Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
  • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone

Definitions

• the present invention relates to a composition for oral use based on active ingredients of natural origin such as pea proteins and tamarind seed polysaccharides.
• the present invention also relates to a composition for oral use based on active ingredients of natural origin, such as pea proteins and tamarind seed polysaccharides, and to its use in therapy, particularly for use in a method for the treatment and/or prevention of oral mucosa disorders, particularly but not limited to stomatitis, aphthae and oral ulcers.
• Topical agents such as local antibiotics, local antiseptics, topical NSAIDs, and topical corticosteroids are generally prescribed to relieve symptoms.
• Various approved drug formulations such as pills, mouthwashes, sprays, and pastes, such as vitamin B12, chlorhexidine mouthwash, steroid tablets, and local anesthetics are mainly suggested for the treatment of aphthous ulcers.
• These topical symptomatic treatments, such as corticosteroids can reduce the severity of ulceration but are not always able to stop recurrences; moreover, longterm side effects are known.
• dentists are often asked to recommend safe and rapid therapy to relieve the pain caused by mouth ulcers.
• Patents EP3131568 B1 and EPO3348273 B1 describe a combination of xyloglucan extracted from tamarind and pea protein for the treatment of diarrhea and other intestinal infections and/or inflammation.
• Patent application WO2017207223 A1 describes the use of protein-polysaccharide conjugates in the treatment of intestinal disorders.
• composition of the invention in therapy particularly, but not only, in the prevention and treatment of mouth disorders, such as stomatitis, mouth ulcers, and aphthae.
• Figure 1 Results of the in vitro study: mucoadhesivity on reconstructed human oral epithelium.
• FIG. 2 Labeling index (LI) of BrdU-labeled cell; values are shown as mean ⁇ SEM. *** P ⁇ 0.001.
• Figure 5, 5A and 5B Effect of the composition of the invention on histopathological changes caused by phenol- induced oral ulcer and cytokine production in phenol-induced oral ulcer.
• an association or combination of specific active ingredients of natural origin, and particularly of plant origin is particularly effective in the treatment and/or prevention of oral disorders, particularly disorders of the oral mucosa, preferably, but not limited to, stomatitis, aphthae, and ulcers.
• an oral topical composition comprising a mixture (I) that comprises or, alternatively, consists of:
• Pea protein (or isolated pea protein) (I) occurs, for example, as a light cream-colored powder derived from Pisum sativum.
• PPs are commercially available in powder form, generally light cream in color and with a bland odor and characteristic taste.
• PPs of the invention are high quality non-GMO.
• PPs are known to be a good source of essential amino acids and fiber and are highly digestible.
• PPs are depot proteins composed mainly of albumins, legumins, and vicilins and are characterized by high solubility at alkaline pH, minimal solubility at the isoelectric point, and moderate solubility in acidic environments.
• globulins and vicilins impart gelling properties to PPs.
• Plant protein products are approved as food additives according to FDA (21CFR ⁇ 170.3), and PPs received Generally Recognized Safe (GRAS) status in 2015 (GRN 608 Axiom Foods GRN 581 and GRN 788) after evaluation of absorption, distribution, excretion, bioavailability studies, toxicity, and mutagenicity.
• GRAS Generally Recognized Safe
• Tamarind seed polysaccharide also called “xyloglucan”
• xyloglucan is a hemicellulose that comes from the kernel, or endosperm, of the seeds of the tamarind tree (Tamarindus indica) and is produced by separating and refining the polysaccharide component.
• Xyloglucan is commercially available as a white/whitish powder.
• Xyloglucan (also referred to here as "XG”) is a nonionic, neutral, branched polysaccharide consisting of a cellulose-like backbone known to have high viscosity in aqueous solution, broad pH tolerance and high solubility in water. Its gel form can occur when xyloglucan is in aqueous phase under certain conditions.
• Xyloglucan is known to be noncytotoxic and not absorbed in the gastrointestinal tract. In addition, xyloglucan has also received Generally Recognized Safe (GRAS) status.
• GRAS Generally Recognized Safe
• tamarind seed extracts rich in xyloglucans are on the market from Indena SpA (Xilogel®) and DSP Gokyo Food & Chemical (Japan) (Glyloid®).
• the molecular weight of xyloglucans contained in said products is preferably from 400,000 to 650,000 daltons.
• compositions comprising pea protein and xyloglucan exert a significant beneficial effect on mucous membranes, particularly the oral mucosa.
• composition of the invention has been shown to be very useful and effective in the treatment of stomatitis, aphthae, and mouth ulcers.
• composition of the invention has been shown to be safe and nontoxic in cytotoxicity, sensitization and irritation tests, and it has a remarkable ability to adhere to the mucous membranes of the mouth, as detailed in the experimental section below.
• composition of the invention thus represents an effective and natural therapeutic answer to oral cavity disorders, providing immediate pain relief through the formation of a protective barrier that also prevents the adhesion and proliferation of pathogens.
• composition of the invention is also highly effective for the treatment of oral disorders such as stomatitis, aphthae and ulcers, due to its properties of promoting mucosa integrity and enabling the restoration of physiological functions of epithelial cells, while protecting them from further external insults and thereby attenuating the inflammatory response associated with mouth injuries.
• composition of the invention exerts rapid action for the healing of stomatitis, aphthae and oral ulcers and oral mucosa disorders in general.
• the subject to be treated with the composition of the invention is preferably a mammal, more preferably a human being.
• composition of the invention is non-toxic and can also be administered to children, pregnant women, the elderly and frail individuals.
• composition of the invention is therefore formulated for oral topical use, such as in the form of a paste, ointment, gel or other suitable preparation, preferably in the form of a gel suitable for application to the oral mucosa.
• the oral composition of the invention comprises at least one vehicle and/or excipient of conventional pharmaceutical or food grade.
• the types of excipients and vehicles of pharmaceutical or food grade used for preparing the composition of the invention, the content ratios of additives to active ingredients, and the methods for preparing the composition may be appropriately chosen by the expert in the field.
• Organic or inorganic substances, or solid or liquid substances may be used as excipients and vehicles, as long as they are of pharmaceutical or food grade.
• excipients used in the preparation of gel form compositions include, for example, one or more excipients and vehicles selected from gums, e.g., xanthan gum; oils, e.g., castor oil; polymers, e.g., polyvinyl pyrrolidone; chelating agents, e.g., EDTA salts; organic acids, e.g., lactic acid; antiseptic agents, e.g., benzalkonium chloride; and the like.
• gums e.g., xanthan gum
• oils e.g., castor oil
• polymers e.g., polyvinyl pyrrolidone
• chelating agents e.g., EDTA salts
• organic acids e.g., lactic acid
• antiseptic agents e.g., benzalkonium chloride
• composition may also contain sweetening agents, preservatives agents, and flavoring agents.
• Sweeteners can be one or more naturally occurring sugars, optionally reduced, such as sucrose, dextrose, xylitol, mannitol, or sorbitol, or a synthetic product such as sodium saccharin, aspartame, acesulfame k, or sucralose.
• Flavoring agents are pharmaceutically acceptable flavors and tastes of synthetic oils or natural oils, the latter extracted from plants, flowers, fruits, and combinations thereof, such as cinnamon, mint, anise, and citrus leaves, bitter almonds, citrus fruits, especially orange and/or lemon oils, linden, vanilla, chocolate, and grapefruit. Flavorings of chocolate, vanilla or eucalyptus and fruit essences, especially apple, pear, peach, strawberry, apricot, orange, lemon and grape, can also be used advantageously.
• composition of the invention can be packaged in dosage units or multi-dose packages, according to conventional methods well known to the expert in the field.
• the composition of the invention is in the form of a gel to be applied to the injured parts of the oral mucosa, such as by means of a dropper.
• the composition of the invention comprises an amount by weight from 0.1% to 5% of pea protein, preferably an amount by weight from 0.5% to 3% , more preferably from 0.8% to 2%, even more preferably from 1.0% to 1.1%, advantageously about 1.05%; and an amount by weight from 0.01% to 3% of xyloglucan, preferably an amount by weight from 0.1% to 1%, more preferably from 0.2% to 0.8%, even more preferably from 0.3% to 0.5%, advantageously about 0.45%; preferably together with at least one conventional vehicle and/or excipient, said percentages being expressed by weight relative to the total weight of the composition.
• the composition of the invention For its use, it is possible to apply to apply a few drops of the composition of the invention, preferably in gel form, once or more times a day. Preferably, it is best to refrain from eating or drinking for a few minutes, such as from 5 to 20 minutes, preferably from 10 to 15 minutes, after application to allow the composition to form the protective barrier.
• composition of the invention is nontoxic, and treatment can therefore be continued for as long as necessary, generally a few days, e.g., from 2 to 10 days, more generally from 3 to 5 days, until symptoms disappear.
• the treatment can be repeated in case of recurrent oral disorders.
• an oral composition according to the invention for use in therapy.
• an oral composition according to the invention for use in a method for the prevention and/or treatment of stomatitis, aphthae and oral ulcers, and oral mucosa disorders in general.
• Example 1 also referred to as “sample” or “MD,” was tested.
• Example 1 The mucoadhesivity of the composition of the tested Example 1 (also referred to here as “sample”) was evaluated by measuring its ability to inhibit lectin-glycoprotein binding on reconstructed human oral epithelium (HOE).
• HOE human oral epithelium
• One set of tissues was untreated (NO, negative control), one set was treated with a substance with known mucoadhesive activity (PC, positive control), and one set was treated with the test sample as such.
• the purpose of the test is to evaluate whether the tested product has mucoadhesive properties in a reconstructed human oral epithelium (HOE) model.
• Bioadhesion can be defined as the condition in which two materials, at least one of which is biological in nature, are held together for long periods of time by interfacial forces. When one of the two adhesive surfaces is mucus or a mucous membrane, the phenomenon is called mucoadhesion.
• Mucous membranes are the moist surfaces lining the walls of body cavities such as the gastrointestinal tract, vagina and urinary tract, lung, eye, etc. They are generally hydrophilic in that they contain high percentages of water (about 95%).
• the epithelial layer of the mucosa contains specific structures (specialized cells or glands depending on the type of epithelium) that secrete mucus directly onto the epithelial surfaces.
• Glycoproteins are the most important component of mucus, to which they impart the characteristic gel-like properties of cohesion and adhesion.
• the mucoadhesivity of a product intended for topical application to mucous membranes promotes a prolongation of contact time with the site of application itself.
• the mucoadhesivity of a product intended for mucosa treatment can be determined by evaluating the percentage of inhibition of lectin-glycoprotein binding.
• biotinylated lectin (concanavalin-A), a protein found in some leguminaceous plants (Canavalia ensiformis) that has a high affinity for glucoside and mannoside residues in membrane glycoproteins, is used.
• the binding sites are then detected by the use of streptavidin peroxidase, which, due to its high affinity for biotin, forms the protein-glucosolectin-biotin-streptavidin-peroxidase complex.
• the complex is quantified due to the presence of peroxidase by the ortho-phenylenediamine oxidation reaction in the following reaction:
• the intensity of the yellow-orange coloration of the solution is proportional to the amount of glycoprotein-lectin bonds and thus to the amount of available sites (glycoproteins) for mucoadhesion.
• the decrease in absorbance value compared to the untreated control is found to be proportional to the ability of the test substance to "mucoadhere.”
• HOE tissue is an epithelium obtained from cells derived from oral mucosa squamous cells carcinoma cultured on an inert polycarbonate filter at the liquid/air interface in a specific medium. This model forms an epithelial tissue without stratum corneum, histologically similar to the mucosa of the oral cavity.
• Tissues were allowed to equilibrate at room temperature for about 15 min and then transferred in growth medium and incubated (37°C, 5% CO2) overnight.
• the tissues were treated for 30 min with 70 pl of the tested sample or 70 pl of the controls.
• the optical density (OD) reading was performed at 450 nm.
• Example 1 for oral use for the treatment of stomatitis (aphthae) has a MUCOADHESIVE effect in reconstructed human oral epithelium.
• LLNA Local lymph node assay
• the local lymph node assay is a mouse model developed to assess the skin sensitization potential of chemicals.
• the LLNA is an alternative approach to traditional guinea pig methods and, in comparison, offers important animal welfare advantages.
• the test is based on the measurement of induced events during the induction phase of skin sensitization, particularly lymphocyte proliferation in draining lymph nodes, which is a hallmark of the skin sensitization response.
• the LLNA has been successfully validated and incorporated worldwide into regulatory guidelines.
• the LLNA when conducted according to published guidelines, provides a robust method for skin sensitization tests that not only provides reliable hazard identification information but also the data needed for effective risk assessment and management.
• LLNA Local Lymph Node Assay
• the residual sample was not preserved because it no longer had the characteristics of the initial sample, was no longer sterile, and its packaging was no longer sealed.
• MD was applied intradermally to the outer surface of the ear at a dose of 200mg/ml as suggested by 18010993-12:2021
• the animals were kept in a quarantine area for one week. At the end of the quarantine week, the animals were carefully examined to assess their suitability for the study.
• mice were injected intradermally on the outer surface of the ear with the test samples and controls (positive and negative) once a day for three consecutive days (25pl for each ear). Twenty hours after the last treatment, the mice were treated intraperitoneally with a 10 mg/ml BrdU solution. Twenty-four hours after injection, the mice were sacrificed and the auricular lymph nodes were harvested. Throughout the in vivo study, animals were monitored for signs of systemic toxicity (described in Table 2). Mice in each group were weighed before the first application of the test and controls, and also on day 6, before being scarified. The animals were monitored daily for signs of local (irritation and/or necrosis at the application site) and systemic toxicity.
• the animals were sedated and their body weights were recorded. Draining (auricular) lymph nodes were excised, mechanically disaggregated and resuspended in sterile saline containing 0.9% NaCI. BrdU incorporation into cells was measured using the ELISA kit (Cell proliferation ELISA, BrdU (colorimetric) version 13.0 cat no 11647229001) and recorded as the mean of BLI (BrdU labeling index)/n mice treated with the sample, positive control and negative control. Each extract was loaded and processed in triplicate by ELISA KIT. A stimulation index (SI), relative to the vehicle, was obtained for each sample.
• SI stimulation index
• the labeling index (LI) of the BrdU-labeled cell in the lymph node was calculated by the number of stained spots BrdU-positive cells per 100 cells counted in the cortex, paracortex and marrow. In each lymph node, we counted cells at each standardized site in three sites of cortex, paracortex and marrow without prior knowledge of treatment.
• the LI of BrdU-labeled cells in the epidermis was calculated by the number of BrdU-positive cells stained per 100 epidermal cells counted from the epidermis for each section (Table 3), according to ISO 10993- 10.
• the stimulation index (SI) was calculated by the fold increase in the total number of BrdU-positive LNCs in a lymph node compared with that in the respective vehicle control groups (Table 4).
• the finished product MD should be considered NON-TOXIC.
• the product was tested at a dose of 0.2g as suggested by ISO 10993-12:2021 .
• Murine fibroblasts L-929 NOTO clone 929 [L cell, L929, L strain derivative] (ATCC® CCL1 TM ) JUSTIFICATION FOR THE ASSAY CHOICE
• murine fibroblasts L-929 were used as reported in ISO 10993-5:2009. Identification and maintenance of test strains. Cultures of a cell line were kept frozen at -80°C in the corresponding culture medium containing DMSO 5% as cryoprotectant. Before use, DMSO was removed using complete culture medium and centrifuged. Using the complete culture medium corresponding to the cell line, sufficient numbers of cells were prepared to complete the assay, as described in Experimental Design.
• the experimental design included a 96-well plate containing a monolayer of subconfluent cells (about 88% confluence) divided into the following groups:
• Viable cells with active metabolism convert MTT to a purple formazan product with a maximum absorbance near 570 nm. When cells die, they lose the ability to convert MTT to formazan; therefore, the color formation serves as a useful and convenient indicator of viable cells only.
• Cell viability (%) was expressed as a percentage compared with untreated control cells. The lower the Viab. % value, the greater the cytotoxic potential of the test item is. If the viability is reduced to ⁇ 70% of the blank, it has cytotoxic potential.
• the 50 % extract of the test sample should have a viability at least equal to or greater than that of the 100 % extract otherwise the test should be repeated.
• Example 1 A biological evaluation was carried out on a sample of the composition of Example 1 to identify potential irritant effects using the following tests: -irritation of the oral mucosa according to ISO 10993-23:2021 (Special irritation test Annex D (D.3)).
• Sex male Weight: 25-30 g at the beginning of the test.
• Rat models provide a convenient means to study pathogen attachment and effusion and simulate all the events observed in natural infections (2).
• the animals were fed a standard complete pelleted diet provided by the licensed breeder.
• the 90% phenol solution causes oral ulcers in rats, representing a convenient means to study the beneficial properties of the test item on oral ulcer.
• Wistar rats (180-220 g), purchased from Harlan (Milan, Italy), were used for the experiment.
• the animals were fed a standard complete pelleted diet provided by the licensed breeder.
• a numbered tag placed across the edge of the right ear identified the animals selected for the study.
• the animals were kept in a quarantine area for one week. During this period, they were observed daily. At the end of the quarantine week, the animals were carefully examined to assess their suitability for the study.
• mice Male Wistar rats (weight, 180-220 g) (Harlan, Milan, Italy) were housed in a controlled environment (22 ⁇ 2 °C, 55 ⁇ 15% relative humidity, 12-h light/dark cycle). After acclimatization for one week, the rats were fed a standard diet and water. The animal experiments complied with Italian legislation on the protection of animals used for experimental and other scientific purposes (DM 116) experimental and other scientific purposes (DM 116192) and with EU regulations (OJEC L 358/1, 18.12.1986).
• the animals used for this study were randomly selected from suitable animals available at that time.
• a small cotton ball was placed at one end of a glass tube with a diameter of 3 mm and then having the cotton ball immersed in a 900 g/l solution of pieno, burning the rats for 60 s on the left cheek. After 24 h, all rats had 3-mm-diameter ulcers on the oral mucosa.
• the oral ulcer tissues (left cheek pockets) of each group was processed for morphological, histological and biochemical analysis.
• Wistar rats were randomly divided into six groups:
• Topical application on oral mucosa once daily for 2, 4 and 5 days.
• Phase III Phase III: RESULTS.
• the effects of MD on morphology, edema and hyperemia in the oral ulcer model were evaluated macroscopically, based on the maximum diameter of ulcer tissue observed ( ⁇ 1 mm, judgment of healing, >1 mm, judgment of nonhealing), edema and degree of hyperemia around the ulcer, according to Miao M et al., 2019 (1): Grade I, hyperemia diameter ⁇ 1 mm; grade II, hyperemia diameter 1-2 mm; grade III, hyperemia diameter 2-3 mm; grade IV, hyperemia diameter >3 mm.
• Scoring was as follows: score 1, normal epithelium and connective tissue without vasodilatation, absence of cellular infiltration or discrete cellular infiltration and absence of hemorrhagic areas, ulcerations or abscesses; score 2, discrete vasodilatation or areas of re-epithelialization, discrete inflammatory infiltration with mononuclear predominance and absence of hemorrhagic areas, ulcerations or abscesses; score 3, moderate vasodilatation, areas of hydropic epithelial degeneration, inflammatory infiltration with mononuclear prevalence hydropic epithelial degeneration, inflammatory infiltration with neutrophil prevalence, presence of hemorrhagic areas, edema and possible ulceration and absence of hemorrhagic areas, edema and possible ulceration and absence of abscesses; score 4, severe vasodilatation and inflammatory infiltration with neutrophil prevalence.
• the oral mucosa epithelium was intact and loose connective tissue in the control groups was restored (groups Sham + veh and MD +veh), while in the injured group (group Phenol + veh), the oral mucosa and squamous epithelium were found to be covered by necrotic tissue and infiltrated by a large number of inflammatory cells.
• group Phenol + veh the degree of injury was significantly reduced and positive staining of inflammatory cells decreased in animals treated with MD for 4 and 5 days (Phenol + MD, 4 days and Phenol + MD, 5 days).
• Cytokine investigations reflect aphthae healing and local inflammatory cell infiltration in the oral ulcer. Specifically, the process observed in oral mucositis is likely initiated by antigenic stimulation of mucosal keratinocytes, causing secretion of T-cell activating cytokines and tumor necrosis factor-alpha (TNF-o), which causes inflammation by endothelial cell adhesion and neutrophil chemotaxis. TNF-o induces inflammation by stimulating the release of interleukins, such as interleukin-2 (IL-2), a glycoprotein that plays a critical role in regulating both cells and neutrophils. Binding of IL-2 to the IL-2 receptor on T lymphocytes induces cell proliferation and increased lymphokine secretion.
• TNF-o tumor necrosis factor-alpha
• TNF-o and IL-2 were performed in oral mucosa tissue.
• TNF-o and IL-2 levels were found to be in damaged group (group Phenol + veh) compared with controls (groups Sham + veh and MD + veh) ( Figure 5A and 5B, respectively), confirming the role of TNF-o and IL_2 as important mediators in oral mucosa pathogenesis.
• treatment with MD at 4 and 5 days significantly reduced both TNF-o and IL-2 expression (groups Phenol + MD, 4 days and Phenol + MD, 5 days) ( Figures 5A and 5B, respectively), suggesting a protective effect to counteract the inflammatory component related to aphthae.
• MD administered for only 2 days was unable to reduce the levels of pro- inflammatory cytokines and TNF-o ( Figures 5A and 5B, respectively).
• TNF-o levels were significantly increased in phenol-damaged rats compared with control (A). These increased cytokine levels were significantly reduced by MD treatment for 4 and 5 days (A), in contrast to MD treatment for 2 days (A). IL-2 levels were significantly increased in phenol-injured rats, compared with control (B). Treatment with MD for 4 and 5 days (B), in contrast to treatment with MD for 2 days (B) significantly protected rats from injury. *** p ⁇ 0.001 vs. Sham + veh; ## p ⁇ 0.01 vs. Phenol + veh. *** p ⁇ 0.001 vs. Sham + veh; ## p ⁇ 0.01 vs. Phenol + veh.
• the present study demonstrates the ability of MD to treat oral ulcer by reducing the degree of hyperemia and edema in the cheek tissue, significantly decreasing the levels of inflammatory cytokines edema in the cheek tissue, significantly decreasing the levels of inflammatory cytokines when administered for 4 and 5 days. This study highlights the role of MD in counteracting phenol-induced oral ulcer.

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