Field of the invention.

The present invention relates to sanitary-veterinary products. It particularly relates to compositions formulated based on two plant extracts, in which the content of active ingredients is appropriately standardized in a mixture exerting synergistic antimicrobial activity over the range of microorganisms that cause halitosis, gingivitis, and periodontal disease in animals, more particularly to compositions containing standardized extracts of Psidii pu mil urn var. guadalupense (D. C.) and Houpoea officinalis {Render & E.H. Wilson) for the treatment of periodontal disease and/or animal halitosis. The objective of the present invention is to provide suitable compositions and methods for the treatment of periodontitis in animals. Background of the invention.

In the market there are various products, which are used to treat oral microorganisms that cause oral disease in toothed animals. However, their application is complicated and risky because of the toxicity degree of these products when they are swallowed. Halitosis or bad breath is not unique to humans. Generally, toothed animals with are susceptible to suffer from it when microbiota of the oral cavity is altered producing the growth of pathogenic microorganisms that are hosted on the gingiva, teeth, tongue and oral mucosa.

The oral cavity is the gateway of food, where the process of transformation of nutrients is started by chewing (grinding the food with the teeth to reduce it to chemically attackable particles) and bolus formation (impregnating the food with water and enzymes from salivary glands and its modeling, by the tongue, of the shape and size) for swallowing it into the stomach. During this physiochemical process it is impossible to avoid the occurrence of accumulation of food residue on teeth and gums, tongue and mucous membranes lining the oral cavity.

Residues in the transformation process by the enzymatic action of the saliva become an ideal substrate for feeding millions of microorganisms commonly found in the oral cavity in addition to those acquired by food itself and opportunistic pathogens.

Physiological and biochemical responses happen in the oral cavity to combat the presence of pathogenic microorganisms; this occurs through various mechanisms, such as changes in volume and saliva enzyme content, changes in the pH medium, increasing the activity of immune cells (macrophages, mainly), the production of bacteriocins (compounds produced by other bacteria), and biochemical reactions that hinder the adhesion of certain strains of the mucous membranes etc. These responses seek to maintain the equilibrium established between the oral microbiota and the host. When this set of responses does not achieve its purpose, the microbiotic equilibrium is upset starting the colonization, which involves the establishment of the infectious process.

Residues adhered to the tongue, teeth and mucous facilitate the retention and growth of microorganisms in the oral cavity, and the accumulation of bacteria that in the medium term will produce a chronic infectious process beginning with halitosis, generalized gingival inflammation or gingivitis, and ultimately, periodontitis. Also, cavities develop and, in later stages, produce the loss of bone and the loosening of teeth.

Initially, the presence of residues leads to the formation of the so-called "dental plaque." This occurs when the teeth present a transparent pellicle formed by saliva glycoproteins, polysaccharides, epithelial cells, macrophages, leukocytes, and water whereto multiple bacteria adhere overlying it permanently and making it an ideal medium for the adherence of some bacteria. This is what properly constitutes dental plaque.

If not removed, the plaque increases in volume, elevating to cover the teeth and colonizing the gingiva. The primary immune response to this aggression is inflammation of the gums or gingivitis. If the inflammatory defense process is not effective and a macrobiotic imbalance is created in the system, colonization continues to advance destroying gum tissue, retracting the gingiva, making it prone to bleeding and producing a purulent infection in form of periodontal pockets.

Once the local immune barrier is retracted, the structure of the marginal gingiva is lost, allowing bacterial growth underneath in direction to the bone (subgingival plaque). In this last phase occurs a bone destruction and separation of the teeth.

The most common pathogenic microorganisms in this process are Streptococcus mutans and Fusobacterium nucleatum, associated with other bacteria such as Escherichia coli, Staphylococcus aureus, Staphylococcus fecalis, yeasts such as Candida albicans, and opportunistic actinomycetes such as Trichophyton rubrum, and Trichophyton mentagrophytes, among others.

The pathogenesis of the more virulent strains is related to their capacity to adhere to the biofilm that constantly forms on the surface of the teeth and mucous membranes of the oral cavity. The microorganisms find in this layer the ideal feeding conditions and metabolizing sucrose in organic acids and polysaccharides. In this enriched medium proliferates Gram-positive and Gram-negative bacteria, resulting in multiple colonization of microorganisms.

All this pathological process should be understood as a rupture of the microbiotic equilibrium in the oral cavity because of the unrestrained proliferation of microorganism colonies with a high capacity of adherence to the tissue.

Halitosis in animals is the first symptom of microbiota imbalance of the oral cavity resulting from the accelerate reproduction of various microbial strains, generating sulfur gases produced by the microorganisms because of its metabolism; these are incorporated to the breath, which consequently acquires an unpleasant odor (halitosis). In subsequent stages, the animals suffer primary gingivitis, periodontal pocket formation (larger than 3 mm in dogs, or 5 mm in cats), in which the infectious material accumulates, and they eventually suffer the loss of teeth.

It is important to consider several predisposing factors to periodontal disease in the smaller species, such as, for example, the age of the animal, the shape of the head and occlusive pattern, the size of the species, diet, constant open mouth breathing, and chewing habits.

Some conditions such as uremia, hypothyroidism, feline leukemia, feline immunodeficiency, pemphigus vulgaris, and lupus erythematosus are associated with halitosis and gingivitis, which highlights the intimate relationship between the immune status of the animal and the equilibrium of its oral microbiota.

In humans, the brush cleaning of the denture after each meal remains the paradigmatic principle of prophylaxis of periodontal disease as it allows the removal of food debris on teeth and gums by the mechanical action of the brushing. The use of toothpastes, mouthwashes with chemical antiseptics and other abrasives help to prevent the formation of plaque as long as frequency of use does not cause toxic effects or abrasion, and gingival and mucosal lesions.

In major domestic species (e.g., domestic dogs and cats) this prophylaxis procedure is difficult to apply because when required, teeth cleaning and sub gingival curettage are done under general anesthesia of the animal as these procedures require an ultrasonic scaler or curette along with manual scraping picks and curettes. Therefore, thorough cleaning should be performed by a veterinarian because it demands the proper management of general anesthesia, depending on the severity of the case, the administration of antibiotics to the animal for long periods of time, often leading to poisoning and various undesirable effects. Accordingly, the treatment is expensive and dangerous, but it is especially ineffective in preventing the evolution process of periodontal disease.

In the search for mechanisms to prevent or delay the development of microbial colonies in the oral cavity of animals, following effects have been investigated:

1 . Bactericidal effect. The destruction of microorganisms by the action of specific chemical compounds altering the permeability of their membrane;

2. Bacteriostatic effect. The interference of certain compounds, particularly enzymes, with the metabolism of microorganisms;

3. Buffer effect. The pH medium alteration to improve the resistance of tissues and retard microbial reproduction; and

4. Anti-adherent effect. The prevention of adhesion of microorganisms to the biofilm. The significant global growth of herbal medicine in preventive and curative programs in dental care has stimulated scientific research to substantiate the antimicrobial activity of various plant extracts to help in controlling plaque and decreasing caries incidence and periodontal disease.

For addressing these health issues, two opposing hypotheses on the pathogenesis of caries and periodontal disease have been suggested; these hypotheses directly influence the pharmacological and treatment solutions that can be proposed.

The first hypothesis assumes the universal presence of potentially pathogenic microorganisms in dental plaque and all accumulation of it is pathogenic, giving rise to the hypothesis of non-specific plaque hypothesis. Based on this theory a model of surgical management of dental treatment was created in which the diagnosis was not considered important since all teeth were affected by a plaque of odonto-pathogenic bacteria and therefore, all individuals were prone to develop the disease. The problem with this hypothesis is that it implies the need to completely remove the plaque in all patients. This goal is unrealistic and difficult to achieve even in better-disposed individuals. Its main weakness, however, lies in the fact that it minimizes the evidence that different microbial ecosystems are always present in the oral cavity. So, to the decade of the nineties of the last century this hypothesis was widely questioned.

The second hypothesis is based on the observation that bacterial plaque is not always accompanied by alterations of the dental enamel, assuming that the plaque is pathogenic only when there are other changes in the ecosystem, since only a limited number of microorganisms, mainly bacteria, are capable of causing dental caries and periodontal disease, and that in the plaque are other microorganisms present in equilibrium with the host. This is the specific plaque hypothesis. Treatment based on this second hypothesis is aimed at eliminating specific pathogens, not all the plaque, for which the pre-clinical microbiological diagnosis is essential. The goal of the treatment is to remove the cariogenic plaque and replace it with plaque free from pathogenic microorganisms, achieving this by applying "soft" antibacterial measures but hard enough to procure some sterility in the tooth surface for short periods of time. Thus, in the new plaque, harmless strains of, e.g., Streptococcus sanguis and Streptococcus mitis should predominate over smaller proportions of Streptococcus mutans.

The application of the treatment derived from this hypothesis led years later to the expansion of the factors considered, surging what is known as the "Keyes Triad", i.e. the identification of three combined factors affecting the production of dental caries. For this pathological process to develop, it is said that following factors must not only be present, but also interact in optimal conditions: a) a host with "susceptible" tissues; b) to maintain a diet rich in sucrose; and c) to be colonized by microbiota with high cariogenic potential. According to researchers in this field, based on local "altered" conditions dental plaques dominated by pathogenic bacteria can develop, which after some time will cause caries, independently from the dental hygiene practiced. This proposal recognizes the existence of other "secondary factors" such as chemical modification of the saliva and an excessive fluoride exposure, which would decrease the tooth resistance and increase the cariogenic potential of microbiota. In other words, until recently researchers began to explore the possibility of modulating the microbiota activity by modifying the ecosystem in which it is located, leaving behind the previous idea of exterminating every bacteria existing in the oral cavity and whose "asepsis" was impossible to achieve.

In recent years, progress has been made in understanding the molecular biochemistry that determines the formation of dental plaque. We know now that dental plaque is always produced, even after thoroughly brushing the teeth; its formation begins within a few minutes after the brushing when an acellular mucinous layer free of bacteria, called dental cuticle, or acquired dental pellicle (APD), or biofilm. Immediately after its release, various forms of bacteria adhere to it by ionic, electrolyte or ligand-lectin type bonds, colonizing it and producing dextran, a high-weight extracellular polysaccharide, which is viscous and very adhesive and is produced exclusively from the transformation of food into sucrose that is performed by some bacteria.

Streptococcus mutans and Fusobacterium nucieatum are particularly known to be capable of adhering to the oral mucosa and teeth, because they produce dextran. In turn, these adherent bacteria have special receivers to the viscose matrix of dextran, allowing them to agglutinate tightly with each other. Once they are fixed, these pioneer microorganisms proliferate and extend laterally forming a layer on the tooth surface, similar to a mat, and allowing the agglutination of other microbes. Subsequent bacterial growth is, therefore, of mixed colonies of different microorganisms. The resulting combined layer allows other weaker microorganisms to concatenate, such as filamentous and spiral bacteria, yeasts, and fungi, which could not otherwise fix themselves. Accordingly, the formation of a mature plaque involves a series of molecular exchange between the cumulative microbiota, which, initially, will depend on the preparatory stage of the local conditions modified by the bacteria with adhesive capacity. The formation of a mature dental plaque takes an average of 4 to 5 days.

The search for plant extracts that modify or prevent the adhesion of the major pathogens mentioned above proves to be the starting point for the design of preventive phyto medications for periodontitis. In recent years, a new group of drugs of natural origin have appeared, developed from medicinal plants, which are called phyto medicines, herbal medicines, or phyto pharmaceuticals, whose greatest strength is safety and low toxicity in general, compared with chemical-pharmaceutical products developed from pure chemical compounds. They are obtained from medicinal plants under growing conditions controlling the use of fertilizers, pesticides, and nutrients to produce pharmaceutical grade complex extracts.

There are numerous herbal extracts that exhibit antimicrobial activity both as bactericide and antiviral and anti-fungal. In dental research, the first studies in this direction described the effect on dental plaque of chewable Nigerian plants. It was observed that some of these extracts influenced the adherence of Streptococcus mutans and Fusobacterium nucieatum to the tooth surface, significantly reducing the presence of these bacteria in dental plaque. Later, studies were published on the antimicrobial action of other extracts (black tea and green tea) against Staphylococcus and Streptococcus along with some other known bacteria present in the oral cavity, which suggested that these plants contain compounds that could be effective in reducing periodontal disease and halitosis. Various papers have been recently published, supporting the existence in the extracts of medicinal plants of several common chemical groups (phenols, flavonoids, anthocyanins, etc.) with diverse antimicrobial capacity, antibiotic potency, and specificity, having the important advantage of not producing toxic effects whose presence in complex extracts was not properly valued before.

In this regard, documents relating to the treatment of oral diseases are found in the Patent application WO2007133721 , which discloses a composition that includes natural ingredients including the use of pomegranate extract, green tea extract, rosemary, quercetin, amla extract, and kakadu extract.

The patent US626501 1 relates to compositions for treating and preventing oral disease in animals and humans, consisting of 8 to 12 parts by weight of whole wheat flour, 4 to 6 parts of oat bran, 2 to 3 parts of dry nonfat milk, and 2 to 3 parts of whole rice flour. The composition further includes water, vanilla extract, parsley, alfalfa powder, vegetable oil, peppermint extract, tarragon, mint leaf and green tea powder. The vegetal oil includes an antioxidant, preferably sunflower oil.

The patent document EP0951838 discloses a cat food to prevent oral malodor containing 0.03 to 0.1 % by weight of green tea extract in which said extract contains 40 to 50% by weight of tannin.

The document ES2052406 discloses a hygiene product for oral deodorization of domestic animals comprising an assimilable protein carrier impregnated with a deodorant substance associated with an inhibitor of proteolytic enzymes and a natural antimicrobial product.

The patent application US2006/0106274 describes a composition comprising Salvadora persica used in coating products such as toothbrush for oral care of pets.

The document US3936527 describes a method for treating oral malodor in dogs and cats daily administering to the animal 50 to 500 mg of methionine in tablet form, further containing concentrated proteins, among which is a mixture of soy and fish flour.

Finally, the document CN 102327594 describes a composition for the treatment of periodontitis and halitosis in dogs, which consists of 10 parts of dried orange peel, 5 to 30 parts of ginger, 5 to 30 parts of odor clove, 5 to 30 parts of mint, and 5 to 30 parts of Aucklandia root, which is applied as an ointment, spray, tincture, or mixture.

However, previous solutions have the disadvantage of only treating oral malodor in pets affected by halitosis, only neutralizing malodors caused by their oral health condition without having any major effect on the treatment of diseases that cause oral malodor, such as periodontitis and/or gingivitis. So, it is necessary to have effective solutions for the treatment of oral diseases in animals; e.g. , smaller species comprising domestic animals such as dogs and/or cats, avoiding invasive treatments of low efficiency and high health risks, such as surgical treatment. Brief description of the invention.

The present invention provides equibiotic compositions whose formulation is based on two extracts obtained from plants (Psidii pumilum var. guadalupense (D. C.) and Houpoea officinalis (Rehder & E.H. Wilson)) whose content is appropriately standardized into a synergistic mix possessing an increased antimicrobial activity on the range of microorganisms that cause halitosis, gingivitis, and periodontal disease in animals. Strains of the most common microorganisms present in the formation of dental plaque and gingivitis in domestic animals, were used; e.g. , Streptococcus mutans, Fusobacterium nucleatum, Escherichia coli, Staphylococcus aureus, Staphylococcus fecalis, Candida albicans, Trichophyton rubrum, and Trichophyton mentagrophytes, to evaluate the antimicrobial activity of the compositions of the invention comprising two concentrated extracts: 1 ) a standardized concentration mixture of glycosyl benzochromones obtained from Psidii pumilum var. guadalupense (D. C.) (Extract A), and 2) a standardized concentration of dialkylphenols obtained from Houpoea officinalis (Rehder & E.H. Wilson) (Extract B) by the method of diffusion in agar plate. The extracts were dissolved in DMSO (dimethyl sulfoxide) centrifuged and filtered (0.22 pm) to achieve its sterility and obtain greater homogeneity when mixed with the culture medium. Also, the compositions of the invention were clinically tested in domestic animals (dogs) afflicted with periodontitis with excellent results in the treatment of this disease.

Brief description of the figures.

Figure 1. Shows the different stages of periodontal infection in dogs. Shows (a) initiation of plaque formation; (b) gingivitis; (c) retraction of the gum; and d) periodontitis with dental attachment loss.

Figure 2. Shows the effect of the composition of the present invention on 8 cases of halitosis in dogs. Time 1 corresponds to 8 days of treatment.

Figure 3. Shows the effect of the composition of the present invention on 8 cases of gingivitis in dogs. Timel corresponds to 8 days of treatment. Detailed description of the invention.

The present invention provides equibiotic compositions whose formulation is based on two extracts obtained from plants (Psidii pumilum var. guadalupense (D. C.) and Houpoea officinalis (Rehder & E.H. Wilson)), whose content of active principles is appropriately standardized in a synergistic mixture possessing an increased antimicrobial activity on the range of microorganisms that cause halitosis, gingivitis, and periodontal disease in animals, for example in domestic species such as, for example, domestic cats and dogs.

The compositions of the present invention comprise two extracts of plants: one of them obtained from Folium Psidii pumilum var. guadalupense (D. C.) (Extract A), whose content of glycosyl benzochromones was standardized; and the other obtained from Cortex Houpoea officinalis (Rehder & E.H. Wilson) (Extract B) whose content of dialkylphenols was standardized; both of them form a mixture having a synergistic antimicrobial activity on the range of microorganisms that cause halitosis, gingivitis, and periodontal disease in animals, wherein the above extracts can be present as fluid and/or dry extracts.

Preferably, the extract A is obtained from leaves (Folium) of Psidii pumilum var. guadalupense (D.C), while the extract B is obtained from bark of Houpoea officinalis (Rehder & E.H. Wilson), although other parts of the plants mentioned above can be used to obtain the active principles mentioned for each (glycosyl benzochromones for extract A and dialkylphenols for extract B), and the proper amount of such actives to standardize both extracts.

According to the present invention, standardized extracts of Psidii pumilum var. guadalupense (D. C.) and Houpoea officinalis (Rehder & E.H. Wilson) are provided, so these extracts contain determined and standardized quantities of the active principles responsible for their therapeutic efficacy. This characteristic of the standardized extracts of the invention allows its use for obtaining frequent and controlled pharmaceutical compositions, by that allowing to control the quantity of active principles in such compositions. Accordingly, it is possible to obtain pharmaceutical compositions with known predetermined doses, which can be administered by different routes to the subject in need of treatment in a right volume. Also, by administering such compositions it is assured that the subject will receive controlled and constant quantities of the active principles contained for different treatment regimes. Furthermore, these extracts allow the physician to design controlled treatments for the subjects suffering from, e.g., periodontal disease, gingivitis, and/or halitosis, as it is possible to administer controlled doses of the drug and a suitable follow up treatment.

So, standardized extracts of the invention allow to easily combining multiple compounds useful for manufacturing various conventional dosage forms, either in powder or liquid depending on the needs and administration and therapeutic regimen. The extracts retain their pharmacological properties, despite the process that is subjected for obtaining suitable dosage forms, which allows great versatility in their management. The standardized extracts described herein contain predetermined quantities of their standardized active principles such as glycosyl benzochromones for Psidii pumilum var. guadalupense (D. C.) extracts and of Houpoea officinalis (Rehder & E.H. Wilson) for dialkylphenol extracts, using the quantitative determination of said active principles by methods known in the art for identifying molecules, such as; e.g., high performance liquid chromatography (HPLC).

Pharmaceutical compositions using the extracts of the invention or combinations thereof as therapeutic agents can be prepared according to any of the methods and procedures known in the art. In preparing such compositions the active ingredient is mixed or diluted with a carrier, or is preferably included within a carrier, which may be in various forms, such as capsules, bags, or in other packings. When the carrier serves as a diluent, it can be a solid, semisolid, or liquid material, which acts as a vehicle, excipient, or medium for the active ingredient. Thus, the compositions can be in a tablet, pill, powder, bag, elixir, suspension, emulsion, gel, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder, and the like.

In this regard, examples of pharmaceutical carriers, excipients, and diluents suitable are for example, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, hydroxymethyl benzoates, hydroxyprophyl benzoates, talc, magnesium stearate, and mineral oil, and others known in the art for the same purpose. The compositions may additionally include fillers, anti- agglutinative agents, flavoring agents, lubricants, wetting agents, emulsifiers, preservatives, and the like. Also, the pharmaceutical compositions of the invention may be formulated to provide quick, sustained, or delayed release of the active ingredients after administration to a mammal by employing any of the procedures and/or methods well known in the art. Synergistic pharmaceutical compositions of the present invention contain an amount of 0.4 to 1 .91 mg of glycosyl benzochromones from extract A, and an amount of 300 to 463 mg of dialkylphenols from extract B. For the various pharmaceutical forms that can be obtained using the standardized extracts disclosed herein; the concentration of the active principles can vary within the ranges mentioned depending on the pharmaceutical in question.

The pharmaceutical compositions of the invention can be administered topically, administering them directly to the gums in subjects suffering for example, periodontal disease, gingivitis, and/or halitosis, in a dose containing at least 0.4 mg of glycosyl benzochromones from extract A in combination with at least 300 mg of dialkylphenols from extract B. Such doses may be administered as a single dose or daily in divided doses up to the complete disappearance of clinical symptoms. The extract amount of the compositions to be administered to the subject in treatment may vary according to age, sex, body weight, severity of the disease, and the formulation used.

One embodiment of the present invention is to provide a semi-solid equibiotic product such as gel, which is formulated based on two plant extracts whose content of active principles is appropriately standardized in a synergistic mixture having antimicrobial activity on the range of microorganisms causing halitosis, gingivitis, and periodontal disease in animals.

A second modality of the present invention is to provide a veterinary hygiene product for major species, such as domestic dogs and cats, to eliminate bad breath, the formation of dental plaque, and combat and prevent gingivitis and periodontal diseases, which are difficult to treat in animals.

The compositions disclosed in the present invention can be in various dosage forms; however, gel is the preferred form of the product, because in contrast to other products manufactured with the purpose of eliminating halitosis and decrease inflammation of the gums, such as, e.g., solutions and antibiotic ointments, antiseptic mouthwashes, abrasive toothpastes, etc., the gel form of the compositions in the present invention is easy to use, and its pleasant taste makes dogs and cats to distribute it with their own tongue in the oral cavity. Additionally, the gel form increases the permanence of the actives of the formulation in the site of action (oral cavity). For purposes of the invention, the application of the pharmaceutical gel form after each meal guarantees its effect for more than 12 hours. Regarding phytomedicines, our research group has discovered "Equibiotics"; that is, products derived from the mixture of particular plant extracts, which allowed us to obtain, along with an antimicrobial synergistic activity, the preservation and strengthening of the equilibrium of the physiological conditions characteristic of the system where they are applied.

Equibiotics act simultaneously in two modality, such as auferobiotic and alerebiotic. In the auferobiotic modality the antimicrobial and/or antibiotic activity is developed, whereas in the alerebiotic modality the restoration of the microbiota is developed. Equibiotics are unique in that their action influences and strengthens the immunological regulatory capacity of the host itself.

Thanks to this double action equibiotics notably reduce the toxic effects on the host, because its action mechanism allows to essentially regaining the biological equilibrium characteristic of tissues and systems in which they operate, such as skin, mucous membranes, gastrointestinal tract, respiratory tract, etc.

The active principles of equibiotics propel bactericidal modalities and pharmacological properties that interfere with the pH medium, inhibit bacterial adhesion, reduce the inflammation, and restore tissues and mucous, among other properties, what distinguishes and constitutes them a superior alternative than traditional antibiotics and simple phytomedicines. Additionally, being complex extracts of medicinal plants simplifies or significantly improves the absorption, diffusion, transport, and general bioavailability of the active compositions present in the original medium.

A further embodiment of the present invention is to provide an equibiotics-based compositions, which are effective and safe as topical treatment to significantly reduce the symptoms of halitosis and gingivitis in animals.

The compositions of the invention in its gel form comprise an equibiotic product formulated based on two plant extracts (Extract A and Extract B), whose active principles content are appropriately standardized in a synergistic mixture showing antimicrobial activity on the range of microorganisms causing halitosis, gingivitis, and periodontal disease.

In studies and tests performed by our research group with the compositions of the present invention it was clearly proved for example, the in vitro growth inhibition of the major bacteria, yeasts, and fungi responsible of periodontal disease in animals, confirming their efficiency in the treatment of periodontal disease and halitosis in animals. Also, clinical tests were conducted on a group of domestic dogs proving its rapid effect to eliminate halitosis and reduce gum inflammation according to the treatment received. So, it is proposed as a preventive product for periodontitis in domestic species, such as cats and dogs. Aside from being an effective and safe equibiotic treatment for 8 days of topical application for dramatically reduce the symptoms of halitosis and gingivitis in dogs.

For purposes of the invention, the compositions here disclosed can be administered topically in the oral cavity of the animal at least one time per day, preferably after food ingestion. For cases of severe periodontal disease the compositions of the invention can be administered 2 to 3 times daily up to observing the reduction of the disease and according to the attending veterinarian who can subsequently decrease the rate of administration at a lower dose; for example 1 time per day to complete the treatment. The extracts used for obtaining the equibiotic compositions of the present invention are obtained for example by hydroalcoholic percolation and subsequent concentration up to obtaining fluid extracts for extract A and dry extracts for extract B. However, other solvents known in the art to obtain the active principles in extracts A and B can be used. During the production and purification of the extract critical variables are controlled, such as temperature and time of extraction, solvent extraction ratio, and initial and final content of active ingredients in the plant material and the final extract, respectively.

For purposes of the invention, extract A must contain at least 0.5% (w/v) of glycosyl benzochromones, while for extract B the contents of dialkylphenols must be of at least 80% w/w.

Separate quality control of each extract and the mixture thereof in the bulk and in the finished product (FP/gel) is generally performed by analytical methods known in the art; for example, by HPLC (high performance liquid chromatography), employing gradients of mobile phases consisting of acidified aqueous mixtures of an organic modifier, although other methods known in the art to allow the characterization of the active principles can also be used.

The compositions of the invention comprising mixtures of the extracts A and B in a w/w interval of 10:90 to 40:60 respectively, showed a significant synergistic activity in the antimicrobial effect, allowing to enhance their effect when applied in animal models; for example; in domestic dogs previously diagnosed with periodontal disease, gingivitis, and/or halitosis, wherein after their application in gel form we observed dramatic effects of improvement in dental and oral conditions of the animals treated, eliminating the halitosis and significantly reducing the periodontal disease in the period of clinical evaluation.

Although for purposes of the present invention the mixtures of extracts A and B that showing a synergistic effect in their antimicrobial activity can be used effectively for the treatment of periodontal disease, is preferred to use the mixtures of the A and B extracts at an w/w interval of 10:90 to 30:70 respectively, and more preferably at a ratio of 30:70 w/w of the A and B extracts respectively. Also, at the proportions indicated in the above mentioned mixtures, the concentration of the extract glycosyl benzochromones in extract A is in an interval of 0.4 to 1 .91 mg, while the dialkylphenols concentration in extract B is in an interval of 300 to 463 mg.

As seen below in the examples, the equibiotic activity of the compositions of the present invention is evident when used in the treatment of periodontal disease in animals. Aside from showing an effective antimicrobial activity at a very low concentration of their active principles because to their synergistic activity, they also restore oral microbiota equilibrium in animals, influencing and strengthening its immunological regulation capacity, so that the animal recovers efficiently and quickly. Also, the compositions described herein are safe and non-toxic to animals treated, substantially improving their health condition.

The following examples are presented for the sole purpose of illustrating the present invention without implying any limitation on its scope.

Example 1. Obtaining and standardization of the extracts A and B of the invention.

Extracts of Folia Psidii pumilum var. guadalupense (D. C.) (extract A) and of Cortex Houpoea officinalis (Rehder & E.H. Wilson) (extract B) were obtained by hydroalcoholic percolation and subsequent concentration from leaves (extract A) and bark (extract B) to obtain fluid extracts (extract A) and dry extracts (extract B). During the obtaining and purification of the extracts, critical variables such as temperature and time of extraction, extraction solvent ratio, initial and final content of active principles were controlled in the plant material and the final extract.

The extraction was performed to obtain an extract A with a content of at least 0.5% (w/v) of glycosyl benzochromones and extract B with a contents of at least 80% (w/w) of dialkylphenols.

Separate quality control of each extract and the mixture of both extracts in bulk and finished product (FP/gel) was performed employing analytical methods by the method of HPLC (high performance liquid chromatography) with the use of mobile phases gradients, consisting of mixtures of spiked acidified aqueous organic modifier.

Through the analysis of the mixtures using a diode array detector (DAD) we performed a scanning of wavelengths between 190 to 400 nm, determining the spectral purity of the samples. For the actives (chemical markers) of extract A we obtained two peaks at wavelengths between 250 to 270 nm and 350 to 380 nm. We selected the wavelength of 370 nm for quali-quantitative analysis of the formulation actives of the invention. The quantification in extract B was performed at 288 nm. Example 2. Mixtures of extracts A and B and evaluation of their antimicrobial effect.

We made mixtures of extracts A and B in w/w ratios 1 : 1 , 1 :2, 1 :3, 2: 1 , 3: 1 , 2:2, 2:3, ...n, etc. , respectively (table 1 ) for detecting their synergistic antibiotic capacity.

To evaluate the effectiveness of the compositions of the present inventions, we used strains of the microorganisms most commonly present in dental plaque formation and responsible for causing gingivitis and periodontal disease, such as Streptococcus mutans, Fusobacterium nucleatum, Escherichia coli, Staphylococcus aureus, Staphylococcus fecalis, Candida albicans, Trichophyton rubrum, and Trichophyton mentagrophytes. We evaluated the antimicrobial activity of a fluid extract standardized in its contents of glycosyl benzochromones of Folia Psidii pumilum var. guadalupense (D. C.) (extract A) and a fluid extract standardized in its contents of dialkylphenols of Cortex Houpoea officinalis (Rehder & E.H. Wilson) (extract B) by the method of diffusion in agar plate. The extracts were dissolved in DMSO, centrifuged, and filtered (0.22 pm) to achieve their sterility and obtain greater homogeneity by mixing the culture medium. Initially, of each of the extracts we tested concentrations from 3.0 to 0.2 mg/ml_ considered in the range of antimicrobial activity of natural products for in vitro tests. The Minimal Inhibitory Concentration (MIC) was determined in each case, defined as the lowest concentration able to inhibit 100% the growth and development of colonies. The reading was performed at 48 and 72 hrs of incubation, conducting triplicate incubation assays.

The two extracts (A) and (B) exhibited antimicrobial activity in vitro, preventing the growth of all the strains used since the first 48 hours of incubation with MIC values ranging from 0.03 and 2.0 mg/ml_, depending on the sensitivity of each strain, being Trichophyton the most sensitive and Fusobacterium the most resistant. Some of the mixtures under test demonstrated a strong synergistic effect producing complete growth inhibition of microorganisms with up to 1 ,000 times less than the concentration quantity required of each one of the extracts separately, obtaining, for example, MIC values for mixture A+B (30:70) equivalent to those described for pure antibiotics used as controls (table 1 ).

The compositions formed by mixing extracts A and B in a w/w proportion of 10:90 to 40:60 respectively showed a synergistic effect in antimicrobial activity in the disclosed assay, compared to the extracts tested alone. Also, the concentration of the active principles in the synergistic mixture A+B (30:70) was 1.43 mg for the extract A (glycosyl benzochromones) and 360 mg for the extract B (dialkylphenols) per kilogram of final product.

Example 3. Composition of de invention in gel form.

With the extracts selected combination (A+B, 30:70) we proceeded to formulate the topical product of the present invention, whose antimicrobial action in vitro was assessed by the same procedure described in example 2.

Table 1. Effect of extracts (A) and (B) and the synergistic mixture A+B (30:70) with their respective values for 100% inhibition growth (MIC) in vitro of the major organisms responsible for periodontal disease

The topical formulation was prepared by dispersing the standardized extracts A and B in a mixture of glycerin/propylene glycol previously hydrated; we integrated the mixture of the extracts with the carbomer, and finally added the rest of the excipients to produce the formulation. Table 2 shows the results obtained with such a formulation.

In these experiments, the fluid extracts prepared from a mixture of dialkylphenols and glycol benzochromones have strong antimicrobial activity in vitro on major organisms responsible for periodontal disease. As shown in tables 1 and 2, the combination A+B (30:70) has synergistic properties, which increase their antibiotic potency dramatically. Also, the gel formulations of said mixture (A+B, 30:70) retain its antimicrobial properties and synergistic effect. Example 4. Treatment of periodontal disease in dogs with the compositions of the invention.

In this experiment we selected 8 domestic dogs of different breeds and ages, all with a history of halitosis among the clientele of a veterinary clinic in Mexico City. We obtained from the owners of the animals a written authorization in which they accepted to participate in the study.

Table 2. Product effect of the composition of the present invention compared with the synergistic mixture A+B (30:70) and the combined extracts A and B. The values are expressed in millimeters of the inhibition halo of the plate cultures

NT. Not tested. We designed the research protocol, and a questionnaire was developed in collaboration with the treating veterinarians, establishing the signs and symptoms to record, using a scale to assess gingival inflammation and other typical parameters of periodontal disease. It was determined that the duration of the treatment would be 8 days, using the composition of the invention in gel form described in example 3, 1 to 2 times a day depending on the possibility of dog owners of applying the gel in gums 10 to 15 min after the dog's meal.

At time 0 we qualified the halitosis and gingivitis degree (with or without bleeding), the apparent formation of dental plaque, and photographs were taken of the gums. On that same consultation the gel described in example 3 was given to the owners with the application instructions and how to fill out the questionnaire for time 1 at the end of the treatment. The second consultation happened after 9-10 days of treatment to assess the status of each animal and photographing again the gums.

The entire group of animals was clearly diagnosed with halitosis with an average time of evolution of the disease of about 5 years. The halitosis degree was qualified by each owner in a scale of 0 to 5, giving a range of 3 to 5. The average obtained for the group was 4.5 at the beginning of the study (time 0). Regarding gingivitis, the initial values ranged from 1 to 4 on the scale of 0 to 5, with 2.62 as an average value.

After the treatment (time 1 ) the reported results showed a clear decrease of halitosis in all cases (mean = 1 .5 in the scale of 0 to 5), i.e. , almost 70% improvement in the group. In 50% of the cases, halitosis completely disappeared, reporting grade 0. Gingivitis decreased at an average value of 0.62 at the end of treatment; i.e. , it decreased 76%. In five of the eight cases reported inflammation disappeared completely. No changes were observed in the plaque's appearance. No animal was excluded from the study, and none had abnormal or side effects resulting from the composition of the invention under test.

The opinion of the dogs' owners and the treating veterinarians agreed that the composition of the invention under test is effective in reducing and eliminating halitosis since the early days of the treatment and that gingivitis decreased significantly. The safety and nontoxicity of the product were a unanimous opinion (table 3, and figures 2 and 3).

As can be seen from the above, the composition of the present invention proved to be a safe and effective equibiotic as an 8-day treatment of topic application to clearly reduce the symptoms of halitosis and gingivitis in dogs. Table 3. Effect of the composition of the present invention in gel form on halitosis in dogs (n=8)

Dental

Halitosis Inflammation Bleeding gum

plaque

Dog

TO T1 TO T1 TO T1 TO T1

01 5 3 2 0 2 1 0 0

02 5 0 3 1 5 5 0 0

03 5 1 1 0 5 4 0 0

04 3 0 2 0 3 1 0 0

05 5 2 4 2 3 3 1 0

06 4 1 4 1.5 3 3 1 0

07 4 3 3 0.5 3 3 0 0

08 5 2 2 0 4 4 0 0

Media 4.5 1.5 2.62 0.62 3.5 3.0 0.25 0