Polysaccharides extracted from onion leaves with an Anti-gastric ulcer effect, method for their production and use thereof

Abstract

The invention relates to polysaccharides extracted from the leaves of yellow onion (Allium cepa L.), which have an anti-gastric ulcer effect. The invention also relates to a method for producing said polysaccharides, compositions comprising the polysaccharides according to the invention, and their use for the prevention or auxiliary treatment of gastric ulcer.

Description

[0001] POLYSACCHARIDES EXTRACTED FROM ONION LEAVES WITH AN ANTI-GASTRIC

[0002] ULCER EFFECT, METHOD FOR THEIR PRODUCTION AND USE THEREOF

[0003] FIELD OF THE INVENTION

[0004] The invention relates to polysaccharides extracted from the leaves of yellow onion (Allium cepa L.), which have an anti-gastric ulcer effect. The invention also relates to a method for producing said polysaccharides, compositions comprising the polysaccharides according to the invention, and their use for the prevention or auxiliary treatment of gastric ulcer.

[0005] TECHNICAL BACKGROUND

[0006] Yellow onion (Allium cepa L.) is a well-known plant that is widely cultivated and consumed worldwide. It is an important food plant, but many beneficial medicinal effects are also attributed to it. It is an important herb in traditional therapeutic systems, it is used in Ayurveda, Chinese and Egyptian medicine for a variety of indications, such as the treatment of fever, chronic bronchitis, dyspnea, cough, bronchial obstruction, colds, dysentery and arthritis.

[0007] The vast majority of varieties belonging to the Allium cepa L. species belong to the common onion group (Allium cepa var. cepa), the so-called "basic species", they are usually referred to simply as onions. The various onion varieties differ in color and taste, the onion can be white, yellow, purple, red or green, with a sweet or less sweet taste. The Allium cepa species also includes other varieties, such as Egyptian onion (A. cepa var. viviparum) and shallots (A. cepa var. aggregatum).

[0008] Yellow onion contains many phytonutrients, vitamins (A, B, C and E) and minerals, and its other valuable substances belong to the group of polyphenols, flavonoids and sulfur-containing compounds. Among its phenolic substances, benzoic and cinnamic acid derivatives, such as protocatechuic acid, p-coumaric acid, ferulic acid and catechol, should be highlighted. Its flavonoids deserve attention due to their antioxidant effect, especially quercetin, isorhamnetin, kaempferol and their glycosides, myricetin, luteolin and genistein. The characteristic colorants of red onions are anthocyanins, such as cyanidin glycosides, peonidin glycosides and pelargonidin glycosides.

[0009] The substance responsible for the typical onion smell is propylene-L-cysteine sulfoxide, other important sulfur-containing substances are onionin A, C-glutamyl peptides, cysteine sulfoxides such as cepaene, cycloalliin, isoalliin, S-allyl-cysteine sulfoxide, propenyl-cysteine sulfoxide, glutamyl-S-propenyl- cysteine sulfoxide, as well as diallyl disulfide, allyl methyl sulfide, allyl propyl disulfide, which play a role in the antimicrobial and anti-inflammatory effects of onions. The fructan content of the underground organ is also known, and the polysaccharide fractions that can be produced by gradual extraction from the onion - with a hot buffer solution, a chelating agent, dilute and concentrated alkali - have also been studied, the dominant sugar components of which were mannose and galactose.

[0010] However, the leaves of yellow onion are considered as waste in agriculture and are accumulated in significant quantities. There is no widespread solution for the utilization of green onion leaves, the disposal of large amounts of agricultural organic waste represents additional costs for producers.

[0011] Several articles have been written about the beneficial effects of Allium cepa leaves. Nausheen et al. (Ann. Exp. Biol. 2014; 2 (2):37-42) reported the cardioprotective and antioxidant effect of the methanol extract of the leaf. Upadhyay (Int. J. Green Pharm. 2016; 10(1): 46-64) also described in his review that the leaves of Allium cepa may have antioxidant and cardioprotective effects, and also disclosed that its cold water concoction was used against diarrhea and gastroenteritis, in the form of a green paste it reduces cholesterol and triglyceride levels in the blood, its oil is anti -coagulant, and it can also be used as ahot syrup against cough and constipation. Marrelli et al. (Molecules. 2019; 24(1): 119) investigated the potential beneficial effects of onion against obesity, and in their article it is stated that the water- alcohol extract of the leaf inhibited pancreatic lipase to a small extent (<40%). Khan et al. (Indian J. Pharm. Educ. Res. 2020; 54(1): 143-149.) investigated the effect of the flavonoid-rich extract of the leaf prepared using methanol against neuropathic pain in rats; the observed effect was attributed to strong antioxidant activity. According to Kianian et al. (Iran. J. Pharm. Res. 2021; 20(2): 107-134.), the leaf is beneficial against vasodilation. According to W02011118067A1, an immunostimulatory compound can be extracted from the leaves of Allium cepa.

[0012] There are relatively few references in the literature for the utilization of leaves that become waste during onion cultivation. Most articles that discuss the possible use of materials that have become "redundant" during industrial onion cultivation focus primarily on the skin and outer layers of the onion bulb, as well as on bulbs with an unaesthetic appearance (e.g. Benitez et al. '. Plant Foods Hum. Nutr. 2011; 66( 1 ) : 48 -57. ; Cebin et al. : Engineering power: bulletin of the Croatian Academy of Engineering. 2020; 15(3): 7-13; Sagar et al. : Food Frontiers. 2022; 3(3): 380-412.). Little data is available on the potential uses of onion leaves. Rita T. Dela Cruz (Agriculture Magazine (Philippines), 2020. 24(1): 10-12.) in an article appearing in the columns of an agricultural magazine, reports on a program launched in the Philippines in 2017, the aim of which was to utilize onion leaves. It presents several possibilities of use: dried onion leaves can be used as a tea, powdered onion leaves can be used as a spice, and pickled onion leaves, vacuum-dried onion leaves and onion leaves extract have also been prepared. The latter is in the form of puree or juice, but neither its production nor its possible beneficial effects were described. In addition, CN101455331A proposes a use of onion leaves waste, in which onion oil is produced from the leaves. It is therefore necessary to develop methods that can be used to produce valuable products from the leaves which are considered to be waste.

[0013] The polysaccharides of the bulb (bulbus) of the yellow onion are mentioned in several articles. According to Zhu et al. (Int. J. Biol. Macromol. 2017; 105 (Pt 1): 1192-1201.) and Ma et al. (Int. J. Biol. Macromol. 2018; 111:92-101.), these polysaccharides can be used in the food industry, and due to their antioxidant and antibacterial effects, they can also be used in functional foods. Zhao et al. (Front. Nutr., 2021; 8:669805) observed an antimicrobial effect of the polysaccharides. Among patent documents, CN113980150A claims polysaccharide extracted from onions for skin care and as a functional food. EP2701714B1 claims polysaccharides extracted from vegetables (e.g. onions) against cold and flu, and as food. However, none of the listed articles mentions that polysaccharides could be extracted from onion leaves.

[0014] BRIEF DESCRIPTION OF THE INVENTION

[0015] We found that polysaccharides with valuable physiological effects can be extracted from the leaves of yellow onion. The extracted polysaccharides have been proven to have an anti-ulcer effect.

[0016] Accordingly, the invention relates to a polysaccharide extract obtained from the leaves of yellow onion (Allium cepa L.), which comprises the following, based on the total weight of the extract:

[0017] Dry matter: 92-98%,

[0018] Sugars: 25-45%,

[0019] Uronic acid: 5-15%,

[0020] Proteins: 13-23%.

[0021] In an embodiment, the polysaccharides of the extract consist of the following monosaccharides:

[0022] Glucose: 30-50%

[0023] Galactose: 15-35%

[0024] Arabinose: 10-30%

[0025] Rhamnose: 5-25% wherein the percentages are expressed as the mass ratio of the monosaccharides that make up the polysaccharides.

[0026] The invention also relates to a method for extracting polysaccharides from the leaves of yellow onion (Allium cepa L.), characterized in that a) an extract is prepared from the onion leaves with water, then a solid / liquid separation is carried out, b) the polysaccharides are precipitated from the aqueous solution with acetone or alcohols to obtain raw polysaccharide, and c) the raw polysaccharide is purified to obtain a purified polysaccharide.

[0027] In a preferred embodiment, in step b), the polysaccharide is precipitated with acetone.

[0028] In an embodiment, in step a), the aqueous extract is prepared by stirring dried, chopped yellow onion leaves in a volume of water 5-50 times, preferably 5-30 times its weight, at 15-60 °C, preferably at ambient temperature, for 1-5 hours, preferably for 1-2 hours, then solid / liquid separation is carried out.

[0029] In an embodiment, step b) is carried out by adding 1-5 equivalent, preferably 1.5-3 equivalent, more preferably 1.8-2.5 equivalent volumes of cold, preferably -20°C to +8°C acetone to the aqueous extract obtained in step a), then solid / liquid separation is carried out.

[0030] In an embodiment, in step c), the purification is carried out by dissolving the raw polysaccharide in water, boiling it, then cooling it, and filtering the precipitated material from the cooled solution, then precipitating the polysaccharides from the filtrate with acetone or alcohols, preferably with acetone, separating, and then optionally drying.

[0031] The invention also relates to a polysaccharide extract obtained from the leaves of yellow onion (Allium cepa L.), which is obtainable by the method according to the invention.

[0032] The polysaccharide extract obtainable by the method according to the invention preferably comprises the following, based on the total weight of the extract:

[0033] Dry matter: 92-98%

[0034] Sugars: 25-45%

[0035] Uronic acid: 5-15%

[0036] Proteins: 13-23%.

[0037] The polysaccharides of the polysaccharide extract obtainable by the method according to the invention preferably consist of the following monosaccharides:

[0038] Glucose: 30-50%

[0039] Galactose: 15-35%

[0040] Arabinose: 10-30%

[0041] Rhamnose: 5-25% wherein the percentages are expressed as the mass ratio of the monosaccharides that make up the polysaccharides.

[0042] The invention also relates to the polysaccharide extract obtained from the leaves of yellow onion (Allium cepa L.) according to the invention for use in the prevention or auxiliary treatment of gastric ulcer.

[0043] In an embodiment, the gastric ulcer is an alcohol-induced gastric ulcer. In another embodiment, the gastric ulcer is a gastric ulcer induced by non-steroidal anti-inflammatory drug.

[0044] The invention also relates to a composition, preferably a pharmaceutical composition, medical device, dietary supplement, foodstuff, medicinal food, food for special medical purposes or fortified food, which comprises the polysaccharide extract obtained from the leaves of yellow onion (Allium cepa L.) according to the invention.

[0045] The invention also relates to a composition, preferably a pharmaceutical composition, medical device, dietary supplement, foodstuff, medicinal food, food for special medical purposes or fortified food, which comprises the polysaccharide extract obtained from the leaves of yellow onion (Allium cepa L.) according to the invention, for use in the prevention or auxiliary treatment of gastric ulcer.

[0046] EXPLANATION OF DEFINITIONS AND ABBREVIATIONS CONTAINED IN THE DESCRIPTION

[0047] The yellow onion (Allium cepa L.) is a cultivated plant belonging to the amaryllis family (Amaryllidaceae), which does not occur in the wild. As a result of breeding, many varieties and variants of onions of different sizes and shapes were produced and brought into cultivation. The shape of the onion can vary from elongated to flat and round, and the color of the skin can be white, yellow, orange or dark red. Better known variants are A. cepa var. aggregatum (scallions, also known as shallots, previously known as a separate species, A. ascalonicum), A. cepa var. aggregatum (potato onion) and A. cepa var. viviparum (tree onion). Chives (A. schoenoprasum)'. serpent garlic (A. sativum var. ophioscorodori), leeks (A. porrum) and Welsh onions (A. fistulosum) form separate species.

[0048] The species Allium cepa is preferably used in the context of the invention. Preferably, variants or varieties belonging to the Allium cepa group are used. Preferably, the Allium cepa basic species is used. In the context of the invention, yellow onion is preferably used.

[0049] Fortified foods are foods to which vitamins, minerals, or other nutritionally important substances have been added during processing in order to increase their nutritional value or biological value, regardless of whether the given substance is originally found in the food or not.

[0050] Auxiliary treatment (also called supportive treatment or supportive therapy) means treatment or therapy carried out in addition to, in parallel with, a therapy for the treatment of a given disease or condition. Auxiliary treatment in itself does not replace traditional medical procedures, but it promotes and / or enhances their effect, or can alleviate, for example, accompanying symptoms or side effects caused by traditional medical treatment, thereby improving the patient's quality of life.

[0051] BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the effect of temperature on the amount of extracted polysaccharide.

[0052] Figure 2 shows the effect of the amount of extractant on the amount of extracted polysaccharide.

[0053] Figure 3 shows the effect of extraction time on the amount of extracted polysaccharide.

[0054] Figure 4 shows the effect of the amount of precipitant on the amount of extracted polysaccharide.

[0055] Figure 5 shows the effect of methylcellulose, carbenoxolone, and onion polysaccharide on ethanol- induced experimental ulceration, in case of a single treatment. **: p<0.01 compared to control

[0056] Figure 6 shows the stomachs of animals treated with test substances and ethanol, in case of a single treatment. The test substance was distilled water (control), methylcellulose, carbenoxolone or onion extract.

[0057] Figure 7 shows the effect of methylcellulose, carbenoxolone, and onion polysaccharide on experimental ulceration induced by salicylic acid, in case of a single treatment. ** and *** mean p<0.01 and p<0.001, respectively, compared to the control.

[0058] Figure 8 shows the stomachs of animals treated with test substances and salicylic acid, in case of a single treatment. The test substance was distilled water (control), methylcellulose, carbenoxolone or onion extract.

[0059] Figure 9 shows the effect of chronically applied methylcellulose, carbenoxolone, and onion polysaccharide on experimental ulceration induced by salicylic acid. ** and *** mean p<0.01 and p<0.001, respectively, compared to the control.

[0060] Figure 10 shows the stomachs of animals treated with test substances and salicylic acid, in case of chronic use. The test substance was distilled water (control), methylcellulose, carbenoxolone or onion extract.

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] Our aim was to develop a method and product that can be used to utilize the leaves of yellow onion, which are considered agricultural waste. More specifically, our aim was to develop a method suitable for the production of polysaccharides from yellow onion (Allium cepa L.) leaves, which are considered agricultural waste. An important aspect of the development of the extraction method was that the method should be sufficiently economical, high-yielding and industrially feasible. The aim was to produce a product that is colorless or pale in color, and in which the flavor and smell of onions are not noticeable, thus which can be used as a food additive. When developing the technology, we paid attention to the fact that solvents and materials approved in the food industry are used in all its stages.

[0063] Preparation of onion leaves Dried, chopped onion leaves are preferably used as the starting material for the method. Although the extraction can also be done from fresh leaves, in this case a lot of chlorophyll will get into the extract, as well, and the final product will also be green, thus it is not recommended to use fresh leaves.

[0064] For preparation, it is advisable to chop the onion leaves, generated as waste, into 1-5 cm pieces, for this e.g. "chaffing" may be suitable. The chopped leaves are dried until they are air-dry at room temperature in a well-ventilated place, spread out in a thin layer, and turned often. After drying, processing should be done as soon as possible.

[0065] Method suitable for the extraction of polysaccharide

[0066] The essence of the method suitable for extracting the polysaccharide is to prepare an aqueous extract from dried, chopped green leaves of yellow onion, filter the extract, and then precipitate it with alcohol or acetone. The precipitated polysaccharide is then dried and subjected to further purification steps. The method is described in detail below.

[0067] Aqueous extraction

[0068] In the first step of the method, an aqueous extract is prepared from the dried, chopped green leaves of yellow onion. Preferably, deionized water is used.

[0069] The extraction can be carried out at 15-60 °C, preferably at ambient temperature. We found that increasing the temperature slightly decreases the amount of extracted polysaccharide, during our tests we were able to extract the largest amount of polysaccharide at about 25°C. This is particularly beneficial because it means that it is not necessary to invest energy in heating the system to prepare the extract.

[0070] The amount of extractant can be e.g. about 5-50 times, preferably 5-30 times the weight of the dried onion leaves. We found that the use of an amount of solvent of 15-30 times results in roughly the same amount of polysaccharide, thus from the point of view of the cost-benefit ratio, the extraction ratio of 1: 15 is preferable. Although the amount of extractable polysaccharides is slightly reduced by using a solvent amount below the ratio of 1: 15, due to the cost reduction, it may still be advantageous to work in this range, i.e. using an amount of solvent of 5-15 times, for example 5-10 times, especially on a large scale.

[0071] At the end of the extraction process, the onion leaves are separated from the aqueous extract, e.g. by filtration and pressing the leaves.

[0072] Precipitation

[0073] Polysaccharides can theoretically be precipitated from the aqueous extract e.g. with alcohols or acetone. However, the use of methanol is avoided, as this solvent is not suitable for food industry purposes. The choice of ethanol is less expedient from a cost point of view, thus acetone is preferably used. The precipitant is added to the aqueous extract cold, preferably at a temperature of -20 °C to +8 °C, for example at -20 °C.

[0074] The amount of the precipitant based on the amount of the aqueous extract can be e.g. 1-5 equivalent volumes, preferably 1.5-3 equivalent volumes, particularly preferably 1.8-2.5, for example 2 equivalent volumes. Although the amount of precipitated polysaccharides can be slightly increased by using larger volumes, smaller amounts are preferable due to economic considerations. Another advantage of precipitating with about two equivalents is that it was possible to extract the purest, lightest colored polysaccharide. The precipitant is preferably added at a slow rate, because in this way the polysaccharide separates out in large pieces.

[0075] The precipitated product is recovered from the mixture by solid / liquid separation, such as fdtration or centrifugation.

[0076] Purification

[0077] Since the raw polysaccharide extracted from the leaves of yellow onion also comprises other contaminants, we also aimed to purify the extracted dry material. Possible contaminants of the precipitated polysaccharide are proteins that can also be precipitated with acetone, thus our primary goal was to remove these foreign substances.

[0078] For purification, the precipitated raw polysaccharide is re-dissolved in water, then the solution is heated to 100 °C and boiled, preferably for about 1-15, for example 5 minutes, in order to precipitate the proteins. After filtering and cooling to room temperature, the polysaccharide is precipitated with cold acetone. For precipitation, the conditions described in the previous step are preferably used.

[0079] The resulting precipitate is separated from the liquid, e.g. by filtration or centrifugation, preferably by centrifugation, and then dried.

[0080] We note that when purifying the product - primarily due to economic considerations - we did not aim to purify the polysaccharide to a high degree. In addition to a significant amount of polysaccharides, the product also comprises other substances, but the anti-ulcer effect of the product, which was proven in animal experiments, is basically attributed to the polysaccharide.

[0081] Recovery of acetone

[0082] A part of the acetone used can be recovered from the aqueous acetone solution obtained after solid / liquid separation at the end of precipitation and / or purification. For this we can use e.g. rotary vacuum evaporator. We found that the acetone recovered in this way has a strong onion smell. The recovered acetone can be recycled, thus industrial costs can be reduced.

[0083] Characterization of the purified polysaccharide

[0084] Tests used in polysaccharide analysis were performed with the purified polysaccharide. The purified polysaccharide typically comprises the following:

[0085] Loss on drying (moisture content): 2-8%, preferably 3.5-7%;

[0086] Total sugar content by phenol -sulfuric acid method: 25-45%, expressed as glucose;

[0087] Uronic acid content by carbazole sulfuric acid method: 5-15%, preferably 7-14%, expressed as glucuronic acid.

[0088] The monosaccharide components of the polysaccharide expressed as glucose: glucose 30-50%; galactose 15-35%, preferably 18-32%; arabinose 10-30%, preferably 13-24%; and rhamnose 5-25%, preferably 10-20%.

[0089] The protein content is 13-23% by the Biuret method, expressed as human serum albumin.

[0090] The remaining part may contain, among other things, minerals, amino acids, vitamins absorbed to the polysaccharide or present as inclusions.

[0091] The composition is a light grayish beige colored, odorless, water-soluble, solid, amorphous substance. The presence of onion-smelling isothiocyanates, characteristic of onions, is not detectable. The taste is slightly salty and sweetish.

[0092] Anti-ulcer effect of the purified polysaccharide

[0093] The anti-ulcer effect of polysaccharide obtained by acetone precipitation from yellow onion (Allium cepa L.) leaves was proven in animal experiments. The polysaccharides according to the invention can be used to prevent the formation of ulcers, and as an auxiliary treatment in the treatment of already formed ulcers. The goal of therapy for peptic ulcers is to reduce the acidity of gastric juice, which helps ulcers heal and prevents further damage. Medications used to treat ulcers include proton pump inhibitors (PPIs) and H2 receptor antagonists (H2RAs). In case of auxiliary treatment, the polysaccharide according to the invention can be used in addition to these. The protective effect of the polysaccharide is also beneficial in combination with other drug treatments. Its mode of action differs from that of drug treatment, thus a more complex effect can be ensured for effective treatment.

[0094] If the polysaccharide according to the invention is used as an auxiliary treatment, its use does not interfere with the treatment. In theory, polysaccharides slow down the absorption of active pharmaceutical ingredients, so as a precaution, it is advisable to avoid their simultaneous use. Undesirable interaction beyond this is not expected. Interventions acting in different ways can improve the efficiency and effectiveness of anti-ulcer treatment. Since contact with the mucous membrane for as long as possible is beneficial in terms of the effect of the polysaccharide, it is advantageous if the composition comprising the polysaccharide is used several times a day. Use

[0095] Owing to its biological activity, the polysaccharide according to the invention can be used as a bioactive additive of fortified food products (called earlier "functional foods") and in health-protective foods.

[0096] The polysaccharide according to the invention can be used as an ingredient in various compositions, e.g. in foodstuffs, medicinal foods, foods for special medical purposes, fortified foods, pharmaceutical compositions, medical devices or dietary supplements.

[0097] In an embodiment, the foodstuffs, medicinal foods, foods for special medical purposes and / or fortified food products also comprise other food ingredients.

[0098] In another embodiment, the medicinal foods, foods for special medical purposes, fortified food products and / or dietary supplements also comprise an excipient acceptable from a food industry or nutritional point of view.

[0099] The polysaccharide according to the invention is colorless or pale in color, the flavor and smell of onion are not noticeable in it, thus it can be added to other foods without any disturbing taste effect. It can be combined particularly well with dairy products and meat products. For example, it can be used to make products such as ham, cold meats, yogurt, soft cheese, and cream cheese, but it can also be used in baked goods.

[0100] The pharmaceutical compositions, medical devices and dietary supplements according to the invention may comprise the polysaccharide extract alone or together with excipients customary in the field and chosen according to the desired administration method. In these cases, in order to achieve accurate dosing, the polysaccharide e.g. can be packaged in single -dose pouches or e.g. can be dosed as tablets or capsules.

[0101] EXAMPLES

[0102] EXAMPLE 1: Development of a method for the extraction of polysaccharide

[0103] Experiment 1: Examination of the temperature of the extractant

[0104] In this experiment, we investigated the effect of the temperature of the extractant on the amount of extracted polysaccharides. In the experiment, 10.0 g of dried leaves of yellow onion were extracted with 100.0-100.0 mL of distilled water at different temperatures for 1 hour while stirring, then filtered and pressed through four to five layers of gauze sheet. We used 30 mL of the extract thus obtained. The polysaccharides were precipitated from the extract portions using 60 mL of acetone of -20 °C. The extracted polysaccharide was filtered through a G4 glass filter, then dried at room temperature for a day, and its weight was then measured. The experiments were performed by carrying out three parallel measurements. Both cold alcohols and cold acetone are suitable for the extraction of polysaccharides, however, due to the high price of ethanol and the limitation of cheaper methanol in the food industry due to its physiological hazards, we chose acetone. The results of the preliminary experiments are presented in Figure 1.

[0105] As a result of the experiment, it can be said that there was no significant difference in the amount of extracted polysaccharides in the temperature range of 25-60°C. The most effective extraction temperature was room temperature of 25 °C. From an industrial point of view, it is advantageous that in order to prepare the extract, it is not necessary to invest energy in heating the system, and a temperature of 15-20 °C can also be suitable.

[0106] Experiment 2: Examination of the amount of extractant to be used

[0107] In the second preliminary experiment, we examined the effect of changing the amount of extractant on the amount of extracted polysaccharide. In the experiment, 10.0-10.0 g of dried onion leaves were extracted with 100.0, 150.0, 300.0 and 500.0 m of water at a temperature of 25 °C for one hour, then filtered and pressed through five layers of gauze sheet. Proportional parts of the extracts were then treated with two equivalents of acetone with a temperature of -20 °C in order to precipitate the polysaccharides. The extracted material was filtered using a G4 glass filter, dried at room temperature for a day, and then it was weighed. The experiments were performed by carrying out three parallel measurements. The results of the experiments are summarized in Figure 2.

[0108] Based on the results, it can be said that the tested range is suitable for the extraction of polysaccharides; at the same time, the largest amount of polysaccharides could be extracted with 150.0 and 300.0 m of distilled water, however, the difference in the amounts of extracted polysaccharides between the two methods is very small, thus from a cost-benefit ratio point of view, the extraction with 150 mb, i.e. 1: 15 ratio, is preferable considering the further purification steps. Using less extractant reduces the amount of polysaccharides that can be extracted, but this reduction is not drastic. Therefore, prioritizing cost reduction, less extractant, e.g. a ratio of 1: 10 or 1:5 can also be used.

[0109] Experiment 3: Examination of extraction time

[0110] In our third preliminary experiment, we investigated the effect of extraction time. 10.0-10.0 g of dried green onion leaves were extracted with 100.0-100.0 mb of distilled water at room temperature while stirring for different time periods, then filtered and pressed through gauze sheet. 30.0-30.0 mb of the extracts thus prepared were treated with two equivalent amounts of acetone of -20 °C in order to extract the polysaccharides. The precipitated polysaccharides were filtered through a G4 glass filter, dried for a day, and then weighed. The experiments were performed by carrying out three parallel measurements.

[0111] The results of the measurements are presented in Figure 3.

[0112] As a result of the experiments, it can be said that the extraction lasting one hour proved to be the most effective. The decrease experienced during longer extraction can presumably be explained by the enzymatic processes taking place in the aqueous medium.

[0113] Experiment 4: Examination of the amount of precipitant

[0114] In continuation of our series of preliminary experiments, we examined the amount of acetone that can be used for the precipitation of polysaccharides. 10.0-10.0 g of dried onion leaves were extracted with 100.0-100.0 m of distilled water while stirring at 25 °C for 1 hour. After filtering and pressing, a 30 m portion of the extract was used. 1, 2, 3, 4 and 5 equivalent amounts of acetone of -20 °C were added to the extracts in order to precipitate the polysaccharides. The precipitated product was filtered through a G4 glass filter, dried for a day, and then weighed. The experiments were performed in three parallel measurements. The results of the experiments are presented in Figure 4.

[0115] Considering the results of the experiments, it can be said that although the largest amount of polysaccharide could be extracted with 5 equivalent amounts of acetone, taking into account the extracted dry matter masses, the percentage of their amounts and the cost -benefit relationships, the precipitation with two equivalents of acetone is the most effective. Another advantage of precipitating with two equivalents is that it was possible to extract the purest, lightest colored polysaccharide. An important experience is that it is advisable to add acetone slowly, because this way the polysaccharide separates in large pieces.

[0116] Experiment 5: Purification of the polysaccharide

[0117] Since the raw polysaccharide extracted from the leaves of yellow onion also comprises other contaminants, we also aimed to purify the extracted dry material. Possible contaminants of the precipitated polysaccharide are mono- and oligosaccharides that can also be precipitated with acetone, as well as proteins, and plant acids and minerals that aggregate as inclusions, thus our primary goal was to remove these foreign substances.

[0118] In our experiment, 1500 mg of polysaccharide extracted from leaves of yellow onion was re-dissolved in 200.0 mb of water, and then, in order to precipitate the proteins, the solution was heated to 100 °C and boiled for 5 minutes. After filtering and cooling to room temperature, the polysaccharide was precipitated with two equivalent amounts of acetone of -20 °C. The resulting precipitate was purified using a centrifuge (0.5 L, 2000 rpm), dried at room temperature for a day, and then it was weighed, which was 640.9 mg (42.73% of the initial amount).

[0119] The color of the starting material was medium brown, while after purification we obtained a pale gray material.

[0120] EXAMPLE 2: Scaled-up production of polysaccharide of green onion leaf

[0121] 10 kg of fresh green onion leaves were chopped, dried at room temperature (3 kg) and extracted by shaking with 15 L of distilled water at room temperature. The extract was filtered and the remaining plant part was squeezed out, then the resulting 5 L of filtrate was precipitated with 10 L of cooled (-20 °C) acetone. The precipitate (raw polysaccharide) was filtered through a Buchner funnel and then dried. Afterwards, the raw polysaccharide was re-dissolved in 2 L of hot distilled water and the solution was boiled for 5 minutes. After cooling, the solution was filtered and again precipitated with acetone (purification). The precipitated material was filtered through a Buchner funnel and dried at room temperature. The mass of the thus obtained purified polysaccharide was 14.5 g.

[0122] Determination of the main analytical characteristics of the extracted polysaccharide (P.S.).

[0123] Determination of total sugar content using phenol-sulfuric acid method

[0124] Execution: a 0.1% solution was prepared from the polysaccharide produced according to Example 2 with water, then 1.00 mb of this was taken out with an automatic pipette and measured into a sample vial. To this, 1.00 mb of 2.5% phenol solution and then 5 mb of concentrated H2SO4 were added.

[0125] Extinction was measured with a spectrophotometer at 480 nm against a blank sample after waiting thirty minutes. The obtained value was projected onto a calibration line made from glucose.

[0126] 1.0000 g P.S. sample comprises 346.46±4.36 mg of total sugar.

[0127] Expressed as a percentage: 34.65±0.44%

[0128] Determination of uronic acid content

[0129] To 1.00 mb of a 0.01% sample (solvent: water), 5 mb of reagent (0.25 M borax concentrated sulfuric acid) was added in small portions under salted ice cooling at -5 °C, then the solution was thermostated at 85 °C for 20 minutes, then at 25 °C for 5 minutes. Then 200 ph of 0.5% carbazole reagent was added to the sample. The absorbance was measured at 530 nm after 15 minutes. Blank sample was a sample solution prepared with borax sulfuric acid, to which no carbazole solution was added (methanol was used instead).

[0130] By subtracting the obtained extinctions from each other, we got an extinction difference, which was projected onto a calibration line made from glucuronic acid.

[0131] 1.0000 g P.S. sample comprises 106.39±1.069 mg of total uronic acid.

[0132] Expressed as a percentage: 10.64±0.19%,

[0133] Determination and quantitative measurement of the monosaccharide components of the polysaccharide by layer chromatography

[0134] The P.S. was hydrolyzed with trifluoroacetic acid (TFA), using 400 mg of P.S. + 4 m of TFA (99%) under reflux for 2 hours. Then, 1-1 m of methanol was added in three parts, and the methanol and TFA were evaporated with N2.

[0135] The sample was diluted to 5 mb, then 1.00 mb of the sample was fdtered through 2.00 g of AI2O3 (Merck, neutral 1.01077.2000). The resulting solution was evaporated with N2, then dissolved in 1.00 mb of water. Using a Hamilton pipette, 1-1 ph of this was applied to a thin-layer chromatogram (Merck silica gel 60 F254, 1055540001), on which the calibration points made from glucose were also applied.

[0136] The concentration range of the calibration line made from glucose was 2-7 pg, the developing mixture was acetone-n-butanol-H2O 8: 1: 1 + 1% CC.NH3, thymol-sulfuric acid reagent was used as a developer, the evaluation was carried out after heating at 110 °C for 5 minutes. The quantitative measurement was done with the CP Atlas software in blue mode.

[0137] Percentage distribution of the sample's 4 monosaccharides identified by layer chromatography in relation to each other, expressed in glucose:

[0138] Glucose: 41.64%

[0139] Galactose: 24.92%

[0140] Arabinose: 18.38%

[0141] Rhamnose: 15.06% Determination of protein content by Biuret method against HSA standard

[0142] 3 mL of Biuret reagent was added to 1 mL of 0.1% aqueous P.S. sample, and after 30 minutes the light absorption was measured against a calibration line made from human serum albumin (HSA) (Sigma) at 555 run on a spectrophotometer. The HSA standard calibration line was prepared with solutions with a concentration of 5-50 mg / 10 mL.

[0143] 1.0000 g P.S. sample comprises 179.3±3.56 mg of total protein expressed as HSA.

[0144] Expressed as a percentage: 17.93±0.36%

[0145] Determination of loss on drying (moisture content): the value of loss on drying was determined by drying at 105 °C to constant mass, the value of loss on drying was 5.18%.

[0146] COMPARATIVE EXAMPLE: Examination of the occurrence of onion leaf polysaccharide in onion bulb

[0147] The purpose of the examination was to evaluate whether the polysaccharide extracted from the leaf is present in the onion bulb.

[0148] Starting material: 314 g peeled yellow onion.

[0149] Sample preparation: The cleaned onion was cut into small pieces (approx. 1 cm pieces) and frozen at -80 °C, then dried using a lyophilizer at 0.08 mbar. The weight of the resulting product was 31 g (10% of the fresh onion), the product was odorless and had a sweet taste. The product was extracted in the same way used for the extraction of leaf P.S. : it was extracted with 465 mL of deionized water for 1 hour at 25 °C. The extracted onion was pressed, the total volume of the extract was 400 mL. To this extract, 800 mL of acetone of -20 °C was added, it was allowed to aggregate, and then centrifuged. Even after centrifugation, no measurable amount of aggregate could be collected from the solution (based on the polysaccharide prepared from onion leaves, the expected amount would have been 5 g).

[0150] Overall, it can be concluded that using the extraction protocol used for the onion leaf, only a negligible amount of the polysaccharide obtained from the leaf can be obtained from the onion bulb.

[0151] EXAMPLE 3: Pharmacological investigation of polysaccharide of green onion leaf for anti-ulcer effect

[0152] A) Characterization of the applied animal experimental models

[0153] The animal experiments were carried out with the permission of the Scientific Ethics Council for Animal Experimentation (IV. / 01758-2 / 2008). Mixed-sex Sprague -Dawley rats weighing 170-210 g were used for all experiments. We randomized 8 animals to each group. The conditions of the animal experiments complied with the relevant animal protection rules and general requirements. In all animal experimental models, the animals received the test substance or the reference substance, then damage to the gastric mucosa was induced with an ulcer-causing agent. In order to quantitatively characterize the degree of damage, the stomachs of the euthanized animals were removed, and then the condition of the mucous membrane was characterized using the following scoring system.

[0154] The scores for gastric mucosal injuries were totaled per animal, the resulting scores were averaged per group, and the results were statistically evaluated (ANOVA test using the GraphPad Prism 4.0 program).

[0155] B) Effect of a single treatment on alcohol-induced ulcer

[0156] We investigated the protective effect of onion polysaccharide on an ulcer model induced by concentrated alcohol. The animals, starved for 16 hours, were treated per os with vehicle (5 mL / kg distilled water), methylcellulose (250 mg / kg), carbenoxolone (125 mg / kg), or with onion polysaccharide (250 mg / kg). During the last 3 hours of the starvation period, the animals did not receive water. After that, the test substances were administered, and 60 minutes later, the experimental ulcer was induced with 5 mL / kg of 96% ethanol. After another 60 minutes, the animals were euthanized, the stomachs were removed, and the ulcer formed was evaluated by scoring (Figures 5 to 6).

[0157] We found that only carbenoxolone had a positive and significant effect on the alcohol -induced ulcer at the doses used. The stomachs of animals treated with methylcellulose practically did not differ from those of rats randomized to the control group, while the onion polysaccharide did cause a modest decrease in the score, but it did not reach statistical significance. C) Effect of a single treatment on salicylic acid-induced ulcer

[0158] In this experiment, the gastric protective effect of the onion extract was evaluated on an ulcer model induced by salicylic acid. The animals, starved for 16 hours, were treated per os with vehicle (5 mL / kg distilled water), methylcellulose (250 mg / kg), carbenoxolone (125 mg / kg), or with onion polysaccharide (250 mg / kg). During the last 3 hours of the starvation period, the animals did not receive water. After 60 minutes, the animals received 400 mg / kg of salicylic acid suspended in 0.25% methylcellulose, again at a treatment volume of 5 mL / kg. After another 60 minutes, the animals were euthanized with isoflurane, bled, the stomachs were removed, and the ulcer formed was evaluated by scoring (Figures 7 to 8).

[0159] We found that all three treatments considerably and significantly reduced the intensity of the induced ulceration. The effect of the onion polysaccharide was practically the same as the effect of methylcellulose, causing an inhibition of about 45% in the overall score. The effect of carbenoxolone used as a reference was more pronounced.

[0160] D) Effect of chronic treatment on salicylic acid-induced ulcer

[0161] In the chronic study, the test substances were used for 7 days without fasting. The test substances and their doses were as follows: 5 mL / kg distilled water (vehicle), methylcellulose (125 mg / kg), carbenoxolone (200 mg / kg), and onion polysaccharide (250 mg / kg). The dosage volume was 5 mL / kg in all cases. We carried out the last treatment on the 8th day, after 16 hours of starvation, in the last 3 hours of which the animals were also deprived of water. 60 minutes after the administration of the test substances, acute gastric ulcer was induced with 450 mg / kg of salicylic acid (suspended in 0.25% methylcellulose, in a dosage volume of 5 mL / kg), and after another 60 minutes the ulceration formed was evaluated (Figures 9 to 10).

[0162] We found that methylcellulose reduced the severity of the formed ulcers, but the reduction was not significant. In contrast, both the carbenoxolone used as a reference and the onion polysaccharide significantly reduced the intensity of the formed lesions.

[0163] Summary

[0164] Based on the results of our animal experiments, it can be concluded that the polysaccharide prepared from yellow onion has agastric protective effect and reduces mucosal damage caused by local irritation. The protective effect against ulcer is greater in the case of ulcers induced by non-steroidal antiinflammatory drugs than in ulcerations induced by ethanol. The effect of onion polysaccharide applied chronically (for 7 days) on the experimental ulceration induced by salicylic acid further confirms that the onion polysaccharide significantly reduces the intensity of the inflammatory lesions that have developed, and thus e.g. could serve as the basis for the development of a health-protective food product family.

[0165] INDUSTRIAL APPLICABILITY OF THE INVENTION

[0166] According to the invention, polysaccharide with valuable biological activity can be produced from yellow onion (Allium cepa L.) leaves, which are considered agricultural waste. The polysaccharide has a stomach-protecting, anti -ulcer effect, and stomach ulcers can be prevented with its use. The polysaccharide according to the invention e.g. can be used as bioactive additive of fortified food products (previously called "functional foods").

[0167] REFERENCES

[0168] Benitez, V., Molla, E., Martin-Cabrejas, M. A., Aguilera, Y., Lopez-Andreu, F. J., Cools, K., Terry, L. A., & Esteban, R. M. (2011). Characterization of industrial onion wastes (Allium cepa L.): dietary fibre and bioactive compounds. Plant foods for human nutrition (Dordrecht, Netherlands), 66(1 ), 48-57. https: / / doi.Org / 10.1007 / sl l l30-011-0212-x

[0169] Cebin et al. (2020). Onion Solid Waste as a Potential Source of Functional Food Ingredients. Engineering power: bulletin of the Croatian Academy of Engineering, 15(3): 7-13.

[0170] Dela Cruz, Rita T. (2020). From waste to wealth: Developing new products from onion leaves. Agriculture Magazine (Philippines), 24(1)'. 10-12.

[0171] Khan et al. (2020). Ameliorative Potential of Allium cepa Lam. Leaves on Diabetes Induced and Chronic Constriction Injury Induced Neuropathic Pain in Experimental Rats. Indian Journal of Pharmaceutical Education and Research, 54(1): 143-149.

[0172] Kianian, F., Marefati, N., Boskabady, M., Ghasemi, S. Z., & Boskabady, M. H. (2021). Pharmacological Properties of Allium cepa, Preclinical and Clinical Evidences; A Review. Iranian journal of pharmaceutical research: IJPR, 20(2), 107-134. https: / / doi.org / 10.22037 / ijpr.2020.112781.13946

[0173] Ma, Y. L„ Zhu, D. Y„ Thakur, K„ Wang, C. H„ Wang, H„ Ren, Y. F„ Zhang, J. G„ & Wei, Z. J. (2018). Antioxidant and antibacterial evaluation of polysaccharides sequentially extracted from onion (Allium cepa L.). International journal of biological macromolecules, 111, 92-101. https: / / doi.Org / 10.1016 / j.ijbiomac.2017.12.154

[0174] Marrelli, M., Amodeo, V., Statti, G., & Conforti, F. (2018). Biological Properties and Bioactive Components of Allium cepa L.: Focus on Potential Benefits in the Treatment of Obesity and Related Comorbidities. Molecules (Basel, Switzerland), 24(1), 119. htps: / / doi.org / 10.3390 / molecules24010119

[0175] Nausheen et al. (2014). Cardioprotective and Antioxidant activity of Onion (Allium cepa) Leaves Extract in Doxorubicin Induced Cardiotoxicity in Rats. Annals of Experimental Biology 2014, 2(2): 37-42. Sagar et al. (2022). Onion (Allium cepa L.) bioactives: Chemistry, pharmacotherapeutic functions, and industrial applications. Food Frontiers, 3(3): 380-412.

[0176] Upadhyay (2016). Nutraceutical, pharmaceutical and therapeutic uses of Allium cepa: A review. International Journal of Green Pharmacy 70(l):46-64.

[0177] Zhao, Xin-Xin et al. (2021). Recent Advances in Bioactive Compounds, Health Functions, and Safety Concerns of Onion (Allium cepa L.). Front. Nutr. 8:669805. doi: 10.3389 / fnut.2021.669805

[0178] Zhu, D. Y„ Ma, Y. L„ Wang, C. H„ Wang, H„ Ren, Y. F„ Zhang, J. G„ Thakur, K„ & Wei, Z. J. (2017). Insights into physicochemical and functional properties of polysaccharides sequentially extracted from onion (Allium cepa L.). International journal of biological macromolecules, 105 (Pt 1), 1192-1201. htps: / / doi.Org / 10.1016 / j.ijbiomac.2017.07.164

Claims

CLAIMS1. A polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves, which comprises the following, based on the total weight of the extract:Dry matter: 92-98%,Sugars: 25-45%,Uronic acid: 5-15%,Proteins: 13-23%.

2. The polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves according to claim1, wherein the polysaccharides of the extract consist of the following monosaccharides:Glucose: 30-50%Galactose: 15-35%Arabinose: 10-30%Rhamnose: 5-25% wherein the percentages are expressed as the mass ratio of the monosaccharides that make up the polysaccharides.

3. A method for extracting polysaccharides from yellow onion (Allium cepa L.) leaves, characterized in that a) an extract is prepared from the onion leaves with water, then a solid / liquid separation is carried out, b) the polysaccharides are precipitated from the aqueous solution with acetone or alcohols to obtain raw polysaccharide, and c) the raw polysaccharide is purified to obtain a purified polysaccharide.

4. The method according to claim 3, wherein in step b) the polysaccharide is precipitated with acetone.

5. A polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves, which is obtainable by the method according any one of claims 3 to 4.

6. The polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves according to claim5, which comprises the following, based on the total weight of the extract:Dry matter: 92-98%Sugars: 25-45%Uronic acid: 5-15%Proteins: 13-23%.

7. The polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves according to claim6, wherein the polysaccharides of the extract consist of the following monosaccharides:Glucose: 30-50%Galactose: 15-35%Arabinose: 10-30%Rhamnose: 5-25% wherein the percentages are expressed as the mass ratio of the monosaccharides that make up the polysaccharides.

8. The polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves according to any one of claims 1 to 2 and 5 to 7 for use in the prevention or auxiliary treatment of gastric ulcer.

9. The polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves for use according to claim 8, wherein the gastric ulcer is an alcohol -induced gastric ulcer.

10. The polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves for use according to claim 8, wherein the gastric ulcer is a gastric ulcer induced by non-steroidal anti-inflammatory drug.

11. A composition, preferably a pharmaceutical composition, medical device, dietary supplement, foodstuff, medicinal food, food for special medical purposes or fortified food, which comprises the polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves according to any one of claims 1 to 2 and 5 to 7.

12. A composition, preferably a pharmaceutical composition, medical device, dietary supplement, foodstuff, medicinal food, food for special medical purposes or fortified food, which comprises the polysaccharide extract obtained from yellow onion (Allium cepa L.) leaves according to any one of claims 1 to 2 and 5 to 7, for use in the prevention or auxiliary treatment of gastric ulcer.

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