours.

Figure 8: Bacillus subtilis seeded in Petri dishes in Trypticase Soya Agar aerosol propellant sprayed with honey and control, incubated at 37 ° C for 24 hours.

Figure 9: Bacillus cereus seeded in Petri dishes on agar medium Soya Tryptic aspen tuples spray honey, propellant and control incubated at 37 ° C for 24 hours Figure 10: Escherichia coli seeded in Petri dishes on agar medium Soya Tryptic sprayed with spray honey and control propellant, incubated at 37 ° C for 24 hours

Figure 11: Staphylococcus aureus seeded in Petri plates in Trypticase Soy Agar incubated at 37 ° C for 24 hours.

Figure 12: microwell plates incubated for 5 days at 20 ° C, used to determine the MIC of extracts of honey unifloral ulmo on Penicillium sp. (P sp), Mucor sp. (M sp), Aspergillus flavus (A f), Aspergillus niger (A n), Aspergillus terrens (A t), Aspergillus fumigatus (Af) and Rhizopus sp. (R sp), the keys indicate the minimum dilution honey extract inhibits the in vitro growth of microorganisms.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is the health, domestic and therapeutic use in topical or systemic in animals and man of an extract rich in phenolic compounds unifloral Ulmo honey, either dissolved in water or another solvent, in spray or aerosol propellants, used in both cleaning products and disinfectants for human use as pharmaceutical and cosmetic formulations such as soaps (liquid, solid and / or gel), creams, dressings, sanitary towels, and household goods such as detergents and cleaners in general.

Preparation of extract of phenolic compounds of unifloral honey ulmo (Eucryphia cordifolia)

The assets to be used in the preparation of a sanitizing and disinfecting composition ingredient is obtained by extraction of phenolic compounds of unifloral honey ulmo {Eucryphia cordifolia), which will be described later.

Phenolic acids and flavonoids (phenolic compounds) are compounds derived from secondary metabolism of plants, and are located in the photosynthetic cells. These compounds are listed in significantly with defense mechanisms inherent to the plants, such as barriers against pests. Furthermore, the prior art discloses the antibacterial activity of phenolic compounds (Wahdan, 1998, Cushnie and Lamb, 2003), which are inherited to honey from the nectar collected by bees that

The extract phenolics unifloral ulmo honey used in the present invention is produced by the following method (shown diagrammatically in Figure 2). The process steps for obtaining extracts phenolics unifloral ulmo honey are:

a) Load feeder pond with honey: In this Step 4 kilos of honey used 20 liters of water acidified with HC1 to pH 2. This charging process takes 30 minutes, time in which honey is dissolved and fills the pond.

b) Adsorption of phenols: The contents of the collecting drum is circulated and recirculated through the columns of PVC containing resin Amberlite XAD-2, a continuous recirculating flow of 148ml / minute for 135 minutes, remaining phenolic compounds retained in the resin .

c) Cleaning sugars from the chromatographic column PVC: To this are circulated 10 liters of distilled water acidified to pH 2 from its respective tank, the columns of PVC, which then returns to the pond feeder. This process takes 68 minutes.

d) Elution of distilled water from the chromatographic column PVC: chromatographic columns are cleaned with 20 liters of distilled water for approximately 130 minutes. The liquid returns to the pond with acidified water.

e) phenolic compounds Desorption retained on the column chromatography: 10 liters of 96% ethanol for elution of these compounds are collected in the collecting pond flavonoids are used. Process time 68 minutes In all the steps described above are performed drainages with vacuum for 5 minutes. Flavonoid concentration: The extract is concentrated at a temperature of 45 ° C and vacuum concentrating the extract volume from 10 liters to 4 liters. The ethanol is recovered by the system refrigerant passing from the gaseous to liquid, to finally collect it in the pool of ethanol. Process that lasts approximately 300 minutes.