FORMULATION BASED ON ETHANOL EXTRACT OF HIBISCUS SABDARIFFA CALYCES FOR USE AS A MOUTHWASH.

Abstract

The present invention describes a mouthwash formulation comprising a synergistic combination of the antimicrobial agent ethanolic extract of hibiscus calyxes with a surfactant, a preservative, and a fluoride ion-providing agent, and at least one or more orally acceptable additives or excipients. In a preferred embodiment, the mouthwash formulation of the present invention is routinely used for oral care and hygiene, for aesthetic, hygienic, and therapeutic purposes. For example, the therapeutic purpose is achieved by the antimicrobial agent inhibiting bacterial growth in the oral cavity, preventing the formation of dental plaque, gingivitis, and periodontal disease associated with bacterial growth.

Description

FORMULATION BASED ON ETHANOL EXTRACT OF HIBISCUS SABDARIFFA CALYXES FOR USE AS A MOUTHWASH TECHNICAL FIELD The present invention belongs to the technical fields of everyday needs and to chemistry. Particularly to the fields of Health Sciences and Pharmaceuticals. More specifically to the technical field of preparations for medical, hygiene, and cosmetic use. BACKGROUND Dental caries is the most prevalent disease in humans. The costs associated with conventional treatment of caries and periodontal disease are high. Numerous studies have attempted to determine the role of bacteria in the development of caries and periodontitis, focusing on the microbiome present in supragingival and subgingival dental plaque formed on the tooth surface (Manji et al., 2018). Dental caries is a dynamic, non-communicable, and multifactorial disease that results in mineral loss of hard dental tissues, mediated by diet and the production of dental biofilm (Fejerskov, 1997; Pitts et al., 2017).Signs of carious demineralization are seen in the hard dental tissues, but the disease process begins within the bacterial biofilm (dental plaque) that covers the surface of a tooth. The disease is initially reversible and can be stopped at any stage, even when some dentin or enamel is destroyed (cavitation), as long as enough dental biofilm can be removed (Struzycka, 2014; Yu, et al., 2017). The oral cavity is known to harbor slightly more than 700 bacterial species (Kilian, 2018). Bacteria belonging to the genus Streptococcus are the primary inhabitants, which can be acquired immediately after birth and thus play an important role in establishing the oral microbiota (Abranches et al., 2018). Bacteria within the mouth primarily live by forming complex polymicrobial biofilms. Dental biofilms are essential etiological factors for dental caries and periodontal diseases, but they have also been linked to diseases outside the oral cavity, such as heart disease (Valen et al., 2018). Controlling the accumulation of bacterial biofilms on teeth is a crucial factor in preventing periodontitis and dental caries. Various chemical agents are used as adjuvants in the prevention and / or control of biofilm or plaque formation (Jafer et al., 2016). Among the chemical agents used to control dental biofilm are antimicrobial substances, such as chlorhexidine and triclosan, which are included in the formulations of some mouthwashes, toothpastes, medications, and dental disinfectants. However, the antimicrobial effect of these compounds or mouthwashes containing these or other antimicrobials is limited or ineffective and can also have toxic effects on patients' health (De Rossi et al., 2014; Saleem et al., 2016). It's also important to mention that most mouthwashes contain alcohol (especially ethanol). Besides having antiseptic properties, the alcohol in mouthwashes helps break down or dissolve active ingredients and preserve the formula's components, although it doesn't directly contribute to the effective control of biofilm and gingivitis. However, there are some contraindications for using alcohol-based mouthwashes, such as their use by infants, pregnant women, people with alcohol use disorder, and patients with mucosal lesions. There are also some undesirable effects, such as burning or a painful sensation, or a feeling of dryness in the mouth in patients with existing soft tissue lesions. On the other hand, the resistance of bacteria to antimicrobials, such as those contained in mouthwashes, must be considered within this problem. In a report published in November 2016 by the World Health Organization (WHO), concern is expressed about the advance of bacterial resistance to antimicrobials, with the fatal consequences that humanity is already experiencing, and with the even greater consequences that could arise if bacteria resistant to current antimicrobials develop and there are no alternatives for their control. Consequently, there is a need for the discovery or development of new products against antibiotic-resistant bacteria, as well as the discovery of new sites of action in the cell of bacterial pathogens that serve as a basis for the development of new active compounds. Therefore, it is necessary to have products that are more effective against both bacteria resistant and non-resistant to current antimicrobials, and that the antimicrobial products are not toxic. Plants are a potential source of antimicrobials with possible applications in dental medicine. Plants used in traditional medicine produce a variety of compounds with known therapeutic properties (Kankariya et al., 2016; Sidhu et al., 2018). However, there is limited information available on the use of medicinal plants as a source of antimicrobials in support of dental medicine. It is a fact that plants produce a variety of secondary metabolites, many of them nonspecific, which serve as a defense against bacterial infections (Radulovic et al., 2013). These antimicrobial compounds are frequently complex mixtures of different structures or molecules synthesized at an early stage of the host-pathogen interaction. However, the mechanisms of action of these active molecules are not fully understood. Nevertheless, there is evidence suggesting that these active molecules synthesized by plants interact with infectious agents through different mechanisms, such as those described by Radulovic et al., 2013): a) by interfering with cell-cell mutual recognition, b) by inhibiting cell-cell interaction, preventing the internalization of the pathogen into the host cell, c) by damaging the bacterial membrane (membrane rupture, increased permeability, increased fluidity, protein damage, enzyme inhibition, etc.), d) by affecting bacterial genetic material, e) by inhibiting ATPase activity and the release of intracellular ATP and other constituents, f) by inhibiting bacterial respiration. The exact mechanisms and sites of action of natural antimicrobials are generally unknown or not well defined. Furthermore, it is difficult to identify a specific site of action where many interacting reactions occur simultaneously. For example, membrane-disrupting compounds have been observed to also cause leakage of cellular contents, interfere with active transport, act on metabolic enzymes, or dissipate cellular energy in the form of ATP (Radulovic et al., 2013). Consequently, it is possible that certain plants can naturally synthesize compounds or molecules that affect the viability of bacteria pathogenic to humans, and that also act on both antibiotic-resistant and antibiotic-sensitive bacterial pathogens. Currently, a variety of plants are used worldwide as alternative treatments for illnesses, such as those caused by bacteria. However, most of these plants have not been evaluated to determine if they actually have any beneficial effect on humans. Furthermore, the mechanism or mechanisms by which the effect is produced are generally unknown, as are the specific molecules or compounds in the plants responsible for the observed effect. Therefore, plants have become a valuable resource for research into molecules or compounds that are alternatives to antibiotics. In fact, it has been reported that several natural plant compounds have a potentially antibiotic effect against multidrug-resistant pathogens and also exhibit a sufficient spectrum of activity, are economically feasible to manufacture, and are cost-effective to implement. Traditional Mexican medicine uses different plants to treat gastrointestinal diseases, specifically infectious diarrhea, one of the top ten causes of death in rural areas. It is possible that some of the plants used in traditional medicine in Mexico contain chemical compounds with antimicrobial activity against various pathogenic microorganisms, both antibiotic-resistant and non-resistant. These plants could be used as sources of antimicrobials for medicinal purposes, either as a base for preparing disinfectant solutions or suspensions for surfaces, or for developing drugs for direct use in humans or animals, for preparing mouthwash solutions, or for treating oral cavity diseases caused by bacteria. Currently, a variety of plants are used worldwide in herbal medicine as an alternative treatment for ailments, such as those caused by bacteria. However, most of these plants have not been evaluated to determine if they actually have any beneficial effect on humans. Furthermore, the mechanism or mechanisms by which the effect occurs are generally unknown, as are the specific molecules or compounds in the plants responsible for the observed effect. Therefore, plants are becoming a valuable resource for research into molecules or compounds that are alternatives to antibiotics.In fact, it has been reported that different natural plant compounds have a potentially antibiotic effect against multidrug-resistant pathogens and also show a sufficient spectrum of activity, are economically feasible to manufacture and economically feasible to implement (Radulovic et al., 2013). There are some studies on the antimicrobial effect of plants on oral bacteria. For example, cranberry juice (Vaccinium macrocarpon Ait., Ericaceae) and crude hydroalcoholic extracts affect the formation of oral bacterial biofilms and bacterial adhesion to the enamel surface in vitro. The cranberry fruit is a unique and rich source of various classes of potentially bioactive phenolic compounds, such as flavonoids. The combination of the cranberry flavonoids myricetin and procyanidin A2 affected both acid production from sugar degradation and acid tolerance by S. mutans. The combined effects of cranberry flavonols and proanthocyanidins on acid production and acid tolerance by S. mutans make them compounds of interest for understanding their anti-biofilm and canostatic properties in vivo (Gregoire et al., 2007). In two studies published by Furiga et al. (2008) and (2013), it was reported that polyphenols from red wine, grape pomace, and grape seed inhibited both the formation of multi-species biofilms composed of oral bacteria (S. mutans, S. sobrinus, Lactobacillus rhamnosus, Actinomyces viscosus, Porphyromonas gingivalis, and Fusobacterium nucleatum) and the synthesis of insoluble glucan. The most effective was grape seed extract, which contains mainly the polyphenols catechin and epicatechin. In a study by Mallery et al. (2008), anthocyanins from freeze-dried black raspberry compounds were found to have a chemoprotective effect. Salivary samples were analyzed using liquid chromatography and spectrophotometry, revealing anthocyanin retention with the use of black raspberry mouthwashes, particularly those containing protocatechuic acid, for up to four hours after use. Furthermore, the authors suggest that oral microflora, salivary enzymes, and the surface oral epithelium may contribute to the intraoral bioactivation of anthocyanins through β-glucosidase activity (Mallery et al., 2011). Ethanolic extracts of Capsicum sp., Gaultheria reticulata, Gaultheria sp., and Guanera sp., when tested on methicillin-resistant Staphylococcus aureus, showed an antibacterial effect against this strain. The fraction obtained from Guanera sp. flowers showed the largest inhibition diameter (26.4 mm), while the smallest was that of Capsicum sp. (14.9 mm) (Piña-Iturbe, 2014). Pimentel et al. (2015) analyzed the antimicrobial effect of Origanum vulgare (oregano), Tagetes elliptica (chincho), and Tagetes minuta (huacatay) against Porphyromonas gingivalis and Lactobacillus acidophilus. Oregano contains thymol and carvacrol, which belong to the phenol functional group; it also contains τ-terinene and π-cimene, which are ketones. These active principles are attributed to its effectiveness against Gram-negative bacteria, demonstrating an antibacterial effect against Porphyromonas gingivalis in their study (Milos et al., 2000). Huacatay contains ocimene, cymenone, tagetone, estragole, flavonoids, sesquiterpenes, and aromatic compounds. These active principles are also attributed to its effectiveness against Gram-negative bacteria. However, it showed no effectiveness against Lactobacillus acidophilus (Tereschuk, et al., 1997) Among the chemical compounds of chincho there are flavonoids, -thienols and terpenoids, these active principles attribute its antibacterial effectiveness in Gram positive bacteria; showing in this study effectiveness against Lactobacillus acidphilus (Gram positive) and Porphyoromonas gingivalis (Gram negative) (Tereschuk, et al., 1997; Pineda, et al.. 2007). Avinash et al. (2016) reported that the Shiitake mushroom is effective in treating gingivitis. Its low molecular weight fractions interfere with the binding of S. mutans cells to hydroxyapatite and of Prevotella intermedia to gingival cells, causing the detachment of pathogenic bacteria from preformed biofilms of the tooth and periodontium. Furthermore, the antimicrobial action has been evaluated using the minimum inhibitory concentration. This antibiotic action of the low molecular weight fraction inhibits DNA synthesis and partially (50%) RNA protein synthesis, leading to inhibition of cell division, cell elongation in S. mutans, and elongation of Prevotella intermedia filaments. Tea polyphenols, nisin and chitosan and their combinations, showed an antimicrobial effect; nisin inhibited the growth of Gram-positive bacteria, while chitosan and tea polyphenols showed an antimicrobial effect for Gram-positive and Gram-negative bacteria (He, et al. 2016). In a study conducted in Korea by Choi et al. (2017), 37 plant species were selected to assess their antimicrobial activity against S. mutans. Methanolic extracts of Sparganium stoloniferum, Pollia japonica, Carex siderosticta, Chelidonium majus var. asiaticum, Firmiana simplex, Thuja orientalis, and Aralia continentalis showed high antibacterial activity (>80% growth inhibition) against S. mutans. Extracts of Camellia japonica and Thuja orientalis inhibited cell growth by more than 70% against this pathogenic microorganism in broth. Camellia japonica, Geranium sibiricum, and Thuja orientalis showed distinct growth inhibition zones at 0.5 mg against Streptococcus mutans. In particular, Thuja orientalis revealed a growth inhibition zone at a concentration of 0.1 mg against S mutans (Choi, et al., 2017). In another study by Hagghgoo, et al. (2017) on the antibacterial effect of Althaea officinalis, it was observed that the root extract had an antimicrobial effect against Lactobacillus acidophilus and S mutans, the effect of the extract increased as the concentration increased. Dandekar et al. (2017) observed that dried cranberry extract at a concentration of 1 mg / mL showed antibacterial activity against Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia using the gel diffusion technique with paper discs (12, 8, 12, and 12 mm inhibition zones, respectively). They further observed that increasing the extract concentration to 5 mg / mL resulted in activity against P. gingivalis (8 mm inhibition zone), while at 10 mg / mL, an effect was also observed against P. gingivalis and T. forsythia (10 mm). However, at a concentration of 15 mg / mL, no antibacterial activity was observed against any of the four periodontopathogens. However, there are no reports in the art or in the technique on the effect of solutions or formulations containing extracts or compounds obtained from calyces of the plant known in Mexico as jamaica (Hibiscus sabdariffa) for use as a mouthwash and that also has an antimicrobial effect. Recently, our research group discovered and reported that the calyces of the hibiscus flower (Hibiscus sabdariffa) contain natural compounds that are effective against bacteria pathogenic to humans (Gutiérrez-Alcántara et al., 2016a; 2016b). Based on this knowledge, we have developed disinfectants for fruits and vegetables and antimicrobials for food using compounds from hibiscus calyces, which we have filed patent applications for. Some of these patents have already been granted, for example: Patent MX 369176 B describes the use of formulations containing mixtures of ethanolic extract of Jamaica calyxes, acetic acid and sodium hypochlorite developed to specifically disinfect seeds intended for the production of sprouts for human consumption. Patent MX 369177 B describes the use of formulations containing mixtures of a methanolic extract of Jamaica calyces, acetic acid, sodium hypochlorite and polysorbate 80 and which have been developed to disinfect tomatoes. Patent MX 369178 B describes the use of mixtures of acetomic extract of Jamaica calyces, acetic acid and sodium hypochlorite developed to disinfect green chilies. Patent MX 369179 B describes the use of mixtures of acetone extract of Jamaica calyxes, acetic acid, sodium hypochlorite and polysorbate 80 that have been developed to disinfect apples. Patent MX 369180 B describes the use of mixtures of an ethanolic extract of Jamaica calyces, acetic acid, sodium hypochlorite and polysorbate 80 that have been developed to disinfect lettuce. Patent MX 369181 B describes the use of different formulations: a) mixtures of a methanolic extract of Jamaica calyces, a chromatographic fraction obtained from an acetone extract of Jamaica calyces, acetic acid, sodium hypochlorite and polysorbate 80; b) mixtures of a methanolic extract of Jamaica calyces, a chromatographic fraction obtained from a methanolic extract of Jamaica calyces, acetic acid, sodium hypochlorite and polysorbate 80. These formulations have been developed to disinfect cilantro. Patent MX 369182 B describes the use of mixtures of a specific chromatographic fraction obtained from an acetone extract of Jamaica calyces mixed with a specific fraction obtained from a methanolic extract of Jamaica calyces in mixture, acetic acid, sodium hypochlorite or calcium hypochlorite and polysorbate 80. These formulations have been developed to disinfect different fruits, such as tomatoes or chili peppers. Patent MX 376290 B describes the use of hibiscus acid and its derivatives as an antimicrobial against antibiotic-resistant and non-resistant bacteria on any type of surface or material, whether living or inert. Patent application MX / a / 2015 / 017440 describes the use of mixtures of aqueous extract, organic acids (such as acetic acid), chlorine derivatives (such as sodium hypochlorite), and surfactants (such as polysorbate 80); these formulations have been developed to disinfect and preserve strawberries and mangoes. Application MX / a / 2015 / 017441 describes the use of mixtures of hibiscus acid derivatives (such as mono- and di-methyl ester of ethanolic extract, mono- and di-ethyl ester of hibiscus acid, mono-, di- and tri-ethyl ester of hydroxycitric acid), with an organic acid (such as acetic acid), a chlorine compound (such as sodium hypochlorite) and a surfactant (such as Polysorbate 80); these formulations have been prepared to disinfect and preserve avocado. However, these inventions do not describe a product formulated from extracts or chemical compounds obtained from Jamaican calyces with a demonstrable effect for use as a mouthwash or for the treatment of infectious diseases of the oral cavity caused by bacteria resistant or not resistant to antibiotics. OBJECT OF THE INVENTION The present invention addresses the technical problem of the lack of antimicrobial drugs or efficient compounds for the control or treatment of infectious diseases caused by antibiotic-resistant bacteria, for use in humans. Currently, bacterial resistance to antibiotics is a global problem. A report published in April 2021 by the World Health Organization (WHO) expresses concern about the advance of antibiotic resistance in bacteria, with the fatal consequences it is having on humanity, and the even greater consequences that could arise if bacteria resistant to all antibiotics were to develop and there were no alternatives for their control. Hence, the WHO states that “Antibiotic resistance is now one of the greatest threats to global health, food security and development, and also…” QMS insists on the need to invest in the research and development of new antibiotics or antimicrobials, as support in the fight against antibiotic-resistant bacteria. Consequently, there is a need for the development of new products against antibiotic-resistant bacteria, as well as the discovery of new sites of action in the cell of bacterial pathogens that serve as a basis for the development of new active compounds. The present invention contributes to solving this problem by providing formulations that are useful for counteracting infections with multidrug-resistant bacteria. Specifically, it discloses compositions comprising an antimicrobial agent of plant origin and at least one pharmaceutically acceptable additive. In a preferred embodiment, the formulation comprises: a) a plant extract of hibiscus (Hibiscus sabdariffa), for example, an ethanolic extract, and b) at least one pharmaceutically acceptable additive. DESCRIPTION OF THE INVENTION The present invention discloses pharmaceutical formulations comprising a combination of a plant-based antimicrobial agent and at least one pharmaceutically acceptable additive. In a preferred embodiment, the antimicrobial agent is an ethanolic extract of hibiscus (Hibiscus sabdariffa) calyces, or antimicrobial compounds derived from such an extract, and at least one pharmaceutically acceptable additive. For the purposes of the present invention, the formulations described herein comprise: a) an antimicrobial agent of plant origin; and b) at least one pharmaceutically acceptable additive. In a preferred embodiment, the formulation comprises: a) Extracts obtained with different solvents from hibiscus (Hibiscus Sabdariffa) calyces alone or in combinations thereof: for example, aqueous extracts of hibiscus calyces, and b) at least one pharmaceutically acceptable additive. According to the present invention, the plant-based antimicrobial agent is an acetone extract obtained from Jamaica (Hibiscus sabdariffa) calyces as shown below: a) Macerating dried Jamaican calyces in a polar solvent, which may be in a ratio ranging from 1:1 to 1:10 (w / v). In a preferred embodiment, 100 g of dried plant material is placed in contact with 900 ml of a polar solvent and left to stand at room temperature for at least 1 day, preferably at least 5 days, and even more preferably between 5 and 10 days. In a preferred embodiment, the polar solvent is ethanol. It is well known in the prior art that various modifications can be made to the process for obtaining the extract without significantly affecting the composition of the resulting extract, which is the essential part of the procedure. For example, a larger volume of solvent can be used to improve extraction efficiency, or the resting times can be shortened by increasing the temperature. A mild temperature, between 20°C and 40°C, could be used without significantly affecting the composition of the extract, but the resting times would be reduced. All these variations or adjustments to the conditions and parameters of the extraction process fall within the scope of the present invention. b) Recover the solvent extract; preferably the plant matter is removed and the resulting extract is recovered; c) Sieve the extract to remove solid residues, one or more sieves can be used, starting with the lowest number and increasing until the desired number is reached, preferably a No. 200 sieve is used; d) Remove the solvent from the extract using any appropriate technique, preferably rotary evaporation at a temperature of 40°C to 55°C, a rotation speed of 60 rpm to 80 rpm and a vacuum pressure of 50 mbar to 72 mbar: e) Remove the solvent completely using any appropriate technique, preferably in an oven or stove under recirculating air for 24 hours at 50 ± 2 C: f) Recover the dry extract; g) Optionally, a 50% solution of the dry Jamaica extract can be prepared with water, sterilized and stored under refrigeration until use. The extract may be present in a concentration of 0.1% to 10%, preferably between 0.5 and 5%, in another, equally preferred form, it is found in a concentration of 2% (w / w). The excipients or vehicles considered pharmaceutically acceptable for use in the compositions of the present invention may include those commonly known, such as sucrose, mannitol, sorbitol, xylitol, sodium fluoride, sodium benzoate, menthol, hydrogenated castor oil (PEG-40), ethanol, glycerol, water, and others (including combinations thereof). Remington's Pharmaceutical Sciences is a reference work on this subject. The pharmaceutically acceptable additive should be chosen based on the desired pharmaceutical form, for example, if a solid form is desired. For example, a freeze-dried powder and its respective reconstituting solution; or a liquid form. Remington's Pharmaceutical Sciences is a reference work on this subject. In this sense, at least one additive can be selected from among an osmoregulator, a bulking agent, a pH regulator, a solubilizing agent, a matrix forming agent, a diluting agent, a stabilizing agent, a protective agent, an antioxidant agent, a binding agent, among others. For the present invention, the filler agents for preparation in lyophilized forms comprise, but are not limited to: sugars, such as mannitol, xylitol, sucrose, trehalose, sorbitol or raffinose; amino acids, such as arginine, glycine and histidine; polymers, such as dextrans and polyethylene glycol. The pH regulator can be selected from any suitable acid-base pair, depending on the desired pH value. Compounds suitable for freeze-dried formulations include citric acid, potassium citrate, sodium citrate, tartaric acid, sodium phosphate, Tris base, Tris HCl, Tris acetate, hydrochloric acid, and sodium hydroxide, among others. The solubilizing agent can be selected from a complexing agent, such as EDTA, cyclodextrins; surfactants, such as polysorbates; cosolvents, such as alcohols, glycerol, among others. The matrix forming agent can be selected from gelatin, dextrans, alginates, and maltodextrins, among others. For the purposes of the present invention, the osmoregulating agent refers to a composition comprising sodium fluoride in a w / w concentration of 0% to 0.2%, preferably 0.05%; sodium benzoate in a w / w concentration of 0% to 1%, preferably 0.1%; xylitol in a w / w concentration of 0% to 5%, preferably 2.5%; menthol in a w / w concentration of 0% to 1%, preferably 0.2%; PEG 40 in a w / w concentration of 0% to 10%, preferably 6.59%; and sorbitol in a w / w concentration of 0% to 10%, preferably 6.59%. The composition may be conveniently presented in unit-dose form and may be prepared by any of the well-known methods in pharmaceutical technique. All methods include the step of combining a polar extract of Jamaica and at least one pharmaceutically acceptable additive or salt, ester, prodrug, or solvate thereof (the “active ingredient”) with the vehicle, which constitutes one or more additional ingredients. In general, formulations are prepared by uniformly and intimately combining the active ingredient with liquid vehicles or finely divided solid vehicles, or both, and then, if necessary, shaping the product into the desired formulation. The methods for preparing various pharmaceutical compositions with a specific amount of active compound are known, or will be evident, to those skilled in this art. For the purposes of the present invention, the formulations described herein are administered directly to humans. EXAMPLES The following processes related to the materials for carrying out the experimental tests are included below, with the sole purpose of illustrating the present invention, without implying any limitation to its scope. Extract For obtaining the antibacterial extract, hibiscus (H. sabdariffa) was used. The sample was handled under aseptic conditions and transported to the laboratory on the day of purchase. The plant was dehydrated in an oven at 40 ± 2°C for 7 days, placed in a sterile plastic bag, and stored at room temperature until use. In one modality, the Tecoanapa variety of Jamaica from Guerrero, in the state of Oaxaca, Mexico, was used. An ethanolic extract solution of H. sabdariffa was prepared from a previously obtained and dried extract. Autoclaved extract was used. Distilled water was used as the solvent for the dried extract; the test solution was prepared in a 1:10 extract:diluent ratio (final concentration: 100 mg / mL). The extract solution was sterilized in an autoclave (Yamato Scientific, Japan) at 121°C (15 lb / in²) for 15 minutes. After the solution cooled to room temperature, its antimicrobial effect against the test bacteria was determined. Preparation of mouthwash The mouthwash formulation of the present invention comprises a synergistic combination of an antimicrobial agent, which is an ethanolic extract of Jamaican calyces, and at least one or more additive agents or excipients.In one embodiment, at least one additive or excipient agent is selected from among a surfactant, a fluoride ion provider, a preservative, a vehicle, a solvent, a sweetener, an anticaries agent, a flavoring agent, a fragrance agent, a cooling agent, a coloring agent, a wetting agent, a bleaching agent, a palatability enhancer, a pH regulator, a thickening or viscosifying agent, and any combination of such agents, among others suitable for this type of oral formulation. For the purposes of the present invention, the term “an agent” shall be interpreted in its broadest sense, without limitation of number; for example, the term “an additive agent” means that the composition comprises any one, two, or more additive agents. The purpose of an anti-caries agent or dental protector is to decrease the solubility of dental enamel in acid and to protect teeth against cavities. Consequently, any anti-caries agent is appropriate for the present invention, as long as it does not interfere with the activity of the other components of the invention. Among the most important anti-caries agents are those that provide fluoride ions, such as sodium fluoride, potassium fluoride, potassium stannous fluoride (SnF2-KF), sodium stannous hexafluoro, stannous chlorofluoride, sodium fluorozirconate, and sodium monofluorophosphate, among many others. The purpose of the solvent agent is to improve the homogeneity of the solution and the overall solubility of the formulation components. Consequently, any solvent agent compatible with the components of the present invention is useful, as long as it does not interfere with the activity of the other components of the invention. Some of the solvent agents include water, ethanol, ethyl acetate, or acetone, and combinations thereof. The surfactant can be non-ionic or ionic in nature, and within the latter, it can be anionic, cationic, or amphoteric, such that any anticaries agent can be used for the present invention, provided it does not interfere with the activity of the other components of the invention. Some surfactants include macrogol, polysorbates, and polyethylene glycol. In some embodiments, the surfactant also functions as a wetting agent or solvent. The wetting agent for the present invention may be any orally acceptable wetting agent, provided it does not interfere with the activity of the other components of the invention; some of the wetting agents include polyhydric alcohols such as glycerin, propylene glycol, sorbitol, xylitol, or low molecular weight polyethylene glycol. The flavoring agent is intended to impart a pleasant taste to the user. Consequently, any flavoring agent is suitable for the present invention, provided it does not interfere with the activity of the other components of the invention. Some flavoring agents include flavoring oils such as mint, peppermint, eucalyptus, camphor, lemon, orange, grapefruit, thyme, chamomile, spearmint, wintergreen, and cinnamon, among many others. In some embodiments, the flavoring agent includes a cooling agent, which is intended to impart a cooling sensation to the mouth, nose, and intestinal tract. Some cooling agents useful in the present invention are menthol, menthol derivatives, menthyl acetate, menthyl lactate, acyclic and / or cyclic carboxamides, N-substituted paramenthane carboxamides, phosphine oxides, substituted p-menthanes, menthoxypropane, alpha-ketoenamine derivatives, and p-menthane carboxamide.N-substituted, menthyl semi-ester derivatives and combinations thereof; a refreshing flavoring combination useful for the present invention is the combination of menthol and methyl salicylate. The purpose of the sweetening agent is to provide a sweet taste; consequently, any sweetening agent is appropriate for the present invention, provided it does not interfere with the activity of the other components of the invention. Some of the sweetening agents are xylitol, sorbitol, mannitol, maltitol, lacitol, sucralose, saccharin, acesulfame, aspartame, cyclamate, neosperidin, thaumatin, and any combination thereof, among many others. The bleaching agent is intended to whiten teeth; consequently, any bleaching agent is appropriate for the present invention, provided it does not interfere with the activity of the other components of the invention. Some of the bleaching agents are peroxide compounds, such as hydrogen peroxide, alkali or alkaline earth metal peroxides, organic peroxide compounds and peroxyacids and salts thereof; chlorine dioxide; chlorites and hypochlorites. The purpose of the coloring agent is to adjust the color of the formulation to improve its appeal to the consumer; consequently, any orally acceptable coloring agent is useful, as long as it does not interfere with the activity of the other components of the invention; some of the coloring agents include pigments, inks, lacquers, and combinations thereof. In a preferred embodiment, the mouthwash formulation of the present invention is routinely used for oral care and hygiene, for aesthetic, hygienic, and therapeutic purposes. For example, the therapeutic purpose is achieved with the antimicrobial agent that inhibits bacterial growth in the oral cavity, preventing the formation of dental plaque, gingivitis, and periodontal disease. The mouthwash formulation of the present invention comprises an antimicrobial agent and at least one or more additive or excipient agent. The mouthwash formulation of the present invention is characterized in that it comprises a synergistic combination of an ethanolic extract of hibiscus with a surfactant, a preservative, and an agent that provides fluoride ions. In a preferred embodiment of the present invention, the mouthwash formulation of the present invention comprises a synergistic combination of an ethanolic extract of hibiscus with a surfactant, a preservative, and an agent that provides fluoride ions, and also comprises at least one or more orally acceptable additives or excipients. For the present invention, the at least one orally acceptable additive or excipient is selected from among a surfactant, a fluoride ion provider, a preservative, a vehicle, a solvent, a sweetener, an anticancer agent, a flavoring agent, a fragrance agent, a cooling agent, a coloring agent, a wetting agent, a bleaching agent, a palatability enhancer, a pH regulator, a thickening or viscosifying agent, and any combination thereof, among others suitable for this type of oral formulation. For the present invention, the term 'an agent' shall be interpreted in its broadest sense, without limitation of number; for example, the term 'an additive' means that the composition comprises any one, two, or more additive agents. In another, equally preferred embodiment of the present invention, the mouthwash formulation comprises the synergistic combination of an ethanolic extract of hibiscus, a surfactant, a preservative, and a fluoride ion provider; and at least one or more additive agents or excipients. The orally acceptable additive agent or excipient is selected from a vehicle, solvent, sweetener, anticaries agent, flavoring agent, aromatizing agent, refreshing agent, coloring agent, humectant, bleaching agent, palatability enhancer, pH regulator, thickening or viscosifying agent, and any combination thereof. In another, equally preferred embodiment of the present invention, the mouthwash formulation comprises an ethanolic extract of hibiscus as an antimicrobial agent, polyethylene glycol as a surfactant, sodium benzoate as a preservative, and sodium fluoride as a fluoride ion provider. In another, equally preferred embodiment of the present invention, the mouthwash formulation comprises: ethanolic extract of hibiscus, polyethylene glycol 40, sodium fluoride, sodium benzoate, sodium fluoride, and sweetening and refreshing agents. In another, equally preferred embodiment of the present invention, the mouthwash formulation comprises ethanolic extract of hibiscus in an amount of 0.1 to 10%, preferably 0.5 to 5%; in another, equally preferred embodiment, the ethanolic extract of hibiscus is present in a concentration of 2% (w / w); the surfactant is present in an amount of 0.1 to 15%, preferably 1 to 10%; the preservative is present in an amount of 0.01 to 5%, preferably 0.01 to 1%; and the fluoride ion provider is present in an amount of 0.01 to 1%, preferably 0.01 to 0.5%. In another, equally preferred embodiment of the present invention, the mouthwash formulation comprises ethanolic extract of Jamaica (hibiscus) in an amount of 2%; polyethylene glycol 40 in an amount of 6.59%; sodium benzoate in an amount of 0.10%; sodium fluoride in an amount of 0.05%; and at least one or more orally acceptable additives or excipients selected from a flavoring agent and a sweetening agent. The flavoring agent is menthol, present in an amount of 0.2%; the sweetening agents are xylitol and sorbitol, with xylitol present in an amount of 2.5% and sorbitol present in an amount of 6.59%. The rinse of the present invention was prepared with the ingredients and proportions described in Table 1. Table 1. Ingredients and proportions of the evaluated mouthwash Mouthwash formulation component % (w / v) Ethanolic extract of hibiscus 2 sodium fluoride 0.05 ! Sodium benzoate 0.1 PEG 40 6.59 j Xylitol 2.5 | Menthol 0.2 Sorbitol 6.59 ' Bacterial strains The following strains of pathogenic bacteria from the oral cavity were used: Streptococcus mutans (ATCC 25175), Staphylococcus aureus (ATCC 25923), Streptococcus sanguinis (ATCC 15300) and Capnocytophaga gingivalis (ATCC) 33624. For the monoculture test, S. mutans (ATCC UA159) and S. aureus (ATCC 12600) were used. Example 1. Antimicrobial effect in culture medium. Commercial mouthwash solutions. Commercial mouthwashes were purchased at a pharmacy: Astringosol®, Colgate Plax Infinity®, Crest Pro Health®, Dental Max®, Equate®, and Listerine Zero®. Inoculation of the culture plates with the bacteria under study. The antimicrobial effect of the ethanolic extract was evaluated separately from a comparative study against the antibiotic nalidixic acid. These studies were conducted against Streptococcus mutans (ATCC 25175), Staphylococcus aureus (ATCC 25923), Streptococcus sanguinis (ATCC 15300), and Capnocytophaga gingivalis (ATCC 33624). The four multidrug-resistant bacterial strains were incubated in 3 mL of tryptic soy broth (TSB) at 35 ± 2°C for 24 h. The bacterial culture density at 24 h was approximately 1 x 10⁹ CFU / mL. All 24 h cultures were washed three times in isotopic saline and resuspended in peptone diluent (approximate concentration 1 x 10⁹ CFU / mL). A decimal dilution was prepared from each washed bacterial culture. From this dilution, 100 pL aliquots were inoculated onto Standard Methods Agar (SMA) plates. The inoculum was spread onto the agar using the surface spreading technique. Subsequently, paper discs (Whatman No. 5, 6 mm in diameter) were placed on the agar surface. The agar diffusion technique was used. Aliquots of 20 pL of the antibiotic (30 pg / mL on a disc) and of the ethanolic extract (2 mg per disc) were placed separately on the paper discs.Three replicates were performed for each type of mouthwash and ethanolic extract. Once the liquid from the discs was absorbed by the agar, the plates were incubated for 24 h ± 2 UC. The diameter (mm) of the resulting inhibition zones was measured for the antibiotic and the ethanolic extract, and an average value was calculated for each (CLSI, 2012). Statistical Analysis All experiments were replicated three times. An exploratory data analysis was performed to verify the assumptions of equal variances and normal distribution of errors in the results obtained for the in vitro antimicrobial activity of commercial extracts and mouthwashes based on H. sabdariffa. The results were analyzed using the Statgraphics statistical software for one-way analysis of variance (ANOVA). Mean comparisons were performed using Tukey's test for each experimental group. All analyses were conducted with a significance level of p < 0.05. Results The results of the antimicrobial effect of the ethanolic extract and of commercial mouthwashes and the mouthwash of the present invention are shown in Table 2. Table 2. Antimicrobial effect of commercial mouthwashes, ethanolic extract of hibiscus calyces and mouthwash based on hibiscus calyces Strain IA 1 BCDEFGH 8 mutans 9 7±0 2 7 4±0 6 7 4±0 2 0 0 8 0±0 1 9.7+0.1 10 5±0 2 12.7±0.6 ii s aureus 9 4+0.3 9.0+0.2 8.5+0.3 i , 0 0 0.0 ! 10.3±0.1 ! ' 10 1 ±0.1 13.2±0.3 s bloods 8 1+0 7 11.5±0 7 10 0±0 5 nn ' 7 5±0 2 9.4±0 3 u. u ί II 9 4±0 1 11.3±0.6 c gingivalis : 9 7±0.2 7 9+0 2 9 1±0 7 I 0Q ! 7 4±0 1 । 9 2±0 2 9 7+0 2 11.5+0.5 A= Astringosol B= Colga te plax Ice mfinitum, C l I ! = Crest pro-health, D= Dental max E= Equate, F= Listerine Zero. G = 10% ethanolic extract, H = Mouthwash formulation containing 2% hibiscus extract. Based on the results shown in Table 2, it can be observed that the various commercial mouthwashes exhibited, on average, a lower antimicrobial effect than the treatment using only hibiscus extract (G); in fact, the treatment indicated in column D showed no effect against any of the bacteria analyzed (Table 2). Furthermore, surprisingly, the greatest antimicrobial effect was observed with the mouthwash formulation containing hibiscus extract (H); this effect was completely unexpected since the mouthwash formulation used only a 2% concentration of dry hibiscus extract, compared to treatment (G) with the control hibiscus solution, which contains a higher concentration of hibiscus extract (10%).These results conclusively demonstrate, without limiting themselves to any particular theory or mechanism, that a synergistic effect exists between the components of the mouthwash formulation with hibiscus extract. This results in an 18% to 31% increase in antimicrobial activity in the mouthwash, even when the amount of hibiscus extract used was reduced by up to five times (Table 3). This synergistic effect could be explained, without limiting themselves to any particular theory or mechanism, by the combination of the ethanolic hibiscus extract with one or more of the selected agents, such as a fluoride ion source, a surfactant, or a preservative. Table 3. Increase (%) in the antimicrobial effect of the mouthwash based on hibiscus calyxes (2% extract) compared to the control solution of aqueous hibiscus extract alone (10% extract) Ethanolic extract of hibiscus. Mouthwash with hibiscus extract. Percentage increase. 10 5±0.2 12.7±0.6 20 10.1 ±0.1 13.2±0.3 30 1 9 4±0 1 11.3±0.6 20.2 9 7±0.2 11.5±0.5 18 It is also important to note that, as any technician in the field knows, in the case of plant extracts or novel plant-derived compounds that exhibit antimicrobial effects against bacteria in culture media, there is no national or international standard for determining resistance or sensitivity. Therefore, if an inhibition zone is observed on the culture medium when examining the effect of a plant extract or novel plant-derived compound, it is assumed that the extract or compound has an antimicrobial effect, regardless of the size of the inhibition zone. Of course, it should also be understood that a longer inhibition zone is considered to indicate a greater antimicrobial effect of the plant extract or plant-derived compound.Therefore, in accordance with the above, it is considered that the mouthwash formulated with ethanolic extract of hibiscus presented a potent antimicrobial effect against the 4 bacteria of study (Table 2), an antimicrobial effect that is greater than that presented by the commercial formulations tested. Example 2. Effect of mouthwash based on aqueous extract of hibiscus on biofilm formation (dental plaque) and on tooth hardness. Biofilm obtained by monoculture Inoculum preparation The pathogenic bacteria S. mutans and S. aureus were streaked onto BHI (Brain Heart Infusion) agar plates and incubated at 37°C for 24 h. Subsequently, colonies were taken and resuspended in sterile distilled water at a McFarland scale level of 0.5, which corresponds to approximately 1.5 x 10⁵ CFU / mL. Biofilm formation in monoculture The tests were performed according to the methodology described by Peralta et al. (2015). A plastic plate containing 24 sterile wells (KASVI Laboratories LTDA) was used, and sterile titanium sheets (1 cm) were used as the test surface for dental biofilm formation (Figure a). A titanium sheet was placed in each well, 1.8 mL of BHI broth (brain heart infusion) was added, and 180 pL of S. mutans or S. aureus suspension was added. This system was incubated at 37°C for 72 h to induce biofilm formation. Every 24 h, the titanium sheets were removed from the wells and washed with sterile 0.9% NaCl solution. The culture medium was removed from the wells by decantation, and each well was washed with the 0.9% NaCl solution. New BHI broth was added to each washed well and the washed titanium plates were placed in the wells. The experiment was performed in triplicate. At the end of 72 h, the titanium foils were removed, washed with NaCl solution, transferred to 1 mL of 0.9% NaCl solution, and sonicated for 30 s at 30 W (S500 Sonicator). Subsequently, decimal dilutions were made using the surface spreading technique on BHI agar and incubated at 37°C for 2 h. The mean colony-forming units (CFU) plus the standard deviation of the three replicates were reported. Action of rinses and chlorhexidine on monoculture biofilms A mouthwash formulation containing ethanolic and ethanolic extracts of Hibiscus sabdariffa criolla from Oaxaca and Tecoanapa de Guerrero was used. Positive controls were 0.12% chlorhexidine and Listerine. For this test, a modification was made to the methodology of Peralta et al. (2015). After the first 48 hours of strain growth in the agar trays and adhesion to the titanium plates, the plates were washed in 0.9% NaCl solution, immersed in the mouthwash or chlorhexidine solutions for 5 minutes, and then washed again in NaCl solution. To evaluate the effect of each mouthwash, a negative control for each microorganism was used, meaning it was not exposed to the test solutions. After this period, the microorganisms were counted according to the methodology described by Peralta et al. (2015) using BHI agar and incubating at 37°C for 24 h. Biofilm produced in a microcosm Experimental design and conditions The microcosm biofilm model described by Filoche et al. (2007, 2008) was used with modifications. Human saliva was used as inoculum and bovine enamel as substrate. The growth medium was a defined mucin-enriched medium (DMM), which contains ions, mucin, a basal mixture of amino acids, vitamins, and growth factors (Wong and Sissons 2007). Biofilm formation was induced for 7 days and occurred under a semi-continuous regime or under DMM, alternately with and without sucrose. The factors under evaluation were mouthwash containing 2% aqueous extract of Hibiscus sabdariffa, 0.12% chlorhexidine as a positive control, Listerine mouthwash, and a second control (mouthwash formulation without extract). Eight biofilms formed for each control and treatment (n = 8).For all experiments, the response variables were CFU and the percentage loss of enamel minerals due to the change in surface hardness (% SHC). Enamel discs Freshly extracted bovine central incisors were used as dental samples, and enamel discs (5 mm in diameter and 2 mm thick) were obtained from them. The cuts were made perpendicular to the buccal surface of the central incisors using a cylindrical drill with a diamond-coated cutter (trephine). The dentin and enamel surfaces were polished with silicon carbide sandpaper of grits 400, 600, 800, 1200, 1500, and 2000, respectively. Both surfaces of the enamel disc were flat and parallel. Nail polish was applied around the enamel discs, leaving only the buccal enamel surface exposed. The coated discs were sterilized in an autoclave and stored at 48°C in a humidified atmosphere until use (Filoche et al., 2007; Filoche et al., 2008). Saliva collection For each experimental run, fresh stimulated saliva was collected from a healthy individual (female, 32 years old) who had not been on antibiotic therapy for 6 months. Saliva was collected in the morning (after fasting), and the volunteer abstained from oral hygiene for 24 hours prior to collection. An aliquot of the saliva was taken to determine the baseline microbial concentration (CFU mL1). Inoculation, biofilm formation, and treatment with the test solutions Sterile 24-well plastic plates (TPP - Techno Plástic Products, Trasadingen, SU) were used to induce biofilm formation on enamel discs. Biofilm formation was carried out over 96 h. Under aseptic conditions, each enamel disc was placed in a well of the plate, and 0.4 mL of fresh, homogenized saliva was added to each well. After 1 h at room temperature (22-25°C), the saliva was aspirated, and 1.8 mL of growth medium (DMM) with sucrose was added. The plates were then incubated in an atmosphere of 5-10% CO₂ and 51% O₂ (Anaerobic - Probac do Brasil produtos Bacteriológicos Ltda, Santa Cecilia, SP, Brazil), created in anaerobic flasks (Probac do Brasil produtos Bacteriológicos Ltda) at 37°C for 6 h. Subsequently, the growth medium (DMM) with sucrose from the wells in which the discs were located was discarded, and the discs were washed with sterile NaCl solution.The discs were placed in a new sterile 24-well plate and new sucrose-free growth medium (DMM) was added. The plate was incubated again in an atmosphere of 5-10% CO2 and 51% O2 for 18 h at 37°C for a total of 96 h. Every 24 h, for 4 days, the same procedure was repeated. Treatments with the test and control solutions (0.9% NaCl solution) began on the second day of the experiment and were applied before feeding the wells with sucrose-containing growth medium (DMM). Discs were taken from the wells and separately immersed completely in the test and control solutions for 1 min. After treatment, the discs were washed by immersion for 10 s in sterile saline solution and placed in wells containing the growth medium. Treatments with the test solutions were applied on the second, third, and fourth days of the experiment. At the end of 96 h, the discs were removed from the wells, washed in a 0.9% NaCl solution, transferred to tubes containing 1 mL of 0.9% NaCl solution, and sonicated for 10 s at 30 W. Subsequently, decimal dilutions were made from each tube in 0.1% peptone diluent, and the microorganisms were counted using the surface spreading technique on blood agar, mitis salivarius agar, and MRS agar. The inoculated culture plates were incubated for 72 h in an atmosphere of 5–10% CO2 and 51% O2 (Anaerobac - Probac do Brasil produtos Bacteriológicos Ltda, Santa Cecilia, SP, Brazil) at 37°C for 24–48 h. The discs that were sonicated (for microbial counting) were subsequently cleaned with a soft bristle brush and distilled water, then placed individually in Eppendorf-type plastic tubes and kept at 48°C in a humid atmosphere until the enamel hardness analysis was performed (Filoche, et al., 2007; 2008). Enamel hardness The surface hardness of the enamel discs was measured before (sound enamel) (SH) and after the experiments (SH2) (Micro Hardness Tester FM 700 - Future-Tech Corp., Kawasaki, Japan). The percentage change in surface hardness was calculated using the following formula: [% SHC -100 (SH2 - SH) / SH]. Statistical Analysis All experiments were replicated three times. An exploratory data analysis was performed to verify the assumptions of equal variances and normal distribution of errors for the results obtained from the biofilm assays produced by monoculture and microcosm culture, the percentage change in enamel surface hardness (% SHC), and the logarithms of colony-forming units (CFU). These results were analyzed using the Statgraphics statistical software for one-way analysis of variance (ANOVA). Mean comparisons were performed using Tukey's test for each experimental group. All analyses were conducted with a significance level of p < 0.05. Results Effect of rinses and chlorhexidine on biofilm obtained in monoculture In this experiment, 0.12% chlorhexidine (positive control) completely inhibited biofilm formation by both S. mutans and S. aureus, unlike any of the other solutions tested (Tables 4 and 5). However, the mouthwash containing ethanolic extract of H. sabdariffa significantly reduced the concentration of microorganisms in the biofilm compared to the second control and the Listerine mouthwash (Tables 4 and 5). It is worth noting that this is the first report in the scientific literature evaluating the effect of mouthwashes on a biofilm obtained using the monoculture technique. Table 4. Mean viable bacteria (log CFU / cm2) in biofilm produced by monoculture for S. mutans (ATCC UA159) in the efficacy of mouthwashes containing Hibiscus sabdariffa extracts and commercial mouthwashes Treatment Average log CFU concentration 104 Formulation Rinse without extract1 7.9610.55 Rinse with H. sabdariffa extract 6.6410.4 Chlorhexidine 0.12 % 0.0 Commercial rinse Listerine 7.4210.22 Water 8.1210.13 '6 replicate mean of log UFC / cm2+ standard deviation Table 5. Mean viable bacteria (log CFU / cm2) in biofilm produced by monoculture for S. aureus ATCC 12600 in the efficacy of mouthwashes containing Hibiscus sabdariffa extracts and commercial mouthwashes. Treatment Median log CFU concentration 104 Formulation Rinse without extract1 7.55±0.5 Rinse with H. sabdariffa extract 618±0.1 Chlorhexidine 0.12 % 0.0 Listerine 7.76±0.2 Control 8.05±0.1 'Mean of 6 replicates of log UFC / cm2± standard deviation. Effect of rinses and chlorhexidine on biofilm obtained by microcosm In the case of the biofilm obtained by microcosm, chlorhexidine did not inhibit biofilm formation (Tables 6 and 7), unlike the biofilm obtained by monoculture (Tables 4 and 5). The main explanation is that the monoculture model contains only one type of microorganism and possibly a less resistant biofilm than the one formed in the microcosm model. In the latter, different genera and species of oral bacteria (because the biofilm was induced using saliva from a volunteer) participate in the formation of a more resistant biofilm. Furthermore, the biofilm may contain microorganisms resistant to chlorhexidine (natural or acquired resistance). In this case, the biofilm obtained by the microcosm model would more closely resemble what occurs naturally in the human oral cavity. Table 6. Mean total anaerobes (log CFU / mL) in biofilm produced by microcosms in the efficacy of mouthwashes containing Hibiscus sabdariffa extract and commercial mouthwashes. Treatment Average log CFU concentration 10.5 Formulation Rinse without extract1 8.27±0.12c Ethanolic rinse H. sabdariffa 7.1±0.10bc Chlorhexidine 0.12 % 8.18±0.11bí: Listerine 8.27±0.08i: 'Mean of 8 replicates of log CFU / mL ± standard deviation Regarding mouthwashes, the one containing H. sabdariffa extract showed a greater effect on preventing biofilm formation than the commercial mouthwash Listerine, and some of them were more effective than chlorhexidine (positive control) (Tables 6 and 7). Chlorhexidine showed a similar effect on the concentration of total anaerobes as the mouthwashes containing ethanolic extracts of hibiscus (Table 6). As for the total amount of Streptococcus sp., there was no significant difference in the antimicrobial effect between chlorhexidine and the ethanolic mouthwashes of H. sabdariffa. However, the lowest concentrations of Streptococcus sp. were obtained with the chlorhexidine treatments and the mouthwashes containing ethanolic extracts, which is interpreted as a greater effect in preventing or delaying biofilm formation (Table 7).The Listerine mouthwash treatment resulted in the highest concentration of Streptococcus sp, which can be interpreted as having a limited or no effect on biofilm formation (Table 7). Table 7. Mean total Streptococcus sp. (log CFU / mL) in biofilm produced by microcosms in the efficacy of mouthwashes containing Hibiscus sabdariffa extract and commercial mouthwashes. Treatment Average log CFU concentration 104 Formulation Rinse without extract1 7.24+0.37 Rinse with H. sabdariffa extract 6.15+0.43 Chlorhexidine 0.12 % 6.31+0.71 Listerine 7.72+0.53 'Mean of 8 replicates of log CFU / mL ± standard deviation Surface hardness of dental enamel after biofilm treatment with the test solutions The enamel hardness of the teeth used in the study was reduced with all rinse treatments, chlorhexidine, and the control solutions. The percentage of loss ranged from 60 to 80%. However, no significant difference was observed in the reduction of enamel hardness between treatments or compared to the control (Table 8). Table 8. Surface hardness of dental enamel after treatment of biofilm produced by microcosms in the effectiveness of mouth rinses containing Hibiscus sabdariffa extracts and commercial rinses. Treatment Percentage of hardness loss Formulation Rinse without extract1 74±3.65a Ethanolic rinse H. sabdariffa 86± 10.32a Chlorhexidine 0.12 % 67±8.41a Listerine 77+9.33a 'Mean of 4 replicates of percentage of mean loss of tooth hardness ± standard deviation. Letter value per treatment expresses significant difference with a = 0.05, Tukey test. There was no statistically significant difference between the groups in the loss of hardness (p=0.0976) The results above show that a formulation containing ethanolic extract and an acceptable pharmaceutical additive or vehicle has a beneficial effect against infections and diseases caused by pathogenic bacteria affecting the oral cavity or teeth; and that the effect is potentiated when aqueous extract of hibiscus calyces is included. This also means that a lower concentration of ethanolic extract can be used in the mixture, which significantly reduces the risk of potential toxicity caused by commercial mouthwash solutions or drugs such as chlorhexidine, whether due to the frequency or high concentration at which they are used. Finally, although the foregoing description was made taking into account the preferred embodiments of the invention, those skilled in the art should bear in mind that any modification of form and detail will be considered within the spirit and scope of the present invention. The terms in which this specification has been drafted should always be taken in a broad and non-restrictive sense. The materials, form, and description of the elements may be varied provided that this does not alter the essential characteristics of the model. LITERATURE Castro-Rosas, J., EML Santos, CA Gómez-Aldapa, CAR González, JR Villagomez-lbarra, AJ Gordillo-Martínez, A. Villarruel-López, and M R. Torres-Vitela. 2010. Incidence and behavior of Salmonella and Escherichia coli on whole and sliced ​​zucchini squash (Cucurbita pepo) fruit. J. Food Prot. 73:1423-1429. 2. Gutiérrez-Alcántara EJ, Gómez-Aldapa CA, Román-Gutiérrez AD. Rangel-Vargas E, González-Olivares LG, Castro-Rosas J. 2016a. Antimicrobial activity of roselle Hibiscus sabdariffa calyx extracts on culture media and carrots against multidrugresistant Salmonella strains isolated from raw carrots. Journal Food Safety doi:10 1111 / jfs. 12259. Gutiérrez-Alcántara EJ, Rangel-Vargas E, Gómez-Aldapa CA, Falfan-Cortes RN, Rodriguez-Marín ML, Godínez-Oviedo A, Cortes-López H, Castro-Rosas J. 2016b Antibacterial effect of roselle extracts (Hibiscus sabadariffa), sodium hypochlorite and acetic acid against multidrug-resistant Salmonella strains isolated from tomatoes. Letters in Applied Microbiology. 62:177-184. 4. WHO (World Health Organization). (2016). Antimicrobial resistance, https: / / www.who.int / es / news-room / fact-sheets / detail / resistenciaa-los-antibi%C3%B3ticos 5. Radulovic, N. S., Blagojevic, P. D., Stojanovic-Radic, Z. Z., Stojanovic, N. M. 2013. Antimicrobial plant metabolites: structural diversity and mechanism of action. Current medicinal chemistry, 20(7), 932-952.

Claims

1. A mouthwash formulation characterized in that it comprises a synergistic combination of ethanolic extract of hibiscus, with a surfactant, a preservative, and an agent providing fluoride ions; and at least one or more orally acceptable additives or excipients.

2. The mouthwash formulation according to claim 1 further characterized in that the hibiscus extract is present in an amount of 0.1 to 10%, preferably 0.5 and 5% (w / v).

3. The mouthwash formulation according to claim 1 further characterized in that the hibiscus extract is present in an amount of 2% (w / v).

4. The mouthwash formulation according to any of claims 1 to 45, further characterized in that the ethanolic extract of hibiscus is present in an amount of 2%; the surfactant is present in an amount of between 0.1% and 15%, preferably between 1% and 10%; the preservative is present in an amount of between 0.01% and 5%, preferably between 0.01% and 1%; and the fluoride ion-providing agent is present in an amount of between 0.01% and 1%, preferably between 0.01% and 0.5%.

5. The mouthwash formulation according to any of claims 1 to 4 further characterized in that the surfactant is polyethylene glycol, the preservative is sodium benzoate, and the fluoride ion-providing agent is sodium fluoride.6 The mouthwash formulation according to any of the preceding claims, further characterized in that at least one or more orally acceptable additives or excipients are selected from among a vehicle, a solvent, a sweetener, an anticaries agent, a flavoring agent, a fragrance agent, a refreshing agent, a coloring agent, a wetting agent, a bleaching agent, a palatability enhancer, a pH regulator, a thickening or viscosifying agent, and any combination thereof. 7 The mouthwash formulation according to any of claims 1 to 7, further characterized in that the ethanolic extract of hibiscus is present in an amount of 2%; polyethylene glycol 40 is present in an amount of 6.59%; sodium benzoate is present in an amount of 0.10%; sodium fluoride is present in an amount of 0.05% and at least one or more orally acceptable additives or excipients are selected from a flavoring agent and a sweetening agent.

8. The mouthwash formulation according to the preceding claim, further characterized in that the flavoring agent is menthol, and is present in an amount of 0.2%; the sweetening agent is xylitol and sorbitol, the xylitol being present in an amount of 2.5% and the sorbitol being present in an amount of 6.59%.