The present invention relates to compositions and their uses in eradicating or ameliorating unwanted infestations. More particularly the invention relates to compositions and their uses which comprise a flavanoid component and an oleuropein component. Such compositions are aptly employed to eliminate or reduce the presence of unwanted bacteria or other undesirable organisms. A variety of flavanoids have been suggested as anti-microbial agents. PCT/GB2007/002756 and PCT/GB2007/002758 describe particularly effective compositions containing flavanoids. However, the effectiveness of known compositions of bioflavanoids would benefit from enhancement. The present invention is based on the finding that certain combinations of agents are particularly effective in eliminating or reducing unwanted bacteria and other undesirable organisms.

The present invention provides a pharmaceutical composition which comprises a first component being a flavanoid component and a second component being an oleuropein component.

The first component will be a one or more flavanoids of Formula (I) :

wherein wherein R ¹ is hydroxyl or methoxyl and R ² is hydrogen, hydroxyl or methoxyl and X is hydrogen or a saccharide. The second component will be oleuropein or an acetylated derivative thereof. Oleuropein is (4S,5E,6S)-4-[2-[2-(3,4-dihydroxyphenyl)ethoxy]- 2-oxoethyl]- 5-ethylidene-6-[[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)- 2-tetrahydropyranyl]oxy]-4H-pyran-3-carboxylic acid, methyl ester.

Aptly in the first component R ² is hydrogen and R ¹ is in the 3- or 4- position . Alternatively, aptly in the first component R ² is 3-hydroxy and R ¹ is 4-methoxyl .

Suitably X in a compound of the Formula (I) is H .

Suitably X in a compound of Formula (I) is a saccharide.

Favourably X is a disaccharide.

Suitable disaccharides include combinations of two monosaccharide, suitably pyranoses, linked by a glycosidic bond, for example rhamnose and glucose, for example L-rhamnose and D-glucose.

Suitable disaccharides can have the structure:

wherein one of R ³ and R ⁴ is H and the other OH or both are H or both are OH . Aptly R ³ is H and R ⁴ is OH so that the disaccharide is rutinose. Favoured aglycones of flavonoids for use in this invention are the disaccharides 6-0-(alpha-L-rhamnopyranosyl)-beta-D-glucopyranose, also known as rutinose, and 2-0-(alpha-L-rhamnopyra-nosyl)-beta-D- glucopyra-rose.

It is presently believed that the first component very suitably comprises narangin or neohesperidin or mixtures thereof. Mixtures of one or both of narangin and neohesperidin with for example, one, two or three other flavanoids of the Formula I are presently believed particularly favoured for use in this invention. Such mixtures can be obtained from extraction from bitter oranges.

Suitable compounds of Formula (I) include Neoeriocitrin, Isonaringin, Narangin, Hesperidin, Neohesperidin, Neodiosmin, Naringenin, Poncirin and Rhiofolin.

Favoured compositions for use include those which comprise either of naringin and neohespiridin or both.

Particularly aptly the invention will contain naringin and neohesperidin and other flavonoids of the Formula (I).

The mixture of flavonoids may aptly contain more than one of neohesperidin, naringin, isocriocrin, isonaringin, naringin, hesperidine, neohesperidin, neocliomin, naringenin, poncrin and rhiofolin. Such a mixture of flavonoids can be obtained from bitter oranges, see for example PCT/GB2007/002756. Suitable mixtures can include 2, 3, 4, 5, 6, 7, 8, 9 or more compounds of formula (I). Thus a mixture comprising 2, 3, 5, 6, 7, 8 or 9 of the above named flavonoids is apt, for example containing 3, or containing 4, or containing 5, or containing 6, or containing 7, or containing 8 or containing 9 of said flavonoids.

It is presently believed that mixtures of such flavonoids have advantages over the use of a single flavonoid. It is particularly advantageous that extract of bitter oranges may be employed without the need for isolating individual flavonoids if desired. The use of the composition generally comprising biomass enhances solubility of the flavanoids. Generally the flavanoids are present in mixtures with biomass by about 10% to 75%, more aptly 30% to 60%, for example 40% to 50%, preferably about 45%. The biomass comprises pectins and other sugar derived materials. Typically about 40% of low molecular weight pectins are present.

If it is desired to avoid biomass, other solubilising agents such as dextrins, for example cyclodextrin, may be employed if desired .

Aptly the mixture of flavonoids will comprise at least 25%, more suitably at least 40% and preferably at least 50% of narangin. More aptly the mixture will contain up to 65% of narangin.

Aptly the mixture of flavonoids will comprise at least 15%, more suitably at least 20% and preferably at least 25% of neohesperidin. More aptly the mixture will contain up to 35% of neohesperidin. In a favoured form the mixture will contain at least 75% of neohesperidin and narangin.

It is presently believed that particularly suitably the second component is oleuropein. Aptly this is obtained from extraction from the leaf of the olive, for example olea europea. Such extracts typically contain 5% to 80% wt/wt, more aptly 10 to 70%, for example 20% wt/wt.

The other components of the extract will often contain pectins and the like.

A particular advantage of many compositions of the invention is that they may employ compounds of natural origin. Thus, for example, it is preferred to employ a first component which may be obtained from bitter oranges and a second component which may be obtained from the olive. However synthetically or semi-synthetically obtained compounds may be employed if desired instead of the ones directly extracted from natural sources although this tends to be less favourable in view of cost.

The compositions of this invention may show synergistic anti-microbial effectiveness. Such synergistic compositions are preferred .

It is presently believed that best synergy occurs wherein the first component and the second component are present in ratios (wt/wt of flavanoids to oleuropein) of 5 : 1 to 1 :4, more aptly 2 : 1 to 1 : 2, favourably 1 : 2 to 1 : 1 and preferably 3 : 2.

Such compositions may desirably contain a first component which is a mixture of flavanoids of Formula (I) wherein 50% - 100% (wt/wt) is composed of narangin and/or neohesperidin. It is presently believed that the first component may favourably contain a mixture of flavanoids containing 60% - 100% of narangin and neohesperidin, for example 65 - 75% of narangin and neohesperidin.

Such compositions may preferably contain 3,4-dihydroxyphenylethanol as the second component or a mixture of compounds of formula II in which 3,4-dihydroxyphenylethanol is present by at least 50% (wt/wt), and more aptly at least 80% (wt/wt).

It has been found that compositions of this invention are particularly effective in the presence of a third component, which is one or more organic acids.

A surprisingly effective third component is salicylic acid or its pharmaceutically acceptable salt optionally together with a further organic acid or pharmaceutically acceptable salt.

It is believed favoured that the salicylic acid is present in the acid rather than its salt. Similarly, a further organic acid if present is similarly in the form of the acid rather than its salt. Suitable further organic acids include acids of up to 8 carbon atoms which are monobasic (i.e. 1 C0 ₂ H group), di-basic or tri-basic acid which optionally contain 1, 2 or 3 hydroxyl groups. Such further organic acid may be one or more of citric acid, malic acid, latic acid, tartaric acid, fumaric acid and the like.

Such compositions can provide an approximately neutral or acid pH, when used, for example of from 3-8, more aptly 3.5-7, for example 4-5.

At present it is preferred to employ salicylic acid and citric acid in compositions.

In compositions containing a first component, a second component and a third component, the weight/weight ratio of the compound(s) of Formula (I) plus the compound(s) of Formula (II) to salicylic acid or pharmaceutically acceptable salt thereof is 2 : 1 to 1 : 7, more aptly 1 : 1 to 1 : 5, favourably 2 : 3 to 1 : 3 and preferably is 2 : 5. When the third component also comprises a further organic acid (such as those named above), the weight/weight ratio of salicylic acid to further organic acid may be 2 : 1 to 1 : 5, more aptly 1 : 1 to 1 : 3, for example 3 : 5.

Such compositions may include a solubilising agent for the salicylic acid, for example a dextrin such as cyclodextrin.

Compositions of the invention may be adapted for application to external surfaces including external surfaces of plants or animals, or for internal administration to an animal. Often the animal is a human.

The compositions of the invention show activity against a wide range of organisms including gram positive bacteria, gram negative bacteria, fungi, virus, protazoans and insect parasites. Particularly surprising the compositions may be employed against difficult bacteria such as methicillin resistant staphylococcus aureus (MRSA), Clostridium difficile (C.diff) helicobacter pyroli (H.py), and vancomycin resistant enterobacteria. The compositions of this invention may also be used against norovirus and other pathogens whereby transmission is by contact or air.

The compositions may be administered systemically or locally if an animal is to be treated . Suitable animals include humans and food and companion animals such as cows, pigs, horses, chickens, sheep, goats, dogs and cats. Hence the compositions may be formulated for oral administration, topical administration, injection or the like as required by the medical practitioner. Particularly suitably such compositions are suitable for use to treat humans.

Suitable oral and topical compositions include those analogous to those described in PCT/GB2007/002756 and PCT/GB2007/002758. Injectable forms may be put up in conventional manner, for example as aqueous solutions or dispersions.

The compositions of the invention are also useful for use in conjunction with a further antibacterial agent. Thus for example, if used to treat a patient suffering from MRSA, the compositions may be used in conjunction with a β-lactam or other antibiotic or antibacterial agent. Suitable β- lactam antibiotics include, a penicillin such as methicillin, flucloxacillin, ampicillin, amoxicillin optionally with clavulanic acid, a cephalosporin, or a carbopenem such as imipenem or the like. The composition as a single composite form or as two or three forms, for example one of dosage form will contain the β-lactam or other antibiotic and another composition will comprise the first and second components. Hence the β-lactam antibiotic can be administered separately from component I and II (and if desired first and second components may also be administered separately from the further antibacterial agent). The compositions of the invention containing the first and second components adapted for oral administration and the further antibacterial agent adopted for oral administration (for orally administrable antibiotic such as amoxicillin) or for injection (for injected antibiotics such as methicillin or imipenem) may also be used together with vancomycin.

Patients suffering with infections caused by C. diff and H.py may be treated similarly. The preferred route of administration of all three agents is orally when the β-lactam or other antibiotic is orally effective, especially for the treatment as gastric infections of H.py. However, if an antibiotic is employed that is best administered by injection that may be done in conjunction with the oral administration of the compositions of the invention.

Since the compositions of the invention are particularly effective for sterilizing surfaces, they are very suitably formulated in a composition useful for external use.

These may be in the form of solutions, gels, soaps, body wash, shampoo, dusting powders and aerially dispersible powder and liquids and the like.

Such compositions may be used to reduce the bacterial count on body surfaces, clothing and in the general environment particularly in hospitals, ambulances, nursing homes especially for the elderly or the like where it is particularly desirable to reduce the presence of bacteria such as MRSA, C. diff or the like. Compositions suitable for washing the hands are particularly useful.

Such compositions may also be employed to wash stethoscopes.

The substantivity of the compositions (as opposed to rapid diminution of effectiveness of ethanol) is an advantage.

If external surfaces of enclosed spaces, such as ambulances, operating theatres, wards, kitchens (and even mortuaries) and so on are to be treated, it is particularly suitable to do so by "misting". In this a fine aerial dispersion of powder or microdroplets of composition are dispersed within the enclosed space. This can then offer a non-toxic alternative to the presently employed methods which often employ noxious gases. Since the compositions of the invention have such low toxicity they may be employed on patients and their visitors and associated clothing, linen and the like by "misting". Such "misting" is of use in vehicles such as ambulances which are required to be free of pathogens but likewise free of residual odors that are typically left following the use of noxious gases. This equally applies to other areas requiring treatment. Compositions used in this way may be in the form of a dispersible liquid, for example akin to the soap or shampoo or skin foaming compositions described hereinafter. These can also be used to what the surfaces to similar ends.

A particular use includes antibacterial soaps, detergents, lotions and the like for treating inter alia human skin and hair in order to reduce or eliminate undesired organisms.

Thus it is possible by using compositions of this invention prevented in such forms, to treat hands, the face and skin generally and the hair, both on the head and elsewhere. This can be employed to reduce bacterial count and so help to reduce the spread of methicillin resistant staphylococcus aureas, Clostridium difficile and other bacteria. Similarly, the composition may be used to reduce microorganisms associated with acne, body odor or the like. A further benefit is that such compositions may be used to reduce viral transmission, for example for influenza virus, which can occur by hand contamination. Other virus that may be on the skin or membranes include HIV, herpes and the like which are also minimized by use of the compositions of the invention adapted for administration to the skin or membranes. Parasite infestation may be treated with compositions of the invention. Such parasites include internal parasites such as protozoa which can lead to diseases of humans such as malaria, leishumaniasis and trypanosomiasis and various diarrhoeas. Other internal parasites that may be treated include flukes. External parasites that may be treated include lice, especially head lice, and scabies and fleas. Soaps and shampoos are favoured for such external application although solutions, lotions and gels are also particularly suitable.

Fungi for treatment include those responsible for dandruff, thrush, athlete's foot and the like, for example, Candida albicans. Conditions such as dandruff may be treated with soaps and shampoos but other formulation types named herein may also be used . Athlete's foot may also be treated with dusting powder. Candida albicans or other infecting agents in the vagina may be put up in the form or a pessary.

A particular use for the compositions of the invention includes mouthwashes, toothpastes and other forms suitable for use in the buccal cavity. Such forms may be formulated as indicated in the PCT applications referred to herein before.

The compositions of the invention may also be used for the prophylaxis or treatment of dandruff or the like.

Compositions of the invention may therefore also suitably contain a pharmaceutically acceptable salt of choline such as choline chloride. This can enhance effectiveness further against organisms such as c. diff.

Formulations may be composed of conventional carriers as long as they are compatible with the first, second and optionally third component of the compositions herein.

Thus soaps, shampoos and the like may aptly contain surfactants. Many conventional surfactants may be employed but it appears certain effective formulations will employ non-ionic surfactants. Particularly effective non- ionic surfactants include alkyl polycyclosides and/or alkenyl polyglycosides (APGs) such as those containing up to 10 sugar residues coupled to a hydrocarbon chain. Oligomerisation of up to about 4 sugar residues can be desirable. Such surfactants are available under the trade name "Plantacare" for example from Henkel as "Plantacare 2000". In some compositions minor amounts of typical anionic surfactants may be employed together with the non-ionic surfactant. Amphoteric surfactants may also be present, for example and preferably, with the non-ionic surfactants, for example those having secondary or tertiary amino and water solubilising anionic groups, such as sulphate, phosphate, phosphonate or carboxylate groups. Such amphoteric surfactants include those available under trade names such as Miranol (of Rhone-Poulenc) and Betain, such as Dehyton from Henkel. The compositions of the invention may optionally comprise thickening agents. Suitable thickening agents include polysaccharide thickeners such as xanthan gums, gellan gums, pectins, carageenans and the like. An apt thickening agent is xanthan gum such as Keltrol CG which is a high molecular weight polysaccharide produced by microbial fermentation. Viscosity may also be selected by use of an amphoteric surfactant such as a cocamido-propyl betain or Tego Betain F50 as a thickening as well as surfactant agent.

The compositions of the invention may be employed for the treatment of food stuffs to reduce or eliminate unwanted pathogens or organisms leading to reduction in storage life of food stuffs. Thus vegetable, fruits and meat may be treated, for example lettuce, tomatoes, cucumbers, peppers, cereals such as wheat and maize, fruit such as apples, grapes, pears and figs, and meats such as beef, pork, lamb, bacon and the like. Methods of treatment include washing, misting and the like.

The hand sterilization test method used in the Examples was as follows:

Experimental procedure:

1) Application of the contamination fluid.

Each of the twelve subjects was asked to wash their hands for one minute in soft soap to remove natural commensal organisms and dried thoroughly on a paper towel. The hands were then contaminated with very large numbers of bacteria well in excess of that experienced in normal everyday occurrence. The hands were immersed in the contamination fluid (containing an overnight culture of the test organism in this instance E. coli at a concentration of approximately 10 ⁸ cfu per ml) in a suitable sized container for five seconds. The hands were removed from the contamination fluid and surplus liquid allowed to drain back into the container. This time the hands were allowed to air dry for approximately three minutes holding them horizontally with fingers spread out and rotating them to and fro to avoid the formation of droplets.

2) Prevalues.

Immediately after drying, each of the twelve subjects was asked to rub their fingertips, including the thumbs for one minute on the base of a petri dish, using a separate petri dish for each hand, containing 10ml of maximum recovery diluent (MRD) without neutraliser, in order to assess the release of test organisms before treatment of the hands. Dilutions of these sample fluids were prepared to 10 ^"3 and 10 ^"4 . A 1ml aliquot of each dilution for each hand was placed in a separate petri dish 10 - 15ml of Tryptone Soy Agar sterilised and cooled to 45°C added and mixed thoroughly. Plates were allowed to set and incubated at 37°C for twenty four hours. Each plate was then examined for growth of the test organism. 3) Hygienic Handwash procedure.

Each of the twelve subjects was asked to pour 3ml of soft soap into the cupped hands pre-moistened with tap water and wash their hands in accordance with a standard handwash procedure. This comprises five strokes backwards and forwards palm to palm, right palm over left dorsum and left palm over right dorsum, palm to palm with fingers interlaced, back of fingers to opposing palms with fingers interlocked, rotational rubbing of right thumb clasped in left palm and left thumb clasped in right palm, rotational rubbing with clasped fingers of right hand in palm of left hand and clasped fingers of left hand in right palm using as much lukewarm water as is needed to produce a lather. After sixty seconds the hands are rinsed under running tap water for fifteen seconds from distal to proximal with fingertips upright. 4) Handwash procedure with test product (P).

After first wetting the hands and arms with water approximately 3ml of a composition of the Examples was applied and rubbed thoroughly onto these areas using the technique described above and this is then rinsed off with running water. The total washing time is limited to sixty seconds and the time for final rinse is not included in the washing time. To avoid recontamination of the sample area (i.e. the fingertips) subjects are requested to hold their hands such that the fingertips are pointing upwards. Wrists and forearms are wiped dry by another person.

Example 1

Surgical Sanitizer Water (481.5g; 96.3%) was added to a beaker and stirring commenced . Keltrol CG-SFT (9.0g; 1.8%) was added and stirring continued until dissolved . Citrox HXT powder (2.5g; 0.5%) was added and stirring continued until dissolved. White willow bark extract (2.0g; 0.4%) was added and stirring continued until dissolved. Glycerol (5.0g; 1.0%) was added and stirring continued until dissolved.

The resulting viscous gel was de-aerated . The pH was 4-5. The viscosity 7000-10000 cp at 20°C (spindle 4/0 rpm). The pH may be adjusted with citric acid if required to bring it within the stated range.

Citrox HXT powder comprises on a wt/wt basis 7.5% HPLC 45%, citric acid 30%, willow bark extract 50% and olea Europeae extract 12.5%.

HPLC-45% contained 45% by weight of a mixed of flavanoids together with residues of extraction from bitter oranges. The Willow Bark extract contains 90% of salicylic acid. The Olea Europeae extract contains 20% of oleuropein.

The mixture of flavanoids in HPLC-45 comprises:

Example 2

Liquid Hand Soap

Water (362g, 72.4%) was added to a beaker and stirring commenced . Keltrol CG-SFT (9g, 1.8%), Plantacare 2000 (67.8g, 13.6%), Tego Betain F50 (lOg, 2%), glycerol (lOg, 2%) and Citrox HXT powder were sequentially added with stirring until complete dissolution occurred prior to adding subsequent ingredients. The product was a clear viscous gel, pH 4.8 to 5 was a viscosity of about 4000 cp at 20°C (spindle 4/10 rpm). Citrox HXT powder and Keltrol CG-SFT were as described in Example 1.

Plantacare 2000 is an aqueous solution containing 6.78g of the surfactant agent. Tego Betain F50 is an aqueous solution containing 3.22g of the surfactant agent.

Example 3 Hand Foam Composition

This was prepared by mixing as described in Example 1.

When tested against test organism according to BS EN 13727 under dirty conditions (interfering substances bovine albumin and sheep erythrocytes), a five minute contact time at 20°C, satisfactory bactericidal activity was observed when the test organism was (i) MRSA (ATCC33591) and Clostridium difficile (NCTC 11209). When tested against spores of this C. difficile according to En 13704, satisfactory sporicidal activity was found with a 15 minute contact time at 20°C. Example 4

Sanitizing Gel

This was prepared by mixing as described in Example 1.

When tested by the methods detailed in Example 1, the gel provided satisfactory bactericidal activity against the MRSA and satisfactory sporidical activity against the C. difficile.

Example 5

Liquid Soap

Olive leaf extract 0.0625%

Citric acid 0.15%

Salicylic acid 0.25%

Dermosoft GMCY 1.0%

Water 72.66%

When used herein HPLC 45% means a mixture containing 45% of bioflavanoids and 55% of other matter from extraction of bitter oranges. The bioflavanoids comprised narangin (about 52%), neohesperidin 28%, poncirin (4%), naringenin (3%), hesperidin (3%), neodiosmin (3%), isonaringin (3%), isocriocrin (2%), other minor to 100%.

Example 6 Gel

Example 3 may be repeated replacing HPLC 45% with an equal weight of a 1 : 1 mixture of narangin and nehesperidin. Example 7

Aerially Dispersible Form

The hand foam composition of Example 3 is used in a commercial misting device to produce a mist for disinfection of surfaces.

Example 8

A commercial hand held misting device is used to direct mist at the surfaces in an ambulance and to the air space. The misting is continued until the operative is satisfied surfaces have been thoroughly treated .

The ambulance may be occupied twenty minutes after the completion of the misting.