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WO2012001347A1 - Produits à effets bénéfiques sur la santé bucco-dentaire - Google Patents

Produits à effets bénéfiques sur la santé bucco-dentaire Download PDF

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Publication number
WO2012001347A1
WO2012001347A1 PCT/GB2011/000969 GB2011000969W WO2012001347A1 WO 2012001347 A1 WO2012001347 A1 WO 2012001347A1 GB 2011000969 W GB2011000969 W GB 2011000969W WO 2012001347 A1 WO2012001347 A1 WO 2012001347A1
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WIPO (PCT)
Prior art keywords
sub
fraction
lmm
composition
fractions
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Application number
PCT/GB2011/000969
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English (en)
Inventor
Gabriella Gazzani
Itzhak Ofek
Carla Pruzzo
Peter Lingstrom
Egija Zaura
Pietro Canepari
Paula Cawkill
Michael Wilson
Jonathan Pratten
David Spratt
Original Assignee
Ucl Business Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB1010948.6A external-priority patent/GB201010948D0/en
Priority claimed from GBGB1019867.9A external-priority patent/GB201019867D0/en
Application filed by Ucl Business Plc filed Critical Ucl Business Plc
Publication of WO2012001347A1 publication Critical patent/WO2012001347A1/fr

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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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • 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
    • 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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/362—Polycarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/365—Hydroxycarboxylic acids; Ketocarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/368—Carboxylic acids; Salts or anhydrides thereof with carboxyl groups directly bound to carbon atoms of aromatic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses

Definitions

  • the present invention relates generally to agents derived from natural products and compounds related thereto which have benefits in promoting oral health, for example in controlling or reducing the risks of dental caries and periodontal diseases when presented in oral preparations or foodstuffs.
  • Caries is one of the most prevalent chronic diseases of humans. It is an endogenous infection of the calcified tissues of the teeth and is a result of their demineralisation by organic acids produced by those plaque bacteria that ferment dietary carbohydrates.
  • the most common aetiological agents of enamel caries are considered to be Streptococcus mutans and Streptococcus sobrinus; additional aetiological agents are lactobacilli and actinomyces, the former being considered as secondary invaders, while the latter are responsible for root surface caries (3-6).
  • the pathogenesis of dental caries is dependent upon the presence of fermentable sugars in the diet and the presence of cariogenic bacterial species.
  • the disease can be considered to involve three main stages: 1 ) adhesion of bacteria to the tooth; 2) production of a glycocalyx resulting in the formation of a bacterial community embedded in the glycocalyx (i.e. a biofilm) which is known as dental plaque, 3) fermentation of sucrose within the biofilm to, mainly, lactic acid which demineralises the enamel resulting in the production of the caries lesion.
  • the main virulence properties of S. mutans and S. sobrinus are, therefore, their ability to adhere to the tooth surface together with their rapid metabolism of sucrose to organic acids and to extra-cellular
  • GTF glycosyltransferase
  • Extracts obtained from different teas and their polyphenol components have been investigated thoroughly for their anti-cariogenic activity.
  • Polyphenols in tea have been shown to reduce caries development in animals because they decrease the cell surface hydrophobicity of S. mutans and its ability to synthesize adherent water-insoluble glucan from sucrose (13-17).
  • Propolis (18) has been shown to possess both antimicrobial and GTF-inhibitory activities.
  • High molecular weight components of hop bract inhibit adherence of and water-insoluble glucan synthesis by, S. mutans (21 ).
  • the cariostatic activity of cacao mass extract has been observed in vitro and in animal experiments.
  • high molecular weight polyphenolic compounds and unsaturated fatty acids were shown to be the active constituents.
  • the former which showed strong anti-GTF activity, were polymeric epicatechins in an acetylated form.
  • the latter showed bactericidal activity against S. mutans (22, 23).
  • An interesting antibacterial activity has been detected in coffee that is effective against S. mutans as well as other Gram-positive bacteria and some Gram-negative species (24-26).
  • roasted coffee interferes with streptococcal sucrose-independent adsorption to hydroxyapatite (HA) beads.
  • HA hydroxyapatite
  • Such activity may be due to small molecules occurring naturally, such as trigonelline, nicotinic and chlorogenic acids, but also to coffee components containing condensed polyphenols or melanoidins that occur during the roasting process (27).
  • Periodontal diseases are a heterogeneous group of inflammatory conditions that involve the supporting tissues of the teeth. They include gingivitis, in which only the gingivae are involved, and the various forms of periodontitis in which destruction of alveolar bone occurs. Characteristically, in these diseases, the junctional epithelial tissue at the base of the gingival crevice migrates down the root of the tooth with the result of the formation of a periodontal pocket. The initiation and progression of periodontal diseases is attributed to the presence of elevated levels of pathogenic bacteria within the gingival crevice. Any of several hundred bacterial species may inhabit the gingival crevice; however, it has been shown that only a few play a significant role in the aetiology of the various periodontal diseases. Indeed, it is generally accepted that a consortium of bacteria, not a single species, is involved in these diseases.
  • gingivitis is extremely common and most adults demonstrate some loss of bony support and loss of probing attachment (28). Brown ef al. (29) has shown that only 15% of the adult population studied had either gingivitis or periodontitis. Gingivitis without periodontitis occurred in 50%, periodontitis (pockets 4 mm or deeper) in 33% and advanced and end-stage periodontal destruction in 8% and 4%, respectively. It is widely accepted that sex, socio-economic and educational status, age and marital quality determine periodontal status (30). These diseases are more prevalent among the less economically and educationally privileged groups in the social hierarchy (31). Finally, tobacco smoking has been shown to be one of the main risk factors for periodontal disease (32-34).
  • Gingivitis is the most prevalent form of periodontal disease and a disease which can be prevented and alleviated by the topical application of suitable agents in, for example, oral hygiene products such as toothpastes, mouthwashes etc.
  • suitable agents such as toothpastes, mouthwashes etc.
  • Accumulation of dental plaque at gingival margins due to inadequate dental hygiene leads to the inflammation of the gingivae, defined as gingivitis (35). It can be defined as 'a non-specific inflammatory process of the gingivae (gums) without destruction of the supporting tissues'. This is a reversible condition as a return to meticulous dental hygiene practices will restore gingival health (36).
  • the bacteria and their extracellular products present within the plaque biofilm on the surfaces of teeth at the gum margin can cause inflammation.
  • Several bacterial species have been implicated as aetiological agents of this disease. These include
  • the high molecular weight material of cranberry juice is effective in inhibiting coaggregation between different representative bacteria and Fusobacterium nucleatum (40). Adhesion of streptococci is inhibited by hop bract polyphenols (41) and by several tea materials (16,42) that have also been shown to inhibit water-insoluble glucan synthesis and bacterial amylases. An interesting antibacterial activity has been detected in coffee (24-27).
  • the present inventors have evaluated a number of natural products (foods and
  • JP2010077028 A20100408 apparently relates to a water-soluble compound, apparently being a protein .
  • the identification of these active fractions forms the basis for various novel materials (e.g. agents, functional foods, and oral healthcare products) and methods for improving oral health.
  • the identification of active constituents may be used to inform selective breeding of the parent plant to increase the content of the active constituents could be achieved.
  • composition comprising an extract of one or more of Shiitake mushroom, chicory, or raspberry to promote oral health.
  • a method of promoting oral health in an individual which method comprises use of a composition comprising of an extract of one or more of Shiitake mushroom, chicory, or raspberry.
  • composition for promoting oral health which composition comprises an extract of one or more of Shiitake mushroom, chicory, or raspberry. • Use of an extract of one or more of Shiitake mushroom, chicory, or raspberry in the manufacture of a composition to promote oral health.
  • the extract will preferably be used in ameliorating, controlling or reducing the risk of periodontal disease such as gingivitis or periodontitis (e.g. to inhibition of the activities of one or more target organisms associated with gingivitis) and ⁇ or will be used as a low molecular mass (LMM) fraction as described below.
  • the "composition” may be an oral preparation or foodstuff (which may by way of non- limiting example be a dietary supplement) - for example a beverage, chewing gum, toothpaste, mouthwash or so on.
  • the foodstuff may be a "functional food” by which is meant both simple foods and food products (meaning technologically-treated foods) in which their chemical composition and, therefore, their organoleptic, nutritional or biological characteristics have been changed. Foods depleted in, or enriched with, specific components, are also considered to be functional foods.
  • oral composition means a composition that is delivered to the oral surfaces.
  • the oral composition is a product, which in the ordinary course of usage, is not intentionally swallowed for purposes of systemic administration of particular therapeutic agents, but is rather retained in the oral cavity for a time sufficient to contact substantially all of the dental surfaces and/or oral tissues for purposes of oral activity.
  • extract is meant a composition comprising a preparation of the components of a substance (here Shiitake mushroom, chicory, or raspberry) typically in concentrated form, prepared by treatment of the substance either mechanically (e.g., by pressure treatment) or chemically (e.g., by distillation, precipitation, enzymatic action or high salt treatment) or using chromatography or other separation or fractionation techniques.
  • a substance here Shiitake mushroom, chicory, or raspberry
  • extract may also be a "fraction” where it has been prepared by fractionation of the original substance.
  • the present inventors have identified a number of fractions and sub-fractions with beneficial activities and these are discussed in more detail hereinafter
  • the composition may consist or consist essentially of the extract. In this embodiment the extract itself may be a "functional food".
  • “Promoting oral health” herein may include one or more of ameliorating, controlling or reducing the risk of dental caries (e.g. by controlling or reducing plaque build up);
  • promoting oral health may include treating dental caries or periodontal disease. It may also include preventing dental caries or periodontal disease. It may also include ameliorating dental caries or periodontal disease.
  • An anti-caries or anti-gingivitis oral preparation comprising, consisting of, or
  • the present inventors have identified particular compounds in these extracts, and shown activity for these isolated compounds in assays relevant to the promotion of oral health. These compounds include: quinic acid, succinic acid, trans-aconitic acid and inosine. Of these quinic acid and succinic acid are preferred. Other compounds identified by the present inventors as having activity in these assays are Shikimic acid; oxalic acid;
  • adenosine uridine
  • cis-aconitic acid epicatechin
  • compositions comprising, consisting, or consisting essentially of one or more of these compounds may also be used in the various aspects of the present invention, in place of the defined extracts obtained from the relevant plants.
  • the level of these compounds may be artificially increased in a foodstuff or oral composition (e.g. by adding the pure compound to supplement the foodstuff or orally acceptable
  • the compound will be present in the composition as an active ingredient. In certain embodiments it may be the sole active ingredient in respect of the promotion of oral health.
  • the manner by which oral health may be promoted may be by inhibition of the activities of one or more of target organisms associated with caries or gingivitis, for example one or more organisms selected from the list consisting of:
  • compositions of the invention will generally have one or preferably more than one of the following counteractive effects against one or more of the target organisms. These counteractive effects include the ability to:
  • acidogenicity e.g. as measured following sucrose rinse.
  • oral surface is meant the hard and soft tissues of the oral cavity.
  • hard tissues refers to tissues such as the teeth and periodontal support in an oral cavity, such as that of a mammal.
  • Soft tissues refers to tissues such as the gums, the tongue, the surfaces of the buccal cavity and the like.
  • the composition contacts the dental enamel and ⁇ or gingival surface.
  • compositions of the invention may be applied with the purpose of reducing inflammation or disease (gingivitis, periodontitis) in soft tissues or hard tissues (e.g. caries and/or sensitivity) and ⁇ or inhibiting or preventing the accumulation of calculus, plaque, and tartar on the hard tissues e.g. dental enamel.
  • compositions may be those which are effective in these activities where used on a daily basis.
  • Compositions and extracts for use in the aspects and embodiments of the present invention may be prepared, by way of non-limiting disclosure, as described in the
  • a preferred extract for use in the present invention may be that obtainable by
  • a so-called low molecular mass (LMM) fraction for use in the present invention may be one less than around 1 , 2, 3, 4, or 5 kDa.
  • a less than about 5 kDa LMM fraction of mushroom homogenate may be that obtainable by ultrafiltration (e.g. using the Vivaflow 200 complete system with a 5,000 MW cut-off filter and retaining the diafiltrate).
  • Such LMM fractions will typically exclude enzymes, for example.
  • a so-called high molecular mass (HMM) fraction for use in the present invention may be one greater than around 1 , 2, 3, 4, or 5 kDa.
  • a >5 kDa HMM fraction of mushroom homogenate may be that obtainable by ultrafiltration (e.g. using the Vivaflow 200 complete system with a 5,000 MW cut-off filter and retaining the retentate) followed by dialysis with a 5,000 MW cut-off to further eliminate the LMM components.
  • a preferred fraction is an LMM fraction.
  • Preferred sub-fractions of the LMM fraction for use in the present invention may be those obtainable by gel filtration (see e.g. Example 8).
  • preferred sub-fractions may be one or more of those obtainable by use of a system with the following dimensions: 300 mm x 10 mm, with a stationary phase having exclusion limits 100-10000 Da and particle size 45 micron and a mobile phase of water (flow rate, 1.0 mL min-1 ).
  • Preferred sub-fractions are or correspond to M4 and M5 obtainable in this manner.
  • Preferred sub-sub-fractions of the LMM fraction for use in the present invention may be those obtainable by reverse phase HPLC semi-preparative chromatography (see e.g. Example 8).
  • preferred sub-sub-fractions may be one or more of those obtainable from sub-fractions M4 and M5 by use of a semipreparative HPLC column such as the C18 LiChrospher ® 250 * 10 mm, 10 ⁇ , using conditions: volume injected, 1 mL; column temperature, 25 °C; UV spectra were recorded in the 190- 600 nm range, and chromatograms were acquired at 210 nm. Based on such a system sub-sub-fractions may be identified with reference to Figures 6 and 7. Sub-fraction M4 produced 8 sub-sub-fractions (M4.1-8) with dry masses as shown in Example 8, section 8.3.
  • a preferred sub-sub-fraction is or corresponds to M4.7.
  • Sub-fraction M5 produced 11 sub-sub-fractions (M5.1 -1 1 ) with dry masses as shown in Example 8, section 8.3
  • a preferred sub-sub-fraction is or corresponds to M5.6.
  • M4.7 aconitic acid, adenosine and oxalic acid
  • Example 1 1 such compounds have been shown by the present inventors to have activities relevant to the improvement of oral health.
  • a preferred extract for use in the present invention may be that obtainable by
  • a so-called low molecular mass (LMM) fraction for use in the present invention may be one less than around 1 , 2, 3, 4, or 5 kDa.
  • a less than about 5kDa LMM fraction of chicory homogenate may be that obtainable by ultrafiltration (e.g. using the Vivaflow 200 complete system with a 5,000 MW cut-off filter and retaining the diafiltrate).
  • a so-called high molecular mass (HMM) fraction for use in the present invention may be one greater than around 1 , 2, 3, 4, or 5 kDa.
  • a >5 kDa HMM fraction of chicory homogenate may be that obtainable by ultrafiltration (e.g. using the Vivaflow 200 complete system with a 5,000 MW cut-off filter and retaining the retentate) followed by dialysis with a 5,000 MW cut-off to further eliminate the LMM components.
  • a preferred fraction is an LMM fraction.
  • Preferred sub-fractions of the LMM fraction for use in the present invention may be those obtainable by gel filtration (see e.g. Example 8).
  • preferred sub-fractions may be one or more of those obtainable by use of a system with the following dimensions: 300 mm x 10 mm, with a stationary phase having exclusion limits 100-10000 Da and particle size 45 micron and a mobile phase of water (flow rate, 1.0 mL min-1 ). Based on such a system the following six sub-fractions may be identified with reference to Figure 8:
  • a preferred sub-fraction is or corresponds to C1 obtainable in this manner.
  • Sub-sub-fractions Preferred sub-sub-fractions of the L M fraction for use in the present invention may be those obtainable by reverse phase HPLC semi-preparative chromatography (see e.g. Example 8).
  • preferred sub-sub-fractions may be one or more of those obtainable from sub-fractions M4 and M5 by use of a semipreparative HPLC column such as the C18 LiChrospher ® 250 * 10 mm, 10 pm, using conditions: volume injected, 1 ml_; column temperature, 25 °C; UV spectra were recorded in the 190- 600 nm range, and chromatograms were acquired at 210 nm.
  • Sub-fraction C1 produced 15 sub-sub-fractions respectively (C1.1-15) with dry masses as shown in Example 9, section 9.3
  • a preferred sub-sub-fraction is or corresponds to C1.7.
  • any one or more of these compounds may be utilised in the aspects of the invention described herein. As shown in Example 11 such compounds have been shown by the present inventors to have activities relevant to the improvement of oral health.
  • a preferred extract for use in the present invention may be that obtainable by
  • a base e.g. NaOH
  • pH e.g. to around or between pH 4 or 5
  • a so-called low molecular mass (LMM) fraction for use in the present invention may be one less than around 1 , 2, 3, or 3.5 kDa.
  • a less than about 3.5kDa LMM fraction of raspberry homogenate may be that obtainable by dialysis e.g. using dialysis material with a cut off 3,500 Da. In this way, a dialysate containing all the compounds with molecular masses lower than 3,500 Da can be obtained.
  • a so-called high molecular mass (HMM) fraction for use in the present invention may be one greater than around 1 , 2, 3, or 3.5 kDa.
  • a >3.5 kDa HMM fraction of raspberry homogenate may be that obtainable as the retentate from the dialysis described above.
  • a preferred fraction is an LMM fraction.
  • a base e.g. NaOH
  • a base may be added to the fraction to increase pH (e.g. to around or between pH 4 or 5).
  • Preferred sub-fractions of the LMM fraction for use in the present invention may be those obtainable by solid-phase extraction (see e.g. Examples below).
  • preferred sub-fractions may be one or more of those obtainable by:
  • R 2 obtainable by eluting afterwards with 25 ml of ethyl acetate
  • R 3 was obtainable by eluting afterwards with 25 ml of a methanol- Millipore grade water mixture (20%-80%)
  • R 4 was obtainable by eluting afterwards with 25 ml of a methanol- Millipore grade water mixture (50%-50%)
  • R 5 was obtainable by eluting afterwards with 25 ml of methanol A preferred sub-sub-fraction is or corresponds to R1.
  • Preferred sub-sub-fractions of the LMM fraction for use in the present invention may be those obtainable by as follows: (i) dilute 5 ml aliquot of R1 was diluted with HCI (0.01 M) and adjusted to pH 3.0 with HCI solution (4M).
  • R1 a consists of the most polar substances not retained by the Sep-Pak ® Vac 20cc (5 g) tC18 cartridge.
  • R1 b was obtained by eluting afterwards with 25 mL of Millipore grade water,
  • R1 c was obtained by eluting afterwards with 25 ml of methanol.
  • the invention this further provides processes for obtaining a composition as described above.
  • Such processes may be those used as described herein, or alternative methods of chromatography or fractionation such as will occur to those skilled in the art in the light of these processes i.e. which provide the same fractions, but by different means.
  • the process comprises: (i) providing an extract which is a less than 5kDa LMM sub fraction of Shiitake mushroom by use of chromatography to sub-fractionate the LMM fraction into sub-fractions and selecting sub-fractions corresponding to sub-fraction M4 or M5 shown in Figure 5;
  • the process comprises providing an extract which is a less than 3.5 kDa LMM sub fraction of raspberry by:
  • LMM fractions of mushroom and chicory were generally the most active in the assays relevant to gingivitis. Such activities, for example compositions of the present invention, are demonstrated in Example 3, Example 7, Example 9 and Example 10. Of the mushroom LMM sub-fractions, M4 and M5 were considered to be the most promising in the anti-gingivitis activity battery of tests.
  • C1 was considered to be the most promising in the anti- gingivitis activity battery of tests.
  • sub-sub-fraction C1.7 was found to be the most active.
  • the main constituents in sub-sub-fraction C1.7 were identified by HPLC and mass spectroscopy as: oxalic acid and quinic acid.
  • compositions for use in the invention will preferably demonstrate one or more of the following activities relevant to the promotion of oral health, and more specifically relevant to the ameliorating, controlling or reducing the risk of gingivitis. Such activities will be compared to a negative control (typically a diluent, buffer or solute) which corresponds generally to that used for the composition of the invention, but from which the active (extracts, fraction or compound) is absent.
  • a negative control typically a diluent, buffer or solute
  • Target organisms selected from the list consisting of: V. dispar, F. nucleatum, A. naeslundii, P. intermedia.
  • Preferred targets are A. naeslundii and ⁇ or P. intermedia, a most preferred target is P. intermedia.
  • Inhibitory activity against one or both of the target organisms A. naeslundii, P.
  • a preferred target is A. naeslundii.
  • Target organisms F. nucleatum, A. naeslundii, P. intermedia
  • epithelial cells Gaingival fibroblast KB cell line
  • Preferred targets are A. naeslundii and ⁇ or P. intermedia.
  • compositions of the present invention are demonstrated in Examples 4 and 6 and 8.
  • LMM fractions of mushroom and raspberry were generally the most active in the assays relevant to caries.
  • M4 and M5 were considered to be the most promising in the anti-caries activity battery of tests.
  • M4.7 aconitic acid, adenosine and oxalic acid
  • compositions for use in the invention will preferably demonstrate one or more of the following activities relevant to the promotion of oral health, and more specifically relevant to the ameliorating, controlling or reducing the risk of caries.
  • a preferred target organism is S. mutans.
  • Inhibitory activity against one or more of the target organisms L. casei, S. mutans, S. sanguinis, A. naeslundii.
  • S. mutans Ability to disrupt biofilms of one of both of the target organisms: S. mutans, L. casei.
  • a preferred target organism is S. mutans.
  • succinic acid and oxalic acid both had a beneficial effect.
  • preferred compounds are quinic acid, succinic acid, trans-aconitic acid and inosine, with quinic acid and succinic acid being most preferred.
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as ⁇ 3 .
  • suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
  • suitable substituted ammonium ions are those derived from:
  • ethylamine diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
  • a salt may be formed with a suitable anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous,
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic (which itself may be an active in the composition, as described above) sulfanilic, tartaric, toluenesulf
  • methods of the invention include: A method for assessing the potential oral health benefits of a plant or fungus, the method comprising assessing the level of a compound in the plant or fungus, wherein the compound is selected from the list consisting of: quinic acid; adenosine; inosine; shikimic acid; trans-aconitic acid; oxalic acid; adenosine; uridine; cis-aconitic acid and succinic acid.
  • the compound is selected from the list consisting of: quinic acid; succinic acid, trans-aconitic acid; inosine.
  • quinic acid Preferably it is quinic acid or succinic acid.
  • plants or fungi including enhanced levels of the compound are selected, and may then be consumed or used to prepare compositions according to the present invention.
  • Assessment of the compounds may be done by conventional methods - for example by HPLC and mass spectroscopy. Description of certain aspects and embodiments
  • aspects of the invention include a composition for promoting oral health, which composition is a functional food or oral preparation which comprises one or more extracts of a natural product selected from the list consisting of: Shiitake mushroom, chicory, or raspberry.
  • the composition is for one or more of ameliorating, controlling or reducing the risk of dental caries or periodontal disease, which periodontal disease is optionally gingivitis or periodontitis.
  • the extract may be obtained or obtainable as described above by homogenization of the natural product and filtering to remove solid matter.
  • a preferred extract is of a low molecular mass (LMM) fraction of the natural product comprising compounds of less than around 1 , 2, 3, 3.5, 4, 4.5 or more preferably 5 kDa.
  • the extract is of a less than 5kDa LMM sub fraction of Shiitake mushroom obtainable by gel filtration in a 300 mm x 10 mm column, with a stationary phase having exclusion limits 100-10000 Da and particle size 45 micron and a mobile phase of water having a flow rate, 1.0 ml/min, such as to sub-fractionate the LMM fraction into 5 sub-fractions as follows and selecting the sub- fractions M4 and M5 peaking at about 106 mins or about 154 mins.
  • a preferred extract is of an LMM sub-sub-fraction of Shiitake mushroom obtainable from sub-fraction M4 by use of a semipreparative HPLC column with the following properties: C18, 250 x 10 mm, 10 ⁇ and conditions: volume injected, 1 mL; column temperature, 25°C; UV spectra recorded in the 190-600 nm range, and chromatograms were acquired at 210 nm, such as to sub-fractionate the LMM fraction into 8 sub-fractions in accordance with Figure 6 and selecting the sub-sub-fraction M4.7.
  • Another preferred extract is of an LMM sub-sub-fraction of Shiitake mushroom obtainable from sub-fraction M5 by use of a semipreparative HPLC column with the following properties: C18, 250 * 10 mm, 10 ⁇ and conditions: volume injected, 1 mL; column temperature, 25°C; UV spectra recorded in the 190-600 nm range, and chromatograms were acquired at 210 nm, such as to sub-fractionate the LMM fraction into 11 sub- fractions in accordance with Figure 7 and selecting the sub-sub-fraction M5.6.
  • the extract is of a less than 5kDa LMM sub fraction of chicory obtainable by gel filtration in a 300 mm x 10 mm column, with a stationary phase having exclusion limits 100-10000 Da and particle size 45 micron and a mobile phase of water having a flow rate, 1.0 ml/min, such as to sub-fractionate the LMM fraction into 5 sub-fractions as follows and selecting the sub-fraction C1 peaking at between about 37 mins and 41 mins.
  • a preferred extract is of an LMM sub-sub-fraction of chicory obtainable from sub-fraction C1 by use of a semipreparative HPLC column with the following properties: C18, 250 * 10 mm, 10 pm and conditions: volume injected, 1 mL; column temperature, 25°C; UV spectra recorded in the 190-600 nm range, and chromatograms were acquired at 210 nm, such as to sub-fractionate the LMM fraction into 15 sub-fractions in accordance with Figure 9 and selecting the sub-sub-fraction C1.7.
  • the extract is of a less than 3.5 kDa LMM sub fraction of raspberry obtainable by:
  • compositions for promoting oral health may be a functional food or oral preparation which has been supplemented with a compound selected from the list consisting of: quinic acid; adenosine; inosine; shikimic acid; trans- aconitic acid; oxalic acid; adenosine; uridine; cis-aconitic acid and succinic acid.
  • a preferred list consists of: quinic acid; succinic acid, trans-aconitic acid; inosine.
  • Preferred compounds are quinic acid and succinic acid.
  • the compositions will generally demonstrate 1 or more (2, 3, 4, 5, 6, or 7) of the following activities relevant to the promotion of oral health:
  • a preferred target is A. naeslundii;
  • inhibitory activity against one or more of the target organisms L casei, S. mutans, S. sanguinis, A. naeslundii;
  • extracts or other relevant compounds of the present invention may be provided in convenient formats for direct use, or for incorporation into functional foods or other orally acceptable form. As demonstrated in the Examples below, one convenient format may be a so-called
  • microbead in which the relevant compound is mixed with a polymer solution, or polymer- forming solution, which is then cross-linked with the compound microencapsulated in situ (e.g. via a process such as ionotropic gelation - see e.g. J Microencapsul. 2001 Mar- Apr; 18(2):237-45. ) Such beads can be filtered and dried and incorporated into orally acceptable compositions. As demonstrated in the Examples below, they can bind well to mucosal membranes and hydrozyapatite.
  • Preferred beads are prepared from chitosan or an alginate. These are available commercially e.g. in the ProtanalTM range. Preferred ratios and compositions are described in Example 14 below, especially Tables 1 1-13.
  • Preferred beads are between 100 and 10000 pm in diameter e.g. in a population of dried beads having an average size of between 500 and 2000.
  • extracts and active constituents could be successfully encapsulated into micro-beads which were resistant to mechanical damage and showed good adhesion to mucosa and to hydroxyapatite. These could be incorporated into functional foods (such as chewing gum) and released in the buccal cavity.
  • the composition may be a functional food or oral preparation supplemented with the extract or compound in pure form such as to promote the oral health benefits or effects of the oral composition.
  • the extract or compound may be present as the sole active ingredient in respect of the promotion of oral health, or other compounds (known to those skilled in the art) may be present to promote this effect.
  • compositions include those selected from: a foodstuff, beverage, chewing gum, toothpaste, mouthwash, mouth rinse, toothpowder or tooth gel. Examples of such compositions (absent the extracts or compounds of the invention) are well known to those skilled in the art.
  • Formulations or functional foods containing succinic acid would preferably contain equal to or at least 0.5 pg/ml of succinic acid.
  • Formulations or functional foods containing quinic acid would preferably contain equal to or at least 3.5 mg/ml of quinic acid.
  • Formulations or functional foods containing trans-aconitic acid would preferably contain equal to or at least 5.0 g/ml of trans-aconitic acid.
  • Formulations or functional foods containing inosine acid would preferably contain equal to or at least 1.0 mg/ml of inosine.
  • Formulations or functional foods containing adenosine would preferably contain equal to or at least 2.0 mg/ml of adenosine.
  • Formulations or functional foods containing epicatechin would preferably contain equal to or at least 2.5 mg/ml of epicatechin.
  • Formulations or functional foods containing cis-aconitic acid would preferably contain equal to or at least 50 pg/ml of cis-aconitic acid.
  • Formulations or functional foods containing uridine would preferably contain equal to or at least 5.0 mg/ml of uridine.
  • Formulations or functional foods containing oxalic acid would preferably contain equal to or at least 0.6 Mg/ml of oxalic acid.
  • Formulations or functional foods containing shikimic acid would preferably contain equal to or at least 0.7 g/ml of shikimic acid.
  • extracts may be provided as described above.
  • these may be further concentrated for commercial use - e.g. 2, 3, 4, or 5 times the LMM fraction concentration obtained using the extraction procedure described above.
  • a preferred mouthwash used in the Examples below includes an extract or compound of the invention (e.g. LMM shiitake mushroom extract) and other ingredients such as sodium fluoride, as well as colouring and flavorings, such as artificial sweetener.
  • Lozenge Tablet e.g. LMM shiitake mushroom extract
  • other ingredients such as sodium fluoride, as well as colouring and flavorings, such as artificial sweetener.
  • Typical ingredients will include a "gum base” with added sweeteners, softeners, and flavoring.
  • Chewing gum base is typically a complex mixture of ingredients: elastomers, resin plasticizers, minerals, waxes, lipids, and emulsifiers. Elastomers provide elasticity and texture. They include natural rubber, natural gums, and styrene-butadiene rubber and polyvinylacetate. Plasticizers act as softening agents and along with minerals, regulate cohesiveness. A non-limiting example is given in WO0042861 (Wrigley). The resulting product is to be chewed in the usual manner.
  • chewing gums including active ingredients of the invention may be prepared using standard techniques.
  • the gum base may be was softened by heating and optionally glycerol was added.
  • Micro-beads or another suitable formal may be uniformly incorporated into the softened gum-glycerol mixture.
  • the resulting chewing gums obtained can then be molded into a desired shape (e.g. rod) and cooled to room temperature.
  • a chewing gum unit may for example be between 200 and 1000mg e.g. around 500 mg.
  • the ratio of active ingredient to gurmplasticiser may optionally be between 0.1 - 1 : 1 , depending on the strength of the active and its loading into the relevant format (e.g. micro-beads).
  • the chewing gum will release the active ingredient efficiently on chewing e.g. >50% within 5 minutes, and most preferably >90 or 95% within 20 mins.
  • micro- beads are used these will preferably be largely resistant to mechanical damage such as to permit them to be released intact during chewing. As shown in the Examles, micro- beads of the invention could be released undamaged within 20 minutes of chewing (a generally accepted measure of time that chewing gum is typically retained in the oral cavity).
  • beneficial counteractive effects of the compounds described herein compared to controls in some cases may only be achieved after use of the compositions over a treatment period of days or weeks - for example a period of equal to or exceeding 1 , 2, 3, 4, 5, 6, 7 days, or 1 , 2, 3, 4 weeks and so on.
  • Figure 2 Effect of chicory homogenate and mushroom homogenate on IL-6 release induced by F. nucleatum supernatant.
  • Figure 3 Effect of chicory homogenates on IL-6 release induced by F. nucleatum supernatant.
  • Figure 4 Effect of mushroom homogenates on IL-6 release induced by F. nucleatum supernatant.
  • Figure 7 Sub-fractionation of mushroom homogenate fraction 5 to sub-sub-fractions respectively (M5.1-5.1 ) by HPLC.
  • Figure 8 Fractionation of LMM chicory homogenate by gel filtration to fractions C1 -C6.
  • Figure 9 Sub-fractionation of chicory homogenate fraction C1 to sub-sub-fractions respectively (C1.1-1.15) by HPLC.
  • Figure 1 1a) - j) - Biological activities against A. naeslundii of isolated compounds identified in mushroom and chicory extracts:
  • Figure 12 shows a schematic schedule of a randomized, double-blind controlled trial on the effects of a mouthrinse containing shiitake mushroom extract on dental plaque, plaque acidogenicity and the plaque microbiota
  • Figure 15 shows changes in plaque-pH after a mouthrinse with 10% sucrose after baseline and the three washout periods (after Shiitake, after Water and after Meridol).
  • Figure 17 shows the proportions of total streptococci vs total flora (left) respective mutans streptococci vs total streptococci (right) in saliva expressed as % (mean) after the three test periods (Shiitaki, Water, Meridol) and the baseline and three washout periods (after Shiitake, after Water and after Meridol).
  • n 30.
  • Figure 18 shows mean protein concentration ( ig) in resting (pre-sucrose rinse) and fermenting (post-sucrose rinse) after the three test periods (Shiitaki, Water, Meridol) and the baseline and three washout periods (after Shiitake, after Water and after Meridol).
  • n 28.
  • Figure 19 shows mean amount of A) acetate, B) lactate and C) minor acids in resting
  • Figure 21 shows the distribution of plaque scores among the various groups during the course of the study described in Example 13.
  • the order is (left to right) scores PS1 , PS2, PS3, with the leftmost on each day being mushroom, then negative control, then positive control.
  • Figure 22 shows the mean plaque scores of the various groups at different time points in the study of Example 13.
  • Figure 23 shows the gingival index scores for the various groups during the study (bars ordered as per Figure 21).
  • Figure 24 shows the mean gingival index scores of the various groups at different time points in the study.
  • Figure 25 shows the percentages of F. nucleatum in plaque samples from the negative control group (pink, squares ⁇ ), the positive control group (yellow triangles, A) and the group administered mushroom extract (blue diamonds, 0). The same coding is used in Figs. 26-32 below.
  • Figure 26 shows the percentages of L casei in plaque samples from the negative control group (pink), the positive control group (yellow) and the group administered mushroom extract (blue).
  • Figure 27 shows the percentages of V. dispar in plaque samples from the negative control group (pink), the positive control group (yellow) and the group administered mushroom extract (blue).
  • Figure 28 shows the percentages of N. subflava in plaque samples from the negative control group (pink), the positive control group (yellow) and the group administered mushroom extract (blue).
  • Figure 29 shows the percentages of A. naeslundii in plaque samples from the negative control group (pink), the positive control group (yellow) and the group administered mushroom extract (blue).
  • Figure 30 shows the percentages of P. intermedia in plaque samples from the negative control group (pink), the positive control group (yellow) and the group administered mushroom extract (blue).
  • Figure 31 shows the percentages of S. sanguinis in plaque samples from the negative control group (pink), the positive control group (yellow) and the group administered mushroom extract (blue).
  • Figure 32 shows the percentages of S. mutans in plaque samples from the negative control group (pink), the positive control group (yellow) and the group administered mushroom extract (blue).
  • Figure 33 shows a scheme for preparation of micro-beads containing active constituents as described in Example 14.
  • Figure 34 shows the evaluation of in vitro mucoadhesion ability of various quinic acid- containing micro-beads. The results are expressed as the percentage of micro-beads adhering to the model membrane (pork mucosa) after 4 hours. The Batches in the list are shown left to right in the chart.
  • Figure 35 show the evaluation of the ability of various quinic acid-containing micro-beads to adhere to hydroxyapatite. The results are expressed as the percentage of micro-beads adhering to 23 mm diameter hydroxyapatite discs after 4 hours. The Batches in the list are shown left to right in the chart.
  • Figure 36 shows the quinic acid release from the various quinic acid-containing micro- beads
  • Figure 37 shows the adhesion of micro-beads containing LMM shiitake mushroom extract to pork mucosa and hydroxyapatite. The results are expressed as the percentage of micro-beads adhering after 4 hours.
  • Figure 38 shows a photograph of chewing gum containing micro-beads loaded with LMM shiitake mushroom extract. Examples
  • the present inventors have evaluated a number of plant-derived foods and beverages not previously identified as having anti-caries and/or anti-gingivitis potential using relevant bioassays. Promising foods/beverages have been fractionated and re-tested in these assays in an iterative process which has resulted in the identification of the active constituents.
  • Suitable types/strains of the chosen foods/beverages were selected and providers of these identified. Homogenates/extracts of the selected foods/beverages were prepared and chemically analysed to provide material suitable for subsequent investigations by the other partners.
  • Protein determination The Kjeldahl method was used. This is the standard method of nitrogen determination (AOAC official methods) consisting of three different steps:
  • Carbohydrate determination glucose, fructose, and sucrose were determined using an enzymatic assay (D-glucose concentration was determined before and after the enzymatic hydrolysis of sucrose; D-fructose was determined subsequent to the determination of D-glucose).
  • the other sugars mannose, ramnose, maltose, xylose, etc
  • the results obtained from these analyses are shown below:
  • Mushroom juice was prepared by homogenisation of frozen fungi
  • Beer Guinness beer was de-alcoholated For each food/beverage an operative protocol was prepared. This enabled the identification of the critical points that could influence the chemical composition of the extracts and therefore their biological properties.
  • the juice after separation from the solid part, was filtered (paper filter). Ultrafiltration was impossible (due to obstruction of the membrane) but, because of the acidic pH value, the juice was microbiologically uncontaminated. Given the highly acid nature of the juice an appropriate volume of NaOH was added in order to obtain a higher pH value (4.82), freeze-dried and tested for microbiological contamination.
  • Mushroom aliquots (400 g) of frozen fungi were homogenized (for 2 minutes) and centrifuged (for 10 minutes at 8000 rpm). The juice, after separation from the solid part, was filtered (paper filter) and then sterilised by ultrafiltration. The sterile juice was tested for microbiological contamination, freeze-dried and re-tested for microbiological contamination.
  • Red chicory aliquots (500 g) of fresh vegetable were homogenized (for 1 min) and centrifuged (for 10 minutes at 8000 rpm). The juice, after separation from the solid part, was filtered on paper filter and then sterilised by ultrafiltration. The sterile juice was tested for microbial contamination, freeze-dried and re-tested for microbiological contamination. Beer: aliquots (325 ml) of Guinness beer were submitted to elimination of C0 2 (AOAC official methods) and de-alcoholated (bath temperature: 50°C, vacuum: 30 bar for 20 min). The resulting beverage was tested for microbiological contamination, freeze-dried and re-tested for microbiological contamination.
  • Protein, sucrose, glucose, and fructose content and minor components were determined in the homogenates/extracts as described above. The results obtained from these analyses are shown below
  • Lactobacillus case/) or under anaerobic conditions P. intermedia, A. naeslundii and V. dispar.
  • Bacterial suspensions were prepared in the appropriate growth medium containing different concentrations of the test material (pH adjusted to 7). The final concentration of bacteria was either 3-5 x 10 5 cfu ml_ '1 (S. mutans, S. sanguinis, L. casei, V. dispar and A. naeslundii) or 5-8 x 10 6 cfu ml "1 (P. intermedia). Aliquots (200 ⁇ ) of the cell suspensions were inoculated into the wells of 96-well polystyrene microtiter plates.
  • test material-untreated controls were included. Plates were then incubated at 37°C up to one week in either 5% C0 2 /air (S. mutans, Streptococcus sanguinis and Lactobacillus casei) or anaerobic conditions (P. intermedia, A. naeslundii and V. dispa , with incubation media changed every 24 h and every 48 h for aerobic and anaerobic bacteria, respectively. Biofilm formation was quantified after 48 h and 7 day incubation. To this end, the growth medium was removed by aspiration, wells were gently washed with water and air dried; adherent bacteria were then stained with 0.01 % crystal violet (100 ⁇ ).
  • Biofilm formation was quantified by measuring the absorbance of the solution at 540 nm. Biofilm inhibitory activity was evaluated as a proportion of untreated controls (100%).
  • Bacteria were grown in 5 ml tubes at 37°C either aerobically at ambient air or under anaerobic conditions (GasPack Anaerobic System, Becton and Dickinson) in Brain Heart Infusion (BHI) broth. After overnight growth, the bacterial culture was diluted in broth to contain 10 5 cfu/mL. Two-fold dilutions of test samples and fractions in 0.1 ml of BHI broth were placed into wells of flat-bottomed microtitre plates (Nunc 96-well flat-bottomed microtitre plates; Nunc, Roskilde, Denmark). A 10 ⁇ volume of bacterial culture was then added.
  • the MICs were determined. The MICs were recorded as the lowest concentration or dilution of test sample or fraction that completely inhibited visible growth of the bacteria,
  • Coaggregation is an important factor when complex biofilm communities are being studied. Important relationships exist between certain strains which allow aggregation and biofilm formation. All combinations of the strains used were tested for co-aggregation activity and the following were used in subsequent assays; S. sanguinis & P. intermedia, S. sanguinis & F. nucleatum, N. subflava & F. nucleatum.
  • the homogenates/extracts were assayed for their ability to inhibit co-aggregation as described below: Bacteria were grown in 5 ml tubes at 37°C either aerobically at ambient air or under anaerobic conditions (GasPack Anaerobic System, Becton and Dickinson) in BHI broth . After overnight growth, cells were harvested, washed with coaggregating buffer (1 millimolar tris (hydroxy-methyl) aminomethane; 0.1 mmol/L magnesium chloride; 0.1 mmol/L sodium chloride; 0.02 percent sodium azide adjusted to pH, 8.0), adjusted to an optical density of 1.5 at 400 nanometers ( UV-Vis. Spectrophotometer) and stored at 4°C until use.
  • coaggregating buffer (1 millimolar tris (hydroxy-methyl) aminomethane; 0.1 mmol/L magnesium chloride; 0.1 mmol/L sodium chloride; 0.02 percent sodium azide adjusted to pH, 8.0
  • test sample or fraction to inhibit co-aggregation of selected pairs of bacteria was tested by adding equal volumes (0.05ml) of bacterial suspension of one pair to equal volume of serial two fold dilution of test sample or fraction in co-aggregating buffer followed by adding equal volume of the bacterial suspension of the other co-aggregating member in 12x75 millimeter test tube. After vigorous vortex of the mixture and further incubation at room temperature for 2 minutes co-aggregation was scored. The last dilution of the sample causing complete inhibition of co-aggregation was recorded and expressed either as final concentration (w/v) or as percent of undiluted sample.
  • Biofilms which build-up in low-shear environments such as those in interproximal regions and plaque within gingival margins are able to become well established climax communities. These mature biofilms are more resistant to antimicrobials and antibiotics than biofilms forming in high-shear systems.
  • Mature biofilms of each of the test organisms were grown on cellulose nitrate membrane filters and incubated with the test compounds for 1 minute. The number of live and dead cells disrupted from the biofilm was assessed as well as the number of live and dead cells remaining. The protocol used is described below:
  • Membrane filters were carefully transferred into 750 ⁇ of the selected test or control agent (in triplicate) in separate microfuge tubes and incubated for 1 min.
  • the positive control consisted of 1.75 mM sodium dodecyl sulphate (SDS) and the negative of reduced transport fluid (RTF). After a contact time of 1 minute was achieved, membrane filters were transferred to 1 ml Neutralising Broth (diluted to a final concentration of 10 % v/v in RTF) and agitated at 200 rpm for 20 s. Membrane filters were transferred to 1 ml RTF and vortexed at full speed for 1 min.
  • SDS sodium dodecyl sulphate
  • RTF reduced transport fluid
  • the original suspension in Neutralising Broth was centrifuged at 13000 rpm for 1 min and the pellet resuspended in 1 ml RTF.
  • BacLight LIVE/DEAD viability stain (Molecular Probes) was used to distinguish between viable and non-viable cells as follows according to manufacturer's instructions. 5 ⁇ dye suspension was placed onto a glass slide beneath a 13 mm circular coverslip in preparation for counting. Both live (green) and dead (red) cells were counted at five locations on the slide to determine the mean number of cells. From this data the total number of live and dead cells attached or detached from the nitrocellulose filter was calculated.
  • the table shows the proportion (%) of cells disrupted from the biofilm after 1 minute incubation with the extracts. Numbers in parentheses indicate the percentage of dead cells. Chicory Mushroom Beer Raspberry
  • homogenates/extracts showed some ability to disrupt biofilms of the target organisms. In many cases, a high proportion of the disrupted organisms were killed by the homogenates/extracts.
  • BHIB Brian heart Infusion broth
  • Streptococcus mutans Streptococcus mutans which was grown in BHIB (0.5x) supplemented with sucrose (final concentration, 0.2%).
  • Cultures were incubated at 37"C in 5% C0 2 /air (S. mutans, S. sanguinis and L casei) or under anaerobic conditions (P. intermedia, A. naeslundii and V. dispar).
  • 10 ⁇ [methyl- 3 H]thymidine 25 Ci mmol "1 ) mL "1 were added to the growth medium.
  • Cells were harvested at stationary phase by centrifugation (5,000 x g for 10 min at 4°C), and washed twice with 10 mM phosphate buffer (PB), pH 7.0; pellets were re-suspended in either 10 mM PB, pH 7.0, or BHIB or phosphate buffered saline (PBS: 0.1 M Na 2 HP0 4 , 0.1 M KH 2 P0 4 , 0.15 M NaCI, pH 7.2 to 7.4), depending on the test to be performed. Cell bound radioactivity was quantified with a liquid scintillation counter. Cell labeling efficiency (number of bacteria per count per min) was then determined.
  • PB phosphate buffer
  • BHIB phosphate buffered saline
  • Gingival fibroblast KB cell line (accession number ICLC HTL96014) obtained from Cell bank Interlab Cell Line Collection (ICLC) of IST-lstituto dei Tumori di Genova (Genoa, Italy) was cultured in a complete medium consisting of Dulbecco's Modified Eagle's Medium (DMEM)- high glucose, with 4500 mg L ' glucose and sodium bicarbonate supplemented with 10% fetal calf serum, penicillin (100 U mL '1 ), streptomycin (100 ig mL " 1 ), and 2 mM L-glutamine. Cells were incubated at 37°C in a 5% C0 2 atmosphere to about 90% confluence and used after 5-10 passages. For bacterial adherence
  • KB monolayers were prepared in 96 well, flat bottom microtiter plates, using Dulbecco's Modified Eagle's Medium (DMEM)- high glucose prepared as described above without antibiotics; before the assay, monolayers were washed twice with PBS (0.1 M Na 2 HP0 4 , 0.1 M KH 2 P0 4 , 0.15 M NaCI, pH 7.2 to 7.4).
  • DMEM Dulbecco's Modified Eagle's Medium
  • Suspensions of labeled bacteria were prepared in PBS containing different concentrations of test materials (pH adjusted to 7) (final bacterial concentration, 4-6 x 10 8 cfu mL "1 ). Aliquots (100 ⁇ ) of the bacterial suspensions were added to KB monolayers, and incubated at 37°C for 1 h in 5% C0 2 atmosphere with gentle shaking. For each strain, untreated controls were included. After incubation, cells were disrupted by adding 200 ⁇ of cold distilled water, and lysates were transferred to PICO-FLUORTM 15 scintillation fluid (Packard Instruments
  • the tested substances did not show cytotoxicity with the exception of mushroom. This was therefore used at 0.5x concentration (sub-cytotoxic).
  • pretreat. pretreat. a) pretreat. pretreat.
  • pretreat. pretreat. a) pretreat. pretreat.
  • homogenates/extracts (mushroom was an exception) were able to inhibit adherence of the target organisms to epithelial cells.
  • KB monolayers were prepared in 16 mm well of 24-well tissue culture plates, in
  • Dulbecco's Modified Eagle's Medium (DMEM)- high glucose prepared as above without antibiotics; before the assay, monalayers were washed twice with PBS.
  • suspensions (P. intermedia and A. naeslundii) were prepared in KB cell growth medium without antibiotics, containing different concentrations of the test materials (pH adjusted to 7) (final bacterial concentration, 6-8 x 10 7 cfu mL and added (1 ml) to monolayers.
  • the inhibitory activity of the test materials was gauged by comparing fraction treated samples to the respective untreated controls. Each strain was tested in three separate assays on different days; each assay represented the average of triplicate wells. Controls without bacteria were always included to evaluate KB cell viability by Trypan blue assay in the presence of the test materials. The P. intermedia strain, in control tests, presented a low internalization capability or no internalization at all making it impossible to evaluate the effect of substances.
  • the pro-inflammatory cytokines (IL-1 , IL-6 and TNF) released by host cells in response to sub-gingival bacteria are considered to be the primary mediators of the inflammation accompanying gingivitis. Compounds able to prevent such cytokine production will, therefore, help to maintain the gingival tissues in a healthy state.
  • monoMac 6 cells a human monocytic cell line
  • the ability of the test materials to inhibit cytokine production by monoMac 6 cells (a human monocytic cell line) in response to bacteria (S. sanguinis, A. naeslundii, F. nucleatum and P. intermedia) was evaluated.
  • the myelomonocytic cell line Mono-Mac-6 was maintained in RPMI-1640 medium containing 2 mM L-glutamine, 5% heat-inactivated FCS, insulin (9 mg/ml), oxaloacetic acid (1 mM), sodium pyruvate (1 mM) and nonessential amino acids (0.1 mM, Sigma).
  • Mono-Mac-6 cells were centrifuged at 1500rpm for 5 min and resuspended in media with 2% (v/v) FCS.
  • Viable cells were dispensed into 24 well tissue culture plates at 2x10 6 /500 ⁇ /well.
  • test or control agent in triplicate was then added to cells neat and at dilutions of 1 :10 and 1 :100.
  • Bacterial strains were inoculated into 10ml of the appropriate broth and grown in appropriate conditions
  • Bacterial cultures were then diluted in fresh broth and grown to exponential growth stage, as determined spectrophotometrically.
  • Mono-Mac-6 cell numbers for each experiment were determined by centrifugation of contents of tissue culture plate well and the cells counted using a haemocytometer.
  • Bacteria were centrifuged onto the monolayer at 2000rpm for 10 minutes at room temperature and then plates incubated at 37°C in an atmosphere containing 5% C0 2 for 5 h.
  • cytokine mRNA was extracted from stimulated cells using the Qiagen RNeasy Protect Cell Mini Kit, as per manufacturer's instructions.
  • RNA in 15 ⁇ of DEPC-treated water from each sample was used.
  • OligodT (Sigma Genosys, UK) were added and samples heated to 70°C for 10 minutes to denature the RNA.
  • master mix I After addition of master mix I, samples were transferred to 42°C and incubated for 2 minutes. 5 ⁇ of master mix II; 0.5 ⁇ Superscript II reverse transcriptase (Gibco BRL, UK) and 4.5 ⁇ DEPC-treated water were then be added and the samples incubated at 42°C for a further hour.
  • transcriptase then diluted 1 :4 with DEPC water and stored at -20°C.
  • Tubes will be gently vortexed and placed into an automated DNA thermal cycler with a heated lid (Eppendorf Mastercycler).
  • the gel will be cooled to hand temperature and 10 ⁇ of ethidium bromide (500 ⁇ g/ml (Sigma, UK) added. The gel will carefully be poured into a gel casting unit, and the appropriate sized comb then placed into the unit and the gel allowed to set for 1 hour.
  • sample buffer Promega
  • PCR product 20 ⁇ will be loaded into the wells and run for 60 minutes at 100 volts (50 mAmps). Gels will be visualised and photographed under UV illumination, using an Alphalmager photo system (Alpha Innotech, Cannock, UK).
  • nucleatum displayed the greatest IL-6 inducing activity and therefore was used in subsequent experiments.
  • homogenate (at a 1 : 10 dilution) were able to inhibit IL-6 release induced by the F.
  • the capability of the selected homogenates/extracts to prevent bacterial adhesion to HA beads was evaluated following three experimental approaches: a) the tested compound and the radiolabelled bacterial suspensions were added simultaneously to saliva coated beads; b) saliva coated beads were pretreated with the tested compounds; c) labeled bacteria grown in THB supplemented with the test material (at 1 ⁇ 2 MIC) were added to the beads.
  • BHIB Brain heart Infusion broth
  • Streptococcus mutans Streptococcus mutans which was grown in BHIB (0.5x) supplemented with sucrose (final concentration, 0.2%). Cultures were incubated at 37°C in 5% C0 2 /air (S. mutans, Streptococcus sanguinis and Lactobacillus casei)
  • 0 ⁇ [methyl- 3 H]thymidine (25 Ci mmol "1 ) mL "1 were added to the growth medium.
  • spheroidal HA beads Fifty mg aliquots of spheroidal HA beads (Sigma Aldrich, UK, code 21223) were washed with 1 mM PB, pH 7.0, in glass tubes and autoclaved. Beads were collected by centrifugation (100x g, 1 min, 4°C) and equilibrated in 1 mM PB, pH 7.0 (1 h at room temperature). HA was then treated (1 h at room temperature) with 200 ⁇ _ undiluted saliva, which was collected from un-stimulated donors, clarified by centrifugation (15,000 x g for 30 min at 4°C), and sterilized through 0.22 ⁇ nitrocellulose membrane filters. Beads were then collected by centrifugation as above, and washed with 10 mM PB, pH 7.0.
  • Untreated control samples were included. Other samples were included to assess total HA-bound bacteria, as described above. Immediately after beads re-suspension (time zero) and after 1 and 2 h incubation, the mixtures were centrifuged (200 x g, 5 min, 4°C), and the supematants were transferred to PICO-FLUORTM 15 scintillation fluid. Radioactivity was assayed in a liquid scintillation counter and, on the basis of cell labeling efficiency, the number of bacteria present in the supernatant, corresponding to detached cells, was evaluated. The percent of detached vs. total HA-bound bacteria was determined. The effect of the tested compounds was evaluated by comparing treated samples with the untreated controls. Experiments were run in triplicate and were performed at least twice.
  • the capability of the homogenates/extracts to prevent biofilm formation was evaluated by the microtitre plate assay.
  • Bacterial suspensions were prepared in the appropriate growth medium containing different concentrations of the test material (pH adjusted to 7). The final concentration of bacteria was either 3-5 x 10 5 cfu ml. '1 (S. mutans, S. sanguinis, L. casei Aliquots (200 ⁇ ) of the cell suspensions were inoculated into the wells of 96-well polystyrene microtiter plates. For each strain, test material-untreated controls were included. Plates were then incubated at 37°C up to one week in 5% C0 2 /air (S. mutans, S. sanguinis and L. casei)), with incubation media changed every 24 h . Biofilm formation was quantified after 48 h and 7 day incubation.
  • the growth medium was removed by aspiration, wells were gently washed with water and air dried; adherent bacteria were then stained with 0.01% crystal violet (100 ⁇ ). After 15 min incubation at room temperature, wells were gently washed with water, and bound dye was extracted from stained cells by adding 200 ⁇ of ethanol: acetone (8:2). Biofilm formation was quantified by measuring the
  • Biofilm inhibitory activity was evaluated as a proportion of untreated controls (100%). Experiments were run in triplicate and were performed at least twice.
  • Coaggregation is an important factor when complex biofilm communities are being studied. Important relationships exist between certain strains which allow aggregation and biofilm formation. All combinations of the strains used were tested for coaggregation activity and the following were used in the assays: S. mutans & L casei, S. mutans & S. sanguinis
  • chicory, mushroom, beer and cranberry inhibited co-aggregation of at least one pair of organisms to some extent.
  • Tri-Reagent Sigma-Aldrich, St. Louis, MO, USA (Tarn et al. 2006 J Antimicrob Chemother. 57(5): 865-71).
  • the bacteria were disrupted with the aid of glass beads (Sigma-Aldrich) in a Fast Prep cell disrupter (Bio 101 , Savant Instrument Inc., NY, USA).
  • the suspensions obtained were centrifuged and the RNA-containing supernatant was transferred to a new microcentrifuge tube.
  • the homogenate was supplemented with BCP-phase separation reagent (Molecular Research Center, Cincinnati, OH, USA), and the upper aqueous phase, containing the RNA, was precipitated with isopropanol.
  • RNA pellet was washed with ethanol, centrifuged and the purified RNA was resuspended in diethyl pyrocarbonate-treated water (Invitrogen, Carlsbad, CA, USA).
  • RNA concentration was determined spectrophotometrically according to the
  • RNA sample was incubated at 65°C for 5 min and then placed on ice. 3.
  • the reaction mix (20 ⁇ ) containes 1 ⁇ of the cDNA sample and 0.5 ⁇ of the appropriate PCR primer.
  • the cycle profile was as follows: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 1 min, 30 cycles at 95°C for 15 sec, and at 60°C for 1 min, following a dissociation stage: a 15 sec hold at 95°C for, and at 20 sec for 20 sec, and a slow ramp (20 min) from 60 to 95°C.
  • the critical threshold cycle (C r ) was defined as the cycle at which fluorescence becomes detectable above the background and is inversely proportional to the logarithm of the initial number of template molecules.
  • a standard curve was plotted for each primer set with C T values obtained from amplification of known quantities of cDNA from S. mutans GS5. The standard curves was used for transformation of the C T values to the relative number of cDNA molecules.
  • the comC/D/E primers were designed by using the algorithms provided in Primer Express (Applied Biosystems). For each set of primers a standard amplification curve was drawn. Only curves with slope ⁇ -3 were accepted as reliable primers.
  • the primer set 16S-F/R corresponding to the 16S rRNA gene of S. mutans (Acc. No. X58303) was designed to correspond to the expression of the housekeeping gene.
  • Biofilms which build-up in low-shear environments such as those in interproximal regions and plaque within gingival margins are able to become well established climax communities. These mature biofilms are more resistant to antimicrobials and antibiotics than biofilms forming in high-shear systems.
  • mature biofilms of each of the test organisms were grown on cellulose nitrate membrane filters and incubated with the test compounds for 1 minute. The number of live and dead cells disrupted from the biofilm were assessed as well as the number of live and dead cells remaining.
  • the table shows cells disrupted from the biofilm after 1 minute incubation with the extracts. Numbers in parenthesis indicate the percentage of dead cells.
  • Caries is a multifactorial disease with low pH as a driving force for mineral dissolution.
  • Plaque pH is lowered by organic acids (e.g., lactate, acetate and propionate) that are released by oral bacteria as fermentation products.
  • organic acids e.g., lactate, acetate and propionate
  • the ideal anti-caries therapeutic agent would inhibit fermentation activity (acid production) of oral microorganisms, especially those that are known to be involved in caries aetiology (e.g. S. mutans).
  • Biofilms were grown in 10% H 2 , 10% C0 2 in N 2 at 37°C.
  • biofilms as a neat and 1/10 solution in water for 5 minutes. Controls included a negative (RTF; Syed and Loesche Applied Microbiology 1972; 24:638-644) and a positive (0.05% chlorhexidine solution).
  • biofilms were incubated in 0.2 ml of 0.5% glucose solution at 37°C for 3 h in 10% H 2 , 10% CO 2 in N 2 .
  • biofilms were suspended in the incubation fluid and sampled into pre-cooled eppendorf tubes. The tubes were set on ice until further processing within one hour.
  • Streptococcus LabM Incubate in 5%C0 2 at 37°C in a plastic pouch (with rnutans C0 2 GEN Compact Atmosphere Generation System,
  • Lactobacillus Code CD0020, Oxoid Ltd.
  • DIFCO Vitamin K menadione
  • 1 mg/mL final concentration
  • Vitamin K (menadione), 1 mg/mL (final concentration).
  • Acid production was either unaffected by the homogenates/extracts or increased.
  • the mushroom homogenate was fractionated into low and high molecular weight fractions by ultrafiltration using the Vivaflow 200 complete system (Vivascience AG, Feodor-Lynen-Strasse 21 , 30625 Hannover, Germany) comprising a pump (240V), tubing, 500ml sample/diafiltration reservoir, and a membrane 5,000 MWCO PES for ultradiafiltration (Vivasience).
  • a diafiltrate i.e. a low molecular mass fraction (LMM) containing all the compounds with molecular masses less than 5000 Da
  • a retentate i.e. a high molecular mass fraction (HMM) containing all the compounds with molecular masses greater than 5000 Da.
  • the chicory homogenate was subjected to ultrafiltration exactly as described above. 50% of the components originally present remained in the retentate - this was verified by Gel Filtration Chromatographic analysis of the dialysate and retentate. As the retentate contained some components with a molecular mass lower than 5,000 Da, the HMM fraction was subjected to dialysis using dialysis membrane tubing with a 5,000 MW cut-off to eliminate the LMM components. The diafiltrate and retentate were sterilized using a 0.20 pm membrane (Vivascience) and then freeze-dried.
  • the diafiltrate and retentate were sterilized using a 0.20 ⁇ membrane (Vivascience) and then freeze-dried.
  • Example 6 Determination of anti-caries activities of the H M and LMM fractions of mushroom, chicory and raspberry
  • the effects of the HMM and LMM fractions of the three test materials on organisms associated with caries and health in assays specifically relevant to this disease were carried out.
  • the assays aimed to assess the ability of each test material to:
  • the LMM fractions of mushroom, raspberry and chicory generally had greater inhibitory effect against the target organisms than the HMM fractions
  • the LMM fractions of mushroom, raspberry and chicory were generally more effective at inhibiting co-aggregation than the HMM fractions
  • the table shows cells disrupted from the biofilm after 1 minute incubation with the extracts.
  • the LMM fractions of mushroom and chicory were generally more effective at disrupting biofilms of the target organisms than the HMM fractions.
  • Example 7 Determination of anti-gingivitis activities of the HMM and LMM fractions of mushroom, chicory and raspberry
  • the assays aimed to assess the ability of each test material to:
  • the LMM fractions of mushroom and chicory were more effective at disrupting biofilms of the target organisms than the HMM fractions.
  • the HMM fraction was the most effective.
  • the bacteria and the tested compounds were added simultaneously in the assay.
  • both the LMM and HMM fractions of raspberry and chicory were able to inhibit adhesion of the target organisms to epithelial cells.
  • Neither the LMM or HMM fractions of mushroom displayed inhibitory activity.
  • ⁇ CH 1 : 10 LMM chicory at a dilution of 1 : 10
  • ⁇ ME 1 :10 LMM mushroom at a dilution of 1 :10
  • the crude raspberry extract and the LMM and HMM fractions were not able to inhibit IL-6 production induced by the F. nucleatum supernatant (results not shown).
  • the LMM fraction of mushroom was fractionated further by gel filtration chromatography.
  • the Agilent Chemstation software was used for control of the system and data processing GFC separation of the LMM fractions (MW ⁇ 5000 Da) was performed using a Superformance Universal giasscartridge system (300 mm x 10 mm) (Merck, Darmstadt, Germany).
  • the eluant was collected as six fractions as shown in Figure 5.
  • Sub-fractions M4 and M5 were further fractionated using reverse phase HPLC semi- preparative chromatography. All experiments were performed using an Agilent 1100 HPLC system (Agilent, Waldbronn, Germany) equipped with a gradient quaternary pump, a thermostatted column compartment, and a DAD. The Agilent Chemstation software was used for control of the
  • HPLC system The semipreparative HPLC column was a C18 LiChrospher ® 250 * 10 mm, 10 L/m (Merck, Darmstadt, Germany) Chromatographic conditions for gradient elution were as follows:
  • sub-fraction M4 produced 8 sub-sub-fractions respectively (M4.1- 8).
  • dry masses of these sub-sub-fractions are shown below: dry mass
  • sub-fraction M5 produced 1 1 sub-sub-fractions (M5.1-11 ).
  • dry masses of these sub-sub-fractions are shown below: dry mass
  • M4.7 aconitic acid, adenosine and oxalic acid
  • Example 9 Further fractionation of LMM mushroom and chicory homogenates and identification of constituents with anti-gingivitis activity
  • the LMM fractions of mushrooms and chicory were fractionated further using gel filtration chromatography as described previously.
  • the chromatogram of the LMM chicory is shown in Figure 8.
  • the eluant was collected as six fractions.
  • the LMM mushroom and chicory extracts each generated six sub-fractions (M1 -6, C1-6) and these were subjected to the following anti-gingivitis assays.
  • Sub-fractions M4 and M5 were further fractionated using reverse-phase HPLC (see Example 8, Section 8.3). Sub-fraction M4 generated 8 sub-sub-fractions (M4.1-8; Figure 6) and sub-fraction M5 generated 1 1 sub-sub-fractions (M5.1 -11 ; Figure 7).
  • M4.7 aconitic acid, adenosine and oxalic acid
  • target organisms A >1:2 >1:2 >1:2 1:2 1:2 1:2 naeslundii
  • sub-fraction C1 was further fractionated using reverse-phase HPLC and this generated 15 sub-sub-fractions (C1.1-15).
  • sub-sub-fraction C1.7 was found to be the most active taking into account the dry masses of the various fractions and, consequently, its specific activity.
  • the main constituents in sub-sub-fraction C1.7 were identified by HPLC and mass spectroscopy as oxalic acid and quinic acid.
  • Example 10 Further fractionation of LMM fraction of raspberry homogenate and assessment of its anti-caries and anti-gingivitis activities
  • a Sep-Pak ® Vac 20cc (5g) tC18 cartridge (Waters, Milford, MA) was conditioned with methanol (10 ml), Millipore grade water (2 x 10 ml), and PB (pH 7.0, 10 ml).
  • the sample was passed through the conditioned cartridge at a flow rate ⁇ 2 ml/min and the following sub-fractions were obtained:
  • R 3 was obtained by eluting afterwards with 25 ml of a methanol- Millipore grade water mixture (20%-80%)
  • R 4 was obtained by eluting afterwards with 25 ml of a methanol- Millipore grade water mixture (50%-50%)
  • the five sub-fractions were freeze-dried and the residues were dissolved in 5 ml of Millipore grade water.
  • the R1 sub-fraction was then passed through the conditioned cartridge at a flow rate ⁇ 2 ml/min.
  • R1A consists of the most polar substances not retained by the Sep-Pak® Vac 20cc (5g) tC18 cartridge.
  • the further sub-sub-fractions were obtained as follows: R1B was obtained by eluting afterwards with 25 ml of Millipore grade water,
  • R1 C was obtained by eluting afterwards with 25 ml of methanol.
  • the three sub-sub-sub-fractions were freeze dried and the residues were dissolved in 5 ml of Millipore grade water.
  • RTF reduced transport fluid
  • Streptococcus mutans biofiims were disrupted by all of the agents tested, though most of the agents did not cause a significant kill of the bacteria.
  • quinic acid did achieve significant kills of S. mutans.
  • Succinic acid resulted in the disruption of approximately 80 % of cells.
  • Oxalic acid resulted in disruption similar to that of the positive control agent at the two lower concentrations, and in 95 % disruption at the highest concentration. 11.1.2 Actinomyces naeslundii as the target organism
  • adenosine and quinic acid exhibited some ability to inhibit the adhesion of S. mutans to hydroxyapatite at mg/ml concentrations.
  • Low concentrations (Mg/ml) of oxalic acid, cis- and trans-aconitic acids also inhibited adhesion but to a limited degree.
  • Adenosine, quinic acid and epicatechin were able to inhibit biofilm formation by the organism at mg/ml concentrations whereas succinic acid also inhibited at much lower concentrations (Mg/ml).

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Abstract

La présente invention concerne de nouvelles compositions pour favoriser la santé bucco-dentaire par exemple, pour améliorer, contrôler ou réduire le risque de carie dentaire ou de maladie parodontale telle que la gingivite ou la parodontite. Les compositions sont généralement des aliments fonctionnels ou des préparations orales qui comportent un ou des extraits, par exemple des extraits particuliers de faible poids moléculaire, de produits naturels (champignons Shiitake, la chicorée et/ou la framboise). L'invention concerne également des compositions utilisant l'acide quinique; l'adénosine; l'inosine; l'acide trans-aconitique; l'acide oxalique; l'adénosine; l'acide cis-aconitique et l'acide succinique, ou les sels de ceux-ci, pour favoriser la santé bucco-dentaire.
PCT/GB2011/000969 2010-06-29 2011-06-28 Produits à effets bénéfiques sur la santé bucco-dentaire WO2012001347A1 (fr)

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US9326925B1 (en) 2015-02-26 2016-05-03 Johnson & Johnson Consumer Inc. Compositions comprising combinations of organic acids
US9326924B1 (en) 2015-02-26 2016-05-03 Johnson & Johnson Consumer Inc. Compositions comprising combinations of organic acids
WO2016138217A1 (fr) 2015-02-26 2016-09-01 Johnson & Johnson Consumer Inc. Compositions comprenant des combinaisons d'acides organiques
US10383902B2 (en) * 2014-09-17 2019-08-20 Hokuto Corporation Agent for promoting growth of nonpathogenic oral indigenous bacteria or agent for improving oral bacterial flora, and composition for oral use
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