A cationic derivative of a beta-glucan of cereal origin for use in treating fungi

Abstract

The first subject of the invention is a use of cationically modified cereal beta-glucan for inhibition or prevention of fungal growth in a mammalian patient, especially a human, wherein the degree of cationic modification of the beta-glucan ranges from 70% to 100%. The invention also relates to the use of said beta-glucan as an antifungal agent for plant protection, an additive to cosmetics, preferably as a preservative, or a dietary supplement.

Description

[0001] A use of a cationic derivative of a beta-glucan of cereal origin for combating pathogenic fungi

[0002] The subject of the invention is a use of cationically modified cereal beta-glucan for use in inhibiting or preventing the growth of fungi.

[0003] The scientific publication "The Use of a Barley-Based Well to Define Cationic Betaglucan to Study Mammalian Cell Toxicity Associated with Interactions with Biological Structures" (Pharmaceutics 2023, 15(7), 2009) describes the synthesis of a polycation based on barley beta-glucan. Additionally, the paper presents the results of studies on its toxicity to mammalian cells and the mechanism responsible for this phenomenon. Scientific publication "Preliminary Studies on the Mechanism of Antifungal Activity of New Cationic 6-Glucan Derivatives Obtained from Oats and Barley” (ACS Omega 2022 7 (44), 40333-40343) describes an alternative method for obtaining cationic barley and oat beta-glucan polymers and the results of studies on their antifungal properties.

[0004] The aim of the invention is therefore to provide macromolecular compounds for use in preparations intended to prevent the growth of fungi and to combat already growing fungi, in various applications, both as preparations for administration to humans and animals for the prevention and treatment of certain fungal infections, including as additives to cosmetics, e.g. acting as preservatives, and as fungicides for use in protecting plants against certain fungal diseases and combating fungal diseases in plants.

[0005] The subject of the invention is a cationically modified beta-glucan obtained from a cereal selected from the group comprising barley, oats, or rye for use in inhibiting or preventing the growth of fungi in a mammalian patient, especially a human, wherein the beta-glucan is modified with glycidyltrimethylammonium chloride, wherein the degree of cationic modification (GTMAC), defined as the number of GTM AC molecules attached per 100 polymer mers, ranges from 70% to 100%.

[0006] Preferably, the cereal beta-glucan is selected from the group comprising barley beta-glucan, oat beta-glucan, or rye beta-glucan. In a preferred embodiment of the invention, the degree of modification of barley beta -glucan ranges from 71% to 100%, most preferably 100%.

[0007] In the next preferred embodiment of the invention, the degree of modification of oat betaglucan is 87%.

[0008] In another preferred embodiment of the invention, the degree of modification of rye betaglucan is 100%.

[0009] In a different preferred embodiment of the invention, beta-glucan is obtained from wholemeal flour.

[0010] Preferably, the cationica lly modified beta-glucan has a zeta potential of not less than 42 mV, preferably from 42 mV to 48 mV.

[0011] Preferably, the fungus is selected from the Candida genus, preferably Candida tropicalis or Candida parapsilosis, the Cryptococcus genus, preferably Cryptococcus neoformans, the Malassezia genus, preferably Malassezia furfur, Malassezia slooffiae, Malassezia japonica or Malassezia pachydermatis, the Fusarium genus, preferably Fusarium graminearum, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani or Fusarium verticillioides, Scopulariopsis brevicaulis, Scedosporium apiospermum, the Lomentospora genus, preferably Lomentospora prolificans.

[0012] The second subject of the invention is a use of cationica lly modified beta-glucan specified in the first subject of the invention as an antifungal agent for plant protection, an additive to cosmetics, preferably as a preservative, or a dietary supplement. Preferably, the cereal betaglucan is selected from the group comprising barley beta-glucan, oat beta-glucan, or rye betaglucan.

[0013] The essence of the invention is the use of cationic polymers with a high degree of modification, obtained in a reaction with glycidyltrimethylammonium chloride (GTMAC, glycidyitrimethylammonium chiaride, CAS: 3033-77-0). The obtained results indicate that the origin of beta-glucans (the substrate from which they were isolated) and the degree of cationization (modification) are necessary conditions for achieving the effect of inhibiting the growth of certain fungal species. The studies on antifungal properties have been conducted on species that are known pathogens of humans and animals, as well as plant pests, hence having an effective tool for limiting the growth of these organisms is important from the point of view of medicine, cosmetology, and agriculture.

[0014] Beta-glucans isolated from cereals, i.e., barley, oats, and rye, were subjected to a cationization process using glycidyltrimethylammonium chloride (GTMAC), obtaining positively charged polymers. The polymers were characterized from a chemical point of view and their antifungal activity against fungi causing diseases in humans, animals, and plants was tested in vitro. Antifungal activity has been demonstrated, which manifested in the inhibition of the growth of fungi and low values of minimum inhibitory concentrations (MIC) inhibiting their growth. Antimycotic activity was observed for fungi of the following species: Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Scopulariopsis brevicaulis, Scedosporium apiospermum, Lomentospora prolificans, which can cause infections in humans and animals, as well as species: Fusarium graminearum, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Fusarium verticillioides, which cause infections in humans and animals, as well as plant diseases. Barley beta-glucan has also been found to be active against fungi of the Malassezia genus, which cause pityriasis versicolor in humans and contribute to other skin conditions, including dandruff, atopic dermatitis, and seborrheic dermatitis. The remaining barley beta-glucan derivatives with lower degrees of modification and obtained from other sources did not demonstrate such good activity (low MIC values). In practice, this means that in the case of confirming the antifungal properties of modified beta-glucans from cereals, with a high degree of cationization, a system with uniquely desirable properties would be available. The use of beta-glucan derivatives with a high degree of cationization may be applied in preparations intended to prevent the growth of fungi and to combat already growing fungi, in various applications, both as preparations for administration to humans and animals for the prevention and treatment of certain fungal infections, including as additives to cosmetics, and as fungicides for use in protecting plants against certain fungal diseases and combating fungal diseases in plants.

[0015] It has been demonstrated that not all cationic beta-glucan derivatives obtained in reactions with GTMAC have satisfactory antifungal properties. This effect is closely related to the degree of modification - cationization - which should be understood as the number of quaternary amine groups or GTMAC molecules on average per one mer in a polymer. This parameter was determined by H1NMR measurements of solutions of the obtained polymers. The method of determining the degree of cationization (modification) was described in the scientific publication "The Use of a Barley-Based Well to Define Cationic Betaglucan to Study Mammalian Cell Toxicity Associated with Interactions with Biological Structures" (Pharmaceutics 2023, 15, 2009). The second condition for obtaining a GTMAC-modified polycation that has optimal antifungal activity is to use as a substrate a cereal material of barley, oat, or rye origin, preferably from wholemeal flour. Macromolecules with an optimal degree of modification may only be obtained under specific conditions of conducting the reaction (sequential, twofold repetition of modification in case of barley beta-glucan derivative or increasing the amount of cationizing agent in case of a derivative obtained from oats and rye), which requires a suitable proportion of reagents and a scheme of performing the synthesis. The synthesis of GTMAC-modified beta-glucans of barley origin was described in publication Pharmaceutics 2023, 15(7), 2009. The synthesis of cationic derivatives of oat and rye beta-glucans has not been described in the literature.

[0016] Furthermore, it has been shown that the degree of cationization of beta-glucans of cereal origin is a significant factor affecting their antifungal activity. The higher the degree of cationization, the better the antifungal activity, manifested by lower concentrations inhibiting the growth of fungi. Therefore, cationic derivatives of beta-glucans of cereal origin with a low degree of cationization (below 70%) will not exhibit protective properties against pathogenic fungi and will not be effective in combating them, as they will not inhibit or prevent the growth of these fungi.

[0017] Embodiments for carrying out the invention have been illustrated in the figures, where

[0018] Fig. 1 shows the FTIR-ATR spectrum of cationically modified rye beta-glucan. Table 1 below shows the assigned vibrations:

[0019] Fig. 2 shows the nuclear magnetic resonance spectrum of cationically modified rye betaglucan in D2O. Example 1. Obtaining and physicochemical characteristics of beta-glucan polycations

[0020] Beta-glucan polycations, which are the subject of the invention, were obtained and modified as described below.

[0021] Beta-glucans of cereal origin: from barley (JG), oats (OG), rye (ZG) were isolated from wholemeal flours, respectively: barley, oat, and rye flour. The method of isolating barley betaglucan was described in publication Pharmaceutics 2023, 15(7), 2009. In short, the procedure went as follows. 20 g of barley flour was suspended in 200 ml of water. The pH of the suspension was adjusted to the value of 7.6 using a 10% potassium carbonate solution, after which the mixture was heated for 30 minutes at a temperature of 45°C and centrifuged for 30 minutes at a temperature of 4°C at 4940 G. The supernatant obtained was adjusted to pH 4.5 using 2 M HCI and centrifuged for 30 minutes at 4940 G at 4°C. The beta-glucan present in the supernatant was precipitated with ethanol (volume ratio 1:1 supernatant:ethanol) and left overnight at a temperature of 8°C. The mixture was then centrifuged for 10 min at 3780 G, and the resulting precipitate was dried under reduced pressure at 40°C. The isolation of oat and rye beta-glucans was conducted in a similar manner, using wholemeal oat flour and wholemeal rye flour, respectively, wherein the pH of the supernatant was brought to 3.5 using 2 M HCI.

[0022] Beta-glucan from Alcaligenesfaecalis bacteria is commercially available (Curdlan, CAS: 54724- 00-4).

[0023] In the case of beta-glucan from Pleurotus ostreatus, the isolation was carried out as follows: fruiting bodies of the fungi were frozen in liquid nitrogen and crushed in a mortar into the smallest possible fragments, after which they were washed with ethanol. 100 cm3of 80% ethyl alcohol was added to the fruiting bodies of the fungi and heated at 70°C for 10 minutes. The mixture was centrifuged at 3000 rpm for 5 minutes and the supernatant was removed. This procedure was carried out fourtimes. The mixture was centrifuged and the insoluble fractions were dried in a dryer (under reduced pressure at a temperature of approximately 60°C).The mixture was heated in an aqueous solution of potassium carbonate with a pH of 10 (10% potassium carbonate solution diluted with water so as to obtain a pH = 10). The warm mixture was then mechanically mixed for 5 min at 10,000 rpm. The mixture was heated for an additional 30 min at a temperature of 90°C and filtered through a sieve. The precipitate was heated in 100 cm3of waterfor40 min, filtered again through a sieve, and centrifuged at 10,000 rpm for 5 min. Beta-glucan was precipitated using cold (4°C) ethanol (volume ratio 1:1 supernatant:ethanol).

[0024] In the case of beta-glucan from Saccharomyces cerevisiae: 10g of dried baker's yeast was suspended in 30 ml of water and shaken for 1 h at 37°C. The resulting suspension was then mixed with 30 ml of PBS of pH=7.4 containing 0.2% Triton X100 surfactant and subjected to an immersion ultrasonic homogenizer with a power of 20 W for 30 minutes. The vessel was placed in an ice bath to dissipate the excess generated heat. In the next step, the resulting suspension was centrifuged at 10,000 rpm for 5 min to separate the precipitate containing beta-glucans from the supernatant. The precipitate was resuspended in 40 ml of ethanol and shaken for 30 min, then centrifuged again as before. The process of purifying the material was repeated 3 more times. The resulting material containing crude beta-glucans was dried under reduced pressure.

[0025] The cationization of beta-glucans isolated according to the above description was carried out as described in the publication by Kaminski K. et al. (ACS Omega 2022 7 (44), 40333-40343). In short, the beta-glucan obtained according to the above description was suspended in 50 cm3of water, followed by the addition of 200 mg of NaOH and GTMAC (exact amounts: 6 ml for barley polymer JG50 and polycation from Alcaligenes faecalis, 12 ml for barley polymer JG71, oat polymer OG87, and rye polymer ZG100). The reaction was carried out at 60°C for 4 hours, after which the solution was transferred to a dialysis tube and dialyzed against water for 4 days while changing the water to fresh one once a day. The obtained mixture was centrifuged for 5 min at 10,000 rpm, and the supernatant was freeze-dried. In the case of JG100 synthesis, JG71 was used as the substrate, and the remaining steps were carried out analogously to those described above, using 200 mg of NaOH and 6 ml of GTMAC. Reaction time, temperature, purification, and isolation were carried out analogously.

[0026] The figures show FT-IR (Fig. 1) and nuclear magnetic resonance (Fig. 2) spectra for modified rye beta-glucan.

[0027] The synthesis of polycation from Pleurotus ostreatus was carried out as follows: 50 mg of crude beta-glucan isolated according to the description in the previous paragraph was suspended in an aqueous solution of potassium carbonate with a pH of 10 (10% potassium carbonate solution diluted with water so as to obtain pH = 10) while mixing for 30 min, and then the mixture was centrifuged for 5 min at 10,000 rpm. 200 mg of NaOH and 6 ml of GTMAC were added to the supernatant obtained, and the reaction was carried out with continuous mixing at 60°C for 4 hours, after which the solution was transferred to a dialysis tube and dialyzed against water for 4 days while changing the water to fresh one once a day. The obtained mixture was centrifuged for 5 min at 10,000 rpm, and the supernatant was freeze- dried in order to isolate the polycation. The synthesis of polycation from Saccharomyces cerevisiae consisted of suspending 2 g of crude beta-glucan isolated as described in the previous paragraph in 100 ml of water with 0.4 g of NaOH and mixing at room temperature for 30 min. After this time, 12 ml of GTMAC was added and the reactions were carried out for 1 hour at 60°C. After this time, the residues of unreacted substrate were first removed by centrifugation at 10,000 rpm for 5 minutes, and then by dialysis against water for 4 days with daily changing of the water. The solution thus obtained was freeze-dried to isolate the polycation. The physicochemical properties of modified beta-glucans are listed in Table 2 below.

[0028] Table 2. Physicochemical properties of cationic beta-glucans

[0029] * measurement for a polymer solution of 2 g / l in water at 25°C. Example 2. In vitro antifungal activity of a cationic derivative of barley polymer

[0030] The antifungal activity of modified beta-glucans was tested using the microdilution method in liquid culture media in 96-well titration plates. Fungal cells were incubated in the presence of various concentrations of polymers (ranging from 0.49 mg / l to 250 mg / l, using a series of ten twofold dilutions) and MIC (Minimum Inhibitory Concentration) values were determined, which were an indicator of antimycotic properties. MIC values were determined visually as complete inhibition of fungal growth visible to the naked eye, after obtaining fungal growth in the growth control (without the addition of polymer). Table 3 shows the results obtained for four fungal species and beta-glucans isolated from different substrates and differing in the degree of cationization. When comparing MIC values, it may be observed that beta-glucans derived from barley, oats, and rye inhibit fungal growth, and this property depends on the degree of cationization. In the case of barley beta-glucan with the lowest degree of cationization (JG5O), no antifungal activity was observed, while intermediate, higher, and the highest degree of cationization (JG71 and JG100) resulted in inhibition of fungal growth, with a stronger effect visible as lower MIC values for the polymer with the highest degree of modification -JG100.

[0031] Table 3. Values of minimum inhibitory concentrations (MIC) inhibiting the growth of selected fungal species obtained for cationic polymers derived from cereals (barley, oats, rye) and other sources. nd - not determined in the tested range of polymer concentrations, i.e., higher than 250 mg / l or no antifungal activity *symbols JG50, JG71 and JG100 indicate the degree of polymer cationization in % using GTMAC, i.e. the number of GTMAC molecules attached per 100 mers of the polymer. The lower the number, the lower the degree of polymer cationization

[0032] 1polycation isolated from bacteria

[0033] 2polycation isolated from fungi

[0034] Selected cationic polymers from barley, oats, and rye were tested on a larger number of fungal species. The species for which the antifungal activity of polymers was assessed and an attempt was made to determine the MIC values of these substances are listed in Table 4. The studies have shown that these polymers inhibit the growth of selected pathogenic fungi, having an identical antifungal spectrum and comparable MIC values. Among the fungi that have been found to be sensitive to low concentrations of polymers, there are species described in the literature as causing skin and nail fungal infections (Scopulariopsis brevicaulis, Candida species), infections of other tissues in humans and animals (Fusarium species, Scedosporium apiospermum, Lomentospora prolificans, Cryptococcus neoformans, Candida species), as well as plant diseases (Fusarium species). For the tested Malassezia species causing skin conditions in humans, i.e., pityriasis versicolor, seborrheic dermatitis, and otitis in animals (Malassezia pachydermatis), the MIC values of barley polymer were slightly higher than for other sensitive fungi and ranged from 7.81 mg / l to 31.25 mg / l.

[0035] Table 4. Values of minimum inhibitory concentrations (MIC) inhibiting the growth of the tested fungal strains obtained for barley, oat, and rye polymers with the highest possible degrees of cationization.

[0036] Example 3. In vitro comparison of the antifungal activity of cationic beta-glucan and antifungal drugs

[0037] The antifungal activity of the cationic derivative of barley polymer with the highest degree of cationization (JG1OO) was compared with that of antifungal drugs: terbinafine, ciclopirox, natamycin, amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole. The study involved determining the minimum inhibitory concentrations (MIC) inhibiting the growth of fungi, using the microdilution method in liquid culture media in 96-well titration plates. The polymer was tested at a concentration range of 0.49-250 mg / l, whereas the drugs were tested in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) procedure for fungi. The experiment used 7 strains of Scopulariopsis brevicaulis and two strains of Fusarium solani. The obtained MIC values are shown in Table 5.

[0038] It has been shown that barley polymer with the highest degree of cationization (JG100) exhibits lower or comparable MIC values to the tested antifungal drugs against 5. brevicaulis strains. In the case of tests carried out for F. solani, the MIC values of the polymer were lower than those obtained for ciclopirox, voriconazole, and itraconazole, and 2-8 times higher than for amphotericin B.

[0039] Table 5. Comparison of MIC values for barley polycation with the highest degree of cationization (JG100) and antifungal drugs.

[0040] TER - terbinafine, CPX - ciclopirox, NAT - natamycin, POZA - posaconazole, IZA - isavuconazole, VCZ - voriconazole, ITR - itraconazole, AMB - amphotericin B, nt - not tested

[0041] Example 4. Antifungal activity of modified beta-glucan as an antifungal agent for plant protection The experiments were conducted under field conditions on three winter cereal species: barley, triticale, and wheat. The studies were carried out in accordance with the methodology of the European and Mediterranean Plant Protection Organization (EPPO). The combat effectiveness was calculated using the Abbott formula - it is a standard mathematical method used in agriculture and phytopathology for objective assessment of the actual effectiveness of plant protection products according to the formula: Abbott's formula (% infection on the protected object - % infection on the control object) / 100 - % infection on the control object = Y x 100). The oat beta-glucan used according to the invention showed antifungal effectiveness of not less than 30%, as shown in Tables 6-8. Considering other beta-glucans, i.e., the ones derived from barley and rye, and their similarities to oat beta-glucan, it may be expected that they will also be effective as antifungal agents in plant protection. Table 6. Control and effectiveness of pathogen combating in winter triticale crops - field experiments.

[0042] Table 7. Control and effectiveness of pathogen combating in winter barley crops - field experiments Table 8. Effectiveness of combating Blumeria graminis in winter wheat crop - field experiments

Claims

Claims1. A cationically modified beta-glucan obtained from a cereal selected from the group comprising barley, oats, or rye for use in inhibiting or preventing the growth of fungi in a mammalian patient, especially a human, wherein the beta-glucan is modified with glycidyltrimethylammonium chloride (GTMAC), wherein the degree of cationic modification, defined as the number of GTMAC molecules attached per 100 polymer mers, ranges from 70% to 100%.

2. Cationically modified cereal beta-glucan according to claim 1 or 2, characterised in that the degree of modification of barley beta-glucan ranges from 71% to 100%, most preferably 100%.

3. Cationically modified cereal beta-glucan according to claim 1 or 2, characterised in that the degree of modification of oat beta-glucan is 87%.

4. Cationically modified cereal beta-glucan according to claim 1 or 2, characterised in that the degree of modification of rye beta-glucan is 100%.

5. Cationically modified cereal beta-glucan according to any of the previous claims, characterised in that the beta-glucan is obtained from wholemeal flour.

6. Cationically modified cereal beta-glucan according to claim 1, 2, 3 or 4, characterised in that the cationically modified beta-glucan has a zeta potential of not less than 42 mV, preferably from 42 mV to 48 mV.

7. Cationically modified cereal beta-glucan according to claim 1, characterised in that the fungus is selected from the Candida genus, preferably Candida tropicalis or Candida parapsilosis, the Cryptococcus genus, preferably Cryptococcus neoformans, the Malassezia genus, preferably Malassezia furfur, Malassezia slooffiae, Malassezia japonica or Malassezia pachydermatis, the Fusarium genus, preferably Fusarium graminearum, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani or Fusarium verticillioides, Scopulariopsis brevicaulis, Scedosporium apiospermum, the Lomentospora genus, preferably Lomentospora prolificans.

8. A use of cationically modified beta-glucan specified in claim 1 as an antifungal agent for plant protection, an additive to cosmetics, preferably as a preservative, or a dietary supplement.

9. The use according to claim 8, characterized in that the cereal beta-glucan is selected from the group comprising barley beta-glucan, oat beta-glucan, or rye beta-glucan.

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