ARISTOTELIA CHILENSIS EXTRACTS FOR THE TREATMENT OF FUNGAL INFECTIONS.

Abstract

This invention provides a natural and safe agent for the prevention and treatment of fungal candidiasis infections of the digestive and urogenital systems, both prophylactic and therapeutic, containing as an active ingredient the extract of Aristotelia chilensis fruit or extracts of other fruits, also acting as a filamentous inhibition mechanism in systemic diseases caused by fungi of the genus Candida, particularly Candida albicans. Other fruits may include aronia (chokeberry), cranberry, blueberry, currant, or other berry fruits.

Description

1 TARIFF ARISTOTELIA CHILENSIS FOR THE TREATMENT OF FUNGAL INFECTIONS EXTRACTS TECHNICAL FIELD This invention belongs to the field of pharmaceutical products and is based on a natural preparation, Maqui berry. high-quality products obtained from the fruits of plants such as Aristotelia chilensis. an extract of plant fruits containing a certain amount of anthocyanin delphinidin, Candida fungal infections caused by various fungi, particularly Candida albicans. It relates to its use for the treatment or prophylaxis of infection. According to the IPC, The invention belongs to classes A61K36 / 00 and A61P31 / 10. TECHNICAL PROBLEM Fungi are a group of 15 organisms that are part of the normal human microbiome. It belongs to microorganisms and is found in mucous membranes, skin, genitals, and also They live in the digestive system, where they aid in food absorption and digestion. One of the best-known types of fungi found in humans is the sac fungus. It is Candida (Candida albicans) from the (Ascomycetes) group. It depends on various factors. as a result of excessive proliferation, in the oral cavity, digestive system, vagina and other 20 areas of the body one of the most common fungal infections of mucous membranes and even skin It causes candidiasis. Candidiasis in the affected areas leads to inflammation. and causes itching, tingling and swelling. Candida, seemingly a successful... Antifungal treatment can suppress the infection, but it can recur and become chronic. It can develop into an infection. If the human immune system is undamaged and exactly 25 If it is functioning with its potential, this type of infection rarely leads to a more serious pathological outcome. This situation develops. However, if the immune system is compromised, Candida It migrates from the site of primary infection and can seriously, even life-threateningly, infect the person. It spreads to the internal organs, putting them at risk. In the treatment of candidiasis, with well-known therapies... The following technical problems remain unresolved: 30 - Candida is difficult to treat. - Candida easily and rapidly develops resistance to antifungal agents. - Known drugs are toxic. - Known medications are not effective enough. 2 - Candida returns or cannot be eliminated from the body. Known antifungal agents either stop Candida from growing or kill it. This forces resistance to build. Due to the reasons mentioned above, 5 days before the growth of Candida yeasts... both preventive and effective against persistent chronic fungal infections caused by An effective and harmless natural preparation is needed for its treatment. PREVIOUS TECHNIQUE Polyphenols are a large group of bioactive compounds found in plants and plant-based foods. It is a group of polyphenols that modulate various biochemical / signaling pathways. and thus vasoprotective effects, improving cognitive abilities and muscle performance. involved in improving the beneficial properties of herbal preparations, including anthocyanins are a large group of compounds belonging to the flavonoids. It is known that they also have anticancer activities. Anthocyanins, 15 Anthocyanidin glycosides are commonly found in fruits. They are responsible for the colors. These non-toxic, natural pigments are water-soluble. They are compounds and are divided into 6 classes: malvidin, elphinidin, petunidin, pelargonidin, cyanidin and peonidine. The position and number of hydroxyl and methyl groups in the basic structure, free It is crucial for their ability to eliminate radicals. 20 Delphinidin is a very potent enzyme from plants of the genus Delphinium, which has approximately 300 species. Anthocyanidin, a primary plant pigment known as an antioxidant, is found in northern regions. 25 They are perennial flowering plants in the Ranunculaceae family. Delphinidin is blue. It gives color to the flowers of plants in the genera Viola and Delphinium. Additionally, the grapes used in Cabernet Sauvignon production have a bluish-red color. and, as with other fruits, cranberries and red grapes (Concord grape), 3 It can also be found in blueberries. Delphinidin, like most other anthocyanidins, It is pH sensitive, meaning it turns red in acidic environments and blue in basic environments. It is a pH indicator. Several glycosides derived from delphinidin are known, and these are from the French. Myrtillaine (Delfinidin-3-O-glucoside) and tulipanin (delphinidin-5) are found in grape pulp. 3-O-rutinocyte), Violdelphin is responsible for the light purple hue of the Aconitum chinense flower. (Delphinidin 3-rutinoside-7-0-(6-0-(4-(6-0-(4-hydroxybenzoyl)-bD-glucosyl)- (oxybenzoyl)-bD-glucoside) and Nasunin, which gives the eggplant its purple peel its color. (Delphinidin-3-(p-coumaroyl routinoside)-5-glucoside). 10 The fruits are rich in anthocyanins, their glycosides, and delphinidin. There are many varieties of strawberries that are rich in nutrients. Today, one of these plants is Aristotelia. It is the maqui berry (chilensis) and its fruits are the richest in delphinidin. It is already reliably known that this is the case. Also known as Maqui or Chilean vine. It is a well-known, very hardy plant. Aristotelia chilensis is native to South America and has 15 species. It is a species of tree in the Elaeocarpaceae family and grows in the temperate rainforests of Chile. It lives in the forests and neighboring regions of southern Argentina. This is sold on the market. This type of fruit mostly comes from the wild and is organic, which makes it different from other cultivated fruits. That's a huge advantage. It has long been used by indigenous people of Chile to treat diarrhea, inflammation, and fever. It is used as a traditional medicine. Japanese scientists have found a cure for dry eyes. isolating the active ingredients, demonstrating that it is very effective in treating the syndrome. They were the first people to discover that the extract of the Aristotelia chilensis fruit was used for various eye diseases. different in treatment and also in regulating blood sugar levels There are numerous articles showing their activities. 25 The small, purplish-black Maqui berries taste similar to blackberries and Studies on the qualitative composition of polyphenols have revealed that anthocyanins... It contains eight glucoside pigments, including delphinidin and cyanidin, and is the main anthocyanin. Delphinidin is 3-sambubioside-5-glucoside (34% of total anthocyanins). has shown. 30 Maqui is a representative of fruits rich in anthocyanins. Its fruits are known to have numerous advantages over other similar fruits. In addition to being an excellent antioxidant, Maqui berries help combat aging. helping to neutralize free radicals, which are the main agents responsible. 4 It is also rich in polyphenols, anthocyanins, and other active ingredients. The most powerful... As a natural antioxidant, it neutralizes free radicals, detoxifies, and cleanses the skin. It protects against the harmful effects of sunlight, strengthens bones and joints, and boosts immunity. It improves the system, enhances energy levels, and inhibits the oxidation of LDL cholesterol. reduces. 5 According to the Chilean National Institute of Food and Technology (INTA), this rich soil supports plants. It is a unique bioclimatic area with aggressive climatic conditions in which its vegetation has developed, and The Andes Mountains experience high levels of solar radiation and large temperature variations. It is located between cold Humboldt currents. Plants that grow under these conditions, It contains unique phytochemical profiles found nowhere else on the planet. 10 These plants include aronia (chokeberry) and blueberry, which grow in other countries. Compared to similar products like black currant, it has 30-40% more bioactive content. It contains molecules. The climate in this region improves the quality of the fruits, thus benefiting human health. It ensures a higher concentration of beneficial molecules in native fruits, Following ancient traditions preserved for centuries, wild 15 They are carefully selected and collected from forests and fields. According to US Department of Agriculture ORAC data from May 2010, Maqui berry It is the most powerful natural antioxidant, 7 times more potent than acai berry and wormwood. It is 9 times more potent than goji berry. Its anthocyanin composition is approximately 25%. It is cyanidin and up to 75% delphinidin. Both anthocyanins and delphinidin 20 It has been extensively researched and its many health benefits have been proven. The dominance of maqui berry activity over other related plants and fruits, This is due to its high delphinidin content. According to a USDA source from September 2015, Maqui berries weigh 100g. It contains 1,260.00 mg of delphinidin per unit, while the closest competitor is the American 25 with 97.59 mg. It is a blueberry (Vaccinummyrtilus). Maqui berry is therefore considered the most powerful antioxidant superfruit. And it is also called a very nutritious grape. Clinical studies have shown that Aristotelia chilensis fruit extract has anti-inflammatory properties. 30 preventive and curative treatments for diseases, oxidative stress, and metabolic disorders. It has demonstrated its effects. Due to its various beneficial effects on general human health, In this study, Aristotelia chilensis fruit extract was selected to test the activity, and It is standardized and contains the highest amount of delphinidin as the most active anthocyanin. Because of its composition, it is the most powerful among all other similar extracts. It has antioxidant effects. Maqui berry - Aristotelia contains anthocyanins known as delphinidins. based on naturally derived active principles such as plant extracts of fruits like chilensis 5 for the treatment and prevention of candidiasis and other fungal infections By reviewing existing patents and scientific literature on pharmaceuticals, the following were determined: It has been found: Patent EP2344154, among others, targets delphinidin found in fruits and Anthocyanidins rich in delphinidins, including Aristotelie chilensis. It describes combinations. However, the described compositions are 10 Additional andrographolides, such as those found in plants of the genus Andrographis or Vaccinium myrtilline, quercetin, or caffeoyl, as found in the genus of plants Combinations of quinic (caffeoyl quinic) derivatives and proanthocyanidins It contains compounds. Patent RS59259 Bl, EP3209315 B1, at least one double-flowered Indian chrysanthemum 15 (Chrysanthellum indicum) extract and a Sunaga scolymus extract and an American blueberry (Viccinium myrtillus) extract and synthetic pipeline and / or pepper containing at least one compound of molecules from the pipeline containing the extract It describes a preparation involving combinations, where one such compound molecule It includes the following: At least one of the following molecules: apigenin-7-O-20 glucuronide, chrysanthemum A, chrysanthemum B, caffeic acid, luteolin, maritimein, eriodictyol, isocanin, apigenin-8-aL-arabinoside-6-CPD-glucoside (shaftoside), apigenin-6,8- C-di-PD-glucopyranoside (vicenin-2) and at least one of the following molecules: monocaffeoyl acid, delphinidin-3-galactoside, Delphinidin -3-glucoside, cyanidin-3- galactoside, Delphinidin-3- arabinoside, cyanidin-3-glucoside, petunidin-3-25 galactoside, cyanidin-3-arabinoside, petunidin-3-glucoside, peonidin-3-galactoside, peonidin-3-galactoside, petunidin-3-arabinoside, peonidin-3-glucoside, peonidin-3- glucoside, malvidin, malvidin-3-glucoside, malvidin-3-arabinoside i-piperine. The aforementioned The combinations described here are not the subject of the patent solution. This invention, It regulates carbohydrate and fat metabolism in humans and animals. It is directed towards. Application W02017113032A1 is from in vitro cultured A. chilensis seedlings. The resulting extract, rich in phenolic compounds, has natural antibacterial properties. and describes a discovery related to antioxidant composition. Specifically, the described 6 the method without the need to use materials grown under natural conditions The goal is to create plant biomass in a sustainable way, which in turn allows species to... It reduces the impact on exploitation. This patent is not inconsistent with the existing invention. Patent JP2013107825B describes an antioxidant compound containing maqui fruit. It defines. 5 Patent application TW201709920A, Maqui fruit and antioxidant. It describes a composition containing more fruits and berries, with a higher capacity. Patent application US2014377338A1, very high oxygen radical absorption. It describes a Maqui berry extract containing anthocyanins with an ORAC (Opportunity Accuracy) rating. and is related to topical application in preventing skin aging. 10 Patent R0131883 describes the antifungal activity of chitosan isolated from A. chilensis. It describes. Patent application US20050158396 describes plant anthocyanins. It describes their antioxidant properties and uses in cosmetics. However, the closest state of the technology is described in three scientific papers. It includes: 15 1) Antioxidant and antimicrobial extracts of Aristotelia chilensis and Ugnimolinae and Article titled "Applications as preservatives in cosmetic products" by Marcia AVELLO * and others, extracts of Aristotelia chilensis and Ugnimolinae leaves against Candida It describes its effects on albicans. 2) "The Powerful Color of Maqui" by Carolina Fredesa, * and Paz Robertb 20 The article titled "Anthocyanins Have Anti-inflammatory and Anti-Diabetic Effects" discusses the effects of anthocyanins on inflammation and diabetes. This explains their performance as antioxidants. 3) "Magnolol and honokiol inhibit Candida albicans adhesion, yeast-hyphal transmission, and Effects on biofilm formation (2015) PlosOne, 0 (2): eOl 17695, Lingmei Sun The article titled "25 isolated from the bark of the magnolia tree (Magnolia officinalis)" magnolol and honokiol, which are natural products obtained, are effective in preventing Candida virulence. It explains the effects. From what has been stated above, in the literature and the known state of the art, Fruit extracts of plants including Aristotelia chilensis Candida albicans its use or extracts with high delphinidin content (30 in this fruit) (as one of the anthocyanins found) regarding its use in the treatment of candidiasis. It is clear that there are no articles or patents. Furthermore, based on the technical information above, it refers to the fruit of Aristotelia chilensis (Maqui berry). Its extract has antioxidant and anti-inflammatory effects and is used for diabetes, eye problems. 7 diseases, cosmetic purposes, hair care, skin care, atopic dermatitis treatment, skin whitening, rosacea (rosacea), as a pain reliever, in the treatment of menstrual syndrome, It is used as a dietary supplement and also in the treatment of hepatitis C, leukemia, and cancer. It is known that candidiasis (thrush) can be treated using extracts of this or other fruits. or any article relating to the treatment of other fungal infections 5 It could not be found. LIST OF FIGURES Figure 1. Disk diffusion test results of Aristotelia chilensis plant extract. antimicrobial activity 10 Figure 2. Aristotelia chilensis extract on various solid substrates. Inhibitory effect on Candida albicans CS5314 filamentation Figure 3. Maqui berry extract (EMB) at 0.5, 0.25 and 0.125 mg / mL. concentrations of nystatin at MIC, ½ MIC and ¼ MIC Filamentation of Candida albicans CS5314 at concentrations 15 combined effect on Figure 4. Maqui berry extract (EMB) applied alone or in combination with other extracts. Nystatin was detected in zebrafish embryos 24 hours after infection. Growth and filamentation of Candida albicans SC5314 in the body its effect on. Filamentation is indicated by an arrow and the yeast-shaped 20 The cells are inside a circle. DETAILED DESCRIPTION OF THE INVENTION Candida albicans and other species of the genus Candida (e.g., C. glabrata, C. krusei, C. tropicalis) - collectively referred to as Candida species, best in warm and humid conditions 25°C They are fungi that survive on surfaces and are often part of the normal flora. as they live on the skin and mucous membranes of the digestive and reproductive organs They are found there. The most common cause of fungal infections (mycosis) in humans is the intestines. naturally occurring in small numbers on the surface of the fold and rapidly changing under various influences. Candida 30, which can multiply unnaturally and lead to pathological conditions. Candida albicans is the most common factor affecting the overgrowth of Candida in the body. antibiotic use, oral contraceptives, hormone therapy, diabetes, as well as This can be due to a weakened immune system or the use of immunosuppressive drugs. 8 Candida albicans is a part of the normal physiological flora found in 80% of the human population. It is part of the fungus and while it does not cause harmful effects, excessive growth of this fungus and Its reproduction manifests itself with the onset of candidiasis. Candidiasis is oral. It can affect the bladder, digestive system, genital system, and, as in pregnancy... It is not uncommon in diabetic patients. Candidiasis is actually a 5% disruption of the normal flora. It is a disorder. There are many reasons why this fungus can grow excessively. These include: A diet rich in sugar, processed carbohydrates, and this yeast. Alcohol consumption, which contributes to its reproduction, and an inappropriate lifestyle are among the factors involved. The oral, intestinal, and vaginal flora, which provide ideal conditions for Candida growth, are 10 It harms your health. Smoking, stress, alcohol, sugar, consumption of yeast-based doughs, and many others. Daily habits alter the pH level in our bodies, leading to excessive Candida growth. This creates the necessary conditions for it. As in oncological, hematological and HIV patients, there is a serious increase in leukocyte count. severe damage to the immune system manifesting as a decline 15 in cases such as in premature babies, the immune system If immature, mushrooms can penetrate the bloodstream and internal organs. can. Broad-spectrum antibiotics are used to treat bacterial infections. It is used. Long-term use or repeated use of several antibiotics 20 After use, the body can become a breeding ground for fungi. Oral This risk is increased in women who use contraceptives. Asthma treatment using inhaled corticosteroids can cause this fungus in the mouth. the risk of developing infection and rapid systemic spread of Candida It increases. One third of patients receiving radiation therapy or chemotherapy reach 25 at some point. They develop an invasive Candida infection. This is because of both chemotherapy and radiation kills good bacteria as well as cancer cells And thus, it allows Candida to grow. In type 1 and type 2 diabetes patients. People have high levels of sugar in their mouths. Candida feeds on sugar. It is yeast, and diabetic patients are at high risk of contracting this fungal infection. They are people. Infants, children, the elderly, cancer patients, people living with HIV, and those with autoimmune diseases. People with weakened immune systems, including those with illnesses, or The risk of any compromised person contracting this fungal infection 9 It is increasing. The preferred drugs for local infections are applied locally, directly. These are agents that act on the infected area. The development of antifungal resistance in fungi leads to treatment. It depends on the type of medication used, and the most common treatment for oropharyngeal candidiasis is... azoles such as miconazole, clotrimazole, fluconazole and itraconazole, which are used as... Resistance is very common in immunocompromised patients. This occurs in individuals who receive long-term prophylactic antifungal treatment. One of the downsides of clinically approved antifungals is that, in addition to their toxicity, In short, it involves killing fungi that further disrupt the normal flora. Proper nutrition and probiotic cultures can be a treatment for mild forms of candidiasis. It could be a solution, but the fundamental problem with these conditions is that 10 every time the conditions are met. Candida infection suppressed in digestive system or oral cavity pockets It is a repetition. This invention is found in the Chilean Maqui, called Maqui Berry, by Aristotelia. The most abundant in chilensis, and rich in vitamins, minerals and anthocyanins. 15 obtained from the fruit extraction of anthocyanin-rich fruits A new biological product was unexpectedly discovered from the natural products obtained. its activity, among which delphinidin is the most active and most frequent, and also Other medicinal alkaloids are antioxidants. This new biological activity is attributed to the Candida genus. It manifests itself in preventing the filamentation of fungi. Filamentation is the process by which a non-pathogenic (yeast) form of Candida forms filaments in epithelial tissues. which is very dangerous for its integrity and has a weak effect on immune system cells. a morphogenetic transformation in which it transforms into an invasive, pathogenic (filamentous) form It is a process. Preventing filamentation reduces virulence and candida with antifungal agents and This makes them an easy target for immune system cells. In the biological activity study, 25 standardized specimens of Aristotelia chilensis were used. The extract was used because of the high anthocyanin content in these fruits, blueberries. It is 3 to 4 times higher than cranberries or other fruits, and this extract is an effective It has antioxidant effects and is a nutritional supplement that is both therapeutic and preventive. It can be used as such. This invention is based on the tested extract of the fruit of Aristotelia chilensis. An unexpected 30-year study suggests that Candida fungi inhibit the filamentation process. This represents a discovery. The surprising effect observed in this study is tested. The fact that Aristotelia chilensis extract does not behave as a fungicide, i.e., normally It does not eliminate Candida, which disrupts the microflora, but rather the non-pathogenic form. Its function is to prevent it from transforming into a pathogenic form. THE BEST WAY TO IMPLEMENT THE INVENTION The extract and its components, which are the subject of this invention, belong to the genus Candida. effective in preventing and / or treating infections caused by fungi It has been shown that the invention is related to fungi of the genus Candida, especially Candida albicans. In the prevention and treatment of infections caused by Aristotelia chilensis 5 natural preparations isolated from the fruits of the plant, and also the fruit It involves using plants that are similar in terms of the composition of their extracts. Of those mentioned above, this fungus is currently an unresolved problem. a safe method that will have a long-term effect in eliminating the reproductive risk It is clear that there is a need to find natural preparations. Drugs in clinical use are immediately effective. and as soon as some of the above factors suitable for reproduction are obtained, new A fungal overgrowth occurs because Candida is resistant to clinical antifungal agents. when treated, the hidden parts of the canal remain active and do not have access to therapeutics. It is being filmed. This patent application is for a fungicidal agent that has no fungicidal effect but is naturally occurring in 15 by preventing the filamentation of Candida, which then develops into an aggressive and pathogenic form. the discovery of using a natural and safe preparation that prevents its passage It places. During the process of testing the biological activity of mulberry fruit extracts, this Although all types of fruit contain similar active ingredients, the 20 most potent are... standardized as an antioxidant and due to its high delphinidin content Aristotelia chilensis extract was used. Results of the experimental research. This applies to all plant extracts containing cyanidin and delphinidin. Aristotelia chilensis Testing the biological activities of fruit extract on different models. During its development, this invention unexpectedly led to the creation of one of the models, the Zebra 25. The fish (Danio rerio) model revealed a lack of Candida hyphae formation. This has resulted in its in vivo filamentation and virulence, that is, its transition to a pathogenic form. It is a laboratory method for monitoring plant extracts and purified substances. The biomedical application of its components is primarily based on the minimum effective (therapeutic) dose. It depends on the range between minimum and toxic doses. For such an assessment, 30 will evaluate both very important effects for use in therapeutic purposes Choosing a suitable model is important. The zebrafish model is used to detect biological activities and to interact with plant extracts and other substances. A very reliable experimental animal for evaluating the toxicity of natural products. 11 This reliability has been proven in extensive scientific literature as a model. This reliability is evident in the zebra pattern. High molecular-genetic, physiological and immune differences between fish and humans in addition to their similarity, the high degree of correlation between them in terms of drug response. It is possible thanks to 5 found in fruits and plants of the Ranunculaceae genus Aristotelia chilensis was selected as one of the anthocyanin-delphinidin sources. Bioactivity and toxicity study of standardized extract of the plant. During this process, the primary objective was to explore all possibilities for its medical application. zebrafish model to evaluate biological safety (toxicity) We used it. 10 All experiments carried out on the zebrafish model were in accordance with regulation 2010 / 63 / EU. Recommendations of the European Directive and the Molecular Research Institute of Belgrade University (Belgrade, Serbia) Institute of Genetics and Genetic Engineering, Care of Laboratory Animals and In accordance with the ethical guidelines of the User Manual, for up to 120 hours only. This was performed on embryos. Standardized 15 anthocyanins content, delphinidin - toxicity and bioactivity of Aristotelia chilensis extract during laboratory tests to evaluate an experimental zebrafish model Standard tests were performed on it and the expected results were obtained, meaning this The safety of the extracts has been confirmed. Unexpectedly, the tests conducted The extract inhibits filamentation, growth, and 20% of Candida albicans. It has been shown to prevent its spread. The invention, for example, is not limited to these. This will be shown in more detail in the regulations. Example 1 - Assessment of Lethal and Teratogenic Potential Acute toxicity analysis was performed on zebrafish embryos - 25 FET (Fish Embryo Toxicity assay). Aristotelia The survival and development of zebrafish embryos using chilensis berry extract. its impact on the OECD 2013 guidelines for testing chemicals and examined according to the protocols described earlier. 6 hours after fertilization. (hpf), zebrafish embryos were treated with Aristotelia 30 aqueous solution at nine concentrations as follows: Sent to chilensis berry extract: 10; 25; 50; 100; 250; 500; 1000; 2000 and 2500 pg / mL (minimum 2.5; 6.25; 12.5; 25; 62.5; 125; 250; 500 and 625 pg / mL delphinidin concentrations and minimums of 3.5; 8.75; 17.5; 35; 87.5; 175; 350; 700 and corresponds to total anthocyanin concentrations of 875 pg / mL). 12 Embryos were exposed to the given concentrations during the period up to 120 hpf. left and survival and teratogenic malformations every 24 hours Its formation was investigated. Distilled water was used as a negative control. Experiments This was performed three times using 30 embryos per concentration. Toxicity Different endpoints were used at 24, 48, 72, 96, and 120 hpf to evaluate the results. (Table 1), followed by the LC50 value as standard toxicological parameters. (concentration that kills 50% of embryos) and EC50 value (of embryos The concentration affecting 50% has been determined. Table 1 - 10 different stages after fertilization in zebrafish embryos (Danio rerio) Lethal and teratogenic effects observed in HPF over time. Data obtained from the FET test show that Aristotelia chilensis berry extract at the highest dose administered, 2.5 mg / mL (LC50 > 2.5 mg / mL) or minimum Even with a delphinidin dose of 625 pg / mL, which is a minimum of 875 pg / mL total anthocyanins, 15 It is non-toxic, and all treated embryos survived up to 120 hpf. and no signs of cardiotoxicity, hepatotoxicity, or developmental disorders. It has been shown that they develop without it. In addition, the treated embryos The melanocytes had developed normally and became pigmented during treatment. This The results show that Aristotelia chilensis berry extract is toxic at high (milligram) doses. It has shown that it is not and has suggested its potential use in treatment. 13 Example 2. Antimicrobial activity As part of the research on antimicrobial activity, Aristotelia chilensis The extract is effective against various bacterial and fungal infections associated with the oral and oropharyngeal cavities in humans. pathogens (Staphylococcus aureus, Streptococcus mutans, 5 against Streptococcus agalactiae, Streptococcus pyogenes and Candida albicans) It has also been tested for antimicrobial activity. Activity disk diffusion test. Tested using Aristotelia chilensis extract at a dose of 0.125 mg to 2 discs. It was administered in mg concentrations (minimum 31.25 pg to 500 pg delphinidin). (or 43.75 pg to 700 pg total anthocyanins per disc). As shown in Figure 1. The results showed no antibacterial or potentiating effect against any of the tested strains at the administered doses. It has shown antifungal activity. Example 3 - Antifungal activity As part of a detailed study on antifungal efficacy, Aristotelia The extract of Candida chilensis had a minimum of 15% efficacy against three clinical isolates of Candida albicans. Inhibitory concentration (MIC) was determined using the standard microdilution method. The extract contains a minimum of 2.5 mg / mL delphinidin and 3.5 mg / mL total anthocyanin. Even at the highest concentration tested, 10 mg / mL, none of the isolates... It was found to have no inhibitory effect on its growth. In addition to its antimicrobial effect, Maqui berry extract has been clinically studied in 20 The efficacy of approved antifungal drugs (AFL) nystatin, clotrimazole, and micafungin. Its effect was investigated using a "checkerboard experiment". The test involved saliva. in a non-irritable RPMI environment (standard conditions) and in the presence of artificial saliva This was carried out. The antifungal concentrations tested were 0.0312 pg / mL to 2 The tested Maqui berry extract concentrations ranged between pg / mL and 25. range 0.0156 mg / mL (3.9 pg / mL delphinidin and 5.46 pg / mL, total anthocyanins) between 4 mg / mL (1 mg / mL delphinidin or 1.4 mg / mL total anthocyanins) It has changed. Combination therapy (simultaneous use of medications and Maqui berry extract) (treatment) Regarding the effect of Candida clinical isolates on growth. The study results showed that the presence of the extract did not affect the effectiveness of the tested drugs, 30 That is, under standard conditions, synergistic or antagonistic in the presence of artificial saliva. It has been shown to have no effect; this suggests that the use of Maqui berry extract is not anti- Studies have shown that it does not reduce the effectiveness of Candida medications. 14 Example 4 - In vitro inhibition of Candida filamentation Candida albicans is part of the normal oral microflora and is weakened. In immune conditions, it can cause candidiasis, which is the name given to the filamentation of yeast. It triggers morphogenetic transition to hyphae in a given process. Filamentation, C. It is an important virulence factor in albicans and is present in the epithelial and subepithelial parts of fungi. 5 It allows it to penetrate tissues and colonize internal organs. Therefore, Aristotelia chilensis extract combats different types of Candida albicans that it encounters in vivo. In terms of filament inhibition in different environments reflecting environmental conditions. Tested with: uridine sucrose (UPS + uridine), N-acetyl-glucosamine (GlcNAc) or Presence of serum proteins (Spider + FBS). Extract, minimum 12.5; 50 and 10 respectively. 500 pg / mL delphinidin corresponds to 17.5; 70 and 700 pg / mL total anthocyanin doses. The incoming doses were 50, 200 and 2000 pg / mL. As shown in Figure 2, filamentation of Aristotelia chilensis extract. It prevented and distorted the morphology of the fymgal colonies from wrinkling in each test medium. (pathogenic state) to smooth (less pathogenic or non-pathogenic state) 15 This has changed the way in which Candida albicans' virulence is prevented by different environmental factors. It shows that it possesses significant characteristics under certain conditions. In some cases, Aristotelia chilensis extract was active at lower concentrations. 50 filamentation occurred under all experimental conditions at concentrations of pg / mL and 2 mg / mL. It completely inhibited it. These results also show that Aristotelia chilensis extract inhibited Candida 20. Different signals trigger filamentation in albicans in response to various environmental stimuli. Maqui berry has been shown to contain active ingredients that are likely to affect pathways. extract and one of the most commonly applied topical antifungal drugs The inhibitory effect of nystatin on Candida filamentation is like a checkerboard pattern. In the experiment, it was investigated in vitro in three clinical isolates in RPMI medium. Extract 25 In the concentration range of 0.125; 0.25 and 0.5 mg / mL (31.25; 62.5 and 125 pg / mL Delphinidine (43.75, 87.5 and 175 pg / mL total anthocyanins, respectively), and nystatin (2 pg / mL). Tested in concentration ranges of 1 pg / mL (MIC), 1 pg / mL (½MIC), and 0.5 pg / mL (¼MIC). This test was performed in the presence of the extract at a concentration of 0.5 mg / mL. filamentation is reduced, and at lower concentrations, the inhibitory effect is 30 It has been shown that no growth was observed. Growth of isolates was reduced at the MIC dose of nystatin. While no filamentation was observed, isolates grew at ½MIC and ¼MIC doses and It showed filamentation. However, the combination treatment (Maqui berry) (extract and nystatin) Inhibitory effect on Candida filamentation, individual doses at which treatments (extract only or nystatin only) have no effect including all extract and antifungal agent concentrations tested It was obtained. In the presence of small doses of extract (0.125 mg / mL and 0.25 mg / mL), The tested strains did not form filaments even at 1 / 4MIC and 1 / 2MIC doses of nystatin. This also shows that under these experimental conditions, Maqui berry extract increased the effectiveness of nystatin. 5 This is shown (Figure 3). According to literature data, in vitro experimental conditions in Candida those that do not induce oxidative stress, but are activated by oxidative stress. Because it stimulates filamentation by activating biochemical pathways outside of the system, the results The performance mechanism of maqui berry extract is related to oxidative stress. This shows that it is not through the inhibition of filamentation. 10 Example 5 - In vivo inhibition of Candida filamentation. Candida albicans, as described in the literature, and the brain of a zebrafish embryo. infection (hindbrain) model, due to the optical transparency of the embryonic body The applied therapeutics promote replication, filamentation, and host activity of fungi. 15 Examining the effect of zebrafish embryo spreading throughout its body. It is an animal model of an infection system that provides monitoring capabilities. 32-34 hpf 50- 75 Candida albicans SC5314 cells were injected into the brain via the optic vesicle. Candida inoculum was prepared in a 5% polyvinylpyrrolidone (PVP) solution. 20 Two hours after Candida microinjection, infected embryos contained 0.0625 mg / mL. Maqui berry extract at concentrations of 0.125 mg / mL and 0.25 mg / mL and 1 pg / mL The animals were treated with nystatin at concentrations of 0.5 pg / mL (½MIC) and 0.5 pg / mL (¼MIC). In addition to individual applications, infected embryos were treated with all the specified doses. 25 exposed to a combined application (nystatin and extract together) in combinations Only embryos injected with 5% PVP represented the control group. After the treatments were applied, zebrafish embryos died over the next 4 days. They were grown at a temperature of 31 °C throughout and not only survived but also fluorescent. Daily observations of Candida filamentation and spread using a microscope. It has been monitored as follows: 30 The results of this in vivo study show that the extract was effective after the start of treatment. At a concentration of 0.0625 mg / mL, it effectively inhibits Candida filamentation within the first 24 hours. Studies have shown that it inhibits Candida in some way. At a dose of 0.0625 mg / mL of the extract, Candida The cells are few in number and only yeast-like in the brains of infected embryos. 16 This was observed in most embryos at a concentration of 0.125 mg / mL of the extract. Candida cells were not detected. Since the extract has no antifungal effect... The absence of Candida cells is one of the factors affecting the extract's immune system. This may result in (immunomodulatory effect). These results are obtained at 0.5 pg / mL (¼MIC) and 1 pg / mL Treatment with nystatin at a dose of (½MIC) resulted in the treatment of 5 untreated cases of infection (candidiasis). It shows a significant reduction compared to infection in embryos. However, Filamentation was present at both nystatin concentrations. However, in the bodies of infected embryos treated with nystatin and Maqui berry extract No Candida cells were detected, which indicates that combination therapy does not reduce filamentation. and that it is very effective in inhibiting Candida growth, and that Maqui berry extract is 10 It did not reduce the in vivo effect of nystatin, but it did not completely eliminate the infection. It shows that it contributed to its removal. The results obtained using the zebrafish model are consistent with Aristotelia chilensis. that the extract is a non-toxic, plant-derived product and has antimicrobial and Infectious tests have shown that Aristotelia chilensis plant extract is a novel agent against candidiasis. 15 It has been shown that it can be used as such. What is important is that the above experimental results... As the results show, the Aristotelia chilensis extract immediately after application then it is not washed off the mucous membranes, therefore its use against Candida Prolonged exposure of tissue colonized with albicans to the active components in the extract It is clear that he will allow it to stay. 20 30

Claims

17 SYSTEMS 1. The pathogenicity of Candida albicans due to filament (hyphae) formation. It is a preparation used for the purpose of suppression; the said preparation The active ingredient is obtained from the fruit of the Aristotelia chilensis plant, 5 It is an agent containing a standardized extract, and its characteristic feature is that it targets Candida albicans. without affecting the viability, reproduction, or growth of their cells, without exhibiting fungicidal or fungistatic effects, Candida albicans Antiviral preparation that selectively inhibits filamentation It is characterized by being 10 2. The preparation according to claim 1, its characteristic is; Aristotelia chilensis fruit extract, It inhibits Candida albicans filamentation but does not stimulate fungal growth. The concentration range that it does not affect is between 0.0625 mg / mL and 0.25 mg / mL. It is characterized by being 15 3. The preparation is in accordance with claims 1 and 2, and its characteristic is that it is used with or without antifungal agents. When used alone, it enhances the fungicidal effect of antifungal agents. by suppressing the filamentation of Candida albicans without altering it including powders, lozenges, gels or solutions that provide antiviral effect, but including those under 20 years of age. in any pharmaceutically acceptable form, including but not limited to It is characterized by its... 30

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